Response of the Nervous System to Ionizing Radiation Response of the Nervous System to Ionizing Radiation Proceedings of an International Symposium held at Northwestern University Medical School Chicago, Illinois, September 7-9, 1960 Edited by THOMAS J. HALEY Laboratory of Nuclear Medicine and Radiation Biology School of Medicine University of California Medical Center Los Angeles, California RAY S. SNIDER Northwestern University Medical School Chicago, Illinois Assistant Editor SHIRLEY MOTTER LINDE 1962 ACADEIVIIC PRESS - New York and London Copyright © 1962, by Academic Press Inc. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS. ACADEMIC PRESS INC. Ill Fifth Avenue New York 3, N. Y. United Kingdom Edition Published by ACADEMIC PRESS INC. (London) Ltd. Berkeley Square House Berkeley Square, London W. 1 Library of Congress Catalog Card Number 61-18880 s ^ printed in the united STATES OF AMERICA CONTRiBUTORS Bengt Andersson, Gustaf Werner Institute for Nuclear Chemistry and the Institute of Anatomy, University of Uppsala, Uppsala, Sweden William J. Arnold, University of Nebraska, Lincoln, Nebraska George Austin, University of Oregon Medical School, Portland, Oregon C. S. Bachofer, University of Notre Dame, Notre Dame, Indiana Orville T. Bailey, University of Illinois College of Medicine, Chicago, Illinois Charles P. Baker, Brookhaven National Laboratory, Upton, New York John S. Barlow, Massachusetts General Hospital, Boston, Massachusetts Albert Behar, Armed Forces Institute of Pathology, Washington, D. C. Leslie R. Bennett, University of California, Los Angeles Medical Center, Los Angeles, California A. Breit, Deutsche Forschungsanstalt fiir Psychiatric, Max-Planck-Institut, Munich, Germany K. R. Brizzee, University of Utah College of Medicine, Salt Lake City, Utah Daniel G. Brown, University of Tennessee — Atomic Energy Commission, Agricultural Research Laboratory, Oak Ridge, Tennessee VV. Lynn Brown, University of Texas, Austin, Texas Robert H. Brownson, Medical College of Virginia, Richmond, Virginia Tor Brustad, Norwegian Radium Hospital, Oslo, Norway W. G. Calvo, Brookhaven National Laboratory, Upton, New York Berry Campbell, College of Medical Evangelists, Los Angeles, California A. Carsten, Brookhaven National Laboratory, Upton, New York Carmine D. Clemente, University of California, Los Angeles Medical Center, Los Angeles, California Howard J. Curtis, Brookhaven National Laboratory, U pton. New York Roger T. Davis, University of South Dakota, Vermillion, South Dakota Donald Duncan, University of Texas Medical Branch, Galveston, Texas L. E. Farr, Brookhaven National Laboratory, Upton, New York C. T. Gaffey, Donner Laboratory, University of California, Berkeley, California Edgar L. Gasteiger, University of Rochester, School of Medicine and Den- tistry, Rochester, New York N. I. Gr,\shchenkov, Institute of the Higher Nervous Activity, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R. H. Hager, Deutsche Forschungsanstalt fiir Psychiatric, Max-Planck-Institut, Germany Harry F. Harlow, University of Wisconsin, Madison, Wisconsin W. Haymaker, Brookhaven National Laboratory, Upton, New York vi CONTRIBUTORS Julian Henry, Donner Laboratory, University of California, Berkeley, California Paul S. Henshaw, Division of Biology and Medicine, Atomic Energy Com- mission, Washington, D. C. Samuel P. Hicks, Harvard Medical School and New England Deaconess Hospital, Boston, Massachusetts Wolfgang Hirschberger, German Research Institute of Psychiatry, Max- Planck-Institute, Munich, Germany J. R. M. Innes, Brookhaven National Laboratory, Upton, New York Walter Isaac, University of Washington School of Medicine, Seattle, Washington* L. A. Jacobs, University of Utah College of Medicine, Salt Lake City, Utah Peter Janssen, Donner Laboratory, University of California, Berkeley, California and Armed Forces Institute of Pathology, Washington, D. C.f Larry P. Jones, University of Tennessee — Atomic Energy Commission, Agricultural Research Laboratory, Oak Ridge, Tennessee Sylvan J. Kaplan, Texas Technological College, Lubbock, Texas X. Kharetchko, University of Utah College of Medicine, Salt Lake City, Utah Donald J. Kimeldorf, U. S. Naval Radiological Defense Laboratory, San Francisco, California Igor Klatzo, National Institute of Neurological Diseases and Blindness, National Institutes of Health, Bethesda, Maryland Ruth Kleinfeld, Ohio State University, Columbus, Ohio Harold Koenig, Veterans Administration Research Hospital and North- western University Medical School, Chicago, Illinois Lawrence Kruger, University of California Medical Center, Los Angeles, California P. Krupp, University of Basel, Basel, Switzerland BoRjE Larsson, Gustaf Werner Institute for Nuclear Chemistry and the Institute of Anatomy, University of Uppsala, Sweden Robert W. Leary, University of Washington School of Medicine, Seattle, Washington^ Lars Leksell, Gustaf Werner Institute for Nuclear Chemistry and the Institute of Anatomy, University of Uppsala, Sweden Billey Levinson, University of Buffalo, Buffalo, New York Leo E. Lipetz, Institute for Research in Vision, Ohio State University, Columbus, Ohio S. W. LipPiNCOTT, Brookhaven National Laboratory, Upton, New York * Present address: Emory University, Atlanta, Georgia t Present address: Institut Neurologique Beige, Brussels, Belgium Present address: University of Oregon, Eugene, Oregon CONTRIBUTORS vii John Lvman, Donncr Laboratory, University of California, Berkeley, California Arnold A. McDowell, University of Texas, Austin, Texas William Mair, Gustaf Werner Institute for Nuclear Chemistry and the Institute of Anatomy, University of Uppsala, Uppsala, Sweden Leonard I. Malis, Mt. Sinai Hospital, New York, New York Dorothea Starbuck Miller, University of Chicago, Chicago, Illinois Jaime Miquel, National histitute of Neurological Diseases and Blindness, National Institutes of Health, Bethesda, Maryland Marcel Monnier, University of Basel, Basel, Switzerland Werner K. Noell, Roswell Park Memorial Institute, Buffalo, New York* Nancy Ragan, University of California School of Medicine, Los Angeles, California Bror Rexed, Gustaf Werner Institute for Nuclear Chemistry and the In- stitute of Anatomy, University of Uppsala, Uppsala, Sweden Harold E. Richardson, Jr., Donner Laboratory, University of California, Berkeley, California Arthur J. Riopelle, Yerkes Laboratories of Primate Biology, Orange Park, Florida Jerzy E. Rose, The University of Wisconsin, Madison, Wisconsin T. G. Ruch, University of Washington School of Medicine, Seattle, Washington Roberts Rugh, Radiological Research Laboratory, Columbia University, New York, New York Daniel P. Sasmore, University of Tennessee — Atomic Energy Commission, Agricultural Research Laboratory, Oak Ridge, Tennessee Makoto Sato, University of Oregon Medical School, Portland, Oregon Mary Elmore Sauer, University of Texas Medical Branch, Galveston, Texas Dante G. Scarpelli, Department of Pathology, The Ohio State University, Columbus, Ohio O. A. Schjeide, University of California School of Medicine, Los Angeles, California Wolfgang Schlote, German Research Institute of Psychiatry, Max- Planck-Institute , Munich, Germany WiLLiBALD ScHOLZ, German Research Institute of Psychiatry, Max-Planck- Institute, Munich, Germany Norbert Schummelfeder, histitute of Pathology, University of Bonn, Germany J. G. Sharp, University of Utah College of Medicine, Salt Lake City, Utah •Present address: University of Buffalo Medical School, Buffalo, New York viii CONTRIBUTORS Sue Simons, University of California School of Medicine, Los Angeles, California Patrick Sourander, Gustaf Werner Institute for Nuclear Chemistry and the Institute of Anatomy, University of Uppsala, Uppsala, Sweden Walter R. Stahl, Oregon State College, Corvallis, Oregon and University of Oregon Medical School, Portland, Oregon E. E. StickleYj Brookhaven National Laboratory, Upton, New York Cornelius A. Tobias, Donner Laboratory, University of California, Berke- ley, California D. C. Van Dyke, Donner Laboratory, University of California, Berkeley, California F. Stephen Vogel, New York Hospital, Cornell University Medical Cen- ter, New York, New York Y. L. Yamamoto, Brookhaven National Laboratory, Upton, New York James N. Yamazaki, University of California, Los Angeles Medical Center, Los Angeles, California Wolfgang Zeman, Indiana University Medical School, Indianapolis, Indiana FOREWORD The assembly of groups of people of divergent views for the purpose of educating each other is a goal which is not too often attained. However, these directions were the ones given to the Symposium and program chair- men by the committee that had decided it was time to look into effects of ionizing radiations on the nervous system. This was a new approach, because many had said that the nervous system was insensitive to radiation, but the undercurrent arriving from many laboratories indicated that the statement was only partly true. If we wished to understand the radiation syndrome itself it would be necessary to consider the ner\ous system in our o\er-all outlook. Upon this basis it was decided that a beginning should be made by reviewing neonatal aspects, histopathological effects, ablation of specific central nervous system areas by particulate irradiation, ev^aluation of functional changes, and last but not least, the psychological effects of irradiation on animal performance. To further these goals, investigators from many parts of the world joined with their colleagues in the United States to present the material contained in the following pages. We do not believe that all available information on the subject is contained herein, but we have strived honestly for a beginning in order that investigators will not only know what has been done and is being done, but also what the future may cause to have done. This symposium was made possible by research grants to Northwestern University from the Institute of Neurology and Blindness, National Insti- tutes of Health, Bethesda, Maryland, and the U. S. Atomic Energy Com- mission, Washington, D. C. Special thanks go to the Neurology Study Sec- tion of National Institutes of Health for the necessary services it rendered. We hope that this modest beginning will inspire others to assist all to a better understanding of the various effects produced by irradiation of the nervous system. Thomas J. Haley Program Chairman Ray S. Snider Symposium Chairman PREFACE This is a meeting of two groups of minds, the basic neurologists and the basic radiobiologists. It is the first meeting of its kind and, if successful, we hope there will be subsequent meetings. It should not be necessary to point out that this select group has a double responsibility, that of pointing up not only what we know, but, equally important, what we don't know about radiation effects on the nervous system. This is a long neglected field, and we are all students with much to learn. On some of the points there is enough information for general agreement; on other points, general agree- ment is impossible. Perhaps the frustrated feelings will be so annoying that you will go back to your laboratories, design better experiments, and come to the next svTnposium with even better scientific papers. The orientation of this meeting is the result of months of planning. There are five major topics of discussion. Each topic is being handled by a chairman, who is a specialist in the field, and is being introduced by a general survey speaker, who will cover much of the literature. The scientific papers are followed by discussion of the subject, which then is summarized by the individual chairman. The physical aspects of radiation and clinical studies will be discussed in a subsequent meeting. Our present task is a noble one, i.e., a mental cross-pollination of neurologists and radiobiologists interested in basic mechanisms. So without further comment, I w-elcome all of you and now ask you to capture your protons, your electrons, dendrons, axons, and neurons and orbit into new frontiers of learning. Ray S. Snider Symposium Chairman CONTENTS Contributors v Foreword ix Preface xi \ Part I. Effects of Ionizing Radiation on the Developing Nervous System Introduction to Part I By Samuel P. Hicks 1 Major Radiobiolotjical Concepts and Effects of Ionizing Radiations on the Embryo and Fetus By Roberts Rugh 3 Quantitative Histologic and Behavioral Studies on Effects of Fetal X-Irradiation in Developing Cerebral Cortex of White Rat By K. R. Brizzee, L. A. Jacobs, X. Kharetchko, and J. C. Sharp 27 Structural and Behavioral Alteration in the Rat Following Cumula- tive Exposure of the Central Nervous System to X-Irradiation By Robert H. Brownson 41 Behavioral and Histologic Effects of Head Irradiation in Newborn Rats By James N. Yamazaki, Leslie R. Bennett, and Car- mine D. Clemente 59 Cytoplasmic Inclusions Containing Deoxyribonucleic Acid in the Neural Tube of Chick Embryos Exposed to Ionizing Radiation By Mary Elmore Sauer and Donald Duncan 75 Biochemical Effects of Irradiation in the Brain of the Neonatal Rat By O. A. Schjeide, J. N. Yamazaki, C. D. Clemente, Nancy Ragan, and Sue Simons 95 Some Effects of Nucleic Acid Antimetabolites on the Central Nerv- ous System of the Cat By Harold Koenig 109 Geographic Distribution of Multiple Sclerosis in Relation to Geomagnetic Latitude and Cosmic Rays By John S. Barlow 123 xiii xiv CONTENTS General Discussion 151 Summation by Chairman By Samuel P. Hicks 157 Part II. Histopathological Changes Resulting from the Irradiation of the Nervous System Basic Problems in the Histopathology of Radiation of the Central Nervous System By Orville T. Bailey 165 Sequence of X-Radiation Damage in Mouse Cerebellum By NoRBERT Schummelfeder 191 Morphological EfTect of Repeated Low Dosage and Single High Dosage AppHcation of X-Irradiation to the Central Nerv- ous System By Willibald Scholtz, Wolfgang Schlote, and Wolf- gang HiRSCHBERGER 211 A Demyelinating or Malacic Myelopathy and Myodegeneration — Delayed Effect of Localized X-LTadiation in Experimental Rats and Monkeys By J. R. M. Innes and A. Carsten 233 EflFects of High-Dose Gamma Radiation on the Brain and on Individual Neurons By F. Stephen Vogel 249 Electron Microscope Observations on the X-Irradiated Central Nervous System of the Syrian Hamster By H. Hager, W. Hirschberger, and A. Breit 261 Bioelectric EfTects of High Energy Irradiation on Nerve By C. T. Gaffey 277 Morphologic and Pathophysiologic Signs of the Response of the Nervous System to Ionizing Radiation By N. I. Grashchenkov : 297 General Discussion 315 Chairman's Summation By Webb Haymaker 321 Part III. Particle Irradiation of the Central Nervous System The Use of Accelerated Heavy Particles for Production of Radiole- sions and Stimulation in the Central Nervous System By Cornelius A. Tobias 325 CONTENTS XV Effect of Local Irradiation of the Central Nervous System with High Energy Protons By Bengt Andersson, Borje Larsson, Lars Leksell, William Mair, Bror Rexed, and Patrick Sourander 345 Production of Laminar Lesions in the Cerebral Cortex by Deuteron Irradiation By Leonard I. Malis, Jerzv E. Rose, Lawrence Kru- GER, and Charles P. Baker 359 Fluorescein as a Sensitive, Semiquantitative Indicator of Injury Following Alpha Particle Irradiation of the Brain By D. C. Van Dyke, P. Janssen, and C. A. Tobias 369 Pathologic Changes in the Brain from Exposure to Alpha Particles from a 60 Inch Cyclotron By Peter Janssen, Igor Klatzo, Jaime Miquel, Tor Brustad. Albert Behar, Webb Haymaker, John Lyman, Julian Henry, and Cornelius Tobias 383 Some Observations of Radiation Effects on the Blood-Brain Barrier and Cerebral Blood Vessels By Carmine D. Clemente and Harold E. Richardson, Jr 411 Chemical and Enzymatic Changes in Nerve Cells Irradiated with High Energy Deuteron Microbeams By Wolfgang Zeman, Howard J. Curtis, Dante G. Scarpelli, and Ruth Kleinfeld 429 Tolerance of Central Nervous System Structures in Man to TheiTnal Neutrons By L. E. Farr, W. G. Calvo, Y. L. Yamamoto, E. E. Stickley, W. Haymaker, and S. W. Lippincott 441 General Discussion 459 Review of Neurophysiologic and Psychologic Research on Irradia- tion Injury in the U.S.S.R. By Walter R. Stahl 469 Part IV. Functional Changes in the Nervous System Resulting from Radiation Exposure General Survey — Functional Changes in the Nervous System In- duced by Ionizing Radiations By Paul S. Henshaw 489 Acute Central Nervous System Syndrome of Burros By Daniel G. Brown, Daniel P. Sasmore, and Larry R Jones 503 xvi CONTENTS Effects of Low Level Radiation on Audiogenic Convulsive Seizures in Mice By Dorothea Starbuck Miller 5L3 Effects of Ionizing Radiation on Visual Function By Leo E. Lipetz 533 X-Irradiation Studies on the Mammalian Retina By Werner K. Noell 543 The Effects of Ionizing Radiation on Spinal Cord Neurons By Makoto Sato, George Austin, and Walter Stahl 561 Radiation Effects on Bioelectric Activity of Nerves By C. S. Bachofer 573 Alteration of Mammalian Nerve Compound Action Potentials by Beta Irradiation By Edgar L. Gasteiger and Berry Campbell 597 Action of Gamma Radiation on Electrical Brain Activity By Marcel Monnier and P. Krupp 607 General Discussion 621 Part V. Psychological Effects of Ionizing Radiation Effects of Radiation on the Central Nervous System and on Be- havior — General Survey By Harry F. Harlow 627 Learning Behavior of Rats Given Low Level X-Irradiation in Utcro on Various Gestation Days By Sylvan J. Kaplan 645 Effects of Neonatal Irradiation on Learning in Rats By BiLLEY Levinson 659 Behavioral Effects of Cranial Irradiation of Rats By William J. Arnold 669 Radiation-Conditioned Behavior By Donald J. Kimeldorf 683 Behavioral and Correlated Hematologic Effects of Sublethal Whole Body Irradiation By T. C. RucH, Walter Isaac, and Robert W. Leary 691 Performance of Monkeys before and after Irradiation to the Head with X-Rays By Roger T. Davis and Arnold A. McDowell 705 Some Behavioral Effects of Ionizing Radiation on Primates By Arthur J. Riopelle 719 CONTENTS xvii Some Effects of Radiation on Psychologic Processes in Rhesus Monkeys By W. Lynn Brown and Arnold A. McDowell 729 General Discussion 747 Author Index 753 Subject Index 767 PART I Effects of Ionizing Radiation on the Developing Nervous System INTRODUCTION TO PART I The past 10 years and especially the last few have seen a great increase in interest in what ionizing radiation may do to the nervous system, both in respect to the malforming effects induced during early development and the functional and structural changes occurring in later stages. Formerly, it was said — and sometimes still is — that the embryo brain is very radio- sensitive and the adult brain will stand almost anything. Unqualified, these phrases are almost meaningless today. We try to specify what dose of radia- tion produces a given effect, because one type of cell in the adult or embryonic nervous system may respond quite differently from another cell. It makes a difference whether one gives 200 r of conventional 250 kv x-rays to a fetal or neonatal rat in divided doses or in a single dose, or whether one gives a single dose of 700 r. E\en the difference in effects between 200 r and 300 r on the fetal and neonatal cortex, cerebellum, and retina can sometimes be remarkable. The term radiosensitivity now is used more carefully, because it has meant quite different things to different workers. We can no longer say, "this stage of embryonic life is the most radiosensitive" or "that enzyme system is the most radiosensitive" without qualification. To a geneticist, radiosensitivity means that a chromosome can be changed easily; to a pathologist, it has often meant that a tumor cell is easily killed; and to an endocrinologist, it may suggest that a cells hormone production can be easily stopped. Many laboratories, including our own, are interested in dis- co\ering subtle radiation changes in neurons. For example, we are attempt- ing to demonstrate changes in nucleic acids in adult rat cortical neurons by ultraviolet microscopy following exposure to 200 or 400 r of conven- tional 250-kv x-rays. I note this because it reflects the growing attitude that we ought to be looking for obscure and perhaps totally unexpected changes in the nervous system and other tissues following radiation. Certainly, among the most attractive areas for research are those which relate to the roles that DNA and RNA have in both the development and, later, the function of neurons. A diverse array of approaches to problems of developmental radio- biology is represented in these papers on the effects of ionizing on the developing nervous system, including effects of successive small doses of radiation on neuron differentiation, chemical and histochemical changes in irradiated nerve cells of all ages, studies on nucleic acids of ner\e cells 2 INTRODUCTION TO PART I by the use of nucleic acid antimetabolites, and considerations of disturb- ances of function that radiation may cause. Development is not restricted to embryonic life, but continues through the life of the organism, and we will hear not only what radiation may do long before the embryo has a brain, but also what man-made radiation and radiation from outer space may be doing during adult life. Samuel P. Hicks Major Radiobiological Concepts and Effects of Ionizing Radiations on the Embryo and Fetus Roberts Rugh Radiological Research Laboratory, Columbia University, New York, New York Introduction As a radiobiologist, it is appropriate to initiate this symposium witti a few general statements regarding the biological eflfects of ionizing radiations. We are concerned with ionizing radiations, not ultraviolet or infrared radiations. Ionizing radiations result in the excitation or loss of an electron from an atom, causing an unstable situation, whether it be in an atom of an inani- mate or animate object. Such imbalance can be brought about by alpha or beta particles, by x-rays or gamma rays, or secondarily by neutrons. The ensuing physical imbalance results in a chemical change which, in turn, may effect a demonstrable biologic adjustment. While the ionization may be immediate or instantaneous, and is in most cases undetectable by the nervous system, the evidence of the biologic adjustment may take decades. Radiations are so potent that the ionization of one molecule in 10,000,000 is sufficient to kill almost all organisms. It matters not whether the ionization is brought about by any of the different qualities of radiation, the basic biologic reactions are the same. However, the total dose absorbed and the dose rate are indeed important. There is a total absorbed dose which cannot be sui-\'ived, and there is a dose rate so small that it can be tolerated. In all properly controlled radiobiologic experiments the ion density, the dose rate, and the total absorbed dose must be determined and recorded. In reporting radiobiologic experiments it should be made clear what specific organism, organ, tissue, or even cells are concerned, because tolerance is not uniform. This does not contradict the statement that the biologic reactions to ionizing radiations are basically the same. It does emphasize the fact that tolerance, restitution, and repair are properties which vary with the large variety of differentiated tissues. Organisms vary during their lifetime in their reaction to absorbed ionizing radiations, even as do cells during their process of dif- 4 ROBERTS RUGH ferentiation. The radiosensitivity of a single cell may vary 1,000 times from its undiflferentiated to its differentiated state. The time has long since passed when data were adequately presented in terms of milligram hours of radium without consideration of the actual absorbed dose (rads), as well as the organ, tissue, or cell exposed. There is a confusion of teiTns, such as threshold, safe, permissible, or toler- able doses of ionizing radiations. If by threshold one means an exposure below which nothing happens, it is very doubtful that such a level exists. A single ionization occurring at a critical point on a chromosome may not affect its bearer, but may have permanent and drastic effects on its progeny, which explains the extreme caution of the geneticists. At the level of the atom, any effect brought about by ionizations is probably all-or-none. It is another matter whether such an effect is detectable and, if detectable, whether it is tolerable to the biologic system. Certainly some changes in the central nervous system of the embryo or of the adult are both detectable and tolerable, if by tolerable we mean that the individual is able to survive. At the cellular level the changes brought about by the absorption of ionizing radiations are irreversible, irrevocable, and irreparable, but they may still be tolerated by the cell. If the cell survives and is a germ cell, it may contribute a new mutant to its succeeding generations of cells, both detectable and tolerable, but not likely of any benefit. If the cell survives as a somatic cell, it may tolerate the damage and reproduce by mitosis for many years, ultimately to blossom out in two or three decades as a center of a malignancy. The ability to tolerate absorbed radiations is good for sur- vival, but possibly not good for progenies. The term permissible dose is used largely in Civil Defense directives and radiologic centers, and its dose level is generally somewhat lower than the tolerable. There is still another aspect of the tolerable dose, and this relates to the ability of the adult to repair or regenerate replacements for damaged tissue, usually scar tissue, or of the embi-yo to redirect its schedule and pattern of differentiation. The words have a wider meaning, therefore, and include reparative processes above the cellular level. A certain amount of radiation may be tolerated by an organism if the remaining tissue is sufficient for survival and can replace the void with protoplasmic mass. This ability is more evident in the embi-yo where undifferentiated cells are being directed and redirected after irradiation toward differentiation along the various routes which result in recognized tissues. Once this is completed, as in the adult, replacement of kind is not usual. A corollary of this discussion is the matter of cumulative effects. Certainly one can see how a primitive germ cell, successively exposed to ionizing radia- tions, might well accumulate mutants at various points along its chromo- somes. In the same way, a somatic cell may be bombarded successively by IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 5 ionizino; radiations, and. as Ions; as it survives, it too should accumulate effects until an intolerable composite of the damasje would result in its death. In the somatic cell, exposure effects short of lethality may not be so s^raphi- cally demonstrable as those produced by a mutated oerm cell. Such a change in the somatic cell might never be detected. There is as yet no direct proof, but it may be conjectured that squamous cell carcinoma of the skin is more likely to follow repeated, accumulated, but tolerable exposures, than a single exposure. This presumption is based on the knowledge that squamous cell carcinoma, which can be produced by ionizing radiations, can result from tolerated exposures. The human embryo or fetus, and to some extent the adult, have powers of sloughing off the undesirable or dead cells so that the only place that cumula- tive effects can be detected is in the progeny of surviving somatic and germ cells. This is why the low and tolerable exposures are so important. To kill is clear cut. To maim for the duration of life may be biologically tolerable, but psychologically and sociologically intolerable. Finally, one must emphasize the difference between the somatic and the genetic effects. Since ionizing radiations can alter the central nervous system either through the germ cells or through direct irradiation, we are concerned with both genetic and somatic efTects. Neither is apt to be immediate; both can be subtle and long delayed. When the embryo or fetus is inadiated it must be realized that both the de\eloping central nervous system and the developing gonads of the organism may have been exposed. Congenital effects resulting from direct irradiation of the de\eloping organ primordia cannot have genetic corollaries except by coincidence, which is \ery unlikely. Congenital effects following direct irradiation cannot be inherited. However, concomitant with somatic exposure there may be germ cell exposure which may well result in different and possibly e\en more severe effects, but ones which cannot become apparent until they appear in a succeeding generation. Somatic exposures alone may alter the soma of one generation, but germ cell exposures may alter all suc- ceeding progenies. Effects on the Embryo or Fetus In analyzing the embryonic effects one must have in mind lour special situations: 1. The medium of the embryo is acjuatic. and there is reason to believe that this enhances its radiosensiti\ity. 2. The embryo is a mosaic of acti\ely differentiating centers with con- stantly changing but high mitotic indexes, both conditions enhancing radio- sensitivity. ROBERTS RUGH ¥■ ^ CVJ t'M \ jail IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS F (T> Hit LctL '^BHUHKlUi^ CM 8 ROBERTS RUGH 3. The embryo can be killed by irradiations which for any species are less than the lethal dose for the adult, and consjenital effects may be produced by exposures of about 2^,r that of the LD/50/30 level (Figs. 1-27). 4. If the embryo survives the irradiation, it has powers of topographic repair which are not known to the adult. But it cannot step up cell produc- tion to replace cells lost trom radiation necrosis, so that the net result is a deficient embryo or fetus — deficient in those cells or those tissues which are most damaged at the time of irradiation (Figs. 28-35). Every one of its systems may be affected by exposure of the embryo or fetus to ionizing radiations, but the most obvious effects appear to be on the central nervous and the skeletal systems. Probably the most common and graphic effect, one most frequently reported from Hiroshima and Nagasaki, as well as in experimental radiobiology, is microcephaly. This is not an isolated condition, and all those so affected undoubtedly exhibit other anomalies. There is often stunting, microphthalmia, and loss or reduction of other parts indicating deficits (Figs. 21-35). But anomalies designated as congenital may be caused by irradiation at times other than during differentiation. First, exposure of the sperm cell or its precursor may produce the anomaly in all succeeding generations due to chromosomal effects. Second, irradiation of the ovary may cause the anomaly to appear in successive generations. Third, the embryo is most likely to de- velop specific anomalies if the differentiating organ concerned is irradiated directly. Fourth, the embryo at any time from the moment of fertilization of the egg through the completion of organogenesis may be caused to develop the same type of anomaly. Once organogenesis is completed, congenital anomalies can no longer be caused by irradiation (Figs. 1-12 and 17-20). Thus, congenital anomalies involving the central nervous system may be caused by irradiation of either germ cell, of the actively differentiating organism, or of any stage prior to this. In our studies we have concentrated on the cerebral hernia or exen- cephalous condition where the midbrain protrudes through the cranial roof. This is a graphic and readily observable maldevelopment, and any fetus exhibiting this condition is presumed also to have other, possibly less graphic, but even more serious effects. Since this anomaly is readily observable, it has been a convenient marker of severe irradiation damage to the developing embryo (Fig. 21 and Table I). Exencephalia has been produced by the irradiation of the mouse testis or ovaiy and has appeared in successive generations following a single exposure. True, its frequency is very low, but it is a severe and lethal anomaly which can be genetically produced. It has also been produced by exposing the mouse embryo at any time from fertilization through gestation day 8.5, and in the earlier stages with doses of as little as 15 r. Exposures of 5 r at certain IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 9 periods have increased the intrauterine mortality by lO'^r, so that in the early embryo we are probably dealing with the most radiosensitive stage in ontogeny. No exencephalies ha\e appeared among thousands of unirradiated control embryos or in those irradiated after completion of organogenesis. It has been produced, however, by other traumatic conditions (Figs. 19, 20. 22.24). The term low dose should be defined here. Green ( 1959), the geneticist, says: "There is no totally sate dose of radiation," so that to him there is no TABLE I Malformations .Among Normal Offspri.ng of CFi X CFi Mice ' Offspring Xumber Per cent Pregnancies 61 Total embryos 630 Normal embryos 591 94 Dead embrvos 2 0.3 Resorptions 37 5.7 Exenccphal les * This table shows that among 63(1 unirradiated CFi mouse embiyo>, not a single exencephaly (brain hernial developed. However, note that there were almost 6% resorptions and two dead embryos. This is an expected ratio and may be due to genetic causes. \t no time in our experience, while examining thousands of mouse embryos. ha\e we found the congenital anomaly of e.xencephaly among the control mice. Figs. 1-12 are on pages 6 and 7. Figs. 1 and 2. These are normal mouse eggs seen during the first 24 hours after conception. Fig. 1 shows the egg at the moment of sperm entrance and Fig. 2 shows the two pro-nuclei and the first polar body. Highest percentage of resorption follows irradiation at this stage. Fig. 3. This shows a group of normal mouse embryos at 1.5 days in the 2-cell stage. This is probably the most radiosensiti\e period with respect to the production of irradiation congenital anomalies. Fig. 4. This is a mouse egg at 1.5 days which had recei\ed 50 r at 0.5 days and shows a hyperchromatic nucleus. Figs. 5 to 8. These show various stages in the disintegration of the mouse embryos following exposure to 15 r at 1.5 days. Note that in some cases the pro-nuclei are being extruded from the cytoplasmic mass. It is unlikely that the residual cellular material could survive. Howc\er. the majority of eggs exposed at this time and to this level of irradiation would survive. Figs. 9 to 12. These are all irradiated embryos exposed to 15 r at 1.5 days and examined at 2.5 days. Note pyknosis, hyperchromatism, and fragmentation of the embryos. 10 ROBERTS RUGH IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 11 HP « 12 ROBERTS RUGH such thing as a low dose, every exposure is too high. In our times this may be impractical and unrealistic, though still a concept to be respected. In Copenhagen, Dr. Hammer- Jacobson (1959) states: "Fetal doses of less than about 1 r are presumed to cause no noticeable injury. . . . Fetal doses between 1 r and about 10 r are assumed in some instances to cause injuries in the form of diseases, malformations, slow development, or reduced resistance, especially when the irradiation occurs between the 2nd and the 6th week. ... If there are additional indications, therapeutic abortion should be assumed advisable. . . . Fetal doses above about 10 r are assumed to involve a rather great probability of fetal injury. In such cases induction of abortion should therefore be the general rule." (See Table II.) In all of this, reference is made to the somatic efTects, but certainly the geneticist would concur. A whole body exposure of as little as 12 r will cause some lymphopenia in the adult; and Brues (1959) refers to 25 r as a "low dose" for somatic effects, probably because there are always at least some hematologic changes. Thus, a dose of 5 r may not have measurable conse- quences for the somatic tissues of the adult, but would be seriously damaging to the embryo or to its progeny through effects on the gametes. The embryo or fetus is not simply a miniature of the adult and must be regarded as a dynamic, tirelessly changing mosaic of differentiating areas all integrated into an over-all pattern under organismic influences which appear themselves to be immune to ionizing radiations. As long as there are undam- aged building units for development which are adequate in number and basically intact, these influences will attempt to organize them into a topo- graphically normal, balanced embryo. But the undamaged cells cannot replace those that were killed by irradiation. Any stimulus to excess cell pro- duction is cancerogenic, so that the embryo may be topographically well Figs. 13-20 are on pages 10 and 11. Fig. 13. This shows 3 normal mouse blastulae suspended within the uterus at 3.5 days. Fig. 14. This shows the normal mouse embryos at the moment of implantation at 4.5 days. Figs. 15 and 16. Mouse embryos at 4.5 days (time of implantation), but following x-irradiation with 15 r at 1.5 days. They show pyknotic nuclei, discarded (necrotic) cells within the blastocoel, and failure at implantation. These embryos might survive to give rise to deficient fetuses. Fig. 17. This embryo was likewise exposed to 15 r at 1.5 days and exhibits a giant ceil with prominent chromosomes at 4.5 days. This is a common irradiation sequela. Fig. 18. This is a mouse embryo treated as that of Fig. 17, showing a prominent cell with vacuolization. This is a frequent irradiation consequence. Figs. 19 and 20. These are members of litters dissected at 18.5 days showing stunting, anencephaiy, and exencephaly, while other members of the litter appear superficially to be normal. When compared with controls they, too, are shown to be stunted. IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS TABLE II Anomalies Reported Following Himan Fetal X-Irradiation ^ 13 1. Microcephaly (most frequent) 2. Hydrocephalus 3. Poroncephaly 4. Mental deficiency 5. Mongolian 6. Idiocy 7. Head ossification defects 8. Skull malformations 9. Micromelia 10. Microphthalmus 11. Microcornea 12. Coloboma 13. Strabismus 14. Cataract 15. Chorioretinitis 16. Nystagmus 17. Stillbirth increase 18. Decrease live birth weight 19. Neonatal and infant death increase 20. Ear abnormalities 21. Spina bifida 22. Cleft palate 23. Deformed amis 24. Clubfeet 25. Hypophalagism 26. Syndactyly 27. Hypermetropia 28. Amelogenesis 29. Odontogenesis imperfecta 30. Gf-nital deformalities ■'' This table lists thirty coiigcnital anomalies found in humans following fetal .\-ii radiation. .Vote that the most frequent type of anomaly relates to the central nervous system. Most, if not all of these anomalies, have been produced in experimental animals by exposure during embryonic development. '' It must be remembered that the levels of irradiation which are hazardous for the embryo or fetus aie very much lower than those foi the somatic tissues of the adult organism. It is, therefore, obvious that extreme caution should be exerted where either the reproductive organs or the developing embryo might be involved. We do not yet know the extent or the duration of radiation effects on the fetus or the germ cells. TABLE III Effect of Low-Dose X-ravs on the Early MorsE Embryo ' '' ^ S o o 9.6 94.0 5.7 0.3 0.0 10.0 85.0 15.0 0.0 0.0 Controls 5 r at 1 .5 dav 630 80 » This table presents data following 5 r exposure of the mou.se embryo at !'/> days post conception. At this time, the mouse embryo is in the 2 cell stage. Eighty such embryos exposed to 5 r gave 15% resorptions which was almost a W7( increase over the expected 5.70^ of the controls. No exencephaly appeared. *> .\n inciease of ').W, in intrauterine deaths caused by 5 r exposure at 1-2 cell stage. 14 ROBERTS RUGH IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 15 16 ROBERTS RUGH TABLE IV X-Irradiation of the Early Mouse Embryo * ^ c< iii i^ 0.5 52 58 42 1.5 90 95 5 2.5 95 73 24 3 3.5 76 88 9 3 4.5 53 92 8 5.5 37 77 17 6 6.5 77 92 8 7.5 25 96 4 8.5 51 96 4 9.5 12 90 10 568 85.7 12.3 2.0 " This table gives data from an extensive study of the effect of 50 r x-iays on the mouse embryo at various days from 0.5 to 9.5. In any somatic study 50 r would be considered a low level exposure, but from this study, when such an exposure kills 42% of the embryos at 0.5 days and large percentages at 2.5, 5.5, and 9.5 days, it is obvious that 50 r to the early developing mouse embryo is a high level of exposure. Of course, exencephalia was produced, the largest per cent being at 5.5 days. Figs. 21-27 are on pages 14 and 15. Fig. 21. An enlarged \'iew of exencephaly (brain hernia) in the mouse. This is a protrusion of the mesencephalon through the cranial roof. Fig. 22. Three members constituting an entire littt r, all .showing severe exencephalic maldevelopment. This followed exposure of 50 r at 2.5 days. Fig. 23. Note the same group of 3 congenital anomalies in a field including a normal control mouse fetus of the same age. This demonstrates that in addition to congenital anomalies there is often a stunting of the irradiated embryos. Fig. 24. This shows an entire litter, as found in the bicornate uterus of the mouse at 18.5 days, following an exposure of 200 r at 8,5 days. Note that 5 of the 11 litter members exhibit exencephalia. Fig. 25. This shows 4 members of a litter exposed to 50 r at 3.5 days. These are to be compared with a single control above. Note not only congenital anomalies but stunting of every member. Figs. 26 and 27. These mice were exposed to 50 r fractionated to 25 r each at two times during embryonic development, one exposure occurring before implantation and the second after implantation but before the completion of neurogenesis. Note the bizarre form of the extruded mesencephalon. The 2 litter members appear to be normal but are stunted. IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 17 balanced, but at the same time may exhibit sross deficiencies. The brain may appear to be grossly normal, but when compared with the control brain may be seen to be microcephalous. Once the neuron is differentiated, it is then almost completely radioresistant, but neurosenesis is not completed by the time of birth. Thus, irradiation effects on the central ner\ous system extend from the serm cell throus^h the completion of neurogenesis of the next generation, at least. Put more succinctly, ionizing radiation should be respected by germ cells at all times and by all undifferentiated cells i Tables III and IV). In treatment of central nervous system malignancies, doses ot 10.000 r are sometimes accumulated. If killing a tumor by irradiation results in the saving of a life, it is certainly justified. If it results in the prolonging of a life with concomitant and permanent injury and possible germ cell exposiuc. the procedure may be questioned. When the indi\idual is beyond the reproduc- tive age, there is no place for this discussion. The emphasis here is on the germ cells which might be used, and on the embryo or fetus which should never be exposed if it can be avoided. Any exposure of the germ cells or early embno is undesirable. Gentry ct al. (1959i ha\"e found a correlation between the areas in New York State of high congenital malformations and geographic concentrations of natural materials of relatively high levels of radioactivity, such as igneous or black shale rock. These may include C'*, K^", Ra--''. Th" ■-', and U- •\ and their decay products. The a\erage exposure of indi\iduals was estimated at 2.1 to 3.2 r per 30 years. The highest record for any single town was 66.7 congenital malformations per 1,000 births, and in an area particularly high in natural radioacti\ity. This was considerably above the average in the "unlikely areas" of 12.9 per 1,000 live births. There were some towns with no anomalies in low radiation areas. A reduction in birth weights also showed a correlation with increasing radioacti\ity. Detailed maps of radioactive concentrations fitted perfectly those of higher incidence of congenital mal- formations. Of all malformations. 15''r involved the central ner\ous svstem. and of these 94.5''r caused death. While some may doubt the conclusions of this study, it cannot be ignored. A somewhat similar study has been made by Wesley i 1960) in which he. as a statistician, finds that "96'' r of all deaths due to congenital malforma- tions are caused by background radiation, and x-rays have caused a 6''r increase in congenital malformations in the United States in the last 30 years."" There was a low incidence of congenital anomalies in southeastern .Asia and a high one in northern Ireland, correlated with background con- centrations. There is no way of determining how manv of the 5.000.000 mentally retarded United States citizens are products of irradiation injury. Hicks ct al. i 1959) made the following statements, all of which emphasize 18 ROBERTS RUGH IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 19 20 ROBERTS RUGH that fetal irradiation of the rat results in a neurologically deficient embryo: "The cerebral hemispheres and diencephalon were a a^ood deal smaller than normal. . . . The neocortex was seriously deficient, and about half as thick as normal at the vertex to about 2^ normal thickness laterally. . . . Small pallium. . . . The anterior commissure was a little less compact. . . . The midbrain was smaller in total cross area than normal due to somewhat flattened superior colliculi. . . . The cerebellum was altosjether a little smaller than normal. . . . The lower medulla showed a slight reduction in total o\er-all size. . . . The lower brain stem and cerebellum were a little smaller than normal. . . . The cords were a little smaller in cross section than normal. . . . Most of the cells in the 13-day retina were killed by radiation." Anomalies mentioned in this excellent study included: "Radiation-killed cells in the periependymal primitive matrix threw the mitotic layer into rosettes which continued to proliferate brain, nonetheless. The result was an anomalous mass of ectopic cortex. . . . No corpus cal- losum. . . . Bizarre bundles of fibers. . . . Disorderly array of all sorts of cortical neurons. . . . The neurons were jumbled, scattered, and they were often upside down or pointed sideways." Figs. 28-35 are on pages 18 and 19. Figs. 28, 29, and 30. These represent members of litters from 3 successive genera- tions following a single exposure of the ovary of the mother of those in Fig. 28 to 100 r. It was whole-body e.xposure, but we have reason to believe that the somatic eflFects of this exposure had nothing to do with these congenital anomalies. The fact that a single exposure of the ovary caused this brain anomaly to appear in three suc- cessive generations is genetically significant, even though its incidence was very low. Fig. 31. These represent an entire litter from a grandfather who had received high- level exposure of his testis. The first generation appeared normal, were viable and fertile. This brain anomaly of exencephalia appeared in the next generation. One might expect it to appear in yet succeeding generations. Fig. 32. The four embryos to the right show the variety of anomalies which ap- peared in the second generation following testis exposure. All were stunted, some died as fetuses (late in development). The single member to the left is a control of the same age. Fig. 33. When mouse embryos are exposed after organogenesis to high but tolerable levels of irradiation, the eflFects are largely skeletal. Note particularly the variations in size within the single litter. One member of the litter is almost as large as the controls. The explanation of this is probably genetic. Fig. 34. This is one litter, all of whom were exposed at the same time to the same irradiation, but which exhibit a wide range of difference in size. Fig. 35. This shows photographs of Spalteholz's preparations of two mice at birth, the upper one being the control, the lower one x-irradiated at 13.5 days. Note that the irradiated embryo appears to be topograhically normal but obviously is very much stunted. The developmental processes have been able to reorganize the undamaged cells to provide an apparently normally proportioned but stunted mouse. IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 21 All of these anomalies could be attributed to deficiencies during develop- ment caused by ionizing radiations. Their explanations of these central nervous system malformations included the following direct statements: "There was a selective extirpation effect on certain primiti\e cells. . . . A patchy deficiency of cells. . . . Numerous dead cells spilled into the ventricles. . . . Virtually all of the primitive migratory cells in transit were killed. . . . The residual mitotic colony of lining cells was thrown into dis- order because their support, the matrix of radiosensiti\e cells forming much of the wall, was gone." The entire emphasis of this study seemed to be on the deficiencies follow- ing fetal irradiation. The appearance of rosettes has often been described in both the neural retina and in the de\eloping cortex following fetal irradiation. The presence of rosettes is proof that cells have been desegregated and that those still viable attempt neural organization. In the case of irradiation, the desegrega- tion is due to the killing of radiosensitive cells, which are then removed, leaving loosely scattered, but \iable cells. It has been shown recently (Mos- cona, 1960) that presumptive nerve, cartilage, and liver cells of mouse and chick embryos may be desegregated (disaggregated) by trypzinization and mixed together, only to reaggregate with respect to whether they were nervous, cartilage, or liver, and irrespective of whether they came from the mouse or the chick. In other words, presumptive ner\e cells show an aflfinity for eacli other, regardless of their genetic source. When they come together without sustentacular materials, they tend to form rosettes which are an expression of disorganization. The rosettes are therefore not a peculiarity of post irradiation, nor of the mouse or rat, but rather of neural disorganization. A single rosette has been formed of neuroblasts from both the mouse and the chick embiyos. These structures, usually temporary in the irradiated and developing embryo, simply represent a stage in the reorganization of viable nerve cells which are inadequate in number to accomplish structural normality (Figs. 36-43 and Table V). Our current studies are utilizing low doses to determine the effect of ionizing radiations on the developing central nervous system as demonstrated by beha\ ior, electroencephalographic records at \ arious stages of maturation, and electron microscope and neuropathologic studies of the postnatal brain. It may develop that it will be the experimental psychologist who will spot the specific developmental stages most drastically affected by ionizing radia- tions. If our society is primarily concerned with the function of the central nervous system, we may be dealing with radiation changes which are beyond analysis by the conventional neuropathologic techniques or by the electron microscope. We expect to have information on this during the next year. 22 ROBERTS RUGH IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 23 ♦ • / SVvC*r>^/2^, •- '9^-^- •p*ystii.^-^ 24 ROBERTS RUGH TABLE V Effect of Fetal X-Irradiation on Mouse Eyes ' (Measurements at 6 weeks of age) Average diameter (in mm) '' Relative volume (%) Controls 3.555 100 150 r at 12.5 days gestation 2.970 69.6 250 r at 12.5 days gestation 2.670 50.6 " X-irradiatioii f>f tlic developing mouse embryo seems to result in cellular deficiencies because tlic damaged cells aie removed. When these ii radiations occur early, before the development of a specific organ system, the deficiency resulting from the elimination of the necrotized cells results in a reduction of organ size. The data of Table IV show that uith increasing irradiation at 12.5 days, there is a decrease in the relative volume of the diameters of the mouse eyes at 6 weeks of age. An exposure of 250 r reduced the volume to approximately 50'J^. There are no studies thus far relative to the visual acuity of these eyes. '' Minimum of 8 diameters of fixed eyes taken foi each a\eiage. Summary The early embryo is more radiosensitive than is the organism at any other time in its entire life cycle. The earlier the stage, the more sensitive, with re- gard to both survival and the development of anomalies. At the cellular level, there is no such thing as "recovery" from irradiation damage, meaning a return to the preirradiated state. Since embryonic cells are precursors of all cells of the adult, irreparable damage to surviving cells results in such damage to all descendant cells of the adult organism. Ionizing radiations represent a very potent tool. Figs. 36-43 are on pages 22 and 23. Fig. 36. When mouse embryos at 6.5 days are exposed to x-rays, 24 hours thereafter they show the sloughing off of cells into the central cavity as seen here. The inner neurectoderm will be deficient to the extent of this cellular loss. Fig. 37. This shows the neural groove at the level of the brain of 8.5-day embryos 24 hours after exposure to x-rays. Note the many pyknotic nuclei and the sloughed off cells into the neural groove. Fig. 38. This is similar to Fig. 37 except it is at the level of somites. Fig. 39. In this figure note the many phagocytes posterior to the developing retina, each of which contains a number of necrotic neurectoderm cells. This occurs about 24 hours after x-irradiation, but the retina will be deficient to the extent of this cellular loss. Fig. 40. This is an enlarged view of the retina 4 hours after irradiation, showing many pykotic nuclei. Fig. 41. This is an enlarged view of single phagocyte containing 14 dead neurecto- derm cells from the x-irradiated retina. Figs. 42 and 43. These are enlarged views of the retina of the control Fig. 42 and the irradiated Fig. 43 to show slight thinning of the various layers in the x-irradiated eye of the mouse. IONIZING RADIATIONS: EFFECTS ON EMBRYO, FETUS 25 The embryo, in contrast with the aduh, has powers of reoroaniziny; its re- sidual and surviving cells so that topographic normality may be achieved. However, every such indi\idual will be deficient, either in parts or in the stunting of the whole. Deficiencies are seldom similar in litter mates, owing to the submicro- scopic nature of ionizing radiations, the genetic \ariations in individuals, the \arying abilities for restitution, and probably other factors. Irradiation of the embryo is the only way to produce irradiation congenital anomalies, but such anomalies may be produced by other traumatic means. Following organogenesis, irradiation efTects are similar to those one expects in the adult. The embryo after a certain stage possesses gonad primordia or developing gonads, and these are subject to irradiation effects which may not be evident for generations. Central nervous system anomalies may be produced by irradiation of the mature gamete of either sex, the fertilized egg, or any stage in development prior to completion ot neurogenesis. Some formati\e cells are present even after the birth of the mammal. The range of radiosensitivity of gamete to formed organism is such that discussion of threshold is meaningless. We can- not now state the extent or the duration of irradiation damage to the de- veloping central nervous system. There may well be subtle effects to be revealed by population studies o\er generations. Any exposure of the early embryo should be regarded as too much. Finally. I would like to make four specific requests: 1 . l^hat we insist on better and more adequate controls in radiobiology. 2. That radiation dosimetry in all radiobiologic experiments be checked by a qualified radiophysicist and be fully reported. 3. That there be a pool of research information on neurologic effects, in- cluding critically reviewed information fiom the U.S.S.R. because of the language barrier. 4. That symposia of this sort be organized as frequently as the accelerat- ing accumulation of data demands. References Brues, .\. M. (ed.) 1959. Low-k-\el irradiation. Publ. Am. A'isoc. Advance. Sci. 59. Gentry. J. T.. Parkhurst, E., and Bulin, G. V. 1959. .\n epidemiological study of con- genital malformations in New York State. Am. J. Public Health 49, 1-22. Green, E. L. 1959. Genetic efTects in low-lc\fl irradiation. Pub!. Am. A^wc. Advance. Sci. 59. Hammer-Jacobson. E. 1959. Therapeutic abortion on account of x-ray examination during pregnancy. Danish Med. Bull. 6, 113-121. 26 ROBERTS RUGH Hicks, S. P., DAmato. C. J., and Lowe. M. J. 1959. The development of the mam- malian nervous system. I. Malformations of the brain, especially the cerebral cortex, induced in rats by radiation. II. Some mechanisms of the malformations of the cortex. /. Comp. Neurol. 113, 435-469. Moscona, A. 1960. Private communication, in press. Rugh. R. 1959a. Vertebrate radiobiology (embryology). Ann. Rev. Nuclear Sci. 9. 493-522. Rugh, R. 1959. Ionizing radiations: Their possible relation to the etiology of some congenital anomalies and human disorders. Military Med. 124, 401-416. Rugh, R., and Grupp, E. 1959a. X-irradiation exencephaly. An^. J. Roentgenol., Ra- diurn Therapy Nuclear Med. 81, 1026-1052. Rugh, R., and Grupp, E. 1959b. Exencephalia following x-irradiation of the pre- implantation mammalian embryo. /. Neuropathol. Exptl. Neurol. 18, 468-481. Rugh, R., and Grupp, E. 1959c. Response of the very early mouse embryo to low levels of ionizing radiations. /. Exptl. Zool. 141, 571-587. Rugh, R., and Grupp, E. 1960. Protection of the embryo against the congenital and lethal effects of x-irradiation. Atompraxis 6. 209-217. Runner, M. N. 1959. Metabolic Mechanisms of Teratogenic Agents During Morpho- genesis, Natl. Cancer Inst. Monograph No. 2 (Symposium on Normal and Ab- normal DiflFerentiation and Development). Wesley, J. P. 1960. Background radiation as the cause of fatal congenital malforma- tions. Intern. ]. Radiation Biol. 2, 97-1 18. Quantitative Histologic and Behavioral Studies on Effects of Fetal X-lrradiation in Developing Cerebral Cortex of White Rat * K. R. Brizzee. L. a. Jacobs. X. Kharetchko. and J. C". Sharp University of Utah College of Medicine, Salt Lake City, Utah Introduction Recent experimental studies on effects of fetal irradiation on nervous tis- sues have clearly showns (Hicks, 1954. and Hicks it al., 1957 i that primiti\e neuroblasts and spongioblasts are selecti\ely damaged by ionizing i adiation. It has also been demonstrated in this work that rather specific and predictable anomalies are produced in nervous tissues in postnatal life in the rat by radia- tion exposure on any given day in the gestation period between the 9th day and birth. At the same time, some beha\ioral studies i Levinson. 1952: Furchtgott and Echols. 1958a. bi have demonstrated serious beha\ioral deficits in rats irradiated as fetuses. While considerable attention has been given in the histopathologic studies to the regenerati\e ability and reco\ery of nervous tissues from such radiation exposure (Hicks. 1957) little effort has been devoted to analyzing the capacity of the cells sur\iving irradiation exposure for normal growth or the specific effects of the irradiation on cell growth. Further, the behavioral studies carried out thus far have not emphasized the possible relationships between cytologic deficits and beha\ ioral deficits. It has been our purpose, therefore, in initiating the present series of investiga- tions to analyze effects of fetal x-irradiation administered in fractionated and single doses on early postnatal growth of sur\i\ing cells in cerebral cortex and to determine what relationships may exist between alterations in normal growth patterns or cytologic deficits and beha\ioral abnormalities appearing later in life. The present report is concerned with our preliminan- findings with fractionated doses administered during the latter half of the testation jx-riod. * Supported in part by research grants from the National Institute of Neurological Diseases and Blindness. National Institutes of Health, and the University of Utah Research Fund. 27 28 BRIZZEE, JACOBS, KHARETCHKO AND SHARP Materials and Methods Three groups of rats of the Sprague-Dawley strain were exposed in utero to fractionated doses of total body x-irradiation. Two of the groups received 12.5 and 25 r per day at the rate of 60 r per minute on gestation days 10 through 17 giving total doses of 100 and 200 r, respectively. A third group (Brizzee et al., 1961) received a total dose of 300 r given at 60 r per day on gestation days 10 through 14. These animals and a series of control animals treated in the same manner as the above groups, except for the exjx)sure to radiation, were grouped according to age at 1,5, 10, and 20 days with from 4 to 6 animals per group and the sexes equally divided. The tissues were fixed and stained as reported previ- ously (Brizzee and Jacobs, 1959; Brizzee et al., 1961) and subjected to quantitative histologic analysis. The parameters studied were neuron packing density, neuron nuclear, cytoplasmic, and soma volume, nucleocytoplasmic ratio, gray cell coefficient, glial packing density, and the glia/neuron index in area 2 (Krieg, 1946). In addition, total brain weight was determined and cortical thickness measured in areas 2, 4, 41, and 17 (Krieg, 1946). All of the volumetric, density, and thickness determinations were confined to the submolecular layers only. Methods employed in the quantitative histologic determinations have been described in earlier publications (Brizzee and Jacobs, 1959; Brizzee r^ ai, 1961). In the behavioral studies, 9 pregnant rats were divided into three equal groups: a full-body group, a half-body group in which the lower half of the dam was shielded by lead, and a control group which received no irradiation. Irradiation took place each day from the 10th through the 17th days of gestation. Each irradiated animal received 40 r per day for a total of 320 r (60 r per min). For the half-body group, a shield made of blocks of lead 2 X 4X 8 in. was constructed so that only the thorax, neck, and head were exposed to radiation. To assess the duration of the effects of x-irradiation on locomotor coordi- nation, the three groups of rats were divided into three subgroups to be tested at different ages. Group one was tested at age 40 days, group two at age 90 days, and group three at age 140 days. The test of locomotor coordi- nation required the rats to traverse a bridge made of two parallel rods. At 115 days of age the rats in the 90- to 140-day groups were given 2 trials in a simple L-shaped water maze. The next day all the rats were run in a 14-unit multiple-T water maze patterned after the Stone design (Heron, 1930; Sharp, in press). At age 50 days 6 rats from each group were sacrificed, and their cerebral cortexes studied in the same manner as in the first three groups. In plotting the values of the various parameters in Figs. 1-6 the vertical FETAL X-RAY EFFECTS: CEREBRAL CORTEX 29 lines indicate the maa;nitude of the standard errors of the mean for the con- trols. Standard errors for the irradiated s^roups were not plotted in the interest of clarity in the figures. Results Neuron packing density in all groups i Fig. 1 ) was seen to decrease very rapidly between the 1- and 5-day stages, with a less notable decrease between the 5th and 10th days, and approached normal adult levels on the 20th day. The values for all irradiated groups and controls were in close agreement and showed no significant differences at anv asje level. 10.0 9.0 ro RD t- E If) O 7.0 X • — V 1- 6.0 00 2 III o o 5.0 z :^ (J S 4.0 ?- o 3.0 UJ 2.0 1.0 I- CONTROL ^ - lOOr = 200r o = 300r 5 10 AGE (DAYS) Fig. 1. Early postnatal changes in neuron packing density. 20 30 BRIZZEE, JACOBS, KHARETCHKO AND SHARP The mean values for neuroglial packing density in the control groups decreased from 45,000 cells/mm^ at 1 day to 27,000 at 20 days, but the dif- ferences between the various age levels in this series are not significant owing to a rather large variance in counts. The mean neuroglial packing density for all four age groups in the nonirradiated animals was 34,000 cells/mm^. The neuroglial density in the 100 and 200 r groups did not differ significantly from the controls, but in the 300 r group, the value for neuroglial density in 1 -day-old animals was significantly higher (80,000 cells/mm'; /; < .05) than in the nonirradiated groups. In later stages the differences were not significant. The neuroglia/neuron index (Fig. 2), almost entirely as a result of the 0.40 0.38 0.36 034 0.32 030 0.26 xO-26 UJ 00.24 z 022 z S020 D UJ z 0.18 < 0.16 §0.14 010 0.08 0.06 0.04 0.02 1= CONTTROL A^ lOOr o-200r o- 300r 10 AGE (DAYS) 20 Fig. 2. Increase in neuroglia/neuron index from 1st to 20th postnatal day. FETAL X-RAY EFFECTS: CEREBRAL CORTEX 31 changes in the neuronal packing density, increased fairly rapidly in controls from the 1st to the 10th day and more slowly from the 10th to the 20th day. Differences in values for the neuroglia/ neuron index between irradiated and control groups were not significant at the 5-, 10-, and 20-day stages. In the 300 r group, however, the neuroglia/neuron index in the 1 -day-old rats is significantly higher ip < .05) than that in the non-irradiated rats of the same age. In 20-day animals the value for the index in the 200 r group is considerably higher than in the controls, but due to a large variance it is not statistically significant at the 0.05 le\el. It is noteworthy that the levels for the neuroglia/neuron index at all ages studied are very low as compared with adult values in some other species as, for example, in man (1.78; Hawkins and Olszewski, 1957) or in the horse (1.24; Friede, 1954), although the index is comparable in our 20-day animals to the average values derived from Friede's data for the cerebral cortex in the mouse (.35) and the rabbit (.42). Neuron nuclear, cytoplasmic, and soma (nucleus -\- perikaryon) volumes (Fig. 3) increased steadily from the earliest to the latest stage examined with the values in all groups in fairly close agreement. As in our preliminary studies describing the 300 r group ( Brizzee ct oL, 1961), however, the 1400 1200 :jlOOO ^ 800 o > 600 6 400 cr 3 200 I - CONTROL A = lOOr ° = 200r 300r 10 AGE (DAYS) 20 Fig. 3. .\lterations in neuron soma volume in early postnatal stages. 32 BRIZZEE, JACOBS, KHARETCHKO AND SHARP 2.0 ( \ L8 ,\\\ I = CONTROL • \aA a = lOOr ^W ° " 200r 1.6 ^^^\ o = 300r 1.4 ^^. L2 ^^^^ 1.0 ^^^ 0.8 \^ 0.6 10 AGE (DAYS) 20 Fig. 4. Decrease in nucleocytoplasinic ratio between 1st and 20th postnatal days. values for neuron soma volume (nucleus + parikaryon ) in the 100 and 200 r series are lower than in the controls in the first three developmental stages and increase to values above the control level on the 20th day. The group receiving the highest dose of irradiation (300 r) diverged more markedly from control values than the animals given lower doses, but the diflferences are within the range of error of the methods employed and are not statisti- cally significant. The nucleocytoplasmic ratio (Fig. 4), reflecting the chang- ing relationships between nuclear and cytoplasmic (perikaryon) volume through the four stages of development, was seen to decrease steadily in all groups with no significant differences appearing among irradiated or control groups. In contrast to the above findings, marked differences were observed in cortical thickness in area 2 (Fig. 5. Table I) between the controls and animals irradiated at 200 and 300 r {p < .01), and between the 200 and 300 r groups themselves {p<.0\). No significant differences were noted between the 100 r groups and nonirradiated rats. FETAL X-RAY EFFECTS: CEREBRAL CORTEX 33 1600 1400 1200 < 800 o CE O o 60d 400 I - CONTROL t. = lOOr D = 200r o = 300r AGE (DAYS) 20 Fig. 5. Comparison of cortical thickness in area 2 in irradiated and control groups in early postnatal period. It is particiilaily noteworthy that the cufves illustratinr=5.8 ff^ = 3.10 Roentgens delivered in air. ' Benign encapsulated salivary gland (Sclerosing angioma). « Post-radiation time ' Low grade carcinoma. s .Animals not utilizfd foi behavioral studies; all otlui animals utilized in behavioral studies. 46 ROBERT H. BROWNSON 90 120 150 TIME IN DAYS 240 Fig. 1. Behavior reaction pattern demonstrated by control and experimental animals for food reward during 45 minute test periods. Slope of line to right indicates a gradual extinguishment of learned reaction. Bar [jresses are plotted against time in days. bar piessins^ acti\ ity for food reward (Fis^. 1). Those animals which re- cei\ed 1.000 r showed only minor deviation and after 228 days equaled the control animals. The 2,000 r animal group showed a more striking initial decrease but they. too. ecjualed the control animal performance by 228 days. Animals receiving the accumulated doses of 3,000 and 4,000 r both demon- strated a severe decrease in activity which reached the lowest performance rates 7 days after receixing their last exposmes. Dinging the remaining sur- vival period the 3,000 and 4,000 groups demonstrated little tendency to increase from this low level acti\ ity. The obvious decline of all animal performance o\ er the 5 weeks of testing, including controls, was attributed to gradual extinguishment of learned behavior. Purkinje cells and granule cells of the cerebellum appeared to have imder- gone certain similar changes at the various le\els of exposiue and time intervals. These changes appear to have in\oIved a mild to se\ere loss of Purkinje and granule cells. Hyperchromatic neiuons and pyknosis were prevalent throughout most exposure levels. These alterations (Figs. 2 and 3) were scattered throughout the cerebellum. Cerebral neurons demonstrated pyknosis and hyperchromatosis in scat- tered areas throughout all le\els of radiation. Mild neurofibrillarv or axonal X-RAY INDUCED C\S CHANGES 47 changes, characterized by beading and swelling, were noted in all groups I Fig. 4). Occasional areas of cell necrosis were observed in the 5.000 r level. Hypothalamic neurons demonstrated occasional evidence of swelling, chromatolysis, pyknosis, and hyperchromatic staining qualities, but these changes were largely inconsistent. The presence of PAS-positi\e globules within the extraneuronal tissue was clearly evident over the entire dosage range of radiation, demonstrating no specific alteration. Brain stem neurons were not observed to ha\e undergone major changes at any level of radiation. Those changes that were obser\ed were mild to moderate chromatolysis. pyknosis. and hyperchromatism. In the 5.000 r level maintained 228 days, hyperchromatic neurons were in excess with no chromatolysis observed. The neuroglial cells underwent slight but consistent degrees of scattered hyperplasia in the subpial cortex ( Fig. 5 1 . These changes were somewhat localized and similar at the 1,820, 2,000, and 3,000 r levels. Astrocytes seem to have been the most regularly reactive cells and in inany instances could be termed gemestocytic. Glial cell hyperplasia and hypertrophy became quite noticeable in the cortex at the 4,000 and 5,000 r level, especially in the group surviving 228 days. Small areas of infarction (Fig. 6) were in the cortex and subcoitical white matter in all groups of animals. There seems to have been a more intensive cellular and fibrillary gliosis about these areas in the 4,000 and 5,000 r animals. The ependymal and subependymal cells were significantly reactive at all levels of radiation. There appeared to ha\e been a thinning of cells in all groups. The pyknosis and hyperchromatic cells were scattered throughout the varied exposuie le\els in different \entricular areas as well as within the same areas. It was not uncommon to find nests of hyperchromatic subependymal cell foci i Fig. 7). P.\S-positive accumulations of globules both within the basal position of the ependymal cell and subependymal areas were increasingly evident as the le\ el ot radiation was increased. Heavy deposits of such materials were noted in the 4.000 and 5,000 r groups. Meninges, specifically the leptomeninges. and the pial-glial membranes underwent changes Fig. 5i. \arying from mild thickening at 1,820 and 2.000 r to moderately severe in the 5.000 r range. Such changes were noted to have been scattered o\er the brain in local areas. Throughout all of the \aried dosage levels occasional cellular and fibrillary infiltrations were present in these foci. .Astrocytes were gemestocytic in such areas along the piaglial membrane. Blood vessels underwent \arious alterations at the different dosage le\els. Vessels most often in\ol\ ed were capillaries and small arteries. Mild to mod- erate hypertrophy and hyperplasia of endothelium and increase in adventitia were noted in animals receiving 1.820, 2,000 and 3.000 r (Fig. 8) and were 48 ROBERT H. BROWNSON Iff' -^**Sa. 'tf*^^'^*^!^# > * .2 I ^ if V / ti • X-RAY INDUCED CNS CHANGES 49 severe at 5,000 r. Some petechial hemorrhages were seen. The animals that underwent 4.000 r exposure showed a more severe reaction. Apparently there was a consistent increase in connective tissue, presumably peri\ascular, from mild to se\ere within the hypothalamus from 1.820 to 5.000 r (Figs. 9, 10. and 11 j. Discussion There were no remarkable or otherwise consistently observable neurologic deficits in animal activity during the 228 days following irradation. If any such alteration could be detected by observation alone, one might describe malaise. The animals" physical appearances were altered by cataracts and varying degrees of hair loss about the head. There was complete epilation of hair immediately surrounding the eyes in a circumscribing area approximately 3 mm wide. In most instances the hair o\er the remaining portion of the rat's body lost its usual healthy sheen and became ruffled. Some of the animals had inflamed margins of the eyes which might be described as blepharitis. Analysis of weights indicated that beginning with group B at 2,000 r le\ el there was a decline in animal body weight. This was in the approximate magnitude of I2''r of control animal weight at the time of sacrifice 228 days after initial exposure. Additional cumulative exposure to x-ray revealed a similar weight loss in the animals at sacrifice. The per cent of difference between the remaining groups of control and experimental animal weights became increasingly greater with increasing dosage. The greatest factor of difTerence was at the 5.000 r level where a loss of 29'^r was noted. However, the percentage weight differences indicated were taken only from weight differences at the time of sacrifice for the group as an a\"erage. In Fig. 2. Cerebellar granule cell pyknosis and hyperchromatic staining reaction 228 days following 5,000 r. NissI: X 320. Fig. 3. Cerebellar granule cells demonstrating normal staining reaction. Nissl; X 320. Fig. 4. Cerebral corte.x focal area of pyknotic and hyperchromatic stained neurons 228 days following 5,000 r. Nissl; X 130. Fig. 5. Cerebral cortex cellular gliosis and thickened pial-glial membrane with some cellular and fibrous infiltration of subarachnoid space 228 days following 5.000 r. Hematoxylin and eosin ; X 130. Fig. 6. Cerebral cortical infarct. Small with slight encapsulation of necrotic area 228 days following 1.820 r. Verhoeff; X 320. Fig. 7. Ependymal cells and area of subependymal cell pyknosis and hyperchromatic staining reaction 228 days following 5,000 r. Nissl; X 320. 50 ROBERT H. BROWNSON '<'• 10 m&^^^ Fig. 8. Cerebral cortical arteriole demonstrating hypertrophy and hyperplasia of ad- vcntitia with large hyperchroniatic nuclei 228 days following 1.820 r. VerhoeflP: X 64. Fig. 9. Normal rat hypothalamus and third \entricle. VerhoefF; X 130. Fig. 10. Hypothalamus with increased vascularity and perivascular connective tissue. Increased numbers of nuclei 228 days following 2,000 r. VerhoeflF; X 130. Fig. 11. Hypothalamus with increased vascularity and perivascular connective tis- sue. Numbers of nuclei present less than demonstrated in Fig. 9 228 days following 5,000 r. VerhoeflP: X 130. nearly all cases there was a favorable increase in the \veia;hts approximately one week followino; the last exposure administered to that group. One notable exception was in oioup G after reaching the 5,000 r level. This group failed to show any tendency to increase weight imtil after 120 days. X-RAY INDUCED CNS CHANGES 51 800 700 600 500 E J. 400 5 300 200 100 WEIGHT ANALYSIS Total Cumulative Dose • — • A 1820 r K « B 2000 r o C 3000 r » * D 4000 r Control ttt t ABC D Radiation Exposure (r) J I 30 60 90 120 150 TIME IN DAYS 180 210 240 Fig. 12. .Analysis ot average animal weights in grams for control and Lxperimental animals plotted against time in days. In no instance were the experimental animals in s,roups B. C, and D capable of equaling their control litter mates" weight record dininsj the 228-day interval before sacrifice. Animals in the control and e.xperimental cjroups underwent 5 weeks of behavioral studies which recjuired a substantial de- crease in food intake durin? observation. Dininq this period of food dep- rivation the control animal weights continued to rise (Fig. 12). empha- sizing that the decrease in body weight and life span of the rat following x-irradiation ot the central nervous system is further magnified bv forced reduction in available nutritional requirements. Cumulatixe exposure ol the rat head to x-irradiation has been noted to cause certain physical and histologic alterations. In the total accumulated dosages 1,820, 2.000. 3.000, 4.000 and 5.000 r these alterations were strik- ingly similar in character and appeared to show a Cjuantitative relationship to time and accumulated dose level. The cell changes, although quite similar in appearance, did display cei tain specific sensitivity. For example, in the cerebellimi both Purkinje and granule cell neurons underwent the severest reactions in rats receiving 5.000 r after 1 16 days. This may be contrasted to those lesser cellular changes that were moderate in rats recei\ing 5.000 r and sur\i\in<; 228 days. It is possible that the inter\ening time between 1 16 and 228 days was sufficient to allow cell recovery. The cytologic changes were noticeably more severe in 52 ROBERT H. BROWNSON TABLE II Cumulative Effects of X-irradiation Physical data Microscopic Neurons Head Rats dosage PRT Purkinje Granule Pyramidal Tuber no. (r)"^ (days) cerebellum cerebellum cortex hypothalamus Large brain stem ;a) (b) (C) (D) (E) (f; (g; (H) 1820 228 Mild hyper- chromatic Mild scat- tered pyknotic, hyper- chromatic Scattered hyper- chromatic, pyknotic, mild neuro- fibrillary changes Mild to mod- — erate, (PAS) positive globules, mild hyper- chromatic pyknotic 2000 228 Mild hyper- chromatic, pyknotic Mild scat- tered pyknotic, hyper- chromatic Scattered pyknotic, mild neuro- fibrillary beading, hyper- chromatic Heavy, (PAS) positive globules, hyper- chromatic, pyknotic Mild chromatolysis, hyper- chromatic, pyknotic 3000 165 Mild hyper- chromatic, pyknotic Moderate scattered pyknotic, hyper- chromatic Scattered pyknotic, mild neuro- fibrillary brading hyper- chromatic Moderate Mild (PAS) posi- chromatolysis, tive globules, hyper- mild pyknotic, chromatic, hyper- pyknotic chromatic 4000 158 Mild hyper- Moderate chromatic, scattered pyknotic pyknotic hyper- chromatic Scattered Moderate pyknotic, (P.^S) positive mild, neuro- globules fibrillary beading, hyper- chromatic moderate hyper- chromatic, pyknotic Moderate chromatolysis, hyper- chromatic, pyknotic 5000 116 Severe Severe Scattered Heavy, (PAS) Moderate hyper- scattered mild neuro- positive chromatolysis, chromatic, pyknotic, fibrillary globules, hyper- pyknotic hyper- beading, hyper- chromatic, chromatic cell chromatic, pyknotic necrosis pyknotic 5000 228 Moderate to severe hyper- chromatic, pyknotic Mild scattered pyknotic hyper- chromatic Scattered Heavy, (PAS) Moderate pyknotic, positive to mild, mild neuro- globules, hyper- fibrillary hyper- chromatic, beading, chromatic, pyknotic cell necrosis pyl-^notic " Roentgens delivered in air. '' Animals utilized in behavioral studies. <• Animals not utilized in behavioral studies. X-RAY INDUCED CNS CHANGES TABLE II (Continued) CiMULATivE Effects of X-irradiatiox 53 Microscopic Glia Oligo- Astro- MicTo- Non-neural Ependyma sub- ependyma Meninges Blood vessels (I) (J) (k; (L) :mi (N) Scattered areas of Mild Mild cellular gliosis in pyknotic, scattered sub-pial cortex (PAS) thickening, gemestocytic astrocytes. positi\e cellular occasional small infarct globules infiltration Moderate increase in perivascular connective tissue and blood vessels in hypothalamus, hypertrophy, hyperplasia of endothelium and ad\entitia Occasional small Heavy Mild Moderate increase in infarct scattered (PAS) scattered perivascular connecti\e cellular gliosis in positive thickening, tissue and blood vessels in sub-pial cortex globules cellular hypothalamus, hypertrophy, infiltration hyperplasia of endotheliimi and adventitia Scattered areas of Hyper- Mild cellular gliosis in chromatic, scattered sub-pial cortex pyknotic, (PAS) positive globules thickening Mild cellular and Heavy Mild fibrillary gliosis (PAS) scattered around infarcts in positl\-e thickening white matter globules Mild increase in peri\ascular connective tissue and blood vessels in hypothalamus, mild hyper- trophy and hyperplasia of endotheliiun and ad\'entitia Moderate increase in perivascular connective tissue and blood \-essels in hypothalamus, mild hyper- trophy and hyperplasia of endothelium and ad\entitia Mild cellular and Heavy Moderate fibrillary gliosis (P.AS) scattered around infarcts in positive thickening white matter globules Moderate increase in perivascular connective tissue and blood vessels in hypothalamus, moderate hypertrophy and hyperplasia of endothelium and adventitia Moderate cellular Heavy Moderate and fibrillary (PAS) scattered gliosis in sub-pial positive thickening, cortex, gemestocytic globules cellular astrocytes, small infiltration infarcts in white matter Heavy increase in perivascular connective tissue and blood vessels in hypothalamus, severe hypertrophy and hyperplasia of endothelium and adventitia 54 ROBERT H. BROWNSON the granule cells than in Purkinje cells, appearing in both as nuclear pyknosis and hyperchromatic staining. There appeared to be an appreciable decrease in cellularity most severely affecting the 5,000 r level at 1 16 days. Wilson ( 1960) irradiated monkeys with whole-body exposure to cobalt-60 (gamma) from 400 to 40,000 r and stressed the changes that occurred in animals dying during the first 54 hours post-irradiation. Cerebellar granule cells underwent nuclear pyknosis especially within the innermost layers with a decreased cellularity explained on the basis of decreased nuclear area. Vogel ( 1959) administered massive doses of gamma radiation to the head of rabbits and dogs sacrificed at intervals up to 10 days. He reported that the altered granule cells of the cerebellum demonstrated pyknotic and hyper- chromatic nuclei most notably within the first 24 hours after exposure. His opinion was that the recovery phase was completed by 72 hours after radiation. The tissues studied in this project are decidedly those of chronic classifi- cation sacrificed up to 228 days after exposure. The results from oiu' study indicate that x-irradiation of the rat head will elicit slight changes in cere- bellar neurons after 228 days and that a dose as high as 5,000 r is capable of causing severe cell change during the 1 16 days. In all instances the changes described through and including 228 days after radiation were accompanied by decreased cellularity. It would only be speculation to propose cell loss. The only absolute measure of this would ha\e to incorporate a quantitative analysis. Wilson (1960) and Vogel's (1959) findings that the granule cells in the cerebellum undergo acute changes followed by reco\ery during a slightly later period after radiation, would seem to indicate the existence of secondary effects. This effect noted months after exposure may further demonstrate its recovery phase at a still later date. Neurons in the cerebral cortex, hypothalamus, and brain stem seldom ap- peared to be necrotic and were probably of reversible nature. Such changes were described as shrinkage in both cytoplasm and karyoplasm or pyknosis. It is suspected that the decrease in cell or nuclear volume or both is para- mount to the hyperchromatic staining quality, as well as to the appearance of decreased cellularity. Peculiar to these alterations was the obvious scat- tering of cell changes within identical structiue, thus indicating certain differences in radiosensitivity within the particular structure imder observa- tion. In view of the perfusion-fixation methods utilized for this study, it is felt that these observations are reasonably free from artifact. These cell changes were present in varying amounts throughout all radiation levels, which seemingly indicates little or no direct qualitati\e relationship between dose and time within the parameters of this study. Only at the 5,000 r level were there evidences of significant neuronal necrosis. The absence of major X-RAY INDUCED CNS CHANGES 55 structural alterations amon ^ "ra -2 P i""" c O rt '— cJ « O tA t/. o — ci »< o o < p i; ^ Ic O ' — o ■a 03 n3 •-». o •:: CC c« o ^ Oj c S re -+■' i/j .2 ^ ^ Oj " T3 "S O w ^ O "L' o -iS E lit " "" O '-;- < CL = 68 YAMAZAKI, BENNETT AND CLEMENTE thickenino, there invariably was some thickening in the walls of the medium sized and smaller arteries of the pial layers. Discussion The neurologic findings observed in the rats in this study are similar to the findings of other investigators in their studies on irradiated newborn and young guinea pigs, rabbits, kittens, and dogs (Danysz, 1903: Turner and George. 1910; Brunner, 1920; Nemenov, 1934; Demel 1926; Mogilnitzky and Podljaschuk, 1930). Tremors, clonic twitching, epileptoid seizures, pa- ralysis of extremities, retardation of head and body size, and poor mental performance have been reported. Previously, however, a systematic sequen- tial age-dose relationship during the neonatal period had not been demon- strated. The eflfect of radiation on the developing human brain is not as well docu- mented as are the animal studies. The occurrence of microencephaly, mental defectives, hydrocephaly, ossification defects of the cranial bones, eye defects, and skeletal abnormalities in children born after maternal pelvic irradiation has been reported (Murphy, 1929; Goldstein and Murphy, 1929). Intrau- terine exposure of the fetus to atomic radiation in sufficient amount to cause acute radiation eflfects on the mother has resulted in children with signifi- cantly smaller head circumferences than in the control group (Plummer, 1952; Yamazaki ct al., 1954). Information concerning irradiation of the head alone in infants and children is scanty. Children irradiated for scalp lesions developed epilation in three weeks, and, after a year or so, hemi- paresis and siezures developed in one patient (Lorey and Schaltenbrand, 1932) and a subdural hematoma was reported. In contrast to this, over 3,000 persons, many of whom were children, recei\ing epilating doses of ra- diation for treatment of tinea capitis had no evidence of injury to the brain (Mackee and Cipollaro, 1946) . In another report, children over 3 years who received radiation to the head for a similar condition did not develop any abnormal neurologic findings (Macleod, 1909). Children under 3 years were not radiated to avoid any possibility of radiation injury. Periods of somno- lence lasting from 4 to 14 days were noted in 30 out of 1,100 children epi- lated by radiation therapy for ringworm of the scalp (Druckmann, 1929). A similar type of reaction was noted in adult human volunteers who received approximately 150 r to the diencephalic area (Birkner and Trautmann, 1953). Disturbances of sleep-wake patterns and changes in gonadal function also were noted. In this regard, radiosensitivity of the hypothalamus and brain stem has been demonstrated in recent studies by Clemente and Hoist (1954) and Arnold and his associates (1954a,b,c). A report on the sur- vivors of the atomic bomb explosions who exhibited no sign of burns. EFFECTS OF HEAD IRRADIATION IN NEWBORN RATS 69 trauma, or oeneralized radiation illness, but who were assumed to have been radiated to the head alone, revealed that the most extensive involvement of the brain was in children ( Uchimura and Shiraki, 1952). The relatively abrupt change in the degree of radiosensitivity of the neo- natal rat brain occurs at a time when unique morphologic, functional, and biochemical changes are taking place, and this seems to present an interest- ing temporal correlation with the findings reported in this study (Waelsch, 1955: Richter. 1957). The general growth rate of the neonatal rat brain is reflected by the five- fold increase in weight by the end of the 2nd postnatal week and this repre- sents almost 809r of the weight of the adult brain (Potter ct al., 1945; Folch- Pi. 1955). The cerebral cortex is gaining weight proportional to the growth of the brain as a whole, but the cerebellum is gaining nearly three times as much weight, and the brain stem is accumulating only one-half its birth weight during the same period ( Sugino, 1917). However, the vascularity of the brain undergoes little change during the first 5 days, but between the 5th and 10th day a definite increase in vascular richness occurs. After the 10th day the density of the capillary bed increases rapidly, and a concomitant richness of the capillary bed increases in oxidase content and mitochondria simultaneously i Campbell, 1939). An example of the neuronal difTerentia- tion taking place during the neonatal period is presented by the change in the packing density of the neurons in the cerebral cortex. This density de- creased rapidly between the 3rd and 4th day after birth and then more slowly, "no change taking place after the 17th day" ( Haddara, 1956). This would indicate an increase in cytoplasmic constituents along with the devel- opment of a more elaborate cortical dendritic system. It is during the first 2 week period postnatally that the electroencephalo- gram becomes more regular and assumes the characteristics seen in adult rats 1 Grain, 1952). The ability of the rat to withstand anoxia is greatest in the immediate postnatal period, and shortly after birth during the first 5 to 6 days there is a loss of tolerance to anoxia (Fazekas ct al., 1941 i. Immature budding vessels may well be more differentially sensitive to nox- ious agents than fully developed ones. It has been shown that growing retinal vessels during the first postnatal week, but not the choroidal vessels, constrict when exposed to oxygen and may e\en be obliterated with prolonged expo- sure ' Ashton and Cook. 1954). .\s the vessel reaches maturity, which takes place at about the 8th postnatal day in the rat. it gradually loses its ability to constrict when exposed to oxygen. In this regard, it is felt that the oxygen ef- fect is an appropriate example since oxygen enhances ionizing reactions (Dowdy et al., 1950) . Moreo\er, the best protectors against x-rays, i.e.. cystea- mine and cysteine, sulfhydryl containing amino acids, are also the best protec- tors of mammals against oxygen poisoning (Bacq and .Alexander. 1955) . How- 70 YAMAZAKI, BENNETT AND CLEMENTE ever, it remains to be demonstrated whether radiation actually affects ma- turing cerebral vessels in a like manner. The capillaries have been demon- strated to be the most radiosensiti\e of the blood vessels, and the degree of vascularity would seem to have a bearing on the pathologic picture. The degree of vascularity varies widely in the rat brain; for example, the globus pallidus has been demonstrated to be strikingly low in vascularity (Craigie, 1945). The globus pallidus was one of the areas where necrosis was most frequently involved. These physiologic and morphologic changes in the rat brain during this initial postnatal 2 weeks when radioresistance is rapidly developing are also associated with dynamic biochemical transformation. Thus, oxygen ixptake increases rapidly as does the lactic acid production ( Greengard and Mc- Ilwain, 1955; Tyler and Van Harreveld, 1942). Marked increase in enzyme activities occur; there is a threefold increase in the respiratory enzymes suc- cinic dehydrogenase and cytochrome oxidase during the initial 2 weeks and a fivefold increase of adenosine triphosphase activity, believed to be involved in making energy available to the cell to accomplish its differential growth (Potter, ct al., 1945; Flexner, 1953). Increased cholinesterase and pseudo- cholinesterase activity is similarly observed i Elkes and Todrick, 1955). The water content decreases significantly and levels of proteins and phosphatides are beginning to approach adult levels. Howe\er, the cerebrosides which are importantly related to myelin formation accimiulate only 119^ of the adult weight by the 19th day (Folch-Pi, 1955). Deoxyribonucleic acid content which continues to accumulate during this period increases fourfold between the 2nd and 16th postnatal days, after which further increase is hardly no- ticeable (Mandel and Bieth, 1951 ). These examples amply demonstrate that the x-irradiation may well alter these manifold biochemical changes occur- ring in the rat biain shortly after birth. Summary Newborn rats ranging in age from 8 hours to 15 days received single doses of x-radiation to the head only. Doses of 125 r, 300 r, 500 r, and 1.000 r were administered. One group of animals was sacrificed at 48 to 72 hours after irradiation, and another group was autopsied at ages ranging from 14 days to 15 months. The study demonstrated the development of a marked radioresistance in the brain by the 3rd postnatal week, compared to the easily damaged brain of the rat during the first postnatal week. The development of this relative radioresistance following the first week of life was abrupt. The radiosensitiv- ity was manifested by an increased mortality, retarded growth, retarded brain size, production of cataracts, and abnormal neurologic signs in the EFFECTS OF HEAD IRRADIATION IN NEWBORN RATS 71 radiated animals. The neuropatholosic findings correlated closely with the behavioral disturbances. Prominent among the early microscopic findings was damage to capillaries. Later histopathologic findings included perivas- culitis, progressive thickening of the \essel walls and a narrowing of the vessel lumen, meningitis, inflammation of the choroid plexus, ventricular en- largement, delayed cerebral cortical and cerebellar de\elopment. focal ne- crosis, and gliosis. References Arnold, A., Bailey, P.. and Harvey. R. .A. 1954a. Intolerance of the primate brainstem and hypothalamus to con\entional and high energy radiation. Neurology 4. 575-585. Arnold. A.. Bailey. P.. Har\ey R. A. . Hass.. L. I., and Laughlin, J. S. 1954b. 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Cerebral injuries caused by atomic bombard- ment. /. Nerrowi Mental Disease 116, 654-671. VVachowski, T. J., and Chenault, H. 1945. Degenerative effects of large doses of roentgen rays on the human brain. Radiology 45, 227-246. Waelsch, H. (ed.) 1955. "Biochemistry of the Developing Nervous System." Aca- demic Press, New York. Warkany, J., and Schraffenberger, E. 1947. Congenital malformations induced in rats by roentgen rays; skeletal changes in offspring following single irradiation of mother. Am. J. Roentgenol. Radium Therapy 57, 455-463. Wilson, J. G. 1954. Diffenntiation and the reaction of rat embryos to radiation. /. Cellular Comp. Physiol. 43, Suppl. 1, 11-37. Yamazaki, J. N.. Bennett. L. R., McFall, R. .A., and Clemente, C. D. 1960. Brain radiation in the newborn rats and differential effects on increased age. 1. Clinical obser\ation. Neurology 10, 530-536. Yamazaki, J. N.. Wright. S. W., and Wright, P. M. 1954. Outcome of pregnancy in women exposed to the atomic bomb in Nagasaki. .4//?. /. Diseases Children 87, 448-468. Cytoplasmic Inclusions Containing Deoxyribonucleic Acid in the Neural Tube of Chick Embryos Exposed to Ionizing Radiation"^ Mary Elmore Saier and Donald Dincax University of Texas Medical Branch. \ Galveston, Texas Introduction The identification of deoxyribonucleic acid ' DNA i with chromatin nor- mally confines it to the nucleus of the cell. Nevertheless, reports of the occurrence ot DXA in the cytoplasm are sufficiently numerous to suggest that its presence there may be a phenomenon of wide distribution, even thous:h the origin and destiny of such DNA may remain obscure. Perhaps not sufficiently appreciated is the large amount of cytoplasmic DNA which makes its appearance in young embryos a few hours tollowing moderate irradiation. The striking picture presented by the numerous Feul- ^en positi\"e bodies in the neural tube of early chick embryos that had received moderate doses of ionizing radiation prompted the present investi- gation. This enlarges on a preliminary report t Sauer. 1957) dealing with irradiated chick embryos. Since similar bodies ha\e a normal occurrence Gliicksman. 1951: \on Sallmann ct al., 1957). their interpretation as an injury response demands special caution. In the chick, the large number and wide distribution ot these in irradiated embryos tar exceed any normal appearance. Materials and Methods Chick embryos ranged from 2 to 4/2 days in incubation age at the time of treatment, most being about 60 hours old. * Investigation supported in part by the U.S. Public Health Service. The authors gratefully acknowledge the cooperation of Dr. \Iartin Schneider, who supervised the .\-irradiation. and of Dr. Bruce E. Walker, who carried out the technique with the tritium-labeled enibrvos. C. Drew Sanders gave technical assistance. 76 MARY ELMORE SAUER AND DONALD DUNCAN Embryos received either 200 or 500 r of x-rays through the unopened shell and were returned to the incubator. Control embryos were subjected to all treatment given the others, except the actual irradiation. Individual embryos were fixed at frequent intervals following irradiation, ranging from 1 to 76 hours. The following factors were employed in the irradiation: 250 kv, 30 ma, added filter 0.25 mm Cu plus 1.0 mm Al. 50 cm FOD, average intensity of 145 r/min. Other embryos were labeled with tritium ( Sauer and Walker, 1961). Some received only thymidine-H'*, usually 50 mc per embryo, specific activ- ity 1.6 c/mmole. Others had received 200 r x-rays immediately preceding treatment with thymidine-H'\ The embryos were fi.xed in Newcomer's (1953), Carnoy's. or Serra's fixative, or in 10 or 50 Cf neutral formalin, embedded in paraffin and sec- tioned at 3 to 6 IX. Stains used included hematoxylin and eosin, toluidine blue O, May-Griinwald and Giemsa, Feulgen's, and methyl green-pyronin. The May-Griinwald and Giemsa stain following Newcomer fixation was preferred for general cytologic study. Kurnick's ( 1955a,b) procedure was followed for DNase and RNase. The enzymes were purchased from Worth- ington Biochemical Corporation, Freehold, New Jersey. For electron micro- scopy, pieces of embryos were fixed in Dalton's osmic-dichromate mixture, embedded in methacrylate, and sectioned with a Porter-Blum microtome. Results Numerous cells in the irradiated embryos have a striking appearance in that one or more bodies approximately 2-5 /x in diameter lie within their cytoplasm (Figs. 2-12). Such bodies occur in small numbers in normal chick embryos but greatly increase in number following irradiation. In 2- to 3-day- old chick embryos, they may begin to appear in the 2nd hour after exposure. In the early hours after treatment the cells containing the inclusions may be few or many, depending on the susceptibility of the particular embryo. The distribution of the afTected cells at this time is wide but irregular: while some fields show many of the bodies, extensive regions may contain none or few. The bodies appear most constantly in the neural tube, especially in the brain. Their number increases with time, so that a 60 hour embryo that has received 200 r will 9 hours later contain large numbers of the inclusions in every system (Figs. 3-6). The body lies within the cytoplasm of an appar- ently normal cell. Its position adjacent to the nucleus is characteristic (Fig. 6) and it often flattens or indents the nucleus at the area of contact. In the series receiving 200 r, the inclusions are \ery numerous in embryos fixed at 9 to 22 hours following irradiation. The number of inclusions then decreases rapidly. Only small numbers of the cytoplasmic bodies remain at CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 77 the end of 34 hours in any of the 200 r series, and by 57 hours recovery seems to be complete. Ahhough a variable amount of degeneration occurs even at this lower dosage, manifested in the neural tube as scattered nuclear fragments or as areas with an indistinct luminal margin, there is no whole- sale degeneration, and most nuclei remain essentially nonnal in appearance (Figs. 2-8). To what extent recovery consists of reversal of the process in the individual cell rather than replacement by unaffected cells can not be answered by this study. Quite different results follow a dosage of 500 r. At 20 hours after exposure, numerous inclusions fill many cells, and there is considerable cell death. Debris is present in the \entricles of the brain and in the central canal of the neural tube. This series shows considerable reduction in the mitotic rate during the 20 to 30 hour period following treatment, while no such sec- ondaiy delayed period of mitotic arrest follows the 200 r dosage. The series receiving 500 r shows complete recoxery at 76 hours. From the small series of older embryos i4 to 5 days i receiving the same amount of radiation as the two groups just described, it was determined that age is not a factor in the mere appearance of inclusions, although their distribution and time of maximum development differ in the two stages. Table I summarizes the staining reactions of most of the cytoplasmic inclusions. Each body commonly displays a deep staining region, or center. (Figs. 2 and 10) which is strongly basophilic and appears to contain a high concentration of DNA. Both with Feulgen's stain and methyl green-pyronin, the centers give a strongly positive reaction for DNA ; the negative reaction when DNase precedes the staining confirms the DN.A. content of the centers. Alfert I 1955; Vendrely ct al., 1958 i showed that reaction to methyl green is not a reliable indicator of the degree of polymerization of DNA, as held by Kurnick 1955a.b!. The intimate imion of DNA and protein in normal chromatin keeps many groups imaxailable tor dye binding. Although the DN.\ of pyknotic nuclei does not decrease until the fragmentation stage, the intense yreen which pyknotic nuclei display can not reflect the amount of DNA present, but only means that autolytic changes accompanying pyk- nosis have unmasked stainable groups. The staining reactions carried out ( Table I ) support the concept that these bodies also contain ribonucleic acid (RNA ) . With Kurnick"s 1955a, b) modification of the methyl green-pyronin stain, for which he claims speci- ficity for each of the types of nucleic acid, the body stains with a green center surrounded by a red periphery. The color resembles the deep green of the metaphases rather than the lighter green of other nuclei. The red color of the periphery might mean either RN.A or depolymerized DNA. The negative Feulgen stain and the absence of red color when the stain is applied 78 MARY ELMORE SAUER AND DONALD DUNCAN TABLE I Staining Reactions of the Bodies Which Appear in THE Cytoplasm After X-Irradiation Method Ceiitei Periphery Color Significance Color Significance Hematoxylin-eosin Toliiidine blue Feulgen's Omitting hydrolysis After DNase After RNase Methyl green- pyronin After DNase After RNase May-Griinwald- Giemsa After DNase After RNase Purple Basophilic Deep blue Presimiably nucleic acid Deep red No red No red Deep red Deep green No green on slide Deep green DNA DNA (polymerized? Black Black Mostly red, but a few remain black DNA and RNA? Red Acidophilic Pale blue — Green Green ) Not DNA Green Red Red ) RNA (not de 1 polymerized No red ! DNA, for Feulgen- negative) Black or — gray Gray — after digestion with RNase indicate that it is RNA. The deep purplish black staining of the center or often of the entire body with the May-Griinwald and Giemsa stain would be consistent with this conclusion. Jacobson and Webb ( 1952) found that chromatin changes in its reaction to this stain dur- ing the sequence of mitosis from red in the interphase to black during meta- phase. These authors apparently demonstrate that black indicates presence of RNA in addition to DNA, but the specificity of this has been questioned (Swift, 1953; Theorell, 1955). In our hands black of the metaphase was more resistant to each type of nuclease than were the other cellular elements and that of the inclusions was even more resistant than were the metaphases. Following DNase, the bodies stained as black as before; following RNase, digestion was incomplete, some resistant bodies always remaining. The response of neural tube cells of embryos exposed for as long as 12 hours to tritiated thymidine resembled the response to x-ray (Sauer and Walker, 1961). However, there was no cessation of mitosis in the tritium- CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 79 tieated embryos, wiiile mitosis was inhibited for IV2 and 3 hours fohowing the 200 and 500 r exposures respectively. Tritium, once added to the es:g, remains axailable over a prolonged period, so that a high degree of incor- poration results (Sauer and Walker, 1959. 1961 i. Assuming that the mitotic stages are the ones most sensitive to radiation, x-ray afTects only those in mitosis at the time of treatment. In the tritium-labeled embryos, howe\er, within the course of a few hours practically e\ery neural tube cell would undergo mitosis and thus be exposed to radiation at its sensitive stage. Those embryos exposed to the combined action of tritiated thymidine and 200 r did not appear to show much greater injury than those that had received thymi- dine alone. With only light microscopy, there remained the possibility of error as to the actual cytoplasmic location of the bodies. The electron microscope pro\ed in\aluable in demonstration of electron dense bodies of complex form not found in normal material 'Fig. 12 j. Discussion ^ Feulgen-staining granules located outside the nucleus ha\e been described in many species under a wide variety of conditions. They occur normally, especially in embryonic development 1 Gliickmann, 1951; Chang, 1940'. but also in the adult in certain locations (Corner. 1932) : pathologically fol- lowing cell death (Barthels and Voit, 1931) and in association with \iruses (Leuchtenberger et al., 1956) : and experimentally in tissue cultmes of nor- mal vertebrate embryos (Maximow. 1925) and in iriadiated material ' .'M- berti and Politzer. 1924: \on Sallmann ct al., 1957 . Wherever encountered, they resemble in their deep staining the chromatin of mitotic stages and are surrounded by a portion of cytoplasm more deep staining than the remainder. E.xtranuclear chromatin bodies assume prominence in developmental stages in both plants and animals in connection with death of superfluous cells, as in regression of transient structures or following excessive cell pro- duction. The latter is probably an almost uni\ersal growth phenomenon. In the intensely studied field of insect dexelopment. Feulgen-positive bodies regularly occur both within the cytoplasm and e.xtracellularly. Wig- glesworth (1942) in an extensive review established two facts: a) whole nuclei break down, and b ) the granules arc often intracellular. They are most numerous during active mitosis, when excess cells would be formed. In- corporation of the remnants of a dead cell by a neighboring cell seemed a distinct possibility in epidermis with its intercellular connections: also, in rapid cell division, of one of the daughter nuclei died before the cytoplasm had divided, the dead cell would remain as a cytoplasmic inclusion. In one 80 MARY ELMORE SAUER AND DONALD DUNCAN known situation, nuclear division regularly occurs without cytoplasmic divi- sion, and one of the daughter nuclei degenerates to become a DNA-contain- ing cytoplasmic inclusion. Since origin from degenerating nuclei in this case is undisputed, Wigglesworth inferred such origin for all cases. Linder's (1956; Linder and Anderson, 1956) more recent observations support this conclusion. Gliicksmann (1951) in his classic review listed numerous descriptions of prominent, dark-staining bodies usually interpreted as degenerating cells in normal embryos. The Feulgen stain, whenever carried out, indicated nuclear material. Gliicksmann concluded that the bodies in all cases represented nu- clear degeneration which typically began with pyknosis, further changes occurring either in the isolated remnant or inside a neighboring cell that had resorbed it. Chang (1940) pointed out the large number and wide dis- tribution of the bodies in mouse embryos. He held that the bodies are within the cytoplasm, being phagocytized fragments of dead cells. According to Hamburger and Levi-Montalcini (1949), the entire body resembles a macro- phage in its reaction to vital stains. Nucleic acid normally moves from nucleus to cytoplasm by submicroscopic particles, but the same function may occasionally be accomplished by trans- port of large bodies. Here probably belong the examples of cytoplasmic DNA granules in certain plants (Sparrow and Hammond, 1947; Chayen and Nor- ris, 1953). A number of species of nematodes and insects undergo a chro- matin diminution process in connection with the segregation of the germ cells from somatic cells, whereby the somatic cells regularly cast out into the cytoplasm what may be a large part of the chromosomes (Wilson, 1934; Painter, 1959). Extranuclear DNA in nonexperimental pathologic states is usually inter- preted as degenerating nuclear remnants (Barthels and Voit, 1931). Other possibilities, especially in malignant cells, are nuclear buds which become enclosed in the cytoplasm, explained as an adjustment of the nuclear-cyto- plasmic surface ratio, and a direct extrusion of chromatin into the cytoplasm, leaving a hypochromatic nucleus (Ludford, 1942). Von Sallmann et al. (1955) in studying radiation-induced changes in the lens of laboratory ani- mals, where extranuclear Feulgen-positive bodies apparently are the pre- dominant pathologic finding, pointed out the striking analogy with age- induced changes. They considered the bodies to be extruded from the nu- cleus. Loewenthal (1957) interpreted the Feulgen-positive bodies found in large numbers in chick embryos homozygous for the "creeper" mutation as degenerating nuclei. Cytoplasmic inclusions containing DNA characterize a number of virus diseases. Leuchtenberger et al. (1956) recently applied electron microscopy and quantitative measurements of the DNA to the bodies constantly present in rectal polypoid tumors and concluded that they were viral. CYTOPLASMIC DNA IN IRRADIATED xNEURAL TUBE 81 Maximow (1925) recognized that the peculiar granules occurring in tissue cultures of young rabbit embryos were inclusions within the cytoplasm of otherwise normal appearing cells, and that, except for their enormous in- crease in number, they were identical wdth the inclusions of normal em- bryos. They were especially abundant in the neural tube and mesenchyme and were usually more numerous in the central part of an explant. He stated that they always first appeared as small granules in close proximity to the nucleus, and he assumed that the large granules resulted from the growth of small ones. Temperature changes ( Chevremont it ai, 1958 1 and chemical agents (Dustin. 1947; McLeish, 1954: Chevremont ct ai, 1958) may evoke DNA- containing cytoplasmic bodies. Explanations have varied. Dustin (1947) saw evidence that dividing cells respond to colchicine and a series of other mitotic poisons by nuclear pyknosis. followed by fragmentation and engulf- ing of the debris by the cytoplasm of surrounding cells. "Micronuclei" may result from chromosome breakage and be the source of DNA-containing bodies in the cytoplasm. Chromosome breakage may occur from intracellular metabolic disturbances resulting from changes in temperature or o.xygen ten- sion (Roller, 1954) and following exposure to chemicals (McLeish, 1954; Frederic it al. 1959). Chevremont vt ai, (1958; Baeckeland it ai. 1957) found that fibroblasts cultivated in the presence of DNase contained numerous Feulgen-positive granules in their cytoplasm. This DNA, which may amount to 90''r of the normal diploid nuclear value, was newly syn- thesized, as demonstrated by labeling with tritiated thymidine (Chevremont ('/ al., 1959). Since other nonphysiologic agents, including chilling to 20°C, gave similar results (Chevreinont irt al.. 1958), and in \iew of the evidence that cytoplasmic DNA may mean only that dead nuclear fragments have been phagocytized, the authors" interpretation that the bodies are altered mitochondria must be accepted with caution. Extranuclear DNA following irradiation may be the result of cell death by nuclear pyknosis, ''micronuclei" resulting from chromosome breakage, or a manifestation of other mechanisms. According to Spear and Gliicksmann i 1938) and Gliicksmann i 1951 ). cell death from radiation injury is by pyknosis, and the Feulgen-positive bodies represent pyknotic degeneration, which they subdivided into three stages: chromatopycnosis, consisting of the separation of the chromatic from the nonchromatic material and the precipitation and coalescence of the chro- matin into granules; hyperchromatosis of the nuclear membrane, in which the chromatin, having united into a single body, lies against the nuclear membrane as a deeply staining rim or partial rim; and chromatolysis, with loss of the Feulgen reaction. The entire process may take place in about an hour. Since onset of prophase in itself effects separation of chromatic from nonchromatic elements, cells degenerating in mitosis omit the first stage. 82 MARY ELMORE SAUER AND DONALD DUNCAN This behavior furnishes a means of distinguishing, on the basis of the Feul- gen-positive granules, between death of mitotic and of interphase stages. The granules become cytoplasmic when resorbed by a neighboring cell. In Fig. 1, based on the neural tube of the irradiated tadpole during the prolonged prophase extending from 11 to 24 hours, the low metaphase count means that heavy casualties occur on completing prophase. These casualties 5 ^5 V O o 50 o S 25 2 50 « 25 I Prophase Xi^ ^ V 1 Metaphase "^ -— ~__^ '•■. \ , — ^ •.\^.-' Telophase / \ II / B. Hyperchromatosis 'A \ C. 'Lysis> /K / ^^"^-^ '^ — ^^._. /;% ',^^/ A. Pyknosis "^»^,^ 8 40 48 16 24 32 Hours after irradiation MODIFIED FROM SPEAR & GLUCKSMANN, (1938) Fig. 1 . Chart showing the number of mitotic and degenerate cells in the brain and retina of young tadpoles for 48 hours after exposure to gamma radiation (268 r). \. Mitosis. Key: prophase, ; metaphase, ; anaphase and telophase, Although prophases are normally fewer than metaphases, from 11 to 24 hours after radiation the number of prophases greatly exceeds the number of metaphases, indi- cating a prolongation of the prophase period. The subsequent decrease in the number of prophases without change in the number of metaphases indicates degeneration of many of the prophases. II. Degeneration: Three stages, as applied to the nucleus. Stage A. Chromatopycnosis, . The chromatic material separates from the non- chromatic, with the chromatin material appearing as scattered granules. Stage B. Hyperchromatosis of the nuclear membrane, . The chromatin granules have united into a single, deeply staining mass sitting as a cap on the nuclear membrane. Stage C. Chromatolysis, The chromatin breaks into fragments, and loses its Feulgen staining properties. Since the mechanism of prophase effects separation of chromatic from non-chromatic material, a cell which reaches prophase before degenerating omits the first stage and passes directly into hyperchromatosis. The high increase in hyperchromatosis, be- ginning shortly after the peak of the prophase curve, indicates that the greatest num- ber of casualties occurs after the cell reaches prophase. (Modified from Spear and Gliicksmann) . CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 83 cause a rapid rise in the degenerate cell count, beginning at about 13 hours, and since the dying cells are mitotic stages, this rise is in the hyperchromatic type of granule. The smaller rise in the pyknotic stage shows that some cells also break down before beginning division. So-called micronuclei are a well known and classic effect of radiation. Although first seen by Koernicke ( 1905) in irradiated roots, it remained for Alberti and Politzer ( 1924) to show in the corneal epithelium of salamander lar\ae what becomes of a piece broken from a chromosome if it does not rejoin. Lacking a centromere, it does not move to one of the poles in ana- phase, but lags on the spindle and is usually not included in either daughter nucleus at telophase. Remaining in the cytoplasm, it becomes spherical and lies beside the chief nucleus. They named these Teilkerne or partial nuclei. Se\eral isolated chromosome fragments may unite into a single larger micro- nucleus ( Ohnuki and Makino, 1960). The great majoritv of so-called micronuclei are nonliving, spherical, deep stainins; bodies about 2 /x in diameter. Those few acentric fragments that contain both an adecjuatc amount of heterochromatin and a nucleolar or- ganizer may continue to li\e, however, playing an active part in cell metabo- lism and dividing synchronously with the main nucleus for several subse- cjuent life cycles i LaC^our, 1953: McLeish, 1954'). The completeness of the chromosome set is supposedly necessary for the normal functioning of the cell; consequently, the daughter cell with the deficient chromatin, presum- ablv the one in whose cytoplasm the micronucleus became enclosed, has been assumed to be short-lived. However, Ohnuki and Makino (1960) pro\ed sur\i\al throughout at least one mitotic cycle, and Hornsey 1956, 1960) showed that their ma.ximum number appeared at the end of the first mitotic cycle ( prolonged by irradiation ) and that their subsequent decrease was exponential, depending only on dilution by further cell di\ ision. Chromosome breaks may occur trom exposure ot a cell to irradiation in any stage of its life cycle. However, interphases in which the chromosomes are widely dispersed show a special resistance to chromosome breakage as compared to cells irradiated in the premitotic and mitotic stages when the chromosomes are tightly condensed. Muller (1954) reviews the factors in- \ol\ed. Lagging chromosome fragments are the chief change resulting from irradiation in interphase, being few in early stages and becoming more abun- dant as interphase progresses. Chromosomes irradiated in late prophase to early telophase, even though eflfectively broken, give no e\idence of being broken at the time, for when in the condensed condition they are held as if by some enveloping material and can not fall apart into fragments. How- ever, when the chromosomes recondense at the next mitosis, after an inter- xenina: interphase has elapsed, more structural changes appear than would ha\e followed irradiation in the interphase. Consequently, micronuclei are 84 MARY ELMORE SAUER AND DONALD DUNCAN 3 ^ '^ms CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 85 rare until cells ha\e undergone at least one mitosis subsequent to irradiation. Micronuclei are more prominent in some material than in others (La Cour, 1953). Both the number of fragments per cell and the number of cells with fragments increase with the dose ( Roller. 1947) . At their height, micronuclei may occur in lO^r or more of cells (Gray and Scholes, 1951 : von Sallmann, et al, 1957; Friedkin, 1959). Most ideas in the literature as to the nature of the bodies in the cytoplasm are based on interpretations of a static picture rather than on direct obser- vation. Of indisputable origin are micronuclei. Recordings with time-lapse photography have been made of the formation of micronuclei in irradiated cells and of their movement into the cytoplasm (Bajer, 1958; Bloom ct al., 1955: Ohnuki and Makino, 1960). The origin of a chromatin body in the cytoplasm through degeneration of a sister nucleus in a cell which did not complete division is well founded in a restricted field ( VVigglesworth, 1942) and has also been observed in irradiated tissue cultures (Stroud and Brues, 1954). Direct extrusion of nuclear material into the cytoplasm has ofen been postulated as the method of formation of these bodies, but apparently has not often been observed in irradiated material, nor has the gradual growth of a cytoplasmic body de novo in the cytoplasm, nor actual phagocytosis of degenerated nuclei. Our material confirms the observations of others (Butler. 1936; Schneller, 1951) working with irradiated chick embryos. Attention has been centered on the large amount of extranuclear Feulgen-positive material present, espe- cially in the neural tube. Apparently the presence of DNA in the cytoplasm is not a universal reaction to irradiation. Mitchell !l942) found the cyto- plasm of irradiated tumor cells consistenly negative to the Feulgen reaction. Our material justifies the conclusion that many of the bodies lie within the cytoplasm (Figs. 2-1 1 ) . This could often be demonstrated with the light microscope with Zeiss stereoscopic eye caps to give an exaggerated view of depth. The electron microscope pictures are indisputable (Fig. 12) Wanko etal.,\959. Spear and Gliicksmann's i^l938j and Glucksmann's (^1951) distinction be- tween two types of chromatin bodies, depending on whether death of the cell occurred in interphase or in mitosis, aids in identification of certain Fig. 2. Feulgen stain of the neural tube of a 3-day chick embryo which had re- ceived 200 r of x-rays 7 hours previously. Many of the cytoplasmic bodies are of com- pound nature (see arrows) containing one or several Feulgen-positive centers, accom- panied by Feulgen-negative material. Compare Fig. 10. Newcomer fixative. X 1200. Fig. 3. Brain of a 2'/2- to 3-day chick embryo which had received 200 r of x-rays 9 hours previously. The lumen is at the top of the figure. This field shows only minor change compared to much of the embryo. Carnoy fixative : May-Griinwald and Giemsa stain. X 1000. Figs. 4-6. From the same brain as Fig. 3 (2/2- to 3-day chick embryo, 200 r, 9 hours). Carnoy fixative; May-Griinwald and Giemsa stain. Fig. 4. The lumen is at the top of the figure. X 1100. Fig. 5. The lumen is at the top of the figure. X 800. Fig. 6. Enlargement of cell indicated in Fig. 5. X 3000. 86 Fig. 7. Brain of a 2yi>-day chick embryo whicti had received 1^00 r of x-rays 1 1 Va hours preceding fixation. Lumen is at the right. Newcomer fixative; May-Griinwald and Giemsa stain. X 900. Fig. 8. Neural tube of a 2'/;- to 3-day chick embr>'o which had received 200 r of x-rays H'/j hours previously. Newcomer fixative: May-Griinwald and Giemsa stain. X 1000. 87 88 MARY ELMORE SAUER AND DONALD DUNCAN bodies. Those which are dish-shaped, lens-shaped, broken ring-shaped, and possibly also those consisting of several granules, are features of the neuro- epithelial layer during the period of highest mitotic activity (8 to 15 hours after 200 r) when numerous cells break down in division. This period is approximately coextensive with the first mitotic period following irradiation. Since these bodies occur at all depths of the neuroepithelial layer, while dead cells are chiefly observed adjacent to the lumen, the bodies must have been moved into the depths of the wall following cell death. Although some are cast into the central canal, it may be speculated that others are resorbed at the lumen into the cytoplasm of adjacent nuclei which are beginning their postmitotic migration peripherally (Sauer, 1935; Sauer and Chitten- den, 1959; Sauer and Walker, 1959: Sidman et al, 1959; Watterson et al, 1956). Evidence for the actual process of phagocytosis is completely lacking in sectioned material; however, to one who has marveled at the antics of cells in tissue cultures, as demonstrated with time-lapse photography, rapid absorption of degenerated cell remnants seems plausible. The many chro- matin bodies surrounded by only intact cells can be explained readily in this way. In addition to the irregular bodies which seem to be degenerated mitotic cells, after irradiation there are also larger, Feulgen-positive bodies more nearly approaching a cell nucleus in size. After 500 r these are the first bodies to appear in the neuroepithelial layer. They begin about the time of resumption of mitosis and become numerous 4 to 5 hours following irra- diation (Fig. 9) . The picture is clean-cut, in contrast to the same region seen overlain with small fragments at 10-14 hours following 200 r. Similar large, spherical bodies occur in enormous numbers in the mantle layer of our older irradiated chick embryos and of irradiated Chinese hamster embryos of com- parable age. Their large sizes suggests whole pyknotic nuclei. It is assumed that they are differentiating cells which have died a pyknotic type of death in interphase. Since they begin to appear even in the period of mitotic arrest, they are unrelated to mitosis. (Hicks, 1958 and Hicks et al., 1959) has studied this radiosensitive form extensively. Some in the neuroepithelial layer are definitely cytoplasmic. In the mantle layer, few intact cells remain. Those Feulgen-positive bodies definitely enclosed in mitotic stages at the lumen are considered to be micronuclei. In some material they are rather numerous. As the nuclei of these mitotic cells migrate into the depths of the wall in their postmitotic period, their micronuclei will be drawn with them. Consequently, some of the mitotic bodies deep in the wall must also be micronuclei. How many of the numerous, spherical bodies, about 2 /x in diameter and apparently cytoplasmic, are actually micronuclei is unknown. That some may represent a direct extrusion from the nucleus is a possibility. This might CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 89 9-. ■• 11 90 MARY ELMORE SAUER AND DONALD DUNCAN Fig. 12. Electron microscope picture of a tangential section of the neural tube. X 15.000. Line indicates 1 micron. not represent e.ssential DNA but an excess formed in a period of derant^ed metabolism or the reversal of a synthesizinc; cell to its presynthetic stage. A related idea is an accumulation in the cytoplasm of nuclear material which difTuses freely but is normally present in only small amount. RNA increases in the cytoplasm followin" irradiation (Mitchell, 1942). Consequently, more DNA from the nucleus is transforming into RNA of the cytoplasm. Some of the cytoplasmic bodies might result from a derangement in this process. This has been a fascinating study, but it has raised more questions than it has answered. What is the potentiality of early neural tube cells for resoip- FiGs. 9-11 are on page 89. Fig. 9. Feulgcn stain of the brain of a 4J/j-day chick embryo iriadiated with 500 r 3 hours previously. The lumen is at the top of the figure. Newcomer fixative. X 750. Fig. 10. Feulgen stain of the brain of a 3- to 3'/; -day chick embryo which had been irradiated with 500 r 3J/2 hours pre\iously. The lumen is at the top of the figure. Most of the bodies contain both Feulgen-positive and negati\e regions. Carnoy fixative. X 1450. Fig. 11. Neural tube of a 2J/2-day chick embryo irradiated 8 hours previously with 500 r. The lumen is at the left. Newcomer fi.xative; May-Griinwald and Giemsa stain. X 1400. CYTOPLASMIC DNA IN IRRADIATED NEURAL TUBE 91 tion of contieuons solid material? What use is made of all of this cytoplasmic DNA? Surely a ready made supply of its own brand of DNA is a material too valuable to rapidly di\idino cells to be wasted. What is the role of DNase, or its absence, in connection with cytoplasmic DNA? Finally, there remains the unsolved problem of the great radiosensitivity of the differentiat- ing neinoblast. What change has suddenly come over this cell, probably still with mitotic potentiality, to increase its sensitivity and, with neurofibril ap- pearance, to lea\e it again to make it among the most resistant of cells? Summary The material consisted of 2- to 4-day chick embryos subjected to 200 to 500 r of x-irradiation or labeled with thymidine-H"* of high specific acti\ity. Moderate doses of radiation led to a structural change in the cytoplasm in numerous cells of the early neiual tube. A striking featvne a few hoins following exposure to ionizing radiation was the presence within the cyto- plasm of one or more dense, basophilic bodies approximately 2-5 fx in diam- eter. These typically consisted of one or several Feulgen-positive centers, surrounded by an RNA-containing rim. The centers were digested by DNase. They represent a relatively large amount of extranuclear DNA. Electron microscopy demonstrated their great density and confirmed their cyto- plasmic location. At the lower dosage of x-ray, the process was completely reversible, without an inteivening period of degeneration. The bodies were not confined to the neural tube, although they attained great prominence there, but were widely distributed throughout the embryo. It is concluded that the bodies are of se\eral types. It is possible to dis- tinguish between those resulting from degeneration of mitotic stages and those of interphases on the basis of their morphology. 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"The Cell in Development and Heredity," 3rd ed., pp. 323-328. Macmillan, New York. Biochemical Effects of Irradiation in the Brain of the Neonatal Rat* O. A. ScHjEiDE. J. N. Yamazaki, C. D. Clemente. Nancy Ragan, and Sue Simons University of California School of Medicine, Los Angeles; and V.A. Hospitals, Los Angeles and Sepidveda. California. Introduction From several perspccti\es the brain of the neonatal rat may be regarded as embryonic. The cortex and the cerebellum are relatively small compared to the brain stem (the more basic and primitive structure), and even in the brain stem the nerve tracts are incompletely myelinated ( Folch-Pi, 1955). The cell nuclei are lartjer and more hydrated than adult nuclei, and the neurons have not yet assumed their characteristic elongate and dendrite configurations. During the first 2 weeks, extensive cell division takes place in the cerebellum and cortex. In the brain stem cells increase greatly in total volume, but there is less cell division. Hicks I 1952 I has shown that neuroblasts in the brains of newborn rats may be exposed to more than an hour of total anoxia without breakdown, a finding that indicates a well developed glycolytic mechanism and relates these structures metabolically to other embnonic tissues (Gal rt al., 1952). Aside from purely practical applications of studies on irradiation damage to brain tissue in young animals, these embryonic properties of the brains of neonatal rats make them useful tools in the assay of radiation effects on cell mechanisms in general (and on ner\ous tissue in particular). The neo- natal brain presents a continuously changing aspect over a period of weeks. During this time radiation-produced lesions may manifest themselves in various ways. C'.onversely. certain mechanisms of difTerentiation may be further elucidated by the changes induced by radiations given soon after birth. The present work is a preliminary sur\ey of the effects of sublethal doses of x-irradiation to the heads of newborn rats. Several aspects of brain mor- phology, biochemistry, and metabolism have been followed up to 40 days after birth. * These studies were partially supported l)y the .\tomic Energy Commission. 95 96 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS Materials and Methods Litters of Wistar strain rats were di\ided into two "roups. The individuals of one group received 750 r of x-irradiation to the head only (the pituitary was irradiated as well as the brain ) at 2 days of age and were kept for vary- ing lengths of time before sacrifice. Individuals of the other group served as litter-mate controls. At sacrifice (by decapitation), the brains of all rats were separated into three precisely resolvable parts: brain stem, cerebellum, and cortex. Each sample was frozen and stored in a freezer. On thawing, the total wet weights of the organs were determined, as were total water contents, total solids, total nitrogens, total lipids, and total phospholipids. Selected samples were analyzed for wet volumes of nuclei, mitochondria, and microsomal fractions; total lipids were extracted and their fatty acid profiles obtained by gas chromatography. Water content was determined by disintegrating the thawed tissue in a piston-type disintegrator, adding acetone to a weighed sample of the wet mash, evaporating thrice under a stream of nitrogen at room temperature (Sperry, 1955), and then drying in an Abderhalden apparatus over boiling water for 3 hours in the presence of PO5. Water loss was measured gravi- metrically. Total lipid was extracted from the dried solids by addition of 10 ml of a 2:1 chloroform-methanol mixture. The extract was pvnified by Sperry's modification of the procedure of Folch-Pi rf al. ( Spen7, 1955), the total lipid being measured gravimetrically. Total phospholipids were deter- mined gravimetrically by extracting the total lipids with 15.0 ml of acetone to which one drop of saturated MgClj had been added. Nitrogen analyses on the solid residue remaining after lipid extraction was done by the micro- Kjeldahl technique. Subcellular components were isolated by differential centrifugation, and measurements of wet volumes of the fractions were carried out as detailed in a previous publication ( Schjeide, d al., 1960). Gas chromatography of the fatty acids derived from nuclei and mito- chondria^ was performed on the Model 10 Barber-Coleman apparatus, using a 50-in. column packed with commercially obtained ethylene glycol suc- cinate at 170°C. The detector was of the argon-ionization type. A 3 /aI aliquot of 2.5% in petroleum ether solution was injected into the column for each analysis. ' The methyl esters of these fatty acids were prepared by solubilizing the total lipid in 1 ml of dried benzene, placing this in a 15-ml glass-stoppered centrifuge tube to which was added 2 ml of 4% anhydrous methanolic HCl, refluxing 4 hours over methanol, extracting the pentane-soluble lipids in 10 ml of pentane, washing thrice with distilled H2O, shaking with magnesium sulfate (to remove residual H:;0), and finally absorbing away the nonesterified lipids on powdered alumina. BIOCHEMICAL CHANGES IN NEONATAL BRAIN 97 Results As the brains of youno or irradiated rats were removed from their brain cases, they appeared less firm and more hydrated than the brains of old or nonirradiated rats. Some of these tissues were analyzed immediately after removal from the animal. The brains of the youngest rats ( 7 days ) contained an average of 88.0^/h water. No modifying effect of x-irradiation was noted. By the time the rats were 40 days old, the cerebellums and cerebral cortexes contained 80.0*^0 water and the brain stems only TS.O^r. X-irradiation elevated the water content to SO.Orf in the brain stems. Only slight hydration was noted in irradiated cerebellum and cortex. Figures 1, 2, and 3 illustrate the efTects of radiation on the total dry weights of brain stem, cortex, and cerebellum. During the first 2 weeks after 750 r of x-irradiation, enlargement of the brain stem followed a normal course, but then the growth rate became some- what depressed in all groups of animals ( Fig. 1 ) . The dry weights of irradi- ated cerebral cortex were comparable to those of control rats until 16 to 20 90 80 Mgs. • Control O X-Ray t X-Ray 15 Days of Age 30 Fig. 1. Dry weights of brain stems from control and x-irradiated neonatal rats. days after exposure. At this time they fell off markedly (Fig. 2) . Cerebellums were profoimdly affected by radiations, retardation in growth being evident 98 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS 4 170 • / / 160 ■ / • r 150 ■ 140 • 130 ■ o • / 120 • • / O < 110 Mgs. • / o 100 / 90 • • / 80 • 70 • o < 60 • X* 50 / 40 / / 30 / • o control X-Ray 20 10 1 1 X-Ray 15 Days of Age 20 Fig. 2. Dry weights of cortexes from control and x-irradiated neonatal rats. during the first week after x-irradiation (Fig. 3). Figures 4 and 5 show that the amounts of total Hpid per unit dry weight of cortex and cerebeUum increased at the same rates with increasing age despite irradiation. Obviously, both total lipid and phospholipid were re- duced per whole brain in those animals that displayed decreases in brain BIOCHEMICAL CHANGES IN NEONATAL BRAIN 99 Mgs 15 Days of Age Fig. 3. Dry weights of cerebellums from control and x-irradiated neonatal rats. dry weights following radiation exposure. Preliminary analyses for total phospholipid revealed no consistent differences either among the different brain com{X)nents or between control and irradiated animals (phospholipid averaged approximately 80^r of the total brain lipid in both cases). How- ever, the unit lipid content of irradiated brain stern was decreased signifi- cantly at 16-20 days of age (Fig. 6). This inhibition of lipid synthesis was closely proportional to a retardation of normally occurring dehydration of the whole brain stem tissue. 100 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS O • • • 40 O • O O • O • < O 09 in >, BIOCHEMICAL CHANGES IN NEONATAL BRAIN 101 o • o* go 9b o 3 O GO •8 o • 102 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS • O Nitrogen values for lipid-extracted brain solids were obtained for rats aged 11-18 days. In nineteen groups, no significant differences could be demonstrated either as a function of age or of x-irradiation. There was an average of 83% protein and amino acid in the lipid-extracted solid material. The potentially most interesting stages with respect to variations in intra- BIOCHEMICAL CHANGES IN NEONATAL BRAIN 103 cellular nitrogen (and DNA), namely rats less than 1 week of age, have not yet been studied. Proportions of centrifugally isolated nuclei, mitochondria, and microsomes for cortices and brain stems of various age groups (with and without prior x-irradiation) are presented in Table I. Mitochondria in the cortex ap- peared to increase per unit cellular volume with increasing age (and devel- opment). The only consistent changes following irradiation appeared to be decreases in wet volumes of mitochondria in some of the younger (8- to 9-day) and older (28- to 30 day rats). However, since electron microscope examination of brain mitochondrial fractions obtained by the technique of Schneider and Hogeboom (1950) reveal the presence of materials other than mitochondria, the present results can only be regarded as tentative. Gas chromatograms of fatty acids from nuclei and mitochondria in TABLE I Wet V'olu.mes of Intracellvlar Components of Cortex and Brain Stem, with and withott Irradiation Sumber of Age (days) Organ Treatment (in r) V olurnes (Ml/4 ; ml)" animals Nuclei Mitochondria I Microsomes 6 8 cortex control 0.20 1.24 0.92 9 8 cortex 750 0.21 1.04 0.80 5 8 cortex control 0.48 2.84 1.60 5 8 cortex 750 0.30 1.80 2.00 5 8 brain stem control 1.20 4.80 .56 5 8 brain stem 750 0.40 2.24 2.64 11 9 cortex control 0.39 1.07 1.00 12 9 cortex 750 0.34 1 .00 .80 5 16 cortex control 0.76 2.80 1.96 4 16 cortex 750 0.80 2.96 1.80 4 20 cortex control 0.30 2.42 1.60 5 20 cortex 750 0.42 2.40 1.90 4 20 brain stem control 0.75 2.66 1.50 3 20 brain stem 750 0.40 2.60 0.68 6 28 cortex control 0.78 4.80 1.91 4 28 cortex 750 1.36 3.73 1.60 4 30 cortex control 0.32 4.05 2.02 3 30 cortex 750 0.27 2.68 3.46 30 cortex control 0.30 1.80 30 cortex 750 0.36 1.44 *MI per inl4 of starting tissue. In some ccises the sum of the wet volumes of the intracellular com- ponents exceeds that of the starting tissue. This phenomenon is due to uncontrolled hydration of the solids and inefficient packing in the volumeti ic tubes. 104 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS cerebral cortices of rats aged 8-9 days to 30 days appear in Fig. 7. The most prominent fatty acids in these cross sections include palmitic, stearic, oleic, arachadonic, and linoleic, in that order. Experience with fatty acids from the developing livers of chicken embryos has shown that, as the cell matures, the percentage of palmitic acid in the nucleus decreases and the percentage of oleic acid increases markedly (Schjeide, 1960). X-irradiation retards the adjustment of these fatty acid ratios, and it may that this is a reflection of inhibition of differentiation in the nucleus. Re-enforcing this finding is the observation that the fatty acid profiles of mitochondria from irradiated chicken embryos contain an increased percentage of oleic acid. Although the nuclear fatty acids of neonatal rat brains failed to show changes as dramatic as those observed in the embryonic livers, there was generally a decrease in the ratio of palmitic acid to stearic acid and oleic acid in nuclei as a function of increasing age (Fig. 8). In cortices of all stages the decrease of this ratio was retarded by a dose of 750 r (Fig. 8). The decrjease in ratio was also retarded in nuclei of irradiated brain stems (Fig. 8). Significantly, control fatty acid ratios of the nuclei in brain stems (the more mature portion of the brain at this early age) were those assumed to be more characteristic of adult-type nuclei (Fig. 8). Linoleic acid in brain lipids decreased with age, reflecting the develop- ment of the "blood brain barrier." Irradiation did not appear to have any consistent effect on the percentage of linoleic acid in the brain lipids. Discussion Although the foregoing represents a purely introductory survey of bio- chemical effects of radiation on maturing brain, three points of interest stand out at this early stage. First, per unit dry weight of cortex and cerebellum, the total lipid, total phospholipid, and total nitrogen appear relatively unchanged by exposure of the heads of rats to 750 r of x-irradiation. Thus, in these respects, the irra- diated neonatal cortex and cerebellum can be considered essentially as miniatures of their control counterparts, difTering primarily in having fewer total cells per organ and having these cells poorly arranged in the greater structural context. A similar situation appears to exist in the deformed skele- tons of rat embryos irradiated between 12 and 16 days of gestation (Russell, 1954). As far as is known, calcification mechanisms of the structurally-poor skeletons are unimpaired by the radiations that initiated the deformity; i.e., the enzymes involved in calcification appear to be present in sufficient quan- tity in irradiated bone. Contrasting with the lack of biochemical effects of radiations hereto ob- served on cerebellum and cerebral cortex is the inhibition of lipid synthesis BIOCHEMICAL CHANGES IN NEONATAL BRAIN 105 .jJk,^ Fig. 7(a) Fatty acid profiles of nuclei from cells of 8-day cortices. Solid line = control pattern. Dotted line=:pattern from x-irradiated cortices. Fig. 7(b). Fatty acid profiles of mitochondria from cells of 8-day cortices. Solid line=control pattern. Dotted line=pattern from x-irradiated cortex. 106 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS 1:1.30 ■ A ^ Brain / stem 1:1.40 / A Ratio 1:1.30 •^. 1:1.20 ^^ ,© 1: 1.10 .--'' • • .--?-t>- -'"' A« Control 1:1.00 o 1:0.90 ©""" A© X-Ray 1.0.70 16 17 20 Days of Age Fig. 8. Ratios of palmitic acid to combinL-d total of stearic and oleic acids in nuclei of control and x-irradiated neonatal rat cortices and brain stems. in the brain stem. Because of the important relationship that has been (in- directly) established between the neural cells and closely adjacent oligoden- droglia cells with respect to the production of myelin (lipid), it would appear to be important to determine histologically the ratio of oligodendrog- lia to neural cells in irradiated brain stem, for in this organ complex, not only does the inhibition of lipid synthesis correlate closely with the retarda- tion of dehydration, but the inhibition is first noted (ca 16 days) at a point in development when a large increase in myelination processes takes place. These results point up the fact that the various sections of the brain are by no means homogeneous tissues and offer the possibility that in brain stems the oligodendroglia are vulnerable to radiations at a time when the strictly neural elements may be relatively less so. (Such vulnerability could be ex- pressed either as outright destruction of the cells or alteration of their syn- thetic abilities.) Histological studies currently being carried out by one of the authors of this paper (C D. Clemente) should help in the elucidation of this issue. A second point of interest concerns the radiation-induced decreases in mitochondrial populations as obserxed in the cortices of some younger and BIOCHEMICAL CHANGES IN NEONATAL BRAIN 107 older rats. Such an eflfect is seen in cells of the embryonic chicken liver (Schjeide, 1960) and is thought to be indirect because of the time required (3 days) for the fall in population. It remains to be determined whether the decrease in mitochondria observed in the neonatal rat cortex is consistent and due to direct effects, to damage to a controlling mechanism in the nucleus, or to elicitation of toxic blood-borne factors. A third interesting result which may be a harbinger of better things to come with respect to elucidation of radiation effects on cell organelles, is the inhibition of change in fatty acid ratios of nuclei in all irradiated rats. The changes in nuclear fatty acid ratios that occur normally as a function of age have tentatively been interpreted as reflecting an advance in the ma- turity of these organelles. Thus, one of the interpretations for the inhibition of these shifts in ratios following irradiation is that maturation (or diflFeren- tiation) of the nucleus has been retarded due to injury by oxidizing radicals. However, in a heterogenous tissue, such as brain, changes in organelle fatty acid ratios may merely reflect changes in the proportions of resident cells. The importance of good histology as an adjunct to biochemical studies in these tissues is thus emphasized. In most animals receiving irradiatic^n to the head only, there was a de- crease in total body weight and the weights of such organs as liver, spleen, kidney and heart. Although the changes in weights of the various brain components did not appear to correlate closely with the weight changes of the above organs, the influence of irradiation on the pituitary (and hence a probable change in output of certain hormones) is a possible factor in- fluencing the observed results. However, a personal communication from Dr. Van Dyke, of the University of California at Berkeley, indicated that growth hoiTnone administered to irradiated neonatal rats has no effect in delaying the onset — or modifying the intensity — of neurological aberrations. Summary All three major divisions of the brain (brain stem, cortex, and cere- bellum) were inhibited in growth following irradiation (750 r) to the head at 2 days of age. Growth of brain stem was not retarded until about the 16th day, and due to a relatively slow rate of growth in the control animals, the difference in dry weight of this part of the brain at 4 weeks postirradiation was not great. In corte.x. the inhibition of growth was also first discernible at about 16 days, but due to a relatively fast rate of increase in the control animals, the differential between control and irradiated cortices at 4 weeks was very significant. Cerebellum was most profoundly affected by x-irradia- tion, the decrease in size being quite apparent early in the 2nd week follow- ing exp)csure. 108 SCHJEIDE, YAMAZAKI, CLEMENTE, RAGAN AND SIMONS Although in this preliminary survey no difTerences could be detected be- tween irradiated and control cortices (and cerebellums) in terms of relative lipid, relative phospholipid, and relative nitrogen, irradiated brain stem apf)eared to contain relatively less total lipid beginning at about 14 days fol- lowing exposure. The inhibition of lipid synthesis in the brain stem (failure of myelination) was accompanied by a parallel retardation of the dehydra- tion normally occurring as a function of age. Some tissues from irradiated brain revealed decreases in wet volumes of mitochondria. In no case was there a significant increase of mitochondria in irradiated tissue. The ratio of palmitic acid to stearic acid and to oleic acid in the nuclei decreased as a function of age. The de\elopment of this ratio was retarded in the irradiated tissues examined. References Folch-Pi, J. 1955. Composition of brain in relation to maturation. In "Biochemistry of the Developing Nervous System" (H. Waelsch, ed.), pp. 121-136. .Academic Press, New York. Gal, E. M., Fung, F. H., and Greenberg, D. M. 1952. Studies on the biological action of malononitriles, II. Distribution of rhodanese ( transulfurase) in the tissues of normal and tumor-bearing animals and the eflPect of malononitriles thereon. Can- cer Research 12, 574-579. Hicks, S. P. 1952. Some effects of ionizing radiation and metabolite inhibition on the developing mammalian nervous system. /. Pediat. 40, 489. Russell, L. B. 1954. Effects of radiation on mammalian prenatal development. In "Radiation Biology," (A. Hollaender, ed.). Vol. I, pp. 861-918. McGraw-Hill, New York. Schjeide, O. A. 1960. Unpublished data. Schjeide, O. A.. Ragan, N.. McCandless. R. G.. and Bishop, F. C. 1960. Effect of x-irradiation on cellular inclusions In chicken embryo livers. Radiation Research 13, 205-213. Sperry, W. M. 1955. Lipid analysis. Methods of Biochem. Anal. 2. 83-111. Some Effects of Nucleic Acid Antimetabolites on the Central Nervous System of the Cat* Harold Koemg Veterans Administration Research Hospital and Xorthuestern University Medical School. Chicago, Illinois Amont; the biolooic effects imputed to ionizini; radiation is a disturbance in nucleic acid metabolism. The deleterious effect of ionizin" radiation on DNA. particularly in proliferating tissues, is well known (Seed. 1960 i. Its influence on RNA metabolism has received less attention, lliat it is not without effect on the latter. howe\er. is suijgested by several recent studies (Krogh and Bersjeder. 1957; Schummelfeder. 1957). It may be sci'mane to this symposium to describe some of the effects of certain nucleic acid anti- metabolites on the mammalian central nervous system. These studies had their inception in obser\ations made earlier with the aid of taooed precursors which showed that neurons, olioodendroolia. and certain other cells are site of acti\e RNA and protein turno\er i Koeni',;. 1958a,b). A slow labeling of DNA also occurs among these cells, neurons excepted, which probably indicates cell di\ision. We ha\e attempted to interlere with these metabolic activities through the use of nucleic acid antimetabolites. Intrathecal administration was used to circum\ent the blood-brain barrier and to attain adequate local concentration of antimetabolities in the nervous system without damaging hematopoietic and other susceptible tissues. Of many purine and pyrimidine analogs tested, several ffuorinated pyrimidines were tound to produce interesting neurologic disorders i Koenig, 1958ci. The neurotoxic antimetabolites were 5-fluoroorotic acid i FO i , the analog of orotic acid > the natural precursor of pyrimidines i and the ribosides. 5-fluoro- uridine FUR) and 5-fluorocytidine FCR ) (Fig. 1 i. The pyrimidine bases, 5-fluorouracil • FU ) and 5-fluorocytosine i FC ) , were without overt effect, even in large doses. The clinical, pathologic, and biochemical effects of these analogs, particularly FO. on the feline neuraxis ha\e been imder inxestiga- tion for several years. .Although their biochemical effects ha\e not been * Supported in part by grants from The U.S. Public Health Service and the .Atomic Energy Commission. 109 110 HAROLD KOENIG OH 1 OH A i HO^ ^N-^ ^COOH HO ^N ^CO( Orotic ac id 5-Fluoroorotic acid (FO) Fig. 1. Structural formulas of orotic acid and 5-fluoroorotic acid. worked out completely, sufficient data have been collected to warrant the conclusion that the neurologic eflfects of these compounds are attributable, directly or indirectly, to disturbances in pyrimidine nucleotide or nucleic acid metabolism or both. Intracisternal Administration Intracisternal administration of 5-15 mg of sodium salt of FO (or 2-5 mg FUR) in cats produces a progressive rhombencephalopathy and, in some animals, a cervical myelopathy ( Koenig, 1958c). Signs of nemal dysfunction appear on the 4th to 6th day after injection. The first indication of disease is a mild clumsiness of gait, which worsens in time. The gait becomes broad- based, unsteady and dysmetric. Decomposition of movement, oscillation of trunk and limbs, and reeling gait complete the picture of cerebellar ataxia. This usually becomes so disabling that the animal is incapable of locomotion or alimentation by the 2nd or 3rd week after injection of the antimetabolites. Many animals have signs of neuronal irritation, including fasciculations of facial musculature, myoclonic jerks of lorelimbs, and various tonic and runnings seizures. Animals die of inanition, seizures, or bulbar failure by the 3rd week. Outstanding pathologic distmbance is a depletion of Nissl sub- stance in Purkinjc neurons of the cerebellum and in neurons of the brain stem and cervical spinal cord. Intraspinal Injection Injection of FO into the lumbar subarachnoid space ( 10-15 mg divided into two doses and injected 3-4 hours apart ) in cats produces a progressive myelopathy, which becomes evident on the 2nd or 3rd day (Koenig, 1960). FUR and FCR produce a similar disorder in doses of 2-4 mg. Signs of neuronal irritation appear first. These consist of muscle fasciculations, hyper- esthesia, and sometimes myoclonic jerks in the hindquarters (Fig. 2) and are associated with clumsiness, mild weakness, enhanced stretch reflexes, and NUCLEIC ACID ANTIMETABOLITES AND CNS 111 J^loo// V I— I = 25 m^ed. Fig. 2. Fasciculations recorded electromyographically in the hamstrings 3 days after FO. hypertonia of flexor muscles in the hindlimbs. In some animals this may not progress beyond a hyperreflexic paraparesis. In more severely affected animals, the signs of neuronal irritation diminish and paraplegia with loss ot muscle tone and stretch reflexes ensues. Sensation becomes obtunded, and the sphincters are paralyzed. These signs indicate a loss of neuronal function. Denerxation atrophy with fibrillary potentials may appear in the 2nd week, presumablv because some motoneurons are destroyed. Animals with a mild myelopathy, i.e.. a hyperreflexic paraparesis, after a period of stability or improvement enter a second stage of illness during the 3rd to 4th week. Their condition worsens, and within a day or two they exhibit an extensor paraplegia with dulling of sensibility in the hindquarters and acute urinary retention. Spastic weakness of the forelimbs sometimes occurs later. In most severely afflicted animals, flaccid paraplegia appears in 4 to 5 days. The forelimbs are affected early, and death occurs from respiratory failure by 5 to 7 days. In general, the larger the dose of the analog, the more severe is the myelopathy. The histopathology of the myelopathy produced by FO has been carefully in\estigated ( Koenig, 1960). The changes initially are confined to neurons. Inflammatory or vascular lesions do not occur. The nucleoli of nerve cells become small and less basophilic. Within 3 or 4 days a fragmentation and loss of peripherally situated Nissl substance is seen in spinal motoneurons and interneinons i Fig. 3 ) . Depletion of Nissl substance progresses to involve much of the perikaryon by 7 to 10 days i Fig. 4). Signs of recovery then ap- pear in \iable neurons. These consist of a striking hypertrophy and an in- crease in basophilia of nucleoli i Fig. 5 ) followed by increasing amounts of Nissl substance. Regeneration is well ad\anced by 35 to 50 days, and some neurons are even chromophilic. White matter is structurally intact for 2 to 3 weeks, but thereafter it almost always exhibits some spongy or mycrocystic degeneration with \arying Cjuantities of neural fat, either free or in macro- phages I Fig. 6 j . A thinning of oligodendroglia is seen in white matter at this 112 HAROLD KOENIG Fig. 3. Peripheral chromatolysis with reduction in size and basophiha of nucleoli in neurons of L-7 three days after FO. Thionin, X 700. Fig. 4. Generalized chromatolysis of lumbar motoneuron seven days after FO. Gallocyanin, X 800. Fig. 5. Recovering lumbar motoneuron 21 days after FO. Note hypertrophy and increased basophilia of nucleolus. Gallocyanin, X 700. stage. The white matter lesion occurs at a time when neurons are recovering and probably results from oligodendroglial disease. Signs of neuronal irritability are correlated with a minimal to moderate depletion of Nissl substance. Loss of neuronal function is associated with severe loss of Nissl substance. In most severelv affected animals, a necrosis of 113 Fig. 6. White matter, L-1, 50 days after FO. Note status spongiosiis. Weil stain, X 195. gray matter appears early, but this is not common unless 20 ma, or more of FO are used. The delayed, subacute deterioration in spinal cord function is associated with spongy deseneration of white matter. Intracerebral Administration The injection of 4-6 mg FO i 2-3 ms: FUR) into the sensorimotor cortex of the cat (3-5 mm imder the pial smface through a 26 gauge needle) pro- duces a focal cortical encephalopathy i Kurth (7 al., 1960). A moderate hemiparesis, propriocepti\e deficit, and impaired contact placing reaction appear in the contralateral limbs 3 or 4 days after injection. Focal motor seizures are sometimes seen. The disorder progresses for several davs and then becomes stationary or impro\es slightly. Neural dysfunction persists for several months. A focal electroencephalographic defect is demonstrable in the \icinity of the injection. Normal ihythms are replaced in part by slow wa\es, sharp waxes, and high \oltage spikes ( Figs. 7 and 8 ) . Focal seizure actixity appears spontaneously or may be provoked by joint movement or skin pinching of the contralateral limbs. Biochemical studies ha\e rexealed a high incorporation of FO-2-C" into RNA near the injection site. Indeed, there is a close correlation between the presence of electroencephalogiaphic abnormalities and a high uptake of FO into RNA of affected cortex. The introduction of FO or FUR into the temporal lobe of cats may pro- duce alterations in personality and epileptiform seizures (Koenig ct al., 1960bi. Some animals become withdrawn, imfriendly. hostile, and e\en aggressive, with periods of confusion, stupor, and other disturbances of be- havior indicative of temporal lobe seizures. Sometimes the contralateral pupil 114 HAROLD KOENIG Rt. frontal lobe Transverse frontal Vl Fig. 7. EEG 16 days after injection of 6 mg FO into right frontal lobe. Note numerous spikes. Pentobarbital anesthesia. Mit¥ Rl. fronUl loll,. V/'-'v^vv-i Ll. frontal lobe /^"^'L^l'l' Fig. 8. EEG 15 days after injection of 6 mg FO into right frontal lobe. Note paroxysm of high voltage activity. Pentobarbital anesthesia. beconie.s dilated, and the nictitating membrane retracts. Focal electroen- cephalographic defects appear 2 to 4 days after injection, with slow waves, sharp waves, and spikes (Figs. 9 and 10). Some of these abnormal rhythms are propagated to the opposite temporal lobe. Spontaneous focal and general- ized electrical seizures occur, even though animals are under pentobarbital anesthesia. Cytopathologic changes occur in cortical neurons which are simi- lar in character to those observed when FO is administered elsewhere in the central nervous system. However, the changes are mild and often unrecog- nizable when neuronal dysfunction is present. NUCLEIC ACID ANTIMETABOLITES AND CNS 115 = s z -^ o ,^_^ cTi JJ Cl, "(5 o o « o __ > n1 ii Sm *^ ^ o j:: ITl c b/1 ^ ^ >, -- o C .^ "t;^ (U 8; a- n, oi ^' S o- n1 ^ -a W li^ rt ' ' — 1 > C O rt rt ^ o ^ a-; - _2 c ,- rt tjH 'C Metabolism of FO The metabolism of taoged FO (FO-2-C") in the spinal cord of cats has been inxestigated by autoradiographic and biochemical methods (Koenig and Young, 1960; Koenig et al., 1960a). Column and paper chromatography have been used for nucleotide analysis. FO is metabolized similarly to orotic 116 HAROLD KOEiNIG _h h K. Rt. temporal lobe ^MA/n/j/i.v'Y^ Ll. temporal lobe Transverse temporal Fig. 10. EEG 6 days after 12 mg FUR into right temporal lobe. Note seizure ac- tivity. Pentobarbital anesthe.sia. acid, the natural precursor of pyriniidiiies in the nervous system. These studies have disclosed that FO is efficiently converted into the followina, acid- soluble nucleotides in nervous tissue: fluorouridine monophosphate, diphos- phate, and triphosphate: fluorouridine diphosphate-2,lucose and diphosphate- acetyl2,lucosamine, and fluorocytidine monophosphate (Fig. 11). FO is in- corporated into RNA as fluorouridine monophosphate, but is not incorporated into DNA. Unlike FO, 5-fluorouracil (FU), is not conxerted efficiently into acid-soluble and RNA nucleotides in the feline neiuaxis. Uracil itself also is poorly metabolized by this tissue. These observations suggest that nucleoside phosphorylase, the enzyme which converts pyrimidine bases to their ribosides, is present in scanty amounts in the central nervous system of the cat. Poor anabolic conversion NUCLEIC ACID ANTIMETABOLITES AND CNS 117 PUMP ♦•PCDP FCMP 70 FUDP +FCTP POMP i FUDPAG .FUDPG FUTP 100 150 200 Tube No. 2 50 300 Fig. 11. Chromatogram of acid-soluble fraction of spinal cord obtained by extended gradient elution with formic acid-ammonium formate from Dowe.x 1 column. Tissue removed 4 hours after the intraspinal injection of FO-2-C". Key: — D 260 um: Radioactivity of C". of FU and FC probably accounts for failure of these analogs to produce neurologic distiubances. Similar anabolic conversions of these fluorinated pyrimidines ha\e been described in other organs i Harbers ct al., 1959). The morphologic distribution of incorporated FO has been e.xamined by high resolution autoradiography ( Koenig and Young. 1960). FO is taken up into RNA of neurons and oligodendroglia and leptomeningeal. ependymal, and Schwann cells. Initially FO is incorporated into nuclear RNA. Some labeling of cytoplasmic RNA is discerned in nerve cells after a day or so; however, FO persists in nuclear RNA for a number of weeks. Metabolic Effects of FO The histopathologic changes produced by FO suggest a depletion of neuronal RNA. The results of biochemical analysis corroborate this inference (Koenig ct ai, 1960a) . The concentration of RNA in gray and white matter of spinal cord diminishes by 30-50% after 1 week. A greater depression in RNA concentration occurs in animals with areflexic paraplegia than in those with hyperactive reflexes. The incorporation of labeled orotic acid and 118 HAROLD KOEMG adenine into RNA in vivo is depressed by FO (Fig. 12). FO evidently inter- feres with the biosynthesis of RNA in neural tissue. The mechanism by which FO brings about a depletion of RNA, however, has not been ascertained at the time of this writing. The well-known participation of RNA in protein synthesis has led us to investigate the uptake of labeled amino acids into protein. Initially, FO does not depress the incorporation of tagged methionine and lysine into neural protein. A depression of 50-85% is observed after 1 week, however. Signifi- cantly, the greatest depression in uptake is observed in cases of severe neu- ronopathy, i.e., when areflexic paraplegia is present. Thus, a loss of neuronal function is associated with a greater depletion of RNA and a severe defect in protein biosynthesis in afTected nerve cells (Fig. 13). The formation of spurious RNA molecules also could contribute to the defect in protein synthesis. The role of pyrimidine nucleotides as cofactors in lipid and polysaccharide biosynthesis and in interconversion of sugars has been recognized recently (Henderson and LePage, 1958). The formation of spurious fluoropyrimidine nucleotides suggests that disturbances in lipid and carbohydrate metabolism may be partly responsible for the neurologic disorders that are produced by the fluorinated pyrimidines. This possibility is being investigated. The bio- chemical basis for the neuronal hyperirritability also remains to be elucidated. If- ■4 Fig. 12. Autoradiographs showing depressed uptake of orotic-6-C" into RNA of lumbar motoneuron 2 days after FO. Control on left, experimental on right. X 700. NUCLEIC ACID ANTIMETABOLITES AND CNS 119 Summary > ? .• < Intrathecal administration of the fluorinated pyrimidines, FO. FUR, and FCR, results in interesting neurologic disorders, the nature of which depends on the injection site. Myelopathy, rhombencephalopathy, and cortical en- cephalopathy are produced by the intralumbar, intracisternal, and intracere- bral routes, respectively. An asymptomatic "incubation'' period precedes the appearance of neural dysfunction. Signs of neuronal hyperirritability ap{3ear *^«• J ■• * D FO HYPERREFLEXIC P.\RAPARESIS ■■'■ .■■••.' jk s ^Bfl^^^^B.* * t-^B^^^KKBK^^^^ * ^p,. £: ■ - '■#* 7 D FO in'PERREFLEXIC PARAPARESIS 7 D FO HYPOREFLEXIC PARAPLEGIA Fig. 13. .Autoradiographs of L-7 segment showing uptake of methionine-S"^ into neuronal protein. Note reduction in blackening over experimental neurons three and seven days after FO, most marked in animal with hyporeflexic paraplegia. X 700. 120 HAROLD KOENIG first. In more severe intoxications, loss of neuronal function follows the stage of neuronal irritation. Alterations in neuronal structure accompany the neurologic disorder. RNA-containing structures, i.e., nucleoli and NissI bodies, are conspicuously aflfected. The neuronopathy may be reversible or may result in necrobiosis, depending on the severity of intoxication. Spongy degeneration of white matter occurs later, probably caused by oligoden- droglial disease. FO undergoes conversion to acid-soluble nucleotides and is incorporated into RNA in the feline neuraxis. Indeed, anabolic con\ersion of the fiu- orinated pyrimidines seems to be a requirement for the production of neural dysfunction. A depletion of RNA and a depression in protein biosynthesis appear later and are most marked in neurons that become inexcitable. Dis- turbances in other metabolic spheres may e.xist, but have not been demon- strated. Many points of similarity, both physiologic and pathologic, can be discerned between the disorders described and some virus infections and degenerative diseases of the nervous system. It seems possible, therefore, that derangements in pyrimidine nucleotide or nucleic acid metabolism may be present in some of the latter disorders. The fiuorinated pyrimidines are useful tools for the production of focal neuronal disease. Their use may provide experimental models for some of the degenerati\e neuronal diseases that afflict man. References Harbcrs, E.. Chaudhuri, N. K.. and Heidtlberger, C. 1959. Studies in fluorinated pyrimidines: VIII. Further biochemical and metabolic investigations. /. Biol. Chem. 234, 1255-2162. Henderson, J. F., and LePage, G. .\. 1958. Naturally occurring acid-soluble nucleotides. Chem. Revs. 58, 645-687. Koenig, H. 1958a. Incorporation of adenine-8-C" and orotic-6-C" acid into nucleic acids of the feline neuraxis. Proc. Soc. Expl. Biol. Med. 97, 255-260. Koenig, H. 1958b. An autoradiographic study of nucleic acid and protein turnover in the mammalian neuraxis. /. Biophys. Biochem. Cytol. 4, 785-792. Koenig, H. 1958c. Production of injury to the feline central nervous system with a nucleic acid antimetabolite. Science 127, 1238-1239. Koenig, H. 1960. Experimental myelopathy produced with a pyramidine analogue. A.M.A. Arch. Neurol, 2. 463-475. Koenig, H. and Young, I. J. 1960. .Autoradiographic studies of nucleoprotein metab- olism in the neuronopathy produced by a pyrimidine analog. Anat. Record 136, 224. Koenig, H., Gaines, D., Wells, W., Young, I. J., and Muniak, S. 1960a. Unpublished data. Koenig, H., Young, I. J., and Kurth, L. E. 1960b. Unpublished data. Krogh, E. v., and Bergeder, H. D. 1957. Experimental irradiation damage of the cerebellum demonstrated by gallocyanin-chromalum staining method. /" Cong. NUCLEIC ACID ANTIMETABOLITES AND CNS 121 intern. Sci. Neurol., Brussels, 1957: 3' Congr. iniern. Neuropathol. pp. 287-294. Acta Medica Belgica, Brussels. Kurth, L. E., Koenig, H., and Freyre. J. 1960. Frontal lobe encephalopathy with focal seizures produced with pyrimidine analogs. Trans. Am. Neurol. Assoc. 85, 215-216. Schiimmelfeder, N. 1957. Fluoreszenzniikroskopische und cytochcmische untersuchun- gen iiber Friihschaden am Kleinhirn der Maus nach Rontgenbestrahlung. /"" Congr. intern. Sci. Neurol, Brussels, 1957: .1' Congr. intern. Neuropathol. pp. 295-308. Acta Medica Belgica, Brussels. Seed, J. 1960. Inhibition of nucleic acid synthesis caused by x-irradiation of the nucleolus. Proc. Roy. Soc. B152, 387-395. Geographic Distribution of Multiple Sclerosis in Relation Geomagnetic Latitude and Cosmic Rays* John S. Barlow Massachusetts General Hospital, Boston, Massachusetts Introduction Despite the fact that multiple sclerosis has been known as a clinical entity for over a century, its etiology is still an enigma (Schumacher, 1960). Moreover, there is no specific treatment for the disease, which in its later stages often results in severe crippling. The disability results from interfer- ence with the processes of electrical conduction along nerve fibers in the brain and spinal cord as the myelin sheath of the fibers degenerates in local- ized regions; the term "demyelinating disease" is accordingly used. One of the interesting aspects of the disease is its geographic distribution. It has been a clinical impression for some years (Steiner, 1938) that multiple sclerosis does not have a uniform distribution throughout the world, and several epidemiologic surveys have been undertaken to clarify this distribu- tion (McAlpine et al., 1955: Hyllested. 1956: Kurland ct ai, 1957). The disease appears to be appreciably more common in northern than in southern latitudes in North .America and in Europe, but uncommon in the Orient, South America. Africa, and the tropics and subtropics. There have been several possible explanations ad\anced for this geo- graphic distribution, some of which I have re\iewed elsewhere (Barlow, 1960), but none has appeared to be consistent with all of the a\ailable data for the distribution. More recently, Acheson et al. (1960) have found that the geographic distribution of multiple sclerosis among veterans in the United States correlates strongly in an inverse manner with the average solar radiation of place of birth, and in particular with the December solar radia- tion, the implication being that this agent may in some way act as a preven- tive or protective agent against multiple sclerosis. When the distribution by residence at onset of symptoms was examined, the correlation appeared best * This work was supported by the National Institute of Neurological Diseases and BHndnesSj U.S. Public Health Service. 123 124 JOHN S. BARLOW with geographic latitude. Since, according to these authors, the isolines for winter solar radiation follow the lines of geographic latitude fairly closely, it is not clear that the relationship observed between December solar radiation and distribution of multiple sclerosis by birthplace would prevail in a similar manner for other areas of the world, for geographic latitude itself does not appear to be a good correlate when data for the disease from different areas of the world are examined (Barlow, 1960). It may well be, however, that the geographic distribution of the disease is deteiTnined by several factors, and the importance of each of these may vary in different areas. The possibility that geologic factors, perhaps in trace elements, may have a role in the distribution of multiple sclerosis recently has been reiterated by Warren (1959). Latitude Distribution of Multiple Sclerosis In the United States in recent years. Dr. Leonard T. Kurland of the Epi- demiology Branch of the National Institute of Neurological Diseases and Blindness has been particularly concerned with surveys on multiple sclerosis, and it was as a result of his epidemiologic summary at the First International Congress of Neurological Sciences in Brussels in 1957 that the present ap- proach had its origin. At that congress, Kurland presented data for North America which indicated that the frequency of the disease is strongly dependent on latitude, and he suggested that any satisfactory explanation of the etiology must take the geographic factor into account (Kurland et al., 1957), a view also held by other observers (McAlpine et al., 1955). The variation of the disease with latitude particularly interested me, and I undertook to determine if there were any similarity between this latitude effect and the intensity of cosmic rays, whose distribution is well known to be dependent in part on latitude (Barlow, 1959). The variation of cosmic ray flux is determined by the earth's magnetic field and therefore is related to geomagnetic latitude rather than to geographic latitude. A map indicating geomagnetic latitude in relation to geographic latitude is reproduced in Fig. 1. The lines of constant geomagnetic latitude are skewed with respect to those of constant geographic latitude, derived from the fact that the earth's magnetic axis is inclined at an angle of about 10° with respect to its axis of rotation. For the eastern United States, the geomagnetic latitude for a given location is about 10° greater than its geo- graphic latitude; for western Europe, the two are approximately the same, and for eastern Asia, the geomagnetic latitude for a particular location is about 10° less than its geographic latitude. It is apparent then that the two latitudes may differ from one another by an amount up to plus or minus 10°, a total rans;e of about 20°. GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 125 y >_ ?•- » — t J^-^ > T ^ ^ ^ .K^, \ — ■ ri^-., ^T— --^^^r \Z ^ v- \\ T ^\^'^ :^: ;>^' iV^ A i:^^ Jr+T 11 _^ jk /'''^ %< ^Vt^ =T i^Xi:^^ \ i"^ ^ m-^^^M V^ \ b-\B=Ht'Mr V -^\ „.,--^ \ yK i __T 1 V y^ y^^ o ^ \_-«4k:-^ J \ — •^^! r' .-4-^ ^ -W >^^5^gl— J — M — / -- -^ f- — ^ Pi ^Md — V w'^^^^^jC/^ 4-^ /^ ~ J :«^»!^,i ^1 " j ^^^y^*-f ^^ ^ ~~~~-i^ ^!/>">* -L / -+ In \^ W\^j^r^ -Xi / Xx -iL \ )^ \ ~"^a7^ /i/ i / ■|^ N A "^//k^ ^^^ 1 i /_r~~ yX y^K ^ -;>^^^r\^ V ^ ^~~~7 ...,,_^ /""^i- .i- r /^' ^/^ 2^ X/ ^^ii > S #/^Tr-- XX ^ ^ \i "^ r-^ / r" fX5E -^ - 7^1, ; -^i ' t i^' jIk^ ^L-, X^i- c^^'^'i^ p^ — ^i f-A A-X* . ^ \ ~=^ ■^ ^ fl XT'' ^1 \ ^' ■ — ^?iri X T --f ——J t J ■^ V. J V \^ ■ 3 •- - tf^ • . ^--T^ \ \^ r_J^ -H • — 1 ( J___ \^% \^- r- \r" V-^T t \ ^ \^» ^^^ ^ L, h K \^-pr~. ' ' r i^ * < -A^l \\ u^ Prevalence Surveys To study the latitude effect for multiple sclerosis, several independent sets of statistics for the United States and other areas in the world were examined (Barlow, 1960). These included mortality data, prevalence data, (i.e. data 126 JOHN S. BARLOW concerning the total number of cases in a population at a particular time), incidence data (i.e. the number of new cases appearing in a population per year), and statistics concerning hospital admissions for the disease. Since multiple sclerosis is a chronic disease (the average case has a dura- tion of approximately 20 years after onset of symptoms) , it appears that the most reliable indication of its distribution may be obtained from prevalence data. Table I shows results from a series of surveys in the United States and TABLE I Multiple Sclerosis Prevalence Ratios for the White Population IN Selected Communities in the United States and Canada" Prevalence per Geogra phic Geomagnetic 100,000 Prevalence rel- City latitude rN) latitude CN) population '' ative ' to Winnipeg Winnipeg 50 60 42 (40) 1.0 (0.95) Boston 42 53 41 0.97 Denver 40 48 38 0.90 San Francisco 37 43 30 0.71 Charleston, S. C. 32 43 18 (12) 0.43 (0.29) New Orleans 30 40 13 (6) 0.31 (0.14) " Data from Kurland el at., 1957. * Values in parentheses are corrected values on "clinical review" of the reported cases. Canada. In the present study, emphasis is laid on relative rather than abso- lute frequencies of occurrence of the disease in diflferent areas, and the table indicates the prevalence in each city relative to that for Winnipeg. Relative 1.0 0.8 0.6 0.2 - OL- 20 30 40 50 30 40 50 60 Geographic latitude °N Geomagnetic latitude °N Fig. 2. Relative prevalence of multiple sclerosis in selected communities in the United States and Canada (see Table I). GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 127 prevalence is plotted against geographic and geomagnetic latitude in Fig. 2, and the points suggest a sigmoid curve. A normal sigmoid curve (i.e., the integral curve for a normal distribution) has been included in each plot, and it is evident that it is not possible to conclude from these data drawn from a limited range of longitude whether geomagnetic latitude is a better param- eter than geographic latitude. The general trend shown by the points between approximately 40° and 50° has been confirmed from several other types of data for the United States (Barlow, 1960; Acheson, 1959) , although the presence of a "knee" at about 50° geomagnetic latitude is not clear from these latter data. Evidence that a rapid rise in the frequency of the disease between geomagnetic latitudes of approximately 40° and 50° appears for other areas of the world in both the northern and southern hemispheres has previously been presented (Barlow, 1960). For further examination of the latitude distribution, prevalence data from recently conducted surveys presented at the Geomedical Conference in Copenhagen in June, 1959 (Hyllested, 1960) are listed in Table II. It also includes the data from Table I and results of other prevalence surveys. As in Table I, the mean prevalence ratio for locations of 50° or greater geo- magnetic latitude was determined, and prevalences relative to this standard (48 cases per 100,000) are shown. These data are plotted against geographic and geomagnetic latitude in Fig. 3. Since a wide range of longitudes is repre- sented in these plots, the sigmoid curve for the geomagnetic plot of Fig. 2 is reproduced in both the geographic and the geomagnetic plot of Fig. 3, and it will be reproduced in subsequent plots for comparative purposes. It is apparent from inspection of the two plots that at any given latitude the scatter of the p>oints is greater for the geographic plot than for the geo- magnetic plot, at least for latitudes of less than 50°. The scatter of the points above 50° is such that a "knee" or leveling off is not as clearly suggested as in Fig. 2. There is some indication, however, that the rapid increase of prevalence between 40° and 50° geomagnetic latitude does not continue upward in the same manner beyond 50°. Several independent surveys are represented in Fig. 3; therefore it is not possible to state how much of the scatter of points above 50° geomagnetic latitude is due to differences in survey procedures and how much is due to real differences in prevalence among the population groups. It is probably unlikely that the scatter is entirely due to differences in survey procedures. Even if uniform survey procedures were used, such a scatter of points could conceivably occur if the prevalence ratios among different population groups formed a normal distribution about a mean value, and the scatter might further be accentuated by differences in the size of the population groups. Particularly if the population is very small, chance variations in the observed prevalence ratios may be pronounced for occasional communities (Deacon et al, 1959). 128 JOHN S. BARLOW TABLE II Prevalence of Multiple Sclerosis at Various Latitudes oi O O C/D b. M W U ffi H H < (1. O z 73 o z en U5 s Pi < O C/2 •~ ^ CO _e ■*) ^ r~- "S S 3 o Qi s O oi ^ 0^ c5 tii S 3 U ^ ^: CO i GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 133 c o O > < Xi E "(5 < 1/5 be "o s s -^ 2 -^ c a D. ,^ O (« c ~ a o s •~; "o x: O u n ^ « ^ 1 c^ > S V -^ c j= ■a c 5 11 o .2 Ic 2 2 c a . £ — o >2.| ■r ^ o CO w 5 a CO a u o . Q OC -" c 134 JOHN S. BARLOW world, but their trend with latitude can be compared, and hence the plots shown in Fig. 5 were constructed from the last column of Table IV. Although there is little to choose between the geographic and the geo- magnetic plots with respect to the better fit by the sigmoid curves that have been included, superimposition on the plots of Figs. 2, 3, and 4 is possible only if the geomagnetic parameter is chosen. From their data, Grashchenkov and his collaborators (1960) separately examined the effect of maritime climate on the frequency of multiple sclerosis by comparison of the statistics obtained for several maritime cities in the TABLE V Relative Hospital Admission Rates for Multiple Sclerosis IN Selected Maritime Cities in the Soviet Union * Geographic G eomagnetic Percentage of R dative number City latitude latitude cases with M.S. of cases ** Archangel 65 59 3.2 L06 Riga 57 55 2.8 0.90 Odessa 47 43 L6 0.53 Sochi & Su khumi 43 38 0.58 0.19 Baku 40 34 0.45 0.15 " Data from Grashchenkov et al. (1960). '' 1.0 corresponds to the mean of the percentages for the two cities of greater than 50° geomagnetic latitude (i.e., 3.0%). i 1.2 - 1.0- 0.8 0.0--- 20 y 30 40 50 60 70 GEOGRAPHIC LATITUDE 'N 20 30 40 50 60 70 GEOMAGNETIC LATITUDE 'N Fig. 6. Relative number of hospital admissions for multiple sclerosis in selected maritime cities of the Soviet Union (see Table V). GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 135 Soviet Union. These data (Table V) also clearly indicate a greater fre- quency in the northern regions. Again, geomagnetic latitude is the better parameter if the results plotted in Fig. 6, are superimposed on those in Figs. 2-4. Predicted Geographic Distribution of Multiple Sclerosis To permit comparison between the data represented in the preceding figures and those from future surveys or from other sources, geomagnetic latitudes corresponding to relative prevalences of 0.1 and 0.9 were deter- mined from the sigmoid curve in Fig. 2 and are approximately 38° and 48°, respectively. These geomagnetic coordinates are indicated as isoprevalence lines by solid lines in Fig. 7. For inhabited areas of geomagnetic latitude less than 38° (i.e., the area between the two 0.1 lines), relatively low prevalence ratios of the order of 4—6 per 100,000 population are predicted. North and south of the 0.9 lines in the northern and southern hemispheres, respectively, relatively high prevalence ratios of the order of 40-60 are predicted. A rapid increase of prevalence with increasing geomagnetic latitude is predicted to lie between the 0.1 and 0.9 lines in both hemispheres. The predictions from such a map appear to be in reasonably good agree- ment with much of the available data concerning the distribution of multiple sclerosis (Barlow, 1960). The general distribution being relatively common in northern Europe and in northern North America, but relatively uncom- mon in the Orient, South America, Africa, and the tropics appears to be in accord with the map. Comparison of Multiple Sclerosis with Hodgkin's Disease The geographic distribution of multiple sclerosis has been contrasted with that for Hodgkin's disease on several occasions (Ulett, 1948; Kurland, 1952; McAlpine et al., 1955), since Hodgkin's disease, at least in the United States and Canada, appears to show little geographic variation. It is of interest to compare statistics for the two diseases from the present standpoint. For this purpose, mortality statistics for the two diseases in several localities through- out the world, compiled by McAlpine et al. (1955), are compared in Table VI and are plotted in Fig. 8. The statistics have limitations and may be subject to considerable sampling error since only one year is represented for each locality. Nonetheless, it is apparent that the latitude trend of rela- tive mortality for multiple sclerosis is somewhat similar to that suggested by the prevalence data in Fig. 3. Moreover, there is possibly less scatter of the points about the sigmoid curve (reproduced from the geomagnetic plot of Fig. 2) for the geomagnetic plot than for the geographic plot. For Hodgkin's 136 JOHN S. BARLOW GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 137 Qi c^ QjJ~« O — 'O OO-^ OOO OOOO OO OO— •OO— '-^-^— ' CNJ O — ^ O — ' O O -^ OOOO -- -^' o o ^ O ^ -H -^ -^ ^i c^io_ o^— iin -^^^co o— '— 'in co-^ oindiooi — r^iOiD o" O OOO o o o o o o o" o o o o' o o o -^ — — < -^ mo i^coo iMcor^ ooco-H JO — CO rn-t- -t--+'Tf co'-h-i- -f -V en 'T *+-+ cocnLO-f-t--t-in-t-Tf' C/D t/3 C/D C/D C/D C/2 c/l ^=0 "S.^"^ ^,oco cnooo-, o' I— j-a u , «« 3 ,^ T3 N jO rt o.i 4J -i:^ C c >,' !N rt iJ a. 3 5 • c 03 ?3 i-c .5 15 -o -2 ^ ^ COG,, rt jG eg bo e-o C c3 ■r.S^>^-c-=-^ 5 0) = o. ■£ •S 8 o . ^ .«; - s c - S g- 138 JOHN S. BARLOW MULTIPLE SCLEROSIS 1.0 « I.ei DEATHS PER 100,000 POP. PER YEAR GEOGRAPHIC LATITUDE °N OR S 20 30 40 50 GEOMAGNETIC LATITUDE °N OR S HODGKIN'S DISEASE l.O = 1.45 DEATHS PER 100,000 POP. PER YEAR 10 20 30 40 50 60 70 CtOCRAPnC LATITUDE °N OR S K> 20 30 40 so 60 70 CEOMACNETIC LATFTUDS °N OR ( Fig. 8. Latitude distribution of multiple sclerosis and Hodgkin's disease by relative mortality rates (see Table VI). GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 139 disease, however, the latitude trend is considerably more poorly defined, although there is some suggestion of an increasing mortality with increasing latitude. Further, there is little to choose between the geographic and the geomagnetic plots for Hodgkin's disease. Latitude Effect for Cosmic Rays The statistics for multiple sclerosis that have been examined here, as well as those considered previously (Barlow, 1960), appear to suggest that the geographic distribution of this disease is better correlated with geomagnetic latitude than with geographic latitude. Since the phenomenon of cosmic radiation is the only one known to be related to geomagnetic latitude,^ it is appropriate to examine the latitude eflfect for various cosmic ray parameters for comparison with the latitude effect for multiple sclerosis. Variation with altitude must also be considered. For multiple sclerosis the available data do not indicate any clear variation with altitude (Barlow, 1960), whereas for cosmic rays the altitude effect is generally large compared to the latitude effect. Such is the case for the ionization produced by cosmic rays as deter- mined by an ionization chamber (Fig. 9). At sea level, the latitude effect between 0° and 50° is only 14%, a much smaller effect than is apparent in Figs. 2-6. Between 40° and 50°, it is even smaller. The altitude effect of 50°, however, is such that there is about 70% more ionization at an altitude of 2,000 meters (6,500 feet) than at sea level. The latitude effect for multiple sclerosis thus is not in accord with that for the ionization produced by cosmic rays, except some of the data for multiple sclerosis are suggestive of a "knee" at about 50°, and a "knee" at this lati- tude is apparent in Fig. 9. Neither the meson component nor the nucleonic comp)onent (protons and neutrons) of cosmic rays near sea level had a latitude effect as pronounced as multiple sclerosis apparently has. More pronounced latitude effects are found at the top of the atmosphere (i.e. at about 80 kilometers or 50 miles), at heights where the atmosphere has not yet exerted its filtering and diffusing effects on the incoming cosmic ray flux. Thus, the total number of particles incident vertically at high lati- tudes is some ten times greater than the number incident at the geomagnetic equator (Fig. 10). Such a curve represents the flux for primary particles of all energies, and if specific energy ranges are examined, even more pro- nounced latitude effects are apparent, as indicated in the theoretically com- puted curves reproduced in Fig. 11. The sigmoid curve from Figs. 2-6 has been included in Fig. 11, and a close parallel is seen between this curve and that for primary cosmic ray protons of 4.5 Bev energy. This order of energy " The aurora borealis and australis appear to be indirectly related to cosmic radiation and the earth's magnetic field through the intermediary of the van Allen radiation behs (van Alien, 1959). 140 JOHN S. BARLOW 7.5 7.0 -i~ / 6.5 6.0 5.5 5.0 4.5 4.Q 2 3.5 g Barometer 45cm ; Altitude. 4360m/ / « / 1.72 ions ' 33 per cent D 2. -,tr^ _fl__ _«__ - Barom eter € Ocm o -J3W r 3.0 c ^ 2.5 2.0 A TITUQc c.\jyj\ji\\ U D A n^ ^ 8 .56 22p ons er ce nt ■) cm - ^^ \fK , n r^m hr— "2 , .22 ions 1.5 1.0 0.5 I J\ , ^Je^ L 10 20 30 40 50 60 70 80 90 Geomagnetic latitude Fig. 9. The latitude effect for the ionization produced by cosmic rays at different altitudes. The altitudes 2,000 and 4,360 meters correspond to 6,500 and 14,000 feet, respectively. (From Janossy, 1950, and adapted from Compton, 1933.) is somewhat more than twice the energy that corresponds to the rest mass of the proton or neutron and hence is in the range of the threshold energy for production of nucleon-antinucleon pairs (Segre, 1958). It is also in the same energy range as that required for a primary proton to penetrate the atmosphere and come to the end of its path in a thickness of some 10-60 cm of water equivalent (Aron et al, 1949), i.e., to come to rest in the brain or spinal cord of a human in the open air. The greatest biologic effect of GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 141 0.8 0.4 20 30 40 Geomagnetic latitude Fig. 10. Vertical flux of cosmic ray particles near the top of the atmosphere. (After Curtis, 1956, and from Puppi and Dallaporta, 1952.) e48.3 100^==* Fig. 11. Dependence of primary cosmic ray intensity on magnetic latitude (\). The ordinate represents the relative intensity for each of the various energies {p = energy of protons in billions of electron-volts; other parameters shown on the cur\'es are not relevant to the present discussion), expressed as the per cent of the maximum possible intensity for that particular energy. (From Richtmyer et al., 1955, and adapted from Lemaitre and Vallarta. 1933.) protons appears just before the end of their trajectory (Malis et al., 1957). However, the number of protons of a similar terminal energy at the surface of the earth which have been produced by the complex interaction of primary cosmic rays with the atmosphere is far greater than the number of 142 JOHN S. BARLOW primary protons in this category (Wilson and Wouthuysen, 1958), and the latitude effect for such secondary protons is much less than that for primary protons (their altitude effect is also appreciable). For these reasons, the striking similarity between the two curves in Fig. 11 must be considered fortuitous. Corrections to the Geomagnetic EflFect Since a rather narrow range of geomagnetic latitudes appears to corre- spond to the zone of rapid increase in prevalence of multiple sclerosis, further clarification of the details of the exact relationship between the lati- tude effect for cosmic rays and the earth's magnetic field possibly may be of importance for the present study (Katz et al., 1958). Thus, corrections for local variations in the earth's magnetic field may be necessary to provide a better fit for observed cosmic ray phenomena than geomagnetic latitude per se. The dashed lines in Fig. 7 indicate isoprevalence lines constructed on this basis and determined from data for cosmic rays published by Quenby and Webber (1959). There is little difference in the location of the two sets of isoprevalence lines in inhabited areas of the world except in central Asia, and the circles in Fig. 5 indicate the corrections that would occur on this basis for the two locations in this area represented in the figure. Discussion Cosmic radiation has been implicated as a factor in human disease in other studies (Morris and Nickerson, 1948; Wesley, 1960), but the marked latitude effect for multiple sclerosis appears to provide a greater possibility for distinguishing between geographic and geomagnetic latitude as a param- eter, a test which originally firmly established the relationship between cosmic rays and the earth's magnetic field (Compton, 1933). The present data and that examined previously (Barlow, 1960) appear to suggest that cosmic radiation in some way may be related to the occurrence of multiple sclerosis. Since at a given location multiple sclerosis apparently is distributed ran- domly among the susceptible population (Kurland et al., 1955) and since plaques of demyelination of this disease are largely randomly distributed in the white matter (Adams and Kubik, 1952; McAlpine et al., 1955), it would be an attractive possibility to attempt to relate a randomly occurring cosmic ray event to the trigger mechanism that Lumsden (1951) suggests may occur in the initiation of plaques of demyelination. Several considera- tions militate against such a direct relationship, if there is any relationship at all between cosmic radiation and multiple sclerosis. Among these consid- GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 143 erations are the generally greater level of radiation from terrestrial sources of naturally occurring radioactivity- as compared with that from cosmic radiation (Libby, 1955; Neher, 1957; Solon et al., 1960) and the lack of an altitude effect for multiple sclerosis. Since direct effects of cosmic rays at the earth's surface cannot be impli- cated, it is of interest to examine the latitude distribution of atomic nuclei made radioactive by cosmic ray events. These radionuclides include T (the hydrogen isotope, tritium), Be^ Be^", C\ Na^^ P^-, P^^ S^^ and CF^ (Suess, 1958). The production rate for cosmic ray-induced radionuclides is said to be greater by a factor of four at the poles than at the geomagnetic equator, following the latitude effect for the neutron component of cosmic rays (Suess, 1958; Kaufman and Libby, 1954; Simpson, 1951). Their concentration at the surface of the earth, however, is dependent on several additional factors, including half-life, diffusion in the atmosphere and the oceans, and meteoro- logic factors. Thus, the half-life of C^* (5,570 years) is so long that diffusion processes tend to minimize any variation of its concentration with latitude. Diffusion effects should be relatively small for elements with short half-lives, P^- for example (14.5 days). In any event, it would not appear possible that the latitude distribution of any cosmic ray-induced radionuclide would be as pronounced as that for multiple sclerosis. A latitude effect for the distribution of fall-out from heavy cosmic ray primary nuclei might be considered additionally (Wesley, 1960). Consideration of the problem of the latitude distribution of radioactive nuclei associated with cosmic rays is even further complicated by the fact that some of these nuclei are produced in the explosion of atomic and hydro- gen bombs. Thus, the amount of T that has been produced by hydrogen bombs is comparable to or larger than the total inventory of natural T on the surface of the earth, and artificially produced C^* had by 1957 increased the C^* content of atmospheric CO2 by about 10% (Suess, 1958). Although there is a pronounced distribution with latitude for fall-out debris, at least as indicated by Sr^" in the northern hemisphere (Fig. 12), the variation is with geographic latitude and not with geomagnetic latitude. It should of course be remembered that multiple sclerosis was well-known as a clinical entity long before the era of bomb testing. Linear vs. Nonlinear Dose-Response Relationships The above considerations concerning comparisons of the geomagnetic latitude distribution of multiple sclerosis with those for various cosmic ray ^Gentry et al. (1959) have recently found that areas with increased rates of con- genital malformations in New York State appear to be associated with increased levels of background terrestrial radiation. 144 JOHN S. BARLOW 1.0 - t^ 0.8 - 20 30 40 50 60 GEOGRAPHIC LATITUDE 1.0 - ?: 0.8 - o'-r 30 40 50 60 GEOGRAPHIC LATITUDE Fig. 12. Relative concentration of Strontium-90 versus geographical latitude. a. In the stratosphere, averaged over the period November 1957-November 1958. 1.0 = 12 mc per square mile. (Data from Feely, 1960). b. On the ground in November, 1958. 1.0 = 4.1 mc per square mile. (Redrawm from Wexler, 1960.) parameters are based on the assumption of a one-to-one {i.e., linear) corre- spondence between the exposure and the number of cases of the disease. Should a nonlinear relationship obtain, it theoretically would be possible for a more pronounced latitude distribution for the disease to result from a cosmic ray phenomenon of a given latitude distribution. (The altitude effect GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 145 for such a phenomenon would still have to be small compared with its latitude effect.) Such a nonlinear relationship between inciting agent and disease can obtain, for example, if the susceptibility of individuals in the population is distributed in a normal (Gaussian) manner. A normal distribu- tion for susceptibility and the concomitant nonlinear relationship between per cent incidence and dose rate at low doses appears to obtain for the occurrence of tumors in mice following exposure to ultraviolet light, and it has been suggested that a similar relationship may occur for the appearance of cancer in man following exposure to ionizing radiation (Blum, 1959a,b). Duration of Exposure to Inciting Agent Whatever is the cause of multiple sclerosis, a most interesting question arises in connection with the incubation period for the disease. Acheson et al. (1960) have pointed out that if the inciting or protective agents are prolonged in their effect, then the important factor in the history of indi- vidual cases will be that of place of residence over an extended period; alternatively, only the place of residence early in life might be the important factor. These workers suggest that a long incubation period may be impli- cated by their own findings as well as by the observation of Dean (1949) that multiple sclerosis is almost unknown in persons of European stock born and raised in the Union of South Africa, whereas it is more frequently described in persons born in Europe who have emigrated to South Africa. An analogous observation in Israel by Rozansky (1952) is being verified by Alter (1960). The present approach does not help in elucidation of this question, for it is conceivable that radiation could have either a single-shot effect, perhaps analogous in animals to the graying of hair produced by heavy cosmic ray nuclei at very high altitudes (Chase and Post, 1956), or alternatively it might have a cumulative effect, as does the carcinogenic property of ultra- violet radiation. Possible Experimental Approaches Despite the difficulties of establishing more than a correlative (and there- fore possibly fortuitous) relationship between cosmic radiation and multiple sclerosis, it is perhaps in order to consider briefly some possible experimental approaches to the problem, particularly if additional carefully collected epi- demiologic data substantiate the results so far obtained. These experimental approaches difTer somewhat according to whether it is the place of residence in adult life, or in early life, that is established as being the important factor in the geography of the disease. 146 JOHN S. BARLOW The early plaques of demyelination in multiple sclerosis appear pre- ponderantly around small veins (Adams and Kubik, 1952), and a similar localization appears for the demyelinative lesions of experimental allergic encephalomyelitis in some animals (Mc Alpine et al., 1955; Waksman, 1960). Further, induction of the lesions of the latter disease in predetermined loca- tions in the brain by use of physical agents has been reported by Clark and Bogdanove (1955). It might be of interest to determine whether lesions of experimental allergic encephalomyelitis could also be induced in predeter- mined locations by low doses of radiation from well focused beams of high energy particles (for example, of appropriately filtered high energy protons). Perivenous staining with trypan blue has been rep>orted in the demyelinative plaques of multiple sclerosis (Broman, 1949) and in lesions of experimental allergic encephalomyelitis in animals (Barlow, 1956; Waksman, 1960) ; a similar focal staining might be looked for following focused irradiation at low doses, since it is a known finding with much larger doses (Clemente and Hoist, 1954) . Should positive results be found from either of these experi- ments, a possible protective eflfort might be explored for some of the agents (e.g., cysteine) which are known to lessen the biological effects of ionizing radiation. If place of residence early in life is the imp>ortant factor, then possible relationships (direct or indirect) might be explored between radiation and immunochemical processes in early life which might underlie demyelinative processes in adult life. In connection with the experiments outlined, it should be noted that if cosmic radiation is at all related to multiple sclerosis, the mechanism of its action is likely to be different from the biologic effects of cosmic rays at high altitudes (Yagoda, 1957; Schaefer, 1958). Finally, the possibility should be kept in mind that some other factors (for example, certain types of infections, perhaps not known) might act as intermediaries between cosmic rays and multiple sclerosis. Summary and Conclusions Multiple sclerosis appears to exhibit a fairly well marked geographic dis- tribution, being appreciably more frequent in northern than in southern latitudes in certain areas of the world. It is generally agreed that this geo- graphic distribution must be taken into account in any satisfactory theory of etiology of the disease. Possible correlates of the geographic distribution which previously have been advanced have not appeared entirely satisfactory; certainly geographic latitude itself appears to be a poor correlate. Several independent sets of statistics on the distribution of the disease were examined from the standpoint of geomagnetic latitude, a parameter that is related to GEOGRAPHIC DISTRIBUTION OF MULTIPLE SCLEROSIS 147 the earth's magnetic axis in the same way that geographic latitude is related to the earth's axis of rotation. That the distribution of multiple sclerosis might be examined in this way was suggested by the fact that a variation with geomagnetic latitude is well known for the intensity of cosmic rays. The frequencies of the disease in widely separated areas were found to vary in a systematic manner with geomagnetic latitude; therefore, the possibility arises that cosmic rays in some way might be a factor in the occurrence of the disease. The latitude distribution for the disease bears some resemblance to that for a particular component of the primary cosmic radiation at the top of the atmophere; no similarly good correlate is apparent, however, among the various cosmic ray components at the surface of the earth, hence the similarity may be entirely fortuitous. Some possible intermediary factors examined appear to offer no great promise. 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Acta Medica Belgica, Brussels. GENERAL DISCUSSION Paul Henshaw (U. S. Atomic Energy Commission, IVashingtoti, D. C): I am indeed interested in the information Dr. Rugh presented with his spectacular slides of early embryos. Seeing evidence of deteriorating embryos at an early stage is to be expected in view of what is known about the quality of germ cells and the uterine bed in some situations. Certainly, degeneration is inevitable as a consequence of some of these conditions. Dr. Rugh has called attention to the kinds of ab- normalities that occur following different levels of exposure to germ cells and to early embryos, and it was particularly interesting that abnormalities show in organisms exposed to doses of 5-15 r. This is an extremely low level of irradiation, and I am sure it will be quoted repeatedly. I feel, therefore, that we should ask questions about the confidence he has in the findings. Dr. Rugh has shown abnor- malizes which indeed do show in samples of organisms that have been exposed to low doses of radiation, but such abnormalities will show as a consequence of other agents as well. I would be pleased if Dr. Rugh would cite the specific evidence he has that permits him to say the low level changes are due to radiation. The second point pertains to the abnormality that showed in the third generation. Were these actually due to radiation change involved in a germ cell? We know something about how cerebral hernias will result from damage to neural fold primordia, the failure to close the neural crest, which permits the brain to turn inside out. This is a developmental abnormality not connected with germ cell damage. If the same can come from irradiation of germplasm, this is exceedingly interesting. I would like Dr. Rugh to indicate whether he feels there is evidence that cerebral hernias may result from mutations in germ cells. Third, Dr. Rugh has emphasized that low levels of radiation produce developmental abnormalities and went so far as to call attention to the possibility that a large portion of the naturally occurring developmental abnormalities may be due to background radia- tion. If I were a physician, I think I would go along with Dr. Rugh's warning and be cautious about any exposure of embryos or germ cells, but I am a laboratory man. I would like Dr. Rugh to give his strongest evidence that environmental radiation is having a significant effect. Does he feel that background radiation can or does account for a substantial proportion of the abnormalities, having knowledge as we do that many things can produce the kind of abnormalities des- cribed? Roberts Rugh (Columbia University): In regard to your first question con- cerning whether we are dealing with low level effects of radiation or possibly other traumatic effects: We are currently studying the embryos found in 98 pairs of uteri of mice exposed to 15 r at 1/2 days, because we felt this sort of statement had to be quantitative and proven. We will have statistical data from over 1,000 embryos which received 15 r at 1/2 days with an appropriate number of controls. It is true that we get anomalies without irradiation, that is, without superimposed 151 152 GENERAL DISCUSSION irradiation from laboratory sources in addition to background irradiation; and it is also true, as you saw from the first slide, that we get about 7% death and resorp- tion of embroys in the controls. This may be due to cosmic radiation or natural background radiation, or even may be due to genetic causes. We do not know. We have been dealing with exencephaly as an anomaly that was definitely pro- duced by irradiation. We have never seen this anomaly except following irradiation, and we have produced this at the low level of 15 r in the early embryo. The second question dealt with genetic effects. I did not state that the three generations of exencephaly found following irradiation of the ovary or the testes were due to 15 r. It was due to a much larger dose as for instance, 100 r to the ovary of the great- grandmother. The important point was that the effect was carried through three generations. Obviously, it would be necessary to determine the statistical frequency. However, that it occurs at all following irradiation of the ovary is of concern to every potential mother. The point to emphasize is that this anomaly was produced by irradiation of the germ cells and was found in three successive generations. It therefore had a genetic origin. Snell showed about 1935 that x-irradiating the testes and having the male mate with a normal female produced in the second generation something like 35% of such anomalies. We have carried it from both the sperm and the egg through several generations, and we were simply empha- sizing that this anomaly appears to be similar, whether it is derived from an effect on the chromosome or an effect directly on neurogenesis. How it is produced genetically we do not know, but having had considerable experience in experimental embryology with amphibia and chicks, it seems to me it is probable that the damage was to chromosomes which at the time of gastrulation caused such dis- ruption in organogenesis that any gross change could follow. This happens to be one that is simply produced due to failure of closure of the cranial roof and loss of neuroblasts and probably osteoblasts during development. The third question related to the dangers of cosmic radiation. Like taxes, we are all faced with cosmic and natural radiation, and there is nothing much we can do about it. This may actually be good for evaluation! I think, however, in line with the last paper and those of Drs. Gentry, Wesley, and others, it may be proven statistically that there is some correlation between the amount of background radiation and the incidence of congenital anomaHes. If our thesis is correct, at this early stage of development the embryo is so extremely radiosensitive that 5 r causes a 10% increase of resorptions and 15 r causes exencephaly in the embryos which develop later. Percfval Bailey (University of Illinois): This afternoon I am handicapped by lack of intimate knowledge of the embryonic cerebral cortex and by the quantity of the material presented. You cannot really judge histologic material by a few projections. The material presented here seemed inadequate for any fine cytologic study. I suppose I should be happy that it is so, because that leaves an oppor- tunity for somebody else to make a good cytologic study of the effects of radiation on the cells of the cerebral cortex, with more adequate cytologic technic. Orville Bailey (University of Illinois): Dr Brownson, were you radiating the whole animal or the head only? And did you conclude that fractional doses of radiation produced less or more effect than the same total dose at one time? How long did the animals live? GENERAL DISCUSSION 133 Robert H. Brownson (Medical College of Virginia): To answer the first question, this was total head x-ray. Second, this was a cumulative exposure, and we have not compared the total radiation effect accumulated at one time with it. Concerning the effects of radiation, the alteration was cumulative in the direction of change which was more quantitative than qualitative. The effects we saw did not seem to show more severity in themselves individually, but more intensity through the actual quantity of such change. The total picture which we saw at the end, beginning with 2,000 r as a minimum dose, demonstrated individual changes at 228 days similar to those changes that we could see using the 5,000 r level following a shorter postirradiation. The problem was to have these animals survive. Many did not survive 228 days, probably due to being stressed with an additional nutritional deficiency. In testing these animals psychologically, we had to deprive them of some food, and this influenced the death rate which accelerated with the increasing cumulative radiation. The group which exhibited the greatest change was the 5,000 r cumulative group, which were not subject to any type of psychologic testing and went through a relatively normal span of 228 days. Much of the probelm in correlating the changes of one of the animals with 18-20 r with 5,000 r was that these animals had gone through 228 days normally while the 5,000 r animals did not survive. Most of the changes were quantitative and in general increased in direct proportion to cumulative dosage and, to a degree, to time after exposure. Orville Bailey: In the terms I ordinarily use it seems as the intensity of the radiation goes down, the amount of damage per total dose also falls. One can build up almost grotesque amounts of x-ray dosage without damage if given slowly enough over a long period of time. Most of the lantern slides which Dr. Brownson showed were in the acute phase of the reaction which is difficult to evaluate. The focal neuronal changes that were described seemed like small foci of "dark neurons," the change which Dr. Gammermeyer has studied. They are artifact or at least, reflect some terminal state of activity in that particular cell. Most of the changes in the Purkinje cells, as Dr. Vogel and associates have demonstrated, are quite frequently found in control monkeys. E. C. Alvord (University of Washington School of Medicine): I would like to stress one minor theme that was developed by Dr. Rugh and has recurred in rather low notes through most of these papers. This is the concept that the body as a whole is made up of a mosaic of many structures, each of which has vastly different sensitivities to radiation. This concept of a mosaic also applies within a part of the body, namely the nervous system itself. There are a number of syndromes that have been delineated, particularly by Maisin of Belgium, on the basis of survival times following various doses of x-rays to various parts of the body. He speaks of a "delayed head syndrome," which occurs in rats after 1,000 r to the head, the rats dying about 5 months later, and of an "oropharyngeal syndrome," which suddenly appears at 1,500 r and cuts the survival time down to 10 days. I would like to ask Dr. Brownson to define the exact site of the irradia- tion to the heads of his rats. I doubt that this included the whole head, since Maisin and others have found it difficult for rats to live beyond 10 days following irradiation of the whole head with 1,500 r or more. This "oropharyngeal syndrome" has been found to be due to the inclusion of the oral pharynx, tongue, and lower 154 GENERAL DISCUSSION jaw of the animal, damage to these particularly sensitive areas causing death by means that are not clearly defined. It would be particularly interesting if repeated doses of 1,000 r can avoid this syndrome and allow as much as 5,000 r to be given, but I would suggest that Dr. Brownson has irradiated only the forebrain so that he sees relatively little of the change in the cerebellum because of a slight rostral advancement of the posterior margin of his x-irradiation. My own experience is only with the adult animal. I am sure that in the adult guinea pig one has to include the cerebellum and has to go well below the cerebellum to produce neurologic signs and death of the guinea pig in a short time. This leads me to Dr. Schjeide's paper, in which, unfortunately, the wet weights are not available. I would predict that, when the wet weights become available, the most striking chemical change will be in the degree of hydration with marked edema of the cerebellum. Dr. Sauer, have you with tritiated thymidine been able to apply this at certain times after the irradiation, with the idea of establishing whether these DNA bodies are dead or still metabolically active? Wolfgang Zeman (Indiana University Medical School): I think we should strive for a more accurate definition of cumulative or fractionated doses. In 1949, I tried to arrive at an understanding as to the radiobiologic effectiveness of frac- tionated doses as compared to a single dose. My data at that time were rather scanty, but in the meantime Lindgren (Stockholm) arrived at a simple formula for converting fractionated doses into single exposures. He determined the x-ray dose which was necessary to produce radiation-induced brain damage in 50% of rabbits. He used various single and cumulative dosages and found that in plot- ting the morbidity dose (50%) in r logarithmically against the total amount of days over which this dose was given, also logarithmically, a straight line results which has a slope of about 0.26 for the adult rabbit brain. In other words, a dose of say 2,000 r given in one day, compares to a dose of 2,000 r times lO^-^'^ given over 10 days. For the human brain the slope has been shown to be about 0.34, and it stands to reason that each different species does have a specific slope. I would predict that within one species, the slope might be dependent on the develop- mental stage. I wonder whether Dr. Brownson would be good enough to convert his data into terms which would make for an easy comparison to the radiobiologic effectiveness of cumulative doses with single dose exposures. L. J. Peacock (University of Georgia): I would like to ask Dr. Brownson whether the decline in response rate in his rats was due to an error in their timing behavior or to a decrease in motivation. That is, was there a decline in the over- all food intake of these animals, or was it a matter of their not being able to properly time the intervals and schedule? Robert H. Brownson: Dr. Alvord, the radiation instrumentation was conducted by our physicists in the biophysics department. Each animal was prepared by placing it in a cage in which the head was elevated out of the cage and held by a clamp with the remainder of the animal shielded. A Victoreen Chamber R-Meter was used to monitor the dosage. Each animal received 1,000 r delivered at the rate of 237 r per minute. The animal was given 1,000 r per week, so that it accumulated as scheduled per week the desired total roentgen dosage. Our ex- perience with guinea pigs has indicated that they are more liable to radiation GENERAL DISCUSSION 155 death than rats. Our rats did well with 5,000 r cumulative total head exposure at the end of 228 days, provided they were not stressed as they were when they were placed in the Skinner box and tested for positive food reward. To reply to Dr. Peacock's question: to our way of thinking this change in the animal's behavior as related to its performance in the Skinner box was one that seemed to be motiva- tion. We tested our animals between and after each dosage and following the end of the first 5 weeks on food intake. The food intake of the x-irradiated animals versus the controls was not so different as to make us think that this was the whole picture. The controls when placed on a deprivation diet maintained their normal weight, which tended to climb slowly. We believe there is a definite food problem involved, but there may have been one of motivation also. We are now utilizing shock avoidance and positive food reward together in anticipation that this will help clarify the matter. John L. Falk (Harvard School of Public Health, Boston, Massachusetts): I was happy to see that Dr. Brownson was using long-term testing. Short-term tests involving food intake or various food-motivated performances might cause radia- tion sickness. Did Dr. Brownson use a variable interval schedule? We have been making readings in medial nuclei of the hypothalamus and getting increases in bar-pressing rates on variable interval schedules. Since there did seem to be some hypothalamic involvement in Dr. Brownson 's animals, I wonder if perhaps there was more involvement in the lateral nuclei, possibly indicating classical aphagia? Robert H. Brownson : I think the only answer we will have to this question is dependent on the results obtained with shock avoidance. We anticipate improved testing methods in our future plans. The schedule was aperiodic, and the tapes were run for 45-minute intervals. The periods ran 4-224 seconds apart with an average of 62 seconds between reinforcements. Cornelius A. Tobias (University of California, Berkeley, California): I had the pleasure of discussing with Dr. Barlow his interesting statistical findings and trying to encourage him and other people who are taking a similar approach to the explanation of some diseases similar to this as to etiology. However, there are several aspects that need further study and improvement. It seems to me that if multiple sclerosis is due to radiation, it is most likely that it should be due to early effects in the embryologic or even germ cell stage. It appears that neither the studies by Dr. Hicks nor Dr. Rugh show that multiple sclerosis is a frequent occurrence in animals developing from irradiated embryos or germ cells. Secondly, findings such as these should be correlated with other findings which were also discussed in Dr. Rugh's paper. For example, those by Gentry, who finds that in certain areas of the United States with high natural radioactivity background congenital malformations occur more frequently than elsewhere. If the Barlow and Gentry studies do not correlate, then it would seem that it is not the low ionizing component of cosmic rays that would cause the effect in multiple sclerosis, but some other component of cosmic radiation that occurs perhaps only rarely. There are some cosmic ray phenomena for which further work may be needed before correlations can be established. For example, the so-called Auger showers which are energetic showers arrive at ground level from primary cosmic rays which can give well measurable doses to a single individual. On the average one such 156 GENERAL DISCUSSION shower passes through the body of an individual once a year. Another possibility is that a small percentage of the primaries that come in would come down to ground level and would produce highly ionizing secondaries. If highly ionizing particles are more eflFective than x-rays, one could perhaps understand the lack of results in x-irradiated animals. One would still expect to find an altitude effect; for example, multiple sclerosis incidence in Denver should be higher than in New York. Secondly, one would expect that in some way the incidence of multiple sclerosis would correlate with the 11-year cycle. The primary cosmic radiation events near the North Pole change radically in this 11 -year sunspot variation period, and perhaps babies bom in periods of low sunspot activity might exhibit a higher statistical incidence of multiple sclerosis. If heavy particles should cause multiple sclerosis, this hypothesis could be tested by exposing embryos to radiations produced in accelerators, such as the heavy alpha particles and other nuclei. John S. Barlow (Massachusetts General Hospital): The points made by Dr. Tobias are well taken; I might make a few additional comments. Multiple sclerosis per se is known only in man. There are naturally occurring demyelinating diseases in animals, but as far as I know these exhibit no latitude effect. At a given latitude, there are regional variations in the occurrence of the diseases, and it is an inter- esting suggestion to determine whether these are correlated with variations in background terrestrial radiation. The Auger showers may well be of biologic importance, but I think that the energy of the original cosmic ray particles giving rise to such showers is so great that no latitude variation for the showers would be expected. Those few primaries that do reach the earth's surface certainly would have a high relative biologic effectiveness, but they apparently are far over- shadowed in numbers by similar secondary particles for which the latitude effect is not very pronounced between 40° and 50° geomagnetic latitude and for which an appreciable altitude effect is present. The question of variations with the 11-year solar cycle merits further investigation. For several of the recent surveys of mul- tiple sclerosis, data for a 10-year period were collected, and no systematic fluctua- tion with time is apparent from these data. They have not, however, been exam- ined, as far as I know, from the standpoint of year of birth of the patients, as suggested by Dr. Tobias. Dr. John A. Simpson, of the University of Chicago, has informed me that the solar variation at 50° geomagnetic north is about 25% and decreases to some 6% at the geomagnetic equator; an effect of this size might well be obscured in statistics for the disease collected for localities of latitudes of 50° or less. Studies in localities further north would be of particular interest from the standpoint of the solar cycle. SUMMATION BY CHAIRMAN* Samuel P. Hicks Harvard Medical School and New England Deaconess Hospital, Boston, Massachusetts The investigators have given summaries, and the discussants have added to this invaluably by emphasizing important points and questioning others. I will comment on matters that were not discussed and add information about the mechanisms of retinal, cortical, and cerebellar malformations produced in fetal and neonatal rats by 150-300 r of 250 kv conventional x-rays, a dose range widely used because of its selective effect and tolerability. The malformations are of prime importance to many of the experimental and behavioral studies to be reported later. In presenting his experiments Dr. Rugh has raised the important question of whether low doses (a few r) may impair the development of the embryo long before it begins to differentiate a nervous system. It is well-known that such early embryos have extraordinary powers of regulation and restitution, and a substantial number of cells may be lost; yet apparently normal individuals result. Dividing early embryos into two parts may result in two apparently norma! individuals. What we don't know is whether such embr)'os are really normal, although they have been reported to be so. The work Dr. Rugh and others are doing aims at exploring one aspect of this. At the morphologic level, the problem remains one of establishing a statistically sound relationship between the presence of necrotic cells in early embr^^os and an effect of low doses of radiation. There is no problem with higher doses — cells are killed. "Spontaneous" cell death has a way of turning up in a variety of circumstances during development, sometimes as a necessary normal process. As the dose of conventional x-rays is increased above 20 r at almost any stage of embryonic life, the number of dead cells increase, yet it is difficult to show that development has been impaired. In some later stages, for example when the neural folds are forming, the rat embr>'o can recover to a remarkable degree from excessive cell loss after 100 r or even 200 r, and it does so in a manner such as that described long ago in amphibians by Harrison and by Detweiler. Cpnira.ry to \:t:hatjp.r^ Rugh said, injuredpartsdo catch up successfully. In the face of this remarkable capacity for ernbryos toregulalestnicture after cell loss, we may have to look at other parameters when the resulting animal looks normal, but does not measure up in some aspects of function or behavior. Mutations and chromosome aberrations in the early embryonic somatic cells, alterations in other organ systems that indirectly affect the brain, and the indirect effects that irradiation of the mother has on her fetuses are some things to be taken into account. Dr. Brizzee's approach to the study of the effects of radiation on cortical growth is a new and promising one because some of the divided doses he gives probably kill few cells. Sixty r will kill some of the primitive cells. The single doses around * This research was supported by the A. E.G., the U.S. Public Health Ser\'ice, and the United Cerebral Palsy Association. 157 158 SUMMATION BY CHAIRMAN 200 r employed by us and others extirpate certain classes of primitive proliferative and migratory cells and at a given stage produce specific deficits which are translated by ensuing morphogenetic sequences into adult patterns of cortical and other malformations. Dr. Brizzee's effects seem to be different and involve sub- lethal alterations of the differentiating neurons and glia. He reports that the over all cytoarchitectural pattern in the cortex is well organized in rats treated, say, with 25 r successively on fetal days 10 to 17, yet there are significant cytologic deviations from normal. These include alterations of the neuron, of the size of the nucleus in relation to cytoplasm, and in how closely packed the cells are. Some neurons were much too large. Closer packing of cells may have reflected subnormal proliferation of dendrites or other deficiencies in fiber development. What controls the size of a cortical neuron? How much influence do afferent fibers have on determining cortical cell types, and what alterations in DNA and RNA might lead to these cytologic abnormalities? Dr. Brizzee's further studies on the morphogenetic sequences of events may tell. Dr. Sauer, extending the studies of the late Prof. F. S. Sauer on the nature of the proliferative neuroepithelium, has come up with a new concept of partially destructive injury to the nucleus of the radiosensitive primitive neural cells. We had always thought that the quickly destructive effect of 200 r on the postmitotic migratory and other sensitive cells was an all-or-none phenomenon. Histologic studies of a series of embryos removed by Cesarean section at hourly or 2 hour intervals up to 9 hours after exposure still confirms this for the most part, but there is probably no discrepancy in our findings. In the chick, 200 r kills relatively fewer cells than in the rat, and Dr. Sauer clearly showed that after this lesser injury sublethal effects occurred. What happens to these partially incapacitated cells? Do they grow up to be abnormal neurons like those in Dr. Brizzee's rats? Some of his doses in rats may have produced effects corresponding to those following 200 r in the chick. The malforming effect of radiation on the developing mammalian central nervous system and retina depends on factors that include the stage of develop- ment, the individual growth characteristics of the species, and the doses of radia- tion. The dose largely determines what cells are killed and, therefore, what kind of malformative sequences of growth will be set in motion. Considerable data are now at hand on the morphology, the mechanisms, and the reproducibility of the malformations induced in albino rats by 200 r of conventional 250 kv x-rays when given at any stage from the 9th day of embryonic life to more than a week after birth. The acute extirpative effect of this exposure seems to be chiefly limited to the post-mitotic and primitive migratory neural cells, but obviously some chromo- somal damage with delayed cell death must also occur. Figure 1 indicates in schematic form how different doses kill cells in the young brain. The cerebral vesicle of a 17 day fetal rat is represented and shows that the neuroepithelial zone is a thick pseudostratified layer of tadpole-shaped cells which replicate their chromosomes in the outer part of this zone and slide in to mitose in the lining. This was demonstrated by F. C. Sauer in 1935 and confirmed by Watterson et al. (1956), M. E. Sauer and Chittenden (1959), Sidman et al. (1959; Sidman, 1961), and by Hicks et al. (1961a, b). Postmitotic cells are shaded, and they are the SUMMATION BY CHAIRMAN 159 DEVELOPING CEREBRAL WALL INJ THE RAT 6^& 20 DAYS Fig. 1. Schematic representation of the neuroepithelium. (Adapted from Hicks and D'Amato, 1960a.) principal radiosensitive ones, we believe. By radiosensitive we mean killed and visibly necrotic in 2 or 8 hours. The threshold of this effect is about 20-30 r. Above 200 r the selectivity is gradually lost, and more and more mature cells are killed. Also, the time after radiation that they die may lengthen from hours to a day or more. A good many young cortical neurons are killed by doses of several hundred r, but most of them escape for a while. Even after 800 r some members of the proliferative cell colony remain, and for the few days that an embryo so exposed may live, these residual cells actually go on proliferating brain or other neural structures as best they can. The little figures at the right in Fig. 1 emphasize the changing ratios of mature to immature cells as the fore- brain develops. At 13 days most of the primitive cells are involved in the pro- liferative cycle, and a cell no sooner divides into two postmitotic cells than the daughter cells enter the premitotic stage of the cycle again. This frenzied growth sub.sides by term, although a series of bursts of mitotic activity occurs in the fore- brain proliferative colonies between 13 days and birth, complicating the assess- ment of just which cells are radiosensitive. A detailed account of the brain malformations, especially in the cortex, and the mechanisms of their formation can be found in Hicks (1958), Hicks et al. (1954, 1959), and Hicks and D'Amato (1960a). The patterns of cortex of a rat irradiated with 200 r on day 13 is so completely difTerent from one irradiated on day 20, or day 16, that from the neurologic standpoint, lumping such animals together in behavioral experiments as "prenatally irradiated" would be absolutely meaningless. Considerable data on the patterns of malformation of the retinas 160 SUMMATION BY CHAIRMAN and the mechanisms involved in their formation, as well as similar information about the cerebellum, are now available (Hicks and D'Amato, 1960a, b) ; Hicks et al., 1959). There is no simple formula for the eye and retinal malformations, and radiation on any day from the 9th fetal to the 8th postnatal day presents its own problem, as Fig. 2 shows. Proliferation in the retina of the albino rat ceases about 8 days after birth. Like the forebrain, the eye passes through a series of stages in which radiation does a variety of things. Restitution seems to be complete or almost complete, after the severe destruction that 200 r causes on day 13, and rosettes do not form. Rosettes are the cell balls that resemble distorted neural tubes and form from residual neuroepithelial cells after destruction of surrounding cells by radiation or other agents. Such distorted neuroepithelium continues to proliferate brain, cord, or retina, as the case may be. On day 20, for complex reasons, the retina is not very radiosensitive in the sense used here, yet on day 19 or 21, mostly because there are greater numbers of sensitive migratory cells present, permanent changes occur. Of special interest for behavioral experiments is the great severity of the malformations that characterize the retinas of rats irradiated on the first few days after birth. From the morphogenetic standpoint, some immature bipolar cells are already present at birth, but when radiation destroys much of the adjacent layer of primitive migratory cells, these infant bipolar cells are suddenly stimulated to grow and sprout fibers, forming a preco- cious plc.xifonn zone. When the residual neuroepithelium begins to catch up, it spawns another layer of bipolar cells and itself forms rosettes as it differentiates into the rod cell layer. (Fig. 2). Malformations of the cerebellum are just as complex in their mechanisms as those of the cortex, thalamus, spinal cord, or retina. Figure 3 shows drawings of EFFECT5 OF 150to200p ON THE DEVELOPING RAT RETINA HEAD OEFGCTi RosETres REDuCfftrJ "I Fig. 2. The spectrum of eye malformations produced by 200 r 2122 23 I 2 3fJS OM THE DAY 5HoWN IIttlE BRAIM5 SUOwJ 5rA,C>E Ar Tirf\e OF RADIATION ON 7 He 3«o DAY On thc Ist (teSTHATAL PAV <:& Radiatiom om THE ZiiT FerAL DAY Fig. 3. The spectrum of cerebellar malformations produced by 200 r fror fetal to neonatal life. (.Adapted from Hicks and D'.\mato, 1960b.) late actual brains representing the gross patterns of cerebellar malformation in the albino rat that result from 200 r on the days indicated. In the gray rat, as in the mouse, the cerebellum is a little more advanced in its schedule of development than in the albino rat. The little brains, drawn to scale, show the fetal or neonatal brain as it was when radiation was given. Malformations induced at earlier stages are described by Hicks et al. (1959). In certain respects there is a front to back sequence of maturation of folia and a lesser center to lateral sequence, which is reflected in a corresponding sequence of deformations. To press this generality further would be misleading, because difTerent regions, and even parts of folia, wax and wane in their growth patterns. While the folial pattern is maturing, the cytologic characteristics of the cerebellum are also being unfolded, and the cyto- architectural malformations do not at all parallel the folial malformations. The malformations induced in late fetal life are characterized by a normal cyto- architecture, while those representing the end of the 1st week are characterized by an ectopic, extra granule cell layer, outside rather than deep to the Purkinje layer. Damage to the neuroepithelium, which in the developing cerebellum comes to lie on its surface instead of lining a ventricle, results at this stage in a preco- cious coarse growth of the Purkinje dendrites. This, we think, blocks the further migration of the primitive cells which would complete the granule layer. The late comers simply stop in the molecular layer. Irradiation on the first days after 162 SUMMATION BY CHAIRMAN birth presents still other cytoarchitectural anomalies, including irregular ectopias of both granule and Purkinje cells. In summary, a certain amount of radiation delivered at given stages of early development results in unique patterns of response. The resultant morphologic patterns are distinctive for each stage, and it follows that the corresponding be- havior patterns must also be distinctive. References Hicks, S. P., 1958. Radiation as an experimental tool in mammalian developmental neurology. Physiol. Revs. 38, 337-356. Hicks, S. P., and D'Amato, C. J. 1960a. How to design £md build abnormal brains using radiation during development. In "Disorders of the Developing Nervous System," Charles C Thomas, Springfield, Illinois. Hicks, S. P., and D'Amato, C. J. 1960b. Malformation and regeneration of the mam- malian retina following experimental radiation. In "Symposium on Phakomatoses Cerebrale," (M. Feld, ed.), Salpetriere Hospital, Paris. Hicks, S. P., O'Brien, R. C, and Newcomb, E. C, 1954. Mechanisms of radiation anencephaly, anophthalmia and pituitary anomalies. Repair in the mammalian embryo. A.M.A. Arch. Pathol. 57, 363-378. Hicks, S. P., D'Amato, C. J., and Lowe, M. J. 1959. Development of the mammalian nervous system. I. Malformations of the brain, especially the cerebral cortex, in- duced in rats by radiation. II. Some mechanisms of the malformations of the cortex. /. Comp. Neurol. 113, 435-469. Hicks, S. P., D'Amato, C. J., and Joftes, D. L. 1961a. The nature of the radio- sensitive cells in the developing nervous system. In "Symposium on Effects of Radiation on the Nervous System" (B. Gross and V. Zeleny, eds. ). Intern. Atomic Energy Agency, Vienna, in press. Hicks, S. P., D'Amato, C. J., Coy, M. A., O'Brien, E. D., Thurston, J. M., and Joftes, D. L. 1961b. Some migratory cells in the developing nervous system studied by their radiosensitivity and tritiated thymidine uptake. Brookhaven Symposia in Biol. 14, in press. Sauer, F. C, 1935. Cellular structure of the neural tube. /. Comp. Neurol. 63, 12-23. Sauer, M. E., and Chittenden, A. C. 1959. Deoxyribonucleic acid content of cell nuclei in the neural tube of the chick embryo: Evidence for intermitotic migration of nuclei. Exptl. Cell Research. 16, 1-6. Sidman, R. L., 1961. Histogenesis of mouse retina studied with thymidine-Ha. In "The Structure of the Eye," Symposium, 7th Intern. Congr. Anatomists, New York, 1960 (G. K. Smelser, ed.). p. 487. Academic Press, New York. Sidman, R. L., Miale, I. L.^ and Feder, N. 1959. Cell proliferation and migration in the primitive ependymal zone; an autoradiographic study of histogenesis in the nervous system. Exptl. Neurol. 1, 322-333. Watterson, R. L., 'Veneziano, P., and Bartha, A. 1956. Absence of a true germinal zone in neural tubes of young chick embryos as demonstarted by colchicine tech- nique (Abstract). Anat. Record 124, 379. PART 11 Histopathological Changes Resulting from the Irradiation of the Nervous System Basic Problems in the Histopathology of Radiation of the Central Nervous System Orville T. Bailey University of Illinois College of Medicine, Chicago, Illinois This survey of the histopathologic chans^es resuhins; from radiation of the central nervous system will be confined to those detected by light microscopy in postnatal experimental animals and largely to the effects of gamma and roentgen radiation. Its purpose is to state some of the problems in tissue reaction following radiation as they are met by the neuropathologist and to illustrate rathei than offer solutions for these questions. Materials and Methods A series of experiments were carried out over many years in the Neuro- surgical Research Laboratory of the Children's Medical Center. Boston, under the direction of Dr. Franc D. Inoraham. in association with Drs. E. A. Bering, Jr.. R. L. McLaurin and others. Many results of these studies have been published Bailey ct al., 1957, 1958; Bering ft ai. 1955: McLaurin ct al., 1955 1. but the histologic findinss. especially in the spinal cord, ha\e not been described in detail. The experiments were of three types. In the first, tantalum'"-' wires co\- ered with polyethylene were inserted into the cerebral cortex of 40 monkeys (Macaca mulatta and Atelcs geoffroy). 1.5-2.0 mm posterior to the motor strip of the right cerebral hemisphere. The wires were removed after 2.5- 4.770 r had been deli\ered. Monkeys were allowed to sur\ive from 2 hoius to 33 months after completion of radiation. The polyethylene encasement was regarded as sufficient to pre\ent any tissue effects of beta radiation from the activated tantalum wire. In the second series of experiments, a piece of tantalum wire acti\ated in the atomic pile at Oak Ridge was encased in polyethylene and placed on the dorsal surface of the spinal cord or, in a few animals, beneath the skin over- lying the spine and fixed in place to the paravertebral fascia ! . Experiments were carried out in 18 Macaca mulatta monkeys with dosages varying from 165 166 ORVILLE T. BAILEY 208 r to 55,000 r, measured at the center of the cord. Survival times were from 1 day to 36 months. The third series was concerned with the eflfects of roentgen radiation in 17 Macaca mulatta monkeys receiving 138 r per minute to the lower dorsal region while under Pentothal or Nembutal anesthesia. Dosages varied from 4,000 to 54,500 r, and survival times from 5 days to 18 weeks. In one addi- tional monkey, both Ta'^- and roentgen radiation were used. Time Factor in Tissue Responses The influence of time on the appearance, extent, and character of the histologic lesions produced by roentgen radiation has been recognized for over 50 years, the early studies being based on the skin of those engaged in therapeutic use of this agent (Wolbach, 1909). Using dogs Nemenow (1934a, b) studied physiologically in regard to conditioned reflexes, Lyman et al. (1933) have demonstrated the increase in the intensity of histologic lesions in the brain caused by roentgen radiation as the interval between the end of radiation and sacrifice is increased. This paper also contains a thor- ough review of the literature to 1933, as does tliat of Warren ( 1943). The eflfects of radiation in experimental animals has been turther consid- ered by Scholz ( 1934, 1935). In fully grown animals, the immediate reaction is not detectable microscopically, but if the animals survive from 4/2 weeks to 1 year alter ladiation, histologic lesions are stiiking. In a study of delayed lesions of brain and spinal cord in the dog, Davidoff ct al. (1938) pointed out that the rapidity with which clinical exidence of spinal cord injury ap- peared is proportional to the dosage and that disabilities in the monkeys tend to be progressive. The importance of time as a crucial factor in the appearance and interpretation of cerebral changes induced by roentgen radiation was carefully studied by Russell it al. ( 1949). They ha\e shown that with dosages of 2,850 r in rabbits no histologic changes were found before 82 days, but were present after that in all but one animal (of 7). Behavioral changes and abnormal neurologic signs did not appear until about 100 days after radia- tion, yet three rabbits killed before these changes appeared (82, 85, and 90 days) showed well defined lesions. The brains of rabbits killed earlier than 82 days after radiation showed no changes detectable by light microscopy. They also found that reducing the dose of radiation lengthened the latent interval. These and other substantial contributions have clearly established that the intensity of the histologic changes induced by roentgen radiation increase with time interval between radiation and sacrifice or death of the experi- mental animal. There are indications that the character of the lesions also alters with time. HISTOPATHOLOGY OF CNS RADIATION 167 Such proiiiession of lesions apparently does not take place in response to beta radiation, at least not to the same decree as with roentijen radiation (Campbell and Novick, 1949: Edwards and Bao;t>;. 192'3). More recently, interest in the neuropatholo2;y of radiation has tended to be focused on the acute phase of the reaction (Haymaker ft al., 1958; Voy;el ct al., 1958). By the use of relatively large doses of gamma radiation, it has been possible to characterize the acute lesion as seen by conventional methods of light microscopy as one of acute inflammatory changes and de- generati\e alterations in the cerebellum ( Vogel it al., 1958). These changes are the direct eflfect of radiation on the brain, since they do not appear when the whole body of the animal is radiated and the head shielded. While the acute changes are definite, they are mild in comparison with those which develop as time after radiation is increased. In personal studies, the experimental procedure precludes critical evalua- tion ot the acute phases of radiation reaction. 1 antalum'''- deli\ers radiation at a rate such that se\eral days are recjuired to accumulate the dosages necessary. When Ta'^- wire, shielded with polyethylene to prevent beta radiation fiom complicating the picture, is inserted into the cerebrum of Fig. 1. tloronal section of monkey brain, showing representati\e lesion induced by insertion of Ta"'" wire. Dosage. 600 r in 5^ days. Left hemiplegia developed aftei 3 months. Sacrifice 1 year after radiation. Lesion measured 7x4 mm. 168 ORVILLE T. BAILEY monkeys posterior to the motor strip, an area of necrosis is produced (Fig. 1 ) . Observation of such animals gives some indication of progression in the lesions. Hemiparesis which developed in 6 of 21 monkeys did not appear until 3 weeks to 20 months after radiation, the animals previously being neurologically normal (Bailey ct al., 1958). Electroencephalograms a few days after radiation showed decreased voltage and some slow waves on the radiated side, while on the opposite side there was a predominant frecjuency faster than the one before radiation. This condition remained and was most prominent 6 to 8 weeks after radiation. The EEG pattern then reverted almost to normal. At longer intervals, slow waves again appeared on the radiated side, and many records demonstrated voltage asymmetry with decreased voltage on that side. Two years after radiation, 2 animals developed runs of spikes and fast activity localized to the region of radiation. One of these developed generalized clonic and tonic seizures 30 months after completion of radiation (Bailey et al., 1958). There was thus some EEG evidence of progression. These results are in fair agree- ment with those of Ross ct al. ( 1954), though the conditions of radiation are so different that direct comparison is difficult. The histologic changes in these animals became more striking as the interval between radiation and sacrifice was lengthened. There was more evidence of irregular streaks of injury extending out from the necrotic region in which the Ta^^- wire had originally been placed (Fig. 1 ) . Studies of radiation efTects in the spinal cord of experimental animals have not been numerous (Cairns and Fulton, 1930; DavidofF ct al., 1938; McLaurin ct al, 1955; Pendergrass ct al, 1922; Peyton, 1934; Sicard and Bauer, 1907). However, the spinal cord has pro\ed a \ery favorable area for the study of radiation efTects. In personal studies, clinical observations ot monkeys gave .some evidence of the time factor as an important consideration. With Ta^""- radiation of 18 monkeys, 2 developed transitory paraplegia with complete recovery, 6 perma- nent paraplegia, and 1 early paraplegia, then recovery followed by permanent paraplegia after 6 weeks. When roentgen radiation was used, 6 of 1 7 animals developed paraplegia. In some monkeys dying with complete paraplegia within a few days after either form of radiation, there were no histologic changes or only scattered vacuolation of myelin in white fiber tracts. In view of the striking alterations described in monkeys surviving for long periods after radiation, there is good evidence that the histologic changes become progressively more obvious, at least to light microscopy, as the inter\al be- tween completion of radiation and sacrifice is lengthened. The time factor thus becomes a dominant consideration in defining dosage effective in causing histologic change and especially in determining the ulti- mate effect on a living organism subjected to ionizing radiation. HISTOPATHOLOGY OF CNS RADIATION 169 High Energy versus Low Energy Radiation In pre\ious experiments, the tolerance of the spinal cord of Macaca mulatta for gamma radiation was approximately 135 r per hour and about 125 kv r for roentgen radiation ( McLaurin et al., 1955). Radiation from activated Ta has a much higher energy than the roentgen radiation used in our studies. These results suggest that, under the experimental condi- tions used, low energy radiation is slightly more effective in producing para- plegia than high energy radiation. This agrees with the findings of Arnold et al. (1954a). However, other investigators (Hicks it al., 1956) ha\e found that central nervous system tissues are more sensiti\e to high energy radiation. It seems difficult to reconcile these dixergent results. Among the studies, there are data from difTerent animals, and sources of radiation also \ary somewhat. E\en so. when both types of radiation ha\e been carried out in the same laboratory under conditions as nearly controlled as possible, contradic- tions in results remain. The situation is no clearer in regard to the sensiti\ity of tissues outside the central nervous system. The cjuestion remains a signifi- cant one ior finthei' inxestigation. Effect of Intensity of Radiation The intensity of radiation has emertied as an important factor in the tissue response in the nervous system (Hicks et al., 1956; McLaurin et al., 1955). Intensity as a factor in other organs has produced more equixocal results (Brunschwig and Perry, 1936; Pack and Quimby, 1932). The effect of the rate at which a given dose of radiation is administered is strikingly demonstrated by 2 monkeys in personal material. Each received the equivalent of 55,000 r of gamma radiation to the spinal cord, at the rate of 4.000 r per day in one and at 1,870 r per day in the other. The first animal developed a flaccid paralysis in the 2nd week after radiation, which pro- gressed steadily in severity until death at 2 months. The second monkey showed no neurologic deficit until its death irom an independent cause 4 months after completion of radiation. In the series in general, it was found that 7.500 r as a single dose were required to produce paraplegia, but two doses of 5.000 r were necessary (McLaurin (/ al.. 1955). These results are somewhat diflferent from those of Davidoflf <7 al. 1938). who found 5.000 r sufficient to cause paraplegia. However, they used onlv 1 animal at that dosage. Differences in Tissue Reaction with Age There is e\idence that changes in young animals are different Irom those in adults of the same species. In young animals, smaller doses of radiation are required to produce behavioral and histologic changes in the brain than in 170 ORVILLE T. BAILEY fully grown ones, and the period between radiation and overt tissue degen- eration is shortened (Clemente et al., 1960; Mogilnitzky and Podljaschuk, 1928, 1929; Scholz, 1934, 1935; Yamazaki ct al, 1960; Ziminern and Chavany, 1931 ) . Clemente et al. (1960) found that as little as 125 r of roentgen radiation to the head may result in microcephaly and cataracts if gi\en to rats 8 hours old, while 300 r produces abnormal neurologic signs and histologic changes in most rats at 1 day and 4 days of age, but not at 7 days. They feel that resistance of the brain to radiation becomes significantly increased toward the end of the 2nd week of postnatal life. Their histologic studies indicate a high degree of correlation between abnormal neurologic signs and histologic lesions in these immature mice. While some changes, predominantly vascular, are found in rats sacrificed at 48 72 hours, they state, "It seemed as though processes were under way which would result in larger necrotic sites, espe- cially in rats administered 1,000 r, had these animals been allowed to live for longer periods. This assumption seems especially \alid since other animals radiated at the same postnatal time and sacrificed 1 to 14 months later showed larger lesions in the brain." (Clemente ct al., 1960). This work is in some way reminiscent of Hicks's ( 1953, 1954) results in antenatal development. The occiurence of cerebellar changes and micro- cephaly is in accord with his timetable. The eye defects are of a different type from those Hicks produced by radiation early in pregnancy. For this reason, the results of Clemente it al. (1960: Yamazaki et al., 1960) in some ways may correspond to late prenatal radiation in certain other species of animals in which brain development is more advanced at birth. However, they are of particular interest because they are quantitative studies in brains with no, or restricted, regenerative capacity. They are also in accord with results in puppies reported by others (Lyman et al., 1933; Scholz, 1934,1935). It seems reasonable to conclude that the nervous system of young animals is considerably more sensitive to radiation than that of adult animals of the same species, that the time inter\al between radiation and o\ert histologic evidence of degenerative changes is less in young animals, and that abnormal neurologic signs, behavioral changes, and histologic lesions are regularly produced with lower dosages of radiation in young animals than in old ones. The particular alterations that accompany maturation in the neuron so that it changes its response to ionizing radiation in these ways remains an important problem. Vascular Responses to Radiation One characteristic of the reaction to radiation in all tissues is the develop- ment of degenerative and occlusive changes in blood \essels. The central HISTOPATHOLOGY OF CNS RADIATION 171 nervous system is no exception. There is general agreement that aherations in vessel walls are found in all phases of radiation reaction in the brain and spinal cord. The sequences in\olved and the importance of these changes in the total picture of radiation injury are still not entirely established. Clemente ct al. (1960) consider the earliest and most constant vascular reaction to be a swelling- of the cytoplasm of endothelial cells and an increase in the intensity of basic staining in the nucleus. They feel that this process may be reversible or arrested or may progress to capillary rupture and in- flammatoiy cell infiltration. Polymorphonuclear leucocytes appear soon after radiation, as early as 6 hours (Clemente and Hoist, 1954), and tend to dis- appear after about fi\e days (Clemente and Hoist, 1954; Haymaker ct al.. 1958). Rachmanow 1926) has demonstrated accumulation of trypan blue in the endothelial cells at this stage. These early reactions are most con- spicuous in capillaries, where they may be associated with detectable necrosis of the wall. Such lesions of capillaries would accoimt for the frequent occur- rence of minute hemorrhages in the acute phase of radiation reaction (Alquier and Faure-Beaulieu. 1909; Clemente and Hoist. 1954; Haymaker rt al., 1958 1 . In animals surviving longer after radiation, hemosiderin deposits mark the location of pre\ious small hemorrhages i Fig. 1 ) . Larger hemor- rhages rarely ha\e been described (Rachmanow, 1926: Scholz, 1935) and usually in animals surviving 3 or 4 weeks after radiation. C'apillary changes may be related to the increased permeability of the blood-brain barrier (Clemente and Hoist. 1954; Mogilnitzki and Podljaschuk, 1930) and per- haps less directly to brain swelling noted by sexeral authors ( Gerstner rt al., 1954; Ross <■? a/., 1953). Damage to larger \essels becomes more obvious at longer inter\als after radiation. Severe necrosis with disruption of vessel walls occurs in the brain, spinal cord and meninges. This is accompanied by cellular infiltration which presumably is at first polymorphonuclear, but at the stage usually seen is predominantly lymphocytic with a component of macrophages. In well marked examples, few vestiges of the structure of the \asculai wall remain (Fig. 2). The line of the endothelium and its basement membrane are sepa- rated from an adventitia which is hea\ily infiltrated with inflammatory cells, but is not necrotic i McLaurin ct al.. 1955). With time, there is repair and reshaping of the vascular wall, the media retaining its form, but being com- posed mostly or entirely of fibrous tissue i Fig. 3 i. Vascular occlusion is often completed by an organi"ed thrombus filling the lumen. This process as a general phenomenon of \ascular repair has certain analogies with the repair of vessels in hypersensiti\ity reactions i Hawn and Janeway. 1947, especially their Fig. 12). Such similarities should not be interpreted as indicating a related pathogenesis, but as reparatixe phenomena in ncciosis of similar distribution. 172 ORVILLE T. BAILEY Fig. 2. Vascular lesion in small leptomeningeal vessel near an area of myelomalacia. There is heavy inflammatory cellular infiltration of the necrotic wall, but the en- dothelial layer appears intact. Gallocyanin-van Gieson X250. Dosage 5,606 r Ta'*" in 3 months. Paraplegia at end of radiation: death 1 week later. Autopsy: myelomalacia 10th thoracic to 2nd lumbar segments. There is at least one other type of vascular occlusion in the central nervous system of chronic animals. This occurs when there has been further necrosis of the collagenous tissue produced in repair followed by secondary collagen- ous response, eventually producing enlarged, bizarre vascular channels with tiny lumina (Fig. 4) or none at all. These striking changes in vessel walls of chronic animals are not nearly so widespread in the area of radiation as in the vasculitis of smaller vessels in the acute phase. There is considerable evidence that the late vascular changes are segmental. Hence more vessels may have an occluded segment at some point than would be inferred from a single microscopic section or even from several sections of one tissue block. HISTOPATHOLOGY OF CNS RADIATIOxX 173 Fig. 3. Vascular lesion in small leptomeningeal vessel near. an area of myelomalacia. End result of radiation change with fibrosis of wall and complete obliteration of the lumen. Hematoxylin-eosin X250. Dosage 4,440 r Ta'''"' in 29 hours. No neurologic deficit. Sacrifice 4 months after radiation. Autopsy: partial myelomalacia at 12th thoracic and 1st lumbar segments. One of the most perple.xins; problems in the histopatholo<;y of radiation reactions is the relation of such lesions to other chano;es induced by this agent. As long ago as 1921, Bagg stated that x-ray injury to the brain is secondary to vascular change. Since that time, workers have been di\ided as to whether the changes in the parenchyma are ischemic and infarctive or whether they are direct eflfects of ionizing radiation without mediation through vascidar mechanisms. The opinion of the author, based on personal material, is in agreement with Arnold's et al. ( 1954b) that the effects on the nervous system are direct effects. The occlusion of vessels could account for areas of complete infarction in the region of their distribution. Such effects, however, are only a minor part of the response of the central ner\ous system to ionizins radiation. Effects of Radiation on the Neuron Largely through the work of Hicks (1953, 1954), it has become generally recognized that the developing neuron is highly susceptible to ionizing radia- 174 ORVILLE T. BAILEY Fig. 4. Marked vascular Irsions after intracerebral implantation of Ta""". Weil's method X30. Dosage 553 r in 13 days; sacrifice 16 days after completion of radiation. No neurologic deficit. Lesion measured 8 mm in diameter. tion. Resistance to such injury incieases in older animals. There is less gen- eral agreement as to whether the adult neurons are directly injured or whether the nerve degeneration is secondary to vascular lesions. In the acute phase of the radiation reaction to gamma and roentgen rays. the changes visible by light microscopy in the nerve cells are not striking. Alvord and Brace ( 1957) found that there is pyknosis of granule cells in the cerebellum, which is probably reversible and coincides with a period of clinical neiuologic dysfimction. This is maximal at 8 hoius after 7,500 r of whole body radiation. Similar changes are produced if only the hindbrain and cerebellar regions are radiated, but no alterations occur when this area is shielded. The same type of pyknosis in the cerebellar granule cells has been produced by Vogel et al. ( 1958) using cobalt''" ( 10,000 r), and they also feel that this effect is transient and reversible. The results of Hicks it al. ( 1956) are in agreement. In none ot these studies is there evidence of vascular HISTOPATHOLOGY OF CNS RADIATION 175 changes in close association with the cerebellar lesions: the alterations in the grannie cells are regarded as direct effects of radiation. Campbell ct al. (1946), on the other hand. ha\e found early changes in Purkinje cells. Haymaker et al. { 1958) have noted somewhat similar changes in Purkinje cells, but also have found these types of alteration in a control monkey. Nerve cell bodies in other regions of the ner\ous system are little affected in the acute phase of the radiation reaction. Brownson (1960), in determining whether any effects on these structures can be detected by changes in the perineuronal satellite cells, found no statistically significant alteration in neuron-neuroglia relationship ot the cerebral cortex alter 1,600 r. Though less extensi\ely studied, beta radiation apparently exerts an effect directly on the nerve cells (Campbell and No\ick. 1949). Delayed necrosis of the para\entricular and supraoptic nuclei of the hypo- thalamus has been demonstrated by Arnold 1 1 al. ( 1954b) . This is a specific effect and is produced by doses of 3.000 r or less, no radioselectivity being noted with larger doses. CMemente and Hoist (1954) also have encountered consistent inxohement of the hypothalamus. There is a high degree of radioselectivity for the white matter ' Arnold et al., 1954b I. The neuronal necrosis becomes progressively more e\ident as the interval between radiation and sacrifice of the animal is lengthened. 4'his delayed reaction is one of the most striking differences in histology between the acute and late phases of radiation change. An extensixe literatiuc is in almost complete agreement that the brainstem is the most sensiti\e region (Arnold et al.. 1954a, b. c: Colwell and Glad- stone. 1937: Demel. 1926: Ellinger. 1942: Ellinger and Davison. 1942: Mogilnitzky and Podljaschuk. 1928). Hicks and Montgomery (1952) also describe special sensiti\ity in parts of the ""oltactory brain."" At least in the dosages generallv used, the injurv tf) the brainstem in\ol\es both white and gray matter. In fact. C'.olwell and Gladstone i 1937 ) emphasize ner\e cell changes in these regions and in the central gray matter of the cerebrum. Personal material, using the monkey spinal cord, is in accord with the literature cited in regard to late effects on the neuron. The nerve cell bodies in both anterior and posterior horns show no detectable changes after extra- dural application of Ta'""-' or roentgen ladiation. except when in an area of total necrosis. Nerve fiber necrosis in the posterior portion of the spinal Cf:)rd is iiregular. but widely distributed, and extends little beyond the immediate zone ot radiation, either with Ta'^- or roentgen radiation. In the monkeys sacrificed in less than 2 weeks after completion of radiation, the only change found was occasional myelin degeneration in isolated segments. In later stages, the <'xtent of mvelin defeneration is somewhat greater than that of demonstrable 176 ORVILLE T. BAILEY Fig. 5. Longitudinal section through posterior (.oluinns ol spinal cord to show myelin sheath degeneration. Gallocyanin-van Gieson X250. Dosage 6,220 r Ta"" in 43/2 hours. Slight weakness of legs and loss of sphincter control 3 days after radiation: gradual recovery over 10 weeks; monkey then normal 1 month; gradual onset of paraparesis, persisting until sacrifice 9 months after radiation. Compare with Fig. 6. neuron disintegration, a finding in agreement with Reynolds (1946), who compares this effect with that obtained with multilayer films of lecithin radiated on a water surface. Doses as low as 600 r destroy the normal molecu- lar arrangement. In embedded sections, this myelin degeneration appears as scattered vacu- oles (Fig. 5), corresponding in distribution with droplets stained with oil red O (Fig. 6). The areas which fail to take such stains for the demonstra- tion of myelin as Weil's method are considerably larger than shown by the other two techniques. Reasons for this difference are not clear. The nerve fibers themselves are interrupted by irregular zones of necrosis affecting individual fibers (Fig. 7). Adjacent nerve fiber segments are swollen or partially fragmented. While the distribution of such injured nerve fibers corresponds closely to the area of radiation, it is possible with smaller HISTOPATHOLOGY OF CNS RADIATION 177 I'u:. h. Longitudinal section through posterior cohnnns of spinal eord stained with oil red 0, X250, to show the similarity in distribution of droplets stained by this method and vacuoles seen in Gallocyanin-van Gieson (Fig. 5). Same monkey as Fig. 5. doses or at the edye of the zone of reaction to find patches of degenerated fibers ( Fis;. 8 ) . The distribution, time of demonstration by histologic methods, and the relation to vascular changes all support the vievs- that the effects of gamma and roentgen radiation are direct effects on the parenchyma of the central nervous system and are not mediated through vascular insufficiency. The long latent period before these changes become apparent to the light micro- scopist is a time when the more quickly demonstrable vascular changes dominate the histologic picture. But this does not imply that the vascular lesions are causati\e. It suggests that changes at a submicroscopic, possibly molecular, le\el have been initiated at the time of radiation, changes which are compatible with preservation of morphologic structure for weeks or months. An endpoint must be reached not only before the light microscope can detect the changes, but also before the cellular sequences of repair are initiated. 178 ORVILLE T. BAILEY tj» ^ ''-^^ ■ - ^s^»^^ . '> -«'»«%■ •» ^» J^^ -i-^' - ;-;:,• Ar>. ^^^ ••--'^iVwlr" "Jr ..>^ HISTOPATHOLOGY OF CNS RADIATION 179 There is evidence ( Bailey <"/ «/., 1958: Gerstner c/ ai, 1955; McLaurin ct ai, 1955; Yamazaki ft ai, 1960) that in this latent period there may be func- tional changes related to the radiation injury before histologic change is established. The demonstration and definition of this latent interval is the role of the light microscopist ; the electron microscopist and chemist must elucidate the processes durino- that time. Such enzyme studies as that of Clammermeyer and Haymaker ( 1958) are promisino;. Effects of Radiation on Glia Arnold and P. liailey (1954) have pointed out that the response to \-radiation involves damage to all types of adult glial cells, depending on the total dose of radiation, the intensity of dose administration, the uniformitv of dose distribution, and the time inter\als between radiation and sacrifice of the animals. The oligodendroglia are early and se\erely affected, with swelling of the cytoplasm (Arnold and Bailey, 1954) followed by pyknosis and disintegra- tion of the cells (Hicks and Montgomery, 1952; Hicks ft ai, 1956). In \iew of the generally accepted role of oligodendroglia in the maintenance of the myelin sheath, this finding is of considerable interest because of the relatively early disintegration of myelin in radiated areas (Hicks ft «i ;'Xv-:: ^^:'$.^M^-:: Fig. 11. Area of necrosis in the spinal cord with connccti\e tissue proliferation and coni]30i.uid granular corpuscle formation. l)ut without astrocytosis. Gallocyanin-\an Gieson X 2'_'5. Same monkey as Fi-^. 7. insertion because of tlie possible participation of meningeal collagenous tissue. However, in the depths of the lesions at a few weeks or months after radiation, the same proliferation of collagenous tissue without admixture ol astrocytes is present (Fig. 12). Occasionally, it can be seen that collagenous tissue is growing into regions of fibrin deposition (Fig. 12). but usually such a relationship cannot be established. When astrocytic proliferation is re- sumed, these areas of collagenous scar remain lor the most part intact. An attempt has been made to hnd out whether there is secondary degen- eration of the collagen and regrowth of new collagen, as has been described in radiation reactions in the skin (Wolbach, 1909). Occasionally, hyalinized collagen fibers into which secondary proliferation of collagenous tissue has taken place, can be seen, but this is uncommon. Some vessel walls (Fig. 4) have appearances suggestive of repeated collagenous repair. 184 ORVILLE T. BAILEY Fig. 12. Clollagen production in the depths of an area of radiation necrosis at a stage before proliferation of astrocytes has begun. Gallocyanin-van Gieson X250. Dosage 600 r Ta'"" in 8 days to right corona radiata. Mild hemiparesis developed. Sacrifice 20 days after completion of radiation. Area of necrosis 7 mm in diameter. Radiation Reactions in Other Structures In the acute phase of the radiation reaction in menins^es, inflammatory changes with polymorphonuclear leucocytic infiltration are conspicuous with doses of 300 r or larger. The process is localized with smaller doses, but tends to spread more widely as the dose is increased. At longer intervals after radiation, the cellular infiltrate becomes mononuclear with some connective tissue proliferation. Vascular lesions occur within the meninges and undergo the same secjuences as those within the brain parenchyma (Clemente ct al., 1960; Haymaker et al., 1958; personal material). In the choroid plexus inflammatory changes similar to those in the menin- ges are present in the acute phase, occasionally accompanied by small hemor- rhages. The end result of these changes is a small fibrous scar. All portions of HISTOPATHOLOGY OF CNS RADIATION 185 the choroid plexus are about equally affected (Clemente et al., 1960; Hay- maker et al., 1958) . Dilatation of the ventricular system has been seen after radiation (Clemente et al, 1960; Demel, 1926), but this is variable from animal to animal. It is greatest in young animals. In the pituitaiy, both anterior and posterior lobes are severely damaged in head or whole-body radiation f Haymaker et al, 1958; Mogilnitzky and Podljaschuk, 1928: Vogel et al., 1958). Comment In spite of numerous disagreements in the results of various workers, a general pattern of degeneration and repair is beginning to be defined in the tissue reactions of the central nervous system to gamma and roentgen radia- tion. It is quite possible that further advances can be made by particular study of points where contradictions in results now exist. The series of studies re\iewed ha\e been carried out on widely dixergent species of animals, ranging from goldfish to monkey. It is not certain how many of the contradictory results are dependent on species variation or to what characteristics of indi'.idual species these differences arc related. The demonstration of certain constant features in a wide range of experimental animals suggests that species differences may be more related to details than to the general pattern of response. The high degree of sensitivity of the brainstem in animals far apart in the phylogenetic scale is an instance in point. In nearly all experiments re\iewed and in personal material, there are e\en more puzzling variations from animal to animal when radiation source, experimental conditions, and other technical aspects have been kept as constant as possible. Special studies of the animals which are particularly sensitive or resistant in a given set of experimental conditions may suggest factors not well recognized. Some such factors may involve the biologic state of the animal at the time of radiation. Rugh ( 1958) has stated, "Probably the most effective physical factors which influence irradiation sensitivity at any biological level are: (a) state of hydration, (b) degree of oxygenation, and (c) amount of activity or movement." The abolition of early cerebellar effects by bar- biturate anesthesia, as demonstrated by Alvord and Brace (1957'i, may be related to such factors. The apparently simple question of what is the minimum dose of radiation which produces damage to the central nervous system is actually one of great complexity. 186 ORVILLE T. BAILEY Summary A review of the literature related to the effects of gamma and roentgen radiation of the central nervous system is presented and compared with personal material, with a few comments on the effects of beta radiation. The reaction of the central nervous system hours or a few days after radia- tion is an acute inflammatory one. dominated by \asculitis, meningitis, and choroid plexitis. Regressive changes, apparently mostly reversible, are present in the granide cells of the cerebellum. Young animals are more susceptible to radiation damage than adults, but histologic effects can be produced at any age with sufficient dosage. Different and more extensive degenerative processes become evident as the time interval between radiation and sacrifice is lengthened. This interval can be reduced by increasing the dose of radiation, increasing the intensity of radiation, and possibly by other factors. In late stage of radiation reaction, there is extensive damage to neurons, with selectixity for the white matter and particidar sensitivity of the brain stem and hypothalamus. Degeneratixe changes in blood vessels, sometimes with complete occlusion, can be demonstrated at this stage. The effects on neurons are considered by most, but not all, workers to be direct effects and not mediated through \ascular insufficiency. All types of glia show degenerative changes after radiation, and there is a prolonged inhibition of glial response in repair. Proliferation of collagenous tissue is important in the first stages of repair. C'ompound granular corpuscle formation is resumed before astrocytic proliferation begins. Some months after radiation, astrocytosis becomes exuberant. Despite many contradictions in results and interpretation, a basic pattern of degeneration and repair in response to gamma and roentgen radiation is becoming apparent by light microscopy. The most striking single featme is the progressive increase in evidence of damage as time after radiation is lengthened. It is impossible to predict end results from any characteristics of the acute response. Investigations by all techniques will be recjuired to explain the processes going on in the interval, and studies of this type may- well have biologic implications beyond the field of radiation pathology. References .\lquRr and Faure-Beaulicu, M. 1909. L'action du radium sur les tissus du nevraxe. Nouv. iconog.Salpet.22, 109-113. Alvord E. C, and Brace, K. C. 1957. X-ray induced pyknosis of cerebellar granule cells in guinea pigs and its suppression by barbiturate anesthesia. /. Neuropathol. Exptl. Neurol. 16, 3-17. HISTOPATHOLOGY OF CNS RADIATION 187 Arnold, A., and Bailey, P. 1954. Alterations in the glial cells following irradiation of the brain in primates. A.M. A. Arch. Pathol. 57. 383-391. Arnold, A.. Bailey, P.. and Harvey, R. A. 1954a. Intolerance of the primate brainstem and hypothalamus to con\entional and high energy radiations. Xeurology 4, 575- 585. .Arnold. A.. Bailey. P.. Harvey, R. .\. Haas. L. I., and Laughlin. J. S. 1954b. Changes in the central ner\ous system following irradiation with 23-me\' x-rays from the betatron. Radiology 62. 37-44. Arnold, .■\.. Bailey, P., and Laughlin. J. S. 1954c. EfTects of betatron radiations on the brain of primates. 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Sequence of X-Radiation Damage in Mouse Cerebellum NORBERT SCHUMMELFEDER Institute of Pathology University of Bonn, Germany Since the time of Brunner and Schwartz 1918: Brunner, 1920, 1921), who in 1918 were the first to observe that the cerebellar o;ranule cells of young- dogs and cats could be easily injured by x-radiation, little attention was directed to the radioxulnerability of this part of the brain until the past few years. Our work in this field was concerned with the effect of x-rays on the cerebellum of mice ( Schiimmelfeder 1957, 1959a, b; Schiimmel- feder ct al, 1957; Krogh and Bergeder, 1957). We used single x-ray doses and studied the irradiated tissue by morphologic, histochemical, and fluo- rescence techniques. The x-ray dosage ranged from 250 to 60,000 r. Fields of the cerebellum .3 X '^ "ini and 0.5 X 2 mm were irradiated by a half-wave x-ray unit (50 kv, 20 ma. focal distance 6 cm. 0.12 mm Al filter) at 3,000 r per min to the surface of the cerebellum. The irradiated animals were sacrificed at interxals up to 6 months. Observations Morphologically demonstrable radiation effects were seen in the range of 2,000 to 60,000 r. T\w latent period between irradiation and the first mor- phologic changes decreased correspondingly. At 2,000 r, damage was first noted at the end of 4 months, while at 60,000 r, changes were observed in 30 minutes. Irradiation doses less than 2,000 r induced no morphologic changes in the cerebellum during the observation time ot 6 months. A few typical experiments will indicate the nature, se\erity, and sequence of damage after exposure of the cerebellum of mice to different x-ray doses. ToT.\L Necrosis of Cerebellar Tissue Following Exposure to X-rav Doses Ranging from 60,000 to 10,000 r In the range from 60,000 to 10,000 r. x-irradiation induced total necrosis of cerebellar tissue within a time depending on the dosage. 191 192 NORBERT SCHUMMELFEDER In 30 minutes following exposure to 60,000 r, swelling of nuclei and cytoplasm occurred in granule cells, Purkinje cells, and basket cells in deeper parts of the molecular layer. The interstitium of the molecular layer exhib- ited slight vacuolation. In 1 hour after 60,000 r, there was greater swelling of the various nerve cells, and the granular layer was loosened. Vacuolation of the molecular layer was increased. The chromatin in the swollen nuclei of the granule cells was condensed in the form of irregularly shaped coarse bodies located on the nuclear membrane. The swollen nerve cells within the molecular layer had a clear space around their nuclei. These spaces contained small, thread-like, ragged or flaky, cytoplasmic residues, often attached to the nuclei (acute cell swelling). The degree of nerve cell swelling was slight in upp)er parts of the molecular layer and increased progressively down to the Purkinje cells. Within the molecular layer, the nuclei of glial cells were slightly swollen or occasionally pyknotic. Vacuolation (status spongiosus) of the molecular layer was evident ; the vacuoles were at first round, then oval, and increased in size downward from the cerebellar surface to the Purkinje cell layer. Occasionally, the vacuoles were arranged in vertical columns. The vacuoles seemed to contain a protein-free aqueous solution, because even with special staining methods and histochemical reactions no other material could be demonstrated. Henceforth, the Purkinje cells, in particular their nuclei, showed hydropic swelling. Within the swollen cyto- plasm the Nissl bodies had usually disappeared. In other cells, the NissI substance was dispersed as dust-like particles over the entire cytoplasm. The Bergmann glial cells occasionally had swollen nuclei, but they seldom dis- played any nuclear pyknosis. At 2 hours after 60,000 r, the regressive changes were still more advanced (Figs. 1 and 2). Within the lower part of the molecular layer, the vacuola- tion had progressed to tissue sponginess. Within the uppermost parts of the granular layer, the tissue looseness had also increased. Some nuclei of the irradiated granule cells were no longer swollen, but were shrunken and pyknotic. Correlated with the decrease of the x-ray dose with distance traversed, there was an upper zone composed mostly of pyknotic nuclei, then a transitional zone with pyknotic as well as swollen nuclei, and a lower zone containing solely swollen nuclei. Apparently as a consequence of the pressure from the swelling of the molecular and granular layers, many Purkinje cells within the center of the damaged area were deformed. These cells were oval, and their longitudinal axes were parallel to the gran- ular layer. In the lateral part of the irradiated field, several Purkinje cells exhibited acute swelling. Near the border of the damaged area, which was evident because of the changes in the granular layer, the Purkinje cells showed slight or no morphologic alterations. 7^ Figs. 1 and 2. Radiation damage 2 hours after exposure to 60,000 r. Plane of the section is parallel to the direction of the x-ray beam. Vacuolation of molecular layer, shrinkage of Purkinje cells, and pyknosis of the nuclei of granule cells occur in the most severely damaged area of the granular layer, and nuclear swelling in the lower part. Fig. 1 : X 40. Hematoxylin-eosin. Fig. 2: X 170. v. Gieson-stain. 193 194 NORBERT SCHUMMELFEDER At 4 to 5 hours after x-irradiation with 60.000 r. nearly all the granule cells in the irradiated part of the granular layer were shrunken and their nuclei pyknotic (Fig. 3). These cells were surrounded by a clear space. In the molecular layer, the nuclei of the swollen nerve cells were now pyknotic, while the degree of vacuolation in this part of the irradiated cerebellar cortex was the same as before. The morphologic changes in the molecular and granular layers were strictly localized, limited to the irradi- ated field. The border betw-een the irradiated and the unchanged cere- bellar tissue was sharp, as though drawn with a ruler. During this time interval, the white matter of the cerebellum was slightly, if at all, modified by the irradiation. There was neither detectable edema in the white matter nor any substantial change in the axis cylinders or in glial cells. Within the first hours after irradiation, no e\idence of a conspicuous Fig. 3. At 4 hours after irradiation with 60,000 r, showing severe vacuolation of the molecular layer, shrinkage of Purkinje cells, and pyknosis of granule ceil nuclei. X 780. Hematoxylin-eosin. SEQIENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 195 change in the blood vessels was observed. Occasionally a slight dilatation of the capillaries was seen as an expression of hyperemia. In 5 to 6 hovns after x-irradiation of a 3 X 3 mm field with 60,000 r the mice died. Further evolution of radiation damage could be followed only after use of a dose of 60,000 r through a smaller aperture 0.5 X 2 mm) or by using lower x-ray doses (40,000 to 20,000 ri. At 12 to 14 hours after 60.000 r. using a 0.5 X 2 mm field, the pyknotic nuclei of the granular layer underwent disintegration, mostly as karyorrhexis. The damaged area was still limited to the irradiated field, as a section cut transversely to the direction of the x-ray beam illustrates ( Fig. 4 ) . The Purkinje cells also exhibited signs of disintegration. Some of these cells had greatly swollen cytoplasm and nuclei and showed lytic changes ( Fig. 5 ) . Within other Purkinje cells, the nuclear chromatin was initially condensed, simulating pyknosis, and then the cytoplasm and nucleus underwent lysis. At 20 to 30 hours after irradiation with 60.000 to 40.000 r. necrosis was completely established in the superficial part of the cerebellar folia, i.e.. nearest the radiation soiuce ( Fis.. 6 ) . The necrosis was strictly limited to 'J:rM - Fig. 4. Radiation damage 12 hours after exposure to 60.000 r (0.5 X 2 mm field). Plane of section is trans\erse to direction of the x-ray beam. Pyknosis and disinte- gration of nuclei in the granular layer. X 4(1. Hematoxylin-eosin. Fig. 5. Different types of nerve cell change after irradiation: (a) condensation of the nuclear chromatin, (b) swelling and partial vacuolation of the cytoplasm, (c) severe swelling of the cytoplasm, (d) severe nuclear and cytoplasmic swelling, in- cipient lysis of the cytoplasm. X 1440. Gallocyanin-chromalum stain. 196 SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 197 198 NORBERT SCHUMMELFEDER i Hi • 1^* .^•^^ • H T •:] Fig. 6. At L!U hours alter irradiation with lU.OOU r, showing the honiogenization type of nerve cell necrosis. X 700. Hematoxylin-eosin. the irradiated field and was sharply demarcated from the nonirradiated part of the cerebellum. The molecidar layer showed granidar and clumped areas of disintegration. Only some of the nerve and glial cells were preserved. Most of them contained pyknotic nuclei and showed all stages of disinte- gration or lysis. Within the necrotic granular layer enlarged by edema, the preserved nuclei were pyknotic, and between them nuclear debris was frequently found. Hemorrhages and extravasations of plasma proteins were remarkable neither within the necrotic granidar layer nor within the white matter. The nuclei of the glial cells in the white matter of the more super- ficial part of the cerebellar folia (nearest the radiation source) were pyk- notic, and in deeper parts of the cerebellum they were swollen. Some blood vessels in the necrotic area were preserved, but dilated. Frequently, they were surroimded by a hollow space, the ground membrane had often under- gone hyaline thickening (hyalinosis) , and there was swelling of endo- thelial and adventitial cells. Within the irradiated field the Purkinje cells had, in part, disappeared. Most of the preserved Purkinje cells showed homogenization. They con- tained pyknotic nuclei and exhibited a strong cytoplasmic eosinophilia. Many were in all stages of disintegration, including cell shadows. At 90 hours after irradiation^ necrosis was completely established in all SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 199 parts of the irradiated cerebellar tissue (Fig. 7). Within the necrotic area there were sca\enger cells and gitter cells as well as proliferating and naked glial cells. The nuclei of most altered granule cells were disintegrated and lysed. Only segregated groups of pyknotic nuclei together with nuclear debris were observed within the irradiated field. In contrast to the animals of shorter survival, edema was seen within the Bergmann layer and in adjacent parts of the molecular and granular layers. Frequently, the edematous process extended a short distance into the nonirradiated part of the Bergmann layer and was associated with hydropic swelling of adjacent Purkinje cells. Homogenization of Pinkinje cells was no longer found, as cells which had suffered this change seemed to have been removed. When the survival period was extended by reducing the x-ray doses to 20,000 to 10,000 r, resorption and repair occurred in the irradiated cere- bellar tissue in the same manner as in necrosis of brain tissue resulting from other causes. The final stage consisted of cystic licjuefaction of the necrotic brain tissue. By 20 days after irradiation with 16.000 r much of the necrotic cerebellar tissue had been removed. The resulting cyst-like areas contained remnants of necrotic debris and were traversed by partly preserved blood vessels. The processes of resorption and repair were e\ident at the margin ^ri; Fig. 7. Total tissue necrosis 90 hours after irradiation with 70.000 r (0.5 X 2 mm field). X 35. Hematoxylin-eosin. 200 NORBERT SCHUMMELFEDER of the damaged brain tissue in the form of numerous gitter cells. Within the preserved tissue only slight glial reaction had occurred. Partial Necrosis of Cerebellar Tissue Following Exposure to X-ray Doses from 10,000 to 4,000 r At radiation doses from 10,000 r down to 4,000 r, there was necrosis only of the granular layer and a loss of single nerve cells in the other parts of the cerebellar cortex. The latent period between irradiation and the develop- ment of morphologic changes was longer than with higher doses. Thus, with a dose of 5,000 to 4,000 r, the first morphologic changes appeared at approximately 12 hours. At this time only a few scattered pyknotic nuclei of granular cells were observed. The other parts of the irradiated cerebellar tissue were not modified. At 5 days the more superficial part of the granular layer had undergone partial dissolution. Only adjacent to the Purkinje cell layer was there a zone of preserved pyknotic granule cell nuclei, and it was narrow. Occupy- ing regions in which granule cells had undergone dissolution were numerous gitter cells and i.solated pigment-bearing scavenger cells. Purkinje cells were destroyed only in the most severely damaged regions of the irradiated field. In their place, proliferated glia were seen. Other Purkinje cells were being phagocytized. The cerebellar white matter was loosened strikingly, and in some foci the white matter was completely destroyed. Some of the blood vessels, especially the capillaries, were greatly dilated and were surrounded by a mantle of mononuclear cells including a few neutrophilic leucocytes. Endothelial cells of occasional blood vessels were swollen. At 10 days after irradiation the destroyed Purkinje cells were replaced by glial shrubbeiies ( Gliastrauchwerk) extending from the Bergmann layer into the molecular layer. The uppermost part of the molecular layer showed decided shrinkage, as did other parts of the cortex (Fig. 8). At 20 days after exposure to 5,000 r, extensive perivascular hen:iorrhages were often found in damaged cerebellar tissue. Shrinkage, pronounced glial proliferation, and cicatrization (gliosis) occurred in the irradiated tissue. The glial fibers within the damaged molecular layer, particularly those of the Bergmann cells, were thickened remarkably. These coarse fibers could easily be demonstrated by Lendrum's (1947) method. Persisting Piukinje cells and nerve cells of the inolecular layer often showed regressive changes, e.g., swelling of cytoplasin and nucleus or chromatolysis. Even at this time interval, some of the granule cells in the irradiated area were pyknotic. Such pyknosis had probably developed in the course of the radiation damage. The periphery of the most severely damaged area was marked off by pronounced vascularisation. The vessels SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 201 Fig. 8. Radiation damage ID days after irradiation witli 5.01)0 r. Dissolution of central parts of the granular layer and destruction of Purkinje cells within the upper- most layer followed by glial proliferation extending into the shrimken molecular layer. X 215. Gallocvanin-chronialiun. were irreoiilaily dilated, and their walls showed remarkable hyalinosis. Circumscribed aneurysmal distention of smaller blood vessels, previously described by Scholz ( 1934a, b. 1937) in brain tisstie damaged by irradiation, was noted at the 20-day sta^e. At 40 days after irradiation, the ylial reaction in the irradiated cerebellar tisstie had progressed further. The entire damaged area was shrimken. Some sjranule cells were preserved, but it was difficidt at times to distinguish them from proliferated glial cells. As at shorter time intervals, hemosiderin- containing sca\enger cells were often seen. Hemosiderin indicated previous hemorrhage. In contrast to higher x-ray doses, 5,000 to 4,000 r produced only partial necrosis of the cerebellar tissue. The molecular layer and the white matter of the cerebellar folia were little affected. All the well known processes of resorption and repair took place in the irradiated cerebellar tissue in the same manner as in partial cerebellar necrosis due to other cause. Glial scarring was the final outcome. 202 NORBERT SCHUMMELFEDER Single Cell Necrosis Within the Cerebellum After Exposure to X-RAv Doses Below 4,000 r X-ray doses below 4,000 r resulted in loss only of single cells or small cell groups, not in necrosis of larger areas of the irradiated cerebellar tissue. The latent period between irradiation and the appearance of damage was increased. Following an x-ray dose of 3,000 r, morphologic changes within the irradiated cerebellar tissue were not observed in less than 45 days. At this time, focal loss of granule cells and destruction of some Purkinje cells were noted in the most superficial cerebellar folia, i.e., those nearest the radiation source. The better preserved Purkinje cells often showed regressive changes, which were of differing types. Cell shadows were occasionally observed. Together with fresh hemorrhages within the damaged brain tissue were residues of older ones in the form of hemosiderin in scavenger cells. The blood vessels were dilated, and their walls often showed hyalinosis. Perivascular infiltrates of mononuclear cells were found over the entire irradiated field. At 4 months after irradiation with 3,000 r the various components of the irradiated cerebellar folia were relatively well preserved. The granular layer showed patchy looseness due to disseminated loss of granule cells. This layer contained proliferated glial elements and was traversed by capillaries. Hyaline thickening was found in the wall of blood vessels in the lepto- meninges and brain tissue. The lumens of many vessels were dilated. Some Purkinje cells were destroyed and replaced by glial nodules from which glial shrubberies extended into the molecular layer. The preserved Purkinje cells seemed imaltered. Early Changes in Cellular Nucleoproteins of Irradiated Cerebellum All these observations indicate that local irradiation of the cerebellum of mice with sufficiently high doses of x-rays results in marked alterations of cerebellar tissue which are limited to the irradiated field. One of the most remarkable changes is the pyknosis of nuclei of granule cells. Another im- portant observation is that during the course of the radiation damage dif- ferent types of regressive changes are observed in the Piukinje cells. We have applied the fluorescence technique, using the basic fluorochrome acridine orange, to study alterations in nerve cells. This histochemical method is especially suitable for investigation of cytoplasmic and nuclear nucleic acids (Schiimmelfeder et al., 1958). Using buffered acridine orange solutions, pH 4.0 to 7.0, all ribonucleic acid (RNA ) -containing material, e.g., the cytoplasm of Purkinje cells, fluoresces bright orange or red. In SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 203 contrast, the highly polymerized deoxyribonucleic acid (DNA) of the nuclei shows an intense yellow or yellow-green fluorescence. Other elements of the brain tissue present a weak green fluorescence. The diff"erence in the staining raction of RNA and DNA is due to the high degree of polymeriza- tion of the nuclear DNA in comparison with the cytoplasmic RNA. After depolymerization, e.g., by placing the tissue slices in boiling water or hydrochloric or perchloric acid, the DNA stained with acridine orange shows a red fluorescence. The depolymerized DNA behaves, therefore, like RNA, which is always more weakly polymerized. The acridine orange method is more sensitive than the methyl green, which can, however, bring out wide differences in the degree of polymerization in DNA. The occur- I'ence of depolymerization in DNA thus can be proved by using the acridine orange fluorescence technique. We tried to determine with this method whether alterations occur in the structural organization of the DNA in the pyknotic nuclei of irradiated granule cells. Many other cells besides those of the cerebellar granular layer show clumping or other changes in the nuclear chromatin. In an histo- chemical study of nuclear changes in the superficial epithelium of the tongue of mice that had received radioactive chromic phosphate, Burstone (1953) observed enlargement and hyperchromasia of the nuclei. In contrast to nonirradiated controls, treatment with deoxyribonuclease (DNase) re- sulted in decreased staining capacity of these nuclei to the Feulgen reaction and to the methyl-green-pyronin method. Burstone (1953) believed that this decreased resistance of the irradiated nuclei is due to a somewhat decreased aggregation of the nuclear DNA. Based on such observations and on radiation experiments on DNA solutions which show a splitting of DNA molecules i Scholcs and Weiss, 1952) some still believe that the clumping of chromatin after irradiation is a result of depolymerization. If such depolymerization of DNA should occm- in the pyknotic nuclei of irradiated cerebellar granule cells as a primary effect or a secondary reaction to ioniz- ing radiation, then it should be demonstrable with acridine orange by the nuclei exhibiting red fluorescence. We ha\e ne\er found this. Disregarding the point that pyknosis causes a higher density which provokes a somewhat more intense fluorescence, no difference in the fluorescent color of these pyknotic nuclei compared to normal nuclei has been obser\ed. The fact that the DNA of these pyknotic nuclei is not significantly depolymerized can also be shown by using methyl green. This dye stains only highly polymerized DNA (Kurnik. 1950. 1952; Kurnik and Forster, 1950: Kurnik and Mirsky, 1950; Pollister and Leuchtenberger, 1949; Ver- cauteren. 1950). The pyknotic nuclei should not be stainable with methyl green if there had been depolymerization of nuclear DNA. But, as compared with nonirradiated cells, no substantial diflference in the staining capacity 204 NORBERT SCHCMMELFEDER of the pyknotic nuclei was observed. This result corresponds to that of Sparrow et al. (1952) on Trillium nuclei. Analogous to Burstone's (1953) observations, Kaufmann et al. (1955) have shown in experiments on meristematic cells of onion roots that DNA in irradiated cells is more easily dissoKed by DNase than in nonirradiated controls. In contrast, similar experiments on grasshopper embryos showed a higher resistance of the irradiated nuclei, i.e., by their DNA. to enzymatic hydrolysis. These different results stimulated us to seek information on whether the de\elopment of pyknosis of granule cell nuclei following ir- radiation alters their response to depolymerizing and hydrolyzing agents. The results of these experiments showed that pyknotic nuclei of granule cells are more resistant than nonpyknotic nuclei to treatment with depoly- merizing agents, e.g. to boiling water or to hydrochloric or perchloric acid. Pro\ided the reaction conditions are favorable, only in the nonirradiated nuclei did exposure to these agents result in depolymerization. These nuclei showed red fluorescence after staining with buffered solutions of acridine orange, pH 5.0 to 7.0, whereas the pyknotic nuclei still fluoresced bright yellow owing to retained high polymerization of DNA. The difference in color and intensity ot the fluorescence was so conspicuous that each indi- vidual pyknotic nucleus could easily be obser\ed. Methyl green, which stains only highly polymerized DNA, has yielded the same results in companion sections. The pyknotic nuclei were still stain- able with methyl green, whereas unaltered granule cell nuclei could not be stained after pretreatment with depolymerizing agents. Further experiments showed that enzymatic breakdown of DNA, using DNase, occurs more slowly in pyknotic than in intact nuclei. This result is similar to that ob.served by Kaufmann ct al. (1955) in irradiated grass- hopper embryos. Hydrolysis with hydrochloric or perchloric acid removed DNA from the nuclei of unaltered granule cells, whereas DNA of the pyknotic nuclei in irradiated granule cells was only slightly depolymerized. Using favorable conditions of hydrolyzation. it is easy to demonstrate selectively the pyk- notic nuclei after staining with acridine orange, whereas nonpyknotic nuclei, which are not altered by the irradiation and which are deprived of the DNA by the foregoing hydrolysis, remain imstained. In our experimental study we were unable to determine whether increased resistance of the irradiated pyknotic nuclei to DNase and to depolymerizing and hydrolysing chemical agents is an immediate and specific effect due to the action of ionizing radiation on the DNA of the nuclei. The observations of Kaufmann ct al. (1955) have indicated that the structural organization of the nucleoproteins in irradiated nuclei is changed. On the other hand Yakar (1952) has demonstrated in plant cells that the speed of enzymatic SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 205 hydrolysis of chromatin decreases if pyknosis is induced by chemical asents. It is conceivable that the increase in resistance of the pyknotic nuclei which we found is attributable to greater density of the nuclear mass in that the increased density reduces depolymerization and hydrolysis. We have emphasized that when RNA-containinsi material, e.g., the cyto- plasm of the Purkinje cells, is stained with acridine oranse. pH 4.0 to 7.0, it takes on a bright orange or red fluorescence. Since different types of regressive changes can be observed in Purkinje cells during the course of radiation damage, we have used the acridine orange method to study the behavior of their cytoplasmic nucleic acids. Because of regressive changes in these cells and because of the nuclear pyknosis in granule cells, the irradi- ated area of the cerebellar tissue can easily be demonstrated by this method. Since regressive changes in the Purkinje cells usually occur more strikingly in the center of the irradiated area than along its margins, the red fluo- rescence exhibited by the more peripheral cells gradually decreases in in- tensity toward the center of the irradiated zone. The cytoplasm of unaltered Purkinje cells fluoresces bright orange-red. Acute shrunken nei-ve cells show the same fluorescence because their cytoplasm contains abimdant RNA. Immediately following" irradiation, the swollen and \acuolated Purkinje cells give off a slightly decreased orange fluorescence, but after sufficient time has passed the cytoplasm exhibits only yellow or yellow-green fluorescence because the RNA content of their altered cytoplasm is decreased. Purkinje cells showing the homogenization type of necrosis have a green fluorescence because they have lost all cytoplasmic RNA. Since color and intensity of fluorescence in these Purkinje cells is similar to that of the neuroglia of the molecular layer, it is somewhat difficult to recognize necrotic and homog- enated Purkinje cells. It bears emphasis that the nuclear pyknosis in Purkinje cells as well as in granule cells is not associated with depolvmerization of DNA, since, when stained with acridine orange, the pyknotic nuclei still fluoresce yellow-green. Discussion There ha\e been apparently conflicting reports in the literature as to the primacy of irradiation damage in the central nervous system, whether in vessels or in nerve cells. The cerebellum seems especially suitable for in\esti- gation of this problem. .Some other workers already ha\e directed attention to the radio\ulnerability of this part of the brain. In macaque monkeys, Hay- maker et al. ( 1958) ha\-e studied the effect on the central nervous system of whole-body BA'^"-LA"" i gamma i radiation. E\ idence of ner\e cell damage in the cerebrum was scanty, but granule cells of the cerebellum were pyknotic within a dose range of 5,000 to 30,000 r. The pyknosis occurred earlier and 206 NORBERT SCHUMMELFEDER was more severe at higher dose levels. In other experiments on the macaque, in which 10,000 r Co*'° (gamma) radiation to the head alone or to the whole body was used, Vogel et al. (1958) also observed pyknosis of granule cells in the cerebellum. Both Haymaker et al. (1958) and Vogel et al. (1958), whose animals survived no longer than one week, observed that the pyknosis of granule cells was reversible by about the 3rd day. In a subsequent study of the effects of cobalt'"^ (gamma) radiation on the cerebellum of macaque monkeys at the same dose levels, Wilson (1960) confirmed the observation that under these conditions the granule cell pyknosis is transitory and reversible, but he found that pyknosis was somewhat briefer than reported by the other authors. Similar results have been obtained in rabbits after exposed to a Co"" source (Vogel, 1959) and in guinea pigs after x-irradiation (Alvord and Brace, 1957). Vogel (1959) noted that after a dose of 15,000 r gamma radiation from a Co''" source, granule cell pyknosis was evident in 15 hours and that by the 10th day practically all the granule cells of exposed folia had disappeared. According to Hicks (1953; Hicks and Montgomery, 1952; Hicks and Wright, 1954; Hicks et al.. 1956) the same holds for rats and mice. In these animals they found that nerve cells or cerebellar tissue could readily be damaged by x-rays, depending on the dose. Our observations coincide with those of Hicks et al. { 1956) on the mouse. We have shown that sufficiently intense x-irradiation of the cerebellar tissue results in primary tissue changes. Incidence, pattern, and course of these changes are clearly related to the x-ray dose. Under the conditions of oiu' experiments, nuclear pyknosis in irradiated granule cells of adult mice is a sign of irreversible cellular change leading to cellular necrosis. In this respect our observations on mice do not coincide with those of Haymaker et al. (1958), Vogel et al. (1958), and Wilson (1960) on macaque monkeys. We are imablc to explain this difTerence, but points to be taken into considera- tion are species and age of the animals, nature of irradiation, radiation energy and rate of dosage. There is still little knowledge of the earliest histopathologic and histo- chemical changes occurring in the cerebellum following irradiation or in the sequence in which the changes develop. Our histochemical investigations show that during early radiation damage of cerebellar tissue following high x-irradiation dose, changes occur in the nucleic-acid-containing components of granule and Purkinje cells. Particularly in Purkinje cells, alterations occur in the cytoplasmic RNA that are secondary eflfects due to regressive cellular alterations, since swelling of the nerve cells was observed initially and de- crease in cytoplasmic nucleic acid content later on. The change in the structural organization of the nuclear DNA in the pyknotic granular cells is possibly also a secondary effect due to the increased density of the pyknotic nuclei. But it could also represent a primary change in the physicochemical SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 207 quality of the DNA caused by the action of ionizino radiation. Further in\estigations are necessary to clarify this problem. As brought out by the acridine orange fluorescence technique, the increased resistance of pyknotic nuclei to depolymerizing and hydrolysing agents allow clear identification of damaged irradiated pyknotic cell nuclei. Summary Dexelopment and course of radiation lesions in the cerebellum in mice were studied after exposure to single doses of x-rays, 250 to 60,000 r, applied to one cerebellar hemisphere through apertures 3 X ^ mm or 0.5 X 2 mm in diameter. Degree and sexerity of radiation damage in the cerebellum was correlated in terms of tinre-intensity relationships with the x-ray dose. After a dose of 60.000 r. morphologic changes of different cerebellar struc- tures (e.g.. nerve cells of the molecidar layer and Purkinje and granule cells) were visible as early as 30 minutes following exposine and were fully dcxel- oped at 60 minutes. Within 90 hours, complete necrosis of the irradiated cerebellar area with concomitant resorptive and reparative changes was observed. C'ystic liquefaction eventually occurred. After less intense x-ray doses, 60,000 to 10,000 r. similar radiation lesions were observed, but the latent period of their inception was longer. Exposure to doses below 10,000 r down to 4,000 r residted in necrosis of the granidar layer and in destruction of single nene cells in other parts of the cerebellar cortex. The latent time was more prolonged. Radiation damage finally resulted in the formation of a glial scar. At x-ray doses below 4,000 r. only single nerve cells or cell groups were damaged, and the latent period was correspondingly lengthened. In such lesions, proliferation of glial elements and capillaries ultimately occurred, and hyalinosis developed in the walls of distended blood vessels. Since no evi- dence of vascular alterations was observed before development of nerve cell lesions, the visible radiation damage was interpreted as due to a direct effect of the ionizing radiation on the cellular elements of cerebellar tissue. The acridine orange fluorescence tcchniciue was used to determine whether alterations occin- in the structural organization of the DNA in pyknotic nuclei of irradiated granule cells and in the RNA content of Purkinje cells. Our results indicated that after irradiation DNA of pyknotic nuclei in the granular layer is not depolymerized to a noticeable degree. These pyknotic nuclei were more resistant than normal to treatment with depolymerizing and hydrolysing chemical agents and DNase. Without fiuther experimenta- tion it is difficult to say whether this increased resistance of irradiated, pyknotic nuclei is an immediate and specific effect due to the action of ioniz- ing radiation on the DNA or whether it is a nonspecific effect due to in- 208 NORBERT SCHCMMELFEDER creased density of the nuclear mass. Within the region damaged by irradia- tion, Purkinje cells in a state of regression showed remarkable decrease or loss of cytoplasmic RNA which was obviously secondary to cellular regression. Acknowledgment The author wishes to acknowledge, with appreciation, the assistance rendered in these studies by Dr. Bergeder and Dr. Ebschner, University of Bonn, Germany, and the late Dr. E. Krogh, University of Aarhus, Denmark. References Alvord, E. C, and Brace, K. C. 1957. X-ray induced pyknosis of cerebellar granule cells in guinea pigs and its suppression by barbiturate anesthesia. /. Neuropathol. Exptl. Neurol. 16, 3ff. Brunner, H. 1920. Ijber den Einfluss der Rontgenstrahlen auf das Gehirn, I. Arch, klin. Chir., Langenbecks 114, 332-372. Brunner, H. 1921. t)ber den Einfluss der Rontgenstrahlen auf das Gehirn. II. Der Einfluss der Rontgenstrahlen auf die Regenerationsvorgange im Gehirn niit be- sonderer Beriicksichtigung der Neuroglia. Arch. klin. Chir., Langenbecks 116, 489-509. 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Variation of pathological responses to radia- tion with time-intensity factors. Am. J. Clin. Pathol. 24, 77. Hicks, S. P., Wright, K. A., and Leigh, K E. 1956. Time-intensity factors in radia- tion response, I. The acute effects of megavolt electrons (cathode rays) and high- and low-energy x-rays. A.M. A. Arch. Pathol. 61, 226-238. Kaufmann, B. P., McDonald, M. R., and Bernstein, M. H. 1955. Cytochemical studies of changes induced in cellular material by ionizing radiations. Ann. N. Y. Acad. Sci. 41, 553-566. Krogh, E. v., and Bergeder, H. D. 1957. Experimental irradiation damage of the cerebellum demonstrated by Einarson's gallocyanin-chromalum staining method. SEQUENCE OF X-RAY DAMAGE IN MOUSE CEREBELLUM 209 /"" Congr. inteni. Set. Neurol., Brussels, 1957: 3' Congr. intern. Xeuropathol. pp. 287-294. Acta Medica Belgica, Brussels. Kurnik, N. B. 1950. Mcthyl-green-pyronin. L Basis of sclecti\e staining of nucleic acids. /. Gen. Physiol. 33. 243-264. Kurnik, N B. 1952. The basis for the specifity of methyl-green staining. Exptl. Cell Research 3, 649-651. Kurnik, N. B., and Forster. M. 1950. Methyl-green. III. Reaction with desoxyribonu- clcic acid, stoichiometry. and beha\ ior of the reaction product. /. Gen. Physiol. 34, 147-159. Kurnik. N. B., and Mirsky, .\. E. 1950. Methyl-green-pyronin. II. Stoichiometry of reaction with nucleic acids. /. Gen. Physiol. 33, 265-274. Lendrum, .\. C. 1947. The phloxin-tartrazin method as general histological stain for the demonstration of inclusion bodies. /. Pathol. Bacterial. 59, 399-404. Pollistcr, A. VV., and Leuchtenbcrger, C. 1949. The nature of the specifity of methyl green for chromatin. Proc. Natl. Acad. Sci. U. S. 35. 111-116. Scholes, G., and Weiss, J. 1952. Chemical action of x-rays on nucleic acids and related substances in aqueous systems. Exptl. Cell Research Suppl. 2, 219-244. Scholz, W. 1934a. Experimentelle Untersuchungen iiber die Einwirkung \on Rontgen- strahlen auf das reife Gehirn. Z. ges. Neurol. Psychiat. 150, 765. Scholz, W. 1934b. Die morphologischen Veriinderungen des Hirngewebes unter dcni Einfluss \en Rontgen- und Radiumstrahlen. 7' Congr. intern. Elettro-radio-biologia 2, 1051. Scholz, VV. 1957. Discussion. /"' Congr. intern. Sci. Neurol. , fi/«s\c/s, 1957: 3' Congr. intern Neuropathol. pp. '2T1-'11~ . Acta Medica Belgica, Brussels. Schummelfeder, N. 1957. Fluoreszenzmikroskopische und cytochemische Unter- suchungen liber Friihschadcn am Kleinhirn der Maus nach Rontgenbestrahlung. /"" Congr. intern. Sci. Neurol., Brussels, 1957: 3' Congr. intern. Neuropathol. Schiimmelfeder, N. 1959a. Der W-rlauf der experimentellen Strahlenschadigung des Hirngewebes. Verhandl. deut. Ges. Pathol. 42, 244-250. Schiimmelfeder, N. 1959b. Experimental irradiation damage of the brain. Proc. 2nd. U. A'. Intern. Conf. on Peaceful Uses of Atomic Energy, Geneva, 1958, 22, 287-291. Schummelfeder, N., Krogh, E.. and Bergeder, H. D. 1957. Morphologische und histochemische L^ntcrsuchimgcn zur experimentellen Strahlenschadigung des Hirn- gewebes (Tagg. nord- u. westdeut. Pathologen. Bad Pyrmont. 1956.) Zentr. allgem. Pathol, u. Pathol. Anat. 96, 409. Schummelfeder. N., Krogh, E., and Ebschner, K. J 1958. Farbungsanalysen zur Acridinorange-Fluorochromierung. Vergleichende histochemische und fluoreszenz- mikroskopische Untersuchungen am Kleinhirn der Maus mit Acridinorange- und Gallocyanin-Chromalaun-Farbungen. ///,\<0(:/7f'/n;V (Histochemie) 1, 1-18. Sparrow. A. H., Moses, M. J., and Dubow, R J. 1952. Relationships between ioniz- ing radiation, chromosome breakage, and certain other nuclear disturbances. Exptl. Cell Research Suppl. 2, 245-262. V'ercauteren. R. 1950. The structure of desoxyribose nucleic acid in relation to the cytochemical significance of the methyl green-pyronin staining. Enzymologia 14. 134-140. Vogel, F. S. 1959. Changes in the fine structure of cerebellar neurons following ionizing radiation. /. Neuropathol. Exptl. Neurol. 18, 580-589. Vogel. F. S.. Hoak. C. G.. Sloper. J. C. and Haymaker, W. 1958. The induction of 210 NORBERT SCHUMMELFEDER acute morphological changes in the central nervous system and pituitary body of macaque monkeys by cobalt"" (gamma) radiation. /. Neuropathol. Exptl. Neurol. 17, 138-150. Wilson, S. G., 1960. Radiation induced central nervous system death. A study of the pathologic findings in monkeys irradiated with massive doses of cobalt™ (gamma) radiation. /. Neuropathol. Exptl. Neurol. 19, 195-215. Yakar, N. 1952. Cytochemical studies on pyknolic root-tip cells. Botan. Gaz. 114, 72-79. Morphological Effect of Repeated Low Dosage and Single High Dosage Application of X-lrradiation to the Central Nervous System * WiLLIBALD SCHOLZ. WoLFGANG ScHLOTE. AND WOLFGANG HiRSCHBERGER Deutsche Forschungsanstalt fur Psychiatrie Max-Planck-Institut, Munich, Germany This preliminary presentation deals with the effects of x-rays on the tissue of the central ner\oiis system and the ensuinij pathogenesis. Although we shall refer repeatedly to x-ray dosage, its relation to the time of manifestation of tissue changes, and the se\erity of these changes, it is not our intention to determine an exact time-dose relationship as this has been done by Berg and Lindgren (1958). We ha\e tried to find a connection between the delayed x-ray lesions seen after moderate single or fractionated doses and the acute tissue necroses occurring within hours after a single application of massi\e doses of hiyh intensity up to 80.000 r. It is now almost universally accepted that the delayed lesions originate from changes ot the vessels. A breakdown of the hematoencephalic barrier has been considered significant since Mogilnitzky and Podljaschuk ( 1930) described the passage of trypan blue into repeatedly irradiated central nervous tissue. When we made our first investigations and experiments with dogs in 1932-1935 (Lyman et al., 1933; Scholz. 1935), the histologic picture was dominated to such a degree by plasmatic transudations with and without erythrodiapedesis into the central nervous tissue, it was difficult to admit any direct influence of the x-rays on ner\ous tissue constituents. While it was true that the x-ray doses applied through different portals to the skull ranged from 4,400 r to about 8.000 r, the intensity of irradiation was so low that to apply 12 skin erythema doses corresponding to about 6.600 r. a radiation time of 6 hours was required. Since that time, x-ray technicjue and the accurate measurement of dosage has improved so considerably that it seemed worthwhile to re-examine oiu" former results with new experimental material and methods. * This project was supported by the School of .\\iation Medicine of the .^ir Re- search and Development Command. U.S. An Force, through its European Office. 211 212 W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER Accordinoly, since 1956, we have studied the neuropathology of x-ray lesions produced by irradiation of the spinal cord of rabbits.^ A 3 x 6 cm field on the back, corresponding to the upper thoracic segments of the cord, was irradiated. The technical conditions were: 180-200 kv, 18 mA, 0.95- 1.12. Cu half value layer, filter 0.5 Cu. 60 r per min, focus — skin distance 50 cm, and pendulum angle 70° on both sides. The average dose was 250 r daily; total doses were 3,000 to 1 1,000 r given over 12 to 40 days. All animals acquired paralysis of the hind legs and loss of sphincter function within 4 to 33 weeks after the beginning of irradiation. This investigation was published in Psychiatria ct Nciirologia Japonica, 1959, and only the important features will be mentioned here. The white matter of the spinal cord was much more affected than the gray substance and showed more or less circum- scribed areas of disintegration following the radial distribution of the spinal vessels entering from the vasocorona along the whole periphery of the cord. Regressive changes of the small vessels with slight perivascular astrocytic reaction within the focal lesions suggest the transudation of a fluid histo- logically not demonstrable, causing swelling and disintegration of the myelin fibers. Only in a few cases did the gray substance participate in the changes. Here, plasmatic extravasation, partly with erythrodiapedesis, could be ob- served, followed by an astrocytic reaction and some regressive changes in some nerve cells. Although a reaction of the neuroglia was observed, it remained rather scanty, especially in the white matter. Regardless of the fact that some lesions were 3 to 4 weeks old, sudanophilic material was not observed. In both white and gray matter, plasmatic disintegration of the walls of larger vessels was encoimtered occasionally, but plasmatic exudation was seen only in the gray substance. Thus the morphologic feature patho- genetically pointed to the primacy of processes of transudation, exudation, and erythrodiapedesis, that is, to a breakdown of the barrier function of the spinal vessels. This concept was further supported by intravital injection of tiypan blue (Fig. 1). There are blue stained foci in the white matter Fig. 1. Delayed lesion of the spinal cord of a rabbit after fractionated x-irradiation, intravitally stained with trypan blue. Beneath some foci of disintegration in the white matter, the whole gray substance although demonstrating no disintegration, has taken the blue color. ' We received the material from the radiologist Dr. Breit, who was interested in questions of tolerance dose, dose fractioning, and concentric application by a pendulum x-ray machine. MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 213 caused by destruction of tissue. The remarkable fact is the sharply limited blue stainino' of the whole gray matter, which demonstrates no disintegration at all. These investigations have been continued in 21 additional adult rabbits. Equivalent changes in the spinal cord could be produced using the same technique with fractionated doses totaling 3,000 r. No exact relation between the amount of applied r and the length of the interval can be stated, but on the average the intervals became longer with diminution of the total dose. The minimum single dose sufficient to produce severe changes in the cord after 5 months was 2.000 r. Some figines demonstrating the pathologic findings in this material show again the important role which the breakdown of the blood-brain barrier plays in the pathogenesis of changes in the nervous tissue. Thus we see in Fig. 2 that the numerous focal changes in the white matter follow the distribution of the small arteries entering the cord from the vasocorona. In this case, a single dose of 3,500 r was followed by paralysis 15 weeks later. Two small vessels in the white matter (Fig. 3) Fig. 2. Delayed lesion of the spinal cord of a rabbit, 3% months after a local single x-ray dose of 3,500 r. Numerous areas of disintegration in the white matter fol- low the radial direction of the entering vessels of the vasocorona. Myelin stain (Schroder). show a swelling and disintegration of their walls cau.sed by infiltration with a plasmatic material which is stained yellow in van Gieson preparations. 214 W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER Fig. 3. Plasmatic swelling and disorganization of the walls of two small \essels in the white matter, still without effect on the neighboring tissue, 5 months after a single dose of 2,000 r. van Gieson. No change of the neighboring myelin fibers can be demonstrated. These changes occurred 5 months after the application of a single x-ray dose of 2,000 r. Within an area of spongy dissolution of the white matter in the same case, plasmatic material with red blood corpuscles spreads out from such vessels (Fig. 4). These conditions are demonstrated more distinctly with the Mallory method in Fig. 5. The fibrinoid disorganization of the vessel walls is here followed by erythrodiapedesis, hemorrhages, and fibrin- containing fluid in the gray matter. In all places where the plasmatic fluid spreads into the tissue, oxygen diff"usion is inhibited and the cellular ele- ments become necrotic. On the whole, the findings in this second series confirm and complete the results of our first investigation on x-ray changes in the spinal cord of adult rabbits. The pathogenic mechanism seems the same as in the brains of dogs, observed more than 20 years ago. We have not seen any proof for a primary effect of ionizing radiation on the neuronal constituents of the tissue. Whenever a damage of neuronal constituents could be observed, it was accompanied and often preceded by changes in the barrier function. The focal lesions of the white matter are distributed irregularly within the field of x-irradiation of the cord. Some sections are filled with areas of demyelina- tion, whereas at other levels not a single one can be seen. This may account MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 215 Fig. 4. Plasmatic mhltration and erythrodiapedesis into the tissue in an area of disintegration of the white matter from the same case as Fig. 3. van Gieson. for the fact that in routine in\estiiiations of single cases, no morphologic changes may be observed, although the animals are paralyzed. In the second series, transformation of the tissue debris into sudanophilic material could not be observed, although in many cases the clinical symp- toms indicated that morpholo2,ic changes were 3 to 4 weeks old. In addition to these experiments on the spinal cord of rabbits, the brains of approximately 100 Syrian hamsters were irradiated to examine the effect of a high and intensi\e single x-ray dose using a special Siemens x-ray machine with a beryllium tube. The animals were fixed on a small table (Fig. 6 I, and the whole body was covered v,ith a half tube of lead, except for the head which was held in position by two metal clips. A small brass cylinder, 1 cm in diameter, was used for local application of x-rays and Fig. 5. The same case as Fig. li. Fibrinoid ( plasmatic] ciisorgamzation of greatly enlarged angioectatic vessels with erythrodiapedesis and transudation of fibrin- containing fluid into the tissue of the posterior horn producing a necrotizing effect. Masson stain. Fig. 6. (see text) 216 MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 217 positioned on the mid-dorsal skull. Technical conditions included: 40 kv, 25 ma, filter 0.3 mm Al, and focus-skin distance 5.5 cm. To determine more exactly the actual intracerebral radiation, the x-ray dosage was measured by a Siemens dosimeter at a level to include skin, bone, and 1 mm of brain substance, which means that within a distance of 2.5 mm about 50% of the surface dose was measured. Further \alues were obtained by using 1 mm plates of a phantom material, Cellon, which has the same absorption value as brain tissue. This procedure indicated a diminution of the dose in dif- ferent regions (Fig. 7). EflFective x-radiation values of 1,000 to 80,000 r were administered to the cerebral cortex with application times of 28 sec to 37 min 34 sec, respectively. Doses of 20,000 r and more were badly tolerated by the animals; generally they died spontaneously 2 or 3 days later. Young animals seemed to be more sensiti\e than older ones, and on the average showed more sexere morphologic changes. With the application of 30,000 r, sharply limited zones of total necroses, sometimes containing numerous small hemorrhages, could be produced and were fully de\eIoped after 67 to 68 ^ ^ ^ ^ ^ 90 80j 7n ^ fc 60 50 4o\ ^ •>o . 20 V ^ ^ / ^% J 1 Cellon mm 10 10 20 30 40 50 (mm) Fig. 7. Equi\alent doses for special tube used with soft x-ray radiation. The diagram demonstrates the diminution of the x-ray dose at different levels in the tissue. The figures represent the effecti\e dose in percentages of the surface dose. The abscissa demonstrates the diffusion of the x-ray beam within the tissue. 218 W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER Fig. 8. Sharply limited total radionccrosis of semicircular shape in the brain of a Syrian hamster, following the application of 30,000 r after 67 hours of survival. Numerous diapedetic hemorrhages, some of them far from the necrotic zone in the thalamus and midbrain. H. and E. hours (Fig. 8). It was not possible to identify the different types of cells in the necrotic zone, which included the dorsal part of the thalamus. The perikaryon had disappeared and all nuclei, including numerous polymorpho- nuclear leucocytes, were in a state of pyknosis or rhexis (Fig. 9). A fairly large number of small hemorrhages could be observed at some distance from the necrotic zone. No progressive interstitial reaction of the glial or mesenchymal tissue was seen. It does not seem possible to determine the pathogenesis of these necroses, which have been designated as anemic by Russel ct al. (1949) because of their pale appearance. We did not find occlusions of pial vessels or larger arteries, and certainly the necrosis does not involve a particular region of arterial irrigation. Rather, it is restricted to just the irradiated field with a semicircular penetration into the depths of the cerebral tissue. To exclude an ischemic condition as the cause of the necrosis, India ink was injected into the left ventricle of the heart of living anesthetized animals. The freely circulating blood carried the indicator substance throughout the capillary bed of the area that had received 20,000 r 50 hours before (Fig. 10). In some places, where erythrodiapedesis had occurred, the India ink pene- y » ^^V"'- • *.., fej.% ^.» ...^ -^^ **- .*/, V •'r. . M' :»'* Fig. 9. Multiple hemorrhages, pyknosis. and rhexis ol tissue cells and of emigrated polymorphonuclear leucocytes in a necrotic region of the same type as in Fig. 8 and produced by the same x-ray dose. H. and E. Fig. 10. Nearly complete representation of the capillary bed of the cerebal cortex by India ink, 50 hours after local irradiation of a Syrian hamster with 20.000 r. 219 220 W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER trated into the tissue. This region was completely stained blue with trypan blue injected subcutaneously shortly after the irradiation. To clarify these observations, the experimental technique was modified in two diflferent ways: (1) we diminished the x-ray dose to 1,000 r, and (2) we shortened the survival time to 1 hour, using x-ray doses large enough to produce complete tissue necroses. With the first of these methods, the size of the necrotic area decreased, so that with 5,000 r only a small tan- gential zone of the cortex including the first and second layer was affected. These lesions developed within about 8 days. The nerve cells and glia cells lost their cytoplasm, and their nuclei were shrunken. A few polymorpho- nuclear leucocytes were scattered throughout the necrotic tissue. This small necrotic zone was surrounded by a broad region of spongy tissue containing numerous tiny hemorrhages. Within 6 days, an application of 10,000 r produced a larger zone of necrosis extending through the whole cortex (Fig. 11). In these cases the first reactive processes had begun, and several fat granular cells and progressive glial cells were found at the borders of the necrotic tissue, especially near the pia. However, within the center of the necrosis nothing seemed viable. Here again, the zone of hemorrhages was considerably more extensive than the necrotic area. The necrotic zone, i^ Fig. 11. Large, sharply bordered areas of acute radionecrosis invohing the whole cortex and corpus callosum and containing numerous, partly confluent, diapedetic hemorrhages; 10,000 r with 6 days survival. Azan stain. MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 221 Fig. 12. Intravital trypan blue staining of the necrotic region of the same brain as in Fig. 11. even when small, was surrounded by a broad shell of sponoy tissue where the cellular elements showed only minor chanoes. To demonstrate the bar- rier function, a series of animals recei\ed a subcutaneous injection of 1 cc of Kr trypan blue solution from 1 to 6 hours after irradiation. As is well known, the brain remains unstained under normal conditions. With disinte- gration of tissue and destruction of vessels, the dye may enter the tissue. Figure 1 1 demonstrates such a necrotic zone which had developed within 6 days after x-irradiation with 10,000 r and which involves the whole thickness of the cerebral cortex. From the numerous hemorrhages in the necrotic zone, trypan blue spread diffusely all through the destroyed tissue (Fig. 12) . These cases do not ser\e to illustrate a disrupted barrier function unless the blue staining siupasses the necrosis and extends to the surrounding spongy area where only minor cellular changes are seen. This could be obsened in some cases, but it is difficult to demonstrate. Bv shortening the sur\i\al time of the animals, we attempted to observe the earliest stages of tissue necroses. A standard x-ray dose of 20.000 r was applied within 10 min 36 sec. This dose proved sufficient to produce clear necrosis within 24 horns. Surprisinyly. we could produce lesions ot similar size with a considerable diminution of the x-ray dose. After 24 to 26 hours, a large semicircular zone of obvious necrosis covering the irradiated field could be seen extending from the dorsal surface of the cerebral cortex to the corpus callosum. With shorter survival times and in older animals, the depth of the necrotic area flattened (Fig. 13). Here again, a sponginess of Fig. 13. Earlier stage of radionecrosis in the brain of a Syrian hamster, 24 hours after the local application of 20,000 r. The tangentially situated zone of clear necrosis has a spongy character and is demarcated by a strip of even more pronounced spongi- ness. Azan stain. r % '7. # ^ ♦ 4 ' » ■■ * » • * . • V ,♦ • \ ^ f « • % V* *. • ' T • « 1 'y « , - :r. * ^\' • • • " * I • < « ♦ * i •* K ^' ,, > -• • ♦ \ «> » "" * 6 # • ' ¥ IV ^ » * ' . * •^ 4 >K • 4 •• ' - 4 lA « • «»• ♦ • • h * • > \ ^ «.■"*» « • * •1 • a ♦ "1 • 1 ♦ ►♦ •; • ^ % «.. » «>' «i * Fig. 14. The same case as in Fig. 13. In the necrotic zone, the cellular constituents can scarcely be diflferentiated, with only a few exceptions. The plasma has become un- stainable : the nuclei are pyknotic. Some emieiated polymorphonuclear leucocyte;, are also seen. Gallocyanin. 222 MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 223 the bordering zone with granular breakdown of the astrocytic processes could be observed in Cajal preparations. In the necrotic zone, it was difficult to identify the different types of cells Fig. 14). Only a few nerve cells could still be recognized by their acidophilic cytoplasm. In all other cells, the perikaryon became unstainable. Almost all cells, including glial elements, demonstrated pyknotic nuclei. Some polymorphonuclear leucocytes could also be recognized. With lurther shortening of sia\i\al time to 7 hours, only two small zones of spongy loosening of the tissue were seen, situated almost symmetrically in both hemispheres (^Fig. 15 j. They were rather sharply \» ' { \ L i ^ ''•^^•^ I :^l»i^^ /j*"^ ' j^. Fig. 15. Two sharply limited small foci of spongy loosening, symmetrically and rather superficially located in the cortex. This is an early stage, 7 hours after local application of 20.000 r. H. and E. limited by intact nerxous tissue. The cellular elements in the spongy zone exhibited only minor changes, such as shrinkage with a clear nuclear struc- ture (Fig. 16). Sometimes, however, groups of nerve cells showed signs of dissolution, including \acuolization of the cell cytoplasm. After local irradi- ation with 45.000 r and a survival time of 6 hours, small areas of sponginess of tissue of the same type and size reaching from the surface to the 3rd cortical layer could be obser\ed Fig. 17). The nerve cells exhibited only minor chances, such as diminished affinity for gallocyanin. and leucocytes were absent. With low power, this deviation can be more clearly seen as a r t i ^ #f ',* 'vf % » .-« » * , ■• f ' ^ .T ;• ^' i M t ^^^^^ < \ * -,' > .1 • % ' ^ •# ' •* 1 ^* ^. ' *i '. ^*-. - • * 4» « ^^ \ , f^J'^ d # ^ ^ ^*^ - ^ ' s ... * ** j" ^ -. . %^ i ds *^V/ .. ,- ^^' .,#■ •gr- c>^«.; # . _ ' ~ ' , , 4^» Fig. 16. The same preparation as in Fig. 15. Bordering zone of the spongy area, with tissvic cells showing only miner changes. H. and E. ^ i'' '. ' '' . •' ■'' '-.'•. -a'^',"' r Fig. spheres method 17. Sytimicti K aii\- and raciially ariangetl spdiiniiu-ss v.\ tissue in lioth hemi- following local x-irradiation of 45,000 r after 6 hours. W. and E. . In this zone, the cells appear less stained. 224 MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 225 slight pallor in the cortex (Fio. 18) . Hii^h power demonstrates disappearance of Nissl bodies in the large pyramidal cells so that the perikaryon is generally pale and difTusely stained, whereas the nuclei of the smaller nerve cells contain rather coarse chromatin particles (Fig. 19). It is extremely difficult to judge the whole tissue situation from deviations in appearance of the cortical nerve cells alone. The significance of such deviations seems to re- c}uire a special cell study as performed by Krogh and Bergeder ( 1957) using the o^allocvanin stain of Einarson and bv Schiimmelfeder (1957) in his .^. «•'■.,• rii.'.t.i...'' i. ■,.■>•- * -.,', ,' , ••• '.•,•- • J, ■<'■■■ ;- ■'' : t -* '. .<- >• •'*..;» ";•^ i-v/vV.".".?.' is ':..'.■- •- > -,• '■ „ :• . .. .... ■■.;.-•".'*, ' '/^•'.■' h-.,' x'-'^ • ' Fig. 18. EquivaK-nt of the spongy region in Fig. 17, stained with the gallocyanin method. In this zone, the cells appear less stained. histochemical investigations with fluorescent acridin-orange on the nerve cells of the cerebellar cortex. With regard to the different types of nerve cell changes after irradiation of 5,000 to 20.000 r, they made similar obser- vations on cortical nerve cells, and they considered this to be an expression of cell necrosis. Certainly, it requires a high level of experience to decide from the appearance of the cell alone that cell shrinking with dark staining of the perikaryon and a pyknotic nucleus is not reversible and must inev- itably lead to necrosis. In cresyl \iolet and gallocyanin preparations, we have seen such cells distributed over wide regions including the cortex of control animals which had ne\er been irradiated. Their significance is not 226 W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER Fig. 19. Detail of Fig. 18. The large nerve cells are of normal shape; their peri- karyon is lightly stained, and the nucleus is not strikingly altered. The nuclei of the smaller nerve cells contain rather coarse chromatin particles. known, but the experiments of Scharrer (1933) and of Cammermeyer (1960) suggest that they can be produced artificially by simple mechanical pressure on the cortex. A high degree of swelling and vacuolization of the cytoplasin with loss of ribonucleic acids and the occurrence of coarse chromatin particles within a nonpyknotic nucleus seem to constitute evidence of necrosis. But again, the nerve cell nuclei of rodents may normally have rather coarse chromatin particles. The third variety of nerve cell alteration referred to by these authors seems to resemble the so-called ischemic type of nerve cell change. Certainly, as soon as a breakdown of the nucleus is established, death of the cell must be accepted if postmortal processes can be excluded. We have observed the cytoplasm of such cells to be acidophilic, staining with eosin and even with azocarmine and acid fuchsin, but we have had no opportunity to make coinparisons with the results obtained by Schiimmelfeder (1957) with acridine-orange. Krogh and Bergeder (1957) did not state whether the pyknosis of the granular cells is due to a direct influence of irradiation, or if edema of the granular layer may play an intermediary role. Schiimmelfeder ( 1957) favors a direct influence of ionizing rays. However, it is not easily imderstood why the same cause in the same quantity elicits a shrinking one time and a high MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 227 grade swelling of the nerve cells another. In approaching this question, it seems necessary to consider the condition of the whole tissue concerned and not solely the nerve cells, since they are only a part of the tissue, and a necrosis of nerve cells alone does not cause tissue disintegration as seen, for example, in anoxic selective neuronal necrosis. Only if the oligoglia and astrocytic glia are also destroyed, does the continuity of the tissue break down. As we ha\e seen in Cajal preparations, astroglia processes show a granular decay rather quickly, long before regressive changes in the vessel walls are demonstrable. Schiimmelfeder (1957) also mentions disap- pearance of ribonucleic acid in glia cells. This would indicate a much more severe lesion of the neivous tissue: however, definite tissue disintegration needs a certain time to become manifest with clear structural changes. In initial stages, when necrosis is not yet clearly apparent, the most reliable histologic phenomenon seems to be the immigration of single leucocytes into the tissue and the beginning of erythrodiapedesis. We have tried to study the earliest stages of cortical necrosis in regard to the function of the hematoencephalic barrier. We did not succeed in staining intravitally with trypan blue during initial stages of spongy tissue transformation (Figs. 15-19). when seemingly only minor alterations such as moderate swelling and dissolution of the Nissl substance without significant changes of the nuclei were seen. A positive result was attainable, however, before distinct symptoms of tissue disintegration became e\ident. Thus, the blue staining with trypan blue was complete as soon as single diapedetic hemorrhages of small size in the zone of irradiation could be observed or single polymorpho- nuclear leucocytes had immigrated into the nervous tissue. This is demon- strated by a section stained with the azocarmin Malloiy method of Heiden- hain (Fig. 20) and, for comparison, by a macro photo of the blue stained field on the siufacc of the brain i Fig. 21). Discussion We must emphasize that the delayed radionecroses of the spinal cord are not significantly diflferent from those in the brain. We obserxe the same phenomena of altered vascular permeability which in the white matter results chiefly in transudation and in the gray matter chiefly in plasmatic infiltration, often with erythrodiapedesis. It now seems established beyond doubt that these processes are primary to all other destruction of nervous tissue in delayed radionecrosis. although in many cases structural changes of the vessels are not evident. The same pathogenic mechanism in delayed radionecrosis is valid in the human brain, as has been demonstrated by Fischer and Holfelder (1933), Markiewicz (1935), Scholz and Hsu (1938), Zeman (1949, 1955) and others. In a necrotic zone, e\'en after many years 228 W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER Fig. 20. Early and incomplete radionecrosis, 20 hours after local x-irradiation with 20,000 r. Only small single hemorrhages and slight diminution of stainability of the cortical tissue at both sides of the median fissure are visible. Fig. 21. Intravital staining of the irradiated zone of the brain from which Fig. 20 is taken. MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 229 the plasmatic material can be found as an amyloid-like substance, resistant to absorption and behavins; like a foreign body. This substance has not been demonstrated in large amounts in the central nervous system of experimental animals. Whereas most investigators, including in recent years Berg and Lindgren ( 1958). and Zeman (1955), acknowledge this pathogenic mecha- nism of the delayed radiolesions, it is much more difficult to explain the pathogenesis of the acute radionecrosis occurring within hours following the single application of extremely large doses of ionizing radiation of high intensity. We did not see convincing morphologic changes either with 80,000 r after 134 hours or with 45,000 r after 1 /a hours; however, necroses were fully developed with 30,000 r after 67 hoins. They cover exactly the field of irradiation, are apparently not related to regions of arterial irrigation, and do not depend on vascular occlusions. As to the restriction of the lesions to the field of irradiation and the morphologic appearance of the nerve cell changes, our results are largely in accordance with the observations of Krogh and Bergeder (1957) and of Schiimmelfeder (1957), though there was some difference in the application and measurement of the x-irradiation. It is true that fully developed acute radionecroses closely resemble anemic infarctions, but we think that such a designation, used by Russel ct al. ( 1949), is not justified, since during the development of the necrosis, the free passage through the capillary bed has been made exident by India ink. In these cases, we find hemorrhages not only in the region of neciosis but also at some distance from the necrotic zone, invading the unaltered nervous tissue which was exposed to a lower intensity of irradiation and indicating a distiubance in the permeability of the blood vessels. Usually, the centers of the necroses are surrounded by a broad shell of spongy tissue which raises the question of whether we deal simply with a demarcation zone as seen around every more or less complete necrosis. However, the initial stages of such necroses produced by an irradiation of 20.000 and 45,000 r after 7 and 6 hours, respectively, consist only of such spongy loosened tissue, suggesting a local edema and demonstrating no distinct signs of cellular necrosis. Efforts to stain these earliest lesions intravitally with trypan blue were not successful, probably because the disorder of the blood-brain barrier is still incomplete in this stage. But already in the next stage, when only minor changes of the cellular constituents of the tissue manifest themselves and only a few vessels show beginning diapedesis, we find a distinct blue coloration, pointing to the important role of permeability disorders in the pathogenesis of the acute radionecroses also. As soon as distinct necrosis is present, the trypan blue staining spreads far beyond the necrotic center throughout the whole spongy zone. It may therefore be reasonable to consider the sponginess of the tissue as well as the multiple hemorrhages within and out of the necrotic center as being due to a disturbance of penneability of the blood-brain barrier, 230 W. SCHOLZ, VV. SCHLOTE AND W. HIRSCHBERGER primarily caused by irradiation. Krogh and Bergedcr (1957), who produced cerebellar lesions by a high x-ray dosage, did not decide whether the break- down of the granular layer is secondary to edema or is a primary lesion. In experiments with Co''", Vogel et al. (1958) produced a reversible pyknosis of the same gramdar cells. Since we know that the granular layer of the cerebellar cortex is rather sensitive to edema, we must consider the possibility that it is the radiation-induced edema that produces the granular cell changes. However, this does not mean that all tissue changes shoidd be con- sidered secondary to the edema-like loosening of the tissue. In the cerebral cortex, nerve cells remain resistant to a simple edema for a long time. More- over, we failed to find in acute radionecroses the dangerous plasmatic infil- tration of tissue that can inhibit oxygen difTusion. As all cellular elements demonstrate an early and rapid structural breakdown, the assumption may be justified that x-irradiation of high dosage and intensity may cause a co- ordinated breakdown of the hcmatoencephalic barrier and a primary destruc- tion of all other tissue constituents as well. It seems that in delayed lesions, the latent period becomes progressively shorter with an increase of x-ray dose and intensity, and the hcmatoencephalic barrier, in common with other tissue constituents, is at last simultaneously affected by the increased ionizing radiation. Conclusions X-irradiation of the spinal cords of 36 rabbits produced results similar to the previously reported delayed x-ray lesions in the brains of dogs. The fractionated application of doses up to 1 1 ,000 r within 40 days and single doses of 2,000 r were followed by focal zones of disintegration, mainly situ- ated in the white matter, but also affecting the gray substance. These changes occurred after latent periods of from 4 to 33 weeks. The fibrinoid disor- ganization of the vessel walls, erythrodiapedesis, and infiltration of plasmatic material into the central nervous tissue seem to be the primary lesions and demonstrate a breakdown of the hcmatoencephalic barrier. All other changes of the tissue, including demyelination and breakdown of cellular constituents, were preceded by permeability distiubance and may be considered secondary. In the brains of approximately 100 Syrian hamsters, acute radionecroses were produced by single applications of large x-ray doses of high intensity. In animals receiving 5,000 r, necroses were fully developed within 8 days; within 6 days after 10,000 r, and within 3 days after 45,000 r. Several animals receiving 20,000 r showed distinct disintegration of tissue in the field of irradiation after 24 hours, and initial stages in these cases could be detected after only 7 hours. The sharply limited semicircularly shaped lesions covered exactly the field of irradiation and decreased in depth with the diminution MORPHOLOGICAL EFFECT OF X-RAYS TO THE CNS 231 of the x-ray dose and abbre\iation of the sur\i\al time. In fully developed necroses, all cellular elements were broken down, and erythrodiapedesis from more or less numerous \essels occurred not only within, but also out of the necrotic zone. Initial stages demonstrated a spong\' transformation of the nervous tissue with only minor changes of the cellular constituents, such as plasma and nuclear shrinkage. Here, erythrodiapedesis was still lacking. A disorder of the hematoencephalic barrier in this initially spongy territory could not be demonstrated by intra\ital trypan blue staining, while a blue coloration of a large area including the surrounding region of spongy loosen- ing became visible before a distinct disintegration of tissue, indicated by signs of cell necrosis, were demonstrable. Solitary small diapedetic hemorrhages and the immigration of single leucocytes seem to indicate a degree of perme- ability disorder sufficient to allow an intraxital staining with trypan blue. This beha\ior and the appearance of more or less small hemorrhages at the borders of. and sometimes far from, the necrotic foci point to the significance of an early disorder of the blood-brain barrier. Since ner\e cells have been shown to be rather resistant within zones of edematous loosening of tissue, and astrocytes even may become progressi\e, the whole process of necrosis cannot be considered as solely secondary to the permeability disorder. From the morphologic tacts, it seems justified to admit that in acute radionecrosis from large x-ray doses ot hiizh intensity a direct effect on the nervous and astrocytic tissue constituents may occur simultaneously with \ascular damage. Processes of transformation, resorption, and organization de\elop slowlv and are established regularly onlv at the borders of the necrotic zones. References Berg. N. O., and Lindgren. M. 1958. Time-dose relationship and morphologv- of delayed radiation lesions of the brain in rabbits. Acta Radiol. Suppl. 167. Cammermeyer, J. 1960. A critique of neuronal hyperchromatosis. /. Xeuropathol. Exptl. Neurol. 19, 141-142. Fischer, A.. W., and Holfelder. H. 1933. Lokales .Amyloid im Gehirn. Deut. Z. Chir. 227, 475. Krogh, E. v., and Bergeder. H. D. 1957. Experimental irradiation damage of the cerebellum demonstrated by Einarson's Gallocyanin-chromalum staining method. /"" Congr. intern. Sci. Neurol., Brussels, 1957: 3' Congr. intern. Neuropathol. pp. 287-294. -Acta Medica Belgica, Brussels. Lyman, R. S., Kupalov, P. S., and Scholz, VV. 1933. Effect of Roentgen rays on the central nervous system. A.M. A. Arch. Neurol. Psychiat. 29, 56-87. Markiewicz, T. 1935. Uber Spatschiidigungen des menschlichen Gehirns durch Ront- genstrahlen. Z. ges. Neurol. Psychiat. 152, 548-568. Mogilnitzky. B. N., and Podljaschuk, L. D. 1930. Rontgenstrahlen und sogen. '"hama- toenzephalische Barricre." Fortschr. Gehiete Rontgenstrahlen 41, 66. Russell. D. S.. Wilson. C. \\'., and Tansley, K. 1949. Experimental radionecrosis of the brain in rabbits. /. Neurol. Neurosurg. Psychiat. 12. 187. 232 W. SCHOLZ, W. SCHLOTE AND W. HIRSCHBERGER Scharrer, E. 1933. Bemerkungen zur Frage der "sklcrotischen" Zellen im Tiergehirn, Z. ges. Neurol. Psychiat. 148, 773-777. Scholz, W. 1935. Uber die Empfindlichkeit des Gehirns fiir Rontgen- und Radium- strahlen. Klin. Wochschr. 14, 189-193. Scholz, W., and Hsii, Y. K. 1938. Late damage from Roentgen irradiation of the human brain. A.M. A. Arch. Neurol. Psychiat. 40, 928-936. Scholz, W., Ducho, E.-G., and Breit, A. 1959. Experimentelle Rontgenspjitschaden am Riickenmark des erwachsenen Kaninchens. Psychiat. et Neurol. Japan. 61, 417-442. Schiimmelfeder, N. 1957. Fluoreszenzmikroskopische und cytochemische Untersuchun- gen iiber Friihschaden am Kleinhirn der Maus nach Rontgcnbestrahlung. 1" Congr. intern. Sci Neurol., Brussels, 1957: 3' Congr. intern. Neuropathol. pp. 295-308. Acta Medica Bclgica, Brussels. Vogel, F. S., Hoak, C. G., Sloper, J. C., and Haymaker, W. 1958. The induction of acute morphological changes in the central nervous system and pituitary body of macaque monkeys by cobalt'" (gamma) radiation. /. Neuropathol. E.xptl. Neurol. 17, 138-150. Zeman, W. 1955. Elektrische Schadigungen und Vcranderungen durch ionisierende Strahlcn. In "Handbuch der speziellen pathologischen Anatomie und Histologic" (O. Lubarsch et al, eds.). Vol. XIII, Part 3, pp. 327-362. Springer. Berlin. Zeman, W. 1949. Zur Frage der Rontgenstrahlenwirkurg am tumorkranken Gehim. Arch. Psychiat. Nervenkrankh. 182, 713-730. A Demyelinating or Malacic Myelopathy and Myodegeneration— Delayed Effect of Localized X-irradiation In Experimental Rats and Monkeys J. R. M. Innes and a. Carsten Brookhaven National Laboratory Upton, Long Island, New York Introduction For many years the central nervous system was considered hislily resistant to radiation damaiie — and statements to this effect occasionally still appear. It is manifest that there must be some ciualification by reference to the part of the system exposed, the conditions and dosimetry of irradiation, and the species and age of the animals used. The earlier experimental irradiation work on the normal nervous system was reviewed by Warren (1943) and Hicks f 1952 1. We are concerned here with oiu initial experimental irradi- ation studies on the spinal cord of rats and with some observations on experimental monkeys. In man the hazard attached to x-irradiation ot the brain or spinal cord, whether by deliberate design for radiotherapy or unaxoidably when extra- neural sites must be exposed, is well established. Late or delayed irradiation effects on the nerxous system are different from acute massixe radionecrosis which follows extremely high doses. The problems associated with the two types of damage are multiple and complex, and the literature was reviewed by Zeman ( 1955) and by Zollinger 1960) . Many original papers on human cases have been perused, e.g., Lyman ct al. (1933), Stevenson and Eckhart (1945), Pennybacker and Russell (1948). Greenfield and Stark (1948), Boden (1948), Friedman i 1954 1, Itabashi et al. (1957), and Dynes and Smedal ( I960). It is not necessary to deal with these contributions individ- uallv. but we can reiterate the patholologic problems to be faced. The number of reported cases in the literature is an inde.x neither to the inci- dence of delayed irradiation lesions in the spinal cord, nor to the importance of the hazard, as is evident from discussions with neurologists, neuropathol- 233 234 J. R. M. INNES AND A. CARSTEN ogists and radiotherapeutists. As Itabashi ct al. (1957) pointed out, when a neoplasm has been the target for irradiation, neurologic signs which might appear later have usually been attributed either to metastasis or extension of the primary lesion, especially when there has been some apparent im- provement. Further, many human cases may not be followed up after x-irradiation of the spine; in others, autopsies may not be possible when death occurs years later, or it may not be possible to examine the spinal cord at autopsy. Some papers on delayed irradiation myelopathy are based largely on clinical data, and the lesions have not been comprehensively studied. Dynes and Smedal's series included 10 therapy cases, and Friedman (1954) estimated that the incidence of delayed neurologic damage was 10% in 100 patients with testicular carcinoma whose spinal cord had received 5,000 rads or more. In all cases, subsequent to irradiation, there is a latent period ranging from many months to many years, during which there are no neurologic signs or symptoms due to irradiation. The clinical onset can be abrupt or insidious, with a variable neurologic syndrome leading to paraplegia and inevitably to death, although some patients have lived for years with paraplegia (Dynes and Smedal, 1960). Lesions in the spinal cord in such cases have been variously reported as radionecrosis, postirradiation myelitis, or myelopathy; but whatever the designation, the damage can be devastating and appears no different from that described by us (cf. Itabashi et al., 1957, one of the few papers describ- ing spinal cord damage in man). In the delayed postirradiation process, it is not the most superficial layers of nervous tissue which are most radiosensitive, but the white matter in both brain and spinal cord. Neuroglial response has varied according to different reports, but it can be absent or negligible, and in some late lesions neuroglia must have been destroyed at a rate equal to the damage to white matter or rendered incapable of response. In both the human brain and cord, observers have commented on the difficulty of separating the damage done specifically by the irradiation on tissue already traumatized by another cause, such as a tumor. The fundamental question, still not answered, is whether the damage is a direct effect of the radiation or an indirect one caused by a primary change in vascular walls which leads to interference of the normal blood supply, possibly then emanating from chronic hypoxia or ischemia. If it were a direct effect, then the question arises as to what happens to the neural tissue in the latent period before the eventual neurologic signs and symptoms. Years ago, Scholz (Scholz et al., 1959) and Zeman (1955) ob- served in such late lesions the deposition in and around vessels of an "amyloid or paramyloid material," the nature of which has never been unequivocally established by histochemical methods. The thickening of vessel walls and subsequent constriction of lumina were thought to cause X-IRRADIATION AND DELAYED MYELOMALACIA 235 hypoxia or dynamic alterations in the \ascular walls resultins: in increased capillary permeability and seeping through of plasma. This was considered sufficient to account for the lesions in areas of neural substance supplied by the afi'ected vessels. These themes have been discussed repeatedly (see Zeman, 1955, and the most recent paper by Scholz et al., 1959). It is doubtful if experimental work has helped greatly, whether we are dealing with brain or spinal cord ( Malamud vt al., 1954; ( Pennybacker and Russell, 1948: Warren. 1943; Davidoff et al, 1938; Clemente and Hoist, 1954; McLaurin rt «/., 1955; Scholz ct al., 1959). Nor are the reasons hard to find. The acute necrosis produced by massi\e doses of x-rays does not help to explain the pathogenesis of the late delayed damage. After x-irradiation, experimentalists ha\e noted the utterly unpredictable variations (a) of the reaction in difl'erent animals of the same species and age, some animals remaining unscathed under the same experimental conditions and dosimetry which causes marked late lesions in others, lb) in the latent period before nemologic signs de\elop. and c ) in the occurrence of the \ascular changes, because thickening or deposition of "amyloid material" has not always been observed to be associated with the neural lesion. An experimental approach with any animal species brings out that it is different than working with established transmissible neurotropic infec- tions which can be reasonably controlled — the dose of causal agent related to a regular incubation period and a specific pathologic effect. The \ arying latent periods (Table I) set a formidable barrier in designing an experiment on a quantitative basis. Many animals may not develop neurologic signs or lesions under the same conditions of experiment, and to determine this with certainty, it might be necessary to wait much longer than 1 year. Little progress toward the solution of the problem may be expected until a lesion can be produced consistently in small animals, under controlled conditions related to dosimetry and exact localization of exposure. Monkeys and dogs can hardly be used in large numbers because of the expense invoked, although delayed cerebral and spinal cord lesions have been produced in both species, and more meticulous clinical obser\ations are f)ossible with the larger animals. If this were possible, it might open the way for study of the pathogenesis of the myelopathv by examination of a large series of animals from a few days postirradiation up to a year or more. Of ecjual importance would be more accurate determination of the minimal pathologic dose to effect the spinal cord damage. Our obser\ations were made with these facts in mind, and their importance may lie in the consistency of production of spinal lesions in rats, together with the continuing study of the pathogenesis. The work was initiated by obser\ations on rats exposed to upper body irradiation in which some clinical signs suggested the animals might be suffering from myelitic pain, evidenced by irritability, sensiti\ity to touch, 236 J. R. M. INNES AND A. CARSTEN and changes in beha\ior. Some animals developed paralysis of the hind legs many months after irradiation. After identification of the demyelinating myelopathy in a few rats, a series of experiments were planned in which, by adequate shielding, only the vertebral column from about sixth cervical to second thoracic segment was irradiated. The first experiments were con- cerned with definition of the pathologic process and its topographic neuro- anatomic distribution. Techniques White female rats were housed two in a cage and given food and water ad libitum throughout the experiment. They were 3 to 6 months of age at irradiation. Before and after irradiation, they were weighed and examined daily. Rats were anesthetized with intraperitoneal injections of sodium pento- barbital, 45 mg per kg of body weight. In the groups exposed to upper body irradiation, the animals were marked with dye at the xiphoid process and placed in 2-in. diameter lucite tubes. A ^4 -in. -thick cylindrical lead shield was placed around the lower half of the tube at the dye mark, so that the upper body and head of the animal were exposed to the x-ray beam. In rats receixing thoracic exposure, a second lead shield was placed over the head end of tlie lucite tube at the manubrium. Animals indicated as spine exposed were completely surrounded by ^ in. lead shields containing a small hole in a position so that only the spine from sixth cervical to second thoracic segment was exposed (Table I). Tlie irradiations were made with a General Electric 250 kV Maxitron x-ray machine. The radiation factors were: 250 k\p. 30 ma. 0.5 mm Cu filter. Aluminum parabolic filter for field uniformity, target to skin distance from 6.5 to 9 in., dose rate of 200 to 350 rad per minute measured in a tissue ecjuixalent phantom, half \alue layer of 2.15 mm of copper. All animals rats and monkeys) received 3500 rads. Clinical Findings After irradiation, a latent period varying from 5 to 9/2 months occurred in rats. The first sign of untoward involvement of the spinal cord was in- continence of urine, which persisted to some degree in some rats for weeks before any motor weakness or incoordination of the hind legs developed. Thereafter, the rats became obviously unsteady on their hind feet and were ataxic, and the tail lost its tonicity. This syndrome progressed imtil the animal's hind quarters were completely immobilized, without the tail or limbs becoming completely flaccid. An afTected animal could move around, X-IRRADIATION AND DELAYED MYELOMALACIA TABLE I Experimental Rats 237 Duration of clinical Irradiation Latent period signs until sacrifice Rat number (3500 rads) (months) (days) 1 (42/59) thorax 5 3 2 (70/59) thorax ^y. 24 3 (96/59) spine VA 2 4 (9/60) spine 5 4 5 (11/60) spine 5 28 6 (17/60) thorax 7 2 7 (18/60) thorax 5'/. 2 8 (21/60) spine 7 No but clinical signs, lesions in spinal cord 9 (27/60) spine 6'/. 8 10 (67/60) thorax 9 28 11 (81/60) / spine 9'/. No clinical signs pulling itself by its forelimbs with the hind limbs cliat:L;inu helplessly behind. Complete loss of sphincter control of bladder and rectum resulted in the hind charters becoming permanently wet and soiled. The "paralyzed"" rats were allowed to surxive for a few days up to 28 days beiore being sacrificed for neuropatholosic study lable I ' . Pathologic Findings As a routine survey for lesions and their extent and distribution, the spinal cord was fixed in situ in the \ertebral cokmin with its sin rounding skeletal muscles. Subsequent to fixation and decalcification, the column was sliced trans\ersely throughout its entire length in 1-2 mm pieces. The caudal surface of each slice was sectioned and stained with hematoxylin and eosin. Usually this amounted to 28 or more blocks i including usually 3 of the brain i being cut from each rat. The distribution of the lesions was plotted for each rat as in Fig. 5. We do not include any detailed reference to examination of the spinal cord of the many rats irradiated and killed at inter\als from 1 dav to 1 month, or to extensive histologic and histochemical work on embedded and frozen sections. The spinal cords from nonirradiated control rats were also studied. As might be expected from the variability of the latent period and the time allowed between on.set of signs and sacrifice, there was some varia- bility in extent of damage to the spinal cord. 238 J. R. M. INNES AND A. CARSTEN r^' X-IRRADIATIOX AND DELAYED MYELOMALACIA 239 At the level in the cord where the lesion was seen in its fullest develop- ment (Figs. 1, 2. and 3), the process had de\eloped into acute malacia terminating in severe liquefaction of the white matter. To some extent, the ventrolateral white columns were more damaged than the dorsal columns, for although dorsal and \entral areas were affected in some rats ' e.g. Fig. 1 ) , the dorsal columns were never selectively changed. In paraffin sections, the afTected areas were spongy or reduced to holes and cystic spaces, bridging across which were scattered skeins of glial and reticular fibrils and minute vessels. As Figs. 1-3 show, the process constantly appeared more severe under the leptomeninx, gradually decreasing in intensity inwards. In most rats the gray matter was intact and never showed acute softening as in the white substance. In the spongy and cystic areas, there were no gitter cells. or so few as not to be noticeable, nor was there any astrocytic or fibrillary response — glial or reticulai' > Fig. 4 ) . Parts of fragmented axis cylinders were scattered throughout the malacic focus, sometimes within what was presumed to be ballooned and liquefied myelin sheaths. There was no meningeal reaction, no hemorrhage, and the spinal nerves and ganglia in the same area were undamaged. The lesion described corresponded in its regional distribution to the irradiated area of the body. For e.xample. in animals in which the thorax had been irradiated, only the thoracic cord was afliected. In others, where the spine was selectively irradiated from about sixth cer\ical to second thoracic segment, only that area showed the acute damage. Above and below the focus, secondary iWallerian) degeneration was clearly e\ident. depicted in the pariffin sections by numerous "holes" — i.e., liquefied myelin sheaths and axis cylinders. As an example of distribution of the lesions in a thorax-irradiated rat. Fig. 5 is a diagram of slices of the spinal cord cut at different le\els. As sections are studied starting at the first cervical and proceeding in series to the sacral level, normal spinal cord and \ertebral marrow is lound until the irradiated area is reached. The lesion then may start on one side, then the other, and continues until it merges into an irreyular funicular focus of Fig. 1. Spinal cord, rat. spine irradiated. Latent period 6' _• months, sacrificed after 8 days duration of neuroparalysis. Severe myelomalacia of all parts of white matter — dorsal and ventrolateral columns. Fatty marrow. Lesions in muscle (upper left) not too clear at this magnification (See Figs. 7.A and 7B). Hemato.xylin-eosin. .\bout X 16. (.Area marked by arrow shown in Figure 4). Fig. 2. -Another case, rat (17/60). thorax irradiated. Latent period 7 months, duration of signs 2 days. Set' Fig. 5 for distribution of lesions. Malacia confined at this level to lateral column of white matter on one side. Fatty marrow. Hematoxylin- eosin. X 16. 240 J. R. M. INNES AND A. CARSTEN K'. .•■; n /' ■■ 4e i fc,:** '.■'■ '■> . z;^ '/f? ?/V 'C'^ (5- c,.* '^'m&^^M.'- X-IRRADIATION AND DELAYED MYELOMALACIA 241 malacia. This is related to the well known irradiation damage which affects the marrow of the surrounding vertebrae. Two rats that showed no clinical signs were killed at 7 and 9/2 months after irradiation, and small lesions (Fig. 6) were found in the white matter at an early stage of development. Severe myodegeneration and necrosis of the veitebral skeletal muscles in the same area as the spinal cord damage was almost a constant concomitant finding (Figs. 7 A and 7B). Such changes have not been reported in experi- mental studies by others or in human cases of postirradiation myelopathy. Experimental work on x-irradiated monkeys Concurrently with the rat studies, comparable experiments were carried out on adult monkeys, and they will be reported upon separately (neuro- pathologic studies made in collaboration with Webb Haymaker, M.D., Armed Forces Institute of Pathology, Washington, D.C.). These can be briefly summarized. Five monkeys were irradiated by the same method and exposures (3500 rads) were restricted to the same area of the vertebral column as in the rats, i.e. to include the area from the sixth cervical to second thoracic segment of the spinal cord. One monkey died from pneiniionia 3 months 3 days after irradiation without showing neurologic signs, and no lesions were found in the irradi- ated part of the spinal cord. One monkey developed neurologic signs 5 months 13 days after the irradiation; motor weakness of the lower limbs started and progressed until there was complete paralysis without the legs or tail being flaccid. There was also loss of sphincter control. In spite of this, the monkey remained very agile and climbed around its cage and tree by use of the arms alone. The animal was sacrificed for study of the nervous system after a clinical course of 4 weeks. A malacic myelopathy similar to that in experimental rats was found in the irradiated part of the spinal cord. Two of the monkeys developed neurologic signs between 6 and 61/2 months postirradiation. Another monkey developed signs 8 months 13 days after irradiation, but the paralytic course was thereafter very acute and the animal was killed when moribund, 4 days after the onset of clinical signs. Fig. 3. Another rat. Latent period 5yi months, duration of cHnical signs 2 days before sacrifice. At this level, the lesion is more pronounced on one side of ventro- lateral column than the other. Hematoxylin-eosin X 16. Fig. 4. High magnification from area in Fig. 1 marked by arrow. Pia mater and nerve roots on right; gray matter, left. Almost complete tissue dissolution of white matter witli no neuroglial response. Hematoxylin-eosin. X 100. 13 14 Fig. 5. Same case as Fig. 2, showing distribution of lesions in the cord. Malacia represented by white areas. Secondary degeneration above and below shown by smaller scattered white blobs, from No. 5 to about No. 9. Lesion starts about No. 9 and develops to its maximum intensity about No. 12, fading out about section No. 14. Numbers 1-8 roughly represent the cervical cord and its segmentation; the remainder, thoracic cord. 242 X-IRRADIATION AND DELAYED MYELOMALACIA 243 Fig. 6. Spinal cord of rat spine irradiated. Killed alter 7 months with no neuro- logic signs, .\bout C 8-T 1. Early stage of small malacic focus about C 8-T 1 on the left. Fatty marrow. Hematoxylin-eosin. X 16. Discussion A severe myelopathy, localized to the area of irradiation, can be produced with some consistency in rats, at a dose le\el within the therapeutic range used in man. and in which neuroparalytic accidents have occurred after deliberate or accidental exposure of the spine. In rats, there is also the par- allel of an unpredictable latent period, sometimes many months before the onset of progressive neurologic signs. The e.xtent and localization of the myelomalacia in the rats is thus responsible for the syndrome starting as motor weakness of the hind legs and progressing to ataxia and paralysis. The clinical pictiue is thus what is commonly called "posterior paralysis" in animals, which bv itself means little. Neurologic examination of small laboratory animals is restricted to obser\ations on a few cardinal objective signs, and may be no indication of what is ultimately found after neuro- pathologic studies. For example, in the course of this experimental work and by virtue of extensive histologic work, we found pituitary chromophobe adenoma in 3 rats, one spinal ependymoma, one sarcoma, and more recently 244 J. R. M. INNES AND A. CARSTEN Fic. 7. A. \ ritflual skeletal muscle from same rat as P'ig. li, shdwiiiy nccio^is, waxy degeneration, loss of muscle nuclei in center, and some proliferation at periphery of lesion. B. Another case with waxy degeneration, calcification and early fibrosis. Hematoxylin-eosin, X 80. a spinal oli,s;odendros;lionia (Innes and Borner, 1961). Apart from some head tilting and circling in the rats with the cranial tumors, all showed virtually a comparable clinical picture. The malacic lesion occurring after a long latent period and due to X-IRRADIATION AND DELAYED MYELOMALACIA 245 x-irradiation of the spinal cord in experimental rats is different from the ravaging necrobiosis wreaked on all areas of the spinal cord (gray and white matter) when the animals are exposed to extremely high doses, with survival from a few days to a week. From the few neuropathologic studies on human cases (Itabashi ct ai, 1957), delayed irradiation myelopathy in rats appears to be a similar process, with the exception of the absence in rats of unmistak- able changes in the wall of vessels. In the spinal cord, white matter seems more sensitive than gray. Irradiations could possibly ha\e some direct effect on myelin sheaths and axis cylinders, aside from any changes caused in the endothelium or walls of vessels. This might be suggested by the work of Leboucq (1934). on the inhibitory effect of irradiation on the developing myelin of baby rats. There might be some concern about the definition of the process, whether it should be designated a demyelinating or malacic one. It is demyelinating in the sense of predilection for attack on the white matter, and no doubt it could start as such. However, it seems that once the lesion starts it is a rapidly progressive one and then it cannot be regarded as anything but a \ery severe liquefacti\e process. A large variety of histochemical methods on sections through such a lesion failed to identify with precision any specific degradation products of myelin breakdown. The almost negligible glial reac- tion is of importance, and in the white matter the oligodendroglia seem to disappear as fast as the myelin sheaths. Regarding the changes in the walls of vessels, which ha\e been depicted as hyaline, amyloid, or para-amyloid degeneration in human postirradiation myelopathy (Zeman. 1955: Scholz ft ai, 1959: Pennybacker and Russell, 1948) but which were not found by O'Connell and Brunschwig, (1937) it is important to note there was no evidence in the rat lesions of the depo- sition of any imusual degenerati\e substance in or around vessels. Whether dynamic alterations in capillary permeability are responsible for an irre\o- cable destruction of white matter is another problem. A spontaneous demyelinating disease of the spinal cord ! and not the brain) in two rats was reported by Pappenheimer i 1952). From his descrip- tions and illustrations, the condition cannot be distinguished clinically or pathologically from experimental postirradiation myelopathy. We know that comparable types of disease processes can be produced in the ner\ous system by divergent types of causal agents; but that spontaneous demyelinating myelopathy can also occur in rats, should be recognized. Pappenheimer was unable to transmit the disease to other rats or mice: the cause was never ascertained, nor has any similar disorder been reported by others. Despite this, we cannot relegate into the limbo of forgotten things the remote feasa- bility that Pappenheimer's murine disease was caused by a virus and that irradiation might not light up some latent neurotropic infection. There remains to be considered the degeneration and necrosis of the 246 J. R. M. INNES AND A. CARSTEN vertebral muscles in the direct vicinity of the spinal cord damage, i.e., in the same irradiated area. The specificity of this myodegeneration in that it was caused by the irradiation is undoubted for the lesion is certainly not artifact or traumatic due to handling the rats. The changes are no different in kind or severity from those seen in types of myodegeneration of man, domestic, or laboratory animals, and which can be produced by a multi- plicity of causes, perhaps most characteristically in natural and experimental alpha-tocopherol deficiency (Hadlow, 1961 ). That some of the lesions were old chronic ones was e\ident by the frecjuency of calcareous depositions, and again no changes in the walls of arteries supplying aflfected muscles were seen. Skeletal muscle is regarded as radio-resistant, but there are few obser- \ations on muscle in concurrent studies of any more deep-seated process which follows irradiation of the nervous system. The study is being continued using both rats and monkeys, with the ]3articular aim of seeking clues to determination of the early stage of damage to the nervous system and thus to a better understanding of patho- genesis. The clinico-pathologic studies on monkeys along with serial EEG recordings is but part of this study. In rats, groups of animals have also been irradiated in the lumbar enlargements of the spinal cord. Finally, as such experimental work has clear medical radiotherapeutic implications, groups of animals are now being irradiated with the same dose (3500 rads), but in divided doses following patterns used in radiotherapeutic treatment of lumian beings. ACKNOWLEDGMRNT.S Our thanks are due to Mr. R. F. Smith, Photographic Division, Brook- haven National Laboratory, for the photographs; to Miss Claire M. Lallier, for technical assistance and care of the experimental rats: and to Miss Ruth Wright, for her part in the extensive histological work involved in the pathologic studies on the rats. Refp:rences Boden, G. 1948. Radiation myelitis of the cervical spinal cord. Brit. J. Radiol. 21. 464-469. Clementc, C. D., and Hoist, E. A. 1954. Pathological changes in neurons, neuroglia, and blood-brain barrier induced by x-irradiation of heads of monkeys. A.M. A. Arch. Neurol. Psychiat. 71, 66-79. DavidofT, L. M., Dyke, C. G., Elsberg, C. A., and Tarlov, I. M. 1938. The eflfect of radiation applied directly to the brain and spinal cord I. Experimental investiga- tions on Macacus rhesus monkeys. Radiology 31, 451-463. Dynes, J. B., and Smedal, M. I. 1960. Radiation myelitis, Am. ] . Roentgenol., Radium Therapy Nuclear Med. 83, 78-87. X-IRRADIATION AND DELAYED MYELOMALACIA 247 Friedman. M. 1954. Calculated risks of radiation injury of normal tissues in treat- ment of cancer of the testis. Proc. 2nd \atl. Cancer Conf. L 390-400. Greenfield. M. M.. and Stark. F. \L 1948. Po:t-irradiation neuropathy. Am. J. Roent- genol. Radium Therapy 60, 617-622. Hadlow, \V. J. 1961. In "Comparative Neuropathology" (J. R. M. Innes and L. Z. Saunders, eds.). Chapter 5. Academic Press. New ^'ork. in press. Hicks. S. P. 1953. Effects of ionizing radiation on adult and embryonic nervous sys- tem. Research Pubis. Assoc. Research Nerrous Mental Disease 32. 439-462. Innes. J. R. M., and Borner, G. 1961. Tumors cf the central nervous system of rats: with rejjorts of two tumors of the spinal cord and comments on posterior jaaralysis. /. Xatl. Cancer Inst. 26. 719-726. Innes, J. R. \I., and Saunders, L. Z. 1961. ■■Comparati\c Neuropathology," Chapter 23. Academic Press. New ^'ork. in press. Itabashi, H. H., Bebin, J., and Dejong, R. N. 1957. Postirradiation cer\ical myelo- pathy, report of two cases. Neurology 7, 844-852. Leboucq. G. 1934. Actions des rayons x sur la formation de la mycline chcz le rat blanche. Rev. beige sci. med. 6, 383-387. Lyman. R. S., Kupalov. P. S., and Scholz. \V. 1933. Effect of roentgen rays on the central ner\ous system. A.M. A. Arch. Seurol. Psychiat. 29. 56-87. McLaurin. R. L.. Bailey. O. T.. Harsh. G. R,. HI. and Ingraham. F. D. 1955. The effect of gamma and roentgen irradiation on the intact s])inal cord of the monkey. Am. ]. Roentgenol., Radium Therapy Nuclear Med. 7.3. 827-835. Malamud.N., Boldrey, E. B., Welch. W. K.. and Fadell. E.J. 1954. Necrosis of !irain and spinal cord following x-ray therapy. /. Neurosurg. 11, 353-362. OConnell, J. E. .\.. and Brunschwig. \. 1937. Obser\ations on the roentgen treat- ment of intracranial gliomata with especial reference to the effects of irradiation upon the surroimding brain. Brain 60, 230-258. Pappenheimer, .-X. M. 1952. Sjjontaneous demyelinating disease of adult rats. Am. J. Pathol. 28, 347-355. Pennybacker. J., and Russell, D. S. 1948. Necrosis of the brain due to radiation therapy. /. Neurol. Neurosurg. Psychiat. INS.] 11. 183-198. Scholz, \V.. rjucho, E.-G.. and Breit. .\. 1959. Exprrimentelle Rontgenj]jatschaden am Riickenmark des erwachsenen Kaninchens: Ein weiterer Beitrag zur wirkungs- weise ionisierende Strahlen auf das Zentralnervose Gewebe. Psychiat et Neurol. Japan 61. 417-442 Stevenson, L. D.. and Eckhart. R. E. 1945. My(4omalacia of the cervical portion of the sjjinal cord, [probably the result of roentgen therapy. A.Al.A. Arch Pathol. 39. 109-112. Warren. S. 1943. Effects of radiation on normal tissue \'III. Effects on the gonads IX. Effects on the nervous system. A. MA. Arch. Pathol. 35. 121-139. Zeman. W. 1955. Electrische Schadigungen und Veriinderungen durch ionisierende Strahlen. Erkrankungen des zentralen Ner\ensystems. In "Handbuch der speziellen pathologischen .Anatomic und Histologic" (O. Lubarsch et al., eds.). Vol XIII, Part 3, [jp. 327-362. Springer. Berlin. Zollinger, H. U. 1960. Radiohistologie und Radio- Histopathologic. In "Handbuch der .Allgemeinen Pathologic. Strahlung und Wetter" ( F. Biichner et al.. eds.), \'ol. X. Part 1. I)]). 127-287. Springer. Berlin. Effects of High-Dose Gamma Radiation on the Brain and on Individual Neurons* F. Stephen Vogel Nezr York Hospital — Cornell University Medical Center, Xeiv York, Xe;c York Massive doses of ionizins, radiation reoularly and promptly brina; about characteristic morpholosic alterations in certain neural tissues and in the mesenchymal structures in and around the brain (Arnold et al., 1954; Hay- maker <'^ al, 1958; Voa;el ct al., 1958: Wilson, 1960). Most notable among these chanoes are contraction and pyknosis of the nuclei of the granule cells of the cerebellum and leiicocytic infiltration into the walls of the cerebral blood vessels, in the leptomeninges, and choroid plexuses. These were conspicuous in monkeys exposed to large doses of gamma radiation from Ba""-La^^" and Co''" sources, as have been described elsewhere (Haymaker ct al.. 1958; Vogel rt al., 1958). There is much e\ idence that the pyknotic change in the cerebellar granule cells in cats i Briinner. 1920), monkeys Vogel ft al., 1958), rabbits (Gerstner ct al., 1956), guinea pigs ( AKord and Brace, 1957), mice, and rats (Hicks and Wright, 1954) is transitory, and ancillary studies indicate that similar transient structural changes occur in these cells grown in tissue culture and exposed to ionizing radiation. Ne\ertheless, recent observations have made it clear that in dogs this cellular response, although initially characterized by nuclear contraction, is often followed promptly by karyor- rhexis with cellular death (Vogel, 1959 i. When examined with the electron microscope, the pyknotic and kaiyorrhetic cells regularly show distinctive alterations in intracellular fine structure i Vogel, 1959). These pro\ide in- * These studies were conducted at the Uni\ersity of California. .-Xrmed Forces Institute of Pathology, Washington, D.C., Randolph .\ir Force Base. Randolph Field, Texas, and at the University of Texas and the U.S. .\ir Force, Austin, Texas. They were supported by funds provided by the U.S..\.F. School of .Aviation Medicine, Ran- dolph .\ir Force Base. Texas, by the Medical Research and Development Board, Office of the Surgeon General, U.S. .\rmy. and by a research grant from the National Insti- tute of Neurological Disease and Blindness of the National Institutes of Health, U. S. Public Health Ser\ice. The technical assistance of Mrs. Margarete Markey in preparing the tissue cultures is gratefully acknowledged. 249 250 F. STEPHEN VOGEL formation about the pathogenesis of transitory and lethal cellular responses of neurons to ionizing radiation. Methods Most procedures employed in these studies have been described in detail in individual publications. Total body radiation was administered to 67 young, 2- to 4-year-old, male Macacus rhesus monkeys, in graded doses from 1.000 to 30,000 r from a Ba^^"-La^^" source, at 1,000 r per minute. Detailed morphologic examina- tions were made of all animals promptly after death (Haymaker ct al., 1958). Gamma radiation, in total doses of 10,000 r, was administered at 1,000 r per minute from a Go''" somce to 48 young male Macacus rhesus monkeys, divided into 3 groups of 16 animals each. One group received radiation to the head with the body shielded ; another, to the body with the head shielded; and a third, to the entire animal. At periodic intervals up to 96 hours later, pairs of animals from each group were killed (Vogel ct al., 1958). Explants of cerebellar tissue from mice, 3 days old, were grown on slides with prepared media (Gillete and Findley, 1958) in Garrel flasks and Maximow chambers. Gamma radiation was administered in doses of 10,000 r from a Go"" Ticker machine at 160.5 r per minute at a distance of 30 cm. Irradiated and nonirradiated cultures were examined periodically by the phase microscope, and tissues were remoxed from the cultmes, stained by hematoxylin and eosin, and examined by light microscopy. Young healthy mongrel dogs and adult white albino rabbits were exposed to 15,000 r of gamma radiation from a Go''" Ticker machine over the superior cerebellar region with a field 5 cm in anterior-posterior dimension and 7 cm across. The rate was 160 r per minute with a source to skin dis- tance of 30 cm and a half value layer of 1 1 mm of lead. Tissues were taken from the cerebellum of anesthetized animals, fixed immediately in 1% osmium tetroxide solution, and prepared by standard methods for electron microscopy (Vogel, 1959). Observations Morphologic Effects of Gamma Radiation on the Brain as viewed WITH THE Light Microscope Exposure of animals to massive doses ol ionizing radiation is followed promptly by conspicuous morphologic alterations in the brain and mesen- chyma, notably by a pyknosis of the cerebellar granule cells and inflamma- EFFECTS OF HIGH-DOSE RADIATION ON BRAIN 251 tion of the cerebral blood vessels, leptomeninties, and choroid plexuses. These lesions were not evident in monkeys exposed to 1,000 r. They were present in some animals, but equixocally or in minimal intensities, after exposine to 2,500 r. They were found with increasing frecjuency and in- tensity after doses of 5,000 and 10,000 r. They showed slight increases with £;reater dosages up to 30,000 r. The severity of the lesions differed appreci- ably from animal to animal. e\en when ex]30sure and sur\i\al conditions were essentially identical. The cytologic chanoes were well established within 2 hours after radi- ation. They were dynamic, for their intensity increased rapidly within the next 8 to 24 hours and then regressed precipitously, the lesions being min- imal or absent 96 hours after exposine. The pathologic alterations were ol the same character and intt'nsity whether the head and body or only the head was irradiated. They did not occm- when the radiation was applied to the body with the head shielded, the findings pro\idecl e\idence that these cytologic responses were induced by the ionizing rays acting directly upon the intracranial tissues, neither being initiated nor enhanced by exposure of other regions of the body. Granule Cp:ll Change The morphologic appearance ot the affected cells was notably sin:iilar in monkeys and rabbits, being regularly characterized by a reduction in the diameter of the nucleus to as much as one-half the normal, with marked condensation of the intranuclear chromatic material. Narrow margins of basophilic cytoplasm were visible about some of the contracted nuclei, and these often stained deeply with pyronin. Up to 50''r of the granule cells of a single animal were severely altered: most others remained normal: few showed intermediate degrees of change. Usually the affected cells were haphazardly distributed throughout all portions of the internal granular layer and cerebellum. In some animals, there was preferential localization, the vermis and deeper portions of the granular layer being most often so inxolved. Golgi and Purkinje cells were regularly spared. The pyknotic cells were more widely separated from one another than nonpyknotic ones. The appearance resembled that caused by extracellular edema, although regu- larly unaccompanied by coagulated fluid. The widened intercellular spaces were infiltrated by only a few leucocytes and macrophages and rarely con- tained hemorrhages. Neuronophagia was absent. Perivascular cuffing by leukocytes was minimal in the cerebellar cortex, particularly so in the granular layer. The initial cytologic changes in the granule cells of dogs was also charac- terized by contraction of the nucleus, but this was accompanied by nuclear 252 F. STEPHEN VOGEL ' ^mtit %'t'i Fig. la. Granular layer of the cerebellum of a normal dog. The granule cells have uniform, rounded nuclei with a nucleolus and fine chromatin material, but no visible cytoplasm. Hematoxylin and eosin stain. X 400. Fig. lb. Granular layer of a dog 15 hours after exposure of the head to 15,000 r of gamma radiation from a Co'" source. Many nuclei are shrunken and hypcrchro- matic. Some show karyorrhexis. Hematoxylin and eosin stain. X 400. EFFECTS OF HIGH-DOSE RADIATION ON BRAIN 253 fragmentation in some (Figs, la, b). Macrophages, with phagocytized cellu- lar debris, were present. Notable losses of granule cells became evident in animals killed 8 days after exposure, and a mild astrocytic proliferation was present at this time. Golgi and Purkinje cells remained intact i Fig. 2). Vasculitis An exudate of leucocytes appeared promptly in all layers of the cerebral blood vessels. Veins and arteries were invoked about equally. Vessels of all sizes were affected. The vessels in the cerebral nuclear masses were generally more intensely involved than those in the cerebral cortex, while those in the white matter shared in the process, but to lesser degree. The vessels of the brain stem, cerebellum, and spinal cord were similarly affected, also in lesser intensity. The leucocytes rarely penetrated into the surrounding neural sub- stance, but usually concentrated in the ad\entitia and perivascular spaces. Hemorrhage was rare. Vessels stained specifically for collagen and elastic tissue regularly showed no notable alterations in these components. With Fig. 2. The rarified granular layer of a dog 10 days after exposure to lo.OOO r of gamma radiation contains CJolgi cells and an increased number of astrocytes, but is largely devoid of granule cells. Hemato.xyiin and eosin stain. X 55. 254 F. STEPHEN VOGEL the passage of 72 to 96 hours, the inflammatory cells lysed and the exudate lessened, but with residual perithelial edema and scant numbers of macro- phages and lymphocytes still present at the latter times. Meningitis Initially the exudate was perivascular and spotty. It persisted as such in some animals, but disseminated over the gyri and spread into the sulci in most. The cellular composition varied with duration after exposure, but also difTered somewhat in animals with identical postirradiation states. Earlier polymorphonuclear leucoctyes predominated in great numbers; later, with decreases in the cellular concentrations, lymphocytes and macrophages were relatively more abimdant. Generally, macrophages persisted and with their contents of cellular debris constituted the inflammatory residue in animals killed 96 hours after exposure. Choroid Plexitis The choroid plexuses in the lateral, 3rd, and 4th ventricles were equally involved. The intensity and cellular composition of the inflammatory exu- date generally paralleled that in the meninges and in the cerebral blood vessels. Edema of the fibrous tissue stroma usually antedated the exudation of cells into these regions. The choroidal epithelium was generally spared, but ulceration followed on frons with imusually heavy exudates. The epi- thelium was reconstituted, and the inflammation had regressed in most animals by 96 hoius after exposure. Morphologic Effects of Gamma Radiation on Granule Cells in Tissue Culture The cells in explants of cerebellum from new born mice proliferated rapidly and migrated in sheets centrifugally onto the glass. Many of these cells had rounded nuclei with one or several small nucleoli, finely particulate chromatin material, a well defined nuclear membrane, and scant or un- detectable cytoplasm. Slender, short processes radiated from the perikaryon of some. These cells with distinctive cytologic features as .seen with the phase microscope and in sections stained with hematoxylin and eosin were considered granule cells (Fig. 3). Also abundant in cultures 5 to 10 days old were cells with more elongated nuclei, coarse chromatin material, and bipolar or diffusely radiating cytoplasmic strands. These resembled fibro- blasts derived from other tissue sources and were identified as such with phase and light microscopy. Some with similar appearances were viewed as EFFECTS OF HIGH-DOSE RADIATION OX BRAIN 255 Fig. 3. Granule cells of the cerelH-Uuin of a 3-day-old mouse grown for lU days in tissue rulturc. The cells ha\c rounded nuclei with extremely scant perikaryon. Phase Microscopy. X 1200. astrocytes. Fewer cells had a lars^er nucleus, usually with a prominent nucleolus, and abimdant perikaryon that formed a sinole dominant process and occasionally lesser ones. These cells were identified as neurons derived from the cerebellar cortex from regions other than the s;ranular layer. Preliminary studies have made it clear that only cells with the cytologic characteristics attributed to the granule cells showed notable structural changes in the immediate postirradiation period. These changes closely resembled those noted pre\iously in histologic preparations of the irradiated cerebellar cortex, being characterized by a contraction of the nuclei to approximately ~/i normal size. The perikaryon that was normally scant about the granide cells in tissue culture became conspicuously wnder and the over-all dimensions of many cells increased (Fig. 4). As noted in sections stained by hematoxylin and eosin. the nuclei were contracted and the chromatin material compressed and hyperchromatic. The perikaryon was more abimdant than normal, stained intensely with eosin, and it was often foamy and \acuolated. Altered cells were most abimdant 24 hours after exposuie. Periodic examinations ot the cultures with the phase microscope 256 F. STEPHEN VOGEL Fig. 4. Granule tells, as in Fig. 3, 24 hours after exposure to 10,000 r of gamma radiation from a Co"" source. The nuclei are markedly contracted and the nuclear membranes are serrated. The cytoplasmic spaces arc enlarged. The over-all size of these cells is somewhat less than normal; in others, it was greater. Phase Microscopy. X 1200. and in stained sections made it clear that these cytologic changes were transitory. The cells in individual clumps survived and became normal in appearance. Cells in nonirradiated cultures did not undergo these cytologic changes. Morphologic Effects of Radiation on Granule Cells as Viewed with Electron Microscope The fine structure of nonirradiated granule cells of rabbits was indis- tinguishable from that of the cells in dogs, and in each species they were strikingly uniform. The cells possessed a large, spherical nucleus with uni- formly distributed, finely granular, abundant intranuclear granules. The dual nuclear membranes were uninterrupted and lay parallel except for a rare out-folding of the external one. The cytoplasm was regularly scant, but clearly visible about the entire nucleus with expansions at the axon hillock. EFFECTS OF rilGH-DOSE RADIATION ON BRAIN 257 Mitochondria were small and sparse, rarely more than 6 in a single cross section of a cell. Their cristae were delicate and inconspicuous. The endo- plasmic reticulum was also scant, but was most abimdant at the axon hillock. A Golgi apparatus frequently occupied this region. Many granule cells lay side by side with cytoplasmic membranes in apposition. Others were encased in part or totally by dendritic processes (Fig. 5). The earliest recognizable cytologic changes, abundantly evident in the tissues of rabbits examined 24 hours after exposme, were characterized by a contraction of the nucleus and clumping of the intranuclear granules, with increased serration of the nuclear membranes and broadening of the cytoplasmic space with dispersion of the cioplasmir constituents. With ■^^ ;* Fig. 5. \ normal granule cell of a rabbit is encasrd in dendrites and has scant cyto