SELECTIVE, ACUTE DESTRUCTION OF GRANULE-CELL PRECURSORS IN THE DENTATE GYRUS BY PROTON IRRADIATION LIGHT AND ELECTRON MICROSCOPIC STUDY ROBERT H. BROWNSON, PH.D.,* AND LARRY W. MCDONALD, M.D. From the Division of Medical Physics, Donner Laboratory, University of California, Berkeley, Calif. Undifferentiated, maturing cells in general are considered to be highly sensitive to ionizing radiation. From this conclusion, Altman 1 suggested that a population of undifferentiated granule cells of the mammalian nervous system, many of which are formed after birth in various regions of the brain (at least in the rat), would be selectively damaged in matur- ing animals exposed to ionizing radiation. Altman and Das 2,3 have shown, in a series of autoradiographic experiments employing tritiated thy- midine, that labeled brain cells (neurons and neuroglia) underwent mi- gration, multiplication, and differentiation when traced in serial sections through the neonatal brain. They further pointed out that small neurons, designated as microneurons, form discrete granule-cell layers in a variety of brain regions in rodents, notably in the cerebellar cortex, the olfactory bulb, and the dentate fascia of the hippocampus. The dentate fascia, to which this study is directed, derives its name from the tooth-like configuration of granular and molecular layers and the hilus which is embraced by them within the hippocampus. One of the most striking features in this area is the closely packed multilayer of large oval or short pyramidal cells.4 These neurons in the rat brain are believed to be derived partly from cells multiplying at a rapid rate after birth in the ependymal and subependymal layers of the forebrain ventricles and, to a lesser extent, from cells multiplying regionally in the internal molecular layer of the dentate gyrus. From these 2 regions the newly formed cells apparently migrate into the granule-cell layer and become fully differentiated there.1"3 The time sequence for precursor granule cells (microneurons of Altman) to differentiate into adult gran- Supported by Grant NBOI289 from the National Institutes of Health, U. S. Public Health Service, and the Atomic Energy Commission. Presented at a meeting of the American Association of Anatomists, San Francsco, Calif., Apr. 6-8, I966. Accepted for publication Dec. 22, I966. * National Institutes of Health Special Postdoctoral Fellow 2FII NBr362 NSRB. Present address: Department of Anatomy, Medical College of Virginia, Richmond, Va. 927 928 BROWNSON AND MC DONALD Vol. 50, No. 5 ule cells in the dentate fascia of the hippocampus in rat brain extends well beyond the usual maturation time for neurons. During this pro- longed period the precursor cells have been shown actively to incorporate thymidine, presumably during the synthetic stage of DNA replication. It would not be presumptuous, therefore, to expect that this particular cell would have a similarly prolonged period of increased radiosensitiv- ity. In order to understand better the role played by granule cells in the cell turnover and maturation of the rodent hippocampus, we must first establish certain fine-structure details concerning the precursor granule cells (microneurons) both in normal and pathologic reactions. For this purpose we have selectively sought to induce acute degenerative reac- tions among the precursor granule cells during differentiation and/or multiplication by means of ionizing radiation. The absorbed tissue dose and postirradiation time sequence are based on a previous study which showed an intense selective destruction of cells in this area as early as 6 hr. after exposing young rat brain to 5000 R or less of X-irradiation.5 METHODS Six i8-day-old male rats were anesthetized and given a single 5-sec. exposure to 4.5 krad of cyclotron-accelerated 55-Mev protons limited to the head. The beam was directed to one cerebral hemisphere, using the plateau of the Bragg curve. Two irradiated animals and I nonirradiated animal were sacrificed at I.5, 3, and 6.5 hr. post exposure. Each animal was anesthetized by an intraperitoneal injection of pentobarbital, 0.047 mg./gm. of body weight, given a mixture of O.I5 ml. of I :iooo heparin and i% sodium nitrite by intracardiac injection, and sacrificed by perfusion- fixation with 3% redistilled glutaraldehyde buffered with o.i M sodium cacodylate. Tissue was removed from the dentate fascia of the dorsal hippocampi of irradiated, nonirradiated (shielded), and control hemispheres with the aid of a low-power dis- secting microscope. The tissues for electron microscopy were postfixed in osmium tetroxide and embedded in Epon0. The tissues for light microscopy were postfixed in neutral buffered formalin and embedded in paraffin. It is recognized that a dose of 4.5 krad is excessive and a lower dose would probably produce the desired effect among actively differentiating, radiosensitive cells such as those in this study. However, in order to be reasonably optimistic about locating the small precursor granule cells undergoing degeneration, we attempted to involve maximum numbers and thereby increase the odds for a successful recovery in ultrathin sections of the dentate fascia area. RESULTS Light microscopy on thick-sectioned, Epon-embedded blocks and con- ventionally sectioned paraffin-embedded blocks revealed that the granule cells of the dentate gyrus were undergoing morphologic changes as early as 3 hr. post exposure to 4.5 krad proton irradiation (Fig. i and 2). In ultrathin sections examined by electron microscopy, cellular alterations were noted at I.5 hr. (Fig. 3-5). These changes were found to be pro- gressive, terminating in what appeared to be irreversible pyknosis and May I967 GRANULE CELL RADIOSENSITIVITY 929 cytoplasmic degeneration in 3-6.5 hr. post exposure (Fig. 6 and 7). The pyknotic cells were concentrated chiefly at the inferior or internal border of the granule cells at the polymorph junction. There also appeared to be a number of large, irregularly shaped, darkly stained cells present in this area by i.5 hr. post exposure. Such cells had large round nuclei and generous, polymorphic cytoplasm, rich in ribosomes and concentric ar- rays of rough-surfaced endoplasmic reticulum. Although similar cells were observed in controls, they were far fewer in number. By 3 hr. fol- lowing irradiation, one of the earliest cell changes involved the nucleus, which appeared to shrink and increase in density, beginning at the nu- clear membrane. The cytoplasm, in contrast to the nucleus, became dis- organized and increasingly electron-lucid. Densely stained tissue frag- ments and enlarged clear spaces were observed among the degenerating cells (Fig. 6). At 6.5 hr. post exposure the intensity of cellular necrosis appeared to have reached a plateau. Large round osmiophilic globules, resembling pyknotic nuclei in variable dense masses of protoplasm, were abundant. Granule cells bordering the necrotic zone were frequently im- plicated in partial cellular changes notably involving liquefaction of the cytoplasm and decrease in nuclear density (Fig. 7). Granule cells distal to the internal necrotic layer of cells bordering the hilus were conspicu- ously normal in appearance. In addition to cell necrosis, a fair number of degenerating cells or fragments appeared to be undergoing phagocytosis as early as I.5 hr. post exposure. In each instance the cell or its remnant was seen to be engulfed by a watery-like cytoplasm. The nucleus of the engulfing cell was generally oval and moderate in chromatin; and when fibrils were present in the cytoplasm, as they frequently were, a positive identification of astrocyte was made (Fig. 4 and 5). The granule cells in the dentate gyrus of the nonirradiated, shielded hemisphere and con- trol brains appeared entirely normal. The absence of necrotic cells in the dentate gyrus of the shielded half of the brain indicated that there was no effective cross scatter of secondary ionizing irradiation or transfer of secondary effects from the irradiated hemisphere. DISCUSSION The hippocampal area has long been known for its low tolerance to a variety of noxious insults such as asphyxia, encephalitis, and whole- head ionizing irradiation.>8 More recently Schoenbrun, Campeau, and Adey9 have demonstrated changes in motor behavior and brain electrical activity in cats 30-60 min. following either unilateral or bilateral focal- beam (8 mm.) exposure of the dorsal hippocampus to as little as 400- 500 R of 250 kvp X-rays. Hicks and Montgomery10 first called attention to the acute localized destruction of the granule cells in the dentate 930 BROWNSON AND MC DONALD Vol. 50, No. 5 gyrus of mice and rats after I50, 250, and 400 kvp X-rays, 2.5 Mev electrons, and 3 Mev X-rays acutely following high dose and high-dose- rate studies. This phenomenon of acute granule cell destruction was again observed by Brownson, Suter, and Diller 5in young rat brain ex- posed to 500-IO,OOO R X-rays. However, in contrast to the findings of Hicks and Montgomery,10 it was noted that the granule-cell destruction was limited entirely to a narrow margin of cells bordering the hilus of the dentate fascia. Although the degenerating cells were incorrectly identi- fied at that time as neuroglial elements, we now believe that the differing accounts in degree of destruction in these 2 reports of granule-cell radio- necrosis6"10 were probably related to differences in dose, dose rates, and age which were of a different magnitude in the earlier study.'0 A survey of the literature relating to the morphology of the hippo- campus and, in particular, the dentate fascia revealed surprisingly few papers on normal or abnormal ultrastructure, particularly in comparison to the large number of electrophysiological or behavioral studies involv- ing this area of the brain. Studies by Blackstad"1 and Blackstad and Dahl 12 were perhaps the first comprehensive coverage of the quantitative analysis of normal granule-cell ultrastructure. In these reports the authors noted 2 features pertinent to this investigation-namely, that there were virtually no oligodendroglia present among granule cells and that a very high percentage of the granule-cell soma was invested in astroglial cytoplasm.