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Postnatal development of the rat CNS following in utero exposure to a low dose of ionizing radiation
Exp Toxic Pathol 1993 ; 45: 223 - 231 Gustav Fischer Verlag Jena
The University of Manitoba, Department of Anatomy'), Winnipeg, Canada Hubei Medical University, Department of Anatomy 2), Wuhan, Peoples Republic of China Health Sciences Centre, Manitoba Cancer Treatment and Research Foundation 3), Winnipeg, Canada
Postnatal development of the rat eNS following in utero exposure to a low dose of ionizing radiation J. E . BRUNII) , T . V. N. PERSAUD 1) , W. HUANG 2 ) , and G . FROESE 3 ) With 9 figures Received: October 17, 1992; Accepted: November 17, 1992
Address for correspondence: Dr. J. E . BRUNI, Department of Anatomy, Faculties of Medicine and Dentistry, The University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada, R3E OW3.
Key words: Gamma-radiation, in utero; Postnatal development, CNS; Ionizing radiation; In utero ionizing radiation.
Summary In utero exposure to iomzlllg radiation is of importance because of its potential health risks . The developing nervous system is particularly vulnerable and the consequences of exposure to low levels of radiation (:::; 1 Gy) are not well established. The developmental effects of maternal exposure to 50 cGy gamma-radiation on gestational days (GD) 9.5, 15, and 18 were investigated in Sprague Dawley rats . Rats exposed on GD-9 .5 along with approriate controls were killed at 4 h, 48 h, and 10 days post-irradiation while those irradiated on GD-15 and GD-18 were killed postnatally (PN) on days 7 and 26. All were examined for developmental anomalies and representative samples of brains were processed for microscopic study. No significant developmental differences were observed between irradiated and control embryos killed 48 h after irradiation on GD 9.5. However, in irradiated fetuses a larger number of developmental anomalies were observed at term. Defects of the eye and of spinal curvature were the most common malformations encountered. Mitoses were reduced within the neuroepithelium of embryos irradiated on GD-9. 5 and evidence of pyknosis and necrosis was seen 4 h after irradiation. The capacity of surviving primitive neural cells for repair, however, was such that by 48 h after exposure the irradiated nervous system no longer differed from controls. Rats irradiated on GD-15 and GD-18 and examined on PN-26 exhibited clusters of small, dark, pyknotic neurons within the hippocampal and dentate gyri, often bilaterally. They were found with an incidence of 57 % and 33 % in rats irradiated on G-15 and G-18, respectively. Significant cerebellar lesions were also seen in 14 % and 22 % of rats irradiated on GD- 15 and GD- 18 respectively, and killed on PN-26 . In these rats the granular cell layer of the cortex harbored numerous lucent islands containing reduced numbers of granule cells . The vulnerability of the hippocampal neurons can be explained by their morphogenesis being coincident with the period of irradiation. However, since cerebellar microneurons are largely acquired postnatally, radiation in this case likely
affected the precursors, proliferative and/or migratory cells of the external granular layer. Alternatively, it may have affected the normal differentiation of Purkinje cells which in tum removed or altered some guiding influence they provide on the normal development of internal granule cells.
Introduction There is increasing interest in the potential health effects of in utero exposure to ionizing radiation for the purpose of estimating risk and determining the extent of radiation protection. Studies in laboratory animals have shown that prenatal exposure to ionizing radiation causes resorptions, intrauterine growth retardation and a wide spectrum of developmental defects (23,26,41,44). Central nervous system lesions are a most consistent pathological finding in the offspring following maternal exposure to radiation during gestation (11, 23, 30, 42, 44, 49) . The nervous system is susceptible to radiation damage, although differentially, throughout the embryonic and fetal periods of development. Both the severity of damage and vulnerability to radiation are a function of dose, dose-rate, and time of exposure. Prenatal radiation exposure to large doses (2:: 1 Gy) produces a variety of well documented structural alterations in the CNS of laboratory animals (12 , 15 , 19, 24, 28, 36). These include faulty neuralation , neuronal loss , failure of dendritic maturation and spine formation, aberrant synpatic circuits, to list a few, which likely form the basis for observed behavioural, mental and physical impairments that have also been reported (15, 24, 32,35,36,38,39,45), including in the human (10, 40, 42) . The consequences of prenatal exposure to low level (:::; 1 Gy) radiation, however, are less familiar. For this reason, we Exp Toxic Pathol 45 (1993) 4
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have investigated the effects of in utero exposure to a single dose of 50 cGy gamma-radiation on the development of the rat nervous system at different post-irradiation intervals. This paper reports our findings in rats 4 h , 48 h, and 10 days folowing exposure on GO-9.5 and in 7 and 26 day old rats following irradiation during late gestation , on GO-IS and GO-18.
Material and Methods Sexually mature female Sprague-Dawley rats, 8-10 wks of age, were used in this study. They were maintained under conditions of controlled temperature (20 ± 2 DC) and illumination (12 h light/dark cycles, 2000-8000 dark) and allowed access to food and water ad libitum . All animals were cared for in accordance with the Candian Council on Animal Care guidelines. Timed pregnancies were obtained by housing one male with three females overnight. The following day females were checked for vaginal plugs and the presence of spermatozoa. If present, the animals were designated as day I of gestation (GD-l) and randomly allocated to either a treatment or a control group . Pregnant rats (1- 3/cage) were then placed in specially constructed clear plexiglas cages and exposed to 50 cGy of 60Co radiation from a Theratron F, cobalt radiotherapy unit. The animals were exposed to two parallel opposed radiation fields positioned 75 cm above and below the treatment table so that the dose was uniformly distributed throughout the irradiated volume.
Irradiation time varied from 14-17 sec and the total time of confinement amounted to 20- 30 min. Control rats were identically treated except that they were not irradiated. Groups of pregnant rats (N = 12-15/group) were irradiated on GD-9 .5 and killed prenatally at 4 h, 48 h, and to days postirradiation. Additional groups (N = 11-13/group) along with appropriate controls (N = 10/ group) were irradiated on GO-IS or 18 and killed on post-natal days (PN) 7 or 26. With the latter groups, all litters were culled to 8 neonates at birth. At the time of sacrifice, all fetuses were recovered and fixed by immersion in 2.0% paraformaldehyde - 2.5 % glutaraldehyde in 0.12 M phosphate buffer with 0.02 mM CaCI added. Seven and 26 day old rats were anesthetized and fixed by intracardiac perfusion with the same fixative. Brains were removed, examined for gross anomalies and then stored in fresh fixative at 4°C pending processing for microscopic examination. Only a representative number of brains (2= 5 per group) randomly selected from the large number recovered, were processed in the conventional manner for LM or TEM examination. Although the CNS was examined in its rostrocaudal extent, several areas selected a priori were the focus of particular attention . The areas were chosen because of their unique cytoarchitecture andlor known sensitivity to radiation damage . The areas included the rhinencephalon, fronto-parietal cortex, caudate-putamen, hippocampal formation, amygaloid nuclear complex, and cerebellum. The cellular and fiber architecture of these regions was assessed primarily for heterotopias and evidence of dysplastic changes .
Fig. 1. Embryo removed from a control rat showing the extent of development of the nervous system on GO-9.5. BP, cranial brain plate; arrow, caudal neural folds. x 120. Fig. 2. Transverse section through the cephalic neural folds (open arrows) of an embryo that received 50 cGy on GO-9.5 and was killed 4 h post-irradiation. Development of the nervous system is rudimentary. A single pseudo stratified layer of mitotic and intermitotic neuroepithelial cells form the walls. Solid arrow , caudal neural folds. Methylene blue - Azure II, X 240. 224
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Fig. 3. Higher magnification of a segment of the cephalic neural folds shown in fig. 2. Compared to controls mitotic figures are reduced within the columnar neuroepithelium and clumped remnants of basophilic material are present (arrows). Methylene blue Azure II, x 2400.
Results The embryo at GD-9.5 is in a very rudimentary stage of development (fig. 1). The nervous system consists of a single pseudostratified layer of mitotic and interrnitotic columnar neuroepithelial cells (fig. 2). In embryos irradiated at this gestational time, mitoses were reduced within the neuroepithelium; pyknosiS and some necrosis of cells was apparent 4 h after irradiation (fig. 3). No significant developmental differences were observed between irradiated and control embryos killed 48 h after irradiation on GD 9.5. Controls, however, did show a greater range in the extent of development of the forebrain, olfactory system, midbrain , hindbrain , and caudal neural tube . In all cases they showed evidence of slower development of these features than their irradiated counterparts. At this gestational period the nervous system has reached the 5 brain vesicle stage of development. An inner cell layer of actively mitotic cells , an intermediate primitive differentiating zone and an acellular marginal zone constitute its wall s. The nervous system of embryos irradiated on day 9.5 and examined 48 h later could not be distinguished from controls . Neuropathological changes such as deformity of structure , ectopic growth, retarded development , arrest of proliferation and/or migration of cells were not seen . The nervous system on GD-I9.5 has attained a more advanced state of development than the primitive 5 vesicle
stage of GD-II. 5 of embryogenesis. The olfactory components of each cerebral hemisphere are discernible as are many nuclei of the telencephalon and diencephalon. In the cerebrum and cerebellum migrating neuroblasts have invaded the peripheral marginal zone to establish the rudiments of an outer gray cortex. The development of the cerebrum is more advanced than the cerebellum at this stage; the latter having evolved only a external granular cell layer. Only a primitive state of development is also the case with the hippocampal formation . In fetu ses irradiated on GD-9.5 and killed 10 days later, a higher incidence of developmental abnormalities was observed compared to controls. Defects of the eye and of spinal curvature were the most common malformations encountered. With this exception, however, no gross defects or significant differences in the development of their nervous systems were observed. Similarly , no significant differences in physical appearance or in behavior were observed between rats irradiated on GD15 or 18 and controls killed on either PN-7 or 26. The nervous system of fetuses irradiated on GD-I5 or 18 and killed on PN7 was also not dissimilar. Differences in numbers of mitoses, pyknotic celis, or in the development of the subependymal zone , or the glomerular, mitral , internal and external granular layers of the olfactory bulb between irradiated and control animals were not seen. Irradiated animals failed to show any discernible difExp Toxic Pathol 45 (1993) 4
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Fig. 4. Segment of the CAl region of hippocampus from a rat irradiated on GD-18 and killed on PN-26 contrasting normal (outside arrows) with abnormal regions (within arrows) of cells within the pyramidal layer. x 512. Fig. 5. Cerebellar folia in a 26 day-old rat irradiated on GD-15. Lesions in the form of characteristic lucent islands (arrows) may be seen scattered throughout an otherwise normal appearing granular layer of cortex. pc, Purkinje cells; m, molecular layer; c, medullary center; p, pia. x 87.
ferences in the prominent ependymal or subependymal regions of the telencephalon, compared to controls at one week after birth. No residual effects of irradiation were detected in the fronto-parietal neocortex or in the proliferating primitive archicorticals cells of the hippocampal formation. On PN-7, the cortex of the developing cerebellum consists of a prominent subpial external ganular layer whose outer zone is proliferative and inner non-mitotic zone is migratory. Beneath this is the developing acellular molecular layer subjacent to which is a layer of maturing Purkinje cells. The developing inner granular cell layer, beneath the Purkinje cells contains differentiating granule cells as well as dividing glial cells and Purkinje cell associated satellites. At this stage of development which contrasts markedly with the cerebellum on PN-26 (fig. 5) no radiation induced changes were detected. In contrast, animals irradiated on either GD-15 or 18, and killed on PN-26, exhibited anomalous development of the hippocampal formation. In 33 % of rats irradiated on GD-18 and 57 % of those irradiated on GD-15 clusters of pyknotic neurons were observed scattered amongst the normal pyramidal cells of the hippocampus and/or granule cells ofthe dentate gyri (fig. 4). This occurrence was often bilateral and without a discernible rostrocaudal predilection. In some cases, the same regions also showed a reduction in cell numbers. 226
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The most pronounced radiation-induced changes were observed in the cerebellum of irradiated rats killed on PN-26. The cerebellum in the 26 days-old rat has attained the 3 layer adult form; consisting of an outer molecular layer and a inner granular layer between which is the single celled Purkinje layer. The granule cells are typically small, densely packed and well differentiated (figs. 5, 8). The cerebellum in 22 % of those irradiated on GD-18 and only 14 % of those irradiated on GD-15 was found to exhibit characteristic radiationinduced lesions. The lesions were similar in both groups of irradiated rats but tended to be more pronounced in those irradiated on GD-15. Although the charcteristic lamination of the cerebellar cortex was preserved in irradiated animals, multiple cell poor areas were observed within the otherwise normal granular cell layer of cortex (figs. 5, 6). These lucent islands were well circumscribed, of variable size, and distributed ubiquitously throughout the granular cell layer of the cerebellum in irradiated animals. They consisted of reduced numbers of cells (figs. 6,9) and contrasted markedly with the surrounding population of normal granule cells (figs. 5, 6). Only occasionally, however, did the granule cells or surrounding Purkinje cells themselves appear abnormal (fig. 7) in the irradiated animals.
Fig. 6. Granular cell layer from the cerebellum of a 26 day old rat irradiated on GD-I5 showing one circumscribed area (arrow) immediately beneath the Purkinje cells (pc) of reduced granule cell number. m, molecular layer. x 545. Fig. 7. Pyknotic Purkinje (open arrows) and granule cells (solid arrows) seen occasionally in irradiated rats. Taken from a 26 day old rat irradiated on GD-I5. x l 090.
Discussion In this study, exposure to a single dose of 50 cGy produced significant observable neuropathologic lesions that were a function of gestational age at the time of irradiation. Embryos irradiated on GD-9.5 showed reduced mitoses, as well as pyknosis and necrosis of primitive neural cells, within 4h of irradiation. This is consistent with observations of HICKS (21) and others (27 , 29 , 33) who found that primative differentiating neural cells undergo necrosis within 4 h of maternal exposure to :5 50 ron GD-9-1O. Our study revealed that the capacity of surviving cells for repair was such that within 48 h of exposure, the nervous system of irradiated embryos no longer differed morphologically from controls . The study did not reveal, however, whether embryos exposed to radiation at this early stage of morphogenesis would exhibit residual neurophysiological or behavioral deficits in adulthood. Although radiosensitive , the undifferentiated nervous system at this early stage of development , has great recuperative capacity and therefore would not be expected to exhibit morphological anomalies or major CNS defects on exposure to radiation (31,33). Consistent with this view, rats irradiated on GD-9 with 0.1-0.6 Gy X-rays failed to exhibit a significant incidence of malformations , intrauterine death or alterations in physiological development postnatally (31 , 32) . Even offsprings of rats that received a relatively large dose (250 r) of X-rays during the period from GD-0-8 showed no histolog-
ical abnormalities in the CNS (14). There are other studies, however, although few in number, that have reported severe damage to the nervous system following exposure to low doses of radiation at early stages of development (49). WILSON et aI., (46, 47,48) exposed exteriorized rat embryos to 25 and 50 r X-rays on GD-8-11. After exposure to 25 r on GD-9, microphthalmia was observed in 6 % of the embryos and ocular maldevelopment and anophthalmia in 72 % after 50 r. The brain and spinal cord were also malformed in 9 % and 3 % of embryos, respectively after 50 r. On GD-lO day, only the eye was affected in 11 % of embryos and only after 50r. Unlike the embryonic period, our study revealed that exposure to radiation during the fetal period (GD-15 or 18) resulted in abnormalities that persisted and were evident in adulthood . Malformations of the nervous system and behavioral deficits have been regularly noted in offspring of rats exposed to radiation ~ 1 Gy on GD-13 to PN-3 (12, 14, 15,20,24, 36). These have included reduced size of cerebral hemispheres, decreased brain weight, defective cortical cytoarchitecture, heterotopias , spinal cord lesions, commissural agensis and altered locomotion. There are also reports of specific cortical vulnerability in rats; neuronal cell loss, malformed cortical patterns , abnormal organization, orientation and synaptogenesis of neurons following exposure to radiation ~ 1 Gy on GD-13-21 (9, 19, 20, 43) . Although exposure to a minimum of 1 Gy appears to be needed, morphologically descernible alterations in the histogenesis of Exp Toxic Pathol 45 (1993) 4
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Fig. 8. Nonnal granule cells (gr) closely clustered beneath the Purkinje cell (pc) layer in a 26 day old rat irradiated on GD-15. Granule cells are characterized by round heterochromatic nuclei and scanty cytoplasm. Glomeruli (arrows) consisting of arrays of pre- and post-synaptic elements comprise the intervening neurophil. Compare with the abnonnal neuropil shown in figs. 6 & 9. X 4500.
the cerebral cortex have been occasionally reported with exposure to::5 0.75 Gy on GD-15, 16, 18,22 and PN-l (20, 22, 37, 38, 39). We have been unable to confirm the latter observations in this study. Unlike the changes in thickness of the cortical laminae in the hippocampus in monkeys that received 2 Gy on GD-75 (13), or the hippocampal ectopias in SID rats exposed to 2 Gy on GD-16 (19) only circumscribed areas of focal hippocampal necrosis were observed in our study. Fifty seven percent and 33 % of rats irradiated on either GD-15 or GD-18, respectively, exhibited scattered clusters of pyknotic neurons and sometimes a reduction in cell numbers within the hippocampal formation. This vulnerabilitiy of hippocampal neurons, in contrast to neurons of other cortical areas is likely due to their morphogenesis being coincident with the period of irradiation. Cells in all mitotic phases as well as post-mitotic undifferentiated migratory cells are known to be radiosensitive (3, 6, 23), Hippocampal pyramidal cells are formed entirely prenatally in the mouse on GD-I 0-18 (6) and on GD16-19 in the rat (7 , 25). The granule cell population of the dentate gyrus, however, is largely (72 %) produced postnatally from PN-I-16 (with a peak on PN-2) and 13 % from PN-16 on into the adult period (7, 8, 35). Only 15 % of granule cells are produced prenatally with initiation thought to occur on GD-15-16 (8, 25) . The most significant radiation induced pathological changes were observed in the cerebellum of 26 days old rats. 228
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The lesions were the same regardless of whether irradiation took place on GD-15 or 18 although the former were more profound. Interestingly, there have been numerous studies of radiation effects on the developing cerebellum, using larger doses than in the present study, in which the anomalous development of the internal granular layer as seen in our study either did not occur or was not reported. Das (16, 17), for example, irradiated rat fetuses at the same stage of gestational development as in our study but used 170 r X-rays. He reported that the mature cerebellum in fetuses irradiated on G-15 was 50 % of normal size, exhibited reduced cortical areas and thickness, and abnormal foliation in the anterior cerebellum (16). There were also 32 - 50 % fewer Purkinje cells, 60 % fewer granule cells and 27 % fewer granule cells per Purkinje cell. When the same dose of radiation was administered on GD-18 similar but less severe consequences were observed; cerebellar size was reduced 7 -19 %. Purkinje and granule cell numbers were 93-98 % and 85 % of normal respectively, and the granule to Purkinje cell ratio was only 12 % of normal (17). These developmental anomalies were attributed to direct radiation effects on migratory neuroblasts of Purkinje cells which caused their death and reduced or altered the inductive influences of surviving Purkinje cells on the mitotic activity of the external granular layer. In contrast, there are also studies, although few in number, in which cell free foci as reported in our study have been
Fig. 9. Granular cell layer of the cerebellum in a 26 day old rat irradiated on GD-15 showing a lesioned area consisting of a loosely organized array of granule cells. Although the granule cells present appear normal, the number is reduced and the surrounding neuropil greatly thinned. X 4500.
observed within the mature cerebellum of rats exposed to large doses of X-ray radiation (2: 200 r) , both prenatally (14) and postnatally (2, 4). No cerebellar anomalies were found in rats that received 250 r X-rays on GD-15; however, at all subsequent intervals anomalies were present in the rostral portions of the cerebellum (14). Disturbances were mild with exposure on GD-1618 and were manifest by decreased size and abnormal orientation of folia and sulci but normal cortical architecture. Cerebellar defects were most severe with exposure from GD19 to PN-l. In addition to an exaggeration of changes already mentioned, ectopias of granule or Purkinje cells in the molecular layer or folial white matter occurred. Various defects in the internal granular layer such as reduced numbers of cells, narrowing, absence of segments of this lamina and the presence of cell free foci were observed. No explanation was offered for the appearance of cell free foci within the internal granular layer of the cerebellum. In a similar study, rats that received 3 - 5 successive daily doses of 200 r of X-rays beginning on PN-\ also exhibited anterior cerebellar abnormalities at maturity (2,4). Notable among them were abnormal foliation, reduction in granule cell numbers, thinning of the molecular layer, mis-shapen Purkinje cells and significantly the presence of radial streaks and circular islands of cell-sparse areas within the internal granular layer (2). The latter observation is similar to our own and that of COWEN and GELLER (14) but was obtained with
postnatal exposure to a considerably larger dose of radiation 3-10 x 200 r. ALTMAN and ANDERSON (2) referred to these cell sparse areas as extensions of the molecular layer in which Purkinje cell dendrites were located. They attributed this anomaly to disoriented growth of Purkinje cell dendrites that became embedded in the internal granular layer taking with them cell free islands of molecular layer; a conclusion not supported by our data. Although Purkinje cells themselves were not destroyed or reduced in number by either single or multiple doses of 200 r, their normal cytological differentiation was affected. This was attributed to direct damage of the external granular layer (the precursors of interneurons) and removal of some guiding influence they provide on the normal development of the Purkinje cells . There is evidence that Purkinje cells are among the earliest formed prenatally (day 13-16) but mature largely postnatally (5, 18) whereas precursors of cerebellar microneurons (granule, basket and stellate cells) are acquired and differentiate during the early (1- 3rd wk) postnatal period (1, 5, 18, 33). Irradiation of the cerebellum at birth and postnatally with successive daily doses of 0.5-2 Gy of x-rays produced dose dependent reduction in granule cells but Purkinje cells were unaffected (2, 3). In view of these observations, we conclude from our data that the single dose of low level radiation on GD-15 or 18 may have directly affected the proliferative external granular layer of the developing cerebellum, precursors thereof and/or Exp Toxic Pathol 45 (1993) 4
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postmitotic migratory cells of this layer. Alternatively, because prenatally forming neurons may have a organizing or regulating influence on the small neurons of postnatal origin, (2, 18) the normal cytological differentiation of the Purkinje cells may have been directly affected by the prenatal radiation. This in tum removed or altered some guiding influence which Purkinje cells provide on the normal development of internal granule cells. Acknowledgements: The authors acknowledge with gratitude the excellent technical assistance provided by Mr. G SEYOUM, PM PERUMAL, R SIMPSON and Ms. L SNELL. We also thank the Unsolicited Proposals Fund, Department of Supply and Services; Atomic Energy Control Board and Health and Welfare, Canada for funding this investigation.
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The University of Manitoba, Department of Anatomy'), Winnipeg, Canada Hubei Medical University, Department of Anatomy 2), Wuhan, Peoples Republic of China Health Sciences Centre, Manitoba Cancer Treatment and Research Foundation 3), Winnipeg, Canada
Postnatal development of the rat eNS following in utero exposure to a low dose of ionizing radiation J. E . BRUNII) , T . V. N. PERSAUD 1) , W. HUANG 2 ) , and G . FROESE 3 ) With 9 figures Received: October 17, 1992; Accepted: November 17, 1992
Address for correspondence: Dr. J. E . BRUNI, Department of Anatomy, Faculties of Medicine and Dentistry, The University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada, R3E OW3.
Key words: Gamma-radiation, in utero; Postnatal development, CNS; Ionizing radiation; In utero ionizing radiation.
Summary In utero exposure to iomzlllg radiation is of importance because of its potential health risks . The developing nervous system is particularly vulnerable and the consequences of exposure to low levels of radiation (:::; 1 Gy) are not well established. The developmental effects of maternal exposure to 50 cGy gamma-radiation on gestational days (GD) 9.5, 15, and 18 were investigated in Sprague Dawley rats . Rats exposed on GD-9 .5 along with approriate controls were killed at 4 h, 48 h, and 10 days post-irradiation while those irradiated on GD-15 and GD-18 were killed postnatally (PN) on days 7 and 26. All were examined for developmental anomalies and representative samples of brains were processed for microscopic study. No significant developmental differences were observed between irradiated and control embryos killed 48 h after irradiation on GD 9.5. However, in irradiated fetuses a larger number of developmental anomalies were observed at term. Defects of the eye and of spinal curvature were the most common malformations encountered. Mitoses were reduced within the neuroepithelium of embryos irradiated on GD-9. 5 and evidence of pyknosis and necrosis was seen 4 h after irradiation. The capacity of surviving primitive neural cells for repair, however, was such that by 48 h after exposure the irradiated nervous system no longer differed from controls. Rats irradiated on GD-15 and GD-18 and examined on PN-26 exhibited clusters of small, dark, pyknotic neurons within the hippocampal and dentate gyri, often bilaterally. They were found with an incidence of 57 % and 33 % in rats irradiated on G-15 and G-18, respectively. Significant cerebellar lesions were also seen in 14 % and 22 % of rats irradiated on GD- 15 and GD- 18 respectively, and killed on PN-26 . In these rats the granular cell layer of the cortex harbored numerous lucent islands containing reduced numbers of granule cells . The vulnerability of the hippocampal neurons can be explained by their morphogenesis being coincident with the period of irradiation. However, since cerebellar microneurons are largely acquired postnatally, radiation in this case likely
affected the precursors, proliferative and/or migratory cells of the external granular layer. Alternatively, it may have affected the normal differentiation of Purkinje cells which in tum removed or altered some guiding influence they provide on the normal development of internal granule cells.
Introduction There is increasing interest in the potential health effects of in utero exposure to ionizing radiation for the purpose of estimating risk and determining the extent of radiation protection. Studies in laboratory animals have shown that prenatal exposure to ionizing radiation causes resorptions, intrauterine growth retardation and a wide spectrum of developmental defects (23,26,41,44). Central nervous system lesions are a most consistent pathological finding in the offspring following maternal exposure to radiation during gestation (11, 23, 30, 42, 44, 49) . The nervous system is susceptible to radiation damage, although differentially, throughout the embryonic and fetal periods of development. Both the severity of damage and vulnerability to radiation are a function of dose, dose-rate, and time of exposure. Prenatal radiation exposure to large doses (2:: 1 Gy) produces a variety of well documented structural alterations in the CNS of laboratory animals (12 , 15 , 19, 24, 28, 36). These include faulty neuralation , neuronal loss , failure of dendritic maturation and spine formation, aberrant synpatic circuits, to list a few, which likely form the basis for observed behavioural, mental and physical impairments that have also been reported (15, 24, 32,35,36,38,39,45), including in the human (10, 40, 42) . The consequences of prenatal exposure to low level (:::; 1 Gy) radiation, however, are less familiar. For this reason, we Exp Toxic Pathol 45 (1993) 4
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have investigated the effects of in utero exposure to a single dose of 50 cGy gamma-radiation on the development of the rat nervous system at different post-irradiation intervals. This paper reports our findings in rats 4 h , 48 h, and 10 days folowing exposure on GO-9.5 and in 7 and 26 day old rats following irradiation during late gestation , on GO-IS and GO-18.
Material and Methods Sexually mature female Sprague-Dawley rats, 8-10 wks of age, were used in this study. They were maintained under conditions of controlled temperature (20 ± 2 DC) and illumination (12 h light/dark cycles, 2000-8000 dark) and allowed access to food and water ad libitum . All animals were cared for in accordance with the Candian Council on Animal Care guidelines. Timed pregnancies were obtained by housing one male with three females overnight. The following day females were checked for vaginal plugs and the presence of spermatozoa. If present, the animals were designated as day I of gestation (GD-l) and randomly allocated to either a treatment or a control group . Pregnant rats (1- 3/cage) were then placed in specially constructed clear plexiglas cages and exposed to 50 cGy of 60Co radiation from a Theratron F, cobalt radiotherapy unit. The animals were exposed to two parallel opposed radiation fields positioned 75 cm above and below the treatment table so that the dose was uniformly distributed throughout the irradiated volume.
Irradiation time varied from 14-17 sec and the total time of confinement amounted to 20- 30 min. Control rats were identically treated except that they were not irradiated. Groups of pregnant rats (N = 12-15/group) were irradiated on GD-9 .5 and killed prenatally at 4 h, 48 h, and to days postirradiation. Additional groups (N = 11-13/group) along with appropriate controls (N = 10/ group) were irradiated on GO-IS or 18 and killed on post-natal days (PN) 7 or 26. With the latter groups, all litters were culled to 8 neonates at birth. At the time of sacrifice, all fetuses were recovered and fixed by immersion in 2.0% paraformaldehyde - 2.5 % glutaraldehyde in 0.12 M phosphate buffer with 0.02 mM CaCI added. Seven and 26 day old rats were anesthetized and fixed by intracardiac perfusion with the same fixative. Brains were removed, examined for gross anomalies and then stored in fresh fixative at 4°C pending processing for microscopic examination. Only a representative number of brains (2= 5 per group) randomly selected from the large number recovered, were processed in the conventional manner for LM or TEM examination. Although the CNS was examined in its rostrocaudal extent, several areas selected a priori were the focus of particular attention . The areas were chosen because of their unique cytoarchitecture andlor known sensitivity to radiation damage . The areas included the rhinencephalon, fronto-parietal cortex, caudate-putamen, hippocampal formation, amygaloid nuclear complex, and cerebellum. The cellular and fiber architecture of these regions was assessed primarily for heterotopias and evidence of dysplastic changes .
Fig. 1. Embryo removed from a control rat showing the extent of development of the nervous system on GO-9.5. BP, cranial brain plate; arrow, caudal neural folds. x 120. Fig. 2. Transverse section through the cephalic neural folds (open arrows) of an embryo that received 50 cGy on GO-9.5 and was killed 4 h post-irradiation. Development of the nervous system is rudimentary. A single pseudo stratified layer of mitotic and intermitotic neuroepithelial cells form the walls. Solid arrow , caudal neural folds. Methylene blue - Azure II, X 240. 224
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Fig. 3. Higher magnification of a segment of the cephalic neural folds shown in fig. 2. Compared to controls mitotic figures are reduced within the columnar neuroepithelium and clumped remnants of basophilic material are present (arrows). Methylene blue Azure II, x 2400.
Results The embryo at GD-9.5 is in a very rudimentary stage of development (fig. 1). The nervous system consists of a single pseudostratified layer of mitotic and interrnitotic columnar neuroepithelial cells (fig. 2). In embryos irradiated at this gestational time, mitoses were reduced within the neuroepithelium; pyknosiS and some necrosis of cells was apparent 4 h after irradiation (fig. 3). No significant developmental differences were observed between irradiated and control embryos killed 48 h after irradiation on GD 9.5. Controls, however, did show a greater range in the extent of development of the forebrain, olfactory system, midbrain , hindbrain , and caudal neural tube . In all cases they showed evidence of slower development of these features than their irradiated counterparts. At this gestational period the nervous system has reached the 5 brain vesicle stage of development. An inner cell layer of actively mitotic cells , an intermediate primitive differentiating zone and an acellular marginal zone constitute its wall s. The nervous system of embryos irradiated on day 9.5 and examined 48 h later could not be distinguished from controls . Neuropathological changes such as deformity of structure , ectopic growth, retarded development , arrest of proliferation and/or migration of cells were not seen . The nervous system on GD-I9.5 has attained a more advanced state of development than the primitive 5 vesicle
stage of GD-II. 5 of embryogenesis. The olfactory components of each cerebral hemisphere are discernible as are many nuclei of the telencephalon and diencephalon. In the cerebrum and cerebellum migrating neuroblasts have invaded the peripheral marginal zone to establish the rudiments of an outer gray cortex. The development of the cerebrum is more advanced than the cerebellum at this stage; the latter having evolved only a external granular cell layer. Only a primitive state of development is also the case with the hippocampal formation . In fetu ses irradiated on GD-9.5 and killed 10 days later, a higher incidence of developmental abnormalities was observed compared to controls. Defects of the eye and of spinal curvature were the most common malformations encountered. With this exception, however, no gross defects or significant differences in the development of their nervous systems were observed. Similarly , no significant differences in physical appearance or in behavior were observed between rats irradiated on GD15 or 18 and controls killed on either PN-7 or 26. The nervous system of fetuses irradiated on GD-I5 or 18 and killed on PN7 was also not dissimilar. Differences in numbers of mitoses, pyknotic celis, or in the development of the subependymal zone , or the glomerular, mitral , internal and external granular layers of the olfactory bulb between irradiated and control animals were not seen. Irradiated animals failed to show any discernible difExp Toxic Pathol 45 (1993) 4
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Fig. 4. Segment of the CAl region of hippocampus from a rat irradiated on GD-18 and killed on PN-26 contrasting normal (outside arrows) with abnormal regions (within arrows) of cells within the pyramidal layer. x 512. Fig. 5. Cerebellar folia in a 26 day-old rat irradiated on GD-15. Lesions in the form of characteristic lucent islands (arrows) may be seen scattered throughout an otherwise normal appearing granular layer of cortex. pc, Purkinje cells; m, molecular layer; c, medullary center; p, pia. x 87.
ferences in the prominent ependymal or subependymal regions of the telencephalon, compared to controls at one week after birth. No residual effects of irradiation were detected in the fronto-parietal neocortex or in the proliferating primitive archicorticals cells of the hippocampal formation. On PN-7, the cortex of the developing cerebellum consists of a prominent subpial external ganular layer whose outer zone is proliferative and inner non-mitotic zone is migratory. Beneath this is the developing acellular molecular layer subjacent to which is a layer of maturing Purkinje cells. The developing inner granular cell layer, beneath the Purkinje cells contains differentiating granule cells as well as dividing glial cells and Purkinje cell associated satellites. At this stage of development which contrasts markedly with the cerebellum on PN-26 (fig. 5) no radiation induced changes were detected. In contrast, animals irradiated on either GD-15 or 18, and killed on PN-26, exhibited anomalous development of the hippocampal formation. In 33 % of rats irradiated on GD-18 and 57 % of those irradiated on GD-15 clusters of pyknotic neurons were observed scattered amongst the normal pyramidal cells of the hippocampus and/or granule cells ofthe dentate gyri (fig. 4). This occurrence was often bilateral and without a discernible rostrocaudal predilection. In some cases, the same regions also showed a reduction in cell numbers. 226
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The most pronounced radiation-induced changes were observed in the cerebellum of irradiated rats killed on PN-26. The cerebellum in the 26 days-old rat has attained the 3 layer adult form; consisting of an outer molecular layer and a inner granular layer between which is the single celled Purkinje layer. The granule cells are typically small, densely packed and well differentiated (figs. 5, 8). The cerebellum in 22 % of those irradiated on GD-18 and only 14 % of those irradiated on GD-15 was found to exhibit characteristic radiationinduced lesions. The lesions were similar in both groups of irradiated rats but tended to be more pronounced in those irradiated on GD-15. Although the charcteristic lamination of the cerebellar cortex was preserved in irradiated animals, multiple cell poor areas were observed within the otherwise normal granular cell layer of cortex (figs. 5, 6). These lucent islands were well circumscribed, of variable size, and distributed ubiquitously throughout the granular cell layer of the cerebellum in irradiated animals. They consisted of reduced numbers of cells (figs. 6,9) and contrasted markedly with the surrounding population of normal granule cells (figs. 5, 6). Only occasionally, however, did the granule cells or surrounding Purkinje cells themselves appear abnormal (fig. 7) in the irradiated animals.
Fig. 6. Granular cell layer from the cerebellum of a 26 day old rat irradiated on GD-I5 showing one circumscribed area (arrow) immediately beneath the Purkinje cells (pc) of reduced granule cell number. m, molecular layer. x 545. Fig. 7. Pyknotic Purkinje (open arrows) and granule cells (solid arrows) seen occasionally in irradiated rats. Taken from a 26 day old rat irradiated on GD-I5. x l 090.
Discussion In this study, exposure to a single dose of 50 cGy produced significant observable neuropathologic lesions that were a function of gestational age at the time of irradiation. Embryos irradiated on GD-9.5 showed reduced mitoses, as well as pyknosis and necrosis of primitive neural cells, within 4h of irradiation. This is consistent with observations of HICKS (21) and others (27 , 29 , 33) who found that primative differentiating neural cells undergo necrosis within 4 h of maternal exposure to :5 50 ron GD-9-1O. Our study revealed that the capacity of surviving cells for repair was such that within 48 h of exposure, the nervous system of irradiated embryos no longer differed morphologically from controls . The study did not reveal, however, whether embryos exposed to radiation at this early stage of morphogenesis would exhibit residual neurophysiological or behavioral deficits in adulthood. Although radiosensitive , the undifferentiated nervous system at this early stage of development , has great recuperative capacity and therefore would not be expected to exhibit morphological anomalies or major CNS defects on exposure to radiation (31,33). Consistent with this view, rats irradiated on GD-9 with 0.1-0.6 Gy X-rays failed to exhibit a significant incidence of malformations , intrauterine death or alterations in physiological development postnatally (31 , 32) . Even offsprings of rats that received a relatively large dose (250 r) of X-rays during the period from GD-0-8 showed no histolog-
ical abnormalities in the CNS (14). There are other studies, however, although few in number, that have reported severe damage to the nervous system following exposure to low doses of radiation at early stages of development (49). WILSON et aI., (46, 47,48) exposed exteriorized rat embryos to 25 and 50 r X-rays on GD-8-11. After exposure to 25 r on GD-9, microphthalmia was observed in 6 % of the embryos and ocular maldevelopment and anophthalmia in 72 % after 50 r. The brain and spinal cord were also malformed in 9 % and 3 % of embryos, respectively after 50 r. On GD-lO day, only the eye was affected in 11 % of embryos and only after 50r. Unlike the embryonic period, our study revealed that exposure to radiation during the fetal period (GD-15 or 18) resulted in abnormalities that persisted and were evident in adulthood . Malformations of the nervous system and behavioral deficits have been regularly noted in offspring of rats exposed to radiation ~ 1 Gy on GD-13 to PN-3 (12, 14, 15,20,24, 36). These have included reduced size of cerebral hemispheres, decreased brain weight, defective cortical cytoarchitecture, heterotopias , spinal cord lesions, commissural agensis and altered locomotion. There are also reports of specific cortical vulnerability in rats; neuronal cell loss, malformed cortical patterns , abnormal organization, orientation and synaptogenesis of neurons following exposure to radiation ~ 1 Gy on GD-13-21 (9, 19, 20, 43) . Although exposure to a minimum of 1 Gy appears to be needed, morphologically descernible alterations in the histogenesis of Exp Toxic Pathol 45 (1993) 4
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Fig. 8. Nonnal granule cells (gr) closely clustered beneath the Purkinje cell (pc) layer in a 26 day old rat irradiated on GD-15. Granule cells are characterized by round heterochromatic nuclei and scanty cytoplasm. Glomeruli (arrows) consisting of arrays of pre- and post-synaptic elements comprise the intervening neurophil. Compare with the abnonnal neuropil shown in figs. 6 & 9. X 4500.
the cerebral cortex have been occasionally reported with exposure to::5 0.75 Gy on GD-15, 16, 18,22 and PN-l (20, 22, 37, 38, 39). We have been unable to confirm the latter observations in this study. Unlike the changes in thickness of the cortical laminae in the hippocampus in monkeys that received 2 Gy on GD-75 (13), or the hippocampal ectopias in SID rats exposed to 2 Gy on GD-16 (19) only circumscribed areas of focal hippocampal necrosis were observed in our study. Fifty seven percent and 33 % of rats irradiated on either GD-15 or GD-18, respectively, exhibited scattered clusters of pyknotic neurons and sometimes a reduction in cell numbers within the hippocampal formation. This vulnerabilitiy of hippocampal neurons, in contrast to neurons of other cortical areas is likely due to their morphogenesis being coincident with the period of irradiation. Cells in all mitotic phases as well as post-mitotic undifferentiated migratory cells are known to be radiosensitive (3, 6, 23), Hippocampal pyramidal cells are formed entirely prenatally in the mouse on GD-I 0-18 (6) and on GD16-19 in the rat (7 , 25). The granule cell population of the dentate gyrus, however, is largely (72 %) produced postnatally from PN-I-16 (with a peak on PN-2) and 13 % from PN-16 on into the adult period (7, 8, 35). Only 15 % of granule cells are produced prenatally with initiation thought to occur on GD-15-16 (8, 25) . The most significant radiation induced pathological changes were observed in the cerebellum of 26 days old rats. 228
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The lesions were the same regardless of whether irradiation took place on GD-15 or 18 although the former were more profound. Interestingly, there have been numerous studies of radiation effects on the developing cerebellum, using larger doses than in the present study, in which the anomalous development of the internal granular layer as seen in our study either did not occur or was not reported. Das (16, 17), for example, irradiated rat fetuses at the same stage of gestational development as in our study but used 170 r X-rays. He reported that the mature cerebellum in fetuses irradiated on G-15 was 50 % of normal size, exhibited reduced cortical areas and thickness, and abnormal foliation in the anterior cerebellum (16). There were also 32 - 50 % fewer Purkinje cells, 60 % fewer granule cells and 27 % fewer granule cells per Purkinje cell. When the same dose of radiation was administered on GD-18 similar but less severe consequences were observed; cerebellar size was reduced 7 -19 %. Purkinje and granule cell numbers were 93-98 % and 85 % of normal respectively, and the granule to Purkinje cell ratio was only 12 % of normal (17). These developmental anomalies were attributed to direct radiation effects on migratory neuroblasts of Purkinje cells which caused their death and reduced or altered the inductive influences of surviving Purkinje cells on the mitotic activity of the external granular layer. In contrast, there are also studies, although few in number, in which cell free foci as reported in our study have been
Fig. 9. Granular cell layer of the cerebellum in a 26 day old rat irradiated on GD-15 showing a lesioned area consisting of a loosely organized array of granule cells. Although the granule cells present appear normal, the number is reduced and the surrounding neuropil greatly thinned. X 4500.
observed within the mature cerebellum of rats exposed to large doses of X-ray radiation (2: 200 r) , both prenatally (14) and postnatally (2, 4). No cerebellar anomalies were found in rats that received 250 r X-rays on GD-15; however, at all subsequent intervals anomalies were present in the rostral portions of the cerebellum (14). Disturbances were mild with exposure on GD-1618 and were manifest by decreased size and abnormal orientation of folia and sulci but normal cortical architecture. Cerebellar defects were most severe with exposure from GD19 to PN-l. In addition to an exaggeration of changes already mentioned, ectopias of granule or Purkinje cells in the molecular layer or folial white matter occurred. Various defects in the internal granular layer such as reduced numbers of cells, narrowing, absence of segments of this lamina and the presence of cell free foci were observed. No explanation was offered for the appearance of cell free foci within the internal granular layer of the cerebellum. In a similar study, rats that received 3 - 5 successive daily doses of 200 r of X-rays beginning on PN-\ also exhibited anterior cerebellar abnormalities at maturity (2,4). Notable among them were abnormal foliation, reduction in granule cell numbers, thinning of the molecular layer, mis-shapen Purkinje cells and significantly the presence of radial streaks and circular islands of cell-sparse areas within the internal granular layer (2). The latter observation is similar to our own and that of COWEN and GELLER (14) but was obtained with
postnatal exposure to a considerably larger dose of radiation 3-10 x 200 r. ALTMAN and ANDERSON (2) referred to these cell sparse areas as extensions of the molecular layer in which Purkinje cell dendrites were located. They attributed this anomaly to disoriented growth of Purkinje cell dendrites that became embedded in the internal granular layer taking with them cell free islands of molecular layer; a conclusion not supported by our data. Although Purkinje cells themselves were not destroyed or reduced in number by either single or multiple doses of 200 r, their normal cytological differentiation was affected. This was attributed to direct damage of the external granular layer (the precursors of interneurons) and removal of some guiding influence they provide on the normal development of the Purkinje cells . There is evidence that Purkinje cells are among the earliest formed prenatally (day 13-16) but mature largely postnatally (5, 18) whereas precursors of cerebellar microneurons (granule, basket and stellate cells) are acquired and differentiate during the early (1- 3rd wk) postnatal period (1, 5, 18, 33). Irradiation of the cerebellum at birth and postnatally with successive daily doses of 0.5-2 Gy of x-rays produced dose dependent reduction in granule cells but Purkinje cells were unaffected (2, 3). In view of these observations, we conclude from our data that the single dose of low level radiation on GD-15 or 18 may have directly affected the proliferative external granular layer of the developing cerebellum, precursors thereof and/or Exp Toxic Pathol 45 (1993) 4
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postmitotic migratory cells of this layer. Alternatively, because prenatally forming neurons may have a organizing or regulating influence on the small neurons of postnatal origin, (2, 18) the normal cytological differentiation of the Purkinje cells may have been directly affected by the prenatal radiation. This in tum removed or altered some guiding influence which Purkinje cells provide on the normal development of internal granule cells. Acknowledgements: The authors acknowledge with gratitude the excellent technical assistance provided by Mr. G SEYOUM, PM PERUMAL, R SIMPSON and Ms. L SNELL. We also thank the Unsolicited Proposals Fund, Department of Supply and Services; Atomic Energy Control Board and Health and Welfare, Canada for funding this investigation.
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