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X-irradiation causes a prolonged reduction in cell proliferation in the dentate gyrus of adult rats
X-rays and subgranular zone cell proliferation
Pergamon
PII: S0306-4522(00)00151-2
Neuroscience Vol. 99, No. 1, pp. 33±41, 2000 33 q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
X-IRRADIATION CAUSES A PROLONGED REDUCTION IN CELL PROLIFERATION IN THE DENTATE GYRUS OF ADULT RATS E. TADA,* J. M. PARENT,² D. H. LOWENSTEIN²³§ and J. R. FIKE*k¶ Departments of *Neurosurgery, ²Neurology, ³Anatomy, k Radiation Oncology, and §Program in Neuroscience, University of California, San Francisco, CA 94143, USA
AbstractÐThe effects of X-irradiation on proliferating cells in the dentate subgranular zone were assessed in young adult Fisher 344 rats exposed to a range of X-ray doses and followed for up to 120 days. Apoptosis was quanti®ed using morphology and endlabeling immunohistochemistry, and cell proliferation was detected using antibodies against the thymidine analog BrdU and the cyclin-dependent kinase p34 cdc2. Radiation-induced apoptosis occurred rapidly, with maximum morphological and end-labeling changes observed 3±6 h after irradiation. Twenty-four hours after irradiation cell proliferation was signi®cantly reduced relative to sham-irradiated controls. The number of apoptotic nuclei increased rapidly with radiation dose, reaching a plateau at about 3 Gy. The maximum number of apoptotic nuclei was substantially higher than the number of proliferating cells, suggesting that nonproliferating as well as proliferating cells in the subgranular zone were sensitive to irradiation. Subgranular zone cell proliferation was signi®cantly reduced relative to age-matched controls 120 days after doses of 5 Gy or higher. These ®ndings suggest that neural precursor cells of the dentate gyrus are very sensitive to irradiation and are not capable of repopulating the subgranular zone at least up to 120 days after irradiation. This may help explain, in part, how ionizing irradiation induces cognitive impairments in animals and humans. q 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: hippocampus, rat, irradiation, apoptosis, precursor cell, proliferation.
The brain is exposed to irradiation under a variety of clinical situations, and radiation injury to normal brain tissue is a dose-limiting factor. Radiation brain injury has many effects, and a number of reviews has documented its clinical and morphological character. 6,10,20,27,52,53,63 Changes involving signi®cant tissue destruction generally occur only after aggressive treatment protocols using relatively high doses of irradiation. However, less severe morphological injury can also occur after radiotherapy, which may lead to cognitive dysfunction, particularly in children. 1,7,15,18,33,64 Much of the available clinical literature suggests that the cognitive effects of ionizing irradiation re¯ect impaired attention and memory more than low intellectual performance. 13,29,51 Based on such data it has been suggested that the hippocampus, a critical component of the medial temporal lobe memory system, 59 may be involved in the cognitive changes observed after irradiation. Indirect evidence of the role of hippocampal damage in this cognitive dysfunction comes from clinical studies of head and neck cancer in which patients received high radiation doses to the inferomedial portion of the temporal lobes. Those patients reported subjective memory impairment and, relative to non-irradiated controls, showed poorer performance on measures of nonverbal recent memory. 37 Within the hippocampus, memory functions are associated with the principal cells of the hippocampal formation, i.e. the pyramidal cells and the granule cells of the dentate gyrus. 11 Studies on the radiation response of the brain in late gestational or neonatal mice have shown that damage to
the granule cell layer of the hippocampus or substantial reductions in dentate granule cell number are associated with cognitive impairments. 14,41,42,57,65 The proliferative potential of neuronal precursor cells in the adult mammalian brain was recognized many years ago, 2,3,38,48,58 but only recently has there been a concerted effort to identify and understand the biology and function of those cells. 21,22,30,40,50 In the forebrain of adult rodents, stem/ precursor cells have been identi®ed in two major sites, the subependyma and the subgranular zone of the dentate gyrus (SGZ). In the SGZ of rodents, new neurons are continuously generated, 4,9,23,24,31 migrate into the granule cell layer, develop granule cell morphology and neuronal markers and connect with their target area, CA3. 60 Recent data also show that neurogenesis occurs in the adult human hippocampus beyond the age of 70. 19 Although many new neurons are produced in the rodent SGZ, their signi®cance has not been fully elucidated. 34 However, recently published data suggest a direct association between hippocampus-dependent learning and survival of neurons generated in the mature hippocampal formation. 23 Proliferating cells in the subependyma of rats are particularly sensitive to ionizing irradiation, undergoing apoptosis after doses as low as 1 Gy. 5,54 Furthermore, stem cells in that region are depleted in a dose-dependent fashion, and cell proliferation and total cellularity of the subependyma remain depressed for a prolonged period of time. 62 We have also shown that neural precursor cells of the SGZ are sensitive to irradiation, undergoing apoptosis after low doses of X-rays, 45 A recent report by Peibner et a1. 47 also showed that immature progenitor cells in the rat hippocampus undergo apoptosis after a single 10 Gy dose of X-rays. Because of the potential involvement of the hippocampus in radiation-induced cognitive changes, we were interested in determining the acute radiation response of proliferating SGZ cells after a range of X-ray doses, and to
¶To whom correspondence should be addressed. Tel.: 11-415-476-4453; fax: 11-415-502-0613. E-mail address: j®[email protected] (J. R. Fike). Abbreviations: BrdU, 5-bromo-2 0 -deoxyuridine; GFAP, glial ®brillary acidic protein; SGZ, subgranular zone of the dentate gyrus; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. 33
34 E. Tada et al. Fig. 1. Photomicrograph of the dentate gyrus of a rat that was irradiated 6 h earlier with 15 Gy. Apoptotic nuclei appeared as brown TUNEL-positive nuclei (red arrow) and TUNEL-negative nuclei (blue) that were fragmented (arrowhead) or pyknotic (black arrow). Clusters and inidividual apoptotic nuclei were observed all along the superior and inferior blades of the dentate gyrus. dgc: dentate granule cells; h: hilus. Scale bar 40 mm.
35
X-rays and subgranular zone cell proliferation
determine any subsequent regeneration of that proliferating population. EXPERIMENTAL PROCEDURES
Young adult (eight to 10 weeks) male Fisher 344 rats, weighing approximately 200 g, were used; many of these rats were used in previously published studies of the radiation response of the subependymal region. 54,62 Rats were housed and cared for in accordance with the United States Department of Health and Human Services Guide for the Care and Use of Labotatory Animals, and institutional guidelines for care and handling of laboratory animals. Rats were anesthetized for irradiation and perfusion procedures. For studies of apoptosis, anesthesia consisted of an intraperitoneal (i.p.) injection of sodium pentobarbital (60 mg/kg). In studies of the longer term effects of irradiation (i.e. seven to 120 days), anesthesia consisted of an i.p. injection of ketamine hydrochloride (80 mg/kg) and xylazine (5 mg/kg) dissolved in normal saline. Proliferating cells were labeled using an antibody against the thymidine analog 5-bromo-2 0 -deoxyuridine (BrdU; Sigma, St Louis, MO). In all radiation studies a single i.p. injection of BrdU (60 mg/kg) was given 1 h before rats were killed. To label all proliferating cells in the SGZ of non-irradiated rats, groups of four animals were injected with BrdU every 2 h for 24 h starting between 8.00 and 9.00 a.m., and brain tissue was collected at 2 h intervals, 30 min after a ®nal BrdU injection. The total number of proliferating cells was also estimated using the cyclin-dependent kinase p34 cdc34 (Santa Cruz Biotechnology, Santa Cruz, NM). 62 Irradiations were done using a Phillips orthovoltage X-ray system as previously described. 54 In brief, each rat was irradiated individually with its head centered in a 5 £ 6 cm treatment ®eld and the eyes and body were shielded with lead. Dosimetry was done using a Keithly electrometer ionization chamber calibrated using lithium ¯uoride thermal luminescent dosimeters. The corrected dose rate was approximately 175 cGy/min at a source to skin distance of 21 cm. The time course for radiation-induced apoptosis was determined by irradiating groups of three to six rats with a single dose of 15 Gy and collecting tissues at various times up to 48 h later. Four rats were killed 6 h following sham irradiation. For tissue collection, rats were re-anesthetized, and 350 ml of a 10% buffered formalin solution was infused into the ascending aorta using a mechanical pump (Master¯ex Model 7014; Cole Parmer, Chicago, IL). After 5 min, rats were decapitated and the brain was removed and immersed in a 10% buffered formalin solution for seven days. The radiation dose response for apoptosis in the SGZ was determined 6 h after irradiation. Whole brain doses of 0, 1, 3, 5, 15 and 30 Gy were given to groups of four to six rats that were killed 6 h later. The perfusion and tissue preparation techniques were performed as described above. To study the longer term effects of radiation on proliferating cells within the SGZ, groups of four rats were given doses of 0, 2, 5, 10 and 15 Gy. At one, seven, 14, 30, 60, and 120 days posttreatment, rats were given a single i.p. injection of BrdU and 1 h later were anesthetized and perfused with formalin as described. After one week of ®xation, brains were placed in a Rat Brain Matrix (Large Adult Rat, Type C, Harvard Apparatus, Natick, MA) which facilitated reproducible transverse sectioning of whole brain. A single 5-mm-thick section containing the hippocampus was placed in a plastic tissue holder and processed for paraf®n embedding. Five-micrometerthick coronal sections were taken from three different brain levels and placed on polylysine-coated glass microscope slides. The brain levels were approximately 125±150 mm apart; the most rostral corresponded to a point 3.6 mm behind the bregma, i.e. plate 32 as de®ned in the atlas of rat brain anatomy. 46 The detection of cells undergoing apoptosis was done using nuclear morphology and immunohistochemical staining. Morphological changes included nuclear fragmentation, which was de®ned as the compaction of chromatin into two or more dense, lobulated masses, and pyknosis, which was de®ned as small, round, darkly stained nuclei (Fig. 1). To avoid including any normal cell pro®les (i.e. glia), if cytoplasm was observed in conjunction with a small, dense nucleus, that cell was not counted as apoptotic. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells appeared as highly stained, brown nuclei against the blue background of the counterstain (Fig. 1). For the TUNEL procedure, all reagents, including buffers, were part of a kit (ApopTag, Oncor,
Gaithersburg, MD). Procedures were carried out as previously described. 54,62 Gill's hematoxylin was used as a counterstain. Immunohistochemical labeling of proliferating cells was done using monoclonal antibodies against BrdU (anti-human BrdU antibody, IU-4, Caltag, San Francisco, CA) and p34 cdc2 (Santa Cruz Biotechnology, Santa Cruz, CA). Binding of biotinylated secondary antibody was detected using an avidin±biotinylated peroxidase complex system (ABC; Vector, Burlingame, CA). To quench endogenous peroxidase activity, deparaf®nized specimens were soaked for 20 min in 0.3% H2O2 (Sigma, St Louis, MO) in double deionized distilled water containing 0.1 M sodium azide (Sigma, St Louis, MO). After the primary and the secondary antibodies were applied, the specimens were incubated with the ABC reagent for 30 min and developed with 0.025% 3,3 0 -diaminobenzidine (DAB, Sigma, St Louis, MO) dissolved in phosphate-buffered saline (PBS) containing 0.005% H2O2. Sections were then counterstained with Gill's hematoxylin (Sigma, St Louis, MO), deydrated and mounted. Due to the length of the longer term radiation studies, tissues from different post-treatment times were not necessarily processed together. To insure that immunohistochemical results were comparable among treatment groups, as a positive control we always included tissues from unirradiated young adult rats (12 weeks of age) that had received a pulse injection of BrdU. Furthermore, to insure that factors such as microwave antigen retrieval (see below) did not affect results, for each follow-up time, tissues from all radiation dose groups and positive controls were processed simultaneously. 5-Bromo-2 0 -deoxyuridine After deparaf®nization and quenching of endogenous peroxidase, tissue sections were treated for 5 min at 378C with 20 mg/ml proteinase K (Sigma, St Louis, MO) in PBS. Sections were then soaked in 5% acetic acid for 1 h and then for 10 min in 4 N HCl. Sections were treated for 20 min with 5% normal horse serum in PBS, and after excess horse serum was removed, incubated for 1 h with primary antibody diluted 1:1000 in PBS containing 5% normal horse serum. Sections were washed with PBS and incubated for 30 min with biotinylated horse anti-mouse secondary antibody diluted 1:200 in PBS. All sections were then incubated for 30 min with ABC reagent, developed with DAB and counterstained as described. p34 cdc2 After deparaf®nization and quenching the endogenous peroxidase as described above, sections were soaked in 0.01 M citrate buffer (pH 6.0), and microwaved for 10 min under full power in a 15 kW microwave oven to enhance antigen binding. Sections were left in the buffer for an additional 15 min, washed with PBS, then incubated with 5% normal horse serum for 15 min. After the horse serum was removed, the sections were incubated for 30 min at room temperature with mouse anti-p34 cdc2 antibody diluted 1:500 with PBS. After washing, the sections were incubated for 30 min with biotin-conjugated ratabsorbed horse anti-mouse IgG diluted 1:200 with PBS. Finally, the specimens were incubated with ABC reagent, developed with DAB and counterstained as described. Glial ®brillary labeling
acidic
protein-5-bromo-2 0 -deoxyuridine
double
After deparaf®nization and quenching of endogenous peroxidase, rehydrated sections were incubated in 5% normal goat serum in PBS for 10 min, and then incubated for 30 min in anti-rabbit glial ®brillary acidic protein (GFAP) antibody (Dako Corp., Carpinteria, CA) diluted 1:500 with PBS. Sections were then incubated for 30 min with biotinconjugated goat anti-rabbit IgG diluted 1:200 with PBS, followed by treatment for 30 min with ABC±horseradish peroxidase. Sections were washed in PBS and then treated as described above for BrdU labeling, except that incubation with the primary antibody was overnight at 48C. Blocking sera and secondary antibody counterstaining were as described. Sections were then incubated with ABC±alkaline phosphatase reagent for 30 min followed by an additional 15 min with Vector Blue Alkaline Phosphatase Substrate Kit III (Vector, Burlingame, CA). The number of proliferating cells or cells showing speci®c characteristics of apoptosis was scored blind using a histomorphometric approach. Three 5-mm-thick brain sections were analysed for each rat, and quanti®cation was made of all positively labeled cells within the SGZ of the suprapyramidal and infrapyramidal blades of the
36
E. Tada et al.
Fig. 2. (A) Photomicrograph of the dentate gyrus of a non-irradiated rat after cumulative labeling with BrdU. Brown BrdU-positive cells (arrows) were observed in the subgranular zone, often in clusters of four or more cells. (B) Photomicrograph of the dentate gyrus of a non-irradiated rat after labeling with an antibody against p34 cdc2. The brown p34 cdc2-positive cells represent proliferating cells. dgc: dentate granule cells; h: hilus. Scale bars 25 mm.
dentate gyrus. Because of the high numbers of apoptotic cells after irradiation (see below), the total numbers of pyknotic cells, cells exhibiting TUNEL-positive nuclei or nuclei showing a distinct pattern of nuclear fragmentation were determined in one dentate gyrus on each tissue section. The total number of apoptotic cells was determined by summing the values from all three tissue sections from a given brain. For all studies involving BrdU and p34 cdc2 labeling, the total number of positively labeled cells in both dentate gyri was determined using the three tissue sections and the numbers were summed for a single value for each animal; there were no systematic differences between right and left dentate gyrus in terms of total numbers of labeled cells (not shown). For all endpoints used in this study, values from three to six rats were averaged and standard errors of the mean (S.E.M.) were calculated. Differences in cell number as a function of dose relative to sham-irradiated controls were analysed using Spearman's correlation coef®cient, and numbers of proliferating cells in sham-irradiated rats at 0 and 120 days were compared using an unpaired t-test. RESULTS
Proliferating SGZ cells were found along the suprapyramidal and infrapyramidal blades of the dentate gyrus, and only an occasional BrdU-positive cell was observed in the hilus. After a pulse injection of BrdU, positively labeled cells generally occurred singly or in clusters of two or three
cells; a total of 23.5 ^ 2.63 (mean ^ S.E.M., n 4) SGZ cells was labeled in the three 5-mm-thick tissue sections. In rats that received multiple injections of BrdU, the numbers of BrdU-positive cells increased continuously over the 24 h labeling period (not shown). After cumulative labeling with BrdU, in addition to positively labeled single cells and small clusters, there were groups of BrdU-positive cells comprised of ®ve or more cells (Fig. 2A). Qualitatively, p34 cdc2-positive cells were distributed similarly to cells showing BrdU immunoreactivity (Fig. 2B). For the three tissue sections assessed, the total number of p34 cdc2-positive cells was 78.5 ^ 6.6 (n 4). Between 3 and 12 h after irradiation, the qualitative distributions of TUNEL-positive cells and cells showing morphological characteristics of apoptosis were similar, with cells appearing in the SGZ along the suprapyramidal and infrapyramidal blades of the dentate gyrus. Apoptotic cells appeared singly or in groups (Fig. 1), some of which were made up of ®ve or more cells. Isolated apoptotic nuclei were occasionally seen in the hilar region. In sham-irradiated rats, the total number of apoptotic nuclei detected averaged 3.0 ^ 1.7 (n 4). After 15 Gy, the numbers
37
X-rays and subgranular zone cell proliferation
Fig. 3. Time-course for apoptotic changes in the dentate subgranular zone of young adult rats after a single dose of 15 Gy. Numbers of cells characterized by three distinct criteria of apoptosis, TUNEL immunoreactivity, nuclear fragmentation and pyknosis were determined 3±48 h after irradiation. Maximum apoptosis (TUNEL, fragmented and pyknotic nuclei combined) was seen 6 h after irradiation. Three tissues sections were obtained 125±150 mm apart; the numbers of apoptotic cells within one dentate subgranular zone from each section were determined and the three values were summed for each animal. Each datum point represents four to six rats and error bars are S.E.M.
of pyknotic, fragmented and TUNEL-positive cells increased sharply with time, reaching maximum values 3 h after irradiation for fragmentation and pyknosis endpoints and 6 h after irradiation for the TUNEL endpoint (Fig. 3). The maximum number of apoptotic cells (fragmentation, pyknosis and TUNEL-positive cells combined) was 149 ^ 13.2 (n 6), and was seen 6 h after irradiation. There was a decrease in the number of apoptotic cells by 12 h and from 24 to 48 h, the total numbers of apoptotic cells were similar to those observed in sham-irradiated rats. To determine the dose±response characteristics for SGZ cell apoptosis, the numbers of pyknotic, fragmented and TUNEL-positive cells were measured 6 h after irradiation (Fig. 4). There was a steep increase in apoptosis between 0 and 3 Gy, but there were no statistically signi®cant changes in the number of apoptotic nuclei after higher radiation doses. A statistically signi®cant decrease (P , 0.001) in BrdU labeling was seen 24 h after radiation doses ranging from 2 to 15 Gy (Fig. 5) due to apoptosis of dividing cells. Seven days after irradiation there were subsequent increases in the numbers of BrdU-positive cells after all doses, and at that time, the extent of BrdU labeling was radiation dose dependent (Fig. 6). Double labeling with GFAP and BrdU was done to determine whether the acute increase in BrdU labeling seven days after 2 Gy involved astrocytic cells. Although there were some double-labeled cells in the SGZ, usually in clusters of two to three cells, there were no double-labeled cells in the hilus. Later time points (14±30 days) showed a few doublelabeled cells in the SGZ and in the hilus. After seven days the number of BrdU-positive cells detected in the SGZ of all treatment groups, including the sham-irradiated controls decreased. The average number of BrdU-positive cells observed in sham-irradiated rats at 120 days, 14.9 ^ 9 (n 4), was signi®cantly less (P , 0.01) than that seen at day 0 (23.5 ^ 2.6, n 4). With the exception of 2 Gy, the
Fig. 4. Total number of apoptotic cells in the subgranular zone of young adult rats as a function of radiation dose. Tissue was collected 6 h after irradiation, and for each animal, three tissue sections were obtained 125± 150 mm apart. The numbers of TUNEL-positive, fragmented and pyknotic nuclei within one dentate subgranular zone from each section were determined and the three values were summed for each animal. Each datum point represents three to six rats and error bars are S.E.M.
BrdU labeling was below that obtained for age-matched controls and was maintained or fell continuously from 30 to 120 days (Fig. 6). At 120 days, the extent of BrdU labeling was signi®cantly lower (P , 0.001) in all treatment groups that received greater than 2 Gy. Because a pulse of BrdU only labeled a fraction of the proliferating cell population and the number of BrdU-positive cells was very low, particularly after long follow-up times and higher doses, we used p34 cdc2 immunoreactivity to provide an estimate of the total number of proliferating cells 120 days after irradiation. The total number of p34 cdc2-positive cells in sham-irradiated controls decreased signi®cantly over the experimental period from 78.5 ^ 6.6 (n 4) at day 0 to 23.5 ^ 2.6 (n 4) at 120 days (P , 0.001). After 5, 10 and 15 Gy, the average numbers of p34 cdc2-positive cells in the SGZ were 4.5 ^ 2.3 (n 4), 2.5 ^ 2 (n 4) and 8 ^ 0.9 (n 4), respectively. Those values were signi®cantly lower (P , 0.001) than age-matched controls. DISCUSSION
The main ®ndings of this study are that: (i) X-ray doses of 1±30 Gy induce signi®cant apoptosis in the SGZ of the hippocampus; (ii) proliferative activity within the SGZ is substantially depressed one day after irradiation; (iii) there is a subsequent and transient increase in the number of proliferating cells seven to 14 days post-irradiation; and (iv) after 30 days there is a prolonged and substantial decrease in the proliferating population of the SGZ after doses above 2 Gy. Similar X-ray sensitivity and long-term depression of proliferative activity have been seen in another proliferative zone in the brain, the subependyma, 5,54,62 suggesting a common responsiveness to ionizing irradiation among stem/ precursor cells of the rodent forebrain. Previous animal studies addressing the effects of ionizing irradiation on hippocampal structure and function have generally involved irradiation of prenatal or neonatal
38
E. Tada et al.
Fig. 5. Total number of BrdU-positive cells in the dentate subgranular zone of young adult rats 24 h after irradiation. A single intraperitoneal injection of BrdU (60 mg/kg) was given 1 h prior to tissue collection. Three tissues sections were obtained 125±150 mm apart, and the total numbers of BrdUpositive cells within the dentate subgranular zones from both sides of the brain were summed for each animal. The decreases in the numbers of BrdU-positive cells after all doses, relative to sham-irradiated rats, were statistically signi®cant (P , 0.001). Each bar represents four rats, and error bars are S.E.M.
animals. 41,42,49,55±57,65 Those studies have shown varying degrees of morphological change within the granule cell layer, including a reduction in the number of granule cells, and it has been suggested that various cognitive de®cits were probably mediated by lesions to the hippocampal formation. In older animals, cognitive effects have been shown to be induced by radiation doses that did not lead to the development of necrosis 26 or after treatment that resulted in no apparent histological changes. 35 In neither of the latter studies was cell proliferation or cell death in the SGZ or the granule cell layer of the hippocampal formation assessed directly. Studies of adult rats by our group 45 and others 47 have recently reported radiation-induced apoptosis of the progenitor population of the SGZ after selected doses of X-rays. In the present study, apoptosis was detected 3±12 h after irradiation, and by 24 h there were few if any apoptotic nuclei remaining in the SGZ. This time course is similar to that of radiation-induced death of cells in the adult rat spinal cord 39 and of precursor cells in the forebrain of adult rats. 5,54 Because there can be considerable temporal separation between nuclear morphological changes and DNA fragmentation, it has been suggested that the detection of apoptosis can be improved when in situ assays of DNA fragmentation such as TUNEL are combined with the assessment of morphological features. 12 We therefore quanti®ed total apoptosis at speci®c times after irradiation by summing three distinct criteria: TUNEL reactivity, pyknosis and nuclear fragmentation (Fig. 1). Our data showed that the time-course for immunohistochemical and distinct morphological changes differed slightly (Fig. 3). However, regardless of the speci®c criteria assessed, the overall onset, peak and resolution of all changes were rapid, leading to a signi®cant loss of BrdUpositive cells 24 h after irradiation (Fig. 5). Since apoptosis occurs rapidly and we made our measurements only at speci®ed time intervals, the values we obtained are probably an underestimation of the true extent of acute cell death in the SGZ. Regardless, our data show that the SGZ is particularly
Fig. 6. The number of BrdU-positive cells in the dentate subgranular zone of young adult rats changes with time after irradiation. Three tissue sections were obtained 125±150 mm apart, and the total numbers of BrdU-positive cells within the dentate subgranular zones from both sides of the brain were summed for each animal. Before irradiation, the number of BrdU-positive averaged 23.5 ^ 2.63; in sham-irradiated rats there was a statistically signi®cant reduction at 120 days (P , 0.01). Twenty-four hours after irradiation there were signi®cant reductions in the number of BrdU-positive cells due to apoptosis. The magnitude of the subsequent increase at seven days was radiation dose dependent, but by 120 days there were no apparent differences in the numbers of BrdU-positive cells after doses higher than 2 Gy. At 120 days the numbers of BrdU-positive cells after 5, 10 and 15 Gy were signi®cantly reduced relative to agematched controls (P , 0.001). Each datum point represents four rats and error bars are S.E.M.
sensitive to irradiation, with a steep dose response from 0 to 3 Gy and no additional apoptosis after doses greater than 3 Gy. The plateau in number of apoptotic cells after doses of 3±30 Gy implies the existence of an apoptotic sensitive population(s) in the SGZ of the hippocampus. 16,61 A major component of the population is the proliferating cells, since 90±95% of BrdU-positive cells within the SGZ are lost 24 h after irradiation (Fig. 4). However, non-proliferating cells or cells in a different phase of the cell cycle must also be dying via apoptosis, because the total number of apoptotic nuclei detected in our time course study (Fig. 3) is much greater than the total number of proliferating cells in our designated counting area as determined by p34 cdc2 immunoreactivity. Although the identity of those cells was not speci®cally addressed in this study, a previous investigation by us suggested that X-ray doses of 1±5 Gy depleted non-cycling immature neurons as well as proliferating neural precursor cells. Peibner et al. 47 also reported that cells dying from radiation-induced apoptosis belonged to the progenitor population of the rat hippocampus. We did not see any evidence that fully differentiated neurons underwent apoptosis after doses up to 30 Gy. After 24 h post-irradiation, there was a vigorous but transient increase in the number of BrdU-positive cells (Fig. 6). This was most apparent after lower doses, and seven days after 2 Gy the number of proliferating cells was actually higher than that seen in sham-irradiated rats (Fig. 5). Whether increased proliferation occurs in a subpopulation of precursor
X-rays and subgranular zone cell proliferation
cells that is apoptotic resistant or represents activation of a population of relatively quiescent stem cells similar to what has been observed in the subependyma 43 is not known. Our study did suggest that seven days after 2 Gy some of the proliferating cells in SGZ were astrocytes. Curiously, however, at that time we did not see any double-labeled cells in the hilus, where there are large numbers of GFAPpositive cells. The signi®cance of this ®nding is unclear, and at later time points we saw few double-labeled cells in the SGZ. The small increase in GFAP±BrdU double-labeled cells seen later in the hilus agrees with our previous observation 45 that brain irradiation produces a mild proliferative response of glia in the dentate gyrus. However, studies of the radiation response of the other forebrain site of neurogenesis, the subependyma, suggest that six months after X-irradiation there is a dose-related decrease in astrocytes. Transient increases in proliferating cells have previously been reported after irradiation of brain and other tissues 28 and may re¯ect abortive cell divisions of lethally irradiated cells, changes in cell proliferation rate, or within a stem/precursor cell population, a change from asymmetrical to symmetrical division. 17 We did not address changes in rate of cell proliferation, and we did not see any morphological evidence of abortive cell divisions or increased cell death, e.g., apoptosis (not shown), although increased mitotic-linked cell death may have occurred after seven days. After irradiation, regeneration of certain tissues can occur in part due to a change from asymmetrical to symmetrical division that repopulates the stem cell pool. 17 The apparent lack of reconstitution of the normal number of proliferating SGZ cells up to 120 days following irradiation suggests that such a change in mode of division did not occur. It remains to be determined whether neural progenitors of the SGZ are unable to divide symmetrically or whether they did not receive the appropriate signal to initiate such a change after exposure to irradiation. In our study, the number of proliferating SGZ cells was determined primarily using BrdU immunoreactivity to label cells in the S-phase of the cell cycle. However, a pulse of BrdU only labels a fraction of the total number of proliferating cells, and after higher doses of irradiation (10±15 Gy), values near zero were seen at 120 days. To ascertain a more
39
accurate measure of the total number of proliferating cells remaining after irradiation, we used an antibody against p34 cdc2, an endogenous protein present in proliferating cells 25 but with levels that have been reported to be low in quiescent cells. 36 In the CNS, the expression of p34 cdc2 changes as cells mature, and a signi®cant decrease in p34 cdc2 has been shown to coincide with the time when proliferating precursor cells differentiate into neurons. 25,44 We have previously shown in the rat subependyma that there is excellent agreement between the numbers of cells cumulatively labeled with BrdU and the number of p34 cdc2-positive cells. 62 In our long-term study, p34 cdc2 labeling showed that 120 days after X-irradiation there were statistically signi®cant reductions in the proliferating cell population relative to sham-irradiated controls. This, together with our BrdU data (Fig. 6), clearly suggests a prolonged radiation-induced reduction in neurogenesis in the dentate gyrus. Although the use of proliferation alone to de®ne neurogenesis is less precise in adult tissue than in developing tissue 32 we have previously shown that low-dose X-irradiation reduces TOAD-64 (turned-on-after-division protein, 64 kDa) and polysialylated-neural cell adhesion molecule (PSA±NCAM) labeling, proteins which are expressed speci®cally in immature neurons in the dentate gyrus and are used as measures of neurogenesis. In the present study, the reduction in SGZ cell proliferation was substantial, leaving only small numbers of p34 cdc2-positive cells remaining in the SGZ after 10± 15 Gy. The fate or potential of those surviving cells in terms of maintaining neurogenesis and cellularity within the dentate granule cell layer is not yet known, nor is it known what radiation dose would be required to completely eradicate all neurogenesis in the hippocampus. Because cell proliferation/neurogenesis in the SGZ is affected after radiation doses that do not result in gross tissue changes or necrosis, 8 our data may have signi®cant implications with respect to certain long-term effects of therapeutic irradiation such as cognitive impairment. AcknowledgementsÐThe authors would like to thank Dr Nobuo Tamesa, Ms Cynthia Yang, Ms Delores Dougherty and Mr King Chiu for technical assistance and Dr Kathleen Lamborn for statistical consultation. This work was supported in part by NIH grants CA 13525, R01 CA76141, NS02006, R01 NS32062, ROI NS35628 and the March of Dimes Birth Defects Foundation.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Abayomi O. K. (1996) Pathogenesis of irradiation-induced cognitive dysfunction. Oncology 35, 659±663. Altman J. (1962) Are neurons formed in the brain of adult mammals? Science 135, 1127±1128. Altman J. (1966) Proliferation and migration of undifferentiated precursor cells in the rat during postnatal gliogenesis. Expl Neurol. 16, 263±278. Bayer S. A. (1982) Changes in the total number of dentate granule cells in juvenile and adult rats: a correlated volumetric and 3H-thymidine autoradiographic study. Expl Brain Res. 46, 315±323. Bellinzona M., Gobbel G. T., Shinohara C. and Fike J. R. (1996) Apoptosis is induced in the subependyma of young adult rats by ionizing irradiation. Neurosci. Lett. 208, 163±166. Burger P. C., Mahaley M. S. J., Dudka L. and Vogel F. S. (1979) The morphologic effects of radiation administered therapeutically for intracranial gliomas. A postmortem study of 25 cases. Cancer 44, 1256±1272. Butler R. W., Hill J. M., Steinherz P. G., Meyers P. A. and Finlay J. L. (1994) Neuropsychologic effects of cranial irradiation, intrathecal methotrexate, and systemic methotrexate in childhood cancer. J. clin. Oncol. 12, 2621±2629. Calvo W., Hopewell J. W., Reinhold H. S. and Yeung T. K. (1988) Time- and dose-related changes in the white matter of the rat brain after single doses of X rays. Br. J. Radiol. 61, 1043±1052. Carneron H. A., Woolley C. S., McEwen B. S. and Gould E. (1993) Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56, 337±344. Caveness A. T. (1977) Pathology of radiation damage to the normal brain of the monkey. NCI Monogr. 46, 57±76. Collier T. J., Quirk G. J. and Routtenberg A. (1987) Separable roles of hippocampal granule cells in forgetting and pyramidal cells in remembering spatial information. Brain Res. 409, 316±328. Collins J. A., Schandl C. A., Young K. K., Vesely J. and Willingham M. C. (1997) Major DNA fragmentation is a late event in apoptosis. J. Histochem. Cytochem. 45, 923±934.
40
E. Tada et al.
13. Crossen J. R., Garwood D., Glatstein E. and Neuwelt E. A. (1994) Neurobehavioral sequelae of cranial irradiation in adults: a review of radiation-induced encephalopathy. J. clin. Oncol. 12, 627±642. 14. Czurko A., Czeh B., Seress L., Nadel L. and Bures J. (1997) Severe spatial navigation de®cit in the Morris water maze after single high dose of neonatal X-ray irradiation in the rat. Proc. natn. Acad. Sci. USA 94, 2766±2771. 15. Dennis M., Spiegler B. J., Obonsawin M. C., Maria B. L., Cowell C., Hoffman H. J., Hendrick E. B., Humphreys R. P., Bailey J. D. and Elirlich R. M. (1992) Brain tumors in children and adolescents-III. Effects of radiation and hormone status on intelligence and on working, associative and serial-order memory. Neuropsychologia 30, 257±275. 16. Dewey W. C., Ling C. C. and Meyn R. E. (1995) Radiation-induced apoptosis: relevance to radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 33, 781±796. 17. DoÈrr W. (1997) Three A's of repopulation during fractionated irradiation of squamous epithelia: asymmetry loss, acceleration of stem±cell divisions and abortive divisions. Int. J. Radiat. Biol. 72, 635±643. 18. Ellenberg L., McComb J. G., Siegel S. E. and Stowe S. (1987) Factors affecting intellectual outcome in pediatric brain tumor patients. Neurosurgery 21, 638±644. 19. Eriksson P. S., Per®lieva E., Bjork-Eriksson T., Alborn A. M., Nordborg C., Peterson D. A. and Gage F. H. (1998) Neurogenesis in the adult human hippocampus. Nat. Med. 4, 1313±1317. 20. Fike J. R. and Gobbel G. T. (1991) Central nervous system radiation injury in large animal models. In Radiation Injury to the Nervous System (eds Gutin P. H., Leibel S. A. and Sheline G. E.), pp. 113±135. Raven, New York. 21. Gage F. H., Kempermann G., Palmer T. D., Peterson D. A. and Ray J. (1998) Multipotent progenitor cells in the adult dentate gyrus. J. Neurobiol. 36, 249±266. 22. Gage F. H., Ray J. and Fisher W. (1995) Isolation, characterization, and use of stem cells from the CNS. A. Rev. Neurosci. 18, 159±192. 23. Gould E., Beylin A., Tanapat P., Reeves A. and Shors T. L. (1999) Learning enhances adult neurogenesis in the hippocampal formation. Nat. Neurosci. 2, 260±265. 24. Gould E., McEwen B. S., Tanapat P., Galea L. A. and Fuchs E. (1997) Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation. J. Neurosci. 17, 2492±2498. 25. Hayes T. E., Valtz N. L. and McKay R. D. G. (1991) Downregulation of CDC2 upon terminal differentiation of neurons. New Biol. 3, 259±269. 26. Hodges J., Katzung N., Sowinski P., Hopewell J. W., Wilkinson J. H., Bywaters T. and Rezvani M. (1998) Late behavioral and neuropathological effects of local brain irradiation in the rat. Behav. Brain Res. 91, 99±114. 27. Hopewell M. (1979) Late radiation damage to the central nervous system: a radiobiological interpretation. Neuropath. appl. Neurobiol. 5, 329±343. 28. Hopewell J. W. and Cavanagh J. B. (1972) Effects of X irradiation on the mitotic activity of the subependymal plate of rats. Br. J. Radiol. 45, 461±465. 29. Imperato J. P., Paleologos N. A. and Vick N. A. (1990) Effects of treatment on long-term survivors with malignant astrocytomas. Ann. Neurol. 28, 818± 822. 30. Johansson C. B., Mornma S., Clarke D. L., Risling M., Lendahl U. and Frisen J. (1999) Identi®cation of a neural stem cell in the adult mammalian central nervous system. Cell 96, 25±34. 31. Kempermann G., Kuhn H. G. and Gage M. (1997) More hippocampal neurons in adult mice living in an enriched environment. Nature 386, 493±495. 32. Kempermann G., Kuhn H. G. and Gage M. (1998) Experience-induced neurogenesis in the senescent dentate gyrus. J. Neurosci. 18, 3206±3212. 33. Kramer J. H., Crittenden M. R., Halberg F. E., Wara W. M. and Cowan M. J. (1992) A prospective study of cognitive functioning following low-dose cranial radiation for bone marrow transplantation. Pediatrics 90, 447±450. 34. Kuhn H. G., Dickinson-Anson H. and Gage M. (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J. Neurosci. 16, 2027±2033. 35. Lamproglou I., Chen Q. M., Boisserie G., Mazeron J.-J., Poisson M., Baillet F., Le Poncin M. and Delattre J.-V. (1995) Radiation-induced cognitive dysfunction: an experimental model in the old rat. Int. J. Radiat. Oncol. Biol. Phys. 31, 65±70. 36. Lee M. G., Norbury C. J., Spurr N. K. and Nurse P. (1988) Regulated expression and phosphorylation of a possible mammalian cell-cycle control protein. Nature 333, 676±679. 37. Lee P. W., Hung B. K., Woo E. K., Tai P. T. and Choi D. T. (1989) Effects of radiation therapy on neuropsychological functioning in patients with nasopharyngeal carcinoma. J. Neurol. Neurosurg. Psychiat. 52, 488±492. 38. Lewis P. D. (1968) A quantitative study of cell proliferation in the subependymal layer of the adult rat brain. Expl Neurol. 20, 203±207. 39. Li Y., Jay V. and Wong C. S. (1996) Oligodendrocytes in the adult rat spinal cord undergo radiation-induced apoptosis. Cancer Res. 56, 5417±5422. 40. McKay R. (1997) Stem cells in the central nervous system. Science 276, 66±71. 41. Mickley G. A., Ferguson J. L., Mulvihill M. A. and Nemeth T. L. (1989) Progressive behavioral changes during the maturation of rats with early radiation-induced hypoplasia of fascia dentata granule cells. Neurotoxicol. Teratol. 11, 385±393. 42. Moreira R. C. M., Moreira M. V., Bueno J. L. O. and Xavier G. F. (1997) Hippocampal lesions induced by ionizing radiation: a parametric study. J. Neurosci. Meth. 75, 41±47. 43. Morshead C. M., Reynolds B. A., Craig C. G., McBurmey M. W., Staines W. A., Morassutti D., Weiss S. and van der Kooy D. (1994) Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 13, 1071±1082. 44. Okano K. T., Pfaff D. W. and Gibbs R. B. (1993) RB and Cdc2 expression in brain: correlations with 3H-thymidine incorporation and neurogenesis. J. Neurosci. 13, 2930±2938. 45. Parent J. M., Tada E., Fike J. R. and Lowenstein D. H. (1999) Inhibition of dentate granuale cell neurogenesis with brain irradiation does not prevent seizure-induced mossy ®ber synaptic reorganization in the rat. J. Neurosci. 19, 4508±4519. 46. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic, San Diego. 47. Peissner W., Kocher M., Treuer H. and Gillardon F. (1999) Ionizing radiation-induced apoptosis of proliferating stem cells in the dentate gyrus of the adult rat hippocampus. Brain Res. Molec. Brain Res. 71, 61±68. 48. Privat A. and Leblond C. P. (1972) The subependymal layer and neighbouring region in the brain of the young rat. J. comp. Neurol. 146, 277±302. 49. Represa A., Dessi F., Beaudoin M. and Ben-Ari Y. (1991) Effects of neonatal g-irradiation on rat hippocampusÐI. Postnatal maturation of hippocampal cells. Neuroscience 42, 137±150. 50. Reynolds B. A. and Weiss S. (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707±1710. 51. Roman D. D. and Sperduto P. W. (1995) Neuropsychological effects of cranial radiation: current knowledge and future directions. Int. J. Radiat. Oncol. Biol. Phys. 31, 983±998. 52. Schultheiss T. E., Kun L. E., Ang K. K. and Stephens L. C. (1995) Radiation response of the central nervous system. Int. J. Radiat. Oncol. Biol. Phys. 31, 1093±1112. 53. Sheline G. E., Wara W. M. and Smith V. (1980) Therapeutic irradiation and brain injury. Int. J. Radiat. Oncol. Biol. Phys. 6, 1215±1228. 54. Shinohara C., Gobbel G. T., Lambom K. R., Tada E. and Fike J. R. (1997) Apoptosis in the subependyma of young adult rats after single and fractionated doses of X-rays. Cancer Res. 57, 2694±2702. 55. Sienkiewicz Z. J., Haylock R. G. and Saunders R. D. (1994) Prenatal irradiation and spatial memory in mice: investigation of dose-response relationship. Int. J. Radiat. Biol. 65, 611±618. 56. Sienkiewicz Z. J., Haylock R. G. and Saunders R. D. (1999) Differential learning impairments produced by prenatal exposure to ionizing radiation in mice. Int. J. Radiat. Biol. 75, 121±127.
X-rays and subgranular zone cell proliferation
41
57. Sienkiewicz Z. J., Saunders R. D. and Butland B. K. (1992) Prenatal irradiation and spatial memory in mice: investigation of critical period. Int. J. Radiat. Biol. 62, 211±219. 58. Smart I. (1961) The subependymal layer of the mouse brain and its cell production as shown by radioautography after thymidine-H3 injection. J. comp. Neurol. 116, 325±338. 59. Squire L. R. and Zola-Morgan S. (1991) The medial temporal lobe memory system. Science 253, 1380±1386. 60. Stan®eld B. B. and Trice J. E. (1988) Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections. Expl Brain Res. 73, 399±406. 61. Stephens L. C., Hunter N. R., Ang K. K., N®las L. and Meyn R. E. (1993) Development of apoptosis in irradiated murine tumors as a function of time and dose. Radiat. Res. 135, 75±80. 62. Tada E., Yang C., Gobbel G. T., Lamborn K. R. and Fike J. R. (1999) Long term impairment of subependymal repopulation following damage by ionizing irradiation. Expl Neurol. 160, 66±77. 63. van der Kogel A. J. (1991) Central nervous system radiation injury in small animal models. In Radiation Injury to the Nervous System (eds Gutin P. H., Leibel S. A. and Sheline G. E.), pp. 91±111. Raven, New York. 64. Waber D. P., Tarbell N. J., Kahn C. M., Gelber R. D. and Sallan S. E. (1992) The relationship of sex and treatment modality to neuropsychologic outcome in childhood acute lymphoblastic leukemia. J. clin. Oncol. 10, 810±817. 65. Wallace R. B., Graziadei R. and Werboff J. (1981) Behavioral correlates of focal hippocampal X-irradiation in rats H. Behavior related to adaptive function in a natural setting. Expl Brain Res. 43, 207±212. (Accepted 24 March 2000)
Pergamon
PII: S0306-4522(00)00151-2
Neuroscience Vol. 99, No. 1, pp. 33±41, 2000 33 q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
X-IRRADIATION CAUSES A PROLONGED REDUCTION IN CELL PROLIFERATION IN THE DENTATE GYRUS OF ADULT RATS E. TADA,* J. M. PARENT,² D. H. LOWENSTEIN²³§ and J. R. FIKE*k¶ Departments of *Neurosurgery, ²Neurology, ³Anatomy, k Radiation Oncology, and §Program in Neuroscience, University of California, San Francisco, CA 94143, USA
AbstractÐThe effects of X-irradiation on proliferating cells in the dentate subgranular zone were assessed in young adult Fisher 344 rats exposed to a range of X-ray doses and followed for up to 120 days. Apoptosis was quanti®ed using morphology and endlabeling immunohistochemistry, and cell proliferation was detected using antibodies against the thymidine analog BrdU and the cyclin-dependent kinase p34 cdc2. Radiation-induced apoptosis occurred rapidly, with maximum morphological and end-labeling changes observed 3±6 h after irradiation. Twenty-four hours after irradiation cell proliferation was signi®cantly reduced relative to sham-irradiated controls. The number of apoptotic nuclei increased rapidly with radiation dose, reaching a plateau at about 3 Gy. The maximum number of apoptotic nuclei was substantially higher than the number of proliferating cells, suggesting that nonproliferating as well as proliferating cells in the subgranular zone were sensitive to irradiation. Subgranular zone cell proliferation was signi®cantly reduced relative to age-matched controls 120 days after doses of 5 Gy or higher. These ®ndings suggest that neural precursor cells of the dentate gyrus are very sensitive to irradiation and are not capable of repopulating the subgranular zone at least up to 120 days after irradiation. This may help explain, in part, how ionizing irradiation induces cognitive impairments in animals and humans. q 2000 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: hippocampus, rat, irradiation, apoptosis, precursor cell, proliferation.
The brain is exposed to irradiation under a variety of clinical situations, and radiation injury to normal brain tissue is a dose-limiting factor. Radiation brain injury has many effects, and a number of reviews has documented its clinical and morphological character. 6,10,20,27,52,53,63 Changes involving signi®cant tissue destruction generally occur only after aggressive treatment protocols using relatively high doses of irradiation. However, less severe morphological injury can also occur after radiotherapy, which may lead to cognitive dysfunction, particularly in children. 1,7,15,18,33,64 Much of the available clinical literature suggests that the cognitive effects of ionizing irradiation re¯ect impaired attention and memory more than low intellectual performance. 13,29,51 Based on such data it has been suggested that the hippocampus, a critical component of the medial temporal lobe memory system, 59 may be involved in the cognitive changes observed after irradiation. Indirect evidence of the role of hippocampal damage in this cognitive dysfunction comes from clinical studies of head and neck cancer in which patients received high radiation doses to the inferomedial portion of the temporal lobes. Those patients reported subjective memory impairment and, relative to non-irradiated controls, showed poorer performance on measures of nonverbal recent memory. 37 Within the hippocampus, memory functions are associated with the principal cells of the hippocampal formation, i.e. the pyramidal cells and the granule cells of the dentate gyrus. 11 Studies on the radiation response of the brain in late gestational or neonatal mice have shown that damage to
the granule cell layer of the hippocampus or substantial reductions in dentate granule cell number are associated with cognitive impairments. 14,41,42,57,65 The proliferative potential of neuronal precursor cells in the adult mammalian brain was recognized many years ago, 2,3,38,48,58 but only recently has there been a concerted effort to identify and understand the biology and function of those cells. 21,22,30,40,50 In the forebrain of adult rodents, stem/ precursor cells have been identi®ed in two major sites, the subependyma and the subgranular zone of the dentate gyrus (SGZ). In the SGZ of rodents, new neurons are continuously generated, 4,9,23,24,31 migrate into the granule cell layer, develop granule cell morphology and neuronal markers and connect with their target area, CA3. 60 Recent data also show that neurogenesis occurs in the adult human hippocampus beyond the age of 70. 19 Although many new neurons are produced in the rodent SGZ, their signi®cance has not been fully elucidated. 34 However, recently published data suggest a direct association between hippocampus-dependent learning and survival of neurons generated in the mature hippocampal formation. 23 Proliferating cells in the subependyma of rats are particularly sensitive to ionizing irradiation, undergoing apoptosis after doses as low as 1 Gy. 5,54 Furthermore, stem cells in that region are depleted in a dose-dependent fashion, and cell proliferation and total cellularity of the subependyma remain depressed for a prolonged period of time. 62 We have also shown that neural precursor cells of the SGZ are sensitive to irradiation, undergoing apoptosis after low doses of X-rays, 45 A recent report by Peibner et a1. 47 also showed that immature progenitor cells in the rat hippocampus undergo apoptosis after a single 10 Gy dose of X-rays. Because of the potential involvement of the hippocampus in radiation-induced cognitive changes, we were interested in determining the acute radiation response of proliferating SGZ cells after a range of X-ray doses, and to
¶To whom correspondence should be addressed. Tel.: 11-415-476-4453; fax: 11-415-502-0613. E-mail address: j®[email protected] (J. R. Fike). Abbreviations: BrdU, 5-bromo-2 0 -deoxyuridine; GFAP, glial ®brillary acidic protein; SGZ, subgranular zone of the dentate gyrus; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. 33
34 E. Tada et al. Fig. 1. Photomicrograph of the dentate gyrus of a rat that was irradiated 6 h earlier with 15 Gy. Apoptotic nuclei appeared as brown TUNEL-positive nuclei (red arrow) and TUNEL-negative nuclei (blue) that were fragmented (arrowhead) or pyknotic (black arrow). Clusters and inidividual apoptotic nuclei were observed all along the superior and inferior blades of the dentate gyrus. dgc: dentate granule cells; h: hilus. Scale bar 40 mm.
35
X-rays and subgranular zone cell proliferation
determine any subsequent regeneration of that proliferating population. EXPERIMENTAL PROCEDURES
Young adult (eight to 10 weeks) male Fisher 344 rats, weighing approximately 200 g, were used; many of these rats were used in previously published studies of the radiation response of the subependymal region. 54,62 Rats were housed and cared for in accordance with the United States Department of Health and Human Services Guide for the Care and Use of Labotatory Animals, and institutional guidelines for care and handling of laboratory animals. Rats were anesthetized for irradiation and perfusion procedures. For studies of apoptosis, anesthesia consisted of an intraperitoneal (i.p.) injection of sodium pentobarbital (60 mg/kg). In studies of the longer term effects of irradiation (i.e. seven to 120 days), anesthesia consisted of an i.p. injection of ketamine hydrochloride (80 mg/kg) and xylazine (5 mg/kg) dissolved in normal saline. Proliferating cells were labeled using an antibody against the thymidine analog 5-bromo-2 0 -deoxyuridine (BrdU; Sigma, St Louis, MO). In all radiation studies a single i.p. injection of BrdU (60 mg/kg) was given 1 h before rats were killed. To label all proliferating cells in the SGZ of non-irradiated rats, groups of four animals were injected with BrdU every 2 h for 24 h starting between 8.00 and 9.00 a.m., and brain tissue was collected at 2 h intervals, 30 min after a ®nal BrdU injection. The total number of proliferating cells was also estimated using the cyclin-dependent kinase p34 cdc34 (Santa Cruz Biotechnology, Santa Cruz, NM). 62 Irradiations were done using a Phillips orthovoltage X-ray system as previously described. 54 In brief, each rat was irradiated individually with its head centered in a 5 £ 6 cm treatment ®eld and the eyes and body were shielded with lead. Dosimetry was done using a Keithly electrometer ionization chamber calibrated using lithium ¯uoride thermal luminescent dosimeters. The corrected dose rate was approximately 175 cGy/min at a source to skin distance of 21 cm. The time course for radiation-induced apoptosis was determined by irradiating groups of three to six rats with a single dose of 15 Gy and collecting tissues at various times up to 48 h later. Four rats were killed 6 h following sham irradiation. For tissue collection, rats were re-anesthetized, and 350 ml of a 10% buffered formalin solution was infused into the ascending aorta using a mechanical pump (Master¯ex Model 7014; Cole Parmer, Chicago, IL). After 5 min, rats were decapitated and the brain was removed and immersed in a 10% buffered formalin solution for seven days. The radiation dose response for apoptosis in the SGZ was determined 6 h after irradiation. Whole brain doses of 0, 1, 3, 5, 15 and 30 Gy were given to groups of four to six rats that were killed 6 h later. The perfusion and tissue preparation techniques were performed as described above. To study the longer term effects of radiation on proliferating cells within the SGZ, groups of four rats were given doses of 0, 2, 5, 10 and 15 Gy. At one, seven, 14, 30, 60, and 120 days posttreatment, rats were given a single i.p. injection of BrdU and 1 h later were anesthetized and perfused with formalin as described. After one week of ®xation, brains were placed in a Rat Brain Matrix (Large Adult Rat, Type C, Harvard Apparatus, Natick, MA) which facilitated reproducible transverse sectioning of whole brain. A single 5-mm-thick section containing the hippocampus was placed in a plastic tissue holder and processed for paraf®n embedding. Five-micrometerthick coronal sections were taken from three different brain levels and placed on polylysine-coated glass microscope slides. The brain levels were approximately 125±150 mm apart; the most rostral corresponded to a point 3.6 mm behind the bregma, i.e. plate 32 as de®ned in the atlas of rat brain anatomy. 46 The detection of cells undergoing apoptosis was done using nuclear morphology and immunohistochemical staining. Morphological changes included nuclear fragmentation, which was de®ned as the compaction of chromatin into two or more dense, lobulated masses, and pyknosis, which was de®ned as small, round, darkly stained nuclei (Fig. 1). To avoid including any normal cell pro®les (i.e. glia), if cytoplasm was observed in conjunction with a small, dense nucleus, that cell was not counted as apoptotic. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells appeared as highly stained, brown nuclei against the blue background of the counterstain (Fig. 1). For the TUNEL procedure, all reagents, including buffers, were part of a kit (ApopTag, Oncor,
Gaithersburg, MD). Procedures were carried out as previously described. 54,62 Gill's hematoxylin was used as a counterstain. Immunohistochemical labeling of proliferating cells was done using monoclonal antibodies against BrdU (anti-human BrdU antibody, IU-4, Caltag, San Francisco, CA) and p34 cdc2 (Santa Cruz Biotechnology, Santa Cruz, CA). Binding of biotinylated secondary antibody was detected using an avidin±biotinylated peroxidase complex system (ABC; Vector, Burlingame, CA). To quench endogenous peroxidase activity, deparaf®nized specimens were soaked for 20 min in 0.3% H2O2 (Sigma, St Louis, MO) in double deionized distilled water containing 0.1 M sodium azide (Sigma, St Louis, MO). After the primary and the secondary antibodies were applied, the specimens were incubated with the ABC reagent for 30 min and developed with 0.025% 3,3 0 -diaminobenzidine (DAB, Sigma, St Louis, MO) dissolved in phosphate-buffered saline (PBS) containing 0.005% H2O2. Sections were then counterstained with Gill's hematoxylin (Sigma, St Louis, MO), deydrated and mounted. Due to the length of the longer term radiation studies, tissues from different post-treatment times were not necessarily processed together. To insure that immunohistochemical results were comparable among treatment groups, as a positive control we always included tissues from unirradiated young adult rats (12 weeks of age) that had received a pulse injection of BrdU. Furthermore, to insure that factors such as microwave antigen retrieval (see below) did not affect results, for each follow-up time, tissues from all radiation dose groups and positive controls were processed simultaneously. 5-Bromo-2 0 -deoxyuridine After deparaf®nization and quenching of endogenous peroxidase, tissue sections were treated for 5 min at 378C with 20 mg/ml proteinase K (Sigma, St Louis, MO) in PBS. Sections were then soaked in 5% acetic acid for 1 h and then for 10 min in 4 N HCl. Sections were treated for 20 min with 5% normal horse serum in PBS, and after excess horse serum was removed, incubated for 1 h with primary antibody diluted 1:1000 in PBS containing 5% normal horse serum. Sections were washed with PBS and incubated for 30 min with biotinylated horse anti-mouse secondary antibody diluted 1:200 in PBS. All sections were then incubated for 30 min with ABC reagent, developed with DAB and counterstained as described. p34 cdc2 After deparaf®nization and quenching the endogenous peroxidase as described above, sections were soaked in 0.01 M citrate buffer (pH 6.0), and microwaved for 10 min under full power in a 15 kW microwave oven to enhance antigen binding. Sections were left in the buffer for an additional 15 min, washed with PBS, then incubated with 5% normal horse serum for 15 min. After the horse serum was removed, the sections were incubated for 30 min at room temperature with mouse anti-p34 cdc2 antibody diluted 1:500 with PBS. After washing, the sections were incubated for 30 min with biotin-conjugated ratabsorbed horse anti-mouse IgG diluted 1:200 with PBS. Finally, the specimens were incubated with ABC reagent, developed with DAB and counterstained as described. Glial ®brillary labeling
acidic
protein-5-bromo-2 0 -deoxyuridine
double
After deparaf®nization and quenching of endogenous peroxidase, rehydrated sections were incubated in 5% normal goat serum in PBS for 10 min, and then incubated for 30 min in anti-rabbit glial ®brillary acidic protein (GFAP) antibody (Dako Corp., Carpinteria, CA) diluted 1:500 with PBS. Sections were then incubated for 30 min with biotinconjugated goat anti-rabbit IgG diluted 1:200 with PBS, followed by treatment for 30 min with ABC±horseradish peroxidase. Sections were washed in PBS and then treated as described above for BrdU labeling, except that incubation with the primary antibody was overnight at 48C. Blocking sera and secondary antibody counterstaining were as described. Sections were then incubated with ABC±alkaline phosphatase reagent for 30 min followed by an additional 15 min with Vector Blue Alkaline Phosphatase Substrate Kit III (Vector, Burlingame, CA). The number of proliferating cells or cells showing speci®c characteristics of apoptosis was scored blind using a histomorphometric approach. Three 5-mm-thick brain sections were analysed for each rat, and quanti®cation was made of all positively labeled cells within the SGZ of the suprapyramidal and infrapyramidal blades of the
36
E. Tada et al.
Fig. 2. (A) Photomicrograph of the dentate gyrus of a non-irradiated rat after cumulative labeling with BrdU. Brown BrdU-positive cells (arrows) were observed in the subgranular zone, often in clusters of four or more cells. (B) Photomicrograph of the dentate gyrus of a non-irradiated rat after labeling with an antibody against p34 cdc2. The brown p34 cdc2-positive cells represent proliferating cells. dgc: dentate granule cells; h: hilus. Scale bars 25 mm.
dentate gyrus. Because of the high numbers of apoptotic cells after irradiation (see below), the total numbers of pyknotic cells, cells exhibiting TUNEL-positive nuclei or nuclei showing a distinct pattern of nuclear fragmentation were determined in one dentate gyrus on each tissue section. The total number of apoptotic cells was determined by summing the values from all three tissue sections from a given brain. For all studies involving BrdU and p34 cdc2 labeling, the total number of positively labeled cells in both dentate gyri was determined using the three tissue sections and the numbers were summed for a single value for each animal; there were no systematic differences between right and left dentate gyrus in terms of total numbers of labeled cells (not shown). For all endpoints used in this study, values from three to six rats were averaged and standard errors of the mean (S.E.M.) were calculated. Differences in cell number as a function of dose relative to sham-irradiated controls were analysed using Spearman's correlation coef®cient, and numbers of proliferating cells in sham-irradiated rats at 0 and 120 days were compared using an unpaired t-test. RESULTS
Proliferating SGZ cells were found along the suprapyramidal and infrapyramidal blades of the dentate gyrus, and only an occasional BrdU-positive cell was observed in the hilus. After a pulse injection of BrdU, positively labeled cells generally occurred singly or in clusters of two or three
cells; a total of 23.5 ^ 2.63 (mean ^ S.E.M., n 4) SGZ cells was labeled in the three 5-mm-thick tissue sections. In rats that received multiple injections of BrdU, the numbers of BrdU-positive cells increased continuously over the 24 h labeling period (not shown). After cumulative labeling with BrdU, in addition to positively labeled single cells and small clusters, there were groups of BrdU-positive cells comprised of ®ve or more cells (Fig. 2A). Qualitatively, p34 cdc2-positive cells were distributed similarly to cells showing BrdU immunoreactivity (Fig. 2B). For the three tissue sections assessed, the total number of p34 cdc2-positive cells was 78.5 ^ 6.6 (n 4). Between 3 and 12 h after irradiation, the qualitative distributions of TUNEL-positive cells and cells showing morphological characteristics of apoptosis were similar, with cells appearing in the SGZ along the suprapyramidal and infrapyramidal blades of the dentate gyrus. Apoptotic cells appeared singly or in groups (Fig. 1), some of which were made up of ®ve or more cells. Isolated apoptotic nuclei were occasionally seen in the hilar region. In sham-irradiated rats, the total number of apoptotic nuclei detected averaged 3.0 ^ 1.7 (n 4). After 15 Gy, the numbers
37
X-rays and subgranular zone cell proliferation
Fig. 3. Time-course for apoptotic changes in the dentate subgranular zone of young adult rats after a single dose of 15 Gy. Numbers of cells characterized by three distinct criteria of apoptosis, TUNEL immunoreactivity, nuclear fragmentation and pyknosis were determined 3±48 h after irradiation. Maximum apoptosis (TUNEL, fragmented and pyknotic nuclei combined) was seen 6 h after irradiation. Three tissues sections were obtained 125±150 mm apart; the numbers of apoptotic cells within one dentate subgranular zone from each section were determined and the three values were summed for each animal. Each datum point represents four to six rats and error bars are S.E.M.
of pyknotic, fragmented and TUNEL-positive cells increased sharply with time, reaching maximum values 3 h after irradiation for fragmentation and pyknosis endpoints and 6 h after irradiation for the TUNEL endpoint (Fig. 3). The maximum number of apoptotic cells (fragmentation, pyknosis and TUNEL-positive cells combined) was 149 ^ 13.2 (n 6), and was seen 6 h after irradiation. There was a decrease in the number of apoptotic cells by 12 h and from 24 to 48 h, the total numbers of apoptotic cells were similar to those observed in sham-irradiated rats. To determine the dose±response characteristics for SGZ cell apoptosis, the numbers of pyknotic, fragmented and TUNEL-positive cells were measured 6 h after irradiation (Fig. 4). There was a steep increase in apoptosis between 0 and 3 Gy, but there were no statistically signi®cant changes in the number of apoptotic nuclei after higher radiation doses. A statistically signi®cant decrease (P , 0.001) in BrdU labeling was seen 24 h after radiation doses ranging from 2 to 15 Gy (Fig. 5) due to apoptosis of dividing cells. Seven days after irradiation there were subsequent increases in the numbers of BrdU-positive cells after all doses, and at that time, the extent of BrdU labeling was radiation dose dependent (Fig. 6). Double labeling with GFAP and BrdU was done to determine whether the acute increase in BrdU labeling seven days after 2 Gy involved astrocytic cells. Although there were some double-labeled cells in the SGZ, usually in clusters of two to three cells, there were no double-labeled cells in the hilus. Later time points (14±30 days) showed a few doublelabeled cells in the SGZ and in the hilus. After seven days the number of BrdU-positive cells detected in the SGZ of all treatment groups, including the sham-irradiated controls decreased. The average number of BrdU-positive cells observed in sham-irradiated rats at 120 days, 14.9 ^ 9 (n 4), was signi®cantly less (P , 0.01) than that seen at day 0 (23.5 ^ 2.6, n 4). With the exception of 2 Gy, the
Fig. 4. Total number of apoptotic cells in the subgranular zone of young adult rats as a function of radiation dose. Tissue was collected 6 h after irradiation, and for each animal, three tissue sections were obtained 125± 150 mm apart. The numbers of TUNEL-positive, fragmented and pyknotic nuclei within one dentate subgranular zone from each section were determined and the three values were summed for each animal. Each datum point represents three to six rats and error bars are S.E.M.
BrdU labeling was below that obtained for age-matched controls and was maintained or fell continuously from 30 to 120 days (Fig. 6). At 120 days, the extent of BrdU labeling was signi®cantly lower (P , 0.001) in all treatment groups that received greater than 2 Gy. Because a pulse of BrdU only labeled a fraction of the proliferating cell population and the number of BrdU-positive cells was very low, particularly after long follow-up times and higher doses, we used p34 cdc2 immunoreactivity to provide an estimate of the total number of proliferating cells 120 days after irradiation. The total number of p34 cdc2-positive cells in sham-irradiated controls decreased signi®cantly over the experimental period from 78.5 ^ 6.6 (n 4) at day 0 to 23.5 ^ 2.6 (n 4) at 120 days (P , 0.001). After 5, 10 and 15 Gy, the average numbers of p34 cdc2-positive cells in the SGZ were 4.5 ^ 2.3 (n 4), 2.5 ^ 2 (n 4) and 8 ^ 0.9 (n 4), respectively. Those values were signi®cantly lower (P , 0.001) than age-matched controls. DISCUSSION
The main ®ndings of this study are that: (i) X-ray doses of 1±30 Gy induce signi®cant apoptosis in the SGZ of the hippocampus; (ii) proliferative activity within the SGZ is substantially depressed one day after irradiation; (iii) there is a subsequent and transient increase in the number of proliferating cells seven to 14 days post-irradiation; and (iv) after 30 days there is a prolonged and substantial decrease in the proliferating population of the SGZ after doses above 2 Gy. Similar X-ray sensitivity and long-term depression of proliferative activity have been seen in another proliferative zone in the brain, the subependyma, 5,54,62 suggesting a common responsiveness to ionizing irradiation among stem/ precursor cells of the rodent forebrain. Previous animal studies addressing the effects of ionizing irradiation on hippocampal structure and function have generally involved irradiation of prenatal or neonatal
38
E. Tada et al.
Fig. 5. Total number of BrdU-positive cells in the dentate subgranular zone of young adult rats 24 h after irradiation. A single intraperitoneal injection of BrdU (60 mg/kg) was given 1 h prior to tissue collection. Three tissues sections were obtained 125±150 mm apart, and the total numbers of BrdUpositive cells within the dentate subgranular zones from both sides of the brain were summed for each animal. The decreases in the numbers of BrdU-positive cells after all doses, relative to sham-irradiated rats, were statistically signi®cant (P , 0.001). Each bar represents four rats, and error bars are S.E.M.
animals. 41,42,49,55±57,65 Those studies have shown varying degrees of morphological change within the granule cell layer, including a reduction in the number of granule cells, and it has been suggested that various cognitive de®cits were probably mediated by lesions to the hippocampal formation. In older animals, cognitive effects have been shown to be induced by radiation doses that did not lead to the development of necrosis 26 or after treatment that resulted in no apparent histological changes. 35 In neither of the latter studies was cell proliferation or cell death in the SGZ or the granule cell layer of the hippocampal formation assessed directly. Studies of adult rats by our group 45 and others 47 have recently reported radiation-induced apoptosis of the progenitor population of the SGZ after selected doses of X-rays. In the present study, apoptosis was detected 3±12 h after irradiation, and by 24 h there were few if any apoptotic nuclei remaining in the SGZ. This time course is similar to that of radiation-induced death of cells in the adult rat spinal cord 39 and of precursor cells in the forebrain of adult rats. 5,54 Because there can be considerable temporal separation between nuclear morphological changes and DNA fragmentation, it has been suggested that the detection of apoptosis can be improved when in situ assays of DNA fragmentation such as TUNEL are combined with the assessment of morphological features. 12 We therefore quanti®ed total apoptosis at speci®c times after irradiation by summing three distinct criteria: TUNEL reactivity, pyknosis and nuclear fragmentation (Fig. 1). Our data showed that the time-course for immunohistochemical and distinct morphological changes differed slightly (Fig. 3). However, regardless of the speci®c criteria assessed, the overall onset, peak and resolution of all changes were rapid, leading to a signi®cant loss of BrdUpositive cells 24 h after irradiation (Fig. 5). Since apoptosis occurs rapidly and we made our measurements only at speci®ed time intervals, the values we obtained are probably an underestimation of the true extent of acute cell death in the SGZ. Regardless, our data show that the SGZ is particularly
Fig. 6. The number of BrdU-positive cells in the dentate subgranular zone of young adult rats changes with time after irradiation. Three tissue sections were obtained 125±150 mm apart, and the total numbers of BrdU-positive cells within the dentate subgranular zones from both sides of the brain were summed for each animal. Before irradiation, the number of BrdU-positive averaged 23.5 ^ 2.63; in sham-irradiated rats there was a statistically signi®cant reduction at 120 days (P , 0.01). Twenty-four hours after irradiation there were signi®cant reductions in the number of BrdU-positive cells due to apoptosis. The magnitude of the subsequent increase at seven days was radiation dose dependent, but by 120 days there were no apparent differences in the numbers of BrdU-positive cells after doses higher than 2 Gy. At 120 days the numbers of BrdU-positive cells after 5, 10 and 15 Gy were signi®cantly reduced relative to agematched controls (P , 0.001). Each datum point represents four rats and error bars are S.E.M.
sensitive to irradiation, with a steep dose response from 0 to 3 Gy and no additional apoptosis after doses greater than 3 Gy. The plateau in number of apoptotic cells after doses of 3±30 Gy implies the existence of an apoptotic sensitive population(s) in the SGZ of the hippocampus. 16,61 A major component of the population is the proliferating cells, since 90±95% of BrdU-positive cells within the SGZ are lost 24 h after irradiation (Fig. 4). However, non-proliferating cells or cells in a different phase of the cell cycle must also be dying via apoptosis, because the total number of apoptotic nuclei detected in our time course study (Fig. 3) is much greater than the total number of proliferating cells in our designated counting area as determined by p34 cdc2 immunoreactivity. Although the identity of those cells was not speci®cally addressed in this study, a previous investigation by us suggested that X-ray doses of 1±5 Gy depleted non-cycling immature neurons as well as proliferating neural precursor cells. Peibner et al. 47 also reported that cells dying from radiation-induced apoptosis belonged to the progenitor population of the rat hippocampus. We did not see any evidence that fully differentiated neurons underwent apoptosis after doses up to 30 Gy. After 24 h post-irradiation, there was a vigorous but transient increase in the number of BrdU-positive cells (Fig. 6). This was most apparent after lower doses, and seven days after 2 Gy the number of proliferating cells was actually higher than that seen in sham-irradiated rats (Fig. 5). Whether increased proliferation occurs in a subpopulation of precursor
X-rays and subgranular zone cell proliferation
cells that is apoptotic resistant or represents activation of a population of relatively quiescent stem cells similar to what has been observed in the subependyma 43 is not known. Our study did suggest that seven days after 2 Gy some of the proliferating cells in SGZ were astrocytes. Curiously, however, at that time we did not see any double-labeled cells in the hilus, where there are large numbers of GFAPpositive cells. The signi®cance of this ®nding is unclear, and at later time points we saw few double-labeled cells in the SGZ. The small increase in GFAP±BrdU double-labeled cells seen later in the hilus agrees with our previous observation 45 that brain irradiation produces a mild proliferative response of glia in the dentate gyrus. However, studies of the radiation response of the other forebrain site of neurogenesis, the subependyma, suggest that six months after X-irradiation there is a dose-related decrease in astrocytes. Transient increases in proliferating cells have previously been reported after irradiation of brain and other tissues 28 and may re¯ect abortive cell divisions of lethally irradiated cells, changes in cell proliferation rate, or within a stem/precursor cell population, a change from asymmetrical to symmetrical division. 17 We did not address changes in rate of cell proliferation, and we did not see any morphological evidence of abortive cell divisions or increased cell death, e.g., apoptosis (not shown), although increased mitotic-linked cell death may have occurred after seven days. After irradiation, regeneration of certain tissues can occur in part due to a change from asymmetrical to symmetrical division that repopulates the stem cell pool. 17 The apparent lack of reconstitution of the normal number of proliferating SGZ cells up to 120 days following irradiation suggests that such a change in mode of division did not occur. It remains to be determined whether neural progenitors of the SGZ are unable to divide symmetrically or whether they did not receive the appropriate signal to initiate such a change after exposure to irradiation. In our study, the number of proliferating SGZ cells was determined primarily using BrdU immunoreactivity to label cells in the S-phase of the cell cycle. However, a pulse of BrdU only labels a fraction of the total number of proliferating cells, and after higher doses of irradiation (10±15 Gy), values near zero were seen at 120 days. To ascertain a more
39
accurate measure of the total number of proliferating cells remaining after irradiation, we used an antibody against p34 cdc2, an endogenous protein present in proliferating cells 25 but with levels that have been reported to be low in quiescent cells. 36 In the CNS, the expression of p34 cdc2 changes as cells mature, and a signi®cant decrease in p34 cdc2 has been shown to coincide with the time when proliferating precursor cells differentiate into neurons. 25,44 We have previously shown in the rat subependyma that there is excellent agreement between the numbers of cells cumulatively labeled with BrdU and the number of p34 cdc2-positive cells. 62 In our long-term study, p34 cdc2 labeling showed that 120 days after X-irradiation there were statistically signi®cant reductions in the proliferating cell population relative to sham-irradiated controls. This, together with our BrdU data (Fig. 6), clearly suggests a prolonged radiation-induced reduction in neurogenesis in the dentate gyrus. Although the use of proliferation alone to de®ne neurogenesis is less precise in adult tissue than in developing tissue 32 we have previously shown that low-dose X-irradiation reduces TOAD-64 (turned-on-after-division protein, 64 kDa) and polysialylated-neural cell adhesion molecule (PSA±NCAM) labeling, proteins which are expressed speci®cally in immature neurons in the dentate gyrus and are used as measures of neurogenesis. In the present study, the reduction in SGZ cell proliferation was substantial, leaving only small numbers of p34 cdc2-positive cells remaining in the SGZ after 10± 15 Gy. The fate or potential of those surviving cells in terms of maintaining neurogenesis and cellularity within the dentate granule cell layer is not yet known, nor is it known what radiation dose would be required to completely eradicate all neurogenesis in the hippocampus. Because cell proliferation/neurogenesis in the SGZ is affected after radiation doses that do not result in gross tissue changes or necrosis, 8 our data may have signi®cant implications with respect to certain long-term effects of therapeutic irradiation such as cognitive impairment. AcknowledgementsÐThe authors would like to thank Dr Nobuo Tamesa, Ms Cynthia Yang, Ms Delores Dougherty and Mr King Chiu for technical assistance and Dr Kathleen Lamborn for statistical consultation. This work was supported in part by NIH grants CA 13525, R01 CA76141, NS02006, R01 NS32062, ROI NS35628 and the March of Dimes Birth Defects Foundation.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Abayomi O. K. (1996) Pathogenesis of irradiation-induced cognitive dysfunction. Oncology 35, 659±663. Altman J. (1962) Are neurons formed in the brain of adult mammals? Science 135, 1127±1128. Altman J. (1966) Proliferation and migration of undifferentiated precursor cells in the rat during postnatal gliogenesis. Expl Neurol. 16, 263±278. Bayer S. A. (1982) Changes in the total number of dentate granule cells in juvenile and adult rats: a correlated volumetric and 3H-thymidine autoradiographic study. Expl Brain Res. 46, 315±323. Bellinzona M., Gobbel G. T., Shinohara C. and Fike J. R. (1996) Apoptosis is induced in the subependyma of young adult rats by ionizing irradiation. Neurosci. Lett. 208, 163±166. Burger P. C., Mahaley M. S. J., Dudka L. and Vogel F. S. (1979) The morphologic effects of radiation administered therapeutically for intracranial gliomas. A postmortem study of 25 cases. Cancer 44, 1256±1272. Butler R. W., Hill J. M., Steinherz P. G., Meyers P. A. and Finlay J. L. (1994) Neuropsychologic effects of cranial irradiation, intrathecal methotrexate, and systemic methotrexate in childhood cancer. J. clin. Oncol. 12, 2621±2629. Calvo W., Hopewell J. W., Reinhold H. S. and Yeung T. K. (1988) Time- and dose-related changes in the white matter of the rat brain after single doses of X rays. Br. J. Radiol. 61, 1043±1052. Carneron H. A., Woolley C. S., McEwen B. S. and Gould E. (1993) Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56, 337±344. Caveness A. T. (1977) Pathology of radiation damage to the normal brain of the monkey. NCI Monogr. 46, 57±76. Collier T. J., Quirk G. J. and Routtenberg A. (1987) Separable roles of hippocampal granule cells in forgetting and pyramidal cells in remembering spatial information. Brain Res. 409, 316±328. Collins J. A., Schandl C. A., Young K. K., Vesely J. and Willingham M. C. (1997) Major DNA fragmentation is a late event in apoptosis. J. Histochem. Cytochem. 45, 923±934.
40
E. Tada et al.
13. Crossen J. R., Garwood D., Glatstein E. and Neuwelt E. A. (1994) Neurobehavioral sequelae of cranial irradiation in adults: a review of radiation-induced encephalopathy. J. clin. Oncol. 12, 627±642. 14. Czurko A., Czeh B., Seress L., Nadel L. and Bures J. (1997) Severe spatial navigation de®cit in the Morris water maze after single high dose of neonatal X-ray irradiation in the rat. Proc. natn. Acad. Sci. USA 94, 2766±2771. 15. Dennis M., Spiegler B. J., Obonsawin M. C., Maria B. L., Cowell C., Hoffman H. J., Hendrick E. B., Humphreys R. P., Bailey J. D. and Elirlich R. M. (1992) Brain tumors in children and adolescents-III. Effects of radiation and hormone status on intelligence and on working, associative and serial-order memory. Neuropsychologia 30, 257±275. 16. Dewey W. C., Ling C. C. and Meyn R. E. (1995) Radiation-induced apoptosis: relevance to radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 33, 781±796. 17. DoÈrr W. (1997) Three A's of repopulation during fractionated irradiation of squamous epithelia: asymmetry loss, acceleration of stem±cell divisions and abortive divisions. Int. J. Radiat. Biol. 72, 635±643. 18. Ellenberg L., McComb J. G., Siegel S. E. and Stowe S. (1987) Factors affecting intellectual outcome in pediatric brain tumor patients. Neurosurgery 21, 638±644. 19. Eriksson P. S., Per®lieva E., Bjork-Eriksson T., Alborn A. M., Nordborg C., Peterson D. A. and Gage F. H. (1998) Neurogenesis in the adult human hippocampus. Nat. Med. 4, 1313±1317. 20. Fike J. R. and Gobbel G. T. (1991) Central nervous system radiation injury in large animal models. In Radiation Injury to the Nervous System (eds Gutin P. H., Leibel S. A. and Sheline G. E.), pp. 113±135. Raven, New York. 21. Gage F. H., Kempermann G., Palmer T. D., Peterson D. A. and Ray J. (1998) Multipotent progenitor cells in the adult dentate gyrus. J. Neurobiol. 36, 249±266. 22. Gage F. H., Ray J. and Fisher W. (1995) Isolation, characterization, and use of stem cells from the CNS. A. Rev. Neurosci. 18, 159±192. 23. Gould E., Beylin A., Tanapat P., Reeves A. and Shors T. L. (1999) Learning enhances adult neurogenesis in the hippocampal formation. Nat. Neurosci. 2, 260±265. 24. Gould E., McEwen B. S., Tanapat P., Galea L. A. and Fuchs E. (1997) Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation. J. Neurosci. 17, 2492±2498. 25. Hayes T. E., Valtz N. L. and McKay R. D. G. (1991) Downregulation of CDC2 upon terminal differentiation of neurons. New Biol. 3, 259±269. 26. Hodges J., Katzung N., Sowinski P., Hopewell J. W., Wilkinson J. H., Bywaters T. and Rezvani M. (1998) Late behavioral and neuropathological effects of local brain irradiation in the rat. Behav. Brain Res. 91, 99±114. 27. Hopewell M. (1979) Late radiation damage to the central nervous system: a radiobiological interpretation. Neuropath. appl. Neurobiol. 5, 329±343. 28. Hopewell J. W. and Cavanagh J. B. (1972) Effects of X irradiation on the mitotic activity of the subependymal plate of rats. Br. J. Radiol. 45, 461±465. 29. Imperato J. P., Paleologos N. A. and Vick N. A. (1990) Effects of treatment on long-term survivors with malignant astrocytomas. Ann. Neurol. 28, 818± 822. 30. Johansson C. B., Mornma S., Clarke D. L., Risling M., Lendahl U. and Frisen J. (1999) Identi®cation of a neural stem cell in the adult mammalian central nervous system. Cell 96, 25±34. 31. Kempermann G., Kuhn H. G. and Gage M. (1997) More hippocampal neurons in adult mice living in an enriched environment. Nature 386, 493±495. 32. Kempermann G., Kuhn H. G. and Gage M. (1998) Experience-induced neurogenesis in the senescent dentate gyrus. J. Neurosci. 18, 3206±3212. 33. Kramer J. H., Crittenden M. R., Halberg F. E., Wara W. M. and Cowan M. J. (1992) A prospective study of cognitive functioning following low-dose cranial radiation for bone marrow transplantation. Pediatrics 90, 447±450. 34. Kuhn H. G., Dickinson-Anson H. and Gage M. (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J. Neurosci. 16, 2027±2033. 35. Lamproglou I., Chen Q. M., Boisserie G., Mazeron J.-J., Poisson M., Baillet F., Le Poncin M. and Delattre J.-V. (1995) Radiation-induced cognitive dysfunction: an experimental model in the old rat. Int. J. Radiat. Oncol. Biol. Phys. 31, 65±70. 36. Lee M. G., Norbury C. J., Spurr N. K. and Nurse P. (1988) Regulated expression and phosphorylation of a possible mammalian cell-cycle control protein. Nature 333, 676±679. 37. Lee P. W., Hung B. K., Woo E. K., Tai P. T. and Choi D. T. (1989) Effects of radiation therapy on neuropsychological functioning in patients with nasopharyngeal carcinoma. J. Neurol. Neurosurg. Psychiat. 52, 488±492. 38. Lewis P. D. (1968) A quantitative study of cell proliferation in the subependymal layer of the adult rat brain. Expl Neurol. 20, 203±207. 39. Li Y., Jay V. and Wong C. S. (1996) Oligodendrocytes in the adult rat spinal cord undergo radiation-induced apoptosis. Cancer Res. 56, 5417±5422. 40. McKay R. (1997) Stem cells in the central nervous system. Science 276, 66±71. 41. Mickley G. A., Ferguson J. L., Mulvihill M. A. and Nemeth T. L. (1989) Progressive behavioral changes during the maturation of rats with early radiation-induced hypoplasia of fascia dentata granule cells. Neurotoxicol. Teratol. 11, 385±393. 42. Moreira R. C. M., Moreira M. V., Bueno J. L. O. and Xavier G. F. (1997) Hippocampal lesions induced by ionizing radiation: a parametric study. J. Neurosci. Meth. 75, 41±47. 43. Morshead C. M., Reynolds B. A., Craig C. G., McBurmey M. W., Staines W. A., Morassutti D., Weiss S. and van der Kooy D. (1994) Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 13, 1071±1082. 44. Okano K. T., Pfaff D. W. and Gibbs R. B. (1993) RB and Cdc2 expression in brain: correlations with 3H-thymidine incorporation and neurogenesis. J. Neurosci. 13, 2930±2938. 45. Parent J. M., Tada E., Fike J. R. and Lowenstein D. H. (1999) Inhibition of dentate granuale cell neurogenesis with brain irradiation does not prevent seizure-induced mossy ®ber synaptic reorganization in the rat. J. Neurosci. 19, 4508±4519. 46. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates. Academic, San Diego. 47. Peissner W., Kocher M., Treuer H. and Gillardon F. (1999) Ionizing radiation-induced apoptosis of proliferating stem cells in the dentate gyrus of the adult rat hippocampus. Brain Res. Molec. Brain Res. 71, 61±68. 48. Privat A. and Leblond C. P. (1972) The subependymal layer and neighbouring region in the brain of the young rat. J. comp. Neurol. 146, 277±302. 49. Represa A., Dessi F., Beaudoin M. and Ben-Ari Y. (1991) Effects of neonatal g-irradiation on rat hippocampusÐI. Postnatal maturation of hippocampal cells. Neuroscience 42, 137±150. 50. Reynolds B. A. and Weiss S. (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707±1710. 51. Roman D. D. and Sperduto P. W. (1995) Neuropsychological effects of cranial radiation: current knowledge and future directions. Int. J. Radiat. Oncol. Biol. Phys. 31, 983±998. 52. Schultheiss T. E., Kun L. E., Ang K. K. and Stephens L. C. (1995) Radiation response of the central nervous system. Int. J. Radiat. Oncol. Biol. Phys. 31, 1093±1112. 53. Sheline G. E., Wara W. M. and Smith V. (1980) Therapeutic irradiation and brain injury. Int. J. Radiat. Oncol. Biol. Phys. 6, 1215±1228. 54. Shinohara C., Gobbel G. T., Lambom K. R., Tada E. and Fike J. R. (1997) Apoptosis in the subependyma of young adult rats after single and fractionated doses of X-rays. Cancer Res. 57, 2694±2702. 55. Sienkiewicz Z. J., Haylock R. G. and Saunders R. D. (1994) Prenatal irradiation and spatial memory in mice: investigation of dose-response relationship. Int. J. Radiat. Biol. 65, 611±618. 56. Sienkiewicz Z. J., Haylock R. G. and Saunders R. D. (1999) Differential learning impairments produced by prenatal exposure to ionizing radiation in mice. Int. J. Radiat. Biol. 75, 121±127.
X-rays and subgranular zone cell proliferation
41
57. Sienkiewicz Z. J., Saunders R. D. and Butland B. K. (1992) Prenatal irradiation and spatial memory in mice: investigation of critical period. Int. J. Radiat. Biol. 62, 211±219. 58. Smart I. (1961) The subependymal layer of the mouse brain and its cell production as shown by radioautography after thymidine-H3 injection. J. comp. Neurol. 116, 325±338. 59. Squire L. R. and Zola-Morgan S. (1991) The medial temporal lobe memory system. Science 253, 1380±1386. 60. Stan®eld B. B. and Trice J. E. (1988) Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections. Expl Brain Res. 73, 399±406. 61. Stephens L. C., Hunter N. R., Ang K. K., N®las L. and Meyn R. E. (1993) Development of apoptosis in irradiated murine tumors as a function of time and dose. Radiat. Res. 135, 75±80. 62. Tada E., Yang C., Gobbel G. T., Lamborn K. R. and Fike J. R. (1999) Long term impairment of subependymal repopulation following damage by ionizing irradiation. Expl Neurol. 160, 66±77. 63. van der Kogel A. J. (1991) Central nervous system radiation injury in small animal models. In Radiation Injury to the Nervous System (eds Gutin P. H., Leibel S. A. and Sheline G. E.), pp. 91±111. Raven, New York. 64. Waber D. P., Tarbell N. J., Kahn C. M., Gelber R. D. and Sallan S. E. (1992) The relationship of sex and treatment modality to neuropsychologic outcome in childhood acute lymphoblastic leukemia. J. clin. Oncol. 10, 810±817. 65. Wallace R. B., Graziadei R. and Werboff J. (1981) Behavioral correlates of focal hippocampal X-irradiation in rats H. Behavior related to adaptive function in a natural setting. Expl Brain Res. 43, 207±212. (Accepted 24 March 2000)