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Cranial irradiation of young rats impairs later learning and growth
Physiology& Behavior,Vol. 23, pp. 179--184.Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Cranial Irradiation of Young Rats Impairs Later Learning and Growth J. B. O V E R M I E R , *z M. E. C A R R O L L , t
R. P A T T E N *
Departments of Psychology* and Psychiatryt W . K R I V I T $ A N D T. H . K I M §
Departments of Pediatrics~ and Therapeutic Radiology§ University of Minnesota, Minneapolis, M N 55455 R e c e i v e d 2 F e b r u a r y 1979 OVERMIER, J. B., M. E. CARROLL, R. PATTEN, W. KRIVIT AND T. H. KIM. Cranial irradiation of young rats impairs later learning and growth. PHYSIOL. BEHAV. 23(1) 179-184, 1979.--Young rats (26 days) were exposed to ionizing radiation of the head of 0, 1200, 2400, or 3000 rads total in 200 rads/day doses. The subsequent growth of irradiated rats was permanently impaired: such impairment was positively related to amount of irradiation. Beginning in adolescence, rats were trained on a horizontal/vertical visual discrimination in a runway task, and although all four groups mastered the discrimination, they differed in their patterns of acquisition. These results indicate long term effects are associated with a cranial irradiation regimen similar to that given to children suffering acute lymphocytic leukemia (ALL). Radiation
Irradiation
X-ray
Growth
Learning
A D R A M A T I C improvement in the prognosis of acute lymphocytic and myelogenous leukemia (ALL) in children has come about in the past decade. This is a result of a combination of chemotherapeutic and radiotherapeutic efforts, ineluding prophylactic craniospinal radiation, commonly using fractionated doses totalling 2400 rads [19, 24, 33]. (Total dose in rads, D: D=NSDxN°.24×T °.11 where NSD is the nominal standard dose in rats, N is the number of fractions, and T is the elapsed time in days for Cr°+linear accelerator+betatron machines.) Large fractionated doses of cranial irradiation in humans (6000 rads) are clearly pathogenic [30], while the behavioral effects of the typical prophylactic doses (2400 rads) are uncertain [11, 23, 24, 33]. However, it is well known that behavioral and psychological disturbances can result even in the absence of identifiable brain pathology (e.g. [13,21]. There are numerous reports of behavioral deficits in animals resulting from exposures to ionizing radiation using single and fractionated doses to the head and whole body [14, 15, 21]. Overall, there has been a tendency to focus upon acute effects resulting in a paucity of data on the longer term
Leukemia
effects of irradiation on behavior. Several acute studies in which fractionated doses of cranial radiation have been given to neonatal rats have reported behavioral sequelae of alternations in activity and aggression, exaggerated reactions to stressful stimuli, changes in avoidance behavior, and deficits in response alternation [3, 6, 29, 33, 38]. Neurological sequelae observed in these studies included hippocampal lesions with cell losses in the dentate nuclei. However, while these experiments constitute an important context, they are not directly relevant to an experimental model of the therapeutic treatments because "developmental ages" of the animals at irradiation (1-20 days during CNS cellular neurogenesis and myelinization [10]), time of testing (3--10 days later), dosages, and irradiated foci (cerebellum or hippocampus) were not comparable to those of children in the clinical setting. The purpose of the present study was to determine whether radiation to the central nervous system of rats had adverse effects upon later learning and performance when dosimetry and developmental age are comparable to that used in prophylactic treatment of the central nervous system in children suffering acute lymphocytic and myelogenous leukemia. The experiment incorporated in separate groups a range of doses of radiation that spanned the common therapeutic doses. Behavioral assays were of learning,
tThis research was supported by grants to J. B. Overmier by NIMH (MH-13558), to W. Krivit by NCI (CA-21731), and by grants to the Center for Research in Human Learning (Minnesota) by NSF (BNS-03816) and NICHHD (HD-01136). M. E. Carroll was a NIDA postdoctoral fellow (DA-05068) during the conduct of this project. We thank J. Michael Flanigan, Robert D. Koppes and Stephanie J. Reynolds for their technical assistance. 2Requests for reprints should be addressed to: J. Bruce Overmier, Center for Research in Human Learning, 205 Elliott Hall, University of Minnesota, Minneapolis, MN 55455.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.bO031-9384/79/070179-06502.00/O
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exploration, and reaction to a stressful stimulus. Physical analyses focused upon indices of growth and maturation. The use of animals, such as rats, as an experimental model eliminates some of the human motivational confounds with respect to the assessment of behavioral deficits. In addition, the shorter life-span of rats makes possible assessment, within a reasonable time frame, of deficits that might not appear in treated children until puberty or maturity [20, 34, 35, 37! METHOD Animals
Seventeen male albino Wistar rats obtained from Bio-Lab Co., St. Paul, Minnesota, served to completion in this experiment. These rats were 18 days old upon arrival and averaged 40 g in weight. The rats were housed in individual cages in a constantly illuminated room. When the rats were 26 days old, they were randomly assigned to one of four radiation treatment groups. This age was chosen because the rat brain develops to near adult levels in terms of neurogenesis, synaptic junctions, myelinization, and neurochemical systems by 26 days of age [1, 10, 12], yet it antedates "adolescence" (testicular descent occurs about 40 days of age with copulatory capacity beginning about 60 days of age and reproductive maturity at 80--90 days). Until the beginning of the behavioral tests, all rats had unlimited access to food (Purina Laboratory Chow or wet mash made from it). During the behavioral testing, the daily free access to food was reduced to 12 hours per day. Weights were taken weekly throughout the experiment. A schedule of experimental events is given in Fig. 1. Irradiation Apparatus
A 222-kVp 15-mA X-ray machine with added filter of 0.25 mm C u + l mm AI giving H V L of 0.85 mm Cu was used to produce a radiation dose rate of 258 rads/min. Prior to each irradiation, the rats were anesthetized with 60 mg/kg of Diabutal. During irradiation each rat was placed in a Plexiglas and lead shielding chamber. The top of the chamber was covered by a 2 mm thick lead shield which overlapped by 3 cm the sides of the chamber. This shield had a hole centered so as to expose only the dorsal head over the brain region while protecting the eyes, nares, ears, and all parts of the body. The shape and size of the hole ranged from an ovate 1.6× .2.2 mm (first) to a circle 2.3 cm in diameter (last) in five steps to accommodate growth of the rats during the radiation treatment phase. Preliminary measures of dissected neonatal brains were used to determine the size and shape of the hole, and X-rayed photographic plates confirmed that only the brain regions were being irradiated. Irradiation Procedure
There were three irradiation groups of four rats each. The rats were 26 days old and weighed 54--73 g at the onset of the radiation treatments. The groups received 1200, 2400, or 3000 rads cranially in fractionated doses of 200 rads/day on successive days. A control group of five rats received no irradiation (0 rads): they were anesthetized 6 (n=2), 12 ( n = l ) , or 15 (n=2) times to match the irradiated groups. After the last treatment day, all rats were allowed to recover from any acute effects until they were 60 days old, at which
time behavioral testing began. A recovery period is necessary before testing in an appetitive task, because some degree of aphagia and/or adipsia occurs in the days immediately following exposure to radiation [21,261.
Behavioral Testing Discrimination apparatus. The discrimination apparatus consisted of two parallel, side-by-side, runway alleys, each with inside dimensions of 154 cm (length), 13 cm (width), and 10 cm (depth). The only difference between the two alleys of the runway was in the pattern of alternating l-cm wide black and white stripes lining them: one runway was lined by horizontal (lengthwise) stripes and the other by vertical stripes. The runways were lighted equally from beneath through translucent Plexiglas by 40-W fluorescent tubes, each extending the length of the runway. A pattern of black stripes, appropriate for each runway alley was on the Plexiglas which in turn was located approximately 5 cm beneath the white hardware cloth floor of the runway alleys. A single gray-painted startbox served both alleys. The startbox was 28 cm (length) ×13 cm (width) and could be moved into position in front of each alley. A clear Plexiglas guillotine-type sliding door separated the startbox from the runway. Running latencies in each alley were recorded by an electric timer, which was started with the raising of the startbox and stopped when the rat passed a photobeam 122 cm further down the runway. The startbox door was raised 10 sec after the rat was placed in the startbox. Discrimination training. The rats were maintained on unlimited access to food and water in their home cages for 12 hours a day with the access period beginning after the last daily discrimination training trial. The daily discrimination training sessions began at 12 midnight. The rats were not exposed to the discrimination apparatus prior to the initiation of discrimination training. After 2 days of adaptation to handling and exposures to the 0.4 ml of a glucose (3%) plus saccharin (0.125%) solution to be used as the reinforcer, discrimination training began. For two rats in each of the four groups, the vertical bars were associated with the reinforcer liquid (S+) and the horizontal bars were associated with nonreward ( S - ) . The S + and S - conditions were reversed for the other rats in the group. Four discrimination training trials were given daily. At the beginning of each trial the rat was placed in the gray startbox, which had been moved in front either the S+ or S - alley, then after 10 see the startbox door was opened. The four daily trials were arranged according to a fixed sequence that repeated every six days. The four-trial sequence of rewarded (+) or nonrewarded ( - ) trials varied over the six days through all possible permutations of two rewarded and two nonrewarded trials. Each rat was randomly assigned a starting permutation. Each rat was run at approximately the same time each day and in the same order. All rats completed one trial before the next was started: the intertrial interval was between 20 and 30 rain, with the longer values during the early stages of discrimination training. If a rat took longer than 60 sec to traverse the runway on any trial, an arbitrary maximum latency of 60 sec was recorded for that trial but the animal was still allowed to enter the goalbox and receive reward or nonreward before being removed from the apparatus. The discrimination training continued for a total of 45 days and the rats were 105 days old at the end of this phase.
CRANIAL IRRADIATION AND GROWTH
181
Exploration Apparatus A simple version of a Dashiell maze [8] was used. This exploration arena was a square 65 cm on a side. The arena was interrupted by four columns 13 cm square located 13 cm from the walls and from each other. All vertical walls and columns were 25 cm high. The wooden maze was illuminated from 2.4 overhead by diffused light from 40-W fluorescent tubes. A 20-cm speaker was centered 0.9 m above the arena: this speaker was served by a Grason-Stadler (Model 901B) white noise generator.
Exploration Sessions The exploration sessions began on the next day after the last discrimination training day. The rats were 106 days old at the start of the exploration sessions. Each rat received three consecutive daily opportunities to explore the maze/arena with each daily session being 12 min long. The number of 13-cmx 13-cm squares traversed by each rat in each session was recorded on an Esterline-Angus event recorder. During the second exploration session, behavioral disruption of exploration by a startling, aversive stimulus was tested. After the sixth minute of the second exploration session, a 30-sec 100-dB white noise stimulus was presented and the effects of this stimulus upon exploration were observed in the last 6 min of the session. This test has previously been shown to enhance any tendencies of adult rats toward differential exploration arising from differing developmental histories [16,22].
Growth Observations In addition to the regular weekly weighings, which began after the recovery period, other indices of growth and development were obtained. At 175 days of age, the rats were sacrificed and their testicles excised and weighed individually. EXPERIMENT SCHEDULE
Age(days) Irradiation
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Learning Test Exploration Tests Growth Meesures
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FIG. 1. Schedule of events of the experiment in terms of the ages of the rats. RESULTS
Discrimination Learning The individual times required to traverse the alleys were converted to running speeds by reciprocal transformation of running latencies and individual daily mean running speeds to each stimulus (S+ and S - ) were computed as performance indices for each of the 45 days. These were combined into means of 5-day blocks and the data are presented in Fig. 2. These data were subjected to a repeated measures analysis of variance (GroupsxStimulus TypexBlocks×Subjects: 4x2x9xn0.
All groups increased their mean running speeds over successive blocks of training [Blocks F(8,104)=15.52, p<0.01: Blocks x Groups F(24,104)= 1.35, p>0.05], and they learned the discrimination, responding more rapidly to S+ than to S - [Trial Type F(1,13)=36.08, p<0.01]. This differentiation itself developed as a result of the training [Blocks xTrial Type F(8,11M)=26.55, p<0.01]. A most important finding was that the groups differed in their performances [Groups F(3,13)=3.54, p<0.05]. In particular, while the three irradiated groups did not offer among themselves (all ts< 1], the 0-tad control group ran significantly faster than all the irradiated groups taken together [t(13)=3.07, p<0.01]. (All pairwise tests of group differences are two-tailed. This contrast is also significant when restricted to pairwise comparison between the 0-rad control group and the 2400-rad group.) With respect to individual stimuli (S+ or S - ) , again, the irradiated groups did not differ among themselves [all ts
Exploration The number of squares of the Dashiell maze entered (explored) were cumulated for each animal, and daily means of the two 6-min periods were computed. A summary of these data are presented in Fig. 3. All groups showed a similar amount of exploration on Day 1 and the first half of Day 2 (i.e., before the loud noise presentation). The noise presentation was followed by a significant reduction in rate of exploration for each group [Us= 1(5/5), 0(4/4), 0(4/4), 0(4/4), all ps<0.05]. All groups showed recovery of exploratory activity on the third day. In no case did the exploratory activity for the control group differ significantly from that of the combined irradiated groups, although the percentage reduction following the noise stimulus for the control group was less than that of the irradiated groups combined [U = 12(5,12), p <0.05, one-tailed].
Growth Before the series of irradiation treatments began, the randomly assigned groups did not differ with respect to body weights (Studentized Range F=8.36, Qr(4,13)=4.09, n.s. [39]). The group mean weekly weights throughout the experiment are shown in Fig. 4. It is immediately clear that weights varied as a function of the treatment group. The weight data were subject to a Groups x Weeks x Subjects repeated measures analysis of variance which confirmed that the groups differed [Groups F(3,13)--9.63, p<0.01]. All groups showed weight gains over the successive
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O V E R M I E R ET AL
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BLOCKS OF FIVE DAYS FIG. 2. Discriminative learning by the control and irradiated animals in terms of group mean running speeds to the S+ (rewarded) and the S - (unrewarded) stimuli over nine blocks of days. Each point represents 40 trials for the irradiated groups and 50 trials for the control group.
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C R A N I A L IRRADIATION AND GROWTH
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FIG. 4. Growth of the groups as reflected by weekly weights beginning 5 days after the last irradiation treatment day and continuing for 15 weeks (until 150 days of age). The last point taken at the time of testicular excision at 175 days of age is included for information but was not included in the ANOVA. The vertical bars represents ___1 standard error of the mean. weeks [Weeks F(14,182)=245.48, p<0.01] but they differed in their rates of growth [GroupsxWeeks F(42,182)=4.46, p<0.01], Overall, the control group (0 rads) was heavier than the 1200 tad [t(13)=2.54, p<0.05], the 2400 rad [t(13)=3.85, p<0.01], and the 3000 rad [t(13)=5.05, p<0.01] groups. At the end of 5 months, the control group was 129%, 150%, and 173% of the weights of the 1200, 2400, and 3000 rad groups, respectively. The weights of the irradiated groups differed among themselves. The 1200 tad group was heavier than the 3000 tad group (t(13)=2.83, p<0.05), but the 2400 rad group was not significantly different from either the 1200 tad or the 3000 rad groups. These differences emerged slowly over the growth period. The four groups did not differ significantly at 45 days of age (i.e., post-irradiation), but by 59 days of age and thereafter, the control and 3000 rad group differed [at the p<0.01 level]. By 73 days of age and thereafter, the 1200 rad group differed [atp<0.01 level] from the 3000 rad group. By 87 days of age, the control group differed [atp<0.01] from all of the irradiated groups and this persisted until the end of the growth measurement period at 150 days of age. At time of sacrifice, the testicles of each rat were excised and weighed. The mean testicle weights were 1.30 g, 1.71 g, 1.69 g, and 1.79 g for the 3000, 2400, 1200, and 0 rad groups, respectively. The data were subjected to a Groups×Testicle ×Subject repeated measures analysis of variance which indicated that the groups differed significantly [Groups F(3,12)=4.45, p<0.05]. Follow-up comparisons revealed that the 3000 tad group lagged the 2400, 1200, and 0 tad groups in testicular development [all t(12)s>2.60, ps<0.05]. DISCUSSION
Many studies have shown that cranial irradiation to neonatal organisms can have serious behavior development and growth consequences [2, 3, 35, 36, 37], but these studies
have applied the radiation within the first 21 days of life during brain cellular neurogenesis and mitosis. Because the cellular effects of cranial irradiation applied to rats later than 21 days of age are minimal [7, 18, 40], with older rats being labelled "radio-resistant" (Law of Bergonie and Tribondeau), it is unlikely that our animals suffered lasting physical damage to the brain. Indeed, it has been reported that changes in cell characteristics of rats cranially irradiated with 2500 rads at 30 days of age completely dissipated within 14 days of irradiation [5,34]. The present results showed the consequences of irradiation on dimensions other than brain pathology. The reduction in asymptotic body size/weight positively related to radiation dose is the most striking fact demonstrated in the present experiment. It is not likely that the reduction in weight gains seen here is simply the result of the stress of treatments, because even severe stressing with electric shocks [27] during a period of 24--45 days of age only reduced weight gain during the exposure period with a total weight loss of less than 26 g, and this disappeared after cessation of the stress treatment. In contrast, the four groups in the present experiment did not differ in body weights immediately prior to or shortly after the irradiation treatments, but very large differences soon developed and were maintained. The testicular measurements also suggested delayed sexual maturation at the highest treatment dose. This highest treatment dose is within the range of those used for prophylaxis (e.g. [9]), while the 2400 rad dose is becoming more common [19, 23, 24]. It is well known that whole body irradiation in young male children retards body growth [321. What the present study suggests is that prophylactic cranial irradiation of leukemic children may also produce stunting and delayed sexual maturation. Whether these effects are attributable to altered pituitary or thyroid function is a question for future study. In the present experiment we observed an effect of radiation treatments upon discrimination learning. It is important to note that the effect was not upon rate of learning the discrimination or upon asymptotic discrimination performances but upon the way in which mastery of the discrimination was achieved. It is not likely that these differences between irradiated groups and the control group were due to generally slower speeds or lack of activity in the irradiated groups, as no differences were found in locomotor behavior during the exploration tests. The difference while behaviorally significant is a subtle one, and single indices of discrimination learning (e.g., a relative differentiation index) would not have detected the group differences. It is important to note that the pattern of discrimination learning of substantial initial generalization of excitation from S+ to S as shown by the 0 rad group is that reported for normal animals [17,281 and that shown by the irradiated groups could be considered abnormal. This suggests that irradiated animals may differ in the learning strategies used, rather than in the degree of success achieved. If task difficulty interacts with strategy this would account for why some have reported that irradiated rats learn some tasks faster than normals [4] while others have reported that irradiated rats when tested in other tasks learn more poorly [6,33]. The present experiment provides evidence that cranial irradiation of 1200 to 3000 rads in fractionated doses to young rats has significant effects upon learning and growth. These doses of cranial irradiation are in the range of those used prophylactically for children suffering acute lymphocy-
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tic l e u k e m i a . W h i l e c o m b i n a t i o n s of c h e m o t h e r a p y a n d prophylactic cranial irradiation for children with l e u k e m i a are n o w r e c o g n i z e d as p a t h o g e n i c [9, 23, 241, c o n c e r n h a s fo-
c u s e d upon the c h e m o t h e r a p e u t i c a g e n t s [25]. T h e p r e s e n t s t u d y suggests t h a t the radiation t r e a t m e n t itself h a s long t e r m d e l e t e r i o u s c o n s e q u e n c e s for y o u n g p a t i e n t s .
REFERENCES I. Aghajanian, G. K. and F. E. Bloom. The formation of synaptic junctions in developing rat brain: A quantitative electron microscopic study. Brain Res. 6: 716--727, 1%7. 2. Altman, J., W. J. Anderson and M. Strop. Retardation of cerebeUar and motor development of focal X-irradiation during infancy. Physiol. Behav. 7: 143-150, 1971. 3. Bayer, S. A., R. L. Brunner, R. Hine and J. Altman. Behavioral effects of interference with postnatal acquisition of hippocampal granule cells. Nature New Biol. 242: 222-224, 1973. 4. Blair, W. C. The effects of cranial X-irradiation on maze acquisition in rats. J. comp. physiol. Psychol. 51: 175--177, 1958. 5. Brownson, R. H., D. C. Price, M. Z. Jones and G. P. Welch. Studies of normal and radiation-induced cell characteristics in postnatal rat hippocampus. In: Radiation Biology o f the Fetal and Juvenile Mammal, edited M. R. Sikov and D. D. Mahlum. (Hanford Biology Symposium). Oak Ridge: U. S. Atomic Energy Commission, 1%9, pp. 799-821. 6. Brunner, R. L., S. J. Haggbloom and R. H. Gazzara. Effects of hippocampal X-irradiation-produced granuel-cell agenesis on instrumental runway performance in rats. Physiol. Behav. 13: 485-494, 1974. 7. Clemente, C. D., J. N. Yamazaki, L. R. Bennett and R. A. McFall. Brain radiation in newborn rats and differential effects of increased age: II. Microscopic observations. Neurology 10: 66%675, 1960. 8. Dashiell, J. Direction and orientation in maze running by the white rat. Comp. Psychol. Monogr. 7:72 pp., 1930. 9. DeVivo, D. C., D. M. Ross, J. J. Nelson and V. J. Land. Leukoencephalopathy in childhood leukemia. Neurology 27: 609-613, 1977. 10. Eayers, J. T. and B. Goodbead. Postnatal development of the cerebral cortex of the rat. J. Anata. 93: 385-402, 1959. 11. Eiser, C. and R. Landsdown. Retrospective study of intellectual development in children treated for acute lymphoblastic leukemia. Arch. Dis. Child. 52: 525-529, 1977. 12. Fibiger, H. C., L. D. Lytle and B. A. Campbell. Cholinergic modulation of adrenergic arousal in the developing rat. J. comp. physiol. Psychol. 72: 384--389, 1970. 13. Furchtgott, E. Behavioral effects of ionizing radiations. Psychol. Bull. 60: 157-199, 1%3. 14. Haley, T. J. and R. S. Snider. Response o f the Nervous System to Ionizing Radiation: First International Symposium. New York: Academic Press, 1962. 15. Haley, T. J. and R. S. Snider. Response o f the Nervous System to Ionizing Radiation: Second International Symposium. Boston: Little-Brown, 1963. 16. Haywood, H. C. and T. D. Wachs. Effects of arousing stimulation upon novelty preferences in rats. Br. J. Psychol. 58: 77-84, 1967. 17. Henderson, K. Within-subjects partial-reinforcement effects in acquisition and in later discrimination learning. J. exp. Psychol. 72: 704-713, 1%6. 18. Hicks, S. P. Effects of ionizing radiation on the adult and embryonic nervous system. Ass. Res. nerv. ment. Dis. Proc. 32: 43%462, 1953. 19. Hustu, H. O., R. J. A. Aur, M. S. Verrosa, J. V. Simone and D. Pinkel. Prevention of central nervous system leukemia by irradiation. Cancer 32: 585-597, 1973. 20. Kaplan, S. Learning behavior of rats given low level X-irradiation in utero on various gestation days. In: Response o f the Nervous System to Ionizing Radiation, edited by T. J. Haley and R. S. Snider. New York: Academic Press, 1%2, pp. 5%73.
21. Kimeldorf, D. J. and E. L. Hunt. Ionizing Radiation: Neural Function and Behavior. New York: Academic Press, 1965. 22. Levitsky, D. A. and R. H. Barnes. Effect of early malnutrition on the reaction of adult rats to aversive stimuli. Nature 225: 468-469, 1970. 23. Mclntosh, S. and G. T. Aspnes. Encephalopathy following CNS prophylaxis in childhood lymphoblastic leukemia. Pediatrics 52: 612-615, 1973. 24. Mclntosh, S., E. H. Klatskin, R. T. O'Brien, G. T. Aspens, B. L. Krammerer, C. Snead, S. M. Kalavsky and H. A. Pearson. Chronic neurologic disturbance in childhood leukemia. Cancer 37: 853-857, 1976. 25. Meadows, A. T. and A. E. Evans. Effects of chemotherapy on the central nervous system. Cancer 37: 107%i985, 1976. 26. Mitchell, R. B. The antibody response of animals exposed to irradiation. U.S.A.F. School Aviation Medicine Project Reports. Proj. No. 21-47-002, 1950. 27. Mogenson, G. J. and D. J. Ehrlicb. Effects of early gentling and shock on growth and behaviour in rats. Can. J. Psychol. 12: 165-170, 1958. 28. Patten, R. L. and R. M. Latta. Frustration effect in discrimination: Effect of extended training. J. exp. Psychol. 1113:831-836, 1974. 29. Peters, P. J. and R. L. Brunner. Increased running-wheel activity and dyadic behavior of rats with hippocampal granule cell deficits. Behav. Biol. 16: 91-97, 1976. 30. Rottenberg, D. A., N. L. Chernick, M. D. Deck, F. Ellis and J. B. Posner. Cerebral necrosis following radio therapy of extracranial neoplasms. Ann. Neurol. 1: 33%357, 1977. 31. Soni, S. S., G. W. Marten, S. E. Pitner, D. A. Duenas and M. Powazek. Effects of central-nervous-system irradiation on neuropsychologic functioning of children with acute lymphocytic leukemia. N. Engl. J. Med. 293: 113--!18, 1975. 32. Sutow, W. W., R. A. Conrad and K. M. Griffith. Growth status of children exposed to fallout radiation on Marshall Islands. Pediatrics. 36: 721-741, 1965. 33. Tsai, L. S. and F. Chandler. Effects of cranial X-irradiation upon one-trial reversal learning in white rats. Psychol. Rep. 29: 1327-1334, 1971. 34. Van Cleave, C. D. Late Somatic Effects o f Ionizing Radiation. Oak Ridge: U. S. Atomic Energy Commission, 1%8. 35. Wallace, R. B. and J. Altman. Behavioral effects of neonatal irradiation of the cerebellum. If. Quantitative studies in youngadult and adult rats. Devl Psychobiol. 2: 266-272, 1970a. 36. Wallace, R. B. and J. Altman. Behavioral effects of neonatal irradiation of the cerebellum. I. Qualitative observations in infant and adolescent rats. Devl Psychobiol. 2: 257-266, 1970b. 37. Wallace, R. B., C. E. Daniels and J. Altman. Behavioral effects of neonatal irradiation of the cerebellum. Ill. Qualitative observations in aged rats. Devl Psychobiol. 5: 35-41, 1972. 38. Wallace, R. B., R. F. Kaplan and J. Werboff. Behavioral correlates of focal hippocampal X-irradiation in rats. Expl Brain Res. 24: 343-349, 1976. 39. Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1%2. 40. Yamazaki, J. N. A review of the literature on the radiation dosage required to cause manifest central nervous system disturbances from in utero and postnatal exposure. Pediatrics Suppl. 37: 877-903, 1966.
Cranial Irradiation of Young Rats Impairs Later Learning and Growth J. B. O V E R M I E R , *z M. E. C A R R O L L , t
R. P A T T E N *
Departments of Psychology* and Psychiatryt W . K R I V I T $ A N D T. H . K I M §
Departments of Pediatrics~ and Therapeutic Radiology§ University of Minnesota, Minneapolis, M N 55455 R e c e i v e d 2 F e b r u a r y 1979 OVERMIER, J. B., M. E. CARROLL, R. PATTEN, W. KRIVIT AND T. H. KIM. Cranial irradiation of young rats impairs later learning and growth. PHYSIOL. BEHAV. 23(1) 179-184, 1979.--Young rats (26 days) were exposed to ionizing radiation of the head of 0, 1200, 2400, or 3000 rads total in 200 rads/day doses. The subsequent growth of irradiated rats was permanently impaired: such impairment was positively related to amount of irradiation. Beginning in adolescence, rats were trained on a horizontal/vertical visual discrimination in a runway task, and although all four groups mastered the discrimination, they differed in their patterns of acquisition. These results indicate long term effects are associated with a cranial irradiation regimen similar to that given to children suffering acute lymphocytic leukemia (ALL). Radiation
Irradiation
X-ray
Growth
Learning
A D R A M A T I C improvement in the prognosis of acute lymphocytic and myelogenous leukemia (ALL) in children has come about in the past decade. This is a result of a combination of chemotherapeutic and radiotherapeutic efforts, ineluding prophylactic craniospinal radiation, commonly using fractionated doses totalling 2400 rads [19, 24, 33]. (Total dose in rads, D: D=NSDxN°.24×T °.11 where NSD is the nominal standard dose in rats, N is the number of fractions, and T is the elapsed time in days for Cr°+linear accelerator+betatron machines.) Large fractionated doses of cranial irradiation in humans (6000 rads) are clearly pathogenic [30], while the behavioral effects of the typical prophylactic doses (2400 rads) are uncertain [11, 23, 24, 33]. However, it is well known that behavioral and psychological disturbances can result even in the absence of identifiable brain pathology (e.g. [13,21]. There are numerous reports of behavioral deficits in animals resulting from exposures to ionizing radiation using single and fractionated doses to the head and whole body [14, 15, 21]. Overall, there has been a tendency to focus upon acute effects resulting in a paucity of data on the longer term
Leukemia
effects of irradiation on behavior. Several acute studies in which fractionated doses of cranial radiation have been given to neonatal rats have reported behavioral sequelae of alternations in activity and aggression, exaggerated reactions to stressful stimuli, changes in avoidance behavior, and deficits in response alternation [3, 6, 29, 33, 38]. Neurological sequelae observed in these studies included hippocampal lesions with cell losses in the dentate nuclei. However, while these experiments constitute an important context, they are not directly relevant to an experimental model of the therapeutic treatments because "developmental ages" of the animals at irradiation (1-20 days during CNS cellular neurogenesis and myelinization [10]), time of testing (3--10 days later), dosages, and irradiated foci (cerebellum or hippocampus) were not comparable to those of children in the clinical setting. The purpose of the present study was to determine whether radiation to the central nervous system of rats had adverse effects upon later learning and performance when dosimetry and developmental age are comparable to that used in prophylactic treatment of the central nervous system in children suffering acute lymphocytic and myelogenous leukemia. The experiment incorporated in separate groups a range of doses of radiation that spanned the common therapeutic doses. Behavioral assays were of learning,
tThis research was supported by grants to J. B. Overmier by NIMH (MH-13558), to W. Krivit by NCI (CA-21731), and by grants to the Center for Research in Human Learning (Minnesota) by NSF (BNS-03816) and NICHHD (HD-01136). M. E. Carroll was a NIDA postdoctoral fellow (DA-05068) during the conduct of this project. We thank J. Michael Flanigan, Robert D. Koppes and Stephanie J. Reynolds for their technical assistance. 2Requests for reprints should be addressed to: J. Bruce Overmier, Center for Research in Human Learning, 205 Elliott Hall, University of Minnesota, Minneapolis, MN 55455.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.bO031-9384/79/070179-06502.00/O
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exploration, and reaction to a stressful stimulus. Physical analyses focused upon indices of growth and maturation. The use of animals, such as rats, as an experimental model eliminates some of the human motivational confounds with respect to the assessment of behavioral deficits. In addition, the shorter life-span of rats makes possible assessment, within a reasonable time frame, of deficits that might not appear in treated children until puberty or maturity [20, 34, 35, 37! METHOD Animals
Seventeen male albino Wistar rats obtained from Bio-Lab Co., St. Paul, Minnesota, served to completion in this experiment. These rats were 18 days old upon arrival and averaged 40 g in weight. The rats were housed in individual cages in a constantly illuminated room. When the rats were 26 days old, they were randomly assigned to one of four radiation treatment groups. This age was chosen because the rat brain develops to near adult levels in terms of neurogenesis, synaptic junctions, myelinization, and neurochemical systems by 26 days of age [1, 10, 12], yet it antedates "adolescence" (testicular descent occurs about 40 days of age with copulatory capacity beginning about 60 days of age and reproductive maturity at 80--90 days). Until the beginning of the behavioral tests, all rats had unlimited access to food (Purina Laboratory Chow or wet mash made from it). During the behavioral testing, the daily free access to food was reduced to 12 hours per day. Weights were taken weekly throughout the experiment. A schedule of experimental events is given in Fig. 1. Irradiation Apparatus
A 222-kVp 15-mA X-ray machine with added filter of 0.25 mm C u + l mm AI giving H V L of 0.85 mm Cu was used to produce a radiation dose rate of 258 rads/min. Prior to each irradiation, the rats were anesthetized with 60 mg/kg of Diabutal. During irradiation each rat was placed in a Plexiglas and lead shielding chamber. The top of the chamber was covered by a 2 mm thick lead shield which overlapped by 3 cm the sides of the chamber. This shield had a hole centered so as to expose only the dorsal head over the brain region while protecting the eyes, nares, ears, and all parts of the body. The shape and size of the hole ranged from an ovate 1.6× .2.2 mm (first) to a circle 2.3 cm in diameter (last) in five steps to accommodate growth of the rats during the radiation treatment phase. Preliminary measures of dissected neonatal brains were used to determine the size and shape of the hole, and X-rayed photographic plates confirmed that only the brain regions were being irradiated. Irradiation Procedure
There were three irradiation groups of four rats each. The rats were 26 days old and weighed 54--73 g at the onset of the radiation treatments. The groups received 1200, 2400, or 3000 rads cranially in fractionated doses of 200 rads/day on successive days. A control group of five rats received no irradiation (0 rads): they were anesthetized 6 (n=2), 12 ( n = l ) , or 15 (n=2) times to match the irradiated groups. After the last treatment day, all rats were allowed to recover from any acute effects until they were 60 days old, at which
time behavioral testing began. A recovery period is necessary before testing in an appetitive task, because some degree of aphagia and/or adipsia occurs in the days immediately following exposure to radiation [21,261.
Behavioral Testing Discrimination apparatus. The discrimination apparatus consisted of two parallel, side-by-side, runway alleys, each with inside dimensions of 154 cm (length), 13 cm (width), and 10 cm (depth). The only difference between the two alleys of the runway was in the pattern of alternating l-cm wide black and white stripes lining them: one runway was lined by horizontal (lengthwise) stripes and the other by vertical stripes. The runways were lighted equally from beneath through translucent Plexiglas by 40-W fluorescent tubes, each extending the length of the runway. A pattern of black stripes, appropriate for each runway alley was on the Plexiglas which in turn was located approximately 5 cm beneath the white hardware cloth floor of the runway alleys. A single gray-painted startbox served both alleys. The startbox was 28 cm (length) ×13 cm (width) and could be moved into position in front of each alley. A clear Plexiglas guillotine-type sliding door separated the startbox from the runway. Running latencies in each alley were recorded by an electric timer, which was started with the raising of the startbox and stopped when the rat passed a photobeam 122 cm further down the runway. The startbox door was raised 10 sec after the rat was placed in the startbox. Discrimination training. The rats were maintained on unlimited access to food and water in their home cages for 12 hours a day with the access period beginning after the last daily discrimination training trial. The daily discrimination training sessions began at 12 midnight. The rats were not exposed to the discrimination apparatus prior to the initiation of discrimination training. After 2 days of adaptation to handling and exposures to the 0.4 ml of a glucose (3%) plus saccharin (0.125%) solution to be used as the reinforcer, discrimination training began. For two rats in each of the four groups, the vertical bars were associated with the reinforcer liquid (S+) and the horizontal bars were associated with nonreward ( S - ) . The S + and S - conditions were reversed for the other rats in the group. Four discrimination training trials were given daily. At the beginning of each trial the rat was placed in the gray startbox, which had been moved in front either the S+ or S - alley, then after 10 see the startbox door was opened. The four daily trials were arranged according to a fixed sequence that repeated every six days. The four-trial sequence of rewarded (+) or nonrewarded ( - ) trials varied over the six days through all possible permutations of two rewarded and two nonrewarded trials. Each rat was randomly assigned a starting permutation. Each rat was run at approximately the same time each day and in the same order. All rats completed one trial before the next was started: the intertrial interval was between 20 and 30 rain, with the longer values during the early stages of discrimination training. If a rat took longer than 60 sec to traverse the runway on any trial, an arbitrary maximum latency of 60 sec was recorded for that trial but the animal was still allowed to enter the goalbox and receive reward or nonreward before being removed from the apparatus. The discrimination training continued for a total of 45 days and the rats were 105 days old at the end of this phase.
CRANIAL IRRADIATION AND GROWTH
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Exploration Apparatus A simple version of a Dashiell maze [8] was used. This exploration arena was a square 65 cm on a side. The arena was interrupted by four columns 13 cm square located 13 cm from the walls and from each other. All vertical walls and columns were 25 cm high. The wooden maze was illuminated from 2.4 overhead by diffused light from 40-W fluorescent tubes. A 20-cm speaker was centered 0.9 m above the arena: this speaker was served by a Grason-Stadler (Model 901B) white noise generator.
Exploration Sessions The exploration sessions began on the next day after the last discrimination training day. The rats were 106 days old at the start of the exploration sessions. Each rat received three consecutive daily opportunities to explore the maze/arena with each daily session being 12 min long. The number of 13-cmx 13-cm squares traversed by each rat in each session was recorded on an Esterline-Angus event recorder. During the second exploration session, behavioral disruption of exploration by a startling, aversive stimulus was tested. After the sixth minute of the second exploration session, a 30-sec 100-dB white noise stimulus was presented and the effects of this stimulus upon exploration were observed in the last 6 min of the session. This test has previously been shown to enhance any tendencies of adult rats toward differential exploration arising from differing developmental histories [16,22].
Growth Observations In addition to the regular weekly weighings, which began after the recovery period, other indices of growth and development were obtained. At 175 days of age, the rats were sacrificed and their testicles excised and weighed individually. EXPERIMENT SCHEDULE
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Discrimination Learning The individual times required to traverse the alleys were converted to running speeds by reciprocal transformation of running latencies and individual daily mean running speeds to each stimulus (S+ and S - ) were computed as performance indices for each of the 45 days. These were combined into means of 5-day blocks and the data are presented in Fig. 2. These data were subjected to a repeated measures analysis of variance (GroupsxStimulus TypexBlocks×Subjects: 4x2x9xn0.
All groups increased their mean running speeds over successive blocks of training [Blocks F(8,104)=15.52, p<0.01: Blocks x Groups F(24,104)= 1.35, p>0.05], and they learned the discrimination, responding more rapidly to S+ than to S - [Trial Type F(1,13)=36.08, p<0.01]. This differentiation itself developed as a result of the training [Blocks xTrial Type F(8,11M)=26.55, p<0.01]. A most important finding was that the groups differed in their performances [Groups F(3,13)=3.54, p<0.05]. In particular, while the three irradiated groups did not offer among themselves (all ts< 1], the 0-tad control group ran significantly faster than all the irradiated groups taken together [t(13)=3.07, p<0.01]. (All pairwise tests of group differences are two-tailed. This contrast is also significant when restricted to pairwise comparison between the 0-rad control group and the 2400-rad group.) With respect to individual stimuli (S+ or S - ) , again, the irradiated groups did not differ among themselves [all ts
Exploration The number of squares of the Dashiell maze entered (explored) were cumulated for each animal, and daily means of the two 6-min periods were computed. A summary of these data are presented in Fig. 3. All groups showed a similar amount of exploration on Day 1 and the first half of Day 2 (i.e., before the loud noise presentation). The noise presentation was followed by a significant reduction in rate of exploration for each group [Us= 1(5/5), 0(4/4), 0(4/4), 0(4/4), all ps<0.05]. All groups showed recovery of exploratory activity on the third day. In no case did the exploratory activity for the control group differ significantly from that of the combined irradiated groups, although the percentage reduction following the noise stimulus for the control group was less than that of the irradiated groups combined [U = 12(5,12), p <0.05, one-tailed].
Growth Before the series of irradiation treatments began, the randomly assigned groups did not differ with respect to body weights (Studentized Range F=8.36, Qr(4,13)=4.09, n.s. [39]). The group mean weekly weights throughout the experiment are shown in Fig. 4. It is immediately clear that weights varied as a function of the treatment group. The weight data were subject to a Groups x Weeks x Subjects repeated measures analysis of variance which confirmed that the groups differed [Groups F(3,13)--9.63, p<0.01]. All groups showed weight gains over the successive
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O V E R M I E R ET AL
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C R A N I A L IRRADIATION AND GROWTH
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FIG. 4. Growth of the groups as reflected by weekly weights beginning 5 days after the last irradiation treatment day and continuing for 15 weeks (until 150 days of age). The last point taken at the time of testicular excision at 175 days of age is included for information but was not included in the ANOVA. The vertical bars represents ___1 standard error of the mean. weeks [Weeks F(14,182)=245.48, p<0.01] but they differed in their rates of growth [GroupsxWeeks F(42,182)=4.46, p<0.01], Overall, the control group (0 rads) was heavier than the 1200 tad [t(13)=2.54, p<0.05], the 2400 rad [t(13)=3.85, p<0.01], and the 3000 rad [t(13)=5.05, p<0.01] groups. At the end of 5 months, the control group was 129%, 150%, and 173% of the weights of the 1200, 2400, and 3000 rad groups, respectively. The weights of the irradiated groups differed among themselves. The 1200 tad group was heavier than the 3000 tad group (t(13)=2.83, p<0.05), but the 2400 rad group was not significantly different from either the 1200 tad or the 3000 rad groups. These differences emerged slowly over the growth period. The four groups did not differ significantly at 45 days of age (i.e., post-irradiation), but by 59 days of age and thereafter, the control and 3000 rad group differed [at the p<0.01 level]. By 73 days of age and thereafter, the 1200 rad group differed [atp<0.01 level] from the 3000 rad group. By 87 days of age, the control group differed [atp<0.01] from all of the irradiated groups and this persisted until the end of the growth measurement period at 150 days of age. At time of sacrifice, the testicles of each rat were excised and weighed. The mean testicle weights were 1.30 g, 1.71 g, 1.69 g, and 1.79 g for the 3000, 2400, 1200, and 0 rad groups, respectively. The data were subjected to a Groups×Testicle ×Subject repeated measures analysis of variance which indicated that the groups differed significantly [Groups F(3,12)=4.45, p<0.05]. Follow-up comparisons revealed that the 3000 tad group lagged the 2400, 1200, and 0 tad groups in testicular development [all t(12)s>2.60, ps<0.05]. DISCUSSION
Many studies have shown that cranial irradiation to neonatal organisms can have serious behavior development and growth consequences [2, 3, 35, 36, 37], but these studies
have applied the radiation within the first 21 days of life during brain cellular neurogenesis and mitosis. Because the cellular effects of cranial irradiation applied to rats later than 21 days of age are minimal [7, 18, 40], with older rats being labelled "radio-resistant" (Law of Bergonie and Tribondeau), it is unlikely that our animals suffered lasting physical damage to the brain. Indeed, it has been reported that changes in cell characteristics of rats cranially irradiated with 2500 rads at 30 days of age completely dissipated within 14 days of irradiation [5,34]. The present results showed the consequences of irradiation on dimensions other than brain pathology. The reduction in asymptotic body size/weight positively related to radiation dose is the most striking fact demonstrated in the present experiment. It is not likely that the reduction in weight gains seen here is simply the result of the stress of treatments, because even severe stressing with electric shocks [27] during a period of 24--45 days of age only reduced weight gain during the exposure period with a total weight loss of less than 26 g, and this disappeared after cessation of the stress treatment. In contrast, the four groups in the present experiment did not differ in body weights immediately prior to or shortly after the irradiation treatments, but very large differences soon developed and were maintained. The testicular measurements also suggested delayed sexual maturation at the highest treatment dose. This highest treatment dose is within the range of those used for prophylaxis (e.g. [9]), while the 2400 rad dose is becoming more common [19, 23, 24]. It is well known that whole body irradiation in young male children retards body growth [321. What the present study suggests is that prophylactic cranial irradiation of leukemic children may also produce stunting and delayed sexual maturation. Whether these effects are attributable to altered pituitary or thyroid function is a question for future study. In the present experiment we observed an effect of radiation treatments upon discrimination learning. It is important to note that the effect was not upon rate of learning the discrimination or upon asymptotic discrimination performances but upon the way in which mastery of the discrimination was achieved. It is not likely that these differences between irradiated groups and the control group were due to generally slower speeds or lack of activity in the irradiated groups, as no differences were found in locomotor behavior during the exploration tests. The difference while behaviorally significant is a subtle one, and single indices of discrimination learning (e.g., a relative differentiation index) would not have detected the group differences. It is important to note that the pattern of discrimination learning of substantial initial generalization of excitation from S+ to S as shown by the 0 rad group is that reported for normal animals [17,281 and that shown by the irradiated groups could be considered abnormal. This suggests that irradiated animals may differ in the learning strategies used, rather than in the degree of success achieved. If task difficulty interacts with strategy this would account for why some have reported that irradiated rats learn some tasks faster than normals [4] while others have reported that irradiated rats when tested in other tasks learn more poorly [6,33]. The present experiment provides evidence that cranial irradiation of 1200 to 3000 rads in fractionated doses to young rats has significant effects upon learning and growth. These doses of cranial irradiation are in the range of those used prophylactically for children suffering acute lymphocy-
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tic l e u k e m i a . W h i l e c o m b i n a t i o n s of c h e m o t h e r a p y a n d prophylactic cranial irradiation for children with l e u k e m i a are n o w r e c o g n i z e d as p a t h o g e n i c [9, 23, 241, c o n c e r n h a s fo-
c u s e d upon the c h e m o t h e r a p e u t i c a g e n t s [25]. T h e p r e s e n t s t u d y suggests t h a t the radiation t r e a t m e n t itself h a s long t e r m d e l e t e r i o u s c o n s e q u e n c e s for y o u n g p a t i e n t s .
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