radiation , as a monotherapy or as a component of a polytherapeutic approach , is used to treat malignant cancers due to its high degree of efficacy . unfortunately , radiation does not distinguish among the types of cells it destroys , and so healthy tissues in and around the cancerous ones are also compromised . thus , survival carries with it a risk of deleterious side effects as a result of radiation - induced normal brain tissue injury . this presents a unique challenge with regard to the pediatric population , wherein cranial radiotherapy ( crt ) is part of treatment for acute lymphoblastic leukemia ( all ) and solid tumors of the brain , which comprise a significant proportion of malignant cancers . the nonspecific radiation - induced perturbations to the developing brain can have adverse consequences , both acute and long - term , for the brain and body [ 39 ] . clinical imaging studies provide evidence at the functional network level that crt has detectable effects on cerebral integrity [ 1012 ] , connectivity [ 1315 ] , and volume [ 11 , 1621 ] . numerous studies have found changes in metabolites and/or diffusion tensor parameters in the brain after irradiation in rodents . using magnetic resonance spectroscopy changes in n - acetylaspartate ( naa ) , glutamate , choline , lactate , taurine , and myo - inositol have been measured in different brain regions after radiation exposure [ 2224 ] . in addition , diffusion tensor imaging ( dti ) parameters have been measured in a variety of brain regions , such as the fimbria , external capsule , and corpus callosum , after brain irradiation [ 2326 ] . in this study , we employ these imaging measurements to investigate the effect of crt on adolescent rats . crt also disrupts endocrine function by suppressing growth hormone expression [ 2729 ] resulting in growth retardation [ 9 , 30 ] and significant alterations in body composition and weight are often observed in survivors of radiation - treated childhood cancers [ 7 , 31 , 32 ] . although there is a great deal of individual variability within survivors in terms of diagnosis , sex , age at treatment , and type , dose , and regimen of treatment , childhood crt is recognized as being the biggest risk factor for negative neurocognitive and psychosocial outcomes in adulthood , leading to a poorer quality of life overall [ 33 , 34 ] . as such , it is imperative to characterize the scope and precise mechanisms of crt - induced damage in order to determine how best to minimize the cost - benefit ratio of crt and establish effective interventions to mitigate late - onset neurocognitive deficits that are generally irreversible . it is widely accepted that the adverse response of the normal tissue in the brain to single dose irradiation occurs early , while its response to fractionated irradiation is late . differences in the expression levels of inflammatory molecules , between single and fractionated doses , have been reported . however , most of these studies have focused on the early phase of radiation damage and little has been done to elucidate the long - term difference between these two regimens . animal models of pediatric radiotherapy provide the opportunity to examine radiation effects at the cellular , structural , and circuit levels in the developing brain . animal studies of single dose radiation effects on neurogenesis show a dramatic decrease [ 3638 ] , but the effects of fractionated irradiation of the adolescent brain on hippocampal cell genesis have been less studied . in this work , we investigated the effects of single dose and fractionated crt in adolescent rats on body weight , brain weight , ki67 ( a marker of cell proliferation ) , and fractional anisotropy ( a measure of functional integrity of fiber tracts ) in the fimbria fornix as well as metabolite changes in the hippocampus . the hippocampus and its primary efferent projection ( the fimbria fornix ) are critical for learning and memory [ 39 , 40 ] . indeed , it is thought that the cognitive impairments associated with cancer treatment are due in large part to effects on this structure [ 4143 ] . in this paper , we will present data on proliferation , microstructural , and metabolite changes measured in these two structures . animal care was in accordance with guidelines set by the national institutes of health guide for the care and use of laboratory animals ( 2011 ) and all procedures were approved by the institutional animal care and use committee of baylor college of medicine ( houston , tx ) . rats were 24 days old upon arrival and were housed in pairs in a temperature - controlled vivarium under a 12-hour dark / light cycle ( lights off 7:00  am7:00  pm ) with unrestricted access to food and water . body weights were recorded daily during the course of radiation and then weekly and prior to euthanasia for brain harvest . their teeth were trimmed under isoflurane anesthesia once every week until termination of the experiment . the overgrowth of the incisors prevented irradiated animals from being able to consume the standard , hard , food pellets . therefore , all rats , including shams , were given fresh , water - softened food pellets in their home cages , every day , throughout the course of the experiment . this was initiated immediately after the first session of crt to ensure that the drop in body weight during the course of radiation was not due to a crt - induced inability to chew the standard pellets or fatigue / illness that would prevent only the irradiated animals from accessing the pellets on top of their home cages . at 28 days of age , animals were anesthetized using isoflurane and randomly assigned to either the radiotherapy ( crt ) or sham group . animals in the crt group were individually irradiated at a dose rate of 128  cgy / min using a rs 2000 biological x - ray irradiator 150  kvp , 25  ma ( rad source technologies , inc . , suwanee , ga ) . each rat was placed prone and lead shielding was used to ensure that only the region of the head beginning behind the eyes and extending to approximately 5  mm behind the ears received radiation , so that the whole brain ( cerebrum and cerebellum ) , but not the eyes , would be within the field of radiation . approximately half of the crt animals received a single dose of 20  gy ( n = 19 ) and the other half received the same dose divided into 4  gy fractions across 5 consecutive days ( n = 18 ) . animals in the sham group ( n = 18 ) were anesthetized for the same length of time as those in the crt group but did not receive radiation . animals from each group were sacrificed 1  d ( n = 6 per group ) , 5  d ( n = 6 per group ) , and 3.5  mo ( n = 6/7 per group ) following start of crt ( figure 1 ) . animals euthanized at the 3.5  mo after crt interval were first imaged approximately between 3 and 3.5  mo after crt ( figure 1 ) . a 9.4 t biospec mri scanner ( bruker , ma ) with a 20  cm bore and a quadrature rat brain array ( bruker , ma ) a respiratory pillow was placed under the abdomen of the animals and a rectal probe was used to monitor respiration and temperature , respectively . a t2-rapid acquisition with relaxation enhancement ( t2-rare ) scan with the following parameters , te = 20  ms , tr = 2600  ms , # of averages = 1 , 3  cm field of view , 12 slices , 1  mm slice thickness , 1.1  mm interslice distance , and 256  256 matrix size , was then performed and used to localize the placement of the spectroscopy voxel . voxel placement was in the hippocampus ( 2  mm height , 4  mm width , 3  mm anterior to posterior ) . prior to mr spectroscopy a fastmap sequence was performed to correct for local field inhomogeneity by adjusting first- and second - order shim coil currents . all mr spectra acquired using a stimulated echo ( steam ) sequence with the following parameters , tr = 2  s , te = 2.22  ms , # of averages = 512 , and voxel size = 24  mm , were executed . water suppression was achieved using variable pulse power and optimized relaxation delay ( vapor ) . for quantification purposes , metabolites were excluded when they did not pass the criterion of n  4/group and cramr - rao lower bounds < 20% . following spectroscopy , dti was performed ( n = 6 - 7/group ) using a spin echo planar imaging ( epi ) sequence with a tr = 500  ms , te = 33.2  ms , 2 repetitions , slice thickness = 12.8  mm , b value = 800  s / mm , # of directions 30 , and matrix size 128  256  16 giving a spatial resolution of 150  m  150  m  800  m . total imaging time , for spectroscopy and dti , was approximately 1 hour per animal . fractional anisotropy ( fa ) maps were processed through medinria and analyzed using inveon research workstation ( siemens , washington , dc ) . briefly , in slices in which the fimbria fornix was visible , a region of interest ( roi ) was drawn around the fimbria fornix ( left and right sides ) and a fa threshold value of 0.6 and 0.75 applied and mean fa calculated . mean fa values obtained were averaged between left and fight fimbria and between slices for each animal . first , total fimbria volume ( tfv ) was calculated using a 0.5  fa threshold , to isolate the entire fimbria ( figure 4(a ) ) , in each imaging slice in which the fimbria was visible . second , a fa threshold value of 0.6 and 0.75 was applied to the tfv and a resulting thresholded volume ( tv0.6 , tv0.75 ) was calculated ( figure 4(b ) ) . third , the tvx was calculated and averaged between left and fight fimbria and between slices for each animal to arrive at tvx . finally , a ratio of tvx / tfv was calculated ( figure 4(c ) ) . animals were given an overdose of ketamine / xylazine ( i.p . ) and transcardially perfused with 4% paraformaldehyde at 1  d , 5  d , or 3.5  mo after the start of crt ( figure 1 ) . brains were removed , postfixed for 24  h , and stored in 30% sucrose until sectioned . brains ( forebrain and cerebellum only ) were weighed and then cut into 50  m serial , coronal sections on a freezing - stage sliding microtome ( leica microsystems , sm2000r , nussloch , germany ) and stored in cryoprotectant in 96-well microtiter plates at 20c . every 6th section throughout the rostrocaudal extent of the hippocampus was processed by standard immunohistochemical procedure as reported previously   to label proliferating cells . briefly , sections were rinsed in 0.1  m tris - buffered saline ( tbs ) , quenched for 30  min at room temperature in 0.6% hydrogen peroxide , and rinsed in tbs again . next , sections were pretreated in 10  mm sodium citrate buffer ( ph 8.5 ) for 30  min in a 80c water bath , allowed to cool down to room temperature , and rinsed in tbs before incubation in 3% normal donkey serum ( sigma - aldrich , st . louis , mo ) for 1  h. this was followed by incubation at 4c for 72  h in primary antibody ( rabbit , polyclonal anti - ki67 , 1  :  1800 ; vector laboratories , burlingame , ca ) . sections were then rinsed in tbs , blocked in 3% normal donkey serum for 15  min , and incubated overnight at room temperature in secondary antibody ( biotinylated donkey , anti - rabbit , 1  :  250 ; jackson immunoresearch laboratories , west grove , pa ) . next , sections were rinsed in tbs , treated for 90  min in avidin - biotin complex ( abc , vector laboratories ) , and rinsed again . sections were reacted and visualized with diaminobenzidine , rinsed before being mounted onto gelatin - coated slides , and allowed to dry overnight . sections were then counterstained with methyl green , cleared in xylene , and cover - slipped using permount mounting medium ( fisher scientific , pittsburgh , pa ) . ki67 + cells were quantified in the subgranular zone ( sgz ) of the dentate gyrus ( dg ) using a nikon eclipse 80i upright microscope in conjunction with stereoinvestigator ( microbrightfield , williston , vt ) . approximately , 10 - 11 sections per animal were analyzed for ki67 + cells at 40x magnification throughout the septotemporal axis of the hippocampus by an experimenter blind to the conditions . since ki67 staining was sparse , cells were counted nonstereologically , and the number of cells was summed across sections for each animal . ki67 + cell counts in the sgz ( 1  d , 5  d , and 3.5  mo after crt ) , body weights ( recorded just before euthanasia at each interval ) , and normalized brain weights ( 24  h and 3.5  mo after the end of crt ) were analyzed by two - way anovas with crt and post - crt interval as factors . one - sample t - tests were used to compare differences between mean brain weight per group ( at each interval ) and shams ( i.e. , a value of 100 ) . mean fa values , fimbria volume - ratios per applied threshold , and hippocampal metabolite levels were analyzed by one - way anovas . data on body weight , body length , and body weight / length collected from a small , separate cohort of animals ( see figure 6 ) were analyzed by one - way repeated measures anovas . there was a significant main effect of crt [ f(2,46 ) = 59.72 , p < 0.0001 ] , post - crt interval [ f(2,46 ) = 20.13 , p < 0.0001 ] , and interaction [ f(4,46 ) = 17.81 , p < 0.0001 ] on the mean number of ki67 + cells in the sgz of the hippocampus ( figure 2 ) . post hoc analyses revealed that single dose crt as well as fractionated crt significantly reduced the number of ki67 + cells relative to shams at the 1  d and 5  d post - crt intervals . however , due to the substantial drop in ki67 + cells in shams due to age alone [ 1  d versus 5  d : p < 0.0001 , 5  d versus 3.5  mo : p = 0.001 ] , this difference did not reach statistical significance in adulthood . in total , 13 metabolites were successfully measured ( n  4/group and 20% cramr - rao lower bound ) in all experimental groups . however , only two metabolites , myo - inositol and n - acetylaspartate + n - acetylaspartylglutamate ( naa + naag ) , had measureable significant change . there was a significant effect of crt on myo - inositol [ f(2,16 ) = 8.3 , p < 0.01 ] and naa + naag levels [ f(2,15 ) = 8.75 , p = 0.003 ] at 3  mo after crt ( figure 3 ) . post hoc analyses showed that myo - inositol was significantly lower in single dose crt animals compared to shams [ p < 0.01 ] . naa + naag levels were significantly higher in single dose crt animals relative to shams [ p < 0.01 ] and fractionated crt animals [ p < 0.05 ] . at 3  mo after crt , no changes in mean fa were found in the fractionated crt animals . however , single dose crt animals demonstrated a significant reduction compared to shams [ p < 0.05 ] in mean fa values within voxels that were equal to or greater than the set threshold of 0.6  fa ( figure 4(b ) ) . further , post hoc analyses revealed that , relative to shams , only single dose crt animals had a significant reduction in the ratio of the fimbria volume above fa thresholds of 0.6 [ p < 0.01 ] and 0.75 [ p < 0.05 ] ( figure 4(c ) ) . furthermore , the ratio of the volume of the fimbria above fa thresholds of 0.6 and 0.75 in fractionated crt was also reduced , albeit not significantly . there was a significant main effect of crt [ f(2,46 ) = 56.55 , p < 0.001 ] and post - crt interval [ f(2,46 ) = 556.9 , p < 0.001 ] on body weight as well as a significant interaction between crt and post - crt interval [ f(4,46 ) = 39.14 , p < 0.001 ] . post hoc tests revealed that , at 3.5  mo after crt , both fractionated and single dose - treated animals had significantly reduced body weights relative to age - matched shams [ p < 0.0001 ] ( figure 5 ) . in addition , single dose crt suppressed body weight even relative to fractionated crt [ p < 0.001 ] . at 5  d after crt , that is , 24  h after the last fractionated dose of radiation , only the single dose crt group had significantly reduced body weight relative to shams [ p < 0.05 ] . we also measured differences in body length and body weight / length ratios ( figure 6 ) . brain weights of crt groups were normalized to that of age - matched shams and compared at 24  h and 3.5  mo following end of treatment ( figure 7(a ) ) . there was a significant effect of interval on brain weight [ f(1,21 ) = 141.1 , p < 0.0001 ] and a significant interaction between crt and interval [ f(1,21 ) = 108.6 , p < 0.0001 ] . post hoc analyses showed that , the day after end of treatment , single dose crt significantly increased brain weight relative to shams ( 9.2% increase , [ p < 0.01 ] ) and fractionated crt ( 15.7% increase , [ p < 0.0001 ] ) . however , in the fractionated crt group , mean brain weight was significantly lower than sham ( 6.5% reduction , [ p = 0.0001 ] ) . 3.5  mo after end of treatment , single dose crt significantly reduced brain weight relative to shams ( 18.9% reduction , [ p < 0.0001 ] ) and fractionated crt ( 10.6% reduction , [ p < 0.0001 ] ) . the mean brain weight of the fractionated crt group was also significantly reduced relative to sham ( 8.4% reduction , [ p = 0.0001 ] ) . mean brain weight of the single dose crt group was reduced 28.1% at 3.5  mo relative to 24  h [ p < 0.0001 ] . crt - induced suppression of cell proliferation in the sgz was evident the day after the start of radiation and persisted into adulthood ( figure 2 ) . there were no significant differences between single dose and fractionated regimens at any interval , although there were more ki67 + cells at the 1  d post - crt interval in the animals that received a single 4  gy fraction relative to those that received a single 20  gy dose while this pattern was reversed at 5  d. it may be that there was some recovery in the single dose animals 5 days after their last exposure to radiation or that this is simply an artifact of within - group variance . if assessed independently at each interval , the differences between single dose and fractionated crt reach statistical significance only at the 1  d interval which is not surprising as at this point the comparison is between doses of 20  gy and 4  gy . note that while proliferation in neither irradiated group differed significantly across intervals , the number of ki67 + cells in shams dropped significantly as a function of age . our observations concur with those of others reporting a comparable number of ki67 + cells in the sgz of nave rodents of similar age [ 38 , 46 ] and a significant drop in neurogenesis between adolescence and adulthood [ 4749 ] . the decrease in proliferation in shams from 1  d to 5  d may in part be a function of error variance and/or due to repeated isoflurane exposure which is reported to suppress hippocampal neurogenesis in juvenile but not in adult rodents [ 50 , 51 ] . however , studies on isoflurane - induced neurotoxicity typically use long durations of anesthesia , whereas , in our experiment , animals received , on average , 1015  min ( 13% , 0.51 furthermore , as sham animals were matched to irradiated animals for isoflurane exposure , the differences in proliferation between the irradiated groups and age - matched sham groups can not be attributed to isoflurane . our results agree with others [ 38 , 52 , 53 ] , as independent anovas for each post - crt interval reveal a significant main effect of crt even at 3.5  mo after crt . however , when the data are analyzed as a whole , the significance is lost . we chose a factorial analysis to control for familywise error rate , but the fact remains that , in the absence of data at earlier time points , we would have detected a statistically significant difference at this time point . in summary , our results indicate that 20  gy of radiation , delivered either as a single dose or in 5 daily fractions , suppresses hippocampal cell proliferation acutely relative to controls , and there is no change in proliferation from the acute to early delayed phase after radiation in both crt groups . the sgz , one of the primary neurogenic niches of the brain , contains progenitor cells which proliferate and subsequently migrate to the adjacent granule cell layer whereupon they differentiate and mature . the survival of these neurons is typically determined 1116 days after mitosis ; therefore , any damage sustained during this period could disrupt normal brain development resulting in adverse long - term effects . our data show that a dose of radiation as low as 4  gy is sufficient to suppress cell proliferation to approximately 6% of control levels , 24 hours after exposure . this is consistent with evidence that the majority of apoptosis among progenitor cells in the sgz occurs within 24 hours after radiation   and other reports of a long - lasting decrease in proliferation or neurogenesis in the rodent hippocampus following cranial radiation in early development [ 5558 ] . disruption of hippocampal neurogenesis during development , especially if persistent , may well contribute to the decrease in brain size that we have observed [ 59 , 60 ] ( figure 7(b ) ) as well as late cognitive sequelae of childhood crt [ 43 , 61 , 62 ] . to assess radiation - induced damage , we used dti , a noninvasive mri technique commonly used to measure the diffusion of water within the brain , which can be altered by organizational or microstructural changes in tissue matter . a decrease in fa value ( an increase in unrestricted water movement ) in the brain has been correlated with edema , inflammation , and a decrease in neuronal myelination . in our model , a significant decrease in mean fa was measured in the fimbria of animals treated with single but not fractionated dose crt , implying increased radiation - induced damage compared to fractionated crt . our findings are consistent with those of others who have reported that fa decreases after single dose radiation exposure in the fimbria [ 23 , 65 ] and other fiber tracts [ 24 , 26 , 65 , 66 ] but is not detectable following fractionated treatment . histological studies also report white matter damage following a single exposure to radiation [ 23 , 6769 ] . we also found that the ratio of fimbria volume was decreased in the single dose - treated animals at both thresholds applied , whereas the fractionated crt animals showed no reduction at either threshold . another study using adolescent mice also reported that volumetric differences in the fimbria emerge as early as 1 week after single dose crt and show limited subsequent recovery . given that the fimbria fornix comprises a bundle of afferent and efferent myelinated projections to and from the hippocampus , radiation - induced damage to this white matter tract may contribute to late cognitive effects . using magnetic resonance spectroscopy , we found that , in the single dose , but not in the fractionated , crt group , myo - inositol decreased significantly , while naa + naag increased significantly relative to shams . these two metabolites are involved in astrocyte metabolism ; myo - inositol , which is an osmolyte and is also involved in membrane turnover [ 71 , 72 ] , is more abundant in astrocytes than in neurons , and the production and metabolism of naa + naag are through a cycle that involves neurons , astrocytes , and oligodendrocytes [ 56 , 57 ] . coupling these metabolites findings with our observed reduction in cell proliferation ( figure 2 ) seems to indicate that in the case of severe radiation - induced injury , as demonstrated by the single dose crt , the onset of gliosis and demyelination could offer an explanation for these changes . we have previously reported an increase in gliosis following single dose cranial irradiation that persists for months after treatment [ 74 , 75 ] . further , we and others have observed demyelination onset at around three months after irradiation [ 76 , 77 ] . further experimentation and maybe the use of nmr spectroscopy of brain tissue samples would be needed to confirm our observation . within the first few days after the start of radiation , body weights of irradiated animals were lower than those of age - matched shams ( figure 5(a ) ) . while both regimens suppressed growth , only the difference between single dose and sham reached statistical significance at the 5  d post - crt interval , but , by 3.5  mo , all three groups differed significantly from one another . we have observed the retarding effect of crt on body weight in other strains of rats as well ( long - evans hooded and fisher 344 , data not shown ) . we also replicated our findings of crt - induced overall reduction in body weight in a small , separate cohort of animals ( figure 6(a ) ) . note that this cohort was followed over time and when data for 5  d after crt ( i.e. , day 4 on x - axis , figure 6(a ) ) are analyzed by one - way anova , shams weigh significantly more than both single dose [ p < 0.001 ] and fractionated crt [ p < 0.01 ] animals . additionally , we documented significant effects of crt , post - crt interval , and crt x post - crt interval on body length ( figure 6(b ) ) and the ratio of body weight to body length ( figure 6(c ) ) . all groups differed significantly from each other in terms of body length and body weight / length ratio and single dose crt had the most severe impact . furthermore , while growth eventually plateaued with age for all groups , irradiated animals never recovered to sham levels . however , note that while crt decreased body weight and length , the rate of weight gain and increase in length was similar across groups . post - irradiation weight loss in rodents has been reported in mice and rats irradiated with different doses and at different ages [ 25 , 7881 ] . in order to mitigate the effects of possible damage to the salivary gland , we provided softened food to the animals inside their home cages on a daily basis , starting immediately after the first session of radiation . our brain weight data at 3.5  mo after radiation show that both single dose and fractionated crt stunted brain growth ( figure 7(a ) ) . once again , a single dose of crt suppressed growth to a significantly larger extent ( 18.9% decrease ) than fractionated did ( 8.4% decrease ) . brains of single dose crt animals weighed , on average , 10.6% less than those of fractionated crt animals , which is visible to the naked eye ( figure 7(b ) ) . other studies have also reported radiation - induced suppression of brain growth in rodents [ 25 , 49 , 79 , 82 , 83 ] . in contrast , 24  h after the end of crt , that is , when both groups had received a total dose of 20  gy , brains of single dose - treated animals were , on average , heavier compared to shams and fractionated crt animals , whereas brains of fractionated crt animals weighed less compared to shams . the initial increase in brain weight among single dose - treated animals may be a result of acute inflammation - induced edema which is absent in the fractionated crt group , because they receive radiation over 5 days which allows for bouts of recovery between exposures that are 1/5th the dose . collectively , our findings show that whole brain radiation has long - lasting consequences for growth , paralleling the clinical literature [ 8486 ] , and that despite some differences , primarily related to inflammation , crt delivered in childhood / adolescence causes growth stunting of the brain and body and severe suppression of proliferation . indeed , hypopituitarism is frequently observed in both adults [ 8791 ] and children [ 84 , 88 , 9294 ] treated with cranial radiation even for nonpituitary cancers . animal studies also show that cranial radiation disrupts endocrine function in a dose - dependent manner [ 78 , 95 ] . we are currently investigating to what extent our findings can be explained by the interaction of radiation and the endocrine system . to our knowledge , this is the first report of the acute and long - term effects of crt delivered during childhood / adolescence in an animal model which measures proliferation , microstructural metabolites ( using translational imaging tools ) , and phenotype changes . importantly , the observed early radiation - induced alterations were present 3.5  mo after exposure coinciding with the early delayed phase of radiation injury . ongoing investigations in our lab are aimed at evaluating the effects of fractionated crt on cognition and the inhibition of the radiation - induced inflammatory response as a possible mechanism for mitigating the long - term sequelae .
cranial radiotherapy ( crt ) increases survival in pediatric brain - tumor patients but can cause deleterious effects . 
 this study evaluates the acute and long - term impact of crt delivered during childhood / adolescence on the brain and body using a rodent model . 
 rats received crt , either 4  gy fractions  5  d ( fractionated ) or a cumulative dose of 20  gy ( single dose ) at 28  d of age . 
 animals were euthanized 1  d , 5  d , or 3.5  mo after crt . 
 the 3.5  mo group was imaged prior to euthanasia . at 3.5  mo , we observed significant growth retardation in irradiated animals , versus controls , and the effects of single dose on brain and body weights were more severe than fractionated . acutely single dose 
 significantly reduced body weight but increased brain weight , whereas fractionation significantly reduced brain but not body weights , versus controls . 
 crt suppressed cell proliferation in the hippocampal subgranular zone acutely . 
 fractional anisotropy ( fa ) in the fimbria was significantly lower in the single dose versus controls . 
 hippocampal metabolite levels were significantly altered in the single dose animals , reflecting a heightened state of inflammation that was absent in the fractionated . 
 our findings indicate that despite the differences in severity between the doses they both demonstrated an effect on cell proliferation and growth retardation , important factors in pediatric crt .