Original Research

Neuroimaging Assessment of Memory-Related BrainStructures in a Rat Model of Acute Space-LikeRadiationLei Huang, MD,1 Anna Smith, BS,1 Peter Cummings, MD,3 Edward J. Kendall, PhD,4 andAndre Obenaus, PhD1,2*

Purpose: To investigate the acute effects on the centralnervous system (CNS) of 56Fe radiation, a component ofhigh-energy charged particles (HZE) in space radiation,using quantitative magnetic resonance imaging (MRI) non-invasively.

Materials and Methods: Sprague–Dawley rats were ex-posed to whole-brain 56Fe (0, 1, 2, and 4 Gy). At 1 weekpostirradiation, MRI scans were made using T2-weighted(T2WI), diffusion-weighted (DWI), and contrast enhancedT1-(CET1) imaging. T2 relaxation time and apparent diffu-sion coefficient (ADC) values were obtained from memory-related brain regions of interest (ROIs). Histopathology wascorrelated using ex vivo tissues.

Results: No overt abnormalities were visualized using T2WIand DWI at 1 week postradiation. CET1 values did not differsignificantly between the irradiated and control animals.Compared to 0 Gy, there were significant prolongations inT2 values and reductions in ADC after irradiation. In theabsence of evident neuronal pathology, immunohistochem-istry revealed astrocytic activation in 4 Gy animals.

Conclusion: At 1 week after whole-brain 56Fe exposure, T2and ADC values can differentiate radiosensitivity in regionscritical for hippocampal-related memory. MRI may providenoninvasive assessment of the initial molecular/cellulardisturbances in vivo after HZE irradiation.

Key Words: T2 relaxation time; apparent diffusion coeffi-cient; hippocampus; entorhinal cortex; thalamus; 56Fe-ir-radiationJ. Magn. Reson. Imaging 2009;29:785–792.© 2009 Wiley-Liss, Inc.

HIGH-ENERGY CHARGED PARTICLES (HZE) are bothan important component of space radiation and an ex-panding resource of radiotherapy for patients. There-fore, the radiobiological effect of HZE particles on thecentral nervous system (CNS) is a subject of consider-able research. Exposure to high-energy 56Fe particles,the densest HZE ion (1) in cosmic rays, have beenshown to induce neuronal and behavioral deficits, in-cluding cognitive impairments (2,3). In recent studiesthe progressive reduction in neurogenesis within thehippocampal formation after 56Fe-irradiation has beenshown to be responsible for the accelerated degenera-tion of hippocampal-dependent memory (3,4). Histori-cally, radiation-induced acute CNS reactions are ex-pressed days to weeks after exposure (3). The changesthat occur in the early period after irradiation may beresponsible for long-term effects seen months or yearslater. It would be advantageous to identify the initialdisturbances in 56Fe-irradiated brain tissue in vivo,which would allow monitoring of the temporal patho-physiology processes associated with late cognitive con-sequences. In this regard, magnetic resonance imaging(MRI) is ideal because of its ability to noninvasivelyidentify changes in the intrinsic properties of waterprotons in the brain (5).

MRI depictions of altered anatomical structures andunderlying pathology have been well documented inbrain injury caused by cranial radiotherapy (6–9). Vi-sual assessment of images generated by conventionalMR provides diagnostic information about chronic CNSinjuries induced by high-dose radiotherapy. The sub-sequent blood–brain barrier (BBB) rupture, tissueedema, and necrosis, can be readily visualized on con-trast-enhanced T1-weighted (CET1) (6,7) and T2-weighted (T2WI) images (7–9). Indeed, the abnormalsignal intensity seen on MR images results from alter-ations in physical tissue parameters induced by a vari-ety of histological, pathologic, and physiological factors.There are additional benefits gained from quantitativelyanalyzing the MR signal in the injured tissue. T2 relax-ation times are considered to be related to the dynamicstate of water at microscopic tissue levels and are sen-sitive to water binding (10). Evaluation of relaxationtimes offers a method for investigating neurophysiologyand neuropathology in vivo (8,9,11). Diffusion-weighted

1Department of Radiation Medicine, Loma Linda University School ofMedicine, Loma Linda, California.2Department of Radiology, Loma Linda University School of Medicine,Loma Linda, California.3Department of Pathology, Division of Neuropathology, University ofVirginia, Charlottesville, Virginia.4Diagnostic Imaging, Janeway Child Health Center, Memorial Univer-sity of Newfoundland, St. John’s, Newfoundland, Canada.Contract grant sponsor: NASA; Contract grant number: NNJ04HD80G(to A.O.).*Address reprint requests to: A.O., 11175 Campus St., CSP A1010,Loma Linda, CA 92354. E-mail: aobenaus@dominion.llumc.eduReceived May 27, 2008; Accepted October 8, 2008.DOI 10.1002/jmri.21661Published online in Wiley InterScience (www.interscience.wiley.com).

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imaging (DWI) complements the classical anatomicalMRI techniques and are sensitive to changes in intra-cerebral structure and function at early stages of braininjury (12–14). The quantitative DWI parameter, appar-ent diffusion coefficient (ADC), is used to characterizewater mobility in local tissues (15). Currently, MRI hasnot been used to evaluate hyperacute tissue distur-bances following HZE brain irradiation.

In the present rat model of whole-brain-only 56Feirradiation, multimodal MRI sequences, includingT2WI, DWI, T1-weighted imaging (T1WI), and contrast-enhanced T1 (CET1), were performed at 1 week after56Fe exposure. We hypothesized that quantitative MRI(T2 and ADC values) can assess the initial stages of thedeveloping pathophysiological processes induced by56Fe irradiation in vivo and are sensitive enough todetect the initial molecular disturbances in memory-related structures that may be responsible for the evo-lution of future cognitive decrements.

MATERIALS AND METHODS

All protocols were approved by the Animal Health andSafety Committees of Loma Linda University (LLU) andBrookhaven National Laboratory (BNL) and were incompliance with Federal regulations.

Animals

Twenty-four adult male Sprague–Dawley rats (�290 g,2 months old), were delivered to BNL from the vendor(Harlan, Oregon, WI). Prior to irradiation, all animalswere allowed to equilibrate for 5 days. On the day ofirradiation rats were transported to the NASA SpaceRadiation Laboratory (NSRL) beamline at BNL (Fig. 1).Each animal was anesthetized with isoflurane in air(4% induction, 1.5% maintenance) and placed in cus-tom-designed stereotaxic cradles to stabilize the headposition and facilitate insertion of the animals into thebeamline (Fig. 1A). Total time for anesthesia and irra-diation was less than 15 minutes. Controls were treatedidentically without being inserted into the beamline.Animals were randomly assigned to experimental andcontrol groups (n � 6). Twenty-four hours after irradi-ation the animals were shipped back to LLU via specialcourier.

56Fe Irradiation

As previously described, whole-brain-only irradiationwas accomplished using a 600 MeV/n iron (56Fe) beamat BNL (16). Briefly, a design using a three-ply collima-tor with two equidistant apertures allowed whole-brain-only exposure. The animal holder was positioned be-hind the collimator, giving a 1.5 � 1.0 cm field forirradiating the brain of the rat (Fig. 1B). Radiation wasdelivered in a single fraction at a dose rate of �1–3Gy/min for a total dose of 0 (controls), 1, 2, and 4 Gy.

MRI

MRI data collection and quantification were finalizedusing previously established protocols (16). One week(7 days) after irradiation, MRI was acquired using aBruker 4.7T 30 cm horizontal bore instrumentequipped with 250mT/m microgradients (slew rate1000 mT/s) and a 116 mm (I.D.) quadrature receivercoil optimized for rat brain assessment. Images wereacquired in 256 � 256 matrices and a 3-cm field of view,producing an in-plane pixel resolution of 0.117 mm.Twenty horizontal slices, 1 mm thick, provided coverageof the entire brain. The imaging protocol consisted of aseries of sequences designed to capture multiple con-trast levels, including: T2WI (TR/TE � 2850/20 msec),DWI (TR/TE � 3000/50 msec, b � 0, 78 mT/m), T1WI(TR/TE � 850/50 msec), and contrast-enhanced T1WI(CET1, TR/TE � 850/50 msec). For CET1, a singlebolus (3 mL over 2 min) of Gd-DTPA (287 mg/mL, Am-ersham Health, Oslo, Norway) was injected via a can-nulated tail vein and the CET1 data was collected 10minutes later. Total imaging time was 2 hours. Duringthe MRI scan, animals were anesthetized with 1%isoflurane. Vital signs including heart and respiratoryrate and body temperature were simultaneously moni-tored with a physiological monitoring system.

T2 and ADC maps were computed from T2WI andDWI, respectively. Regions of interest (ROIs) for quan-tifying T2WI and DWI included the bilateral hippocam-pus, entorhinal cortex, and thalamus (Fig. 2A,B). Thehippocampal ROIs included the dentate gyrus, thecornu ammonis (fields CA1–CA3), and portions of thesubiculum; all are involved in memory functions.Cheshire image processing software (Hayden Image

Figure 1. The NASA Space Ra-diation Laboratory (NSRL)beamline for 56Fe irradiation.Animals were placed in thebeamline (a) in specialized ste-reotactic cradles (b) that immo-bilized the animals usingisoflurane anesthesia. The col-limator (a) was optimized forwhole-brain-only irradiation(b, arrow). Dosimetry was per-formed using a digital parallelplate ion chamber (c).

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Processing Group, Waltham, MA) was used to outlinethe anatomical ROIs, which were then superimposedonto corresponding T2 and ADC maps (Fig. 2C,D).Computed regional statistics of the mean, standard de-viation, and number of pixels in the area of the ROIswere extracted for each animal. Digital subtraction ofT1WI and CET1 was performed on all slices for theentire brain and visually examined for location of signalhyperintensity on subtracted images, as an indicator ofBBB perturbations.

Histopathology

After the final imaging data collection, animals wereeuthanized and perfused intracardially with a fixativesolution of 4% paraformaldehyde (PFA) in 0.12 M Mil-lonig’s phosphate buffer (1 mL/g of body weight, pH7.3). Following perfusion, the brains were left in situ for1 hour at 4°C, after which the whole brain was removedfrom the skull and further postfixed for 1 hour in 4%PFA. The fixed brain tissue was rinsed three times withMillonig’s buffer and cryoprotected in 30% sucroseprior to being frozen in optimal cutting temperaturecompound (O.T.C., Tissue Tek; Sakura Fine Tek, Tor-rance, CA). The brain was sliced sequentially in an

inferior to superior horizontal direction on a cryostat(Leica CM1850, Leica Microsystems, Wetzlar, Germany)at a thickness of 30 �m. Sections were mounted ongelatin-chrome-alum coated slides. Cresyl violet ace-tate (0.5%) stain was used to determine the presence ofneuronal damage. Every tenth horizontal section overthe extent of the three ROIs was included for staining.

For immunohistochemical staining of astrocytes andmicroglia, four animals from each group were randomlyselected and three corresponding sections (100 �mapart) per animal were used to match the same hori-zontal level at which MRI datasets were quantified (seeFig. 2). Immunohistochemistry of glial fibrillary acidicprotein (GFAP) was utilized to detect astrocyticchanges. The staining protocol was performed concur-rently on selected tissue samples in animals in eachdose group. Briefly, sections were incubated in phos-phate-buffered saline (PBS) containing H2O2 (1%) toblock endogenous peroxidase activity for 15 minutesand subsequently in PBS containing 2% goat serum,0.3% Triton X-100, and 0.05% Tween for 2 hours atroom temperature to block nonspecific staining. There-after, sections were incubated for 24 hours at 4° C withGFAP primary antibody solution (Dako, Carpinteria,CA) at a dilution of 1:1000, followed by secondary an-tirabbit antibodies conjugated to biotin (Vector Labora-tories, Burlingame, CA) at a dilution of 1:200 for 2hours at room temperature. Sections were rinsed inPBS and incubated with avidin-biotinylated horserad-ish peroxidase (HRP) conjugate (Vector Laboratories) for1 hour, and the HRP reaction visualized by incubationin diaminobenzidine (DAB) (Invitrogen, Carlsbad, CA)for 6 minutes. Sections were rinsed in PBS briefly tostop the reaction and then mounted on slides and air-dried prior to mounting. Slides were permanently cov-erslipped using Permount mounting medium (ElectronMicroscopy Sciences, Hatfield, PA).

F4/80, a macrophage-associated antibody, was usedto detect activated microglial cells (17). Similar immu-nostaining protocols were performed in which sectionswere incubated with F4/80 (Serotec, Raleigh, NC) at adilution of 1:2000 for 24 hours at 4°C. After rinsing inPBS, sections were incubated with biotinylated goatantirat IgG (1:200 dilution in PBS containing 5% nor-mal goat serum; Vector) for 2 hours at room tempera-ture. After washing in PBS the peroxidase was visual-ized using DAB (Invitrogen) for 15 minutes. Thereaction was stopped by washing in PBS. Sections werethen mounted as described above.

Statistical Analysis

Student’s t-test comparisons of MRI data betweenhemispheres revealed no significant differences; conse-quently, data from these regions were combined. One-way analysis of variance (ANOVA) was followed by thepost-hoc Student-Newman-Keuls test to compare thequantitative measurements of the four dose groups.Data were presented as mean � SEM. Statistical signif-icance was accepted at P � 0.05.

Figure 2. Quantitative T2 and ADC analysis. a,b: Represen-tative T2WI and DWI images on which regions of interest(ROIs) were outlined. The ROIs included bilateral thalamus(Thal), hippocampus (HP) and entorhinal cortex (EC). c,d: T2and ADC maps were computed from corresponding T2WI (A)and DWI (B) images and ROIs translated for data extraction.T2 Map, T2 relaxation map; ADC Map, apparent diffusioncoefficient map.

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RESULTS

All animals survived the whole-brain-only 56Fe expo-sure and recovered without adverse effects. There wasno significant difference in body weights between theirradiated and control animals at the end of the 1-weekobservation period (310 � 22 g vs. 300 � 25 g).

T2-Weighted Imaging (T2WI)

In animals exposed to 56Fe irradiation, no overt abnor-malities in signal intensities were visualized at anydose. Compared to animals receiving 0 Gy, prolonged

T2 values were found for all regions in all animals ex-posed to 56Fe irradiation, suggestive of increased watercontent in local tissues (Fig. 3). The dose effects werenot uniform within the three ROIs. In the absence ofdose-dependent effects within hippocampi, the mostdominant increase in T2 (18%) was associated with 2Gy irradiation (Fig. 3A). Dose dependence, however,was found in the entorhinal cortex (Fig. 3B) and thethalamus (Table 1), with 30% and 25% maximal in-crease at 4 Gy, respectively.

DWI

Visual examination of the DW images also did not re-veal any changes in gross morphology for the animalsthat received 56Fe irradiation. Irradiation exposure sig-nificantly compromised water mobility in the hip-pocampal formation, with ADC values maximally re-duced at 1 Gy in comparison to 0 Gy controls (Fig. 4A).In the irradiated entorhinal cortex, there was an inversedose-dependent decrease in ADC values (Fig. 4B), inwhich 1 Gy exposure resulted in the greatest (45%)decrease in water mobility compared to control ani-mals. The ADC decrement increased with dose suchthat there was only an 8% decrease at 4 Gy with an ADCvalue (127 � 3.2 � 10�6 mm2/s) which was still signif-icantly less than that found for 0-Gy animals (139 �2.5 � 10�6 mm2/s). In comparison to 0 Gy, ADC valuesin the thalamus were significantly decreased only in theanimals that received 2- and 4-Gy irradiation, althoughthere was a general trend of decreasing values withincreasing radiation dose (Table 1).

CET1

The incidence of visible Gd-DTPA contrast enhance-ment was 50% at 0 Gy, 83% at 1-Gy, 75% at 2 Gy, and80% at 4 Gy. The locations of enhancement were mainlydistributed within the ventricular system. Cerebellarenhancement was also observed in 80% of animals at 4Gy and 50% at 0, 1, and 2 Gy. No focal contrast-en-hanced lesions were found in the parenchyma. Overall,the restricted enhancement suggested the absence ofovert BBB disruptions at 1 week after 56Fe irradiation.

Histopathological Observations

Cresyl violet staining did not disclose significant neu-ronal histological changes after irradiation. The princi-pal cell layers of the hippocampus, including CA1, CA3,and DG granule cell layers, were intact. Neurons withinthe entorhinal cortex and thalamus also had normalpyramidal appearances.

GFAP immunohistochemistry did not reveal any overtincrease in numbers of astrocytes after irradiation (1

Figure 3. Increased T2 relaxation times within the hippocam-pus and entorhinal cortex at 1 week after 56Fe irradiation. A:In the hippocampus, 2 Gy exposure resulted in the largestincrease in T2 values compared to 0 Gy controls. T2 values inboth 1 Gy and 4 Gy irradiated animals were also significantlylonger. B: In the entorhinal cortex there was a significantdose-dependent increase in T2 values when compared to 0 Gycontrols, with the most prolonged T2 value in the 4 Gy group.*P � 0.05, **P � 0.01 vs. 0 Gy; ##P � 0.01 vs. 2 Gy; &P � 0.05,&&P � 0.01 vs. 4 Gy.

Table 1T2 and ADC Values in Thalamus (Mean � SEM)

Parameter 0 Gy 1 Gy 2 Gy 4 Gy

T2 (ms) 61.19 � 0.99 66.64 � 0.82**††§§ 72.67 � 2.24**§§ 79.50 � 1.72**ADC (X 106 mm2/s) 109 � 5.43 107 � 3.51††§§ 97 � 5.27**§§ 82.9 � 3.89**

**p � 0.01 vs 0 Gy; ††p � 0.01 vs 2 Gy; §§p � 0.01 vs 4 Gy

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and 2 Gy), suggesting the absence of evident prolifera-tion (gliosis) early after irradiation. Within the hip-pocampus and the entorhinal cortex, however, in-creased staining intensities were observed in 50% of theanimals at 4 Gy. The morphological changes includedcellular hypertrophy and increased process thickness(Fig. 5). Quantification in staining intensities did notreveal significant differences. A similar pattern of GFAPstaining also was observed in the thalamic region (datanot shown).

No overt microglial activation was identified by F4/80after irradiation. The lack of macrophagic–microglialactivation or/and peripheral macrophage intrusionsuggest that inflammation was not yet evident at thisearly timepoint.

DISCUSSION

In this ground-based study of space HZE irradiation,we demonstrated that quantitative MRI of the brain at 1

week after whole-brain 56Fe exposure revealed region-specific tissue modifications. Prior to evident histologi-cal abnormalities we report four outcomes. First, sig-nificantly prolonged T2 relaxation times are seen withinthe irradiated hippocampus that is maximal at 2 Gy.Dose-dependent increases in T2 values were also foundwithin the entorhinal cortex and the thalamus. Second,there is a significant ADC reduction within the hip-pocampus, entorhinal cortex, and thalamus. An in-verse dose-dependent effect was observed in the ento-rhinal cortex but not in the thalamus or hippocampus.Third, no significant disruption of the BBB occurred atthis early timepoint of postirradiation. Fourth, histopa-thology was not evident except for changes in astrocyticmorphology in 50% of 4-Gy animals.

Because of the unique feature of a high-energycharge, 56Fe particles are able to exert direct effects onneuronal function (3,18). The high toxicity of low dosesof 56Fe particles leads to shortened latency for age-likecognitive deficits, in contrast to chronic cognitive im-pairments induced by clinical brain irradiationschemes (19,20). At 1 month after 1.5 Gy whole-body56Fe irradiation, analysis of the Morris water maze be-havioral tests in rats revealed deterioration of spatiallearning and memory (3). Our MRI results also demon-strate that there are measurable alterations in the re-gions important to memory function, suggestive of earlyaltered tissue characteristics. The entorhinal cortex-hippocampus-thalamic system plays an important rolein memory acquisition and consolidation in which: 1)hippocampus critical for memory and spatial naviga-tion; 2) the entorhinal cortex connecting primarily thehippocampus and neocortex; and 3) the thalamus inte-grating and consolidating hippocampal memory (21–23). In a longitudinal human study, MRI-identifiablealterations in those regions related to higher cognitiveprocesses preceded clinical symptoms of mild cognitiveimpairment (24). Similarly, the present findings mayprovide a glimpse into the initial pathogenesis respon-sible for future cognitive impairment induced by space-like irradiation. Our quantitative protocol is readilytranslatable to the clinical context.

MRI descriptions of cranial radiation-induced lesionshave been identified as increased T2WI signals in braintissue (7–9,25). However, T2 relaxation times are thedominant feature of T2W images; thus, irradiation-in-duced early increases in T2 values can occur soonerthan visible T2WI lesions (8,9). In agreement with pre-vious findings (8,9), our results showed that T2 relax-ation times were increased in 56Fe-irradiated hip-pocampi, entorhinal cortex, and thalamus withoutvisible abnormal signal intensities on T2WI.

Radiotherapy-induced prolongation of T2 relaxationtimes usually reflects the underlying histopathologicalprocess of tissue edema and necrosis (7–9). Consideringthe early postirradiation timepoint and relative low-dose exposure in the present study, we would not ex-pect significant edema or evident tissue pathology. In-deed, no significant histopathological changes exceptfor reactive morphological changes in astrocytes wereobserved in 50% of 4-Gy animals. Radiation has beenshown to alter the dynamic structures of water boundto biomolecules, including oxidatively damaged DNA,

Figure 4. Decreased water mobility was observed in the hip-pocampus and entorhinal cortex at 1 week after 56Fe irradia-tion. A: In the hippocampus, ADCs were significantly de-creased in animals exposed to 1, 2, and 4 Gy irradiationcompared to 0 Gy controls. B: Within the entorhinal cortex, thesignificant decreases in ADC were associated with irradiatedgroups in an inverse dose-dependent manner. Animals irradi-ated by 1 Gy had the lowest ADC (restricted water mobility)compared to controls. **P � 0.01 vs. 0 Gy; ##P � 0.01 vs. 2 Gy;&&P � 0.01 vs. 4 Gy.

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proteins, and lipids (26–28). Thus, the mechanismsmediating 56Fe irradiation-induced early increases inT2 relaxation times likely reflect alterations in the cellultrastructure. CET1 analysis indicated an intact BBB,consistent with this putative mechanism.

While the present study did not find any overt glialproliferation after irradiation, we did note that 50%animals in the highest dose group (4 Gy) had increasedGFAP staining that accompanied astrocytic hypertro-phy in brain tissue. As a member of the cytoskeletalprotein family, GFAP is important in modulating astro-cytic motility and shape by providing structural stabil-ity to astrocytic processes (29). In previous studies ofbrain irradiation with high doses, increased GFAPstaining intensity suggested an active response of as-trocytes in favor of their neuroprotective roles (30,31).Therefore, the current upregulation of GFAP may implythe initiation of a protective response. Instead of prolif-eration, the increased arborization of the processesconstitutes the response of astrocytes to low-dose irra-diation. Further experiments will be needed to assessthe significance of astrocytic responses.

Notably, if mild vasogenic edema evolves with a dose-dependent injury in the 56Fe-irradiated brain, extrava-gated fluid could shift to the cytoplasm of the reactiveastrocytes without significant enlargement of the extra-cellular space (32). The increase of net water contentmay be responsible for T2 prolongation in animals ex-posed to high doses of 56Fe particles.

Overt microglial activation, assessed by F4/80 (17),was not found at the acute postirradiation phase in thepresent study. In a recent report, whole-brain exposureto 10 Gy 137Cs resulted in activation of microglia at 1week that was relatively mild in young adults (8 monthsold) compared to older animals (33). Considering theage (2 months) we used, radiation-induced microglialalterations are likely age-dependent, although the un-derlying mechanism is not clear.

ADC values are influenced by a host of intracellularand extracellular constituents that vary with the natureof cellular barriers (32). A variety of brain pathologiescan be identified using DWI, including stroke (14) andseizures (13), in which the initial decrease in ADC oc-curs simultaneously with glial cell proliferation, neuro-degeneration, or cytotoxic edema (13,14). At 1 weekafter 56Fe exposure, there was a significant drop inbrain ADC values in the absence of evident histopathol-ogy except for the subtle astrocytic hypertrophy at 4 Gy.It is likely that the changes in astrocytic morphologyconstitute the cellular basis for water accumulation asobserved by dose-dependent prolongation in T2 relax-ation times. However, the underlying mechanism forthe early inverse dose-dependent reduction of watermobility in the current study is unclear. Within theextracellular space of the brain after 56Fe irradiation,the complex interaction among a variety of cellular con-stituents may affect overall tortuosity, whose influenceon ADC values has been well documented (13,34).

Cellular radiosensitivity is dictated by both the typeof DNA damage and the efficacy of the repair mecha-nisms, whereas the repair efficiency is influenced bycell and tissue types involved in the actual recoveryprocesses (35). Therefore, differential cellular composi-tions may be responsible for the region-specific charac-teristics of our quantitative MRI findings within thehippocampus, entorhinal cortex, and thalamus. Thecomposition of multipotent neural precursors in thedentate gyrus (36) may in part account for the differentpattern of T2 changes within the hippocampus. Thepredominant alterations in T2 relaxation times andADC associated with the entorhinal cortex suggested agreater vulnerability at an early stage after irradiation.Similarly vulnerabilities have been described for epi-lepsy (37).

In studies of cranial radiation treatment, therapeuticdoses resulted in prolonged T2 values that are associ-

Figure 5. Astrocyte immunohisto-chemistry revealed responsive astro-cytes within the hippocampus andentorhinal cortex. Representative im-ages from the dentate gyrus of 0- and4-Gy irradiated animals illustratingthe increased hypertrophy and thickprocesses of the astrocytes (see in-sets). A similar observation wasfound in the entorhinal cortex com-paring the 0- and 4-Gy groups. Thesquare on each micrograph indicatesthe expanded astrocytes in the in-sets. However, these findings wereonly observed in 50% of the irradi-ated animals. Scale bar � 30 �m.

790 Huang et al.

ated with increased ADC (increased water mobility),reflecting an increase in water content (vascular edema)within the tissues (7–9). In our study of low-dose brainirradiation, the decrease in ADC instead did not mirrorthe increase in T2 relaxation times within the radiosen-sitive entorhinal cortex (Fig. 6), although both shareddose-dependent increases. These intriguing findingsimplicate different pathological processes most likelyinvolving dose-dependent modifications at subcellularand cellular levels at 1 week postirradiation. If the dy-namic structure of molecular water binding is the pre-sumable basis for early T2 changes in tissues exposedto low dose, the effects on ADC may not be directlylinked and/or comparable to that of altered tissue tor-tuosity. In support of this conjecture, the lowest dose of1 Gy resulted in the smallest prolongation in T2 but thelargest (45%) decrease in ADC (water mobility). Alsowith increasing radiation dose there was an apparentincrease in tissue pathology. The appearance of astro-cytic hypertrophy at 4 Gy reflected the increasing netwater content, which may counteract and even overridethe cellular components responsible for restricting wa-ter mobility. Accordingly, the largest T2 prolongationwas reflected by the smallest ADC changes at 4 Gy. Thevariable time course of T2 and ADC responses revealsthe underlying tissue pathologic processes. In the set-ting of cerebral ischemia, previous investigations haveshown that the decrease in ADC often returns to normalvalues (pesduonormalization) while T2 values increaseat the 7-day postinjury timepoint (14). In light of ourpresent findings, the neuroimaging interpretations maynot be uniform and should be addressed within thecontext of the triggering pathology.

Despite the convincing imaging data, the lack of evi-dent histopathological changes precludes the interpre-tation of the underlying cellular mechanisms. It is pos-sible that current immunohistochemical stainingtechniques are not sensitive enough to detect subtledifferences at sub- or cellular levels between the controland experimental animals. GFAP staining seen within50% of the animals in the 4-Gy group may also reflectbystander effects or the effects of secondary particles onindividual astrocytes. In addition, the current animalmodel of a single-fraction radiation dose does not com-pletely reflect the chronic low-dose exposure which anastronaut might encounter in space. Nevertheless, thecurrent MRI characteristics, at least in part, provide aninsight into tissue modifications after acute HZE irra-diation in vivo, which may be used as the basis fornoninvasive evaluation of radiation effects in patientsfollowing proton or high-linear-energy-transfer (LET)radiotherapy.

In conclusion, at 1 week after whole-brain-only 56Feexposure, quantitative analysis of T2WI and DWI datarevealed a differential radio-response pattern within theregions critical for hippocampal-related memory func-tion. Temporal monitoring of these quantitativechanges can demonstrate their significance in thepathological evolution of HZE radiation-induced loss ofneurogenesis and subsequent cognitive impairments.

ACKNOWLEDGMENTS

The authors thank the animal care staff at BNL andGeorge Asberry for histochemistry. We also thank GregNelson, PhD, and Karen Tong, MD, for helpful discus-sions regarding the underlying mechanisms of the im-aging findings.

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