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. Author manuscript; available in PMC: 2011 Sep 10.
Published in final edited form as: Brain Res. 2010 Jul 1;1351:23–31. doi: 10.1016/j.brainres.2010.06.049

Differential Effects of Radiation and Age on Diffusion Tensor Imaging in Rats

Ann M Peiffer 1, Lei Shi 2, John Olson 3, Judy K Brunso-Bechtold 2
PMCID: PMC2934908  NIHMSID: NIHMS219283  PMID: 20599817

Abstract

Greater than 50% of adults and ∼100% of children who survive >6 months after fractionated partial or whole-brain radiotherapy develop cognitive impairments. Noninvasive methods are needed for detecting and tracking the radiation-induced brain injury associated with these impairments. Using magnetic resonance imaging, we sought to detect structural changes associated with brain injury in our rodent model of fractionated whole-brain irradiation (fWBI) induced cognitive impairment and to compare those changes with alterations that occur during the aging process. Middle aged rats were given a clinically relevant dose of fWBI (40 Gy: two 5 Gy fractions/wk for 4 wk) and scanned approximately one year post-irradiation to obtain whole-brain T2 and diffusion tensor images (DTI); control groups of sham-irradiated age-matched and young rats were also scanned. No gross structural changes were evident in the T2 structural images, and no detectable fWBI-induced DTI changes in fractional anisotropy (FA) were found in heavily myelinated white matter (corpus callosum, cingulum, and deep cortical white matter). However, significant fWBI-induced variability in FA distribution was present in the superficial parietal cortex due to an fWBI-induced decline in FA in the more anterior slices through parietal cortex. Young rats had significantly lower FA values relative to both groups of older rats, but only within the corpus callosum. These findings suggest that targets of the fWBI-induced change in this model may be the less myelinated or unmyelinated axons, extracellular matrix, or synaptic fields rather than heavily myelinated tracts.

Keywords: Fractionated Whole Brain Irradiation, Diffusion Tensor Imaging, Aging, White Matter, Brain

Introduction

Fractionated whole-brain irradiation (fWBI) is widely used for the treatment of primary and metastatic brain tumors (Coleman et al., 2004; Monje et al., 2002; Stone et al., 2004; Tofilon and Fike, 2000). As many as 200,000 patients receive fWBI each year (Auperin et al., 1999; Fife et al., 2004; Soffietti et al., 2002; Varlotto et al., 2003). Improvements in cancer therapy and health care have expanded the population of long-term cancer survivors with 62% of adult cancer patients now surviving beyond 5 years (Coleman et al., 2003; Jemal et al., 2009; Sheline et al., 1980; Stone et al., 2002; Walker et al., 1979). For approximately 12 million long-term survivors, late effects from treatments have emerged as a major risk factor (Leibel et al., 1989; Sheline et al., 1980). In particular, the progressive cognitive impairment that occurs in up to 50% of cancer patients who survive 6 months or longer following partial or fWBI (Crossen et al., 1994; Imperato et al., 1990; Johannesen et al., 2003) can significantly diminish the quality of life for cancer survivors.

Radiation-induced brain injury can be divided into 3 phases – acute, early delayed and late delayed (Tofilon and Fike, 2000). Acute injury occurs days to weeks after radiation therapy and is uncommon under current radiotherapy protocols. Early delayed injury occurs from 1 to 6 months after irradiation and may include transient demyelination with somnolence. These injuries are normally reversible and resolve spontaneously. Late delayed effects begin 6 months or more after brain irradiation, have been reported to be irreversible and progressive, and are the main contributors to the morbidity and mortality of fWBI-induced brain injury. Brain injury during this late period can be characterized by a variety of gross pathological findings including vascular damage, lacunar lesions, generalized cerebral atrophy and demyelination that ultimately lead to white matter necrosis (Belka et al., 2001; Eiser, 1991; Hopewell and van der Kogel, 1999; Tofilon and Fike, 2000). However, radiation-induced cognitive impairment has been observed frequently in animals and humans without gross histological changes (Akiyama et al., 2001; Robbins and Diz, 2006; Rola et al., 2004). Taken together, these data suggest that cognitive impairment precedes fWBI-induced changes in brain structure or that cognitive impairment can arise without gross structural change (Hodges et al., 1998; Shi et al., 2009; Yoneoka et al., 1999).

Further complicating the relationship between fWBI, white matter changes, and cognitive impairment in clinical studies is the cancer itself (Armstrong et al., 2004). Patients' brains are not only exposed to the tumor microenvironment, but also receive multimodality therapy, including surgery, radiosurgery, and chemotherapy. Although rodent studies are free from these clinical complexities, most have assessed the effects of large single doses of WBI (Atkinson et al., 2003; Kim et al., 2004; Wang et al., 2009) that may not accurately predict the central nervous system response to fWBI (Gaber et al., 2003) and are rarely used in the clinic. Moreover, those studies almost exclusively have assessed the effects of irradiating the brains of young rats, although brain metastases most commonly result from cancers such as breast and lung which increase in incidence at middle age (Auperin et al., 1999; Fife et al., 2004; Soffietti et al., 2002; Varlotto et al., 2003). Accordingly, the present study investigated the effect of fWBI in an established rat model of middle-age fWBI (Shi et al., 2006; Shi et al., 2008, Shi et al., in review; Shi et al., 2009). One year following fWBI at middle age, studies in this model have shown that fWBI induced impairments in spatial learning and memory correlate with subtle changes in glutamate receptor subunits (Shi et al., 2006), but have shown no loss of neurons (Shi et al., 2008), detectable white matter changes (Shi et al., 2009), or magnetic resonance spectroscopy change in the hippocampus (Shi et al., in review). Absence of changes on these structural measures in rats exhibiting fWBI-induced cognitive impairment indicates the need for more sensitive neurobiological measures to elucidate the neural changes underlying the impairment. Enhanced imaging techniques may provide the sensitivity to detect more subtle alterations in brain structure associated with the onset and progression of fWBI-induced cognitive impairment.

In the present study, we have evaluated fWBI-induced changes in the rat brain using diffusion tensor imaging (DTI), a non-invasive neuroimaging technique primarily used to evaluate white matter (Song et al., 2002; Song et al., 2005). DTI measures the diffusion of water using strong directional gradients. If water is free to diffuse in any direction, such as in cerebral spinal fluid, it will have isotropic diffusion and a low FA or fractional anisotropy value (Le Bihan and van Zijl, 2002). If water diffusion is constrained due to myelin, axonal flow, cytoskeletal structures or extracellular matrix composition, the FA value will be high and indicate directional diffusion, such as in white matter fiber tracts. Disruption in the integrity of axons or other elements of brain structure can disrupt directional diffusion of the water, and thus decrease the FA value even when necrosis or white matter disease is absent from that spatial location. To study fWBI-induced changes in DTI of normal brain tissue, we selected several different regions of interest (ROIs; see Figure 1A) representing different types of white matter tracts (e.g., inter-hemispheric, intra-hemispheric) and amount of myelination (e.g. deep versus superficial cortex). These ROIs were compared between rats who received fWBI or sham irradiation at middle age consistent with previous protocols (Shi et al., 2006; Shi et al., 2008, Shi et al., in review; Shi et al., 2009). In addition, these ROI's were further investigated for age-related differences by comparing the sham-irradiated group to an unirradiated group of young rats. We hypothesized that fWBI and age would result in similar changes to white matter tracts as measured with DTI, since the free radical oxidation hypothesis of aging is comparable to radiation associated damage by free radical production. The results revealed age-related changes to heavily myelinated white matter, but fWBI-induced changes limited to the least heavily myelinated ROI in the study.

Fig. 1. Region of Interest Locations.

Fig. 1

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ROI overlays of Slice 2 are shown on images used to elucidate borders. A: ROIs of Bilateral Parietal Cortex are composed of Superficial (SCtx) and Deep (DCtx) Parietal Cortex and were defined on the unsmoothed EPI image as described in the methods. B: ROIs of Bilateral Cingulum (Cg), Corpus Callosum (CC) and Bilateral Deep Cortical White Matter (wm) were defined on the color-coded FA maps. C: For comparison the myelin-stained section corresponds anatomically to the cortical ROI diagram and illustrates the various amounts of myelin in the investigated ROIs. Superficial cortex (SCtx) has less myelin stain than deep cortical ROI (DCtx), which is in turn less stained than deep cortical white matter (wm).

Results

All ROI mean DTI measurement values are presented in Table 1.

Table 1. Mean ROI Values of DTI Measures for Groups.

ROI DTI Measure Young Sham fWBI
Parietal Cortex FA 0.190 +/-0.0194 0.169 +/- 0.0121 0.152 +/- 0.0202
Trace 2.39 +/- 0.154 2.42 +/- 0.0807 2.39 +/- 0.0949
λ1 0.949 +/- 0.0470 0.951 +/- 0.0385 0.916 +/- 0.0527
λ2 0.780 +/- 0.0539 0.788 +/- 0.0238 0.788 +/- 0.0261
λ3 0.684 +/- 0.0546 0.685 +/- 0.0222 0.681 +/- 0.0225
Superficial Parietal Cortex FA 0.189 +/- 0.0208 0.184 +/- 0.0184 0.164 +/- 0.0207
Trace 2.50 +/- 0.165 2.43 +/-0.169 2.56 +/- 0.127
λ1 0.993 +/- 0.0596 0.964 +/- 0.0751 1.00 +/- 0.0581
λ2 0.806 +/- 0.0595 0.785 +/- 0.0518 0.833 +/- 0.0403
λ3 0.697 +/- 0.0521 0.682 +/- 0.0465 0.729 +/- 0.0362
Deep Parietal Cortex FA 0.188 +/-0.0205 0.172 +/- 0.0113 0.173 +/- 0.0202
Trace 2.40 +/- 0.192 2.40 +/- 0.0536 2.33 +/- 0.0694
λ1 0.949 +/- 0.0601 0.942 +/- 0.0262 0.914 +/- 0.0436
λ2 0.787 +/- 0.0682 0.782 +/- 0.0153 0.769 +/- 0.0180
λ3 0.663 +/-0.0647 0.672 +/- 0.0174 0.651 +/-0.0169
Corpus Callosum FA* 0.203 +/- 0.0142 0.265 +/- 0.0121 0.250 +/- 0.0117
Trace 2.55 +/- 0.0367 2.61 +/- 0.0213 2.64 +/- 0.0268
λ1* 1.04 +/- 0.0179 1.13 +/- 0.0150 1.12 +/- 0.0139
λ2 0.820 +/- 0.0148 0.824 +/- 0.00945 0.829 +/- 0.0114
λ3 0.689 +/- 0.0163 0.663 +/- 0.0117 0.697 +/- 0.0145
Cingulum FA 0.267 +/- 0.00502 0.257 +/- 0.00556 0.256 +/- 0.00630
Trace 2.40 +/- 0.0207 2.42 +/- 0.0399 2.40 +/- 0.0146
λ1 0.999 +/- 0.00778 1.01 +/- 0.0121 0.993 +/- 0.00944
λ2*† 0.827 +/- 0.00783 0.855 +/- 0.00810 0.821 +/- 0.00588
λ3* 0.573 +/- 0.00833 0.592 +/- 0.00700 0.582 +/-0.00634
Deep Cortical White Matter FA 0.261 +/- 0.0108 0.264 +/- 0.0110 0.261 +/- 0.0116
Trace 2.40 +/- 0.0389 2.44 +/- 0.0227 2.41 +/- 0.0153
λ1 1.02 +/- 0.0152 1.05 +/- 0.0117 1.02 +/- 0.0118
λ2 0.762 +/-0.0147 0.768 +/- 0.00952 0.765 +/- 0.00673
λ3 0.615 +/- 0.0153 0.624 +/- 0.0112 0.622 +/- 0.00991
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All data are presented as mean ± SEM with the units μm2/ms.

*

significant difference when Sham are compared to Young rats (p<0.05).

†

significant difference when fWBI are compared to Sham rats (p<0.05).

Parietal Cortex ROIs

The rodent model used here demonstrates both fWBI-induced (Shi et al., 2006) and age-related (Shi et al., in review Neuro-Oncology) impairments in spatial learning and memory, Thus, this ROI was selected as a region of cerebral cortex that has been closely associated with spatial learning and memory function (Save and Poucet, 2009) and was partitioned into two distinct gray matter regions – superficial cortex with limited infiltration of myelinated axons and deep cortex with considerably more myelinated axons (see Figure 1B). No significant group differences in FA were seen for radiation or age on any mean DTI measure, either for parietal cortex as a whole or when partitioned; however, a significant difference in the standard deviation of FA was detected for superficial cortex between the radiation groups. Specifically, Sham rats had significantly more variability than fWBI rats in the standard deviation of FA among the voxels included in the superficial parietal cortex ROI (F1,14=5.06, p<0.05; see Table 2). The follow-up within-subject ANOVA indicated that the variance in means was being driven by a significant group difference between the slices (F3,42 = 11.35, p<0.001), not between the hemispheres (F1,14 = 1.11, p = n.s.). The most anterior slice had a significant fWBI-induced FA difference between groups, with fWBI rats being significantly lower than Sham rats (F1,30=6.44, p<0.02); all other slices did not reveal significant differences between groups (see Figure 3).

Table 2. ROI Standard Deviation Values of DTI Measures for Groups.

ROI DTI Measure Young Sham fWBI
Parietal Cortex FA 0.0644 +/-0.0103 0.0619 +/- 0.00936 0.0532 +/- 0.00478
Trace 0.323 +/- 0.105 0.235 +/- 0.0423 0.252 +/- 0.0438
λ1 0.139 +/- 0.0354 0.108 +/- 0.0154 0.111 +/- 0.0179
λ2 0.116 +/- 0.0341 0.0819 +/- 0.0125 0.0908 +/- 0.0162
λ3 0.112 +/-0.0318 0.0900 +/- 0.0160 0.0919 +/-0.0142
Superficial Parietal Cortex FA† 0.0617 +/-0.0125 0.0677 +/- 0.00673 0.0544 +/- 0.00957
Trace 0.411 +/- 0.155 0.339 +/- 0.0610 0.283 +/- 0.0761
λ1 0.164 +/- 0.0573 0.134 +/- 0.0265 0.115 +/- 0.0267
λ2 0.143 +/- 0.0480 0.113 +/- 0.0159 0.102 +/- 0.0256
λ3 0.134 +/- 0.0493 0.119 +/- 0.0184 0.100 +/- 0.0201
Deep Parietal Cortex FA 0.0571 +/- 0.00921 0.0530 +/- 0.00857 0.0548 +/- 0.00831
Trace 0.171 +/- 0.0597 0.129 +/-0.0199 0.145 +/- 0.0199
λ1 0.0870 +/- 0.0184 0.0741 +/- 0.00971 0.0795 +/- 0.0104
λ2 0.0648 +/- 0.0178 0.0513 +/- 0.00545 0.0586 +/- 0.00654
λ3 0.0703 +/- 0.0175 0.0584 +/- 0.00862 0.0653 +/- 0.00875
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All data are presented as mean ± SEM with the units μm2/ms.

*

significant difference when Sham are compared to Young rats (p<0.05).

†

significant difference when fWBI are compared to Sham rats (p<0.05).

Fig. 3. DTI Slice Prescription.

Fig. 3

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The anterior commissure was centered in the 3rd slice for each animal to align the anterior to posterior slice acquisition. ROIs were drawn on slices 2-5 of the color-coded directional FA maps and prescribed as detailed in figure 1.

Corpus Callosum ROI

This ROI was selected as the inter-hemispheric white matter tract. For the corpus callosum ROI, radiation and age demonstrated different effects on the DTI measures evaluated in the present study. No significant fWBI-induced effects were seen on FA, Trace or λ1, although a marginally significant interaction was detected for fWBI and λtrans (i.e., λ2 & λ3; p<0.07, F1,62=3.534) which was driven by a marginal increase in λ3 of fWBI relative to Sham. In contrast, FA differed significantly by age (p<0.002, F1,62 = 11.08) with the Young group having lower FA values relative to older Sham rats (0.203 versus 0.265, respectively). Reduced FA in the Young rats was driven by a significantly smaller λ1 (p<0.002, F1,62 = 13.11; Young = 1.04 and Sham = 1.13).

Cingulum ROI

This ROI was selected as the intra-hemispheric white matter tract. Radiation and age had significant, but opposite effects on λtrans (i.e., fWBI < Sham > Young rats); radiation showed a decrease in λtrans while age showed an increase. No significant effects of radiation or age were seen in FA, Trace or λ1. In the transverse plane, radiation showed a main effect (p<0.02, F1,126 = 6.004) with lower values for both λ2 and λ3 in fWBI versus higher values in Sham rats. Radiation also interacted with λtrans (p<0.004, F1,126 = 9.351), which was driven by a significant decrease for irradiated rats in λ2 (p<0.002, F1,127 = 11.67; fWBI = 0.821 and Sham = 0.855), but not in λ3. The λtrans main effect of age was seen (p<0.03, F1,126 = 5.241) with older Sham rats having larger λ2 and λ3 values than Young rats.

Deep Cortical White Matter ROI

This ROI was selected based on the report of Wang and colleagues that a gradual reduction in FA occurred between 2 and 40 weeks following a single dose of radiation at 3 months of age relative to sham irradiated age-matched controls (Wang et al., 2009). No significant differences were detected between Sham and fWBI rats on any DTI measure here, and no significant age effects were seen.

Discussion

The present results indicated distinct differences between the effects of fWBI and age on DTI measures in the parietal cortex and major white matter tracts. Effects of fWBI were minimal or absent within the corpus callosum, cingulum, and deep cortical white matter, but fWBI resulted in significantly lower FA values in the superficial parietal cortex, the ROI with the lowest density of myelinated fibers. These results have important implications for both detecting brain injury associated with fWBI-induced cognitive impairment and identifying the underlying mechanisms of that injury. In contrast, significant age-related changes in DTI measures were only observed in white matter tracts, specifically increased FA and λ1 in corpus callosum as well as increased λ2 and λ3 in cingulum.

FA is calculated from all three λ measurements and higher FA values indicate greater directional diffusion of water in the primary direction (λ1) relative to diffusion in the transverse plane (λ2 and λ3). Surprisingly, no fWBI-induced changes were detected one year following irradiation at middle age in any of the major white matter ROIs included in the present study. Previous clinical studies have reported DTI changes consistent with demyelination in radiation therapy patients (Nagesh et al., 2008). Wang and colleagues (Wang et al., 2009) also reported such changes in a white matter area corresponding to our deep cortical white matter ROI; although in that study, younger rats (i.e., 1 month old) than those used here received a single high-dose radiation exposure known to produce a molecular response different from the molecular response of the clinically relevant fWBI protocol used here (Gaber et al., 2003; Yuan et al., 2003; Yuan et al., 2006). The stability of dense highly-myelinated white matter tracts one year following fWBI at middle age observed here is consistent with the absence of morphological change in brain commissures observed in this model at both the light and electron microscopic level (Shi et al., 2009), and may be indicative of a reduced vulnerability at middle age in the rat to irradiation damage. There are two potential explanations for the difference in the observed white matter changes between these rats and brain tumor patients after fWBI. First, the rat is inherently more radioresistant; the whole body lethal dose that kills 50% of humans (LD50) with no intervention is approximately 4.0 Gy while the (LD50) for rats is 7.0 Gy (Hall, 1994). Thus, a higher total fractionated dose may be required to produce the same white matter effect in rats as seen in patients. However, this explanation is unlikely given that a higher dose of WBI, 45 Gy delivered in 9 fractions, to middle age rats produced the same cognitive impairments with no measurable white matter changes (Shi et al., 2006; Shi et al., 2009). Second, the presence of a tumor and the edema that increases intracranial pressure in patients treated with fWBI could influence the development of cognitive impairment, white matter changes, and/or DTI measurements. Ongoing experiments in our laboratories will explore these possibilities.

Interestingly, the only fWBI-induced FA change observed in the present study involved the anterior-most superficial parietal cortex. As defined here, superficial parietal cortex is comprised approximately of cell layers 1, 2, and 3 and includes far fewer heavily myelinated axons than the deep parietal cortex which includes the remainder of the cortical layers, cell layers 4, 5, and 6. Thus, diffusion estimates include areas containing not only some axons like those that predominate in the major white matter tracts, but also less heavily myelinated and unmyelinated axons, extracellular matrix and synaptic fields. It is further important to note the observed heterogeneity of FA differences within the superficial parietal cortex. This heterogeneity suggests that averaging across voxels within ROIs instead of relying on voxel-by-voxel assessment of DTI measures may mask localized differences among groups. Comparisons between ROI analysis methods and voxel-by-voxel assessment of DTI measures have indicated that the ROI analysis will fail to detect areas of significant difference. This failure occurs, in part, because not all brain areas are evaluated and averaging across heterogeneous voxels reduces the true effect (Furutani et al., 2005; Kanaan et al., 2006; Snook et al., 2007). Furthermore, detection of the FA differences within the most anterior slice evaluated in the DTI sequence suggests a propensity for more anterior cortical areas to be most affected in this model of fWBI. Ongoing studies in our laboratories will address the application of voxel-by-voxel DTI analysis versus ROI in this rodent model of fWBI-induced cognitive impairment and evaluate the entire cerebral cortex to investigate an anterior-posterior gradient in radiosensitivity.

Unlike fWBI, aging demonstrated a significant impact on both the FA and λ1 in the corpus callosum, the largest of the major white matter pathways studied here. Interestingly, counter to the decreased FA that has been reported in the corpus callosum of aging humans (Bhagat and Beaulieu, 2004; Head et al., 2004; Pfefferbaum et al., 2000; Pfefferbaum et al., 2005; Sullivan et al., 2006), FA of the corpus callosum in our sample of rats showed an increase with age. This finding, however, corroborates the report of Yates and Juraska (Yates and Juraska, 2007) of age-related increases in size and myelinated area of the rat corpus callosum over a similar time in the life span. Since increased myelination restricts the random diffusion of water, FA values would be expected to increase with increased myelination. However, most studies of tracking DTI measures in the maturing brain have been performed during early life in the rat (i.e. between 1 month and 4 months of life) and tissue properties that drive DTI measures during early myelination may not be the same as those at middle age. In addition, recent developments in the methodology of DTI indicate that the b-value selected within an experimental model may bias the detection of parallel and perpendicular diffusivity differences (Hui et al., 2010). However, this sensitivity to b-value is based on known histological evidence of early brain development suggesting the importance of further research characterizing the neurobiological features underlying the DTI measures at multiple time points in middle aged rat models. The b-value used in this experiment (1.0 ms/μm2) favors detection of parallel diffusivity differences over perpendicular (Hui et al., 2010), and thus could be contributing to this partial finding.

In the cingulum, both λ2 and λ3 in the transverse plane demonstrate age-related increases; although these differences were not sufficient to alter the Trace or FA values. Song and colleagues have related transverse plane (i.e., λ2 and λ3) increases with no λ1 difference to demyelination of axonal fibers (Song et al., 2002; Song et al., 2005). This finding is consistent with greater changes to myelinated axons in the cingulum than the corpus callosum after middle age in the rat. It is important to note, however, that the present study is a cross-sectional investigation and thus, the observed age-related differences could also reflect group differences between the cohorts. Therefore, a longitudinal investigation is currently underway to assess the effects of aging on DTI measures across life span in the adult rat. Finally, the λ2 reduction in the cingulum of fWBI rats was unexpected and suggested increased integrity of the white matter tract relative to Shams. However, once again, this change was not associated with overall changes in Trace or FA values, and will be further investigated in ongoing studies.

In summary, a total fWBI dose of 40 Gy at middle age resulted in reduced FA within anterior-most superficial parietal cortex, but not within larger white matter tracts one year post-irradiation. Targets of the fWBI-induced change in healthy brain tissue in this model may involve less myelinated or unmyelinated axons, extracellular matrix, or synaptic fields. The spatial variability in the cortical DTI measurements suggests that a voxel-wise comparison will provide more accurate assessments of differences between irradiated and sham groups. Age-related FA effects in rodents appear to support increasing myelination that is maintained into old age, unlike the decrease that is seen in human aging. Longitudinal studies will be necessary to understand the full implications of this observation on the use of rats as models of the aging process in humans.

Experimental Procedure

Subjects

A total of 24 Fischer 344×Brown Norway (F344×BN) F1 hybrid male rats were used in this experiment. Eight young rats (Young) were scanned between 5 and 6 months of age. Sixteen rats were 30 months of age at the time of scanning; 18 months previously they had received fWBI (fWBI; n=8) or sham irradiation (Sham; n=8). All protocols in this study were approved by the Animal Care and Use Committee at Wake Forest University School of Medicine and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were singly housed in a climate-controlled environment on a 12h light-dark cycle with food and water available ad libitum. A weekly weighing was used to monitor health throughout the experiment.

Irradiation Procedure

The fWBI rats received a total of 40 Gy delivered to the whole brain as 8 fractions of 5 Gy delivered twice weekly over 4 weeks. As described in detail previously (Brown et al., 2005), rats were lightly anesthetized (26.5mg/kg ketamine and 5.4mg/kg xylazine) and irradiated in a 312-TBq self-shielded 137Cs irradiator with lead and Cerrobend shielding devices to collimate the beam to irradiate the whole brain. Dosimetry was previously performed using, i] thermoluminescent dosimeters inserted in and around the brains of dead rats, and ii] ionization chambers in playdough phantoms. By delivering alternate fractions to alternate sides of the head, the dose across all areas of the brain varied by <5 %, with the midline of the brain receiving a slightly higher dose than the periphery. Sham rats were anesthetized, but not irradiated.

MR Imaging Methods

All MR imaging was performed in a Bruker Biospin 7.0 Tesla 30 cm I.D. horizontal bore magnet (Bruker Biospin Ettlingen, Germany) equipped with a Bruker S116 actively shielded gradient coil. A 38mm ID quadrature Litzcage RF transceiver volume coil (Doty Scientific, Columbia SC), tuned to 300.2MHz was used for signal transmission and reception. A Linux workstation running ParaVision 4.0 interfaced the user with the scanner. Anesthesia was induced in an induction chamber (Surgivet, Waukesha, WI) with isoflurane (3%) and oxygen (4 L/min). Once anesthesia was induced, isoflurane and oxygen flow to the rat were reduced (1.5% and 1 L/min) and maintained via an MR compatible nose cone. The rat's respiration and body temperature were monitored throughout the scan (SAI Instruments, Stoney Brook, NY). Respiration was maintained between 35 and 45 breaths per minute by adjusting isoflurane levels. Body temperature was maintained at 37° C by blowing thermostatically controlled warm air into the bore of the magnet.

Each animal was positioned with the brain in the isocenter of the magnet and RF coil. A three-plane localizer scan [Rapid Acquisition with Relaxation Enhancement (RARE) pulse sequence with parameters: Repetition Time (TR) = 1500ms, Echo Time (TE) = 30ms, Field of View (FOV) = 3.5cm, slice thickness = 2mm, matrix = 256×256, number of averages (NEX) = 1] was acquired and used to precisely center the animal's brain in the system. Once the animal was correctly positioned, a set of high resolution sagittal T2-weighted structural images were acquired, using a RARE pulse sequence with parameters: TR = 3700ms, TE = 60ms, FOV = 3.0cm, slice thickness = 1mm, matrix = 256×56, NEX = 4.

Diffusion Tensor Imaging (DTI)

A 2-shot 2D spin-echo, echo planar imaging (EPI) pulse sequence was used to acquire DTI data. A slab of 10 contiguous coronal slices through the forebrain was positioned using the high resolution T2-weighted images. The EPI slice package was positioned so that slice number 3 (of 10) was centered on the anterior commissure (see Figure 3). The EPI DTI parameters were: TR=3000ms, TE=37.8ms, FOV=4.0cm, matrix=128×128 (in-plane resolution = 313μm), slice thickness=1.0mm diffusion gradient duration (δ) = 4ms, diffusion gradient separation (Δ) = 20ms, b-value = 1.0 ms/μm2 b-max = 2.9ms/μm2 in 30 noncolinear directions (with 5 b0 images acquired) and 8 averages. EPI images were converted to compressed nifti format using MRIcron (http://www.sph.sc.edu/comd/rorden/mricron/dcm2nii.html) and processed using MedINRIA 1.7.1 (http://www-sop.inria.fr/asclepios/software/MedINRIA/). Fractional Anisotropy (FA), Apparent Diffusion Coefficient (ADC) and three eigenvalues (λ1, λ2 and λ3) were obtained for 4 regions of interest (ROI): corpus callosum and bilateral parietal cortex, deep cortical white matter, and cingulum. MedINRIA's ADC label is equal to the sum of the three eigenvalues and is known in the literature as Trace (labeled as such in this manuscript).

Due to heterogeneous nature of cortex, the parietal cortex ROI was delineated in the following manner, illustrated in the T2 image in Figure 1A and schematically in a Heidenhain myelin-stained section from a comparable brain section in Figure 1B (green contours). A line parallel to the corpus callosum (red line) and extending the entire width of the brain in coronal section established the dorsomedial corner of the ROI at its intersection with the border between cortex and the underlying deep cortical white mater. The medial border of the ROI followed the contour, and the dorsal border extended perpendicular to the cortical layers from the underlying white matter to the cortical surface which formed the lateral border. The ventral border was set parallel to the dorsal border at a position five voxels away from the dorsal border. The parietal cortex ROI was further separated into superficial and deep cortex as illustrated in the T2 image in Figure 1A (see high magnification inset), and corresponding to layers 1-3 and 4-6, respectively. Superficial cortex was lightly myelinated compared to deep cortex and to the deep cortical white matter as can be seen in the myelin-stained section (Figure 1B). Exact volume and variability of the voxels within these cortical ROIs were assessed by analyzing the standard deviation tabulated when arriving at the mean ROI value for each DTI measure. ROIs were manually traced in a blinded manner; using anatomical landmarks in four adjacent color-coded FA map slices (slice 2 through 5, see slices in Figure 3 and ROI placement in Figure 1A). ROIs for deep cortical white matter, corpus callosum, and cingulum are also shown in Figure 1A and were drawn using the color-coded FA map to delineate the main structural body of the anatomical tract.

Statistical Analyses

All analyses were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL). Each slice and hemisphere contributed an average ROI value to the analysis resulting in 64 measurements per cortical, deep cortical white matter, and cingulum ROI and 32 measurements for the corpus callosum ROI. Cortical ROIs also considered the standard deviation value from each slice and hemisphere to assess cortical variability within the DTI measures. Age effects were tested between the Young and older Sham groups, and radiation effects were tested between Sham and fWBI rats only. One-way ANOVAs were used to evaluate FA, Trace, and λ1 independently within each ROI. Instead of calculating λtrans by averaging λ2 and λ3, we evaluated the transverse plane using a within subject repeated-measure General Linear Model of λ2 and λ3 within each ROI. Finally, to allow for direct comparison between superficial and deep parietal cortical ROIs, analyses were performed using a within subject repeated-measure General Linear Model of Layer ROI (Superficial or Deep) using FA, Trace, or λ1 independently as the dependent measure. For analysis of the transverse plane two within subject variables were used Layer (Superficial or Deep) and Lambda (λ2 and λ3). Significant interactions between treatment group and DTI measurements were explored using post-hoc t-test analyses.

Fig. 2. Mean FA values in Superficial Parietal Cortex within each slice.

Fig. 2

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Slice 2 is the most anterior section and fWBI have significantly less FA than Sham rats (F1,30 = 6.44, *p<0.02). The heterogeneity between these slices call for voxel-wise analysis of cortical DTI measures to accurately evaluate radiation-induced changes.

Acknowledgments

Research funding was provided by the National Cancer Institute: CA119990 to JKB-B, LS, and the Center for Biomolecular Imaging to acquire and process data. Salary support to AMP was provided by National Institute of Health grant NS#544722. The authors would like to thank Dr. Kenneth T. Wheeler, Jr., PhD for helpful editorial assistance.

Abbreviations

fWBI

Fractionated Whole-Brain Irradiation

DTI

Diffusion Tensor Imaging

FA

Fractional Anisotropy

ADC

Apparent Diffusion Coefficient

ROI

Region of Interest

Footnotes

Conflict of Interest Notification: None of the authors have any conflict of interests to report.

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