Hippocampal atrophy is a well-documented consequence of traumatic brain injury1 (TBI) with hippocampus associated learning and memory deficits as hallmarks of brain trauma. Astrocytes and microglia, cells of the CNS, are considered key players in initiating an inflammatory response post injury (PI). These cellular changes can influence the local microenvironment and thus determine the extent of damage and subsequent repair. The present study focuses on the hippocampal microstructural and inflammatory alterations following TBI, alongwith it’s role in hippocampal damage PI.

55 adult, male sprague-dawley rat(250gms) were used during the study. Based on 40-50% mortality rate, out of the 55 rats, 27 rats survived during the study period. Of these 07 rats were used for MR imaging at day 0 (each rat served as its own control) and at 4h, D1 and D5 PI. The remaining 20 rats were divided in groups of 5 for Iba-1 and GFAP analysis at 4h, D1, D5 PI and control group. Closed head moderate injury was induced by freely dropping 450 gm(1cm diameter) brass rod from a height of 50cm above the sagittal midway of the rat brain2.

All MR imaging was performed at a Bruker Biospec 7.0 Tesla system. A rapid acquisition with relaxation enhancement (RARE) sequence was used to acquire T2 weighted images of the rat brain with parameters of 256 Х 256 matrix size, FOV of 4 Х 4 cm, repetition time echo time (TR)/ (TE) = 2,000/13 msec, 1 mm slice thickness with no interslice gap, and 15 slices. DTI images were acquired using a multislice, multiple-shot spin echo EPI sequence with the following parameters: TR/ TE = 5,000 msec/34.46 msec, 46 gradient encoding directions, and b = 672 sec mm-2, acquisition matrix = 128 Х 128, field-of-view = 4 cm x 4 cm, slice thickness = 1 mm, 15 slices. The DTI was processed as described in detail elsewhere (3) to compute the DTI metrics such as the mean diffusivity (MD), fractional anisotropy (FA), radial diffusivity (RD), axial diffusivity (AD) from hippocampus region by placing two bilateral ROIs. Control rats (day0) and injury groups (4hrs, 1day, 3days, 5 days PI) were sacrificed and rat brains were postfixed for immunostaing for Iba-1 and GFAP. 2 FOVs bilaterally on hippocampus were taken at 10× magnification to calculate the mean number of microglia/astrocytes in each rat. Counting was performed manually using a imageJ analysis system.

A repeated measure ANOVA was performed to assess alterations in diffusion indices with time in hippocampus region of the brain. Differences in immunohistological counts of Iba-1+/GFAP+ cells at day0, 4h, D1, D5 PI was analysed using one way ANOVA. p values ≤ 0.05 was considered statistically significant (indicated by an asterisk marks).DTI shows injury induced alterations in diffusivites and anisotropy in hippocampus region PI. MD and RD were significantly reduced as compared to control at all timepoints PI. Fractional anisotropy levels significantly increased as early as 4h and remain elevated till D5 (Fig1). Histological analysis showed significantly increased microglial cells as Iba-1+ cells at D1 and D5 PI. Significantly increased GFAP+ cells were seen at D5 PI(Fig2).The present study investigates moderate TBI induced acute hippocampal neuroimflammatory and microstructural alterations. Injury induced cascade may include blood brain barrier (BBB) breakdown, edema formation, subsequent infiltration of blood cells leading to elevated levels of inflammatory cytokines and activation of resident glial cells4. Due to stretching of axons, cells and oligodendrocytes during mechanical trauma, there is decline in the function of Na+-K+ ATPase pump. This leads to an influx of fast moving extracellular water to the diffusion restricted intracellular region resulting in a net decrease in MD values. Also, trauma induced cytotoxic edema reduces extracellular space, further depleting the regional MD values. Astrocytes are thought to regulate the extracellular concentrations of water, potassium, glutamate and other neurotransmitters5 thereby regulating the diffusivity indices of the brain. Conclusively, the present study demonstrates DTI may predict acute hippocampal damage induced through injury (when hippocampus is not the site of injury) and subsequent neuroinflammatory response. This study may help in understanding the underlying pathology in regions remote to injury site.