It is estimated that over 500,000 children ranging in age from 0-14 years incur a traumatic brain injury (TBI) every year in the United States¹. The majority of these TBIs are mild with no loss of consciousness in 81-92 % of these cases². Most of these patients do not seek medical advice and therefore are at an increased risk of repeat TBI.

We recently developed a closed skull repeated mild (rm) TBI model in postnatal day (PND) 18 rats. The rats displayed deficits in short-term memory retention and long-term associative learning along with axonal injury without overt neuronal death³. We hypothesized that MRS and DTI can detect early microstructural changes of brain and metabolite changes in the hippocampus which might play a role in neurobehavioral deficits observed after rmTBI in PND18 rats.

PND18 male Sprague-Dawley rats were divided into three groups and subjected to either three sham insults (n=8), one mTBI and two sham insults (n=9, mTBI), or three rmTBIs (n=10), each 24 hours apart. The mild TBI was induced as follows, rats were anesthetized using 2% isoflurane and (2:1) N₂O/O₂ and an incision was made along the scalp. A rubber ball (9.5mm in diameter) was mounted into a bowl shaped metal tip. This rubber tip was lowered at 23° and measured to strike at 1.8mm caudal to bregma and 3.0mm left of the midline at a 1.0 mm depth, a velocity of 4.0 m/s ± 0.2 and a 50 ms duration.

MRS studies were performed on a 7-Tesla Bruker Biospec AVIII spectrometer 7 days after the last impact. Rats were anesthetised via nose cone with 2% Isoflurane and (1:1) N₂O/O₂. Single voxel ¹H spectroscopy was carried out using water signal suppression with variable power radiofrequency (RF) pulses with optimized relaxation delays (VAPOR)⁴. Outer volume suppression combined with point-resolved spectroscopy (PRESS)⁵ sequence from a 2×2×2 mm³ voxel placed in the hippocampal area was used for signal acquisition, with TR/TE = 1800/40 ms, spectral bandwidth = 3 kHz, number of data points = 2048, number of averages = 576. The 1D proton spectra were analyzed off-line using MestReNova 10.0 software (Mestrelab Research SL, Spain).

A DTI data set covering the entire brain was collected using a multislice spin echo sequence with 1 reference and 20 non-collinear diffusion-weighted images with the following parameters: TE/TR = 25/4000 ms, 4 averages, matrix size = 256 x 256, field of view = 36 x 36 mm, 11 slices, slice thickness = 1 mm, b-value = 1200 s/mm²,and Δ/δ = 12/6 ms. DSI Studio (http://dsi-studio.labsolver.org/) was used to analyze DTI data. Diffusion imaging data was fitted to yield fractional anisotropy (FA), axial and radial diffusivity (AD and RD respectively). Data were analyzed using ANOVA and expressed as Mean and Std Dev.

After mTBI and rmTBI, the NAA/Cr ratio was sig­nifi­cantly re­duced (p=0.03, p<0.0001, respectively), and the Ins/Cr ratio sig­nifi­cantly in­creased (p=0.017, p=001, respectively) when com­pared to sham controls (Figure 1). There was a small, but sig­nifi­cant, further re­duction (p=0.01) in the NAA/Cr ratio after rmTBI verus mTBI. mTBI resulted in significant changes in FA, RD and AD for several brain regions such as the corpus callosum, hippocampus and external capsule. Repetitive injury (rmTBI) produced significantly decreased FA values when compared with the single injury (mTBI) (Figure 2).

¹H-MRS and DTI can identify metabolic and structural alterations, 7 days after mTBI and rmTBI in the hippocampus which appear normal on histological analysis and conventional MR images and show up as subtle changes with silver staining and microglial immunohistochemistry. Alteration in NAA and Ins after mTBI and rmTBI likely reflect neuro­axonal damage and glial pro­liferation, respectively. Reduced FA and increased AD in the white matter reflects a loss of integrity an indication of damage to myelin/axonal membranes and/or demyelination³.

Future studies combining longitudinal advanced neuroimaging with behavioral testing could further our understanding of pathophysiology of mTBI and assess the vulnerable period for worsened injury after rmTBI in the immature brain.