Effects of antioxidant treatment on blast-induced brain injury

Abstract

Blast-induced traumatic brain injury has dramatically increased in combat troops in today's military operations. We previously reported that antioxidant treatment can provide protection to the peripheral auditory end organ, the cochlea. In the present study, we examined biomarker expression in the brains of rats at different time points (3 hours to 21 days) after three successive 14 psi blast overpressure exposures to evaluate antioxidant treatment effects on blast-induced brain injury. Rats in the treatment groups received a combination of antioxidants (2,4-disulfonyl α-phenyl tertiary butyl nitrone and N-acetylcysteine) one hour after blast exposure and then twice a day for the following two days. The biomarkers examined included an oxidative stress marker (4-hydroxy-2-nonenal, 4-HNE), an immediate early gene (c-fos), a neural injury marker (glial fibrillary acidic protein, GFAP) and two axonal injury markers [amyloid beta (A4) precursor protein, APP, and 68 kDa neurofilament, NF-68]. The results demonstrate that blast exposure induced or up-regulated the following: 4-HNE production in the dorsal hippocampus commissure and the forceps major corpus callosum near the lateral ventricle; c-fos and GFAP expression in most regions of the brain, including the retrosplenial cortex, the hippocampus, the cochlear nucleus, and the inferior colliculus; and NF-68 and APP expression in the hippocampus, the auditory cortex, and the medial geniculate nucleus (MGN). Antioxidant treatment reduced the following: 4-HNE in the hippocampus and the forceps major corpus callosum, c-fos expression in the retrosplenial cortex, GFAP expression in the dorsal cochlear nucleus (DCN), and APP and NF-68 expression in the hippocampus, auditory cortex, and MGN. This preliminary study indicates that antioxidant treatment may provide therapeutic protection to the central auditory pathway (the DCN and MGN) and the non-auditory central nervous system (hippocampus and retrosplenial cortex), suggesting that these compounds have the potential to simultaneously treat blast-induced injuries in the brain and auditory system.

Figure 1. Examples of 4-HNE immunofluorescence images obtained from the dorsal hippocampal commissure of the hippocampus from either NC (A), 24H-B (B) or 24H-B/T (C) groups.

No positive 4-HNE staining was observed in the NC group. Many 4-HNE positive cells were observed in the 24H-B group (arrows in B), and a reduced number of 4-HNE positive cells were observed in the 24H-B/T group (arrows in C). 4-HNE positive cells in the hippocampus were quantified and statistically analyzed (D). Four to six sections from each rat brain (6 rats in each group) were used in this analysis. Significantly increased numbers of 4-HNE-positive cells were found in the 24H-B group compared to the NC group (p < 0.001). Significant differences were also found between the 24H-B and 24H-B/T groups (p < 0.001), suggesting that antioxidant treatment reduced 4-HNE production in the hippocampus at this time point after blast exposure. *** indicate p < 0.001. Error bars represent standard error of the means. Scale bar in C = 10 µm for A-C.

Figure 2. Examples of c-Fos immunostaining images obtained from the granular RC of the NC (A), 3H-B, (B), and 3H-B/T (C) groups by light microscopy.

Few c-fos-positive cells were observed in layer three in the cortex of the NC group (A). Numerous c-fos-positive stained cells were seen in layer three in the cortex of the 3H-B group (B), and decreased numbers of c-fos-positive cells were seen in the cortex of the 3H-B/T group relative to the 3H-B group (C). C-fos-positive cells in the RC were quantified and statistically analyzed (D). Four to six sections (8-12 images) from each rat brain (6 rats in each group) were used in this analysis. Significantly increased numbers of c-fos-positive cells were found in the 3H-B and 7D-B groups compared to the NC group (all p < 0.001). Significant differences were also found between the 3H-B and 3H-B/T groups (p < 0.001), suggesting that antioxidant treatment suppressed c-fos upregulation in the cortex at this time point after blast exposure. However, no significant difference was found between the 7D-B and 7D-B/T groups (p > 0.05). C-fos-positive cells in the DCN were quantified and statistically analyzed (E). Significantly increased numbers of c-fos-positive cells were found in the lateral region of the DCN in the 3H-B and 3H-B/T groups compared to the NC group (all p < 0.05), however no antioxidant treatment effect was observed in the DCN at this time point (all p > 0.05). ### indicates p < 0.001 compared to normal controls. *** indicate p < 0.001 compared to the blast only group. Error bars represent standard error of the means. Scale bar in C = 500 µm for A-C.

Figure 3. A low magnification image of the DCN is shown in A.

The DCN is divided into three parts (dashed lines in A): medial, middle, and lateral. The squares in A indicate where images were collected for cell counting. Examples of GFAP staining (arrows in B-D) in the middle region of the DCN of the NC (B), 21D-B (C) and 21D-B/T (D) groups. ML, FCL, and DL in B demarcate the molecular layer, fusiform cell layer, and deep layer, respectively. Scale bar in A = 500 µm, in D = 200 µm in for B-D.

Figure 4. GFAP-positive cells in the DCN of the NC, 21D-B, and 21D-B/T (A) or 7D-B, and 7D-B/T (B) groups were quantified and statistically analyzed.

Two to three DCN sections from each rat brainstem (6-7 rats in each group) were used in these analyses. Significantly increased GFAP expression is observed in the lateral and middle regions of the 21D-B and 7D-B groups compared to the NC group (all p < 0.05). Decreased numbers of GFAP-positive cells were observed in these two regions in the 21D-B/T group compared to the 21D-B group (A, all p < 0.05), suggesting a treatment effect in these regions at this time point after blast exposure. However, no treatment effect was observed in GFAP expression in these regions 7 days after blast exposure (B, all p > 0.05). No significant difference was observed in the medial region of the DCN of the 21D or 7D groups compared to the NC group (all p > 0.05). Error bars represent standard error of the means. * indicate p < 0.05.

Figure 5. Examples of NF-68 expression (arrows in B-G) in the MGN (B-D) and MoDG of the hippocampus (E-G) from the NC (B and E), 21D-B (C and F) and 21D-B/T (D and G) cohorts.

A low magnification image of the relevant brain region for NF-68 staining is shown in A. The squares in A indicate where images were collected from the MoDG and the MGN for NF-68-positive axon counting and statistical analyses (H). Four to five images were taken from each MGN section and 3-4 MGN sections from each rat brain were used. One image was taken from each MoDG section and six MoDG sections from each rat brain (6 rats in each group) were used in these analyses. Significantly increased numbers of NF-68-positive axons were observed in the MGN and MoDG of the 21D-B group compared to the NC group (all p < 0.001). Significantly decreased numbers of NF-68-positive axons were observed in the MGN and MoDG in the 21D-B/T group relative to the 21D-B group (p < 0.001 or 0.05). Error bars represent standard error of the means. Scale bar = 50 µm in G for B-G, = 500 µm in A.

Figure 6. Examples of APP immunolabeling in the hippocampus of the NC (A), 24H-B (B) and 24H-B/T (C) groups.

No positive APP staining was observed in the hippocampus of normal controls (A). Strong positive APP labeling was observed in the hippocampus of the 24H-B group (arrows in B). Decreased APP expression was observed in the hippocampus of the 24H-B/T group relative to the 24H-B group (arrows in C). APP-positive labeling in the hippocampus was quantified and statistically analyzed (D). Two to three hippocampal sections from each rat brain (6 rats in each group) were used in these analyses. Significantly increased APP expression was observed in the hippocampus of the 24H-B group compared to the NC group (p < 0.001). An antioxidant treatment effect was found at 24 hours after blast exposure (p < 0.001), however no significant difference was observed between the treated and untreated groups 7 days after blast exposure (7D-B v.s. 7D-B/T, all p > 0.05). Error bars represent standard error of the means. Scale bar = 10 µm in C for A-C. *** indicate p < 0.001.