Increase in blood-brain barrier permeability, oxidative stress, and activated microglia in a rat model of blast-induced traumatic brain injury

Abstract

Traumatic brain injury (TBI) as a consequence of exposure to blast is increasingly prevalent in military populations, with the underlying pathophysiological mechanisms mostly unknown. In the present study, we utilized an air-driven shock tube to investigate the effects of blast exposure (120 kPa) on rat brains. Immediately following exposure to blast, neurological function was reduced. BBB permeability was measured using IgG antibody and evaluating its immunoreactivity in the brain. At 3 and 24 hr postexposure, there was a transient significant increase in IgG staining in the cortex. At 3 days postexposure, IgG immunoreactivity returned to control levels. Quantitative immunostaining was employed to determine the temporal course of brain oxidative stress following exposure to blast. Levels of 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT) were significantly increased at 3 hr postexposure and returned to control levels at 24 hr postexposure. The response of microglia to blast exposure was determined by autoradiographic localization of (3) H-PK11195 binding. At 5 days postexposure, increased binding was observed in the contralateral and ipsilateral dentate gyrus. These regions also displayed increased binding at 10 days postexposure; in addition to these regions there was increased binding in the contralateral ventral hippocampus and substantia nigra at this time point. By using antibodies against CD11b/c, microglia morphology characteristic of activated microglia was observed in the hippocampus and substantia nigra of animals exposed to blast. These results indicate that BBB breakdown, oxidative stress, and microglia activation likely play a role in the neuropathology associated with TBI as a result of blast exposure.

Fig. 1

Effect of BOP on acute neurological functioning immediately following exposure. Exposure to blast (full columns) significantly delayed the return of corneal and paw flexion reflexes compared to control (open columns). The data represents the mean duration of suppression ± SEM. Significance (t-test) is denoted as follows: #p<0.05, *p<0.01 vs. control.

Fig. 2

Representative images of H & E stained brain sections of control (A) and at 3 days post blast 120 kPa (B). There were no overt signs of tissue disruption or cell loss. Scale bar denotes 1 mm.

Fig. 3

Effect of 120 kPa BOP on brain IgG immunoreactivity. The images are representative for A - Control animals; B - 0.5 h post blast; C - 3 h post blast; D - 24 h post blast; E - 3 days post blast. Increased IgG immunoreactivity in layer I of the cortex was observed at 3 and 24 h following exposure. Although not significant there was increased staining in the area surrounding the lateral ventricles (4 out of 6 brains) at 0.5 h post blast. There was no difference in IgG staining in brains 3 days post exposure compared with controls. Scale bar denotes 1 mm.

Fig. 4

Quantification of IgG immunoreactivity in brain sections after exposure to blast. Significant increases in IgG staining were observed at 3 and 24 h after exposure compared to control. The data represents the percentage of brain area stained for IgG ± SEM. Significance with ANOVA followed by post hoc analysis (SNK) as follows: *p<0.01 vs. control; #p<0.05 vs. 72 hr.

Fig. 5

Quantification of 4HNE (A) and 3NT (B) levels in brain sections after exposure to blast. Significant increases in 3NT and 4HNE levels were observed at 3 h after exposure compared to control. At 24 h after exposure, 3 NT and 4HNE levels returned to control values. The data represents the mean fluorescence ± SEM. Significance with ANOVA followed by post hoc analysis (SNK) as follows: *p<0.05.

Fig. 6

Effect of exposure to 120 kPa BOP on TPSO expression. Pseudocolored autoradiogram of brain sections labeled with [³H]PK11195 in control and in 5 or 10 days post exposure animals. Four different levels are shown: A - anterior striatum; B - dorsal hippocampus; C - dorsolateral thalamus; D - ventral hippocampus. Brain regions abreviated as follows: primary motor cortex (M1), striatum (St), dentate gyrus (DG), dorsolateral thalamus (DT), ventral hippocampus (VH), and substantia nigra (SN). Prominent areas of high TPSO density in control animals are restricted to ventricular/choroid plexus regions. High density areas of TPSO in exposed animals are the dentate gyrus and substantia nigra. The scale shown at bottom shows the range from low to high expression.

Fig. 7

Effect of 120 kPa BOP on microglia morphology in selected brain loci. In order to confirm PK11195 autoradiographic data demonstrating microglial activation in the dentate gyrus and substantia nigra, antibodies against CD11b/c were employed to visualize microglial morphology. In sham animals all regions that were evaluated contained microglia with resting morphology characterized by highly ramified processes (A-C). In the cortex of animals exposed to blast microglia also displayed resting morphology (D). Exposure to blast resulted in activated microglial morphology characterized by microglial clustering in the hippocampus (dentate gyrus, E). In addition activated microglia morphology in the substantia nigra is revealed by microglia with rounded cell bodies and reduced ramifications (F). Scale bar = 50 μm.