Lack of chronic neuroinflammation in the absence of focal hemorrhage in a rat model of low-energy blast-induced TBI

Abstract

Blast-related traumatic brain injury (TBI) has been a common cause of injury in the recent conflicts in Iraq and Afghanistan. Blast waves can damage blood vessels, neurons, and glial cells within the brain. Acutely, depending on the blast energy, blast wave duration, and number of exposures, blast waves disrupt the blood-brain barrier, triggering microglial activation and neuroinflammation. Recently, there has been much interest in the role that ongoing neuroinflammation may play in the chronic effects of TBI. Here, we investigated whether chronic neuroinflammation is present in a rat model of repetitive low-energy blast exposure. Six weeks after three 74.5-kPa blast exposures, and in the absence of hemorrhage, no significant alteration in the level of microglia activation was found. At 6 weeks after blast exposure, plasma levels of fractalkine, interleukin-1β, lipopolysaccharide-inducible CXC chemokine, macrophage inflammatory protein 1α, and vascular endothelial growth factor were decreased. However, no differences in cytokine levels were detected between blast-exposed and control rats at 40 weeks. In brain, isolated changes were seen in levels of selected cytokines at 6 weeks following blast exposure, but none of these changes was found in both hemispheres or at 40 weeks after blast exposure. Notably, one animal with a focal hemorrhagic tear showed chronic microglial activation around the lesion 16 weeks post-blast exposure. These findings suggest that focal hemorrhage can trigger chronic focal neuroinflammation following blast-induced TBI, but that in the absence of hemorrhage, chronic neuroinflammation is not a general feature of low-level blast injury.

Fig. 1

Three low-level 74.5-kPa blast exposures do not result in microglial activation. Hippocampal microglia in Vibratome-cut sections visualized by Iba1-peroxidase immunohistochemistry as described. Control (a); blast (b). Scale bar, 10 μm

Fig. 2

Similar local densities of microglia and microglial subtypes in the hippocampus and prefrontal cortex 6 weeks following blast exposure. Estimated densities of total microglia and microglial subtypes are shown for the hippocampus (a) and prefrontal cortex (b). Panel (c) shows examples of microglial subtypes. Error bars indicate the standard error of the mean (SEM). There was no statistically significant difference between blast and control

Fig. 3

Lack of activated proinflammatory Iba1⁺ MHCII⁺ microglia in the hippocampus of blast-exposed animals (6 weeks post-blast exposure). Iba1, green; MHCII, red; DAPI, blue. Similar negligible presence of MHCII⁺ microglia (<1% of Iba1⁺ cells) was observed in the hippocampus of blast exposed (a-c) and control animals (d-f). Merged images correspond to Panels c and f, respectively. Scale bar, 100 μm. Panels g-i show MHCII⁺ cells residing in the meninges surrounding the motor cortex of a blast-exposed animal (positive control). Merged image is shown in Panel i. Scale bar, 20 μm

Fig. 4

Selected cytokines in the brains of control and blast-exposed animals (6 weeks post-blast exposure). Shown are levels of IL-1β, IL-6 and IL-10 in the left (L) or right (R) hemispheres of the indicated brain regions. Error bars indicate the standard error of the mean (SEM). Statistical differences indicated represent unpaired t-tests

Fig. 5

Selected cytokines in the brains of control and blast-exposed animals (40 weeks post-blast exposure). Shown are levels of IL-1β, IL-6 and IL-10 in the left (L) or right (R) hemispheres of the indicated brain regions. Error bars indicate the standard error of the mean (SEM). Statistical differences indicated represent unpaired t-tests

Fig. 6

Focal tear and hemorrhage associated with microglia activation in the rat brain 16 weeks post-blast exposures. HE staining (a, b). Black arrows indicate location of a blood clot. Scale bars: (a), 500 μm; (b), 200 μm. Brain sections were stained with Iba1 (c) and GFAP (d) and counterstained with DAPI (e). Arrows in Panel (c) indicate the areas next to the focal tissue tear devoid of microglia. Letters inside Panel (c) indicate the relative location of areas illustrated in Fig. 7. Merged image (f). Scale bar, 200 μm

Fig. 7

Distribution of microglia in the vicinity of a blast-induced hemorrhagic focal tear. The relative localization of the different panels in the brain of the blast-exposed animal is indicated in Fig. 6 (e). Panels (a-c), Iba 1 (green) and GFAP (red) immunostaining. Absence of microglia (green) in the molecular layer of the dentate gyrus immediately next to the blast-induced tear (a). Presence of reactive and ameboid microglia in the molecular layer of the dentate gyrus (b) and in the stratum lucidum (c) of the hippocampus, away from the tear. Microglial activation gradient, Iba 1 (green) immunostaining (d-f). Primed and reactive (types 2–3) microglia in the cortex (d, e). Ameboid (type 4) microglia in the region associated with the molecular layer of the dentate gyrus away from the tear (f). Scale bar, 50 μm