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. 2018 May 15;35(10):1192-1203.
doi: 10.1089/neu.2017.5401. Epub 2018 Jan 24.

Rat Model of Brain Injury to Occupants of Vehicles Targeted by Land Mines: Mitigation by Elastomeric Frame Designs

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Free PMC article

Rat Model of Brain Injury to Occupants of Vehicles Targeted by Land Mines: Mitigation by Elastomeric Frame Designs

Flaubert Tchantchou et al. J Neurotrauma. .
Free PMC article

Abstract

Many victims of blast traumatic brain injury (TBI) are occupants of vehicles targeted by land mines. A rat model of under-vehicle blast TBI was used to test the hypothesis that the ensuing neuropathology and altered behavior are mitigated by vehicle frame designs that dramatically reduce blast-induced acceleration (G force). Male rats were restrained on an aluminum platform that was accelerated vertically at up to 2850g, in response to detonation of an explosive positioned under a second platform in contact with the top via different structures. The presence of elastomeric, polyurea-coated aluminum cylinders between the platforms reduced acceleration by 80% to 550g compared with 2350g with uncoated cylinders. Moreover, 67% of rats exposed to 2850g, and 20% of those exposed to 2350g died immediately after blast, whereas all rats subjected to 550g blast survived. Assays for working memory (Y maze) and anxiety (Plus maze) were conducted for up to 28 days. Rats were euthanized at 24 h or 29 days, and their brains were used for histopathology and neurochemical measurements. Rats exposed to 2350g blasts exhibited increased cleaved caspase-3 immunoreactive neurons in the hippocampus. There was also increased vascular immunoglobulin (Ig)G effusion and F4/80 immunopositive macrophages/microglia. Blast exposure reduced hippocampal levels of synaptic proteins Bassoon and Homer-1, which were associated with impaired performance in the Y maze and the Plus maze tests. These changes observed after 2350g blasts were reduced or eliminated with the use of polyurea-coated cylinders. Such advances in vehicle designs should aid in the development of the next generation of blast-resistant vehicles.

Keywords: acceleration; blast; blood–brain barrier; inflammation; synapses.

Conflict of interest statement

No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
Experimental design. Detailed timeline of behavioral tests and end-points of experimental procedures starting on study day 0 (blast or sham blast exposure). YM, Y-maze test; PM, plus-maze test; H, histology; WB, Western blots; E, euthanasia; TC, tissue collection.
<b>FIG. 2.</b>
FIG. 2.
Coating of cylinders with polyurea is necessary for mitigation of behavioral deficits following under-vehicle blast. Rats were restrained on the top of a simulated vehicle that incorporated either uncoated or polyurea-coated aluminum cylinders, resulting in maximal acceleration of 2350g and 550g, respectively. (A) Polyurea-coated cylinders protected rats from loss of spontaneous alternation in the Y maze at day 0 (*,#p < 0.05, ##p < 0.01; n = 10–16) and at day 6 (##p < 0.01, ***p < 0.001). (B) Polyurea-coated cylinders were also effective at protecting against anxiety behavior, which was inversely related to the time rats spent in the open arms of the elevated Plus maze. (***p < 0.001, #p < 0.05, *p < 0.05; n = 10–16).
<b>FIG. 3.</b>
FIG. 3.
Increased perivascular immunoglobulin (Ig)G effusion in the brain post-blast and by incorporation of polyurea-coated cylinders. (A) Representative microscopic images exhibiting IgG immunoreactivity in different brain regions at 24 h post-blast or post-sham blast. Scale bar is 50 μm. (B) Quantification of the percentage of the cerebral cortex area that was immunopositive for IgG. There was a significant, 700% increase in rat cortical IgG immunostaining following blasts generating 2300g compared with sham animals (**p < 0.01, n = 5) and a 100% increase compared with the area present following blasts with vehicles that incorporated polyurea-coated cylinders (*p < 0.05; n = 6).
<b>FIG. 4.</b>
FIG. 4.
Increased intercellular adhesion molecule 1 (ICAM-1) expression in the brain following under-vehicle blast and mitigation by incorporation of polyurea-coated cylinders. (A) Representative microscopic images expression of ICAM-1 (green) and glucose transporter 1 (GluT-1) (red) in immunostained rat brain sections at 24 h post-blast. Size bar is 100 μm. (B) Quantification of the area of ICAM-1 immunoreactivity indicating significantly increased levels of ICAM-1 in the brains (n = 5) of rats subjected to blast intensity with incorporation of uncoated cylinders compared with shams (**p < 0.01; n = 6). ICAM-1 immunostaining was not significantly different between shams and after blasts that incorporated the polyurea-coated cylinders.
<b>FIG. 5.</b>
FIG. 5.
Activated microglia/microphages following under-vehicle blasts and mitigation by incorporation of polyurea coated cylinders. (A) Representative fluorescent images of F4/80 immunopositive cells (green) showing overlap with inducible nitric oxide synthase (iNOS) immunopositive cells (red) and 4′,6-diamidino-2-phenylindole (DAPI) as counterstain (blue). Scale bar is 100 μm. (B) Quantification of the F4/80 immunopositive area in the cortex suggests dramatic increase of activated microglia/microphages in the brains of rats subjected to lethal blast intensity with no polyurea coating compared with sham rats (n = 5, **p < 0.01). Levels of activated microglia/microphages were significantly reduced (n = 6; *p < 0.05) in the brains of rats exposed to blast with polyurea coating mitigation system.
<b>FIG. 6.</b>
FIG. 6.
Synaptic protein density decreased in rat dendate gyrus subgranular layer (SGL) 24 h post-injury, protected by the advanced hull design system. (A) Representative fluorescent images demonstrating the presence of the pre-synaptic protein Bassoon (red) and the post-synaptic protein Homer-1 (green), and 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei in the dendate gyrus SGL of the rat hippocampus. Scale bar is 100 μm. (B) Quantitation of Bassoon, Homer-1, and Bassoon/Homer-1 co-localized puncta density indicated a significant decrease in Homer-1 and Bassoon/Homer-1 co-localized puncta in the SGL of rats exposed to lethal blast intensity with no polyurea coating compared with sham (n = 5; *p < 0.05 and **p < 0.01). Blast-induced impairment in synaptic proteins expression was significantly prevented by the polyurea coating mitigation system (n = 6; #p < 0.05).
<b>FIG. 7.</b>
FIG. 7.
Synaptic protein density decreased in the rat cornu ammonis (CA)2 region 24 h post-injury, protection by the advanced hull design system. (A) Representative fluorescent images demonstrating the presence pre-synaptic protein bassoon (red) and post-synaptic protein Homer-1 (green) and 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei in the CA2 region of the rat hippocampus. Scale bar is 100 μm. (B) Quantitation of Bassoon, Homer-1, and Bassoon/Homer-1 co-localized puncta density indicated a significant decrease in Homer-1 and Bassoon/Homer-1 co-localized puncta in the CA2 region of rats exposed to lethal blast intensity with no polyurea coating compared with sham (n = 5; *p < 0.05 and **p < 0.01). Blast-induced impairment in synaptic proteins expression was relatively prevented by the polyurea coating mitigation system (n = 6).
<b>FIG. 8.</b>
FIG. 8.
Cleaved caspase 3 immuno-positive cells present in rat hippocampus. (A) Representative fluorescent images showing cleaved caspase 3 immunopositive cells (green) overlapping mostly with NeuN immunoreactive cells (red), and 4′,6-diamidino-2-phenylindole (DAPI) for nuclei counterstaining. Scale bar is 100 μm. (B) Quantitation indicated significant increase in cleaved caspase 3 immunoreactive cells in the dentate gyrus and cornu ammonis (CA)2/3 region of rats subjected to blast with no polyurea coating compared with sham animals (n = 5; *p < 0.05, and ***p < 0.001). The advanced hull design significantly reduced the blast-initiated hippocampal apoptotic cell loss (n = 6; #p < 0.05).
<b>FIG. 9.</b>
FIG. 9.
Decreased expression of cell survival promoting proteins and increased expression of pro-apoptotic protein in rat hippocampus 24 h post-blast, and mitigation by the shock-absorbing hull design system. (A,B) Representative immunoblots illustrating the expression of cell survival promoting protein phospho-extracellular signal-regulated kinase (ERK), anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and pro-apoptotic marker α II spectrin in rat hippocampus. (C–E) Quantitation of protein band signal intensity showed, compared with sham rats, a significant decrease in phospho-ERK and Bcl-2 (n = 6, **,##p < 0.01), and a drastic increase in α ii spectrin (n = 6, ***p < 0.001) in the hippocampus of rats exposed to lethal blast intensity with no polyurea coating compared with sham rats. Rats subjected to blast with advanced hull mitigation design still showed significantly lower Bcl-2 expression levels than shams (n = 6; #p < 0.05). However, the advanced hull mitigation design abrogated the deleterious effect of blast exposure on the expression phospho-ERK and α II spectrin (n = 6; *p < 0.05, and **p < 0.01, respectively).

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