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. 2012 Jan 20;29(2):385-93.
doi: 10.1089/neu.2011.2053. Epub 2011 Oct 26.

Ghrelin Prevents Disruption of the Blood-Brain Barrier After Traumatic Brain Injury

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Free PMC article

Ghrelin Prevents Disruption of the Blood-Brain Barrier After Traumatic Brain Injury

Nicole E Lopez et al. J Neurotrauma. .
Free PMC article

Abstract

Significant effort has been focused on reducing neuronal damage from post-traumatic brain injury (TBI) inflammation and blood-brain barrier (BBB)-mediated edema. The orexigenic hormone ghrelin decreases inflammation in sepsis models, and has recently been shown to be neuroprotective following subarachnoid hemorrhage. We hypothesized that ghrelin modulates cerebral vascular permeability and mediates BBB breakdown following TBI. Using a weight-drop model, TBI was created in three groups of mice: sham, TBI, and TBI/ghrelin. The BBB was investigated by examining its permeability to FITC-dextran and through quantification of perivascualar aquaporin-4 (AQP-4). Finally, we immunoblotted for serum S100B as a marker of brain injury. Compared to sham, TBI caused significant histologic neuronal degeneration, increases in vascular permeability, perivascular expression of AQP-4, and serum levels of S100B. Treatment with ghrelin mitigated these effects; after TBI, ghrelin-treated mice had vascular permeability and perivascular AQP-4 and S100B levels that were similar to sham. Our data suggest that ghrelin prevents BBB disruption after TBI. This is evident by a decrease in vascular permeability that is linked to a decrease in AQP-4. This decrease in vascular permeability may diminish post-TBI brain tissue damage was evident by decreased S100B.

Figures

FIG. 1.
FIG. 1.
(A) Histological examination of tissue 200 μm medial to the cortical impact site reveals that compared to sham animals, traumatic brain injury (TBI) caused significant neuron degeneration as evidenced by neuronal contraction (solid arrows) in the neocortex with increased vacuolization (open arrows) and axonopathy in the neuropil. Ghrelin-treated animals (TBI/G) had decreased cortical degeneration and axonopathy. (B) Hippocampal regions, CA1, and dentate gyrus show TBI-induced degenerating neurons (solid arrows) and vacuolization (open arrows). Administration of ghrelin also blunted neuronal degeneration in these areas.
FIG. 2.
FIG. 2.
Mice were injected with 70-kDa FITC-dextran permeability tracer, and vascular permeability was measured. Representative images of a 1-mm-thick brain section are shown for sham, for TBI, TBI/ghrelin (TBI/G). Vascular permeability (VP) to 70-kD FITC-dextran was quantified. TBI increases VP compared to sham animals (mean difference 5.81E+08; *p<0.001), and ghrelin significantly reduced arbitrary fluorescent intensity compared to TBI alone (mean difference 4.30E+08, #p<0.01; +p=NS versus sham). Error bars represent standard deviation.
FIG. 3.
FIG. 3.
Immunohistochemical staining of injured sections using an anti-CD31 antibody (red) for endothelial cells, and anti-aquaporin-4 (AQP-4) antibody (green) to localize vessel-associated astrocytic end-feet is shown. There is increased AQ4 immunoreactivity in traumatic brain injury (TBI) mice compared to exposure-matched sham images. This increase is mitigated by treatment with ghrelin (TBI/G).
FIG. 4.
FIG. 4.
Quantification of aquaporin-4 (AQP-4) immunoreactivity in injured area of cortex is shown. AQP-4 immunoreactivity is significantly increased in traumatic brain injury (TBI) mice compared to sham animals (mean difference 1.65; *p<0.05). The ghrelin-treated TBI (TBI/G) group had significantly reduced fluorescence compared to TBI alone (mean difference 2.35, #p<0.005; +p=NS versus sham). Error bars represent standard deviation.
FIG. 5.
FIG. 5.
(A) Immunohistochemical staining of injured hemisphere using DAPI (blue) to locate cell nuclei, and GFAP (red) to identify reactive astrocytes. At 6 h following TBI astrocytes in the neocortex of the ipsilateral hemisphere show equivocal increases in GFAP staining such that GFAP in the neocortex of all three groups are similar. (B) Immunohistochemical staining of injured hemisphere using DAPI (blue) to locate cell nuclei, and GFAP (red) to identify reactive astrocytes. At 6 h following TBI astrocytes in the corpus callosum of the ipsilateral hemisphere display a marked increase in GFAP staining. This reactive astrogliosis is attenuated by treatment with ghrelin (TBI/G, TBI with ghrelin; GFAP, glial fibrillary acidic protein; DAPI, 4′,6-diamidino-2-phenylindole).
FIG. 6.
FIG. 6.
(A) Immunoblotting of injured hemisphere for S100B, a marker of astrocyte injury, with β-actin control. (B) Quantification of relative band density of S100B. There is a fourfold increase in brain S100B expression following TBI (*p<0.05). Animals treated with ghrelin (TBI/G) exhibited S100B levels similar to sham animals (#p<0.05 versus TBI; +p=NS versus sham). Error bars represent standard deviation.

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