Activation of P2X7 promotes cerebral edema and neurological injury after traumatic brain injury in mice.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Cerebral edema, the abnormal accumulation of fluid within the brain parenchyma, contributes to elevated intracranial pressure (ICP) and is a common life-threatening neurological complication following TBI. Unfortunately, neurosurgical approaches to alleviate increased ICP remain controversial and medical therapies are lacking due in part to the absence of viable drug targets. In the present study, genetic inhibition (P2X7-/- mice) of the purinergic P2x7 receptor attenuated the expression of the pro-inflammatory cytokine, interleukin-1β (IL-1β) and reduced cerebral edema following controlled cortical impact, as compared to wild-type mice. Similarly, brilliant blue G (BBG), a clinically non-toxic P2X7 inhibitor, inhibited IL-1β expression, limited edemic development, and improved neurobehavioral outcomes after TBI. The beneficial effects of BBG followed either prophylactic administration via the drinking water for one week prior to injury or via an intravenous bolus administration up to four hours after TBI, suggesting a clinically-implementable therapeutic window. Notably, P2X7 localized within astrocytic end feet and administration of BBG decreased the expression of glial fibrillary acidic protein (GFAP), a reactive astrocyte marker, and attenuated the expression of aquaporin-4 (AQP4), an astrocytic water channel that promotes cellular edema. Together, these data implicate P2X7 as a novel therapeutic target to prevent secondary neurological injury after TBI, a finding that warrants further investigation

Brain expression of P2X7.

<p>(<b>A</b>) Representative Western blots (top panel) of P2X7 in the cerebral cortex of mice following sham injury, TBI, or TBI +50 mg/kg BBG. Tissue was collected at 12h or 24h after TBI. Blots were normalized to β-actin to control for equal protein loading between lanes. Data are representative of six mice/group. Densitometric analysis of Western blots (bottom panel) is presented as normalized P2X7 expression. (<b>B</b>) Cellular localization of P2X7 in the mouse cerebral cortex by dual immunfluorescence. Brains were immunolabeled for P2X7 (green) and AQP4 (red), a marker of astrocytic endfeet. Confocal images (top panel, 25x objective; bottom panel, 40x objective) were obtained from the pericontusional cortex. Scale bar  = 20 µm.</p

Distribution of BBG after TBI.

<p>(<b>A</b>) Photograph of representative mice following an intravenous administration of placebo (left) or BBG (50 mg/kg; right). Note the blue appearance in the skin, eyes, ears, paws and tail. (<b>B</b>) BBG accumulates in the contused cortex after TBI. Photographs of brains taken from a sham-operated mouse administered placebo (left panel), a mouse administered placebo at 0.5h after TBI (middle panel), or a mouse administered 50 mg/kg BBG via the tail vein at 0.5h post-TBI.</p

Genetic inhibition of P2X7 attenuates cerebral edema after TBI.

<p>(<b>A</b>) P2X7−/− mice exhibited a significant reduction in brain water content, as compared to wild-type mice, when assessed at 24h post-TBI. Comparisons within each hemisphere between different treatments groups were done using a one-way ANOVA followed by Dunnett's post-hoc test (* p<0.05 vs. the ipsilateral hemisphere in sham-operated mice). No significant differences in cerebral edema were observed between groups in the contralateral hemisphere. (<b>B</b>) P2X7−/− mice displayed attenuated cerebral edema, as compared to wild-type mice, when assessed by MRI. The top panels depict a representative wild-type and a P2X7−/− mouse imaged at 24h post-TBI. Bottom panels represent the mean edemic volume of mice imaged by MRI. Data are represented as the mean ± SEM from six mice/group and were analyzed using a t-test (p<0.01 vs. wild-type).</p

Effect of P2X7 inhibition on cortical lesion volume after TBI.

<p>Quantification of cortical lesion volume following placebo or BBG (50 mg/kg, i.p) treatment. Lesion volume is expressed as mm³. Data were analyzed using a t-test (*p<0.05 vs. placebo).</p

Antagonism of P2X7 reduces cerebral edema after TBI.

<p>(<b>A</b>) A single intravenous bolus of 50 mg/kg BBG provided 15 minutes prior to TBI, significantly reduced the development of cerebral edema at 24h post-TBI, as measured by brain water content. (<b>B</b>) A single intravenous bolus of 50–100 mg/kg BBG administered 0.5h after TBI significantly reduced cerebral edema at 24h post-TBI. (<b>C</b>) Administration of a single intravenous bolus of 50 mg/kg BBG reduced cerebral edema when administered 1h or 4h after injury. This effect was lost if post-treatment was delayed beyond 8h from the time of injury. (<b>D</b>) Prophylactic treatment with BBG in the drinking water for 7 days reduced edema at 24h post-TBI at a concentration of 25 mg/ml but not 10mg/ml. Comparisons within each hemisphere between different treatments groups were done using a one-way ANOVA followed by Dunnett's post-hoc test (*p<0.05, **p<0.01, ***p<0.001 vs. the ipsilateral hemisphere in sham-operated mice). No significant differences in cerebral edema were observed between groups in the contralateral hemisphere. Data are represented as the mean ± SEM from 5–6 mice/group.</p

BBG attenuates glial activation.

<p>(<b>A</b>) Representative Western blot (left panel) of cortical GFAP expression taken at 12h or 24h after sham injury, TBI, or TBI +50 mg/kg BBG. (<b>B</b>) Representative Western blot (left panel) of AQP4 in the cerebral cortex of mice at 12h following sham injury, TBI, or TBI +50 mg/kg BBG. Densitometric analysis of Western blots (right panels) is presented as either GFAP or AQP4 expression following normalization to β-actin, which was used to control for equal protein loading. Data (mean ± SEM) are representative of six mice/group from three independent experiments (n = 3/group in each experiment) and are expressed as % change vs. sham. Data were analyzed by One-Way ANOVA followed by Dunnett's post-hoc test (* p<0.05, ** p<0.01 vs. sham operated mice).</p

Inhibition of P2X7 attenuates post-traumatic IL-1β expression.

<p>A single intravenous bolus of 50–100 mg/kg BBG administered 0.5h after TBI significantly reduced peri-contusional IL-1β expression, as assessed by (<b>A</b>) EIA and by (<b>B</b>) Western blotting at 12h or 24h post-injury. (<b>C</b>) IL-1β was quantified by EIA at 24h post-injury in wild-type or P2X7−/− mice. In panels A and C, data are represented as IL-1β expression as a % of sham expression levels. In panel B, data was normalized to β-actin to control for equal protein loading between lanes. Data are representative of 6–8 mice/group. Data were analyzed with One-Way ANOVA followed by Dunnett's post-hoc test (* p<0.05, ** p<0.01 vs. sham operated mice).</p