P2X7 receptor antagonism inhibits p38 mitogen-activated protein kinase activation and ameliorates neuronal apoptosis after subarachnoid hemorrhage in rats

Abstract

Objectives: Brilliant blue G, a selective P2X7 receptor antagonist, exhibits neuroprotective properties. This study examined whether brilliant blue G treatment ameliorates early brain injury after experimental subarachnoid hemorrhage, specifically via inhibiting p38 mitogen-activated protein kinase-related proapoptotic pathways.

Design: Controlled in vivo laboratory study.

Setting: Animal research laboratory.

Subjects: One hundred fifty-four adult male Sprague-Dawley rats weighing 280-320 g.

Interventions: Subarachnoid hemorrhage was induced in rats by endovascular perforation. Experiment 1 implemented sham-operated rats (sham) and subarachnoid hemorrhage animals, which received vehicle (subarachnoid hemorrhage + vehicle), brilliant blue G (subarachnoid hemorrhage + brilliant blue G), or brilliant blue G plus 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) (subarachnoid hemorrhage + brilliant blue G + BzATP). The animals were intraperitoneally treated with brilliant blue G (30 mg/kg) at 30 minutes after subarachnoid hemorrhage. BzATP (50 μg/rat), a P2X7 receptor agonist, was intracerebroventricularly administered. Experiment 2 implemented sham-operated rats (sham) and subarachnoid hemorrhage animals, which received vehicle (subarachnoid hemorrhage + vehicle), scramble small interfering RNA (subarachnoid hemorrhage + scramble small interfering RNA), or P2X7 receptor small interfering RNA (subarachnoid hemorrhage + P2X7 receptor small interfering RNA). Subarachnoid hemorrhage grading, neurobehavioral score, and brain edema were evaluated at 24 and 72 hours after surgery. The expression of phosphorylated p38 mitogen-activated protein kinase, phosphorylated extracellular signal-regulated kinases, phosphorylated c-Jun N-terminal kinases, P2X7 receptor, Bcl-2, and cleaved caspase-3 in the left cerebral hemisphere were determined by Western blot. Neuronal apoptosis was examined by double immunofluorescence staining using P2X7 receptor, terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling, and neuronal nuclei.

Measurements and main results: Brilliant blue G significantly improved neurobehavioral function and ameliorated brain water content at 24 and 72 hours after subarachnoid hemorrhage. BzATP reversed these treatment effects. Brilliant blue G attenuated neuronal apoptosis in the subcortex, which was associated with decreased expression of phosphorylated p38 mitogen-activated protein kinase and cleaved caspase-3 and an increased expression of Bcl-2 in the left cerebral hemisphere. The beneficial effects of P2X7 receptor small interfering RNA were also mediated by a p38 mitogen-activated protein kinase pathway.

Conclusions: Inhibition of P2X7 receptor by brilliant blue G or P2X7 receptor small interfering RNA can prevent early brain injury via p38 mitogen-activated protein kinase after subarachnoid hemorrhage.

Figure 1

Effects of Brilliant blue G (BBG) treatment on subarachnoid hemorrhage (SAH) grading, Modified Garcia and Beam balance score, and Brain water content at 24 hours after SAH. A: Similar SAH grading was observed in the SAH+vehicle group (n=24), SAH+BBG group (n=21) and SAH+BBG+ BzATP (n=5). B and C: BBG treatment increased modified Garcia and beam balance score at 24 hours after SAH (*p<0.05 and **p<0.01 vs. sham; ^#p<0.05 vs. vehicle) respectively, BzATP tended to reversed the effect of BBG (p>0.05). D: BBG treatment decreased brain water content significantly in the left hemisphere at 24 hours after SAH, BzATP aggravated post-SAH brain edema in cerebellum (*p<0.05 and **p<0.01 vs. sham; ^#p<0.05 vs. vehicle; ^@p<0.05 vs. BBG, sham: n=8, vehicle: n=8, BBG: n=6, BBG+BzATP: n=5). A and D: error bar=SEM, B and C: error bar=25th–75th interquarile percentiles

Figure 2

Effects of Brilliant blue G (BBG) treatment on subarachnoid hemorrhage (SAH) grading, Modified Garcia and Beam balance score, and Brain water content at 72 hours after SAH. A: Similar SAH grading was observed in the SAH+vehicle group and SAH+BBG group. B and C: modified Garcia score decreased at 72 hours after surgery. BBG treatment significantly increased modified Garcia score, but not beam balance test (*p<0.05 and **p<0.01 vs. sham; ^#p<0.05 vs. vehicle). D: BBG treatment significantly decreased brain water content in the left hemisphere. (*p<0.05 and **p<0.01 vs. sham; ^#p<0.05 vs. vehicle, n=9). A and D: error bar=SEM, B and C: error bar=25th–75th interquarile percentiles.

Figure 3

Effects of Brilliant blue G (BBG) treatment on phosphorylated p38 MAPK (A), phosphorylated JNK (B), phosphorylated ERK (C) and Bcl-2 (D) expressions in the left cerebral hemisphere at 24 hours after subarachnoid hemorrhage (SAH). BBG reduced the level of p38 MAPK (A) and increased Bcl-2 (D) (*p<0.05 and **p<0.01 vs. sham; ^#p<0.05 vs. vehicle, n=6). error bar=SEM.

Figure 4

Effects of Brilliant blue G (BBG) treatment on Cleaved caspase-3 expression and Neuronal Apoptosis at 24 hours after subarachnoid hemorrhage (SAH). A: Representative photographs of immunofluorescence staining for P2X7R (red) and neuron (NeuN, red) in the subcortical area at 24 hours following SAH. Scale bar: 30μm. B: western blotting showed BBG reduced the level of cleaved caspase-3 in the left hemisphere at 24 hours after SAH (*p<0.05 and **p<0.01 vs. sham; ^#p<0.05 vs. vehicle, n=6). C and D: Neuronal apoptosis quantification representative microphotographs showing colocalization of NeuN (Texas Red/red) and TUNEL (green). BBG treatment decreased TUNEL-positive neuron (n=4 or 5). Scale bar: 50μm. error bar=SEM.

Figure 5

Effects of P2X7 recptor (P2X7R) siRNA on subarachnoid hemorrhage (SAH) grading, Modified Garcia and Beam balance score at pre-SAH and 24 hours after SAH. A: Similar subarachnoid hemorrhage (SAH) grading was observed in the SAH+vehicle group, SAH+scramble siRNA group and SAH+P2X7R siRNA (p>0.05). B and C: P2X7R siRNA treatment increased modified Garcia score and beam balance at 24 hours after SAH (*p<0.05 and **p<0.01 vs. sham; ^#p<0.05 vs. vehicle; ^@ p<0.05 vs. scramble siRNA, n=6) respectively. A: error bar=SEM, B and C: error bar=25th–75th interquarile percentiles.

Figure 6

Effects of P2X7 recptor (P2X7R) siRNA on P2X7R, phosphorylated p38 MAPK, and Cleaved caspase-3 in the left cerebral hemisphere at 24 hours after subarachnoid hemorrhage (SAH). P2X7R siRNA reduced the level of P2X7R (A), phosphorylated p38 MAPK (B) and cleaved caspase-3 (C) (*p<0.05 and **p<0.01 vs. sham; ^#p<0.05 vs. vehicle; ^@ p<0.05 vs. scramble siRNA, n=6). error bar=SEM.