Dexmedetomidine alleviates blood-brain barrier disruption in rats after cerebral ischemia-reperfusion by suppressing JNK and p38 MAPK signaling

Abstract

Dexmedetomidine displays multiple mechanisms of neuroprotection in ameliorating ischemic brain injury. In this study, we explored the beneficial effects of dexmedetomidine on blood-brain barrier (BBB) integrity and neuroinflammation in cerebral ischemia/reperfusion injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1.5 h and reperfusion for 24 h to establish a rat model of cerebral ischemia/reperfusion injury. Dexmedetomidine (9 􀁐g/kg) was administered to rats 30 min after MCAO through intravenous injection, and SB203580 (a p38 MAPK inhibitor, 200 􀁐g/kg) was injected intraperitoneally 30 min before MCAO. Brain damages were evaluated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, Nissl staining, and brain water content assessment. BBB permeability was examined by Evans blue staining. Expression levels of claudin-5, zonula occludens-1, occludin, and matrix metalloproteinase-9 (MMP-9) as well as M1/M2 phenotypes-associated markers were assessed using immunofluorescence, RT-qPCR, Western blotting, and gelatin zymography. Enzyme-linked immunosorbent assay was used to examine inflammatory cytokine levels. We found that dexmedetomidine or SB203580 attenuated infarct volume, brain edema, BBB permeability, and neuroinflammation, and promoted M2 microglial polarization after cerebral ischemia/reperfusion injury. Increased MMP-9 activity by ischemia/reperfusion injury was inhibited by dexmedetomidine or SB203580. Dexmedetomidine inhibited the activation of the ERK, JNK, and p38 MAPK pathways. Moreover, activation of JNK or p38 MAPK reversed the protective effects of dexmedetomidine against ischemic brain injury. Overall, dexmedetomidine ameliorated brain injury by alleviating BBB permeability and promoting M2 polarization in experimental cerebral ischemia/reperfusion injury model by inhibiting the activation of JNK and p38 MAPK pathways.

Keywords: Blood-brain barrier; Dexmedetomidine; Matrix metalloproteinase 9; Microglia; Middle cerebral artery occlusion; p38 mitogen-activated protein kinases.

Fig. 1. Dexmedetomidine reduces infarct volume and neurological deficits.

(A) Schematic of the experiment protocols. (B, C) Effects of dexmedetomidine or SB203580 on the infarct volume were detected by TTC staining. (D) Representative photomicrographs of H&E staining in the rat brain in the four groups. Scale bar = 100 μm. (E) Representative images of Nissl staining in the rat brain. Scale bar = 100 μm. (F) Effects of dexmedetomidine or SB203580 on the neurological deficit scores. N = 8. Results are presented as mean ± SD, and statistical significance was performed by one-way ANOVA. MCAO, middle cerebral artery occlusion; i.p., intraperitoneal; i.v., intravenous; TTC, 2,3,5-triphenyltetrazolium chloride. ***p < 0.001. ^###p < 0.001.

Fig. 2. Dexmedetomidine alleviates BBB disruption in MCAO rats.

(A) BBB permeability was detected by EB staining in the brain. (B) Effects of dexmedetomidine or SB203580 on the brain water content. (C) Western blotting was performed to measure ZO-1, claudin-5, occluding, and AQP-4 protein levels. (D) Immunofluorescence staining of claudin-5, ZO-1, and occludin protein levels. Scale bar = 100 μm. N = 8. Results are presented as mean ± SD, and statistical significance was performed by one-way ANOVA. BBB, blood-brain barrier; MCAO, middle cerebral artery occlusion; EB, Evans blue. ***p < 0.001. ^###p < 0.001.

Fig. 3. Dexmedetomidine reduces MMP-9 levels in MCAO rats.

(A) Western blotting of MMP-9 protein levels. (B) The MMP-9 activity was detected by Gelatin zymography. N = 8. Results are presented as mean ± SD, and statistical significance was performed by one-way ANOVA. MMP-9, matrix metalloproteinase-9; MCAO, middle cerebral artery occlusion. ***p < 0.001. ###p < 0.001.

Fig. 4. Dexmedetomidine promotes M2-type microglial polarization.

(A-D) iNOS, CD86, CD206 and Arg-1 mRNA levels were measured by RT-qPCR. (E, F) iNOS, CD86, Arg-1 and CD206 protein levels were measured by Western blotting. (G, H) TNF-α and IL-1β levels were examined by ELISA. N = 8. Results are presented as mean ± SD, and statistical significance was performed by one-way ANOVA. iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor-α; IL, interleukin; MCAO, middle cerebral artery occlusion. ***p < 0.001. ^###p < 0.001.

Fig. 5. Dexmedetomidine or SB203580 reduces p38 MAPK phosphorylation.

(A) The ratio of p-p38/p38 was evaluated by Western blotting. (B) The ratios of p-ERK1/2/ERK1/2 and p-JNK/JNK were assessed by Western blotting. N = 8. Results are presented as mean ± SD, and statistical significance was performed by one-way ANOVA. MCAO, middle cerebral artery occlusion. ***p < 0.001. ^###p < 0.001.

Fig. 6. Activation of JNK and p38 MAPK reverses the protective effects of dexmedetomidine in brain injury.

(A, B) Infarct volume was detected by TTC staining. (C) BBB permeability was detected by EB staining in the brain. (D) Brain water content in each group. N = 8. Results are presented as mean ± SD, and statistical significance was performed by one-way ANOVA. MCAO, middle cerebral artery occlusion; BBB, blood-brain barrier; EB, Evans blue. ***p < 0.001. ^###p < 0.001. ^&&&p < 0.001.