Hydrogen Sulfide Donor Promotes Angiogenesis And Functional Recovery After Cerebral Ischemia

Aim: We investigated whether post-ischemic administration of ADT-OH, a hydrogen sulfide donor, promotes functional recovery after cerebral ischemia by increasing alternative polarization(M2) of microglia/macrophages and angiogenesis in the ischemic brain, which working on angiogenesis.Methods: Daily intraperitoneal injection of ADT-OH(50 mg/kg/day) or vehicle was given to adult male mice at 24 h after 1 hour middle cerebral artery occlusion(MCAO). Modified Neurological Severity Scores, Corner test and Rota-Rod test were used to assess the function recovery of the mice over the 30 days after MCAO. Western Blot was used to examine the effects of ADT-OH on cerebral AMPK activation(phosphorylation) following MCAO. Real-time PCR was preformed to assess the m RNA expression of M1 and M2 types cytokines in the ischemic brain. ELISA was performed to determine the protein expression of IL-10 and v EGF. Post-MCAO brain histological injury was examined with TTC staining and by assessing brain atrophy. Iimmunohistochemistrical analysis of the markers of microvessels was performed to characterize ADT-OH effects on post-MCAO angiogenesis in the ischemic brain. Finally, by using two vitro models, in which M1 polarization of BV2 microglia were induced by LPS or conditioned medium collected from primary neurons treated with oxygen-glucose deprivation(OGD), we investigated in vitro if ADT-OH promoted b End 3 cells(mouse brain microvessel endothelia cell line)-mediated angiogenesis in a M2 polarization-dependent manner.Results: The three behavioral tests showed that mice treated with ADT-OH displayed remarkably better functional recovery than the mice receiving vehicle. Western Blot results showed that ADT-OH administered after MCAO enhanced AMPK activation in the ischemia brains at 3 and 14 days after MCAO. As shown by real-time PCR results, the m RNA expression levels of M1 type cytokines( CD32、IL-1β 、TNF-α) in the ischemia cortex were lower while and the expression levels of M2 type cytokines(CD206、Arginase 1) were higher in the ADT-OH-treated mice. vehicle-treated mice. ELISA results showed at the protein level that the the expression of IL-10, a M2 signature cytokine, was enhanced in ischemia cortex of ADT-OH-treated mice. However, ADT-OH did not reduce acute infarct damage at 4 days after MCAO, as indicated by TTC staining results.. Immunohistochemical examination showed that ADT-OH increased post-MCAO agiogenesis as evidenced the fact that ADT-OH enhanced SMA-positive, CD31-positive and lectin-positive cells in the infarct border zones of the ADT-OH-treated mice at 30 days following MCAO, which is accompanied with the enhanced v EGF protein expression in the ischemic brain as assessed with a ELISA kit. In vitro models, co-treatment of microglia with by LPS or OGD-conditioned neuronal medium plus ADT-OH, but not ADT-OH treatment alone, promoted b End 3 cells to form vascular structures.Conclusions: Our results suggest that the hydrogen sufide ADT-OH promoted post-MCAO functional recovery and brain repair by acting through AMPK activation to induce M2 polarization of micrglia/macrophages in the ischemic brain.