Hydrogen Sulfide Reduces TPA-Induced Cerebral Hemorrhage After Ischemic Stroke

Objective: The thrombolytic agent tissue plasminogen activator(t PA) remains the only drug approved by Food and Drug Administration(FDA) for the treatment of ischemic stroke. However, very few patients could benefit from it since it has narrow time window and increases the risk of cerebral hemorrhage. Hydrogen sulfide(H2S) has been recognized as the third gaseous signal molecule in addition to nitric oxide(NO) and carbon monoxide(CO). We investigated whether H2 S alleviated cerebral hemorrhage transformation associated with t PA therapy following stroke.Methods: C57/BL6 J male mice were subjected to middle cerebral artery occlusion(MCAO). Glucose(6 ml/kg, 50%w/v) was intraperitoneally injected 15 min before MCAO. t PA(10 mg/kg) or saline(4 ml/kg) was administered from femoral vein immediately after reperfusion, followed by intraperitoneal injection of H2 S donors or vehicle. The mice were divided into six groups: Sham,control group, t PA group, t PA+Na HS group, t PA+ADT group, t PA +ADT-OH group. Cerebral hemorrhage was examined by assessing hemoglobin content in the brain with a commercial kit at 24 hours after reperfusion. Infarct volumes were assessed by 2, 3, 5-triphenyltetrazolium chloride(TTC) histology at 1 day and 7 days after MCAO. Blood-brain barrier permeability was evaluated by assessing extravasated evans blue(EB), and brain water content was examined with the dry-wet weight method to indicate cerebral edema at 24 hours after MCAO. Neurological deficits were assessed with modified neurological severity scores(m NSS), corner test and Rotarod test over 7 days after MCAO. MMP-9 activity was measured with gelatinase zymography, and the levels of H2 S in blood were measured with methylthionine chloride method.Results: Compared to Sham mice and control mice subjected MCAO, mice receiving MCAO and t PA displayed enhanced hemoglobin content in the ischemic brain, indicating that t PA enhanced cerebral hemorrhage after MCAO. Hydrogen sulfide donors Na HS, ADT and ADT-OH reduced t PA-induced cerebral hemorrhage as indicated by decreased hemoglobin content in the ischemic brain. ADT-OH further reduced t PA-enhanced EB extravasation into the ischemic brain and edema as compared to vehicle treatment at 24 hours after MCAO, suggesting that the hydrogen sulfide donor protected against BBB damage induced by t PA following MCAO. In mice treated with MCAO and t PA, co-treatment with ADT-OH had no effect on infarction at 1 day after MCAO but decreased t PA-enhanced infarct volumes at 7 days after MCAO as compared to vehicle co-treatment. Moreover, ADT-OH co-treatment alleviated neurological deficits of mice receiving t PA over 7 days following MCAO. Gelatinase zymography assays showed that ADT-OH attenuated t PA-enhanced MMP-9 activity in the ischemic brain at 24 hours after MCAO.Innovation: For the first time, we showed that H2 S donors protected against t PA-enhanced cerebral hemorrhagic transformation following ischemia.Conclusions: H2 S donors reduced t PA-enhanced cerebral hemorrhagic transformation and improved behavioral and histological outcomes. H2 S donors have the potential to be developed into a novel drug therapy to reduce cerebral hemorrhagic transformation induced by t PA following cerebral ischemia.