Effects And Mechanisms Of Hydrogen Sulfide On Focal Cerebral Ischemia Injury In Rats

Strokes caused by ischemia (lack of blood flow), blockage or hemorrhageare major causes of mortality and long-term disability. The dangerous factorsof cerebral ischemia injury include Ca2+overload, free radical, nitric oxide(NO), cell apoptosis, cytokines and so on. Therefore, the prevention,mechanism and treatment of cerebral ischemic injury are the points of medicalresearch at present. Among the various rodent models of stroke, middlecerebral artery occlusion (MCAO) is a simple surgical approach with minimaltrauma and is widely used to reconstruct focal cerebral ischemic model.During the past decade, increasing lines of evidence have shown thathydrogen sulfide (H₂S) has important physiological functions, especially inthe nervous system. Now H₂S is recognized as the third endogenous signalinggasotransmitter, following NO and carbon monoxide (CO). H₂S has beenshown to attenuate hepatic and myocardial ischemia-reperfusion injuries viaantioxidant and anti-apoptotic signaling. In the present study, we used MCAOas focal cerebral ischemic model to evaluate the effects of H₂S on focalcerebral ischemia in rats and explored the possible mechanisms.Part1Effects of hydrogen sulfide on focal cerebral ischemia injury inratsObjective: To investigate the effects of hydrogen sulfide on focalcerebral ischemia injury in rats.Methods: Eighty male SD rats (weigh250-280g) were randomly dividedinto five groups (n=16): sham group, ischemia group, NaHS low, middle andhigh dose groups. Rats were anesthetized by10%chloral hydrate at350mg/kg. Focal cerebral ischemia model was reconstructed by inserting craniallya nylon thread with rounded tip into internal carotid artery. Rats were onlyunderwent a surgical operation but without ischemia in the sham group. The NaHS (0.7mg,1.4mg and2.8mg/kg) were respectively administrated at3hafter ischemia in rats. The equal volume of saline was administrated in thesham and the ischemia groups. Rats were sacrificed at24h after ischemia.Half of the rats in each group were used for measurement of the infarctvolumes by TTC staining. The others in each group were used fordetermination of the content of H₂S and the activity of3MST in the braintissue.Results:Compared with those of the group sham, the infarct volume wassignificantly increased, the content of H₂S and the activity of3MST in thebrain were significantly decreased in those of the ischemia group (P<0.01).Compared with those of the ischemia group, the infarct volume wassignificantly decreased, the content of H₂S and the activity of3MST weresignificantly increased in those of the NaHS middle and high dose groups (P<0.05, P <0.01).Conclusion: Administration of NaHS could decrease the infarct volume,increase the activity of3MST and the content of H₂S. It could be concludedthat H₂S may play the protective role against focal cerebral ischemic injury.Part2Effects of hydrogen sulfide on oxidative stress in focal cerebralischemic injury in ratsObjective: To investigate the effects of H₂S on focal cerebral ischemicinjury in rats and explore the possible mechanisms.Methods: Forty male SD rats (weigh250-280g) were randomly dividedinto five groups (n=8): sham group, ischemia group, NaHS low dose, middledose and high dose groups. Rats were anesthetized by10%chloral hydrate at350mg/kg. Focal cerebral ischemia model was reconstructed by insertingcranially a nylon thread with rounded tip into internal carotid artery. Rats wereonly underwent a surgical operation but without ischemia in the sham group.The NaHS (0.7mg,1.4mg and2.8mg/kg) were respectively administrated at3h after ischemia in rats. The equal volume of saline was administrated in thesham and the ischemia groups. Rats were sacrificed at24h after ischemia. The content of malondialdehyde (MDA), and the activities of superoxidedismutase (SOD) and glutathione peroxidase (GSH-PX) in the brain tissuewere respectively measured. The ultrastructure changes of neurons wereobserved by transmission electron microscope.Results:1The activities of SOD and GSH-PX in the brain tissue weresignificantly decreased, the content of MDA in the brain tissue weresignificantly increased in the ischemia group compared with those of the shamgroup (P<0.01). The activities of SOD and GSH-PX in the brain tissue weresignificantly increased, the content of MDA in the brain were significantlydecreased in the NaHS middle and high dose groups compared with those ofthe ischemia group (P<0.01).2Transmission electron microscope showed the neuronal cytoplasm andthe mitochondria are normal in the sham group. The neuronal cytoplasm andthe mitochondria swelled, the cristae of mitochondria disrupted, dissolved ordisappeared, the amounts of organelles are significantly reduced in theischemia group compared with those of the sham group. The above injurieswere significantly ameliorated in the NaHS middle and high dose groupscompared with those of the ischemia group.Conclusion: Administration of NaHS could increase the activities ofSOD and GSH-PX and decrease the content of MDA. It could be concludedthat the protection role of H₂S on focal cerebral ischemic tissue is related todiminishing oxidative stress.Part3Effects of hydrogen sulfide on apoptosis in focal cerebral ischemiainjury in ratsObjective: To study the effects of H₂S on neruronal apoptosis in focalcerebral ischemic injury in rats and explore the possible mechanism.Methods: Forty male SD rats (weigh250-280g) were randomly dividedinto five groups (n=8): sham group, ischemia group, NaHS low, middle andhigh dose groups. Rats were anesthetized by10%chloral hydrate at350mg/kg. Focal cerebral ischemia model was reconstructed by inserting cranially a nylon thread with rounded tip into internal carotid artery. Rats were onlyunderwent a surgical operation but without ischemia in the sham group. TheNaHS (0.7mg,1.4mg and2.8mg/kg) were respectively administrated at3hafter ischemia in rats. The equal volume of saline was administrated in thesham and the ischemia groups. Rats were sacrificed at24h after ischemia. Thepathological changes of brain tissue were observed with light microscope byHE staining. The neuronal apoptosis were assayed by TUNEL detection. Theexpressions of Bcl-2and Bax in the brain tissue were respectively detected byimmunohistochemisty. The expression of Caspase-3in the brain tissue wasanalyzed by Western blot.Results:1HE staining showed that the neurons are normal in the sham group. Thevascular dilatation, some of neurons cellular swelling, cell nucleusconcentrating,cytoplasmic rarefaction, staining weakly, vacuole formation inthe ischemia group. The pathological alterations were obviously amelioratedin the NaHS middle and high dose groups compared with those of theischemia group.2The apoptotic rate of neurons was significantly increased in theischemia group compared with that of the sham group (P<0.01). Comparedwith that of the ischemia group, the apoptotic rate of neurons was significantlydecreased in the NaHS middle and high dose groups (P<0.05or P<0.01).3Immunohistochemisty showed the expression of Bcl-2wassignificantly decreased, the expression of Bax was significantly increased inthe ischemia group compared with those of the sham group (P<0.01). Theexpression of Bcl-2was significantly increased, the expression of Bax wassignificantly decreased in the NaHS middle and high dose groups comparedwith those of the ischemia group (P<0.05or P<0.01).4Western blot showed that there was only a small amount of expressionof Caspase-3in the sham group. The expression of Caspase-3wassignificantly increased in the ischemia group compared with that of the shamgroup. The expression of Caspase-3was significantly decreased in the NaHS middle and high dose groups compared with that of the ischemia group.Conclusion: Administration of NaHS could decrease the apoptotic rateof neurons, attenuate the expression of Bax and Caspase-3, and increase theexpression of Bcl-2in brain tissue in focal cerebral ischemic injury in rats.The results showed that H₂S may play a protective role against focal cerebralischemic injury by inhibiting neuronal apoptosis.Part4Effects of hydrogen sulfide on inflammatory factors in focalcerebral ischemia injury in ratsObjective: To investigate the effects of H₂S on focal cerebral ischemicinjury in rats and explore the possible mechanisms.Methods: Forty male SD rats (weigh250-280g) were randomly dividedinto five groups (n=8): sham group, ischemia group, NaHS low, middle andhigh dose groups. Rats were anesthetized by10%chloral hydrate at350mg/kg. Focal cerebral ischemia model was reconstructed by inserting craniallya nylon thread with rounded tip into internal carotid artery. Rats were onlyunderwent a surgical operation but without ischemia in the sham group. TheNaHS (0.7mg,1.4mg and2.8mg/kg) were respectively administrated at3hafter ischemia in rats. The equal volume of saline was administrated in thesham and the ischemia groups. Rats were sacrificed at24h after ischemia. Thecontents of TNF-α, IL-1β and IL-10were respectively measured byenzyme-linked immunosorbent assay (ELISA). The transposition of nuclearfactor-κB (NF-кB) in nucleus was detected by immunohistochemisty. Theexpression of NF-кB in the brain tissue was detected by Western blot.Results:1The contents of TNF-α and IL-1β in serum and brain tissue weresignificantly increased in the ischemia group in rats compared with those ofthe sham group (P<0.01). The contents of IL-10in serum and brain tissuewere significantly decreased in the ischemia group in rats compared with thoseof the sham group (P<0.01). The contents of TNF-α and IL-1β in serum andbrain tissue were significantly decreased. The content of IL-10in serum andbrain tissue were significantly increased in the NaHS middle and high dose groups compared with those of the ischemia group (P<0.05or P<0.01).2The NF-κB was significantly translocated from the neurons cytoplasminto the nucleus and the expression of NF-κB was significantly increased inthe ischemia group compared with those of the sham group(P<0.05). In theNaHS middle and high dose groups, the NF-κB translocation was markedlyinhibited and the expression of NF-κB in the nuclei was obviously decreasedcompared with those of the ischemia group (P﹤0.05).Conclusion: Administration of NaHS could inhibit NF-κB activation,decrease the expression of TNF-α and IL-1β, increase the expression of IL-10,which may be one of the molecular mechanisms of its neuroprotection.Summary:1Administration of NaHS could decrease the infarct volume, increase theactivity of3MST and the content of H₂S. H₂S may play the protective roleagainst focal cerebral ischemic injury.2Administration of NaHS could increase the activities of SOD andGSH-PX and decrease the MDA content. The protection role of H₂S on focalcerebral ischemic tissue may be related to diminishing oxidative stress.3Administration of NaHS could decrease the apoptotic rate of neurons,attenuate the expression of Bax and Caspase-3, and increase the expression ofBcl-2in brain tissue in focal cerebral ischemic injury in rats. H₂S may play aprotective role against focal cerebral ischemic injury by inhibiting neuronalapoptosis.4Administration of NaHS could inhibit NF-κB activation, decrease theexpression of TNF-α and IL-1β, increase the expression of IL-10, which maybe one of the molecular mechanisms of its neuroprotection.