Protective Effect Of Hydrogen Sulfide Against Global Cerebral Ischemia

Object: Cerebral ischemic diseases are seriously endangering humanhealthy. Mortality and morbidity of cerebral ischemic diseases are growing inrecent years.. The development of effective prevention and treatment of drugs,improving the neurons resistant to ischemic damage is an important strategy inthe treatment of these diseases. Hydrogen sulfide (H₂S) is a gaseous signalmolecule in vivo following the discovery of nitric oxide (NO) and carbonmonoxide (CO). The endogenous production of H₂S in mammals is fromcystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) and a newlyidentified enzyme, namely3-mercaptopyruvate sulfurtransferase (3MST).Numerous studies confirmed that H₂S not only play an important role in thecardiovascular system, but also be regarded as an important neuromodulator inthe central nervous system. H₂S are also involved in the occurrence anddevelopment of a variety of neurological diseases. ATP-sensitive potassiumchannel (ATP-sensitive potassium channels, KATP) is the main target of H₂S inthe cardiovascular system, and KATPchannels are also widely distributed in thebrain, defend the brain injury in hypoxic-ischemic condition. Wetherendogenous H₂S is involved in cerebral protective effect on brain ischemia ornot, and could exogenous administration of NaHS protect the nourons in brainischemia or not? All of these are not clearly and need to be elucidated.Establishing a stable, reliable, global cerebral ischemia model in theimplementation of the ischemic brain is essential. This study firstly evaluatedtwo different modeling methods of global cerebral ischemia, established ahigh successful rate, and low mortality model by global cerebral ischemiathrough direct vision with surgical microscope. The relationship betweenexogenous H₂S and cerebral ischemia-reperfusion damage, and the expressionof KATPchannels kir6.1and SUR2B subunit protein after H₂S administration are observed. These results will provide a theoretical basis for futureprevention and treatment of cerebral ischemic diseases.Methods:160healthy male Wistar rats (280³20g) were randomlydivided into the following three parts:1Compared two different global cerebral ischemia models and to evaluatedthe successful rate, morbidity, mortalityGrouped as follows:①Traditional model group (n=30): produced themodel of global cerebral ischemia with the traditional four-vessel occlusionmethod (four-vessel of occlusion,4-VO);②Look directly model group (n=30): produced the model of global cerebral ischemia with the surgicalmicroscope. Rats in different group were occluded bilateral vertebral arteriesfor8min,48h after clipping bilateral common carotid artery, and then restoreblood flow reperfusion. Record the number of postoperative animals ofhemiplegia, the number of animals of hematuria, the number of intraoperativeand postoperative death of the animal in different group. And7days after thelast surgery, animals are decapitated, hippocampus thionin staining to evaluateneuropathology. Almost all of the CA1region of hippocampus neurons deathbeing criteria to determine the successful model, record the number ofsuccessfulanimals in each group model.2The protective effect of sodium hydrosulfide preconditioning on globalcerebral ischemia in rat hippocampal CA1pyramidal neuronsUsing four-vessel occlusion rat cerebral ischemia model,effects ofdifferent dosing schedules, through different administration methods, anddifferent doses of sodium hydrosulfide on global cerebral ischemia wereobserved in hippocampal CA1neurons.Methods:40male Wistar rats were randomly divided into the followinggroups (n=5):①Sham group: Rats only received sham surgery, only condensate thevertebral artery not subjected to global cerebral ischemia insult.②Ischemic insult (II) group:8min induced occlusion of bilateralcommon carotid artery global cerebral ischemia,after48h permanently occluded bilateral vertebral artery.③Sodium hydrosulfide intracerebroventricular injection group:Administration of injection in the lateral ventricle before ischemia, inaccordance with the administration of different concentrations, and furtherdivided into four subgroups (Table1).④Sodium drosulfide intraperitoneal injection group: Administration ofintraperitoneal injection, in accordance with the different dosage,administration time point, and further divided into two subgroups (Table1)Table1NaHS in③,④group administration, dosage, administration timepointDose of sodium hydrosulfide in each group, time of administration, modeof administration the seventh day after global cerebral ischemia, the rats weredecapitated, and on the conventional brain tissue sections (5μm), thioninstaining. DND situation of the hippocampal CA1region with the lightmicroscope to determine the histological grade((histological grade, HG: grade 0: no neuronal death; grade I: scattered neuronal death; grade II: a piece ofneuronal death; grade III: almost all of the neuronal death)and neuronaldensity (of neuronal density, ND: count he number of pyramidal neuron withcharacter of membrane integrity, full nucleus, clear nucleolus in thehippocampal CA1region per lmm, and count three sections in each slice withbilateral hippocampal, then take the average neuronal density).3The variety of SUR2B protein and Kir6.1protein expression in the processof sodium hydrosulfide pretreatment against cerebral ischemia andreperfusion.Grouping as follow:①NaHS+II group(n=15);②saline+II group (n=15).30minutes before ischemia and reperfusion, immediately intraperitonealinjection of NaHS14μmol/kg or an equal volume of saline, and thenclamping the bilateral common carotid artery for8min.After the last ischemia for6h,12h,24h, take the hippocampus (at eachtime point, n=5), and application by western blot observed SUR2B proteinand Kir6.1protein expression, to explore SUR2B protein and Kir6.1proteinexpression affected by giving sodium hydrosulfide pretreatment before brainischemia.Results:1Compared two different methods of global cerebral ischemia model,evaluated the survival and the successful rate1.1rat survival rateThe number of rat survival for7days is26in traditional model group①,the survival rate is86.7%(26/30); the number of rat survival for7days is28in look directly model group②, the survival rate is93.3%(28/30). Basiccondition of rat after operation will be seen(Fig.2)1.2Successful rate of global cerebral ischemia-reperfusion modelSeven days after global cerebral ischemia, almost all of hippocampalCA1neurons death is criteria to determine the model of successful. Traditionalmodel group①, with a successful rate of60%; look directly model group②with a successful rate of80%, significantly higher than that of group①. 2The protective effect of sodium hydrosulfide pretreatment on globalcerebral ischemia in rat hippocampal CA1pyramidal neuronsHippocampal CA1pyramidal neurons are2-3layers, arranged in neat,clear and complete nucleus and nucleolus, full of Nissl bodies in the shamgroup (Fig.3B). In cerebral ischemia group, the hippocampal CA1pyramidalcells are almost all died, and compared to sham group HG was significantlyhigher, while the ND is significantly reduced (p<0.05). Group1and2oflateral ventricle administration of sodium hydrosulfide and group1ofintraperitoneal administration, compared with the global cerebral ischemiagroup, the morphology of the hippocampal CA1region, HG and ND showedno significant difference (p>0.05). Group3of lateral ventricle administrationof sodium hydrosulfide it could be seen that part of the pyramidal neuronsurvival, compared with the global cerebral ischemia group HG issignificantly lower, while ND is significantly higher (p<0.05), In the sodiumhydrosulfide intraperitoneal administration group2, most of the pyramidal cellsurvival, cell arranged neatly compared with the global cerebral ischemiagroup. HG is significantly lower, while ND is significantly higher (p<0.05).Group4of lateral ventricle administration of hydrosulfide sodium (lateralventricle of administration2000μΜ), compare with II group, it could be seenthat significant tissue damaged and piece of neuron lossed and ND wassignificantly lower (p<0.05).It could be seen that the hippocampal CA1neurons death was aggravatedwhen high concentrations of NaHS given by the lateral ventricles. Whileintraperitoneal administration30min before ischemia and reperfusion for14μmol/kg NaHS each time, could play a protective role of neuron.3The variety of SUR2B protein and Kir6.1protein expression in theprocess of sodium hydrosulfide pretreatment against cerebral ischemia andreperfusionWestern blot analysis showed that SUR2B protein in6h,12h,24h timepoint with time were significantly raised in NaHS pretreatment group andcerebral ischemia group after8min severe ischemia,(p<0.05); compared with group SN+II SUR2B protein expression in6h,12h has no significantchanges (p>0.05), however SUR2B protein expression was significantlyupregulated in the24h time point (p<0.05).These results indicated thatSUR2B protein expression in24h time point was significantly upregulated inthe progress of NaHS pretreatment in global cerebral ischemia.Kir6.1protein in6h and12h time point had no significant changeregardless of global cerebral ischemia group and NaHS pretreatment groupafter global cerebral ischemia (p>0.05), but compared with the first two timepoints in24h time point its expression were raised. Compared with groupSN+II, Kir6.1protein expression was no significant difference in NaHSpretreatment group at each time point (p>0.05). These results indicated thatNaHS pretreatment didn't affect the expression of Kir6.1protein after globalcerebral ischemia.Conclusion:1Established a new global cerebral ischemia models by occludedbilateral vertebral artery through direct vision with steromicroscope, thesurvival of rats and successful rate can be significantly improved.2The way of the intraperitoneal injection of the rat14μmol/kg NaHS30min before the cerebral brain ischemia and immediately before reperfusioncould protect neurons of hippocampal CA1region against8min ischemicinjury. The way of lateral ventricle administration with2000μΜ,10μl couldaggravate the neuronal injury.3The expression of SUR2B protein in24h time point is upregulatedwith NaHS pretreatment in global cerebral ischemia.4NaHS pretreatment does not affect the expression of Kir6.1protein in6h,12h,24h time point after whole brain ischemia.