Neuroprotection Of Hydrogen Sulfide After Hypoxic-ischemic Brain Damage In Neonatal Rats

BackgroundHypoxic-ischemic brain damage (HIBD) is one of the important reasons of neonate acute death and chronic nervous system damage. The legacy of cerebral palsy, learning disabilities, mental retardation, epilepsy, psychomotor retardation and nervous system sequela. Survivors nearly25%exist nervous system disorder. The study found that0.2～0.4%full-term children asphyxia in childbirth,15～20%death for secondary neonatal anaerobic ischemic brain injury, nearly25%of survivors had secondary nervous system disorders becaused of neonatal hypoxic-ischemic brain injury. With the establishment of the Neonatal Intensive Care Unit, and the improvement of the level of medical technology, the survival rate of severe neonatal suffocation with increased significantly, but the incidence of neurological sequel of them remains high. Study the HIBD damage and find a way of effective treatment to improve the survival of HIBD and reduce the neurological sequel of survival children.The HIBD core complex pathogenesis is a lack of oxygen due, the main lesion is cortex, hippocampus, basal ganglia and thalamus parts, various of mechanisms lead to hypoxic-ischemic biochemical chain reaction:①The early stage of hypoxia brain hemodynamic changes.②the changes in the energy metabolism of brain cells.③the neurotoxic effects of excitatory amino acid.④oxygen free radicals.⑤Ca2+influx and reperfusion injury.⑥NO, inflammatory factors and cytokines.⑦neurons apoptosis. Apoptosis of neuronal cells play a critical role in hypoxic-ischemic brain injury in the pathogenesis have demonstrated.Hydrogen sulfide (H2S) is the third small molecular weight gases signaling molecules after the nitric oxide (NO) and carbon monoxide (CO), H2S earliest awareness as a toxic gas, and the late of1990s found that was a new gaseous signal molecule in physiological concentrations, the researchers found that endogenous H2S is widely present in mammalian tissues and organs, play a role via variety of signal transduction forms and regulate in pathophysiological in physiological concentrations. Abe first confirmed that endogenous H2S is a neuro-active molecules involved in neural regulation and signal transduction process in1996. Two key enzyme regulating endogenous H2S generation in vivo, cystathionine-β-synthase (CBS) and cystathionine-y-lyase (CSE).1/3the form of H2S gas exists in vivo,2/3the form of sodium hydrosulfide (NaHS) hydrolysis of the Na+and HS-,HS-and H+combine into H2S in vivo. CBS is mainly distributed in the nervous system, such as the hippocampus, cortex, cerebellum, brainstem. CSE is distributed the outside of nervous system. Studies have shown that in rat brain endogenous H2S concentrations up to50～160μmol/L, and the free of H2S physiological concentration in rat brain approximately14±3.0μmol/L, the physiological functions:①Selectively enhance NMDA receptor-mediated excitatory current and strengthen neural response effect.②Regulate synaptic activities affect hippocampal long-term potentiation (LTP).③Regulate neuroendocrine function via the hypothalamic-pituitary-adrenal axis.④Affect the nervous system function via regulate vascular endothelial cells and smooth muscle cells control the blood supply in the cerebral.⑤By up regulating y-amino butyric acid (GABA) B receptors thereby regulating neurotransmitter release and balance excitability.⑥Regulating Ca2+in the intracellular and extracellular.⑦By CL-/HCO3-, Na+/H+ion channels on the microglia and astrocyte cell membrane to regulate intracellular pH value.⑧Protect neurons against oxidative stress by enhancing the vitality of the y-glutamylcysteine synthetase (y-GCS) to improve intracellular y-glutamylcysteine (y-GC) content, and at the same time strengthen the cysteine transporter thereby increasing the efficiency of intracellular glutathione (GSH) levels.⑨Anti-apoptotic effects. NO is a gas signaling molecule too, play a role in signal transduction and neurotransmitter. The amount of NO regulation of vascular tone,immune function,nerve conduction and attack tumor cells, kill pathogenic microorganisms; Excessive NO have neurotoxic, lead to neuron apoptosis.Interaction between H2S and NO, H2S can inhibit NOS activity, reduce NO production, to protect the neuron.The7days newborn SD rat brain tissue is similar to neonatal brain development, and the SD rat Willis of carotid artery and the brain blood supply similar to humans, and the pathological physiological changes of hemodynamic changes, energy metabolism and the secondary energy failure after hypoxic-ischemic similar to neonate hypoxic-ischemic brain injury. Combined with a relatively simple model with rats and the relatively low mortality rate,7olds SD rat is extensive used and reference to the Rice method to prepare HIBD model in neonatal rats. Rencai Li found that the concentration of H2S dynamic changes increased firstly and then decreased in cerebral cortex of newborn rats after HIBD. A large number of studies have shown that H2S could reduce cerebral ischemia-reperfusion injury and protect neuron. Therefore, to explore the impact of H2S on neuronal apoptosis after HIBD and understanding apoptosis through interventions on this basis, explore a new intervention method.Apoptosis process is factors trigger the cell death program that lead to cells death in vitro and in vivo, is different form of cell death from necrosis. Apoptosis plays a vital role in the normal development of the body, growth, and maintain the body environment stability, apoptosis disorder leads to the pathogenesis of many diseases and threats to human health.Caspase family began to cell programmed death with C. elegans (C.elegans), is the related cysteine protease present in the cytoplasmic, and the common is specific fracture the back of aspartic acid residue peptide bond. Caspase-1,4,11, etc. mainly involve in the activation of the precursor; Caspase-2,8,9,10involve in apoptosis initiation; Caspase-3,6,7involve in the execution of apoptosis. Caspase-3was named in1996, is the key terminal shear enzyme in the apoptotic process, is also one of the important part of the the CTL cell killing mechanisms. Caspase-3is the intersection point of the variety of downstream effector of the apoptosis pathway, play a role in the death receptor, is the only way of apoptotic protein cascade.This study establish the HIBD model in neonatal rats to observe the dynamic change of endogenous H2S and NO, and study the correlation of both. Change the levels of endogenous H2S and NO by giving the exogenous H2S donor sodium hydrosulfide(NaHS) and CBS inhibitor hydroxylamine(HA), observe the the pathological changes of brain tissue,use TUNEL method detect neuronal apoptosis, and use immunohistochemical method to detect the expression of caspase-3, the purpose to explore H2S may play a protection role in the development of HIBD.Specific studies include the following two parts:Part I The concentration of H2S and NO in brain tissue levels change after hypoxic-ischemic brain damage in neonatal rats ObjectiveObserved the hydrogen sulfide(H2S) and NO levels in brain tissue, and H2S in serum after establishment of hypoxic-ischemic brain damage model,to explore the H2S dynamic changes in brain tissue and serum, the corredation between H2S and NO.Methods124seven-day-old newborn SD rats are randomly divided into control group (n=4), sham-operation group (n=30),HIBD group (n=30), HIBD+NaHS group (n=30) and HIBD+HA group (n=30), reference to the Rice method of preparation of the newborn rat HIBD model, sham group only separate the left carotid artery and do not hypoxia. After HIBD30min give NaHS(14μmol/kg) or HA(12.5mg/kg) which dilute with saline by intraperitoneal injection.Use a biochemical reaction method to detect H2S levels in brain cortex and serum at the6h,12h,24h,48h and72h after HIBD and intervention separately, use nitrale reduetase method detect NO levels, sham group and control group detect same to HIBD group. Analyse dynamic change of H2S and NO, and evaluate the correlation.ResultsThe H2S levels in serum and brain tissue at6h increased significantly, reached peak at12h, and then began to decrease; the NO levels in brain tissue increased at6h gradually. Give the H2S donor of NaHS, the H2S was significantly higher than the HIBD group in different time, conversely the NO decreased; Given the inhibitors of HA, the H2S significantly reduced, the NO increased. In serum, sham-operation group and HIBD+HA group were not statistically significant at72hours(p>0.05). Between6h and24h among HIBD group, HIBD+NaHS group and HIBD+HA group, between48h and72in HIBD group, between24h and48h in HIBD+NaHS group were not statistically significant at72hours(p>0.05); In brain tissue, there had statistically significant among four group(P<0.05). Between6h and12h, between12h and another time were statistically significant(P<0.05), between6h and24h, between48h and72h in HIBD+NaHS group, between6h and48h,72h,between12h and24h in HIBD+HA group were not statistically significant(P>0.05); The NO levels between sham-operation group and HIBD+NaHS group at6hours were not statistically significant(P>0.05). Between6h and12h in HIBD+NaHS group were not statistically significant(P>0.05). The correlation of the HIBD group and NaHS group was negative(r=-0.537,-0.838, P<0.05).ConclusionsThe H2S may participate in the pathophysiological process of HIBD in neonatal rats. Give the NaHS and HA, the H2S and NO dynamic change, suggest that exogenous intervention may change the endogenous H2S and NO levels, it may providing a new method of treatment for clinical. Second Part Ⅱ Effect of hydrogen sulfide on neurons apoptosis in newborn rats after hypoxia-ischemia brain damageObjectiveExplore the effect of H2S on neurons apoptosis in neonatal rat after HIBD.Methods124seven-day-old newborn SD rats were randomly divided into control group (n=4), sham-operation group (n=30),HIBD group (n=30), HIBD+NaHS group (n=30) and HIBD+HA group (n=30), took the cerebral cortex and hippocampus respectively at the6h,12h,24h,48h and72h after HIBD and intervention, observed of brain tissue pathological change by HE staining, detected neurons apoptotic index and the averageoptical density of caspase-3expression.ResultsSham-operation group with HE staining cells arranged in neat rows,the body was basically normal, clear nucleolus, nerve cells loss; the neurons in left brain tissue in HIBD group degenerated, disorganized, significantly reduced, neurons were necrosis; HIBD+NaHS group’s degeneration was decreased than HIBD group, necrosis of neurons reduced; no significant difference between HIBD+HA group and HIBD group. A small amount of apoptosis positive cells in hippocampus and cortex in sham-operation group. In HIBD group,apoptosis cells begin to rise at6h,gradually reduce after48hours. In HIBD+NaHS group,begin to rise at6h also,begin to reduce after peak at48h, but lower compair with HIBD group. In HIBD+HA group, apoptosis cells begin to increase and then reduce after48h also, and higher compair with HIBD group. In HIBD group, immune response positive cells caspase-3begin to increase, and gradually increase in12-24h, then reduce after the peak at48h. In HIBD+NaHS group, begin to rise at6h, a slight decline at12h, at24h rise again and reach the peak at48h, and then reduce at72h, the number and stain are lower compair with HIBD group. In HIBD+HA group, begin to increase and then reduce also, the number and stain are higher compair with HIBD group. The neurons apoptotic index: in hippocampus, sham-operation group and HIBD+NaHS group at6h,12h,24h and48h were not statistically significant(P>0.05); between6h and12h,24h,72h, between12h and24h,72h, between24h and72h were not statistically significant(P>0.05). In the cerebral cortex, between24h and72h in HIBD group, between6h and24h,between24h and72h in HIBD+NaHS group, between12h and24h,between24h and72h, between48h and72h in HIBD+HA group were not statistically significant(P>0.05). The caspase-3expression:in hippocampus, sham-operation group and HIBD+NaHS group, HIBD group and HIBD+NaHS group at6h, sham-operation group and HIBD+NaHS group at12h were not statistically significant(P>0.05); Between6h and12h,between12h and24h,72h, between24h and72h in HIBD group, between6h and12h,24h,72h,between24h and48h,72h in HIBD+NaHS group, between6h and12h,between12h and24h,between24h and 72h,between48h and72h in HIBD+HA group were not statistically significant(P>0.05). In the cerebral cortex, between sham-operation group and HIBD group,HIBD+NaHS group, between HIBD group and HIBD+NaHS group,HIBD+HA group at6h, sham-operation group and HIBD+NaHS group at12h,24h and72h, HIBD group and HIBD+HA group at24h and72h were not statistically significant(P>0.05); between6h and12h,between12h and24h,between24h and48h,72h,between48h and72h in HIBD group, between6h and12h,between24h and72h,between48h and72h in HIBD+NaHS group,between12h and24h,72h,between24h and72h,between48h and72h in HIBD+HA group were not statistically significant(P>0.05).ConclusionsH2S participate in HIBD neurons apoptosis process, administration of exogenous H2S donor NaHS can inhibit apoptosis of neurons, promoted neurons apoptosis after given inhibitor HA.