Effects And Mechanisms Of CBS/H₂S System On Alleviating Early Brain Injury And Secondary Cognitive Defecits Following Subarachnoid Hemorrahge

BackgroundSubarachnoid hemorrhage is a severe subtype of stroke that can insult multiple neurological damage and systematic disorders.The average incidence of SAH is approximately 9/10,0000 throughout the world which leads to high mortality and mobility each year.Rupture of intracranial aneurysm is the primary cause of SAH occurrence.Blood rapidly flushes into subarachnoid space and disrupts the hemostasis of central nervous system,resulting in fatal symptoms and complications.Despite the fact that developments of surgical or interventional techniques have significantly alleviated fatal cases of post-SAH in recent years,there are still 20%-30%patients survived SAH suffering from long-term neurological dysfunctions,especially cognitive deficits,for years.These persistent damage compromises patients’ recovery and hinders them from returning to normal social work.For decades,vasospasm was considered as the major cause of poor outcome post-SAH.Frequently initiated by hemoglobin or endothelin from bleeding,visible narrowing of arteries occurred due to vasospasm and finally introduced Delayed Cerebral Ischemia,the most critical complications that would lead to critical clinical signs involving brain ischemia or even infarction.Management of post-SAH treatment focused conventionally on ways to prevent vasospasm or delayed cerebral ischemia but were revealed no long-term benefits to the recovery of SAH patients by several large-scale clinical cohort studies.However,early pathological changes before vasospasm appears have been reported to play more important roles in predicting and determining long-term neurological deficits.Researchers on neuroprotection field are now switching their interest to a new concept-Early Brain Injury(EBI),referring to acute adverse events within 72h after SAH which commerced immdiately.Evidence shows that elevation of intracranial pressure,impaired blood flow,cell necrosis,apoptosis,inflammatory response and oxidative stress are involved in this stage.Since more and more researches have indicated EBI severity as a single and critical factor to predict impact of bleeding following aneurysm rupture and the progression of DCI,investigations on effective interventions for EBI are clinically promising.Hydrogen sulfide(H2S)is basically a type of toxic gas with a smell of rotten eggs.Each year numerous fatal accidents are involved in H2S inhalation.However,since production of endogenous H2S was firstly uncovered and measured in 1990,physiological roles of H2S have rapidly been revealed.Three main enzymes are reported to contribute to endogenous H2S production in mammalian cells:Cystathionine β-synthase(CBS)and Cystathionine γ-lyase(CSE)are recognized as traditional ways to generate H2S using L-Cysteine as substrate,and 3-mercaptopyruvate sulfurtransferase(3-MST)is newly found in recent studies located in mitochondria of 3-mercaptopyruvate metabolism dependent neurons.Perception of H2S has now changed into a critical biological signaling molecular following NO and CO.Penetrating membranes without transporters or energy consumption enables H2S to pass through blood-brain barriers(BBB)smoothly.Additionally,expression of CBS in brain overweights CSE and 3-MST quantitively while its activity to produce H2S is also 6-9 fold high.All of these characteristics have indicated that CBS/H2S is mainly responsible for physiological functions in CNS among the three H2S-producing systems.H2S exhibits multiple functions under pathological circumstances.In response to oxidative stress,H2S could markedly increase glutathione to inhibit free radical damage.Moreover,H2S could reduce necrosis and apoptosis by suppressing ion channels on cell membranet which blocks Ca2+ overload in cytoplasm.Although previous research has observed that application of exogenous H2S was able to promote proliferation of neural stem cell and protect hippocampus from ischemia and reperfusion injury induced by cerebral ischemic stroke,whether it could present neuroprotective effects in hemorrhagic stroke including SAH and its underlying mechanisms still remains obscure.We hypothesized that H2S could play critical roles in alleviating early brain injury and improve long-term cognitive dysfunction after SAH.These beneficial effects were proposed to be initiated by supplement of L-Cysteine,which is mainly catalyzed by CBS.We operated autologous blood injection into cistern magna to set experimental SAH model in rats.General evaluation showed decreased mortality and improved neurological behavior after NaHS,donor of exogenous H2S,were intraperitoneally given to the SAH rats.The following histological analysis proved that H2S could markedly inhibit prefrontal cerebral(PFC)edema and neuronal apoptosis in early brain injury stage post SAH,which was consistent to better performance in Morris Water Maze test.Both the expression and activity of CBS were severely compromised by SAH.As a major substrate chemically corresponding to CBS in CNS,application of L-Cysteine could not only exert neuron protection,but attenuate synaptic dysfuntions via stabilizing the structural and molecular changes.All these effects were abrogated when CBS specific inhibitor AOAA were added,which might indicate that such neuroprotective activities of L-Cysteine heavily relied on "CBS/H2S" regulation.This study exhibits a therapeutic value of H2S and present promising clinical application of L-Cysteine via exploring its molecular mechanisms.Part I Neuroprotective Effects of Hydrogen Sulfide Against Early Brain Injury and Cognitive Dysfunctions After Subarachnoid Hemorrhage1.1 Aims(1)Use experimental SAH model in rats to explore basic pathological events in EBI following SAH and detect the improvement induced by the treatment of H2S on SAH rats.(2)To investigate potential molecular changes and related pathways which were involved in EBI and H2S application.(3)To identify that H2S releasing from its donor is beneficial to performance in Cognitive and behavioral test and to reveal an up-stream molecular mechanism.1.2 Methods(1)Set up experimental SAH modelMale Wistar rats weighting from 280 g to 350 g were subjected to blood injection method,of which autologous blood from right femoral artery was injected into cistern magna.(2)H2S donorNaHS is a compound which is automatically releasing H2S when dissolved into solutions.Low dose of NaHS has been proved to be bio-friendly to in vivo studies.It is safe and effective to play as H2S donor in current research.(3)Groups,drug administration and euthanasia of ratsBased on experimental design,rats were randomly assigned into 4 groups:Sham+vehicle,Sham+NaHS,SAH+vehicle,SAH+NaHS.NaHS was dissolved into saline and intraperitoneally given at 2h,6h,24h,46h after SAH.At 48h post SAH,they were under euthanasia through over-dose aspiration of isoflurane.(4)General description of mortality and neurological evaluationBefore execution at 48h after SAH,record mortality of each group and observe neurological deficits using Yamaguchi neurological scores system.(5)Investigation on pathological eventsParts of each group were selected to detect brain water content which indicates levels of brain edema.Hematoxylin-Eosin staining were used to show basic structural and pathological changes including edema,necrosis and infiltration of inflammatory cells in pre-frontal cortex(PFC)after SAH.Ultrastructural alterations of cells in the PFC at 48h were detected by Transmission Electron Microscopy(TEM).TUNEL and Immunofluorescence imaging were aimed at testing apoptosis in PFC and activity of caspase 3,the key initiator in apoptotic pathway.All the counting were processed by 2"blinded" investigators.(6)Detection of molecular expressionTotal RNA were extracted from PFC and amplified by Reverse Transcription-Polymerase Chain Reaction(RT-PCR)to explore gene expression post SAH at 48h.Western blot were used to show protein expression that were related to EBI and H2S treatment.Several molecular pathways were also verified by Western blot.(7)Cognitive deficits evaluationLearning and memory competence were taken as main markers to reflect cognitive function after SAH and they were tested in Morris Water Maze system.Rats of each group were going through Navigation Probe Test for consecutive 5 days and Spatial Probe Test commenced at 6th day.Swimming traces and time spent were used to statistical analysis.1.3 Results(1)Rats that subjected to SAH embraced higher mortality than Sham groups.However,this rate presented significant decreasing with administration of NaHS.This is also consistent to the neurological test,NaHS-treated SAH rats got lower scores than those in vehicle-treated groups,which indicates better outcomes.Moreover,compared to Sham groups,brain water content significantly expanded when SAH occurred while after NaHS application,it was markedly alleviated at 48 post SAH.(2)Pale zone which indicates cell edema was sharply observed in PFC of SAH rats on HE staining slides,necrosis and pathological alteration of different cell were mixed.Outline of these cell were obscure and they are sparsely arranged.Giving NaHS to the SAH rats could rescue such negative events as shrunk edema region and decreased cell death.Layers and neurons exhibited clear borderline in SAH+NaHS group.Detection via TEM imaging revealed disruption of endoplasmic reticulum,ribosome and mitochondria as well as the nuclei of neurons in PFC after SAH insult,treatment of SAH with NaHS reduced these morphological injuries.(3)Accumulating apoptosis was displayed in PFC at 48h after SAH.TUNEL-positive cells were notably increased.This is consistent to the high level of cleaved-caspase 3 in neurons,which indicates the activation of caspase-dependent pathway.Expression of Bax,a typical pro—apoptotic protein,was markedly simulated while anti-apoptotic protein Bcl-2 shrunk.Administration of NaHS could suppress excessive apoptosis by inhibiting activation of caspase 3 as well as reversing the changes of Bax and Bcl-2,lowering the Bax/Bcl-2 ratio.Single injection of NaHS to rats subjected to Sham could not induce significant apoptosis and Bax/Bcal-2 change.(4)Phosphorylation of ERK1/2 and Akt were blocked when SAH occurred accompanied by augment of PTEN,the negative regulator of PI3K/Akt pathway.NaHS treatment was effective in abolishing the suppression of ERK1/2 and Akt signaling.However,expression of PTEN were not altered compared to the SAH group.(5)MWM test revealed that SAH rats undergone application of NaHS performed better than vehicle-treated SAH rats,as shortening their escape latency and swimming distance in navigation probe and presenting more time and bias in target quadrant in Probe trial.Exposure to SAH inhibited expression of brain-derived neurotrophic factor(BDNF)and activation of cAMP-response element binding protein(CREB)in PFC,while NaHS was able to rescue the regression on CREB-BDNF pathway.1.4 Conclusions(1)H2S could significantly decrease mortality and ameliorate neurological deficits in EBI stage following SAH occurrence.These neuroprotective effects are dependent on reducing brain edema,necrosis and neuron apoptosis.(2)ERK1/2 and Akt pathways are potential targets which H2S relies on to exert anti-apoptotic roles in SAH model.However,PTEN was not changed by interference of H2S,which hints that there might be other mechanisms to regulate ERK1/2 and Akt activation.(3)H2S enables improvement on cognitive dysfunction following SAH via stimulating CREB-BDNF related mechanism.PartⅡ L-Cysteine Attenuates Early Brain Injury and Improves Synaptic Density via CBS/H2S Pathway Following Subarachnoid Hemorrhage in Rats 2.1 Aims(1)Explore physiological changes on CBS expression as well as activation after SAH and verify its key role in producing endogenous H2S in CNS.(2)Secure the bio-safety of L-Cysteine and AOAA administrated to rats and assess whether L-Cysteine exerts its neuroprotective roles by H2S released from CBS catalyzing.Find out molecular alterations induced by L-Cysteine in the EBI stage,(3)Evaluate L-Cysteine’s role in improving pathological events to synapse which are closely related to cognitive deficits following SAH and reveal molecular mechanism.2.2 Methods(1)Male adult Wistar rats were randomly divided into 5 groups according to experimental design:Sham,Sham+L-Cysteine,SAH,SAH+L-Cysteine,SAH+L-Cysteine+AOAA.L-Cysteine was dissolved into saline(100 nM)and given to the rats 30min after SAH through cerebroventricular injection.Meanwhile,AOAA(5 mg/kg)was intraperitoneally administrated to rats as soon as the L-Cysteine injection ended.There are additional rats to test bio-safety of AOAA.(2)At 48h post SAH,modified Garcia scores system was introduced to assess neurological function of each rat before euthanasia.Brains were quickly removed to determine water content.(3)Immunohistochemistry was used to detect expression of CBS in PFC.Its activity was measured following instructions from CBS assay kit.Traditional methylene blue method was applied to quantify the alteration of endogenous H2S production.(4)Apoptosis were measured by TUNEL staining and double labeled immunofluorescence was used to explore activation of caspase-dependent signaling pathway.(5)Molecular changes were quantified from gene to protein expression using RT-PCR and Western blot.(6)Structural alterations on synapses in PFC were observed through TEM.Related proteins that indirectly reflect integrity and function of the synapses were tested by RT-PCR and Western blot.2.3 Results(1)Solely administration of L-Cysteine or AOAA stimulated no significant edema or neuron death in PFC.(2)As shown in immunohistochemistry result,CBS were highly expressed on astrocytes rather than neurons.Application of L-Cysteine through intracerebroventricular way did not alter CBS expression,but notably accelerated the enzymatic activity,which promoted the production of endogenous H2S.AOAA blocked these effects.(3)L-Cysteine could markedly ameliorate neurological and behavioral dysfunctions resulting from SAH via suppressing cortex edema and reducing cell apoptosis as well as its downstreaming molecular expression,which were consistent with our previous research on the role of H2S.(4)There are vigorous loss of synapse in PFC following SAH occurrence.Structure altered as presynaptic and post synaptic membranes were vague.Dense deposits which were dark stained accumulated in synaptic cleft,disordering the well-fined border of synapse.Two synaptic density-related proteins exhibited opposite results:synaptophysin was disrupted while PSD presented an up-going trajectory when SAH blast.L-Cysteine application could dramatically improve the damage induced by SAH with increasing the number of normal synapse and reverse the expression of synaptophysin and PSD95.The use of AOAA abrogated these beneficial effects led by L-Cysteine.(5)Suspended by SAH,the CREB-BDNF pathway was rescued as L-Cysteine was given.2.4 Conclusions(1)L-Cysteine is a CNS-friendly amino acid and is safe to be administrated.Being catalyzed by CBS expressed on astrocytes and releasing endogenous H2S is the main pathway L-cysteine exerts its neuroprotective roles after SAH,which reduces edema and apoptosis in Early Brain Injury stage.(2)As a substrate,L-Cysteine could not only be catalyzed to H2S but significantly stimulate the activation of CBS.There might be a potential feedback in the interaction of CBS and L-Cysteine.(3)L-Cysteine could protect synapse in PFC from suffering loss and structural damage post SAH.Pre-and postsynaptic proteins are reliable targets in which L-Cysteine plays roles to regulate cognitive functions.