The Role And Mechanism Of Astrocytic Glutamate Transporter In Delayed Cognitive Impairment After SAH

Background and ObjectiveSubarachnoid hemorrhage（SAH）is a kind of severe acute hemorrhagic cerebrovascular disease caused by a variety of causes（ruptured cerebral aneurysm,trauma,tumor,coagulation dysfunction,congenital vascular malformation,etc.）,accounting for about 5%of all stroke subtypes,with a high mortality rate（more than 60%）.Among the surviving patients,more than 20%have long-term and severe cognitive impairment,such as depression,anxiety,memory impairment and so on.It not only greatly affects the life quality of patients,but also brings a heavy burden to the family and society.However,at present,there is a lack of effective treatment for this in clinic.How to prevent and treat delayed cognitive dysfunction after SAH early is a scientific problem that needs to be solved urgently in this field.At present,the pathogenesis of delayed cognitive impairment after SAH is not clear,but the neurotransmitter system,especially the excitatory glutamatergic transmitter system disorder,is highly valued in cognitive and mental diseases.A large number of studies have shown that the levels of glutamate in cerebrospinal fluid and peripheral plasma are abnormally increased after stroke,suggesting that the disturbance of glutamate metabolism and abnormal glutamatergic nerve transmission may be an important mechanism of cognitive impairment after SAH,but the specific molecular mechanism needs to be further discussed.Focusing on the mechanism of cognitive impairment after SAH,this study used mouse SAH model,combined with cognitive behavioral analysis,gene expression detection,neurotransmitter detection and target pharmacological intervention,to carry out in-depth mechanism and functional verification research,in order to reveal the pathogenesis and molecular mechanism of delayed cognitive impairment after SAH,and through the behavioral verification of key target intervention.It provides a new idea for the early targeted treatment of this kind of disease.Materials and Methods1.Analysis of the correlation between astrocyte function and delayed cognitive impairment after SAH:using the SAH model of internal carotid artery puncture in mice,the depression was evaluated by forced swimming and sugar water preference test,and the learning and memory function of mice was tested by Morris water maze at 1 week before modeling and 2 weeks,4 weeks,8 weeks and 12 weeks after modeling.Eight weeks after modeling,the differentially expressed genes in hippocampus of SAH group and sham group were analyzed by gene transcriptome sequencing,and the enrichment analysis of gene function was carried out,and the cerebrospinal fluid of sham and SAH mice were collected by microdialysis,and the concentration of glutamate in cerebrospinal fluid was detected by HPLC-MS/MS.Astrocytes in the hippocampus of sham and SAH mice were sorted with anti-ACSA-1 magnetic beads at 8 weeks after modeling.Glutamate reuptake experiments were performed after primary culture to analyze the glutamate reuptake ability of astrocytes after SAH.;Western blot was used to detect the expression and phosphorylation of glutamate receptors Glu N2B and Glu A1 in the hippocampus of sham and SAH mice 1 week before and 8 weeks after modeling,and the expression levels of GLT-1 and GLAST in astrocytes.Behavioral experiments were conducted to analyze the effect of GLT-1 agonist treatment on cognitive impairment in SAH mice.2.Molecular mechanism of abnormal regulation of GLT-1 expression during cognitive impairment after SAH:real-time quantitative PCR was used to detect the m RNA level of GLT-1 in the hippocampus of sham and SAH mice at 1 week before modeling and 2 weeks,4 weeks,8 weeks and 12 weeks after modeling,and the difference of histone acetylation in hippocampal astrocytes of sham and SAH mice was detected by acetylated labelfree protein spectrum at 8 weeks after modeling.Western blot was used to detect the expression of Ac-H3（K18）and HDAC1-6 in the hippocampus of mice sorted by magnetic beads at 1 week before modeling and 2 weeks,4 weeks,8 weeks and 12 weeks after modeling.The expression of GLT-1 and HDAC2 was detected by transfection of sh HDAC2 plasmid into primary astrocytes,Western blot of postnatal mice.;Western blot,q PCR and luciferase reporter gene analysis were used to analyze the effect of inhibition of HDAC2 on GLT-1 gene transcription after SAH.3.Study on the mechanism of targeted inhibition of HDAC2 on delayed cognitive impairment after SAH:Hdac2 conditioned knockout mice(Hdac2^fl/fl)and hippocampal astrocyte-specific Hdac2 knockout mice(Gfap Hdac2^-/-)were used here.The expression of GLT-1 was detected Western blot after SAH.behavior to detect the cognitive function of sham group and SAH group;behavioral pharmacology experiments to evaluate the effects of HDAC2 inhibitors and GLT-1 inhibitors on the cognitive function of SAH mice.Research results and conclusions.1.Analysis of the correlation between astrocyte function and delayed cognitive impairment after SAH:（1）the characteristics and rules of delayed cognitive impairment in SAH mice were clarified.Depression and memory impairment lasted for more than 12weeks and were the most serious at 8 weeks.（2）Gene sequencing showed that hippocampal genes were generally down-regulated 8 weeks after SAH,which was mainly related to cognition,learning and memory,neurotransmitter transport and synaptic plasticity,and the differential changes of these genes may be related to the function of astrocytes.（3）it is confirmed that abnormal accumulation of glutamate in hippocampal interstitial space occurs in cognitive impairment after SAH,which may be related to the decrease of glutamate uptake in astrocytes.（4）after SAH,the phosphorylation level of excitatory glutamate receptor in hippocampal neurons decreased significantly,and the function of excitatory glutamate receptor decreased significantly.（5）GLT-1 in astrocyte-specific EAATs in hippocampal tissue decreased significantly after SAH,but GLAST did not change significantly.（6）treatment with specific GLT-1 activator can effectively alleviate delayed depression and memory impairment after SAH.Based on the above results,we analyzed the characteristics and rules of delayed cognitive impairment in SAH mice,and combined with gene sequencing,glutamatergic function detection,target intervention and behavioral verification,we confirmed that down-regulation of GLT-1rather than GLAST on astrocytes may be an important mechanism of delayed cognitive impairment after SAH.2.Study on the molecular mechanism of abnormal regulation of GLT-1 expression during cognitive impairment after SAH:（1）the transcriptional expression of GLT-1glutamate transporter in hippocampal astrocytes was down-regulated after SAH,but there was no significant change in GLAST.（2）the level of H3 acetylation（especially Ac-H3（K18））in hippocampal astrocytes decreased significantly after SAH,which may be related to the down-regulation of GLT-1 transcription.（3）the expression of HDAC2 in astrocytes was significantly increased after SAH.（4）inhibition of HDAC2 could significantly up-regulate the expression of GLT-1 in astrocytes and reverse the down-regulation of GLT-1 induced by SAH and Oxy Hb in vitro.Through the above studies,it is preliminarily confirmed that the down-regulation of GLT-1 protein expression after SAH is caused by its gene transcriptional inhibition,and its mechanism may be due to the increased specific expression of HDAC2 in astrocytes after SAH,which mediates the acetylation of H3 in astrocytes（especially the acetylation of lysine K18）,resulting in the inhibition of GLT-1gene transcriptional activity.These findings suggest the molecular mechanism of the decreased expression of GLT-1 in HDAC2-Ac H3-GLT1 pathway during the development of cognitive impairment after SAH,and HDAC2 may be an upstream target for the regulation of GLT-1 expression and may play an important role in the intervention of related diseases.3.The mechanism of inhibition of HDAC2 on delayed cognitive impairment after SAH:（1）HDAC2 mice with specific knockout of hippocampal astrocytes can maintain the expression level of GLT-1 after SAH,and their delayed cognitive impairment is significantly less than that of wild mice;（2）efficient and selective inhibitors of HDAC2can effectively improve delayed cognitive impairment after SAH,which may depend on the activity of GLT-1.The above results confirm that the specific inhibitors targeting HDAC2 play a significant role in the treatment of delayed cognitive impairment secondary to SAH,and the related target drugs may have potential clinical significance.In summary,our study analyzed the pathological mechanism of delayed cognitive impairment after SAH from the perspective of astrocytes,and provided new experimental evidence for targeted therapy of related cognitive impairment.