Study Of High Concentration Glutamate On Delayed Cerebral Ischemia Prediction After Subarachnoid Hemorrhage And MGluR1 Regulating Neurological Function

BackgroundSubarachnoid hemorrhage(SAH)is a serious life-threatening disease caused by the rupture of intracranial aneurysm(I A),its incidence accounts for about 5%-10%of the incidence of cerebrovascular diseases,and about 85%of spontaneous subarachnoid hemorrhage.The onset age of aSAH patients is relatively young(mostly between 25 and 64 years old),and the mortality rate is as high as 40%,survivors often have short-term or permanent neurological dysfunction,which require long-term rehabilitation care,and cause a heavy burden on families and society.With the gradual improvement of clinical treatment and the concept of intensive care,the risk of secondary rupture of aneurysm and the mortality and disability have decreased.However,SAH is still a serious threat to people’s health,and it is a difficult problem for researchers and clinical medical workersDelayed cerebral ischemia is a serious complication after aSAH.Studies have confirmed that DCI is closely related to poor prognosis after SAH.DCI seriously reduces the survival rate and quality of life of patients after SAH.Many studies have shown that early treatment of DCI,is the key to reduce mortality and improve prognosis after SAH.The main diagnostic criteria of DCI were as follows:1)the new focal neurological deficit lasted for at least 1 hour and showed changes in neurological function score;2)new infarct lesions were found by CT(computed tomography,CT).CT examination of cerebral infarction is a retrospective observation,so the clinical diagnosis of DCI is limited,which may lead to delayed diagnosis and insufficient treatment of DCI.Therefore,detecting early potential predictors related to DCI occurrence is a method to prevent or early intervention in DCI.Studies have shown that glutamate concentration in cerebrospinalfluid significantly increases after SAH,which is related to the occurrence of cerebral vasospasm and edema.However,it is not clear whether early high glutamate concentration in CSF is related to DCI occurrence after SAH,and whether it can be used as a possible potential predictor of DCI.Glutamate is an excitatory neurotransmitter of the central nervous system,which acts on postsynaptic glutamate receptors.Metabolic glutamate receptor 1(mGluR1)is a G protein coupled receptor that binds to Gq and activates phospholipase C,resulting in phosphatidylinositol-4-diphosphate hydrolysis,calcium release from endoplasmic reticulum and slow excitatory postsynaptic potentialThe previous animal experiment results of the research group showed that inhibiting mGluR1 can reduce the calcium overload and neuronal apoptosis mediated by high concentration of glutamate,and the blood-brain barrier damage and brain edema induced by high concentration of glutamate after experimental SAH.Provides neuroprotection in early brain injury(EBI)after experimental SAH.But it is unclear whether mGluR1 is related to long-term neurological dysfunction after SAH.The negative allosteric regulator JNJ16259685 of mGluR1 can selectively bind to the allosteric binding site of the seven-helix transmembrane domain of the receptor and inhibit the activity of the receptor,showing the characteristics of non-competition and negative regulation,and JNJ16259685 effectively crossing the blood-brain barrier However,the effect of negative allosteric regulator JNJ16259685 on long-term neurobehavioral function of rats after SAH and its possible mechanism(cerebral blood flow decrease,cerebral vasospasm,microthrombosis and neuron injury)are still unclear,and need further study and explorationObjective1.To detect the early concentration of glutamate in CSF samples of patients with aSAH.2.To analyze the correlation between high concentration of glutamate in the early CSF of aSAH patients and DCI,and to explore whether high concentration of glutamate in early CSF can be used as an early potential predictor of DCI.3.Based on the results of clinical research,animal experiments adopt intravascular puncture to establish rat SAH model4.To analyze the effect of JNJ16259685,a negative allosteric regulator of mGluR1,on the long-term neurofunction of experimental SAH and explore the related mechanismMethods1.Approved by the Ethics Committee of Jining first people’s Hospital and Baotou Central Hospital.From January 2019 to October 201 9,61 patients with aSAH were enrolled in the study,and the control group was eight patients with idiopathic normal pressure hydrocephalus.Hunt-Hess grade and WFNS grade were evaluated according to the neurological function of aSAH patients on admission,and the bleeding severity after SAH was graded by modified Fisher score.CSF samples were collected within 48 hours after SAH by extraventricular drainage or lumbar puncture The concentration of glutamate in CSF was determined by enzyme-linked immunosorbent assay.Univariate Logistic model regression was used to analyze the correlation between glutamate concentration in CSF and DCI in patients with SAH The adjusted OR value and 95%confidence interval were used to reflect the effect of high concentration glutamic acid on DCI.P<0.05 is the difference with statistical significance2.To explore the effect of mGluR1 negative allosteric regulator JNJ 16259685,on long-term neurological function after SAH and its possible mechanism.SAH model of SD rats was established by intravascular puncture.After the model was successful,the femoral artery blood of rats was taken for arterial blood gas analysis The local and whole brain blood flow of rats was detected by laser Doppler blood flow meter and lmin average weight cerebral blood flow value.HE staining was used to observe the morphological changes of cerebral basilar artery and measure the cross-sectional area of cerebral basilar artery in SAH rats.Double staining of CD31 and fibrin immunofluorescence was used to observe the formation of micro vessels and microthrombosis in the basal cortex of rats.The number of normal neurons in hippocampal CA1 region of rats was observed by NeuN immunofluorescence.ELISA was used to determine the content of cortical microvascular fibrin.TUNEL staining was used to observe neuronal apoptosis.Fluoro-Jade C staining was used to analyze the degenerated neurons qualitatively and quantitatively.The expression of p-eNOS,eNOS,VASP,P-VASP protein in basilar artery was detected by westernblot,and the protein levels of NeuN,Bcl-2,Bax,active caspase-9 and caspase-3 in basal cortex and hippocampal CA1 region were detected.Modified Garcia score was used to evaluate the neurobehavioral function of rats.Foot error test,forelimb placement test and rotation axis test were used to detect long-term sensorimotor and sensorimotor coordination function.Water maze test was used to test the learning and memory functions of spatial position and direction in rats.Results1.Prediction of high concentration glutamate in cerebrospinal fluid on delayed cerebral ischemia after subarachnoid hemorrhage(1)Compared with the control group,the age and gender of aSAH patients had no statistical significance.(2)In aSAH patients,there is no significant difference in gender,age,history of hypertension,location of aneurysm and treatment of aneurysm between patients with DCI and patients without DCI(3)Glutamate concentration in CSF of SAH patients at early stage was higher than that of control group,and early CSF glutamate concentration in CSF of patients with DCI was higher than that of patients without DCI.The data of both groups were statistically significant.(4)The proportion of Hunt-Hess grade 4-5,WFNS grade Ⅳ-Ⅴ and modified Fisher score 3-4 in patients with DCI after SAH is higher than that of patients without DCI after SAH,and the data has statistical significance.Early glutamate concentration in CSF of Hunt-Hess grade 4-5 patients was higher than that of patients with grade 1-3,the early concentration of glutamate in CSF of patients with grade Ⅳ-V of WFNS grade was higher than patients of grade Ⅰ-Ⅲ,and the early concentration of glutamate in CSF of patients with modified Fisher score of 3-4 was higher than patients with grade 1-2.The data obtained were statistically significant(5)Univariate regression model analysis showed that there was a significant correlation between the high concentration of glutamate in early CSF and the occurrence of DCI in aSAH patients,under the control of the WFNS classification,OR=1.75(95%CI:1.19-2.57),under the control of Hunt-Hess classification and modified Fisher score,OR=1.85(95%CI:1.69-2.03)2.Negative Allosteric Modulator of mGluR1 Improves Long-term Neurologic Deficits after Experimental Subarachnoid Hemorrhage(1)Compared with Vehicle(control group,sterile water treatment group containing 5%DMSO),there was no significant difference in mortality,weight change within 14 days and 24h SAH score in JNJ16259685 group(2)Compared with Sham(sham operation group),SAH rats showed obvious sensory,motor and memory deficits in modified Garcia score,foot error test,forelimb placement test,rotating rod test and water maze test.Compared with the vehicle group,the sensorimotor memory function of rats of JNJ 16259685 group improved.(3)There was no significant difference in arterial blood gas indexes among Sham group,Vehicle group and JNJ 1 6259685 group.There was no significant difference in intracranial pressure,local cerebral blood flow in cortex and average weight cerebral blood flow in 1 hour between the Vehicle group and JNJ 16259685 group.On the 7th day after SAH,the average weight cerebral blood flow in 1 minute between the Vehicle group and JNJ 16259685 group was statistically significant(4)Compared with Sham group,the cross-sectional area of basilar artery lumen in Vehicle group decreased significantly,while the lumen cross-sectional area in JNJ 16259685 group was larger than that in Vehicle group.Both groups of data were statistically significant.(5)On the 7th day after SAH,the fibrin in the cortical microvascular lumen of rats in the Vehicle group increased significantly,and decreased after administration of JNJ16259685.Both groups of data were statistically significant(6)On the 7th day after SAH,TUNEL and FJC positive cells in basal cortex increased significantly,but decreased significantly after administration of JNJ16259685.In hippocampal CA1,NeuN positive neurons decreased and FJC positive cells increased,which improved after administration of JNJ16259685.the data were statistically significant.(7)On the 7th day after SAH,the levels of p-eNOS,eNOS,VASP,p-VASP protein in basilar artery decreased significantly,and improved after administration of JNJ16259685.(8)On the 7th day after SAH,the protein levels of NeuN and Bcl-2 in basal cortex and hippocampal CA1 region of rats decreased,while he protein levels of bax,active caspase-9 and active caspase-3 increased,and improved after administration of JNJ16259685.The data were statistically significant.Conclusions1.Prediction of high concentration glutamate in cerebrospinal fluid on delayed cerebral ischemia after subarachnoid hemorrhage(1)The concentration of glutamate in CSF of patients with aSAH was increased in the early stage,and the concentration of glutamate in CSF of patients with DCI was also higher than that of patients without DCI.(2)The concentration of glutamate in CSF of aSAH patients increased with the increase of Hunt-Hess grade,WFNS grade and modified Fisher score.(3)The high concentration of glutamate in the early CSF of aSAH patients has a certain predictive effect on the occurrence of DCI,and can be used as a potential early predictor of DCI.2.Negative Allosteric Modulator of mGluR1 Improves Long-term Neurologic Deficits after Experimental Subarachnoid Hemorrhage(1)JNJ16259685 improves long-term neurobehavioral function after experimental SAH(2)JNJ16259685 attenuates delayed decrease of cerebral blood flow,cerebral vasospasm and microthrombosis induced by SAH(3)JNJ16259685 inhibits SAH-induced long-term neuronal death and degeneration.