| Subarachnoid Hemorrhage(SAH)is a hemorrhagic disease with very high lethality and high disability.Early barin injury(EBI)caused by SAH is the key to poor prognosis.Factors related to intracranial pressure,autonomic dysregulation of cerebral blood flow,ion homeostasis and energy imbalance,glutamate excitotoxicity,neuroinflammation,neuronal apoptosis,microcirculatory disorders,etc.,their specific roles and each other The mechanism of influence is not very clear,and in these changes,cerebral edema,blood-brain barrier destruction and neuronal apoptosis are considered to be key pathophysiological changes.Excitoxicity refers to the post-synaptic neurotoxic effects caused by excitatory amino acids(mainly glutamate)in the brain acting on their receptors.Glutamate homeostasis is necessary to maintain the normal physiological function of the body.Studies have shown that cerebrospinal fluid in patients with clinical SAH and experimental SAH models has an increased glutamate concentration,excessive release of glutamate,and a large number of activated glutamate receptors.Such as NMDAR NR 2B receptor and m Glu R1 receptor),causing brain damage.The active glutamate receptor 1(m Glu R1)is expressed on the surface of cerebral vascular endothelial cells.After activation,m Glu R 1 activates phospholipase C(PLC)to act on phosphatidylinositol diphosphate(PIP2)to produce two A second messenger,one is inositol triphosphate(IP3+)and the other is diglyceride(DAG),IP3+ activates neuroprotective PI3K/AKT signaling pathway,causing a large amount of Ca2+ ions in the endoplasmic reticulum Release,affecting the release of endogenous glutamate and regulating and facilitating the binding of NMDA receptors.Over-release of glutamate causes Ca2+ overload,activates calmodulin kinase,activates m Glu R1,aggravates neurotoxic excitotoxicity,highly stimulates the release of Ca2+ in the endoplasmic reticulum of cells,and mediates Ca2+ influx through mutual complementation and enhancement of NMDA receptors.The cascade activates the apoptotic pathway.JNJ16259685 is a highly potent and non-competitive inhibitory effect on m Glu R 1,selectively binds to the aminoacyl allosteric site carried by glutamate receptors,inhibits receptor activity,decreases glutamate concentration,and is able to cross the blood-brain barrier.Allosteric agents that selectively bind to receptor allosteric sites can avoid ligand interference and can distinguish receptors of different subtypes,and have very good clinical application prospects.This study intends to use JNJ16259685 to interfere with the excessive activation of m Glu R 1 induced by high concentration of glutamate after SAH,and to detect and analyze the brain edema,blood-brain barrier destruction and neuronal apoptosis induced by high concentration of glutamate in rats after JHJ16259685 The protective effect and mechanism of action were evaluated by the neurological deficit score after SAH,SAH grade classification,brain tissue water content and aquaporin AQP4 changes,blood-brain barrier permeability change,TUNEL staining,immunofluorescence analysis,etc.to evaluate JNJ16259685 Neuroprotective effect,explore its mechanism of brain damage protection in the EBI stage after SAH,and find new targets for clinical treatment.Experiment purpose:(1)SAH model was established by intracranial intravascular puncture,and the imp-rovement of neurological deficits in rats after SAH was studied by JNJ16259685.(2)To study the effect and mechanism of JNJ16259685 on the expression of m Glu R1 in endothelial cells,endothelial cell injury,blood-brain barrier and cerebral edema in rats with EBI;(3)Study on the reduction of apoptosis and its mechanism of action after SAH in rats,and the mechanism of exerting neuroprotection.Research methods:(1)Select SPF male SD rats(body weight 270-320 g,9-10 weeks old),raise room temperature 23-25 ° C,relative humidity(60-65),caged,environmentally ventilated,food And water intake on time,circadian rhythm.The experimental animals were randomly divided into 3 groups: sham group(n=18),subarachnoid hemorrhage group(SAH group),and SAH group were divided into 2 subgroups according to the time phase: 24 h and 72 h groups.: SAH+Vehicle group(n=36),SAH+JNJ16259685 group(n=36).The SAH experimental animal model was established by internal carotid artery puncture.The SAH group was modeled by intravascular puncture.The sham operation group introduced the suture to stop the resistance.The other surgical procedures were consistent with the above methods.Experimental animals were randomly assigned to each group,and 6 rats in each group were individually subjected to individualized experiments,supplemented according to the condition of the experimental animals and the postoperative mortality.DMSO or JNJ16259685(1 mg/Kg)was intraperitoneally injected 2 h after the successful modeling.(2)24 hours after SAH,the modified Garcia scale was used for neurological function score,and the brain was decapitated for SAH grade classification to assess the severity of subarachnoid hemorrhage,and whether the severity of hemorrhage and the score of neurological function were correlated.Group standard rat models were subjected to individualized experiments.(3)After the successful modeling,the severity of SAH grade bleeding was evaluated at 24 h and 72 h,and the neurological deficit was assessed by Garcia scale.The expression of m Glu R1 in endothelial cells was detected dynamically.Brain edema was measured by dry and wet weight method,Evans blue and albumin.FITC method was used to assess the degree of blood-brain barrier destruction.Western blot was used to detect aquaporin(AQP4)and blood-brain barrier tight junction proteins(ZO-1,Occludin,Claudin-5)and apoptosis-related signaling molecules.The blood-brain barrier was observed by immunofluorescence.The damage of basal cortical neurons and endothelial cells to further confirm the specific role of m Glu R1 in EBI and related mechanisms,providing a reliable basis for clinical treatment.Results:1.JNJ16259685 can significantly improve the SAH neurological deficit after 24 h and72h of subarachnoid hemorrhage in rats.2.JNJ16259685 can significantly improve the blood-brain barrier and brain edema after subarachnoid hemorrhage in rats for 24 h and 72 h.3.JNJ16259685 can significantly improve the apoptosis of rat subarachnoid hemorrhage after 24 h,72h.Conclusion:1.We used the internal carotid artery puncture method to make the subarachnoid hemorrhage model,successfully simulated the subarachnoid hemorrhage caused by the spontaneous rupture of the aneurysm,and graded the severity of subarachnoid hemorrhage through SAH grade.The modified Garcia scale was used for neurological function scoring,and the animal models that met the requirements were screened for individualized experiments.2.According to SAH grade,the model was successfully established,and JNJ16259685(1mg/Kg)was injected intraperitoneally.Two time points were selected for24 h and 72 h for individualized experiments.The results showed that JNJ16259685 can significantly improve the neurological deficit after SAH.In order to reduce the apoptosis of neurons,reduce the damage of the blood-brain barrier,improve the permeability of the blood-brain barrier,thereby reducing vasogenic cerebral edema.3.JNJ16259685 play a neuroprotective effect after SAH may be achieved by reducing damage to brain vascular endothelial cells. |
