Roles Of ZO-land Claudin-5 In Early Brain Injury After Subarachnoid Hemorrhage In Rats

ObjectiveAneurysmal subarachnoid hemorrhage (SAH) affects 10 per 100000 population in the Western world. For survivors of the initial hemorrhage, cerebral vasospasm and early brain injury are major causes of subsequent morbidity and mortality,12.4% sudden death before receiving medical attention, and up to 40%to 60%of patients will die within the first 48 hours because of the initial bleeding and about 35%within 24 hours after SAH. Although considerable advances have been made in endovascular techniques, diagnostic methods, and surgical and perioperative management paradigms, outcome for patients with SAH still remains poor. Many survivors of SAH experience persistent cognitive deficits, with an effect on functional status and quality of life. The vasospasm is delayed in onset, usually 4-5days after SAH. This shows that patients with early death were not due to delayed vasospasm after SAH. Recently, the term "early brain injury" has been generated referring to the immediate injury within 72 h and includes secondary events to SAH before the development of cerebral vasospasm. Early brain injury is characterized by an acute increase in intracranial pressure (ICP) and subsequent reductions of cerebral perfusion pressure (CPP) and cerebral blood flow (CBF) resulting in global cerebral ischemia. These primary mechanisms trigger secondary phenomena such as brain edema formation, acute vasoconstriction, and, on the molecular level, anaerobic cellular respiration, energy depletion, excitotoxicity, and the formation of free radicals. Early brain injury comprising blood-brain barrier (BBB)disruption, brain edema, and global ischemia is important in the pathophysiology of subarachnoid hemorrhage (SAH); however, the mechanisms are not clearly understood. The study demonstrated tight junction(TJ) is the inportant factor to maintain blood-brain barrier(BBB) integrity. TJs constituted the barrier of ion and molecule passing membrance transport through the paracellular pathway. TJ was constituted by a group of molecular protein elements, such as claudins, occludin. Claudin-5 is the brain microvascular endothelial cell-specific protein in the TJ. Study found that claudin-5 was the necessary and suficient condition in the constitution of TJ. ZO-1 is the first protein identified in TJs, and more and more evidence shows that the lack of ZO-1 may lead to TJs broken.In present study, we established endovascular perforation model of SAH in rats as described previously and observed blood-brain barrier ultrastructual changes by transmission electron microscopy (TEM), meanwhile examined the tight junction proteins ZO-1 and Claudin-5 expression in cerebral hemisphere by Western blot, and to further explore the roles of which in early brain injury after subarachnoid hemorrhage in rats.Materials and methodsThe endovascular perforation SAH model was used as previously described in adult male Sprague Dawley rats. One hundred and twenty animals weighing 300mg to 350mg were were randomly divided into 6 groups:control(n=20), sham(n=20), SAH(n=20), SAH+DMSO(n=20), SAH+SP600125 (10mg/kg) (n=20)and SAH+ SP600125 (30mg/kg) (n=20). SP600125(a inhibitor of JNK) was intraperitoneally injected 1 hour before and 6 hour after the induction of SAH which was dissolved in DMSO. The same volume of DMSO without the inhibitor was injected in rats as the SAH+DMSO group.Results1. The tight junctions of brain microvessel endothelial cells were open to different degrees 24 h after SAH, tight junction associated protein ZO-1 and Claudin-5 expression levels:Sham group compared with the Control group was not significant (ANOVA, P> 0.05); SAH group and SAH+DMSO group compared with the Sham group had significant statistical difference (ANOVA, P<0.05); SAH group compared with the SAH+DMSO group was not significant (ANOVA, P> 0.05).2. The blood brain microvascular endothelial cell tight junctions (TJs) open lower, and SAH+10mg/kg(SP600125) group with SAH group and SAH+DMSO group was not statistically significant (ANOVA, P> 0.05); SAH+30mg/kg(SP600125) group with SAH group and SAH+DMSO group was statistically significant differences (ANOVA, P<0.05); SAH+30mg/kg(SP600125) group with SAH+ 10mg/kg(SP600125) group was statistically significant differences (ANOVA, P<0.05).3. JNK inhibitor SP600125 group improved the neurological deficits 24 h after subarachnoid hemorrhage; the mortality at 24 h after SAH was 30%in SAH group, 35%in SAH+DMSO group,25%in SAH+SP600125(10mg/kg)group and 15%in SAH+SP600125(30mg/kg)group respectively,and sham group had zero mortality,no significant differences were observed between the groups (x2 test, P>0.05).Conclusions1. Blood-brain barrier damage after subarachnoid hemorrhage is one of manifestations in early brain injury, in which the tight junctions associated protein ZO-1 and Claudin-5 play an important role.2. JNK inhibitor SP600125 can amend the damage of blood-brain barrier in early brain injury after subarachnoid hemorrhage.