Blood-brain Barrier Permeability Change And Regulation Mechanism After Subarachnoid Hemorrhage

ObjectiveSpontaneous subarachnoid hemorrhage (SAH) is characterized by the extravasation of blood into the spaces covering the central nervous system that are filled with cerebrospinal fluid. Overall incidence of SAH is about around 6 per 100 000 persons per year. Overall mortality rate of SAH is 32-67%, SAH accounts for about 25% of all stroke deaths. Community-and population-based epidemiological survey showed that about 50% of subarachnoid hemorrhage deaths caused by aneurysm rupture occures within 30 days of initial bleeding.20-30% of survivors were left disabled. Ruptured intracranial aneurysm is main cause of subarachnoid hemorrhage, the mortality of SAH has decreased significantly due to the rapid development of microsurgical technique and endovascular treatment. Case mortality is around 20～50% (including pre-hospital deaths) and about 30% of patients died within the first few days after subarachnoid hemorrhage. Subarachnoid hemorrhage is a major reason for sudden death, About 12% of patients with ruptured anterior circulation aneurysms and 45% of patients with ruptured posterior circulation aneurysms suffered sudden death. The mechanism of brain injury after subarachnoid hemorrhage remain unclear, and therefore there is no specific treatment.Acute brain injury caused by Subarachnoid hemorrhage is a key factor of poor prognosis, However, due to subarachnoid hemorrhage involving the brain parenchyma blood vessels diameter is much smaller than the resolution of conventional imaging techniques, the injury mechanism is not yet entirely clear. subarachnoid hemorrhage cause ed Blood-brain barrier damage and brain edema formation,These processes were caused by vascular endothelial contraction and decomposition of tight junctions and increase in vascular permeability. Intracranial pressure has been found increased rapidly after subarachnoid hemorrhage both in patients with subarachnoid hemorrhage and experimental animal models, resulting in significantly decreased regional cerebral blood flow and metabolic changes. Early brain damage after subarachnoid hemorrhage mainly for the blood-brain barrier damage and brain edema formation, Recent results show that microcirculatory injury caused by diameter changes and continuity of the microcirculation damage,increase of vascular permeability,and fluid, fibrin and red blood cell extravasation into the brain substancecan occur in very early stage of Subarachnoid hemorrhage. It is reported that increase in microvascular permeability after subarachnoid hemorrhage interrelated with delayed cerebral ischemia secondary and poor prognosis, Apoptosis of neurons and cerebral vascular endothelial cells may be a key factor in the blood-brain barrier damage and formation of brain edema.In this study, rat model of subarachnoid hemorrhage was established by endovascular filament perforation technique,We use immunohistochemistry and Molecular biology methods to find blood brain barrier change and molecular mechanism of brain damage after Subarachnoid hemorrhage, we also And to explore the time phase of microvascular injury and tight junction protein expression after subarachnoid hemorrhage, provide a theoretical basis for the Clinical target in the treatment of acute brain injury after subarachnoid hemorrhage。Materials and methods1. The establishment of subarachnoid hemorrhage model:SAH was induced by endovascular filament perforation technique,. The common carotid artery was exposed via a midline incision,and the external carotid artery (ECA) was isolated and ligated. A 0.26mm monofilament nylon suture was placed into the ECA and advanced into the internal carotid artery(ICA). The suture was advanced to the anterior cerebral artery (ACA)-middle cerebral artery (MCA) bifurcation,where resistance was encountered. The suture was then advanced 2-3 mm, perforating the ACA, and then immediately withdrawn, allowing reperfusion. In sham-operated mice,the suture was advanced only until the point of resistance, but arterial perforation was not performed.2. Regional cerebral blood flow (rCBF) measurements:The animals were anesthetized with 10% chloral hydrate (3.5 ml/kg) by peritoneal injection. The skin over the brain was snipped with shears. Fixed the animal in stereotaxic apparatus and cut off a 2 cm incision in center, strip periost inactively. The craniotomy was performed to remove a bone of cranium (3×3mm) so that the ultrasound pressure amplitude could be accurately estimated in the brain. The skin over the craniotomy was sutured and allowed to heal completely about 5 days before the irradiation. Measurements was performed through the bone window with Doppler flowmeter measurement.3. Measurement of blood-brain barrier permeability:The blood-brain barrier (BBB) permeability was quantitatively evaluated by extravasation of Evans blue (EB) as a marker of albumin extravasation. Briefly,2% EB in saline (2 ml/kg) was injected intravenously at each time point.One hour after EB injection, rats of all groups were deeply anesthetized with chloral hydrate transcardially perfused until colorless perfusion fluid was obtained from the right atrium. After decapitation, brain tissues were weighed and immersed into formamide (1 ml/100 mg) at 60℃for 24 h. The supernatant was obtained, and its optical density was determined with a spectrophotometer (at 620 nm). The quantitative calculation of the dye content in the brain was based on the external standards dissolved in the same solvent.4. Transmission electron microscopy was used to detect the change of in BMECs in BBB after experimental. subarachnoid hemorrhage.5. Tight junction protein clauding5, Occludin, Zo-1 and caveolin-1 distribution and expression change detected by application of immunohistochemistry.6. Detection of tight junction protein clauding5, Occludin, Zo-1 and caveolin-1 protein expression by application of Western blot at different time points after subarachnoid hemorrhage.7. Detection of change in Akt, P-Akt and Foxo1A protein expression within 12h after subarachnoid hemorrhage by application of Western blot.Results1. A rat model of subarachnoid hemorrhage was successfully established. 2. Regional cerebral blood flow drops sharply in 1 minute after subarachnoid hemorrhage, then rise slowly, close to baseline at 24h,48～72h regional cerebral blood flow decreased again, 1w return to the near baseline once again.3. BBB permeability increased in early stage of subarachnoid hemorrhage, reach the first peak at 3 hoursafter SAH,24-hour recovery close to baseline levels of permeability. Blood-brain barrier permeability increased again in 48 to 72h,and reach the second peak at 72h after SAH, the blood-brain barrier permeability resumes again 1w after SAH.4. Blood-brain barrier tight junction opening can be seen in transmission electron microscopy after subarachnoid hemorrhage, the largest opened obviously at 3h and 72h after subarachnoid hemorrhage. There is no increase in the quantity of pinocytotic vesicles.5. The tight junction associated protein Zo-1 and Occludin expression decreased after subarachnoid hemorrhage,the lowest point is at 3h and 72h after SAH, respectively. There is no change in Claudin-5 and in caveolin-1 expression.6. P-Akt expression reduced in the early stage after subarachnoid hemorrhage, and then Foxo1A and P-Akt expression increased, then returned to baseline levels.Conclusion1. Blood-brain barrier permeability showed two peaks of time phase changes after subarachnoid hemorrhage, and related to regional cerebral blood flow in the time phase change.2. The mechanism of blood-brain barrier opening after subarachnoid hemorrhage is related to the damage of tight junction.3. Downregulated of tight junction protein Zo-1 and Occludin expression is molecular basis of tight junction opening after subarachnoid hemorrhage.4. There is no increase in the number of pinocytosis vesicles, Caveolin-1 expression did not change after subarachnoid hemorrhage5. Blood-brain barrier permeability increased significantly Within 12h after SAH, Tight junction protein Occludin and Zo-1 protein expression decreased significantly, P-Akt protein expression decreased significantly at first then increased significantly, Foxol A expression was significantly transient increased.6. Blood-brain barrier permeability change, tight junction protein expression patterns and expression of P-Akt and Foxol A associated closely in time course after subarachnoid hemorrhage, indicate that Akt/Foxo signaling pathway may involved in the regulation of tight junction opening and the blood-brain barrier permeability changes in the early stage after subarachnoid hemorrhage.