The Strategy And Mechanism Of Vascular Neural Network Preservation In Early Brain Injury After Subarachnoid Hemorrhage

IntroductionSubarachnoid hemorrhage(SAH) is one of the most common cerebrovascular emergency and severe cases in clinical, mainly from the ruptured intracranial aneurysm. With the progress of neuroimaging and microsurgery, the direct mortality of the hemorrhage after aneurysm rupture significantly decreased, but almost 50% of the survivors after initial bleeding were left with limb paralysis, emotional or memory dysfunction, and the loss of self-care ability to work and life. Therefore, in order to reduce morbidity and improve the prognosis of SAH patients, the prevention of secondary brain injury after SAH has become increasingly important and the hot spot of recent researches.The study of the mechanisms of secondary brain injury after SAH has lasted for decades, and has not been fully elucidated. So far, most of the researches focus on the intracranial vasospasm after SAH. They consider the intracranial vasospasm as the most serious complications after SAH. This comes from the observations of cerebral angiogram in the patients with SAH, which showed the presence of intracranial artery narrowing(generally refers to: ≥200 microns in diameter), and at the same time SAH patients clinically suffered with ischemic symptoms. Intracranial vasospasm, therefore, has long been considered to be the main cause of death or disability after SAH. In recent years, however, its relationship with secondary brain injury after SAH is in doubt. A clinical research with selective endothelin A receptor antagonist Clazosentan treatment on SAH patients showed that Clazosentan can significantly reduce the intracranial vasospasm, but does not improve the prognosis of SAH patients. In fact, at the same time of ruptured intracranial aneurysm, secondary brain damage has occurred. At the aneurysm rupture moment, intracranial pressure significantly increases, even reach the level of artery systolic blood pressure. SAH patients, therefore, suffered with the most common complaint ‘thunder headache’. Animal experiments confirmed that within 10 minutes after SAH, excitatory amino acid release, neuronal apoptosis, and neuronal cell death has been activated. Based on the clinical and experimental research results, Prof. John H. Zhang from Loma Linda University defined the brain injury in the first 72 hours SAH is Early Brain Injury(EBI). Early brain injury of SAH patients with late survivors of acute death and neural function defect, the important factors are the important factors influencing the late-onset vasospasm, for Early Brain Injury intervention can obviously improve the patients’ survival rate and prognosis.In recent years, however, clinical trials failed to protect neurons in patients, persuading us pay more attentions the neuronal damage after SAH, as well as the connection between all kinds of cells in vascular neural network, including: endothelial cells, astrocytes, pericytes, neurons and the vascular smooth muscle cells, endothelial and nerve fiber directly attached to the capillaries, intracranial microcirculation and its upstream arteries and downstream veins. Due to the fast communication among various cells, protecting other cells in the vascular neural network is equally important as protecting neuron. The blood brain barrier is the most typical neurovascular unit in this vascular neural network, and also blood brain barrier damage is thought to be one of the main pathophysiological processes in SAH patients. In this case, repairing blood brain barrier has been considered as one of the most promising therapy strategies in rescuing and repairing brain injury after SAH. Thus, as the key factor of blood brain barrier structural proteins, the tight junction proteins become the high interested repairing targets.Therefore, based on the previous work, the present study focuses on the key factors of the vascular neural network after SAH, neuron and blood brain barrier, to explore the pathophysiological mechanisms of early brain injury after SAH, which may lay a foundation for furfure clinical translational study. In addition, the present study intend to explore the role of the downstream venous system after intracranial microcirculation in the development of early brain injury after SAH, which may shedding new lights on the clinical treatment for patients with SAH.Material and Methods1. SAH model was induced by endovascular perforation..2. BPV(pic) treatment(0.2 mg/kg) was evaluated for effects on neurological score, brain water content, Evans blue extravasation, hippocampal neuronal death and AMPA receptor subunits alterations after SAH.3. Frizzled 4 small interfering RNA(siRNA) was injected intracerebroventricularly 48 hours before SAH. Norrin was administrated intracerebroventricularly 3 hours after SAH. SAH grade, neurologic scores, brain water content, Evans blue extravasation, western blots and immunofluorescence were employed to study the mechanisms of Norrin and its receptor regulation protein TSPAN12, as well as neurological outcome.4. Hemisphere swelling ratio, hemorragical volume were evaluated at 24 hours after SAH. And the correlationship between hemorragical volume and SAH grade, modified Garcia scores, hemisphere swelling ratio, respectively, were analysised. Then the effects of left cortical superficial vein occlusion and left internal jugular vein occlusion towards brain tissues after SAH were illustrated.Results1. We found that BPV(pic) is effective in attenuating BBB disruption, lowering edema, reducing hippocampal neural death and improving neurological outcomes. In addition, the AMPA receptor subunit Glu R1 protein expression at cytomembrane was downregulated, whereas the expression of GluR2 and GluR3 were upregulated after BPV(pic) treatment.2. Endogenous Norrin and TSPAN12 expression were increased after SAH, and Norrin was colocalizated with astrocytes marker GFAP in cortex. Exogenous Norrin treatment significantly alleviated neurobehavioral dysfunction, reduced brain water content and Evans blue extravasation, promoted β-catenin nuclear translocation and increased Occludin, VE-Cadherin and ZO-1 expressions. These effects were abolished by Frizzled 4 si RNA pretreated before SAH.3. At 24 hours after SAH, the ipsilateral/left hemisphere showed larger swelling than right hemisphere, and the hemorrhagical volume in left hemisphere was also increased after SAH by comparing to the sham group. Furthermore, the hemrrhagical volume of left hemisphere was positively correlated with hemisphere swelling ratio, and negatively correlated with modified Garcia scores, but had no correlations with SAH grade. After left cortical superficial vein occlusion, the drainage area of brain cortex showed significant swelling than solely SAH rats, whereas the superficial cortex vein were markedly dilated after left internal jugular vein occlusion in SAH rats.Conclusion1. BPV(pic) attenuates early brain injury after SAH via glutamate AMPA receptor subunit alterations following PTEN inhibition. Because BPV(pic) exhibits inhibitory potency and specificity at low concentrations where no significant cytotoxicity observed, it may be a good candidate for the therapeutic treatment of early brain injury after human SAH.2. Norrin protected BBB integrity and improved neurological outcome after SAH, and the action of Norrin seemed mediated by Frizzled 4 receptor activation which promoted β-catenin nuclear translocation, which then enhanced Occludin, VE-Cadherin and ZO-1 expression. Norrin might have potential to protect BBB after SAH.3. There was blood sedimentation in the brain after SAH, which was more significant in ipsilateral hemisphere than contralateral hemisphere. Left cortical superficial vein occlusion and left internal jugular vein occlusion would aggravate the brain swelling and dilate the cerebral venous system at their drainage areas. Therefore, restoration of the balance between arterials and veins after SAH might be a promising way to alleviate early brain injury after SAH.4. Vascular neural network plays a vital role in maintaining the hemostasis of central nervous system. By targeting the pathophysiological changes of vascular neural network, these strategies could significantly alleviate the early brain injury after SAH, and eventually improve the outcomes of SAH patients. In conclusion, the clinical translational researches based on vascular neural network may provide a new way to treat the SAH patients.