Role And Mechanism Of Pericytes In Microvascular Spasm And Microcirculatory Disturbance After Subarachnoid Hemorrhage In Rats

Background and ObjectiveSubarachnoid hemorrhage(SAH) is a class of common clinical cerebrovascular accident, characterized by high mortality, high morbidity, and susceptible to young, which seriously threat to human health. Recent studies suggest that subarachnoid hemorrhage secondary to brain injury(Early brain injury, EBI) may be the main reason for the high mortality and disability rates. So studies on reduction of early brain injury After SAH will improve survival and prognosis in patients with subarachnoid hemorrhage.Early brain injury denotes a series of complex pathophysiological process after subarachnoid hemorrhage early(within 72 hrs), which includes the increased intracranial pressure(ICP), decreased perfusion pressure(CPP) and cerebral blood flow(CBF), dropped hydrocephalus, disterbtion of ion homeostasis, cell death, blood-brain barrier damage, cerebral edema, all of which leading to hypoperfusion of cerebral microcirculation, cerebral ischemia and hypoxia, eventually cause microcirculation dysfunction. Microcirculatory disturbance was the most serious complications after subarachnoid hemorrhage. Micro-thrombosis, microvascular spasm, decreased cerebral blood flow and auto-regulation contributed to microcirculatory disturbance. Especially, microvascular spasm, which showed thickening in microvascular wall and stenosis in lumen, blocked red blood flow, directly hinder blood supply to brain tissues, leading to cerebral ischemic and hypoxic damage. Therefore, it is an effective way to deduce mortality and disability rates after subarachnoid hemorrhage through alleviating microvascular spasm, due to reducing the microcirculation dysfunction and early brain injury.Pericytes, which are attached to microvascular endothelial cells, possess pluripotent function, including regulation of cerebral blood flow, maintain the integrity of the blood brain barrier, and angiogenesis. In recent years, studies on the role of pericytes in regulation of microcirculation under pathological conditions attract attention. Previous works have shown that pericytes evoke microvascular contraction under hypoxic or induced by electrical stimulation, blocking the flow of red blood cells, causing microcirculatory disturbance. Properties of Pericytes in regulating microcirculation play a critical role in the pathogenesis of hypertension, cerebral ischemia, myocardial infarction, due to pericytes express actin(such as α-SMA) with a systolic function. However, the role of pericytes in subarachnoid hemorrhage is still elusive. In addition, endothelial nitric oxide synthase(e NOS) is an important molecular in subarachnoid hemorrhage induced vasospasm and circulatory disorders, and nitric oxide(NO) signal is a critical mechanism for moderating vascular tone. Besides, the release of hemoglobin after subarachnoid hemorrhage can reduce nitric oxide content. Other studies show that nitric oxide mediated inflammation by activation of pericytes, while pericytes may respond to nitric oxide signal and cause phenotypic transformation, subsequently regulate vascular function. Therefore, we speculate that, nitric oxide signal is suppressed after subarachnoid hemorrhage, activated pericytes phenotypic conversion, pericytes mediated microvascular spasm, leading microcirculation and eventually cause early brain injury after subarachnoid hemorrhage.To verify this hypothesis, this study divided into two parts. The first part:rats undergoing subarachnoid hemorrhage through endovascular perforation, were treated with e NOS inhibitor L-NNA and its agonists scutellarin, then microvascular spasm、pericytes expression、 mortality、 weight loss、 neurological impairment 、 brain water content、blood-brain barrier permeability damage 、 and the distribution of hemoglobin after subarachnoid hemorrhage, were also detected. The aim of first part was to determine whether pericytes mediated microvascular spasm after subarachnoid hemorrhage, further deterioration of the microcirculation, and whether intervention with scutellarin unregulated e NOS-derived NO and alleviate early brain injury after SAH. The second part: establishment of pericytes culture system and rat brain slices model of microcirculation research, with a hemoglobin-treated cells and brain slices, nitric oxide scavenger PTIO as well as its donor DETA / NO were incubated. Using RT- PCR and western blot(WB), pericytes α-SMA expression at various time points after SAH were detected. Using upright fluorescence microscope, microvascular contraction at pericytes sites were also detected. The aim of second part was to further illustrate the role of nitric oxide signal suppression in pericytes Phenotype conversion, and hemoglobin-induced microvascular spasm.MethodsPart Ⅰ(In vivo):The SD rat model of subarachnoid hemorrhage was established using intravascular puncture method; animals were randomly divided into four groups: sham operation group(Sham), subarachnoid hemorrhage(SAH + Vehicle), L-NNA intervention group(SAH + L-NNA) and scutellarin intervention group(SAH + scutellarin).1. Microvascular spasm and microcirculatory disturbance was detected by in vivo fluorescence imaging to the surface microvessels of cerebral cortex after SAH;2. The mortality, SAH grade, body weight, neurological deficits, brain water content and blood-brain barrier permeability post SAH, were detected;3. The e NOS activity and NO content were detected by a biochemical method; the expression of nitric oxide synthase isoforms was detected by WB;4. Histological examination: the pericytes expression of PDGFRβ and α-SMA after SAH were assessed by immunofluorescence; The distribution of hemoglobin after SAH was detected by H&E and immunohistochemical staining;Part Ⅱ(In vitro):To directly assess the effect of hemoglobin on microvascular spasm, the rat brain slices model was established, and superfused with artificial cerebrospinal fluid, hemoglobin, PTIO, L-NNA and DETA / NO, respectively; To assess the effect of hemoglobin and NO signaling on pericytes α-SMA expression, the rat brain pericytes cultured model was established, and cells were divided into 4 groups: control group(control), hemoglobin treatment group(Hb), PTIO intervention group(Hb + PTIO) and DETA / NO intervention group(Hb + DETA / NO).1. After treatment with different drugs on brain slices, the microvascular contraction and local microcirculation was observed using an infrared upright microscope;2. The pericytes α-SMA protein and mRNA expression in each group were detected using WB and RT-PCR;3. Nitric oxide content of the medium was detected by a biochemistry method;4. After cells were respectively incubated with Hb and PTIO for 0h, 3h, 6h and 12 h, the α-SMA expression at protein and mRNA levels were also detected by using WB and RT-PCR;5. After cells were incubated with PTIO for 6 hrs and followed by 3 hrs of different concentrations(0mM, 0.1mM, 1mM, 10mM) of DETA / NO and 8-Br-c GMP, respectively, pericytes α-SMA expression was observed by WB;ResultsPart Ⅰ(In vivo):1. Microvascular spasm and Microcirculatory Disturbance were observed at 3 hrs after subarachnoid hemorrhage. Treatment with scutellarin alleviated microvascular spasm and decreased spastic numbers. Treatment with L-NNA aggravated microvascular spasm but not significant affect spastic numbers.2. No significant difference was found in mortality and SAH grade in each group after subarachnoid hemorrhage. Treatment with scutellarin attenuated body weight, neurological deficits, blood-brain barrier impairment and brain edema. Treatment with L-NNA aggravated neurological deficits but not significantly affected body weight, blood-brain barrier impairment and brain edema.3. Subarachnoid hemorrhage induced deduction of e NOS expression and activity and NO content. Treatment with scutellarin enhanced e NOS expression and activity, and inhibited the SAH induced sharp decline in NO levels. Treatment with L-NNA aggravated the drop of e NOS expression, eNOS activity and NO levels after SAH.4. Hemoglobin penetrated into brain parenchyma at 6 hrs after subarachnoid hemorrhage. In parallel, enhanced expression of PDGFRβand α-SMA was also observed.Part Ⅱ(In vitro):1. Hemoglobin evoked microvascular spasm at pericytes sites, and was attenuated by perfusion with DETA / NO and ASCF. Perfusion with PTIO and L-NNA also provoked microvascular contraction at pericyte-like sites, respectively.2. Hemoglobin enhanced α-SMA expression in pericytes, and its effects were abolished by NO donor DETA/NO and increased by NO scavenger PTIO.3. Hemoglobin increased NO content in culture mediums, and its effects were deteriorated by PTIO and reversed by DETA/NO.4. Hemoglobin and PTIO up-regulated pericytesα-SMA expression at protein and mRNA levels within 12 hrs.5. Removing NO in mediums by Pretreatment with PTIO, the pericytes α-SMA expression was decreased by administration with NO donor DETA/NO or cGMP analogue 8-Br-cGMP.Conclusion1. Microvascular spasm deteriorate microcirculation after subarachnoid hemorrhage related to high expression of α-SMA, deduction of e NOS expression, e NOS activity and NO concentration after SAH.2. Scutellarin alleviates SAH-induced microvascular spasm and early brain injury through increasing eNOS-derived NO after subarachnoid hemorrhage.3. Hemoglobin penetrate into brain parenchyma early after SAH and provoke microvascular contraction at the pericytes sites by scavenging NO, inhibiting NO / cGMP signal, and regulating pericytes α-SMA expression.4. DETA / NO attenuate the hemoglobin-induced microvascular contraction at pericytes sites via increasing the NO levels and blocking hemoglobin-induced high expression of α-SMA in pericytes.