The Effect Of Thrombin In ICH Induced Brain Damage And Recovery

Spontaneous intracerebral hemorrhage (ICH) is a common and often fatal stroke subtype. Community based studies have indicated a mortality of more than 40%, and many survivors are left with significant neurological deficits.The mechanism of ICH induced brain damage is very complicated, there're four main influence factors:1) the initial physical damage and hematoma effect,2) thrombin, 3) hemoglobin and its degradation products,4) inflammatory mediators and complement. PAR1 is one of thrombin receptors, and has a close relation with ICH induced brain damage. Meanwhile, thrombin and its receptor PAR1 and are thought to be tied to rehabilitation after brain injury. But these are still lack more direct evidence and deep research.This article has a further study in vivo and in vitro:1) thrombin cause brain cell death including autophagy,2) PAR1 knockout mice are used to get the direct evidence of PAR1 in thrombin caused brain damage,3) thrombin and its receptor in brain rehabilitation after damage, especially in cerebrovascular regeneration. Through these researches, we expected to further clarify the role of thrombin and its receptor PAR1 after ICH and provide adequate theories and evidence-based guidelines for clinical work.Part 1The role of thrombin in intracerebral hemorrhage induced autophagyThrombin contributed to intracerebral hemorrhage (ICH) induced brain injury and cell death. We demonstreated that autophagy occurred after ICH and iron plays a role in ICH mediated autophagy. In this study, we are going to investigate the role of thrombin in the autophagy.There were two parts in this study. Part 1, in vivo study. (1) Male, adult Sprague-Dawley rats received infusion of autologus blood with hirudin (thrombin inhibitor) or vehicle into the right caudate nucleus, the rats were sacrificed at day 7. (2) Rats received an infusion of rat thrombin or vehicle into the right caudate nucleus and were killed 1,3 or 7 days later. Brains were used for Western Blot, immunohistochemistry, Electron microscopy analyses. Part 2, in vitro study. Primary cultured cortex astrocytes from rat pups were exposed to rat thrombin or thrombin±3-MA (an autophagy inhibitor) and cells were collected for Western Blot and immunocytochemistry.We found that the conversion of LC3-Ⅰto LC3-Ⅱ, cathepsin D and beclin-1 levels in ipsilateral basal ganglia were upregulated after ICH and it was blocked by hirudin treatment. The conversion of LC3-Ⅰto LC3-Ⅱ, cathepsin D and beclin-1 expression, and vacuole formation were increased in the ipsilateral basal ganglia 3 day after thrombin infusion. Astrocytes with thrombin added had an enhancement in the conversion of LC3-Ⅰto LC3-Ⅱand the immunoreactivity of beclin-1. MDC labeling in cultured astrocytes showed a peaked accumulation of MDC at 24 hours.3-MA suppressed the thrombin induced activation of autophagy and promoted the cell death.Part 2The Role of PAR-1 in Thrombin Mediated Brain InjuryThe serine proteases tissue plasminogen activator, plasmin, and thrombin and their receptors were involved in brain damage in certain pathological situations. Here we investigated whether mice lacking protease-activated receptor-1 (PAR-1) have less thrombin mediated brain injury and neuronal death in vivo and in vitro.There were two parts in this study. Part 1, in vivo study. Wild type (WT), PAR-1+/- and PAR1-/- mice received an infusion of thrombin (0.5 U) into the right caudate nucleus and were killed 1 or 3 days later. Behavior tests were performed at day 1 and mice mortalities were determined at day 3 after intracaudated injection. Brains were used for Western Blot analyses and stainings of Fluoro-Jade C (F-J C), PANT and TUNEL. Part 2, in vitro study. Primary cultured cortex neurons from WT or PAR1-/-mice were exposed to thrombin (5 or 10 U/ml) for 24 hours and the culture medium was collected for LDH assay.PAR1-/-mice had better behavior scores at day-1 after thrombin injection. The 3 days mortality was lower in PAR1-/-mice than that in PAR1+/- and WT mice. F-J C, PANT and TUNEL positive cells in ipsilateral basal ganglie were significantly less in PAR1-/- mice at both day-1 and -3 day vs. that in WT mice. There was lower F-J C positive cells at day-1 in PAR1+/- than that in WT mice. The protein levels of activated P44/42 MAPK were higher in PAR1-/- than that in WT and PAR1+/- mice at day-1, but the levels of Bcl2 were lower in PAR1+/- and PAR1-/- than that in WT mice at day-3 after thrombin injection. The LDH levels in the neurons of WT mice were significantly higher than that in the neurons of PAR1-/- mice after treated with human thrombin at both 5 and 10 U/ml for 24 hours.Part 3Thrombin stimulates VEGF secretion in astrocytesVEGF (vascular endothelial growth factor) is a potent stimulator of angiogenesis and activated through PAR-1 (protease-activated receptor-1) and p44/42 MAPK (mitogen-activated protein kinase) pathway has been associated with VEGF production. The present study examined whether thrombin regulates VEGF levels in primary cultured astrocytes and the role of PAR-1, p44/42 MAPKs pathway.This study was divided into two parts; (1) Astrocytes from rat's cortex were treated with vehicle, thrombin with or without PD98059 (p44/42 MAPKs inhibitor), YC-1 (HIF-la inhibitor) and TRAG (PAR-1 agonist). (2) Astrocytes wild type (WT) or PAR-1 knockout (KO) mice were treated with vehicle or thrombin. The culture medium was collected for VEGF levels determination with enzyme-linked immunosorbent assay (ELISA) kit and cells were used for immunocytochemistry, Western Blot assays and HIF-1αELISA.The proteins levels of VEGF and activated p44/42 MAPKs were upregulated by thrombin and TRAG stimulation in rat astrocytes, PD98059 blocked the thrombin induced p44/42 MAPKs activation and VEGF up-regulation in rat astrocytes. YC-1 couldn't block VEGF up-regulation in rat astrocytes by thrombin. There were less upregulation of VEGF and activation of p44/42 in astrocytes from PAR-1 KO mice than that from WT mice after treated with thrombin. The levels of HIF-1α(hypoxia inducible factor-1α) showed no different between the WT and PAR-1 KO mice astrocytes.Conclusion1. Thrombin plays an important role in ICH induced autophayr. Autophagy protectes thrombin related astrocytes death.2. PAR-1 knockout attenuates thrombin mediated brain injury and neuronal death. It is regulated by P44/42 MAPK.3. Thrombin induces VEGF upregulation through PAR 1 and p44/42 MAPK pathway but not HIF-1αin astrocytesThrombin and its receptor PAR1 have an important and complicated effection during ICH induced brain injury. To investigate the effection will make us a more accurate and correct drug control to ease brain injury and promote brain recovery..