Mechanism Of Recombinant ADAMTS13 On Brain Injury And Edema After Intracerebral Hemorrhage

Background and purpose:Stroke has two main kinds of sub-styles:brain ischemia and hemorrhagic stroke. Brain ischemia accounts for about 80% of all strokes, intracerebral hemorrhage accounts for approximately 15% of all strokes. Spontaneous intracerebral hemorrhage results from blood rushing into brain parenchyma after rupture of brain blood vessels without surgery or trauma. Intracerebral hemorrhage is a devastating disease that ICH-induced death occurs during acute phase and very few patients who suffer from ICH could live independently in later life.Tissue plasminogen activator (tPA) is the only drug approved by the U.S. Food and Drug Administration (FDA) for acute ischemic stroke. However, tPA-induced thrombolytic bleeding is a major side effect and leading to increase of death.Von Willebrand factor (vWF) is a blood glycoprotein that plays a pivotal role in coagulation and thrombosis, additionally it also acts as a mediator of inflammation to improve leukocytes’adhesion and infiltration. Previous study suggests that VWF knock out reduces infarct volume after ischemic stroke in mice. Accumulating evidences show that subarachnoid hemorrhage (SAH) accompanies with up-regulation of vWF in blood, which could lead to delayed cerebral ischemia and vasospasm after SAH. Clinical researches reveal that higher level of vWF predicts a worse clinical outcome after SAH. ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) is a vWF specific protease that cleaves ultra-large von willebrand factor into smaller and less active multimer. We applied two mice stroke models(MCAO and ICH) to investigate the relationship between VWF and stroke and probe the effect of VWF specific excisionase on brain ischemia and intracerebral hemorrhage.Methods:This experiment is comprised of two parts, the first part is: Mechanism of recombinant ADAMTS13 on brain injury and edema after intracerebral hemorrhage; The second one is:rADAMTS13 reduces tPA-induced thrombolytic bleeding after stroke through VWF. The methods of first part:1.The mice intracerebral hemorrhage model was induced by double-injection of autologous blood. 2.We measure the level of vWF in plasma 24h after intracrebral hemorrhage by ELISA.3.We detect the injury volume 72h after intracerebral hemorrhage by H&E staining.4.We apply the dry/wet weight method to evaluate the water content 72h after intracerebral hemorrhage.5.We survey the degenerating neurons number by Fluoro-jade B staining 24h after ICH.6.Evans blue is used to test the blood brain barrier permeability 24h after intracerebral hemorrhage.7. We survey the level of IL-6 by ELISA 24h after intracerebral hemorrhage.8. MPO experiment is operated for the evaluation of MPO activity 24h after intracerebral hemorrhage 9. Immunofluorescence technique is utilized to probe infiltration of neutrophil and activated microglia 24h after intracerebral hemorrhage.10.Western blot is used to quantify the expression of ICAM-1、VCAM-1 24h after intracerebral hemorrhage. 11.We utilize zymography to measure the activity of MMP-9 24h after intracerebral hemorrhage. The methods of the second part:1.The mice intracerebral hemorrhage model was induced by double-injection of autologous blood.2.We measure the level of vWF in plasma 24h after intracrebral hemorrhage by ELISA.3.We detect the injury volume 72h after intracerebral hemorrhage by H&E staining.4.We apply the dry/ wet weight method to evaluate the water content 72h after intracerebral hemorrhage. 5.We survey the degenerating neurons number by Fluoro-jade B staining 24h after ICH.6.Evans blue is used to test the blood brain barrier permeability 24h after intracerebral hemorrhage.7.We survey the level of IL-6 by ELISA 24h after intracerebral hemorrhage.8. MPO experiment is operated for the evaluation of MPO activity 24h after intracerebral hemorrhage 9. Immunofluorescence technique is utilized to probe infiltration of neutrophil and activated microglia 24h after intracerebral hemorrhage.10.Western blot is used to quantify the expression of ICAM-1、VCAM-1 24h after intracerebral hemorrhage.11.We utilize zymography to measure the activity of MMP-9 24h after intracerebral hemorrhage.Results:The results of first part:1. ELISA quantitation indicates that vWF level in plasma elevated 24h after intracerebral hemorrhage.2. Recombinant ADAMTS13 can decrease injury volume、brain edema and degenerating neurons compare with vehicle treatment.3. Lateral ventricle administration of recombinant ADAMTS13 could significantly extenuates blood brain barrier permeability 24h after intracerebral hemorrhage compare with vehicle treatment.4. Elisa analysis reveals that IL-6 was decreased by Lateral ventricle administration of recombinant ADAMTS13 after ICH. 5. MPO activity after ICH was significantly inhibited by recombinant ADAMTS13 compare with vehicle.6.Recombinant ADAMTS13 can apparently down-regulate infiltration of neutrophil、activation of microglia after ICH compare with vehicle.7. Recombinant ADAMTS13 is able to lower the expression of ICAM-1 compare with vehicle treatment after ICH.8. The zymography results show that the MMP-9 is negatively regulated by Lateral ventricle administration of recombinant ADAMTS13 after ICH. The results of the second part:1. ELISA quantitation indicate that vWF level in plasma elevate 24h after intracerebral hemorrhage.2. Recombinant ADAMTS13 can decrease injury volume^ brain edema and degenerating neurons compare to PBS treatment.3. Lateral ventricle administration of recombinant ADAMTS13 could significantly extenuate blood brain barrier permeability 24h after intracerebral hemorrhage compare to PBS treatment.4. Elisa analysis reveals that IL-6 is decreased by Lateral ventricle administration of recombinant ADAMTS13 after ICH.5. MPO activity after ICH is significantly inhibited by recombinant ADAMTS13 compare to vehicle 6. Compare to PBS treatment recombinant ADAMTS13 can apparently down-regulate infiltration of neutrophil-. activation of microglia after ICH.7. Recombinant ADAMTS13 is able to lower the expression of ICAM-1 compare to PBS treatment after ICH.8. The zymography result shows that the MMP-9 is negatively regulated by Lateral ventricle administration of recombinant ADAMTS13 after ICH.Conclusions:Recombinant ADAMTS13 is able to decrease expression of mediators of inflammation、neutrophil infiltration and activation of microglia which contribute to mitigate blood brain barrier breakdown, ultimately lead to extenuate brain damage and brain edema.