The Early Use Of Hemocoagulase Agkistrodon To Prevent Hematoma Expansion And Its Neuroprotective Mechanism In Experimental Intracerebral Hemorrhage

objective: By using the collagenase-induced experimental intracerebral hemorrhage of rats,the different doses of hemocoagulase agkistrodon(HCA)were intravenous therapy.Hematoma volume,neurological functions,blood-brain barrier permeability,were observed at different time points after theearly injection of different doses of Agkistrodon hemocoagulase.and whether it can reduce the Hematoma expansion,improve the neurological symptoms and reduces prognosis,and to explore the neuroprotective mechanism of HCA.Finally,according to our research,providing the basis for the clinical treatment of HCA in the early hematoma enlargement and nerve protection function for ICH.Methods:1.Groups of experimental: A total of 192 adult male SD rats was randomly assigned into six groups,including the sham group(n=42),ICH group(n=22),ICH+vehicle group(n=42),ICH+HCA0.1mg/Kg group(n=22),ICH+HCA 0.2mg/Kg group(n=22)and ICH+HCA0.4mg/Kg group(n=42);2.Model making and drug treatment:Intracerebral hemorrhage was induced by stereotactic injection of collagenase Type VII(0.5 U)into the right striatum of rats.(coordinates: 0.2 mm anterior,3.5 mm lateral,and 5.5mm ventral to the bregma),and the administration of HCA were intravenous by tail vein at 1h after surgery;3.Neurological deficit: m NSS score and Corner turn test were adopted to evaluate neurologic impairment at 24 h and 72 h post surgery,with higher scores implying greater neurological injury;4.Blood coagulation and biochemical function:To estimate whether blood coagulation and biochemical function is different in each group,the blood from inferior vena cava were analysis at 24 h post ICH;5.Hemorrhage volume:At 24 h after the induction of ICH,hemorrhage volume was quantified with the spectrophotometric assay and MRI;6.The blood-brain barrier permeability: BBB permeability was evaluatedusing Evans blue staining at 24 h after ICH;7.Brain water content : 72 h after ICH,dry wet weight method was used to detect brain water content;8.Apoptosis detection: 72 h after ICH with TUNEL method to detect apoptosis;9.ELISA detection of inflammatory factors and fibrinogen: ELISA was used to detect the inflammatory factor and fibrinogenin perihematoma after 24h;10.Western blot : Using the Western Blot method to analyze the change trends of MMP-9,Claudin-5 and ZO-1 in perihematoma;11.Immunofluorescence staining: Microglia was detected by immunofluorescence staining at 24 h after intracerebral hemorrhage.Results: According to our research,we found that coagulation and biochemical function tests were not difference between each group in the reserach at 24 h post ICH(P<0.05).Firstly,compared with Sham group,the neurological deficit in ICH group and ICH+Vehicle group were obvious(P<0.001),on the other hand,HCA can improve the neurological function,significant differences were found between high dose group and control group at 24 h and 72 h post ICH.HCA can also reduces hematoma enlargementat at 24 h post ICH(P<0.05),and the effect is dose-dependent.Thirdly,compared with contrast group,the Evans blue content and brain water content were significant reduce in middle and high dose treatment groups after operation(P<0.05).Fourthly,HCA can significantly reduce nerve apoptosisin in perihematoma at 24 h post ICH(P<0.05).In addition,The levels of fibrinogen,Microglia,cytokines and MMP-9around the hematoma were increased in the group of Vehicle at 24 h after ICH,and these levels was significantly decreased in HCA group(P<0.05).Finally,HCA can significantly improve the level of Claudin-5 and ZO-1 in perihematoma(P<0.05).Conclusion: 1.Intravenous injection of Agkistrodon hemocoagulase on experimental intracerebral hemorrhage rats do not effects of blood coagulation function and biochemical function;2.The hemostatic effect of HCA is exact.It can reduce the hematoma volume and attenuates hematoma expansion after intracerebral hemorrhage in experimental rats,with dose effect relationship;3.By reduced the fibrinogen deposition around the hematoma and inhibit the activation of microglia,HCA significantly reduces inflammatory reaction and neuronal apoptosis around hematoma,and downregulates of MMP-9 levels,and protect the permeability of blood-brain barrier by upregulating the expression of Claudin-5 and ZO-1,with improvement of neural function of experimental intracerebral hemorrhage in rats.