Effect Of Neuroserpin On Blood-brain Barrier Damage Caused By Intracerebral Hemorrhage

Spontaneous intracerebral hemorrhage (ICH) accounts for about30%of acute cerebral vascular disease in our country with no specific therapy. Patients with ICH show the worst outcome of all stroke subtypes with acute phase mortality rate of30%～40%. It is characteristic of high morbidity, mortality, disability rate. Blood-brain barrier breakdown is the most important mechanism of edema formation after ICH while brain edema is the major cause of the deterioration and death. Control edema is one of key of cerebral hemorrhage treatment.Zhang et al demonstrated that aministration of neuroserpin after stroke is neuroprotective, reduced BBB leakage, brain edema, and ischemic lesion volume. There are NSP related studies on neurological diseases such as ischemic stroke, epilepsy. But no research on its effect in ICH has been reported.We aim to explore effect of NSP on BBB permeability in ICH and its possible mechanism. Attempt to develop new treatment strategy for ICH.We first studied NSP expression around hematoma at24h/48h/72h after ICH compared with intact and sham group by immunofluorescence and western blot. Analysed change trend over time. Then measured brain water content by dry-wet weight ratio method and detected BBB permeability with the use of Evans blue. Compared ICH with NSP injected group with ICH group at24h/48h/72h after ICH to analyse BBB permeability change trend. Further more, we observed and compared expression of tight junction (TJ) protein occludin around hematoma in ICH+NSP group with ICH group at48h after ICH by immunofluorescence and western blot.We discuss the regulation of NSP expression in ICH. Study its effect on BBB and further explored the mechanism. Attempt to develop new treatment strategy for ICH.PART I Study NSP expression around hematoma after ICH and analyse change trend over time Objective:To study NSP expression around hematoma at24h/48h/72h after ICH compared with intact and sham group and analyse the change trend over time.Methods:C57BL/6J mice were randomly divided into three groups:intact group, sham group and ICH group. ICH group divided into24h,48h,72h subgroups. Intact group mice were killed without any intervention. Sham operation group mice were killed24h after intervention. Use double autologous blood injection method to establish ICH models. ICH subgroup mice were killed at corresponding time after intervention. All mice were scored for neurological deficits before been killed. Observed NSP expression around normal striatum or hematoma at24h/48h/72h after ICH compared with intact and sham group by immunofluorescence and western blot and analyse the change trend over time.Results:1. Neurological deficits score showed scores of ICH subgroups increased compared with intact and sham groups(p<0.01), Socres of ICH48h subgroup ncreased compared with ICH24h subgroup(p<0.05);2. Immunofluorescence results showed NSP expression around hematoma of ICH subgroups increased compared with intact group and sham group, especially obvious in superficial cortex of hematoma and at48h after intervention.3. Western blot results showed NSP expression around hematouma increased in ICH subgroups compared with intact and sham groups(p<0.01). No statistical differences among ICH subgroups although mean value of ICH48h group is the highest.Conclusion:1. Neurological function of mice suffered from intracerebral hemorrhage was damaged obviously.2. NSP expression around hematoma increased significantly in mice suffered from intracerebral hemorrhage without obvious change trend over time. PART Ⅱ Study effect of NSP local injection on brain edema and BBB permeability in ICH and analyse change trend over timeObjective:Measure brain water content by dry-wet weight ratio method and detect BBB permeability with the use of Evans blue. Compare ICH with NSP injected group with ICH group at24h/48h/72h after ICH to analyse BBB permeability change trend.Methods:C57BL/6J mice were randomly divided into three groups:sham group, ICH group, ICH+NSP group. ICH group and ICH+NSP group divided into24h,48h,72h subgroups separately. Sham operation group mice were killed24h after intervention. Use double autologous blood injection method to establish ICH models and ICH+NSP group with NSP solution local injection. ICH and ICH+NSP subgroup mice were killed at corresponding time after intervention. All mice were scored for neurological deficits before been killed. Measure brain water content by dry-wet weight ratio method and detect BBB permeability with the use of Evans blue. Analyse effect of NSP injection on brain edema and BBB permeability in ICH and analyse change trend over time.Results:1. Neurological deficits score showed scores of ICH and ICH+NSP subgroups increased compared with sham group (p<0.01); Socres of ICH+NSP48h subgroup decreased obviously compared with ICH48h subgroup (p<0.01); Socres of ICH+NSP72h subgroup decreased obviously compared with ICH72h subgroup (p<0.01); Socres of ICH+NSP48h subgroup decreased obviously compared with ICH48h subgroup (p<0.01); Scores with no statistical differences between ICH+NSP24h and ICH24h subgroups (p=0.076).2. Dry-wet weight ratio method results showed brain edema content of ICH24h/48h/72h subgroups and ICH+NSP48h/72h subgroups increased obviously compared with sham operation group (p<0.01); Brain edema content of ICH+NSP24h subgroup decreased significantly compared with ICH24h subgroup (p<0.05); Brain edema content of ICH+NSP48h/72h subgroup decreased significantly compared with ICH48h/72h subgroup (p<0.01);3. Evans blue method to detect BBB permeability showed brain EB content of ICH subgroups and ICH+NSP subgroups increased obviously compared with sham operation group (p<0.01); Brain EB content of ICH24h/48h subgroups increased obviously compared with ICH+NSP24h/48h subgroups (p<0.01); Brain EB content of ICH72h subgroup increased obviously compared with ICH+NSP72h subgroup (p<0.05).Conclusion:1. Neurological function of mice suffered from intracerebral hemorrhage was damage, which could be improved obviously through NSP local injection.2. NSP local injection immediately after ICH could decrease brain edema content and relieve encephaledema.3. NSP local injection immediately after ICH could decrease brain EB content and reduce BBB permeability. PART Ⅲ Study effect of NSP local injection on expression of tight junction protein occludin at48h after ICHObjective:To study and compare expression of tight junction protein occludin around hematoma in ICH with NSP local injection mice with ICH mice at48h after ICH.Methods:C57BL/6J mice were randomly divided into three groups:sham group, ICH group and ICH+NSP group. Sham group mice established sham operation models. Use double autologous blood injection method to establish ICH models and ICH+NSP group with NSP solution local injection. All mice were scored for neurological deficits at48h after intervention and then killed. Observed and compared occludin expression around normal striatum or hematoma at48h after intervention by immunofluorescence and western blot.Results:1. Neurological deficits score showed scores of ICH48h group and ICH+NSP48h group increased compared with sham groups (p<0.01); Socres of ICH+NSP48h group decreased compared with ICH48h group (p<0.01);2. Immunofluorescence results showed tight junction protein occludin distributed disorderly and expression decreased significantly around microvessel in ICH group compared with sham group. These conditions were improved in ICH with NSP local injection mice. Occludin expression around microvessel increased significantly.3. Western blot results showed no significant differences of occluding expression among sham, ICH and ICH+NSP groups at48h after intervention (p>0.05).Conclusion:1. Neurological function of mice suffered from intracerebral hemorrhage was damage, which could be improved obviously through NSP local injection.2. Tight junction protein occludin distributed disorderly and expression decreased significantly around microvessel after ICH which can be improved with NSP local injection immediately after ICH.3. Expression of total occludin protein48h after ICH with NSP local injection or not did not change obviously. Summary:NSP expression around hematoma increased significantly in mice suffered from intracerebral hemorrhage. NSP local injection immediately after ICH could decrease brain edema and decrease BBB permeability. Tight junction protein occludin distributed disorderly and expression decreased significantly around microvessel after ICH which could be improved with NSP local injection immediately after ICH.