The Protective Effect Of Necrostatin-1 On The Peripheral Tissues Of The Hematoma In The Mouse Model Of Intracerebral Hemorrhage

Objective Studying whether programmed necrosis involved in the pathological process after cerebral hemorrhage, programmed necrosis specific inhibitor Necrostatin-1’ effect on the surrounding tissues of the hematoma, and its relationship with the recovery of neurological function.Methods 1 In vivo experiment:ICR mice were randomly divided into normal group, sham operation group, cerebral hemorrhage group, Necrostatin-1 group and Dmso group. The cerebral hemorrhage model was made by injecting 1 μl into the left basal ganglia of the type Ⅳ collagenase, and the sham operation group was injected with the same amount of normal saline. Necrostatin-1 group, Dmso group first in the left lateral ventricle were injected Necrostatin-1 μl,Dmsol μl pretreatment,5 minutes after the injection of collagenase in the basal ganglia 1 μl. Western blot method was used to detect the changes of RIP1 and RIP3 expression in bilateral striatum lday,3day,5day and 7day after operation in each group.The change of water content of 24 hours brain tissue was determined by wet and dry weight method, and the content of hemoglobin in the cerebral hemisphere of the two sides of the brain was detected by the detection kit of hemoglobin after 24 hours;Using modified Garcia score, rotation test, and transfer rod test to evaluate the neurological deficits of mice;Tissue immunofluorescence was used to observe the changes of the glial cells in the peripheral tissues of the hematoma of the 3D after the operation.2 In vitro experiment:Fetal rats were taken out from the uterus of the C57/BL6 pregnant mouse uterus of 13.5 or 14.5 days of pregnancy, and the cortical cells of fetal rat were taken out and cultured in 5% carbon dioxide and 37 degree.48-72 hours later, according to 1:2 passage to 24 pore plate, two days later, divided into the normal group and red blood cell lysate group, the red blood cell lysate group added lysate 10 ml, on the third day under the inverted microscope observation effect of red blood cell lysate on oligodendrocyte cell morphology.The cultured oligodendroglial cells were divided into normal group, erythrocyte lysate group, Necropstatin-1 group and Dmso group,1 day after treatment, pyridine iodide (propidium iodide, PI) labeled necrotic cells and fluorescence microscopy observation groups oligodendrocyte cell changes.Results 1 In vivo experiment① Expression of RIP1 and RIP3 protein in bleeding side:The expression of RIP 1 protein in the normal group is low, and the expression of the sham operation group was slightly higher than that of the normal group. The expression of RIP1 protein in the cerebral hemorrhage group was significantly increased at 1 days, reached a peak at 3 days, and gradually decreased at 5 and 7 days. RIP1 protein expression in Necrostatin-1 group was higher than that in normal group and sham operation group at the corresponding time point, but lower than that in cerebral hemorrhage group. Dmso group RIP1 protein expression is also very high, higher than the sham operation group, Necrostatin-1 group, compared with the cerebral hemorrhage group, the difference was not significant.The expression changes of RIP3 at the corresponding time point was the same as that of RIP 1, but the expression of RIP3 was not higher than that of RIP 1.② Brain water content:The average water content of normal brain tissue was about 72%, and the sham operation group was about 73.1%, there was no significant difference between the normal group and the sham group (P>0.05).The cerebral water content in the cerebral hemorrhage group was significantly increased, compared with the sham operation group, the difference was statistically significant (P<0.05). The water content in Necrostatin-1 group was about 75.5%, which was also increased (P<0.05) compared with sham operation group, but it was significantly less than that in cerebral hemorrhage group (P<0.05). The water content in Dmso group was significantly increased, and there was difference compared with the sham operation group (P<0.05), and there was no significant difference with the cerebral hemorrhage group (P>0.05).③ Hemoglobin content of brain tissue:The hemoglobin content of normal group was very low, with an average of about 0.1 μg/mg, and the sham operation group was about 0.35 μg/mg. There was no significant difference between the normal group and the normal group (P>0.05).There was a significant increase in blood hemoglobin levels of the cerebral hemorrhage group compared with the sham operation group, the difference was statistically significant(P<0.05).The blood hemoglobin content of Necrostatin-1 group was about 0.8 μg/mg, wnich was higher than that in the sham operation group (P<0.05), but less than that in the cerebral hemorrhage group (P<0.05).There was significant difference in the blood hemoglobin content in the Dmso group than in the sham operation group (P>0.05), but there was no significant difference between the Dmso group and the cerebral hemorrhage group.④ Changes of oligodendrocyte after cerebral hemorrhage:In normal group, there was very little death of the cells in the normal group, and there was a large number of death of the cerebral hemorrhage group. There was a significant difference between the cerebral hemorrhage group and the normal group (P<0.01). Necrostatin-1 group and cerebral hemorrhage group compared, the less glial cell death (P<0.05). There was also a large number of deaths in the Dmso group, and there was no significant difference with cerebral hemorrhage group (P>0.05).⑤ Nerve function defect:Mouse in each group of modified Garcia score in 20 minutes or so, there was no significant difference, significantly decreased 1 day after operation in cerebral hemorrhage group,Dmso group, Necrostatin-1 group, the differences among the groups without statistically significant. Necrostatin-1 group in 4 days,7 days scores increased, and the difference was statistically significant compared with cerebral hemorrhage group(P<0.05).Before operation, the number of rotation of the mice in each group were no significant difference,1 day after operation, mice in each group to the injured side (left) corner number were increased, beginning on day 4, Necrostatin-1 group to the ipsilateral rotation number decreased, the difference was statistically significant compared with cerebral hemorrhage group (P< 0.05).Preoperative mice on the wheel and there is no difference in the duration,1 day, all mice were in the round of the time was shortened, the differences among the groups without statistically significant.2 days to 7 days, Necrostatin-1 group in the round time prolonged, the difference was statistically significant compared with cerebral hemorrhage group(P< 0.05). 2 In vitro experiment In normal group, there was a lot of processes and the cell shape was full. After the addition of red blood cell lysis, the cell processes were significantly decreased and cell shrinkage occurred. Using PI tag dead cells,PI positive cell number of normal group is less, cell lysis cell lysis solution group can be observed a large number of positive cells.The number of PI positive cells in Necrostatin-1 group was much higher than that in normal group (P<0.05), but it was significantly lower than that of cell lysis solution group (P<0.05). There were also a large number of PI positive cells in the Dmso group, and there was a significant difference between the Dmso group and the normal group (P<0.01), and there was no significant difference with cell lysis solution group (P>0.05).Conclusions ① Programmed necrosis may be involved in the process of pathological damage after cerebral hemorrhage. The expression of RIP3 and RIP1 in the hemorrhagic site after cerebral hemorrhage is increased, and Necrostatin-1 can reduce the expression of RIP 1 and RIP3 protein after cerebral hemorrhage.② After cerebral hemorrhage, the cerebral tissue water content and hemoglobin content increased, Necrostatin-1 can reduce the brain tissue water content, hemoglobin content.③ After intracerebral hemorrhage, there was a lot of death in the area surrounding the hematoma.Necrostatin-1 can decrease the number of the necrotic cells perihematoma, decrease the necrosis of the cells affected by the red cell lysis solution.The red blood cell lysis solution may be related to the programmed necrosis of the brain after intracerebral hemorrhage.④ Nerve function injury after intracerebral hemorrhage in mice,Necrostatin-1 can promote the recovery of neurological function after intracerebral hemorrhage.