| Objective: To explore the possible mechanism in the formation of secondary cerebral edema after intracerebral hemorrhage and the effect of butylphthalide on secondary injury after cerebral hemorrhage and its possible protective mechanism,we observe the effect of butylphthalide(NBP)on neurologic deficit,brain water content,evans blue content,pathological change of brain and inflammatory response,the expression of aquaporin4(APQ4)and claudin-5 in perihematoma tissue in rats with intracerebral hemorrhage.To provide a theoretical basis and a new direction for the prevention and treatment of edema after intracerebral hemorrhage.Methods:(1)Sprague-Dawley male rats were randomly divided into sham operation(Sham)group,intracerebral hemorrhage(ICH)group,low dose butyrophthalide(SNBP)group,high-dose butylphthalide(LNBP)group.The above four groups were divided into five subgroups: 6h,1d,3d,5d and 7d according to the different time after ICH.(2)All the other groups but the Sham group created the rat model of intracerebral hemorrhage by injecting 50 ul blood to the right caudate nucleus,which were got from caudal vein.And the sham group only cranial hole without the needle in.(3)The NBP group was injected with the corresponding dose of butyphthalide intraperitoneally twice aday immediately after modeling till the corresponding time point,and the sham group and the ICH group injected the same amount of normal saline.(4)Garcia score were used to observe the neurological deficit,EB were used to detect the permeability of blood-brain barrier,dry-wet method was used to measure the water content of brain tissue,and HE was used to observe the pathological changes and inflammatory response around the hematoma.And the immunohistochemical method was used to observe the expression of APQ4 and claudin-5 after the successful establishment of the model.(5)Finally,all data were analyzed with SPSS20.0,and the experimental results are "mean standard deviation"(s),and P<0.05 means the result is statistically significant(P < 0.05).Results:(1)Neurological deficit score: Compared with Sham group,the scores of nerve function in ICH group and NBP group were significantly decreased at each time point,the difference was statistically significant(P<0.05),and the neurological deficit score was the lowest at 3 d group after ICH.The score of each time point in NBP group was significantly higher than that in ICH group,and the difference was statistically significant(P<0.05);The score of LNBP group was significantly higher than that of SNBP group except for the 6 h time points,and the difference was statistically significant(P<0.05).(2)Water content of brain tissue: the water content of brain tissue in ICH group and NBP group was significantly higher than that in Sham group except at 7d group,the difference was statistically significant(P<0.05),and the water content of brain tissue was the highest at the3 d group after ICH.The water content of brain tissue in NBP group was significantly lower than that in ICH group at all time points except 7days(P<0.05);The water content of brain tissue in NBP group was significantly lower than that in SNBP group at 1 day,3 day and 5 day(P<0.05).(3)EB content:the EB content in ICH group and NBP group was significantly higher than that in Sham group at each time point,the difference was statistically significant(P<0.05),and the content of EB in brain tissue was the highest at the time of 3 days after ICH.EB content in NBP group was significantly lower than that in ICH group at all time points(P<0.05);The EB content of LNBP group was significantly lower than that of SNBP group at all time points except at 6 h in NBP group,and there was significant difference in EB content between LNBP group and SNBP group(P<0.05).(4)Pathological changes and inflammatory response around the hematoma: the neurons in the brain tissue of the group of Sham were intact and the cytoplasm and nucleus were stained evenly.In ICH group,neuronal swelling was observed at 6 h.The swelling and necrosis of neuronal cells,the pyknosis of nucleus,the widening of tissue space,the infiltration of neutrophilic granulocytes were observed over time,and it was most obvious at 1 day to 3 day.At 5 day,we can observed the swelling was alleviated and a few glial cell proliferation.At 7 day,we can observed the swelling of neurons were alleviated,the amount of inflammatory cells were decreased and the amount of glial cells are increased.At 6h and 1day,the degree of cell swelling and inflammatory response in NBP group wassimilar to the ICH group,and at 3 d,5 d and 7 d,which were lower than that in ICH group.The swelling degree and inflammatory response of neurons in LNBP group were lighter than those in SNBP group.(5)AQP4: ICH group and NBP group was significantly higher than that in Sham group at all time points,the difference was statistically significant(P<0.05),and the expression of AQP4 reached the peak at 3 days in those bleeding groups.The expression of AQP4 in NBP group were significantly lower than that in ICH group,the differences were statistically significant(P<0.05);In NBP group,the AQP4 expression in LNBP group was lower than that in SNBP group,except at 6 h time point.The differences were statistically significant(P<0.05).(6)claudin-5:ICH group and NBP group was significantly lower than that in Sham group(P<0.05).In those bleeding group,the difference was statistically significant(P<0.05),and the expression of claudin-5 was the least in 1 day.The expression of claudin-5 in NBP group was significantly higher than that in ICH group at all time points(P<0.05).The expression of claudin-5 in LNBP group was significantly higher than that in SNBP group at other time points except at 6 h in NBP group(P<0.05).(7)There is a positive correlation between brain water content and EB content after intracerebral hemorrhage(r=0.702,P<0.001).Expression of AQP4 have a similar trend with EB content(r=0.812,P<0.001)and Water content(r=0.782,P<0.001)in brain tissue,and showing a significant positive correlation,and a negative correlation with the neurological deficit score(r=-0.796,P<0.001);Claudin-5 content have anegative correlation with brain water content(r=-0.520,P<0.001)and EB content(r=-0.472,P<0.001);and have a positive correlation with neurological impairment score(r=0.537,P<0.001);There was a negative correlation between AQP4 and claudin-5 expression(r=-0.482,P<0.001).Conclusion:(1)After intracerebral hemorrhage,the expression of AQP4 increased and the expression of claudin-5 decreased around the hematoma.Both of them were related to the increase of blood-brain barrier permeability,involved in the formation of brain edema,resulting in neurological dysfunction.(2)NBP can reduce the expression of AQP4 and increase the expression of claudin-5,stabilizing the blood-brain barrier,alleviating brain edema,and eventually protect the nerve function. |
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