The Influence Of Erythrocytes On Aqp-4 In Spontaneous Intracerebral Hemorrhage And The Relationship Between AQP-4 And With Blood Brain Barrier Permeability

Background: Spontaneous intracerebral hemorrhage(SICH) refers to bleeding that arises in the brain parenchyma in the absence of trauma or surgery. It is associated with higher morbidity and mortality rate than other nervous system disease. SICH accounted for 20% ~ 30% of stroke in Asian countries. One of the main contributing factors for its high mortality rate is cerebral edema. In SICH, deterioration is mainly caused by the enlargement of cerebral hematoma. So far, the pathophysiological mechanism of post SICH cerebral edema is still not fully elucidated. Study shows that the main component of cerebral hematoma which is red blood cells in this study is closely related with the formation of cerebral edema in different stages. Aquaporin 4(AQP-4) water channels plays a very important role in water homeostasis in the brain. This study is conducted in order to provide experimental and theoretical evidence for the preventive treatment of post SICH cerebral edema. It studies the effect of red blood cells on the formation process of cerebral edema as well as on the expression of AQP-4 in spontaneous intracerebral hemorrhage and the relationship between AQP4 and blood brain barrier permeability. It has not been reported in the domestic and abroad.Objective:1. To elucidate the effect of red blood cells on the expression of AQP-4 in SICH;2. Explore the relationship between AQP-4 expression and the BBB permeability during the formation process of cerebral edema in SICH.Methods: This is an experimental study, all the sample rats are provided by Henan University of Science and Technology laboratory animal centre. Healthy male and Male total 195 Sprague Dawley(SD) rats are selected, each rat weighing about 210-300 g. SD rats were randomly divided into four groups: Normal controls(NC), Sham operation group(SO), whole blood group(WB), Red blood cellgroup(RBC).For the SO group, WB group and RBC group, each group is further divided in to four subgroups depends on the sacrificial time(6 hours, 1 day, 3 day, 5 day). Each subgroup includes a number of 15 rats. The laboratory test point for each subgroup include measuring of cerebral fluid volume, HE staining and immunohistochemistry, And BBB permeability. So each laboratory test point allotted 5 rats. Each group of SD rats are injected with autologous WBC or RBC to the right caudate nucleus by brain stereotaxic apparatus, So these rats become SICH models. Rats in each subgroup are sacrificed at the designed time. And specimen will be taken.Formamide immersion method will be used to test the Evan blue(EB) content in BBB permeability, Cerebral fluid volume, And HE staining and immunohistochemical distribution of positive AQP-4 cells around the cerebral edema.Results:1. EB content in cerebral tissue: Compare among RBC, WB, SO and NC groups’ 6h, 1d, 3d, 5d subgroup, the RBC volume in 3d subgroup is much higher than 6h, 1d and 5d subgroup. Compare among 6h, 1d, 3d and 5d subgroups, the EB blue content in RBC group shows increase in 6h subgroup, significantly higher in 1d subgroup, reach to the peak(P<0.01) in 3d subgroup and shows decrease but still higher than normal range(P<0.05) in 5d subgroup. The EB blue content shows the same varying pattern in WB groups’ four subgroups, but basically all less than RBC groups’ subgroups, SO group and NC group don’t show any difference(P>0.05).2. For the cerebral fluid volume test, compare among the subgroups in the RBC group, cerebral fluid volume on the third day is much higher than the other subgroups(P<0.01), and compare among the subgroups in RBC and WB groups, the cerebral fluid volume in WB group shows the similar trend, but the cerebral fluid volume in WB group is lower than RBC group in each same sacrifice time subgroup.3. RT-PCR to test AQP-4 expression: compare among four subgroups in RBC group, 3d subgroup is obviously higher than 6h, 1d, and 5d subgroups(P<0.01), in RBC group, the positive expression of AQP-4 in cerebral tissue start increasing in 6h subgroup, reach peak level(P<0.01) in 3d subgroup, and gradually decline after 3d subgroup, but still higher than normal(P<0.01) in 5d subgroup. The positive expression of AQP-4 shows similar in WB and RBC groups, but WB group shows lessor than RBC group.4. AQP-4 expression is detected by immunohistochemistry, and result shows that the expression of AQP-4 is present in the glial cells and their podocyte foot processes in both RBC and WB subgroup 3d. The cytoplasm is brown-yellow.Conclusions:1. Cerebral fluid volume reach to the highest in 3d subgroup in both RBC group and WB group shows that RBC plays an important role in the formation of cerebral edema on the 3d after SICH.2. The positive expression of AQP-4 reach to the highest at the 3d in both RBC and WB group. It suggested that RBC can stimulate the expression of AQP-4.3. The BBB permeability in RBC group and WBC group is the highest, and both start increasing at 6 hour, reach the peak on the third day shows that RBC can affect the BBB permeability in SICH.4. In general, the positive AQP-4 expression is positively related with cerebral fluid volume and BBB permeability, which can see from RBC and WB groups’ 3d subgroup, the cerebral fluid volume and EB content reach the peak, and the positive AQP-4 expression shows the strongest. The AQP-4 expression, cerebral fluid volume and EB blue content show the same trend suggest that the deterioration of cerebral edema on the third day in SICH is closely related with increasing BBB permeability and positive AQP-4 expression.