| Objective: Diffuse axonal injury (DAI) is a common type of brain injury, characterized by severe symptoms, a long time coma and high rate of disability and mortality. The formation of DAI included a series of pathophysiological changes. Except the primary injury caused by shear stress directly, the secondary brain injury caused by changes such as ischemia, hypoxia, edema were more serious. The blood-brain barrier (BBB) damage Accompanied with DAI led to cerebral edema and other secondary nerve damage, which were the main reason of the poor prognosis. The Aquaporin-4(AQP4) discovered In recent years had close relations with the formation of brain edema after DAI, but it is still unclear that if AQP4 caused the changes of the permeability of BBB, and Ca2+ have relations with them. The study shows that in the model cerebral hemorrhage rat, Ca2+ antagonist nimoton can decrease the expression of AQP4 mRNA in brain tissue, lower the BBB permeability, so as to inhibit brain edema. Though dynamically observing the brain water content, BBB permeability, AQP4 expression after DAI, and the changes after the use of Nimoton, this research discussed the mechanism of cerebral edema after DAI, so as to provide a theoretical basis for prevention and cure of DAI.Methods: 110 adult male SD rats, each animal weight 350-375g, afforded by the Hebei Medical University Laboratory Animal Centre. These animals were divided into three groups according to the principle of completely randomized: the control group, the DAI Group and the nimoton treatment group. The control group had 10 rats, while the DAI group and the nimoton treatment group each 50. The DAI group and the nimoton treatment group were randomly divided into 6 h, 1d, 3d, 5d, 7d five sub-groups, each sub-groups had 10 rats. The DAI Group, in accordance with the Marmarou's production method to cause DAI model; The nimoton treatment group were intraperitoneal injected of nimoton (0.5mg/kg body weight), after 20 min, according to Marmarou's production method to cause DAI model, then injected nimoton once every 24 h (the same dose as before); The control rats sutured directly after incising scalp and exposing skull. The rats ate normaly after having been injured. Selected five rats from each sub-groups and the control group randomly. According to the scheduled time, after being reperfused by 4% paraformaldehyde solution decapitated and took out brain, The brain tissue will be divided into two parts along the median sagittal line. The left brain tissue was used to determinate the brain water content immediately. The right brain tissue were fixed, dewatered, transparented, paraffin imbeded for staining and observing.Using the wet-dry weighting method to measure the water content of the left brain tissue, as the representative of the extent of brain edema.Using spectrophotometric to measure the EB content in brain of the remaining rats at every time point in order to quantitatively evaluate the functional damage of BBB.In light microscopy, specimens after HE staining was to detect the changes of brain edema, such as cell edema, the extent of cell gap and vascular peripheral gap widened. specimens after immunohistochemical staining was to detect the AQP4 protein expression which the cell membrane was yellow or brown. Using Motic 6.0 digital medical image analysis system for quantitative analysis of each field of the positive reaction target's average optical density (OD), on behalf of the intensity of AQP4 expression.Applying SPSS 13.0 statistical software, the data are mean±standard deviation ( x±s). Between the two groups using the two-sample t-test, multiple comparisons using single-factor analysis of variance, when there are significant differences using analysis of variance, for further testing by q comparison. Based on the experimental requirements, as well as for linear correlation analysis. To P<0.05 as a judge if there was significant difference between the standards.Results: 1. Naked eye and light microscopy observationNaked eye: We can see leptomeningeal blood vessel dilated and congested, scattered subarachnoid hemorrhage point in the DAI group and the nimoton treatment group, but there was no obvious brain contusion and brain hemorrhage; HE staining: In the DAI group, we can see brain tissue osteoporosis and edema, cell vacuolar changes, cell clearance and the gap around vascular widened. The nimoton treatment group's pathological changes was lighter than the DAI group; The control group did not have these pathological changes; Immunohistochemical staining: In the DAI group, we can see AQP4 expression which astrocytes membrane were yellow or deep brown in ependymal, leptomeningeal and the vessels surrounding; The nimoton treatment group's AQP4 expression was lighter than the DAI group, but stronger than the control group. The control group showed weak AQP4 positive expression.2. The changes of brain water contentThe brain water content of the DAI group and the nimoton treatment group has a tendency from low to high, and reached its peak before gradually reducing process. The brain water content began to increase from 6h and reach a peak 1d, then started to decline, 7d brain water content is still higher than normal. Compared with the control group, The DAI group and the nimoton treatment group have significant differences (P <0.05). The nimoton treatment group reached its peak the same time as the DAI Group, but at 6h, 1d, 3d, 5d time point the brain water content were lower than the DAI group (P<0.05). The 7d brain water content had no significant difference (P>0.05).3. The changes of BBB permeability (The changes of brain EB content)The BBB permeability of the DAI group and the nimoton treatment group has a tendency from low to high, and reached its peak before gradually reducing process. The BBB permeability began to increase from 6h and reach its peak 1d-3d( 1d compared with 3d had no significant differences), then started to decline, 7d BBB permeability is still higher than normal. Compared with the control group, The DAI group and the nimoton treatment group have significant differences (P <0.05). The nimoton group reached its peak the same time as the DAI Group, but each time point BBB permeability were lower than the DAI group(P<0.05).4. The changes of brain AQP4 expressionThe brain AQP4 expression of the DAI group and the nimoton treatment group has a tendency from low to high, and reached its peak before gradually reducing process. The brain AQP4 expression began to increase from 6h and reach its peak 3d, then started to decline, 7d brain AQP4 expression is still higher than normal. Compared with the control group, the DAI group and the nimoton treatment group have significant differences (P<0.05). The nimoton treatment group reached its peak the same time as the DAI Group, but each time point brain AQP4 expression were lower than the DAI group (P<0.05).5. the correlation analysis of brain water content, EB content and AQP4 expressionThe brain water content, EB content and AQP4 expression had the same rule, correlation analysis showed that brain water content and AQP4 expression was positively correlated (r= 0.824,P<0.01); EB content and AQP4 expression was positively correlated (r=0.871,P<0.01).Conclusion: 1. the brain water content, BBB permeability, AQP4 expression has a tendency from low to high, and reached its peak before gradually reducing process in rats after DAI. The AQP4 expression have a positive correlation with the brain water content and the BBB permeability. It was confirmed that the AQP4 changes in the level of protein have a positive correlation with the changes of brain water content and the extent of damage on BBB.2. Given nimoton treatment, compared with the DAI group, the brain water content, BBB permeability, AQP4 expression in the corresponding time points were significant differences (P<0.05). It was confirmed that nimoton can lower BBB permeability and reduce cerebral edema by reducing the AQP4 expression after DAI. It was certain that Nimoton play a role in the process of BBB restoration after DAI.
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