| Objective1. By several methods:transfection siRNA of aquaporin-4(AQP4), inhibition of Na+-K+-Cl2- cotransport (NKCC) and the mitogen activated protein kinase (MAPK), to reducing the reaction of AQP4 when cultured astrocytes are being damaged by different degrees of pressurized gas. Observations have shown a reduction in damage of the astrocytes when the edema is at its peak.2. Research involving the inhibition of AQP4 reactions of MAPK activity in mice of varying stages of focal cerebral ischemia-reperfusion damage from brain edemas, neurological functional defect scores and cell apoptosis conditions.Methods1. Cultured primary Sprague Dawley rats cortical astrocytes. Grouping cultured astrocytes were randomly divided into 4 groups:pressure (1.OMPa,1.5MPa, 2.0MPa and 2.5MPa). Observation of the cell survival and AQP4 reaction 5 minutes after pressure damage, and detection 3 hours after damage finished. Choose two suitable pressures for further experiments. Every pressure damage subject was divided into four groups:The positive control group (T), siRNA-treated group, bumetanide-treated group (BU) and p38-MAPK inhibition agent (SB203580, SB) treatment groups. Build the siRNA silencing AQP4 mRNA transiently transfected plasmid rat astrocytes.48 hours after the success of transfection, damage astrocytes by air pressure. Bumetanide and p38-MAPK inhibitors were pretreated astrocytes 30 minutes before being pressure damaged. When the cell edema peaked 3 hours after being damaged, using the forward side scatter (FSC) of flow cytometry to detect cell volume because the intensity of light is proportional with the cells volume. Stain with Annexin-V/PI to detect the degree of apoptosis and death. Using western blot to detect the presence of AQP4.2.90 Healthy Kunming mice with weight between 18g to 22g were randomly divided into negative control group (C), ischemic damage 1.5h and 2.5h groups, and then into the positive control group (T1.5, T2.5) and p38-MAPK inhibitor group (SB203580, SB1.5, SB2.5). SB group, an injection of SB203580 20mg/kg 30 minutes before testing were given an intraperitoneal. Right middle cerebral artery embolization established a model of focal cerebral ischemia in mice.12 mice put to death in each group at 24h, the brain edema during peak post-operation had brain tissue removed.6 for the detection of brain moisture content by dry wet weight method.6 mice used the Western blot detection the expression of AQP4 and Caspase-3. The remaining 6 mice were observed and neurological damage was recorded daily after the ischemia. SB group received an intraperitoneal injection of SB203580 once every day. On the seventh day the mice were put to death for pathological observation of brain tissue injury.Results1. Primary cultured rat astrocytes grew well, the AQP4 siRNA transfected significantly reduced the AQP4 appearance of astrocytes. Using an optical microscope the astrocytes of gas pressure injury group were observed to have significantly more swelling than the normal control group after 1.5MPa, 2.0MPa injury 3 hours. Used FSC to detect the cell size, after 1.5MPa and 2.0MPa pressure damage, the positive control group were significantly large than the control group (P<0.05). The cell volume in 2.OMPa positive group was significantly larger than in 1.5MPa positive group size (P<0.05). The cell volume in every interpolation group was smaller than the positive control group. In addition to the siRNA group and SB group after 1.5MPa damage, other groups’ cell volume were larger than the negative control group, and the 2.0M?a positive group increased obviously (P<0.01). After 1.5MPa pressure damage the siRNA interference group, BU group and SB group, the astrocyte edema lightened compared with the positive control group (P<0.05), but the apoptotic cells increased compared with the positive control group (P<0.05). After 2.0MPa pressure injury finished 3 hours, cells in each group were substantial death. Western blot analysis AQP4 expression found siRNA interference group, BU group and SB group were lower than their positive control group (P<0.05).2. Mice after 1.5h and 2.5h ischemic brain injury, at 24h the p38-MAPK inhibitor group (SB1.5, SB2.5) when compared with the positive control group(T1.5, T2.5), the brain moisture content and AQP4 expression were significantly lower(P<0.05), but the appearance of Caspase-3 was higher. No significant difference was found in the brain moisture content and the AQP4 appearance between the SB 1.5 and the negative control group (P> 0.05), but other groups showed an increase over the negative control group and the difference showed a statistical significance. In the 7 days of neurological damage points, there was no significant difference in the comparison of 7 days general neurological damage score in T 1.5 and SB 1.5, but both were higher than the negative control group (P<0.05). T1.5 group in the 4th and 5th day the neurological damage score decreased, and were lower than the SB 1.5 group (P0.05). The neurological damage score of SB 1.5 group decreased at the 7th day mark, compared with the T1.5 group, there was no significant difference (P> 0.05). T2.5 group 7 days total neurological damage points were higher than that of the SB2.5 group (P<0.05).Conclusions1. The AQP4 presence upregulated after astrocytes damage finished 3 hours by 1.5MPa pressure. AQP4 showed upregulating and cell edema. By siRNA interference, inhibit NKCC and p38-MAPK signaling pathway could reduce the appearance of AQP4 and mitigate astrocytes edema. But AQP4 also plays an important role in astrocyte migration and repair, inhibiting the expression of AQP4 in reducing edema it also affects the reparation process. The cell swelling itself is an adaptive response to injury, by diluting the intracellular ion concentration increased due to damage. So it is inferred that because of a reduced AQP4 level, they also blocked the adaptive changes in cells, delayed the repair of astrocytes and mediated apoptosis. In addition, as the surrounding cells are not fixed, when the cell edema did not make surrounding pressure progress rise and progressively damaged the cells. When 2.0MPa pressure injury is a serious injury, it is difficult to withstand, directly led to large amount of cell necrosis, no significant difference showed in the 2.0MPa pressure injury groups. In this experiment the reduced regulation of AQP4 can reduce the edema of cells, but also promotes apoptosis and cell death, analysis of cell survival did not show a protective effect.2. After focal cerebral ischemia in mice, through inhibition NKCC activity and p38-MAPK signaling pathway, can reduce the appearance of AQP4 and reduce cerebral edema. It may not cause severe ischemic brain edema in the case of 1.5 hours, so the positive control group restored neurological function after the edema. The SB 1.5 group reduced the edema and also led to partial apoptosis and necrosis, at same time may inhibit the astrocyte migration and repair function. AQP4 can be mediated, so the recovery is slow, this showed the role of aggravated damages to nerve function to a certain degree. However, in the case of ischemic 2.5 hour, because the heavier damaged parts of brain area caused a severe edema, increased intracranial pressure, and then caused edema in the surrounding brain tissue forming a vicious cycle. So neurological damage was extremely high in the positive group. At this point SB2.5 group by inhibiting the expression of AQP4 reduced cerebral edema and showed relief for the secondary damage it also plays a protective role. |
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