A Study On Effects Of Aquaporin-4 Expression In Brain Edema Formation After ICH And The Regulation Mechanism Of Aquaporin-4

Part IAnalysis of the expression of aquaporin-4 in experimental intracerebralhemorrhage ratsObjective: To study the expression of AQP4(aquaporin-4) in order to find the pathologic effect of AQP4 protein on brain edema formation after hematoma formation.Methods: Intracerebral hemorrhage (ICH) models were established by infusing autologous caudate artery blood into caudate-putamen nucleus (CPu), in addition, 15 U thrombin was injected into CPu. the expression of AQP4 protein was detected by immunohistochemical technique.Results: 6 hour after ICH, the expression of AQP4 protein in astrocytic end-feet adjacent to capillaries around the hematoma obviously increased(P<0.01), especially on the 1th day and 3th days(P<0.01), and went down to the level which is above the norml level gently on the 7th day(P<0.01). In thrombin treated rats, AQP4 protein expression increased occurred at 6th hour, especially on 1th day and the 3th day (P<0.01). From the 5th day, AQP4 protein expression decreased to normal level (P>0.05).Conclusions: AQP4 protein played a very important role in the formation of brain edema after ICH, expression of AQP4 protein may be induced by thrombin.Part II Thrombin induced aquaporin-4 expression in rat primary astrocytesObjective: To study the biologic effects of various concentrations of thrombin on the aquaporin-4(AQP4) expression in rat primary cultured astrocytes, and explore the regulation mechanism of transmembrane water transportation in astrocytes after intracerebral hemorrhage (ICH).Methods: Primary cultured astrocytes were incubated in culture medium containing various concentrations of thrombin for 24 h and harvested for determination of AQP4 mRNA and AQP4 protein expression by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical technique. Cell apoptosis was detected by TdT-mediated dUTP nick end labeling ( TUNEL ) technique. Cell morphology was observed by phase contrast microscope, and cell viability was assayed by MTT.Results: AQP4 mRNA and protein showed a weak expression in normal astrocytes. The expression of AQP4 mRNA and AQP4 protein significantly increased in astrocytes following the higher concentrations of thrombin (100 U/ml, 200 U/ml)treatment, which resulted in markedly astrocyte swelling. The number of TUNEL positive cells significantly increased. Otherwise, the downregulated AQP4 mRNA and AQP4 protein occurred in the low concentrations thrombin(0.5 U/ml, 1 U/ml) treated astrocytes, cell volume did not increase in astrocytes. Few TUNEL positive cells were observed.Conclusions: The AQP4 overexpression induced by high concentrations of thrombin causes a increase in astrocytic membrane permeability to water. On the contrary, the decreased AQP4 expression refers to the neuroprotective mechanism of low concentrations of thrombin, and avoids atrocyte volume increase and apoptosis in the cases of the brain insults. Part IIIaquaporin-4 expression and the permeability of blood-brain barrierObjective: To investigate the relationship between aquaporin-4(AQP4) expression and the permeability of blood-brain barrier (BBB), explore the possibility for V_1a receptor (V_1aR antagonist)antagonist in therapy of brain edema .Methods: Intracerebral hemorrhage (ICH)models were established by injection of autologous caudate atery blood into the caudate-putamen nucleus (CPu). After successful surgergy, in treated group 0.1μg V_1aR antagonist was injected into lateral cerebral ventricle. In ICH group, the rats received intracerebroventricular injection of the equivalent artificial CSF. Immunohistochemistry method was adopted to analyse the expression of AQP4 protein around the microvessels. The BBB permeability was evaluated quantitatively by measuring Evans blue dye extravasations.Results: After experimental intracerebral hemorrhage, the AQP4 protein performed significant enhanced level in the ICH group, especially at 1 d, 3 d after ICH(P<0.01). In addition, the expression of AQP4 was positively correlated with the permeability of BBB. When the rats received V_1aR antagonist treatment, AQP4 protein showed a lower expression than that in the control group(P<0.01). The BBB permeability showed a significant decrease at every time point in the treated group comparing with the control group(P<0.05).Conclusions: There is a close relationship between AQP4 expression and permeability of BBB. Furthermore V_1aR antagonists may protect the BBB and reduce brain edema in the perihematoma region after ICH by downregulating the expression of AQP4 protein. Part IVEffect of arginine vasopressin on aquaporin-4 expression in primarycultured astrocytesObjective: To investigate the effects of arginine vasopressin (AVP) on the aquaporin-4(AQP4) expression and cell volume in primary cultured astrocytes.Methods: Astrocytes were purified to >98% by passage from new-born rat cerebral cortex. Primary cultured astrocytes were incubated in culture medium containing 500 nM AVP or 500 nM V_1a receptor (V_1aR) antagonist for 1, 6, 12, 24 h, harvested for determination of AQP4 protein and AQP4 mRNA expression by immunohistochemical technique and reverse transcription polymerase chain reaction (RT-PCR). Brain microvascular endothelial cells were cultured in vitro and treated with AVP. Cell morphology was observed by a phase contrast microscope.Results: After astrocytes were treated with 500 nM AVP for 6 h, the expression of AQP4 mRNA began to increase(P<0.01), at 12 h reached expression peak(P<0.01), at 24 h the AQP4 mRNA still maintained the higher expression(P<0.01). The similar change was showed for AQP4 protein. AVP treatment resulted in significant astrocyte volume increase at 24 h, V_1aR antagonist inhibited upregulation of AQP4 expression and cell swelling. After AVP treatment, the morphology of endothelial cells remained as the normal.Conclusions: The results suggest high level of AVP may induce overexpression of AQP4 mRNA and AQP4 protein by activation of V_1aR, which increases water permeability of astrocyte membrane, and makes astrocyte swell. The morphology of endothelial cell was not affected by AVP. Part Varginine vasopressin induced expression of aquaporin-4 and astrocyte apoptosis by the way of p38 MAPKObjective: To determine the role of p38 MAPK in the aquaporin-4(AQP4) expression induced by arginine vasopressin(AVP), investigate the effect of p38 MAPK on apoptosis in cultured rat astrocytes.Methods: Primary cultured astrocytes were treated with AVP V_1aR antagonist and SB 203580, harvested for determination of AQP4 mRNA expression by reverse transcription polymerase chain reaction (RT-PCR). p38 MAPK phosphorylation and protein expression of Caspase-3 P20 were assessed by Western blot analysis. Cell viability was assayed by MTT, Cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL ) technique.Results: After astrocytes treated with 500 nM AVP for 6 h, the expression of AQP4 mRNA began to increase(P<0.01), at 12 h, reached expression peak(P<0.01), at 24 h, the AQP4 mRNA still maintained the higher expression(P<0.01). This course were not exhibited after SB 203580 treatment. p38 MAPK phosphorylation increased 15 min after AVP treatment, remained elevated level even at 120min. Protein expression of Caspase-3 were upregulated 6 h after AVP treatment(P<0.01), it reached the peak at 12 h(P<0.01). AVP treatment resulted in significantly apoptosis at 6 h, 12 h and 24 h (P<0.01). V_1aR antagonist and SB 203580 inhibited this change.Conclusions: AVP may induce higher expression of AQP4 mRNA and apopotosis through stimulation of p38 MAPK in astrocytes, SB 203580 and V_1aR antagonist could inhibit upregulation of AQP4 mRNA in the AVP environment, protect astrocyte from apoptosis.