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Objective To explore the expression of aquaporin 4 (AQP4) in primary and secondary cultured rat astrocytes. Methods Rat cortical astrocytes from a newborn (one day) Wistar rat were cultured. Astrocytes were identified with immunofluorescence staining of glial fibrilillary acidic protein (GFAP). The expression of AQP4 was determined with real-time quantitative polymerase chain reaction and immu-nofluorescence staining three, five, seven and nine days of primary culture, and nine days of secondary culture. Results The purity of GFAP-positive cells was more than 95％. The expression of AQP4 mRNA was found three days of primary culture, remained unchanged five days of primary culture (P>0.05), and increased seven and nine days of primary culture (P0.05). AQP4 immunofluorescence staining showed the same trend of AQP4 mRNA. Conclusion AQP4 may express since three days of primary culture in rat astrocytes in vitro, and increase slowly until nine days of primary culture.
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Objective To investigate the expression changes of astrocytic syntrophin in hippocampus from human mesial temporal lobe epilepsy (MTLE). Methods From April, 2015 to July, 2016, 17 cases of hippocampus, collected from temporal lobectomy, were divided into MTLE group (n=13) and non-MTLE group (n=4) according to hematoxylin and eosin staining, glial fibrillary acidic protein and neuronal nu-clei immunohistochemical staining. Immunofluorescence double labeling and immunofluorescence histochemistry were used to observe the expression of syntrophin. Results The proliferation of astrocytes increased and neurons reduced in the hippocampus of MTLE group. Syntro-phin was found in the membrane and foot processes of astrocyte, that was enriched along perivascular astrocyte end-feet domain in non-MTLE group, but lost in MTLE group. While the whole expression of syntrophin was more in MTLE group than in non-MTLE group (t=5.421, P<0.001). Conclusion The distribution of syntrophin in hippocampus astrocytes may be related to the development of MTLE.
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Objective To compare the differences between the cell swelling of cultured astrocytes ( AST) from Wistar and Sprague-Dawley ( SD) rats after incubation with glutamate.Methods Primary cultured AST derived from the cerebral cortex of one-day-old Wistar or SD rats were prepared.The cultured AST received 1 or 10 mmol/L glutamate treat-ment for 48 h on the tenth day after subculture.The viability of AST was determined by lactate dehydrogenase ( LDH) kit to assess the cell injury, and the perimeter of AST was measured using Image Pro Plus software after glial fibrillary acidic protein immunofluorescence staining to evaluate the astrocyte swelling.Then, the expression of aquaporin 4 ( AQP4 ) in cultured AST was detected by quantitative reverse transcription polymerase chain reaction.Results No significant differ-ence was found in the LDH release after the glutamate treatment in cultured AST from these two strains (P>0.05).The perimeter of AST from normal Wistar rats was shorter than that from SD rats, but was longer after the treatment of glutamate (P<0.05).Meanwhile, AQP4 expression in the Wistar rats was significantly higher than that from SD rats after incuba-tion with 1 mmol/L glutamate ( P<0.05 ) .Conclusions These results suggeste that cultured AST from Wistar rats are more susceptible to glutamate-induced swelling than that from SD rats, and there are differences between the effects of glu-tamate on AQP4 expression in astrocytes of Wistar and SD rats.
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Objective To investigate the protective effect of methylene blue (MB) on blood-brain barrier (BBB) injury after focal cere-bral ischemia-reperfusion in rats. Methods 18 male Sprague-Dawley rats were randomly divided into sham-operated group (n=6), model group (n=6) and MB treatment group (n=6). The left middle cerebral arteries were occluded for 1 hour and reperfused. MB was infused intra-venously immediately after reperfusion (3 mg/kg) and again 2 hours post-reperfusion (1.5 mg/kg), while normal saline was administered in the model group. The sham-operated group was treated as same as the model group without occlusion and infusion. HE staining was used to observe the histological injury in the cortex around the infarcted region 47 hours after reperfusion, while albumin immunohistochemistry was used to evaluate the permeability of the BBB, and immunohistochemistry and double immunofluorescence staining were used to exam-ine the expressions of glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP-4). Results HE staining showed that cells and blood ves-sels were not intact in the cortex around the infarcted region in the model group and they were better in the MB treatment group. The expres-sions of the albumin, GFAP and AQP-4 were higher in the model group than in the sham-operated group (P<0.01), and were lower in MB treatment group than in the model group (P<0.05). The double immunofluorescence staining showed the colocalization of GFAP and AQP-4 in the astrocytes. Conclusion MB may ameliorate the BBB disruption induced by focal cerebral ischemia-reperfusion through reducing glio-cyte proliferation and down-regulation of AQP-4 expression in rats.
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@#Objective To examine the sensitivity of human glioblastoma multiforme cell line BT325 to 5 antineoplastic drugs, including cisplatin (DDP), teniposide (VM26), nimustine (ACNU), temozolomide (TMZ) and vincristine (VCR). Methods BT325 cells were incubated in DMEM with 10% or 20% fetal bovine serum (FBS) or without FBS respectively. The cell numbers were counted at 24 h, 48 h, 72 h,96 h, 120 h, and 144 h, then platting and growth curve were drafted. Cell counting kite-8 was used to detect the influence of 5 drugs with different concentrations on human glioma cell line BT325. Results DDP and VM26 significantly suppressed BT325 cells(>75%) viability in a dose-dependent manner, while VCR inhibited BT325 cells (50%) growth without dose-effect relationship. In contrast, ACNU and TMZwere not effective on the viability of BT325 cells. Conclusion BT325 cells were very sensitive to chemotherapeutic drugs DDP amd VM26.
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@# ObjectiveTo determine whether glutamate induces the alteration of aquaporin 4 (AQP4) expression in cultured rat astrocytes. MethodsThe secondary cultured astrocytes were treated with 1 mmol/L L-glutamate for 1 h, 3 h, 6 h, 12 h, 24 h and 48 h. The morphologic changes of astrocytes were observed through microscope after GFAP immunostaining and AQP4 mRNA expression were detected with real-time PCR. ResultsThe astrocytes swelled when exposed to glutamate for 1 h and remained with prolonged treatment. Meanwhile, the AQP4 mRNA expression were early down-regulated and subsequently up-regulated, featured with the lowest AQP4 mRNA level at 12 h after treatment (P<0.01) and higher at 48 h (P<0.05). ConclusionAquaporin 4 may be involved in the occurrence and development of astrocyte swelling induced by glutamate.
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@#Objective To reproduce a scratch-wound model in cultured rat astrocytes (AST).Methods The secondary cultured AST prepared from newborn Wistar rat cerebral cortex were scratched with plastic pipette tips. The morphologic change of AST was observed through microscope at 10 min before and 1, 3, 6, 12, and 24 h after injury, meanwhile the lactate dehydrogenase (LDH) leakages in the cultured medium were determined.Results Immediately after injury the edge of the scratch was lined with irregularly shaped cell. 6 h after injury the AST processes began extending to cell-free area, and elongated further at 12 and 24 h after injury, with presented of new generated cells in the denuded area. At different times after injury, the LDH leakages of the experiment group were higher than that before injury ( P<0.05), and were higher than that of the control group ( P<0.05).Conclusion According to observed AST morphologic changes and determined LDH leakages in culture medium, the scratch-wound model in cultured rat AST is successfully reproduced.
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@#The expression of brain aquaporin 4 (AQP4) is different at the different stages of cellular differentiation, and distribution of brain AQP4 is affected by the existence of endothelial cells. Other factors including osmolality, ammonia, hypoxia, temperature, hormone, C-type natriuretic peptide, lead, complement inhibitor, lipopolysaccharide can also influence AQP4 expression in astrocytes. However, the regulating mechanisms for brain AQP4 expression are not clear. Protein interaction, protein kinase C (PKC) through protein phosphorylation, mitogen-activated protein kinase signal pathway, Ca2+ signaling cascade signal pathway and transcription factor pathway have been proposed, among which phosphorylation of PKC for the inhibition of AQP4 is frequently studied.