Oxygen Glucose Deprivation-Induced Apoptosis In Oligodendrocytes Through TRPC3

Background:With the aging of population,the incidence of neurodegenerative diseases is increasing,however,there is no effective measures which can control the progression of the disease. Swiss researchers found that demyelination is involved in triggering neurodegenerative diseases,further studies have shown that Oligodendrocyte apoptosis is involved in triggering demyelination. Oligodendrocytes display a number of features that render them more vulnerable to hypoxia-ischemia than other CNS glial cells,and in certain brain regions and stages of development,more vulnerable than neurons.Therefore,it have proposed that hypoxic-ischemic injury in oligodendrocytes is an important reason for neurodegenerative diseases.Calcium is an essential intracellular messenger and serves critical cellular functions,alterations in Ca2+homeostasis have been suggested in the onset/progression of cell death which is involved in hypoxia-ischemia. Ca2+influx can also be initiated by second manners such as voltage-dependent and non-voltage-dependent calcium channels,Transient receptor potential channel protein (TRPC) is a non-voltage-dependent transmembrane protein which is important to maintain Ca2+homeostasis, TRPCproteins are more highly expressed and is involved in critical physiological functions in CNS.Overexpression of TRPC3is involved in neurodegenerative diseases,howerer,whether TRPC3 involved in the injury of hypoxia-ischemia in oligodendrocytes is not clear.In this study,To explore whether TRPC3 plays a role in the apoptosis of oligodendrocytes induced by Oxygen glucose deprivation(OGD),Control group were established by primary cultured oligodendrocytes of SD rats,model group were established by primary cultured oligodendrocytes of SD rats which exposed to OGD2h,treated group were established by model group+Pyr3. Western blotting were used to detect the expression of TRPC3 at protein levels,The viability of the oligodendrocytes was determined,and the apoptosis of oligodendrocytes were measured,intracellular Ca2+ concentration were measured.We aim to explore whether TRPC3 plays a role in the apoptosis of oligodendrocytes induced by OGD and provide a further understanding for the neurodegenerative diseases.Methods:Part Ⅰ1.Neonatal Sprague-Dawley rat cortex were used,the oligodendrocytes were cultured and isolated with different cell proliferation methods (cytokines and B104 conditioned medium) and different isolation/purification methods (shaking and EDTA digestion mechanical pipetting). A:cytokines proliferation+shaker shock B:B104CM(the conditioned medium prepared from B104 neuroblastoma cells,B104CM) proliferation+shaker shock C:EDTA digestion mechanical pipetting proliferation+cytokineD:B104CM proliferation+EDTA digestion mechanical pipetting.Morphological were observed under an inverted microscope.2.Immune-staining of A2B5 and myelin basic protein were applied to identify oligodendrocytes and assess the culture purity.3. Methods to induce oxygen glucose deprivation(OGD)of oligodendrocytes:oligodendrocytes were washed with DMEM for two times.Then were incubated for a different period(1,2,3,and0h)in a tri-gas incubator with an atmosphere of 1%O2,5%CO2and94%N2,98% humidity at 37℃.And then these cells were removed from the anaerobic incubator,washed,and added normal medium.4.viability assay and apoptosis assay:The viability of the oligodendrocytes was determined by MTT assay,and the apoptosis of oligodendrocytes were measured by flow cytometry after AnnexinV-FITC staining.Part II1.Methods to induce OGD of oligodendrocytes:We induced oxygen glucose deprivation (OGD) of oligodendrocytes to mimic a hypoxic-ischemic injury insult.2.Western blotting were used to detect the expression of TRPC3 at protein levels.3.Groups:Control group were established by primary cultured oligodendrocytes of SD rats,model group were established by primary cultured oligodendrocytes of SD rats which exposed to OGD2h,treated group were established by model group+Pyr3.4.The viability of the oligodendrocytes was determined by MTT assay,and the apoptosis of oligodendrocytes were measured by flow cytometry after AnnexinV-FITC staining.5.Intracellular Ca2+concentration were measured by flow cytometry and Laser Confocal Scamfing Microscope(LCSM)Results:l.The number of cells harvested by B104 conditioned medium proliferation and EDTA digestion isolation was higher than the cells obtained by other three methods (P< 0.05).2.A2B5 and myelin basic protein specific markers were positive and the purification was more than 95%.3.MTT assay showed that, cell viability was decreased after oxygen-glucose deprivation for 1 hour and reoxygenation for 24 hours (P< 0.05), and cell viability decreased with prolonged hypoxia time (P< 0.05). Flow cytometry results showed that,the apoptotic index in the normal control group was lower than that after exposure to oxygen-glucose deprivation for 1,2,3 hours (P< 0.05).4.A significant increase in the expression of TRPC3 in cultured oligodendrocytes.5.The viability of OGD oligodendrocytes (54.34±6.55)% was obviously decreased and the apoptosis rate was increased (24.24±0.86)% when compared with the control cells (P<0.05).Pyr3 significantly increased the survival rate (72.26±5.41)% and decreased the apoptosis rate (14.82±0.28)% (P<0.05).6.Calcium concentration measured by confocal results showed fluorescence intensity of the normal control group (28.89±4.43)%,OGD group fluorescence intensity (58.05±5.80)%,the fluorescence intensity of treatment group (42.26±5.44)%,the difference was statistical significance (P<0.05).Flow cytometry measurement of intracellular calcium concentration results showed that the fluorescence intensity of the control group (4.15±0.65)%,OGD group fluorescence intensity (23.99±1.22)%,(he fluorescence intensity of treatment group (10.54±0.73)%,the difference was statistically significant (P<0.05).Two sets of results showed elevated levels of intracellular calcium in the OGD group,but after adding TRPC3 blockers Pyr3, intracellular calcium increase significantly suppressed.Conclusions:1.The method of B104 conditioned medium proliferation plus EDTA digestion and mechanical pipetting is a simple and reliable technique for the in vitro primary culture of rat oligodendrocytes.2.We have established a simple, stable and reliable model of OGD of oligodendrocytes in vitro successfully by means of tri-gas incubator and Earle’s balanced salt solution without glucose inducing oxygen glucose deprivation.3.The increased expression of TRPC3 which mediated intracellular calcium level may be one of the important reasons for OGD oligodendrocyte apoptosis.