| Objectives: To explore the effects of mild hypothermia and NeuroD1 overexpression on spinal cord reactive astrocytes after the injury of astrocytes.Methods:1 In vitro,the culture of primary spinal cord astrocytes from neonatal SD rats: extracted and purified astrocytes from neonatal 1 ~ 3 d SD rat spinal cord tissue,astrocytes purity was detected by immunofluorescence and flow cytometry,for the preparation of reactive astrocytes.2 RAS was prepared by scratch test: using 10 ul sterile pipette tip along "well" words scratches in culture plates,controlling the same power and speed.After scratch injury,the medium was changed and using immunofluorescence to detect RAS purity.3 Effect of mild hypothermia on RAS after scratch injury: experimental cells were divided into 4 groups: control group,scratches group,mild hypothermia group and scratches + mild hypothermia group.Control cells did not accept scratches and was cultured at 37℃ incubator,scratches group cells accepted scratches and was cultured at 37℃ incubator,mild hypothermia group cells did not accept scratches and was cultured at 33℃ incubator,scratches + mild hypothermia group cells accepted scratches and was cultured at 33℃ incubator.Cell morphology was observed in each group at the corresponding time points,and using immunofluorescence to detect Nestin positive rate,cell activity was detected by MTT colorimetric assay,the degree of apoptosis was detected by PI staining method.4 RAS was prepared by stretch test: the stretch test experimental device consists Bioflex cell culture plate,CIC Ⅱ type cell damage meter,high purity nitrogen.Setting the metering valve pressure to 12 PSI,the peak pressure would be 3 PSI after be hit.After stretch injury,the medium was changed and using immunofluorescence to detect RAS purity.5 Effect of mild hypothermia on RAS after stretch injury: experimental cells were divided into 4 groups: control group,stretch group,mild hypothermia group and stretch + mild hypothermia group.Control cells did not accept stretch and was cultured at 37℃ incubator,stretch group cells accepted stretch and was cultured at 37℃ incubator,mild hypothermia group cells did not accept stretch and was cultured at 33℃ incubator,stretch + mild hypothermia group cells accepted stretch and was cultured at 33℃ incubator.Cell morphology was observed in each group at the corresponding time points,and cell activity was detected by MTT colorimetric assay,the degree of cell damage was detected by LDH release.6 Effect of NeuroD1 overexpression on RAS after scratch injury: experimental cells were divided into 3 groups: Blank group(NV group),virus control group(GFP group)and NeuroD1 virus group(NeuroD1 group).After scratch test,NV group cells did not accept the virus-infected,GFP group cells were infected retrovirus carrying GFP gene,while Neuro D1 group cells were infected retrovirus carrying the GFP gene and NeuroD1 gene.Cell morphology was observed in each group at the corresponding time points.DCX positive rate were detected at 7 d after infection and NeuN positive rate were detected at 14 d after infection by using immunofluorescence staining method.Results:1 The purity of primary spinal astrocytes: After 3rd passaged using GFAP immunofluorescence,GFAP positive astrocytes rate reached more than 95%.The flow cytometry purity can reach 93.94%.2 The purity testing of RAS after scratches or stretch: Using Nestin / GFAP immunofluorescence double staining tested purity RAS after scratches or stretch injury,RAS purity can be expressed by Nestin positive rate,Nestin positive rate reached more than 80%.3 Optical microscopy observed cell morphology after scratch injury: After the cells were damaged by scratch,cell body became hypertrophy,projections began to gradually increase and extension,the cytoplasm became rich.In the mild hypothermia environment,cells changed slowly and the increase of projections was not obvious,and the time of cells gradually grew into scratches area was short.4 Optical microscopy observed cell morphology after stretch injury: Astrocyte activated into reactive astrocytes,cell body enlargement,surrounding projections and connecting multiple fractured,and the cells became activated proliferation.In the mild hypothermia environment,cells did not change significantly reduce and the required time for recovery breaks was short.5 After scratch injury detecting Nestin positive rate of cells in each group to reflect the degree of activation: Compared with the control group and mild hypothermia group,scratches and scratches + mild hypothermia group were significantly higher,indicating that damage caused by scratches astrocyte activation and the result was statistically significant(P <0.01).Compared with the scratches group,scratches + mild hypothermia group was lower,indicating that mild hypothermia can inhibit the activation of astrocytes and the result was statistically significant(P <0.01).6 After scratch injury detecting PI positive rate of cells in each group to reflect the degree of apoptosis: Compared with scratches and scratches + mild hypothermia group,the control group and mild hypothermia group were significantly lower,indicating that damage caused by scratches astrocyte apoptosis and the result was statistically significant(P <0.01).Compared with the scratches group,scratches + mild hypothermia group was lower,indicating that mild hypothermia can inhibit the apoptosis of astrocytes and the result was statistically significant(P <0.01).7 After scratch injury detecting MTT values of cells in each group to reflect the growth rate: Compared with scratches and scratches + mild hypothermia group,Control group and mild hypothermia group was significantly decreased after the 3 d,indicating that damage caused by scratches cells proliferation.Compared with the scratches group,scratches + mild hypothermia group was lower,indicating that mild hypothermia can inhibit the cells proliferation and the result was statistically significant(P <0.01).8 After stretch injury detecting MTT values of cells in each group to reflect the growth rate: Stretch group compared with the control group and stretch + mild hypothermia group compared with mild hypothermia group,the rate of former is higher than the latter after the 3 d or 5 d,indicating that damage caused by scratches cells proliferation.Stretch group compared with stretch + mild hypothermia group,the rate of former is higher than the latter after the 3 d,indicating that mild hypothermia can inhibit the cells proliferation and the result was statistically significant(P <0.01).9 After stretch injury detecting LDH values of cells in each group to reflect the degree of damage: Stretch group compared with the control group and stretch + mild hypothermia group compared with mild hypothermia group,indicating that RAS were damaged,and in the 2 d,the LDH release rate of injury is greater than the value at 1 d,indicating stretch group RAS were further damaged.Stretch group compared with stretch + mild hypothermia group,the rate of former is higher than the latter after the 2 d,indicating that mild hypothermia can inhibit the degree of damage and the result was statistically significant(P <0.01).10 Optical microscopy observed cell morphology after retrovirus infected cells: With in 7 d after infection,cells morphological in each group changed,nucleus became obviously full,cytoplasm became reduced,the surrounding protrusions gradually reduced and extend.Compared with NeuroD1 group,the surrounding projections of NV group and GFP group were short and more branches,the nucleus did not change significantly.After infection 7 d,the cell morphology of NV group and GFP group gradually restored to its former shape,but NeuroD1 group maintained in its current form.11 After retrovirus infected cells 7 days,testing DCX positive rate: Compared with the positive rate of NV group and GFP group,DCX positive rate of NeuroD1 group increased significantly,and the result was statistically significant(P <0.01).12 After retrovirus infected cells 14 days,testing NeuN positive rate: Compared with the positive rate of NV group and GFP group,NeuN positive rate NeuroD1 group increased significantly,and the result was statistically significant(P <0.01).Conclusions:1 After scratch injury,astrocytes were activated to be reactive astrocytes and hyperplasia.Mild hypothermia significantly inhibited spinal cord reactive astrocytes overgrowth and restrain astrocytes apoptosis.2 After stretch injury,reactive astrocytes projections and cell junctions fracture occurred,and cell became hypertrophic significantly.Besides mild hypothermia significantly inhibited proliferation and activation of reactive astrocytes,and can inhibited the further damage.3 In vitro culture,the overexpression of NeuroD1 can mediate the transdifferentiation for spinal reactive astrocytes into neurons. |
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