Effect Of Mild Hypothermia On The Expression Of Aquaporin4 In Astrocytes After Lack Of Nutrition/Restore Nutrition

Objective:Discussion on lack of nutrition / restore of nutrition impact on astrocytes AQP4 expression and in the mild hypothermia under the condition of AQP4 expression with the corresponding time points, is proposed to study the pathogenesis of brain edema in diseases of the nervous system, and to clarify the cellular and molecular the mechanism for the clinical treatment of cerebral edema open up new strategies. Methods:1. Directly swing shaking culture, purification and amplification astrocytes; Using immunohistochemical identification of astrocytes, astrocytes(AC) can be divided into normal control group(C), lack of nutrition/restore of nutrition group(L/R), lack of nutrition/restore nutrition hypothermia group(L/R+S). With serum-free medium at room temperature cells lack of nutrition production model, record lack of nutrition injury time, lack of nutrition group time was 4h, 8h, 12 h, 16 h, 24 h, the control group without treatment. Using trypan blue staining method to judge the lack of nutrition cell vitality in different time.2. Lack of nutrition were adjusted after 12 hours Set temperature 37? and 32 ?, began to record restore of nutrition time. In each category restore of nutrition 4h, 8h, 12 h, 16 h a total of four time points, establish a lack of nutrition / restore of nutrition normothermia group(L / R), lack of nutrition / restore of nutrition hypothermia group(L / R + S) model. Using PCR analysis of the expression of AQP4, immunofluorescence staining AQP4 expression. Results:1. Based on the original generation of astrocytes in the separation and purification, then through GFAP immunohistochemical identification of the third generation of astrocytes in more than 95% purity.2. At room temperature astrocytes lack of nutrition in after 12 h cell viability compared with the control group(P <0.05), and keep the good condition after the cells restore of nutrition, can satisfy the requirement of next experiment.3. Take lack of nutrition 12 h cells were divided into lack of nutrition / restore of nutrition normothermia group(L / R), lack of nutrition / restore of nutrition hypothermia group(L / R + S), each set up restore of nutrition 4h, 8h, 12 h, 16 h,a total of four time points. Using PCR analysis of the expression of AQP4. Observation of AQP4 by Immunofluorescence staining. And PCR analysis of the expression level compared with the control group(P <0.05), at normothermia group and mild hypothermia group experimental results were statistically compared with the control group(P < 0.05).4. Immunofluorescence showed that mild hypothermia group AQP4 protein in the restore of nutrition 12 h, 16 h expression strength were lower than the same time point normothermia group set of results. Conclusions:Astrocytes lack of nutrition in the state with time, to express their AQP4 m RNA decline, AQP4 immunofluorescence gray also drops. When astrocytes lack of nutrition 12 h after its restore of nutrition AQP4 expression increased over the corresponding time points, In mild hypothermic conditions, the expression of AQP4 magnitude smaller than the normothermia group, reducing the edema state of astrocytes. In brain edema formation stage use the mild hypothermic treatment and AQP4 blocker, can effectively relieve clinical symptoms of brain edema, to remove cerebral ischemia and hypoxia gain time.