Value of measuring the volume of rat spinal astrocytes by live cell imaging system after oxygen-glucose deprivation/reoxygenation injury

English Keywords:Live cell imaging system  Cell volume  Injury  Edema  Aquaporin 4

English Abstract:

  【Abstract】 Objectives： To investigate the effect of oxygen-glucose deprivation/reoxygenation(OGD/R) by live cell imaging system on the volume of rat spinal cord astrocytes. Methods: The neonatal Sprague-Dawley rats within 24 hours were taken and the rat spinal cords were collected. Rat spinal cord astrocytes were isolated by trypsinization and differential adhesion ways and cultured in vitro. The fourth generation mature astrocytes were conducted with OGD/R and divided into four groups: group A(normal astrocytes), group B(OGD6h/R12h), group C(OGD6h/R24h), group D(OGD6h/R 48h) group. The astrocyte volumes were measured and calculated by live cell imaging system, and the morphological and ultrastructural changes of the astrocytes were observed by light microscopy and transmission electron microscopy, and the expression of AQP4 protein was measured by Western blot protein to further testify the accuracy of live cell imaging system in measuring cell volume. Results: Compared with group A (2810.19±306.11 μm3), the cell volume of group B (4311.53±407.73 μm3) was significantly increased(P<0.01), and the maximum increase was observed in group C (6248.86±702.11 μm3)(P<0.01). The astrocytes volume in group D (4541.33±503.17 μm3) was reduced, and it was higher than group A(P<0.01). The astrocyte swelling and increased refraction were observed in group B with light microscope. The cells swelling reached the peak in group C and was obvious in group D although the swelling was alleviated. The swelling of astrocyte organelles was observed in group B under transmission electron microscope. The organelles swelling was more significant and the breakage of mitochondria was observed in group C. The swelling was slightly relieved, but it was evident in group D. The Western blot results showed that the AQP4 expression in group A was(12.37±1.25)%, the AQP4 expression in group B was (35.50±1.08)%, the AQP4 expression in group C was (43.57±1.75)%, the AQP4 expression in group D was (32.63±0.81)%. Compared with that in group A, the AQP4 expression was significantly increased in group B, group C and group D(P<0.01). Compared with that in group C, the AQP4 expression was reduced in group B and group D(P<0.01). Conclusions: Live cell imaging system is used to measure the swelling volume of rat spinal cord astrocytes after oxygen glucose deprivation/reoxygenation, and this method can be used as a routine detection method in the measurement of astrocytes volume. It can also be applied in the detection of other cell volumes.