Fig. 4

Long-term isoflurane anesthesia inhibits GJs-Cx43 expression, impairs gap junctions function, and causes inflammation in primary astrocytes. a A significant reduction of total Cx43 protein was observed in isoflurane-treated astrocytes. Upper panel showed representative immunoblot and lower panel were quantifications (n = 6/group). Three representative samples per group are shown. Cx43-soluble protein fractions was increased (b), while Cx43-insoluble protein fractions was decreased (c), in control astrocytes compared with isoflurane-treated astrocytes (n = 6/group). Three representative samples per group are shown. d Representative Fluorescent punctas represent GJs-Cx43 of control and isoflurane-treated astrocytes. Scale bar = 20 μm. e Quantification of the proportion of GJs-Cx43 punctas (n = 6/group). f The schematic diagram of scrape-loading dye transfer. The lucifer yellow can specifically stain cells through the GJs-Cx43, therefore, the longer the distance of lucifer yellow diffusion indicates the stronger the function of the gap junctions. g Representative lucifer yellow diffusion images in scrape-loading dye transfer. Scale bar = 200 μm. h The intensity of lucifer yellow and diffusion distance after long-term isoflurane exposure (n = 6/group). The dashed line represents the mean value of lucifer yellow fluorescence intensity at a specific distance, the shaded part represents SEM, and the solid line represents the fitted curve of lucifer yellow fluorescence intensity versus diffusion distance. i The yellow fluorescence diffusion distances of long-term isoflurane-treated astrocytes decreased noticeably (n = 6/group). Data are presented as the mean ± SEM. Student’s t-test was used for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001