Cells (green) correspond to MG in cultures of astrocytes alone (Astrocytes, a1) and in astrocyte-MG cocultures ( Microglia; a), both treated or not with LPS (10 ng/ml; 24 h). Scale channels in cultured astrocytes (Meme bar, 50 m. a , Representative photographs for SL/DT with LY (a) and34 uptake (a) to measure the functional states of ^ EthBr 52 58 9 2 et al., 2006), we studied no matter whether LPS- GJC or hemichannels, respectively. Experiments had been performed with similar culture models and Bak review remedies as described above. activated MG may also affect the activity Scale bar, 100 m. b, c, Graphs corresponding to the quantification of LY diffusion by means of astrocytic gap junctions (b) plus the of Cx43 hemichannels. For this objective, EthBr uptake (c) either in enriched astrocyte cultures (Astrocytes) or in astrocyte-MG cocultures ( Microglia). Each culture EthBr uptake was investigated in astro- models were treated (LPS), or not (C, control), with LPS (10 ng/ml) for 24 h. Each plotted number corresponds to the mean SEM cytes cocultured with MG. Immuno- of three independent experiments. p 0.01, p 0.001; n.s., no substantial distinction compared with handle astrocyte staining with astrocyte and MG markers group; p 0.001, compared with the astrocyte-LPS group. (GFAP and isolectin B4, respectively) in1a8,b). This reduction was also statistically substantial compared dicated that major cultures of astrocytes had been composed by with the weak reduction induced by LPS in astrocytes cultures 98.four 0.1 and 1.six 0.1 GFAP-positive (red cells) and alone ( p 0.001). isolectin B4-positive (green cells) cells, respectively (n three for In parallel, and as demonstrated previously (Contreras et al., each), whereas in cocultures, this proportion reached to 2002; Retamal et al., 2006, 2007), EthBr uptake was taken as an 88.4 0.1 and 11.six two.six (n 3 for each), respectively (Fig. index of the activity of Cx hemichannels in astrocytes. Under 1a14). control situations, in the presence of external calcium, only a couple of The SL/DT approach was used to quantify the gap junctioncells from highly enriched astrocyte cultures (pure cultures) exmediated intercellular diffusion of LY in astrocytes. Under conhibited prominent EthBr uptake (18.3 4.4 EthBr cells/field; n trol circumstances, pure astrocyte cultures showed a fluorescent area ten) (Fig. 1a9) and 24 h therapy with LPS (10 ng/ml) PAK3 Compound increased of 48.3 2.5 AU (a representative approach to quantify the dye couby around twofold the number of cells that present EthBr pling) (Fig. 1a5,b, white bar). Twenty four hours just after LPS (ten uptake (84 10 ; n three; p 0.01) (Fig. 1a10,c). Addition of ng/ml), addition to astrocyte cultures dye coupling was lowered MG on confluent astrocyte cultures and cocultured for 48 h did 24 2 (n three; p 0.01) (Fig. 1a6,b). In MG-astrocyte coculnot induce dye uptake boost in astrocytes (five 1 high over tures, under control conditions (resting MG), no substantial manage worth; n 3, n.s.) (Fig. 1a11,c). On the other hand, remedy with adjustments in dye coupling have been detected (7 4 reduction; n 3; LPS (ten ng/ml) for 24 h substantially enhanced EthBr uptake in p 0.05) (Fig. 1a7,b), compared with pure astrocyte cultures. astrocytes cocultured with MG (439 20) (n 3; p However, 24 h soon after the addition of LPS (ten ng/ml) to these 0.001) (Fig. 1a12,c). cocultures, the dye transfer was considerably decreased by 56 7 (n three; p 0.001) compared with untreated cocultures (Fig. To determine irrespective of whether the detected astrocyte permeabiliza-Retamal et al. C.
