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 two et al., 2006), we studied whether or not LPS- GJC or hemichannels, respectively. Experiments were performed with similar culture models and therapies as described above. activated MG may also affect the activity Scale bar, one hundred m. b, c, Graphs corresponding towards the quantification of LY diffusion through astrocytic gap junctions (b) and the of Cx43 hemichannels. For this objective, EthBr uptake (c) either in enriched astrocyte cultures (Astrocytes) or in astrocyte-MG cocultures ( Microglia). Both culture EthBr uptake was investigated in astro- models were treated (LPS), or not (C, control), with LPS (ten ng/ml) for 24 h. Each plotted number corresponds for the imply SEM cytes cocultured with MG. Immuno- of three independent experiments. p 0.01, p 0.001; n.s., no considerable distinction CETP Inhibitor web compared with manage astrocyte staining with astrocyte and MG markers group; p 0.001, compared together with the astrocyte-LPS group. (GFAP and isolectin B4, respectively) in1a8,b). This reduction was also statistically substantial compared dicated that key cultures of astrocytes were composed by using the weak reduction induced by LPS in astrocytes cultures 98.4 0.1 and 1.6 0.1 GFAP-positive (red cells) and alone ( p 0.001). isolectin B4-positive (green cells) cells, respectively (n 3 for In parallel, and as demonstrated previously (Contreras et al., every), 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 2.six (n three for each and every), respectively (Fig. index of your activity of Cx hemichannels in astrocytes. Under 1a14). control situations, inside the presence of external calcium, only a handful of The SL/DT method was made use of to quantify the gap junctioncells from very enriched astrocyte cultures (pure cultures) exmediated intercellular diffusion of LY in astrocytes. Beneath conhibited prominent EthBr uptake (18.3 four.four EthBr cells/field; n trol PKCĪ± Biological Activity conditions, pure astrocyte cultures showed a fluorescent location ten) (Fig. 1a9) and 24 h therapy with LPS (10 ng/ml) elevated of 48.3 2.5 AU (a representative method to quantify the dye couby about twofold the number of cells that present EthBr pling) (Fig. 1a5,b, white bar). Twenty four hours right after LPS (10 uptake (84 10 ; n 3; 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 enhance in astrocytes (five 1 high more than tures, below manage circumstances (resting MG), no important manage value; n 3, n.s.) (Fig. 1a11,c). Having said that, therapy with alterations in dye coupling were detected (7 4 reduction; n 3; LPS (10 ng/ml) for 24 h significantly increased EthBr uptake in p 0.05) (Fig. 1a7,b), compared with pure astrocyte cultures. astrocytes cocultured with MG (439 20) (n three; p Even so, 24 h right after the addition of LPS (10 ng/ml) to these 0.001) (Fig. 1a12,c). cocultures, the dye transfer was substantially decreased by 56 7 (n 3; p 0.001) compared with untreated cocultures (Fig. To decide irrespective of whether the detected astrocyte permeabiliza-Retamal et al. C.