FIGURE 5.

Cultured cortical astroglia isolated from VPA animals showed an inflammatory profile that was revealed in vivo at PND35. (A) Mixed cortical glial cultures were prepared from postnatal day (PND) 3 control and VPA rats to obtain astroglial-enriched cultures, which were processed [day in vitro (DIV) 2] for flow cytometry, RT-PCR, and immunofluorescence. Siblings were behaviorally evaluated. At PND3 and 35, immunofluorescence (IF) was performed on brains from control and VPA animals. (B) The PFC of control and VPA rats immunostained for GFAP at PND3 and 35. At PND3, the immunostaining pattern corresponded to radial glia and at PND35 to astrogliosis in VPA animals. (C) Quantification at PND35 confirmed an increase in GFAP relative immunoreactive area in the PFC of VPA animals. (D) Cortical astrocytes in culture isolated from VPA animals showed an increase in cell size and internal complexity when analyzed by flow cytometry. (E) Representation of astrocyte morphology in culture immunostained for GFAP and its corresponding categorization. Astrocytes isolated from VPA animals displayed a reduction in the proportion of polygonal cells with a concomitant increase in stellate astrocytes. (F) Astrocytes isolated from VPA animals showed increased expression of IL1β and IL6 but lowered TNFα RNA levels. (G) The expression of Drosha and Dicer was diminished in astrocytes isolated from VPA rats. Results are expressed as mean values (±SD). (C) Control, n = 5 animals; VPA, n = 6 animals. (D) A representative experiment. (E) Control, n = 47 photomicrographs; VPA, n = 47 photomicrographs from 3 independent cultures. (F,G): 4 independent cultures, **p < 0.01, ***p < 0.001 between groups by Student’s t-test and ns: non-significant; #p < 0.05; ##p < 0.01 between groups by Mann–Whitney U test. Scale bar: 50 µm.