Fig. 4
From: β-adrenergic signaling triggers enteric glial reactivity and acute enteric gliosis during surgery
NE triggers acute primary enteric gliosis and glial reactivity via β-adrenergic receptors. A Schematic of primary EGC cultures from Sox10iCreERT2/Rpl22HA/+/Ai14fl/fl mice. B ELISA analysis for IL-6 (mean ± SEM) from conditioned medium of cultured EGCs after stimulation with vehicle (PBS) or NE (3 h; 10 µM, 100 µM) (n = 8 distinct cell culture wells per condition; multiple unpaired t-tests, comparison to vehicle, *** < 0.001). C qPCR analysis (mean ± SEM) of Sox10iCreERT2/Rpl22HA/+ RiboTag mRNA and RNA from cultured primary EGCs for different adrenergic receptors (2−ΔΔCT, Tubb4/Actb/PGK/GAPDH, n = 5 distinct cell culture wells per condition). D ELISA analysis for IL-6 and CCL2 (mean ± SEM) from conditioned medium of cultured primary EGCs after stimulation (3 h) with vehicle (PBS), adrenergic agonists (against: α2a, β3, pan-β (isoprenaline)), and NE (all 10 µM); CCL2: n = 6 distinct cell culture wells per condition; IL-6: n = 9–21 distinct cell culture wells per condition; multiple unpaired t-tests, comparison to vehicle, *** < 0.001). E qPCR analysis (mean ± SEM) of cultured primary EGCs for acute gliosis genes after vehicle (PBS), isoprenaline (100 µM), or NE (100 µM) treatment (2−ΔΔCT, 18S, Naïve; n = 4–7 distinct cell culture wells per condition; multiple unpaired t-tests, comparison to vehicle, * < 0.05). F Immunohistochemistry of cryo-embedded intestinal specimens stained for GFAP+ enteric glia (magenta) and ADRβ1 (green) in the ME; Hoechst was used to detect cell nuclei (white). Arrows indicate double-positive cells. Scale bar (50 µm). G Western blot and corresponding densitometry (mean ± SEM) of lysates of primary EGC cultures treated with vehicle, isoprenaline (10 µM), or forskolin (10 µM) stained for phosphorylated cAMP-dependent protein kinase (pPKA) (multiple bands) and β-actin (~ 42 kDa) as loading control (n = 4 cultures from 4 different animals treated with the compounds or vehicle; representative blot shows three technical replicates of one biological repeat per condition); multiple unpaired t-tests, comparison to vehicle, ** < 0.01)