Activation of myenteric glia during acute inflammation in vitro

ABSTRACT: Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammator microenvironment. In this study we isolated GFAP-positive myenteric glia from FVB/hGFAP-eGFP transgenic postnatal day 7 mice. Following cell sorting for the eGFP reporter, GFAP-positive EGCs were cultured for 3 weeks to generate neurosphere-like bodies. This cell culture was stimulated with LPS for 48 h and cells were employed for gene expression profiling. LPS-stimulated cell cultures were compared to untreated control cell cultures. Enriched GFAP+ EGC cultures secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Further, in vitro cultures were compared to GFAP-eGFP-positive cells directly analyzed after cell sorting of small intestinal LMMP digests (in vivo) to assess alterations in transcriptomic profiles due to the in vitro culture. In vivo data and in vitro data were collected in three independent replicates. For each replicate one litter of FVB/hGFAP-eGFP transgenic mice at postnatal day 7 was employed. GFAP-eFP-positive small intestines were digested enzymatically and from the single cell suspensions eGFP-positive GFAP-expressing cells were sorted by fluorescence-activated cell sorting. For the in vivo data the cells were directly sorted into lysis buffer and further processed . For the in vitro data GFAP-eGFP cells were seeded onto coated plastic dishes for adherent growth and cultured in DMEM/F12-medium supplemented with antibiotics, N2, B27, bFGF and EGF. In the first passage cells were divided into two uncoated six well dishes to promote spheroid growth. One well was supplemented with LPS (100 µg/ml, from E. coli O26:B6, Sigma Aldrich, potency 3 EU/ng) for 48 h, the corresponding second well was left untreated and used as respective control. After 48 h, cells were processed for total RNA isolation.

ORGANISM(S): Mus musculus

SUBMITTER: Corinna Rosenbaum 

PROVIDER: E-GEOD-78015 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress