This component focuses on hemorrhagic shock (HS) and resuscitation (R)-induced derangement in intestinal mucosal barrier function. Two critical determinants of gut barrier function are the structural integrity of the epithelial sheet and the status of the tight junctions between adjacent enterocytes. Our hypothesis is that HS/R leads to the formation of reactive oxygen and nitrogen intermediates (ROIs and RNIs, respectively), which are capable of initiating structural and functional damage to the epithelium and thereby degrading intestinal barrier function.
Aim I is to assess the importance of gut-derived microbes or microbial products as agents that promote of amplify HS/R-induced inflammatory processes. Experiments will determine the response to HS/R in gnotobiotic mice as compared to conventional mice. The primary read-outs will consist of markers of inflammation to be studied throughout this proposal: namely up-regulation of pro-inflammatory transcription factors (e.g., NF-kappaB and NF-IL6) and genes (i.e., iNOS, COX-2, TNF, and IL-6) in the intestinal mucosa and muscularis propria and in the liver, a distant organ. Subsequent experiments will focus on the role of microbial products rather than viable organisms. For these studies, we will use gnotobiotic mice, but some animals will be enterally contaminated with various pro-inflammatory substances of microbial origin, including endotoxin, peptidoglycan, and formyl-methionyl-leucyl phenylalanine.
Aim II is to investigate the hypothesis that down- regulation of protein tyrosine phosphatase activity is an effector mechanism contributing to gut mucosal hyperpermeability caused by ROIs and RNIs generated during HS or HS/R. Experiments will be performed in a parallel fashion suing both cultured enterocytic monolayers and animals subjected to HS/R or sham HS/R, We will use both pharmacological and genetic approaches to manipulate protein tyrosine phosphorylation or the levels of ROIs and RNIs generated within the mucosa.
Aim III is to investigate the importance and regulation of intestinal epithelial apoptosis as a mechanism contributing to barrier dysfunction after HS/R. These studies will employ standard morphological and biochemical assays for apoptosis (e.g., TUNEL, caspase activation) as well as indices of gut barrier function (incidence of BT to MLNs and permeability to FITC-conjugated dextran). As in the previous aim, we will use both genetic and pharmacological approaches in these studies.
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