Ight junction disruption in intestinal mucosa, consistent with instances recently described in the literature [30,31]. To determine whether TLR expression on gut epithelial cells is one mechanism by which morphine modulates barrier function, we implanted mice with placebo or morphine pellets for 24 hours and isolated epithelial cells from the small intestines as described previously [25]. Total RNA was isolated from these cells and processed for qPCR. For flow cytometery, the isolated cells were gated by cytokeratin as an epithelial marker [32] (Figure 4A).Results showed 24 hours of morphine treatment upregulated both mRNA (Figure 4B) and protein levels (Figure 4C?F) of TLR2 and TLR4. In addition, the messenger RNA levels of TLR2 and TLR4 in colonic epithelial cells following morphine treatment was determined by gel-based PCR (Figure S5). The results showed that neither TLR2 nor TLR4 was significantly upregulated by morphine in the colonic epithelium in contrast to the observation in the small intestinal epithelium.Chronic morphine induces inflammation and disrupts organization of tight junction proteins Title Loaded From File between epithelial cells in small intestineTo investigate the effects of morphine on the morphology of the intestinal epithelium, small Xpressed the Ste2p in relatively low expression manner [13], our result intestine and colon were excised and fixed in a formalin solution for hematoxylin and eosin (H E) staining. Histological analysis showed injured epithelium and increased inflammatory infiltrates in small intestinal villi of morphine-treated mice (Figure 2). In contrast, no morphological change was observed in the colon of morphine-treated mice, suggesting a differential effect by morphine on small intestinal and colonic epithelium. Our findings of morphine-induced microbial translocation and barrier compromise in the small intestine of mice prompted us to study the tight-junction organization of the intestinal epithelium. Wild-type mice were implanted with placebo or 75 mg morphine pellet for 24 hours. Then parts of the small intestine were excised, frozen and 5 mm sections were cut. The sections were stained for occludin and zona occludens 1 (ZO-1), two proteins integral to the formation of epithelial tight-junction. In placebo treated mice, the trans-membrane protein occludin localized to the apical side of epithelium with a continuous and intact organization (Figure 3A). Images showed that occludin colocalized with the well-organized F-actin on the membrane of epithelial cells of placebo-treated mice (Figure 3A). In contrast, morphine treated mice showed disrupted localization of occludin, suggesting impaired recruitment of the protein to the membrane (Figure 3A). Similar to occludin, the paracellular tight junction protein ZO-1 also localized with F-actin on 23977191 the apical side of the membrane in placebo-treated mice, and its organization was seen to be disrupted following 24 hours of morphine treatment (Figure 3C). Morphine treatment did not change the expression levels of occludin or ZO-1 (Figure S2), suggesting that morphine modulated the distribution of tight junction proteins, resulting in increased intestinal permeability. Quantification of yellow fluorescence (indicating the co-localization of red and green) also showed significant reduction in the co-localization of tight junction and Factin in morphine-treated mice (Figure 3B and D). In MORKO mice, consistent with our bacterial translocation data, morphine did not have any effect on occludin and ZO-1 organization in the small intestine, indicat.Ight junction disruption in intestinal mucosa, consistent with instances recently described in the literature [30,31]. To determine whether TLR expression on gut epithelial cells is one mechanism by which morphine modulates barrier function, we implanted mice with placebo or morphine pellets for 24 hours and isolated epithelial cells from the small intestines as described previously [25]. Total RNA was isolated from these cells and processed for qPCR. For flow cytometery, the isolated cells were gated by cytokeratin as an epithelial marker [32] (Figure 4A).Results showed 24 hours of morphine treatment upregulated both mRNA (Figure 4B) and protein levels (Figure 4C?F) of TLR2 and TLR4. In addition, the messenger RNA levels of TLR2 and TLR4 in colonic epithelial cells following morphine treatment was determined by gel-based PCR (Figure S5). The results showed that neither TLR2 nor TLR4 was significantly upregulated by morphine in the colonic epithelium in contrast to the observation in the small intestinal epithelium.Chronic morphine induces inflammation and disrupts organization of tight junction proteins between epithelial cells in small intestineTo investigate the effects of morphine on the morphology of the intestinal epithelium, small intestine and colon were excised and fixed in a formalin solution for hematoxylin and eosin (H E) staining. Histological analysis showed injured epithelium and increased inflammatory infiltrates in small intestinal villi of morphine-treated mice (Figure 2). In contrast, no morphological change was observed in the colon of morphine-treated mice, suggesting a differential effect by morphine on small intestinal and colonic epithelium. Our findings of morphine-induced microbial translocation and barrier compromise in the small intestine of mice prompted us to study the tight-junction organization of the intestinal epithelium. Wild-type mice were implanted with placebo or 75 mg morphine pellet for 24 hours. Then parts of the small intestine were excised, frozen and 5 mm sections were cut. The sections were stained for occludin and zona occludens 1 (ZO-1), two proteins integral to the formation of epithelial tight-junction. In placebo treated mice, the trans-membrane protein occludin localized to the apical side of epithelium with a continuous and intact organization (Figure 3A). Images showed that occludin colocalized with the well-organized F-actin on the membrane of epithelial cells of placebo-treated mice (Figure 3A). In contrast, morphine treated mice showed disrupted localization of occludin, suggesting impaired recruitment of the protein to the membrane (Figure 3A). Similar to occludin, the paracellular tight junction protein ZO-1 also localized with F-actin on 23977191 the apical side of the membrane in placebo-treated mice, and its organization was seen to be disrupted following 24 hours of morphine treatment (Figure 3C). Morphine treatment did not change the expression levels of occludin or ZO-1 (Figure S2), suggesting that morphine modulated the distribution of tight junction proteins, resulting in increased intestinal permeability. Quantification of yellow fluorescence (indicating the co-localization of red and green) also showed significant reduction in the co-localization of tight junction and Factin in morphine-treated mice (Figure 3B and D). In MORKO mice, consistent with our bacterial translocation data, morphine did not have any effect on occludin and ZO-1 organization in the small intestine, indicat.
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