Nociceptive and Barrier changes in Mouse intestines after Parasitic Infection

• Main Authors: Hsiu-Wei Wu, 吳秀薇
• Other Authors: Chia-Hui Yu
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/06939394873428160421

碩士 === 國立臺灣大學 === 生理學研究所 === 101 === Backgrounds and Aims: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by chronic abdominal pain and altered bowel habits without macroscopic abnormality and presence of pathogen. The pathogenesis of IBS is still unclear and most studies are based on patient samples while animal models are not widely recognized. Symptoms of IBS may begin following stressful events or infectious gastroenteritis termed post-infectious (PI) IBS. A recent study in Norway showed that 40-80 % of patients developed IBS symptoms after a parasite Giardia infection. Previous animal studies have shown that psychological stress increases abdominal pain and intestinal barrier disruption. The aim is to establish a IBS model using post-giardiasis combined with water avoidance stress (WAS) to evaluate abdominal pain and gut barrier function. Materials and Methods: Mice were inoculated with Giardia lamblia trophozoites, and the trophozoites in the small intestine were enumerated. The number of colonized Giardia peaked on day 4-7 which was termed “colonization phase”; the parasites were cleared by day 14, and therefore, day 21-49 was denoted “post-clearance phase”. Further water avoidance stress (WAS) or nonhandled (NH) protocols were performed post-giardiasis on PI day 35. Gut barrier function and visceral hypersensitivity are experimented in “colonization V phase” and “post-clearance phase”. Besides, pain-related molecules expression in intestines and dorsal root ganglia (DRG) are analyzed. Results: Intestinal colonization of Giardia trophozoites peaked on day 4-7 and increased macromolecular permeability and tight junction occludin cleavage was seen along with commensal bacterial overgrowth. During the post-clearance phase, increased visceral motor response (VMR) was observed after low-volume colorectal distension, suggesting that post-infection alone triggers allodynia. On the other hand, increased VMR by high-volume distension was noted after WAS, indicating stress alone caused hyperalgesia. Dual factors caused synergistic effects of hyperalgesia and allodynia. Moreover, small intestinal tight junction disruption and increased epithelial permeability were seen in post-giardiasis with or without stress and augmented by dual factors of post-infection and stress. Fluorescence in situ hybridization and gentamycin resistance assay revealed the presence of bacteria in lamina propria and increased endocytosed bacteria in mucosal cells in mice post-giardiasis, which was potentiated by stress. Furthermore, the expression of pain-related molecules including CCK, CCK-1R (AR), CCK-2R (BR) and mast cell tryptase in intestine and dorsal root ganglia were analyzed by Western blotting. In both single and dual factors treated mice, we found increased CCK expression in large intestine and increased CCK-1R VI expression in small intestine, while post-giardiasis augmented the stress effect. Intestinal CCK-2R levels did not change among group. The levels of mast cell tryptase increased in both small and large intestine in post-giardiasis plus stress. The CCK, CCK-1R and CCK-2R levels in dorsal root ganglia were comparable among groups. Conclusion: Post-infection combined with stress caused visceral hypersensitivity, intestinal barrier dysfunction and commensal bacteria invasion. Intestinal CCK, CCK-1R and mast cell tryptase may be involved in the mechanisms of the IBS-like pathology.