Unknown,Transcriptomics,Genomics,Proteomics

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Genes involved in cholestatic intestinal injury

ABSTRACT: In this study, we examined C57BL/6J and AJ mice who received either sham surgery or cholestatic intestinal injury. Mice were anesthetized by inhaled isoflurane anesthesia. The abdomen was clipped and then prepared in sterile fashion with 70% ethyl-ethanol followed by betadine. A transverse upper abdominal incision was performed. The CBD was dissected away from the portal vein and was ligated near its junction with the duodenum using aneurysm clips engineered with a precisely standardized opening/closing mechanism. The abdominal wall was then closed in a two-layer fashion using absorbable sutures. Sham-operated mice were treated identically but without dissection or ligation of the CBD. Male AJ and C57BL6J mice, 8 weeks of age, were randomly assigned to surgery or sham (n=3 microarrays per strain/condition).

ORGANISM(S): Mus musculus

SUBMITTER: Susan Dorsey

PROVIDER: E-GEOD-47099 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Gut dysbiosis and host genetics are implicated as causative factors in inflammatory bowel disease, yet mechanistic insights are lacking. Longitudinal analysis of ulcerative colitis patients following total colectomy with ileal anal anastomosis (IPAA) where >50% develop pouchitis, offers a unique setting to examine cause vs. effect. To recapitulate human IPAA, we employed a mouse model of surgically-created blind self-filling (SFL) and self-emptying (SEL) ileal loops. SFL exhibit fecal stasis due to directional peristalsis motility oriented towards away from the loop end, whereas SEL remain empty. In wild type mice, SFL, but not SEL, develop pouch-like microbial communities without accompanying active inflammation. However, in genetically susceptible IL-10-/- deficient mice, SFL, but not SEL, exhibit severe inflammation and mucosal transcriptomes resembling human pouchitis. Germ-free IL10-/- mice conventionalized with wild type SFL, but not SEL, microbiota, develop severe colitis. These data demonstrate an essential role for fecal stasis, gut dysbiosis, and genetic susceptibility and offer insights into human pouchitis and ulcerative colitis. All animal protocols were approved by IACUC at the University of Chicago. All animals were C57Bl/6 mice that were bred and housed under standard 12:12 light/dark conditions at the University of Chicago. Female mice aged 6-8 weeks were fed ad libitum gel diet 76A (Cat# 72-07-5022, Clear H20, Portland, ME) for 5-days prior to surgery to prevent obstruction at the anastomosis. Animals were anesthetized with ketamine/xylazine. Aseptic surgery was performed to resect 2.5cm of ileum 3cm proximal to the ileal-cecal value with anastomosis to the ileum using 8-0 suture (Figure 1a). The abdominal wall was closed with interrupted 4-0 silk suture and skin was closed with staples. Analgesics (betanorphine mg/kg BW) were provided post-operatively. After 5 weeks, mice were humanely euthanized. Intestinal loops were collected for RNA, protein, and histology. Loop, sham ileum, and sham colon contents were collected and snap frozen at −80°C for microbiota analysis. Human biopsies and stool samples were obtained under IRB approval and privacy protocols were followed. Our initial work demonstrated up regulation of TLR4 signaling in the mucosa of self-filling ileal loops. We hypothesized that TLR4 may be in-part responsible for mediating the metaplasia and inflammatory responses observed. Therefore, TLR4 KO mice were used to test this hypothesis and subsequently demonstrated attenuated responses in these parameters.

Project description:Astrogliosis is a hallmark of the response to brain ischemia, comprised of changes in gene expression and morphology. Hsp72 protects from cerebral ischemia, and although several mechanisms of protection have been investigated, effects on astrocyte activation are unknown. To identify potential mechanisms of protection, gene expression was assessed in mice subjected to middle cerebral artery (MCAO) or sham surgery, of either wildtype (WT) or Hsp72-overexpressing (Hsp72Tg) mice. After stroke, both genotypes exhibited genes related to cell death, stress response, and immune response. Furthermore, genes indicative of astrocyte activation, including cytoskeletal proteins and cytokines, were upregulated. To measure astrocyte activation after stroke, detailed histological and morphological analyses were performed in the cortical penumbra after stroke using unbiased stereology. Consistent with other reports, we observed a marked and persistent increase in glial fibrillary acidic protein (GFAP ) as soon as 3 hours after MCAO. In contrast, vimentin immunoreactivity appeared 12-24 hours after stroke, peaked at 72 hours, and returned to baseline after 30 days. Surprisingly, no change in overall astrocyte number was observed based on glutamine synthetase (GS) immunoreactivity. To determine if Hsp72Tg mice exhibited altered astrocyte activation compared to WT controls, morphological evaluation by fractal analysis was used. Overexpression of Hsp72 reduced astrocyte cell area, arbor area, and to a lesser extent fractal dimension, 72 hours following stroke. In conclusion, in vivo overexpression of Hsp72 alters gene expression following stroke, including genes involved in astrocyte activation, and decreases astrocyte activation acutely following MCAO. Thus, modulation of astrogliosis may be a neuroprotective mechanism exerted by Hsp72 after ischemia. A total of 10 samples were analyzed, with 5 of each genotype, wildtype (WT) and Hsp72-overexpressing (Hsp72Tg) mice. Of the 5 in each group, 3 received middle cerebral artery occlusion (MCAO) and 2 received a sham surgery. The sham samples serve as the controls for the MCAO samples in each genotype. All samples were taken from the ischemic or control hemisphere 24 hours after surgery.

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Genes involved in cholestatic intestinal injury

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