Date of Award

January 2017

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Guadalupe Garcia-Tsao

Abstract

We hypothesized that the oral administration of obeticholic acid vs. placebo in a rat model of cirrhosis with ascites would decrease gut bacterial translocation and lead to changes in the intestinal microbiome, specifically the number and type of bacteria.

Male Harlan Sprague-Dawley rats weighing 100-125g underwent the induction of cirrhosis via inhalation of carbon tetrachloride (CCl4) three times a week until the development of ascites. Animals with ascites were randomized to placebo (methylcellulose solution) or obeticholic acid, administered by orogastric tube at a dose of 5mg/kg/day for 14 days. Stool samples were collected at weeks 1, 2, 4, and 7; at the development of ascites; 1 week into treatment; and at the time of sacrifice. At the end of the 14-day treatment period, animals underwent non-survival surgery with general anesthesia (isoflurane open-drop, followed by ketamine (75mg/kg)/xylazine (5mg/kg) intramuscularly) and strict aseptic conditions. Ascites fluid, blood, and mesenteric lymph nodes were cultured to assess for bacterial growth, a sign of bacterial translocation. Samples from the intestine and colon underwent 16S amplification and sequencing to assess the microbiome. The liver was fixed for histopathological analysis.

A first group of 20 rats was used to establish the experimental model of cirrhosis. These animals completed the induction process with 20-22 weeks of exposure to carbon tetrachloride by inhalation per Dr. Iwakiri’s lab, Lopez-Novoa1 and Garcia-Tsao et al2. However, the dose of CCl4 administered was too low to yield meaningful ascites; the rats consistently gained weight and did not exhibit any of the physical or behavioral hallmarks of animals with decompensated cirrhosis. Timepoint stool microbiome studies were completed on these animals, showing decreases in some beneficial species, such as Bacteroides, but not others, such as Lactobacillus and Bifidobacterium; and insignificant changes in Proteobacteria. After some troubleshooting, we pursued a more aggressive approach to exposing the animals to carbon tetrachloride by inhalation, involving exposing them to enough gas to anesthetize them starting week 4 of the induction process. At the time of this writing, 11 of 40 animals had developed ascites, 6 were undergoing treatment, and 1 had undergone laparotomy and sample collection.

We have so far established a more efficient way of inducing cirrhosis with ascites compared to the one recommended by Dr. Iwakiri’s lab. The induction of cirrhosis by carbon tetrachloride inhalation in rats requires escalating doses of the gas to reach the amount necessary to cause narcosis, in order to produce ascites. The effect of obeticholic acid on bacterial translocation has yet to be completed.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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