UVM Theses and Dissertations
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Print
Author:
Roberts, Jane Anstee
Dept./Program:
Neuroscience Graduate Program
Year:
2013
Degree:
Ph. D.
Abstract:
Inflammatory Bowel Disease (IBD) is a common and devastating disorder that affects the lives of hundreds of thousands of Americans. Most morbidity in IBD is related to altered gut function, but the underlying pathophysiology is not well understood, and as a result, treatment options are limited. We have previously demonstrated that colitis is associated with a number of alterations in the neural circuitry of the colon, and a recently identified change that could contribute to colonic dysmotility is altered neuromuscular transmission. Specifically, there is a decrease in the strength ofthe purinergic inhibitory junction potential (IJP). Therefore, the overall objectives of this dissertation were to identify the mechanisms responsible for disrupted purinergic transmission, and to determine whether these changes can be prevented.
We first tested the hypothesis that colitis is associated with a decrease in the release of purines from nerve terminals. Previous studies in our lab indicate that the disruption in purinergic neuromuscular transmission does not involve a decrease in the density of inhibitory nerve fibers or interstitial cells of Cajal (ICC) in the muscle layers, or a change in the responsiveness of the muscle to purinergic receptor stimulation. Therefore, we tested whether the colitis-induced decrease in purinergic neuromuscular transmission is associated with a decrease in the release of purines from inhibitory motor nerve terminals. Using high-performance liquid chromatography (HPLC) we found that release of a variety of purines, including ATP, ADP and AMP, is decreased from colon muscularis preparations with colitis as compared to controls.
The second set of experiments in this dissertation tested the hypothesis that the decrease in purine release in the inflamed colon could involve a disruption of mitochondrial function by oxidative stress. Purines are synthesized by mitochondria and transported into synaptic vesicles, and it has been shown that oxidative stress leads to disruptive changes in mitochondrial function. We used histological approaches to show increased levels of oxidative stress in the muscularis of our colitis animals. Furthennore, in intracellular electrophysiological recording studies, we found that the IJP was reduced by disruption of ATP synthesis with ATP synthase inhibitors or by induction of oxidative stress, in vitro, via application of the free radical donor hydrogen peroxide in the normal colon. In other words, inhibition of ATP synthesis or induction of oxidative stress in normal tissue mimicked the effects of inflammation on the IJP.
Finally, we tested the hypothesis that the colitis-induced decrease in purinergic transmission can be prevented or rescued by treatment with free radical scavengers. If the increases in free radicals during inflammation contribute to altered neuromuscular transmission in colitis, treatments that would decrease free radical levels may have a protective effect on the purinergic IJP. In inflamed colon preparations from animals treated in vivo with the free radical scavenger Tempol, the IJP was comparable to that recorded in normal tissue and propulsive motility was improved.
The studies described in this dissertation elucidate the mechanisms responsible for reduced purinergic neuromuscular transmission that occurs in the guinea pig and murine colon using two different models of colitis. These findings open a new realm of potential therapeutic targets for treating dysmotility in patients with IBD, as well as other GI motility disorders with increased oxidative stress, including diabetes, obesity, and aging.
We first tested the hypothesis that colitis is associated with a decrease in the release of purines from nerve terminals. Previous studies in our lab indicate that the disruption in purinergic neuromuscular transmission does not involve a decrease in the density of inhibitory nerve fibers or interstitial cells of Cajal (ICC) in the muscle layers, or a change in the responsiveness of the muscle to purinergic receptor stimulation. Therefore, we tested whether the colitis-induced decrease in purinergic neuromuscular transmission is associated with a decrease in the release of purines from inhibitory motor nerve terminals. Using high-performance liquid chromatography (HPLC) we found that release of a variety of purines, including ATP, ADP and AMP, is decreased from colon muscularis preparations with colitis as compared to controls.
The second set of experiments in this dissertation tested the hypothesis that the decrease in purine release in the inflamed colon could involve a disruption of mitochondrial function by oxidative stress. Purines are synthesized by mitochondria and transported into synaptic vesicles, and it has been shown that oxidative stress leads to disruptive changes in mitochondrial function. We used histological approaches to show increased levels of oxidative stress in the muscularis of our colitis animals. Furthennore, in intracellular electrophysiological recording studies, we found that the IJP was reduced by disruption of ATP synthesis with ATP synthase inhibitors or by induction of oxidative stress, in vitro, via application of the free radical donor hydrogen peroxide in the normal colon. In other words, inhibition of ATP synthesis or induction of oxidative stress in normal tissue mimicked the effects of inflammation on the IJP.
Finally, we tested the hypothesis that the colitis-induced decrease in purinergic transmission can be prevented or rescued by treatment with free radical scavengers. If the increases in free radicals during inflammation contribute to altered neuromuscular transmission in colitis, treatments that would decrease free radical levels may have a protective effect on the purinergic IJP. In inflamed colon preparations from animals treated in vivo with the free radical scavenger Tempol, the IJP was comparable to that recorded in normal tissue and propulsive motility was improved.
The studies described in this dissertation elucidate the mechanisms responsible for reduced purinergic neuromuscular transmission that occurs in the guinea pig and murine colon using two different models of colitis. These findings open a new realm of potential therapeutic targets for treating dysmotility in patients with IBD, as well as other GI motility disorders with increased oxidative stress, including diabetes, obesity, and aging.