Gastrointestinal stasis, as manifested clinically by paralytic ileus, is one of the more common manifestations of traumatic injury. Inflammation, infection, and the need for analgesics such as morphine for pain control and sedation during assisted ventilation further aggravate and prolong the inability of the gut to move nutrients down its lumen. As a consequence, severely injured and critically ill patients often receive total parenteral nutrition and enteral deprivation. Although the exact reasons are not known, it is generally accepted that ileus, the lack of enteral nutrition, and the presence of parenteral nutrition have a negative impact on survival. The long-term goal of this research is to test the HYPOTHESIS that nitric oxide production by both constitutive and inducible isoforms of nitric oxide synthase is altered in conditions in which ileus occurs, and the altered levels of NO produced interfere with the fluid propelling ability of the intestine.
The SPECIFIC AIMS for testing this hypothesis are to: 1) evaluate changes in expression of NOS isoforms in specific regions of the small intestine in response to selected procedures known to cause a reduction in intestinal transit, 2) determine a cause-effect relationship between the altered expression of NOS isoforms and the decreased intestinal transit, 3) determine the alterations in propulsive forces that may be responsible for the altered intestinal transit, and 4) determine the effects of prolonged reduced smooth muscle contractility on the contractile proteins that effect contraction. Rats and mice will be instrumented with catheters for measurement of intestinal transit. Segments of intestine will be taken and subjected to sensitive assays for the production of NO and for the detection of NOS isoforms. The sites of production of NO will be localized by separation of gut layers prior to homogenization, and by immunohistochemistry and in situ hybridization. NOS isoform specific antagonists will be used and mice that are NOS II and/or NOS III deficient will be employed in order to determine the functional roles played by each of the NOS isoforms. Such reagents and techniques will be employed in protocols in which transit will be altered at various times after inducing ischemia/reperfusion injury by temporarily occluding the superior mesenteric artery. A propulsion evaluation system will be used to characterize changes in propulsive forces in intestine isolated from animals that have been treated with LPS or have undergone ischemia/.reperfusion, the same procedures used in the in vivo studies. Derangements in muscle will be assessed by evaluating contractility, changes in contractile protein isoforms, and the nitration of key proteins involved in the excitation-contraction process. Data from these experiments may provide a rational for developing ways to prevent, ameliorate, and/or reverse the ileus that often accompanies traumatic injury and the procedures used to support trauma patients. Hopefully, results from these experiments will be translatable to the study and care of patients as described in project one of this proposal.
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