A. Munoz-Abraham1, G. Torres-Valencia1, T. Alfadda1, C. Jasinski1, R. Patron-Lozano1, M. I. Rodriguez-Davalos1, J. P. Geibel1 1Yale University School Of Medicine,Surgery – Transplant,New Haven, CT, USA
Introduction: Intestinal ischemia remains a major limitation in successful intestinal transplantation. Several animal intestinal ischemia models have been developed and used. However, rats remain as the most commonly used. The fragile balance of the intestinal mucosa relies on stable homeostatic mechanisms. To further understand the disruption of these mechanisms during ischemia/reperfusion injury, we have developed a new method to objectively measure osmotic changes in the intestinal lumen of rats that equate to injury.
Material and
Methods: We used Sprague Dawley rats (401 to 442 grams).The rats were anesthetized and euthanized with isofluorane. 20 cm of distal ileum were taken and stripped from the mesentery. The intestinal lumen was flushed with regular HEPES solution (pH 7.408, mosm 297) to remove any remaining intestinal debris . The intestinal loops were then attached to two custom perfusion chambers that received a constant flow of regular HEPES solution. The chambers were submerged in a bath of deionized water at 37 C . At time 0, a known concentration of 3 ml of 0.001 mM FITC-Inulin was perfused into both lumens. Samples were collected and Relative Fluorescence Units (RFU) were measured using the NanoDrop 3300 Fluorospectrometer. The control intestine remained receiving a flow of regular HEPES, while the experimental intestine was bathed with a flowing solution of 10 µM Forskolin in a perfusion volume of 200 ml regular HEPES. Samples from the lumen were again collected from both intestines at times 25 and 35 minutes, and measured.
Results: A significant increase in luminal secretion was observed after the administration of the 10 µM Forskolin solution by observing the decrease in RFU units by almost half in the experimental intestine compared to the control.
Conclusion: It can be concluded that the use of FITC-Inulin can be an effective and objective method to measure fluid secretion or absorption in the small bowel, thus giving a more accurate estimate of the viability of the organ. By measuring the difference in RFU of a known solution after stop flow in the small bowel we demonstrated that absorption and secretion processes take place depending on the pathophysiological state of the organ. Absorption was observed when the intestine was maintained in physiological-like conditions, while secretion of large amounts of water into the lumen occurred when FSK was administered. In both cases the change in RFU using the method proposed gave accurate reproducible results with small sample variations demonstrating the usesfulness of this model system for assessing intestinal viability.