A new method to measure intestinal secretion using fluorescein isothiocyanate-inulin in small bowel of rats.

BACKGROUND: Small intestine ischemia can be seen in various conditions such as intestinal transplantation. To further understand the pathologic disruption in ischemia-reperfusion injury, we have developed a method to measure fluid changes in the intestinal lumen of rats. METHODS: Two 10-cm rat intestine segments were procured, connected to the terminal apertures of a perfusion device, and continuously infused with 3 mL of HEPES solution (control solution) containing 50 µM of fluorescein isothiocyanate (FITC)-inulin. The perfusion device consists of concentric chambers that contain the perfused bowel segments, which are maintained at 37°C via H2O bath. The individual chamber has four apertures as follows: two fill and/or drain the surrounding HEPES solution on the blood side of the tissue. The others provide flow of HEPES solution containing FITC-inulin through the lumens. The experimental intestine was infused with the same solution with 100 µM of Forskolin. A pump continuously circulated solutions at 6 mL/min. Samples were collected at 15-min intervals until 150 min and were measured by the nanoflourospectrometer. RESULTS: A mean of 6-µM decrease in the FITC-inulin concentration in the Forskolin-treated experimental intestine was observed in comparison with that in the control intestine. The FITC-inulin count dilution in the experimental intestine is a result of an increase of fluid secretion produced by the effect of Forskolin, with P values <0.0001. CONCLUSIONS: We demonstrate that it is possible to measure luminal fluid changes over time using our new modified perfusion system along with FITC-inulin to allow real-time determinations of fluid and/or electrolyte movement along the small intestine.

Use of Fluorescein Isothiocyanate-Inulin as a Marker for Intestinal Ischemic Injury.

BACKGROUND: Intestinal ischemia is observed in conditions such as mesenteric ischemia, or during traumatic events such as intestinal transplantation. Intestinal ischemia leads to pathophysiologic disruptions that present as increased fluid secretion into the intestinal lumen. We propose a novel method to detect real-time ischemic injury that is used in an in vitro model applicable to intestinal transplantation. STUDY DESIGN: Small intestine segments from rats were procured. The segments were attached to customized perfusion chambers. Both intestines were perfused on the vascular side with a Ringer buffer solution. The experimental buffer solution was bubbled with 100% nitrogen to mimic ischemia. Both lumens were perfused with 3 mL HEPES-Ringer solution containing 50 µM fluorescein isothiocyanate (FITC)-inulin. Intraluminal samples were collected at 15-minute intervals to measure FITC-inulin concentration using a nanofluorospectrophotometer. Intestinal tissue samples were processed and evaluated by a blinded pathologist using the Park/Chiu scoring system for grading intestinal ischemia. RESULTS: Samples collected from the ischemic intestine showed a significant decrease in FITC-inulin fluorescence compared with the control intestine, indicating enhanced fluid secretion. Histopathologic samples from the experimental arm exhibited higher scores of ischemic injury in comparison with the control arm, confirming the FITC-inulin as a correlation to ischemia. CONCLUSIONS: Fluorescein isothiocyanate-inulin can be used as a real-time volume marker to monitor the ischemic state of intestinal tissue. A positive correlation between the degree of fluid shift and presence of ischemic injury. The changes in fluorescence signal provide a potential selective method to measure real-time fluid changes inside an intestinal graft to evaluate viability.

Extracorporeal Hypothermic Perfusion Device for Intestinal Graft Preservation to Decrease Ischemic Injury During Transportation.

INTRODUCTION: The small intestine is one of the most ischemia-sensitive organs used in transplantation. To better preserve the intestinal graft viability and decrease ischemia-reperfusion injury, a device for extracorporeal perfusion was developed. We present the results for the first series of perfused human intestine with an intestinal perfusion unit (IPU). METHODS: Five human intestines were procured for the protocol. (1) An experimental segment was perfused by the IPU delivering cold preservation solution to the vascular and luminal side continually at 4 ºC for 8 h. (2) Control (jejunum and ileum) segments were preserved in static cold preservation. Tissue samples were obtained for histopathologic grading according to the Park/Chiu scoring system (0 = normal, 8 = transmural infarction). RESULTS: Jejunal experimental segments scored 2.2 with the Park/Chiu system compared to the control segments, which averaged 3.2. Overall scoring for ileum experimental and control segments was equal with 1.6. CONCLUSION: This data presents proof of concept that extracorporeal intestinal perfusion is feasible. The evidence shows that the IPU can preserve the viability of human intestine, and histopathologic evaluation of perfused intestine is favorable. Our early results can eventually lead to expanding the possibilities of intestinal preservation.

A novel device to preserve intestinal tissue ex-vivo by cold peristaltic perfusion.

In the past two decades, much advancement has been made in the area of organ procurement and preservation for the transplant of kidneys, livers, and lungs. However, small intestine preservation remains unchanged. We propose a new preservation system for intestinal grafts that has the potential to increase the viability of the organ during transport. When experimented with porcine intestine, our device resulted in superior tissue quality than tissue in standard of care.