B. S. Morocho1, N. A. Drucker1, J. P. Te Winkel1, M. J. Ferkowicz1, T. A. Markel1 1Indiana University School Of Medicine,Pediatric Surgery,Indianapolis, IN, USA
Introduction: Human umbilical mesenchymal stromal cells (USC) improve survival and mesenteric perfusion following intestinal ischemia. USCs likely act via the paracrine release of specific key mediators, of which IL-6 may serve an important role. We hypothesized that IL-6 would play a key role in stem cell mediated intestinal protection following ischemia and reperfusion (IR) injury.
Methods: Eight to ten week old C57BL/6J male mice were used: 1) IR + Vehicle, 2) IR + USC, 3) IR + Negative Control siRNA treated USC and 4) IR + IL-6 siRNA treated USC. Mice were anesthetized using isoflurane and a midline laparotomy was performed. Initial perfusion of the small intestine was imaged using laser Doppler imaging. The superior mesenteric artery was then occluded for 60 minute with an atraumatic vascular clamp. After ischemia, the clamp was removed and intestines allowed to recover. Immediately prior to closure, mice were injected intraperitoneally with either 250 ul phosphate buffered saline vehicle, or with USC transfected with siRNA as above. siRNA efficacy was confirmed by RT-PCR. After 24 hours of recovery, perfusion was reassessed, mice were euthanized, and the intestines were harvested for histological analysis. Tissue blocks were H&E stained and sectioned. Mucosal injury was assessed and graded by an established injury grading scale. Perfusion was expressed as percentage of baseline (mean+/-SEM) and analyzed by student’s t test. Histology was presented as injury score (median and IQR) and analyzed by Mann-Whitney. P<0.05 was significant.
Results: IL-6 siRNA effectively knocked down USC IL-6 production. USCs, as well as USCs transfected with a negative siRNA control significantly increased perfusion (A) and improved mucosal injury scores (B) compared to vehicle following injury (Perfusion: Vehicle 42.49+/-9.28%, USC 78.3+/-8.83, siRNA Negative Control: 77.8+/-4.37,p<0.05; Histology: Vehicle: 3(2.75), USC: 1(0.75), siRNA Negative control: 1(1),p<0.05. USCs transfected with IL-6 siRNA yielded significantly lower mesenteric perfusion and worse mucosal injury scores compared to negative control (Perfusion: siRNA Negative control: 77.8+/-4.37, IL-6 siRNA: 28.4+/-5.84,p<0.05; Histology: siRNA Negative control:1(1), IL-6 siRNA: 3.5(2.25),p<0.05).
Conclusion: USC mediated intestinal protection was negatively impacted by the decrease in IL-6 production from USCs. IL-6 likely acts in an anti-inflammatory manner, possibly through the membrane bound IL-6 receptor. Further studies are required to delineate the downstream effects of IL-6 during stem cell mediated intestinal protection.
