Intravitalmikroskopie mit in-vivo intra-pancreatico-duktaler ROS Bestimmung — eine neue Methode zur Abschätzung des Hypoxie-/Ischämie/Reperfusionschadens am Pankreas der Ratte

Abstract

Background: Ischemia/reperfusion injury (IRI) remains to be one of the key factors determining graft survival after pancreas transplantation. Beside several mechanisms, reactive oxygen species (ROS) are considered to be decisive factors in this pathophysiology as well. Until now no in vivo data are available concerning release of ROS from blood or pancreatic dialysates as an organ specific parameter in correlation to microcirculatory parameters. Material and Methods: Male Sprague-Dawley-Rats (n = 5) were anaesthetized. The pancreatic duct was cannulated with a microdialysis probe and ligated. According to continuous infusion from spin label CMH (1-hydroxy-3-methoxycarbonyl- 2,2,5,5-tetramethyl-pyrrolidine), pancreas was arranged for intravital epifluorescence-microscopy. After a stabilization period of 50 min. (I), hypovolamic shock (II) was induced for 50 min by taking arterial blood. Finally resuscitation (III) was introduced by re-substitution of blood. ROS in blood and pancreatic-duct dialysate as well as intravital microscopy parameters were quantified [leucocyteendothelium interaction (LEI), functional capillary density (FCD)]. Result: As expected, stabilization period [I, MAP (mmHg) 91 ± 9] compared to hypovolamic shock period [II, MAP 40 ± 3] demonstrates a significant deteriorated microcirculation [FCD (cm/cm2) 360 ± 13 vs. 258 ± 12; LEI (cells/mm2) 147 ± 23 vs. 310 ± 39]. While free radicals of the pancreatic-duct dialysate rise clearly during shock phase II compared to stabilization phase [208 % ± 134 compared to I], in peripheral blood ROS is only easily increased [108 % ± 23 compared to I]. A recovery of physiological blood pressure conditions [MAP 83 ± 15] leads both in blood and in dialysate to a rise of the radical activity compared to the phase II [120 % ± 49 and 291 % ± 294]. An in vitro analysis of blood results in an increased radicalactive potential [543AU ± 101 vs. 431AU ± 80] during the shock phase compared to the control phase, which decreases again after resuscitation [448AU ± 81]. Conclusion: By the example of a proven experimental shock model in combination with intravital-microscopy and our new method of ROS-detection we demonstrate for the first time that a significantly deteriorated microcirculation after pathological stimulus directly correlates with a rise of free radicals both in blood and dialysate of panreatic-duct. This method probably saves further experimental potential in order to get a more complex understanding of pathophysiological procedures in acute pancreatitis