title: Refinement of tourniquet-induced peripheral ischemia/reperfusion injury in rats: comparison of 2 h vs 24 h reperfusion.
abstract: Prolonged ischemia of skeletal muscle tissue, followed by reperfusion, leads to ischemia/reperfusion injury (IRI), which is a feared local and systemic inflammatory reaction. With respect to the 3Rs, we wanted to determine which parameters for assessment of IRI require a reperfusion time of 24 h and for which 2 h of reperfusion are sufficient. Rats were subjected to 3 h of hind limb ischemia and 2 h or 24 h of reperfusion. Human plasma derived C1 inhibitor was used as a drug to prevent reperfusion injury. For 2 h of reperfusion the rats stayed under anesthesia throughout (severity grade 1), whereas for 24 h they were awake under analgesia during reperfusion (grade 2). The femoral artery was clamped and a tourniquet was placed, under maintenance of venous return. C1 esterase inhibitor was systemically administered 5 min before the induction of ischemia. No differences in local muscle edema formation and depositions of immunoglobulin G and immunoglobulin M were observed between 2 h and 24 h (P > 0.05), whereas lung edema was only observed after 24 h. Muscle viability was significantly lower after 24 h vs 2 h reperfusion (P < 0.05). Increased plasma creatine kinase (CK)-MM and platelet-derived growth factor (PDGF)-bb could be detected after 2 h, but not after 24 h of reperfusion. By contrast, depositions of C3b/c and fibrin in muscle were only detected after 24 h (P < 0.001). In conclusion, for a first screening of drug candidates to reduce IRI, 2 h reperfusions are sufficient, and these reduce the severity of the animal experiment. Twenty-four-hour reperfusions are only needed for in-depth analysis of the mechanisms of IRI, including lung damage.
authors.affiliation: Department of Clinical Research, University of Bern, Switzerland.
authors.forename: C
authors.initials: C
authors.lastname: Duehrkop
authors.forename: R
authors.initials: R
authors.lastname: Rieben
mesh_terms: D000827:Animal Welfare; D000818:Animals; D050718:Complement C1 Inhibitor Protein / Q000494:pharmacology; D051056:Complement Inactivating Agents / Q000494:pharmacology; D004195:Disease Models, Animal; D005263:Femoral Artery / Q000473:pathology; D006614:Hindlimb / Q000293:injuries*; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D008297:Male; D010146:Pain / Q000517:prevention & control; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000209:etiology / Q000503:physiopathology / Q000517:prevention & control*; D013995:Time; D014111:Tourniquets / Q000009:adverse effects
chemical_list: D050718:Complement C1 Inhibitor Protein; D051056:Complement Inactivating Agents
keywords: C1 inhibitor; ischemia/reperfusion injury; rat; refinement; tourniquet