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Detection of free radicals in ischemic and reperfused canine gracilis muscle flaps by use of spin-trapping electron paramagnetic resonance spectroscopy

Brigitte A. Brisson DMV, DVSc1, Craig W. Miller DVM, MVSc2, Guoman Chen PhD3,4, L. Jill McCutcheon DVM, PhD5, and Edward G. Janzen PhD6
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  • 1 Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 2 Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 3 Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 4 Present address is Department of Botany, University of Toronto, Toronto, ON, Canada.
  • | 5 Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 6 Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.

Abstract

Objective—To determine whether free radicals are produced in ischemic and reperfused canine skeletal muscle, whether free radicals can be detected from effluent blood by use of spin-trapping electron paramagnetic resonance (EPR) spectroscopy, and whether free radical-induced skeletal muscle damage is detectable by use of light microscopy.

Animals—6 healthy mixed-breed dogs.

Procedures—Dogs were anesthetized and both gracilis muscles were isolated, leaving only the major vascular pedicle intact. Ischemia was induced in 1 flap for 4 hours; the contralateral flap served as the control. Ischemic flaps were then reperfused for 15 minutes. α-Phenyl-N-tert-butylnitrone, a spin-trapping agent, was administered intravenously to each dog 1 hour prior to reperfusion. Following reperfusion, effluent blood samples from muscle flaps were obtained and processed for EPR spectroscopy. Muscle biopsy specimens were obtained for histologic evaluation, and dogs were euthanatized.

Results—Spin adducts were not detected in blood from control flaps. However, spin adducts were detected in all ischemic-reperfused muscle flaps. Principal signals identified were characteristic of oxygen- and carbon-centered radicals. Significantly more muscle damage was detected in ischemic-reperfused flaps, compared with control flaps.

Conclusions and Clinical Relevance—Free radicals may be an important component of injury induced by ischemia and reperfusion of canine skeletal muscle. Spin-trap adducts of free radicals can be detected in effluent blood of canine muscle flaps by use of spin-trapping EPR spectroscopy. Spin-trapping EPR spectroscopy may be useful for the study of antioxidants and free radical scavengers in attenuating ischemia and reperfusionmediated skeletal muscle damage. (Am J Vet Res 2001;62:384–388)

Abstract

Objective—To determine whether free radicals are produced in ischemic and reperfused canine skeletal muscle, whether free radicals can be detected from effluent blood by use of spin-trapping electron paramagnetic resonance (EPR) spectroscopy, and whether free radical-induced skeletal muscle damage is detectable by use of light microscopy.

Animals—6 healthy mixed-breed dogs.

Procedures—Dogs were anesthetized and both gracilis muscles were isolated, leaving only the major vascular pedicle intact. Ischemia was induced in 1 flap for 4 hours; the contralateral flap served as the control. Ischemic flaps were then reperfused for 15 minutes. α-Phenyl-N-tert-butylnitrone, a spin-trapping agent, was administered intravenously to each dog 1 hour prior to reperfusion. Following reperfusion, effluent blood samples from muscle flaps were obtained and processed for EPR spectroscopy. Muscle biopsy specimens were obtained for histologic evaluation, and dogs were euthanatized.

Results—Spin adducts were not detected in blood from control flaps. However, spin adducts were detected in all ischemic-reperfused muscle flaps. Principal signals identified were characteristic of oxygen- and carbon-centered radicals. Significantly more muscle damage was detected in ischemic-reperfused flaps, compared with control flaps.

Conclusions and Clinical Relevance—Free radicals may be an important component of injury induced by ischemia and reperfusion of canine skeletal muscle. Spin-trap adducts of free radicals can be detected in effluent blood of canine muscle flaps by use of spin-trapping EPR spectroscopy. Spin-trapping EPR spectroscopy may be useful for the study of antioxidants and free radical scavengers in attenuating ischemia and reperfusionmediated skeletal muscle damage. (Am J Vet Res 2001;62:384–388)