E- and L-selectin adhesion molecules in musculocutaneous flap reperfusion injury.
Definition of the elements governing leukocyte adhesion to the microvascular endothelium may lead to new forms of treatment for reperfusion injury. The objectives of this study, employing a porcine latissimus dorsi flap reperfusion model, were (1) to characterize the expression of E- and L-selectin adhesion molecules and (2) to test for a possible benefit of E- and L-selectin blockade in the preceding experimental setting. In experiment 1, full-thickness biopsies were collected sequentially over an 8-hour ischemia and subsequent 6-hour reperfusion period. Immunocytochemistry was performed with monoclonal antibody EL-246, an antibody that crossreacts with both E- and L-selectin. In experiment 2, the binding of EL-246 to L-selectin on circulating porcine neutrophils was determined by flow cytometric analysis. In experiment 3, in situ hybridization was performed using complementary RNA probes for detection of endothelial E-selectin mRNA. In experiment 4, bilateral flaps were elevated in six pigs and subjected to 8 hours of arterial ischemia followed by 20 hours of reperfusion. Flaps on each animal were randomly assigned to receive either treatment with a continuous local intraarterial infusion of EL-246 (1 mg per flap) or solvent vehicle. Muscle and skin survivals were assessed by nitroblue tetrazolium and intravenous fluorescein staining techniques, respectively. Computer digitization permitted quantitation of relative tissue survival. In experiment 1, specific immunostaining of microvascular endothelium was achieved using EL-246. Greater-intensity staining was detected in reperfusion than in baseline or ischemic sections. In experiment 2, flow cytometric analysis indicated specific recognition by EL-246 of isolated peripheral porcine neutrophils (> 45 percent staining) as compared with an isotype-matched control antibody (< 3 percent staining). In experiment 3, in situ hybridization studies demonstrated an early ischemic up-regulation and later reperfusion downregulation of E-selectin mRNA during the reperfusion period. In experiment 4, administration of monoclonal antibody EL-246 afforded a significant augmentation in mean percentage survival of muscle (37.6 versus 18.7 percent, p = 0.015) and skin (48.6 versus 29.3 percent, p = 0.046). In conclusion, it was determined that E-selectin is expressed along the microvascular surface and is upregulated and subsequently downregulated during ischemia-reperfusion conditions. The monoclonal antibody EL-246 appears to recognize porcine L-selectin as well as E-selectin. Blockade of E/L-selectin-mediated leukocyte adhesion significantly reduces musculocutaneous flap reperfusion injury.
Stotland
M A
MA
Microsurgical Laboratories, Royal Victoria Hospital, Montreal Quebec, Canada.
Kerrigan
C L
CL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D002448:Cell Adhesion; D004396:Coloring Agents; D015536:Down-Regulation; D019040:E-Selectin / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D004730:Endothelium, Vascular / Q000473:pathology / Q000503:physiopathology; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015870:Gene Expression; D006085:Graft Survival / Q000187:drug effects; D007091:Image Processing, Computer-Assisted; D007074:Immunoglobulin G / Q000627:therapeutic use; D017403:In Situ Hybridization; D007511:Ischemia / Q000503:physiopathology; D019041:L-Selectin / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D007962:Leukocytes / Q000473:pathology / Q000502:physiology; D008833:Microcirculation; D018482:Muscle, Skeletal / Q000098:blood supply / Q000637:transplantation*; D009504:Neutrophils / Q000473:pathology / Q000502:physiology; D009580:Nitroblue Tetrazolium; D014677:Pharmaceutical Vehicles; D012333:RNA, Messenger / Q000032:analysis; D011897:Random Allocation; D015427:Reperfusion Injury / Q000209:etiology* / Q000503:physiopathology / Q000517:prevention & control; D016038:Skin Transplantation / Q000473:pathology*; D012997:Solvents; D013524:Surgical Flaps / Q000473:pathology*; D013552:Swine; D015854:Up-Regulation
D000911:Antibodies, Monoclonal; D004396:Coloring Agents; D019040:E-Selectin; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D014677:Pharmaceutical Vehicles; D012333:RNA, Messenger; D012997:Solvents; D019041:L-Selectin; D009580:Nitroblue Tetrazolium