Erythropoietin protects critically perfused flap tissue.
OBJECTIVE
The objective of this study was to analyze whether erythropoietin (EPO) protects from necrosis of critically perfused musculocutaneous tissue and the mechanisms by which this protection is achieved.


BACKGROUND
EPO is the regulator of erythropoiesis and is used to treat patients with anemia of different causes. Recent studies suggest that EPO has also other tissue-protective effects, irrespective of its erythropoietic properties.


METHODS
C57BL/6-mice were treated with 3 doses of EPO at 500 IU/kg intraperitoneally. EPO was given either before (preconditioning, n = 7), before and after (overlapping treatment, n = 7), or after (treatment, n = 7) surgery. Animals receiving only saline served as controls (CON). Acute persistent ischemia was induced by elevating a randomly perfused flap in the back of the animals. This critically perfused tissue demonstrates an initial microvascular failure of approximately 40%, resulting in approximately 50% tissue necrosis if kept untreated. Repetitive fluorescence microscopy was performed over 10 days, assessing angiogenesis, functional capillary density, inflammatory leukocyte-endothelial cell interaction, apoptotic cell death, and tissue necrosis. Additional molecular tissue analyses included the determination of inducible nitric oxide synthase, erythropoietin receptor (EPO-R), and vascular endothelial growth factor (VEGF).


RESULTS
EPO preconditioning did not affect hematocrit and EPO-R expression, but increased inducible nitric oxide synthase in the critically perfused tissue. This correlated with a significant arteriolar dilation, which resulted in a maintained functional capillary density (CON: 0 +/- 0 cm/cm(2); preconditioning: 37 +/- 21 cm/cm(2); overlapping treatment: 72 +/- 26 cm/cm(2); P < 0.05). EPO pretreatment further significantly reduced microvascular leukocyte adhesion and apoptotic cell death. Moreover, EPO pretreatment induced an early VEGF upregulation, which resulted in new capillary network formation (CON: 0 +/- 0 cm/cm(2); preconditioning: 40 +/- 3 cm/cm(2); overlapping treatment: 33 +/- 3 cm/cm(2); P < 0.05). Accordingly, EPO pretreatment significantly reduced tissue necrosis (CON: 48% +/- 2%; preconditioning: 26% +/- 3%; overlapping treatment: 20% +/- 3%; P < 0.05). Of interest, EPO treatment was only able to alleviate ischemia-induced inflammation but could not improve microvascular perfusion and tissue survival.


CONCLUSIONS
EPO pretreatment improves survival of critically perfused tissue by nitric oxide -mediated arteriolar dilation, protection of capillary perfusion, and VEGF-initiated new blood vessel formation.
Rezaeian
Farid
F
Division of Plastic, Reconstructive and Aesthetic Surgery, Geneva University Hospitals, Geneva, Switzerland.
Wettstein
Reto
R
Amon
Michaela
M
Scheuer
Claudia
C
Schramm
René
R
Menger
Michael D
MD
Pittet
Brigitte
B
Harder
Yves
Y
D000818:Animals; D001160:Arterioles / Q000473:pathology; D015153:Blotting, Western; D002196:Capillaries / Q000502:physiology; D004108:Dilatation, Pathologic; D004921:Erythropoietin / Q000008:administration & dosage / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008833:Microcirculation / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods; D009336:Necrosis; D018919:Neovascularization, Physiologic / Q000187:drug effects; D052247:Nitric Oxide Synthase Type II / Q000502:physiology; D012039:Regional Blood Flow / Q000187:drug effects; D013524:Surgical Flaps / Q000098:blood supply* / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D042461:Vascular Endothelial Growth Factor A; D004921:Erythropoietin; D052247:Nitric Oxide Synthase Type II