Effects of Systemic Erythropoietin on Ischemic Wound Healing in Rats

Results of in vivo studies have shown erythropoietin (EPO) is associated with anti-inflammatory, anti-apoptotic, and cell protective effects on wound healing. These effects are dose-dependent. The aim of this study was to evaluate whether the duration of EPO treatment affects the healing process in the ischemic wound.

  Forty-two (42) Sprague-Dawley rats were anesthetized, wounded with H-shaped flaps, and randomized to 2 groups; Group 1 received 400 u/kg/day EPO and Group 2 received a saline solution, both via intraperitoneal injection following the wounding. All substances were administered once daily at the same time for up to 10 days after surgery. At days 3, 5, and 10, 7 rats from each group were sacrificed. Skin samples were stained with hematoxylin/eosin, viewed under an optical microscope at 10X and 40X magnification, and analyzed by blinded investigators for re-epithelialization, neovascularization amount and maturation of granulation tissue, inflammatory cells, and ulcer healing using an evaluation scale where 0 = none; 1 = partial; 2 = complete, but immature/thin: and 4 = complete and mature. Blood hemoglobin and hematocrit levels also were measured. Data were analyzed using ANOVA one-way test (P <0.05). Hemoglobin and hematocrit levels rose while subsequent doses of EPO were administered over time, accompanied by a transient surge in healing on day 5, when differences in healing scores were significant. Flap necrosis, ulceration, and abscess were noted on post-wounding day 10 near the pedicle. The study showed EPO therapy can improve wound healing early in the post-wounding period but can reduce wound healing after post-injury treatment day 5. Further research is necessary, particularly to establish how EPO influences the microcirculation and rheology.

Wound healing is a complex reconstructive process that restores damaged tissues and involves cells, mediators, growth factors, and cytokines.¹ Many metabolic and systemic conditions, especially the impairment of tissue perfusion, can interfere with the healing process; inadequate blood perfusion may chronically delay wound healing. In a wound with inadequate tissue perfusion, the degree of the ischemia impacts the likelihood for infection and tissue necrosis.² Clinical and experimental studies and review articles^3-5 documenting the acceleration of wound healing with stem cell-derived growth factors recently have been published. Results of animal studies^6,7 provide evidence that angiogenesis is a primary target for therapeutic interventions to prevent necrosis in ischemic wounds.

  Erythropoietin (EPO) is a mediator that stimulates mitosis, angiogenesis, and erythropoiesis by elevating the plasma levels of some growth factors, including vascular endothelial growth factor (VEGF); stimulation of these factors has been found in experimental studies⁸ to promote wound healing. Experimental studies^9,10 also demonstrate the effects of systemic EPO on wound healing are dose-dependent (frequency of administration). Repetitive low doses but especially single high doses of EPO improve the process of wound healing.¹⁰ However, the effects of the duration of EPO treatment is unknown. The aim of this study is to evaluate the effect of EPO administration duration on the ischemic wound healing process at different time points of treatment after the injury.

Animals and experimental ischemic wound model. The study was reviewed and approved by the ethical committee of Istanbul University Cerrahpasa School of Medicine. All parts of the study were performed in the Experimental Animal Research and Breeding Laboratory, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey.

  Forty-two (42) male adult Sprague-Dawley rats (weight: 250–300 g) were used in the study. All animals were kept under optimal environmental conditions. The rats were fed freely with pellet foods and water. No prophylactic antibiotherapy was given. After general anesthesia with intramuscular 40–50 mg/kg ketamine hydrochloride (Ketalar, Pfizer, Turkey) and 10 mg/kg xylazine hydrochloride (Rompun, Bayer, Turkey), the surgical area was shaved and cleaned with povidone-iodine solution. The skin wounds of the rats were rendered ischemic by the method described by Quirinia et al.¹¹ This model consists of a cranially and caudally based flap 2 cm wide and 4 cm long marked on the dorsal skin. The skin and panniculus carnosus were incised using surgical scissors. After the flaps were raised, perforating branches of the flaps were cut and then sutured back in anatomical position with separate 4/0 silk sutures placed at 1-cm intervals (see Figure 1). The hypoxic and ischemic area was defined as the distal parts of the superior-based and inferior-based flaps.

  The animals were randomly assigned to 2 groups of 21 samples. Group 1 (the EPO group) was provided 400 U/kg/day recombinant human EPO (Janssen-Cilag, Switzerland). Group 2 (the control group) received a 0.9% NaCl solution. The dose and the type (human) of EPO were chosen in agreement with previous experimental data in rats showing that effects of EPO in skin wound healing are dose-related.¹⁰ All medication was administered via intraperitoneal injection (0.1 mL) once daily at the same time of day. Seven rats (7) in each group were sacrificed via decapitation after 3, 5, and 10 days, respectively, at which time skin and blood samples were taken.

  Histological evaluation. At each time point, the distal edges of the superior and inferior portion of the flaps were harvested and their histologic features were assessed by the same specialist (blinded to the treatment) in paraffin-embedded sections using hematoxylin/eosin stains under light microscopy at a magnification of 10X to 40X. Histological wound evaluation scoring included reepithelialization (0 = none; 1 = partial; 2 = complete, but immature/thin; 3 = complete and mature), neovascularization (0 = none; 1 = up to 5 vessels/high-powered magnification field [HMF]; 2 = 6–10 vessels/HMF; 3 = >10 vessels/HMF), amount of granulation tissue (0 = none; 1 = scant; 2 = moderate; 3 = abundant), maturation of granulation tissue (0 = none; 1 = partial; 2 = complete, but immature/thin; and 3 = complete and mature), and inflammatory cells (0 = none; 1 = scant; 2 = moderate; 3 = abundant). The scoring system was modified from Sevimli-Gür et al¹²; ulceration criteria (0 = wide and deep ulcers, abscesses; 1 = wide ulcers; 2 = none or very small; 3 = none) were added as the main histological evaluation criteria (see Table 1).

  Evaluation of hemoglobin and hematocrit levels. The blood samples taken from the rats after decapitation were kept in tubes containing ethylenediaminetetraacetic acid (EDTA, an anticoagulant) (15% K3 EDTA 0.054mL/4.5 mL blood), and hemoglobin and hematocrit levels were measured by an automatic device (ABL 700, Radiometer, Copenhagen, Denmark). Recombinant human EPO administration increased these variables.

  Statistical analysis. Mean wound healing scores (including reepithelialization, neovascularization, amount and maturation of granulation tissue, inflammatory cells, and ulcer evidence of healing) and the mean hemoglobin and hematocrit levels between groups at different time intervals were compared using one-way ANOVA, followed by Bonferroni post hoc tests. The results are expressed as mean ± SD. Data were analyzed using SPSS (Version 21.0, Chicago, IL, USA), and the P value was set at <0.05 for all analyses.

Histopathological results.

  Reepithelialization. Reepithelialization scores were significantly higher in Group 2 (1.80 ± 0.84) than Group 1 (1.00 ± 0.00) on day 3 (P <0.05), but on days 5 and 10, the results were similar in the 2 groups (Group 1: day 5 = 2.00 ± 1.55, day 10 = 1.50 ± 1.73; Group 2; day 5 = 1.00 ± 1.41, day 10 = 2.00 ± 0.89; P >0.05).

  Neovascularization. Neovascularization scores were not significantly higher in either group (Group 1: day 3 = 2.00 ± 0.00, day 5 = 1.33 ± 0.52, day 10 = 1.75 ± 0.50; Group 2: day 3 = 2.50 ± 1.00, day 5 = 1.57 ± 0.54, day 10 = 1.33 ± 0.52; P >0.05) (see Table 2).

  Granulation tissue. Granulation tissue scores of Group 1 on days 3 and 5 were different than Group 2 (Group 1: day 3 = 1.00 ± 0.00; day 5 = 2.67 ± 0.57; Group 2: day 3 = 1.75 ± 0.50; day 5 = 1.57 ± 0.79; P <0.05), but the scores score on day 10 were similar between groups (day 10: Group 1 = 2.25 ± 0.50, Group 2 = 2.00 ± 0.89; P >0.05). Granulation tissue maturation in the EPO group (Group 1) day 5 was significantly higher than Group 2 (Group 1: day 3 = 0.86 ± 0.38, day 5 = 2.50 ± 0.55, day 10 = 2.25 ± 0.50; Group 2: day 3 = 1.25 ± 0.96, day 5 = 1.67 ± 0.79, day 10 = 1.67 ± 0.52; P <0.05).

  Inflammation. The inflammatory cell scores were similar between both groups (Group 1: day 3 = 2.0 ± 0.00, day 5 = 2.50 ± 0.55, day 10 = 2.00 ± 1.41; Group 2: day 3 = 2.40 ± 0.89, day 5 = 1.29 ± 0.76, day 10 = 0.67 ± 1.03; P >0.05). In Group 2, the number of inflammatory cells was significantly higher on day 3 (2.40 ± 0.89) than day 10 (0.67 ± 1.03) (P <0.05).

  Ulcer scores. Ulcer scores were similar between the groups (Group 1: day 3 = 1.0 ± 0.00, day 5 = 2.67 ± 0.52, day 10 = 1.50 ± 1.00; Group 2: day 3 = 1.60 ± 1.14, day 5 = 1.29 ± 1.38, day 10 = 2.00 ± 0.89; P >0.05). In Group 1, the ulcer scores on day 5 were significantly higher than on days 3 and 10 (day 3 = 1.0 ± 0.00, day 5 = 2.67 ± 0.52, day 10 = 1.50 ± 1.00; P <0.05).

  Hemoglobin and hematocrit levels. In Group 1, the hemoglobin (day 3 = 14.27 ± 0.54, day 5 = 15.60 ± 0.92, day 10 = 17.18 ± 2.37; P <0.05) and hematocrit levels (day 3 = 43.77 ± 1.62, day 5 = 47.79 ± 2.77, day 10 = 52.48 ± 7.12; P <0.05) were significantly elevated. In Group 2, the hemoglobin (day 3 = 11.90 ± 2.25, day 5 = 12.33 ± 0.74, day 10 = 12.35 ± 1.62; P >0.05) and hematocrit levels (day 3 = 36.63 ± 6.80, day 5 = 37.97 ± 2.24, day 10 = 37.97 ± 4.89; P >0.05) were within acceptable ranges (see Figure 2).

  Two rats from Group 1 and 1 rat from Group 2 died due to infection and flap necrosis during the study.

 The present in vivo study on ischemic skin wound healing in rats shows EPO causes a decrease in the amount of granulation tissue and wound epithelialization on day 3 and accelerates the amount and maturation of granulation tissue and increased ulcer healing on the wound site at day 5.

  EPO is the main regulator of erythropoiesis but has been shown in experimental study¹³ to induce a variety of biological effects, including anti-apoptosis, anti-inflammation, and cell protection. The hematopoietic activities of EPO include the inhibition of apoptosis and induction of proliferation of erythroid precursor cells. These effects are mediated when EPO binds to EPO receptors (EPORs). According to an in situ study,¹³ EPORs are frequently located on erythroid progenitor cells in the bone marrow and nonerythropoietic receptors present in most tissue. EPO and EPORs have been attributed to antioxidative and anti-inflammatory effects in a variety of nonhematopoietic organs and tissue. In an in vitro model,¹⁵ EPO also has been shown to induce a proangiogenic effect on endothelial cells derived from human adult myocardial tissue.

  The effect of EPO on wound healing is varied. In an in vivo study, Haroon et al¹⁶ found EPO improved the formation of granulation tissue. This was associated with a significant dose-dependent, proangiogenic effect. In an experimental study, Sayan et al¹⁷ demonstrated better reepithelialization and a significantly increased wound breaking strength in the early phase (after 1 week) of wound healing; this was associated with a higher collagen deposition content. In the current study, EPO treatment resulted in better reepithelialization, an acceleration of ulcer healing, increased granulation tissue, and maturation of granulation tissue on day 5, although EPO treatment decreased reepithelialization and granulation tissue amount on day 3.

  In addition to the applied dose of EPO prewounding or postwounding of the initial treatment, the duration of the treatment had an effect on wound healing. Buemi et al⁹ investigated the effect of EPO treatment using the ischemic dorsal flap model in rats and demonstrated improved wound healing in the early (24 hours) and late stages (7 days) of injury. In the same model, Saray et al¹⁸ observed subcutaneous administration of different doses of EPO over a time period ranging from 3 weeks before to 1 week after injury was associated with improved flap survival; the authors showed a low dose (50 IU/kg) and an intermediate dose (100 IU/kg) of EPO improved flap survival. In an in vivo study, Harder and et al¹⁹ started 2 different regimens of EPO treatment (500 IU/kg and 5.000 IU/kg) beginning 1 day before ischemia and continuing until day 3 after ischemia was noted; researchers found EPO treatment administered before ischemia occurred provided myocutaneous tissue with a dose-dependent protection from apoptotic and necrotic cell death and decreased the inflammatory response. Seven days of systemic EPO treatment following ischemic injury had been found to provide a positive effect on wound healing and survival of flap tissue.⁹ In the current study, the best healing score results were noted on day 5. No statistically significant differences were noted between the 2 groups regarding neovascularization at any time point; this supports the idea EPO has a positive effect on wound healing because of its anti-apoptotic, anti-inflammatory, and cell-protective effects, rather than an effect on neovascularization.

  Hemoglobin and hematocrit levels increased in accordance with the EPO treatment. An in vivo study showed EPO not only promotes erythropoiesis, but also markedly enhances platelet and endothelial activation in humans. Whether heightened platelet reactivity and endothelial activation may increase the risk of thromboembolism warrants further research.¹⁹ This effects on blood rheology may impair microcirculation.²⁰ In the current 10-day study, the EPO treatment group had necrosis, ulceration, and abscess formation on the flap after the day 7.

  Two of the rats in the EPO group died due to infection and flap necrosis; the reason for necrosis could be increased hemoglobin and hematocrit levels, impairing microcirculation. Impaired microcirculatory rheology might have a negative effect on wound healing. EPO is both hematopoietic and tissue protective, putatively through interaction with different receptors. Carbamylated EPO (CEPO) or certain EPO mutants did not bind to the classical EPO receptor (EPOR) and did not show any hematopoietic activity in human cell signaling assays or upon chronic dosing in different animal species.²¹ The availability of nonerythropoietic derivatives of EPO such as CEPO that do not trigger (EPOR)² also opens possibilities to distinguish experimentally between EPO’s tissue-protective effects (eg, anti-apoptosis, cytoprotection, neuroprotection) and its potentially detrimental effects (eg, excessive erythropoiesis, thromboembolism, impairing microcirculation).²¹

The main limitation of this study is blood rheology and microcirculation were not measured to support the concluding statement that they were impaired. Also, 2 rats from Group 1 and one rat from Group 2 died during the study, decreasing the sample size and breaking the equality at time points for sets of data, potentially compromising the power of the study to detect statistically significant differences.

Ischemia is a major risk factor for delayed or nonhealing of wounds. The study showed systemic EPO therapy can improve ischemic wound healing early in the postoperative period but can delay healing after the fifth postoperative day. In ischemic wound treatment using EPO, it appears systemic, long-term use of EPO is not advised due to its negative effects. In the meantime, nonerythropoietic derivatives of EPO²¹ may be used more effectively for the anti-apoptosis, anti-inflammatory, and cell-protective effects on ischemic wound treatment. Further research is necessary, particularly to establish how EPO influences microcirculation and rheology.

Dr. M. Arslantas is a consultant, Department of Anesthesiology and Reanimation, Marmara University Pendik Education and Research Hospital, Istanbul, Turkey. Dr. R. Arslantas is a consultant, Department of Anesthesiology and Reanimation, Dr Lutfi Kirdar Kartal Education and Research Hospital, Istanbul, Turkey. Dr. Tozan is a Professor, Department of Anesthesiology and Pain Medicine, Istanbul University Capa Faculty of Medicine, Istanbul, Turkey. Please address correspondence to: Mustafa Kemal Arslantas, MD, Department of Anesthesiology and Reanimation, Marmara University Pendik Education and Research Hospital, Fevzi Cakmak Mah, Muhsin Yazicioglu Cad. No: 10 Ust Kaynarca, Pendik, Istanbul, Turkey; email: mkarslantas@gmail.com.