Adverse Effect of Sildenafil on Healing Ischemic Wounds: Results of an In vivo Study

Phosphodiesterase-5 is an enzyme that inactivates cyclic guanosine monophosphate and regulates the balance of nitric oxide (NO). NO is an important molecule synthesized during wound repair. An in vivo study was conducted to evaluate the effect of sildenafil, known to have a role in regulating the effect of NO in perfusion, on the wound healing process under ischemic conditions in rats. Reepithelialization, neovascularization, inflammatory cells, and amount and maturation of granulation tissue were scored on a scale of 0–3 (none, partial, complete but immature/thin, complete and mature, respectively). Data were analyzed using ANOVA one-way test, with statistical significance determined at P <0.05. Forty-two (42) Sprague-Dawley rats were anesthetized, wounded with H-shaped flaps, and randomized into 2 groups: 1 group received 10 mg/kg sildenafil (dissolved in 1 mL distilled water) orally via orogastric tubes and the other group received a 0.9% NaCl solution via intraperitoneal injection (0.1 mL). On days 3, 5, and 10, 7 rats from each group were sacrificed. Blinded investigators analyzed skin samples for the wound healing evaluating criteria using from hematoxylin/eosin staining under an optical microscope at 10X and 40X magnification. Histopathological analysis showed sildenafil significantly reduced reepithelialization, neovascularization, amount of granulation tissue, and number of inflammatory cells on day 3 and increased inflammatory cells on day 10 (P <0.05). Further research is needed to clarify the potential role of oral or topically applied different doses of sildenafil for ischemic wound healing as well to evaluate its safety and efficacy when administered alone or in combination with other therapies.

Wound healing is a complex reconstructive process to restore damaged tissues that involves soluble mediators, blood cells, extracellular matrix, and parenchymal cells.¹ Stadelmann et al’s² review of impediments to wound healing found insufficient perfusion may cause a wound to become chronic depending on the degree of ischemia. Recently, experimental research studies have been focused on different effects of sildenafil, a phosphodiesterase inhibitor known for its effects on erectile dysfunction, on the wound healing process.³ Phosphodiesterase-5 (PDE5) is an enzyme that has been shown in in vivo studies⁴ to inactivate cyclic guanosine monophosphate (cGMP) and regulate the balance of nitric oxide (NO).⁴ The increase of NO has a positive effect on wound healing with regard to angiogenesis, inflammation, cell proliferation, matrix deposition, and remodeling; however, high levels of NO may be responsible for the increase of apoptosis in cells due to free-radical damage and increased levels of tumor necrosis factor (TNF) in ischemic conditions.⁴

A controlled, single-blinded, in vivo study was conducted to create an ischemic wound healing animal model and explore the possible mechanism and effects of sildenafil on the wound healing process under ischemic conditions in rats.

Animals and experimental ischemic wound model. The study design was approved by the ethical comity of Istanbul University Cerrahpasa School of Medicine (IUCSM), and the study was performed in the animal research laboratory of the IUCSM.

Forty-two (42) male adult Sprague-Dawley rats (250–300 g) were used in the study. All animals were kept under standard environmental conditions. 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 using the method described by Quirinia et al.⁵ This model, consisting of a cranially based and a caudally based flap 2 cm wide by 4 cm long, is marked on the dorsal skin. The cutaneous blood flow in flaps initially decreases to ischemic level but subsequently gradually increases, reaching the flow of intact skin and a normally healing incisional wound. The flaps were sutured back in position with 4/0 silk sutures placed at 1-cm intervals (see Figure 1). The hypoxic and ischemic areas were located at the distal part of the superior and inferior based flaps. The animals were randomly assigned to 2 groups of 21 samples each. Group 1 received 10 mg/kg sildenafil (Viagra®, Pfizer, Istanbul, Turkey), dissolved in 1 mL distilled water, orally via an orogastric tube. Group 2 received a 0.9% NaCl solution via intraperitoneal injection (0.1 mL). All medication was administered daily at the same time. Seven rats in each group were killed by decapitation method on day 3, day 5, and day 10. The distal edges of the superior and inferior portion of the flaps were taken for histological evaluation and were fixed in 10% formalin.

Wound evaluation scale. 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 blinded expert in paraffin-embedded sections using hematoxylin & eosin stains under light microscopy at a magnification of 10X–40X. The histological evaluation was made according to the wound healing evaluation scale. Reepithelialization, neovascularization, inflammatory cells, and amount and maturation of granulation tissue were scored on a scale of 0–3 (none, partial, complete but immature/thin, complete and mature) as shown in Table 1. The scoring system was modified from the one used by Sevimli-Gür et al.⁶

Statistical analysis. The means of the wound healing evaluation scores were analyzed by ANOVA one-way test using the SPSS statistical software (Version 21.0, Chicago, IL, USA). Bonferroni post hoc test was performed for multiple comparisons when appropriate. Data were presented as mean ± SD. Values of P <0.05 were considered statistically significant. 

Two rats from Group 1 and 1 rat from Group 2 died due to wound abscess and sepsis during the study. Reepithelialization was more accelerated in Group 2 than Group 1 on day 3 and day 10 (Group 1 day 3: 0.00 ± 0.00; day 10: 0.00 ± 0.00); Group 2 (day 3: 1.80 ± 0.37; day 10: 2.00 ± 0.89); P <0.05). The neovascularization scores of Group 2 on day 3 were higher than Group 1 (Group 1 day 3: 1.43 ± 0.53; Group 2 day 3: 2.50 ± 1.00; P <0.05). The granulation tissue amount in Group 2 on day 3 was higher than Group 1 (Group 1 day 3: 0.86 ± 0.38; Group 2 day 3: 1.75 ± 0.50; P <0.05). The inflammatory cell scores of Group 1 (1.00 ± 0.00; P <0.05) were observed to be the lowest on day 3, but these scores were increased day 5 and day 10. Group 2 had the highest inflammatory cell scores (2.40 ± 0.89; P <0.05) on day 3, but these scores decreased on day 10 (0.69 ± 1.03; P <0.05). The differences in the other wound healing scores evaluating time points between groups were not statistically significantly different (see Table 2). 

The present in vivo study on ischemic skin wound healing in rats showed sildenafil reduced reepithelialization, neovascularization, and amount of granulation tissue and inflammatory cells on day 3; increased inflammatory cells on day 10; and reduced reepithelialization at the wound site on day 10.

An organism responds to damage to tissue integrity by reestablishing homeostasis, which includes inflammatory, proliferative, and remodeling phases.⁷ After the first insult, the inflammatory phase provides hemostasis by vasoconstriction and thrombus formation. Platelets adhere to the damaged tissue and secrete coagulation factors and growth factors that are in the platelet alpha-granules. Vasoconstriction is followed by vasodilatation, where growth factors have chemotactic effects on macrophages and neutrophils.⁸ Macrophages and neutrophils try to move bacteria and necrotic tissue away from the injury.⁹ Substances such as NO are rapidly transmitted to the tissue, directly affect vascular cells, and play an active role in inflammation and other wound healing phases.¹⁰

NO is synthesized from arginine by NO synthase (NOS). NOS has 3 genetically different isoforms: eNOS (endothelial NO synthase) is produced in small amounts and facilitates the vascular tonus; nNOS (neuronal-type NO synthase) provides synaptic signaling events and neurotransmission; and iNOS (inducible NO synthase) is produced in large amounts and plays a role in immune/inflammatory processes and cellular immune response.¹¹ In all acute events (eg, trauma, stress, acute inflammation), the secreted iNOS may provide either a protective or a harmful effect.¹² These harmful effects are caused by the antioxidative effects of NO that inactivate superoxide and produce radicals such as peroxynitrite.¹³ The harmful effects of NO, which is produced in high concentration by biological systems, are actualized by 3 mechanisms.¹⁴ In the first mechanism, NO transfers into the cells like oxygen because it has an electron that is not shared, adheres to transmission metals such as iron that exist in protein structure, and releases free iron to the environment. In the second mechanism, by oxidation NO forms N-nitroso compound and N2O3 (N-nitroso compounds [NOC] including 2 chemical classes, nitrosamines, and nitrosamides, which are formed by the reaction of amines and amides, respectively, with nitrosating agents derived from nitrite) that are harmful to DNA. Lastly, NO reacts with oxygen radicals and produces DNA, proteins, and peroxynitrite, which oxidize membrane lipids.¹⁴

Sildenafil increases the concentration of NO in the environment by inhibiting PDE5; subsequently, the NO concentration in the medium increases. Effects on wound healing in ischemic tissue may be visualized both as protective or damaging to tissue depending on the increase in NO concentration.¹⁵ The different results (improved survival of skin flaps versus decreased viability of skin flaps) in recent animal studies¹⁶ are due to the methodological differences such as route of administration (oral, topical, intraperitoneal), drug dosage (3 mg/kg to 20 mg/kg), and duration of sildenafil treatment (1–21 days). In the current study, an ischemic wound healing model with insufficient perfusion was employed to worsen the wound healing conditions; the authors aimed to observe the already known vasodilation effect of sildenafil on inflammation and proliferation phases of wound healing.

Sarıfakıoğlu et al¹⁷ pioneered a study of the efficacy of sildenafil on wound healing in rats (n = 32) with caudally based skin flaps. The authors found sildenafil administered orally for 7 days at doses of 3, 10, and 20 mg caused a dose-dependent increase in wound healing. Also, sildenafil has been used by different routes and at different doses and durations in various preclinical studies. The results of these studies support the beneficial effects of sildenafil in the early phase of wound healing in various conditions. However, studies in which medications were administered for >7 days demonstrate this beneficial effect disappears and harmful effects are encountered. Hart et al¹⁸ investigated the short-term and long-term effect of intraperitoneal sildenafil on cutaneous flap survival in rats (N = 109). They observed the group receiving sildenafil had lower necrosis and stasis of the flap on day 1 and day 3 as compared to the control group, but this beneficial effect disappeared after day 5. This beneficial effect in the first days may be due to the greater vasodilator effect of sildenafil when applied at the early stages of postoperative period. With this finding, Hart et al suggested sildenafil alone is not sufficient for long-term maintenance of flap viability.

Tsai et al19 conducted a study with sildenafil and vascular endothelium growth factor (VGEF) in rats (N = 123), but this combination therapy revealed no statistically significant benefit as compared to sildenafil alone. This failure may be attributed to the insufficient VGEF dosage. Further studies are needed to assess the most effective amount of VGEF dosage for inducing angiogenic effect. Also, slow-release VGEF formulations for successful late-stage survival may be more useful than a single-shot administration that causes a rapid and short effect; they are yet to be developed.

The presence of excessive NO caused by sildenafil in ischemic tissues may result in free oxygen radical formation and increased apoptosis. In an in vivo study involving rats, Uzun et al20 showed the amount of NO in the anastomosis region after sildenafil application was lower on day 3 in ischemic anastomosis as compared to normal anastomosis. NO level on day 7 was higher in the ischemic group. The authors suggest the effects of sildenafil on normal and ischemic anastomosis may depend on the phase of the inflammation. This scenario also may be valid for ischemic wounds. In the current study, the sildenafil group, according to the duration of medication, developed necrosis and abscess of the flap. Two rats from the group that received sildenafil for 10 days died on day 8 because of sepsis. The increase in inflammatory cells of the wound site on day 10 in the sildenafil group is believed to be due to abscess formation in the wound and sepsis. The excessive secretion of NO into the wound environment may be a significant factor in toxic shock formation and may be associated with increased TNF levels.

This study is limited in that microcirculation and the tissue concentration of NO to support the conclusion they were impaired were not measured. In addition, different routes of drug usage were employed: the study group received sildenafil via the enteral route, and the control group received 0.9% NaCl via the intraperitoneal route. In order to generalize the current results, experimental and clinical studies are needed to define effective and safe dose, route of administration, and treatment duration with sildenafil for ischemic wound healing.

An in vivo study on ischemic skin wound healing in rats showed sildenafil reduces reepithelialization, neovascularization, and the amount of granulation tissue and inflammatory cells on day 3 of administration, increases inflammatory cells on day 10, and reduces reepithelialization at the wound site on day 10. Further research is needed to evaluate the safety, efficacy, and usefulness of oral or topically applied different doses of sildenafil alone or when combined with other therapies on ischemic wound healing. 

Dr. R Arslantaş is a consultant, Department of Anesthesiology and Reanimation, Dr. Lutfi Kirdar Kartal Education and Research Hospital, Istanbul, Turkey. Dr. MK Arslantaş is a consultant, Marmara University Pendik Education and Research Hospital Department of Anesthesiology and Reanimation, Istanbul, Turkey. Please address correspondence to: Mustafa K. Arslantaş, MD, Department of Anesthesiology and Reanimation, Marmara University Pendik Education and Research Hospital, Yahya Kemal Beyatlı Caddesi, No: 11/12 İstanbul Pendik 34912 Istanbul, Turkey; email: mkarslantas@hotmail.com.