Preoperative subcutaneous sildenafil injection increases random flap survival in rats

Purpose: To investigate the effect of subcutaneous sildenafil on random flap survival. Methods: Fourteen Wistar rats, which were divided in to two groups, were used for this experimental study. Rats in the sildenafil group received subcutaneous sildenafil injections daily for seven days before flap elevation. At the end of the treatment period, 9x3 cm dorsal skin flaps were elevated and reinserted back into their place in all of the animals. Necrotic and whole flaps areas were recorded on graph papers. Seven days after the flap elevation samples for histological examination were taken and angiographies were performed to visualize the flap vascularization. Results: The calculated average percentage of necrotic flap areas were 18.29% and 42.26% in the sildenafil and control group respectively.(p=0.0233). In selected angiography images, vessels were found to be more prominent in the sildenafil group. The average number of capillary formations under light microscopy was higher in the sildenafil group (p= 0.0286). Conclusion: The subdermal high dose sildenafil has a positive effect on flap survival.

animals.Povidone-iodine solution was used for skin preparation.Isoflurane (Adeka Ilac ® , Turkey) was used for analgesia.
The animals were divided into two groups with seven animals in each: Group 1 (Sildenafil group): 5 mg/kg of sildenafil in 5 ml of saline solution was divided equally and injected from two points under the proposed flap areas for seven days daily before the flap elevation (Figure 1).Isoflurane inhalation anesthesia was used during the injection.
Group 2 (Control group): These animals were kept under the same conditions with the others without any pretreatment.At the end of the first week caudally based dorsal skin flaps 9x3 cm in size were raised and reinserted back into their place in all of the animals.The anatomical border of the flap was approximately from the scapula at the cephalic border to the posterior iliac crest at the caudal border (Figure 2).The flaps were sutured with polypropylene sutures.After the operation, daily photographs of the flaps were taken with a digital camera (Sony Nex-3N).The photographs at the postoperative seventh day were taken from a standard focus distance

■ Introduction
Random flaps are valuable tools in reconstructive surgery practice.Distal flap necrosis is commonly encountered in random flap surgery which can be caused by various anatomical or hemodynamic factors 1 .Various techniques and pharmacological agents have been studied to increase random flap survival [2][3][4] .Many of these studies have also been adapted to wound healing and tissue perfusion applications.
Sildenafil is a drug which inhibits Phosphodiesterase type 5 (PDE5) enzyme.This inhibition leads to an increase in the intracellular cGMP level which causes dilatation of blood vessels and relaxation of smooth muscles.PDE 5 inhibitors are approved for the treatment of pulmonary hypertension 5 and erectile dysfunction 6 .Various side effects are associated with the systemic use of PDE5 inhibitors including low acute myocardial infarction [7][8][9] , headache, flushing, nasal congestion, hearing loss and optic neuropathy 10 .
Various studies have been made to investigate the effect of PDE5 inhibitors on flap survival [11][12][13][14][15][16] .These studies include oral and intraperitoneal administration of sildenafil.Considering the systemic side effects of sildenafil, in this study we examined the efficiency of subcutaneous sildenafil injection prior to surgery on random flap survival.

■ Methods
The study was performed in the Bezmialem Vakif University Animal Research Laboratory with ethical comity approval.
Fourteen female 300-gram Wistar rats were used in the study.Surgical anesthesia was performed with Diethyl ether induction and intraperitoneal administration of 90 mg/ kg Ketamine (Pfizer ® , New York, USA) and 10 mg/kg Xylazine (Bayer ® , Germany).An electric shaver was used to shave the back skin of with milimetric graph papers and the necrotic/ whole flap areas were calculated using the VistaMetrix software (Version 1.35.0,Skillcrest LLC; USA) (Figure 3) 17,18 .Percentage of necrotic flap areas in each group were compared using Mann-Whitney test with Prism 7 software (La Jolla California USA).Seven days after the flap elevation an incision on the abdominal wall was made to expose aorta and inferior vena cava for angiographic study.Abdominal aorta and inferior vena cava were cannulated with a 22-gauge and 20-gauge cannula respectively.High dose intravenous Pentothal Sodium (Abbott ® , Turkey) was used to sacrifice the rats at this point when the collection of samples was performed from 3 cm distal to the base of the flaps for histological examination.250 ml of saline solution was administrated through aorta cannula to wash vascular bed %40 Barium sulfate (40 ml) (Yeni Ilac ® , Turkey) and 10 ml of %10 gelatin were combined, heated and mixed in a solution of 100 ml serum saline.The final solution was administrated into the circulation from the aorta.Both cannulas were then closed.The whole body of the animal was kept in -18ºC cooler overnight.The day following the operation the dorsal flap area was excised from the bodies of the animals.The flaps were stored in a refrigerator for another night at +4ºC, and following day any remaining irrelevant tissues such as muscles and fascia were removed from the flap and fixed on a hard paper board.The flaps were taken into a mammography device for angiographic imaging (GE mammography machine) 19 (Figure 4).The histological samples they were stained with hematoxylin and eosin.The samples were examined under light microscopy.The number of capillaries and inflammatory cells in the dermis were counted in samples from both groups in three different high-power fields by the same pathologist.The mean of three separate counts are calculated and statistical analysis was performed using Mann-Whitney test with Prism 7 software (La Jolla California USA).

Flap area calculation results
The calculated average percentages of necrotic flap areas were 18.29% and 42.26% in the sildenafil and control group respectively (p=0.0233) (Figure 5).

Evaluation of angiography images
Flaps were fixed on hard paper boards to flatten the flap surface.The angiography images from the mammography device were analyzed on high magnification in the computer for the identification of vessel structures.Vessels formations were marked in all of the magnified images in each group.Statistical analysis was not performed but subjective evaluation of the selected images in the sildenafil group revealed more prominent vessel formations when compared with the control group (Figure 6).

Evaluation of histological images
The number of inflammatory cells were very low in both groups and differences among the groups were insignificant.The average number of capillary formations was were 6.75±0.95 and 4.75±0.5 in the sildenafil and control group respectively (p=0.0286) (Figures 7 and 8).

■ Discussion
Various studies have been conducted to investigate the effects of PDE5 inhibitors on flap survival.Barral et al. 20 and Ayyildiz et al. 21have investigated the effect of subdermal sildenafil injection on flap survival.These studies have a similar design to our study.But in these study sildenafil injection were administrated after the flap elevation.We believe that pretreatment of the flap will yield to more consistent and efficient results to increase the flap survival rate but further comparative studied would be required to prove this hypothesis.Ayyildiz et al. 21have investigated the effect of subdermal injection of 0.5 mg/kg sildenafil on flap survival.This study has showed the positive effects of subdermal sildenafil.We believe that the 6x2 cm flap dimension chosen in this study might have interfered with the results.Barral et al. 20 has shown that 0.5 mg/kg can have a negative effect on flap survival which completely contradicts with the previous results.We have administrated much higher dosage of 5 mg/ kg sildenafil in this study when compared with previous studies in an attempt to augment the effects of this drug.Hart et al. 11 has previously reported even higher dosage of intraperitoneal 9 mg/kg of sildenafil in a previous study.
In our study, the subdermal route of administration was preferred.We have hypothesized that this route would be more efficient to increase flap survival.In previous studies, other routes of sildenafil administration have resulted in confusing results on its effect on flap survival.Kaya et al. 12 have shown that oral administration of sildenafil, tadalafil, and vardenafil have not led to a meaningful increase in the flap survival rate.Sarifakioglu et al. 14 have also reported a similar result with oral sildenafil.On the other hand, Hart et al. 11 have found that intraperitoneal injection of sildenafil could have a positive increase in flap survival on the early postoperative period.
Serious side effects including hypotension, myocardial infarcts, and optic neuropathies have been reported with use of systemic PDE5 inhibitors.In this study, we believe that the use of subdermal injection method compared to other administration methods can reduce the risk of adverse effects reported with PDE5 inhibitor.This approach can allow the administration of higher doses of sildenafil with minimal systemic effects.
In this current study, we have found a considerable increase in flap survival with subdermal sildenafil.Although the angiography results supported these findings, it should be noted that angiography evaluation was subjective and obtaining clear images was highly demanding.To overcome this shortcoming further histological analysis was performed.The results from histological evaluation revealed increased number of capillary formations in the sildenafil group under light microscopy.These findings does further the support flap survival and angiography results.
In this study we preferred the use of a control group without any pretreatment instead of a sham group with saline injection.We hypothesized that the effect of the trauma of the injection could have caused a different amount of increase in the amount of necrosis in each animal in the control group which could been harder to evaluate.The other reason being was to obtain better angiography images in the control groups.

■ Conclusions
The subdermal sildenafil prior to surgery has a positive increase in flap survival.The underlying mechanism under this finding has to be further investigated.Studies involving Doppler investigation rather than angiographies might give better insight to the change in the blood flow.Other effects of a decrease in the cGMP concentrations such alterations in ion channels might have other impacts on microcirculation and flap survival.

Figure 1 -
Figure 1 -Sildenafil injection points are marked under the proposed flap areas.

Figure 3 -
Figure 3 -Sample photographs of seventh-day flap areas.A. Control group; B. Sildenafil group; C. Calculation of necrotic and whole flap areas from graph papers.

Figure 6 -
Figure 6 -Selected angiography images from each group (vessel formations are marked with circles).A. Sildenafil group.B. Control group.

Figure 7 -
Figure 7 -Average number of capillary formations detected in histological analysis for each group (*p<0.05).