Effects of Systemic Tadalafil on Skin Flap Survival in Rats

MATERIALS AND METHODS

All experimentation was performed with the approval of the institutional review board and the Institution's Animal Care and Use Committee and was in compliance with all federal statutes regulating animal experimentation.

Thirty-seven male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing 318 to 400 g were used in this experiment. All rats were allowed to acclimate for at least 1 week prior to surgery, housed in pairs in a standard animal facility with environmental control and a 12-hour light-dark schedule with regular food and water ad libitum. After surgery, the rats were housed individually to prevent injury to the surgical flap.

On the day of surgery, the animals were anesthetized with isoflurane induction via precision vaporizer and an attached scavenger system, 2% to 5% isoflurane by chamber initially, and then 1% to 3% by face mask for maintenance throughout the procedure. A modified McFarlane flap¹⁶ measuring 8 cm × 2.5 cm was raised with sharp dissection using a no. 10 scalpel blade using standard approved surgical procedure. The pedicle was placed caudally at the level of the iliac crests. The flap was then sutured back into its original donor position using 5-0 undyed monofilament sutures (Ethicon, Somerville, NJ) in a running subcuticular fashion. Analgesia was provided once preoperatively and postoperatively every 12 hours for 36 hours via a subcutaneous injection of 0.03 mg/kg buprenorphine.

Rats were divided into a control group, high-dose tadalafil group, and low-does tadalafil group. The control group was given the vehicle solution of 1% hydroxypropyl methylcellulose and 1% tween-80 in water. The test groups were given tadalafil powder (Eli Lilly and Company, Indianapolis, Indiana) mixed in the vehicle solution at either a high-dose concentration of 30 mg/mL for a dose of 20 mg/kg and the low-dose concentration of 15 mg/mL for a dose of 10 mg/kg. These concentrations were calculated on the basis of a 200 µL dose for a standard 300 g rat. Treatment doses were administered immediately before surgery and every 24 hours after surgery for a total of 7 doses. Study medication and control doses were administered by oral gavage using a bulb-tipped curved gavage syringe specifically designed for this purpose. The amount of treatment solution given was determined before each dosing based on the animal's weight at that time.

On postoperative days 7 and 14, the rats were anesthetized for tracing of the flap. A sheet of writable transparency paper was placed over the dorsum of each rat, accounting for the curvature of the dorsal surface, and the flap was traced taking care to draw the line of demarcation between the viable proximal flap tissue and the necrotic distal portion. Flap tracing analysis was performed digitally after each flap tracing was scanned into a computer as an image file. The images were then analyzed using Photoshop (Adobe, San Jose, California) to determine the percentage of necrosis and survival of each flap. A straight line was digitally drawn connecting the tracings representing the caudal ends of the lateral incisions. The areas of the resulting 2 enclosed shapes, representing the healthy and necrotic portions of the flap, were calculated by determining the number of pixels within each outline using the histogram function. Percentage of flap survival was determined by dividing the area of the healthy portion by the sum of the healthy and necrotic areas. Percentage of flap survival in each group was averaged and statistical analysis using the Mann-Whitney test was performed to determine the statistical significance of the data.

RESULTS

All 37 surgeries were successful, and all rats continued to thrive until the end of the 14-day experimental period. By postoperative day 7, all flaps showed clear demarcation between the necrotic distal portion and the viable base.

The data for each of the experimental groups is shown in Table 1 and Figure 1. Control-group rats showed an average flap survival of 77% ± 11% at 7 days, and 77% ± 9% at 14 days. Low-dose-group rats showed an average flap survival of 82% ± 10% at 7 days (P = .21) and 81% ± 12% at 14 days (P = 0.41). High-dose-group rats showed an average flap survival of 81% ± 11% at 7 days (P = .45) and 80% ± 12% at 14 days (P = .53). Despite small trends in the data showing increased flap survival in the tadalafil treatment groups at days 7 and 14, these differences are not statistically significant based on analysis of variance with the Mann-Whitney test.

Figure 1. Flap survival rate.

DISCUSSION

Cutaneous flaps based on random blood supply are commonly used for the coverage of skin and soft tissue defects but are at high risk for ischemia and distal flap necrosis consequent to low axial blood flow. Pharmacologic agents that can increase blood flow and thereby improve flap survival could provide a low-risk method of minimizing and preventing such complications.

Phosphodiesterase-5 inhibitors are a family of agents that are most widely used to treat erectile dysfunction. Considering their ability to selectively increase blood flow to smooth muscle through the nitric oxide pathway, it has been hypothesized that they may be useful in improving perfusion to random pattern flaps. The first PDE-5 inhibitor to be tested on random pattern flap survival was sildenafil. It was shown to improve flap survival in a rat model through intraperitoneal administration,^8,9 topically with a fibrin glue,¹⁰ and orally.¹¹

Subsequently developed PDE-5 inhibitor, tadalafil, has a longer half-life and has been shown in studies of erectile dysfunction to be safe and effective with daily oral administration.¹⁷ Tadalafil was tested by Oh et al¹⁵ using a rat model with a local injection of the drug into the flap and demonstrated a significant improvement in random pattern flap survival. While injected administration of these drugs has shown promise, oral administration remains the most convenient method of drug deliverance. Our study showed a trend of increased flap survival in animals that received oral administration of tadalafil, but further studies are warranted to determine significance.

In our experience, we attribute the high standard deviation to several identified problems with the current study and studies like it. First, McFarlane flaps are very difficult to reproduce consistently. The dorsal rat skin is extremely mobile and very thick. The act of incising the skin frequently distorts the flap pattern. Second, administration of tiny doses of tadalafil is very imprecise. The amount of compound that is actually administered to the rats given the large fraction that remains in the dead space of the syringe and gavage needle is inconsistent. Third, transfer of the flap pattern and line of demarcated necrosis onto the transparency paper is approximate at best. Given these limitations, we suggest further study to examine the effect of daily oral administration of tadalafil. While our results are suggestive, they are not conclusive and warrant further investigation into the potential therapeutic use of the PDE-5 inhibitor, tadalafil.