Effect of Natural Hirudin on Random Pattern Skin Flap Survival in a Porcine Model

Abstract

Objective:

The effect of local administration of hirudin on random pattern skin flap survival was investigated in a porcine model.

Methods:

Three random pattern skin flaps (4 × 14 cm) were created on each flank of five Chinese minipigs. The experimental group (10 flaps) received 20 antithrombin units of hirudin, injected subdermally into the distal half immediately after surgery and on days 1 and 2; a control group (10 flaps) was injected with saline and a sham group (10 flaps) was not injected. All flaps were followed for 10 days postoperatively.

Results:

Macroscopically, the congested/necrotic length in the experimental group was significantly decreased compared with the other two groups by day 3. Histopathological evaluation revealed venous congestion and inflammation in the control and sham groups from day 1, but minimal changes in the experimental group. By day 10, the mean ± SD surviving area was significantly greater in the experimental group (67.6 ± 2.1%) than in the control (45.2 ± 1.4%) or sham (48.3 ± 1.1%) groups.

Conclusions:

Local administration of hirudin can significantly increase the surviving area in overdimensioned random pattern skin flaps, in a porcine model.

Introduction

Random pattern skin flaps are frequently used in plastic surgery to reconstruct various defects, but the distal necrosis of the flap associated with this procedure remains a challenging problem, with high morbidity.^1,2 Causes of necrosis can usually be traced to inadequate venous drainage of the flap, but the pathophysiology of the process is complex.^3,4 Studies aimed at improving the survival of the distal portion of random pattern skin flaps have employed a variety of interventions.^4,5 Many have focused on enhancing flap viability through pharmacological manipulations.^{26– 8} Although various agents are beneficial to some extent, the major drawback associated with these drug treatments is the need for systemic application at relatively high doses to achieve significant improvements in flap survival, with the increased possibility of potential systemic side-effects.

In the quest for other promising salvage procedures for imminent flap failure, one of the oldest medical practices – the application of leeches – has been rediscovered.^5,9 The effectiveness of leech therapy in relieving venous congestion is due to both mechanical and biological effects. However, the use of leeches may be associated with problems related to fear and disgust, allergic responses, infection and substantial blood loss. ⁹ Given the limitations and drawbacks of leeches, alternative methods that simulate the benefits of leeches could be very important to the future of flap surgery. Hirudin is a small protein isolated from salivary gland secretions of the leech. Owing to its strong anticoagulant properties, hirudin has the potential to improve flap survival by inhibiting microthrombosis and improving the microcirculation of the distal portion of random pattern skin flaps.^5,10 To our knowledge, no randomized, controlled studies have been undertaken to assess the effect of hirudin on flap viability. In the present study, the effect of local administration of hirudin on random pattern skin flap survival was investigated in a porcine model.

Materials and methods

Animals

Five pure-bred Chinese Bama minipigs weighing 9 – 10 kg and aged 6 – 8 months were used for the study. The pigs were housed in an environmentally controlled room on a 12-h light–dark cycle at a temperature of 24 °C and 55% humidity. They were fed a regular diet and received tap water ad libitum. The pigs were housed together throughout a 1-week acclimation period until surgery, but were housed individually after surgery to prevent injury to the skin flaps from cannibalism or normal social activities. All conditions and procedures were approved by the medical ethics committee of Guangxi Medical University, Guangxi, China (No. 20061201-B) and met the standards of the ethical guidelines of the 1975 Declaration of Helsinki.

Flap Creation and Treatment

The animals were anaesthetized using intravenous ketamine hydrochloride combined with midazolam. With the animal lying on its side, three rectangular flaps were created on each flank, making a total of six flaps on each pig (Fig. 1). Each flap was 14 cm long and 4 cm wide, with its pedicle 2.5 cm from the dorsal midline. The entire flap was undermined below the level of the panniculus carnosus and then sewn back in the same place. The flaps (n = 30) were assigned to experimental (n = 10), control (n = 10) and sham (n = 10) groups.

Figure 1:

Schematic illustration of the three rectangular random pattern skin flaps created on each pig flank, in a study investigating the effect of local administration of hirudin (20 antithrombin units, injected subdermally) on skin flap survival

In the experimental group, 20 antithrombin units of natural hirudin lyophilized powder (Ke Kang Ltd, Nanning, China) suspended in 2 ml phosphate-buffered saline (pH 7.4) was injected subdermally into the distal half of the flap immediately after surgery, and on postoperative days 1 and 2. In the control group, the flaps were injected in the same way as in the experimental group but with 2 ml of saline solution rather than hirudin. The sham group did not receive any injections and were simply followed up. All the flaps were followed up for 10 days postoperatively.

Macroscopic Evaluation

Gross photographs of all 30 flaps were taken immediately after surgery and on postoperative days 3 and 8, to observe the macroscopic changes over time. The length of congested or necrotic tissue on each flap was measured at each time point.

Histopathological Evaluation

On postoperative days 1 and 7, a full-thickness 4-mm punch biopsy was taken from each flap, 4 cm from the distal edge (under general anaesthesia with a standard regimen of ketamine and xylazine). Biopsies were fixed in 10% neutral formalin solution for 1 day and embedded in paraffin wax, before tissue sections 6 mm in thickness were prepared. Tissue sections were stained with haematoxylin and eosin, and the slides were examined using an optical microscope.

Assessment of Surviving Area

Survival of flaps was assessed on postoperative day 10 by measuring the size of viable and necrotic areas using transparent graph paper under direct visualization, which is an easy and reliable method described by many researchers for measuring flap survival.^11,12 The surviving area was defined as the viable area expressed as a percentage of the area of the whole flap.

Statistical Analyses

Results were given as the mean ± SD. One-way analysis of variance and the Student– Newman–Keuls post hoc test were used to compare differences between the experimental, control and sham groups. Two-sided P-values were used for all the statistical tests, and a P-value < 0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS^® software, version 13.0 (SPSS Inc., Chicago, IL, USA).

Results

All five animals survived and no complications (such as haematoma, infection or destruction of the flap or the suture line) developed during the 10-day follow-up period. Local injection of natural hirudin did not cause any local or systemic allergic reactions or any other side-effects.

Macroscopic Evaluation

Immediately after the operation, the distal two-thirds of the flaps in all three groups were slightly congested. There were no significant differences in the length of the congested area between the groups (Fig. 2A). Inspection on postoperative day 3 showed the distal part of the flaps to be swollen and dark due to venous congestion. There were no significant differences in the length of the congested area between the control and sham groups, but the length of the congested area in the experimental group was significantly shorter than that in the other two groups (P < 0.05 for both) (Fig. 2B). By postoperative day 8, the distal congested area of the flaps had become necrotic. There were no significant differences in the length of the necrotic area between the control and sham groups; however, the length of the necrotic area in the experimental group was significantly shorter than that in the other two groups (P < 0.05 for both) (Fig. 2C).

Figure 2:

Comparison of congested or necrotic length of random pattern skin flaps in a porcine model treated with hirudin injections (20 antithrombin units, injected subdermally) (experimental group; n = 10), saline injections (control group; n = 10) or no injections (sham group; n = 10). Representative flaps from each group are shown below each column. (A) Immediately after flap creation; (B) postoperative day 3; (C) postoperative day 8. Data presented as mean (SD). *P < 0.05 versus control and sham groups

Histopathological Evaluation

On postoperative day 1, there were minimal changes in the experimental group (Fig. 3A), whereas moderate to severe venous congestion and inflammation was seen in the control (Fig. 3B) and sham (Fig. 3C) groups. On postoperative day 7, a consistent pattern of exaggerated amounts of granulation tissue in the dermal and subdermal layers and early neovascularization was seen in the experimental group (Fig. 3D), whereas substantial areas of fibrosis and necrosis in all layers of the flaps were observed in the control (Fig. 3E) and sham (Fig. 3F) groups, with no evidence of neovascularization.

Figure 3:

Histopathological sections of random pattern skin flaps in a porcine model treated with hirudin (20 antithrombin units, injected subdermally) (experimental group), saline injections (control group) or no injections (sham group). (A) Experimental group, postoperative day 1; (B) control group, postoperative day 1; (C) sham group, postoperative day 1; (D) experimental group, postoperative day 7; (E) control group, postoperative day 7; (F) sham group, postoperative day 7. Haematoxylin and eosin

Surviving Area

On postoperative day 10, there was a clear demarcation between the necrotic and viable areas of the flaps. The mean ± SD surviving area was significantly greater in the experimental group (67.6 ± 2.1%) compared with the control (45.2 ± 1.4%) and sham (48.3 ± 1.1%) groups (P < 0.05 for both). Although the mean surviving area in the sham group was slightly higher than that in the control group, this difference was not statistically significant.

Discussion

Advances in plastic and reconstructive surgery have led to increasing use of skin flaps for the closure of surgical defects. Random pattern flaps are widely used for this purpose, because flap elevation and transfer are technically simple and their colour and texture mostly match the recipient site tissues.^11,12 However, due to the unpredictable vascularity of these flaps, there is a risk of flap necrosis because vascular flow decreases after flap elevation due to venous congestion, particularly in the distal part of the flap.^4,13,14 Maintaining the viability of flaps is therefore a clinically important goal for plastic surgeons.

Various methods have been investigated for preventing or reversing skin flap necrosis. Pharmacological agents often need to be used systemically, which can increase the possibility of systemic side-effects.^{6 – 8} The leech has long been recognized by surgeons for its ability to reduce venous congestion and improve blood flow in congested tissue. However, leeches are expensive and are often not readily available in an emergency situation. Alternative methods to simulate the benefits of leeches could be very important in the future of flap surgery.

Hirudin, which is isolated from the salivary gland secretions of the leech, is a potent specific inhibitor of thrombin^10,14,15 that acts by forming a 1:1 stoichiometric complex;^9,16 the recombinant protein has a dissociation constant in the picomolar range.^9,16 It is possible that flap tissue microcirculation in the vulnerable distal flap site may be improved by the ability of hirudin to decrease thrombosis and improve blood flow. However, the effects of hirudin on skin flap survival have not been previously reported. ¹⁰

In the present study, the effect of hirudin on random pattern skin flap survival was investigated. Topical application was chosen as the mode of drug delivery; this delivery route can lead to effective therapeutic tissue levels without significant systemic drug distribution, with a high therapeutic index. ² Pigs were chosen for this investigation instead of loose-skinned animals (such as rats, dogs or rabbits) because pig skin more closely approximates human skin, therefore conclusions drawn from such studies are more applicable to humans.

A random pattern skin flap is supplied by a subdermal plexus of small vessels, which does not have an axial orientation. The size of such flaps is limited by the ability to ensure an adequate blood supply. ⁴ The length-to-width ratio associated with good survival of random pattern skin flaps is around 1.5 : 1. The present study used overdimensioned flaps, measuring 14 cm in length and 4 cm in width, with a ratio of more than 3 : 1, so that the degree of congestion or necrosis could be more clearly evaluated in the distal flap areas.

Random pattern skin flaps are commonly used to cover skin defects. Creation of such flaps is an easily performed technique, but the limited length-to-width ratio means that, in many cases, alternative procedures need to be used (such as free tissue transfers, which create additional donor site defects in other parts of the body and are more time-consuming, costly and put a greater strain on the patient). If the length-to-width ratio of random pattern skin flaps could be increased, it would open up a wider range of clinical indications for this simple technique. In the present study, there was a 20% increase in the surviving area using local hirudin injections. This would enable the length-to-width ratio for random pattern skin flaps to be increased from 1.5 :1 to 1.8:1, and therefore may allow a wider range of applications for this simple flap technique.^4,17

In conclusion, data from the present experimental study showed that local administration of natural hirudin could significantly increase the surviving area in overdimensioned random pattern skin flaps, in a porcine model. Natural hirudin may have important therapeutic potential in improving skin flap survival in clinical settings. However, more extensive studies are required to clarify the specific mechanisms and pathways of the protective effect of hirudin.