The effect of recombinant hirudin on rabbit ear flaps with venous insufficiency.

The effect of recombinant hirudin, which is the most powerful antithrombotic agent, on flaps with venous insufficiency was investigated. Oedema and congestion are frequent on flaps, causing necrosis unpredictably. Venous insufficiency and thrombosis are experimentally and clinically more frequent than arterial occlusion. Twenty-one adult New Zealand rabbits were used in this study. Skin flaps (3 × 6 cm) were elevated on a 1-cm-wide pedicle on rabbit ears. The artery, nerve, and vein were exposed and examined with the aid of a surgical microscope. Venous insufficiency was established by cutting the vein and nerve. In the control group, no additional surgical or medical procedures were performed and the ear flap was inset to its original location. Subcutaneous low molecular weight heparin (LMWH; 320 IU/kg) was administered to a second group of rabbits after the same surgery, and recombinant hirudin (2 μg) was administered via the pedicle artery 5 minutes after the vein and nerve were bound and cut in a third group of rabbits. Compared with control and LMWH groups on day 3 and 7, the hirudin-treated group had less hair loss, lower oedema scores and less haematoma formation. Furthermore, a lower size of necrotic areas and an increase in the circulating area on day 7 was found in the hirudin-treated group. In addition, angiography revealed new vessel development (neovascularisation) only in the hirudin group. On histologic sections, hirudin-treated animals had lower oedema, inflammation and congestion scores than animals in the other two groups. Thus, when administered into the ear flap through the pedicle as a pure recombinant preparation, hirudin increased flap survival by its antithrombotic effects and by accelerating neoangiogenesis. Recombinant hirudin may be used in clinical practice to treat flaps with venous problems and to increase survival rates.

INTRODUCTION

Flap failure can be catastrophic in patients.[1] Partial or complete necrosis of skin flaps is also an important problem in reconstructive surgery. Inadequate blood flow, venous insufficiency and ischaemia-reperfusion injury are believed to be the important factors.[1] Research on pharmacological manipulation of ear flap viability has included use of sympatholytics, vasodilators, antithrombotics, antiagregans and free radical scavengers.[2]

Hirudin is a polypeptide consisting of 65 aminoacids that is secreted from parapharyngeal glands of leeches. It is one of the most potent natural thrombin inhibitors known. Recombinant hirudin is also known as desulfato-hirudin due to its lack of a sulphate group on tyrosine 63 that is present in the natural form. Recombinant hirudin specifically inhibits the serine protease activity of thrombin by occupying its active site. The recombinant hirudin forms desirudin and lepirudin that exhibit activity equal to 15,000 antithrombin units (ATU) in 1 milligram (1 ATU hirudin binds to 1 unit of thrombin). Because antithrombotic properties of hirudin are dose independent, haemorrhagic side effects are rare. The advantage of using hirudin rather than heparin is that hirudin does not need ATIII for activity and it is not inhibited by factor IV.

In this study, the effect of recombinant hirudin on ear flaps with venous insufficiency was investigated. We showed that hirudin, administered into the ear flap through the pedicle as a pure recombinant preparation, increases ear flap survival by its antithrombotic effect and by accelerating neoangiogenesis. Recombinant hirudin may be used in clinical practice in flaps with venous problems to increase survival rates.

MATERIALS AND METHODS

Subjects

The study was conducted at the GATA Research Center after its approval by the Local Ethics Committee. Twenty-one adult New Zealand rabbits (3.6-4.5 kg) were selected randomly, kept in separate cages and fed standard rabbit feed. The right ears were operated on [Figures 1–8]; the left ears were used for the fluorescent application. Each of three experimental groups had seven rabbits:

Figure 1

Flap design

Figure 8

Inset flap

Control group: The vein and nerve were dissected from the artery, and were bound and transected. Without any other procedures, the flap was sutured to its original location with 5.0 silk.

LMWH-treated group: After the operation described above, low molecular weight heparin (2850 IU Axa/0.3 ml nadroparine calcique; SANOFİ SYNTHELABO) at a dose of 320 lU/kg was administered by subcutaneous injection.

Hirudin-treated group: The artery in the pedicle was followed proximally for 2 cm and recombinant hirudin (100 units; H-0393 HIRUDIN (Lowry) 11500 U/mg; SIGMA) at a dose of 2 micrograms was administered intra-arterially 5 minutes after the vein and nerve were bound and transected.

Flap artery and vein

Flap artery and vein image under the microscope

Cutting vein

Flap pedicle

Surgical procedures

The same surgeon performed all surgical procedures on the right ear of each rabbit under sterile conditions. A Microsurgeon F 6-branded surgical loop (6× magnification) was used for pedicle dissection and a Zeiss OPMI F 6-branded surgical microscope (30× magnification) was used for arterial drug administration. Rabbit ears were cleaned with povidone iodine solution. After inducing anaesthesia with intramuscular ketamine (35 mg/kg), the rabbits were prepared in a supine position. Axial-pattern skin flaps (3.0 × 6.0 cm) [Figure 1] on rabbit ears were elevated so that the vein, nerve and artery were present in its 1cm-wide pedicle.

ASSESSMENT

Fluorescein technique

Fifteen minutes after completion of all surgical procedures, an average of 0.93 (0.8-11) ml fluorescein (15% fluorescein, 10 ml; GATA CENTER FOR PHARMACEUTIC SCIENCES) was injected into the opposite ear vein. [Figure 2] Five minutes later, the distance from the base of the ear flap to the most distal stained part of the ear flap was measured under ultraviolet light [Figure 3] and the result was recorded as the operation day measurement.

Figure 2

Applying the fluorescein on the rabbit ear

Figure 3

After the fluorescein, under the wood lamb

The incision area and flaps were monitored daily. Oedema, inflammation, congestion, haematoma, hair loss and circulating area were estimated on the day of the operation, and the necrotic area was measured using millimetre scale paper [Table 1. 0-7 day measurements and scale].

Table 1

0 – 7 day measurements and scale

Angiogram

Experimental animals were killed with sodium pentobarbital (120 U/kg) after 7 days. Ear flaps were dissected 2 cm proximal to the pedicle. Barium sulphate (1.5 ml) was injected through the pedicle artery. Injection was sustained until the vascular system of the ear flap filled with barium (until the flap whitened). Angiograms were obtained 30 minutes after the injection of barium sulphate [Figures 9, 10].

Figure 9

Angiogram after hirudin

Figure 10

Angiogram after LMHH

Histology

Transverse sections obtained 3 cm proximal to the distal ear flap were examined histologically with haemotoxylin-eosin staining; inflammation, oedema and congestion were scored.

Statistical analysis

Statistical analyses were performed using SPSS version11.0 (Statistical Package for Social Sciences for Windows). The Kruskal-Wallis test was used for inter-group differences. In addition, the Mann-Whitney U test was used. The results were reported to be statistically significant at a confidence interval of 95% and a significance level of P < 0.05.

FINDINGS

Inflammation and congestion

The group that received recombinant hirudin had lower levels of inflammation and congestion than the control and LMWH groups at days 3 and 7, although the result was notstatistically significant (P > 0.05).

Hair loss

The recombinant hirudin group had lower hair loss than control and LMWH groups at days 3 and 7 (P < 0.05).

Area estimated to survive

The recombinant hirudin group had a higher measurement of areas estimated to survive than control and LMWH groups at days 3 and 7, although the result was not statistically significant (P > 0.05).

Oedema

The recombinant hirudin group had a similar oedemascore at day 0 as compared with control and LMWH groups, butthe scores at day 3 and 7 were significantly lower (P < 0.05).

Haematoma

The recombinant hirudin group had similar haematoma formation at day 0 as compared with the control and LMWH groups while day 3 and 7 haematoma formation was significantly lower (P < 0.05). There was a significant increase in haematoma formation on the day of the operation in the LMWH group (P < 0.05).

Circulating area

There was no significant difference in the circulating area among the three groups at 0 and 3 days. The recombinant hirudin group showed a significant increase in the circulating area as compared with the control and LMWH groups at day 7 (P < 0.05).

Necrotic area

The recombinant hirudin group showed a significantly decreased size of the necrotic area as compared with control and LMWH groups at day 0 and 7 (P < 0.05). There was no significant difference between the three groups at day 3.

Neovascularisation was observed in the hirudin group with no evidence of new blood vessel formations in the control and LMWH groups.

Histologic examination revealed that all three groups exhibited venous dilatation and inflammatory cell infiltration due to congestion. There was nonspecific arteritis in all three groups, which was more pronounced in control and LMWH groups.

When oedema, inflammation and congestion were compared, the recombinant hirudin group showed decreased values relative tocontrol and LMWH groups. In addition, the recombinant hirudin group showed newly formed anastomoses.

All measurement scores are shown in Table 2.

Table 2

Measurement scores

DISCUSSION

The venous congestion model employed in this study has been shown to be aneffective model.[3] Venous congestion was observed with a pedicle width of 1 cm. In addition, postoperative pain and myogenic reflex were prevented andnerve cutting eliminated potential drainage. This congested flap model was a simple and balanced model. Intravenous or intra-arterial urokinase, prostaglandin E 1, I2, topical capsaicin, fibroblast growth factor and vascular endothelial growth factor have been used by others and have shown efficacy in flap survival.[3] These studies focused on antithrombosis, vasodilatation and neovascularisation.

Studies of antithrombotic agents have focused on thrombin because it has a pivotal role in the regulation of thrombus formation. Thrombin regulates thrombocyte activation and aggregation, activates factor V and VIII, increases prothrombin activation, stabilises the thrombus by activating fibrinogen and factor XIII, regulates itself by protein activation and is a growth factor effective on striated muscle cells while it also modulates other growth factors.[6] Heparin is an antithrombotic agent that is still used in clinics for salvation of skin flaps with venous insufficiency. Its use is simple and it is cheap; however, there is a need for coagulation test follow-up. In particular, aPTT and bleeding and clotting times should be monitored, because heparin can cause bleeding problems in patients scheduled for reoperation. Hirudin has no factor Xa activity and prolongs aPTT bleeding and clotting time less effectively than heparin does.[5] In addition, heparin exerts its effects through ATIM and tends to bind free thrombin. Heparin is inactivated by circulating factor VI, vitronectin, fibrin monomer II and fibronectins. In addition, heparin only exerts inhibitory effects on free thrombin through ATIII and has no effect on clot-bound thrombin. Hirudin directly inhibits without the need for a cofactor and inhibits clot-bound thrombin, thereby inhibiting all thrombin-dependent thrombolytic activation by binding to the catalytic and anionicactive site of thrombin. Hirudin may be used to remove resistant thrombi. LMWHs have fewer bleeding side effects than unfractioned heparin.[6] Therefore, studies have employed LMWHs, which were effective in congested skin flaps.[3] Anti-factor Xa activity of LMWH reaches a maximum after 2-4 hours. A clinical study on coronary artery disease also found that LMWH had a better antithrombotic effect than unfractioned heparin.[7] Our study revealed that LMWH was more effective in flaps with venous insufficiency than the control treatment, although no statistical significance was observed. Especially, haematoma formation significantly favoured hirudin over LMWH.

Hair loss is a prominent marker of necrosis that reflects dermal involvement and signifies a full-thickness necrosis. In our study, the lesser amount of hair loss in the recombinant hirudin group showed that the intensity of necrosis was diminished. No studies in the literature were retrieved that described the intraarterial administration of recombinant hirudin through the pedicle in flaps. The primary problem is to determine the dose of hirudin to be administered intraarterially through the pedicle artery. Previous studies on hirudin suggested that a dose of 0.005-0.5 mg/kg should be used for dose-dependent antithrombotic effects.[8] We administered hirudin (2 μg)intraarterially through the pedicle artery. It has been reported that a single dose of intravenous hirudin (2 mg/kg) showed antithrombotic effects for 30 minutes.[9] Time needed for vicious cycle will be exceeded. Recombinant hirudin was directly administered to the region where it must exert its action and its antithrombotic effect was investigated. As a result, venous insufficiency was milder in animals treated with hirudinthan in control and LMWH animals at all points of assessment. In addition to recombinant, hirudin treatments led to increases in superoxide dismutase level and decreases in malondialdehyde and endothelin levels, which may potentially contribute to the hirudin-mediated effects of improving skin flap survival, via various molecular mechanisms such as anti-inflammatory, anti-thrombotic and anti-coagulatory pathways.[13]

Histology and radiology showed that recombinant hirudin both prevent microembolisms and increase the number and the width of venous anastomoses, between flap and the recipient area by triggering neoangiogenesis; however, the mechanism is not understood. The most important disadvantage of hirudin is the lack of a treatment for a rare haemorrhage that can occur upon its systemic administration. This haemorrhagic effect does not occur when hirudin is used locally for its powerful antithrombotic effects.

Medical leeches have long been used for skin flap salvation. The main disadvantages of their use are predisposition to infection, discomfort, psychological nervousness in patients and problems with their availability and administration. More importantly, patients continue to lose blood upon treatment with leeches and haemoglobin amounts drop by about 2 grams. As a result, blood transfusions are often needed, which increases risks to patients.

Although third generation cephalosporins are used to treat infections, such drugs are expensive. Hirudin is the most important substance needed for flap salvation. It is produced from E. coli strains using recombinant DNA technology and is ideal for treatment of skin flaps with venous insufficiency. The most important factor limiting its use is the cost involved with recombinant production methods.[4]

Some researchers recommend multiple venous anastomoses to prevent venous insufficiency. Futran and Stackreported no difference between single and double venous anastomoses in terms of skin flap survival.[10] Alternatively, anastomosis to separate venous systems has been implemented. Vascular endothelial growth factor (VEGF) has been used in skin flaps showing venous insufficiency[11] to prevent congestion by increasing neovascularisation. However, it is imperative to restore venous return quickly and other studies show that the combined use of VEGF with leech and/or hyperbaric oxygen therapy is more effective than VEGF alone. Others report hyperbaric oxygen and medical leech administration in skin flaps with venous insufficiency.[12] Since arterial blood flow is uninterrupted in skin flaps with venous insufficiency, tissues can be damaged by free oxygen radicals. In addition, patients who undergo operative processes such as free tissue transfer show problems in mobilisation and some are unable to tolerate hyperbaric oxygen therapy.

As with most medications, recombinant hirudin is not without drawbacks. Just as heparin can induce an immunological response in 1 to 6.5% of patients, recombinant hirudin is associated with antibody formation in 10% of recipients, although these antibodies do not appear to be inhibitory; recombinant hirudin potency is not affected by their presence. Additionally, unlike heparin-induced antibodies, the development of these antibodies has no proven impact on deep vein thrombosis, allergic reactions or haemorrhage. Moreover, a large randomised controlled study of orthopaedic patients reported no cases of thrombocytopenia.[14] Having demonstrated that recombinant hirudin significantly decreases the rate of microvenous thrombosis in a rat tuck model, we believe that this medication may have future expanded indications and utility in patients undergoing free tissue transfer. As already discussed, current free flap survival rates are high, and aggressive drug prophylaxis would unlikely be warranted in all patients undergoing free tissue transfer. Yet, there is a potential role for pharmacologic intervention with an agent like recombinant hirudin in patients with a higher risk for potential flap failure. This would include patients with a history of prior local radiation therapy, vascular insufficiency or with the intraoperative findings of challenging pedicle geometry or anastomoses. Recombinant hirudin is a unique anticoagulant in that its mechanism of action is both prophylactic and therapeutic, widening its applicability to those patients with clinical indications of flap anastomosis compromise.[14]

Such techniques have only been used in animal studies and have not been implemented in clinical practice. When viewed from this perspective, bedside recombinant hirudin preparations may be a wise approach for the treatment of skin flaps.

CONCLUSION

Flap loss caused by venous insufficiency poses a challenge for microsurgery in clinical practice. Currently, many agents are used experimentally and clinicallyto retrieve skin flaps with venous insufficiency. Our study suggests that recombinant hirudin might be useful as a therapeutic agent to increase flap survival. The data showed that this therapeutic approach increases survival of flaps with venous insufficiency, provides an efficient antithrombotic effect on thrombosis that can develop during venous insufficiency and increases the amount of microanastomosis due to neovascularisation. Recombinant hirudin could potentially increase skin flap survival and accelerate neoangiogenesis. Under the light of the results of our study, we reached the conclusion that use of hirudin for flaps with venous insufficiency is encouraging for clinical implementation.