Investigating the therapeutic potential of medical leech and leech saliva extract in flap survival: an in vivo study using rats

Introduction

Wounds, characterized by skin or tissue integrity disruption, often result from accidents, trauma, or surgery, leading to structural and functional deterioration [1]. Wound healing unfolds through successive, overlapping phases – homeostasis, inflammation, proliferation, and remodelling. Initiated after tissue injury, physiological wound healing establishes a sophisticated signalling network among different cell types and skin compartments [2]. In addressing such wounds caused by tumour removal, burns, or cuts, dermatological surgery frequently employs skin flaps, which consist of skin and subcutaneous tissue with a robust vascular supply [3]. Sometimes, applying these flaps becomes necessary to treat and promote the healing of such complex wounds effectively. Despite their widespread use, random skin flaps, especially in distal regions, are prone to necrosis, posing a risk of flap loss. Consequently, research globally focuses on methods to reduce necrosis rates and enhance flap survival, with complementary and supportive treatments at the forefront of investigation [4], 5].

Medicinal Leech Therapy (MLT), also known as hirudotherapy, has been employed for centuries to treat various diseases [6]. Leech saliva contains over a hundred bioactive peptides and proteins, exhibiting anti-coagulant, anti-microbial, anti-inflammatory, and analgesic effects [7]. The therapeutic efficacy of medicinal leeches is primarily attributed to the rich biochemical composition of their secretions [8]. Hirudin, a potent thrombin inhibitor, plays a key role in anticoagulation, while other molecules such as calins inhibit platelet aggregation; bdellins and eglins act as protease inhibitors with anti-inflammatory potential. Additionally, hyaluronidase facilitates tissue permeability, destabilase contributes to fibrin degradation, and acetylcholine and histamine-like substances cause vasodilation [9]. MLT is recognized for its effectiveness in increasing perfusion and promoting rapid wound healing. Numerous experimental animal studies in the literature have explored the impact of medicinal leech application on wound healing [10], 11].

Although previous studies have explored the use of medicinal leeches in various wound models, there is a notable lack of research directly evaluating the effects of isolated leech saliva extract (LSE) in an in vivo flap model. Therefore, the main aim of this study is to investigate and compare the therapeutic effects of both medicinal leech therapy (MLT) and direct subcutaneous injection of LSE on skin flap survival and wound healing. By integrating histopathological, immunohistochemical (VEGF), and biochemical analyses, this study seeks to provide novel insight into the in vivo efficacy of leech saliva constituents. To the best of our knowledge, this is the first study to assess LSE in a random skin flap model in rats.

Materials and methods

Experimental design

In this experimental study, 18 animals were randomly assigned to three groups, each comprising six animals (Figure 1). It is important to note that an unfortunate incident occurred during the experiments, resulting in two deaths in the Medicinal Leech Therapy (MLT) and Medicinal Leech Saliva Extract (LSE) experimental groups.

Figure 1:

Quantitative comparison of flap necrosis area (%) among the three experimental groups on postoperative day 7. Group I (control): flap surgery without treatment. Group II (MLT): treated with medicinal leeches applied to the flap center. Group III (LSE): treated with subcutaneous injection of leech saliva extract.

On the seventh post-operative day, rats were euthanized through intra-cardiac blood collection under anaesthesia. Dorsal skin flaps were excised proximally and distally around the suture line for histopathological evaluation. Some tissues were preserved in containers with 10 % buffered formaldehyde, while the remaining samples were frozen in liquid nitrogen for biochemical analysis and stored at −80 °C. A homogeniser with a metal blade was used for the tissue homogenization. It was performed to obtain homogenates from the proximal and distal tissues of the flap areas in the experimental groups (Heidolph, SilentCrusher).

Random flap model

All rats were anesthetized under appropriate conditions by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). After anaesthesia, the hair of the rats was shaved with a razor. The surgical operation was performed under aseptic conditions under general anaesthesia. The dorsal random flap model was applied to all rats in all groups. After the anatomical borders of the flaps (6 × 2 cm) were determined, the random dorsal flap was elevated over the deep muscle fascia. All flaps were then repositioned in their original positions with simple sutures.

Results

MLT and LSE application increased the flap survival rate by decreasing the rate of necrosis in the flap area

There was a statistically significant difference between the groups regarding necrosis area (%) (p=0.028). The flap necrosis area (%) in the MLT-treated rats (12.04 ± 0.90) and LSE–treated rats (16.25 ± 0.92) were found to be significantly lower than the control group (Figure 1). Although the flap necrosis area (%) in the LSE–treated rats was lower than in the MLT–treated rats, no statistically significant difference was detected between these two groups (Figure 2). It was determined that the rate of necrosis area (%) was the highest in the control group (respectively *p=0.026, 0.046).

Figure 2:

Comparison of flap necrosis area in the experimental groups.

Histological evaluation of flap tissues for epithelial regeneration and granulation formation

In this study, comprehensive histological evaluations were undertaken to investigate the impact of MLT and LSE applications on cellular wound repair mechanisms in rats with a random flap model (Figure 3). Notably, in the proximal flap tissue, rats treated with LSE exhibited a significant enhancement in epithelial regeneration and the development of well-formed granulation tissue compared to the control group (p<0.05). Furthermore, in the distal flap tissue, rats subjected to both MLT and LSE treatments demonstrated a noteworthy increase in epithelial regeneration and the presence of robust granulation tissue when compared to the control group (p<0.05). These findings highlight the positive effects of both MLT and LSE in promoting epithelial regeneration and facilitating the formation of well-developed granulation tissue.

Figure 3:

Representative histological images of flap tissues from each group. (A, B) Sections stained with Hematoxylin & Eosin (H&E) to assess epithelial structure, granulation, inflammatory infiltration, and neovascularization. (C, D) Sections stained with Masson’s trichrome to visualize collagen deposition and connective tissue organization. (A, C) Proximal regions of the flap. (B, D) Distal regions of the flap. Arrows and symbols indicate key histological findings: ➔ epithelial regeneration, ❖ granulation tissue, ⟡ fibroblast proliferation, ► neovascularization, ⧓ presence of inflammatory cells, ⧱ decreased collagen density, Ν necrosis. Group I: control; group II: MLT; group III: LSE.

MLT and LSE increases VEGF (+) cell (%) in the flap area

MLT and LSE-treated rats showed an increase in VEGF (+) cells (%) for both proximal and distal flap tissue when compared with the control group (p<0.05) (Figure 4A and B). Although the VEGF (+) cell (%) was observed at the highest level in group III, no significant difference was detected between groups II and III (p<0.05).

Figure 4:

Immunohistochemical analysis for VEGF expression in proximal (A) and distal (B) flap tissue. VEGF-positive endothelial cells are indicated by ⧪, and VEGF-negative cells by ⧨. Increased VEGF staining is evident in groups II and III, reflecting enhanced angiogenic activity. Group I: control; group II: MLT; group III: LSE.

Discussion

Today, MLT and products obtained from medicinal leeches are used worldwide. The mechanism of action of medicinal leech saliva, which contains more than 100 bioactive substances, is still a matter of curiosity, and various scientific studies are carried out on medicinal leeches and medicinal leech saliva. This study aimed to investigate the effects of MLT and LSE on wound healing in the flap area via biochemical and histological methods. In our research, sterile medicinal leeches of H. verbana were used.

In the initial stage, the flap necrosis area was compared to the total flap area, revealing that the control group had the highest necrosis area (%). However, Groups II and III, treated with medicinal leech and medicinal leech extract, exhibited significantly lower necrosis areas compared to the control group. These results imply that the application of medicinal leech and its extract increased flap survival rates by reducing necrosis area (%). Anti-coagulant substances in medicinal leech saliva, such as hirudin and destabilase, and vasodilator substances, like acetylcholine, enhance blood flow. Therefore, our study suggests that the decreased flap necrosis area (%) and the improved flap survival rates are attributed to the enhanced blood flow in the treated areas [19].

In the second stage of our study, histopathological evaluation of tissue samples revealed that Groups II and III, treated with medicinal leech and its extract, exhibited significantly higher epithelial regeneration, granulation tissue thickness, and neovascularization compared to the control group, with Group III showing the most pronounced effects. These results indicate a positive contribution of LSE to the wound-healing process. Notably, the low inflammatory cell count in the LSE-treated group suggests a potential anti-inflammatory effect.

Similar positive effects of MLT on wound healing were observed in studies by Darestani et al. [20] and Zakian et al. [21], where MLT promotes faster wound closure and reduces inflammation. In another study by Mousavi et al., MLT demonstrated efficacy in reducing inflammatory cells, necrosis, and tissue damage in a rat model of acute venous congestion attributed to anti-inflammatory components in medicinal leech saliva [22]. Consistent with these findings, Bilden et al. also reported improved healing outcomes in rats with incisional wounds following MLT application, highlighting the broad therapeutic potential of leech therapy in different wound models [23].

Contrasting our study design, Schlaudraff et al. investigated MLT’s effects on flap survival in a different model, exposing random flaps to ischemia and applying H. medicinalis [24]. Their findings suggested that multi-session leech application might not be suitable in cases of insufficient arterial support. In our research, the improved flap survival and wound healing in both MLT and LSE-treated groups could be linked to adequate arterial support.

In summary, our study and previous research highlight the positive impact of medicinal leech and its extract on various aspects of wound healing, including epithelial regeneration, tissue thickness, neovascularization, and anti-inflammatory effects.

In the third phase of our study, we assessed the percentage of VEGF-positive cells in proximal and distal tissue samples using immunohistochemistry. VEGF plays a crucial role in wound healing, promoting angiogenesis by stimulating endothelial cell activities. Unlike most tissues, areas with active angiogenesis, such as the skin, ovaries, and uterus, exhibit increased VEGF levels during wound healing [25], 26].

In a study by Yingxin et al., the impact of natural hirudin, obtained from leech secretion, on VEGF gene expression in flap-modelled rats was investigated. The study revealed a significant increase in VEGF gene expression with natural hirudin compared to control and recombinant hirudin groups, improving flap survival by reducing necrosis [27]. In contrast to their PCR-based gene expression evaluation, our study focused on VEGF-positive cell percentages. Compared to the control group, it increased proximal and distal flap tissue in rats treated with Medicinal Leech Therapy (MLT) and Medicinal Leech Extract (LSE).

Hirudin, a potent natural thrombin inhibitor found in medicinal leech secretion, is believed to enhance VEGF levels, which are crucial for angiogenesis. Additionally, the literature suggests in vitro studies showing that LSE increases VEGF expression. In a study by Ünal et al., LSE’s effects on a healthy cell line, HUVEC, demonstrated increased VEGF gene expression and cell migration without inducing apoptosis or necrosis [28]. Consistent with these findings, a recent study by Ünal et al. further reported that lyophilized leech saliva extract exerted anti-proliferative effects on breast cancer cells while promoting migration in healthy endothelial cells, supporting its dual potential in cancer and wound healing contexts [29].

Our study consistently found elevated VEGF-positive cell counts, enhanced epithelial regeneration, and increased neovascularization. These findings indicate that the applied methods in our flap model positively influence the wound-healing process, promoting the formation of new blood vessels.

In the last stage of our study, serum samples were taken from all animals in all groups. In addition, tissue homogenates were obtained by taking tissue samples from the proximal and distal parts of the flap regions. HIF-1-α, TGF-α, and IGF-1 parameters were analyzed by ELISA test from all these samples. However, no statistically significant differences in these growth factor concentrations were observed among the experimental groups.

HIF-1-α, TGF-α, and IGF-1 play crucial roles in different stages of wound healing, including proliferation and angiogenesis, with mitogenic effects. HIF-1-α, particularly important for cell survival under hypoxic conditions, regulates various aspects of wound healing, such as cell division, growth factor release, and matrix synthesis. TGF-α, a key growth factor in routine healing, is highly expressed in skin tissue. At the same time, IGF-1 acts as a chemotactic agent for endothelial cells and promotes fibroblast and keratinocyte proliferation and migration [30], [31], [32], [33]. In the intricate orchestration of wound healing, HIF-1-α and TGF-α emerge as pivotal players, steering the early stages of the reparative journey. Yet, within this biological tapestry, the roles of TGF-β and the orchestrated motility of macrophages weave additional threads into the narrative of recovery [34], 35]. It remains crucial to underscore that the landscape of wound healing is not a static tableau but rather a dynamic, intricate process. Despite significant strides in understanding, the precise mechanisms governing this complex biological phenomenon are still under scrutiny in ongoing scientific exploration.

Our study is distinctive in the literature, representing the first instance of using H. verbana leeches and direct leech saliva injection under in vivo conditions. The findings suggest that medicinal leech extract application can be an effective agent for wound healing. Our research significantly contributes to the existing literature by exploring the in vivo application of leech saliva extract and examining a variety of parameters in experimental animals.

While our findings offer promising evidence supporting the use of medicinal leech therapy and its extract in wound healing, there are several limitations to consider. First, the study involved a relatively small sample size, and two animal losses during the experiment reduced statistical power. Second, although histological and immunohistochemical analyses were performed, molecular investigations (e.g., VEGF mRNA expression, angiogenic gene panels) were not included. Third, the dose of 0.5 mL LSE at 50 μg/mL protein concentration was chosen to assess the effects of the crude extract. The optimal concentration has not yet been established, as protein content can vary considerably depending on the individual leech and collection season. Furthermore, the exact concentration and individual effects of the active compounds in LSE were not isolated or quantified. Future studies should incorporate proteomic profiling of LSE, explore dose-dependent responses, and examine longer-term outcomes. Additionally, translational research into human tissue models and clinical settings may pave the way for therapeutic applications of LSE in reconstructive surgery.