Biochemical and Histopathological Effects of Hirudotherapy in a Rat Model of Chronic Exercise-Induced Physiological Stress

Summary

This protocol establishes a rat model of chronic exercise-induced physiological stress to investigate stress responses. It enables controlled evaluation of hirudotherapy–exercise interactions through serum biochemical profiling and histopathological analysis. It provides a reproducible framework for studying post-exercise recovery mechanisms.

Abstract

Chronic exercise can induce physiological stress characterized by muscle damage, oxidative imbalance, and hormonal alterations, potentially impairing recovery and performance. Hirudotherapy, a traditional biotherapeutic intervention that contains anti-inflammatory, antioxidant, and circulation-enhancing components, has attracted interest as a potential natural recovery strategy. This study aimed to evaluate the biochemical and histopathological effects of hirudotherapy following chronic exercise in a rat model of exercise-induced physiological stress. Thirty-two male rats were assigned to four groups: control, hirudotherapy, exercise, and hirudotherapy plus exercise. Serum biochemical parameters and oxidative stress markers were analyzed, and skeletal muscle and liver tissues were examined histopathologically to assess exercise-induced alterations and potential modulatory effects of hirudotherapy. Rats exposed to chronic exercise exhibited biochemical and structural indicators of physiological stress. Hirudotherapy was associated with partial attenuation of these alterations, particularly in histopathological findings, while changes in oxidative stress markers were not statistically significant. These findings suggest that hirudotherapy may exert a modulatory effect on post-exercise recovery processes; however, further studies are required to clarify its impact on oxidative stress pathways.

Introduction

Traditional medical practices have been used in a wide range of clinical contexts for the management of various acute and chronic conditions^1,2. Among these, medicinal leech therapy (hirudotherapy) has a long history of use across different cultures and medical systems. In recent years, this traditional approach has regained scientific interest due to its potential therapeutic effects in various clinical conditions, including inflammatory diseases, osteoarthritis, and vascular disorders^1,2,3. These effects are primarily attributed to the complex mixture of bioactive molecules present in leech saliva, which exhibit anticoagulant, anti-inflammatory, and antimicrobial properties^3,4,5. In addition, experimental studies in animal models have demonstrated that hirudotherapy may exert protective effects against tissue injury through antioxidant, anti-inflammatory, and anti-apoptotic mechanisms⁶.

The saliva of Hirudo verbana, the species most commonly used in therapy, contains a diverse array of bioactive components, including hirudin, bdellin, eglin, and calin, as well as enzymes such as hyaluronidase and various low-molecular-weight metabolites. These components exhibit anticoagulant, anti-inflammatory, antioxidant, fibrinolytic, analgesic, and anticancer activities^2,7. This broad biological activity profile suggests that leech saliva (LS) may serve as a complementary therapeutic tool in modern biomedical applications; however, its systemic effects under specific physiological stress conditions remain incompletely understood³.

Intense or prolonged physical activity can induce microstructural damage to muscle membranes, leading to significant increases in serum levels of enzymes such as creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST). Alanine aminotransferase (ALT) may also increase in response to muscle injury, although typically to a lesser extent than AST^5,8,9. In addition, high-intensity or long-duration exercise triggers elevations in cortisol, a key stress hormone, with responses varying according to exercise intensity, duration, and individual physiological status^10,11. Post-exercise increases in blood glucose may also occur, largely driven by cortisol-mediated gluconeogenesis, representing a hallmark of the metabolic stress response¹². Collectively, these findings demonstrate that both biochemical markers of muscle damage and indicators of metabolic stress tend to rise following strenuous exercise⁵.

Various strategies, including massage, cold-water immersion, and antioxidant-rich nutritional supplements (e.g., polyphenols, tart cherry extract, and curcumin), have been investigated for their potential to support post-exercise recovery. Systematic reviews suggest that these interventions may influence oxidative stress and muscle damage parameters; however, their effectiveness varies considerably among individuals and may not consistently provide sufficient biochemical protection^13,14,15. Accordingly, there is growing interest in exploring alternative or complementary approaches that may provide additional benefits under conditions of exercise-induced physiological stress. In this context, bioactive compounds present in leech saliva have been reported to modulate oxidative stress and inflammatory responses⁷. In addition, clinical observations suggest that hirudotherapy may reduce pain and inflammation, particularly in musculoskeletal and vascular conditions¹.

Several compounds identified in LS, including flavonoids, phenolic molecules, and other bioactive constituents, contribute to the attenuation of inflammatory responses through modulation of oxidative stress pathways. These antioxidant properties may act synergistically with other salivary components, enhancing the overall anti-inflammatory potential of hirudotherapy^7,16. In addition, protease inhibitors such as bdellin and eglin have been shown to reduce the activity of neutrophil-derived enzymes, thereby limiting tissue damage⁴. Furthermore, various peptides and lipid-derived molecules identified in leech tissues have been reported to exert inhibitory effects on inflammatory signaling pathways¹⁷.

Despite these findings, the potential role of hirudotherapy in exercise-induced physiological stress remains insufficiently explored, particularly within controlled experimental models^1,2,4. Although previous studies have demonstrated its anti-inflammatory, anticoagulant, and microcirculatory effects, and exercise is well known to induce muscle damage and oxidative stress responses¹⁸, studies integrating biochemical, hormonal, and histopathological outcomes within a single experimental framework are limited.

Therefore, the present study was designed to provide a comprehensive experimental evaluation of hirudotherapy in a controlled model of exercise-induced physiological stress by integrating biochemical, hormonal, and histopathological outcomes. This multidimensional approach aims to contribute to the understanding of the potential modulatory effects of hirudotherapy and to provide a reproducible framework for evaluating complementary biotherapeutic interventions.

Protocol

All experimental procedures were conducted in accordance with the ARRIVE guidelines¹⁹. The study protocol was reviewed and approved by the Kırşehir Ahi Evran University Local Ethics Committee for Animal Experiments (Approval No: 06/03/2025-05-1, Türkiye). All procedures complied with institutional guidelines for the care and use of laboratory animals. The reagents and the equipment used are listed in the Table of Materials.

1. Animal selection and housing

Thirty-two male Wistar albino rats (RRID: RGD_2308852; 8–12 weeks old, 200–250 g) were used. Animals were housed individually in polycarbonate cages (40 cm × 25 cm × 20 cm) with wood-shaving bedding. Environmental conditions were maintained at 22–24 °C with a 12 h light/12 h dark cycle. Food and water were provided ad libitum.

A 1-week acclimation period was performed prior to experimentation. During this period, animals were monitored daily for food intake, hydration status, grooming behavior, and locomotor activity. Only animals showing normal physiological behavior were included in the study.

2. Experimental groups and randomization

Animals were randomly assigned to four experimental groups (n = 8 per group) using a computer-generated randomization sequence. Allocation concealment was ensured by assigning animals numerical identifiers prior to group allocation. The experimental groups consisted of the control group (C), which received no intervention; the exercise group (E), which underwent treadmill exercise only; the hirudotherapy group (H), which received hirudotherapy alone three times per week; and the combined hirudotherapy plus exercise group (HE), which underwent both interventions according to the study protocol. All interventions were conducted over a 2-week period. To minimize circadian variability, all procedures were performed between 09:00 and 11:00 AM.

3. Chronic exercise protocol

Exercise was performed using a motorized rodent treadmill equipped with an adjustable electrical stimulation grid. Animals were placed in individual lanes of the treadmill apparatus (Figure 1). A 2-day adaptation phase was conducted at a speed of 15 m/min for 15 min/day. Following the adaptation period, the exercise intensity was increased to 20 m/min, and the animals were subjected to daily running sessions of 45 min/day for 2 weeks. A mild electrical stimulus (1.0 mA) was applied via the grid when necessary to maintain continuous running.

1. Exhaustion criteria
   Exhaustion was defined as the inability to leave the shock grid for three consecutive stimulations (~5 s each). Animals reaching this threshold were immediately removed. This protocol was adapted from previously established rodent treadmill exercise models with defined adaptation, intensity progression, and exhaustion criteria²⁰.
2. Hirudotherapy application
   Medicinal leeches (Hirudo verbana, ~2 g) were obtained from an institutionally certified source. Prior to application, the dorsal region of each animal was shaved with an electric clipper, and the skin was gently cleaned with sterile saline, without antiseptics, to avoid deterring leech attachment. Animals were then placed in a transparent acrylic restrainer tube to limit movement while minimizing stress. A single leech was placed on the dorsal midline, and successful attachment was visually confirmed by oral sucker fixation. Feeding was allowed for 10 min (Figure 2). Following the application period, leeches were detached by exposing the attachment area to 70% ethanol vapor using a cotton applicator held approximately 1–2 cm from the site. After detachment, local bleeding was controlled with sterile gauze using gentle pressure for approximately 1–2 min, and no suturing was applied. Applications were performed three times per week for 2 weeks. This procedure was conducted based on previously described experimental and clinical hirudotherapy protocols with standardized application parameters²¹.

4. Euthanasia and blood collection

At the end of the experiment (24 h after the last intervention)⁶, the animals were anesthetized intraperitoneally with ketamine (75 mg/kg) and xylazine (10 mg/kg). Adequate depth of anesthesia was confirmed by the absence of pedal withdrawal and corneal reflexes. Intracardiac blood collection was then performed using a sterile 5 mL syringe via left ventricular puncture. Euthanasia was completed by exsanguination followed by thoracotomy. Subsequently, the medial gastrocnemius muscle and liver tissues were collected for further analyses. Blood samples were processed within 1 h after collection by transferring them into clot activator tubes and centrifuging at 1,000 × g for 15 min at 4 °C. The separated serum was then aliquoted into 200 µL portions and stored at −80 °C until analysis.

5. Biochemical and ELISA analysis

Biochemical parameters, including ALT, AST, LDH, CK, glucose, electrolytes, and cortisol, were analyzed using an automated biochemical analyzer according to standardized laboratory procedures. All ELISA assays for CAT, SOD, MDA, and GPX were performed in accordance with the manufacturer’s instructions under standardized conditions. Serum samples were diluted 1:5 with sample buffer when required by the kit protocol. Subsequently, 50 µL of standards and 50 µL of serum samples were added to the designated wells and incubated at 37 °C for 60 min. Following incubation, the plates were washed five times using the provided wash buffer to remove unbound components. The detection antibody (50 µL) was then added to each well and incubated at 37 °C for 30 min. Thereafter, the substrate solution was added, and the reaction mixture was incubated for 10–15 min under dark conditions to allow color development. The reaction was terminated by adding 50 µL of stop solution, and the optical density was measured at 450 nm using a microplate reader. All samples were analyzed in duplicate to ensure analytical reliability and reproducibility.

6. Histopathological analysis

For histopathological evaluation, tissue samples were processed according to standard histopathological procedures²². The tissues were initially fixed in 10% neutral buffered formalin for 72 h, followed by dehydration through a graded ethanol series consisting of 50%, 70%, 80%, 96%, and 100% ethanol, with 1 h incubation at each concentration. After dehydration, the tissues were cleared in xylene for two consecutive 30-min periods and subsequently embedded in paraffin blocks. Sections with a thickness of 5 µm were obtained using a rotary microtome and stained with Hematoxylin and Eosin (H&E) and Masson’s Trichrome for histological examination. Finally, the stained slides were examined using a light microscope equipped with a digital imaging system.

7. Statistical analysis

Data analysis was performed using statistical software. Data distribution was assessed using the Kolmogorov–Smirnov test. For normally distributed data, one-way ANOVA followed by Tukey’s post hoc test was applied. For non-normally distributed data, the Kruskal–Wallis test followed by the Mann–Whitney U test was used. A p-value < 0.05 was considered statistically significant.

Results

Muscle damage and fatigue parameters

Serum muscle-damage markers revealed that creatine kinase (CK) levels differed significantly among the groups (p < 0.05). Although LDH, AST, and ALT values showed upward trends in the exercise (E) group, these changes were not statistically significant (p > 0.05). CK concentrations were highest in the E group, whereas the HE group exhibited markedly lower levels, approaching those of the control and H groups, indicat...

Discussion

This study demonstrated that hirudotherapy was associated with measurable changes in selected parameters related to exercise-induced physiological stress. The elevations in CK, LDH, AST, and ALT observed in the exercise group reflect sarcolemmal disruption and enzyme leakage, consistent with previous reports describing these markers as indicators of muscular strain^23,24,25. In contrast, the HE group exhibited enzyme levels close...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
-80°C Deep Freezer (Model DF590)	Nüve	DF590	Sample storage at −80°C
Acrylic restrainer tube	N/A	N/A	Transparent acrylic restrainer (6 cm diameter, 20 cm length) used for immobilization during hirudotherapy
Automated Biochemical Analyzer (Alinity)	Abbott	N/A	Used for serum biochemical parameter analysis
Automatic Tissue Processor	Leica Biosystems	N/A	Tissue dehydration and paraffin infiltration
Centrifuge (Model FC5706)	OHAUS Corporation	FC5706	Max speed: 6000 rpm (≈ 4,000 × g, depending on rotor radius)
Digital Microscope Camera (Axiocam 208 color)	Carl Zeiss	Axiocam 208 color	Image acquisition for histopathology
Eosin Y (yellowish)	Merck / Sigma-Aldrich	1.15935.0025	H&E staining (eosin component)
Ethanol Absolute (99.5%)	Tekkim Kimya	TK.200655.05001	Used for ethanol dehydration series
Formaldehyde Solution (~37%)	Merck / Sigma-Aldrich	1.04002.2500	Used for tissue fixation
Harris Hematoxylin Solution	BESTLAB	BS-002	H&E staining (hematoxylin component)
Ketamine hydrochloride	Pharmaceutical grade (commercial source not specified)	N/A	Used for intraperitoneal anesthesia (75 mg/kg)
Light Microscope (Primo Star)	Carl Zeiss	Primo Star	Histopathological imaging
Masson’s Trichrome Staining Kit	GBL	REF 5022	Used for connective tissue staining
Motorized Rodent Treadmill	Columbus Instruments	Exer-3/6	Used for standardized treadmill exercise with controlled speed and electrical stimulation
Rat Catalase (CAT) ELISA Kit	BT LAB	E0869Ra	96-well plate; serum CAT activity measurement
Rat Glutathione Peroxidase (GPX1) ELISA Kit	Reed Biotech	RE2557R	96-well plate; serum CAT activity measurement
Rat Malondialdehyde (MDA) ELISA Kit	BT LAB	E0156Ra	96-well plate; serum CAT activity measurement
Rat Superoxide Dismutase (SOD) ELISA Kit	BT LAB	E0168Ra	96-well plate; serum CAT activity measurement
Rotary Microtome (Autocut)	Leica Biosystems	RM Autocut	Cutting 5 μm paraffin sections
Slide Warmer	Leica Biosystems	HI1210	Drying histology slides
Xylazine hydrochloride	Pharmaceutical grade (commercial source not specified)	N/A	Used for intraperitoneal anesthesia (10 mg/kg)
Xylene (mixture of isomers)	Tekkim Kimya	TK.090270.05003	Tissue clearing before paraffin embedding