Volume 27 Number 2

Key Messages

• Background This study investigates the therapeutic potential of botulinum toxin A in improving delayed wound healing, a major complication of diabetes mellitus.
• Objective The aim was to evaluate both macroscopic and microscopic effects of botulinum toxin A on secondary full-thickness skin defect healing in a streptozotocin-induced diabetic rat model.
• Results Botulinum toxin A significantly accelerated wound closure, enhanced angiogenesis, increased fibroblast proliferation and collagen deposition, and reduced inflammatory cell infiltration compared to controls, indicating improved rate and quality of wound healing.

Introduction

Diabetes mellitus is a chronic metabolic condition characterised by elevated blood glucose due to impaired insulin secretion or resistance.¹ One of its major complications is delayed wound healing, which can lead to persistent ulcers, infections, and increased healthcare burden.² The pathophysiology of diabetic wound healing is multifactorial, involving vascular insufficiency, neuropathy and chronic inflammation. Hyperglycemia alters cytokine production, interferes with collagen synthesis, and leads to accumulation of advanced glycation end-products (AGEs), all of which contribute to impaired tissue repair.^3,4

In diabetic wounds, disrupted angiogenesis and reduced growth factor activity compromise each phase of healing from coagulation and inflammation to proliferation and remodeling.^5-7 Endothelial dysfunction and excessive leukocyte infiltration further disturb the local tissue environment. As a result, healing is prolonged and often incomplete.⁸

Botulinum toxin type A (BTX-A) has emerged as a novel agent that may support wound healing. It works by temporarily inhibiting acetylcholine release at neuromuscular junctions, which reduces local muscle tension and mechanical stress at wound margins. Recent studies have reported enhanced angiogenesis, increased collagen deposition, and improved wound healing outcomes following BTX-A application.^9-15

Given the complexity of diabetic wound repair and the need for effective adjunctive treatments, this study aimed to evaluate both the macroscopic and microscopic effects of BTX-A on full-thickness skin wound healing in a diabetic rat model.

Materials and methods

Ethical approval

All experimental procedures involving animals were conducted in accordance with the guidelines of the College of Veterinary Medicine, Shahid Chamran University of Ahvaz, and the National Research Council’s Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, Revised 1985). The study protocol was approved by the Institutional Animal Care and Use Committee (ethical approval code: EE/99.3.02.58127/ scu.ac.ir). Humane care was provided to all animals throughout the study.

Study design and setting

This comparative preclinical study was conducted on 24 male Wistar rats (250–300g) obtained from the Laboratory Animal Reproduction Center at Shahid Chamran University of Ahvaz. Male rats were selected to avoid potential confounding effects of hormonal fluctuations associated with the estrous cycle, which could influence wound healing. Animals were housed under controlled conditions (22–24°C, 12-hour light/dark cycle) with free access to standard food and water.

Induction of diabetes

Experimental diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 60mg/kg, Sigma, Germany) dissolved in 0.1M sodium citrate buffer (pH 4.5).¹⁶ Blood glucose levels were measured three days post-injection using a glucometer via tail-tip blood sampling. Rats with glucose levels >300mg/dL were considered diabetic.¹⁷ Animals were observed for two weeks before grouping, with clinical signs including polyuria, polydipsia and weight loss.¹⁸

Animal grouping and wounding procedure

Rats were randomly divided into two main groups (n=12 each): a diabetic control and diabetic treated with BTX-A. Anesthesia was administered via intraperitoneal injection of ketamine (100mg/kg) and xylazine (20mg/kg). Dorsal skin areas were shaved and disinfected using iodine 10% and surgical scrub. A full-thickness wound (20×20 mm) was excised between the scapulae using a sterile scalpel.^19-21

Intervention and group subdivision

The treatment group received 5IU of botulinum toxin type A (BTX-A), diluted in 1mL distilled water, injected intradermally at and around the wound. The control group received an equal volume of saline.^20,22 To prevent infection and minimise animal suffering, enrofloxacin (10mg/kg) was administered orally for five consecutive days to all experimental groups. The dosage was selected based on previous experimental studies in rats.²³ Animals were housed separately to avoid wound interference. Each main group was further divided into three time-point subgroups (n=4 each) for evaluation on days 7, 14, and 21 post-treatments. Animals in each subgroup were euthanised on their respective evaluation day using an intraperitoneal overdose of ketamine and xylazine, in accordance with the American Veterinary Medical Association (AVMA) guidelines for the humane treatment of laboratory animals. Wound tissue samples were collected immediately post-euthanasia for microscopic analysis.

Macroscopic evaluation

Wound images were taken on days 0, 7, 14, and 21. Wound areas were measured using ImageJ software, and healing percentage was calculated using the following formula.¹⁶

Wound area percent = (wound area in day X ÷ wound area in day 0) ×100

Healing percent on day X=100−wound area percent

Microscopic evaluation

Wound tissue samples were collected immediately after euthanasia from animals in each time-point subgroup (days 7, 14, and 21). Samples were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5μm, and stained with hematoxylin-eosin and Masson’s trichrome. Parameters assessed included re-epithelialisation, angiogenesis, presence of fibroblast and inflammatory cells (neutrophils, macrophages, plasma cells, and lymphocytes) and collagen fiber density. Qualitative analysis was conducted using a 4× objective lens. For quantitative measurements, the number and diameter of blood vessels, and counts of fibroblast and inflammatory cells, were assessed in five random microscopic fields using a 40× lens. Masson’s trichrome staining was specifically employed to evaluate collagen deposition within the granulation tissue.

Statistical analysis

Data were analysed using Prism version 6 (GraphPad, Chicago). Inter-group and intra-group comparisons were conducted using Tukey’s post hoc test. Results are presented as mean ± standard error, with P<0.05 considered statistically significant.

Results

Macroscopic evaluation of wound healing

By day 7, both the BTX-A and control groups exhibited a significant increase in wound size, with the wound area expanding by more than 4cm-squared compared to day 0. This early increase in wound size may be due to the inflammatory response and edema (swelling) associated with the initial stages of wound healing, where tissue expansion often occurs before the healing process accelerates. This phase typically involves the migration of cells to the wound site and fluid accumulation, both of which can temporarily increase the wound area. Despite this early enlargement, examining the macroscopic results revealed that the percentage of wound healing in BTX-A–treated diabetic rats was significantly higher than that in the control group on day 7 (P<0.05). Throughout the study period, both groups showed an increasing trend in wound healing percentages, but this trend was more pronounced in the BTX-A group. Specifically, the healing percentage on day 7 was significantly different from those on days 14 and 21 in the same group (P < 0.001). On day 7, the BTX-A group had an average healing percentage of 42.5%, compared to 27.8% in the control group. By day 21, these values increased to 95.3% and 80.7%, respectively (Figure 1A and 1B).

 

Figure 1. Macroscopic evaluation of wound healing. (A) Comparison of wound healing percentages in diabetic and BTX-A-treated rats over 21 days. Statistical significance: (i) between groups at the same time point; (ii) within the diabetic group; (iii) within the BTX-A group. *P < 0.05, *** P < 0.001,
**** P < 0.0001. (B) Representative photographs showing wound area reduction at each time point in both groups.

Abbreviations: Dia=diabetic group; Dia+BTX=diabetic group treated with botulinum toxin A.

 

Angiogenesis and vascular parameters

According to the microscopic evaluation of the wound tissue sections, angiogenesis was more pronounced in the BTX-A–treated group during the first week post-surgery, especially within the granulation tissue (P<0.001). On day 7, both groups showed a statistically significant increase in the number of blood vessels compared to days 14 and 21 (P<0.001). Moreover, the average vessel diameter in the BTX-A group was significantly greater than that in the control group on day 7 (P<0.01). Temporal analysis within the BTX-A group revealed that vessel diameters peaked on day 7 and were significantly higher than those on days 14 and 21 (P<0.001) (Figure 2A and 2B).

 

Figure 2. Comparison of the number and diameter of blood vessels in diabetic and BTX-A–treated rats. (A) Mean number of blood vessels per microscopic field on days 7, 14, and 21. (B) Mean diameter of blood vessels on days 7, 14, and 21. Statistical significance: (i) between groups at the same time point; (ii) within the diabetic group; (iii) within the BTX-A group. *P < 0.05, **P < 0.01,
*** P < 0.001.

Abbreviations: Dia=diabetic group; Dia+BTX=diabetic group treated with botulinum toxin A.

 

Fibroblast and inflammatory cell response

Fibroblast counts were significantly higher in the BTX-A–treated group compared to controls at all time points (P<0.001). The peak occurred on day 7, followed by a gradual decline by day 21 in both groups, though values remained higher in the BTX-A group throughout (P<0.0001). Inflammatory cell infiltration was initially high in both groups, but significantly greater in controls on days 7, 14, and 21 (P<0.01). Over time, the BTX-A group showed a more rapid and consistent decline in inflammatory cells, with the lowest values observed on day 21 (P<0.0001) (Figure 3A and 3B). These findings indicate that BTX-A promoted early fibroblast proliferation and accelerated resolution of inflammation during the wound healing process.

 

Figure 3. Cellular responses in diabetic and BTX-A–treated rats.  (A) Mean number of fibroblast cells per microscopic field on days 7, 14, and 21. (B) Mean number of inflammatory cells at the same time points. Statistical significance: (i) between groups at the same time point; (ii) within the diabetic group; (iii) within the BTX-A group. *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001.

Abbreviations: Dia = diabetic group; Dia+BTX = diabetic group treated with botulinum toxin A.

 

Histopathological findings

Keratinocyte proliferation was observed at the wound margins in both the BTX-A and control groups (Figure 4A). In the control group, the wound remained in the inflammatory phase, with numerous inflammatory cells present in the granulation tissue. In contrast, the BTX-A group exhibited more advanced wound healing: granulation tissue covered a greater portion of the wound bed, and numerous pink-stained collagen fibers were visible under hematoxylin-eosin staining, which appeared blue-green under Masson’s trichrome (Figure 4B).

 

Figure 4. Microscopic evaluation of wound healing in diabetic and BTX-A–treated rats. (A) Week 1: Granulation tissue (G) is visible in both groups. In the diabetic group, sparse fibroblasts are observed among connective fibers (stars), while in the BTX-A group, enhanced angiogenesis (stars) is evident. (B) Week 2: The diabetic group shows migrating keratinocytes (arrows) and scattered collagen fibers (stars) in granulation tissue. In the BTX-A group, increased collagen deposition is evident—appearing pink in H&E and blue in Masson’s trichrome staining. (C) Week 3: The diabetic group displays dense inflammatory cell infiltration within the granulation tissue. In contrast, the BTX-A group exhibits extensive organized collagen fiber formation, visualised as pink with H&E and blue with Masson’s trichrome staining (stars).

Abbreviations: Dia=diabetic group; Dia+BTX=diabetic group treated with botulinum toxin A; G=granulation tissue; H&E=hematoxylin-eosin.

 

By the third week, wound closure was more complete in the BTX-A group. A substantial portion of the wound surface was covered by newly formed keratinocytes, and mature granulation tissue was present beneath. However, in the control group, wound healing was delayed, with persistent inflammation and abundant inflammatory cells still observed in the tissue (Figure 4C).

Dicussion and conclusion

This study investigated the effects of botulinum toxin A (BTX-A) on wound healing in diabetic rats, aiming to identify alternative treatments with fewer side effects than conventional drugs. The results demonstrated significantly improved wound closure and healing quality in the BTX-A group compared to the diabetic control group, particularly during the early stages of healing.

Our findings align with those of Oryan and Alamzadeh, who reported enhanced wound healing following BTX-A injection due to reduced inflammation and mechanical tension on the wound.²⁴ Similarly, Zhong et al found that BTX-A facilitated healing in chronic wounds by suppressing inflammatory mediators like bradykinin and prostaglandins, reducing vascular permeability, and limiting neutrophil and macrophage infiltration.²⁵ These mechanisms may explain the accelerated wound closure observed in the early phase of healing in our study.

Regarding angiogenesis, our study showed increased blood vessel formation in the BTX-A group on day 7, followed by a decrease by day 21. These changes were consistent with previous research by Kim et al and Roh et al, who attributed angiogenesis to VEGF upregulation after BTX-A injection.^14,26 However, Lee et al did not observe significant vascular differences, possibly due to differing experimental timelines or doses. Camargo et al also observed a similar increase in vessel diameter and vascular density following BTX-A treatment and explained that the neurotoxin prevented the release of neurotransmitters at the nerve-muscle junction and caused vasodilation.¹³

Fibroblast proliferation was elevated in the BTX-A group on day 7 but decreased in later phases, likely due to faster wound closure and reduced tissue demand. Similar patterns were noted by Oryan and Alamzadeh, who associated reduced fibroblast counts with lower mechanical stress.²⁴ Other studies, including those by Lee et al and Kucukkaya et al, confirmed increased collagen deposition after BTX-A treatment, with some disagreement about the timing and magnitude of effects. Differences may relate to the site of injection, model used, or time intervals considered.^15,21

The increase in collagen fibers observed in the BTX-A group during the second and third weeks of healing is particularly noteworthy. This structural improvement in the granulation tissue suggests enhanced matrix remodeling, potentially improving long-term wound integrity. Lee et al also noted elevated collagen synthesis, particularly in later stages of healing, while other studies highlighted qualitative changes such as better fiber alignment and scar organisation.²⁰

Inflammatory cell counts were lower in the BTX-A group across all time points in our study. This is consistent with reports by Lee et al and Oryan and Alamzadeh, who linked reduced inflammation to the immobilisation of subcutaneous muscles and shortened inflammatory phase post-BTX-A injection.^20,24 These effects may contribute to a more organised granulation tissue formation and improved scar quality. Additionally, faster transition from inflammation to proliferation phase may prevent chronic wound formation, which is a common complication in diabetic wounds. It should be noted that prophylactic antibiotic therapy was administered uniformly across all experimental groups to prevent infection in this diabetic wound model. Therefore, any potential influence of enrofloxacin on inflammatory or infection-related parameters would have been consistent across groups and is unlikely to have biased the comparative outcomes observed in this study.

Other investigations support the anti-scar and regenerative effects of BTX-A. In a rabbit hypertrophic scar model, Xiao and Qu reported thinner, better-organised scars following BTX-A injection.²⁷ In vitro, Roh et al observed increased type I and III collagen expression after BTX-A exposure.²⁸ Gassner et al found improved cosmetic outcomes in monkeys and humans, with reduced scar formation after BTX-A application at wound edges.^29,30 These findings underscore the potential translational relevance of our animal-based results.

In conclusion, this study provides compelling evidence that botulinum toxin A (BTX-A) significantly enhances wound healing in diabetic rats by accelerating early wound closure, promoting angiogenesis, reducing inflammation and improving collagen organisation. These findings suggest that BTX-A may offer a novel therapeutic approach for managing diabetic wounds, a common and challenging complication of diabetes mellitus. While the results are promising, further experimental and clinical investigations are required to confirm the safety, efficacy, and long-term benefits of BTX-A in wound healing. Future studies should focus on evaluating the mechanical strength and physiological functionality of the healed tissue, which will be critical in determining the clinical applicability of BTX-A as a routine treatment.

Implications for clinical practice

• Botulinum toxin A may accelerate wound healing in diabetic conditions by enhancing angiogenesis and collagen deposition.
• BTX-A could serve as an adjunctive treatment option for managing chronic diabetic wounds.
• Early use of BTX-A may reduce inflammation and improve overall wound healing quality.

Further research

• Clinical trials are required to confirm safety, efficacy, and optimal dosing of BTX-A in human subjects.
• Long-term studies should assess functional outcomes, scar quality, and tissue strength following BTX-A therapy.
• Comparative studies with other adjunctive treatments are needed to define BTX-A’s relative clinical benefits.

Conflict of interest

The authors declare no conflicts of interest.

Funding

This research was supported by Shahid Chamran University of Ahvaz. No specific grant was received from public, commercial or not-for-profit funding agencies.

Author(s)

Zohre Sabzloun¹ DVM, Ali Ronagh¹* DVSc, Seyed Reza Fatemi Tabatabaei¹ PhD, Anahita Rezaie DVSc
¹Department of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran

*Corresponding author email alironagh@yahoo.com

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