Inflammatory alterations provoked by metronidazole in wounds: an experimental study in rats

Abstracts

CONTEXT: Chronic feet and leg ulcers affect about 2.7% of the Brazilian population, 10% of diabetic patients. The condition represents the second most frequent cause of absence from work in Brazil. This shows the need for a product that promotes healing of these wounds at a low cost. OBJECTIVE: To evaluate the effects of metronidazole on ulcer healing by second intention. METHODS: Eighty male rats divided into two groups of 40 had a wound made on their dorsum. The control group was treated with a 0.9% NaCl solution and the experimental group was treated with 4% metronidazole. On the third, seventh, 14th and 21st days, the healing process was assessed through macroscopical, histological and immunohistochemical parameters. RESULTS: Collagen concentration was higher in wounds in the experimental group in all samples. Concentration of type I collagen was also significant on the 7th (p = 0.020) and 21st (p = 0.016) days. Concentration of type III collagen was similar in both groups in the initial phase, but it was higher in the experimental group on the 21st day (p = 0.005). Angiogenesis, assessed with anti-CD34, revealed a larger number of vessels in the experimental group, with a significant difference on the third (p < 0.001) and 14th (p = 0.003) days. CONCLUSION: Metronidazole contributes to healing wounds by second intention and stimulates collagen production and angiogenesis.

Wound healing; metronidazole; physiological neovascularization; collagen; rats

CONTEXTO: Cerca de 2,7% da população brasileira tem úlceras crônicas nos pés e nas pernas, porcentagem que chega a 10% nos diabéticos e que representa a segunda causa de afastamento do trabalho no Brasil. Isso demonstra a necessidade de se encontrar um produto de baixo custo que favoreça a cicatrização dessas feridas. OBJETIVO: Avaliar os efeitos do metronidazol na cicatrização de feridas por segunda intenção. MÉTODOS: Utilizaram-se 80 ratos machos, em cujos dorsos se produziu uma ferida, distribuindo-se, os animais, em dois grupos de 40. Os ratos do grupo-controle tiveram suas feridas tratadas com solução de NaCl 0,9%, e os pertencentes ao grupo-experimento, com metronidazol 4%. No terceiro, sétimo, 14º e 21º dias, avaliou-se o processo cicatricial por parâmetros macroscópicos, histológicos e imunoistoquímicos. RESULTADOS: A concentração de colágeno foi maior nas cicatrizes dos animais do grupo-experimento em todos os tempos examinados. A concentração de colágeno do tipo I também foi significante no sétimo dia (p = 0,020) e no 21º dia (p = 0,016). O colágeno tipo III mostrou concentração semelhante nos tempos iniciais e apresentou-se com maior concentração no 21º dia (p = 0,005). A angiogênese, avaliada pelo anti-CD34, demonstrou maior número de vasos, no grupo-experimento, com diferença significante no terceiro dia (p < 0,001) e no 14º dia (p = 0,003). CONCLUSÃO: O metronidazol contribui para a cicatrização de feridas por segunda intenção, estimulando a produção de colágeno e a angiogênese.

Cicatrização de feridas; metronidazol; neovascularização fisiológica; colágeno; ratos

ORIGINAL ARTICLE

Inflammatory alterations provoked by metronidazole in wounds: an experimental study in rats

Cláudia Paraguaçu Pupo Sampaio^I; Maria de Lourdes Pessole Biondo-Simões^II; Lilian Cristine Teixeira Trindade^III; Rogério Estevam Farias^IV; Rodrigo Jardim Pierin^V; Rafael César Martins^V

^IMestranda, Programa de Pós-Graduação em Clínica Cirúrgica, Pontifícia Universidade Católica do Paraná (PUCPR), Curitiba, PR, Brazil

^IIProfessora titular, Metodologia Científica, PUCPR, Curitiba, PR, Brazil. Professora adjunta, Departamento de Cirurgia, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazil

^IIIAluna, Programa de Pós-Graduação em Clínica Cirúrgica, PUCPR, Curitiba, PR, Brazil

^IVDoutorado em Patologia, Universidade Federal Fluminense (UFF), Niterói, RJ, Brazil. Professor adjunto, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, MG, Brazil

^VAcadêmico de Medicina, PUCPR, Curitiba, PR, Brazil.

Correspondence

ABSTRACT

Context: Chronic feet and leg ulcers affect about 2.7% of the Brazilian population, 10% of diabetic patients. The condition represents the second most frequent cause of absence from work in Brazil. This shows the need for a product that promotes healing of these wounds at a low cost.

Objective: To evaluate the effects of metronidazole on ulcer healing by second intention.

Methods: Eighty male rats divided into two groups of 40 had a wound made on their dorsum.

The control group was treated with a 0.9% NaCl solution and the experimental group was treated with 4% metronidazole. On the third, seventh, 14th and 21st days, the healing process was assessed through macroscopical, histological and immunohistochemical parameters.

Results: Collagen concentration was higher in wounds in the experimental group in all samples. Concentration of type I collagen was also significant on the seventh (p = 0.020) and 21st (p = 0.016) days. Concentration of type III collagen was similar in both groups in the initial phase, but it was higher in the experimental group on the 21st day (p = 0.005). Angiogenesis, assessed with anti-CD34, revealed a larger number of vessels in the experimental group, with a significant difference on the third (p < 0.001) and 14th (p = 0.003) days.

Conclusion: Metronidazole contributes to healing wounds by second intention and stimulates collagen production and angiogenesis.

Keywords: Wound healing, metronidazole, physiological neovascularization, collagen, rats.

Introduction

Some authors report that 3% of the Brazilian population has some type of chronic lesion. Chronic feet and leg ulcers affect about 2.7% of the Brazilian population, 10% of diabetic patients and represent the second most frequent cause of absence from work in Brazil.¹

Ereno draws attention to the importance of developing affordable bandages for the 4.5 million Brazilians who probably cannot afford the high cost of imported drugs and sophisticated bandages.²

Despite results of isolated studies and because of the few reports available,³ it is probable that statistical data are not accurate in revealing the serious problem of chronic wounds in public health.

There are studies that report that metronidazole has healing properties, which promote rapid contraction and epithelization, culminating in an early repair process.^4-6

After conducting experiments, Mallikarjuna suggested a line between ends of oxidative products and epithelization⁵ because, according to their investigations, any factor that decreased lipid peroxidation would promote healing of burn injuries. Girish & Patil argued that metronidazole had pro-healing properties because it promotes epithelization and keratinization.⁷

Considering that metronidazole is available in the public health system and is a low cost drug, its use in chronic wounds could represent a better treatment option for millions of people. It should be taken into account that public costs are not limited to treating the ulcer; time during which the patient is absent from work and the daily cost of a bed for hospitalized patients are also included.

The aim of the present study is to evaluate inflammatory effects of metronidazole on ulcer healing as compared to effects of saline solution.

Methods

The project of which this study is a part was approved by the Animal Use Research Ethics Committee (Comitê de Ética no Uso de Animais) of Pontifícia Universidade Católica do Paraná (PUCPR) under number 231, according to the Brazilian Federal Law number 6638 and recommendations of the Brazilian College for Animal Experimentation (Colégio Brasileiro de Experimentação Animal -- COBEA).

Eighty male Wistar rats were used (Rattus norvegicus albinus, Rodentia mammalia); each one was 90 days old and weighted between 184.28 g and 255.56 g. Sample size was estimated based on reports available in the literature.^8-10

Rats, from the Central Vivarium of PUCPR, were kept in cages suitable for the species with free access to water and standard chow for the species. Environmental conditions were controlled with temperature at 20.2ºC and light-dark cycle of 12 hours; relative air humidity and noise were not controlled. All the cages were at the same distance from the light source.

All the animals received anesthesia, with intramuscular administration of 0.1mL/100g of body weight with a solution of 1 mL of ketamine (50 mg) and 1 mL of xylazine (20 mg)¹¹ in the right posterior thigh, after which the dorsum was shaven, antisepsis with polyvinylpyrrolidone-iodine was performed and a sterile fenestrated drape was placed over the operative site.

In the center of the shaved area, a circular incision with 2 cm in diameter was made on the skin of each rat using the cutting edge of a punch. The circular skin segment was resected, leaving the muscular fascia of the dorsum exposed.

After surgery, the animals received diclofenac potassium at 10 mg/kg, via intramuscular route, for analgesic and anti-inflammatory purposes.¹¹

The surgical wounds were photographed with a digital camera (Sony Cyber-Shot^® P71, resolution of 3.2 Megapixels) on a tripod at a constant 34 cm distance.

After anesthetic recovery, rats returned to their cages (five in each cage) and they were observed until the day they were sacrificed.

The sample of 80 animals was randomly divided into control group and experimental group. The wounds of rats from the control group were cleaned daily with a 0.9% NaCl solution and those of the rats from the experimental group were also cleaned and, after that, had 4% topical metronidazole applied on them. Ten rats from each group were sacrificed on the third, seventh, 14th and 21st days.

The metronidazole solution used was obtained from the oral suspension presentation (benzoyl metronidazole) 40 mg/mL (4%) in base q.s. The dose administered was 0.3 mL of metronidazole, amounting to 12.5 mg/day. This was established based on the indication for a 50 mg/kg/day as the standard dose of metronidazole.

Immediately after sacrifice, the surgical wounds were photographed and resected with a 1 cm margin of healthy skin around the wound and as deep as the dorsal musculature of the rat. Tissues were fixed in 10% formalin and taken to histopathological exams.

Each block was sectioned in 4-µm thick sections, which were stained with the Picrosirius method (Sirius supra red F3BA). For each histological section, three fields were analyzed, with a 400x magnification over the wounded area.

Analysis of sections stained with Sirius Red revealed that thicker birefringent collagen fibers were orange-red (type I collagen) and thinner more scattered fibers, weakly birefringent, were green (type III collagen).¹² An Olympus^® BX50 microscope coupled with a 3 CCD pro series camera was used. Images were captured with a Sony^® CCD101 camera, transmitted to a Trinitron Sony^® color monitor, frozen and digitalized with the oculus^® TCX board. Image analysis was performed using MediaCybernetics Image-Plus^® 4.5 for Windows^® with a Pentium personal computer. For each section, five fields were analyzed with a 200x magnification on the scar line. For each field, the percentage of areas occupied by red and yellow fibers (type I collagen) and green fibers (type III collagen) were calculated.¹² Since other types of collagen were represented by very few numbers, for practical purposes, the sum of collagen I and III were considered the total of collagen types in scars.

For immunohistochemical studies, the tissue array or microarray technique was chosen, with samples taken from the paraffin block used for hematoxylin-eosin (HE) staining and Picrosirius staining. The sample was taken from the superficial central area of the wound, located in the HE slide, previously marked with an overhead projector marker and used as a mirror image for the punch. Punch number 3 was used. Samples were put into cassettes, as previously planned.

After that, the material was sent for immunohistochemical analysis, with the use of anti-CD34.^13,14 The immunohistochemical technique is based on an antigen-antibody reaction. In order to identify a specific protein (antigen), a particular antibody will recognize a certain domain. The antibody binds to the specific antigen, which is recognized as a secondary antibody to which a streptavidin-biotin-peroxidase complex is bound. Peroxidase transforms a chromogenic substance, usually diaminobenzidine, producing a brownish reaction product.

Anti-CD34 is an endothelial marker, but its results are not satisfactory for large vessels; depending on indication, it is satisfactory as a microvessel marker. For each tissue section, 10 fields were analyzed and the evaluation on the inflammatory process was complemented by a quantitative evaluation of neovascularization. Analysis was done with an Olympus^® BX500 microscope.

Slide analysis (10 fields per slide) was performed by a pathologist blind to the animals to which the tissues belonged.

Student t test was used for independent samples in order to compare control and experimental groups at each sacrifice day. For comparisons within each group, one factor analysis of variance was used. Shapiro-Wilk normality test was used to test variables.¹⁵ For non-normal variables, a transformation was used (square root). To compare the groups in terms of dichotomic variables, Fisher's exact test was used.¹⁶ Values of p < 0.05 were statistically significant. Calculations were performed by Statistica version 7.0.

Results

One animal died because of an accident during the anesthetic procedure; it was not replaced.

In the four times evaluated, a higher collagen concentration was observed in wounds treated with metronidazole, with significant differences on the seventh (p = 0.012) and 21st (0.001) days (Figure 1).

Concentration of type I collagen was higher in the experimental group in all times, but it was only significant on the seventh (p = 0.020) and 21st days (Figure 2).

Concentration of type III collagen was similar in both groups in the initial time, and it was higher in the experimental group on the 21st day (p = 0.005) (Figure 3).

The fact that angiogenesis was more effective in wounds treated with metronidazole was revealed by the presence of more vessels in all times evaluated. However, a significant difference was only observed on the third (p < 0.001) and 14th (p = 0.003) days (Figure 4).

Discussion

Mallikarjuna et al.¹⁷ compared the use of oral metronidazole (180 mg/kg) to topical metronidazole (10 mg/kg) on partial thickness burn wounds. They reported that oral administration promoted epithelization, whereas topical metronidazole depressed it. According to them, systemic metronidazole could be assumed to have scavenged the free radicals and prevented lipid peroxidation, thus promoting the healing. They argued that the inefficacy of topical metronidazole could be attributed to the high concentration, which would be cytotoxic to fibroblasts.

Prasad & Rao⁴ used 160 mg/kg of oral metronidazole. They reported increased wound contraction and accelerated epithelization.

Girish & Patil¹⁸ conducted an investigation using 108 mg of oral metronidazole to evaluate wounds produced on the abdomen of rats. They reported that metronidazole promoted tissue formation and keratinization, increased breaking strength and collagen content, represented by a higher hydroxyproline content.

Differently from other authors, Borden et al.¹⁹ reported that the use of 20/mg/kg of metronidazole predisposes to failure in wound contraction. Girish & Patil¹⁸ argued that the divergent results reported by Borden et al.¹⁸ may be because of the smaller dose of metronidazole used in the latter.

In the present study, wounds treated with metronidazole were observed to have higher collagen concentration than wounds treated with 0.9% NaCl solution in all times; however, this difference was only significant on the seventh and 21st days. When the type of collagen is evaluated, it is observed that it is type I collagen that causes that difference. It should also be considered that if the sample were larger, differences could be also significant in the other times evaluated.

It should also be considered that, while Prasad & Rao,⁴ Mallikarjuna et al.,^5,17 Girish & Patil,¹⁸ and Borden et al.¹⁹ used oral metronidazole, in the present study topical metronidazole was used. Stoltze & Stellfeld²⁰ evaluated the systemic absorption after topical application of metronidazole 25% gel into inflamed periodontal pockets, without inducing high plasma concentrations.

This dosage probably represents the limit for the effect of metronidazole on collagen synthesis. Thus, low doses such as those used by Borden et al.¹⁹ (20 mg/kg) would not promote collagen synthesis; however, high doses, such as those used by Prasad & Rao⁴ and by Mallikarjuna et al.^4,5,17 (160 mg/kg) would have a cytotoxic effect on fibroblasts, thus inhibiting collagen production. However, since administration routes were different, it is not possible to know what the concentration was in the wounds of those who received oral metronidazole.

Healing depends on two foundations: vascularization and ability to produce collagen. Vascularization promotes the formation of new cells in the inflamed site and transportation of nutrients and oxygen. For Reed et al.,²¹ angiogenesis is influenced by the transforming growth factor-beta1 (TGF-beta1) and by the matrix protein. TGF-beta1 enhances cytokine production, which promotes angiogenesis and production of type I collagen, and inhibits production of interstitial collagenase.

Ingber & Folkman²² reported that type I collagen is found among proteins of the matrix that regulates wound repair, thus promoting angiogenesis. Probably the use of metronidazole, in appropriate dose, stimulates fibroblasts to produce collagen, thus promoting an increased neovascularization.

In the present study, metronidazole promoted angiogenesis. Considering that there was an increased number of vessels and collagen production, it can be claimed that granulation tissue formation was stimulated.

Results of the present study suggest the hypothesis that metronidazole acts in different forms during the healing process. Despite the fact that a fibroblast marker was not used, the possibility should be considered that metronidazole reduced the duration of the inflammatory process because it is an antibiotic, thus enabling fibroblasts to be more efficient.

Results indicate an increased collagen production and angiogenesis, resulting in early healing. Poletti et al.²³ and Kalinski et al.²⁴ reported the use of metronidazole, in gel or topical formulation, for wound odor control, based on the eradication of anaerobic infection. Since metronidazole is an antibiotic, it should be considered that it could prevent bacterial replication, thus promoting a better result for wound healing in the experimental group. Although secretion has not been found in any of the wounds, the reduced number of colonizing bacteria could facilitate the healing process.

It should be also considered in the analysis that other substances could promote tissue inflammation, stimulating an increased collagen production and angiogenesis; thus , this is not an exclusive property of metronidazole.

Conclusion

Metronidazole contributes to healing wounds by second intention in rats and stimulates collagen production and angiogenesis.

References

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Publication Dates

• Publication in this collection
  05 Jan 2010
• Date of issue
  Sept 2009

History

• Accepted
  03 Aug 2009
• Received
  25 Mar 2009