INTRODUCTION

Pythiosis is part of a piogranulomatous disease complex, which constitutes a diverse group of mycotic and pseudomycotic conditions that are highly similar from an anatomopathological perspective,which affects skin or subcutaneous tissue in what is called zygomycosis (1). It is caused by Pythium insidiosum, and is phylogenetically far removed from the realm of fungi and more closely related to the realm of algae (2).

Histopathologically speaking, there is a multifocal diffuse eosinophilic granulomatous dermatitis caused by intense piogranulomatous inflammatory infiltration, primarily polymorphonuclear eosinophils, followed by macrophages and neutrophils, multifocal necrotic mass and Splendore Hoeppli phenomenon, which is comprised by hyphae and inflammatory cells, particularly eosinophils and around large amounts of inflammatory exudate that is rich in neutrophils and eosinophils, the hyphae are visible in Grocott Methanamine Silver Staining, and branched structures are observed, septate on occasion, of a dark brown color, their walls smooth and parallel, with a size of 2.6 - 6.4 μm, occasionally forming 90° angles (3).

Triamcinolone acetonide (TA) is a powerful corticosteroid that provides anti-inflammatory, antiallergic and immunological activity, due to the fact that it controls powerful inflammation medators, such as prostaglandins, thromboxanes, prostacyclins, leukotrienes, and hydroxy-ethatrienoic acids, all synthesized from Arachidonic acid, the release of which is catalyzed by the A2 phospholipase enzyme; as a result of this event, it will have anti-inflammatory effects, such as polymorphonuclear leukocyte migration delays and decreased fibrinogenesis (4).

There are no reports of histomorphometric quantification for type I and III cellular and collagen types in veterinary medicine that would support diagnosys and prognosis to assess cutaneous pythiosis recovery in horses treated with TA. Therefore, this study seeks to quantify and characterize cell types and collagen fibers that are present in the granuloma healing process in horses that carry pythiosis, whether or not they were trated with triamcinolone acetonide over time.

MATERIALS AND METHODS

Ethical aspects. To conduct this descriptive study, a non-probabilistic approach was used on convenient animals that were naturally infected with cutaneous pythiosis. This study was approved by the Universidad Federal de Viçosa (UFV) Animal Use Ethics Comission (Comisión de Ética en el uso Animal - CEUA), process No. 74/2012. No animals were subjected to processes that would cause unnecessary pain and/or stress. Animals were immobilized pursuant to the animal handling and restraint technical standards, in compliance with the Universal Declaration of Animal Rights, concerning the CIOMS (Council for International Organizations of Medical Sciences) international principles of biomedical research conducted on animals, established in 1949 by UNESCO (United Nations Educational, Scientific and Cultural Organization) and the WHO (World Health Organization) and Law 84 of October 27, 1989 (Colombian Animal Protection Statute) (5).

Location. This study was carried out in the Caribbean coast of Colombia, in the department of Córdoba, located between Greenwich meridian coordinates latitude 7°23’ and 9°26’ north, and longitude 74°52’ and 76°32’ west, at an elevation of 30 meters above sea level, with annual average temperatures of 28°C, a relative humidity of 82%, an annual rainfall of 1.400 mm and a tropical forest weather. There are two clearly defined seasons (rainy season and dry season) (6).

Animals. 24 horses with cutaneous pythiosis of both sexes and at different ages were used, averaging 380 kg in weight. The inclusion criterion was based on the presence of clinical cutaneous pythiosis, diagnosed in the general physical exam performed during the selection process and confirmed by the histopathology shown in Hematoxylin & Eosin (HE) and Grocott staining. Animals in the experiment were put under the same management conditions they were in at their place of origin and received the same feeding and handling rigor they had before the experiment, in such a way that grass and water were left ad libitum .

A group of 12 animals suffering from pythiosis received an intramuscular treatment consisting of 50 mg of triamcinolone acetonide applied 3 times every 15 days (treated group - TG). The other group of 12 animals suffering from pythiosis received no treatment (control group - CG). Tissue biopsies were collected from the granuloma periphery of all animals in the trial every four days until the granuloma TG was fully cured.

Methodology. Preliminary sedation using Acepromazine (Tranquilán 1%, Zoo® , Colombia) administered intravenously at a dose of 0.05 mg/kg, and anesthesia using vasoconstrictor-free lidocaine solution (Lidocaine 2%, Synthesis® , Colombia). Normal and diseased cutaneous tissue were collected in a biopsy from the periphery of the lesion and throughout its entire thickness with a 6 mm punch (7), and were later analyzed at every stage of the scarring process (inflammatory, granulation, contraction and epithelization). The samples were placed in 10% formalin for 24 hours, then in 70% alcohol, and were later taken to the University of Córdoba Department of Livestock Sciences,Colombia, where they were processed until being included in parafin.

Subsequently, tissue samples were transferred to the Universidad Federal de Viçosa Structural Biology Laboratory of the General Biology Department, where they were processed for histopathological and histochemical assessment, cut to a thickness of 5μm on a microtome (Leica RM2125 RTS®, Japan ) and dyed using Hematoxylin - Eosin (HE), Gomori Trichrome (TG), Picrosirius Red/Polarization (PR/P) and Grocott´s Methanamine Silver (GMS) to perform a hystomorphometric analisis of cell content, blood vessels, total collagen fibers, collagen fibers (type I and III), and hyphae from the oomycete P. insidiosum respectively.

A microscope with a polarization option (Olympus BX-53®, Japan) from the Laboratory of Molecular Systematics/BEAGLE of the Department of Animal Biology at Universidad Federal de Viçosa, Brazil, was used to perform sample analysis. 10 histological fields were selected and photomicrographed with a digital camera (5.0 m) and a magnification of 40x. The fields were selected from left to right and top to bottom and, for each photomicrographed field, two fields were discarded, until reaching a total of ten fields. Images were analyzed with the help of the BioEstat 5.0 software (8), establishing the number of eosinophil, neutrophil, macrophage, blood vessel, fibroblast and collagen (general, types I and III) tissue cells, in addition to the number of hyphae in Pythium throughout the course of this study, by conducting a point counting planimetric assessment (using a 20 x 16 mm grid).

An analysis of variance (ANOVA) was used to compare the data produced between two or more means. The ANOVA was used to verify whether there were significant differences between the two groups (CG and TG) over time. The minimum level of statistical significance used in all tests was 95%. Student’s t test was conducted showing significant differences (p≤0.05) between the two groups (GT and GC). Considering variable reduction to be the response variable in the treatment group over time. Pearson’s simple correlation test was also used to verify the relationship between the characteristics studied. All statistical tests were performed with the aid of the SAS computer program 9.1.3 (9) and Excel 2003 was used for data tabulation and organization.

RESULTS

Grocott Methanamine Silver Staining (GSM). The presence of branched structures, occasionally septated, with a dark brown color and smooth and parallel walls, occasionally forming 90° angles, was observed according to what Márquez et al (3) reported as a diagnostic method.

Figure 1 shows the quantification of the presence of P. insidiosum septated hyphae in biopsies collected from both groups. Therefore, the comparison between the two groups (TG and CG) showed that intralesional hyphae decreased progressively in the TG as the granuloma recovery progresses; furthermore, the t - student test analysis established a highly significant statistical difference (p=0.0001<0.05) between the gradual decrease of tissue hyphae in the GT compared to the CG from day 4 to day 20. The TG had an evident presence throughout the study with a 5.2% increase in intralesional hyphae quantification.

Hematoxylin dyeing - eosin . Multifocal and diffuse piogranulomatous lesions, characterized by an intense proliferation of irregular connective tissue and a marked inflammatory infiltration known as the Splendore-Hoeppli phenomenon were observed in HE-dyed tissues, which correspond to the piogranulomatous eosinophilic and macrophagic reactions with multifocal necrotic lesions increased by Oomycete hyphae, according to reports of the histological features of pythiosis (7,10).

Eosinophils. Eosinophil quantification is shown in Figure 2, the eosinophil breakdown of both groups during the experiment can be observed. Comparing between the two groups (TG and CG), the TG showed a sharp and progressive decrease in the number of eosinophils contained in the lesion. Using the t-student test as an analysis tool, a statistically significant difference (p=0.0001<0.05) was established between the two groups starting at day 4 and until the end of the trial (p<0.0001). The TG experienced a 13.2% increase in intralesional hyphae quantification.

Neutrophils. Neutrophil quantification is described in Figure 3; when comparing the two groups, the TG showed an initial decrease on the 4th and 8th days, followed by a slight increase on days 16 and 24, and then finally decreased progressively until the end of the study. Using the t-student test as an analysis tool, a statistically significant difference (p=0.0001<0.05) was established between the two groups starting at day 4 and until the end of the study (p<0.0001). The TG experienced a 34.6% increase in cell quantification throughout study.

Macrophages. Figure 4 illustrates macrophage quantification for the animals used during the trial. When comparing the two groups, the TG showed an initial decrease on the 4th and 8th days, followed by a slight increase on days 16 and 24, and then finally decreased progressively until the end of the study. Using the t-student test as an analysis tool, a statistically significant difference (p=0.0001<0.05) was established between the two groups starting at day 4 and until the end of the study (p<0.0001). The TG experienced a 22.3% increase in cell quantification throughout study.

Polymorphonuclear (PMN) totals. PMN quantification is illustrated in figure 5, where it can be observed that the number of PMNs sharply and progressively decreased, from day 4 until the end of the study. Using the t-student test as an analysis tool, a statistically significant difference (p=0.0001<0.05) was established between the two groups starting at day 4 and until the end of the study (p<0.0001). The TG experienced a 15.5% increase in cell quantification throughout study.

Blood vessels. Blood vessel quantification is shown in figure 6, where it can be observed that the TG had a slight decrease on days 4 and 8, then an increase on days 16 through 40, and finally a gradual decrease until the end of the study. Using the t-student test as an analysis tool, a statistically significant difference (p=0.0001<0.05) was found between the two groups starting at day 4 and until the end of the study (p 0.0001). The TG experienced a 15.5% increase in cell quantification throughout study

Gpmori trichrome staining (TG). All TG-stained tissues showed a slight presence of fibroblast and collagen fibers, disordered and scarce, as well as small areas with an absence of fibroblasts and collagen fibers, and loss of epithelium with a fibrinoleucocyte layer formation in the area affected by granuloma . Similarly, it is evident that in as the healing process progresses in the TG, the number of fibroblasts and collagen fibers increase progressively in the areas where there were none before, and they are seen to be thicker and nore highly organized from a mild to moderate, and to intense degree, according to the method used to assess the presence fibroblast and collagen fibers in the tissue (11).

Fibroblasts. Fibroblasts quantification is illustrated in Figure 7. Comparing the two groups, the TG showed an initial decrease on the 4th, 8th and 16th days, followed by a sharp increase on days 24, 32 and 40, and then finally decreased progressively until the end of the study. Using the t-student test as an analysis tool, a statistically significant difference (p=0.0001<0.05) was established between the two groups starting at day 8 and until the end of the study (p<0.0001). The TG experienced a 10.6% increase in cell quantification throughout study.

Total collagen fibers. Total collagen fiber percentage quantification is shown in Figure 8, where it can be observed that the TG experienced an initial decrease on days 4 and 8, then a moderate increase on day 16, where it became greater on day 24 , and a sharp increase on days 32 and 40, with a progressive increase that continued until the end of the study. Using the t-student test as an analysis tool, a statistically significant difference (p=0.0001<0.05) was established between the two groups starting at day 4 and until the end of the study (p<0.0001). The TG experienced a 3.6% increase in the total coallagen fiber quantification throughout study.

Dyeing with Picrosirius Red Polarization(PR/P) (PR/P). A homogeneous mix of colors characterized by areas of reddish and green-yellow birefringence was observed, indicating the presence of mature type I collagen (bright red color in polarization) and type III (green-bright yellow color in polarization) where type I collagen was the most prevalent, and type III collagen fibers were thinner and less organized. Similarly, as the healing process progressed in the TG, there was an increase in type III collagen, and the effect of such was seen in the changing color of collagen which moved towards a green-yellow color; thereafter, the type III collagen birefringence areas were gradually replaced by type I collagen, which could be seen in the changing color of collagen which moved towards a reddish yellow. The presence of type I collagen was observed at the end of the healing process, which was demonstrated by the predominance of bright red birefringence, according to the method used to assess the presence of type I and type III collagen fibers in tissue (12).

Type III collagen fibers. The quantification of type III collagen fiber percentages is illustrated in Figure 8; when comparing the two groups, the TG showed an initial decrease on days 4 and 8 days, followed by a sharp increase on days 12 and 16, and then finally decreased progressively until the end of the study. A statistically significant difference (p<0.05) was seen between the two groups on days 4, 8, 32 and 48, and a highly significant difference (p<0.0001) on days 16, 24, 56 and 64. The TG experienced a 5.0% increase in total type III collagen fiber quantification throughout study.

Type I collagen fibers. The total collagen fiber percentage quantification is shown in Figure 8, where it can be observed that the TG experienced an initial decrease on days 4 and 8, then a moderate increase on day 16, where it became greater on day 24 , and a continuous progressive increase on days 32 and 40, with a progressive increase that continued until the end of the study. Using the t-student test as an analysis tool, a statistically significant difference (p=0.0001<0.05) was established between the two groups starting at day 4 and until the end of the study (p<0.0001). The TG experienced a 3.2% increase in total type I collagen fiber quantification throughout study.

Correlation analysis. A simple Pearson’s test was to determine whether a correlation exists between the variables, which are described in table 1. A significant correlation between the neutrophil/macrophage (p<0.01), eosinophil/neutrophile (p<0.03), total collagen fibers/ type I collagen fibers(p<0.01) and total collagen fibers/eosinophils (p<0.03) variables was revealed.

DISCUSSION

No report on the use of triamcinolone acetonide in equine cutaneous pythiosis treatment was found in the literature reviewed, which makes this the first study to evaluate histomorphometry therapeutic response using histopathological and histochemical techniques.

Wound healing times depend on various factors including the extent and anatomical location of the lesion and the presence of local infection. The process to heal wounds in horses is highly resembles that of humans, which is produced by the re-epithelialization of the skin surface with the dermis, which heals through stroma granulation, fibroblast migration and wound contraction. The skin-healing process is divided into 5 well-defined stages, which include the vascular coagulation, inflammatory, proliferative or granulation, epithelization and remodeling or maturation stages, where the granular stage is subdivided into vascular permeability, re-epithelization, fibroplasia, matrix formation and lastly angiogenesis (13).

The results obtained in the present study were satisfactory with a 100% total recovery at 60 ± 3.4 days after applying the treatment to all TG animals. None of the treatments studied provides favorable results in terms of achieving a complete solution to the disease, but they show conflicting results regarding the use of the antifungal drugs assessed (7).

Large amounts of degranulated eosinophils (Splendore-Hoeppli reaction), neutrophils and macrophages around the hyphae are primarily responsible for the extensive and rapid tissue damage and also the main Pythium mechanism of defense, as they release various types of inflammation mediators to destroy the epithelial cells of the host, the interaction between these toxic proteins and cells leads to osmotic and calcium disturbances that cause cell death from necrosis or apoptosis (14).

In the TG showed a significant reduction in Oomycete hyphae, which led to their complete disappearance 16 days after the treatment was applied. The foregoing is explained by the fact that TA decreases the eosinophil by blocking the synthesis of IL-5 and the stimulating factor in granulocyte-macrophage (GM-CSF) colonies, which triggers cell apoptosis in these cells, and thus Pythium is exposed to neutrophils and macrophages, which in turn eliminate tissue hyphae completely and stimulates the physiological healing of wounds. In addition, glucocorticoids extend neutrophil survival and reduce apoptosis (15).

In the TG, PMN behavior was analyzed throughout the study, where a sharp and progressive decrease in PMN cells was observed, especially in the Splendore-Hoeppli reaction (degranulate eosinophils), showing that TA inhibits Th2 profile cytokines, especially IL-5, which is the main factor involved in eosinophil chemotaxis, activation and survival (14). The TG experienced no decrease in PMN cells and the continuous antigenic stimulus of P. insidiosum provided a persistent stimulation of Th2 profile cytokines, primarily IL5 and GM-CSF (granulocyte and macrophage colony stimulating factor), which activate eosinophils, creating the Splendore-Hoeppli phenomenon , and a subsequent piogranulomatous reaction with a high tissue proliferation.

All wounds initially undergo an acute process, characterized by neutrophil infiltration, as a first step in the immune response against pathogens, which acts as the first line of defense in the infectious process, restricting the increase of microorganism production and preventing systemic dissemination. These cells are the main innate immune response component (16), after crossing the endothelium, PMN cells can modulate adaptive immune responses by releasing chemokines and defensins capable of drawing lymphocytes and dendritic cells to the to the inflammation site (17). However, if the the injurious agent remains present in the injury, the continuous exoantigen production will sustain the production of these cells; no neutrophil decrease was observed in the TG, and thus maintaining the chronic active or acute granulomatous characteristics of pythiosis .

A neutrophil cell expression decrease was observed on days 4 and 8 in the TG after TA application, whereas an increase was seen in days 16 and 32 days, due to the fact that the cell survival increases with glucocorticoids, decreasing Apoptosis (15) and therefore assisting the elimination of Pythium hyphae. Considering that neutrophils employ 3 strategies to eliminate invading microorganisms, namely; phagocytosis, through internalization and the subsequent oxidative explosion and antimicrobial compounds that are released in the vacuoles by cytoplasmic granules; degranulation, which involves the release of antimicrobial substances at the inflamation site and the release of DNA network structures and microbicidal proteins into the extracellular space, known as NET (Neutrophil Extracellular Traps), which provide a defense strategy to avoid microbial spread, and therefore concentrating microbicide action and perhaps promoting synergy, which helps to eliminate larger pathogens such as fungal hyphae when phagocytosis is impossible (18).

GT macrophages decreased on days 4 and 8, they decreased significantly from day 8 to day 24 (p <0.05), indicating phagocytic activity, after which macrophage expression increased, which indicates that the antigenic stimulus and the causative agent of pythiosis disappeared from the tissue.

Macrophages are efficient phagocytes that envelop pathogens and cell debris. As opposed top neutrophils, macrophages may remain in the tissue for months or years, acting like true sentinels, but their most relevant participation in inflammation and tissue repair is the production and local release of various growth factors and cytokines , which are crucial to inflammatory reaction maturation and to initiating the wound healing process by fibroblast migration stimulation and collagen production (19).

Macrophages in the wound bed may present different functional phenotypes, divided into two groups: macrophages M1 (MM1 - classically activated) and M2 (MM2 - alternatively activated). MM1 are necessary to eliminate residues and possible invasive pathogens, due to the fact that they possess antimicrobial properties by releasing inflammatory mediators that induce tumor necrosis factor α (TNF α), nitric oxide (NO) and Inter leucine 6 (IL- 6), although this is all important for host to defend itself, if the expressed cytokines are extended, they may induce collateral tissue damage. On the other hand, MM2 is present in new tissue formatio and plays an important role in wound healing, angiogenesis and in the defense against parasitic infections (20).

A significant correlation (p = 0.00018 <0.05) was found between neutrophils and macrophages in the TG, which explains Pythium elimination by neutrophils and MM1, as well as tissue repair process activation by MM2 macrophages.

Lucas et al. (21) demonstrated that a decrease in macrophages during the proliferative stage significantly disrupts the transition stage, which delays repair response. The outcomes of this study highlight the importance of the role of macrophages in resolution during the inflammatory stage and, consequently, in the transition from inflammatory stage to proliferative stage, causing a decrease in TGF-β proliferation, fibroblast reduction and decreased extracellular matrix deposition (EMD).

Fibroblast migration and activation become more intense in the presence of macrophages derived from monocytes and with the production and release of chemical mediators (19). Fibroblasts undergo a series of phenotypic changes, initially adopting a migratory phenotype, then a pro-fibrotic phenotype (as they produce collagen I, III and VI), later adopting the myo-fibroblast phenotype, which is rich in actin microfilaments in the cytoplasmic side of the membrane and establishes cell-cell (adherent) junctions, these cells produce the new EMD necessary for cells that support cell and blood tissue, and provide the nutrients and oxygen necesary for cell growth and proliferation (22). TGF-β cytokines (beta transforming growth factor) and PDGF (Platelet-Derived Growth Factor) stimulate fibroblasts to produce collagen and other EMD components.

Fibroblasts and endothelial cells simultaneously begin to migrate on the provisional matrix in the wound space with the help of MMPs (extracellular matrix metalloproteinases), which are regulated by keratinocytes and macrophages at the edge of the wound. MMP degradation of EMD components is necessary to remove and rearrange provisional matrices and allow migration, promoting the formation of new blood vessels in the wound bed. IL-1 is an important migration-promoting cytokine , which stimulates the release of MMPs and synergistically induces collagenase activity related to IFN-γ and TNF-α. Therefore, increased levels of IL-1 may result in EDM degradation and wound repair delays (23).

In chronic wounds, the healing process is characterized by a more proliferative (fibroblastic) inflammatory response, which is exudative, with disorganized and prolonged inflammation, insufficient cellular matrix deposition, decreasing neovascularization and re-epithelialization, with the presence of transcutaneous oxygen tension, necrotic tissue and delayed healing, which causes an increase in wound size, increased secretion which could become purulent, granulation tissue devitalization, and gradually taking on a a friable consistency (7). Which all TG animals experienced throughout the study.

TG fibroblasts had a highly significant increase 24 days after TA was administered, and they remained that way until the end of the study, this is possibly due to the disappearance of the aggressive agent caused by neutrophils and MM1 and to MM2 macrophage stimulation promoting tissue repair and TGF-β cytokine production, which in turn influence fibroblast function and promote fibrosis (24). In addition to stimulating collagen production, these cytokines reduce wound matrix degradation through collagenase and by increasing MMP inhibition by means of a greater TIMP (MMPs and their natural inhibitors) expression (22 ).

Blood vessels showed a significant decrease in days 4 and 8, caused by corticosteroid action during the vascular phase of the inflammatory process, reducing vasodilation and endothelial permeability with a decrease in vasoactive substances such as serotonin or histamine; however, between days 16 and 40 after TA was administered, a significant increase in neoformed blood vessels, primarily mediated by macrophage-derived VEGF-A, assisting the wound healing process was observed, by restoring oxygen and nutrient flow to the site, after which a gradual decrease in blood vessels began at day 48 and until the end of the study, possibly due to vessel apoptosis which normally takes place during the remodeling phase at the end of the scarring process (25).

Collagen is the primary extracellular matrix structural component and accounts for approximately 25% of total body protein. Type I and type III collagens prevail in normal skin dermis, constituting 87% and 10% of dermal collagen, respectively. Collagen fibers play a vital role in maintaining structural integrity and establishing tissue function. In addition to the fact that collagen adds tensile strength, excessive accumulation is detrimental (26).

Auto-regenerative capacity is universal phenomenon present in living beings and is paramount to their survival. Several cell types are involved in the healing process, whose interrelationships are regulated by cytokines, the extracellular matrix (EMD) and extracellular matrix metalloproteinases MMP). When a wound is inflicted, cutaneous healing enables stratified epithelium (epidermis) reconstruction, epidermal-dermal junction and dermis, in addition the vascularization thereof (27).

Pythiosis is a granulomatous disease with dermal lesions, with a high presence of collagen fibers, especially type I collagen fibers. After the TA treatment was administered to the TG, tissue reconstitution began with fibroblasts accumulating at the center of the wound, followed by collagen synthesis and a provisional extracellular matrix. This study showed an overall initial decrease in collagen fiber starting at days 4 and 8, after which a significant increase in type III collagen fibers was observed until day 24, followed by a final decrease and the onset of a significant increase in Type I collagen fibers from day 24 and until the end of the study. The foregoing coincides with PMN cell expression and a decrease in hyphae starting on day 16, when the physiological repair process in TG animals is activated.

The apoptosis process begins in fibroblasts during the final stages of the healing process, where a transition takes place from a fybroblast-rich scar with granulation tissue to an acellular scar. This explains the significant correlation between fibroblasts and collagen fibers. Collagen is a fundamental component in the tissue repair process, it is synthesized from fibroblasts and it is found abumdantly during the proliferative and wound healing phases (28). Collagen types I and III were the most expressive collagen types found during the healing process. Type III collagen appears primarily in the wound, is characterized as young or immature collagen and confers a disorganized appearance to the fibers. Already type I collagen or mature, it replaces the type III in old scars and is characterized by the greater organization of the fibers (12).

Enoch and Leaper (29) state that, over a period of several months, there are changes in collagen organization in the tissue being repaired, which would slowly increase tissue resistance, and reach a normal tissue ratio of approximately 80%. At the end of the process, cellular activity decreases and conjunctive scar tissue becomes rich in collagen, poor in cells and vessels, with an absence of hair follicles as well as sweat and sebaceous glands.

Young and McNaught (30) state that a period of over two years may elapse before scar tissue reaches maturation; during this phase, there must be a balance between collagen synthesis and degradation through fibroblast collagenases, neutrophils and macrophages, which break the molecule into small fragments, to then be denatured and digested by other proteases.

In conclusion, the cell expression, angiogenesis, fibroblast and collagen fiber histomorphometry analysis results obtained in this study show that there is sufficient evidence to infer that triamcinolone acetonide represents a good alternative in the cutaneous granuloma treatment of horses suffering from pythiosis.

Acknowledgment

In memory of Dr. Álvaro Pío Mendoza, because he who advised me in choosing this investigation that served as doctoral thesis topic in Veterinary Science at expressing concern on the need for further experiments and updated techniques. Dr. Pío, his contribution will last forever in the scientific memory of the Córdoba University “Thank you teacher, for his example and wise advice, I was able to fulfill a dream in a reality that I will thank for all my life (José Cardona).”

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Author notes

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