Periodo de adaptacion

Journal of Surgical Research 149, 84 –93 (2008) doi:10.1016/j.jss.2007.10.015

Nitric Oxide Donor Improves Healing if Applied on Inflammatory and Proliferative Phase Thaís P. Amadeu, Ph.D.,* Amedea. B. Seabra, Ph.D.,† Marcelo G. de Oliveira, Ph.D.,† and Andréa Monte-Alto-Costa, Ph.D.*,1 *Histology and Embryology Department, State University of Rio de Janeiro, Rio de Janeiro, Brazil; and †Chemistry Institute, State University of Campinas, UNICAMP, São Paulo, Brazil Submitted for publication June 27, 2007

re-epithelialization, and granulation tissue organization) is more impressive. © 2008 Elsevier Inc. All rights reserved. Key Words: Nitric oxide; Pluronic F127 hydrogel; Reepithelialization; S-nitrosoglutathione; Skin; Wound healing.

Background. Nitric oxide (NO) is an important molecule synthesized during wound repair. Studies have reported the use of NO donors on cutaneous wound repair, but their effects in different phases of healing are still not elucidated. The aim of this work was to investigate the effects of topical application of a NO donor (S-nitrosoglutathione, GSNO)-containing hydrogel on excisional wounds in the inflammatory ( inf), proliferative ( prol), and inflammatory and proliferative phases ( infⴙprol) of rat cutaneous wound repair. Material and Methods. In each group (control, GSNO inf, GSNO prol, and GSNO infⴙprol), excisional wounds on the dorsal surface were made and wound contraction and re-epithelialization were evaluated. Fourteen days after wounding, wounds and adjacent skin were formalin-fixed and paraffin-embedded. Collagen fibers organization, mast cells, myofibroblasts and vessels were evaluated. Results. Wound contraction of the GSNO infⴙprol group was faster than control, GSNO inf, and GSNO prol groups, 5 and 7 d after wounding. Topical application of GSNO accelerated re-epithelialization 14 d after wounding, mainly in GSNO infⴙprol group. In addition, the GSNO infⴙprol group showed improved collagen fibers maturation and tissue organization, and lower amount of inflammatory cells in the superficial and deep areas of the granulation tissue, compared with the other groups. Conclusions. NO is important in all phases of rat cutaneous wound repair, but if applied on inflammatory and proliferative phases, the improvement in wound healing (accelerating wound closure, wound

INTRODUCTION

Cutaneous wound healing is a dynamic and complex physiological process that represents a response to tissue damage. This process is subdivided into three complementary and overlapping phases: inflammation, granulation tissue formation (proliferative), and remodeling [1]. Wound healing involves cytokines and growth factors, extracellular matrix components, and several cell types [1, 2]. For normal wound healing, all these phases and their behavior have to be strongly coordinated in a spatial and temporal manner [3]. The inflammatory phase initiates with a disruption of blood vessels and extravasation of blood constituents, as well as with the production of provisional extracellular matrix [4]. At the same time, platelets release several vasoactive mediators and chemotactic factors, such as interleukin (IL), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-␤), that recruit inflammatory cells such as neutrophils and macrophages and also fibroblasts and endothelial cells to the site of wound [1, 5, 6]. These stimuli lead to the next phase, granulation tissue formation, characterized by angiogenesis, myofibroblastic differentiation and fibroplasia [1, 4]. During this phase, many cells differentiate into myofibroblasts that synthesize extracellular matrix and generate mechanical force to allow wound closure [7]. Many events are crucial to promote re-

1 To whom correspondence and reprint requests should be addressed at Histology and Embryology Department, State University of Rio de Janeiro (UERJ), Rua Professor Manuel de Abreu, 444, 3 0 andar, 20550-170, Rio de Janeiro, RJ, Brazil. E-mail: amacosta@ uerj.br

0022-4804/08 $34.00 © 2008 Elsevier Inc. All rights reserved.

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epithelialization, which occurs by proliferation and migration of epithelial cells from the edge of the tissue across the site of wound [8]. When this process occurs rapidly, the restoration of skin functions are quicker and, consequently, a reduction of patient morbidity and mortality is observed. After complete reepithelialization, the remodeling phase begins. This phase is characterized by the replacement of granulation tissue by a scar tissue, mainly by the activation of matrix metalloproteinases (MMPs) that degrade the extracellular matrix components [9]. Nitric oxide (NO) is synthesized by three isoforms of nitric oxide synthase (NOS) [neuronal (nNOS), inducible (iNOS), and endothelial (eNOS)] from its substrate, L-arginine [10, 11]. NO plays many physiological functions such as vasodilatation, inhibition of platelet aggregation, and reduction of leukocyteendothelial cell adhesion. Since NO is one of the molecules present in the wound environment [12], its role in cutaneous wound healing has been the focus of great attention [13–17]. NO has been implicated as a regulator of all phases of wound healing. Studies demonstrated the ability of NO to modulate chemoattractant cytokines, such as IL-1, IL-6, IL-8, and TGF-␤1, and to control collagen deposition, angiogenesis, cellular proliferation, and apoptosis [17, 18]. Experimental studies reported beneficial effects of a diet rich in L-arginine on the healing and survival of injured rats [19 –21]. Inhibitors of NOSs have been reported to delay wound healing [14, 19, 20] and the administration of NO donors or transfection of a wound with the gene for iNOS have beneficial effects on wound repair [13, 15, 22–25]. Although the effects of NO in wound healing have already been described, the effects and the importance of NO in different phases of cutaneous wound healing are still not completely elucidated. S-nitrosoglutathione (GSNO) is a S-nitrosothiol endogenously found in mammals, which is known to act as a NO carrier and donor [26]. GSNO-containing pluronic F-127 hydrogel was already applied on the forearm skin of healthy human volunteers and led to an increase in the local blood flow [27]. Recently, our group reported an improvement of cutaneous wound healing (acceleration of wound contraction, reepithelialization, and granulation tissue organization) by topical administration of GSNO-containing hydrogel during the first 5 d of wounding [15]. Considering that the importance of NO in each phase of wound healing is unknown, although some studies suggest that the main activity of NO is during inflammatory phase [28], the purpose of this study was to investigate the effects of topical application of GSNOcontaining hydrogel in different phases of rat exci-

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sional cutaneous wound healing (inflammatory or proliferative or inflammatory and proliferative phases). MATERIALS AND METHODS Pluronic F-127 (poly(ethylene oxide) 99-poly(propylene oxide) 65poly(ethylene oxide) 99, PEO 99-PPO 65-PEO 99, MW ⬃ 12,0000; ICI Corp., Bridgewater, NJ), glutathione (␥-Glu-Cys-Glu, GSH) (Sigma, St. Louis, MO), sodium nitrite (NaNO 2) (Aldrich, St. Louis, MO) were used as received. All of the experiments were carried out using analytical grade water from a Millipore Milli-Q gradient filtration system (Barueri, SP, Brazil).

GSNO Synthesis GSNO was synthesized as described previously [29]. In brief, reduced glutathione was reacted with an equimolar quantity of sodium nitrite in aqueous HCl solution, under stirring, in an ice bath for 40 min. The final solution was precipitated with acetone, filtered, and washed with cold water and acetone. The final precipitate was further freeze-dried for 24 h. GSNO was stored at freezer temperature (⫺20°C) protected from light.

Preparation of Pluronic F-127 Hydrogels Hydrogels of pluronic F-127 (25 wt%) containing GSNO (100 ␮mol L –1) were prepared as previously described [30]. In brief, solid pluronic F-127 was added to cold water (5°C). The solution was allowed to attain dissolution equilibrium at 5°C overnight. An appropriate volume of GSNO solution (0.35 mmol L –1) was added to the pluronic F-127 solution under gentle stirring in an ice bath for homogenization. Control hydrogel was prepared by the same procedure, using water in the place of GSNO solution. The hydrogels were used immediately after preparation.

Wounding and Hydrogel Application This study was approved by the Ethical Committee for Animal Use of the State University of Rio de Janeiro. Male Wistar rats weighing 250 to 300 g were anesthetized with intraperitoneal injection of ketamine (5 mg/kg) and xylazine (2 mg/kg). The dorsal surface was shaved, a full-thickness excisional wound (1 cm 2) was made on the back of each rat by removing the skin (epidermis and dermis) and exposing Paniculus carnosus. The wounds were not sutured and were allowed to heal by second intension, as already described [14, 31]. Four groups (n ⫽ 6 in each group) were separated as follow: (A) control group (control): in which the hydrogel without GSNO was topically applied daily on wound bed, beginning in the day of lesion until the sixth day after wounding; (B) GSNO in inflammatory phase (GSNO inf), in which GSNO-containing hydrogel was topically applied daily on wound bed, beginning on the day of lesion until the second day after wounding. In the next 4 d of application, only the hydrogel without GSNO was topically applied on the wound; (C) GSNO in proliferative phase (GSNO prol), in which the hydrogel without GSNO was topically applied daily on wound bed on the day of lesion until the third day after wounding. In the next 3 d of application, GSNOcontaining hydrogel was topically applied; (D) GSNO in inflammatory and proliferative phases (GSNO inf ⫹ prol), in which GSNOcontaining hydrogel was topically applied daily on wound bed on the day of lesion until the sixth day after wounding. Two additional groups were created to evaluate if results obtained in GSNO inf ⫹ prol group were due to longer exposition to GSNO; these two groups are (A) control 2 where hydrogel without GSNO was topically applied from day 3 until day 8 after wounding and (B) GSNO prol longer where hydrogel containing-GSNO was topically applied from day 3 until day 8 after wounding. All wounds were covered with 80 ␮L of cold liquid F-127 solution, and after its application on the skin, liquid

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F-127 solution underwent a rapid gelation yielding hydrogel. The concentration of GSNO in the hydrogel was 100 ␮mol L –1, which corresponds to a dose of 8 nmol of GSNO per application. GSNO incorporated in the hydrogel is quite stable during the 24 h periods of application at the temperature of the wound lesion (36°C), with decomposition smaller than 2%, as evaluated spectrophotometrically (data no shown). After each daily application of the hydrogel (with or without GSNO), wound beds were covered with an occlusive wound dressing, until the sixth day after wounding. From the seventh day after wounding, the wounds were left uncovered in all groups. In the groups control 2 and GSNO prol longer wounds were kept without dressing until the beginning of treatment (day 3) and after the end of treatment (day 9). The animals were housed in individual cages with free access to food and water. Wound contraction and re-epithelialization were evaluated; for that, lesion tracings (total lesion perimeter and, 14 d after lesion, also non-re-epithelialized region perimeter) were performed on a transparent sheet on the day of lesion and 5, 7, and 14 d after wounding, as already described [16]. Lesion tracings were digitalized and analyzed using Zeiss image-processing system KS400 (ZeissVision; Oberkochen, Germany). Data are expressed as percentage of the initial wound area and as percentage of re-epithelialized wound area (mean ⫾ standard deviation).

Wound Tissue Processing and Staining All animals were killed in a CO 2 chamber 14 d after wounding. The lesions and the adjacent nonwounded skin were removed, formol-fixed (pH 7.2), and paraffin-embedded. Sections (5 ␮m) were stained with hematoxylin-eosin for histological evaluation and quantification of epidermis and neoepidermis thickness with Sirius red for collagen analysis and with toluidine blue for mast cells evaluation.

Inflammatory and Mast Cells Quantification To evaluate the inflammatory infiltrate, the amount of inflammatory cells was evaluated in HE-stained tissue sections. To quantify inflammatory cells, 10 random fields (0.02 mm 2) were counted using ⫻100 objective (Olympus CBA; Olympus Optical Co., Ltd., Tokyo, Japan). All analyses were performed blindly and repeated without any difference between the replicates. The number of mast cells and the percentage of degranulating mast cells were evaluated in toluidine blue stained tissue sections. Fifteen random fields (0.119 mm 2) were counted using a ⫻40 objective (Olympus BH-2, Olympus Optical Co., Ltd.) and the average cell count per field was calculated for each animal. All analyses were done blindly and repeated, without significant differences among them. Results are expressed as mean ⫾ standard deviation.

Quantification of Epidermis and Neoepidermis Thickness The epidermis and neoepidermis thickness were evaluated in hematoxylin-eosin stained sections. Five random images of epidermis and neoepidermis were captured for each animal using ⫻20 objective. For analysis, a videomicroscopic system (Axiolab Zeiss microscope and a JVC video camera) and KS400 image program were used. The epidermis and neoepidermis thickness (excluding the cornea layer) was measured in three different points for each image. The average of epidermis and neoepidermis thickness was calculated for each animal, data are expressed as mean ⫾ standard deviation.

Immunohistochemistry and Quantification For assessment of blood vessels and myofibroblasts, mouse monoclonal antibody against alpha-smooth muscle actin (␣-SM actin) was used and for proliferating cells, mouse monoclonal antibody against proliferating cellular nuclear antigen (PCNA) (DAKO; Carpinteria,

CA) was used, as already described [31]. Briefly, after antigen retrieval (using trypsin digestion or citrate buffer pH 6.0), tissue sections were incubated with primary antibody. For revelation the Envision system (DAKO) was used. Diaminobenzidine was used as chromogen, and nuclei were stained with hematoxylin. Negative controls were performed replacing primary antibody by nonimmune serum and no labeling was observed. The distribution of blood vessels and myofibroblasts in granulation tissue was evaluated using a stereological method test system with cycloid arcs as already described [32]. Vessels were identified by the presence of blood cells in their lumens or positive staining for ␣-SM actin in the wall (to confirm the presence of smooth muscle cells or pericytes) [33]. Five random fields were analyzed in superficial and deep regions of granulation tissue for each animal using ⫻20 objective for vessels and ⫻40 for myofibroblasts. For analysis, a videomicroscopic system (Axiolab Zeiss microscope, a JVC videocamera and Sony Trinitron monitor; Sony, Pencoed, United Kingdom) was used. Volume density of vessels was estimated in superficial and deep regions and the volume density of myofibroblasts was estimated in superficial region of granulation tissue. The number of PCNA-positive cell nuclei was counted in neoepidermis. In neoepidermis, all basal cells were counted with a ⫻40 objective (Olympus BH-2; Olympus Optical Co. Ltd) and the result is expressed as percentage of PCNA-positive basal cells. The data are expressed as mean ⫾ standard deviation. All analysis were done blindly and repeated, without significant difference among them.

Statistical Analysis Analysis of wound contraction and re-epithelialized wound area and inflammatory cells quantification were done using parametric Unpaired t-test with Welch correction. Mast cell quantification, epidermis and neoepidermis quantification and stereology evaluation were analyzed with a nonparametric Mann-Whitney test. In all cases, P ⬍ 0.05 was considered statistically significant. Statistical analysis was done using the software Graph Pad Instat version 3.01 (GraphPad Software Inc., San Diego, CA).

RESULTS Macroscopic Evaluation

Scab was not observed along the experiment. Figure 1 shows the macroscopic aspect of the wound areas in the control, GSNO inf, GSNO prol, GSNO infl⫹prol, and GSNO prol longer groups. A reduction in the wound area and an improved re-epithelization was observed in all groups, however these effects were more evident in the GSNO infl⫹prol group (Fig. 1). The signs of reepithelialization were observed only 14 d after wounding in all groups. However, at this time point only 16.6% of animals in the control, GSNO inf, and GSNO prol groups presented complete re-epithelialization while in the GSNO inf⫹prol group, 50% of animals presented complete re-epithelialization (data not shown). Analysis of the lesion areas showed that the wound contraction was improved only in the GSNO inf⫹prol group compared with the control group (Fig. 2A). Two days after wounding, the percentage of initial wound area did not present differences among the groups (Fig. 2A). Five and 7 d after wounding, wound areas of the GSNO inf and GSNO prol groups were similar to the control group (Fig. 2A). Five days after wounding, the lesion area was 18% higher in the control group com-

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FIG 1. Macroscopic evaluation of wound in control and in GSNO treated groups. Photographs of wounds in control group, GSNO inf, GSNO prol, GSNO inf⫹prol, and GSNO prol longer treated groups; 5 (d5), 7 (d7), and 14 (d14) days after wounding.

pared with the GSNO inf⫹prol group (P ⬍ 0.05) and the wound area of the GSNO prol group was 22% higher than the GSNO inf⫹prol group (P ⬍ 0.01) (Fig. 2A). Seven days

after wounding, the wound area was 36% higher in the control compared with GSNO inf⫹prol group (P ⬍ 0.01) and the wound area of the GSNO prol group was 35%

FIG 2. Wound contraction and re-epithelialization in control and in GSNO treated groups. (A) Percentage of initial wound area in control, GSNO inf (treated in inflammatory phase), GSNO prol (treated in proliferative phase), GSNO inf⫹prol (treated in both phases), and GSNO prol longer (treated in longer period of proliferative phase) groups; 5 (d5), 7 (d7), and 14 (d14) d after wounding (mean ⫾ standard deviation). (B) Percentage of wound re-epithelialization in control and in GSNO treated groups (GSNO inf, GSNO prol, GSNO inf⫹prol, and GSNO prol longer) 14 d after wounding (mean ⫾ standard deviation).

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FIG 3. Percentage of PCNA-positive epithelial basal cells in wound area of control and GSNO treated groups (GSNO inf, GSNO prol, and GSNO inf⫹prol) 14 (d14) d after wounding (mean ⫾ standard deviation).

higher than the GSNO inf⫹prol group (P ⬍ 0.01) (Fig. 2A). At the end of the experiment (14 d after wounding), wound areas of all groups did not present differences (Fig. 2A). Fourteen days after wounding, the wound re-epithelialization was more advanced in the GSNO inf (⫹45%), GSNO prol (⫹37%), and GSNO inf⫹prol (⫹59%) groups compared with the control group (P ⬍ 0.05, P ⬍ 0.05 and P ⬍ 0.01, respectively), confirming the macroscopic results (Fig. 2B). There were no differences among control 2 and control regarding wound contraction or re-epithelialization in all time points studied. The results of wound contraction and re-epithelialization in the group GSNO prol longer were not different from those observed in group GSNO prol, showing that a longer exposition to GSNO was not beneficial; so histological analysis was not performed in control 2 and GSNO prol longer groups. To evaluate epithelial cell proliferation, immunohistochemistry against PCNA was performed. Figure 3 shows the percentage of PCNA-positive epithelial basal cells in wound areas of all groups. It was observed that 14 d after wounding, epithelial cell proliferation was higher in the control group compared with GSNO inf (P ⬍ 0.05), GSNO prol (P ⬍ 0.01), and GSNO inf-prol (P ⬍ 0.01) groups. This result shows that keratinocyte proliferation was already reduced in all GSNO-treated groups since re-epithelialization was more advanced in those groups, while in the control group keratinocyte were still proliferating. Evaluation of Granulation Tissue

Figure 4 shows the granulation tissue pattern of the control and the GSNO treated groups, 14 d after wounding. It can be observed that the GSNO inf group (Fig. 4B) presented an intense cellularity, including the presence of inflammatory cells and “fibroblast-like” cells, in superficial and deep areas of granulation tissue, compared with the control group (Fig. 4A) and to the others GSNO treated groups. The GSNO prol group

(Fig. 4C) presented a decrease in cellularity, as well as an increase of “fibroblast-like” cells, mainly in deep area of the granulation tissue, in comparison with the control group (Fig. 4A). The GSNO inf⫹prol group (Fig. 4D) presented a decrease in the amount of inflammatory cells and an increase in the amount of “fibroblast-like” (fusiform and parallel to surface) cells compared with the control, GSNO inf and GSNO prol groups. Fourteen days after wounding, the number of inflammatory cells per field in the superficial area of granulation tissue was elevated in control group (41 ⫾ 23) compared with GSNO inf (18 ⫾ 12), GSNO prol (17 ⫾ 9), and GSNO inf⫹prol (14 ⫾ 8) groups (P ⬍ 0.0001, for all). The distribution of inflammatory cells in GSNO inf group was not different when compared with GSNO prol group. The number of inflammatory cells was higher in GSNO inf and GSNO prol groups than GSNO inf⫹prol (P ⬍ 0.05, for both). In the deep area of granulation tissue, no difference was observed among the groups studied. Figure 5 shows the collagen organization in wound areas of the control and GSNO treated groups, 14 d after wounding. Collagen arrangement was analyzed in Sirius red stained sections observed under polarization. The control group (Fig. 5A) showed, in superficial and deep areas of granulation tissue, thicker yellowred collagen fibers arranged parallel to surface. In deep area of GSNO inf group (Fig. 5B) reduced collagen fiber density and the presence of immature collagen fibers (thin yellow-greenish collagen fibers) were observed. The GSNO prol group (Fig. 5C) presented immature (thin yellow-greenish collagen fibers arranged perpendicularly to surface) and mature organized collagen fibers (thicker red-yellow collagen fibers arranged parallel to surface). In deep area of GSNO inf⫹prol group (Fig. 5D), a prevalence of organized mature fibers (thick red-yellow collagen fibers) was observed. Figure 6 shows the immunohistochemistry against alpha-smooth muscle actin (␣-SMa) in the granulation tissue of control group and GSNO treated groups, 14 d after wounding. It can be observed that the vascularization patterns of control and GSNO treated groups were different. In control group (Fig. 6A), high amount of vessels was observed, some of the vessels presented a thick wall. The GSNO inf group (Fig. 6B) presented a decrease in the amount of vessels in granulation tissue and these vessels were smaller than in the control group. In granulation tissue of GSNO prol group, a decrease in the amount of vessels was observed compared with the control and to the GSNO inf groups (Fig. 6C). The GSNO inf⫹prol group showed a decrease in vessel amount compared with all groups (Fig. 6D). In superficial region of granulation tissue, the volume density of blood vessels of control group (22.5% ⫾ 8.1) was elevated compared with GSNO inf (8.8% ⫾ 8.0), GSNO prol (12.2% ⫾ 8.0), and GSNO inf⫹prol (5.8% ⫾ 7.3) groups (P ⫽ 0.0001, P ⬍ 0.005, P ⬍ 0.0001, respec-

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FIG 4. Granulation tissue in control group and in GSNO treated groups 14 d after wounding. Control group (A) presents a higher amount of inflammatory cells in granulation tissue compared with all GSNO treated groups (B)–(D); in GSNO inf (B) an intense cellularity is observed; GSNO prol group (C) presents decrease in cellularity compared with other groups; in GSNO inf⫹prol group (D) an increase in the amount of fibroblastic cells as well as of the granulation tissue organization are observed. HE stained. Bar – 40 ␮m.

tively). The volume density of vessels of GSNO inf group did not present difference compared with GSNO prol and GSNO inf⫹prol, but GSNO prol presented the volume density of blood vessels elevated compared with GSNO inf⫹prol (P ⬍ 0.05). In the deep region of granulation tissue, the volume density of blood vessels of control group (23.3% ⫾ 16.4) was elevated only compared with GSNO prol (11.9% ⫾ 7.0) and GSNO inf⫹prol (7.1% ⫾ 7.4) groups (P ⬍ 0.05, P ⫽ 0.001, respectively). The volume density of vessels of GSNO inf group (16.3% ⫾ 11.4) did not present difference compared with GSNO prol group, but was higher than GSNO inf⫹prol group (P ⬍ 0.05). The volume density of blood vessels of GSNO prol group was higher than GSNO inf⫹prol group (P ⬍ 0.05). In control group (Fig. 6A) and in the GSNO inf group (Fig. 6B) few myofibroblasts were present. In granulation tissue of GSNO prol group (Fig. 6C) a decrease in the

amount of myofibroblasts was observed, and in GSNO inf⫹prol group myofibroblasts were absent (Fig. 6D). In superficial area of granulation tissue, the volume density of myofibroblasts was higher in control group (10.8% ⫾ 11.7) than GSNO inf (3.4% ⫾ 3.8), GSNO prol (6.9% ⫾ 5.7) and GSNO inf⫹prol (1.3% ⫾ 2.6) groups, but it was only statistically significant when compared with GSNO prol group (P ⬍ 0.05). No differences among the other groups were observed. Mast Cell Analysis

In all groups, mast cells were localized mainly in deep area of granulation tissue. The majority of them were ovoid and localized near to blood vessels. Fourteen days after wounding, the total number of mast cells and the percentage of degranulated mast cell in

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FIG 5. Collagen organization in wounds of control group and GSNO treated groups, 14 d after wounding. Control group (A) collagen fibers are thicker yellow-red, and in GSNO inf group (B) collagen fibers are thin yellow-greenish. In the same time point, GSNO prol group (C) presents thin yellow-greenish collagen fibers with parallel arrangement, and GSNO inf⫹prol group (D) presents thin and thicker yellow-red collagen fibers with an organized pattern. Sirius red stained (A)–(H). Bar – 45 ␮m.

deep area of granulation tissue did not present differences among the groups (data not shown). Epidermis and Neoepidermis Thickness

Figure 7 shows the epidermis and neoepidermis thickness of control and GSNO treated groups, 14 days

FIG 7. Epidermis and neoepidermis thickness in control and in GSNO treated groups (GSNO inf, GSNO prol, and GSNO inf⫹prol) 14 d after wounding (mean ⫾ standard deviation).

after wounding. Epidermis thickness did not show differences among all groups (Fig. 7). However, the neoepidermis was 83% thicker (P ⬍ 0.01) in GSNO inf group and 58% (P ⬍ 0.005) in GSNO prol group compared with the control group (Fig. 7). The neoepidermis thickness did not present differences between GSNO inf⫹prol group and control group (Fig. 7). By comparing GSNO inf, GSNO prol, and GSNO inf⫹prol groups neoepidermis thickness was similar between the first two groups, however it was 45 and 36% thicker in GSNO inf and in GSNO prol groups compared with the GSNO inf⫹prol group, respectively (P ⬍ 0.05; for both) (Fig. 7). DISCUSSION

FIG 6. Immunohistochemistry against alpha-smooth muscle actin (␣-SMa) in granulation tissue of control and GSNO treated groups 14 d after wounding. Control group (A) presents a high amount of vessels, some of them with thick wall (arrows). GSNO inf group (B) presents small vessels in granulation tissue, myofibroblasts may be observed (arrow heads). GSNO prol group (C) presents decreased amount of vessels and of myofibroblasts compared with the control group and to the GSNO inf group. GSNO inf⫹prol group (D) presents small amount of vessels and myofibroblasts are not observed. Bar – 70 ␮m.

In this work, a hydrogel containing GSNO was topically applied on wound beds of rat excisional cutaneous lesions during different phases of the wound healing. Its effects on wound contraction, reepithelialization and granulation tissue formation were evaluated. Data showed that the topical application of GSNO in both inflammatory and proliferative phases is the more efficient treatment to improve rat cutaneous wound repair, since the improvement of wound contraction, re-epithelialization, and granulation tissue development was more evident. Functional role of NO on cutaneous wound repair is still not completely understood. Studies have demonstrated that NO is able to regulate several events involved in cutaneous wound repair, such as collagen deposition, angiogenesis, cellular proliferation, and apoptosis [17, 18]. Wound repair is subdivided into three complementary and overlapping phases: inflammatory, granulation tissue formation (proliferative), and remodeling [1]. In all phases, several cell types in the

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wound site are able to produce and release NO [34 –36]. During inflammation, NO acts to kill microorganisms and controls the release of cytokines and growth factors (IL-1, IL-8, TGF-␤, for example) [18]. Since these cytokines are potent chemoattractants, the modulation of these cytokines by NO may also be involved in the next phase of wound healing (proliferative phase), where angiogenesis, recruitment, proliferation, and migration of keratinocytes, fibroblasts, and endothelial cells, as well as myofibroblastic differentiation is observed [28, 37, 38]. NO also seems to stimulate extracellular matrix (mainly collagen) synthesis and deposition; the link between NO and collagen deposition has been described in vivo and in vitro, but it is still controversial [17, 18]. Previous studies have described that closure of rat wounds was improved by administration of NO donors [39 – 42], which suggests an important role of NO in wound closure. However, these works did not distinguish between contraction and reepithelialization. The beneficial effects of NO on cutaneous wound healing have already been reported [12, 14, 43, 44], but the importance of this molecule in different phases of wound repair have still not been investigated and elucidated. This is the first study that reports the effects of a hydrogel containing a NO donor (GSNO) topically applied in different phases of wounding (inflammatory phase or proliferative phase and in both phases). Several parameters were used to evaluate the wound repair process. Wound contraction was one of these parameters [31, 45]. Other studies have demonstrated that administration of NO donors improve wound closure [40, 41], similarly to our results. In the present study, an enhancement of wound contraction 5 and 7 d after wounding in the group where hydrogelcontaining GSNO was applied in both phases of wounding was shown. The enhancement of wound contraction is probably due to an increase in myofibroblastic differentiation during granulation tissue formation, since studies reported that NO stimulates inflammatory cells to secrete a larger amount of growth factors, such as the TGF-␤1 [46], which is known to stimulate myofibroblastic differentiation [7, 18, 47, 48]. Another important parameter that should be analyzed in wound healing process is the re-epithelialization [1]. Several studies reported that wound re-epithelialization is NO-dependent and may be regulated by several growth factors and cytokines, including TGF-␤1, VEGF, EGF, and IL-1 and IL-8 [3, 49 –52]. In accordance with our previous study [15], GSNO accelerates wound re-epithelialization 14 d after wounding in all GSNO-treated groups. As reepithelialization is accomplished by keratinocyte proliferation and migration, keratinocyte proliferation was also evaluated. The results showed that GSNOcontaining hydrogel applied only on inflammatory

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phase, or only on proliferative phase and on both phases presented a decrease of PCNA positive epithelial basal cells 14 d after wounding compared with the control group; at this phase re-epithelialization is almost finished and proliferation of epithelial cells is consequently reduced. This reduction was more prominent in the last group suggesting that, in this group, the proliferation of epithelial basal cells was enhanced and accelerated, allowing the keratinocyte migration and, consequently, leading to the rapid epithelialization, as observed. The fact that neoepidermis thickness is the same among control and GSNO inf⫹prol group can be explained if we imagine that in controls proliferation peak has just started, and in GSNO inf⫹prol group neoepidermis is already been remodeled. Considering that humans heal mainly by re-epithelialization and rats by contraction [53], our results are very important if we assume that GSNO will be used for the treatment of wounds in humans. Inflammatory phase stimulates the next phases of healing: proliferative and remodeling. In proliferative phase, synthesis and deposition of collagen as well as angiogenesis occur. The increase in collagen content during wound repair may be attributed to an increase of collagen synthesis and/or proliferation of fibroblasts [28, 54]. In accordance to other experimental studies [12, 55, 56], our results showed an increase in the amount of fibroblast as well as an increase of collagen deposition, due to the topical GSNO application, mainly when applied in both inflammatory and proliferative phases. The results also showed a more organized granulation tissue in the group where GSNO was applied in the two phases, compared with the other groups. It suggests that the complete reepithelialization observed in GSNO (inf⫹prol) leads to an improvement of granulation tissue development, leading to an acceleration of events of healing. In remodeling phase, the complete re-epithelialization determines the remodeling of vessels as well as apoptosis of myofibroblasts. It confirms and explains the presence of a few vessels and the absence of myofibroblasts observed in GSNO applied in both phases 14 d after wounding. Recently, Weller and Finnen [42] reported that the topical application of acidified nitrite (NO 2 –) cream promote wound healing in normal and diabetic mice. These authors showed that applications starting from day 0 impair wound healing while an acceleration of wound closure is observed when applications start from days 1, 2, or 4. The possible causes for the different results reported in this work may reside in the fact that Weller and Finnen used acidified nitrite in much higher concentrations (in the molar range), compared with the GSNO concentration used in the present work (100 ␮M). In addition, the different chemical nature of the nitrosating species (mainly HONO from the acidified NO 2 – cream and GSNO in the F-127 hydrogel) may

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also lead to different results. Other studies that administrated NO donors suggest that exogenous NO has an important and positive effect on cutaneous wound healing [2, 13, 43, 44, 56, 57]. The biological effect of NO is highly depending on its concentration. High doses of NO increase clot formation and reactive tissue, as well as impair collagen organization, which contribute to cellular toxicity and to delayed wound repair [56]. On the other hand, low doses of NO (in the range 25–100 ␮M) are reported to increase the collagen synthesis, which allows wound repair [58]. For this reason, the concentration of 100 ␮M of GSNO was used in this study. In summary, the results presented in this study demonstrate that the topical application of GSNO on both inflammatory and proliferative phases improves rat cutaneous wound repair by enhancing wound contraction and, mainly by accelerating reepithelialization, show that NO is crucial in both phases. Further studies should be performed to investigate the effects of topical application of GSNOcontaining hydrogel in models of impaired cutaneous wound repair and its influence on the expression of several molecules such as TGF-␤1, EGF, VEGF, and IL-1 to better understand the exact role of NO on cutaneous wound repair and to propose the use of this formulation for the treatment of abnormal wound healing.

9.

10. 11. 12. 13.

14. 15.

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17. 18. 19.

20.

21.

ACKNOWLEDGMENTS This study was partially supported by CNPq, FAPERJ, and FAPESP. TPA held a postgraduate fellowship from CAPES, and ABS from Fundaçao de Amparo à Pesquisa do Estado de Sao Paulo (FAPESP), project 01/07868-9.

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