Comparative analysis of the effects of Copaifera multijuga oil-resin and nitrofurazona in the cutaneous wound healing process

Objectives: to evaluate, histologically and macroscopically, the influence of Copaifera multijuga (Copaíba) oil-resin on the healing process of cutaneous wounds, comparing it with nitrofurazone. Methods: we divided 36 rats into three groups of 12 animals, according to the treatment to be administered. Group SL (control) received saline on the lesion; the Group OIL received topical treatment with Copaíba oil; and the Group NITRO was treated with Nitrofurazone. We inflicted a circular wound of 8mm in diameter on the back of each animal. We subdivided each of the three groups of 12 animals into three subgroups, according to treatment time and euthanasia (7, 14 and 21 days). All animals received the proposed treatment daily. We photographed the lesions for area measurement, as well as for evaluation of macroscopic aspects. We resected and stained the scars to quantify and qualify elastic fibers, collagen, degree of epithelization, neovascularization and inflammation. Results: although the saline solution provided a faster wound closure in its initial phase, after 14 days the wound size of the three groups tested was the same. Levels of inflammation and neovascularization were similar in all three groups. The amount of collagen and elastic fibers was higher in the Nitrofurazone and Copaíba oil groups. Conclusion: in male Wistar rats, Copaifera multijuga oil-resin positively influences the healing process, but it is less effective than nitrofurazone in healing by secondary intention.

an important molecule involved in the cellular activation process of the innate immune response 13 .
Nitrofurazone is an antibactericidal agent of the furans family, whose mechanism of action is the inactivation of ribosomal proteins and other macromolecules, with consequent inhibition of proteins, DNA, RNA and cell wall synthesis, blocking the aerobic metabolism of bacterial cells.It can be used as adjuvant in the healing process of cutaneous wounds, since in addition to the antimicrobial activity, it interferes in the formation of granulation tissue.Its topical use is adequate, since it does not suffer significant absorption through whole or burned skin, nor through the mucosa 14,15 .It is found commercially as an ointment (30mg), at a concentration of 2mg/g.The objective of this research was to histologically and macroscopically evaluate the influence of Copaifera multijuga oil-resin on wound healing of rats, comparing it with nitrofurazone.

METHODS
We used 36 adult, male rats of the same age, Rattus norvegicus albinus rodentia mammalia, We divided the sample into three groups of 12 animals each, according to the proposed treatment: the Control group (Group SL), received only 0.9% saline solution on the lesion; the Test Group (Group OIL) received topical treatment with pure Copaíba oil, at the dose of 0.3ml/day; the Comparison group (Group NITRO) was treated with topical Nitrofurazone (2mg/ ml) 0.3ml daily.
We weighed and identified all animals.We subdivided each of these groups into three cages, each with four animals, according to the time they would be submitted to euthanasia, seven, 14 and 21 days 16 .
With the animal duly anesthetized with inhalational isofluorane in anesthetic bell until the deep plane, tricotomy was done on the back of the animal attached to a plank in the ventral decubitus position, with scissors and disposable razor 2 .Then, we performed the site antisepsis with 70% alcohol 3 and made a circular wound using a dermatological punch, measuring 8mm in diameter, removing skin and subcutaneous, without damaging the underlying aponeurosis.Hemostasis was achieved by digital compression and gauze 16 .
All animals received the proposed topical treatment for each group daily, respecting the 24hour interval between the applications.In each of these applications, a gauze dressing was made around the animal with micropore tape, so that the animals were not subject to limitation of respiratory incursions and did not have direct contact with their wounds and the wounds of the other animals of the cage.We managed postoperative pain with tramadol 50mg/ml at a dose of 5mg/day, intramuscularly for three days.
At the end of each pre-established period (7,

RESULTS
We compared the groups at each moment by testing the null hypothesis of equal results in the three groups versus the alternative hypothesis of different results.When comparing the areas of the wounds to each other within the same moment of the experiment, we observed no statistical significance (p>0.05).
Although the groups SL and OIL had better results in the first seven days, on the 14th day of the experiment   all the animals had wounds of very similar area, remaining in this tendency until complete healing.Figure 1 shows the statistics of the area variable as a function of time within each group.
We analyzed inflammation, epithelization and neovascularization based on histopathological analysis of the HE-stained slides (Tables 1, 2 and 3).
By analyzing and quantifying types III and I collagen fibers in each group and comparing with the time of analysis, we obtained the data shown in Figures 2 and 3.
We observed that saline had the worst performance, not stimulating the production of collagen in the same proportion as the other tested compounds.Copaíba oil was more capable of converting type III (young) collagen to type I (mature) collagen, a fact that has a positive effect, since the higher the amount of mature collagen, the greater the wound's mechanical resistance.
Applting the same method used to measure the collagen fibers (except for the staing used, the Weigert's), we obtained an estimate of the amount of elastic fibers in each wound (Figure 4).Elastic fibers are essential in that they give elasticity to the tissue, but as the latter gets more mature, the tendency is for some of them to join the collagen fibers, thus making the tissue more resistant.of data available in the literature on the characteristics of the skin and the cicatricial process of these animals, as well as the great resistance they display to infectious processes and surgical aggressions, besides being easy to obtain and to handle.The animals selected for the study were necessarily male, so that there was no interference of the hormonal variation due to females' estrous cycle, which could interfere in the tissue repair process 16 .

DISCUSSION
We observed no significant difference between the mean areas of the wounds, except that the wounds of the nitrofurazone group had a smaller tendency to reduce size on the 7th day when compared with the two other groups, a difference that we did not observe at 14 and 21 days, a fact corroborated by other studies 18 .
The findings of inflammation, epithelialization and neovascularization were similar to those observed by Teixeira 18 , and we could observe a greater presence of inflammatory component in the Copaíba group, with evolution to the disappearance of the inflammatory process at day 21 in all groups.All the wounds showed an excellent capacity of neoangiogenesis and, in light microscopy, a rich capillary network with endothelial and red blood cells, which is in agreement with Estevão's findings 19 .
In the quantification of collagen fibers, we found that at day seven, Copaíba oil was more effective than the other two compounds in the induction of collagen formation, predominantly of type I.This differs from the work of Vieira et al. 3 , who found that the animals of the Copaíba oil group presented less collagen fibers when compared with the animals of the saline group.This divergence of data may be because they used Copaifera reticulata instead of Copaifera multijuga, used in this study.
At 14 days, there was an expressive increase in the amount of collagen of both types I and III in the wounds of the nitrofurazone group, reaching levels close to those of healthy skin (4:1 ratio).There was not a significant increase in the number of such fibers in the Copaíba group, the same happening in the saline group.
With 21 days of experiment, we could observe that both the OIL and NITRO groups reached good levels of collagen fibers, enough to maintain tissue resistance and retraction force.Saline did not prove to be a good agent inducing the formation of collagen fibers, with approximately 20% less collagen fibers than the other groups.
By the Weigert coloration, we could measure the amount of elastic fibers in each wound.These give greater elasticity to the tissue, besides interspersing with the collagen, conferring greater resistance.
Nitrofurazone proved to be the best inducer of elastic fiber formation, but was closely monitored by the other two groups until the 14th day.On day 21, there was a large drop in elastic fiber levels in the SL group, and the NITRO and OIL groups finished the experiment with very similar levels.

R E S U M O
from the Wistar line, from the TECPAR vivarium.All animals were acclimated and kept in the laboratory of the Paraná Evangelical Faculty (FEPAR), receiving water and chow for the species ad libitum, and respecting the ethical principles of animal handling and experimentation defined by the Animal Experiment Ethics Committee and the Brazilian Legislation on Animal Experimentation, Federal Law N o 6638, of 1979.The research project was submitted and approved by the Ethics Committee on the Use of Animals of FEPAR (protocol number 004988/2012).
14 and 21 days), we again weighed four animals from each group and killed them by anesthetic overdose with inhaled isofluorane.The animals were then attached to the surgical board, where the lesions were analyzed macroscopically by highresolution photographs (8mp) obtained from a fixed camera on a pedestal, with an auxiliary light focus and a millimeter scale present in the field.We submitted the obtained images to software analysis (AutoCad 2013), and accurately measured the area of each wound16 .We resected the dead animals' cutaneous scars respecting a margin of at least 3mm from the edge of the lesion, and immediately put them in previously identified flasks with buffered formalin.Rev. Col. Bras.Cir.2016; 43(6): 445-451 The FEPAR Histotechnical laboratory provided the preparation of the slides with Hematoxylin and Eosin (HE) staining for morphometric analysis (epithelization, classification and degree of inflammatory process, and vascularization), Sirius-red staining (quantified and qualified collagen, types I or III) and Weigert staining for analysis of elastic fibers.The same pathologist evaluated all HE slides with a optical microscopy, without being aware of the group they belonged to.For the evaluation of the amount and type of collagen fibers, as well as the amount of elastic fibers, we captured five images from each wound, with the aid of a microscope with coupled camera and polarized light lens.We then submitted the images to the ImagePro 2013 software, which counted the amount of type I and type III collagen fibers in each slide, as well as counting the elastic fibers17 .For the description of quantitative variables, we considered the mean, median, minimum value, maximum value and standard deviation.For the description of qualitative variables, we used frequencies and percentages.We used the non-parametric Kruskal-Wallis test to compare independent groups (groups at each moment and moments within each group), with p-values lower than 0.05 indicating statistical significance.We analyzed the data with the statistical software Statistica, v.8.0.All the results received statistical treatment, adopting p<0.05 as the level of significance.

Figure 1 .
Figure 1.Wound area as a function of experiment time.

Table 1 -
Wound epithelialization by group according to the day of analysis

Table 3 -
Wound neovascularization by group according to the day of analysis