Materials and Methods
This experimental study was conducted on 42 adult male Sprague Dawley rats from the same colony, with each rat weighing between 250 g and 350 g. The rats were obtained from the Experimental Animals Laboratory of the Karadeniz Technical University in Trabzon, Turkey. The purpose of using rats was due to their availability, being safe to use, and resistance to infections and surgical procedures when compared with other experimental animals, as well as having a high ratio of repeating the experiment and having substantial data in the literature on the characteristics of rats' skin and the cicatricial process.[10,11] Male rats were used to ensure there would be no interference in hormonal variation due to females' estrous cycle, which could impede in the tissue repair process.
The study was approved by the local ethics committee at the Karadeniz Technical University Faculty of Medicine, Animal Care and Use Committee. The rats were handled in accordance with the 8th edition of the Guide for the Care and Use of Laboratory Animals.
The rats were randomly divided into 2 groups: group A (control group; n = 21) and group B (nitrofurazone group; n = 21). In both groups, circular full-thickness skin defects of about 1 cm x 1 cm in size were formed in the left thoracoabdominal regions of the rats. On days 3, 7, and 10, 7 rats from each group were euthanized, and their defected regions were resected (Figure 1).
Macroscopic images of the skin defects created in the rats in group A are seen in the upper images at (A) baseline, (B) day 3, (C) day 7, and (D) day 10. Those in group B are seen in the lower images at (E) baseline, (F) day 3, (G) day 7, and (H) day 10.
Group A. In the control group, with the exception of application of local saline to the wounds once daily to prevent the wound from drying, no treatment was given to the rats.
Group B. In the nitrofurazone group, a thin layer of cream containing nitrofurazone (Furacin Pomade, 0.2%; Zentiva) was applied to the wounds on the rats once daily.
Based on previous experiment methodology, the rats were kept under special pathogen-free conditions to prevent infections and were placed separately in a light-controlled room with a 12-hour light/12-hour dark cycle. The temperature was kept at 22°C ± 0.5°C, and the relative humidity remained between 65% and 70%. Care was taken to avoid unnecessary stress during the study. The rats were given standard laboratory rodent chow and water. The animals had neither been used in another study nor been given any drugs previously. Their feeding was discontinued 12 hours before the experiment began, but they were allowed to drink water.
Technical and Surgical Procedures
All of the rats were anesthetized by administering ketamine hydrochloride (Ketalar; Eczacıbaşı) 50 mg/kg and xylazine hydrochloride (Rompun; Bayer) 3 mg/kg intraperitoneally. The anesthetic deepness was assessed with extremity pulling response, and additional doses were administered when necessary. The rats were placed in the supine position during the procedures, which allowed spontaneous breathing under sterile conditions. A heating pad was used to keep their body temperature at 37.0°C in order to prevent the effects of hypothermia and maintain the stability of hemodynamic parameters. The left thoracoabdominal regions of the rats were shaved without harming the skin, and the area was cleaned with 10% povidone-iodine solution (Baticon 10% Solution; Adeka Pharmaceuticals). A circular full-thickness wound tissue measuring 1 cm x 1 cm was formed by using a no. 15 surgical blade to remove the skin and subcutaneous tissue without damaging the underlying aponeurosis. Firm pressure was immediately applied to the defect areas to control bleeding.
No complications occurred in the rats, and none were lost during the surgical procedure. Wound care for all 42 rats was performed daily.
The defect sizes of all the rats were photographed at baseline and days 3, 7, and 10 of the experiment. The reductions in wound size were measured macroscopically on the computer using the metric system. The Formula was used to calculate the healing rates based on the present data.
When the aforementioned times mentioned in the study protocol expired, the rats were euthanized with high doses of the anesthetic drug administered intraperitoneally, and their defect areas were resected as full thickness, retaining at least 1 cm of unharmed tissue around the defect. The specimens were promptly fixed in 10% formalin and processed for paraffin embedding. Then, tissue sections of 5 μm in thickness were obtained with a microtome. Light microscopy (CX41 Upright Microscope; Olympus) was used for histopathological analysis of the hematoxylin and eosin-stained and Masson's trichrome-stained sections. The histopathological assessment was carried out by the same pathologist, who did not know which tissue specimen belonged to which group; the assessment was conducted through random selection. The histopathological examination was performed according to the scoring of the wound healing assessment (Table 1); inflammatory cells, collagen accumulation, granulation tissue formation, reepithelization, and features of skin defect (eg, what layers of the skin were affected by it, defect size, any abscess-necrosis) were assessed in the specimens.
In this scale, the parameters were scored from 0 to 3 and were recorded separately for the rats in each group. For inflammatory cells, the following points occurred: 0 points for no inflammation; 1 point for a dispersed, small amount of mixed inflammation; 2 points for moderate mixed inflammation concentrating around vessels; and 3 points for intensive mixed inflammation concentrating around vessels and forming clusters. For collagen accumulation, scores included: 0 points for none; 1 point for patch-like collagenization in the form of short strips; 2 points for strip-like, thin collagenization; and 3 points for strip-like, coarse, thick collagenization. For granulation tissue formation, scores included: 0 points for none; 1 point for involving less than 10 new vessel formations in 1 high power field (HPF); 2 points for involving 11 to 20 new vessel formations in 1 HPF; and 3 points for much and mature granulation tissue involving more than 20 new vessel formations in 1 HPF. For reepithelization, scores included: 0 points for none; 1 point for partial reepithelization in less than one-third of the tissue; 2 points for thin reepithelization in one-third to two-thirds of the tissue; and 3 points for mature reepithelization in the entire tissue. For features of skin defect, scores included: 0 points for none, 1 point for an ulcer limited to the epidermis and smaller than 0 cm to 0.4 cm in diameter microscopically, 2 points for an ulcer limited to the epidermis and papillary dermis of 0.4 cm to 0.6 cm in diameter microscopically, and 3 points for an ulcer in the epidermis and extending to reticular dermis, wider than 0.7 cm microscopically or involving necrosis-abscess formation (Table 1).
All statistical data analyses were performed using the SPSS Statistics Version 15.0 for Windows (SPSS Inc.). The histopathological scores of the results obtained for the groups at days 3, 7, and 10 were individually compared within group A and group B, as well as between the 2 groups, using the nonparametric Friedman and Wilcoxon signed-rank tests. The results of the healing rates obtained for group A and group B were compared using the independent samples t test. Statistical significance was set at P < .05.
Wounds. 2020;32(5):134-141. © 2020 HMP Communications, LLC