Animals and experimental protocol
The study used 48 male Wistar Albino rats (Kobay Deney Hayvanları Laboratuarı San. Tic. A.S., Ankara, Turkey), each weighing 250 g to 300 g (age, 5–6 months) and raised under the same environmental conditions. Rats were housed at 20°C to 21°C with a 12-hour light/dark cycle and free access to water until 2 hours before the anesthesia procedure. Animals were divided randomly into 4 groups, each containing 12 rats.
After an overnight fast, rats were given an intraperitoneal injection of streptozotocin (Sigma-Aldrich, St Louis, MO) at a dose of 60 mg/kg body weight in 0.05 mol/L sodium citrate buffer (pH 4.5). Streptozotocin possesses a diabetogenic property characterized by selective destruction of pancreatic islet β-cells. It causes hyperglycemia, insulin deficiency, polyuria, and polydipsia, all of which mimic human type 1 diabetes mellitus.14 Blood glucose concentration was measured with a OneTouch glucometer (LifeScan, Milpitas, CA) on samples taken from the tail vein. The development of diabetes was confirmed by measurement of blood glucose > 200 mg/dL at 3 days and 1 week post streptozotocin injection.
A full-thickness wound measuring 2 cm x 1 cm, involving the panniculus carnosus, was created on the back of each animal by surgical excision. In all groups, the wounds were cleaned daily with 0.9% sodium chloride (NaCl). In group 1 (control) and group 3 (systemic NAC), the wounds were covered with 0.9% NaCl-treated sterile gauze. In group 2 (topical NAC) and group 4 (topical + systemic NAC), the wounds were covered with sterile gauze treated with 3 mL (300 mg) of NAC (Asist 10%, 3 mL, 300 mg, intravenous form, Husnu Arsan, Turkey). The animals in groups 3 and 4 received an additional NAC through an orogastric tube at a dose of 200 mg/kg daily for 14 days. All animals were euthanized on day 14 by using high-dose anesthetic.
Evaluation of wound contraction rates
The wounded areas were observed for 14 days. On days 1 (1 day post wound creation) and 14, the wounds were drawn onto acetate paper. After scanning the drawings, the surface area was calculated using a scientific image processing program (ImageJ, Version 1.45; National Institutes of Health, Bethesda, MD).
Histopathological evaluation
The wounded areas, excised together with the surrounding scar tissue just until healthy tissue outside scarring on day 14, were fixed in 10% phosphate-buffered formaldehyde solution and kept at room temperature for 24 hours. Histopathological examinations were performed by a pathologist blinded to the groups. The prepared specimens were washed under running tap water and dehydrated through a series of graded concentrations of alcohol (70%-40 min, 75%-40 min, 80%-45 min, 85%-45 min, 90%-50 min, 96%-50 min). After dehydration, specimens were placed into xylene to obtain transparency and embedded in paraffin. Then, 5-µm thick tissue sections were cut with a microtome (Leica RM 2125 RT; Leica Biosystems, Buffalo Grove, IL) from the paraffin blocks. These preparations were stained with hematoxylin and eosin and Masson’s trichrome. The degrees of epithelialization, inflammation, and fibrosis were evaluated using semiquantitative scoring systems as shown in Table 1, Table 2, and Table 3.
Evaluation of oxidative stress parameters
The oxidative stress parameters of malondialdehyde (MDA), fluorescent oxidation products (FOP), and total oxidative stress (TOS) were measured in the tissue samples. Levels of MDA were measured using the spectrofluorometric method defined by Wasowicz et al.15 Total sulfhydryl (SH) groups were measured spectrophotometrically using the Sedlak and Lindsay method.16 Tissue homogenates extracted using ethanol-ether for FOP measurements were calculated with a spectrofluorometer at wavelengths of 360/430 nm.17 The TOS measurement was performed using the calorimetric method based on the cumulative oxidation of the molecules from ferrous ion to ferric ion. Data were stated as µmol H2O2 equivalent per L.18
Statistical analysis
All data analyses were applied using SPSS for Windows version 15.0 software (SPSS Inc, Chicago, IL). All variables were found to be distributed normally around the mean, and the data were given as mean ± standard deviation. In the present study, nonparametric tests were used because the number of rats in the groups was < 30. Kruskal-Wallis variance analysis was used for evaluation of the differences between the groups. If the P values obtained from the variance analysis were statistically significant, the Mann-Whitney U multiple comparison test was applied to determine from which group the difference originated. A value of P < .05 was considered statistically significant.