Comparative evaluation of propolis and Persea americana creams for epidermal wound healing in rats

The body weights of all experimental rats were monitored daily using a high-precision digital balance (accuracy ± 0.01 g). Weighing was conducted at the same time each day to minimize diurnal fluctuations related to feeding and metabolic activity. Prior to the experimental procedures, all animals underwent a one-week acclimatization period under controlled laboratory conditions, with unrestricted access to water and a nutritionally balanced standard diet composed of carbohydrates, proteins, fats, fiber, and essential minerals. Throughout the experiment, weight gain was calculated for each animal to evaluate the general health status and any potential systemic effects of the applied formulations. Weight gain was determined using the following equation²⁰:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{The}}\;{\text{weight}}\;{\text{gain}}\left( {\text{g}} \right) = \frac{{{\text{Weight}}\left( {{\text{Cw}}} \right) - {\text{Weight}}\left( {{\text{Lw}}} \right)}}{{{\text{Weight}}\left( {{\text{Lw}}} \right)}}$$\end{document}where: Weight(Cw): The body weight of the rat in the current week, Weight(Lw): The body weight of the rat in the preceding week.

The resulting data were analyzed to assess temporal changes and evaluate the influence of treatment conditions over the course of the experiment.

Prior to wound induction, the rats were anesthetized using intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg), as recommended in previous protocols²¹. Dorsal hair was carefully shaved using an electric razor⁵, and the skin was cleaned with 70% ethanol. A full-thickness excisional wound measuring approximately 1 cm in diameter was then created on the dorsum of each rat using a sterile disposable surgical blade (size 10) to ensure standardized wound margins. Gentle dissection with surgical scissors was performed to separate the skin from the underlying tissue³.

For all wounded groups (2nd, 3rd, and 4th Groups), the wound site was sterilized with Betadine prior to treatment application. The treatment lasted for 15 consecutive days, during which the wounds were visually monitored, and their surface area was measured. The percentage of wound contraction was calculated using the following formula^23–26:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{Contraction}}\;{\text{of}}\;{\text{wound}}\;{\text{area}}\left( \% \right) = \frac{{{\text{W}}\left( 0 \right) - {\text{W}}\left( {\text{x}} \right)}}{{{\text{W}}\left( 0 \right)}} \times { }100$$\end{document}Where W(0): Initial wound area (on Day 0), W(x): Wound area on the day of assessment (Day x).

At the end of the experimental period, all rats were fasted for 24 h and then anesthetized using an intraperitoneal injection of ketamine (50 mg/kg) combined with xylazine (5 mg/kg)²¹. While under deep anesthesia, blood samples were collected from the saphenous vein²⁷. Approximately 1.5–2.0 mL of blood was collected per animal, sufficient for all hematological and biochemical assays. Following blood collection, euthanasia was performed via cervical dislocation using a sharp surgical blade, ensuring rapid and humane termination.

Whole blood samples were centrifuged at 3000 rpm for 10 min at 4 °C to ensure proper serum separation without degradation of labile components to separate plasma, which was used for the assessment of coagulation parameters. Prothrombin time (PT) and partial thromboplastin time (PTT) were measured using a CoData 504 analyzer (Siemens, Germany), employing standard PT and PTT reagents provided by the manufacturer.

For serum biochemical analysis, samples were allowed to clot, then centrifuged, and the supernatant serum was collected. Serum albumin was determined using a colorimetric assay kit (Biosystems, Spain), while glucose concentration was measured with a commercial kit (Agappe Diagnostics, India). Both were analyzed on a fully automated chemistry analyzer (Mindray BC-2800Vet, Mindray Bio-Medical Electronics Co., Ltd., China), and calibration was conducted prior to use. Blood samples were collected in EDTA-coated tubes and analyzed within 30 min to ensure accuracy and sample integrity.

Hemoglobin levels were measured using the Sahli’s method. Complete blood count (CBC), including white blood cell (WBC) count, red blood cell (RBC) count, and hematocrit (HCT), was performed on whole blood using an automated hematology analyzer (Mindray BS-600 m). The mean corpuscular volume (MCV) was calculated using the formula²⁸: MCV = Fl/Cell. All measurements were conducted under standardized laboratory conditions, and proper sample handling protocols were followed to ensure data reliability and reproducibility.

After completing the experimental period, skin tissue samples were collected and immediately fixed in 10% neutral buffered formalin for 48 h. Following fixation, tissues were dehydrated through a graded series of ethyl alcohol concentrations, cleared with xylene, and then infiltrated and embedded in paraffin wax to prepare tissue blocks. Sections were cut using a rotary microtome (RM2125, Leica, Germany) and mounted onto glass slides. The sections were then dewaxed and rehydrated through a descending alcohol series. Staining was performed using hematoxylin and eosin (H&E) as well as Masson’s trichrome (MT) stains. Finally, the stained slides were examined under a light microscope (BX51TF, Olympus, Japan) for histopathological evaluation^29–31.

All statistical analyses were carried out using SPSS software version 21. Results are presented as mean values ± standard error (M ± SE). Before conducting ANOVA, the normality of data distribution was assessed using the Shapiro–Wilk test to ensure the appropriateness of parametric tests. One-way ANOVA was then applied to compare differences between experimental groups. Further pairwise comparisons were performed using Tukey’s and Duncan’s post-hoc tests to identify specific group differences. Statistical significance was set at P < 0.05, while P < 0.01 was considered highly significant. Data visualization through graphs and tables was used to clearly illustrate the experimental outcomes^21,32.

Red Blood Cell (RBC) Count (million/mm³): Among all groups, the Persea americana cream-treated group (4th Group) recorded the lowest RBC count (8.95 ± 0.59), followed by the negative control group (1st Group) with (9.21 ± 0.60). The untreated wounded group (2nd Group) and the propolis-treated group (3rd Group) demonstrated slightly lower values (8.53 ± 0.36 and 8.94 ± 0.19, respectively). These differences were not statistically significant (P > 0.05), suggesting a modest impact on erythropoiesis across treatment groups (Fig. 11A).

Hemoglobin (HGB) (g/dL): The highest hemoglobin concentration was observed in the 1st Group (10.05 ± 0.48), while the remaining groups showed slightly reduced values: 2nd Group (9.47 ± 0.78), 3rd Group (9.77 ± 0.34), and 4th Group (9.79 ± 0.63). Although the differences were minor, the relatively stable HGB levels in the 4th Group may reflect a preserved oxygen-carrying capacity (Fig. 11B).

Hematocrit (HCT) (%): Statistical analysis revealed a significantly higher hematocrit level in the 4th Group (50.19 ± 0.98) compared to the other groups (P < 0.05), indicating an improved red blood cell mass in response to Persea americana cream treatment (Fig. 11C).

Mean Corpuscular Volume (MCV) (fL/Cell): The MCV values were markedly elevated in the 4th Group (56.47 ± 3.14), suggesting an increase in red cell volume. In contrast, the 1st and 2nd Groups showed relatively lower values (51.83 ± 2.97 and 52.19 ± 2.63, respectively), which may indicate a more active hematopoietic response in the Persea americana cream group (Fig. 11D).

White Blood Cell (WBC) Count (thousand/mm³): The 2nd Group displayed the highest WBC count (12.03 ± 0.46), followed by the 3rd and 4th Groups (9.80 ± 0.46 and 9.23 ± 0.18, respectively). These values were significantly different (P < 0.01), possibly reflecting the persistence of inflammation in the untreated group and a moderated immune response in the treated groups (Fig. 11E).

Glucose (Glu) (mg/dl): The 2nd Group exhibited the highest glucose concentration (171.33 ± 2.33 mg/dL) compared to all other groups. In contrast, the 4th Group maintained a stable and significantly lower glucose level (136.00 ± 2.31 mg/dL), indicating better glycemic control during the healing process (P < 0.01) (Fig. 13A).

The wound healing process in the experimental groups showed distinct histological features corresponding to the classical phases of inflammation, granulation tissue formation, contraction, and remodeling. In the control group (1st Group), the skin section displayed intact epidermal layers with a normal dermal architecture (Fig. 14A). In contrast, the 2nd Group (Fig. 14B), representing injured untreated skin, exhibited a pronounced inflammatory phase, characterized by marked erosion of the epidermal layers and infiltration of inflammatory cells, including neutrophils and Langerhans cells. This prolonged inflammation was accompanied by impaired granulation, reflected in the presence of weakly stained collagen fibers in the dermis.

Progression into the remodeling phase was noted, with keratinocyte proliferation leading to the formation of a new epidermal layer, reduced inflammatory infiltrate, enhanced angiogenesis, and gradual restoration of collagen fibers with normalized pink staining. These regenerative changes were most prominent in the 4th Group (Fig. 14D), where both epidermal and dermal layers displayed near-normal architecture. Moreover, this group showed a marked increase in glandular structures, hair follicles, and associated appendages, suggesting that Persea americana cream promoted hair regeneration and comprehensive tissue repair compared to the 2nd and 3rd Groups (Fig. 14B, C, respectively).

In the control group (1st Group), the skin section exhibited normal architecture with well-organized collagen fibers (Fig. 14E). In contrast, in the wounded skin of the 2nd Group (Fig. 14F), the healing process appeared incomplete. The presence of a persistent scab on the wound surface, coupled with insufficient collagen fiber deposition and an abundance of inflammatory cells, such as Langerhans cells and neutrophils, indicated a delayed transition from the inflammatory to the granulation phase. Sensory cells also appeared compromised, further supporting the observation of impaired healing.