Ethical clearance

The study began following ethics approval from the Animal Research Ethics Committee at the University of the Witwatersrand (AREC 2020/11/02C). Animal handling and treatment adhered to the committee's established standards and principles.

Animal husbandry

Twenty-four rats (12 males and 12 females) aged seven weeks were placed into either the alcohol-exposed (n = 12) or pair-fed control group (n = 12), at 7 weeks of age weighing approximately 175 g-199 g. All study animals were bred and kept at the University of the Witwatersrand Research Animal Facility (WRAF), Parktown Campus. These animals were maintained under controlled, pathogen-limited conditions, in a temperature-controlled environment (26-28°C) with a 12-hour light/dark cycle. Rats were housed in pairs in plastic cages (2 male rats per cage or 2 female rats per cage) (cage dimensions: 43 mm length, 220 mm width, and 200 mm height), with free movement within the cages. All the animals in the study were fed a standard rodent diet and tap water was provided ad libitum.

Treatment with alcohol or maltose dextrin

The animals (n = 24) were placed into either the pair-fed control (n = 12) or the alcohol-exposed group (n = 12) (Fig.1). They were exposed to alcohol for 1 week. Alcohol was administered via oral gavage of a 20 % (vol/vol) alcohol solution (Associated Chemical Enterprises, South Africa) at a dose of 3 g/kg, chosen to achieve peak blood alcohol concentrations (BACs) of approximately 100 mg/dL (88. Bhika A, Pillay D, Ihunwo AO. Effect of a Dose-Dependent Administration of Binge Alcohol on the Mandible in the Adolescent S prague Dawley Rat. Int J Morphol. 2024;1(2):607-613.
-1010. Walker BM, Ehlers CL. Age-related differences in the blood alcohol levels of Wistar rats. Pharmacol Biochem Behav. 2009;91(4):560-5.
). Alcohol was given 3 days/week (on every alternate day as a single dose). No alcohol was administered during the remaining 4 days of the week.

As a caloric equivalent control, there was a pair-fed rat that was matched individually to an alcohol-fed rat based on initial body weight. One gram of alcohol yields 7 kilocalories (kcal), while 1 gram of maltose dextrin yields 3.89 kcal. Consequently, if 1 gram of alcohol equals 7 kcal/g, then a 20 % alcohol solution at a dose of 1.5 g/kg provides 1.4 kcal/g (1111. Bertola A, Mathews S, Ki SH, Wang H, Gao B. Mouse model of chronic and binge ethanol feeding (the niaaa model). Nat Protoc. 2013;8(3):627-637.
). Due to alcohol being calorie dense, a pair-fed model was used to control potential weight changes resulting from caloric intake rather than the effects of alcohol itself. The pair-fed group was given an isocaloric equivalent of maltose dextrin (Sigma-Aldrich, USA) which was also administered via oral gavage. Oral gavage for both groups was performed with a metal, curved, 16-gauge rounded/bulb-tipped gavage needle.

Measuring blood alcohol concentration levels

Blood alcohol concentrations (BACs) were tested an hour after exposure by drawing blood using the tail prick method. Blood was stored in heparinized microcapillary tubes (75µl) (Marienfeld, Germany). The estimated blood volume in adult animals is 55 to 70 ml/kg body weight. If repeated blood samples are needed at short intervals, up to 0.6 ml/kg/day, 1.0 % of the animal's total blood volume can be taken every 24 hours (1212. Parasuraman S, Raveendran R. Biological sample collection from experimental animals. Introduction to Basics of Pharmacology and Toxicology: Volume 3: Experimental Pharmacology: Research Methodology and Biostatistics. 2022;16:45-63. Singapore: Springer Nature Singapore.
). Only 75 µl were drawn per animal, 3 times per week (on exposure days) as this amount was within the acceptable amount, caused the least possible discomfort to the animals, and was sufficient to perform BAC analysis.

Blood was transferred from the microcapillary tubes into sterile 15 ml conical culture tubes and then centrifuged in a Hettich Rotofix 32A centrifuge (Germany) at 4000 rpm for 10 minutes. Plasma was collected, and its alcohol concentrations were tested using an Alcohol Colorimetric Assay Kit from Sigma-Aldrich (USA), following the manufacturer’s instructions. Readings were taken using an iMark Bio-Rad Microplate Absorbance Reader from Bio-Rad Laboratories Inc, USA, at a wavelength of 540nm. All reactions and readings were conducted in an alcohol-free environment.

Skeletal harvesting

The animals were terminated on day 7 by a lethal pentobarbital intraperitoneal injection (200 mg/kg). Skin incisions were made, and mandibles were dissected out carefully, making sure to remove the muscles before removing the bones. Each mandible was then stored individually in 10 % buffered formalin for further fixation and processing for cytoarchitecture and immunohistochemistry analysis.

Tissue processing for histological and immunohistochemical techniques

Mandibles were removed and fixed in 10 % buffered formalin for at least 14 days. The right hemi-mandible was used for Haematoxylin and Eosin (H&E) staining and immunohistochemistry (IHC). Formalin-fixed samples were decalcified in 14 % w/v ethylenediaminetetraacetic acid (EDTA) (pH 7.4) (Sigma-Aldrich, USA) in an incubator with continuous gentle shaking at 45°C for 7 days. Subsequently, the bones were rinsed, and the region of interest was isolated by cutting through the mandible anteriorly, anterior to the 1st molar tooth, and posteriorly, posterior to the third molar tooth. These bone samples were then placed in 10 % buffered formalin 24 hours before processing. The samples were then processed through ascending grades of alcohol overnight using an automatic tissue processor (Citadel 2000, Thermo Fisher Scientific, USA), before embedding in paraffin. During embedding, special care was taken to orient the bone samples longitudinally, with the buccal aspect of the bone-facing the cutting surface of the paraffin block to minimize variations in the sectioning angle. Serial sections were cut with a microtome (Leica RM2125 RTS; Leica Biosystems, Germany) at 5μm thickness and placed on Epredia SuperfrostTM plus adhesion microscope slides (Netherlands). Every third section in the sequence was stained with Hematoxylin and Eosin (H&E) to evaluate mandibular cytoarchitecture or subjected to immunolocalization of the Ki-67 protein (a cell proliferation marker) or anti-alkaline phosphatase (ALP) (an osteoblast maker).

Immunohistochemistry for Ki-67 and ALP

Immunolocalization of mandibular cells expressing the Ki-67 protein and the alkaline phosphatase (ALP) enzyme was performed using the anti-Ki-67 antibody (NB 500-170) (Whitehead Scientific, South Africa) and the anti-ALP antibody (Ab95462) (Abcam, UK) respectively. In summary, the sections were deparaffinized, rehydrated and subsequently immersed in citrate buffer solution (pH=6), kept in a water bath set at a temperature of 60°C for 2 hours to allow for antigen retrieval. Tissue sections were then cooled down to room temperature for 20 minutes before washing for 5 minutes in a phosphate buffer solution (PBS) (pH=7.4). Endogenous peroxidase was blocked with 1 % hydrogen peroxide (H2O2), washed with PBS for 3×5 minutes, incubated with a goat serum protein block (Ab156024) (Abcam, UK) for 20 minutes, and washed in PBS for 3×5 minutes. The tissue sections were then incubated with primary antibody (anti-Ki-67 in 1:200 or anti-ALP in 1:1000 dilution factor) overnight at 4°C. On the second day, sections were allowed to reach room temperature and washed in PBS for 4×5 minutes. A biotinylated secondary antibody (biotinylated goat anti-rabbit IgG [H+L] [ab64256]) (Abcam, UK) was applied for 10 minutes and then washed in PBS for 4×5 minutes. To reduce nonspecific background staining while enhancing antibody binding, sections were incubated with streptavidin horseradish peroxidase for 10 minutes (ab64269) (Abcam, UK) and washed in PBS for 4×5 minutes. For visualization under a light microscope, sections were incubated with 3-3’di-aminobenzidine working solution for 5 minutes and then rinsed in running tap water for 5 minutes. The sections were then dehydrated through alcohol grades, cleared in xylene, and mounted in Entellen.

Photomicrograph acquisition and cell quantification

A light microscope (Zeiss Axioscope 2 plus, Germany) fitted with an axiocam HRC digital camera was used to acquire photomicrographs of the stained slides. These were taken under ×5 (H&E) and ×10 (IHC) magnification. Photomicrographs were transferred into Fiji Image-J software and the region of interest was identified and delineated based on the morphology of the roots of the 1st and 2nd molar teeth. For the quantification of cells, a manual counting method from the images was employed.

Data analysis

The data was managed in Microsoft Excel 365, 2023 (Microsoft Corporation) and analyzed using the Statistical Package for Social Service (SPSS®) version 28 (IBM Co.). The descriptive data of the number of chondrocytes and quantity of Ki-67 and ALP immunopositive cells were reported as means and standard deviation. The Shapiro-Wilk test was used to assess the normality of the data. The Independent Samples t-test was used to analyze normally distributed data, comparing means between the pair-fed control and alcohol-exposed groups. Non-parametric data was analyzed using the Mann-Whitney U test. The threshold for statistical significance was set at p < 0,05.

Blood alcohol concentration

Mean blood alcohol concentration (BAC) was 108.04 mg/dL (± 16.60) in the alcohol group, whereas the pair-fed control had negligible amounts.

Mandibular histology and immunohistochemistry