In this study, forty male Wistar albino rats were obtained from the Tekirdag Namik Kemal University Experimental Animals Application and Research Centre. All rats were housed at a room temperature of 22 ± 2 °C, with 60 ± 10% humidity, a 12-hour light/dark cycle, and in cages specifically designed for rats. The rats were fed with standard pellet feed and tap water ad libitum. This research was approved by the Tekirdag Namik Kemal University Animal Experiments Ethics Committee, Tekirdag, Turkey (TNKUHADYEK T2019-360, Decision No. 6, Date: 11.11.2019).

Experimental design

Forty male Wistar albino rats (10 weeks old)were categorized into four distinct groups, each consisting of ten rats. The groups were as follows:

I.

Group C (Control) (n = 10).

The rats were administered 3.15 ml/kg saline via intragastric gavage (i.g.) for two consecutive days, followed by a two-day break. Additionally, we administered 1 ml/kg of saline intraperitoneally (i.p.) to the rats every three days.

II.

Group E (Ethanol) (n = 10).

The rats were administered 3 g/kg 20% ethanol (3.15 ml/kg) solution (Cat.No: 1009832500, Merck, Darmstadt, Germany) via i.g. for two consecutive days, followed by a two-day break (Lamarão-Vieira et al. 2019) and injected with 1 ml/kg saline i.p. every three days.

III.

Group DA (Darbepoetin Alpha) (n = 10).

The rats were injected with 0.25 µg/kg darbepoetin alpha (Aranesp 10 µg, Thousand Oaks/USA) i.p. every three days (Ozkurt et al. 2018), and administered 3.15 ml/kg saline via i.g. for two consecutive days, followed by a two-day break.

IV.

Group E + DA (Ethanol + Darbepoetin Alpha) (n = 10).

The rats were injected with 0.25 µg/kg darbepoetin alpha i.p. every three days and administered 3 g/kg 20% ethanol (3.15 ml/kg) solution via i.g. for two consecutive days, followed by a two-day break.

During the 30-day experiment period, ethanol and an equal volume of saline were administered for two consecutive days, followed by a two-day break. Also, darbepoetin alpha and an equal volume of saline were administered every third day.

No rats died during the experimental period. At the end of the experiment, the rats were anesthetized with 90 mg/kg ketamine and 10 mg/kg xylazine via i.p. Blood samples were collected by intracardiac puncture, and the rats were sacrificed by hypovolemia induced by intracardiac blood collection.

Biochemical methods

Blood samples were centrifuged at 1000 RPM for 10 min in a centrifuge at + 4 °C (Hettich, Tuttlingen, Germany), and supernatants were obtained, placed into tubes, and stored at − 80 °C. The left hemispheres of the brains were removed, taking care to maintain the cold chain. After washing with physiological saline, their weights were recorded, and the samples were placed into tubes and stored at − 80 °C until used. The brain tissues were homogenized in phosphate-buffered saline (PBS) (Thermo Fisher Scientific, Cat.No: 003002) containing 10 mM phosphate and 150 mM sodium chloride (pH 7.4) using a homogenizer (Next Avance Bullet Blender, Troy/New York/USA). 0.2 ml of PBS buffer was added for every 100 mg of tissue. The homogenates were then centrifuged at 10,000 RPM for 10 min at + 4 °C using a centrifuge (Sigma 3-18KS, An der Unteren Söse/Osterode am Harz/Germany), and the supernatants were collected for analysis.

Spectophotometric analyses

We used the spectrophotometric method to determine SOD enzyme activity and malondialdehyde (MDA) levels in tissue homogenate samples using a spectrophotometer device (Shimadzu UV-1800, Tokyo/Japan).

Determination of SOD enzyme activity

The activity of the SOD enzyme was determined based on the methodology originally described by Sun et al. (1988) and subsequently modified by Durak et al. (1993).This method relies on the reduction of nitroblue tetrazolium (NBT) through the superoxide anion (O₂˙⁻), generated by the xanthine-xanthine oxidase system. The absence or low activity of SOD results in the formation of blue-violet formazan with maximum absorbance at 560 nm. One unit of SOD is defined as the enzyme activity that inhibits the reduction of NBT by 50%.

Determination of MDA levels

MDA levels in brain tissue were measured using a previously described method (Buege and Aust 1978). This method relies on the reaction of MDA, a lipid peroxidation product, with thiobarbituric acid (TBA) to form a pink complex exhibiting maximum absorbance at 532–535 nm. To 250 µl of sample, 1.5 ml of 0.75% TBA solution, 1 ml of 30% trichloroacetic acid, and 0.2 ml of 5 M HCl solution were added. The mixture was boiled at 95 °C for 15 min, cooled, and the absorbance was measured at 532 nm. The MDA concentration was determined by utilizing the absorbance coefficient of the MDA-TBA complex (1.56 × 10⁵ cm⁻¹ M⁻¹).

ELİSA measurements

S100-β, NSE, catalase (CAT), GR levels and GPx enzyme activity in serum and tissue homogenate samples were analyzed by ELISA kits (Cat.No: E1360Ra, E0541Ra, E0869Ra, E1085Ra, E1759Ra, respectively; Bioassay Technology Laboratory, Zhejiang/China). ELISA results were obtained using Biotek Elx800 device (Santa Clara, California, USA).

Histopathological evaluation

The hippocampus is the brain region most affected by ethanol toxicity (Mira et al. 2019), so this region was selected for histopathological evaluation. The right hemispheres of the rat brains were fixed in 10% neutral buffered formaldehyde. After routine histological tissue processing and embedding the tissue samples in paraffin wax, 5 μm thick sections were cut. Horizontal sections of the dentate gyrus region of the hippocampus (approximately between the interaural 6.48–6.00 mm interval) were obtained using the rat brain atlas of Paxinos (Paxinos and Watson 2006). The sections were examined under a light microscope after being stained with hematoxylin-eosin (H-E) and photographed at 200X and 400X magnification (Olympus CX41-Japan).

In brain tissue, the presence of hyperchromasia, pycnotic changes, absence of nucleolus, and marked loss of nuclear border were indicative of cell death (Mutch et al. 1993; Seymen et al. 2013). The cells with diffusely eosinophilic cytoplasm were considered dead, while cells containing Nissl substance and showing basophilic stippling were considered viable(Auer et al. 1984). Based on these criteria, the ratio of dead neurons in the dentate gyrus was calculated. Neurodegeneration was scored as follows: 3 = severe neurodegeneration (more than 40% dead neurons), 2 = moderate neurodegeneration (20–30% dead neurons), 1 = mild neurodegeneration (less than 20% dead neurons), and 0 = no neurodegeneration. Evaluations were performed by two pathologists blinded to the groups and each other’s findings.

Statistical analysis

Statistical analysis was performed using the IBM SPSS Statistics V21 software (SPSS Inc., Chicago, IL, USA). All data were reported as the mean ± SEM. Group distributions were analyzed using the Shapiro–Wilk test. Depending on the normality and homogeneity of the data distribution, the independent sample t-test or Mann-Whitney U test was used for the statistical comparison of two groups. Statistical significance was considered at p < 0.05.