This study involved twenty young-adult female Wistar rats (Rattus norvegicus), with five animals assigned to each experimental group. The subjects were sourced from the animal facility at the Faculty of Medicine, University of Porto (FMUP). Sample size determination was based on a prior power analysis (n = 5, power of 0.08, and type I error rate of 0.05) and in accordance with the legal determinations regarding replacement, reduction, and refinement that demand the usage of the minimum possible number of animals (Portuguese Act 113/13). The rats were kept in a controlled environment with a 12 h light/dark cycle, an ambient temperature of 21 ± 1 °C, relative humidity of 45 ± 5%, and free access to a standard solid diet and water. Regular monitoring included food and water intake, as well as body weight. Reproductive viability was confirmed through vaginal lavage, with only those exhibiting at least four complete estrous cycles being included in the study. All the procedures adhered to the European Communities Council Directive 2010/63/EU and Portuguese Act 113/13 and were approved by FMUP’s ethical review board (ORBEA).

Following a three-week acclimatization period, eight-week-old rats were anesthetized with ketamine (4 mg/kg) and medetomidine (0.05 mg/kg) before undergoing gonadectomy. Some of the rats received atipamezole (1 mg/kg) to counteract anesthetic effects, which was chosen because of its short-lasting action (two to three hours), which is less than the duration of the recovery phase. No antibiotic treatment was needed after surgery. A two-week recovery phase preceded experimental treatments. Two compounds, THC and estradiol benzoate (EB), were dissolved in 0.1 mL sesame oil and administered subcutaneously at 14:00 h; THC was administered every day, while EB was administered once every 4 days. The time of drug administration was chosen because that is the schedule that best mimics the physiological surge of estradiol [45]; THC was given at the same time to avoid extensive manipulation and distress to the animals. Half of the subjects received daily THC injections (1 mg/kg) throughout the study. This subcutaneous THC dose was selected for its translational relevance to both recreational and therapeutic human exposure and is consistent with previous reports [46]. Five days after THC administration began, the rats were further divided into two groups: one receiving sesame oil injections (Oil and THC groups) and the other receiving 10 μg of EB (EB and THC-EB groups) every four days from day six onward, spanning three hormonal cycles. The hormone doses were proven to mimic the physiological hormone values of female rats, as discussed in previous studies by the authors [47,48].

At the study’s conclusion (day 18, counted from the first injection of THC), the rats were euthanized thirty hours after the last EB administration and six hours after the last THC administration via intracardiac perfusion with 4% paraformaldehyde; their brains and uteri were extracted, with the uterine weight assessed to confirm hormone exposure. The schedule of animal sacrifice took place during the first third of the night, a moment when the estrogenic action is of relevance for female neuroendocrinology [34]. Serial 40 μm thick coronal sections were obtained from the brains and kept in storage boxes, each with twenty-four compartments, and preserved in Olmos solution [30% (w/v) sucrose, 30% (v/v) ethylene glycol, and 1% (w/v) polyvinylpyrrolidone-40 in phosphate-buffered saline (PBS)] at −20 °C until further analysis.