*2.1. Animals*

Experimentally naive, adult male Wistar rats were provided by the Biobase of the George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures. Before the experiments, all animals were subjected to a 7-day habituation period, when acclimatization to single housing, daily handling, and standard environmental conditions (12 h light–dark cycle, 20 ± 2 ◦C temperature, 60% ± 10% humidity) were carried out. Standard rodent pellet chow ("Cantacuzino" National Institute of Research and Development) and tap water were provided ad libitum. Body weight was recorded once weekly, and their health status and well-being were monitored daily. The applied procedures were in accordance with European Directive 2010/63/EU and approved by the

Ethics Committee for Scientific Research of the George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures (approval no. 63/2018).

This study was designed to evaluate the long-term effects of CBD in the PTZ-kindling model of epilepsy. Therefore, to study the protective effects of CBD (60 mg/kg, oral), the drug was administered either 24 h before or 1 h after PTZ injections. The PTZ-kindled animals were randomly divided into two groups, a control group (orally received the vehicle, 1 mL/kg, *n* = 12) and a CBD-treated group (orally administered CBD, 60 mg/kg, *n* = 12). All 24 animals underwent the PTZ-kindling procedure, i.e., intraperitoneal injection of a subconvulsive dose of 35 mg/kg PTZ every other day for 50 days. Both the control and the CBD-treated group underwent the same care and injection protocol, and they were evaluated equally. The third group of animals (sham, *n* = 8) was used to compare the eventual cellular alterations observed by immunohistochemistry; these animals were, every other day, administered i.p. injections of the vehicle instead of PTZ, and they were not treated with CBD.

#### *2.2. Drugs and Reagents*

Crystalline cannabidiol (99.5% purity from Trigal Pharma GmbH, Wien, Austria), dissolved in extra virgin olive oil (Salov S.p.A., Massarosa, Italy) was administered to the animals. The individually calculated CBD dose based on the previously measured body weight (60 mg/kg body weight) was administered daily by adsorbing CBD oil onto food pellets. Pellets that were not loaded with CBD, but which were coated with olive oil, were administered to the control group. CBD treatment was initiated at day 0, before the first PTZ injection. The dose of CBD was chosen based on previously published results showing that doses below 50 mg/kg body weight did not exhibit anticonvulsant effects [18,37]. An overview of the experimental design is illustrated in Figure 1.

**Figure 1.** Timeline illustration of the experimental model. Cannabidiol was administered orally in a dose of 60 mg/kg each day, starting from day 0 until achieving a kindled state. Abbreviations: OF, open field test; NOR, novel object recognition test.

The plasma concentration and brain penetration ratio of the formulation (i.e., CBD dissolved in virgin olive oil and adsorbed on food pellets) used in this study was determined using plasma and brain samples obtained from anesthetized animals (ketamine–xylazine 100 mg/kgbw and 10 mg/kgbw, respectively) at two preliminarily determined time points (1 h and 24 h) after administration, corresponding to Cmax and Cmin, respectively. Each interval group consisted of 5 animals. Serum and brains were collected, frozen, and kept at −20 ◦C until analysis. Ketamin hydrochloride (Bela-Pharm GmbH & Co. KG, Vechta, Germany) and xylazine hydrochloride (Bioveta, Ivanovice na Hané, Czech Republic) were used for anesthesia.

#### *2.3. Determination of Plasma and Brain Levels of CBD*

To ensure the proper selectivity accompanied with high sensitivity for the quantification of CBD in rat plasma and homogenized brain tissue samples, liquid chromatography, coupled with mass spectrometric detection (LC–MS), was used. The system was an Agilent 1100 chromatograph equipped with an Agilent Triple Quadrupole MS detector (Agilent G6410A1, Technologies, Santa Clara, CA, USA), realizing the separation by using a Kinetex Polar C18 (100 × 4.6 mm, 2.6 μm, Phenomenex, Torrance, CA, USA) stationary phase with a flowrate of 0.5 mL/min, at 30 ◦C, with a total runtime of 4.5 min. To support speci-

ficity, negative electrospray ionization with multiple-reaction monitoring (MRM) mode was applied, quantifying the ion with 245 *<sup>m</sup>*/*<sup>z</sup>*, derived from 313 *<sup>m</sup>*/*<sup>z</sup>*. The potential fragmentation pathway of cannabidiol and the characteristic MRM mass spectrum are presented on Supplementary Figure S1, while the detailed chromatographic conditions are summarized in Supplementary Table S1. The method was partially validated in accordance with the ICH Q2(R2) guideline, demonstrating the specificity, linearity, accuracy, and limit of quantification. The results of the validation procedure are presented in Supplementary Table S2. The obtained chromatograms for the limit of quantification, and representative chromatograms of the plasma and homogenized brain tissue samples are presented on Supplementary Figure S2.

Methanol (Sigma-Aldrich, Steinheim, Germany), acetonitrile (Merck, Darmstadt, Germany), formic acid (Scharlau Chemie, Sentmenat, Spain), and water (Millipore Direct Q10, Merck Millipore, Burlington, MA, USA) used for analytical procedures were of HPLC grade.

Brain and plasma samples were collected on the last day of the experiment (day 70 and day 71, respectively) and underwent the same processing steps as described previously [38]. Briefly, blood obtained via cardiac puncture in K3 EDTA tubes was centrifuged at 3000 *g* for 10 min at 4 ◦C within 2 h of collection. The plasma samples were frozen at −20 ◦C until further pre-analytical processing. Following blood collection, the animals were perfused with approximately 30 mL of normal saline until the entire volume of blood was removed. This technique ensures that the measured concentrations reflect the concentrations in brain tissue by eliminating contributions from brain vasculature. The brains were removed within 7 min of when the thorax was opened. Tissue samples were weighed, homogenized in 5 mL phosphate buffer (PBS) in a grinding ball mill (UltraTurrax Tube Drive, IKA, Königswinter, Germany) for 10 min, and stored at −70 ◦C until analysis.

Plasma samples were diluted with blank plasma if necessary and mixed with 3 volumes of methanol to induce plasma protein precipitation. The mixture was vortexed for 10 s and then centrifuged (Sigma 2–15 centrifuge, Sigma, Osterode am Harz, Germany) for 10 min at 9167 g. The supernatant was diluted with a mobile phase and injected into the LC-MS/MS system. Homogenized brain tissue samples underwent the same procedures as plasma samples.

#### *2.4. Pentylenetetrazole Induced Kindling Model (PTZ-Kindling)*

Pentylenetetrazole (PTZ, Sigma Aldrich, St. Louis, MO, USA) was dissolved in 0.9% saline at a concentration of 35 mg/mL and injected intraperitoneally (i.p.) in a volume of 1.0 mL/kg at a sub-convulsive dose of 35 mg/kg according to the previously published schedule [39,40]. Seizure scoring was performed in real-time, followed by two blinded observers' confirmation of the registered data. Briefly, after each PTZ injection, rats were housed singly in transparent plexiglass cages and monitored for 1 h. Seizure intensities were rated by one experienced observer according to a modified Racine scale as follows: 0 = no response; 1 = ear and facial twitching; 2 = myoclonic jerks without rearing; 3 = myoclonic jerks with rearing; 4 = turning over into side position with tonic-clonic seizures; 5 = turning over into back position, generalized tonic-clonic convulsions, and loss of balance and falling. The same scoring scale was used by the two blinded observers who analyzed the video registrations offline. The final seizure score was established by combining the scores given by each observer.

An animal was considered kindled when it had experienced stage 4 or 5 seizures on two consecutive trials. At the beginning of the experiment, the sensitivity to the convulsant action of PTZ was assessed, and animals having two consecutive stage 5 seizures after the first two PTZ doses were not included in the study.
