*2.3. Experimental Design*

Group C: control group; Group CCL4: exposure to CCL4; Group Q: exposure to quercetin only; Group Q + CCL4: treated with both quercetin and CCL4. Each group consisted of six animals. Sunflower oil (vehicle) was used to dissolve CCL4 and then administered to specific groups of animals. To the control group, only the sunflower oil was administered. Due to CCL4's non-polar nature and high volatility, it was necessary to dissolve it in sunflower oil (3:1) to maintain a consistent, effective dose. Neurotoxicity in mice occurs with a single dose of CCL4 (1 mL/kg bw) [5]. To the CCL4 Group, the CCL4 dose was administered by oral gavage. To the quercetin-only group (Group Q), quercetin was given as a single dose of i.p injection of quercetin (100 mg/kg bw) [17–19]. In the combination group, quercetin (100 mg/kg bw) was administered 2 h before the assault by CCL4 (1 mL/kg bw). The Cmax of quercetin is 2–3 h; therefore, quercetin was administered to obtain protective concentration levels of quercetin in systemic circulation before the CCL4 assault [10]. The animals were sacrificed by carbon dioxide asphyxiation after 24 h of their respective treatments bw) [17–20]. The brains of the studied groups were removed and processed immediately for biochemical assays. The harvested brains were weighed immediately for determining the organ/body weight ratio. The harvested brains were homogenized in 1X PBS, and slices from the cortex were cut and stored in formalin for histopathology.

The samples were processed as follows: fixing the specimens in a 10% neutral buffered formalin solution (Loba Chemie, Colaba, India) block preparation in paraffin (Leica Biosystems, Wetzlar, Germany) cutting sections of 5–6 µm thick, and the sections stained with hematoxylin-eosin stain (Leica Biosystems, Wetzlar, Germany). The sections were photographed and analyzed using an electron microscope (Leica Biosystems, Wetzlar, Germany) by an expert pathologist who was not informed about the sample assigned to the experimental groups.
