4.4. Paraclinical Evaluation: Biochemistry Assay and Immunohistochemistry Analysis

All the mice were euthanized (neck dislocation under anesthesia) after the completion of the behavioral tests, and a cardiac puncture was performed to sample 1 mL of terminal blood in 3 mL clot activator vacutainer tubes for biochemistry profiling and the acetylcholinesterase activity assay. In each group, six mouse brains (three male and three females) were collected. Each brain was sampled and fixed in 10% formalin for a detailed immunohistochemical stain analysis.

Biochemistry analysis was used to investigate the implications of SGLT2i canagliflozin and donepezil, under separate or combined 21-day treatment, on the primary organs responsible for drug metabolism (e.g., kidney and liver). A series of biochemical parameters (creatinine, aspartate transaminase (AST), alanine transaminase (ALT), total cholesterol, glucose, albumin, urea and total protein) were used, as previously described by our team [74].

Moreover, 30 min after harvesting, the vacutainer tubes were centrifuged at 1500× g for 15 min at 4 °C; the separated serum samples were then subjected to biochemistry analysis using an ACCENT-200 analyzer (PZ Cormay, Warsaw, Poland). The acetylcholinesterase activity was conducted, as mentioned by Al-Hazmi et al. [46], and measured by commercially available kits (Colorimetric, ab138871). All the experimental steps were carried out according to the manufacturer’s protocol, using a microplate reader to measure the absorbance at their respective absorption wavelengths.

The immunohistochemical (IHC) staining was performed according to previously described protocols [75,76] with modifications, using the antibodies listed in Table 3. The brain from each mouse was processed using the ExcelsiorTM AS Tissue Processor (Epredia Holdings Ltd., Portsmouth, NH, USA) and embedded in a single paraffin wax block. All the embedded paraffin blocks were then sectioned using a semi-automatic microtome CUT 5062 (SLEE medical GmbH, Nieder-Olm, Germany), at a 4 μm cutting thickness. Three sections for each animal were then transferred onto a microscope slide and stained using hematoxylin and eosin (H&E) standard staining protocol. Subsequently, all the H&E-stained tissue microscope slides were examined using light microscopy using an Aperio AT2 DX slide scanner (Leica 557 GmBh, Berlin, Germany), at a 400× magnification scale. Photomicrographs were then analyzed and compared to the control by a veterinary histopathologist.

Primary and secondary antibodies, with the related dilution used in immunohistochemical analysis.

Tissues were used for the detection of muscarinic acetylcholine receptor (M1 mAChR) expression, vascular endothelial growth factor A (VEGFA), cyclooxygenase-2 (COX2), the mammalian target of rapamycin (mTOR), the glial fibrillary acidic protein (GFAP), major histocompatibility complex class II (MHCII), the cluster of differentiation 68 (CD68), nuclear factor erythroid 2-related factor 2 (Nrf2) and p65.