Chemicals
All compounds, unless otherwise indicated, were purchased from Sigma Aldrich (St Louis, MO). The other chemicals were all of analytical-grade.
Animal grouping and treatment
Forty-eight adult male Wistar rats (weighing between 150 and 200g apiece) were obtained from the Animal Holdings of Anatomy Department, College of Medicine, Faculty of Basic Medical Sciences, University of Nigeria, Enugu Campus (UNEC). Six groups were created from the Forty-eight rats that were randomly divided. The animals were kept in neat plastic cages in groups of three to four rats, with natural light and dark cycles, temperature (27oC – 30oC) for proper acclimatization. The animals were provided with unlimited access to food and water in a conventional laboratory setting. The treated groups received either 40 mg/kg of cobalt chloride (CoCl2) alone, 240 mg/kg of curcumin alone, 40 mg/kg CoCl2 plus 120 mg/kg or 240 mg/kg of curcumin, and 40 mg/kg CoCl2 plus 100 mg/kg vitamin C (vitC), while the control group received an equivalent volume of distilled water. For four weeks, all dosages were given by oral gavage only. All animals were treated strictly in accordance with the National Institute of Health's recommendations for the handling and use of laboratory animal (Albus 2012) and approved by the local Institutional Research Committee. Doses of CoCl2, curcumin and Vitamin C were based on previous studies that have established the rat model of Cobalt Chloride-induced neurotoxicity (Oria 2022), curcumin (Abubakar et al. 2019) and Vitamin C (Ajibade et al. 2017) treatments.
Neurobehavioral studies
Rats were housed overnight in the behavioral testing room to allow for appropriate acclimatization. After that, all behavioral tests were administered between the hours of 10 am and 3 pm in a quiet room. In order to eliminate bias brought on by the odor of previous animals, all apparatus was cleaned with an ethanol solution between trials. The tests were recorded with a digital camera, and trained blind observers thereafter scored the behaviors.
Test for memory performance
The Y-maze test battery was performed as previously described (Prieur and Jadavji 2019; Rao et al. 2021). The short-term spatial memory of the rats is evaluated using this test. The arms of the Y-maze, which were 75 cm long and 15 cm wide with a 120° angle between them, were used to confine the animals. The rats were put on a predetermined start arm and allowed to wander the maze for five minutes. The result was determined by arm entry (both hind limbs entirely in the arm). When the animal successfully completed each of the three arms of the maze in each exploration triad, the alternation was confirmed to be correct (i.e., entering all three arms in the overlapping triplet sets) (For example, XYZ, ZXY, or YZX). After two arms per exploration triad (e.g., XYX, ZXZ, YXY) were explored, it was regarded as an incorrect alternation. The percentage of spontaneous alternation was calculated as follows: (successive triplet sets/(total number of arm entries – 2) × 100.
Biochemical assays
The rats were sacrificed and their brains were removed twenty-four hours following the neurobehavioral test. Right and left hemispheres of whole brains were separated; one was prepared for histology and the other was used for biochemical tests. In eight volumes of 50 mM Tris-HCl buffer (pH 7.4) containing 1.15% potassium chloride, brain samples from each rat in each group were separately homogenized. The homogenate was then centrifuged at 10,000 rpm for 15 min at 4o C. The supernatant was extracted and then subjected to analyses of protein content using BCA kit and other biochemical analyses as previously described (Ebokaiwe et al. 2019; Ebokaiwe et al. 2020).
Assessment of brain superoxide dismutase (SOD), Catalase (CAT), glutathione S-transferase (GST) activities and LPO levels.
SOD activity was assessed using Misra and Fridovich (1972) methods, which are based on the enzyme's ability to suppress epinephrine autoxidation. The test is carried out in a solution containing 0.3mM adrenaline (Acros Organics, New Jersey, NJ), 0.05M carbonate buffer (pH 10.2), and 20mL of brain homogenate. The results are given in units/mg protein. Additionally, CAT activity, was measured using Claiborne (1985) methods. The assay is performed in a solution of 50mL of brain homogenate, 19 mM hydrogen peroxide (H2O2), and 50 mM phosphate buffer. The values are given in mmole H2O2 consumed/min/mg protein. GST was measured using the protocols developed by Habig et al. (1974) with the use of the substrate 1-Chloro-2,4-dinitrobenzene (CDNB). The assay was carried out in a mixture of 20mL of brain homogenates, 30 mmol/L of CDNB, and 100 mmol/L of phosphate buffer (pH 6.5). By using an extinction coefficient of 9.6L/(mmol/cm), the amount of CDNB/GSH conjugate generated in 1 minute per mg of protein served as an indicator of enzyme activity. LPO was quantified as malondialdehyde (MDA) using the Farombi et al. (2000) method. The assay is carried out in a mixture of 10% TCA and 0.75 percent TBA in 0.1 mol/L of HCl, 5% (w/v) butylated hydroxytoluene (BHT), and brain homogenate. MDA is thus calculated using the following equation: å = 1.56 x 105 L/mol/cm (where å denotes the extinction coefficient)
Immunohistochemistry of GFAP and Nrf2
Sections of conventional paraffin with a thickness of 5µm were deparaffinized, heated in a citrate-based antigen unmasking solution with a pH of 6.0 (Vector Labs, CA, USA) for 30 minutes, and then allowed to cool on the bench at room temperature for 30 minutes. Endogenous peroxidase blocking was carried out in PBS with a pH of 7.4 and 0.3% hydrogen peroxide for 10 minutes. For 20 minutes, sections were blocked in animal-free serum and blocker (Vector Labs, USA). After that, sections were probed for 2 hours at room temperature with primary rabbit antibodies: GFAP and Nrf2 (ThermoFisher, USA; #16825-1-AP and #PA1-38312, respectively). ImmPRESSTM HRP Anti-Rabbit IgG (Peroxidase) Polymer Reagent, produced in horse serum (Vector Labs, USA), was incubated with sections after sections had been washed in PBS. DAB Peroxidase (HRP) Substrate Kit (Vector Labs, USA) was used to develop the color, and hematoxylin was used as a counterstain on the sections (Erukainure et al. 2019; Ijomone et al. 2018).
Image analysis and cell count
The sections were photographed with a digital brightfield microscope. Non-overlapping micrographs were created at x400 magnification and used in Image J program for image analysis (NIH, USA). GFAP reactive cells (astrocytosis) were counted using the Image J program's Cell Counter function to assess positive immunoreactive cells (Akingbade et al. 2021), whilst Nrf2 immunoreactivity was quantified using the ImmunoRatio function, which separates and measures the percentage of DAB (positive immunoreactivity) using digital color deconvolution (Tuominen et al. 2010)
Statistical analysis
Results were presented as mean ± standard mean error (SEM). The statistical analysis was done using GraphPad Prism program (Version 9, GraphPad Inc., USA). One-way Analysis of Variance (ANOVA) was performed to analyze the data, and Tukey's test was used post hoc. Results were declared statistically significant at p< 0.05.