Neuroprotective Effects of Alpha-Lipoic Acid Against Behavioral Toxicity, Oxidative and Inflammatory Damage Caused by Titanium Dioxide Nanoparticles methods
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Experimental Design
Experimental procedures followed the Alexandria University Institutional Animal Care and Use Committee guidelines (ALEXU-IACUC, 120/2022). Twenty-four male rats were allocated randomly into four groups (six each): (1) negative control group obtained distilled water IP together with corn oil orally; (2) TiO 2 -NP-treated group administered TiO 2 -NPs (150 mg/kg bwt) IP; (3) ALA treated group given ALA (50 mg/kg bwt) orally; (4) TiO 2 -NP- and ALA-treated group was given both drugs. The treatment was administered for 28 days on alternate days [, ]. After the treatment, the rats'memory and learning ability were evaluated by neurobehavioral tests. Afterward, rats were humanely euthanized by decapitation, and brain samples were collected. The brain was split into two halves. One half was kept at - 80 °C for antioxidants, inflammatory biomarkers, and gene expression, while...
Elevated Plus Maze (EPM)
This test evaluates animals' anxiety levels. The maze comprises two open arms (50 × 10 cm) and two closed arms of similar dimensions, with high side walls (40 cm). A central platform connects the arms, measuring 10 × 10 cm. A high edge about 0.5 cm surrounded the open arms, and the maze was 50 cm above the ground. Each rat was positioned in the central area, facing an open arm, and allowed 5 min to explore before being returned to its cage. An entry into the central region was considered when two paws were on it, while all four paws were considered for arm entry. Time elapsed to enter open or closed arms, time present in open and closed arms, open and closed arms entries (N), and time and entries in open and closed arms percent were recorded [ ].
Morris Water Maze (MWM)
The MWM analyzes spatial learning and memory. The apparatus consisted of a spherical tank (diameter of 120 cm and a height of 60 cm), with water filling to 45 cm depth maintained at 23 ± 1 °C. A non-toxic brown dye was applied to the water to obscure visibility. Four imaginary quadrants divided the tank (1, 2, 3, and 4), with the center of quadrant 3 (the target quadrant) having a hidden platform (5 cm in diameter) located 1-2 cm below the water 's surface. The test was conducted in two phases (place navigation and spatial probe), and it was performed on two consecutive days. All rats in all groups were tested on the same day for each phase. The first rats were taught to escape the hidden platform in the place navigation phase. Each rat was liberated into the water facing the tank wall and underwent four trials starting from a different quadrant in a clockw...
Evaluation of Apoptotic and Inflammatory Proteins in Brain Tissue
To analyze inflammatory and apoptotic proteins in brain tissue, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and caspase-3 were evaluated using ELISA kits. The corresponding kits were obtained from My BioSource (San Diego, CA, USA) under the catalog numbers MBS267737 (TNF-α), MBS764577 (IL-6), and MBS700575 (caspase-3). Each sample was analyzed in triplicate according to the procedures provided by the manufacturer.
Real-Time PCR
According to the manufacturer's guidelines, a Trizol reagent (iNtRON Biotechnology, Inc., Korea) was used to isolate total RNA from 100 mg of brain tissue. A NanoDrop spectrophotometer (UV-Vis, Q5000/Quawell, USA) was used to assess RNA concentration and purity. The extracted RNA was used to synthesize complementary DNA (cDNA) using the SensiFAST™ cDNA synthesis kit (Bioline, UK), following the manufacturer's instructions. The synthesized cDNA samples were kept at - 20 °C until further investigation. Gene-specific primers and β-actin (used as an internal control) were designed based on accession numbers retrieved from GenBank (NCBI) (Table ). Quantitative real-time PCR (RT-PCR) was performed to evaluate mRNA expression levels using the SYBR Green method (SensiFAST SYBR Lo-ROX kit, Bioline). The thermal cycling conditions included an...
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Experimental procedures followed the Alexandria University Institutional Animal Care and Use Committee guidelines (ALEXU-IACUC, 120/2022). Twenty-four male rats were allocated randomly into four groups (six each): (1) negative control group obtained distilled water IP together with corn oil orally; (2) TiO 2 -NP-treated group administered TiO 2 -NPs (150 mg/kg bwt) IP; (3) ALA treated group given ALA (50 mg/kg bwt) orally; (4) TiO 2 -NP- and ALA-treated group was given both drugs. The treatment was administered for 28 days on alternate days [, ]. After the treatment, the rats'memory and learning ability were evaluated by neurobehavioral tests. Afterward, rats were humanely euthanized by decapitation, and brain samples were collected. The brain was split into two halves. One half was kept at - 80 °C for antioxidants, inflammatory biomarkers, and gene expression, while the other half was soaked in formalin solution for histopathological examination.
This test evaluates animals' anxiety levels. The maze comprises two open arms (50 × 10 cm) and two closed arms of similar dimensions, with high side walls (40 cm). A central platform connects the arms, measuring 10 × 10 cm. A high edge about 0.5 cm surrounded the open arms, and the maze was 50 cm above the ground. Each rat was positioned in the central area, facing an open arm, and allowed 5 min to explore before being returned to its cage. An entry into the central region was considered when two paws were on it, while all four paws were considered for arm entry. Time elapsed to enter open or closed arms, time present in open and closed arms, open and closed arms entries (N), and time and entries in open and closed arms percent were recorded [ ].
The MWM analyzes spatial learning and memory. The apparatus consisted of a spherical tank (diameter of 120 cm and a height of 60 cm), with water filling to 45 cm depth maintained at 23 ± 1 °C. A non-toxic brown dye was applied to the water to obscure visibility. Four imaginary quadrants divided the tank (1, 2, 3, and 4), with the center of quadrant 3 (the target quadrant) having a hidden platform (5 cm in diameter) located 1-2 cm below the water 's surface. The test was conducted in two phases (place navigation and spatial probe), and it was performed on two consecutive days. All rats in all groups were tested on the same day for each phase. The first rats were taught to escape the hidden platform in the place navigation phase. Each rat was liberated into the water facing the tank wall and underwent four trials starting from a different quadrant in a clockwise sequence. The platform 's position remained fixed in the target quadrant for all trials. Each trial terminated when the rat reached the platform or after 60 s. If the rat did not locate the platform within 60 s, it was guided to it, allowed to remain on it for 5-10 s, and then return...
To analyze inflammatory and apoptotic proteins in brain tissue, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and caspase-3 were evaluated using ELISA kits. The corresponding kits were obtained from My BioSource (San Diego, CA, USA) under the catalog numbers MBS267737 (TNF-α), MBS764577 (IL-6), and MBS700575 (caspase-3). Each sample was analyzed in triplicate according to the procedures provided by the manufacturer.
According to the manufacturer's guidelines, a Trizol reagent (iNtRON Biotechnology, Inc., Korea) was used to isolate total RNA from 100 mg of brain tissue. A NanoDrop spectrophotometer (UV-Vis, Q5000/Quawell, USA) was used to assess RNA concentration and purity. The extracted RNA was used to synthesize complementary DNA (cDNA) using the SensiFAST™ cDNA synthesis kit (Bioline, UK), following the manufacturer's instructions. The synthesized cDNA samples were kept at - 20 °C until further investigation. Gene-specific primers and β-actin (used as an internal control) were designed based on accession numbers retrieved from GenBank (NCBI) (Table ). Quantitative real-time PCR (RT-PCR) was performed to evaluate mRNA expression levels using the SYBR Green method (SensiFAST SYBR Lo-ROX kit, Bioline). The thermal cycling conditions included an initial denaturation at 95 °C for 10 min, followed by 40 amplification cycles of 95 °C for 15 s, 60 °C for 30 s, and a final extension step at 85 °C for 1 min. Relative gene expression levels were normalized to β-actin and calculated using the 2^ - Δ...
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