Animals
All procedures in this study adhered to the "Guidelines for the Care and Use of Laboratory Animals" as outlined by the U.S. National Institutes of Health and received approval from the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine (IACUC Approval No: 20220519). We used Wistar rats weighing between 250 and 300 grams (approximately 3 months old). These rats were housed in groups of three per Plexiglas cage, with unrestricted access to food and water. The housing room was maintained at a controlled temperature (22 ± 3 ℃) and followed a 12/12-hour light/dark cycle, with lights turning on at 7:00 am. All behavioral assessments were conducted during the animals' active phase, specifically from 19:00 to 23:00 hours.
Drugs
Rotenone (RTN) ((2R,6aS,12aS)-1,2,6,6a,12,12a-hexahydro-2-isopropenyl-8,9-dimethoxychromeno[3,4-b]furo(2,3-h)chromen-6-one ≥ 95%; Sigma, St. Louis, Missouri, USA) was prepared in a 1% DMSO solution and administered subcutaneously (s.c.). Berberine (BBR) (9,10-Dimethoxy-7,8,13,13a-tetradehydro-2′H-[1,3]dioxolo[4′,5′:2,3]berbin-7-ium ≥ 95%; Sigma, St. Louis, Missouri, USA) was dissolved in double-distilled water and given orally (p.o.). Trigonelline (TGN) (1-Methylpyridin-1-ium-3-carboxylate; Sigma, St. Louis, Missouri, USA) was mixed in normal saline and delivered intraperitoneally (i.p.). Each drug solution was freshly prepared prior to administration. Initial trials with BBR at a low dose of 3 mg/kg showed no noticeable effects; therefore, the dose was incrementally increased to a maximum of 300 mg/kg to achieve statistically significant results. For this study, dosages of 30 and 100 mg/kg were selected based on their effectiveness. All drug dosages, adapted from earlier studies (Akbar et al. 2021; Garabadu and Agrawal 2020; Kadir et al. 2022; Tseng et al. 2020), were administered at a volume of 2 ml/kg body weight.
Experimental Groups and Drug Treatment
The experimental rats were randomly assigned into groups (n = 8 each, of either sex) according to Table 1. Prior to RTN or DMSO injection, administration of BBR or distilled water occurred 60 minutes earlier. TGN or normal saline injection preceded BBR administration by 30 minutes. On the 21st day, 8 hours post-RTN or DMSO injection, all animals underwent behavioral assessment. The experimental design is depicted in Fig. 1. To minimize subjective bias, each animal received an arbitrary identification number. Behavioral assessments were conducted independently by two experienced coworkers who were unaware of the treatment given to each animal. Each experimenter performed tests and evaluations separately. Animals were euthanized approximately 1 hour after behavioral assessments. During the study, nine rats died naturally for reasons unknown.
Measurement of Body Weight
The body weight of the animals was recorded before ROT administration (1st day) and on the final day of the study (21st day). To determine the percentage change in body weight, the following formula was used: Change in bodyweight = bodyweight (1st day-21st day)/1st day body weight x 100.
Assessment of Behavioral Parameters
Open Field Test
To assess spontaneous locomotor activity, we employed an open field test using a wooden, rectangular apparatus measuring 100 x 100 x 40 cm, with a light brown color. The floor of the apparatus was divided into 25 rectangular squares using pencil lines. The experimental room was illuminated by a 40 W white bulb positioned 150 cm above the test apparatus. During the test, each animal was placed in the center of the apparatus for 12 minutes, and the number of squares crossed during the last 10 minutes was recorded. A square crossing was only counted when all four paws of the animal entered another square. After each trial, the apparatus was meticulously cleaned before the next animal was tested (Thangarajan et al. 2014).
Bar Catalepsy Test
Cataleptic behavior was assessed using the bar test, as described by Ferro et al. (2005). Rats were placed in a standing position with their forelimbs resting on a 10 cm high bar, and the latency for each rat to remove one forelimb from the bar was measured in three consecutive trials. A maximum cut-off time of 60 seconds was set for each trial. The sum of the latencies across all three trials was calculated to evaluate cataleptic behavior.
Beam-crossing Task
To assess motor coordination, a beam-crossing task was employed, requiring animals to traverse a narrow wooden beam. The beam consisted of two platforms (8 cm in diameter) connected by a wooden beam measuring 0.5 mm in thickness, 2.0 cm in width, and 120 cm in length, elevated 50 cm above the ground. A box filled with sawdust was positioned below the beam to cushion potential falls. Rats were given 5 minutes to acclimate to the elevated beam before training. During a training trial, a rat was placed at one end of the platform and encouraged to walk across the beam to the other end. The number of slips and the time taken to cross the beam in each trial were recorded (Kumar et al. 2011).
Rotarod Activity
Motor coordination and grip performance were assessed using a rotarod performance test. Prior to testing, rats underwent a training session to familiarize them with the task. During the test, rats were placed on a rotating rod with a diameter of 7 cm, rotating at a speed of 25 rpm. The time taken for each rat to fall from the rod was recorded, with a maximum cut-off time of 180 seconds (Kumar et al. 2011).
Grip Strength Test
Neuromuscular strength was assessed using a grip strength test following standard procedures (Moran et al. 1995). The apparatus consisted of a 90 cm long metal wire with a diameter of 1 mm, horizontally fixed between two vertical supports and elevated 50 cm above a flat surface. The animal was suspended by its forepaws at the central position of the wire and evaluated based on the following scale:
0—fall off;
1—hangs onto the wire with two forepaws;
2—same as 1, but attempts to climb on the wire;
3—hangs onto the wire with two forepaws and one or both hind paws;
4—hangs onto the wire with all forepaws plus the tail wrapped around the wire;
5—escapes from the apparatus and falls onto the flat surface.
Measurement of Biochemical Parameters
Dissection and Homogenization
On the 21st day, approximately 1 hour after behavioral assessment, rats were euthanized. The brain was swiftly removed, and the striatum was carefully separated, placed on ice, weighed, and homogenized using a 0.1M phosphate buffer (pH 7.4). The homogenate was then centrifuged at 10,000 g for 15 minutes, and aliquots of the resulting supernatant were collected and utilized for biochemical estimations.
Measurement of Oxidative Stress Parameters
Estimation of Nitrite
To assess the accumulation of nitrite in the striatum supernatant, indicative of nitric oxide (NO) production, a colorimetric assay was conducted using Greiss reagent, as outlined by Green et al. (Green et al. 1982). Equal volumes of the supernatant and Greiss reagent (comprising 0.1% N-(1-naphthyl) ethylene diamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid) were mixed and incubated for 10 minutes at 25°C in the dark. The absorbance was then measured at 540 nm using a Shimazdu spectrophotometer (Kyoto, Japan). The concentration of nitrite in the supernatant was determined from a standard curve and expressed in µg/ml.
Assessment of Lipid Peroxidative Indices
The concentration of lipid peroxides was assessed using the thiobarbituric acid reactive substances (TBARS) assay, adapted from Ohkawa et al. (Ohkawa et al. 1979), following the procedures outlined by Hashimoto et al. (Hashimoto et al. 2005). The concentration was measured in nmol malondialdehyde per milligram of protein. Malondialdehyde levels were subsequently normalized to a standard preparation of 1,1,3,3-tetraethoxypropane.
Measurement of Glutathione (GSH)
GSH levels were determined using the method described by Ellman (Ellman 1959). To the homogenate, 10% trichloroacetic acid was added along with 1.0 mL of Ellman's reagent, containing 19.8 mg of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in 100 mL of 1.0% sodium citrate and 3 mL of phosphate buffer (pH 8.0). The mixture was then centrifuged, and the absorbance of the resulting solution was measured at 412 nm. The results were expressed in nmol GSH per milligram of tissue.
Measurement of Superoxide Dismutase (SOD) Activity
The assay for determining SOD activity relied on SOD's ability to inhibit the spontaneous oxidation of adrenaline to adrenochrome (Misra and Fridovich 1972). In this assay, 0.05 mL of the supernatant was combined with 2.0 mL of carbonate buffer and 0.5 mL of EDTA. The reaction was initiated by adding 0.5 mL of epinephrine. The auto-oxidation of adrenaline (3×10− 4 M) to adrenochrome at pH 10.2 was monitored by measuring the optical density at 480 nm. The change in optical density was recorded every minute and normalized to a blank reagent. SOD activity was expressed in units (milligrams per protein), with one unit of SOD activity causing approximately 50% inhibition of adrenaline. The results were further expressed in nmol SOD units per milligram of tissue.
Measurement of Catalase (CAT) Activity
The CAT activity assay employed in this study was adapted from the method described by Beers and Sizer (Beers and Sizer 1952). In brief, a reaction mixture was prepared by combining 2 mL of phosphate buffer (pH 7.0), 0.95 mL of hydrogen peroxide (0.019 M), and 0.05 mL of supernatant, resulting in a total volume of 3 mL. The absorbance was then measured at 240 nm every 10 seconds for 1 minute. One unit of catalase (CAT) activity was defined as the amount of enzyme required to decompose 1 mmol of peroxide per minute at 25°C and pH 7.0. CAT activity was expressed in units per milligram of protein. Activity units were determined from a standard curve of H2O2. The results were reported as catalase units per milligram of tissue.
Measurement of Mitochondrial Function
Mitochondria were isolated via a differential centrifugation method with slight modifications as previously outlined (Moreadith and Fiskum 1984). Initially, a 10% homogenate of the striatum was prepared in ice-cold Tris-Sucrose buffer (0.25 M, pH 7.4) using a glass-Teflon grinder at 4°C. The homogenate was then centrifuged at 1000 g for 10 minutes at 4°C to yield the nuclear pellet. The resulting supernatant underwent further centrifugation at 10,000 g for 20 minutes at 4°C to obtain the mitochondrial pellet along with the cytosol. The pellet was washed thrice in Mannitol–Sucrose–HEPES buffer (pH 7.4) and subsequently resuspended in the same buffer. The activity of succinate dehydrogenase (SDH) was determined following the method described by Pennington (1961) with minor adjustments. In brief, mitochondrial protein (0.05 mg) was incubated with 50 mM potassium phosphate (pH 7.4) containing sodium succinate (0.01 mol/L) and p-iodonitrotetrazolium violet (2.5 µg/mL) for 10 minutes. The reaction was terminated by adding 10% trichloroacetic acid (TCA). The resulting color was extracted using ethyl acetate:ethanol:trichloroacetic acid (5:5:1, v:v:w) and measured at 490 nm. SDH activity was expressed as the optical density (OD) value at 490 nm per milligram of protein. Total ATPase activity was determined by measuring the liberation of inorganic phosphate from ATP, as described by Prasad and Muralidhara (2013). The reaction was initiated by adding cytosolic protein (50 µg) to a reaction mixture containing Tris HCl buffer (0.02 M, pH 7.4), NaCl (100 mM), KCl (20 mM), and MgCl2 (5 mM), and incubated for 15 minutes at 37°C. The reaction was terminated by adding 20% TCA. After centrifugation (15,009g; 10 minutes), the phosphate content in the protein-free supernatant was estimated. Enzyme activity was expressed as micrograms of inorganic phosphate liberated per milligram of protein. NADH-cytochrome C reductase (complex I-III) and succinate-cytochrome C reductase (complex II-III) activities were determined following standard procedures (Navarro et al. 2004).
Measurement of Neuroinflammatory Markers
The quantification of TNF-α, IL-1β, and IL-6 levels was conducted using an immunoassay kit (KRISHGEN BioSystem, Ashley Ct, Whittier, CA). Specifically, a Quantikine rat TNF-α, IL-1β, and IL-6 immunoassay kit was utilized. This kit employs a 4.5-hour solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of rat TNF-α, IL-1β, and IL-6 levels, utilizing a microplate reader. The concentrations of TNF-α, IL-1β, and IL-6 were determined from standard curves and expressed in picograms per milliliter of protein.
Measurement of Apoptotic Marker
Caspase-3 Colorimetric Assay
Caspase-3, also known as CPP-32, Yama, or Apopain, is an intracellular cysteine protease. Normally, caspase-3 is present as an inactive pro-enzyme but becomes activated during the apoptotic cascade. To assess the protease activity in tissue lysates/homogenates, a caspase-specific peptide linked to the color reporter molecule p-nitroaniline (pNA) is added. Caspase cleaves this peptide, releasing the chromophore pNA, which can be quantified spectrophotometrically at 405 nm. The colorimetric kit used for evaluating caspase activity in this study was obtained from GeneTex Inc., Hsinchu, Taiwan. The results were reported as nanomoles of pNA per milligram of protein.
Determination of Protein
The protein content of both the cytosol and mitochondria was determined using bovine serum albumin (Sigma, St. Louis, MI, USA) as a standard reference (Lowry et al. 1951).
Statistical Analysis
All results were presented as mean ± standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 8.3.0 (GraphPad Software Inc., San Diego, CA, USA). One-way ANOVA was performed followed by post hoc Tukey’s test for all behavioral and biochemical estimations. A p-value of less than 0.05 was considered statistically significant, indicating significant differences between groups.