Drugs and chemicals
Polyclonal rabbit anti-cyclooxygenase-2 (COX-2), anti-inducible nitric oxide synthase (iNOS), and anti-glial fibrillary acidic protein (GFAP) antibodies were purchased from Abcam, Cambridge, MA, USA. Anti-ionized calcium-binding adaptor molecule-1 (Iba-1) polyclonal rabbit antibody was procured from Wako Chemicals, Richmond, VA, USA. Polyclonal rabbit anti-tyrosine hydroxylase antibody was obtained from Novus Biologicals, Littleton, CO, USA. Alexa fluor 488 conjugated secondary goat anti-rabbit antibodies were purchased from Life Technologies, Grand Island, NY, USA. Biotinylated secondary anti-rabbit antibody was purchased from Jackson Immunoresearch, West Grove, PA, USA. Nerolidol was purchased from Santa Cruz Biotechnology Inc., CA, USA. ROT and the assay kit for reduced glutathione (GSH) and other reagents of analytical grade were purchased from Sigma-Aldrich, St. Louis, MO, USA.
We used male Wistar rats at 6–7 months old (weighing 280–300 g) from the Animal Research Facility of the College of Medicine and Health Sciences, United Arab Emirates University, UAE. Prior to the start of the experiment, a maximum of 4 rats were housed per cage, and rats were acclimatized for 1 week to the laboratory conditions. The cages of the animals were changed twice a week. The animals were housed and kept under standard laboratory light and dark cycle conditions. The animals were fed with commercially available rodent food and water ad libitum. All experiments were conducted between 0900 and 1500 hours. The Animal Ethics Committee of the United Arab Emirates University, UAE, approved the experimental protocol for animal experimentation.
To induce the PD in rats, ROT (2.5 mg/kg body weight) was administered intraperitoneally (i.p.) once daily for 4 weeks. The regimen used in the current study for the induction of Parkinsonism in rats was adopted as reported earlier . Briefly, ROT was first dissolved in dimethyl sulfoxide (DMSO) to prepare a 50X stock solution and stored at −80 °C for further use. Before injection, the stock solution of ROT was thawed and further diluted in sunflower oil to obtain a final concentration of 2.5 mg/mL. To evaluate its neuroprotective efficacy, NRD was initially diluted in olive oil to obtain a final concentration of 50 mg/2 mL, and injected i.p. 30 min prior to ROT administration at a dose of 50 mg/kg of body weight once daily for 4 weeks. The dose of NRD was selected based on dose–response studies (data not shown) as well as earlier reports [15, 23]. Rats that were used as controls received an equivalent volume of vehicle only. The animals were sacrificed 48 h after the last injection of NRD or ROT or both in combination to completely eliminate these drugs from the body. The rats were divided into 4 experimental groups where each group consisted of 8 animals; the groups were named as follows:
Group I, the vehicle-injected control group (CONT).
Group II, the rotenone-injected and vehicle-treated group (ROT).
Group III, the rotenone-injected and NRD-treated group (ROT + NRD).
Group IV, the NRD-only injected group (NRD).
At the end of the experiment, the animals from all groups were anaesthetized with pentobarbital (40 mg/kg of body weight) and a cardiac perfusion was performed using 0.01 M of phosphate-buffered saline (PBS) pH 7.4 to completely clear the blood. The skull of the rat was open to quickly isolate the brain. The brain was placed on an ice-plate and cut along the midline to separate the 2 cerebral hemispheres. For biochemical assay, the midbrain and striatum regions of the brain were isolated from 1 hemisphere and immediately fresh frozen in liquid nitrogen for further use. The other hemisphere of the brain was incubated in 4 % paraformaldehyde solution for 48 h and subsequently exchanged with 10 % sucrose solution containing 0.1 M of phosphate buffer (PB) 3 times daily for 3 consecutive days at 4 °C prior to cryostat sectioning.
Sample preparation for biochemical assay
For biochemical assay, the midbrain tissue of each animal was homogenized in KCl buffer (10-mM Tris–HCl, 140-mM-NaCl, 300-mM KCl, 1-mM EDTA, and 0.5 % Triton X-100) at pH 8.0 supplemented with protease and phosphatase inhibitor, keeping the samples on ice. The tissue homogenates of each sample were centrifuged at 14,000×g for 20 min at 4 °C to obtain the post-mitochondrial supernatant (PMS) fraction. This PMS fraction was used to estimate the levels of the antioxidant enzyme GSH, lipid peroxidation product, and proinflammatory cytokines using spectrophotometric measurements and an enzyme-linked immunosorbent assay (ELISA) following a standard protocol, as reported earlier .
Assay for lipid peroxidation
A malondialdehyde (MDA) assay kit procured from Northwest Life Science (Vancouver, WA, USA) was used to determine the amount of lipid peroxidation, as reported earlier . Briefly, the samples or standards (250 µL) were incubated in the presence of acid reagent (250 µL) and thiobarbituric acid (250 µL) and vortexed vigorously. Samples were further incubated for 60 min at 60 °C followed by centrifugation at 10,000×g for 2–3 min. The reaction mixture was then aseptically transferred without disturbing the pellet to a cuvette and the absorbance was recorded at 532 nm. The concentration of MDA was calculated using a standard curve and expressed as µM MDA/mg protein.
Estimation of GSH
A GSH kit was used to estimate the GSH level, as reported earlier . Briefly, the samples were first deproteinized with 5 % 5-sulfosalicylic acid solution and centrifuged to remove the precipitated protein. The supernatant was used to measure the GSH level. Samples and standards of different concentrations (10 µL) were added in each well of a 96-well plate and incubated for 5 min with the working mixture (150 µL; assay buffer + 5,5′-dithiobis (2-nitrobenzoic acid) + glutathione reductase). Diluted NADPH solution (50 µL) was added to each well and mixed properly. The absorbance of the samples was measured at 412 nm with kinetics capability for 5 min using a microplate reader. The results were expressed as µM GSH/mg protein.
Assay for antioxidant enzymes activity
The activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were determined following the manufacturer’s instructions of a kit (Cayman Chemicals Company, Ann Arbor, MI, USA), as reported earlier . The CAT activity was expressed as nmol/min/mg protein and the SOD activity was expressed as U/mg protein.
Assay for pro-inflammatory cytokines
Commercially available ELISA kits for IL-1β, IL-6, and TNF-α were purchased from R&D systems, Minneapolis, MN, USA. The level of IL-1β, IL-6, and TNF-α were estimated as described earlier . The results were expressed as pg/mg protein.
Immunohistochemistry for tyrosine hydroxylase (TH) expression
The one hemisphere of the brains collected from each animal were serially cut after fixation and processed for immunohistochemical analysis. Briefly, 14 μm-thick coronal brain sections were cut at the level of the striatum and SNc using a cryostat (Leica, Wetzlar, Germany) for the immunohistochemical analysis of TH. Sections were washed twice with 0.01 M of PBS, pH 7.4, and then incubated with blocking reagent (10 % normal goat serum in PBS containing 0.3 % Triton-X 100) for 1 h. Next, the sections were incubated overnight with a primary polyclonal rabbit antibody against TH (1:500) at 4 °C. Sections were washed and incubated with biotinylated secondary anti-rabbit (1:1000) antibody for 1 h at room temperature. The brain sections were incubated with avidin–biotin complex (Vector Laboratories Ltd. Burlingame, CA, USA) and 3,3′ diaminobenzidine (DAB) to visualize and analyze the TH immunoreactivity. Finally, the sections were coverslipped using DPX mounting medium. The slides were then viewed under a light microscope (Olympus, Hamburg, Germany) and images were acquired for analysis.
Immunofluorescence staining of GFAP and Iba-1
Immunofluorescence staining was performed using 14 µm-thick striatum sections to quantify the number of activated GFAP-positive astrocytes and Iba-1-positive microglia. The brain sections were first washed twice with PBS and incubated with blocking reagent (10 % normal goat serum in PBS containing 0.3 % Triton-X 100) for 1 h. The sections were then incubated overnight with the primary polyclonal rabbit antibodies ant-GFAP (1:1000) and anti-Iba-1 (1:1000) at 4 °C. The sections were washed and incubated with fluorescent secondary anti-rabbit Alexa fluor 488 antibody for 1 h at room temperature. Sections were then washed, mounted on slides, and coverslipped using the mounting medium Fluoroshield (Sigma Aldrich, St. Louis, MO, USA). The images were captured using a fluorescence microscope EVOS FL (Thermo Fisher Scientific, Waltham, MA, USA).
Assessment of TH-immunopositive (TH+) dopamine (DA) neurons in the SNc and TH-immunoreactive (TH-ir) DA nerve fibers in the striatum
To evaluate the ROT-induced neurodegeneration and neuroprotective effect of NRD, the total number of TH+ DA neurons at 3 different anatomical levels of the SNc (−4.8, −5.04, and −5.28 mm of the bregma) were counted. The average number of TH+ neurons were calculated and converted as a percentage with reference to the control. The loss of striatal fibers was evaluated by measuring the intensity of TH-ir dopaminergic fibers in the striatum using Image J software (NIH, Bethesda, MD, USA). The intensity of the TH-immunoreactive nerve fibers in 3 different fields of brain sections (3 sections per animal) within the striatal region (adjacent to 0.3 mm of the bregma) was measured to examine the DA nerve fibers loss. An average of the 3 sections was calculated and presented as a percentage with reference to the values of the control group. The intensity of the overlying cortex area was used as the background measurement and subtracted from the value generated from the striatum. An investigator that was ‘blind’ to the experimental groups counted the TH+ DA neurons and determined the immunoreaction intensity of the TH fibers.
Assessment of activated astrocytes and microglia in the striatum
Three coronal sections from a comparable anatomical level of striatum from each animal were used to analyze and count the number of activated astrocytes and microglia. Activated astrocytes and microglia were considered for counting based on the intense immunoreactivity of GFAP- and Iba-1-labeled cells, whose characteristic morphological features include hypertrophied and extended glial processes. The total number of activated astrocytes and microglia were counted from 3 randomly chosen fields of an equal area in each section using the Image J software (NIH, Bethesda, MD, USA), and the results were presented as a percentage.
Western blot analysis of COX-2 and iNOS expression
Western blot analysis was carried out to measure the level of COX-2 and iNOS expression in different groups of animals following the protocol, as reported earlier . Briefly, striatal tissues isolated from each animal were homogenized in radioimmuno-precipitation buffer with protease and phosphatase inhibitors. The cell lysates were then centrifuged at 15,000 rpm for 20 min. The supernatant containing cytoplasmic fractions was isolated, and protein concentration was measured as mentioned below. The cytoplasmic fraction containing equal amounts of protein (35 μg) were loaded and separated using 10 % SDS–polyacrylamide gel electrophoresis. The proteins were then transferred onto a PVDF membrane and incubated overnight at 4 °C with specific primary rabbit polyclonal antibodies against COX-2 (1:1000) and iNOS (1:500). The membrane was washed and then incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody. The protein recognized by the antibody was visualized using an enhanced chemiluminescence Pico kit (Thermo Fisher Scientific, Rockford, IL, USA). The blots were stripped and re-probed for β-actin (1:5000; monoclonal mouse, Millipore, MA, USA) as a loading control. The intensity of the bands was measured using densitometry and quantified using Image J software (NIH, Bethesda, USA).
The concentration of protein in each sample was estimated using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA) following the manufacturer’s instructions.
The data were expressed as the mean value ± SEM. The data were analyzed with Graph Pad (InStat software, La Jolla, CA, USA) using a one-way analysis of variance (ANOVA) followed by a Tukey’s test to determine the statistical significance between various groups. For all of the tests, the criterion for any statistically significant difference was set at p < 0.05.