Female Sprague-Dawley (SD) rats weighing 180–220 g (6–10 weeks) were used, since female rats are more prone to quick development of arthritis as compare to their male counterpart due to their sex chromosome and estrogen hormone association . The animals were housed at Animal Research Facility, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi. The housing facility was controlled at ambient room temperature (21 ± 2°C), relative humidity of 50% ± 10% in a 12-hr light/dark cycle. All procedures were conducted according to the institutional guidelines of Scientific Advisory Committee on Animal Care, Use and Standards by International Center for Chemical and Biological Sciences (ICCBS), University of Karachi (protocol # 2018-0005) and guideline of United States National Institutes of Health guide for the care and use of laboratory animals (NIH Publications, 2011 edition: eight (8)).The group size was determined as the minimum number of rats for valid statistical analyses based on a pilot study. The group size of 12 has an 80% power to detect differences in the mean. Animals were divided into five different groups (1) normal control (2) arthritis only (3) indomethacin (4) gabapentin and (5) low dose combination of gabapentin + indomethacin treated. All experiments were repeated at least three times independently.
2.2 Induction of arthritis:
Lyophilized Mycobacterium tuberculosis H37Ra (MT H37Ra) was purchased from BD™ Difco™ Laboratories (Detroit, USA). A suspension of 10 mg/ml of fresh adjuvant was prepared using mineral oil at the beginning of experiment for the arthritis induction. Intradermal injection of 0.1 ml of suspension was injected at the base of rat tail using a sterile hypodermic needle under a combination of ketamine/xylazine (20 mg/kg/5 mg/kg) anesthesia [28, 29]. Treatment was initiated on the same day of adjuvant injection and count as day 0 and all values were taken as triplicate to get better precision of data set.
Gabapentin was purchased from Sigma-Aldrich (USA) and indomethacin was obtained from MP Biomedicals (USA). Phosphate-buffered saline (PBS) was used to prepare the suspension of indomethacin (5mg/kg and 2.5 mg/kg) and gabapentin (5mg/kg and 1.5 mg/kg). The gabapentin and indomethacin were injected intra-peritoneally on daily basis, alternatively on the right and left peritoneum region to avoid any bruises and tissue damage. Once the full-blown arthritis was established, the animals were humanely euthanized to collect brain and serum samples for further processing.
2.4 Clinical assessment of the adjuvant- induced arthritis:
The paw volume assessment is a method to quantify the edema of joints, and was done through plethysmometer (Model # 7140; Ugo Basile, Italy). The advantage of using this method over diameter measurements of tibio-tarsal joint is that it can measures the limb in three dimensions and therefore takes into account any variability of the pattern of swelling of individual limbs. The volume of a hind paw is reported as the mean ± of standard error in milliliters.
The clinical severity of inflammation through arthritis like symptoms was also determined by quantitating the change in the body weights to assess vulnerability of disease . All measurements were made at the same time of the day. Weight variation and plantar test were performed to analyze the severity and effect of pain on weight gaining and nociception respectively and paw edema volume test is used to confirm the swelling and inflammation of joints.
2.5 Nociceptive assay / Thermal hyperalgesia:
All groups of rats were evaluated for nociception assay  by using plantar test apparatus (UGO Basile, Italy). Animals were placed in transparent Lucite cubicle and were acclimatized for at least 20 minutes before taking readings. Sensitivity to noxious heat was assessed by using a plantar stimulator, which was positioned under a glass substrate, directly beneath the hind paws. The time was recorded as paw withdrawal latency (PWL) from the initiation of the radiant heat until the paw withdrawal. A maximal time of 15 seconds was used to prevent tissue damage. Each paw was tested three times and the mean withdrawal latency was calculated.
2.6 Total protein
Total protein was also estimated in plasma of control and test groups. Various studies suggested that pro-inflammatory markers like tumor necrosis factor alpha (TNF-α) and other markers of inflammation such as leukotriene B4, prostaglandin E2 and platelet activating agents may increase the total protein concentration in plasma [32–34] reported that, total concentration of protein in non-painful and effusion free joints is significantly lower than total concentration of protein in painful joints and joints effusion [35–37] due to the fact that healthy synovial membrane is impermeable to the high molecular weight plasma proteins . The permeability of plasma proteins increases with the gradual increase in the inflammation and perforation of synovial membrane . But the content and amount of total protein might differ in each joint due to the degree of inflammation and permeability of blood vessels .
2.7 Estimation of Oxidative Stress Parameters in Serum
2.7.1 Nitric Oxide (NO) and Peroxide (PO)
The NO and PO are free radicals normally detoxified by antioxidants in healthy condition, but generated extensively in inflammatory condition like arthritis and other inflammatory diseases. Evidences also suggested that the implication of high level of NO and PO in the development of chronic pain . Estimation of PO and NO was determined by Quantichrome DINO-250 Nitric oxide assay kit and peroxide assay kit (Bioassay systems, USA) according to the user manual. Optical density was taken at 540 nm and data was calculated by standard curve method.
2.7.2 Superoxide Dismutase (SOD) Assay
Superoxide dismutase (SOD) is a part of antioxidant mechanism of biological system in inflammatory condition. Whenever antioxidant level drops, it increases the level of free radicals (NO, PO). For the quantification of SOD, Superoxide Dismutase Assay Kit (Caymon Chemical Company, USA) was used. All experiments were performed according to the manufacturer’s protocol. The optical density was taken at 450 nm and results were calculated through standard curve method.
2.8 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis of BDNF mRNA Expression:
Following cardiac perfusion with heparin-PBS solution, animals were decapitated and the brain samples were collected. The region of interests (ROIs) i.e., amygdala, cortex, hippocampus and thalamus were dissected-out and placed in TRIzol™ reagent (Invitrogen, USA) and stored at -80°C for subsequent RNA extraction. Total RNA was purified using TRIzol™ reagent and the manufacturer’s protocol was followed. Briefly, frozen tissue sections prefilled with TRIzol according 100 mg: 1 ml ratio at the time of dissection and incubated for few minutes at room temperature and then homogenized. The homogenates were subjected to phase separation through chloroform, aqueous layer precipitation by propane-2-ol. RNA pellet was washed with 75% absolute ethanol and re-suspended in 30 µl of diethyl pyrocarbonate (DEPC) treated water. The purity and the quantification of the RNA were determined through NANODROP™ 2000 (Thermo fisher Scientific, USA). Samples exhibiting an absorbance ratio (A260/A280) greater than 1.75 or equal to ~ 2.0 were stored at -80°C for cDNA synthesis.
For cDNA synthesis, reverse transcription of RNA samples was performed using Superscriptase III First-Strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA, USA). Rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (product size: 450bp) gene was used as an internal standard. The cDNA was then amplified through PCR GoTaq® Green Master Mix (Promega Corporation, Madison, WI, USA) with BDNF primer sequence of (F: 5´-GGGTGAAACAAAGTGGCTGT-3´) (R: 5´-ATGTTGTCAAACGGCACAAA-3´); and GAPDH (F: 5´-GGAAAGCTGTGGCGTGATTGG-3´) (R: 5´-GTAGGCCATGAGGTCCACCA-3´) (product size: 175).
The amplified PCR products were resolved in 1% agarose gel with ethidium bromide as a DNA intercalating dye. Samples were carefully loaded to avoid the cross mixing between the wells and the electrophoresis was run for 70 minutes at 70 mV. Along with samples, ladder was also run to confirm the product size. The gel bands were visualized through CCD under the UV Gel-Doc System (Alpha Innotech) and quantified through NIH ImageJ software.
2.9 Immunohistochemistry of BDNF on Brain Sections
At the end of each experiment, animals were trans-cardially perfused under anesthesia with cold phosphate-buffered saline (PBS) containing a 100 U/ml of heparin. Once animals were decapitated, brain samples were removed and post fixed in 4% paraformaldehyde (PFA) and then cryoprotected overnight in 30% sucrose in PBS at 4°C. Optimal cutting temperature (OCT) media was used to make blocks for cryo-sectioning and placed in the − 20oC until the OCT media was frozen. The sample blocks were placed in the cryostat to equilibrate at -20°C prior to cryo-sectioning for at least one hour. The coronal sections of 35 µm were cut and directly transferred onto the poly-L-lysine coated slides and stored overnight in a humid chamber at ambient temperature. Next day, the sections were re-hydrated by rinsing with PBS followed by incubation in blocking solution containing 2% BSA, 2% normal goat plasma and 0.1% Tween-20 for 1 hr at 37°C. The immunostaining was performed by incubating sections with purified BDNF (N-20) rabbit polyclonal IgG primary antibody (Santa Cruz, USA) for 2 hr at 37°C. After washing with PBS, the tissue sections were incubated with secondary antibody i.e., Cy3® anti-rabbit (IGg ABCAM) for 1 hr. at room temperature. The slides were examined by fluorescence microscope (Nikon 90i, Japan). Images were taken through CCD camera using NIS Element AR 3.2 software and further processed through NIH ImageJ and adobe Photoshop software.
2.10 Statistical Analysis
The data was analyzed by using SPSS 20 and represented as a mean ± standard error of mean (SEM) of multiple experiments (n = 3). Each group was compared by using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test analysis. The p-value of ≤ 0.05 was considered as significant for comparing all groups.