Complete Freund’s Adjuvant (CFA)-Induced Arthritis Model

After validating the antiarthritic potential of prazosin and screening the topmost antiarthritic dose, the CFA-induced arthritis model was employed to get a deep insight into the antiarthritic mechanism of prazosin. Animals were alienated into four groups with six rats in each group. Group I was kept as the vehicle-treated group, group II was the arthritic control group, group III received the standard drug piroxicam (10 mg/(kg day)), and group IV received the test drug prazosin (20 mg/(kg day)) for 28 days. CFA (0.1 mL) was injected via the subplantar route on day 1 only.

All animals included in different treatment classes were carefully observed for arthritic manifestations. Rats inoculated with CFA developed arthritis manifestations after 4–5 days, as is evident from paw inflammation. Each paw was observed visually, and a score was given according to the criteria mentioned in Table 4. The maximum score for the arthritic index was 16/animal.⁴¹

Paw volume was appraised on a weekly basis with a digital plethysmometer to elucidate the extent of paw edema provoked by CFA injection and suppressed by different treatments. Percentage inhibition of CFA-provoked arthritis was estimated according to the following formula:⁴¹

where V_c is the paw volume of the control group and V_t is the paw volume of the treatment-provided groups.

Body weight was estimated before inducing arthritis and later weekly for all rats included in different treatment groups to assess the change in body weight induced by CFA. Body weight recorded on a specific day was subtracted from body weight of the same animal on day 0 to find out the mean change in body weight.

RNA was extracted from blood samples of all animals included in different treatment groups according to the TRIZOL method. cDNA was then synthesized according to instructions provided along with the kit by the manufacturer (ThermoScientifc). Real-time polymerase chain reaction was executed using the Bio-Radsystem to amplify and quantify the PCR product. Primer sequences for GAPDH and TNF-α were used from a previously conducted study, and they were prepared by a local manufacturer.⁴² The housekeeping gene GAPDH was utilized for normalization of the target gene, TNF-α.⁴³

The TNF-α level was estimated by employing ELISA as per the instructions provided by the manufacturer (Abcam; ab101784—TNF-α Rat ELISA Kit). A total of 100 μL of the standard as well as sample solution was added in appropriate wells, followed by incubation for 2.5 h at room temperature. The solution was then discarded from each well and washed with 1× wash solution (300 μL) with 4 times repetition. Afterward, any residual wash buffer was removed by aspirating or decanting. The plate was inverted and blotted against clean paper towels. Then, 100 μL of 1× biotinylated TNF-α detection antibody was poured into each well and incubated for 1 hour at room temperature with gentle shaking. The solution was discarded, and the wash step was repeated again. Then, 100 μL of a 1× HRP-streptavidin solution was added to each well and heated in an incubator for 45 min at room temperature with gentle shaking. Again, the solution was discarded and washed. TMB one-step substrate reagent (100 μL) was added to each well, and the plate was incubated for 30 min at room temperature in the dark with gentle shaking. Subsequently, 50 μL of stop solution was added to each well and read at 450 nm immediately.⁴⁴

Hematological parameters including RBCs, WBCS, platelets, Hb, and ESR levels were estimated by an automated hemocytometer (Sysmex XT-1800i). Similarly, biochemical parameters such as ALT, AST, urea, and creatinine levels were estimated in the serum of sacrificed animals by employing an autoanalyzer (Humalyzer 3500). The manufacturer’s protocol provided along with commercially obtainable kits was followed for analyzing the above-mentioned parameters (Analyticon Biotechnologies AG, Germany). Furthermore, serum RF and CRP level were also measured using commercially available kits.

Rats ankle joints excised after the completion of the study were taken for radiographic investigation and later for histological assessment.^41,43 Radiographic examination was done with a computerized radiographic system (Toshiba 630 MA, DS-TA-5A). For histopathological assessment, ankle joints were placed in 10% formalin for fixation; later on, after five days, they were decalcified using a decalcification solution. The tissues were then processed for paraffin embedding. Afterward, tissue slices of about 5 μm thickness were prepared using microtome and then stained with hematoxylin and eosin (H&E). Bone erosion, inflammatory cell infiltration, and pannus formation were examined by a blinded histopathologist.⁴⁰