Effects of different doses of complete Freund's adjuvant on nociceptive behaviour and inflammatory parameters in polyarthritic rat model mimicking rheumatoid arthritis

Abstract

Complete Freund's adjuvant (CFA) has been used to develop the arthritic or inflammatory condition in the animal, but there is a lack of information concerning high CFA doses on nociceptive behaviour and inflammatory parameters. This study aimed to compare the effects of different high doses of CFA in rat to closely mimic nociceptive and inflammatory parameters of rheumatoid arthritis (RA) in humans. Twenty-four male Sprague-Dawley rats were randomly divided into four groups (n = 6): Control (C), CFA-induced polyarthritic groups at 5.0 mg/mL (CFA 5.0), 7.5 mg/mL (CFA 7.5) and 10.0mg/mL (CFA 10.0). The rats' right hindpaw was inoculated with CFA intradermally and developed into a polyarthritic state within 20 days. Nociceptive behavioural assessments, including von Frey and hot plate tests and spontaneous activities, were conducted on day 0, 7, 15 and 20. Bilateral ankle joints diameter and circumference, full blood count, joints and paw histological examinations were also conducted throughout the study period. Based on the results, CFA 5.0 and CFA 7.5 groups showed a significant increase in spontaneous activities and development of thermal hyperalgesia but no change in body weight and food intake, no development of tactile allodynia and haematological indices, and no significant morphological changes of joints histology. Meanwhile, CFA 10.0 group demonstrated significant and constant changes in all nociceptive and inflammatory parameters investigated. In conclusion, CFA at the dose of 10mg/mL has the most potential and reliable dosage to develop polyarthritis in a rat model to mimic RA condition in humans.

Fig 1. Percentage of body weight changes in CFA treatment groups (n = 6).

The data are presented as mean ± standard error of the mean (SEM). *p < 0.05 statistical comparison between the group receiving CFA injection at 10 mg/mL to the control group.

Fig 2. Total food intake in the groups for 20 days of experimentation.

No significant changes were detected between the groups.

Fig 3. Spontaneous activities as indicated by (a) standing paw pressure and (b) walking paw pressure in the groups on day 0, 7, 15 and 20 (n = 6).

The data are presented as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001 Statistical comparison to control group, ^###p < 0.001 Significant comparison to CFA 5.0 group and ^§§p < 0.05, ^§§§p < 0.001 Significant comparison to CFA 7.5 group. The CFA 10 group showed the most consistent and the highest score for both standing and walking paw pressure compared to the control group, especially on day 7, 15 and 20.

Fig 4. Paw withdrawal threshold at the (a) ipsilateral (CFA-inoculated) hind paw and (b) contralateral (non-CFA-inoculated) hind paw in the treatment groups on day 0, 7, 15 and 20 (n = 6).

The values are presented as mean ± standard error of the mean (SEM). *p < 0.05 Statistical comparison to control group and ^#p < 0.05 Statistical comparison to CFA 7.5. CFA 10.0 group showed a consistent significant reduction in paw withdrawal threshold compared to the control group, indicating the development of tactile allodynia in the rat, beginning with the ipsilateral followed by contralateral hind paws.

Fig 5. Thermal withdrawal threshold in the treatment groups on day 0, 7, 15 and 20 (n = 6).

The values were expressed as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01 and ***p < 0.001 Significant comparison to control group. #p < 0.05 Significant comparison to CFA 5.0 group. CFA 10.0 group demonstrated a significant gradual reduction in thermal withdrawal threshold, indicating the gradual development of thermal hyperalgesia throughout the experiment.

Fig 6. Changes in rats’ ankle joint (a) circumferences and (b) diameter at the ipsilateral (CFA-inoculated) hind paw in the treatment groups on day 0 to 20 (n = 6).

The data are expressed as mean ± standard error of the mean (SEM). *p < 0.05 and **p < 0.001 significant comparison to control group. ^###p < 0.001 significant comparison to CFA 5.0 group and ^§§§p < 0.001 significant comparison to CFA 7.5 group. CFA 10.0 group demonstrated the most reliable and constant gradual increase in the ankle joint oedema at the ipsilateral side compared to other doses of CFA inoculation.

Fig 7. Ankle joint (a) circumference and (b) diameter at the contralateral (non-CFA- inoculated) hind paw in the treatment groups (n = 6).

The values are presented as mean ± standard error of mean (SEM). *p < 0.05, **p < 0.01 and ***p < 0.001 significant comparison to control group. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 significant comparison to CFA 5.0 group. CFA 10.0 group showed a constant increase of inflammatory oedema at the contralateral (non-injected hind paw), indicating the development of chronic polyarthritis following CFA inoculation.

Fig 8. Histological sections of the CFA-inoculated hind paw tissues in (A) control, (B) CFA 5.0, (C) CFA 7.5 and (D) CFA 10.0 subjected to haematoxylin and eosin (H&E) staining at 400x magnification.

The control group demonstrated no significant lesion appearance in the mineral oil-injected hind paw. Meanwhile, extensive inflammatory cells infiltration was observed in CFA 10.0 group, (D) mild and moderate level of inflammatory cells infiltration were detected in CFA 5.0 (B) and CFA 7.5 (C), respectively. The arrow indicates the extensive inflammatory cells infiltration at the injected hind paw tissue.

Fig 9. Representative histological sections of ipsilateral ankle joint by haematoxylin and eosin (H&E) staining at 100x magnification in control (A), polyarthritic rat inoculated with CFA at 5.0 mg/mL (CFA 5.0) (B), 7.5 mg/mL (CFA 7.5) (C) and 10.0 mg/mL (CFA 10.0) (D).

(A) In the control group, smooth articulation of the joint cartilage surface, regular joint space (JS) with the normal connective tissue of the synovial membrane were observed. Meanwhile, ipsilateral ankle joint of rats in CFA 5.0 (B) and CFA 7.5 (C) groups showed mild penetration of inflammatory cells into the synovial cavity, indicating suppressed joint pathology and less soft tissue swelling. Additionally, the synovial hyperplasia with extensive inflammatory cells infiltration (as shown by the purplish stains), bone resorption and severe cartilage destruction indicating the narrowing of the joint with inflammatory cell infiltration into the synovial cavity (arrow) were found in the ipsilateral ankle joints of CFA 10.0 group.

Fig 10. Histological sections of contralateral ankle joint by haematoxylin and eosin (H&E) staining at 40x magnification in control (A), polyarthritic rat inoculated with CFA at 5.0 mg/mL (CFA 5.0) (B), 7.5 mg/mL (CFA 7.5) (C) and 10.0 mg/mL (CFA 10.0) (D).

(A) Like the ipsilateral ankle joint, the control rat exhibited normal joint cartilage structure, regular joint space with the normal connective tissue of the synovial membrane. CFA 5.0 group did not demonstrate prominent inflammatory cells penetration in the regular joint space. Meanwhile, the inflammatory cells infiltration was identified between the joint space (arrow) of the CFA 7.5 (C) and CFA 10.0 (D) groups, although it was not as extensive as the ipsilateral ankle joint.