Understanding Joint Pain: Temporal Changes in Pathological Structure in Collagen Conditions

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects joints, causing redness, swelling, heat, and pain. The underlying mechanism of RA is the inflammation of the joint synovium, leading to cartilage destruction, bone damage, dysfunction, and deformity. RA impacts about 1% of the global population and significantly increases disability and economic burden.

Pain is a major concern for RA patients. It often results from inflammation in affected joints. Current treatments, including non-steroidal anti-inflammatory drugs and biological therapies, can only partially relieve this pain. Many RA patients still experience moderate pain. A clinical study showed that 41% of RA patients have pain associated with central sensitization. In the early stages of RA, pain is mainly due to pro-inflammatory cytokines released in the joints. As RA progresses, persistent pain activates several pain pathways, causing changes in the central nervous system and impairing pain regulation mechanisms.

Research employs various models to study RA, such as collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA), collagen antibody-induced arthritis (CAIA), and K/BxN serum-transfer arthritis (STA). These models reveal a relationship between synovial inflammation and joint pain, indicating a potential state of post-inflammatory hyperalgesia. Studies found that CIA rats display different degrees of ankle redness and swelling over time, but pain trends remain unclear. Additionally, joint synovial inflammation can decrease bone mass and damage bone structure, increasing fracture risk and leading to secondary osteoporosis. RA patients have double the incidence of osteoporosis compared to healthy individuals, and brittle fractures occur significantly more frequently in RA patients. CIA model rats show reduced bone density and altered bone structure, reflecting the relationship between RA and osteoporosis, although the mechanism of bone destruction in this model is still unclear.

Materials and Methods

Establishment and Grouping of CIA Model Rats

Thirty-five Sprague-Dawley (SD) rats, aged 7–8 weeks and weighing around 200 g, were obtained from BEIJING HFK BIOSCIENCE. They were housed at Shanxi University of Traditional Chinese Medicine in controlled conditions (temperature 22–24°C, humidity 55–60%, 12h light/dark cycle). The Experimental Animal Ethics Committee approved the study protocols. Six rats formed a control group. The remaining 29 rats received subcutaneous injections of complete Freund’s adjuvant and type II collagen. After 21 days, 24 rats with an arthritis index of 4 or higher were selected as CIA models and divided into four groups for observation.

Ethology

Weekly assessments of body weight, arthritis score, and paw swelling were performed. The von Frey filament method measured the mechanical withdrawal threshold (PWT) in the left hind paw. The open field analysis system measured activity levels.

Micro-CT

The micro-CT scanned the left hind paw, focusing on the calcaneus for quantitative analysis of bone microstructure, including bone volume fraction and density.

Biomechanical of Femur

Mechanical tests evaluated the tension and compression of femoral samples using an electronic universal tester.

ELISA

Blood was taken from the abdominal aorta, with serum cytokines IL-1β and TNF-α levels measured by ELISA kits.

HE and TRAP Staining

Paraformaldehyde-fixed ankle joints underwent HE staining for synovial hyperplasia assessment. TRAP staining was used to identify osteoclasts.

Western Blotting

Spinal cord tissues were processed for protein analysis via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and PVDF membrane transfer. Antibodies for c-Fos and CGRP were used for detection.

Immunofluorescence Staining

Paraffin-embedded spinal cord tissues were sectioned and treated for immunofluorescence with anti-c-Fos and DAPI.

Statistical Analysis

Statistics were computed using GraphPad Prism 8. Two-way ANOVA assessed weight and arthritis scores, while one-way ANOVA analyzed other results, ensuring a robust analysis of data significance.

Results

Temporal Change of Weight and Joint in CIA Rats

The weight of CIA rats significantly decreased, while arthritis scores and paw swelling showed marked increases over time, indicating the impact of the CIA model on these metrics.

Temporal Change of Pain and Central Hyperalgesia in CIA Rats

CIA model rats reduced activity levels. The paw withdrawal threshold indicated changes in pain tolerance over time. The expression of pain-related proteins c-Fos and CGRP was elevated in the spinal cord.

Temporal Change of Inflammatory Response in CIA Rats

Inflammatory factors IL-1β and TNF-α in serum showed fluctuations during the modeling period. HE staining revealed a decrease in synovial inflammation over time.

Temporal Change of Bone Destruction in CIA Rats

All model groups exhibited significant bone destruction. Analysis revealed reduced bone volume fraction, density, and trabecular thickness, indicating severe secondary osteoporosis in CIA rats.

Temporal Change of Bone Biomechanics in CIA Rats

The maximum load capacity of the femur decreased significantly in model groups compared to controls, highlighting the effects of bone weakening.

Discussion

This study observed inflammation changes in CIA model rats and their links to pain and osteoporosis. Persistent inflammation may lead to neuropathic pain, suggesting different treatment strategies are needed over the disease course. Early RA treatment should prioritize anti-inflammatory medications, while later stages may require anti-neuropathic pain drugs. Continuous bone destruction in CIA rats underlines the need for prompt intervention following a diagnosis of RA.

Conclusion

The research underscores the need for targeted treatment strategies in RA, considering both inflammatory and bone health aspects to improve patient outcomes provided by further understanding of these interlinked mechanisms.