Alteration of sensory neurons and spinal response to an experimental osteoarthritis pain model

Abstract

Objective: To verify the biologic links between progressive cellular and structural alterations within knee joint components and development of symptomatic chronic pain that are characteristic of osteoarthritis (OA), and to investigate the molecular basis of alterations in nociceptive pathways caused by OA-induced pain.

Methods: An animal model of knee joint OA pain was generated by intraarticular injection of mono-iodoacetate (MIA) in Sprague-Dawley rats, and symptomatic pain behavior tests were performed. Relationships between development of OA with accompanying pain responses and gradual alterations in cellular and structural knee joint components (i.e., cartilage, synovium, meniscus, subchondral bone) were examined by histologic and immunohistologic analysis, microscopic examination, and microfocal computed tomography. Progressive changes in the dynamic interrelationships between peripheral knee joint tissue and central components of nociceptive pathways caused by OA-induced pain were examined by investigating cytokine production and expression in sensory neurons of the dorsal root ganglion and spinal cord.

Results: We observed that structural changes in components of the peripheral knee joint correlate with alterations in the central compartments (dorsal root ganglia and the spinal cord) and symptomatic pain assessed by behavioral hyperalgesia. Our comparative gene expression studies revealed that the pain pathways in MIA-induced knee OA may overlap, at least in part, with neuropathic pain mechanisms. Similar results were also observed upon destabilization of the knee joint in the anterior cruciate ligament transection and destabilization of the medial meniscus models of OA.

Conclusion: Our results indicate that MIA-induced joint degeneration in rats generates an animal model that is suitable for mechanistic and pharmacologic studies on nociceptive pain pathways caused by OA, and provide key in vivo evidence that OA pain is caused by central sensitization through communication between peripheral OA nociceptors and the central sensory system. Furthermore, our data suggest a mechanistic overlap between OA-induced pain and neuropathic pain.

Fig. 1. Behavioral pain assessments of the MIA-induced rat OA model

A rat model for joint pain was generated by intra-articular injection of MIA (from 0.125 mg to 2 mg) or saline as sham control followed by sequential pain behavioral tests (n=12). A. Knee pressure hyperalgesia B. Knee extension hyperalgesia. C. Mechanical allodynia (von Frey) D. Edema, injected knee.

Fig. 2. Histological, morphological examination and gene expression in the rat OA model

Rats received a single intra-articular injection of MIA (0.5 or 2 mg per knee) or saline (sham) were euthanized at the specified time-points (2 and 5 weeks). Each rat knee shown is representative for a group of n=12. A. Histological assessment for proteoglycan depletion by Safranin-O staining (X40). B. Microscopic analyses for the gross appearance of the distal femur articular cartilage surfaces. C. Architectural changes in subchondral bone structures analyzed by mCT. D. Real-time PCR analyses using cartilage of 2 & 5 wks post-MIA injection. The expression levels were normalized by b-actin level.

Fig. 3. Histological and immunohistological assessment of synovium tissues in the MIA-induced rat OA model compared with human OA synovium

Rats injected with MIA (0.5 and 2 mg per knee) or saline (sham) were euthanized at week 5 time point. Structural changes in knee joint synovium with increased neovascularization were assessed by H&E staining in rats (n=4) (Panel A). Increased nerveingrowth were detected by increased immunoreactivity of anti-NF-M antibody in MIA-induced knee joint synovium (n=4) (Panel C). H&E staining of age-matched normal and end-stage OA synovium tissues from patients with knee joint pain (n=7) (Panel B).

Fig. 4. Gene expression analysis in DRGs in the rat OA model

Rats received a single intra-articular injection of MIA (2 mg) or saline (sham) were euthanized at week 5. Bilateral lumbar DRGs at levels of 3/4, 4/5 and 5/6 were harvested and relative expression of target gene mRNA were analyzed using real-time PCR. A. Analysis of pro-inflammatory cytokine (IL-1and TNF- α). B. Analysis of pain mediators and neuropeptides (CGRP, Substance P, neuropeptide Y and Galanin). C. Comparison analyses of gene expression patterns in DRG (L4/L5) harvested from established OA animal models of ACLT and DMM. Values are the mean and SEM. ACLT, anterior cruciate ligament transection; DMM, surgical destabilization of the medial meniscus.

Fig. 5. Cytokine profiling in lumbar spinal cord in the MIA-induced rat OA model

Lumbar spinal cords were harvested from rats that received a single intra-articular injection of MIA (2mg) or saline at week 2 and 5. A. Antibody array membranes incubated with the spinal cord tissue lysates (week 5) of the sham (upper panel) and animal with knee joint pain (lower panel) groups. B. The histogram shows relative levels of selected cytokines; note that only the cytokines with altered levels in the experimental group are shown. Each cytokine level is represented as the relative fold-induction by assigning the sham level value of “1”. * <0.05, ** p<0.01, *** p<0.001. D. The spinal dorsal horn was harvest and relative expression of TNF-α mRNA was analyzed using real-time PCR. Values are the mean and SEM.