Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Oct 1.
Published in final edited form as: Arthritis Rheum. 2010 Oct;62(10):2995–3005. doi: 10.1002/art.27608

Alteration of Sensory Neurons and Spinal Response To An Experimental Osteoarthritis Pain Model

Hee-Jeong Im 1,2,3,4,*, Jaesung Kim 1,*, Xin Li 1, Naomi Kotwal 5, Dale R Sumner 5, Andre J van Wijnen 6, Francesca J Davis 1, Dongyao Yan 1, Brett Levine 3, James L Henry 7, Jacques Desevré 8, Jeffrey S Kroin 9
PMCID: PMC2952041  NIHMSID: NIHMS214387  PMID: 20556813

Abstract

OBJECTIVE

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

METHODS

An animal model for knee joint OA pain was generated by intra-articular injection of monosodium iodoacetate (MIA) in Sprague Dawley rats followed by symptomatic behavioral pain tests. The corroborating 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 histology, immunohistology, microscopic examination and μCT imaging. Progressive changes in the dynamic interrelationships between peripheral knee joint tissues and central components of nociceptive pathways by OA-induced pain were examined by focusing on cytokine production and expression in sensory neurons of the dorsal root ganglion and spinal cord.

RESULTS

Our results indicate that intra-articular injection of MIA-induced joint degeneration in rats generates an animal model that is suitable for mechanistic and pharmacological studies on nociceptive pain pathways caused by OA. Our results 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.

INTRODUCTION

Chronic pain is a prominent symptom of OA but few studies have examined its role in the etiology of the disease (1). While OA is already a major public health problem, aging of the U.S. population will substantially increase the occurrence of OA-related disability over the next decade. It remains to be established what causes pain in OA, and there is no effective way to relieve the pain induced by OA.

Nociceptors are located throughout the joint in the capsule, ligaments, menisci, periosteum and subchondral bone(2). Data from previous OA pain studies suggest that cellular and structural changes within the peripheral joint components may be the sources of heightened nociception within the arthritic joint (3). The mechanical stimulation of peripheral knee joint components promotes biochemical sensitization and may activate nociceptors located on the afferent nerve fibers of either primary lesions in the cartilage (4, 5, 6) or subchondral bone (7). Nevertheless, the cause of pain in knee OA remains elusive because cartilage, which is the primary site of pathology in OA, lacks pain fibers (8).

Our prior studies indicate that synovial fluid, synovium, joint capsular tissues and cartilage from OA patients with severe pain, highly expresses multiple inflammatory cytokines, and neuropeptides (13). These inflammatory cytokines may further increase biological function of pain mediators by stimulating their cognate receptors, contributing to clinically symptomatic pain perception (13–16). Investigation of OA-related pain mechanisms may elucidate how peripheral tissue injury transmits pain-provoking signals caused by OA and alters nociceptive pathways.

Several OA animal models, including spontaneous, surgically or non-surgically induced OA animal models, have provided biological insights into OA-induced progressive pathological changes in knee joint structures (17) and OA-associated behavioral pain analyses (18). Comparative gene array data demonstrate substantial difference between the MIA-induced OA animal model and human OA (19). Nevertheless, many other investigators have shown that intra-articular injection of MIA, which is an inhibitor of glycolysis that disrupts metabolism in chondrocytes, causes joint tissue damage that may mimic clinical OA in patients (20–29). In this study, we verified the MIA-induced OA animal model to pursue our investigation of the nociceptive pathways caused by OA. We determined the correlations between progressive cellular and structural alterations within the knee joint components and symptomatic chronic pain development by behavioral tests in this animal model using a range of different concentrations of MIA in a time course (0.125 to 2 mg per knee joint over 5 weeks). Using this verified animal model, we examined whether defective peripheral OA region can alter central components of nociceptive pathways in the dorsal root ganglia (DRG) and the dorsal horn of the spinal cord. Our results suggest that activation of peripheral nociceptors is centrally sensitized, stimulating inflammatory cytokines at the level of sensory neurons and spinal cord. Furthermore, our results indicates that pain caused by MIA-induced OA exhibits sensory neuronal responses that are similar to those observed in neuropathic pain model, suggesting mechanistic overlap in these two pain pathways.

MATERIALS AND METHODS

Induction of osteoarthritis

Sprague Dawley rats (weight 200–220g) were housed under standard laboratory conditions (in a temperature-controlled (21±1°C) room with a normal 12-h light/dark cycle). The procedures used in this study were in agreement with the guidelines of the Rush Institutional Animal Care and Use Committee (IACUC). Animals retained full mobility and continued to grow normally. For induction of MIA-induced arthritis, rats were anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL, USA) in oxygen and given a percutaneous single intra-articular injection of 0.125, 0.25, 0.5 and 2.0 mg of monosodium iodoacetate (MIA; Sigma, St. Louis, MO, USA; cat #I2512) or saline vehicle through the infrapatellar ligament of the left knee (N=8 for each group). MIA was dissolved in physiologic saline and administered in a volume of 25 μl using a 26-gauge, 0.5-inch needle. The right contra-lateral knee was used as a behavioral and histological control. Anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgical instability models of OA were induced as previously described (50).

Animal behavioral tests

Thermal hyperalgesia

Responses to noxious thermal stimuli were determined using a thermal plantar device according to the procedure described by Hargreaves before and at defined times during the 31 day period post-MIA injection (31). Rats were placed in opaque plastic chambers (22 cm in width × 17 cm in length × 14 cm in height), above a glass plate, for 10 minutes prior to the start of the each experiment. The animals were allowed to adjust to their new environment before testing. A movable infrared radiant heat source was placed directly under the glass plate aimed at the plantar surface of the hind paw, and the time taken for hind paw withdrawal was monitored. A cut-off time of 20 seconds was used to avoid tissue damage. Two tests were carried out at 10 minutes intervals and then the mean value taken as the nociceptive threshold.

Mechanical allodynia

The method of testing for mechanical allodynia followed that of Chaplan et al. (32). After allowing rats to accommodate for 15 min on a wire mesh grid, a calibrated set of von Frey filaments (Stoelting, Wood Dale, IL) was applied from below to the plantar hind paw to determine the 50% force withdrawal threshold using an iterative method. The filament forces ranged from 0.04 to 15 g, beginning with 2.0 g. The filament was applied to the skin with enough pressure to buckle, and was maintained for up to 6 sec. A brisk lifting of the foot was recorded as a positive response. If no response was observed, the filament with the next highest force was applied, while the filament with the next lowest force was applied upon a positive response.

Knee extension test was performed for both legs by starting with knee in resting position (slightly flexed). While holding thigh, the knee was extended. Number of vocalizations was counted in 5 extensions.

Knee squeeze was performed for both legs by holding knee between thumb and forefinger in medial-lateral direction. The knee was squeezed firmly (but not so strong that un-injected leg responds), and number of vocalizations was counted in 5 squeezes.

Knee Edema

The cross-sectional area of the injected knee and the contralateral control knee were measured with calipers to determine the time course of edema.

Computerized incapacitance meter system

The Dynamic Weight Bearing (DWB) (Bioseb Co. Vitrolles, France) is a computerized incapacitance meter system in which behavioral observations are made in free ambulation on four paws which is consistent with the use of quadrupeds, thus, there is no bias of observation. It is an important aspect for basic and pre-clinical research due to the weight redistribution on other body parts (i.e. the front limbs). Decreased paw surface was quantified as OA induced impairments in paw positioning. During the data capture, the raw data for each paw were synchronized with the images from a video camera and the averaged values were encrypted and recorded on a computer with a sampling rate of 10 Hz, providing accurate and non-biased pain assessment. The weight distribution of the animal, per limb, was then shown in the result window, for each time period with the mean and the variation coefficient.

General tissue preparation

Human articular synoviual tissues from knees were obtained from tissue donors through the Gift of Hope Organ and Tissue Donor Network (normal tissue specimens) and Rush Orthopedic Depository Studies (surgically removed OA tissues). For normal tissues, each donor specimen was graded for gross degenerative changes based on a modified version of the 5-point scale of Collins (33). At 2 and 5 weeks post-MIA or saline injection, the animals were euthanized with halothane anaesthesia. The entire knee joints were then dissected for histology, immunohistochemistry, microscopic analyses and μCT imaging evaluation. Bilateral lumbar DRGs and dorsal horn of the spinal cords were harvested under the light microscope for further analyses.

Histology

The animals were sacrificed and the each knee was dissected aseptically, and fixed in 4% paraformalin, then decalcified in EDTA, which was changed every 5 days. The decalcified knee was cut in the mid-sagital plane, and paraffin-embedded. Serial knee sections of exact 5 μm thickness from the middle part of the knee were obtained to prepare slides. Safranin-O Fast Green stain was performed to assess general morphology and the loss of proteoglycan in cartilage ground substance Human synovium and rat knee joints were stained with hematoxylin and eosin (H&E) stain to assess general morphology and neovascularization. For immuno-histochemistry of neurofilament, medium chain, rabbit polyclonal antibody (Thermo Scientific, Rockford, IL, Cat# PA1-84587) which specifically recognizes both human and rat proteins was used in 1:500 dilution. All samples from both knees were stained, and examined independently by two observers.

Microscopic analyses and μCT

Structural alterations of articular cartilage surface and subchondral bone architecture were evaluated by microscopic examination and μCT scan. Freshly dissected tibias and femurs were immediately fixed in 10 % formalin followed by μCT imaging analyses in the Rush Imaging Core Facility, using a Scanco Model 40 Desktop μCT. Microscopic analyses were performed to examine structural alterations in anterior and inferior articular cartilage using a Nikon SMZ1000 (Model #3.2.0, Diagnostic Instrument, Inc. Sterling Heights, MI). Subchondral bone was assessed in animal groups with and without OA-induced knee joint pain. Two different concentrations of MIA (0.5 and 2.0mg per leg), and two time courses (2 and 5 weeks) were assessed.

Cytokine Antibody Array and quantification

Entire rat spinal cords were ejected and the dorsal horn was dissected under light microscopy. Tissues were lysed by homogenization in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS and 50 mM Tris, pH 7.5) with protease cocktail inhibitors (Sigma). The total protein concentrations of cell lysates were determined by a bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). An array for cytokine proteins (Cytokine Array, RayBio, Norcross, GA) was used to determine relative alterations in the level of cytokines. Membranes with immobilized antibodies were incubated for 14 h with either 500 μg total protein of the control tissues or experimental tissues (degenerated), followed by biotin-conjugated antibodies, then further incubated with horseradish-peroxidase (HRP)-conjugated streptavidin. Immunoreactivity was visualized using the ECL system (Amersham Biosciences, Piscataway, NJ, USA) and the Signal Visual Enhancer system (Pierce), which magnifies the intensity of the signal. Densitomtric measurement was performed by calculating the integrated density values for each spot (area relative intensity) by using Molecular Imager Versadoc MP 4000 System (Bio-Rad, Hercules CA) and Quantity One-4.5.0 Basic 1-D Analysis Software (Bio-Rad). The positive control signals on each membrane were used in normalization of signal intensity.

Total RNA isolation and Reverse transcription and real-time PCR

Lumbar DRGs and spinal dorsal horn were disrupted and homogenized. Total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA) following the instructions provided by the manufacturer. Reverse transcription (RT) was carried out with 1μg total RNA using ThermoScript TM RT-PCR system (Invitrogen) for first strand cDNA synthesis. For real-time PCR, cDNA was amplified using MyiQ Real-Time PCR Detection System (Bio-Rad Hercules, CA). Beta-actin was used as internal control. The deviations in samples represent three different donors in three separate experiments. The primers employed were: (forward) 5′-TCTAGTGTCACTGCCCAGAAGAGA-3′, (reverse) 5′-GGCACAAAGTTGTCCTTCACCACA-3′ for CGRP (NCBI reference # NM_001033956.1); (forward) 5′-AGATCCAGCCCTGAGACACTGATT-3′ and (reverse) 5′-TGGAAGGGTCTTCAAGCCTTGTTC-3′ for Neuropeptid Y (NCBI reference # M15793.1); (forward) 5′-TTCCCACCACTGCTCAAGATG-3′ (reverse), 5′-TGGCTGACAGGGTTGCAA-3′ for Galanin (NCBI reference # NM_033237.1 ); (forward ) 5′-TCTGTGCCTCAGCCTCTTCTCATT-3′, (reverse) 5′-TTGGGAACTTCTCCTCCTTGTTGG-3′ for TNF-α (NCBI reference # NM_012675.3 ); (forward) 5′-TCATCTTTGAAGAAGAGCCCGTCC-3′, (reverse) 5′-TGCAGTGCAGCTGTCTAATGGGAA-3′ for IL-1 (NCBI reference # NM_031512.2); (forward) 5′-TGGTCAGATCTCTCACAAAGG-3′ and (reverse) 5′-TGCATTGCGCTTCTTTCATA-3′ for substance P (NCBI reference #NM_012666.2), (forward) 5′-TGTCACCAACTGGGACGATATGGA-3′ and (reverse) 5′-AGCACAGGGTGCTCCTCA-3′ for β-actin (NCBI reference # NM_031144).

Statistical analysis

All results are expressed as mean values ± S.E.M. (standard error of mean). Repetitive testing of paw withdrawal was analyzed using repeated measures general linear model with a post-hoc Student-Newman-Keuls test with P<0.05 accepted as statistically significant (SPSS 11.5 software, Chicago, IL). The evaluation of real-time PCR data was done by one-way ANOVA with a post-hoc Tukey’s test using 2−ΔΔct values of each sample. A value of P<0.05 was considered significant.

RESULTS

Articular-injection of MIA dose-dependently increases knee joint discomfort in rats

A rat model for knee joint pain was generated by intra-articular injection of MIA followed by behavioral tests. Sequential pain behavioral tests reveal that animals administered with concentrations of 0.5 and 2.0 mg per knee (ipsilateral) express significant increases in joint discomfort in response to MIA. The response occurs in a concentration-dependent manner as defined by increased vocalization to knee squeeze (p<0.001), and to knee extension (p<0.001) compared to the contralateral knee and lower concentrations of MIA (0.125, 0.25 mg/joint, N=8 rats per group; representative of two separate experiments) (Fig. 1A&B). Interestingly, MIA (0.5 mg/knee joint)-injected rats display a time-dependent knee joint pain behavior with phases of early-OA (2–3 weeks), and end-stage OA (>4 weeks) after the initial period of inflammation (day 7–10) (Fig. 1A). Consistently, dose-dependent decreases in latency to heat (p<0.001) (thermal hyperalgesia, data not shown), and increases in mechanical allodynia (von Frey) (p<0.001) are also observed 35 days post-injection, when compared to the contra-lateral side (Fig. 1C). This symptomatic joint discomfort is sustained during the entire joint pain assessment period (>8 weeks, data not shown), suggesting the development of chronic pain. No significant edema in the injected knee is observed after 7–10 days, indicating that MIA-induced knee joint pain is OA-like chronic pain, and inflammatory responses are not a major component to the sustained pain (Fig. 1D). These behavioral pain assessments are further verified by dynamic weight bearing measurements using a computerized incapacitance meter system (Bioseb Co. Vitrolles, France) in which behavioral observations are made during free quadrupedal ambulation. These measurements assess consistent use of all four paws and whether there is observational bias due to weight redistribution to other body parts (i.e. the front limbs). Our results revealed significant differences (p<0.03) in the weight distribution during rearing (g) between 5 weeks post-MIA (0.5mg)-injected knee (ipsilateral) and contralateral side (data not shown).

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

Fig. 1

Open in a new tab

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.

MIA-induced joint pain is correlated with alterations in the structural components of the knee joints, subchondral bone and chondrocyte gene expression

To evaluate the interrelationship between the symptomatic pain and joint pathology in the MIA-induced knee joint pain model, alterations in structural components of the knee joint tissues were examined by histology. We observed dose-, and time-dependent (2 and 5 weeks) loss of proteoglycan in the MIA-injected knee joint cartilage and meniscus by Safranin-O staining (Fig. 2A). These histological changes correlate with altered gross appearance in both anterior and inferior articular cartilage surfaces in a time- and dose-dependent manner (Fig. 2B). Similarly, the three-dimensional μCT scans (Fig. 2C) demonstrate structural alterations in the subchondral bone of MIA-induced animal knee joints, as seen in human OA pathology (33). The surface rendering of the subchondral plate indicates that MIA-injected joint surface topography is considerably altered in a time-, and concentration-dependent manner. Administration of 2 mg MIA per knee on week 5 results in severely heaved and sunken surface (Fig. 2C.k, l) compared to a lower dose of MIA (0.5 mg) at an earlier time-point (week 2) (Fig. 2C. c, d). Saline-injected control knees are not altered and maintain structural integrity of the knee joint (Fig. 2B, 2C. a,b,g,h). In the coronal and sagittal planes of the ipsilateral joints, pathological changes are evident in large regions beneath the subchondral plates, reflected by the formation of cysts, osteophytes, and structural changes in bone shapes especially in knee joints injected with 2 mg MIA (Fig. 2C. k, l). Collectively, these results suggest that MIA-induced joint pain is correlated with structural alterations in knee joint components and subchondral bone that resemble pathological alterations characteristic of human OA.

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

Fig. 2

Open in a new tab

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.

In order to examine the changes in early- (2 wks) and end-stage of OA (>4 wks), we further analyzed altered gene expression for early- and late-stage of disease progression. Our real-time PCR results suggest the similar patterns of altered gene expression as the disease progresses. These genes include, downregulation of MMP-13 and upregulation of IL-1β, TNFα, nerve growth factor (NGF) in late-OA compared to early-OA (Fig. 2D). These results are consistent with those previously reported (34).

MIA-induced knee joint OA is associated with angiogenesis in the peripheral knee joint tissues and mimics pathological changes in human end-stage OA

Synovial angiogenesis may be stimulated during the inflammatory response that accompanies the pathological progression of OA (35). We examined whether MIA-induced knee joint synovium is associated with neovascularization as is frequently seen in OA patients’ synovium with knee joint pain history. Interestingly, there is a clear evidence of neovascularization in synovial tissues in H&E stained sections from MIA-injected knee joint tissues at week 5 (Fig. 3A). This angiogenic feature in OA-induced animal model mimics changes within the human synovium from symptomatic OA patients (Fig. 3B). Angiogenesis promotes ingrowth of new sensory nerves into peripheral knee joint tissues exposed to damage and inflammation, and can contribute to persistent pain, even after inflammation has subsided (36). Indeed, in our OA model, we observed a significant increase in sensory innervation as reflected by increased neuroflament-M (NF-M) immunoreactivity in MIA-injected synovium, when compared to saline-injected and/or contralateral knee joint tissue (Fig. 3C).

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

Fig. 3

Open in a new tab

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).

Alterations in DRG sensory neurons in the MIA-induced OA pain model: comparison studies with a neuropathic pain model

To understand the nociceptive pathways that mediate knee joint pain, we investigated peripheral sensory neuronal responses in the MIA-induced knee OA model. The expression levels of cytokines and pain mediators in the L3-5 DRGs were investigated using real-time PCR. Analyses using each bilateral lumbar section of DRG (L1-L6), there were no difference in a panel of representative inflammation or pain-related phenotypic gene levels in the bilateral DRGs [L1/L2 (L1), L2/L3 (L2), L3/L4 (L3), L4/L5 (L4), L5/L6 (L5) and S1 (L6/S1)] (data not shown). Therefore, subsequent analyses were focused on L3-L5 DRGs, and examined selected inflammatory cytokines (Fig. 4A), or pain-mediators and neuropeptides (Fig. 4B). These include IL-1β, TNF-α, CGRP (calcitonin gene related protein), substance P, Neuropeptide Y, and galanin. These pain-associated genes are significantly up- or downregulated in other pain pathways, such as neuropathic and inflammatory pain (37). Alterations in sensory neurons in DRGs from animals with MIA-induced knee joint OA pain were compared with neuronal changes in animals with neuropathic pain. Interestingly, the results show that gene expression patterns for the two pain pathways are strikingly similar, suggesting that pain provoked by OA may be mediated in part through neuropathic pain mechanisms. To ensure that the MIA-induced rat pain model is appropriate for translational OA pain studies, we also compared gene expression patterns in DRGs, using destabilization surgery by anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) which are alternative established OA animal models (Fig. 4C). As summarized in Table 1, our data suggest that all three rat models for OA-induced pain (i.e., ACLT, DMM and intra-articular injection of MIA models) exhibit similar gene expression patterns in DRGs. Taken together, neuropathic pain mechanisms may be fundamental to the etiology of OA independent of the biological models in which OA is initiated.

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

Fig. 4

Open in a new tab

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.

Table 1.

Altered gene expression in the sensory neurons in DRGs in a neuropathic animal model compared to rat models for OA-induced knee joint pain including ACLT, DMM and the intra-articular injection of MIA-induced OA model. Upregulated (↑) or down-regulated (↓) expression patterns relative to sham control are indicated by arrows. SNL, Spinal nerve ligation; ACLT, anterior cruciate ligament transection; DMM, surgical destabilization of the medial meniscus.

PAIN: NEUROPATHIC (SNL) OA KNEE JOINT PAIN
MODEL: MIA-induced ACLT DMM
DREAM ↑ ↑ ↑ ↑
TNFα ↑ ↑ ↑ ↑
CGRP ↓ ↓ ↓ ↓
Substance P ↓ ↓ ↓ ↓
Galanin ↑ ↑ ↑ ↑
Neuropeptide Y ↑ ↑ ↑ ↑
IL-1α,β ↑ ↑ ↑ ↑
Open in a new tab

Spinal response to the experimental MIA-induced knee OA pain model

Peripheral sensitization is characterized by the interplay between peripheral nociceptors and inflammation, and may initiate an osteoarthritic joint pain (38,39) via central sensitization. Because central sensitization involves the enhanced excitability of neurons, we examined spinal responses provoked by MIA-induced chronic knee joint pain Protein expression profiling was performed by using a Cytokine Antibody Array that permits simultaneous quantitative detection of anti- and pro-inflammatory cytokines. Spinal dorsal horn tissue protein extracts were prepared from carefully dissected ipsilateral dorsal horn of the lumbar spinal cord from animals treated with 2 mg MIA and sham animals receiving saline-injections. Tissue lysates from the dorsal horn of the spinal cord were prepared after 5 weeks, and incubated with array membranes to monitor altered cytokine levels (Fig. 5A). Quantitative changes in cytokine protein levels were measured by densitometric analyses and were graphically depicted as histograms (Fig. 5B). The results revealed significant increases in the levels of multiple pro-inflammatory cytokines and chemokines (e.g., IL-1β and α, RANTES, CINC2α/β, IL-17, Thymus chemokines (TC), TNFα, L-Selectin and VEGF). Unexpectedly, we observed a reduction of fractalkine an acute spinal injury pain-associated chemokine (40) and GM-CSF (p<0.05, respectively). We also found a robust reduction in anti-inflammatory factors such as IL-4 (p<0.05) and IL-10 (p<0.05). However, the anti-inflammatory factor IL-13 is increased at the spinal level in OA-induced animals.

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

Fig. 5

Open in a new tab

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.

To assess whether cytokine protein levels correlate with corresponding changes in mRNA levels within the cellular components of the spinal cord (i.e. glia and neurons), we examined TNFα mRNA as a representative pain-associated cytokine which is highly upregulated at the protein level in the spinal cord due to OA-induced knee joint pain. Real-time PCR results demonstrate that the expression of TNFα is substantially increased at the late stages of OA-induced pain period (week 5), but not during an earlier stages (week 2) (Fig. 5C). Our results suggest that modulation in the expression of TNFα and perhaps other cytokines contribute to chronic pain development in OA.

DISCUSSION

Our study provides new insights into molecular mechanisms by which nociceptive pathways associated with chronic pain in OA. Our results verified that the intra-articular-injected MIA-induced OA rat animal model is suitable for mechanistic and pharmacological studies on symptomatic pain caused by OA. We correlated alterations in the central compartments (DRGs and the spinal cord) with structural changes in components of the peripheral knee joint and symptomatic pain. Our results provide key in vivo evidence that OA pain is caused by central sensitization of the nociceptive pathways. Our data suggest that the affected peripheral OA region transduces nociceptive signals to central compartments to overproduce inflammatory cytokines and pain-mediators at the level of the sensory neurons and spinal cord.

Animal models have provided useful tools for investigating neuropathic or inflammatory pain (17). Generation of animal models for OA pain studies has been more challenging because it requires gradual structural changes associated with the sustained chronic pain while avoiding severe inflammation or direct nerve damage. The MIA-induced OA animal model was first described by Kalbhen (41), and is highly reproducible with predictable behavioral pain responses. The model demonstrates a clear interrelationship between structural knee joint components, including cartilage, synovium, and subchondral bone and corresponding pain behavior. The histopathology of the knee joint after intra-articular injection of MIA mimics features similar to that seen in human OA (20–29, 41,42). Our present data corroborate these previous reports. Furthermore, our dose response- and time-course studies suggest specifically that intra-articular injection with 0.5 mg of MIA per knee joint could be representative of early-OA at 2 weeks, and end-stage OA at >4 weeks post-injection, similar to the structural changes observed in human OA. However, Barve et al (2007) suggested substantial difference between the MIA-induced knee joint pain model and human OA. It is not clear at this point whether this difference is resulted from differences in two species therefore, it need to be more careful to translate the data obtained from MIA-induced knee joint OA pain analyses (19).

Blood vessels formation is remarkably increased in synovium of knee joints in OA patients compared to normal human tissues obtained from normal (Collin’s grade 0 or 1) (33). In the current study, MIA-induced OA knee joint pain is associated with the increased neovascularization which mimics human end-stage OA. The increase in angiogenesis is significant in the animal model at week 5 time point, however we did not observe prominent angiogenic features in rats at 2 weeks after injection with MIA, suggesting that angiogenesis progresses slowly. It is possible that extensive angiogenesis, that is characteristic of advanced human OA, may not develop until 4–5 weeks after the administration of MIA in the animal model (end-stage OA). Slow progression of angiogenesis in our animal model may resemble early stages of human OA in which we have not been able to detect significant increases in angiogenesis in human knee joint synovium derived from early-OA patients (Collin’s grade 2 or 3) (33). Synovial neovascularization is frequently accompanied by inflammation (‘synovitis’) in human OA (37) and largely driven by secretion of angiogenic factors (i.e. bFGF, VEGF) and inflammatory cytokines by lymphocytes that initiate a positive feedback loop (14, 37). Thus, it is possible that angiogenesis may facilitate the sustained chronic pain in OA as proposed previously (37).

Our comparison of several OA animal models including the MIA-injected knee OA model and neuropathic pain models reveals that OA pain pathways may, at least in part, overlap with neuropathic pain mechanisms. Our results suggest that OA-induced pain is associated with central sensitization, perhaps via neuronal and glial cellular activity. MIA-induced alterations of sensory neuronal responses to OA pain is also observed upon destabilization of the knee joint by anterior cruciate ligament transection (ACLT), another well-established OA animal model. Our results are consistent with the previous observation that MIA-injected rats become responsive to medication that is normally prescribed for chronic and neuropathic pain states (44–49).

Peripheral sensitization, which is the result of increased activity of peripheral nociceptors by inflammation, leads to central sensitization, which involves enhanced excitability of cellular components of spinal cord (i.e. glia and neurons) (49). Expression profiling using Cytokine Antibody Arrays and real-time PCR analyses reflects the responses of cellular components of the spinal cord by modulating levels of multiple cytokines in response to OA-induced pain. Some of the cytokines that are elevated in the OA-induced pain are well-characterized pain-mediators that promote central sensitization in other pain animal models, such as neuropathic pain (47–49). We do not know yet which cytokines and what signaling components triggered by these cytokines are rate-limiting targets that control central sensitization caused by OA. Continuation of studies using the MIA-induced OA pain model, which is a useful tool for the investigation of onset and progression of chronic nociceptive pain mechanisms, may ultimately lead to the potential development of a new class of molecular medicines that can effectively alleviate knee joint pain caused by degenerative joint diseases such as OA. The development of ‘OA-specific analgesic drugs’ as a future objective will build on the principal ramification of the current study that the dynamic interactions between peripheral knee joint tissues and central sensitization may occur through nociceptive pain pathways.

Acknowledgments

We would like to thank the tissue donors, Dr. Arkady Margulis, and the Gift of Hope Organ and Tissue Donor Network for normal human joint tissue samples, and also thank Dr. Gabriella Cs-Szabo and Orthopedic Tissue Repository Studies for OA tissues. We would like to thank Dr. Carol Muehleman for her thoughtful discussions on microscopic observations and morphological changes in structural knee joint components, Dr. John Cavanaugh (Wayne State University) for generously sharing his expertise in sensory innervations and musculoskeletal pain. This work was supported by grants (to HJI) from NIH NIAMS (R01AR053220), the Arthritis Foundation, and the National Arthritis Research Foundation. We also appreciate the support from the Department of Anesthesiology at Rush to allow us access to the institutional animals and surgery facility. We express sincere thank to Dr. Jinyuan Li and Boris Jozic for their help during animal surgery and pain assessments.

References

  • 1.Sprangers MA, de Regt EB, Andries F, van Agt HM, Bijl RV, de Boer JB, et al. Which chronic conditions are associated with better or poorer quality of life? J Clin Epidemiol. 2000;53(9):895–907. doi: 10.1016/s0895-4356(00)00204-3. [DOI] [PubMed] [Google Scholar]
  • 2.Felson DT. The sources of pain in knee osteoarthritis. Curr Opin Rheumatol. 2005;17(5):624–8. doi: 10.1097/01.bor.0000172800.49120.97. [DOI] [PubMed] [Google Scholar]
  • 3.Schuelert N, McDougall JJ. Electrophysiological evidence that the vasoactive intestinal peptide receptor antagonist VIP6-28 reduces nociception in an animal model of osteoarthritis. Osteoarthritis Cartilage. 2006;14(11):1155–62. doi: 10.1016/j.joca.2006.04.016. [DOI] [PubMed] [Google Scholar]
  • 4.Schaible HG, Grubb BD. Afferent and spinal mechanisms of joint pain. Pain. 1993;55(1):5–54. doi: 10.1016/0304-3959(93)90183-P. [DOI] [PubMed] [Google Scholar]
  • 5.Just S, Pawlak M, Heppelmann B. Responses of fine primary afferent nerve fibres innervating the rat knee joint to defined torque. J Neurosci Methods. 2000;103(2):157–62. doi: 10.1016/s0165-0270(00)00310-1. [DOI] [PubMed] [Google Scholar]
  • 6.Broom N, Chen MH, Hardy A. A degeneration-based hypothesis for interpreting fibrillar changes in the osteoarthritic cartilage matrix. J Anat. 2001;199(Pt 6):683–98. doi: 10.1046/j.1469-7580.2001.19960683.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Arlet J, Ficat P, Mazieres B. Coxarthroses due to ischemia. Ischemic coxarthropathy. Rev Rhum Mal Osteoartic. 1978;45(10):549–60. [PubMed] [Google Scholar]
  • 8.Dye SF, Vaupel GL, Dye CC. Conscious neurosensory mapping of the internal structures of the human knee without intraarticular anesthesia. Am J Sports Med. 1998;26(6):773–7. doi: 10.1177/03635465980260060601. [DOI] [PubMed] [Google Scholar]
  • 9.Creamer P, Hunt M, Dieppe P. Pain mechanisms in osteoarthritis of the knee: effect of intraarticular anesthetic. J Rheumatol. 1996;23(6):1031–6. [PubMed] [Google Scholar]
  • 10.Dieppe PA, Lohmander LS. Pathogenesis and management of pain in osteoarthritis. Lancet. 2005;365(9463):965–73. doi: 10.1016/S0140-6736(05)71086-2. [DOI] [PubMed] [Google Scholar]
  • 11.Hannan MT, Felson DT, Pincus T. Analysis of the discordance between radiographic changes and knee pain in osteoarthritis of the knee. J Rheumatol. 2000;27(6):1513–7. [PubMed] [Google Scholar]
  • 12.Ji RR, Gereau RWt, Malcangio M, Strichartz GR. MAP kinase and pain. Brain Res Rev. 2009;60(1):135–48. doi: 10.1016/j.brainresrev.2008.12.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Im HJ, Li X, Muddasani P, Kim GH, Davis F, Rangan J, et al. Basic fibroblast growth factor accelerates matrix degradation via a neuro-endocrine pathway in human adult articular chondrocytes. J Cell Physiol. 2008;215(2):452–63. doi: 10.1002/jcp.21317. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 14.Im HJ, Muddasani P, Natarajan V, Schmid TM, Block JA, Davis F, et al. Basic fibroblast growth factor stimulates matrix metalloproteinase-13 via the molecular crosstalk between the mitogen-activated protein kinases and protein kinase Cdelta pathways in human adult articular chondrocytes. J Biol Chem. 2007;282(15):11110–21. doi: 10.1074/jbc.M609040200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Li X, Ellman M, Muddasani P, Wang JH, Cs-Szabo G, van Wijnen AJ, et al. Prostaglandin E2 and its cognate EP receptors control human adult articular cartilage homeostasis and are linked to the pathophysiology of osteoarthritis. Arthritis Rheum. 2009;60(2):513–23. doi: 10.1002/art.24258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Muddasani P, Norman JC, Ellman M, van Wijnen AJ, Im HJ. Basic fibroblast growth factor activates the MAPK and NFkappaB pathways that converge on Elk-1 to control production of matrix metalloproteinase-13 by human adult articular chondrocytes. J Biol Chem. 2007;282(43):31409–21. doi: 10.1074/jbc.M706508200. [DOI] [PubMed] [Google Scholar]
  • 17.Bendele A, McComb J, Gould T, McAbee T, Sennello G, Chlipala E, et al. Animal models of arthritis: relevance to human disease. Toxicol Pathol. 1999;27(1):134–42. doi: 10.1177/019262339902700125. [DOI] [PubMed] [Google Scholar]
  • 18.Neugebauer V, Han JS, Adwanikar H, Fu Y, Ji G. Techniques for assessing knee joint pain in arthritis. Mol Pain. 2007;3:8. doi: 10.1186/1744-8069-3-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Barve Ra, Minnerly JC, Weiss DJ, Meyer DM, Aguiar DJ, Sullivan PM, Weinrich SL, Head RD. Transcriptional profiling and pathway analysis of monosodium iodoacetate-induced experimental osteoarthritis in rats: relevance to human disease. Osteoarthritis Cartilage. 2007;15:1190. doi: 10.1016/j.joca.2007.03.014. [DOI] [PubMed] [Google Scholar]
  • 20.Bendele AM, Hulman JF. Spontaneous cartilage degeneration in guinea pigs. Arthritis Rheum. 1988;31(4):561–5. doi: 10.1002/art.1780310416. [DOI] [PubMed] [Google Scholar]
  • 21.Gustafson SB, Trotter GW, Norrdin RW, Wrigley RH, Lamar C. Evaluation of intra-articularly administered sodium monoiodoacetate-induced chemical injury to articular cartilage of horses. Am J Vet Res. 1992;53(7):1193–202. [PubMed] [Google Scholar]
  • 22.Pond MJ, Nuki G. Experimentally-induced osteoarthritis in the dog. Ann Rheum Dis. 1973;32(4):387–8. doi: 10.1136/ard.32.4.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Moskowitz RW, Davis W, Sammarco J, Martens M, Baker J, Mayor M, et al. Experimentally induced degenerative joint lesions following partial meniscectomy in the rabbit. Arthritis Rheum. 1973;16(3):397–405. doi: 10.1002/art.1780160317. [DOI] [PubMed] [Google Scholar]
  • 24.Combe R, Bramwell S, Field MJ. The monosodium iodoacetate model of osteoarthritis: a model of chronic nociceptive pain in rats? Neurosci Lett. 2004;370(2–3):236–40. doi: 10.1016/j.neulet.2004.08.023. [DOI] [PubMed] [Google Scholar]
  • 25.Guzman RE, Evans MG, Bove S, Morenko B, Kilgore K. Mono-iodoacetate-induced histologic changes in subchondral bone and articular cartilage of rat femorotibial joints: an animal model of osteoarthritis. Toxicol Pathol. 2003;31(6):619–24. doi: 10.1080/01926230390241800. [DOI] [PubMed] [Google Scholar]
  • 26.Piscaer TM, Waarsing JH, Kops N, Pavljasevic P, Verhaar JA, van Osch GJ, et al. In vivo imaging of cartilage degeneration using microCT-arthrography. Osteoarthritis Cartilage. 2008;16(9):1011–7. doi: 10.1016/j.joca.2008.01.012. [DOI] [PubMed] [Google Scholar]
  • 27.Clements KM, Ball AD, Jones HB, Brinckmann S, Read SJ, Murray F. Cellular and histopathological changes in the infrapatellar fat pad in the monoiodoacetate model of osteoarthritis pain. Osteoarthritis Cartilage. 2009;17(6):805–12. doi: 10.1016/j.joca.2008.11.002. [DOI] [PubMed] [Google Scholar]
  • 28.Kobayashi K, Imaizumi R, Sumichika H, Tanaka H, Goda M, Fukunari A, et al. Sodium iodoacetate-induced experimental osteoarthritis and associated pain model in rats. J Vet Med Sci. 2003;65(11):1195–9. doi: 10.1292/jvms.65.1195. [DOI] [PubMed] [Google Scholar]
  • 29.Bove SE, Calcaterra SL, Brooker RM, Huber CM, Guzman RE, Juneau PL, et al. Weight bearing as a measure of disease progression and efficacy of anti-inflammatory compounds in a model of monosodium iodoacetate-induced osteoarthritis. Osteoarthritis Cartilage. 2003;11(11):821–30. doi: 10.1016/s1063-4584(03)00163-8. [DOI] [PubMed] [Google Scholar]
  • 30.Ferreira-Gomes J, Adaes S, Castro-Lopes JM. Assessment of movement-evoked pain in osteoarthritis by the knee-bend and CatWalk tests: a clinically relevant study. J Pain. 2008;9(10):945–54. doi: 10.1016/j.jpain.2008.05.012. [DOI] [PubMed] [Google Scholar]
  • 31.Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 1988;32(1):77–88. doi: 10.1016/0304-3959(88)90026-7. [DOI] [PubMed] [Google Scholar]
  • 32.Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994;53(1):55–63. doi: 10.1016/0165-0270(94)90144-9. [DOI] [PubMed] [Google Scholar]
  • 33.Muehleman C, Bareither D, Huch K, Cole AA, Kuettner KE. Prevalence of degenerative morphological changes in the joints of the lower extremity. Osteoarthritis Cartilage. 1997;5(1):23–37. doi: 10.1016/s1063-4584(97)80029-5. [DOI] [PubMed] [Google Scholar]
  • 34.Editorial Review. Is the responseof cartilage to injury relevant to osteoarthritis? Arthritis Rheum. 2008;58(5):1207–10. doi: 10.1002/art.23443. [DOI] [PubMed] [Google Scholar]
  • 35.Appleton CT, McErlain DD, Pitelka V, Schwartz N, Bernier SM, Henry JL, et al. Forced mobilization accelerates pathogenesis: characterization of a preclinical surgical model of osteoarthritis. Arthritis Res Ther. 2007;9(1):R13. doi: 10.1186/ar2120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.McDougall JJ, Watkins L, Li Z. Vasoactive intestinal peptide (VIP) is a modulator of joint pain in a rat model of osteoarthritis. Pain. 2006;123(1–2):98–105. doi: 10.1016/j.pain.2006.02.015. [DOI] [PubMed] [Google Scholar]
  • 37.Bonnet CS, Walsh DA. Osteoarthritis, angiogenesis and inflammation. Rheumatology (Oxford) 2005;44(1):7–16. doi: 10.1093/rheumatology/keh344. [DOI] [PubMed] [Google Scholar]
  • 38.McDougall JJ. Arthritis and pain. Neurogenic origin of joint pain. Arthritis Res Ther. 2006;8(6):220. doi: 10.1186/ar2069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Harvey VL, Dickenson AH. Behavioural and electrophysiological characterisation of experimentally induced osteoarthritis and neuropathy in C57Bl/6 mice. Mol Pain. 2009;5:18. doi: 10.1186/1744-8069-5-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Verge GM, Milligan ED, Maier SF, Watkins LR, Naeve GS, Foster AC. Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions. Eur J Neurosci. 2004;20(5):1150–60. doi: 10.1111/j.1460-9568.2004.03593.x. [DOI] [PubMed] [Google Scholar]
  • 41.Kalbhen DA. Chemical model of osteoarthritis--a pharmacological evaluation. J Rheumatol. 1987;14(Spec No):130–1. [PubMed] [Google Scholar]
  • 42.Janusz MJ, Hookfin EB, Heitmeyer SA, Woessner JF, Freemont AJ, Hoyland JA, et al. Moderation of iodoacetate-induced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors. Osteoarthritis Cartilage. 2001;9(8):751–60. doi: 10.1053/joca.2001.0472. [DOI] [PubMed] [Google Scholar]
  • 43.Collins DH. The pathology of articular and spinal diseases. London: Edward Arnold; 1949. pp. 76–9. [Google Scholar]
  • 44.Vonsy JL, Ghandehari J, Dickenson AH. Differential analgesic effects of morphine and gabapentin on behavioural measures of pain and disability in a model of osteoarthritis pain in rats. Eur J Pain. 2009;13(8):786–93. doi: 10.1016/j.ejpain.2008.09.008. [DOI] [PubMed] [Google Scholar]
  • 45.Ivanavicius SP, Ball AD, Heapy CG, Westwood FR, Murray F, Read SJ. Structural pathology in a rodent model of osteoarthritis is associated with neuropathic pain: increased expression of ATF-3 and pharmacological characterisation. Pain. 2007;128(3):272–82. doi: 10.1016/j.pain.2006.12.022. [DOI] [PubMed] [Google Scholar]
  • 46.Fernihough J, Gentry C, Malcangio M, Fox A, Rediske J, Pellas T, et al. Pain related behaviour in two models of osteoarthritis in the rat knee. Pain. 2004;112(1–2):83–93. doi: 10.1016/j.pain.2004.08.004. [DOI] [PubMed] [Google Scholar]
  • 47.Cheng HY, Pitcher GM, Laviolette SR, Whishaw IQ, Tong KI, Kockeritz LK, et al. DREAM is a critical transcriptional repressor for pain modulation. Cell. 2002;108(1):31–43. doi: 10.1016/s0092-8674(01)00629-8. [DOI] [PubMed] [Google Scholar]
  • 48.Xiao HS, Huang QH, Zhang FX, Bao L, Lu YJ, Guo C, et al. Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain. Proc Natl Acad Sci U S A. 2002;99(12):8360–5. doi: 10.1073/pnas.122231899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Gardell LR, Wang R, Ehrenfels C, Ossipov MH, Rossomando AJ, Miller S, et al. Multiple actions of systemic artemin in experimental neuropathy. Nat Med. 2003;9(11):1383–9. doi: 10.1038/nm944. [DOI] [PubMed] [Google Scholar]
  • 50.Glasson SS, Blanchet TJ, Morris EA. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthrithis Cartilage. 2007;15:1061–9. doi: 10.1016/j.joca.2007.03.006. [DOI] [PubMed] [Google Scholar]

RESOURCES