Neurophysiological assessment of joint nociceptors in the rat medial meniscus transection model of post-traumatic osteoarthritis.

OBJECTIVE: Meniscal injury is a common prelude to post-traumatic osteoarthritis (PTOA). Joint nerves can become damaged in arthritic joints leading to the manifestation of neuropathic pain. Both PTOA and neuropathic pain are more common in females; however, it is unknown whether the neural processing of joint pain is sex-specific. DESIGN: Male and female Wistar rats (230-286g) underwent unilateral medial meniscus transection (MMT) and allowed to recover for 28 days. Pain development was assessed over the time course by von Frey hair algesiometry and dynamic weight bearing. Recordings from joint primary afferents was carried out by electrophysiology at end-stage disease. Nerve damage and ß-endorphin levels were also compared between MMT and sham operated animals. RESULTS: Male MMT rats exhibited significant pain behaviour compared to sham control. Evoked afferent firing rate was heightened in male MMT animals. Female PTOA rats did not show signs of pain behaviour on each of the test days and the neurophysiological properties of their nociceptors was not different from control. Peripheral neuropathy was observed in about 30% of axons from male MMT animals compared to 15% in females. Systemic ß-endorphin levels in female PTOA rats was 91.0 ± 10.4 pg/mL and only 49.0 ± 5.0 pg/mL in males. CONCLUSIONS: Secondary allodynia and joint pain were observed in male but not female MMT rats. Joint nociceptors were sensitized in PTOA males but not in females. This lack of pain in females may be due to the absence of a peripheral neuropathy and greater endogenous opioid production.

Synovial nerve fiber density decreases with naturally-occurring osteoarthritis in horses.

OBJECTIVE: To measure the nerve fiber density in synovial membranes from healthy and OA equine joints and to investigate the relationship between synovial innervation and OA severity, synovial vascularity and synovitis. DESIGN: Twenty-five equine metacarpophalangeal joints were collected post-mortem. The joints were dissected and the macroscopic lesions of the articular cartilage were scored. Synovial membrane specimens (n = 50) were harvested, fixed, sectioned and scored histologically. Immunohistochemical staining and immunofluorescence with S-100 protein, that identifies nerve fibers, and ⍺-actin, that stains vascular smooth muscle, were also performed on site-matched specimens and the relationships between these tissues was interrogated. RESULTS: The nerve fiber density was higher in the superficial layer (≤200 µm) of the synovium when compared to the deeper layer in control equine joints (mean difference (95% C.I.): 0.054% (0.018%, 0.11%)). In osteoarthritic joints, synovial innervation decreased in the superficial layer with increasing macroscopic OA score (ß (SEM), 95% C.I.: -0.0061 (0.00021), -0.0011, -0.00017). The blood vessel density was also higher in the superficial layer of the synovium compared to the deep layer in the control (mean difference (95% C.I.): 1.1% (0.36%, 2.3%)) and OA (mean difference (95% C.I.): 0.60% (0.22%, 1.2%)) equine joints. Moreover, considering all synovial specimens, higher nerve fiber density in the deep layer positively correlated with blood vessel density (ß (SEM), 95% C.I.: 0.11 (0.036), 0.035, 0.18). CONCLUSION: The reduction in nerve fiber density with advanced cartilage degeneration suggests that peripheral neuropathy is associated with equine OA. Whether this link is associated with neuropathic pain, requires further investigation.

Lysophosphatidic acid provides a missing link between osteoarthritis and joint neuropathic pain.

OBJECTIVE: Emerging evidence suggests that osteoarthritis (OA) has a neuropathic component; however, the identity of the molecules responsible for this peripheral neuropathy is unknown. The aim of this study was to determine the contribution of the bioactive lipid lysophosphatidic acid (LPA) to joint neuropathy and pain. DESIGN: Male Lewis rats received an intra-articular injection of 50 µg of LPA into the knee and allowed to recover for up to 21 days. Saphenous nerve myelination was assessed by g-ratio calculation from electron micrographs and afferent nerve damage visualised by activation transcription factor-3 (ATF-3) expression. Nerve conduction velocity was measured electrophysiologically and joint pain was determined by hindlimb incapacitance. The effect of the LPA antagonist Ki-16425 was also evaluated. Experiments were repeated in the sodium monoiodoacetate (MIA) model of OA. RESULTS: LPA caused joint nerve demyelination which resulted in a drop in nerve conduction velocity. Sensory neurones were ATF-3 positive and animals exhibited joint pain and knee joint damage. MIA-treated rats also showed signs of demyelination and joint neuropathy with concomitant pain. Nerve damage and pain could be ameliorated by Ki-16425 pre-treatment. CONCLUSION: Intra-articular injection of LPA caused knee joint neuropathy, joint damage and pain. Pharmacological blockade of LPA receptors inhibited joint nerve damage and hindlimb incapacitance. Thus, LPA is a candidate molecule for the development of OA nerve damage and the origin of joint neuropathic pain.

A commentary on modelling osteoarthritis pain in small animals.

OBJECTIVE: To describe the currently used animal models for the study of osteoarthritis (OA) pain, with an emphasis on small animals (predominantly mice and rats). OUTLINE: Narrative review summarizing the opportunities and limitations of the most commonly used small animal models for the study of pain and pain pathways associated with OA, and discussing currently used methods for pain assessment. Involvement of neural degeneration in OA is briefly discussed. A list of considerations when studying pain-related behaviours and pathways in animal models of OA is proposed. CONCLUSIONS: Animal models offer great potential to unravel the complex pathophysiology of OA pain, its molecular and temporal regulation. They constitute a critical pathway for developing and testing disease-specific symptom-modifying therapeutic interventions. However, a number of issues remain to be resolved in order to standardize pre-clinical OA pain research and to optimize translation to clinical trials and patient therapies.

Cathepsin K inhibition reduces CTXII levels and joint pain in the guinea pig model of spontaneous osteoarthritis.

Cathepsin K is a cysteine proteinase which is believed to contribute to osteoarthritis (OA) pathogenesis. This brief report evaluates the effect of the novel selective cathepsin K inhibitor AZ12606133 on cartilage metabolism in the Dunkin-Hartley guinea pig model of spontaneous OA. In parallel, electrophysiological studies were performed to determine whether acute and chronic treatment with the cathepsin K inhibitor could alter joint nociception. Acute treatment of OA knees with AZ12606133 had no effect on joint afferent nerve activity; however, prolonged (1 month) administration of the cathepsin K inhibitor delivered via a chronically implanted osmotic pump significantly reduced mechanosensitivity in response to both non-noxious and noxious joint movements. Urinal concentrations of the cartilage breakdown products cross-linked C-telopeptides of type II collagen (CTXII) were also reduced by chronic cathepsin K inhibition. These data suggest that prolonged AZ12606133 administration can reduce cartilage turnover and joint nociception in the Dunkin-Hartley guinea pig model of spontaneous OA.

Paradoxical effects of the cannabinoid CB2 receptor agonist GW405833 on rat osteoarthritic knee joint pain.

OBJECTIVE: The present study examined whether local administration of the cannabinoid-2 (CB(2)) receptor agonist GW405833 could modulate joint nociception in control rat knee joints and in an animal model of osteoarthritis (OA). METHOD: OA was induced in male Wistar rats by intra-articular injection of sodium monoiodo-acetate with a recovery period of 14 days. Immunohistochemistry was used to evaluate the expression of CB(2) and transient receptor potential vanilloid channel-1 (TRPV1) receptors in the dorsal root ganglion (DRG) and synovial membrane of sham- and sodium mono-iodoacetate (MIA)-treated animals. Electrophysiological recordings were made from knee joint primary afferents in response to rotation of the joint both before and following close intra-arterial injection of different doses of GW405833. The effect of intra-articular GW405833 on joint pain perception was determined by hindlimb incapacitance. An in vitro neuronal release assay was used to see if GW405833 caused release of an inflammatory neuropeptide (calcitonin gene-related peptide - CGRP). RESULTS: CB(2) and TRPV1 receptors were co-localized in DRG neurons and synoviocytes in both sham- and MIA-treated animals. Local application of the GW405833 significantly reduced joint afferent firing rate by up to 31% in control knees. In OA knee joints, however, GW405833 had a pronounced sensitising effect on joint mechanoreceptors. Co-administration of GW405833 with the CB(2) receptor antagonist AM630 or pre-administration of the TRPV1 ion channel antagonist SB366791 attenuated the sensitising effect of GW405833. In the pain studies, intra-articular injection of GW405833 into OA knees augmented hindlimb incapacitance, but had no effect on pain behaviour in saline-injected control joints. GW405833 evoked increased CGRP release via a TRPV1 channel-dependent mechanism. CONCLUSION: These data indicate that GW405833 reduces the mechanosensitivity of afferent nerve fibres in control joints but causes nociceptive responses in OA joints. The observed pro-nociceptive effect of GW405833 appears to involve TRPV1 receptors.

Proteinase activated receptor (PAR) involvement in mediating arthritis pain and inflammation.

Proteinase activated receptors (PARs) are a newly identified family of G-protein-coupled receptors that are activated by proteinases released into tissues during inflammation. Evidence has accumulated which shows that PARs can exhibit both anti- and pro-inflammatory properties in different organ systems. During arthritis, proteinases are known to be released into the joint where they orchestrate tissue remodelling and degeneration. By activating PARs, these proteinases have the potential to modulate joint inflammation and pain by a highly targeted and selective receptor pathway. The recent identification of PARs on synovial fibroblasts, neutrophils, macrophages and mast cells is further evidence that this intriguing family of receptors could play a role in the pathogenesis and symptoms of arthritis. This review article provides an overview of the current knowledge regarding PARs and their emerging role in arthritis.

Central neural activation of hyperdynamic circulation in portal hypertensive rats depends on vagal afferent nerves.

BACKGROUND: Hyperdynamic circulation in portal hypertensive rats depends on central neural c-fos gene expression, but how the portal hypertensive signal activates central c-fos expression remains obscure. AIM: To elucidate the afferent pathway from the gut to the central cardiovascular regulatory nuclei in portal hypertensive rats. METHODS: Cervical vagus nerves and the coeliac ganglion were treated with topical capsaicin to denervate the sensory afferents. Surgical portal vein stenosis (PVS) was performed 3 weeks after denervation. Effectiveness of vagal afferent denervation was confirmed by absent oesophagus to brainstem transfer of the neural tracer cholera toxin-conjugated horseradish peroxidase. Fos, the protein product of the c-fos gene, was detected by immunohistochemistry in central cardiovascular regulatory nuclei. Vagal nerve activity was measured after acute portal hypertension induced by an inflatable cuff around the portal vein. Cardiac output and mean arterial pressure were recorded. RESULTS: In vagal capsaicin-treated rats, no horseradish peroxidase was detected in the brainstem after oesophageal injection. In vagal capsaicin-treated rats subjected to PVS, Fos expression was significantly decreased in both the solitary tract nucleus and paraventricular nucleus compared with untreated PVS rats. Acute portal hypertension stimulated vagal nerve electrical activity. The hyperdynamic circulation was completely abrogated in vagal capsaicin-treated PVS rats. Capsaicin induced no neural or haemodynamic changes in either sham-operated controls or coeliac ganglion-treated PVS rats. CONCLUSION: In portal hypertensive rats, central cardiovascular regulatory nuclei initiate hyperdynamic circulation in response to a gut signal associated with portal hypertension. Portal hypertension signals to the central nuclei via capsaicin-sensitive vagal afferent nerves.

In vivo effects of CB2 receptor-selective cannabinoids on the vasculature of normal and arthritic rat knee joints.

BACKGROUND AND PURPOSE: Cannabinoids (CBs) are known to be vasoactive and to regulate tissue inflammation. The present study examined the in vivo vasomotor effects of the CB2 receptor agonists JWH015 and JWH133 in rat knee joints. The effect of acute and chronic joint inflammation on CB2 receptor-mediated responses was also tested. EXPERIMENTAL APPROACH: Blood flow was assessed in rat knee joints by laser Doppler imaging both before and following topical administration of CB2 receptor agonists. Vasoactivity was measured in normal, acute kaolin/carrageenan inflamed and Freund's complete adjuvant chronically inflamed knees. KEY RESULTS: In normal animals, JWH015 and JWH133 caused a concentration-dependent increase in synovial blood flow which in the case of JWH133 was blocked by the selective CB2 receptor antagonist AM630 as well as the transient receptor potential vanilloid-1 (TRPV1) antagonist SB366791. The vasodilator effect of JWH133 was significantly attenuated in both acute and chronically inflamed knees. Given alone, AM630 had no effect on joint blood flow. CONCLUSION AND IMPLICATIONS: In normal joints, the cannabinomimetic JWH133 causes hyperaemia via a CB2 and TRPV1 receptor mechanism. During acute and chronic inflammation, however, this vasodilatatory response is significantly attenuated.

Comparison of cannabinoids with known analgesics using a novel high throughput zebrafish larval model of nociception.

It has been established that both adult and larval zebrafish are capable of showing nociceptive responses to noxious stimuli; however, the use of larvae to test novel analgesics has not been fully explored. Zebrafish larvae represent a low-cost, high-throughput alternative to traditional mammalian models for the assessment of product efficacy during the initial stages of drug development. In the current study, a novel model of nociception using zebrafish larvae is described. During the recovery from an acute exposure to low levels of acetic acid, larvae display innate changes in behaviour that may be indicative of nociception. To assess the usefulness of this model for testing potential analgesics, three known synthetic pain medications were assessed (ibuprofen, acetaminophen and tramadol) along with three naturally occurring products (honokiol, tetrahydrocannabinol and cannabidiol). When the effect of each compound on both the acetic acid recovery and control activity was compared there appeared to be both similarities and differences between the compounds. One of the most interesting effects was found for cannabidiol which appeared to oppose the activity change during the recovery period of AA exposed larvae while having a nominal effect on control activity. This would appear to be in line with current research that has demonstrated the nociceptive properties of cannabidiol. Here we have provided a novel model that will complement existing zebrafish models and will expand on the potential use of zebrafish larvae for studying both nociception and new analgesics.

Spatial heterogeneity of the effects of calcitonin gene-related peptide (CGRP) on the microvasculature of ligaments in the rabbit knee joint.

1. Experiments were performed in anaesthetized rabbits to examine the effects of calcitonin gene-related peptide (CGRP) and the CGRP antagonist CGRP8-37 on blood flow to the medial collateral ligament of the knee joint. 2. Topical application of CGRP (10(-13) to 10(-9) mol) to the exposed external surface of eight knee joints resulted in dose-dependent dilatation of vessels in both the ligament and the joint capsule. The magnitude of this response varied significantly in different regions of the medial collateral ligament, with the 10(-9) mol dose of CGRP giving the maximum response (101.5 +/- 25.3% increase) at the femoral insertion site of the medial collateral ligament and lowest (23.1 +/- 8.8%) at the tibial insertion site. 3. Topical application of CGRP8-37 (0.1, 1 and 10 nmol) produced dose-dependent constriction of vessels in the ligament and the joint capsule in five knees, with a trend towards the greatest effect occurring at the femoral insertion site (45.8 +/- 8.1% reduction in blood flow). With the 10 nmol dose, the vasoconstrictor response at the femoral insertion site differed significantly (P<0.05) from the responses obtained at the tibial insertion and joint capsule sites. 4. Topical application of CGRP8-37 (0.1, 1 and 10 nmol) to four chronically denervated knees produced substantially smaller vasoconstrictor responses at all sites. At the femoral insertion site, where 10 nmol CGRP8-37 normally produces a 45.8 +/- 8.1% reduction in blood flow (n=8), ten days following denervation this response was reduced to 6.5 +/- 6.1%, this difference being significant (P=0.01). 5. Adrenaline was applied topically to augment blood vessel tone, in order to establish how effectively co-administration of CGRP would offset this increase in tone. Adrenaline (10(-10) mol) produced vasoconstriction at all sites (n=6). In the capsule this vasoconstriction was virtually abolished when CGRP (10(-9) mol) was co-administered with adrenaline but in the ligament vasodilatation occurred at all sites. This vasodilatation was significantly greater at the femoral insertion site compared to the tibial insertion and mid ligament sites (P<0.05 for both) and the capsule (P<0.01). 6. Topical application of substance P (10(-10) or 10(-9) mol) failed to elicit dilatation of ligament blood vessels. 7. These results suggest that endogenous CGRP may play an important role in regulating blood flow to different structures in and around the knee joint.

Endomorphin-1 causes synovial hypoaemia in rat knee joints via a capsaicin-sensitive neural pathway.

In joints, synthetic mu-opioids reduce inflammatory changes such as protein extravasation and associated oedema formation. However, the effect of endogenous opioid peptides on other inflammatory processes such as altered tissue blood flow has not been investigated. The present study examined the peripheral effects of the endogenous mu-opioid ligand endomorphin-1 (EM-1) on rat knee joint blood flow using laser Doppler perfusion imaging. Topical application of EM-1 (10(-16)-10(-9) mol) to exposed rat knee joints resulted in a dose-dependent increase in synovial vascular resistance with a maximum rise of 56% occurring with the 10(-9) mol dose. Destruction of unmyelinated articular afferents by capsaicin treatment completely abolished the hypoaemic effects of EM-1. These findings suggest that EM-1 acts peripherally in knee joints to decrease synovial blood flow, and this hypoaemic response is dependent on the presence of capsaicin-sensitive nerves.

Peripheral modulation of rat knee joint afferent mechanosensitivity by nociceptin/orphanin FQ.

The peripheral effects of nociceptin were examined in normal and acutely inflamed rat knee joints by analyzing single unit recordings from articular primary afferents in response to normal and extreme rotation of the knee. Bolus close intraarterial injection of nociceptin (0.01, 1 and 100 microM) caused a sensitization of normal and inflamed knee joint afferents in response to movements in the normal working range of the joint. When the joint was hyper-rotated, nociceptin had no significant effect on afferent discharge rate in normal knees, however, in inflamed joints the top dose of the neuropeptide caused a decrease in articular mechanosensitivity. These findings suggest that nociceptin seems to be involved in the control of peripheral nociceptive mechanisms, although the behaviour of the peptide is dependent upon the inflammatory status of the tissue.

Alteration of substance P-mediated vasodilatation and sympathetic vasoconstriction in the rat knee joint by adjuvant-induced inflammation.

The effects of nerve stimulation and topical administration of substance P (SP) on the blood flow supplying the rat knee joint were measured using laser Doppler perfusion imaging. A comparison was made between the responses found in normal knees and those observed in a group of animals with unilateral chronic inflammation induced by intra-articular injection of Freund's adjuvant, 1 week prior to experimentation. In control knees, nerve stimulation produced a frequency-dependent vasoconstriction over the range of 5-30 Hz and application of SP caused a dose-dependent vasodilatation. Chronically inflamed joints showed virtually no response to either nerve stimulation or SP application, suggesting a radical alteration in sympathetic and neuropeptidergic actions.

Adaptation of post-traumatic angiogenesis in the rabbit knee by apposition of torn ligament ends.

Apposition of torn ligament ends has been shown to have a beneficial effect on healing of the medial collateral ligament; however, the mechanism underlying this improved recovery is unclear. Excessive post-traumatic angiogenesis, an inherent component of soft-tissue regeneration, may be functionally detrimental in relatively hypovascular tissues such as ligaments. The present study therefore examined the relationship between contact of transected ligament ends and vascular remodeling. Female New Zealand White rabbits were subjected to a gap injury, Z-plasty apposition, or sham operation to the midsubstance of the medial collateral ligament. Six weeks after treatment, the volume of vessels supplying the healing zone of the medial collateral ligament, as well as the ipsilateral lateral collateral ligament, posterior cruciate ligament. menisci, and medial capsule, was quantified by carmine red vascular casting. The volume of vessels supplying the neoligamentous scar formed by gap injury to the medial collateral ligament was found to be twice that of ligaments that had undergone the sham operation, and lateral collateral ligament and meniscal vascularity was also augmented in the injured joint. The medial collateral ligaments that underwent Z-plasty apposition exhibited a level of vascularity comparable with that of the control ligaments that had undergone the sham procedure, whereas meniscal and lateral collateral ligament vascularities remained elevated in this group. Capsular and posterior cruciate ligament vascularities were unaffected by gap injury or Z-plasty to the ipsilateral medial collateral ligament. These findings indicate that injury to the medial collateral ligament not only stimulates angiogenesis in the healing ligament, but other ipsilateral soft tissues also undergo vascular remodeling. Furthermore, apposition of an injured medial collateral ligament modifies these pro-angiogenic events, and this may partly explain why contact of torn ligament ends is beneficial for post-traumatic recovery of an injured joint.

Dynamic measurement of bone blood perfusion with modified laser Doppler imaging.

Although the mechanisms are not clearly defined, blood flow may play an important role in moderating skeletal adaptation. Most techniques currently available to measure blood flow in bone are time-consuming and require destruction of the tissue, but laser Doppler technology offers a less invasive method. The present study assessed whether laser Doppler perfusion imaging could detect changes in perfusion in cortical bone. By use of modified laser Doppler perfusion imaging with an adjustable, incorporated, near infrared-laser gain photodetection system, perfusion of blood in the mid-diaphyseal tibial cortex of New Zealand White rabbits (n = 5) was measured before, during, and after occlusion of the femoral artery. During occlusion, perfusion decreased 69% compared with control levels; removal of the arterial clip caused flux values to return to near normal. Laser Doppler perfusion imaging provides a two-dimensional image related to blood flow, and the results of this pilot study suggest that it may be an effective technique for imaging in vivo dynamic changes in perfusion in cortical bone.

Evaluation of laser Doppler imaging to measure blood flow in knee ligaments of adult rabbits.

Laser Doppler imaging (LDI) is investigated as a novel method for in vivo ligament tissue blood flow determination. LDI output signal is obtained from surgically exposed rabbit medial collateral ligaments (MCL). The LDI signal is compared with simultaneously determined, coloured microsphere (CM)-derived standardised MCL blood flow. Correlation of LDI output with the CM flow data and a linear regression of 17 data points in nine rabbits (joint injured to provoke an acute vascular response in the tissues) indicate that LDI provides a reasonable estimate of MCL blood flow, at least over the ranges assessed. If properly calibrated, and given enough tissue-specific data points, LDI may have advantages over conventional, but more invasive, techniques. The potential clinical application of LDI technology to joint injury and arthritis research is discussed.

Activation of PAR(2) receptors sensitizes primary afferents and causes leukocyte rolling and adherence in the rat knee joint.

BACKGROUND AND PURPOSE: The PAR(2) receptors are involved in chronic arthritis by mechanisms that are as yet unclear. Here, we examined PAR(2) activation in the rat knee joint. EXPERIMENTAL APPROACH: PAR(2) in rat knee joint dorsal root ganglia (DRG) cells at L3-L5, retrogradely labelled with Fluoro-gold (FG) were demonstrated immunohistochemically. Electrophysiological recordings from knee joint nerve fibres in urethane anaesthetized Wistar rats assessed the effects of stimulating joint PAR(2) with its activating peptide, 2-furoyl-LIGRLO-NH(2) (1-100 nmol·100 µL(-1) , via close intra-arterial injection). Fibre firing rate was recorded during joint rotations before and 15 min after administration of PAR(2) activating peptide or control peptide. Leukocyte kinetics in the synovial vasculature upon PAR(2) activation were followed by intravital microscopy for 60 min after perfusion of 2-furoyl-LIGRLO-NH(2) or control peptide. Roles for transient receptor potential vanilloid-1 (TRPV1) or neurokinin-1 (NK(1) ) receptors in the PAR(2) responses were assessed using the selective antagonists, SB366791 and RP67580 respectively. KEY RESULTS: PAR(2) were expressed in 59 ± 5% of FG-positive DRG cells; 100 nmol 2-furoyl-LIGRLO-NH(2) increased joint fibre firing rate during normal and noxious rotation, maximal at 3 min (normal; 110 ± 43%, noxious; 90 ± 31%). 2-Furoyl-LIGRLO-NH(2) also significantly increased leukocyte rolling and adhesion over 60 min. All these effects were blocked by pre-treatment with SB366791 and RP67580 (P < 0.05 compared with 2-furoyl-LIGRLO-NH(2) alone). CONCLUSIONS AND IMPLICATIONS: PAR(2) receptors play an acute inflammatory role in the knee joint via TRPV1- and NK(1) -dependent mechanisms involving both PAR(2) -mediated neuronal sensitization and leukocyte trafficking.

Involvement of transient receptor potential vanilloid 1 receptors in protease-activated receptor-2-induced joint inflammation and nociception.

Protease-activated receptor-2 (PAR-2) is a G-protein-coupled receptor activated through proteolytic cleavage. It is localized on epithelial, endothelial and inflammatory cells, as well as on transient receptor potential vanilloid 1 (TRPV1) receptor-expressing neurones. It plays an important role in inflammatory/nociceptive processes. Since there are few reports concerning PAR-2 function in joints, the effects of intraarticular PAR-2 activation on joint pain and inflammation were studied. Secondary hyperalgesia/allodynia, spontaneous weight distribution, swelling and inflammatory cytokine production were measured and the involvement of TRPV1 ion channels was investigated in rats and mice. Injection of the PAR-2 receptor agonist SLIGRL-NH(2) into the knee decreased touch sensitivity and weight bearing of the ipsilateral hindlimb in both species. Secondary mechanical allodynia/hyperalgesia and impaired weight distribution were significantly reduced by the TRPV1 antagonist SB366791 in rats and by the genetic deletion of this receptor in mice. PAR-2 activation did not cause significant joint swelling, but increased IL-1beta concentration which was not influenced by the lack of the TRPV1 channel. For comparison, intraplantar SLIGRL-NH(2) evoked similar primary mechanical hyperalgesia and impaired weight distribution in both WT and TRPV1 deficient mice, but oedema was smaller in the knockouts. The inactive peptide, LRGILS-NH(2), injected into either site did not induce any inflammatory or nociceptive changes. These data provide evidence for a significant role of TRPV1 receptors in secondary mechanical hyperalgesia/allodynia and spontaneous pain induced by PAR-2 receptor activation in the knee joint. Although intraplantar PAR-2 activation-induced oedema is also TRPV1 receptor-mediated, primary mechanical hyperalgesia, impaired weight distribution and IL-1beta production are independent of this channel.