Purpose: Cartilage oligomeric matrix protein (COMP) is a member of the thrombospondin family. COMP interacts with extracellular matrix proteins in cartilage including aggrecan, matrilin-3, and type IX collagen, and as such is thought to serve as an adaptor protein to mediate interactions between type II collagen and aggrecan. Despite this important structural role in cartilage, Comp null mice display no obvious defects in skeletal development and showed no overt differences from wild-type littermates.

Purpose: Knee osteoarthritis (OA) is characterized by progressive joint damage, including cartilage degradation, bone remodeling, subchondral bone sclerosis, and synovitis. Knee joints are densely innervated by sensory afferents, mostly pain-sensing neurons (nociceptors). We previously reported that the intra-articular nociceptive innervation of the medial compartment of the murine knee joint displays profound neuroplasticity 16 weeks after destabilization of the medial meniscus (DMM). In particular, we observed sprouting of nociceptors in the medial synovium and meniscus, as well as NaV1.8+ nociceptors in subchondral bone channels, closely recapitulating what has been described in human knees with late-stage OA.

Purpose: Osteoarthritis (OA) is one of the leading causes of chronic pain and disability. Yet, management of OA pain remains poor, and often relies on analgesics with limited efficacy. Recent literature points to the emerging role of innate immunity in mediating OA pain. We previously found increased levels of F4/80+ macrophages in the knee-innervating dorsal root ganglia (DRG), 8 weeks after destabilization of the medial meniscus (DMM) in the mouse knee, coinciding with onset of behaviors indicative of persistent pain.

Purpose: Notch signaling plays a fundamental role in neurogenesis and neuroplasticity. Aberrant Notch signaling also contributes to cartilage and bone damage in osteoarthritis (OA) joints. Our previous data showed that Notch signaling is activated in knee-innervating dorsal root ganglia (DRG) after surgical destabilization of the medial meniscus (DMM) in mice, and in vitro studies using DRG cell culture suggested that Notch signaling may interact with toll-like receptor (TLR) signaling to promote synthesis of the pro-algesic chemokine, C-C motif chemokine ligand 2 (CCL2).

Purpose: The knee joint is highly innervated by sympathetic neurons and sensory afferents, most of which are nociceptors (high threshold pain-sensing neurons). NaV1.8 is a voltage-gated sodium channel mainly expressed in nociceptive neurons, the cell bodies of which are located in dorsal root ganglia (DRG). To study the mechanisms of osteoarthritis (OA)-associated pain, we have used NaV1.8-tdTomato reporter mice to document the sensory innervation of murine knee joints. We also reported that systemic chemogenetic inhibition of NaV1.8-expressing neurons decreases knee hyperalgesia in the early stages of experimental OA induced by destabilization of the medial meniscus. The purpose of this study was to (1) evaluate the effects of chemogenetic excitation of NaV1.8-expressing cells on knee hyperalgesia, and (2) determine if NaV1.8-expressing knee-innervating neurons can be selectively ablated by intra-articular (i.a.) injection of adeno-associated virus (AAV) carrying diphtheria toxin A (dtA). Methods: Designer receptors exclusively activated by a designer drug (DREADD) technology allows neuron activation upon binding the synthetic ligand clozapine noxide (CNO). NaV1.8Cre-excitatory (exc) DREADD mice were generated by crossing DREADD hM3D(Gq) mice with NaV1.8Cre mice to express the excitatory DREADD receptor in nociceptors. At age 17-23 weeks, male NaV1.8Cre-Exc-DREADD mice were given CNO via intraperitoneal (i.p.) injection at doses of 0.1, 1, or 10 mg/kg. Age-matched wild type mice were used as controls. Hyperalgesia was assessed on the right knees at different time points before and after CNO injection (0-24 h) using a Pressure Application Measurement (PAM) device by an experimenter blinded to the groups. pAAV-EF1a-mCherry-flex-dtA (AAV-dtA) were packaged into AAV2retro or AAV.PHPS serotypes by Canadian Neurophotonics Platform Viral Vector Core Facility. pAAV-EF1a-flex-GFP was used as a control (AAV-control). AAVs were administered i.a. in 3 μl at a dose of 1013 or 1012 viral particles (vp)/mL into the right knees of 7-week old male NaV1.8Cre-GCaMP6 mice. Three weeks later, these mice were perfused with 4% paraformaldehyde, and their ipsilateral L4 DRG were cryosectioned. AAV transduction efficiency was assessed by calculating the percentages of GFP positive neurons. Another set of male NaV1.8Cre mice were injected i.a. with 1012 vp/mL of AAV.PHP.S-dtA at age 7 weeks. Three weeks later, the Toll-like receptor 2 (TLR2) ligand, Pam3CSK4 (3 μg), was injected into the same knees to detect the effect of AAV-dtA on Pam3CSK4-induced knee hyperalgesia. Results: In the excitatory DREADD study, i.p. injection of CNO at 10 mg/kg in naive NaV1.8Cre-Exc-DREADD mice significantly decreased the knee withdrawal threshold, 1 and 2 h after injection, compared to pre-injection (n=3, p<0.001 and p<0.05, respectively) (Fig. 1). A lower dose of CNO (1 mg/kg) also induced a decreased pain threshold at 2 h after injection compared to pre-injection (n=3, p<0.01). No hyperalgesia was induced by 0.1 mg/kg of CNO in NaV1.8Cre-Exc-DREADD mice. In naive wild type mice, CNO injection induced no hyperalgesia at all given doses (n=2 at each time point). These results indicate that short-term activation of NaV1.8-nociceptors elicited acute knee hyperalgesia. In L4 DRG of NaV1.8Cre-GCaMP6 mice, GFP positive neurons representing NaV1.8 positive neurons were counted to evaluate AAV transduction efficiency. Percentages of NaV1.8 neurons were reduced by 23.8% and 26.3% after i.a. injection of 1013 vp/mL of AAV2retro-dtA and AAV.PHP.S-dtA, respectively (n=3, p<0.05 vs controls) (Fig. 2A). Injection with 1012 vp/mL of AAV.PHP.S-dtA also resulted in a 38.5% reduction of NaV1.8 cells (n=3, p<0.05 vs control). NaV1.8Cre mice injected with AAV-control 3 weeks earlier responded to TLR2 stimulation, showing decreased knee pain thresholds 4 h and 6 h after i.a. injection of Pam3CSK4 (n=3, p<0.05 vs pre-injection). In contrast, mice injected with AAV-dtA had no response to Pam3CSK4 (n=3, NS vs pre-injection) (Fig. 2B), indicating successful ablation of the knee-innervating nociceptors with AAV-dtA injection into the knee joint. Conclusions: These results show successful selective ablation of NaV1.8-expressing DRG neurons through retrograde intra-articular delivery of AAV carrying dtA. Furthermore, the findings in these studies, where NaV1.8-neurons are either excited or ablated, suggest that nociceptors mediate knee hyperalgesia. Future work will use different Cre lines to target subsets of NaV1.8 nociceptors in order to further dissect their roles in mediating this behavior. Overall, this work demonstrates that this approach provides a useful tool in studying joint pain in mouse models.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Purpose: Osteoarthritis pain is often associated with mechanical stimuli, but the way that sensory neurons detect these painful mechanical stimuli remains unknown. The mechanically sensitive ion channel, Piezo2, has been implicated in mediating proprioception and detecting light touch on the skin, as well as mechanical allodynia in a model of nerve injury. To study the role of Piezo2 in osteoarthritis pain, we have created nociceptor-specific Piezo2 conditional knock-out mice. We have previously shown that these mice do not have proprioception deficits and osteoarthritis joint damage develops similar to wild-type mice. In this study, we use in vivo calcium imaging of the lumbar dorsal root ganglia (DRG), which allows for imaging of sensory neuron responses to stimuli applied to the hind limbs of anesthetized mice. We use this technique to investigate the role of Piezo2 in nociceptor responses to different types of mechanical stimuli applied to the hind limb. In addition, we explore whether deletion of Piezo2 in nociceptors affects development of knee hyperalgesia after DMM surgery. We also use chemogenetics to determine whether therapeutic targeting of Piezo2+ neurons can reduce knee hyperalgesia after DMM surgery. Methods: All animal experiments were approved by IACUC committees. In vivo calcium imaging: 35-week old male naïve NaV1.8 cre-GCaMP6s loxp mice (n=5) or NaV1.8-GCaMP6s-Piezo2 loxp+/- mice (n=4) were used for this study. These mice express the fluorescent calcium indicator, GCaMP6s, in nociceptors by using the voltage-gated sodium channel 1.8 (NaV1.8) as a marker, since this channel is expressed by >90% of C-fiber nociceptors (pain-sensing neurons), and the latter mice are heterozygote conditional Piezo2 knock-out mice. Mice were deeply anesthetized using isoflurane, a laminectomy from vertebrae L2-L6 was performed, and the right-side L4 dorsal root ganglion (DRG) was exposed. This DRG contains sensory neurons that innervate both the mouse knee joint and the paw. The mouse was positioned under a Bruker Ultima In Vivo two-photon microscope. Anesthesia was maintained using isoflurane during imaging. A series of stimuli were applied to each animal’s right hind limb while imaging was performed: (1) 50, 100, or 200 g paw pinch; (2) paw dynamic brush; (3) 30 or 100 g knee pinch. For each mouse, the change in fluorescence over time was calculated using a custom ImageJ macro in order to identify responding sensory neurons. DMM surgery was performed in the right knee of 10-week old C57BL/6 male NaV1.8 cre+/- (n=5) or NaV1.8 cre-Piezo2 loxp+/+ (n=7) mice. Knee hyperalgesia was measured 4 and 8 weeks after surgery. DMM surgery was performed in the right knee of 10-week old C57BL/6 male Piezo2-cre x RC::PDi DREADD mice. These mice express the engineered G protein-coupled receptor used for neuronal silencing, hM4D, with selectivity for the biologically inert synthetic ligand, clozapine-N-oxide (CNO), in Piezo2-expressing sensory neurons. Seven weeks after surgery, baseline knee hyperalgesia was tested. Subsequently, CNO (n=3 mice) was administered by intra-articular injection (10 mg/kg) and knee hyperalgesia was assessed 1, 2, and 4 hours post injection. Two weeks later, the same mice were tested after intra-articular injection of saline as a vehicle control. Knee hyperalgesia was measured using a Pressure Application Measurement (PAM) device (Ugo Basile). Mice were restrained by hand and the PAM device was used to press against the ipsilateral knee. The PAM software guided the user to apply an increasing amount of force at a constant rate (30 g/s), up to a maximum of 450 g. If the mouse tried to withdraw its knee, the force at which this occurred was recorded. If the mouse did not try to withdraw, the maximum possible force of 450 g was assigned. Two measurements were taken per knee and the withdrawal force data were averaged. Results: In naïve mice, in vivo calcium imaging was used to assess the role of Piezo2 in nociceptor responses to mechanical stimuli applied to the hind limb of anesthetized mice. Conditional deletion of Piezo2 on nociceptors reduced the responsiveness of these sensory neurons to mechanical stimuli. In particular, fewer neurons responded to non-noxious dynamic brush applied to the hind paw (p=0.09), 30g knee pinch (p=0.04), and 100g knee pinch (p=0.04) in the conditional KO mice compared to the controls (Fig 1). In wild-type mice, we have previously shown that DMM surgery induces knee hyperalgesia to develop by 4 weeks after surgery and continue through 8 weeks after surgery, compared to sham surgery. Here we show that conditional deletion of Piezo2 from nociceptors by using Na V1.8 cre reduces knee hyperalgesia by 8 weeks after DMM compared to NaV1.8 cre controls (p=0.01, Fig 2). Seven weeks following DMM surgery, mice expressing the inhibitory DREADD receptor on Piezo2+ neurons developed knee hyperalgesia in the ipsilateral knee joint, similar to wild-type mice. Intra-articular injection of the synthetic ligand CNO acutely reversed knee hyperalgesia for up to 2 hours after injection (pre-injection, mean±SEM: 351±28 g; 1 hour post: 443±4 g (p=0.006 vs pre); 2 hours post: 419±5 g (p=0.03 vs pre)), and by 4 hours after injection, knee hyperalgesia had returned (357±5 g) (Fig 3A). Intra-articular vehicle injection had no effect on knee hyperalgesia (Fig 3B). Conclusions: Deletion of the mechanically-sensitive ion channel, Piezo2, specifically in nociceptors, resulted in a significant reduction of responses to noxious and non-noxious mechanical stimuli applied to the hind limbs of naïve mice and reduced knee hyperalgesia following DMM surgery. In addition, silencing of intra-articular nerves expressing Piezo2 was able to acutely reverse knee hyperalgesia after DMM surgery. These data suggest that Piezo2 may be involved in the transmission of mechanical pain in osteoarthritis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

In this Special Issue on Prevention of Osteoarthritis (OA), Whittaker and colleagues thoughtfully discuss their views on strategies for preventing this common form of arthritis, which would likely have a high impact on reducing its associated individual and societal burden, given the paucity of available treatment options1. The authors highlight the fact that most of the focus in this arena has been on prevention of risk factors for the ‘disease’ of OA, i.e. structural features of joint pathology, while few clinical studies to date have considered the risk factors for the accompanying ‘illness’, i.e.

Purpose: Nerve growth factor (NGF) is under active investigation as a promising target for treating osteoarthritis pain. Sensitization is a key process in many chronic pain states (including OA) that are characterized by exaggerated responses to innocuous or mildly noxious stimuli, and it is well known that NGF exerts its potent pro-algesic effects through sensitization of nociceptors. However, the precise biological mechanisms underlying the pain-producing effects of NGF in the knee joint are incompletely understood. One of the objectives of this study, therefore, was to test whether intra-articular injection of NGF into the healthy mouse knee induced sensitization. In addition, destabilization of the medial meniscus (DMM) surgery was used to test whether mice become sensitized to intra-articular injection of NGF in experimental osteoarthritis. Methods: All animal experiments were approved by IACUC committees. DMM surgery was performed in the right knee of 10-week old male NaV1.8 cre-GCaMP6s loxp mice. In vivo calcium imaging: Twenty-week old male naïve NaV1.8 cre-GCaMP6s loxp mice (n=4) or NaV1.8-GCaMP6s loxp mice 20 weeks after DMM surgery (n=4) were used for this study. These mice express the fluorescent calcium indicator, GCaMP6s, in nociceptors by using the voltage-gated sodium channel 1.8 (NaV1.8) as a marker, since this channel is expressed by >90% of C-fiber nociceptors (pain-sensing neurons). Mice were deeply anesthetized using isoflurane, a laminectomy from vertebrae L2-L6 was performed, and the right-side L4 dorsal root ganglion (DRG) was exposed. This DRG contains most of the sensory neurons that innervate the mouse knee joint. The mouse was positioned under a Bruker Ultima In Vivo two-photon microscope. Anesthesia was maintained using isoflurane during imaging. A series of compounds were injected intra-articularly (i.a.) (5-7 uL per compound) into the right knee while imaging was performed: (1) Saline; (2) 2.5S NGF (100 ug/mL). For each injection, a 30 G needle was inserted into the intra-articular space of the knee joint, imaging was started, the compound was injected from frame 15-20, and imaging continued until frame 100. For each mouse, the change in fluorescence over time was calculated using a custom ImageJ macro in order to identify responding sensory neurons. The area under the peak was calculated using GraphPad Prism. Knee hyperalgesia: Three uL of NGF (100 ng/uL) (R&D systems, # 1156-NG-100) (n=4 mice) or 3 uL of vehicle control (0.1% BSA) (Bovine serum albumin, 05470, Sigma) (n=4 mice) was injected i.a. in the right knee of 10-week old male C57BL/6 naïve mice. Knee hyperalgesia was assessed prior to injection and 30 mins, 2, 4, and 24 hours after injection using a Pressure Application Measurement device by a person blinded to treatment. Results: Injection of NGF into the knee joints of anesthetized NaV1.8-GCaMP6s mice elicited intracellular calcium increases in a similar number of neurons in naïve and DMM mice (mean±SEM; naïve : 2.5±1.3%; DMM: 1.9±0.7%; p=0.7), suggesting that the NGF receptor is expressed by a similar number of cells at this time point after DMM. Example responses are shown in Figure 1A,B. As a comparison, we have previously shown that 15% of the L4-DRG neurons innervate the intra-articular space of the knee joint in healthy mice. Examining the intracellular calcium responses in each neuron (Figure 1C), we found that, although NGF induced responses in a similar number of neurons in naïve and DMM mice, the responses were greater in DMM mice compared to naïve mice, as assessed by peak area under the curve (naïve: 0.7±0.2 peak AUC; DMM: 1.6±0.2 peak AUC; p=0.03), suggesting that the DMM neurons expressing the NGF receptor have become sensitized to NGF. We are using in situ hybridization with RNAscope to further analyze the expression of TrkA in the DRG (Figure 1D). The neuronal response to NGF was reflected in the behavioral response, where i.a. NGF caused rapid onset of knee hyperalgesia in naïve mice. Intra-articular administration of NGF, but not vehicle, induced knee hyperalgesia in naïve wild-type mice by 4 hours after injection (NGF: 311±16 g; vehicle: 439±5 g; p=0.01). Conclusions: Injection of NGF into the knee joints of anesthetized NaV1.8-GCaMP6s mice elicited intracellular calcium increases in a subset of NaV1.8+ L4-DRG neurons. These responses indicate nerve activity and suggest that NGF directly binds to neuronal receptors in the intra-articular space of the knee joint. DMM mice were sensitized to i.a. NGF, as measured by intracellular calcium responses. In naïve mice, i.a. NGF was able to induce sensitization to mechanical stimuli, as assessed by measuring knee hyperalgesia. It will be important to determine if and how NGF contributes to the pain-related behaviors induced by mechanical stimuli known to accompany pre-clinical models of osteoarthritis through studies using anti-NGF ab.

In this narrative review, we discuss the emerging role of innate immunity in osteoarthritis (OA) joint pain. First, we give a brief description of the pain pathway in the context of OA. Then we consider how neuro-immune signaling pathways may promote OA pain. First, activation of neuronal Pattern Recognition Receptors by mediators released in a damaged joint can result in direct excitation of nociceptors, as well as in production of chemokines and cytokines. Secondly, indirect neuro-immune signaling may occur when innate immune cells produce algogenic factors, including chemokines and cytokines, that act on the pain pathway. Neuro-immune crosstalk occurs at different levels of the pathway, starting in the joint but also in the innervating dorsal root ganglia and in the dorsal horn. Synovitis is characterized by recruitment of immune cells, including macrophages, mast cells, and CD4+ lymphocytes, which may contribute to nociceptor sensitization and OA pain through production of algogenic factors that amplify the activation of sensory neurons. We discuss examples where this scenario has been suggested by findings in human OA and in animal models. Overall, increasing evidence suggests that innate immune pathways play an initiating as well as facilitating role in pain, but information on how these pathways operate in OA remains limited. Since these innate pathways are eminently targetable, future studies in this area may provide fruitful leads towards a better management of symptomatic OA.

Objectives: To document the nociceptive innervation of the normal and osteoarthritic murine knee. Methods: Knees were collected from naive male C57BL/6 Na(V)1.8-tdTomato reporter mice aged 10, 26, and 52 weeks (n = 5/group). Destabilization of the medial meniscus (DMM) or sham surgeries (n = 5/group) were performed in the right knee of 10-week old male Na(V)1.8-tdTomato mice, and knees were harvested 16 weeks later. Twenty 20-mu m frozen sections from a 400-mu m mid-joint region were collected for confocal microscopy. Integrated density of the tdTomato signal was calculated using Image J by two independent observers blinded to the groups. Consecutive sections were stained with hematoxylin & eosin. C57BL/6-Pirt-GCaMP3 mice (n = 5/group) and protein gene product 9.5 (PGP9.5) immunostaining of C57BL/6 wild type (WT) mice (n = 5/group) were used to confirm innervation patterns. Results: In naive 10-week old mice, nociceptive innervation was most dense in bone marrow cavities, lateral synovium and at the insertions of the cruciate ligaments. By age 26 weeks, unoperated knees showed a marked decline in nociceptors in the lateral synovium and cruciate ligament insertions. No further decline was observed by age 1 year. Sixteen weeks after DMM, the medial compartment of OA knees exhibited striking changes in Na(V)1.8+ innervation, including increased innervation of the medial synovium and meniscus, and nociceptors in subchondral bone channels. All results were confirmed through quantification, also in Pirt-GCaMP3 and PGP9.5-immunostained WT mice. Conclusions: Nociceptive innervation of the mouse knee markedly declines by age 26 weeks, before onset of spontaneous OA. Late-stage surgically induced OA is associated with striking plasticity of joint afferents in the medial compartment of the knee. (C) 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Purpose: Osteoarthritis pain is often associated with mechanical stimuli, but the way that sensory neurons detect these painful stimuli remains unknown. The recently discovered mechanically sensitive ion channel, Piezo2, has been implicated in mediating proprioception and detecting light touch on the skin, as well as mechanical allodynia in a model of nerve injury. Those studies were performed by generating mice where Piezo2 was deleted on all sensory neurons or on proprioceptors, and thus the mice had strongly altered proprioception such that they could not move their limbs normally. The role of Piezo2 in mediating pain-related behaviors through nociceptors, however, is unknown, and the role in osteoarthritis in particular has not been studied. The purpose of this study was to create a nociceptor-specific knock-out mouse for Piezo2 in order to test the effects of Piezo2 on pain-related behaviors following destabilization of the medial meniscus (DMM) surgery. Methods: Nociceptor-specific Piezo2 knock-out mice were created by using the voltage-gated sodium channel, NaV1.8, as a marker for nociceptors (NaV1.8 cre). By performing two crosses with Piezo2 loxp mice, the following genotypes were created: mice homozygous for Piezo2 deletion (n=7), heterozygous for Piezo2 deletion (n=5), and mice without Piezo2 deletion (n=5). In order to confirm Piezo2 deletion, L3-L5 dorsal root ganglia (DRG that contain sensory neurons from the knee) were collected and pooled from each mouse, RNA was extracted, and qPCR for Piezo2 and Gapdh was performed. Destabilization of the medial meniscus (DMM) surgery was performed in the right knee of 10-week old male littermate mice. Four and 8 weeks after surgery, knee hyperalgesia and mechanical allodynia were tested by a blinded observer. Knee hyperalgesia was measured using a Pressure Application Measurement (PAM) device (Ugo Basile). Mice were restrained by hand and the PAM device was used to press against the ipsilateral knee. The PAM software guided the user to apply an increasing amount of force at a constant rate (30 g/s), up to a maximum of 450 g. If the mouse tried to withdraw its knee, the force at which this occurred was recorded. If the mouse did not try to withdraw, the maximum possible force of 450 g was assigned. Two measurements were taken per knee and the withdrawal force data were averaged. Mechanical allodynia in the ipsilateral hind paw was measured using von Frey fibers and the up-down staircase technique. Results: qPCR confirmed that both homozygous and heterozygous deletion of Piezo2 in nociceptors by using NaV1.8-cre resulted in decreased expression compared to mice without Piezo2 deletion (0.5 fold-change for homozygotes; 0.62 fold-change for heterozygotes). We did not expect complete deletion of Piezo2 in the DRG since this channel is also expressed by proprioceptors, which do not express NaV1.8. Following DMM surgery, mice without Piezo2 deletion developed knee hyperalgesia and mechanical allodynia of the ipsilateral hind paw by 4 weeks after surgery, as has been reported previously. Heterozygous deletion of Piezo2 resulted in less mechanical allodynia of the hind paw at week 4 after DMM surgery (0.09±0.015 g), compared to mice without Piezo2 deletion (0.05±0.004 g) (p=0.03). Knee hyperalgesia was unaffected (p=0.33). There was no difference in either measure at week 8. Homozygous deletion of Piezo2 resulted in less knee hyperalgesia at week 4 after DMM surgery (428±11 g), compared to mice without Piezo2 deletion (391±12 g) (p=0.05). In addition, homozygous deletion resulted in less mechanical allodynia of the hind paw at weeks 4 and 8 after DMM surgery (week 4: 0.15±0.039 g; p=0.01; week 8: 0.17±0.059 g; p=0.05) compared to controls (week 4: 0.05±0.004 g; week 8: 0.05±0.012 g). Histological evaluation of knees is in progress. Conclusions: Deletion of the mechanically-sensitive ion channel, Piezo2, specifically in nociceptors, resulted in a significant reduction of knee hyperalgesia and mechanical allodynia of the hindpaw in the early stage of the DMM model. These data suggest that Piezo2 may be involved in the transmission of mechanical pain in osteoarthritis.

Purpose: Intracellular calcium mobilization is known to be a downstream signaling response to different types of mechanical loading in a variety of cells, including chondrocytes. Osteoarthritis involves aberrant loading, however, the calcium responses of chondrocytes to the mechanical loading changes induced by osteoarthritis have not been examined. Therefore, the purpose of this study was to generate novel methods to begin to address this question using the destabilization of the medial meniscus (DMM) mouse model of osteoarthritis. By better understanding how chondrocyte response to loading is altered in osteoarthritis, novel targets for therapies may be uncovered. Methods: We created a novel line of mice by crossing Collagen II cre mice with GCaMP6s loxp mice. GCaMP6s is an ultra-sensitive fluorescent calcium reporter that can be genetically expressed in specific cell types using cre-loxp recombination (in this case, chondrocytes). In these Collagen II-GCaMP6s mice, when intracellular calcium increases in chondrocytes, an increase in green fluorescence can be measured. DMM surgery in these mice causes joint damage similar to wild-type mice. To apply mechanical loading to mouse femoral condyle cartilage while performing calcium imaging, we built a system using a Zeiss Axio Observer inverted fluorescent microscope and an Aurora Scientific dual-mode lever system (300C) in order to apply compressive strain with 1 μm resolution (Fig 1). This setup enables us to compress murine medial femoral cartilage against a glass coverslip while imaging from below. We verified that our applied displacement resulted in accurate movement of our sample by an Eddy sensor (Micro-Epsilon ES04) with a resolution of 0.04 μm. DMM (n=4) or sham (n=3) surgery was performed in the right knee of 10-week old male Collagen II-GCaMP6 mice. Sixteen weeks after surgery, each femur was freshly dissected and prepared for calcium imaging. Calcium Imaging and Data Analysis: A defined sodium-calcium buffer, commonly used for calcium imaging, was used for all imaging and was slowly perfused through the chamber during each experiment. Images were taken at a frequency of 1 Hz, with an exposure time of 20 ms, using a GFP filter set. For each femur, baseline images were acquired without loading for 5 min, loading using a square waveform with amplitude 7 μm and frequency 0.05 Hz was applied for 5 min, 5 min of imaging was acquired post loading with the femur out of contact, and 8 min of imaging was performed while a 50% hypo-osmotic buffer was perfused past the femur. Images were processed using custom macros in Fiji to correct for minor movement as well as to calculate (F-Fo)/Fo, where Fo = the first 2-20 frames imaged. Responses in individual chondrocytes during the different imaging phases were compared between sham and DMM using unpaired, two-tailed t-tests (GraphPad Prism 6), where p<0.05 was considered statistically significant. Results: Seven microns of compression (∼18% strain; our sham mouse medial condyle cartilage was on average 39 μm thick) in medial femoral condyles resulted in greater numbers of chondrocytes generating intracellular calcium responses in sham mice compared to DMM mice 16 weeks after surgery (3.8-fold higher; p=0.03) (Fig 2). However, on average, DMM chondrocyte calcium responses were greater in amplitude and duration than sham chondrocyte responses (2.5-fold higher; p=0.03) (Fig 2). The average peak forces observed during compression were similar for sham (13.4±2.6 mN) and DMM mice (16.3±1.6 mN) (p = 0.35). Exposure to hypo-osmotic salt solution has been used previously as a positive control for inducing intracellular calcium responses in chondrocytes as it causes cells to stretch. Here, we found that 50% hypo-osmotic solution induced similar numbers of chondrocyte responses in both sham (54±10) and DMM (42±11; p=0.46) mice 16 weeks after surgery, and similar amplitude and duration of responses (sham: 6.9±0.2 ΔF/Fo x seconds; DMM: 9.7±2.6 ΔF/Fo x seconds; p=0.36). Conclusions: Here, we have generated a novel strain of mice and a novel mechanical stimulation and imaging system in order to image real-time responses of chondrocytes to mechanical loading within intact mouse femoral condyles after DMM surgery. Our results suggest that after DMM surgery, femoral condyle chondrocytes are still able to generate intracellular calcium responses to changes in osmolarity, but they have become dysregulated in response to mild levels of cartilage compression. Future work will seek to understand which calcium signaling pathways may be mediating these changes and how these changes may contribute to other pathological cartilage changes in osteoarthritis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Purpose: Surgical destabilization of the medial meniscus (DMM) in the mouse knee results in slowly progressive osteoarthritis (OA) in the operated knee and is associated with behaviors indicative of pain. Mice develop mechanical allodynia in the ipsilateral but not the contralateral hind paw by week 4 after DMM surgery and this allodynia is maintained through week 16. In addition, hyperalgesia develops in the ipsilateral but not the contralateral knee by 4 weeks after surgery. In nerve injury models, there is increasing evidence that unilateral nerve injury induces changes in both the ipsilateral and the contralateral dorsal root ganglia (DRG), where the cell bodies of sensory neurons reside; in particular, upregulation of the pro-algesic neurotrophin, nerve growth factor (NGF). We have recently reported that 8 and 16 weeks after DMM surgery in the right knee, microglial activation occurs both in the ipsi- and in the contralateral dorsal horn of the spinal cord. Therefore, we aimed to explore whether the molecular changes and macrophage infiltration we have observed in the ipsilateral DRG after DMM surgery also occur in the contralateral DRG. Methods: DMM or sham surgery was performed on the right knees of 10-week old male C57BL/6 mice. At 8 and 16 weeks after surgery, bilateral L3-L5 DRGs from DMM and age-matched naïve mice were collected for qRT-PCR of NGF, monocyte chemoattractant protein (MCP)-1 and its receptor, CCR2. To assess macrophage infiltration, DMM and age-matched sham and naïve controls were perfused transcardially with 4% paraformaldehyde and bilateral L4 DRGs were collected and cryosectioned. DRG sections were immunostained with an antibody against the macrophage marker, F4/80, and images were captured with a confocal microscope. ImageJ was then used to count the total number of F4/80 expressing macrophages in each DRG section. The counts for 3 DRG sections were averaged per mouse. Results: Previously, we have reported that mRNA for NGF, MCP-1 and CCR2 was significantly increased in the ipsilateral DRGs 8 weeks after DMM but not sham surgery, compared to age-matched naïve controls. Interestingly, we now found that NGF, MCP-1 and CCR2 mRNA are all similarly upregulated in the contralateral DRGs from 8-week DMM mice compared to naïve controls. Moreover, 16 weeks after DMM surgery, mRNA expression of NGF, MCP-1 and CCR2 were attenuated bilaterally. Eight and 16 weeks after DMM surgery, there were significant increases in the number of macrophages in both the ipsilateral and contralateral DRGs, compared to age-matched naïve and sham-operated controls (see figure below) Conclusions: After DMM surgery, OA joint damage only occurs in the operated knee and pain behaviors, including mechanical allodynia and knee hyperalgesia, are exhibited exclusively in the operated limb (in a 16-week follow-up period). In contrast, there are neuroimmune changes in the peripheral nervous system that occur bilaterally. These findings are concordant with our previous findings that microglial activation in the dorsal horn also occurs bilaterally. The biological significance of this observation needs further exploration. These findings also suggest that when using the DMM model, the use of the contralateral side as a control should be avoided.

Purpose: The horizontal ladder assay was developed to assess balance, limb co-ordination, and movement deficits. It has been used to study these types of behavioral changes in rodent models of stroke, Parkinson's disease, and spinal cord injury. The purpose of this study was to investigate whether mice develop deficits in the horizontal ladder assay after destabilization of the medial meniscus surgery. In addition, the role of nociceptors and proprioceptors in mediating the ability to traverse the horizontal ladder was evaluated in naïve mice by using DREADD (Designer Receptor Activated by a Designer Drug) technology to selectively inhibit either nociceptors (using NaV1.8 as a marker) or proprioceptors (using parvalbumin as a marker). Methods: A horizontal ladder was constructed following previous protocols such that metal rungs were placed at 2 cm intervals in a straight line, attached to plexiglass walls spaced 4 cm apart. The ladder is 60 cm long. A high definition video camera was used to film the crossings from the side. In addition, a mirror was placed beneath the ladder in order to also capture the movements from below. Destabilization of the medial meniscus (DMM) surgery was performed in the right knee of 10-week old male C57BL/6 mice. Age-matched naïve and sham mice were used as controls. Ten, 12 or 15 weeks after surgery, mice (n = 5/treatment) were trained to cross the horizontal ladder on day one, and were tested on day two. Two types of naïve DREADD mice, 16–20 weeks of age, were also used. These mice express the engineered G protein-coupled receptor used for neuronal silencing, Pdi, with selectivity for the biologically inert synthetic ligand, CNO. To silence nociceptors, the Pdi receptor was expressed in NaV1.8-expressing nociceptors (NaV1.8-Pdi mice). To silence proprioceptors, the Pdi receptor was expressed in parvalbumin-expressing neurons (PV-Pdi mice). Mice were pre-trained to cross the ladder, and subsequently injected with either CNO or vehicle (PBS) by i.p. injection (10 mg/kg). One hour post injection, mice were tested and recorded while crossing the ladder. Data were averaged for 2 crossings/mouse. The experimenter was blinded to the groups. Two independent experiments were performed using different litters of mice (n = 6–10 mice/strain/treatment). The following measurements were recorded: 1) The time (in seconds) the mouse needed to walk across the ladder (mean ± SEM); and 2) the number of errors made in placing the right hind paw on a ladder rung (median, IQR). Qualitatively, the use of the tail while crossing the ladder was observed. Results: Ten and 12 weeks after surgery, naïve, sham, and DMM mice crossed the ladder in a similar amount of time. By 15 weeks after surgery, DMM mice took longer to cross the ladder (12 ± 2 s) compared to naïve mice (5 ± 0.6 s) (P = 0.02), while sham mice fell in between the two groups (8.5 ± 1 s). Ten weeks after surgery, one naïve and one sham mouse made one error in ipsilateral hind paw placement (0, 0–0.5) while three DMM mice made 1–2 errors (1, 0–1.5). By 12 weeks after surgery, errors in all groups increased, with DMM mice making (3, 2.5–3.5) errors compared to (1, 0–2.5) errors by naïve mice and (1, 0.5–2.5) errors by sham mice (P = 0.08 DMM vs naïve). In contrast to time to cross, by 15 weeks after surgery, the number of errors dissipated for all treatment groups. In order to parse out the contributions of different sets of sensory neurons, the ladder test was performed in naïve NaV1.8-Pdi and PV-Pdi mice one hour after CNO or PBS was injected in order to silence nociceptors or proprioceptors, respectively. In two separate trials, NaV1.8-Pdi mice given either CNO or PBS performed similarly with respect to time to cross and number of errors made. In contrast, PV-Pdi mice given CNO took longer to cross the ladder and made more errors while crossing compared to PV-Pdi mice given PBS, providing evidence that this assay assesses proprioceptive function instead of nociceptive function. Conclusions: Chemogenetic silencing of proprioceptors and not nociceptors resulted in deficits in the horizontal ladder assay. Following DMM surgery, mice also developed deficits in this assay indicating that the horizontal ladder assay may provide information on proprioceptive input in the DMM model.

Purpose: Recent advances in molecular biology have revealed distinct sensory neuron subpopulations that display unique patterns of molecular markers and selectively encode different aspects of sensation. Proprioceptors express parvalbumin (PV) and the majority of nociceptors express the voltage-gated sodium channel, NaV1.8. These NaV1.8+ neurons can be further classified into distinct functional classes, including peptidergic TRPV1+ C-fibers that sense noxious heat, non-peptidergic mechanosensitive Mrgprd+ C-fibers, and a subset of C-fibers known as C-low threshold mechanoreceptors (C-LTMRs), which express tyrosine hydroxylase (TH) and Piezo2 receptors. It is known that more than 80% of sensory nerves in the knee are proprioceptors and nociceptors, but the exact distribution of neuronal subpopulations is unknown. Hence, we aimed to develop new methods to enable detailed anatomical characterization of sensory innervation of the mouse knee. Methods: We have used a variety of Cre/Flp drivers to produce diverse lines of C57BL/6 mice where distinct subsets of nociceptive, mechanoreceptive or proprioceptive neurons are labeled with either TdTomato or EGFP, including Mrgprd-EGFPf. Since TH+ nerves in the knee could be either sensory dorsal root ganglia (DRG) neurons or sympathetic neurons, we have used an intersectional approach (crossing TH-flpo mice with NaV1.8-cre mice) to prepare mice in which only TH nerves in the DRG are labelled (THCLTMR mice). In addition, we have parvalbumin (PV)-TdTomato mice to visualize proprioceptors. We are using these mice to trace labelled neurons in the DRG and in the knee. At age 10 weeks, male mice were perfused transcardially with paraformaldehyde, and the DRGs and knees were collected. Individual ipsilateral L3-L5 DRG were embedded with OCT (Tissue-Tek), frozen with dry ice, and cut into 12-μm sections. The right knees were post fixed and decalcified. Twenty-μm thick frozen sections were collected at mid-joint level. Both DRG and knee sections were imaged using confocal microscope. Results: Fig. 1 illustrates the diversity of cell bodies in the DRG of these different lines of mice: Mrgprd cell bodies are small diameter neurons, consistent with C-fibers. PV+ cell bodies in the DRG are large cells, consistent with proprioceptors. Finally, by crossing TH-flpo mice with NaV1.8-cre mice, the TH+ subset is specifically labeled in the DRG but not in the sympathetic ganglia (SCG). We have now started analyzing the knees of these mice. In the knees of 10-week old Mrgprd-EGFPf mice, we detected GFP signal in a pattern that was similar to our previous findings in NaV1.8-TdTomato mice, with dense innervation of the bone marrow cavities, the lateral synovium, and the attachment areas of the cruciate ligaments. Other structures, such as the medial synovium and the mid-portion of the cruciate ligaments, were less densely innervated. These findings suggest that non-peptidergic, mechanosensitive C-fibers innervate the intra-articular structures of the knee. In contrast, the knees of PV-TdTomato reporter mice showed that proprioceptors are predominantly innervating the insertion sites of the cruciate ligaments (as well as the peri-articular muscles). The other intra-articular structures showed less Tomato signal. Conclusions: Genetically engineered mice can be used to label specific subsets of sensory neurons, including proprioceptors and functionally distinct subpopulations of nociceptors. These mice are useful for studying the sensory innervation of the mouse knee. Our initial data show a distinct pattern of innervation by proprioceptors, mainly at the insertion of the cruciate ligaments. Further, we detected Mrgprd+ non-peptidergic fibers in the knee (which is different from what has been reported before using IB4 as a marker). We expect that these mice will provide valuable tools for the study of murine joint neuroanatomy, in healthy joints as well as in mouse models of osteoarthritis.

Blockade of nerve growth factor (NGF) with antibodies is a promising strategy for treatment of chronic pain associated with osteoarthritis (OA). This narrative review describes the current status of NGF-blockade for the treatment of OA pain. We summarise briefly current evidence for the efficacy and risks of anti-NGF blockade. Two anti-NGF antibodies, tanuzemab and fasinumab, are in active development, with tanuzemab close to completing Phase 3 trials in preparation for an application for approval for clinical use.

Objective: The role of inflammation in s tructural and symptomatic osteoarthritis (OA) remains unclear. One key mediator of inflammation is the chemokine CCL2, primarily responsible for attracting monocytes to sites of injury. We investigated the role of CCL2 and its receptor CCR2 in experimental OA.Design: OA was induced in 10 weeks old male wild type (WT), Ccl2(-/-) and Ccr2(-1-) mice, by destabilisation of the medial meniscus (DMM). RNA was extracted from whole joints at 6 h and 7 days post surgery and examined by reverse transcription polymerase chain reaction (RT-PCR). Gene expression changes between naive and DMM-operated mice were compared. Chondropathy scores, from mice at 8, 12, 16 and 20 weeks post DMM were calculated using modified Osteoarthritis Research Society International (OARSI) grading systems. Changes in hind paw weight distribution, as a measure of pain, were assessed by Linton incapacitance.Results: Absence of CCL2 strongly suppressed (>90%) selective inflammatory response genes in the joint 6 h post DMM, including arginase 1, prostaglandin synthase 2, nitric oxide synthase 2 and inhibin A. IL6, MMP3 and tissue inhibitor of metalloproteinase 1 were also significantly suppressed. Similar trends were also observed in the absence of CCR2. A lower average chondropathy score was observed in both Ccl2(-/-) and Ccr2(-1-) mice at 12, 16 and 20 weeks post DMM compared with WT mice, but this was only statistically significant at 20 weeks in Ccr2(-/-) mice. Pain -related behaviour in Cc12(-/-) and Ccr2(-/-) mice post DMM was delayed in onset.Conclusion: The CCL2/CCR2 axis plays an important role in the development of pain in murine OA, but contributes little to cartilage damage. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

Purpose: Genome-wide association studies (GWAS) have been useful in identifying genes that may predispose for osteoarthritis (OA) and for pain, but they demand large patient cohorts. To improve the power of smaller cohorts, we developed a novel approach by combining data generated from a mouse model of OA pain with clinical GWAS data from cohorts with specific OA pain phenotypes. We used the destabilization of the medial meniscus (DMM) mouse model, which results in slowly progressive joint damage accompanied by pain-related behaviors. The development of chronic pain is characterized by molecular changes in the DRG, where the cell bodies of sensory afferent neurons reside. Thus, we performed microarrays on dorsal root ganglia (DRG) cells during the persistent pain period following DMM. Human homologues of the top regulated murine genes were compared to clinical GWAS datasets to see if there were SNPs in these genes that were associated with clinical OA pain phenotypes. Methods: Approval for these studies was obtained from each institution. DMM or sham surgery was performed in the right knee of 10-week old male C57BL/6 mice. Age-matched naïve mice were also included. Eight and 16 weeks after surgery, ipsilateral L3-L5 DRG (DRG that innervate the knee) were collected and pooled from each mouse, RNA was extracted, and Affymetrix Mouse Transcriptome 1.0 Arrays were performed. A total of 3 mice (3 arrays) were used for each treatment per time point. Two out of three sham 8-week samples did not amplify well and were excluded. To identify genes associated with persistent pain, data pooled from DMM 8- and 16-week samples were compared to data pooled from naïve and sham 8- and 16-week samples by Student’s t-test. The top differentially expressed genes were identified by p < 0.001. These top genes were investigated for single nucleotide polymorphisms (SNPs) and association with clinical OA pain phenotypes (p < 0.05) using three different patient GWAS datasets: (1) Symptomatic vs asymptomatic knee OA; (2) Neuropathic pain symptoms post total joint replacement; (3) Disturbed sleep (potentially related to chronic pain) post total joint replacement. For phenotype (1), genotyping with the Exome BeadChip array was carried out in 458 knee OA cases with a K/L score of 2 or higher in the tibiofemoral compartment. Of these, 212 were asymptomatic, reporting no knee pain. The remaining 246 reported pain in the knee at least 15 days during the past month. For phenotypes (2) and (3), genotyping with the Illumina 610k array was carried out in patients recruited post-total hip or knee replacement for OA (n = 613). Individuals were assigned a phenotype by classifying them according to their scores on the painDETECT questionnaire. Scores >12 were classified as “possible neuropathic pain.” Sleep scores from the Medical Outcomes Survey (MOS) were available for these same individuals. We classified the bottom tertile of the MOS sleep subscale as “disturbed sleep.” Results: A total of 36 genes were differentially regulated in the DRG in the persistent pain phase of the DMM mouse model, according to our cutoff of p < 0.001. Sixteen genes were upregulated in DMM samples compared to controls, and 20 genes were downregulated. Of the 36 differentially regulated murine genes, 27 genes had human homologues, and 34 SNPs within 11 of these genes were associated with clinical OA pain phenotypes (p < 0.05). These 11 genes have functions that include roles in circadian rhythm, apoptosis signaling, complement system, autophagy, regulation of cell signaling, and transcriptional regulation. Only 1 of the 11 genes, a cysteine proteinase, has been associated with a pain phenotype in other experimental models. SNPs within 8 genes were associated with disrupted sleep, SNPs within 5 genes were associated with neuropathic pain, and SNPs within 2 genes were associated with symptomatic OA. The most significant signal (p = 9.97 × 10−4) was a SNP on chromosome 11 within a gene that is implicated in transcriptional regulation and was downregulated after DMM. This SNP had an odds ratio of 1.538 in association with disrupted sleep. Other SNPs in the same gene were found to be nominally associated with symptomatic OA (p < 0.032) and with neuropathic pain (p < 0.005). Conclusions: This study suggests that it may be possible to combine microarray data from well-characterized translational models of osteoarthritis pain and GWAS data from well-characterized patient cohorts in order to refine the results of both types of studies. Future work will investigate the roles of these genes in experimental OA, will address the functional implications of these SNPs, and will seek to replicate the findings in other cohorts.

Purpose: The aim of the study was to document the nociceptive innervation in the healthy murine knee joint, and to document age-related changes in the innervation pattern. Methods: We used male NaV1.8-TdTomato reporter mice on a C57BL/6 background. These mice express a bright red fluorescent tomato reporter in all neurons that express the sodium-gated voltage channel, NaV1.8. This channel is expressed by approximately 75% of dorsal root ganglion (DRG) sensory neurons, including >90% of C-nociceptors (pain-sensing neurons) and C-low-threshold mechanoreceptors, as well as a lower percentage of Aδ-nociceptors and Aβ afferents. At age 10 weeks (n = 5) and at age 26 weeks (n = 5), mice were perfused transcardially with paraformaldehyde, and the right knees were collected, post fixed and decalcified. Twenty-μm thick frozen sections were collected at mid-joint level. Consecutive sections were stained with hematoxylin & eosin. Age-matched heterozygous C57BL/6 Pirt-GCaMP3 mice were used to confirm innervation patterns. These mice express the green fluorescent calcium indicator, GCaMP3, in ∼90% of all sensory DRG neurons (including the Nav1.8 population), and not in other peripheral or central tissues, through the Pirt promoter. Results: Examination of the knees of 10-week old NaV1.8-TdTomato mice revealed areas of dense innervation by NaV1.8-expressing sensory fibers, most notably the bone marrow, the lateral synovium, and the connective tissue layer (epiligament) surrounding the cruciate ligaments, including the areas of attachment. Other structures, such as the medial synovium and the collagenous substance of the cruciate ligaments, were less densely innervated. NaV1.8 nociceptors were also present in the outer third of the lateral meniscus. The articular cartilage, the inner two thirds of the lateral meniscus, and the medial meniscus did not show innervation. Figure 1 shows an example of these features in one mouse - but these findings were remarkably reproducible in n = 5 mice. Assessment of NaV1.8 signal in knees of 26-week old mice revealed marked changes in innervation density (not shown). Compared to 10-week old knees, 26-week old knees showed a dramatic decline in NaV1.8-expressing nociceptors in the lateral synovium, as well as in the epiligament and attachment areas of the cruciate ligaments. Similar age-related changes in the innervation were also detected in the knees of 26-week old Pirt-GCaMP3 mice compared to 10-week old knees, providing independent evidence that the chosen markers are specific for nerve fibers. Conclusions: This study reproducibly shows, for the first time, that the nociceptive innervation of specific murine knee tissues dramatically declines with age. Remarkably, this occurs quite early on in the life of the mouse, where we find dense innervation at 10 weeks and a marked decline by 26 weeks. Ongoing studies are aimed at monitoring innervation with more advanced age. The biological significance of these findings needs to be explored, as well as the relationship with pathogenesis of osteoarthritis.