A NOVEL INTERVENTION TO PREVENT POST-TRAUMATIC OSTEOARTHRITIS FOLLOWING KNEE JOINT INJURY

The knee joint is the most commonly injured body part in the human body. Injuries as a result of participation in sports, or other recreational activities, often leads to damage to the anterior cruciate ligament (ACL) and meniscus. Injury to these tissues is strongly associated with subsequent knee post-traumatic osteoarthritis (PTOA), which is considered a serious disease because it greatly impacts a patient’s quality of life and significantly increases their risk of premature death. To return stability to the joint, the current clinical treatment is to perform reconstruction of the torn ACL and a meniscal debridement, or meniscectomy, when needed. However, long-term studies have shown that surgical intervention does not prevent the onset or progression of PTOA. It is believed that reconstruction surgery fails to address the occult damage to the soft tissues within the knee joint at the cellular level. Recently, a single dose of the amphipathic and nonionic surfactant Poloxamer 188 (P188) has been shown to be an effective inhibitor of acute cell death and inhibit pathways associated with inflammation in articular cartilage in both in-vitro and in-vivo studies. This project strives to test if combined surgical and P188 treatment following injury to the ACL will help prevent PTOA in an in-vivo closed-joint traumatic injury model. It is hypothesized that combined treatment will mitigate or delay the progression of the disease by returning normal knee kinematics and restoring the integrity of damaged cell membranes in the soft tissues. Thus, the aims of the proposed project are to: 1) Determine the efficacy of single and multiple temporal administrations of P188 in inhibiting cell death following traumatic impact in meniscal tissue. 2) Assess morphological, functional, and histological changes in the meniscus 3) Evaluate chronic changes in subchondral cortical and trabecular bone of femurs and tibias 4) Determine the presence of biomarkers associated with PTOA in the synovial fluid Finding solutions to prevent the onset of PTOA, in conjunction with ACL reconstructive surgery, would provide a novel treatment to improve the quality of life for millions of people and decrease their risk of premature death

Mechanisms of Peripheral Sensitization in Inflammatory Knee Pain

Arthritis affects millions of people and costs billions to the global economy with painful, inflamed joints being the major clinical symptoms. Knee joints are innervated by the distal ends of dorsal root ganglion (DRG) neurons and hyperexcitability of knee-innervating DRG neurons (knee neurons), through a process called peripheral sensitization, underlies arthritic knee pain. My research goal was to elucidate the mechanisms of knee neuron peripheral sensitization by developing and utilizing several in vitro and in vivo disease models. Since experimental animal models of arthritis do not fully recapitulate the human disease, I developed a novel, in vitro translational model of arthritic pain by incubating mouse knee neurons with human synovial fluid obtained from osteoarthritic patients. Results from this disease model provide proof-of-concept that synovial fluid is a key modulator of arthritic pain. In order to reconcile the behavioral and neural correlates of arthritic pain, I utilized the mouse model of complete Freund’s adjuvant (CFA)-induced knee inflammation to establish digging behavior as an ethologically relevant spontaneous pain measure. I observed that digging is reduced after knee inflammation and that it occurs concomitant with an increase in knee neuron excitability. After inflammation, knee neurons also showed increased expression of the nociceptive ion channel transient receptor potential vanilloid 1 (TRPV1), possibly mediated by nerve growth factor, and systemic administration of a TRPV1 antagonist normalized digging behavior in mice. Interactions between non-neuronal cells and neurons are critical in peripheral sensitization and I examined the role of fibroblast-like synoviocytes (FLS, non-neuronal joint cell) in mediating knee neuron hyperexcitability by establishing a mouse FLS/DRG neuron co-culture system. A pro-inflammatory phenotype was produced in FLS after stimulation with tumor necrosis factor-$\alpha$ (TNF-$\alpha$ a cytokine that is upregulated in CFA mouse models, TNF-FLS); and in co-culture with TNF-FLS or their secreted mediators knee neurons became hyperexcitable and showed altered TRP channel function. These results suggest that specifically controlling the excitability of knee neurons could provide pain relief in arthritis, one possibility for achieving such control is to deliver excitatory or inhibitory genes via adeno-associated virus (AAV). However, delivering genes into DRG neurons by injection of AAVs into the peripheral organs has had limited success due to the large distances involved. Here I show that the newly engineered serotype, AAV-PHP.S, can deliver functional excitatory (Gq) and inhibitory (Gi) designer receptors activated by designer drugs (DREADDs) into knee neurons to bi-directionally control excitability in vitro and that Gi- DREADD activation in vivo can reverse a knee inflammation-induced decrease in digging behavior. Findings from this thesis highlight multiple ways to identify drivers of inflammatory knee pain and open the door for peripheral organ targeted gene therapy to alleviate arthritic pain.Gates Cambridge Trus

Pain in rheumatoid arthritis : bone and neuroinflammation-associated mechanisms

Pain is often the primary reason patients with rheumatoid arthritis (RA) seek medical care. Despite effective disease control with currently available disease modifying antirheumatic drugs (DMARDs), there are still hurdles to overcome as a significant proportion of patients still report continuous pain. This suggests that the relationship between joint inflammation and pain severity is not linear. Currently few effective pain treatments for RA are available, which leads to individual and societal burden. Understanding the regulation of chronic pain in RA is thus vital to identify new drug targets and improve therapeutical strategies. This thesis explores alternative mechanisms of pain in RA with a specific focus on bone and neuroinflammationassociated mechanisms. In Paper I, the contribution of osteoclasts to pain mechanisms was characterized in the collagen antibody-induced arthritis (CAIA) model. As previously reported, CAIA induces transient joint inflammation and persistent mechanical hypersensitivity that outlasts active inflammatory state. Herein, local bone erosion was detected in the calcaneus during both inflammatory and late phases of the CAIA model. Interestingly, while osteoclast activity was prominently increased during the inflammatory phase, pain-like behavior was reversed by two different osteoclast inhibitors in the late phase. In order to understand the contribution of osteoclast activity in nociceptive mechanisms, bone vascularization and innervation were examined. Both vascular and nerve densities were increased in the calcaneus during inflammation, but surprisingly remained elevated in the late phase despite resolution of joint inflammation. Notably, the CAIA-induced changes in bone, vascular and nerve densities in the late phase were attenuated by osteoclast-blocking agents correlating with suppression of osteoclast-derived angiogenic and neurogenic factors, such as netrin-1. Blockade of netrin-1 activity reversed CAIA-induced hypersensitivity in the late phase. Collectively, these findings suggest that the pronociceptive role of osteoclasts is not entirely dependent of their resorbing actions and that osteoclast inhibitors are effective in alleviating pain during the refractive phase of RA. Janus kinase/signal transducers and activators of transcription (JAK/STAT) inhibitors represent a new class of DMARDs. In Paper II, the antinociceptive effects of the JAK1/2 inhibitor baricitinib on the CAIA model as well as the underlying mechanisms were determined. In this study, baricitinib produced reversal effects on CAIA-induced pain-like behavior, which was more pronounced in the late phase of the model. Importantly, the antinociceptive properties of baricitinib in the CAIA model do not completely covary with its anti-inflammatory effects. Intriguingly, no sign of JAK/STAT activation was detected in the dorsal root ganglia (DRGs) or spinal cords of CAIA-subjected mice, thus prompting other signaling pathways targeted by baricitinib to be explored. The effect of baricitinib on AAK1 activity was examined as this pathway was recently identified to be an additional target of baricitinib. mRNA levels of Aak1 and its downstream target Ap2m1 as well as phosphorylation and total protein of AP2M1 were upregulated in DRGs from the late phase of the CAIA model. Baricitinib treatment was able to normalize phosphorylation and total protein levels of AP2M1. Taken together, our data suggest that baricitinib may exert its antinociceptive effects through modulation of AAK1 rather than JAK/STAT signaling in the phase of refractive arthritisinduced pain. Paper III and IV delineated the role of peripheral and spinal high mobility group box 1 (HMGB1) in arthritis-induced pain and if the pronociceptive actions of HMGB1 is sexdependent. In Paper III, blocking the activity of HMGB1 in the periphery was shown to alleviate CAIA-induced pain-like behavior in male but not female mice. Interestingly, local injection of Toll-like receptor (TLR)4-activating disulfide HMGB1 induced mechanical hypersensitivity in both sexes, but was associated with more pronounced contribution of immune cells in males compared to females. CAIA induction has been associated with activation of microglia in the spinal cord. In Paper IV, disrupting microglial activity was shown to prevent development of disulfide HMGB1-induced pain-like behavior in male but not female mice. To further explore sex-specific differences, global spinal protein expression was examined using liquid chromatography-mass spectrometry. Surprisingly, several antinociceptive and anti-inflammatory proteins were elevated in only male mice that received the microglial inhibitor minocycline, some of which modulate protein activation cascade that converges on proteinase-activated receptor (PAR)2. Targeting the identified proteins individually, however, did not produce robust antinociceptive effects as minocycline. Overall, these studies demonstrate the important aspects of sex and cellular location in the contribution of peripheral and spinal HMGB1 and TLR4 to arthritis-induced pain. In summary, this thesis has described three additional mechanisms of RA-induced pain. The findings suggest the involvement of osteoclasts, AAK1/AP2M1 and HMGB1 in mediating CAIA-induced hypersensitivity, particularly in the refractive state of the model. In addition, this work has highlighted the importance of mapping sex dimorphism and the prospective that pain relief is achieved differently in different sexes. Although more research are warranted in order to decipher the exact mechanisms that drive and maintain chronic pain in RA, this thesis has provided interesting mechanistic insights with respect to the bone environment and neuroinflammatory factors

Osteoarthritis: From Molecular Pathways to Therapeutic Advances

In this book, we have reported the most recent discoveries and updates regarding molecular pathways in osteoarthritis. In particular, the advances regarding therapeutical options, from a molecular point of view, are largely discussed

The Roles of S100A8 and S100A9 in Cartilage: Degradation and Formation

Osteoarthritis (OA) is a debilitating joint disease that increases in prevalence with age, with latest figures show that around 2.4 million Australians suffer from OA. There are currently no disease-modifying drugs available and due to poor management options, the number of joint replacement surgeries is increasing by nearly 10% per annum in Australia. S100A8 and S100A9 calcium-binding proteins are expressed by immune cells, and are involved in inflammatory situations, including inflammatory diseases such as rheumatoid arthritis, where there is an increase in circulating and local (synovial fluid) levels of S100A8 and S100A9. They are present in the body preferentially as a heterodimer (S100A8/S100A9) or less commonly as homodimers. Recently, S100A8 and S100A9 were found in bone and cartilage cells; however their roles in these tissues are still unknown. The aim of this research therefore was to determine the role(s) of S100A8 and S100A9 in cartilage degeneration and OA. The results presented in this thesis have provided insight into the role(s) of S100A8 and S100A9 in cartilage and in OA. S100A8 and S100A9 act as primers of cartilage degradation, through TLR4 and MAPK signalling pathways, but require a second messenger in order to achieve full biochemical breakdown. S100A8 is abundant in chondrocytes of human OA joints, but does not appear to be involved in non-inflammatory mouse models. S100A8, and to a lesser degree S100A9, are also abundant at joint margins and in osteophytes from human OA patients, and may play a role in chondrogenesis. The results presented in this thesis have determined some of the mechanisms of action of S100A8 and S100A9 in cartilage, however, the exact role of S100A8 and S100A9 in OA remain unclear, and further work will need to be performed to fully determine the significance of S100A8 and S100A9 in OA

Caractérisation d'un modèle animal de douleur articulaire associée à l'arthrose du genou chez le rat Sprague-Dawley

La douleur articulaire associée à l’arthrose est un problème clinique majeur, spécialement chez les personnes âgées. L’intensité de la douleur est souvent amplifiée lors de mouvement de l’articulation et principalement lors du soutien de la charge corporelle sur le membre lésé. Malheureusement, les traitements pharmacologiques proposés sont trop souvent associés à des effets secondaires néfastes et à une inefficacité pour le soulagement de la douleur à long terme. Divers modèles murins sont utilisés en laboratoire de recherche pour des études précliniques de molécules aux propriétés analgésiques. Une évaluation comparative de la réponse comportementale douloureuse des animaux d’un modèle d’instabilité articulaire induit par le sectionnement du ligament croisé antérieur accompagné d’une méniscectomie partielle (le modèle ACLT+pMMx) et d’un modèle de dégénérescence articulaire induite par le monoiodoacetate (le modèle MIA) a permis de sélectionner un modèle approprié pour la continuité du projet. Les deux modèles ont démontré des lésions tissulaires, mais le modèle MIA a démontré une réponse douloureuse plus prononcée que le modèle ACLT+pMMx. Par l’analyse de la démarche, le modèle MIA a démontré une boiterie claire dans le patron de la démarche des animaux qui est associée à une lésion unilatérale. Le modèle MIA a donc été choisi pour la suite du projet. La problématique principale dans la recherche sur la douleur associée à l’arthrose est une compréhension incomplète des mécanismes de douleur responsables de l’induction et du maintien de l’état de douleur. Il devient donc nécessaire d’améliorer nos connaissances de ces mécanismes en effectuant une caractérisation plus approfondie des modèles animaux employés pour l’évaluation de stratégies pharmacologiques analgésiantes. Afin de bien comprendre le modèle MIA, une caractérisation des événements moléculaires centraux lors de la progression du processus dégénératif des structures articulaires de ce modèle s’est effectuée aux jours 3, 7, 14, 21 et 28 post injection. Des mécanismes hétérogènes qui modulent l’information nociceptive en fonction de la progression temporelle de la pathologie ont été observés. Les changements du contenu i spinal des neuropeptides sélectionnés (substance P, CGRP, dynorphine A et Big dynorphine) ont débuté sept jours suivant l’injection de MIA. L’observation histologique a démontré que les dommages structuraux les plus importants surviennent entre les jours 14 et 21. C’est entre les jours 7 et 21 que les lésions démontrent le plus de similarités à la pathologie humaine. Cela suggère que lors d’une évaluation préclinique d’un traitement pharmacologique pour pallier la douleur articulaire utilisant le modèle MIA, l’étude doit tenir compte de ces événements afin de maximiser l’évaluation de son efficacité. Puisque les traitements pharmacologiques conventionnels proposés pour le soulagement de la douleur ne font pas l’unanimité en terme d’efficacité, d’effets non désirés et de coûts monétaires parfois onéreux, les molécules de dérivés de plante deviennent une alternative intéressante. L’eugénol, le principal constituant de l’huile de clou de girofle, a été administré oralement pour une période de 28 jours chez des rats ayant reçu l’injection intra-articulaire de MIA afin d’évaluer son efficacité pour le traitement de la douleur articulaire. L’eugénol à une dose de 40 mg/kg s’est révélé efficace pour l’amélioration du patron de la démarche des animaux ainsi que pour la diminution de l’allodynie mécanique secondaire. De plus, les concentrations spinales de neuropeptides pronocicepteurs ont diminué chez les animaux traités. Par une évaluation histopathologique, l’eugénol n’a démontré aucune évidence d’effets toxiques suite à une administration per os quotidienne pour une période prolongée. Ces résultats suggèrent le potentiel thérapeutique complémentaire de la molécule d’eugénol pour le traitement de la douleur articulaire.Pain is the most predominant clinical symptom associated with osteoarthritis (OA), mostly among older people. Joint movement and weight bearing often increase the pain intensity. Unfortunately, the proposed pharmacological treatments are frequently associated with side effects and ineffective for pain alleviation for long time periods. Many murine models are used in laboratories for preclinical studies evaluating analgesic compounds. A comparative evaluation of the behavioral pain responses of animals with a joint instability model induced by the transection of the anterior cruciate ligament followed by a partial menisectomy (the ACLT+pMMx model) and of an articular degenerative model induced by an intra-articular injection of monoiodoacetate (the MIA model) was conducted to select an appropriate model for the continuation of the project. Both models demonstrated articular lésions, however the MIA model demonstrated a clearer behavioral pain response over the ACLT+pMMx model. The gait pattern of the MIA model revealed a clear limping gait similar to that observed with unilateral OA in humans. The MIA model was chosen for the subsequent studies. An unresolved issue in pain OA research is the lack of understanding of the pain mechanisms responsible for the induction and maintenance of the pain. Therefore, there is an urgent clinical need to improve the characterization of animal models to effectively discover novel pain relief pharmacological treatment stratégies for OA patients. A characterization of the spinal pain molecular events during the progression of the joint degenerative process in the MIA model was performed on days 3, 7, 14, 21 and 28 post injection. Heterogeneous nociceptive central molecular events were observed in respect to the time course of the pathology’s progression. Changes in selected spinal neuropeptide content (substance P, CGRP, dynorphin A, Big dynorphin) began 7 days following the MIA injection. Most severe joint structural damage on histology occured between days 14 and 21 post injection. These results suggest that preclinical drug evaluation employing this model should be conducted between 7 and 21 days post injection when the lesions resemble most those of human OA. iii As current pharmacological therapy for the alleviation of joint pain does not achieve the unanimity in respect to efficacy, side effects and cost, plant derivate compounds are now interesting alternatives to improve the situation. Eugenol, the main constituent of clove oil, was evaluated for its efficacy for alleviation of joint pain in rats who previously received an intra-articular injection of mono-iodoacetate to induce the MIA model. Eugenol, administered orally for 28 consecutive days at a dose of 40 mg/kg, improved gait pattern and reduced secondary mechanical allodynia. Furthermore, spinal concentrations of pronociceptive neuropeptides were also decreased in the treated animals. No toxic effects of the compoud were identified on histopathological assessment of the various tissues. These results suggest that eugenol could be a potential therapeutic option for alleviating OA joint pain

La force de réaction au sol verticale maximale comme témoin d'effets fonctionnels et structuraux chez des modèles canins d'arthrose : potentiel envers le développement thérapeutique

Les modèles animaux d’arthrose permettent d’évaluer le potentiel d’agents thérapeutiques en phase préclinique de développement. Le présent ouvrage tient compte du chien comme modèle d’arthrose naturelle (chez l’animal de compagnie) ou expérimentale (par sectionnement chirurgical du ligament croisé crânial). Au sein des expérimentations, la force de réaction au sol verticale maximale, mesurée lors de l’analyse cinétique de la locomotion, est proposée comme témoin d’effets fonctionnels et structuraux sur ces modèles d’arthrose. Sur un modèle canin d’arthrose naturelle, le seuil de changement minimal détectable a été déterminé. Les changements au dysfonctionnement locomoteur peuvent désormais être cernés en s’affranchissant de la marge d’erreur inhérente à la mesure de la force verticale maximale. Il en découle l’identification de répondants lors d’essais cliniques entrepris chez le chien arthrosique. Une analyse rétrospective a, par la suite, déterminé un taux de répondants de 62.8% et d’une taille d’effet de 0.7 pour des approches thérapeutiques actuellement proposées aux chiens arthrosiques. Cette analyse détermina également que la démonstration d’une réponse thérapeutique était favorisée en présence d’un fort dysfonctionnement locomoteur. Sur un modèle canin d’arthrose par sectionnement chirurgical du ligament croisé crânial, la force verticale maximale a démontré une relation inverse avec certains types de lésions arthrosiques évaluées à l’aide d’imagerie par résonance magnétique. Également, la sensibilité de la force verticale maximale a été mise en évidence envers la détection d’effets structuraux, au niveau de l’os sous-chondral, par un agent anti-résorptif (le tiludronate) sur ce même modèle. Les expérimentations en contexte d’arthrose naturelle canine permettent de valider davantage les résultats d’essais cliniques contrôlés utilisant la force verticale maximale comme critère d’efficacité fonctionnelle. Des évidences cliniques probantes nécessaires à la pratique d’une médecine basée sur des faits sont ainsi escomptées. En contexte d’arthrose expérimentale, la pertinence d’enregistrer le dysfonctionnement locomoteur est soulignée, puisque ce dernier est en lien avec l’état des structures. En effectuant l’analyse de la démarche, de pair avec l’évaluation des structures, il est escompté de pouvoir établir la répercussion de bénéfices structurels sur l’inconfort articulaire. Cet ouvrage suggère qu’une plateforme d’investigations précliniques, qui combine le modèle canin d’arthrose par sectionnement chirurgical du ligament croisé crânial à un essai clinique chez le chien arthrosique, soit un moyen de cerner des bénéfices structuraux ayant des impacts fonctionnels. Le potentiel inférentiel de ces modèles canins d’arthrose vers l’Homme serait ainsi favorisé en utilisant la force verticale maximale.Animal models of osteoarthritis are useful to evaluate the potential of osteoarthritis therapeutics at the preclinical stage of development. In this thesis, the dog is used as a model of naturally-occurring (i.e. companion animal) and experimentally induced (i.e. by surgical transection of the cranial cruciate ligament) osteoarthritis. The peak of the vertically-oriented ground reaction force, which is measured during kinetic gait analysis, is proposed to be an indicator of structural and functional benefits in these models of osteoarthritis. In a canine model of naturally-occurring osteoarthritis, the threshold of the minimal detectable change in peak vertical force was determined. An improvement in the locomotor disability can now be identified according to the measurement error (noise) of the peak vertical force. This allows the identification of responders when the peak vertical force is used as an outcome measure of functional benefits. A retrospective analysis later determined that current therapeutic approaches provided a responder rate of 62.8% with an effect size of 0.7 in dogs with naturally-occurring osteoarthritis. This analysis also determined that the therapeutic response is favored in cases of severe locomotor disability.   In a canine model of osteoarthritis induced by surgical transection of the cranial cruciate ligament, the peak vertical force demonstrated an inverse relationship with different types of structural changes, as evaluated upon magnetic resonance imaging. The sensitivity of the peak vertical force to detect structural benefits on the subchondral bone was also shown in this model using an antiresorptive agent (i.e. tiludronate). The experiments conducted in dogs with naturally-occurring osteoarthritis further validate findings from clinical trials in which the peak vertical force is used as an outcome measure of functional benefits. The practice of an evidence-based medicine is then expected. The experiments conducted in dogs with surgically-induced osteoarthritis support the recording of the locomotor disability, being in line with the level of the structural changes. By performing gait analysis in addition to structural evaluations, it is expected to establish the impact of structural benefits on joint discomfort This thesis suggests that a platform for preclinical investigations, which combines the canine model of osteoarthritis induced by surgical transection of the cranial cruciate ligament and a clinical trial in dogs with naturally-occurring osteoarthritis, offers the opportunity to discern structural benefits having functional impacts. A better prediction of outcomes for human clinical trials is expected by using the peak vertical force