The cholinergic system plays a major anti-inflammatory role in many diseases through acetylcholine (Ach) release after vagus nerve stimulation. Osteoarthritis (OA) is associated with local low-grade inflammation, but the regulatory mechanisms are unclear. Local Ach release could have antiinflammatory activity since articular cells express Ach receptors involved in inflammatory responses. Using the 3DISCO clearing protocol that allows whole-sample 3-dimensional (3D) analysis, we cleared human OA cartilage-subchondral bone samples to search for cholinergic nerve fibres able to produce Ach locally. We analysed 3 plugs of knee cartilage and subchondral bone from 3 OA patients undergoing arthroplasty. We found no nerves in the superficial and intermediate articular cartilage layers, as evidenced by the lack of Peripherin staining (a peripheral nerves marker). Conversely, peripheral nerves were found in the deepest layer of cartilage and in subchondral bone. Some nerves in the subchondral bone samples were cholinergic because they coexpressed peripherin and choline acetyltransferase (ChAT), a specific marker of cholinergic nerves. However, no cholinergic nerves were found in the cartilage layers. It is therefore feasible to clear human bone to perform 3D immunofluorescence. Human OA subchondral bone is innervated by cholinergic fibres, which may regulate local inflammation through local Ach release. Osteoarthritis (OA) is the most common joint disease and is a considerable burden on quality of life due to joint pain, stiffness and loss of function 1. OA is mostly characterized by cartilage degradation but is now considered a whole-joint disease associated with subchondral bone remodelling and synovitis. Articular cartilage is composed of several layers, and the deepest layer, called calcified cartilage, is located on top of subchondral bone. Articular cartilage is a unique tissue that is not innervated or vascularized. Communication between subchondral bone and calcified cartilage is needed for cartilage homeostasis. Such communication is also involved in the OA process because of the circulation of soluble mediators from subchondral bone to cartilage undergoing alterations 2. Interplay between subchondral bone and deep cartilage may involve micro-cracks at the junction between the two tissues as well as neoangiogenesis of the calcified cartilage, which could be directly involved in OA pathophysiology 3-5. Additionally, neurogenesis may play a role in OA pathophysiology. Walsh and colleagues previously showed that during OA, peripheral sensory and sympathetic nerves grow in calcified cartilage and may be involved in OA pain 6,7. Beyond sensory mediators and sensory nerves, the parasympathetic system has been reported to have anti-inflammatory properties through the action of its mediator, acetylcholine (Ach), on nicotinic Ach receptors and, in particular, the alpha7 nicotinic receptor 8,9. Ach production is controlled by choline acetyltransferase (ChAT), which is mainly recognized as a marker of cholinergic nerves. Bec...

Purpose: Beyond its role on the autonomic nervous system, the main parasympathetic neuromediator acetylcholine (Ach) has anti-inflammatory properties through the activation of the nicotinic alpha-7 receptor (Chrna7, homopentamer of 5 alpha-7 subunits) expressed by non-neuronal cells. Little is known on its role in joints in general and in OA in particular. We determined 1) the presence of neuronal cholinergic fibers in human and murine joints tissues 2) whether chondrocytes and osteoblasts have the ability to produce Ach (non-neuronal production) 3) the expression and the biological role of Chrna7 in chondrocytes and osteoblasts 4) and the in vivo involvement of Chrna7 using medial meniscus destabilization model of osteoarthritis (OA-DMM). Methods: In order to explore the presence of neuronal cholinergic fibers in joints, we took advantage of a recent sophisticated method that uses a whole joint immunolabeling protocol: after 3Disco clearing, in toto 3D immunofluorescence was performed to evaluate the colocalization of peripherin (nerve marker) and choline acetyltransferase (ChAT, cholinergic fiber marker) in human OA bone and cartilage samples, obtained from OA patients undergoing knee arthroplasty, and from 6-day-old C57Bl6 mice. Acquisitions were performed using an ultramicroscope and analyzed using IMARIS. Primary cultures of murine osteoblasts and chondrocytes were obtained from 6-day-old C57Bl6 mice calvaria and cartilage knees and femoral heads respectively. Primary cultures of human OA chondrocytes were obtained from human OA cartilage obtained from OA patients undergoing knee arthroplasty. At confluence, RNA was extracted to determine by PCR the expression of the molecular actors of production, transport and degradation of Ach as well as the nicotinic subunits alpha 1 to 7, alpha 9 and beta 1 to 4. In vitro, WT and KO for Chrna7 (Chrna7 -/ -) chondrocytes and WT osteoblasts were treated with IL1b. In order to study the role of nicotinic receptors in cell activation, murine chondrocytes and osteoblasts were pretreated with nicotine at 1; 10 or 100 mM. After 24 hours, we collected mRNA and medium to quantify the expression of cytokines (IL6, IL8-Kc) and metalloproteinases (MMP3) along with RANK-ligand and osteoprotegerin expression for osteoblasts using quantitative PCR. DMM was performed on the right knees of 12-week-old WT (n¼4) and KO Chrna7 -/mice (n¼4 per group). After 9 weeks, histological analysis of the knees was performed using safranin and light green coloration to determine the OARSI score on femoral and tibial compartments (from 0 to 12). Results: Cholinergic fibers were present in subchondral bone of all 3 OA joints explants analyzed coming from 3 different patients (Figure 1). No cholinergic nerves were found in cartilage. We also confirmed the presence of cholinergic fibers in murine bone, located into the cortical bone. Human OA and murine chondrocytes as well as murine osteoblasts express the whole system needed for the production (carnityl acetyltransferase), the transport (vesicular tran...