Abstract
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 anti-inflammatory 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.
Highlights
The cholinergic system plays a major anti-inflammatory role in many diseases through acetylcholine (Ach) release after vagus nerve stimulation
We first used one sample to establish that plugs of a particular size could be analysed by microscopy, and we confirmed that all cartilage layers and sufficient subchondral bone were present for analysis (Fig. 1A,B)
We report for the first time that cholinergic fibres are present in human OA subchondral bone
Summary
The cholinergic system plays a major anti-inflammatory role in many diseases through acetylcholine (Ach) release after vagus nerve stimulation. 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. Human OA subchondral bone is innervated by cholinergic fibres, which may regulate local inflammation through local Ach release. Improvements in light sheet microscopy and the clearing protocol have made it possible to readily immunolabel a whole tissue sample in a straightforward manner. This technique is of interest for the study of small structures, such as nerves. The objective of our study was to determine the presence of cholinergic nerves in cartilage and subchondral OA human samples using 3D immunolabelling
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