Sorbonne Université, Paris, France.
INSERM UMRS_938, CRSA, Paris, France.
Sci Rep. 2020 Jun 1;10(1):8852. doi: 10.1038/s41598-020-65873-6.
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.
胆碱能系统通过迷走神经刺激后释放乙酰胆碱（Ach），在许多疾病中发挥主要的抗炎作用。骨关节炎（OA）与局部低度炎症有关，但调节机制尚不清楚。由于关节细胞表达参与炎症反应的 Ach 受体，局部 Ach 释放可能具有抗炎活性。使用允许整个样本 3 维（3D）分析的 3DISCO 清除方案，我们清除了人类 OA 软骨-软骨下骨样本，以寻找能够局部产生 Ach 的胆碱能神经纤维。我们分析了 3 名接受关节置换术的 OA 患者的 3 个膝关节软骨和软骨下骨样本。我们发现，在表层和中层关节软骨层中没有神经，外周蛋白（一种周围神经标志物）的缺乏证明了这一点。相反，在软骨的最深层和软骨下骨中发现了周围神经。软骨下骨样本中的一些神经是胆碱能的，因为它们共同表达了外周蛋白和胆碱乙酰转移酶（ChAT），这是胆碱能神经的特异性标志物。然而，在软骨层中没有发现胆碱能神经。因此，清除人骨以进行 3D 免疫荧光是可行的。人 OA 软骨下骨由胆碱能纤维支配，这些纤维可能通过局部 Ach 释放来调节局部炎症。
