Microglia in the rostral ventromedial medulla mediate synaptic pruning via the C1q/C3-CR3 signaling pathway – a mechanism for the chronic orofacial pain

The mechanism of chronic orofacial pain was investigated by examining the interaction between activated microglia, C1q and neurons in RVM of rats with orofacial pain caused by temporomandibular joint injection of CFA. The results demonstrated that the pain threshold in CFA group exhibited a continuous decline, reaching its lowest point on the third day. During the modeling process, administered daily stereotactic injections of ANX-005 and minocycline into the RVM, which resulted in a notable recovery in the rats' pain threshold and a significant increase in C1q/C3 and microglia in RVM of CFA rat. The application of ANX-005 or minocycline resulted in a reduction in the expression of C1q/C3 and microglia. Notably, the expression of excitatory presynaptic membrane markers reduced and the length and density of dendritic spines decreased on neurons in RVM. Additionally, C1q was abundantly localized on excitatory presynaptic membranes and expressed in microglial lysosomes. Treatment with ANX-005 or minocycline resulted in a reduced number of immunofluorescence colocalizations and an elevated dendritic spine density. These findings indicate that initial orofacial pain induced by CFA, microglia in RVM are involved in the pruning of excitatory presynaptic membranes through the complement C1q/C3-CR3 signaling pathway. This process results in a reduction in the proportion of excitatory synapses and a disruption in the physiological balance between RVM descending facilitation and descending inhibition. This leads to the predominance of descending facilitation in pain transmission in the RVM, which in turn facilitates the chronification of orofacial pain.本研究通过探究颞下颌关节注射完全弗氏佐剂（Complete Freund's Adjuvant, CFA）诱发大鼠口面部疼痛后，其延髓头端腹内侧区（Rostral Ventromedial Medulla, RVM）内活化小胶质细胞、C1q与神经元之间的相互作用，解析慢性口面部疼痛的发病机制。结果显示，CFA组大鼠的疼痛阈值呈持续性下降趋势，并于造模后第3日降至最低水平；造模期间，每日向大鼠RVM区域立体定向注射ANX-005与米诺环素（minocycline），可显著提升CFA模型大鼠的疼痛阈值，同时其RVM内C1q/C3表达水平与小胶质细胞数量显著升高。单独给予ANX-005或米诺环素干预，则可下调C1q/C3的表达并抑制小胶质细胞活化；值得注意的是，RVM内神经元的兴奋性突触前膜标志物表达量降低，树突棘的长度与密度均出现下降。此外，C1q大量定位于兴奋性突触前膜，并在小胶质细胞溶酶体中表达；经ANX-005或米诺环素治疗后，免疫荧光共定位的数量减少，而树突棘密度则有所升高。上述研究结果表明，在CFA诱导的初始口面部疼痛中，RVM内的小胶质细胞可通过补体C1q/C3-CR3信号通路介导兴奋性突触前膜的修剪过程，该过程会降低兴奋性突触的占比，破坏RVM下行易化与下行抑制之间的生理平衡，最终导致下行易化在RVM疼痛传递中占据主导地位，进而促进口面部疼痛的慢性化进程。