Introduction: Modic changes are usually associated with degenerative disc diseases (DDD) and vertebral endplate subchondral changes, which can be the major cause of discogenic low back pain. However, the sensory innervation, spinal hypersensitivity and underlying mechanisms remain still unclear. The presence and association of osteoclast and NETRIN1 in the endplate changes has not been well studied.

Methods: Firstly, we detected the osteoclasts, the expression of NETRIN1, and nerve ingrowth in the specimens from the human lumbar endplate Modic changes and P. acnes-induced rat lumbar. Secondly, to clarify P. acnes-associated pain, calcium imaging and flux was used to show the neuronal calcium traces from dorsal root neurons (DRGs) exposed to P. acnes. We then investigated whether NETRIN1 was derived from the osteoclasts by ELISA, Western Blot and immunofluorescence (IF) in vitro and in vivo. Further, we detected the calcitonin gene-related peptide (CGRP) or substance P-positive nerve fibers in lumbar endplates, and the c-Fos-positive neurons in lumbar DRGs in the P. acnes-induced animal model in vivo. Finally, by the subcutaneous injection of denosumab (5mg/kg), we examined the formation of osteoclasts and proceeded the pain assessment to observe the pain sensation in the P. acnes-induced model in vivo.

Results: By TRAP staining and IF assays, we firstly found the increased osteoclasts and NETRIN1 in P. acnes-induced rat lumbar and the human lumbar endplate Modic changes, especially type I Modic changes. Western blot and IF data showed that osteoclasts could secret a large amount of NETRIN1 rather than NETRIN2 or NETRIN3, and TRAP/NETRIN1 were co-localized in vivo, indicating that NETRIN1 was derived from osteoclasts. By the co-culture system (20% conditioned medium), we clarified that osteoclast-derived NETRIN1 promoted the axon elongation of DRGs in vitro. Secondly, we detected the increased calcium and influx in the DRGs exposed to P. acnes, implying that P. acnes were positively associated with pain. Then, we discovered the increasing CGRP-positive nerve fibers in lumbar endplates and the c-Fos-positive neurons in lumbar DRGs in the P. acnes-induced animal model in vivo, compared to the control group. Further, we inhibited the in vivo formation of osteoclasts and revealed the less pain in the P. acnes-induced animal model with denosumab treatment. Finally, by IF assay, the CGRP or substance P-positive nerve fibers in lumbar endplates and the c-Fos-positive neurons in lumbar DRGs were decreased in the P. acnes-induced animal model with denosumab treatment. Sensory innervation by NETRIN1 from osteoclasts mediates spinal hypersensitivity in lumbar endplate Modic changes.

Discussion: Modic changes are strongly related with low back pain, especially type I Modic changes. Many researches showed the increased sensory innervation in lumbar endplate region, whereas its precise mechanism remained unknown. We showed the NETRIN1 from osteoclasts in lumbar endplate guided the axon elongation of DRGs to mediate spinal pain hypersensitivity. By denosumab management, CGRP-positive nerve fibers were suppressed in lumbar endplates. Meanwhile, c-Fos-positive neurons were reduced in lumbar DRGs. These data indicated that osteoclast might be the therapeutic target for lumbar endplate Modic changes.