Life sciences · 2024
Relief of pain in mice by an antibody with high affinity for cell adhesion molecule 1 on nerves.
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Takeuchi Fuka, Hagiyama Man, Yoneshige Azusa, Wada Akihiro, Inoue Takao, Hosokawa Yoichiroh, Ito Akihiko
Аннотация
AIMS: Cell adhesion molecule 1 (CADM1) is a member of the immunoglobulin superfamily and is abundantly expressed on nerve fibers. Recently, the anti-CADM1 ectodomain antibody 3E1 has proven useful as a drug delivery vector for CADM1-expressing cells in vitro. When injected subcutaneously into mice, whether 3E1 accumulates on nerve fibers and serves as an analgesic was examined.
MAIN METHODS: Injected 3E1 was detected by immunohistochemistry and double immunofluorescence. Analgesic effects were verified by a formalin-induced chemical-inflammatory pain test and video-recorded behavior analysis that were performed 6, 12, and 24 h after antibody injection. Primary cultures of mouse dorsal root ganglion (DRG) cells were incubated with 3E1 and expressions of CADM1 and its key downstream molecules were examined by Western blot analyses and live cell imaging. DRG cells were loaded with a Ca2+ fluorescent indicator Fluo-8 and a femtosecond laser pulse was irradiated near the cell body to mechanically stimulate the nerves.
KEY FINDINGS: Subcutaneously injected 3E1 was widely localized almost exclusively on peripheral nerve fibers in the dermis. In formalin tests, 3E1-injected mice exhibited less pain-related behavior than control mice. When 3E1 was added to DRG cell cultures, it localized to neurites and resulted in decreased expression of CADM1, increased phosphorylation of Src and Akt, and CADM1-3E1 complex formation. Femtosecond laser-induced stimulation transmission along neurites was clearly visualized by Fluo-8 fluorescence in control cells, whereas it was markedly suppressed in 3E1-treated cells.
SIGNIFICANCE: 3E1 was suggested to be a potential long-acting analgesic based on its high affinity for CADM1.
MAIN METHODS: Injected 3E1 was detected by immunohistochemistry and double immunofluorescence. Analgesic effects were verified by a formalin-induced chemical-inflammatory pain test and video-recorded behavior analysis that were performed 6, 12, and 24 h after antibody injection. Primary cultures of mouse dorsal root ganglion (DRG) cells were incubated with 3E1 and expressions of CADM1 and its key downstream molecules were examined by Western blot analyses and live cell imaging. DRG cells were loaded with a Ca2+ fluorescent indicator Fluo-8 and a femtosecond laser pulse was irradiated near the cell body to mechanically stimulate the nerves.
KEY FINDINGS: Subcutaneously injected 3E1 was widely localized almost exclusively on peripheral nerve fibers in the dermis. In formalin tests, 3E1-injected mice exhibited less pain-related behavior than control mice. When 3E1 was added to DRG cell cultures, it localized to neurites and resulted in decreased expression of CADM1, increased phosphorylation of Src and Akt, and CADM1-3E1 complex formation. Femtosecond laser-induced stimulation transmission along neurites was clearly visualized by Fluo-8 fluorescence in control cells, whereas it was markedly suppressed in 3E1-treated cells.
SIGNIFICANCE: 3E1 was suggested to be a potential long-acting analgesic based on its high affinity for CADM1.