Kentucky Spinal Cord Injury Research Center, University of Louisville, School of Medicine, Louisville, KY 40202, USA; Anatomical Sciences and Neurobiology, University of Louisville, School of Medicine, Louisville, KY 40202, USA.
Kentucky Spinal Cord Injury Research Center, University of Louisville, School of Medicine, Louisville, KY 40202, USA; Departments of Neurological Surgery, University of Louisville, School of Medicine, Louisville, KY 40202, USA.
Neurobiol Dis. 2020 Dec;146:105123. doi: 10.1016/j.nbd.2020.105123. Epub 2020 Oct 1.
Secondary axonal loss contributes to the persistent functional disability following trauma. Consequently, preserving axons following spinal cord injury (SCI) is a major therapeutic goal to improve neurological outcome; however, the complex molecular mechanisms that mediate secondary axonal degeneration remain unclear. We previously showed that IPR-mediated Ca release contributes to axonal dieback and axonal loss following an ex vivo laser-induced SCI. Nevertheless, targeting IPR in a clinically relevant in vivo model of SCI and determining its contribution to secondary axonal degeneration has yet to be explored. Here we used intravital two-photon excitation microscopy to assess the role of IPR in secondary axonal degeneration in real-time after a contusive-SCI in vivo. To visualize Ca changes specifically in spinal axons over time, adult 6-8 week-old triple transgenic Avil-Cre:Ai9:Ai95 (sensory neuron-specific expression of tdTomato and the genetic calcium indicator GCaMP6f) mice were subjected to a mild (30 kdyn) T12 contusive-SCI and received delayed treatment with the IPR blocker 2-APB (100 μM, intrathecal delivery at 3, and 24 h following injury) or vehicle control. To determine the IPR subtype involved, we knocked-down IPR3 using capped phosphodiester oligonucleotides. Delayed treatment with 2-APB significantly reduced axonal spheroids, increased axonal survival, and reduced intra-axonal Ca accumulation within dorsal column axons at 24 h following SCI in vivo. Additionally, knockdown of IPR3 yielded increased axon survival 24 h post-SCI. These results suggest that IPR-mediated Ca release contributes to secondary axonal degeneration in vivo following SCI.
继发性轴突损失是创伤后持续功能障碍的原因。因此，保护脊髓损伤（SCI）后的轴突是改善神经功能预后的主要治疗目标；然而，介导继发性轴突退化的复杂分子机制仍不清楚。我们之前的研究表明，IPR 介导的 Ca2+释放有助于体外激光诱导 SCI 后的轴突退变和轴突丢失。然而，在 SCI 的临床相关体内模型中靶向 IPR 并确定其对继发性轴突退化的贡献尚未得到探索。在这里，我们使用活体双光子激发显微镜实时评估 IPR 在体内撞击性 SCI 后继发性轴突退化中的作用。为了随时间推移特异性地可视化脊髓轴突中的 Ca2+变化，成年 6-8 周龄三转基因 Avil-Cre:Ai9:Ai95（感觉神经元特异性表达 tdTomato 和遗传钙指示剂 GCaMP6f）小鼠接受轻度（30 kdyn）T12 撞击性 SCI，并在损伤后 3 小时和 24 小时接受 IPR 阻滞剂 2-APB（100 μM，鞘内给药）或载体对照处理。为了确定涉及的 IPR 亚型，我们使用加帽的磷酸二酯寡核苷酸敲低了 IPR3。延迟用 2-APB 治疗可显著减少轴突球，增加轴突存活，并减少体内 SCI 后 24 小时背柱轴突内的轴内 Ca2+积累。此外，IPR3 的敲低在 SCI 后 24 小时增加了轴突存活。这些结果表明，IPR 介导的 Ca2+释放有助于体内 SCI 后继发性轴突退化。
