Spinal cord injury (SCI) induces a secondary degenerative response that causes the loss of spared axons and worsens neurological outcome. The complex molecular mechanisms that mediate secondary axonal degeneration remain poorly understood. To further our understanding of secondary axonal degeneration following SCI, we assessed the spatiotemporal dynamics of axonal spheroid and terminal bulb formation following a contusive SCI in real-time in vivo. Adult 6-8 week old Thy1YFP transgenic mice underwent a T12 laminectomy for acute imaging sessions or were implanted with a custom spinal cord imaging chamber for chronic imaging of the spinal cord. Two-photon excitation time-lapse microscopy was performed prior to a mild contusion SCI (30 kilodyne, IH Impactor) and at 1-4 h and 1-14 days post-SCI. We quantified the number of axonal spheroids, their size and distribution, the number of endbulbs, and axonal survival from 1 h to 14 days post-SCI. Our data reveal that the majority of axons underwent swelling and axonal spheroid formation acutely after SCI resulting in the loss of ~70% of axons by 1 day after injury. In agreement, the number of axonal spheroids rapidly increased at 1 h after SCI and remained significantly elevated up to 14 days after SCI. Furthermore, the distribution of axonal spheroids spread mediolaterally over time indicative of delayed secondary degenerative processes. In contrast, axonal endbulbs were relatively sparse and their numbers peaked at 1 day after injury. Intriguingly, axonal survival significantly increased at 7 and 14 days compared to 3 days after SCI revealing a potential endogenous axonal repair process that mirrors the known spontaneous functional recovery after SCI. In support, ~43% of tracked axonal spheroids resolved over the course of observation revealing their dynamic nature. Furthermore, axonal spheroids and endbulbs accumulated mitochondria and excessive tubulin polyglutamylation suggestive of disrupted axonal transport as a shared mechanism. Collectively, this study provides important insight into both degenerative and recoverable responses of axons following contusive SCI in real-time. Understanding how axons spontaneously recover after SCI will be an important avenue for future SCI research and may help guide future clinical trials.

中文翻译：

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反复进行鼠体内脊髓活体成像，可以发现挫伤性脊髓损伤后实时显示脊髓轴突的变性和修复反应。

脊髓损伤（SCI）引起继发性退行性反应，从而导致多余的轴突丢失，并使神经功能恶化。介导继发性轴突变性的复杂分子机制仍然知之甚少。为了进一步了解SCI后继发的轴突变性，我们实时评估了挫伤性SCI后轴突球体的时空动力学和末端球形成。6-8周大的成年Thy1YFP转基因小鼠经过T12椎板切除术以进行急性成像，或植入定制的脊髓成像室进行脊髓的慢性成像。在轻度挫伤SCI（30千达因，IH Impactor）之前以及在SCI后1-4小时和1-14天进行双光子激发延时显微镜。我们量化了轴突球体的数量，脊髓损伤后1小时到14天，它们的大小和分布，鳞茎的数量和轴突存活率。我们的数据显示，大多数脊髓损伤的轴突在SCI后会急剧肿胀并形成轴突球体，导致损伤后1天损失约70％的轴突。一致的是，在脊髓损伤后1小时，轴突球体的数量迅速增加，直到脊髓损伤后14天仍显着升高。此外，轴突球体的分布随时间在中外侧散布，表明继发性退化过程延迟。相反，轴突终生相对稀疏，其数量在受伤后1天达到峰值。有趣的是 与SCI后3天相比，在7天和14天时，轴突存活率显着增加，揭示了潜在的内源性轴突修复过程，反映了SCI后已知的自发功能恢复。在支持下，约43％的跟踪轴突球体在观察过程中分解，揭示了它们的动态性质。此外，轴突球体和球根积累线粒体和过多的微管蛋白聚谷氨酰化提示轴突运输受阻是一种共同的机制。总的来说，这项研究为挫伤性SCI实时提供了对轴突变性和可恢复反应的重要见解。了解SCI后轴突如何自发恢复将是未来SCI研究的重要途径，并可能有助于指导未来的临床试验。在观察过程中，约有43％的追踪轴突椭球体得以分辨，从而揭示了它们的动态特性。此外，轴突球体和球根积累线粒体和过多的微管蛋白聚谷氨酰化提示轴突运输受阻是一种共同的机制。总的来说，这项研究为挫伤性SCI实时提供了对轴突变性和可恢复反应的重要见解。了解SCI后轴突如何自发恢复将是未来SCI研究的重要途径，并可能有助于指导未来的临床试验。在观察过程中，约有43％的追踪轴突椭球体得以分辨，从而揭示了它们的动态特性。此外，轴突球体和球根积累线粒体和过多的微管蛋白聚谷氨酰化提示轴突运输受阻是一种共同的机制。总的来说，这项研究为挫伤性SCI实时提供了对轴突变性和可恢复反应的重要见解。了解SCI后轴突如何自发恢复将是未来SCI研究的重要途径，并可能有助于指导未来的临床试验。这项研究提供了重要的见解实时挫伤性脊髓损伤后轴突的退化和可恢复的反应。了解SCI后轴突如何自发恢复将是未来SCI研究的重要途径，并可能有助于指导未来的临床试验。这项研究为挫伤性SCI实时提供了对轴突变性和可恢复反应的重要见解。了解SCI后轴突如何自发恢复将是未来SCI研究的重要途径，并可能有助于指导未来的临床试验。