Spinal cord injury (SCI) is a devastating condition lacking a cure. Research on axon damage and regeneration in SCI has been hampered by the lack of suitable in vivo imaging tools. Microglia, the resident immune cells of the central nervous system (CNS), are known to play crucial roles in brain development, homeostasis, and neurological disorders. In healthy CNS, microglia exhibit a ramified morphology, constantly surveying their environment. Upon CNS damage, microglia rapidly activate, changing their morphology and biochemistry. Growing evidence shows direct microglia-neuron contact modulates neurogenesis, synaptic plasticity, neuronal firing, neurodegeneration, and regeneration. Direct physical contact between microglia and neurons provides a highly efficient, precisely controlled, and versatile way to regulate neuronal activity and fate. This contact can be diverse and complex, varying across neuronal compartments. Beyond the well-studied synaptic interactions, specialized microglia-neuron junctions have been identified. Microglia-axon contact, also observed widely, supports axonal function in the developing and adult CNS, particularly at Nodes of Ranvier (NR), short unmyelinated segments along myelinated nerve fibers. These NRs are sites for microglia-axon communication and contribute to remyelination. Previous research suggested NR as a hub regulating axonal function and fate through microglial interaction. Rodent models of SCI are widely used to study axonal degeneration and regeneration, but conventional SCI induction methods (transection, crush, contusion) create complex microenvironments hindering systematic examination of specific variables. Laser axotomy, guided by two-photon imaging, provides a more precisely defined method of single axon injury. In vivo two-photon imaging with localized laser axotomy allows investigation of microglia-node interactions in response to controlled injury. The researchers used an optically cleared intervertebral window for in vivo spinal cord imaging to examine microglia-neuron interactions at NR and microglia's role in axonal degeneration following single spinal cord axon injury.

Extensive research has explored the multifaceted roles of microglia in the CNS, highlighting their involvement in development, homeostasis, and various neurological disorders. Studies have revealed the dynamic nature of microglia, constantly extending and retracting their processes to monitor the surrounding microenvironment. While microglia's involvement in inflammatory responses and neurodegeneration is well-established, the specific mechanisms of microglia-neuron interaction remain incompletely understood. Several studies have demonstrated the existence of direct physical contacts between microglia and neurons, impacting various neuronal functions. The Nodes of Ranvier, unmyelinated regions along myelinated axons, have emerged as critical sites for microglia-axon interaction, particularly in the context of injury and repair. The existing literature suggests that these interactions at the Nodes of Ranvier are crucial for maintaining axonal health and promoting remyelination following demyelinating insults. However, a comprehensive understanding of the dynamic nature of these interactions, especially under controlled injury conditions, was lacking before this study. Previous studies, using techniques like two-photon microscopy, have shown some aspects of microglia-axon interactions. Yet, the precise mechanisms by which these interactions influence axonal fate during injury remain elusive, making this research crucial in clarifying the protective role of microglia.

This study used double-crossed CX3CR1^+/GFP/Thy1-YFPH transgenic mice to visualize microglia (GFP) and myelinated axons (YFP) in the spinal dorsal column. An optically cleared intervertebral window provided access to the spinal cord without introducing surgical artifacts. In vivo two-photon excited fluorescence (TPEF) and stimulated Raman scattering (SRS) imaging were used to visualize microglia-axon interactions at Nodes of Ranvier (NR). Immunostaining with Caspr confirmed NR identification using SRS. Three-hour time-lapse imaging showed microglial processes dynamically contacting NR. Microglial contact was characterized as continuing or intermittent, based on the duration and components (soma, primary processes, secondary processes) involved. Laser axotomy, guided by two-photon imaging, induced single axon injury. Time-lapse imaging tracked axon-microglia dynamics after injury. To investigate the role of microglial P2Y12 receptors, a selective inhibitor (PSB0739) was administered intrathecally. To assess the role of ATP released through axonal volume-activated anion channels (VAAC), a VAAC inhibitor (NPPB) was used. Microglia depletion was achieved using PLX3397. The effects of voltage-gated sodium channel (NaV) inhibition (TTX) and two-pore-domain potassium (K2P) channel inhibition (TPA) were also examined. Immunohistology was used to assess microglial density after PLX3397 treatment. Image processing and analysis involved image registration to compensate for motion artifacts, quantification of microglial density and morphology, and analysis of microglia-node contact dynamics and axonal degeneration.

Under normal physiological conditions, microglia contact nodes in a random scanning pattern, modulated by neuronal activity. Most contacts are intermittent, initiated by secondary processes. Continuing contacts are usually initiated by soma or primary processes. Following axonal injury, microglia rapidly establish a wrapping contact at the nodes nearest to the lesion, halting acute axonal degeneration. This wrapping behavior is dependent on P2Y12 receptors. Inhibition of P2Y12 receptors almost abolishes the wrapping response and allows axonal degeneration. Inhibition of VAAC, likely involved in ATP release from injured axons, also abolishes the wrapping response. Microglia depletion resulted in increased axonal degeneration beyond the nodes. Inhibition of voltage-gated sodium channels (NaV) with TTX, while not affecting physiological microglia-node interactions, delayed axonal degeneration even in the absence of wrapping contact. Inhibition of K2P channels reduced wrapping contact after axonal injury. At secondary branch points, where axons bifurcate, injury to one branch did not cause degeneration beyond the branch point with or without microglial wrapping. However, injury to both branches led to degeneration beyond the branch point.

The findings demonstrate a crucial neuroprotective role for microglia in the acute phase of single spinal cord axon injury. Microglia's rapid wrapping response at Nodes of Ranvier effectively prevents axonal degeneration from extending beyond these critical points. This protective mechanism is mediated by P2Y12 receptors, suggesting a purinergic signaling pathway is involved. The involvement of NaV and K2P channels further highlights the intricate interplay between neuronal activity and microglial response to injury. The protective effects of microglia are particularly significant in the acute phase of injury, highlighting the importance of timely intervention. The results also highlight the importance of considering different types of branch points in understanding axonal degeneration, with secondary branch points exhibiting resilience to degeneration, possibly due to the presence of intact branches. The study provides a new perspective on the complex dynamics of neuron-glia interaction and its impact on axonal fate during injury. The findings have implications for the development of novel therapeutic strategies targeting microglia to promote axonal protection and regeneration.

This study demonstrates that microglia play a vital neuroprotective role in acute spinal cord axon injury through their rapid formation of wrapping contacts at Nodes of Ranvier. This protective mechanism is dependent on P2Y12 receptors and involves voltage-gated sodium and potassium channels. Future research should explore the precise mechanisms underlying this protective effect and investigate whether similar microglia-mediated protection occurs in other types of axon injuries and in chronic phases of injury and regeneration.

The study used a localized laser axotomy model of spinal cord injury, which may not fully recapitulate the complex microenvironment of large-scale SCI. The effects of long-term anesthesia on neuronal activity might have influenced the results of the electrical stimulation and TTX experiments. The limited field of view of the imaging technique prevented simultaneous visualization of multiple nodes along the same axon. Further research is needed to validate these findings in other SCI models and to translate these findings to clinical settings.