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Stirling et al. use two-photon excitation, confocal spectral microscopy and a novel ex vivo whole spinal cord model to assess the microglial response to spinal cord injury in real-time. Stimulation of microglia with a synthetic toll-like receptor 2 agonist, Pam2CSK4, modulated the microglial response and reduced white matter injury.Improving neurological outcome after spinal cord injury is a major clinical challenge because axons, once severed, do not regenerate but ‘dieback’ from the lesion site. Although microglia, the immunocompetent cells of the brain and spinal cord respond rapidly to spinal cord injury, their role in subsequent injury or repair remains unclear. To assess the role of microglia in spinal cord white matter injury we used time-lapse two-photon and spectral confocal imaging of green fluorescent protein-labelled microglia, yellow fluorescent protein-labelled axons, and Nile Red-labelled myelin of living murine spinal cord and revealed dynamic changes in white matter elements after laser-induced spinal cord injury in real time. Importantly, our model of acute axonal injury closely mimics the axonopathy described in well-characterized clinically relevant models of spinal cord injury including contusive-, compressive- and transection-based models. Time-lapse recordings revealed that microglia were associated with some acute pathophysiological changes in axons and myelin acutely after laser-induced spinal cord injury. These pathophysiological changes included myelin and axonal spheroid formation, spectral shifts in Nile Red emission spectra in axonal endbulbs detected with spectral microscopy, and ‘bystander’ degeneration of axons that survived the initial injury, but then succumbed to secondary degeneration. Surprisingly, modulation of microglial-mediated release of neurotoxic molecules failed to protect axons and myelin. In contrast, sterile stimulation of microglia with the specific toll-like receptor 2 agonist Pam2CSK4 robustly increased the microglial response to ablation, reduced secondary degeneration of central myelinated fibres, and induced an alternative (mixed M1:M2) microglial activation profile. Conversely, Tlr2 knock out: Thy1 yellow fluorescent protein double transgenic mice experienced greater axonal dieback than littermate controls. Thus, promoting an alternative microglial response through Pam2CSK4 treatment is neuroprotective acutely following laser-induced spinal cord injury. Therefore, anti-inflammatory treatments that target microglial activation may be counterintuitive after spinal cord injury. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) induces a robust inflammatory response and the extravasation of leukocytes into the injured tissue. To further knowledge of the functions of neuroinflammation in SCI in mice, we depleted the early arriving neutrophils using an anti-Ly6G/Gr-1 antibody. Complete blood counts revealed that neutrophils increased~3-fold over uninjured controls and peaked at 6 -12 h after injury, and that anti-Ly6G/Gr-1 treatment reduced circulating neutrophils by>90% at these time points. Intravital and spinning disk confocal microscopy of the exposed posterior vein and postcapillary venules showed a significant reduction in rolling and adhering neutrophils in vivo after anti-Ly6G/Gr-1 treatment; this was accompanied by a parallel reduction in neutrophil numbers within the injured spinal cord at 24 and 48 h as determined by flow cytometry. The evolution of astrocyte reactivity, a wound healing response, was reduced in anti-Ly6G/Gr-1-treated mice, which also had less spared white matter and axonal preservation compared with isotype controls. These histological outcomes may be caused by alterations of growth factors and chemokines important in promoting wound healing. Importantly, anti-Ly6G/Gr-1 treatment worsened behavioral outcome as determined using the Basso Mouse Scale and subscores. Although the spectrum of cells affected by anti-Ly6G/Gr-1 antibody treatment cannot be fully ascertained at this point, the correspondence of neutrophil depletion and worsened recovery suggests that neutrophils promote recovery after SCI through wound healing and protective events that limit lesion propagation. [ABSTRACT FROM AUTHOR]

Minocycline has been demonstrated to be neuroprotective after spinal cord injury (SCI). However, the cellular consequences of minocydine treatment on the secondary injury response are poorly understood. We examined the ability of minocycline to reduce oligodendrocyte apoptosis, microglial/macrophage activation, corticospinal tract (CST) dieback, and lesion size and to improve functional outcome after SCI. Adult rats were subjected to a C7-C8 dorsal column transection, and the presence of apoptotic oligodendrocytes was assessed within the ascending sensory tract (AST) and descending CST in segments (3-7 mm) both proximal and distal to the injury site. Surprisingly, the numbers of dying oligodendrocytes in the proximal and distal segments were comparable, suggesting more than the lack of axon- cell body contiguity played a role in their demise. Minocycline or vehicle control was injected into the intraperitoneal cavity 30 min and 8 hr after SCI and thereafter twice daffy for 2 d. We report a reduction of apoptotic oligodendrocytes and microglia within both proximal and distal segments of the AST after minocycline treatment, using immunostaining for active caspase-3 and Hoechst 33258 staining in combination with cell-specific markers. Activated microglial/macrophage density was reduced remote to the lesion as well as at the lesion site. Both CST dieback and lesion size were diminished after minocycline treatment. Footprint analysis revealed improved functional outcome after minocycline treatment. Thus, minocycline ameliorates multiple secondary events after SCI, rendering this clinically used drug an attractive candidate for SCI treatment trials. [ABSTRACT FROM AUTHOR]

Ryanodine receptors (RyRs) mediate calcium release from calcium stores and have been implicated in axonal degeneration. Here, we use an intravital imaging approach to determine axonal fate after spinal cord injury (SCI) in real-time and assess the efficacy of ryanodine receptor inhibition as a potential therapeutic approach to prevent intra-axonal calcium-mediated axonal degeneration. Adult 6-8 week old Thy1YFP transgenic mice that express YFP in axons, as well as triple transgenic Avil-Cre:Ai9:Ai95 mice that express the genetically-encoded calcium indicator GCaMP6f in tdTomato positive axons, were used to visualize axons and calcium changes in axons, respectively. Mice received a mild SCI at the T12 level of the spinal cord. Ryanodine, a RyR antagonist, was given at a concentration of 50 μM intrathecally within 15 min of SCI or delayed 3 h after injury and compared with vehicle-treated mice. RyR inhibition within 15 min of SCI significantly reduced axonal spheroid formation from 1 h to 24 h after SCI and increased axonal survival compared with vehicle controls. Delayed ryanodine treatment increased axonal survival and reduced intra-axonal calcium levels at 24 h after SCI but had no effect on axonal spheroid formation. Together, our results support a role for RyR in secondary axonal degeneration. [ABSTRACT FROM AUTHOR]

The vagus nerve is comprised primarily of non-myelinated sensory neurons whose cell bodies are located in the nodose ganglion (NG). The vagus has widespread projections that supply most visceral organs, including the bladder. Due to its non-spinal route, the vagus nerve itself is not directly damaged from spinal cord injury (SCI). Because most viscera, including bladder, are dually innervated by spinal and vagal sensory neurons, an impact of SCI on the sensory component of vagal circuitry may contribute to post-SCI visceral pathologies. To determine if SCI, in male Wistar rats, might impact neurochemical characteristics of NG neurons, immunohistochemical assessments were performed for P2X3 receptor expression, IB4 binding, and Substance-P expression, three known injury-responsive markers in sensory neuronal subpopulations. In addition to examining the overall population of NG neurons, those innervating the urinary bladder also were assessed separately. All three of the molecular markers were represented in the NG from non-injured animals, with the majority of the neurons binding IB4. In the chronically injured rats, there was a significant increase in the number of NG neurons expressing P2X3 and a significant decrease in the number binding IB4 compared to non-injured animals, a finding that held true also for the bladder-innervating population. Overall, these results indicate that vagal afferents, including those innervating the bladder, display neurochemical plasticity post-SCI that may have implications for visceral homeostatic mechanisms and nociceptive signaling. [ABSTRACT FROM AUTHOR]

Historically, the immune response after spinal cord injury was considered largely detrimental owing to the release of neurotoxic factors. While there is validity to this view, there is much greater heterogeneity of immune cells than was previously realized. Associated with this heterogeneity of immune cell subtypes, there is diversity of functions of immune cells that is still poorly understood after spinal cord injury. Modulating the immune system requires improved understanding of the major players: those immune cell subtypes that are more detrimental than beneficial and those that are important in repair. In this review we will discuss the early findings that supported the use of various anti-inflammatory medications as well as the evolving concept that not all immune subtypes are detrimental and some might even be beneficial. In the last section we will highlight the need to characterize better the role of immune cell subsets in the hopes of developing potential therapeutic targets for the future. [ABSTRACT FROM AUTHOR]

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 IP 3 R-mediated Ca2+ release contributes to axonal dieback and axonal loss following an ex vivo laser-induced SCI. Nevertheless, targeting IP 3 R 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 IP 3 R in secondary axonal degeneration in real-time after a contusive-SCI in vivo. To visualize Ca2+ 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 IP 3 R blocker 2-APB (100 μM, intrathecal delivery at 3, and 24 h following injury) or vehicle control. To determine the IP 3 R subtype involved, we knocked-down IP 3 R3 using capped phosphodiester oligonucleotides. Delayed treatment with 2-APB significantly reduced axonal spheroids, increased axonal survival, and reduced intra-axonal Ca2+ accumulation within dorsal column axons at 24 h following SCI in vivo. Additionally, knockdown of IP 3 R3 yielded increased axon survival 24 h post-SCI. These results suggest that IP 3 R-mediated Ca2+ release contributes to secondary axonal degeneration in vivo following SCI. • Intravital imaging following contusive SCI reveals changes in spinal axons in real-time. • IP3R contribute to secondary axonal degeneration. • Delayed application of the IP3R antagonist, 2-APB increased axonal survival. • Delayed application of 2-APB reduced axonal spheroid formation. • Knockdown of IP3R subtype 3 increased axonal survival. [ABSTRACT FROM AUTHOR]

Axonal degeneration plays a key role in the pathogenesis of numerous neurological disorders including spinal cord injury. After the irreversible destruction of the white matter elements during the primary (mechanical) injury, spared axons and their supporting glial cells begin to breakdown causing an expansion of the lesion site. Here we mechanistically link external sources of calcium entry through axoplasmic reticulum calcium store depletion that contributes to secondary axonal degeneration through a process called store-operated calcium entry. There is increasing evidence suggesting that store-operated calcium entry impairment is responsible for numerous disorders. Nevertheless, its role following spinal cord injury remains poorly understood. We hypothesize that store-operated calcium entry mediates secondary white matter degeneration after spinal cord injury. We used our previously published model of laser-induced spinal cord injury to focally transect mid cervical dorsal column axons from live 6–8-week-old heterozygous CNPaseGFP/+: Thy1 YFP+ double transgenic murine spinal cord preparations (five treated, eight controls) and documented the dynamic changes in axons over time using two-photon excitation microscopy. We report that 1 hour delayed treatment with YM-58483, a potent inhibitor of store-operated calcium entry, significantly decreased intra-axonal calcium accumulation, axonal dieback both proximal and distal to the lesion site, reduced secondary axonal "bystander" damage acutely after injury, and promoted greater oligodendrocyte survival compared to controls. We also targeted store-operated calcium entry following a clinically relevant contusion spinal cord injury model in vivo. Adult, 6–8-week-old Advillin-Cre: Ai9 mice were subjected to a mild 30 kdyn contusion and imaged to observe secondary axonal degeneration in live animals. We found that delayed treatment with YM-58483 increased axonal survival and reduced axonal spheroid formation compared to controls (n = 5 mice per group). These findings suggest that blocking store-operated calcium entry acutely is neuroprotective and introduces a novel target to prevent pathological calcium entry following spinal cord injury using a clinically relevant model. • Live imaging of a laser-induced SCI model reveals changes in spinal axons in real-time. • Delayed SOCE inhibition reduces axonal dieback and axonal degeneration post-LiSCI. • Delayed SOCE inhibition reduces Ca2+ concentrations in degenerating axons post-LiSCI. • Delayed SOCE inhibition increases axonal survival post contusive-SCI in vivo. [ABSTRACT FROM AUTHOR]

Ryanodine receptors (RyRs) are highly conductive intracellular Ca 2+ release channels and are widely expressed in many tissues, including the central nervous system. RyRs have been implicated in intracellular Ca 2+ overload which can drive secondary damage following traumatic injury to the spinal cord (SCI), but the spatiotemporal expression of the three isoforms of RyRs (RyR1-3) after SCI remains unknown. Here, we analyzed the gene and protein expression of RyR isoforms in the murine lumbar dorsal root ganglion (DRG) and the spinal cord lesion site at 1, 2 and 7 d after a mild contusion SCI. Quantitative RT PCR analysis revealed that RyR3 was significantly increased in lumbar DRGs and at the lesion site at 1 and 2 d post contusion compared to sham (laminectomy only) controls. Additionally, RyR2 expression was increased at 1 d post injury within the lesion site. RyR2 and -3 protein expression was localized to lumbar DRG neurons and their spinal projections within the lesion site acutely after SCI. In contrast, RyR1 expression within the DRG and lesion site remained unaltered following trauma. Our study shows that SCI initiates acute differential expression of RyR isoforms in DRG and spinal cord. [ABSTRACT FROM AUTHOR]

Microglia/macrophage activation and recruitment following spinal cord injury (SCI) is associated with both detrimental and reparative functions. Stimulation of the innate immune receptor Toll-like receptor-2 (TLR2) has shown to be beneficial following SCI, and it increases axonal regeneration following optic nerve crush. However, the mechanism(s) remain unclear. As microglia express high levels of TLR2, we hypothesized that modulating the microglial response to injury using a specific TLR2 agonist, Pam3CSK4, would prevent secondary-mediated white matter degeneration following SCI. To test this hypothesis, we documented acute changes in microglia, axons, and oligodendroglia over time using two-photon excitation and an ex vivo laser-induced SCI (LiSCI) model. We utilized double transgenic mice that express GFP in either microglia or oligodendroglia, and YFP in axons, and we applied the lipophilic fluorescent dye (Nile Red) to visualize myelin. We found that treatment with Pam3CSK4 initiated one hour after injury induced a significant increase in the extent and timing of the microglial response to injury compared to vehicle controls. This enhanced response was observed 2 to 4 h following SCI and was most prominent in areas closer to the ablation site. In addition, Pam3CSK4 treatment significantly reduced axonal dieback rostral and caudal to the ablation at 6 h post-SCI. This protective effect of Pam3CSK4 was also mirrored when assessing secondary bystander axonal damage ( i.e. , axons spared by the primary injury that then succumb to secondary degeneration), and when assessing the survival of oligodendroglia. Following these imaging experiments, custom microarray analysis of the ex vivo spinal cord preparations revealed that Pam3CSK4-treatment induced an alternative (mixed M1:M2) microglial activation profile. In summary, our data suggest that by providing a second “sterile” activation signal to microglia through TLR2/TLR1 signaling, the microglial response to injury can be modulated in situ and is highly neuroprotective. [ABSTRACT FROM AUTHOR]

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 Thy1 YFP 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. • Intravital imaging following contusive SCI reveals changes in spinal axons in real-time. • Axons predominantly undergo spheroid formation acutely after contusive SCI. • Axonal spheroids spread mediolaterally over time indicative of secondary injury. • Axonal spheroids are highly dynamic and can resolve spontaneously. • Disrupted axonal transport likely underlies spheroid and endbulb formation. [ABSTRACT FROM AUTHOR]