RESUMO
The formation of axonal spheroid is a common feature following spinal cord injury. To further understand the source of Ca2+ that mediates axonal spheroid formation, we used our previously characterized ex vivo mouse spinal cord model that allows precise perturbation of extracellular Ca2+. We performed two-photon excitation imaging of spinal cords isolated from Thy1YFP+ transgenic mice and applied the lipophilic dye, Nile red, to record dynamic changes in dorsal column axons and their myelin sheaths respectively. We selectively released Ca2+ from internal stores using the Ca2+ ionophore ionomycin in the presence or absence of external Ca2+. We reported that ionomycin dose-dependently induces pathological changes in myelin and pronounced axonal spheroid formation in the presence of normal 2 mM Ca2+ artificial cerebrospinal fluid. In contrast, removal of external Ca2+ significantly decreased ionomycin-induced myelin and axonal spheroid formation at 2 hours but not at 1 hour after treatment. Using mice that express a neuron-specific Ca2+ indicator in spinal cord axons, we confirmed that ionomycin induced significant increases in intra-axonal Ca2+, but not in the absence of external Ca2+. Periaxonal swelling and the resultant disruption in the axo-myelinic interface often precedes and is negatively correlated with axonal spheroid formation. Pretreatment with YM58483 (500 nM), a well-established blocker of store-operated Ca2+ entry, significantly decreased myelin injury and axonal spheroid formation. Collectively, these data reveal that ionomycin-induced depletion of internal Ca2+ stores and subsequent external Ca2+ entry through store-operated Ca2+ entry contributes to pathological changes in myelin and axonal spheroid formation, providing new targets to protect central myelinated fibers.
RESUMO
Most spinal cord injury (SCI) research programs focus only on the injured spinal cord with the goal of restoring locomotor function by overcoming mechanisms of cell death or axon regeneration failure. Given the importance of the spinal cord as a locomotor control center and the public perception that paralysis is the defining feature of SCI, this "spinal-centric" focus is logical. Unfortunately, such a focus likely will not yield new discoveries that reverse other devastating consequences of SCI including cardiovascular and metabolic disease, bladder/bowel dysfunction and infection. The current review considers how SCI changes the physiological interplay between the spinal cord, the gut and the immune system. A suspected culprit in causing many of the pathological manifestations of impaired spinal cord-gut-immune axis homeostasis is the gut microbiota. After SCI, the composition of the gut microbiota changes, creating a chronic state of gut "dysbiosis". To date, much of what we know about gut dysbiosis was learned from 16S-based taxonomic profiling studies that reveal changes in the composition and abundance of various bacteria. However, this approach has limitations and creates taxonomic "blindspots". Notably, only bacteria can be analyzed. Thus, in this review we also discuss how the application of emerging sequencing technologies can improve our understanding of how the broader ecosystem in the gut is affected by SCI. Specifically, metagenomics will provide researchers with a more comprehensive look at post-injury changes in the gut virome (and mycome). Metagenomics also allows changes in microbe population dynamics to be linked to specific microbial functions that can affect the development and progression of metabolic disease, immune dysfunction and affective disorders after SCI. As these new tools become more readily available and used across the research community, the development of an "ecogenomic" toolbox will facilitate an Eco-Systems Biology approach to study the complex interplay along the spinal cord-gut-immune axis after SCI.