A Novel Lysolecithin Model for Visualizing Damage in vivo in the Larval Zebrafish Spinal Cord.

Background: Lysolecithin is commonly used to induce demyelinating lesions in the spinal cord and corpus callosum of mammalian models. Although these models and clinical patient samples are used to study neurodegenerative diseases, such as multiple sclerosis (MS), they do not allow for direct visualization of disease-related damage in vivo. To overcome this limitation, we created and characterized a focal lysolecithin injection model in zebrafish that allows us to investigate the temporal dynamics underlying lysolecithin-induced damage in vivo. Results: We injected lysolecithin into 4-6 days post-fertilization (dpf) zebrafish larval spinal cords and, coupled with in vivo, time-lapse imaging, observed hallmarks consistent with mammalian models of lysolecithin-induced demyelination, including myelinating glial cell loss, myelin perturbations, axonal sparing, and debris clearance. Conclusion: We have developed and characterized a lysolecithin injection model in zebrafish that allows us to investigate myelin damage in a living, vertebrate organism. This model may be a useful pre-clinical screening tool for investigating the safety and efficacy of novel therapeutic compounds that reduce damage and/or promote repair in neurodegenerative disorders, such as MS.

Transmission electron microscopy of zebrafish spinal motor nerve roots.

BACKGROUND: Spinal motor nerves are essential for relaying information between the central and peripheral nervous systems. Perturbations to cell types that comprise these nerves may impair rapid and efficient transmission of action potentials and alter nerve function. Identifying ultrastructural changes resulting from defects to these cellular components via transmission electron microscopy (TEM) can provide valuable insight into nerve function and disease. However, efficiently locating spinal motor nerves in adult zebrafish for TEM is challenging and time-consuming. Because of this, we developed a protocol that allows us to quickly and precisely locate spinal motor nerve roots in adult zebrafish for TEM processing. RESULTS: Following fixation, a transverse slab of adult zebrafish dissected from the trunk region was mounted in embedding media, sectioned, and secondary fixation with osmium tetroxide performed. Transverse sections containing motor nerves were selected for TEM ultrathin sectioning and imaging. CONCLUSIONS: We developed an efficient protocol for locating spinal motor nerves in adult zebrafish to allow for ultrastructural characterization. Although our work focuses on spinal motor nerves, this protocol may be useful for efficiently identifying other discrete, repeated structures within the developing and mature nervous system that are difficult to find via traditional, whole organism TEM processing. Developmental Dynamics 246:956-962, 2017. © 2017 Wiley Periodicals, Inc.

Perineurial Glial Plasticity and the Role of TGF-ß in the Development of the Blood-Nerve Barrier.

Precisely orchestrated interactions between spinal motor axons and their ensheathing glia are vital for forming and maintaining functional spinal motor nerves. Following perturbations to peripheral myelinating glial cells, centrally derived oligodendrocyte progenitor cells (OPCs) ectopically exit the spinal cord and myelinate peripheral nerves in myelin with CNS characteristics. However, whether remaining peripheral ensheathing glia, such as perineurial glia, properly encase the motor nerve despite this change in glial cell and myelin composition, remains unknown. Using zebrafish mutants in which OPCs migrate out of the spinal cord and myelinate peripheral motor axons, we assayed perineurial glial development, maturation, and response to injury. Surprisingly, in the presence of OPCs, perineurial glia exited the CNS normally. However, aspects of their development, response to injury, and function were altered compared with wildtype larvae. In an effort to better understand the plasticity of perineurial glia in response to myelin perturbations, we identified transforming growth factor-ß1 as a partial mediator of perineurial glial development. Together, these results demonstrate the incredible plasticity of perineurial glia in the presence of myelin perturbations.SIGNIFICANCE STATEMENT Peripheral neuropathies can result from damage or dysregulation of the insulating myelin sheath surrounding spinal motor axons, causing pain, inefficient nerve conduction, and the ectopic migration of oligodendrocyte progenitor cells (OPCs), the resident myelinating glial cell of the CNS, into the periphery. How perineurial glia, the ensheathing cells that form the protective blood-nerve barrier, are impacted by this myelin composition change is unknown. Here, we report that certain aspects of perineurial glial development and injury responses are mostly unaffected in the presence of ectopic OPCs. However, perineurial glial function is disrupted along nerves containing centrally derived myelin, demonstrating that, although perineurial glial cells display plasticity despite myelin perturbations, the blood-nerve barrier is compromised in the presence of ectopic OPCs.

Contact-mediated inhibition between oligodendrocyte progenitor cells and motor exit point glia establishes the spinal cord transition zone.

Rapid conduction of action potentials along motor axons requires that oligodendrocytes and Schwann cells myelinate distinct central and peripheral nervous system (CNS and PNS) domains along the same axon. Despite the importance of this arrangement for nervous system function, the mechanisms that establish and maintain this precise glial segregation at the motor exit point (MEP) transition zone are unknown. Using in vivo time-lapse imaging in zebrafish, we observed that prior to myelination, oligodendrocyte progenitor cells (OPCs) extend processes into the periphery via the MEP and immediately upon contact with spinal motor root glia retract back into the spinal cord. Characterization of the peripheral cell responsible for repelling OPC processes revealed that it was a novel, CNS-derived population of glia we propose calling MEP glia. Ablation of MEP glia resulted in the absence of myelinating glia along spinal motor root axons and an immediate breach of the MEP by OPCs. Taken together, our results identify a novel population of CNS-derived peripheral glia located at the MEP that selectively restrict the migration of OPCs into the periphery via contact-mediated inhibition.

Cold plasma technology: bactericidal effects on Geobacillus stearothermophilus and Bacillus cereus microorganisms.

INTRODUCTION: Cold plasma, also known as Low Temperature Atmospheric Pressure Plasma (LTAPP) is a novel technology consisting of neutral and charged particles, including free radicals, which can be used to destroy or inactivate microorganisms. Research has been conducted regarding the effect of cold plasma on gram-positive bacteria; however, there is limited research regarding its ability to inactivate the spore-formers Geobacillus stearothermophilus and Bacillus cereus. PURPOSE: The purpose of this study was to determine if cold plasma inactivates G. stearothermophilus and B. cereus vegetative cells and spores. METHODS: Nine hundred eighty-one samples were included in this study (762 experimental and 219 controls). Experimental samples were exposed indirectly or directly to cold plasma, before plating and incubating for 16 hours. Control samples were not exposed to cold plasma. The percentage-kill and cell number reductions were calculated from Colony Forming Units (CFU). Data were statistically analyzed at the .05 level using one-way ANOVA, Kruskal Wallis and Tukey's tests. RESULTS: There was a statistically significant difference in the inactivation of G. stearothermophilus vegetative cells receiving indirect and direct exposure (p=0.0001 and p=0.0013, respectively), as well as for B. cereus vegetative cells and spores (p=0.0001 for direct and indirect). There was no statistically significant difference in the inactivation of G. stearothermophilus spores receiving indirect exposure (p=0.7208) or direct exposure (p=0.0835). CONCLUSION: Results demonstrate that cold plasma exposure effectively kills G. stearothermophilus vegetative cells and B. cereus vegetative cells and spores; however, G. stearothermophilus spores were not significantly inactivated.