V2a neurons restore diaphragm function in mice following spinal cord injury.

The specific roles that different types of neurons play in recovery from injury is poorly understood. Here, we show that increasing the excitability of ipsilaterally projecting, excitatory V2a neurons using designer receptors exclusively activated by designer drugs (DREADDs) restores rhythmic bursting activity to a previously paralyzed diaphragm within hours, days, or weeks following a C2 hemisection injury. Further, decreasing the excitability of V2a neurons impairs tonic diaphragm activity after injury as well as activation of inspiratory activity by chemosensory stimulation, but does not impact breathing at rest in healthy animals. By examining the patterns of muscle activity produced by modulating the excitability of V2a neurons, we provide evidence that V2a neurons supply tonic drive to phrenic circuits rather than increase rhythmic inspiratory drive at the level of the brainstem. Our results demonstrate that the V2a class of neurons contribute to recovery of respiratory function following injury. We propose that altering V2a excitability is a potential strategy to prevent respiratory motor failure and promote recovery of breathing following spinal cord injury.

Developmental stage of transplanted neural progenitor cells influences anatomical and functional outcomes after spinal cord injury in mice.

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for replacing lost neurons following spinal cord injury (SCI). However, how graft cellular composition influences regeneration and synaptogenesis of host axon populations, or recovery of motor and sensory functions after SCI, is poorly understood. We transplanted developmentally-restricted spinal cord NPCs, isolated from E11.5-E13.5 mouse embryos, into sites of adult mouse SCI and analyzed graft axon outgrowth, cellular composition, host axon regeneration, and behavior. Earlier-stage grafts exhibited greater axon outgrowth, enrichment for ventral spinal cord interneurons and Group-Z spinal interneurons, and enhanced host 5-HT+ axon regeneration. Later-stage grafts were enriched for late-born dorsal horn interneuronal subtypes and Group-N spinal interneurons, supported more extensive host CGRP+ axon ingrowth, and exacerbated thermal hypersensitivity. Locomotor function was not affected by any type of NPC graft. These findings showcase the role of spinal cord graft cellular composition in determining anatomical and functional outcomes following SCI.

Hydrophobic and Hydrogel-Based Methods for Passive Tissue Clearing.

Optical tissue clearing enables the precise imaging of cellular and subcellular structures in whole organs and tissues without the need for physical tissue sectioning. By combining tissue clearing with confocal or lightsheet microscopy, 3D images can be generated of entire specimens for visualization and large-scale data analysis. Here we demonstrate two different passive tissue clearing techniques that are compatible with immunofluorescent staining and lightsheet microscopy: PACT, an aqueous hydrogel-based clearing method, and iDISCO+, an organic solvent-based clearing method.

Navigating the Light-Sheet Image Analysis Software Landscape: Concepts for Driving Cohesion From Data Acquisition to Analysis.

From the combined perspective of biologists, microscope instrumentation developers, imaging core facility scientists, and high performance computing experts, we discuss the challenges faced when selecting imaging and analysis tools in the field of light-sheet microscopy. Our goal is to provide a contextual framework of basic computing concepts that cell and developmental biologists can refer to when mapping the peculiarities of different light-sheet data to specific existing computing environments and image analysis pipelines. We provide our perspective on efficient processes for tool selection and review current hardware and software commonly used in light-sheet image analysis, as well as discuss what ideal tools for the future may look like.

Passive Clearing and 3D Lightsheet Imaging of the Intact and Injured Spinal Cord in Mice.

The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here, we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.

Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss.

The muscular dystrophy X-linked mouse (mdx) is the most commonly used preclinical model for Duchenne muscular dystrophy. Although disease progression in the mouse does not perfectly model the human disease, it shares many pathological features. Early characterizations of the model reported severe pathology through early adulthood followed by disease stabilization. As a result, research in the mdx mouse has largely focused on early adulthood. The overarching goal of this study is to improve the understanding of the mdx mouse model by tracking pathological features of the disease throughout life. We performed a thorough characterization of myofiber pathology in mdx mice from 2 weeks to 2 years of age. We report that individual mdx muscle fibers undergo progressive hypertrophy that continues through the lifespan. Despite massive hypertrophy on the myofiber level, we report no hypertrophy on the muscle level. These seemingly contradictory findings are explained by previously underappreciated myofiber loss in mdx mice. We conclude that due to myofiber loss, in combination with the progressive nature of other pathological features, aged mdx muscle tissue provides reliable benchmarks for disease progression that may be valuable in testing the efficacy of therapeutics for Duchenne muscular dystrophy.