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.

Spleen tyrosine kinase facilitates neutrophil activation and worsens long-term neurologic deficits after spinal cord injury.

BACKGROUND: Spinal cord injury elicits widespread inflammation that can exacerbate long-term neurologic deficits. Neutrophils are the most abundant immune cell type to invade the spinal cord in the early acute phase after injury, however, their role in secondary pathogenesis and functional recovery remains unclear. We have previously shown that neutrophil functional responses during inflammation are augmented by spleen tyrosine kinase, Syk, a prominent intracellular signaling enzyme. In this study, we evaluated the contribution of Syk towards neutrophil function and long-term neurologic deficits after spinal cord injury. METHODS: Contusive spinal cord injury was performed at thoracic vertebra level 9 in mice with conditional deletion of Syk in neutrophils (Sykf/fMRP8-Cre). Hindlimb locomotor recovery was evaluated using an open-field test for 35 days following spinal cord injury. Long-term white matter sparing was assessed using eriochrome cyanide staining. Blood-spinal cord barrier disruption was evaluated by immunoblotting. Neutrophil infiltration, activation, effector functions, and cell death were determined by flow cytometry. Cytokine and chemokine expression in neutrophils was assessed using a gene array. RESULTS: Syk deficiency in neutrophils improved long-term functional recovery after spinal cord injury, but did not promote long-term white matter sparing. Neutrophil activation, cytokine expression, and cell death in the acutely injured spinal cord were attenuated by the genetic loss of Syk while neutrophil infiltration and effector functions were not affected. Acute blood-spinal cord barrier disruption was also unaffected by Syk deficiency in neutrophils. CONCLUSIONS: Syk facilitates specific neutrophil functional responses to spinal cord injury including activation, cytokine expression, and cell death. Long-term neurologic deficits are exacerbated by Syk signaling in neutrophils independent of acute blood-spinal cord barrier disruption and long-term white matter sparing. These findings implicate Syk in pathogenic neutrophil activities that worsen long-term functional recovery after spinal cord injury.

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.