Tissue optical clearing techniques have provided important tools for large‐volume imaging. Aqueous‐based clearing methods are known for good fluorescence preservation and scalable size maintenance, but are limited by long incubation time, insufficient clearing performance, or requirements for specialized devices. Additionally, few clearing methods are compatible with widely used lipophilic dyes while maintaining high clearing performance. Here, to address these issues, m‐xylylenediamine (MXDA) is firstly introduced into tissue clearing and used to develop a rapid, highly efficient aqueous clearing method with robust lipophilic dyes compatibility, termed MXDA‐based Aqueous Clearing System (MACS). MACS can render whole adult brains highly transparent within 2.5 days and is also applicable for other intact organs. Meanwhile, MACS possesses ideal compatibility with multiple probes, especially for lipophilic dyes. MACS achieves 3D imaging of the intact neural structures labeled by various techniques. Combining MACS with DiI labeling, MACS allows reconstruction of the detailed vascular structures of various organs and generates 3D pathology of glomeruli tufts in healthy and diabetic kidneys. Therefore, MACS provides a useful method for 3D mapping of intact tissues and is expected to facilitate morphological, physiological, and pathological studies of various organs.

Acute spinal cord injury (SCI) induces secondary hemorrhage and initial blood-spinal cord barrier (BSCB) disruption. The transient receptor potential melastatin 4 (Trpm4) together with sulfonylurea receptor 1 (Sur1) forms the Sur1-Trpm4 channel complex. The up-regulation of Sur1-Trpm4 after injury plays a crucial role in secondary hemorrhage, which is the most destructive mechanism in secondary injuries of the central nervous system (CNS). The matrix metalloprotease (MMP)-mediated disruption of the BSCB leads to an inflammatory response, neurotoxin production and neuronal cell apoptosis. Thus, preventing secondary hemorrhage and BSCB disruption should be an important goal of therapeutic interventions in SCI.Methods: Using a moderate contusion injury model at T10 of the spinal cord, flufenamic acid (FFA) was injected intraperitoneally 1 h after SCI and then continuously once per day for one week.Results: Trpm4 expression is highly up-regulated in capillaries 1 d after SCI. Treatment with flufenamic acid (FFA) inhibited Trpm4 expression, secondary hemorrhage, and capillary fragmentation and promoted angiogenesis. In addition, FFA significantly inhibited the expression of MMP-2 and MMP-9 at 1 d after SCI and significantly attenuated BSCB disruption at 1 d and 3 d after injury. Furthermore, we found that FFA decreased the hemorrhage- and BSCB disruption-induced activation of microglia/macrophages and was associated with smaller lesions, decreased cavity formation, better myelin preservation and less reactive gliosis. Finally, FFA protected motor neurons and improved locomotor functions after SCI.Conclusion: This study indicates that FFA improves functional recovery, in part, due to the following reasons: (1) it inhibits the expression of Trpm4 to reduce the secondary hemorrhage; and (2) it inhibits the expression of MMP-2 and MMP-9 to block BSCB disruption. Thus, the results of our study suggest that FFA may represent a potential therapeutic agent for promoting functional recovery.

The developed optical clearing methods show great potential for imaging of large-volume tissues, but these methods present some nonnegligible limitations such as complexity of implementation and long incubation times. In this study, we tried to screen out rapid optical clearing agents by means of molecular dynamical simulation and experimental demonstration. According to the optical clearing potential of sugar and sugar-alcohol,we further evaluated the improvement in the optical clearing efficacy of mouse brain samples, imaging depth, fluorescence preservation, and linear deformation. The results showed that drops of sorbitol, sucrose, and fructose could quickly make the mouse brain sample transparent within 1 to 2 min, and induce about threefold enhancement in imaging depth. The former two could evidently enhance the fluorescence intensity of green fluorescent protein (GFP) and prodium iodide (PI) nuclear dye. Fructose could significantly increase the fluorescence intensity of PI, but slightly decrease the fluorescence intensity of GFP. Even though the three agents caused some shrinkage in samples, the contraction in horizontal and longitudinal directions are almost the same.

Advanced glycation end-products (AGEs) can induce rabbit annulus fibrosus (AF) cell apoptosis and mitochondrial pathway may be involved in AGEs-mediated cell apoptosis. The antioxidant N-acetyl-L-cysteine significantly reversed AGE-induced AF cell apoptosis. This research may provide a theoretical basis for diabetic intervertebral disc degeneration.

Taxol has been clinically approved as an antitumor drug, and it exerts its antitumor effect through the excessive stabilization of microtubules in cancer cells. Recently, moderate microtubule stabilization by Taxol has been shown to efficiently promote neurite regeneration and functional recovery after spinal cord injury (SCI). However, the potential for the clinical translation of Taxol in treating SCI is limited by its side effects and low ability to cross the blood-spinal cord barrier (BSCB). Self-assembled peptide hydrogels have shown potential as drug carriers for the local delivery of therapeutic agents. We therefore hypothesized that the localized delivery of Taxol by a self-assembled peptide scaffold would promote axonal regeneration by stabilizing microtubules during the treatment of SCI. In the present study, the mechanistic functions of the Taxol-releasing system were clarified in vitro and in vivo using immunofluorescence labeling, histology and neurobehavioral analyses. Based on the findings from the in vitro study, Taxol released from a biological functionalized SAP nanofiber scaffold (FGLmx/Taxol) remained active and promoted neurite extension. In this study, we used a weight-drop contusion model to induce SCI at T9. The local delivery of Taxol from FGLmx/Taxol significantly decreased glial scarring and increased the number of nerve fibers compared with the use of FGLmx and 5% glucose. Furthermore, animals administered FGLmx/Taxol exhibited neurite preservation, smaller cavity dimensions, and decreased inflammation and demyelination. Thus, the local delivery of Taxol from FGLmx/Taxol was effective at promoting recovery after SCI and has potential as a new therapeutic strategy for SCI.

Knowledge regarding the relationship between muscles and the corresponding motor neurons would allow therapeutic genes to transport into specific spinal cord segments. Retrograde tracing technique by targeting the motor endplate (MEP), a highly specialized structure that offers direct access to the spinal motor neurons, has been used to elucidate the connectivity between skeletal muscles and the innervating motor neuron pools. However, current injection strategies mainly based on blind injection or the local MEP region might lead to an underestimation of the motor neuron number due to the uneven distribution of MEP in skeletal muscles. In this work, we proposed a novel intramuscular injection strategy based on the 3D distribution of the MEPs in skeletal muscles, applied the 3D intramuscular injection to the gastrocnemius and tibialis anterior for retrograde tracing of the corresponding motor neurons, and compared this with the existing injection strategy. The intramuscular diffusion of the tracer demonstrated that 3D injection could maximize the retrograde transport by ensuring a greater uptake of the tracer by the MEP region. In combination with optical clearing and imaging, we performed 3D mapping and quantification of the labeled motor neurons and confirmed that 3D injection could label more motor neurons than the current injection method. It is expected that 3D intramuscular injection strategy will help elucidate the connective relationship between muscles and motor neurons faithfully and becomes a promising tool in the development of gene therapy strategies for motor neuron diseases.

The development of the neuromuscular system, including muscle growth and intramuscular neural development, in addition to central nervous system maturation, determines motor ability improvement. Motor development occurs asynchronously from cephalic to caudal. However, whether the structural development of different muscles is heterochronic is unclear. Here, based on the characteristics of motor behavior in postnatal mice, we examined the 3D structural features of the neuromuscular system in different muscles by combining tissue clearing with optical imaging techniques. Quantitative analyses of the structural data and related mRNA expression revealed that there was continued myofiber hyperplasia of the forelimb and hindlimb muscles until around postnatal day 3 (P3) and P6, respectively, as well as continued axonal arborization and neuromuscular junction formation until around P3 and P9, respectively; feature alterations of the cervical muscle ended at birth. Such structural heterochrony of muscles in different body parts corresponds to their motor function. Structural data on the neuromuscular system of neonatal muscles provide a 3D perspective in the understanding of the structural status during motor development.

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