Spinal cord injuries (SCI) often lead to persistent neurological dysfunction due to failure in axon regeneration. Unfortunately, currently established treatments, such as direct drug administration, ...do not effectively treat SCI due to rapid drug clearance from our bodies. Here, we introduce a three-dimensional aligned nanofibers-hydrogel scaffold as a bio-functionalized platform to provide sustained non-viral delivery of proteins and nucleic acid therapeutics (small non-coding RNAs), along with synergistic contact guidance for nerve injury treatment. A hemi-incision model at cervical level 5 in the rat spinal cord was chosen to evaluate the efficacy of this scaffold design. Specifically, aligned axon regeneration was observed as early as one week post-injury. In addition, no excessive inflammatory response and scar tissue formation was triggered. Taken together, our results demonstrate the potential of our scaffold for neural tissue engineering applications.
The clearance of damaged myelin sheaths is critical to ensure functional recovery from neural injury. Here we show a previously unidentified role for microvessels and their lining endothelial cells ...in engulfing myelin debris in spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). We demonstrate that IgG opsonization of myelin debris is required for its effective engulfment by endothelial cells and that the autophagy-lysosome pathway is crucial for degradation of engulfed myelin debris. We further show that endothelial cells exert critical functions beyond myelin clearance to promote progression of demyelination disorders by regulating macrophage infiltration, pathologic angiogenesis and fibrosis in both SCI and EAE. Unexpectedly, myelin debris engulfment induces endothelial-to-mesenchymal transition, a process that confers upon endothelial cells the ability to stimulate the endothelial-derived production of fibrotic components. Overall, our study demonstrates that the processing of myelin debris through the autophagy-lysosome pathway promotes inflammation and angiogenesis and may contribute to fibrotic scar formation.
We developed a simple yet powerful technique to visualize neuronal morphology in human brain tissues. By ballistically shooting DiI (1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine ...perchlorate)-coated tungsten particles to randomly label neurons, then clearing tissues with OPTIClear, we demonstrated the tracing of branched dendritic trees and spines in three dimensions. High-resolution imaging revealed dendrites up to 300 μm long and spine necks down to 200 nm across. Quantitative analyses of 1304 dendritic spines showed no decrease in spine density with imaging depth, indicating excellent clearing and tracing. Segmentation and modeling of dendritic spines enabled morphological characterization. This technique enables assumption-free, high-resolution and cost-efficient visualization of neuronal morphology in human tissues. Combined with immunohistochemistry and electron microscopy, it could provide new perspectives for studying human neuroanatomy and pathology.
This method illustrates optical 3D imaging of dendritic spines over a large tissue volume via combination of ballistic lipophilic tracing and tissue clearing with OPTIClear, a detergent-free, nondestructive clearing agent.
Modern clearing techniques for the three-dimensional (3D) visualisation of neural tissue microstructure have been very effective when used on rodent brain but very few studies have utilised them on ...human brain material, mainly due to the inherent difficulties in processing post-mortem tissue. Here we develop a tissue clearing solution, OPTIClear, optimised for fresh and archival human brain tissue, including formalin-fixed paraffin-embedded material. In light of practical challenges with immunostaining in tissue clearing, we adapt the use of cresyl violet for visualisation of neurons in cleared tissue, with the potential for 3D quantification in regions of interest. Furthermore, we use lipophilic tracers for tracing of neuronal processes in post-mortem tissue, enabling the study of the morphology of human dendritic spines in 3D. The development of these different strategies for human tissue clearing has wide applicability and, we hope, will provide a baseline for further technique development.
Newly developed tissue clearing techniques can be used to render intact tissues transparent. When combined with fluorescent labeling technologies and optical sectioning microscopy, this allows ...visualization of fine structure in three dimensions. Gene-transfection techniques have proved very useful in visualizing cellular structures in animal models, but they are not applicable to human brain tissue. Here, we discuss the characteristics of an ideal chemical fluorescent probe for use in brain and other cleared tissues, and offer a comprehensive overview of currently available chemical probes. We describe their working principles and compare their performance with the goal of simplifying probe selection for neuropathologists and stimulating probe development by chemists. We propose several approaches for the development of innovative chemical labeling methods which, when combined with tissue clearing, have the potential to revolutionize how we study the structure and function of the human brain.
Tissue clearing techniques are novel methodologies that turn intact tissues transparent. However, probing these thick tissues has remained a significant challenge. In this article, Lai et al. comprehensively review available chemical labeling approaches and their working principles, facilitating probe selection and development by researchers working with tissue clearing techniques.
Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined ...an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed, we report that the cells survive and differentiate upon transplant into newborn rat brain.
Spinal cord injury (SCI) can cause severe traumatic injury to the central nervous system (CNS). Current therapeutic effects achieved for SCI in clinical medicine show that there is still a long way ...to go to reach the desired goal of full or significant functional recovery. In basic medical research, however, cell transplantation, gene therapy, application of cytokines, and biomaterial scaffolds have been widely used and investigated as treatments for SCI. All of these strategies when used separately would help rebuild, to some extent, the neural circuits in the lesion area of the spinal cord. In light of this, it is generally accepted that a combined treatment may be a more effective strategy. This review focuses primarily on our recent series of work on transplantation of Schwann cells and adult stem cells, and transplantation of stem cell-derived neural network scaffolds with functional synapses. Arising from this, an artificial neural network (an exogenous neuronal relay) has been designed and fabricated by us—a biomaterial scaffold implanted with Schwann cells modified by the neurotrophin-3 (NT-3) gene and adult stem cells modified with the TrkC (receptor of NT-3) gene. More importantly, experimental evidence suggests that the novel artificial network can integrate with the host tissue and serve as an exogenous neuronal relay for signal transfer and functional improvement of SCI.
Abstract Self-assembly peptide nanofibrous scaffold (SAPNS), such as RADA16-I, has been shown to reduce acute brain injury and enhance functional recovery in rat intracerebral hemorrhage (ICH) ...models. The acidic property of RADA16-I, however, limits its application in patients. In the present study, by using a modified neutral SAPNS (the RADA16mix) in collagenase IV induced ICH mice, we detected there were less microglial and apoptotic cells in mice injected with RADA16mix, meanwhile, more cells survived in this group. In addition, behavioral tests indicated that mice treated with RADA16mix showed better functional recovery than RADA16-I. Local delivery of RADA16mix reduces acute brain injury by lowering the number of apoptotic cells, decreasing glial reaction, reducing inflammatory response and, therefore promotes functional recovery. Moreover, new nerve fibers have grown into this new SAPNS, which indicates RADA16mix is able to serve as a bridge for nerve fibers to grow through. From the Clinical Editor Acute brain injury, such as intracerebral hemorrhage is a serious problem. In this work, self-assembly peptide nanofibrous scaffold (SAPNS) were tested in a rat model to aid functional recovery. Several items have been considered, such as histology, brain water content, hematoma volume, cell death and survival, inflammatory response, and nerve fiber growth. The positive data generated should pave the way towards better treatment options.
Current treatment approaches toward spinal cord injuries (SCI) have mainly focused on overcoming the inhibitory microenvironment that surrounds lesion sites. Unfortunately, the mere modulation of the ...cell/tissue microenvironment is often insufficient to achieve desired functional recovery. Therefore, stimulating the intrinsic growth ability of injured neurons becomes crucial. MicroRNAs (miRs) play significant roles during axon regeneration by regulating local protein synthesis at growth cones. However, one challenge of using miRs to treat SCI is the lack of efficient delivery approaches. Here, a 3D fiber‐hydrogel scaffold is introduced which can be directly implanted into a spinal cord transected rat. This 3D scaffold consists of aligned electrospun fibers which provide topographical cues to direct axon regeneration, and collagen matrix which enables a sustained delivery of miRs. Correspondingly, treatment with Axon miRs (i.e., a cocktail of miR‐132/miR‐222/miR‐431) significantly enhances axon regeneration. Moreover, administration of Axon miRs along with anti‐inflammatory drug, methylprednisolone, synergistically enhances functional recovery. Additionally, this combined treatment also decreases the expression of pro‐inflammatory genes and enhance gene expressions related to extracellular matrix deposition. Finally, increased Axon miRs dosage with methylprednisolone, significantly promotes functional recovery and remyelination. Altogether, scaffold‐mediated Axon miR treatment with methylprednisolone is a promising therapeutic approach for SCI.
A novel approach of targeting axon local protein synthesis to promote the intrinsic growth ability of neurons by scaffold‐mediated microRNA delivery is introduced. The biofunctional 3D fiber‐hydrogel scaffold provides topographical cues that direct axon regrowth and a novel microRNA cocktail (miR‐132/miR‐222/miR‐431), which in the presence of methylprednisolone, enhances both nerve regeneration and functional recovery after spinal cord injury.
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