3D culture of the spinal cord with roots as an ex vivo model for comparative studies of motor and sensory nerve regeneration.

Motor and sensory nerves exhibit tissue-specific structural and functional features. However, in vitro models designed to reflect tissue-specific differences between motor and sensory nerve regeneration have rarely been reported. Here, by embedding the spinal cord with roots (SCWR) in a 3D hydrogel environment, we compared the nerve regeneration processes between the ventral and dorsal roots. The 3D hydrogel environment induced an outward migration of neurons in the gray matter of the spinal cord, which allowed the long-term survival of motor neurons. Tuj1 immunofluorescence labeling confirmed the regeneration of neurites from both the ventral and dorsal roots. Next, we detected asymmetric ventral and dorsal root regeneration in response to nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF), and we observed motor and sensory Schwann cell phenotypes in the regenerated ventral and dorsal roots, respectively. Moreover, based on the SCWR model, we identified a targeted effect of collagen VI on sensory nerve fasciculation and characterized the protein expression profiles correlating to motor/sensory-specific nerve regeneration. These results suggest that the SCWR model can serve as a valuable ex vivo model for comparative study of motor and sensory nerve regeneration and for pharmacodynamic evaluations.

Nerve bundle formation during the promotion of peripheral nerve regeneration: collagen VI-neural cell adhesion molecule 1 interaction.

The formation of nerve bundles, which is partially regulated by neural cell adhesion molecule 1 (NCAM1), is important for neural network organization during peripheral nerve regeneration. However, little is known about how the extracellular matrix (ECM) microenvironment affects this process. Here, we seeded dorsal root ganglion tissue blocks on different ECM substrates of peripheral nerve ECM-derived matrix-gel, Matrigel, laminin 521, collagen I, and collagen IV, and observed well-aligned axon bundles growing in the peripheral nerve ECM-derived environment. We confirmed that NCAM1 is necessary but not sufficient to trigger this phenomenon. A protein interaction assay identified collagen VI as an extracellular partner of NCAM1 in the regulation of axonal fasciculation. Collagen VI interacted with NCAM1 by directly binding to the FNIII domain, thereby increasing the stability of NCAM1 at the axolemma. Our in vivo experiments on a rat sciatic nerve defect model also demonstrated orderly nerve bundle regeneration with improved projection accuracy and functional recovery after treatment with 10 mg/mL Matrigel and 20 µg/mL collagen VI. These findings suggest that the collagen VI-NCAM1 pathway plays a regulatory role in nerve bundle formation. This study was approved by the Animal Ethics Committee of Guangzhou Medical University (approval No. GY2019048) on April 30, 2019.

Recombinant COL6 α2 as a Self-Organization Factor That Triggers Orderly Nerve Regeneration Without Guidance Cues.

Collagen VI (COL6) in the microenvironment was recently identified as an extracellular signal that bears the function of promoting orderly axon bundle formation. However, the large molecular weight of COL6 (≈2,000 kDa) limits its production and clinical application. It remains unclear whether the smaller subunit α chains of COL6 can exert axon bundling and ordering effects independently. Herein, based on a dorsal root ganglion (DRG) ex vivo model, the contributions of three main COL6 α chains on orderly nerve bundle formation were analyzed, and COL6 α2 showed the largest contribution weight. A recombinant COL6 α2 chain was produced and demonstrated to promote the formation of orderly axon bundles through the NCAM1-mediated pathway. The addition of COL6 α2 in conventional hydrogel triggered orderly nerve regeneration in a rat sciatic nerve defect model. Immunogenicity assessment showed weaker immunogenicity of COL6 α2 compared to that of the COL6 complex. These findings suggest that recombinant COL6 α2 is a promising material for orderly nerve regeneration.

Decellularization optimizes the inhibitory microenvironment of the optic nerve to support neurite growth.

Allogeneic or homologous tissue transplantation is an effective strategy to repair tissue injury. However, the central nervous tissues like the brain, spinal cord, and optic nerve are not ideal materials for nervous tissue regeneration due to the excessive axonal inhibitor cues in their microenvironments. In the present study, we found that decellularization optimizes the function of the adult optic nerve in supporting the oriented outgrowth of dorsal root ganglion (DRG) neurites. The neurites growing on the decellularized optic nerve (DON) showed longer extension distances than those growing on the normal optic nerve (ON). Neurite branching was also significantly increased on the DON compared to on the ON. Decellularization selectively removed some axon-inhibitory molecules such as myelin-associated glycoprotein (basically not detected in DON) and chondroitin sulfate proteoglycans (detected in DON at a level less than 0.3 fold that in ON) and preserved some axon-promoted extracellular matrix (ECM) proteins, including collagen IV and laminin (detected at levels 6.0-fold higher in DON than in ON). Furthermore, collagen IV and laminin were shown to be preserved in DON, and their binding activities with integrin α1 were retained to promote the extension of DRG neurites. Together, the findings provide a feasible way to optimize the axon-inhibited microenvironment of central nervous tissues and establish a theoretical basis for the application of DON scaffolds in repairing central nervous injury.

Covering the proximal nerve stump with chondroitin sulfate proteoglycans prevents traumatic painful neuroma formation by blocking axon regeneration after neurotomy in Sprague Dawley rats.

OBJECTIVE: Neuropathic pain caused by traumatic neuromas is an extremely intractable clinical problem. Disorderly scar tissue accumulation and irregular and immature axon regeneration around the injury site mainly contribute to traumatic painful neuroma formation. Therefore, successfully preventing traumatic painful neuroma formation requires the effective inhibition of irregular axon regeneration and disorderly accumulation of scar tissue. Considering that chondroitin sulfate proteoglycans (CSPGs) can act on the growth cone and effectively inhibit axon regeneration, the authors designed and manufactured a CSPG-gelatin blocker to regulate the CSPGs' spatial distribution artificially and applied it in a rat model after sciatic nerve neurectomy to evaluate its effects in preventing traumatic painful neuroma formation. METHODS: Sixty female Sprague Dawley rats were randomly divided into three groups (positive group: no covering; blank group: covering with gelatin blocker; and CSPG group: covering with the CSPG-gelatin blocker). Pain-related factors were evaluated 2 and 8 weeks postoperatively (n = 30). Neuroma growth, autotomy behavior, and histological features of the neuromas were assessed 8 weeks postoperatively (n = 30). RESULTS: Eight weeks postoperatively, typical bulb-shaped neuromas did not form in the CSPG group, and autotomy behavior was obviously better in the CSPG group (p < 0.01) than in the other two groups. Also, in the CSPG group the regenerated axons showed a lower density and more regular and improved myelination (p < 0.01). Additionally, the distribution and density of collagenous fibers and the expression of α-smooth muscle actin were significantly lower in the CSPG group than in the positive group (p < 0.01). Regarding pain-related factors, c-fos, substance P, interleukin (IL)-17, and IL-1ß levels were significantly lower in the CSPG group than those in the positive and blank groups 2 weeks postoperatively (p < 0.05), while substance P and IL-17 remained lower in the CSPG group 8 weeks postoperatively (p < 0.05). CONCLUSIONS: The authors found that CSPGs loaded in a gelatin blocker can prevent traumatic neuroma formation and effectively relieve pain symptoms after sciatic nerve neurotomy by blocking irregular axon regeneration and disorderly collagenous fiber accumulation in the proximal nerve stump. These results indicate that covering the proximal nerve stump with CSPGs may be a new and promising strategy to prevent traumatic painful neuroma formation in the clinical setting.

Spatial distribution affects the role of CSPGs in nerve regeneration via the actin filament-mediated pathway.

CSPGs are components of the extracellular matrix in the nervous system, where they serve as cues for axon guidance during development. After a peripheral nerve injury, CSPGs switch roles and become axon inhibitors and become diffusely distributed at the injury site. To investigate whether the spatial distribution of CSPGs affects their role, we combined in vitro DRG cultures with CSPG stripe or coverage assays to simulate the effect of a patterned substrate or dispersive distribution of CSPGs on growing neurites. We observed neurite steering at linear CSPG interfaces and neurite inhibition when diffused CSPGs covered the distal but not the proximal segment of the neurite. The repellent and inhibitory effects of CSPGs on neurite outgrowth were associated with the disappearance of focal actin filaments on growth cones. The application of an actin polymerization inducer, jasplakinolide, allowed neurites to break through the CSPG boundary and grow on CSPG-coated surfaces. The results of our study collectively reveal a novel mechanism that explains how the spatial distribution of CSPGs determines whether they act as a cue for axon guidance or as an axon-inhibiting factor. Increasing our understanding of this issue may promote the development of novel therapeutic strategies that regulate the spatial distributions of CSPGs to use them as an axon guidance cue.