Schwann Cell Migration Research Articles

MiR-140 and miR-200 regulate the migratory heterogeneity of location-specific Schwann cell population.

Schwann cells are functional cells in nerve regeneration and are commonly used as seed cells in tissue engineering. Enhanced Schwann cell migration capacity improves recovery effects, and thus, the identification of Schwann cells with greater migration ability is of great importance. In the present study, we examined the biological activities of Schwann cells collected from rat sciatic nerves (SN) and dorsal root ganglia (DRG). Observations from transwell migration assay and wound healing assay demonstrate that DRG Schwann cells migrate at a faster speed as compared with SN Schwann cells. Sequencing and bioinformatics suggest that differentially expressed genes between SN and DRG Schwann cells are associated with cell motility and migration. miR-140 and miR-200, two microRNAs (miRNAs) that are highly expressed in SN Schwann cells negatively influence Schwann cell migration and thus may be key regulators of Schwann cell phenotype. Igsf10, Plxna2, and Lcp1 are screened as candidate downstream targets of miR-140 and miR-200 based on bioinformatic analysis and their expression correlation with miRNAs. Our comparative analysis displays the unique characteristics of Schwann cells in different anatomical localizations and demonstrates that DRG Schwann cells are suitable seed cells for tissue engineering and regenerative medicine.

Post-hatching developmental changes in the adrenal gland of the Japanese quail (Coturnix coturnix japonica): Histological, immunohistochemical, and electron microscopic studies.

The adrenal glands are paired abdominal endocrine organs vital to the bird's health. The present research aimed to provide a comprehensive examination of the histological, ultrastructural, and immunohistochemical investigations of the adrenal gland in Japanese quail during the post hatching period. The current study was performed on 21 healthy Japanese quail chicks at different post hatching periods. Our results showed the adrenal gland is surrounded by a connective tissue capsule, which consists of dense collagen fibers containing large blood vessels, chromaffin cells, autonomic ganglia, fibroblasts, and migrating Schwann cells. The zonation of the adrenal gland is composed of a subcapsular layer, a peripheral zone, and a central zone, which gets more pronounced with age. At the ultrastructural level, the interrenal cells take the steroid-secreting cells characters; they have varying amounts of lipid droplets and abundant mitochondria. Adrenal medullary chromaffin cells showed positive immunoreactivity to the NSE. With increasing age, the chromaffin tissue's Sox10 immunoreactivity increased. β-catenin is expressed within the plasmalemma and the cytoplasm of the interrenal and chromaffin cells and its reactivity increased with age, especially in the chromaffin cells. Our results indicate the adrenal gland undergoes significant morphological changes during the postnatal life. Overall, the postnatal period is an important time for the development and maturation of the adrenal glands.

Branched-chain aminotransferase 1 promotes Schwann cell migration and proliferation to accelerate facial nerve regeneration through the Twist/FoxC1 and Sox2 pathways

Facial paralysis caused by injury to the facial nerve is common clinical presentation resulting in significant physical and psychological damage. In addition, due to the lack of understanding about the mechanisms of injury and repair and the lack of effective treatment targets, the clinical treatment outcomes for such patients remain poor. Schwann cells (SCs) have a central role in the regeneration of nerve myelin. In a rat model of facial nerves crush injury, we found that branched-chain aminotransferase 1 (BCAT1) was upregulated after injury. Moreover, it had a positive role in nerve repair. Using intervention methods such as gene knockdown, overexpression, and protein-specific inhibitors, combined with detection methods such as CCK8, Transwell, EdU, and flow cytometry, we demonstrated that BCAT1 significantly enhanced the migration and proliferation of SCs. It affected SC cell migration by regulating the Twist/Foxc1 signal axis and promoted cell proliferation by directly regulating the expression of SOX2. Similarly, animal experiments demonstrated that BCAT1 promotes facial nerve repair, improving nerve function and myelin regeneration by activating both the Twist/Foxc1 and SOX2 axes. In sum, BCAT1 promotes SC migration and proliferation, suggesting its potential as a key molecular target for improving the outcome of facial nerve injury repairs.

Composite Hydrogel Conduit Incorporated with Platelet-Rich Plasma Improved the Regenerative Microenvironment for Peripheral Nerve Repair.

Peripheral nerve regeneration and functional recovery remain major challenges in clinical practice. Nerve guidance conduits (NGCs) which can regulate the regenerative microenvironment are beneficial for peripheral nerve repair. Platelet-rich plasma (PRP) can secrete multiple growth factors to regulate the regenerative microenvironment. However, current administration methods of PRP are rapidly activated followed by the burst release of growth factors, causing low therapeutic efficiency in vivo. To overcome these disadvantages, a composite nerve conduit was fabricated by incorporating PRP into a gelatin methacrylate (GelMA) and sodium alginate (SA) hydrogel. The GelMA/SA-3/PRP-20 NGCs possess optimal mechanical properties, degradation rate, and superior biological performance. Importantly, GelMA/SA-3/PRP-20 NGCs achieved the sustained release of two major growth factors (VEGF-A, PDGF-BB) from PRP. Moreover, the GelMA/SA-3/PRP-20 NGCs significantly promoted the migration of Schwann cells and the neovascularization of endothelial cells in vitro. While bridging 10 mm rat sciatic nerve defects, the GelMA/SA-3/PRP-20 NGCs promoted axonal regeneration and functional recovery of peripheral nerves. Therefore, the GelMA/SA-3/PRP-20 NGCs could regulate the regenerative microenvironment by sustained release of growth factors from PRP and shed new light on the clinical application of PRP in peripheral nerve repair.

Matrilin-2 within a three-dimensional lysine-modified chitosan porous scaffold enhances Schwann cell migration and axonal outgrowth for peripheral nerve regeneration.

Background: Matrilin-2 is a key extracellular matrix protein involved in peripheral nerve regeneration. We sought to develop a biomimetic scaffold to enhance peripheral nerve regeneration by incorporating matrilin-2 within a chitosan-derived porous scaffold. We hypothesized that the use of such a novel biomaterial delivers microenvironmental cues to facilitate Schwann cell (SC) migration and enhance axonal outgrowth during peripheral nerve regeneration. Materials and Methods: The effect of matrilin-2 on SC migration was evaluated with agarose drop migration assay on matrilin-2 coated dishes. SC adhesion was determined with SCs cultured atop tissue culture dishes coated with matrilin-2. Various formulations of chitosan vs matrilin-2 in scaffold constructs were examined with scanning electron microscopy. The effect of the matrilin-2/chitosan scaffold on SC migration in the collagen conduits was determined by capillary migration assays. Neuronal adhesion and axonal outgrowth were evaluated with three-dimensional (3D) organotypic assay of dorsal root ganglions (DRG). DRG axonal outgrowth within the scaffolds was determined by immunofluorescence staining of neurofilaments. Results: Matrilin-2 induced SC migration and enhanced its adhesion. A formulation of 2% chitosan with matrilin-2 demonstrated an optimal 3D porous architecture for SC interaction. Matrilin-2/chitosan scaffold enabled SCs to migrate against gravity within conduits. Chemical modification of chitosan with lysine (K-chitosan) further improved DRG adhesion and axonal outgrowth than the matrilin-2/chitosan scaffold without lysine modification. Conclusion: We developed a matrilin-2/K-chitosan scaffold to mimic extracellular matrix cues and provide a porous matrix to enhance peripheral nerve regeneration. Taking advantage of matrilin-2's capability to stimulate SC migration and adhesion, we formulated a porous matrilin-2/chitosan scaffold to support axongal outgrowth. Chemical modification of chitosan with lysine further improved matrilin-2 bioactivity in the 3D scaffold. The 3D porous matrilin-2/K-chitosan scaffolds have high potential for enhancing nerve repair by stimulating SC migration, neuronal adhesion, and axonal outgrowth.

Electrohydrodynamic Printing of Microfibrous Architectures with Cell‐Scale Spacing for Improved Cellular Migration and Neurite Outgrowth (Small 19/2023)

Microfibrous Architectures In article number 2207331, Jiankang He and co-workers propose an electrohydrodynamic printing strategy for the fabrication of orderly parallel microfibers with cell-scale spacing. The resultant microfibrous architectures with cell-scale spacing can accelerate the directional outgrowth of neurites and collective migration of Schwann cells to form highly-aligned and densely-organized neurite constructs.

Aligned core–shell fibrous nerve wrap containing Bletilla striata polysaccharide improves functional outcomes of peripheral nerve repair

Peripheral nerve injuries are commonly encountered in extremity traumas. Their motor and sensory recovery following microsurgical repair is limited by slow regeneration speed (<1 mm/d) and subsequent muscle atrophy, which are consequently correlated with the activity of local Schwann cells and efficacy of axon outgrowth. To promote post-surgical nerve regeneration, we synthesized a nerve wrap consisting of an aligned polycaprolactone (PCL) fiber shell with a Bletilla striata polysaccharide (BSP) core (APB). Cell experiments demonstrated that the APB nerve wrap markedly promoted neurite outgrowth and Schwann cell migration and proliferation. Animal experiments applying a rat sciatic nerve repair model indicated that the APB nerve wrap restored conduction efficacy of the repaired nerve and the compound action potential as well as contraction force of the related leg muscles. Histology of the downstream nerves disclosed significantly higher fascicle diameter and myelin thickness with the APB nerve wrap compared to those without BSP. Thus, the BSP-loaded nerve wrap is potentially beneficial for the functional recovery after peripheral nerve repair and offers sustained targeted release of a natural polysaccharide with good bioactivity.

The activation of gastric inhibitory peptide/gastric inhibitory peptide receptor axis via sonic hedgehog signaling promotes the bridging of gapped nerves in sciatic nerve injury.

Schwann cells play an essential role in peripheral nerve regeneration by generating a favorable microenvironment. Gastric inhibitory peptide/gastric inhibitory peptide receptor (GIP/GIPR) axis deficiency leads to failure of sciatic nerve repair. However, the underlying mechanism remains elusive. In this study, we surprisingly found that GIP treatment significantly enhances the migration of Schwann cells and the formation of Schwann cell cords during recovery from sciatic nerve injury in rats. We further revealed that GIP and GIPR levels in Schwann cells were low under normal conditions, and significantly increased after injury demonstrated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Wound healing and Transwell assays showed that GIP stimulation and GIPR silencing could affect Schwann cell migration. In vitro and in vivo mechanistic studies based on interference experiment revealed that GIP/GIPR might promote mechanistic target of rapamycin complex 2 (mTORC2) activity, thus facilitating cell migration; Rap1 activation might be involved in this process. Finally, we retrieved the stimulatory factors responsible for GIPR induction after injury. The results indicate that sonic hedgehog (SHH) is a potential candidate whose expression increased upon injury. Luciferase and chromatin immunoprecipitation (ChIP) assays showed that Gli3, the target transcription factor of the SHH pathway, dramatically augmented GIPR expression. Additionally, in vivo inhibition of SHH could effectively reduce GIPR expression after sciatic nerve injury. Collectively, our study reveals the importance of GIP/GIPR signaling in Schwann cell migration, providing a therapeutic avenue toward peripheral nerve injury.

Schwann cell-specific RhoA knockout accelerates peripheral nerve regeneration via promoting Schwann cell dedifferentiation.

Our previous studies indicated that RhoA knockdown or inhibition could alleviate the proliferation, migration, and differentiation of Schwann cells. However, the role of RhoA in Schwann cells during nerve injury and repair is still unknown. Herein, we developed two lines of Schwann cells conditional RhoA knockout (cKO) mice by breeding RhoAflox / flox mice with PlpCre -ERT2 or DhhCre mice. Our results indicate that RhoA cKO in Schwann cells accelerates axonal regrowth and remyelination after sciatic nerve injury, which enhances the recovery of nerve conduction and hindlimb gait, and alleviates the amyotrophy in gastrocnemius muscle. Mechanistic studies in both in vivo and in vitro models revealed that RhoA cKO could facilitate Schwann cell dedifferentiation via JNK pathway. Schwann cell dedifferentiation subsequently promotes Wallerian degeneration by enhancing phagocytosis and myelinophagy, as well as stimulating the production of neurotrophins (NT-3, NGF, BDNF, and GDNF). These findings shed light on the role of RhoA in Schwann cells during nerve injury and repair, indicating that cell type-specific RhoA targeting could serve as a promising molecular therapeutic strategy for peripheral nerve injury.

Effects of ECM proteins (laminin, fibronectin, and type IV collagen) on the biological behavior of Schwann cells and their roles in the process of remyelination after peripheral nerve injury.

Introduction: It is important to note that complete myelination and formation of myelinated fibers are essential for functional nerve regeneration after peripheral nerve injury (PNI). However, suboptimal myelin regeneration is common and can hinder ideal nerve regeneration. Therefore, it is important to closely monitor and support myelin regeneration in patients with PNI to achieve optimal outcomes. Methods: This study analyzed the effects of three extracellular matrix (ECM) proteins on Schwann cells (SCs) in the nerve regeneration environment, including their adhesion, proliferation, and migration. The study also explored the use of composite sodium alginate hydrogel neural scaffolds with ECM components and investigated the effects of ECM proteins on remyelination following peripheral nerve injury. Results: The results showed that laminin (LN), fibronectin (FN), and collagen Ⅳ (type IV Col) promoted the early adhesion of SCs in 2-dimensional culture but the ratios of early cell adhesion were quite different and the maintenance of cells' morphology by different ECM proteins were significantly different. In transwell experiment, the ability of LN and FN to induce the migration of SCs was obviously higher than that of type IV Col. An vitro co-culture model of SCs and dorsal root ganglia (DRG) neurons showed that LN promoted the transition of SCs to a myelinated state and the maturation of the myelin sheath, and increased the thickness of neurofilaments. Animal experiments showed that LN had superior effects in promoting myelin sheath formation, axon repair, and reaching an ideal G-ratio after injury compared to FN and Col IV. The situation of gastrocnemius atrophy was significantly better in the LN group. Notably, the thickness of the regenerated myelin sheaths in the type IV Col group was the thickest. Conclusion: In this experiment, we analyzed and compared the effects of LN, FN, and type IV Col on the biological behavior of SCs and their effects on remyelination after PNI and further clarified their unique roles in the process of remyelination. Further research is necessary to explore the underlying mechanisms.

Sox2ot /miR-9 /Cthrc1 Promote Proliferation and Migration of Schwann Cells Following Nerve Injury

The effects of traditional treatments for peripheral nerve injury (PNI) are not ideal, which has prompted the identification of new therapeutic strategies. As unique glial cells in the peripheral nervous system, Schwann cells (SCs) play an important role in the repair of PNI. Recent studies have demonstrated that long noncoding RNAs (lncRNAs) are involved in the regulation of nerve repair after PNI. In this study, we used microarray technology to detect mRNA and lncRNA expression profiles at different time points after PNI and identified lncRNA Sox2ot-miR-9-Cthrc1 as a competitive endogenous RNA (ceRNA) for further investigation. Expression of lncRNA Sox2ot was increased after PNI, and overexpression of Sox2ot promoted SCs migration and proliferation. Mechanistic analyses confirmed that Sox2ot can regulate the expression of Cthrc1 through competitive adsorption of miR-9 in SCs, ultimately affecting SCs migration and proliferation. Our findings reveal the key role of lncRNA Sox2ot in nerve regeneration and provide a new direction for PNI treatment.

Enhancing Functional Recovery after Segmental Nerve Defect Using Nerve Allograft Treated with Plasma-Derived Exosome.

Nerve injuries can result in detrimental functional outcomes. Currently, autologous nerve graft offers the best outcome for segmental peripheral nerve injury. Allografts are alternatives, but do not have comparable results. This study evaluated whether plasma-derived exosome can improve nerve regeneration and functional recovery when combined with decellularized nerve allografts. The effect of exosomes on Schwann cell proliferation and migration were evaluated. A rat model of sciatic nerve repair was used to evaluate the effect on nerve regeneration and functional recovery. A fibrin sealant was used as the scaffold for exosome. Eighty-four Lewis rats were divided into autograft, allograft, and allograft with exosome groups. Gene expression of nerve regeneration factors was analyzed on postoperative day 7. At 12 and 16 weeks, rats were subjected to maximum isometric tetanic force and compound muscle action potential. Nerve specimens were then analyzed by means of histology and immunohistochemistry. Exosomes were readily taken up by Schwann cells that resulted in improved Schwann cell viability and migration. The treated allograft group had functional recovery (compound muscle action potential, isometric tetanic force) comparable to that of the autograft group. Similar results were observed in gene expression analysis of nerve regenerating factors. Histologic analysis showed no statistically significant differences between treated allograft and autograft groups in terms of axonal density, fascicular area, and myelin sheath thickness. Plasma-derived exosome treatment of decellularized nerve allograft may provide comparable clinical outcomes to that of an autograft. This can be a promising strategy in the future as an alternative for segmental peripheral nerve repair. Off-the-shelf exosomes may improve recovery in nerve allografts.

Hypoxic culture of umbilical cord mesenchymal stem cell-derived sEVs prompts peripheral nerve injury repair.

Repair and regeneration of the peripheral nerve are important for the treatment of peripheral nerve injury (PNI) caused by mechanical tears, external compression injuries and traction injuries. Pharmacological treatment can promote the proliferation of fibroblasts and Schwann cells (SCs), which longitudinally fill the endoneurial canal and form Bungner's band, helping the repair of peripheral nerves. Therefore, the development of new drugs for the treatment of PNI has become a top priority in recent years. Here, we report that small extracellular vesicles (sEVs) produced from umbilical cord mesenchymal stem cells (MSC-sEVs) cultured under hypoxia promote repair and regeneration of the peripheral nerve in PNI and may be a new therapeutic drug candidate. The results showed that the amount of secreted sEVs was significantly increased in UC-MSCs compared with control cells after 48 h of culture at 3% oxygen partial pressure in a serum-free culture system. The identified MSC-sEVs could be taken up by SCs in vitro, promoting the growth and migration of SCs. In a spared nerve injury (SNI) mouse model, MSC-sEVs accelerated the recruitment of SCs at the site of PNI and promoted peripheral nerve repair and regeneration. Notably, repair and regeneration in the SNI mouse model were enhanced by treatment with hypoxic cultured UC-MSC-derived sEVs. Therefore, we conclude that hypoxic cultured UC-MSC-derived sEVs may be a promising candidate drug for repair and regeneration in PNI.

Crystallinity and Stiffness of Spider Silk Govern the Migration of Schwann Cells

Introduction: Nerve guidance conduits are a promising alternative to autologous nerve transplantation and conduits filled with fibrous materials have shown great potential in bridging nerve gaps. The application of spider silk as a filament material has led to results comparable to nerve autograft.1 However, the use of spider silk has been phenomenological so far and the reasons for its success are still not identified. The aim of this study is to elucidate the material properties of spider silk leading to its unique medical performance. This knowledge enables a targeted production of synthetic alternatives such as recombinant silk. Materials and Methods: In this work, various silk types were investigated.2 Schwann cells were seeded on the spider silks and monitored by live cell imaging and immunofluorescent staining. In addition, the silk fibers were inspected with an electron microscopy, Raman spectroscopy wide angle X-ray scattering, and tensile tests to determine morphology, secondary protein structure, crystallinity and stiffness, respectively. Results: It was found that the secondary protein structure and specifically the high content of β-sheets in the direction of silk’s axis gives the fiber its ability to act as a guiding element for Schwann cells. In addition, the results show that the crystallinity of the silk does not affect the proliferation of Schwann cells, while the directed movement of cells is affected by this material property. Conclusion: This direct comparison demonstrated the crucial role of the secondary protein structures and crystallinity of spider silk for the guidance properties of fibers during nerve regeneration. The crystalline structure of silk affects its stiffness, which in turn influences the directed migration of Schwann cells. These results should be considered during the targeted production of synthetic fibrous materials for nerve regeneration. 1. Kornfeld T, Nessler J, Helmer C, et al. Spider silk nerve graft promotes axonal regeneration on long distance nerve defect in a sheep model. Biomaterials 2021;271:120692. 2. Naghilou A, Pöttschacher L, Millesi F, et al. Correlating the secondary protein structure of natural spider silk with its guiding properties for Schwann cells. Mater Sci Eng C. 2020;116:111219.

A Fibrous and Porous Nature of Hydrogels Promotes Directed Migration of Schwann Cells

Introduction: Hydrogel fillings have been successful in enhancing artificial conduits for nerve injuries, but the ideal hydrogel is yet to be found. Its discovery is impeded by an overflow of available products as well as a lack of in vitro experiments that fully analyze the effects of these hydrogels on cells crucial for nerve regeneration. Materials and Methods: This study investigated various hydrogels advertised for peripheral nerve regeneration. Schwann cells were seeded on the hydrogels and their morphology, viability, proliferation and migration were examined in vitro. Results: Our results demonstrated that the basement membrane extract hydrogel Cultrex® 3D Culture Matrix® (Trevigen) promoted elongated morphology and directed migration in SCs. To elucidate the possible reasons behind this, detailed material characterizations of the hydrogels were conducted and identified highly bundled networks of fibres in Cultrex® 3D Culture Matrix® as well as strain-stiffening. In a next step, we further analyzed two more hydrogels consisting of solely one component of Cultrex® 3D Culture Matrix® respectively namely laminin and collagen and found that the SCs also migrated more directed in the laminin hydrogel but not in the collagen hydrogel. Interestingly, out of the two hydrogels only the laminin hydrogel showed a similar fibrous surface roughness to Cultrex® 3D Culture Matrix® and also exhibited strain-stiffening. This indicates a correlation between a fibrous nature of hydrogels, strain-stiffening and directed migration in Schwann cells. Conclusion: These experiments provide a unique systematic assessment of various hydrogels in respect to their material properties and their effect on cells thereby paving the path for the future manufacturing of tailored biomaterials.

LncRNA HAGLR promotes the proliferation, migration, and neurotrophic factor production of Schwann cells via miR-204/CDK5R1 after sciatic nerve injury.

Peripheral nerve injury induces motor and sensory defects and has serious impacts on patients' quality of life. Schwann cells (SCs) are the major glial cells in the peripheral nervous system and play important roles in the repair and regeneration of peripheral nerves. Long noncoding RNA HAGLR has been reported to be highly expressed in neurons and to promote neuronal differentiation but its expression decreases after nerve injury, suggesting that HAGLR may be involved in the process of nerve injury repair. This study aimed to investigate the role and mechanism of HAGLR in neural repair functions of SCs. We found that HAGLR promoted SC proliferation and migration and facilitated the secretion of neurotrophic factors. Furthermore, HAGLR functions as a competing endogenous RNA to regulate CDK5R1 expression via sponging miR-204. Overexpression of miR-204 or silencing of CDK5R1 partially abolished the promoting effect of HAGLR on SCs. Moreover, overexpression of HAGLR promoted the functional recovery of sciatic nerve crush (SNC) model rats. In summary, HAGLR promoted SC proliferation, migration, neurotrophic factor production, and facilitated functional recovery of SNC rats via miR-204/CDK5R1. Therefore, it may provide a potential therapeutic target for peripheral nerve repair and regeneration.

Aligned Polyhydroxyalkanoate Blend Electrospun Fibers as Intraluminal Guidance Scaffolds for Peripheral Nerve Repair.

The use of nerve guidance conduits (NGCs) to treat peripheral nerve injuries is a favorable approach to the current "gold standard" of autografting. However, as simple hollow tubes, they lack specific topographical and mechanical guidance cues present in nerve grafts and therefore are not suitable for treating large gap injuries (30-50 mm). The incorporation of intraluminal guidance scaffolds, such as aligned fibers, has been shown to increase neuronal cell neurite outgrowth and Schwann cell migration distances. A novel blend of PHAs, P(3HO)/P(3HB) (50:50), was investigated for its potential as an intraluminal aligned fiber guidance scaffold. Aligned fibers of 5 and 8 μm diameter were manufactured by electrospinning and characterized using SEM. Fibers were investigated for their effect on neuronal cell differentiation, Schwann cell phenotype, and cell viability in vitro. Overall, P(3HO)/P(3HB) (50:50) fibers supported higher neuronal and Schwann cell adhesion compared to PCL fibers. The 5 μm PHA blend fibers also supported significantly higher DRG neurite outgrowth and Schwann cell migration distance using a 3D ex vivo nerve injury model.

Low-intensity pulsed ultrasound ameliorates erectile dysfunction induced by bilateral cavernous nerve injury through enhancing Schwann cell-mediated cavernous nerve regeneration.

Cavernous nerve injury-induced erectile dysfunction caused by pelvic surgery or trauma is refractory to conventional medications and required an alternative treatment. Low-intensity pulsed ultrasound is a noninvasive mechanical therapy that promotes nerve regeneration. To investigate the therapeutic effect and potential mechanism of low-intensity pulsed ultrasound in the treatment of neurogenic erectile dysfunction. Thirty rats were randomly divided into the sham-operated group, bilateral cavernous nerve injury group, and bilateral cavernous nerve injury + low-intensity pulsed ultrasound group. The erectile function was assessed 3weeks after daily low-intensity pulsed ultrasound treatment. The penile tissues and cavernous nerve tissues were harvested and subjected to histologic analysis. Primary Schwann cells and explants were extracted from adult rats. The effects of low-intensity pulsed ultrasound on proliferation, migration, and nerve growth factor expression of Schwann cells and axonal elongation were examined in vitro. RNA sequencing and western blot assay were applied to predict and verify the molecular mechanism of low-intensity pulsed ultrasound-induced Schwann cell activation. Our study showed that low-intensity pulsed ultrasound promoted Schwann cells proliferation, migration, and neurotrophic factor nerve growth factor expression. Meanwhile, low-intensity pulsed ultrasound exhibits a stronger ability to enhance Schwann cells-mediated neurite outgrowth of major pelvic ganglion neurons and major pelvic ganglion/cavernous nerve explants in vitro. In vivo experiments demonstrated that the erectile function of the rats in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group was significantly higher than those in the bilateral cavernous nerve injury groups. Moreover, the expression levels of smooth muscle and cavernous endothelium also increased significantly in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group. In addition, we observed the higher density and number of cavernous nerve regenerating axons in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group, indicating that low-intensity pulsed ultrasound promotes axonal regeneration following cavernous nerve injury in vivo. RNA sequencing analysis and bioinformatic analysis suggested that low-intensity pulsed ultrasound might trigger the activation of the PI3K/Akt pathway. Western blot assay confirmed that low-intensity pulsed ultrasound activated Schwann cells through TrkB/Akt/CREB signaling. Low-intensity pulsed ultrasound promoted nerve regeneration and ameliorated erectile function by enhancing Schwann cells proliferation, migration, and neurotrophic factor nerve growth factor expression. The TrkB/Akt/CREB axis is the possible mechanism of low-intensity pulsed ultrasound-mediated Schwann cell activation. Low-intensity pulsed ultrasound-based therapy could be a novel potential treatment strategy for cavernous nerve injury-induced neurogenic erectile dysfunction.

Electrohydrodynamic Printing of Microfibrous Architectures with Cell-Scale Spacing for Improved Cellular Migration and Neurite Outgrowth.

Electrohydrodynamic (EHD) printing provides unparalleled opportunities in fabricating microfibrous architectures to direct cellular orientation. However, it faces great challenges in depositing orderly microfibers with cell-scale spacing due to inherent fiber-fiber electrostatic interactions. Here a finite element method is established to analyze the electrostatic forces induced on the EHD-printed microfibers and the relationship between the fiber diameter and spacing for parallel deposition of EHD-printed microfibers is revealed theoretically and experimentally. It is found that uniform fiber arrangement can be achieved when the fiber spacing is five times larger than the fiber diameter. This finding enables the successful printing of parallel fibrous architectures with a fiber diameter of 4.9 ± 0.1µm and a cell-scale fiber spacing of 25.6 ± 1.9µm. The resultant microfibrous architectures exhibit unique capability to direct cellular alignment and enhance cellular density and migration as the fiber spacing decreases from 100 to 25µm. The EHD-printed parallel microfibers with cell-scale spacing are found to improve the outgrowth length of neurites and accelerate the migration of Schwann cells from Dorsal Root Ganglion spheres, which facilitate the formation of densely-arranged and highly-aligned cellular constructs. The presented method is promising to produce biomimetic microfibrous architectures for functional nerve regeneration.

METTL3-Mediated N6-Methyladenosine Modification of lncRNA D26496 Suppresses the Proliferation and Migration of Schwann Cells after Sciatic Nerve Injury.

Previous reports showed that LncRNA D26496 was downregulated and N6-methyladenosine (m6A) methyltransferase METTL3 was upregulated in sciatic nerve injury (SNI). YTH-Domain Family Member 2 (YTHDF2) regulated RNA degradation through recognizing m6A sites. However, whether METTL3-mediated m6A of D26496 plays a role in development of SNI is unknown. Therefore, in this study, we established a rat SNI model and a H2O2-induced Schwann cell injury model to investigate the role of D26496 in modulating SNI and how the expression of D26496 was regulated during this process. D26496 expression was downregulated in both models. Rats with SNI displayed severe oxidative stress, manifested as increased MDA production and decreased SOD and GSH activity. Moreover, overexpression of D26496 alleviated H2O2-induced Schwann cell injury likely by promoting cell proliferation and migration and suppressing cell apoptosis and oxidative stress. Mechanism studies found that METTL3 expression was upregulated after SNI, and silencing METTL3 reduced the D26496 m6A level, but upregulated D26496 expression. Subsequent studies found that YTHDF2 was upregulated after SNI, and abundant m6A modified D26496 in the precipitated protein-RNA complexes by anti-YTHDF2 antibody, whereas silencing YTHDF2 promoted D26496 expression but had no effect on m6A levels of D29496. Silencing D26496 reversed the protective effect of knocking down METTL3 or knocking down YTHDF2 on H2O2-induced cell damage. In vivo, D26496 overexpression alleviated SNI-induced neuropathic pain and oxidative stress. In conclusion, our results suggested that D26496 m6A modification mediated by METTL3 and recognition of D26496 m6A sites by YTHDF2 induced D26496 degradation, thereby participating in the progression of SNI.