Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro.

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse Electric Field (nsPEF) is an emerging method applicable in nerve electrical stimulation that has been demonstrated to be effective in stimulating cell proliferation and other biological processes. Aiming to assess whether SCs undergo significant changes under nsPEF and help explore the potential for new peripheral nerve regeneration methods, cultured RSC96 cells were subjected to nsPEF stimulation at 5 kV and 10 kV, followed by continued cultivation for 3-4 days. Subsequently, some relevant factors expressed by SCs were assessed to demonstrate the successful stimulation, including the specific marker protein, neurotrophic factor, transcription factor, and myelination regulator. The representative results showed that nsPEF significantly enhanced the proliferation and migration of SCs and the ability to synthesize relevant factors that contribute positively to the regeneration of peripheral nerves. Simultaneously, lower expression of GFAP indicated the benign prognosis of peripheral nerve injuries. All these outcomes show that nsPEF has great potential as an efficient treatment method for peripheral nerve injuries by stimulating SCs.

Sox10 Activity and the Timing of Schwann Cell Differentiation Are Controlled by a Tle4-Dependent Negative Feedback Loop.

The HMG-domain containing transcription factor Sox10 plays a crucial role in regulating Schwann cell survival and differentiation and is expressed throughout the entire Schwann cell lineage. While its importance in peripheral myelination is well established, little is known about its role in the early stages of Schwann cell development. In a search for direct target genes of Sox10 in Schwann cell precursors, the transcriptional co-repressor Tle4 was identified. At least two regions upstream of the Tle4 gene appear involved in mediating the Sox10-dependent activation. Once induced, Tle4 works in tandem with the bHLH transcriptional repressor Hes1 and exerts a dual inhibitory effect on Sox10 by preventing the Sox10 protein from transcriptionally activating maturation genes and by suppressing Sox10 expression through known enhancers of the gene. This mechanism establishes a regulatory barrier that prevents premature activation of factors involved in differentiation and myelin formation by Sox10 in immature Schwann cells. The identification of Tle4 as a critical downstream target of Sox10 sheds light on the gene regulatory network in the early phases of Schwann cell development. It unravels an elaborate regulatory circuitry that fine-tunes the timing and extent of Schwann cell differentiation and myelin gene expression.

Design and Development of a Polymeric-Based Curcumin Nanoparticle for Drug Delivery Enhancement and Potential Incorporation into Nerve Conduits.

Peripheral nerve injuries (PNI) impact millions of individuals in the United States, prompting thousands of nerve repair procedures annually. Nerve conduits (NC) are commonly utilized to treat nerve injuries under 3 cm but larger gaps still pose a challenge for successful peripheral nerve regeneration (PNR) and functional recovery. This is partly attributed to the absence of bioactive agents such as stem cells or growth factors in FDA-approved conduits due to safety, harvesting, and reproducibility concerns. Therefore, curcumin, a bioactive phytochemical, has emerged as a promising alternative bioactive agent due to its ability to enhance PNR and overcome said challenges. However, its hydrophobicity and rapid degradation in aqueous solutions are considerable limitations. In this work, a nanoscale delivery platform with tannic acid (TA) and polyvinylpyrrolidone (PVP) was developed to encapsulate curcumin for increased colloidal and chemical stability. The curcumin nanoparticles (CurNPs) demonstrate significantly improved stability in water, reduced degradation rates, and controlled release kinetics when compared to free curcumin. Further, cell studies show that the CurNP is biocompatible when introduced to neuronal cells (SH-SY5Y), rat Schwann cells (RSC-S16), and murine macrophages (J774 A.1) at 5 µM, 5 µM, and 10 µM of curcumin, respectively. As a result of these improved physicochemical properties, confocal fluorescence microscopy revealed superior delivery of curcumin into these cells when in the form of CurNPs compared to its free form. A hydrogen peroxide-based oxidative stress study also demonstrated the CurNP's potential to protect J774 A.1 cells against excessive oxidative stress. Overall, this study provides evidence for the suitability of CurNPs to be used as a bioactive agent in NC applications.

Electrical aligned polyurethane nerve guidance conduit modulates macrophage polarization and facilitates immunoregulatory peripheral nerve regeneration.

Biomaterials can modulate the local immune microenvironments to promote peripheral nerve regeneration. Inspired by the spatial orderly distribution and endogenous electric field of nerve fibers, we aimed to investigate the synergistic effects of electrical and topological cues on immune microenvironments of peripheral nerve regeneration. Nerve guidance conduits (NGCs) with aligned electrospun nanofibers were fabricated using a polyurethane copolymer containing a conductive aniline trimer and degradable L-lysine (PUAT). In vitro experiments showed that the aligned PUAT (A-PUAT) membranes promoted the recruitment of macrophages and induced their polarization towards the pro-healing M2 phenotype, which subsequently facilitated the migration and myelination of Schwann cells. Furthermore, NGCs fabricated from A-PUAT increased the proportion of pro-healing macrophages and improved peripheral nerve regeneration in a rat model of sciatic nerve injury. In conclusion, this study demonstrated the potential application of NGCs in peripheral nerve regeneration from an immunomodulatory perspective and revealed A-PUAT as a clinically-actionable strategy for peripheral nerve injury.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Enhancing intraneural revascularization following peripheral nerve injury through hypoxic Schwann-cell-derived exosomes: an insight into endothelial glycolysis.

BACKGROUND: Endothelial cell (EC)-driven intraneural revascularization (INRV) and Schwann cells-derived exosomes (SCs-Exos) both play crucial roles in peripheral nerve injury (PNI). However, the interplay between them remains unclear. We aimed to elucidate the effects and underlying mechanisms of SCs-Exos on INRV following PNI. RESULTS: We found that GW4869 inhibited INRV, as well as that normoxic SCs-Exos (N-SCs-Exos) exhibited significant pro-INRV effects in vivo and in vitro that were potentiated by hypoxic SCs-Exos (H-SCs-Exos). Upregulation of glycolysis emerged as a pivotal factor for INRV after PNI, as evidenced by the observation that 3PO administration, a glycolytic inhibitor, inhibited the INRV process in vivo and in vitro. H-SCs-Exos more significantly enhanced extracellular acidification rate/oxygen consumption rate ratio, lactate production, and glycolytic gene expression while simultaneously suppressing acetyl-CoA production and pyruvate dehydrogenase E1 subunit alpha (PDH-E1α) expression than N-SCs-Exos both in vivo and in vitro. Furthermore, we determined that H-SCs-Exos were more enriched with miR-21-5p than N-SCs-Exos. Knockdown of miR-21-5p significantly attenuated the pro-glycolysis and pro-INRV effects of H-SCs-Exos. Mechanistically, miR-21-5p orchestrated EC metabolism in favor of glycolysis by targeting von Hippel-Lindau/hypoxia-inducible factor-1α and PDH-E1α, thereby enhancing hypoxia-inducible factor-1α-mediated glycolysis and inhibiting PDH-E1α-mediated oxidative phosphorylation. CONCLUSION: This study unveiled a novel intrinsic mechanism of pro-INRV after PNI, providing a promising therapeutic target for post-injury peripheral nerve regeneration and repair.

p62/sequestosome-1 as a severity-reflecting plasma biomarker in Charcot-Marie-Tooth disease type 1A.

Autophagy is a self-degradation system for recycling to maintain homeostasis. p62/sequestosome-1 (p62) is an autophagy receptor that accumulates in neuroglia in neurodegenerative diseases. The objective of this study was to determine the elevation of plasma p62 protein levels in patients with Charcot-Marie-Tooth disease 1A (CMT1A) for its clinical usefulness to assess disease severity. We collected blood samples from 69 CMT1A patients and 59 healthy controls. Plasma concentrations of p62 were analyzed by ELISA, and we compared them with Charcot-Marie-Tooth neuropathy score version 2 (CMTNSv2). A mouse CMT1A model (C22) was employed to determine the source and mechanism of plasma p62 elevation. Plasma p62 was detected in healthy controls with median value of 1978 pg/ml, and the levels were significantly higher in CMT1A (2465 pg/ml, p < 0.001). The elevated plasma p62 levels were correlated with CMTNSv2 (r = 0.621, p < 0.0001), motor nerve conduction velocity (r = - 0.490, p < 0.0001) and disease duration (r = 0.364, p < 0.01). In C22 model, increased p62 expression was observed not only in pathologic Schwann cells but also in plasma. Our findings indicate that plasma p62 measurement could be a valuable tool for evaluating CMT1A severity and Schwann cell pathology.

Biomimetic bone-periosteum scaffold for spatiotemporal regulated innervated bone regeneration and therapy of osteosarcoma.

The complexity of repairing large segment defects and eradicating residual tumor cell puts the osteosarcoma clinical management challenging. Current biomaterial design often overlooks the crucial role of precisely regulating innervation in bone regeneration. Here, we develop a Germanium Selenium (GeSe) co-doped polylactic acid (PLA) nanofiber membrane-coated tricalcium phosphate bioceramic scaffold (TCP-PLA/GeSe) that mimics the bone-periosteum structure. This biomimetic scaffold offers a dual functionality, combining piezoelectric and photothermal conversion capabilities while remaining biodegradable. When subjected to ultrasound irradiation, the US-electric stimulation of TCP-PLA/GeSe enables spatiotemporal control of neurogenic differentiation. This feature supports early innervation during bone formation, promoting early neurogenic differentiation of Schwann cells (SCs) by increasing intracellular Ca2+ and subsequently activating the PI3K-Akt and Ras signaling pathways. The biomimetic scaffold also demonstrates exceptional osteogenic differentiation potential under ultrasound irradiation. In rabbit model of large segment bone defects, the TCP-PLA/GeSe demonstrates promoted osteogenesis and nerve fibre ingrowth. The combined attributes of high photothermal conversion capacity and the sustained release of anti-tumor selenium from the TCP-PLA/GeSe enable the synergistic eradication of osteosarcoma both in vitro and in vivo. This strategy provides new insights on designing advanced biomaterials of repairing large segment bone defect and osteosarcoma.

[Experimental study on promotion of peripheral nerve regeneration by selenium-methylselenocysteine].

Objective: To investigate the feasibility of selenium-methylselenocysteine (SMC) to promote peripheral nerve regeneration and its mechanism of action. Methods: Rat Schwann cells RSC96 cells were randomly divided into 5 groups, which were group A (without any treatment, control group), group B (adding 100 µmol/L H 2O 2), group C (adding 100 µmol/L H 2O 2+100 µmol/L SMC), group D (adding 100 µmol/L H 2O 2+200 µmol/L SMC), group E (adding 100 µmol/L H 2O 2+400 µmol/L SMC); the effect of SMC on cell proliferation was detected by MTT method, and the level of oxidative stress was detected by immunofluorescence for free radicals [reactive oxygen species (ROS)] after determining the appropriate dose group. Thirty-six 4-week-old male Sprague Dawley rats were randomly divided into 3 groups, namely, the sham operation group (Sham group), the sciatic nerve injury group (PNI group), and the SMC treatment group (SMC group), with 12 rats in each group; the rats in the PNI group were fed with food and water normally after modelling operation, and the rats in the SMC group were added 0.75 mg/kg SMC to the drinking water every day. At 4 weeks after operation, the sciatic nerves of rats in each group were sampled for neuroelectrophysiological detection of highest potential of compound muscle action potential (CMAP). The levels of inflammatory factors [interleukin 17 (IL-17), IL-6, IL-10 and oxidative stress factors catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA)] were detected by ELISA assay. The luxol fast blue (LFB) staining was used to observe the myelin density, fluorescence intensity of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) was observed by immunofluorescence staining, and myelin morphology was observed by transmission electron microscopy with measurement of axon diameter. Western blot was used to detect the protein expressions of p38 mitogen-activated protein kinases (p38MAPK), phosphorylated p38MAPK (p-p38MAPK), heme oxygenase 1 (HO-1), and nuclear factor erythroid 2-related factor 2 (Nrf2). Results: MTT assay showed that the addition of SMC significantly promoted the proliferation of RSC96 cells, and the low concentration could achieve an effective effect, so the treatment method of group C was selected for the subsequent experiments; ROS immunofluorescence test showed that group B showed a significant increase in the intensity of ROS fluorescence compared with that of group A, and group C showed a significant decrease in the intensity of ROS fluorescence compared with that of group B ( P<0.05). Neuroelectrophysiological tests showed that the highest potential of CMAP in SMC group was significantly higher than that in PNI and Sham groups ( P<0.05). ELISA assay showed that the levels of IL-6, IL-17, and MDA in PNI group were significantly higher than those in Sham group, and the levels of IL-10, SOD, and CAT were significantly lower; the levels of IL-6, IL-17, and MDA in SMC group were significantly lower than those in PNI group, and the levels of IL-10, SOD, and CAT were significantly higher ( P<0.05). LFB staining and transmission electron microscopy showed that the myelin density and the diameter of axons in the SMC group were significantly higher than those of the PNI group and the Sham group ( P<0.05). Immunofluorescence staining showed that the fluorescence intensity of GFAP and MBP in the SMC group were significantly stronger than those in the PNI group and Sham group ( P<0.05). Western blot showed that the relative expressions of Nrf2 and HO-1 proteins in the SMC group were significantly higher than those in the PNI group and Sham group, and the ratio of p-p38MAPK/p38MAPK proteins was significantly higher in the PNI group than that in the SMC group and Sham group ( P<0.05). Conclusion: SMC may inhibit oxidative stress and inflammation after nerve injury by up-regulating the Nrf2/HO-1 pathway, and then inhibit the phosphorylation of p38MAPK pathway to promote the proliferation of Schwann cells, which ultimately promotes the formation of myelin sheaths and accelerates the regeneration of peripheral nerves.

Exploring miR-21 as a key regulator in two distinct approaches of bone marrow stromal cells differentiation into Schwann-like cells.

The differentiation of bone marrow stromal cells (BMSCs) into Schwann-like cells (SCLCs) has the potential to promote the structural and functional restoration of injured axons. However, the optimal induction protocol and its underlying mechanisms remain unclear. This study aimed to compare the effectiveness of different induction protocols in promoting the differentiation of rat BMSCs into SCLCs and to explore their potential mechanisms. BMSCs were induced using two distinct methods: a composite factor induction approach (Protocol-1) and a conditioned culture medium induction approach (Protocol-2). The expression of Schwann cells (SCs) marker proteins and neurotrophic factors (NTFs) in the differentiated cells was assessed. Cell proliferation and apoptosis were also measured. During induction, changes in miR-21 and Sprouty RTK signaling antagonist 2 (SPRY2) mRNA were analyzed. Following the transfection of BMSCs with miR-21 agomir or miR-21 antagomir, induction was carried out using both protocols, and the expression of SPRY2, ERK1/2, and SCs marker proteins was examined. The results revealed that NTFs expression was higher in Protocol-1, whereas SCs marker proteins expression did not significantly differ between the two groups. Compared to Protocol-1, Protocol-2 exhibited enhanced cell proliferation and fewer apoptotic and necrotic cells. Both protocols showed a negative correlation between miR-21 and SPRY2 expression throughout the induction stages. After induction, the miR-21 agomir group exhibited reduced SPRY2 expression, increased ERK1/2 expression, and significantly elevated expression of SCs marker proteins. This study demonstrates that Protocol-1 yields higher NTFs expression, whereas Protocol-2 results in stronger SCLCs proliferation. Upregulating miR-21 suppresses SPRY2 expression, activates the ERK1/2 signaling pathway, and promotes BMSC differentiation into SCLCs.

Cellular and molecular alterations to muscles and neuromuscular synapses in a mouse model of MEGF10-related myopathy.

Loss-of-function mutations in MEGF10 lead to a rare and understudied neuromuscular disorder known as MEGF10-related myopathy. There are no treatments for the progressive respiratory distress, motor impairment, and structural abnormalities in muscles caused by the loss of MEGF10 function. In this study, we deployed cellular and molecular assays to obtain additional insights about MEGF10-related myopathy in juvenile, young adult, and middle-aged Megf10 knockout (KO) mice. We found fewer muscle fibers in juvenile and adult Megf10 KO mice, supporting published studies that MEGF10 regulates myogenesis by affecting satellite cell differentiation. Interestingly, muscle fibers do not exhibit morphological hallmarks of atrophy in either young adult or middle-aged Megf10 KO mice. We next examined the neuromuscular junction (NMJ), in which MEGF10 has been shown to concentrate postnatally, using light and electron microscopy. We found early and progressive degenerative features at the NMJs of Megf10 KO mice that include increased postsynaptic fragmentation and presynaptic regions not apposed by postsynaptic nicotinic acetylcholine receptors. We also found perisynaptic Schwann cells intruding into the NMJ synaptic cleft. These findings strongly suggest that the NMJ is a site of postnatal pathology in MEGF10-related myopathy. In support of these cellular observations, RNA-seq analysis revealed genes and pathways associated with myogenesis, skeletal muscle health, and NMJ stability dysregulated in Megf10 KO mice compared to wild-type mice. Altogether, these data provide new and valuable cellular and molecular insights into MEGF10-related myopathy.

Comparative study of susceptibility to methylmercury cytotoxicity in cell types composing rat peripheral nerves: a higher susceptibility of dorsal root ganglion neurons.

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

The effects of exosomes originating from different cell sources on the differentiation of bone marrow mesenchymal stem cells into schwann cells.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can differentiate into Schwann cells (SCs) during peripheral nerve injury; in our previous research, we showed that SC-derived exosomes (SC-exos) played a direct induction role while fibroblast-derived exosomes (Fb-exos) had no obvious induction role. The induction role of neural stem cell (NSC)-derived exosomes (NSC-exos) has also been widely confirmed. However, no studies have compared the induction effects of these three types of cells at the same time. Therefore, by investigating the effect of these three cell-derived exosomes upon the induction of BMSCs to differentiate into SCs, this study explored the role of different exosomes in promoting the differentiation of stem cells into SCs cells, and conducted a comparison between the two groups by RNA sequencing to further narrow the range of target genes and related gene pathways in order to study their related mechanisms. MATERIALS AND METHODS: We extracted exosomes from SCs, fibroblasts (Fb) and neural stem cells (NSC) and then investigated the ability of these exosomes to induce differentiation into BMSCs under different culture conditions. The expression levels of key proteins and gene markers were detected in induced cells by fluorescence immunoassays, western blotting and polymerase chain reaction (PCR); then, we statistically compared the relative induction effects under different conditions. Finally, we analyzed the three types of exosomes by RNA-seq to predict target genes and related gene pathways. RESULTS: BMSCs were cultured by three media: conventional (no induction), pre-induction or pre-induction + original induction medium (ODM) with exosomes of the same cell origin under different culture conditions. When adding the three different types of exosomes separately, the overall induction of BMSCs to differentiate into SCs was significantly increased (P < 0.05). The induction ability was ranked as follows: pre-induction + ODM + exosome group > pre-induction + exosome group > non-induction + exosome group. Using exosomes from different cell sources under the same culture conditions, we observed the following trends under the three culture conditions: RSC96-exos group ≥ NSC-exos group > Fb-exos group. The overall ability to induce BMSCs into SCs was significantly greater in the RSC96-exos group and the NSC-exos group. Although there was no significant difference in induction efficiency when comparing these two groups, the overall induction ability of the RSC96-exos group was slightly higher than that of the NSC-exos group. By combining the differentiation induction results with the RNA-seq data, the three types of exosomes were divided into three comparative groups: RSC vs. NSC, RSC vs. Fb and NSC vs. Fb. We identified 203 differentially expressed mRNA target genes in these three groups. Two differentially expressed genes were upregulated simultaneously, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). We did not identify any co-upregulated target genes for the miRNAs, but did identify one target gene of the lncRNAs, namely ENSRNOG00000065005. Analysis identified 90 GO terms related to nerves and axons in the mRNAs; in addition, KEGG enrichment and GASA analysis identified 13 common differential expression pathways in the three groups. CONCLUSIONS: Our analysis found that pre-induction + ODM + RSC96/NSC-exos culture conditions were most conducive with regards to induction and differentiation. RSC96-exos and NSC-exos exhibited significantly greater differentiation efficiency of BMSCs into SCs. Although there was no statistical difference, the data indicated a trend for RSC96-exos to be advantageous We identified 203 differentially expressed mRNAs between the three groups and two differentially expressed target mRNAs were upregulated, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). 90 GO terms were related to nerves and axons. Finally, we identified 13 common differentially expressed pathways across our three types of exosomes. It is hoped that the efficiency of BMSCs induction differentiation into SCs can be improved, bringing hope to patients and more options for clinical treatment.

Dual growth factor methacrylic alginate microgels combined with chitosan-based conduits facilitate peripheral nerve repair.

Treating severe peripheral nerve injuries is difficult. Nerve repair with conduit small gap tubulization is a treatment option but still needs to be improved. This study aimed to assess the use of microgels containing growth factors, along with chitosan-based conduits, for repairing nerves. Using the water-oil emulsion technique, microgels of methacrylic alginate (AlgMA) that contained vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) were prepared. The effects on rat Schwann cells (RSC96) and human umbilical vein endothelial cells (HUVECs) were evaluated. Chitosan-based conduits were fabricated and used in conjunction with microgels containing two growth factors to treat complete neurotmesis in rats. The results showed that the utilization of dual growth factor microgels improved the migration and decreased the apoptosis of RSC96 cells while promoting the growth and formation of tubes in HUVECs. The utilization of dual growth factor microgels and chitosan-based conduits resulted in notable advancements in the regeneration and myelination of nerve fibers, recovery of neurons, alleviation of muscle atrophy and recovery of neuromotor function and nerve conduction. In conclusion, the use of dual growth factor AlgMA microgels in combination with chitosan-based conduits has the potential to significantly improve the effectiveness of nerve repair.

Metformin improves diabetic neuropathy by reducing inflammation through up-regulating the expression of miR-146a and suppressing oxidative stress.

PURPOSE: Diabetic neuropathy (DN) is a notable complication of diabetes mellitus. The potential involvement of miR-146a in DN regulation is presently under investigation. Metformin, a commonly prescribed medication for diabetes, is the primary therapeutic intervention. This study aimed to unveil the potential protective effects of metformin on diabetic neuropathy and explore the mechanisms underlying its action. METHOD: Six-weeks male Sprague Dawley rats (n = 40) were randomly divided into 5 groups. The rat model of diabetic neuropathy (DN) was established by administering streptozotocin (STZ). To investigate the effects on the sciatic nerve and resident Schwann cells (RSCs), metformin and miR-146a mimics were administered, and our research explored the potential underlying mechanism. RESULT: The sciatic nerve samples obtained from diabetic rats exhibited noticeable morphological damage, accompanied by decreased miR-146a expression (2.61 ± 0.11 vs 5.0 ± 0.3, p < 0.01) and increased inflammation levels (p65: 1.89 ± 0.04 vs 0.82 ± 0.05, p < 0.01; TNF-α: 0.93 ± 0.03 vs 0.33 ± 0.03, p < 0.01). Notably, the administration of metformin effectively ameliorated the structural alterations in the sciatic nerve by suppressing the inflammatory pathway (p65: 1.15 ± 0.05 vs 1.89 ± 0.04, p < 0.01; TNF-α: 0.67 ± 0.04 vs 0.93 ± 0.03, p < 0.01) and reducing oxidative stress (NO: 0.062 ± 0.004 vs 0.154 ± 0.004umol/mg, p < 0.01; SOD: 3.08 ± 0.09 vs 2.46 ± 0.09 U/mg, p < 0.01). The miR-146a mimics intervention group exhibited comparable findings. CONCLUSION: This study's findings implied that metformin can potentially mitigate diabetic neuropathy in rats through the modulation of miR-146a expression.

Forskolin-mediated cAMP activation upregulates TNF-α expression despite NF-κB downregulation in LPS-treated Schwann cells.

Although Schwann cells have been found to play a key role in inflammation and repair following nerve injury, the exact pathway is still unknown. To explore the mechanism by which Schwann cells exert their effects in the neuron microenvironment, we investigated two main inflammatory pathways: the NF-κB and cAMP pathways, and their downstream signaling molecules. In this study, lipopolysaccharide (LPS), a bacterial endotoxin, was used to activate the NF-κB pathway, and forskolin, a plant extract, was used to activate the cAMP pathway. The rat RT4-D6P2T Schwann cell line was treated with 0.1, 1, or 10 µg/mL of LPS, with or without 2 µM of forskolin, for 1, 3, 12, and 24 hours to determine the effects of elevated cAMP levels on LPS-treated cell viability. To investigate the effects of elevated cAMP levels on the expression of downstream signaling effector proteins, specifically NF-κB, TNF-α, AKAP95, and cyclin D3, as well as TNF-α secretion, RT4-D6P2T cells were incubated in the various treatment combinations for a 3-hour time period. Overall, results from the CellTiter-Glo viability assay revealed that forskolin increased viability in cells treated with smaller doses of LPS for 1 and 24 hours. For all time points, 10 µg/mL of LPS noticeably reduced viability regardless of forskolin treatment. Results from the Western blot analysis revealed that, at 10 µg/mL of LPS, forskolin upregulated the expression of TNF-α despite a downregulation of NF-κB, which was also accompanied by a decrease in TNF-α secretion. These results provide evidence that cAMP might regulate TNF-α expression through alternate pathways. Furthermore, although cAMP activation altered AKAP95 and cyclin D3 expression at different doses of LPS, there does not appear to be an association between the expression of AKAP95 or cyclin D3 and the expression of TNF-α. Exploring the possible interactions between cAMP, NF-κB, and other key inflammatory signaling pathways might reveal a potential therapeutic target for the treatment of nerve injury and inflammation.

Dental Pulp Stem Cell-Derived Exosomes Promote Sciatic Nerve Regeneration via Optimizing Schwann Cell Function.

Repair strategies for injured peripheral nerve have achieved great progresses in recent years. However, the clinical outcomes remain unsatisfactory. Recent studies have found that exosomes secreted by dental pulp stem cells (DPSC-exos) have great potential for applications in nerve repair. In this study, we evaluated the effects of human DPSC-exos on improving peripheral nerve regeneration. Initially, we established a coculture system between DPSCs and Schwann cells (SCs) in vitro to assess the effect of DPSC-exos on the activity of embryonic dorsal root ganglion neurons (DRGs) growth in SCs. We extracted and labeled human DPSC-exos, which were subsequently utilized in uptake experiments in DRGs and SCs. Subsequently, we established a rat sciatic nerve injury model to evaluate the therapeutic potential of DPSC-exos in repairing sciatic nerve damage. Our findings revealed that DPSC-exos significantly promoted neurite elongation by enhancing the proliferation, migration, and secretion of neurotrophic factors by SCs. In vivo, DPSC-exos administration significantly improved the walking behavior, axon regeneration, and myelination in rats with sciatic nerve injuries. Our study underscores the vast potential of DPSC-exos as a therapeutic tool for tissue-engineered nerve construction.

Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R.

AIMS: This study aimed to investigate the potential therapeutic applications of stigmasterol for treating neuropathic pain. METHODS: Related mechanisms were investigated by DRG single-cell sequencing analysis and the use of specific inhibitors in cellular experiments. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, CCI group, ibuprofen group, and stigmasterol group. We performed behavioral tests, ELISA, H&E staining and immunohistochemistry, and western blotting. RESULTS: Cell communication analysis by single-cell sequencing reveals that after peripheral nerve injury, Schwann cells secrete IL-34 to act on CSF1R in macrophages. After peripheral nerve injury, the mRNA expression levels of CSF1R pathway and NLRP3 inflammasome in macrophages were increased in DRG. In vitro studies demonstrated that stigmasterol can reduce the secretion of IL-34 in LPS-induced RSC96 Schwann cells; stigmasterol treatment of LPS-induced Schwann cell-conditioned medium (L-S-CM) does not induce the proliferation and migration of RAW264.7 macrophages; L-S-CM reduces CSF1R signaling pathway (CSF1R, P38MAPK, and NFκB) activation, NLRP3 inflammasome activation, and ROS production. In vivo experiments have verified that stigmasterol can reduce thermal and cold hyperalgesia in rat chronic compressive nerve injury (CCI) model; stigmasterol can reduce IL-1ß, IL-6, TNF-α, CCL2, SP, and PGE2 in serum of CCI rats; immunohistochemistry and western blot confirmed that stigmasterol can reduce the levels of IL-34/CSF1R signaling pathway and NLRP3 inflammasome in DRG of CCI rats. CONCLUSION: Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R axis.

Postoperatively Noninvasive Optogenetic Stimulation via Upconversion Nanoparticles Enhancing Sciatic Nerve Repair.

The efficacy of electrical stimulation facilitating peripheral nerve regeneration is evidenced extensively, while the associated secondary damage resulting from repeated electrode invasion and indiscriminate stimulation is inevitable. Here, we present an optogenetics strategy that utilizes upconversion nanoparticles (UCNPs) to convert deeply penetrating near-infrared excitation into blue emission, which activates an adeno-associated virus-encoding ChR2 photoresponsive ion channel on cell membranes. The induced Ca2+ flux, similar to the ion flux in the electrical stimulation approach, efficiently regulates viability and proliferation, secretion of nerve growth factor, and neural function of RSC96 cells. Furthermore, deep near-infrared excitation is harnessed to stimulate autologous Schwann cells in situ via a UCNP-composited scaffold, which enhances nerve sprouting and myelination, consequently promoting functional recovery, electrophysiological restoration, and reinnervation of damaged nerves. This developed postoperatively noninvasive optogenetics strategy presents a novel, minimally traumatic, and enduring therapeutic stimulus to effectively promote peripheral nerve repair.

Fabry disease Schwann cells release p11 to induce sensory neuron hyperactivity.

Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a mechanism we believe to be novel in which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observed in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrated that Fabry rat sensory neurons exhibited pronounced hyperexcitability. Schwann cells probably contributed to this finding because application of mediators released from cultured Fabry Schwann cells induced spontaneous activity and hyperexcitability in naive sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells released elevated levels of the protein p11 (S100A10), which induced sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media caused hyperpolarization of neuronal resting membrane potentials, indicating that p11 may contribute to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that sensory neurons from rats with Fabry disease exhibit hyperactivity caused in part by Schwann cell release of the protein p11.