Characterization of sciatic nerve myelin sheath during development in C57BL/6 mice.

Myelin sheath plays important roles in information conduction and nerve injury repair in the peripheral nerve system (PNS). Enhancing comprehension of the structure and components of the myelin sheath in the PNS during development would contribute to a more comprehensive understanding of the developmental and regenerative processes. In this research, the structure of sciatic nerve myelin sheath in C57BL/6 mice from embryonic day 14 (E14) to postnatal 12 months (12M) was observed with transmission electron microscopy. Myelin structure appeared in the sciatic nerve as early as E14, and the number and thickness of myelin lamellar gradually increased with the development until 12M. Transcriptome analysis was performed to show the expressions of myelin-associated genes and transcriptional factors involved in myelin formation. The genes encoding myelin proteins (Mag, Pmp22, Mpz, Mbp, Cnp and Prx) showed the same expression pattern, peaking at postnatal day 7 (P7) and P28 after birth, whereas the negative regulators of myelination (c-Jun, Tgfb1, Tnc, Cyr61, Ngf, Egr1, Hgf and Bcl11a) showed an opposite expression pattern. In addition, the expression of myelin-associated proteins and transcriptional factors was measured by Western blot and immunofluorescence staining. The protein expressions of MAG, PMP22, MPZ, CNPase and PRX increased from E20 to P14. The key transcriptional factor c-Jun co-localized with the Schwann cells Marker S100ß and decreased after birth, whereas Krox20/Egr2 increased during development. Our data characterized the structure and components of myelin sheath during the early developmental stages, providing insights for further understanding of PNS development.

The telocytes relationship with satellite cells: Extracellular vesicles mediate the myotendinous junction remodeling.

The peripheral nerve injury (PNI) affects the morphology of the whole locomotor apparatus, which can reach the myotendinous junction (MTJ) interface. In the injury condition, the skeletal muscle satellite cells (SC) are triggered, activated, and proliferated to repair their structure, and in the MTJ, the telocytes (TC) are associated to support the interface with the need for remodeling; in that way, these cells can be associated with SC. The study aimed to describe the SC and TC relationship after PNI at the MTJ. Sixteen adult Wistar rats were divided into Control Group (C, n = 8) and PNI Group (PNI, n = 8), PNI was performed by the constriction of the sciatic nerve. The samples were processed for transmission electron microscopy and immunostaining analysis. In the C group was evidenced the arrangement of sarcoplasmic evaginations and invaginations, the support collagen layer with a TC inside it, and an SC through vesicles internally and externally to then. In the PNI group were observed the disarrangement of invaginations and evaginations and sarcomeres degradation at MTJ, as the disposition of telopodes adjacent and in contact to the SC with extracellular vesicles and exosomes in a characterized paracrine activity. These findings can determine a link between the TCs and the SCs at the MTJ remodeling. RESEARCH HIGHLIGHTS: Peripheral nerve injury promotes the myotendinous junction (MTJ) remodeling. The telocytes (TC) and the satellite cells (SC) are present at the myotendinous interface. TC mediated the SC activity at MTJ.

Effect of peripheral blood mononuclear cells on ischemia-reperfusion injury of sciatic nerve of adult male albino rat: histological, immunohistochemical, and ultrastructural study.

Ischemia/reperfusion (I/R) injury of sciatic nerve is a serious condition that results in nerve fiber degeneration, and reperfusion causes oxidative injury. Peripheral blood mononuclear cells (PBMNCs) have neuroregenerative power. This study was carried out to evaluate the potential ameliorative effect of PBMNCs on changes induced by I/R injury of the sciatic nerve. Fifty adult male albino rats were divided into donor and experimental groups that were subdivided into four groups: group I (control group), group II received 50 µL PBNMCs once intravenously via the tail vein, group III rubber tourniquet was placed around their Rt hind limb root for 2 hours to cause ischemia, group IV was subjected to limb ischemia as group III, then they were injected with 50 ul PBMNCs as group II before reperfusion. I/R injury showed disorganization of nerve fascicles with wide spaces in between nerve fibers. The mean area of collagen fibers, iNOS immunoexpression, and number of GFAP-positive Schwann cells of myelinated fibers showed a highly significant increase, while a highly significant reduction in the G-ratio and neurofilament immunoexpression was observed. Myelin splitting, invagination, evagination, and myelin figures were detected. PBMNC-treated group showed a marked improvement that was confirmed by histological, immunohistochemical, and ultrastructural findings.

Ninj2 regulates Schwann cells development by interfering laminin-integrin signaling.

Rationale: Myelin sheath is an important structure to maintain normal functions of the nerves. Nerve Injury-Induced Protein 2 (Ninj2) was found upregulated in Schwann cells (SC) upon injury. However, whether and how Ninj2 plays a role in myelination remain unknown. Methods: In this study, we use transmission electron microscope imaging, immunofluorescent imaging, and behavioral tests to show the effects of Ninj2 on myelination and remyelination in peripheral nervous system (PNS) of SC-specific Ninj2 knockout mice (Dhhcre/+;Ninj2fl/fl ). For mechanism studies, we use RNA-Seq analysis to show the Ninj2-related pathways, and co-immunoprecipitation/mass-spectrometry to identify the Ninj2-interacting proteins in SCs. Furthermore, we evaluate the effect of integrin inhibitor GRGDSP during remyelination. Results: Ninj2 negatively regulates SC development. Ninj2-deficient mice exhibit precocious myelination phenotype, as well as the accelerated remyelination process after sciatic nerve injury. Loss of Ninj2 promotes myelination by promoting SC proliferation to augment its population. Mechanistically, Ninj2 interacted with ITGB1 on SC membrane, which inhibits laminin-integrin signaling. Removal of Ninj2 induces the activity of laminin-integrin signaling, resulting in the improved myelination in the Dhhcre/+;Ninj2fl/fl mice. Inhibition of laminin-integrin signaling by integrin inhibitor GRGDSP sufficiently delays the remyelination process in the Dhhcre/+;Ninj2fl/fl mice with sciatic nerve injury. Conclusion: Our study found Ninj2 as a negative regulator in the network controlling myelination in the PNS.

Super-resolved fluorescence imaging of peripheral nerve.

Traditional histopathologic evaluation of peripheral nerve employs brightfield microscopy with diffraction limited resolution of ~ 250 nm. Though electron microscopy yields nanoscale resolution of the nervous system, sample preparation is costly and the technique is incompatible with living samples. Super-resolution microscopy (SRM) comprises a set of imaging techniques that permit nanoscale resolution of fluorescent objects using visible light. The advent of SRM has transformed biomedical science in establishing non-toxic means for investigation of nanoscale cellular structures. Herein, sciatic nerve sections from GFP-variant expressing mice, and regenerating human nerve from cross-facial autografts labelled with a myelin-specific fluorescent dye were imaged by super-resolution radial fluctuation microscopy, stimulated emission depletion microscopy, and structured illumination microscopy. Super-resolution imaging of axial cryosections of murine sciatic nerves yielded robust visualization myelinated and unmyelinated axons. Super-resolution imaging of axial cryosections of human cross-facial nerve grafts demonstrated enhanced resolution of small-caliber thinly-myelinated regenerating motor axons. Resolution and contrast enhancement afforded by super-resolution imaging techniques enables visualization of unmyelinated axons, regenerating axons, cytoskeleton ultrastructure, and neuronal appendages of mammalian peripheral nerves using light microscopes.

Quercetin reduces inflammation in a rat model of diabetic peripheral neuropathy by regulating the TLR4/MyD88/NF-κB signalling pathway.

Neuroinflammation contributes significantly to the pathogenesis of diabetic peripheral neuropathy (DPN). Quercetin reportedly exerts neuroprotective effects in DPN. Here, we aimed to evaluate the potential anti-inflammatory effects of quercetin in a DPN rat model. Eight weeks after streptozotocin administration, diabetic rats were treated with quercetin (30 and 60 mg/kg/day orally) for 6 weeks. We assessed the mechanical withdrawal threshold (MWT), nerve conduction velocity (NCV) and morphological changes in sciatic nerves. Additionally, we measured the levels of tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, and IL-6 by ELISA and the expression of TLR4, MyD88, and NF-κB in sciatic nerves by western blotting and immunohistochemical assays. Our results revealed that blood glucose levels and body weight were unaltered following quercetin treatment. However, quercetin improved MWT (p < 0.05), NCV (p < 0.05), and pathological changes in the sciatic nerves of DPN rats. Quercetin significantly alleviated the increased expression of TNF-α (p < 0.05) and IL-1ß (p < 0.001). Furthermore, high-dose quercetin administration significantly downregulated the expression of TLR4 (p < 0.001), MyD88 (p < 0.001), and NF-κB (p < 0.001) in sciatic nerves of DPN rats. Our findings revealed that quercetin could reduce the levels of inflammatory factors in DPN rats, possibly mediated via the downregulation of the TLR4/MyD88/NF-κB signalling pathway. Collectively, these results suggest that although quercetin did not decreased blood glucose levels or reversed the reduced body weight, it showed anti-inflammatory and neuroprotective effects, which was beneficial for the treatment of DPN.

Protective Effects of PACAP in a Rat Model of Diabetic Neuropathy.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide with a widespread occurrence and diverse effects. PACAP has well-documented neuro- and cytoprotective effects, proven in numerous studies. Among others, PACAP is protective in models of diabetes-associated diseases, such as diabetic nephropathy and retinopathy. As the neuropeptide has strong neurotrophic and neuroprotective actions, we aimed at investigating the effects of PACAP in a rat model of streptozotocin-induced diabetic neuropathy, another common complication of diabetes. Rats were treated with PACAP1-38 every second day for 8 weeks starting simultaneously with the streptozotocin injection. Nerve fiber morphology was examined with electron microscopy, chronic neuronal activation in pain processing centers was studied with FosB immunohistochemistry, and functionality was assessed by determining the mechanical nociceptive threshold. PACAP treatment did not alter body weight or blood glucose levels during the 8-week observation period. However, PACAP attenuated the mechanical hyperalgesia, compared to vehicle-treated diabetic animals, and it markedly reduced the morphological signs characteristic for neuropathy: axon-myelin separation, mitochondrial fission, unmyelinated fiber atrophy, and basement membrane thickening of endoneurial vessels. Furthermore, PACAP attenuated the increase in FosB immunoreactivity in the dorsal spinal horn and periaqueductal grey matter. Our results show that PACAP is a promising therapeutic agent in diabetes-associated complications, including diabetic neuropathy.

Macroscopic detection of demyelinated lesions in mouse PNS with neutral red dye.

Lysophosphatidylcholine (LPC)-induced demyelination is a versatile animal model that is frequently used to identify and examine molecular pathways of demyelination and remyelination in the central (CNS) and peripheral nervous system (PNS). However, identification of focally demyelinated lesion had been difficult and usually required tissue fixation, sectioning and histological analysis. Recently, a method for labeling and identification of demyelinated lesions in the CNS by intraperitoneal injection of neutral red (NR) dye was developed. However, it remained unknown whether NR can be used to label demyelinated lesions in PNS. In this study, we generated LPC-induced demyelination in sciatic nerve of mice, and demonstrated that the demyelinated lesions at the site of LPC injection were readily detectable at 7 days postlesion (dpl) by macroscopic observation of NR labeling. Moreover, NR staining gradually decreased from 7 to 21 dpl over the course of remyelination. Electron microscopy analysis of NR-labeled sciatic nerves at 7 dpl confirmed demyelination and myelin debris in lesions. Furthermore, fluorescence microscopy showed NR co-labeling with activated macrophages and Schwann cells in the PNS lesions. Together, NR labeling is a straightforward method that allows the macroscopic detection of demyelinated lesions in sciatic nerves after LPC injection.

Comprehensive Analysis of Age-related Changes in Lipid Metabolism and Myelin Sheath Formation in Sciatic Nerves.

To investigate the molecular changes related to myelin formation and lipid metabolism in the sciatic nerve in Sprague Dawley (SD) rats during aging. Thirty-six healthy male SD rats were divided into five groups according to age: 1 week, 1 month, 6 months, 12 months, and 24 months. Sciatic nerves were collected from 1-month-old and 24-month-old SD rats (n = 3) to perform next-generation sequencing (NGS) and bioinformatics analysis. Specimens from each group were harvested and analyzed by qPCR, Western blotting, and transmission electron microscopy (TEM). Protein-protein interaction (PPI) networks of differentially expressed mRNAs (DEmRNAs) related to myelin and lipid metabolism were constructed. DEmRNAs in subnetworks were verified using qPCR. A total of 4580 DEmRNAs were found during aging. The top enriched GO biological processes were primarily clustered in cholesterol and lipid metabolism, including the cholesterol biosynthetic process (RF = 3.16), sterol biosynthetic process (RF = 3.03), cholesterol metabolic process (RF = 2.15), sterol metabolic process (RF = 2.11), fatty acid biosynthetic process (RF = 2.09), and lipid biosynthetic process (RF = 1.79). The mRNA levels of MBP, PMP22, and MPZ were downregulated during aging, while the protein expression of MBP showed an increasing trend. The TEM results showed thin myelin sheaths and an increased number of unmyelinated axons in the 1-week-old rats, and the sheaths became thickened with degenerated axons appearing in older animals. Forty PPI subnetworks related to lipid metabolism were constructed, including one primary subnetwork and two smaller subnetworks. The hub genes were mTOR in sub-network 1, Akt1 in sub-network 2, and SIRT1 in sub-network 3. No gene expression was found consistent with the sequencing results, while in the downregulated genes, AKT1, CEBPA, LIPE, LRP5, PHB, and Rara were significantly downregulated in 24-month-old rats. Lipid metabolism might play an important role in maintaining the structure and physiological function in sciatic nerves during aging and could be candidates for nerve aging research.

Efficacy of Large Groove Texture on Rat Sciatic Nerve Regeneration In Vivo Using Polyacrylonitrile Nerve Conduits.

Physical guidance cues play an important role in enhancing the efficiency of nerve conduits for peripheral nerve injury repair. However, very few in vivo investigations have been performed to evaluate the repair efficiency of nerve conduits with micro-grooved inner textures. In this study, polyacrylonitrile nerve conduits were prepared using dry-jet wet spinning, and micro-grooved textures were incorporated on the inner surface. The nerve conduits were applied to treat 10 mm sciatic nerve gaps in Sprague-Dawley (SD) rats. Sixteen weeks following implantation, nerve function was evaluated based on heat sensory tests, electrophysiological assessments and gastrocnemius muscle mass measurements. The thermal latency reaction and gastrocnemii weight of SD rats treated with grooved nerve conduits were almost 25% faster and 60% heavier than those of SD rats treated with smooth nerve conduits. The histological and immunohistochemical stain analyses showed the repair capacity of inner grooved conduits was found to be similar to that of autografts. These results suggest that grooved nerve conduits with groove width larger than 300 µm significantly improve peripheral nerve regeneration by introducing physical guidance cues. The obtained results can support the design of nerve conduits and lead to the improvement of nerve tissue engineering strategies.

Deletion of the Gene Encoding the NMDA Receptor GluN1 Subunit in Schwann Cells Causes Ultrastructural Changes in Remak Bundles and Hypersensitivity in Pain Processing.

Abnormalities in interactions between sensory neurons and Schwann cells (SCs) may result in heightened pain processing and chronic pain states. We previously reported that SCs express the NMDA receptor (NMDA-R), which activates cell signaling in response to glutamate and specific protein ligands, such as tissue-type plasminogen activator. Herein, we genetically targeted grin1 encoding the essential GluN1 NMDA-R subunit, conditionally in SCs, to create a novel mouse model in which SCs are NMDA-R-deficient (GluN1- mice). These mice demonstrated increased sensitivity to light touch, pinprick, and thermal hyperalgesia in the absence of injury, without associated changes in motor function. Ultrastructural analysis of adult sciatic nerve in GluN1- mice revealed increases in the density of Aδ fibers and Remak bundles and a decrease in the density of Aß fibers, without altered g-ratios. Abnormalities in adult Remak bundle ultrastructure were also present including aberrant C-fiber ensheathment, distances between axons, and increased poly-axonal pockets. Developmental and post radial sorting defects contributed to altered nerve fiber densities in adult. Uninjured sciatic nerves in GluN1- mice did not demonstrate an increase in neuroinflammatory infiltrates. Transcriptome profiling of dorsal root ganglia (DRGs) revealed 138 differentially regulated genes in GluN1- mice. One third of the regulated genes are known to be involved in pain processing, including sprr1a, npy, fgf3, atf3, and cckbr, which were significantly increased. The intraepidermal nerve fiber density (IENFD) was significantly decreased in the skin of GluN1- mice. Collectively, these findings demonstrate that SC NMDA-R is essential for normal PNS development and for preventing development of pain states.SIGNIFICANCE STATEMENT Chronic unremitting pain is a prevalent medical condition; however, the molecular mechanisms that underlie heightened pain processing remain incompletely understood. Emerging data suggest that abnormalities in Schwann cells (SCs) may cause neuropathic pain. We established a novel mouse model for small fiber neuropathy (SFN) in which grin1, the gene that encodes the NMDA receptor (NMDA-R) GluN1 subunit, is deleted in SCs. These mice demonstrate hypersensitivity in pain processing in the absence of nerve injury. Changes in the density of intraepidermal small fibers, the ultrastructure of Remak bundles, and the transcriptome of dorsal root ganglia (DRGs) provide possible explanations for the increase in pain processing. Our results support the hypothesis that abnormalities in communication between sensory nerve fibers and SCs may result in pain states.

In Vivo Imaging of Anterograde and Retrograde Axonal Transport in Rodent Peripheral Nerves.

Axonal transport, which is the process mediating the active shuttling of a variety cargoes from one end of an axon to the other, is essential for the development, function, and survival of neurons. Impairments in this dynamic process are linked to diverse nervous system diseases and advanced ageing. It is thus essential that we quantitatively study the kinetics of axonal transport to gain an improved understanding of neuropathology as well as the molecular and cellular mechanisms regulating cargo trafficking. One of the best ways to achieve this goal is by imaging individual, fluorescent cargoes in live systems and analyzing the kinetic properties of their progression along the axon. We have therefore developed an intravital technique to visualize different organelles, such as signaling endosomes and mitochondria, being actively transported in the axons of both motor and sensory neurons in live, anesthetized rodents. In this chapter, we provide step-by-step instructions on how to deliver specific organelle-targeting, fluorescent probes using several routes of administration to image individual cargoes being bidirectionally transported along axons within the exposed sciatic nerve. This method can provide detailed, physiologically relevant information on axonal transport, and is thus poised to elucidate mechanisms regulating this process in both health and disease.

Ultrasound therapy with optimal intensity facilitates peripheral nerve regeneration in rats through suppression of pro-inflammatory and nerve growth inhibitor gene expression.

BACKGROUND: Therapeutic ultrasound (US) is a promising physical therapy modality for peripheral nerve regeneration. However, it is necessary to identify the most effective US parameters and clarify the underlying mechanisms before its clinical application. The intensity of US is one of the most important parameters. However, the optimum intensity for the promotion of peripheral nerve regeneration has yet to be determined. OBJECTIVES: To identify the optimum intensity of US necessary for the promotion of peripheral nerve regeneration after crush injuries in rats and to clarify the underlying mechanisms of US by mRNA expression analysis. METHODS: We inflicted sciatic nerve crush injuries on adult Lewis rats and performed ultrasound irradiation using 4 different US intensities: 0 (sham stimulation), 30, 140, and 250 mW/cm2 with frequency (5 days/week) and duration (5 min/day). We evaluated peripheral nerve regeneration by quantitative real-time PCR one week after injury. Histomorphometric analyses and motor function analysis were evaluated 3 weeks after injury. RESULTS: US stimulation enhanced re-myelination as well as sprouting of axons, especially at an intensity of 140 mW/cm2. mRNA expression revealed that US suppressed the expression of the inflammatory cytokines TNF and IL-6 and the axonal growth inhibitors SEMA3A and GSK3ß. CONCLUSIONS: An intensity of 140 mW/cm2 was optimal to support regeneration of the sciatic nerve after a crush injury in rats by, in part, the suppression of pro-inflammatory and nerve growth inhibitor gene expression.

CMT2Q-causing mutation in the Dhtkd1 gene lead to sensory defects, mitochondrial accumulation and altered metabolism in a knock-in mouse model.

Charcot-Marie-Tooth disease (CMT) is a group of inherited neurological disorders of the peripheral nervous system. CMT is subdivided into two main types: a demyelinating form, known as CMT1, and an axonal form, known as CMT2. Nearly 30 genes have been identified as a cause of CMT2. One of these is the 'dehydrogenase E1 and transketolase domain containing 1' (DHTKD1) gene. We previously demonstrated that a nonsense mutation [c.1455 T > G (p.Y485*)] in exon 8 of DHTKD1 is one of the disease-causing mutations in CMT2Q (MIM 615025). The aim of the current study was to investigate whether human disease-causing mutations in the Dhtkd1 gene cause CMT2Q phenotypes in a mouse model in order to investigate the physiological function and pathogenic mechanisms associated with mutations in the Dhtkd1 gene in vivo. Therefore, we generated a knock-in mouse model with the Dhtkd1Y486* point mutation. We observed that the Dhtkd1 expression level in sciatic nerve of knock-in mice was significantly lower than in wild-type mice. Moreover, a histopathological phenotype was observed, reminiscent of a peripheral neuropathy, including reduced large axon diameter and abnormal myelination in peripheral nerves. The knock-in mice also displayed clear sensory defects, while no abnormalities in the motor performance were observed. In addition, accumulation of mitochondria and an elevated energy metabolic state was observed in the knock-in mice. Taken together, our study indicates that the Dhtkd1Y486* knock-in mice partially recapitulate the clinical phenotypes of CMT2Q patients and we hypothesize that there might be a compensatory effect from the elevated metabolic state in the knock-in mice that enables them to maintain their normal locomotor function.

Neuroprotective Effect of Salvianolic Acid A against Diabetic Peripheral Neuropathy through Modulation of Nrf2.

Oxidative stress has been recognized as the contributor to diabetic peripheral neuropathy (DPN). Antioxidant strategies have been most widely explored; nevertheless, whether antioxidants alone prevent DPN still remains inconclusive. In the present study, we established an in vitro DPN cell model for drug screening using Schwann RSC96 cells under high glucose (HG) stimulation, and we found that salvianolic acid A (SalA) mitigated HG-induced injury evidenced by cell viability and myelination. Mechanistically, SalA exhibited strong antioxidative effects by inhibiting 1,1-diphenyl-2-picrylhydrazyl (DPPH) and reducing reactive oxygen species (ROS), malondialdehyde (MDA), and oxidized glutathione (GSSG) content, as well as upregulating antioxidative enzyme mRNA expression. In addition, SalA significantly extenuated neuroinflammation with downregulated inflammatory factor mRNA expression. Furthermore, SalA improved the mitochondrial function of HG-injured Schwann cells by scavenging mitochondrial ROS, decreasing mitochondrial membrane potential (MMP), and enhancing ATP production, as well as upregulating oxidative phosphorylation gene expression. More importantly, we identified nuclear factor-E2-related factor 2 (Nrf2) as the upstream regulator which mediated protective effects of SalA on DPN. SalA directly bound to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) and thus disrupted the interaction of Nrf2 and Keap1 predicted by LibDock of Discovery Studio. Additionally, SalA significantly inhibited Nrf2 promoter activity and downregulated Nrf2 mRNA expression but without affecting Nrf2 protein expression. Interestingly, SalA upregulated the nuclear Nrf2 expression and promoted Nrf2 nuclear translocation by high content screening assay, which was confirmed to be involved in its antiglucotoxicity effect by the knockdown of Nrf2 in RSC96 cells. In KK-Ay mice, we demonstrated that SalA could effectively improve the abnormal glucose and lipid metabolism and significantly protect against DPN by increasing the mechanical withdrawal threshold and sciatic nerve conduction velocity and restoring the ultrastructural impairment of the injured sciatic nerve induced by diabetes. Hence, SalA protected against DPN by antioxidative stress, attenuating neuroinflammation, and improving mitochondrial function via Nrf2. SalA may be prospective therapeutics for treating DPN.

Circular RNA profile in diabetic peripheral neuropathy: analysis of coexpression networks of circular RNAs and mRNAs.

Aim: To study the expression pattern of circular RNAs in diabetic peripheral neuropathy. Materials & methods: Transmission electron microscopy was used to observe the ultrastructure of sciatic nerves and dorsal root ganglion (DRGs). circRNAs in DRGs were identified with high-throughput RNA sequencing. Whole-genome mRNAs were detected by a chip scan. Results: The ultrastructure of sciatic nerves and DRGs in diabetes mellitus mice changed significantly. A total of 11,004 circRNAs and 15 differentially expressed circRNAs, as well as 35,368 mRNAs and 133 differentially expressed mRNAs were identified in DRGs between wild-type and diabetes mellitus mice. 11 circRNAs and 14 mRNAs have a significant correlation using strict coexpression analysis. The expression of circRNA.4614 was validated to be upregulated significantly. Conclusion: Our study suggested that circRNAs might be involved in the regulation of mRNA expressions in diabetic peripheral neuropathy.

Tumors Provoke Inflammation and Perineural Microlesions at Adjacent Peripheral Nerves.

Cancer-induced pain occurs frequently in patients when tumors or their metastases grow in the proximity of nerves. Although this cancer-induced pain states poses an important therapeutical problem, the underlying pathomechanisms are not understood. Here, we implanted adenocarcinoma, fibrosarcoma and melanoma tumor cells in proximity of the sciatic nerve. All three tumor types caused mechanical hypersensitivity, thermal hyposensitivity and neuronal damage. Surprisingly the onset of the hypersensitivity was independent of physical contact of the nerve with the tumors and did not depend on infiltration of cancer cells in the sciatic nerve. However, macrophages and dendritic cells appeared on the outside of the sciatic nerves with the onset of the hypersensitivity. At the same time point downregulation of perineural tight junction proteins was observed, which was later followed by the appearance of microlesions. Fitting to the changes in the epi-/perineurium, a dramatic decrease of triglycerides and acylcarnitines in the sciatic nerves as well as an altered localization and appearance of epineural adipocytes was seen. In summary, the data show an inflammation at the sciatic nerves as well as an increased perineural and epineural permeability. Thus, interventions aiming to suppress inflammatory processes at the sciatic nerve or preserving peri- and epineural integrity may present new approaches for the treatment of tumor-induced pain.

microRNA-192-5p is involved in nerve repair in rats with peripheral nerve injury by regulating XIAP.

Objective: MicroRNAs (miRNAs) have been demonstrated to engage in the nerve injury, while the effect of microRNA-192-5p (miR-192-5p) on the nerve repair has not yet been well understood. This study is performed to investigate how miR-192-5p affects nerve repair in rats with peripheral nerve injury by regulating X-linked inhibitor of apoptosis protein (XIAP).Methods: The rat model of left sciatic nerve injury was established, and the expression of miR-192-5p was then detected. A series of experiments were conducted to investigate the role of miR-192-5p on nerve repair in rats with peripheral nerve injury. The expression of apoptosis-related proteins (Caspase-3, Bax and Bcl-2) and nerve repair factors (NGF, BDNF, and GAP-43) was measured. Bioinformatics analysis and dual-luciferase reporter gene assay confirmed the targeting relationship between miR-192-5p and XIAP.Results: MiR-192-5p inhibition promoted the recovery of sensory function and the recovery and regeneration in rats with sciatic nerve injury. MiR-192-5p inhibition promoted the recovery of muscle atrophy caused by nerve injury. MiR-192-5p inhibition inhibited neuronal apoptosis by affecting the expression of apoptosis-related proteins and promoted the recovery of nerve function by elevating the expression of nerve repair factors induced by peripheral nerve injury. Bioinformatics analysis and dual-luciferase reporter gene assay confirmed that XIAP was a target gene of miR-192-5p.Conclusion: This study demonstrates that miR-192-5p inhibition can up-regulate the expression of XIAP, decrease the apoptosis of nerve cells, and promote the repair and regeneration of peripheral nerve injury.

Neural Selective Cryoneurolysis with Ice Slurry Injection in a Rat Model.

BACKGROUND: Postoperative pain caused by trauma to nerves and tissue around the surgical site is a major problem. Perioperative steps to reduce postoperative pain include local anesthetics and opioids, the latter of which are addictive and have contributed to the opioid epidemic. Cryoneurolysis is a nonopioid and long-lasting treatment for reducing postoperative pain. However, current methods of cryoneurolysis are invasive, technically demanding, and are not tissue-selective. This project aims to determine whether ice slurry can be used as a novel, injectable, drug-free, and tissue-selective method of cryoneurolysis and resulting analgesia. METHODS: The authors developed an injectable and selective method of cryoneurolysis using biocompatible ice slurry, using rat sciatic nerve to investigate the effect of slurry injection on the structure and function of the nerve. Sixty-two naïve, male Sprague-Dawley rats were used in this study. Advanced Coherent anti-Stokes Raman Scattering microscopy, light, and fluorescent microscopy imaging were used at baseline and at various time points after treatment for evaluation and quantification of myelin sheath and axon structural integrity. Validated motor and sensory testing were used for evaluating the sciatic nerve function in response to ice slurry treatment. RESULTS: Ice slurry injection can selectively target the rat sciatic nerve. Being injectable, it can infiltrate around the nerve. The authors demonstrate that a single injection is safe and selective for reversibly disrupting the myelin sheaths and axon density, with complete structural recovery by day 112. This leads to decreased nocifensive function for up to 60 days, with complete recovery by day 112. There was up to median [interquartile range]: 68% [60 to 94%] reduction in mechanical pain response after treatment. CONCLUSIONS: Ice slurry injection selectively targets the rat sciatic nerve, causing no damage to surrounding tissue. Injection of ice slurry around the rat sciatic nerve induced decreased nociceptive response from the baseline through neural selective cryoneurolysis.

Histological and ultrastructural study of AflatoxinB1 induced neurotoxicity in Sciatic nerve of adult male Albino rats.

Aflatoxins are mycotoxins produced by Aspergillus spp. which is a common contaminant of food items such as corn, spices, rice, nuts, and flour. Aflatoxin contamination of foods is a worldwide problem. Chronic aflatoxin exposure is found to be associated with Sciatic nerve damage. In vivo study was carried out to evaluate the toxic effect of aflatoxin B1 (AFB1) on the Sciatic nerve. Twenty-one adult male rats were included and divided equally into 3 groups (7 rats each): Group I (control group), group II (olive oil group) and group III: (AflatoxinB1 group). The rats received AFB1 (250 µg/kg B.W./day) orally by gastric tube 5 days/week for 4 weeks. Sciatic nerve specimens were prepared, and semi-thin sections were stained with Toluidine blue, examined by light microscope and photographed. Ultrathin sections (50-80 nm) from selected areas of the trimmed blocks were made, examined and photographed by transmission electron microscopy (JEOL-JSM-1011) in King Saud University Electron Microscopy Unit. The findings indicate that the administration of AFB1 to rats' results in degeneration in the sciatic nerve in the form of Wallerian degeneration in the myelin sheath. Macrophages appear to engulf the degenerated myelin and neutrophils.