Myelinated Axons Research Articles

Electrospun Piezoelectric Scaffold with External Mechanical Stimulation for Promoting Regeneration of Peripheral Nerve Injury.

Safe and efficient provision of electrical stimulation (ES) for nerve repair and regeneration is a problem that needs to be addressed. In this study, a silk fibroin/poly(vinylidene fluoride-co-hexafluoropropylene)/Ti3C2Tx (SF/PVDF-HFP/MXene) composite scaffold with piezoelectricity was developed by electrospinning technology. MXene was loaded to the scaffold to enhance the piezoelectric properties (Output voltage reaches up to 100 mV), mechanical properties, and antibacterial activity. Cell experiments demonstrated piezoelectric stimulation under external ultrasonication for promoting the growth and proliferation of Schwann cells (SCs) cultured on this electrospun scaffold. Further in vivo study with rat sciatic nerve injury model revealed that the SF/PVDF-HFP/MXene nerve conduit could induce the proliferation of SCs, enhance the elongation of axon, and promote axonal myelination. Under the piezoelectric effect of this nerve scaffold, the rats with regenerative nerve exhibited a favorable recovery effect of motor and sensory function, indicating a safe and feasible method of using this SF/PVDF-HFP/MXene piezoelectric scaffold for ES provision in vivo.

Two different isoforms of osteopontin modulate myelination and axonal integrity.

Abnormal myelination underlies the pathology of white matter diseases such as preterm white matter injury and multiple sclerosis. Osteopontin (OPN) has been suggested to play a role in myelination. Murine OPN mRNA is translated into a secreted isoform (sOPN) or an intracellular isoform (iOPN). Whether there is an isoform-specific involvement of OPN in myelination is unknown. Here we generated mouse models that either lacked both OPN isoforms in all cells (OPN-KO) or lacked sOPN systemically but expressed iOPN specifically in oligodendrocytes (OLs-iOPN-KI). Transcriptome analysis of isolated oligodendrocytes from the neonatal brain showed that genes and pathways related to increase of myelination and altered cell cycle control were enriched in the absence of the two OPN isoforms in OPN-KO mice compared to control mice. Accordingly, adult OPN-KO mice showed an increased axonal myelination, as revealed by transmission electron microscopy imaging, and increased expression of myelin-related proteins. In contrast, neonatal oligodendrocytes from OLs-iOPN-KI mice compared to control mice showed differential regulation of genes and pathways related to the increase of cell adhesion, motility, and vasculature development, and the decrease of axonal/neuronal development. OLs-iOPN-KI mice showed abnormal myelin formation in the early phase of myelination in young mice and signs of axonal degeneration in adulthood. These results suggest an OPN isoform-specific involvement, and a possible interplay between the isoforms, in myelination, and axonal integrity. Thus, the two isoforms of OPN need to be separately considered in therapeutic strategies targeting OPN in white matter injury and diseases.

Lee's "Transmembrane Electrostatically-Localized Proton" model does NOT offer a better understanding of neuronal transmembrane potentials.

Recently, in a paper entitled "Protonic conductor: Better understanding [sic] neural resting and action potential," Lee applied his Transmembrane Electrostatically-Localized Protons (TELP) hypothesis to neuronal signaling. He stated that Hodgkin's cable theory "could not fully explain the different conductive patterns in unmyelinated and myelinated nerves," whereas his TELP hypothesis "enables much better understanding of neural resting/action potential and [the biological significance of] axon myelination…" However, Lee's TELP hypothesis predicts that under resting conditions, the neuron "accumulat[es] excess negative charge (anions) inside," whereas resting chloride gradients actually feature excess Cl- outside of the cell. Experiments on the neuron have shown that raising external [K+] and decreasing external [Cl-] cause membrane potential depolarization, which is predicted by the Goldman equation, but opposite to TELP hypothesis predictions. Finally, based on his TELP hypothesis, Lee predicted that the main purpose of myelin is to insulate the axonal plasma membrane specifically against proton permeability. However, he cited literature showing that myelin contains proteins that may "serve as a proton conductor with the localized protons." Thus, we show here that Lee's TELP hypothesis is highly problematic, and does NOT offer a "better understanding" of neuronal transmembrane potentials.NEW & NOTEWORTHY In this manuscript I critique a 2020 J. Neurophysiol. paper by James W. Lee. His TELP hypothesis 1) mispredicts the resting neuron's excess of external chloride; 2) predicts the preponderance of surface H+ over Na+ using ΔG° rather than ΔG; 3) mispredicts the dependence of the neuronal resting potential on external [Na+], [K+], and [Cl-]; 4) neither cites experimental results nor proposes experiments to test his hypothesis; and 5) presents a problematic characterization of the purpose of myelin.

Transcription Factor BCL11A Regulates Schwann Cell Behavior During Peripheral Nerve Regeneration.

Nerve injury-induced Schwann cell dedifferentiation helps to construct a favorable microenvironment for axon growth. Transcription factors regulate cell reprogramming and thus may be critical for Schwann cell phenotype switch during peripheral nerve regeneration. Here, we show that transcription factor B-cell lymphoma/leukemia 11A (BCL11A) is up-regulated in Schwann cells of injured peripheral nerves. Bcl11a silencing suppresses Schwann cell viability, decreases Schwann cell proliferation and migration rates, and impairs the debris clearance ability of Schwann cells. Reduced Bcl11a in injured peripheral nerves results in restricted axon elongation and myelin wrapping, leading to recovery failure. Mechanistically, we demonstrate that BCL11A may mediate Schwann cell activity through binding to the promoter of nuclear receptor subfamily 2 group F member 2 (Nr2f2) and regulating Nr2f2 expression. Collectively, we conclude that BCL11A is essential for Schwann cell activation and peripheral nerve regeneration, providing a potential therapeutic target for the treatment of peripheral nerve injury.

Advances in Large Gap Peripheral Nerve Injury Repair and Regeneration with Bridging Nerve Guidance Conduits.

Peripheral nerve injury is a common complication of accidents and diseases. The traditional autologous nerve graft approach remains the gold standard for the treatment of nerve injuries. While sources of autologous nerve grafts are very limited and difficult to obtain. Nerve guidance conduits have been widely used in the treatment of peripheral nerve injuries as an alternative to nerve autografts and allografts. However, the development of nerve conduits does not meet the needs of large gap peripheral nerve injury. Functional nerve conduits can provide a good microenvironment for axon elongation and myelin regeneration. In this review, we summarize the manufacturing methods and different design types of functional bridging nerve conduits for nerve conduits combined with electrical or magnetic stimulation and loaded with Schwann cells, etc. It summarizes the literature and finds that the technical solutions of functional nerve conduits with electrical stimulation, magnetic stimulation and nerve conduits combined with Schwann cells can be used as effective strategies for bridging large gap nerve injury and provide an effective way for the study of large gap nerve injury repair. In addition, functional nerve conduits provide a new way to construct delivery systems for drugs and growth factors in vivo. This article is protected by copyright. All rights reserved.

Toll-like receptor 4-mediated microglial inflammation exacerbates early white matter injury following experimental subarachnoid hemorrhage.

Neuroinflammation has been reported to be associated with white matter injury (WMI) after subarachnoid hemorrhage (SAH). As the main resident immune cells of the brain, microglia can be activated into proinflammatory and anti-inflammatory phenotypes. Toll-like receptor 4 (TLR4), expressed on the surface of the microglia, plays a key role in microglial inflammation. However, the relationship between TLR4, microglial polarization, and WMI following SAH remains unclear. In this study, a total of 121 male adult C57BL/6 wild-type (WT) mice, 20 WT mice at postnatal day 1 (P1), and 41 male adult TLR4 gene knockout (TLR4-/-) mice were used to investigate the potential role of TLR4-induced microglial polarization in early WMI after SAH by radiological, histological, microstructural, transcriptional, and cytological evidence. The results indicated that microglial inflammation was associated with myelin loss and axon damage, shown as a decrease in myelin basic protein (MBP), as well as increase in degraded myelin basic protein (dMBP) and amyloid precursor protein (APP). Gene knockout of TLR4 revised microglial polarization toward the anti-inflammatory phenotype and protected the white matter at an early phase after SAH (24 h), as shown through reduction of toxic metabolites, preservation of myelin, reductions in APP accumulation, reductions in white matter T2 hyperintensity, and increases in FA values. Cocultures of microglia and oligodendrocytes, the cells responsible for myelin production and maintenance, were established to further elucidate the relationship between microglial polarization and WMI. In vitro, TLR4 inhibition decreased the expression of microglial MyD88 and phosphorylated NF-κB, thereby inhibiting M1 polarization and mitigating inflammation. Decrease in TLR4 in the microglia increased preservation of neighboring oligodendrocytes. In conclusion, microglial inflammation has dual effects on early WMI after experimental SAH. Future explorations on more clinically relevant methods for modulating neuroinflammation are warranted to combat stroke with both WMI and gray matter destruction.

Microfluidic Synthesis of Ultrasmall Chitosan/Graphene Quantum Dots Particles for Intranasal Delivery in Alzheimer's Disease Treatment.

Nanoparticles (NPs) based therapies for Alzheimer's disease (AD) attract interest due to their ability to pass across or bypass the blood-brain barrier. Chitosan (CS) NPs or graphene quantum dots (GQDs) are promising drug carriers with excellent physicochemical and electrical properties. The current study proposes the combination of CS and GQDs in ultrasmall NP form not as drug carriers but as theranostic agents for AD. The microfluidic-based synthesis of the CS/GQD NPs with optimized characteristics makes them ideal for transcellular transfer and brain targeting after intranasal (IN) delivery. The NPs have the ability to enter the cytoplasm of C6 glioma cells in vitro and show dose and time-dependent effects on the viability of the cells. IN administration of the NPs to streptozotocin (STZ) induced AD-like models lead to a significant number of entrances of the treated rats to the target arm in the radial arm water maze (RAWM) test. It shows the positive effect of the NPs on the memory recovery of the treated rats. The NPs are detectable in the brain via in vivo bioimaging due to GQDs as diagnostic markers. The noncytotoxic NPs localize in the myelinated axons of hippocampal neurons. They do not affect the clearance of amyloid β (Aβ) plaques at intercellular space. Moreover, they showed no positive impact on the enhancement of MAP2 and NeuN expression as markers of neural regeneration. The memory improvement in treated AD rats may be due to neuroprotection via the anti-inflammation effect and regulation of the brain tissue microenvironment that needs to be studied.

Adult-onset depletion of sulfatide leads to axonal degeneration with relative myelin sparing.

3-O-sulfogalactosylceramide (sulfatide) constitutes a class of sphingolipids that comprise about 4% of myelin lipids in the central nervous system. Previously, our group characterized a mouse with sulfatide's synthesizing enzyme, cerebroside sulfotransferase (CST), constitutively disrupted. Using these mice, we demonstrated that sulfatide is required for establishment and maintenance of myelin, axoglial junctions, and axonal domains and that sulfatide depletion results in structural pathologies commonly observed in Multiple Sclerosis (MS). Interestingly, sulfatide is reduced in regions of normal appearing white matter (NAWM) of MS patients. Sulfatide reduction in NAWM suggests depletion occurs early in disease development and consistent with functioning as a driving force of disease progression. To closely model MS, an adult-onset disease, our lab generated a "floxed" CST mouse and mated it against the PLP-creERT mouse, resulting in a double transgenic mouse that provides temporal and cell-type specific ablation of the Cst gene (Gal3st1). Using this mouse, we demonstrate adult-onset sulfatide depletion has limited effects on myelin structure but results in the loss of axonal integrity including deterioration of domain organization accompanied by axonal degeneration. Moreover, structurally preserved myelinated axons progressively lose the ability to function as myelinated axons, indicated by the loss of the N1 peak. Together, our findings indicate that sulfatide depletion, which occurs in the early stages of MS progression, is sufficient to drive the loss of axonal function independent of demyelination and that axonal pathology, which is responsible for the irreversible loss of neuronal function that is prevalent in MS, may occur earlier than previously recognized.

Pulsed Electromagnetic Fields Improved Peripheral Nerve Regeneration After Delayed Repair of One Month.

The goal of this study was to determine if postoperative pulsed electromagnetic fields (PEMFs) could improve the neuromuscular rehabilitation after delayed repair of peripheral nerve injuries. Thirty-sixSprague-Dawley rats were randomly divided into sham group, control group, and PEMFs group. The sciatic nerves were transected except for the control group. One month later, the nerve ends of the former two groups were reconnected. PEMFs group of rats was subjected to PEMFs thereafter. Control group and sham group received no treatment. Four and 8 weeks later, morphological and functional changes were measured. Four and eight weeks postoperatively, compared to sham group, the sciatic functional indices (SFIs) of PEMFs group were higher. More axons regenerated distally in PEMFs group. The fiber diameters of PEMFs group were larger. However, the axon diameters and myelin thicknesses were not different between these two groups. The brain-derived neurotrophic factor and vascular endothelial growth factor expressions were higher in PEMFs group after 8 weeks. Semi-quantitative IOD analysis for the intensity of positive staining indicated that there were more BDNF, VEGF, and NF200 in PEMFs group. It's concluded that PEMFs have effect on the axonal regeneration after delayed nerve repair of one month. The upregulated expressions of BDNF and VEGF may play roles in this process. © 2023 Bioelectromagnetics Society.

P.145 Uncovering differences in oligodendrogenesis between human and rodent spinal cord stem cells

Background: Spinal cord regeneration in pre-clinical rodent studies is feasible by promoting oligodendrocyte regrowth, which is necessary for axon myelination. It is uncertain whether human neural stem/progenitor cells (NSPCs) are capable of differentiation into oligodendrocytes, similar to rat. In this study, we compare the functional and transcriptional features of primary spinal cord NSPCs from adult humans and rats. Methods: Oligodendrogenesis between human & rat NSPCs from adult donors and rats was cultured using the Neurosphere assay. NSPCs were exposed to 1% FBS to trigger differentiation & PDGF-AA to promote oligodendrocyte formation. Immunocytochemistry & RNA sequencing compared transcriptomes and analyzing differentially expressed genes. Results: Human NSPCs showed a reduced potential for oligodendrocyte generation compared to rat NSPCs (0.013±0.01% and 0.029±0.01% O4+ after one and two weeks in humans; 4.9±0.4% and 6.3±0.6% O4+ after one and two weeks in rats). PDGF-AA treatment at 40 ng/µL for one week was able to effectively promote oligodendrocyte differentiation in rat NSPCs, but had a minimal effect on human NSPCs (8.5±1.4 fold increase in O4+). OLIG1/2, SOX10, and CNP were enriched in rat NSPCs. Conclusions: We compared oligodendrogenesis potential between human and rat NSPCs and found significantly lower capacity in human NSPCs, possibly hindering successful myelinating techniques.

Fiber counts and architecture of the human dorsal penile nerve

The human penis transmits behaviorally important sensory information via the dorsal penile nerve, which is required for initiation and maintenance of erection. The human penis differs from the penes of other hominids. The lack of a baculum makes the human penis dependent on erectile tissue, which is under control of neural signals activated by tactile stimulation. Accordingly, the penile sensory innervation is crucial for human sexual behavior. To clarify penile innervation, we analyzed the architecture of the dorsal penile nerve of five male subjects who donated their body. We stained the sensory fibers in the penile dorsal nerve with anti-neurofilament H antibody, and identified myelinated axons with Luxol fast blue staining. Furthermore, we visualized nerve bundles as they travel along the shaft of the penis by performing microfocus computed tomography scans after counterstaining penes with iodine. Our results show that the dorsal penile nerve is organized in 25–45 loosely packed nerve bundles, running mediodorsally in the shaft of the penis. This organization corresponds to that in penes of other mammalian species, but differs from the organization of the other peripheral sensory nerves. Around half of the dorsal penile nerve fibers were myelinated and a human hemipenis contained a total of 8290 ± 2553 (mean ± SD) axons. Thus, the number of sensory axons in the human dorsal penile nerve is higher than in other species described so far. The large fraction of unmyelinated nerve fibers suggests that the conduction speed is not a crucial aspect of penile sensory transmission.

Alzheimer's Disease-Related Phospho-Tau181 Signals Are Localized toDemyelinated Axons of Parvalbumin-Positive GABAergic Interneurons in an App Knock-In Mouse Model of Amyloid-β Pathology.

The tau protein phosphorylated at Thr181 (p-tau181) in cerebrospinal fluid and blood is a sensitive biomarker for Alzheimer's disease (AD). Increased p-tau181 levels correlate well with amyloid-β (Aβ) pathology and precede neurofibrillary tangle formation in the early stage of AD; however, the relationship between p-tau181 and Aβ-mediated pathology is less well understood. We recently reported that p-tau181 represents axonal abnormalities in mice with Aβ pathology (AppNLGF). However, from which neuronal subtype(s) these p-tau181-positive axons originate remains elusive. The main purpose of this study is to differentiate neuronal subtype(s) and elucidate damage associated with p-tau181-positive axons by immunohistochemical analysis of AppNLGF mice brains. Colocalization between p-tau181 and (1) unmyelinated axons positive for vesicular acetylcholine transporter or norepinephrine transporter and (2) myelinated axons positive for vesicular glutamate transporter, vesicular GABA transporter, or parvalbumin in the brains of 24-month-old AppNLGF and control mice without Aβ pathology were analyzed. The density of these axons was also compared. Unmyelinated axons of cholinergic or noradrenergic neurons did not overlap with p-tau181. By contrast, p-tau181 signals colocalized with myelinated axons of parvalbumin-positive GABAergic interneurons but not of glutamatergic neurons. Interestingly, the density of unmyelinated axons was significantly decreased in AppNLGF mice, whereas that of glutamatergic, GABAergic, or p-tau181-positive axons was less affected. Instead, myelin sheaths surrounding p-tau181-positive axons were significantly reduced in AppNLGF mice. This study demonstrates that p-tau181 signals colocalize with axons of parvalbumin-positive GABAergic interneurons with disrupted myelin sheaths in the brains of a mouse model of Aβ pathology.

α7 Nicotinic Acetylcholine Receptors May Improve Schwann Cell Regenerating Potential via Metabotropic Signaling Pathways.

Schwann cells (SCs) are glial cells involved in peripheral axon myelination. SCs also play a strategic role after peripheral nerve injury, regulating local inflammation and axon regeneration. Our previous studies demonstrated the presence of cholinergic receptors in SCs. In particular, the α7 nicotinic acetylcholine receptors (nAChRs) are expressed in SCs after peripheral axotomy, suggesting their involvement in the regulation of SC-regenerating properties. To clarify the role that α7 nAChRs may play after peripheral axon damage, in this study we investigated the signal transduction pathways triggered by receptor activation and the effects produced by their activation. Both ionotropic and metabotropic cholinergic signaling were analyzed by calcium imaging and Western blot analysis, respectively, following α7 nAChR activation. In addition, the expression of c-Jun and α7 nAChRs was evaluated by immunocytochemistry and Western blot analysis. Finally, the cell migration was studied by a wound healing assay. Activation of α7 nAChRs, activated by the selective partial agonist ICH3, did not induce calcium mobilization but positively modulated the PI3K/AKT/mTORC1 axis. Activation of the mTORC1 complex was also supported by the up-regulated expression of its specific p-p70 S6KThr389 target. Moreover, up-regulation of p-AMPKThr172, a negative regulator of myelination, was also observed concomitantly to an increased nuclear accumulation of the transcription factor c-Jun. Cell migration and morphology analyses proved that α7 nAChR activation also promotes SC migration. Our data demonstrate that α7 nAChRs, expressed by SCs only after peripheral axon damage and/or in an inflammatory microenvironment, contribute to improve the SCs regenerating properties. Indeed, α7 nAChR stimulation leads to an upregulation of c-Jun expression and promotes Schwann cell migration by non-canonical pathways involving the mTORC1 activity.

Morphological Distribution Patterns and Neuroimmune Communication of Ganglia in Molly Fish (Poecilia sphenops, Valenciennes 1846)

Twenty-four adult molly fish (Poecilia sphenops, Valenciennes 1846) were collected to study the morphology and distribution of ganglia using histological, immunohistochemical, and electron microscopy and focusing on their relation to the immune cells. The ganglia were classified spatially into cranial and spinal, and functionally into sensory and autonomic. Spinal ganglia (dorsal root ganglia, DRG) contained large close ganglionic cells, enclosed by satellite cells, as well as bundles of both myelinated and non-myelinated nerve fibers. There are glial cells, immune cells and telocytes close to the ganglion. In addition, oligodendrocytes were closely related to myelinated axons. Glial fibrillary acidic protein (GFAP) expression was confined to the glia cells and the nerve fibers in the cervical ganglia next to the gills, and surprisingly, in the large ganglionic cells of the DRG. The vestibular ganglia were large, connected to the hind brain, and contained numerous neurons packed in columns. The cervical ganglia were large and observed around the pseudobranch, head kidney, and thymus. Their neurons are randomly distributed, and nerve fibers are peripherally situated. CD3-positive T-lymphocytes, dendritic cells, and CD68-positive macrophages were in close contact with the ganglia. Furthermore, the ganglia around the head kidney showed positive Iba1-expressing cells. Most ganglion cells and nerve fibers in the DRG, autonomic, and vestibular ganglia showed moderate to strong S-100 immunoreactivity. The enteric glia, CD68-expressing macrophages, and acetylcholine (Ach)-expressing neurons were observed along the muscular layer of the intestinal wall. In conclusion, different ganglia of molly fish displayed direct communication with immune cells which support and maintain healthy ganglionic cells.

Mechanism of Bisphenol F Affecting Motor System and Motor Activity in Zebrafish.

Bisphenol F (BPF; 4,4'-dihydroxydiphenylmethane) is one of the most frequently used compounds in the manufacture of plastics and epoxy resins. Previous studies have demonstrated that BPF affects locomotor behavior, oxidative stress, and neurodevelopment in zebrafish. However, its neurotoxic effects are controversial, and the underlying mechanisms are unclear. In order to determine whether BPF affects the motor system, we exposed zebrafish embryos to BPF and assessed behavioral, histological, and neurochemical changes. Spontaneous locomotor behavior and startle response were significantly decreased in BPF-treated zebrafish larvae compared with control larvae. BPF induced motor degeneration and myelination defects in zebrafish larvae. In addition, embryonic exposure to BPF resulted in altered metabolic profiles of neurochemicals, including neurotransmitters and neurosteroids, which may impact locomotion and motor function. In conclusion, exposure to BPF has the potential to affect survival, motor axon length, locomotor activity, myelination, and neurochemical levels of zebrafish larvae.

(001) How many Nerve Fibers Innervate the Human Clitoris? A Histomorphometric Evaluation of the Dorsal Nerve of the Clitoris

Abstract Introduction It is frequently quoted in mainstream media that the clitoris has “8000 nerve endings”. However, no study has yet quantified the number of nerve fibers (axons) innervating the human clitoris. The dorsal nerve of the clitoris (DNC) is the primary source of somatic clitoral innervation. Reporting the number of axons in the DNC is an important step in our understanding of clitoral innervation and sexual response with implications for many fields of medical practice. Reconstruction following iatrogenic injuries to the dorsal clitoral nerve and restoration following cases of genital mutilation will benefit from a better understanding of the DNC. This knowledge will also be an important factor contributing to the optimization of nerve selection and sensory outcomes in gender-affirming genital surgery. Objective The purpose of this study is to quantify the number of nerve fibers in the human dorsal nerve of the clitoris (DNC) and report the approximate number of nerve fibers that innervate the human clitoris. Methods Following institutional approval and informed consent, dorsal clitoral nerve samples were obtained from 7 transmasculine patients undergoing gender-affirming phalloplasty surgery. In phalloplasty one of the two dorsal clitoral nerves is used to coapt to a sensory nerve in the phallus in order to provide for erogenous phallic sensation. At the time of nerve coaptation, a small excess of dorsal clitoral nerve (5 mm) was collected for analysis at the proximal level of the clitoral body, just after appearance of the dorsal clitoral nerve from underneath the pubic symphysis. The samples were placed into 3% glutaraldehyde fixative, post-fixed in 1% osmium tetroxide and serially dehydrate in ethanol and toluene. They were then embedded in araldite, sectioned on ultramicrotome into 1 μm cross sections and counter-stained with 1% toluidine blue. Histomorphometric evaluation was performed at 1000x magnification using a Leitz Laborlux S microscope and image analysis software (Clemex Vision Professional, Longueuil, Quebec) in order to obtain a nerve fiber count from each dorsal clitoral nerve specimen. Descriptive statistics were performed to yield a mean and range of the number of nerve fibers in the DNC. Assuming anatomic symmetry between bilateral DNC, the total number of somatic nerve fibers innervating the human clitoris was obtained by doubling the mean count of the DNC. Results 7 samples of the DNC were collected. The mean number of nerve fibers in the human dorsal clitoral nerve was 5140 with a range of 4926 to 5543. The mean number of myelinated nerve fibers innervating the human clitoris was 10281 (range 9852 to 11086). Conclusions This study is the first to report the number of myelinated nerve fibers in the human dorsal clitoral nerve and approximate the number of nerve fibers innervating the human clitoris. Findings demonstrate high and consistent myelinated axon counts and density. When taking into account the presence of uncounted unmyelinated axons, it is clear that far more than 8000 fibers innervate the human clitoris. Disclosure No

Global DNA Methylation and Hydroxymethylation Levels in PBMCs Are Altered in RRMS Patients Treated with IFN-β and GA-A Preliminary Study.

Multiple sclerosis (MS) is a chronic disease affecting the central nervous system (CNS) due to an autoimmune attack on axonal myelin sheaths. Epigenetics is an open research topic on MS, which has been investigated in search of biomarkers and treatment targets for this heterogeneous disease. In this study, we quantified global levels of epigenetic marks using an ELISA-like approach in Peripheral Blood Mononuclear Cells (PBMCs) from 52 patients with MS, treated with Interferon beta (IFN-β) and Glatiramer Acetate (GA) or untreated, and 30 healthy controls. We performed media comparisons and correlation analyses of these epigenetic markers with clinical variables in subgroups of patients and controls. We observed that DNA methylation (5-mC) decreased in treated patients compared with untreated and healthy controls. Moreover, 5-mC and hydroxymethylation (5-hmC) correlated with clinical variables. In contrast, histone H3 and H4 acetylation did not correlate with the disease variables considered. Globally quantified epigenetic DNA marks 5-mC and 5-hmC correlate with disease and were altered with treatment. However, to date, no biomarker has been identified that can predict the potential response to therapy before treatment initiation.

ADAMTS4 Enhances Oligodendrocyte Differentiation and Remyelination by Cleaving NG2 Proteoglycan and Attenuating PDGFRα Signaling.

Although NG2 is known to be selectively expressed in oligodendrocyte precursor cells (OPCs) for many years, its expressional regulation and functional involvement in oligodendrocyte differentiation have remained elusive. Here, we report that the surface-bound NG2 proteoglycan can physically bind to PDGF-AA and enhances PDGF receptor alpha (PDGFRα) activation of downstream signaling. During differentiation stage, NG2 protein is cleaved by A disintegrin and metalloproteinase with thrombospondin motifs type 4 (Adamts4), which is highly upregulated in differentiating OPCs but gradually downregulated in mature myelinating oligodendrocytes. Genetic ablation of Adamts4 gene impedes NG2 proteolysis, leading to elevated PDGFRα signaling but impaired oligodendrocyte differentiation and axonal myelination in both sexes of mice. Moreover, Adamts4 deficiency also lessens myelin repair in adult brain tissue following Lysophosphatidylcholine-induced demyelination. Thus, Adamts4 could be a potential therapeutic target for enhancing oligodendrocyte differentiation and axonal remyelination in demyelinating diseases.SIGNIFICANCE STATEMENT NG2 is selectively expressed in OPCs and downregulated during differentiation stage. To date, the molecular mechanism underlying the progressive removal of NG2 surface proteoglycan in differentiating OPCs has been unknown. In this study, we demonstrate that ADAMTS4 released by differentiating OPCs cleaves surface NG2 proteoglycan, attenuates PDGFRα signaling, and accelerates oligodendrocyte differentiation. In addition, our study also suggests ADAMTS4 as a potential therapeutic target for promoting myelin recovery in demyelinating diseases.

Compromised Myelin and Axonal Molecular Organization Following Adult-Onset Sulfatide Depletion.

3-O-sulfogalactosylceramide, or sulfatide, is a prominent myelin glycosphingolipid reduced in the normal appearing white matter (NAWM) in Multiple Sclerosis (MS), indicating that sulfatide reduction precedes demyelination. Using a mouse model that is constitutively depleted of sulfatide, we previously demonstrated that sulfatide is essential during development for the establishment and maintenance of myelin and axonal integrity and for the stable tethering of certain myelin proteins in the sheath. Here, using an adult-onset depletion model of sulfatide, we employ a combination of ultrastructural, immunohistochemical and biochemical approaches to analyze the consequence of sulfatide depletion from the adult CNS. Our findings show a progressive loss of axonal protein domain organization, which is accompanied by axonal degeneration, with myelin sparing. Similar to our previous work, we also observe differential myelin protein anchoring stabilities that are both sulfatide dependent and independent. Most notably, stable anchoring of neurofascin155, a myelin paranodal protein that binds the axonal paranodal complex of contactin/Caspr1, requires sulfatide. Together, our findings show that adult-onset sulfatide depletion, independent of demyelination, is sufficient to trigger progressive axonal degeneration. Although the pathologic mechanism is unknown, we propose that sulfatide is required for maintaining myelin organization and subsequent myelin-axon interactions and disruptions in these interactions results in compromised axon structure and function.

Drop-off in axonal regeneration along the length of a cross-face nerve graft: An experimental study in rats.

The average nerve graft length utilized in cross-face nerve grafting for reconstruction of facial nerve palsy is 20-22 cm. While the graft length is thought to be one of the greatest determinants of muscle strength, the mechanism through which this happens remains unknown. We studied changes in axonal regeneration along the length of a 2 cm cross-face nerve graft in a rat model. The hypothesis was that axon count would decrease along the length of the graft. A 2 cm nerve graft (sciatic nerve) was used as a cross-face nerve graft in 16 adult female, 210-250 g, Sprague Dawley rats. Thirteen weeks later, 5 mm nerve biopsies were taken at four sites: the facial nerve trunk (control), proximal graft, midpoint of graft (1 cm distal to coaptation) and distal graft (2 cm distal to coaptation). Retrograde nerve labeling with FluoroGold was performed at the biopsied nerve site and the facial motor nucleus was taken 1 week later. Microscopic imaging and manual counting of axons and labeled motor nuclei was performed. Retrograde-labeled motor neuron counts were decreased at the midway point of the graft compared to the facial trunk (1517 ± 335 axons, Δ% = 92.5, p = .01) and even further decreased at the distal end of the graft (269 ± 293 axons, Δ% = 175.5, p = .006). Analysis of the nerve biopsies demonstrated no significant differences in myelinated axon count between the nerve trunk and over the length of the nerve graft (range 6207-7179 axons, Δ% = 14.5, p = .07). In a rat model, the number of regenerating motor neurons drops off along the length of the graft and axon count is preserved due to axon sprouting. How this pattern correlates to ultimate muscle strength remains unknown, but this study provides insight into why shorter grafts may afford better outcomes.