Non-myelinating Schwann Cells Research Articles

Gap junction protein connexin 43 and its distribution in damaged nerve cells

BACKGROUND: Connexins, particularly connexin 43, form gap junctions that mediate neuron-glial communication. To date, the expression of connexin 43 in phenotypically distinct neurolemmocytes, in particular non-myelinating (Remak cells) and repair Schwann cells, has not been studied. AIM: To determine the presence of connexin 43 in Schwann cells of the rat sciatic nerve under normal conditions and after mechanical damage by applying a ligature. MATERIALS AND METHODS: The study was carried out on Wistar rats (n = 10). In experimental rats, the sciatic nerve was damaged by applying a ligature (40 s). 7 days after surgery, sciatic nerve segments were isolated for subsequent immunohistochemical study using antibodies to connexin 43 and glial fibrillary acidic protein (GFAP). In the control group rats, segments of intact sciatic nerves were isolated in a similar way. RESULTS: It has been shown that in the endoneurium of the intact rat sciatic nerve there are no cells expressing connexin 43. It was found that 7 days after injury, a large number of connexin-43-immunopositive cells of irregular shape with several processes were identified in the endoneurium of the damaged nerve. There was a lack of expression of connexin 43 in GFAP-containing Schwann cells. CONCLUSIONS: It can be stated that nerve damage leads to active synthesis of the studied protein by endoneurium cells; however, the origin of the cells expressing connexin 43 remains to be elucidated.

Nageotte nodules in human DRG reveal neurodegeneration in painful diabetic neuropathy.

Diabetic neuropathy is frequently accompanied by pain and loss of sensation attributed to axonal dieback. We recovered dorsal root ganglia (DRGs) from 90 organ donors, 19 of whom had medical indices for diabetic painful neuropathy (DPN). Nageotte nodules, dead sensory neurons engulfed by non-neuronal cells, were abundant in DPN DRGs and accounted for 25% of all neurons. Peripherin-and Nav1.7-positive dystrophic axons invaded Nageotte nodules, forming small neuroma-like structures. Using histology and spatial sequencing, we demonstrate that Nageotte nodules are mainly composed of satellite glia and non-myelinating Schwann cells that express SPP1 and are intertwined with sprouting sensory axons originating from neighboring neurons. Our findings solve a 100-year mystery of the nature of Nageotte nodules linking these pathological structures to pain and sensory loss in DPN.

Cilostazol Ameliorates Motor Dysfunction and Schwann Cell Impairment in Streptozotocin-Induced Diabetic Rats.

This study investigated the effects of cilostazol on motor dysfunction, spinal motor neuron abnormalities, and schwannopathy in rats with diabetes. Diabetes mellitus (DM) was induced in rats via femoral intravenous streptozotocin (STZ) injection (60 mg/kg). After successful DM induction, cilostazol was administered on day 15 via oral gavage (100 mg/kg/day) for 6 weeks until sacrifice. Behavioral assays, including motor function, were performed weekly. The sciatic nerve, L5 spinal cord, and spinal ventral root were collected to evaluate the expression of the glial fibrillary acidic protein (GFAP), myelin protein zero (P0), and choline acetyltransferase (ChAT) by immunofluorescence and Western blotting. DM rats displayed decreased running speeds, running distances, and toe spread but increased foot pressure. In addition, loss of non-myelinating Schwann cells and myelin sheaths was observed in the sciatic nerve and L5 spinal ventral root. Reduced numbers of motor neurons were also found in the L5 spinal ventral horn. Cilostazol administration significantly potentiated running speed and distance; increased hind paw toe spread; and decreased foot pressure. In the sciatic nerve and L5 spinal ventral root, cilostazol treatment significantly improved non-myelinated Schwann cells and increased myelin mass. ChAT expression in motor neurons in the spinal ventral horn was improved, but not significantly. Cilostazol administration may protect sensorimotor function in diabetic rats.

Role of Piezo2 in Schwann Cell Volume Regulation and Its Impact on Neurotrophic Release Regulation.

Tactile perception relies on mechanoreceptors and nerve fibers, including c-fibers, Aβ-fibers and Aδ-fibers. Schwann cells (SCs) play a crucial role in supporting nerve fibers, with non-myelinating SCs enwrapping c-fibers and myelinating SCs ensheathing Aβ and Aδ fibers. Recent research has unveiled new functions for cutaneous sensory SCs, highlighting the involvement of nociceptive SCs in pain perception and Meissner corpuscle SCs in tactile sensation. Furthermore, Piezo2, previously associated with Merkel cell tactile sensitivity, has been identified in SCs. The goal of this study was to investigate the channels implicated in SC mechanosensitivity and the release process of neurotrophic factor secretion. Immortalized IFRS1 SCs and human primary SCs generated two distinct subtypes of SCs: undifferentiated and differentiated SCs. Quantitative PCR was employed to evaluate the expression of differentiation markers and mechanosensitive channels, including TRP channels (TRPV4, TRPM7 and TRPA1) and Piezo channels (Piezo1 and Piezo2). To validate the functionality of specific mechanosensitive channels, Ca2+ imaging and electronic cell sizing experiments were conducted under hypotonic conditions, and inhibitors and siRNAs were used. Protein expression was assessed by Western blotting and immunostaining. Additionally, secretome analysis was performed to evaluate the release of neurotrophic factors in response to hypotonic stimulation, with BDNF, a representative trophic factor, quantified using ELISA. Induction of differentiation increased Piezo2 mRNA expression levels both in IFRS1 and in human primary SCs. Both cell types were responsive to hypotonic solutions, with differentiated SCs displaying a more pronounced response. Gd3+ and FM1-43 effectively inhibited hypotonicity-induced Ca2+ transients in differentiated SCs, implicating Piezo2 channels. Conversely, inhibitors of Piezo1 and TRPM7 (Dooku1 and NS8593, respectively) had no discernible impact. Moreover, Piezo2 in differentiated SCs appeared to participate in regulatory volume decreases (RVD) after cell swelling induced by hypotonic stimulation. A Piezo2 deficiency correlated with reduced RVD and prolonged cell swelling, leading to heightened release of the neurotrophic factor BDNF by upregulating the function of endogenously expressed Ca2+-permeable TRPV4. Our study unveils the mechanosensitivity of SCs and implicates Piezo2 channels in the release of neurotrophic factors from SCs. These results suggest that Piezo2 may contribute to RVD, thereby maintaining cellular homeostasis, and may also serve as a negative regulator of neurotrophic factor release. These findings underscore the need for further investigation into the role of Piezo2 in SC function and neurotrophic regulation.

Microstructure white matter changes and serum GFAP among different phenotypes of multiple sclerosis

Background. Multiple sclerosis (MS) is an autoimmune disease that targets the central nervous system (CNS). A patient’s degree of demyelination and axonal degeneration can only be roughly estimated based on clinical symptoms, neurochemical analysis or standard clinical MRI. Magnetic resonance diffusion tensor imaging (DTI) may provide more information on MS pathology than T1- and T2-weighted MRI alone. Glial fibrillary acidic protein (GFAP) uniquely found in astrocytes in the (CNS), non-myelinating Schwann cells in the peripheral nervous system (PNS), and enteric glial cells. GFAP is postulated to be a biomarker of astrocytic damage and reactive astrogliosis. Methods. A total of 60 patients with MS was categorized into three equal groups according to The Multiple Sclerosis Progression Discussion Tool (MSProDiscuss tool): RRMS, SPMS, RRMS with high risk to become SPMS and 20 healthy controls. Baseline clinical characteristics and detailed medical and neurological history were taken into consideration, as well as time of onset of MS, delay in diagnosis, initial symptoms, relapses features and behavior, EDSS and disease modifying therapy. They were subjected to DTI-MRI and blood sampling for GFAP. Results. DTI was able to differentiate between different MS phenotypes and was able to detect progression when we evaluated DTI changes in NAWM in different brain areas as low FA and high MD were associated with progression and increasing disability (p value =0.001). Serum GFAP differs significantly between patients with SPMS or patients in transition, also, it was higher in patients in transition than RRMS or control group (P value <0.001). There was a significant correlation between serum GFAP and DTI changes in NAWM as higher titres of GFAP were associated with lower FA and higher MD values in NAWM of frontal, temporal lobes, and CC body. Conclusion. Serum GFAP in addition to DTI measurable microdamage in NAWM can give us a wide scope of view about potential progression in MS pathology and related astrocytopathy.

Revisiting roles of mast cells and neural cells in keloid: exploring their connection to disease activity.

Mast cells (MCs) and neural cells (NCs) are important in a keloid microenvironment. They might contribute to fibrosis and pain sensation within the keloid. However, their involvement in pathological excessive scarring has not been adequately explored. To elucidate roles of MCs and NCs in keloid pathogenesis and their correlation with disease activity. Keloid samples from chest and back regions were analyzed. Single-cell RNA sequencing (scRNA-seq) was conducted for six active keloids (AK) samples, four inactive keloids (IK) samples, and three mature scar (MS) samples from patients with keloids. The scRNA-seq analysis demonstrated notable enrichment of MCs, lymphocytes, and macrophages in AKs, which exhibited continuous growth at the excision site when compared to IK and MS samples (P = 0.042). Expression levels of marker genes associated with activated and degranulated MCs, including FCER1G, BTK, and GATA2, were specifically elevated in keloid lesions. Notably, MCs within AK lesions exhibited elevated expression of genes such as NTRK1, S1PR1, and S1PR2 associated with neuropeptide receptors. Neural progenitor cell and non-myelinating Schwann cell (nmSC) genes were highly expressed in keloids, whereas myelinating Schwann cell (mSC) genes were specific to MS samples. scRNA-seq analyses of AK, IK, and MS samples unveiled substantial microenvironmental heterogeneity. Such heterogeneity might be linked to disease activity. These findings suggest the potential contribution of MCs and NCs to keloid pathogenesis. Histopathological and molecular features observed in AK and IK samples provide valuable insights into the mechanisms underlying pain and pruritus in keloid lesions.

TEAD1 is crucial for developmental myelination, Remak bundles, and functional regeneration of peripheral nerves.

Previously we showed that the hippo pathway transcriptional effectors, YAP and TAZ, are essential for Schwann cells (SCs) to develop, maintain and regenerate myelin . Although TEAD1 has been implicated as a partner transcription factor, the mechanisms by which it mediates YAP/TAZ regulation of SC myelination are unclear. Here, using conditional and inducible knockout mice, we show that TEAD1 is crucial for SCs to develop and regenerate myelin. It promotes myelination by both positively and negatively regulating SC proliferation, enabling Krox20/Egr2 to upregulate myelin proteins, and upregulating the cholesterol biosynthetic enzymes FDPS and IDI1. We also show stage-dependent redundancy of TEAD1 and that non-myelinating SCs have a unique requirement for TEAD1 to enwrap nociceptive axons in Remak bundles. Our findings establish TEAD1 as a major partner of YAP/TAZ in developmental myelination and functional nerve regeneration and as a novel transcription factor regulating Remak bundle integrity.

Cell type evolution reconstruction across species through cell phylogenies of single-cell RNA sequencing data.

The origin and evolution of cell types has emerged as a key topic in evolutionary biology. Driven by rapidly accumulating single-cell datasets, recent attempts to infer cell type evolution have largely been limited to pairwise comparisons because we lack approaches to build cell phylogenies using model-based approaches. Here we approach the challenges of applying explicit phylogenetic methods to single-cell data by using principal components as phylogenetic characters. We infer a cell phylogeny from a large, comparative single-cell dataset of eye cells from five distantly related mammals. Robust cell type clades enable us to provide a phylogenetic, rather than phenetic, definition of cell type, allowing us to forgo marker genes and phylogenetically classify cells by topology. We further observe evolutionary relationships between diverse vessel endothelia and identify the myelinating and non-myelinating Schwann cells as sister cell types. Finally, we examine principal component loadings and describe the gene expression dynamics underlying the function and identity of cell type clades that have been conserved across the five species. A cell phylogeny provides a rigorous framework towards investigating the evolutionary history of cells and will be critical to interpret comparative single-cell datasets that aim to ask fundamental evolutionary questions.

Severe asthma in horses is associated with increased airway innervation.

Altered innervation structure and function contribute to airway hyperresponsiveness in human asthma, yet the role of innervation in airflow limitation in asthma in horses remains unknown. To characterize peribronchial innervation in horses with asthma. We hypothesized that airway innervation increases in horses with asthma compared with controls. Formalin-fixed lung samples from 8 horses with severe asthma and 8 healthy horses from the Equine Respiratory Tissue Biobank. Ante-mortem lung function was recorded. Blinded case-control study. Immunohistochemistry was performed using rabbit anti-s100 antibody as a neuronal marker for myelinating and non-myelinating Schwann cells. The number and cumulative area of nerves in the peribronchial region and associated with airway smooth muscle were recorded using histomorphometry and corrected for airway size. Both the number (median [IQR]: 1.87 × 10-5 nerves/μm2 [1.28 × 10-5 ]) and the cumulative nerve area (CNA; 1.03 × 10-3 CNA/μm2 [1.57 × 10-3 ]) were higher in the peribronchial region of horses with asthma compared with controls (5.17 × 10-6 nerves/μm2 [3.76 × 10-6 ], 4.14 × 10-4 CNA/μm2 [2.54 × 10-4 ], Mann-Whitney, P = .01). The number of nerves within or lining airway smooth muscle was significantly higher in horses with asthma (4.47 × 10-6 nerves/μm2 [5.75 × 10-6 ]) compared with controls (2.26 × 10-6 nerves/μm2 [1.16 × 10-6 ], Mann-Whitney, P = .03). Asthma in horses is associated with greater airway innervation, possibly contributing to airway smooth muscle remodeling and exacerbating severity of the disease.

Transcriptional profiles of non-neuronal and immune cells in mouse trigeminal ganglia.

Non-neuronal cells constitute 90%-95% of sensory ganglia. These cells, especially glial and immune cells, play critical roles in the modulation of sensory neurons. This study aimed to identify, profile, and summarize the types of trigeminal ganglion (TG) non-neuronal cells in naïve male mice using published and our own data generated by single-cell RNA sequencing, flow cytometry, and immunohistochemistry. TG has five types of non-neuronal cells, namely, glial, fibroblasts, smooth muscle, endothelial, and immune cells. There is an agreement among publications for glial, fibroblasts, smooth muscle, and endothelial cells. Based on gene profiles, glial cells were classified as myelinated and non-myelinated Schwann cells and satellite glial cells. Mpz has dominant expression in Schwann cells, and Fabp7 is specific for SCG. Two types of Col1a2+ fibroblasts located throughout TG were distinguished. TG smooth muscle and endothelial cells in the blood vessels were detected using well-defined markers. Our study reported three types of macrophages (Mph) and four types of neutrophils (Neu) in TG. Mph were located in the neuronal bodies and nerve fibers and were sub-grouped by unique transcriptomic profiles with Ccr2, Cx3cr1, and Iba1 as markers. A comparison of databases showed that type 1 Mph is similar to choroid plexus-low (CPlo) border-associated Mph (BAMs). Type 2 Mph has the highest prediction score with CPhi BAMs, while type 3 Mph is distinct. S100a8+ Neu were located in the dura surrounding TG and were sub-grouped by clustering and expressions of Csf3r, Ly6G, Ngp, Elane, and Mpo. Integrative analysis of published datasets indicated that Neu-1, Neu-2, and Neu-3 are similar to the brain Neu-1 group, while Neu-4 has a resemblance to the monocyte-derived cells. Overall, the generated and summarized datasets on non-neuronal TG cells showed a unique composition of myeloid cell types in TG and could provide essential and fundamental information for studies on cell plasticity, interactomic networks between neurons and non-neuronal cells, and function during a variety of pain conditions in the head and neck regions.

Morphological features of basal laminas of enteral nervous system structures in chronic slow transit constipation

BACKGROUND: Studies on basal laminas in the tissues of the enteric nervous system are few and are carried out on experimental models in vivo and in vitro, performed on animals. The morphological features of the structure and localization of BL, their cellular sources of origin in various tissues of the gastrointestinal tract in normal and pathological conditions remain poorly studied.
 AIM: Study of morphological features and distribution of basal laminas in human colon tissues and their changes in pathology.
 MATERIALS AND METHODS: Fragments of the large intestine, obtained as a result of surgery for chronic slow-transit constipation, performed in S. M. Kirov Military Medical Academy were studied. The selective marker of basal laminas, type IV collagen, as well as neuronal and glial immunohistochemical markers (PGP 9.5, GFAP, S100 proteins) were used in the work
 RESULTS: It has been shown that the greatest immunoreactivity within the intestinal wall is observed in the myenteric membrane, weak in the vessels of the submucosa, locally expressed in the subepithelial region of the mucous membrane (in the upper sections of the crypts). In smooth muscle, basal laminas were found around the smooth muscle cells of the longitudinal and concentric layers of the muscular membrane, mucous membrane, veins and arteries, as well as in the endothelium. It has been shown that the ganglionic Auerbachs plexus is delimited from closely adjacent muscle layers of a continuous basal lamina, similar to the basal plate (glia limitans) of the brain and spinal cord of the CNS. It is clearly defined by its appearance it has the form of a continuous hollow tubular structure. The sources of the formation of the basal plate around the ganglia of Auerbach and Meissners plexuses are various glial elements: in the first plexus, astrocyte-like and non-myelinated Schwann cells (both types located in the neuropil), and in the second plexus, glia of the autonomic nervous system (satellite cells of neurons and neurolemmocytes of postganglionic nerve fibers).
 CONCLUSIONS: For the first time, the transition of basal lamina from the Auerbachs plexus to numerous basal plates of neurolemmocytes of the Remakov fibers of the main terminal nerve plexus, which are involved in the innervation of the smooth muscle cells of the muscular membrane, is shown. Signs of dystrophic changes in the basal lamina structure associated with pathological changes in chronic slow transit constipation (tissue edema, inflammatory reactions, manifestations of agangliosis, gliosis, focal denervation of muscle cells, degeneration of nerve endings of neuromuscular and ganglionic plexuses) are shown in the work.

Characterisation of GFAP-Expressing Glial Cells in the Dorsal Root Ganglion after Spared Nerve Injury.

Satellite glial cells (SGCs), enveloping primary sensory neurons' somas in the dorsal root ganglion (DRG), contribute to neuropathic pain upon nerve injury. Glial fibrillary acidic protein (GFAP) serves as an SGC activation marker, though its DRG satellite cell specificity is debated. We employed the hGFAP-CFP transgenic mouse line, designed for astrocyte studies, to explore its expression within the peripheral nervous system (PNS) after spared nerve injury (SNI). We used diverse immunostaining techniques, Western blot analysis, and electrophysiology to evaluate GFAP+ cell changes. Post-SNI, GFAP+ cell numbers increased without proliferation, and were found near injured ATF3+ neurons. GFAP+ FABP7+ SGCs increased, yet 75.5% of DRG GFAP+ cells lacked FABP7 expression. This suggests a significant subset of GFAP+ cells are non-myelinating Schwann cells (nmSC), indicated by their presence in the dorsal root but not in the ventral root which lacks unmyelinated fibres. Additionally, patch clamp recordings from GFAP+ FABP7-cells lacked SGC-specific Kir4.1 currents, instead displaying outward Kv currents expressing Kv1.1 and Kv1.6 channels specific to nmSCs. In conclusion, this study demonstrates increased GFAP expression in two DRG glial cell subpopulations post-SNI: GFAP+ FABP7+ SGCs and GFAP+ FABP7- nmSCs, shedding light on GFAP's specificity as an SGC marker after SNI.

Integration of single-nuclei RNA-sequencing, spatial transcriptomics and histochemistry defines the complex microenvironment of NF1-associated plexiform neurofibromas

Plexiform neurofibroma (PN) is a leading cause of morbidity in children with the genetic condition Neurofibromatosis Type 1 (NF1), often disfiguring or threatening vital structures. During formation of PN, a complex tumor microenvironment (TME) develops, with recruitment of neoplastic and non-neoplastic cell types being critical for growth and progression. Due to the cohesive cellularity of PN, single-cell RNA-sequencing is difficult and may result in a loss of detection of critical cellular subpopulations. To bypass this barrier, we performed single-nuclei RNA-sequencing (snRNA-seq) on 8 frozen PN samples, and integrated this with spatial transcriptomics (ST) in 4 PN samples and immunohistochemistry to provide morphological context to transcriptomic data. SnRNA-seq analysis definitively charted the heterogeneous cellular subpopulations in the PN TME, with the predominant fraction being fibroblast subtypes. PN showed a remarkable amount of inter-sample homogeneity regarding cellular subpopulation proportions despite being resected from a variety of anatomical locations. ST analysis identified distinct cellular subpopulations which were annotated using snRNA-seq data and correlated with histological features. Schwann cell/fibroblast interactions were identified by receptor/ligand interaction analysis demonstrating a high probability of Neurexin 1/Neuroligin 1 (NRXN1/NLGN1) receptor-ligand cross-talk predicted between fibroblasts and non-myelinated Schwann cells (NM-SC) and subtypes, respectively. We observed aberrant expression of NRXN1 and NLGN1 in our PN snRNA-seq data compared to a normal mouse sciatic nerve single-cell RNA-seq dataset. This pathway has never been described in PN and may indicate a clear and direct communication pathway between putative NM-SC cells of origin and surrounding fibroblasts, potentially driving disease progression. SnRNA-seq integrated with spatial transcriptomics advances our understanding of the complex cellular heterogeneity of PN TME and identify potential novel communication pathways that may drive disease progression, a finding that could provide translational therapy options for patients with these devastating tumors of childhood and early adulthood.

Comprehensive analysis of intercellular communication in the thermogenic adipose niche

Brown adipose tissue (BAT) is responsible for regulating body temperature through adaptive thermogenesis. The ability of thermogenic adipocytes to dissipate chemical energy as heat counteracts weight gain and has gained considerable attention as a strategy against obesity. BAT undergoes major remodeling in a cold environment. This remodeling results from changes in the number and function of brown adipocytes, expanding the network of blood vessels and sympathetic nerves, and changes in the composition and function of immune cells. Such synergistic adaptation requires extensive crosstalk between individual cells in the tissue to coordinate their responses. To understand the mechanisms of intercellular communication in BAT, we apply the CellChat algorithm to single-cell transcriptomic data of mouse BAT. We construct an integrative network of the ligand-receptor interactome in BAT and identify the major signaling inputs and outputs of each cell type. By comparing the ligand-receptor interactions in BAT of mice housed at different environmental temperatures, we show that cold exposure enhances the intercellular interactions among the major cell types in BAT, including adipocytes, adipocyte progenitors, lymphatic and vascular endothelial cells, myelinated and non-myelinated Schwann cells, and immune cells. These interactions are predicted to regulate the remodeling of the extracellular matrix, the inflammatory response, angiogenesis, and neurite growth. Together, our integrative analysis of intercellular communications in BAT and their dynamic regulation in response to housing temperatures provides a new understanding of the mechanisms underlying BAT thermogenesis. The resources presented in this study offer a valuable platform for future investigations of BAT development and thermogenesis.

Nucleoporin Seh1 maintains Schwann cell homeostasis by regulating genome stability and necroptosis

Schwann cells play critical roles in peripheral neuropathies; however, the regulatory mechanisms of their homeostasis remain largely unknown. Here, we show that nucleoporin Seh1, a component of nuclear pore complex, is important for Schwann cell homeostasis. Expression of Seh1 decreases as mice age. Loss of Seh1 leads to activated immune responses and cell necroptosis. Mice with depletion of Seh1 in Schwann cell lineage develop progressive reduction of Schwann cells in sciatic nerves, predominantly non-myelinating Schwann cells, followed by neural fiber degeneration and malfunction of the sensory and motor system. Mechanistically, Seh1 safeguards genome stability by mediating the interaction between SETDB1 and KAP1. The disrupted interaction after ablation of Seh1 derepresses endogenous retroviruses, which triggers ZBP1-dependent necroptosis in Schwann cells. Collectively, our results demonstrate that Seh1 is required for Schwann cell homeostasis by maintaining genome integrity and suggest that decrease of nucleoporins may participate in the pathogenesis of periphery neuropathies.

Single-cell RNA-seq uncovers novel metabolic functions of Schwann cells beyond myelination.

Schwann cells (SCs) support peripheral nerves under homeostatic conditions, independent of myelination, and contribute to damage in prediabetic peripheral neuropathy (PN). Here, we used single-cell RNA sequencing to characterize the transcriptional profiles and intercellular communication of SCs in the nerve microenvironment using the high-fat diet-fed mouse, which mimics human prediabetes and neuropathy. We identified four major SC clusters, myelinating, nonmyelinating, immature, and repair in healthy and neuropathic nerves, in addition to a distinct cluster of nerve macrophages. Myelinating SCs acquired a unique transcriptional profile, beyond myelination, in response to metabolic stress. Mapping SC intercellular communication identified a shift in communication, centered on immune response and trophic support pathways, which primarily impacted nonmyelinating SCs. Validation analyses revealed that neuropathic SCs become pro-inflammatory and insulin resistant under prediabetic conditions. Overall, our study offers a unique resource for interrogating SC function, communication, and signaling in nerve pathophysiology to help inform SC-specific therapies.

In vitro myelination using explant culture of dorsal root ganglia: An efficient tool for analyzing peripheral nerve differentiation and disease modeling.

Peripheral nerves conducting motor and somatosensory signals in vertebrate consist of myelinated and unmyelinated axons. In vitro myelination culture, generated by co-culturing Schwann cells (SCs) and dorsal root ganglion (DRG) neurons, is an indispensable tool for modeling physiological and pathological conditions of the peripheral nervous system (PNS). This technique allows researchers to overexpress or downregulate molecules investigated in neurons or SCs to evaluate the effect of such molecules on myelination. In vitro myelination experiments are usually time-consuming and labor-intensive to perform. Here we report an optimized protocol for in vitro myelination using DRG explant culture. We found that our in vitro myelination using DRG explant (IVMDE) culture not only achieves myelination with higher efficiency than conventional in vitro myelination methods, but also can be used to observe Remak bundle and non-myelinating SCs, which were unrecognizable in conventional methods. Because of these characteristics, IVMDE may be useful in modeling PNS diseases, including Charcot Marie Tooth disease (CMT), in vitro. These results suggest that IVMDE may achieve a condition more similar to peripheral nerve myelination observed during physiological development.

Mesenchymal stem cell therapy to promote functional recovery in a limb transplant model

Abstract Introduction: Battlefield trauma leads to limb amputations. We investigated whether Mesenchymal Stem Cell (MSC) therapy can improve nerve regeneration and functional outcome in limb transplantation (Tx). Methods: We performed orthotopic syngeneic hind-limb Tx in Lewis rats. Following Tx rats received syngeneic MSC (2×10 6; passage ≤6) or vehicle i.v. and locally via hydrogel. Limb sensory function (SF) and motor function (MF) were assessed weekly and at study endpoint (16 weeks) limb tissues were harvested for cell/molecular analysis. Results: SF in the tibial, peroneal/sural and saphenous nerve boundaries recovered as early as 5, 6 and 8 weeks, respectively. Total limb SF did not vary (P>0.05) between MSC and control groups; recovery was gradual at week 8 (Grade 1.4), week 12 (Grade 2.3) and week 16 (2.6) on a scale of Grade 0–3 (0, no function; 3, optimal function). A novel grading system was used to assess MF based on walking track footprints: Grade 0–4 (0, no footprint; 4, heel and 4–5 toe prints). MF was significantly (P<0.05) improved with MSC compared to controls at week 8 (Grade 1.33 Vs 0.5) and at week 16 (Grade 2.66 Vs 0.6). Ex vivoexpanded rat MSCs were CD29 +, CD31 −, CD34 −, CD44 +, CD45 low, CD90 +, MHC Class I +, Class II −and pluripotent. Neurofilament M (axonal marker), S100 and Krox20 (myelinating Schwann cells), Oct6 (Pre-myelinating Schwann cells) and GAP43 (regeneration marker) expressions were increased (P<0.05) and GFAP (non-myelinating Schwann cells) were reduced (P<0.05) in the distal portion of the sciatic nerve in MSC treated rats compared to controls. Conclusion: MSC administration following limb Tx appears to promote limb function recovery and is attributed to enhanced nerve regeneration/myelination/innervation. DoD –CDMRPL-19-0-DM190329 grant (PI, Salgar)

Schwann Cells Are Key Regulators of Corneal Epithelial Renewal.

Corneal sensory nerves protect the cornea from injury. They are also thought to stimulate limbal stem cells (LSCs) to produce transparent epithelial cells constantly, enabling vision. In other organs, Schwann cells (SCs) associated with tissue-innervating axon terminals mediate tissue regeneration. This study defines the critical role of the corneal axon-ensheathing SCs in homeostatic and regenerative corneal epithelial cell renewal. SC localization in the cornea was determined by in situ hybridization and immunohistochemistry with SC markers. In vivo SC visualization and/or ablation were performed in mice with inducible corneal SC-specific expression of tdTomato and/or Diphtheria toxin, respectively. The relative locations of SCs and LSCs were observed with immunohistochemical analysis of harvested genetically SC-prelabeled mouse corneas with LSC-specific antibodies. The correlation between cornea-innervating axons and the appearance of SCs was ascertained using corneal denervation in rats. To determine the limbal niche cellular composition and gene expression changes associated with innervation-dependent epithelial renewal, single-cell RNA sequencing (scRNA-seq) of dissociated healthy, de-epithelized, and denervated cornea limbi was performed. We observed limbal enrichment of corneal axon-associated myelinating and non-myelinating SCs. Induced local genetic ablation of SCs, although leaving corneal sensory innervation intact, markedly inhibited corneal epithelial renewal. scRNA-seq analysis (1) highlighted the transcriptional heterogenicity of cells populating the limbal niche, and (2) identified transcriptional changes associated with corneal innervation and during wound healing that model potential regulatory paracrine interactions between SCs and LSCs. Limbal SCs are required for innervation-dependent corneal epithelial renewal.

Schwann cells and myelin in human peripheral nerve: Major protein components vary with age, axon size and pathology.

We examined major protein components of Schwann cells (SCs) and myelin in normal and diseased human peripheral nerves. We evaluated distributions of neural cell adhesion molecule (NCAM), P0 protein (P0) and myelin basic protein (MBP) in frozen sections of 98 sural nerves. Non-myelinating SC in normal adults contained NCAM, but not P0 or MBP. With chronic axon loss, SC without associated axons (Büngner band cells) often co-stained for both NCAM and P0. Onion bulb cells also co-stained for both P0 and NCAM. Infants had many SC with MBP but no P0. All myelin sheaths contained P0. Myelin around large, and some intermediate-sized, axons co-stained for both MBP and P0. Myelin on other intermediate-sized axons had P0, but no MBP. Regenerated axons often had sheaths with MBP, P0 and some NCAM. During active axon degeneration, myelin ovoids often co-stained for MBP, P0 and NCAM. Demyelinating neuropathy patterns included SC (NCAM) loss, and myelin with abnormally distributed, or reduced, P0. Peripheral nerve SC and myelin have varied molecular phenotypes, related to age, axon size and nerve pathology. In normal adult peripheral nerve, myelin has two different patterns of molecular composition. MBP is mostly absent from myelin around a population of intermediate-sized axons, whereas P0 is present in myelin around all axons. Denervated SCs have a molecular signature that differs from normal SC types. With acute denervation, SCs may stain for both NCAM and MBP. Chronically denervated SCs often stain for both NCAM and P0.