Remak Bundles Research Articles

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.

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.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Sortilin Modulates Schwann Cell Signaling and Remak Bundle Regeneration Following Nerve Injury.

Peripheral nerve regeneration relies on the ability of Schwann cells to support the regrowth of damaged axons. Schwann cells re-differentiate when reestablishing contact with the sprouting axons, with large fibers becoming remyelinated and small nociceptive fibers ensheathed and collected into Remak bundles. We have previously described how the receptor sortilin facilitates neurotrophin signaling in peripheral neurons via regulated trafficking of Trk receptors. This study aims to characterize the effects of sortilin deletion on nerve regeneration following sciatic crush injury. We found that Sort1–/– mice displayed functional motor recovery like that of WT mice, with no detectable differences in relation to nerve conduction velocities and morphological aspects of myelinated fibers. In contrast, we found abnormal ensheathment of regenerated C-fibers in injured Sort1–/– mice, demonstrating a role of sortilin for Remak bundle formation following injury. Further studies on Schwann cell signaling pathways showed a significant reduction of MAPK/ERK, RSK, and CREB phosphorylation in Sort1–/– Schwann cells after stimulation with neurotrophin-3 (NT-3), while Schwann cell migration and myelination remained unaffected. In conclusion, our results demonstrate that loss of sortilin blunts NT-3 signaling in Schwann cells which might contribute to the impaired Remak bundle regeneration after sciatic nerve injury.

The Role of Schwann Cells in the Onset of Neuropathic Pain in Neurofibromatosis-1

Neurofibromatosis type 1 (NF1) is a genetic disorder that predisposes individuals to benign tumors in the peripheral nervous system (PNS). Pain is a primary cause of decreased quality of life in NF1 patients. Specific loss of Nf1 in SCs and SC precursors is responsible for neurofibroma formation, but the role of SCs in nociception and pain development in NF1 is not known. Here, we wanted to determine whether Schwann cells (SCs), sensory neurons, or both contributed to neuropathic pain-like behaviors in an animal model of NF1. We performed behavioral assessments of mechanical sensitivity, and single unit electrophysiological recordings of sensory neurons using our ex vivo preparation from mice with SC specific (DhhCre;Nf1f/f) or sensory neuron specific (PirtCre;Nf1+/f) deletion of Nf1 and compared results to Nf1+/- mice and littermate controls. We then performed similar assessments in animals' wildtype for Nf1 with chemogenetic manipulation of SC calcium activity. Data was correlated with DRG gene expression using realtime PCR and nerve structure using electron microscopy. Findings show that Nf1 deletion in SCs produced mechanical hypersensitivity, while Nf1 deletion in sensory neurons did not. Behavioral hypersensitivity in the SC specific Nf1 mutants, correlated with sensitization of myelinated A-fiber nociceptors and unmyelinated polymodal C-fibers (CPM) to mechanical stimuli. This occurred with Remak bundle disruption but not tumor formation. Chemo-genetic manipulation of SC calcium activity induced mechanical hypersensitivity at the behavioral level and produced sensitization of CPM neurons to mechanical stimuli compared to CNO treated controls. The observed peripheral sensitization correlated with elevation of cytokines and growth factors in the L2/L3 DRGs. This suggests that alterations in SCs are directly responsible for changes in specific sensory neuron subtypes that possibly modulate neuropathic pain development in NF1. This study may help to identify novel cell-specific treatment strategies to ameliorate pain in NF1 patients.

P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis.

Neurofibromatosis type 1 (NF1) is characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. We identified the G-protein-coupled receptor (GPCR) P2ry14 in human neurofibromas, neurofibroma-derived SC precursors (SCPs), mature SCs, and mouse SCPs. Mouse Nf1-/- SCP self-renewal was reduced by genetic or pharmacological inhibition of P2ry14. In a mouse model of NF1, genetic deletion of P2ry14 rescued low cAMP signaling, increased mouse survival, delayed neurofibroma initiation, and improved SC Remak bundles. P2ry14 signals via Gi to increase intracellular cAMP, implicating P2ry14 as a key upstream regulator of cAMP. We found that elevation of cAMP by either blocking the degradation of cAMP or by using a P2ry14 inhibitor diminished NF1-/- SCP self-renewal in vitro and neurofibroma SC proliferation in in vivo. These studies identify P2ry14 as a critical regulator of SCP self-renewal, SC proliferation, and neurofibroma initiation.

A genetic compensatory mechanism regulated by Jun and Mef2d modulates the expression of distinct class IIa Hdacs to ensure peripheral nerve myelination and repair.

The class IIa histone deacetylases (HDACs) have pivotal roles in the development of different tissues. Of this family, Schwann cells express Hdac4, 5, and 7 but not Hdac9. Here, we show that a transcription factor regulated genetic compensatory mechanism within this family of proteins, blocks negative regulators of myelination ensuring peripheral nerve developmental myelination and remyelination after injury. Thus, when Hdac4 and 5 are knocked-out from Schwann cells in mice, a JUN-dependent mechanism induces the compensatory overexpression of Hdac7 permitting, although with a delay, the formation of the myelin sheath. When Hdac4, 5, and 7 are simultaneously removed, the myocyte-specific enhancer-factor d (MEF2D) binds to the promoter and induces the de novo expression of Hdac9, and although several melanocytic lineage genes are misexpressed and Remak bundle structure is disrupted, myelination proceeds after a long delay. Thus, our data unveil a finely tuned compensatory mechanism within the class IIa Hdac family, coordinated by distinct transcription factors, that guarantees the ability of Schwann cells to myelinate during development and remyelinate after nerve injury.

Human organ donor-derived vagus nerve biopsies allow for well-preserved ultrastructure and high-resolution mapping of myelinated and unmyelinated fibers

The vagus nerve provides motor, sensory, and autonomic innervation of multiple organs, and electrical vagus nerve stimulation (VNS) provides an adjunctive treatment option for e.g. medication-refractory epilepsy and treatment-resistant depression. The mechanisms of action for VNS are not known, and high-resolution anatomical mapping of the human vagus nerve is needed to better understand its functional organization. Electron microscopy (EM) is required for the detection of both myelinated and unmyelinated axons, but access to well-preserved human vagus nerves for ultrastructural studies is sparse. Intact human vagus nerve samples were procured intra-operatively from deceased organ donors, and tissues were immediately immersion fixed and processed for EM. Ultrastructural studies of cervical and sub-diaphragmatic vagus nerve segments showed excellent preservation of the lamellated wall of myelin sheaths, and the axolemma of myelinated and unmyelinated fibers were intact. Microtubules, neurofilaments, and mitochondria were readily identified in the axoplasm, and the ultrastructural integrity of Schwann cell nuclei, Remak bundles, and basal lamina was also well preserved. Digital segmentation of myelinated and unmyelinated axons allowed for determination of fiber size and myelination. We propose a novel source of human vagus nerve tissues for detailed ultrastructural studies and mapping to support efforts to refine neuromodulation strategies, including VNS.

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 Aβ 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.

Peripheral nerve pathology in sickle cell disease mice.

Introduction:Many patients with sickle cell disease (SCD) suffer from chronic pain, which is often described as neuropathic in nature. Although vascular and inflammatory pathology undoubtedly contribute to the SCD pain experience, the nociceptive signals that ultimately drive symptoms are detected and transmitted by peripheral sensory neurons. To date, no systematic histological examination of peripheral nerves has been completed in patients or mouse models of SCD to diagnose disease-related neuropathy.Objectives:In this brief report, we compared peripheral nerve morphology in tissues obtained from Berkeley transgenic SCD mice and control animals.Methods:Sciatic nerves were visualized using light and transmission electron microscopy. Myelin basic protein expression was assessed through Western blot. Blood–nerve barrier permeability was measured using Evan's blue plasma extravasation.Results:Peripheral fibers from SCD mice have thinner myelin sheaths than control mice and widespread myelin instability as evidenced by myelin sheath infolding and unwrapping. Deficits are also observed in nonmyelinating Schwann cell structures; Remak bundles from SCD nerves contain fewer C fibers, some of which are not fully ensheathed by the corresponding Schwann cell. Increased blood–nerve barrier permeability and expression of myelin basic protein are noted in SCD tissue.Conclusions:These data are the first to characterize Berkeley SCD mice as a naturally occurring model of peripheral neuropathy. Widespread myelin instability is observed in nerves from SCD mice. This pathology may be explained by increased permeability of the blood–nerve barrier and, thus, increased access to circulating demyelinating agents at the level of primary sensory afferents.

Glial-derived growth factor and pleiotrophin synergistically promote axonal regeneration in critical nerve injuries

The repair of nerve gap injuries longer than 3 cm is limited by the need to sacrifice donor tissue and the morbidity associated with the autograft gold standard, while decellularized grafts and biodegradable conduits are effective only in short nerve defects. The advantage of isogenic nerve implants seems to be the release of various growth factors by the denervated Schwann cells. We evaluated the effect of vascular endothelial growth factor, neurotrophins, and pleiotrophin (PTN) supplementation of multi-luminal conduits, in the repair of 3 and 4 cm nerve gaps in the rabbit peroneal nerve. In vitro screening revealed a synergistic regenerative effect of PTN with glial-derived neurotrophic factor (GDNF) in promoting sensory axon density, and motor axonal growth from spinal cord explants. In vivo, pleiotrophins were able to support nerve regrowth across a 3 cm gap. In the 4 cm lesions, PTN-GDNF had a modest effect in the number of axons distal to the implant, while increasing the mean axon diameter (1 ± 0.4; p ≤ 0.001) over PTN or GDNF alone (0.80 ± 0.2, 0.84 ± 0.5; respectively). Some regenerated axons reinnervated muscle targets as indicated by neuromuscular junction staining. However, many were wrapped in Remak bundles, suggesting a delay in axonal sorting, explaining the limited electrophysiological function of the reinnervated muscle, and the modest recovery in toe spreading in the PTN-GDNF repaired animals. These results support the use of synergistic neurotrophic/pleiotrophic growth factors in long gap repair and underscore the need for re-myelination strategies distal to the injury site. Statement of SignificanceNerve injuries due to trauma or tumor resection often result in long gaps that are challenging to repair. The best clinical option demands the use of autologous grafts that are associated with serious side effects. Bioengineered nerves are considered a good alternative, particularly if supplemented with growth factors, but current options do not match the regenerative capacity of autografts. This study revealed the synergistic effect of neurotrophins and pleiotrophins designed to achieve a broad cellular regenerative effect, and that GDNF-PTN are able to mediated axonal growth and partial functional recovery in a 4 cm nerve gap injury, albeit delays in remyelination. This report underscores the need for defining an optimal growth factor support for biosynthetic nerve implants.

MTORC1 Activation by Loss of Tsc1 in Myelinating Glia Causes Downregulation of Quaking and Neurofascin 155 Leading to Paranodal Domain Disorganization.

Mutations in human tuberous sclerosis complex (TSC) genes TSC1 and TSC2 are the leading causes of developmental brain abnormalities and large tumors in other tissues. Murine Tsc1/2 have been shown to negatively regulate the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in most tissues, and this pathway has been shown to be essential for proper oligodendrocytes/Schwann cell differentiation and myelination. Here, we report that ablation of Tsc1 gene specifically in oligodendrocytes/Schwann cells activates mTORC1 signaling resulting in severe motor disabilities, weight loss, and early postnatal death. The mutant mice of either sex showed reduced myelination, disrupted paranodal domains in myelinated axons, and disorganized unmyelinated Remak bundles. mRNA and protein expression analyses revealed strong reduction in the RNA–binding protein Quaking (Qk) and the 155 kDa glial Neurofascin (NfascNF155). Re-introduction of exogenous Qk gene in Tsc1 mutant oligodendrocytes restored NfascNF155 protein levels indicating that Qk is required for the stabilization of NfascNF155 mRNA. Interestingly, injection of Rapamycin, a pharmacological mTORC1 inhibitor, to pregnant mothers increased the lifespan of the mutant offspring, restored myelination as well as the levels of Qk and NfascNF155, and consequently the organization of the paranodal domains. Together our studies show a critical role of mTORC1 signaling in the differentiation of myelinating glial cells and proper organization of axonal domains and provide insights into TSC-associated myelinated axon abnormalities.

Protective role of the lipid phosphatase Fig4 in the adult nervous system.

The signaling lipid phosphatidylinositol 3,5-bisphosphate, PI(3,5)P2, functions in vesicular trafficking through the endo-lysosomal compartment. Cellular levels of PI(3,5)P2 are regulated by an enzyme complex comprised of the kinase PIKFYVE, the phosphatase FIG4, and the scaffold protein VAC14. Mutations of human FIG4 cause inherited disorders including Charcot-Marie-Tooth disease type 4J, polymicrogyria with epilepsy, and Yunis-Varón syndrome. Constitutive Fig4-/- mice exhibit intention tremor, spongiform degeneration of neural tissue, hypomyelination, and juvenile lethality. To determine whether PI(3,5)P2 is required in the adult, we generated Fig4flox/-; CAG-creER mice and carried out tamoxifen-induced gene ablation. Global ablation in adulthood leads to wasting, tremor, and motor impairment. Death follows within 2 months of tamoxifen treatment, demonstrating a life-long requirement for Fig4. Histological examinations of the sciatic nerve revealed profound Wallerian degeneration of myelinated fibers, but not C-fiber axons in Remak bundles. In optic nerve sections, myelinated fibers appear morphologically intact and carry compound action potentials at normal velocity and amplitude. However, when iKO mice are challenged with a chemical white matter lesion, repair of damaged CNS myelin is significantly delayed, demonstrating a novel role for Fig4 in remyelination. Thus, in the adult PNS Fig4 is required to protect myelinated axons from Wallerian degeneration. In the adult CNS, Fig4 is dispensable for fiber stability and nerve conduction, but is required for the timely repair of damaged white matter. The greater vulnerability of the PNS to Fig4 deficiency in the mouse is consistent with clinical observations in patients with Charcot-Marie-Tooth disease.

Schwann cells regulate sensory neuron gene expression before and after peripheral nerve injury.

Sensory neurons in the PNS demonstrate substantial capacity for regeneration following injury. Recent studies have identified changes in the transcriptome of sensory neurons, which are instrumental for axon regeneration. The role of Schwann cells (SCs) in mediating these changes remains undefined. We tested the hypothesis that SCs regulate expression of genes in sensory neurons before and after PNS injury by comparing mice in which LDL Receptor-related Protein-1 (LRP1) is deleted in SCs (scLRP1-/- mice) with wild-type (scLRP1+/+ ) littermates. LRP1 is an endocytic and cell-signaling receptor that is necessary for normal SC function and the SC response to nerve injury. scLRP1-/- mice represent a characterized model in which the SC response to nerve injury is abnormal. Adult DRG neurons, isolated from scLRP1-/- mice, with or without a conditioning nerve lesion, demonstrated increased neurite outgrowth when cultured ex vivo, compared with neurons from wild-type mice. Following sciatic nerve crush injury, nerve regeneration was accelerated in vivo in scLRP1-/- mice. These results were explained by transcriptional activation of RAGs in DRG neurons in scLRP1-/- mice prior to nerve injury. Although the presence of abnormal SCs in scLRP1-/- mice primed DRG neurons for repair, nerve regeneration in scLRP1-/- mice resulted in abnormalities in ultrastructure, principally in Remak bundles, and with the onset of neuropathic pain. These results demonstrate the importance of SCs in controlling RAG expression by neurons and the potential for this process to cause chronic pain when abnormal. The SC may represent an important target for preventing pain following PNS injury.

Sustained MAPK/ERK Activation in Adult Schwann Cells Impairs Nerve Repair.

The MAPK/ERK pathway has a critical role in PNS development. It is required for Schwann cell (SC) differentiation and myelination; sustained embryonic MAPK/ERK activation in SCs enhances myelin growth overcoming signals that normally end myelination. Excess activation of this pathway can be maladaptive as in adulthood acute strong activation of MAPK/ERK has been shown to cause SC dedifferentiation and demyelination. We used a mouse model (including male and female animals) in which the gain-of-function MEK1DD allele produces sustained MAPK/ERK activation in adult SCs, and we determined the impact of such activation on nerve repair. In the uninjured nerve, MAPK/ERK activation neither impaired myelin nor reactivated myelination. However, in the injured nerve it was detrimental and resulted in delayed repair and functional recovery. In the early phase of injury, the rate of myelin clearance was faster. Four weeks following injury, when nerve repair is normally advanced, myelinated axons of MEK1DD mutants demonstrated higher rates of myelin decompaction, a reduced number of Cajal bands. and decreased internodal length. We noted the presence of abnormal Remak bundles with long SCs processes and reduced numbers of C-fibers/Remak bundle. Both the total number of regenerating axons and the intraepidermal nerve fiber density in the skin were reduced. Sustained activation of MAPK/ERK in adult SCs is therefore deleterious to successful nerve repair, emphasizing the differences in the signaling processes coordinating nerve development and repair. Our results also underline the key role of SCs in axon regeneration and successful target reinnervation.SIGNIFICANCE STATEMENT The MAPK/ERK pathway promotes developmental myelination and its sustained activation in SCs induced continuous myelin growth, compensating for the absence of essential myelination signals. However, the strength of activation is fundamental because acute strong induction of MAPK/ERK in adulthood induces demyelination. What has been unknown is the effect of a mild but sustained MAPK/ERK activation in SCs on nerve repair in adulthood. This promoted myelin clearance but led to abnormalities in nonmyelinating and myelinating SCs in the later phases of nerve repair, resulting in slowed axon regeneration, cutaneous reinnervation, and functional recovery. Our results emphasize the distinct role of the MAPK/ERK pathway in developmental myelination versus remyelination and the importance of signaling between SCs and axons for successful axon regeneration.

Sustained activation of ERK1/2 MAPK in Schwann cells causes corneal neurofibroma.

Recent studies have shown that constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in Schwann cells (SCs) increases myelin thickness in transgenic mice. In this secondary analysis, we report that these transgenic mice develop a postnatal corneal neurofibroma with the loss of corneal transparency by age six months. We show that expansion of non-myelinating SCs, under the control of activated ERK1/2, also drive myofibroblast differentiation that derives from both SC precursors and resident corneal keratocytes. Further, these mice also harbor activated mast cells in the central cornea, which contributes to pathological corneal neovascularization and fibrosis. This breach of corneal avascularity and immune status is associated with the growth of the tumor pannus, resulting in a corneal stroma that is nearly four times its normal size. In corneas with advanced disease, some axons became ectopically myelinated, and the disruption of Remak bundles is evident. To determine whether myofibroblast differentiation was linked to vimentin, we examined the levels and phosphorylation status of this fibrotic biomarker. Concomitant with the early upregulation of vimentin, a serine 38-phosphorylated isoform of vimentin (pSer38vim) increased in SCs, which was attributed primarily to the soluble fraction of protein-not the cytoskeletal portion. However, the overexpressed pSer38vim became predominantly cytoskeletal with the growth of the corneal tumor. Our findings demonstrate an unrecognized function of ERK1/2 in the maintenance of corneal homeostasis, wherein its over-activation in SCs promotes corneal neurofibromas. This study is also the first report of a genetically engineered mouse that spontaneously develops a corneal tumor.

Chronically denervated distal nerve stump inhibits peripheral nerve regeneration.

Chronically denervated distal nerve stump inhibits peripheral nerve regeneration.

(424) Cross-linked hyaluronic acid in the treatment of lower extremity neuropathic pain

Persistent neuropathic pain presents a special challenge to the clinician as current treatment regimens include opioids, anti-neuropathic adjuvants and nerve blocks provide only modest pain control, and are hindered by dose limiting side effects. While the literature describes the use of cross-linked hyaluronic (CL-HA) acid as a dermal filler, our aim is to assess its safety and efficacy in treating lower extremity neuropathic pain. A 36-month retrospective chart review revealed: 11 patients (4 female/7 male); Average: Pain duration-79.5 mos. (4-240); Age-54.7 yrs. (27-82). Patients underwent targeted, neural matrix antinociception injection of CL-HA: Buttocks, 1 (9%); Thigh, 1 (9%); Feet, 9 (82%). Patients approved and consented to the use of their clinical data. FDA approved CL-HA dermal filler agents use was off-label. Average: Dose given-0.15 cc (0.05-0.2); Time to relief-24 hrs. (0-48); Relief duration-11.7 mos. (2.5 -24); VAPS-1.5/10 (0-3.5); Untoward effects: 0; Results assessed by degree and duration of pain relief from a single injection. Mechanisms underlying these findings are likely multifactorial and require additional study include: 1. CL-HA as a physical, protective shielding and compartment: Blunting activation of spontaneous activity in: C-fiber and Remak bundle afferency, and aberrant nociceptive ephapse; 2. CL-HA anionic depolarization: Polyanionic nature results in a sustained action potential refractory state; 3. CL-HA LMW/HMW-HA mismatch correction and ICAM-1 inflammatory modulation. This results in TSG-6 modulation of the regional, inflammatory activation, thereby permitting CD44/RHAMM interactions to proceed with HMW-HA, stabilizing and restoring the extracellular neural matrix (ECNM) post-injury, immuno-neural cross-talk dysregulation at the level of the ECNM, which resulted in the chronification of pain in the first place. We conclude that targeted, neural matrix antinociception injection of CL-HA is a safe and effective method of treating lower extremity neuropathic pain. Its routine use should be considered early in the treatment of this patient group.

Structural reorganization of the sciatic, peroneal, and tibial nerves during osteosynthesis of lower leg fracture and after fracture consolidation (an experimental study)

The study purpose was to identify histological changes in the peripheral nerves and determine functional significance of subclinical neuropathy in experimental simulation of lower leg fractures. We simulated a tibial shaft fracture in 24 dogs under anesthesia and performed osteosynthesis using an Ilizarov apparatus. After 7, 14, 20, 35–37, and 50 days of fixation using the apparatus and 30, 60–90, and 120 days after removal of the apparatus, the dogs were euthanised. Electromyography was performed before fracture and within the main phases of the experiment. Samples of the sciatic, peroneal, and tibial nerves were studied histologically using computer morphometry of semithin araldite sections. After 37 days of fixation, the amplitude of M-responses of the tibialis anterior and gastrocnemius muscles decreased by 70% compared to the baseline value, increased after removal of the apparatus, but not restored. There were no sighs of nerve damage by bone fragments or wires. The fraction of degenerating myelinated fibers in the studied nerves was less than 13%. Reorganization of the Remak bundles led to a temporary increase in the number density of nerve fibers. Along with axonal atrophy, the peroneal nerve was characterized by demyelination-remyelination, and the tibial nerve was characterized by hypermyelination. The dynamics of numerical density of endoneurial blood vessels was also different. Despite minor sharp degenerative changes in myelinated fibers, persistent axonal atrophy, demyelination, and retrograde changes were not compensated for up to 120 days after removal of the apparatus. Correlations between the morphometric parameters of myelinated fibers and the M-response amplitude indicated a causal role of subclinical neurological changes in functional outcomes of fractures.

The effects of tibial fracture and Ilizarov osteosynthesis on the structural reorganization of sciatic and tibial nerves during the bone consolidation phase and after fixator removal.

Reactive and adaptive changes in mechanically uninjured nerves during fracture healing have not been studied previously although the status of innervation is important for bone union and functional recovery. This study explores whether subclinical nerve fibre degeneration occurs in mechanically uninjured nerves in an animal fracture model and to quantify its extent and functional significance. Twenty-four dogs were deeply anaesthetized and subjected to experimental tibial shaft fracture and Ilizarov osteosynthesis. Before fracture and during the experiment, electromyography was performed. In 7, 14, 20, 35–37 and 50 days of fixation and 30, 60–90 and 120 days after fixator removal, the dogs were euthanized. Samples from sciatic, peroneal and tibial nerves were processed for semithin section histology and morphometry. On the 37th postoperative day, M-response amplitudes in leg muscles were 70 % lower than preoperative ones. After fixator removal, these increased but were not restored to normal values. There were no signs of nerve injuries from bone fragments or wires from the fixator. The incidence of degenerated myelin fibres (MFs) was less than 12 %. Reorganization of Remak bundles (Group C nerve fibres—principally sensory) led to a temporal increase in numerical nerve fibre densities. Besides axonal atrophy, the peroneal nerve was characterized with demyelination–remyelination, while tibial nerve with hypermyelination. There were changes in endoneural vessel densities. In spite of minor acute MF degeneration, sustained axonal atrophy, dismyelination and retrograde changes did not resolve until 120 days after fracture healing. Correlations of morphometric parameters of degenerated MF with M-response amplitudes from electromyography underlie the subclinical neurologic changes in functional outcomes after tibial fractures even when nerves are mechanically uninjured.