Specific paucity of unmyelinated C-fibers in cutaneous peripheral nerves of the African naked-mole rat: comparative analysis using six species of Bathyergidae

In mammalian peripheral nerves, unmyelinated C-fibers usually outnumber myelinated A-fibers. By using transmission electron microscopy, we recently showed that the saphenous nerve of the naked mole-rat (Heterocephalus glaber) has a C-fiber deficit manifested as a substantially lower C:A-fiber ratio compared with other mammals. Here we determined the uniqueness of this C-fiber deficit by performing a quantitative anatomical analysis of several peripheral nerves in five further members of the Bathyergidae mole-rat family: silvery (Heliophobius argenteocinereus), giant (Fukomys mechowii), Damaraland (Fukomys damarensis), Mashona (Fukomys darlingi), and Natal (Cryptomys hottentotus natalensis) mole-rats. In the largely cutaneous saphenous and sural nerves, the naked mole-rat had the lowest C:A-fiber ratio (～1.5:1 compared with ～3:1), whereas, in nerves innervating both skin and muscle (common peroneal and tibial) or just muscle (lateral/medial gastrocnemius), this pattern was mostly absent. We asked whether lack of hair follicles alone accounts for the C-fiber paucity by using as a model a mouse that loses virtually all its hair as a consequence of conditional deletion of the β-catenin gene in the skin. These β-catenin loss-of function mice (β-cat LOF mice) displayed only a mild decrease in C:A-fiber ratio compared with wild-type mice (4.42 compared with 3.81). We suggest that the selective cutaneous C-fiber deficit in the cutaneous nerves of naked mole-rats is unlikely to be due primarily to lack of skin hair follicles. Possible mechanisms contributing to this unique peripheral nerve anatomy are discussed.     

Phantom limb pain: peripheral neuromodulatory and neuroprosthetic approaches to treatment

Post-amputation phantom limb pain (PLP) is a widespread phenomenon that can have physical, psychological, and functional impacts on amputees who experience the condition. The varying presentations and mechanisms of PLP make it difficult to effectively provide long-term pain relief. Multiple neuromodulatory approaches to treating PLP have focused on electrical stimulation of the peripheral nervous system, with varying degrees of success. More recently, research has been done to study the effects of neuroprosthetic approaches on PLP. Neuroprosthetics combine the use of a functional prosthetic with stimulation to the peripheral nerves in the residual limb. Although many of the neuroprosthetic studies focus on improving function, several have shown preliminary evidence for the reduction of severity of PLP. In this review we provide an overview of the current understanding of the neurological mechanisms that initiate and sustain PLP, as well as the neuromodulatory and neuroprosthetic approaches under development for treatment of the condition. Muscle Nerve 59 :154–167, 2019     

LITAF (SIMPLE) regulates Wallerian degeneration after injury but is not essential for peripheral nerve development and maintenance: Implications for Charcot-Marie-Tooth disease

Missense mutations affecting the LITAF gene (also known as SIMPLE) lead to the dominantly inherited peripheral neuropathy Charcot‐Marie‐Tooth disease type 1C (CMT1C). In this study, we sought to determine the requirement of Litaf function in peripheral nerves, the only known affected tissue in CMT1C. We reasoned that this knowledge is a prerequisite for a thorough understanding of the underlying disease mechanism with regard to potential contributions by Litaf loss of function. In addition, we anticipated to obtain valuable information about the basic function of the Litaf protein in peripheral nerves. To address these issues, we generated mice without Litaf expression using gene disruption in embryonic stem cells and analyzed Litaf‐deficient peripheral nerves during development, in maintenance, and after injury. Our results show that Litaf function is not absolutely required for peripheral nerve development and maintenance. In injured nerves, however, we found that lack of Litaf led to increased numbers of macrophages during Wallerian degeneration, accelerated myelin destruction, and the emergence of more axonal sprouts. Consistent with these data, the migration of Litaf‐deficient macrophages was increased upon chemokine stimulation. We conclude that loss of Litaf function is unlikely to be a major contributor to CMT1C, but modulating effects of macrophages need to be considered in the etiology of the disease. © 2012Wiley Periodicals, Inc.     

Cell cycle inhibitors p21 and p16 are required for the regulation of Schwann cell proliferation

Regulated cell proliferation is a crucial prerequisite for Schwann cells to achieve myelination in development and regeneration. In the present study, we have investigated the function of the cell cycle inhibitors p21 and p16 as potential regulators of Schwann cell proliferation, using p21- or p16-deficient mice. We report that both inhibitors are required for proper withdrawal of Schwann cells from the cell cycle during development and following injury. Postnatal Schwann cells express p21 exclusively in the cytoplasm, first detectable at postnatal day 7. This cytoplasmic p21 expression is necessary for proper Schwann cell proliferation control in the late development of peripheral nerves. After axonal damage, p21 is found in Schwann cell nuclei during the initiation of the proliferation period. This stage is critically regulated by p21, since loss of p21 leads to a strong increase in Schwann cell proliferation. Unexpectedly, p21 levels are upregulated in this phase suggesting that the role of p21 may be more complex than purely inhibitory for the Schwann cell cycle. However, inhibition of Schwann cell proliferation is the overriding crucial function of p21 and p16 in peripheral nerves as revealed by the consequences of loss-of-function in development and after injury. Different mechanisms appear to underlie the inhibitory function, depending on whether p21 is cytoplasmic or nuclear.     

Follow-up evaluation with ultrasonography of peripheral nerve injuries after an earthquake

Published data on earthquake-associated peripheral nerve injury is very limited. Ultrasonography has been proven to be efficient in the clinic to diagnose peripheral nerve injury. The aim of this study was to assess the role of ultrasound in the evaluation of persistent peripheral nerve injuries 1 year after the Wenchuan earthquake. Thirty-four patients with persistent clinical symptoms and neurologic signs of impaired nerve function were evaluated with sonography prior to surgi- cal repair. Among 34 patients, ultrasonography showed that 48 peripheral nerves were entrapped, and 11 peripheral nerves were disrupted. There was one case of misdiagnosis on ultrasonogra- phy. The concordance rate of ultrasonographic findings with those of surgical findings was 98%. A total of 48 involved nerves underwent neurolysis and the symptoms resolved. Only five nerves had scar tissue entrapment. Preoperative and postoperative clinical and ultrasonographic results were concordant, which verified that ultrasonography is useful for preoperative diagnosis and postoperative evaluation of injured peripheral nerves.     

Application of neurotrophic and proangiogenic factors as therapy after peripheral nervous system injury

The intrinsic ability of peripheral nerves to regenerate after injury is extremely limited,especially in case of severe injury.This often leads to poor motor function and permanent disability.Existing approaches for the treatment of injured nerves do not provide appropriate conditions to support survival and growth of nerve cells.This drawback can be compensated by the use of gene therapy and cell therapy-based drugs that locally provide an increase in the key regulators of nerve growth,including neurotrophic factors and extracellular matrix proteins.Each growth factor plays its own specific angiotrophic or neurotrophic role.Currently,growth factors are widely studied as accelerators of nerve regeneration.Particularly noteworthy is synergy between various growth factors,that is essential for both angiogenesis and neurogenesis.Fibroblast growth factor 2 and vascular endothelial growth factor are widely known for their proangiogenic effects.At the same time,fibroblast growth factor 2 and vascular endothelial growth factor stimulate neural cell growth and play an important role in neurodegenerative diseases of the peripheral nervous system.Taken together,their neurotrophic and angiogenic properties have positive effect on the regeneration process.In this review we provide an in-depth overview of the role of fibroblast growth factor 2 and vascular endothelial growth factor in the regeneration of peripheral nerves,thus demonstrating their neurotherapeutic efficacy in improving neuron survival in the peripheral nervous system.     

Current strategies in peripheral nerve repair

The paper presents main research trends focused on peripheral nerves regeneration. Modern surgery techniques in humans require dissection of the nerve bridges, leading to extra mutilation of a patient. The recovery of motor and sensory functions is also unsatisfactory. Therefore scientists are looking for new materials and techniques capable of replacing injured nerve trunks as well as of improving the reinnervation of target tissues. In this kind of experiments chambers joining proximal and distal stumps of transected peripheral nerve are very useful. Such chambers may also contain growth-promoting factors or glial cells supporting the regrowth. Progress in molecular biology and biomaterials engineering will be probably the most critical step to therapeutic intervention in nerve regeneration.     

Current landscape in motoneuron regeneration and reconstruction for motor cranial nerve injuries

The intricate anatomy and physiology of cranial nerves have inspired clinicians and scientists to study their roles in the nervous system. Damage to motor cranial nerves may result from a variety of organic or iatrogenic insults and causes devastating functional impairment and disfigurement. Surgical innovations directed towards restoring function to injured motor cranial nerves and their associated organs have evolved to include nerve repair, grafting, substitution, and muscle transposition. In parallel with this progress, research on tissue-engineered constructs, development of bioelectrical interfaces, and modulation of the regenerative milieu through cellular, immunomodulatory, or neurotrophic mechanisms has proliferated to enhance the available repertoire of clinically applicable reconstructive options. Despite these advances, patients continue to suffer from functional limitations relating to inadequate cranial nerve regeneration, aberrant reinnervation, or incomplete recovery of neuromuscular function. These shortfalls have profound quality of life ramifications and provide an impetus to further elucidate mechanisms underlying cranial nerve denervation and to improve repair. In this review, we summarize the literature on reconstruction and regeneration of motor cranial nerves following various injury patterns. We focus on seven cranial nerves with predominantly efferent functions and highlight shared patterns of injuries and clinical manifestations. We also present an overview of the existing reconstructive approaches, from facial reanimation, laryngeal reinnervation, to variations of interposition nerve grafts for reconstruction. We discuss ongoing endeavors to promote nerve regeneration and to suppress aberrant reinnervation and the development of synkinesis. Insights from these studies will shed light on recent progress and new horizons in understanding the biomechanics of peripheral nerve neurobiology, with emphasis on promising strategies for optimizing neural regeneration and identifying future directions in the field of motor cranial neuron research.     

Schwann cells express erythropoietin receptor and represent a major target for Epo in peripheral nerve injury

Erythropoietin (Epo) expresses potent neuroprotective activity in the peripheral nervous system; however, the underlying mechanism remains incompletely understood. In this study, we demonstrate that Epo is upregulated in sciatic nerve after chronic constriction injury (CCI) and crush injury in rats, largely due to local Schwann cell production. In uninjured and injured nerves, Schwann cells also express Epo receptor (EpoR), and its expression is increased during Wallerian degeneration. CCI increased the number of Schwann cells at the injury site and the number was further increased by exogenously administered recombinant human Epo (rhEpo). To explore the activity of Epo in Schwann cells, primary cultures were established. These cells expressed cell-surface Epo receptors, with masses of 71 and 62 kDa, as determined by surface protein biotinylation and affinity precipitation. The 71-kDa species was rapidly but transiently tyrosine-phosphorylated in response to rhEpo. ERK/MAP kinase was also activated in rhEpo-treated Schwann cells; this response was blocked by pharmacologic antagonism of JAK-2. RhEpo promoted Schwann cell proliferation, as determined by BrdU incorporation. Cell proliferation was ERK/MAP kinase-dependent. These results support a model in which Schwann cells are a major target for Epo in injured peripheral nerves, perhaps within the context of an autocrine signaling pathway. EpoR-induced cell signaling and Schwann cell proliferation may protect injured peripheral nerves and promote regeneration.     

Activated RHOA and peripheral axon regeneration

The regeneration of adult peripheral neurons after transection is slow, incomplete and encumbered by severe barriers to proper regrowth. The role of RHOA GTPase has not been examined in this context. We examined the expression, activity and functional role of RHOA GTPase and its ROK effector, inhibitors of regeneration, during peripheral axon outgrowth. We used qRT-PCR, quantitative immunohistochemistry, and assays of RHOA activation to examine expression in sensory neurons of rats with sciatic transection injuries. In vitro, we exposed dissociated adult sensory neurons, not grown on inhibitory substrates, to a RHOA-ROK inhibitor HA-1077 and measured neurite initiation and outgrowth. In vivo, we exposed early regenerating axons and Schwann cells directly to HA-1077 in a conduit connecting the proximal and distal stumps of transected sciatic nerves. Intact adult dorsal root ganglia sensory neurons expressed RHOA and ROK 1 mRNAs and protein and there were rises in RHOA after injury. Activated GTP-bound RHOA, undetectable in intact ganglia, was dramatically upregulated in both neurons and axons after injury. Adult rat sensory neurons in vitro demonstrated a dose-related increase in the initiation of neurite outgrowth, and in the proportion with long neurites when they were exposed to a ROK antagonist. Regenerative bridges that were directly exposed to the ROK inhibitor had a dose-related rise in the extent and distance of in vivo axon and partnered Schwann cell regrowth within them. RHOA activation and signaling are features of adult peripheral axon regeneration within its own milieu, independent of myelin. Inhibition of its activation may benefit peripheral axon lesions.     

Regeneration of sciatic nerves of adult rats induced by extracts from distal stumps of pre-degenerated peripheral nerves

Despite numerous experimental and clinical attempts to reconstruct injuries of peripheral nerves, the methods developed until now have not been sufficiently effective. We examined the influence of extracts (postmicrosomal fractions) obtained from non-pre-degenerated or 7-day-pre-degenerated distal segments of peripheral nerves on the regeneration of injured sciatic nerves of male adult rats. The extracts were introduced to the site of injury with autologous connective tissue chambers filled with fibrin. Reference groups were treated with brain-derived neurotrophic factor (BDNF) or fibrin only. We examined DiI-labeled motoneurons, toluidine blue-labeled myelinated fibers in the mid-part of the chambers, and AChE-positive nerve endings to assess the regeneration intensity. In addition, the length of fibers regrowing within the chambers was measured. We found that extracts obtained from distal stumps of 7-day-pre-degenerated peripheral nerves enhanced nerve regeneration as strongly as BDNF.     

Disruption of laminin in the peripheral nervous system impedes nonmyelinating Schwann cell development and impairs nociceptive sensory function

The mechanisms controlling the differentiation of immature Schwann cells (SCs) into nonmyelinating SCs is not known. Laminins are extracellular matrix proteins critical for myelinating SC differentiation, but their roles in nonmyelinating SC development have not been established. Here, we show that the peripheral nerves of mutant mice with laminin‐deficient SCs do not form Remak bundles, which consist of a single nonmyelinating SC interacting with multiple unmyelinated axons. These mutant nerves show aberrant L1 and neural cell adhesion molecule (N‐CAM) expression pattern during development. The homophilic and heterophilic interactions of N‐CAM are also impaired in the mutant nerves. Other molecular markers for nonmyelinating SCs, including Egr‐1, glial fibrillary acidic protein, and AN2/NG2, are all absent in adult mutant nerves. Analysis of expression of SC lineage markers demonstrates that nonmyelinating SCs do not develop in mutant nerves. Additionally, mutant mice are insensitive to heat stimuli and show a decreased number of C‐fiber sensory neurons, indicating reduced nociceptive sensory function. These results show that laminin participates in nonmyelinating SC development and Remak bundle formation and suggest a possible role for laminin deficiency in peripheral sensory neuropathies. © 2008 Wiley‐Liss, Inc.     

Ultrasonography of the peripheral nervous system in the early stage of Guillain‐Barré syndrome

Ultrasonography can be used to visualize peripheral nerve abnormalities in immune‐mediated neuropathies. The objective of this study was to prove the role of ultrasonography (US) in acute phase of Guillain‐Barré syndrome (GBS). Systematic ultrasonic measurements of several peripheral nerves including the vagal nerve as well as the sixth cervical nerve root were performed in 18 patients with GBS at days 1–3 after symptom onset and compared to 21 healthy controls. Nerve conduction studies (NCS) of corresponding nerves were undertaken. Consequently, significant differences between the groups were found in compound muscle action potential amplitudes, F‐wave latency, and persistency. Ultrasonic cross‐sectional areas (CSAs) showed significant enlargement in all nerves except of the ulnar nerve (upper arm) and the sural nerve compared to healthy controls, most prominent in proximal and middle median nerve (p < 0.01). The vagal nerve also showed enlargement compared to controls (p < 0.05), which was most pronounced in patients with autonomic dysfunction compared to patients without (p < 0.05). C6 root diameter showed a significant correlation to the amount of cerebrospinal fluid (CSF)‐protein (Pearson correlation, p < 0.05). US shows nerve enlargement in several peripheral nerves including vagal nerve and C6 root in acute phase of GBS and could be an additional diagnostic tool for example, in GBS of atypical onset and autonomic dysfunction.     

A contemporary overview of peripheral nerve research from the Cleveland Clinic Microsurgery Laboratory

Abstract

Despite our better understanding of the pathophysiology of peripheral nervous system and advancements in microsurgical repair techniques, peripheral nerve injuries are still considered as a reconstructive challenge for all surgeons. For achieving a better nerve regeneration and better end organ reinnervation, advanced microsurgical manipulations are parallel with molecular biological discoveries. The field of peripheral nerve research is still developing and includes more sophisticated approach at the basic science level. In our Microsurgery Research Laboratory we have been working on different nerve repair techniques, including sleeve neurorrhaphy, sleeve grafts, single and polyfascicular nerve grafting techniques and studies on nerves in diabetic rats, in addition to the roles of different growth factors and pharmacological agents on peripheral nerve regeneration. New approaches for filling nerve gaps with nerve allografts and tolerance inducing strategies with their effect on nerve regeneration are included into our research armamentarium. In this overview wewill summarize our 15-year experience in peripheral nerve research.     

A contemporary overview of peripheral nerve research from the Cleveland Clinic microsurgery laboratory

Despite our better understanding of the pathophysiology of peripheral nervous system and advancements in microsurgical repair techniques, peripheral nerve injuries are still considered as a reconstructive challenge for all surgeons. For achieving a better nerve regeneration and better end organ reinnervation, advanced microsurgical manipulations are parallel with molecular biological discoveries. The field of peripheral nerve research is still developing and includes more sophisticated approach at the basic science level. In our Microsurgery Research Laboratory we have been working on different nerve repair techniques, including sleeve neurorrhaphy, sleeve grafts, single and polyfascicular nerve grafting techniques and studies on nerves in diabetic rats, in addition to the roles of different growth factors and pharmacological agents on peripheral nerve regeneration. New approaches for filling nerve gaps with nerve allografts and tolerance inducing strategies with their effect on nerve regeneration are included into our research armamentarium. In this overview we will summarize our 15-year experience in peripheral nerve research.     

Ultrasonography of peripheral nerves

During sonographic examination of the extremities using high frequency "small-parts" equipment, peripheral nerves may be identified in virtually all patients. Peripheral nerves have a typical ultrasonographic pattern that correlates well with histologic structure and facilitates differentiation between nerves and tendons. The ability of this technique to depict peripheral nerves makes it possible, in many instances, to study nerve abnormalities in trauma, entrapment syndromes and tumors. Ultrasound can enable differentiation of an endoneural from an extraneural space-occupying lesion and evaluation of the extent and consistency of the lesion, as well as the integrity and dynamic behavior of the nerve involved at follow-up study. The purpose of this review article is to describe the normal ultrasonographic appearance of peripheral nerves and to discuss the potential role of this technique to image nerve lesions noninvasively. A series of paradigmatic ultrasound images of diverse pathologic processes involving peripheral nerves is presented. Although the ultrasound study of peripheral nerves remains in its infancy, with further refinement of ultrasound technology and a more precise knowledge of the ultrasound appearance of the extremities we may be optimistic to the future impact of this technique on diagnosis, treatment and prognosis in patients clinically suspected to have a nerve lesion.     

Cold storage of peripheral nerves: An in vitro assay of cell viability and function

The development of a nerve bank as a source of donor material to repair large defects in peripheral nerve injuries requires an understanding of the influence of cold storage on cell viability and function in these potential nerve grafts. Segments of peripheral nerves from both human and rat were stored in University of Wisconsin Cold Storage Solution (UW) at 4°C for < 12 h, 3 days, and 1, 2, or 3 weeks. Cellular viability was initially assessed by the degree of cellular outgrowth from explants of the stored nerves placed in culture, and then further quantitated by dissociating the cultured nerve explants and calculating the type and number of cells per milligram of peripheral nerve. Rat Schwann cells (SCs) obtained from the stored (control and 1 and 2 weeks) nerves were tested for their functional ability to myelinate dorsal root ganglion (DRG) neurons in culture. Our findings indicate that human and rat peripheral nerves contain few viable SCs and fibroblasts after 3 weeks of cold storage with the quantity of viable cells within the human cold stored peripheral nerves decreasing significantly after 1 week of cold storage. Despite their reduced number, some SCs from rat nerves stored up to 2 weeks are capable of myelinating DRG axons in culture. These results suggest that short intervals (< 1 week) of cold storage will result in potential peripheral nerve grafts containing large populations of functional cells, while long-term (≥ 3 weeks) cold stored peripheral nerves will contain few viable cells. © 1994 Wiley-Liss, Inc.     

Evidence for macrophage-mediated myelin disruption in an animal model for Charcot-Marie-Tooth neuropathy type 1A

Charcot-Marie-Tooth neuropathy type 1A (CMT 1 A) is the most common inherited neuropathy in humans and is mostly caused by a 1.5-Mb tandem duplication of chromosome 17 comprising the gene for the peripheral myelin protein 22-kDa (PMP 22). Although there are numerous studies on the functional role of PMP 22, the mechanisms of myelin degeneration under PMP 22-overexpression conditions have not yet been fully understood. We have shown previously that in mouse mutants hetero- or homozygously deficient for two other myelin components, P0 and C x 32, respectively, immune cells contribute to the demyelinating neuropathy. To test this possibility for PMP 22 overexpression, we investigated a putative mouse model for CMT 1 A, i.e., the mouse strain C 6 1 mildly overexpressing human PMP 22 in peripheral nerves. Electron microscopic and electrophysiologic investigations revealed that this mouse strain develops pathologic features similar to those found in CMT 1 A patients. A novel finding, however, was the upregulation of CD8- and F4/80-positive lymphocytes and macrophages, respectively, in peripheral nerves. The observation that macrophages enter endoneurial tubes of the mutants and obviously phagocytose morphologically normal myelin strongly suggests that the myelin degeneration is mediated at least partially by these phagocytic cells. By gene array technology and quantitative RT-PCR of peripheral nerve homogenates from PMP 22 mutants, monocyte chemoattractant protein-1 (MCP-1; cc l2) could be identified as a putative factor to attract or activate macrophages that attack myelin sheaths in this model of CMT 1 A.