Injury-Induced HSP27 Expression in Peripheral Nervous Tissue Is Not Associated with Any Alteration in Axonal Outgrowth after Immediate or Delayed Nerve Repair

We investigated injury-induced heat shock protein 27 (HSP27) expression and its association to axonal outgrowth after injury and different nerve repair models in healthy Wistar and diabetic Goto-Kakizaki rats. By immunohistochemistry, expression of HSP27 in sciatic nerves and DRG and axonal outgrowth (neurofilaments) in sciatic nerves were analyzed after no, immediate, and delayed (7-day delay) nerve repairs (7- or 14-day follow-up). An increased HSP27 expression in nerves and in DRG at the uninjured side was associated with diabetes. HSP27 expression in nerves and in DRG increased substantially after the nerve injuries, being higher at the site where axons and Schwann cells interacted. Regression analysis indicated a positive influence of immediate nerve repair compared to an unrepaired injury, but a shortly delayed nerve repair had no impact on axonal outgrowth. Diabetes was associated with a decreased axonal outgrowth. The increased expression of HSP27 in sciatic nerve and DRG did not influence axonal outgrowth. Injured sciatic nerves should appropriately be repaired in healthy and diabetic rats, but a short delay does not influence axonal outgrowth. HSP27 expression in sciatic nerve or DRG, despite an increase after nerve injury with or without a repair, is not associated with any alteration in axonal outgrowth.     

PVDF and P(VDF-TrFE) Electrospun Scaffolds for Nerve Graft Engineering: A Comparative Study on Piezoelectric and Structural Properties,and In Vitro Biocompatibility

Polyvinylidene fluoride (PVDF) and its copolymer with trifluoroethylene (P(VDF-TrFE)) are considered as promising biomaterials for supporting nerve regeneration because of their proven biocompatibility and piezoelectric properties that could stimulate cell ingrowth due to their electrical activity upon mechanical deformation. For the first time, this study reports on the comparative analysis of PVDF and P(VDF-TrFE) electrospun scaffolds in terms of structural and piezoelectric properties as well as their in vitro performance. A dynamic impact test machine was developed, validated, and utilised, to evaluate the generation of an electrical voltage upon the application of an impact load (varying load magnitude and frequency) onto the electrospun PVDF (15–20 wt%) and P(VDF-TrFE) (10–20 wt%) scaffolds. The cytotoxicity and in vitro performance of the scaffolds was evaluated with neonatal rat (nrSCs) and adult human Schwann cells (ahSCs). The neurite outgrowth behaviour from sensory rat dorsal root ganglion neurons cultured on the scaffolds was analysed qualitatively. The results showed (i) a significant increase of the β-phase content in the PVDF after electrospinning as well as a zeta potential similar to P(VDF-TrFE), (ii) a non-constant behaviour of the longitudinal piezoelectric strain constant d₃₃, depending on the load and the load frequency, and (iii) biocompatibility with cultured Schwann cells and guiding properties for sensory neurite outgrowth. In summary, the electrospun PVDF-based scaffolds, representing piezoelectric activity, can be considered as promising materials for the development of artificial nerve conduits for the peripheral nerve injury repair.     

Gellan-Xanthan Hydrogel Conduits with Intraluminal Electrospun Nanofibers as Physical,Chemical and Therapeutic Cues for Peripheral Nerve Repair

Optimal levels of functional recovery in peripheral nerve injuries remain elusive due to the architectural complexity of the neuronal environment. Commercial nerve repair conduits lack essential guidance cues for the regenerating axons. In this study, the regenerative potential of a biosimulated nerve repair system providing three types of regenerative cues was evaluated in a 10 mm sciatic nerve-gap model over 4 weeks. A thermo-ionically crosslinked gellan-xanthan hydrogel conduit loaded with electrospun PHBV-magnesium oleate-N-acetyl-cysteine (PHBV-MgOl-NAC) nanofibers was assessed for mechanical properties, nerve growth factor (NGF) release kinetics and PC12 viability. In vivo functional recovery was based on walking track analysis, gastrocnemius muscle mass and histological analysis. As an intraluminal filler, PHBV-MgOl-NAC nanofibers improved matrix resilience, deformation and fracture of the hydrogel conduit. NGF release was sustained over 4 weeks, governed by Fickian diffusion and Case-II relaxational release for the hollow conduit and the nanofiber-loaded conduit, respectively. The intraluminal fibers supported PC12 proliferation by 49% compared to the control, preserved up to 43% muscle mass and gradually improved functional recovery. The combined elements of physical guidance (nanofibrous scaffolding), chemical cues (N-acetyl-cysteine and magnesium oleate) and therapeutic cues (NGF and diclofenac sodium) offers a promising strategy for the regeneration of severed peripheral nerves.     

Blood–Nerve Barrier (BNB) Pathology in Diabetic Peripheral Neuropathy and In Vitro Human BNB Model

In diabetic peripheral neuropathy (DPN), metabolic disorder by hyperglycemia progresses in peripheral nerves. In addition to the direct damage to peripheral neural axons, the homeostatic mechanism of peripheral nerves is disrupted by dysfunction of the blood–nerve barrier (BNB) and Schwann cells. The disruption of the BNB, which is a crucial factor in DPN development and exacerbation, causes axonal degeneration via various pathways. Although many reports revealed that hyperglycemia and other important factors, such as dyslipidemia-induced dysfunction of Schwann cells, contributed to DPN, the molecular mechanisms underlying BNB disruption have not been sufficiently elucidated, mainly because of the lack of in vitro studies owing to difficulties in establishing human cell lines from vascular endothelial cells and pericytes that form the BNB. We have developed, for the first time, temperature-sensitive immortalized cell lines of vascular endothelial cells and pericytes originating from the BNB of human sciatic nerves, and we have elucidated the disruption to the BNB mainly in response to advanced glycation end products in DPN. Recently, we succeeded in developing an in vitro BNB model to reflect the anatomical characteristics of the BNB using cell sheet engineering, and we established immortalized cell lines originating from the human BNB. In this article, we review the pathologic evidence of the pathology of DPN in terms of BNB disruption, and we introduce the current in vitro BNB models.     

Sciatic nerve regeneration with collagen type I hydrogel containing chitosan nanoparticle loaded by insulin

Abstract

The current study was conducted to promote the extent of peripheral nerve regeneration using a collagen type I hydrogel containing chitosan nanoparticle loaded by insulin. The developed hydrogel increased the proliferation rate of Schwann cells as shown by tetrazolium salt-based assay. For functional analysis, the hydrogel was administrated in the sciatic nerve crush injury of Wistar rats. The results of the sciatic functional index, hot plate latency, compound muscle action potential amplitude and weight-loss percentage of the wet gastrocnemius muscle, indicated that the prepared hydrogel could be considered as a potential material for peripheral nerve regeneration.     

Nerve Demyelination Increases Metabotropic Glutamate Receptor Subtype 5 Expression in Peripheral Painful Mononeuropathy

Wallerian degeneration or nerve demyelination, arising from spinal nerve compression, is thought to bring on chronic neuropathic pain. The widely distributed metabotropic glutamate receptor subtype 5 (mGluR5) is involved in modulating nociceptive transmission. The purpose of this study was to investigate the potential effects of mGluR5 on peripheral hypersensitivities after chronic constriction injury (CCI). Sprague-Dawley rats were operated on with four loose ligatures around the sciatic nerve to induce thermal hyperalgesia and mechanical allodynia. Primary afferents in dermis after CCI exhibited progressive decreases, defined as partial cutaneous denervation; importantly, mGluR5 expressions in primary afferents were statistically increased. CCI-induced neuropathic pain behaviors through the intraplantar injections of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective mGluR5 antagonist, were dose-dependently attenuated. Furthermore, the most increased mGluR5 expressions in primary afferents surrounded by reactive Schwann cells were observed at the distal CCI stumps of sciatic nerves. In conclusion, these results suggest that nerve demyelination results in the increases of mGluR5 expression in injured primary afferents after CCI; and further suggest that mGluR5 represents a main therapeutic target in developing pharmacological strategies to prevent peripheral hypersensitivities.     

Nanostructured Guidance for Peripheral Nerve Injuries: A Review with a Perspective in the Oral and Maxillofacial Area

Injury to peripheral nerves can occur as a result of various surgical procedures, including oral and maxillofacial surgery. In the case of nerve transaction, the gold standard treatment is the end-to-end reconnection of the two nerve stumps. When it cannot be performed, the actual strategies consist of the positioning of a nerve graft between the two stumps. Guided nerve regeneration using nano-structured scaffolds is a promising strategy to promote axon regeneration. Biodegradable electrospun conduits composed of aligned nanofibers is a new class of devices used to improve neurite extension and axon outgrowth. Self assembled peptide nanofibrous scaffolds (SAPNSs) demonstrated promising results in animal models for central nervous system injuries, and, more recently, for peripheral nerve injury. Aims of this work are (1) to review electrospun and self-assembled nanofibrous scaffolds use in vitro and in vivo for peripheral nerve regeneration; and (2) its application in peripheral nerve injuries treatment. The review focused on nanofibrous scaffolds with a diameter of less than approximately 250 nm. The conjugation in a nano scale of a natural bioactive factor with a resorbable synthetic or natural material may represent the best compromise providing both biological and mechanical cues for guided nerve regeneration. Injured peripheral nerves, such as trigeminal and facial, may benefit from these treatments.     

Heat Shock Protein 27 Injection Leads to Caspase Activation in the Visual Pathway and Retinal T-Cell Response

Heat shock protein 27 (HSP27) is one of the small molecular chaperones and is involved in many cell mechanisms. Besides the known protective and helpful functions of intracellular HSP27, very little is known about the mode of action of extracellular HSP27. In a previous study, we showed that intravitreal injection of HSP27 led to neuronal damage in the retina and optic nerve after 21 days. However, it was not clear which degenerative signaling pathways were induced by the injection. For this reason, the pathological mechanisms of intravitreal HSP27 injection after 14 days were investigated. Histological and RT-qPCR analyses revealed an increase in endogenous HSP27 in the retina and an activation of components of the intrinsic and extrinsic apoptosis pathway. In addition, an increase in nucleus factor-kappa-light-chain-enhancer of activated B cells (NFκB), as well as of microglia/macrophages and T-cells could be observed. In the optic nerve, however, only an increased apoptosis rate was detectable. Therefore, the activation of caspases and the induction of an incipient immune response seem to be the main triggers for retinal degeneration in this intravitreal HSP27 model.     

Peripheral Nerve Regeneration and Muscle Reinnervation

Injured peripheral nerves but not central nerves have the capacity to regenerate and reinnervate their target organs. After the two most severe peripheral nerve injuries of six types, crush and transection injuries, nerve fibers distal to the injury site undergo Wallerian degeneration. The denervated Schwann cells (SCs) proliferate, elongate and line the endoneurial tubes to guide and support regenerating axons. The axons emerge from the stump of the viable nerve attached to the neuronal soma. The SCs downregulate myelin-associated genes and concurrently, upregulate growth-associated genes that include neurotrophic factors as do the injured neurons. However, the gene expression is transient and progressively fails to support axon regeneration within the SC-containing endoneurial tubes. Moreover, despite some preference of regenerating motor and sensory axons to “find” their appropriate pathways, the axons fail to enter their original endoneurial tubes and to reinnervate original target organs, obstacles to functional recovery that confront nerve surgeons. Several surgical manipulations in clinical use, including nerve and tendon transfers, the potential for brief low-frequency electrical stimulation proximal to nerve repair, and local FK506 application to accelerate axon outgrowth, are encouraging as is the continuing research to elucidate the molecular basis of nerve regeneration.     

Bioactive Nanofiber-Based Conduits in a Peripheral Nerve Gap Management—An Animal Model Study

The aim was to examine the efficiency of a scaffold made of poly (L-lactic acid)-co-poly(ϵ-caprolactone), collagen (COL), polyaniline (PANI), and enriched with adipose-derived stem cells (ASCs) as a nerve conduit in a rat model. P(LLA-CL)-COL-PANI scaffold was optimized and electrospun into a tubular-shaped structure. Adipose tissue from 10 Lewis rats was harvested for ASCs culture. A total of 28 inbred male Lewis rats underwent sciatic nerve transection and excision of a 10 mm nerve trunk fragment. In Group A, the nerve gap remained untouched; in Group B, an excised trunk was used as an autograft; in Group C, nerve stumps were secured with P(LLA-CL)-COL-PANI conduit; in Group D, P(LLA-CL)-COL-PANI conduit was enriched with ASCs. After 6 months of observation, rats were sacrificed. Gastrocnemius muscles and sciatic nerves were harvested for weight, histology analysis, and nerve fiber count analyses. Group A showed advanced atrophy of the muscle, and each intervention (B, C, D) prevented muscle mass decrease (p < 0.0001); however, ASCs addition decreased efficiency vs. autograft (p < 0.05). Nerve fiber count revealed a superior effect in the nerve fiber density observed in the groups with the use of conduit (D vs. B p < 0.0001, C vs. B p < 0.001). P(LLA-CL)-COL-PANI conduits with ASCs showed promising results in managing nerve gap by decreasing muscle atrophy.     

Involvement of the miR-363-5p/P2RX4 Axis in Regulating Schwann Cell Phenotype after Nerve Injury

Although microRNAs (miRNAs or miRs) have been studied in the peripheral nervous system, their function in Schwann cells remains elusive. In this study, we performed a microRNA array analysis of cyclic adenosine monophosphate (cAMP)-induced differentiated primary Schwann cells. KEGG pathway enrichment analysis of the target genes showed that upregulated miRNAs (mR212-5p, miR335, miR20b-5p, miR146b-3p, and miR363-5p) were related to the calcium signaling pathway, regulation of actin cytoskeleton, retrograde endocannabinoid signaling, and central carbon metabolism in cancer. Several key factors, such as purinergic receptors (P2X), guanine nucleotide-binding protein G(olf) subunit alpha (GNAL), P2RX5, P2RX3, platelet-derived growth factor receptor alpha (PDGFRA), and inositol 1,4,5-trisphosphate receptor type 2 (ITPR2; calcium signaling pathway) are potential targets of miRNAs regulating cAMP. Our analysis revealed that miRNAs were differentially expressed in cAMP-treated Schwann cells; miRNA363-5p was upregulated and directly targeted the P2X purinoceptor 4 (P2RX4)-UTR, reducing the luciferase activity of P2RX4. The expression of miRNA363-5p was inhibited and the expression of P2RX4 was upregulated in sciatic nerve injury. In contrast, miRNA363-5p expression was upregulated and P2RX4 expression was downregulated during postnatal development. Of note, a P2RX4 antagonist counteracted myelin degradation after nerve injury and increased pERK and c-Jun expression. Interestingly, a P2RX4 antagonist increased the levels of miRNA363-5p. This study suggests that a double-negative feedback loop between miRNA363-5p and P2RX4 contributes to the dedifferentiation and migration of Schwann cells after nerve injury.     

Use of Natural Neural Scaffolds Consisting of Engineered Vascular Endothelial Growth Factor Immobilized on Ordered Collagen Fibers Filled in a Collagen Tube for Peripheral Nerve Regeneration in Rats

The search for effective strategies for peripheral nerve regeneration has attracted much attention in recent years. In this study, ordered collagen fibers were used as intraluminal fibers after nerve injury in rats. Vascular endothelial growth factor (VEGF) plays an important role in nerve regeneration, but its very fast initial burst of activity within a short time has largely limited its clinical use. For the stable binding of VEGF to ordered collagen fibers, we fused a collagen-binding domain (CBD) to VEGF through recombinant DNA technology. Then, we filled the ordered collagen fibers-CBD-VEGF targeting delivery system in a collagen tube to construct natural neural scaffolds, which were then used to bridge transected nerve stumps in a rat sciatic nerve transection model. After transplantation, the natural neural scaffolds showed minimal foreign body reactions and good integration into the host tissue. Oriented collagen fibers in the collagen tube could guide regenerating axons in an oriented manner to the distal, degenerating nerve segment, maximizing the chance of target reinnervation. Functional and histological analyses indicated that the recovery of nerve function in the natural neural scaffolds-treated group was superior to the other grafted groups. The guiding of oriented axonal regeneration and effective delivery systems surmounting the otherwise rapid and short-lived diffusion of growth factors in body fluids are two important strategies in promoting peripheral nerve regeneration. The natural neural scaffolds described take advantage of these two aspects and may produce synergistic effects. These properties qualified the artificial nerve conduits as a putative candidate system for the fabrication of peripheral nerve reconstruction devices.     

Advances and Future Applications of Augmented Peripheral Nerve Regeneration

Peripheral nerve injuries remain a significant source of long lasting morbidity, disability, and economic costs. Much research continues to be performed in areas related to improving the surgical outcomes of peripheral nerve repair. In this review, the physiology of peripheral nerve regeneration and the multitude of efforts to improve surgical outcomes are discussed. Improvements in tissue engineering that have allowed for the use of synthetic conduits seeded with neurotrophic factors are highlighted. Selected pre-clinical and available clinical data using cell based methods such as Schwann cell, undifferentiated, and differentiated stem cell transplantation to guide and enhance peripheral nerve regeneration are presented. The limitations that still exist in the utility of neurotrophic factors and cell-based therapies are outlined. Strategies that are most promising for translation into the clinical arena are suggested.     

Modified Hyaluronic Acid-Laminin-Hydrogel as Luminal Filler for Clinically Approved Hollow Nerve Guides in a Rat Critical Defect Size Model

Hollow nerve guidance conduits are approved for clinical use for defect lengths of up to 3 cm. This is because also in pre-clinical evaluation they are less effective in the support of nerve regeneration over critical defect lengths. Hydrogel luminal fillers are thought to improve the regeneration outcome by providing an optimized matrix inside bioartificial nerve grafts. We evaluated here a modified hyaluronic acid-laminin-hydrogel (M-HAL) as luminal filler for two clinically approved hollow nerve guides. Collagen-based and chitosan-based nerve guides were filled with M-HAL in two different concentrations and the regeneration outcome comprehensively studied in the acute repair rat sciatic nerve 15 mm critical defect size model. Autologous nerve graft (ANG) repair served as gold-standard control. At 120 days post-surgery, all ANG rats demonstrated electrodiagnostically detectable motor recovery. Both concentrations of the hydrogel luminal filler induced improved regeneration outcome over empty nerve guides. However, neither combination with collagen- nor chitosan-based nerve guides resulted in functional recovery comparable to the ANG repair. In contrast to our previous studies, we demonstrate here that M-HAL slightly improved the overall performance of either empty nerve guide type in the critical defect size model.     

New Frontiers in Peripheral Nerve Regeneration: Concerns and Remedies

Topical advances in studying molecular and cellular mechanisms responsible for regeneration in the peripheral nervous system have highlighted the ability of the nervous system to repair itself. Still, serious injuries represent a challenge for the morphological and functional regeneration of peripheral nerves, calling for new treatment strategies that maximize nerve regeneration and recovery. This review presents the canonical view of the basic mechanisms of nerve regeneration and novel data on the role of exosomes and their transferred microRNAs in intracellular communication, regulation of axonal growth, Schwann cell migration and proliferation, and stromal cell functioning. An integrated comprehensive understanding of the current mechanistic underpinnings will open the venue for developing new clinical strategies to ensure full regeneration in the peripheral nervous system.     

Multi-channel chitosan–polycaprolactone conduits embedded with microspheres for controlled release of nerve growth factor

Nerve growth factor (NGF)-loaded chitosan microspheres were prepared via emulsification method using sodium tripolyphosphate as a crosslinker. Some selected chitosan microspheres were embedded into chitosan–polycaprolactone (CH–PCL) multi-channel conduits that can potentially be used for long-gap nerve repair. PCL percentages in the CH–PCLs were optimized into a region changing from around 30 to 42 wt.% while the porosity and average channel diameter of the resulting multi-channel conduits were selected as about 80% and 200 μm, respectively. SEM images confirmed that the channels inside the conduits were longitudinally arrayed with an approximately parallel arrangement, and the NGF-loaded microspheres appeared to be embedded into the wall of channels without blocking. The compressive properties in wet state and in vitro degradation rates of CH–PCL multi-channel conduits were found to be mainly manipulated by the PCL content in the CH–PCLs whereas the cumulative amount of released NGF from the conduits could be independently regulated by altering the initial NGF load inside the embedded microspheres. The optimal microsphere-embedded CH–PCL multi-channel conduits with a dimension of around 6 mm in outer diameter and 30 mm in length were able to administrate bioactivity-preserved NGF release in a sustained and controlled manner without significant initial burst release, and the release rates of the conduits could be maintained with approximately linear characteristic over a period of time longer than 6 weeks.     

Chitosan Micro-Grooved Membranes with Increased Asymmetry for the Improvement of the Schwann Cell Response in Nerve Regeneration

Peripheral nerve injuries are a common condition in which a nerve is damaged, affecting more than one million people every year. There are still no efficient therapeutic treatments for these injuries. Artificial scaffolds can offer new opportunities for nerve regeneration applications; in this framework, chitosan is emerging as a promising biomaterial. Here, we set up a simple and effective method for the production of micro-structured chitosan films by solvent casting, with high fidelity in the micro-pattern reproducibility. Three types of chitosan directional micro-grooved patterns, presenting different levels of symmetricity, were developed for application in nerve regenerative medicine: gratings (GR), isosceles triangles (ISO) and scalene triangles (SCA). The directional patterns were tested with a Schwann cell line. The most asymmetric topography (SCA), although it polarized the cell shaping less efficiently, promoted higher cell proliferation and a faster cell migration, both individually and collectively, with a higher directional persistence of motion. Overall, the use of micro-structured asymmetrical directional topographies may be exploited to enhance the nerve regeneration process mediated by chitosan scaffolds.     

Myelin and multiple sclerosis

Myelin is a lipid rich membrane system elaborated by oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system to provide axons with a high resistance but discontinuous sheath. The node internode structure that results allows very fast saltatory nerve impulse conduction. Myelin is rich in cholesterol, phospholipids and glycolipids. However, the ability of the brain to synthesise cholesterol determines its capacity to elaborate myelin and as a result cholesterol is an essential nutrient for brain function. Multiple sclerosis is an autoimmune disease of the brain and nervous system resulting in inflammation leading to demyelination and eventually to axonal degeneration. The discovery of remyelination a regenerative process offers some hope that by boosting its capacity more effective treatments for multiple sclerosis will be developed in the future.     

Potential Therapeutic Strategies and Substances for Facial Nerve Regeneration Based on Preclinical Studies

Despite advances in microsurgical technology and an improved understanding of nerve regeneration, obtaining satisfactory results after facial nerve injury remains a difficult clinical problem. Among existing peripheral nerve regeneration studies, relatively few have focused on the facial nerve, particularly how experimental studies of the facial nerve using animal models play an essential role in understanding functional outcomes and how such studies can lead to improved axon regeneration after nerve injury. The purpose of this article is to review current perspectives on strategies for applying potential therapeutic methods for facial nerve regeneration. To this end, we searched Embase, PubMed, and the Cochrane library using keywords, and after applying exclusion criteria, obtained a total of 31 qualifying experimental studies. We then summarize the fundamental experimental studies on facial nerve regeneration, highlighting recent bioengineering studies employing various strategies for supporting facial nerve regeneration, including nerve conduits with stem cells, neurotrophic factors, and/or other therapeutics. Our summary of the methods and results of these previous reports reveal a common feature among studies, showing that various neurotrophic factors arising from injured nerves contribute to a microenvironment that plays an important role in functional recovery. In most cases, histological examinations showed that this microenvironmental influence increased axonal diameter as well as myelination thickness. Such an analysis of available research on facial nerve injury and regeneration represents the first step toward future therapeutic strategies.