Study Of Peripheral Nerve Regeneration Research Articles

EZH2-dependent myelination following sciatic nerve injury

Abstract Demyelination and remyelination have been major focal points in the study of peripheral nerve regeneration following peripheral nerve injury. Notably, the gene regulatory network of regenerated myelin differs from that of native myelin. Silencing of enhancer of zeste homolog 2 (EZH2) hinders the differentiation, maturation, and myelination of Schwann cells in vitro. To further determine the role of EZH2 in myelination and recovery post-peripheral nerve injury, conditional knockout mice lacking Ezh2 in Schwann cells (Ezh2fl/fl;Dhh-Cre and Ezh2fl/fl;Mpz-Cre) were generated. Our results show that a significant proportion of axons in the sciatic nerve of Ezh2-depleted mice remain unmyelinated. This highlights the crucial role of Ezh2 in initiating Schwann cell myelination. Furthermore, we observed that 21 days after inducing a sciatic nerve crush injury in these mice, most axons had remyelinated at the injury site in the control nerve, while Ezh2fl/fl;Mpz-Cre mice had significantly fewer remyelinated axons compared with their wild-type littermates. This suggests that the absence of Ezh2 in Schwann cells impairs myelin formation and remyelination. In conclusion, EZH2 has emerged as a pivotal regulatory factor in the process of demyelination and myelin regeneration following peripheral nerve injury. Modulating EZH2 activity during these processes may offer a promising therapeutic target for the treatment of peripheral nerve injuries.

Comprehensive Development of a Cellulose Acetate and Soy Protein-Based Scaffold for Nerve Regeneration.

The elaboration of biocompatible nerve guide conduits (NGCs) has been studied in recent years as a treatment for total nerve rupture lesions (axonotmesis). Different natural polymers have been used in these studies, including cellulose associated with soy protein. The purpose of this report was to describe manufacturing NGCs suitable for nerve regeneration using the method of dip coating and evaporation of solvent with cellulose acetate (CA) functionalized with soy protein acid hydrolysate (SPAH). The manufacturing method and bacterial control precautions for the CA/SPAH NGCs were described. The structure of the NGCs was analyzed under a scanning electron microscope (SEM); porosity was analyzed with a degassing method using a porosimeter. Schwann cell (SCL 4.1/F7) biocompatibility of cell-seeded nerve guide conduits was evaluated with the MTT assay. The method employed allowed an easy elaboration and customization of NGCs, free of bacteria, with pores in the internal surface, and the uniform wall thickness allowed manipulation, which showed flexibility; additionally, the sample was suturable. The NGCs showed initial biocompatibility with Schwann cells, revealing cells adhered to the NGC structure after 5 days. The fabricated CA/SPAH NGCs showed adequate features to be used for peripheral nerve regeneration studies. Future reports are necessary to discuss the ideal concentration of CA and SPAH and the mechanical and physicochemical properties of this biomaterial.

Ultrastructural Observation of Axon Complex of Epithelial Cells after Nerve Implantation into Denervated Fingers of Monkey

The recognition of visual shape in monkeys depends on a multi-layer pathway from primary visual cortex to lower temporal cortex. Visual stimulation is received by retina and then projected to the primary visual cortex VI region through lateral geniculate nucleus. There are a large number of neurons activated by linear stimulation such as short side and line segment. This paper mainly studies the ultrastructural observation of axon complex of epithelial cells after nerve implantation in monkey nerve loss fingers. Through the ultrastructural view of epithelial axon complex, we can master the changes of nerve regeneration function to skin cells and solve the problems caused by nerve defects, this paper mainly studies the method of nerve implantation, uses the neural interface model and the algorithm of nerve electrode, and compares the experiment with the monkey without nerve implantation, and then observe the synaptic ultrastructure under the electron microscope after the experiment to find that nerve implantation can promote the skin sensory organs. The results showed that the repair of sensory cells was faster than that of the skin sensory cells after nerve implantation in the ultrastructures of epithelial axon complex after nerve implantation in monkeys. 90% of the cells implanted with nerve were very fast to repair, which could provide useful information for the study of peripheral nerve regeneration in the nervous system. Nerve implantation regeneration has been a medical research the research focus of the topic,medical researchers hope to find an effective method of nerve implantation for skin cell repir.

Preclinical study of peripheral nerve regeneration using nerve guidance conduits based on polyhydroxyalkanaotes.

Nerve guidance conduits (NGCs) are used as an alternative to the “gold standard” nerve autografting, preventing the need for surgical intervention required to harvest autologous nerves. However, the regeneration outcomes achieved with the current NGCs are only comparable with autografting when the gap is short (less than 10 mm). In the present study, we have developed NGCs made from a blend of polyhydroxyalkanoates, a family of natural resorbable polymers. Hollow NGCs made from a 75:25 poly(3‐hydroxyoctanoate)/poly(3‐hydroxybutyrate) blend (PHA‐NGCs) were manufactured using dip‐molding. These PHA‐NGCs showed appropriate flexibility for peripheral nerve regeneration. In vitro cell studies performed using RT4‐D6P2T rat Schwann cell line confirmed that the material is capable of sustaining cell proliferation and adhesion. PHA‐NGCs were then implanted in vivo to repair 10 mm gaps of the median nerve of female Wistar rats for 12 weeks. Functional evaluation of the regenerated nerve using the grasping test showed that PHA‐NGCs displayed similar motor recovery as the autograft, starting from week 7. Additionally, nerve cross‐sectional area, density and number of myelinated cells, as well as axon diameter, fiber diameter, myelin thickness and g‐ratio obtained using the PHA‐NGCs were found comparable to an autograft. This preclinical data confirmed that the PHA‐NGCs are indeed highly promising candidates for peripheral nerve regeneration.

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.

In vitro models for peripheral nerve regeneration.

The study of peripheral nerve repair and regeneration is particularly relevant in the light of the high clinical incidence of nerve lesions. However, the clinical outcome after nerve lesions is often far from satisfactory and the functional recovery is almost never complete. Therefore, a number of therapeutic approaches are being investigated, ranging from local delivery of trophic factors and other molecules to bioactive biomaterials and complex nerve prostheses. Translation of the new therapeutic approaches to the patient always requires a final pre-clinical step using in vivo animal models. The need to limit as much as possible animal use in biomedical research, however, makes the preliminary use of in vitro models mandatory from an ethical point of view. In this article, the different types of in vitro models available today for the study of peripheral nerve regeneration have been ranked by adopting a three-step stair model based on their increasing ethical impact: (i) cell line-based models, which raise no ethical concern; (ii) primary cell-based models, which have low ethical impact as animal use, although necessary, is limited; and (iii) organotypic ex vivo-based models, which raise moderate ethical concerns as the use of laboratory animals is required although with much lower impact on animal wellbeing in comparison to in vivo models of peripheral nerve regeneration. This article aims to help researchers in selecting the best experimental approach for their scientific goals driven by the 'Three Rs' (3Rs) rules (Replacement, Reduction or Refinement of animal use in research) for scientific research.

International symposium on peripheral nerve repair and regeneration and 2nd club Brunelli meeting

The International Symposium "Peripheral Nerve Repair and Regeneration and 2nd Club Brunelli Meeting" was held on December 4-5, 2009 in Turin, Italy (Organizers: Bruno Battiston, Stefano Geuna, Isabelle Perroteau, Pierluigi Tos). Interest in the study of peripheral nerve regeneration is very much alive because complete recovery of nerve function almost never occurs after nerve reconstruction and, often, the clinical outcome is rather poor. Therefore, there is a need for defining innovative strategies for improving the success of recovery after nerve lesion and repair and this meeting was intended to discuss, from a multidisciplinary point of view, some of today's most important issues in this scientific field, arising from both basic and clinical neurosciences.

Mesenchymal Stem Cells in a Polycaprolactone Conduit Promote Sciatic Nerve Regeneration and Sensory Neuron Survival after Nerve Injury

Despite the fact that the peripheral nervous system is able to regenerate after traumatic injury, the functional outcomes following damage are limited and poor. Bone marrow mesenchymal stem cells (MSCs) are multipotent cells that have been used in studies of peripheral nerve regeneration and have yielded promising results. The aim of this study was to evaluate sciatic nerve regeneration and neuronal survival in mice after nerve transection followed by MSC treatment into a polycaprolactone (PCL) nerve guide. The left sciatic nerve of C57BL/6 mice was transected and the nerve stumps were placed into a biodegradable PCL tube leaving a 3-mm gap between them; the tube was filled with MSCs obtained from GFP+ animals (MSC-treated group) or with a culture medium (Dulbecco's modified Eagle's medium group). Motor function was analyzed according to the sciatic functional index (SFI). After 6 weeks, animals were euthanized, and the regenerated sciatic nerve, the dorsal root ganglion (DRG), the spinal cord, and the gastrocnemius muscle were collected and processed for light and electron microscopy. A quantitative analysis of regenerated nerves showed a significant increase in the number of myelinated fibers in the group that received, within the nerve guide, stem cells. The number of neurons in the DRG was significantly higher in the MSC-treated group, while there was no difference in the number of motor neurons in the spinal cord. We also found higher values of trophic factors expression in MSC-treated groups, especially a nerve growth factor. The SFI revealed a significant improvement in the MSC-treated group. The gastrocnemius muscle showed an increase in weight and in the levels of creatine phosphokinase enzyme, suggesting an improvement of reinnervation and activity in animals that received MSCs. Immunohistochemistry documented that some GFP+ -transplanted cells assumed a Schwann-cell-like phenotype, as evidenced by their expression of the S-100 protein, a Schwann cell marker. Our findings suggest that using a PCL tube filled with MSCs is a good strategy to improve nerve regeneration after a nerve transection in mice.

Evaluation of myelin sheath and collagen reorganization pattern in a model of peripheral nerve regeneration using an integrated histochemical approach

Peripheral nerves are complex histological structures that can be affected by a variety of conditions with different degree of axonal degeneration and demyelination. For the study of peripheral nerve regeneration in pathology and tissue engineering, it is necessary to evaluate the regeneration, remyelination and extracellular matrix reorganization of the neural tissue. Currently, different histochemical techniques must be used in parallel, and a correlation among their findings should be further performed. In this work, we describe a new histochemical method for myelin and collagen fibers based on luxol fast blue and picrosirius methods, for the evaluation of the morphology, the myelin sheath and the collagen fiber reorganization using a model of peripheral nerve regeneration. Whole brain, normal sciatic nerve and regenerating peripheral nerve samples were fixed in 10% neutral buffered formalin and paraffin-embedded, for the performance of the hematoxylin-eosin stain, the Luxol fast blue method and the new histochemical method for myelin and collagen. The results of this technique revealed that this new histochemical method allowed us to properly evaluate histological patterns, and simultaneously observe the histochemical reaction for myelin sheath and collagen fibers in normal tissue, and during the regeneration process. In conclusion, this new method combines morphological and histochemical properties that allowed us to determine with high accuracy the degree of remyelination and collagen fibers reorganization. For all these reasons, we hypothesize that this new histochemical method could be useful in pathology and tissue engineering.

Gene Therapy for the Peripheral Nervous System: A Strategy to Repair the Injured Nerve?

Peripheral nerve injury in humans often leads to incomplete functional recovery. In this review we discuss the potential for gene therapy to be used as a strategy alongside surgical repair techniques for the study of peripheral nerve regeneration in rodent models and with a view to its eventual use for the promotion of successful regeneration in the clinic. Gene therapy vectors based on herpes simplex virus, adenovirus, lentivirus and adeno-associated virus have been developed to deliver genes to the neurons of the peripheral nervous system, i.e. primary sensory neurons in the dorsal root ganglia and primary motor neurons. Adenoviral and lentiviral vectors have also been used to transduce Schwann cells and fibroblasts in the injured nerve. We present an overview of these vectors, their application so far in the peripheral nervous system, their potential as vectors for enhancing peripheral nerve repair, and the successful interventions that have been demonstrated in animal models. We also discuss some of the limitations of current vectors and how they may be overcome. While the technology for gene delivery is approaching a state of readiness for clinical translation, the current range of therapeutic genes for the repair of the traumatically injured peripheral nerve is mostly limited to neurotrophic factors delivered to neurons, Schwann cells or possibly the target organs. Finally, therefore, we consider what type of therapeutic transgene may be desirable to enhance nerve regeneration in the future.

The Response of Dorsal Root Ganglion Axons to Nerve Growth Factor Gradients Depends on Spinal Level

Directed sensory axon regeneration has the potential to promote functional recovery after peripheral nerve injury. Using a novel guidance assay to generate precisely controllable nerve growth factor gradients, we show for the first time that the guidance and outgrowth response of rat dorsal root ganglion neurons to identical nerve growth factor gradients depends on the rostrocaudal origin of the dorsal root ganglion explant. These findings have implications for the study of peripheral nerve regeneration in response to exogenous neurotrophins such as nerve growth factor, and provide new insight into the clinical potential of nerve growth factor in the treatment of nerve injury.

Peripheral Nerve Anatomy for Regeneration Studies in Pigs

The aim of the study was to provide information about the feasibility of peripheral nerve studies in a large animal model for future research. Seven Yorkshire pigs (25-30 kg) were used in a cadaver study. The accessibility, surgical approach, maximum length of possible nerve defect, diameter, and histologic characteristics of the sural, ulnar, laryngeal, and facial nerve were evaluated. All nerves are easily accessible and demonstrate desirable histologic qualities. The differences in nerve length and diameter vary among the nerves studied and a maximum nerve gap of up to 9.62 +/- 0.93 cm for the right sural nerve is recommended considering the interposition of a nerve graft or tissue engineered nerve construct. The facial nerve demonstrates the largest diameter and the highest number of axons. The sural and laryngeal nerve display significantly less myelination compared with the facial and ulnar nerve. The porcine model for the studies of peripheral nerve regeneration is an attractive alternative to other large animal models.

Standardized crush injury of the mouse median nerve

The employment of transgenic mouse models for peripheral nerve regeneration studies is continuously increasing. In this paper, we describe a standardized method for inducing a crush lesion in mouse median nerve using a non-serrated clamp exerting a crush compression force of 20.43MPa for a duration of 30s. Quantitative assessment of posttraumatic functional recovery by grasping test showed that recovery was very fast and mice returned to baseline performance already after 20 days only. Stereological analysis of nerve fibers distal to the crush lesion showed the presence of axons with a significantly smaller size and thinner myelin sheath in comparison to controls. This experimental nerve injury model is highly reproducible and the impact on animal well-being is minimal. Its employment can be particularly indicated for exploring the basic neurobiological mechanisms of peripheral nerve regeneration.

Proteomic analysis of microdissected facial nuclei of the rat following facial nerve injury

Recent studies using molecular and genetic techniques just have started to elucidate the complex process that drives successful peripheral nerve regeneration. Introducing proteomics to this field, we unilaterally performed a facial nerve axotomy in 13 adult Wistar rats. Seven days later, a total of 40 20-μm coronary cryostat sections of the operated and contralateral unoperated nucleus facialis were microdissected. On the one hand, microdissected areas were pooled for each side, lysed and applied to ProteinChip Arrays. On the other hand, one microdissected area from the right and left facial nucleus each was directly placed on the affinity chromatographic array. Facial motoneurons were lysed in situ and released their proteins to spatially defined points. 215 laser addressable distinct positions across the surface of the spot enabled a high spatial resolution of measured protein profiles for the analysed tissue area. Protein profiles of the single positions were plotted over the used tissue section to visualize their distribution.The comparative analysis of the protein lysates from operated and normal nuclei facialis revealed, for both approaches used, differentially expressed proteins. Although by direct application of one cryostat section only a few hundred motoneurons were analysed, results comparable to these using lysates were obtained. Additionally, the applied technique revealed differences in the intensity distribution of several proteins of unknown function in the lesioned in comparison to the contralateral normal facial nucleus.This proteomic analysis with ultra high sensitivity paired with potential for a spatial resolution is a promising methodology for peripheral nerve regeneration studies.

Strain Differences in the Branching of the Sciatic Nerve in Rats

The sciatic nerve in the rat is the site most often used for peripheral nerve regeneration studies. The length of sciatic nerve available for research, however, depends on the point at which the sciatic nerve divides into the peroneal and tibial nerves. In the present study, the hind limbs of 150 adult male rats of five different strains (Sprague-Dawley, Fischer 344, Wistar-Han, Lewis and Nude) were analysed with regard to femur length, the point at which the sciatic nerve divides into the tibial and peroneal nerves, and where these are surrounded by the same epineurium, and the point at which they are encased in individual epineurial sheaths. The results indicate that the lengths of sciatic nerve are fairly constant in all strains of rats. In absolute terms, they amount to about one-third of the length of the femur for stretches of undivided sciatic nerve, and up to nearly half of the femur length for stretches where the tibial and peroneal nerves are already present, but are still enclosed by the same epineurium. In 61.7% of the hind limbs examined in Fischer rats, however, no sciatic nerve could be seen as such, but only in the form of its successors surrounded by the separate epineuria. This makes it highly advisable not to use male adult Fischer rats in peripheral nerve regeneration studies with the sciatic nerve as the point of focus.

Implantable biomedical microsystems for neural prostheses

In the following article, the technologies to fabricate polyimide-based thin and flexible substrates with monolithically integrated electrode arrays and printed circuit boards (PCB) for hybrid electronic assemblies as well as an assembling technique that connects bare electronic dice with flexible PCBs are presented. The concept of modular, flexible biomedical microsystems as neural prostheses is introduced in general and described in detail in three examples. A cuff electrode with integrated multiplexer circuitry and standard implantable cables represents the combination of microtechnology with precision mechanics; a sieve electrode used as an implant in peripheral nerve regeneration studies demonstrates the next level of integration density but still uses a cable connection; and last, joint effort to fabricate the demonstrator of a vision prosthesis that is completely implantable in the eye with a wireless link for energy supply and data transmission is presented. System design, hybrid assembling technology, and flexible multilayer encapsulation using parylene and silicone rubber are the key components for creating a new generation of neural prostheses for complex and challenging new applications.

Optimal Degradation Rate for Collagen Chambers Used for Regeneration of Peripheral Nerves over Long Gaps

The experimental study of peripheral nerve regeneration has depended heavily on the use of a nerve chamber in which the stumps of the transected nerve are inserted. A large variety of chamber fillings and chamber types have been used in an effort to induce a higher quality of regeneration across the gap initially separating the two stumps. In this study we studied the morphology of nerves regenerated across a 15 mm gap following implantation of a series of five chambers. The chambers were fabricated from type I collagen and possessed identical pore volume fractions as well as average pore diameters, but differed in cross-link density continuously along the series. The residual mass of the implanted chambers at 9 weeks was observed to increase continuously with increasing cross-link density along the series, indicating a continuous decrease in degradation rate. The quality of regenerated nerves, determined by the number of large diameter fibers (A-fibers) per nerve, the average diameter of all axons and the ratio of area occupied by axons (N-Ratio), was superior at an intermediate level of chamber degradation rate. The maximal quality of peripheral nerve regeneration corresponded to an optimal degradation rate with an estimated chamber half-life of approximately 2–3 weeks following implantation. A speculative mechanistic explanation of the observed optimum focuses on the hypothetical role of cell and cytokine traffic that may take place through holes in the chamber generated by the degradation process. The data show the presence of a hitherto unreported optimal chamber degradation rate that leads to regenerated nerves of maximum quality.

2-16-04 Electrophysiological studies of peripheral nerve regeneration across tissue gaps in the monkey

2-16-04 Electrophysiological studies of peripheral nerve regeneration across tissue gaps in the monkey

An electron microscopic study of peripheral nerve regeneration with pulsing electromagnetic fields.

An electron microscopic study of peripheral nerve regeneration with pulsing electromagnetic fields.

The rat brachial plexus and its terminal branches: an experimental model for the study of peripheral nerve regeneration.

Despite the introduction of microsurgical techniques into clinical practice, the results of surgical procedures involving the brachial plexus and peripheral nerves are still far from spectacular. We therefore studied the rat brachial plexus and its terminal branches in 203 rats. Detailed anatomic and morphologic analyses of the biceps brachii and musculocutaneous nerve, finger flexors, flexor carpi radialis, and the median nerve were performed. Various sources of conventional and vascularized nerve grafts were explored. After musculocutaneous nerve section or median nerve section, there were no articular contractures or automutilations, which constitutes an advantage for these experimental models over the sciatic nerve model. The brachial plexus and its terminal branches provide a good experimental model which can be used to assess the development and normal control of muscle function, examine the mechanisms underlying functional recovery, and test the effects of treatments to enhance recovery.