Exploring an innovative decellularization protocol for porcine nerve grafts: a translational approach to peripheral nerve repair.

Introduction: Peripheral nerves are frequently affected by lesions caused by traumatic or iatrogenic damages, resulting in loss of motor and sensory function, crucial in orthopedic outcomes and with a significant impact on patients' quality of life. Many strategies have been proposed over years to repair nerve injuries with substance loss, to achieve musculoskeletal reinnervation and functional recovery. Allograft have been tested as an alternative to the gold standard, the autograft technique, but nerves from donors frequently cause immunogenic response. For this reason, several studies are focusing to find the best way to decellularize nerves preserving either the extracellular matrix, either the basal lamina, as the key elements used by Schwann cells and axons during the regenerative process. Methods: This study focuses on a novel decellularization protocol for porcine nerves, aimed at reducing immunogenicity while preserving essential elements like the extracellular matrix and basal lamina, vital for nerve regeneration. To investigate the efficacy of the decellularization protocol to remove immunogenic cellular components of the nerve tissue and to preserve the basal lamina and extracellular matrix, morphological analysis was performed through Masson's Trichrome staining, immunofluorescence, high resolution light microscopy and transmission electron microscopy. Decellularized porcine nerve graft were then employed in vivo to repair a rat median nerve lesion. Morphological analysis was also used to study the ability of the porcine decellularized graft to support the nerve regeneration. Results and Discussion: The decellularization method was effective in preparing porcine superficial peroneal nerves for grafting as evidenced by the removal of immunogenic components and preservation of the ECM. Morphological analysis demonstrated that four weeks after injury, regenerating fibers colonized the graft suggesting a promising use to repair severe nerve lesions. The idea of using a porcine nerve graft arises from a translational perspective. This approach offers a promising direction in the orthopedic field for nerve repair, especially in severe cases where conventional methods are limited.

The Impact of the Molecular Weight of Degradation Products with Silicon from Porous Chitosan-Siloxane Hybrids on Neuronal Cell Behavior.

Silicon (Si) is an essential trace element in the human body and it exists in connective tissue as aqueous orthosilicic acid. Porous chitosan-3-glycidoxypropyltrimethoxysilane (GPTMS) hybrids can regenerate nerve tissue and recover sensor and motor functions. However, the structures and roles of the degradation products with Si extracted from the hybrids in nerve regeneration are not clear. In this study, we prepared porous chitosan-GPTMS hybrids with different amounts of GPTMS to amino groups of chitosan (chitosan:GPTMS = 1:0.5 and 1:1 molar ratios). The structures of the degradation products with Si from the hybrids were examined using time-of-flight mass spectrometry, and biological assessments were conducted in order to evaluate their potential use in the preparation of devices for nerve repair. Glial and motor cell lines and ex vivo explants of dorsal root ganglia were used in this study for evaluating their behavior in the presence of the different degradation products with Si. The structure of the degradation products with Si depended on the starting composition. The results showed that glial cell proliferation was lower in the medium with the higher-molecular-weight degradation products with Si. Moreover, motor cell line differentiation and the neurite outgrowth of dorsal root ganglion explants were improved with the lower-molecular-weight degradation products with Si. The results obtained could be useful for designing a new nerve regeneration scaffold including silicon components.

Biological effects of sub-lethal doses of glyphosate and AMPA on cardiac myoblasts.

Introduction: Glyphosate is the active compound of different non-selective herbicides, being the most used agriculture pesticide worldwide. Glyphosate and AMPA (one of its main metabolites) are common pollutants of water, soil, and food sources such as crops. They can be detected in biological samples from both exposed workers and general population. Despite glyphosate acts as inhibitor of the shikimate pathway, present only in plants and some microorganisms, its safety in mammals is still debated. Acute glyphosate intoxications are correlated to cardiovascular/neuronal damages, but little is known about the effects of the chronic exposure. Methods: We evaluated the direct biological effects of different concentrations of pure glyphosate/AMPA on a rat-derived cell line of cardiomyoblasts (H9c2) in acute (1-2 h) or sub-chronic (24-48 h) settings. We analyzed cell viability/morphology, ROS production and mitochondrial dynamics. Results: Acute exposure to high doses (above 10 mM) of glyphosate and AMPA triggers immediate cytotoxic effects: reduction in cell viability, increased ROS production, morphological alterations and mitochondrial function. When exposed to lower glyphosate concentrations (1 µM-1 mM), H9c2 cells showed only a slight variation in cell viability and ROS production, while mitochondrial dynamic was unvaried. Moreover, the phenotype was completely restored after 48 h of treatment. Surprisingly, the sub-chronic (48 h) treatment with low concentrations (1 µM-1 mM) of AMPA led to a late cytotoxic response, reflected in a reduction in H9c2 viability. Conclusion: The comprehension of the extent of human exposure to these molecules remains pivotal to have a better critical view of the available data.

Morphological Methods to Evaluate Peripheral Nerve Fiber Regeneration: A Comprehensive Review.

Regeneration of damaged peripheral nerves remains one of the main challenges of neurosurgery and regenerative medicine, a nerve functionality is rarely restored, especially after severe injuries. Researchers are constantly looking for innovative strategies for tackling this problem, with the development of advanced tissue-engineered nerve conduits and new pharmacological and physical interventions, with the aim of improving patients' life quality. Different evaluation methods can be used to study the effectiveness of a new treatment, including functional tests, morphological assessment of regenerated nerve fibers and biomolecular analyses of key factors necessary for good regeneration. The number and diversity of protocols and methods, as well as the availability of innovative technologies which are used to assess nerve regeneration after experimental interventions, often makes it difficult to compare results obtained in different labs. The purpose of the current review is to describe the main morphological approaches used to evaluate the degree of nerve fiber regeneration in terms of their usefulness and limitations.

Neurodynamic Treatment Promotes Mechanical Pain Modulation in Sensory Neurons and Nerve Regeneration in Rats.

BACKGROUND: Somatic nerve injuries are a rising problem leading to disability associated with neuropathic pain commonly reported as mechanical allodynia (MA) and hyperalgesia. These symptoms are strongly dependent on specific processes in the dorsal root ganglia (DRG). Neurodynamic treatment (NDT), consisting of selective uniaxial nerve repeated tension protocols, effectively reduces pain and disability in neuropathic pain patients even though the biological mechanisms remain poorly characterized. We aimed to define, both in vivo and ex vivo, how NDT could promote nerve regeneration and modulate some processes in the DRG linked to MA and hyperalgesia. METHODS: We examined in Wistar rats, after unilateral median and ulnar nerve crush, the therapeutic effects of NDT and the possible protective effects of NDT administered for 10 days before the injury. We adopted an ex vivo model of DRG organotypic explant subjected to NDT to explore the selective effects on DRG cells. RESULTS: Behavioural tests, morphological and morphometrical analyses, and gene and protein expression analyses were performed, and these tests revealed that NDT promotes nerve regeneration processes, speeds up sensory motor recovery, and modulates mechanical pain by affecting, in the DRG, the expression of TACAN, a mechanosensitive receptor shared between humans and rats responsible for MA and hyperalgesia. The ex vivo experiments have shown that NDT increases neurite regrowth and confirmed the modulation of TACAN. CONCLUSIONS: The results obtained in this study on the biological and molecular mechanisms induced by NDT will allow the exploration, in future clinical trials, of its efficacy in different conditions of neuropathic pain.

The Potential Benefits of Dietary Polyphenols for Peripheral Nerve Regeneration.

Peripheral nerves are frequently affected by lesions caused by trauma (work accidents, car incidents, combat injuries) and following surgical procedures (for instance cancer resection), resulting in loss of motor and sensory function with lifelong impairments. Irrespective of the intrinsic capability of the peripheral nervous system for regeneration, spontaneous or surgically supported regeneration is often unsatisfactory with the limited functional success of nerve repair. For this reason, many efforts have been made to improve the regeneration process. Beyond innovative microsurgical methods that, in certain cases, are necessary to repair nerve injuries, different nonsurgical treatment approaches and adjunctive therapies have been investigated to enhance nerve regeneration. One possibility could be taking advantage of a healthy diet or lifestyle and their relation with proper body functions. Over the years, scientific evidence has been obtained on the benefits of the intake of polyphenols or polyphenol-rich foods in humans, highlighting the neuroprotective effects of these compounds in many neurodegenerative diseases. In order to improve the available knowledge about the potential beneficial role of polyphenols in the process of peripheral nerve regeneration, this review assessed the biological effects of polyphenol administration in supporting and promoting the regenerative process after peripheral nerve injury.

The Peptide A-3302-B Isolated from a Marine Bacterium Micromonospora sp. Inhibits HSV-2 Infection by Preventing the Viral Egress from Host Cells.

Herpesviruses are highly prevalent in the human population, and frequent reactivations occur throughout life. Despite antiviral drugs against herpetic infections, the increasing appearance of drug-resistant viral strains and their adverse effects prompt the research of novel antiherpetic drugs for treating lesions. Peptides obtained from natural sources have recently become of particular interest for antiviral therapy applications. In this work, we investigated the antiviral activity of the peptide A-3302-B, isolated from a marine bacterium, Micromonospora sp., strain MAG 9-7, against herpes simplex virus type 1, type 2, and human cytomegalovirus. Results showed that the peptide exerted a specific inhibitory activity against HSV-2 with an EC50 value of 14 µM. Specific antiviral assays were performed to investigate the mechanism of action of A-3302-B. We demonstrated that the peptide did not affect the expression of viral proteins, but it inhibited the late events of the HSV-2 replicative cycle. In detail, it reduced the cell-to-cell virus spread and the transmission of the extracellular free virus by preventing the egress of HSV-2 progeny from the infected cells. The dual antiviral and previously reported anti-inflammatory activities of A-3302-B, and its effect against an acyclovir-resistant HSV-2 strain are attractive features for developing a therapeutic to reduce the transmission of HSV-2 infections.

Effect of unacylated ghrelin on peripheral nerve regeneration.

Ghrelin is a circulating peptide hormone released by enteroendocrine cells of the gastrointestinal tract as two forms, acylated and unacylated. Acylated ghrelin (AG) binds to the growth hormone secretagogue receptor 1a (GHSR1a), thus stimulating food intake, growth hormone release, and gastrointestinal motility. Conversely, unacylated GHR (UnAG), through binding to a yet unidentified receptor, protects the skeletal muscle from atrophy, stimulates muscle regeneration, and protects cardiomyocytes from ischemic damage. Recently, interest about ghrelin has raised also among neuroscientists because of its effect on the nervous system, especially the stimulation of neurogenesis in spinal cord, brain stem, and hippocampus. However, few information is still available about its effectiveness on peripheral nerve regeneration. To partially fill this gap, the aim of this study was to assess the effect of UnAG on peripheral nerve regeneration after median nerve crush injury and after nerve transection immediately repaired by means of an end-to-end suture. To this end, we exploited FVB1 Myh6/Ghrl transgenic mice in which overexpression of the ghrelin gene (Ghrl) results in selective up-regulation of circulating UnAG levels, but not of AG. Regeneration was assessed by both functional evaluation (grasping test) and morphometrical analysis of regenerated myelinated axons. Results obtained lead to conclude that UnAG could have a role in development of peripheral nerves and during more severe lesions.

Elovl5 is required for proper action potential conduction along peripheral myelinated fibers.

Elovl5 elongates fatty acids with 18 carbon atoms and in cooperation with other enzymes guarantees the normal levels of very long-chain fatty acids, which are necessary for a proper membrane structure. Action potential conduction along myelinated axons depends on structural integrity of myelin, which is maintained by a correct amount of fatty acids and a proper interaction between fatty acids and myelin proteins. We hypothesized that in Elovl5-/- mice, the lack of elongation of Elovl5 substrates might cause alterations of myelin structure. The analysis of myelin ultrastructure showed an enlarged periodicity with reduced G-ratio across all axonal diameters. We hypothesized that the structural alteration of myelin might affect the conduction of action potentials. The sciatic nerve conduction velocity was significantly reduced without change in the amplitude of the nerve compound potential, suggesting a myelin defect without a concomitant axonal degeneration. Since Elovl5 is important in attaining normal amounts of polyunsaturated fatty acids, which are the principal component of myelin, we performed a lipidomic analysis of peripheral nerves of Elovl5-deficient mice. The results revealed an unbalance, with reduction of fatty acids longer than 18 carbon atoms relative to shorter ones. In addition, the ratio of saturated to unsaturated fatty acids was strongly increased. These findings point out the essential role of Elovl5 in the peripheral nervous system in supporting the normal structure of myelin, which is the key element for a proper conduction of electrical signals along myelinated nerves.

The neurodynamic treatment induces biological changes in sensory and motor neurons in vitro.

Nerves are subjected to tensile forces in various paradigms such as injury and regeneration, joint movement, and rehabilitation treatments, as in the case of neurodynamic treatment (NDT). The NDT induces selective uniaxial repeated tension on the nerve and was described to be an effective treatment to reduce pain in patients. Nevertheless, the biological mechanisms activated by the NDT promoting the healing processes of the nerve are yet still unknown. Moreover, a dose-response analysis to define a standard protocol of treatment is unavailable. In this study, we aimed to define in vitro whether NDT protocols could induce selective biological effects on sensory and motor neurons, also investigating the possible involved molecular mechanisms taking a role behind this change. The obtained results demonstrate that NDT induced significant dose-dependent changes promoting cell differentiation, neurite outgrowth, and neuron survival, especially in nociceptive neurons. Notably, NDT significantly upregulated PIEZO1 gene expression. A gene that is coding for an ion channel that is expressed both in murine and human sensory neurons and is related to mechanical stimuli transduction and pain suppression. Other genes involved in mechanical allodynia related to neuroinflammation were not modified by NDT. The results of the present study contribute to increase the knowledge behind the biological mechanisms activated in response to NDT and to understand its efficacy in improving nerve regenerational physiological processes and pain reduction.

Mesenchymal Stem Cell Treatment Perspectives in Peripheral Nerve Regeneration: Systematic Review.

Traumatic peripheral nerve lesions affect hundreds of thousands of patients every year; their consequences are life-altering and often devastating and cause alterations in movement and sensitivity. Spontaneous peripheral nerve recovery is often inadequate. In this context, nowadays, cell therapy represents one of the most innovative approaches in the field of nerve repair therapies. The purpose of this systematic review is to discuss the features of different types of mesenchymal stem cells (MSCs) relevant for peripheral nerve regeneration after nerve injury. The published literature was reviewed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A combination of the keywords "nerve regeneration", "stem cells", "peripheral nerve injury", "rat", and "human" were used. Additionally, a "MeSH" research was performed in PubMed using the terms "stem cells" and "nerve regeneration". The characteristics of the most widely used MSCs, their paracrine potential, targeted stimulation, and differentiation potentials into Schwann-like and neuronal-like cells are described in this paper. Considering their ability to support and stimulate axonal growth, their remarkable paracrine activity, their presumed differentiation potential, their extremely low immunogenicity, and their high survival rate after transplantation, ADSCs appear to be the most suitable and promising MSCs for the recovery of peripheral nerve lesion. Clinical considerations are finally reported.

Preclinical Validation of SilkBridgeTM for Peripheral Nerve Regeneration.

Silk fibroin (Bombyx mori) was used to manufacture a nerve conduit (SilkBridgeTM) characterized by a novel 3D architecture. The wall of the conduit consists of two electrospun layers (inner and outer) and one textile layer (middle), perfectly integrated at the structural and functional level. The manufacturing technology conferred high compression strength on the device, thus meeting clinical requirements for physiological and pathological compressive stresses. As demonstrated in a previous work, the silk material has proven to be able to provide a valid substrate for cells to grow on, differentiate and start the fundamental cellular regenerative activities in vitro and, in vivo, at the short time point of 2 weeks, to allow the starting of regenerative processes in terms of good integration with the surrounding tissues and colonization of the wall layers and of the lumen with several cell types. In the present study, a 10 mm long gap in the median nerve was repaired with 12 mm SilkBridgeTM conduit and evaluated at middle (4 weeks) and at longer time points (12 and 24 weeks). The SilkBridgeTM conduit led to a very good functional and morphological recovery of the median nerve, similar to that observed with the reference autograft nerve reconstruction procedure. Taken together, all these results demonstrated that SilkBridgeTM has an optimized balance of biomechanical and biological properties, which allowed proceeding with a first-in-human clinical study aimed at evaluating safety and effectiveness of using the device for the reconstruction of digital nerve defects in humans.

Role of neurotrophic factors in enhancing linear axonal growth of ganglionic sensory neurons in vitro.

Neurotrophins play a major role in the regulation of neuronal growth such as neurite sprouting or regeneration in response to nerve injuries. The role of nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor in maintaining the survival of peripheral neurons remains poorly understood. In regenerative medicine, different modalities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. This study was to investigate the influence of nerve growth factor, neurotrophin-3 and brain-derived neurotrophic factor on the growth of neurites using two in vitro models of dorsal root ganglia explants and dorsal root ganglia-derived primary cell dissociated cultures. Quantitative data showed that the total neurite length and tortuosity were differently influenced by trophic factors. Nerve growth factor and, indirectly, brain-derived neurotrophic factor stimulate the tortuous growth of sensory fibers and the formation of cell clusters. Neurotrophin-3, however, enhances neurite growth in terms of length and linearity allowing for a more organized and directed axonal elongation towards a peripheral target compared to the other growth factors. These findings could be of considerable importance for any clinical application of neurotrophic factors in peripheral nerve regeneration. Ethical approval was obtained from the Regione Piemonte Animal Ethics Committee ASLTO1 (file # 864/2016-PR) on September 14, 2016.

Dextran-based tube-guides for the regeneration of the rat sciatic nerve after neurotmesis injury.

In this work, dextran-based nerve tube-guides were prepared, characterized and used in a standardized animal model of neurotmesis injury. Non-porous and porous transparent tube-guides were obtained by photocrosslinking of two co-macromonomers based on dextran and poly(ε-caprolactone) (PCL). Swelling capacity of the tube-guides ranged from 40-60% with no visible constriction of their inner diameter. In vitro hydrolytic degradation tests showed that the tube-guides maintained their structural integrity up to 6 months. The in vivo performance of the tube-guides was evaluated by entubulation of the rat sciatic nerve after a neurotmesis injury, with a 10 mm-gap between the nerve stumps. The results showed that the tube-guides were able to promote the regeneration of the nerve in a similar manner to what was observed with conventional techniques (nerve graft and end-to-end suture). Stereological analysis proved that nerve regeneration occurred, and both tube-guides presented fibre diameter and g-ratio closer to healthy sciatic nerves. The histomorphometric analysis of Tibialis anterior (TA) skeletal muscle showed decreased neurogenic atrophy in the porous tube-guides treated group, presenting measurements that are similar to the uninjured control.

New basic insights on the potential of a chitosan-based medical device for improving functional recovery after radical prostatectomy.

OBJECTIVES: To evaluate: (i) the neuro-regenerative potential of chitosan membrane (CS-Me) on acutely axotomised autonomic neurones in vitro; (ii) to exclude the possibility that a pro-regenerative biomaterial could interfere with the proliferation activity of prostate cancer cell lines; (iii) to provide an in vivo proof of the biocompatibility and regeneration promoting effect of CS-Me in a standardised rat model of peripheral nerve injury and repair; (iv) finally, to evaluate the tissue reaction induced by the degrading material; as previous studies have shown promising effects of CS-Me for protection of the neurovascular bundles for potency recovery in patients that undergo nerve-sparing radical prostatectomy (RP). MATERIALS AND METHODS: Addressing aim (i), the neuro-regenerative potential, organotypic cultures derived from primary sympathetic ganglia were cultured on CS-Me over 3 days and neurite extension and axonal sprouting were evaluated. Addressing aim (ii), effects of CS on cancer cells, different human prostate cancer cell lines (PC3, DU-145, LN-Cap) were seeded on CS-coated plates or cultured in the presence of CS-Me dissolution products. Addressing aims (iii) and (iv), functional recovery of peripheral nerve fibres and tissue reaction with the biomaterial, CS-Me and CS nerve guides were used to repair a median nerve injury in the rat. Functional recovery was evaluated during the post-recovery time by the behavioural grasping test. RESULTS: CS-Me significantly stimulated axon elongation from autonomic ganglia in comparison to control conditions in organotypic three-dimensional cultures. CS coating, as well as the dissolution products of CS-Me, led to a significantly lower proliferation rate of prostate cancer cell lines in vitro. Tissue reaction towards CS-Me and standard CS nerve guides was similar in the rat median nerve model, as was the outcome of nerve fibre regeneration and functional recovery. CONCLUSION: The results of this study provide the first experimental evidence in support of the clinical safety of CS-Me and of their postulated effectiveness for improving functional recovery after RP. The presented results are coherent in demonstrating that acutely axotomised autonomic neurones show increased neurite outgrowth on CS-Me substrate, whilst the same substrate reduces prostate cancer cell line proliferation in vitro. Furthermore, CS-Me do not demonstrate any disadvantage for peripheral nerve repair in a standard animal model.

Mice harbouring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity.

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.

Strategies to improve nerve regeneration after radical prostatectomy: a narrative review.

Peripheral nerves are complex organs that spread throughout the entire human body. They are frequently affected by lesions not only as a result of trauma but also following radical tumor resection. In fact, despite the advancement in surgical techniques, such as nerve-sparing robot assisted radical prostatectomy, some degree of nerve injury may occur resulting in erectile dysfunction with significant impairment of the quality of life. The aim of this review was to provide an overview on the mechanisms of the regeneration of injured peripheral nerves and to describe the potential strategies to improve the regeneration process and the functional recovery. Yet, the recent advances in bio-engineering strategies to promote nerve regeneration in the urological field are outlined with a view on the possible future regenerative therapies which might ameliorate the functional outcome after radical prostatectomy.

Evaluation of Vascular Endothelial Growth Factor (VEGF) and Its Family Member Expression After Peripheral Nerve Regeneration and Denervation.

Vascular endothelial growth factor (VEGF) represents one of the main factors involved not only in angiogenesis and vasculogenesis but also in neuritogenesis. VEGF plays its function acting via different receptors: VEGF receptor1 (VEGFR-1), VEGF receptor2 (VEGFR-2), VEGF receptor3 (VEGFR-3), and co-receptors Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2). This study reports on the first in vivo analysis of the expression of VEGF and VEGF family molecules in peripheral nerve degeneration and regeneration: for this purpose, different models of nerve lesion in rat were adopted, the median nerve crush injury and the median nerve transaction followed or not by end-to end microsurgical repair. Results obtained by real time polymerase chain reaction showed that VEGF and VEGF family molecules are differentially expressed under regenerating and degenerating condition, furthermore, in order to study the modulation and involvement of these factors in two different regenerative models, crush injury and end-to-end repair, protein expression analysis was evaluated. In addition, immunohistochemical analysis allowed to state a glial localization of VEGF and VEGFR-2 after peripheral nerve crush injury. Finally in vitro assay on primary Schwann cells culture show that VEGF165 stimulation increases Schwann cells migration, a major process in the promotion of neurite outgrowth. Anat Rec, 301:1646-1656, 2018. © 2018 Wiley Periodicals, Inc.

Possible effects of some agents on the injured nerve in obese rats: A stereological and electron microscopic study.

PURPOSE: The main aim of this study was to research new treatments following peripheral nerve injury involving melatonin (Mel), acetyl-l-carnitine (ALCAR), and leptin (Lep) using updated unbiased methods at the stereological and electron microscopic levels. MATERIALS AND METHODS: Wistar albino rats were randomly divided into nine equal groups; control (Cont), obese control (OG), obese group exposed to sciatic nerve resection (Gap) (OGG), obese group injected intraperitoneally (i.p.) with Mel (OMG), obese group injected with Mel i.p. with gap (OMGG), obese group injected with Lep i.p. (OLG), obese group injected with Lep i.p. with gap (OLGG), obese group injected with ALCAR i.p. (OAG), and obese group injected with ALCAR i.p. with gap (OAGG). Electromyography (EMG) procedures were performed. Following routine histological procedures, stereological analysis was performed for each group. RESULTS: In terms of the number of myelinated axons, high significant increase in OGG was observed compared to OG and Cont (p < 0.01). In addition, a highly significant increase in axon surface area and myelin thickness of OGG compared to OG and Cont (p < 0.01) was noted. A significant decrease in myelin thickness/axon diameter ratio of OGG was found in comparison with the other groups. In terms of latency, there was a highly significant decrease in OGG compared to Cont and OG (p < 0.01). Myelinated axon numbers in OAGG, OMGG and OLGG increased highly significantly compared to other groups (p < 0.01). Latency in OMGG, a highly significant increase, was determined in OMG compared to Cont (p < 0.01). In addition, latency values in OGG were highly significantly greater than in OAC and OAGG (p < 0.01). CONCLUSION: In particular, administration of Lep, Mel and ALCAR as neuroprotective agents may make a positive contribution to regeneration and myelination in obese rats.

Generation of new neurons in dorsal root Ganglia in adult rats after peripheral nerve crush injury.

The evidence of neurons generated ex novo in sensory ganglia of adult animals is still debated. In the present study, we investigated, using high resolution light microscopy and stereological analysis, the changes in the number of neurons in dorsal root ganglia after 30 days from a crush lesion of the rat brachial plexus terminal branches. Results showed, as expected, a relevant hypertrophy of dorsal root ganglion neurons. In addition, we reported, for the first time in the literature, that neuronal hypertrophy was accompanied by massive neuronal hyperplasia leading to a 42% increase of the number of primary sensory neurons. Moreover, ultrastructural analyses on sensory neurons showed that there was not a relevant neuronal loss as a consequence of the nerve injury. The evidence of BrdU-immunopositive neurons and neural progenitors labeled with Ki67, nanog, nestin, and sox-2 confirmed the stereological evidence of posttraumatic neurogenesis in dorsal root ganglia. Analysis of morphological changes following axonal damage in addition to immunofluorescence characterization of cell phenotype suggested that the neuronal precursors which give rise to the newly generated neurons could be represented by satellite glial cells that actively proliferate after the lesion and are able to differentiate toward the neuronal lineage.