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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the feasibility and the safety of applying chitosan membrane (ChiMe) on the neurovascular bundles (NVBs) after nerve‐sparing robot‐assisted radical prostatectomy (NS‐RARP). The secondary aim of the study was to report preliminary data and in particular potency recovery data. Patients and Methods: This was a single‐centre, single‐arm prospective study, enrolling all patients with localised prostate cancer scheduled for RARP with five‐item version of the International Index of Erectile Function scores of >17, from July 2015 to September 2016. All patients underwent NS‐RARP with ChiMe applied on the NVBs. The demographics, perioperative, postoperative and complications data were evaluated. Potency recovery data were evaluated in particular and any sign/symptom of local allergy/intolerance to the ChiMe was recorded and evaluated. Results: In all, 140 patients underwent NS‐RARP with ChiMe applied on the NVBs. Applying the ChiMe was easy in almost all the cases, and did not compromise the safety of the procedure. None of the patients reported signs of intolerance/allergy attributable to the ChiMe and potency recovery data were encouraging. Conclusion: In our experience, ChiMe applied on the NVBs after NS‐RARP was feasible and safe, without compromising the duration, difficulty or complication rate of the ‘standard’ procedure. No patients had signs of intolerance/allergy attributable to the ChiMe and potency recovery data were encouraging. A comparative cohort would have added value to the study. The present paper was performed before Conformité Européene (CE)‐mark achievement. [ABSTRACT FROM AUTHOR]

The therapeutic effect of three polyvinyl alcohol (PVA) membranes loaded with electrically conductive materials - carbon nanotubes ( PVA-CNTs) and polypyrrole ( PVA-PPy) - were tested in vivo for neuro-muscular regeneration after an axonotmesis injury in the rat sciatic nerve. The membranes electrical conductivity measured was 1.5 ± 0.5 × 10−6 S/m, 579 ± 0.6 × 10−6 S/m, and 1837.5 ± 0.7 × 10−6 S/m, respectively. At week-12, a residual motor and nociceptive deficit were present in all treated groups, but at week-12, a better recovery to normal gait pattern of the PVA-CNTs and PVA-PPy treated groups was observed. Morphometrical analysis demonstrated that PVA-CNTs group presented higher myelin thickness and lower g-ratio. The tibialis anterior muscle, in the PVA-PPy and PVA-CNTs groups showed a 9% and 19% increase of average fiber size area and a 5% and 10% increase of the 'minimal Feret's diameter,' respectively. No inflammation, degeneration, fibrosis or necrosis were detected in lung, liver, kidneys, spleen, and regional lymph nodes and absence of carbon deposits was confirmed with Von Kossa and Masson-Fontana stains. In conclusion, the membranes of PVA-CNTs and PVA-PPy are biocompatible and have electrical conductivity. The higher electrical conductivity measured in PVA-CNTs membrane might be responsible for the positive results on maturation of myelinated fibers. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1267-1280, 2017. [ABSTRACT FROM AUTHOR]

Introduction: Perineural scar formation is responsible for pain and loss of function after surgical procedures. Neurolysis and application of anti-adhesion gels are required to restore a gliding surface. We tested a carboxymethylcellulose (CMC) and polyethylene oxide (PEO) gel on mouse sciatic nerve to describe its safety and efficacy.Methods: Adult mice underwent a surgical procedure in which we burned the muscular bed of the sciatic nerve bilaterally (Burned group) and applied anti-adhesion gel to 1 of the nerves (Burned+gel group). After 3 weeks, we studied scar tissue by biomechanical and histological evaluation.Results: Both histological and biomechanical analysis showed that the gel reduced perineural scarring. The difference between the Burned and Burned+gel groups was statistically significant.Conclusions: CMC-PEO gel can reduce perineural scar tissue. In histological section, scar tissue was present in both groups, but in the Burned+gel group a gliding surface was identified between scar and nerve. [ABSTRACT FROM AUTHOR]

The peripheral nervous system has an intrinsic capability to regenerate, crucially related to the ability of Schwann cells (SC) to create a permissive environment, for example, through production of regeneration-promoting neurotrophic factors. Survival, proliferation, migration and differentiation of SC into a myelinating phenotype during development and after injury is regulated by different Neuregulin1 (NRG1) isoforms. This study investigates the expression of different NRG1 isoforms and of their ErbB receptors in distal rat median nerve samples under regenerating conditions after a mild (crush) and more severe (end-to-end repair) injury and under degenerating condition. The expression of the NRG1/ErbB system was evaluated at mRNA and protein level, and demonstrated to be specific for distinct and consecutive phases following nerve injury and regeneration or the progress in degeneration. For the first time a detailed analysis of expression profiles not only of soluble and transmembrane NRG1 isoforms, but also of alpha and beta as well as type a, b and c isoforms is presented. The results of mRNA and protein expression pattern analyses were related to nerve ultrastructure changes evaluated by electron microscopy. In particular, transmembrane NRG1 isoforms are differentially regulated and proteolytically processed under regeneration and degeneration conditions. Soluble NRG1 isoforms alpha and beta, as well as type a and b, are strongly upregulated during axonal regrowth, while type c NRG1 isoform is downregulated. This is accompanied by an upregulation of ErbB receptors. This accurate regulation suggests that each molecule plays a specific role that could be clinically exploited to improve nerve regeneration. [ABSTRACT FROM AUTHOR]

Quantitative estimation of myelinated nerve fiber number, together with fiber size parameters, is one of the most important tools for nerve regeneration research. In this study we used a design-based stereological method to evaluate the regenerative process in two experimental paradigms: crush injury and autograft repair. Samples were embedded in resin and morphometric counting and measurements were performed using both light and electron microscopes. Results show a significant difference in myelinated fiber number estimation between light and electron microscopes, especially after autograft repair; light microscope significantly underestimates the number of fibers because of the large number of very small axons that can be detected only in electron microscope. The analysis of the size parameters also shows a higher number of small fibers in electron microscopic analysis, especially in regenerated nerves. This comparative study shows that the integration of data obtained in light microscope with those obtained in electron microscope is necessary in revealing very small myelinated fibers that cannot be detected otherwise. Moreover, the difference in the estimation of total number of myelinated fibers between light and electron microscopes must be considered in data analysis to ensure accurate interpretation of the results. [ABSTRACT FROM AUTHOR]

Neuromuscular recovery after peripheral nerve lesion depends on the regeneration of severed axons that re-establish their functional connection with the denervated muscle. The aim of this study was to determine the effects of electrical stimulation (ES) on the neuromuscular recovery after nerve crush injury in rats. Electrical stimulation was carried out on the tibialis anterior (TA) muscle after sciatic nerve crush injury in a rat model. Six ES sessions were administered every other day starting from day 3 postinjury until the end of the experiment (day 14). The sciatic functional index was calculated. Muscle excitability, neural cell adhesion molecule (N-CAM) expression, and muscle fiber cross-sectional area (CSA) were accessed from TA muscle. Regenerated sciatic nerves were analyzed by light and confocal microscopy. Both treated (crush+ES) and untreated (crush) groups had their muscle weight and CSA decreased compared with the normal group ( P < 0.05). Electrical stimulation accentuated muscle fiber atrophy more in the crush+ES than in the crush group ( P < 0.05). N-CAM expression increased in both crush and crush+ES groups compared with the normal group ( P < 0.05). Regenerated nerves revealed no difference between the crush and crush+ES groups. Nevertheless, functional recovery at day 14 post-injury was significantly lower in crush+ES group compared with the crush group. In addition, the crush+ES group had chronaxie values significantly higher on days 7 and 13 compared with the crush group, which indicates a decrease in muscle excitability in the crush+ES animals. The results of this study do not support a benefit of the tested protocol of ES during the period of motor nerve recovery following injury. Muscle Nerve, 2010 [ABSTRACT FROM AUTHOR]

Abstract: The toxic effect of non-steroidal anti-inflammatory drugs (NSAIDs) during development has been widely investigated. While it has been shown that these drugs impair central nervous development and compromise the neural activity, the effects of these substances on the development of peripheral nerves are still not clarified. In the present study, sciatic nerves withdrawn from three experimental groups of 4-week-old rats, prenatally exposed to either saline solution, or diclofenac sodium, and controls not exposed to any substance, were evaluated in terms of axon number, cross-sectional area of axon and myelin sheet thickness as well as of the ultrastructure of nerve fibers. Comparisons of stereological estimations among these three groups showed that axon number and mean axon cross-sectional area, but not average myelin sheet thickness, were significantly decreased in rats that were exposed to both diclofenac sodium and also to the saline solution, in comparison of the control group. Electron microscope analysis revealed, in both treated groups, deterioration of myelin sheaths that was more pronounced in rats that were exposed to diclofenac sodium. Altogether, these findings show that the prenatal administration of both diclofenac sodium and saline solution impairs peripheral nervous system development, thus suggesting that this potential teratogenic effect should be also taken into consideration in the clinical use of these substances in pregnant patients. [Copyright &y& Elsevier]

Experimental investigation of peripheral nerve fiber regeneration is attracting more and more attention among both basic and clinical researchers. Assessment of myelinated nerve fiber morphology is a pillar of peripheral nerve regeneration research. The gold standard for light microscopic imaging of myelinated nerve fibers is toluidine blue staining of resin-embedded semithin sections. However, many researchers are unaware that the dark staining of myelin sheaths typically produced by this procedure is due to osmium tetroxide postfixation and not due to toluidine blue. In this article, we describe a simple pre-embedding protocol for staining myelin sheaths in paraffin-embedded nerve specimens using osmium tetroxide. The method involves immersing the specimen in 2% osmium tetroxide for 2 h after paraformaldeyde fixation, followed by routine dehydration and paraffin embedding. Sections can then be observed directly under the microscope or counterstained using routine histological methods. Particularly good results were obtained with Masson's trichrome counterstain, which permits the imaging of connective structures in nerves that are not detectable in toluidine blue-stained resin sections. Finally, we describe a simple protocol for osmium etching of sections, which makes further immunohistochemical analysis possible on the same specimens. Taken together, our results suggest that the protocol described in this article is a valid alternative to the conventional resin embedding-based protocol: it is much cheaper, can be adopted by any histological laboratory, and allows immunohistochemical analysis to be conducted. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Little is known on the early homing features of transplanted mesenchymal stem cells (MSCs).We used the isolated rat heart model to study the homing of MSCs injected in the ventricular wall of a beating heart. In this model all types of cells and matrix elements with their interactions are represented, while external interferences by endothelial/neutrophil interaction and neurohormonal factors are excluded.We studied the morphology and marker expression of MSCs implanted in normal hearts and in the border-zone of infarcted myocardium. Early morphological adaptation of MSC homing differs between normal and infarcted hearts over the first 6 hrs after transplantation. In normal hearts, MSCs migrate very early through the interstitial milieu and begin to show morphological changes.Yet, in infarcted hearts MSCs remain in the site of injection forming clusters of round-shaped cells in the border-zone of the infarcted area. Both in normal and infarcted hearts, immunohistochemistry and confocal imaging showed that, besides the proliferative marker proliferating cell nuclear agent (PCNA), some transplanted cells early express myoblastic maker GATA-4, and some of them show a VWF immunopositivity. Moreover, a few hours after injection connexin-43 is well evident between cardiomyocytes and injected cells. This study indicates for the first time that the isolated beating heart is a good model to study early features of MSC homing without external interferences. The results show (i ) that MSCs start to change marker expression few hours after injection into a beating heart and (ii ) that infarcted myocardium influences transplanted MSC morphology and mobility within the heart. [ABSTRACT FROM AUTHOR]

Denervation induces muscle fiber atrophy and changes in the gene expression rates of skeletal muscle. Electrical stimulation (ES) is a procedure generally used to treat denervated muscles in humans. This study evaluated the effect of ES based on chronaxie and rheobase on the expression of the myoD and atrogin-1 genes in denervated tibialis anterior (TA) muscle of Wistar rats. Five groups were examined: (1) denervated (D); (2) D+ES; (3) sham denervation; (4) normal (N); and (5) N+ES. Twenty muscle contractions were stimulated every 48 h using surface electrodes. After 28 days, ES significantly decreased the expression of myoD and atrogin-1 in D+ES compared to the D group. However, ES did not prevent muscle-fiber atrophy after denervation. Thus, ES based on chronaxie values and applied to denervated muscles using surface electrodes, as normally used in human rehabilitation, was able to reduce the myoD and atrogin-1 gene expressions, which are related to muscular growth and atrophy, respectively. The results of this study provide new information for the treatment of denervated skeletal muscle using surface ES. [ABSTRACT FROM AUTHOR]

Increasing attention is being given to the use of adult rather than embryonic stem cells, both for research and for the development of transplantation treatments for human disease. In particular, mesenchymal bone marrow stem cells have been studied extensively because of their ability to self-renew and to give rise to various differentiated cell types, and because of the relative ease with which they can be obtained and cultured. In addition, the possibility of labelling stem cells with green fluorescent protein before transplantation has opened new and promising perspectives for their use in basic research. Because no structural or ultrastructural description of adult mesenchymal stem cells is available in the literature, this paper describes their morphology as revealed by light, confocal and electron microscopy, focusing on cells that are particularly suitable for transplantation studies, i.e. those derived from rat bone marrow transfected with green fluorescent protein. The results provide a basis for experimental studies of the differentiation of these cells in normal and pathological tissues. [ABSTRACT FROM AUTHOR]

Twenty years have passed since the publication of the seminal paper enunciating the disector principle by an author using the pseudonym D.C. Sterio. During this time, methods based on the revolutionary principle of counting 'tops' have become progressively better known and have been included in several commercially available systems for quantitative morphology. Analysis of the number of published studies citing Sterio's paper on the ISI Web of Knowledge® database showed that its scientific 'impact factor' has almost continuously risen since its publication, indicating the growing knowledge about disector-based methods in the various scientific fields where morphological quantification is required. This report briefly reviews the first two decades of disector use, pointing to its advantages as well as to shortcomings that have recently been addressed in critical papers and have given rise to a lively debate on the role of counting tops in quantitative morphology today. Microsc. Res. Tech. 66:270-274, 2005. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The issue of postnatal neurogenesis has gained great importance over the last few years and the recent amazing scientific advancements, changing our viewpoint on the long-lasting “no new neurons” dogma, have opened promising new perspectives on the treatment of the damaged nervous system. While most of the researchers have focused on the central nervous system, the peripheral nervous system has received little attention so far with respect to postnatal histogenesis. To attract scientific attention on this issue, the present article was written with the aim of reviewing the body of literature on postnatal histogenesis in the various districts of the peripheral nervous system, from the historical roots to the most recent reports. [Copyright &y& Elsevier]