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Optimal recovery of muscle function after proximal nerve injuries remains a complex and challenging problem. After a nerve injury, alterations in the affected muscles lead to atrophy, and later degeneration and replacement by fat-fibrous tissues. At present, several different strategies for the preservation of skeletal muscle have been reported, including various sets of physical exercises, muscle massage, physical methods (e.g. electrical stimulation, magnetic field and laser stimulation, low-intensity pulsed ultrasound), medicines (e.g. nutrients, natural and chemical agents, anti-inflammatory and antioxidants, hormones, enzymes and enzyme inhibitors), regenerative medicine (e.g. growth factors, stem cells and microbiota) and surgical procedures (e.g. supercharge end-to-side neurotization). The present review will focus on methods that aimed to minimize the damage to muscles after denervation based on our present knowledge.

Templeton World Charity Foundation, Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

The accumulating evidence linking bacteria in the gut and neurons in the brain (the microbiota–gut–brain axis) has led to a paradigm shift in the neurosciences. Understanding the neurobiological mechanisms supporting the relevance of actions mediated by the gut microbiota for brain physiology and neuronal functioning is a key research area. In this review, we discuss the literature showing how the microbiota is emerging as a key regulator of the brain’s function and behavior, as increasing amounts of evidence on the importance of the bidirectional communication between the intestinal bacteria and the brain have accumulated. Based on recent discoveries, we suggest that the interaction between diet and the gut microbiota, which might ultimately affect the brain, represents an unprecedented stimulus for conducting new research that links food and mood. We also review the limited work in the clinical arena to date, and we propose novel approaches for deciphering the gut microbiota–brain axis and, eventually, for manipulating this relationship to boost mental wellness., Templeton World Charity Foundation, Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

Biosynthetic nerve grafts are developed in order to complement or replace autologous nerve grafts for peripheral nerve reconstruction. Artificial nerve guides currently approved for clinical use are not widely applied in reconstructive surgery as they still have limitations especially when it comes to critical distance repair. Here we report a comprehensive analysis of fine-tuned chitosan nerve guides (CNGs) enhanced by introduction of a longitudinal chitosan film to reconstruct critical length 15 mm sciatic nerve defects in adult healthy Wistar or diabetic Goto-Kakizaki rats. Short and long term investigations demonstrated that the CNGs enhanced by the guiding structure of the introduced chitosan film significantly improved functional and morphological results of nerve regeneration in comparison to simple hollow CNGs. Importantly, this was detectable both in healthy and in diabetic rats (short term) and the regeneration outcome almost reached the outcome after autologous nerve grafting (long term). Hollow CNGs provide properties likely leading to a wider clinical acceptance than other artificial nerve guides and their performance can be increased by simple introduction of a chitosan film with the same advantageous properties. Therefore, the chitosan film enhanced CNGs represent a new generation medical device for peripheral nerve reconstruction.

Michela Morano,1,* Sandra Wrobel,2,3,* Federica Fregnan,1 Ofra Ziv-Polat,4 Abraham Shahar,4 Andreas Ratzka,2 Claudia Grothe,2,3 Stefano Geuna,1 Kirsten Haastert-Talini2,3 1Department of Clinical and Biological Sciences, Università Degli Studi di Torino, Orbassano, Piemonte, Italy; 2Institute of Neuroanatomy, Hannover Medical School, Hannover, Lower-Saxony, Germany; 3Center for Systems Neuroscience (ZSN), Hannover, Lower-Saxony, Germany; 4NVR Research Ltd, Ness-Ziona, Israel *These authors contributed equally to this work and share first authorship Purpose: Innovative nerve conduits for peripheral nerve reconstruction are needed in order to specifically support peripheral nerve regeneration (PNR) whenever nerve autotransplantation is not an option. Specific support of PNR could be achieved by neurotrophic factor delivery within the nerve conduits via nanotechnology or stem cell engineering and transplantation.Methods: Here, we comparatively investigated the bioactivity of selected neurotrophic factors conjugated to iron oxide nanoparticles (np-NTFs) and of bone marrow-derived stem cells genetically engineered to overexpress those neurotrophic factors (NTF-BMSCs). The neurite outgrowth inductive activity was monitored in culture systems of adult and neonatal rat sensory dorsal root ganglion neurons as well as in the cell line from rat pheochromocytoma (PC-12) cell sympathetic culture model system.Results: We demonstrate that np-NTFs reliably support numeric neurite outgrowth in all utilized culture models. In some aspects, especially with regard to their long-term bioactivity, np-NTFs are even superior to free NTFs. Engineered NTF-BMSCs proved to be less effective in induction of sensory neurite outgrowth but demonstrated an increased bioactivity in the PC-12 cell culture system. In contrast, primary nontransfected BMSCs were as effective as np-NTFs in sensory neurite induction and demonstrated an impairment of neuronal differentiation in the PC-12

Michela Morano,1,* Sandra Wrobel,2,3,* Federica Fregnan,1 Ofra Ziv-Polat,4 Abraham Shahar,4 Andreas Ratzka,2 Claudia Grothe,2,3 Stefano Geuna,1 Kirsten Haastert-Talini2,3 1Department of Clinical and Biological Sciences, Università Degli Studi di Torino, Orbassano, Piemonte, Italy; 2Institute of Neuroanatomy, Hannover Medical School, Hannover, Lower-Saxony, Germany; 3Center for Systems Neuroscience (ZSN), Hannover, Lower-Saxony, Germany; 4NVR Research Ltd, Ness-Ziona, Israel *These authors contributed equally to this work and share first authorship Purpose: Innovative nerve conduits for peripheral nerve reconstruction are needed in order to specifically support peripheral nerve regeneration (PNR) whenever nerve autotransplantation is not an option. Specific support of PNR could be achieved by neurotrophic factor delivery within the nerve conduits via nanotechnology or stem cell engineering and transplantation.Methods: Here, we comparatively investigated the bioactivity of selected neurotrophic factors conjugated to iron oxide nanoparticles (np-NTFs) and of bone marrow-derived stem cells genetically engineered to overexpress those neurotrophic factors (NTF-BMSCs). The neurite outgrowth inductive activity was monitored in culture systems of adult and neonatal rat sensory dorsal root ganglion neurons as well as in the cell line from rat pheochromocytoma (PC-12) cell sympathetic culture model system.Results: We demonstrate that np-NTFs reliably support numeric neurite outgrowth in all utilized culture models. In some aspects, especially with regard to their long-term bioactivity, np-NTFs are even superior to free NTFs. Engineered NTF-BMSCs proved to be less effective in induction of sensory neurite outgrowth but demonstrated an increased bioactivity in the PC-12 cell culture system. In contrast, primary nontransfected BMSCs were as effective as np-NTFs in sensory neurite induction and demonstrated an impairment of neuronal differentiation in the PC-12

Biosynthetic nerve grafts are desired as alternative to autologous nerve grafts in peripheral nerve reconstruction. Artificial nerve conduits still have their limitations and are not widely accepted in the clinical setting. Here we report an analysis of fine-tuned chitosan tubes used to reconstruct 10 mm nerve defects in the adult rat. The chitosan tubes displayed low, medium and high degrees of acetylation (DAI: similar to 2%, DA: similar to 5%, DAIII: similar to 20%) and therefore different degradability and microenvironments for the regenerating nerve tissue. Short and long term investigations were performed demonstrating that the chitosan tubes allowed functional and morphological nerve regeneration similar to autologous nerve grafts. Irrespective of the DA growth factor regulation demonstrated to be the same as in controls. Analyses of stereological parameters as well as the immunological tissue response at the implantation site and in the regenerated nerves, revealed that DAI and DAIII chitosan tubes displayed some limitations in the support of axonal regeneration and a high speed of degradation accompanied with low mechanical stability, respectively. The chitosan tubes combine several pre-requisites for a clinical acceptance and DAII chitosan tubes have to be judged as the most supportive for peripheral nerve regeneration. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker (R) staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration., Included in Cristina Mantovanis thesis in manuscript form.

Estimation of cerebellar Purkinje cell number is an important evaluation parameter for the study of the development of the cerebellum as well as for investigating the changes occurring in various experimental and physiological conditions. The introduction of stereological techniques in biological and biomedical research has made quantification of Purkinje cells not only more reliable, because independent from morphology-related sources of bias, but also easier and less time consuming. In this paper, we will briefly overview the stereological techniques that have been used so far for estimation of Purkinje cell number in different animal species and we will describe a simple method (the confocal physical disector) for Purkinje cell quantification on confocal microscope optical slices, based on a variation of the physical disector technique that can be adopted without the need of any dedicated stereological workstation., SCOPUS: ar.j, info:eu-repo/semantics/published

Estimation of cerebellar Purkinje cell number is an important evaluation parameter for the study of the development of the cerebellum as well as for investigating the changes occurring in various experimental and physiological conditions. The introduction of stereological techniques in biological and biomedical research has made quantification of Purkinje cells not only more reliable, because independent from morphology-related sources of bias, but also easier and less time consuming. In this paper, we will briefly overview the stereological techniques that have been used so far for estimation of Purkinje cell number in different animal species and we will describe a simple method (the confocal physical disector) for Purkinje cell quantification on confocal microscope optical slices, based on a variation of the physical disector technique that can be adopted without the need of any dedicated stereological workstation., SCOPUS: ar.j, info:eu-repo/semantics/published

Cell transplantation therapy has raised a great interest in the perspective of its employment for nerve tissue repair. Among the various cell populations proposed, olfactory ensheathing glial cells have raised great interest over recent years, especially in the perspective of their employment for neural repair because of their homing capacity in both central and peripheral nervous system. This paper is aimed to provide an in vitro characterization of the NOBEC (neonatal olfactory bulb ensheathing cell) line that was obtained from primary cells dissociated from rat neonatal olfactory bulb (OB) and immortalized by retroviral transduction of SV40 large T antigen. Light and electron microscopy investigation showed that NOBECs are a homogeneous cell population both at structural and ultrastructural level. RT-PCR, Western blotting and immunocytochemistry showed that NOBECs express the glial markers S100, GFAP (Glial Fibrillar Acid Protein) and p75NGFR as well as NRG1 (neuregulin-1) and ErbB1-2-3 receptors; while they are negative for ErbB4. Yet, NOBECs exhibit a high proliferation and migration basal activity and can be transducted with vectors carrying GFP (green fluorescent protein) and NRG1 cDNA. Functional stimulation by means of NRG1-III-beta3 overexpression through viral transduction induced a significant increase in cell proliferation rate while it had no effect on cell migration. Altogether, these results show that NOBEC cell line retain glial features both morphologically and functionally, responding to the NRG1/ErbB-mediated gliotrophic stimulus, and represents thus a good tool for in vitro assays of glial cell manipulation and for in vivo experimental studies of glial cell transplantation in the central and peripheral nervous system.

The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks), and significant (24 weeks) PC loss. Only in mice with significant cell loss, grafted NPCs migrated into the cerebellar cortex. These animals showed improved motor skills compared with sham-treated controls. No grafted cell adopted the morphological and immunohistochemical characteristics of PCs, but the cerebellar cortex in NPC-grafted SCA1 mice had a significantly thicker molecular layer and more surviving PCs. Perforated patch-clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. We postulate that a similar neuroprotective effect of NPCs may be applicable to other cerebellar degenerative diseases., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks), and significant (24 weeks) PC loss. Only in mice with significant cell loss, grafted NPCs migrated into the cerebellar cortex. These animals showed improved motor skills compared with sham-treated controls. No grafted cell adopted the morphological and immunohistochemical characteristics of PCs, but the cerebellar cortex in NPC-grafted SCA1 mice had a significantly thicker molecular layer and more surviving PCs. Perforated patch-clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. We postulate that a similar neuroprotective effect of NPCs may be applicable to other cerebellar degenerative diseases., info:eu-repo/semantics/published

The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks), and significant (24 weeks) PC loss. Only in mice with significant cell loss, grafted NPCs migrated into the cerebellar cortex. These animals showed improved motor skills compared with sham-treated controls. No grafted cell adopted the morphological and immunohistochemical characteristics of PCs, but the cerebellar cortex in NPC-grafted SCA1 mice had a significantly thicker molecular layer and more surviving PCs. Perforated patch-clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. We postulate that a similar neuroprotective effect of NPCs may be applicable to other cerebellar degenerative diseases., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks), and significant (24 weeks) PC loss. Only in mice with significant cell loss, grafted NPCs migrated into the cerebellar cortex. These animals showed improved motor skills compared with sham-treated controls. No grafted cell adopted the morphological and immunohistochemical characteristics of PCs, but the cerebellar cortex in NPC-grafted SCA1 mice had a significantly thicker molecular layer and more surviving PCs. Perforated patch-clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. We postulate that a similar neuroprotective effect of NPCs may be applicable to other cerebellar degenerative diseases., info:eu-repo/semantics/published