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.

Discrepancies in quantitative assessment of normal and regenerated peripheral nerve fibers between light and electron microscopy.

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.

Role of inflammatory cytokines in peripheral nerve injury.

Inflammatory events occurring in the distal part of an injured peripheral nerve have, nowadays, a great resonance. Investigating the timing of action of the several cytokines in the important stages of Wallerian degeneration helps to understand the regenerative process and design pharmacologic intervention that promotes and expedites recovery. The complex and synergistic action of inflammatory cytokines finally promotes axonal regeneration. Cytokines can be divided into pro- and anti-inflammatory cytokines that upregulate and downregulate, respectively, the production of inflammatory mediators. While pro-inflammatory cytokines are expressed in the first phase of Wallerian degeneration and promote the recruitment of macrophages, anti-inflammatory cytokines are expressed after this recruitment and downregulate the production of all cytokines, thus determining the end of the process. In this review, we describe the major inflammatory cytokines involved in Wallerian degeneration and the early phases of nerve regeneration. In particular, we focus on interleukin-1, interleukin-2, interleukin-6, tumor necrosis factor-ß, interleukin-10 and transforming growth factor-ß.

Plasticity and regeneration in the peripheral nervous system.

While in the central nervous system plasticity (in response to stimulus) and regeneration (in response to injury) are mainly based on adaptive changes in neural circuitries and synaptic reorganization, in the peripheral nervous system they are predominantly based on axonal (re)growth and neuron addition. In this paper, we will briefly overview the main investigation lines on plasticity and regeneration in the peripheral nervous system that have been carried out at the Laboratory of Human Anatomy of the Department of Clinical and Biological Sciences at the "San Luigi Gonzaga" Medical Faculty of the University of Turin. This body of research was mainly focused on the identification of the adaptive changes occurring to the sensory and autonomic neurons as a consequence of exceptional stimuli and/or damage at their periphery, as well as on the identification of effective new strategies for improving post-traumatic peripheral nerve fiber regeneration. These studies are in line with the long standing tradition on peripheral nervous system investigation carried out by the Anatomical School at the University of Turin since the times of Giuseppe Levi and we are honoured to have the occasion to present the results of our research on occasion of the appointment of Giovanni Orlandini as Emeritus Professor of the University of Florence.

Morphological and biomolecular characterization of the neonatal olfactory bulb ensheathing cell line.

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.

Neuron-specific Hu proteins sub-cellular localization in primary sensory neurons.

The Hu family of RNA-binding proteins is involved in many post-transcriptional mechanisms for the development and maintenance of the nervous system. Three members of the Hu family (HuB, HuC and HuD) are neuron-specific proteins. In this study, we present data using light and electron microscopy to show the sub-cellular localization of neuron-specific Hu proteins in rat primary sensory neurons taken from dorsal root ganglia (DRG). Using these techniques we morphologically revealed the presence of neuron-specific-Hu proteins in the nucleus and in the cytoplasm and discriminated the presence of Hu proteins within different cellular organelles, specifically mitochondria and Golgi apparatus, thus supporting previous ideas that NS-Hu proteins enable RNA interactions with sub-cellular organelles and may be involved in mRNA cellular localization.