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Chitosan conduits have demonstrated to allow a good regeneration of an injured peripheral nerve (1), but an empty chitosan conduit is successful only for short nerve gap. For gap longer than 10mm it is necessary to enrich the conduit in order to create inside it an environment that allows and improves the regeneration of a damaged peripheral nerve. The conduit enrichment could be achieved by neurotrophic factor (NTFs) delivery within the nerve conduits via nanotechnology or cell engineering and transplantation. Here, we investigated in vitro the bioactivity of selected NTFs conjugated to iron oxide nanoparticles (IONP) and of bone marrow-derived stem cells genetically engineered to overexpress those NTFs. Moreover we reported preliminary in vivo experiments aimed to test the effect of free versus conjugated NTFs on regeneration of the rat sciatic nerve after a sever segment loss. In vitro experiments demonstrated that IONP-NTFs have a long-term bioactivity, even superior to free NTFs, while engineered NTF-cell proved to be less effective in induction of sensory neurite outgrowth but demonstrated an increased bioactivity in the PC-12 cell culture system (2). In vivo experiments, after the functional analysis and the morphometrical analysis of regenerated nerves, lead to conclude that after 5 months no differences are detectable between rats treated with free versus conjugated NTFs., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Promoting neuromuscular recovery after neural injury is a major clinical issue. While techniques for nerve reconstruction are continuously improving and most peripheral nerve lesions can be repaired today, recovery of the lost function is usually unsatisfactory. This evidence claims for innovative nonsurgical therapeutic strategies that can implement the outcome after neural repair.Although no pharmacological approach for improving posttraumatic neuromuscular recovery has still entered clinical practice, various molecules are explored in experimental models of neural repair. One of such molecules is the circulating peptide hormone ghrelin. This hormone has proved to have a positive effect on neural repair after central nervous system lesion, and very recently its effectiveness has also been demonstrated in preventing posttraumatic skeletal muscle atrophy. By contrast, no information is still available about its effectiveness on peripheral nerve regeneration although preliminary data from our laboratory suggest that this molecule can have an effect also in promoting axonal regeneration after nerve injury and repair.Should this be confirmed, ghrelin might represent an ideal candidate as a therapeutic agent for improving posttraumatic neuromuscular recovery because of its putative effects at all the various structural levels involved in this regeneration process, namely, the central nervous system, the peripheral nerve, and the target skeletal muscle. © 2013 Elsevier Inc.

The evidence of neurons generatedex novoin 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.

Skeletal muscle atrophy following denervation may not allow a good functional recovery if the muscle is not quickly re-innervated by the regenerating nerve fibers. In this regard, both improvement of peripheral nerve regeneration and prevention of skeletal muscle atrophy are important to achieve a satisfactory functional recovery after nerve injury. Local gene transfer could represent a promising strategyto reach this goal. Various genes are candidates in fostering peripheral axon regeneration and in preventing muscle atrophy, among these, the vascular endothelial growth factor (VEGF). The main goals of our study were twofold: (i) to accelerate axonal regeneration along muscle-vein-combined conduits used to bridge a 1-cm-long rat median nerve gap, and (ii) to prevent denervation-related atrophy of Fingers Flexor muscle after complete rat median nerve transection. Results showed that the over-expression of VEGF led to a worse nerve regeneration in the muscle-vein-combined guide in comparison to LacZ controls, whereas in the muscle it led to a significantly lower progression of atrophy. Altogether, our results suggest that VEGF direct local injection in denervated skeletal muscle significantly attenuates denervation-related atrophy thus representing a promising strategy for improving the outcome of posttraumatic neuromuscular recovery after nerve injury and repair., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

There is abundant evidence suggesting that growth factors may play a key role in the healing process, especially in the early stages of inflammation. Despite the reported clinical successes with the use of growth factors there is still a lack of knowledge on the biological mechanism underlying the activity of platelet-rich plasma during the process of muscle healing. The aim of this study was to analyse the early effects of platelet- rich plasma in an easily reproducible animal model.Wistar male adult rats (n=102) were used in this study. The muscle lesion was created with a scalpel in the flexor sublimis muscles. Platelet-rich plasma was applied immediately after surgery. Treated, untreated and contralateral muscles were analysed by morphological evaluation and western blot assay.Leucocyte infiltration was significantly greater in muscles treated with platelet-rich plasma than in both untreated and contralateral muscles. The latter showed greater leucocyte infiltration when compared to the untreated muscles. Platelet-rich plasma treatment also modified the cellular composition of the leucocyte infiltration leading to increased expression of CD3, CD8, CD19 and CD68 and to decreased CD4 antigen expression in both platelet-rich plasma treated and contralateral muscles. Blood vessel density and blood vessel diameters were not statistically significantly different between the three groups analysed.The results of this study showed that treatment with platelet-rich plasma magnified the physiological early inflammatory response following a muscle injury, modifying the pattern of cellular recruitment. Local platelet-rich plasma treatment may exert a direct or, more plausibly, indirect systemic effect on healing processes, at least in the earliest inflammatory phase.

Objective: Standard methods to evaluate the functional regeneration after injury of the rat median nerve are insufficient to identify any further differences of axonal nerve regeneration after restitution of motor recovery is completed. An important complementary method for assessing such differences is a histomorphometric analysis of the distal to lesion nerve fibers. Recently, an electrophysiological method has been proposed as a sensitive method to examine the quality of axonal nerve regeneration. Methods: A linear regression analysis has been performed to correlate histomorphometric and neurographic data originating from 31 rats subjected to neurotmesis and immediate reconstruction of their right median nerve. Results: A significant linear correlation between the velocity of neuromuscular conduction and the total number of nerve fibers (P = .037) as well as between the amplitude of compound muscle action potential and the total number of nerve fibers (P = .026) has been identified. Interestingly, a significant correlation between the velocity of neuromuscular conduction and the square root of the cross-sectional area of the nerve could be found (P = .008). This corresponds to a linear correlation between the velocity of neuromuscular conduction and the radius of the nerve. Conclusion: These results contribute in a better interpretation of morphological predictors of nerve regeneration and verify the previously described electrophysiological assessment in the median nerve rat model as a valid method.

The possibility that posttraumatic regeneration may eventually lead rat peripheral nerve fibers back to normal is still under debate. While it has been shown that, after a sufficiently long posttraumatic time, the number of regenerated rat peripheral nerve fibers can return to normal levels and the animal can regain normal pre-lesion function, no information regarding long-term changes in size parameters of regenerated nerve fibers is still available. To fill this gap, 24-week posttraumatic changes in myelinated axon and nerve fiber diameter, myelin thickness and g-ratio (axon diameter/fiber diameter), distal to a nerve crush (axonotmesis lesion) of the rat median nerve were assessed by stereology. Results showed that, while as expected fiber number returned to normal values at week-24, both axon and fiber diameter and myelin thickness were still significantly lower at week-24 in comparison to pre-lesion values. Finally, g-ratio, which persisted unmodified along the regeneration process, eventually resulted to be significantly reduced at week-24 in comparison to pre-lesion value. Based on these results, the hypothesis that regenerated rat peripheral nerve fibers are able to spontaneously return to normal, provided that a sufficiently long time recovery post-axonotmesis is allowed, is rejected., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011