S. Nicolino

Schwann cell behavior after nerve repair by means of tissue‐engineered muscle‐vein combined guides

Schwann cells play a critical role in peripheral nerve regeneration. When a non‐nervous conduit is used to bridge a nerve defect, the conduit is soon colonized by a number of Schwann cells that make a pathway for regrowing axons. By using electron microscopy, immunohistochemistry, and reverse transcriptase‐polymerase chain reaction analysis, we have investigated the behavior of migratory glial cells along a particular type of autologous tissue‐engineered conduit made of a vein filled with fresh skeletal muscle, using the rat sciatic nerve model. With this particular type of autograft, our data show that many Schwann cells soon take up a close relationship with grafted muscle fibers, and especially with their basal lamina, which appears to serve as a migration pathway for them. The early and massive colonization of the conduit is sustained by both Schwann cell migration and proliferation, as demonstrated by PCNA immunostaining. Later, as they meet regenerating axons, Schwann cells become closely associated with them and eventually lose their connections with grafted muscle fibers because of the formation of perineurial envelopes. Because previous studies showed that α2a‐2b NRG1 is overexpressed at early stages along the muscle‐vein combined tubes, we have also investigated mRNA expression of its two receptors, erbB2 and erbB3. Both messengers are overexpressed, although with different time courses. Overall, our results provide some morphological and biochemical bases for explaining the effectiveness of fresh muscle‐vein combined nerve guides and throw an interesting light on the possible role of α2a‐2b NRG1 through the erbB2/erbB3 heterodimer receptor for nerve regeneration inside non‐nervous conduits. J. Comp. Neurol. 489:249–259, 2005.

Neuronal intermediate filament expression in rat dorsal root ganglia sensory neurons: An in vivo and in vitro study

Qualitative and quantitative examination was performed to evaluate the expression of peripherin and 200 kDa neurofilament in the sensory compartment of the peripheral nervous system of the rat both in vivo and in a new in vitro model. Under physiological conditions, these two neuronal intermediate filaments show different expression patterns in sensory neurons. To have a more complete comprehension of the role of these intermediate filaments and to fill in the blanks left in previously reported literature, we demonstrate in vivo using a morphological approach that peripherin and 200 kDa neurofilament define two distinct subpopulations within the dorsal root ganglia sensory neurons. Moreover, peripherin is specifically expressed in unmyelinated fibers while 200 kDa neurofilament is expressed in myelinated fibers. Additionally, in vitro analysis of RNA taken from dorsal root ganglia explants suggested that 200 kDa neurofilament is downregulated and peripherin is transiently expressed throughout sensory fiber regrowth. In particular, both neuronal intermediate filaments are downregulated immediately after sensory fiber axotomy thus suggesting that neither peripherin nor 200 kDa neurofilament has a role in the first steps of fiber regrowth. However, the upregulation of peripherin a few days after the beginning of fiber regrowth in vitro suggests that low levels of peripherin may be require to carry on the sequence of events involved in the correct regeneration and direction of sensory fibers.

Functional and morphological assessment of a standardized crush injury of the rat median nerve

The availability of effective experimental models for investigating nerve regeneration and designing new strategies for promoting this unique repair process is important. The aim of this study was to standardize a rat median nerve crush injury model using a non-serrated clamp exerting a compression force of 17.02 MPa for a duration of 30s. Results showed that functional recovery, evaluated by grasping test, was already detectable at day-12 and progressively increased until day-28 after which animal performance plateaued until the end of testing (day-42), reaching a range of 75-80% of pre-operative values. Morphological analysis on the median nerve segments, distal to the crush lesion, which were withdrawn at the end of the experiment showed that regenerated nerve fibers are significantly more numerous and densely packed; they are also smaller and have a thinner myelin sheath compared to controls. Together, these results provide a baseline characterization of the crush median nerve injury experimental model for its employment in the investigation of nerve regeneration research, especially when a reproducible regeneration process is required, such as for the study of biological mechanisms of peripheral nerve fiber regeneration or development of new therapeutic agents for promoting posttraumatic nerve repair.

Employment of the mouse median nerve model for the experimental assessment of peripheral nerve regeneration.

The experimental investigation of nerve regeneration after microsurgical repair is usually carried out in rats, rather than mice, because of the larger sized peripheral nerves. Today however, the availability of genetically modified mice makes the use of this laboratory animal very intriguing for investigating nerve regeneration at a molecular level. In this study we aimed to provide a standardization of the experimental model based on microsurgical direct repair, by 12/0 suture, of the left median nerve in adult male mice. Postoperative recovery was regularly assessed by the grasping test. At day-75 postoperative, regenerated median nerve fibers were analyzed by design-based quantitative morphology and electron microscopy. Yet, sections were immuno-labelled using two axonal antibodies commonly employed for rat nerve fibers. Results indicated that functional recovery begun at day-15 and progressively increased reaching values not significantly different from normal by day-50. Quantitative morphology showed that, at day-75, the number of regenerated nerve fibers was not significantly different in comparison to controls. In contrast, differences were detected in fiber density, mean axon and fiber diameter and myelin thickness which were all significantly lower than controls. Immunohistochemistry showed that axonal markers commonly used for rat nerves studies are effective also for mouse nerves. Similar to the rat, the mouse median nerve model is superior to sciatic nerve model for the minimal impact on animal well-being and the effectiveness of the grasping test for motor function evaluation. The main limitation is the small nerve size which requires advanced microsurgical skills for performing 12/0 epineurial suturing.

Poly(ester urethane) Guides for Peripheral Nerve Regeneration.

A biocompatible and elastomeric PU was synthesized from low-molecular-weight PCL as macrodiol, CMD as chain extender and HDI as chain linker for applications in the field of peripheral nerve repair. PU cast films supported in vitro attachment and proliferation of NOBEC. The in vitro adhesion and proliferation of S5Y5 neuroblastoma cells on the inner surface of uncoated, gelatin- and PL-coated PU guides were compared. Due to their superior in vitro performance, PL-coated PU guides were tested in vivo for the repair of 1.8 cm-long defects in rat sciatic nerves. The progressive regeneration was confirmed by EMG and histological analysis showing the presence of regenerating fibers in the distal stumps.

Denervation and reinnervation of adult skeletal muscle modulate mRNA expression of neuregulin-1 and ErbB receptors.

Skeletal muscle atrophy represents one of the main causes of poor outcome of microsurgical nerve reconstruction. Recent studies have pointed to the importance of the neuregulin/ErbB signaling pathway in the development and regeneration of the neuromuscular system. Here, we show by immunohistochemistry, RT‐PCR, and Western blotting analyses, in an in vivo model of adult skeletal muscle denervation/reinnervation, that expression of Neuregulin1 (NRG1) and ErbB receptors is regulated by the innervation condition. We found out that a significant upregulation of the α‐, but not β‐, isoform of NRG1, as well as of ErbB2, ErbB3, and ErbB4‐cyt1 isoform occurs as a consequence of denervation of flexor digitorum muscles of the rat forelimb by median nerve transection. Moreover, after tubulization median nerve repair, and consequent muscle reinnervation, all messengers of the NRG1/ErbB system are promptly downregulated. Therefore, our results suggest the existence of a α‐NRG1‐mediated autocrine and/or paracrine trophic loop in skeletal muscles that is activated after denervation and promptly deactivated after nerve reconstruction. This myotrophic loop is a promising therapeutic target for the prevention of muscle atrophy. Yet, the recent demonstration of a similar α‐NRG1‐mediated gliotrophic loop in denervated Schwann cells provides a possible explanation for the effectiveness of muscle conduits for tubulization nerve repair.

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.

ErbB receptors modulation in different types of peripheral nerve regeneration

ErbBs are a family of receptors involved in the trophic maintenance of Schwann cells. Little is known about their expression changes during peripheral nerve regeneration. The aim of this study was thus to investigate variations in ErbBs after end-to-end and end-to-side nerve regeneration in the rat median nerve model. Expression of ErbBs was assessed at 7, 14, and 28 days postoperatively by real-time PCR. Results showed that expression of ErbB1 and ErbB4 mRNAs was downregulated, whereas ErbB3 mRNA was upregulated. No significant changes in ErbB2 mRNA were detected. Our results suggest that ErbBs changes are involved in the molecular response to peripheral nerve injuries.

Functional, morphological and biomolecular assessment of posttraumatic neuro-muscular recovery in the rat forelimb model.

Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization (using both straight and Y-shaped guides). The outcome of nerve repair at different postoperative times was assessed by functional, morphological and biomolecular analysis. Results showed that both end-to-side and tubulization repair of rat median and ulnar nerves led to successful axonal regeneration along the severed nerve trunk as well as to a partial recovery of the lost function as assessed by grasping test. Biomolecular analysis by means of reverse transcription polymerase chain reaction (RT-PCR) demonstrated early overexpression during nerve regeneration of the gliotrophic factor NRG1 and two of its receptors: erbB2 and erbB3. Finally, our experience also suggests that the rat forelimb experimental model is particularly appropriate for the study of microsurgical reconstruction of major mixed nerve trunks. Furthermore, since the forelimb model is less compromising for the animal, it should be preferred to the hindlimb model for many research purposes.