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.

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.

Melt-extruded guides for peripheral nerve regeneration. Part I: poly(epsilon-caprolactone).

Melt-extruded guides for peripheral nerve repair based on poly(epsilon-caprolactone) (PCL) were realised and their physico-chemical properties were evaluated. Preliminarily, PCL cast films were found to support the attachment and proliferation of Neonatal Olfactory Bulb Ensheating Cells (NOBEC). S5Y5 neuroblastoma cells were cultured inside PCL guides in their uncoated form or coated with a non-specific adhesion protein (gelatin) and a specific peptide for nerve regeneration (poly(L-lysine)). Coating increased cell density (gelatin) and/or the cell density rate on substrates (poly(L-lysine); gelatin) as compared to uncoated guides. Various in vivo tests were carried out for the repair of small (0.5 cm), medium (1.5 cm) and long (4.5 cm) size defects in the peripheral nerves of Wistar rats. For the small nerve defects, uncoated and coated PCL guides were tested. Results from in vivo tests were subjected to histological examination after 45 days, 6 and 8 months postoperative for small, medium and large defects, respectively. Regeneration was found for small and medium size defects. For 0.5 cm defects, the coating did not affect regeneration significantly. Grip-tests also evidenced functional recovery for the 1.5 cm-long defects treated with PCL guides, after 6 months from implantation. On the other hand, mechanical stiffness of PCL conduits impaired the repair of 4.5 cm-long defects in 8-month period: the lack of flexibility of the guide to rat movements caused its detachment from the implant site. The research showed that PCL guides can be used for the successful repair of small and medium size nerve defects, with possible improvements by suitable bio-mimetic coatings.

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 alpha-, but not beta-, 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 alpha-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 alpha-NRG1-mediated gliotrophic loop in denervated Schwann cells provides a possible explanation for the effectiveness of muscle conduits for tubulization nerve repair.

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.

Denervation-related changes in acetylcholine receptor density and distribution in the rat flexor digitorum sublimis muscle.

Reorganization of the muscle endplate structures is an important parameter for the study of posttraumatic neuromuscular recovery. The aim of this study was to investigate the changes and the distribution of the acetylcholine receptors (AChRs) in flexor digitorum sublimis muscle after 30 days of denervation in the rat forelimb experimental model. In young male rats, the median and ulnar nerves of the right forelimb were surgically transected and a 1-cm-long segment was removed to avoid spontaneous regeneration. Along the postoperative, the presence of complete functional loss was assessed by the grasping test. After 30 days, rats were sacrificed and flexor digitorum sublimis muscles of both limbs were explanted. The muscles were analysed by light microscopy, to assess the degree of muscle atrophy, and by immunofluorescence after rodhamine-conjugated alpha-bungarotoxin incubation to investigate the reorganization of endplates. The occurrence of muscle denervation was established, prior to sacrifice, by complete loss of the grip function. Light microscopy showed that 30-day denervation is sufficient to induce severe muscle fiber atrophy. Fluorescence analysis at low resolution showed that background fluorescence was higher in denervated muscles possibly because of the presence of extrajunctional AChR. At higher resolution, the endplates were clearly visible as ribbon-like structures. In control fibres, AChR formed a compact and bright structure while in denervated samples it appeared more diffuse and dimmer. Quantitative analysis showed that endplate area was larger in denervated muscles than in control samples. A corresponding decrease in fluorescence intensity was observed after subtracting the basal fluorescence. In conclusion, results of the present study demonstrate that 30 days of denervation induce severe atrophy in rat flexor digitorum sublimis muscle that is accompanied by significant changes in acetylcholine receptor density and distribution. These results also suggest that the rat median nerve denervation experimental model can be an excellent approach for the study of the progression of endplate re-organization after muscle denervation, and reinnervation, considering also its relatively low impact on animal well being in comparison to other experimental models.

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 alpha(2a-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 alpha(2a-2b) NRG1 through the erbB2/erbB3 heterodimer receptor for nerve regeneration inside non-nervous conduits.

Expression of alpha2a-2b neuregulin-1 is associated with early peripheral nerve repair along muscle-enriched tubes.

Using RT-PCR, we have investigated expression of isoforms beta1 (the axonal isoform) and alpha2a-2b (the mesenchymal isoform) of neuregulin-1, one of the most important known trophic factors for Schwann cells, in the rat sciatic nerve repaired by muscle-enriched non-nervous conduits (made by a vein filled with fresh skeletal muscle). Repaired nerves were harvested 2, 6 and 13 days post-operatively. Results showed that while muscle-vein combined grafts were enriched in mRNA coding for alpha2a-2b since the very early regeneration stages, isoform beta1 mRNA was not detectable inside the tubes at day 2 and 6 post-operatively while its expression at day 13 was very slight. These results suggest that Schwann cell survival and activity inside a fresh muscle-enriched non-nervous conduit graft (a key factor for successful nerve regeneration along the graft) may be supported by the mesenchymal isoform of neuregulin-1 during very early repair phases, i.e. when axons are still not present along the tube.