Aims: Multiple nerve repair by means of a Y-shaped nerve guide represents a good model for studying the specificity of peripheral nerve fiber regeneration. Here we have employed this model for investigating the specificity of axonal regeneration in mixed nerves of the rat forelimb model. Specificity of nerve regeneration can be defined as the ability of the nerve fibers of a peripheral nerve, after a lesion. Tree types of specificity on nerve regeneration has been postulated: “tissue specificity” (the preferential reinnervation of distal nerve tissue versus other types of tissue), topographic specificity (regenerating nerve fibers are preferentially attracted by analogous distal pathways (e.g. preferential regeneration along tibial nerve pathways by tibial nerve fibers), and end-organ specificity, which is the hypothesis that distal end-organs (muscle vs. sensory targets) specifically attracts the respective (motor vs. sensory) regenerating nerve fibers. Exists no agreement regarding the presence and features of the two last type of specificity.

Methods: The left median and ulnar nerves, in adult female rats, were transected and repaired with a 14-mm Y-shaped conduit. The proximal end of the Y-shaped conduit was sutured to the proximal stump of either the median nerve or the ulnar nerve. Ten months after surgery, rats were tested for functional recovery of each median and ulnar nerve. Quantitative morphology of regenerated myelinated nerve fibers was then carried out by the two-dimensional disector technique.

Results: Results showed that partial recovery of both median and ulnar nerve motor function was regained in all experimental groups. Performance in the grasping test was significantly lower when the ulnar nerve was used as the proximal stump. Ulnar test assessment showed no significant difference between the two Y-shaped repair groups. The number of regenerated nerve fibers was significantly higher in the median nerve irrespectively of the donor nerve, maintaining the same proportion of myelinated fibers between the two nerves (about 60% median and 40% ulnar). On the other hand, nerve fiber size and myelin thickness were significantly larger in both distal nerves when the median nerve was used as the proximal donor nerve stump. G-ratio and myelin thickness/ axon diameter ratio returned to normal values in all experimental groups.

Conlusions: These results demonstrate that combined Y-shaped-tubulization repair of median and ulnar nerves permits the functional recovery of both nerves, independently from the proximal donor nerve employed, and that tissue, and not topographic, specificity guides nerve fiber regeneration in major forelimb mixed nerves of rats.

Aims: The combination of microsurgical techniques with engineering of pseudo-nerves has recently seen an increased employment for the repair of peripheral nerve defects. Over the last ten years, we have investigated a particular type of bio-engineered nerve guide, the muscle-vein-combined tube, which is made by filling a vein with skeletal muscle. However, the basic mechanism underlying the effectiveness of this surgical technique are still unclear and yet an experimental study on its efficacy on functional recovery compared to traditional nerve autografts is still lacking in the literature. The aim of the present study was thus to fill this gap.

Methods: In rats, 10-mm-long median nerve defects were repaired using either traditional autografts or fresh muscle-vein-combined bioengineered scaffolds. Posttraumatic nerve recovery was assessed by grasping test. The samples were collected at different times after surgery: 5, 15, 30 days and 6 months. Analysis was carried out by light and electron microscopy. In addition, reverse transcription polymerase chain reaction (RT-PCR) was used to investigate the expression of mRNAs coding for glial markers as well as glial growth factor (NRG1) and its receptors (erbB2 and erbB3).

Results: Results showed that both types of nerve repair techniques 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. Rats operated on by traditional nerve autografts performed better in the grasping test. Biomolecular analysis by RT-PCR demonstrated early overexpression during nerve regeneration of the gliotrophic factor NRG1 and two of its receptors: erbB2 and erbB3.

Conclusions: Our results confirmed that use of muscle-vein-combined tissue-engineered conduits is a good approach for bridging peripheral nerve defects in selected cases when traditional autografts are not employable and disclosed one of the basic biological mechanism that support the effectiveness of this surgical technique. Our experience also suggested 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.

Aims: Biosynthetic scaffolds made of degradable bio-materials enriched with cultured cells holds promise for peripheral nerve repair after complex traumatic injuries. In the perspective of future transplantation applications, the aim of this study was to investigate how cultures of olfactory ensheathing cells (OECs), in particular neonatal olfactory ensheathing cells (NOBECs), grow up in vitro on degradable polymeric films made with polycap-rolactone matrices and multi-block polyesterurethane respectively. In addition, since several transplantation studies use green fluorescent protein (GFP) positive cells so that they can be easily located in the receiving tissues, the cDNA encoding for GFP was cloned in expression vector and transfected in NOBECs.

Methods: To characterize NOBECs we employed electron microscopy, immunohistochemistry, RT-PCR and western blotting analyses. Moreover the proliferative ratio of NOBECs and the ability of the cells to migrate in a three dimensional environment were evaluated under basal and experimental culture conditions. Finally, the GFP-positive NOBEC were seeded on two types of synthetic films and their behaviour was analyzed to determine cell adhesion, survival and proliferation.

Results: We examined the expression of glial markers and NRG1/ErbB system in the NOBEC cell line at RNA and protein level. Results showed that NOBECs express both glial markers (GFAP and S-100), ErbB receptors (ErbB1, ErbB2 and ErB3) and different isoforms of NRG1. NOBECs exhibited a remarkable proliferation activity and a high basal migration activity. GFP positive NOBECs showed no significant difference in their behaviour as compared to untransfected parental cells. Finally, both normal and GFP-NOBECs showed good cell adhesion, survival and proliferation properties when seeded on both films employed in this study.

Conclusion: Taken together, results of our study showed that the glial cell line has similar biochemical properties as primary cultures of OECs. Moreover, we showed that NOBECs survive, proliferate and migrate on two different types of synthetic films that were prepared in the perspective of build up nerve scaffolds. Therefore, our results indicated that the NOBECs produce growth-promoting proteins and possess regeneration-promoting capabilities that make them a potentially good transplant material to enhance axonal regeneration inside synthetic tubes used to bridge nerve lesion with substance loss.

Aims: the recent ten years have seen a growing interest in termino-lateral (end-to-side) neurorrhaphy; this interest mainly originates from the prospected possibility to recover the function of a damaged nerve without loosing the function of a donor nerve.

We investigated voluntary control recovery after termino-lateral neurorrhaphy in the upper limb of mixed rat nerves (median and ulnar nerves) to assess functional recovery and nerve fibers regeneration.

Methods: We made a termino-lateral neurorrhaphy between median (lesioned) and ulnar nerve (donor) on left upper limb of 24 Wistar female rats. After 6 months, functional recovery of the limb was investigated using grasping test and electrical stimulation; then, rats were sacrificed and we studied morphological changes in muscles and regenerated nerves with light microscopy and stereology.

Results: We observed a functional recovery up to 15 % ± 5% of the normal at grasping test, while electrostimulation was positive in all cases; muscle trophism was good (40 % > than denervated muscles).

On microscope median nerve presented the typical structure of a regenerated nerve; in ulnar nerve some slight signs of degeneration can be detected distally to the site of suture. At the point of suture, nerve fibers of ulnar nerve sprouts into median nerve (collateral sprouting).

Conclusions: Termino-lateral neurorrhaphy induces a collateral sprouting from the donor nerve (as described in the literature); fiber regeneration in the severed nerve leads to a recovery of voluntary functional control.

In the donor ulnar nerve distally to the suture site we found some slight signs of damage, but they do not impair motor function.

In conclusion, termino-lateral neurorrhaphy can be used to repair peripheral nerve lesions with large substance loss where other types of repair strategies cannot be attempted.