Bruno Battiston

Nerve repair by means of vein filled with muscle grafts I. Clinical results

Peripheral nerve lesions with a long segment defect need a grafting conduit to heal. Although autogenous nerve grafting is still considered the best method for bridging nerve defects, several alternative types of conduits (biological and synthetic) have been studied. We have demonstrated in previous experimental research in rats that a graft made using a vein (providing a guide for nerve regeneration) filled with fresh skeletal muscle (to prevent vein collapse and support axon regeneration) gave similar results to traditional nerve grafts. On this basis, we decided to use the muscle‐vein‐combined grafts in clinical cases. From 1993 to 1997, this technique was applied for bridging both sensory and mixed nerve defects (21 cases). We report good results in 85% of our cases with a minimum follow‐up of 14 months. These results, obtained on nerve defects ranging from 0.5 to 6 cm in length, seem to be superior to those reported with other kinds of artificial or biological conduits.

Methodological issues in size estimation of myelinated nerve fibers in peripheral nerves

Size estimation of myelinated nerve fibers in peripheral nerves is a very common task in neuromorphology and different dedicated morpho-quantitative procedures have been devised and used to date. Unfortunately, many reports on experimental nerve studies lack comprehensive information on the procedures that have been designed and applied for myelinated fiber size estimation. This paper addresses the issue in the light of the recent advances in quantitative morphology that have recognized the concept of unbiased estimates as the key methodological issue to be addressed in morpho-quantitative studies. The potential foundations of bias at various study levels are analysed together with indications on how to cope with them. In addition, the issue of the precision of size estimates is addressed and the various geometrical parameters that can be selected for myelinated nerve fiber size assessment are outlined. Taken together, information provided in this paper is expected to help investigators conduct an appropriate preliminary study design phase, the key step for setting up the most adequate morpho-quantitative procedure for any given research goal.

Verification of the two-dimensional disector, a method for the unbiased estimation of density and number of myelinated nerve fibers in peripheral nerves

Quantification of the number of myelinated fibers in peripheral nerves is a common requirement in quantitative morphology. This parameter provides important information on the consequences of various physiological, pathological and experimental conditions on the nerve structure and is one of the main indicators of success of peripheral nerve repair. In this paper, the theoretical rationale for the application of stereological principles to obtain unbiased estimates of the density and total number of myelinated fibers in peripheral nerves is discussed and a simple stereological method is described. The method is applied together with a systematic random sampling scheme, that was optimized for the purposes of the present study, and with sampling scheme analysis by calculating the coefficient of error (CE). The stereological method, which consists of a two-dimensional variation of the classical disector procedure (two-dimensional disector), and the sampling scheme are verified by comparing estimates with the true density and total number of myelinated fibers in peripheral nerve trunks where true values have been accurately determined by extensive counting. The verification of the 2-D disector method, both of normal and regenerated nerves, showed that estimates of density and total number of myelinated nerve fibers are unbiased. The method also proved to be efficient (time-saving): Estimation of density and total number of myelinated fibers in a single nerve takes about 2-3 hours.

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.

On the use of the grasping test in the rat median nerve model: a re-appraisal of its efficacy for quantitative assessment of motor function recovery.

The quantitative assessment of motor function is an important requirement for studies on peripheral nerve injury and repair. So far, most studies on peripheral nerves have been performed on the sciatic nerve model using walking track analysis for assessing motor function. Alternatively, the employment of the median nerve model, which allows motor function evaluation by means of a simple behavioural test named grasping test (GT), have been more recently proposed. In this paper, the efficacy of the GT for the quantitative assessment of motor function recovery is re-appraised and a modified device for its carrying out is described. Finally, the rationale for the employment of the median nerve model as an alternative to the sciatic nerve model is critically discussed.

Chapter 11: Tissue engineering of peripheral nerves.

Tissue engineering of peripheral nerves has seen an increasing interest over the last years and, similarly to many other fields of regenerative medicine, great expectations have risen within the general public to its potential clinical application in the treatment of damaged nerves. However, in spite of the scientific advancements, applications to the patients is still very limited and it appears that to optimize the strategy for the tissue engineering of the peripheral nerves in the clinical view, researchers have to strive for a new level of innovation which will bring together (in a multitranslational approach) the main pillars of tissue engineering: namely (1) microsurgery, (2) cell and tissue transplantation, (3) material science, and (4) gene transfer. This review paper provides an overview of these four key approaches to peripheral nerve tissue engineering. While some of these issues will also be specifically addressed in other papers in this special issue on peripheral nerve regeneration of the International Review of Neurobiology, in this paper we will focus on an example of successful translational research in tissue engineering, namely nerve reconstruction by muscle-vein-combined nerve scaffolds.

Bridging peripheral nerve defects with muscle-vein combined guides.

Abstract Various tubulization techniques can be used to bridge peripheral nerve lesions with substance loss. Among the different materials that have been used so far in alternative to traditional fresh nerve autografts, fresh muscle-vein combined conduits (made by a vein segment filled with fresh skeletal muscle) proved to be particularly effective. In this study, nerve repair of 10-mm long nerve defects by means of muscle-vein combined tubes was compared with repair by means of traditional nerve autografts in the rat sciatic nerve experimental model. Results did not reveal any significant difference between the two groups of regenerated nerves with respect to the total number, mean density and mean size of myelinated nerve fibers. In addition, we also report the results of an experimental study in the rabbit sciatic nerve model, which showed that fresh skeletal muscle enrichment of the vein segment made it possible to bridge 55-mm long nerve gaps. These results provide further evidence of the effectiveness of fresh muscle-vein combined grafts and support the view that this type of conduit can be used also for repairing long nerve gaps.

Perspectives in regeneration and tissue engineering of peripheral nerves

Peripheral nerve injury is a common casualty and although peripheral nerve fibers retain a considerable regeneration potential also in the adult, recovery is usually rather poor, especially in case of large nerve defects. The aim of this paper is to address the perspectives in regeneration and tissue engineering after peripheral nerve injury by reviewing the relevant experimental studies in animal models. After a brief overview of the morphological changes related to peripheral nerve injury and regeneration, the paper will address the evolution of peripheral nerve tissue engineering with special focus on transplantation strategies, from organs and tissues to cells and genes, that can be carried out, particularly in case of severe nerve lesions with substance loss. Finally, the need for integrated research which goes beyond therapeutic strategies based on single approaches is emphasized, and the importance of bringing together the various complimentary disciplines which can contribute to the definition of effective new strategies for regenerating the injured peripheral nerve is outlined.

Morphological analysis of peripheral nerve regenerated by means of vein grafts filled with fresh skeletal muscle.

Clinical data have shown that a vein segment filled with fresh skeletal muscle can be considered a good autologous grafting conduit for the repair of peripheral nerve lesions. In this study, the long-term morphological organization of rat sciatic nerve fibers regenerated along a muscle-vein-combined graft conduit is further analysed by light and electron microscopy. Regenerated nerve fibers were organized into fascicles of various sizes that were clearly delimited by perineurial-like shells made by long and thin cytoplasmic processes of perineurial-like bipolar cells and by densely packed collagen fibrils. Grafted skeletal muscle fibers were still detectable among nerve fiber fascicles. However, in spite of the persistence of skeletal muscle along the graft, regenerated nerve fibers showed a good morphological pattern of regeneration, providing further evidence that the muscle-vein-combined grafting technique represents an effective surgical alternative to the classical fresh nerve autograft for the repair of peripheral nerve defects.

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.