Can regenerated nerve fibers return to normal size? A long-term post-traumatic study of the rat median nerve crush injury model.

Whether post-traumatic regeneration can eventually result in rat peripheral nerve fibers regaining their pretrauma size is still an open question. While it has been shown that, after a sufficient duration in post-traumatic time, the number of regenerated rat peripheral nerve fibers can return to pretrauma numbers and the animal can regain normal prelesion function, no information regarding long-term changes in the size parameters of the regenerated nerve fibers is available. To fill this gap, we have investigated the post-traumatic changes in myelinated axon and nerve fiber diameter, myelin thickness, and g-ratio (the ratio of the inner axonal diameter to the fiber diameter) at three different time points following nerve injury: week-6, week-8, and week-24. A standardized nerve crush injury of the rat median nerve obtained using a nonserrated clamp was used for this study. The results showed that, consistent with previous studies, fiber number returned to normal values at week-24, but both axon and fiber diameter and myelin thickness were still significantly lower at week-24 than prelesion, and the g-ratio, which remained unchanged during the regeneration process, was significantly reduced at week-24 in comparison to the prelesion value. On the basis of these results, the hypothesis that regenerated rat peripheral nerve fibers are able to return spontaneously to their normal pretrauma state, provided there is a sufficiently long recovery time postaxonotmesis, is not supported.

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.

Functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp.

Peripheral nerve researchers frequently use the rat sciatic nerve crush as a model for axonotmesis. Unfortunately, studies from various research groups report results from different crush techniques and by using a variety of evaluation tools, making comparisons between studies difficult. The purpose of this investigation was to determine the sequence of functional and morphologic changes after an acute sciatic nerve crush injury with a non-serrated clamp, giving a final standardized pressure of p = 9 MPa. Functional recovery was evaluated using the sciatic functional index (SFI), the extensor postural thrust (EPT) and the withdrawal reflex latency (WRL), before injury, and then at weekly intervals until week 8 postoperatively. The rats were also evaluated preoperatively and at weeks 2, 4, and 8 by ankle kinematics, toe out angle (TOA), and gait-stance duration. In addition, the motor nerve conduction velocity (MNCV) and the gastrocnemius-soleus weight parameters were measured just before euthanasia. Finally, structural, ultrastructural and histomorphometric analyses were carried out on regenerated nerve fibers. At 8 weeks after the crush injury, a full functional recovery was predicted by SFI, EPT, TOA, and gait-stance duration, while all the other parameters were still recovering their original values. On the other hand, only two of the histomorphometric parameters of regenerated nerve fibers, namely myelin thickness/axon diameter ratio and fiber/axon diameter ratio, returned to normal values while all other parameters were significantly different from normal values. The employment of traditional methods of functional evaluation in conjunction with the modern techniques of computerized analysis of gait and histomorphometric analysis should thus be recommended for an overall assessment of recovery in the rat sciatic nerve crush model.

Bridging peripheral nerve defects with muscle-vein combined guides.

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.

Repairing nerve gaps by vein conduits filled with lipoaspirate-derived entire adipose tissue hinders nerve regeneration.

In spite of great recent advancements, the definition of the optimal strategy for bridging a nerve defect, especially across long gaps, still remains an open issue since the amount of autologous nerve graft material is limited while the outcome after alternative tubulization techniques is often unsatisfactory. The aim of this study was to investigate a new tubulization technique based on the employment of vein conduits filled with whole subcutaneous adipose tissue obtained by lipoaspiration. In adult rats, a 1cm-long defect of the left median nerve was repaired by adipose tissue-vein-combined conduits and compared with fresh skeletal muscle tissue-vein-combined conduits and autologous nerve grafts made by the excised nerve segment rotated by 180°. Throughout the postoperative period, functional recovery was assessed using the grasping test. Regenerated nerve samples were withdrawn at postoperative month-6 and processed for light and electron microscopy and stereology of regenerated nerve fibers. Results showed that functional recovery was significantly slower in the adipose tissue-enriched group in comparison to both control groups. Light and electron microscopy showed that a large amount of adipose tissue was still present inside the vein conduits at postoperative month-6. Stereology showed that all quantitative morphological predictors analyzed performed significantly worse in the adipose tissue-enriched group in comparison to the two control groups. On the basis of this experimental study in the rat, the use of whole adipose tissue for tissue engineering of peripheral nerves should be discouraged. Pre-treatment of adipose tissue aimed at isolating stromal vascular fraction and/or adipose derived stem/precursor cells should be considered a fundamental requisite for nerve repair.

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.

Chapter 4: Methods and protocols in peripheral nerve regeneration experimental research: part I-experimental models.

This paper addresses several basic issues that are important for the experimental model design to investigate peripheral nerve regeneration. First, the importance of carrying out adequate preliminary in vitro investigation is emphasized in light of the ethical issues and with particular emphasis on the concept of the Three Rs (Replacement, Reduction, and Refinement) for limiting in vivo animal studies. Second, the various options for the selection of the animal species for nerve regeneration research are reviewed. Third, the two main experimental paradigms of nerve lesion (axonotmesis vs. neurotmesis followed by microsurgical reconstruction) are critically outlined and compared. Fourth, the various nerve models that have most commonly been employed are overviewed focusing in particular on forearm mixed nerves and on behavioural tests for assessing their function: the ulnar test and the grasping test which is useful for assessing both median and radial nerves in the rat. Finally, the importance of considering the influence of various factors and diseases which could interfere with the nerve regeneration process is emphasized in the perspective of a wider adoption of experimental models which more closely mimic the environmental and clinical conditions found in patients.

Chapter 5: Methods and protocols in peripheral nerve regeneration experimental research: part II-morphological techniques.

This paper critically overviews the main procedures used for carrying out morphological analysis of peripheral nerve fibers in light, confocal, and electron microscopy. In particular, this paper emphasizes the importance of osmium tetroxide post-fixation as a useful procedure to be adopted independently from the embedding medium. In order to facilitate the use of any described techniques, all protocols are presented in full details. The pros and cons for each method are critically addressed and practical indications on the different imaging approaches are reported. Moreover, the basic rules of morpho-quantitative stereological analysis of nerve fibers are described addressing the important concepts of design-based sampling and the disector. Finally, a comparison of stereological analysis on myelinated nerve fibers between paraffin- and resin-embedded rat radial nerves is reported showing that different embedding procedures might influence the distribution of size parameters.

Morphology of nerve fiber regeneration along a biodegradable poly (DLLA-epsilon-CL) nerve guide filled with fresh skeletal muscle.

Previous morphological and morphometrical studies showed that fresh-skeletal-muscle-enriched vein segments are good conduits for leading peripheral nerve regeneration. In the present study, we investigated the morphological features of peripheral nerve fibers regenerated along a 10-mm-long biodegradable poly (DLLA-epsilon-CL) nerve guide enriched with fresh skeletal muscle, comparing them to nerve fiber regeneration along 10-mm-long phosphate-buffered saline (PBS)-enriched poly (DLLA-epsilon-CL) tubes. Repaired nerves were analyzed at weeks 6 and 24 postoperatively. Structural and ultrastructural observation showed that good nerve fiber regeneration occurred in both PBS-enriched and fresh-skeletal-muscle-enriched nerve guides, and histomorphometrical analysis of regenerated myelinated fibers revealed no statistically significant differences between the two experimental groups at week 24 after surgery. The employment of fresh-muscle-enriched conduits for the repair of nerve defects is critically discussed in the light of these results.