Maria G. Giacobini-Robecchi

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.

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.

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.

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.

Adult stem cells and neurogenesis: Historical roots and state of the art

Over the last few years, an impressive number of papers have addressed the stem cell issue. However, as often occurs when a scientific subject undergoes a period of fast growth, some confusion is generated. To help reduce the existing uncertainty, this paper focuses on the concept of adult stem cells in relation to the classification of cell populations on the basis their proliferative behavior. Particular attention is dedicated to adult neural stem cells, an issue that has recently seen the most amazing advances. Finally, the concept of adult stem cells is differentiated from that of developmental stem cells in relation to the employment of stem cells for transplantation therapies. Anat Rec (New Anat) 265:132–141, 2001.

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.