Giulia Ronchi

Chapter 3: Histology of the peripheral nerve and changes occurring during nerve regeneration.

Peripheral nerves are complex organs that can be found throughout the body reaching almost all tissues and organs to provide motor and/or sensory innervation. A parenchyma (the noble component made by the nerve fibers, i.e., axons and Schwann cells) and a stroma (the scaffold made of various connective elements) can be recognized. Although morphological analysis is the most common approach for studying peripheral nerve regeneration, researchers are not always aware of several histological peculiarities of these organs. Therefore, the aim of this review is to describe, at a structural and ultrastructural level, the main features of the parenchyma and the stroma of the normal undamaged nerve as well as the most important morphological changes that occur after nerve damage and during posttraumatic nerve regeneration. The paper is aimed at providing the reader with the basic framework information on nerve morphology. This would enable the correct interpretation of morphological data obtained by many experimental studies on peripheral nerve repair and regeneration such as those outlined in several other papers included in this special issue of the International Review of Neurobiology.

Acylated and unacylated ghrelin impair skeletal muscle atrophy in mice

Cachexia is a wasting syndrome associated with cancer, AIDS, multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite, and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1a-independent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3Kβ-, mTORC2-, and p38-mediated pathways in myotubes. Upregulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsr-deficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner.

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.

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.

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.

Calibration of the stereological estimation of the number of myelinated axons in the rat sciatic nerve: A multicenter study

Several sources of variability can affect stereological estimates. Here we measured the impact of potential sources of variability on numerical stereological estimates of myelinated axons in the adult rat sciatic nerve. Besides biological variation, parameters tested included two variations of stereological methods (unbiased counting frame versus 2D-disector), two sampling schemes (few large versus frequent small sampling boxes), and workstations with varying degrees of sophistication. All estimates were validated against exhaustive counts of the same nerve cross sections to obtain calibrated true numbers of myelinated axons (gold standard). In addition, we quantified errors in particle identification by comparing light microscopic and electron microscopic images of selected consecutive sections. Biological variation was 15.6%. There was no significant difference between the two stereological approaches or workstations used, but sampling schemes with few large samples yielded larger differences (20.7+/-3.7% SEM) of estimates from true values, while frequent small samples showed significantly smaller differences (12.7+/-1.9% SEM). Particle identification was accurate in 94% of cases (range: 89-98%). The most common identification error was due to profiles of Schwann cell nuclei mimicking profiles of small myelinated nerve fibers. We recommend sampling frequent small rather than few large areas, and conclude that workstations with basic stereological equipment are sufficient to obtain accurate estimates. Electron microscopic verification showed that particle misidentification had a surprisingly variable and large impact of up to 11%, corresponding to 2/3 of the biological variation (15.6%). Thus, errors in particle identification require further attention, and we provide a simple nerve fiber recognition test to assist investigators with self-testing and training.

Employment of the mouse median nerve model for the experimental assessment of peripheral nerve regeneration.

The experimental investigation of nerve regeneration after microsurgical repair is usually carried out in rats, rather than mice, because of the larger sized peripheral nerves. Today however, the availability of genetically modified mice makes the use of this laboratory animal very intriguing for investigating nerve regeneration at a molecular level. In this study we aimed to provide a standardization of the experimental model based on microsurgical direct repair, by 12/0 suture, of the left median nerve in adult male mice. Postoperative recovery was regularly assessed by the grasping test. At day-75 postoperative, regenerated median nerve fibers were analyzed by design-based quantitative morphology and electron microscopy. Yet, sections were immuno-labelled using two axonal antibodies commonly employed for rat nerve fibers. Results indicated that functional recovery begun at day-15 and progressively increased reaching values not significantly different from normal by day-50. Quantitative morphology showed that, at day-75, the number of regenerated nerve fibers was not significantly different in comparison to controls. In contrast, differences were detected in fiber density, mean axon and fiber diameter and myelin thickness which were all significantly lower than controls. Immunohistochemistry showed that axonal markers commonly used for rat nerves studies are effective also for mouse nerves. Similar to the rat, the mouse median nerve model is superior to sciatic nerve model for the minimal impact on animal well-being and the effectiveness of the grasping test for motor function evaluation. The main limitation is the small nerve size which requires advanced microsurgical skills for performing 12/0 epineurial suturing.

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.

Future Perspectives in Nerve Repair and Regeneration

After peripheral nerve injuries, the process of nerve regeneration and target reinnervation is very complex and depends on many different events occurring not only at the lesion site but also proximally and distally to it. In spite of the recent scientific and technological advancements, the need to find out new strategies to improve clinical nerve repair and regeneration remains. To reach this goal, the therapeutic strategy should thus exert its effects at different levels in order to simultaneously potentiate axonal regeneration, increase neuronal survival, modulate central reorganization, and inhibit or reduce target organ atrophy. It is expected that this multilevel approach might lead to significant improvement in the functional outcome and thus the quality of life of the patients suffering from peripheral nerve injury.

Early homing of adult mesenchymal stem cells in normal and infarcted isolated beating hearts

Little is known on the early homing features of transplanted mesenchymal stem cells (MSCs). We used the isolated rat heart model to study the homing of MSCs injected in the ventricular wall of a beating heart. In this model all types of cells and matrix elements with their interactions are represented, while external interferences by endothelial/neutrophil interaction and neurohormonal factors are excluded. We studied the morphology and marker expression of MSCs implanted in normal hearts and in the border‐zone of infarcted myocardium. Early morphological adaptation of MSC homing differs between normal and infarcted hearts over the first 6 hrs after transplantation. In normal hearts, MSCs migrate very early through the interstitial milieu and begin to show morphological changes. Yet, in infarcted hearts MSCs remain in the site of injection forming clusters of round‐shaped cells in the border‐zone of the infarcted area. Both in normal and infarcted hearts, immuno‐histochemistry and confocal imaging showed that, besides the proliferative marker proliferating cell nuclear agent (PCNA), some transplanted cells early express myoblastic maker GATA‐4, and some of them show a VWF immunopositivity. Moreover, a few hours after injection connexin‐43 is well evident between cardiomy‐ocytes and injected cells. This study indicates for the first time that the isolated beating heart is a good model to study early features of MSC homing without external interferences. The results show (i) that MSCs start to change marker expression few hours after injection into a beating heart and (ii) that infarcted myocardium influences transplanted MSC morphology and mobility within the heart.