Luisa Muratori

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.

The effect of bioartificial constructs that mimic myocardial structure and biomechanical properties on stem cell commitment towards cardiac lineage

Despite the enormous progress in the treatment of coronary artery diseases, they remain the most common cause of heart failure in the Western countries. New translational therapeutic approaches explore cardiomyogenic differentiation of various types of stem cells in combination with tissue-engineered scaffolds. In this study we fabricated PHBHV/gelatin constructs mimicking myocardial structural properties. Chemical structure and molecular interaction between material components induced specific properties to the substrate in terms of hydrophilicity degree, porosity and mechanical characteristics. Viability and proliferation assays demonstrated that these constructs allow adhesion and growth of mesenchymal stem cells (MSCs) and cardiac resident non myocytic cells (NMCs). Immunofluorescence analysis demonstrated that stem cells cultured on these constructs adopt a distribution mimicking the three-dimensional cell alignment of myocardium. qPCR and immunofluorescence analyses showed the ability of this construct to direct initial MSC and NMC lineage specification towards cardiomyogenesis: both MSCs and NMCs showed the expression of the cardiac transcription factor GATA-4, fundamental for early cardiac commitment. Moreover NMCs also acquired the expression of the cardiac transcription factors Nkx2.5 and TBX5 and produced sarcomeric proteins. This work may represent a new approach to induce both resident and non-resident stem cells to cardiac commitment in a 3-D structure, without using additional stimuli.

Role of inflammatory cytokines in peripheral nerve injury

Inflammatory events occurring in the distal part of an injured peripheral nerve have, nowadays, a great resonance. Investigating the timing of action of the several cytokines in the important stages of Wallerian degeneration helps to understand the regenerative process and design pharmacologic intervention that promotes and expedites recovery. The complex and synergistic action of inflammatory cytokines finally promotes axonal regeneration. Cytokines can be divided into pro- and anti-inflammatory cytokines that upregulate and downregulate, respectively, the production of inflammatory mediators. While pro-inflammatory cytokines are expressed in the first phase of Wallerian degeneration and promote the recruitment of macrophages, anti-inflammatory cytokines are expressed after this recruitment and downregulate the production of all cytokines, thus determining the end of the process. In this review, we describe the major inflammatory cytokines involved in Wallerian degeneration and the early phases of nerve regeneration. In particular, we focus on interleukin-1, interleukin-2, interleukin-6, tumor necrosis factor-β, interleukin-10 and transforming growth factor-β.

Generation of New Neurons in Dorsal Root Ganglia in Adult Rats after Peripheral Nerve Crush Injury

The evidence of neurons generated ex novo in sensory ganglia of adult animals is still debated. In the present study, we investigated, using high resolution light microscopy and stereological analysis, the changes in the number of neurons in dorsal root ganglia after 30 days from a crush lesion of the rat brachial plexus terminal branches. Results showed, as expected, a relevant hypertrophy of dorsal root ganglion neurons. In addition, we reported, for the first time in the literature, that neuronal hypertrophy was accompanied by massive neuronal hyperplasia leading to a 42% increase of the number of primary sensory neurons. Moreover, ultrastructural analyses on sensory neurons showed that there was not a relevant neuronal loss as a consequence of the nerve injury. The evidence of BrdU-immunopositive neurons and neural progenitors labeled with Ki67, nanog, nestin, and sox-2 confirmed the stereological evidence of posttraumatic neurogenesis in dorsal root ganglia. Analysis of morphological changes following axonal damage in addition to immunofluorescence characterization of cell phenotype suggested that the neuronal precursors which give rise to the newly generated neurons could be represented by satellite glial cells that actively proliferate after the lesion and are able to differentiate toward the neuronal lineage.

Evaluation of Vascular Endothelial Growth Factor (VEGF) and Its Family Member Expression After Peripheral Nerve Regeneration and Denervation: VASCULAR ENDOTHELIAL GROWTH FACTOR

Vascular endothelial growth factor (VEGF) represents one of the main factors involved not only in angiogenesis and vasculogenesis but also in neuritogenesis. VEGF plays its function acting via different receptors: VEGF receptor1 (VEGFR‐1), VEGF receptor2 (VEGFR‐2), VEGF receptor3 (VEGFR‐3), and co‐receptors Neuropilin‐1 (NRP1) and Neuropilin‐2 (NRP2). This study reports on the first in vivo analysis of the expression of VEGF and VEGF family molecules in peripheral nerve degeneration and regeneration: for this purpose, different models of nerve lesion in rat were adopted, the median nerve crush injury and the median nerve transaction followed or not by end‐to end microsurgical repair. Results obtained by real time polymerase chain reaction showed that VEGF and VEGF family molecules are differentially expressed under regenerating and degenerating condition, furthermore, in order to study the modulation and involvement of these factors in two different regenerative models, crush injury and end‐to‐end repair, protein expression analysis was evaluated. In addition, immunohistochemical analysis allowed to state a glial localization of VEGF and VEGFR‐2 after peripheral nerve crush injury. Finally in vitro assay on primary Schwann cells culture show that VEGF165 stimulation increases Schwann cells migration, a major process in the promotion of neurite outgrowth. Anat Rec, 301:1646–1656, 2018.

Mice harboring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity.

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but signs of ataxia were detectable by beam test at 18 months. Cerebellar pathology was negative; electrophysiological analysis showed increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls, although not statistically significant. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was also altered, in line with greatly reduced expression of Opa1 fusogenic protein L-isoforms. The mitochondrial alterations observed in MEFs were also detected in cerebella of 18-month-old heterozygous mutants, suggesting they may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.

The guidance of stem cell cardiomyogenic differentiation by bioartificial scaffolds mimicking myocardium structure and biomechanics

Scientific objectives Despite enormous progresses in the treatment of coronary artery disease, it remains the most common cause of heart failure and the leading cause of death in the Western countries. New translational therapeutic approaches based on personalized and regenerative medicine explore cardiomyogenic differentiation of various types of stem cells by electrical stimulation, biochemical inducers, or cell co-culturing [1-3]. In this study we fabricated bioartificial constructs mimicking anisotropic structure and mechanical properties of the myocardium [4].

Direct muscle neurotization after end-to end and end-to-side neurorrhaphy: An experimental study in the rat forelimb model.

The need for the continuous research of new tools for improving motor function recovery after nerve injury is justified by the still often unsatisfactory clinical outcome in these patients. It has been previously shown that the combined use of two reconstructive techniques, namely end-to-side neurorrhaphy and direct muscle neurotization in the rat hindlimb model, can lead to good results in terms of skeletal muscle reinnervation. Here we show that, in the rat forelimb model, the combined use of direct muscle neurotization with either end-to-end or end-to-side neurorrhaphy to reinnervate the denervated flexor digitorum muscles, leads to muscle atrophy prevention over a long postoperative time lapse (10 months). By contrast, very little motor recovery (in case of end-to-end neurorrhaphy) and almost no motor recovery (in case of end-to-side neurorrhaphy) were observed in the grasping activity controlled by flexor digitorum muscles. It can thus be concluded that, at least in the rat, direct muscle neurotization after both end-to-end and end-to-side neurorrhaphy represents a good strategy for preventing denervation-related muscle atrophy but not for regaining the lost motor function.

A long-term posttraumatic study in the rat median nerve crush injury model

The possibility that posttraumatic regeneration may eventually lead rat peripheral nerve fibers back to normal is still under debate. While it has been shown that, after a sufficiently long posttraumatic time, the number of regenerated rat peripheral nerve fibers can return to normal levels and the animal can regain normal pre-lesion function, no information regarding long-term changes in size parameters of regenerated nerve fibers is still available. To fill this gap, 24-week posttraumatic changes in myelinated axon and nerve fiber diameter, myelin thickness and g-ratio (axon diameter/fiber diameter), distal to a nerve crush (axonotmesis lesion) of the rat median nerve were assessed by stereology. Results showed that, while as expected fiber number returned to normal values at week-24, both axon and fiber diameter and myelin thickness were still significantly lower at week-24 in comparison to pre-lesion values. Finally, g-ratio, which persisted unmodified along the regeneration process, eventually resulted to be significantly reduced at week-24 in comparison to pre-lesion value. Based on these results, the hypothesis that regenerated rat peripheral nerve fibers are able to spontaneously return to normal, provided that a sufficiently long time recovery post-axonotmesis is allowed, is rejected.

Adaptive changes following crush injury of brachial plexus terminal branches in adult rats

In the present study, we investigated the adaptive changes after a nerve crush lesion applied to the median, ulnar and radial nerves, both downstream and upstream to the lesion site. Animals were sacrificed at different time points after the injury and the nerves and the corresponding C5-T1 DRGs were extracted Distal to the crush lesion, morphological analysis showed that axonal regeneration and maturation was very fast. Regenerated nerve fibers were significantly more numerous and densely packed. On the other hand axons were smaller and with a thinner myelin sheath compared to controls. Proximal to the crush lesion, morphological analysis of DRGS showed an unusual number of small size cells different from the glial satellite cells. Neurogenesis in the DRGs was then investigated by injecting rats with bromodeoxyuridine (BrDU). Most of the BrDU positive cells belong to the glial family although, some BrDU colocalized with neuronal markers (nestin, Sox-2) suggesting that neurogenesis occurs in adult DRGs neurons that undergo peripheral nerve injury. Finally, a stereological analysis, using the physical dissector method, showed a significant increase in number (42%) of DRGs sensory neurons 1 month after nerve-crush injury compared to control. All together our data support the idea that the population of DRG’s neurons increased as a consequence of the nerve damage. Evidence of morphological changes in the population of cells surrounding neurons and the immunopositivity for neuronal progenitor markers, suggested the hypothesis that the increased number of neurons is due to undifferentiated precursors localized within the adult DRG. Taken together, these results provide further information about the adaptation of the nervous system to a axonal damage and suggest that nerve regeneration is supported by neurogenesis, at least in the sensory compartment.