Silmara de Lima

Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors.

The mature optic nerve cannot regenerate when injured, leaving victims of traumatic nerve damage or diseases such as glaucoma with irreversible visual losses. Recent studies have identified ways to stimulate retinal ganglion cells to regenerate axons part-way through the optic nerve, but it remains unknown whether mature axons can reenter the brain, navigate to appropriate target areas, or restore vision. We show here that with adequate stimulation, retinal ganglion cells are able to regenerate axons the full length of the visual pathway and on into the lateral geniculate nucleus, superior colliculus, and other visual centers. Regeneration partially restores the optomotor response, depth perception, and circadian photoentrainment, demonstrating the feasibility of reconstructing central circuitry for vision after optic nerve damage in mature mammals.

Bone marrow mesenchymal stem cell transplantation for improving nerve regeneration.

Although the peripheral nervous system has an inherent capacity for regeneration, injuries to nerves still result in considerable disabilities. The persistence of these disabilities along with the underlying problem of nerve reconstruction has motivated neuroscientists worldwide to seek additional therapeutic strategies. In recent years, cell-based therapy has emerged as a promising therapeutic tool. Schwann cells (SCs) are the main supportive cells for peripheral nerve regeneration; however, there are several technical limitations regarding its application for cell-based therapy. In this context, bone marrow mesenchymal stem cells (BM-MSCs) have been used as alternatives to SCs for treating peripheral neuropathies, showing great promise. Several studies have been trying to shed light on the mechanisms behind the nerve regeneration-promotion potential of BM-MSCs. Although not completely clarified, understanding how BM-MSCs exert tissue repair effects will facilitate their development as therapeutic agents before they become a clinically viable tool for encouraging peripheral nerve regeneration.

A Combination of Schwann-Cell Grafts and Aerobic Exercise Enhances Sciatic Nerve Regeneration

Background Despite the regenerative potential of the peripheral nervous system, severe nerve lesions lead to loss of target-organ innervation, making complete functional recovery a challenge. Few studies have given attention to combining different approaches in order to accelerate the regenerative process. Objective Test the effectiveness of combining Schwann-cells transplantation into a biodegradable conduit, with treadmill training as a therapeutic strategy to improve the outcome of repair after mouse nerve injury. Methods Sciatic nerve transection was performed in adult C57BL/6 mice; the proximal and distal stumps of the nerve were sutured into the conduit. Four groups were analyzed: acellular grafts (DMEM group), Schwann cell grafts (3×105/2 µL; SC group), treadmill training (TMT group), and treadmill training and Schwann cell grafts (TMT + SC group). Locomotor function was assessed weekly by Sciatic Function Index and Global Mobility Test. Animals were anesthetized after eight weeks and dissected for morphological analysis. Results Combined therapies improved nerve regeneration, and increased the number of myelinated fibers and myelin area compared to the DMEM group. Motor recovery was accelerated in the TMT + SC group, which showed significantly better values in sciatic function index and in global mobility test than in the other groups. The TMT + SC group showed increased levels of trophic-factor expression compared to DMEM, contributing to the better functional outcome observed in the former group. The number of neurons in L4 segments was significantly higher in the SC and TMT + SC groups when compared to DMEM group. Counts of dorsal root ganglion sensory neurons revealed that TMT group had a significant increased number of neurons compared to DMEM group, while the SC and TMT + SC groups had a slight but not significant increase in the total number of motor neurons. Conclusion These data provide evidence that this combination of therapeutic strategies can significantly improve functional and morphological recovery after sciatic injury.

Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration

Significance The inability of CNS pathways to regenerate after injury can lead to devastating, life-long losses in sensory, motor, and other functions. We report that after injury to the optic nerve, a widely studied CNS pathway that normally cannot regenerate, mobile zinc (Zn2+) increases rapidly in the processes of retinal interneurons (amacrine cells) and then transfers via vesicular release to retinal ganglion cells (RGCs), the injured projection neurons. Eliminating Zn2+ leads to both persistent RGC survival and substantial axon regeneration with a broad therapeutic window. These findings show that signaling between interneurons and RGCs contributes to regulating the fate of RGCs after optic nerve injury, and that Zn2+ chelation may provide a potent therapeutic approach. Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn2+) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn2+ increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn2+ accumulation in amacrine cell processes involves the Zn2+ transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn2+ chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosphatase and tensin homolog (pten). Importantly, the therapeutic window for Zn2+ chelation extends for several days after nerve injury. These results show that retinal Zn2+ dysregulation is a major factor limiting the survival and regenerative capacity of injured RGCs, and point to Zn2+ chelation as a strategy to promote long-term RGC protection and enhance axon regeneration.

Reassembly of Excitable Domains after CNS Axon Regeneration

Action potential initiation and propagation in myelinated axons require ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Disruption of these domains after injury impairs nervous system function. Traditionally, injured CNS axons are considered refractory to regeneration, but some recent approaches challenge this view by showing robust long-distance regeneration. However, whether these approaches allow remyelination and promote the reestablishment of AIS and nodes of Ranvier is unknown. Using mouse optic nerve crush as a model for CNS traumatic injury, we performed a detailed analysis of AIS and node disruption after nerve crush. We found significant disruption of AIS and loss of nodes within days of the crush, and complete loss of nodes 1 week after injury. Genetic deletion of the tumor suppressor phosphatase and tensin homolog (Pten) in retinal ganglion cells (RGCs), coupled with stimulation of RGCs by inflammation and cAMP, dramatically enhanced regeneration. With this treatment, we found significant reestablishment of RGC AIS, remyelination, and even reassembly of nodes in regions proximal, within, and distal to the crush site. Remyelination began near the retina, progressed distally, and was confirmed by electron microscopy. Although axons grew rapidly, remyelination and nodal ion channel clustering was much slower. Finally, genetic deletion of ankyrinG from RGCs to block AIS reassembly did not affect axon regeneration, indicating that preservation of neuronal polarity is not required for axon regeneration. Together, our results demonstrate, for the first time, that regenerating CNS axons can be remyelinated and reassemble new AIS and nodes of Ranvier. SIGNIFICANCE STATEMENT We show, for the first time, that regenerated CNS axons have the capacity to both remyelinate and reassemble the axon initial segments and nodes of Ranvier necessary for rapid and efficient action potential propagation.

Mice lacking GD3 synthase display morphological abnormalities in the sciatic nerve and neuronal disturbances during peripheral nerve regeneration.

The ganglioside 9-O-acetyl GD3 is overexpressed in peripheral nerves after lesioning, and its expression is correlated with axonal degeneration and regeneration in adult rodents. However, the biological roles of this ganglioside during the regenerative process are unclear. We used mice lacking GD3 synthase (Siat3a KO), an enzyme that converts GM3 to GD3, which can be further converted to 9-O-acetyl GD3. Morphological analyses of longitudinal and transverse sections of the sciatic nerve revealed significant differences in the transverse area and nerve thickness. The number of axons and the levels of myelin basic protein were significantly reduced in adult KO mice compared to wild-type (WT) mice. The G-ratio was increased in KO mice compared to WT mice based on quantification of thin transverse sections stained with toluidine blue. We found that neurite outgrowth was significantly reduced in the absence of GD3. However, addition of exogenous GD3 led to neurite growth after 3 days, similar to that in WT mice. To evaluate fiber regeneration after nerve lesioning, we compared the regenerated distance from the lesion site and found that this distance was one-fourth the length in KO mice compared to WT mice. KO mice in which GD3 was administered showed markedly improved regeneration compared to the control KO mice. In summary, we suggest that 9-O-acetyl GD3 plays biological roles in neuron-glia interactions, facilitating axonal growth and myelination induced by Schwann cells. Moreover, exogenous GD3 can be converted to 9-O-acetyl GD3 in mice lacking GD3 synthase, improving regeneration.

Wallerian Degeneration in Injury and Diseases: Concepts and Prevention

The axon is a highly specialized compartment of neurons. Besides their basic function connecting neurons to their targets, axons play key roles in the nervous system. They are involved in the transport of several molecules indispensable to neuronal activity, act as sensors to guidance cues during development and regeneration, and are essential to maintain normal glial cell functions and myelin sheath assembly (Nave & Trap 2008). Recent evidence indicates that mRNA and Schwann cells-delivered ribosomes can be found within the axoplasm, suggesting that axons may be capable of synthesizing specific proteins (Court et al., 2008). However, most axonal structural proteins are synthesized in the neuronal cell body and transported along the length of the axon. Interruption of this supply leads to a degenerative process known as Wallerian degeneration (WD) in the distal portion of the axon (Coleman, 2005). WD is triggered by intrinsic degenerative pathways that are not correlated to cellular apoptosis (Finn et al., 2000). Axon degeneration is a final common pathway observed not only after a traumatic nerve injury, but also in many neurodegenerative disorders (e.g., Parkinson`s and Alzheimer`s diseases) and in demyelinating diseases such as multiple sclerosis (Coleman, 2005; Coleman & Freeman, 2010). Uncovering the mechanisms that trigger and control axon degeneration is extremely relevant, as such knowledge may offer novel tools to treat severed or damaged axons as well as several neurodegenerative disorders in which WD takes place. In this chapter, we will review the basic concepts of WD, with emphasis on the mechanisms that control axon degeneration following trauma. Next, we will address the issue whether or not current antidegenerative strategies are efficient and can be envisioned to be applied to humans in the near future.

Past, present and future of preserving and restoring function in the visual system: removing galectin-3 as a promising treatment

Great advances in retinal ganglion cells survival (RGCs), optic nerve preservation and regeneration have been made in the past 15 years. Nowadays, we know that RGCs are capable of regenerating the full length of the optic nerve, cross the chiasm, enter the brain and reinnervate visual targets. In order to obtain successful regeneration, RGCs need to activate signaling pathways related to cell survival and turn on their intrinsic growth capacity. Studies that aimed at blocking cell death and inhibiting apoptosis by B-cell lymphoma 2 (Bcl-2) overexpression showed an increase in cell survival, but these approaches were not sufficient to promote axon regeneration, even when axons were put in an permissive environment, as peripheral nerve graft (Inoue et al., 2002). Neither stimulating axon regeneration by intraocular inflammation, nor delaying axon degeneration by overexpression of Wlds protein, or inhibition of calpain activation (Figure 1C) (de Lima et al., 2016) showed any increase in cell survival. Nevertheless, Park et al. (2008) showed that phosphatase and tensin homolog (PTEN) gene deletion on RGCs stimulates both cell survival and axon regeneration (Benowitz et al., 2016 for review). The scenario revealed by these studies indicates that different mechanisms regulate RGC survival and axon regeneration. From these evidences, investigators started to combine different treatments, focusing on cell survival, axon regeneration, or both. The rationale behind this approach is that one would be able to stimulate both RGCs survival and axon regeneration at the same time, and possibly get additional effect after a lesion to the optic nerve. For instance, specific single treatments, such as conditional deletion of the PTEN gene in RGCs resulted in 45% of cell survival after optic nerve crush (ONC), and also promoted modest axon regeneration (Park et al., 2008). However, when combined with intraocular inflammation, a RGC survival rate of 54% was achieved as well as a 10 fold increase in axon regeneration, resulting in brain reinnervation (de Lima et al., 2012) (Figure 1E). Therefore, a combination of treatments can be a powerful tool to stimulate recovery of visual pathway. Thus, researchers are focusing their efforts on identifying potential candidates that can be more effective in one aspect (cell survival), in the other (axon regeneration), or both, so they can combine those candidates and boost brain target reinnervation.

Injection of bone marrow mesenchymal stem cells by intravenous or intraperitoneal routes is a viable alternative to spinal cord injury treatment in mice

In spite of advances in surgical care and rehabilitation, the consequences of spinal cord injury (SCI) are still challenging. Several experimental therapeutic strategies have been studied in the SCI field, and recent advances have led to the development of therapies that may act on the inhibitory microenvironment. Assorted lineages of stem cells are considered a good treatment for SCI. This study investigated the effect of systemic transplantation of mesenchymal stem cells (MSCs) in a compressive SCI model. Here we present results of the intraperitoneal route, which has not been used previously for MSC administration after compressive SCI. We used adult female C57BL/6 mice that underwent laminectomy at the T9 level, followed by spinal cord compression for 1 minute with a 30-g vascular clip. The animals were divided into five groups: sham (anesthesia and laminectomy but without compression injury induction), MSC i.p. (intraperitoneal injection of 8 × 105 MSCs in 500 µL of DMEM at 7 days after SCI), MSC i.v. (intravenous injection of 8 × 105 MSCs in 500 µL of DMEM at 7 days after SCI), DMEM i.p. (intraperitoneal injection of 500 µL of DMEM at 7 days after SCI), DMEM i.v. (intravenous injection of 500 µL of DMEM at 7 days after SCI). The effects of MSCs transplantation in white matter sparing were analyzed by luxol fast blue staining. The number of preserved fibers was counted in semithin sections stained with toluidine blue and the presence of trophic factors was analyzed by immunohistochemistry. In addition, we analyzed the locomotor performance with Basso Mouse Scale and Global Mobility Test. Our results showed white matter preservation and a larger number of preserved fibers in the MSC groups than in the DMEM groups. Furthermore, the MSC groups had higher levels of trophic factors (brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and neurotrophin-4) in the spinal cord and improved locomotor performance. Our results indicate that injection of MSCs by either intraperitoneal or intravenous routes results in beneficial outcomes and can be elected as a choice for SCI treatment.

Peripheral Nervous System: Regenerative Therapies

There is a general belief that regeneration in the Peripheral Nervous System (PNS) is a successful event, however complete functional regeneration is seldom achieved in patients that have suffered a nerve traumatic injury. In fact, what is clinically observed is that these patients live with permanent disabilities that interfere negatively in their daily routine activities. In injuries where there is tissue loss a direct neurorraphy is not possible without causing nerve tension and, therefore, another repair technique is needed. Clinically, these lesions are repaired by nerve autograft, a technique that requires a second surgery to harvest a segment of a donor nerve, a disadvantage of the method. Also, the area covered by the donor nerve becomes denervated and its function is lost. Other techniques that are used by surgeons when the proximal stump is not available are end-to-side coaptation and nerve transfer. Experimental studies aiming at developing alternative strategies that can improve nerve regeneration have increased over the last decades. Particularly, the search for nerve guiding conduits that can be used to bridge the nerve defect has received much attention by researchers all over the world. These conduits can be made by either synthetic or biological materials, but ideally, they should be biodegradable and biocompatible, have adequate permeability so as to allow the entrance of nutrients into the tube lumen and yet avoid the passage of cells that can interfere negatively in the regeneration processes, such as fibroblasts and inflammatory cells. Other therapeutic strategies such as gene, cell and molecular therapies as well as physical therapies (exercise, electrical and LASER therapy) have also been tested in experimental studies with positive results. In this chapter we review the literature covering all these strategies in terms of experimental studies and existing clinical trials.