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The vascular and the nervous systems share similarities in addition to their complex role in providing oxygen and nutrients to all cells. Both are highly branched networks that frequently grow close to one another during development. Vascular patterning and neural wiring share families of guidance cues and receptors. Most recently, this relationship has been investigated in terms of peripheral nervous system (PNS) regeneration, where nerves and blood vessels often run in parallel so endothelial cells guide the formation of the Büngner bands which support axonal regeneration. Here, we characterized the vascular response in regenerative models of the central and peripheral nervous system. After sciatic nerve crush, followed by axon regeneration, there was a significant increase in the blood vessel density 7 days after injury. In addition, the optic nerve crush model was used to evaluate intrinsic regenerative potential activated with a combined treatment that stimulated retinal ganglion cells (RGCs) regrowth. We observed that a 2-fold change in the total number of blood vessels occurred 7 days after optic nerve crush compared to the uncrushed nerve. The difference increased up to a 2.7-fold change 2 weeks after the crush. Interestingly, we did not observe differences in the total number of blood vessels 2 weeks after crush, compared to animals that had received combined treatment for regeneration and controls. Therefore, the vascular characterization showed that the increase in vascular density was not related to the efficiency of both peripheral and central axonal regeneration., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The retina is the sensory tissue responsible for the first stages of visual processing, with a conserved anatomy and functional architecture among vertebrates. To date, retinal eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, glaucoma, and others, affect nearly 170 million people worldwide, resulting in vision loss and blindness. To tackle retinal disorders, the developing retina has been explored as a versatile model to study intercellular signaling, as it presents a broad neurochemical repertoire that has been approached in the last decades in terms of signaling and diseases. Retina, dissociated and arranged as typical cultures, as mixed or neuron- and glia-enriched, and/or organized as neurospheres and/or as organoids, are valuable to understand both neuronal and glial compartments, which have contributed to revealing roles and mechanisms between transmitter systems as well as antioxidants, trophic factors, and extracellular matrix proteins. Overall, contributions in understanding neurogenesis, tissue development, differentiation, connectivity, plasticity, and cell death are widely described. A complete access to the genome of several vertebrates, as well as the recent transcriptome at the single cell level at different stages of development, also anticipates future advances in providing cues to target blinding diseases or retinal dysfunctions.

Some types of peripheral nerve injury lead to limb deafferentation, which leads to remodeling of body representation areas in different parts of the brain, such as in the primary motor cortex and primary sensory cortex. This plasticity is a consequence of several cellular events, such as the emergence and elimination of synapses in these areas. Beside neurons, microglial cells are intimately involved in synapse plasticity, especially in synaptic pruning. In this study, we investigated the transient changes in synaptic density in the primary motor and sensory cortex after different types of peripheral nerve injury, as well as the behavior of microglial cells in each scenario. Male C57/B6 mice were divided into a control group (no injury), sciatic-crush group, and sciatic-transection group, and treated with PBS or minocycline daily for different time points. Both types of sciatic lesion led to a significant decrease of synaptophysin and PSD-95 positive puncta counts compared to control animals 4 days after lesion (DAL), which recovered at 7 DAL and was sustained until 14 DAL. The changes in synaptic puncta density were concomitant with changes in the density and morphology of microglial cells, which were significantly more ramified in the primary motor cortex of injured animals at 1 and 4 DAL. Although the decreased synaptic puncta density overlapped with an increased number of microglial cells, the number of lysosomes per microglial cell did not increase on day 4 after lesion. Surprisingly, daily administration of minocycline increased microglial cell number and PSD-95 positive puncta density by 14 DAL. Taken together, we found evidence for transient changes in synaptic density in the primary motor, related to peripheral injury with possible participation of microglia in this plasticity process., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Neocortex development comprises of a complex series of time- and space-specific processes to generate the typical interconnected six-layered architecture of adult mammals. Axon growth is required for the proper establishment of cortical circuits. Malformations in axonal growth and pathfinding might lead to severe neuropathologies, such as corpus callosum dysgenesis. Cenpj, a microcephaly gene, encodes a scaffold protein that regulates centrosome biogenesis and microtubule stabilization. During corticogenesis, Cenpj regulates progenitor division and neuronal migration. Since microtubule stabilization is crucial for axon extension, we investigated the role of Cenpj in axon growth during cortical development in a mouse model. Through loss- and gain-of-function assays ex vivo and in utero, we quantified callosal axonal length, branching, and growth cone size compared to controls. We observed that silencing Cenpj results in an increased axonal length. Ex vivo, we assessed the number of branches, the area of growth cones and the stability of microtubules. In silenced Cenpj axons, there were more branches, larger growth cone area, and more stable microtubules. Rescue experiments confirmed that neurons present axonal length comparable to controls. Here we propose that Cenpj regulates axon growth by destabilizing microtubules during cortical development. Finally, our findings suggest that Cenpj might be a novel target for axonal regeneration., (© 2022 International Society for Neurochemistry.)

Neural-immune interactions are related to the synapse plasticity and other dynamic processes in the nervous system. The absence or dysfunction of cellular/molecular elements from the immune system lead to impairments in the central and peripheral nervous system with behavior consequences such as cognitive, sensory, and locomotor deficits as well as social disabilities and anxiety disturbances. Cellular interactions between immune cells such as macrophages, microglia, and neutrophils with glial or neuronal cells have been of increasing interest over the last years. However, little is known about the role of immune-derived soluble factors in the context of homeostasis of the nervous system. Leukotrienes (LTs) are lipid mediators derived from the oxidation of arachidonic acid by 5-lipoxygenase (5-LO), and are classically involved in inflammation, allergies, and asthma. Here, we demonstrated that adult mice lacking 5-LO (5-LO -/- ) showed motor deficits in rotarod test and increased repetitive behavior (marble burying test). These behavioral changes are accompanied by increased levels of synapse proteins (PSD95 and synaptophysin) at the motor cortex and hippocampus, but not with BDNF alterations. No changes in microglial cell density or morphology were seen in the brains of 5-LO -/- mice. Furthermore, expression of fractalkine receptor CX3CR1 was increased and of its ligand CX3CL1 was decreased in the cortex of 5-LO -/- mice. Here we provide evidence for the involvement of 5-LO products structuring synapses network with motor behavior consequences. We suggest that the absence of 5-LO products lead to modified microglial/neuron interaction, reducing microglial pruning., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Like the seasons of the year, all natural things happen in stages, going through adaptations when challenged, and Schwann cells are a great example of that. During maturation, these cells regulate several steps in peripheral nervous system development. The Spring of the cell means the rise and bloom through organized stages defined by time-dependent regulation of factors and microenvironmental influences. Once matured, the Summer of the cell begins: a high energy stage focused on maintaining adult homeostasis. The Schwann cell provides many neuron-glia communications resulting in the maintenance of synapses. In the peripheral nervous system, Schwann cells are pivotal after injuries, balancing degeneration and regeneration, similarly to when Autumn comes. Their ability to acquire a repair phenotype brings the potential to reconnect axons to targets and regain function. Finally, Schwann cells age, not only by growing old, but also by imposed environmental cues, like loss of function induced by pathologies. The Winter of the cell presents as reduced activity, especially regarding their role in repair; this reflects on the regenerative potential of older/less healthy individuals. This review gathers essential information about Schwann cells in different stages, summarizing important participation of this intriguing cell in many functions throughout its lifetime.

In an injury to the peripheral nervous system, the spinal cord and brain structure reorganize connections to optimize the function of the remaining parts. Many cell events are triggered in the spinal cord to support changes in the synaptic connections around motoneurons, where old connections are removed, and new ones created. Microglial cells are primitive macrophages that invade the central nervous system in early stages of neurodevelopment and have several functions, such as eliminating synapses. We investigated the synaptic plasticity after different types of peripheral (sciatic) nerve injury (crush or total transection), as well as the behavior of microglial cells for 2 weeks after a peripheral lesion. As expected, sciatic-nerve injury reduced motor performance in mice, but crushed animals regained partial motor control. Because of sciatic-nerve injury, pre-synaptic inputs decreased around the motoneurons in the ventro-lateral horn, while microglial cells increased around these cells. Microglial cells also exhibited altered morphology in both types of peripheral lesion, indicating a similar underlying mechanism of plasticity. To investigate the involvement of microglia in this scenario, microglial activation was modulated by daily administration of minocycline. The minocycline treatment directly affected the microglial response and impacted the synapse rearrangement in the spinal cord. Together, these results demonstrate that microglia cells are involved in synaptic plasticity in the lumbar spinal cord in both nerve-injury scenarios., Summary of Statement: Here, we demonstrated that acute plasticity in the lumbar spinal cord (LSC) did not differ between crush and transection of peripheral nerve, and that microglial reactivity in the LSC was important after both injury types., (© 2021 The Authors.)

Unlike the central nervous system, peripheral nerves can regenerate after injury. However, depending on the size of the lesion, the endogenous regenerative potential is not enough to replace the lost nerve tissue. Many strategies have been used to generate biomaterials capable of restoring nerve functions. Here, we set out to investigate whether adsorbing the extracellular matrix protein, laminin (LM), to poly-ℇ-caprolactone (PCL) filaments would enhance functional nerve regeneration. Initial in vitro studies showed that explants of dorsal root ganglia (DRGs) of P1 neonate mice exhibited stronger neuritogenesis on a substrate of LM that had been previously polymerized (polylaminin [polyLM]) than on ordinary LM. On the other hand, when silicone tubes filled with PCL filaments were used to bridge a 10-mm sciatic nerve gap in rats, only filaments coated with LM improved tissue replacement beyond that obtained with empty tubes. Motor function recovery correlated with tissue replacement as only LM-coated filaments consistently improved motor skills. Finally, analysis of the lateral gastrocnemius muscle revealed that the LM group presented twice the amount of α-bungarotixin-labeled motor plates. In conclusion, although polyLM was more effective in stimulating growth of sensory fibers out of DRGs in vitro , LM adsorbed to PCL filaments exhibited the best regenerative properties in inducing functional motor recovery after peripheral injury in vivo ., (© 2019 Published by Elsevier Ltd.)

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are dynamic cells that can sense the environment, adapting their regulatory functions to different conditions. Accordingly, the therapeutic potential of BM-MSCs can be modulated by preconditioning strategies aimed at modifying their paracrine action. Although rat BM-MSCs (rBM-MSCs) have been widely tested in preclinical research, most preconditioning studies have employed human and mouse BM-MSCs. Herein, we investigated whether rBM-MSCs modify their phenotype and paracrine functions in response to Toll-like receptor (TLR) agonists. The data showed that rBM-MSCs expressed TLR3, TLR4, and MDA5 mRNA and were able to internalize polyinosinic-polycytidylic acid (Poly(I:C)), a TLR3/MDA5 agonist. rBM-MSCs were then stimulated with Poly(I:C) or with lipopolysaccharide (LPS, a TLR4 agonist) for 1 h and were grown under normal culture conditions. LPS or Poly(I:C) stimulation did not affect the viability or the morphology of rBM-MSCs and did not modify the expression pattern of key cell surface markers. Poly(I:C) did not induce statistically significant changes in the release of several inflammatory mediators and VEGF by rBM-MSCs, although it tended to increase IL-6 and MCP-1 secretion, whereas LPS increased the release of IL-6, MCP-1, and VEGF, three factors that were constitutively secreted by unstimulated cells. The neurotrophic activity of the conditioned medium from unstimulated and LPS-preconditioned rBM-MSCs was investigated using dorsal root ganglion explants, showing that soluble factors produced by unstimulated and LPS-preconditioned rBM-MSCs can stimulate neurite outgrowth similarly, in a VEGF-dependent manner. LPS-preconditioned cells, however, were slightly more efficient in increasing the number of regrowing axons in a model of sciatic nerve transection in rats. In conclusion, LPS preconditioning boosted the production of constitutively secreted factors by rBM-MSCs, without changing their mesenchymal identity, an effect that requires further investigation in exploratory preclinical studies., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper.

Neurospheres prepared from multipotent progenitors in the retina obtained from postnatal mice differentiate into neurons and Müller glia (De Melo Reis et al., in Cell Mol Neurobiol 31:835-846, 2011). Here, we investigated whether neurospheres prepared from adult chickens (ciliary marginal zone, CMZ) or (ciliary body) retina could also lead to differentiated neurons and glia. Neurospheres were prepared from post-hatched chickens or from adult mice after 7 days in the presence of mitogenic factors (FGFb, insulin, and EGF), generating neurons and glial cells. In addition, Müller (2M6 or glutamine synthetase positive cells) derived from post-hatch chicken CMZ neurospheres displayed the dopaminergic phenotype. Furthermore, we observed that Müller cells derived from adult chickens and mice retina neurospheres released significant amounts of dopamine as well as of its metabolites. Taken together, our data lead us to conclude that as for embryonic (chick) or newborn (mouse), the dopaminergic phenotype is a default condition of Müller glial cells obtained from neurospheres prepared from mature retina. Our data raise the possibility that Müller cells from differentiated tissue could be used to ameliorate neurodegenerative diseases involving dopaminergic dysfunction as in Parkinson's disease as shown previously (Stutz et al., in J Neurochem 128:829-840, 2014).

Adult peripheral nerves in vertebrates can regrow their axons and re-establish function after crush lesion. However, when there is extensive loss of a nerve segment, due to an accident or compressive damage caused by tumors, regeneration is strongly impaired. In order to overcome this problem, bioengineering strategies have been employed, using biomaterials formed by key cell types combined with biodegradable polymers. Many of these strategies are successful, and regenerated nerve tissue can be observed 12 weeks after the implantation. Mesenchymal stem cells (MSCs) are one of the key cell types and the main stem-cell population experimentally employed for cell therapy and tissue engineering of peripheral nerves. The ability of these cells to release a range of different small molecules, such as neurotrophins, growth factors and interleukins, has been widely described and is a feasible explanation for the improvement of nerve regeneration. Moreover, the multipotent capacity of MSCs has been very often challenged with demonstrations of pluripotency, which includes differentiation into any neural cell type. In this study, we generated a biomaterial formed by EGFP-MSCs, constitutively covering microstructured filaments made of poly-ε-caprolactone. This biomaterial was implanted in the sciatic nerve of adult rats, replacing a 12-mm segment, inside a silicon tube. Our results showed that six weeks after implantation, the MSCs had differentiated into connective-tissue cells, but not into neural crest-derived cells such as Schwann cells. Together, present findings demonstrated that MSCs can contribute to nerve-tissue regeneration, producing trophic factors and differentiating into fibroblasts, endothelial and smooth-muscle cells, which compose the connective tissue.

The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB 1 and CB 2 ), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB 1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB 1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB 2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB 2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB 1 and CB 2 were found both in neurons and glia cells, but MAGL was only expressed in Müller glia. Older retinas (PE7) show CB 1 positive cells mainly in the INL and co-expression of CB 1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB 1 co-localized with TH and was heavily associated to D 1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D 1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212-2 (WIN) in a CB 1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB 1 limits cAMP accumulation via D 1 receptor activation and may influence embryological parameters during avian retina differentiation.

Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1-10 mM) showed that 5-10 mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50 mM KCl (labeled as βIII tubulin positive cells). BBG 100 nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70 μM and MK-801 20 μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5 mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit.

Introduction: Peripheral nerves may fail to regenerate across tube implants because these lack the microarchitecture of native nerves. Bone marrow mesenchymal stem cells (MSC) secrete soluble factors that improve the regeneration of the peripheral nerves. Also, microstructured poly-caprolactone (PCL) filaments are capable of inducing bands of Büngner and promote regeneration in the peripheral nervous system (PNS). We describe here the interaction between PCL filaments and MSC, aiming to optimize PNS tubular implants., Methods: MSC were plated on PCL filaments for 48 h and the adhesion profile, viability, proliferation and paracrine capacity were evaluated. Also, Schwann cells were plated on PCL filaments covered with MSC for 24 h to analyze the feasibility of the co-culture system. Moreover, E16 dorsal root ganglia were plated in contact with PCL filaments for 4 days to analyze neurite extension. Right sciatic nerves were exposed and a 10 mm nerve segment was removed. Distal and proximal stumps were reconnected inside a 14-mm polyethylene tube, leaving a gap of approximately 13 mm between the two stumps. Animals then received phosphate-buffered saline 1×, PCL filaments or PCL filaments previously incubated with MSC and, after 12 weeks, functional gait performance and histological analyses were made. Statistical analyses were made using Student's unpaired t-test, one-way analysis of variance (ANOVA) or two-way ANOVA followed by Bonferroni post-test., Results: MSC were confined to lateral areas and ridges of PCL filaments, aligning along the longitudinal. MSC showed high viability (90 %), and their proliferation and secretion capabilities were not completely inhibited by the filaments. Schwann cells adhered to filaments plated with MSC, maintaining high viability (90 %). Neurites grew and extended over the surface of PCL filaments, reaching greater distances when over MSC-plated filaments. Axons showed more organized and myelinized fibers and reinnervated significantly more muscle fibers when they were previously implanted with MSC-covered PLC filaments. Moreover, animals with MSC-covered filaments showed increased functional recovery after 12 weeks., Conclusions: We provide evidence for the interaction among MSC, Schwann cells and PCL filaments, and we also demonstrate that this system can constitute a stable and permissive support for regeneration of segments of the peripheral nerves.

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.

Background: The ability of S. pneumoniae to generate infections depends on the restrictions imposed by the host's immunity, in order to prevent the bacterium from spreading from the nasopharynx to other tissues, such as the brain. Some authors claim that strains of S. pneumoniae, which fail to survive in the bloodstream, can enter the brain directly from the nasal cavity by axonal transport through the olfactory and/or trigeminal nerves. However, from the immunological point of view, glial cells are far more responsive to bacterial infections than are neurons. This hypothesis is consistent with several recent reports showing that bacteria can infect glial cells from the olfactory bulb and trigeminal ganglia. Since our group previously demonstrated that Schwann cells (SCs) express a functional and appropriately regulated mannose receptor (MR), we decided to test whether SCs are involved in the internalization of S. pneumoniae via MR., Results: Immediately after the interaction step, as well as 3 h later, the percentage of association was approximately 56.5%, decreasing to 47.2% and 40.8% after 12 and 24 h, respectively. Competition assays by adding a 100-fold excess of mannan prior to the S. pneumoniae infection reduced the number of infected cells at 3 and 24 h. A cytochemistry assay with Man/BSA-FITC binding was performed in order to verify a possible overlap between mannosylated ligands and internalized bacteria. Incubation of the SCs with Man/BSA-FITC resulted in a large number of intracellular S. pneumoniae, with nearly complete loss of the capsule. Moreover, the anti-pneumococcal antiserum staining colocalized with the internalized man/BSA-FITC, suggesting that both markers are present within the same endocytic compartment of the SC., Conclusions: Our data offer novel evidence that SCs could be essential for pneumococcal cells to escape phagocytosis and killing by innate immune cells. On the other hand, the results also support the idea that SCs are immunocompetent cells of the PNS that can mediate an efficient immune response against pathogens via MR.

Background: Among the essential biological roles of bone marrow-derived cells, secretion of many soluble factors is included and these small molecules can act upon specific receptors present in many tissues including the nervous system. Some of the released molecules can induce proliferation of Schwann cells (SC), satellite cells and lumbar spinal cord astrocytes during early steps of regeneration in a rat model of sciatic nerve transection. These are the major glial cell types that support neuronal survival and axonal growth following peripheral nerve injury. Fibroblast growth factor-2 (FGF-2) is the main mitogenic factor for SCs and is released in large amounts by bone marrow-derived cells, as well as by growing axons and endoneurial fibroblasts during development and regeneration of the peripheral nervous system (PNS)., Results: Here we show that bone marrow-derived cell treatment induce an increase in the expression of FGF-2 in the sciatic nerve, dorsal root ganglia and the dorsolateral (DL) region of the lumbar spinal cord (LSC) in a model of sciatic nerve transection and connection into a hollow tube. SCs in culture in the presence of bone marrow derived conditioned media (CM) resulted in increased proliferation and migration. This effect was reduced when FGF-2 was neutralized by pretreating BMMC or CM with a specific antibody. The increased expression of FGF-2 was validated by RT-PCR and immunocytochemistry in co-cultures of bone marrow derived cells with sciatic nerve explants and regenerating nerve tissue respectivelly., Conclusion: We conclude that FGF-2 secreted by BMMC strongly increases early glial proliferation, which can potentially improve PNS regeneration.

Mycobacterium leprae (ML), the etiologic agent of leprosy, mainly affects the skin and peripheral nerves, leading to demyelization and loss of axonal conductance. Schwann cells (SCs) are the main cell population infected by ML in the nerves, and infection triggers changes in the SC phenotype from a myelinated to a nonmyelinated state. In the present study, we show that expression of 9-O-acetyl GD3, a ganglioside involved in cellular anti-apoptotic signaling and nerve regeneration, increases in SCs following infection with ML. Observation by confocal microscopy together with coimmunoprecipitation suggested that this ganglioside participates in ML attachment and internalization by SC. Immunoblockage of 9-O-acetyl GD3 in vitro significantly reduced adhesion of ML to SC surfaces. Finally, we show that activation of the MAPK (ERK 1/2) pathway and SC proliferation, two known effects of ML on SCs that result in demyelization, are significantly reduced when the 9-O-acetyl GD3 ganglioside is immunoblocked. Taken together, these data suggest the involvement of 9-O-acetyl GD3 in ML infection on SCs.

Peripheral nerve lesions are considered the most relevant symptoms of leprosy, a chronic infectious disease caused by Mycobacterium leprae. The strategies employed by M. leprae to infect and multiply inside Schwann cells (SCs), however, remain poorly understood. In this study, it is shown that treatment of SCs with M. leprae significantly decreased cell death induced by serum deprivation. Not displayed by Mycobacterium smegmatis or Mycobacterium bovis BCG, the M. leprae survival effect was both dose dependent and specific. The conditioned medium (CM) of M. leprae-treated cultures was seen to mimic the protective effect of the bacteria, suggesting that soluble factors secreted by SCs in response to M. leprae were involved in cell survival. Indeed, by quantitative RT-PCR and dot blot/ELISA, it was demonstrated that M. leprae induced the expression and secretion of the SC survival factor insulin-like growth factor-I. Finally, the involvement of this hormone in M. leprae-induced SC survival was confirmed in experiments with neutralizing antibodies. Taken together, the results of this study delineate an important strategy for the successful colonization of M. leprae in the nerve based on the survival maintenance of the host cell through induction of IGF-I production.

Peripheral human nerves fail to regenerate across longer tube implants (>2 cm), most likely because implants lack the microarchitecture of native nerves, including bands of Büngner. Bands of Büngner comprise longitudinally aligned Schwann cell strands that guide selectively regrowing axons. We aim to optimize tubular implants by integrating artificial bands of Büngner. Three principle strategies for inducing the formation of bands of Büngner were investigated: (a) an aligned extracellular matrix, (b) polarizing differentiation factors, and (c) microstructured biomaterial filaments. In vitro oriented collagen and a combination of differentiation factors (NGF, neuregulin-1, TGF-beta) induced Schwann cell alignment to some extent. The most pronounced Schwann cell alignment was evident on ultrathin, endless poly-epsilon-caprolactone (PCL) filaments with longitudinal microgrooves. Precoated PCL filaments proved to be non-cytotoxic, displayed good cell attachment, and supported Schwann cell proliferation as well as guided axonal outgrowth. In vitro on PCL filaments Schwann cells displayed a polarized expression of the cell adhesion molecule L1 similar to that seen in vivo in bands of Büngner after sciatic nerve crush in adult rats. In summary, the integration of bioengineered bands of Büngner based on microstructured polymer filaments in nerve conduits promises to be the most valuable approach to initiating a more efficient regeneration across longer nerve lesions.

A rat model of complete sciatic nerve transection was used to evaluate the effect of bone marrow mononuclear cells (BMMC) transplanted to the injury site immediately after lesion. Rats treated with BMMC had both sensory and motor axons reaching the distal stump earlier compared to untreated animals. In addition, BMMC transplantation reduced cell death in dorsal root ganglia (DRG) compared to control animals. Transplanted BMMC remained in the lesion site for several days but there is no evidence of BMMC differentiation into Schwann cells. However, an increase in the number of Schwann cells, satellite cells and astrocytes was observed in the treated group. Moreover, neutralizing antibodies for nerve growth factor (NGF) (but not for brain-derived neurotrophic factor and ciliary-derived neurotrophic factor) added to the BMMC-conditioned medium reduced neurite growth of sensory and sympathetic neurons in vitro, suggesting that BMMC release NGF, improve regeneration of the sciatic nerve in the adult rat and stimulate Schwann and satellite cell proliferation or a combination of both.

Evidence accumulates suggesting that 9-O-acetylated gangliosides, recognized by a specific monoclonal antibody (Jones monoclonal antibody), are involved in neuronal migration and axonal growth. These molecules are expressed in rodent embryos during the period of axon extension of peripheral nerves and are absent in adulthood. We therefore aimed at verifying if these molecules are re-expressed in adult rats during peripheral nerve regeneration. In this work we studied the time course of ganglioside 9-O-acetyl GD3 expression during regeneration of the crushed sciatic nerve and correlated this expression with the time course of axonal regeneration as visualized by immunohistochemistry for neurofilament 200 in the nerve. We have found that the ganglioside 9-O-acetyl GD3 is re-expressed during the period of regeneration and this expression correlates spatio-temporally with the arrival of axons to the lesion site. Confocal analysis of double and triple labeling experiments allowed the localization of this ganglioside to Schwann cells encircling growing axons in the sciatic nerve. Explant cultures of peripheral nerves also revealed ganglioside expressing reactive Schwann cells migrating from the normal and previously crushed nerve. Ganglioside 9-O-acetyl GD3 is also upregulated in DRG neurons and motoneurons of the ventral horn of spinal cord showing that the reexpression of this molecule is not restricted to Schwann cells. These results suggest that ganglioside 9-O-acetyl GD3 may be involved in the regrowth of sciatic nerve axons after crush being upregulated in both neurons and glia.