Spinal cord injury (SCI) is a devastating condition that may cause permanent functional loss below the level of injury, including paralysis and loss of bladder, bowel, and sexual function. Patients are rarely treated immediately, and this delay is associated with tissue loss and scar formation that can make regeneration at chronic time points more challenging. Herein, we investigated regeneration using a poly(lactide-
Many bacteria build elaborate molecular machines to import DNA via natural competence, yet this activity is often not identified until strains have been handled and domesticated in laboratory settings. For example, one of the best studied Gram-positive model organisms, Bacillus subtilis, has a non-transformable ancestor. Transformation in the ancestral strain is inhibited by a transmembrane peptide, ComI, which is encoded on an extrachromosomal plasmid. Although ComI was shown to be necessary and sufficient to inhibit transformation when produced at high levels under an inducible promoter, the mechanism by which ComI inhibits transformation is unknown. Here, we examine the native regulation and mechanism of transformation inhibition by ComI. We find that under native regulation, ComI expression is restricted in the absence of the plasmid. In the presence of the plasmid, we find that ComI is preferentially expressed in cells that are differentiating into a competent state. The subcellular localization of ComI, however, does not depend on any other competence proteins and permeabilization activity is concentration dependent. Thus over time, the competent cells gradually producing ComI, are permeabilized and killed. Based on these observations we propose a new model for the mechanism of ComI, suggesting a response to competence activation that selectively eliminates the competent subpopulation.
Abstract Neuropathic pain is a critical source of comorbidity following spinal cord injury (SCI) that can be exacerbated by immune-mediated pathologies in the central and peripheral nervous systems. In this article, we investigate whether drug-free, biodegradable, poly(lactide- co -glycolide) (PLG) nanoparticle treatment mitigates the development of post-SCI neuropathic pain in female mice. Our results show that acute treatment with PLG nanoparticles following thoracic SCI significantly reduces tactile and cold hypersensitivity scores in a durable fashion. Nanoparticles primarily reduce peripheral immune-mediated mechanisms of neuropathic pain, including neuropathic pain-associated gene transcript frequency, transient receptor potential ankyrin 1 nociceptor expression, and MCP-1 (CCL2) chemokine production in the subacute period after injury. Altered central neuropathic pain mechanisms during this period are limited to reduced innate immune cell cytokine expression. However, in the chronic phase of SCI, nanoparticle treatment induces changes in both central and peripheral neuropathic pain signaling, driving reductions in cytokine production and other immune-relevant markers. This research suggests that drug-free PLG nanoparticles reprogram peripheral proalgesic pathways subacutely after SCI to reduce neuropathic pain outcomes and improve chronic central pain signaling.
Spinal cord injury (SCI) causes permanent paralysis below the damaged area. SCI is linked to neuronal death, demyelination, and limited ability of neuronal fibers to regenerate. Regeneration capacity is limited by the presence of many inhibitory factors in the spinal cord environment. The use of poly(lactide-co-glycolide) (PLG) bridges has demonstrated the ability to sustain long-term regeneration after SCI in a cervical hemisection mouse model. Critically, imaging of regenerating fibers and the myelination status of these neuronal filaments is a severe limitation to progress in SCI research. We used a transgenic mouse model that selectively expresses fluorescent reporters (eGFP) in the neuronal fibers of the spinal cord. We implanted a PLG bridge at C5 vertebra after hemisection and evaluated in live animals’ neuronal fibers at the bridge interface and within the bridge 8 weeks postimplant. These in vivo observations were correlated with in situ evaluation 12 weeks postimplantation. We sectioned the spinal cords and performed fluorescent bioimaging on the sections to observe neuronal fibers going through the bridge. In parallel, to visualize myelination of regenerated axons, we exploited the characteristics of the third-harmonic generation arising from the myelin structure in these fixed sections.
One million estimated cases of spinal cord injury (SCI) have been reported in the United States and repairing an injury has constituted a difficult clinical challenge. The complex, dynamic, inhibitory microenvironment postinjury, which is characterized by proinflammatory signaling from invading leukocytes and lack of sufficient factors that promote axonal survival and elongation, limits regeneration. Herein, we investigated the delivery of polycistronic vectors, which have the potential to coexpress factors that target distinct barriers to regeneration, from a multiple channel poly(lactide-co-glycolide) (PLG) bridge to enhance spinal cord regeneration. In this study, we investigated polycistronic delivery of IL-10 that targets proinflammatory signaling, and NT-3 that targets axonal survival and elongation. A significant increase was observed in the density of regenerative macrophages for IL-10+NT-3 condition relative to conditions without IL-10. Furthermore, combined delivery of IL-10+NT-3 produced a significant increase of axonal density and notably myelinated axons compared with all other conditions. A significant increase in functional recovery was observed for IL-10+NT-3 delivery at 12 weeks postinjury that was positively correlated to oligodendrocyte myelinated axon density, suggesting oligodendrocyte-mediated myelination as an important target to improve functional recovery. These results further support the use of multiple channel PLG bridges as a growth supportive substrate and platform to deliver bioactive agents to modulate the SCI microenvironment and promote regeneration and functional recovery. Impact statement Spinal cord injury (SCI) results in a complex microenvironment that contains multiple barriers to regeneration and functional recovery. Multiple factors are necessary to address these barriers to regeneration, and polycistronic lentiviral gene therapy represents a strategy to locally express multiple factors simultaneously. A bicistronic vector encoding IL-10 and NT-3 was delivered from a poly(lactide-co-glycolide) bridge, which provides structural support that guides regeneration, resulting in increased axonal growth, myelination, and subsequent functional recovery. These results demonstrate the opportunity of targeting multiple barriers to SCI regeneration for additive effects.
An important role of neural stem cell transplantation is repopulating neural and glial cells that actively promote repair following spinal cord injury (SCI). However, stem cell survival after transplantation is severely hampered by the inflammatory environment that arises after SCI. Biomaterials have a demonstrated history of managing post-SCI inflammation and can serve as a vehicle for stem cell delivery. In this study, we utilize macroporous polyethylene glycol (PEG) tubes, which were previously found to modulate the post-SCI microenvironment, to serve as a viable, soft substrate for injecting mouse embryonic day 14 (E14) spinal progenitors 2 weeks after tube implantation into a mouse SCI model. At 2 weeks after transplantation (4 weeks after injury), 4.3% of transplanted E14 spinal progenitors survived when transplanted directly into tubes compared to 0.7% when transplanted into the injury alone. Surviving E14 spinal progenitors exhibited a commitment to the neuronal lineage at 4 weeks post-injury, as assessed by both early and late phenotypic markers. Mice receiving tubes with E14 spinal progenitor transplantations had on average 21 ± 4 axons/mm2 regenerated compared to 8 ± 1 axons/mm2 for the injury only control, which corresponded with a significant increase in remyelination compared to the injury only control, while all conditions exhibited improved forelimb control 4 weeks after injury compared to the injury only. Collectively, we have demonstrated the feasibility of using PEG tubes to modify the implantation site and improve survival of transplanted E14 spinal progenitors.
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