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Electrical activity underpins all life, but is most familiar in the nervous system, where long range electrical signalling is essential for function. When this is lost (e.g., traumatic injury) or it becomes inefficient (e.g., demyelination), the use of external fields can compensate for at least some functional deficits. However, its potential to also promote biological repair at the cell level is underplayed despite abundant in vitro evidence for control of neuron growth. This perspective article considers specifically the emerging possibility of achieving cell growth through the interaction of external electric fields using conducting materials as unwired bipolar electrodes, and without intending stimulation of neuron electrical activity to be the primary consequence. The use of a wireless method to create electrical interactions represents a paradigm shift and may allow new applications in vivo where physical wiring is not possible. Within that scheme of thought an evaluation of specific materials and their dynamic responses as bipolar unwired electrodes is summarized and correlated with changes in dynamic nerve growth during stimulation, suggesting possible future schemes to achieve neural growth using bipolar unwired electrodes with specific characteristics. This strategy emphasizes how nerve growth can be encouraged at injury sites wirelessly to induce repair, as opposed to implanting devices that may substitute the neural signals.
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Estimulación Eléctrica , Electrodos , Tecnología Inalámbrica , Humanos , Animales , Neuronas/fisiologíaRESUMEN
STUDY DESIGN: Systematic review and meta-analysis of preclinical literature. OBJECTIVES: To assess the effects of biomaterial-based combination (BMC) strategies for the treatment of Spinal Cord Injury (SCI), the effects of individual biomaterials in the context of BMC strategies, and the factors influencing their efficacy. To assess the effects of different preclinical testing paradigms in BMC strategies. METHODS: We performed a systematic literature search of Embase, Web of Science and PubMed. All controlled preclinical studies describing an in vivo or in vitro model of SCI that tested a biomaterial in combination with at least one other regenerative strategy (cells, drugs, or both) were included. Two review authors conducted the study selection independently, extracted study characteristics independently and assessed study quality using a modified CAMARADES checklist. Effect size measures were combined using random-effects models and heterogeneity was explored using meta-regression with tau2, I2 and R2 statistics. We tested for small-study effects using funnel plot-based methods. RESULTS: 134 publications were included, testing over 100 different BMC strategies. Overall, treatment with BMC therapies improved locomotor recovery by 25.3% (95% CI, 20.3-30.3; n = 102) and in vivo axonal regeneration by 1.6 SD (95% CI 1.2-2 SD; n = 117) in comparison with injury only controls. CONCLUSION: BMC strategies improve locomotor outcomes after experimental SCI. Our comprehensive study highlights gaps in current knowledge and provides a foundation for the design of future experiments.
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Traumatismos de la Médula Espinal , Regeneración de la Medula Espinal , Animales , Humanos , Traumatismos de la Médula Espinal/terapia , Materiales Biocompatibles/uso terapéutico , Modelos Animales de Enfermedad , Procedimientos NeuroquirúrgicosRESUMEN
The cilium of a cell translates varied extracellular cues into intracellular signals that control embryonic development and organ function. The dynamic maintenance of ciliary structure and function requires balanced bidirectional cargo transport involving intraflagellar transport (IFT) complexes. IFT172 is a member of the IFT complex B, and IFT172 mutation is associated with pathologies including short rib thoracic dysplasia, retinitis pigmentosa and Bardet-Biedl syndrome, but how it underpins these conditions is not clear. We used the WIM cell line, derived from embryonic fibroblasts of Wimple mice (carrying homozygous Leu1564Pro mutation in Ift172), to probe roles of Ift172 and primary cilia in cell behavior. WIM cells had ablated cilia and deficiencies in directed migration (electrotaxis), cell proliferation and intracellular signaling. Additionally, WIM cells displayed altered cell cycle progression, with increased numbers of chromatids, highlighting dysfunctional centrosome status. Exposure to a physiological electric field promoted a higher percentage of primary cilia in wild-type cells. Interestingly, in situ hybridization revealed an extensive and dynamic expression profile of Ift172 in both developing and adult mouse cortex. In vivo manipulation of Ift172 expression in germinal regions of embryonic mouse brains perturbed neural progenitor proliferation and radial migration of post-mitotic neurons, revealing a regulatory role of Ift172 in cerebral morphogenesis. Our data suggest that Ift172 regulates a range of fundamental biological processes, highlighting the pivotal roles of the primary cilium in cell physiology and brain development.
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The factors and signals driving T cell activation and polarisation during immune responses have been studied mainly at the level of cells and chemical mediators. Here we describe a physical driver of these processes in the form of physiological-strength electric fields (EFs). EFs are generated at sites where epithelium is disrupted (e.g. wounded skin/bronchial epithelia) and where T cells frequently are present. Using live-cell imaging, we show human primary T cells migrate directionally to the cathode in low strength (50/150 mV/mm) EFs. Strikingly, we show for the first time that EFs significantly downregulate T cell activation following stimulation with antigen-activated APCs or anti-CD3/CD28 antibodies, as demonstrated by decreased IL-2 secretion and proliferation. These EF-induced functional changes were accompanied by a significant dampening of CD4+ T cell polarisation. Expression of critical markers of the Th17 lineage, RORγt and IL-17, and the Th17 polarisation mediator phospho-STAT3 were reduced significantly, while STAT1, ERK and c-Jun phosphorylation were comparatively unaffected suggesting STAT3 modulation by EFs as one mechanism driving effects. Overall, we identify electrical signals as important contributors to the co-ordination and regulation of human T cell functions, paving the way for a new research area into effects of naturally occurring and clinically-applied EFs in conditions where control of T cell activity is paramount.
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Campos Electromagnéticos , Activación de Linfocitos/efectos de la radiación , Subgrupos de Linfocitos T/efectos de la radiación , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/efectos de la radiación , División Celular/efectos de la radiación , Movimiento Celular , Polaridad Celular/efectos de la radiación , Células Cultivadas , Citocinas/biosíntesis , Electrodos , Endotoxinas/farmacología , Humanos , Interleucina-2/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Fosforilación , Procesamiento Proteico-Postraduccional , Factor de Transcripción STAT3/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Células Th17/inmunología , Células Th17/efectos de la radiaciónRESUMEN
Natural direct current electric fields (DC EFs) within tissues undergoing angiogenesis have the potential to influence vessel formation, but how they affect endothelial cells is not clear. We therefore quantified behaviours of human umbilical vein endothelial cells (HUVEC) and human microvasculature endothelial cells (HMEC) stimulated by EFsin vitro. Both cell types migrated faster and toward the cathode; HUVECs responded to fields as low as 50mV/mm, but the HMEC threshold was 100 mV/mm. Mitosis was stimulated at 50 mV/mm for HMEC and at 150 mV/mm for HUVECs, but the cleavage plane was oriented orthogonal to the field vector at 200 mV/mm for both cell types. That different field strengths induced different cell responses suggests distinct underlying cellular mechanisms. A physiological electric field also upregulated expression of CXCR4 and CXCR2 chemokine receptors and upregulated phosphorylation of both chemokines in HUVEC and HMEC cells. Evidence that DC EFs direct endothelial cell migration, proliferation and upregulate chemokines involved in wound healing suggests a key role for electrical control of capillary production during healing. Our data contribute to the molecular mechanisms by which DC EFs direct endothelial cell behaviour and present a novel signalling paradigm in wound healing, tissue regeneration and angiogenesis-related diseases.
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Movimiento Celular , Proliferación Celular , Estimulación Eléctrica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Microvasos/metabolismo , Neovascularización Fisiológica , Receptores CXCR4/metabolismo , Receptores de Interleucina-8B/metabolismo , Células Cultivadas , Humanos , Microvasos/citología , Transducción de Señal , Regulación hacia ArribaRESUMEN
Innovative neurostimulation therapies require improved electrode materials, such as poly(3,4-ethylenedioxythiophene) (PEDOT) polymers or IrOx mixed ionic-electronic conductors and better understanding of how their electrochemistry influences nerve growth. Amphibian neurons growing on transparent films of electronic (metal) conductors and electronic-ionic conductors (polymers and semiconducting oxides) are monitored. Materials are not connected directly to the power supply, but a dipole is created wirelessly within them by electrodes connected to the culture medium in which they are immersed. Without electrical stimulation neurons grow on gold, platinum, PEDOT-polystyrene sulfonate (PEDOT-PSS), IrOx , and mixed oxide (Ir-Ti)Ox , but growth is not related to surface texture or hydrophilicity. Stimulation induces a dipole in all conductive materials, but neurons grow differently on electronic conductors and mixed-valence mixed-ionic conductors. Stimulation slows, but steers neurite extension on gold but not on platinum. The rate and direction of neurite growth on PEDOT-PSS resemble that on glass, but on IrOx and (Ir-Ti)Ox neurites grow faster and in random directions. This suggests electrochemical changes induced in these materials control growth speed and direction selectively. Evidence that the electric dipole induced in conductive material controls nerve growth will impact electrotherapies exploiting wireless stimulation of implanted material arrays, even where transparency is required.
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Conductividad Eléctrica , Polímeros/química , Materiales Biocompatibles/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Óxidos/químicaRESUMEN
BACKGROUND: Primary microglial cultures have been used extensively to facilitate the development of therapeutic strategies for a variety of CNS disorders including neurodegeneration and neuropathic pain. However, existing techniques for culturing these cells are slow and costly. NEW METHOD: Here, we report a refined protocol based on our previously published methods described by Clark et al., which reduces in the time, reagents and the number of animals used for each culture whilst yielding high number and excellent quality microglial cells. RESULTS: Our refined protocol offers an isolation of >96% microglia from a mixed glial culture after only four days of incubation. It results in a high yield of microglia, in excess of one million cells per cortex with predominantly resting morphology and a low level of cell activation. COMPARISON WITH EXISTING METHOD(S): Compared to conventional procedures our refined protocol requires only one third of the time to prepare high quality microglial cultures, cuts the cost more than four-fold, and significantly reduces the number of animals used per culture. CONCLUSION: Our consistent, reliable, and time/cost effective microglial culture protocol is crucial for efficient in vitro screening of potential therapeutics. By dramatically reducing the culture time from 2 weeks to just 4â¯days and increasing the laboratory research output it has implications for the Reduction, Refinement and Replacement policies endorsed by many government funding agencies and animal research regulatory bodies.
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Microglía/fisiología , Cultivo Primario de Células/economía , Cultivo Primario de Células/métodos , Animales , Animales Recién Nacidos , Células Cultivadas , Corteza Cerebral/citología , Femenino , Masculino , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The intricate patterns of cell migration that are found throughout development are generated through a vast array of guidance cues. Responding integratively to distinct, often conflicting, migratory signals is probably crucial for cells to reach their correct destination. Pax6 is a master transcription factor with key roles in neural development that include the control of cell migration. In this study, we have investigated the ability of cells derived from cortical neurospheres from wild-type (WT) and Pax6-/- mouse embryos to integrate diverging guidance cues. We used two different cues, either separately or in combination: substratum nanogrooves to induce contact guidance, and electric fields (EFs) to induce electrotaxis. In the absence of an EF, both WT and Pax6-/- cells aligned and migrated parallel to grooves, and on a flat substrate both showed marked electrotaxis towards the cathode. When an EF was applied in a perpendicular orientation to grooves, WT cells responded significantly to both cues, migrating in highly oblique trajectories in the general direction of the cathode. However, Pax6-/- cells had an impaired response to both cues simultaneously. Our results demonstrate that these neurosphere derived cells have the capacity to integrate diverging guidance cues, which requires Pax6 function.
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This study shows that the core planar cell polarity (PCP) genes direct the aligned cell migration in the adult corneal epithelium, a stratified squamous epithelium on the outer surface of the vertebrate eye. Expression of multiple core PCP genes was demonstrated in the adult corneal epithelium. PCP components were manipulated genetically and pharmacologically in human and mouse corneal epithelial cells in vivo and in vitro. Knockdown of VANGL2 reduced the directional component of migration of human corneal epithelial (HCE) cells without affecting speed. It was shown that signalling through PCP mediators, dishevelled, dishevelled-associated activator of morphogenesis and Rho-associated protein kinase directs the alignment of HCE cells by affecting cytoskeletal reorganization. Cells in which VANGL2 was disrupted tended to misalign on grooved surfaces and migrate across, rather than parallel to the grooves. Adult corneal epithelial cells in which Vangl2 had been conditionally deleted showed a reduced rate of wound-healing migration. Conditional deletion of Vangl2 in the mouse corneal epithelium ablated the normal highly stereotyped patterns of centripetal cell migration in vivo from the periphery (limbus) to the centre of the cornea. Corneal opacity owing to chronic wounding is a major cause of degenerative blindness across the world, and this study shows that Vangl2 activity is required for directional corneal epithelial migration.
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PURPOSE: Patients with a heterozygous mutation in the gene encoding the transcription factor, PAX6, have a degenerative corneal opacity associated with failure of normal radial epithelial cell migration across the corneal surface and a reported wound healing defect. This study investigated the guidance mechanisms that drive the directed migration of corneal epithelial cells. METHODS: In vivo corneal epithelial wounding was performed in adult wild-type and Pax6(+/-) mice, and the healing migration rates were compared. To investigate the control of the cell migration direction, primary corneal epithelial cells from wild-type and Pax6(+/-) mice were plated on grooved quartz substrates, and alignment relative to the grooves was assayed. A reconstructed corneal culture system was developed in which dissociated wild-type and genetically mutant corneal epithelial cells could be cultured on a de-epithelialized corneal stroma or basement membrane and their migration assayed with time-lapse microscopy. RESULTS: The Pax6(+/-) cells efficiently re-epithelialized corneal wounds in vivo but had mild slowing of healing migration compared to the wild-type. Cells aligned parallel to quartz grooves in vitro, but the Pax6(+/-) cells were less robustly oriented than the wild-type. In the reconstructed corneal culture system, corneal epithelial cells continued to migrate radially, showing that the cells are guided by contact-mediated cues from the basement membrane. Recombining wild-type and Pax6 mutant corneal epithelial cells with wild-type and Pax6 mutant corneal stroma showed that normal Pax6 dosage was required autonomously in the epithelial cells for directed migration. Integrin-mediated attachment to the substrate, and intracellular PI3Kγ activity, were required for migration. Pharmacological inhibition of cAMP signaling randomized migration tracks in reconstructed corneas. CONCLUSIONS: Striking patterns of centripetal migration of corneal epithelial cells observed in vivo are driven by contact-mediated cues operating through an intracellular cAMP pathway, and failure to read these cues underlies the migration defects that accompany corneal degeneration in patients with mutations in PAX6.
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Movimiento Celular/fisiología , Lesiones de la Cornea/fisiopatología , Células Epiteliales/fisiología , Adhesiones Focales/fisiología , Factor de Transcripción PAX6/fisiología , Cicatrización de Heridas/fisiología , Animales , Fosfatidilinositol 3-Quinasa Clase Ib/fisiología , Sustancia Propia/citología , AMP Cíclico/fisiología , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Repitelización/fisiología , Transducción de Señal/fisiologíaRESUMEN
The GTPase ARL13B is localized to primary cilia; small cellular protrusions that act as antennae. Its defective ARL13B hennin (HNN) variant is linked causally with Joubert Syndrome, a developmental ciliopathy attributed to poor sensing of extracellular chemical gradients. We tested the hypothesis that impaired detection of extracellular voltage gradients also contributes to the HNN phenotype. In vitro, extracellular electric fields stimulated migration of wild type (WT) and HNN fibroblasts toward the cathode but the field only increased the migration speed of WT cells. Cilia on WT cells did not align to the field vector. HNN cells divided more slowly than WT cells, arresting at the G2/M phase. Mechanistically, HNN cells had reduced phospho-ERK1/2 signaling and elevated levels of Suppressor of Fused protein. These suggest that cells may not be able to read extracellular chemical cues appropriately, resulting in deficits in cell migration and proliferation. Finally, an increase in tubulin stabilization (more detyrosinated tubulin) confirmed the general stagnation of HNN cells, which may further contribute to slower migration and cell cycle progression. We conclude that Arl13b dysfunction resulted in HNN cell stagnation due to poor growth factor signaling and impaired detection of extracellular electrical gradients, and that the role of Arl13b in cell proliferation may be understated.
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Factores de Ribosilacion-ADP/metabolismo , Ciclo Celular , Movimiento Celular , Cilios/metabolismo , Animales , Recuento de Células , Proliferación Celular , Electricidad , Electrodos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fase G2 , Inmunohistoquímica , Ratones , Microtúbulos/metabolismo , Mitosis , Modelos Biológicos , Proteínas Represoras , Fase S , Tubulina (Proteína)/metabolismoRESUMEN
Macrophages are key cells in inflammation and repair, and their activity requires close regulation. The characterization of cues coordinating macrophage function has focused on biologic and soluble mediators, with little known about their responses to physical stimuli, such as the electrical fields that are generated naturally in injured tissue and which accelerate wound healing. To address this gap in understanding, we tested how properties of human monocyte-derived macrophages are regulated by applied electrical fields, similar in strengths to those established naturally. With the use of live-cell video microscopy, we show that macrophage migration is directed anodally by electrical fields as low as 5 mV/mm and is electrical field strength dependent, with effects peaking â¼300 mV/mm. Monocytes, as macrophage precursors, migrate in the opposite, cathodal direction. Strikingly, we show for the first time that electrical fields significantly enhance macrophage phagocytic uptake of a variety of targets, including carboxylate beads, apoptotic neutrophils, and the nominal opportunist pathogen Candida albicans, which engage different classes of surface receptors. These electrical field-induced functional changes are accompanied by clustering of phagocytic receptors, enhanced PI3K and ERK activation, mobilization of intracellular calcium, and actin polarization. Electrical fields also modulate cytokine production selectively and can augment some effects of conventional polarizing stimuli on cytokine secretion. Taken together, electrical signals have been identified as major contributors to the coordination and regulation of important human macrophage functions, including those essential for microbial clearance and healing. Our results open up a new area of research into effects of naturally occurring and clinically applied electrical fields in conditions where macrophage activity is critical.
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Electricidad , Macrófagos/citología , Macrófagos/metabolismo , Citoesqueleto de Actina/metabolismo , Calcio/metabolismo , Movimiento Celular , Citocinas/metabolismo , Electrodos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Espacio Intracelular/metabolismo , Fagocitosis , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Regulación hacia ArribaRESUMEN
PURPOSE: To investigate the roles of intracellular signaling elicited by Hedgehog (Hh) ligands in corneal maintenance and wound healing. METHODS: The expression of Hedgehog pathway components in the cornea was assayed by immunohistochemistry, western blot and reverse-transcription polymerase chain reaction (RT-PCR), in wild-type mice and mice that were heterozygous null for the gene encoding the transcription factor, paired box gene 6 (Pax6). Corneal epithelial wound healing and cell migration assays were performed after pharmacological upregulation and downregulation of the hedgehog pathway. Reporter mice, mosaic for expression of the gene encoding ß-galactosidase (LacZ), were crossed to Pax6(+/-) mice, mice heterozygous for the gene encoding GLI-Kruppel family member GLI3, and Pax6(+/-)Gli3(+/-) double heterozygotes, to assay patterns of cell migration and corneal epithelial organization in vivo. RESULTS: Corneal epithelial wound healing rates increased in response to application of Sonic hedgehog (Shh), but only in mice with wild-type Pax6 dosage. Downregulation of Hedgehog signalling inhibited corneal epithelial cell proliferation. Pax6(+/-) corneal epithelia showed increased proliferation in response to exogenous Shh, but not increased migration. Desert hedgehog (Dhh) was shown to be the major endogenous ligand, with Shh detectable only by RT-PCR and only after epithelial wounding. The activity of phosphatidylinositol-3-OH kinase-γ (PI3Kγ) was not required for the increased migration response in response to Shh. Nuclear expression of the activator form of the transcription factor Gli3 (which mediates Hh signalling) was reduced in Pax6(+/-) corneal epithelia. Pax6(+/-)Gli3(+/-) double heterozygotes showed highly disrupted patterns of clonal arrangement of cells in the corneal epithelium. CONCLUSIONS: The data show key roles for endogenous Dhh signalling in maintenance and regeneration of the corneal epithelium, demonstrate an interaction between Pax6 and Hh signalling in the corneal epithelium, and show that failure of Hh signalling pathways is a feature of Pax6(+/-) corneal disease that cannot be remedied pharmacologically by addition of the ligands.