Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 75
Filtrar
1.
J Neurochem ; 168(9): 3095-3107, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39018376

RESUMO

Microglia, the immune cells of the central nervous system, are dynamic and heterogenous cells. While single cell RNA sequencing has become the conventional methodology for evaluating microglial state, transcriptomics do not provide insight into functional changes, identifying a critical gap in the field. Here, we propose a novel organelle phenotyping approach in which we treat live human induced pluripotent stem cell-derived microglia (iMGL) with organelle dyes staining mitochondria, lipids, lysosomes and acquire data by live-cell spectral microscopy. Dimensionality reduction techniques and unbiased cluster identification allow for recognition of microglial subpopulations with single-cell resolution based on organelle function. We validated this methodology using lipopolysaccharide and IL-10 treatment to polarize iMGL to an "inflammatory" and "anti-inflammatory" state, respectively, and then applied it to identify a novel regulator of iMGL function, complement protein C1q. While C1q is traditionally known as the initiator of the complement cascade, here we use organelle phenotyping to identify a role for C1q in regulating iMGL polarization via fatty acid storage and mitochondria membrane potential. Follow up evaluation of microglia using traditional read outs of activation state confirm that C1q drives an increase in microglia pro-inflammatory gene production and migration, while suppressing microglial proliferation. These data together validate the use of a novel organelle phenotyping approach and enable better mechanistic investigation of molecular regulators of microglial state.


Assuntos
Complemento C1q , Células-Tronco Pluripotentes Induzidas , Microglia , Fenótipo , Microglia/metabolismo , Complemento C1q/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Organelas/metabolismo , Mitocôndrias/metabolismo , Microscopia/métodos , Células Cultivadas
2.
Proc Natl Acad Sci U S A ; 116(30): 14947-14954, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31285339

RESUMO

Traumatic primary spinal cord injury (SCI) results in paralysis below the level of injury and is associated with infiltration of hematogenous innate immune cells into the injured cord. Methylprednisolone has been applied to reduce inflammation following SCI, yet was discontinued due to an unfavorable risk-benefit ratio associated with off-target effects. In this study, i.v. administered poly(lactide-coglycolide) nanoparticles were internalized by circulating monocytes and neutrophils, reprogramming these cells based on their physicochemical properties and not by an active pharmaceutical ingredient, to exhibit altered biodistribution, gene expression, and function. Approximately 80% of nanoparticle-positive immune cells were observed within the injury, and, additionally, the overall accumulation of innate immune cells at the injury was reduced 4-fold, coinciding with down-regulated expression of proinflammatory factors and increased expression of antiinflammatory and proregenerative genes. Furthermore, nanoparticle administration induced macrophage polarization toward proregenerative phenotypes at the injury and markedly reduced both fibrotic and gliotic scarring 3-fold. Moreover, nanoparticle administration with the implanted multichannel bridge led to increased numbers of regenerating axons, increased myelination with about 40% of axons myelinated, and an enhanced locomotor function (score of 6 versus 3 for control group). These data demonstrate that nanoparticles provide a platform that limits acute inflammation and tissue destruction, at a favorable risk-benefit ratio, leading to a proregenerative microenvironment that supports regeneration and functional recovery. These particles may have applications to trauma and potentially other inflammatory diseases.


Assuntos
Imunomodulação , Metilprednisolona/administração & dosagem , Monócitos/imunologia , Nanopartículas/metabolismo , Neutrófilos/imunologia , Traumatismos da Medula Espinal/terapia , Animais , Feminino , Imunidade Inata , Injeções Intravenosas , Metilprednisolona/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Traumatismos da Medula Espinal/imunologia
3.
Biotechnol Bioeng ; 116(1): 155-167, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30229864

RESUMO

Spinal cord injury (SCI) results in paralysis below the injury and strategies are being developed that support axonal regrowth, yet recovery lags, in part, because many axons are not remyelinated. Herein, we investigated strategies to increase myelination of regenerating axons by overexpression of platelet-derived growth factor (PDGF)-AA and noggin either alone or in combination in a mouse SCI model. Noggin and PDGF-AA have been identified as factors that enhance recruitment and differentiation of endogenous progenitors to promote myelination. Lentivirus encoding for these factors was delivered from a multichannel bridge, which we have previously shown creates a permissive environment and supports robust axonal growth through channels. The combination of noggin+PDGF enhanced total myelination of regenerating axons relative to either factor alone, and importantly, enhanced functional recovery relative to the control condition. The increase in myelination was consistent with an increase in oligodendrocyte-derived myelin, which was also associated with a greater density of cells of an oligodendroglial lineage relative to each factor individually and control conditions. These results suggest enhanced myelination of regenerating axons by noggin+PDGF that act on oligodendrocyte-lineage cells post-SCI, which ultimately led to improved functional outcomes.


Assuntos
Proteínas de Transporte/administração & dosagem , Terapia Genética/métodos , Bainha de Mielina/efeitos dos fármacos , Regeneração Nervosa , Fator de Crescimento Derivado de Plaquetas/administração & dosagem , Medicina Regenerativa/métodos , Traumatismos da Medula Espinal/terapia , Animais , Proteínas de Transporte/genética , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Vetores Genéticos , Lentivirus/genética , Camundongos , Fator de Crescimento Derivado de Plaquetas/genética , Resultado do Tratamento
4.
J Immunol ; 199(3): 1069-1085, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28687659

RESUMO

Inflammatory processes play a key role in pathophysiology of many neurologic diseases/trauma, but the effect of immune cells and factors on neurotransplantation strategies remains unclear. We hypothesized that cellular and humoral components of innate immunity alter fate and migration of human neural stem cells (hNSC). In these experiments, conditioned media collected from polymorphonuclear leukocytes (PMN) selectively increased hNSC astrogliogenesis and promoted cell migration in vitro. PMN were shown to generate C1q and C3a; exposure of hNSC to PMN-synthesized concentrations of these complement proteins promoted astrogliogenesis and cell migration. Furthermore, in vitro, Abs directed against C1q and C3a reversed the fate and migration effects observed. In a proof-of-concept in vivo experiment, blockade of C1q and C3a transiently altered hNSC migration and reversed astroglial fate after spinal cord injury. Collectively, these data suggest that modulation of the innate/humoral inflammatory microenvironment may impact the potential of cell-based therapies for recovery and repair following CNS pathology.


Assuntos
Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Complemento C1q/biossíntese , Complemento C3a/biossíntese , Células-Tronco Neurais/fisiologia , Neutrófilos/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Complemento C1q/antagonistas & inibidores , Complemento C1q/genética , Complemento C1q/imunologia , Complemento C3a/antagonistas & inibidores , Complemento C3a/genética , Complemento C3a/imunologia , Meios de Cultivo Condicionados , Humanos , Imunidade Inata , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/imunologia , Neutrófilos/imunologia , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/fisiopatologia
5.
Methods ; 133: 81-90, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29050826

RESUMO

Neural stem cell (NSC) cultures have been considered technically challenging for time-lapse analysis due to high motility, photosensitivity, and growth at confluent densities. We have tested feasibility of long-term live-cell time-lapse analysis for NSC migration and differentiation studies. Here, we describe a method to study the dynamics of cell cycle, migration, and lineage selection in cultured multipotent mouse or human NSCs using single-cell tracking during a long-term, 7-14 day live-cell time-lapse analysis. We used in-house made PDMS inserts with five microwells on a glass coverslip petri-dish to constrain NSC into the area of acquisition during long-term live-cell imaging. In parallel, we have defined image acquisition settings for single-cell tracking of cell cycle dynamics using Fucci-reporter mouse NSC for 7 days as well as lineage selection and migration using human NSC for 14 days. Overall, we show that adjustments of live-cell analysis settings can extend the time period of single-cell tracking in mouse or human NSC from 24-72 h up to 7-14 days and potentially longer. However, we emphasize that experimental use of repeated fluorescence imaging will require careful consideration of controls during acquisition and analysis.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Neurais/citologia , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/métodos , Linhagem da Célula/fisiologia , Movimento Celular/fisiologia , Rastreamento de Células/métodos , Humanos , Células-Tronco Neurais/fisiologia
6.
Mol Ther ; 26(7): 1756-1770, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29778523

RESUMO

Trauma to the spinal cord and associated secondary inflammation can lead to permanent loss of sensory and motor function below the injury level, with the resulting environment serving as a barrier that limits regeneration. In this study, we investigate the localized expression of anti-inflammatory cytokines IL-10 and IL-4 via lentiviral transduction in multichannel bridges. Porous multichannel bridges provide physical guidance for axonal outgrowth with the cytokines hypothesized to modulate the neuroinflammatory microenvironment and enhance axonal regeneration. Gene expression analyses indicated that induced IL-10 and IL-4 expression decreased expression of pro-inflammatory genes and increased pro-regenerative genes relative to control. Moreover, these factors led to increased numbers of axons and myelination, with approximately 45% of axons myelinated and the number of oligodendrocyte myelinated axons significantly increased by 3- to 4-fold. Furthermore, the combination of a bridge with IL-10 and IL-4 expression improved locomotor function after injury to an average score of 6 relative to an average score of 3 for injury alone. Collectively, these studies highlight the potential for localized immunomodulation to decrease secondary inflammation and enhance regeneration that may have numerous applications.


Assuntos
Anti-Inflamatórios/metabolismo , Citocinas/metabolismo , Imunomodulação/fisiologia , Lentivirus/metabolismo , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/terapia , Animais , Axônios/metabolismo , Axônios/fisiologia , Linhagem Celular , Feminino , Células HEK293 , Humanos , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Bainha de Mielina/fisiologia , Regeneração Nervosa/fisiologia , Oligodendroglia/metabolismo , Oligodendroglia/fisiologia , Medula Espinal/metabolismo , Medula Espinal/fisiologia , Traumatismos da Medula Espinal/metabolismo
7.
J Neurosci ; 37(38): 9269-9287, 2017 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-28847814

RESUMO

The interaction of transplanted stem cells with local cellular and molecular cues in the host CNS microenvironment may affect the potential for repair by therapeutic cell populations. In this regard, spinal cord injury (SCI), Alzheimer's disease, and other neurological injuries and diseases all exhibit dramatic and dynamic changes to the host microenvironment over time. Previously, we reported that delayed transplantation of human CNS-derived neural stem cells (hCNS-SCns) at 9 or 30 d post-SCI (dpi) resulted in extensive donor cell migration, predominantly neuronal and oligodendrocytic donor cell differentiation, and functional locomotor improvements. Here, we report that acute transplantation of hCNS-SCns at 0 dpi resulted in localized astroglial differentiation of donor cells near the lesion epicenter and failure to produce functional improvement in an all-female immunodeficient mouse model. Critically, specific immunodepletion of neutrophils (polymorphonuclear leukocytes) blocked hCNS-SCns astroglial differentiation near the lesion epicenter and rescued the capacity of these cells to restore function. These data represent novel evidence that a host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population, and support targeting the inflammatory microenvironment in combination with cell transplantation after SCI.SIGNIFICANCE STATEMENT The interaction of transplanted cells with local cellular and molecular cues in the host microenvironment is a key variable that may shape the translation of neurotransplantation research to the clinical spinal cord injury (SCI) human population, and few studies have investigated these events. We show that the specific immunodepletion of polymorphonuclear leukocyte neutrophils using anti-Ly6G inhibits donor cell astrogliosis and rescues the capacity of a donor cell population to promote locomotor improvement after SCI. Critically, our data demonstrate novel evidence that a specific host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population.


Assuntos
Regeneração Nervosa/imunologia , Células-Tronco Neurais/transplante , Neurogênese/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Animais , Comunicação Celular , Diferenciação Celular/imunologia , Movimento Celular , Feminino , Camundongos , Camundongos SCID , Células-Tronco Neurais/imunologia , Recuperação de Função Fisiológica , Nicho de Células-Tronco
8.
J Neurosci ; 35(10): 4332-49, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25762679

RESUMO

Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI.


Assuntos
Axônios/efeitos dos fármacos , Complemento C1q/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Neuritos/fisiologia , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Arginase/genética , Arginase/metabolismo , Células Cultivadas , Complemento C1q/genética , Complemento C1q/metabolismo , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Feminino , Proteína GAP-43/metabolismo , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/fisiologia , Glicoproteína Associada a Mielina/metabolismo , Neuritos/efeitos dos fármacos , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/tratamento farmacológico , Neuropatia Ciática/patologia , Traumatismos da Medula Espinal/patologia
9.
Immun Ageing ; 11: 15, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25512759

RESUMO

BACKGROUND: Although the incidence of spinal cord injury (SCI) is steadily rising in the elderly human population, few studies have investigated the effect of age in rodent models. Here, we investigated the effect of age in female rats on spontaneous recovery and repair after SCI. Young (3 months) and aged (18 months) female rats received a moderate contusion SCI at T9. Behavioral recovery was assessed, and immunohistocemical and stereological analyses performed. RESULTS: Aged rats demonstrated greater locomotor deficits compared to young, beginning at 7 days post-injury (dpi) and lasting through at least 28 dpi. Unbiased stereological analyses revealed a selective increase in percent lesion area and early (2 dpi) apoptotic cell death caudal to the injury epicenter in aged versus young rats. One potential mechanism for these differences in lesion pathogenesis is the inflammatory response; we therefore assessed humoral and cellular innate immune responses. No differences in either acute or chronic serum complement activity, or acute neutrophil infiltration, were observed between age groups. However, the number of microglia/macrophages present at the injury epicenter was increased by 50% in aged animals versus young. CONCLUSIONS: These data suggest that age affects recovery of locomotor function, lesion pathology, and microglia/macrophage response following SCI.

10.
Pain ; 165(1): 92-101, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-37463227

RESUMO

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.


Assuntos
Neuralgia , Traumatismos da Medula Espinal , Feminino , Camundongos , Animais , Hiperalgesia/metabolismo , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Neuralgia/metabolismo , Citocinas/metabolismo , Medula Espinal/metabolismo
11.
Sci Rep ; 14(1): 25186, 2024 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-39448736

RESUMO

Spinal cord injury creates an inflammatory microenvironment that regulates the capacity of transplanted human Neural Stem Cells (hNSC) to migrate, differentiate, and repair injury. Despite similarities in gene expression and markers detected by immunostaining, hNSC populations exhibit heterogeneous therapeutic potential. This heterogeneity derives in part from the epigenetic landscape in the hNSC genome, specifically methylation (5mC) and hydroxymethylation (5hmC) state, which may affect the response of transplanted hNSC in the injury microenvironment and thereby modulate repair capacity. We demonstrate a significant up-regulation of methylcytosine dioxygenase 2 gene (TET2) expression in undifferentiated hNSC derived from human embryonic stem cells (hES-NSC), and report that this is associated with hES-NSC competence for differentiation marker expression. TET2 protein catalyzes active demethylation and TET2 upregulation could be a signature of pluripotent exit, while shaping the epigenetic landscape in hES-NSC. We determine that the inflammatory environment overrides epigenetic programming in vitro and in vivo by directly modulating TET2 expression levels in hES-NSC to change cell fate. We also report the effect of cell fate and microenvironment on differential methylation 5mC/5hmC balance. Understanding how the activity of epigenetic modifiers changes within the transplantation niche in vivo is crucial for assessment of hES-NSC behavior for potential clinical applications.


Assuntos
Diferenciação Celular , Metilação de DNA , Proteínas de Ligação a DNA , Dioxigenases , Epigênese Genética , Células-Tronco Neurais , Proteínas Proto-Oncogênicas , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Diferenciação Celular/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Células-Tronco Pluripotentes/metabolismo , Inflamação/genética , Inflamação/patologia , Inflamação/metabolismo , Animais , Camundongos
12.
NPJ Regen Med ; 9(1): 12, 2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38499577

RESUMO

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected the ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery. These data identify a successful strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.

13.
Adv Healthc Mater ; : e2302498, 2023 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-37768019

RESUMO

Spinal cord injury (SCI) is a life-altering event, which often results in loss of sensory and motor function below the level of trauma. Biomaterial therapies have been widely investigated in SCI to promote directional regeneration but are often limited by their pre-constructed size and shape. Herein, the design parameters of microporous annealed particles (MAPs) are investigated with tubular geometries that conform to the injury and direct axons across the defect to support functional recovery. MAP tubes prepared from 20-, 40-, and 60-micron polyethylene glycol (PEG) beads are generated and implanted in a T9-10 murine hemisection model of SCI. Tubes attenuate glial and fibrotic scarring, increase innate immune cell density, and reduce inflammatory phenotypes in a bead size-dependent manner. Tubes composed of 60-micron beads increase the cell density of the chronic macrophage response, while neutrophil infiltration and phenotypes do not deviate from those seen in controls. At 8 weeks postinjury, implantation of tubes composed of 60-micron beads results in enhanced locomotor function, robust axonal ingrowth, and remyelination through both lumens and the inter-tube space. Collectively, these studies demonstrate the importance of bead size in MAP construction and highlight PEG tubes as a biomaterial therapy to promote regeneration and functional recovery in SCI.

14.
Res Sq ; 2023 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-37502943

RESUMO

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery vs. control and hNSC transplant alone. These data identify a successful novel strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.

15.
J Neuroinflammation ; 9: 137, 2012 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-22721265

RESUMO

The complement system, a major component of the innate immune system, is becoming increasingly recognised as a key participant in physiology and disease. The awareness that immunological mediators support various aspects of both normal central nervous system (CNS) function and pathology has led to a renaissance of complement research in neuroscience. Various studies have revealed particularly novel findings on the wide-ranging involvement of complement in neural development, synapse elimination and maturation of neural networks, as well as the progression of pathology in a range of chronic neurodegenerative disorders, and more recently, neurotraumatic events, where rapid disruption of neuronal homeostasis potently triggers complement activation. The purpose of this review is to summarise recent findings on complement activation and acquired brain or spinal cord injury, i.e. ischaemic-reperfusion injury or stroke, traumatic brain injury (TBI) and spinal cord injury (SCI), highlighting the potential for complement-targeted therapeutics to alleviate the devastating consequences of these neurological conditions.


Assuntos
Lesões Encefálicas/imunologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Ativação do Complemento/imunologia , Doenças Neurodegenerativas/imunologia , Traumatismos da Medula Espinal/imunologia , Animais , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Sistema Nervoso Central/patologia , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia
16.
Lancet Neurol ; 21(7): 659-670, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35569486

RESUMO

Spinal cord injury is a severely disabling neurological condition leading to impaired mobility, pain, and autonomic dysfunction. Most often, a single traumatic event, such as a traffic or recreational accident, leads to primary spinal cord damage through compression and laceration, followed by secondary damage consisting of inflammation and ischaemia, and culminating in substantial tissue loss. Patients need appropriate timely surgical and critical care, followed by neurorehabilitation to facilitate neuronal reorganisation and functional compensation. Although some neurological function might be regained, most patients with initially complete lesions have severe, irreversible neurological impairment. Cell-based and stem-cell-based therapies are recognised as promising candidates to promote functional recovery. However, no trials of these therapies in patients have yet provided reproducible evidence for clinical efficacy, challenged by small effect sizes, low immune suppression, and low sensitivity study designs. Nevertheless, in the past decade, clinical trials have shown the feasibility and long-term safety of cell transplantation into the injured spinal cord. This crucial milestone has paved the way to consider refinements and combined therapies, such as the use of biomaterials to augment the effects of cell transplantation. In the future, emerging cell types, scaffolding, and cell engineering might improve cell survival, integration, and therapeutic efficiency.


Assuntos
Reabilitação Neurológica , Traumatismos da Medula Espinal , Humanos , Neurônios/patologia , Recuperação de Função Fisiológica/fisiologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia
17.
Brain ; 133(Pt 2): 433-47, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20085927

RESUMO

Traumatic injury to the central nervous system results in the disruption of the blood brain/spinal barrier, followed by the invasion of cells and other components of the immune system that can aggravate injury and affect subsequent repair and regeneration. Although studies of chronic neuroinflammation in the injured spinal cord of animals are clinically relevant to most patients living with traumatic injury to the brain or spinal cord, very little is known about chronic neuroinflammation, though several studies have tested the role of neuroinflammation in the acute period after injury. The present study characterizes a novel cell preparation method that assesses, quickly and effectively, the changes in the principal immune cell types by flow cytometry in the injured spinal cord, daily for the first 10 days and periodically up to 180 days after spinal cord injury. These data quantitatively demonstrate a novel time-dependent multiphasic response of cellular inflammation in the spinal cord after spinal cord injury and are verified by quantitative stereology of immunolabelled spinal cord sections at selected time points. The early phase of cellular inflammation is comprised principally of neutrophils (peaking 1 day post-injury), macrophages/microglia (peaking 7 days post-injury) and T cells (peaking 9 days post-injury). The late phase of cellular inflammation was detected after 14 days post-injury, peaked after 60 days post-injury and remained detectable throughout 180 days post-injury for all three cell types. Furthermore, the late phase of cellular inflammation (14-180 days post-injury) did not coincide with either further improvements, or new decrements, in open-field locomotor function after spinal cord injury. However, blockade of chemoattractant C5a-mediated inflammation after 14 days post-injury reduced locomotor recovery and myelination in the injured spinal cord, suggesting that the late inflammatory response serves a reparative function. Together, these data provide new insight into cellular inflammation of spinal cord injury and identify a surprising and extended multiphasic response of cellular inflammation. Understanding the role of this multiphasic response in the pathophysiology of spinal cord injury could be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions.


Assuntos
Estudos de Avaliação como Assunto , Mediadores da Inflamação/fisiologia , Traumatismos da Medula Espinal/patologia , Vértebras Torácicas/patologia , Doença Aguda , Animais , Doença Crônica , Feminino , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Peptídeos Cíclicos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/fisiologia , Traumatismos da Medula Espinal/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , Vértebras Torácicas/metabolismo , Fatores de Tempo
18.
Neurotherapeutics ; 18(1): 503-514, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33051853

RESUMO

In mammals, spinal cord injuries often result in muscle paralysis through the apoptosis of lower motor neurons and denervation of neuromuscular junctions. Previous research shows that the inflammatory response to a spinal cord injury can cause additional tissue damage after the initial trauma. To modulate this inflammatory response, we delivered lentiviral anti-inflammatory interleukin-10, via loading onto an implantable biomaterial scaffold, into a left-sided hemisection at the C5 vertebra in mice. We hypothesized that improved behavioral outcomes associated with anti-inflammatory treatment are due to the sparing of fine motor circuit components. We examined behavioral recovery using a ladder beam, tissue sparing using histology, and electromyogram recordings using intraspinal optogenetic stimulation at 2 weeks post-injury. Ladder beam analysis shows interleukin-10 treatment results in significant improvement of behavioral recovery at 2 and 12 weeks post-injury when compared to mice treated with a control virus. Histology shows interleukin-10 results in greater numbers of lower motor neurons, axons, and muscle innervation at 2 weeks post-injury. Furthermore, electromyogram recordings suggest that interleukin-10-treated animals have signal-to-noise ratios and peak-to-peak amplitudes more similar to that of uninjured controls than to that of control injured animals at 2 weeks post-injury. These data show that gene therapy using anti-inflammatory interleukin-10 can significantly reduce tissue damage and subsequent motor deficits after a spinal cord injury. Together, these results suggest that early modulation of the injury response can preserve muscle function with long-lasting benefits.


Assuntos
Terapia Genética/métodos , Interleucina-10/genética , Neurônios Motores/fisiologia , Traumatismos da Medula Espinal/terapia , Animais , Vértebras Cervicais , Eletromiografia , Feminino , Lentivirus , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Optogenética , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia
19.
J Neurotrauma ; 38(19): 2731-2746, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34130484

RESUMO

Human neural stem cells (hNSCs) have potential as a cell therapy after traumatic brain injury (TBI). While various studies have demonstrated the efficacy of NSCs from ongoing culture, there is a significant gap in our understanding of freshly thawed cells from cryobanked stocks-a more clinically relevant source. To address these shortfalls, the therapeutic potential of our previously validated Shef-6.0 human embryonic stem cell (hESC)-derived hNSC line was tested after long-term cryostorage and thawing before transplant. Immunodeficient athymic nude rats received a moderate unilateral controlled cortical impact (CCI) injury. At four weeks post-injury, 6 × 105 freshly thawed hNSCs were transplanted into six injection sites (two ipsi- and four contra-lateral) with 53.4% of cells surviving three months post-transplant. Interestingly, most hNSCs were engrafted in the meninges and the lining of lateral ventricles, associated with high CXCR4 expression and a chemotactic response to SDF1alpha (CXCL12). While some expressed markers of neuron, astrocyte, and oligodendrocyte lineages, the majority remained progenitors, identified through doublecortin expression (78.1%). Importantly, transplantation resulted in improved spatial learning and memory in Morris water maze navigation and reduced risk taking in an elevated plus maze. Investigating potential mechanisms of action, we identified an increase in ipsilateral host hippocampus cornu ammonis (CA) neuron survival, contralateral dentate gyrus (DG) volume, and DG neural progenitor morphology as well as a reduction in neuroinflammation. Together, these findings validate the potential of hNSCs to improve function after TBI and demonstrate that long-term biobanking of cells and thawing aliquots before use may be suitable for clinical deployment.


Assuntos
Lesões Encefálicas Traumáticas/psicologia , Lesões Encefálicas Traumáticas/terapia , Lesão Encefálica Crônica/psicologia , Lesão Encefálica Crônica/terapia , Cognição/fisiologia , Células-Tronco Neurais/transplante , Animais , Bancos de Espécimes Biológicos , Criopreservação , Modelos Animais de Doenças , Humanos , Masculino , Neurogênese , Ratos , Ratos Nus , Nicho de Células-Tronco , Transplante de Células-Tronco
20.
Mol Ther ; 17(2): 318-26, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19050701

RESUMO

The regeneration of tissues with complex architectures requires strategies that promote the appropriate cellular processes, and can direct their organization. Plasmid-loaded multiple channel bridges were engineered for spinal cord regeneration with the ability to support and direct cellular processes and promote gene transfer at the injury site. The bridges were manufactured with a gas foaming technique, and had multiple channels with controllable diameter and encapsulated plasmid. Initial studies investigating bridge implantation subcutaneously (SC) indicated transgene expression in vivo for 44 days, with gene expression dependent upon the pore size of the bridge. In the rat spinal cord, bridges implanted into a lateral hemisection supported substantial cell infiltration, aligned cells within the channels, axon growth across the channels, and high levels of transgene expression at the implant site with decreasing levels rostral and caudal. Immunohistochemistry revealed that the transfected cells at the implant site were present in both the pores and channels of the bridge and were mainly identified as Schwann cells, fibroblasts, and macrophages, in descending order of transfection. This synergy between gene delivery and the scaffold architecture may enable the engineering of tissues with complex architectures.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Plasmídeos/genética , Traumatismos da Medula Espinal/terapia , Engenharia Tecidual/métodos , Transgenes/genética , Animais , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Ratos , Ratos Long-Evans , Traumatismos da Medula Espinal/fisiopatologia
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa