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1.
Glia ; 70(5): 842-857, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-34978340

RESUMEN

In amyotrophic lateral sclerosis (ALS) caused by SOD1 gene mutations, both cell-autonomous and noncell-autonomous mechanisms lead to the selective degeneration of motoneurons (MN). Here, we evaluate the therapeutic potential of gene therapy targeting mutated SOD1 in mature astrocytes using mice expressing the mutated SOD1G93A protein. An AAV-gfaABC1 D vector encoding an artificial microRNA is used to deliver RNA interference against mutated SOD1 selectively in astrocytes. The treatment leads to the progressive rescue of neuromuscular junction occupancy, to the recovery of the compound muscle action potential in the gastrocnemius muscle, and significantly improves neuromuscular function. In the spinal cord, gene therapy targeting astrocytes protects a small pool of the most vulnerable fast-fatigable MN until disease end stage. In the gastrocnemius muscle of the treated SOD1G93A mice, the fast-twitch type IIB muscle fibers are preserved from atrophy. Axon collateral sprouting is observed together with muscle fiber type grouping indicative of denervation/reinnervation events. The transcriptome profiling of spinal cord MN shows changes in the expression levels of factors regulating the dynamics of microtubules. Gene therapy delivering RNA interference against mutated SOD1 in astrocytes protects fast-fatigable motor units and thereby improves neuromuscular function in ALS mice.


Asunto(s)
Esclerosis Amiotrófica Lateral , Superóxido Dismutasa-1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/terapia , Animales , Astrocitos/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Interferencia de ARN , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genética
2.
J Neurosci Res ; 93(1): 43-55, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25131829

RESUMEN

In spinal cord injury (SCI), absence of functional recovery and lack of spontaneous axonal regeneration are attributed, among other factors, to the formation of a glial scar that forms both physical and chemical barriers. The glial scar is composed mainly of reactive astrocytes that overexpress two intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin (VIM). To promote regeneration and sprouting of spared axons after spinal cord trauma and with the objective of translation to clinics, we designed an original in vivo gene transfer strategy to reduce glial scar formation after SCI, based on the RNA interference (RNAi)-mediated inhibition of GFAP and VIM. We first show that direct injection of lentiviral vectors expressing short hairpin RNA (shRNA) against GFAP and VIM in a mouse model of SCI allows efficient and specific targeting of astrocytes. We then demonstrate that the lentiviral-mediated and stable expression of shGFAP and shVIM leads to a strong reduction of astrogliosis, improves functional motor recovery, and promotes axonal regrowth and sprouting of spared axons. This study thus examplifies how the nonneuronal environment might be a major target within the lesioned central nervous system to promote axonal regeneration (and sprouting) and validates the use of lentiviral-mediated RNAi in SCI.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/terapia , Vimentina/metabolismo , Análisis de Varianza , Animales , Astrocitos/metabolismo , Axones/fisiología , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/fisiología , Proteína Ácida Fibrilar de la Glía/genética , Lentivirus/genética , Locomoción/fisiología , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Serotonina/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Vimentina/genética
3.
Neural Regen Res ; 19(11): 2354-2364, 2024 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-38526271

RESUMEN

Spinal cord injury results in significant sensorimotor deficits, currently, there is no curative treatment for the symptoms induced by spinal cord injury. Basic and pre-clinical research on spinal cord injury relies on the development and characterization of appropriate animal models. These models should replicate the symptoms observed in human, allowing for the exploration of functional deficits and investigation into various aspects of physiopathology of spinal cord injury. Non-human primates, due to their close phylogenetic association with humans, share more neuroanatomical, genetic, and physiological similarities with humans than rodents. Therefore, the responses to spinal cord injury in nonhuman primates most likely resemble the responses to traumatism in humans. In this review, we will discuss nonhuman primate models of spinal cord injury, focusing on in vivo assessments, including behavioral tests, magnetic resonance imaging, and electrical activity recordings, as well as ex vivo histological analyses. Additionally, we will present therapeutic strategies developed in non-human primates and discuss the unique specificities of non-human primate models of spinal cord injury.

4.
BMC Neurosci ; 12: 99, 2011 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-21985235

RESUMEN

BACKGROUND: The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin+ Sox2+ neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium). RESULTS: Here we report the isolation and long term propagation of another population of Nestin+ cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. CONCLUSION: Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.


Asunto(s)
Calcificación Fisiológica/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Cultivo Primario de Células/métodos , Médula Espinal/irrigación sanguínea , Médula Espinal/metabolismo , Adulto , Adhesión Celular/fisiología , Separación Celular/métodos , Proteínas de Homeodominio/sangre , Humanos , Proteínas de Filamentos Intermediarios/sangre , Miocitos del Músculo Liso/citología , Proteínas del Tejido Nervioso/sangre , Nestina , Médula Espinal/citología
5.
Front Aging Neurosci ; 13: 769548, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34899275

RESUMEN

The glial scar that forms after traumatic spinal cord injury (SCI) is mostly composed of microglia, NG2 glia, and astrocytes and plays dual roles in pathophysiological processes induced by the injury. On one hand, the glial scar acts as a chemical and physical obstacle to spontaneous axonal regeneration, thus preventing functional recovery, and, on the other hand, it partly limits lesion extension. The complex activation pattern of glial cells is associated with cellular and molecular crosstalk and interactions with immune cells. Interestingly, response to SCI is diverse among species: from amphibians and fishes that display rather limited (if any) glial scarring to mammals that exhibit a well-identifiable scar. Additionally, kinetics of glial activation varies among species. In rodents, microglia become activated before astrocytes, and both glial cell populations undergo activation processes reflected amongst others by proliferation and migration toward the injury site. In primates, glial cell activation is delayed as compared to rodents. Here, we compare the spatial and temporal diversity of the glial response, following SCI amongst species. A better understanding of mechanisms underlying glial activation and scar formation is a prerequisite to develop timely glial cell-specific therapeutic strategies that aim to increase functional recovery.

6.
Front Pharmacol ; 12: 614949, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33643047

RESUMEN

In traumatic spinal cord injury, the initial trauma is followed by a cascade of impairments, including excitotoxicity and calcium overload, which ultimately induces secondary damages. The sigma-1 receptor is widely expressed in the central nervous system and is acknowledged to play a key role in calcium homeostasis. Treatments with agonists of the sigma-1 receptor induce beneficial effects in several animal models of neurological diseases. In traumatic injury the use of an antagonist of the sigma-1 receptor reversed several symptoms of central neuropathic pain. Here, we investigated whether sigma-1 receptor activation with PRE-084 is beneficial or detrimental following SCI in mice. First, we report that PRE-084 treatment after injury does not improve motor function recovery. Second, using ex vivo diffusion weighted magnetic resonance imaging completed by histological analysis, we highlight that σ1R agonist treatment after SCI does not limit lesion size. Finally, PRE-084 treatment following SCI decreases NeuN expression and increases astrocytic reactivity. Our findings suggest that activation of sigma-1 receptor after traumatic spinal cord injury is detrimental on tissue preservation and motor function recovery in mice.

7.
Brain Sci ; 11(12)2021 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-34942945

RESUMEN

Microglia are major players in scar formation after an injury to the spinal cord. Microglia proliferation, differentiation, and survival are regulated by the colony-stimulating factor 1 (CSF1). Complete microglia elimination using CSF1 receptor (CSF1R) inhibitors worsens motor function recovery after spinal injury (SCI). Conversely, a 1-week oral treatment with GW2580, a CSF1R inhibitor that only inhibits microglia proliferation, promotes motor recovery. Here, we investigate whether prolonged GW2580 treatment further increases beneficial effects on locomotion after SCI. We thus assessed the effect of a 6-week GW2580 oral treatment after lateral hemisection of the spinal cord on functional recovery and its outcome on tissue and cellular responses in adult mice. Long-term depletion of microglia proliferation after SCI failed to improve motor recovery and had no effect on tissue reorganization, as revealed by ex vivo diffusion-weighted magnetic resonance imaging. Six weeks after SCI, GW2580 treatment decreased microglial reactivity and increased astrocytic reactivity. We thus demonstrate that increasing the duration of GW2580 treatment is not beneficial for motor recovery after SCI.

8.
Theranostics ; 11(18): 8640-8659, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34522204

RESUMEN

No curative treatment is available for any deficits induced by spinal cord injury (SCI). Following injury, microglia undergo highly diverse activation processes, including proliferation, and play a critical role on functional recovery. In a translational objective, we investigated whether a transient pharmacological reduction of microglia proliferation after injury is beneficial for functional recovery after SCI in mice and nonhuman primates. Methods: The colony stimulating factor-1 receptor (CSF1R) regulates proliferation, differentiation, and survival of microglia. We orally administrated GW2580, a CSF1R inhibitor that inhibits microglia proliferation. In mice and nonhuman primates, we then analyzed treatment outcomes on locomotor function and spinal cord pathology. Finally, we used cell-specific transcriptomic analysis to uncover GW2580-induced molecular changes in microglia. Results: First, transient post-injury GW2580 administration in mice improves motor function recovery, promotes tissue preservation and/or reorganization (identified by coherent anti-stokes Raman scattering microscopy), and modulates glial reactivity. Second, post-injury GW2580-treatment in nonhuman primates reduces microglia proliferation, improves motor function recovery, and promotes tissue protection. Finally, GW2580-treatment in mice induced down-regulation of proliferation-associated transcripts and inflammatory associated genes in microglia that may account for reduced neuroinflammation and improved functional recovery following SCI. Conclusion: Thus, a transient oral GW2580 treatment post-injury may provide a promising therapeutic strategy for SCI patients and may also be extended to other central nervous system disorders displaying microglia activation.


Asunto(s)
Microglía/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Animales , Anisoles/farmacología , Proliferación Celular/efectos de los fármacos , Cheirogaleidae , Modelos Animales de Enfermedad , Expresión Génica/genética , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Neurogénesis , Enfermedades Neuroinflamatorias , Pirimidinas/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/efectos de los fármacos , Recuperación de la Función/efectos de los fármacos , Transcriptoma/genética
9.
Stem Cells ; 27(11): 2722-33, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19785035

RESUMEN

In humans and rodents the adult spinal cord harbors neural stem cells located around the central canal. Their identity, precise location, and specific signaling are still ill-defined and controversial. We report here on a detailed analysis of this niche. Using microdissection and glial fibrillary acidic protein (GFAP)-green fluorescent protein (GFP) transgenic mice, we demonstrate that neural stem cells are mostly dorsally located GFAP(+) cells lying ependymally and subependymally that extend radial processes toward the pial surface. The niche also harbors doublecortin protein (Dcx)(+) Nkx6.1(+) neurons sending processes into the lumen. Cervical and lumbar spinal cord neural stem cells maintain expression of specific rostro-caudal Hox gene combinations and the niche shows high levels of signaling proteins (CD15, Jagged1, Hes1, differential screening-selected gene aberrative in neuroblastoma [DAN]). More surprisingly, the niche displays mesenchymal traits such as expression of epithelial-mesenchymal-transition zinc finger E-box-binding protein 1 (ZEB1) transcription factor and smooth muscle actin. We found ZEB1 to be essential for neural stem cell survival in vitro. Proliferation within the niche progressively ceases around 13 weeks when the spinal cord reaches its final size, suggesting an active role in postnatal development. In addition to hippocampus and subventricular zone niches, adult spinal cord constitutes a third central nervous system stem cell niche with specific signaling, cellular, and structural characteristics that could possibly be manipulated to alleviate spinal cord traumatic and degenerative diseases.


Asunto(s)
Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismo , Nicho de Células Madre/citología , Nicho de Células Madre/metabolismo , Células Madre/citología , Actinas/metabolismo , Animales , Proliferación Celular , Proteína Doblecortina , Regulación del Desarrollo de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Transgénicos , Neuronas/citología , Neuronas/metabolismo , Células Madre/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc
10.
J Neurosci Res ; 87(2): 403-7, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18798282

RESUMEN

It has now been established that functional recovery after spinal cord injury (SCI) depends on several parameters, including animal strain. Here we demonstrate that rats from the same strain (Wistar) but from two independent commercial suppliers present different motor, sensory, and autonomic outcomes after a standard model of SCI, the so-called compression model. Recovery is correlated with the extension of the lesion, and we show that the vertebral canal diameter varies between the two suppliers. To substantiate this point, we carried out another set of experiments, with the so-called contusion model, which requires bone ablation and thus whose extension is not related to vertebral canal diameter. We show that there is no difference between the two suppliers. The purpose of our communication is to alert researchers on how crucial it is to control experimental parameters as closely as possible and to establish a standard for animal experiment in order to avoid unexpected biases.


Asunto(s)
Experimentación Animal/normas , Investigación Biomédica/normas , Traumatismos de la Médula Espinal/patología , Animales , Modelos Animales de Enfermedad , Ratas , Ratas Wistar , Recuperación de la Función
11.
Stem Cell Reports ; 12(5): 1159-1177, 2019 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-31031189

RESUMEN

Anamniotes, rodents, and young humans maintain neural stem cells in the ependymal zone (EZ) around the central canal of the spinal cord, representing a possible endogenous source for repair in mammalian lesions. Cell diversity and genes specific for this region are ill defined. A cellular and molecular resource is provided here for the mouse and human EZ based on RNA profiling, immunostaining, and fluorescent transgenic mice. This uncovered the conserved expression of 1,200 genes including 120 transcription factors. Unexpectedly the EZ maintains an embryonic-like dorsal-ventral pattern of expression of spinal cord developmental transcription factors (ARX, FOXA2, MSX1, and PAX6). In mice, dorsal and ventral EZ cells express Vegfr3 and are derived from the embryonic roof and floor plates. The dorsal EZ expresses a high level of Bmp6 and Gdf10 genes and harbors a subpopulation of radial quiescent cells expressing MSX1 and ID4 transcription factors.


Asunto(s)
Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Médula Espinal/metabolismo , Células Madre/metabolismo , Animales , Células Madre Embrionarias/citología , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Femenino , Humanos , Factor de Transcripción MSX1/genética , Factor de Transcripción MSX1/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Persona de Mediana Edad , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , ARN/metabolismo , Médula Espinal/citología , Nicho de Células Madre , Células Madre/citología , Adulto Joven
12.
Neurotherapeutics ; 15(3): 751-769, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29181770

RESUMEN

Spinal cord injuries (SCI) lead to major disabilities affecting > 2.5 million people worldwide. Major shortcomings in clinical translation result from multiple factors, including species differences, development of moderately predictive animal models, and differences in methodologies between preclinical and clinical studies. To overcome these obstacles, we first conducted a comparative neuroanatomical analysis of the spinal cord between mice, Microcebus murinus (a nonhuman primate), and humans. Next, we developed and characterized a new model of lateral spinal cord hemisection in M. murinus. Over a 3-month period after SCI, we carried out a detailed, longitudinal, behavioral follow-up associated with in vivo magnetic resonance imaging (1H-MRI) monitoring. Then, we compared lesion extension and tissue alteration using 3 methods: in vivo 1H-MRI, ex vivo 1H-MRI, and classical histology. The general organization and glial cell distribution/morphology in the spinal cord of M. murinus closely resembles that of humans. Animals assessed at different stages following lateral hemisection of the spinal cord presented specific motor deficits and spinal cord tissue alterations. We also found a close correlation between 1H-MRI signal and microglia reactivity and/or associated post-trauma phenomena. Spinal cord hemisection in M. murinus provides a reliable new nonhuman primate model that can be used to promote translational research on SCI and represents a novel and more affordable alternative to larger primates.


Asunto(s)
Modelos Animales de Enfermedad , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Investigación Biomédica Traslacional/métodos , Animales , Proteínas de Unión al Calcio , Cheirogaleidae , Proteínas de Unión al ADN/metabolismo , Conducta Exploratoria , Femenino , Estudios de Seguimiento , Lateralidad Funcional , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Proteínas de Microfilamentos , Microglía/patología , Persona de Mediana Edad , Fuerza Muscular/fisiología , Unión Neuromuscular/patología , Desempeño Psicomotor/fisiología , Especificidad de la Especie , Médula Espinal/patología , Factores de Tiempo , Tritio
13.
J Neurotrauma ; 35(24): 2924-2940, 2018 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-29877129

RESUMEN

Spinal cord injuries (SCI) are disastrous neuropathologies causing permanent disabilities. The availability of different strains of mice is valuable for studying the pathophysiological mechanisms involved in SCI. However, strain differences have a profound effect on spontaneous functional recovery after SCI. CX3CR1+/eGFP and Aldh1l1-EGFP mice that express green fluorescent protein in microglia/monocytes and astrocytes, respectively, are particularly useful to study glial reactivity. Whereas CX3CR1+/eGFP mice have C57BL/6 background, Aldh1l1-EGFP are in Swiss Webster background. We first assessed spontaneous functional recovery in CX3CR1+/eGFP and Aldh1l1-EGFP mice over 6 weeks after lateral spinal cord hemisection. Second, we carried out a longitudinal follow-up of lesion evolution using in vivo T2-weighted magnetic resonance imaging (MRI). Finally, we performed in-depth analysis of the spinal cord tissue using ex vivo T2-weighted MRI as well as detailed histology. We demonstrate that CX3CR1+/eGFP mice have improved functional recovery and reduced anxiety after SCI compared with Aldh1l1-EGFP mice. We also found a strong correlation between in vivo MRI, ex vivo MRI, and histological analyses of the injured spinal cord in both strain of mice. All three modalities revealed no difference in lesion extension and volume between the two strains of mice. Importantly, histopathological analysis identified decreased gliosis and increased serotonergic axons in CX3CR1+/eGFP compared with Aldh1l1-EGFP mice following SCI. These results thus suggest that the strain-dependent improved functional recovery after SCI may be linked with reduced gliosis and increased serotonergic innervation.


Asunto(s)
Gliosis/patología , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Estudios Longitudinales , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL
14.
Neuroreport ; 18(14): 1463-8, 2007 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-17712275

RESUMEN

In this study, we have grafted neural stem cells (NSCs) into the lumbar spinal cord of a mouse mutant that has a specific loss of motoneurons (progressive motor neuronopathy/pmn). A small number of grafted cells ( approximately 3000) increased the life span of the mice by 56%. The improved survival was accompanied by a rescue of host motoneurons, a stabilization in the weight and an increase in the size of the muscle fibers. The grafted NSCs were small and round and exhibited no neural markers, suggesting that they remained in an undifferentiated state. Thus grafting of NSCs in a mouse model with motoneuron degeneration exerts a neuroprotective effect.


Asunto(s)
Diferenciación Celular/fisiología , Chaperonas Moleculares/genética , Enfermedad de la Neurona Motora , Neuronas Motoras/fisiología , Médula Espinal/patología , Trasplante de Células Madre/métodos , Análisis de Varianza , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Mutantes , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/patología , Enfermedad de la Neurona Motora/cirugía , Fibras Musculares Esqueléticas/patología , Fosfopiruvato Hidratasa/metabolismo , Recuperación de la Función/fisiología , Médula Espinal/fisiopatología , Médula Espinal/cirugía , Factores de Tiempo
15.
Front Aging Neurosci ; 9: 227, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28785215

RESUMEN

Over the last decade, microglia have been acknowledged to be key players in central nervous system (CNS) under both physiological and pathological conditions. They constantly survey the CNS environment and as immune cells, in pathological contexts, they provide the first host defense and orchestrate the immune response. It is well recognized that under pathological conditions microglia have both sequential and simultaneous, beneficial and detrimental effects. Cell-specific transcriptomics recently became popular in Neuroscience field allowing concurrent monitoring of the expression of numerous genes in a given cell population. Moreover, by comparing two or more conditions, these approaches permit to unbiasedly identify deregulated genes and pathways. A growing number of studies have thus investigated microglial transcriptome remodeling over the course of neuropathological conditions and highlighted the molecular diversity of microglial response to different diseases. In the present work, we restrict our review to microglia obtained directly from in vivo samples and not cell culture, and to studies using whole-genome strategies. We first critically review the different methods developed to decipher microglia transcriptome. In particular, we compare advantages and drawbacks of flow cytometry and laser microdissection to isolate pure microglia population as well as identification of deregulated microglial genes obtained via RNA sequencing (RNA-Seq) vs. microarrays approaches. Second, we summarize insights obtained from microglia transcriptomes in traumatic brain and spinal cord injuries, pain and more chronic neurological conditions including Amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD) and Multiple sclerosis (MS). Transcriptomic responses of microglia in other non-neurodegenerative CNS disorders such as gliomas and sepsis are also addressed. Third, we present a comparison of the most activated pathways in each neuropathological condition using Gene ontology (GO) classification and highlight the diversity of microglia response to insults focusing on their pro- and anti-inflammatory signatures. Finally, we discuss the potential of the latest technological advances, in particular, single cell RNA-Seq to unravel the individual microglial response diversity in neuropathological contexts.

16.
Front Aging Neurosci ; 9: 230, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28769787

RESUMEN

Central nervous system (CNS) injury has been observed to lead to microglia activation and monocytes infiltration at the lesion site. Ex vivo diffusion magnetic resonance imaging (diffusion MRI or DWI) allows detailed examination of CNS tissues, and recent advances in clearing procedures allow detailed imaging of fluorescent-labeled cells at high resolution. No study has yet combined ex vivo diffusion MRI and clearing procedures to establish a possible link between microglia/monocytes response and diffusion coefficient in the context of spinal cord injury (SCI). We carried out ex vivo MRI of the spinal cord at different time-points after spinal cord transection followed by tetrahydrofuran based clearing and examined the density and morphology of microglia/monocytes using two-photon microscopy. Quantitative analysis revealed an early marked increase in microglial/monocytes density that is associated with an increase in the extension of the lesion measured using diffusion MRI. Morphological examination of microglia/monocytes somata at the lesion site revealed a significant increase in their surface area and volume as early as 72 hours post-injury. Time-course analysis showed differential microglial/monocytes response rostral and caudal to the lesion site. Microglia/monocytes showed a decrease in reactivity over time caudal to the lesion site, but an increase was observed rostrally. Direct comparison of microglia/monocytes morphology, obtained through multiphoton, and the longitudinal apparent diffusion coefficient (ADC), measured with diffusion MRI, highlighted that axonal integrity does not correlate with the density of microglia/monocytes or their somata morphology. We emphasize that differential microglial/monocytes reactivity rostral and caudal to the lesion site may thus coincide, at least partially, with reported temporal differences in debris clearance. Our study demonstrates that the combination of ex vivo diffusion MRI and two-photon microscopy may be used to follow structural tissue alteration. Lesion extension coincides with microglia/monocytes density; however, a direct relationship between ADC and microglia/monocytes density and morphology was not observed. We highlighted a differential rostro-caudal microglia/monocytes reactivity that may correspond to a temporal difference in debris clearance and axonal integrity. Thus, potential therapeutic strategies targeting microglia/monocytes after SCI may need to be adjusted not only with the time after injury but also relative to the location to the lesion site.

17.
Sci Rep ; 7(1): 9367, 2017 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-28839165

RESUMEN

Nociceptors are a particular subtype of dorsal root ganglion (DRG) neurons that detect noxious stimuli and elicit pain. Although recent efforts have been made to reveal the molecular profile of nociceptors in normal conditions, little is known about how this profile changes in pathological conditions. In this study we exploited laser capture microdissection to specifically collect individual injured and non-injured nociceptive DRG neurons and to define their gene profiling in rat spared nerve injury (SNI) model of neuropathic pain. We found minimal transcriptional changes in non-injured neurons at 7 days after SNI. In contrast, several novel transcripts were altered in injured nociceptors, and the global signature of these LCM-captured neurons differed markedly from that the gene expression patterns found previously using whole DRG tissue following SNI. Pathway analysis of the transcriptomic profile of the injured nociceptors revealed oxidative stress as a key biological process. We validated the increase of caspase-6 (CASP6) in small-sized DRG neurons and its functional role in SNI- and paclitaxel-induced neuropathic pain. Our results demonstrate that the identification of gene regulation in a specific population of DRG neurons (e.g., nociceptors) is an effective strategy to reveal new mechanisms and therapeutic targets for neuropathic pain from different origins.


Asunto(s)
Neuralgia/etiología , Nociceptores/metabolismo , Piel/lesiones , Nervios Espinales/lesiones , Transcriptoma , Animales , Biopsia , Caspasa 6/metabolismo , Biología Computacional , Modelos Animales de Enfermedad , Ganglios Espinales , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Ratones Noqueados , Neuralgia/metabolismo , Neuralgia/patología , Nociceptores/patología , Paclitaxel/efectos adversos , Ratas
18.
Front Mol Neurosci ; 10: 90, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28420963

RESUMEN

Neurons have inherent competence to regrow following injury, although not spontaneously. Spinal cord injury (SCI) induces a pronounced neuroinflammation driven by resident microglia and infiltrating peripheral macrophages. Microglia are the first reactive glial population after SCI and participate in recruitment of monocyte-derived macrophages to the lesion site. Both positive and negative influence of microglia and macrophages on axonal regeneration had been reported after SCI, raising the issue whether their response depends on time post-lesion or different lesion severity. We analyzed molecular alterations in microglia at several time-points after different SCI severities using RNA-sequencing. We demonstrate that activation of microglia is time-dependent post-injury but is independent of lesion severity. Early transcriptomic response of microglia after SCI involves proliferation and neuroprotection, which is then switched to neuroinflammation at later stages. Moreover, SCI induces an autologous microglial expression of astrocytic markers with over 6% of microglia expressing glial fibrillary acidic protein and vimentin from as early as 72 h post-lesion and up to 6 weeks after injury. We also identified the potential involvement of DNA damage and in particular tumor suppressor gene breast cancer susceptibility gene 1 (Brca1) in microglia after SCI. Finally, we established that BRCA1 protein is specifically expressed in non-human primate spinal microglia and is upregulated after SCI. Our data provide the first transcriptomic analysis of microglia at multiple stages after different SCI severities. Injury-induced microglia expression of astrocytic markers at RNA and protein levels demonstrates novel insights into microglia plasticity. Finally, increased microglia expression of BRCA1 in rodents and non-human primate model of SCI, suggests the involvement of oncogenic proteins after CNS lesion.

19.
PLoS Negl Trop Dis ; 11(9): e0005913, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28873445

RESUMEN

In the last decade, the number of emerging Flaviviruses described worldwide has increased considerably. Among them Zika virus (ZIKV) and Usutu virus (USUV) are African mosquito-borne viruses that recently emerged. Recently, ZIKV has been intensely studied due to major outbreaks associated with neonatal death and birth defects, as well as neurological symptoms. USUV pathogenesis remains largely unexplored, despite significant human and veterinary associated disorders. Circulation of USUV in Africa was documented more than 50 years ago, and it emerged in Europe two decades ago, causing massive bird mortality. More recently, USUV has been described to be associated with neurological disorders in humans such as encephalitis and meningoencephalitis, highlighting USUV as a potential health threat. The aim of this study was to evaluate the ability of USUV to infect neuronal cells. Our results indicate that USUV efficiently infects neurons, astrocytes, microglia and IPSc-derived human neuronal stem cells. When compared to ZIKV, USUV led to a higher infection rate, viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology related to USUV infection in order to anticipate the potential threat of USUV emergence.


Asunto(s)
Astrocitos/virología , Virus de la Encefalitis Japonesa (Subgrupo)/fisiología , Células-Madre Neurales/virología , Neuroglía/virología , Neuronas/virología , Tropismo Viral , Animales , Astrocitos/fisiología , Encéfalo/virología , Células Cultivadas , Virus de la Encefalitis Japonesa (Subgrupo)/crecimiento & desarrollo , Ratones , Células-Madre Neurales/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Virus Zika/crecimiento & desarrollo , Virus Zika/fisiología
20.
Brain ; 128(Pt 4): 854-66, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15689362

RESUMEN

Wallerian degeneration in the CNS and PNS consists of degradation and phagocytosis of axons and their myelin sheath distal to the site of injury. This process of degeneration, which requires an effective macrophage response, occurs rapidly in peripheral nerves but is slow in the CNS. Rapid Wallerian degeneration in peripheral nerves may contribute to subsequent axonal regeneration. We show that there is a marked increase in mRNA expression of three pro-inflammatory molecules, the chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha), and the cytokine interleukin-1beta (IL-1beta), in the mouse sciatic nerve but not in the spinal cord undergoing Wallerian degeneration. Neutralizing MCP-1, MIP-1alpha and IL-1beta in the lesioned sciatic nerve with function-blocking antibodies suppressed macrophage responses and myelin clearance. Injecting recombinant MCP-1, MIP-1alpha or IL-1beta into the normal, uninjured spinal cord triggered the expression of a number of chemokines and cytokines. Furthermore, injecting recombinant MCP-1/MIP-1alpha or IL-1beta into the dorsal column of the spinal cord undergoing Wallerian degeneration triggered rapid macrophage/microglial activation and myelin clearance. These findings provide direct evidence that MCP-1, MIP-1alpha and IL-1beta are important regulators of macrophage responses that lead to rapid myelin breakdown and clearance in Wallerian degeneration.


Asunto(s)
Citocinas/fisiología , Traumatismos de la Médula Espinal/metabolismo , Degeneración Walleriana/metabolismo , Animales , Quimiocina CCL2/fisiología , Quimiocina CCL3 , Quimiocina CCL4 , Citocinas/genética , Femenino , Expresión Génica , Interleucina-1/fisiología , Activación de Macrófagos/efectos de los fármacos , Proteínas Inflamatorias de Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Microinyecciones , Microscopía Electrónica , Vaina de Mielina/metabolismo , Fagocitosis/efectos de los fármacos , ARN Mensajero/genética , Proteínas Recombinantes/farmacología , Nervio Ciático/inmunología , Nervio Ciático/lesiones , Nervio Ciático/ultraestructura , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/patología , Degeneración Walleriana/etiología , Degeneración Walleriana/patología
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