RESUMEN
Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Fibrinógeno/antagonistas & inhibidores , Enfermedades Neurodegenerativas/inmunología , Animales , Epítopos , Humanos , Inflamación/inmunología , Ratones , RatasRESUMEN
Alzheimer's disease (AD), the most common form of dementia, is characterized by the abnormal accumulation of amyloid plaques and hyperphosphorylated tau aggregates, as well as microgliosis. Hemizygous missense variants in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) are associated with elevated risk for developing late-onset AD. These variants are hypothesized to result in loss of function, mimicking TREM2 haploinsufficiency. However, the consequences of TREM2 haploinsufficiency on tau pathology and microglial function remain unknown. We report the effects of partial and complete loss of TREM2 on microglial function and tau-associated deficits. In vivo imaging revealed that microglia from aged TREM2-haploinsufficient mice show a greater impairment in their injury response compared with microglia from aged TREM2-KO mice. In transgenic mice expressing mutant human tau, TREM2 haploinsufficiency, but not complete loss of TREM2, increased tau pathology. In addition, whereas complete TREM2 deficiency protected against tau-mediated microglial activation and atrophy, TREM2 haploinsufficiency elevated expression of proinflammatory markers and exacerbated atrophy at a late stage of disease. The differential effects of partial and complete loss of TREM2 on microglial function and tau pathology provide important insights into the critical role of TREM2 in AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer , Haploinsuficiencia , Hemicigoto , Glicoproteínas de Membrana , Microglía/metabolismo , Mutación Missense , Receptores Inmunológicos , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Microglía/patología , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismoRESUMEN
Frontotemporal dementia (FTD) is the second most common dementia before 65 years of age. Haploinsufficiency in the progranulin (GRN) gene accounts for 10% of all cases of familial FTD. GRN mutation carriers have an increased risk of autoimmune disorders, accompanied by elevated levels of tissue necrosis factor (TNF) α. We examined behavioral alterations related to obsessive-compulsive disorder (OCD) and the role of TNFα and related signaling pathways in FTD patients with GRN mutations and in mice lacking progranulin (PGRN). We found that patients and mice with GRN mutations displayed OCD and self-grooming (an OCD-like behavior in mice), respectively. Furthermore, medium spiny neurons in the nucleus accumbens, an area implicated in development of OCD, display hyperexcitability in PGRN knockout mice. Reducing levels of TNFα in PGRN knockout mice abolished excessive self-grooming and the associated hyperexcitability of medium spiny neurons of the nucleus accumbens. In the brain, PGRN is highly expressed in microglia, which are a major source of TNFα. We therefore deleted PGRN specifically in microglia and found that it was sufficient to induce excessive grooming. Importantly, excessive grooming in these mice was prevented by inactivating nuclear factor κB (NF-κB) in microglia/myeloid cells. Our findings suggest that PGRN deficiency leads to excessive NF-κB activation in microglia and elevated TNFα signaling, which in turn lead to hyperexcitability of medium spiny neurons and OCD-like behavior.
Asunto(s)
Demencia Frontotemporal/metabolismo , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Microglía/metabolismo , FN-kappa B/metabolismo , Trastorno Obsesivo Compulsivo/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Modelos Animales de Enfermedad , Femenino , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Granulinas , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Ratones , Ratones Noqueados , Microglía/patología , FN-kappa B/genética , Trastorno Obsesivo Compulsivo/genética , Trastorno Obsesivo Compulsivo/patología , Progranulinas , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Trombina/metabolismo , Animales , Axones/patología , Factores de Coagulación Sanguínea/química , Conexina 30 , Conexinas/genética , Enfermedades Desmielinizantes/etiología , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Fibrina/metabolismo , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Básica de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/toxicidad , Fragmentos de Péptidos/toxicidad , Poli I-C/toxicidad , Trombina/químicaRESUMEN
Insulin resistance is a key factor in the etiology of type 2 diabetes. Insulin-stimulated glucose uptake is mediated by the glucose transporter 4 (GLUT4), which is expressed mainly in skeletal muscle and adipose tissue. Insulin-stimulated translocation of GLUT4 from its intracellular compartment to the plasma membrane is regulated by small guanosine triphosphate hydrolases (GTPases) and is essential for the maintenance of normal glucose homeostasis. Here we show that the p75 neurotrophin receptor (p75(NTR)) is a regulator of glucose uptake and insulin resistance. p75(NTR) knockout mice show increased insulin sensitivity on normal chow diet, independent of changes in body weight. Euglycemic-hyperinsulinemic clamp studies demonstrate that deletion of the p75(NTR) gene increases the insulin-stimulated glucose disposal rate and suppression of hepatic glucose production. Genetic depletion or shRNA knockdown of p75(NTR) in adipocytes or myoblasts increases insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, overexpression of p75(NTR) in adipocytes decreases insulin-stimulated glucose transport. In adipocytes, p75(NTR) forms a complex with the Rab5 family GTPases Rab5 and Rab31 that regulate GLUT4 trafficking. Rab5 and Rab31 directly interact with p75(NTR) primarily via helix 4 of the p75(NTR) death domain. Adipocytes from p75(NTR) knockout mice show increased Rab5 and decreased Rab31 activities, and dominant negative Rab5 rescues the increase in glucose uptake seen in p75(NTR) knockout adipocytes. Our results identify p75(NTR) as a unique player in glucose metabolism and suggest that signaling from p75(NTR) to Rab5 family GTPases may represent a unique therapeutic target for insulin resistance and diabetes.
Asunto(s)
Glucosa/metabolismo , Homeostasis , Resistencia a la Insulina , Receptor de Factor de Crecimiento Nervioso/metabolismo , Adipocitos/metabolismo , Secuencia de Aminoácidos , Animales , Peso Corporal , Transportador de Glucosa de Tipo 4/metabolismo , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Células Musculares/metabolismo , Músculo Esquelético/citología , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptor de Factor de Crecimiento Nervioso/química , Receptor de Factor de Crecimiento Nervioso/deficiencia , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Microglia, the resident immune cells of the central nervous system, exist in either a "resting" state associated with physiological tissue surveillance or an "activated" state in neuroinflammation. We recently showed that ATP is the primary chemoattractor to tissue damage in vivo and elicits opposite effects on the motility of activated microglia in vitro through activation of adenosine A2A receptors. However, whether systemic inflammation affects microglial responses to tissue damage in vivo remains largely unknown. Using in vivo two-photon imaging of mice, we show that injection of lipopolysaccharide (LPS) at levels that can produce both clear neuroinflammation and some features of sepsis significantly reduced the rate of microglial response to laser-induced ablation injury in vivo. Under proinflammatory conditions, microglial processes initially retracted from the ablation site, but subsequently moved toward and engulfed the damaged area. Analyzing the process dynamics in 3D cultures of primary microglia indicated that only A2A , but not A1 or A3 receptors, mediate process retraction in LPS-activated microglia. The A2A receptor antagonists caffeine and preladenant reduced adenosine-mediated process retraction in activated microglia in vitro. Finally, administration of preladenant before induction of laser ablation in vivo accelerated the microglial response to injury following systemic inflammation. The regulation of rapid microglial responses to sites of injury by A2A receptors could have implications for their ability to respond to the neuronal death occurring under conditions of neuroinflammation in neurodegenerative disorders.
Asunto(s)
Corteza Cerebral/lesiones , Inflamación/fisiopatología , Microglía/fisiología , Neuroinmunomodulación/fisiología , Animales , Técnicas de Cultivo de Célula , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Femenino , Factores Inmunológicos/farmacología , Inflamación/tratamiento farmacológico , Inflamación/patología , Rayos Láser/efectos adversos , Lipopolisacáridos , Masculino , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/patología , Neuroinmunomodulación/efectos de los fármacos , Pirimidinas/farmacología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A3/metabolismo , Sepsis/tratamiento farmacológico , Sepsis/patología , Sepsis/fisiopatología , Triazoles/farmacologíaAsunto(s)
Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Fibrinógeno/metabolismo , Inflamación/metabolismo , Esclerosis Múltiple/metabolismo , Animales , Encéfalo/patología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Humanos , Esclerosis Múltiple/patologíaRESUMEN
OBJECTIVE: Vessels in brain arteriovenous malformations are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of brain arteriovenous malformation than the general population. We tested the hypothesis that vascular endothelial growth factor impairs vascular integrity in the Alk1-deficient brain through reduction of mural cell coverage. METHODS AND RESULTS: Adult Alk1(1f/2f) mice (loxP sites flanking exons 4-6) and wild-type mice were injected with 2×10(7) PFU adenovious-cre recombinase and 2×10(9) genome copies of adeno-associated virus-vascular endothelial growth factor to induce focal homozygous Alk1 deletion (in Alk1(1f/2f) mice) and angiogenesis. Brain vessels were analyzed 8 weeks later. Compared with wild-type mice, the Alk1-deficient brain had more fibrin (99±30×10(3) pixels/mm(2) versus 40±13×10(3); P=0.001), iron deposition (508±506 pixels/mm(2) versus 6±49; P=0.04), and Iba1(+) microglia/macrophage infiltration (888±420 Iba1(+) cells/mm(2) versus 240±104 Iba1(+); P=0.001) after vascular endothelial growth factor stimulation. In the angiogenic foci, the Alk1-deficient brain had more α-smooth muscle actin negative vessels (52±9% versus 12±7%, P<0.001), fewer vascular-associated pericytes (503±179/mm(2) versus 931±115, P<0.001), and reduced platelet-derived growth factor receptor-ß expression. CONCLUSIONS: Reduction of mural cell coverage in response to vascular endothelial growth factor stimulation is a potential mechanism for the impairment of vessel wall integrity in hereditary hemorrhagic telangiectasia type 2-associated brain arteriovenous malformation.
Asunto(s)
Receptores de Activinas Tipo I/deficiencia , Vasos Sanguíneos/enzimología , Encéfalo/irrigación sanguínea , Neovascularización Patológica , Pericitos/enzimología , Telangiectasia Hemorrágica Hereditaria/enzimología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Actinas/metabolismo , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo II , Animales , Becaplermina , Vasos Sanguíneos/patología , Dependovirus/genética , Modelos Animales de Enfermedad , Fibrina/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Hierro/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Microglía/patología , Pericitos/patología , Proteínas Proto-Oncogénicas c-sis/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Telangiectasia Hemorrágica Hereditaria/genética , Telangiectasia Hemorrágica Hereditaria/patología , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The first line of defense for the central nervous system (CNS) against injury or disease is provided by microglia. Microglia were long believed to stay in a dormant/resting state, reacting only to injury or disease. This view changed dramatically with the development of modern imaging techniques that allowed the study of microglial behavior in the intact brain over time, to reveal the dynamic nature of their responses. Over the past two decades, in vivo imaging using multiphoton microscopy has revealed numerous new functions of microglia in the developing, adult, aged, injured, and diseased CNS. As the most dynamic cells in the brain, microglia continuously contact all structures and cell types, such as glial and vascular cells, neuronal cell bodies, axons, dendrites, and dendritic spines, and are believed to play a central role in sculpting neuronal networks throughout life. Following trauma, or in neurodegenerative or neuroinflammatory diseases, microglial responses range from protective to harmful, underscoring the need to better understand their diverse roles and states in different pathological conditions. In this chapter, we introduce multiphoton microscopy and discuss recent advances in structural and functional imaging technologies that have expanded our toolbox to study microglial states and behaviors in new ways and depths. We also discuss relevant mouse models available for in vivo imaging studies of microglia and review how such studies are constantly refining our understanding of the multifaceted role of microglia in the healthy and diseased CNS.
Asunto(s)
Microglía , Microglía/metabolismo , Microglía/patología , Animales , Humanos , Microscopía de Fluorescencia por Excitación Multifotónica , Encéfalo/diagnóstico por imagen , Enfermedades Neuroinflamatorias/diagnóstico por imagen , Enfermedades Neuroinflamatorias/patología , Enfermedades Neurodegenerativas/diagnóstico por imagen , Enfermedades Neurodegenerativas/patologíaRESUMEN
Microglia, the resident immune cells of the central nervous system, are intimately involved in the brain's most basic processes, from pruning neural synapses during development to preventing excessive neuronal activity throughout life. Studies have reported both helpful and harmful roles for microglia at the blood-brain barrier (BBB) in the context of disease. However, less is known about microglia-endothelial cell interactions in the healthy brain. To investigate the role of microglia at a healthy BBB, we used the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia and analyzed the BBB ultrastructure, permeability, and transcriptome. Interestingly, we found that, despite their direct contact with endothelial cells, microglia are not necessary for the maintenance of BBB structure, function, or gene expression in the healthy brain. However, we found that PLX5622 treatment alters brain endothelial cholesterol metabolism. This effect was independent from microglial depletion, suggesting that PLX5622 has off-target effects on brain vasculature.
Asunto(s)
Barrera Hematoencefálica , Encéfalo , Colesterol , Células Endoteliales , Microglía , Microglía/metabolismo , Microglía/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Animales , Colesterol/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Ratones , Encéfalo/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Ratones Endogámicos C57BL , Masculino , Compuestos OrgánicosRESUMEN
We present a method to form an optical window in the mouse skull that spans millimeters and is stable for months without causing brain inflammation. This enabled us to repeatedly image blood flow in cortical capillaries of awake mice and determine long-range correlations in speed. We also repeatedly imaged dendritic spines, microglia and angioarchitecture, as well as used illumination to drive motor output via optogenetics and induce microstrokes via photosensitizers.
Asunto(s)
Cráneo/anatomía & histología , Animales , Velocidad del Flujo Sanguíneo , Cementos para Huesos , Isquemia Encefálica/fisiopatología , Corteza Cerebral/fisiología , Circulación Cerebrovascular/fisiología , Cerebro/anatomía & histología , Cerebro/fisiología , Mamíferos , Ratones , Microscopía Confocal/métodos , Cráneo/fisiología , Cráneo/cirugía , VigiliaRESUMEN
Several microglia-expressed genes have emerged as top risk variants for Alzheimer's disease (AD). Impaired microglial phagocytosis is one of the main proposed outcomes by which these AD-risk genes may contribute to neurodegeneration, but the mechanisms translating genetic association to cellular dysfunction remain unknown. Here we show that microglia form lipid droplets (LDs) upon exposure to amyloid-beta (Aß), and that their LD load increases with proximity to amyloid plaques in brains from human patients and the AD mouse model 5xFAD. LD formation is dependent upon age and disease progression and is more prominent in the hippocampus in mice and humans. Despite variability in LD load between microglia from male versus female animals and between cells from different brain regions, LD-laden microglia exhibited a deficit in Aß phagocytosis. Unbiased lipidomic analysis identified a substantial decrease in free fatty acids (FFAs) and a parallel increase in triacylglycerols (TAGs) as the key metabolic transition underlying LD formation. We demonstrate that DGAT2, a key enzyme for the conversion of FFAs to TAGs, promotes microglial LD formation, is increased in microglia from 5xFAD and human AD brains, and that inhibiting DGAT2 improved microglial uptake of Aß. These findings identify a new lipid-mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD.
RESUMEN
Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as "resting versus activated" and "M1 versus M2." This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.
Asunto(s)
MicroglíaRESUMEN
Deposition of the blood coagulation factor fibrinogen in the central nervous system is a hallmark of neurological diseases with blood-brain barrier disruption. We describe in vivo two-photon imaging of microglial responses and neuronal spine elimination to either intracortical microinjection of fibrinogen in healthy mice or to endogenously labeled fibrinogen deposits in Alzheimer's disease mice. This protocol allows the longitudinal study of glial and neuronal responses to blood proteins and can be used to test drug efficacy at the neurovascular interface. For complete details on the use and execution of this protocol, please refer to Davalos et al. (2012), Ryu et al. (2018), and Merlini et al. (2019).
Asunto(s)
Encéfalo/metabolismo , Espinas Dendríticas/metabolismo , Fibrinógeno/metabolismo , Microglía/metabolismo , Microscopía/métodos , Enfermedad de Alzheimer/metabolismo , Animales , Barrera Hematoencefálica , Modelos Animales de Enfermedad , Colorantes Fluorescentes/química , Ratones , FotonesRESUMEN
BACKGROUND: Glioblastoma (GBM) is the most aggressive primary brain tumor and has a dismal prognosis. Previously, we identified that junctional adhesion molecule A (JAM-A), a cell adhesion molecule, is highly elevated in human GBM cancer stem cells and predicts poor patient prognosis. While JAM-A is also highly expressed in other cells in the tumor microenvironment, specifically microglia and macrophages, how JAM-A expression in these cells affects tumor growth has yet to be determined. The goal of this study was to understand the role of microenvironmental JAM-A in mediating GBM growth. METHODS: Male and female wild-type (WT) and JAM-A-deficient mice were transplanted intracranially with the syngeneic glioma cell lines GL261 and SB28 and were assessed for differences in survival and microglial activation in tumors and in vitro. RNA-sequencing was performed to identify differentially regulated genes among all genotypes, and differences were validated in vitro and in vivo. RESULTS: We found that JAM-A-deficient female mice succumbed to GBM more quickly compared with WT females and JAM-A-deficient and male WT mice. Analysis of microglia in the tumors revealed that female JAM-A-deficient microglia were more activated, and RNA-sequencing identified elevated expression of Fizz1 and Ifi202b specifically in JAM-A-deficient female microglia. CONCLUSIONS: Our findings suggest that JAM-A functions to suppress pathogenic microglial activation in the female tumor microenvironment, highlighting an emerging role for sex differences in the GBM microenvironment and suggesting that sex differences extend beyond previously reported tumor cell-intrinsic differences.
Asunto(s)
Glioblastoma , Animales , Línea Celular Tumoral , Femenino , Glioblastoma/genética , Humanos , Molécula A de Adhesión de Unión , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía , Microambiente TumoralRESUMEN
Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury.
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Adenosina Trifosfato/metabolismo , Lesiones Encefálicas/metabolismo , Quimiotaxis/fisiología , Gliosis/metabolismo , Microglía/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/antagonistas & inhibidores , Animales , Apirasa/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Quimiotaxis/efectos de los fármacos , Conexinas/antagonistas & inhibidores , Conexinas/metabolismo , Gliosis/patología , Gliosis/fisiopatología , Proteínas Fluorescentes Verdes , Ratones , Ratones Transgénicos , Microglía/citología , Microglía/efectos de los fármacos , Fagocitosis/fisiología , Antagonistas del Receptor Purinérgico P2 , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Receptores Purinérgicos P2Y1 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Imaging the dynamic interactions between immune cells, glia, neurons and the vasculature in living rodents has revolutionized our understanding of physiological and pathological mechanisms of the CNS. Emerging microscopy and imaging technologies have enabled longitudinal tracking of structural and functional changes in a plethora of different cell types in the brain. The development of novel methods also allowed stable and longitudinal optical access to the spinal cord with minimum tissue perturbation. These important advances facilitated the application of in vivo imaging using two-photon microscopy for studies of the healthy, diseased, or injured spinal cord. Indeed, decoding the interactions between peripheral and resident cells with the spinal cord vasculature has shed new light on neuroimmune and vascular mechanisms regulating the onset and progression of neurological diseases. This review focuses on imaging studies of the interactions between the vasculature and peripheral immune cells or microglia, with emphasis on their contribution to neuroinflammation. We also discuss in vivo imaging studies highlighting the importance of neurovascular changes following spinal cord injury. Real-time imaging of blood-brain barrier (BBB) permeability and other vascular changes, perivascular glial responses, and immune cell entry has revealed unanticipated cellular mechanisms and novel molecular pathways that can be targeted to protect the injured or diseased CNS. Imaging the cell-cell interactions between the vasculature, immune cells, and neurons as they occur in real time, is a powerful tool both for testing the efficacy of existing therapeutic approaches, and for identifying new targets for limiting damage or enhancing the potential for repair of the affected spinal cord tissue.
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Neuroimagen/métodos , Médula Espinal , Animales , Barrera Hematoencefálica/patología , Médula Espinal/inmunología , Médula Espinal/patología , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/patologíaRESUMEN
Cerebrovascular alterations are a key feature of Alzheimer's disease (AD) pathogenesis. However, whether vascular damage contributes to synaptic dysfunction and how it synergizes with amyloid pathology to cause neuroinflammation and cognitive decline remain poorly understood. Here, we show that the blood protein fibrinogen induces spine elimination and promotes cognitive deficits mediated by CD11b-CD18 microglia activation. 3D molecular labeling in cleared mouse and human AD brains combined with repetitive in vivo two-photon imaging showed focal fibrinogen deposits associated with loss of dendritic spines independent of amyloid plaques. Fibrinogen-induced spine elimination was prevented by inhibiting reactive oxygen species (ROS) generation or genetic ablation of CD11b. Genetic elimination of the fibrinogen binding motif to CD11b reduced neuroinflammation, synaptic deficits, and cognitive decline in the 5XFAD mouse model of AD. Thus, fibrinogen-induced spine elimination and cognitive decline via CD11b link cerebrovascular damage with immune-mediated neurodegeneration and may have important implications in AD and related conditions.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Disfunción Cognitiva/metabolismo , Espinas Dendríticas/metabolismo , Fibrinógeno/metabolismo , Microglía/metabolismo , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/fisiología , Encéfalo/fisiopatología , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Disfunción Cognitiva/patología , Disfunción Cognitiva/fisiopatología , Espinas Dendríticas/patología , Modelos Animales de Enfermedad , Humanos , Imagenología Tridimensional , Ratones , Placa Amiloide/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In vivo imaging has revolutionized our understanding of biological processes in brain physiology and pathology. However, breathing-induced movement artifacts have impeded the application of this powerful tool in studies of the living spinal cord. Here we describe in detail a method to image stably and repetitively, using two-photon microscopy, the living spinal tissue in mice with dense fluorescent cells or axons, without the need for animal intubation or image post-processing. This simplified technique can greatly expand the application of in vivo imaging to study spinal cord injury, regeneration, physiology and disease.
Asunto(s)
Anestesia/métodos , Proteínas Fluorescentes Verdes/genética , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Confocal/métodos , Médula Espinal/citología , Coloración y Etiquetado/métodos , Acepromazina/farmacología , Anestésicos/farmacología , Animales , Axones/fisiología , Axones/ultraestructura , Capilares/citología , Capilares/fisiología , Movimiento Celular/fisiología , Ketamina/farmacología , Laminectomía/métodos , Ratones , Ratones Transgénicos , Microcirculación/citología , Microcirculación/fisiología , Microglía/citología , Microglía/fisiología , Procedimientos Neuroquirúrgicos/métodos , Fenómenos Fisiológicos Respiratorios/efectos de los fármacos , Médula Espinal/irrigación sanguínea , Técnicas Estereotáxicas/instrumentación , Uretano/farmacología , Xilazina/farmacologíaRESUMEN
Oligodendrocytes myelinate axons in the central nervous system and develop from oligodendrocyte precursor cells (OPCs) that must first migrate extensively during brain and spinal cord development. We show that OPCs require the vasculature as a physical substrate for migration. We observed that OPCs of the embryonic mouse brain and spinal cord, as well as the human cortex, emerge from progenitor domains and associate with the abluminal endothelial surface of nearby blood vessels. Migrating OPCs crawl along and jump between vessels. OPC migration in vivo was disrupted in mice with defective vascular architecture but was normal in mice lacking pericytes. Thus, physical interactions with the vascular endothelium are required for OPC migration. We identify Wnt-Cxcr4 (chemokine receptor 4) signaling in regulation of OPC-endothelial interactions and propose that this signaling coordinates OPC migration with differentiation.