Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.322
Filtrar
1.
Curr Protoc ; 1(10): e256, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34610200

RESUMO

Understanding the kinetics and spatiotemporal interactions of living cells within the tissue environment requires real-time imaging. The introduction of two-photon microscopy has substantially boosted the power of live intravital imaging, making it possible to obtain information of individual cells in near-physiologic conditions within intact tissues nondestructively. Intravital imaging of the liver has proved useful in understanding its 3D structure, function, and dynamic cellular interactions. Recently we have shown that integrity of the blood-bile barrier in different physiologic and pathophysiologic conditions can be imaged in real time using intravital microscopy. Here we discuss the real-time intravital imaging method to visualize blood-bile barrier integrity in the murine liver. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Live imaging in the mouse liver Support Protocol: Monitoring vital signs of the mouse during live liver imaging Basic Protocol 2: Visualizing blood and bile transport using intravital microscopy.


Assuntos
Microscopia Intravital , Fígado , Animais , Bile , Diagnóstico por Imagem , Testes Diagnósticos de Rotina , Fígado/diagnóstico por imagem , Camundongos
2.
J Vis Exp ; (175)2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34661568

RESUMO

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery. After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.


Assuntos
Leucócitos , Neovascularização Fisiológica , Animais , Artéria Femoral , Membro Posterior , Microscopia Intravital , Camundongos
3.
J Vis Exp ; (175)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34633361

RESUMO

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.


Assuntos
Hemodinâmica , Microscopia Intravital , Animais , Cóclea , Cobaias , Camundongos , Microcirculação , Microscopia de Fluorescência
4.
Nat Commun ; 12(1): 5739, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34667166

RESUMO

Protein aggregates associated with neurodegenerative diseases have the ability to transmit to unaffected cells, thereby templating their own aberrant conformation onto soluble homotypic proteins. Proteopathic seeds can be released into the extracellular space, secreted in association with extracellular vesicles (EV) or exchanged by direct cell-to-cell contact. The extent to which each of these pathways contribute to the prion-like spreading of protein misfolding is unclear. Exchange of cellular cargo by both direct cell contact or via EV depends on receptor-ligand interactions. We hypothesized that enabling these interactions through viral ligands enhances intercellular proteopathic seed transmission. Using different cellular models propagating prions or pathogenic Tau aggregates, we demonstrate that vesicular stomatitis virus glycoprotein and SARS-CoV-2 spike S increase aggregate induction by cell contact or ligand-decorated EV. Thus, receptor-ligand interactions are important determinants of intercellular aggregate dissemination. Our data raise the possibility that viral infections contribute to proteopathic seed spreading by facilitating intercellular cargo transfer.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Vesículas Extracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Agregação Patológica de Proteínas/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope Viral/metabolismo , Adulto , Idoso , Encéfalo/patologia , Estudos de Casos e Controles , Linhagem Celular , Endocitose , Feminino , Humanos , Microscopia Intravital , Masculino , Pessoa de Meia-Idade , Príons/metabolismo , Agregação Patológica de Proteínas/patologia , Dobramento de Proteína , Proteínas tau/metabolismo
5.
Kidney Int ; 100(5): 978-980, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34688386

RESUMO

Podocyte loss is a key element underlying glomerulosclerosis. Various mechanisms have been proposed for cell loss. These include local hemodynamic effects and local stresses on cell and hemodynamic changes, which together may contribute to detachment of podocytes, leading to sclerosis. Elegant studies based on classic observations add state-of-the-art imaging, modeling, intravital microscopy, and ultrastructural geometry analyses and provide new insights into potential mechanisms for injury and loss of this key cell.


Assuntos
Glomerulosclerose Segmentar e Focal , Nefropatias , Podócitos , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Microscopia Intravital , Nefropatias/patologia , Podócitos/patologia , Esclerose/patologia
6.
Nat Commun ; 12(1): 5712, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588436

RESUMO

Animals make decisions under the principle of reward value maximization and surprise minimization. It is still unclear how these principles are represented in the brain and are reflected in behavior. We addressed this question using a closed-loop virtual reality system to train adult zebrafish for active avoidance. Analysis of the neural activity of the dorsal pallium during training revealed neural ensembles assigning rules to the colors of the surrounding walls. Additionally, one third of fish generated another ensemble that becomes activated only when the real perceived scenery shows discrepancy from the predicted favorable scenery. The fish with the latter ensemble escape more efficiently than the fish with the former ensembles alone, even though both fish have successfully learned to escape, consistent with the hypothesis that the latter ensemble guides zebrafish to take action to minimize this prediction error. Our results suggest that zebrafish can use both principles of goal-directed behavior, but with different behavioral consequences depending on the repertoire of the adopted principles.


Assuntos
Aprendizagem da Esquiva/fisiologia , Comportamento Animal/fisiologia , Neocórtex/fisiologia , Recompensa , Peixe-Zebra/fisiologia , Animais , Microscopia Intravital , Microscopia de Fluorescência por Excitação Multifotônica , Neocórtex/citologia , Redes Neurais de Computação , Neurônios/fisiologia , Estimulação Luminosa/métodos , Técnicas Estereotáxicas , Realidade Virtual
7.
Nat Commun ; 12(1): 5700, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588437

RESUMO

Bacterial biofilms are aggregates of surface-associated cells embedded in an extracellular polysaccharide (EPS) matrix, and are typically stationary. Studies of bacterial collective movement have largely focused on swarming motility mediated by flagella or pili, in the absence of a biofilm. Here, we describe a unique mode of collective movement by a self-propelled, surface-associated biofilm-like multicellular structure. Flavobacterium johnsoniae cells, which move by gliding motility, self-assemble into spherical microcolonies with EPS cores when observed by an under-oil open microfluidic system. Small microcolonies merge, creating larger ones. Microscopic analysis and computer simulation indicate that microcolonies move by cells at the base of the structure, attached to the surface by one pole of the cell. Biochemical and mutant analyses show that an active process drives microcolony self-assembly and motility, which depend on the bacterial gliding apparatus. We hypothesize that this mode of collective bacterial movement on solid surfaces may play potential roles in biofilm dynamics, bacterial cargo transport, or microbial adaptation. However, whether this collective motility occurs on plant roots or soil particles, the native environment for F. johnsoniae, is unknown.


Assuntos
Biofilmes , Flavobacterium/fisiologia , Locomoção , Simulação por Computador , Microscopia Intravital , Técnicas Analíticas Microfluídicas , Raízes de Plantas/microbiologia , Microbiologia do Solo , Imagem com Lapso de Tempo
8.
Methods Mol Biol ; 2388: 149-156, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34524670

RESUMO

The immune response in the liver is a highly dynamic process involving the recruitment of many types of immune cells. As a powerful imaging technique, intravital microscopy has been widely used for real-time observation and quantification of cell movements in living animals. Here we describe the use of an in vivo half-dissociated preparation method combined with intravital confocal microscopy to observe the dynamic activities of invariant natural killer T cells in the liver of CXCR6GFP/+ transgenic mice. We believe that this method will enable researchers to explore the dynamics of many other types of immune cells in the liver.


Assuntos
Células T Matadoras Naturais , Animais , Diagnóstico por Imagem , Microscopia Intravital , Fígado , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Receptores CXCR6
9.
Nat Commun ; 12(1): 5364, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508074

RESUMO

Ribosomes comprise a large (LSU) and a small subunit (SSU) which are synthesized independently in the nucleolus before being exported into the cytoplasm, where they assemble into functional ribosomes. Individual maturation steps have been analyzed in detail using biochemical methods, light microscopy and conventional electron microscopy (EM). In recent years, single particle analysis (SPA) has yielded molecular resolution structures of several pre-ribosomal intermediates. It falls short, however, of revealing the spatiotemporal sequence of ribosome biogenesis in the cellular context. Here, we present our study on native nucleoli in Chlamydomonas reinhardtii, in which we follow the formation of LSU and SSU precursors by in situ cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). By combining both positional and molecular data, we reveal gradients of ribosome maturation within the granular component (GC), offering a new perspective on how the liquid-liquid-phase separation of the nucleolus supports ribosome biogenesis.


Assuntos
Nucléolo Celular/metabolismo , Ribossomos/metabolismo , Nucléolo Celular/ultraestrutura , Chlamydomonas reinhardtii , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Microscopia Intravital/métodos , Organogênese , Ribossomos/ultraestrutura , Análise Espaço-Temporal
10.
Nat Commun ; 12(1): 5380, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508092

RESUMO

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.


Assuntos
Anáfase/genética , Cromatina/metabolismo , Reparo do DNA , Pontos de Checagem da Fase M do Ciclo Celular/genética , Rad51 Recombinase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Segregação de Cromossomos , DNA/biossíntese , Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Humanos , Microscopia Intravital , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo
11.
Nat Commun ; 12(1): 5363, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508093

RESUMO

The activity of epiphyseal growth plates, which drives long bone elongation, depends on extensive changes in chondrocyte size and shape during differentiation. Here, we develop a pipeline called 3D Morphometric Analysis for Phenotypic significance (3D MAPs), which combines light-sheet microscopy, segmentation algorithms and 3D morphometric analysis to characterize morphogenetic cellular behaviors while maintaining the spatial context of the growth plate. Using 3D MAPs, we create a 3D image database of hundreds of thousands of chondrocytes. Analysis reveals broad repertoire of morphological changes, growth strategies and cell organizations during differentiation. Moreover, identifying a reduction in Smad 1/5/9 activity together with multiple abnormalities in cell growth, shape and organization provides an explanation for the shortening of Gdf5 KO tibias. Overall, our findings provide insight into the morphological sequence that chondrocytes undergo during differentiation and highlight the ability of 3D MAPs to uncover cellular mechanisms that may regulate this process.


Assuntos
Condrócitos/fisiologia , Fator 5 de Diferenciação de Crescimento/metabolismo , Lâmina de Crescimento/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Diferenciação Celular , Proliferação de Células , Embrião de Mamíferos , Feminino , Fator 5 de Diferenciação de Crescimento/economia , Lâmina de Crescimento/citologia , Lâmina de Crescimento/diagnóstico por imagem , Imageamento Tridimensional , Microscopia Intravital , Camundongos Knockout , Modelos Animais , Tíbia/citologia , Tíbia/efeitos dos fármacos , Tíbia/crescimento & desenvolvimento , Microtomografia por Raio-X
12.
Clin Hemorheol Microcirc ; 79(1): 137-147, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34487026

RESUMO

BACKGROUND: Stroke, traumatic brain injury, or other forms of central nervous system (CNS) injury initiate a local inflammatory response. Compensatory anti-inflammatory pathways are activated to limit secondary damage due to inflammation. The associated release of immunosuppressing neuromodulators can result in system-wide immune dysregulation (CNS injury-induced immune-depression syndrome -CIDS). OBJECTIVE: To establish an experimental stroke model where CIDS can be studied by intravital microscopy (IVM). METHODS: We used the photothrombotic stroke (PTS) model in C57BL/6 mice and studied its effects on peripheral immunity following challenge with lipopolysaccharide (LPS). Leukocyte activation, as well as capillary perfusion of the microcirculation, were assessed using intestinal intravital microscopy (IVM). RESULTS: PTS caused a significant reduction in the number of adhering leukocytes in submucosal venules of the terminal ileum of mice challenged with LPS compared to LPS-challenged animals without stroke. Leukocyte rolling was also impacted by PTS in the submucosal venules. Following stroke, we also observed decreased mucosal functional capillary density (FCD). CONCLUSIONS: Our results suggest that PTS with subsequent LPS challenge poses as a viable model to further study CIDS using intravital microscopy of the intestinal microcirculation.


Assuntos
Endotoxemia , Animais , Adesão Celular , Modelos Animais de Doenças , Microscopia Intravital , Leucócitos , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação , Ratos , Ratos Endogâmicos Lew
13.
Nat Commun ; 12(1): 5660, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580289

RESUMO

Small Open Reading Frames (smORFs) coding for peptides of less than 100 amino-acids are an enigmatic and pervasive gene class, found in the tens of thousands in metazoan genomes. Here we reveal a short 80 amino-acid peptide (Pegasus) which enhances Wingless/Wnt1 protein short-range diffusion and signalling. During Drosophila wing development, Wingless has sequential functions, including late induction of proneural gene expression and wing margin development. Pegasus mutants produce wing margin defects and proneural expression loss similar to those of Wingless. Pegasus is secreted, and co-localizes and co-immunoprecipitates with Wingless, suggesting their physical interaction. Finally, measurements of fixed and in-vivo Wingless gradients support that Pegasus increases Wingless diffusion in order to enhance its signalling. Our results unveil a new element in Wingless signalling and clarify the patterning role of Wingless diffusion, while corroborating the link between small open reading frame peptides, and regulation of known proteins with membrane-related functions.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos/metabolismo , Asas de Animais/crescimento & desenvolvimento , Proteína Wnt1/metabolismo , Animais , Animais Geneticamente Modificados , Microscopia Intravital , Peptídeos/genética , Imagem com Lapso de Tempo
14.
PLoS One ; 16(8): e0255204, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34351959

RESUMO

Advances in intravital microscopy (IVM) have enabled the studies of cellular organization and dynamics in the native microenvironment of intact organisms with minimal perturbation. The abilities to track specific cell populations and monitor their interactions have opened up new horizons for visualizing cell biology in vivo, yet the success of standard fluorescence cell labeling approaches for IVM comes with a "dark side" in that unlabeled cells are invisible, leaving labeled cells or structures to appear isolated in space, devoid of their surroundings and lacking proper biological context. Here we describe a novel method for "filling in the void" by harnessing the ubiquity of extracellular (interstitial) fluid and its ease of fluorescence labelling by commonly used vascular and lymphatic tracers. We show that during routine labeling of the vasculature and lymphatics for IVM, commonly used fluorescent tracers readily perfuse the interstitial spaces of the bone marrow (BM) and the lymph node (LN), outlining the unlabeled cells and forming negative contrast images that complement standard (positive) cell labeling approaches. The method is simple yet powerful, offering a comprehensive view of the cellular landscape such as cell density and spatial distribution, as well as dynamic processes such as cell motility and transmigration across the vascular endothelium. The extracellular localization of the dye and the interstitial flow provide favorable conditions for prolonged Intravital time lapse imaging with minimal toxicity and photobleaching.


Assuntos
Meios de Contraste/química , Microscopia Intravital , Animais , Automação , Medula Óssea/diagnóstico por imagem , Feminino , Corantes Fluorescentes/química , Linfonodos/diagnóstico por imagem , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fluxo Sanguíneo Regional , Fatores de Tempo
15.
J Vis Exp ; (174)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34459808

RESUMO

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.


Assuntos
Coração , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Microscopia Intravital , Microscopia de Fluorescência
16.
Nat Commun ; 12(1): 5165, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34453049

RESUMO

Bariatric surgery improves both insulin sensitivity and secretion and can induce diabetes remission. However, the mechanisms and time courses of these changes, particularly the impact on ß cell function, are difficult to monitor directly. In this study, we investigated the effect of Vertical Sleeve Gastrectomy (VSG) on ß cell function in vivo by imaging Ca2+ dynamics in islets engrafted into the anterior eye chamber. Mirroring its clinical utility, VSG in mice results in significantly improved glucose tolerance, and enhanced insulin secretion. We reveal that these benefits are underpinned by augmented ß cell function and coordinated activity across the islet. These effects involve changes in circulating GLP-1 levels which may act both directly and indirectly on the ß cell, in the latter case through changes in body weight. Thus, bariatric surgery leads to time-dependent increases in ß cell function and intra-islet connectivity which are likely to contribute to diabetes remission.


Assuntos
Cálcio/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/cirurgia , Células Secretoras de Insulina/metabolismo , Animais , Cirurgia Bariátrica , Glicemia/metabolismo , Diabetes Mellitus/diagnóstico por imagem , Feminino , Gastrectomia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Insulina/metabolismo , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estômago/cirurgia
17.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445151

RESUMO

Subarachnoid hemorrhage (SAH) is associated with acute and delayed cerebral ischemia. We suggested spasms of pial arterioles as a possible mechanism; however, it remained unclear whether and how pial microvasospasms (MVSs) induce cerebral ischemia. Therefore, we used in vivo deep tissue imaging by two-photon microscopy to investigate MVSs together with the intraparenchymal microcirculation in a clinically relevant murine SAH model. Male C57BL/6 mice received a cranial window. Cerebral vessels and leukocytes were labelled with fluorescent dyes and imaged by in vivo two-photon microscopy before and three hours after SAH induced by filament perforation. After SAH, a large clot formed around the perforation site at the skull base, and blood distributed along the perivascular space of the middle cerebral artery up to the cerebral cortex. Comparing the cerebral microvasculature before and after SAH, we identified three different patterns of constrictions: pearl string, global, and bottleneck. At the same time, the volume of perfused intraparenchymal vessels and blood flow velocity in individual arterioles were significantly reduced by more than 60%. Plugging of capillaries by leukocytes was observed but infrequent. The current study demonstrates that perivascular blood is associated with spasms of pial arterioles and that these spasms result in a significant reduction in cortical perfusion after SAH. Thus, the pial microvasospasm seems to be an important mechanism by which blood in the subarachnoid space triggers cerebral ischemia after SAH. Identifying the mechanisms of pial vasospasm may therefore result in novel therapeutic options for SAH patients.


Assuntos
Encéfalo/irrigação sanguínea , Leucócitos/patologia , Microvasos/patologia , Hemorragia Subaracnóidea/patologia , Vasoespasmo Intracraniano/patologia , Animais , Encéfalo/patologia , Circulação Cerebrovascular , Microscopia Intravital , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica
19.
Methods Mol Biol ; 2350: 145-156, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331284

RESUMO

Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment-the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.


Assuntos
Imunofluorescência/métodos , Microscopia Intravital/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Algoritmos , Animais , Linhagem Celular , Análise de Dados , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Intravital/instrumentação , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação
20.
Sci Rep ; 11(1): 14986, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294763

RESUMO

Irreversible electroporation (IRE) is a non-thermal tissue ablative technology that has emerging applications in surgical oncology and regenerative surgery. To advance its therapeutic usefulness, it is important to understand the mechanisms through which IRE induces cell death and the role of the innate immune system in mediating subsequent regenerative repair. Through intravital imaging of the liver in mice, we show that IRE produces distinctive tissue injury features, including delayed yet robust recruitment of neutrophils, consistent with programmed necrosis. IRE treatment converts the monocyte/macrophage balance from pro-inflammatory to pro-reparative populations, and depletion of neutrophils inhibits this conversion. Reduced generation of pro-reparative Ly6CloF4/80hi macrophages correlates with lower numbers of SOX9+ hepatic progenitor cells in areas of macrophage clusters within the IRE injury zone. Our findings suggest that neutrophils play an important role in promoting the development of pro-reparative Ly6Clo monocytes/macrophages at the site of IRE injury, thus establishing conditions of regenerative repair.


Assuntos
Regeneração Hepática , Fígado/diagnóstico por imagem , Macrófagos/metabolismo , Neutrófilos/metabolismo , Animais , Antígenos Ly , Eletroporação , Feminino , Microscopia Intravital , Fígado/imunologia , Masculino , Camundongos , Medicina Regenerativa , Fatores de Transcrição SOX9/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...