RESUMO
The importance of mechanosensory transduction pathways in cellular signalling has prominently come to focus in the last decade with the discovery of the Piezo ion channel family. Mechanosignaling involving Piezo1 ion channels in the function of the heart and cardiovascular system has only recently been identified to have implications for cardiovascular physiology and pathophysiology, in particular for heart failure (i.e., hypertrophy or dilative cardiomyopathy). These results have emphasized the need for higher throughput methods to study single-cell cardiovascular mechanobiology with the aim of identifying new targets for therapeutic interventions and stimulating the development of new pharmacological agents. Here, we present a novel method to assess mechanosignaling in adherent cardiac cells (murine HL-1 cell line) using a combination of isotropic cell stretch application and simultaneous Ca2+ fluorescence readout with quantitative analysis. The procedure implements our IsoStretcher technology in conjunction with a single-cell- and population-based analysis of Ca2+ signalling by means of automated image registration, cell segmentation and analysis, followed by automated classification of single-cell responses. The method is particularly valuable for assessing the heterogeneity of populations with distinct cellular responses to mechanical stimulation and provides more user-independent unbiased drug response classifications.
Assuntos
Canais Iônicos , Mecanotransdução Celular , Camundongos , Animais , Canais Iônicos/metabolismo , Transdução de Sinais , Coração , Linhagem CelularRESUMO
OBJECTIVE: Mucosal T cells play a major role in inflammatory bowel disease (IBD). However, their immunometabolism during intestinal inflammation is poorly understood. Due to its impact on cellular metabolism and proinflammatory immune cell function, we here focus on the enzyme ATP citrate lyase (ACLY) in mucosal T cell immunometabolism and its relevance for IBD. DESIGN: ACLY expression and its immunometabolic impact on colitogenic T cell function were analysed in mucosal T cells from patients with IBD and in two experimental colitis models. RESULTS: ACLY was markedly expressed in colon tissue under steady-state conditions but was significantly downregulated in lamina propria mononuclear cells in experimental dextran sodium sulfate-induced colitis and in CD4+ and to a lesser extent in CD8+ T cells infiltrating the inflamed gut in patients with IBD. ACLY-deficient CD4+ T cells showed an impaired capacity to induce intestinal inflammation in a transfer colitis model as compared with wild-type T cells. Assessment of T cell immunometabolism revealed that ACLY deficiency dampened the production of IBD-relevant cytokines and impaired glycolytic ATP production but enriched metabolites involved in the biosynthesis of phospholipids and phosphatidylcholine. Interestingly, the short-chain fatty acid butyrate was identified as a potent suppressor of ACLY expression in T cells, while IL-36α and resolvin E1 induced ACLY levels. In a translational approach, in vivo administration of the butyrate prodrug tributyrin downregulated mucosal infiltration of ACLYhigh CD4+ T cells and ameliorated chronic colitis. CONCLUSION: ACLY controls mucosal T cell immunometabolism and experimental colitis. Therapeutic modulation of ACLY expression in T cells emerges as a novel strategy to promote the resolution of intestinal inflammation.
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Colite , Doenças Inflamatórias Intestinais , Linfócitos Intraepiteliais , Humanos , Animais , Linfócitos Intraepiteliais/metabolismo , ATP Citrato (pro-S)-Liase/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Colite/metabolismo , Inflamação/metabolismo , Butiratos , Mucosa Intestinal/metabolismo , Sulfato de Dextrana , Modelos Animais de DoençasRESUMO
Immune cell activity is a major factor for disease progression in inflammatory bowel diseases (IBD). Classifying the type and functional state of immune cells is therefore crucial in clinical diagnostics of IBD. Label-free optical technologies exploiting NADH and FAD autofluorescence, such as multiphoton microscopy, have been used to describe tissue morphology in healthy and inflamed colon samples. Nevertheless, a strategy for the identification of single immune cell subtypes within the tissue is yet to be developed. This work aims to initiate an understanding of autofluorescence changes depending on immune cell type and activation state. For this, NADH and FAD autofluorescence signals of different murine immune cell subtypes under native conditions, as well as upon in vitro stimulation and cell death, have been evaluated. Autofluorescence was assessed using flow cytometry and multiphoton microscopy. Our results reveal significantly increased NADH and FAD signals in innate immune cells compared to adaptive immune cells. This allowed identification of relative amounts of neutrophils and CD4+ T cells in mixed cell suspensions, by using NADH signals as a differentiation marker. Furthermore, in vitro stimulation significantly increased NADH and FAD autofluorescence in adaptive immune cells and macrophages. Cell death induced a significant drop in NADH autofluorescence, while FAD signals were hardly affected. Taken together, these results demonstrate the value of autofluorescence as a tool to characterize immune cells in different functional states, paving the way to the label-free clinical classification of IBD in the future.
Assuntos
Flavina-Adenina Dinucleotídeo , Doenças Inflamatórias Intestinais , Animais , Biomarcadores , Colo/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Doenças Inflamatórias Intestinais/diagnóstico , Camundongos , NAD/metabolismoRESUMO
BACKGROUND & AIMS: Intestinal fibrosis is a long-term complication in inflammatory bowel diseases (IBD) that frequently results in functional damage, bowel obstruction, and surgery. Interleukin (IL) 36 is a group of cytokines in the IL1 family with inflammatory effects. We studied the expression of IL36 and its receptor, interleukin 1 receptor like 2 (IL1RL2 or IL36R) in the development of intestinal fibrosis in human tissues and mice. METHODS: We obtained intestinal tissues from 92 patients with Crohn's disease (CD), 48 patients with ulcerative colitis, and 26 patients without inflammatory bowel diseases (control individuals). Tissues were analyzed by histology to detect fibrosis and by immunohistochemistry to determine the distribution of fibroblasts and levels of IL36R ligands. Human and mouse fibroblasts were incubated with IL36 or control medium, and transcriptome-wide RNA sequences were analyzed. Mice were given neutralizing antibodies against IL36R, and we studied intestinal tissues from Il1rl2-/- mice; colitis and fibrosis were induced in mice by repetitive administration of DSS or TNBS. Bone marrow cells were transplanted from Il1rl2-/- to irradiated wild-type mice and intestinal tissues were analyzed. Antibodies against IL36R were applied to mice with established chronic colitis and fibrosis and intestinal tissues were studied. RESULTS: Mucosal and submucosal tissue from patients with CD or ulcerative colitis had higher levels of collagens, including type VI collagen, compared with tissue from control individuals. In tissues from patients with fibrostenotic CD, significantly higher levels of IL36A were noted, which correlated with high numbers of activated fibroblasts that expressed α-smooth muscle actin. IL36R activation of mouse and human fibroblasts resulted in expression of genes that regulate fibrosis and tissue remodeling, as well as expression of collagen type VI. Il1rl2-/- mice and mice given injections of an antibody against IL36R developed less severe colitis and fibrosis after administration of DSS or TNBS, but bone marrow cells from Il1rl2-/- mice did not prevent induction of colitis and fibrosis. Injection of antibodies against IL36R significantly reduced established fibrosis in mice with chronic intestinal inflammation. CONCLUSION: We found higher levels of IL36A in fibrotic intestinal tissues from patients with IBD compared with control individuals. IL36 induced expression of genes that regulate fibrogenesis in fibroblasts. Inhibition or knockout of the IL36R gene in mice reduces chronic colitis and intestinal fibrosis. Agents designed to block IL36R signaling could be developed for prevention and treatment of intestinal fibrosis in patients with IBD.
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Colite Ulcerativa/metabolismo , Colágeno Tipo VI/metabolismo , Colo/patologia , Doença de Crohn/metabolismo , Interleucina-1/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Receptores de Interleucina-1/metabolismo , Actinas/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Estudos de Casos e Controles , Células Cultivadas , Colite/induzido quimicamente , Colite/patologia , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Sulfato de Dextrana , Fibroblastos/efeitos dos fármacos , Fibrose , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Interleucina-1/farmacologia , Ligantes , Camundongos , Camundongos Knockout , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/genética , Transdução de Sinais , Transcriptoma , Ácido TrinitrobenzenossulfônicoRESUMO
BACKGROUND & AIMS: It is not clear how regulation of T-cell function is altered during development of inflammatory bowel diseases (IBD). We studied the mechanisms by which geranylgeranyltransferase-mediated prenylation controls T-cell localization to the intestine and chronic inflammation. METHODS: We generated mice with T-cell-specific disruption of the geranylgeranyltransferase type I, beta subunit gene (Pggt1b), called Pggt1bΔCD4 mice, or the ras homolog family member A gene (Rhoa), called RhoaΔCD4 mice. We also studied mice with knockout of CDC42 or RAC1 and wild-type mice (controls). Intestinal tissues were analyzed by histology, multiphoton and confocal microscopy, and real-time polymerase chain reaction. Activation of CDC42, RAC1, and RHOA were measured with G-LISA, cell fractionation, and immunoblots. T cells and lamina propria mononuclear cells from mice were analyzed by flow cytometry or transferred to Rag1-/- mice. Mice were given injections of antibodies against integrin alpha4beta7 or gavaged with the RORC antagonist GSK805. We obtained peripheral blood and intestinal tissue samples from patients with and without IBD and analyzed them by flow cytometry. RESULTS: Pggt1bΔCD4 mice developed spontaneous colitis, characterized by thickening of the intestinal wall, edema, fibrosis, accumulation of T cells in the colon, and increased expression of inflammatory cytokines. Compared with control CD4+ T cells, PGGT1B-deficient CD4+ T cells expressed significantly higher levels of integrin alpha4beta7, which regulates their localization to the intestine. Inflammation induced by transfer of PGGT1B-deficient CD4+ T cells to Rag1-/- mice was blocked by injection of an antibody against integrin alpha4beta7. Lamina propria of Pggt1bΔCD4 mice had increased numbers of CD4+ T cells that expressed RORC and higher levels of cytokines produced by T-helper 17 cells (granulocyte-macrophage colony-stimulating factor, interleukin [IL]17A, IL17F, IL22, and tumor necrosis factor [TNF]). The RORC inverse agonist GSK805, but not antibodies against IL17A or IL17F, prevented colitis in Pggt1bΔCD4 mice. PGGT1B-deficient CD4+ T cells had decreased activation of RHOA. RhoAΔCD4 mice had a similar phenotype to Pggt1bΔCD4 mice, including development of colitis, increased numbers of CD4+ T cells in colon, increased expression of integrin alpha4beta7 by CD4+ T cells, and increased levels of IL17A and other inflammatory cytokines in lamina propria. T cells isolated from intestinal tissues from patients with IBD had significantly lower levels of PGGT1B than tissues from individuals without IBD. CONCLUSION: Loss of PGGT1B from T cells in mice impairs RHOA function, increasing CD4+ T-cell expression of integrin alpha4beta7 and localization to colon, resulting in increased expression of inflammatory cytokines and colitis. T cells isolated from gut tissues from patients with IBD have lower levels of PGGT1B than tissues from patients without IBD.
Assuntos
Alquil e Aril Transferases/deficiência , Quimiotaxia de Leucócito , Colite/enzimologia , Colo/enzimologia , Integrinas/metabolismo , Linfócitos T/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Imunidade Adaptativa , Alquil e Aril Transferases/genética , Animais , Estudos de Casos e Controles , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/patologia , Colo/imunologia , Colo/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/patologia , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTPAssuntos
Colite Ulcerativa/diagnóstico , Colonoscopia/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Colo/diagnóstico por imagem , Colo/efeitos dos fármacos , Colo/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Fluoresceína/administração & dosagem , Humanos , Mucosa Intestinal/diagnóstico por imagem , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Camundongos , Camundongos KnockoutAssuntos
Colite/diagnóstico por imagem , Colo/diagnóstico por imagem , Neoplasias do Colo/diagnóstico por imagem , Imagem Óptica/métodos , Técnicas Fotoacústicas , Ultrassonografia/métodos , Animais , Modelos Animais de Doenças , Processamento de Imagem Assistida por Computador , Camundongos , Valor Preditivo dos TestesRESUMO
The CAS3D image processing method intuitively applies a combination of Fourier space and real space 3D analysis algorithms to volumetric images of single skeletal muscle fiber Myosin II Second Harmonic Generation (SHG) XYZ image data. Our developed tool automatically quantifies the myofibrillar orientation in muscle samples by determining the cosine angle sum of intensity gradients in 3D (CAS3D) while determining the mean sarcomere length (SL) and sample orientation. The expected CAS3D values could be reproduced from ideal artificial data sets. Applied random noise in artificial images lowers the detected CAS3D value, and for noise levels below 20%, the correlation can be approximated by a linear function with a slope of -0.006 CAS3D/noise%. The deviations in SL and orientation detection were determined on ideal and noisy artificial data sets and were statistically indistinguishable from 0 (null hypothesis t-test P > 0.1). The software was applied to a previously published data set of single skeletal muscle fiber volumetric SHG image data from a rat intensive care unit (ICU) model of ventilator-induced diaphragm dysfunction (VIDD) with treatment regimens involving the small anti-inflammatory molecules BGP-15, vamorolone, or prednisolone. Our method reliably reproduced the results of the previous work and improved the standard deviation of the cosine angle sum detection in all sample groups from a mean of 0.03 to 0.008. This improvement is achieved by applying analysis algorithms to the whole volumetric images in 3D in contrast to the previously common method of slice-wise XY analysis.
Assuntos
Imageamento Tridimensional , Fibras Musculares Esqueléticas , Animais , Ratos , Imageamento Tridimensional/métodos , Algoritmos , SoftwareRESUMO
The pore size of biopolymer networks governs their mechanical properties and strongly impacts the behavior of embedded cells. Confocal reflection microscopy and second harmonic generation microscopy are widely used to image biopolymer networks; however, both techniques fail to resolve vertically oriented fibers. Here, we describe how such directionally biased data can be used to estimate the network pore size. We first determine the distribution of distances from random points in the fluid phase to the nearest fiber. This distribution follows a Rayleigh distribution, regardless of isotropy and data bias, and is fully described by a single parameter--the characteristic pore size of the network. The bias of the pore size estimate due to the missing fibers can be corrected by multiplication with the square root of the visible network fraction. We experimentally verify the validity of this approach by comparing our estimates with data obtained using confocal fluorescence microscopy, which represents the full structure of the network. As an important application, we investigate the pore size dependence of collagen and fibrin networks on protein concentration. We find that the pore size decreases with the square root of the concentration, consistent with a total fiber length that scales linearly with concentration.
Assuntos
Biopolímeros/química , Microscopia , Colágeno/química , Fibrina/química , PorosidadeRESUMO
Cell viability of many cell types strongly relies on their ability to adjust to mechanical conditions and alterations. Cellular mechanisms for sensing and responding to mechanical forces and pathophysiological variations in these processes have become an emerging research field in recent years. An important signaling molecule involved in mechanotransduction as in many cellular processes is Ca2+. New experimental methods to probe cellular Ca2+ signaling live under conditions of mechanical stimulation facilitate new insights into previously overlooked aspects of mechanical regulation of cells.Here, we describe a protocol for using Ca2+ imaging in combination with a cell stretching device, the IsoStretcher. Cells grown on elastic membranes can be isotopically stretched in-plane, and their intracellular Ca2+ level can be accessed online on the single cell level using fluorescent calcium indicator dyes. We show a protocol for functional screening of mechanosensitive ion channels and related drug screenings using BJ cells, a foreskin fibroblast cell line that strongly reacts to acute mechanical stimulation.
Assuntos
Fenômenos Mecânicos , Mecanotransdução Celular , Mecanotransdução Celular/fisiologia , Linhagem Celular , Transdução de Sinais , Canais Iônicos/metabolismo , Corantes Fluorescentes , Cálcio/metabolismoRESUMO
BACKGROUND: Closing mucosal defects to reach mucosal healing is an important goal of therapy in inflammatory bowel disease (IBD). Among other cells, monocyte-derived macrophages are centrally involved in such intestinal wound healing. We had previously demonstrated that the anti-α4ß7 integrin antibody vedolizumab blocks the recruitment of non-classical monocytes as biased progenitors of wound healing macrophages to the gut and delays wound healing. However, although important for the interpretation of disappointing results in recent phase III trials in IBD, the effects of the anti-ß7 antibody etrolizumab on wound healing are unclear so far. METHODS: We analyzed the expression of etrolizumab targets on human and mouse monocyte subsets by flow cytometry and assessed their function in adhesion and homing assays. We explored wound-associated monocyte recruitment dynamics with multi-photon microscopy and compared the effects of etrolizumab and vedolizumab surrogate (-s) antibodies on experimental wound healing and wound-associated macrophage abundance. Finally, we investigated wound healing macrophage signatures in the large intestinal transcriptome of patients with Crohn's disease treated with etrolizumab. RESULTS: Human and mouse non-classical monocytes expressed more αEß7 integrin than classical monocytes and were a target of etrolizumab-s, which blocked non-classical monocyte adhesion to MAdCAM-1 and E-Cadherin as well as gut homing in vivo. Intestinal wound healing was delayed on treatment with etrolizumab-s along with a reduction of peri-lesional wound healing macrophages. Wound healing macrophage signatures in the colon of patients with Crohn's disease were substantially down-regulated on treatment with etrolizumab, but not with placebo. CONCLUSIONS: Combined blockade of αEß7 and α4ß7 with etrolizumab seems to exceed the effect of anti-α4ß7 treatment on intestinal wound healing, which might help to inform further investigations to understand the recent observations in the etrolizumab phase III trial program.
Assuntos
Fármacos Gastrointestinais , Doenças Inflamatórias Intestinais , Integrinas , Macrófagos , Cicatrização , Animais , Humanos , Camundongos , Doença de Crohn/tratamento farmacológico , Doença de Crohn/imunologia , Doença de Crohn/patologia , Fármacos Gastrointestinais/imunologia , Fármacos Gastrointestinais/farmacologia , Fármacos Gastrointestinais/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Integrinas/antagonistas & inibidores , Integrinas/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/patologia , Cicatrização/efeitos dos fármacos , Cicatrização/imunologiaRESUMO
Deep learning (DL) shows notable success in biomedical studies. However, most DL algorithms work as black boxes, exclude biomedical experts, and need extensive data. This is especially problematic for fundamental research in the laboratory, where often only small and sparse data are available and the objective is knowledge discovery rather than automation. Furthermore, basic research is usually hypothesis-driven and extensive prior knowledge (priors) exists. To address this, the Self-Enhancing Multi-Photon Artificial Intelligence (SEMPAI) that is designed for multiphoton microscopy (MPM)-based laboratory research is presented. It utilizes meta-learning to optimize prior (and hypothesis) integration, data representation, and neural network architecture simultaneously. By this, the method allows hypothesis testing with DL and provides interpretable feedback about the origin of biological information in 3D images. SEMPAI performs multi-task learning of several related tasks to enable prediction for small datasets. SEMPAI is applied on an extensive MPM database of single muscle fibers from a decade of experiments, resulting in the largest joint analysis of pathologies and function for single muscle fibers to date. It outperforms state-of-the-art biomarkers in six of seven prediction tasks, including those with scarce data. SEMPAI's DL models with integrated priors are superior to those without priors and to prior-only approaches.
Assuntos
Inteligência Artificial , Aprendizado Profundo , Redes Neurais de Computação , Algoritmos , MúsculosRESUMO
OBJECTIVE: Muscle biomechanics is set by the spacing of repetitive striation patterns of individual sarcomeres within single muscle fibres of stacked myofibrils. Sarcomere lengths (SL) are rather unequally distributed than of equal distance. This non-uniformity may affect both, force production as well as passive-elastic deformation. However, online recording of SL during axially imposed strains is cumbersome due to a lack of compact technologies. METHODS: To fuse SL pattern recognition with restoration force assessments during quasi-static axial stretch, we implemented live tracking of SL distributions simultaneous to voice-coil actuated stretch and restoration force recordings in our MyoRobot 2.0 automated biomechatronics platform. Both were obtained online during stretch-relaxation cycles of murine single muscle fibres. RESULTS: Under quasi-static stretch conditions ( â¼ 1 µm/s fibre length changes), almost no apparent hysteresis was detected in single fibres. SL showed a non-uniform distribution. While mean SL varied between 2.6 µm and 3.4 µm upon 140% stretch, two populations of fibres were noticed: one showing a minor change in SL distribution with stretch, and one becoming more equally distributed upon stretch. CONCLUSION: A roughly 5% SL variability under rest either diminishes or remains almost unaltered upon elastic axial deformation. This may reflect differential impact of mostly extra-sarcomeric components to stretch in this stretch range. SIGNIFICANCE: The augmented functionality of the MyoRobot 2.0 towards online sarcomere analyses within single fibres shall provide a valuable tool for the muscle community to study the contribution of serial elastic and force producing elements in health and disease models.
Assuntos
Fibras Musculares Esqueléticas , Sarcômeros , Animais , Elasticidade , Camundongos , Contração Muscular , Relação Estrutura-AtividadeRESUMO
Group 3 innate lymphoid cells (ILC3s) are crucial mediators of immunity and epithelial barrier function during immune responses against extracellular bacteria. Here, we identify Interferon regulatory factor 1 (IRF-1), a transcription factor previously associated with type 1 immunity, as an essential regulator of intestinal ILC3 accumulation and effector cytokine production. We demonstrate that IRF-1 is upregulated in the context of infection with the enteropathogen Citrobacter rodentium and that its presence is central for anatomical containment and prevention of pathogen dissemination. We furthermore show that IRF-1 is required in order for intestinal ILC3s to produce large amounts of the protective effector cytokine IL-22 early in the course of infection. On a molecular level, our data indicate that IRF-1 controls ILC3 numbers and their activation by direct transcriptional regulation of the IL-12Rß1 chain, thereby allowing ILCs to physiologically respond to IL-23 stimulation.
Assuntos
Citrobacter rodentium , Infecções por Enterobacteriaceae , Citocinas , Humanos , Imunidade Inata , Fator Regulador 1 de Interferon/genética , Interleucina-23 , LinfócitosRESUMO
Inflammatory fibrotic tissue remodeling can lead to severe morbidity. Histopathology grading requires extraction of biopsies and elaborate tissue processing. Label-free optical technologies can provide diagnostic readout without preparation and artificial stainings and show potential for in vivo applications. Here, we present an integration of Raman spectroscopy (RS) and multiphoton microscopy for joint investigation of the bio-chemical composition and morphological features related to cellular components and connective tissue. Both modalities show that collagen signatures were significantly increased in a murine fibrosis model. Furthermore, autofluorescence signatures assigned to immune cells show high correlation with disease severity. RS indicates increased levels of elastin and lipids. Further, we investigated the effect of joint data sets on prediction performance in machine learning models. Although binary classification did not benefit from adding more features, multi-class classification was improved by integrated data sets.
Assuntos
Microscopia de Fluorescência por Excitação Multifotônica , Análise Espectral Raman , Animais , Tecido Conjuntivo , Pulmão , Aprendizado de Máquina , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Análise Espectral Raman/métodosRESUMO
Cancer cells facilitate tumor growth by creating favorable tumor micro-environments (TME), altering homeostasis and immune response in the extracellular matrix (ECM) of surrounding tissue. A potential factor that contributes to TME generation and ECM remodeling is the cytoskeleton-associated human death-associated protein kinase 1 (DAPK1). Increased tumor cell motility and de-adhesion (thus, promoting metastasis), as well as upregulated plasminogen-signaling, are shown when functionally analyzing the DAPK1 ko-related proteome. However, the systematic investigation of how tumor cells actively modulate the ECM at the tissue level is experimentally challenging since animal models do not allow direct experimental access while artificial in vitro scaffolds cannot simulate the entire complexity of tissue systems. Here, we used the chorioallantoic membrane (CAM) assay as a natural, collagen-rich tissue model in combination with all-optical experimental access by multiphoton microscopy (MPM) to study the ECM remodeling potential of colorectal tumor cells with and without DAPK1 in situ and even in vivo. This approach demonstrates the suitability of the CAM assay in combination with multiphoton microscopy for studying collagen remodeling during tumor growth. Our results indicate the high ECM remodeling potential of DAPK1 ko tumor cells at the tissue level and support our findings from proteomics.
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BACKGROUND: Clinical challenges in inflammatory bowel diseases require microscopic in vivo evaluation of inflammation. Here, label-free imaging holds great potential, and recently, our group demonstrated the advantage of using in vivo multiphoton endomicroscopy for longitudinal animal studies. This article extends our previous work by in-depth analysis of label-free tissue features in common colitis models quantified by the multiphoton colitis score (MCS). METHODS: Fresh mucosal tissues were evaluated from acute and chronic dextran sulfate sodium (DSS), TNBS, oxazolone, and transfer colitis. Label-free imaging was performed by using second harmonic generation and natural autofluorescence. Morphological changes in mucosal crypts, collagen fibers, and cellularity in the stroma were analyzed and graded. RESULTS: Our approach discriminated between healthy (mean MCSâ =â 2.5) and inflamed tissue (mean MCSâ >â 5) in all models, and the MCS was validated by hematoxylin and eosin scoring of the same samples (85.2% agreement). Moreover, specific characteristics of each phenotype were identified. While TNBS, oxazolone, and transfer colitis showed high cellularity in stroma, epithelial damage seemed specific for chronic, acute DSS and transfer colitis. Crypt deformations were mostly observed in acute DSS. CONCLUSIONS: Quantification of label-free imaging is promising for in vivo endoscopy. In the future, this could be valuable for monitoring of inflammatory pathways in murine models, which is highly relevant for the development of new inflammatory bowel disease therapeutics.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Camundongos , Animais , Sulfato de Dextrana , Oxazolona , Modelos Animais de Doenças , InflamaçãoRESUMO
High-definition endoscopy is one essential step in the initial diagnosis of inflammatory bowel disease (IBD) characterizing the extent and severity of inflammation, as well as discriminating ulcerative colitis (UC) from Crohn's disease (CD). Following general recommendations and national guidelines, individual risk stratification should define the appropriate surveillance strategy, biopsy protocol and frequency of endoscopies. Beside high-definition videoendoscopy the application of dyes applied via a spraying catheter is of additional diagnostic value with a higher detection rate of intraepithelial neoplasia (IEN). Virtual chromoendoscopy techniques (NBI, FICE, I-scan, BLI) should not be recommended as a single surveillance strategy in IBD, although newer data suggest a higher comparability to dye-based chromoendoscopy than previously assumed. First results of oral methylene blue formulation are promising for improving the acceptance rate of classical chromoendoscopy. Confocal laser endomicroscopy (CLE) is still an experimental but highly innovative endoscopic procedure with the potential to contribute to the detection of dysplastic lesions. Molecular endoscopy in IBD has taken application of CLE to a higher level and allows topical application of labeled probes, mainly antibodies, against specific target structures expressed in the tissue to predict response or failure to biological therapies. First pre-clinical and in vivo data from label-free multiphoton microscopy (MPM) are now available to characterize mucosal and submucosal inflammation on endoscopy in more detail. These new techniques now have opened the door to individualized and highly specific molecular imaging in IBD in the future and pave the path to personalized medicine approaches. The quality of evidence was stated according to the Oxford Center of evidence-based medicine (March 2009). For this review a Medline search up to January 2021 was performed using the words "inflammatory bowel disease," "ulcerative colitis," "crohn's disease," "chromoendoscopy," "high-definition endoscopy," "confocal laser endomicroscopy," "confocal laser microscopy," "molecular imaging," "multiphoton microscopy."
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Rationale: Structural remodeling or damage as a result of disease or injury is often not evenly distributed throughout a tissue but strongly depends on localization and extent of damaging stimuli. Skeletal muscle as a mechanically active organ can express signs of local or even systemic myopathic damage, necrosis, or repair. Conventionally, muscle biopsies (patients) or whole muscles (animal models) are mechanically sliced and stained to assess structural alterations histologically. Three-dimensional tissue information can be obtained by applying deep imaging modalities, e.g. multiphoton or light-sheet microscopy. Chemical clearing approaches reduce scattering, e.g. through matching refractive tissue indices, to overcome optical penetration depth limits in thick tissues. Methods: Here, we optimized a range of different clearing protocols. We find aqueous solution-based protocols employing (20-80%) 2,2'-thiodiethanol (TDE) to be advantageous over organic solvents (dibenzyl ether, cinnamate) regarding the preservation of muscle morphology, ease-of-use, hazard level, and costs. Results: Applying TDE clearing to a mouse model of local cardiotoxin (CTX)-induced muscle necrosis, a complete loss of myosin-II signals was observed in necrotic areas with little change in fibrous collagen or autofluorescence (AF) signals. The 3D aspect of myofiber integrity could be assessed, and muscle necrosis in whole muscle was quantified locally via the ratios of detected AF, forward- and backward-scattered Second Harmonic Generation (fSHG, bSHG) signals. Conclusion: TDE optical clearing is a versatile tool to study muscle architecture in conjunction with label-free multiphoton imaging in 3D in injury/myopathy models and might also be useful in studying larger biofabricated constructs in regenerative medicine.
Assuntos
Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Músculo Esquelético/metabolismo , Necrose/diagnóstico , Animais , Cardiotoxinas/farmacologia , Colágeno/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Imageamento Tridimensional/métodos , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Miofibrilas/metabolismo , Miosina Tipo II/metabolismo , Necrose/induzido quimicamente , Necrose/metabolismo , Compostos de Sulfidrila/farmacologiaRESUMO
The second harmonic generation (SHG) signal intensity sourced from skeletal muscle myosin II strongly depends on the polarization of the incident laser beam relative to the muscle fiber axis. This dependence is related to the second-order susceptibility χ((2)), which can be described by a single component ratio γ under generally assumed symmetries. We precisely extracted γ from SHG polarization dependence curves with an extended focal field model. In murine myofibrillar preparations, we have found two distinct polarization dependencies: With the actomyosin system in the rigor state, γ(rig) has a mean value of γ(rig) = 0.52 (SD = 0.04, n = 55); in a relaxed state where myosin is not bound to actin, γ(rel) has a mean value of γ(rel) = 0.24 (SD = 0.07, n = 70). We observed a similar value in an activated state where the myosin power stroke was pharmacologically inhibited using N-benzyl-p-toluene sulfonamide. In summary, different actomyosin states can be visualized noninvasively with SHG microscopy. Specifically, SHG even allows us to distinguish different actin-bound states of myosin II using γ as a parameter.