RESUMO
Fluid flow shear stresses are strong regulators for directing the organization of vascular networks. Knowledge of structural and flow dynamics information within complex vasculature is essential for tuning the vascular organization within engineered tissues, by manipulating flows. However, reported investigations of vascular organization and their associated flow dynamics within complex vasculature over time are limited, due to limitations in the available physiological pre-clinical models, and the optical inaccessibility and aseptic nature of these models. Here, we developed laser speckle contrast imaging (LSCI) and side-stream dark field microscopy (SDF) systems to map the vascular organization, spatio-temporal blood flow fluctuations as well as erythrocytes movements within individual blood vessels of developing chick embryo, cultured within an artificial eggshell system. By combining imaging data and computational simulations, we estimated fluid flow shear stresses within multiscale vasculature of varying complexity. Furthermore, we demonstrated the LSCI compatibility with bioengineered perfusable muscle tissue constructs, fabricated via molding techniques. The presented application of LSCI and SDF on perfusable tissues enables us to study the flow perfusion effects in a non-invasive fashion. The gained knowledge can help to use fluid perfusion in order to tune and control multiscale vascular organization within engineered tissues.
Assuntos
Circulação Sanguínea , Vasos Sanguíneos/fisiologia , Imagem Óptica/métodos , Engenharia Tecidual/métodos , Animais , Vasos Sanguíneos/diagnóstico por imagem , Vasos Sanguíneos/crescimento & desenvolvimento , Embrião de Galinha , Eritrócitos/fisiologia , Imagem Multimodal/métodos , Músculos/irrigação sanguínea , Músculos/diagnóstico por imagem , Neovascularização FisiológicaRESUMO
BACKGROUND: The glycocalyx is an extracellular layer lining the lumen of the vascular endothelium, protecting the endothelium from shear stress and atherosclerosis and contributes to coagulation, immune response and microvascular perfusion. The GlycoCheck system estimates glycocalyx' thickness in vessels under the tongue from perfused boundary region (PBR) and microvascular perfusion (red blood cell (RBC) filling) via a camera and dedicated software. OBJECTIVES: Evaluating reproducibility and influence of examination conditions on measurements with the GlycoCheck system. METHODS: Open, randomised, controlled study including 42 healthy smokers investigating day-to-day, side-of-tongue, inter-investigator variance, intraclass-correlation (ICC) and influence of examination conditions at intervals from 0-180 minutes on PBR and RBC filling. RESULTS: Mean (SD) age was 24.9 (6.1) years, 52% were male. There was no significant intra- or inter-investigator variation for PBR or RBC filling nor for PBR for side-of-tongue. A small day-to-day variance was found for PBR (0.012µm, p = 0.007) and RBC filling (0.003%, p = 0.005) and side-of-tongue, RBC filling (0.025%, p = 0.009). ICC was modest but highly improved by increasing measurements. Small significant influence of cigarette smoking (from 40-180 minutes), high calorie meal intake and coffee consumption was found. The latter two peaking immediately and tapering off but remained significant up to 180 minutes, highest PBR changes for the three being 0.042µm (p<0.05), 0.183µm (p<0.001) and 0.160µm (p<0.05) respectively. CONCLUSIONS: Measurements with the GlycoCheck system have a moderate reproducibility, but highly increases with multiple measurements and a small day-to-day variability. Smoking, meal and coffee intake had effects up to 180 minutes, abstinence is recommended at least 180 minutes before GlycoCheck measurements. Future studies should standardise conditions during measurements.
Assuntos
Doenças Cardiovasculares/diagnóstico , Técnicas de Diagnóstico Cardiovascular/instrumentação , Endotélio Vascular/diagnóstico por imagem , Microvasos/diagnóstico por imagem , Soalho Bucal/irrigação sanguínea , Adolescente , Adulto , Doenças Cardiovasculares/fisiopatologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiopatologia , Eritrócitos/fisiologia , Feminino , Glicocálix/fisiologia , Humanos , Masculino , Microcirculação/fisiologia , Microvasos/citologia , Microvasos/fisiopatologia , Soalho Bucal/diagnóstico por imagem , Reprodutibilidade dos Testes , Fumantes , Software , Adulto JovemRESUMO
Many useful therapeutic bio-effects can be generated using ultrasound-induced cavitation. However, cavitation is also capable of causing unwanted cellular and vascular damage, which should be monitored to ensure treatment safety. In this work, the unique opportunity provided by passive acoustic mapping (PAM) to quantify cavitation dose across an entire volume of interest during therapy is utilised to provide setup-independent measures of spatially localised cavitation dose. This spatiotemporally quantifiable cavitation dose is then related to the level of cellular damage generated. The cavitation-mediated destruction of equine red blood cells mixed with one of two types of cavitation nuclei at a variety of concentrations is investigated. The blood is placed within a 0.5-MHz ultrasound field and exposed to a range of peak rarefactional pressures up to 2 MPa, with 50 to 50,000 cycle pulses maintaining a 5% duty cycle. Two co-planar linear arrays at 90° to each other are used to generate 400-µm-resolution frequency domain robust capon beamforming PAM maps, which are then used to generate estimates of cavitation dose. A relationship between this cavitation dose and the levels of haemolysis generated was found which was comparable regardless of the applied acoustic pressure, pulse length, cavitation agent type or concentration used. PAM was then used to monitor cellular damage in multiple locations within a tissue phantom simultaneously, with the damage-cavitation dose relationship being similar for the two experimental models tested. These results lay the groundwork for this method to be applied to other measures of safety, allowing for improved ultrasound monitoring of cavitation-based therapies.
Assuntos
Eritrócitos/fisiologia , Terapia por Ultrassom/efeitos adversos , Terapia por Ultrassom/métodos , Acústica , Animais , Hemólise , Cavalos , Modelos AnimaisRESUMO
BACKGROUND: Mild hemolysis is difficult to determinate by traditional methods, and its role in Gilbert's syndrome (GS) is unclear. The main aims were to inspect the erythrocyte (RBC) survival in GS by using Levitt's carbon monoxide (CO) breath test and to assess its contribution to unconjugated hyperbilirubinemia. METHODS: Fifty subjects with GS and 1 with type-II Crigler-Najjar syndrome (CN2) received RBC lifespan measurement with Levitt's CO breath test. Mean RBC lifespan was compared with normal referral value. Correlations of serum total bilirubin (TB) with RBC lifespan, blood panel data, demographic factors, and uridine diphosphate glucuronosyltransferase (UGT1A1) mutation load were calculated by Spearman analysis. Susceptibility factors for mild hemolysis were analyzed by multivariate regression analysis. RESULTS: The mean RBC lifespan of the GS subjects was significantly shorter than the normal reference value (95.4â±â28.9 days vs 126 days; tâ=â-7.504, Pâ<â.01), with 30.0% below the lower limit of the normal reference range (75 days). The RBC lifespan of the participant with CN2 was 82 days. Serum TB correlated positively with UGT1A1 mutation load (γâ=â0.281, Pâ=â.048), hemoglobin (γâ=â.359, Pâ=â.010) and hematocrit (γâ=â0.365, Pâ=â.010), but negatively with RBC lifespan (γâ=â-0.336, Pâ=â.017). No significant susceptibility factors for mild hemolysis were found. CONCLUSIONS: The results indicate that mild hemolysis indeed, exists in a portion of patients with GS and might serve as an important contributor to unconjugated hyperbilirubinemia in addition to UGT1A1 polymorphism. Further studies on the mechanism and the potential risks in various medical treatments might be wanted.
Assuntos
Monóxido de Carbono/análise , Doença de Gilbert/complicações , Hemólise , Hiperbilirrubinemia/etiologia , Adulto , Testes Respiratórios/instrumentação , Eritrócitos/fisiologia , Feminino , Humanos , Hiperbilirrubinemia/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
Red blood cells (RBCs) transport oxygen to tissues and remove carbon dioxide. Diffuse optical flowmetry (DOF) assesses deep tissue RBC dynamics by measuring coherent fluctuations of multiply scattered near-infrared light intensity. While classical DOF measurements empirically correlate with blood flow, they remain far-removed from light scattering physics and difficult to interpret in layered media. To advance DOF measurements closer to the physics, here we introduce an interferometric technique, surmounting challenges of bulk motion to apply it in awake humans. We reveal two measurement dimensions: optical phase, and time-of-flight (TOF), the latter with 22 picosecond resolution. With this multidimensional data, we directly confirm the unordered, or Brownian, nature of optically probed RBC dynamics typically assumed in classical DOF. We illustrate how incorrect absorption assumptions, anisotropic RBC scattering, and layered tissues may confound classical DOF. By comparison, our direct method enables accurate and comprehensive assessment of blood flow dynamics in humans.
Assuntos
Circulação Cerebrovascular/fisiologia , Modelos Biológicos , Córtex Pré-Frontal/fisiologia , Espalhamento de Radiação , Animais , Emulsões/administração & dosagem , Emulsões/farmacocinética , Eritrócitos/fisiologia , Emulsões Gordurosas Intravenosas/administração & dosagem , Estudos de Viabilidade , Humanos , Injeções Intravenosas , Interferometria/instrumentação , Interferometria/métodos , Luz , Camundongos , Método de Monte Carlo , Fosfolipídeos/administração & dosagem , Fosfolipídeos/farmacocinética , Córtex Pré-Frontal/irrigação sanguínea , Fluxo Sanguíneo Regional/fisiologia , Óleo de Soja/administração & dosagem , Óleo de Soja/farmacocinéticaRESUMO
Ferrite nanoparticles (NPs) have gained attention in biomedicine due to their many potential applications, such as targeted drug delivery, their use as contrast agents for magnetic resonance imaging and oncological treatments. The information about the risk effects of ferrite NPs in human blood cells is, however, scarce. To assess their potential toxicity, in vitro studies were carried out with magnetite and zinc, nickel and nickelzinc ferrites NPs at different concentrations (50, 100 and 200⯵g·ml-1). The toxicity of the ferrite NPs was evaluated in humans by determining red blood hemolysis, by measuring the content of total proteins, and by assaying catalase and glutathione-S-transferase activities. Our results show that nickelzinc ferrite lead to hemolysis, and that magnetite, zinc and nickelzinc ferrites increase glutathione-S-transferase activity. No significant changes in human peripheral blood mononuclear cells viability were observed after the treatment with the four different ferrite NPs in vitro.
Assuntos
Eritrócitos/efeitos dos fármacos , Compostos Férricos/toxicidade , Óxido Ferroso-Férrico/toxicidade , Leucócitos Mononucleares/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Níquel/toxicidade , Compostos de Zinco/toxicidade , Adulto , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Eritrócitos/fisiologia , Glutationa Transferase/metabolismo , Humanos , MasculinoRESUMO
INTRODUCTION: Optical coherence tomography (OCT) is a novel high-resolution imaging technique capable of visualizing in vivo structures at a resolution of ~10 µm. We have developed specialized OCT-based approaches that quantify diameter, speed, and flow rate in human cutaneous microvessels. In this study, we hypothesized that OCT-based microvascular assessments would possess comparable levels of reliability when compared with those derived using conventional laser Doppler flowmetry (LDF). METHODS: Speckle decorrelation images (OCT) and red blood cell flux (LDF) measures were collected from adjacent forearm skin locations on 2 d (48 h apart), at baseline, and after a 30-min rapid local heating protocol (30°C-44°C) in eight healthy young individuals. OCT postprocessing quantified cutaneous microvascular diameter, speed, flow rate, and density (vessel recruitment) within a region of interest, and data were compared between days. RESULTS: Forearm skin LDF (13 ± 4 to 182 ± 31 AU, P < 0.05) and OCT-derived diameter (41.8 ± 6.6 vs 64.5 ± 6.9 µm), speed (68.4 ± 9.5 vs 89.0 ± 7.3 µm·s), flow rate (145.0 ± 60.6 vs 485 ± 132 pL·s), and density (9.9% ± 4.9% vs 45.4% ± 5.9%) increased in response to local heating. The average OCT-derived microvascular flow response (pL·s) to heating (234% increase) was lower (P < 0.05) than the LDF-derived change (AU) (1360% increase). Pearson correlation was significant for between-day local heating responses in terms of OCT flow (r = 0.93, P < 0.01), but not LDF (P = 0.49). Bland-Altman analysis revealed that between-day baseline OCT-derived flow rates were less variable than LDF-derived flux. CONCLUSIONS: Our findings indicate that OCT, which directly visualizes human microvessels, not only allows microvascular quantification of diameter, speed, flow rate, and vessel recruitment but also provides outputs that are highly reproducible. OCT is a promising novel approach that enables a comprehensive assessment of cutaneous microvascular structure and function in humans.
Assuntos
Microcirculação/fisiologia , Pele/irrigação sanguínea , Tomografia de Coerência Óptica/métodos , Arteríolas/anatomia & histologia , Arteríolas/fisiologia , Velocidade do Fluxo Sanguíneo/fisiologia , Eritrócitos/fisiologia , Antebraço , Humanos , Reprodutibilidade dos TestesAssuntos
Anemia Ferropriva , Eritrócitos , Ferro da Dieta , Ferro , Anemia Ferropriva/tratamento farmacológico , Anemia Ferropriva/metabolismo , Anemia Ferropriva/fisiopatologia , Criança , Côte d'Ivoire , Eritrócitos/química , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritrócitos/fisiologia , Hepcidinas/metabolismo , Hepcidinas/fisiologia , Humanos , Ferro/sangue , Ferro/metabolismo , Ferro/fisiologia , Ferro da Dieta/administração & dosagem , Ferro da Dieta/metabolismo , Ferro da Dieta/uso terapêuticoRESUMO
Transfusion of packed red blood cells (PRBC) to patients in critical states is often accompanied by post-transfusion complications. This may be related with disturbance of properties of PRBC and their membranes during long-term storage in the hemopreservative solution. The purpose of our work is the study of transformation of morphology, membranes stiffness and nanostructure for assessment of PRBC quality, in vitro. By atomic force microscopy we studied the transformation of cell morphology, the appearance of topological nanodefects of membranes and by atomic force spectroscopy studied the change of membrane stiffness during 40 days of storage of PRBC. It was shown that there is a transition period (20-26 days), in which we observed an increase in the Young's modulus of the membranes 1.6-2 times and transition of cells into irreversible forms. This process was preceded by the appearance of topological nanodefects of membranes. These parameters can be used for quality assessment of PRBC and for improvement of transfusion rules.
Assuntos
Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Forma Celular , Elasticidade , Eritrócitos/citologia , Eritrócitos/ultraestrutura , Armazenamento de Medicamentos , Eritrócitos/fisiologia , Voluntários Saudáveis , Humanos , Microscopia de Força Atômica , Federação Russa , Fatores de TempoRESUMO
BACKGROUND: Accurate prediction of 24-hour energy expenditure (24EE) relies on knowing body composition, in particular fat-free mass (FFM), the largest determinant of 24EE. FFM is closely correlated with hematologic volumes: blood volume (BV), red cell mass (RCM), and plasma volume (PV). However, it is unknown whether predicted hematologic volumes, based on easily collected variables, can improve 24EE prediction. OBJECTIVE: The aim was to develop and validate equations to predict 24EE based on predicted BV, RCM, and PV and to compare the accuracy and agreement with models developed from FFM and with the Mifflin-St Jeor equation, which is recommended for clinical use by the Academy of Nutrition and Dietetics. DESIGN: Participants had body composition measured by underwater weighing and 24EE by respiratory chamber. BV, RCM, and PV were calculated from five published equations. PARTICIPANTS/SETTING: Native American and white men and women were studied (n=351). Participants were healthy adults aged 18 to 49 years from the Phoenix, AZ, metropolitan area. MAIN OUTCOME MEASURE: Accuracy to within ±10% of measured 24EE and agreement by Bland-Altman analysis. STATISTICAL ANALYSIS: Regression models to predict 24EE from hematologic and body composition variables were developed in half the dataset and validated in the other half. RESULTS: Hematologic volumes were all strongly correlated with FFM in both men and women (r≥0.94). Whereas the accuracy of FFM alone was 69%, four hematologic volumes were individually more accurate (75% to 78%) in predicting 24EE. Equations based on hematologic volumes plus demographics had mean prediction errors comparable to those based on body composition plus demographics; although the Mifflin-St Jeor had modestly better mean prediction error, body composition, hematologic, and Mifflin-St Jeor models all had similar accuracy (approximately 80%). CONCLUSIONS: Prediction equations based on hematologic volumes were developed, validated, and found to be comparable to Mifflin-St Jeor and body composition models in this population of healthy adults.
Assuntos
Volume Sanguíneo/fisiologia , Composição Corporal , Metabolismo Energético , Modelos Teóricos , Adiposidade , Adolescente , Adulto , Índice de Massa Corporal , Calorimetria Indireta , Eritrócitos/fisiologia , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Haemorheology has been long identified as an early biomarker of a wide range of diseases, especially cardiovascular diseases. This study investigates for the first time the suitability of Photoplethysmography (PPG) as a non-invasive diagnostic method for haemorheological changes. The sensitivity of both PPG components (AC and DC) to changes in haemorheology were rigorously investigated in an in vitro experimental setup that mimics the human circulation. A custom-made reflectance PPG sensor, a pressure transducer and an ultrasonic Doppler flowmeter were used to map changes in flow dynamics and optical responses in an arterial model. The study investigated the effect of shear rates by varying fluid pumping frequencies using 4 set-points and the effect of clot formation using a chemical trigger. Both PPGAC amplitudes and PPGDC levels showed significant (p < 0.001) changes during the increase in shear rates and an immediate change after thromboplastin activation. The findings highlight that PPG has the potential to be used as a simple non-invasive method for the detection of blood characteristics, including disaggregation, radial migration and cross-linking fibrin formations. Such capability will enable the assessment of the effects of clotting-activators and anticoagulants (including non-pharmacological methods) and might aid in the early non-invasive assessment of cardiovascular pathologies.
Assuntos
Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , Hemorreologia , Fotopletismografia/instrumentação , Fotopletismografia/métodos , Animais , Coagulação Sanguínea , Eritrócitos/fisiologia , CavalosRESUMO
In recent years the development of portable microscopes, which enable the noninvasive bedside evaluation of the sublingual microcirculation in critically ill patients, has expanded the clinical research on this level of the cardiovascular system. Several semi-quantitative scores have been defined in order to provide researchers with a standardized framework for the offline assessment of the microcirculation status. Among those, space-time diagrams (STDs) constitute an established method for obtaining an estimate of the red blood cells (RBCs) flow velocity in capillaries. However, STDs have the drawback of being time-consuming, inherently subjective, and difficult to manage when the flow is not regular. OBJECTIVE: In this work we propose an automated method for calculating erythrocyte flow speed, aiming to provide a fast and objective tool for the evaluation of peripheral blood perfusion. APPROACH: The proposed method exploits an image segmentation module for estimating the positions of candidate flowing cells. A multi-object tracking algorithm based on Kalman filters analyzes and matches the positions corresponding to specific erythrocytes within consecutive frames. Thus, the output of the filter enables to estimate the displacement of each cell, yielding their instantaneous speed. MAIN RESULTS: The method has been validated against the results obtained by the manual analysis of STDs, proving a good agreement for speeds up to 300 µm s-1. At higher speeds, RBC tracking becomes unstable due to the currently limited video acquisition rate (25 Hz) of state-of-the-art devices, that makes the matching between objects appearing in consecutive frames very challenging.
Assuntos
Eritrócitos/citologia , Processamento de Imagem Assistida por Computador , Microcirculação , Microscopia , Velocidade do Fluxo Sanguíneo , Eritrócitos/fisiologia , HumanosRESUMO
Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs. In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection on hepatic hemodynamics. This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug's effect on inflammation by quantifying its effect on blood flow.
Assuntos
Microscopia Intravital/métodos , Leishmania donovani , Leishmaniose/fisiopatologia , Fígado/irrigação sanguínea , Animais , Eritrócitos/parasitologia , Eritrócitos/patologia , Eritrócitos/fisiologia , Feminino , Hemodinâmica , Processamento de Imagem Assistida por Computador/métodos , Leishmaniose/sangue , Fígado/parasitologia , Circulação Hepática , Camundongos , Camundongos Endogâmicos C57BLRESUMO
We have developed a new fiber-optic system that combines diffuse reflectance spectroscopy (DRS) and laser Doppler Flowmetry (LDF) for a multi-modal assessment of the microcirculation. Quantitative data is achieved with an inverse Monte Carlo algorithm based on an individually adaptive skin model. The output parameters are calculated from the model and given in absolute units: hemoglobin oxygen saturation (%), red blood cell (RBC) tissue fraction (%), and the speed resolved RBC perfusion separated into three speed regions; 0-1mm/s, 1-10mm/s and above 10mm/s (% mm/s). The aim was to explore microcirculatory parameters using the new optical method, integrating DRS and LDF in a joint skin model, during local heating of the dorsal foot and venous and arterial occlusion of the forearm in 23 healthy subjects (age 20-28years). There were differences in the three speed regions in regard to blood flow changes due to local heating, where perfusion for high speeds increased the most. There was also a high correlation between changes in oxygenation and changes in perfusion for higher speeds. Oxygen saturation at baseline was 44% on foot, increasing to 83% at plateau after heating. The larger increase in perfusion for higher speeds than for lower speeds together with the oxygenation increase during thermal provocation, shows a local thermoregulatory blood flow in presumably arteriolar dermal vessels. In conclusion, there are improved possibilities to assess microcirculation using integrated DRS and LDF in a joint skin model by enabling both oxygenation and speed resolved blood flow assessment simultaneously and in the same skin site. Output parameters in absolute units may also yield new insights about the microcirculatory system.
Assuntos
Fluxometria por Laser-Doppler/métodos , Microcirculação/fisiologia , Oxigênio/sangue , Espectrofotometria/métodos , Adulto , Algoritmos , Velocidade do Fluxo Sanguíneo , Eritrócitos/fisiologia , Feminino , Tecnologia de Fibra Óptica , Temperatura Alta , Humanos , Masculino , Modelos Cardiovasculares , Método de Monte Carlo , Fluxo Sanguíneo Regional , Pele/irrigação sanguínea , Adulto JovemRESUMO
Extracellular trypanosomes can cause a wide range of diseases and pathological complications in a broad range of mammalian hosts. One common feature of trypanosomosis is the occurrence of anemia, caused by an imbalance between erythropoiesis and red blood cell clearance of aging erythrocytes. In murine models for T. brucei trypanosomosis, anemia is marked by a very sudden non-hemolytic loss of RBCs during the first-peak parasitemia control, followed by a short recovery phase and the subsequent gradual occurrence of an ever-increasing level of anemia. Using a newly developed quantitative pHrodo based in vitro erythrophagocytosis assay, combined with FACS-based ex vivo and in vivo results, we show that activated liver monocytic cells and neutrophils as well as activated splenic macrophages are the main cells involved in the occurrence of the early-stage acute anemia. In addition, we show that trypanosomosis itself leads to a rapid alteration of RBC membrane stability, priming the cells for accelerated phagocytosis.
Assuntos
Anemia/fisiopatologia , Eritrócitos/fisiologia , Medições Luminescentes/métodos , Fagocitose/fisiologia , Tripanossomíase/complicações , Tripanossomíase/fisiopatologia , Anemia/etiologia , Animais , Citometria de Fluxo , Concentração de Íons de Hidrogênio , Fígado/citologia , Fígado/metabolismo , Macrófagos/fisiologia , Camundongos , Monócitos/fisiologia , Parasitemia/fisiopatologia , Baço/fisiologiaRESUMO
Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.
Assuntos
Linhagem da Célula/fisiologia , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/fisiologia , Fosfolipases A2 Secretórias/metabolismo , Animais , Apoptose/fisiologia , Plaquetas/metabolismo , Plaquetas/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Eritrócitos/metabolismo , Eritrócitos/fisiologia , Citometria de Fluxo/métodos , Genitália Masculina/metabolismo , Genitália Masculina/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Timócitos/metabolismo , Timócitos/fisiologiaRESUMO
Turbidity variation is one of the major limitations in Raman spectroscopy for quantifying blood components, such as glucose, non-invasively. To overcome this limitation, we have developed a Raman scattering simulation using a photon-cell interactive Monte Carlo (pciMC) model that tracks photon migration in both the extra- and intracellular spaces without relying on the macroscopic scattering phase function and anisotropy factor. The interaction of photons at the plasma-cell boundary of randomly oriented three-dimensionally biconcave red blood cells (RBCs) is modeled using geometric optics. The validity of the developed pciMCRaman was investigated by comparing simulation and experimental results of Raman spectroscopy of glucose level in a bovine blood sample. The scattering of the excitation laser at a wavelength of 785 nm was simulated considering the changes in the refractive index of the extracellular solution. Based on the excitation laser photon distribution within the blood, the Raman photon derived from the hemoglobin and glucose molecule at the Raman shift of 1140 cm(-1) = 862 nm was generated, and the photons reaching the detection area were counted. The simulation and experimental results showed good correlation. It is speculated that pciMCRaman can provide information about the ability and limitations of the measurement of blood glucose level.
Assuntos
Análise Química do Sangue/métodos , Animais , Glicemia , Bovinos , Forma Celular , Simulação por Computador , Eritrócitos/fisiologia , Modelos Biológicos , Método de Monte Carlo , Análise Espectral Raman/métodosRESUMO
Based on the effects of n-3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on reduction of blood viscosity, we theorized that PUFA could improve aerobic performance by increasing oxygen supply to tissues. Twenty male subjects were randomly divided into two groups (n = 10): a fish oil group (FG) and a control (CG). Maximal oxygen uptake and oxygen uptake during submaximal exercise were measured using a cycle ergometer. For 8 weeks, the FG then ingested capsules containing 3.6 g/day of EPA-rich fish oil, while the CG took 3.6 g/day of a medium-chain triglyceride. After supplementation, erythrocyte EPA and DHA in the FG were significantly increased. In the FG, a negative linear correlation was detected in the change between erythrocyte EPA and whole oxygen uptake during submaximal exercise pre- and post-supplementation. The present study showed that EPA-rich fish oil supplementation improves exercise economy in humans.
Assuntos
Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Óleos de Peixe/administração & dosagem , Esforço Físico/efeitos dos fármacos , Administração Oral , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Eritrócitos/química , Eritrócitos/efeitos dos fármacos , Eritrócitos/fisiologia , Exercício Físico/fisiologia , Óleos de Peixe/química , Humanos , Masculino , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Esforço Físico/fisiologia , Adulto JovemRESUMO
A number of adherent mycoplasmas have developed highly complex polar structures that are involved in diverse aspects of the biology of these microorganisms and play a key role as virulence factors by promoting adhesion to host cells in the first stages of infection. Attachment activity of mycoplasma cells has been traditionally investigated by determining their hemadsorption ability to red blood cells and it is a distinctive trait widely examined when characterizing the different mycoplasma species. Despite the fact that protocols to qualitatively determine the hemadsorption or hemagglutination of mycoplasmas are straightforward, current methods when investigating hemadsorption at the quantitative level are expensive and poorly reproducible. By using flow cytometry, we have developed a procedure to quantify rapidly and accurately the hemadsorption activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve erythrocyte and mycoplasma cells by their different size and fluorescence. This method is very reproducible and permits the kinetic analysis of the obtained data and a precise hemadsorption quantification based on standard binding parameters such as the dissociation constant K d. The procedure we developed could be easily implemented in a standardized assay to test the hemadsorption activity of the growing number of clinical isolates and mutant strains of different mycoplasma species, providing valuable data about the virulence of these microorganisms.
Assuntos
Hemadsorção/fisiologia , Mycoplasma/metabolismo , Mycoplasma/fisiologia , Benzotiazóis , Biomassa , Diaminas , Eritrócitos/metabolismo , Eritrócitos/fisiologia , Citometria de Fluxo/métodos , Fluorescência , Hemadsorção/genética , Hemaglutinação/genética , Hemaglutinação/fisiologia , Mutação/genética , Mycoplasma/genética , Compostos Orgânicos/metabolismo , Quinolinas , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Although Doppler optical coherence tomography techniques have enabled the imaging of blood flow in mid-sized vessels in biological tissues, the generation of velocity maps of capillary networks remains a challenge. To better understand the origin and information content of the Doppler signal from small vessels and limitations of such measurements, we used joint spectral and time domain optical coherence tomography to monitor the flow in a model, semitransparent microchannel device. The results obtained for Intralipid, whole blood, as well as separated red blood cells indicate that the technique is suitable to record velocity profiles in vitro, in a range of microchannel configurations.