Reproducibility of Non-Invasive Assessment of Skin Endothelial Function Using Laser Doppler Flowmetry and Laser Speckle Contrast Imaging

Background

Endothelial dysfunction precedes atherosclerosis. Vasodilation induced by acetylcholine (ACh) is a specific test of endothelial function. Reproducibility of laser techniques such as laser-Doppler-flowmetry (LDF) and Laser-speckle-contrast-imaging (LSCI) to detect ACh vasodilation is debated and results expressions lack standardization. We aimed to study at a 7-day interval (i) the inter-subject reproducibility, (ii) the intra-subjects reproducibility, and (iii) the effect of the results expressions over variability.

Methods and Results

Using LDF and LSCI simultaneously, we performed two different ACh-iontophoresis protocols. The maximal ACh vasodilation (peak-ACh) was expressed as absolute or normalized flow or conductance values. Inter-subject reproducibility was expressed as coefficient of variation (inter-CV,%). Intra-subject reproducibility was expressed as within subject coefficients of variation (intra-CV,%), and intra-class correlation coefficients (ICC). Fifteen healthy subjects were included. The inter-subject reproducibility of peak-ACh depended upon the expression of the results and ranged from 55% to 162% for LDF and from 17% to 83% for LSCI. The intra-subject reproducibility (intra-CV/ICC) of peak-ACh was reduced when assessed with LSCI compared to LDF no matter how the results were expressed and whatever the protocol used. The highest intra-subject reproducibility was found using LSCI. It was 18.7%/0.87 for a single current stimulation (expressed as cutaneous vascular conductance) and 11.4%/0.61 for multiple current stimulations (expressed as absolute value).

Conclusion

ACh-iontophoresis coupled with LSCI is a promising test to assess endothelial function because it is reproducible, safe, and non-invasive. N°: {"type":"clinical-trial","attrs":{"text":"NCT01664572","term_id":"NCT01664572"}}NCT01664572.     

How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles similar to how laser Doppler analyzes frequency shifts to determine particle speed. Because it can be used to monitor the movement of red blood cells, LSCI has become a popular tool for measuring blood flow in tissues such as the retina, skin, and brain. It has become especially useful in neuroscience where blood flow changes during physiological events like functional activation, stroke, and spreading depolarization can be quantified. LSCI is also attractive because it provides excellent spatial and temporal resolution while using inexpensive instrumentation that can easily be combined with other imaging modalities. Here we show how to build a LSCI setup and demonstrate its ability to monitor blood flow changes in the brain during an animal experiment.Download video file.^{(31M, mov)}     

Laser Flow Cytofluorometric Analysis of Htc Cells Synchronized With Hydroxyurea, Nocodazole and Aphidicolin

The technique of laser flow cytofluorometry has been used to monitor the arrival in G₁ and the subsequent progression through the cell cycle of HTC cells accumulated in metaphase with colcemid alone or after treatment with hydroxyurea and Nocodazole. Under the experimental conditions used in this study, the latter procedure gives much better results, avoiding in particular the extensive formation of micronucleated cells. Aphidicolin, an inhibitor of DNA polymerase, in combination with Nocodazole, provides a useful method to tightly synchronize these cells at the G₁/S border.     

Alteration of Blood Flow in a Venular Network by Infusion of Dextran 500: Evaluation with a Laser Speckle Contrast Imaging System

This study examined the effect of dextran-induced RBC aggregation on the venular flow in microvasculature. We utilized the laser speckle contrast imaging (LSCI) as a wide-field imaging technique to visualize the flow distribution in venules influenced by abnormally elevated levels of RBC aggregation at a network-scale level, which was unprecedented in previous studies. RBC aggregation in rats was induced by infusing Dextran 500. To elucidate the impact of RBC aggregation on microvascular perfusion, blood flow in the venular network of a rat cremaster muscle was analyzed with a stepwise reduction of the arterial pressure (100 → 30 mmHg). The LSCI analysis revealed a substantial decrease in the functional vascular density after the infusion of dextran. The relative decrease in flow velocity after dextran infusion was notably pronounced at low arterial pressures. Whole blood viscosity measurements implied that the reduction in venular flow with dextran infusion could be due to the elevation of medium viscosity in high shear conditions (> 45 s^-1). In contrast, further augmentation to the flow reduction at low arterial pressures could be attributed to the formation of RBC aggregates (< 45 s^-1). This study confirmed that RBC aggregation could play a dominant role in modulating microvascular perfusion, particularly in the venular networks.     

电针对小鼠肝脏血流灌注量影响的激光散斑成像显示

目的：应用激光散斑成像技术连续监测电针过程小鼠肝脏表面血流灌注图像,研究电针不同穴位对肝脏血流灌注量的影响,探讨激光散斑技术在针灸效应研究中的应用价值。方法：采用Moor．FLPI激光散斑血流成像系统分别对足三里组、曲泉组、非经非穴组正常小鼠电针30min以及不电针对照组连续观察30min过程中肝脏表面血流灌注量变化进行观察,分析电针不同穴位、各个时点肝脏血流变化的规律。结果：（1）肝脏激光散斑图显示电针后各电针组肝脏表面整体血流灌注均增加,肝门附近区域灌注量增加幅度大于肝脏边缘区域;（2）电针各时点各电针组肝脏血流灌注量均出现增加,电针0～20min灌注量增加率为足三里组〉曲泉组〉非经非穴组;电针25～30min为足三里组〉非经非穴组〉曲泉组。结论：激光散斑血流成像技术能够精确记录显示电针过程肝脏表面的微循环变化情况,电针可以增强正常小鼠肝脏血流灌注量,电针增加肝脏血流灌注的效应存在穴位特异性。     

Quantification of the Blood Platelet Reactivity in the ADP-Induced Model of Non-Lethal Pulmonary Thromboembolism in Mice with the Use of Laser Doppler Flowmetry

IntroductionThe paper describes an alternative method for quantification of in vivo ADP-induced thromboembolism. The aim of the studies was to develop a method of quantification which would not require either extravasation or labelling of platelets. Our proposed approach is based on the monitoring of changes of blood flow with the use of laser Doppler flowmetry.ResultsThe injection of ADP resulted in a dose-dependent reduction of the blood flow in the mesentery. These responses were fully attributable to blood platelet aggregation, as shown by the lack of the effect in platelet-depleted mice, and significantly reduced responses in mice pretreated with cangrelor and eptifibatide. No platelet aggregate formation in mesenteric vessels was revealed by intravital microscopy, while ex vivo imaging showed accumulation of fluorescent labelled platelets in the lung.ConclusionsInjection of ADP to the venous system results in the formation of platelet aggregates predominantly in the lung. This results in reversible blood flow cessation in peripheral blood vessels. The measurement of this blood flow cessation in the mesentery allows indirect measurement of ADP-induced pulmonary thromboembolism. We suggest that this approach can be useful for in vivo screening for antiplatelet drug candidates.     

Monitoring Hypoxia Induced Changes in Cochlear Blood Flow and Hemoglobin Concentration Using a Combined Dual-Wavelength Laser Speckle Contrast Imaging and Doppler Optical Microangiography System

A synchronized dual-wavelength laser speckle contrast imaging (DWLSCI) system and a Doppler optical microangiography (DOMAG) system was developed to determine several ischemic parameters in the cochlea due to a systemic hypoxic challenge. DWLSCI can obtain two-dimensional data, and was used to determine the relative changes in cochlear blood flow, and change in the concentrations of oxyhemoglobin (HbO), deoxyhemoglobin (Hb) and total hemoglobin (HbT) in mice. DOMAG can obtain three-dimensional data, and was used to determine the changes in cochlear blood flow with single vessel resolution. It was demonstrated that during a hypoxic challenge there was an increase in the concentrations of Hb, a decrease in the concentrations of HbO and cochlear blood flow, and a slight decrease in the concentration of HbT. Also, the rate of change in the concentrations of Hb and HbO was quantified during and after the hypoxic challenge. The ability to simultaneously measure these ischemic parameters with high spatio-temporal resolution will allow the detailed quantitative analysis of several hearing disorders, and will be useful for diagnosing and developing treatments.     

Noninvasive Optical Measurement of Cerebral Blood Flow in Mice Using Molecular Dynamics Analysis of Indocyanine Green

In preclinical studies of ischemic brain disorders, it is crucial to measure cerebral blood flow (CBF); however, this requires radiological techniques with heavy instrumentation or invasive procedures. Here, we propose a noninvasive and easy-to-use optical imaging technique for measuring CBF in experimental small animals. Mice were injected with indocyanine green (ICG) via tail-vein catheterization. Time-series near-infrared fluorescence signals excited by 760 nm light-emitting diodes were imaged overhead by a charge-coupled device coupled with an 830 nm bandpass-filter. We calculated four CBF parameters including arrival time, rising time and mean transit time of a bolus and blood flow index based on time and intensity information of ICG fluorescence dynamics. CBF maps were generated using the parameters to estimate the status of CBF, and they dominantly represented intracerebral blood flows in mice even in the presence of an intact skull and scalp. We demonstrated that this noninvasive optical imaging technique successfully detected reduced local CBF during middle cerebral artery occlusion. We further showed that the proposed method is sufficiently sensitive to detect the differences between CBF status in mice anesthetized with either isoflurane or ketamine–xylazine, and monitor the dynamic changes in CBF after reperfusion during transient middle cerebral artery occlusion. The near-infrared optical imaging of ICG fluorescence combined with a time-series analysis of the molecular dynamics can be a useful noninvasive tool for preclinical studies of brain ischemia.     

Spatiotemporal Dynamics of Dilute Red Blood Cell Suspensions in Low-Inertia Microchannel Flow

Microfluidic technologies are commonly used for the manipulation of red blood cell (RBC) suspensions and analyses of flow-mediated biomechanics. To enhance the performance of microfluidic devices, understanding the dynamics of the suspensions processed within is crucial. We report novel, to our knowledge, aspects of the spatiotemporal dynamics of RBC suspensions flowing through a typical microchannel at low Reynolds number. Through experiments with dilute RBC suspensions, we find an off-center two-peak (OCTP) profile of cells contrary to the centralized distribution commonly reported for low-inertia flows. This is reminiscent of the well-known “tubular pinch effect,” which arises from inertial effects. However, given the conditions of negligible inertia in our experiments, an alternative explanation is needed for this OCTP profile. Our massively parallel simulations of RBC flow in real-size microfluidic dimensions using the immersed-boundary-lattice-Boltzmann method confirm the experimental findings and elucidate the underlying mechanism for the counterintuitive RBC pattern. By analyzing the RBC migration and cell-free layer development within a high-aspect-ratio channel, we show that such a distribution is co-determined by the spatial decay of hydrodynamic lift and the global deficiency of cell dispersion in dilute suspensions. We find a cell-free layer development length greater than 46 and 28 hydraulic diameters in the experiment and simulation, respectively, exceeding typical lengths of microfluidic designs. Our work highlights the key role of transient cell distribution in dilute suspensions, which may negatively affect the reliability of experimental results if not taken into account.     

颈动脉瘤个体化模型血流数值分析

动脉瘤日益受到广泛关注,介入术成为动脉瘤的理想化治疗方法,但现实临床实践缺少理论支撑,所以数值模拟成为研究动脉瘤血流动力学参数的关键技术。为了研究动脉瘤的血液动力特性,进而探讨不同参数对动脉瘤的血液动力学特性影响因素,本研究基于1例个体化动脉瘤的影像数据,通过影像提取的方法建立了个体化动脉瘤模型,并根据双向流固耦合的方法进行血流场分析(流线,壁面变形分布,壁面剪切力分布)。通过数值分析结果表明,在一个心动周期的不同时刻动脉瘤瘤腔内的涡流强度先增强再减弱并在0.1 s达到最强。动脉瘤壁面的壁面切应力(wall shear stress, WSS)的分布也随着时间的变化,同样在0.1 s时刻壁面WSS分布梯度较大,到0.22 s时刻以及后各个时刻达到均匀无梯度分布。而从壁面变形来看,一个心动周期前0.3 s时刻动脉瘤壁的最大变形位置均为瘤顶处并呈现由小到大再到小的变化(0.1 s时刻达到最大变形量),0.3 s时刻的最大变形量位置发生改变,为瘤颈中间部位,但变形量很小为0.01 mm。     

大鼠小肠肠系膜微血管血流随温度变化的光学检测

利用CCD显微成像技术和激光散斑技术,对局部加热（41℃-54℃,30min）下大鼠小肠肠系膜微血管的管径（直径约15μm-50μm)和血流速率的变化进行实时,在体监测,并由二者计算血流量的变化。结果表明：41℃,43℃,45℃,46℃各温度点30min的加热过程中,血流速度变化平缓,血管管径和血流量明显增加,最终均逐渐趋趋于恒定。49℃时,血流速率仅在前12min内增加,30min时已降至低于初始值,血管管径及血流量在14min时达最大后开始减小,加热停止时仍旧高于初始值。温度高于49℃时（51℃,54℃）血流速率,管径,血流量呈现先升高后下降的变化趋势,在30min时,三者均低于初始值,由此可知,加热时间为30min,大鼠肠系膜微血管的临界温度为49℃;在相同的时间条件下,热损伤速率随温度升高而增加。     

The Impact of Increased Bladder Blood Flow on Storage Symptoms after Holmium Laser Enucleation of the Prostate

In order to investigate how holmium laser enucleation of the prostate (HoLEP) improves urinary storage symptoms, we assessed blood flow in the urinary bladder mucosa of patients with benign prostatic hyperplasia (BPH) before and after laser surgery. Seventy-four consecutive patients with BPH (median age 69 years, range; 53–88) underwent HoLEP at our institution and are included in this study. We prospectively assessed the International Prostate Symptom Score (IPSS), IPSS-QOL Score, the Overactive Bladder Symptom Score (OABSS), uroflowmetry, and blood flow in the urinary bladder, before and after surgery. Blood flow in the bladder mucosa was measured using the OMEGA FLOW (OMEGAWAVE, Tokyo, Japan) laser Doppler flowmeter. The median volume of the enucleated adenomas was 45.0 g (range: 25.0 to 83.2). The median IPSS improved significantly from 20 (range: 6–35) to 3 (0–22) (p<0.001; Wilcoxon signed-rank test), as did the storage symptoms score, which decreased from 13 (2–20) to 3 (1–8) (p<0.001). Median bladder blood flow increased at the trigone from 9.57±0.83 ml/sec to 17.60±1.08 ml/sec. Multiple regression analysis for the improved storage symptom score eliminated all explanatory variables except increased bladder perfusion. The data suggest that HoLEP improves blood flow in the bladder mucosa, which independently leads to the improvement of storage symptoms.     

Relationships between Cerebral Blood Flow Dynamics and Bioelectric Activity of the Human Brain in Experimental Acute Hypoxia

The relationships between the parameters of oxygen content in the body (hemoglobin saturation with oxygen and trancutaneous oxygen tension), central hemodynamics (cardiac output), and cerebral hemodynamics (cerebral blood flow rate) were studied during a hypoxic test (inhalation of an oxygen–nitrogen mixture containing 8% oxygen for 15 min). Special attention was paid to the relationships between the dynamics of cerebral blood flow and cerebral bioelectric activity measured by EEG parameters. It was demonstrated that the trancutaneous oxygen tension decreased to a greater extent than the hemoglobin saturation with oxygen and the cerebral blood flow increased to a greater extent than the cardiac output. The increase in cerebral blood flow and the increase in the indices and power of and EEG waves in the course of hypoxia were strongly positively correlated with each other in most subjects. However, if these parameters were considered in the series of subjects, the degree of the increase in the indices and power of and waves in different subjects was negatively correlated with the increase in the cerebral blood flow. The results are explained in terms of redistribution of blood flow in the body to provide a better oxygen supply to the brain and optimization of the ratios between the cerebral oxygen consumption and the functional load on the system of oxygen supply.     

Analysis of Flow Cytometry Data by Matrix Relevance Learning Vector Quantization

Flow cytometry is a widely used technique for the analysis of cell populations in the study and diagnosis of human diseases. It yields large amounts of high-dimensional data, the analysis of which would clearly benefit from efficient computational approaches aiming at automated diagnosis and decision support. This article presents our analysis of flow cytometry data in the framework of the DREAM6/FlowCAP2 Molecular Classification of Acute Myeloid Leukemia (AML) Challenge, 2011. In the challenge, example data was provided for a set of 179 subjects, comprising healthy donors and 23 cases of AML. The participants were asked to provide predictions with respect to the condition of 180 patients in a test set. We extracted feature vectors from the data in terms of single marker statistics, including characteristic moments, median and interquartile range of the observed values. Subsequently, we applied Generalized Matrix Relevance Learning Vector Quantization (GMLVQ), a machine learning technique which extends standard LVQ by an adaptive distance measure. Our method achieved the best possible performance with respect to the diagnoses of test set patients. The extraction of features from the flow cytometry data is outlined in detail, the machine learning approach is discussed and classification results are presented. In addition, we illustrate how GMLVQ can provide deeper insight into the problem by allowing to infer the relevance of specific markers and features for the diagnosis.     

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions ^1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes ³, the complete development of the parasite in the skin ⁴, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream ⁵. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria ⁶. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line ⁷. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. Ethics statement All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.     

Numerical Modeling of Interstitial Fluid Flow Coupled with Blood Flow through a Remodeled Solid Tumor Microvascular Network

Modeling of interstitial fluid flow involves processes such as fluid diffusion, convective transport in extracellular matrix, and extravasation from blood vessels. To date, majority of microvascular flow modeling has been done at different levels and scales mostly on simple tumor shapes with their capillaries. However, with our proposed numerical model, more complex and realistic tumor shapes and capillary networks can be studied. Both blood flow through a capillary network, which is induced by a solid tumor, and fluid flow in tumor’s surrounding tissue are formulated. First, governing equations of angiogenesis are implemented to specify the different domains for the network and interstitium. Then, governing equations for flow modeling are introduced for different domains. The conservation laws for mass and momentum (including continuity equation, Darcy’s law for tissue, and simplified Navier–Stokes equation for blood flow through capillaries) are used for simulating interstitial and intravascular flows and Starling’s law is used for closing this system of equations and coupling the intravascular and extravascular flows. This is the first study of flow modeling in solid tumors to naturalistically couple intravascular and extravascular flow through a network. This network is generated by sprouting angiogenesis and consisting of one parent vessel connected to the network while taking into account the non-continuous behavior of blood, adaptability of capillary diameter to hemodynamics and metabolic stimuli, non-Newtonian blood flow, and phase separation of blood flow in capillary bifurcation. The incorporation of the outlined components beyond the previous models provides a more realistic prediction of interstitial fluid flow pattern in solid tumors and surrounding tissues. Results predict higher interstitial pressure, almost two times, for realistic model compared to the simplified model.     

Quantitative Analysis of Mechanisms That Govern Red Blood Cell Age Structure and Dynamics during Anaemia

Mathematical modelling has proven an important tool in elucidating and quantifying mechanisms that govern the age structure and population dynamics of red blood cells (RBCs). Here we synthesise ideas from previous experimental data and the mathematical modelling literature with new data in order to test hypotheses and generate new predictions about these mechanisms. The result is a set of competing hypotheses about three intrinsic mechanisms: the feedback from circulating RBC concentration to production rate of immature RBCs (reticulocytes) in bone marrow, the release of reticulocytes from bone marrow into the circulation, and their subsequent ageing and clearance. In addition we examine two mechanisms specific to our experimental system: the effect of phenylhydrazine (PHZ) and blood sampling on RBC dynamics. We performed a set of experiments to quantify the dynamics of reticulocyte proportion, RBC concentration, and erythropoietin concentration in PHZ-induced anaemic mice. By quantifying experimental error we are able to fit and assess each hypothesis against our data and recover parameter estimates using Markov chain Monte Carlo based Bayesian inference. We find that, under normal conditions, about 3% of reticulocytes are released early from bone marrow and upon maturation all cells are released immediately. In the circulation, RBCs undergo random clearance but have a maximum lifespan of about 50 days. Under anaemic conditions reticulocyte production rate is linearly correlated with the difference between normal and anaemic RBC concentrations, and their release rate is exponentially correlated with the same. PHZ appears to age rather than kill RBCs, and younger RBCs are affected more than older RBCs. Blood sampling caused short aperiodic spikes in the proportion of reticulocytes which appear to have a different developmental pathway than normal reticulocytes. We also provide evidence of large diurnal oscillations in serum erythropoietin levels during anaemia.     

Three-Dimensional Numerical Simulations of Biofilm Dynamics with Quorum Sensing in a Flow Cell

We develop a multiphasic hydrodynamic theory for biofilms taking into account interactions among various bacterial phenotypes, extracellular polymeric substance (EPS), quorum sensing (QS) molecules, solvent, and antibiotics. In the model, bacteria are classified into down-regulated QS, up-regulated QS, and non-QS cells based on their QS ability. The model is first benchmarked against an experiment yielding an excellent fit to experimental measurements on the concentration of QS molecules and the cell density during biofilm development. It is then applied to study development of heterogeneous structures in biofilms due to interactions of QS regulation, hydrodynamics, and antimicrobial treatment. Our 3D numerical simulations have confirmed that (i). QS is beneficial for biofilm development in a long run by building a robust EPS population to protect the biofilm; (ii). biofilms located upstream can induce QS downstream when the colonies are close enough spatially; (iii). QS induction may not be fully operational and can even be compromised in strong laminar flows; (v). the hydrodynamic stress alters the biofilm morphology. Through further numerical investigations, our model suggests that (i). QS-regulated EPS production contributes to the structural formation of heterogeneous biofilms; (ii) QS down-regulated cells tend to grow at the surface of the biofilm while QS up-regulated ones tend to grow in the bulk; (iii) when nutrient supply is sufficient, QS induction might be more effective upstream than downstream; (iv) QS may be of little benefit in a short timescale in term of fighting against invading strain/species; (v) the material properties of biomass (bacteria and EPS) have strong impact on the dilution of QS molecules under strong shear flow. In addition, with this modeling framework, hydrodynamic details and rheological quantities associated with biofilm formation under QS regulation can be resolved.