Guillaume Mahé

Assessment of skin microvascular function and dysfunction with laser speckle contrast imaging.

In recent years, skin microcirculation has been considered an easily accessible and potentially representative vascular bed to evaluate and understand the mechanisms of microvascular function and dysfunction.1–3 Vascular dysfunction (including impaired endothelium-dependent vasodilation) induced by different pathologies is evident in the cutaneous circulation.4–7 It has been suggested that the skin microcirculation may mirror generalized systemic vascular dysfunction in magnitude and underlying mechanisms.1 Furthermore, minimally invasive skin-specific methodologies using laser systems make the cutaneous circulation a useful translational model for investigating mechanisms of skin physiology and skin pathophysiology induced either by skin disease itself or by other diseases such as vascular, rheumatologic, and pneumologic. To date, the skin has been used as a circulation model to investigate vascular mechanisms in a variety of diseased states, including hypercholesterolemia,8 Alzheimer disease,9 carpal tunnel syndrome,10 schizophrenia,11 hypertension,6 renal disease,12 type 2 diabetes,13 peripheral vascular disease,14 atherosclerotic coronary artery disease,2 heart failure,15 systemic sclerosis,16 obesity,17 primary aging,18,19 and sleep apnea.20 Assessment of skin microvascular function can be done by both invasive and noninvasive techniques. Among noninvasive techniques, laser systems are mainly used.21 The recent development of the laser speckle contrast imaging (LSCI) technique for monitoring skin microvascular function enables its use as a surrogate end point in clinical trials. LSCI allows for noncontact, real-time, and noninvasive monitoring of cutaneous blood flow changes.22,23 Recent evidence has shown that the LSCI technique dramatically reduces the variability of clinical measurements compared with laser Doppler flowmetry (LDF), making the technique a fascinating tool to facilitate microvascular studies in clinical routine.23,24 In this review, we describe …

Increasing the "region of interest" and "time of interest", both reduce the variability of blood flow measurements using laser speckle contrast imaging.

OBJECTIVE Both spatial variability and temporal variability of skin blood flow are high. Laser speckle contrast imagers (LSCI) allow non-contact, real-time recording of cutaneous blood flow on large skin surfaces. Thereafter, the observer can define different sizes for the region of interest (ROI) in the images to decrease spatial variability and different durations over which the blood flow values are averaged (time of interest, TOI) to decrease temporal variability. We aimed to evaluate the impact of the choices of ROI and TOI on the analysis of rest blood flow and post occlusive reactive hyperemia (PORH). METHODS Cutaneous blood flow (CBF) was assessed at rest and during PORH. Three different sizes of ROI (1mm(2), 10mm(2) and 100mm(2)), and three different TOI (CBF averaged over 1s, 15s, and 30s for rest, and over 1s, 5s and 10s for PORH peak) were evaluated. Inter-subjects and intra-subjects coefficient of variations (inter-CV and intra-CV) were studied. RESULTS The inter-subject variability of CBF is about 25% at rest and is moderately improved when the size of the ROI increases (inter-CV=31%, for 1s and 1mm(2) versus inter-CV=23%, for 15s and 100mm(2)). However, increasing the TOI does not improve the results. The variability of the PORH peak is lower with an inter-CV varying between 11.4% (10s and 100mm(2)) and 21.6% (5s and 1mm(2)). The lowest intra-CV for the CBF at rest was 7.3% (TOI of 15s on a ROI of 100mm(2)) and was 3.1% for the PORH peak (TOI of 10s on a ROI of 100mm(2)). CONCLUSION We suggest that a size of ROI larger than 10mm(2) and a TOI longer than 1s are required to reduce the variability of CBF measurements both at rest and during PORH peak evaluations at the forearm level. Many technical aspects such as comparison of laser speckle contrast imaging and laser Doppler imaging or the effect of skin to head distance on recorded values with LCSI are required to improve future studies using this fascinating clinical tool.

Relevance of Laser Doppler and Laser Speckle Techniques for Assessing Vascular Function: State of the Art and Future Trends

In clinical and research applications, the assessment of vascular function has become of major importance to evaluate and follow the evolution of cardiovascular pathologies, diabetes, hypertension, or foot ulcers. Therefore, the development of engineering methodologies able to monitor noninvasively blood vessel activities-such as endothelial function-is a significant and emerging challenge. Laser-based techniques have been used to respond-as much as possible-to these requirements. Among them, laser Doppler flowmetry (LDF) and laser Doppler imaging (LDI) were proposed a few decades ago. They provide interesting vascular information but possess drawbacks that prevent an easy use in some clinical situations. Recently, the laser speckle contrast imaging (LSCI) technique, a noninvasive camera-based tool, was commercialized and overcomes some of the LDF and LDI weaknesses. Our paper describes how-using engineering methodologies-LDF, LDI, and LSCI can meet the challenging clinician needs in assessing vascular function, with a special focus on the state of the art and future trends.

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°: NCT01664572.

Air movements interfere with laser speckle contrast imaging recordings.

Laser speckle contrast imaging (LSCI) allows the noncontact monitoring of changes in cutaneous blood flow (CBF) [1, 2]. Recent evidence shows that the LSCI technique dramatically reduces the variability in clinical measurements as compared to laser Doppler, making the technique a fascinating tool to facilitate microvascular studies in clinical routine [2, 3]. LSCI records CBF using a camera placed at a distance ranging from 10 cm to 30 cm from the area of interest of the skin [4, 5]. As such, we hypothesized that movements of air particulate matter between the skin and the laser head could interfere with the LSCI backscattered signal during microvascular tests. This is of particular interest because microvascular studies are generally performed in air-conditioned rooms and the fan of the air-conditioner might induce rapid air movements. Further, an open window, or displacement of objects (door opening) or personnel in the room are a few of the events that could induce air movements. We tested the influence of air movements induced by a fan with different CBF levels: at rest, during ischaemia, and during postocclusive reactive hyperaemia (PORH).

Effect of skin temperature on skin endothelial function assessment

PURPOSE Microcirculatory dysfunction plays a key role in the development of sepsis during which core temperature is often disturbed. Skin microvascular assessment using laser techniques has been suggested to evaluate microvascular dysfunction during sepsis, but skin microcirculation is also a major effector of human thermoregulation. Therefore we aimed to study the effect of skin temperature on endothelial- and non-endothelial microvascular responses. METHODS Fifteen healthy participants were studied at different randomized ambient temperatures leading to low (28.0+/-2.0 °C), intermediate (31.6+/-2.1 °C), and high (34.1+/-1.3 °C) skin temperatures. We measured skin blood flow using laser speckle contrast imaging on the forearm in response to vasodilator microvascular tests: acetylcholine (ACh) iontophoresis, sodium nitroprussiate (SNP) iontophoresis, and post-occlusive reactive hyperemia (PORH). The results are expressed as absolute (laser speckle perfusion units, LSPU) or normalized values (cutaneous vascular conductance, CVC in LSPU/mmHg and multiple of baseline). RESULTS Maximal vasodilation induced by these tests is modified by skin temperature. A low skin temperature induced a significant lower vasodilation for all microvascular tests when results are expressed either in absolute values or in CVC. For example, ACh peak was 57.6+/-19.6 LSPU, 66.8+/-22.2 LSPU and 88.5+/-13.0 LSPU for low, intermediate and high skin temperature respectively (p<0.05). When results are expressed in multiple of baseline, statistical difference disappeared. CONCLUSIONS These results suggest that skin temperature has to be well controlled when performing microvascular assessments in order to avoid any bias. The effect of skin temperature can be corrected by expressing the results in multiple of baseline.

Acetylcholine chloride as a potential source of variability in the study of cutaneous vascular function in man.

INTRODUCTION Laser-Doppler flowmetry (LDF) coupled with acetylcholine chloride (ACh) iontophoresis is increasingly recognized as a reliable non-invasive method to study the endothelial function. However, ACh-vasodilation measurement appears highly variable possibly due to the ACh pharmacological properties itself. These problems may be partially overcome by using methacholine chloride (MCh), a more stable synthetic agonist of muscarinic receptors, instead of ACh. Therefore, we first studied the correlation between the two drugs and then the effects of (1) spatial variability (inter-site measurements), (2) temporal variability (inter-day measurements), (3) intra-day variability (morning versus evening), and (4) age on the variability of both ACh-vasodilation and MCh-vasodilation. METHODS The endothelium-dependent vasodilation response to simultaneous iontophoretic applications (4 doses of 10s at 0.1mA with 2min of current-free interval) of ACh (11mM) or MCh (10mM) was studied on the forearm of 40 healthy subjects (36 males, median 28yr, range 21-59yr). The percent change in perfusion (CVCpeak) from baseline and the area under the curve (CVC(AUC)) during iontophoresis were assessed. Inter-site, inter-day and intra-day coefficients of variation (CV) were studied for each drug as well as correlations between drugs and age. RESULTS A linear relationship was found between ACh- and MCh-CVCpeak (r²=0.75, p=0.01) and between ACh- and MCh-CVC(AUC) (r²=0.55, p=0.02). MCh inter-site CV for both CVCpeak (12.2%) and CVC(AUC) (13.8%) was significantly lower than ACh inter-site CV for CVCpeak (15.5%) and CVC(AUC) (15.3%), respectively. MCh inter-day CV for CVCpeak (17.2%) and CVC(AUC) (14.6%) was significantly lower than ACh inter-day CV for CVCpeak (19.7%) and ACh CVC(AUC) (21.2%). For ACh and MCh, the CVCpeak and CVC(AUC) were higher at 16:00pm than at 11:00am (p<0.05 for all). Finally, both ACh- and MCh-CVCpeak exhibited a logarithmic decrease with age (r²=0.61, p<0.01 and r²=0.58, p<0.01). CONCLUSION Although both drugs exhibited circadian and age variability, MCh exhibited less inter-site and interday variabilities than did ACh for the evaluation of cutaneous endothelium-dependent vasodilation. These findings should be taken into account in studies of cutaneous vascular function by iontophoresis coupled with laser Doppler flowmetry.

Multiscale Analysis of Microvascular Blood Flow: A Multiscale Entropy Study of Laser Doppler Flowmetry Time Series

Processes regulating the cardiovascular system (CVS) are numerous. Each possesses several temporal scales. Their interactions lead to interdependences across multiple scales. For the CVS analysis, different multiscale studies have been proposed, mostly performed on heart rate variability signals (HRV) reflecting the central CVS; only few were dedicated to data from the peripheral CVS, such as laser Doppler flowmetry (LDF) signals. Very recently, a study implemented the first computation of multiscale entropy for LDF signals. A nonmonotonic evolution of multiscale entropy with two distinctive scales was reported, leading to a markedly different behavior from the one of HRV. Our goal herein is to confirm these results and to go forward in the investigations on origins of this behavior. For this purpose, 12 LDF signals recorded simultaneously on the two forearms of six healthy subjects are processed. This is performed before and after application of physiological scales-based filters aiming at isolating previously found frequency bands linked to physiological activities. The results obtained with signals recorded simultaneously on two different sites of each subject show a probable central origin for the nonmonotonic behavior. The filtering results lead to the suggestion that origins of the distinctive scales could be dominated by the cardiac activity.

Multiscale Entropy Study of Medical Laser Speckle Contrast Images

Laser speckle contrast imaging (LSCI) is a noninvasive full-field optical imaging technique that gives a 2-D microcirculatory blood flow map of tissue. Due to novelty of commercial laser speckle contrast imagers, image processing of LSCI data is new. By opposition, the numerous signal processing works of laser Doppler flowmetry (LDF) data-that give a 1-D view of microvascular blood flow-have led to interesting physiological information. Recently, analysis of multiscale entropy (MSE) of LDF signals has been proposed. A nonmonotonic evolution of MSE with two distinctive scales-probably dominated by the cardiac activity-has been reported. We herein analyze MSE of LSCI data. We compare LSCI results with the ones of LDF signals obtained during the same experiment. We show that when time evolution of LSCI single pixels is studied, MSE presents a monotonic decreasing pattern, similar to the one of Gaussian white noises. By opposition, when the mean of LSCI pixel values is computed in a region of interest (ROI) and followed with time, MSE pattern becomes close to the one of LDF data, for ROI large enough. LSCI is gaining increased interest for blood flow monitoring. The physiological implications of our results require future study.

Multifractal analysis of heart rate variability and laser Doppler flowmetry fluctuations:comparison of results from different numerical methods

To contribute to the understanding of the complex dynamics in the cardiovascular system (CVS), the central CVS has previously been analyzed through multifractal analyses of heart rate variability (HRV) signals that were shown to bring useful contributions. Similar approaches for the peripheral CVS through the analysis of laser Doppler flowmetry (LDF) signals are comparatively very recent. In this direction, we propose here a study of the peripheral CVS through a multifractal analysis of LDF fluctuations, together with a comparison of the results with those obtained on HRV fluctuations simultaneously recorded. To perform these investigations concerning the biophysics of the CVS, first we have to address the problem of selecting a suitable methodology for multifractal analysis, allowing us to extract meaningful interpretations on biophysical signals. For this purpose, we test four existing methodologies of multifractal analysis. We also present a comparison of their applicability and interpretability when implemented on both simulated multifractal signals of reference and on experimental signals from the CVS. One essential outcome of the study is that the multifractal properties observed from both the LDF fluctuations (peripheral CVS) and the HRV fluctuations (central CVS) appear very close and similar over the studied range of scales relevant to physiology.