Jean-Luc Cracowski

Non-invasive assessment of skin microvascular function in humans: an insight into methods.

Please cite this paper as: Roustit and Cracowski (2012). Non‐invasive Assessment of Skin Microvascular Function in Humans: An Insight Into Methods. Microcirculation 19(1), 47–64.

Excellent reproducibility of laser speckle contrast imaging to assess skin microvascular reactivity.

OBJECTIVE We compared the inter-day reproducibility of post-occlusive reactive hyperemia (PORH) assessed by single-point laser Doppler flowmetry (LDF) and laser speckle contrast analysis (LSCI), and the reproducibility of local thermal hyperemia (LTH) assessed by LDF, laser Doppler imaging (LDI) and LSCI. We also tested whether skin blood flow assessment by LDF and by LSCI are correlated. METHODS Skin blood flow was evaluated during PORH and LTH using LDF, LDI (for LTH only) and LSCI on the forearms of healthy volunteers, at a 7day interval. Data are expressed as cutaneous vascular conductance (CVC), as a function of baseline and scaled to the thermal plateau. Reproducibility is expressed as within subject coefficients of variation (CV, in %) and intra-class correlation coefficients (ICC). RESULTS Twenty-eight healthy participants were enrolled in this study. The reproducibility of the PORH peak CVC was better when assessed with LSCI compared to LDF (CV=8%; ICC=0.76 and CV=30%; ICC=0.54, respectively). Inter-day reproducibility of the LTH plateau was better when assessed with LSCI or LDI than LDF (CV=15%, ICC=0.66; CV=17%, ICC=0.51 and CV=42%, ICC=0.28 respectively). Finally, we observed significant correlation between simultaneous LDF and LSCI measurements of the PORH peak CVC (R=0.54; p=0.001). CONCLUSION The recently developed LSCI technique showed very good inter-day reproducibility for assessing PORH and LTH. Moreover, we showed significant correlation between LSCI and single-point LDF for PORH. However, more data are needed to evaluate the linearity between the LSCI signal and skin blood flow.

Assessment of endothelial and neurovascular function in human skin microcirculation

Peripheral microvascular dysfunction has been described in many physiological and pathological conditions. Owing to its accessibility, the cutaneous microcirculation provides a unique index of microvascular function. Skin microvascular function has therefore been proposed as a prognostic marker or for evaluating the effect of drugs on the microcirculation. Various reactivity tests, coupled with techniques measuring skin blood flux, are used to non-invasively explore both endothelial and neurovascular microvascular functioning in humans. We review the advantages and limitations of the main reactivity tests, including post-occlusive reactive hyperemia, local thermal hyperemia, pressure-induced vasodilation, and iontophoresis of vasodilators, combined with measurement techniques such as laser Doppler and laser speckle contrast imaging. Recent advances in our comprehension of the physiological pathways underlying these reactivity tests, as well as technological developments in microcirculation imaging, have provided reliable and reproducible tools for studying the microcirculation.

Reproducibility and methodological issues of skin post-occlusive and thermal hyperemia assessed by single-point laser Doppler flowmetry

OBJECTIVE The primary objective of this study was to evaluate 1-week reproducibility of post-occlusive reactive hyperemia (PORH) and local thermal hyperemia (LTH) assessed by single-point laser-Doppler flowmetry (LDF) on different skin sites. We also evaluated spatial reproducibility of both tests on the forearm. Finally, we assessed the influence of mental stress and room temperature variations on PORH and LTH. METHODS We performed PORH and LTH assessing skin blood flow on the forearm and on the finger pad with LDF. We repeated the sequence 1 week later. We also performed PORH and LTH during mental stress (Stroop test) and at room temperatures of 21 degrees C and 27 degrees C. Data were expressed as cutaneous vascular conductance (CVC), as a function of baseline and as a function of 44 degrees C vasodilation (%CVC(44)). Reproducibility was expressed as within subject coefficients of variation (CV) and intra-class correlation coefficients (ICC). RESULTS Fourteen Caucasian healthy volunteers were recruited. Median age was 25 (2.7) and 50% were female. Median body mass index was 21.2 (5). PORH was reproducible on the finger, whether expressed as raw CVC (CV=25%; ICC=0.56) or as %CVC(44) (CV=24%; ICC=0.60). However, PORH showed poor reproducibility on the forearm. In the same way, LTH was reproducible on the finger pad when expressed as CVC (CV=17%; ICC=0.81) but not on the forearm. Spatial reproducibility was poor on the forearm. Elevated room temperature (27 degrees C) affected PORH and LTH on the finger pad (p<0.05) but not on the forearm. CONCLUSION Single-point LDF is a reproducible technique to assess PORH and LTH on the finger pad when data are expressed as raw CVC or %CVC(44). On the forearm, however, it shows great inter-day variability, probably due to spatial variability of capillary density. These results highlight the need for alternative techniques on the forearm.

Determination of isoprostaglandin F2α type III in human urine by gas chromatography-electronic impact mass spectrometry. Comparison with enzyme immunoassay

F2-Isoprostanes are stable lipid peroxidation products of arachidonic acid, the quantification of which provides an index of oxidative stress in vivo. We describe a method for analysing isoprostaglandin F2alpha type III (15-F2t-IsoP) in biological fluids. The method involves solid-phase extraction on octadecyl endcapped and aminopropyl cartridges. After conversion to trimethylsilyl ester trimethylsilyl ether derivatives, isoprostaglandin F2alpha type III is analysed by mass spectrometry, operated in electronic impact selected ion monitoring mode. We have compared enzyme immunoassay (EIA; Cayman, Ann Arbor, MI, USA) to this method with 30 human urine aliquots following the same extraction procedure in order to determine the agreement between both methods. Isoprostaglandin F2alpha type III concentrations determined with gas chromatography-mass spectrometry (GC-MS) did not agree with those determined with EIA. Our results suggest that GC-MS and EIA do not measure the same compounds. As a consequence, comparison of clinical results using GC-MS and EIA should be avoided.

Lipid Peroxidation Is Not Increased in Patients With Untreated Mild-to-Moderate Hypertension

Abstract—In contrast with the huge amount of experimental data available, only few and somewhat unconvincing clinical studies support the hypothesis that oxidative stress is involved in the early stages of essential hypertension in humans. Isoprostanes are chemically stable lipid peroxidation products of arachidonic acid, the quantification of which provides a novel approach to the assessment of oxidative stress in vivo. The main objective of this study was to quantify the urinary levels of 15-F2t-IsoP in the early stages of essential hypertension, using gas chromatography/mass spectrometry, by comparing 30 patients with never-treated mild-to-moderate hypertension with 30 gender- and age-paired healthy controls. Urinary 15-F2t-IsoP levels were not significantly different in hypertensive patients (69±36 pmol/mmol creatinine) compared with controls (75±34 pmol/mmol creatinine, 95% confidence intervals on differences: −23 to 13). No significant correlation was found between basal urinary 15-F2t-IsoP levels and age, low-density lipoprotein cholesterol, glucose, clinical pulse pressure, carotid intima-media thickness, left ventricular mass index, or aortic pulse wave velocity. In conclusion, this study shows that lipid peroxidation is not increased in never-treated mild-to-moderate hypertension. This suggests that oxidative stress is not implicated in the pathogenesis of human essential hypertension, at least in the early stages.

Phosphodiesterase-5 inhibitors for the treatment of secondary Raynaud's phenomenon: systematic review and meta-analysis of randomised trials

Introduction Recent controlled trials have assessed the efficacy of phospodiesterase-5 (PDE-5) inhibitors in secondary Raynauds phenomenon (RP). However, the conclusions are conflicting, and whether these drugs are effective remains unclear. The objective of this meta-analysis was to determine the efficacy of PDE-5 inhibitors on Raynauds Condition Score (RCS) and frequency and duration of attacks. Methods A systematic review of articles was performed (sources included Medline, Embase, Web of Science, the Cochrane Central Register of Controlled Trials). Only double-blind, randomised controlled trials (RCTs) were included. Studies were selected independently by two authors using predefined data fields, including study quality indicators. Results Six RCTs were included (one with sildenafil, one with modified-release sildenafil, three with tadalafil and one with vardenafil). PDE-5 inhibitors significantly decreased mean RCS by −0.46 (−0.74 to −0.17) (p=0.002), the daily frequency of ischaemic attacks by −0.49 (−0.71 to −0.28) (p<0.0001), and daily duration of RP attacks by −14.62 (−20.25 to −9.00) min (p<0.0001). Conclusions PDE-5 inhibitors appear to have significant but moderate efficacy in secondary RP. A further large RCT is needed.

Local hyperhemia to heating is impaired in secondary Raynaud's phenomenon

Accurate and sensitive measurement techniques are a key issue in the quantification of the microvascular and endothelial dysfunction in systemic sclerosis (SSc). Thermal hyperhemia comprises two separate mechanisms: an initial peak that is axon reflex mediated; and a sustained plateau phase that is nitric oxide dependent. The main objective of our study was to test whether thermal hyperhemia in patients with SSc differed from that in patients with primary Raynauds phenomenon (RP) and healthy controls. In a first study, we enrolled 20 patients suffering from SSc, 20 patients with primary RP and 20 healthy volunteers. All subjects were in a fasting state. Post-occlusive hyperhemia, 0.4 mg sublingual nitroglycerin challenge and thermal hyperhemia were performed using laser Doppler flowmetry on the distal pad of the third left finger. In a second study, thermal hyperhemia was performed in 10 patients with rheumatoid arthritis and 10 patients with primary RP. The thermal hyperhemia was dramatically altered in terms of amplitude and kinetics in patients with SSc. Whereas 19 healthy volunteers and 18 patients with primary RP exhibited the classic response, including an initial peak within the first 10 minutes followed by a nadir and a second peak, this occurred only in four of the SSc patients (p < 0.0001). The 10 minutes thermal peak was 43.4 (23.2 to 63), 42.6 (31 to 80.7) and 27 (14.7 to 51.4) mV/mm Hg in the healthy volunteers, primary RP and SSc groups, respectively (p = 0.01), while the 44°C thermal peak was 43.1 (21.3 to 62.1), 42.6 (31.6 to 74.3) and 25.4 (15 to 52.4) mV/mm Hg, respectively (p = 0.01). Thermal hyperhemia was more sensitive and specific than post-occlusive hyperhemia for differentiating SSc from primary RP. In patients with rheumatoid arthritis, thermal hyperhemia was also altered in terms of amplitude. Thermal hyperhemia is dramatically altered in patients with secondary RP in comparison with subjects with primary RP. Further studies are required to determine the mechanisms of this altered response, and whether it may provide additional information in a clinical setting.

Comparison between laser speckle contrast imaging and laser Doppler imaging to assess skin blood flow in humans.

OBJECTIVE We tested the linearity between skin blood flux recorded with laser speckle contrast imaging (LSCI) and laser Doppler imaging (LDI), comparing different ways of expressing data. A secondary objective was to test within-subject variability of baseline flux with the two techniques. METHODS We performed local heating at 36, 39, 42, and 44°C on the forearm of healthy volunteers, and measured cutaneous blood flux with LDI and LSCI. Biological zero (BZ) was obtained by occluding the brachial artery. We expressed data as raw arbitrary perfusion units (APUs) and as a percentage increase from baseline (%BL), with and without subtracting BZ. Inter-site variability was expressed as a within subject coefficient of variation (CV). RESULTS Twelve participants were enrolled. Inter-site variability at baseline was lower with LSCI (CV=9.2%) than with LDI (CV=20.7%). We observed an excellent correlation between both techniques when data were expressed as raw APUs or APU-BZ (R=0.90; p<0.001). The correlation remained correct for %BL (R=0.77, p<0.001), but decreased for %BL-BZ (R=0.44, p=0.003). Bland-Altman plots revealed a major proportional bias between the two techniques. CONCLUSION This study suggests that skin blood flux measured with LSCI is linearly related to the LDI signal over a wide range of perfusion. Subtracting BZ does not affect this linearity but introduces variability in baseline flux, thus decreasing the correlation when data are expressed as a function of baseline. Finally, systematic bias makes it impossible to assimilate arbitrary perfusion units provided by the two systems.

Biologically active oxidized lipids (phytoprostanes) in the plant diet and parenteral lipid nutrition

Phytoprostanes (PP) are autoxidation products of α-linolenate that are present in all plant tissues. Several classes of PP with a prostaglandin (PG) F1-, E1-, A1- and B1-like structure were identified and quantified by gas chromatography-mass spectrometry in vegetable oils and parenteral nutrition (intralipid). High levels of PP (0.09 up to 99 mg/l) were found even in apparently fresh vegetable oils. After oral consumption of olive or soybean oil, PPF1 were absorbed, found to circulate in plasma in conjugated form and excreted in free form into urine. Evidence is emerging that certain PP, such as the PPE1, may modulate the function of immune cells in a PG-like fashion. Here, we show that PPA1- and deoxy-PPJ1 display potent anti-inflammatory and apoptosis inducing activities similar to PGA1 and deoxy-PGJ2. Results of this study indicate that PP are novel, biologically active lipids in plant nutrition.