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Assessment of endothelial function by acetylcholine iontophoresis: Impact of inter-electrode distance and electrical cutaneous resistance
Microvascular Research 93 (2014) 114–118
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Assessment of endothelial function by acetylcholine iontophoresis: Impact of inter-electrode distance and electrical cutaneous resistance Cyril Puissant a, Pierre Abraham a,b, Sylvain Durand c, Anne Humeau-Heurtier d, Sébastien Faure e, Georges Leftheriotis a,b, Guillaume Mahé f,g,⁎ a
Laboratory of Vascular Investigations, University Hospital of Angers, France L'UNAM University, University of Angers, Biologie Neurovasculaire et Mitochondriale Intégrée (BNMI), Unité mixte UMR CNRS 6214/INSERM U 1083, Medicine Faculty, Angers, France L'UNAM University, University du Maine, bEA 4334N, Motricity, Interactions, and Performance, Avenue Olivier Messiaen, 72085 Le Mans Cedex 9, France d L'UNAM University, University of Angers, LARIS — Laboratoire Angevin de Recherche en Ingénierie des Systèmes, 62 avenue Notre Dame du Lac, 49000 Angers, France e L'UNAM University, University of Angers, INSERM UMR 1063, Stress oxydant et pathologies métaboliques (SOPAM), Angers, France f CHU Rennes, Imagerie Coeur-Vaisseaux, F-35033 Rennes, France g INSERM Clinical Investigation Center CIC 1414, F-35043 Rennes, France b c
a r t i c l e
i n f o
Article history: Accepted 4 April 2014 Available online 13 April 2014
a b s t r a c t Objectives: Endothelial function can be assessed by acetylcholine (ACh) iontophoresis with single current application. The effect of inter-electrode distance as well as electrical cutaneous resistance (ECR) on ACh dependent vasodilation has never been studied using single current application. The aims of this study are (i) to compare ACh-peak and ECR measured at different inter-electrode distances, (ii) to assess the relationship between ACh-peak and ECR, (iii) and to study the reproducibility of the ECR values. Methods: Fourteen healthy subjects were included. Using laser speckle contrast imaging, ACh-iontophoreses (0.1 mA, 30 s) were performed on the forearm at a 7-day interval with an inter-electrode distance set at 5 cm. Two other inter-electrode distances were also evaluated: 10 cm and 15 cm. ECR was measured during each ACh-iontophoresis as well as the ACh-peak. Results: No statistical difference was found between the ACh-peak values obtained at 5 cm, 10 cm and 15 cm. ECRs were also not statistically different. An inverse relationship (r = −0.60) was found between the ACh-peak and ECR (p b 0.05). The coefficient of variation of the inter-day reproducibility of the ECR values was 9.1% [6.5%–15.1%] with an intra-class-correlation coefficient of 0.93 [0.81–0.98]. Conclusion: Inter-electrode distance ranging from 5 cm to 15 cm changes neither the ACh-peak value nor the ECR value. ECR impacts ACh-peak values. © 2014 Elsevier Inc. All rights reserved.
Introduction Endothelial dysfunction appears as an essential element in the early onset of cardiovascular disease (Deanfield et al., 2007; Turner et al., 2008). It is therefore of interest to develop techniques to assess the endothelial function in order to screen population for estimating the subjects' cardiovascular risk. In recent years, numerous methods have been developed and used to assess endothelial function at different levels: heart level and peripheral level (Flammer et al., 2012; Lekakis et al., 2011). The recently developed laser speckle contrast imaging (LSCI) technique has been shown to be of interest for peripheral endothelial tests (Mahe et al., 2013; Puissant et al., 2013, 2014; Humeau-Heurtier et al., ⁎ Corresponding author at: Imagerie Coeur-Vaisseaux, University Hospital, 2, Rue Henri Le Guilloux, 35033 Rennes Cedex 9, France. Fax: +33 2 99 28 43 64. E-mail addresses: [email protected], [email protected] (G. Mahé).
http://dx.doi.org/10.1016/j.mvr.2014.04.001 0026-2862/© 2014 Elsevier Inc. All rights reserved.
2013 with the following reference:Relevance of laser Doppler and laser speckle techniques for assessing vascular function: state of the art and future trends. Humeau-Heurtier A, Guerreschi E, Abraham P, Mahé G.IEEE Trans Biomed Eng. 2013 Mar;60(3):659-66). LSCI is based on the speckle phenomenon and explores the skin microcirculation up to a depth of nearly 300 μm (Mahe et al., 2012c; O'Doherty et al., 2009). LSCI measurements have a good reproducibility compared with other flowmetry techniques (Roustit et al., 2010; Tew et al., 2011;Puissant et al., 2013). It can be coupled with different pharmacological tests: the microdialysis and the iontophoresis (Mahe et al., 2012c). Microdialysis is an invasive method whereas the iontophoresis is a non-invasive one (Cracowski et al., 2011). Iontophoresis allows transdermal drug delivery using current application during a specific duration (Tesselaar and Sjoberg, 2011). Moreover, iontophoresis is safe and painless since low intensity currents are used. Depending on the charged drug used, different physiological pathways can be assessed (Khan et al., 2004; Mahe et al., 2012c; Tesselaar and Sjoberg, 2011). When acetylcholine (ACh) is used as an iontophoresed drug, the endothelial function can be studied (Cordovil
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et al., 2012; Morris and Shore, 1996; Puissant et al., 2014; Sauvet et al., 2011). It has been shown that this measurement with LSCI has an excellent intra- and inter-observer reproducibility (Humeau-Heurtier et al., 2013). To perform iontophoresis, two electrodes are required. One, socalled the active, contains the drug and the other one closes the current system to allow current delivery. The impact of the distance between both electrodes (inter-electrode distance) on the amplitude of the microvascular response has never been studied. This is of a major interest for the use of the technique in routine. In other words, where do physicians have to place the electrodes when performing an endothelial assessment using ACh iontophoresis? Furthermore, the main limitation of the iontophoresis method is that the delivered drug dose is unknown but depends on the current intensity and its application duration (Kalia et al., 2004; Tesselaar and Sjoberg, 2011). The Ohm's law (U = R × I) defines the relation between voltage U, resistance R and intensity I. When iontophoresis is performed, the intensity I (expressed in Ampere) of the delivered current is set by the operator. The voltage U (expressed in Volt) delivered by the generator is linked to the circuit resistance R (expressed in Ohm). The resistance of the circuit, called Electric Cutaneous Resistance (ECR), would be related to the resistance of the “vehicle” solution diluting the charged molecule, and to cutaneous resistance itself (Khan et al., 2004; Ramsay et al., 2002). Using laser Doppler flowmetry (LDF), Ramsay et al. have shown an inverse relationship between ECR and vasodilatory response to ACh iontophoresis using multiple current applications with intensity from 5 μA to 20 μA with NaCl (0.5%) as vehicle (Ramsay et al., 2002). Although deionized water resistance alone is greater than the resistance of NaCl alone, it has been suggested that the best “vehicle” for ACh iontophoresis would be deionized water because: (i) the resistance of solutions of ACh dissolved in NaCl or in deionized water is similar and (ii) the microvascular response is greater with deionized water (Khan et al., 2004). Different iontophoresis protocols have been published and divided into continuous current application protocols and protocols involving multiple current pulses separated by current-free intervals. We have previously studied the endothelial function using ACh (20g.l-1 iontophoresis with a continuous single current application (0.1 mA and 30 s) (Durand et al., 2004; Puissant et al., 2013). When ACh is dissolved in deionized water, we have shown that the endothelium-dependent vasodilation is biphasic and composed of a rapid peak where muscarinic receptor M3 is involved and a late plateau which involves muscarinic receptor and prostaglandins (Durand et al., 2004). Since it has been suggested that ECR influences the ACh dependent vasodilation using NaCl with multiple current applications, the question remains for iontophoresis of ACh with deionized water and a single continuous current application (Ramsay et al., 2002). Finally, it is still unknown whether the ECR is similar in a 7-day interval using a single current application. In other terms, what is the interday reproducibility of the ECR values? This is of interest because if the ECR influences the ACh dependent vasodilation and if there is a modification of the ECR at a 7-day interval, then the interpretation of the results might be modified with time. Thus the aims of this study are (i) to assess the effect of interelectrode distance on ACh dependent vasodilation, (ii) to assess the relationship between microvascular response to ACh iontophoresis and ECR using a protocol previously validated, (iii) to study the reproducibility of the ECR values at a 7-day interval, and (iii) to compare the ECR obtained at different inter-electrode distances.
were pregnancy, participation in another biomedical study, and the intake of anti-inflammatory drugs during the last 7 days. The fourteen subjects studied aged from 24 to 41 years (6 women, 8 men). One woman was under hormonal contraception. The mean body mass index was 21.61 (2.22) kg/m2, heart rate was 62 (10) beats per minute and rest blood pressure was 113 (11)/70 (5) mm Hg. The general characteristics measured for each protocol are shown in Table 1. Each subject gave his/her written informed consent prior to participation. This study, which received local ethics committee approval and which is conformed to the Declaration of Helsinki, was registered to the American National Institutes of Health database under reference N°: NCT01664572.
Materials and methods
Protocols
Study population
All the subjects had four appointments. Each appointment was separated by at least one day. For each appointment a microvascular recording was performed as follows: Transdermal iontophoresis of ACh (Sigma-Aldrich Corporation, L'Isle d'Abeau, France) has been performed
Fourteen volunteers (aged 18 years or older) without known cardiovascular disease were recruited in this study. Non-inclusion criteria
Microvascular recordings All microvascular tests were performed with subjects resting supine in a temperature-controlled room (23 ± 1 °C) (Abraham et al., 2013; Mahe et al., 2012b). All subjects had an acclimatization period longer than 20 min before the beginning of the cutaneous blood flow (CBF) acquisitions. LSCI recordings of the CBF forearm were performed using a 70-mW system (PeriCam PSI System ®, Perimed, Järfälla, Sweden) having a laser wavelength of 785 nm. The sampling frequency was 18 Hz. The distance between the laser head and skin surface was fixed at 15 cm, as previously validated (Mahe et al., 2011). LSCI recordings were performed avoiding any air movements (Mahe et al., 2012a). Images were stored on a computer and analyzed off-line. The perfusion values from the skin were calculated using the manufacturer's software (PimSoft 1.2.2.0®; Perimed, Järfälla, Sweden) before being exported to an Excel spreadsheet (Excel 2002 V3®, Microsoft, USA). The software expresses recorded values in laser speckle perfusion units (LSPU). Iontophoresis was carried out by manufacturer's iontophoresis device (periIont USB Power Supply®; Perimed, Järfälla, Sweden). Cardiovascular and temperature recordings Blood pressure was recorded from the left middle finger (Nexfin, Bmeye, Amsterdam, Netherlands) with mean arterial pressure (MAP) obtained from the continuous blood pressure signal. Blood pressure was simultaneously recorded during microvascular recordings. The MAP signal was recorded at a sampling rate of 18 Hz using an analogto-digital converter (MP150, Biopac Sys, Goleta, USA) and analyzed offline using the Acknowledge® software V3.5.4 (Biopac Sys, Goleta, USA). Local cutaneous temperature was measured on the right forearm using a surface thermocouple probe connected to an electronic thermometer (BAT-12®, Physitemp Instruments, Inc., Clifton, NJ, USA). Electrical cutaneous resistance (ECR) measurement The PeriIont Micropharmacology System (Perimed, Jarfalla, Sweden) and the dedicated PeriIont Software (Perimed, Jarfalla, Sweden) are new devices that simultaneously allow current delivery and electrical cutaneous resistance (ECR) measurement. The ECR was measured during the whole current application. The ECR follows an exponentially decreasing function during the current application. In what follows, ECR corresponds to the mean of the ECR values obtained during the whole current application (30 s) and expressed in kOhm (Ramsay et al., 2002; Tesselaar and Sjoberg, 2011).
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Table 1 General characteristics during the 4 appointments. D1 means Day 1 and D7 means Day 7.
Inter-electrode distance (cm) Skin temperature (°C) Mean systolic pressure (mm Hg) Mean diastolic pressure (mm Hg) Heart rate (bpm)
Appointment 1 (D1)
Appointment2 (D7)
Appointment 3
Appointment 4
5 32.9 (1.4) 112 (10) 70 (5) 62 (11)
5 33.2 (1.1) 117 (12) 72 (6) 62 (11)
10 33.7 (2.1) 116 (12) 72 (8) 66 (10)
15 32.6 (1.9) 118 (8) 72 (6) 64 (11)
in the right forearm skin of the subject. Two electrodes were randomly placed on the subject's skin: one was the drug-delivery chamber (LI 611, Perimed Jarfalla, Sweden) and the other was an electrode that closes the electric circuit (Kendall, Mansfield, USA). Drug-delivery chamber was filled with ACh (20 g·l−1) dissolved in deionized water and connected to the anode of the PeriIont Micropharmacology System (Perimed, Jarfalla, Sweden). Cathode electrode was stuck at 5 cm from the other. The single current stimulation was set at 0.10 mA for 30 s. All microvascular recordings lasted for 22.5 min. After a 2-minute resting period, a single current stimulation of 30 s was performed. Then, the microvascular blood flow was recorded for 20.5-min to record the late phase of ACh dependent vasodilation. Comparison of the ACh dependent vasodilation at three different interelectrode distances Three different appointments were fixed with the subjects to study whether or not inter-electrode distance induces changes in cutaneous resistance and endothelial dependent values. For each appointment, the inter-electrode distance was randomly set at either 5 cm or 10 cm or 15 cm. Relationship between the maximal ACh dependent vasodilation and the ECR The relationship between the maximal ACh vasodilation and the ECR was studied with the first appointment (inter-electrode distance set at 5 cm). Inter-day reproducibility of ECR The inter-day reproducibility of ECR was assessed with a second microvascular recording with an inter-electrode distance set at 5 cm. This second measurement with an inter-electrode distance set at 5 cm was performed seven days (D7) after the first measurement (Day 1; D1) with this same inter-electrode distance.
electrode distance were compared with ANOVA test for repeated measures. The normality of the distribution for each variable tested (ACh peak, ECR) was assessed by the Shapiro–Wilk test. Results were expressed in mean (standard deviation). The relationship between the ACh peak (in CVC) and the ECR (kOhm) was studied by the Pearson's correlation test. The intra-subject variability between D1 and D7 was evaluated by the typical error of the estimate (TEE) of the ECR as well as by the coefficient of variations (CV) with 95% confidence interval [95% CI] according to the procedure proposed by Hopkins (2000). The lower the CV, the better the reproducibility. The repeatability of measurements was expressed as intra-class correlation coefficients (ICC) (Bland, 2000). ICC values b 0.40; 0.40 to 0.75; and N0.75 correspond to low, fair to good and excellent agreements, respectively (Landis and Koch, 1977). Data are also presented with Bland–Altman plots (Bland and Altman, 1986). Statistical analysis was performed using SPSS v15.0 for Windows (LEAD Tech., Inc., USA). For all statistical tests, a value of p b 0.05 was considered statistically significant.
Results Effect of inter-electrode distance on ACh peak and ECR No statistical difference was found between the ECRs (F = 0.851; p = 0.43), and the ACh peaks (F = 0.421; p = 0.74) obtained at three different inter-electrode distances. For inter-electrode distances of 5 cm (D1), 5 cm (D7), 10 cm and 15 cm, mean ECR values were 143.9 (45.3) kΩ, 147.0 (53.3) kΩ, 138.7 (41.3) kΩ, and 135.6 (43.5) kΩ, respectively. Mean ACh peak values expressed in CVC were 0.86 (0.34) LSPU/mm Hg, 0.82 (0.28) LSPU/mm Hg, 0.86 (0.23) LSPU/mm Hg and 0.80 (0.21) LSPU/ mm Hg, respectively.
Comparison of the ECR at three different inter-electrode distances This comparison was performed during the appointments when the comparison of the ACh dependent vasodilation at three different interelectrode distances was performed. Data analysis CBF was measured over a region of interest (ROI) and a time of interest (TOI) from images recorded by the LSCI. CBF in a ROI is defined as the average of all the pixel perfusion values in the skin area of interest. The size of ROI was 20 mm2. TOI is defined as the average of ROI perfusion value during a given duration. TOI has been set to 5 s for the maximal ACh induced vasodilation (Rousseau et al., 2011). Results were expressed in cutaneous vascular conductance (CVC in LSPU/mm Hg) corresponding to CBF divided by mean blood pressure (MAP). The peak of ACh vasodilation (ACh peak) was the maximal value observed between 1 and 3 min after the end of current application. Statistical analysis Mean ACh peak as well as mean ECR measured at a 5 cm interelectrode distance, 10 cm inter-electrode distance and 15 cm inter-
Fig. 1. Relationship between the acetylcholine peak and electrical cutaneous resistance. LSPU means laser speckle perfusion unit. Electrical cutaneous resistance is expressed in kΩ.
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Relationship between ACh peak and ECR Values of ACh peak as well as values of ECR followed a normal distribution. A statistically significant inverse relationship (r = −0.60) was found between the ACh peak and ECR (Fig. 1) (p b 0.05). The higher the ECR, the lower the ACh peak. Inter-day reproducibility of ECR The CV of the inter-day reproducibility of the ECR was 9.1% [6.5– 15.1] with an ICC of 0.93 [0.81–0.98]. The Bland and Altman plots are presented in Fig. 2. The TEE was 13.8 [10.0–22.3] kΩ. Discussion This study demonstrates that (i) when endothelial function is assessed by ACh iontophoresis (0.1 mA and 30 s) the inter-electrode distance changes neither the measured average ACh peak values, nor the average ECR, (ii) there is an inverse relationship between the ACh peak and the ECR, and (iii) the inter-day reproducibility of the ECR is excellent. The question of the inter-electrode distance is important. Indeed two electrodes need to be placed on the skin to close the current system and allow current delivery for all iontophoresis measurements. We could have supposed that the higher the inter-electrode distance, the higher the ECR, then the lower the ACh dependent vasodilation. Our study shows that contrary to our hypothesis, in a range between 5 cm and 15 cm for the inter-electrode distance, mean values of ACh peak do not differ significantly and cutaneous resistances are not different. We assume that this is probably due to the small distance differences that we studied. However, the distances that we studied are the distances used in practice. These results are interesting because they mean that when an operator performs an ACh iontophoresis, electrodes can be placed wherever the operator wants in the range between 5 cm and 15 cm on the forearm. Ramsay et al. have previously shown that there is an inverse relationship between the vasodilation induced by iontophoresis of ACh distilled in NaCl and the ECR when multiple current applications are performed (Ramsay et al., 2002). Furthermore, all Ferrell's work has been performed using platinum electrodes, not AgCl electrodes as in our study. Using AgCl electrodes, there is no H2O hydrolysis, and therefore no risk of acid/basic change and subsequent chemical burn. Our results demonstrate that the same inverse relationship between the ACh
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peak and the ECR is found when ACh iontophoresis is performed with deionized water and a single current stimulation of 0.1 mA. Therefore, ECR should be monitored when performing ACh iontophoresis either with NaCl and multiple current applications or with deionized water and single current application. In many previous studies, ECR was not monitored because there was a lack of dedicated feature like amperometric biosensor unit, which was needed to perform such measurements (Rossi and Carpi, 2004; Rossi et al., 2009; Rousseau et al., 2009; Trzepizur et al., 2009). Recently, new iontophoresis devices have been developed and allow current delivery and resistance monitoring in the same time. Another original point presented in this study is that the ECR is similar at a 7-day interval for a defined subject whereas ECR between subjects can vary from 44 kΩ to 253 kΩ in our population. This variation is in the same range as the variation found by Ramsay et al. with a different iontophoresis protocol (Ramsay et al., 2002). This important variation of ECR between subjects might be an explanation of the different vasodilation responses found between subjects and could depend on the density of sweat ducts or hair follicles. Cordovil et al. have shown that young women have a higher ACh dependent vasodilation than young men (Cordovil et al., 2012). However we found no statistical difference between men's ECR and women's ECR in this study (data not shown). Due to our small population to study the gender difference effect, this point should be addressed in future works. The excellent reproducibility of the ECR values at a 7-day interval strengthens the idea developed by Ramsay et al. that the cutaneous resistance might rely on a large part from the skin site of measurements and even from the composition of the subject's skin itself. Further, it has been recently shown that ECR increases with age (Millet et al., 2012) and aging impairs electrical conduction along the endothelium of resistance arteries (Behringer et al., 2013). This corroborates the fact that the skin structure might modify the ECR. Indeed, there are many physiological changes in skin with aging such as a decrease of the hydration and modifications on lipid composition. Further, the rate of hair declines, as well as the quantity of eccrine, apocrine, and sebum secretion; the cutaneous vascular supply is also decreased (Balin and Pratt, 1989; Cerimele et al., 1990; Diridollou et al., 2007; Jung et al., 1997). Influence of the modifications of the skin composition with aging, melanin content, or hydration remains to be studied to determine the variables affecting the ECR. Finally, the last interesting question is do we need to take into account the ECR when assessing endothelial function with LSCI? Indeed, it is not known yet if the ECR must be taken into account in the way of expression of the results. Unfortunately, we cannot answer this interesting issue with this study but our work strengthens the fact that additional studies are needed. To date and to our knowledge, no study has assessed the endothelial function taking into account this factor. Several clinical prospective studies have to be performed in this way. Study limitations We only studied the iontophoresis of ACh, but other drugs like sodium nitroprussiate or insulin could have been tested to show whether the role of the ECR is similar from a drug to another enabling a possible standardization of microcirculatory study results. However, we have chosen to study ACh iontophoresis because (i) it is a simple way to assess the endothelial function, which is of interest in the cardiovascular field (Cracowski et al., 2006) and (ii) it is one of the most widely used drugs in the literature (Mahe et al., 2012c; Morris and Shore, 1996; Rossi et al., 2009; Rousseau et al., 2009; Trzepizur et al., 2009). Conclusion
Fig. 2. Representation of Bland and Altman of ECR measured on two separate days: R (D1) and R (D7) correspond to the ECR measured at D1 and D7; the red lines represent the confidence interval of 95%.
Our study shows that (i) inter-electrode distance can range from 5 to 15 cm without affecting ACh peak values and (ii) ECR is associated with the ACh peak value and has to be considered in the context of
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microcirculatory measurements especially when endothelial function is assessed by ACh iontophoresis. Taking into account these results, this study will contribute to standardize the endothelial function assessment using ACh iontophoresis and develop its use in clinical routine. Disclosures The study was supported in part by a grant from the “Institut National de la Santé et de la Recherche Médicale” (INSERM) and was promoted by the University Hospital of Angers. Author contributions CP and GM participated in the acquisition, analysis and treatment of the data. CP, AHH, GL and SD helped develop the project and provided technical and administrative support. SF and GL reviewed the manuscript. PA and GM supervised the project. All authors approved the final version of the manuscript. Acknowledgments The authors thank Lydie Gascoin, Isabelle Albertini and Yoanna Onillon for technical help. References Abraham, P., et al., 2013. Effect of skin temperature on skin endothelial function assessment. Microvasc. Res. Jul;88, 56–60. Balin, A.K., Pratt, L.A., 1989. Physiological consequences of human skin aging. Cutis 43, 431–436. Behringer, E.J., et al., 2013. Aging impairs electrical conduction along endothelium of resistance arteries through enhanced Ca2+-activated K+ channel activation. Arterioscler. Thromb. Vasc. Biol. 33, 1892–1901. Bland, M., 2000. An Introduction to Medical Statistics. Oxford University Press, London. Bland, J.M., Altman, D.G., 1986. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1, 307–310. Cerimele, D., et al., 1990. Physiological changes in ageing skin. Br. J. Dermatol. 122 (Suppl. 35), 13–20. Cordovil, I., et al., 2012. Evaluation of systemic microvascular endothelial function using laser speckle contrast imaging. Microvasc. Res. 83, 376–379. Cracowski, J.L., et al., 2006. Methodological issues in the assessment of skin microvascular endothelial function in humans. Trends Pharmacol. Sci. 27, 503–508. Cracowski, J.L., et al., 2011. Skin microdialysis coupled with laser speckle contrast imaging to assess microvascular reactivity. Microvasc. Res. 82, 333–338. Deanfield, J.E., et al., 2007. Endothelial function and dysfunction: testing and clinical relevance. Circulation 115, 1285–1295. Diridollou, S., et al., 2007. Comparative study of the hydration of the stratum corneum between four ethnic groups: influence of age. Int. J. Dermatol. 46 (Suppl. 1), 11–14. Durand, S., et al., 2004. Prostaglandins participate in the late phase of the vascular response to acetylcholine iontophoresis in humans. J. Physiol. 561, 811–819. Flammer, A.J., et al., 2012. The assessment of endothelial function: from research into clinical practice. Circulation 126, 753–767. Hopkins, W., 2000. Measures of reliability in sports medicine and science. Sports Med. 30, 1–15.
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Assessment of endothelial function by acetylcholine iontophoresis: Impact of inter-electrode distance and electrical cutaneous resistance Cyril Puissant a, Pierre Abraham a,b, Sylvain Durand c, Anne Humeau-Heurtier d, Sébastien Faure e, Georges Leftheriotis a,b, Guillaume Mahé f,g,⁎ a
Laboratory of Vascular Investigations, University Hospital of Angers, France L'UNAM University, University of Angers, Biologie Neurovasculaire et Mitochondriale Intégrée (BNMI), Unité mixte UMR CNRS 6214/INSERM U 1083, Medicine Faculty, Angers, France L'UNAM University, University du Maine, bEA 4334N, Motricity, Interactions, and Performance, Avenue Olivier Messiaen, 72085 Le Mans Cedex 9, France d L'UNAM University, University of Angers, LARIS — Laboratoire Angevin de Recherche en Ingénierie des Systèmes, 62 avenue Notre Dame du Lac, 49000 Angers, France e L'UNAM University, University of Angers, INSERM UMR 1063, Stress oxydant et pathologies métaboliques (SOPAM), Angers, France f CHU Rennes, Imagerie Coeur-Vaisseaux, F-35033 Rennes, France g INSERM Clinical Investigation Center CIC 1414, F-35043 Rennes, France b c
a r t i c l e
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Article history: Accepted 4 April 2014 Available online 13 April 2014
a b s t r a c t Objectives: Endothelial function can be assessed by acetylcholine (ACh) iontophoresis with single current application. The effect of inter-electrode distance as well as electrical cutaneous resistance (ECR) on ACh dependent vasodilation has never been studied using single current application. The aims of this study are (i) to compare ACh-peak and ECR measured at different inter-electrode distances, (ii) to assess the relationship between ACh-peak and ECR, (iii) and to study the reproducibility of the ECR values. Methods: Fourteen healthy subjects were included. Using laser speckle contrast imaging, ACh-iontophoreses (0.1 mA, 30 s) were performed on the forearm at a 7-day interval with an inter-electrode distance set at 5 cm. Two other inter-electrode distances were also evaluated: 10 cm and 15 cm. ECR was measured during each ACh-iontophoresis as well as the ACh-peak. Results: No statistical difference was found between the ACh-peak values obtained at 5 cm, 10 cm and 15 cm. ECRs were also not statistically different. An inverse relationship (r = −0.60) was found between the ACh-peak and ECR (p b 0.05). The coefficient of variation of the inter-day reproducibility of the ECR values was 9.1% [6.5%–15.1%] with an intra-class-correlation coefficient of 0.93 [0.81–0.98]. Conclusion: Inter-electrode distance ranging from 5 cm to 15 cm changes neither the ACh-peak value nor the ECR value. ECR impacts ACh-peak values. © 2014 Elsevier Inc. All rights reserved.
Introduction Endothelial dysfunction appears as an essential element in the early onset of cardiovascular disease (Deanfield et al., 2007; Turner et al., 2008). It is therefore of interest to develop techniques to assess the endothelial function in order to screen population for estimating the subjects' cardiovascular risk. In recent years, numerous methods have been developed and used to assess endothelial function at different levels: heart level and peripheral level (Flammer et al., 2012; Lekakis et al., 2011). The recently developed laser speckle contrast imaging (LSCI) technique has been shown to be of interest for peripheral endothelial tests (Mahe et al., 2013; Puissant et al., 2013, 2014; Humeau-Heurtier et al., ⁎ Corresponding author at: Imagerie Coeur-Vaisseaux, University Hospital, 2, Rue Henri Le Guilloux, 35033 Rennes Cedex 9, France. Fax: +33 2 99 28 43 64. E-mail addresses: [email protected], [email protected] (G. Mahé).
http://dx.doi.org/10.1016/j.mvr.2014.04.001 0026-2862/© 2014 Elsevier Inc. All rights reserved.
2013 with the following reference:Relevance of laser Doppler and laser speckle techniques for assessing vascular function: state of the art and future trends. Humeau-Heurtier A, Guerreschi E, Abraham P, Mahé G.IEEE Trans Biomed Eng. 2013 Mar;60(3):659-66). LSCI is based on the speckle phenomenon and explores the skin microcirculation up to a depth of nearly 300 μm (Mahe et al., 2012c; O'Doherty et al., 2009). LSCI measurements have a good reproducibility compared with other flowmetry techniques (Roustit et al., 2010; Tew et al., 2011;Puissant et al., 2013). It can be coupled with different pharmacological tests: the microdialysis and the iontophoresis (Mahe et al., 2012c). Microdialysis is an invasive method whereas the iontophoresis is a non-invasive one (Cracowski et al., 2011). Iontophoresis allows transdermal drug delivery using current application during a specific duration (Tesselaar and Sjoberg, 2011). Moreover, iontophoresis is safe and painless since low intensity currents are used. Depending on the charged drug used, different physiological pathways can be assessed (Khan et al., 2004; Mahe et al., 2012c; Tesselaar and Sjoberg, 2011). When acetylcholine (ACh) is used as an iontophoresed drug, the endothelial function can be studied (Cordovil
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et al., 2012; Morris and Shore, 1996; Puissant et al., 2014; Sauvet et al., 2011). It has been shown that this measurement with LSCI has an excellent intra- and inter-observer reproducibility (Humeau-Heurtier et al., 2013). To perform iontophoresis, two electrodes are required. One, socalled the active, contains the drug and the other one closes the current system to allow current delivery. The impact of the distance between both electrodes (inter-electrode distance) on the amplitude of the microvascular response has never been studied. This is of a major interest for the use of the technique in routine. In other words, where do physicians have to place the electrodes when performing an endothelial assessment using ACh iontophoresis? Furthermore, the main limitation of the iontophoresis method is that the delivered drug dose is unknown but depends on the current intensity and its application duration (Kalia et al., 2004; Tesselaar and Sjoberg, 2011). The Ohm's law (U = R × I) defines the relation between voltage U, resistance R and intensity I. When iontophoresis is performed, the intensity I (expressed in Ampere) of the delivered current is set by the operator. The voltage U (expressed in Volt) delivered by the generator is linked to the circuit resistance R (expressed in Ohm). The resistance of the circuit, called Electric Cutaneous Resistance (ECR), would be related to the resistance of the “vehicle” solution diluting the charged molecule, and to cutaneous resistance itself (Khan et al., 2004; Ramsay et al., 2002). Using laser Doppler flowmetry (LDF), Ramsay et al. have shown an inverse relationship between ECR and vasodilatory response to ACh iontophoresis using multiple current applications with intensity from 5 μA to 20 μA with NaCl (0.5%) as vehicle (Ramsay et al., 2002). Although deionized water resistance alone is greater than the resistance of NaCl alone, it has been suggested that the best “vehicle” for ACh iontophoresis would be deionized water because: (i) the resistance of solutions of ACh dissolved in NaCl or in deionized water is similar and (ii) the microvascular response is greater with deionized water (Khan et al., 2004). Different iontophoresis protocols have been published and divided into continuous current application protocols and protocols involving multiple current pulses separated by current-free intervals. We have previously studied the endothelial function using ACh (20g.l-1 iontophoresis with a continuous single current application (0.1 mA and 30 s) (Durand et al., 2004; Puissant et al., 2013). When ACh is dissolved in deionized water, we have shown that the endothelium-dependent vasodilation is biphasic and composed of a rapid peak where muscarinic receptor M3 is involved and a late plateau which involves muscarinic receptor and prostaglandins (Durand et al., 2004). Since it has been suggested that ECR influences the ACh dependent vasodilation using NaCl with multiple current applications, the question remains for iontophoresis of ACh with deionized water and a single continuous current application (Ramsay et al., 2002). Finally, it is still unknown whether the ECR is similar in a 7-day interval using a single current application. In other terms, what is the interday reproducibility of the ECR values? This is of interest because if the ECR influences the ACh dependent vasodilation and if there is a modification of the ECR at a 7-day interval, then the interpretation of the results might be modified with time. Thus the aims of this study are (i) to assess the effect of interelectrode distance on ACh dependent vasodilation, (ii) to assess the relationship between microvascular response to ACh iontophoresis and ECR using a protocol previously validated, (iii) to study the reproducibility of the ECR values at a 7-day interval, and (iii) to compare the ECR obtained at different inter-electrode distances.
were pregnancy, participation in another biomedical study, and the intake of anti-inflammatory drugs during the last 7 days. The fourteen subjects studied aged from 24 to 41 years (6 women, 8 men). One woman was under hormonal contraception. The mean body mass index was 21.61 (2.22) kg/m2, heart rate was 62 (10) beats per minute and rest blood pressure was 113 (11)/70 (5) mm Hg. The general characteristics measured for each protocol are shown in Table 1. Each subject gave his/her written informed consent prior to participation. This study, which received local ethics committee approval and which is conformed to the Declaration of Helsinki, was registered to the American National Institutes of Health database under reference N°: NCT01664572.
Materials and methods
Protocols
Study population
All the subjects had four appointments. Each appointment was separated by at least one day. For each appointment a microvascular recording was performed as follows: Transdermal iontophoresis of ACh (Sigma-Aldrich Corporation, L'Isle d'Abeau, France) has been performed
Fourteen volunteers (aged 18 years or older) without known cardiovascular disease were recruited in this study. Non-inclusion criteria
Microvascular recordings All microvascular tests were performed with subjects resting supine in a temperature-controlled room (23 ± 1 °C) (Abraham et al., 2013; Mahe et al., 2012b). All subjects had an acclimatization period longer than 20 min before the beginning of the cutaneous blood flow (CBF) acquisitions. LSCI recordings of the CBF forearm were performed using a 70-mW system (PeriCam PSI System ®, Perimed, Järfälla, Sweden) having a laser wavelength of 785 nm. The sampling frequency was 18 Hz. The distance between the laser head and skin surface was fixed at 15 cm, as previously validated (Mahe et al., 2011). LSCI recordings were performed avoiding any air movements (Mahe et al., 2012a). Images were stored on a computer and analyzed off-line. The perfusion values from the skin were calculated using the manufacturer's software (PimSoft 1.2.2.0®; Perimed, Järfälla, Sweden) before being exported to an Excel spreadsheet (Excel 2002 V3®, Microsoft, USA). The software expresses recorded values in laser speckle perfusion units (LSPU). Iontophoresis was carried out by manufacturer's iontophoresis device (periIont USB Power Supply®; Perimed, Järfälla, Sweden). Cardiovascular and temperature recordings Blood pressure was recorded from the left middle finger (Nexfin, Bmeye, Amsterdam, Netherlands) with mean arterial pressure (MAP) obtained from the continuous blood pressure signal. Blood pressure was simultaneously recorded during microvascular recordings. The MAP signal was recorded at a sampling rate of 18 Hz using an analogto-digital converter (MP150, Biopac Sys, Goleta, USA) and analyzed offline using the Acknowledge® software V3.5.4 (Biopac Sys, Goleta, USA). Local cutaneous temperature was measured on the right forearm using a surface thermocouple probe connected to an electronic thermometer (BAT-12®, Physitemp Instruments, Inc., Clifton, NJ, USA). Electrical cutaneous resistance (ECR) measurement The PeriIont Micropharmacology System (Perimed, Jarfalla, Sweden) and the dedicated PeriIont Software (Perimed, Jarfalla, Sweden) are new devices that simultaneously allow current delivery and electrical cutaneous resistance (ECR) measurement. The ECR was measured during the whole current application. The ECR follows an exponentially decreasing function during the current application. In what follows, ECR corresponds to the mean of the ECR values obtained during the whole current application (30 s) and expressed in kOhm (Ramsay et al., 2002; Tesselaar and Sjoberg, 2011).
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Table 1 General characteristics during the 4 appointments. D1 means Day 1 and D7 means Day 7.
Inter-electrode distance (cm) Skin temperature (°C) Mean systolic pressure (mm Hg) Mean diastolic pressure (mm Hg) Heart rate (bpm)
Appointment 1 (D1)
Appointment2 (D7)
Appointment 3
Appointment 4
5 32.9 (1.4) 112 (10) 70 (5) 62 (11)
5 33.2 (1.1) 117 (12) 72 (6) 62 (11)
10 33.7 (2.1) 116 (12) 72 (8) 66 (10)
15 32.6 (1.9) 118 (8) 72 (6) 64 (11)
in the right forearm skin of the subject. Two electrodes were randomly placed on the subject's skin: one was the drug-delivery chamber (LI 611, Perimed Jarfalla, Sweden) and the other was an electrode that closes the electric circuit (Kendall, Mansfield, USA). Drug-delivery chamber was filled with ACh (20 g·l−1) dissolved in deionized water and connected to the anode of the PeriIont Micropharmacology System (Perimed, Jarfalla, Sweden). Cathode electrode was stuck at 5 cm from the other. The single current stimulation was set at 0.10 mA for 30 s. All microvascular recordings lasted for 22.5 min. After a 2-minute resting period, a single current stimulation of 30 s was performed. Then, the microvascular blood flow was recorded for 20.5-min to record the late phase of ACh dependent vasodilation. Comparison of the ACh dependent vasodilation at three different interelectrode distances Three different appointments were fixed with the subjects to study whether or not inter-electrode distance induces changes in cutaneous resistance and endothelial dependent values. For each appointment, the inter-electrode distance was randomly set at either 5 cm or 10 cm or 15 cm. Relationship between the maximal ACh dependent vasodilation and the ECR The relationship between the maximal ACh vasodilation and the ECR was studied with the first appointment (inter-electrode distance set at 5 cm). Inter-day reproducibility of ECR The inter-day reproducibility of ECR was assessed with a second microvascular recording with an inter-electrode distance set at 5 cm. This second measurement with an inter-electrode distance set at 5 cm was performed seven days (D7) after the first measurement (Day 1; D1) with this same inter-electrode distance.
electrode distance were compared with ANOVA test for repeated measures. The normality of the distribution for each variable tested (ACh peak, ECR) was assessed by the Shapiro–Wilk test. Results were expressed in mean (standard deviation). The relationship between the ACh peak (in CVC) and the ECR (kOhm) was studied by the Pearson's correlation test. The intra-subject variability between D1 and D7 was evaluated by the typical error of the estimate (TEE) of the ECR as well as by the coefficient of variations (CV) with 95% confidence interval [95% CI] according to the procedure proposed by Hopkins (2000). The lower the CV, the better the reproducibility. The repeatability of measurements was expressed as intra-class correlation coefficients (ICC) (Bland, 2000). ICC values b 0.40; 0.40 to 0.75; and N0.75 correspond to low, fair to good and excellent agreements, respectively (Landis and Koch, 1977). Data are also presented with Bland–Altman plots (Bland and Altman, 1986). Statistical analysis was performed using SPSS v15.0 for Windows (LEAD Tech., Inc., USA). For all statistical tests, a value of p b 0.05 was considered statistically significant.
Results Effect of inter-electrode distance on ACh peak and ECR No statistical difference was found between the ECRs (F = 0.851; p = 0.43), and the ACh peaks (F = 0.421; p = 0.74) obtained at three different inter-electrode distances. For inter-electrode distances of 5 cm (D1), 5 cm (D7), 10 cm and 15 cm, mean ECR values were 143.9 (45.3) kΩ, 147.0 (53.3) kΩ, 138.7 (41.3) kΩ, and 135.6 (43.5) kΩ, respectively. Mean ACh peak values expressed in CVC were 0.86 (0.34) LSPU/mm Hg, 0.82 (0.28) LSPU/mm Hg, 0.86 (0.23) LSPU/mm Hg and 0.80 (0.21) LSPU/ mm Hg, respectively.
Comparison of the ECR at three different inter-electrode distances This comparison was performed during the appointments when the comparison of the ACh dependent vasodilation at three different interelectrode distances was performed. Data analysis CBF was measured over a region of interest (ROI) and a time of interest (TOI) from images recorded by the LSCI. CBF in a ROI is defined as the average of all the pixel perfusion values in the skin area of interest. The size of ROI was 20 mm2. TOI is defined as the average of ROI perfusion value during a given duration. TOI has been set to 5 s for the maximal ACh induced vasodilation (Rousseau et al., 2011). Results were expressed in cutaneous vascular conductance (CVC in LSPU/mm Hg) corresponding to CBF divided by mean blood pressure (MAP). The peak of ACh vasodilation (ACh peak) was the maximal value observed between 1 and 3 min after the end of current application. Statistical analysis Mean ACh peak as well as mean ECR measured at a 5 cm interelectrode distance, 10 cm inter-electrode distance and 15 cm inter-
Fig. 1. Relationship between the acetylcholine peak and electrical cutaneous resistance. LSPU means laser speckle perfusion unit. Electrical cutaneous resistance is expressed in kΩ.
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Relationship between ACh peak and ECR Values of ACh peak as well as values of ECR followed a normal distribution. A statistically significant inverse relationship (r = −0.60) was found between the ACh peak and ECR (Fig. 1) (p b 0.05). The higher the ECR, the lower the ACh peak. Inter-day reproducibility of ECR The CV of the inter-day reproducibility of the ECR was 9.1% [6.5– 15.1] with an ICC of 0.93 [0.81–0.98]. The Bland and Altman plots are presented in Fig. 2. The TEE was 13.8 [10.0–22.3] kΩ. Discussion This study demonstrates that (i) when endothelial function is assessed by ACh iontophoresis (0.1 mA and 30 s) the inter-electrode distance changes neither the measured average ACh peak values, nor the average ECR, (ii) there is an inverse relationship between the ACh peak and the ECR, and (iii) the inter-day reproducibility of the ECR is excellent. The question of the inter-electrode distance is important. Indeed two electrodes need to be placed on the skin to close the current system and allow current delivery for all iontophoresis measurements. We could have supposed that the higher the inter-electrode distance, the higher the ECR, then the lower the ACh dependent vasodilation. Our study shows that contrary to our hypothesis, in a range between 5 cm and 15 cm for the inter-electrode distance, mean values of ACh peak do not differ significantly and cutaneous resistances are not different. We assume that this is probably due to the small distance differences that we studied. However, the distances that we studied are the distances used in practice. These results are interesting because they mean that when an operator performs an ACh iontophoresis, electrodes can be placed wherever the operator wants in the range between 5 cm and 15 cm on the forearm. Ramsay et al. have previously shown that there is an inverse relationship between the vasodilation induced by iontophoresis of ACh distilled in NaCl and the ECR when multiple current applications are performed (Ramsay et al., 2002). Furthermore, all Ferrell's work has been performed using platinum electrodes, not AgCl electrodes as in our study. Using AgCl electrodes, there is no H2O hydrolysis, and therefore no risk of acid/basic change and subsequent chemical burn. Our results demonstrate that the same inverse relationship between the ACh
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peak and the ECR is found when ACh iontophoresis is performed with deionized water and a single current stimulation of 0.1 mA. Therefore, ECR should be monitored when performing ACh iontophoresis either with NaCl and multiple current applications or with deionized water and single current application. In many previous studies, ECR was not monitored because there was a lack of dedicated feature like amperometric biosensor unit, which was needed to perform such measurements (Rossi and Carpi, 2004; Rossi et al., 2009; Rousseau et al., 2009; Trzepizur et al., 2009). Recently, new iontophoresis devices have been developed and allow current delivery and resistance monitoring in the same time. Another original point presented in this study is that the ECR is similar at a 7-day interval for a defined subject whereas ECR between subjects can vary from 44 kΩ to 253 kΩ in our population. This variation is in the same range as the variation found by Ramsay et al. with a different iontophoresis protocol (Ramsay et al., 2002). This important variation of ECR between subjects might be an explanation of the different vasodilation responses found between subjects and could depend on the density of sweat ducts or hair follicles. Cordovil et al. have shown that young women have a higher ACh dependent vasodilation than young men (Cordovil et al., 2012). However we found no statistical difference between men's ECR and women's ECR in this study (data not shown). Due to our small population to study the gender difference effect, this point should be addressed in future works. The excellent reproducibility of the ECR values at a 7-day interval strengthens the idea developed by Ramsay et al. that the cutaneous resistance might rely on a large part from the skin site of measurements and even from the composition of the subject's skin itself. Further, it has been recently shown that ECR increases with age (Millet et al., 2012) and aging impairs electrical conduction along the endothelium of resistance arteries (Behringer et al., 2013). This corroborates the fact that the skin structure might modify the ECR. Indeed, there are many physiological changes in skin with aging such as a decrease of the hydration and modifications on lipid composition. Further, the rate of hair declines, as well as the quantity of eccrine, apocrine, and sebum secretion; the cutaneous vascular supply is also decreased (Balin and Pratt, 1989; Cerimele et al., 1990; Diridollou et al., 2007; Jung et al., 1997). Influence of the modifications of the skin composition with aging, melanin content, or hydration remains to be studied to determine the variables affecting the ECR. Finally, the last interesting question is do we need to take into account the ECR when assessing endothelial function with LSCI? Indeed, it is not known yet if the ECR must be taken into account in the way of expression of the results. Unfortunately, we cannot answer this interesting issue with this study but our work strengthens the fact that additional studies are needed. To date and to our knowledge, no study has assessed the endothelial function taking into account this factor. Several clinical prospective studies have to be performed in this way. Study limitations We only studied the iontophoresis of ACh, but other drugs like sodium nitroprussiate or insulin could have been tested to show whether the role of the ECR is similar from a drug to another enabling a possible standardization of microcirculatory study results. However, we have chosen to study ACh iontophoresis because (i) it is a simple way to assess the endothelial function, which is of interest in the cardiovascular field (Cracowski et al., 2006) and (ii) it is one of the most widely used drugs in the literature (Mahe et al., 2012c; Morris and Shore, 1996; Rossi et al., 2009; Rousseau et al., 2009; Trzepizur et al., 2009). Conclusion
Fig. 2. Representation of Bland and Altman of ECR measured on two separate days: R (D1) and R (D7) correspond to the ECR measured at D1 and D7; the red lines represent the confidence interval of 95%.
Our study shows that (i) inter-electrode distance can range from 5 to 15 cm without affecting ACh peak values and (ii) ECR is associated with the ACh peak value and has to be considered in the context of
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microcirculatory measurements especially when endothelial function is assessed by ACh iontophoresis. Taking into account these results, this study will contribute to standardize the endothelial function assessment using ACh iontophoresis and develop its use in clinical routine. Disclosures The study was supported in part by a grant from the “Institut National de la Santé et de la Recherche Médicale” (INSERM) and was promoted by the University Hospital of Angers. Author contributions CP and GM participated in the acquisition, analysis and treatment of the data. CP, AHH, GL and SD helped develop the project and provided technical and administrative support. SF and GL reviewed the manuscript. PA and GM supervised the project. All authors approved the final version of the manuscript. Acknowledgments The authors thank Lydie Gascoin, Isabelle Albertini and Yoanna Onillon for technical help. References Abraham, P., et al., 2013. Effect of skin temperature on skin endothelial function assessment. Microvasc. Res. Jul;88, 56–60. Balin, A.K., Pratt, L.A., 1989. Physiological consequences of human skin aging. Cutis 43, 431–436. Behringer, E.J., et al., 2013. Aging impairs electrical conduction along endothelium of resistance arteries through enhanced Ca2+-activated K+ channel activation. Arterioscler. Thromb. Vasc. Biol. 33, 1892–1901. Bland, M., 2000. An Introduction to Medical Statistics. Oxford University Press, London. Bland, J.M., Altman, D.G., 1986. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1, 307–310. Cerimele, D., et al., 1990. Physiological changes in ageing skin. Br. J. Dermatol. 122 (Suppl. 35), 13–20. Cordovil, I., et al., 2012. Evaluation of systemic microvascular endothelial function using laser speckle contrast imaging. Microvasc. Res. 83, 376–379. Cracowski, J.L., et al., 2006. Methodological issues in the assessment of skin microvascular endothelial function in humans. Trends Pharmacol. Sci. 27, 503–508. Cracowski, J.L., et al., 2011. Skin microdialysis coupled with laser speckle contrast imaging to assess microvascular reactivity. Microvasc. Res. 82, 333–338. Deanfield, J.E., et al., 2007. Endothelial function and dysfunction: testing and clinical relevance. Circulation 115, 1285–1295. Diridollou, S., et al., 2007. Comparative study of the hydration of the stratum corneum between four ethnic groups: influence of age. Int. J. Dermatol. 46 (Suppl. 1), 11–14. Durand, S., et al., 2004. Prostaglandins participate in the late phase of the vascular response to acetylcholine iontophoresis in humans. J. Physiol. 561, 811–819. Flammer, A.J., et al., 2012. The assessment of endothelial function: from research into clinical practice. Circulation 126, 753–767. Hopkins, W., 2000. Measures of reliability in sports medicine and science. Sports Med. 30, 1–15.
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