Assessment of endothelial function by acetylcholine iontophoresis: Impact of inter-electrode distance and electrical cutaneous resistance

Highlights

• ACh iontophoresis with one current stimulation is a test of endothelial function.

• An inverse relationship exists between electrical cutaneous resistance and ACh peak.

• Inter-electrode distance between 5 and 15 cm does not modify the ACh peak.

Abstract

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 < 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.

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, Puissant et al., 2014, Humeau-Heurtier et al., 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 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, so-called 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 inter-day 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 inter-electrode 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.