Acetylcholine iontophoresis in diabetic patients with and without peripheral neuropathy: a potential therapeutic tool

Background Iontophoresis had been widely used as a therapeutic option in the field of dermatology and physiotherapy. In vascular medicine, vasodilating response to acetylcholine (ACh) iontophoresis had been also previously studied in the evaluation of endothelial dysfunction in diabetes mellitus and its microvascular complications but less was published about its therapeutic implementations. The current study aimed at investigating the therapeutic role of ACh iontophoresis in the improvement of endothelial dysfunction seen in diabetic patients with and without peripheral neuropathy (PN). Patients and methods Forty patients with type 2 diabetes mellitus, 20 with and 20 without PN, were subjected to a therapeutic program of ACh iontophoresis three times a week for 2 successive weeks ‘long-term iontophoresis’. Percentage change in perfusion was measured in the two groups using laser Doppler flowmetry. Readings were taken pretreatment and post-treatment on three occasions: at baseline temperature, after local warming to 35°C, and at maximal flow after exposure to ACh iontophoresis ‘short-term iontophoresis’. Results Perfusion significantly improved after 2 weeks of therapy in the two groups with percentage change improvement of 180.65 vs. 131.50% in the baseline, 219.45 vs. 149.40% after local warming to 35°C and 269.60 vs. 236.95% after short-term ACh iontophoresis in patients without and those with PN, respectively, with P values of 0.0004, 0.0005, and 0.049, respectively. Conclusion ACh iontophoresis may be an optional treatment procedure for improving cutaneous perfusion in diabetics for further randomized control studies.


Background
Iontophoresis had been widely used as a therapeutic option in the field of dermatology and physiotherapy. In vascular medicine, vasodilating response to acetylcholine (ACh) iontophoresis had been also previously studied in the evaluation of endothelial dysfunction in diabetes mellitus and its microvascular complications but less was published about its therapeutic implementations. The current study aimed at investigating the therapeutic role of ACh iontophoresis in the improvement of endothelial dysfunction seen in diabetic patients with and without peripheral neuropathy (PN). Patients and methods Forty patients with type 2 diabetes mellitus, 20 with and 20 without PN, were subjected to a therapeutic program of ACh iontophoresis three times a week for 2 successive weeks 'long-term iontophoresis'. Percentage change in perfusion was measured in the two groups using laser Doppler flowmetry. Readings were taken pretreatment and posttreatment on three occasions: at baseline temperature, after local warming to 35°C, and at maximal flow after exposure to ACh iontophoresis 'short-term iontophoresis'.

Results
Perfusion significantly improved after 2 weeks of therapy in the two groups with percentage change improvement of 180.65 vs. 131.50% in the baseline, 219.45 vs. 149.40% after local warming to 35°C and 269.60 vs. 236.95% after short-term ACh iontophoresis in patients without and those with PN, respectively, with P values of 0.0004, 0.0005, and 0.049, respectively. Conclusion ACh iontophoresis may be an optional treatment procedure for improving cutaneous perfusion in diabetics for further randomized control studies.

Introduction
Iontophoresis is the process of delivery of a chemical substance to the skin through an electrical current applied through two adhesive electrodes depending on repulsive electromotive forces [1]. As a therapy, it was studied in the treatment of palmoplantar and axillary hyperhydrosis [2] plantar fasciitis [3], migraine [4], to enhance wound healing [5] and digital ischemia in systemic sclerosis [6].
In diabetes mellitus, acetylcholine (ACh) iontophoresis had been used in the assessment of cutaneous microcirculation and endothelial dysfunction [7][8][9][10] and its complications such as peripheral neuropathy (PN) [11,12] but less was published regarding its therapeutic implementations.

Patients and methods
This observational case-control study was approved by the Research Ethics Committee for experimental and clinical studies at the Faculty of Medicine, Cairo University, Egypt. Patients were asked to give a written informed voluntary consent to participate in the study after full description of all procedures including possible risk of skin irritation during treatment time.
The study included 40 patients with type 2 diabetes mellitus enrolled from Kasr Al-Ainy diabetes outpatient clinics and inpatient wards of the Internal Medicine Department, Cairo University. They were divided into two groups: group I included 20 patients without PN and group II included 20 patients with PN diagnosed according to neuropathy symptom score, neuropathy disability This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. score [13], and the 'yes or no' the Semmes-Weinstein monofilament test [14]. We excluded patients with diabetic autonomic neuropathy, skin allergy or inflammation, implanted cardiac pacemakers, peripheral vascular disease (PVD), unstable angina, and those with chronic renal impairment.
At the beginning, standard pulsed wave Doppler velocimetry was done for all patients using HDI 5000 echograph (ALT USA) color-coded duplex ultrasound to exclude PVD.
All patients were subjected to a three-step procedure: First evaluation procedure (pretreatment) Microcirculation blood flow was assessed using a single-point laser probe placed on the dorsum of the foot and a laser Doppler blood flow monitor (Periflux PF5000; Perimed, Sweden). Blood flow was measured in three situations: at room temperature '30°C' (A) as a baseline measurement, after local warming to 35°C (B) for 3 min, and lastly maximum flow was assessed using short-term ACh iontophoresis (C) delivered through a 'Perilont device' (Perimed, Sweden) apparatus applied at a current intensity of 0.4 mA for 3 min (Figure 1) [15].

Treatment procedure
Iontophoresis apparatus (Phyaction 787; Uniphy, Holland) was used to deliver ACh to the skin through an electrode placed on the medial side of the quadriceps muscle (5 cm above the knee) in addition to another indifferent electrode attached to the medial side of the calf (5 cm below the knee) ( Figure 2). A total of three sessions/week for 2 weeks were applied using a current intensity that ranged from 3 to 5 mA for 15 min per session (therapeutic long-term ACh iontophoresis).
To ensure sufficient safety of this treatment protocol (to avoid pain and burn), the following was considered [16,17]: (1) A current intensity below the pain threshold that was comfortably tolerated by patients was applied (usually below 5 mA).
(3) Decreased current intensity during treatment to accommodate for decrease in skin impedance. (4) The current intensity should be gradually increased. (5) Skin was checked every 3-5 min for any sign of irritation.
Second evaluation procedure (post-treatment) Cutaneous blood flow was reassessed after the treatment protocol using the same laser probe at the same three occasions 'A, B, and C'.

Figure 1
Monitoring of microcirculation with acetylcholine iontophoresis.

Discussion
ACh iontophoresis is a noninvasive technique used to deliver high local drug concentration causing cutaneous vasodilation without systemic side effects [18,19].
According to our results, microcirculation baseline perfusion at room temperature was better in patients without compared with those with PN in line with many previous studies [20][21][22][23][24] that confirms the potential pathogenic role of functional microcirculation abnormalities in the pathogenesis of PN and can be explained by impaired nerve axon reflex-mediated vasodilatation or the Lewis triple flare response noticed in PN patients [21].
We also found that local warming to 35°C, improved the perfusion in both studied groups with again much better response in patients without PN (P<0.0001) that agreed with the results of Arora et al. [23] with two differences; they raised the temperature to 44°C and included patients with combined PN and PVD 'before and after revascularization' as well as healthy individuals to show the importance of neuropathy in impaired cutaneous perfusion despite adequate correction of large vessel blood flow. Of note, both of our work and Arora et al. [23] study showed that improved perfusion after local heating was lesser than that achieved by iontophoresis which was not the case in another study by Christen et al. [25] that they explained by possible release of additional vasodilatory mediators other than nitric oxide in response to local heating considering that the latter study included 26 healthy individuals and non-diabetics.
According to our results, microcirculation perfusion after ACh iontophoresis improved by 269.60 and 236.95% in patients without and with PN, respectively, in line with Brooks and colleagues [16,21,24], who stated that abnormalities of microvascular blood flow as assessed by ACh Procedure of therapeutic acetylcholine iontophoresis.   iontophoresis were evident in diabetics but became more marked with the development of microvascular complications such as PN.
On the other hand, according to Beer et al. [26], both endothelial dependent (to ACh iontophoresis) and independent (to sodium nitroprusside iontophoresis) reactivity were significantly blunted in diabetics compared with healthy controls but regardless of the presence or absence of microvascular or macrovascular complications.
Regarding the therapeutic use of iontophoresis, there are many limitations including unknown and variable delivered drug dose that depends on the current intensity and duration of exposure [27,28] as well as electrical characteristics of human skin [29].
For ACh iontophoresis in particular, there is an additive limitation that includes rapid offset of the drug action once the current was terminated that may be explained by ACh esterase [30].
Despite the above-mentioned limitations, we tried in the current study to use ACh iontophoresis as a therapeutic tool depending on two important facts; first, there are two ways of current application protocols; continuous and multiple current pulses separated by current-free interval protocols [6] and the second thing that change of the current duration, intensity, and frequency as well as the duration of rest time before and in between stimulations may all enhance delivery of the drug [28].
So we used the multiple current pulse protocol, a relative higher current intensity '3-5 mA', for rather longer duration of '15 min' and with repeated frequency of 'three sessions/week for 2 weeks'.
Close to our protocol, Murray et al. [6] have demonstrated that increasing ACh iontophoresis time from 2 to 5 min led to more sustained vasodilation with increased perfusion in patients with systemic sclerosis.One more important note, we believe that applying the iontophoresis process in the thighs in our therapeutic maneuver may decrease the risk of prolonged current application depending on the fact that current density equals the current intensity divided by cross-sectional skin area in contact with the electrodes 'expressed in mA/Cm 2 ' [27].
Even more prolonged current duration and intensity was safely used before in the induction of hypohydrosis by tap-water iontophoresis at an intensity of 15-20 mA applied to each palm or sole for 30 min per session (vs. only 3-5 mA for 15 min in our work) for 10 consecutive days, followed by 1-2 maintenance sessions per week [31].
According to our results, percentage change in the improvement of local skin perfusion after sessions of Pretreatment and post-treatment perfusion in group I.

Figure 5
Percentage change in perfusion in the two groups.

Figure 4
Pretreatment and post-treatment perfusion in group II.
ACh iontophoresis for 2 weeks was close to the results of the studies by Durand and colleagues [32,33], who found improved cutaneous perfusion after ACh iontophoresis with biphasic response with early and transient peak that fade off gradually and reincreased after 10 days.
The rapid peak may be related to endothelium-dependent vasodilation through muscarinic receptor M3 stimulation, whereas the late response involves muscarinic receptor as well as prostaglandins [32] (Figs 4 and 5).

Conclusion
Therapeutic ACh iontophoresis may be a potentially useful tool to enhance cutaneous microcirculation in diabetics and its complications like PN in which local alteration of microcirculation may play a role in its pathogenesis.

Financial support and sponsorship
Nil.

Conflicts of interest
None declared.