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1Laboratory of Vascular Investigations, University Hospital, and 2Laboratory of Physiology, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6188, University of Medicine, Angers, France
Submitted 4 March 2005 ; accepted in final form 17 June 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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prostaglandins; iontophoresis; primary afferent fibers
The vascular response to galvanic current application is suggested to rely on an axon reflex vasodilation with either anodal or cathodal current (9, 13, 24, 46), as observed following transcutaneous electrical nerve stimulation (46). Because it seems quite unlikely that the direction of the current would differently excite nerve fibers, CIV should be the same, whatever the current polarity. However, many differences have been observed between anodal CIV and cathodal CIV, such as their kinetics and their amplitude. Indeed, cathodal CIV appears 90 s following the start of current application, whereas anodal CIV only appears after the current is stopped, suggesting that anodal current interferes with the mechanism leading to vasodilation. Neither break stimulation (7) nor anodal block hypotheses (41) were the sole explanation for the delayed onset of anodal CIV. For the same charge, expressed in millicoulomb [product of current magnitude (mA) by duration (s)], the amplitude of the anodal CIV is weaker than the one observed at the cathode (2, 27), although the reason for this difference is unknown. The exact nature of the excitatory mechanism then remains unclear. The most obvious differences between the anode and the cathode would be the pH change observed, according to current polarity. Nevertheless, although CIV is suggested as being due to the accumulation of protons under the anode, the mechanism remains unknown at the cathodal level.
Many studies have been conducted to investigate CIV at the anode, but, as a result of the differences observed between anodal and cathodal CIV, it is likely that the results previously observed at the anode cannot necessarily be extrapolated to the cathode. However, it is of major interest to study cathodal CIV, because cathodal current is used in various applications, such as sodium nitroprusside iontophoresis to test the integrity of vascular smooth muscle (2, 23, 27, 28) or insulin iontophoresis to investigate the local effect of insulin on skin blood flow (SkBF) (32, 38). In these particular cases, what is the role of cathodal CIV in the total vascular response?
At the anode, our laboratory has shown that, when current of 0.1 mA was applied for at least 1 min, the amplitude of the CIV increases with the duration of current application (5). This was not true at the cathode (5). However, for sodium nitroprusside iontophoresis, cathodal current applications <1 min are widely used to study the integrity of smooth muscle cells (1, 2, 22, 23, 27). Then the relationship between the amplitude of cathodal CIV and the current duration <1 min deserves to be studied.
In the aim to allow for large-molecule delivery or for dose-response studies, some authors have used segmented current application (13, 27), assuming that the vascular effect of repeated current applications, resulting in a defined cumulated charge, would be lower than the vascular effect of the all-at-once current application of the same total charge. However, at the anode, we have shown that segmented current application induced a greater vascular response than the one observed with all-at-once current application corresponding to the same total charge (6). This amplification observed following segmented anodal current application is assumed to occur via sensitization of afferent nerve endings. This sensitization is a long-lasting phenomenon (at least 60 min) and is aspirin sensitive, suggesting a participation of prostaglandins (PG) (6). Last, our laboratory has shown that a single oral high dose of aspirin resulted in a prolonged decrease of the amplified response induced by segmented anodal current application (8), but it had no effect on the slow vasodilation that occurred following the first application. The vascular response induced by segmented current application and its sensitivity to aspirin have not been studied at the cathode.
The aim of the present work was to study the mechanisms involved in the cathodal CIV in human skin. 1) We investigated the effects of different durations of cathodal current (<1 min) on the amplitude of CIV. 2) We hypothesized that segmented cathodal current application would result in an amplified vascular response induced by a prolonged primary afferent fiber sensitization and that this eventual sensitization mechanism would be a long-lived phenomenon, as reported for the anode. For this purpose, we studied whether CIV observed following segmented cathodal application was higher than the CIV observed following the same total charge delivered all at once. We also analyzed the influence of the interstimulation interval on the CIV. 3) Last, we analyzed whether aspirin interferes with the cathodal CIV observed following all-at-once and segmented current applications and the duration of the aspirin effect.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Nonsmoking, healthy volunteers with no clinical sign of, or risk factor for, vascular diseases participated in this study. Anthropometric characteristics of studied subjects are summarized in Table 1.
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Microvascular Investigation
We studied the variations in SkBF in response to 0.1-mA cathodal current application through deionized water on the volar aspect of the forearms, using laser-Doppler flowmetry. The laser-Doppler flowmetry technique has been shown to accurately monitor SkBF continuously (19, 34) and is not influenced by underlying muscle blood flow (35). The technique used has been extensively described elsewhere (5).
In the aim to assess SkBF, laser-Doppler probes connected to laser-Doppler flowmeters (Periflux PF4001, Perimed) were positioned on the volar aspect of the forearm skin. Each probe, also called "active" probe, was fixed to the skin with an adhesive patch designed with a sponge. Before each experiment, the sponge was wetted with 0.2 ml of deionized water and connected to the cathodal terminal of a current intensity-regulated supplier (Periiont, Micropharmacology System, PF382 Perimed, or A395 R linear stimulus isolator, WPI Instruments) for iontophoresis. Each anodal terminal was connected to an Ag-AgCl disposable electrode (Care 610, Kendall, Neustadt, Germany) fixed 5 cm apart from its respective active probe. Finally, connected to the heating system (Peritemp PF4005 Perimed), the active probe allows for local heating of the skin up to 44°C, to attain maximal cutaneous vasodilation capacity (20, 33, 36, 42). The number of active probes was chosen according to the protocol.
Connected to a laser-Doppler flowmeter (Periflux PF5000, Perimed), a reference laser-Doppler probe (PF408, Perimed) was systematically positioned on the volar aspect of the forearm skin in every experiment. This reference probe was used to control the stability of SkBF at an adjacent site without electrical or local heating application. Local cutaneous temperature was measured at another site of the forearm without electrical or local heating application, using a surface thermocouple probe connected to an electronic thermometer (BAT-12, Physitemp Instruments, Clifton, NJ). The surface thermocouple probe was positioned 5 cm from active probes. Systemic arterial blood pressure was monitored with the use of a Finapres 2350 (Ohmeda, Englewood, CO) positioned on the second or third finger of the hand.
Protocols
For each protocol, experiments were performed with the subjects placed supine in a quiet room with the ambient temperature set at 24 ± 1°C. They rested for 15 min before the start of the experiments. On the same subject, at least 1 day elapsed between two consecutive experiments, and at least 1 wk elapsed between two consecutive protocols.
A reference period of 2 min was recorded in resting conditions. After the reference period, we performed the stimulation, according to each protocol. After completion of the electrical stimulations within each protocol, a recovery period of at least 20 min was recorded to study the long-lasting effects of the current applications on SkBF, except for protocol 1, where the recovery period was 10 min. At the end of the recovery period, local heating to 44°C was systematically applied on active probes for 24 min.
Protocol 1: Response to different durations of cathodal current application. The aim of this protocol was to study the effects of different durations of 0.1-mA cathodal application on the amplitude of CIV in 14 subjects. The durations tested were 5, 10, 20, 30, or 40 s in a random order, with one, two, or four active probes, until eight subjects were tested with each duration. Subjects underwent from one to three experiments, but the same duration was never tested twice in the same subject. Zero-current application was performed to test for the possibility of nonspecific effects of the patch and deionized water to the skin.
Protocol 2: Response to 20-s cathodal current application delivered all at once or in two consecutive 10-s applications with different interstimulation intervals. This protocol was performed in two parts.
PART 1. On one of the two active probes used, cathodal current was applied for 20 s and on the other active probe for 10 s followed by a subsequent period of current application of the same duration. All-at-once current application and the first 10 s of the segmented current application were started simultaneously. The second 10-s cathodal current application was started 10 min following the end of the first current application period.
PART 2. Two consecutive periods of 10-s cathodal current application were performed on two active probes with 20- and 40-min interstimulation intervals.
Fourteen subjects were included in protocol 2. Among these 14 subjects, 9 performed part 1, and 9 performed part 2. Only four subjects performed both parts.
Protocol 3: Influence of aspirin pretreatment on the response to 20-s cathodal current application delivered all at once or in two consecutive periods of 10 s. Each subject (n = 8) underwent two series of three consecutive experiments after both aspirin and placebo pretreatment in a random order, resulting in six experiments per subject. At least 3 wk elapsed between pretreatments. Aspirin (Aspegic adulte 1 g; Sanofi-Synthelabo) was dissolved in 125 ml of orange juice to disguise the taste and appearance of aspirin, whereas nothing was added to the orange juice in the placebo experiments. Two hours before the first experiment of each series, subjects drank the 125-ml orange juice, blinded from the presence or not of aspirin in the glass. The experiments were conducted following each pretreatment: at 2 h (H2), day 3 (D3), and day 10 (D10). Each experiment was performed according to protocol 2, part 1.
Measurements
SkBF was assessed with laser-Doppler flowmeter in arbitrary units and recorded on a computer via an analog-to-digital converter (Biopac System) with a sample frequency of 3 Hz. Due to instantaneous variability due to vasomotion, individual recordings were averaged over 1-min intervals throughout each experiment.
Subsequently, SkBF was indexed as cutaneous vascular conductance (CVC), calculated as the ratio of SkBF to mean arterial blood pressure over the same 1-min intervals, to take into account possible changes in systemic hemodynamic conditions.
Finally, normalization of CVC to the maximum achieved in response to local heating (last minute of the heating period) was performed to better reflect changes in SkBF (21, 31). Then results were expressed in percentage of heat-induced maximal CVC (%MVC).
Statistical Analysis
In protocol 1, the relationship between the duration of cathodal current application and the amplitude of the vascular responses was studied with a correlation test (Pearson) at 10 min following the start of current application.
For data analysis and interpretation of protocols 2 and 3, we defined the following points: rest was the last minute of the resting period, and peak was the maximal value recorded in the recovery period.
In protocol 2, comparisons between rest and its respective peak values were performed with a two-tailed paired t-test. Multiple comparisons were performed with one-way ANOVA followed by Dunnett's multiple-comparison tests.
In protocol 3, comparisons between segmented and all-at-once current applications, as well as between placebo and aspirin within the all-at-once (20 s) and within the segmented (10 s + 10 s) current applications, were performed with a two-tailed unpaired t-test. Comparisons between rest and its respective peak values were performed with a two-tailed paired t-test.
Statistical analyses were performed with Prism (Prism 2.01 Graphpad Software). SkBF was recorded in arbitrary units and expressed as means ± SE in %MVC. A P value < 0.05 was considered significant in all statistical analyses.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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There were no significant differences in rest values under the active probes between groups for all protocols.
Protocol 1
SkBF changes recorded following 5-, 10-, 20-, 30-, or 40-s cathodal current application are represented in Fig. 1. The effect of the patch and deionized water on SkBF (called control in Fig. 1) was also recorded.
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Protocol 2
A significant increase in SkBF occurred in response to cathodal current application after both methods of current delivery (P < 0.05 vs. rest). However, the amplitude of the peak vascular response following segmented current application with 10-min interstimulation interval is higher (79.1 ± 8.6% MVC) than the one observed following all-at-once current application (39.5 ± 4.3% MVC, P < 0.05). Following segmented current application, the amplified response was not statistically different among interstimulation intervals (86.0 ± 14.27 and 83.1 ± 8.2% MVC with 20- and 40-min interstimulation intervals, respectively). Thus the difference of cathodal CIV amplitude between all-at-once and segmented applications was observed for all tested interstimulation intervals (Fig. 2).
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Protocol 3
At H2, D3, and D10 following aspirin pretreatment, a significant increase from rest of SkBF occurred in response to both all-at-once and segmented current applications. However, this CIV in response to both methods of current delivery was significantly reduced at H2 compared with placebo (P < 0.05). The difference of cathodal CIV amplitude between all-at-once and segmented cathodal current applications was abolished at H2, still reduced at D3, and present at D10 (P < 0.05).
Under placebo pretreatment, the vascular responses recorded following current application at H2, D3, and D10 mimicked the responses observed in protocol 2 for comparable interstimulation interval. In brief, we observed a significant increase of SkBF in response to cathodal current application after both methods of current delivery (P < 0.05 vs. rest) and a significant difference of cathodal CIV amplitude between all-at-once and segmented application (P < 0.05) (Fig. 3).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Cathodal current applications are widely used to perform iontophoresis of drugs such as sodium nitroprusside (2, 23, 27, 28) or insulin (32, 38). However, in humans, the vascular response recorded following cathodal iontophoresis of drugs does not result only from the delivered drug. Indeed, cutaneous CIV is observed following cathodal iontophoresis performed with deionized water. In a previous study, our laboratory reported that the cutaneous CIVs observed with 0.1-mA cathodal current applied for 1, 3, or 5 min were all close to 75% MVC (5). This study has shown that the amplitude of cathodal CIV was not correlated with the duration of current application when the duration was >1 min. In the present study, we observed that the amplitude of cathodal CIV was correlated with the duration of current application when duration is <1 min. In brief, when delivered all at once, 0.1-mA cathodal application of <1 min resulted in a CIV proportional to the duration of current application. This result is in contrast to the absence of correlation observed with longer duration. We then assume that the maximal cathodal CIV is obtained from 1-min current application. As a result, it is likely that, at the cathode, current application duration of <1 min would be preferred to limit CIV. However, cathodal CIV is observed with duration of current application as short as 10 s and thus remains a pitfall for noninvasive assessments of microvascular response to the drugs delivered.
In an effort to decrease the CIV or test increased doses of drugs, some authors have used segmented cathodal current application (13, 27). However, segmented cathodal current application resulted in an amplified vascular response compared with all-at-once cathodal current application of comparable total charge. Because a pure additive effect should not explain this amplified CIV, this suggests an increased sensitivity of nerve fibers to the electrical current, induced by the first period of current application. Indeed, CIV is assumed to rely on an axon reflex following current-induced primary afferent fiber excitation, since it was abolished following local anesthesia with Emla cream (5). Furthermore, the main fibers involved are C fibers, as CIV was decreased following chronic capsaicin pretreatments (5). Capsaicin-sensitive primary afferent excitation is followed by the release of a large variety of neurotransmitters. Among these neurotransmitters, substance P and calcitonin gene-related peptide are powerful vasodilators involved in neurogenic inflammation (25). Although calcitonin gene-related peptide and/or substance P release could explain the CIV observed following single-current application, it cannot explain the amplification observed following segmented current application. A sensitization of nerve endings should be considered as the underlying mechanism of the amplified cathodal CIV to segmented application in our experiments. Substances such as PG are synthesized and released from small-diameter sensory neurons. In parallel to their direct vasodilator effects, PG can be involved in sensitization mechanisms by binding to specific receptors that are localized on sensory neurons where they lower the firing threshold of these neurons (37). Then, following a single cathodal current application, PG could play a key role, either as direct vasodilator and/or as a potent sensitizer, resulting in an amplified CIV, if a second cathodal current application is performed. Then, using segmented current application, resulting in the same total charge as all-at-once application, increases the CIV. This sensitization results in an amplified response, even with long interstimulation intervals (up to 40 min). Then, as hypothesized, this sensitization is a long-lasting phenomenon.
It has been previously described that the cathodal CIV, recorded following long duration of current application, was sensitive to aspirin. Indeed, Berliner (4) demonstrated that the major vasodilation resulting from cathodal current application was decreased following aspirin treatment. Our laboratory also observed that the CIV resulting from 5-min cathodal application was reduced following aspirin treatment (5). The results of the present study showed the aspirin sensitivity of cathodal CIV recorded following a short duration of current application. Indeed, although there is some vasodilation following 20-s or 10-s + 10-s cathodal current application in the presence of aspirin at H2, Fig. 3 indicates that it was almost abolished. Furthermore, 2 h following aspirin treatment, the cathodal CIV recorded following either all-at-once or segmented applications was reduced compared with placebo. Then, we assumed that, following short duration of current application (all at once or segmented), cathodal CIV development is, in part, an aspirin-sensitive mechanism. Aspirin leads to a direct and irreversible blockade of PG synthesis through the acetylation of cyclooxygenase (COX), wherever these PGs could be synthesized. This principal effect of aspirin could be proposed to explain the inhibition of cathodal CIV induced by 20-s or 10-s + 10-s current application. In addition to its effect on PG synthesis, recent reports suggest that aspirin may exert an inhibition of vanilloid receptors (VR1) (40) and interfere with the function of acid-sensing ion channels (ASIC) (44). VR1 and ASIC can be activated by acidosis. However, as cathodal current application induces an alkalosis (26), proton accumulation could not occur, and VR1 or ASIC inhibition by aspirin pretreatment could not explain the decrease of the vascular response recorded following cathodal current application. Although cathodal CIV is aspirin sensitive, the blockade of PG synthesis decreased but could not totally abolish the CIV resulting from 20-s or 10-s + 10-s cathodal application, since various neuropeptides released at nerve endings may exert direct vasodilator effects, independent of the PG pathway (16, 18, 45). Then the exact mechanisms involved in this nonspecific vasodilation induced by cathodal current remain to be studied.
In addition to its effect on cathodal CIV from either all-at-once or segmented applications, aspirin pretreatment interferes with sensitization mechanisms. In the present study, at H2 and D3 following the single oral high dose of aspirin intake, the difference of cathodal CIV amplitude between all-at-once and segmented current applications was abolished or reduced. The second effect of aspirin in the present study was an inhibition of the sensitization mechanism induced by the first application of current, limiting the CIV amplification and suggesting a major role of PG in this sensitization.
PGs are synthesized in a large variety of cells, including endothelium, smooth muscle (39), nerves (11), and platelets (29). Because a few hours are sufficient for COX to be resynthesized in endothelial or smooth muscle cells (15, 17), PGs of these origins could be involved in the cathodal CIV observed following 20-s or 10-s + 10-s current application. However, although the response at D3 following aspirin pretreatment is not statistically different from placebo, either for all-at-once or segmented cathodal current applications, the normal response to both methods appeared only to be restored at D10. Consistently, a single oral dose of aspirin had a prolonged effect on the difference of cathodal CIV amplitude between all-at-once and segmented applications. Thus, although the results from this study cannot differentiate between the roles of PG as direct vasodilator and/or as a sensitizer, it is unlikely that PGs involved in cathodal CIV are only of endothelial or smooth muscle cell origin. In nerves, COX, as other molecules, is synthesized in the cytoplasm close to the nucleus. Thereafter, molecules are transported to the periphery through active nerve trafficking at a maximal rate of 40 cm/day (12). It would be hypothesized that the time required to supply nerve endings with unblocked resynthesized COX would result in prolonged inhibition of the cathodal CIV and sensitization mechanisms. This hypothesis had been tested without success for sensitization mechanisms at the anode and would require further experiments at the cathode (8). Contrary to nucleated cells, the effect of aspirin in nonnucleated cells, like platelets, which are unable to resynthesized COX, is reversed when cells are replaced (e.g., 10 days after aspirin administration). Although there is no in vivo evidence of platelet-mediated vasodilation in humans, there is in vitro evidence of platelet-mediated vasorelaxation in animal vessels (10, 30). Whether platelets participate in this in vivo human model of CIV is a fascinating but still unproven possibility and also deserves future studies.
In conclusion, previous studies have shown differences between cathodal and anodal CIV, suggesting that anodal CIV mechanisms cannot be extrapolated at cathodal CIV. As observed at the anode, the present study showed that the amplitude of the cathodal CIV increased with the duration of the applied current. This relationship between duration and amplitude was observed for periods of <1 min, since cathodal CIV reached a plateau at 1 min. As reported for anodal CIV, an amplification of cathodal CIV is observed following segmented application, and the difference of amplitude between all-at-once and segmented applications is aspirin sensitive. Indeed, this difference is abolished at H2 and D3 following aspirin treatment, suggesting that sensitization likely relies on mediators other than PGs from endothelial or smooth muscle cells. In contrast to anodal CIV, our data showed that the CIV observed following a short duration of cathodal current application (all at once or segmented) is reduced at H2 or D3 and restored at D10 following aspirin pretreatment. This finding suggests that PG of nonendothelial or smooth muscle cell origin could also be involved in cathodal CIV as direct vasodilator. Because the durations of current application used in this study are usually reported in the literature for cathodal iontophoresis of sodium nitroprusside, aspirin pretreatment could be used to decrease the vasodilation resulting from single and repeated cathodal current applications and study the specific vascular effect induced by the delivered drug.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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