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Local anesthesia reduces the maximal skin vasodilation during iontophoresis of sodium nitroprusside and heating
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Microvascular Research 66 (2003) 134 –139
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Local anesthesia reduces the maximal skin vasodilation during iontophoresis of sodium nitroprusside and heating Antonella Caselli,a,b Luigi Uccioli,b Lalita Khaodhiar,a and Aristidis Vevesa,* a
Joslin-Beth Israel Deaconess Foot Center, Department of Surgery, Microcirculation Laboratory, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA b Department of Internal Medicine, University of Tor Vergata, Rome, Italy Received 9 April 2003
Abstract Aim. To evaluate the effect of local anesthesia on the skin vasodilation induced by the iontophoresis of sodium nitroprusside and heating. Methods. Skin vascular reactivity, in response to iontophoresis of sodium nitroprusside (SNP), was evaluated at the forearm and foot in 13 neuropathic diabetic (DN) and 11 nonneuropathic diabetic (D) patients and 9 healthy, nondiabetic subjects who served as controls (C). The direct (DI) and nerve axon reflex-related (N-V) vasodilation were measured by using two single-point laser Doppler probes. The vasodilation in response to local warming was also assessed. A topical anesthetic was applied on the contralateral forearm and foot and all the measurements were repeated. Results. Dermal anesthesia resulted in a reduction of the direct vasodilation to SNP at the forearm [C: 58.1 ⫾ 16, D: 60.6 ⫾ 11%, and DN: 48.3 ⫾ 37% (postanesthesia percentage of reduction; mean ⫾ SEM), P ⬍ 0.01] and at the foot in all three groups (D: 38.5 ⫾ 12%, P ⬍ 0.01; C: 27.2 ⫾ 14% and DN: 11.3 ⫾ 17.5%, P ⫽ NS). The N-V related vasodilation was very low before and did not change after local anesthesia. The postanesthesia hyperemic response to warming was significantly reduced at low temperatures but did not change at 44°C. Conclusion. The sodium nitroprusside-related vasodilation is reduced after local anesthesia in a similar way in healthy subjects and diabetic patients with and without neuropathy. The response to heating is also reduced at low temperatures. This indicates a stabilizing effect of local anesthesia on the smooth muscle cell. © 2003 Elsevier Inc. All rights reserved. Keywords: Laser Doppler; Skin; Microcirculation; Endothelial function; Vascular reactivity; Smooth muscle cell
Introduction The development of new technologies made it possible to study the blood flow changes in response to various stimuli at the skin level as an index of the microcirculation function (Morris et al., 1995). More specifically, the vasodilator response to acetylcholine (ACh), iontophoretically applied to the skin, is considered to be an index of the maximal endothelium-dependent vasodilation (Furchgott and Zawadzki, 1980). On the other hand, sodium nitroprusside (SNP), which is a nitric oxide donor, is iontophoretically applied to directly induce relaxation of the vascular * Corresponding author. Microcirculation Lab, Palmer 317, West Campus, One Deaconess Rd., Boston, MA 02215, USA. Fax: ⫹1-617-6327090. E-mail address: [email protected] (A. Veves). 0026-2862/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0026-2862(03)00053-0
smooth muscle cells (VSMC) through an increase in cGMP formation (Rang and Dale, 1991). Therefore, SNP-related vasodilation is indicative of the endothelium-nondependent vasodilation or VSMC function. The iontophoresis technique utilizes a small electrical charge (usually 0.2 mA) to allow ions to penetrate in the superficial layers of the skin (Singh and Maibach, 1994). The technique is not invasive and does not have any side effect other than a transient erythema in the area where it is applied. Iontophoresis, combined with laser Doppler skin blood perfusion monitoring, is also attractive because it allows repetitive measurements since it has a relatively low coefficient of variation (ranging from 12% to 40%, depending on which laser Doppler is used; Kubli et al., 2000; Caselli et al., 2002). However, despite its widespread use, this technique may have technical and interpretative limitations.
A. Caselli et al. / Microvascular Research 66 (2003) 134 –139
ACh iontophoresis is not only able to induce a direct endothelium-dependent vasodilation, but also an indirect vasodilation adjacent to the site of its administration. The latter, called nerve axon reflex-related vasodilation, derives from the activation of the local C-nociceptive nerve fibers by ACh, causing the release of a number of vasodilating neuropeptides (Walmsley and Wiles, 1990). We have previously shown that local anesthesia significantly reduces the nerve axon reflex-related vasodilation while it has no effect on the total ACh-related vasodilation (Caselli et al., 2003). This indicates that ACh iontophoresis is a true and reliable index of the skin endothelium-dependent vasodilation and it is not affected by local nerve fiber function. Iontophoretic administration of SNP, as opposed to ACh, needs a cathodal charge. We and others have already shown that the maximal vasodilation achieved during the iontophoresis of sodium nitroprusside contains a nonspecific response that is believed to be related to galvanic effect of the employed constant current (Abou-Elenin et al., 2002; Morris and Shore, 1996). However, little is known regarding the exact nature of this nonspecific vasodilation. Skin blood flow increase in response to local warming is another method to test microcirculation nonspecific vasodilation. This vasodilator response is mediated by local production of nitric oxide, but other mechanisms, such as direct relaxation of the vascular smooth muscle cells in response to heating, may also be involved (Kellogg et al., 1999; Vinik et al., 2001). Therefore, the physiologic mechanisms underlying skin blood flow increase during local heating are still under investigation. In the present study, we have evaluated the effect of local anesthesia on the skin vasodilation that is induced by SNP iontophoresis and local heating.
Research design and methods Patients Thirteen neuropathic diabetic patients (DN), 11 nonneuropathic diabetic patients (D), and 9 healthy control subjects (C) were recruited from our unit. Details about the inclusion and exclusion criteria and population characteristics have already been reported elsewhere (Caselli et al., 2003). Briefly, the inclusion criterion for the two diabetic groups (DN and D) was an established diagnosis of type 1 or 2 diabetes mellitus, while the exclusion criteria were a smoke history during the previous 6 months, diagnosis of cardiovascular disease, stroke or transient ischemic attack, peripheral vascular disease (symptoms of claudication and/or absence of peripheral pulses), or any other serious chronic disease requiring active treatment. The study protocol was approved by the Institutional Review Board (IRB) and a written consent form was obtained from all participating volunteers. The presence of diabetic peripheral neuropathy was de-
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fined for a Neuropathy Disability Score (NDS) greater than 6/10 and a vibration perception threshold (VPT) at big toe greater or equal to 25 V for one foot and greater than 20 V for the other one, as previously described (Young et al., 1993). Laser Doppler iontophoresis Each patient was evaluated after a 20-min acclimatization period in a warm environment (room temperature 23– 24°C). The blood flow response to iontophoresis of 1% acetylcholine chloride and of 1% sodium nitroprusside solutions has been assessed at the volar surface of the forearm and at the dorsum of the foot. ACh induces endotheliumdependent vasodilation and also vasodilation adjacent to the site of its administration by the activation of the nerve axon reflex, whereas SNP induces an endothelium-independent vasodilation, since it acts directly on the VSMC by the local release of nitric oxide. Iontophoresis was performed for 60 s using a constant current of 200 mA. Two single-point laser probes (8 mm apart) and a DRT4 Laser Doppler Blood Flow Monitor (Moor Instruments Ltd., Millwey, Devon, UK) were used. The iontophoresis instrument consists of an iontophoresis delivery vehicle device that contains two chambers that accommodate two singlepoint laser probes. One probe is in touch with the iontophorized solution, thus measuring the direct ACh- or SNPrelated vasodilation (DI), while the other probe is not in touch with the iontophorized solution, thus measuring the indirect or nerve axon reflex-related vasodilation (N-V). The distance between the two probes is 8 mm. The reproducibility of the single-point laser Doppler measurements, tested on 5 volunteers over 10 consecutive days, was 40.1% for the direct response and 33.5% for the indirect response to SNP. Further details about the iontophoresis protocol have been previously described (Hamdy et al., 2001; Caselli et al., 2003). A topical local anesthetic that is an emulsion in which the oil phase consists of a mixture of lidocaine and prilocaine (lidocaine 2.5% and prilocaine 2.5%, EMLA cream) was also applied on the volar surface and dorsum of the contra lateral forearm and foot respectively for 1–2 h under occlusive dressing. When a satisfactory anesthesia was achieved, as indicated by abolition of tactile and pinprick sensation, all the blood flow measurements were repeated. Heating protocol To evaluate the effect of local anesthesia on the vascular response to heating, the blood flow response to heat was assessed in 9 healthy subjects (5 males/4 females, age 35.4 ⫾ 10 years) before and after the application of a topical anesthetic. We employed the DRT4 Laser Doppler Blood Flow Monitor (Moor Instruments Ltd.) to assess the blood flow changes in response to warming. The blood flow was measured at the volar surface of the forearm at the baseline
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A. Caselli et al. / Microvascular Research 66 (2003) 134 –139
Table 1 Demographic characteristics
No. of subjects (males) Age (years) Body Mass Index (BMI) Type 1/2 diabetes Diabetes duration (years) Neuropathy Disability Score (NDS) Vibration Perception Threshold (VPT) Semmes-Weinstein Monofilaments a
Diabetes with neuropathy (DN)
Diabetes without neuropathy (D)
Controls (C)
13 (2) 52.2 ⫾ 10 29.8 ⫾ 5.7 6/7 19.5 ⫾ 14 8.7 ⫾ 2.2a
11 (5) 46.9 ⫾ 11 28.2 ⫾ 8.8 7/4 16 ⫾ 12.5 0.4 ⫾ 1.2
9 (4) 49.3 ⫾ 11 26 ⫾ 1.4 — — 0⫾0
36.8 ⫾ 14.4a
6.0 ⫾ 2.0
5.1 ⫾ 0.8
5.8 ⫾ 0.5a
4.3 ⫾ 0.2
4.3 ⫾ 0.2
DN vs. D and C; P ⬍ 0.05.
temperature (36°C). The skin was then progressively heated at 40°C, 42°C, and finally 44°C, using a small brass heater (Moor Instruments Ltd.). The heating lasted for 6 min at each temperature. The averaged blood flow during the last minute at each temperature was calculated and entered for final analysis. The topical local anesthetic (EMLA cream) was simultaneously applied on the contralateral forearm for 1–2 h until a satisfactory anesthesia was achieved and all the measurements were repeated. Statistical analysis The Minitab statistical package (Minitab, State College, Pennsylvania) for personal computers was used for the statistical analysis. To compare the vasodilator responses before and after the induction of skin anesthesia, we used paired t test. Changes in microvascular blood flow are expressed as the percentage of increase over the baseline blood flow and the mean was used for comparisons. Parametric data are expressed as mean ⫾ SEM. All tests are two-tailed with significance taken as P ⬍ 0.05.
Results
anesthesia at the forearm in all three groups [C: 58.1 ⫾ 16%, D: 60.6 ⫾ 11%, and DN: 48.3 ⫾ 37% (postanesthesia percentage of reduction; mean ⫾ SEM), P ⬍ 0.01 vs. preanesthesia]. A similar reduction was also observed at the foot in all three groups, though it was statistically significant only in the diabetic group without neuropathy (D: 38.5 ⫾ 12%, P ⬍ 0.01; C: 27.2 ⫾ 14% and DN: 11.3 ⫾ 17.5%, P ⫽ NS vs. preanesthesia). In contrast, the nerve axon reflexrelated vasodilation was very low in all three groups at baseline both at the forearm and at the foot levels and did not change in response to local anesthesia. No changes were observed in the direct response after the iontophoresis of ACh before and after local anesthesia in any of the studied groups (data not shown). In Table 2, the indirect or nerve axon reflex-related (N-V) vasodilation after ACh and SNP iontophoresis are compared. In agreement with previous studies, ACh iontophoresis was able to stimulate a significant indirect vasodilation, which derives from the direct stimulation of the C-nociceptive fibers by ACh. In contrast, a negligible neurovascular response was observed after iontophoresis of NSP, which was comparable to the N-V response observed after ACh iontophoresis at the foot of diabetic patients with peripheral neuropathy. The resting blood flow was assessed by employing a Laser Doppler Perfusion Imager (LDP1 Lisca 2.0, Lisca development AB, Linkoping, Sweden), which has a better spatial variation compared to the single-point laser Doppler (Kubli et al., 2000). No changes in the resting blood flow were observed as a consequence of the Emla cream application when all participants were considered as one group [1.1 ⫾ 0.3 vs. 1.1 ⫾ 0.3 at the forearm; 1.3 ⫾ 0.5 vs. 1.4 ⫾ 0.4 at the foot (blood flow, AU); preanesthesia vs. postanesthesia, P ⫽ NS]. Similar results were also observed when analysis was performed for each group separately. Heating protocol After local dermal anesthesia, the skin hyperemic response to warming was significantly reduced at 40°C and 42°C compared to the preanesthesia response. Conversely, no change was observed between the pre- and postanesthesia responses at 44°C (Fig. 2).
Iontophoresis protocol Discussion Table 1 shows the demographic characteristics of the subjects enrolled in this study. Diabetic patients had the same diabetes duration but, as expected from the selection criteria, those with peripheral neuropathy had a higher NDS and VPT. The results of the direct (DI) and the indirect or nerve axon reflex-related (N-V) vascular responses to SNP in all three groups before and after local anesthesia are shown in Fig. 1. Comparing to baseline measurements, a significant reduction of the direct response to SNP was observed after
In the present study, we found that the application of a topical local anesthetic resulted in a reduction of the SNPrelated vasodilation. The nerve axon reflex-related vasodilation, which was very low after SNP iontophoresis, did not change after local anesthesia. Finally, the heat-related vasodilator response was reduced after dermal anesthesia only at low temperatures but not at 44°C. A significant reduction of the total vasodilator response to SNP was observed after local anesthesia. This suggests
A. Caselli et al. / Microvascular Research 66 (2003) 134 –139
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Fig. 1. Direct (DI) and nerve axon reflex-related (N-V) vasodilator response to sodium nitroprusside in healthy subjects (a), nonneuropathic diabetic patients (b), and neuropathic diabetic patients (c) (% change of blood flow over baseline, mean ⫾ SEM). *Pre anesthesia (black columns) vs. post local anesthesia (white columns), P ⬍ 0.05.
that direct stimulation of the vascular smooth muscle cell by nitric oxide is not the only factor that regulates and influences the SNP iontophoresis response. Similar results have
been previously reported by other investigators who attributed the nonspecific vasodilation to the galvanic effect of the employed constant current (Morris and Shore, 1996;
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A. Caselli et al. / Microvascular Research 66 (2003) 134 –139
Table 2 Nerve axon reflex-related (N-V) vasodilation (percentage of blood flow increase over baseline) at the forearm and foot levels in response to acetylcholine (ACh) and Sodium nitroprusside (SNP) iontophoresis in diabetic patients with (DN) and without (D) neuropathy and healthy subjects (C)
Forearm DN D C Foot DN D C
N-V response after ACh iontophoresis (%)
N-V response after SNP iontophoresis (%)
P
108.9 ⫾ 31 162.4 ⫾ 19 170.4 ⫾ 46
28.2 ⫾ 11 39.3 ⫾ 5 24.1 ⫾ 12
⬍0.05 ⬍0.01 ⬍0.01
26.3 ⫾ 9 63.0 ⫾ 11 66.6 ⫾ 12
21.4 ⫾ 6 25.6 ⫾ 15 23.4 ⫾ 5
0.39 0.10 ⬍0.01
Grossmann et al., 1995). This galvanic response can be prevented by employing a 5% saline solution (5% NaCl) as iontophoresis vehicle instead of deionized water (AbouElenin et al., 2002; Asberg et al., 1999). Although the reasons for this are not clear, it has been hypothesized that high molar concentrations of NaCl, by increasing the ionic strength of the solution subjected to iontophoresis, may reduce the total resistance in the electric circuit, resulting in a reduction of the electrical potential applied over the skin. It is also of interest that the use of an anodal current, which is employed for the iontophoresis of ACh, does not induce such a nonspecific vasodilation, indicating that polarity plays a significant role (Berliner, 1997; Durand et al., 2002). One reasonable explanation for the reduction in the SNP response after the application of local anesthesia may be a direct pharmacological action of lidocaine on the smooth muscle cell. In fact, lidocaine acts through the reduction of nerve excitability by interacting with voltage-gated Na⫹ channels, thus decreasing the nerve permeability to Na⫹. Theoretically, lidocaine can have acted not only on the nerve axon membrane but also on the VSMCs, resulting in a reduction of their ability to dilate in response to the cathodal current. Further support to this hypothesis is provided by the fact that local anesthetics can initially produce contraction of the VSMC, especially at low concentrations (Goodman and Gilman, 2001). Another possible mechanism may be related to the local application of the emulsion over the skin. More specifically, the Emla cream may have an effect similar to that of a 5% NaCl solution in reducing the electrical potential applied over the skin. Further studies are needed to fully understand the mechanism(s) responsible for this nonspecific currentinduced vasodilation. However, it should be emphasized that the iontophoresis of SNP is being employed to examine the maximal vasodilation that can be achieved by the direct stimulation of the VSMC. Therefore, we believe that even if pathways other than the generation of nitric oxide by the SNP are involved, the final measurement does represent the
maximal endothelium-independent vasodilation and can be used to assess the VSMC function. It could also be argued that a high charge related to a high current in a small area could be responsible for the observed results. However, this results in a delivery of a 6 mC · cm⫺2 dose, which is minimal. The fact that no galvanic response was observed after the iontophoresis of ACh, which requires reverse polarity, further indicates that the observed vasodilation was not related to very high current that results in skin injury. In agreement with previous studies, we observed a significant nerve axon reflex-related vasodilation after ACh iontophoresis. In contrast, a small N-V response was observed in this report and previous studies after SNP iontophoresis, indicating that SNP or the nonspecific galvanic effect, do not specifically stimulate the C-nociceptive fibers (Kilo et al., 2000). It is worth noticing that the N-V response observed after SNP iontophoresis in all three groups at both the forearm and foot levels was equal to the ACh-induced neurovascular response of diabetic patients with peripheral neuropathy. This further supports the hypothesis that SNP iontophoresis does not stimulate the C-fibers. The heat-related vasodilator response was also reduced after dermal anesthesia at 40°C and 42°C but not at 44°C. Similar findings have been previously reported (Minson et al., 2001) and suggest that stimulation of the small nerve fibers may be involved in the vasodilator response to heat at low temperatures. On the other hand, we believe that the vasoconstrictor effect of local anesthesia itself may have counteracted the vasodilator effect induced by warming at low temperatures. However, higher temperatures have a stronger effect on the VSMC and can achieve maximal vasodilation despite any small fiber activity and/or anesthesia-induced vasoconstriction. In conclusion, the nature of SNP-related nonspecific vasodilation remains unclear even though a role of the C-fibers has been excluded. A direct effect of the electrical stimulation on the VSMCs is proposed as the main contributing factor to this galvanic vasodilation. The skin vasodilator response to warming at 44°C is not affected by small nerve fiber function, indicating that it is mainly dependent on direct relaxation of the VSMC. SNP iontophoresis and local
Fig. 2. Heat-related vasodilator response in healthy subjects [blood flow (AU); *pre vs. post local anesthesia, P ⬍ 0.01].
A. Caselli et al. / Microvascular Research 66 (2003) 134 –139
heating are reliable methods to test the maximal vasodilator ability of the skin VSMCs.
References Abou-Elenin, K., Xydakis, A., Hamdy, O., Economides, P.A., Horton, E.S., Veves, A., 2002. The effect of aspirin and various iontophoresis solution vehicles on skin microvascular reactivity. Microvasc. Res. 63, 91–95. Asberg, A., Holm, T., Vassbotn, T., Andreassen, A.K., Hartmann, A., 1999. Nonspecific microvascular vasodilation during iontophoresis is attenuated by application of hyperosmolar saline. Microvasc. Res. 58, 41– 48. Berliner, M.N., 1997. Skin microcirculation during tap water iontophoresis in humans: cathode stimulates more than anode. Microvasc. Res. 54, 74 – 80. Caselli, A., Bedi, D.S., O’Connor, C., Shah, C., Veves, A., 2002. Assessment of Laser Doppler Perfusion Imager’s in vivo reliability: can it be used for a prospective analysis. J. Laser Appl. 14, 198 –202. Caselli, A., Rich, J., Hanane, T., Uccioli, L., Veves, A., 2003. Role of C-nociceptive fibers in the impairment of nerve axon reflex-related vasodilation In diabetes. Neurology 60, 297–300. Durand, S., Fromy, B., Bouye, P., Saumet, J.L., Abraham, P., 2002. Current-induced vasodilation during water iontophoresis (5 min, 0.10 mA) is delayed from current onset and involves aspirin sensitive mechanisms. J. Vasc. Res. 39, 59 –71. Furchgott, R.F., Zawadzki, J.V., 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373–376. Grossmann, M., Jamieson, M.J., Kellogg Jr., D.L., Kosiba, W.A., Pergola, P.E., Crandall, C.B., Shepherd, A.M., 1995. The effect of iontophoresis on cutaneous vasculature: evidence for current-induced hyperemia. Microvasc. Res. 50, 444 – 452. Hamdy, O., Abou-Elenin, K., LoGerfo, F.W., Horton, E.S., Veves, A., 2001. Contribution of nerve-axon reflex-related vasodilation to the total
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skin vasodilation in diabetic patients with and without neuropathy. Diabetes Care 24, 344 –349. Hardman, J.G., Limbird, L.E. (Eds.), 2001. Goodman & Gilman’s: The Pharmacological Basis of Therapeutics. McGraw–Hill, New York, Edition 10, pp. 373–384. Kellogg, Jr., D.L., Liu, Y., Kosiba, I.F., O’Donnell, D., 1999. Role of nitric oxide in the vascular effects of local warming of the skin in humans. J. Appl. Physiol. 1185–1190. Kilo, S., Berghoff, M., Hilz, M., Freeman, R., 2000. Neural and endothelial control of the microcirculation in diabetic peripheral neuropathy. Neurology 54, 1246 –1252. Kubli, S., Waeber, B., Dalle-Ave, A., Feihl, F., 2000. Reproducibility of laser Doppler imaging of skin blood flow as a tool to assess endothelial function. J. Cardiovasc. Pharmacol. 36, 640 – 648. Minson, C.T., Berry, L.T., Joyner, M.J., 2001. Nitric oxide and neurally mediated regulation of skin blood flow during local heating. J. Appl. Physiol. 91, 1619 –1626. Morris, S.J., Shore, A.C., Tooke, J.E., 1995. Responses of the skin microcirculation to acetylcholine and sodium nitroprusside in patients with NIDDM. Diabetologia 38, 1337–1344. Morris, S.J., Shore, A.C., 1996. Skin blood flow responses to the iontophoresis of acetylcholine and sodium nitroprusside in man: possible mechanisms. J. Physiol. 15, 531–542. Rang, H.P., Dale, M.M., 1991. The heart, in: Pharmacology. Churchill Livingstone, London, pp. 309 –345. Singh, P., Maibach, H.I., 1994. Iontophoresis in drug delivery: basic principles and applications. Crit. Rev. Ther. Drug Carrier Syst. 11, 161–213. Vinik, A.I., Erbas, T., Park, T.S., Stansberry, K.B., Scanelli, J.A., Pittenger, G.L., 2001. Dermal neurovascular dysfunction in type 2 diabetes. Diabetes Care 24, 1468 –1475. Walmsley, D., Wiles, P.G., 1990. Assessment of the neurogenic flare response as a measure of nociceptor C fibre function. J. Med. Eng. Technol. 14, 194 –196. Young, M.J., Boulton, A.J., MacLeod, A.F., Williams, D.R., Sonksen, P.H., 1993. A multicentre study of the prevalence of diabetic peripheral neuropathy in the United Kingdom hospital clinic population. Diabetologia 36, 150 –154.
Microvascular Research 66 (2003) 134 –139
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Local anesthesia reduces the maximal skin vasodilation during iontophoresis of sodium nitroprusside and heating Antonella Caselli,a,b Luigi Uccioli,b Lalita Khaodhiar,a and Aristidis Vevesa,* a
Joslin-Beth Israel Deaconess Foot Center, Department of Surgery, Microcirculation Laboratory, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA b Department of Internal Medicine, University of Tor Vergata, Rome, Italy Received 9 April 2003
Abstract Aim. To evaluate the effect of local anesthesia on the skin vasodilation induced by the iontophoresis of sodium nitroprusside and heating. Methods. Skin vascular reactivity, in response to iontophoresis of sodium nitroprusside (SNP), was evaluated at the forearm and foot in 13 neuropathic diabetic (DN) and 11 nonneuropathic diabetic (D) patients and 9 healthy, nondiabetic subjects who served as controls (C). The direct (DI) and nerve axon reflex-related (N-V) vasodilation were measured by using two single-point laser Doppler probes. The vasodilation in response to local warming was also assessed. A topical anesthetic was applied on the contralateral forearm and foot and all the measurements were repeated. Results. Dermal anesthesia resulted in a reduction of the direct vasodilation to SNP at the forearm [C: 58.1 ⫾ 16, D: 60.6 ⫾ 11%, and DN: 48.3 ⫾ 37% (postanesthesia percentage of reduction; mean ⫾ SEM), P ⬍ 0.01] and at the foot in all three groups (D: 38.5 ⫾ 12%, P ⬍ 0.01; C: 27.2 ⫾ 14% and DN: 11.3 ⫾ 17.5%, P ⫽ NS). The N-V related vasodilation was very low before and did not change after local anesthesia. The postanesthesia hyperemic response to warming was significantly reduced at low temperatures but did not change at 44°C. Conclusion. The sodium nitroprusside-related vasodilation is reduced after local anesthesia in a similar way in healthy subjects and diabetic patients with and without neuropathy. The response to heating is also reduced at low temperatures. This indicates a stabilizing effect of local anesthesia on the smooth muscle cell. © 2003 Elsevier Inc. All rights reserved. Keywords: Laser Doppler; Skin; Microcirculation; Endothelial function; Vascular reactivity; Smooth muscle cell
Introduction The development of new technologies made it possible to study the blood flow changes in response to various stimuli at the skin level as an index of the microcirculation function (Morris et al., 1995). More specifically, the vasodilator response to acetylcholine (ACh), iontophoretically applied to the skin, is considered to be an index of the maximal endothelium-dependent vasodilation (Furchgott and Zawadzki, 1980). On the other hand, sodium nitroprusside (SNP), which is a nitric oxide donor, is iontophoretically applied to directly induce relaxation of the vascular * Corresponding author. Microcirculation Lab, Palmer 317, West Campus, One Deaconess Rd., Boston, MA 02215, USA. Fax: ⫹1-617-6327090. E-mail address: [email protected] (A. Veves). 0026-2862/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0026-2862(03)00053-0
smooth muscle cells (VSMC) through an increase in cGMP formation (Rang and Dale, 1991). Therefore, SNP-related vasodilation is indicative of the endothelium-nondependent vasodilation or VSMC function. The iontophoresis technique utilizes a small electrical charge (usually 0.2 mA) to allow ions to penetrate in the superficial layers of the skin (Singh and Maibach, 1994). The technique is not invasive and does not have any side effect other than a transient erythema in the area where it is applied. Iontophoresis, combined with laser Doppler skin blood perfusion monitoring, is also attractive because it allows repetitive measurements since it has a relatively low coefficient of variation (ranging from 12% to 40%, depending on which laser Doppler is used; Kubli et al., 2000; Caselli et al., 2002). However, despite its widespread use, this technique may have technical and interpretative limitations.
A. Caselli et al. / Microvascular Research 66 (2003) 134 –139
ACh iontophoresis is not only able to induce a direct endothelium-dependent vasodilation, but also an indirect vasodilation adjacent to the site of its administration. The latter, called nerve axon reflex-related vasodilation, derives from the activation of the local C-nociceptive nerve fibers by ACh, causing the release of a number of vasodilating neuropeptides (Walmsley and Wiles, 1990). We have previously shown that local anesthesia significantly reduces the nerve axon reflex-related vasodilation while it has no effect on the total ACh-related vasodilation (Caselli et al., 2003). This indicates that ACh iontophoresis is a true and reliable index of the skin endothelium-dependent vasodilation and it is not affected by local nerve fiber function. Iontophoretic administration of SNP, as opposed to ACh, needs a cathodal charge. We and others have already shown that the maximal vasodilation achieved during the iontophoresis of sodium nitroprusside contains a nonspecific response that is believed to be related to galvanic effect of the employed constant current (Abou-Elenin et al., 2002; Morris and Shore, 1996). However, little is known regarding the exact nature of this nonspecific vasodilation. Skin blood flow increase in response to local warming is another method to test microcirculation nonspecific vasodilation. This vasodilator response is mediated by local production of nitric oxide, but other mechanisms, such as direct relaxation of the vascular smooth muscle cells in response to heating, may also be involved (Kellogg et al., 1999; Vinik et al., 2001). Therefore, the physiologic mechanisms underlying skin blood flow increase during local heating are still under investigation. In the present study, we have evaluated the effect of local anesthesia on the skin vasodilation that is induced by SNP iontophoresis and local heating.
Research design and methods Patients Thirteen neuropathic diabetic patients (DN), 11 nonneuropathic diabetic patients (D), and 9 healthy control subjects (C) were recruited from our unit. Details about the inclusion and exclusion criteria and population characteristics have already been reported elsewhere (Caselli et al., 2003). Briefly, the inclusion criterion for the two diabetic groups (DN and D) was an established diagnosis of type 1 or 2 diabetes mellitus, while the exclusion criteria were a smoke history during the previous 6 months, diagnosis of cardiovascular disease, stroke or transient ischemic attack, peripheral vascular disease (symptoms of claudication and/or absence of peripheral pulses), or any other serious chronic disease requiring active treatment. The study protocol was approved by the Institutional Review Board (IRB) and a written consent form was obtained from all participating volunteers. The presence of diabetic peripheral neuropathy was de-
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fined for a Neuropathy Disability Score (NDS) greater than 6/10 and a vibration perception threshold (VPT) at big toe greater or equal to 25 V for one foot and greater than 20 V for the other one, as previously described (Young et al., 1993). Laser Doppler iontophoresis Each patient was evaluated after a 20-min acclimatization period in a warm environment (room temperature 23– 24°C). The blood flow response to iontophoresis of 1% acetylcholine chloride and of 1% sodium nitroprusside solutions has been assessed at the volar surface of the forearm and at the dorsum of the foot. ACh induces endotheliumdependent vasodilation and also vasodilation adjacent to the site of its administration by the activation of the nerve axon reflex, whereas SNP induces an endothelium-independent vasodilation, since it acts directly on the VSMC by the local release of nitric oxide. Iontophoresis was performed for 60 s using a constant current of 200 mA. Two single-point laser probes (8 mm apart) and a DRT4 Laser Doppler Blood Flow Monitor (Moor Instruments Ltd., Millwey, Devon, UK) were used. The iontophoresis instrument consists of an iontophoresis delivery vehicle device that contains two chambers that accommodate two singlepoint laser probes. One probe is in touch with the iontophorized solution, thus measuring the direct ACh- or SNPrelated vasodilation (DI), while the other probe is not in touch with the iontophorized solution, thus measuring the indirect or nerve axon reflex-related vasodilation (N-V). The distance between the two probes is 8 mm. The reproducibility of the single-point laser Doppler measurements, tested on 5 volunteers over 10 consecutive days, was 40.1% for the direct response and 33.5% for the indirect response to SNP. Further details about the iontophoresis protocol have been previously described (Hamdy et al., 2001; Caselli et al., 2003). A topical local anesthetic that is an emulsion in which the oil phase consists of a mixture of lidocaine and prilocaine (lidocaine 2.5% and prilocaine 2.5%, EMLA cream) was also applied on the volar surface and dorsum of the contra lateral forearm and foot respectively for 1–2 h under occlusive dressing. When a satisfactory anesthesia was achieved, as indicated by abolition of tactile and pinprick sensation, all the blood flow measurements were repeated. Heating protocol To evaluate the effect of local anesthesia on the vascular response to heating, the blood flow response to heat was assessed in 9 healthy subjects (5 males/4 females, age 35.4 ⫾ 10 years) before and after the application of a topical anesthetic. We employed the DRT4 Laser Doppler Blood Flow Monitor (Moor Instruments Ltd.) to assess the blood flow changes in response to warming. The blood flow was measured at the volar surface of the forearm at the baseline
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Table 1 Demographic characteristics
No. of subjects (males) Age (years) Body Mass Index (BMI) Type 1/2 diabetes Diabetes duration (years) Neuropathy Disability Score (NDS) Vibration Perception Threshold (VPT) Semmes-Weinstein Monofilaments a
Diabetes with neuropathy (DN)
Diabetes without neuropathy (D)
Controls (C)
13 (2) 52.2 ⫾ 10 29.8 ⫾ 5.7 6/7 19.5 ⫾ 14 8.7 ⫾ 2.2a
11 (5) 46.9 ⫾ 11 28.2 ⫾ 8.8 7/4 16 ⫾ 12.5 0.4 ⫾ 1.2
9 (4) 49.3 ⫾ 11 26 ⫾ 1.4 — — 0⫾0
36.8 ⫾ 14.4a
6.0 ⫾ 2.0
5.1 ⫾ 0.8
5.8 ⫾ 0.5a
4.3 ⫾ 0.2
4.3 ⫾ 0.2
DN vs. D and C; P ⬍ 0.05.
temperature (36°C). The skin was then progressively heated at 40°C, 42°C, and finally 44°C, using a small brass heater (Moor Instruments Ltd.). The heating lasted for 6 min at each temperature. The averaged blood flow during the last minute at each temperature was calculated and entered for final analysis. The topical local anesthetic (EMLA cream) was simultaneously applied on the contralateral forearm for 1–2 h until a satisfactory anesthesia was achieved and all the measurements were repeated. Statistical analysis The Minitab statistical package (Minitab, State College, Pennsylvania) for personal computers was used for the statistical analysis. To compare the vasodilator responses before and after the induction of skin anesthesia, we used paired t test. Changes in microvascular blood flow are expressed as the percentage of increase over the baseline blood flow and the mean was used for comparisons. Parametric data are expressed as mean ⫾ SEM. All tests are two-tailed with significance taken as P ⬍ 0.05.
Results
anesthesia at the forearm in all three groups [C: 58.1 ⫾ 16%, D: 60.6 ⫾ 11%, and DN: 48.3 ⫾ 37% (postanesthesia percentage of reduction; mean ⫾ SEM), P ⬍ 0.01 vs. preanesthesia]. A similar reduction was also observed at the foot in all three groups, though it was statistically significant only in the diabetic group without neuropathy (D: 38.5 ⫾ 12%, P ⬍ 0.01; C: 27.2 ⫾ 14% and DN: 11.3 ⫾ 17.5%, P ⫽ NS vs. preanesthesia). In contrast, the nerve axon reflexrelated vasodilation was very low in all three groups at baseline both at the forearm and at the foot levels and did not change in response to local anesthesia. No changes were observed in the direct response after the iontophoresis of ACh before and after local anesthesia in any of the studied groups (data not shown). In Table 2, the indirect or nerve axon reflex-related (N-V) vasodilation after ACh and SNP iontophoresis are compared. In agreement with previous studies, ACh iontophoresis was able to stimulate a significant indirect vasodilation, which derives from the direct stimulation of the C-nociceptive fibers by ACh. In contrast, a negligible neurovascular response was observed after iontophoresis of NSP, which was comparable to the N-V response observed after ACh iontophoresis at the foot of diabetic patients with peripheral neuropathy. The resting blood flow was assessed by employing a Laser Doppler Perfusion Imager (LDP1 Lisca 2.0, Lisca development AB, Linkoping, Sweden), which has a better spatial variation compared to the single-point laser Doppler (Kubli et al., 2000). No changes in the resting blood flow were observed as a consequence of the Emla cream application when all participants were considered as one group [1.1 ⫾ 0.3 vs. 1.1 ⫾ 0.3 at the forearm; 1.3 ⫾ 0.5 vs. 1.4 ⫾ 0.4 at the foot (blood flow, AU); preanesthesia vs. postanesthesia, P ⫽ NS]. Similar results were also observed when analysis was performed for each group separately. Heating protocol After local dermal anesthesia, the skin hyperemic response to warming was significantly reduced at 40°C and 42°C compared to the preanesthesia response. Conversely, no change was observed between the pre- and postanesthesia responses at 44°C (Fig. 2).
Iontophoresis protocol Discussion Table 1 shows the demographic characteristics of the subjects enrolled in this study. Diabetic patients had the same diabetes duration but, as expected from the selection criteria, those with peripheral neuropathy had a higher NDS and VPT. The results of the direct (DI) and the indirect or nerve axon reflex-related (N-V) vascular responses to SNP in all three groups before and after local anesthesia are shown in Fig. 1. Comparing to baseline measurements, a significant reduction of the direct response to SNP was observed after
In the present study, we found that the application of a topical local anesthetic resulted in a reduction of the SNPrelated vasodilation. The nerve axon reflex-related vasodilation, which was very low after SNP iontophoresis, did not change after local anesthesia. Finally, the heat-related vasodilator response was reduced after dermal anesthesia only at low temperatures but not at 44°C. A significant reduction of the total vasodilator response to SNP was observed after local anesthesia. This suggests
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Fig. 1. Direct (DI) and nerve axon reflex-related (N-V) vasodilator response to sodium nitroprusside in healthy subjects (a), nonneuropathic diabetic patients (b), and neuropathic diabetic patients (c) (% change of blood flow over baseline, mean ⫾ SEM). *Pre anesthesia (black columns) vs. post local anesthesia (white columns), P ⬍ 0.05.
that direct stimulation of the vascular smooth muscle cell by nitric oxide is not the only factor that regulates and influences the SNP iontophoresis response. Similar results have
been previously reported by other investigators who attributed the nonspecific vasodilation to the galvanic effect of the employed constant current (Morris and Shore, 1996;
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Table 2 Nerve axon reflex-related (N-V) vasodilation (percentage of blood flow increase over baseline) at the forearm and foot levels in response to acetylcholine (ACh) and Sodium nitroprusside (SNP) iontophoresis in diabetic patients with (DN) and without (D) neuropathy and healthy subjects (C)
Forearm DN D C Foot DN D C
N-V response after ACh iontophoresis (%)
N-V response after SNP iontophoresis (%)
P
108.9 ⫾ 31 162.4 ⫾ 19 170.4 ⫾ 46
28.2 ⫾ 11 39.3 ⫾ 5 24.1 ⫾ 12
⬍0.05 ⬍0.01 ⬍0.01
26.3 ⫾ 9 63.0 ⫾ 11 66.6 ⫾ 12
21.4 ⫾ 6 25.6 ⫾ 15 23.4 ⫾ 5
0.39 0.10 ⬍0.01
Grossmann et al., 1995). This galvanic response can be prevented by employing a 5% saline solution (5% NaCl) as iontophoresis vehicle instead of deionized water (AbouElenin et al., 2002; Asberg et al., 1999). Although the reasons for this are not clear, it has been hypothesized that high molar concentrations of NaCl, by increasing the ionic strength of the solution subjected to iontophoresis, may reduce the total resistance in the electric circuit, resulting in a reduction of the electrical potential applied over the skin. It is also of interest that the use of an anodal current, which is employed for the iontophoresis of ACh, does not induce such a nonspecific vasodilation, indicating that polarity plays a significant role (Berliner, 1997; Durand et al., 2002). One reasonable explanation for the reduction in the SNP response after the application of local anesthesia may be a direct pharmacological action of lidocaine on the smooth muscle cell. In fact, lidocaine acts through the reduction of nerve excitability by interacting with voltage-gated Na⫹ channels, thus decreasing the nerve permeability to Na⫹. Theoretically, lidocaine can have acted not only on the nerve axon membrane but also on the VSMCs, resulting in a reduction of their ability to dilate in response to the cathodal current. Further support to this hypothesis is provided by the fact that local anesthetics can initially produce contraction of the VSMC, especially at low concentrations (Goodman and Gilman, 2001). Another possible mechanism may be related to the local application of the emulsion over the skin. More specifically, the Emla cream may have an effect similar to that of a 5% NaCl solution in reducing the electrical potential applied over the skin. Further studies are needed to fully understand the mechanism(s) responsible for this nonspecific currentinduced vasodilation. However, it should be emphasized that the iontophoresis of SNP is being employed to examine the maximal vasodilation that can be achieved by the direct stimulation of the VSMC. Therefore, we believe that even if pathways other than the generation of nitric oxide by the SNP are involved, the final measurement does represent the
maximal endothelium-independent vasodilation and can be used to assess the VSMC function. It could also be argued that a high charge related to a high current in a small area could be responsible for the observed results. However, this results in a delivery of a 6 mC · cm⫺2 dose, which is minimal. The fact that no galvanic response was observed after the iontophoresis of ACh, which requires reverse polarity, further indicates that the observed vasodilation was not related to very high current that results in skin injury. In agreement with previous studies, we observed a significant nerve axon reflex-related vasodilation after ACh iontophoresis. In contrast, a small N-V response was observed in this report and previous studies after SNP iontophoresis, indicating that SNP or the nonspecific galvanic effect, do not specifically stimulate the C-nociceptive fibers (Kilo et al., 2000). It is worth noticing that the N-V response observed after SNP iontophoresis in all three groups at both the forearm and foot levels was equal to the ACh-induced neurovascular response of diabetic patients with peripheral neuropathy. This further supports the hypothesis that SNP iontophoresis does not stimulate the C-fibers. The heat-related vasodilator response was also reduced after dermal anesthesia at 40°C and 42°C but not at 44°C. Similar findings have been previously reported (Minson et al., 2001) and suggest that stimulation of the small nerve fibers may be involved in the vasodilator response to heat at low temperatures. On the other hand, we believe that the vasoconstrictor effect of local anesthesia itself may have counteracted the vasodilator effect induced by warming at low temperatures. However, higher temperatures have a stronger effect on the VSMC and can achieve maximal vasodilation despite any small fiber activity and/or anesthesia-induced vasoconstriction. In conclusion, the nature of SNP-related nonspecific vasodilation remains unclear even though a role of the C-fibers has been excluded. A direct effect of the electrical stimulation on the VSMCs is proposed as the main contributing factor to this galvanic vasodilation. The skin vasodilator response to warming at 44°C is not affected by small nerve fiber function, indicating that it is mainly dependent on direct relaxation of the VSMC. SNP iontophoresis and local
Fig. 2. Heat-related vasodilator response in healthy subjects [blood flow (AU); *pre vs. post local anesthesia, P ⬍ 0.01].
A. Caselli et al. / Microvascular Research 66 (2003) 134 –139
heating are reliable methods to test the maximal vasodilator ability of the skin VSMCs.
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