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Pathophysiology/Complications

ORIGINAL

ARTICLE

Contribution of Nerve-Axon Reflex-Related Vasodilation to the Total Skin Vasodilation in Diabetic Patients With and Without Neuropathy

OSAMA HAMDY, MD KARIM ABOU-ELENIN, MD FRANK W. LOGERFO, MD

EDWARD S. HORTON, MD ARISTIDIS VEVES, MD

OBJECTIVE — To examine the contribution of nerve-axon reflex-related vasodilation to total acetylcholine-induced vasodilation in the skin of normal and diabetic subjects.

RESEARCH DESIGN AND METHODS — The skin microcirculation was evaluated at the forearm level in 69 healthy subjects and 42 nonneuropathic diabetic patients and at the foot level in 27 healthy subjects and 101 diabetic patients (33 with neuropathy, 23 with Charcot arthropathy, 32 with peripheral vascular disease and neuropathy, and 13 without complications). Two single-point laser probes were used to measure total and neurovascular vasodilation response to the iontophoresis of 1% acetylcholine, 1% sodium nitroprusside, and deionized water.

RESULTS — The neurovascular response to acetylcholine was significantly higher than the response to sodium nitroprusside and deionized water (P 0.01). At the forearm level, the contribution of neurovascular response to the total response to acetylcholine was 35% in dia- betic patients and 31% in control subjects. At the foot level, the contribution was 29% in dia- betic patients without neuropathy and 36% in control subjects, while it was significantly diminished in the three neuropathic groups. A significantly lower nonspecific nerve- axon–related vasodilation was observed during the iontophoresis of sodium nitroprusside, which does not specifically stimulate the C nociceptive fibers.

CONCLUSIONS — Neurovascular vasodilation accounts for approximately one-third of the total acetylcholine-induced vasodilation at both the forearm and foot levels. The presence of diabetic neuropathy results in reduction of both the total vasodilatory response to acetylcholine and the percentage contribution of neurovascular vasodilation to the total response. Acetyl- choline and sodium nitroprusside cause vasodilation in the skin microcirculation through dif- ferent pathways.

Diabetes Care 24:344–349, 2001

F unctional abnormalities of the micro-

circulation have gained significant

attention in recent years for their

potential pathogenic role in the develop- ment of diabetic complications, particularly diabetic neuropathy and diabetic foot prob-

lems (1–5). The microvascular tone is reg- ulated by several humoral and neural fac- tors. The vascular endothelium has an important role in controlling the microvas- cular tone by releasing several vasodilator substances such as nitric oxide, prostacyclin

vasodilator substances such as nitric oxide, prostacyclin From the Clinical Research Center (O.H., K.A.-E., E.S.H.),
vasodilator substances such as nitric oxide, prostacyclin From the Clinical Research Center (O.H., K.A.-E., E.S.H.),

From the Clinical Research Center (O.H., K.A.-E., E.S.H.), Joslin Diabetes Center, Department of Medicine, and the Joslin–Beth Israel Deaconess Foot Center and Microcirculation Laboratory (F.W.L., A.V.), Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. Address correspondence and reprint requests to Aristidis Veves, MD, Microcirculation Laboratory, Palmer 317, West Campus, Beth Israel Deaconess Medical Center, One Deaconess Road, Boston, MA 02215. E-mail:

aveves@caregroup.harvard.edu. Received for publication 6 July 2000 and accepted in revised form 19 October 2000. Abbreviations: CV, coefficient of variation. A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.

and endothelium-derived hyperpolarizing factor, and vasoconstrictor substances such as prostaglandins and endothelin (6). Nitric oxide is the most important vasodilator substance responsible for endothelium- dependent vasodilation. After its secretion from the endothelium, it diffuses to the adjacent smooth muscle cells and stimu- lates the guanylate cyclase enzyme to pro- duce cyclic guanosine 3 ,5 -monophos- phate, which, in turn, leads to smooth muscle relaxation and vasodilation (7). The normal neurovascular response conducted through the C nociceptive nerve fibers is another important mechanism for the regulation of the microcirculation. Stimulation of the C nociceptive nerve fibers leads to antidromic stimulation of the adjacent C fibers, which secrete vasodilat- ing substances such as substance P and bradykinin, causing vasodilation at the injured or inflamed skin areas. This vasodi- lating response, also known as the Lewis triple-flare response, is decreased in the presence of diabetic neuropathy. Reduction of local blood flow increases the vulnera- bility of the neuropathic limb to severe dia- betic foot problems (8,9). It has been postulated that the abnormality in the neu- rovascular response in the neuropathic limb further aggravates the abnormalities in the microcirculation, and a vicious cycle may ensue (10). Several recent studies (10–13) have demonstrated reduced endothelium- dependent vasodilation in patients with either type 1 or type 2 diabetes. However, little information is available regarding the contribution of nerve-axon reflex-related vasodilatation to maximal skin vasodila- tion in such patients (8,9). The recent development of noninvasive techniques that can reliably quantify blood flow in the skin microcirculation has made it possible to study changes in microvascular function in patients with diabetes (14,15). In the present study, we have examined the con- tributing role of the nerve-axon reflex- related vasodilation response to the total skin vasodilation at both the forearm and

Hamdy and Associates Table 1— Characteristics of the forearm study subjects   Diabetic nonneuropathic patients

Hamdy and Associates

Table 1—Characteristics of the forearm study subjects

 

Diabetic nonneuropathic patients

Control subjects

n Age (years) Men/women Type 2 diabetes Diabetes duration (years) BMI (kg/m 2 )* HbA 1c (%)* Albumin-to-creatinine ratio Neuropathy symptom score* Neuropathy disability score* Vibration perception threshold*

42

69

54 ± 9

49 ± 9

21/21

33/36

42

27.3 ± 4.3 5.6 ± 0.4 0.02 ± 0.13 0.15 ± 0.62 10.69 ± 6.39

4 ± 5 32.3 ± 6.3 8.0 ± 1.6 30 ± 50 1.43 ± 2.27 0.8 ± 1.45 15.86 ± 9.5

Data are means ± SD. *P 0.001.

foot levels of neuropathic and nonneuro- pathic diabetic patients.

RESEARCH DESIGN AND METHODS

Patients We studied the skin microcirculation at the forearm level in 69 healthy subjects and 42 nonneuropathic diabetic subjects. The fol- lowing exclusion criteria were applied to subjects in all groups: smoking any amount of cigarettes during the previous 6 months, subjects with diagnosed cardio- vascular disease (coronary artery disease, arrhythmia, congestive heart failure), stroke or transient ischemic attack, periph- eral vascular disease (symptoms of claudi- cation and/or absence of peripheral pulses), chronic renal disease, severe dys- lipidemia (triglycerides 600 mg/dl or cholesterol 300 mg/dl), or any other seri- ous chronic disease requiring active treat- ment. Subjects were also excluded if they were on any of the following medications:

any type of antihypertensive drugs, lipid- lowering agents, glucocorticoids, antineo- plastic agents, psychoactive agents, or bronchodilators. In addition, diabetic patients with proliferative retinopathy, peripheral somatic neuropathy, macroal- buminuria (expressed as albumin-to-crea- tinine ratio 300 µg/mg), and/or on insulin or troglitazone were excluded from the study. We also evaluated the skin microvascu- lar reactivity at the foot level in 27 healthy subjects and 101 diabetic patients who were divided into four groups. The first group consisted of 33 diabetic neuropathic patients with a history of foot ulceration but no peripheral vascular disease, the second

group of 23 diabetic patients with Charcot arthropathy, the third group of 32 diabetic patients with peripheral vascular disease and neuropathy, and the fourth group of 13 diabetic patients without any complications. All healthy subjects were free of any ill- ness and did not take any medications. Spe- cial emphasis was given to exclude anyone with a history of hypertension, diabetes, hypercholesterolemia, active tobacco use, history of any systemic illness, or the use of any antihypertensive, cardiac, or hormonal medication. Patients with either type 1 or type 2 diabetes were included. Patients with nephropathy (creatinine 2 mg/l), severe heart failure, or any other serious illness were excluded from the study.

Further details of the characteristics of the study population are shown in Tables 1 and 2. The study was approved by the institutional review board, and consent was obtained from all participants.

Methods A history, physical examination, and fasting plasma glucose measurement were per- formed on all patients. Diabetic neuropathy was diagnosed according to the San Anto- nio Consensus Statement criteria (16). The symptoms were evaluated by using a neu- ropathy symptom score, and the clinical signs were evaluated by using a neuropathy disability score (17). Quantitative sensory testing included the assessment of vibration perception threshold using a Biothesiome- ter and cutaneous perception threshold using Semmes-Weinstein monofilaments (18,19). The diagnosis of Charcot neu- roarthropathy was made when gross destruction of the joints of the mid-foot that resulted in significant foot deformity was present. Patients were characterized as having peripheral vascular disease based on the presence of one or more of the follow- ing clinical features: claudication, absent foot pulses, and/or abnormal invasive and abnormal noninvasive vascular tests. Each participant was studied after a 20- min acclimatization period in a warm envi- ronment (room temperature 23–24°C). We used two single-point laser probes and a DRT4 Laser Doppler Blood Flow Monitor

Table 2—Characteristics of the foot study subjects

 

Charcot

Neuropathy and

Diabetic patients

Control

 

Neuropathy

arthropathy

peripheral vascular

without

subjects

(DN)

(DA)

disease (DI)

complications (D)

(C)

n

33

23

32

13

27

Age (years)* Men/women Type of diabetes (1/2) Diabetes duration BMI (kg/m 2 )

HbA 1c (%) Creatinine (mg/dl) Neuropathy symptom score† Neuropathy disability score† Vibration perception threshold† Semmes-Weinstein monofilaments†

56 ± 9

57 ± 9

60 ± 8

39 ± 10

52 ± 13

24/9

13/10

23/9

9/4

13/14

12/21

5/18

16/16

8/5

27.5 ± 4.9 — — 0.1 ± 0.4

21 ± 12 30.3 ± 6.8 8.7 ± 2.7 1.03 ± 0.3 3.2 ± 2.9

17 ± 11 29.5 ± 4.8 8.7 ± 2.0 1.01 ± 0.4 2.9 ± 2.5

25 ± 13 27.8 ± 4.5 8.9 ± 0.9 1.05 ± 0.031 3.5 ± 2.8

17 ± 7

± 9.9 ± 4.3 1.02 ± 0.03 0.5 ± 1.1

26.8

4.5

19.3 ± 6.1

21.8 ± 3.9

18.7 ± 6.7

0.5 ± 1.2

0.4 ± 1.2

48 ± 5

50 ± 3.4

47 ± 8

11 ± 5

12 ± 6

6.6 ± 0.7

6.9 ± 0.4

6.6 ± 0.5

4 ± 0.5

4 ± 0.5

Data are means ± SD. *DN, DA, and DI vs. D, P 0.001; †DN, DA, and DI vs. D and C, P 0.001.

Nerve-axon reflex and skin vasodilation Table 3— The contribution of nerve-axon reflex-related vasodilation to the

Nerve-axon reflex and skin vasodilation

Table 3—The contribution of nerve-axon reflex-related vasodilation to the total response to acetylcholine, sodium nitroprusside, and deionized water at the forearm level

 

Nonneuropathic diabetic patients

Control subjects

Total response to Ach Nerve-axon–related response to Ach The % contribution of nerve-axon response to the total response to Ach Total response to SNP Nerve-axon–related response to SNP The contribution of nerve-axon response to the total response to SNP Total response to W, anodal mode Nerve-axon–related response to W The % contribution of nerve-axon response to the total response to W Total response to W, cathodal mode Nerve-axon–related response to W The % contribution of nerve-axon response to the total response to W

835 (289–1476)

1,181 (547–2,299)

365 (120–513)

338 (207–706)

35 (16–83)*

31 (17–60)*

525 (307–974)

880 (445–2,178)

77 (22–230)

118 (40–769)

13 (6–34)

10 (3–22)

83 (15–400)

300 (65–897)

14 (1–60)

35 (8–141)

16 (2–54)

17 (4–40)

111 (45–315)

108 (20–252)

11 (1–60)

35 (0–22)

5 (1–35)

6 (0–27)

Data are medians (25th–75th quartiles). Ach, acetylcholine; SNP, sodium nitroprusside; W, deionized water. *Ach vs. SNP and W, P 0.01.

(Moor Instruments, Millwey, Devon, U.K.) to evaluate the skin microcirculation. Fore- arm microcirculatory flow was measured over the flexor surface of the forearm, and foot microcirculatory flow was measured over the dorsum of the foot. The blood flow response was measured in response to ion- tophoresis of each of three substances: 1% acetylcholine chloride solution (a substance that elicits both a neurovascular response and endothelium-dependent vasodilation), 1% sodium nitroprusside (a substance that does not elicit a neurovascular response, but induces endothelium-independent vasodilation), and deionized water (used as

a control during iontophoresis to measure

the vasodilation caused by the direct effect of a constant current flow) (10). Deionized water was iontophoresed in both anodal mode (the same mode in which the ion- tophoresis of acetylcholine is performed) and cathodal mode (the mode that the ion- tophoresis of sodium nitroprusside is per- formed). The difference in these two modes

is the polarity of the iontophoresis chamber:

the chamber serves as the anode for ion- tophoresis of acetylcholine, which has a negative electrical charge, and as the cath- ode for the iontophoresis of sodium nitro- prusside, which has a positive electrical charge. Therefore, the constant current has an opposite direction when the polarity of the chamber is changed. The iontophoresis instrument (MIC1 iontophoresis system; Moor Instruments) consists of an iontophoresis delivery vehicle device that sticks firmly to the skin with the help of adhesive tape. The device contains two chambers that accommodate two sin- gle-point laser probes. One probe is placed

within the chamber containing the ion- tophoresis solution (thus measuring the direct response to acetylcholine or sodium nitroprusside iontophoresis), while the sec- ond probe is placed outside but within proximity (within 5 mm) to the ion- tophoresis solution chamber, thus meas- uring the indirect nerve-axon–related response that results from stimulation of the C nociceptive nerve fibers. A small amount ( 1 ml) of test solution was applied to the iontophoresis chamber. Subsequently, a constant current of 200 µA for 60 s was applied, achieving a dose of 6 mC/cm 2 between the iontophoresis chamber and a second nonactive electrode placed 10–15

cm proximal to the chamber. The two laser

probes recorded changes in skin blood flow. Measurements were obtained for 40 s before the iontophoresis, during the ion- tophoresis, and 90 s after it (10,11). The day-to-day reproducibility of the technique was evaluated in five healthy subjects (four men and one woman, ages 23–39 years) who were repeatedly tested at their foot

and forearm for 10 consecutive working

days. With use of a single-point laser probe,

the coefficient of variation (CV) for the base-

line blood flow before iontophoresis of acetylcholine was 60.6% and for the maxi- mal hyperemic response was 35.2% after

the iontophoresis of acetylcholine.

Statistical analysis The results were recorded and tabulated before revealing the patient category assig- nations. Changes in microvascular blood flow were expressed as the percentage of increase over baseline, where median, first quartile, and third quartile values are used

for comparisons. Parametric data were expressed as means ± SD. Statistical analysis was performed using the Minitab computer software (State College, PA), using both parametric and nonparametric tests. All tests were two-tailed, with significance taken as P 0.05. For between-group comparisons, we used paired t test for parametric data and Kruskal-Wallis test for nonparametric data.

RESULTS

Forearm level The results of the iontophoresis are shown in Table 3. To evaluate the degree of vasodi- lation that is specific to the neurovascular response, we measured the capillary blood flow in a skin area in direct contact with acetylcholine and in an adjacent skin area not in direct contact with it. The latter rep- resents the nerve-axon–related portion of the total response. The percentage contri- bution of the nerve-axon–related response to the total response was similar between nonneuropathic diabetic patients and the control group after the iontophoresis of acetylcholine (35 and 31%, respectively, NS). In both the nonneuropathic diabetic patients and control group, the percentage contribution of the nerve-axon–related response to the total response was signifi- cantly less after the iontophoresis of either sodium nitroprusside (13 and 10%, respec- tively, P 0.01) or deionized water (16 and 17%, respectively, P 0.01). No sig- nificant difference was seen between the percentage contribution of the nerve- axon–related reflex to the total response to sodium nitroprusside and to deionized water both in anodal and cathodal mode in

Hamdy and Associates Table 4— Contribution of nerve-axon reflex-related vasodilation to the total response to

Hamdy and Associates

Table 4—Contribution of nerve-axon reflex-related vasodilation to the total response to acetylcholine, sodium nitroprusside, and deionized water at the foot level

 

Charcot

Neuropathy and

Diabetic patients

Control

 

Neuropathy

arthropathy

peripheral vascular

without

subjects

(DN)

(DA)

disease (DI)

complications (D)

(C)

Total response to Ach Nerve-axon–related response to Ach The % contribution of nerve-axon response to the total response to Ach Total response to SNP Nerve-axon–related response to SNP The % contribution of nerve-axon response to the total response to SNP Total response to W, Anodal mode Nerve-axon–related response to W The % contribution of nerve-axon response to the total response to W Total response to W, Cathodal mode Nerve-axon–related response to W The % contribution of nerve-axon response to the total response to W

90 (15–378)

227 (86–554)

74 (1–212)

578 (152–1,858)

411 (148–541)

4 (0–26)

13 (1–52)

5 (0–52)

118 (19–304)

153 (60–264)

8 (0–31)*

5 (0–27)†

20 (0–70)‡

29 (7–52)§

36 (18–88)§

89 (31–227)

80 (74–400)

86 (7–239)

234 (141–520)

234 (129–590)

10 (0–24)

2 (0–32)

1 (0–12)

27 (8–87)

48 (16–108)

8 (0–31)

10 (0–29)

2 (0–18)

12 (2–35)

9 (4–76)

8 (0–40)

12 (1–50)

19 (0–31)

238 (35–427)

33 (11–107)

0 (0–13)

6 (0–12)

3 (0–10)

23 (7–28)

12 (0–25)

11 (0–100)

43 (6–106)†

26 (0–81)

13 (2–48)

18 (6–111)

4 (0–14)

12 (0–38)

18 (0–42)

28 (10–109)

25 (8–43)

0 (0–9)

1 (0–12)

0 (0–15)

11 (1–42)

3 (0–11)

35 (0–100)

18 (6–106)†

4 (0–81)

41 (2–48)

0 (6–111)

Data are medians (25th–75th quartiles). Ach, acetylcholine; SNP, sodium nitroprusside; W, deionized water. *DN vs. D and C, P 0.01; †DA vs. D and C, P 0.01; ‡DI vs. D and C, P 0.01; §Ach vs. SNP and W, P 0.01.

both the nonneuropathic diabetic patients and control group (NS). This is consistent with the fact that acetylcholine specifically stimulates C nociceptive fibers and the nerve-axon–related reflex, whereas sodium nitroprusside and deionized water do not. The contribution of the neurovascular response to the total response to acetyl- choline is approximately one-third of the total response and is not compromised by diabetes at the forearm level.

Foot level The results of the iontophoresis are shown in Table 4. In response to iontophoresis of acetylcholine, the percentage contribution of the nerve-axon–related response was similar to that seen at the forearm level in both the diabetic patients without complications and the healthy control subjects (29 and 36%, respectively, NS). The diabetic neuropathic patients had a significantly lower median increase of capillary blood flow over baseline in response to acetylcholine compared with the diabetic patients without complications and the control group (P 0.01) (Fig. 1). The neurovascular response was markedly decreased in all three neuropathic groups when compared with diabetic patients with- out complications and the control group. The contribution of the nerve-axon–related response to the total response was 8% in diabetic patients with neuropathy (P 0.01), 5% in diabetic patients with Charcot

arthropathy (P 0.01), and 20% in diabetic patients with neuropathy and peripheral vascular disease (P 0.01). The nerve- axon–related response to sodium nitro- prusside and to the anodal and cathodal iontophoresis of deionized water was simi-

lar to the response observed in the upper extremity.

CONCLUSIONS In the present study, we have shown that in healthy sub- jects and in nonneuropathic diabetic

that in healthy sub- jects and in nonneuropathic diabetic Figure 1— Total and neurovascular (N) change

Figure 1—Total and neurovascular (N) change in skin blood flow in response to acetylcholine at the foot level. The median, first quartile, and third quartile and the range are shown. The total response is significantly lower in neuropathic diabetic patients than it is in control subjects and diabetic patients without neuropathy (P 0.01). The percentage contribution of neurovascular response to the total response is also significantly lower in neuropathic diabetic patients than in control subjects and diabetic patients without neuropathy (P 0.01).

Nerve-axon reflex and skin vasodilation patients, at both the forearm and foot lev- els, the

Nerve-axon reflex and skin vasodilation

patients, at both the forearm and foot lev- els, the microvascular vasodilation that is related to the neurovascular response accounts for approximately one-third of the total vasodilation that is observed after the iontophoresis of acetylcholine. This portion is markedly decreased in the pres- ence of diabetic neuropathy. The total microvascular vasodilation in response to acetylcholine is currently con- sidered to represent the sum of direct stim- ulation of the endothelium by acetylcholine and of the vasodilation that is related to the nerve-axon reflex (20). However, the mag- nitude of the contribution of each of these two factors to the total vasodilation has not been adequately studied, and the currently available data are conflicting. Thus, although some studies have suggested a considerably higher contribution of the neurovascular response, the techniques used did not allow the precise quantification of this contribu- tion (8,21). On the other hand, another study has shown that local sensory inhibi- tion by topical application of lignocaine and prilocaine did not have an effect on the total vasodilatory response to the iontophoresis of acetylcholine (22). The main problem in interpreting these data, though, lies in the fact that there is no evidence that topical application of lignocaine abolishes the nerve-axon reflex, since it may cause local anesthesia via mechanisms that are not affecting the antidromic stimulation of local C nociceptive fibers. This is further empha- sized by the findings of a previous study that showed that deep subcutaneous injec- tion of lignocaine does inhibit the nerve- axon–related vasodilation in response to the iontophoresis of acetylcholine (23). In the present study, we have used a chamber that can accommodate two single- point laser probes that can measure the total and the nerve-axon reflex-related vasodila- tion. This technique can satisfactorily mea- sure the two responses separately, making it possible to evaluate the relative contribution of the neurovascular response to the total response with an adequate reliability. Fur- thermore, we have studied subjects with and without peripheral neuropathy rather than testing with local anesthesia, which, as mentioned previously, has questionable effects on the nerve-axon reflex. Finally, it should be remembered that under condi- tions of stress (such as injury or inflamma- tion), hyperemia is necessary not only in the injured area alone but in a considerably larger area that surrounds the injured site. Because this response depends mainly on a

normal nerve-axon reflex, our findings make the point that this response, under normal conditions, is one-third of the maximal achievable vasodilation and that this is drastically reduced in the presence of dia- betic neuropathy. Single-point laser probe measurements are known to have a considerably high CV, whereas the use of laser scanners reduces this variability (10,16). However, with laser scanners, one cannot evaluate the nerve- axon response, a measurement that can be done only with use of the single-point laser technique. The large number of subjects in each studied group compensates for the high variability and does not affect the valid- ity of the conclusions regarding the contri- bution of nerve-axon response to total vasodilation. On the other hand, the high variability does not allow the direct com- parison of the vasodilatory response among the various studied groups, which makes this study prone to type 2 statistical error. Therefore, it is recommended that for reli- able data regarding these questions, the reader is directed to studies that have specif- ically addressed this question and used the appropriate techniques, including the use of a Laser Scanner Imager (10,11,24–26). In contrast to acetylcholine, sodium nitroprusside causes vasodilation by directly stimulating the vascular smooth muscle cell and does not specifically stim- ulate the C nociceptive fibers. This result can be seen in the present study by the small nerve-axon–related vasodilation achieved with sodium nitroprusside, simi- lar to that achieved by deionized water, which can be attributed to a nonspecific galvanic effect of the constant current that is used for the iontophoresis (27). There- fore, we believe that the presented data also provide further evidence of different pathways through which acetylcholine and sodium nitroprusside cause vasodilation in the skin microcirculation. The iontophoresis of deionized water in the same polarity with that of acetylcholine (i.e., with an anodal constant current) has been previously shown to lead to a small nonspecific galvanic effect (20,21). In con- trast, iontophoresis with a cathodal current, as used for the iontophoresis of sodium nitroprusside, has been reported in one study to result in a significant nonspecific vasodilatory response (22). In the present study, we have not found such an exagger- ated response, and both anodal and cathodal modes elicited very similar responses. The main differences between previous studies

and the present study that may explain this discrepancy are the duration and amplitude of the current used for iontophoresis. Thus, in our unit, we apply 200 µA for 60 s. This produces maximal specific vasodilation with a minimal nonspecific vasodilation. This is in sharp contrast with a previous study in which three rather small pulses of ion- tophoresis were performed over a period of 10 min, raising the question as to whether maximal vasodilation was achieved. In a previous study, we showed that diabetes impairs the total endothelium- dependent and endothelium-independent vasodilation at the forearm level, a skin area that is rarely affected by diabetic neu- ropathy (11). In addition, the present study shows that this reduction is inde- pendent of the nerve-axon–related response. A direct effect of diabetes on endothelium function or smooth muscle cells should therefore be considered as the main cause of the observed impaired vasodilation in response to acetylcholine and sodium nitroprusside. We have previ- ously shown that differences exist between the forearm and foot microcirculation beds, with the foot vasodilatory response being approximately half that of the response at the forearm level (11). Similar results were observed in the present study. Neuropathy has been shown to reduce the vasodilatory response at the foot level, irrespectively of the presence or absence of peripheral vascular disease (10,11). In the present study, the nerve-axon–related response in diabetic patients during specific stimulation of the C fibers with acetylcholine was markedly decreased, being similar to that observed with sodium nitroprusside. Thus, this is another indication that neu- ropathy renders the diabetic foot function- ally ischemic, as blood flow fails to increase under conditions of stress. In summary, we have shown in the present study that the neurovascular vasodi- lation response accounts for approximately one-third of the total acetylcholine-induced vasodilation response at both the forearm and foot levels of healthy subjects and non- neuropathic diabetic patients. The presence of diabetic neuropathy at the lower extrem- ity results in a significant reduction in the total vasodilatory response to acetylcholine and to an even more pronounced reduction in the percentage contribution of the neu- rovascular response to the total skin vasodilatory response to acetylcholine. Acetylcholine and sodium nitroprusside cause vasodilation in the skin microcircula-

tion through different pathways. Finally, the technique used in this study may be partic- ularly

tion through different pathways. Finally, the technique used in this study may be partic- ularly helpful in developing new methods that can objectively evaluate the efficacy of new treatments on small-fiber function.

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