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Functional abnormalities of the micro-circulation have gained significantattention in recent years for theirpotential pathogenic role in the develop-ment of diabetic complications, particularlydiabetic neuropathy and diabetic foot prob-

lems (15). The microvascular tone is reg-ulated by several humoral and neural fac-tors. The vascular endothelium has animportant role in controlling the microvas-cular tone by releasing several vasodilatorsubstances such as nitric oxide, prostacyclin

and endothelium-derived hyperpolarizingfactor, and vasoconstrictor substances suchas prostaglandins and endothelin (6). Nitricoxide is the most important vasodilatorsubstance responsible for endothelium-dependent vasodilation. After its secretionfrom the endothelium, it diffuses to theadjacent smooth muscle cells and stimu-lates the guanylate cyclase enzyme to pro-duce cyclic guanosine 3,5-monophos-phate, which, in turn, leads to smoothmuscle relaxation and vasodilation (7).

The normal neurovascular responseconducted through the C nociceptive nervefibers is another important mechanism forthe regulation of the microcirculation.Stimulation of the C nociceptive nervefibers leads to antidromic stimulation of theadjacent C fibers, which secrete vasodilat-ing substances such as substance P andbradykinin, causing vasodilation at theinjured or inflamed skin areas. This vasodi-lating response, also known as the Lewistriple-flare response, is decreased in thepresence of diabetic neuropathy. Reductionof local blood flow increases the vulnera-bility of the neuropathic limb to severe dia-betic foot problems (8,9). It has beenpostulated that the abnormality in the neu-rovascular response in the neuropathiclimb further aggravates the abnormalities inthe microcirculation, and a vicious cyclemay ensue (10).

Several recent studies (1013) havedemonstrated reduced endothelium-dependent vasodilation in patients witheither type 1 or type 2 diabetes. However,little information is available regarding thecontribution of nerve-axon reflex-relatedvasodilatation to maximal skin vasodila-tion in such patients (8,9). The recentdevelopment of noninvasive techniquesthat can reliably quantify blood flow in theskin microcirculation has made it possibleto study changes in microvascular functionin patients with diabetes (14,15). In thepresent study, we have examined the con-tributing role of the nerve-axon reflex-related vasodilation response to the totalskin vasodilation at both the forearm and

From the Clinical Research Center (O.H., K.A.-E., E.S.H.), Joslin Diabetes Center, Department of Medicine,and the JoslinBeth Israel Deaconess Foot Center and Microcirculation Laboratory (F.W.L., A.V.), Departmentof Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.

Address correspondence and reprint requests to Aristidis Veves, MD, Microcirculation Laboratory, Palmer317, 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 Systme International (SI) units and conversion

factors for many substances.

Contribution of Nerve-Axon Reflex-Related Vasodilation to the TotalSkin Vasodilation in Diabetic PatientsWith and Without Neuropathy

O R I G I N A L A R T I C L E

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

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

RESULTS The neurovascular response to acetylcholine was significantly higher than theresponse to sodium nitroprusside and deionized water (P 0.01). At the forearm level, thecontribution 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 significantlydiminished in the three neuropathic groups. A significantly lower nonspecific nerve-axonrelated 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 thetotal acetylcholine-induced vasodilation at both the forearm and foot levels. The presence ofdiabetic neuropathy results in reduction of both the total vasodilatory response to acetylcholineand 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 24:344349, 2001

OSAMA HAMDY, MDKARIM ABOU-ELENIN, MDFRANK W. LOGERFO, MD

EDWARD S. HORTON, MDARISTIDIS VEVES, MD

P a t h o p h y s i o l o g y / C o m p l i c a t i o n s

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foot levels of neuropathic and nonneuro-pathic diabetic patients.

RESEARCH DESIGN AND METHODS

PatientsWe studied the skin microcirculation at theforearm level in 69 healthy subjects and 42nonneuropathic diabetic subjects. The fol-lowing exclusion criteria were applied tosubjects in all groups: smoking anyamount of cigarettes during the previous 6months, 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 peripheralpulses), chronic renal disease, severe dys-lipidemia (triglycerides 600 mg/dl orcholesterol 300 mg/dl), or any other seri-ous chronic disease requiring active treat-ment. Subjects were also excluded if theywere on any of the following medications:any type of antihypertensive drugs, lipid-lowering agents, glucocorticoids, antineo-plastic agents, psychoactive agents, orbronchodilators. In addition, diabeticpatients with proliferative retinopathy,peripheral somatic neuropathy, macroal-buminuria (expressed as albumin-to-crea-tinine ratio 300 g/mg), and/or oninsulin or troglitazone were excluded fromthe study.

We also evaluated the skin microvascu-lar reactivity at the foot level in 27 healthysubjects and 101 diabetic patients who weredivided into four groups. The first groupconsisted of 33 diabetic neuropathicpatients with a history of foot ulceration butno peripheral vascular disease, the second

group of 23 diabetic patients with Charcotarthropathy, the third group of 32 diabeticpatients with peripheral vascular diseaseand neuropathy, and the fourth group of 13diabetic 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 anyonewith a history of hypertension, diabetes,hypercholesterolemia, active tobacco use,history of any systemic illness, or the use ofany antihypertensive, cardiac, or hormonalmedication. Patients with either type 1 ortype 2 diabetes were included. Patients withnephropathy (creatinine 2 mg/l), severeheart failure, or any other serious illnesswere excluded from the study.

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

MethodsA history, physical examination, and fastingplasma glucose measurement were per-formed on all patients. Diabetic neuropathywas diagnosed according to the San Anto-nio Consensus Statement criteria (16). Thesymptoms were evaluated by using a neu-ropathy symptom score, and the clinicalsigns were evaluated by using a neuropathydisability score (17). Quantitative sensorytesting included the assessment of vibrationperception threshold using a Biothesiome-ter and cutaneous perception thresholdusing Semmes-Weinstein monofilaments(18,19). The diagnosis of Charcot neu-roarthropathy was made when grossdestruction of the joints of the mid-footthat resulted in significant foot deformitywas present. Patients were characterized ashaving peripheral vascular disease based onthe presence of one or more of the follow-ing clinical features: claudication, absentfoot pulses, and/or abnormal invasive andabnormal noninvasive vascular tests.

Each participant was studied after a 20-min acclimatization period in a warm envi-ronment (room temperature 2324C). Weused two single-point laser probes and aDRT4 Laser Doppler Blood Flow Monitor

Table 1Characteristics of the forearm study subjects

Diabetic nonneuropathic patients Control subjects

n 42 69Age (years) 54 9 49 9Men/women 21/21 33/36Type 2 diabetes 42 Diabetes duration (years) 4 5 BMI (kg/m2)* 32.3 6.3 27.3 4.3HbA1c (%)* 8.0 1.6 5.6 0.4Albumin-to-creatinine ratio 30 50 Neuropathy symptom score* 1.43 2.27 0.02 0.13Neuropathy disability score* 0.8 1.45 0.15 0.62Vibration perception threshold* 15.86 9.5 10.69 6.39

Data are means SD. *P 0.001.

Table 2Characteristics of the foot study subjects

Charcot Neuropathy and Diabetic patients ControlNeuropathy arthropathy peripheral vascular without subjects

(DN) (DA) disease (DI) complications (D) (C)

n 33 23 32 13 27Age (years)* 56 9 57 9 60 8 39 10 52 13Men/women 24/9 13/10 23/9 9/4 13/14Type of diabetes (1/2) 12/21 5/18 16/16 8/5 Diabetes duration 21 12 17 11 25 13 17 7 BMI (kg/m2) 30.3 6.8 29.5 4.8 27.8 4.5 26.8 4.5 27.5 4.9HbA1c (%) 8.7 2.7 8.7 2.0 8.9 0.9 9.9 4.3 Creatinine (mg/dl) 1.03 0.3 1.01 0.4 1.05 0.031 1.02 0.03 Neuropathy 3.2 2.9 2.9 2.5 3.5 2.8 0.5 1.1 0.1 0.4symptom score

Neuropathy 19.3 6.1 21.8 3.9 18.7 6.7 0.5 1.2 0.4 1.2disability score

Vibration perception 48 5 50 3.4 47 8 11 5 12 6threshold

Semmes-Weinstein 6.6 0.7 6.9 0.4 6.6 0.5 4 0.5 4 0.5monofilaments

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

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(Moor Instruments, Millwey, Devon, U.K.)to evaluate the skin microcirculation. Fore-arm microcirculatory flow was measuredover the flexor surface of the forearm, andfoot microcirculatory flow was measuredover the dorsum of the foot. The blood flowresponse was measured in response to ion-tophoresis of each of three substances: 1%acetylcholine chloride solution (a substancethat elicits both a neurovascular responseand endothelium-dependent vasodilation),1% sodium nitroprusside (a substance thatdoes not elicit a neurovascular response,but induces endothelium-independentvasodilation), and deionized water (used asa control during iontophoresis to measurethe vasodilation caused by the direct effectof a constant current flow) (10). Deionizedwater was iontophoresed in both anodalmode (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 modesis the polarity of the iontophoresis chamber:the chamber serves as the anode for ion-tophoresis of acetylcholine, which has anegative electrical charge, and as the cath-ode for the iontophoresis of sodium nitro-prusside, which has a positive electricalcharge. Therefore, the constant current hasan opposite direction when the polarity ofthe chamber is changed.

The iontophoresis instrument (MIC1iontophoresis system; Moor Instruments)consists of an iontophoresis delivery vehicledevice that sticks firmly to the skin with thehelp of adhesive tape. The device containstwo chambers that accommodate two sin-gle-point laser probes. One probe is placed

within the chamber containing the ion-tophoresis solution (thus measuring thedirect response to acetylcholine or sodiumnitroprusside iontophoresis), while the sec-ond probe is placed outside but withinproximity (within 5 mm) to the ion-tophoresis solution chamber, thus meas-uring the indirect nerve-axonrelatedresponse that results from stimulation of theC nociceptive nerve fibers. A small amount(1 ml) of test solution was applied to theiontophoresis chamber. Subsequently, aconstant current of 200 A for 60 s wasapplied, achieving a dose of 6 mC/cm2

between the iontophoresis chamber and asecond nonactive electrode placed 1015cm proximal to the chamber. The two laserprobes recorded changes in skin blood flow.Measurements were obtained for 40 sbefore the iontophoresis, during the ion-tophoresis, and 90 s after it (10,11). Theday-to-day reproducibility of the techniquewas evaluated in five healthy subjects (fourmen and one woman, ages 2339 years)who were repeatedly tested at their footand forearm for 10 consecutive workingdays. With use of a single-point laser probe,the coefficient of variation (CV) for the base-line blood flow before iontophoresis ofacetylcholine was 60.6% and for the maxi-mal hyperemic response was 35.2% afterthe iontophoresis of acetylcholine.

Statistical analysisThe results were recorded and tabulatedbefore revealing the patient category assig-nations. Changes in microvascular bloodflow were expressed as the percentage ofincrease over baseline, where median, firstquartile, and third quartile values are used

for comparisons. Parametric data wereexpressed as means SD. Statistical analysiswas performed using the Minitab computersoftware (State College, PA), using bothparametric and nonparametric tests. All testswere two-tailed, with significance taken asP 0.05. For between-group comparisons,we used paired t test for parametric data andKruskal-Wallis test for nonparametric data.

RESULTS

Forearm levelThe results of the iontophoresis are shownin Table 3. To evaluate the degree of vasodi-lation that is specific to the neurovascularresponse, we measured the capillary bloodflow in a skin area in direct contact withacetylcholine and in an adjacent skin areanot in direct contact with it. The latter rep-resents the nerve-axonrelated portion ofthe total response. The percentage contri-bution of the nerve-axonrelated responseto the total response was similar betweennonneuropathic diabetic patients and thecontrol group after the iontophoresis ofacetylcholine (35 and 31%, respectively,NS). In both the nonneuropathic diabeticpatients and control group, the percentagecontribution of the nerve-axonrelatedresponse to the total response was signifi-cantly less after the iontophoresis of eithersodium nitroprusside (13 and 10%, respec-tively, P 0.01) or deionized water (16and 17%, respectively, P 0.01). No sig-nificant difference was seen between thepercentage contribution of the nerve-axonrelated reflex to the total response tosodium nitroprusside and to deionizedwater both in anodal and cathodal mode in

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

Nonneuropathic diabetic patients Control subjects

Total response to Ach 835 (2891476) 1,181 (5472,299)Nerve-axonrelated response to Ach 365 (120513) 338 (207706)The % contribution of nerve-axon response to the total response to Ach 35 (1683)* 31 (1760)*Total response to SNP 525 (307974) 880 (4452,178)Nerve-axonrelated response to SNP 77 (22230) 118 (40769)The contribution of nerve-axon response to the total response to SNP 13 (634) 10 (322)Total response to W, anodal mode 83 (15400) 300 (65897)Nerve-axonrelated response to W 14 (160) 35 (8141)The % contribution of nerve-axon response to the total response to W 16 (254) 17 (440)Total response to W, cathodal mode 111 (45315) 108 (20252)Nerve-axonrelated response to W 11 (160) 35 (022)The % contribution of nerve-axon response to the total response to W 5 (135) 6 (027)

Data are medians (25th75th quartiles). Ach, acetylcholine; SNP, sodium nitroprusside; W, deionized water. *Ach vs. SNP and W, P 0.01.

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both the nonneuropathic diabetic patientsand control group (NS). This is consistentwith the fact that acetylcholine specificallystimulates C nociceptive fibers and thenerve-axonrelated reflex, whereas sodiumnitroprusside and deionized water do not.The contribution of the neurovascularresponse to the total response to acetyl-choline is approximately one-third of thetotal response and is not compromised bydiabetes at the forearm level.

Foot levelThe results of the iontophoresis are shown inTable 4. In response to iontophoresis ofacetylcholine, the percentage contribution ofthe nerve-axonrelated response was similarto that seen at the forearm level in both thediabetic patients without complications andthe healthy control subjects (29 and 36%,respectively, NS). The diabetic neuropathicpatients had a significantly lower medianincrease of capillary blood flow over baselinein response to acetylcholine compared withthe diabetic patients without complicationsand the control group (P 0.01) (Fig. 1).The neurovascular response was markedlydecreased in all three neuropathic groupswhen compared with diabetic patients with-out complications and the control group.The contribution of the nerve-axonrelatedresponse to the total response was 8% indiabetic patients with neuropathy (P 0.01), 5% in diabetic patients with Charcot

arthropathy (P 0.01), and 20% in diabeticpatients with neuropathy and peripheralvascular disease (P 0.01). The nerve-axonrelated response to sodium nitro-prusside and to the anodal and cathodaliontophoresis of deionized water was simi-

lar to the response observed in the upperextremity.

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

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

Charcot Neuropathy and Diabetic patients ControlNeuropathy arthropathy peripheral vascular without subjects

(DN) (DA) disease (DI) complications (D) (C)

Total response to Ach 90 (15378) 227 (86554) 74 (1212) 578 (1521,858) 411 (148541)Nerve-axonrelated response to Ach 4 (026) 13 (152) 5 (052) 118 (19304) 153 (60264)The % contribution of nerve-axon response to the total 8 (031)* 5 (027) 20 (070) 29 (752) 36 (1888)response to Ach

Total response to SNP 89 (31227) 80 (74400) 86 (7239) 234 (141520) 234 (129590)Nerve-axonrelated response to SNP 10 (024) 2 (032) 1 (012) 27 (887) 48 (16108)The % contribution of nerve-axon response to the total 8 (031) 10 (029) 2 (018) 12 (235) 9 (476)response to SNP

Total response to W, Anodal mode 8 (040) 12 (150) 19 (031) 238 (35427) 33 (11107)Nerve-axonrelated response to W 0 (013) 6 (012) 3 (010) 23 (728) 12 (025)The % contribution of nerve-axon response to the total 11 (0100) 43 (6106) 26 (081) 13 (248) 18 (6111)response to W

Total response to W, Cathodal mode 4 (014) 12 (038) 18 (042) 28 (10109) 25 (843)Nerve-axonrelated response to W 0 (09) 1 (012) 0 (015) 11 (142) 3 (011)The % contribution of nerve-axon response to the total 35 (0100) 18 (6106) 4 (081) 41 (248) 0 (6111)response to W

Data are medians (25th75th 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.

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

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patients, at both the forearm and foot lev-els, the microvascular vasodilation that isrelated to the neurovascular responseaccounts for approximately one-third ofthe total vasodilation that is observed afterthe iontophoresis of acetylcholine. Thisportion is markedly decreased in the pres-ence of diabetic neuropathy.

The total microvascular vasodilation inresponse to acetylcholine is currently con-sidered to represent the sum of direct stim-ulation of the endothelium by acetylcholineand of the vasodilation that is related to thenerve-axon reflex (20). However, the mag-nitude of the contribution of each of thesetwo factors to the total vasodilation has notbeen adequately studied, and the currentlyavailable data are conflicting. Thus, althoughsome studies have suggested a considerablyhigher contribution of the neurovascularresponse, the techniques used did not allowthe precise quantification of this contribu-tion (8,21). On the other hand, anotherstudy has shown that local sensory inhibi-tion by topical application of lignocaine andprilocaine did not have an effect on the totalvasodilatory response to the iontophoresis ofacetylcholine (22). The main problem ininterpreting these data, though, lies in thefact that there is no evidence that topicalapplication of lignocaine abolishes thenerve-axon reflex, since it may cause localanesthesia via mechanisms that are notaffecting the antidromic stimulation of localC nociceptive fibers. This is further empha-sized by the findings of a previous studythat showed that deep subcutaneous injec-tion of lignocaine does inhibit the nerve-axonrelated vasodilation in response to theiontophoresis of acetylcholine (23).

In the present study, we have used achamber that can accommodate two single-point laser probes that can measure the totaland the nerve-axon reflex-related vasodila-tion. This technique can satisfactorily mea-sure the two responses separately, making itpossible to evaluate the relative contributionof the neurovascular response to the totalresponse with an adequate reliability. Fur-thermore, we have studied subjects withand without peripheral neuropathy ratherthan testing with local anesthesia, which, asmentioned previously, has questionableeffects on the nerve-axon reflex. Finally, itshould be remembered that under condi-tions of stress (such as injury or inflamma-tion), hyperemia is necessary not only in theinjured area alone but in a considerablylarger area that surrounds the injured site.Because this response depends mainly on a

normal nerve-axon reflex, our findings makethe point that this response, under normalconditions, is one-third of the maximalachievable vasodilation and that this isdrastically reduced in the presence of dia-betic neuropathy.

Single-point laser probe measurementsare known to have a considerably high CV,whereas the use of laser scanners reducesthis variability (10,16). However, with laserscanners, one cannot evaluate the nerve-axon response, a measurement that can bedone only with use of the single-point lasertechnique. The large number of subjects ineach studied group compensates for thehigh variability and does not affect the valid-ity of the conclusions regarding the contri-bution of nerve-axon response to totalvasodilation. On the other hand, the highvariability does not allow the direct com-parison of the vasodilatory response amongthe various studied groups, which makesthis study prone to type 2 statistical error.Therefore, it is recommended that for reli-able data regarding these questions, thereader is directed to studies that have specif-ically addressed this question and used theappropriate techniques, including the use ofa Laser Scanner Imager (10,11,2426).

In contrast to acetylcholine, sodiumnitroprusside causes vasodilation bydirectly stimulating the vascular smoothmuscle cell and does not specifically stim-ulate the C nociceptive fibers. This resultcan be seen in the present study by thesmall nerve-axonrelated vasodilationachieved with sodium nitroprusside, simi-lar to that achieved by deionized water,which can be attributed to a nonspecificgalvanic effect of the constant current thatis used for the iontophoresis (27). There-fore, we believe that the presented dataalso provide further evidence of differentpathways through which acetylcholine andsodium nitroprusside cause vasodilation inthe skin microcirculation.

The iontophoresis of deionized water inthe same polarity with that of acetylcholine(i.e., with an anodal constant current) hasbeen previously shown to lead to a smallnonspecific galvanic effect (20,21). In con-trast, iontophoresis with a cathodal current,as used for the iontophoresis of sodiumnitroprusside, has been reported in onestudy to result in a significant nonspecificvasodilatory response (22). In the presentstudy, we have not found such an exagger-ated response, and both anodal and cathodalmodes elicited very similar responses. Themain differences between previous studies

and the present study that may explain thisdiscrepancy are the duration and amplitudeof the current used for iontophoresis. Thus,in our unit, we apply 200 A for 60 s. Thisproduces maximal specific vasodilation witha minimal nonspecific vasodilation. This isin sharp contrast with a previous study inwhich three rather small pulses of ion-tophoresis were performed over a period of10 min, raising the question as to whethermaximal vasodilation was achieved.

In a previous study, we showed thatdiabetes impairs the total endothelium-dependent and endothelium-independentvasodilation at the forearm level, a skinarea that is rarely affected by diabetic neu-ropathy (11). In addition, the presentstudy shows that this reduction is inde-pendent of the nerve-axonrelatedresponse. A direct effect of diabetes onendothelium function or smooth musclecells should therefore be considered as themain cause of the observed impairedvasodilation in response to acetylcholineand sodium nitroprusside. We have previ-ously shown that differences exist betweenthe forearm and foot microcirculationbeds, with the foot vasodilatory responsebeing approximately half that of theresponse at the forearm level (11). Similarresults were observed in the present study.

Neuropathy has been shown to reducethe vasodilatory response at the foot level,irrespectively of the presence or absence ofperipheral vascular disease (10,11). In thepresent study, the nerve-axonrelatedresponse in diabetic patients during specificstimulation of the C fibers with acetylcholinewas markedly decreased, being similar tothat observed with sodium nitroprusside.Thus, this is another indication that neu-ropathy renders the diabetic foot function-ally ischemic, as blood flow fails to increaseunder conditions of stress.

In summary, we have shown in thepresent study that the neurovascular vasodi-lation response accounts for approximatelyone-third of the total acetylcholine-inducedvasodilation response at both the forearmand foot levels of healthy subjects and non-neuropathic diabetic patients. The presenceof diabetic neuropathy at the lower extrem-ity results in a significant reduction in thetotal vasodilatory response to acetylcholineand to an even more pronounced reductionin the percentage contribution of the neu-rovascular response to the total skinvasodilatory response to acetylcholine.Acetylcholine and sodium nitroprussidecause vasodilation in the skin microcircula-

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

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