Differences in Skin Microcirculation on the Upper and Lower Extremities in Patients with Diabetes Mellitus: Relationship of Diabetic Neuropathy and Skin Microcirculation

Abstract

Introduction:

During recent years, the role of microcirculation has received increasing attention especially for its potential pathogenic role in the development of diabetes complications, particularly diabetic foot syndrome. The aim of this study was to evaluate the differences in the skin microcirculatory reactivity on the upper and lower extremities (UE and LE, respectively) in the patient with type 2 diabetes mellitus (T2DM). We also evaluated the changes in the skin microcirculation independently of the individual test for peripheral diabetic neuropathy (DN) diagnosis (Semmes–Weinstein monofilaments, Bio-Thesiometer [Bio-Medical Instrument Co., Newbury, OH], and Neuropad^® [TRIGOcare International GmbH, Wiehl, Germany]).

Patients and Methods:

Fifty-two patients with T2DM were enrolled. Microvascular reactivity was measured by laser Doppler iontophoresis, using 1% acetylcholine chloride (ACH) and 1% sodium nitroprusside.

Results:

Significant reduction of perfusion was found in LE compared with UE when using ACH. In patients with DN skin microvascular reactivity on LE and UE was reduced, compared with patients without DN. Impaired skin microvascular reactivity to ACH (dominant on LE) was demonstrated in all patients who were positive in at least one of the tests for the presence of DN.

Conclusions:

Reactivity of the skin microcirculation is worse on the foot than on the hand. This study confirmed a close relationship of DN and impaired skin microcirculation. It seems that autonomous neuropathy (assessed using the Neuropad) precedes the manifestation of somatosensory neuropathy.

Introduction

M icrocirculation and its functional changes are of interest for a potential pathogenic role in the development of diabetes complications, particularly diabetic foot syndrome. The main risk factors of diabetic foot syndrome are diabetic neuropathy (DN) and peripheral vascular disease.¹ Both conditions occur more frequently in the lower extremities (LEs). Defects on the upper extremities (UEs) are present only very rarely in patients with diabetes mellitus. The diagnosis of macroangiopathy and DN can be made quite easily on the basis of the patient's history and available examinations. On the other hand, the examination of the microcirculation and early detection of microangiopathy are much more complicated and still unresolved. The examination of microcirculation is not possible with routine clinical tests. There is a mutual association between the changes in microcirculation and the presence of DN. The microvascular bed is regulated by the peripheral and central autonomous nerve systems. On the other hand, microvascular changes can be responsible for the ischemic etiology of DN.²

Skin microcirculation is controlled by several mechanisms:

1. control by the autonomous nerve system, which innervates arteriovenous shunts and thus regulates the amount of blood flowing through arterioles.

2. nerve–axon reflex-related vasodilatation, which is a reaction mediated by C-nociceptive fibers, where antidromic stimulation of neighboring C-fibers occurs on stimulation. This results in release of vasodilator substances (substance P, bradykinin, ATP analog adenosine, calcitonin-generated peptide). This reaction is known as the Lewis triple flare.

3. Sympathetically mediated vasoconstriction reflex (venoarterial reflex), which is activated by gravity. When standing, the hydrostatic pressure is higher in the vessels of the LE; this would soon lead to a tissue edema. An increase in venous pressure activates nerve fibers. This impulse ultimately causes vasoconstriction of precapillary sphincter of an arteriole.³ The extent of vasoconstriction is directly related to the amount of hydrostatic pressure.

4. Role of the endothelium. It is acknowledged that the endothelium plays an important role in microvascular tone control (synthesis of vasodilator [for example, nitric oxide, prostacyclin, endothelium-derived hyperpolarizing factor] and vasoconstrictor [for example, prostaglandins, angiotensin, endothelin] substances).⁴

Microcirculation in patients with diabetes is additionally affected by metabolic factors. These are functional or structural changes. Among the structural impairments that occur in particular are reduction of capillary density, rigidity of precapillary vessels, and thickening of the basement membrane.⁵ Functional changes include endothelium damage, which leads to, inter alia, insufficient vasomotor response to endothelium-dependent stimuli. Pathophysiological basis of this phenomenon is the reduction of nitric oxide availability resulting from endothelial dysfunction.⁶

The object of our interest is the study of skin microcirculation in patients with diabetes mellitus type 2 (T2DM). Most studies evaluate skin microvascular reactivity by laser Doppler flowmetry (LDF) with the use of stimulation tests, such as local thermal hyperemia and postocclusive reactive hyperemia. Fewer studies have used iontophoresis. This method uses various substances to induce changes in microcirculation. The most commonly used substances are 1% acetylcholine chloride (ACH), which induces neurovascular responses and endothelium-dependent vasodilatation, and 1% sodium nitroprusside (SNP), which causes endothelium-independent vasodilatation. It affects directly the smooth muscle cells of the vessels but does not specifically stimulate C-nociceptive fibers.⁷ Functional changes in microcirculation are revealed using the above-mentioned tests as a microvascular response reduction upon use of vasodilator substances.

This study had three objectives:

1. Does the skin microvascular reactivity assessed using LDF iontophoresis differ on the UE and LE in patients with T2DM?

2. Is there a difference in skin microcirculation evaluated using LDF iontophoresis in patients with and without DN? Do the results of LDF iontophoresis vary in groups of patients divided according to the test used for DN diagnosis?

3. Is there a relationship between skin microcirculation and other chronic microvascular complications and cardiovascular risk factors?

Subjects and Methods

Fifty-two patients with T2DM from the Diabetology Centre of the University Hospital Pilsen, Pilsen, Czech Republic, were enrolled in the study. Further characteristics of the group are provided in Table 1. Patients signed an informed consent prior to enrollment in the study, and the protocol had been approved by a local ethics committee.

Table 1.

Patient Characteristics

Characteristic	Median (1 ^st ; 3 ^rd quartile)
Number of patients	52
Male/female	27/25
Age (years)	60 (42; 68)
Duration of DM (years)	13 (9; 19)
DDI (units)	46.3 (32; 70)
BMI (kg/m²)	28.7 (25.8; 32.6)
HbA_1c (mmol/mol)	78 (66; 94)
Hypertension (−/+)	14/38
Hyperlipoproteinemia (−/+)	29/23
Smoking (−/+)	48/4
Retinopathy (−/+)	28/24
Nephropathy (−/+)	18/34

BMI, body mass index; DDI, daily dose of insulin; DM, diabetes mellitus; HbA_1c, glycosylated hemoglobin.

Patients with a defect on an LE or with such defect in their history, as well as patients with Charcot's osteoarthropathy, dermatitis, or history of an allergic reaction to the substances administered were excluded. Among other exclusion criteria were ischemic heart disease (diagnosed coronary insufficiency, arrhythmia, heart failure), peripheral vascular disease (impalpable peripheral pulse, claudications), severe renal disease (classified as glomerular filtration rate under 0.5 mL/s), and the use of glucocorticoids, psychoactive substances, antineoplastic agents, or bronchodilators.

We noted the presence of microvascular complications (retinopathy, nephropathy, neuropathy [see below]) and cardiovascular risk factors (treated hypertension, hyperlipidemia, smoking).

Skin microvascular reactivity was assessed using iontophoresis.

Laser Doppler iontophoresis

Each patient was examined after a 20-min acclimatization in the examining room (at a temperature of 22°C). The measurement was carried out with the patient in the supine position, with LE and UE at the same level. The Periflux system 5000 instrument and Perisoft for Windows version 2.5 PSW software (Perimed AB, Stockholm, Sweden) were used for the examination of the microcirculation. The following substances were used for our measurements: 1% ACH and 1% SNP (see above). The electrode was placed on hairy skin, where the number of arteriovenous shunts is at a minimum⁸: on the dorsum of the foot between the first and second toe on the LE. On the UE, the electrode was placed over the distal part of the antebrachial flexor muscles (ulnar side).⁹

The iontophoresis instrument (MIC1 iontophoresis system; Moor Instruments, Axminster, United Kingdom) consists of an iontophoresis delivery vehicle device that is attached firmly to the skin with double-sided adhesive tape. The device contains two chambers that accommodate two single-point laser probes. We used one probe in this study, placed within the chamber containing the iontophoresis solution (thus measuring the direct response to ACH or SNP iontophoresis). A small quantity (<1 mL) of test solution was placed in the iontophoresis chamber, and a constant current of 200 μA was applied for 60 s, achieving a dose of 6 mC/cm² between the iontophoresis chamber and a second non-active electrode placed 10–15 cm proximal to the chamber. We used the protocol of Droog et al.¹⁰ The current caused a movement of the solution to be iontophoresed into the skin, and the resulting vasodilatation was recorded. The change in perfusion can be assessed as the percentage change from the baseline.

Peripheral DN

We used routine evaluation methods for peripheral DN^11

–14 based on the patient's symptoms and results of the physical exam, which included examinations using monofilaments (Semmes–Weinstein; 5.07/10 g), the Bio-Thesiometer (Bio-Medical Instrument Co., Newbury, OH)-calibrated vibration perception threshold (VPT) meter, and the Neuropad^® (TRIGO-care International GmbH, Wiehl, Germany).

The monofilaments test was for superficial sensation in the thick myelinated (A) fibers. We tested the big toe, the third and fifth metatarsus, and the heel of both LEs. The test is regarded as positive when one or more sites are insensitive.^15
–17

The Bio-Thesiometer test for deep vibration sensation was positive at a vibration perception threshold over 25 V.^16,17 We tested the metatarsophalangeal joint of the big toe on both LEs.

The Neuropad serves to examine sudomotor function (autonomous neuropathy). It evaluates sweating and cholinergic innervation of the foot.^18

–22 The Neuropad plaster is applied to the sole of the feet of both LEs.

The patients were divided into two groups based on these methods: patients without DN (all three tests were negative) (DN⁻) (n=20) and those with DN (at least one of the three described tests was positive) (DN⁺) (n=32). The changes in skin microcirculation were evaluated independently of the individual tests for peripheral DN diagnosis.

Statistical analysis

The following nonparametric tests were used for statistical evaluation: Wilcoxon paired and unpaired tests, Kruskal–Wallis test, Spearman correlation, and multiple regression analysis. The level of significance was set to P<0.05.

Results

In total, 52 patients with T2DM were examined. A statistically significant reduction in skin microvascular reactivity in LE compared with UE after ACH administration was found. The same trend, although statistically insignificant, was observed in the reaction with SNP (Table 2).

Table 2.

Differences Between Lower and Upper Extremities in Skin Microcirculatory Reactivity in the Whole Patient Group (n=52)

	LE	UE	P (LE vs. UE) ^a
ACH
Baseline	10.6 (7.9; 11.9)	11.1 (8.7; 14.7)	NS (P=0.507)
% change	276.1 (184.7; 558.2)	509.3 (316.1; 788.6)	P=0.002
NPS
Baseline	11.0 (8.5; 13.9)	12.9 (9.6; 16.7)	NS (P=0.139)
% change	373.6 (237.7; 539.9)	405.4 (314.2; 821.3)	NS (P=0.122)

Data are median values (first; third quartile).

P<0.01 was taken as the level of significance.

ACH, acetylcholine chloride; LE, lower extremity; NPS, sodium nitroprusside; NS, difference not significant; UE, upper extremity.

Based on the tests for the presence of DN, the patients were divided in a group without DN (DN⁻) and those with a DN present (DN⁺). Patients without DN had a statistically significantly shorter duration of diabetes, were younger, and had a considerably lower insulin daily dose in comparison with the patients with DN. More detailed characteristics of the two groups are provided in Table 3.

Table 3.

Characteristics of Patients With and Without Diabetic Neuropathy

	DN⁻ (n=20)	DN⁺ (n=32)	P^a
Male/female	10/10	17/15	—
Age (years)	41.5 (34; 52.2)	61 (55; 68)	P=0.047
Duration of DM (years)	7 (2; 12.25)	15 (10; 21)	P=0.028
DDI (units)	32 (10; 62)	50 (37; 72)	P=0.049
BMI (kg/m²)	26.75 (24.3; 31.4)	29 (26.5; 32.6)	NS (P=0.285)
HbA_1c (mmol/mol)	8.3 (6.7; 9.1)	7.4 (6.6; 9.5)	NS (P=0.897)

Data are median values (first; third quartile).

P<0.05 was taken as the level of significance.

BMI, body mass index; DDI, daily dose of insulin; DM, diabetes mellitus; DN⁻, absence of diabetic neuropathy; DN⁺, presence of diabetic neuropathy; HbA_1c, glycosylated hemoglobin; NS, difference not significant.

We were able to demonstrate a statistically significant reduction in skin microvascular reactivity on LE and UE in DN⁺ patients compared with the DN⁻ group when using ACH. In DN⁺ patients a statistically significant difference in reactivity was observed on LE compared with UE. No statistically significant differences were found when using SNP (Table 4).

Table 4.

Skin Microcirculatory Reactivity in Patients With and Without Diabetic Neuropathy

	DN⁻			DN⁺			P (DN⁻ vs. DN⁺) ^a
	LE	UE	P (LE vs. UE) ^a	LE	UE	P (LE vs. UE) ^a	LE	UE
ACH
Baseline	11.3 (10.8; 13.1)	12.8 (10.2; 16.4)	NS (P=0.889)	10.2 (7.8; 11.6)	10.9 (7.7; 14.2)	NS (P=0.381)	NS (P=0.184)	NS (P=0.223)
% change	603.7 (318.3; 864.0)	706.3 (469.5; 798.6)	NS (P=0.748)	240.8 (165.6; 337.5)	462.9 (52.5; 788.6)	P=0.0	P=0.001	P=0.013
NPS
Baseline	9.9 (7.3; 11.8)	9.8 (8.8; 15.6)	NS (P=0.327)	11.5 (8.7; 14.9)	12.9 (10.5; 17.3)	NS (P=0.289)	NS (P=0.386)	NS (P=0.184)
% change	380.8 (451.8; 566.5)	514.5 (396.7; 773.3)	NS (P=0.674)	366.0 (206.6; 470.7)	398.3 (308.6; 821.3)	NS (P=0.157)	NS (P=0.507)	NS (P=0.238)

Data are median values (first; third quartile).

P<0.05 was taken as the level of significance.

ACH, acetylcholine chloride; DN⁻, absence of diabetic neuropathy; DN⁺, presence of diabetic neuropathy; LE, lower extremity; NPS, sodium nitroprusside; NS, difference not significant; UE, upper extremity.

Division of the patients according to the individual tests for the presence of DN is provided in Table 5. Basic characteristics of the groups are given in Table 6. Changes in skin microcirculation related to individual tests for DN diagnosis are presented in Table 7.

Table 5.

Patient Division According to Results of Individual Tests for Presence of Diabetic Neuropathy

	Pattern ^a
Number	Monofilaments	Bio-Thesiometer	Neuropad
20	−	−	−
12	−	−	+
1	−	+	−
13	−	+	+
6	+	+	+

+, positive result; −, negative result.

Table 6.

Patient Characteristics According to Results of Individual Tests for Diagnosis of Peripheral Neuropathy (Monofilaments, Bio-Thesiometer, and Neuropad)

	Monofilaments (n=6)	Bio-Thesiometer (n=20)	Neuropad (n=31)
Male/female	4/2	11/9	16/15
Age (years)	60 (57; 60.8)	61.5 (58.5; 67)	60.5 (55; 68)
Duration of DM (years)	18 (13.8; 20.8)	15.5 (10.5; 20.8)	15 (10; 21.8)
DDI (units)	67 (50.1; 71.5)	51.4 (37.3; 76.5)	53.2 (36.8; 72.3)
BMI (kg/m²)	31.2 (29.9; 33.6)	30.5 (28.7; 33.7)	28.9 (26.3; 32.9)
HbA_1c (mmol/mol)	6.85 (6.2; 8.5)	7.7 (6.7; 9.7)	7.7 (6.6; 9.6)

Data are median values (first; third quartile).

BMI, body mass index; DDI, daily dose of insulin; DM, diabetes mellitus; HbA_1c, glycosylated hemoglobin.

Table 7.

Skin Microcirculatory Reactivity According to Results of Individual Tests for Diagnosis of Peripheral Neuropathy (Monofilaments, Bio-Thesiometer, and Neuropad)

	M⁺			B⁺			N⁺			P (M⁺ vs. DN⁻) ^a		P (B⁺ vs. DN⁻) ^a		P (N⁺ vs. DN⁻) ^a
	LE	UE	P^a	LE	UE	P^a	LE	UE	P^a	LE	UE	LE	UE	LE	UE
ACH
Baseline	9.3 (7.8; 16.1)	8.9 (7.9; 13.8)	NS (P=0.969)	9.8 (7.7; 11.8)	12.4 (8.9; 15.3)	NS (P=0.117)	10.1 (7.8; 11.5)	11 (8.6; 14.3)	NS (P=0.374)	NS	NS	NS	NS	NS	NS
% change	237.8 (174.1; 280.5)	397 (206.2; 730.8)	P=0.049	224.1 (170.3; 289.3)	430 (206.2; 606.6)	P=0.041	244.5 (164.4; 355)	510.2 (320.9; 803.1)	P=0.004	P=0.043	NS	P=0.014	NS	P=0.048	NS
NPS
Baseline	16.3 (11.1; 19.7)	11.5 (9.6; 12.8)	NS (P=0.094)	12.9 (9.9; 16.7)	15.1 (11.9; 17.7)	NS (P=0.117)	11.3 (8.7; 14.1)	12.9 (10.4; 16.9)	NS (P=0.131)	NS	NS	NS	NS	NS	NS
% change	370.2 (359; 394.6)	322 (308.6; 419.9)	NS (P=0.531)	366.4 (261.1; 413.6)	319.9 (271.2; 736.7)	NS (P=0.530)	364.4 (204.2; 474.4)	401.9 (311.9; 828.2)	NS (P=0.286)	NS	NS	NS	NS	NS	NS

Data are median values (first; third quartile).

P<0.05 was taken as the level of significance.

ACH, acetylcholine chloride; B⁺, Bio-Thesiometer test positive; DN⁻, absence of diabetic neuropathy; LE, lower extremity; M⁺, monofilament test positive; N⁺, Neuropad test positive; NPS, sodium nitroprusside; NS, difference not significant; UE, upper extremity.

No correlation was found between microvascular reactivity changes and the presence of diabetic retinopathy, DN, or cardiovascular risk factors (Table 8).

Table 8.

Skin Microcirculatory Reactivity and Presence of Diabetic Retinopathy, Diabetic Nephropathy, and Cardiovascular Risk Factors (Hypertension and Hyperlipidemia)

	% change
	Positive		Negative
	LE	UE	LE	UE	P (LE) ^a	P (UE) ^a
Retinopathy
n	24		28
ACH	228.7 (178.8; 520.6)	565.7 (388.9; 832.1)	281.9 (224.4; 569.2)	462.9 (241.9; 736.9)	NS	NS
NPS	395.9 (179.1; 497.1)	409.1 (284.4; 842.1)	366 (257.7; 565.8)	405.4 (319.9; 657.4)	NS	NS
Nephropathy
n	34		18
ACH	269.5 (174; 517)	431.5 (311.5; 793.4)	281.9 (200.6; 558.2)	319.1 (172; 574.1)	NS	NS
NPS	364.4 (203.5; 507.9)	393.4 (320.8; 772.1)	401.6 (356.4; 539.9)	356.4 (269.5; 526.6)	NS	NS
Hypertension
n	38		14
ACH	267.4 (178.8; 454.2)	400.2 (232.6; 647.6)	334.9 (208.1; 673.6)	591.9 (357.3; 874.9)	NS	NS
NPS	364 (199.3; 473.9)	347.9 (309.7; 468.3)	470.7 (315.9; 1,083)	521.3 (386.5; 729.2)	NS	NS
Hyperlipidemia
n	23		29
ACH	245 (168.4; 354.8)	442.3 (206.2; 687.9)	337.4 (212.1; 650.7)	574 (331.4; 994.8)	NS	NS
NPS	369.8 (214.4; 433.5)	377.3 (284.4; 530.7)	380.8 (262.8; 690)	526.6 (330.8; 942.1)	NS	NS

Data are median values (first; third quartile).

P<0.05 was taken as the level of significance.

ACH, acetylcholine chloride; LE, lower extremity; NPS, sodium nitroprusside; NS, difference not significant; UE, upper extremity.

Discussion

In the study we were able to demonstrate worse microcirculation on LEs than UEs. Similar results were reported in some previous studies.^7,23,24 The difference is explained by orthostatic body posture and thus action of physical factors such as gravity, mechanical strain, capillary pressure increase, and shear forces. These factors lead to microvascular changes in the LE, unlike other parts of the body closer to the heart with lower hydrostatic pressure. This explanation is confirmed by the findings of compromised skin microcirculation on the LE even in healthy subjects without diabetes.^23
–25 The described physical factors themselves lead to mechanical damage to the endothelium even without the presence of other pathological factors, such as atherosclerosis, hypertension, or hyperlipidemia. According to some authors a lowered nitric oxide synthesis plays the key role.⁶ Hamdy et al.⁷ described an almost 50% lower response on LE than on UE; our study found similar results. A reduction in response was observed when using ACH, which tests the endothelium-dependent and neurovascular response of the microcirculation. The endothelium-independent response (with SNP) was also reduced on LE in comparison with the reactivity on UE, although the difference was statistically insignificant. This result was in contrast with results of some other studies, which described vasodilatation reduction in microcirculation to endothelium-dependent as well as endothelium-independent stimuli.^26

–29 SNP stimulates the smooth muscle cells directly. They are damaged most frequently by atherosclerosis or already present structural changes of the vessels (decreased elasticity of the vessels, rigidity of precapillary vessels, or thickening of the basement membrane). In our study, however, one of the exclusion criteria was the presence of peripheral vascular disease and severe complications of diabetes. We believe that this is the exact reason why the difference in the reactivity of the microcirculation on LE and UE was not statistically significant when using SNP.

After the division of the patients into groups with and without neuropathy, we found statistically significantly deteriorated microvascular reactivity in patients with neuropathy when using ACH, whereas the finding was more pronounced on LE compared with UE. The difference between the extremities was preserved also in patients without neuropathy but was less significant. When using SNP, a reduced reactivity was also found in patients with neuropathy compared with the group without neuropathy, but this result was not statistically significant. The microvascular change on LE compared with UE was not significant either. Microvascular reactivity reduction in diabetes patients with neuropathy was described in other studies.^{4,7,24,25,30,31} The cause of this phenomenon is considered to be an impairment in the so-called Lewis triple flare reaction described in the introduction of this study. The ratio of neurovascular response to total vasodilatation in ACH in healthy subjects is 1:3. This proportion is reduced when neuropathy is present.^7,30 Sympathetic impairment in a present neuropathy opens the arteriovenous shunts, which results in hypoperfusion of the microcirculation.⁴ Another cause is the decrease in the venoarterial reflex.³ Last but not least, the direct action of diabetes on the function of the endothelium and vascular smooth muscle cells is in play.⁷

We tried to correlate the reactivity of microcirculation with some selected factors. Patients in the group with DN and deteriorated microvascular reactivity were older and had a longer diabetes duration, but their diabetes was not under worse control than that of patients from the group without neuropathy. Forst et al.³² had a different experience; in their study, they described microvascular changes already in the early stages of diabetes mellitus. The study of Karnafel et al.⁹ suggests that provided no severe organ complications were present in patients with diabetes, the duration of diabetes did not have any influence on the reactivity of the microcirculation. Another factor that interferes with the function of microcirculation is insulin.³³ Fysekidis et al.³⁴ explained the action of insulin on the microcirculation in improvement of endothelial or direct myogenic vessel activity. Better reactivity of microcirculation in patients on intensive insulin therapy when using ACH was previously described.³⁵ The results of a recent study³⁶ suggest that strict glycemic control itself leads to an improvement in skin microcirculation. Therefore, it does not depend merely on the effect of insulin. In spite of the fact that the patients with neuropathy in our study were better controlled than the group without neuropathy, the impairment of their microcirculation was more severe. Not even their higher daily insulin dose acted positively.

When comparing skin microvascular reactivity separately with individual tests for diagnosis of peripheral neuropathy (microfilament, VPT, and Neuropad), we found a statistically significant correlation for each test. Impaired skin microvascular reactivity to ACH (dominant on LE) in our study was demonstrated in all patients who had at least one of the tests for the presence of DN positive. In total, 11 patients were positive only with the Neuropad (VPT and monofilaments negative). Even in this group we were able to demonstrate an already present mirocirculatory impairment. The same result was reported in a study comparing impaired skin microcirculation and sudomotor dysfunction.³⁷ The work of Zimny et al.,³¹ who claimed that impaired skin microcirculation is present already in mild neuropathy, is also in agreement with these results.

From these results we could infer that autonomous neuropathy (assessed using the Neuropad) precedes the manifestation of somatosensory neuropathy (assessed using VPT or MF). It thus seems that autonomous neuropathy itself can have a negative influence on microcirculation.³⁸ However, autonomous neuropathy may not be preceding somatosensory neuropathy if a more sensitive indicator had been used (such as nerve conduction studies). The Neuropad may simply correlate with an ealier stage of somatosensory neuropathy better than either VPT or microfilament. On the other hand, the study of Papanas et al.³⁹ showed that the Neuropad test has a validity comparable to that of nerve conduction studies.

The Neuropad is a test with high diagnostic sensitivity for peripheral DN.¹⁹ It is a technically undemanding, fast, and available examination with easy interpretation of results that can be performed at home by the patient him- or herself.

Monofilaments can detect only a more severe degree of neuropathy and are a suitable examination for identification of those patients at high risk of diabetic foot syndrome development.¹⁵ More significant reduction in reactivity (ACH) on UE was found in patients with severe neuropathy (with positive microfilament and VPT tests; n=12) compared with the group in which only the Neuropad was positive or the group without neuropathy. We can speculate that in severe peripheral neuropathy, the microvasular impairment is pronounced even on UE. It is a systemic phenomenon that we assume in the whole body. The worst microvascular impairment remains on LE, where the postural theory also applies.

The association between impaired skin microcirculation and the presence of cardiovascular risk factors is known from several studies.^40
–42 Arterial hypertension, hyperlipoproteinemia, and even smoking are among the risk factors for endothelial dysfunction development.⁶ Logically, we would expect a microvascular reactivity deterioration in the presence of these risk factors. Similarly, the presence of diabetes complications, such as nephropathy (assessed using microalbuminuria) and retinopathy, is significantly associated with the impairment in skin microcirculation.^43,44 The study of Nguyen et al.⁴⁵ also described reduced endothelium-dependent and -independent vasodilatation in patients with diabetic retinopathy. It is surprising that we did not observe the above-described association between microcirculation and other chronic complications (i.e., retinopathy and nephropathy) or cardiovascular risk factors (hypertension and hyperlipidemia) in our study. The reason for the different result in the mentioned studies may be due to other methods of evaluation of microcirculation than in our study. Most of the cited studies used the LDF postocclusive reaction for the evaluation of microcirculation, which is a method used for early detection of microvascular dysfunction. It is considered a sensitive indicator for microangiopathy in patients with diabetes mellitus.^42,46 However, the reaction produced by this method is rather different from the one obtained using iontophoresis used in our study. Postocclusive reactive hyperemia is created by the mechanical effect of a blood wave and nitric oxide release. In iontophoresis the mechanism is different (see Subjects and Methods), and we thus may obtain different results. Additionally, our results were certainly affected by the small number of patients in the groups. The fact that the patients in our study had well-controlled blood pressure in the long term and were normotensive at the time of examination of the microcirculation could also play a role. Their lipidemia was in a normal range within the last 6 months, and the middle value of microalbuminuria was only 110 mg/day. Proliferative retinopathy was present in only one patient; in the other patients only the simple form was present. Additionally, the presence of macroangiopathic and microangiopathic complications was the exclusion criterion of our study. We were unable to find any differences in microcirculation regarding sex of the patients. A previous study⁴⁷ demonstrated a protective effect of estrogens on microcirculation. In our study, however, most women were postmenopausal; thus we did not expect any differences. The relationship with smoking was not evaluated as only four patients were smokers in our study.

Conclusions

This study demonstrated that the reactivity of the skin microcirculation assessed using LDF iontophoresis on the foot is worse than on the hand. The microvascular reactivity deteriorates significantly, and the difference between the hand and the foot increases in DN where, apart from gravity, other risks arising from the neuropathy itself may occur. The study confirmed a close relationship of DN and impaired skin microcirculation. If we want to reduce the number of patients with diabetic foot syndrome, it is necessary to identify the risk group with present incipient neuropathy and impaired microcirculation in a fast and inexpensive way. A test using the Neuropad fulfills the requirement.

Footnotes

Acknowledgments

This research was supported by the Charles University Research Fund (project number P36).

Author Disclosure Statement

No competing financial interests exist.

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