Early Endothelial Dysfunction Severely Impairs Skin Blood Flow Response to Local Pressure Application in Streptozotocin-Induced Diabetic Mice Dominique Sigaudo-Roussel, Claire Demiot, Be´renge`re Fromy, Audrey Koı¨tka, Georges Lefthe´riotis, Pierre Abraham, and Jean Louis Saumet

Pressure-induced vasodilation (PIV) is a mechanism al. (2) demonstrated a major role for vasodilators such as whereby skin blood flow increases in response to pro- the calcitonin gene–related peptide and endothelial vaso- gressive locally applied pressure. Skin blood flow in dilators such as nitric oxide (NO) and prostaglandins in response to applied pressure decreased early in diabetic animal studies. In a recent study, we observed in diabetic patients as a result of vascular and/or neural impair- patients an early decrease of skin blood flow in response ment. This study was designed to determine the effect of to locally applied pressure that could be involved in the vascular changes on PIV in 1-week streptozotocin-in- development of diabetic ulceration (3). However, it was duced diabetic mice. We assessed cutaneous microvas- cular response to local increasing pressure application unclear whether skin blood flow alteration during measured by laser Doppler flowmetry (LDF) and endo- depends directly on vascular and/or neural alterations. thelium-dependent and -independent vasodilation by Diabetes through is widely known to be iontophoretic delivery of acetylcholine and sodium ni- a major factor that leads to microvascular and neural troprusside and sciatic motor nerve conduction velocity complications (4). Indeed, hyperglycemia-induced end- and morphometry. In control mice, LDF increased 34% organ damage in diabetes is associated with increased flux from baseline to 0.2 kPa external pressure, showing PIV of glucose through 1) the polyol metabolic pathway response. In contrast, diabetic mice had no LDF in- (5,6), 2) accumulation of advanced glycation end products crease in response to progressive external pressure. Moreover, after iontophoretic delivery of acetylcholine, (7,8), 3) increased oxidative stress (9,10), and 4) activation endothelium-dependent vasodilation was largely atten- of the protein kinase C pathway (11,12). However, the uated in diabetic mice (25%) compared with control pathogenesis of the vascular complications of diabetes is mice (81%), whereas vasodilation to sodium nitroprus- controversial (13). Several studies in experimental animal side was not different between groups. Nerve function models of diabetes and in human type I and II diabetic as assessed by sciatic nerve conduction velocity and patients revealed impaired endothelium-dependent re- morphometry did not differ between groups. These find- laxation (14,15). However, many in vitro studies have ings suggest that endothelial impairment is sufficient to reported unaltered or enhanced endothelium-dependent severely alter PIV response, which seems to be highly sensitive to endothelial nitric oxide levels. PIV suppres- relaxation (16,17). In addition to these vascular observa- sion could favor diabetes complications such as diabetic tions, Coppey et al. (18) reported as early as 1 week of foot ulcers. Diabetes 53:1564–1569, 2004 diabetes in rats a sciatic nerve blood flow reduction that preceded the slowing of motor nerve conduction velocity (MNCV). Therefore, it seems that vascular dysfunction may be a major factor in the development of diabetic e recently reported a mechanism that allows neuropathy. Indeed, there is evidence that progressive skin blood flow to increase in response to contributes to progressive loss of periph- progressive locally applied pressure in hu- eral and later central neurologic function (19). However, Wmans (1) and rats (2). The pressure applied clinical trials using specific inhibitors of various biochem- is a nonpainful stimulation, but we showed that pres- ical pathways activated by hyperglycemia have shown sure-induced vasodilation (PIV) involved nerve C fibers, modest improvements in neurologic symptoms (20,21). It because it disappeared after chronic treatment with cap- therefore seems to be of importance to focus on early saicin in animals and humans (1,2). In addition, Fromy et vascular alteration, because preventing vascular dysfunc- tion could delay the appearance of . This study was designed to develop a diabetic mouse From the Laboratoire de Physiologie, Faculte´deMe´decine d’Angers, Angers, model that exhibits vascular but not neuropathic changes France. Address correspondence and reprint requests to Prof. Jean Louis Saumet, to study the microvascular response 1) to local pressure Laboratoire de Physiologie, Faculte´deMe´decine d’Angers, 49045 Angers increase and 2) to endothelium-dependent and -indepen- cedex, France. E-mail: [email protected]. Received for publication 23 October 2003 and accepted in revised form dent vasodilators. Nerve function was assessed by sciatic 1 March 2004. nerve conduction velocity and morphometry to verify its ACh, acetylcholine; LDF, laser Doppler flowmetry; MABP, mean arterial integrity. We hypothesized that vascular dysfunction that blood pressure; MNCV, motor nerve conduction velocity; PIV, pressure- induced vasodilation; STZ, streptozotocin. occurs early in diabetes could be sufficient to alter the PIV © 2004 by the American Diabetes Association. response.

1564 DIABETES, VOL. 53, JUNE 2004 D. SIGAUDO-ROUSSEL AND ASSOCIATES

RESEARCH DESIGN AND METHODS TABLE 1 Male Swiss mice (20–30 g) were kept on a 12/12-h light/dark cycle with food Body weight and blood glucose and levels and water available ad libitum. The present investigation was performed in accordance with the guiding principles in the care and use of animals. Body Blood glucose Fructosamine Diabetes was induced by a single intraperitoneal injection of streptozotocin Animals weight (g) (mg/dl) (␮mol/l) (STZ; 200 mg/kg; Sigma) in citrate buffer (pH 4.5) during the fasting state. Control (n ϭ 10) 31 Ϯ 1 161 Ϯ 7 167 Ϯ 9 Control animals received equivalent doses of the citrate buffer solution. ϭ Ϯ Ϯ Ϯ Hyperglycemia occurred 2 days after STZ injection and was verified using an Diabetic (n 9) 22 1* 495 32† 343 23† Accu-Check Active glucometer (Roche, Lyon, France). We considered mice to *P Ͻ 0.01; †P Ͻ 0.001 vs. control. be diabetic when blood glucose was Ն16 mmol/l (normal 5–8 mmol/l), and only mice with blood glucose Ն16 mmol/l both on the second day and 1 week the M-wave (compound muscle action potential) from the tibial-innervated after the STZ injection were included in the experimental diabetic group. dorsal interossei foot muscles. During recording, the temperature of the site Three studies were performed on the diabetic group 1 week after STZ surrounding the nerve was kept constant at 37°C. treatment. Study 1 examined the microvascular response to local pressure Morphometric analysis. The right sciatic nerves were removed and fixed in increase. Study 2 evaluated the endothelium-dependent and -independent 4% glutaraldehyde in 0.1 mol/l phosphate buffer (pH 7.4) overnight at 4°C, and function. Study 3 was concerned with measurements of sciatic nerve function then washed in 0.1 mol/l phosphate buffer. These fixed samples were postfixed and structure as the MNCV and nerve morphometry. in 2% osmium tetroxide in phosphate buffer, dehydrated through an ascending For presenting a hairless area for the skin laser Doppler flow measure- series of ethanol concentrations, embedded in Epon, and polymerized. One- ments, local pressure application, and iontophoretic application, hair from the micron-thick semithin transverse nerve sections were stained with toluidine blue. top of the skull to the back of the animals was removed with a depilatory Morphometric analysis was performed using a computer-assisted image lotion. This was performed 2 days before the experiments to prevent skin analysis (Qwin, Leı¨ca, France) allowing for the determination of myelinated irritation during the experiment from confounding the results. fiber number and size. All counting was duplicated by two different micros- For the experiments, animals were anesthetized by intraperitoneal injec- copists who were blinded to the animal group, and results were averaged. tion of thiopental sodium (65 mg/kg). The level of anesthesia was determined Data analysis. The reproducibility of each technique was first evaluated in by testing eye reflexes and tail pinch. At the end of each experiment, animals healthy nontreated mice using the coefficient of variation. The techniques were killed by an overdose of thiopental. used in study 1 (PIV assessment), in study 2 (ACh and SNP iontophoretic Study 1: assessment of PIV. Microvascular responses to local pressure in deliveries), and in study 3 (MNCV) had coefficient of variation of 0.24 (139 Ϯ ϭ ϭ control (n 10) and diabetic (n 9) mice were measured by laser Doppler 33 arbitrary units [au], mean Ϯ SD), 0.41 (59 Ϯ 24%, ACh), 0.43 (58 Ϯ 25%, flowmetry (LDF). This method was described by Fromy et al. (2) using a SNP), and 0.25 (51 Ϯ 13 m/s), respectively. The power of the study was 95%. weighbridge that was adapted to hold a laser Doppler probe at one end Unpaired t test was used to evaluate the significance between the diabetic and (PF415; Periflux, Perimed, Sweden). The probe was connected to a laser control groups. To test for significant differences among groups, we per- Doppler flowmeter (PF5000 Master; Periflux). formed one-way ANOVA with Dunnett’s multiple comparison over the base- After general anesthesia, animals were settled in an incubator (MMS, line period. The results are presented as mean Ϯ SE, and P Ͻ 0.05 was Chelles, France) warmed to 30°C to maintain stable cutaneous temperature. considered significant. Mice were placed in the prone position, and the head was fixed on a frame followed by a 20-min resting period to stabilize the blood pressure and the cutaneous temperature. Blood pressure was measured using a noninvasive RESULTS system (IITC, Woodland Hills, CA). The weighbridge was equilibrated care- Body weight and blood glucose and fructosamine fully with the probe placed in the middle of the hairless skull of the mouse, levels. One week of diabetes caused a threefold elevation and an external pressure was increased progressively at 2.2 Pa/s through the laser Doppler probe using a syringe pump. of blood glucose (Table 1). Fructosamine levels were sig- The LDF signal was digitized with a 20-Hz sampling frequency using a nificantly increased twofold (P Ͻ 0.001) in diabetic mice computerized acquisition system (Biopac, Santa Barbara, CA). Data collection compared with control mice, showing long-lasting hyper- started with a 1-min control period before the onset of increasing pressure. glycemia. At the time of experimentation, diabetic mice LDF signal and cutaneous temperature were continuously recorded for 30 weighted 30% less than control mice (P Ͻ 0.01). min. Noninvasive blood pressure was recorded once before the onset of increasing pressure and every minute subsequently. The LDF signal was Assessment of PIV. Cutaneous temperature and mean averaged every 30 s to reduce the instantaneous variability of the signals as a arterial blood pressure (MABP) were stable throughout result of vasomotion. the experiment. In the control group, MABP was 87 Ϯ 6 Blood samples were taken at the end of the experiment to measure mmHg at rest, 88 Ϯ 5 mmHg at 0.2 kPa, and 89 Ϯ 6 mmHg fructosamine. Fructosamine was determined by a nitroblue tetrazolium col- orimetric test, based on the ability of the ketoamine group of glycated proteins at the end of the experiment. to reduce tetrazolium salts under alkaline conditions. Mean resting LDF was 113 Ϯ 7 au in control mice (Fig. Study 2: assessment of endothelium-dependent and -independent re- sponses. After general anesthesia, animals were settled in an incubator (MMS, Chelles, France) warmed to 30°C to maintain stable cutaneous temperature. Mice were placed in the prone position followed by a 20-min resting period to stabilize the blood pressure and the cutaneous temperature. Noninvasive blood pressure was recorded before and after iontophoresis application. Cutaneous blood flow was recorded using a laser Doppler multifiber probe (481-1; Perimed) during transcutaneous iontophoresis ap- plied to 1.2-cm2 area on the hairless back of the animals. Endothelium- dependent response was assessed in control (n ϭ 10) and diabetic (n ϭ 10) mice using anodal acetylcholine (ACh) iontophoresis (5.5 mmol/l; Sigma) with a current application of 100 ␮A during 10 s. Endothelium-independent response was assessed in control (n ϭ 10) and diabetic (n ϭ 10) mice using cathodal sodium nitroprusside (SNP) iontophoresis (Nitriate; SERB, Paris, France) (67 mmol/l) with a current application of 100 ␮A for 20 s. In addition, we checked the nonspecific anodal (100 ␮A, 10 s) and cathodal (100 ␮A, 20 s) current effect using deionized water as a vehicle as reported in humans (22). Iontophoresis technique was chosen to assess the in vivo skin microvascular function to avoid any systemic effects. ;10 ؍ F) and control mice (n ;9 ؍ Study 3: assessment of nerve function and structure FIG. 1. PIV in 1-week diabetic mice (n ϭ ϭ MNCV. MNCV was examined in control (n 12) and 1-week diabetic (n 12) Ⅺ). The reduction in LDF is significant at all pressures >0.46 kPa in mice as proposed by Zochodne et al. (23). MNCV in sciatic-tibial fibers was diabetic mice and for all pressures >1.16 kPa in control mice. *P < 0.05 assessed by stimulating at the exposed sciatic notch and knee while recording vs. rest.

DIABETES, VOL. 53, JUNE 2004 1565 PIV ALTERATION IN DIABETIC MICE

TABLE 2 Morphometric data on myelinated fibers in 1-week diabetic mice and control mice Total fiber Mean myelinated Animals number fiber size (␮m2) Control (n ϭ 8) 3,936 Ϯ 122 29.4 Ϯ 0.2 Diabetic (n ϭ 8) 3,980 Ϯ 55 29.7 Ϯ 0.2

the control group, MABP was not different between before (92 Ϯ 4 mmHg) and after (93 Ϯ 3 mmHg) ACh delivery. In the diabetic group, MABP was not different between FIG. 2. Percentage of vasodilation after ACh iontophoresis (A) and before (97 Ϯ 9 mmHg) and after (98 Ϯ 9 mmHg) ACh after SNP iontophoresis (B) in 1-week diabetic mice and control mice. delivery. *P < 0.05 vs. control. SNP iontophoresis produced a significant endothelium- 1). A maximal increase in mean LDF occurred at 0.2 kPa independent vasodilation in both diabetic (67 Ϯ 21%) and (max LDF: 151 Ϯ 15 au; P Ͻ 0.05), demonstrating a vaso- control (54 Ϯ 15%) mice. There was no significant differ- dilation. With further increases in pressure, mean LDF ence between the diabetic and control groups (Fig. 2B). In decreased and first became significantly lower (79 Ϯ 11 au; the control group, MABP was not different between before P Ͻ 0.05) than baseline value at 1.1 kPa (Fig. 1). (93 Ϯ 3 mmHg) and after (92 Ϯ 3 mmHg) SNP delivery. In In the diabetic group, MABP was 87 Ϯ 6 mmHg at rest, the diabetic group, MABP was not different between before 88 Ϯ 7 mmHg at 0.2 kPa, and 87 Ϯ 5 mmHg at the end (90 Ϯ 5 mmHg) and after (92 Ϯ 8 mmHg) SNP delivery. of the experiment. One-week diabetic mice had a mean Assessment of nerve function and structure resting LDF of 123 Ϯ 11 au and did not show a significant MNCV. After 1 week of diabetes, MNCV showed no dif- increase in mean LDF at any time during the experiment ference (47 Ϯ 3 m/s) compared with control mice (48 Ϯ 5 (Fig. 1). Mean LDF in diabetic mice was not changed at 0.2 m/s; Fig. 3). kPa (119 Ϯ 9 au) compared with mean resting LDF (0.2 Sciatic nerve morphometric analysis. The morpho- kPa corresponding to the pressure of the maximal value metric effects of 1-week diabetes in mice are shown in obtained in control mice). With further increases in pres- Table 2. There was no difference in total fiber number or in sure, mean LDF decreased and was first significantly lower mean myelinated fiber size between the diabetic and the (110 Ϯ 10 au; P Ͻ 0.01) than baseline value at 0.46 kPa control groups. Myelinated fiber size distribution was very (Fig. 1). similar between 1-week diabetic and control mice (Fig. 4). Endothelium-dependent and -independent responses. We did not observe any current-induced vasodilation DISCUSSION during iontophoresis of deionized water with anodal (P ϭ It is generally agreed that development is a 0.99) and cathodal (P ϭ 0.99) currents in randomly chosen consequence of neuropathy. In our study, we reported that control and diabetic mice. PIV that is a natural mechanism protecting the skin from In the control group, iontophoresis of ACh induced a pressure loads was completely abolished at the onset of significant endothelium-dependent vasodilation from 65 Ϯ diabetes. This demonstrates that neuropathy is not essen- 16 to 140 Ϯ 33 au (P Ͻ 0.01). In the diabetic group, tial for the development of pressure-induced ulcerations. iontophoresis of ACh also induced a significant vasodila- That is why it is of great importance to prevent the tion from 80 Ϯ 13 to 98 Ϯ 15 au (P Ͻ 0.01). Baseline values development of diabetic foot by detecting as soon as did not differ between diabetic and control mice. However, possible vascular alterations. To go further, we propose a the percentage of endothelium-dependent vasodilation new physiopathological concept of the cutaneous micro- was largely attenuated in diabetic mice (25 Ϯ 4%) com- circulatory control by assessing PIV responses. pared with control mice (81 Ϯ 25%; P Ͻ 0.05; Fig. 2A). In In the present study, we have demonstrated that PIV is abolished in 1-week diabetic mice that exhibit endothelial dysfunction but no neuropathy. The absence of PIV devel-

FIG. 3. MNCV of the sciatic nerve in 1-week diabetic mice and control FIG. 4. Fiber size distribution of myelinated fibers in the sciatic nerve .(Ⅺ ;8 ؍ f) and control mice (n ;8 ؍ mice. of 1-week diabetic mice (n

1566 DIABETES, VOL. 53, JUNE 2004 D. SIGAUDO-ROUSSEL AND ASSOCIATES opment in diabetic mice suggested that NO-dependent fore, it is recognized that an impairment of ACh-dependent response to external pressure is reduced. In addition to relaxation mainly reflects a decrease in NO bioactivity or this result, reduction of NO-dependent response to ACh NO production. Thus, NO-dependent response reduction delivery in diabetic mice supports the idea that back- to ACh delivery in diabetic mice suggests that at 1 week of ground levels of NO were reduced at the early stage of diabetes, there is already a reduction in endothelial NO diabetes. We proposed that NO level is reduced as early as levels, significant enough to impair PIV response, meaning 1-week of diabetes and that this reduction is significant that the process of diabetic endothelium alteration has enough to alter the PIV response in diabetic mice. already started. However, we cannot rule out possible The PIV mechanism allows skin blood flow to increase alterations in other endothelial vasodilatory pathways in response to a nonpainful, locally applied pressure contributing to the ACh response impairment such as stimulation in humans (1) and rats (2). It has been endothelium-derived hyperpolarizing factor and prosta- reported that this cutaneous vasodilation involved C fi- glandins, which have also been proposed to mediate the bers, because it disappeared after chronic treatment with effects of ACh in addition to NO (17). capsaicin in animals and humans. In addition, vasodilators In human studies, the iontophoresis technique has en- such as the calcitonin gene-related peptide as well as abled clinical measurement of the effects of diabetes on endothelium factors prostaglandins and endothelial NO vascular dysfunction. Several studies have demonstrated were involved in the PIV mechanism (2). In a previous reduced endothelium-dependent vasodilation in patients study, we reported an early decrease of skin blood flow at with either type 1 or (32–35) that was low applied pressures in diabetic patients both with and concordant with our study. Vascular studies in animals without clinical neuropathy, compared with control sub- using in vitro–isolated perfused arteries have provided jects (3). In the present study, we observed an early inconsistent results with enhanced, unaltered, or impaired decrease in LDF in response to external pressure applica- endothelium-dependent relaxation during diabetes (13–15) tion in the diabetic group that is similar to the one that might be explained by experimental conditions, in- observed by Fromy et al. (3) in diabetic patients compared cluding the type of artery and the type of vasodilator with control subjects. Indeed, LDF in 1-week diabetic mice examined in a given artery preparation. However, our was reduced beginning at a pressure as mild as 0.46 kPa, result is concordant with Terata et al. (37), who showed whereas LDF in control mice was not reduced until higher that ACh-induced vasodilation by arterioles, providing pressures (1.16 kPa). This clearly demonstrates the inabil- circulation to the region of the sciatic nerve, was impaired ity of the diabetic skin microcirculation to adapt to the early in diabetes. applied pressure; as a result, 1-week diabetic mice exhib- In contrast to ACh, SNP causes vasodilation by directly ited no PIV responses compared with control mice. Under stimulating the vascular smooth muscle cells. The present conditions of diabetic hyperglycemia, it has been sug- data show that vasodilation resulting from iontophoretic gested that three main biochemical pathways—nonenzy- application of SNP was not different between diabetic and matic glycation, redox potential alterations, and the polyol control mice, thus demonstrating intact smooth muscle pathway—decrease NO bioavailability (4,15). In our pres- cell vasodilation capacity. ent study, advanced glycation end product accumulation As expected, 1-week diabetic mice had no nerve alter- was confirmed via fructosamine increase as early as 1 ations as shown by the similar MNCV, total fiber number, week of diabetes induction, suggesting a reduced NO and myelinated fiber size between diabetic and control activity (24,25). Increased flux of the polyol pathway is mice. These results are in agreement with several studies thought to competitively decrease NADPH, leading to performed in rats (18,37,38) and mice (5). As nerve func- impaired production of NO (26–30). Consumption of tion was intact, it could not explain PIV absence in our NADPH may also contribute to decrease the production of mouse model. reduced glutathione and so decrease the antioxidant ca- Some authors (39) have observed a transient MNCV pacity leading to excessive oxidative stress and produc- decrease in 1-week STZ-induced diabetic rats that came tion of peroxinitrite (4,31). Therefore, the reduction in NO back to the onset level at 2 weeks. They suggested that the bioavailability that could occur as early as 1 week of transient MNCV decrease could result from reduced ATP diabetes could explain the absence of PIV, because NO supply or related to changes in myo-inositol and phospho- was shown to be essential for its development (2). inositide metabolism. We did not observe this change, To go further in the assessment of the cutaneous which could be due to the animal model used. Our study microvascular function in 1-week diabetic mice, we used used a diabetic mouse model, and the induction of diabe- the iontophoretic method, which gives the opportunity to tes and complications may be different from diabetic rat study in vivo endothelium-dependent and -independent models. It is recognized that slowing of nerve conduction responses. Although this method is commonly used in is a hallmark of both experimental and human diabetic diabetic patients (32–35), only a few diabetic animal neuropathy, and Coppey et al. (18) suggested that the studies have examined in vivo endothelium-dependent and vascular dysfunction observed in their study was respon- -independent responses (36). We observed a reduced ACh- sible for the nerve disorders associated with the early dependent relaxation in diabetic compared with control onset of diabetes. mice. ACh interacts with the endothelium and mediates its In conclusion, endothelium-dependent relaxation was endothelium-dependent vasodilatory effect through a mus- reduced but still present, whereas PIV was totally abol- carinic receptor on the endothelium surface. This leads to ished in 1-week experimental diabetic mice. These results a rise in intracellular calcium concentration, activation of are in accordance with those obtained in diabetic patients NO synthase, and an increase in NO production. There- (40). These findings point out that PIV response seems to

DIABETES, VOL. 53, JUNE 2004 1567 PIV ALTERATION IN DIABETIC MICE be highly sensitive to endothelial NO levels, and therefore dysfunction and pathogenesis of diabetic . Cardiovasc Res endothelial impairment by itself is sufficient to alter se- 34:55–68, 1997 14. Pieper GM: Review of alterations in endothelial nitric oxide production in verely the PIV response. We suggest that PIV could be a diabetes. Protective role of arginine on endothelial dysfunction. Hyperten- sensitive indicator of endothelium alteration occurring sion 31:1047–1060, 1998 during diabetes. PIV suppression could favor diabetes 15. Chan NN, Vallance P, Colhoum HM: Nitric oxide and vascular responses in complications such as diabetic foot ulcers. Detecting and type I diabetes. Diabetologia 43:137–147, 2000 preventing early vascular dysfunction could delay the appa- 16. Brands MW, Fitzgerald SM: Acute endothelium-mediated vasodilation is rition of diabetic neuropathy and limit diabetic compli- not impaired at the onset of diabetes. Hypertension 32:541–547, 1998 17. Pieper GM: Enhanced, unaltered and impaired nitric oxide-mediated cations. Further experiments are needed to explore the endothelium-dependent relaxation in experimental diabetes mellitus: im- individual involvement of each biochemical pathway in portance of disease duration. Diabetologia 42:204–213, 1999 PIV disappearance. 18. Coppey LJ, Davidson EP, Dunlap JA, Lund DD, Yorek MA: Slowing of motor nerve conduction velocity in streptozotocin-induced diabetic rats is preceded by impaired vasodilation in arterioles that overlie the sciatic ACKNOWLEDGMENTS nerve. Int J Exp Diabetes Res 1:131–143, 2000 19. Flynn MD, Tooke JE: Diabetic neuropathy and the microcirculation. D.S.-R. was supported by a grant from Conseil Re´gional Diabet Med 12:298–301, 1995 des Pays de la Loire. The present study was supported by 20. Greene DA, Arezzo JC, Brown MB: Effect of aldose reductase inhibition on the French National Scientific Institute INSERM and the nerve conduction and morphometry in diabetic neuropathy. Zenarestat Region des Pays de la Loire (ESPRI 2002). Study Group. Neurology 53:580–591, 1999 We thank the local Animal Care Unit of the University of 21. Ziegler D, Hanefeld M, Ruhnau KJ, Meissner HP, Lobisch M, Schutte K, Angers for facilities. We also thank Robert Filmon and Gries FA: Treatment of symptomatic diabetic with the anti-oxidant ␣-lipoic acid. A 3-week multicentre randomized controlled Christine Audrin of the Microscopy-Histology free access trial (ALADIN Study). Diabetologia 38:1425–1433, 1995 platform for valuable advice and technical assistance. We 22. 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