The study was approved by the local committee on animal research and adhered for the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health. Male ZDF (fa/fa) (Charles River Laboratories, Wilmington, MA) rats with a genetically induced form of type 2 diabetes including severe hyperglycemia and their normoglycemic controls, the Zucker lean (ZL) (Charles River Laboratories, Wilmington, MA) rats were studied. Rats were fed Purina 5008 rat chow (Charles River Laboratories) containing 29.2% protein, 7% fat, 52.3% carbohydrates, 4.3% fiber and 7.2% ash. They received tap water ad libitum. Rats were individually housed on a 12-h dark/12-h light cycle. After development of diabetes animals were divided into 7 groups. ZL and ZDF rats received either normal-salt (0.28% NaCl) (ZL, n = 9) (ZDF, n = 9) or high-salt diet (5.5% NaCl in chow) (ZL+S, n = 9) (ZDF+S, n = 8) for 10 weeks starting at an age of 15 weeks. Two additional groups of normal-salt and high-salt treated ZDF rats received eplerenone (100 mg/kg body weight per day in chow; Pharmacia/Pfizer) (ZDF + E, n = 8) (ZDF+S+E, n = 8). To control for effects of blood pressure lowering one group of ZDF rats on high salt diet received orally the vasodilator hydralazine (25 mg/kg body weight per day) (ZDF+S+H, n = 7). Serum glucose was measured weekly from an incision in the tail vein (Accu-Check Sensor, Roche, Mannheim, Germany). Systolic blood pressure (SBP) was measured weekly by the tail cuff method using an automated cuff inflator-pulse system (W+W electronic AG, BPrecorder No. 8005, Basel, Switzerland). The experiment was terminated at week 25 by decapitation of the rats. Blood was collected and plasma samples immediately frozen for further analysis. The mesenteric vasculature was dissected, and one segment was used for preparation of small arteries.
Preparation and Study of Small Arteries
A 3rd order branch of the superior mesenteric artery was carefully dissected and mounted on a pressurized myograph (111 P; Danish Myo Technology, Aarhus, Denmark). Perfusion was performed using oxygenated 37°C Krebs solution (95% O2, 5% CO2) pH 7.4 containing (mmol-1): NaCl (118), KH2PO4 (1.18), KCl (4.7), MgSO4 (1.18), CaCl2 (2.5), D-Glucose (5.5), NAHCO3 (25) and EDTA (0.026) as described previously . Vessels were equilibrated under a constant pressure of 45 mmHg. Vascular reactivity was tested with a single dose of norepinephrine (10-5 mol/L-1). After precontraction with 10-5 mol/L norepinephrine, endothelium dependent relaxation was assessed with acetylcholine (10-4 mol/L). Vascular morphology and mechanics was assessed under resting conditions at three different points along the vessel. For assessment of passive mechanical properties, arteries were deactivated of myogenic tone by calcium-depletion (Ca2+-free KREBS solution containing EDTA 10 mmol/l for 30 min). Then intravascular pressure was increased from 3 to 10, 20, 30, and 40 mmHg and afterward in 20-mmHg steps up to 140 mmHg. The pressure was maintained at each pressure step until stable conditions were reached to allow the vessel to reach a steady-state diameter. The changes in internal diameter as well as media thickness of vessels in response to each increase in intravascular pressure were measured at three points along the vessel with the use of a calibrated video system (111 P, Danish Myo Technology, Aarhus, Denmark). At last vessels were fixed for histological analysis.
Third order mesenteric arteries were pressurized at 45 mmHg, fixed with 2% paraformaldehyde solution at room temperature for 30 minutes, removed from the cannula, and processed for histological analysis. Paraffin embedded tissue sections of mesenteric arteries were stained with Sirius red F3BA (0,5% in saturated aqueous picric acid, Sigma Aldrich chemical Company, Steinheim, Germany). Collagen content in the media was quantified light microscopically with the Northern Eclipse imaging program (EM-PIX Imaging Inc.) and was determined by measuring the relative density per area in each Sirius red-stained section.
Paraffin sections were dewaxed in sequential xylol and isopropyl alcohol to distilled water. Endogenous peroxidase activity was quenched by incubation with 3% H2O2 for 20 min at room temperature. For collagen IV examination sections were incubated with the primary antibody (goat anti-rat polyclonal antibody, 1:100, 37°C for 60 min; Southern biotech) after blocking with normal rabbit serum (30 min, room temperature). After rinsing in PBS, sections were incubated with the second antibody (biotinylated anti-goat, 1:100, 30 min, room temperature; Vector Laboratories, Inc., Burlingame, CA, USA). Antibody-antigen complexes were detected using a Vectastain Elite ABC kit (Vector). Then the label complex was applied (AEC Kit; Vector) for 7.5 min. Colour development was stopped under microscopic control by adding water, and sections were finally counterstained using haemoxylin. For examination of elastin content immunohistochemistry was performed in a similar manner. After blocking with normal goat serum (30 min, room temperature) rabbit anti-rat polyclonal antibody served as primary antibody (1:100 dilution, 37°C for 60 min; Chemicon International, Inc., Temecula, CA, USA). After rinsing in PBS, we used biotinylated anti-rabbit (1:100, 30 min, room temperature; Vector Laboratories, Inc., Burlingame, CA, USA) as second antibody and performed the further investigation steps identical to the collagen IV examination.
Plasma Aldosterone Levels
Blood samples were centrifugated at 4°C, and the plasma was saved for subsequent analysis. Plasma aldosterone levels were measured by radioimmunoassay with the use of commercially available kit (DiaSorin, Dietzenbach, Germany), according to the manufacturer's instructions.
Data are presented as mean ± SEM. For systolic blood pressure levels, mean values of weekly measurements throughout active treatment were calculated for each animal. Circumferential stress which corresponds to wall tension or the distending force on the vessel wall was calculated as σ = (PDi)/(2 M), where P was intraluminal pressure, and Di and M were lumen diameter and media thickness, respectively. Circumferential strain which corresponds to pressure-induced relative increases in lumen diameter was calculated as ε = (D - D0)/D0 where D was the observed lumen diameter at a given intraluminal pressure and D0 was the original diameter measured at 3 mmHg, respectively. The strain-stress relation was fitted to an exponential curve for each vessel and the slope for each curve was determined. Groups were compared using 1-way ANOVA, or 2-way ANOVA for repeated measurements as appropriate. Post-hoc testing was performed using LSD test (1-way ANOVA and 2-way ANOVA for repeated measures). A p-value of < 0.05 was considered significant. Statistical analyses were performed using SigmaStat 3.0 software and SPSS 18 software (SPSS GmbH, Muenchen, Germany).