Animal preparation and experimental design
All experimental procedures and animals handling were reviewed and approved by the Institutional Committee for Animal Care and Use of the School of Medicine of Ribeirão Preto, University of São Paulo.
Twenty-seven male mongrel dogs from kennel of School of Medicine of Ribeirão Preto (18-25 kg, young adult) were studied. After an overnight fast except for ad libitum water, the animals were premedicated with intramuscular injection of ketamine (15 mg/Kg, Ketamin - S(+), Cristália Produtos Químicos Ltda, Itapira, SP, Brazil) associated with xylazine (2 mg/Kg, Dopaser®, Hertape Calier Saúde Animal S/A, Juatuba, MG, Brazil). The anesthesia was maintained with intravenous bolus administration of sodium thiopental (30 mg/kg, Thiopentax, Cristália Produtos Químicos Ltda, Itapira, SP, Brazil). The animals were intubated with an endotracheal tube (8.0 mm, Rüsch, Teleflex Medical, Durham, NC, USA) and ventilated with 100% O2 in a pressure controlled mode (Takaoka 600, K. Takaoka Indústria e Comércio Ltda, São Bernardo do Campo, SP, Brazil). An intravenous catheter was placed in the jugular vein for fluid administration and blood drawn. Maintenance fluid consisted of physiologic solution (NaCl 0.9%) at 2 ml/kg/hr and the blood was drawn at end of the experiment (immediately before the euthanasia) for biochemical assessment. The right carotid artery was cannulated for continuous intra-arterial blood pressure and an electrocardiogram monitor showed the heart rate. A median transperitoneal laparotomy was performed and the abdominal supraceliac aorta was exposed. Then, the animals were randomly allocated in one of the three groups: sham (no clamping, n = 9), ischemia (clamping for 60 minutes, n = 9), and ischemia/reperfusion (clamping for 60 minutes followed by reperfusion for 30 minutes, n = 9). The vascular clamp was applied to the abdominal supraceliac aorta except for sham group. The sham animals were submitted to the same surgical procedures with the omission of vascular occlusion and monitored for 90 minutes. After the desirable protocol for each group, the animals were sacrificed with an overdose of sodium thiopental followed by exsanguinations via carotid. Then, the celiac trunk, renal and superior mesenteric arteries were quickly harvested for vascular reactivity studies. Renal tissue samples were also collected. Plasma and renal samples were stored at -70°C until determination of malondialdehyde (MDA) and nitrite and nitrate (NOx) levels.
Vessel preparation and isometric tension recording
The arterial segments (celiac trunk, renal and superior mesenteric) were carefully dissected free of connective tissue and immersed in a cooled and oxygenated Krebs solution (NaCl: 118.0, KCl: 4.7, CaCl2: 2.5, KH2PO4: 1.2, MgSO4: 1.66, glucose: 11.1, NaHCO3: 25.0 (mM), pH 7.4). The arterial segments were cut in rings of 4-5 mm in length and prepared with great care to avoid touching the intimal surface. In some rings the endothelium was removed by gently rubbing the intimal surface of the blood vessel with a pair of watchmaker's forceps. This procedure removes endothelium but does not affect the ability of the vascular smooth muscle to contract or relax.
The rings were mounted in organ chambers (10 mL) filled with Krebs solution maintained at 37°C and bubbled with 95% O2/5% CO2 (pH 7.4). Each arterial ring was suspended by two stainless steel clips placed through the lumen. One clip was anchored to the bottom of the organ chamber, while the other was connected to a strain gauge for measurement of the isometric force using Grass FT03 (Grass Instrument Company, Quincy, MA, USA). The rings were placed at an optimal length-tension of 10 g (determined in a pilot study) and allowed to equilibrate for 60 min with the bath fluid being changed every 15 to 20 min.
Endothelial integrity was assessed qualitatively by the degree of relaxation caused by acetylcholine (Ach, 10-6 M; Sigma, St. Louis, MO, USA) in the presence of contractile tone induced by prostaglandin F2α (PGF2α, 2.10-6 M; Sigma, St. Louis, MO, USA). For studies of endothelium intact vessels, the ring was discarded if relaxation with Ach was not 80% or greater. For studies of endothelium-denuded vessels, rings were discarded if there was any measurable degree of relaxation. Sequentially, each ring was washed and re-equilibrated for 30 min.
Arterial rings were then precontracted with PGF2α (2.10-6 M), and cumulative concentration-response curves were obtained after a stable plateau was reached. The receptor-dependent and -independent relaxations were evoked by Ach (10-10 - 3.10-5 M) and calcium ionophore (A23187, 10-10 - 3.10-5 M, Sigma, St. Louis, MO, USA), respectively, both in endothelium-intact rings. The endothelium-independent relaxation was evoked by sodium nitroprusside (SNP, 10-10 - 3.10-5 M; Sigma, St. Louis, MO, USA) in denuded rings. All concentration-response curves were accomplished by pre-incubating the arterial rings with indomethacin (2.10-5 M, an unspecific cyclooxygenase inhibitor; Sigma, St. Louis, MO, USA) for 50 minutes.
The changes in vascular wall tension are expressed as percent of relaxation in relation to the maximal contraction achieved following exposure to PGF2α, a convention that corrects inter-animal variability.
Malondialdehyde (MDA) measurement
Blood samples were collected in tubes containing EDTA (1:20 v/v). After blood centrifugation (3000×g, 10 min, 4°C), plasma aliquots were stored at -70°C until MDA measurement.
Renal tissue samples were wrapped and promptly stored at -70°C. For analysis, the renal samples were homogenized in Tris-HCl (20 mM, pH 7.4, 10% w/v), the homogenate was centrifuged (3000×g, 10 min, 4°C), and the supernatant was used for the assay.
Plasmatic and renal MDA concentration was measured using a commercially available kit (Lipid Peroxidation Assay kit, Calbiochem, San Diego, CA, USA). The assay is based on the ability of a chromogenic agent to react with MDA, yielding a stable chromophore with maximal absorbance at 586 nm. Results are expressed in μM.
Nitrite and nitrate (NOx) quantification
Blood samples were collected in tubes containing heparin (1:20 v/v). After blood centrifugation (3000×g, 10 min, 4°C), plasma aliquots were stored at -70°C until NOx measurement.
Renal tissue samples were wrapped and promptly stored at -70°C. For analysis, the renal samples were homogenized in Tris-HCl (20 mM, pH 7.4, 10% w/v), the homogenate was centrifuged (3000×g, 10 min, 4°C), and the supernatant was used for measurement of NOx and total protein by means of the modified biuret reaction .
Plasma and renal samples were analyzed using an ozone-based chemiluminescence assay. Briefly, the samples were treated with cold ethanol (1:2 v/v for 30 min at -20°C) and centrifuged (4000×g, 10 min). NOx levels were measured by injecting 25 μL of the supernatant in a glass purge vessel containing 0.8% of vanadium (III) in HCl (1 N) at 90°C, which reduces NOx to NO gas. A nitrogen stream was bubbled through the purge vessel containing vanadium (III), then through NaOH (1 N), and then into an NO analyzer (Sievers® Nitric Oxide Analyzer 280, GE Analytical Instruments, Boulder, CO, USA). NOx concentration was calculated from a standard curve (sodium nitrate 0.5, 1.5, 10, and 50 mM). NOx concentration is expressed in μM for plasma and in μM/mg protein for renal samples.
The results are expressed as mean ± standard error of the mean (SEM). The dose-response curves to Ach, A23187 and SNP were performed using molar concentrations of these drugs and the figures show logarithm of molar concentration (log [M]). The concentration-response curves were analyzed using two-way repeated-measures analysis of variance (ANOVA) and Bonferroni post-test, and the concentrations of MDA and NOx were analyzed using one-way ANOVA (Prism 4.0, GraphPad Software Inc., San Diego, CA, USA). Values were considered to be statistically significant at p values less than 0.05.