Bacterial translocation in cirrhotic rats stimulates eNOS-derived NO production and impairs mesenteric vascular contractility (original) (raw)

All experimental procedures in this study were conducted according to the American Physiological Society principles for the care and use of laboratory animals.

Induction of liver cirrhosis in rats by CCl4.

Male Sprague-Dawley rats (Harlan Sprague Dawley Inc., Indianapolis, Indiana, USA), weighing 100–125 g, underwent inhalation exposure to CCl4 and had phenobarbital (0.35g/L) added to their drinking water, as described previously by ourselves and others (4, 5). This protocol produces a high yield of micronodular cirrhosis in about 12–16 weeks. Phenobarbital and CCl4 exposure were stopped at least 6 days before the perfusion experiments. Normal sex- and age-matched untreated rats were used as controls.

Assessment of BT.

On the study day, the animals were anesthetized with ketamine hydrochloride (100 mg/kg), and the abdominal skin was shaved and sterilized with an iodine solution. All of the following surgical procedures were performed under strict sterile conditions with sterile instruments. In cirrhotic animals, ascites was quantified. To exclude animals with systemic bacteremia for protocol 1 (see below), 3 cm3 of blood was withdrawn from the inferior vena cava and inoculated into aerobic and anaerobic Bactec culture bottles. The blood was incubated at 35°C, and the growth value (a measurement of CO2 production by bacteria) was continuously monitored for at least 7 days. None of these cultures showed bacterial growth, confirming that, in our laboratory, this model of CCl4-induced liver cirrhosis is free of systemic bacteremia (33). Therefore, we did not test for bacteremia in the following protocols. The caudal and cranial MLNs were removed and weighed with an E400D scale from Ohaus Corp. (Florham Park, New Jersey, USA), which is accurate to ±0.01 g. Tissues were then homogenized in a measured amount of saline, and 0.1-mL aliquots were plated onto blood, MacConkey, and phenylethyl alcohol blood agar plates (BBL Prepared Media; Becton Dickinson Microbiology Systems, Sparks, Maryland, USA). A swab of ascitic fluid from the peritoneal cavity was cultured. If this culture showed bacterial growth, the animal was excluded from the study. Spleen and liver were removed and weighed. Liver slices were fixed in 10% neutral buffered formaldehyde. Solid culture media were examined, and colonies were counted after 24 and 48 hours of aerobic incubation at 35°C. Any positive MLN cultures were considered indicative of BT from the intestinal lumen.

In vitro perfusion.

The in vitro perfusion system used was a partial modification of that originally described by McGregor (48); it has been used extensively in previous studies by our laboratory (4–7, 16, 17). Briefly, the superior mesenteric artery (SMA) was cannulated with a polyethylene PE-60 catheter, and then gently perfused with 15 mL of warm Krebs solution to eliminate blood. After isolating the SMA with its mesentery, the gut was cut off close to its mesenteric border. The arterial vasculature was then transferred to a 37°C water-jacketed container and perfused with oxygenated (95% O2, 5% CO2) 37°C Krebs solution, using a roller pump (Masterflex; Cole-Parmer Instrument Co., Barrington, Illinois, USA). The Krebs solution had the following composition (in mmol/L): NaCl, 118.0; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; disodium EDTA, 0.026; glucose, 11.0 (pH was 7.4). The effluent of the perfused tissue was continuously removed from the perfusing chamber. The perfusion pressure was measured with a H-P pressure transducer (Hewlett Packard, Andover, Massachusetts, USA) mounted on a side arm just before the perfusing cannula. Pressure was recorded continuously on a 7D polygraph inscriber (Grass Instruments, Quincy, Massachusetts, USA).

Experimental protocols

All cirrhotic rats were studied prospectively, because at the time of experimental procedures, it was not known whether the cirrhotic animals were positive or negative for BT. A total of 78 cirrhotic rats with ascites and 26 age-matched control rats were included in this study. In all animals (except in protocol 4), before starting procedures to assess BT, the left femoral artery was exposed and cannulated with a PE-50 catheter. Blood (1.5 cm3) was drawn into a pyrogen-free Vacutainer (Becton Dickinson) and centrifuged at 5,000 rpm for 15 minutes. The separated serum was stored immediately at –70°C until analysis for NO metabolites (NOx) and TNF-α. Mean arterial pressure (MAP) was evaluated by connecting the catheter to the H-P pressure polygraph inscriber, and was recorded on a model 7D inscription recorder (Grass Instruments). Measurements of MAP were performed before blood was drawn (and after its replacement with 1.5 cm3 saline). In the animals used in protocols 2 and 3, additional MLN homogenate was stored at –70°C until measurement of NOx and TNF-α levels.

Protocol 1. In 18 ascitic cirrhotic rats (with BT: n = 10; without BT: n = 8) and 10 control rats, in vitro perfusion of the mesenteric vessel bed was performed. Baseline perfusion at 4 mL/min was established for 60 minutes. At 30, 45, and 60 minutes, the perfusion solution from the mesenteric tissue was collected into glass tubes for 1 minute. Then 2 doses of methoxamine (MTx; 30 and 100 μM) were administered noncumulatively by continuous infusion for 2 minutes (first perfusion cycle), and the pressure response was documented. Three perfusate samples were collected for each pressure-response period, beginning with the increase of perfusion pressure. Each sample was collected for 1 minute, frozen immediately, and stored at –30°C until the NOx assay. After the first perfusion cycle was completed, each vessel preparation was perfused at the basal flow rate of 4 mL/min for 50 minutes (after that time, the perfusate NOx concentration has returned to baseline levels; data not shown).

Then the specific NOS inhibitor _N_ϖ-nitro-L-arginine (l-NNA; 10–4 M; Sigma Chemical Co., St. Louis, Missouri, USA) was added, and a second perfusion cycle was initiated after 20 minutes of incubation time. The NOS inhibitor was present at the same molar concentration in the perfusion system throughout the second perfusion cycle. Before initiation of the experiment, the Krebs solution that was circulated through the perfusion system (including the catheter, but not mesenteric tissue) was collected for use in determining background NOx levels. As shown previously, the perfusion system showed stable basal perfusion conditions and unchanged pressure response for the duration of this experiment (6, 7, 17).

Protocol 2. Endothelial denudation of the mesenteric vasculature was performed in 16 cirrhotic ascitic rats (with BT: n = 8; without BT: n = 8) and in 6 control rats by a combined treatment of cholic acid (sodium salt) and distilled water (4). In brief, after cannulation of the SMA and gentle flushing with 10 mL of warmed Krebs solution to eliminate blood, perfusion with 1.5 mL of 0.5% cholic acid (for 10 seconds) followed by 15 mL of Krebs solution (to eliminate cholic acid) was performed. The preparation was then transferred to a 37°C water-jacketed container and perfused with oxygenated 37°C Krebs solution (4 mL/min for 10 minutes). After the mesenteric vasculature was relaxed, it was perfused with 37°C distilled water for 10 minutes. A period of 45 minutes was allowed before noncumulative concentration-response curves to MTx (1–100 μM) were derived. To assess whether the vessel was completely de-endothelialized and whether smooth muscle function was maintained, the mesenteric preparation was kept preconstricted with MTx (100 μM) at the end of each dose-response curve, while dose-dependent vasorelaxation response to the endothelium-dependent vasodilator acetylcholine (10–8 to 10–6 g; bolus of 0.1 mL) and the endothelium-independent vasodilator sodium nitroprusside (10–6 to 10–5 g; bolus of 0.1 mL) was tested.

Protocols 3 and 4. In protocol 3, a total of 25 cirrhotic ascitic rats and 10 control rats were used for performance of blotting and tetrahydrobiopterin (BH4) measurements in the studied mesenteric vasculature. In protocol 4, a separate group of 19 cirrhotic ascitic animals were used for evaluation of NOS activity in mesenteric resistance vessels (MRVs).

Western blotting. SMA vessels and MRVs were analyzed for the presence of eNOS and iNOS protein. SMA vessels were harvested after removal of MLNs and freeing the artery from surrounding tissue over a length of 3–4 cm, starting at its aortic origin. Tissue for Western blotting of MRVs was harvested from a portion of the capillary bed situated between the MLNs and the small intestine. The tissue was cut off from the SMA at the level of second-order arteries and dissected from the intestine as close as possible to the gut wall. Vessels were washed in PBS and homogenized in a lysis buffer described previously (17). Protein supernatants were quantitated using the Lowry assay, and equal amounts of protein from each sample were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes. Membranes were probed with an mAb recognizing eNOS (Transduction Laboratories, Lexington, Kentucky, USA) and a polyclonal antibody recognizing iNOS (Affinity BioReagents Inc., Golden, Colorado, USA). The specificity of the eNOS and iNOS antibodies for rat tissue was established previously by using quiescent and activated rat sinusoidal endothelial cells, respectively, as positive controls. Enhanced chemiluminescence was used for protein detection.

BH4 assay. Frozen mesenteric vasculature was weighed and homogenized in 3 volumes of 50 mM Tris-HCl (pH 7.4) containing 1 mM DTT and 1 mM EDTA. This mixture was centrifuged, and supernatant BH4 levels were measured by HPLC with fluorescence detection after oxidation, as described previously (49).

NOS activity assay. NOS activity was measured by determining the conversion of 3H-labeled L-arginine to 3H-labeled L-citrulline, using a commercially available NOS assay kit (Calbiochem-Novabiochem Corp., San Diego, California, USA). Briefly, mesenteric tissue was harvested as described above, and then minced with fine scissors and homogenized in lysis buffer. Samples were incubated with a reaction buffer containing 1 mM NADPH, 3 μM BH4, 100 nM calmodulin, 2.5 mM CaCl2, 1 μM FAD, 1 μM FMN, and 1 μCi/μL of [3H]arginine or [14C]arginine. To determine NOS activity, duplicate samples were incubated in the presence and absence of _N_ϖ-nitro-L-arginine methylester (l-NAME; 1 mM) or vehicle. The reaction was continued for 1 hour at 35°C, and then stopped by the addition of 0.4 mL of cold stop buffer (50 mM HEPES and 5 mM EDTA; pH 5.5). After addition of 100 μL of equilibrated resin to each reaction mixture, samples were passed over prepared columns. The eluates were transferred into vials and analyzed using a liquid scintillation counter. Radiolabeled cpm of L-citrulline generation were measured.

Determination of NOx concentration. The NOx concentration of each perfusate and serum sample was measured using the Nitric Oxide Analyzer from Sievers Instruments (Boulder, Colorado, USA), as described previously (16, 17). In brief, this assay is based upon spectrophotometric analysis after a chemiluminescent reaction between NO and ozone. Fifty microliters of each sample was placed into the purge vessel, which contained 3 mL of 0.1 M vanadium chloride in 1 M HCl at 95°C. The NO generated from nitrite and nitrate ions was carried into the analyzer by vacuum through a gas bubbler trap containing 5 mL of 1 M NaOH. This analyzer quantitates dissolved NO and NO2-derived NO that has been generated by acid and stripped from the solution by nitrogen gas. The NO then reacts with analyzer-generated ozone to form excited NO2, which releases light in the red and near-infrared regions of the spectrum and is detected by a thermoelectrically cooled, red-sensitive photomultiplier tube. The lower limit of sensitivity for this machine is below 2 picomoles of NO per second. Because of this high sensitivity, it is necessary to make sure that exogenous contamination does not affect the results. Therefore, we conducted control experiments in which the perfusate was circulated through the perfusion system without tissue for the duration of the experiment. Under these circumstances, NOx concentrations of the collected perfusate were low and practically the same at all timepoints. The displayed NOx concentrations and related NOx production rates were corrected for each experiment by subtracting background NOx concentrations. The net NOx release in response to MTx (Δ NOx concentration) is calculated as the difference between NOx concentration at baseline (without MTx) and after each pressure-response period. NOx production rate was obtained by multiplying the perfusate flow rate by the concentration of NOx in the perfusate, and is expressed in nanomoles per minute. For calculation of the regression coefficients between the amount of NOx production and shear stress, the average NOx concentration was calculated for each experimental period. Shear stress was calculated as described previously using the following equation (16, 17):

(Equation 1)1

where K = (η/2πL3)1/4 or K = 1/2(8η/πL3)1/4 = constant factor.

Assays. Concentrations of TNF-α in serum and MLN homogenate were measured in duplicate with a commercially available rat-specific ELISA (sandwich ELISA) kit (Endogen Inc., Woburn, Massachusetts, USA) according to the protocol supplied by the manufacturer. The lower limit of sensitivity for TNF-α in this assay was 3 pmol/mL.

Statistical analysis. Results were expressed as mean ± SEM. Statistical analysis was performed using 2-way, repeated-measures ANOVA and paired and unpaired Student’s t tests, with statistical significance set at P < 0.05. Each relationship of hemodynamic, perfusate, serum, and lymph node parameters was tested for regression by a simple regression analysis. To increase the sensitivity of the regression analysis, statistical significance for correlations was set at P < 0.01.