All procedures using animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of LSU Health Sciences Center and were performed according to the criteria outlined by National Institutes of Health guidelines. All surgery was performed under ketamine and xylazine anesthesia, and all efforts were made to minimize suffering.
Wild-type (WT) C57/BL6J and B6.B10 ScN-Tlr4lps-del/JthJ (TLR4-/-) mice, weighing 17-22 g, the ages of 6-8 weeks, were obtained from the Jackson Laboratory (Bar Harbor, ME). The mice were housed in a temperature- and humidity-controlled room and were allowed free access to a standard chow diet and water before the experiments.
The following 7 experimental groups were included in this study: 1) saline-treated WT mice (n = 10); 2) WT mice + 10 ml/kg olive oil (n = 12); 3) WT mice + antibiotics (pre-treated with 450 mg/kg/day polymyxin B and 150 mg/kg/day neomycin by gavage for 7 days) + 10 ml/kg olive oil (n = 3); 4) TLR4-/- mice + 10 ml/kg olive oil (n = 3); 5) WT mice + 10 ml/kg corn oil (n = 12) 6). WT mice + antibiotics (pre-treated with 450 mg/kg/day polymyxin B and 150 mg/kg/day neomycin by gavage for 7 days) + 10 ml/kg corn oil; (n = 3) 7) TLR4-/- mice + 10 ml/kg corn oil (n = 3). Intravital microscopy was performed 1.0, 1.5, 2.0, 2.5 hours after oil administration in 2) and 5) groups. In 3) and 4) groups, intravital microscopy was performed only at 2.0 h. In 6) and 7) groups, intravital microscopy was performed just at 1.5 h.
Approximately 0.9 ml of blood was collected from corresponding donor mice through a carotid artery cannula (polyethylene tubing, PE-10) into a polypropylene tube containing 0.1 ml of acid-dextrose buffer (Sigma; St. Louis, MO). The tube was centrifuged at 1,200 rpm for 8 min. The supernatant was transferred into another polypropylene tube and centrifuged again at 1,200 rpm for 3 min to remove any residual RBCs. This supernatant was transferred to another new tube and centrifuged at 3,000 rpm for 10 min to separate the platelets from the plasma. The platelets were suspended in PBS, counted, and divided into aliquots that contained 1-1.5 million platelets. Before injection into recipient mice, the platelets were incubated for 10 min with the fluorochrome CFSE, Molecular Probes, Eugene, OR) at a final concentration of 90 μM. The platelet suspension was then centrifuged at 3,000 rpm for 10 min, resuspended in 250 μl PBS and stored in the dark until injected into mice .
The procedures used to evaluate blood cell adhesion and rolling in the murine intestinal microvasculature have been described previously . Briefly, the animals were anesthetized subcutaneously using a mixture of ketamine and xylazine at a dose of 100 and 5 mg/kg, respectively. The right jugular vein was cannulated with polyethylene tubing (PE-10) for the administration of platelets. Core body temperature was maintained at 35 ± 0.5°C using an external temperature controlled heat lamp.
Following a midline incision, a small portion of the jejunum along with attached mesentery was carefully exteriorized, placed across a viewing cover glass, and superfused on a thin sheet of glass through which the bowel wall could be visualized. After a 20-min stabilization period, the CFSE-labeled platelets were injected via the jugular vein. 5 minutes later, rhodamine-6G (0.02%) was administered via the jugular vein for fluorescent labeling and visualization of leukocytes.
Fluorescently labeled platelets and leukocytes were visualized with an OLYMPUS IX71 inverted microscope equipped with a 75-W XBO xenon lamp and using a 20× objective. Visualization was accomplished using a filter with an excitation of 470-490 nm, a dichroic mirror (510 nm) with images recorded using a SONY DXC-390 video camera for offline evaluation. A 1-min recording of a 200-um length of three to five jejunal venules were obtained from each mouse. The total number of rolling and adherent leukocytes was determined, as well as the number of rolling and adherent platelets. Leukocytes and platelets were counted as ‘rolling’ if they were moving at a velocity significantly slower than the centerline velocity of the microvessel. ‘Adherent’ leukocytes or platelets were defined as cells remaining stationary on the vessel wall for ≥30 seconds and expressed as the number of cells per second per square millimeter of venular surface, calculated from diameter and length, and assuming cylindrical vessel shape. Venular diameter was measured with a video caliper.
Once the venular data were collected, the animals were allowed to stabilize for 20-30 min, and the arterioles with diameters between 15-40 μm and a wall shear rate (WSR) of ≥500/s were chosen for study. The diameters and red blood cell velocities were measured in the chosen sections before and after superfusion with 10-5 M of the endothelium-dependent vasodilator Ach for 5 min. The arterioles were then superfused with BBS and allowed to return to baseline values. Arterioles were then exposed to 10 μM papaverine (an endothelial-independent vasodilator) to determine maximal dilation. Arteriolar vasorelaxation responses to Ach were expressed as the % change in diameter normalized to the baseline.
Western blotting assay
Total protein (100 μg) was extracted from frozen samples, separated on 10% SDS/PAGE gels and transferred to PVDF membranes. Membranes were blotted with anti-TLR4 (Cell Signaling Technology; Danvers, MA), anti-SIRT1 (Santa Cruz Biotechnology), anti- NF-κB p65 (Cell Signaling Technology; Danvers, MA), anti-pNF-κB p65 (Cell Signaling Technology; Danvers, MA) and β-actin (AbD-Serotec) overnight. Target proteins were visualized using ECL-Plus detection reagents (Amersham Biosciences; Piscataway, NJ) in a Chemidox XRS documentation system (Bio-Rad Laboratories; Hercules, CA).
Statistical analyses of the data were performed with using one-way ANOVA with Fisher’s post hoc test. All values are reported as means ± SEM. Statistical significance was set at p < 0.05.