Fig 1.
Effect of renal ischemia on peripheral blood erythrocyte aggregation.
Representative Wright’s stained peripheral smears (A-C) and quantification of systolic blood pressure (D), hematocrit (E), serum creatinine (F), erythrocyte aggregates(G) and fibrinogen levels (H) 48 hours after surgery are presented. There was no difference between sham surgery/vehicle and sham surgery/dipyridamole in the measured parameters and thus only the sham surgery/vehicle values are presented in this and subsequent figures. Each group contained 6 animals. In this and subsequent figures, mean values ± 1 standard error of the mean are presented. Statistical analysis employed ANOVA. *p<0.0001 vs sham (exact p values are in S1 File); isch, ischemia; veh, vehicle; dph, dipyridamole.
Fig 2.
Effect of renal ischemia on leukocyte retention and vascular flow in the brain.
Representative images of brain 48h after renal ischemia or sham surgery and quantification of plasma flow, thrombi, adherent WBC, tissue MPO activity and C3 and ICAM transcript levels are presented. In this and subsequent figures, the vascular space is delineated with fluorescein-conjugated albumin (green) and the nuclei labeled with Hoechst (blue). The arrow shows an example of a nucleated cell (WBC) adherent to the vessel in live images. During intravital imaging, obstruction then reversal of flow was seen at the asterisk. Each group contained 6 animals. For thrombi, percent of total fields are presented (numerator and denominator values for this and subsequent figures are in S1 File). In this and subsequent figures, statistical analysis for plasma flow, number of WBC, MPO, C3 and ICAM employed ANOVA; for categorical data (thrombi), Fisher’s exact test was used. *p<0.0001 and †p<0.004 vs sham; §p<0.001 and #p<0.05 vs ischemia/ vehicle (exact p values are in S1 File); scale bar is 100 μm.
Fig 3.
Effect of renal ischemia on leukocyte retention and vascular flow in the small intestine.
Representative images of intestinal microcirculation 48h after renal ischemia or sham surgery and quantification of plasma flow, microthrombi, adherent WBC, tissue MPO activity and C3 and ICAM transcript levels are presented. The arrows show adherent nucleated leukocytes in live images. Each group contained 6 animals. Statistical analysis for plasma flow, number of WBC, MPO, C3 and ICAM employed ANOVA; for categorical data (thrombi), Fisher’s exact test was used. *p<0.003 and †p<0.05 vs sham; §p<0.001 and #p<0.05 vs ischemia/vehicle (exact p values are in S1 File); scale bar is 100 μm.
Fig 4.
Effect of renal ischemia on leukocyte retention and vascular flow in the mesentery.
Representative images of mesenteric microvasculature 48h after renal ischemia or sham surgery, quantification of plasma flow, rouleaux, adherent leukocytes, tissue MPO activity and C3 and ICAM transcript levels are presented. The arrow points to RBC aggregation and intravascular nuclei (WBC) adherent to vessels in live images. Each group contained 6 animals. Statistical analysis for plasma flow, number of WBC, MPO, C3 and ICAM employed ANOVA; for categorical data (thrombi), Fisher’s exact test was used. *p<0.002, † p<0.04, vs sham; §p<0.05 vs ischemia/vehicle (exact p values are in S1 File); scale bar is 100 μm.
Fig 5.
Effect of renal ischemia on leukocyte retention and vascular flow in the liver.
Representative images of the hepatic microcirculation 48h after renal ischemia or sham surgery, quantification of microvascular plasma flow, erythrocyte aggregates, adherent WBC, tissue MPO activity and transcript levels of C3 and ICAM are presented. Examples of erythrocyte aggregation and intravascular leukocytes adherent to vessels, resulting in retention of the fluorescent dye, are at arrows. Each group contained 6 animals. Statistical analysis for plasma flow, number of WBC, MPO, C3 and ICAM employed ANOVA; for categorical data (thrombi), Fisher’s exact test was used. *p<0.0001, † p<0.005, ‡p<0.05 vs sham, §p<0.005 vs ischemia/vehicle (exact p values are in S1 File); scale bar is 100 μm.
Fig 6.
Effect of renal ischemia on leukocyte retention and vascular flow in the spleen.
Representative images of the microvasculature of the spleen show areas of RBC aggregation and adherent leukocytes (examples at arrows) 48 hours after renal ischemia as compared to sham surgery. Quantification of microvascular flow, RBC aggregates, adherent leukocytes, tissue MPO activity and C3 and ICAM transcription after sham surgery or renal ischemia are also presented. Each group contained 6 animals. Statistical analysis for plasma flow, number of WBC, MPO, C3 and ICAM employed ANOVA; for categorical data (thrombi), Fisher’s exact test was used. *p<0.0002, † p<0.003, ‡p<0.05 vs sham; §p<0.002, #p<0.05 vs ischemia/vehicle (exact p values are in S1 File); scale bar is 100 μm.
Fig 7.
Effect of renal ischemia on leukocyte retention and vascular flow in the kidney.
Representative images of the renal microvasculature show areas of RBC aggregation and adherent leukocytes (arrows) with retention of administered fluorophore 48 hours after renal ischemia as compared to sham surgery. The graphs show quantification of microvascular plasma flow, thrombi, adherent WBC, tissue MPO activity and C3 and ICAM transcript levels. Each group contained 6 animals. Statistical analysis for plasma flow, number of WBC, MPO, C3 and ICAM employed ANOVA; for categorical data (thrombi), Fisher’s exact test was used. *p<0.0001, † p<0.003, ‡p<0.05 vs sham; §p<0.001, #p<0.05 vs ischemia/vehicle (exact p values are in S1 File); scale bar is 100 μm.
Fig 8.
Effect of renal ischemia on the heart.
Representative images of the hematoxylin and eosin and esterase stained heart sections 48 hours after renal ischemia or sham surgery are presented. Arrows show leukocytes and arrowhead RBC aggregates. The graphs show quantification of tissue MPO activity and C3 and ICAM transcript levels. Each group contained 6 animals. Statistical analysis employed ANOVA. *p<0.006 vs sham (exact p values are in S1 File); scale bar is 100 μm.