Discordance between microvascular permeability and leukocyte dynamics in septic inducible nitric oxide synthase deficient mice

Abstract

Introduction: Microvascular dysfunction causing intravascular leakage of fluid and protein contributes to hypotension and shock in sepsis. We tested the hypothesis that abrogation of inducible nitric oxide synthase (iNOS) activation would decrease leukocyte rolling, leukocyte adhesion, and microvascular leakage in sepsis. We compared wild-type mice made septic by cecal ligation and puncture with mice deficient in iNOS.

Methods: Leukocyte dynamics and microvascular permeability were assessed simultaneously by fluorescence intravital microscopy in the cremaster muscle 15 to 20 hours after induction of sepsis by cecal ligation and puncture in C57Bl/6 mice. Rolling and adhesion of leukocytes labeled with rhodamine and leakage of fluorescein isothiocyanate-conjugated albumin was measured in single nonbranching venules (25 to 40 microm) and compared among septic wild-type, septic iNOS-deficient transgenic, and sham-operated control mice.

Results: Leukocyte rolling and adhesion were increased in septic animals (61.6 +/- 14.4 cells/minute and 4.1 +/- 0.6 cells/100 microm per minute, respectively) as compared with control animals (8.5 +/- 2.3 cells/minute and 1.1 +/- 0.2 cells/100 microm per minute, respectively; P < 0.001 for both). Rolling increased in iNOS-deficient septic mice (to 105.5 +/- 30.0 cells/minute, P = 0.048, versus wild-type septic); adhesion was unchanged (5.1 +/- 0.5 cells/100 microm per minute, P = 0.30). Sepsis produced an increase in leakage ratio in wild-type septic mice compared with controls (0.36 +/- 0.05 versus 0.08 +/- 0.01, P < 0.001). Leakage was attenuated in iNOS-deficient septic mice (0.12 +/- 0.02, P < 0.001, versus wild-type septic mice).

Conclusion: Leukocyte adhesion and vascular leakage were discordant in this setting. The finding that septic iNOS-deficient mice exhibited less microvascular leakage than wild-type septic mice despite equivalent increases in leukocyte adhesion suggests an important role for nitric oxide in modulating vascular permeability during sepsis.

Figure 1

Leukocyte rolling. Shown is leukocyte rolling in wild-type control mice (white bar; 8.5 ± 2.3 cells/minute; n = 8), wild-type septic mice (black bar; 61.6 ± 14.4 cells/minute; n = 8), inducible nitric oxide synthase (iNOS)-deficient control mice (light stippled bar; 18.8 ± 6.9 cells/minute; n = 8), iNOS-deficient mice (dark stippled bar; 105.5 ± 30.0; n = 10), and mice treated with the selective iNOS inhibitor 1400W (cross-hatched bar; 74.0 ± 13.3 cells/minute; n = 5). *P < 0.001 versus wild-type control. ^§P < 0.001 versus wild-type septic. KO, iNOS-deficient knockout; WT, wild-type.

Figure 2

Leukocyte adhesion. Shown is leukocyte adhesion in wild-type control mice (white bar; 1.1 ± 0.2 cells/100 μm per minute; n = 8), wild-type septic mice (black bar; 4.1 ± 0.6 cells/100 μm per minute; n = 8); inducible nitric oxide synthase (iNOS)-deficient control mice (light stippled bar; 4.1 ± 0.1 cells/100 μm per minute; n = 8), iNOS-deficient mice (dark stippled bar; 5.1 ± 0.5 cells/100 μm per minute; n = 10), and mice treated with the selective iNOS inhibitor 1400W (cross-hatched bar; 4.9 ± 0.6 cells/100 μm/minute; n = 5). *P < 0.001 versus wild-type control. KO, iNOS-deficient knockout; WT, wild-type.

Figure 3

Microvascular leakage. Shown is microvascular leakage in wild-type control mice (white bar; 0.08 ± 0.01; n = 8), wild-type septic mice (black bar; 0.36 ± 0.52; n = 8), inducible nitric oxide synthase (iNOS)-deficient control mice (light stippled bar; 0.08 ± 0.03; n = 8), iNOS-deficient mice (dark stippled bar; 0.12 ± 0.02; n = 10), and mice treated with the selective iNOS inhibitor 1400W (cross-hatched bar; 0.16 ± 0.05; n = 5). *P < 0.001 versus wild-type control. ^§P < 0.001 versus wild-type septic. KO, iNOS-deficient knockout; WT, wild-type.