Role of LPS in the hepatic microvascular dysfunction elicited by cecal ligation and puncture in mice

Abstract

Background/aims: Sepsis remains a leading cause of death in critically ill patients. Because endotoxemia is viewed as a key mediator of sepsis-induced inflammation, administration of bacterial endotoxin (LPS) is often used to simulate sepsis in experimental animals. This study tests the hypothesis that LPS is a critical determinant of the hepatic microvascular dysfunction in mice made septic by cecal ligation and puncture (CLP).

Methods: Intravital videomicroscopy was used to quantify sinusoidal perfusion, and platelet and leukocyte adhesion in terminal hepatic venules (THV) and sinusoids in LPS-sensitive and LPS-insensitive mice subjected to CLP or LPS (i.p.). mRNA expression of TLR-2, TLR-4, MyD-88, and Ly-96 was also assessed.

Results: While LPS-sensitive mice responded to both CLP and LPS challenges with elevated leukocyte and platelet adhesion in THV and sinusoids, and a reduced sinusoidal perfusion density, LPS-insensitive mice exhibited comparable blood cell adhesion and sinusoidal malperfusion following CLP, but not LPS. Hepatic mRNA of MyD-88 and TLR-2 was elevated in the CLP and LPS groups. Endotoxin was not detectable in the blood of LPS-sensitive mice after CLP, but was elevated after LPS administration.

Conclusions: These findings do not support a major role for LPS in the hepatic microvascular disturbances associated with polymicrobial sepsis.

Figure 1

Firm adhesion of leukocytes (panel A) and platelets (panel B) in terminal hepatic venules following cecal ligation and puncture (CLP) in LPS-sensitive and LPS-insensitive mice. * p < 0.005 versus LPS sens Ctrl; # p < 0.0005 versus LPS insens Ctrl. n=8 for LPS sens Ctrl, n=6 for LPS sens CLP, n=9 for LPS insens Ctrl, n=6 for LPS insens CLP.

Figure 2

Leukocyte (panel A) and platelet (panel B) recruitment in hepatic sinusoids of LPS-sensitive and LPS-insensitive mice after cecal ligation and puncture (CLP). * p < 0.001 versus LPS sens Ctrl; # p < 0.0005 versus LPS insens Ctrl. n=8 for LPS sens Ctrl, n=6 for LPS sens CLP, n=9 for LPS insens Ctrl, n=6 for LPS insens CLP.

Figure 3

Number of nonperfused sinusoids (NPS) in LPS-sensitive and LPS-insensitive mice after cecal ligation and puncture (CLP). * p < 0.001 versus LPS sens Ctrl; # p < 0.0005 versus LPS insens Ctrl. n=8 for LPS sens Ctrl, n=6 for LPS sens CLP, n=9 for LPS insens Ctrl, n=6 for LPS insens CLP.

Figure 4

Leukocyte (panel A) and platelet adhesion (panel B) in terminal hepatic venules of LPS-sensitive and LPS-insensitive mice after graded doses of LPS (50 and 500 μg/kg, respectively) and controls. * p < 0.05 versus corresponding Ctrl; # p < 0.05 versus corresponding LPS.insens group. n=4 for LPS sens Ctrl, n=3 for LPS sens 50g/kg, n=6 for LPS sens 500 g/kg, n=5 for LPS insens Ctrl, n=3 for LPS insens 50g/kg, n=3 for LPS insens 500 g/kg.

Figure 5

Leukocyte (panel A) and platelet (panel B) recruitment in hepatic sinusoids following injection of 50 and 500 μg/kg LPS in LPS-sensitive and LPS-insensitive mice. * p < 0.01 versus corresponding Ctrl; # p < 0.05 versus corresponding LPS insens group. n=4 for LPS sens Ctrl, n=3 for LPS sens 50g/kg, n=6 for LPS sens 500 g/kg, n=5 for LPS insens Ctrl, n=3 for LPS insens 50g/kg, n=3 for LPS insens 500 g/kg.

Figure 6

Sinusoidal perfusion failure (number of nonperfused sinusoids) in LPS-sensitive and LPS-insensitive animals after treatment with 50 and 500 μg/kg LPS. * p < 0.05 versus corresponding Ctrl; # p < 0.05 versus corresponding LPS insens group. n=4 for LPS sens Ctrl, n=3 for LPS sens 50g/kg, n=6 for LPS sens 500 g/kg, n=5 for LPS insens Ctrl, n=3 for LPS insens 50g/kg, n=3 for LPS insens 500 g/kg.

Figure 7

Fold changes in hepatic mRNA expression of MyD-88, Ly96, TLR-2 and TLR-4, measured using RT-PCR in LPS-sensitive mice following sham-surgery, CLP, or administration of either 50 or 500 μg/kg LPS.