A chronic model for intravital microscopic study of microcirculatory disorders and leukocyte/endothelial cell interaction during normotensive endotoxemia

Sepsis-induced microvascular leukocyte/endothelial cell interaction may result in a deterioration of capillary perfusion that finally leads to septic organ dysfunction. The aim of the present study was to characterize a novel, sublethal, two-hit model of chronic systemic sepsis that allows the repeated analysis of microcirculation by intravital microscopy. In Syrian golden hamsters the effect of a single i.v. endotoxin (LPS, 2 mg/kg, E. coli) injection (SH-LPS group, n = 5 animals) vs. a double LPS injection (DH-LPS group, n = 6 animals) was analyzed. After monitoring baseline parameters (t(1)), measurements were performed at 30 min (t(2)), 3 h (t(3)), 8 h (t(4)), 24 h (t(5)), 48 h (t(6)), 56 h (t(7)) and 72 h (t(8)) (both groups) after initial LPS exposure. In DH-LPS animals, a second LPS injection (2 mg/kg) was given at t(6) (48 h). Intravital fluorescence microscopy was performed in a dorsal skin fold chamber preparation and allowed determination of leukocyte/endothelial cell interaction (leukocyte rolling and sticking), and measurement of functional capillary density (FCD), which served as a measure of capillary perfusion. The first LPS injection comparably altered leukocyte/endothelial cell interaction and capillary perfusion in both groups (t(1)-t(6), P > 0.05, MANOVA). Between t(6) and t(8) leukocyte adherence decreased in SH-LPS animals, whereas in DH-LPS animals adherence remained constantly elevated (SH-LPS: -53.0 +/- 6.2% between t(6) and t(8) vs. DH-LPS: -3 +/- 5; P < 0.05). The ongoing inflammatory response in DH-LPS animals was associated with a progressive deterioration of FCD, whereas FCD remained constant in SH-LPS animals (DH-LPS: -71.5 +/- 17% between t(6) and t(8) vs. SH-LPS: 3.0 +/- 13%; P < 0.05). In parallel, coagulatory parameters were found significantly altered only in DH-LPS animals but not in SH-LPS animals. We conclude that "double hit" LPS exposure is an appropriate model (i) to analyze repeatedly over time microcirculatory disorders under conditions of persistent endotoxemia-induced inflammatory response, and (ii) to prove the effectiveness of novel anti-inflammatory strategies.