1) Trauma-hemorrhagic shock induces rapid RBC adhesion to endothelial cells in patients and animals. 2) Increased RBC CD36 expression characterizes the RBC-adhesive phenotype. 3) The RBC phenotypic and functional changes were induced by gut-derived humoral factors. These novel findings may explain the microvascular dysfunction occurring after trauma-hemorrhagic shock, sepsis, and other stress states.
Objective
To test the hypothesis that gut-derived factors carried in trauma-hemorrhagic shock (T/HS) lymph are sufficient to induce red blood cells (RBC) injury, to investigate their potential mechanisms of action, and to define the time post-T/HS that these factors appear in the lymph.
Methods
Mesenteric lymph collected from T/HS or trauma-sham shock (T/SS) rats over different time periods was injected intravenously into male rats at a rate of 1 mL/h for 3 hours. RBC deformability was measured using laser-assisted ektacytometer to calculate the elongation index. From the shear-stress elongation curve, the stress required for the erythrocytes to reach 50% of their maximal elongation was also determined. RBC deformability was measured before lymph infusion and at 1 hour and 3 hours after the initiation of lymph infusion. The effect of the lymph samples (5% v/v) was also determined in vitro by incubating naïve whole blood with the lymph samples. The potential role of T/HS lymph-induced RBC oxidant injury mediated by inducible nitric oxide synthase (iNOS)-generated oxidants and/or white blood cells (WBC) was investigated using iNOS inhibitors and WBC depletion, respectively. In all the in vivo studies, five to seven rats were studied per group.
Results
The intravenous injection of T/HS lymph but not T/SS lymph caused in vivo RBC injury. The biological activity of T/HS lymph varied over time with the RBC-injurious factors being produced only during the first 3 hours postshock. The in vivo inhibition of iNOS did not prevent lymph-induced RBC injury. T/HS lymph incubated in vitro with naïve whole blood resulted in RBC injury, but this injury was not observed in blood depleted of WBC.
Conclusions
These results indicate that T/HS lymph produced during the initial 3-hour postshock period is sufficient to induce RBC injury in otherwise normal rats and that the lymph-induced RBC injury is not dependent on activation of the iNOS pathway but seems to require WBC.
Immune depression after trauma-hemorrhage has been implicated as an important factor in the pathogenesis of sepsis and septic-organ failure. Although recent studies have implicated immune cell apoptosis as an important factor in the evolution of this post-trauma immune suppressed state, neither the initial triggers that induce this response nor the cellular pathways through which these triggering pathways act have been fully defined. Thus, the current study tests the hypothesis that acute splenic and thymic immune cell apoptosis developing after trauma-hemorrhagic shock (T/HS) is due to gut-derived factors carried in intestinal lymph and that this T/HS lymph-induced immune depressed state is mediated through TLR4. The first set of experiments documented that T/HS caused both thymic and splenic immune cell apoptosis as measured by TUNEL and caspase-3 immunohistochemistry and that this increase in apoptosis was totally abrogated by mesenteric lymph duct ligation. In subsequent experiments, mesenteric lymph collected from animals subjected to T/HS or trauma-sham shock (T/SS) were injected into TLR4 deficient (TLR4mut) mice or their wild-type (WT) littermates. T/HS, but not T/SS, lymph caused splenic apoptosis in the WT mice. However, the TLR4mutmice were resistant to T/HS lymph-induced splenic apoptosis. Furthermore, the WT, but not the TLR4mutmice developed splenic apoptosis after actual T/HS. In conclusion, gut-derived factors appear to initiate a sequence of events that leads to an acute increase in splenic and thymic immune cell apoptosis and this process is TLR4-dependent.
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