No abstract

Virtually of the all recent therapeutic interventions for treating sepsis have failed to improve survival. One potential explanation is that the heterogeneity of the immune response to the septic challenge is such that only a portion of the patients die as a result of excessive inflammation. The clinical trials lacked power because traditional measurements do not accurately identify these patients. Previous work has shown that higher levels of interleukin (IL)-6 are found in those mice that die from septic peritonitis; therefore, we sought to determine whether IL-6 measured 6 h after surgery could predict outcome. Adult, female BALB/c mice (n = 79) were subjected to cecal ligation and puncture with a 21-gauge needle and treated with imipenem in D5W every 12 h for 5 days, resulting in a homogenous population at the outset. Six hours after surgery, 20 microL of blood was obtained from the tail vein to measure IL-6. Mortality was followed for 21 days. Overall 3-day survival was 77%, and 21-day mortality was 56%. Plasma IL-6 levels >2,000 pg/mL were determined to predict mortality within the first 3 days with a sensitivity of 58% and specificity of 97%. To further refine the mortality prediction, body weight and a complete blood count were performed 24 hours after cecal ligation and puncture. Discriminate analysis indicated that a weighted formula combining body mass, lymphocyte, and platelet count would predict death with sensitivity of 83% and a specificity of 79%. We tested the value of the IL-6 prediction by surgically resecting the cecum in those animals with IL-6 > 2000 pg/mL, which resulted in a significant improvement in survival. These data demonstrate that IL-6 measured 6 h after injury accurately predicts mortality resulting from experimental sepsis. This measurement may be determined quickly so that therapy may be targeted only to those individuals at significant risk of dying and initiated within sufficient time to be effective.

Previous studies have suggested that interleukin-6 (IL-6) serves as both a marker and a mediator for the severity of sepsis. We tested whether interleukin 6 knockout (IL-6KO) mice were more susceptible to sepsis mortality induced by cecal ligation and puncture. IL-6KO and wild-type (WT) mice were subjected to increasing degrees of sepsis severity. Physiologic support was given with fluids and appropriate antibiotics. Plasma IL-6 levels were determined 6 h after the onset of sepsis, and a complete hematologic profile was performed on day 2. As expected, increasing sepsis severity resulted in greater and more rapid mortality. However, the mortality was nearly identical in the IL-6KO and WT mice. All WT septic mice had high plasma levels of IL-6 6 h after the onset of sepsis, while IL-6KO were near or below the lower limit of detection. Among the WT mice, mortality was significantly higher in mice with plasma IL-6 >3,000 pg/ml. Both IL-6KO and WT mice destined to die in the early stages of sepsis had substantial and nearly identical weight gain in the first 24 h. However, at later stages the WT mice had significantly greater weight loss than the KO mice. The KO mice failed to develop the characteristic hypothermia within the first 24 h of severe sepsis routinely observed in the WT mice. These data demonstrate that IL-6 serves as a marker of disease severity in sepsis and does modulate some physiologic responses, but complete lack of IL-6 does not does not alter mortality due to sepsis.

In biomedical research using animal models, the phrase "humane endpoints" refers to predetermined criteria used to judge when the research animals should be humanely euthanized. The intended goal of humane endpoints is to minimize the distress or suffering of research animals; however, if applied incorrectly, this well-intended concept could lead to premature decisions and inaccurate data, resulting in a waste of animal life. A concensus on specific endpoints for shock and inflammation research is not available but several biochemical, physical and behavioral parameters have been suggested for other research models. In addition, the authors have found, in the studies presented here, that increasing body weight, decreased body temperature, and inability to ambulate are important parameters in a model of cecal ligation and puncture. However, it is clear that the applicability of these endpoints may change with the model of disease, intensity of insults, experimental treatments and other factors. Consequently, humane endpoints should be assigned cautiously and preferably after preliminary studies to prevent aberrant research results. In order to accomplish this, investigators must become aware of certain concepts including: when to implement endpoints, what endpoints to consider, and how to establish the endpoints for their studies. Equipped with the basic principles of humane endpoints, investigators can make informed decisions that meet current standards of animal care while still achieving the scientific goals of their research studies.

In anesthetized BALB/c mice, sampling site readily influences blood counts and neutrophil CD11b. The findings underscore the need to standardize sampling site when measuring these parameters.