Septic shock (dangerously low blood pressure) during invasive bacterial infection is one of the most common causes of morbidity and mortality in patients in medical intensive care units (ICUs). Despite the use of effective antibiotics in combination with cardiovascular support (therapy to increase blood pressure), the mortality rate with septic shock remains high (29%)(1). Myocardial (heart) dysfunction is an important contributor to the pathogenesis of shock during sepsis (1, 2). Understanding the nature of this dysfunction and its treatment will be important for the management of sepsis in the future. Relevant animals models will greatly aid in such research. Although the mouse is one of the most frequently employed animal species for the study of sepsis, a recent literature review has shown that myocardial dysfunction in this model has received little attention (4). In fact, only one published study has assessed myocardial changes in a bacteria challenged mouse sepsis model. That study was limited and did not adequately control for fluid treatment which is commonly employed in septic patients and which is recognized to alter myocardial function (5). The goal of the present study is to employ echocardiography (organ imaging with sound waves) and pressure-conductance techniques (pressure and volume measures using electrical conductance measures or PV-measures) to define the pattern of mycocardial dysfunction occurring during E. coli pneumonia in mice.? ? Myocardial dysfunction in septic patients and large animal models varies based on whether measurements are performed before or after fluid replacement (3). Fluid treatment is an essential part of cardiovascular support in sepsis and has been shown to improve survival in animal sepsis models (3, 6, 7). Prior to fluids, the heart appears small and the overall amount of blood that it pumps is low (i.e. a hypodynamic state) (7). With standard fluid treatment which is given early to almost all patients, the heart dilates, becomes larger than normal, and pumps an increased amount of blood (i.e. a hyperdynamic state). Despite this increased cardiac output, sepsis is characterized by decreased myocardial contractility (reduced pressure generated for a given heart volume) and ejection fraction (EF) is decreased. A reduction in EF is a highly reproducible finding in volume resuscitated patients and large animals and correlates with the severity of sepsis and outcome (7). Whether this pattern of change actually occurs in the mouse during bacterial sepsis has not been clearly tested. If present however, it would add to the relevance of this species for the study of sepsis. In the only published study that assessed myocardial function with bacterial challenge in mice, cardiac output was reported to be increased in fluid treated animals following cecal ligation and puncture (infection in the abdomen). However this study was limited. Uninfected control animals did not receive fluids and there were no infected animals studied not receiving fluid (5). Importantly, ejection fraction, fractional shortening and end systolic volumes were not reported and PV-measures were not performed. ? ? In contrast to this published report, we found that compared to uninfected control mice, intratracheal E. coli challenge was associated with reductions in end systolic and diastolic volumes, increases in EFs and overall reductions in cardiac output (CCM 05-07). All animals in this study including uninfected controls were treated with fluids before cardiac studies, however the volume of fluids employed (1 dose of 25 ml/kg 4 h after bacteria challenge) were not as great as the study by Hollenberg et al (5) (50 ml/kg q6h x 48 h). Thus, whether the hypodynamic changes we noted (decreased end systolic and diastolic volumes and cardiac output and increased EF) were a function of inadequate fluid resuscitation or whether the mouse heart responds to bacterial sepsis in a fundamentally different way from larger species is unknown. ? ? The purpose of the present study is to comprehensively define the myocardial changes and their response to a protective regimen of fluid support during intra-tracheal E. coli challenge in the mouse. This challenge simulates bacterial pneumonia, now recognized to be the most common cause of sepsis and septic shock in medical ICUs (CCM 04-02,1, 2). The study will be performed in three stages. First, to confirm that echocardiography and PV-measurement techniques reliably detect changes in myocardial function and contractility in the mouse, animals will be treated with either dobutamine, a positive-inotropic agent (increases heart function) or esmolol, a negative-inotrope and measures (as described in SECTION F) will be performed. This stage will include an initial group of animals in which techniques to place high-fidelity micro- tip pressure-conductance catheters into the left ventricle via the carotid artery with microscopic assistance are established. The second stage will determine a level of fluid support that improves survival during intra-tracheal E. coli challenge. Although protective fluid regimens have been defined for intraperitoneal infection in the mouse (8), whether they apply to pneumonia, where aggravation of hypoxemia with fluid treatment may worsen outcome, has not been determined. Therefore, in this second stage the effect of increasing doses of normal saline 0, 25, 50, 100 or 200 ml/kg/d subcutaneously either as a single dose (BID) or in 3 divided doses, (TID) on survival will be tested in mice after challenge with intra-tracheal (IT) E. coli producing a mortality rate of 80%. Two different dosing regimens will be tested (BID vs TID) to determine whether one larger fluid bolus will result in comparable survival effects to an equivalent volume divided into 3 doses. The former would be more practical but may not be as effective. In the third part of study, the effects of intra-tracheal E. coli challenge and fluid on myocardial function will be measured. Animals will be randomized to normal saline or E. coli challenge and to no fluid support or the regimen of fluids noted to be most protective in the second part of the study. Animals will then be randomly selected at 24 or 48 h to undergo echocardiography and PV measures. They will then have blood drawn for blood cultures, chemistry, cytokine and nitric oxide measures after which they will be sacrificed for myocardial histologic and mitogen activated protein kinase (MAPK) and nuclear-factor kappa B (NF-KB) studies. All animals in stages 2 and 3 will receive ceftriaxone 100 mg/kg per day for 96 h.
