The Myocardial Effects of E. coli Pneumonia and Sepsis in Mice

大肠杆菌肺炎和败血症对小鼠心肌的影响

• 批准号: 7733617
• 负责人: Peter Eichacker
• 金额: $ 5.4万
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733617
• 关键词: Abdomen; Animal Model; Animals; Antibiotics; Attention; Bacteria; Bacterial Infections; Bacterial Pneumonia; Blood; Blood Pressure; Bolus Infusion; Cardiac; Cardiac Output; Cardiac Volume; Cardiovascular system; Carotid Arteries; Catheters; Ceftriaxone; Chemistry; Control Animal; Dobutamine; Dose; EFRAC; Echocardiography; Electric Conductivity; Escherichia coli; Functional disorder; Future; Goals; Heart; Histologic; Hypotension; Hypoxemia; Image; Infection; Intensive Care Units; Invasive; Left ventricular structure; Ligation; Liquid substance; Measurement; Measures; Medical; Microscopic; Mitogen-Activated Protein Kinases; Modeling; Morbidity - disease rate; Mus; Myocardial; Myocardial dysfunction; NF-kappa B; Nature; Nitric Oxide; Normal saline; Organ; Outcome; Pathogenesis; Patients; Pattern; Pneumonia; Publishing; Pump; Puncture procedure; Purpose; Randomized; Rate; Reporting; Research; Resuscitation; Review Literature; Sepsis; Septic Shock; Severities; Shock; Simulate; Staging; Standards of Weights and Measures; Techniques; Testing; Titrations; Treatment Protocols; base; cytarabine/methotrexate protocol; cytokine; day; esmolol; heart function; improved; intraperitoneal; mortality; pressure; response; septic; sound

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.<br><br>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. <br><br>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. <br><br>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). In studies thus far, protective regimens of fluid have been defined. 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. Thus far techniques have been developed to perform PV loops in the animals and titration studies with dobutamine and esmolol are underway. In addition, fluid regimens have been established that are protective in the model.

侵袭性细菌感染期间的感染性休克（危险的低血压）是重症监护病房（icu）患者发病和死亡的最常见原因之一。尽管使用了有效的抗生素并结合心血管支持（升高血压的治疗），感染性休克的死亡率仍然很高（29%）(1)。心肌（心脏）功能障碍是脓毒症期间休克发病机制的重要因素（1,2）。了解这种功能障碍的性质及其治疗将对脓毒症的管理在未来很重要。相关的动物模型将对此类研究有很大的帮助。尽管小鼠是研究脓毒症最常用的动物物种之一，但最近的一篇文献综述显示，该模型的心肌功能障碍很少受到关注(4)。事实上，只有一项已发表的研究评估了细菌挑战小鼠败血症模型的心肌变化。该研究是有限的，并没有充分控制液体治疗，而液体治疗通常用于脓毒症患者，并被认为可以改变心肌功能(5)。本研究的目的是利用超声心动图（声波器官成像）和压力传导技术（使用电导测量或pv测量的压力和体积测量）来确定小鼠大肠杆菌肺炎期间发生的心肌功能障碍模式。脓毒症患者和大型动物模型的心肌功能障碍因在补液前或补液后进行测量而异(3)。液体治疗是脓毒症中心血管支持的重要组成部分，在动物脓毒症模型中已被证明可以提高生存率（3,6,7）。在输液之前，心脏显得很小，泵出的血液总量很低（即低动力状态）(7)。采用标准的液体治疗（几乎所有患者都在早期接受），心脏会扩张，变得比正常情况更大，并泵出更多的血液（即高动力状态）。尽管心输出量增加，但脓毒症的特征是心肌收缩力降低（给定心脏容积产生的压力降低）和射血分数（EF）降低。在大容量复苏的患者和大型动物中，EF的降低是一个高度可重复的发现，并且与败血症的严重程度和结果相关(7)。在细菌性败血症期间，这种变化模式是否真的发生在小鼠身上还没有得到明确的测试。然而，如果存在，它将增加该物种对败血症研究的相关性。在唯一一项已发表的评估小鼠心肌功能与细菌攻击的研究中，据报道，在盲肠结扎和穿刺（腹部感染）后，经液体处理的动物心输出量增加。然而，这项研究是有限的。未感染的对照动物不接受液体治疗，研究中没有不接受液体治疗的感染动物(5)。重要的是，没有报道射血分数、缩短分数和收缩期末期容积，也没有进行pv测量。与这篇发表的报告相反，我们发现与未感染的对照小鼠相比，气管内大肠杆菌攻击与收缩期和舒张末期体积减少、EFs增加和心输出量总体减少有关（CCM 05-07）。本研究中的所有动物，包括未感染的对照组，在心脏研究之前都进行了液体治疗，然而所使用的液体量（细菌攻击后4小时1剂量25毫升/公斤）并不像Hollenberg等人的研究（50毫升/公斤q6h x 48小时）那么大。因此，我们注意到的低动力变化（收缩期和舒张期末容积和心输出量减少以及EF增加）是液体复苏不足的功能，还是小鼠心脏对细菌性败血症的反应与大型物种的根本不同，目前尚不清楚。本研究的目的是全面确定小鼠气管内大肠杆菌攻击时心肌的变化及其对液体支持保护方案的反应。这个挑战模拟了细菌性肺炎，现在被认为是医疗icu中败血症和感染性休克的最常见原因（CCM 04-02, 1,2）。在迄今为止的研究中，已经确定了液体的保护方案。这项研究将分三个阶段进行。首先，为了确认超声心动图和pv测量技术能够可靠地检测小鼠心肌功能和收缩力的变化，将对动物进行多巴酚丁胺（一种正性肌力剂（增加心脏功能））或艾思洛尔（一种负性肌力剂）治疗，并进行测量（如第F节所述）。这一阶段将包括一组初始的动物，在这些动物中，建立了在显微镜辅助下通过颈动脉将高保真微尖端压力传导导管置入左心室的技术。第二阶段将确定液体支持水平，以提高气管内大肠杆菌攻击期间的存活率。虽然已经为小鼠腹腔感染确定了保护性液体治疗方案(8)，但它们是否适用于肺炎还没有确定，在肺炎中，液体治疗会加重低氧血症，从而使结果恶化。因此，在第二阶段，增加生理盐水0、25、50、100或200 ml/kg/d皮下单次剂量（BID）或分3次剂量（TID）对小鼠存活的影响将在气管内大肠杆菌攻击后进行试验，产生80%的死亡率。将测试两种不同的给药方案（BID vs TID），以确定一个较大的液体丸是否会产生与等量分3次剂量相当的生存效果。前者可能更实际，但效果可能不如前者。在研究的第三部分，将测量气管内大肠杆菌攻击和液体对心肌功能的影响。动物将随机分为生理盐水组或大肠杆菌组，无液体支持组或研究第二部分中被认为最具保护作用的液体方案组。然后在24或48小时随机选择动物进行超声心动图和PV测量。然后，他们将被抽血进行血液培养、化学、细胞因子和一氧化氮的测量，之后他们将被处死，用于心肌组织学和有丝分裂原活化蛋白激酶（MAPK）和核因子κ B （NF-KB）的研究。第2和第3阶段的所有动物将接受头孢曲松100 mg/kg /天，持续96小时。迄今为止，已经开发了在动物体内进行PV循环的技术，多巴酚丁胺和艾司洛尔的滴定研究正在进行中。此外，已经建立的流体方案在模型中具有保护作用。