可视化<em>肺炎链球菌</em>在心脏Microlesions和随后的心脏重构
Summary
Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.
Abstract
期间菌血症肺炎链球菌可在血管内皮转运进入心肌和形成离散菌填充微观病变（microlesions）是显着的，由于缺乏浸润免疫细胞。由于其释放心脏毒性的产品，S.肺炎 microlesions内被认为有助于向心脏衰竭是在成人中雷酸侵袭性肺炎球菌疾病中经常观察到。在此证明一个协议，用于实验的小鼠感染，导致30小时内可重复的心脏microlesion形成。设在microlesion鉴定苏木精 – 伊红染色心脏切片和早期和晚期microlesions之间的形态区别的指令被突出显示。设置在一个协议，用于验证S的指令肺炎使用抗肺炎球菌荚膜多糖和microlesions内免疫荧光显微镜。最后，提供了一种协议，用于抗生素干预拯救感染小鼠和用于检测和瘢痕形成在恢复期小鼠的心脏评估。一起，这些协议将促进肺炎球菌心脏侵袭，心肌细胞死亡，心脏重塑底层的暴露至S.的结果的分子机制的研究肺炎 ，以及在心脏的肺炎球菌的免疫应答。
Introduction
成人社区获得性肺炎（CAP）住院进行了心脏不良事件，有助于死亡率1-4文件化的风险。在最近的一项研究科拉莱斯-Medina 等人 ，发现心脏并发症要关联有和/或负责肺炎相关死亡3的27％。 肺炎链球菌 （肺炎球菌），CAP和败血症5的最常见原因，已直接与多达19％的人承认的成人患者6心脏不良事件相关。与肺炎相关的不良心脏事件包括为了降低频率6的新的或恶化充血性心脏衰竭，心律失常和心肌梗死。
在最近的一个公共科学图书馆病原布朗等人的文章。，肺炎链球菌 ，发现通过心脏血管内皮，进入myocardiu是能够易位米，并形成独立非化脓性病变显微镜（microlesions）侵袭性肺炎球菌疾病（IPD）7时充满细菌侧脑室。观察心脏样本中心脏microlesion形成的证据来自非人类灵长类动物和个人谁曾死于肺炎球菌感染。同样，实验感染小鼠重复开发的心脏microlesions。在小鼠中，microlesion大小和数量呈正的持续时间和菌血症，心肌肌钙蛋白血清水平的严重程度，以及异常心脏电相关。细菌易位入心脏，发现发生通过负责跨越血-脑屏障和肺炎球菌性脑膜炎的发展， 也就是 ，胆碱结合蛋白A介导的血管内皮细胞的浸润在层粘连蛋白受体和血小板的肺炎球菌的易位相同的机制-activating因子受体依赖性8。 Microles离子的形成还需要肺炎球菌成孔毒素肺炎球菌被发现杀死心肌7。
肺炎球菌心脏microlesions是从由其他革兰氏阳性菌，包括金黄色葡萄球菌所引起的化脓性软组织和心脏脓肿鲜明。这些特点是由细菌中性粒细胞和纤维蛋白沉积9,10包围的单一病灶。 Microlesions由S.形成肺炎在尺寸，在整个受影响的心脏分布更小，且缺乏免疫细胞浸润。在感染的早期阶段，microlesions引起由S.肺炎表现为受损或发炎的组织让人想起心肌病的病理征象领域。一些单核细胞可能在此期间观察到，但是他们的存在是短暂的，病变迅速成为坏死在外观，充满了细菌，同时继续GR流的大小，直到动物或抗微生物干预的死亡。重要的是，内抗生素干预后3天，大量的免疫细胞浸润被观察到前者病变部位，这是伴随着强劲的胶原沉积。类似心脏重塑已报道发生对心脏功能11-15持久后果沿着以下梗死。因此，microlesions是心脏相关死亡率的恢复期个体的发病率增加谁生存了疾病发作期间的IPD和可能发生的不良心脏事件的潜在的解释。
这里，指令被设置在IPD与心肌损伤形成和心肌microlesions可视化的实验小鼠模型中，在感染的早期和晚期阶段。已保存的抗菌干预动物的协议检测胶原沉积的证明。本文的目的是至facilitate在这一重要和新颖的肺炎病理其他研究者的研究。
Protocol
Representative Results
Discussion
在这份报告中，高度重复的方法诱导S.肺炎介导的小鼠心脏microlesions IPD期间和技术的可视化展示。小鼠感染剂量的细菌，优于通关免疫系统的门槛，导致高档菌血症，类似这人类败血症期间发生，并导致最终的细菌移位到心肌。对于未知的原因，S.肺炎的心脏是能够通过宿主的免疫反应来复制偏向虎山行。这是正在进行的调查的主题，本出版物可以促进其他研究者在他们的努力?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by grant 13IRG14560023 from the American Heart Association and NIH grants AI078972 and HL108054 to CJO. Support for AOB was from the NIH National Center for Advancing Translational Sciences NIH ULTR001120, and F31 A110417701.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Todd-Hewitt broth | Neogen | 7161A | |
| O.C.T Compound | Tissue-Tek (Sakura Finetek) | 4583 | |
| Triton X-100 | Fisher Scientific (Acros) | 9002-93-1 | |
| Normal Goat Serum | Abcam | ab7481 | |
| anti-serotype 4 pneumococcus antiserum | Statens Serum Institut | 16747 | |
| goat anti-rabbit FITC conjugated antibody | Jackson ImmunoResearch | 111-096-144 | |
| DAPI | Invitrogen | D1306 | |
| Fluorsave | Millipore | 345789 | |
| Permount | Fisher Scientific | S70104 | |
| Ampicillin | Sigma | A9393 | |
| phosphomolybdic acid 0.2% | Electron Microscopy Sciences | 26357-01 | |
| 0.1% Sirius Red in picric acid | Electron Microscopy Sciences | 26357-02 | |
| 0.01 N hydrochloric acid | Electron Microscopy Sciences | 26357-03 | |
| Tissue Tack Microscope Slides | Polysciences, Inc | 24216 | |
| Paralube Vet Ointment | Dechra | 12920060 | |
| Hematoxylin and Eosin Staining Kit | unspeciified | Standard |
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