JoVE Journal > Immunology and Infection
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Introduction
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Immunology and Infection

细菌性心内膜炎小鼠模型中感染参数的内注射与定量

Published: February 06, 2021
doi:
10.3791/61749
, , , , ,
1Department of Microbiology and Immunology,University of Oklahoma Health Sciences Center, 2Department of Ophthalmology,Dean McGee Eye Institute, 3Dean McGee Eye Institute, 4Department of Cell Biology and Department of Family and Preventive Medicine,University of Oklahoma Health Sciences Center

Summary

在这里，我们描述了一种细菌性心内膜炎小鼠模型中的内注射和随后的细菌定量方法。该协议可以扩展用于测量宿主免疫反应和细菌和宿主基因表达。

Abstract

眼内细菌感染对视力有危险。研究人员使用动物模型来研究宿主和细菌因素以及与感染相关的免疫反应途径，以确定可行的治疗靶点，并测试预防失明的药物。体内注射技术用于将生物体、药物或其他物质直接注射到眼后段的玻璃腔中。在这里，我们演示了这种注射技术，以启动小鼠眼感染和量化眼内细菌的技术。 脑杆菌在脑 心输液介质中生长18小时，并重新灌注至浓度为100个菌落形成单位（CFU）/0.5μL。使用氯胺酮和西拉辛的组合对一只C57BL/6J小鼠进行麻醉。使用皮奥利特微注射器和玻璃毛细管针，将0.5μL 的杆 菌悬浮液注射到小鼠眼的中层。反向对照眼要么注射了无菌介质（手术控制），要么没有注射（绝对控制）。感染后10小时，小鼠被安乐死，用无菌手术钳子采集眼睛，并放入含有400μL无菌PBS和1毫米无菌玻璃珠的管子中。对于ELISAs或性内洛西达酶测定，蛋白质酶抑制剂被添加到管子中。对于RNA提取，添加了适当的解解缓冲液。眼睛在组织均质器中均质1-2分钟。在PBS中，同质酸盐被连续稀释10倍，并跟踪稀释到加糖板上。其余同质物储存在-80°C，用于其他测定。板孵育24小时，每只眼睛的CFU被量化。这些技术导致小鼠眼睛的可重复感染，并促进活体细菌的定量，宿主免疫反应，宿主和细菌基因表达的奥米。

Introduction

细菌性内膜炎是一种导致炎症的破坏性感染，如果治疗不当，可能导致视力丧失或失明。心内膜炎的结果是细菌进入眼睛内部1，2，3，4，5。一旦进入眼睛，细菌复制，产生毒素和其他有害因素，并可能导致不可逆转的损害微妙的视网膜细胞和组织。眼部损伤也可以由炎症引起，由于炎症途径的激活导致炎症细胞流入眼内1，5，6。内膜炎可能发生在眼内手术（术后），对眼睛的穿透性损伤（创伤后），或从转移传播细菌到眼睛从不同的解剖位点（内源）7，8，9，10。细菌性内膜炎的治疗包括抗生素、抗炎药或外科介入3、4、11。即使有这些治疗，视力或眼睛本身也可能丢失。细菌性内膜炎的视觉预后一般因治疗效果、表现的视力和感染生物体的毒性而有所不同。

杆菌杆菌（B.cereus）是导致创伤后内膜炎7，12的主要细菌病原体之一。大多数乙型脑膜炎病例有一个快速的过程，这可能会导致失明在几天内。B. cereus 内皮炎的标志包括眼内炎症迅速演变、眼痛、视力迅速丧失和发烧。与其他通常引起眼部感染的细菌相比，脑细胞在眼睛中生长迅速，并且具有许多毒性因素。因此，成功治疗干预的窗口相对较短，即1、2、3、4、5、6、7、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25。 这种感染的治疗通常成功地治疗由其他毒性较低的病原体引起的内膜炎，但乙型脑膜炎通常导致超过70%的患者患有严重的视力丧失。这些病人中约有50%的被感染眼7、16、22、23等眼睛被感染或引化。乙基脑内皮炎的破坏性和快速性要求立即和适当的治疗。最近在辨别疾病发展的基本机制方面的进展已经确定了潜在的干预目标19、26、27。B. cereus 内皮炎的实验小鼠模型在辨别感染机制和测试可能防止视力丧失的潜在治疗方面仍然很有用。

小鼠在脑膜炎28期间的实验眼内感染一直是了解细菌和宿主因素及其相互作用的工具模型。此模型模拟创伤后或术后事件，其中细菌在受伤期间被引入眼睛。这个模型是高度可重复的，并一直有用的测试实验疗法和提供数据，改善护理标准1，6，19，29，30。与许多其他感染模型一样，此模型允许独立控制许多感染参数，并能够高效且可重复地检查感染结果。过去几十年中，对兔子的类似模型的研究已经研究了B.cereus病毒因素对眼睛2、4、13、14、31的影响。通过注射缺乏个体或多种毒性因子的乙型细菌突变菌株，这些毒性因子对疾病严重程度的贡献可以通过结果来衡量，如细菌在感染后不同时间浓度或视觉功能丧失13、14、27、31、32。此外，通过感染缺乏特定炎症宿主因子的敲敲小鼠菌株，该模型中已对宿主因子进行了研究，这些菌株缺乏特定的炎症宿主因子26、29、33、34、35。该模型也可用于测试潜在的治疗方法，通过注射新的化合物到眼睛感染后30，36。在这份手稿中，我们描述了一个详细的方案，其中包括用B.cereus感染老鼠眼，感染后采集眼睛，量化眼内细菌负荷，以及保存标本以检测疾病严重程度的其他参数。

Protocol

所有程序都是按照《实验室动物护理和使用指南》和《眼科和视觉研究动物使用视觉和眼科声明协会》中的建议进行的。协议得到俄克拉荷马大学健康科学中心机构动物护理和使用委员会的批准（协议号15-103、18-043和18-087）。 1. 无菌玻璃针 打开 指针 移液器拉拔器。 调整加热器 旋 钮，直到显示屏显示 12.6。 打开门，通过上部?…

Representative Results

生成可重复的宫内注射过程和准确性是开发微生物内膜炎模型的关键步骤。在这里，我们演示了使用革兰阳性杆菌 的静脉注射程序。我们向5只C57BL6小鼠的中层注射了100个CFU/0.5μL的 B.cereus。 感染后10小时，我们观察到 B.cereus的眼内生长到 大约1.8 x 105 CFU/眼睛。 图1 演示了玻璃针的构造，将细菌送入小鼠眼睛的中游。 图2显示了在 血脂板和?…

Discussion

即使有有效的抗生素、抗炎药和玻璃切除术，细菌性内膜炎也可以使患者失明。临床研究对治疗内膜炎是有益的;然而，眼膜炎的实验模型提供了快速和可重复的结果，可以转化为护理标准的进步，从而为患者提供更好的视觉结果。

小鼠眼的玻璃体积约为7μL40。这种小体积只允许注入有限的材料。不应注入大于 1.0 μL 的体积，以避免眼部损伤。该过程需要特?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢冯丽博士和马克·迪特玛（OUHSC P30活的动物成像核心，美国俄克拉荷马市麦基眼科研究所所长）的帮助。我们的研究得到了国家卫生研究院 R01EY028810、R01EY028066、R01EY025947 和 R01EY024140 的资助。我们的研究还得到了P30EY21725（NIH CORE实时动物成像和分析、分子生物学和细胞成像资助）的支持。我们的研究还得到了 NEI 视觉科学博士前实习生计划 5T32EY023202、长老会健康基金会研究支持补助金以及从研究到预防失明对院长 A. McGee 眼科研究所的无限制资助。

Materials

2-20 µL pipette RANIN L0696003G NA
37oC Incubator Fisher Scientific 11-690-625D NA
Bacto Brain Heart Infusion BD 90003-032 NA
Cell Microinjector MicroData Instrument, Inc. PM2000 NA
Fine tip forceps Thermo Fisher Scientific 12-000-122 NA
Glass beads 1.0 mm BioSpec 11079110 NA
Incubator Shaker New Brunswick Scientific NB-I2400 NA
Microcapillary Pipets 5 Microliters Kimble 71900-5 NA
Micro-Pipette Beveler Sutter Instrument Co. BV-10 NA
Microscope Axiostar Plus Zeiss NA
Microscope OPMI Lumera Zeiss NA
Mini-Beadbeater-16 BioSpec Model 607 NA
Multichannel pipette 30-300 µL Biohit 15626090 NA
Multichannel pipette 5-100 µL Biohit 9143724 NA
Needle/Pipette Puller Kopf 730 NA
PBS GIBCO 1897315 Molecular grade
Protease Inhibitor Cocktail Roche 4693159001 Molecular grade
Reverse action forceps Katena K5-8228 NA
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Tags

Intravitreal InjectionMouse ModelBacterial EndophthalmitisPathogenesisIntraocular InfectionQuantitationMicroorganismsHost Immune ResponsesGene ExpressionTreatment EffectivenessBiosafety Level TwoB. CereusMicroinjectorOphthalmic MicroscopeAnesthetized MouseReverse Action Forceps
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Mursalin, M. H., Livingston, E., Coburn, P. S., Miller, F. C., Astley, R., Callegan, M. C. Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis. J. Vis. Exp. (168), e61749, doi:10.3791/61749 (2021).
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