Name: listeria monocytogenes infection of the brain
Uploaded: 2018-10-02T15:00:00
Description: Watch this Scientific Journal Video about Listeria monocytogenes Infection of the Brain at JoVE.com

这项工作得到美国国立卫生研究院 AI103806 的资助。

所有动物都要保持和处理, 最大限度地照顾, 以尽量减少不适在过程中。该程序将按照机构动物照料和使用委员会以及所有联邦、州和地方法律进行。还请注意, 实验室实验将按照生物安全等级2准则进行。
1. 增殖性李斯特菌在小鼠感染研究中的生长
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高压釜500毫升脑心输液 (BHI) 琼脂培养基在1升锥形瓶中制备固体培养基板。让媒体在56摄氏度的水浴冷却, 然?...

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李斯特菌能在人类身上造成威胁生命的脑炎。先前的研究表明细菌有能力穿过血脑屏障 (BBB), 并使大脑得以殖民化。在感染过程中提出了三种脑侵袭途径: 通过细菌直接穿透血屏障, 由单个核细胞内的细菌进行隐形转运3, 以及轴突迁移.rhombencephalitis15。因为大脑是高度血管化的, 而l.在系统感染期间, 已知的增生性李斯特菌在血液中循环, 测定<em...

革兰氏阳性细菌李斯特菌是一种兼生的胞内病原体, 是世界上最致命的食物传播病原体之一。摄取l-李斯特菌污染的食物会导致人类李斯特菌病, 一种严重的侵袭性疾病, 主要针对孕妇、新生儿、老年人和免疫缺陷患者1人。李斯特菌是美国食品传播病原体死亡的主要原因之一, 李斯特菌病的病死率高达 20–30%, 是所有食物传播病原体的最高2。目前尚无疫苗用于L. 李斯特菌和细菌有效地扩散到远端器官和大脑的能力通过血脑屏障 (BBB) 可能导致危及生命的脑膜炎和大脑的殖民化3,4,5,6. 细菌性脑膜炎通常是严重的;虽然大多数接受治疗的人康复, 感染可能会引起严重的并发症,例如, 脑损伤, 听力丧失, 或学习障碍儿童。据预测, 在美国7的社区获得性脑膜炎中,李斯特菌至少占10%。
细菌向大脑和脑膜传播的主要途径是通过血液。在大脑血管中循环的细菌能够穿过脑屏障引起脑部感染。脑屏障是一种高度血管化的屏障系统, 可以保护大脑不受血液中流动的异物的侵害。内皮细胞构成一层线的血管的内部表面8,9。除了L. 李斯特菌, 多种细菌病原体, 如淋球菌奈瑟,肺炎链球菌,大肠杆菌,流感嗜血杆菌b 型 (Hib) 能够殖民化通过脑屏障3,4,5,6的大脑。然而, 当检查被感染老鼠的大脑中的细菌负担时, 确定细菌是否越过了脑屏障是很重要的, 否则, 大脑中的细菌负担可能代表仍然在大脑血管中的细菌。因此, 在确定脑组织匀浆的成藏单元 (CFU) 之前, 必须灌注所有血液中的小鼠。
在本研究中, 我们描述了在体内方法检查的脑细胞增生性李斯特菌感染。在这里描述的方法, 我们使用了 L. 李斯特菌10403S。10.李斯特菌10403S 是最广泛使用的实验室菌株之一, 研究系统性李斯特菌病在小鼠感染模型中的应用。本协议以标准静脉注射的L. 型李斯特菌为基础, 然后灌注小鼠。图 1显示了小鼠感染协议的示意图。从非灌注或灌注小鼠中采集到的李斯特菌感染的大脑和其他器官, 并确定了细菌的器官负担。这些方法不仅可以确定受感染动物的脑部细菌的全殖化, 还有利于确定细菌是否穿透了体内的脑屏障, 以调停大脑的侵袭。必须强调的是, 这项实验室议定书应在与有关的机构生物安全委员会和动物设施管理部门协商后进行。

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		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Brain Heart Infusion media</td>
			<td style="width:119px;">Becton Dickinson</td>
			<td style="width:119px;">237200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin sulfate&#160;</td>
			<td>Amresco</td>
			<td>0382-50G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>VWR</td>
			<td>97062-832</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">IKA T18 ULTRA-TURRAX Basic Homogenizer</td>
			<td>IKA</td>
			<td>3352109</td>
			<td>Model: T18BS1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrophotometer</td>
			<td>Beckman Coulter</td>
			<td>DU 800 series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BALB/c mice</td>
			<td>Jackson Laboratory</td>
			<td>Model #000651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL syringes</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">26-gauge needles</td>
			<td>Becton Dickinson</td>
			<td>305115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21-gauge butterfly needles</td>
			<td>Becton Dickinson</td>
			<td>367281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>60004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical tubes</td>
			<td>VWR</td>
			<td>21008-918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round-bottom test tubes</td>
			<td>VWR</td>
			<td>60819-546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline&#160;</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel spatula</td>
			<td>VWR</td>
			<td>82027-520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel scissors (6.5 in)</td>
			<td>VWR</td>
			<td>82027-592</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel scissors (4.5 in)</td>
			<td>VWR</td>
			<td>82027-578</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel blunt forceps&#160; (4.5 in)</td>
			<td>VWR</td>
			<td>82027-440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel fine tip forceps&#160; (6 in)</td>
			<td>VWR</td>
			<td>82027-406</td>
			<td></td>
		</tr>
	</tbody>
</table>

listeria monocytogenes infection of the brain

李斯特菌是一种胞内细菌性致病菌, 常与食物传播的感染有关。L. 李斯特的能力, 通过血脑屏障 (BBB) 是有关的, 因为它可能导致危及生命的脑膜炎和脑炎。脑屏障保护大脑微环境的各种有毒代谢物和微生物病原体发现的血液后感染, 从而支持大脑稳态。在血流中存在的l. 型李斯特菌在脑屏障中的作用机制还没有得到充分的理解, 也缺乏一个健壮的模型系统来研究脑部感染的原因.在这里, 我们提出了一个简单的老鼠感染模型, 以确定是否细菌已经越过脑屏障和定量的负担, 细菌已在体内殖民的大脑。在这种方法中, 动物被静脉注射与L. 李斯特菌, 并被人道安乐死的接触到 CO2 , 其次是颈椎脱位。动物的心脏灌注是在收割被感染的器官之前进行的。血液在灌注前采集, 并通过琼脂板上的血液或器官组织匀浆的电镀稀释和计数形成的菌落数来确定每器官或血毫升的细菌数量。该方法可用于研究新的受体配体相互作用, 增强了李斯特菌的脑部感染, 并可方便地适应多种细菌病原体的研究。

大脑是高度血管化的, 而L. 李斯特菌是已知的感染细胞类型存在于血液中3,13。所描述的协议是用来证明L. 李斯特的能力, 通过血脑屏障 (BBB) 导致大脑感染的小鼠。为了确定细菌是否在体内越过了脑屏障, 在确定大脑中的细菌负担之前, 老鼠的血流灌注。否则, 获得的 CFU 可能包括在脑血管中存在的细菌。(图<stro...

Watch this Scientific Journal Video about Listeria monocytogenes Infection of the Brain at JoVE.com

Listeria monocytogenes Infection of the Brain

李斯特菌脑部感染

在感染过程中,李斯特菌可以通过血脑屏障来殖民大脑。在本协议中, 我们演示了如何评估小鼠感染后器官的细菌定植。为具体测定脑实质中的细菌数, 进行全器官灌注的程序。

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

该方法有助于回答细菌发病机制领域有关血液传播细菌能否跨越血脑屏障感染中枢神经系统和大脑的关键问题。通过这项技术，可以确定受感染动物的血液和脑组织内的细菌数量，同时排除大脑血管内的细菌数量。证明这个程序将是帕拉布·戈什，一个来自我实验室的副研究员。感染后72小时，将自动灌注系统设置为4毫升PBS的流量，并辅以每分钟10毫升EDTA。将安乐死动物放在解剖锅的超级位置。确认安乐死缺乏对爪子捏的反应。用75%乙醇淋湿腹部。使用剪刀将腹壁切口切到肋骨笼下方，以暴露隔膜和内脏器官。切开隔膜打开胸腔。双边切割摩擦笼以暴露左心室，并使用钳子轻轻抓住心脏。将连接到灌注系统的 21 粒蝴蝶针插入左心室，并小心地切割右心房，将约 200 微升血液收集到含有 10 毫摩尔 EDTA 的两毫升管中，以防止凝固。然后用 15 到 20 毫升的 PBS 加 EDTA 将动物淋长 4 分钟。完全灌注后，大脑和肝脏会出现发白，确保剩余的细菌来自收获的器官，而不是循环的血液。灌注后，小心地将感兴趣的器官转移到单个无菌的15毫升锥形管中，每管含有5毫升的无菌4摄氏度PBS。要收获斩首后的大脑，用剪刀将头皮皮肤切下眼睛之间的中线，保持尖端压在头骨上，以修剪任何多余的组织，必要时。轻轻地将剪刀尖插入前人大玉中，横向切成两侧的头骨。小心地将头骨切下头骨中线，注意施加横向压力，避免大脑扰动。用钳子打开头骨，露出大脑，在大脑底部和头骨底部之间放置一个铲子，小心地将大脑转移到一个15毫升的管子中，管内含有5毫升4摄氏度的PBS。在生物安全柜中，插入尖端，在单独的 15 毫升锥形管中连续运行 5% 漂白剂、无菌水、75% 乙醇和无菌水溶液，运行组织均质器 10 秒。当尖端被彻底消毒后，在其管中使受感染的大脑均质化。当没有可见的组织碎片时，重新消毒尖端，以防止细菌携带到下一个器官样本，并同化下一个器官。当所有器官均质化时，在无菌PBS中准备每个器官均质的10倍序列稀释，并盘灌脑心输液加糖板上的稀释。然后在37摄氏度下孵育同质稀释培养物，并计算每个牌盘上的菌落数量，以确定每个器官或毫升血液的细菌总数。对受L.单细胞基因感染的小鼠器官的细菌负担的计算表明，感染后灌注不会显著改变受感染小鼠血液或器官的细菌负担，而这一代表性研究对此进行了检查。按照这个程序，李斯特菌单细胞基因感染的器官可以进一步处理，以进行额外的分析，如组织病理学处理，以回答有关炎症细胞渗透到受感染的小鼠器官与未受感染的对照动物相比的其他问题。在尝试此程序时，重要的是要记住避免大脑的扰动，并保持剪刀和脑组织之间的最小接触。

Research

JoVE Journal

Immunology and Infection

Listeria monocytogenes Infection of the Brain

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

科研

免疫与感染

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

选择恶性疟原虫</emCytoadhesion>寄生虫对人脑血管内皮细胞

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

口头传播的<em&gt;单核细胞增生李斯特氏菌</em小鼠通过食入被污染的食物

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³ Responses

实验性感染<em&gt;李斯特菌</em&gt;作为研究主机干扰素γ响应模型

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

弥散性念珠菌感染模型

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...