Name: injections of lipopolysaccharide into mice to mimic entrance of microbial-derived products after intestinal barrier breach
Uploaded: 2018-05-02T15:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach at JoVE.com

JHN 得到瑞士国家基金会 (SNSF 310030_146290) 的支持。

在巴塞尔大学生物医学系 (瑞士巴塞尔) 的动物设施中, 小鼠被饲养并保存在特定的无病原体 (SPF) 条件下。所有的老鼠实验都是按照瑞士联邦和州的规定进行的 (动物协议编号 2816 [巴塞尔莫斯丹塔])。
1. 脂多糖溶液的制备
<ol><li>打开从大肠杆菌中纯化的 LPS 0111: B4 在无菌条件下, 并在水中重新将其重组为5毫克/毫升的浓度. </li>
	<li>稀释含无菌磷酸盐缓冲盐水 (PBS) 的 LPS 库存, 使其工作浓度为0.2 µg/µL。</li>
</ol>2. 腹腔注射 LPS 对小鼠的作用
<ol><li>在层流气流中用30克针吸入0.5 毫升注射器中的 LPS 工作溶液。</li>
	<li>称雌性 C57Bl/6 小鼠 (六岁至10周), 并计算将注射的 LPS 量:10 µL (2 µg)/g 体重。 注: 例如, 如果鼠标重20克, 则20克 x 10 µL = 200 µL LPS 工作溶液需要注射。</li>
	<li>轻轻而坚定地处理鼠标, 一方面抑制动物。确保动物能够正常呼吸, 但不能在克制的时候扭动和转动。</li>
	<li>将鼠标的鼻子稍稍倾斜到地板上, 露出腹部注射。定位腹部中线和从中线左或右低位腹部注射部位。向控制小鼠注入 ip PBS 而不是 LPS。</li>
	<li>用自由的手, 注入适当的体积 (在步骤2.2 中计算的体积) 的 LPS 工作解决方案。确保针进入腹膜腔, 否则, 可能会出现腹腔肌肉层的错误注射。不过, 不要太深的针进入腹腔, 以避免伤害内器官位于该地区。</li>
	<li>把动物小心地送到笼子里, 每笼多达5只老鼠。</li>
</ol>3. 监控小鼠
<ol><li>在注射时对动物进行监测, 每2小时注射8小时, 以检测内毒素血症的发生和严重程度。因此, 使用已从以前发布的手稿24改编的评分表 (表 1)。</li>
	<li>
分数的 LPS 注射小鼠的以下参数表1所列
	<ol>
<li>检查通常光滑的毛皮的外观。如果毛皮出现褶皱, 尖刺或肿胀的皮毛, 表明不适和/或疼痛, 评分为 1, 2, 或3。</li>
		<li>检查鼠标的活动。 注意: 老鼠通常在整个笼子周围自由走动进食, 饮酒, 攀爬, 但抑制或限制活动可能是痛苦的征兆。</li>
		<li>检查意识水平。 注意: 老鼠很好奇, 他们通常在笼子里四处走动调查周围。缺乏或减少运动会提示疼痛和不适。</li>
		<li>检查眼睛 注: 完全睁开眼睛有望在健康的小鼠, 但部分或完全闭合的眼睛, 可能与分泌物, 可以表明疼痛。</li>
		<li>检查呼吸速率。 注意: 健康的老鼠通常有正常和快速的呼吸速率, 降低呼吸率可能是不适的征兆)。</li>
		<li>检查呼吸质量。 注意: 呼吸困难或不喘气是痛苦的征兆。</li>
	</ol>
</li>
</ol>4. 核酸 (RNA) 提取和均匀化的组织取样
<ol><li>准备收集/均匀化管: 添加1毫升单步 RNA 隔离试剂到2毫升管预先填入1.4 毫米陶瓷球。</li>
	<li>在适当的时间点 (前 (0 小时) 和2小时, 4 小时和8小时后, LPS 注射液) 弄死小鼠与麻醉过量 (异氟醚) 后放血和进行解剖。</li>
	<li>用剪刀切开皮肤和肌肉层, 用内脏把腹腔露出来。</li>
	<li>仔细解剖脾脏、肝脏和结肠, 将器官从脂肪中清除, 并将它们放在 PBS 上放在冰上。</li>
	<li>用剪刀或手术刀从每个器官剪下一小块 (大约0.5 厘米长)。 注意: 在每只老鼠 (肝脏的同一裂片、近端或远端结肠) 中, 总是从器官的同一部位取一块, 这一点很重要。</li>
	<li>将纸巾放在纸上, 然后将其放在收集/均匀化管中, 确保它完全浸入 RNA 隔离试剂中。</li>
	<li>融汇高速台式均质体中的组织。对于像脾脏、肝脏和结肠这样的软组织, 在三十年代的速度6.00 中使用1的均匀化循环。</li>
	<li>在室温下孵化样品5分钟。</li>
	<li>小心地将管子放在液氮中, 将组织的裂解液凝固。将快照冷冻裂解液贮存在-80 摄氏度, 直到 RNA 分离。</li>
</ol>5. 从组织中提取 RNA
注意: RNA 分离试剂、氯仿和醇被认为是危险物质。在化学罩下进行 RNA 提取。使用经认证的无 RNase 试剂和设备, 以保持 RNase 的环境。
<ol><li>
相分离
	<ol>
<li>在冰上解冻冰冻组织裂解液。</li>
		<li>离心样品在 1000 x g 5 分钟, 在4°c, 以颗粒的剩余的组织微粒, 可能会留下。</li>
		<li>将上清液转移到新的无核酸酶1.5 毫升管。</li>
		<li>每1毫升 RNA 分离试剂中加入200µL 的冰冷氯仿, 用手用力摇动10-15 秒。</li>
		<li>室温下孵育2-3 分钟。</li>
		<li>离心机在 12, 000 x g 15 分钟在4°c。 注: 样品现在包含3个阶段: 低红苯酚-氯仿相, 界面和上无色水相。RNA 在上部水相存在。</li>
		<li>收集上水相 (总容积的 50%), 并转移到一个新的无核酸酶1.5 毫升管。</li>
	</ol>
</li>
	<li>
RNA 降雨雪
	<ol>
<li>每1毫升 RNA 分离试剂加入0.5 毫升冰冷异丙醇, 并通过反转管5-6 次轻轻混合。</li>
		<li>室温下孵育10-15 分钟。</li>
		<li>离心机在 1.2万 x g 10 分钟在4摄氏度。 注意: RNA 颗粒在这个阶段应该是可见的。</li>
	</ol>
</li>
	<li>
RNA 颗粒的洗涤
	<ol>
<li>完全去除含有异丙醇的上清液, 不干扰颗粒。</li>
		<li>用1毫升冰冷75% 乙醇清洗颗粒, 每1毫升 RNA 分离试剂用于初始裂解。</li>
		<li>简单地涡流样品。</li>
		<li>离心样品在 7500 (或 1.2万) x g 5 分钟在4°c。</li>
	</ol>
</li>
	<li>
溶解 RNA
	<ol>
<li>完全去除上清液中的乙醇而不干扰颗粒。</li>
		<li>将开管置于化学罩下3-5 分钟, 使 rna 颗粒在室温下风干 (不要过度干燥, 因为在这种情况下很难溶解 rna)。</li>
		<li>在20-50 µL 核酸酶水中溶解 RNA 颗粒, 吹打小心上下。</li>
		<li>在55摄氏度孵化样品10-15 分钟, 进一步改善 RNA 的溶液。 注意: 在这个阶段, RNA 可以储存在-80 摄氏度, 直到进一步分析。</li>
	</ol>
</li>
</ol>6. 剩余 DNA 的消化
<ol><li>通过将整除 (2 µl) 吹打到分光光度计中, 测量 RNA 浓度, 并调整到推荐浓度。</li>
	<li>开始消化与最大的污染 DNA。10µg RNA 在50µL 核酸酶无水。</li>
	<li>添加0.1 卷 10x DNase i 缓冲器和1µL rDNase 我每样, 并轻轻地混合吹打上下。</li>
	<li>孵育37°c 20-30 分钟。</li>
	<li>涡流 DNase 灭活剂, 并添加0.1 体积的样品与吸管混合良好。</li>
	<li>有时用手在室温下混合孵育5分钟。</li>
	<li>离心机在 1万 x g 1.5 分钟。</li>
	<li>将含有无 DNA RNA 的上清液转移到新的无核酸酶管中。</li>
	<li>用分光光度法测定 RNA 浓度和质量。</li>
</ol>7. 反向转录
<ol><li>使用商业上可用的脱氧核糖核酸 cDNA 反向转录 (RT) 试剂盒进行反向转录。 注: 在这里, 多达2µg 的 RNA 可以逆转转录。</li>
	<li>计算体积, 其中包含2µg RNA。吸管2µg RNA 在一个新的 PCR 管。</li>
	<li>将核酸酶的水添加到 PCR 管中的样品中, 最终体积为10µL。</li>
	<li>通过混合表 2中列出的下列组件, 准备2x 主混合
</li>
	<li>添加10µL 2x 主混合到10µL RNA 获得1x 主混合在总容量20µL。</li>
	<li>关闭聚合酶链反应 (PCR) 管, 并简单地向下旋转样品。</li>
	<li>将示例放在 PCR Thermocycler 中, 并设置表 3中列出的设置。</li>
	<li>将生成的 cDNA 存储在-20 摄氏度以进行进一步分析。</li>
</ol>8. 定量 PCR (qPCR)
<ol><li>使用商业上可用的工具包执行 qPCR 反应。</li>
	<li>自设计和测试内含子生成底漆, 使用前, 具有不同浓度的模板 cDNA。通过创建标准曲线来分析底漆的功效, 并使用高效、正确的熔融曲线的引物。 注意: 本实验中使用的引物序列列在表 2中。</li>
	<li>用表 4中描述的反应设置, 准备384井板中的定量聚合酶链反应 (qPCR) 反应。</li>
	<li>用铝箔把所有的溶液和盘子放在冰上, 以防在反应混合物准备好的时候使盘子变湿。</li>
	<li>执行 triplicates 中的所有反应。</li>
	<li>使用表 5中列出的设置在 thermocycler 上执行 PCR 反应。</li>
	<li>计算 triplicates 所有反应的平均 Ct 值。通过计算 2 ^ (-ΔCt) 将所有目标基因正常化为 glycerinealdehyde-3-phosphate-dehydrogenase (Gapdh)家政基因的表达水平。 注意: 所有的目标基因, 平均 Ct > 35 被认为是在检测限额。</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneously+colitic+C3H/HeJBir+mice+demonstrate+selective+antibody+reactivity+to+antigens+of+the+enteric+bacterial+flora.">Spontaneously colitic C3H/HeJBir mice demonstrate selective antibody reactivity to antigens of the enteric bacterial flora.</a> J Immunol. 159, 44-52 (1997).</li><li>Macpherson, A. J., McCoy, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardised+animal+models+of+host+microbial+mutualism.">Standardised animal models of host microbial mutualism.</a> Mucosal Immunol. 8, 476-486 (2015).</li><li>Masopust, D., Sivula, C. P., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+Mice,+Dirty+Mice,+and+Men:+Using+Mice+To+Understand+Human+Immunology.">Of Mice, Dirty Mice, and Men: Using Mice To Understand Human Immunology.</a> J Immunol. 199, 383-388 (2017).</li><li>Wirtz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+lethal+septic+peritonitis+by+neutralizing+the+biological+function+of+interleukin+27.">Protection from lethal septic peritonitis by neutralizing the biological function of interleukin 27.</a> J Exp Med. 203, 1875-1881 (2006).</li></ol>

作者没有什么可透露的。

该协议模仿微生物衍生产品入侵后发生的免疫学过程。该协议中的关键步骤是选择鼠标线、小鼠的卫生状况、脂多糖的剂量、监测动物对内毒素血症的发生情况以及实验终止的时间点。最重要的是, 必须考虑到小鼠菌株的遗传背景。不同的小鼠菌株对 LPS 有不同的敏感性。例如, C3H/HeJ 和 C57BL/10ScCr 小鼠对 LPS 诱导的内毒素血症有抵抗力21,25。此外, 动物的?...

An erratum was issued for: <a href="https://www.jove.com/video/57610/injections-lipopolysaccharide-into-mice-to-mimic-entrance-microbial">Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach</a>
One of the authors&#39; names was corrected from:
Katarina Raduolovic
to:
Katarina Radulovic

An erratum was issued for: <a href="https://www.jove.com/video/57610/injections-lipopolysaccharide-into-mice-to-mimic-entrance-microbial">Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach</a>.&#160;An author name was updated.

Erratum: Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

人类肠道被殖民化, 形成了微生物群, 在进化过程中与宿主建立了互惠互利的关系。在这种关系中, 宿主为微生物群提供了一个安全的利基, 而微生物群则提供维生素, 养分消化和保护病原体到宿主, 其中微生物群驻留在1。当宿主和微生物群之间的这种有益关系受到干扰时, 疾病就会发生, 如炎症性肠病 (IBD)。IBD 是由遗传和环境因素引起的肠道慢性炎症性疾病, 发生于两种主要形式, 克罗恩病 (CD) 和溃疡性结肠炎 (UC)。尽管两种 IBD 形式有相似之处, 但它们的特点是炎症修饰的位置和性质存在一定的差异。CD 是一种复发性壁炎症紊乱, 可能扩展到胃肠道的任何部分, 而 UC 是非壁的, 并限于结肠。此外, 核苷酸结合齐聚域含蛋白 2 (NOD2) 的突变, 一种模式识别受体 (PRR), 识别 muramyl 二肽 (MDP), 一个组成部分的细胞壁的大多数革兰氏阳性和阴性细菌, 是与 CD2关联。此外, 大肠杆菌(大肠杆菌)、李斯特菌和链球菌及其产品都在 CD 患者的巨噬细胞中发现, 在屏障破裂3后进入宿主。当细菌或其产品在 CD 的研制过程中进入宿主时, 免疫系统就会产生一种反应, 从而导致生产循环抗菌抗体4。也许, 最有说服力的证据表明, 微生物在 IBD 发病机制中的作用源于老鼠模型。当动物被抗生素治疗, 或当老鼠被保存在无菌 (GF) 条件下, 疾病的严重性在大多数结肠炎模型中减少, 如在 GF 设施中不开发结肠炎的 IL-10-/小鼠5,6。此外, 结肠炎也扰乱了微生物群的组成, 其特点是不平衡的组成和减少的丰富称为 dysbiosis7。IBD 的结果可能是增加肠道通透性, 从而导致微生物和微生物源产品进入宿主。
在动物中, 葡聚糖硫酸钠 (DSS) 的应用诱导肠道上皮破裂, 导致上皮屏障的通透性增强8。门内毒素的浓度在动物中升高, 与 DSS 结肠炎9。有趣的是, 缺乏 C 型凝集素受体特异细胞内黏附分子 molecule-3 抓取 nonintegrin 同源相关 1 (SIGN-R1) 的动物保护不受 DSS 结肠炎和 LPS 诱导的内毒素血症10。为了进一步传播到宿主, 细菌或细菌衍生产品必须通过血管屏障11, 腹膜腔, 在其中的小和大肠, 肠系膜淋巴结和/或肝脏12。为了降低系统的复杂性, 采用了一种定义的细菌衍生化合物。lps, 导致内毒素血症后腹腔 (ip) 或静脉注射 (静脉注射) 注射13注射到小鼠, 研究 interleukins Il6和Ilb和细胞因子Tnfa响应 LPS 的表达。
lps 是一种病原体相关分子模式 (玉兰), 表达为革兰阴性菌的细胞壁成分, 由脂 a (lps 结构中的主要玉兰)、核心寡糖和 O 侧链14组成。由树突状细胞、巨噬素和上皮细胞表达的4类像样受体 (TLR4) 识别 LPS15, 这需要联合受体进行适当的结合。急性期蛋白脂多糖结合蛋白 (LBP) 束缚脂多糖形成一个复杂的转移脂多糖到分化 14 (CD14), 一个 glycosylphosphatidylinositol 定位膜蛋白。CD14 进一步航天飞机 LPS 到淋巴细胞抗原96或也称为 MD-2, 这是与细胞外领域的 TLR4。LPS 与 MD-2 的结合促进了 TLR4/MD-2 的二聚化诱导构象变化, 以吸收细胞内的适配器分子激活下游信号通路14, 其中包括髓系分化初级反应基因 88 (MyD88) 依赖通路和包含适配器诱导干扰素β (TRIF) 依赖通路16。TLR4 对 LPS 的识别然后激活 NF-nf-κb 通路, 诱导炎症细胞因子的表达, 如 TNFα、IL-6 和 IL-1β17。
特别是当 lps 被注射到动物体内时, 应考虑到动物的脂多糖浓度、动物的遗传背景和饮食。高浓度的脂多糖导致感染性休克, 其特点是低血压和多脏器衰竭, 最后到死亡18。与人类相比, 小鼠对 lps 的敏感性较小, 其中脂多糖浓度介于 2-4 ng/千克体重之间 (生物武器) 能够诱发细胞因子风暴19。对于小鼠, 致命剂量 (LD50), 导致死亡的一半的老鼠范围从10-25 毫克/千克体重20取决于使用的鼠标应变。对于常用的小鼠菌株, C57Bl/6 和 BALB/c, 致死剂量 50% (LD50) 为10毫克/千克。相比之下, C3H/HeJ 和 C57BL/10ScCr 的菌株受到 LPS 诱导的内毒素血症的保护, 这是由于Tlr421中的突变造成的。因此, Tlr4-deficient 小鼠 hyporesponsive 注射 LPS22。其他基因修饰的鼠标线, 如 PARP1-/鼠23对 LPS 诱导的毒性休克有抵抗力。
这里描述的老鼠模型使用致死剂量的 lps 进行系统的管理, 以模拟在屏障破坏身体表面后, lps 传播的后果。所选择的 LPS 浓度 (2 毫克/千克) 没有诱发 C56Bl/6 小鼠的死亡率, 而是诱导释放的促炎性细胞因子。

<table><tbody><tr><td>DreamTaq Green PCR Master Mix (2x)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>K1081</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit,</td><td>Applied Biosystems, Foster City, CA, USA</td><td>4368813</td><td></td></tr><tr><td>RNase-Free DNase Set,</td><td>Qiagen, Hilden, Germany</td><td>79254</td><td></td></tr><tr><td>LPS Escherichia coli O111:B4</td><td>Invivogen, San Diego, CA, USA.</td><td>tlrl-eblps</td><td></td></tr><tr><td>Omnican 50 Single-use insulin syringe</td><td>B. Braun Melsungen, Melsungen, Germany</td><td>9151125</td><td></td></tr><tr><td>Bioanalyzer 2100</td><td>Agilent Technologie, Santa Clara, USA</td><td>not applicable</td><td></td></tr><tr><td>Centrifuge 5430</td><td>Eppendorf, Hamburg, Germany</td><td>not applicable</td><td></td></tr><tr><td>Centrifuge Mikro 220R</td><td>Hettich, Kirchlengern, Germany</td><td>not applicable</td><td></td></tr><tr><td>Dissection tools</td><td>Aesculap, Tuttlingen, Germany</td><td>not applicable</td><td></td></tr><tr><td>Fast-Prep-24 5G Sample Preparation System</td><td>M.P. Biomedicals, Santa Ana, CA, USA</td><td>not applicable</td><td></td></tr><tr><td>NanoDrop ND-1000</td><td>NanoDrop Products, Wilmington, DE, USA</td><td>not applicable</td><td></td></tr><tr><td>TRI Reagent</td><td>Zymo Research, Irvine, CA, USA</td><td>R2050-1</td><td></td></tr></tbody></table>

injections of lipopolysaccharide into mice to mimic entrance of microbial-derived products after intestinal barrier breach

肠道上皮屏障将宿主与通常耐受或忽略的微生物群分离开来。这种屏障的破坏导致细菌或细菌来源的产品进入宿主, 进入宿主循环和内脏, 导致炎症性肠病 (IBD) 患者观察到无控制发炎, 即的特点是肠道上皮通透性增加。
为了模拟细菌源性化合物进入宿主体内的入口, 采用了内毒素血症模型, 并将脂多糖 (LPS) 作为革兰氏阴性菌外细胞壁的一部分注入小鼠。在本研究中, 致死剂量的 LPS 被腹腔注射, 并随后监测的小鼠8小时使用疾病评分。此外, 在不同时间点的 LPS 注射后, 在脾、肝、结肠中分别分析了炎症细胞因子Il6、 Il1b、和Tnfa的表达水平。该模型可用于研究在侵入微生物或细菌衍生产品后对机体表面的屏障破坏引起的免疫应答。

为了模拟在肠道屏障破坏后发生的细菌或细菌衍生产品进入后宿主的后果, LPS 在致死剂量 (2 µg/克体重) 中注入 C57Bl/6 小鼠体内。每只老鼠都被监测并得分, 其中包括: 小鼠的出现, 动物的活动, 眼睛的状况, 和呼吸速率和质量 (表 1), 其中含有内毒素血症的发生和评分表中列出的参数。.这些动物显示了疾病的临床症状, 在注射 LPS 后6至10小时达到峰值, 在24小时内?...

Watch this Scientific Journal Video about Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach at JoVE.com

向小鼠注射脂多糖以模拟肠道屏障破坏后微生物衍生产物的进入免疫学和感染|视频

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

本文提出了一种模拟肠道屏障破坏后细菌衍生化合物入口的协议。将低致死剂量的脂多糖系统地注入小鼠体内, 对24小时注射后进行监测。在脾、肝、结肠几个时间点上测定了促炎性细胞因子的表达。

注射脂多糖对小鼠肠道屏障破坏后微生物源产物的模拟进入

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

injections-lipopolysaccharide-into-mice-to-mimic-entrance-microbial

Research

Education

JoVE Journal

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Biology

Immunology and Infection

Human Biology

Immune System

SCIENTISTS IN ACTION

19.4K 次观看.  University of Basel. 本文提出了一种模拟肠道屏障破坏后细菌衍生化合物入口的协议。将低致死剂量的脂多糖系统地注入小鼠体内, 对24小时注射后进行监测。在脾、肝、结肠几个时间点上测定了促炎性细胞因子的表达。

视频: Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&kappa;B Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, as visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse1.
Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration2,3. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-&kappa;B luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-&kappa;B signaling within the LPS-injected gland4.
These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.

Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse.

导管内可视化通过NF-κB记者的转基因小鼠模型的乳腺炎：信号注射LPS

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as ...

科研

医学

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

淋巴免疫治疗和疫苗接种小鼠

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

免疫与感染

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

舌下免疫治疗作为一种替代起到保护作用对急性呼吸道感染

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

通过腹腔注射药物诱导小鼠胰腺损伤

Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological changes associated with SAP using animal models can aid in identifying potential therapeutic targets and exploring novel treatment approaches. Previous studies primarily induced pancreatic injury through retrograde bile duct injection of sodium taviaurocholate, but the impact of surgical damage on the quality of the animal model remains unclear. In this study, we employed various frequencies of intraperitoneal Caerulein injections combined with different doses of LPS to induce pancreatic injury in C57BL/6J mice and compared the extent of injury across five intraperitoneal injection protocols. Regarding inducing acute pancreatitis in mice, an intraperitoneal injection protocol is proposed that results in a mortality rate as high as 80% within 5 days. Specifically, mice received ten daily intraperitoneal injections of Caerulein (50 &#181;g/kg), followed by an injection of LPS (15 mg/kg) one hour after the last Caerulein administration. By adjusting the frequency and dosage of injected medications, one can manipulate the severity of pancreatic injury effectively. This model exhibits strong controllability and has a short replication cycle, making it feasible for completion by a single researcher without requiring expensive equipment. It conveniently and accurately simulates key disease characteristics observed in human SAP while demonstrating a high degree of reproducibility.

作者聚焦:确定开发小鼠严重急性胰腺炎新疗法的治疗靶点

Intraperitoneal drug administration is a safe and effective non-invasive approach for inducing pancreatic injury. This study compared five distinct intraperitoneal injection protocols on mice to induce varying degrees of pancreatic injury and established a model of severe pancreatic injury to investigate the pathological changes and treatment strategies for severe acute pancreatitis (SAP).

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological ...

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables&#160;the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.&#160;P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal&#160;P. gingivalis infection.

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular ...

生物学

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of acute lung injury or acute inflammation. The current most commonly used methods in acute lung injury mouse models are an intraperitoneal injection of LPS and tracheostomy for the tracheal infusion of LPS. However, the former method lacks lung targeting and damages other organs, and the latter method induces operative trauma, infection risk, and a low survival rate. Here, we recommend a noninvasive oropharyngeal endotracheal intubation method for LPS instillation in mice. In this method, LPS is noninvasively introduced into the trachea through the oropharyngeal cavity to be instilled into the lung with the help of an apparatus for endotracheal intubation. This method not only assures lung targeting but also avoids damage and the risk of death in the animals. We expect that this approach will become widely used in the field of acute lung injury.

Here, we propose a protocol for intratracheal lipopolysaccharide (LPS) delivery via noninvasive oropharyngeal endotracheal intubation. This method minimizes the trauma of the surgical procedure for the animal and accurately delivers LPS to the trachea and then to the lungs.

小鼠无创气管内脂多糖滴注

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of ...

注射脂多糖对小鼠肠道屏障破坏后微生物源产物的模拟进入

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通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

小鼠无创气管内脂多糖滴注