我们感谢马修博士在本议定书和项目的发展过程中对树突免疫的手稿和实验室的批判性阅读。这项工作得到了欧洲联盟第七框架方案居里夫人行动 (PCIG11-GA-2012-3221170 和免疫肿瘤学 LabEx (马伊) 的资助。

根据éthique en expérimentation animale 巴黎中心和南方 (动物实验伦理学委员会) 批准的2012-0024 号议定书, 在应用欧洲指令2010/63 时进行了老鼠实验。欧盟.
1. 导管的制备
<ol><li>为每组被感染的小鼠准备一个儿科静脉插管。使用内置弹簧机构, 剥离其针的每个套管, 按照制造商的指示。丢弃针头, 只保留塑料静脉导管。</li>
	<li>在层流罩内消毒导管, 用于一个紫外线 (紫外线) 循环, 通常为 25-30 分钟。 注: 导管可用于多只鼠标, 但应在实验组、性别或细菌菌株之间使用新的导管。</li>
</ol>2. 为感染而制备肾炎的大肠杆菌
<ol><li>至少在感染前两天, 使用无菌接种回路, 条纹 UPEC 从一个冷冻细菌库存到 Luria 肉汤 (LB) 琼脂板, 含有抗生素, 如果适当的。在晚上37° c 孵育。板材可储存在4° c, 最多一周。</li>
	<li>在一个100毫升无菌锥形烧瓶, 接种10毫升磅, 其中含有抗生素, 如果合适的, 一个单一的殖民地从 LB 板和孵化站在37° c 过夜 (约 16-18 小时)。 注意: 站立区域性允许类型1毛的表达式, 这是感染23所必需的。文化的颤抖将有较低的效率毛表达, 因此, 不一致的感染率。</li>
	<li>测量过夜培养的光学密度 (OD)600 , 并使用预先确定的生长曲线计算每毫升细菌的数量。举例来说, 对于 UPEC 应变 UTI89, od600 = 0.35 等于每毫升 2 x 108 CFU, 因此, 具有 od600 = 2.4 的隔夜区域性相当于 13.7 x 108 CFU 每毫升. 根据经验确定OD600和可存活的细菌与每一个新的菌株使用的感染。</li>
	<li>
通过考虑被感染动物的数量来确定所需的细菌数量, 并在50μ l PBS 中每只老鼠接收大约 107菌落形成单位 (CFU)。在计算中包括130µl 死亡体积的导管和注射器的核心和100µl 需要确定接种。
	<ol>
<li>旋转的细菌悬浮在一个桌面离心在 1.7万 x g 为1分钟和重的结果细菌颗粒在 2 x 108 CFU 每毫升在 PBS。连续地稀释这个悬浮和板材的分在 LB 琼脂, 与抗生素, 如果适当, 确定确切的接种为每传染。</li>
	</ol>
</li>
	<li>将细菌接种成1毫升的注射器, 并将导管连接到注射器的末端。在开始灌输前, 轻敲注射器以除去任何空气, 并压低柱塞以填满导管内的死亡空气空间。</li>
</ol>3. 小鼠的制备
<ol><li>麻醉小鼠通过腹腔注射100毫克/千克氯胺酮和5毫克/千克嗪。可提供补充热量。</li>
	<li>确保每只老鼠都通过挤压脚中压来充分镇静。小鼠完全镇静, 当他们没有反应, 并要求 3-5 分钟达到这个状态。</li>
	<li>将小鼠仰卧, 应用中压到下腹, 排空尿液中的膀胱。全膀胱感觉像一个豌豆下的皮肤之间的回肠波峰。 注: 吸入异氟醚已被用于 catheterize 雌性小鼠15,22, 但是, 该方法尚未对雄性小鼠是否已被吸入异氟醚充分麻醉进行了测试。</li>
</ol>4. 经尿道灌注的雌性小鼠
<ol><li>使用非显性的手, 将拇指放在鼠标的腹部和手指上, 并在相反的方向施加柔和的压力, 以使鼠标牢牢地固定到位。</li>
	<li>
将导管的尖端垂直于老鼠的尿道口。用轻柔的压力, 将导管滑入尿道, 直到轮毂碰到尿道口, 同时降低注射器, 使其与工作表面平行。导管不需要润滑。不要将导管推入尿道。导管应平滑滑入尿道, 几乎无阻力。
	<ol>
<li>在灌输接种 (步骤 4.3) 之前, 一旦导管到位, 就用手指从已经在鼠标腹部的非显性手轻轻地将腹部皮肤拉向鼠标的头部 (步骤 4.1)。如果导管在尿道内, 构成尿道口的组织不会移动, 而如果导管不正确放置在阴道内, 则组织会向上和远离导管。这种快速测试应该在每只老鼠身上进行。</li>
	</ol>
</li>
	<li>当导管的中心与尿道口相遇时, 慢慢免除50µl 的细菌接种。缓慢的灌注率将膀胱的回流降到肾脏。慢慢地取出导管以防止渗漏, 超过数5。把老鼠放在笼子里放在仰卧位。</li>
</ol>5. 经尿道灌注的雄性小鼠
<ol><li>放置两个拇指钳 cranially 和尾到鼠标的外生殖器。收回包皮以充分暴露头头的阳具。一旦阳具外部定位, 松开拇指钳。</li>
	<li>重新定位钳, 以稳定突出的生殖器垂直的动物, 轻轻地拿着器官, 但紧。可视化尿道道, 并小心地将导管插入器官尖端的小开口处。将导管轻轻地引导到阳具中, 朝向老鼠的身体, 用镊子保持温和的张力。导管不需要润滑。不要强迫导管进入生殖器;导管应平滑滑入尿道, 指示正确放置。</li>
	<li>一旦导管的中心与生殖器的尖端, 非常缓慢地免除50µl 的接种, 同时保持的位置的小弟弟。根据预先确定的灌输质量分数 (如图 2所示), 此时可以注意到灌输的质量。</li>
	<li>慢慢缩回导管, 超过数 5, 以防止接种的渗漏。把老鼠放在笼子里放在仰卧位。动物应开始恢复 30-45 分钟后的麻醉管理。</li>
</ol>6. 感染器官中 CFU 的测定
<ol><li>在预定的时间点, 牺牲小鼠使用批准的标准操作程序 (例如, 颈椎脱位或二氧化碳过量)。用70% 乙醇彻底滋润腹部, 以减少毛皮污染。</li>
	<li>用剪刀和菌将至少2厘米的小鼠腹部的下三分之一切开, 除去膀胱和其他所需器官, 包括但不限于肾脏、睾丸、精囊或包皮腺体, 到5毫升含有1毫升无菌 PBS 的聚丙烯快盖管。把所有的样品放在冰上。</li>
	<li>用手持式均质器质器官, 直到几乎没有固体组织残留, 大约 15-60 秒取决于器官。脂肪不质好, 容易辨认为闪亮的米色白色组织。在同质化之前或之后丢弃脂肪。在每个实验或器官组之间, 用乙醇清洗匀浆器。 注: 通常使用手持质或珠磨质。虽然所有这些系统都产生了类似的结果, 通过直接比较细菌 CFU 在24小时后感染, 质宿主组织的最佳条件, 同时保留细菌的生存能力, 应在经验前确定例行执行此协议。</li>
	<li>执行均匀化器官悬浮的串联稀释。平板稀释在 LB 琼脂板上, 适当时含有抗生素, 在37° c 时孵育过夜 (约15小时)。UPEC 菌株有非常快的倍倍, 因此, 应采取谨慎不孵化琼脂板太长, 因为这将阻碍能力计数个别殖民地。</li>
</ol>7. 膀胱组织的流动细胞分析
<ol><li>在 PBS 的100µg/毫升, 准备34单位/毫升和 dnasei 的消化缓冲液中含有胶原酶。继续冰每只动物准备一个15毫升的锥管进行分析, 每管分1毫升消化缓冲液。</li>
	<li>在预定的 timepoints, 使用批准的标准操作程序 (例如,颈椎脱位或二氧化碳过量) 牺牲小鼠。用70% 乙醇彻底滋润腹部, 以减少毛皮污染。</li>
	<li>
用剪刀将至少2厘米的小鼠腹部的下三分之一切开, 并取出整个膀胱。用剪刀切碎, 将膀胱切成 2-3 mm2大小的碎片, 通常约8件。
	<ol>
<li>每管含消化缓冲液加一个切碎的膀胱。摇动以将切碎的膀胱组织冲洗到消化缓冲液中。继续冰, 直到所有膀胱被解剖和剁碎。</li>
	</ol>
</li>
	<li>在37° c 下孵育 60-75 分钟的碎膀胱, 用手剧烈摇动5秒, 每15分钟。当组织有玻璃透明的外观, 类似于湿纸巾, 消化是完整的。长时间的消化将从细胞中去除表面蛋白, 以供鉴别, 应避免。</li>
	<li>通过加入 2-3 毫升的冰-冷流式细胞仪缓冲液 (PBS 含有2% 胎牛血清 (FBS) 和0.2 毫米 EDTA) 使消化酶钝化, 并轻轻混合。把管子放在冰上。</li>
	<li>为了确保单个细胞悬浮, 并去除任何结缔组织, 通过100µm 过滤器放置在一个15毫升锥形管管的内容。用注射器柱塞的末端轻轻按压任何剩余的组织。用额外的2毫升流式细胞仪缓冲清洗过滤器。把样品放在冰上。</li>
	<li>用离心法洗涤样品7分钟, 在 200 x g, 4 ° c。重颗粒在100µL 流式细胞仪缓冲包含 Fc 块, 稀释到5µg/毫升和转移到一个96井圆底板。10-15 分钟后, 在每个样本中添加100µL 所需的抗体鸡尾酒。在冰上孵育样品 30-45 分钟, 免受光照。 注: fc 块是一种试剂, 用于防止抗体 fc 部分与表达 fc 受体的细胞结合。其目的是尽量减少非特异性抗体结合。使用的抗体的测定将取决于在实验中测试的假说, 并应选择从文献的输入。在图 3中, 为了测量整体免疫细胞浸润, 采用了一种 anti-CD45 抗体, 它是一种泛免疫细胞蛋白。</li>
	<li>用离心法洗涤样品7分钟, 在 200 x g, 4 ° c。重细胞颗粒在200µL 流式细胞仪缓冲和通过一个40µm 细胞过滤器上的5毫升聚苯乙烯管, 刚刚获得的流量胞内液。</li>
	<li>收集整个样品流式细胞仪。一个良好的消化将产生 20万-40万细胞, 8000-1万 CD45+免疫细胞从天真的小鼠膀胱, 而感染器官将包含更多的细胞16 (图 3)。 注: 为流细胞分析准备的样品不能用于评价 CFU。不建议将膀胱分成两部分, 因为没有证据证明 UPEC 定植或免疫细胞浸润在整个组织中都是均匀的。</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+urinary+tract+infection.">A murine model of urinary tract infection.</a> Nat Protoc. 4 (8), 1230-1243 (2009).</li><li>Wright, K. J., Seed, P. C., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+intracellular+bacterial+communities+of+uropathogenic+Escherichia+coli+depends+on+type+1+pili.">Development of intracellular bacterial communities of uropathogenic Escherichia coli depends on type 1 pili.</a> Cell Microbiol. 9 (9), 2230-2241 (2007).</li></ol>

作者没有什么可透露的。

经尿道电切的雄性小鼠对膀胱黏膜疾病的影响提供了许多新的研究机会, 但也提出了一些挑战。首先, 一个限制是, 灌输可能最初证明是技术上的困难, 导致过度炎症在导管插入。技术的改进可以通过用有色溶液注入死老鼠来实现, 如埃文斯蓝染料。为了证实注入成功, 膀胱应进行视觉检查和灌输的液体的体积可以用结核菌素注射器的提取评估。缓慢的, 温和的运动的重要性不能被夸大: 不应该有阻力...

泌尿道感染是发达国家最常见的感染之一1。在新生儿和老年人中, 女性和男性的感染率相似,2。绝经前的成年女性, 但是, 有一个大大增加的发病率社区获得性尿路感染相比, 男性2,3。鉴于这一疾病主要影响妇女, 基础和临床研究压倒性地集中在女性尿路感染。然而, 在男性泌尿感染是一个重要的和理解的医疗保健挑战4。事实上, 由于男性泌尿系感染与发病率较高有关, 这些传染病通常被定义为复杂的4,5。
随着我们对性别偏见在生理学和病理学中的核心作用的理解, 我们需要新的方法来探索这一以前被忽视的疾病方面。性别差异在免疫和感染中起着至关重要的作用;女性自身免疫性疾病的发病率较高, 而男性更容易受到某些感染, 如结核病、疟疾和 HIV6,7。膀胱癌, 另一个泌尿系病理, 是显着更普遍的男性比妇女, 和几项研究显示了作用的雄激素在发展的恶性肿瘤8,9,10,11 ,12。然而, 值得注意的是, 由于无法反复 catheterize 雄性小鼠13, 对膀胱癌的膀胱灌注治疗的研究完全是在雌性动物身上进行的。
对泌尿学发病机制的研究主要依赖于啮齿动物的感染模型 (如、小鼠和大鼠)。小鼠尿路感染模型可能采用 uropathogens 最初孤立于人类传染病, 如肾炎大肠杆菌(UPEC),克雷伯杆菌,肠球菌,葡萄球菌, 或变形杆菌14。通常情况下, 细菌被引入膀胱通过导尿管的尿道。继感染后, 膀胱和肾脏可以被移除, 以评估特定的感染参数, 如细菌定植、组织损伤或宿主免疫应答15,16。然而, 普遍的, 只有雌性动物被用于泌尿的研究。事实上, 许多研究指出, 由于解剖原因, 男性小鼠的插管是不可能的13,17,18,19。最近, 对男性灌输的外科手术方法已经被描述, 在其中腹部打开, 膀胱是外部流离失所通过施加温和的压力, 在腹部的开放, 细菌被注射到膀胱20。这种方法能使男性感染, 以手术治疗为代价。因此, 这种方法的一个主要的告诫包括炎症的影响, 对感染的结果, 如潜在的诱导抗炎伤口愈合的反应的切口21。由于我们的兴趣包括对疾病的性别偏见的理解, 我们开发了一种在雄性小鼠体内进行细菌膀胱灌注的方法, 它与长期建立的非手术电切法在雌性啮齿动物中的应用更加吻合22.
我们的模型建立在一个既定的方法和提供的能力, 直接比较宿主免疫反应与泌尿系统感染的雌性和雄性动物。这种方法将允许解剖的性别差异的感染, 并可能提供分子和细胞线索的明显差异的敏感性和反应的性别感染。此外, 这一模式的价值超越了泌尿系研究, 允许建立模型, 以调查其他膀胱相关疾病, 如膀胱癌, 前列腺炎, 下或过活动膀胱综合征, 和间质性膀胱炎。

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="45">
			<td height="45" style="height:45px;width:283px;">BD Insyte Autoguard Shielded IV Catheters 24G, 0.7 mm external diameter, 14 mM long</td>
			<td style="width:131px;">BD Medical</td>
			<td style="width:135px;">381811 or 381411*</td>
			<td style="width:260px;">* catalog number is country-dependent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">inoculating loop</td>
			<td style="width:131px;">Greiner Bio-One</td>
			<td style="width:135px;">731171</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">LB Miller broth</td>
			<td style="width:131px;">Difco</td>
			<td style="width:135px;">244620</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">LB Miller agar</td>
			<td style="width:131px;">Difco</td>
			<td style="width:135px;">244520</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cuvettes</td>
			<td style="width:131px;">Bio-rad</td>
			<td style="width:135px;">223-9955</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">syringes</td>
			<td style="width:131px;">B Braun</td>
			<td style="width:135px;">9166017V</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Thumb (Adson) forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">11006-12</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5 mL tubes, polypropylene</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">352063</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tissue Ruptor disposable probes</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:135px;">990890</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">15 mL flip cap tubes</td>
			<td style="width:131px;">Thermo Scientific</td>
			<td style="width:135px;">362694</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">DNAse</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:135px;">18047019</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Liberase TM</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:135px;">5401119001</td>
			<td style="width:260px;">should be aliquotted and stored at -20 &#176;C to avoid repeated freeze-thaw</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">100 &#181;M MACS SmartStrainers&#160;</td>
			<td style="width:131px;">Miltenyi</td>
			<td style="width:135px;">130-098-463</td>
			<td style="width:260px;">compatible with 15 mL tubes, preferred</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">100 &#181;m cell strainers&#160;</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">352360</td>
			<td style="width:260px;">compatible with 50 mL tubes, if MACS Smart Strainers are not available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">96 well plate</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">353077</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Fc block</td>
			<td style="width:131px;">BD Pharmingen</td>
			<td style="width:135px;">553142</td>
			<td style="width:260px;">anti-CD16/CD32</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">anti-mouse CD45 antibody</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">561487</td>
			<td style="width:260px;">many different fluorescent conjugation options are available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5 mL tubes polystyrene with filter cap</td>
			<td style="width:131px;">Falcon</td>
			<td style="width:135px;">352235</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">insulin syringe</td>
			<td style="width:131px;">Terumo</td>
			<td style="width:135px;">BS05M2913</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">hand held homogenizer</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:135px;">9001272</td>
			<td style="width:260px;">TissueRuptor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">flow cytometer</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">N/A</td>
			<td style="width:260px;">Fortessa SORP&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Flow cytometry software</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">N/A</td>
			<td style="width:260px;">Diva</td>
		</tr>
	</tbody>
</table>

urinary tract infection in a small animal model: transurethral catheterization of male and female mice

泌尿道感染在世界范围内非常普遍, 引起严重的发病率和医疗费用。小动物模型, 准确地反映疾病的建立和进展, 允许解剖宿主-病原体相互作用和产生的免疫力感染。在小鼠, 膀胱灌注肾炎的大肠杆菌, 致病剂在85% 以上的社区获得性尿路感染, 重述的许多阶段的感染者观察到的人。然而, 直到最近, 泌尿感染只能以雌性动物为模型。这一限制阻碍了研究泌尿和其他膀胱疾病, 如癌症的性别相关的差异。在这里, 我们描述一种方法来灌输雄性小鼠, 允许直接比较雌性和雄性动物, 并提供一个详细的协议, 以评估膀胱组织的流式细胞仪作为一种手段, 更好地了解宿主对感染的反应。这些方法将有助于确定在泌尿系和其他膀胱相关疾病中所观察到的性别偏见的寄主因素。

在这个协议的发展中, 雌性和雄性 C57Bl/6 小鼠的年龄在6到8周之间被灌输, 并且细菌负担评估在膀胱在时间点范围从1小时到30天。这些研究的结果在别处 (Zychlinsky 沙夫et al., 待定) 进行了详细说明。在这里, 我们提供来自24小时感染的代表性数据。值得注意的是, 24 小时后感染的雄性和雌性小鼠的细菌负担是相等的 (图 1)。在每组24小时感染后观察到的细菌?...

Watch this Scientific Journal Video about Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice at JoVE.com

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

建立的模型, 经尿道小鼠导尿, 允许研究膀胱疾病, 包括泌尿道感染, 但只能在女性进行。一个新的男性经尿道灌注的模式, 在这里提出, 将使研究在一个地区的特点是强的临床和流行病学差异的性别。

小动物模型尿路感染的实验研究: 男性和女性的经尿道插管

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

urinary-tract-infection-small-animal-model-transurethral

Research

JoVE Journal

Immunology and Infection

16.3K 次观看.  Institut Pasteur, INSERM U818. 建立的模型, 经尿道小鼠导尿, 允许研究膀胱疾病, 包括泌尿道感染, 但只能在女性进行。一个新的男性经尿道灌注的模式, 在这里提出, 将使研究在一个地区的特点是强的临床和流行病学差异的性别。

视频: Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

经尿道诱导小鼠尿路感染

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

科研

免疫与感染

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Ecto和实验室大鼠和小鼠体内寄生虫的诊断

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

γHV68感染小鼠的测量

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Transurethral Instillation Procedure in Adult Male Mouse

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into the urinary bladder to either induce animal models of bladder pathologies or evaluate the effectiveness of intravesical treatments. Most rodent models of lower urinary tract (LUT) pathologies are induced in female mice due to ease of intravesical instillation of the substances via the female urethra. However, due to anatomical differences between the female and male LUT, transurethral instillation in a male mouse has been deemed a very challenging procedure, and it has not been previously described. In this manuscript, we provide a detailed description of how to prepare polyethylene (PE) tubing for subsequent insertion into the urethra of a male mouse. In addition, we discuss the ideal types of PE tubing to be used depending on the desired site of inoculation. Furthermore, we describe point by point how to prepare an animal for a successful transurethral instillation to avoid injury to the urethra and ensure the delivery of the solution to the desired location. The procedure is started by retracting the prepuce and the glans to expose the opening of the urethral meatus. Next, the glans are grasped by blunt non-crushing forceps to stabilize the penis and the PE tubing. The PE tubing is first inserted into the urethral meatus parallel to the animal body, then its angle is adjusted by tilting the catheter to maneuver it to follow the natural curvature of the urethra. This technique can be used to induced murine models of bladder pathologies and/or evaluate the effectiveness of intravesical treatments in male mice.

Transurethral instillation is a challenging procedure and has not been well described in the literature. The aim of this manuscript is to describe a technique for transurethral insertion of a catheter for intravesical delivery of liquids with active substances into the urinary bladder and/or prostate in adult male mice.

成年雄性小鼠经尿道灌注术

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

颅内 Subarachnoidal 感染对猪链球菌生物膜在小鼠感染模型中作用的研究

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

尿道感染小鼠模型产生的单细胞内细菌群落的分离, 用于下游单细胞分析

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336&#916;micC by the &#955; Red-based recombination system and the complemented mutant 50336&#916;micC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336&#916;micC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336&#916;micC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336&#916;micC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336&#916;micC and 50336&#916;micC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An &#955;-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

非编码小RNA MicC有助于肠炎 沙门氏菌 外膜蛋白的毒力

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...