这项研究得到了防御威胁减少代办处 (DTRA) 根据合同 HDTRA1-14-C-0116 和卓越的中心在传染基因组学 (DOD #W911NF-11-1-0136)。

这项工作是严格按照国家卫生研究院的实验室动物护理和使用指南中的建议进行的。涉及啮齿动物的动物协议是由德克萨斯大学在圣安东尼奥机构动物保育和使用委员会 (IACUC) 根据协议 MU009 (RA) 批准的. 
 1. 准备导管、气管指示器和 F. tularensis 接种 <ol> <li> 准备导管 (20 g x 2 英寸) <ol> <li> 将 20 g x 2 针切割, 并将针头的尖端磨成钝而平滑的完成旋转工具. 
		注: 钝针的长度应允许金属针尖在完全固定在钝针上时, 将大约3毫米的导管套筒解除。钝针使导管结构刚性, 使导管在气管内放置, 并没有钝针尖端凸出从导管套筒防止损伤的气管. </li>
		<li> 用70% 乙醇清洗钝针和导管, 在紫外线照射下进行15分钟消毒. </li>
	</ol> </li> <li> 准备气管指示器 <ol> <li> 修剪一个1000和 #181; L 吸管尖端, 让尖端坐在导管口, 形成一个密封. </li>
		<li> 从200和 #181 中移除筛选器; l 气溶胶屏障吸管尖和在先前修剪过的1000和 #181 中的位置; l 吸管尖. </li>
		<li> 确保过滤器可以在1000和 #181 内自由移动; 当刀尖尖 tip-down 时, L 吸管尖. </li>
	</ol> </li> <li> f. tularensis 接种 <ol> <li> tularensis 在适当的琼脂板上过夜, 在37和 #176; 刮约100和 #181; 从琼脂板上的细菌草坪, 无菌接种环路和用于接种500毫升锥形烧瓶, 其中含有250毫升适当的液体生长培养基 (Fn: 胰酱油), 辅以 0.1% l-半胱氨酸盐酸盐一水合物, 并在37和 #176 夜间孵育摇晃; C. 
		注意: 视频中使用的 F. novicida 在胰大豆琼脂 (TSA) 板上生长, 并辅以 0.1% l-半胱氨酸盐酸盐一水合物. </li>
		<li> 在室温下将夜间生长的液体培养在 4221 x g 处离心10分钟, 除去上清液而不扰乱细菌颗粒. 
		注意: 请遵循机构和 #39 的生物安全建议, 放弃上清. </li>
		<li> 温和地 re-suspend 25 毫升适当液体培养基 (Fn: 补充 0.1% l-半胱氨酸盐酸盐一水合物) 的细菌颗粒, 使用25毫升吸管. </li>
		<li> 使用1毫升吸管, 分悬浮细菌培养成500和 #181; 等分在瓶子里, 在干冰/乙醇浴中冷冻, 然后在-80 和 #176 储存; C. </li>
		<li> 要确定冷冻培养瓶的效价, 请取出存储在-80 和 #176 上的两个冰冻小瓶; C、在冰上解冻并在磷酸盐缓冲盐水 (PBS) 中执行串行稀释, 然后在适当的培养基上电镀 (Fn: TSA 含有 0.1% l-半胱氨酸盐酸盐)。在37和 #176 中孵育 24-48 h; C 和菌落形成单元 (CFU) 被列举来计算冷冻培养瓶内细菌细胞的数量 (平均两个小瓶). </li>
		<li> 在冰上解冻冰冻小瓶, 然后用 PBS 将培养基稀释到最后浓度为 10 7 CFU/100 和 #181, 准备细菌接种;接种应在100和 #181 的浓度, 将产生期望的 CFU; L (在接种的最后浓度的甘油不能超过 3%, 以防止窒息的大鼠)。冷冻培养瓶储存在-80 和 #176; C 可在准备后六月内使用. </li>
	</ol> </li> </ol> 2。大鼠麻醉 <ol> <li> 将麻醉室连接到麻醉机的正常操作的异氟醚蒸发器. </li>
	<li> 将麻醉箱上的气体清理管连接到气体清除系统. </li>
	<li> 打开麻醉机上的氧气流量为4升/分. </li>
	<li> 将异氟醚蒸发器设置为 5%. </li>
	<li> 将老鼠放在麻醉室里. 
	注意: 我们通常使用 8-10 周大的老鼠 (130-180 克);年轻的老鼠很难通过这种技术接种, 因为小的口腔腔的大小. </li>
	<li> 在进行麻醉诱导时, 保持5% 异氟醚的大鼠, 2 升/分氧作用。这需要大约 3-10 min. </li>
	<li> 根据呼吸和心跳的质量和速率确定麻醉深度, 以及在脚趾夹紧试验中对反射刺激的反应。理想的麻醉深度是由 1-1.5 s 计数之间的每次呼吸. </li>
</ol> 3。气管内接种 <ol> <li> 将大鼠从麻醉室中取出, 并将鼠背放在啮齿动物插管支架上。将大鼠的前牙附着在支架上以保持老鼠的位置. 
	注意: 如果在手术过程中, 老鼠开始苏醒, 导管可以被移除, 老鼠回到麻醉室, 到达更深的麻醉面. </li>
	<li> 用惯用手将舌头移到同一侧, 用宽点修整拇指钳. </li>
	<li> 使用支持的手和喉镜对大鼠和 #39 的下颌, 并可视化动物的气管和食道。当老鼠呼吸时, 气管就会打开和关闭. </li>
	<li> 插入导管, 其中包含20口径钝针, 在步骤1.1 中准备, 放入气管。可能有轻微的阻力, 并插入气管可能和 #34; 颠簸和 #34; 由于导管摩擦气管软骨。由于湿气或浅呼吸, 气管可以覆盖的会厌, 防止可视化的气管。轻轻地触摸会厌软骨的边缘会导致软骨瓣打开并揭开气管. </li>
	<li> 从导管中取出钝针, 同时确保导管仍留在气管内. </li>
	<li> 允许几秒钟, 使老鼠能够在导管插入气管内呼吸. </li>
	<li> 牢牢地将气管指示器固定在导管的开口上。气溶胶屏障的运动将指示导管正确插入气管. </li>
	<li> 确保大鼠放置在插管支架上, 使动物的胸部朝向垂直于插管架的平面. </li>
	<li> 卸下气管指示器, 并提供100和 #181; 包含 tularensis 的接种 (10 7 CFU), 使用200和 #181; l 吸管尖, 将尖端固定在导管口上. </li>
	<li> 将1毫升滑尖结核注射器附在空气中, 并提供300和 #181; L 空气以确保接种到达鼠的肺部. </li>
	<li> 从气管取出导管, 从手术台上取出大鼠. </li>
	<li> 允许老鼠苏醒并回到笼子里。确保呼吸恢复正常. </li>
	<li> 确定 F. tularensis 中的 CFU 接种如步骤1.3.5 所述, 以确认 CFU 在气管内的传递. </li>
	<li> 重复步骤 3.1-3.9 接种 (na 和 #239; ve) 动物与100和 #181; L PBS 代替接种. </li>
</ol>

<ol>
	<li>Ellis, J., Oyston, P. C. F., Green, M., Titball, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tularemia.">Tularemia.</a> Clin Microbiol Rev. 15 (4), 631-646 (2002).</li><li>Lyons, C. R., Wu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Francisella+tularensis+infection.">Animal models of Francisella tularensis infection.</a> Ann N Y Acad Sci. 1105, (2007).</li><li>Hutt, J. A., Lovchik, J. A., Dekonenko, A., Hahn, A. C., Wu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Natural+History+of+Pneumonic+Tularemia+in+Female+Fischer+344+Rats+after+Inhalational+Exposure+to+Aerosolized+Francisella+tularensis+subspecies+tularensis+Strain+Schu+S4.">The Natural History of Pneumonic Tularemia in Female Fischer 344 Rats after Inhalational Exposure to Aerosolized Francisella tularensis subspecies tularensis Strain Schu S4.</a> Am J Pathol. 187 (2), 252-267 (2017).</li><li>Ray, H. J., 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=The+Fischer+344+rat+reflects+human+susceptibility+to+Francisella+pulmonary+challenge+and+provides+a+new+platform+for+virulence+and+protection+studies.">The Fischer 344 rat reflects human susceptibility to Francisella pulmonary challenge and provides a new platform for virulence and protection studies.</a> PloS one. 5, e9952 (2010).</li><li>Jemski, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+tularemia:+comparison+of+selected+routes+of+vaccination+in+Fischer+344+rats.">Respiratory tularemia: comparison of selected routes of vaccination in Fischer 344 rats.</a> Infect Immun. 34 (3), 766-772 (1981).</li><li>Wu, T. H., 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=Vaccination+of+Fischer+344+rats+against+pulmonary+infections+by+Francisella+tularensis+type+A+strains.">Vaccination of Fischer 344 rats against pulmonary infections by Francisella tularensis type A strains.</a> Vaccine. 27 (34), 4684-4693 (2009).</li><li>Mara-Koosham, G., Hutt, J. A., Lyons, C. R., Wu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+contribute+to+effective+vaccination+against+respiratory+infection+by+type+A+Francisella+tularensis+strains.">Antibodies contribute to effective vaccination against respiratory infection by type A Francisella tularensis strains.</a> Infect Immun. 79 (4), 1770-1778 (2011).</li><li>Oyston, P. C., Sjostedt, A., Titball, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tularaemia:+bioterrorism+defence+renews+interest+in+Francisella+tularensis.">Tularaemia: bioterrorism defence renews interest in Francisella tularensis.</a> Nat Rev Microbiol. 2 (12), 967-978 (2004).</li><li>Kingry, L. C., Petersen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+review+of+Francisella+tularensis+and+Francisella+novicida.">Comparative review of Francisella tularensis and Francisella novicida.</a> Front Cell Infect Microbiol. 4, 35 (2014).</li><li>Rohmer, 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=Comparison+of+Francisella+tularensis+genomes+reveals+evolutionary+events+associated+with+the+emergence+of+human+pathogenic+strains.">Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains.</a> Genome Biol. 8 (6), R102 (2007).</li><li>Chu, P., 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=Live+attenuated+Francisella+novicida+vaccine+protects+against+Francisella+tularensis+pulmonary+challenge+in+rats+and+non-human+primates.">Live attenuated Francisella novicida vaccine protects against Francisella tularensis pulmonary challenge in rats and non-human primates.</a> PLoS Pathog. 10 (10), e1004439 (2014).</li><li>Signarovitz, A. 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=Mucosal+Immunization+with+Live+Attenuated+Francisella+novicida+U112&#916;iglB+Protects+against+Pulmonary+F.+tularensis+SCHU+S4+in+the+Fischer+344+Rat+Model.">Mucosal Immunization with Live Attenuated Francisella novicida U112&#916;iglB Protects against Pulmonary F. tularensis SCHU S4 in the Fischer 344 Rat Model.</a> PloS one. 7 (10), e47639 (2012).</li><li>Cunningham, A. 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=Enhancement+of+vaccine+efficacy+by+expression+of+a+TLR5+ligand+in+the+defined+live+attenuated+Francisella+tularensis+subsp.+novicida+strain+U112DiglB::fljB.">Enhancement of vaccine efficacy by expression of a TLR5 ligand in the defined live attenuated Francisella tularensis subsp. novicida strain U112DiglB::fljB.</a> Vaccine. 32 (40), 5234-5240 (2014).</li></ol>

作者没有透露

费希尔344大鼠正在成为兔疫苗开发的重要模型3。通过肺部通道暴露于f. tularensis对于演示对F. tularensis的武器化形式的有效性至关重要, 因为这些方法是作为气溶胶提供的。大鼠的气管内接种有助于大鼠肺部暴露于F. tularensis , 而不需要大型、昂贵和复杂的气溶胶发电设备。使用F. tularensis的选择代理形式的所有实验还需要 BSL3 的包容, 这通常发生在严格限?...

F. tularensis (Ft) 导致人类疾病, 兔。当细菌通过肺通路获得, 这导致肺炎兔, 有高发病率和死亡率1。由于与雾化表单相关的危险, F. tularensis被认为是 biothreat 剂, 目前在美国没有批准用于人类使用的疫苗。目前正在加紧努力, 研制抗肺兔的疫苗和治疗措施, 以保护人类不受非法使用这种细菌 biothreat。
由于小鼠对F. tularensis感染的极度敏感, 以及试剂的流行, 兔的研究大多集中在鼠标模型上。然而, 由于在这一模型2中展示疫苗功效的难度, 老鼠已经证明是疫苗开发的一个困难模型。最近, Fischer 344 鼠已被开发成一个模型的兔疫苗开发3。Fischer 344 大鼠的敏感性对各种各样的F. tularensis亚种模仿人的敏感性4, 并且鼠可以被保护免受F. tularensis肺挑战接种与活疫苗应变已知保护人5,6,7。由于费舍尔344大鼠模型的一些特点, f. tularensis感染的人, 它可能是一个非常有用的模型, 开发的疫苗, 以防止肺部的F. tularensis暴露。
一种有效的疫苗需要保护人类免受肺部接触到F. tularensis。最有可能的肺部暴露从武器化的F. tularensis将雾化细菌吸入肺部8。然而, 气溶胶生成的F. tularensis既危险又繁琐, 需要专门的设备和控制。在大鼠肺部暴露的另一条途径, 可能更适合于多实验室缺乏专门设备是通过气管内接种6。该技术利用喉镜正确放置在麻醉大鼠气管内的导管。在气管内的位置, 而不是食道, 通过一个简单的装置来证实肺部的气流。F. tularensis随后通过导管将注射器传递到肺部, 然后在导管中引入空气, 以确保细菌的肺部传递。相比之下, Jemski5以前报告说, 通过鼻腔通道将F. tularensis接种到 Fischer 344 大鼠体内, 直到3天 post-inoculation 才从肺部培养出来, 表明在大鼠鼻腔接种不会导致细菌直接输送到肺部。
选择f. tularensis的代理窗体 (f. tularensis亚种tularensis, f. tularensis 亚种holarctica) 要求安全级别 3 (BSL3) 控制程序, 这将防止录像。但是, F. novicida (Fn) 由于其在健康人中的无毒而免受选择代理状态的限制, 并且可在生物安全级别 2 (BSL2) 条件910下安全地使用。此外, Fn 还可作为减毒活疫苗的基础, 在通过气管接种的情况下, 可预防F. tularensis肺暴露, 其方法是:11,12,13。这里介绍的技术允许研究通过肺路线发生的感染, 利用大鼠作为人类的模型。这种技术可以在不需要专门的气溶胶产生设备的情况下进行。Fn 被用于这里拍摄的技术。

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			<td height="40" style="width:376px;height:40px;">GreenLine fiber optic blade size 0</td>
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</table>

intratracheal inoculation of fischer 344 rats with francisella tularensis

肺部感染的细菌土 tularensis可能导致严重的和潜在的致命疾病, 兔, 在人类。由于目前没有批准的人类兔疫苗, 研究的重点是利用适当的动物模型开发疫苗。费希尔344大鼠已经成为一个模型, 反映了人类对F. tularensis感染的敏感性, 因此是兔疫苗开发的一个有吸引力的模型。344只大鼠的气管内接种与F. tularensis模拟人肺暴露。成功交付到大鼠气管是至关重要的肺分娩。用带光照的喉镜对麻醉大鼠的气管进行适当插管;正确放置在气管内是由一个简单的装置来检测呼吸。在插管后, F. tularensis培养通过注射器在测量剂量内传递。这项技术规范了大鼠气管内的肺部分娩tularensis , 以评估疫苗的功效。

对大鼠气管内接种的体液反应可以通过酶联免疫吸附试验 (ELISA) 对紫外线灭活细菌进行测定, 如前所述, 11。在14和天 28 (图 1) 中, 对344大鼠的总免疫球蛋白 G (IgG) 反应进行了 post-intratracheal 接种, 并对 Fn (107 CFU 接种) 的减毒菌株进行了评估。模拟接种的大鼠接受 PBS intratracheally。相对于天真的模拟接种动物, 血清抗体滴?...

Watch this Scientific Journal Video about Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis at JoVE.com

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

本议定书描述的是344大鼠气管内接种的土 tularensis.这个过程模仿人的肺暴露对这个潜在的 biothreat 代理并且可以用于测试疫苗和治疗疗效对肺兔。

费希尔344大鼠气管内接种土 tularensis

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the ...

intratracheal-inoculation-fischer-344-rats-with-francisella

Research

JoVE Journal

Immunology and Infection

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

7.1K 次观看.  University of Texas San Antonio. 本议定书描述的是344大鼠气管内接种的土 tularensis.这个过程模仿人的肺暴露对这个潜在的 biothreat 代理并且可以用于测试疫苗和治疗疗效对肺兔。

视频: Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

原位大鼠后肢移植

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

科研

免疫与感染

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

非手术治疗的小鼠气管内滴入肺和肺淋巴结流式细胞仪分析

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

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 ...

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

实验小鼠模型阴道接种和样品采集的协议念珠菌阴道炎

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

完整的胸腺切除术的成年大鼠非侵入气管插管

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

小鼠边缘区 b 细胞剪切流诱导迁移的体外分析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

在小鼠小腿上应用一致的按摩样扰动，并监测由此产生的肌肉压力变化

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...