这项研究是由美国国立卫生研究院/ NIDDK由授予1K01DK079019A2（PT）。

1。小鼠接种细菌的准备工作以下，必须在无菌条件下完成。 <ol><li>拿一个17×100毫米聚丙烯管帽和加入3毫升的新鲜Luria培养基（LB）媒体。使用蒸压提示，采取一些冷冻的细菌甘油CP1 2株，少量的文化转移到管中的LB培养基。更换的管帽，这样的氧气仍然可以进入管 - 文化需要在有氧条件下生长。将管在37℃下摇床中在220rpm过夜。 </li><li>第二天，5微升过夜培养物转移到新的管中，用3毫升的LB培养基，并在37℃的培养箱中培养过夜的静态条件下成长</li><li>在第3天转移到AA 50ml管40μl的文化含40毫升LB媒体。在37°C的静态培养箱中培养过夜地点。 </li><li>打开并设置离心机至4℃。一旦离心机已达到最终温度，每40毫升的文化转移到40毫升的Nalgene离心管中。 </li><li>称取离心管，以平衡 - 调节音量LB媒体。在6,000 rpm，20分钟的自旋管。 </li><li>仔细收集上清，轻轻地暂停细菌沉淀在40毫升无菌PBS。 </li><li>重复步骤1.5 PBS。 </li><li>吸出上清液，并暂停细菌在500μl无菌PBS。将该溶液转移到1.5毫升微量离心管中。取10微升的悬浮液，并在990微升冰冷的PBS中稀释。使用分光光度计读取OD 420 nm处的体积，以确定所需的接种。要确定适当体积的冰冷的PBS中止颗粒:OD 420 nm的数量（毫升）和减去估计量的细菌沉淀前。 OD 420 nm处 = 0.545，颗粒约0.075毫升。 0.545 - 0.075 = 0.470]。 </li><li>取出10μl的细菌悬浮液和二琵琶在990微升冰冷的PBS。外径420纳米 。我们的目标是实现稀释后的悬浮液中1.000±0.010的OD 420 nm处的值。如果您的稀释的数量超过这个目标，增加更多的体积悬浮液中，采取另一种解读。如果数目是低于0.990然后降速的悬浮液，并重复OD 420 nm处读数，直到获得所需的读数。 </li></ol> 2。动物感染小鼠（5〜7周龄，10％组）均购自巴港的杰克逊实验室（ME）。 <ol><li>小鼠麻醉吸入1-4％异氟醚一个有机玻璃室中，被监视，直到卧位。 </li><li>在一个时间的一个鼠标滴注细菌采取导尿用聚乙烯（PE10）的导管连接到的变形30 G针的玻璃Hamilton注射器（长度为1.5-2.0厘米）。导管被插入到轮毂的针。鼠标是放置其背表面麻醉下用鼻锥保持。 </li><li>用于润滑的入口到阴茎尿道，阴茎的鼠标是温和的压力和自由量的润滑剂棉签挤出。 </li><li> 10μl的磷酸盐缓冲生理盐水中含1×10 8个细菌引入到尿道后麻醉小鼠导尿。 </li><li>维持小鼠在麻醉的状态下的细菌引进的有机玻璃室后15分钟，让细菌附着，防止即时排尿。 </li><li>小鼠被放置在笼子里，并在接下来的24小时监控。 </li></ol> 3。行为测试小鼠在感染前（基线）和感染后3，7，14，21和28进行了测试。简称痛觉过敏和触觉异常进行了测试使用von Frey细丝适用于腹部11,12和足底ŕ对旅游区的后掌13。测试perfomed在一个固定的时间的，标准的方法和单实验者测试的所有动物。盲法测试组被用来打击在动物模型的局限性，基于行为的疼痛测试。五个单独的von Frey细丝与力量，0.04，0.16，0.4，1.0和4.0克（Stoelting，美国）被施加到撤回的腹部和频率响应计算。 <ol><li>的驯化后，30分钟内，被放置在个别有机玻璃腔室（6×10×12厘米），在内部用不锈钢线栅地板小鼠。 </li><li>采用von Frey细丝简称痛觉过敏和触觉异常。各灯丝施加为1-2秒的总共有10倍刺激间的间隔为5秒，武力升序顺序和毛发进行了测试。 </li><li>每个灯丝被施加到下腹部区域中的一般的前列腺附近护理刺激不同的区域，在此区域内，以避免的脱敏或"风"的影响。适用于力1-2秒递增的顺序与刺激之间的间隔至少为5秒的10倍，总的细丝。 </li><li>三种类型的行为被认为是一个积极的回应长丝刺激:1）急剧收缩腹部; 2）直接舔或，搔抓的长丝刺激; 3）跳跃。 </li><li>肯定响应（满分10， 例如 ，5的反应10 = 50％）的百分比计算，并响应频率数据被报告为响应频率的平均百分比±SE </li><li>超过25个阳性总基线反应的动物被排除在研究之外。 </li><li>用von Frey细丝的后肢力量，0.04，0.16，0.4，1.0和4.0克的的足底地区的触觉异常性疼痛进行了测试。中位数的50％收回阈值（5）进行了评估使用的上下方法在测试开始0.04克长丝的后掌跖面垂直，直到灯丝稍微弯曲。升序排列，直到一个积极的反应，观察单丝测试。积极应对的灯丝被定义为测试爪爪或舔的大幅撤出。当一个正响应记录下一个较弱施加长丝，如果观察到的否定响应，则下一个较强的长丝。 </li><li>的实验和描述的方法进行了审查，并批准由西北大学动物照顾及使用委员会。 </li></ol>

<ol>
	<li>Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+epidemiology+of+uropathogenic+Escherichia+coli.">Molecular epidemiology of uropathogenic Escherichia coli.</a> Journal of infection and chemotherapy official journal of the Japan Society of Chemotherapy. 13, 68-73 (2007).</li><li>Rudick, C. N., 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=Uropathogenic+Escherichia+coli+induces+chronic+pelvic+pain.">Uropathogenic Escherichia coli induces chronic pelvic pain.</a> Infect. Immun. 79, 628-635 (2011).</li><li>Pontari, M. A., Ruggieri, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+in+prostatitis/chronic+pelvic+pain+syndrome.">Mechanisms in prostatitis/chronic pelvic pain syndrome.</a> The Journal of urology. 172, 839-845 (2004).</li><li>Schaeffer, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Chronic+prostatitis+and+the+chronic+pelvic+pain+syndrome.">Clinical practice. Chronic prostatitis and the chronic pelvic pain syndrome.</a> The New England journal of medicine. 355, 1690-1698 (2006).</li><li>Rudick, C. N., Schaeffer, A. J., Thumbikat, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+prostatitis+induces+chronic+pelvic+pain.+American+journal+of+physiology.">Experimental autoimmune prostatitis induces chronic pelvic pain. American journal of physiology.</a> Regulatory, integrative and comparative physiology. 294, R1268-R1275 (2008).</li><li>Elkahwaji, J. E., Ott, C. J., Janda, L. M., Hopkins, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+for+acute+bacterial+prostatitis+in+genetically+distinct+inbred+strains.">Mouse model for acute bacterial prostatitis in genetically distinct inbred strains.</a> Urology. 66, 883-887 (2005).</li><li>Hultgren, S. J., Porter, T. N., Schaeffer, A. J., Duncan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+type+1+pili+and+effects+of+phase+variation+on+lower+urinary+tract+infections+produced+by+Escherichia+coli.">Role of type 1 pili and effects of phase variation on lower urinary tract infections produced by Escherichia coli.</a> Infect. Immun. 50, 370-377 (1985).</li><li>Jarrell, J., Giamberardino, M. A., Robert, M., Nasr-Esfahani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bedside+testing+for+chronic+pelvic+pain:+discriminating+visceral+from+somatic+pain.">Bedside testing for chronic pelvic pain: discriminating visceral from somatic pain.</a> Pain research and treatment. 2011, 692102 (2011).</li><li>Jarrell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+Cutaneous+Allodynia+in+Association+with+Chronic+Pelvic+Pain.">Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain.</a> J. Vis. Exp. (28), e1232 (2009).</li><li>Giamberardino, M. A., 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=Viscero-visceral+hyperalgesia:+characterization+in+different+clinical+models.">Viscero-visceral hyperalgesia: characterization in different clinical models.</a> Pain. 151, 307-322 (2010).</li><li>Laird, J. M., Martinez-Caro, L., Garcia-Nicas, E., Cervero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+visceral+pain+and+referred+hyperalgesia+in+the+mouse.">A new model of visceral pain and referred hyperalgesia in the mouse.</a> Pain. 92, 335-342 (2001).</li><li>Laird, J. M., Souslova, V., Wood, J. N., Cervero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+visceral+pain+and+referred+hyperalgesia+in+Nav1.8+(SNS/PN3)-null+mice.">Deficits in visceral pain and referred hyperalgesia in Nav1.8 (SNS/PN3)-null mice.</a> J. Neurosci. 22, 8352-8356 (2002).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> Journal of neuroscience. 53, 55-63 (1994).</li><li>Schaeffer, A. J., Schwan, W. R., Hultgren, S. J., Duncan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+type+1+pilus+expression+in+Escherichia+coli+to+ascending+urinary+tract+infections+in+mice.">Relationship of type 1 pilus expression in Escherichia coli to ascending urinary tract infections in mice.</a> Infect. Immun. 55, 373-380 (1987).</li><li>Boehm, B. J., Colopy, S. A., Jerde, T. J., Loftus, C. J., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+bacterial+inflammation+of+the+mouse+prostate.">Acute bacterial inflammation of the mouse prostate.</a> The Prostate. 72, 307-317 (2012).</li><li>Rivero, V. E., Cailleau, C., Depiante-Depaoli, M., Riera, C. M., Carnaud, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-obese+diabetic+(NOD)+mice+are+genetically+susceptible+to+experimental+autoimmune+prostatitis+(EAP).">Non-obese diabetic (NOD) mice are genetically susceptible to experimental autoimmune prostatitis (EAP).</a> Journal of autoimmunity. 11, 603-610 (1998).</li><li>Pakarainen, T., Zhang, F. P., Makela, S., Poutanen, M., Huhtaniemi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testosterone+replacement+therapy+induces+spermatogenesis+and+partially+restores+fertility+in+luteinizing+hormone+receptor+knockout+mice.">Testosterone replacement therapy induces spermatogenesis and partially restores fertility in luteinizing hormone receptor knockout mice.</a> Endocrinology. 146, 596-606 (2005).</li><li>Taguchi, O., Kojima, A., Nishizuka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+prostatitis+after+neonatal+thymectomy+in+the+mouse.">Experimental autoimmune prostatitis after neonatal thymectomy in the mouse.</a> Clinical and experimental immunology. 60, 123-129 (1985).</li></ol>

没有利益冲突的声明。

感染的小鼠前列腺UPEC允许为在体内建模可能涉及在细菌性前列腺炎的发病机制中，CP / CPPS或作为慢性炎症的诱发事件的事件。 UPEC模型描述的方法对细菌制剂和滴注战平后，庞大的身躯文学中女性尿路感染7,14。该模型具有广泛的适用性研究发病机制，检测潜在的候选疫苗和免疫调节机制。疼痛行为的能力，按照可量化的方式使感染引起的疼痛发病机制的研究，并有可能作为测试疼?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>文化管</td><td> BD猎鹰</td><td> 352059 </td></tr><tr><td> LB肉汤米勒</td><td> EMD </td><td> EM1.10285.0500 </td></tr><tr><td> NALGENE离心管</td><td>热</td><td> 3118-0050 </td></tr><tr><td>异氟醚</td><td>巴特勒施恩</td><td> NDC 11695-6776-1 </td></tr><tr><td>导管针30G </td><td>汉密尔顿</td><td> 91030 </td></tr><tr><td> PE10管材</td><td> BD intramedic </td><td> 427400 </td></tr><tr><td> von Frey细丝</td><td> Stoelting </td><td> 58025-31 </td></tr></tbody></table>

measurement of tactile allodynia in a murine model of bacterial prostatitis

致肾盂肾炎大肠杆菌 （UPEC）是病原体中发挥了重要的作用，尿路感染和细菌性前列腺炎1。最近，我们已经显示，UPEC有重要的作用开始在慢性盆腔疼痛2，功能的慢性前列腺炎/慢性盆腔疼痛综合征（CP / CPPS）3,4。感染前列腺的临床相关UPEC可以发起和建立慢性疼痛的机制可能涉及组织损伤和5的自身免疫机制的启动。 了解的统一企业在前列腺癌的发病机制是一个挑战，相对交通不便的前列腺操作。我们利用先前描述的尿道感染的方法提供临床株UPEC雄性小鼠，从而建立一个上行性感染的前列腺。在这里，我们描述的协议规范BActerial接种7以及滴注细菌置管麻醉的雄性小鼠的程序。 CP / CPPS的主要特征是由触觉异常4的存在下。行为测试是基于皮肤痛觉过敏的概念称为内脏痛8-10。易怒集中在内脏器官，减少皮肤的痛阈值，允许夸张的反应，正常的非疼痛刺激（痛觉超敏）。正常的力中的应用皮肤导致异常反应，往往增加与强度的底层的内脏痛。我们描述在的NOD / ShiLtJ小鼠，利用von弗雷纤维中的量化触觉异常性疼痛的随着时间的推移，在响应于一个单一的UPEC细菌感染的方法。

<html>我们审查NOD / ShiLtj的C57BL/6J雄性小鼠的存在细菌感染后的慢性疼痛。雄性小鼠（5至7周龄），用生理盐水或细菌进入尿道（ 图1），灌输。机械性刺激导致的骨盆区，用von Frey细丝的生理盐水注入小鼠和UPEC感染的小鼠在28天的试验过程中（ 图1）的响应频率并没有改变。 UPEC感染的NOD小鼠表现出与此相反，是显着更大的盆腔刺激的反应仍然显着升高，直到第28天（P &lt;0.05...

Watch this Scientific Journal Video about Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis at JoVE.com

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

在调解盆腔疼痛慢性前列腺炎前列腺感染可能是一个促进因素。我们的程序编制标准化的细菌接种的细菌进入尿道的雄性小鼠和方法测量触觉异常的小鼠，随着时间的推移，滴入。

细菌性前列腺炎小鼠模型的测量触觉异常

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

measurement-tactile-allodynia-murine-model-bacterial

Research

JoVE Journal

Immunology and Infection

15.7K 次观看.  Northwestern University Feinberg School of Medicine. 在调解盆腔疼痛慢性前列腺炎前列腺感染可能是一个促进因素。我们的程序编制标准化的细菌接种的细菌进入尿道的雄性小鼠和方法测量触觉异常的小鼠，随着时间的推移，滴入。

视频: Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

在 vivo小鼠创面模型中活性氧的成像

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

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 High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

细菌性前列腺炎小鼠模型的测量触觉异常

摘要

探索更多视频

此视频中的章节

在细菌粘附的研究剪应力

移植物抗宿主病的诱导和

过敏原诱发的哮喘小鼠模型

定量测定的免疫反应和睡眠模式

生成和多表型高内涵筛选

B组的小鼠模型

在 vivo小鼠创面模型中活性氧的成像

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

一种高通量志贺氏菌特异性杀菌试验

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统