Name: measuring local anaphylaxis in mice
Uploaded: 2014-10-14T14:00:00.000+00:00
Duration: 7 min 49 s
Description: Watch this Scientific Journal Video about Measuring Local Anaphylaxis in Mice at JoVE.com

The authors would like to acknowledge Dr. Ellen M. Fox for her work regarding this model of local anaphylaxis. This work was supported by the Uniformed Services University of the Health Sciences grant number R073UE.

1，小鼠敏感<ol><li>通过在1×PBS中稀释的OVA的20毫克/毫升的最终浓度制备OVA原液。冻结下在冷冻管并储存等分在-80℃下以供将来使用。 </li><li>带来的20毫克/毫升OVA股票的一个或多个等分试样至室温。在1X PBS稀释的OVA为1毫克/毫升的最终浓度在5ml的聚苯乙烯圆底管帽。轻轻振荡混匀。 </li><li> 1的比例:在保持含有1毫克/毫升OVA上的旋涡设置在中等速度的聚苯乙烯管中，同时继续涡管，以产生1加彻底匀浆Imject明矾（40毫克/毫升）逐滴加入等体积明矾免疫原。 </li><li>将瓶盖上的管带管的涡流。转涡旋上最低的设置，并允许OVA吸附到明矾30分钟。 <ol><li>不要让大量的时间来准备OVA /明矾和致敏小鼠之间传递。如果增感，不能直接OVA /明矾preparat后完成离子，商店OVA /明矾在室温下。旋涡使用前1分钟。 </li></ol></li><li>装入1 ml胰岛素注射器以OVA /明矾的混合物。顶盖采用27号针头的注射器。 </li><li>注入100μl的OVA /明矾到BALB / c小鼠的腹膜为每只小鼠的50微克的OVA和2mg明矾剂量。 </li><li>为阴性对照，致敏另一组小鼠以PBS中。制备1:明矾和PBS的混合物中同样地在上面列出，省略OVA。 </li><li>敏感的所有小鼠（OVA和PBS对照），每周一次，连续3周，共注射3次。最后一次注射后，请等待至少2周进行局部过敏反应试验之前。 </li></ol> 2，局部过敏反应含量<ol><li>带来的OVA原液至室温。在1.5ml微量离心管中，稀释20毫克/毫升OVA库存到5毫克/毫升，用PBS的终浓度（1:4稀释的OVA在1X PBS）。轻轻振荡混匀。 </li><li>麻醉采用RC 2 &lt;老鼠/ SUP&gt;啮齿动物麻醉系统（或类似的汽化输送系统）。填写水库异氟醚，然后打开氧气气流。小鼠放置在感应腔和异氟醚调节控制转盘至5％，开始麻醉。一旦老鼠被完全麻醉，由于缺乏流动性的指示，调整异氟烷控制转盘至3％，以维持深麻醉。麻醉在此过程的目的是为了固定所述动物。耳和尾静脉注射不会引起显著疼痛或痛苦的动物。 注:异氟醚可以让人类的生殖系统产生负面影响。请务必在通风良好的室内使用，并附上清除罐到感应室中和废气。如果您在使用异氟醚是孕妇，戴上碳滤光罩，以尽量减少风险。 </li><li>负载10微升5毫克/毫升的OVA到3月10日CC胰岛素注射器上铺上一个31 G针。加载另一个3/10毫升的胰岛素注射器，10微升的1X PBS。 </li><li>用解剖显微镜，注入10微升的OVA到麻醉小鼠的右耳为50微克OVA的挑战剂量。其他的剂量，例如20微克，也是可接受的。 <ol><li>为了稳定耳朵注射，包装胶带（粘性的一面朝上），围绕一个15毫升的锥形管。右耳的背侧面固定到磁带上。将针头斜面朝上两叶耳之间。慢慢地注入卵于耳组织的中心，注意不要打任何血管。 </li></ol></li><li>用上述同样的方法，注入10微升PBS中进左耳作为阴性对照。 </li><li>等待3分钟。 <ol><li>在此期间，将鼠标的抑止住在热灯或温水​​浴1分钟，扩张血管的尾巴。 </li></ol></li><li>用1毫升注射器胰岛素盖有27G的针头，慢慢注入200微升0.5％伊文思蓝染料到尾vEIN鼠标。快速注入整个加入200μl之前可能破坏血管结构被施用。如果出现这种情况，试图对尾巴的相对侧染料的剩余体积注入静脉。 </li><li>等待10分钟。 </li><li>安乐死鼠标和删除使用镊子和手术剪两个耳朵。 <ol><li>抓耳的边缘与镊子，使得没有任何压力被施加到注射部位处的耳的中心。去除尽可能多的耳组织尽可能通过切割接近，但不通过，软骨存在于耳朵的底部的脊。少量的皮毛上可能存在的切除耳;这并不会影响该测定。 </li></ol></li><li>分装700微升甲酰胺用1.5 ml离心管中。准备每穗一管（每只小鼠两管）。 注意:甲酰胺是致畸，致突变和刺激性。戴上手套，保护眼睛和面部防护。搬运时，孕妇应谨慎使用Š甲酰胺可能是胎儿中毒。 </li><li>添加耳甲酰胺，盖上盖子，并在干热浴中63℃孵育过夜。耳朵可能仍然会出现孵育后略带蓝色;然而大多数的染料将被提取到甲酰胺。 </li><li>加入300μl各个样品，一式两份，向96孔板中。避免转印皮毛，如果它是存在于样品中。确保没有气泡存在于所述孔中，因为它们会影响到吸光率读数。 </li><li>加入300μl甲酰胺中，一式两份，向96孔板中。这些孔将被用作空白。 </li><li>用分光光度计在620nm处测量吸光度。减甲酰胺井从每个样品的读数。 注:根据批准的统一服务大学实验动物管理和使用委员会的协议进行所有的实验。 </li></ol>

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The authors have nothing to disclose.

许多过敏性疾病的标志物被用于评估下面的变应原激发过敏性反应，包括在气道激发的设置改变循环组胺，Th2型细胞因子的产生和细胞招募的水平进入支气管肺泡灌洗液的强度。而在这些参数监测的变化是研究过敏反应重要的是，生物标记物不总是关联与临床过敏性疾病。在本协议中所描述的地方过敏反应试验提供了本地化的血管通透性为局部过敏性疾病的一个相关可重复的测量。用蓝色染料外渗，该协议使双方的可视化和本地化的过敏反应相对定量。相反，依赖于临床评分过敏测定法，该测定法的（蓝染料外渗通过分光光度法测定吸光度）的主要成果是不容易受到观察者偏倚。另外ADVAntage是动物安乐死的挑战后不久，限制了潜在的过敏原的挑战和染料的管理造成任何不适。替代使用由秋山等人 10所述的手持式分光光度计的允许变化的血管通透性的连续测量。然而，伊文思蓝染料是有毒的，并且注射将最终导致动物的需要被实施安乐死。 虽然此法具有许多优势，但它的特点，是有点技术难度的限制。可靠的结果依赖于一致的，成功的耳和尾静脉注射。如果整个攻击剂量未给药于耳，如果耳注射导致的血管明显穿刺，或者如果伊文思蓝静脉注射是不完整的，该动物应排除分析。在我们的实验室中，研究人员不得不实行皮内注射耳的塞弗拉尔周开发能力之前。此外，对用于测定运行适当的控制是很重要的。具体而言，PBS注射应经常被用来作为内部对照，如动物似乎略有不同引起的针插入损伤作出反应。 PBS的读数表示为诱导注射本身的改变血管通透性的基线量度。代替使用皮内注射耳的，该测定也可以应用到小鼠背部皮肤。调查人员可能发现这条路线要少技术上具有挑战性，并且，此外，它允许多个抗原的同时检测。 局部过敏反应测定是非常通用的，因为攻击剂量可以根据不同的实验来操作。如果有必要看变化过敏性反应，这可能是有益的是使用较低的OVA激发剂量，例如20微克。这允许在吸光度监测变化的敏感性增加，如这将是不太可能的细胞活化的饱和点会到达。 另外，该测定可用于学习被动皮肤过敏反应（PCA）。在此设置中，抗原特异性IgE被注入到一个耳朵和车辆到其他。 24小时后，将动物给予抗原和伊文思蓝染料的静脉挑战。在PCA模型可以减少需要完成该实验中，所用动物的数量的时间，并允许调查者看的IgE交联的具体效果。 尽管需要较长时间来执行，也有一些优势，使用本文所描述的活性皮肤过敏反应（ACA）的模型。通过每周接触致敏抗原模拟由个人开发的过敏性疾病的过程中，由于过敏通常是系统性的，人们一般有两种过敏原特异性IgE和IgG抗体的循环。虽然IgE的交联是马云的主要手段ST细胞脱粒，肥大细胞也响应IgG抗体交联。因此，ACA模型允许研究人员进行调查的过敏性反应的机制，除了仅IgE介导的疾病。最后，虽然增感所用的方法是在研究者的判断，本地过敏测定法是通用的，并且可以与各种增感的方法中使用。 耳肿胀，常用的指标，以评估过敏性反应，也可以OVA激发后测量。我们已发现，耳挑战以OVA具有比耳挑战用PBS在1小时后喷射显著更加膨胀。与此相反的伊文思蓝的试验，我们已经观察到耳厚度测量比染料外渗并呈现研究者之间更大的可变性作为耳厚度可通过耳朵的压缩使用的测量设备时，可以改变较不敏感。 小鼠品系比其他BALB / c小鼠可用于局部过敏反应测定;然而吸光度读数会略有不同取决于所使用的菌株。因为他们的倾向发展强Th2应答的，BALB / c小鼠通常产生可靠的，可重复的读数与该测定。 此测定法可用于局部过敏反应的动物致敏多种抗原的定量。抗原的选择是灵活的，因为该测定法已被成功地与常见的过敏原，如OVA和匙孔血蓝蛋白（KLH）上进行。此外，代替致敏，局部过敏反应，可通过注射抗体引起的幼稚动物的交叉链接的IgE或IgE受体上嗜碱性粒细胞和肥大细胞，或通过对这些细胞如CD200R3 1结扎非IgE抗体激活受体。

I型超敏反应是由IgE介导的肥大细胞和嗜碱性粒细胞的表面上的抗原诱导的交联介导的。这导致细胞脱颗粒和血管活性和炎症介质如组胺，类胰蛋白酶，和血小板活化因子2的释放。以下预成形介体的脱颗粒过程中释放细胞，肥大细胞合成和释放前列腺素和白三烯，这进一步增加血管渗透性，3。最初的临床反应迅速发生，并且被称为"直接反应"。在皮肤中，风团和耀斑响应的抗原激发分钟内很容易地看到。根据这一挑战的剂量，就可以看到一个"晚期反应"几个小时后。后期肿胀是由 ​​于局部水肿和白细胞募集到组织2。组胺，一般被认为是主要的介体参与立即过敏性反应，作用于组胺受体1（HR1）表示对血管和组胺受体2（HR2）表达平滑肌。的联合作用增加了血流量和血管通透性在炎症4的位点。 多种过敏的动物模型已经发展为了研究参与过敏性炎症的机制，包括过敏性哮喘，全身性过敏反应，及本地过敏反应模型。静脉给药染料被用来衡量在动物模型中的局部过敏反应近一个世纪以来，随着出版物描述了这一技术可以追溯到20世纪20年代5。兔和豚鼠分别用于测试速发型超敏反应的第一个动物模型，以及最敏感的反应普遍发现在耳朵5,6。该法后来被证实为7大鼠和小鼠8使用。 从历史上看，各种实验方法已被使用，包括在注射之前，染料，染料注射之前注射抗原的注射抗原，以及同时注射的染料和抗原。静脉内给予染料作为测定过敏性反应的装置是一种多用途的测定法，它可以用于测量有源，无源，和反向被动反应的5,9。许多染料已被利用来评估过敏性反应，包括锥虫蓝，滂胺天蓝，伊文思蓝，嘉基蓝536，和印度油墨5,6,9。 0.5％伊文思蓝溶液是目前用于测定过敏性反应在皮肤上的标准染料。 挑战过敏性反应是短暂的;在10达到最大强度-染料注射15分钟，无反应是可见的，如果染料被施用多于30分钟挑战后，无论使用9的动物物种。染料外渗的定量原创LY获得通过测量风团大小的蓝色染料7-9所示。此外，脱颗粒的肥大细胞的数量可以通过从反应的部位切除的皮肤组织和甲苯胺蓝7染色来定量。肥大细胞脱颗粒经常被用来作为标记物用于皮肤，IgE介导的过敏性反应，如肥大细胞是表达高亲和力IgE受体了FcεRI主本地细胞群。分光光度技术测定染料外渗进入组织的被动皮肤过敏反应（PCA）在大鼠10和鼠标11，在20世纪90年代开发的。 以下本地过敏试验方案是改编自小岛等 。1，并利用鸡蛋卵白蛋白（OVA）作为抗原诱发过敏性反应。然而，可以使用的抗原相比OVA其他如果需要的话。该测定，然后使用伊文思蓝染料以监测变化的血管permeabilit发生由于肥大细胞的IgE交联和组胺释放年。

<table><tbody><tr><td>BALB/c Mice</td><td>The Jackson Laboratory</td><td>651</td><td></td></tr><tr><td>PBS pH 7.4</td><td>Quality Biologic</td><td>114-058-101</td><td></td></tr><tr><td>Ovalbumin</td><td>Sigma</td><td>A5503-10G</td><td></td></tr><tr><td>Imject Alum</td><td>Thermo Scientific</td><td>77161</td><td>Mix thoroughly before use</td></tr><tr><td>Evans Blue Dye</td><td>Sigma</td><td>E2129</td><td></td></tr><tr><td>Formamide</td><td>Sigma</td><td>295876</td><td>99%+ Spectrophotometric grade</td></tr><tr><td>Isoflurane, USP</td><td>Phoenix</td><td>NDC 57319-474-06</td><td></td></tr><tr><td>1cc Insulin syringes</td><td>BD</td><td>329654</td><td></td></tr><tr><td>3/10 cc Insulin syringe with 31 G needle</td><td>Terumo</td><td>NDC 100861</td><td></td></tr><tr><td>27 G Needles</td><td>BD</td><td>305109</td><td></td></tr><tr><td>Forceps</td><td>F.S.T.</td><td>11000-12</td><td></td></tr><tr><td>Surgical scissors</td><td>F.S.T.</td><td>14070-12</td><td></td></tr><tr><td>5 ml Polystyrene round-bottom tubes</td><td>BD Falcon</td><td>352058</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tubes</td><td>Medical Supply Partners</td><td>15-1151</td><td></td></tr><tr><td>15 ml Conical tubes</td><td>BD Falcon</td><td>352097</td><td></td></tr><tr><td>Flat-bottom 96 well plate</td><td>Costar</td><td>3590</td><td></td></tr><tr><td>Scotch tape</td><td></td><td></td><td></td></tr><tr><td>RC&lt;sup&gt;2&lt;/sup&gt; Rodent Anesthesia System</td><td>VetEquip</td><td>922100</td><td></td></tr><tr><td>Vortex Genie 2</td><td>Scientific Industries</td><td>SI-0236</td><td>Model G560 with 3 inch platform</td></tr></tbody></table>

measuring local anaphylaxis in mice

过敏性反应是肥大细胞和嗜碱性粒细胞，并随后释放血管活性和促炎介质的活化的结果。暴露于变应原的致敏个体可能会导致因不同轻微的红斑到危及生命的过敏反应临床症状。在实验室中，各种动物模型已经发展到理解驱动过敏性反应的机制。在这里，我们描述了用于测量变化，血管通透性量化局部过敏性反应的详细方法。局部过敏反应测定最早是在20世纪20年代的报道，并已改编自Kojima 等人发表的技术。在2007年1，在该试验中，敏化，以OVA小鼠左耳用车辆和在右耳用OVA激发。这后面是静脉注射伊文思蓝染料。注射伊文思蓝后十分钟，将动物安乐死，并在已渗出的染料到耳朵在一夜之间甲酰胺提取。所提取的染料的吸光率，然后定量用分光光度计。这种方法确实导致的局部过敏反应的可视化和可量化的表现。

已成功进行了试验动物都会有皮肤和眼睛呈现蓝色。 PBS致敏动物不应反应以PBS或OVA激发，因此两个耳朵应保持白色（ 图1A）。 OVA致敏的动物中，耳朵接收PBS挑战（左）应该是全白或在注射部位的局部的方式轻轻蓝色。耳接收OVA激发（右）应逐渐变暗的蓝色染料施用（ 图1B）之后的10分钟期间成为。 PBS致敏的动物将具有可忽略的吸光度读数为与PBS和OVA挑战（ 图2A）。对OVA致敏的动物中，注射PBS通常导致0.13的平均吸光率读数和0.04的标准偏差。相反，50微克OVA激发产生的0.58的平均吸光度读数和0.18的标准偏差（ 网络gure 2B）。在用于挑战OVA的量，如果必要，可以调整单个实验。例如，有20微克OVA激发的结果在0.30平均吸光度和0.10（ 图2C）的标准偏差，但不到20微克挑战可能不能给出可靠的结果。变化OVA浓度必须优化和使用之前进行验证。 结果可以用图形配对，用PBS吸收相比，OVA吸收。可替换地，结果可以被绘制成一个单一的值，用PBS中的吸光度从OVA吸光度为每个单独的动物中减去。 <img alt="图1" src="/files/ftp_upload/52005/52005fig1highres.jpg" /> 图1:在PBS和OVA致敏动物耳色素沉着。 （一）。在PBS耳色素增感型的动物。左耳（挑战用PBS）和右耳（挑战50微克OVA）显示几乎没有染料外渗（B）。耳色素沉着在OVA致敏动物。有在左耳（挑战以PBS）无过敏性反应，通过不存在的蓝色染料的外渗所指示的。右耳（挑战用50微克OVA）表示指示血管通透性增加，细胞活化中的耳组织的直接结果，一个明亮的蓝色着色。 <img alt="图2" src="/files/ftp_upload/52005/52005fig2highres.jpg" /> 图2:在OVA致敏动物代表的OD值在620nm处采取分光光度测量得到的OD值（A）。 50微克OVA激发的PBS致敏动物。为PBS中，OVA激发的OD值是可忽略的（B）。 50微克OVA挑战OVA致敏动物。 PBS挑战结果OD值&lt;0.2和OVA挑战地区环境部门一道在OD值LTS&gt; 0.3（C）。 20微克OVA挑战。 PBS挑战结果OD值&lt;0.2。右耳朵已经略有减少，由于OVA的挑战较小剂量使用OD值。

Watch this Scientific Journal Video about Measuring Local Anaphylaxis in Mice at JoVE.com

Measuring Local Anaphylaxis in Mice

过敏性反应，其特征在于由肥大细胞和嗜碱性粒细胞的活化，是由交联的IgE和炎症介质的释放驱动。过敏性反应的定量评估可以用伊文思蓝染料监察过敏原刺激后改变血管通透性来实现。

小鼠量度本地过敏反应

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

19.2K 次观看.  Uniformed Services University of the Health Sciences. 过敏性反应，其特征在于由肥大细胞和嗜碱性粒细胞的活化，是由交联的IgE和炎症介质的释放驱动。过敏性反应的定量评估可以用伊文思蓝染料监察过敏原刺激后改变血管通透性来实现。 

视频: Measuring Local Anaphylaxis in Mice

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, ...

科研

免疫与感染

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

局部或全身药物干预后评估的变化小鼠中挥发性全身麻醉药敏感性

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

医学

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

神经科学

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

生物学

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

抗原脂质体用于生成特定于疾病的抗体

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

红外测温仪在被动系统过敏反应和食品过敏评价中的应用

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

用于同时测量疼痛和瘙痒相关行为的切面注射模型

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-&#947;- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified ...

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day "0", mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day "4", the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into na&#239;ve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.

Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.

接触超敏反应作为过敏性接触性皮炎的小鼠模型

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an ...

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a common skin disease that manifests as a local rash, redness, and skin lesions. The occurrence and development of ACD are influenced by both genetic and environmental factors. Although many scholars have constructed a series of models of ACD in recent years, the experimental protocols of these models are all different, which makes it difficult for readers to establish them well. Therefore, a stable and efficient animal model is of great significance to further study the pathogenesis of atopic dermatitis. In this study, we detail a modeling method using 1-fluoro-2,4-dinitrobenzene (DNFB) to induce ACD-like symptoms in the ears of mice and describe several methods for assessing the severity of dermatitis during modeling. This experimental protocol has been successfully applied in some experiments and has a certain promotional role in the field of ACD research.

Here, we describe the methods for inducing allergic contact dermatitis in mouse ears by 1-fluoro-2,4-dinitrobenzene (DNFB) and how to evaluate the severity of allergic contact dermatitis.

用于过敏性接触性皮炎评估的小鼠耳模型

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a ...