作者要感谢MALINDA轻快的技术援助。我们也承认目前和过去​​资金来源：美国国家癌症研究所（R01 CA131075，R01 CA131075-02S1），温迪威尔案例癌症研究基金，马基癌症基金会，儿童奇迹网络和珍妮弗与大卫·狄更斯黑色素瘤研究基金会。

1。从左手石竹（Cohleus毛喉）植物的粗根提取物制剂毛喉素用于局部给药的<ol><li>协议对小鼠实验其次为动物的护理和使用道德行为的准则和批准的机构动物护理和使用委员会在肯塔基大学（协议＃00768M2004）。根提取物是由在40％重量/体积70％乙醇中的一个标准皮肤病学的基础上，30％的丙二醇。 </li><li>权衡将200g粗福斯克林根提取物，并将其转移到烧杯中。制成500毫升40％（重量/体积）溶液，重悬将200g粗福斯克林根提取物，加入大部分但不是全部车辆的体积（70％乙醇，30％丙二醇），并把该溶液至大约450毫升。 </li><li>搅拌一个小时在室温下。该解决方案将有点粘稠，可能需要手动搅拌以“升降机”提取到溶液中之前搅拌棒是能够接管​​。 </li><li>搅拌一小时后，混合物倒入量筒中，并使用车辆已被用于“漂洗”，是用来搅拌的浆料中的烧杯中（最大限度地从烧杯中恢复毛喉素），使体积至500ml 。 </li><li>将浆料转移至50ml聚丙烯离心管中。离心（1,500×g离心，室温，15分钟）使用台式离心机。在这一点上，不溶性材料将是相当密实，使上清液容易被倒出。 </li><li>通过0.22微米乙酸纤维素膜过滤该溶液，以从提取物中除去残留的不溶性物质。我们使用的瓶顶系统设计用于细胞培养，随着使用的预过滤器来与单元以防止从根提取物的不溶性组分的膜过早堵塞。当使大量的提取物，过滤大致100毫升在一个时间，改变了预过滤器的选择吐温每个添加量。 </li><li>当在室温下保存，提取物保持生物学活性高达长达一年。 </li></ol> 2。用于局部治疗制备C57BL / 6 K14-SCF小鼠<ol><li>电动剪切从动物取出背毛皮。简要麻醉动物吸入异氟醚，方便背部皮毛配备0.25mm的手术预备头（飞世尔科技）电热剪的剪切。最好只使用一种类型的麻醉（ 如氯胺酮/甲苯噻嗪），以尽量减少麻醉药过量的风险。饱和吸入室进行职业风险时，通风橱外使用，并提供未知量的麻醉剂的动物。理想的情况是一个精密蒸发器应该被使用。 </li><li>以去除残留的毛茬，善待动物用化学脱毛剂。管理麻醉的动物用腹腔注射氯胺酮40 mg / kg和甲苯噻嗪4毫克/千克</li&gt; <li>一旦动物有足够的麻醉（用脚趾捏来判断），适用的脱毛霜指尖大小的量，用戴手套的手指剪切背侧皮肤。擦眼霜渗入皮肤30-60秒或直到毛发可以清楚地在奶油被看作是它被搬来搬去。离开上霜只对需要的时间脱毛的最小量为长时间暴露引起的皮肤，表皮破裂和死亡的表皮完整性损失的化学燃烧。 </li><li>反复擦拭背部皮肤用清水浸泡过的纱布垫，直到所有的冰淇淋已被删除。用柔软的纸巾，并允许他们在一个温暖的僻静的位置（ 如干净的笼子放置在一个变暖垫）恢复干燥的动物。脱毛的动物，一个接一个，整个手术过程中密切监测。 </li></ol> 3。毛喉素或车辆控制的局部给药<ol><li>动物应该在一个时间进行处理1。与吸入麻醉简要通过将鼠标上的下一个形状吻合尼龙透气式过滤器的顶部ð异氟醚已被放置的异氟醚饱和的纸巾在通风橱中的异氟醚的饱和带盖透明玻璃广口瓶中。暴露的小鼠对异氟烷足够的时间，以抑制自发肌肉运动，但保持自主呼吸（通常是10-20秒）。留在异氟醚的动物过长会导致呼吸抑制而死亡。这是更好地犯错“去光”，不得不重新暴露鼠标简单地多异氟醚，而不是动物过度暴露于药物和死亡风险的一面。 </li><li>从异氟醚室中取出的动物，并放置在干净的吸水垫板凳。 </li><li>使用1,000微升微量配备一次性聚丙烯尖，制定400微升40％的粗提取物毛喉素（车辆控制动物将获得70％的乙醇，独自一人30％丙二醇）。 </li><li>提取物转印到背面的动物通过它滴落到皮肤上，然后，用移液管尖端的侧面，涂抹提取在背部皮肤，直到所有的皮肤已经被覆盖。有没有必要向吸干涂布后的皮肤上。 </li><li>返回鼠标笼子里，并仔细观察，直到它从麻醉中恢复。 </li><li>为了使非色素cAMP的作用不应该混淆紫外线敏感性试验，停止所有的局部治疗前2天的紫外线照射（颜料效果持续数天超越去年局部治疗）。 </li></ol> 4。皮肤颜色测量由反射比色法<ol><li>简要麻醉小鼠吸入异氟醚（见上文）。 </li><li>通过将便携式头所提供的色度计标准化白色表面校准美能达色差。 </li><li>将色度计平齐的便携式测量头与该动物的背部皮肤确保1cm 2的圆形aperture的完全压在皮肤上。采取在背部皮肤的不同区域的至少三个单独的测量。 </li><li>计算平均长*得分±每只动物和每个治疗组的SD。反射色度可以在任何时候在实验中进行。 </li></ol> 5。由“最小红斑剂量”（MED）的计算测定紫外灵敏度<ol><li>使用已预先用载体或如上述毛喉素的动物。麻醉动物腹腔注射氯胺酮和甲苯噻嗪（见上文）的标准混合物。 </li><li>准备一张紫外闭塞磁带MED测试。以产生在带的孔中，使用了重型打孔器以1厘米2的圆形开口（ 图2A和B）。具有在带一个定义尺寸和对称布置的孔便于照射后识别的皮肤变化。多年来在磁带每孔，应用一个小而易于拆卸件<html>磁带，可以在规定的时间紫外线照射过程中被移除，以允许不同的紫外线剂量给药。 </li><li>一旦动物有足够的镇静，将磁带上的背水面。眼部润滑剂应始终在麻醉下利用。 </li><li>打开UV光源包括两个西屋F15T8UV-B灯与313纳米的峰值输出和一系列的280-370纳米。允许通过紫外分光光度计用紫外线传感器测得的灯，以平衡到一个恒定的紫外线输出（通常需要几分钟，让灯预热）。 </li><li>根据如通过UV光度计测得的紫外线透射率，计算出的紫外线照射时间为每个所需的剂量。例如，我们的灯的紫外线输出的措施2.4毫瓦/厘米2。因此，施用5千焦耳/米2，皮肤将需要暴露在208秒（这是3分钟，28秒）的UVB辐射，计算如下： upload/50670/50670eq1.jpg“/&gt; </li><li>将动物镇静剂（每到位闭塞磁带）腹面向下，以确保即使紫外线照射。施用所选择的剂量的紫外线辐射，依次取出小闭塞胶带覆盖该孔以露出1厘米皮肤的2个区域，以正确的剂量的辐射。因此，使用上面的例子，如果40千焦耳/米2是实验中的最大剂量，则该动物将灯泡下为27分钟和47秒的总和皮肤中的40千焦耳/米2的条件就没有覆盖磁带的整个时间。但是，磁带上覆5千焦/米2的条件时，会出现208秒剩余曝光被删除。胶带去除时机应该做的，让每个条件同时结束。 </li><li>紫外线照射后，撕下胶带小心地从背部皮肤，注意不要撕裂突然或过度有力的运动的皮肤。将动物在温暖安静的地方，让麻醉恢复期。 </li><li>监测小鼠为24-48小时，以寻找红斑（发红）或水肿的离散区域（肿胀）对应于暴露于UV辐射的特定剂量的解剖部位。照相记录皮肤的发现。 </li><li> MED值对应于最小剂量的紫外线红斑和/或皮肤的整个暴露圆的水肿定义，导致炎症。需要注意的是皮肤的色素沉着可以挑战的决心MED，然而，红斑和水肿仍然可以大致准确地评估，这要部分归功于在磁带上的小孔中的紫外线照射过程中定义的形状。 </li></ol> 6。统计分析通过单因素方差分析和Bonferroni事后检验（图垫棱镜）。P值 &lt;0.05被认为是统计学显著分析老鼠的同伙之间的数据。

作者宣称，他们有没有竞争的财务权益。

使用皮肤白皙的人的动物模型，我们发现一个毛喉素富含粗根提取物的局部应用强劲，刺激黑色素的生成在皮肤变暗的表皮。表皮黑色素是依赖于干细胞因子在基底表皮的表达，如发生在人类皮肤，但不能在基因未修饰小鼠皮肤。基因未修饰小鼠的背部皮肤缺乏足够数量的滤泡黑色素细胞以赋予颜料在皮肤上。只在黑素细胞生长因子，如干细胞因子（kit配体）或肝细胞生长因子（HGF）的组成型表?...

黑色素瘤的发病率，皮肤癌的最致命的形式，也大幅增加，在过去几十年在美国，尤其是皮肤白皙的人。强烈的分子和流行病学证据牵连紫外线辐射为主要致病环境因素2-5。在阳光下暴晒和晒黑床使用的形式增加紫外线照射很可能是负责大部分的增加黑色素瘤的发病率6-7。黑色素瘤的风险似乎与晒伤8，尤其是那些生活在9-10月初特别的联系。晒伤的风险被链接不仅剂量和紫外线照射的强度，而且还通过影响紫外线辐射的皮肤反应遗传因素。皮肤色素沉着是一种UV灵敏度的最重要的决定因素，晒伤和癌症风险的风险。黑色素瘤约20倍更频繁地发生在浅肤色的人相比，皮肤黝黑的individuaLS 11-13。 黑色素，由黑素细胞在表皮中产生的颜料，是肤色的主要决定因素。黑色素有两个主要品种有：（1）真黑素，黑棕色/黑色颜料在吸收UV辐射的能量有效的，并且（2）褐黑素，红/金色颜料防止紫外线光子的穿透入皮肤效果较差。肤色，紫外线的敏感性和黑色素瘤的风险在很大程度上是由表皮黑色素含量14-15决定。在表皮中的黑色素多，越少的UV光子可以渗透进皮肤。因为真黑素的先天低层次的，皮肤白皙的人对紫外线辐射16-18急性和慢性影响更容易发生。 皮肤色素沉着，黑色素瘤的风险，并有能力“晒黑”的紫外线照射后，所有与关联的黑素皮质素受体​​1（MC1R）的信令能力，一个G S耦合的七次跨膜s对黑素细胞19-22 urface受体。当MC1R结合其同源的高亲和力配体，α-黑素细胞刺激激素（α-MSH），有活化腺苷酸环化酶的第 ​​二信使cAMP 23的制作中。紫外线照射后皮肤的正常生理反应包括表皮生产α-MSH的由角质形成细胞24-29。我们和其他人推测，角质形成细胞源性α-MSH结合MC1R对表皮黑素细胞，通过激活腺苷酸环化酶30的启动下游生产内cAMP第二信使。 cAMP水平控制黑色素细胞分化的许多方面，包括存活途径，DNA修复和颜料的合成。 MC1R信号和cAMP诱导清楚色素酶水平和黑色素的生产。当MC1R信号是否完好及黑素细胞内cAMP水平是强大的，真黑素的产生和皮肤变暗。但是，如果MC1R信号是有缺陷的，并胞浆内cAMP水平仍然很低，褐黑素产生，而不是1。真黑素合成可以通过药理学的提高cAMP水平1,14,31-35剂刺激。 由于MC1R蛋白是在人类36-46黑素瘤风险的主要调节剂，我们感兴趣的是其中MC1R保护皮肤免受紫外线诱导黑素细胞癌变的机制。作为我们研究的基础，我们产生了转基因MC1R基因变体小鼠模型在纯C57BL / 6遗传背景1。在这个模型中，干细胞因子（SCF）的组成型表达在基底表皮和表皮黑素细胞滤泡被保留在整个生命47的皮肤，而相比之下，在非转基因小鼠，其中黑素细胞定位于毛囊真皮。与K14-SCF基因掺入，表皮变得与颜料的特定黑色素着色特性动物1株。K14-SCF小鼠对C57BL / 6遗传背景与野生型MC1R信号已经乌黑的皮肤特点是非常高水平的真黑素色素。毫不奇怪，这些动物是高度抗紫外线。相比之下，基因匹配K14-SCF的C57BL / 6动物怀有一种突变无效的MC1R在表皮几乎没有黑色素。相反，这些“ 延伸 ”的动物（MC1R E / E）具有引起的褐黑素色素（ 图1A）沉积公平肤色和更为紫外线敏感48-49。 与化学特性，使渗透到皮肤药物化合物已被证明直接操作cAMP水平的表皮黑素细胞在皮肤有效地诱导黑色素的扩展名（MC1R E / E）K14-SCF动物模型。在这个模型中黑色素的上调已经Ř通过腺苷酸环化酶激活1以及磷酸二酯酶4抑制35 eported。在这篇文章中，我们展示了准备并在扩展名（MC1R E / E）K14-SCF的动物模型中，皮肤白皙紫外线敏感的人毛喉素局部应用。我们表明，该药物每日两次应用促进加速黑色素，即皮肤变黑是由于黑色素的表皮沉积而引起的表皮黑色素防止紫外线引起的晒伤通过“最小红斑剂量”（MED）48的测量值。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>Coleus Forskoli extract 20%</td>
			<td>Buckton Scott USA Inc. </td>
			<td>n/a</td>
			<td>Princeton, NJ</td>
		 </tr>
		 <tr>
			<td>Isothesia, Isoflurane , USP</td>
			<td>Butler Schein</td>
			<td>NCD 11695-6776-1</td>
			<td>Dublin, OH, USA</td>
		 </tr>
		 <tr>
			<td>Xylazine</td>
			<td>Anased Injection</td>
			<td>LA04612</td>
			<td>Shenandoah, Iowa, USA</td>
		 </tr>
		 <tr>
			<td>Ketamine HCl, USP</td>
			<td>Putney</td>
			<td>NDC 26637-411-01</td>
			<td>St. Joseph, MO, USA</td>
		 </tr>
		 <tr>
			<td>Ethanol</td>
			<td>Decon Labs.</td>
			<td>2705</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Propylene glycol</td>
			<td>Adesco</td>
			<td>05751L</td>
			<td>Solon, OH, USA</td>
		 </tr>
		 <tr>
			<td>Depilatory cream, Nair</td>
			<td>Church &amp; Dwight</td>
			<td>JF-11 4381322</td>
			<td>Priceton, NJ</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Germicidal Hg Lamp UV-B</td>
			<td>Westinghouse</td>
			<td>F15T8UV-B</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Radiometer photometer</td>
			<td>International light</td>
			<td>1LT400A</td>
			<td>Peabody, MA,USA</td>
		 </tr>
		 <tr>
			<td>Chromameter</td>
			<td>Konica Minolta</td>
			<td>CR-400</td>
			<td>Ramsey, NJ, USA</td>
		 </tr>
		 <tr>
			<td>Data Processor for 	Chromameter CR-400</td>
			<td>Konica Monilta</td>
			<td>DR-400</td>
			<td>Ramsey, NJ, USA</td>
		 </tr>
		</tbody>
 </table>

pharmacologic induction of epidermal melanin and protection against sunburn in a humanized mouse model

公平性皮肤，紫外灵敏度和皮肤癌的风险的所有关联与的黑皮质素-1受体的生理功能，黑素细胞的表面上发现了G S耦合信号蛋白。 MC1R刺激腺苷酸环化酶和cAMP的产生，这反过来，上调黑色素细胞黑色素的产生在皮肤上。为了研究由MC1R信令保护皮肤免受紫外线伤害皮肤的机理，本研究依赖于小鼠模型与基于干细胞因子（SCF）的表达表皮“人源化的皮肤”。K14-SCF基因小鼠黑素细胞保留在表皮，因此具有沉积黑色素在表皮的能力。在这个动物模型，在黑色的真黑素色素和UV保护型健壮的沉积野生型MC1R状态结果。与此相反，K14-SCF动物有缺陷的MC1R信令能力表现出红色/金色的色素沉着，很少真黑素在皮肤和一个对紫外线敏感的表型。理由是真黑色素的沉积可能是由局部用药，模仿MC1R信号增强，我们发现直接应用毛喉素提取物对MC1R基因缺陷的皮肤白皙的小鼠的皮肤造成了强大的真黑素诱导和防紫外线1。在这里，我们描述了制备和应用含毛喉素天然根提取物对K14-SCF皮肤白皙小鼠及报告，通过测定最小红斑量（MED）测定紫外灵敏度的方法的方法。使用该动物模型，能够研究的皮肤表皮的cAMP的诱导和黑化如何影响生理反应，以UV曝光。

上结合了K14-SCF基因所描述的（ 图1A）eumelanotic，pheomelanotic或无色素的背景中产生C57BL / 6小鼠。皮肤白皙的扩展同伙（MC1R E / E，酪氨酸+ / +）小鼠，每日两次剂量溶媒（70％乙醇，30％丙二醇）或40％的粗毛喉鞘蕊根提取物（每80微米的局部治疗剂量），连续5天（ 图2B）。对表皮色素沉着的局部治疗效果进行既通过目测和通过反射色度（ ?...

Watch this Scientific Journal Video about Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model at JoVE.com

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

表皮黑色素是由毛喉素局部应用在皮肤白皙的紫外线敏感的人小鼠模型诱导。 cAMP水平的皮肤和表皮发黑免受紫外线介导的炎症（晒斑）强烈保护如由最小红斑量（MED）法测定的药理操纵。

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

pharmacologic-induction-epidermal-melanin-protection-against-sunburn

Research

JoVE Journal

Medicine

18.0K 次观看.  University of Kentucky College of Medicine. 表皮黑色素是由毛喉素局部应用在皮肤白皙的紫外线敏感的人小鼠模型诱导。 cAMP水平的皮肤和表皮发黑免受紫外线介导的炎症（晒斑）强烈保护如由最小红斑量（MED）法测定的药理操纵。 

视频: Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

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

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

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

舌下免疫治疗作为一种替代起到保护作用对急性呼吸道感染

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

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

建立和UTI和CAUTI表征在小鼠模型

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

干细胞基工程免疫抗HIV感染的人性化小鼠模型

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

利用人体肝干细胞人源化小鼠肝脏生成

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导："电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

活体显微镜及血栓诱导在无毛小鼠的耳垂

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

小鼠脑海绵体畸形的诱导及显微 CT 成像

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...