这项工作得到了LSUHSC基金会（PLF）的支持，部分由美国国家卫生研究院国家普通医学科学研究所U54 GM104940支持，该研究所资助路易斯安那州临床和转化科学中心。

所有涉及尾静脉注射和使用加热/抑制装置的动物协议均经过当地机构动物护理委员会 （IACUC） 的审查和批准。
1. 准备
<ol><li>在居住环境中对动物进行至少1周的适应，并允许食物和水。 注:对于这种注射技术的大多数新用户来说，带有白色或浅色毛皮的动物菌株可能更可取，因为尾静脉通过皮肤很容易看到。深色小鼠（例如C57BL/6）或大鼠（例如挪威布朗）的深色菌株有深色染色尾巴，导致与静脉的颜色对比较弱。强烈建议新用户接受充分培训，直到达到熟练程度。</li>
	<li>尾静脉注射剂<ol>
<li>无能地准备所有测试代理和解决方案。在管理生物体或细胞材料时，在加工的所有步骤中采取预防措施，以保持无丙原的条件。</li>
		<li>仅使用普通盐水（0.9% w/v 氯化钠）或平衡盐溶液（如磷酸盐缓冲盐水 （PBS） 作为尾静脉注射的车辆。 注意:切勿使用水、油或粘性溶液，因为存在血管损伤的潜在风险。由于血液的缓冲作用和啮齿动物的快速血流量，广泛的pH值（4.5-8.0）是可以容忍的。然而，高酸性或碱性溶液可能导致注射部位不必要的组织损伤，应避免。</li>
		<li>将注射的体积和频率限制在最低限度。注射前在体温下使用小鼠和大鼠推荐的体积（分别为≤200微升和≤500微升），以尽量减少对动物的压力。</li>
		<li>确保每个注射器和针头的制备液均无溶液中的气泡:如果存在气泡，则完全清除它们，以防止栓塞的风险。 注:通常，1 mL注射器与27G，1/2针是足够的大多数尾静脉注射。</li>
		<li>使用当地 IACUC 要求的适当的个人防护装备 （PPE），至少配备一次性或专用礼服、乳胶或亚硝酸盐手套。在进行尾静脉注射时，强烈建议使用安全眼镜。</li>
	</ol>
</li>
	<li>加热和约束装置<ol>
<li>使用前仔细检查所有组件，以确保设备没有任何缺陷（图1）。</li>
		<li>加热设备初始化 （图 2A）<ol>
<li>将加热装置放在干净的平台式台面上，并打开设备电源。确保恒温器功率指示灯呈绿色照明。将床上用品放在加热室内，以保持区域干燥并保持热量。</li>
		</ol>
</li>
		<li>约束装置设置 （图2B）<ol>
<li>将约束单元与加热单元放在一起，并确定动物的相应锥体大小。如有必要，手动调整柔滑铝锥的基宽，为动物提供足够的约束。或者，用定制的模型替换圆锥体，以容纳不同体型的小鼠或大鼠。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 尾静脉注射
<ol><li>设备调整<ol>
<li>设置内部温度<ol>
<li>使用控制表盘，将恒温器设置在所需的温度下。确保加热器指示灯呈红色，灯泡亮起。灯泡照明（加热）时，小心监控内部温度显示。恒温器在达到目标温度后自动灭活灯泡，大约在 10 到 15 分钟内。 注意:设置高于环境温度的温度会激活加热器。一般来说，建议在标准病毒条件下的住房温度不同，从20至26°C不等，而中性（即舒适）温度的实验鼠被认为是在30至32°C42之间。因此，建议加热室的内部温度略高于热中性，大约在 32°36 °C。 切勿将恒温器设置在体温之上。</li>
		</ol>
</li>
		<li>定位约束平台<ol>
<li>使用高度调整旋钮，将圆锥体高度调整为用户的最佳水平。</li>
		</ol>
</li>
	</ol></li>
	<li>热处理 （图3A）<ol>
<li>一旦达到目标温度（32°36°C），轻轻地将动物从笼子转移到暖室。 注意:5 至 10 分钟的热处理足以诱导血管变异，提高尾静脉的可见性。然而，在手术过程中，动物可以安全地被关在热调节室中（通常为20至30分钟，没有体温过高的迹象）。加热室可以安全地容纳4至6只老鼠或一只老鼠。</li>
		<li>监测动物是否有急性热应激的迹象（例如，快速呼吸、嗜睡、跳跃逃生行为）。 警告:出现体温过高迹象的动物应返回笼中并受到监测，直到它们在重复使用之前恢复正常活动。如果这是因为内部温度超过最佳范围，请确保加热设备关闭。</li>
	</ol></li>
	<li>注射步骤<ol>
<li>将动物从尾巴底部抬起来，从加热室中取出。将动物引入约束单元的锥体开口。 注意:切勿将老鼠从尾端抬起来:这可能导致严重的伤害。其他处理方法应用于肥胖或怀孕的小鼠28。</li>
		<li>当动物用前体抓住圆锥体的远边缘时，轻轻地向后拉尾巴，通过开阔的缝隙穿过尾巴。将动物的后端固定在圆锥体底部，一条后腿从圆锥体中伸出，使横静脉显示在 12 点的位置。任何后腿都可以突出，因为有两个侧静脉，每侧一个（图3B）。</li>
		<li>用拇指和食指之间的非主导手抓住中到三分之二长度的尾巴，在横静脉上施加轻微的张力，以保持尾部定位和血管增生。 注:通过热处理增强扩张静脉的可见性，使用户能够快速确定注射部位以获得最佳效果（图4）。</li>
		<li>用纱布海绵擦拭注射部位的皮肤，或用 70% 酒精滋润垫。尽可能轻柔和快速地清洁，以避免对尾部发炎。 注:这一程序可以由机构IACUC酌情省略。</li>
		<li>用占主导地位的手握住注射器，将针头平行于尾部。将针头插入血流方向，以 10+15° 角（图 5A+B）向上倾斜，通过穿透 2+4 mm（图 5C+D）进一步进入静脉的流明。慢慢注入溶液。 注:如果注射成功，则不应感觉到柱塞上的阻力，并且可以看到液体通过静脉移动。如果在注射部位上方出现阻力或白色水泡，请取出针头，并在原始针位上方的部位尝试第二次注射。不要尝试在初始注射部位下方注射，因为液体会通过初始部位释放。如果注射一个侧静脉不成功，将动物重新定位到另一侧，并在逆静脉上进行更多的尝试。尝试的最大次数将取决于尝试注射沿静脉和肿胀可能发生在错过尝试。咨询国际民航联合会关于误射和相关伤害的机构条例。</li>
		<li>取出针头，用拇指牢牢按压，以防止注射溶液和/或血液回流。继续使用干净的纱布/擦拭或组织进行温和压缩，直到出血停止（图6）。</li>
		<li>将动物送回笼子，并监测至少5分钟。确保动物恢复正常活动，不进一步出血。</li>
	</ol></li>
</ol>3. 真菌血流感染和败血症的穆林模型
<ol><li>鼠标菌株<ol>
<li>根据机构推荐的指南，瑞士雌性韦伯斯特在6周大时繁殖了小鼠。或者，使用近亲繁殖/转基因菌株（例如，C57BL/6 背景）使用此协议与改良的内在球菌（见注）。 注（见讨论细节）:由于深色色素的尾巴，有深色毛皮的小鼠的尾部静脉往往比有较浅毛皮的小鼠更不明显（图4）。不同小鼠菌株对真菌败血症/致命性的影响各不相同。使用瑞士韦伯斯特以外的小鼠菌株可能需要额外的协议优化，考虑可能影响宿主免疫状态的相关因素（例如遗传背景、年龄、性别、体型）。例如，C57BL/6小鼠的致命挑战通常需要更高的脑球菌（高达10倍）才能达到瑞士韦伯斯特小鼠的死亡率水平。</li>
	</ol>
</li>
	<li>微生物<ol>
<li>对于致命的挑战 （败血症）， 条纹冷冻库存 的念珠菌 株 DAY185 （或选择菌株） 到萨博劳德克斯特罗斯阿加， 并在 30 °C 孵育 2 天.</li>
		<li>将单个菌落转移到 10 mL 酵母提取物 - 辣椒 - 德克斯特罗斯肉汤中，并在 30 °C 下摇晃时将培养物转移到 18 小时的静止生长阶段。</li>
	</ol>
</li>
	<li>接种解决方案<ol>
<li>在致命挑战的那天，收集肉汤培养物，用无菌PBS离心（800× 克）清洗颗粒3次。</li>
		<li>通过尝试蓝色染料排除来识别可行的酵母细胞，并使用血细胞计进行列举。在室温下将细胞浓度调整到 1 x10 6 细胞/mL 无菌 PBS 中。 注:每只动物将收到100微升的接种液。准备过量的输液（>500微升），以允许注射过程中的潜在损失。最后的接种是每只小鼠1×105 个细胞。通过相应地调整细胞浓度，可以增加高达 200 μL 的接种量。 警告:注射前必须在室温下保存真菌接种液。将接种液加热到体温可能会诱发从酵母细胞到催眠细胞的形态变化。相反，冷溶液的施用可以迅速降低动物的体温，应避免。</li>
	</ol>
</li>
	<li>静脉接种<ol>
<li>加热动物，并按照第2节的程序诱导血管变异。</li>
		<li>使用 1 mL 注射器和 27 G 1/2 英寸针头将 100 μL 的接种液注射到尾静脉中。</li>
	</ol>
</li>
	<li>接种后监测<ol>
<li>监测动物是否有以下败血症引起的发病率迹象:i） 毛皮方面（例如， 平滑、褶皱）、ii） 活动（例如，自由移动、无反应）、iii） 姿势（如驼背、僵硬）、iv）行为（例如，缓慢、无移位）、v） 胸部运动（如正常呼吸、呼吸困难）、vi）眼睑（如打开、关闭）43。</li>
	</ol>
</li>
	<li>败血症评分<ol>
<li>根据经修改的败血症（M-CASS）小鼠临床评估分数，在每个类别的四分评分表中对观察到的发病率进行评分:0，正常:1、温和:2、中度:3，严重43。</li>
	</ol>
</li>
	<li>可选协议:预防真菌败血症<ol>
<li>在致命挑战前14天，用Canada dubliniensis菌株Wé284或衰减的C.藻类菌株（如+efg1/+cph1突变体（每只小鼠1x105细胞）给小鼠接种疫苗，如第3.2 3.4节（代替C.藻类DAY185）。</li>
		<li>如第3.2-3.4节所述，对接种疫苗的小鼠进行致命挑战，并监测第3.5-3.6节中描述的败血症引起的发病率的迹象。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

一致和准确的剂量是动物模型实验可靠性的关键要求。这一点在注射剂系统生物可用性远高于其他管理途径3的情况下尤为重要。因此，尾静脉注射的错误可能会对研究结果产生有害影响。从历史上看，腹腔注射（即p.）注射，而不是i.v.，由于技术简单和方便，一直是啮齿动物系统获取的最常见方法。然而，当将动物的临床前读数翻译成临床环境时，管理途径变得更加重要。因此，有必要不断改进啮齿动物协议，以促进成功的尾静脉注射。
本协议的关键进展是创新的热调节加热装置，能够有效地诱导啮齿动物的血管消耗，从而显著提高尾静脉和针线的可见性。温度调节不良的加热方法（如灯具）、局部血管消耗剂或皮肤刺激物（如二甲苯）不仅不可靠，而且对动物也不安全，应避免使用。与其他传统方法（如将尾巴浸入温水中）相反，该设备的自动调节能力可以同时安全地对多个动物进行调节。此外，使用设计最优化的约束装置，使动物处于最能显示横向尾静脉的位置，从而进一步加强了此协议。
透明的输卵管格式在许多目前的抑制器中看到，虽然实际上设计精良，需要更多的处理时间与每一个动物，从而延长约束过程45。这可能更成问题的啮齿动物菌株与侵略性特性，提供有限的合作46，47。相比之下，约束装置的半封闭锥形结构允许动物快速定位，并有助于最大限度地缩短约束持续时间。使用创新、高度优化的加热/抑制系统简化的方案共同加快了注射程序，允许快速有效地给大群动物剂量。在我们的实验室中，我们通常使用此协议在 1 小时内完成 30 只小鼠的整个注射过程，从热处理到注射后监测。
尽管具有先进的功能，但该设备有一些明显的缺点:第一个是设备的成本和加热室中常规灯泡更换的成本。但是，除了注射的效率和速度外，该设备还耐用，可重复使用，并且与大多数常见的消毒剂兼容，允许在使用之间彻底清洁设备。这抵消了最初的投资。在工作空间有限的情况下，本协议的缺点可能是要求专用板凳面积足够大，以便在进行注射时并排放置两个单元。但是，由于该设备可以在涉及 i.v. 注射的多个啮齿动物协议中广泛使用，因此该设备有可能作为类似于其他公用病毒设备（如异黄酮蒸发器）的核心仪器。无论如何，这两个单位很容易携带，可以捆绑和存储，而不使用。
本协议中描述的真菌败血症的i.v.致命挑战模型与人类的C.藻类血液感染非常相似，并被广泛用于研究真菌毒性、测试抗真菌疗法的疗效，以及描述宿主对感染的免疫反应37、39、48。为了实现可重复的感染，即通过尾静脉注射接种是协议的最重要步骤，以确保生物体准确输送到血液中。事实上，动物对不同程度的念珠菌（即v.v.挑战）的反应大相径庭:接种过低的接种量会导致不必要的自发恢复，而接受过高剂量的动物会过早死亡。特定生物体的接种量导致败血症/死亡率的特定窗口在很大程度上取决于真菌株和小鼠菌株。
目前使用瑞士韦伯斯特小鼠在1×105野生型C.藻类的接种中的协议可在1天内重复诱发败血症发病，随后逐步死亡，导致100%的致死性5×7天。相比之下，高于1 x 105的脑膜通常会导致加速死亡（即1×106的1×2天，5×105的3×4天），低于1×105的亚致命性。根据文献中的许多报告，使用非阿尔比肯人念珠菌代替C.藻类导致杀伤力显著降低40，49。此外，小鼠菌株的选择，甚至菌落的起源，可以产生相当大的影响，由于小鼠菌株之间的怀疑性不同，如其他人报告39，40，41，50，51，52，53，54，55。因此，在设计实验时应考虑两者。
在致命的i.v.挑战之后，真菌细胞迅速通过血液传播，并开始侵入多个器官，其中受影响最大的是肾脏41。其他受影响的器官是大脑、脾脏和骨髓48，56。无论如何，急性败血症是早期死亡的根本原因37。如代表性结果所示，败血症的严重程度可由小鼠临床评估分数（M-CASS）根据被挑战动物43，57中表现出的败血症症状进行定量评估。在致命败血症的几个代孕标记中，体温过低被认为是临床和实验败血症43、58、59中即将死亡的关键预测。
虽然没有进行正式研究来直接比较本模型中的近亲繁殖小鼠与近亲繁殖小鼠，但从目前使用瑞士韦伯斯特近亲繁殖小鼠的协议获得的数据在各种败血症参数中具有特别可复制性，尽管假定遗传异质性。一般来说，在3至5天内死亡模式是急性败血症的坚定模型，败血症发病率的快速上升和致命后挑战50，51小时内的炎症标记水平就证明了这一点。在更长的生存时间（7至10天）中，死亡可能是微生物负担导致目标器官和中枢神经系统致命组织损伤的结果。选择败血症或微生物负担可以根据需要用于评估免疫功能或对抗炎方案或抗真菌疗法/疫苗的反应，由使用的接种决定。
除了i.v.致命挑战模型外，通过i.p.挑战在小鼠中感染C.藻类的腹内感染也可能导致传播的念珠菌病和随后的败血症，尽管与细菌病原体金黄色葡萄球菌共同接种，与C.藻类单感染51、60、61相比，协同提高了死亡率。在i.p.致命挑战模型中，需要高得多的微生物内皮（1.75 x 107C. 藻类/8 x 107S. aureus每只小鼠）引起多微生物腹膜炎，并将生物体从腹腔传播到血液中。同样，用免疫抑制剂和/或粘膜损伤剂治疗的小鼠中对C.藻类的胃肠道感染会导致真菌细胞转移到血液中，并导致真菌败血症62，63。尽管独特的接种途径，随后的真菌败血症的机制在很大程度上类似于三种疾病模型，涉及一个不受控制的系统性宣泄反应，导致器官衰竭37，51，61。同样，在人类中，正是这种宿主反应的过程，而不仅仅是坦率的血症，导致高发病率/死亡率与血液传播的念珠菌病在卫生保健环境中获得64，65。
使用目前的真菌败血症模型，我们在这里证明，通过即v.免疫前/接种疫苗与C.杜布林西斯（病毒性）或衰减的C.藻类突变体，同时显著减少败血症发病率，可以达到预防致命的C.藻类感染。保护是由先天性的Gr-1-骨髓衍生抑制细胞，似乎诱导在骨髓作为训练有素的先天免疫66，67的一种形式调解。正在努力扩大对这种新的形式的与生俱来的免疫介质保护，防止C.藻类血液感染。
总之，创新的啮齿动物加热/抑制装置有助于提高我们高效、高效地进行大规模多组动物研究的注射能力。因此，我们为设备创造了"鼠标一分钟"一词。设备规格可在请求采购类似设备时从相应作者处获得。这里展示的技术可以作为啮齿动物模型的有用工具，在广泛的研究领域使用尾静脉注射。

使用涉及啮齿动物的动物模型一直是生物医学研究的主要内容。世界各地实验室都有许多近亲繁殖和近亲繁殖的菌株以及转基因系。尾静脉注射是啮齿动物模型中需要静脉注射（即静脉注射）实验剂的基本方法之一。一般来说，即注射比其他管理途径有重大优势，如高吸收率绕过局部组织和消化道和高耐受性溶液的浓度范围广泛或非生理pH值1，2，3，4。在其他可行的即v.路线（例如，鼻窦，复古轨道静脉窦），尾静脉被认为是最安全和最容易获得的血管在啮齿动物2，3，5，6。因此，尾静脉注射已广泛应用于一系列啮齿动物模型，包括传染病模型7，8，9，生物材料移植10，11，临床前治疗评估12，13，毒理学分析14，15。
剂量的一致性和准确性是成功尾静脉注射的关键要求。令人惊讶的是，文献中尾静脉注射的定量和定性评价涉及频繁误注射16，17。一项研究报告说，训练有素的注射者进行的30次注射中，有12次在18号尾部留下了超过10%的注射剂量。此外，接受尾静脉注射的动物的安全性和舒适性应是手术过程中的首要问题。不适当的克制可能导致伤害和一系列与压力相关的病理（例如，体重减轻，免疫反应受损），这可能在样本质量19，20中引入大量变量。这些错误会导致数据变异性增加和可重复性差，从而对研究结果产生负面影响。
由于血管直径小，在动物体内进行尾静脉注射时，通常需要诱导血管扩散，估计在21号小鼠中为300μm。血管吸附可提高尾静脉的可见性，有助于在静脉流明内实现最佳的针静脉对齐。实验室报告了各种方法，如将尾巴浸入温水中22，使用暖窗帘、灯或吹风机23、24将热量施加到尾巴上，或使用加热垫、孵化器或盒子将动物置于温暖的环境中，并结合其中一种热源25。这些设备可以是针对特定目的自行制造的，也可以从商业供应商那里购买。然而，许多人缺乏热调节能力，如果有的话，设备温度维护不善，经常受到室温变化的影响。同样，使用抑制装置是必要的尾静脉注射，因为不建议使用麻醉26，27。已开发了几种类型的实验室特定或商业限制装置。通常，动物被放置在一次性50毫升圆锥管4，槽丛玻璃墙，隧道，或圆锥28，所有这些都允许充分暴露的尾巴，同时限制动物的运动。但是，由于材料的刚性，大多数限制器具有尺寸限制。此外，现代高复杂度设备，尽管实用和复杂的设计，似乎不可行注射涉及大群动物22。
小鼠模型的血液感染和相关的败血症是需要使用这种技术的情况的一个典型例子。在所有严重临床败血症的微生物病理学中，真菌败血症往往是一种致命的疾病，尽管抗真菌疗法29，但死亡率仍为>40%。事实上，由念珠菌感染已被报告为第四大原因的医院获得的血液感染（念珠菌血症）30，31。在腹内念珠菌病中，胃肠道中的微生物可以通过血液传播，导致多微生物败血症，死亡率更高，为32、33、34。由于大多数结膜性贫血病例来自受污染的中线导管或内聚医疗器械35，36，即通过尾静脉注射与C.藻类接种可以密切反映人类败血症的发展，并已成为血吸虫病37，38小鼠模型的主要方法。在这个模型中，通过调整C.藻类，即39，40，41，在数天内发生的死亡率可以延长或缩短。
最近，我们的实验室开发了一个创新方案，使用一种创新设备，配备热调节加热装置，配以可调节约束装置，在一个方便的系统中优化流线型尾静脉注射。该协议允许研究人员准确、及时地进行尾静脉注射，而动物可以安全地进行条件和约束，以最小的痛苦进行手术。这里展示的技术，使用先进的加热和抑制装置，可以作为一个有用的工具，在各种研究领域使用啮齿动物模型。

<table>
	<tbody>
		<tr>
			<td>Candida albicans strain DAY185</td>
			<td>Carnegie Melon University</td>
			<td>N/A</td>
			<td>provided by the laboratory of Aaron Mitchell</td>
		</tr>
		<tr>
			<td>Candida albicans strain efg1&#916;/&#916; cph1&#916;/&#916;</td>
			<td>University of Tennessee Health Sciences Center</td>
			<td>N/A</td>
			<td>provided by the laboratory of Glen Palmer</td>
		</tr>
		<tr>
			<td>Candida dubliniensis strain W&#252;284</td>
			<td>Trinity College, Dublin, Ireland</td>
			<td>N/A</td>
			<td>provided by the laboratory of Gary Moran</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Charles River Laboratories</td>
			<td>551NCICr:SW</td>
			<td>Female Swiss Webster; 6-8 weeks old</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Charles River Laboratories</td>
			<td>556NCIC57BL/6</td>
			<td>Female C57BL/6; 6-8 weeks old</td>
		</tr>
		<tr>
			<td>Needles, 27G, &#189;-in</td>
			<td>Becton Dickinson</td>
			<td>305109</td>
			<td>can be substituted from other vendors</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>GE</td>
			<td>SH30028.02</td>
			<td>can be substituted from other vendors</td>
		</tr>
		<tr>
			<td>Rodent warming and restraining device (Mouse a Minute)</td>
			<td>LSU Health</td>
			<td>custom order</td>
			<td>Mouse a Minute is available for custom ordering from LSU Health</td>
		</tr>
		<tr>
			<td>Sabouraud dextrose agar (SDA)</td>
			<td>Becton Dickinson</td>
			<td>211584</td>
			<td>can be substituted from other vendors</td>
		</tr>
		<tr>
			<td>Syringes, 1 mL</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td>can be substituted from other vendors</td>
		</tr>
		<tr>
			<td>Trypan blue solution</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast peptone dextrose (YPD) broth</td>
			<td>Fisher Scientific</td>
			<td>BP2469</td>
			<td>can be substituted from other vendors</td>
		</tr>
	</tbody>
</table>

a contemporary warming/restraining device for efficient tail vein injections in a murine fungal sepsis model

在啮齿动物模型中，尾静脉注射是实验剂静脉注射的重要方法。尾静脉注射通常涉及动物的温暖，以促进血管炎，这有助于识别血管和定位针头到血管流明，同时安全地约束动物。虽然尾静脉注射是许多协议中的常见程序，如果执行得当，则不被视为高度技术性，但准确和一致的注射对于获得可重复的结果和最大限度地减少变异性至关重要。在尾静脉注射之前诱导血管吸附的传统方法通常取决于热源的使用，如热灯、电/充电热垫或 37 °C 预热水。 尽管在标准实验室环境中很容易获得，但这些工具显然受到热调节能力差/有限的损害。同样，虽然各种形式的限制装置在商业上是可用的，但必须小心使用，以避免对动物造成创伤。这些现有方法的局限性在实验中产生不必要的变量，或导致实验和/或实验室之间产生不同的结果。
在本文中，我们展示了一个改进的协议，使用创新的设备，结合了独立，热调节，加热装置与可调节的约束单元到一个系统，高效流线型尾静脉注射。我们使用的例子是真菌血流感染的静脉注射模型，导致败血症。加热装置由热反射丙烯酸盒组成，该盒配有可调节的自动恒温器，可在预先设定的阈值下保持内部温度。同样，锥体约束装置的宽度和高度可以调整，以安全地适应各种啮齿动物的大小。凭借该设备的先进和多功能功能，此处显示的技术可以成为一个有用的工具，跨越一系列研究领域，涉及啮齿动物模型，采用尾静脉注射。

加热室内的温度由内部传感器持续检测，并由恒温器自动调节。首先，恒温器的控制表盘定位在 78、85、90 或 95 °F（26、29、32 或 95 °C）以选择设定的温度。一旦加热器被激活（图7，黄点），灯泡的热量发射在前5至15分钟迅速提高内部温度，这取决于设定的温度。如果检测到的内部温度超过设定温度（灰点），加热器会灭活灯泡。初始峰值温度应上升到高于所有组设定温度的 5~7 °C，以抵消动物转移期间的温度损失。随后，设备继续自动重复热循环，并在设定温度下保持加热室。
图8显示了使用当前协议成功尾静脉注射获得的实验数据示例。在导致败血症的血液念珠菌病的小鼠模型中，瑞士韦伯斯特小鼠与念珠菌（每只小鼠1×105细胞）的i.v.挑战导致败血症的迅速发作和生物体的传播，导致3×4天内（开放点）的高死亡率（图8A）。相比之下，动物可以通过预先接种即用一种病毒性酵母菌株，坎迪达·都柏林人，在致命的i.v.挑战与毒性C.藻类（固体点）的挑战后，实现>95%的存活率来保护动物免受败血症的侵占。这些在渐进死亡率与疫苗介于媒介的保护中的结果在四个独立的实验中得到了可重复获得（补充图1）。类似的保护可以使用其他可病毒酵母菌株，如衰减的C.藻类突变体（+efg1/+cph1）（数据未显示）。败血症也可以监测，与死亡率相关:未接种疫苗的致命感染动物败血症引起的发病率显著增加，而接种疫苗的群体在致命挑战后表现出的症状最少（图8B）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61961/61961fig01.jpg"> 图1:啮齿动物加热和抑制装置的描述。（A） 显示加热装置的外部视图，包括:
<ol><li>恒温器盖 - 通过手柄向上抬起以暴露恒温器</li>
	<li>电气外壳 - 永久密封以保护</li>
	<li>室盖 - 在动物转移期间向上抬起来/从</li>
	<li>暖室 • 可拆卸，使用前用床上用品盖住地板</li>
	<li>约束装置 - 不使用时可与加热装置一起存放</li>
	<li>电源开关 + 主开/关功能的内联摇动器开关</li>
	<li>电源线 + 电压/电流: 120V/10A</li>
</ol>（B） 显示加热装置的内部:
<ol><li>白炽灯泡 - 100 瓦的光输出</li>
	<li>灯泡保护屏蔽 - 可拆卸灯泡更换</li>
	<li>温度传感器探头 - 位于腔室内</li>
	<li>内部温度计 - 放置在室内以监测温度</li>
</ol>（C） 显示加热装置恒温器的组件:
<ol><li>内部温度计</li>
	<li>温度计 - 自动调节加热器</li>
	<li>设置点控制杆 = 最小/最大值:78 °F/108 °F （25 °C/42 °C）</li>
	<li>恒温器功率指示灯 - 绿灯表示正常运行</li>
	<li>恒温器加热器指示灯 - 加热周期期间亮红色</li>
</ol>（D） 显示约束装置的组件:
<ol><li>锥体 - 柔和铝板专为啮齿动物约束设计</li>
	<li>尾通道 - 形状，允许尾巴平稳定位</li>
	<li>锥体提升平台 - 提供锥体底座的坚固提升</li>
	<li>高度调整旋钮 - 专为手动高度调整而设计</li>
	<li>剪刀千斤顶 - 高度范围从 45°140 mm （1.77-5.52"）</li>
	<li>支持板 - 安装橡胶脚，以提供稳定性 <a href="https://www.jove.com/files/ftp_upload/61961/61961fig01large.jpg" target="_blank">请点击这里查看此图的较大版本。</a>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61961/61961fig02.jpg"> 图2:啮齿动物加热和抑制装置。（A） 使用前，设备的两个部分并排放置在干净的长凳顶部。（B） 加热装置打开后，恒温器激活加热器。灯泡保持亮度并发出热量，直到加热室达到设定温度。加热装置自动重复热循环以保持内部温度。<a href="https://www.jove.com/files/ftp_upload/61961/61961fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61961/61961fig03.jpg"> 图3:放置在加热和抑制装置中的老鼠（C57BL/6）。（A） 接受血管吸附热处理的小鼠。动物（每次治疗4至6只小鼠）从其住房笼转移到设备的加热室，并经过至少5至10分钟的热处理。（B）一只小鼠被限制进行尾静脉注射。鼠标从加热室转移到约束装置的锥体开口处，其尾巴穿过开裂的缝隙。鼠标被轻轻地向后拉到圆锥体的远端，直到尾巴底部到达圆锥体的尖端。当动物被拉向圆锥体底部，轻轻的横向旋转时，一条后腿向上定位，以便它从开缝处突出出来，使横向尾静脉在12点时定位。 <a href="https://www.jove.com/files/ftp_upload/61961/61961fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61961/61961fig04.jpg"> 图4:小鼠横向尾静脉的识别。（A） 未经治疗的瑞士韦伯斯特鼠标的尾巴。鼠标被放置在抑制装置中，无需事先进行血管吸附的热处理。横向尾静脉可以识别为皮肤下的细暗血管。（B） 瑞士韦伯斯特老鼠的尾巴用加热装置处理10分钟。热处理小鼠被限制用于尾静脉注射。由于血管扩张引起的血管直径增大，横向尾静脉通过皮肤很容易看到。（C） 使用加热装置处理的C57BL/6小鼠的尾部10分钟，并抑制尾静脉注射。血管沉着通过深色素的皮肤增强尾静脉的可见性，尽管静脉不像浅色瑞士韦伯斯特小鼠那样容易看到，因为与静脉的颜色对比较弱。红色箭头表示横向尾静脉的位置。 <a href="https://www.jove.com/files/ftp_upload/61961/61961fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61961/61961fig05.jpg"> 图5:在热处理小鼠（瑞士韦伯斯特）中进行尾静脉注射。（A-B）针插入注射部位的横向尾静脉。针头（27 G， 1/2 英寸）与尾静脉平行，向上倾斜，指向血流并插入。（C/D）针头放置在尾静脉和注射。针尖进一步推进2/4毫米到静脉的流明。拇指位于注射器的柱塞上，所需的音量可减轻缓慢而稳定的压力。椭圆形表示注射部位。 <a href="https://www.jove.com/files/ftp_upload/61961/61961fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61961/61961fig06.jpg"> 图6:注射后程序。（A） 注射部位的出血区。注射溶液的出血和回流在取出针头后立即发生。通过用拇指在注射部位上应用坚定的压缩，可以最大限度地减少这种情况。（B） 注射部位的血块形成。用干净的纱布/擦拭轻轻压缩，有助于注射伤口的血液凝固。箭头表示注射位点。 <a href="https://www.jove.com/files/ftp_upload/61961/61961fig06large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61961/61961fig07.jpg"> 图7:使用过程中暖室的内部温度。加热装置在指定的设定温度下被激活以进行加热。设备的加热室被监测到内部的温度和热循环（灯泡/黄点，关闭/灰点）记录超过45分钟。橙色区域表示啮齿动物血管吸附诱导的最佳温度范围。 <a href="https://www.jove.com/files/ftp_upload/61961/61961fig07large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61961/61961fig08.jpg"> 图8:败血症死亡率与疫苗中介保护后，与念珠菌的致命挑战。老鼠（8周大的瑞士韦伯斯特雌性）静脉注射与病毒活的念珠菌Wé284（Cd），其次是致命的静脉注射挑战与野生型C.藻类第185天（1×105细胞每只小鼠）14天后。（A） 在致命挑战后10天内评估死亡率。（B） 动物被监测败血症发病率，并根据经修改的败血症（M-CASS）43的老鼠临床评估分数评分。数据是每组10只小鼠的4个独立实验的累积数据，并使用曼特尔-考克斯日志排名测试进行分析。p < 0.0001。SEM，平均值的标准错误。<a href="https://www.jove.com/files/ftp_upload/61961/61961fig08large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
补充图1:败血症死亡率的可重复性和疫苗介质生存从血液 念珠菌阿尔比肯人 的挑战。 每个面板代表图 8A中显示的累积结果中包含的四个独立实验的数据。每个实验使用每组10只小鼠进行，并使用曼特尔-考克斯日志等级测试进行分析。 卡， 坎迪达阿尔比肯人Cd， 坎迪达 · 都柏林人 p < 0.0001。p < 0.01。 <a href="https://cloudflare.jove.com/files/ftp_upload/61961/supfig01.jpg" target="_blank">请点击这里下载此图。</a>

Watch this Scientific Journal Video about A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model at JoVE.com

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

在这里，我们提出了一个有效和高效的方法，啮齿动物尾静脉注射使用独特的设计的加热/抑制装置。通过简化血管吸附和抑制过程的启动，本协议允许准确和及时地静脉注射大群动物，以最小的痛苦。

在穆林真菌败血症模型中用于高效尾静脉注射的当代加热/抑制装置

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

a-contemporary-warmingrestraining-device-for-efficient-tail-vein

Research

JoVE Journal

Immunology and Infection

13.8K 次观看.  Louisiana State University Health-School of Dentistry. 在这里，我们提出了一个有效和高效的方法，啮齿动物尾静脉注射使用独特的设计的加热/抑制装置。通过简化血管吸附和抑制过程的启动，本协议允许准确和及时地静脉注射大群动物，以最小的痛苦。

视频: A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for establishing new therapeutic approaches in this field. Therefore, animal models are required that are able to closely mimic the human disease and also sufficiently deal with scientific questions. The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. In this model, a small stent is surgically inserted into the ascending colon of mice or rats leading to a continuous leakage of intestinal bacteria into the peritoneal cavity. The procedure results in peritonitis, systemic bacteraemia, organ infection by gut bacteria, and systemic but also local release of several pro- and anti-inflammatory cytokines. The lethality of CASP can be controlled by the diameter of the inserted stent. A variant of this model, the so-called CASP with intervention (CASPI), raises opportunity to remove the septic focus by a second operation according to common procedures in clinical practice. CASP is an easily learnable and highly reproducible model that closely mimics the clinical course of abdominal sepsis. It leads way to study on questions in several scientific fields e.g. immunology, infectiology, or surgery.

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. This article describes the surgical procedure of CASP. The CASP model and its variants allow the systematic investigation of various problems concerning the subject of sepsis.

科隆Ascendens支架腹膜炎（CASP） - 标准化的模式为多种微生物腹部败血症

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

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 ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

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 ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

下腔静脉狭窄: 深静脉血栓形成的小鼠模型

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

高频超声监测小鼠瘀血致深静脉血栓形成

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

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 ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

新生儿多药脓毒症的对照小鼠模型

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...