Name: ferric chloride-induced murine thrombosis models
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This work was supported by the National Heart Lung and Blood Institute (NHLBI) of the National Institutes of Health under award numbers R01 HL121212 (PI: Sen Gupta), R01 HL129179 (PI: Sen Gupta, Co-I: Li) and R01 HL098217 (PI: Nieman). The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

所有的过程和动物的操作都按照美国公共卫生服务政策上的人文关怀和动物的使用 ，以及NIH 指南照顾和批准了克利夫兰诊所的机构动物护理和使用委员会（IACUC） 实验动物使用 。 1.准备: <ol><li> 荧光染料用于标记血小板 <ol><li>制备罗丹明6G溶液，0.5毫克/毫升，在盐水和消毒用0.22微米的过滤器将溶液。 </li></ol></li><li> 三氯化铁3溶液 <ol><li>使30％的FeCl 3新鲜原液（无水，等于1.85 M），在纯水中，并用0.45微米的过滤器进行过滤。制备5毫升的新鲜的FeCl以所需浓度3溶液[ 例如，2.5％（0.154 M），5％（0.308 M），7.5％（0.463 M），10％（0.617 M）和12.5％（0.771 M）[由用水稀释我的30％三氯化铁3溶液呐6厘米组织培养皿，保持盖子关闭。 注:使用培养皿使得它更容易拿起用三氯化铁溶液中3比把它捡起来，从一个狭窄的容器，如一个1.5 ml离心管饱和的滤纸。三氯化铁是非常危险和预防措施，如戴手套，白大褂等个人防护装备，应始终采取。 </li></ol></li><li> 滤纸 <ol><li>切滤纸到1mm×2mm的大小。浸泡切割滤纸足够件在同一培养皿中的FeCl 3溶液。 </li></ol></li><li> 盐酸罂粟碱解决方案 <ol><li>在水中制备罂粟碱盐酸盐溶液（25毫克/毫升）和消毒用0.22微米的过滤器将溶液。 </li></ol></li><li> 琼脂糖凝胶 <ol><li>制备在PBS中的2％琼脂糖凝胶或盐水在6孔组织培养板，约1cm的厚度，和c它向UT斯达康半圈与〜直径3厘米。 </li></ol></li></ol> 2. 氯化铁对颈动脉动脉损伤血栓形成模型<ol><li> 对于血栓形成模型外科手术 <ol><li>使用8 - 19周龄C57BL6小鼠（或其他菌株如必要）。麻醉小鼠通过腹膜内注射氯胺酮（100毫克/千克）/赛拉嗪（10毫克/千克）的混合物中。确认麻醉由脚趾夹住的深度。 注:氯胺酮/赛拉嗪的该量是足以保持在稳定的麻醉动物并没有疼痛（响应于趾夹持）至少1小时。 </li><li>对颈部及上胸部有小动物电推剪删除皮毛。脱毛膏是没有必要的。对眼睛应用中性石油眼药膏，以防止在麻醉过程中干燥。 </li><li>固定鼠标在15厘米组织培养板的盖子仰卧位。使用一个长带（约10厘米），以确保欣ð四肢和下半身，和小磁带（2毫升×0.5厘米），以确保前腿的两件。使用4-0缝合线环绕的门牙，缝线的两端用胶带在盖保持小鼠颈部直（ 图2）。 </li><li>将鼠标头部朝向操作板下手术光源。 </li><li>消毒手术部位用酒精垫。使用微斜角肌挤压钳保持皮肤，用手术剪刀做一个小切口（2 - 3毫米），。 </li><li>保持与镊子切口的皮肤和插入外科剪刀钳口闭合成切口。皮下推剪刀朝柄或下巴，然后打开剪刀摆脱皮下层皮肤。 </li><li>切断与外科剪刀的皮肤，使从柄一个中线切口以舌骨水平（ 图 3A和3B）。说白了剖析薄筋膜（虚线，在<stroNG&gt;图3B）用细止血的颌下腺和格雷夫钳（ 图 3B和3C之间; 蓝色箭头 ）。 注:柄（ 图3C黑色箭头）和气管（T位于图3C）将在此步骤后可以看到。 </li><li>持软组织，并在与格雷夫钳柄的右侧看到的皮肤，插入止血朝向2点钟位置筋膜下（图3C黄色虚线 ），打开止血钳的钳口以释放从周围组织颈静脉。 </li><li>切筋膜，软组织和皮肤向着2点钟位置（ 图3C）用手术剪，以暴露右颈静脉（ 图3D，箭头 ）。 </li><li>绘制大约200 - 使用1毫升注射器300微升罗丹明6G液与22克针，然后改变它至30g针。 注:此保护为30G针头从它的精尖的损坏。绘图直接与各30 g针不会破坏尖端罗丹明6G的解决方案;然而接触容器壁无意中会造成损坏，这可能使注射困难。 </li><li>冲洗慢慢压出罗丹明6G液各30 g针，确保没有气泡留在注射器或针头。 100保持注射微升罗丹明6G的解决方案。 </li><li>弯曲2 -针3毫米尖端90°角与针保持器，这将防止插入针太深的颈静脉，使注入更容易地控制（ 图3D）。 </li><li>注入罗丹明6G溶液到右颈静脉，以标签血小板。以稳定注射器，并保持所述针的位置，用另一只手握住injection.Af期间用一只手将注射器和针与格雷夫钳子之三注射液，夹住注射部位与格雷夫钳，以避免出血。 </li><li>打开止血钳的钳口，并插入它的格雷夫镊子下夹住注射部位的血管壁，然后结扎注射部位用6-0缝合线。 注:由于步骤2.1.15的另一种选择，将压力施加到注射部位2分钟将止血手指;然而，这种方法具有造成进一步的，如果在注射部位的凝块被无意中除去出血的危险。 </li><li>直截了当地解剖软组织和筋膜与周围的止血钳和格雷夫镊子左下颌腺和拉向7点钟位置（ 图3E）以暴露左胸锁乳突肌（ 图3E蓝色箭头）的腺。 </li><li>左胸锁乳突肌与肩胛舌骨肌或sternohy之间直言解剖筋膜（ 图3E，虚线 ）oid的肌肉（ 图3E，绿色箭头 ）位于与止血气管的左侧部位。 </li><li>通过一个针与胸锁乳突肌（ 图3E蓝色箭头）的中间下6-0丝线缝合（约15厘米长），把缝线的两端在一起，横向拉动缝线朝​​10点钟位置。 注:应仔细执行此过程，以避免左颈静脉，它位于胸锁乳突肌之外的损伤。 </li><li>直截了当地分离薄胸骨舌骨肌和/或肩胛舌骨肌抽离胸锁乳突肌后以暴露颈动脉（CA）（ 图3F箭头）。切开胸骨舌骨肌和/或肩胛舌骨肌肉是必要的。用止血钳而不触及CA.分开CA软组织 注:CA伴随迷走神经，白色组织见于<strong&gt;的图3G（绿色箭头），并且在大多数情况下，它是薄的胸骨舌骨肌和/或肩胛舌骨肌（ 图3F黄色虚线之间）下。 </li><li>用细尖镊子拿起身边的CA外侧筋膜同时避免了迷走神经和CA.使用另一个精尖钳猛击对CA和迷走神经的筋膜一个洞。 </li><li>通过孔中穿过挂钩，然后轻轻提起CA，然后将CA.下的精尖镊子移动至钩和镊子以相反的方向沿CA剥离外膜软组织。从周围组织CA（ 图3H，蓝色箭头 ）免费至少约5毫米的长度。 </li><li>要阻止背景荧光，按下一个黑色的塑料咖啡搅拌器平年底，横切咖啡搅拌成3毫米一块，然后沿折叠边纵向切割获得的"U"形的塑料（ 图3H，绿两件箭头 ）。 </li><li&gt;冲洗一块这个"U"形的塑料用生理盐水，并用镊子按住它，并把它放在CA而下的轻轻提起与钩（ 图3H，绿色箭头 ）的CA.立即申请生理盐水2-3滴，以避免干燥的CA. </li></ol></li><li> 实时录制视频 <ol><li>一起传送鼠标和板盖到显微镜阶段和发生在10X水透镜之下的适当位置。填充10X透镜和盐水在CA之间的空间。 </li><li>启动数字视频录像的软件应用，并推出一个新的文件，并据此命名。记录正常血管图像，并在视频录制停止写下帧数。 注:引用的视频录制软件，在电脑记录。我们使用的数字视频记录的软件和下面的描述是基于这样的应用程序。 注:由于机械TRAUMA可以伤害内皮，导致血栓形成20，有必要确认没有手术损伤到之前的FeCl 3损伤血管壁。它也有必要确认有围绕在CA没有剩余的组织，其可以形成屏障并衰减的FeCl 3诱导的损伤的效果。写下记录的帧数会很容易地分析后，根据经过时间的视频。 </li><li>移动钢板盖鼠标移出10X镜头。折叠纸巾的一角，使薄和精尖，并用它小心吸干周围的CA生理盐水（避免触及CA），以及在手术野等地。这是很重要的，以避免使用的FeCl 3溶液稀释。 </li><li>用细尖镊子，拿起用三氯化铁3溶液饱和滤纸片，并直接将其放在CA，并保持它在现场1分钟。 注:tÓ血流和收获的准确数据的援助可视化，接近暴露的CA（ 图3I）的远端放置滤纸，并在近端部位离开一个短段（ 在图3I绿色箭头 ），用于在后期观察血流量阶段（见下文）。 </li><li>取出滤纸（该时间点被定义为开始"之后伤害"）和冲洗用盐水（至少2毫升）的CA. </li><li>把钢板盖回镜头10X鼠标;观察船只，并开始录制视频图像。写下视频帧数在录制第一分钟结束。 注:血栓形成由荧光血小板，这是在实时视频图像观察计算机屏幕上或显微镜下的累积识别。录制视频图像把鼠标背部在显微镜下之后。保持记录到第一分钟的末尾取出的F后ILTER纸。从远端整个容器观察到近端网站以获得关于损伤的信息，然后集中在感兴趣的区域（通常为受伤区域的中心），用于进一步的成像。 </li><li>记录的视频图像的每分钟的第10分钟（ 例如，2:55至3:05），然后10秒每隔一分钟，直到实验结束10秒。当每个记录停止写下帧数。 注:该模型的结束点是:1）时，血流量已停止为&gt; 30秒;或者2）如果闭塞是没有看到在受伤后30分钟。在这种情况下，用于统计分析30分钟。设置视频记录作为开头10毫秒的曝光时间，所以这将是便于观察过血栓的血流量。当血栓变大，荧光饱和相机的传感器，它变得难以识别在血栓的血流量。为了解决这个问题，将成像CENTER到血流仍然可以清楚地观察到近端未受伤的部位（ 图3I，绿色箭头）。我们还增加了暴露时间15或20毫秒聚焦近侧未损伤部位上时，使血流可以更清楚地观察到。当血液流动将会停止，众多的大细胞（白细胞）开始在血栓的近端部位对血管壁滚动。大单元格的外观后3分钟 - 流量通常在2站。记下如果它改变了记录片的曝光时间。它通常需要大约30分钟从麻醉到实验结束时，如果使用正常的野生型C57BL6小鼠。 小心注:请不要使用白色聚集的血小板凝块随着血流停止，不会给出一个准确的数据的符号，它是由曝光时间的影响。白色凝块覆盖管腔并不意味着停止血流量（见代表视频1）。 </li><li&gt;在实验结束后，安乐死使用过量氯胺酮/赛拉嗪（200/20毫克/千克），接着通过颈椎脱位确认没有呼吸和心跳后小鼠。 </li></ol></li></ol> 3. 氯化铁诱导肠系膜动脉/静脉血栓形成模型<ol><li>麻醉8 - 19周龄C57BL6小鼠在2.1.1节中提到。对眼睛应用兽医药膏，以防止在麻醉过程中干燥。 </li><li>注入罂粟碱溶液（总0.4毫克/小鼠）腹膜内抑制肠道蠕动。 </li><li>对颈部和腹部与动物电推剪删除皮毛。脱毛膏是没有必要的。 </li><li>固定鼠标在15厘米组织培养板的盖子仰卧位。使用四件小吊带（2毫升×0.5厘米）的，以确保所有的腿。使用4 -0缝线环绕门齿，缝线的两端用胶带在盖保持颈部直（ 图2）。 </li><li>按照程序2.1.4 -2.1.15暴露颈静脉注射罗丹明6G的标注血小板。 </li><li>执行通过从剑突皮肤中线切开至小腹使用手术剪（ 图4A）。拿起中间腹膜（也称为白线， 图4A，箭头）与格雷夫钳，这是明确的，没有血管，解除约2 - 3mm高确认切割线下无排便，并剪开腹膜开放纵向与细剪刀（ 图4B）。 </li><li>重新固定小鼠右侧卧位。放置半圈琼脂糖凝胶靠近腹部，并轻轻exteriorize肠子和传播它在与格雷夫镊子（ 图4C）的凝胶的顶部。用于血栓实验（ 图4C，箭头 ）的第二分支。 注:使用止血钳或微斜角肌挤压钳帮助exteriorize肠子。以免伤肠和容器中。 </li><li>放置5 - 6滴盐水保持肠和容器中的水分。与板盖一起传送鼠标显微镜台上，在干燥的10X镜头下的适当位置放置。 注:使用干的镜头，因为空肠回肠膜不能持有生理盐水。确保在暴露的组织降盐液中保持湿润。 </li><li>围绕吸干治疗前肠系膜动脉和静脉盐水。 </li><li>用细尖镊子拿起12.5％三氯化铁3溶液饱和滤纸片，并直接将其放在肠系膜动脉和静脉1分钟（ 图4D）。取出滤纸（该时间点被定义为"后损伤"的开始），并用盐水冲洗受伤肠系膜动脉和静脉（至少2毫升）中。 </li><li>把钢板盖的10X镜头干背下鼠标;观察船只，并开始录制在2.2中提到ORD视频图像）。 注意:这个实验的结束点是:1）时，血流量已停止为&gt; 30秒;或者2）如果闭塞未在30分钟内受伤后看到的，在这种情况下使用的统计分析30分钟。 </li><li>在实验结束时，安乐死使用过量氯胺酮/赛拉嗪（200/20毫克/千克），接着通过颈脱位无呼吸和心脏跳动被确认后的小鼠。 </li></ol>

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The authors have nothing to disclose.

所述的FeCl 3诱导的模型是最广泛使用的血栓形成的模型，它不仅可以提供有关对血小板功能和血栓形成7,8,16,19,31-33遗传修饰的有价值的信息中的一个，但也可以是一个有价值的工具用于治疗化合物和战略动脉粥样硬化性疾病11,17,34-37的治疗和预防的评价。在这里，我们已经表明我们的修改和该模型的改进和表明该技术中，这是足够敏感以确定抗凝物（tPA），抗血小板药物?...

动脉血栓形成（血块）是与慢性炎症，包括动脉粥样硬化，糖尿病，肥胖症，癌症和慢性自身免疫性风湿病相关的许多全身性疾病的常见并发症。发生在不适当的血小板活化，聚集和后续机制混凝动脉循环茎，血栓有牵连的心脏发作，中风和肢体损失。血管壁是一个复杂的系统，它包括多种细胞类型和是由外在因素，包括剪切应力多种影响，循环血细胞，激素和细胞因子，以及抗氧化蛋白表达在血管壁。 体外实验不能复制这种复杂的环境，因此， 在使用动物模型的体内研究是至关重要的，以允许更好地理解涉及血栓性疾病的机制。 小鼠已经显示出具有卡ILAR机制对人类中的血栓形成，动脉粥样硬化，炎症和糖尿病1,2-条款。此外，可以创建转基因和基因敲除小鼠测试的特定基因产物的功能在一个复杂的生理或病理环境。这样的研究模仿人类病理学，并且可以提供有关的新途径和疗法，发现重要的机械信息以及在上血栓形成表征药物作用提供重要的信息。 病理动脉血栓的发生是由于内皮层损伤或功能障碍和血液流暴露于内皮下矩阵3,4。各种血栓形成模型已经发展到诱导这种内皮损伤，如机械性损伤，基于孟加拉-光反应性化合物玫瑰氧化损伤和激光损伤5。在这个频谱，氯化铁（ 氯化铁）诱导的血管损伤是血栓形成的一个广泛使用的模型。该试剂时，施加到容器的外表方面诱导对血管细胞6-8氧化损伤，与来自循环血小板和凝血级联组分内皮细胞保护的丧失。所述的FeCl 3模型简单到两个抗凝和抗血小板药物敏感，并已在颈动脉和股动脉，颈静脉和肠系膜和提睾动脉和小静脉在小鼠，大鼠，豚鼠和兔6-15进行。 在此模型中的一个可测量参数是从损伤的经过时间来完成血管闭塞，作为血流停止用多普勒血流计或根 ​​据与活体显微镜6,7,9直接观察来测定。的5至30分钟之间倍一系列已报道在C57BL6小鼠7-10,16不同的研究，表明的FeCl 3浓度，种类麻醉，手术技术，小鼠年龄，基因组背景，测量的B法的lood流，和其他环境变量具有在该模型显著影响。这个很大的可变性使得难以研究从不同的研究小组比较，并且可以使细微的差别检测变得困难。 具有远见尽量减少这种变异，并建立一个在体内模型系统统一重复性好，我们已经完善了氯化铁诱发颈动脉模型，从而以最小的变化6-10,16-19高重现性的数据。在本文中，我们描述和分享技能和报告，可以从这个模式中受益，几个有代表性的实验例。

<table><tbody><tr><td>Surgical Scissors - Tungsten Carbide</td><td>Fine Science Tools&amp;nbsp;</td><td>14502-14</td><td>cut and hold skin</td></tr><tr><td>Micro-Adson Forceps - Serrated/Straight/12 cm</td><td>Fine Science Tools&amp;nbsp;</td><td>11018-12</td><td>cut and hold skin</td></tr><tr><td>Metzenbaum Fino Scissors - Tungsten Carbide/Curved/Blunt-Blunt/14.5 cm</td><td>Fine Science Tools&amp;nbsp;</td><td>14519-14&amp;nbsp;&amp;nbsp;</td><td>to dissect and separate soft tissue</td></tr><tr><td>Ultra Fine Hemostat - Smooth/Curved/12.5 cm</td><td>Fine Science Tools&amp;nbsp;</td><td>13021-12</td><td>to dissect and separate soft tissue</td></tr><tr><td>Graefe Forceps - Serrated/Straight/10 cm</td><td>Fine Science Tools&amp;nbsp;</td><td>11050-10</td><td>to dissect and separate soft tissue</td></tr><tr><td>Dumont #5 Fine Forceps - Biology Tips/Straight/Inox/11 cm</td><td>Fine Science Tools&amp;nbsp;</td><td>11254-20&amp;nbsp;</td><td>Isolate vessel from surounding tissue</td></tr><tr><td>Dumont #5XL Forceps - Standard Tips/Straight/Inox/15 cm</td><td>Fine Science Tools&amp;nbsp;</td><td>11253-10</td><td>Isolate vessel from surounding tissue</td></tr><tr><td>Blunt Hook- 12 cm/0.3 mm Tip Diameter</td><td>Fine Science Tools&amp;nbsp;</td><td>10062-12</td><td>Isolate vessel from surounding tissue</td></tr><tr><td>Castroviejo Micro Needle Holders</td><td>Fine Science Tools&amp;nbsp;</td><td>12061-02</td><td>Needle holders</td></tr><tr><td>Suture Thread 4-0</td><td>Fine Science Tools&amp;nbsp;</td><td>18020-40</td><td>For fix the incisors to the plate</td></tr><tr><td>Suture Thread 6-0</td><td>Fine Science Tools&amp;nbsp;</td><td>18020-60</td><td>For all surgery and ligation</td></tr><tr><td>Kalt Suture Needles</td><td>Fine Science Tools&amp;nbsp;</td><td>12050-03</td><td></td></tr><tr><td>rhodamine 6G&amp;nbsp;</td><td>Sigma</td><td>83697-1G</td><td>To lebel platelets</td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt; (Anhydrous)</td><td>Sigma</td><td>12321</td><td>To induce vessel injury</td></tr><tr><td>Papaverine hydrochloride</td><td>Sigma</td><td>P3510</td><td>To inhibit gut peristalsis.</td></tr><tr><td>Medline Surgical Instrument Sterilization Steam Autoclave Tapes</td><td>Medline</td><td>111625</td><td>To fix the mouse to the plate</td></tr><tr><td>Fisherbrand&amp;trade;&amp;nbsp;Syringe Filters - Sterile 0.22 &amp;micro;m</td><td>Fisher</td><td>09-720-004</td><td>For sterlization of solutions injected to mice</td></tr><tr><td>Fisherbrand&amp;trade;&amp;nbsp;Syringe Filters - Sterile 0.45 &amp;micro;m</td><td>Fisher</td><td>09-719D</td><td>To filter the FeCl&lt;sub&gt;3&lt;/sub&gt; solution</td></tr><tr><td>Sterile Alcohol Prep Pad</td><td>Fisher</td><td>06-669-62</td><td>To sterilize the surgical site</td></tr><tr><td>Agarose&amp;nbsp;</td><td>BioExpress</td><td>E-3120-500</td><td>To make gel stage</td></tr><tr><td>Leica DMLFS fluorescent microscope</td><td>Leica</td><td></td><td>Intravital microscope</td></tr><tr><td>GIBRALTAR Platform and X-Y Stage System with heating plate attached.</td><td>npi electronic GmbH</td><td></td><td>http://www.npielectronic.de/products/micropositioners/burleigh/gibraltar.html</td></tr><tr><td>Streampix version 3.17.2 software</td><td>NorPix</td><td></td><td>https://www.norpix.com/</td></tr></tbody></table>

ferric chloride-induced murine thrombosis models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

颈动脉血栓形成模型 在C57BL6背景小鼠中，我们建议使用7.5％的氯化铁治疗血管1分钟为出发点。下的FeCl 3处理的7.5％，受伤的区域和正常血管壁的边界显微镜下很容易识别（参见在线视频1），这表明内皮层显著损坏。血栓形成，立即经氯化铁处理，损伤后的所有WT C57BL6小鼠1分钟观察。最初形成的血栓是不稳定的，它?...

Watch this Scientific Journal Video about Ferric Chloride-induced Murine Thrombosis Models at JoVE.com

Ferric Chloride-induced Murine Thrombosis Models

我们报告的氯化铁（氯化铁3）颈动脉和肠系膜动脉血栓诱发款精致的程序以及静脉，其特征有效地利用活体显微镜监测的时间来闭塞血栓的形成。

三氯化铁诱导小鼠模型的血栓形成

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

ferric-chloride-induced-murine-thrombosis-models

Research

JoVE Journal

Medicine

21.5K 次观看.  Lerner Research Institute, Cleveland Clinic. 我们报告的氯化铁（氯化铁3）颈动脉和肠系膜动脉血栓诱发款精致的程序以及静脉，其特征有效地利用活体显微镜监测的时间来闭塞血栓的形成。 

视频: Ferric Chloride-induced Murine Thrombosis Models

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential therapeutic compounds (1). New compounds to be utilized in the treatment and prevention of DVT are currently being developed. The delivery of potential therapeutic antagonist compounds to an affected thrombosed vein has been problematic. In the context of therapeutic applications, a model that uses partial stasis and consistently generates thrombi within a major vein has been recently established. The Electrolytic Inferior vena cava Model (EIM) is mouse model of DVT that permits thrombus formation in the presence of continuous blood flow. This model allows therapeutic agents to be in contact with the thrombus in a dynamic fashion, and is more sensitive than other models of DVT (1). In addition, this thrombosis model closely simulates clinical situations of thrombus formation and is ideal to study venous endothelial cell activation, leukocyte migration, venous thrombogenesis, and to test therapeutic applications (1). The EIM model is technically simple, easily reproducible, creates consistent thrombi sizes and allows for a large sample (i.e. thrombus and vein wall) which is required for analytical purposes.

Electrolytic Inferior Vena Cava Model EIM of Venous Thrombosis

The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.

电解下腔静脉静脉血栓形成的模型（EIM）

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential ...

科研

医学

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

鼠标完成血瘀模型的下腔静脉血栓

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

利用X射线计算机断层扫描在小鼠肥胖模型的FAT卷的分割和测量

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

自体血液或梭菌胶原酶脑内注射为脑出血的小鼠模型

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

使用血管夹在小鼠癌症模型中优化静脉血栓形成测定

Venous Thrombosis Assay in a Mouse Model of Cancer

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

作者聚焦：推进啮齿动物模型中的癌症相关血栓形成研究 

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis.

癌症小鼠模型中的静脉血栓形成测定

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis ...

癌症研究

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

三氯化铁诱发血栓形成小鼠模型上的颈动脉和肠系膜血管

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

三氯化铁诱导小鼠模型的血栓形成

摘要

探索更多视频

此视频中的章节

电解下腔静脉静脉血栓形成的模型（EIM）

鼠标完成血瘀模型的下腔静脉血栓

利用X射线计算机断层扫描在小鼠肥胖模型的FAT卷的分割和测量

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

自体血液或梭菌胶原酶脑内注射为脑出血的小鼠模型

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

癌症小鼠模型中的静脉血栓形成测定

癌症小鼠模型中的静脉血栓形成测定

癌症小鼠模型中的静脉血栓形成测定

三氯化铁诱发血栓形成小鼠模型上的颈动脉和肠系膜血管