体内成像是利用IVIS共享资源在科罗拉多大学癌症中心（以出让P30-CA046934支持）进行。在流式细胞仪共享资源，科罗拉多大学癌症中心（以出让P30CA046934支持）进行流式细胞术。这项工作是由美国国立卫生研究院（RO1CA137078到DKG）的部分支持。 ABLS是小儿科学家开发计划，由来自美国儿科学会，美国儿科学会和儿童健康与人类发展（K12-HD000850）的尤尼斯·肯尼迪·施莱佛国立资金支持的资深会员。

所有涉及动物实验其次批准科罗拉多机构动物护理和使用委员会大学的监管标准。该演示方案经科罗拉多机构动物护理和使用委员会的大学。 1。磷酸化蛋白质印迹<ol><li>裂解液的制备<ol><li>培养细胞系中表达感兴趣的受体酪氨酸激酶。收获细胞和板3-5×10在48孔组织培养皿中加入400μl生长培养基6个细胞/样品。在5％的CO 2为2-3小时在37°C。 </li><li>加入100μl的5倍TKI解决方案在生长培养基中孵育60分钟。 </li><li>制备裂解缓冲液，用50mM HEPES（pH值7.5），150 mM氯化钠，10毫摩尔EDTA，10％（V / V）甘油，1％（体积/体积）的Triton X-100和蛋白酶抑制剂。如果培养物将不会与过钒酸盐之前收获的处理，加磷酸酶抑制剂（1 mM的钠orthovanadate和0.1mM钼酸钠），以裂解缓冲液。 </li><li>在使用前配制过钒酸盐（PV）的磷酸酶抑制剂的解决方案。在黑暗的管置于冰上（1:100稀释的30％原液）制备过氧化氢（小心）0.3％的溶液在冷磷酸盐缓冲盐水（PBS）。煮沸的0.2M的正钒酸钠（OV，小心）原液5分钟等分。稀释1:10 PBS中，使在室温（RT）的20mM溶液，OV。混合0.3％过氧化氢和20mM OV 1:1和孵育在黑暗中在RT下20分钟。 </li><li>光伏稀释成完全培养基（60微升PV + 940μL培养基）。加入100μl稀释的PV /孔。在5％CO 2持续3分钟孵育在37℃，然后将板置于冰上。 </li><li>收获细胞在冷的微量离心管在2,500 rpm离心5分钟。吸上清，重悬细胞120μL裂解缓冲液。在冰上孵育15分钟。澄清在寒冷的离心裂解物在6,000 rpm离心3分钟。转让苏佩rnatant到一个新的冷轧管。储存在-80℃。 </li></ol></li><li>免疫沉淀<ol><li>降速蛋白G琼脂糖珠在微量离心以1,000 rpm进行1分钟。除去上清，洗珠2倍1毫升冷PBS。添加PBS等体积，使50％的浆料。初级抗体和20μl的50％蛋白质G珠加至每个样品孔。孵育2小时 - O / N在4℃旋转器上。 </li><li>脉冲下降在寒冷的离心珠在9,000转每分钟。除去上清，洗珠与2倍150微升冷裂解缓冲液。旋在冷离心以1,000 rpm进行1分钟。去除上清，重悬沉淀于20μL2×SDS上样缓冲液与2 - 巯基乙醇（小心）。立即使用或储存于-80°C。 </li><li>煮沸样品5分钟，并运行在SDS-PAGE上tris-甘氨酸凝胶。的蛋白质转移至硝酸纤维素膜，并通过免疫印迹检测磷酸化的蛋白。 </li><li>漂洗印迹用水，然后孵育在2％SDS，62.5毫摩尔Tris（pH值6.8），0。1米的β-巯基乙醇（警告）30分钟，在50°C。漂洗2倍的水，然后洗60-90分钟中的TBS-T 5-6改变，以消除剥离液。 </li><li>通过Western blot检测总蛋白。 </li></ol></li></ol> 2。甲基纤维素含量<ol><li>分装4毫升甲基纤维素人力基础培养基中于50 ml锥形管使用无菌大口径钝端针。 （注：甲基纤维素，可以分装并储存于-20°C解冻在室温O / N在使用前） </li><li>加入500μl的10倍TKI或车辆在生长培养基〜4毫升，持续10秒甲基纤维素和旋涡混合。 </li><li>收获和计数细胞。制备细胞悬浮液中的生长培养基中的浓度，它比最终细胞浓度的10倍以上。加入500μl细胞悬液，以甲基纤维素的混合物。涡旋10秒，让坐10分钟以去除气泡。 </li><li>用5毫升注射器和无菌LARG制定4毫升甲基纤维素混合物Ë孔钝端针。避免绘制了气泡。分配将1ml甲基纤维素混合物分成三个35mm组织培养平板的中央。漩涡的菜，直到底部完全覆盖甲基纤维素。 </li><li>对于每个甲基纤维素板，准备一块10厘米无菌组织培养板有两个额外的35mm组织培养皿充满无菌水，以确保在潮湿的环境。在水文化广场，5％的CO 2为10〜14天套培养箱在37℃。 </li><li> 1-2周后计数菌落。菌落应多于20个细胞和不重叠。菌落可使用倒置显微镜配备有显微镜载物台与电网或使用自动菌落计数器计数。响应于与TKI治疗变化菌落大小和形态也应进行评估。提高检测通过菌落计数器，菌落染色，加入60-100微升（3 - （4,5 - 二甲基-2 - 噻唑基）-2,5 - 二苯基-2H-四唑鎓溴化物溶胶ution（MTT法，在-20°C 5毫克/毫升H 2 O，存储，避光，注意保护）逐滴在每个板的表面，并在5％CO 2孵育8小时，在37℃。 </li></ol> 3。软琼脂测定<ol><li>准备1％琼脂高贵的底层，0.7％琼​​脂高贵的顶层和2x生长培养基。在42℃水浴中平衡2倍的培养液。热1％，并在微波炉中（约1 min/100毫升）0.7％琼​​脂的高贵和平衡在42℃水浴。混合1％琼脂和2x中，分别为0.7％琼​​脂和2倍培养基在50毫升圆锥等份。计划10毫升软琼脂混合/六孔板中每一层的。 </li><li>标签6孔组织培养板中。每次用1.5ml 1％软琼脂混合地填补。电镀过程中避免气泡。干板用盖子关闭在无菌罩30分钟。 </li><li>计数细胞。备中的浓度，它比最终细胞concentrati 500倍以上的细胞悬浮在生长培养基中上。加入细胞悬液，以0.7％的琼脂混合，拌匀并在1％的琼脂层的顶部免除1.5毫升琼脂细胞混合物。让顶部层固化30分钟。 </li><li>制备含TKI或车辆控制1X生长培养基。加入2毫升培养基与TKI的顶部琼脂层的顶部所需的浓度。 </li><li>在5％孵育培养10天，在37℃的CO 2。取出并更换上覆介质，每3天。 </li><li>当菌落可见肉眼，吸覆介质，并加入200微升硝基四氮唑蓝溶液（1毫克/毫升NBT在H 2 O，储存在4℃，避光保存，注意保护）。回到培养箱中培养24-48小时。移动到4℃至少2小时之前计数。彩色板可以被存储在4℃下一个月。用显微镜或自动菌落计数器计数菌落。 </li></ol> 4。评价磷酸化蛋白抑制体内 <ol><li>收集ÇELL的生长对数生长期通过离心在台式离心机中以1500 rpm离心5分钟。用无菌PBS洗涤一次，将细胞重悬在PBS中以适当的浓度（通常为1-5×10 7细胞/ ml）。移植细胞的体积为100微升至6-12周龄的NSG小鼠经静脉注射入尾静脉用28政胰岛素注射器。将动物的抑止，热尾在温水的烧杯扩张的静脉，并清洁尾部与洗必泰棉签注射前。注射后，施加压力，用无菌纱布，直到出血停止注射部位。移植至少3只动物/组。维持对含有1毫克/毫升的硫酸庆大霉素水老鼠。 </li><li>十四天后移植，治疗动物与TKI或仅车辆和收获样本中抑制分析。称量动物和治疗用适当剂量的TKI或车辆（毫克/公斤体重）。管理治疗5毫升/ K的体积g体重腹腔（IP）注射或10毫升/千克体重灌胃。对于腹腔注射，人工抑制小鼠和注射在腹部用29号针头的右下象限。对于灌胃，手动抑制鼠标并按住动物直立。将管饲法在口中在舌头和进入口的后面。适用于轻微的压力通过食道进入胃通管。压下注射器活塞进行救治。如果柱塞不容易压低灌胃针应删除并重新定位。在使用前配制TKI制剂。 </li><li>如果过钒酸盐治疗是理想，收获前准备光伏溶液20分钟以上（步骤1.1.4）所述稀释到培养基中1微米氯化镁和100 U / ml的DNA酶（120微升PV + 880μL培养基）。注意：PV是稳定的稀释后至少1小时。 </li><li>用CO 2麻醉在AP牺牲动物propriate时间（s）后处理。通过去除皮毛，皮肤和肌肉从整个腿部，使骨头都完全暴露解剖股骨。用解剖剪略低于髋关节和再次高于膝关节裁剪取出股骨。转移到培养皿中，并冲洗骨髓用1毫升冷PBS或稀释的光伏解决方案使用注射器和27号针头。如果用光伏治疗，在黑暗中在室温下孵育10分钟。 </li><li>准备裂解液及以上（步骤1.1.6-1.2.5）所示，分析磷酸化蛋白和总蛋白水平。 </li><li>定量的相对磷酸化蛋白和总蛋白水平，使用光密度测定各样品。计算phospho-protein/total-protein相对于载体处理的动物的平均phospho-protein/total-protein值。 </li></ol> 5。评价TKI的所有异种移植模型抗白血病活性<ol><li>如果有必要，暴露小鼠200 cGy的辐射在一个RS-2000辐射1天PRIOr来移植。上面（步骤4.1）所述移植小鼠白血病细胞株。移植至少每组6只小鼠。耳朵打孔识别小鼠。可替换地，一个黑色的标记可以被用于识别小鼠尾巴上的散列标记。添加浓缩到笼子里，以减少在研究期间笼交配侵略的潜力。 </li><li>治疗小鼠上面（步骤4.2）仅作为说明的TKI或车辆。每天通过体检和权重确定监测小鼠的健康状况。白血病的预期症状（降低的活性水平，体重减轻，后肢麻痹，和眼部感染）。减重超过10％-15％，通常用鼠标在两天内死亡有关，通常是死亡按照标准IACUC要求的替代终点。 </li><li>通过监控在体内生物发光成像系统高达2倍每周移植和白血病的发展。在免缝准备一个200×D-荧光素原液（30毫克/毫升）乐水。颠倒轻轻混匀，至D-荧光素完全溶解。立即使用，或分装冷冻在-20°C。在成像之前用体内生物发光成像系统，解冻的D-荧光素的等分试样在室温下和在PBS中稀释1:2到的通过0.2微米的过滤器15毫克/毫升，并过滤灭菌的最终浓度。 </li><li>成像前麻醉小鼠中的氧气1.5％，吸入异氟醚。监视足够麻醉，其特点是肌肉松弛，运动的损失和应对脚趾捏刺激的损失。 </li><li>立即成像之前，管理150毫克/公斤体重D-荧光素腹腔注射（10微升的15毫克/毫升荧光素/克体重）。 </li><li>使用体内生物发光成像系统捕获2系列连续的30，60和90秒曝光，并用120秒的曝光最终图像的图像。成像后，返回小鼠的笼子和监视从麻醉中恢复。取决于生物发光的强度，曝光时间可以变化。最佳曝光时间应预先确定一个给定的模型，并保持这种一致性，对于个别时间点的信号强度是整个研究具有可比性。 </li><li>确定生物发光强度，使用生活3.2图像采集和分析软件每只小鼠。第二通过绘制的权益，每只小鼠相同大小的回报率（ROI）区域确定在光子/测量总光通量值。 </li><li>如果经营不善，参展瘫痪，在大于15％的重量损失的情况下，或在研究结束时的CO 2曝光和颈椎脱位牺牲老鼠。解剖小鼠并记录观察结果（脾，肝，或淋巴结肿大，例如 ，面色苍白骨髓）。用27号针头解剖股骨和冲洗骨髓用冷PBS。 </li><li>在2500 rpm离心5分钟收集在微量细胞。重悬含2％FBS的细胞在PBS中孵育5分钟，在RT。收集细胞并染色用20毫克/毫升的DAPI（4,6 -二脒基-2 -苯基吲哚二盐酸盐，1:1,000）和FITC标记的α-hCD45（1:100）或鼠IgG 1同种型对照抗体（1:100） 。采用流式细胞仪评估白血病细胞植入。门上可存活种群（DAPI负），并确定CD45 +细胞（％骨髓幼稚细胞）的百分比。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

此手稿描述的有效策略在急性白血病的治疗新型酪氨酸激酶抑制剂评价。使用这种方法，生物化学和抗白血病活性，首先在体外基于细胞的测定在体内异种移植模型中进行评估，然 ​​后。免疫印迹分析已成功用于阐明用的TKI治疗后抑制白血病细胞的目标酪氨酸激酶，并直接在细胞比较多个化合物的效力。在这里介绍的协议，过钒酸盐被用于稳定磷酸化蛋白和靶蛋白浓缩，通过免疫...

急性淋巴细胞白血病（ALL）是小儿1,2最常见的恶性肿瘤。总体生存率为小儿B系ALL（B-ALL）是约85％，但具体的生物亚型，包括T细胞ALL（T-ALL），仍然有预后较差，即使与目前的治疗方案。复发性ALL的进一步治疗仍然是一个挑战3。尽管大多数成年患者的急性白血病实现与前期化疗缓解，许多患者仍然遭受复发4。在急性白血病的治疗目前的化疗方案是已知的导致毒性相关的短期和长期副作用。因此，非常需要毒性较低的疗法，专门针对癌细胞与正常组织的影响最小。近年来，重点一直放在新颖，具有特异性的分子靶向药物对肿瘤细胞中，经常利用迭代化学的发展以产生必须随后进行比较，并优先5多种活性化合物。这个手稿描述了一种高效的用于治疗急性白血病，可为了便于药物开发用于单个化合物的评价或多个化合物的直接比较策略为TKI的临床前评估。 这里提出的方法包括四个步骤。第一生化（1）和抗白血病（2）的化合物（S）的活性在细胞培养物进行评估，然后抑制靶的被确认的动物模型（3），和TKI（次）的最终治疗效果在白血病原位移植瘤模型确定（4）。对于这些研究，它可以选择有关的细胞系，这是最常见的生物亚型的代表是重要的。细胞系应选择，这两者表达的利益目标和缺乏兴趣的目标，探讨生物效应是否MED通过抑制靶iated。这是特别相关的小分子抑制剂，其具有的脱靶效应，可能是对于抗肿瘤活性是重要的发展。它也有必要选择的细胞系是依赖于目标为功能性的效果，如增殖或存活。利用RNA干扰或其他特定方式抑制靶目标初步验证研究（在本文的范围之内）可以被用来识别目标相关的细胞系。还希望选择能够形成小鼠异种移植物，以使得细胞培养物的结果可以更直接地转化为体内实验的细胞系。 由白血病细胞介导的​​TKI生化活性的评价，在受体磷酸化的降低，可作为靶抑制的指标。 Western印迹分析或酶联免疫吸附测定法可以采用，这取决于抗体的可用性和特异性。如果是现有抗体具有足够的特异性靶，酶联免疫吸附测定法是优选的，因为它们是更加定量的和有效的。在抗体具有足够的特异性ELISA不可用的情况下，免疫印迹分析可能是必要的。在这种情况下，大量的溶胞产物的免疫沉淀可以用于检测是在低丰度指标是有用的。这种方法是特别相关的磷酸化蛋白质，这可能有一个短的半衰期，以允许快速改变以响应环境刺激信号的测量。一些磷酸化蛋白是非常不稳定的，极有可能为复杂的形成与磷酸酶的结果。鲁棒和一致检测这些磷酸化蛋白，它也可以是可以治疗的细胞与过钒酸盐，一个不可逆的蛋白酪氨酸磷酸酶抑制剂6，对磷酸化蛋白质稳定之前制备的全细胞裂解液。 至确定生化活性是否导致了抗肿瘤效果，目标相关的生物学过程可以在基于细胞的试验来监测。对于白血病细胞株，通过TKIs的介导的抗白血病活性可以用甲基纤维素或软琼脂7进行克隆形成实验进行评估。软琼脂中可能是优选的，因为这是一种固体介质是更适合于操作，如果有一个TKI重复治疗是必要的。虽然许多急性myloid白血病（AML）细胞系将形成在软琼脂中集落，大多数ALL细胞系将只形成菌落在甲基纤维素，它是一种半固体培养基中。虽然这是可能的刷新介质和/或在TKI中的甲基纤维素培养物中，只有少量可直接或与有限的频率。同样，它是更难以染色的菌落，而不破坏它们的甲基纤维素。初步的研究应该定义的合适的细胞系以形成菌落在甲基纤维素和/或软琼脂和t的能力他的细胞在培养的最佳密度，使得菌落是不重叠的，并在足够数量以获得统计学上相关的数据（每35毫米板通常50-200集落）。 虽然在体外实验是强大的和具有成本效益，并比整体动物实验中，治疗性化合物的进步较少的伦理问题，需要有效性和安全性在动物模型中证明。对于体内研究，人急性白血病细胞株可原位移植到NOD.Cg-PRKDC SCID我l2rg tm1Wjl / SZJ（NSG）小鼠和TKIs的可以通过注射或口服灌胃很容易管理。某些细胞系，可能需要NSG小鼠暴露于辐射的低细胞系依赖剂量，以便为异种移植物的建立，并且在此实例中没有一个5-10天的恢复期后照射小鼠可能不能耐受口服灌胃。此手稿描述新一代的B-ALL和T-ALL移植使用特定的细胞系（697和Jurkat）作为例子，但异种移植物可使用多种细胞系建立在NSG小鼠。在该事件的其他细胞系是更适用，因为照射的要求，细胞移植，以及疾病发病和进展的定时的最优数量应通过试验确定。理想情况下，这些车型将拥有完全外显率（每次移植动物开发白血病），一致动力学（白血病的进展同样在所有的动物），以及合理的治疗窗口（处理和清除从研究开始的理想20-30天因疾病） 。可以增加细胞移植的数量，以提高外显率和动力学一致性或减少，如果需要改善的治疗窗口。 要确定是否TKIs的调解体内靶抑制，样品与TKI或仅车辆处理后的小鼠白血病移植收集。理想的情况下，剂量的政府对这些实验Ð时间表是由药代动力学研究，而这往往是由商业实验室进行，而且本文的范围之内引导。如果药动学数据是可用的，下面TKI的单剂量所需的有效靶标抑制在细胞培养和最高血清浓度化合物的浓度可以用来定义用于动物研究的起始剂量。药代动力学研究也可以通知样品采集后处理的定时药效学研究和给药途径。靶的抑制可以在任何受影响的器官来评估，但其是最容易收集和处理组织是优选的。大部分急性白血病细胞系建立在肝脏，骨髓，脾，外周血，和中枢神经系统中，虽然具体的受影响器官和植入在这些器官的范围内的模型之间变化。这里介绍的协议评估phosph邻蛋白抑制在使用Western印迹分析的骨髓，但实质器官可能更容易始终如一地收获并需要很少或没有处理之前冷冻，允许磷酸化蛋白质的样品的收集和处理过程中的降解较少机会。免疫组织化学染色，也可用于评估实体瘤或受白血病器官。 最后，TKI（次）的治疗效果在白血病原位移植瘤模型是确定的。在这些研究中，治疗开始的时间可以是多种多样的，这样的疾病或多或少成立。治疗可移植后，立即开始进行初始研究，然后在随后的研究中被延迟，直到显著疾病负担中检测到更接近一个诊断性治疗的模型。理想的是，这些动物模型也有疾病负担的非侵入性测量的能力。我们已经优化了介绍萤火虫路西法的方法酶基因导入使用的病毒样颗粒白血病细胞株，从而为疾病的发病和进展的骨髓和实体器官疾病负担和评估的非侵入性的，纵向分析。临界这种方法是使用单克隆荧光素标记的细胞系，以防止变异性与使用多克隆细胞系的相关荧光素酶表达细胞白血病的发展和无关同一个TKI 8治疗。 合在一起，可用于单个TKI的评价或多个TKI的直接比较和排序这些步骤。虽然这里介绍的协议集中于TKI的发展，这些方法可以适用于其它的目标和考虑检验发展进行了描述。因而，这种策略可能更广泛地适用于分子靶向药物的临床前评价用于治疗急性白血病。

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			<td height="21" style="height:21px;width:443px;"> </td>
			<td style="width:167px;"> </td>
			<td style="width:233px;"> </td>
			<td style="width:223px;">Reagent/Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Hydrogen Peroxide</td>
			<td style="width:167px;">MP Biomedicals</td>
			<td style="width:233px;">#02194057</td>
			<td style="width:223px;">GHS05, GHS07, H302-H318</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Sodium Orthovanadate</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#S6508</td>
			<td style="width:223px;">GHS07, H302+ H312+H332 </td>
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		<tr height="63">
			<td height="63" style="height:63px;width:443px;">2-Mercaptoethanol </td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#M7522</td>
			<td style="width:223px;">GHS05, GHS06, GHS08, GHS09, H301 + H331-H310-H315-H317-H318-H373-H410 </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">ColonyGel Human Base Medium </td>
			<td style="width:167px;">ReachBio</td>
			<td style="width:233px;">#1101</td>
			<td style="width:223px;"> </td>
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		<tr height="42">
			<td height="42" style="height:42px;width:443px;">3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#M5655</td>
			<td style="width:223px;">GHS07, GHS08, H315-H319-H335-H341 </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Difco Noble Agar </td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:233px;">#214883</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Nitrotetrazolium Blue Chloride</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#N6639</td>
			<td style="width:223px;">GHS07, H302 </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">D-Luciferin Firefly, Potassium Salt</td>
			<td style="width:167px;">PerkinElmer</td>
			<td style="width:233px;">#122796</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">4&#39;,6- Diamidino-2-phenylindole dihydochloride</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#D9542</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">FITC CD45 </td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#347463</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">FITC Mouse IgG1 Isotype control</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#51-35404X-2</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Gentamycin Sulfate</td>
			<td style="width:167px;">Sparhawk</td>
			<td style="width:233px;">#NDC58005-633-04</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Protease Inhibitors </td>
			<td style="width:167px;">Roche </td>
			<td style="width:233px;">#11836153001</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">DNase</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:233px;">#D4263 </td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Protein G Beads</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:233px;">#10-1242</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Isofluran</td>
			<td style="width:167px;">VETONE</td>
			<td style="width:233px;">#NDC13985-030-60</td>
			<td style="width:223px;"> </td>
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			<td height="21" style="height:21px;width:443px;"> </td>
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			<td height="21" style="height:21px;width:443px;"> </td>
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			<td style="width:223px;">Equipment</td>
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			<td height="21" style="height:21px;width:443px;">Cell culture dishes, diam. 35 mm &#215; H 10 mm</td>
			<td style="width:167px;">Nunclon</td>
			<td style="width:233px;">#D7804-500EA</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Cell culture dishes, diam. 100 mm x H 20 mm </td>
			<td style="width:167px;">Nunclon</td>
			<td style="width:233px;">#D8429-1CS</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">6-well plates</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#353046</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">14 gauge x 4 inch blunt-end needles</td>
			<td style="width:167px;">Cadence science</td>
			<td style="width:233px;">#7956</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">5 ml syringe with luer-lok</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#309646</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">GelCount automated colony counter</td>
			<td style="width:167px;">Oxford Optronix</td>
			<td style="width:233px;"> </td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">In vivo bioluminescence imaging system</td>
			<td style="width:167px;">PerkinElmer</td>
			<td style="width:233px;">#IVIS200</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Scout pro portable balances, scale</td>
			<td style="width:167px;">Ohaus</td>
			<td style="width:233px;">#SP202</td>
			<td style="width:223px;"> </td>
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		<tr height="22">
			<td height="22" style="height:22px;width:443px;">Broome style rodent restrainer</td>
			<td style="width:167px;">Plas-labs</td>
			<td style="width:233px;">#551-BSRR</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Ear punch, punch diameter: 2 mm</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#24210-02</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Chlorhexidine swabs, Prevantics</td>
			<td style="width:167px;">PDI</td>
			<td style="width:233px;">#B10800</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Insulin syringe 1 mL (40 Units) 29 G x 1/2</td>
			<td style="width:167px;">Monoject </td>
			<td style="width:233px;">#8881500042</td>
			<td style="width:223px;"> </td>
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		<tr height="42">
			<td height="42" style="height:42px;width:443px;">Plastic feeding needles for rodents (disposable) 20 ga x 38 mm, sterile</td>
			<td style="width:167px;">Instech</td>
			<td style="width:233px;">#FTP-20-38</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">1 mL Luer-Lok disposable syringe</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#309628</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:443px;">Lo-Dose U-100 insulin syringe with 28 G x &#189;, permanently attached needle</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:233px;">#329465</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Extra fine bonn scissors</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#14084-08</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Student fine scissors</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#91460-11</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Moria ultra fine forceps</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#11370-40</td>
			<td style="width:223px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Extra fine graefe forceps</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#11150-10</td>
			<td style="width:223px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Scalpel handle</td>
			<td style="width:167px;">FST</td>
			<td style="width:233px;">#10003-12</td>
			<td style="width:223px;"> </td>
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			<td height="21" style="height:21px;width:443px;">Scalpel blades</td>
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pre-clinical evaluation of tyrosine kinase inhibitors for treatment of acute leukemia

受体酪氨酸激酶有牵连的许多癌症，包括白血病和实体瘤的发生和发展，并且是有吸引力的成药治疗靶点。这里我们描述了一种有效的四步策略的酪氨酸激酶抑制剂（TKIs）在急性白血病的治疗的临床前评估。最初，western印迹分析是用来确认在培养的白血病细胞中抑制靶。功能活性，然后在使用甲基纤维素或软琼脂培养克隆形成实验评价。这证明在细胞培养实验活动的实验化合物在体内的利用NOD-SCID-γ（NSG）的小鼠与人类白血病细胞株原位移植评估。初步的体内药效学研究评估目标抑制白血病细胞从骨髓中分离。这种方法是用来确定管理所需的有效靶点抑制的剂量和时间安排离子。随后的研究中评估的TKI 在体内使用的荧光素酶表达的白血病细胞的功效，从而允许非侵入性生物发光监测白血病负担和治疗反应的评估使用体内生物发光成像系统。这种策略已经生效的TKI的评价在体外和体内 ，可以应用于识别分子靶向药物的具有治疗潜力或多种化合物的直接比较和确定优先次序。

这里介绍的实验评估通过的TKI介导的生化和功能的影响，并可以用来进行排名基于靶抑制在体外和体内的程度的新的化合物，还原的集落形成，并且延迟在白血病发生中NSG小鼠移植了荧光素酶标签的白血病细胞。 免疫印迹分析，使用下列与TKI的治疗，以确定抑制白血病细胞中靶蛋白的活性磷酸化形式的。治疗用的TKI导致剂量依赖性降低磷酸化的蛋白质的量， <str...

Watch this Scientific Journal Video about Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia at JoVE.com

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

受体酪氨酸激酶是异位表达在许多癌症和已被确定为在急性白血病的治疗靶点。此手稿描述为酪氨酸激酶抑制剂对急性白血病的治疗的临床前评价的有效策略。

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

pre-clinical-evaluation-tyrosine-kinase-inhibitors-for-treatment

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Medicine

Unit 1

Signaling Networks of Kinase Receptors

SCIENTISTS IN ACTION

18.0K 次观看.  University of Colorado Anschutz Medical Campus. 受体酪氨酸激酶是异位表达在许多癌症和已被确定为在急性白血病的治疗靶点。此手稿描述为酪氨酸激酶抑制剂对急性白血病的治疗的临床前评价的有效策略。 

视频: Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

挑战的药理功能磁共振成像：在临床前研究使用的5  - 羟色胺系统中的应用

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

科研

医学

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

原位膀胱肿瘤模型和评价治疗膀胱萨纳

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

处理棉鼠的研究为溶瘤病毒的临床前评估

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

人前列腺癌的临床前原位小鼠模型

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

低流量数字麻醉系统的小鼠和大鼠的用途

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

正交心脏移植的临床前猪模型

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

循环死亡后心脏捐献的临床前模型

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

小鼠右心室系统及缺氧诱发肺动脉高血压的侵入性血液动力学评估

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...