笔者想感谢加州大学戴维斯分校的鼠标老鼠繁殖生物学计划。这项研究是从默克公司，德国达姆施塔特的赠款支持。

所有动物的研究和实验是由美国加州大学戴维斯分校机构动物护理和使用管理咨询委员会批准的协议下进行。 1。 MUC1.Tg鼠标育种与繁殖<ol><li>加州大学戴维斯分校鼠标生物学计划（MBP）品种的野生型C57BL / 6雄性小鼠杂合子MUC1.Tg C57BL / 6雌性小鼠建立繁殖。 MUC1.Tg后代交付研究需要。 </li><li> MBP人员夹脚趾的后代鼠标识别定义的模式（0-99），以及适用时夹尾巴。趾部或尾部组织用于基因分型使用标准的DNA提取和聚合酶链反应（PCR）分析处理。 </li></ol> 2。研究设计<ol><li>研究组分配<ol><li>称量每只小鼠单独在一个天平上，并记录每只小鼠的重量以克计。 </li><li>这部分的程序INVolves的方法和设计的研究。对于第一部分的研究中，分配OH-BBN开始诱导膀胱癌与年龄相匹配的MUC1.Tg和野生型小鼠。 8星期后，最后OH-BBN的剂量膀胱肿瘤组织学证实存在小鼠安乐死。 </li><li>对于第二部分的研究中，随机男MUC1.Tg小鼠成相应数量的治疗组和对照组。这些小鼠在诱导肿瘤的发展过程中监测血清细胞因子和T细胞的免疫应答，然后OH-BBN的最后剂量后8周的研究终止。 </li></ol></li><li>膀胱癌的感应<ol><li> OH-BBN是一种致癌物质和剧毒。请阅读并遵循指引的材料安全数据表时，这种化学处理和存储。 </li><li>计算计量溶液的浓度，需要提供相应的剂量（3毫克），在一个体积为100μl，每只小鼠的基础上的平均重量和number的每个研究组的小鼠。 </li><li>在100％乙醇稀释OH-BBN。使终浓度为30毫克/毫升，用无菌水。最后的乙醇:水的浓度应为20:80，V / V的</li><li>管理OH-BBN口服使用不锈钢，20 G每日灌胃针，5天/周，共12周，根据治疗组的分配开始在8周龄。 </li></ol></li><li>接种治疗<ol><li>每一瓶每瓶600微升0.9％的无菌生理盐水彻底重悬绘制解决方案，通过一个0.5英寸的27 G针6X冻干肽疫苗。使用生理盐水的浓度调节，使所需的剂量交付在一个体积为100μl。 </li><li>在第20周开始，OH-BBN最后一次给药后，使用25 G针（周数对应的老鼠的年龄）皮下注射100微升八周的周期由每周一次的基础上接种疫苗。 </li></ol></li><li>监测和样品采集<ol><li>权衡所有小鼠和触诊每星期一次新的肿瘤的存在。安乐死失去的任何小鼠体重≥20％或者有可触知的肿瘤，尿液中的血液和/或尿潴留。 </li><li> OH-BBN的第一剂量之前，然后在此后4个星期的时间间隔，采集全血，通过颌下出血。血清凝血管（BD Microtainer），收集血液，并允许30分钟的血液凝块。 </li><li>在3500×g离心10分钟，离心的血液样品中的微量离心。小心转移的血清旋盖冻存管，用吸管。 </li><li>闪存冷冻在液氮中，并储存于-80°C，直到进一步分析多重。 </li><li>八星期后，最后剂量OH-BBN，安乐死所有小鼠CO 2窒息。 </li><li>每个鼠标放置在夹层板和牵制的四肢。 </li><li>使用镊子和剪刀，马柯水平切口中上腹地区。插入到切口之间的表皮层和腹壁和剪刀的帮助镊子轻轻分开皮肤从下面的组织。 </li><li>使从横向切口后，对鼠标的前端的中间轴的垂直切口。分开皮肤，从左肋，用1毫升注射器和一个22 G针，刺破心脏，并收集血液与平稳的平局。 </li><li>参照步骤2.4.3和2.4.4血清分离和存储。 </li><li>使用镊子和剪刀，剪切和剥离表皮层的其余部分。切透腹壁和腹膜，，无菌取出膀胱肿瘤免疫组化（IHC）和Western blot。 </li><li>收集脾细胞活力分析（缪斯）和酶联免疫斑点法。对于IHC，地方组织卡带和膀胱肿瘤标本固定冰鲜甲醛在室温下过夜。 </lI&gt; <li>次日，用70％乙醇取代福尔马林。 </li><li>肿瘤免疫印迹分析，同质化的肿瘤，补充蛋白质提取缓冲液加暂停蛋白酶抑制剂，转移到1.5毫升离心管中。 </li><li>涡街30-60秒，并保持在冰上5分钟。闪光冻结在液氮环境水解冻。重复该过程，涡旋，冻结和解冻两次。 </li><li>样品以10,000 xg离心10分钟，在4℃下，细胞提取物转移到新的标记的试管中。 </li><li>量化的浓度进行二喹啉酸蛋白质含量（BCA）蛋白质测量。样品储存在-80°C，直到准备Western blot分析。 </li></ol></li></ol> 3。分子生物学/西方下列程序进行验证表达MUC1在小鼠膀胱肿瘤组织使用标准的免疫印迹协议（数据未显示n）的。 <ol><li>通过SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离蛋白提取物，然后转移到PVDF膜用半干法装置。 </li><li>座将蛋白转移膜用5％脱脂牛奶，0.1％Tween-20的磷酸盐缓冲生理盐水（PBS-T）的pH值为7.4，上轨道摇床在室温下进行1小时。 </li><li>用0.1％的PBS-T中的的抗MUC1或抗β-肌动蛋白抗体，在室温下在振荡器上孵育膜1小时。 </li><li>滗出溶液，加入0.1％PBS-T洗膜。旋流膜振动筛上5分钟，然后倾析。重复此步骤两次。 </li><li>用辣根过氧化物酶（HRP）标记的二抗在室温下孵育膜1小时。 </li><li>洗3倍（步骤3.4）。 </li><li>按照协议，增强化学发光（ECL）试剂盒，荧光自激活和读取。 </li></ol> 4。多重荧光微珠免疫 &lt;醇&gt; <li>设置和计算<ol><li>使用96 - 孔的板图，分配到各孔中的空白，标准，控制，和未知。计算分析物的数量和体积，捕捉抗体和链霉亲和素 - 藻红蛋白（SA-PE）所需的检测。 </li><li>允许所有的缓冲区和稀释剂，以平衡至室温。准备的标准和使用一个空的96 - 孔板的样品稀释液的稀释倍数。 </li><li>每瓶血清基质和冻干的标准，根据制造商的方案的是标准的1:5系列稀释液，并用适当的稀释剂中的96 - 孔板。对于空白井，使用适当的稀释剂。 </li><li>所有的控制和未知样品1:2稀释在适当的稀释剂中的其余部分的96 - 孔板。 </li><li>珠混合到15毫升管中，吸液管将所需体积的适当的稀释剂。涡每个珠瓶和吸管20秒到15毫升管所需体积的每个分析物。 </lI&gt; <li>始终保护的珠子光，以避免漂白。不要将96孔板吸水材料，以避免样品的损失通过排汗。 </li></ol></li><li>含量协议<ol><li>预湿用200μl的检测缓冲液的96孔过滤的底板，并允许完全浸泡过滤器。轻轻地使用的96 - 孔板的真空装置中排出，并涂抹的底部板用纸巾干燥。 </li><li>使用多道移液器吸取25微升血清基质的空白，标准和吸管25微升的缓冲液分配的控制和未知的井井分配。 </li><li>移液器25微升的空白，标准，监控，和未知各自分配井。涡珠混合，持续20秒，并转让珠水库。 </li><li>吸取25微升到每口井珠混合。用铝箔或不透明避光板盖盖上板。 </li><l&gt;摇动板的板振动筛上以500rpm的转速在室温下两小时。排水和洗板200微升0.1％吐温20的磷酸盐缓冲液（PBS-T）两次。排水和吸干。 </li><li>准备捕获抗体溶液。用移液管将所需量的0.1％的PBS-T，并在10秒到15毫升管和涡捕获抗体。捕获抗体组合转移到每口井水库和吸管25微升。 </li><li>一小时的板振动筛上，在室温下，在500rpm下摇动板。漏，用200μl0.1％的PBS-T洗板两次。排水和吸干。 </li><li>准备SA-PE解决方案。移液器所需体积的0.1％PBS-T和SA-PE到15毫升管和旋涡，持续10秒。将溶液转移到每口井水库和吸管25微升。 </li><li>以500rpm摇板的板振动筛上，在室温下30分钟。漏，用200μl0.1％的PBS-T洗板两次。排水和b很多干燥。 </li><li>吸取100μl的0.1％PBS-T和屋顶板的一盘摇床至少两分钟，在500 rpm离心悬浮珠粒。阅读和分析Luminex公司LX200机板。 </li></ol></li></ol> 5。 IFN-γ/IL-4酶联免疫斑点法的制备及分析<ol><li>在生物安全柜，通过100微米的尼龙组织处理脾脏筛到5毫升无菌磷酸盐缓冲液（PBS）在无菌培养皿中。到的脾细胞在无菌的15毫升管中的3ml淋巴细胞分离液层。 </li><li>以600×g离心管15分钟，从红血细胞中分离淋巴细胞。以上的梯度分层淋巴细胞转移到新的15毫升无菌管。 </li><li>将音量调整到10毫升用无菌PBS。该悬浮液在600×g离心10分钟沉淀细胞。 </li><li>吸出上清，重悬在1ml的PBS细胞活力的细胞计数缪斯分析仪。按照缪斯计数与活力套件协议。 </li><li>不断的淋巴细胞的系列稀释液1.5毫升螺旋盖离心管中，在1:10的稀释因子，1:20，计数及生存力试剂（最小总体积300微升）和1:40（或根据需要）。移液器每个稀释度上下几次，以混合和分析上的缪斯。 </li><li>准备一盘地图ELISPOT板样品和条件。根据制造商的方案和移液管将100μl的培养基，肽（10微克/毫升），或加扰的肽（10微克/毫升）以每孔准备的酶联免疫斑点法板。 </li><li>吸取100μl的细胞悬浮液，提供1.0×10 6个细胞的各孔中，于37℃培养过夜孵育板。 </li><li>按照制造商的协议酶联免疫斑点法。分析解剖镜下发达的酶联免疫斑点板使用。 </li><li>量化结果通过计算相应的色斑数量纳克在每口井每个待测。这些点对应于各孔中的斑点形成细胞（SFC）的数目。 </li></ol> 6。免疫组织化学（IHC）和苏木精 - 伊红染色（H＆E） <ol><li>上述保存膀胱肿瘤组织（2.4.11节），在4微米，免疫组化分析中嵌入的石蜡和步骤节。 </li><li>执行IHC使用MUC1抗体识别MUC1串联重复区域。动物研究试剂盒使用过氧化物酶的二次小鼠抗体的反应性，以尽量减少与内源性免疫球蛋白存在于组织中。 </li><li>执行H＆E染色，使用标准协议。 </li></ol> 7。统计方法对于多重荧光微珠免疫，使用双尾学生的t检验，观察血清细胞因子治疗组与对照组之间的比较平均。酶联免疫斑点法，使用一个-W唉ANOVA比较斑点形成殖民地之间的媒体控制，炒肽和多肽组。使用Dunnett的多重比较试验，以减少假阳性结果的可能性。 p值≤0.05被认为是显着不同的分析。

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Pathol. 41 (11), 1191-1195 (1988).</li><li>Hughes, O., Denley, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUC1+mucin+expression+in+transitional+cell+carcinoma+of+the+bladder:+a+target+for+diagnosis+and+therapy+with+monoclonal+antibody+C595.">MUC1 mucin expression in transitional cell carcinoma of the bladder: a target for diagnosis and therapy with monoclonal antibody C595.</a> J. Urol. Pathol. 12, 185-197 (2000).</li><li>Rowse, G. J., Tempero, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+and+immunity+to+MUC1+in+a+human+MUC1+transgenic+murine+model.">Tolerance and immunity to MUC1 in a human MUC1 transgenic murine model.</a> Cancer Res. 58 (2), 315-321 (1998).</li><li>Mukherjee, P., Madsen, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucin-1+specific+immunotherapy+in+a+mouse+model+of+spontaneous+breast+cancer.">Mucin-1 specific immunotherapy in a mouse model of spontaneous breast cancer.</a> J. Immunother. 26 (1), 47-62 (2003).</li><li>McCormick, D. L., Ronan, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+total+dose+and+dose+schedule+on+induction+of+urinary+bladder+cancer+in+the+mouse+by+N-butyl-N-(4-hydroxy-butyl)nitrosamine.">Influence of total dose and dose schedule on induction of urinary bladder cancer in the mouse by N-butyl-N-(4-hydroxy-butyl)nitrosamine.</a> Carcinogenesis. 2 (3), 251-254 (1981).</li><li>Wurz, G. T., Gutierrez, A. 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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+human+bladder+carcinogenesis.">Experimental models of human bladder carcinogenesis.</a> Carcinogenesis. 27 (3), 374-381 (2006).</li><li>Samma, S., Uemura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+induction+of+carcinoma+in+situ+in+dog+urinary+bladder+by+sequential+treatment+with+N-methyl-N'-nitrosourea+and+N-butyl-N-(4-hydroxybutyl)-nitrosamine.">Rapid induction of carcinoma in situ in dog urinary bladder by sequential treatment with N-methyl-N'-nitrosourea and N-butyl-N-(4-hydroxybutyl)-nitrosamine.</a> Gann. 75 (5), 385-387 (1984).</li><li>Bornhof, C., Wolfrath, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+urinary+bladder+urothelial+cancers+in+the+rabbit+by+dibutylnitrosamine+with+an+artificial+bladder+calculus+as+cocarcinogen.">Induction of urinary bladder urothelial cancers in the rabbit by dibutylnitrosamine with an artificial bladder calculus as cocarcinogen.</a> Urologe A. 28 (6), 339-343 (1989).</li><li>Irving, C. 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深水铺管起重船，GTW，SMG，中日韩，AMG，GKH声明没有利益冲突。 MWD是从默克公司收到的赠款项目的首席研究员，分子量是默克公司的雇员。

浸润性移行膀胱癌和鳞状细胞在人类MUC1.Tg小鼠成功诱导免疫发展提供了临床前模型。免疫治疗的研究需要使用一个自发的，免疫的完整的模型，以评估随着时间的推移，以及免疫治疗的免疫反应的肿瘤进展的炎症反应。在自发肿瘤的发展模式，在肿瘤微环境保持完好，肿瘤发展一个更具代表性的增长率，允许的抗肿瘤治疗的效果进行评估。此外，免疫系统可以测量和监测通过的生物标志物，可以...

膀胱癌是最常见的一种癌症第四和第八，在美国男性癌症死亡的首要原因。在美国，膀胱癌新发病例估计有72,500和15,000人死亡，预计在2013年1相结合的男性和女性。膀胱癌的发病率是3倍左右高的妇女相比，在人。在美国超过90％的情况下，移行细胞癌（TCC）帐户，而鳞状细胞癌（SCC）的发生率小于2％2。乳头状台泥整体相对5年生存率为91.5％相比，只有30.9％为鳞状细胞癌2。虽然非侵入性乳 ​​头状的部队派遣国占约75％的病例在诊断时，即使处理超过50％的患者会经历复发5年之内，多达30％的患者进展到肌层浸润性疾病3,4 。典型的治疗方案非肌肉INV意味，疾病，包括经尿道前列腺切除术（TUR），然后通过膀胱内灌注化疗。高档Ta或T1肿瘤患者，可能会重复TUR 3,4化疗前。 TUR对于低同类钽复发的或高档的Ta或T1病变的患者，其次是辅助化疗或免疫治疗中的形式，卡介苗（BCG），可以使用3,4。膀胱内BCG已被证明优于膀胱内丝裂霉素C相对于时间复发5。对于T2肌层浸润性疾病，根治性膀胱切除术，化疗或不推荐的过程中，治疗3。鳞状细胞癌的患者，根治性膀胱切除似乎是最有效的治疗6。给予了很高的复发率，尽管最好的治疗，显然是有一个新的，更有效的治疗膀胱癌的需求。 拓展新的免疫疗法bladdER癌是一种可能的方法，可能有希望延长无病生存。从历史上看，BCG一直是唯一有效的免疫治疗膀胱癌。其作用机制被认为涉及的非特异性的T辅助细胞1（Th1细胞）型免疫应答的诱导，通过增加白细胞介素-2（IL-2）和干扰素-γ（IFN-γ）4。蜂窝或Th1免疫，体液，或Th2癌症免疫疗法中是至关重要的，免疫从来没有被证明是有效的抗实体肿瘤，对生长因子受体7抗体的异常。在试图改善后的好处，单用卡介苗，IFN-α2B/BCG组合的免疫进行了评估，第二阶段的临床试验与不确定的结果8。膀胱癌的免疫治疗的另一种方法可能是针对肿瘤相关抗原（塔斯），鉴定，其中的癌症免疫疗法。具体7 </s高达&gt;。 一个这样的TAA是粘蛋白1（MUC1的），它是一种细胞表面糖蛋白的过度表达在许多上皮细胞癌，膀胱癌，乳腺癌，肺癌，胰腺癌9,10。癌变过程中MUC1的表达和修改也被大大地改变，使得underglycosylation公开的氨基酸被称为可变数目的串联重复序列（VNTR）上的肽核心抗原序列。 MUC1的是一个自分子，这些免疫VNTR区域通常不暴露，由于广泛的糖基化作用，因此它们被视为由免疫系统的外国11,12。已分离出的肿瘤引流淋巴结的乳腺癌患者13，以及14,15骨髓瘤患者的血液和骨髓，MUC1的一个潜在的目标为细胞毒性T淋巴细胞（CTL）的特异性识别MUC1的抗原决定簇的细胞免疫应答。本的underglycosylate免疫优势的VNTRCTL识别MUC1的D型，从而导致肿瘤细胞的破坏16-19。癌MUC1的原住民蜂窝网络和/或体液免疫反应，但是，没有强大到足以消除肿瘤。为了加强已经存在的免疫反应弱MUC1，合成免疫肽可以通过接种疫苗，以产生足够强大的CTL反应，临床受益18,20。 ÂMUC1脂质体疫苗已经表明，提高肺癌患者的生存21,22，产生的CTL能杀死MUC1阳性肿瘤细胞，并产生Th1细胞极化的细胞因子反应23,24。 MUC1表达高水平的9,11,25，膀胱癌是检测MUC1定向免疫26,27的一个合乎逻辑的候选。此外，MUC1有潜力的舞台和档次明显伴有膀胱癌28，MUC1表达台泥作为预后因子，转移性TCC已被证明，继续表达MUC1的29。 为了评估MUC1定向免疫治疗膀胱癌的潜在效用，我们开发了免疫完好的人MUC1（hMUC1）表达的转基因小鼠模型（MUC1.Tg）膀胱癌同源C57BL / 6的背景30。人MUC1表示为一个自它自己的启动子控制下的蛋白质，从而在组织中的表达模式与观察人类30,31一致。在诱导小鼠膀胱已知的致癌物质N-丁基-N-（4 -羟丁基）亚硝胺（OH-BBN）32，然后所产生的肿瘤进行了评价的hMUC1的表达与肿瘤的类型和等级。 Th1/Th2型细胞因子水平的肿瘤的发生发展过程中的致癌物质的效果进行评估，定期收集血清样品进行多重分析。然后将小鼠治疗与MUC1的定位的肽疫苗，血清中的细胞因子和免疫反应的评价方法的特德多重荧光微珠免疫分析法和酶联免疫斑点。

<table border="1">
	<tbody>
		<tr>
			<td>Reagent&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN)</td>
			<td>TCI America</td>
			<td>B0938</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G Gavage Needles</td>
			<td>Popper &#38; Sons, Inc.</td>
			<td>7921</td>
			<td>Stainless steel</td>
		</tr>
		<tr>
			<td>Peptide Vaccine</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>investigational agent</td>
		</tr>
		<tr>
			<td>BD Microtainers</td>
			<td>BD</td>
			<td>365957</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Cassettes</td>
			<td>Simport</td>
			<td>M490-12</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Fisher Scientific</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis Buffer</td>
			<td>Pierce</td>
			<td>87787</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease &#38; Phosphatase inhibitor cocktail</td>
			<td>Thermo Scientific</td>
			<td>78444</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Cytokine 20plex Kit</td>
			<td>Invitrogen</td>
			<td>LMC006</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Microsphere Beads</td>
			<td>Luminex</td>
			<td>MC100xx-01</td>
			<td>xx is the bead region</td>
		</tr>
		<tr>
			<td>Anti-mouse TNF- Capture Antibody</td>
			<td>BD Pharmingen</td>
			<td>551225</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse TNF- Detection Antibody</td>
			<td>BD Pharmingen</td>
			<td>554415</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IFN- Capture Antibody</td>
			<td>Abcam</td>
			<td>ab10742</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IFN- Detection Antibody</td>
			<td>Abcam</td>
			<td>ab83136</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Sigma</td>
			<td>P3813-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Buffer</td>
			<td>Millipore</td>
			<td>L-MAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokine Standard</td>
			<td>Millipore</td>
			<td>MXM8070</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-screen HTS 96well filter plates</td>
			<td>Millipore</td>
			<td>MSBVN1210</td>
			<td></td>
		</tr>
		<tr>
			<td>SA-PE</td>
			<td>Invitrogen</td>
			<td>SA10044</td>
			<td></td>
		</tr>
		<tr>
			<td>100 m Nylon Tissue Sieves</td>
			<td>BD</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Splenocyte Separation Media</td>
			<td>Lonza</td>
			<td>17-829E</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF- /IL-4 ELISpot plates</td>
			<td>R&#38;D Systems</td>
			<td>ELD5217</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-MUC1 monoclonal antibody</td>
			<td>Epitomics</td>
			<td>2900-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-actin monoclonal antibody</td>
			<td>Sigma</td>
			<td>A1978</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit HRP antibody</td>
			<td>Promega</td>
			<td>W401B</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse HRP antibody</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td>SC-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>BioRad</td>
			<td>162-0174</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Protean TGX Precast Gels</td>
			<td>BioRad</td>
			<td>456-1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse Count &#38; Viability Kit</td>
			<td>Millipore</td>
			<td>MCH100104</td>
			<td></td>
		</tr>
		<tr>
			<td>MUC1 Antibody</td>
			<td>BD Pharmingen</td>
			<td>550486</td>
			<td>IHC antibody</td>
		</tr>
		<tr>
			<td>Animal Research Peroxidase Kit</td>
			<td>Dako</td>
			<td>K3954</td>
			<td>IHC staining</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore plate vaccum apparatus</td>
			<td>Millipore</td>
			<td>MSVMHTS00</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminex Lx200</td>
			<td>Millipore / Luminex</td>
			<td>40-013</td>
			<td>Manufactured by Luminex, distributed by Millipore</td>
		</tr>
		<tr>
			<td>Luminex Xponent Software</td>
			<td>Millipore / Luminex</td>
			<td>N/A</td>
			<td>Version 3.1; included with Luminex Lx200</td>
		</tr>
		<tr>
			<td>Milliple Analyst Software</td>
			<td>Milliplex / VigeneTech</td>
			<td>40-086</td>
			<td>Version 5.1</td>
		</tr>
		<tr>
			<td>Muse Cell Analyzer</td>
			<td>Millipore</td>
			<td>0500-3115</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse Software</td>
			<td>Millipore</td>
			<td>N/A</td>
			<td>Version 1.1.0.0; included with Analyzer</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Unitron</td>
			<td>Z730</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism Software</td>
			<td>Graphpad Software Inc.</td>
			<td>N/A</td>
			<td>Version 5.1</td>
		</tr>
		<tr>
			<td>Mini Protean Tetra Cell Gel apparatus</td>
			<td>BioRad</td>
			<td>165-8001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans Blot SD Cell and PowerPac</td>
			<td>BioRad</td>
			<td>170-3849</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of invasive transitional cell bladder carcinoma in immune intact human muc1 transgenic mice:  a model for immunotherapy development

浸润性膀胱癌的临床前模型开发的人粘蛋白1（MUC1）转基因小鼠（MUC1.Tg）的评估免疫治疗和/或细胞毒性化疗的目的。为了诱发膀胱癌，C57BL / 6小鼠（MUC1.Tg和野生型）与致癌物质N-丁基-N-（4 - 羟丁基）亚硝胺（OH-BBN）口服3.0毫克/天，5天/周12周。 OH-BBN血清细胞因子在肿瘤发展的影响进行评估，采集全血，通过颌下出血治疗前，每四个星期。另外，MUC1的定位的肽疫苗和安慰剂给药组小鼠每周八周。多重荧光微珠immunoanalyses的血清细胞因子在肿瘤的发展和疫苗接种后进行。终止时，干扰素γ（IFN-γ）/白细胞介素4（IL-4）的酶联免疫斑点法分析MUC1基因特异性T细胞免疫反应和肿瘤类型的病理组织学评价和档次进行。结果表明:（1）在既MUC1.Tg和野生型小鼠的膀胱癌的发病率是67％，（2）以2:1的比例在开发移行细胞癌（TCC）相比，鳞状细胞癌（SCC） （3）炎性细胞因子随时间而增加肿瘤的发展过程中，及（4）给药的肽疫苗诱导的Th1偏振光血清细胞因子的档案中和MUC1的特定的T细胞应答。所有MUC1.Tg小鼠的肿瘤MUC1表达阳性，并侵入所有肿瘤在MUC1.Tg和野生型小鼠的一半。总之，用团队的方式通过协调药理学家，免疫学家，病理学家和分子生物学家的努力，我们已经开发了一个完整的转基因小鼠模型免疫膀胱癌表达hMUC1。

新型免疫疗法和组合的影响，在膀胱癌的临床前评估需要一个合适的动物模型的发展。在我们的转基因小鼠模型，用化学致癌剂诱导OH-BBN导致膀胱癌的发病主要与一些SCC，这类似于在人类膀胱癌TCC率很高。要确定肿瘤的组织学，MUC1表达状态和免疫反应的肽疫苗治疗，21的MUC1.Tg和18个野生型小鼠处死后收集血液，膀胱，脾脏（ 图1）八周OH-BBN诱导（28周）。两个MUC1.Tg（14/21）和野生型小?...

Watch this Scientific Journal Video about Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development at JoVE.com

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

的N-丁基-N-（4 - 羟丁基）亚硝胺的人粘蛋白1（MUC1）测试的目的MUC1基因定向免疫治疗的转基因小鼠中诱导的膀胱癌模型的开发。施用MUC1的目标肽疫苗后，MUC1的细胞毒性T淋巴细胞反应证实，通过测定血清中的细胞因子水平和T细胞的比活性。

浸润性膀胱移行细胞癌中的完整的人MUC1转基因小鼠免疫诱导免疫治疗发展模型

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

induction-invasive-transitional-cell-bladder-carcinoma-immune-intact

Research

JoVE Journal

Medicine

21.2K 次观看.  University of California, Davis. 的N-丁基-N-（4 - 羟丁基）亚硝胺的人粘蛋白1（MUC1）测试的目的MUC1基因定向免疫治疗的转基因小鼠中诱导的膀胱癌模型的开发。施用MUC1的目标肽疫苗后，MUC1的细胞毒性T淋巴细胞反应证实，通过测定血清中的细胞因子水平和T细胞的比活性。

视频: Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

持续时间较长的小鼠全身热疗协议

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

科研

医学

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

的的膀胱测压小老鼠:A膀胱Chemosensation研究的使用

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型:转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

膀胱胀诱发内脏运动反应为膀胱疼痛小鼠模型

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

面神经干切断小鼠:模型，研究运动神经元损伤反应

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

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

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

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

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

小鼠加速动脉硬化的诱导:"电线损伤"模型

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

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

小鼠横主动脉狭窄诱导左心室肥厚的技术研究

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...