Breanne Cuddington holds a fellowship from the Canadian Breast Cancer Foundation. This work was sponsored by operating grants from the Cancer Research Society and the Canadian Cancer Society Research Institute (formerly the Canadian Breast Cancer Research Alliance). We thank Ann Tollefson (Saint Louis University School of Medicine) for LCRT cells and Dr. Kathleen Delaney and Marion Corrick for technical assistance with cotton rat housing and sedation.

注:根据加拿大会议上的动物护理准则使用的协议已经批准了我们的机构动物研究伦理委员会麦克马斯特大学。实验是在麦克马斯特大学动物中心进行设施。 1.培养LCRT细胞<ol><li>培养LCRT细胞在Dulbecco改进的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），2mM的L-谷氨酰胺，100U / ml青霉素和100μg/ ml链霉素。维持细胞在T-150细胞培养瓶中，在37℃和5％的CO 2。传代细胞时它们形成90％的汇合单层（每2-3天， 图1）。 </li><li>预暖1X磷酸盐缓冲盐水（PBS），1×胰蛋白酶和介质在37℃的水浴中之前，分裂细胞10分钟。 </li><li>从烧瓶中吸出培养基并冲洗细胞用5ml的1×PBS中。 </li><li>冲洗后，吸PBS和孵化细胞用2ml的1×胰蛋白酶直到细胞分离从烧瓶（〜2分钟）。 </li><li>重悬的细胞在8ml培养基（总共为10ml的细胞悬浮液），并轻轻地吸液管上下打散细胞团块。 </li><li>播种1毫升细胞悬浮于24毫升培养基（总共每T-150 25ml）和岩石烧瓶轻轻地从一边到另一边维持细胞的T-150烧瓶中。维持细胞在37℃和5％的CO 2，直到下一次分裂。 </li></ol> 2.评估病毒复制和细胞毒性LCRT细胞<ol><li>病毒复制 注:表达荧光标记病毒的结构，如绿色荧光蛋白（GFP），根据内源性病毒启动子控制有利于病毒感染的可视化和推广使用荧光板读者。 <ol><li>种子LCRT细胞进入培养板，留下一个井用于计数。种子细胞，使得它们将是80％-90％汇合的一天之后。使用10 5 CEL浓度LS /毫升（每孔100μl），以在96孔平底板一天后产生所需的汇合。 </li><li>第二天，预暖的1×PBS，1×胰蛋白酶，在37℃水浴中开始实验10分钟，以事先完全和无血清培养基。 </li><li>从计数井和冲洗细胞用5ml的1×PBS的摇动它在井的表面吸出培养基。 </li><li>冲洗后，吸出PBS孵育细胞用2ml 1X胰蛋白酶直到细胞分离从烧瓶（〜2分钟）。 </li><li>将细胞重悬于完全培养基的适当体积，以使用血细胞计数器的可数的范围内，得到的细胞密度。为了确保准确的细胞计数，彻底混合细胞悬浮液通过上下吹打接种血细胞计数仪之前。 </li><li>确定在感染所需复数（MOI）所需的感染病毒原液的体积。 所需帕确形成单位（PFU）=镀细胞的数量* MOI（PFU /细胞） 病毒股票的数量=所需需要PFU /病毒滴度的股票（PFU /毫升） </li><li>制备病毒接种物在管中的无血清培养基。加入接种到细胞前通过涡旋或移液调匀。 </li><li>感染细胞1小时，于37℃，在此之后施加DMEM + 1％FBS的维持覆盖。 </li><li>扫描板1和后两天感染（PI）以可视化的GFP荧光。 </li></ol></li><li>病毒的细胞毒性 注:低光条件下进行的刃天青细胞毒性测定为化合物是光敏的。只有良好的培养基应包括纠正背景荧光。 <ol><li>制备刃天青的5％（体积/体积）中的1×PBS溶液中。混合该溶液通过移液。 </li><li>从细胞中吸出培养基，并应用5％刃天青溶液。包括一个孔含有介质只以校正背景荧光。 </li><li>孵育细胞30分钟，在37 °C，后它使用荧光酶标仪（激发波长530，发射595纳米）读取荧光。 </li><li>分析相对于未感染的控制校正背景荧光数据。 </li></ol></li></ol> 3.住房和处理<ol><li>住房和饮食<ol><li>房子棉鼠分别减少内斗中含灭鼠床上用品（1/8"玉米芯床上用品），PVC管材不超过8英寸，nestlets为充实（ 图2）的部分聚碳酸酯老鼠笼。 </li><li>使用可固定钢篮坐在凌驾笼子，并包含啮齿类动物食物和一瓶水。 注意:此笼设置将允许动物的安全和容易捕捉，与载置对至关重要轿厢的存在的端富集管。 </li></ol></li><li>处理<ol><li>处理棉鼠在早晨之前，轮由动物设施的技术人员，以避免激动人心它们之前的过程。 </li><li>在所有的手续，穿上厚厚的皮手套的保护。 </li><li>作为动物主要停留在富集管，使用它们在常规清洗大鼠转移到一个新的笼子。可替代地，略微打开笼子，以允许处理程序以在到达他们的手，该动物随后可以通过洁肤皮肤只是肩膀上方和向下推抑制。实践中注意不要过度使用武力作为动物可能咬自己的舌头。 </li><li>要有耐心，并且使用一个稳定的手，动物有很强的飞行和战斗回应，并会尽量避免捕捉运行，并跳出笼子。重要的是，不要在尾部处理动物会发生脱套。 </li><li>陷阱在他们的富集管过直接处理的动物。这大大减少伤害和逃避。 </li></ol></li></ol> 4.捕捉和麻醉<ol><li>捕获<ol><li>穿着厚厚的皮手套保护在所有的程序离子。 </li><li>使用带有孔的空气和一个盖，麻醉诱导室足够大，以适应在容器和装在气体输出软管（ 图3）一鼻锥体的大型的透明塑料容器。 </li><li>在对工作，以使该过程更高效，对动物的曝光时间减少到异氟醚，吸入麻醉剂。确保一位研究人员负责开启和更换钢盖在笼子和感应室的盖（处理器＃1）及其联营公司是负责管和运输的感应室动物的捕捉（处理器＃ 2）。 </li><li>将笼子放在一个平面上，并取下外盖。抬起钢送纸托盘稍微缓慢操纵富集管所以它与保持架的两侧，并针对背面平行。如果有必要，使用对象来操纵富集管，而无需打开笼子，避免搅动动物（手柄R·1）。 </li><li>如果动物变得激动，离开管子，以便有足够的时间进行动物放松，在管（处理器＃1）再次定居。 </li><li>慢慢地，故意抬起，从浓缩管钢盖最远的边缘，保持在笼子里接触的另一端。作的空间足够大的塑料容器（处理程序＃2）。 </li><li>在一个平稳，快速运动，充实管凌驾推动塑料容器。保持容器保持器的侧面的接触，捕集该动物在管。作为快速执行步骤4.1.6和4.1.7越好（处理程序＃2）。 </li><li>除去钢馈纸盘并给塑料容器盖，处理程序＃2（处理程序＃1）。滑动的塑料盖的笼和容器的侧面之间，是注意到在这个过程中，被困动物的附属物。不要密封容器，因为这将使下一步更困难（处理器＃2）。 </li></ol></li><li> AnesthesIA <ol><li>确保该容器保持闭合和运输动物到感应腔。快速地放置在腔室中的动物并取出容器盖在一个流体运动（处理程序＃2）。打开并立即更换感应室盖（处理器＃1）。 </li><li>打开异氟烷向感应室（5升/分钟）的流量和监视动物为嗜睡的迹象，在该点迅速从管和容器，无论是从感应腔室移除滑动动物，以促进气体循环。 </li><li>当鼠被完全麻醉，将其移动到工作表面，并放置在鼻子和嘴到鼻锥（ 图3）。当它是反应迟钝有力脚趾捏鼠完全麻醉。 </li><li>放置在动物的眼睛兽医凡士林眼药膏，以防止干燥和擦伤。这是因为棉鼠有大眼睛，可以容易感染，如果伤害发生的重要一步。 </li> &lt;LI&gt;小心地监控和保持恒定的呼吸率，并确保该动物的鼻子保持在鼻锥整个过程中嵌合。适当调整异氟烷的流速。所需的异氟醚的量麻醉各动物会有所不同。 </li><li>程序后，返回动物笼子里，并确保它重新获得充分的流动性和胸骨斜卧。 </li></ol></li></ol> 5.准备LCRT细胞皮下肿瘤形成的注意:一个T-150烧瓶LCRT（90％汇合），得到大约2×10 7个细胞。碱对所需的细胞的总数所需的T-150烧瓶的数目。种子额外烧瓶，以确保得到所需的细胞的总数量，以及容纳电池的制备和那些需要额外的注射过程中丢失。置于冰上细胞尽可能延长细胞活力。 <ol><li>收获细胞，吸出培养基，从该烧瓶中的ð冲洗细胞用5毫升1X PBS的。 </li><li>抽吸PBS孵育细胞用2ml的1×胰蛋白酶直到细胞从烧瓶中（〜2分钟）分离。 </li><li>重悬的细胞在8ml培养基（总共为10ml的细胞悬浮液），并轻轻地吸液管上下打散细胞团块。继续从另外的烧瓶收获细胞。 </li><li>池中的所有细胞悬浮液到一个锥形管，大约4的T-150S每50毫升锥形管中。 </li><li>离心管，在160×g下 10分钟，在4℃。 </li><li>抽吸培养基并悬浮在PBS中的适当体积（10毫升的PBS元T-150）的细胞沉淀到使用血细胞计数器的可数的范围内，得到的细胞密度。为了确保准确的细胞计数，彻底混合细胞悬浮液通过上下吹打加载血细胞计数仪之前。 </li><li>计算细胞总数: 细胞的总数目收获=细胞计数（细胞/ ml）×悬浮体积（ml） </li><li>确定细胞悬浮液所需的所有注射​​体积。使每个实验2-3额外的剂量。总共皮下注射5×10 5 LCRT细胞将形成在3-4天内可触及的肿瘤。 所需的细胞总数= 5×10 5个单元×剂量的总数 体积的细胞悬液需要（毫升）=（要求*总和注射量的总细胞）/（细胞收获的总数） </li><li>吸管细胞悬浮液的所需体积成锥形管含有PBS中并充分混合。等分试样单独注射（100微升）进Eppendorf管中。在注射过程保持在冰管。 </li></ol> 6.注射注:带有两个研究者执行程序，一个用于执行，而其他显示器动物的呼吸速率和一般状况，同时在麻醉下注射。使用胰岛素注射器（29克X 1/2"，0.3毫升）的所有注射​​和一个新的针每只动物。 <ol><li>皮下注射<ol><li>捕捉和麻醉的动物（第4节）。 </li><li>剃须使用快船注射部位。棉鼠毛皮是厚并要求一个尖锐微调以获得一光滑的表面，用于注射。清洁注射部位使用棉棒70％的乙醇，并允许它在继续之前完全蒸发。 </li><li>负载注射器（29克X 1/2"，0.3毫升）与细胞制定缓慢而稳步增长。如果泡沫是很明显的甩尾一些力注射器。一旦气泡在顶部推柱塞直到液体是在针的顶端。 </li><li>解除皮肤注射部位（称为皮肤隆起），并插入针头斜面朝上。确保针头在皮肤下自由移动，以避免肌内注射。 </li><li>排出注射器的内容物均匀和缓慢。下拔出针头斜面的一面。 </li></ol></li><li>瘤内注射<ol><li>捕获ð麻醉的动物（第4节）。 </li><li>清洁注射部位使用棉棒70％的乙醇，并允许它在继续之前完全蒸发。 </li><li>负载注射器（29克X 1/2&#39;，0.3毫升）与病毒接种物由制定缓慢而稳定地握住针在直立位置。如果泡沫是很明显的甩尾一些力注射器。一旦气泡在顶端推然后柱塞直到液体是在针的顶端。 </li><li>插入针头斜面朝上进入肿瘤并排出注射器的内容物均匀和缓慢地移动的同时在针中的风扇状图案，部分撤回针头的每个移动之前，防止肿瘤的裂伤。下拔出针头斜面的一面。 注:皮下LCRT肿瘤生长快，在5-7天达到约100 立方毫米。此外，坏死和出血性中心常常形成肿瘤的表面上在几天内，并要求CAreful监测（ 图4）。 </li></ol></li><li>腹腔注射<ol><li>捕捉和麻醉的动物（第4节）。 </li><li>使用棉签清洁注射部位用70％乙醇，并允许它在继续之前完全蒸发。 </li><li>负载注射器（29克X 1/2"，0.3毫升）与药物通过制定缓慢而稳步增长。如果泡沫是很明显的甩尾一些力注射器。一旦气泡在顶端推然后柱塞直到液体是在针的顶端。 </li><li>针插入腹部的右下象限。向后拉柱塞，以确保血液或粪便不吸移，这表示所述针的不正确放置。如果发生这种情况，撤回的针头和准备一个新的注射器。当针被正确放置，排出注射器的内容物均匀和缓慢。 </li></ol></li></ol> 7.肿瘤切除和剖检<ol><li>收集和清理人升工具用70％乙醇之前，动物安乐死。 </li><li>通过所需的方法安乐死的动物，CO 2吸入（2升/分钟5-10分钟）的建议。检查动物的身体状况有任何异常。 </li><li>将背斜卧的动物上的夹层板，清洁用70％乙醇的动物。 </li><li>使用镊子解除肌肤的小腹。用剪刀通过皮肤和肌肉切割和使内侧切口运行动物的长度（肛门至下巴）。 </li><li>通过使两个切口，1横向向上胸腔的侧面和一个横跨胸骨暴露心脏和肺切的肋笼。检查肺的裂片为任何转移27,29。 </li><li>审查所有器官的异常，并记录在颜色，大小和一致性的任何变化。如果有必要，切割器官用手术刀来检查内部组织。具体来说，检查肝，肾，脾，胃肠道。 </li><li>检查淋巴结转移的膨大和27,29。 </li><li>收集肿瘤，使侧翼切口上面和下面的肿瘤，使得皮肤能够拉离体用镊子。虽然紧紧握住用镊子皮肤，用手术刀在肿瘤和真皮（ 图5）之间切割仔细切除肿瘤。 </li><li>立即将肿瘤中的10％中性缓冲福尔马林的标记的容器。 </li><li>取决于肿瘤的大小，允许1-2天（≤2毫米，小）或5-6天（&gt; 2毫米，大）用于制备切片用于组织学分析（ 图6）之前的固定。 </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+analysis+of+an+oncolytic+herpesvirus+encoding+granulocyte-macrophage+colony+stimulating+factor+for+the+treatment+of+malignant+melanoma.">Critical analysis of an oncolytic herpesvirus encoding granulocyte-macrophage colony stimulating factor for the treatment of malignant melanoma.</a> Journal of Oncolytic Virotherapy. 3, 11-20 (2014).</li><li>Jones, C., Chowdhury, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+biology+of+bovine+herpesvirus+type+1+(BHV-1),+its+role+as+a+cofactor+in+the+bovine+respiratory+disease+complex+and+development+of+improved+vaccines.">A review of the biology of bovine herpesvirus type 1 (BHV-1), its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines.</a> Anim Health Res Rev. 8 (2), 187-205 (2007).</li><li>Jones, C., Chowdhury, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+herpesvirus+type+1+(BHV-1)+is+an+important+cofactor+in+the+bovine+respiratory+disease+complex.">Bovine herpesvirus type 1 (BHV-1) is an important cofactor in the bovine respiratory disease complex.</a> Vet Clin North Am Food Anim Pract. 26 (2), 303-321 (2010).</li><li>Hushur, O., Takashima, Y., Matsumoto, Y., Otsuka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+of+bovine+herpesvirus+1+(BHV-1)+growth+in+non-permissive+cells+beyond+the+expression+of+immediate+early+genes.">Restriction of bovine herpesvirus 1 (BHV-1) growth in non-permissive cells beyond the expression of immediate early genes.</a> J Vet Med Sci. 66 (4), 453-455 (2004).</li><li>Cuddington, B. P., Dyer, A. L., Workenhe, S. T., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+bovine+herpesvirus+type+1+infects+and+kills+breast+tumor+cells+and+breast+cancer-initiating+cells+irrespective+of+tumor+subtype.">Oncolytic bovine herpesvirus type 1 infects and kills breast tumor cells and breast cancer-initiating cells irrespective of tumor subtype.</a> Cancer Gene Ther. 20 (5), 282-289 (2013).</li><li>Cuddington, B. P., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permissiveness+of+Human+Cancer+Cells+to+Oncolytic+Bovine+Herpesvirus+1+Is+Mediated+in+Part+by+KRAS+Activity.">Permissiveness of Human Cancer Cells to Oncolytic Bovine Herpesvirus 1 Is Mediated in Part by KRAS Activity.</a> J Virol. 88 (12), 6885-6895 (2014).</li><li>Small, E. J., 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=A+phase+I+trial+of+intravenous+CG7870,+a+replication-selective,+prostate-specific+antigen-targeted+oncolytic+adenovirus,+for+the+treatment+of+hormone-refractory,+metastatic+prostate+cancer.">A phase I trial of intravenous CG7870, a replication-selective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer.</a> Mol Ther. 14 (1), 107-117 (2006).</li><li>Freytag, S. O., 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=Phase+I+study+of+replication-competent+adenovirus-mediated+double+suicide+gene+therapy+for+the+treatment+of+locally+recurrent+prostate+cancer.">Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer.</a> Cancer Res. 62 (17), 4968-4976 (2002).</li><li>Benjamin, R., Helman, L., Meyers, P., Reaman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I/II+dose+escalation+and+activity+study+of+intravenous+injections+of+OCaP1+for+subjects+with+refractory+osteosarcoma+metastatic+to+lung.">A phase I/II dose escalation and activity study of intravenous injections of OCaP1 for subjects with refractory osteosarcoma metastatic to lung.</a> Hum Gene Ther. 12 (12), 1591-1593 (2001).</li><li>Prince, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cotton+Rat+in+Biomedical+Research.">The Cotton Rat in Biomedical Research.</a> Animal Welfare Information Center Newsletter. 5 (2), 3-5 (1994).</li><li>Tsai, J. C., Garlinghouse, G., McDonnell, P. J., Trousdale, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+animal+model+of+adenovirus-induced+ocular+disease.+The+cotton+rat.">An experimental animal model of adenovirus-induced ocular disease. The cotton rat.</a> Arch Ophthalmol. 110 (8), 1167-1170 (1992).</li><li>Ginsberg, H. 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=A+mouse+model+for+investigating+the+molecular+pathogenesis+of+adenovirus+pneumonia.">A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia.</a> Proc Natl Acad Sci U S A. 88 (5), 1651-1655 (1991).</li><li>Russell, S. J., Peng, K. W., Bell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+virotherapy.">Oncolytic virotherapy.</a> Nat Biotechnol. 30 (7), 658-670 (2012).</li><li>Mossman, K. L., Saffran, H. A., Smiley, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+ICP0+mutants+are+hypersensitive+to+interferon.">Herpes simplex virus ICP0 mutants are hypersensitive to interferon.</a> J Virol. 74 (4), 2052-2056 (2000).</li><li>Mossman, K. L., Smiley, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+ICP0+and+ICP34.5+counteract+distinct+interferon-induced+barriers+to+virus+replication.">Herpes simplex virus ICP0 and ICP34.5 counteract distinct interferon-induced barriers to virus replication.</a> J Virol. 76 (4), 1995-1998 (2002).</li><li>Hummel, J. L., Safroneeva, E., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ICP0-Null+HSV-1+and+interferon+signaling+defects+in+the+effective+treatment+of+breast+adenocarcinoma.">The role of ICP0-Null HSV-1 and interferon signaling defects in the effective treatment of breast adenocarcinoma.</a> Mol Ther. 12 (6), 1101-1110 (2005).</li><li>Papp, Z., Middleton, D. M., Mittal, S. K., Babiuk, L. A., Baca-Estrada, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immunization+with+recombinant+adenoviruses:+induction+of+immunity+and+protection+of+cotton+rats+against+respiratory+bovine+herpesvirus+type+1+infection.">Mucosal immunization with recombinant adenoviruses: induction of immunity and protection of cotton rats against respiratory bovine herpesvirus type 1 infection.</a> J Gen Virol. 78 (11), 2933-2943 (1997).</li><li>Papp, Z., Babiuk, L. A., Baca-Estrada, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+pre-existing+adenovirus-specific+immunity+on+immune+responses+induced+by+recombinant+adenovirus+expressing+glycoprotein+D+of+bovine+herpesvirus+type+1.">The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1.</a> Vaccine. 17 (7-8), 933-943 (1999).</li><li>Mittal, S. K., 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+systemic+and+mucosal+immune+responses+in+cotton+rats+immunized+with+human+adenovirus+type+5+recombinants+expressing+the+full+and+truncated+forms+of+bovine+herpesvirus+type+1+glycoprotein+gD.">Induction of systemic and mucosal immune responses in cotton rats immunized with human adenovirus type 5 recombinants expressing the full and truncated forms of bovine herpesvirus type 1 glycoprotein gD.</a> Virology. 222 (2), 299-309 (1996).</li><li>Steel, J. C., 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=Syngeneic+Cotton+Rat+Cancer+Model+for+Replicating+Adenoviral+Vectors.">Syngeneic Cotton Rat Cancer Model for Replicating Adenoviral Vectors.</a> Molecular Therapy. 13 (1), 123 (2006).</li><li>Toth, K., 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=Cotton+rat+tumor+model+for+the+evaluation+of+oncolytic+adenoviruses.">Cotton rat tumor model for the evaluation of oncolytic adenoviruses.</a> Hum Gene Ther. 16 (1), 139-146 (2005).</li><li>Toth, K., Spencer, J. F., Wold, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocompetent,+semi-permissive+cotton+rat+tumor+model+for+the+evaluation+of+oncolytic+adenoviruses.">Immunocompetent, semi-permissive cotton rat tumor model for the evaluation of oncolytic adenoviruses.</a> Methods Mol Med. 130, 157-168 (2007).</li><li>Steel, J. C., 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=Immunocompetent+syngeneic+cotton+rat+tumor+models+for+the+assessment+of+replication-competent+oncolytic+adenovirus.">Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus.</a> Virology. 369 (1), 131-142 (2007).</li><li>Workenhe, S. 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=Immunogenic+HSV-mediated+oncolysis+shapes+the+antitumor+immune+response+and+contributes+to+therapeutic+efficacy.">Immunogenic HSV-mediated oncolysis shapes the antitumor immune response and contributes to therapeutic efficacy.</a> Mol Ther. 22 (1), 123-131 (2014).</li><li>Sobol, P. 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=Adaptive+antiviral+immunity+is+a+determinant+of+the+therapeutic+success+of+oncolytic+virotherapy.">Adaptive antiviral immunity is a determinant of the therapeutic success of oncolytic virotherapy.</a> Mol Ther. 19 (2), 335-344 (2011).</li><li>Prince, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cotton+Rat+in+Biomedical+Research.">The Cotton Rat in Biomedical Research.</a> Animal Welfare Information Center Newsletter. 5 (2), (1994).</li></ol>

The authors acknowledge there are no financial conflicts of interest related to this research.

Cotton rats are highly excitable and have a strong flight response. Therefore, special care should be taken to minimize any undue stress on the animal. The cage setup described will allow for safe and easy capture of the animals, with the placement of the enrichment tube being of the utmost importance. When setting up cages, ensure that the enrichment tubes meet the size and shape requirements, and are placed in proper orientation in the cage. It is also important to ensure that any technicians who might be aiding in animal maintenance are informed of the specific requirements for housing. Taking these precautions will reduce agitation of the animal prior to performing procedures. It is also advisable that procedures be performed in the morning, before any handling by technicians or animal facility staff. Commencing procedures with calm animals will aid in their capture and handling.
The use of nestlets is highly recommended. In the wild, these rodents make their nests out of cotton so they are more comfortable when given the option to build them in their cages. A nest can also aid in animal capture, such that the presence of the animal in the enrichment tube may no longer be required. In this case, the plastic container may be placed directly on top of the animal from above. This action is of particular use as an experiment progresses as the rats may adapt to avoid the traditional capture method.
When handling cotton rats it is of the utmost importance that the researcher remain calm and make deliberate movements. These animals can sense unease and will respond to the perceived emotional state of the handler. Wearing thick leather gloves, following the procedures as described and approaching them calmly and with confidence will protect from bite injuries.
There are conflicting reports in the literature regarding the response of cotton rats to anesthetics 26,32. Sedation of cotton rats for extended periods of time using isoflurane is not recommended 32. We have found that, with careful monitoring, animals can be sedated using isoflurane for approximately 15-20 minutes without adverse effects. In our experience the rats respond rapidly to isoflurane and are fully sedated within several minutes. Likewise, waking occurs in less than a minute in most cases. However, anesthetizing an animal under intense stress alters their response to isoflurane, such that during waking they experience a lack of hind limb motor control and will stumble around the cage. This effect does not appear to have any lasting impact as the animal can execute proper motor control within several minutes of recovery. 
When preparing the LCRT cells for tumor injections, it is imperative that the cells be kept on ice whenever possible to prolong viability. Mix the cells with a large bore pipet to avoid shearing the cells. Furthermore, to ensure that all tumors grow at a similar rate we recommend staggering the preparation of multiple cell suspensions when more than five animals are being injected. This will avoid slower growth of tumors in animals injected last due to decreased viability of the LCRT cells. 
Excising the tumor tissue from the skin using a scalpel better maintains the tumor architecture and vasculature for histology in comparison to using tweezers to pinch the tumor from the skin. Although perfusion with phenobarbital will best preserve tissues for histopathology, obtaining a license for barbiturates was a limiting factor and was not performed. Thus, necropsies were performed immediately following euthanasia to preserve tissues. In our experience, this allowed for an accurate assessment of the pathology associated with our treatment. 
Overall, the major limiting factor is the requirement for two researchers for handling procedures. Although the procedures can be performed by one handler, the process is greatly facilitated, and the stress of the animals significantly reduced, using two handlers. These procedures require practice and careful planning. Allow sufficient time to carry out methods calmly and deliberately. There exists significant stigma about the aggressive nature of these animals and the difficulty in their handling. This article serves to make these animals more accessible to researchers by detailing a simple, established handling method that effectively minimizes stress and injury to both the researcher and the animals. Using the methods we have described, cotton rats represent an excellent animal model for the study of many immune therapies, including oncolytic virotherapy.

溶瘤病毒（OV）通过利用正常和肿瘤细胞1,2-之间的生化差异选择性地复制在肿瘤细胞中。有两种类型的OVS:那些不需要的突变来实现选择性溶瘤，称为天然存在的野生型病毒和那些必须被改造以实现选择性溶瘤。在给定的肿瘤类型突变的集合确定为一个OV 2的选择生长优势正常细胞的性质。使用OVS的安全和利益已被证明在临床试验中3-7。尽管在溶瘤病毒疗法的领域中的先进存在临床前和临床结果之间的间隙，这表明需要更好的模型来评价OVS的抗肿瘤功效。 牛疱疹病毒1型（BHV-1）是疱疹病毒家族的一个成员，并且Alphaherpesviridae亚科。 BHV-1 initi茨牛呼吸系统疾病综合症在牛，体现在各种各样的症状类似感冒8,9。 BHV-1结合使用的HSV-1的附件和条目受体如硫酸乙酰肝素和的Nectin-1 10。然而，它在Nectin-2的10的地方结合CD155。 BHV-1具有一个非常窄的宿主范围，使得其不能有效地进入并启动复制在正常和转化的鼠细胞3,4,10。这使得使用常规的鼠模型的有问题的。 BHV-1的溶瘤能力已被证明在体外 11,12。 BHV-1已经显示出从各种组织学起源，包括乳腺癌细胞和乳腺癌起始细胞11,12的启动在复制和杀死人肿瘤细胞。然而，BHV-1的抗肿瘤能力，必须在体内的免疫活性宿主的范围内进行评价。 人类腺病毒（广告），对于这有57种血清型鉴定，最常见的导致呼吸系统疾病的人。溶瘤腺病毒载体已被评估为他们的抗肿瘤功效与若干前进进入临床试验阶段13-15。尽管有前途的临床前数据，临床效果已经达到人们的期望。人肿瘤异种移植模型通常用于研究Ad载体的抗肿瘤功效，尽管它们表现出减弱的免疫应答的病毒16,17。此外，同基因小鼠模型是非允许对广告感染，使得使用这些模型不切实际17,18宿主的免疫应答的评估。 宿主免疫系统已被列为最具影响力的机制，OVS引起肿瘤细胞死亡19。耐受和非耐受的肿瘤相关抗原之间的抗肿瘤反应（TAA）的型号而异，并且可以极大地影响OV疗法的成功。在HSV-1 OV KM100（ICP0 N212VP16 在 1814年20）20,21引起肿瘤消退中的80％的荷瘤小鼠中的鼠多瘤中间T抗原乳腺癌模型22。然而，在HER-2 / neu的型号，KM100的抗肿瘤功效在同系小鼠的20％完全消退和肿瘤停滞之间在转基因变化，HER-2耐受小鼠。总之，这些数据突出的利用动物模型最能概括人类的免疫景观，充分了解哪些功能确定治疗的成功充分评估OVS的重要性。 棉鼠（Sigmodon hispidus），原产于北美和南美，是最常用的呼吸道合胞病毒感染模型（如5审查）。棉鼠中也使用抗BHV-1接种研究，他们概括与牛呼吸道疾病6,23相关联的病理。此外，BHV-1感染棉鼠是免疫原性，诱导持续粘膜和系统免疫应答6,23-25。细胞系已被来自自发纤维肉瘤和乳腺（LCRT）和骨（CCRT和VCRT），分别为26的骨肉瘤。棉鼠已被用来评估溶瘤腺病毒载体在体内的效力，因为它们很容易受到感染的广告，并表现出相似的病理学人类27-29。对于OVS的临床前评价使用免疫受损模型不仅少指示对治疗的临床反应，但它们不能顾及在溶瘤病毒疗法30,31免疫系统的作用。因此，同系和乳腺癌和骨肉瘤的肿瘤耐受棉鼠模型是相关的模型，其中，以评估OVS，如BHV-1和Ad的临床前功效不能使用常规的小鼠模型的研究。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium&#160;</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>May use any brand&#160;</td>
		</tr>
		<tr>
			<td>1X Phosphate Buffered Saline&#160;</td>
			<td></td>
			<td></td>
			<td>Can prepare in lab, filter to sterilize</td>
		</tr>
		<tr>
			<td>200 mM L-glutamine</td>
			<td>Gibco</td>
			<td>25030164</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>100x Antibiotic-Antimycotic&#160;</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Quality Biological Inc.</td>
			<td>110-001-101HI</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>T-150cm2 tissue culture flask</td>
			<td>Fisher Scientific</td>
			<td>14-826-80</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>1X TypLE Express</td>
			<td>Life Technologies</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture plate, flat bottom</td>
			<td>Fisher Scientific</td>
			<td>08-772-29</td>
			<td>May use any brand, must be tissue culture treated</td>
		</tr>
		<tr>
			<td>alamarBlue</td>
			<td>Life Technologies</td>
			<td>DAL1025</td>
			<td>May use an alternative reagent for determination of cell viability</td>
		</tr>
		<tr>
			<td>8640 Teklad 22/5 Rodent diet</td>
			<td>Harlan&#160;</td>
			<td>8640</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8&#8221; corncob rodent bedding</td>
			<td>Harlan</td>
			<td>7092</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestlets</td>
			<td>Ancare</td>
			<td>-</td>
			<td>Made of pulped virgin cotton fiber, dust-free and autoclavable</td>
		</tr>
		<tr>
			<td>50 mL Conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td>May use any brand, must be sterile</td>
		</tr>
		<tr>
			<td>Isoflurane USP, 99.9 %, inhalation anesthetic</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
			<td>M60302</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td>Can prepare in lab</td>
		</tr>
		<tr>
			<td>10 % Neutral Buffered Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>NAPCO NapFlow 1200 Class II A/B3 Biosafety Microbiological Safety Cabinet (cell culture hood)</td>
			<td>NAPCO</td>
			<td>Model used not currently available</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific Precision Heated Water Bath</td>
			<td>Fisher Scientific</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Reichert Bright-line Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Typhoon Trio BioAnalyzer&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any fluorescence plate reader</td>
		</tr>
		<tr>
			<td>Tecan Safire2 Multi-detection Microplate Reader</td>
			<td>Tecan</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any fluorescence plate reader</td>
		</tr>
		<tr>
			<td>Allegra 6R benchtop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>366816</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Table Top Anaesthesia machine</td>
			<td>VetEquip</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any brand, must be portable</td>
		</tr>
		<tr>
			<td>Wahl Peanut Mini Clippers</td>
			<td>Wahl</td>
			<td></td>
			<td>May use any brand of small clippers</td>
		</tr>
		<tr>
			<td>Insulin syringes 29 G x 1/2&#39;, 0.3 mL</td>
			<td>BD</td>
			<td>329464</td>
			<td>May use any brand. Insulin syringes are recommended as they make injections easier through the rat&#8217;s tough skin.&#160;</td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>MedPro</td>
			<td>018-425</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Sharp-Pointed Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>8940</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Dissecting Tissue Forceps</td>
			<td>Fisher Scientific</td>
			<td>13-812-41</td>
			<td>May use any brand</td>
		</tr>
	</tbody>
</table>

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.

由于棉鼠的极其激发的性质，是熟悉并利用优化，以减少动物的应力缓和在其作为临床前动物模型中使用的程序。采用适当的处理技术也将最大限度地减少风险，研究员。 当使用棉鼠当务之急是保持冷静。老鼠是非常激动，将试图逃跑的笼子。使用富集管和nestlets将最大限度地减少逃逸尝试。 图2示出了最佳的笼设置在棉鼠，包括富集管安置捕获提供帮助。此外，工作在一个小房间如果可能的话在夺回来帮助。如果发生逃逸，等待动物冷静下来，保持静止，然后覆盖它与明确的捕获容器或盖上戴手套的手，小心不要过度使用武力。 在对比小鼠，棉鼠具有细长吻瓦特HICH需要不同的鼻子嵌合递送麻醉气体。 图3示出了鼻锥工程化以适当地适合棉鼠和最大化递送异氟烷。使用橡胶膜作为嵌合可能导致创伤的大鼠的鼻子。 如果可能的话获得丢弃的动物练习之前，尝试他们在大鼠注射技术（这些并不需要其他研究人员，棉鼠或其他）。这将允许研究者获得熟悉针头和如何安全地处理它们。胰岛素注射器建议在棉鼠注射作为自己的皮肤厚韧相比于鼠标。然而，一个较大的针（21g的值x 1&#39;），可用于在注射肿瘤细胞，以避免对细胞活力的损失是由于在注射过程中细胞剪切。安全注意事项应遵循，如不重新盖上针头和妥善处置进入锐器容器。 该injec存活的肿瘤细胞的灰是正确的肿瘤的形成很重要的。 图1A示出了可以用于注射制备成棉鼠LCRT细胞的健康单层。在比较图1B示出LCRT细胞具有低的可行性，而不应被用于注射。它使用了一种用于注射细胞计数时，以验证肿瘤细胞活力，如台盼蓝染色法是重要的。 从LCRT细胞所形成的肿瘤快速生长和坏死中心常形成（ 图4A）。正因为如此，肿瘤的形成应仔细监测以避免溃疡（ 图4B）。如果发生溃疡的动物应该被牺牲掉，以避免感染，甚至死亡的败血症。 通常最好通过组织学分析研究的抗肿瘤治疗的效果。这需要肿瘤验尸切除。维持肿瘤组织的完整性将水库ULT的样品是在体内的肿瘤的更准确的表示中， 图5示出了切除术通过该肿瘤被小心地从使用手术刀和镊子周围组织分开。通过使用镊子可能破裂的肿瘤或破坏肿瘤组织的完整性，影响正确的组织学分析从周围组织拉强行去除肿瘤。肿瘤的致密且高度血管结构，如在图6中看到的那样，通过该切除术保持。这是在对各种影响肿瘤脉管系统的治疗分析重要的，因为与很多OVS的情况。 <img alt="图1" src="/files/ftp_upload/52232/52232fig1highres.jpg" /> 图1:健康LCRT细胞明视场显微镜图像（A）表型（〜90％存活）LCRT细胞准备准备注入棉鼠。LCRT细胞（B）不良型不适合注入。圆形的细胞是死的或濒死（由箭头表示）。图像被抓获放大10倍;比例尺= 1毫米。 <img alt="图2" src="/files/ftp_upload/52232/52232fig2highres.jpg" /> 图2:笼安装，便于棉鼠捕获的例子对笼子和包容nestlets有助于动物捕获年底富集管的优化配置。 <img alt="图3" src="/files/ftp_upload/52232/52232fig3highres.jpg" /> 图3:麻醉鼻锥递送异氟烷来麻醉棉鼠嵌合制造鼻锥体适合的棉鼠细长吻，以确保准确递送异氟烷气体的无创伤的鼻子。 <i毫克ALT ="图4"SRC ="/文件/ ftp_upload / 52232 / 52232fig4highres.jpg"/&gt; 图4:在一个皮下LCRT肿瘤坏死组织（A）的肿瘤组织的坏死的早期阶段。动物应仔细监测，以避免发展为（B）全开溃疡。应该被牺牲的动物，如果肿瘤溃烂感染和败血症会导致。 <img alt="图5" src="/files/ftp_upload/52232/52232fig5highres.jpg" /> 图5:皮下LCRT肿瘤为组织学上切除一棉鼠的侧翼的皮下肿瘤从皮肤用手术刀以维持肿瘤组织的完整性小心除去，从而提供了用于组织学分析的一个更好的代表性肿瘤架构。 <img alt="图6" src="/files/ftp_upload/52232/52232fig6highres.jpg"/&gt; 图6:从皮下LCRT肿瘤病理组织切片用苏木和伊红（H＆E）石蜡切片检查LCRT肿瘤组织的形态。图像捕捉，放大20倍;比例尺= 1毫米。

Watch this Scientific Journal Video about Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses at JoVE.com

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

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 ...

handling-cotton-rat-studies-for-pre-clinical-evaluation-oncolytic

Universidad San Sebastian

Research

JoVE Journal

Medicine

9.2K 次观看.  McMaster University. 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. 

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

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

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

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

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 ...

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

溶瘤病毒的Transarterial管理局大鼠原位肝癌的局部治疗

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

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 ...

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

分析营养补充剂的有益作用于肠上皮屏障功能在实验性结肠炎

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

大鼠舌的机械和粘弹性的体内评估

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

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. ...