This work was supported by the KU Leuven PF &#8216;IMIR&#8217;, the FWO Research community on biology and ecology of bacterial and fungal biofilms (FWO: WO.026.11N) and by the FWO project G.0804.11. SK gratefully acknowledges KU Leuven for the PDMK 11/089 fellowship and FWO for the postdoctoral fellowship. We are grateful to Nico Vangoethem for his assistance with preparation of the figures. We would like to acknowledge Celia Lobo Romero for technical assistance during in vivo experimental procedures.

注：所有的动物实验批准了鲁汶大学（项目编号090/2013）的伦理委员会。维持动物按照鲁汶大学动物护理准则。 1. C.白色念珠菌生长<ol><li>动物实验开始二十四小时之前，通过加入10克的酵母提取物的造粒，将20g细菌蛋白胨，和15g造粒琼脂制备的YPD平板上。弥补体积至900毫升Milli-Q水和高压釜中。 </li><li>加50毫升无菌的40％的葡萄糖。调匀，倒入培养皿中。离开琼脂板上以冷却和固化。 注：在本研究中，使用两个株，即野生型C.白色念珠菌 SC5314 - GLUC阴性菌株（称为WT）和C.白色念珠菌 SKCA23菌株，它是一种野生型C.白色念珠菌 SC5314转化Clp10 :: Act1p-gLUC59质粒22。在这株（命名为SKCA23- 一CTgLuc）GLUC融合到内源PGA59基因ACT1（肌动蛋白）启动子的控制下（该启动子被激活，在酵母，以及菌丝真菌的生长阶段）。这株可以从教授帕特里克·范·Dijck，鲁汶大学，比利时鲁汶的实验室要求。质粒Clp10 :: Act1p-gLUC59质粒22好心教授C. D&#39;Enfert，巴斯德研究所，法国巴黎的捐赠。 </li><li>在-80℃下保持两个菌株中的甘油的库存和存储。 </li><li>在此之前的任何实验连胜株上的YPD板。孵育板在37℃下过夜。 </li></ol> 2.导管件准备<ol><li>实验前，确定需要多少导管。因此能够植入高达每鼠标器6的导管件（3导管在左侧和3导管在右侧的动物（ 图2A的）。 </li><li>二十四小时之前，动物外科手术，打开包年龄含有生物安全柜下三腔导管。移除所有不必要的部分，用无菌镊子和切断附连到所述导管，用无菌解剖刀的部分。放置标尺下的塑料包，并根据标尺上的刻度（图1）切聚氨酯导管件恰好1厘米。 注意：一提的是这种类型的导管件不是磷光，因此，适合于BLI 18,19是重要的。 </li><li>最多15个切割导管件（步骤2.2）将分为2 ml离心管中。总是准备3件额外，其用于粘附力的时间后，以列举附着念珠菌细胞的装置上的量。 </li><li>补充导管具有约100％的胎牛血清（FBS）的1.8毫升。 </li><li>大力涡和增加额外的100-200微升100％FBS的。完全覆盖所有的导管部分血清。 </li><li>孵育在37℃下过夜。 </li&gt; </ol> 3.动物和抑制免疫系统<ol><li>保持雌性BALB / c小鼠（约8周龄）的独立通风过滤器上笼免费获得食物的标准和水自由采食 。 </li><li>通过加入地塞米松（0.4毫克/升），以动物的饮用水发起抑制动物手术前的免疫系统24小时的。为了避免宿主的任何细菌污染，补充饮用水与抗生素， 例如氨苄西林钠粉末（0.5克/升）。 </li><li>保持整个实验（6天）期间的动物的免疫抑制。 </li></ol> 4. 体外白色念珠菌黏附在FBS涂聚氨酯底材<ol><li>将每个血清涂层的导管件到一个新的1.5 ml离心管。 </li><li>刮去一些C.白念珠菌细胞生长在YPD平板（第1步），并暂停它们在1ml PBS中。 </li><li>稀能迪达细胞（1：100），在一个单独的离心管中。取10微升的稀释样品和其应用在一个细胞计数室。算上至少16个小方格。 </li><li>准备在RPMI 1640培养基念珠菌细胞（每个菌株分别）至5×10 4个细胞/ ml的终浓度。 </li><li>加入1毫升细胞悬液以每血清涂覆导管件。 </li><li>大力旋涡振荡，并确保该导管浸没在介质和不浮在顶部。 </li><li>孵育导管在37℃下90分钟（密合性的周期）。 </li><li>除去导管，用无菌镊子并用1ml的PBS洗两次它们。在该步骤中做出肯定的洗涤流体通过保持导管在垂直位置，同时轻轻地冲洗导管穿过内腔。重要的是，不使用强流动，这可能会导致除去附着的细胞。 </li><li>每次洗导管转移到一个干净的离心管（每一块涂定）。 </li><li>置于冰上，并保持在那里，直到手术。 </li></ol> 5.麻醉<ol><li>通过混合75微升氯胺酮（100毫克/毫升）用100μl美托咪定（1毫克/毫升）和825微升无菌盐水制备麻醉。辖腹膜内（IP）60-80微升每10g体重麻醉鸡尾酒，导致45-60毫克/千克氯胺酮和0.6-0.8毫克/千克美托咪定的剂量。 </li><li>逆转麻醉，稀释50微升阿替美唑（5毫克/毫升）在4.95毫升盐水，施用的ip每10g体重100μl的作为解毒剂，造成了0.5毫克/公斤的剂量。 </li><li>注射麻醉后，将动物到一个单独的笼子里，等待，直到它完全睡着了。 </li><li>确认动物的光皮捏和脚趾捏，不造成对皮肤的损害适当的麻醉。任何观察到的运动指示该动物不能充分麻醉进行外科手术。如果发生这种情况，等待腠分钟的时间，直至该动物的PLE不显示运动后的皮肤或脚趾捏任何迹象。 </li></ol> 6.动物手术<ol><li>转移从一个干净的纸巾盛放在加热垫笼麻醉动物，预先温热至37℃（ 图2A，（1））。 注：一个便宜的替代方法是使用电热毯。另外，也可以使用等温垫，必须升温中之前，动物外科手术的微波。 </li><li>对眼睛应用眼药膏。 </li><li>剃动物的下背部用电动剃刀。删除所有的动物毛发和干净的纸巾转移动物。皮肤消毒（ 例如 ，用1％的碘的异丙醇或70％的乙醇为0.5％氯己定），并离开该消毒区干燥大约1分钟（ 图2A，（2））。 </li><li>做一个小切口，在皮肤（一个在左边，一个在右侧的动物）（约0.5＆＃8211 1厘米）（ 图2A，（3））。 </li><li>剖析皮下组织用剪刀创建两个皮下隧道。每个隧道应该是大约1.5厘米长，1厘米宽。 </li><li>插入三个导管件，先前感染念珠菌 ，在每个隧道。确保该导管处在一个水平布置成彼此相邻并且它们不相互覆盖，使六个导管片段在总的注入（ 图2A，（4））。 </li><li>关闭与缝合切口。或者，使用DERMABOND来闭合伤口。 </li><li>消毒伤口轻轻地用0.5％氯己定在70％的醇或与碘的异丙醇（1％）。 </li><li>应用局部麻醉剂（利多卡因凝胶，2％），直接在伤口上。 </li><li>辖逆转麻醉（协议5，步骤2）：腹膜内100μl的每10g体重。 </li><li>转移动物到一个干净笼子先前放置在加热板上。保持动物独立和温暖，直到动物是完全清醒的。同时，继续进行下一个动物的动作和植入物。一旦所有的操作都​​是动物完全清醒。转移到一个笼子里。定期监测动物。 </li></ol> 7.生物发光成像：腔肠（CTZ）的制备，基材为G. princeps的荧光素酶 <ol><li>通过将5毫克/毫升CTZ在酸化乙醇或根据制造商的说明准备新鲜腔肠素（CTZ）原液。 </li><li>通过稀释在无菌PBS原液1:10的制备1.2毫工作溶液。 注：注入100微升CTZ工作溶液皮下注射在包围导管的区域。使用胰岛素注射器皮下注射CTZ的。 </li><li>始终保持CTZ在黑暗中（ 例如 ，包括含CTZ用铝箔离心管）。储存原液在-80℃的实验的持续时间。 </li></ol&gt; 8.生物发光成像<ol><li>初始化BLI相机。 </li><li>麻醉使用感应框的动物。使用异氟醚的气体混合物中的氧气，N 2 O / O 2或空气中，在2-3％。 </li><li>诱导后，维持麻醉在感应框和在1.5-2％的成像室。 </li><li>开始成像会话之前，将成像板在位置A，其对应于10cm的视场。确保麻醉网点和动物通过放置一个沉睡的动物在框中的右侧位置。 </li><li>采取一些照片，直到动物是在期望的成像位置时，在右视野的镜头下。 </li><li>准备两个胰岛素注射器每片含100微升的CTZ工作液。 </li><li>将一个动物在板凳上，并保持它睡着了鼻锥的方式提供气体麻醉。 </li><li>把注射器针头的地方周围的皮下导管注入所述CTZ同时在导管的顶端。 </li><li>立即注射后，将动物在暖板上在相机盒并启动生物发光图像采集。 </li><li>获得连续扫描以采集时间范围从20至60秒（取决于信号强度），直到最大的信号强度被达到。在采集下一帧，通过这样的ROI放置一个ROI在每个导管三人并测量光子通量测量的先前获得的帧的BLI信号强度。 </li><li>从下一个动物（S）第7步重复。 </li><li>成像后，返回动物的笼子。一个实验的过程中为纵向的，非侵入性的后续的生物膜的形成过程中重复BLI。 </li><li>分析用的图像生活软件BLI数据。放置固定尺寸的矩形的ROI在每个导管三人并测量光子通量（辐射率），通过每个ROI。重复此为每个动物。 </li><li>报告每个导管三人每秒光子通量的BLI信号强度。通过绘制平均值和每秒的光子通量为每一组的SD表示以对数刻度的BLI数据。进行统计分析上的日志10转换后的数据。 </li></ol> 9.导管植<ol><li>准备包含每个导管装置1毫升的PBS，一种离心管，并让他们在冰。 </li><li>通过颈脱位安乐死的动物的（ 图2B，（1））。 </li><li>消毒用0.5％氯己定的回在70％的醇或与碘的异丙醇（1％）的皮肤上。 </li><li>做一个切口（约3厘米）以上的导管。 </li><li>切皮下组织和由一个从下使用无菌镊子皮下组织取出导管片段1（ 图2B，（2））。 </li><li>在垂直位置轻轻处理导管并用1ml无菌PBS两次洗涤。将每个导管片到一个单独的离心管（在步骤1中制备的）。 </li></ol>通过菌落形成单位计数（CFU的）和结果的统计分析10定量生物膜相关细胞<ol><li>声处理导管预先放入1ml的PBS中进行10分钟，以40,000赫兹在水浴超声波仪，把它们在冰上。 </li><li>超声处理后，再次剧烈涡旋30秒并置于冰上。 </li><li>制备含有900微升的PBS中两个额外的离心管。 </li><li>使1:10 1：从原始样本（一个包含导管）100稀释，并将所有离心管冰上。 </li><li>板100微升原始样品的1:10和1：在重复100稀释在YPD琼脂平板上。 </li><li>孵育板2天，在37℃和计数的CFU。 </li><li>计数生长在YPD琼脂板上的菌落（菌落可数量，最大300菌落/板），并通过适当的DILU它们相乘化因子（1X-原来，10倍或100倍稀释）。进一步乘以各导管件10倍，其对应于集落的最终量在1ml试管含有导管。带来菌落的最终金额记录10个细胞/导管件与标准偏差（SD）。表达数据作为平均值±SD表示。 </li><li>放置每个导管和特定组的所有值到电子表格和/或统计程序，以执行上述的计算和统计分析。表明特定群体进行比较， 即 WT 与突变或2 天比。 6天生物膜形成，并与对log10的变换数据图基检测后进行非配对t检验和方差分析。 <ol><li>表达的意义通过的 p值的水平。在这项研究中表明的意义，如果P值&lt;0.05，表示如下：* P &lt;0.05，** P &lt;0.005，*** P &lt;0.0005。 </li></ol></li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+forms+biofilms+on+the+vaginal+mucosa.">Candida albicans forms biofilms on the vaginal mucosa.</a> Microbiology. 156 (12), 3635-3644 (2010).</li><li>Dongari-Bagtzoglou, A., Kashleva, H., Dwivedi, P., Diaz, P., Vasilakos, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characerterization+of+mucosal+Candida+albicans+biofilms.">Characerterization of mucosal Candida albicans biofilms.</a> PloS One. 4, e7976 (2009).</li><li>Ricicov&#225;, M., Kuchar&#237;kov&#225;, S., Tournu, H., Hendrix, J., Bujd&#225;kov&#225;, H., Van Eldere, J., Lagrou, K., Van Dijck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+biofilm+formation+in+a+new+in+vivo+rat+model.">Candida albicans biofilm formation in a new in vivo rat model.</a> Microbiol. 156, 909-919 (2010).</li><li>Kuchar&#237;kov&#225;, S., Tournu, H., Holtappels, M., Van Dijck, P., Lagrou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+efficacy+of+anidulafungin+against+Candida+albicans+mature+biofilms+in+a+novel+rat+model+of+catheter-associated+candidiasis.">In vivo efficacy of anidulafungin against Candida albicans mature biofilms in a novel rat model of catheter-associated candidiasis.</a> Antimicrob Agents Chemother. 54 (10), 4474-4478 (2010).</li><li>Bink, A., 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=The+nonsteroidal+antiinflammatory+drug+diclofenac+potentiates+the+in+vivo+activity+of+caspofungin+against+Candida+albicans+biofilms.">The nonsteroidal antiinflammatory drug diclofenac potentiates the in vivo activity of caspofungin against Candida albicans biofilms.</a> J Infect Dis. 206 (11), 1790-1797 (2012).</li><li>Van de Velde, G., Kuchar&#237;kov&#225;, D., Schrevens, D., Himmelreich, U., Van Dijck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+non-invasive+monitoring+of+pathogen-host+interactions+during+Candida+albicans+biofilm+formation+using+in+vivo+bioluminescence.">Towards non-invasive monitoring of pathogen-host interactions during Candida albicans biofilm formation using in vivo bioluminescence.</a> Cell Microbiol. 16 (1), 115-130 (2014).</li><li>Van de Velde, G., Kuchar&#237;kov&#225;, S., Van Dijck, P., Himmelreich, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+fungal+biofilm+development+in+live+animals.">Bioluminescence imaging of fungal biofilm development in live animals.</a> Methods in Molecular Biology. 1098, 153-167 (2014).</li><li>Gahan, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterial+lux+reporter+system:+applications+in+bacterial+localisation+studies.">The bacterial lux reporter system: applications in bacterial localisation studies.</a> Curr Gene Ther. 12 (1), 12-19 (2012).</li><li>Doyle, T. C., Nawotka, K. A., Kawahara, C. B., Francis, K. P., Contag, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+fungal+infections+in+living+mice+using+bioluminescent+pathogenic+Candida+albicans+strains+transformed+with+the+firefly+luciferase+gene.">Visualizing fungal infections in living mice using bioluminescent pathogenic Candida albicans strains transformed with the firefly luciferase gene.</a> Microb Pathog. 40 (2), 82-90 (2006).</li><li>Enjalbert, B., 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+multifunctional+synthetic+Gaussia+princeps+luciferase+reporter+for+live+imaging+of+Candida+albicans+infections.">A multifunctional synthetic Gaussia princeps luciferase reporter for live imaging of Candida albicans infections.</a> Infect Immun. 77 (11), 4847-4858 (2009).</li><li>Mosci, P., 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+novel+bioluminescence+mouse+model+for+monitoring+oropharyngeal+candidiasis+in+mice.">A novel bioluminescence mouse model for monitoring oropharyngeal candidiasis in mice.</a> Virulence. 4 (3), 250-254 (2013).</li><li>Van Wijngaerden, E., 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=Foreign+body+infection:+a+new+rat+model+for+prophylaxis+and+treatment.">Foreign body infection: a new rat model for prophylaxis and treatment.</a> J. Antimicrob. Chemoth. 44 (5), 669-674 (1999).</li><li>Nobile, C. J., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell-surface+genes+and+biofilm+formation+by+the+C.+albicans+transcription+factor+Bcr1p.">Regulation of cell-surface genes and biofilm formation by the C. albicans transcription factor Bcr1p.</a> Curr. Biol. 15 (12), 1150-1155 (2005).</li></ol>

作者宣称，他们有没有竞争的财务权益。

使用动物模型，尤其是啮齿动物模型的，用于研究致力于微生物生物膜是非常重要的，因为宿主的免疫系统是在生物膜形成， 在体外模型不能解释的一个重要因素。在这项研究中，我们描述了一个相对简单的皮下C.白色念珠菌生物膜的小鼠模型，可以很容易地通过了一个研究实验室，也不需要很强的技术技能。最初开发这种模式来研究大鼠24 表皮葡萄球菌生物膜的形?...

白色念珠菌是一种共生生物，它可以在不同的地点健康个体中找到，例如在皮肤上或作为胃肠道和阴道菌群的一部分。然而，在住院治疗，尤其是免疫功能低下的患者，可能会造成大范围感染1。在这样的个人，削弱免疫系统使念珠菌细胞传播到血液中，并侵入深层组织造成危及生命的感染。此外，非生物基材如中心静脉和导尿管的存在下，人工心脏瓣膜和关节可提供一个利基念珠菌附件2。粘附于这种基材是用于进一步生物膜发展，它代表一个层嵌入在细胞外聚合物材料酵母和菌丝细胞，主要由多糖2的一个先决条件。C.白色念珠菌导管机相关性感染与高死亡率有关。生物膜的一般特征是它们减少的易感性的已知抗真菌剂，如唑类3,4。仅新类抗真菌药物，例如棘白菌素和两性霉素B的脂质体制剂被证明是有效对抗导管相关感染5-7。由于生物膜抵御抗真菌剂，治疗方法非常有限，往往导致拔除尿管及其后续替代作为唯一的解决方案。 我们大部分的电流C.理解白念珠菌生物膜发展来源于在非生物基质如聚苯乙烯，或塑料用于上述装置， 即，有机硅，聚氨酯2的制造体外研究。这些模型是相当先进的，并试图尽可能接近模拟体内的情况成为可能。然而，这些系统不涉及连续血流量和叔他宿主的免疫系统。这导致了在体内模型系统，诸如在中央静脉导管（CVC）模型8-10，口腔念珠菌11义齿口腔炎模型和导管相关念珠菌12小鼠模型的发展。此外，C。白色念珠菌生物膜发展进行了研究在体内的粘膜表面，如那些从阴道13和口腔14上。我们的实验室贡献了建立皮下的C.白念珠菌生物膜模型，这是基于对受感染的导管件上Sprague Dawley大鼠15的背面的植入物。被成功地用于在我们的实验室该模型以测试生物膜易感性氟康唑和棘白菌素药物5,16，以研究双氯芬酸和卡泊芬净17的组合治疗的效果。最近，我们采用了这种系统在BALB / c小鼠18,19使用。在与其它体内模型相比，本皮下模型的主要优点是研究每只动物植入的导管件的管腔内部开发多种生物膜的可能性。 以减少实验动物的数量，我们已经适应了这个模型来研究C的发展白色念珠菌生物膜的非侵入性地用生物发光成像（BLI）18,19。这种方法被证明是一个功能强大的技术，它可以用来通过测量在感兴趣的区域（植入导管的区域中的情况下）特定BLI信号进行量化生物膜，避免动物牺牲。相比于细菌，其可以同时表达该基因和所需的生物发光反应基板由于引入一个特定勒克斯操纵子20的，大多数真核有机体，包括C。白色念珠菌，依赖于荧光素酶基因的异源表达加上特定基底，例如D-萤光素或腔肠素21的外部施用。这可能是由于真菌细胞壁和C的存在下白色念珠菌形态发生，细胞内输送的底物萤光素酶是一种主要挑战21。为了解决这个问题，Enjalbert 等人 22工程化的菌株，其中的合成C.该基因的天然分泌的Gaussia princeps的荧光素酶（GLUC）的白色念珠菌的密码子最优化形式融合到到C.白色念珠菌PGA59基因，GPI-锚定的细胞壁蛋白。因为荧光素酶的细胞壁中存在的，可避免关于基板的胞内可用性问题。这个特殊的系统被用来研究引起C.浅表感染念珠菌 22。最近，BLI也被用来跟踪口咽念珠菌及其possibl的进展Ë治疗23。这样的研究结果支持使用BLI作为有前途的技术，研究引起自由生活的细胞，而且还与设备相关的感染的感染。 在这项研究中，我们描述了C.白念珠菌生物膜发展上的聚氨酯导管件在BALB / c小鼠和其量化使用BLI。我们期间密合性的时期，随后通过植入小鼠和随后的生物膜发展在活的动物提供体外定植聚氨酯导管的详细协议。除了 ​​测量由C.发射的BLI信号白色念珠菌细胞，我们也决定了菌落形成单位与标准技术生物膜真菌负荷的定量比较。

<table border="0" cellpadding="0" cellspacing="0" width="877">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Name of Reagent</td>
			<td style="width:199px;">Company</td>
			<td style="width:113px;">Catalog Number</td>
			<td style="width:236px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Yeast extract granulated</td>
			<td style="width:199px;">Merck</td>
			<td style="width:113px;">MERC1.03753.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Bacteriological peptone</td>
			<td style="width:199px;">Oxoid</td>
			<td style="width:113px;">LP037B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Agar granulated</td>
			<td style="width:199px;">Difco</td>
			<td style="width:113px;">214530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">D-(+)-glucose</td>
			<td style="width:199px;">Fluka</td>
			<td style="width:113px;">49159-5KG</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Phosphate buffered saline&#160;</td>
			<td style="width:199px;">Prepared in the laboratory</td>
			<td style="width:113px;"></td>
			<td>for 1L of 10x PBS: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">RPMI1640 with L-glutamine and without sodium carbonate&#160;</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">R6504-1L</td>
			<td>Prepare according the protocol for Candida albicans drug susceptibility testing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">3-(N-Morpholino)propanesulfonic acid (MOPS)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">M1254</td>
			<td>MOPS is used to adjust the pH of RPMI medium (pH 7.0)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">fetal bovine serum (FBS)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">F7524</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Polyurethane tripe-lumen intravenous catheter piece (2.4 mm diameter, Certofix Trio S730)&#160;</td>
			<td style="width:199px;">BBraun</td>
			<td style="width:113px;">CV-15703</td>
			<td>Polyurethane part cut into 1 cm pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dexamethasone</td>
			<td style="width:199px;">Fagron SAS, France</td>
			<td style="width:113px;">611139</td>
			<td>Immunosuppressant (stock solution 10 mg/ml)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Ampicillin&#160;</td>
			<td style="width:199px;">Duchefa Biochemie, The Netherlands</td>
			<td style="width:113px;">A0104</td>
			<td>Antibacterial prophylaxis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ketamine 1000</td>
			<td>Pfizer</td>
			<td style="width:113px;">804 119</td>
			<td>Anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Domitor</td>
			<td>Pfizer</td>
			<td style="width:113px;">134737-1</td>
			<td>Anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Antisedan</td>
			<td>Pfizer</td>
			<td style="width:113px;">134783-2</td>
			<td>Reversal of anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Xylocaine gel (2%) - this is Linisol</td>
			<td style="width:199px;">AstraZeneca</td>
			<td style="width:113px;">352 1206</td>
			<td>Local anesthetic for the skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terramycin/ polymyxin-b ophthalmic ointment</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>To prevent drying and infection of eyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Coelenterazine&#160;</td>
			<td style="width:199px;">Prolume (Nanolight)</td>
			<td style="width:113px;">NF-CTZ-FB</td>
			<td>Light sensitive agent (must be kept in the dark)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodine isopropanol (1%)</td>
			<td style="width:199px;">3M&#8482; DuraPrep&#8482;&#160;</td>
			<td style="width:113px;"></td>
			<td>Disinfectant for the skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 % chlorhexidine in 70 % alcohol.&#160;&#160;</td>
			<td style="width:199px;">Cedium</td>
			<td style="width:113px;"></td>
			<td>Disinfectant for the skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Equipment</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Cell counting chamber</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Insulin syringes (0.3 ml)</td>
			<td style="width:199px;">Terumo Myjector 29G</td>
			<td style="width:113px;">324826</td>
			<td>For injection of coelenterazine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Electric razor</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>For small animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Sterile surgical tools</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>Scissors, 2 pairs of tweezers, scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Heating pad</td>
			<td style="width:199px;">Leica</td>
			<td style="width:113px;">14042321474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin suture</td>
			<td style="width:199px;">Johnson&#38;Johnson</td>
			<td style="width:113px;">K890H</td>
			<td>Surgical thread, needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath sonicator</td>
			<td>Branson 2210</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">BLI camera (IVIS Spectrum)&#160;</td>
			<td style="width:199px;">Perkin Elmer, Alameda</td>
			<td style="width:113px;">IVISSPE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Living Image software&#160;</td>
			<td style="width:199px;">Perkin Elmer, Alameda</td>
			<td style="width:113px;"></td>
			<td>(version 4.2)&#160;</td>
		</tr>
	</tbody>
</table>

candida albicans biofilm development on medically-relevant foreign bodies in a mouse subcutaneous model followed by bioluminescence imaging

对生物和/或非生物表面白色念珠菌生物膜发展代表对于住院患者特定威胁。到目前为止，C。白念珠菌生物膜已主要研究了在体外 ，但存在用于在体内条件下更好地理解本动力过程的一个关键需求。我们开发了一种在体内的皮下大鼠模型来研究C.白色念珠菌生物膜的形成。在我们的模型中，多个（最多9） -感染念珠菌的设备被植入到该动物的背部的部分。这给了我们一个主要的优势中央静脉导管模型系统，因为它可以让我们学习几个独立的生物膜在一个动物。最近，我们适应这个模型来研究C.白色念珠菌生物膜发展在BALB / c小鼠。在这种模式下，成熟的C。白色念珠菌生物膜内的48小时开发和示范典型的三维生物膜结构。真菌生物膜的量化传统上分析验尸并要求主机牺牲。因为这需要使用许多动物来进行动力学研究，我们采用非侵入性的生物发光成像（BLI）纵向跟进体内成熟C.白色念珠菌生物膜发展在我们皮下的模型。C.白念珠菌细胞被工程化以表达附着到细胞壁的Gaussia princeps的荧光素酶基因（GLUC）。生物发光信号是由所加的基板腔肠素转换成光，可测得的萤光素酶生成。在BLI信号相似，从植导管获得的细胞计数。非侵入性成像的体内生物膜形成定量提供即时申请在体内条件下筛选和抗真菌药验证，以及用于基于宿主-病原体相互作用的研究，在此有助于更好地理解荷兰国际集团的导管相关感染的发病机制。

在这项研究中，我们表明导管植入和植体在小鼠体内的白念珠菌生物膜的发育过程中的外科手术。此外，我们显示成熟生物膜不仅通过经典的CFUs枚举而且通过BLI的量化。 如图1A所示 ，非磷光聚氨酯导管片切成1厘米的设备，并随后涂覆有血清。这个步骤是非常重要的，因为它允许念珠菌细胞更迅速地附着在基板与非血清涂覆的植入物15的比?...

Watch this Scientific Journal Video about Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging at JoVE.com

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

candida-albicans-biofilm-development-on-medically-relevant-foreign

Research

JoVE Journal

Medicine

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

12.9K 次观看.  Institute of Botany and Microbiology, VIB, KU Leuven. We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

视频: Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

科研

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

超声成像引导下腔内注射到患乳腺癌脑转移其次是纵向的MRI的小鼠模型

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

急性脑外伤小鼠其次是纵向双光子成像

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

免疫活性动物模型胶质母细胞瘤的生物发光成像

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...