这项工作是由H＆H李某外科住院医师研究学者项目（扬），支持的AO基金会创业补助金的S-12-03M（以LSM）和健康补助R01-AI078910（以LSM）的国家机构。

道德声明：所有的动物都严格按照中所列的动物福利法（AWA）在联邦法规中定义良好的动物惯例办理，1996年指南的护理和使用实验动物和PHS政策人文善待实验动物和动物所有工作被批准为美国约翰霍普金斯大学动物护理和使用委员会（协议＃：MO12M465）。 1，准备的接种对数中期发光细菌<ol><li>连胜生物发光S。金黄色葡萄球菌菌株Xen29（或另一bioluminscent菌株如Xen36）上的胰蛋白酶大豆琼脂平板上（在琼脂[1.5％]的胰蛋白酶大豆肉汤）中。 注：S。金黄色葡萄球菌 Xen29 21是遗传工程学金黄色葡萄球菌菌株，其中包含从发光光 ，它被集成到这个菌株发现了一个稳定的天然质粒的修改勒克斯操纵子。这些引擎ERED细菌组成发光活和代谢活跃的细胞。 </li><li>通过温育它们在37℃下持续约16小时（O / N）生长在平板上的菌落。 </li><li>在约16小时（O / N）摇晃液体TSB（240转）选择单一细菌菌落形成单位和文化。 </li><li>与1/50稀释的O / N培养，以获得中期对数生长期的细菌（约2小时时长）中进行的亚文化。 </li><li>颗粒，重悬洗细菌3倍的PBS。 </li><li>通过测定光密度的吸光度在600nm处估计的细菌接种物（1×10 3 CFU中加入2μlPBS中）。 </li><li>在平板上培养细菌的O 2 / N后确认菌落形成单位的接种。 </li></ol> 2，鼠标外科手术注：对于这些实验中，使用一个12周大的雄性小鼠LysEGFP。这些小鼠具有增强的绿色荧光蛋白（EGFP）表达的髓样细胞（其中包括米ostly中性粒细胞），20。手术过程中和手术准备用优碘和70％的酒精通过将每只小鼠在无菌的悬垂在坚硬的表面水循环加热垫顶部后维持无菌状态。用长衫，无菌手套，口罩和消毒器械。 <ol><li>采用异氟醚的2％吸入麻醉鼠标。使用对眼睛的兽医药膏，以防止干燥时在麻醉下。通过观察呼吸频率，肌肉紧张，脚趾捏，角膜反射及粘膜色泽评估麻醉的适当水平。涵盖了小鼠无菌手术铺巾带洞手术部位的右膝。鼠标应该得到支持的加热措施，以维持体温，而在麻醉下。温暖的37℃的循环水在温暖的水套毯子或水循环硬塑料加热工作站（ProStation，帕特森，科学）都是很好的方法，以防止体温过低。 </li><li>注buprenorphINE（缓释性制剂）（2.5毫克/千克）经皮下刚刚手术前。额外剂量的缓释丁丙诺啡可以根据需要为镇痛在第3天的间隔给药。 </li><li>剃了膝盖手术，并利用预习用优碘和70％的酒精三个交替磨砂。 </li><li>在皮肤覆盖在右膝关节进行中线切开。延伸的皮肤切口，使得伸肌装置可以很好地限定。 </li><li>执行内侧髌关节切开术，并与ADSON钳侧向半脱位股四头肌，髌骨腱伸膝装置。 注意：这使股骨髁间窝到普通视图。 </li><li>通过将25g针头接着是23政针手工铰髓腔。 注意：为避免损坏股骨，稳定的平台都可以使用。这应该尽量减少股骨的意外断​​裂是重要的技术。 </li><li>将医疗级钛Kirsc维纳尔线（直径0.8mm），通过使用一个压入配合的技术，这需要手动推动它用针夹持器，在逆行方向进入髓腔内。 注：钛克氏针被用作有看到的μCT图像钛克氏针不锈钢克氏针16相比减少文物。 </li><li>切开克氏针线与针切割器的端部，使得克氏针的切断端大约1mm到膝关节空间延伸。 </li><li>使用微量吸管，吸管2微升生物发光S的1×10 3 CFU 葡萄球菌 Xen29到膝关节空间内植入物的前端。 注：更多的数量会导致更广泛的组织和污染少的离散成像。 注：在控制感染的小鼠，加入2微升的无菌生理盐水，没有任何细菌。 </li><li>减少股四头肌，髌骨复杂的回用钳中线和上覆皮下组织和皮肤可吸收合皮内缝合。 注意：不要让动物无人看管，直到它已经恢复了足够的意识来维持胸骨斜卧。不要将已动过手术，以其他动物的公司，直到完全康复的动物。 </li><li>在实验结束后，用安乐死根据约翰霍普金斯大学动物护理和使用委员会的指导方针吸入二氧化碳所有的动物。通过观察动物核实死亡未能在二氧化碳曝光结束，颈椎脱位后10分钟恢复。 </li></ol> 3，二维光学成像（ 体内生物发光和荧光成像） <ol><li>麻醉LysEGFP小鼠（ 如 2％吸入异氟醚），并把它们与腹侧到成像室。 </li><li>进行活体生物发光利用活体成像系统的IVIS光谱光学整个动物成像。首先，检查发光并确认公开f的选择滤波器选择，视角（FOV）C字段 - 12厘米，并滚动曝光时间缩短到自动（自动曝光设置）。自动曝光自动调整采集时间（快门速度），像素合并（数字像素组合），和仪器的光圈（孔径），以优化信号强度，同时避免饱和。然后点击获取捕获生物发光图像。 注：对于活体生物发光成像，图像小鼠之间1 - 5分钟。 </li><li>勾选旁边的框荧光执行顺序体内荧光成像。选择465 nm激发滤光片和520 nm发射滤波器。滚动曝光时间缩短到汽车，选择视野℃（步骤3.2.1）。然后点击获取捕捉荧光图像。 注：对于活体荧光成像，0.5秒之间的图像小鼠。 </li><li>通过第一膨胀的量化使用活影像软件的体内生物发光信号的总通量（光子/秒）中的感兴趣区域（ROI）的区域在工具选项板中的投资回报率工具部分。 <ol><li>选择的圆圈图标和感兴趣区域的数目对应于对象的动物，在视场的数目。调整投资回报率包括利息，即该地区的生物发光扩散模式收集。 </li><li>选择测量的ROI投资回报率的工具，在工具选项板和投资回报率的测量窗​​口将出现。总辐射率（光子/秒）的值表示所产生的ROI内的生物发光的像素的总和。 </li><li>选择全选，并在该窗口的右下角复制标签将信息传递粘贴到剪贴板中，并允许进入随后的程序进行分析。 </li></ol></li><li>使用活影像软件的感兴趣区域（ROI）的圆形区域内定量体内的荧光信号作为总辐射效率（[光子/秒] / [μW/ cm 2的]）。 <ol><li>内的生活图像软件窗口中，展开投资回报率的工具，在工具选项板。选择该圆圈图标和对应于受试动物中的FOV的ROI的数的数量。 </li><li>调整投资回报率涵盖的利益紧密对应，从以前的图像采集的生物发光信号的区域。 </li><li>选择测量的ROI投资回报率的工具，在工具选项板和投资回报率的测量窗​​口将出现。 注：总辐射效率（[光子/秒] / [微瓦/厘米2）表示ROI内的荧光像素的总和。 </li><li>全选并复制标签在此窗口的右下角将信息传递粘贴到剪贴板中，并允许进入随后的程序进行分析。 </li></ol></li></ol> 4μCT图像采集<ol><li>将麻醉LysEGFP小鼠成成像室。 注意：此成像室被设计为适合两个IVIS光谱成像系统和量子FX 体内 μCT成像系统，以允许在配准光学和μCT图像。 </li><li>打开CT软件，选择菜单预置60毫米视野性病从下拉列表中动态。 </li><li>插入大孔盖和转接器臂用于成像穿梭到仪器中。 </li><li>将鼠标成像班车到适配器的手臂，然后将手臂推入孔和关门。打开实时模式（在控制面板上的按钮眼），并使用X轴和Y轴的控制，以集中在X捕获窗口的动物放置在0°和90°龙门位置的问题。然后关闭实时模式通过单击眼扣。 </li><li>通过点击（旁边的实时模式按钮），在CT扫描按钮获得与60毫米视野动态扫描图像。出口以后可以访问的位置在DICOM格式和存储获得的图像。 注：近似的剂量是每次扫描26毫戈瑞。向30mm×视场可以使用，如果更高的分辨率需要。 </li></ol> 5。3D光电图像采集，形成与μCT联合登记<ol><li>将鼠标成像穿梭INSERT INTO的频谱通过定位包含鼠标放到这个插件的成像班车，保证鼠标不动。 </li><li>使用生活图像，收购控制面板中选择影像向导开始安装向导。开始，选择生物发光，然后DLIT，然后从下拉菜单和用于模型的相应发射器的记者“细菌”将被自动选择，在这种情况下，500 - 620纳米。 <ol><li>选择下一页，然后指定采集参数，并在最后一个窗口被摄体信息。具体来说，影像主题将是鼠标，自动设置将被选中允许自动曝光，以最大限度地提高信号质量，同时避免饱和度，视场会被设置为C - 13厘米。 </li><li>在最后的窗口中选择完成及收购事务的序列窗口将是一个utomatically填入DLIT序列。会有每个选择发射滤波器和自动曝光会选择最佳的设置在每个波长收购按成像向导选择一个图像。生成的序列还包括通过表面形貌的工具，下文详述需要受表面生成结构光图像。 </li></ol></li><li>选择采集序列获取DLIT数据。 </li><li>图像采集完成后，产生的表面形貌。首先，扩大在工具选项板的表面形貌标签。 <ol><li>接下来，选择准确反映所面临的IVIS仪器的照相机或顶部动物的一侧的方向。然后单击生成曲面。裁剪包含动物的视场的区域。 </li><li>然后用紫掩模来定义动物的边界。 注意：屏蔽工具使用的颜色对比使动物有黑毛和皮肤不会从S适当地掩盖踏歌。 </li><li>选择完成，表面会自动出现。保存表面形貌选项卡下的结果，然后关闭选项卡，我们将不再需要它。 </li></ol></li><li>重建使用通过扩大DLIT三维重建片生活图像22实施的扩散光学重建算法的3D光源的位置。 <ol><li>收购的DLIT序列图像显示。 注意：该软件会自动验证所获得的数据的质量，并会取消图像视为太暗或者饱和度存在。在底部右侧选择开始。 </li><li>如果需要的话，可以调整阈值通过双击每个生物发光图像，并使用在底部左侧的阈值滑块。 注：这主要包括低强度信号和警告阈值化更高，因为这可以调整最终重建源的整体强度时，应实行。 </li><lI&gt;当与阈值满足，选择重构为自动运行的重建算法，这将导致光源的3D表示。 </li></ol></li><li>通过单击软件（左三）顶部的工具栏上的3D图标，打开DICOM浏览器和搜索以前获得的量子外汇形象。 <ol><li>双击加载这个影像到生活图像三维视图选项卡中导入的文件。 注：基准应该被自动检测到并导致μCT图像被注册的三维光学图像。 </li></ol></li><li>取消选择表面形貌的可视化地图，扩大3D眼镜工具在工具选项板，并取消在表面标签的复选框标记显示被测面。 </li><li>手动创建一个查找表以可视化的骨架和克氏针植入物可见于使用3D多Modali的音量选项卡下的直方图μCT图像在工具选项板的TY工具部分。 <ol><li>直方图表示体素的强度，在三维体积数据和它们的颜色不透明度的分布。以确定所关注的特定组织中是在直方图中，使用滑块工具阈渲染直至组织或结构是可见的。 </li><li>然后，右键单击直方图生成点，形成一个曲线分离直方图的那个区域。这将重复为每个结构 - 骨架接着克氏针植入物和作为查找表的未来的分析可以保存。 </li><li>组件可以是颜色编码的，如果需要的话通过双击在直方图中的任何产生的点，然后从弹出窗口中的所需颜色。 </li></ol></li></ol> 6μCT图像可视化与分析<ol><li>利用量子外汇软件，选择感兴趣的图像并启动浏览器。选择旋转工具并调整图像以visuali泽股骨的纵向轴线。选择测量工具，测量股骨的长度。 </li><li>启动3D浏览器，以产生3D效果。调整阈值，以显示与植入物感染相关的变化的骨解剖结构。 </li><li>适用于剪切平面，使得3D渲染是有限的，在远侧股骨感兴趣区域的所需横截面部分。 </li><li>启动分析11.0软件包。加载用于创建3D渲染的* .VOX文件。 </li><li>启动图像计算器工具。使用“地区盘”的工具，裁剪图像（除去飞机不包括股骨）。 </li><li>启动倾斜部分的工具。使用3个选项，以找到点在股骨，大转子和所述销的端部的中间。提出这些问题的斜面，并产生了新的切片图像。 </li><li>推出利率工具的地区。显示横断切片。调整的最小值和最大值设置s到显示皮质骨。创建轮廓为对应的多个切片（用5片的大致间隔）对应于股骨远端25％的切片的切片。使用“传播区”的工具这些轮廓之间进行插值，并造成利益三维区域。关注这个区域保存为一个对象映射。 </li><li>启动“样本选项”工具。选择刚刚创建的对象映射的复选框，然后选择单选按钮的选项。点击“配置日志统计”按钮，确认“音量”复选框被选中。点击“样照”按钮，使实际测量。 </li><li>出口量测到的数据分析程序。使用公式正常化的外骨量从稍后的时间点，所述第一成像时间点：Δ量（％）=（[音量（天X） - 卷（第2天）] / [音量（第2天）]）×100 。 注意：在这个公式中，变量“X”代表感兴趣的时间点。所得到的数字将代表的外骨体积股骨远端随时间的大小的变化。 </li><li>为了形象地对骨骼的顶部感兴趣的三维区域，装载在CT图像中的“音量渲染”工具。加载包含所感兴趣的三维区域中的对象映射。转到“View&#39;&#39;Objects”，并设置了“原创”是“开”。打开“预览”窗口中。启动“渲染类型”菜单，并选择“对象合成”。 </li><li>点击“阈值”按钮，工具和调整阈值，显示骨骼和对象映射。使用相同的固定阈值范围内的所有时间点。点击“旋转”按钮，设置方向是一个真正的外侧看法。单击“渲染”生成最终渲染。保存渲染从主&#39;卷渲染“窗口。 </li></ol>

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	<li>Dothager, R. 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=Advances+in+bioluminescence+imaging+of+live+animal+models.">Advances in bioluminescence imaging of live animal models.</a> Curr Opin Biotechnol. 20, 45-53 (2009).</li><li>Badr, C. E., Tannous, B. A. <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+progress+and+applications.">Bioluminescence imaging progress and applications.</a> Trends Biotechnol. 29, 624-633 (2011).</li><li>Luker, G. D., Luker, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+current+applications+and+future+directions.">Optical imaging current applications and future directions.</a> J Nucl Med. 49, 1-4 (2008).</li><li>Ntziachristos, V., Ripoll, J., Wang, L. V., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+and+listening+to+light+the+evolution+of+whole-body+photonic+imaging.">Looking and listening to light the evolution of whole-body photonic imaging.</a> Nat Biotechnol. 23, 313-320 (2005).</li><li>Reumann, M. K., Weiser, M. C., Mayer-Kuckuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Musculoskeletal+molecular+imaging+a+comprehensive+overview.">Musculoskeletal molecular imaging a comprehensive overview.</a> Trends Biotechnol. 28, 93-101 (2010).</li><li>Snoeks, T. J., Khmelinskii, A., Lelieveldt, B. P., Kaijzel, E. L., Lowik, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+advances+in+skeletal+imaging+applied+to+bone+metastases.">Optical advances in skeletal imaging applied to bone metastases.</a> Bone. 48, 106-114 (2011).</li><li>Sjollema, 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=The+potential+for+bio-optical+imaging+of+biomaterial-associated+infection+in+vivo.">The potential for bio-optical imaging of biomaterial-associated infection in vivo.</a> Biomaterials. 31, 1984-1995 (2010).</li><li>Del Pozo, J. L., Patel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Infection+associated+with+prosthetic+joints.">Clinical practice. Infection associated with prosthetic joints.</a> N Engl J Med. 361, 787-794 (2009).</li><li>Parvizi, J., Adeli, B., Zmistowski, B., Restrepo, C., Greenwald, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+periprosthetic+joint+infection+the+current+knowledge+AAOS+exhibit+selection.">Management of periprosthetic joint infection the current knowledge AAOS exhibit selection.</a> J Bone Joint Surg Am. 94, e104 (2012).</li><li>Arciola, C. R., Campoccia, D., Speziale, P., Montanaro, L., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+in+Staphylococcus+implant+infections.+A+review+of+molecular+mechanisms+and+implications+for+biofilm-resistant+materials.">Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials.</a> Biomaterials. 33, 5967-5982 (2012).</li><li>Zimmerli, W., Moser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+treatment+concepts+of+orthopaedic+biofilm+infections.">Pathogenesis and treatment concepts of orthopaedic biofilm infections.</a> FEMS Immunol Med Microbiol. 65, 158-168 (2012).</li><li>Cram, 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=Total+knee+arthroplasty+volume+utilization+and+outcomes+among+Medicare+beneficiaries+1991-2010.">Total knee arthroplasty volume utilization and outcomes among Medicare beneficiaries 1991-2010.</a> JAMA. 308, 1227-1236 (2012).</li><li>Wolf, B. R., Lu, X., Li, Y., Callaghan, J. J., Cram, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+outcomes+in+hip+arthroplasty+long-term+trends.">Adverse outcomes in hip arthroplasty long-term trends.</a> J Bone Joint Surg Am. 94, (2012).</li><li>Bernthal, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+post-arthroplasty+Staphylococcus+aureus+joint+infection+to+evaluate+in+vivo+the+efficacy+of+antimicrobial+implant+coatings.">A mouse model of post-arthroplasty Staphylococcus aureus joint infection to evaluate in vivo the efficacy of antimicrobial implant coatings.</a> PLoS ONE. 5, (2010).</li><li>Bernthal, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+of+IL-1beta+against+post-arthroplasty+Staphylococcus+aureus+infection.">Protective role of IL-1beta against post-arthroplasty Staphylococcus aureus infection.</a> J Orthop Res. 29, 1621-1626 (2011).</li><li>Niska, J. 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=Monitoring+bacterial+burden,+inflammation+and+bone+damage+longitudinally+using+optical+and+&#181;CT+imaging+in+an+orthopaedic+implant+infection+in+mice.">Monitoring bacterial burden, inflammation and bone damage longitudinally using optical and &#181;CT imaging in an orthopaedic implant infection in mice.</a> PLoS ONE. 7, e47397 (2012).</li><li>Niska, J. 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=Daptomycin+and+tigecycline+have+broader+effective+dose+ranges+than+vancomycin+as+prophylaxis+against+a+Staphylococcus+aureus+surgical+implant+infection+in+mice.">Daptomycin and tigecycline have broader effective dose ranges than vancomycin as prophylaxis against a Staphylococcus aureus surgical implant infection in mice.</a> Antimicrob Agents Chemother. 56, 2590-2597 (2012).</li><li>Niska, J. 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=Vancomycin-Rifampin+Combination+Therapy+has+Enhanced+Efficacy+Against+an+Experimental+Staphylococcus+aureus+Prosthetic+Joint+Infection.">Vancomycin-Rifampin Combination Therapy has Enhanced Efficacy Against an Experimental Staphylococcus aureus Prosthetic Joint Infection.</a> Antimicrob Agents Chemother. 57, 5080-5086 (2013).</li><li>Pribaz, J. R., 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=Mouse+model+of+chronic+post-arthroplasty+infection+noninvasive+in+vivo+bioluminescence+imaging+to+monitor+bacterial+burden+for+long-term+study.">Mouse model of chronic post-arthroplasty infection noninvasive in vivo bioluminescence imaging to monitor bacterial burden for long-term study.</a> J Orthop Res. 30, 335-340 (2012).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96, 719-726 (2000).</li><li>Kadurugamuwa, J. L., 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=Direct+continuous+method+for+monitoring+biofilm+infection+in+a+mouse+model.">Direct continuous method for monitoring biofilm infection in a mouse model.</a> Infect Immun. 71, 882-890 (2003).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J Biomed Opt. 12, 024007 (2007).</li><li>Kim, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+survival+and+c-kit+-progenitor+proliferation+in+Staphylococcus+aureus-infected+skin+wounds+promote+resolution.">Neutrophil survival and c-kit+-progenitor proliferation in Staphylococcus aureus-infected skin wounds promote resolution.</a> Blood. 117, 3343-3352 (2011).</li><li>Deirmengian, C. A., Lonner, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+new+in+adult+reconstructive+knee+surgery.">What's new in adult reconstructive knee surgery.</a> J Bone Joint Surg Am. 94, 182-188 (2012).</li><li>Ning, X., 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=Maltodextrin-based+imaging+probes+detect+bacteria+in+vivo+with+high+sensitivity+and+specificity.">Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity.</a> Nat Mater. 10, 602-607 (2011).</li><li>Panizzi, 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=In+vivo+detection+of+Staphylococcus+aureus+endocarditis+by+targeting+pathogen-specific+prothrombin+activation.">In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation.</a> Nat Med. 17, 1142-1146 (2011).</li><li>van Oosten, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Realtime+in+vivo+imaging+of+invasive+and+biomaterial+associated+bacterial+infections+using+fluorescently+labelled+vancomycin.">Realtime in vivo imaging of invasive and biomaterial associated bacterial infections using fluorescently labelled vancomycin.</a> Nat Commun. 4, 2584 (2013).</li><li>Kong, Y., 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=Imaging+tuberculosis+with+endogenous+beta+lactamase+reporter+enzyme+fluorescence+in+live+mice.">Imaging tuberculosis with endogenous beta lactamase reporter enzyme fluorescence in live mice.</a> Proc Natl Acad Sci USA. 107, 12239-12244 (2010).</li></ol>

JAM，BNT，EL，新西兰，KPF支付PerkinElmer公司，该公司生产的成像仪器的员工，所提供的Xen29生物发光S。金黄色葡萄球菌菌株，并为此付出了视频文章的出版费用。其余的作者什么都没有透露。

多模态成像如利用体内光学成像与μCT成像相结合的成像技术提供了一种新的技术方法，使三维可视化，量化和生物过程的纵向监测解剖方面1-4。在本研究的协议提供了如何在体内生物发光和荧光成像可以用μCT成像组合在小鼠中的矫形假体植入物感染模型来监测细菌的负担，中性粒细胞炎症和解剖学上的变化在骨无创和纵向上的详细信息时间。两者合计，通过结合光学和结构成像获得的信息是一个重大的技术进步，这可能是特别适用于研究影响肌肉骨骼系统的生物过程和病理状况。 一个利益荷兰国际集团的发现，即应当指出的是，我们观察到EGFP-嗜中性粒细胞的荧光信号降低到背景水平由14-21天，并保持在背景水平为实验的持续时间，尽管发光细菌的存在。这是不太可能的X射线照射的影响嗜中性粒细胞存活，因为我们观察到相似的嗜中性粒细胞的信号的动力学中的非照射小鼠19。在我们以前的工作，涉及S的模型金黄色葡萄球菌感染的伤口，中性粒细胞浸润所涉及的流通稳健中性粒细胞募集，延长生存中性粒细胞的组合，在感染的部位和套装+祖细胞归巢的脓肿，他们在当地引起成熟中性粒细胞23。很可能是类似的过程促进了嗜中性粒细胞浸润，在整形外科植入物S。金黄色葡萄球菌感染模型。虽然它是未知原因的嗜中性粒细胞的信号在骨科下降aedic感染模型，它可能是免疫反应的变化随着时间的推移这种病从急性转为慢性感染的进展，这是未来研究的课题。 有带的整形外科假体关节感染该小鼠模型和体内多模态成像应该注意的限制。首先，这种小鼠模型是用在整形外科手术中24人，实际操作和材料过于简单。然而，这种模式确实重温慢性感染和随之而来的炎症在骨和关节组织被视为人类的骨科植入物感染8,9。此外，为了获得μCT图像，相对低剂量的X射线照射被用于减少感染的过程中，对动物的健康产生任何不良影响。为更好的分辨率骨的，较高剂量的X射线辐射，可用于μCT成像上安乐死Änimals。然而，这将消除的能力，非侵入性和纵向监测在实验的持续时间内的骨变化。 总之，多模态成像涉及活体整体动物光学成像的解剖μCT成像相结合，允许对感染和炎症反应更全面的信息。此外，这些技术已经允许对骨和关节组织的感染和炎症的后果进行评价。未来的工作可能需要多模态成像的优势，评估抗菌疗法，免疫反应，疾病的发病机制，并在骨反应性改变的效果，因为我们已经开始调查14-18。此外，多模态成像可以评估探针和示踪物以诊断感染的存在如先前在动物模型中大腿感染，心内膜炎，肺侵染描述离子和生物材料感染25-28。最后，利用多模态成像可以超越传染病扩大和跨学科，包括骨科，风湿病学和肿瘤学应用，探讨其他条件的影响，肌肉骨骼系统，如骨肿瘤，转移性疾病，骨折和关节炎5-7 。

涉及光学和解剖学信息相结合的多模临床前成像技术使可视化和监视的3D 1-4生物现象。由于μCT成像允许骨解剖的精致可视化，使用μCT成像与光学成像是一个独特的组合，可能是调查涉及的骨生物学5-7进程特别有用结合。一个例子是使用这些技术来研究骨科植入物感染，这代表了一个灾难性的并发症整形外科手术8,9。细菌生物膜的形成促进了细菌的存活作为阻止免疫细胞从访问细菌10,11感应感染，并阻止抗生素的物理屏障植入的异物。联合组织（化脓性关节炎）的慢性和持续性感染ð骨（骨髓炎）诱导骨吸收，导致松动的假体，并最终失败8,9。此得到的假体周围骨溶解时具有增加的发病率和死亡率12,13相关联。 在我们之前的工作中， 在体内生物发光和荧光成像被一起使用的X射线和微计算机断层成像（μCT）在小鼠中14-19整形外科假体关节感染模型。该模型涉及到放置钛克氏针线（K线）以这样的方式延伸，在膝关节从小鼠14-19的股骨植入物的切断端。 金黄色葡萄球菌 （生物发光应变Xen29或Xen36）的接种物，然后用移液管上，在膝关节植入物的表面外科手术部位被关闭14-19前在体内光学成像方法检测和定量的生物发光信号，该信号对应于NUMBER细菌感染的关节和骨组织14-19。另外， 在体内荧光LysEGFP小鼠，其具有荧光嗜中性粒细胞20的成像，来定量嗜中性粒细胞移居到含有克氏针植入物14,19被感染的膝盖关节的数量。最后，解剖成像方式，包括高分辨率的X射线成像和μCT成像，允许各自的2D受影响的骨和三维解剖成像在慢性感染的整个持续时间，这是我们会任意地结束通常为2和6的术后周16之间18。利用该模型，对局部和全身抗生素治疗，保护性免疫应答和在骨病理解剖变化的疗效可评价14-18。在这个手稿，在这个骨科人工关节感染模型的光学和μCT成像方式的具体方案提供了一个representati已经系统来研究生物过程中的骨的解剖上下文。这些包括外科手术过程进行建模的整形外科假体关节感染小鼠中，二维和三维体内光学成像程序（以检测细菌的生物发光信号和荧光信号的嗜中性粒细胞）的3D光学图象用的，μCT成像采集和分析，并共同登记μCT图像。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Xen36 bioluminescent Staphylococcus aureus strain</td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Bioluminescent Staphylococcus aureus strain derived from ATCC 49525 (Wright), a clinical isolate from a bacteremia patient</td>
		</tr>
		<tr>
			<td>Tryptic soy broth</td>
			<td>BD Biosciences, Franklin Lakes, NJ</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Soy Agar</td>
			<td>BD Biosciences, Franklin Lakes, NJ</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>LysEGFP knockin mouse strain</td>
			<td></td>
			<td></td>
			<td>Not commercially available. This strain contains a knockin of enhanced green fluorescence protein (EGFP) into the lysozyme M gene</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue Products, Stamford, CT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kirschner-wire (titanium, 0.8 mm diameter)</td>
			<td>Synthes, West Chester, PA</td>
			<td>492.08</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire Cutter - Duracut T.C.</td>
			<td>H&#38;H Company, Ontario, Canada</td>
			<td>83-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter, Deerfield, IL</td>
			<td>118718</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl 5-0 sutures (P-3 Reverse cutting)</td>
			<td>Ethicon, Summerville, NJ. Purchased through VWR International.</td>
			<td>95056-936</td>
			<td></td>
		</tr>
		<tr>
			<td>Sustained-release Buprenorphine (5 ml - 1 mg/ml)</td>
			<td>Zoopharm, Windsor, CO</td>
			<td></td>
			<td>analgesic</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Product/ID/IVISSPE?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">IVIS Spectrum Imaging System</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>optical in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Quantum%20FX%20%20microCT?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">Quantum FX in vivo &#956;CT system</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>&#956;CT in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Living%20Image%20Software?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">IVIS SpectrumCT Imaging System</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>combined optical and &#956;CT in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Product/ID/119240?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">Living Image Software</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Image analysis software for in vivo optical imaging</td>
		</tr>
	</tbody>
</table>

combined in vivo optical and &#181;ct imaging to monitor infection, inflammation, and bone anatomy in an orthopaedic implant infection in mice

多模态成像已经成为在临床前和临床研究中常用的技术方法。相结合的活体光学和μCT成像先进技术使生物现象的解剖方面的可视化。这种成像方式可能特别有用的学习条件影响骨骼。特别是，整形外科植入物感染在临床整形外科手术的一个重要问题。这些感染是难以治疗的，因为细菌生物膜有关外手术植入的材料形成，从而导致持久性炎症，骨髓炎和植入物周围，从而最终导致假体松动和失败的骨的最终骨溶解。这里，使用了受感染的整形外科假体植入物的小鼠模型涉及克氏针线植入物的手术放置成在股骨的髓内管以这样的方式，植入物e的端xtended到膝关节。在这个模型中，LysEGFP小鼠，小鼠菌株具有EGFP荧光中性粒细胞，分别用在与生物发光的金黄色葡萄球菌菌株，其中自然发射光一起使用。将细菌之前闭合手术部位接种到小鼠的膝关节。 体内生物发光和荧光成像来定量的细菌负担和嗜中性粒细胞的炎症反应，分别。此外，μCT成像在同一小鼠中进行，使得生物发光和荧光的光信号的三维位置可以被共登记了解剖μCT图像。量化的变化，骨随时间推移，所述外骨骼体积远端股骨的测定在使用半自动化的基于轮廓分段过程的特定时间点。两者合计， 在体内生物发光/荧光成像用μCT成像的组合可以是F特别有用或无创监测的感染，炎症反应，随着时间的推移解剖学上的变化在骨。

在体内生物发光和荧光成像在本研究中，该协议描述了用于整形外科假体关节感染小鼠14-19此以前发表的模型，其中涉及钛克氏针植入物从髓腔在股骨延伸到关节的手术放置空间14-19 S。金黄色葡萄球菌的生物发光应变Xen29（1×10 3 CFU中加入2μl的PBS）中以直接关闭手术部位16之前，联合吸在端钛种植体在膝盖上方。可视化和无创定量细菌负担和中性粒细胞涌入麻醉LysEGFP小鼠体内的整个动物光学成像进行顺序图像从细菌的生物发光信号和绿色荧光蛋白的荧光信号使用的 IVIS光谱光学整个动物的嗜中性粒细胞浸润体内三术后天（ 即 2天，14和28）成像系统。 Xen29感染的小鼠的生物发光信号仍高于假感染的小鼠用于实验（ 图1A，C）16的持续时间的背景信号。 我们以前的工作表明， 在体内生物发光信号密切近似的体外 CFU隔离关节/骨组织，并附着于植入物17,18的号码。另外，绿色荧光蛋白的荧光信号比假感染的小鼠更高的早期时间点，但是感染的过程中（ 图2B，C）16中接近背景水平。 体内用μCT图像的光信号的三维配准为了形象在手术后膝关节的解剖范围内的光信号（ 即细菌生物发光和绿色荧光蛋白的荧光信号）IÑ​​3D，用IVIS光谱成像系统所产生的光学图像进行共登记使用量子FXμCT成像系统产生的μCT图像。这个配准可以实现，因为鼠标成像室可插入任一机器，以确保小鼠在完全相同的方向。为了验证这个精度，其结果，比较用的图像采集使用IVIS光谱-CT 的体内成像，集成两种模式进入一台仪器，而不需要该动物的物理搬迁系统执行。要映射的光学数据到三维的μCT图像，我们利用漫反射光学层析重建算法16。所得到的三维重建显示（ 电影1）。 此外，μCT成像允许在质量的相应变化和尺寸的过程中发生的骨的可视化和量化感染（ 图2）16。如先前报道，外骨骼体积股骨远端的基本上随时间增加（ 图2A）16。为了量化这些变化，在股​​骨上的BONEY表面的远端25％进行三维体积图像分析和变更的骨量随着时间的推移被归一化到初始骨体积。相比于假感染的小鼠（ 图2B）16，在感染小鼠的外骨量显着增加。在股骨远端外骨量的增加可能是由于所引起的关节组织和骨骼，这是用μCT成像和组织学分析16观察到的感染骨损伤。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51612/51612fig1highres.jpg" width="500" /> 图1 2D 体内 </str翁&gt; 生物发光和荧光信号。S. 葡萄球菌 Xen29或无细菌（未感染）接种到后克氏针放置和LysEGFP小鼠用IVIS光谱成像系统16拍摄的膝关节（A）的平均数在体内的生物发光信号，如通过总通量（光子/秒）的测定±SEM（B）平均在体内的EGFP荧光信号通过总辐射效率（光子/秒）的测定体内生物发光和荧光信号叠加在黑白照相/（μW/ cm 2）的平均值±SEM（C）代表图像的小鼠。检测使用体内生物发光成像的细菌负荷的极限是在1×10 2和1×10 3 CFU。 * P &lt;0.05，†P &lt;0.01，‡P &lt;0.001 Xen29感染的小鼠与假手术感染的小鼠（学生t检验[双尾]）。请注意，这是一个代表性的人物，其中包括使用Xen29产生成像与IVIS Lumina的XR的成像系统16此前公布的数据， <a href="https://www.jove.com/files/ftp_upload/51612/51612fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51612/51612fig2highres.jpg" width="500" /> Xen29的图2的3DμCT成像。 金黄色葡萄球菌 Xen29或无细菌（未感染）接种到膝关节克氏针置入后与小鼠使用量子FX 体内 μCT系统成像的（A）代表三维μCT效果图-感染的小鼠（上图）和假感染的小鼠（下图）。外骨骼的体积变化（在股骨的远端25％）的（B）的百分比归一化到初始钛我点（平均值±标准差）。 * P &lt;0.05，†P &lt;0.01，‡P &lt;0.001 Xen29感染的小鼠与假手术感染的小鼠（学生t检验[双尾]）。请注意，这是一个代表性的人物，其中包括利用生物发光株S.产生此前公布的数据金黄色葡萄球菌 Xen29和成像的量子FX 在体内的μCT成像系统16。 <a href="https://www.jove.com/files/ftp_upload/51612/51612fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <a href="https://www.jove.com/files/ftp_upload/51612/Movie_1.mpg" target="_blank">影片1</a> 。代表三维的Xen29生物发光信号和EGFP-嗜中性粒细胞的荧光信号的组合与μCT图像的解剖共登记。的图像旋转的垂直轴。

Watch this Scientific Journal Video about Combined In vivo Optical and ÂµCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice at JoVE.com

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

结合光学和μCT成像中的整形外科植入物感染的小鼠模型中，利用金黄色葡萄球菌的生物发光的工程应变，所提供的能力，以非侵入性和纵向监测细菌感染的动力学，以及相应的炎症反应和解剖学上的变化，在骨。

结合<em&gt;在体内</em&gt;光学及μCT成像监视感染，炎症和骨解剖在骨科植入物感染的小鼠

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

combined-vivo-optical-ct-imaging-to-monitor-infection-inflammation

Research

JoVE Journal

Medicine

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

16.9K 次观看.  David Geffen School of Medicine at University of California, Los Angeles (UCLA). 结合光学和μCT成像中的整形外科植入物感染的小鼠模型中，利用金黄色葡萄球菌的生物发光的工程应变，所提供的能力，以非侵入性和纵向监测细菌感染的动力学，以及相应的炎症反应和解剖学上的变化，在骨。 

视频: Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

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 ...

科研

医学

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

支气管肺泡灌洗（BAL）的研究，获得适当的采样率

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

人体实验肺炎球菌运输

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.
In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.
In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

万古霉素滴入硫酸钙和自体骨治疗兔骨感染的改进模型

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant&#039;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

小鼠闭合性股骨骨折的形成: 骨愈合模型的研究

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

使用电子地图的光映射数据的高通量分析

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...