我们感谢 Ms. 索尼娅 Dufentester, Ms. Elke 韦伯和 Mrs. Klaudia Niepagenkämper 为优秀的技术援助。

根据德国动物保护法 (LANUV), 所有动物实验均经 Landesamt Natur、Umwelt 和 Verbraucherschutz (Nordrhein) Westfalen-Tierschutzgesetz。
1. 材料和实验装置
<ol>
	<li>动物护理。
	<ol>
		<li>使用性别和年龄匹配的任何 DSS 敏感株 (例如, C57BL/6) 在20-25 克体重的小鼠。</li>
		<li>根据当地的动物保育指南, 计划每组至少五只小鼠, 并将小鼠置于家中。</li>
		<li>提供标准的鼠粮和蒸压饮用水ad 随意。</li>
		<li>在扫描前至少三天去除标准的 chow, 并将其换成无苜蓿草, 以减少腔荧光。</li>
	</ol>
	</li>
	<li>急性 DSS 诱导的结肠炎。
	<ol>
		<li>溶解2克的 DSS (分子量〜 4万 Da) 在100毫升的蒸压饮用水中获得 2% (瓦特/v 溶液)。</li>
		<li>只用 DSS 解决方案来填满小鼠的饮水供应, 并估计每只老鼠每天5毫升液体。提供相同的饮用水没有 DSS 的控制小鼠10。 注意: 每天监测老鼠直到实验结束。根据当地适用的动物指南, 安乐的老鼠会失去超过20% 的初始体重或变得奄奄一息 (即,持续驼背的姿势, 减少运动, 呼吸吃力, 明显直立的皮毛)福利.</li>
	</ol>
	</li>
	<li>荧光介导的层析成像的制备。
	<ol>
		<li>标签所需的抗体 (例如,大鼠 anti-mouse F4/80) 与荧光染料 (例如, Cyanine7, λ激发: 750 nm, λ发射: 776 nm), 如制造商的协议所述。透析纯化抗体在适当的透析膜 (孔大小和 #60; 50-100 kDa) 反对 1 L 0.15 M 氯化钠为至少 2 h 或隔夜。<ol>
			<li>将抗体转移到0.1 米 NaHCO 的 1 L,3 , 透析至少2小时。</li>
			<li>在二甲基亚砜 (10.8 µL/毫克抗体) 中溶解所需数量的荧光染料, 并将其添加到抗体溶液中。使用荧光染料在20倍的摩尔过剩, 以达到 dye-to-protein 的比例为1:3。</li>
			<li>在黑暗中孵育在4° c 为 1 h. 通过透析对1升0.15 米钠或使用 PD-10 脱盐柱和解决磷酸盐缓冲盐水 (PBS) 中的体内应用去除未标记抗体。</li>
			<li>用分光光度法测定最终抗体浓度和标记比值。</li>
			<li>测量250-330 纳米吸收的蛋白质浓度, 并考虑染料的额外吸收。校正最大吸收在280毫微米 (蛋白质) 由11% 最大吸收在750毫微米 (下步) 为 Cyanine7 标记。</li>
			<li>测量化合物 (通常为 1:10) 在 250-800 nm 的稀释量, 并提取 Cyanine7 在 750 nm 的浓度。</li>
			<li>确定 dye-to-protein 比为: 染料或抗体 = 最大吸收在750毫微米/20万/(最大吸收在280毫微米-0.11 x 最大吸收在750毫微米)/17万。</li>
			<li>将抗体溶液保持在4° c, 并将其从光中屏蔽以避免在注射前漂白。</li>
		</ol>
		</li>
		<li>在注射前立即将所需的抗体溶液容积装入无菌注射器中, 直至使用。</li>
		<li>根据示踪药代动力学确定探针注射的最佳时机和扫描程序。</li>
		<li>麻醉使用 1.5-2.5% 吸入的异氟醚在氧气中, 或将它们安全地放置在一个专门的抑制剂尾静脉注射的标记抗体的小鼠。</li>
		<li>对于全长抗体, 在扫描前注入至少24小时的标记抗体, 如用于小鼠结肠炎的 anti-mouse F4/80 巨噬细胞可视化。通过尾静脉注射小鼠 (静脉注射) 与标记抗体的数量对应的 2.0 nmol 染料。</li>
		<li>使用同样标记的不抗体 (例如,大鼠 IgG 或与主要抗体遗传相对应的另一种同种) 作为同种控制, 剂量相当于特定探针的量。 注: 在注射控制化合物后的体内扫描结果可以作为对特定探针成像数据的解释的参考。</li>
		<li>使用电动剃须刀将动物皮毛剃毛在腹部区域, 以减少对光的反射和吸收。</li>
	</ol>
	</li>
</ol>
2. 技术设备
<ol>
	<li>使用兽医荧光介导的层析成像 (裂变材料) 装置的小动物荧光 (图 4)。</li>
	<li>通过点击 "新研究" 按钮为每个项目创建一个新的研究, 并在研究描述中包括相关的示踪剂, 包括成像参数和剂量供将来参考。</li>
	<li>在本研究中, 通过单击 "新建学习组" 按钮, 根据各自的研究设计 (例如,为特定的跟踪器和不同种控件) 创建研究组。装备每个研究小组与动物的各自数量。</li>
	<li>校准跟踪器构造的系统。<ol>
		<li>对每批示踪剂进行校准, 使标签的变化正常化, 并从 OI 数据中进行定量测量。</li>
		<li>按照仪器制造商的指导, 对各个系统进行校准;在选择 "添加新的跟踪器" 时, 系统将通过步骤提供指南。在探针中提供了应用抗体溶液的稀释和示踪剂的绝对浓度。</li>
		<li>对于裂变材料, 使用一种组织模仿的定义厚度和吸收特性 (类似于重要组织) 的幻像, 并用特定体积的抗体溶液填充。在裂变材料装置上测量这一点。 注: 系统将使用所提供探头的参考测量, 连同给定的浓度, 从未来的活体测量中计算绝对示踪剂浓度。</li>
	</ol>
	</li>
	<li>使用土炕检测盒, 温度为42° c。 注意: 这可以防止小鼠在检查过程中变得体温过低。</li>
</ol>
3. 动物麻醉
<ol>
	<li>使用连续流动的 1.5-2%% 异氟醚 ([2-氯-2-(氟)-11, 1-中和--乙烷]) 和 1.5 L O2/分钟麻醉老鼠。</li></ol>使用专门设计的啮齿动物麻醉吸入系统 (异氟醚蒸发器), 以方便地控制麻醉深度和减少工作人员暴露。
	<li>将鼠标放在防漏感应室中, 并打开用于异氟醚供应的蒸发器 (100% (v/五), 5% 氧, 3 升/分)。监测鼠标, 直到它是横卧和无意识的。</li>
	<li>将它放在检查盒中进行断层扫描, 通过鼻锥连续吸入异氟醚, 剂量为100% 伏/伏, 1.5% 氧, 1.5 升/分钟, 在检查过程中尽量减少运动伪影。对于持续时间超过5分钟的手术, 应将眼膏涂抹于小鼠眼, 以防止角膜损伤。</li>
	<li>通过检查反应来评估麻醉深度。把老鼠放在背上;如果麻醉足够了, 老鼠就不应该转过身来。在脚趾间轻轻地捏老鼠;如果麻醉足够, 腿将不会被撤回 (手术耐受阶段)。</li>

4. 荧光介导的断层扫描
注: 根据需要, 对本研究中使用的裂变材料体系 (见资料表) 中的替代荧光反射成像设备或裂变物质系统, 采用以下具体的细节进行调整。
<ol>
	<li>将麻醉小鼠放回检查盒中。</li>
	<li>执行扫描过程。<ol>
		<li>将卡带插入成像系统并立即关闭, 以确保连续麻醉。从以前创建的研究组中选择适当的示例。从下拉菜单中选择所管理的跟踪器, 以确保正确计算示踪剂浓度值。</li>
		<li>在适当波长 (720 nm Cyanine7) 上获取荧光反射图像, 用于扫描规划, 并通过单击 "获取图像" 按钮来勾勒扫描字段。</li>
		<li>请注意, 扫描字段显示为荧光反射图像上的叠加。将其调整到感兴趣的区域 (例如,冒号或腹部), 避免空气或剩余毛皮的区域。根据图像目标, 通过在右侧的菜单中选择 "粗到中" 和 "精细扫描-字段分辨率", 在扫描字段中设置图像数据点的数目。 注意: 请记住, 一个精细的扫描字段可能提供更好的空间分辨率, 其代价是扫描时间大大延长。</li>
		<li>单击 "扫描", 在选定的波长处启动数据/图像采集。 注: 扫描时间为中细扫描整个腹部将约5分钟;在这段时间内, 每个数据点分别由激励激光照射, 并记录产生的荧光。</li>
		<li>在扫描结束时从成像盒中取出动物, 让动物完全恢复, 然后再将其放回笼子。</li>
		<li>在实验过程中, 如果认为需要在不同的时间点 (例如,天 0, 5, 和 9-10 (实验结束)), 重复的裂变材料, 但考虑体内抗体的积累, 因此增加了背景荧光信号。</li>
	</ol>
	</li>
</ol>
5. 扫描后
<ol>
	<li>将鼠标放在一个单独的笼子里, 放在一盏红光灯下的纸巾上, 监视鼠标是否有不适或苦恼的迹象, 直到完全恢复。当完全清醒时, 将鼠标放回各自的笼子里。</li>
	<li>安乐在实验结束时, 通过将 CO2传递到隔离器, 剂量为 100% (v/v)、100% 和3升/分。不要填充的商会与 co2,作为一个突然暴露在高浓度 co2可能会导致遇险。验证死亡后, 鼠标停止呼吸由随后的第二模式的安乐死, 如快速颈椎脱位。</li>
	<li>通过腹部剖腹术外植体结肠, 并对说明结肠进行活体扫描, 如步骤 4.2.1-4.2.4 所述。在准备进一步的分析之前, 用 Metzenbaum 的外科剪刀纵向打开每个结肠, 用生理盐水彻底冲洗。</li>
	<li>使用手术刀切割从远端结肠0.5 厘米的片段, 并立即放置在1.5 毫升低温管。将其冷冻在液氮中, 贮存在-70 ° c, 直至进一步使用 (例如,过氧化物 (过氧化物酶) 测量)。</li>
	<li>使用木棍和卷起剩余的纵向打开的结肠从远端到近端, 与黏膜向外 ("瑞士卷技术"), 为组织学分析。将准备工作放置在固定剂中 (请参见材料表), 并在-80 ° c14处冻结。</li>
</ol>
6. 数据重建和解释
<ol>
	<li>使用各自成像软件的重建工具, 从原始成像数据中创建3D 荧光分布图;扫描将自动添加到重建工具中, 当扫描时, 会选择 "添加到重建队列" 功能。<ol>
		<li>否则, 从相应的学习和学习组下的下拉菜单中选择扫描, 右键单击扫描, 然后选择 "添加到重建"。</li>
	</ol>
	</li>
	<li>要进一步分析, 请将数据集加载到分析软件中。从顶栏的下拉菜单中选择相应的研究, 然后从右侧的菜单中选择学习组和单个动物;所有对此动物进行的扫描将显示。选择正确的扫描, 然后单击 "加载"。 注: 示踪剂分布的3D 重建将在左侧出现, 作为最初获得的荧光反射图像的叠加。该模型可以旋转和放大, 以便于分析。</li>
	<li>在重建的3D 地图上确定不标签堆积 (例如,肝或尿囊) 的病灶, 并与靶组织 (如肠或肠道) 区分。</li>
	<li>从顶部栏中选择最适合目标的 ROI 形状。通过在分析软件中放置相应的测量工具, 将目标组织标记为感兴趣的区域 (ROI);该软件将提供一个荧光强度的 ROI, 以及 (微微) 摩尔量的示踪剂, 扫描已被校准。</li>
	<li>在适当的 roi 中选择跟踪器的总数量, 将其归为 roi 大小, 作为与炎症浸润 (组织学) 相关的疾病活动评估的最合适的等价物。 注: ROI 的其他功能可以在适当时作为特定模型的代表选择。</li>
</ol>
7。Ex 活体分析
<ol>
	<li>苏木精和红素染色和免疫荧光染色。
	<ol>
		<li>Deparaffinize 部分通过放置在70% 乙醇 (乙醇) 为2分钟;之后用蒸馏水冲洗。染色用苏木精溶液5分钟, 随后用温水冲洗10分钟。</li>
		<li>用蒸馏水冲洗, counterstain 用曙红溶液2分钟, 再用蒸馏水冲洗。</li>
		<li>放置在70% 乙醇, 96% 乙醇, 99% 乙醇, 和二甲苯 (2 次) 为2分钟, 以脱水和清除部分。装载树脂安装介质。</li>
	</ol>
	</li>
	<li>免疫荧光染色。
	<ol>
		<li>使用先前获得的组织切片 ("瑞士卷") 进行免疫荧光染色, 并准备7µm 的冷冻切片。</li>
		<li>在5.0% 只大鼠血清中阻断丙酮固定和冰冻结肠切片10分钟, 并在夜间用稀释的 (1/500 伏/v) 化原 anti-mouse F4/80 抗体进行孵育。在三缓冲盐水 (TBS) 三次洗涤部分和孵育与亲和-FITC (1:100 v/v) 在 PBS 或 BSA (0.1% 瓦特/v) 隔夜在4° c。</li>
		<li>用 4, 6-diamidino-2-吲 (DAPI, 1:1000 伏/v) 来洗涤 TBS 和染色的部分, 以达到对比。在共焦显微镜下分析荧光图像 (见材料表; 40x 放大倍率; 过滤器立方体 N2.1 与励磁过滤器 515-560) 和计数 F4/80-positive 细胞每个大功率领域。 注意: 考虑使用相同的克隆和格式的抗体结合在体内的裂变材料和验尸组织学分析, 如免疫组织化学或免疫荧光染色, 根据预期的目标。为检测 F4/80-positive 巨噬细胞在体内和ex 体内, 使用了不同的格式。</li>
	</ol>
	</li>
	<li>髓过氧化物酶在结肠样品中的测量。
	<ol>
		<li>使用新鲜获得的, PBS 冲洗的结肠样品或解冻的样品进行过氧化物酶测量。在 ELISA 试剂盒中提供的溶解缓冲液中, 称量所有样品和质组织 (见材料表), 每毫克组织的裂解缓冲液量为20µL。</li>
		<li>几种为十五年代 (超声频率:20 赫, 力量:70 W) 和离心样品为10分钟在 200 x g 和4° c。</li>
		<li>根据制造商的描述, 使用商业上可用的 ELISA 试剂盒 (参见材料表)。执行重复的测试。</li>
	</ol>
	</li>
</ol>

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S., Manocha, M. M., Khan, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+intestinal+inflammation+in+DSS-induced+model+of+IBD.">Investigating intestinal inflammation in DSS-induced model of IBD.</a> J Vis Exp. (60), e3678 (2012).</li><li>Dieleman, L. 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=Chronic+experimental+colitis+induced+by+dextran+sulphate+sodium+(DSS)+is+characterized+by+Th1+and+Th2+cytokines.">Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.</a> Clin Exp Immunol. 114 (3), 385-391 (1998).</li><li>Kojouharoff, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralization+of+tumour+necrosis+factor+(TNF)+but+not+of+IL-1+reduces+inflammation+in+chronic+dextran+sulphate+sodium-induced+colitis+in+mice.">Neutralization of tumour necrosis factor (TNF) but not of IL-1 reduces inflammation in chronic dextran sulphate sodium-induced colitis in mice.</a> Clin Exp Immunol. 107 (2), 353-358 (1997).</li><li>Sunderkotter, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subpopulations+of+mouse+blood+monocytes+differ+in+maturation+stage+and+inflammatory+response.">Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.</a> J Immunol. 172 (7), 4410-4417 (2004).</li><li>Willmann, J. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Targeted+Therapies+for+Inflammatory+Bowel+Disease.">Novel Targeted Therapies for Inflammatory Bowel Disease.</a> Trends Pharmacol Sci. , (2016).</li><li>Vermeire, 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=The+mucosal+addressin+cell+adhesion+molecule+antibody+PF-00547,659+in+ulcerative+colitis:+a+randomised+study.">The mucosal addressin cell adhesion molecule antibody PF-00547,659 in ulcerative colitis: a randomised study.</a> Gut. 60 (8), 1068-1075 (2011).</li><li>Terai, T., Nagano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+fluorophores+and+fluorescent+probes+for+bioimaging.">Small-molecule fluorophores and fluorescent probes for bioimaging.</a> Pflugers Arch. 465 (3), 347-359 (2013).</li><li>Ren, W., 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=Dynamic+Measurement+of+Tumor+Vascular+Permeability+and+Perfusion+using+a+Hybrid+System+for+Simultaneous+Magnetic+Resonance+and+Fluorescence+Imaging.">Dynamic Measurement of Tumor Vascular Permeability and Perfusion using a Hybrid System for Simultaneous Magnetic Resonance and Fluorescence Imaging.</a> Mol Imaging Biol. 18 (2), 191-200 (2016).</li><li>Ale, A., Ermolayev, V., Deliolanis, N. 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作者没有什么可透露的。

尽管近年来医学成像技术发展迅速, 但在早期的发育阶段, 我们在检测炎症过程或肿瘤以及其他疾病方面的能力仍然有限。然而, 这对于了解肿瘤的生长, 侵袭, 或转移的发展和细胞过程中的炎症性疾病和退化, 心血管和免疫疾病的发展至关重要。虽然传统的成像技术依赖于物理或生理参数, 分子成像使特定分子标记的可视化在体内20。
对于小动物的成像,...

动物模型被广泛应用于科学研究, 许多非侵入性的程序都存在于监测疾病的活动和生命力, 如体重变化的量化或血液、尿液和粪便的分析。但是, 这些只是间接的代理参数, 也受个体的可变性。它们必须经常通过对组织标本的验尸分析来补充, 防止在重复的时间点进行连续观察, 并直接观察生理或病理过程在体内。成熟的小动物成像技术已经出现, 包括横断面成像, 光学成像和内窥镜, 这使这些过程的直接可视化, 也允许重复分析相同的动物1,2,3. 此外, 在同一动物中反复监测各种疾病的可能性可能会减少所需的动物数量, 从动物伦理学的角度来看, 这可能是可取的。
几种不同的光学成像技术存在于体内荧光成像。最初, 共聚焦成像被用来研究表面和地下荧光事件4,5。然而, 最近, 允许进行定量的三维组织评估的层析系统已经开发成6。这是通过开发荧光探针, 在近红外线 (近红外光谱) 频谱发出光, 提供低吸收, 灵敏的探测器, 单色光源7。虽然传统的切削成像技术, 如计算机断层扫描 (CT), 磁共振成像 (MRI), 或超声 (美国), 主要依赖于物理参数和可视化形态学, 光学成像可以提供额外的信息使用内生或外源荧光探针的基础分子过程8。
分子生物学的进步有助于为越来越多的目标提供智能和有针对性的荧光分子探针的生成。例如, 受体介导的摄取和分布在给定的目标区域可以可视化使用 carbocyanine 衍生物标记抗体9。大量可用的抗体, 可被标记为功能作为特定的示踪剂在其他无法进入的身体区域, 提供了前所未有的洞察力分子和细胞的过程中的模型的肿瘤和神经退行性,心血管、免疫学和炎症性疾病7。
在这项研究中, 我们描述了使用荧光介导的层析成像在小鼠结肠炎模型。葡聚糖硫酸钠 (DSS) 诱导结肠炎是一种标准的化学诱导的小鼠肠道炎症模型, 类似炎症性肠病 (IBD)10。这是特别有用的评估先天免疫系统对发展的肠道炎症的贡献11。由于单核和巨噬细胞的招募、活化和浸润是 IBD 发病机制的关键步骤, 因此它们的招募和渗透动力学的可视化是监测的关键, 例如, 影响潜在的治疗性药物在临床前设置12。我们描述了诱导的 DSS 结肠炎, 并演示了利用荧光分子层析成像技术对巨噬细胞浸润肠黏膜的特性, 为单核/巨噬细胞标记 F4/80 的具体可视化13. 此外, 我们还说明了辅助和补充程序, 如抗体标签;实验装置;对所获得的图像进行分析和解释, 与常规读数相关, 如疾病活动指数、流式细胞仪和组织学分析、免疫组织化学。我们讨论这项技术的局限性和与其他成像方式的比较。

<table border="0" cellpadding="0" cellspacing="0" width="1601">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Reagents</td>
			<td style="width:423px;"></td>
			<td style="width:320px;"></td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alfalfa-free diet</td>
			<td>Harlan Laboritories, Madison, USA</td>
			<td style="width:320px;">2014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bepanthen eye ointment</td>
			<td style="width:423px;">Bayer, Leverkusen, Germany</td>
			<td style="width:320px;">80469764</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran sulphate sodium (DSS)</td>
			<td>TdB Consulatancy, Uppsala, Sweden</td>
			<td>DB001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Eosin</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">E 4382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Ethylenediaminetetraacetic acid (EDTA)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">E 9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Florene 100V/V</td>
			<td>Abbott, Wiesbaden, Germany</td>
			<td style="width:320px;">B506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Haematoxylin&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">HHS32-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">O.C.T. Tissue Tek compound&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:423px;">Sakura, Zoeterwonde, Netherlands</td>
			<td style="width:320px;">4583</td>
			<td>fixative for histological analyses</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Phosphate buffered saline, PBS</td>
			<td style="width:423px;">Lonza, Verviers, Belgium</td>
			<td style="width:320px;">4629</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride 0,9%</td>
			<td>Braun, Melsungen, Germany</td>
			<td style="width:320px;">5/12211095/0411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium bicarbonate powder</td>
			<td>Sigma Aldrich Deisenhofen, Germany</td>
			<td style="width:320px;">S5761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Standard diet</td>
			<td>Altromin, Lage, Germany</td>
			<td style="width:320px;">1320</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-Tek Cryomold</td>
			<td>Sakura, Leiden, Netherlands</td>
			<td style="width:320px;">4566</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemoccult (guaiac paper test)</td>
			<td>Beckmann Coulter, Germany</td>
			<td style="width:320px;">3060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotin rat-anti-mouse anti-F4/80 antibody</td>
			<td>Serotec, Oxford, UK</td>
			<td style="width:320px;">MCA497B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotin rat-anti-mouse anti-GR-1&#160;</td>
			<td>BD Pharmingen, Heidelberg Germany</td>
			<td>553125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptavidin-Alexa546</td>
			<td>Molecular Probes, Darmstadt, Germany</td>
			<td style="width:320px;">S-11225</td>
			<td>excitation/emission maximum:&#160; 556/573nm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD11b rat-anti-mouse antibody TC</td>
			<td>Calteg, Burlingame, USA</td>
			<td style="width:320px;">R2b06</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Purified anti-mouse F4/80 antibody</td>
			<td>BioLegend, London, UK</td>
			<td style="width:320px;">123102</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td style="width:320px;">D9542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FITC-conjugated anti-Ly6C rat-anti-mouse antibody</td>
			<td>BD Pharmingen, Heidelberg, Germany</td>
			<td>553104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS buffer</td>
			<td>BD Pharmingen, Heidelberg, Germany</td>
			<td style="width:320px;">342003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cy7 NHS Ester</td>
			<td>GE Healthcare&#160;Europe, Freiburg, Germany</td>
			<td style="width:320px;">PA17104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MPO ELISA</td>
			<td>Immundiagnostik AG, Bensheim, Germany</td>
			<td>K 6631B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cy5.5 labeled anti-mouse F4/80 antibody</td>
			<td>BioLegend, London, UK</td>
			<td>123127</td>
			<td>ready to use labelled Antibodies (alternative)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Mouse F4/80 Antigen PerCP-Cyanine5.5</td>
			<td>eBioscience, Waltham, USA</td>
			<td>45-4801-80</td>
			<td>ready to use labelled Antibodies (alternative)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO (Dimethyl sulfoxide)</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Isoflurane</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>792632</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumins (BSA)</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>A4612</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris Buffered Saline Solution (TBS)</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>SRE0032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Name</td>
			<td style="width:423px;">Company</td>
			<td style="width:320px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td style="width:320px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS Calibur Flow Cytometry System</td>
			<td>BD Biosciences GmbH, Heidelberg, Germany</td>
			<td style="width:320px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FMT 2000 In Vivo Imaging System</td>
			<td>PerkinElmer Inc., Waltham, MA, USA</td>
			<td style="width:320px;">FMT2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">True Quant 3.1 Imaging Analysis Software</td>
			<td>PerkinElmer Inc., Waltham, MA, USA</td>
			<td style="width:320px;">included in FMT2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leica DMLB Fluorescent Microscope</td>
			<td>Leica,&#160; 35578 Wetzlar, Germany&#160;</td>
			<td>DMLB</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bandelin Sonopuls HD 2070</td>
			<td>Bandelin, 12207 Berlin, Germany</td>
			<td style="width:320px;">HD 2070</td>
			<td>ultrasonic homogenizer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Disposable scalpel No 10</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>Z692395-10EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Metzenbaum scissors 14cm</td>
			<td>Ehrhardt Medizinprodukte GmbH, Geislingen, Germany</td>
			<td>22398330</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">luer lock syringe 5ml</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td style="width:320px;">Z248010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">syringe needles</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>Z192368&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon Tube 50ml</td>
			<td>BD Biosciences, Erembodegem, Belgium</td>
			<td style="width:320px;">352070</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence-mediated tomography for the detection and quantification of macrophage-related murine intestinal inflammation

小鼠疾病模型对科学研究是必不可少的。然而, 许多诊断工具, 如内窥镜或断层成像, 在动物模型中并不经常使用。传统的实验读数通常依赖于宰后和前体分析, 从而防止个体的随访检查, 增加所需的研究动物数量。荧光介导的断层扫描使非侵入性的, 重复的, 定量的, 三维荧光探针的评估。它是高度敏感的, 并允许使用分子制造商, 这使得特定的检测和表征明确的分子目标。特别是, 有针对性的探针代表了一个创新的工具, 分析基因活化和蛋白表达在炎症, 自身免疫疾病, 感染, 血管疾病, 细胞迁移, 肿瘤,等。在这篇文章中, 我们提供了 step-by 的步骤说明, 对这种复杂的成像技术的在体内检测和定性的炎症 (即, F4/80-positive 巨噬细胞浸润) 在广泛使用的小鼠模型肠道炎症。该技术也可用于其他研究领域, 如免疫细胞或干细胞追踪。

结肠炎的评估:
DSS 诱导的结肠炎是一种化学诱导的小鼠肠道炎症模型, 类似于人 IBD, 导致体重下降, 直肠出血, 表面溃疡, 和粘膜损害的敏感的老鼠15。研究先天免疫系统对肠道炎症发育的贡献特别有用10,11。为了能诱发 colitic 炎症, 小鼠在整个实验过程中...

Watch this Scientific Journal Video about Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation at JoVE.com

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

目标特定的探针代表了一种用于分析分子机制的创新工具, 例如各种疾病的蛋白质表达 (例如,炎症、感染和肿瘤发生)。在这项研究中, 我们描述了一个定量的三维层析成像评估肠道巨噬细胞浸润的小鼠结肠炎模型使用 F4/80-specific 荧光介导的断层扫描。

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

fluorescence-mediated-tomography-for-detection-quantification

Research

JoVE Journal

Medicine

8.1K 次观看.  University Hospital Münster. 目标特定的探针代表了一种用于分析分子机制的创新工具, 例如各种疾病的蛋白质表达 (例如,炎症、感染和肿瘤发生)。在这项研究中, 我们描述了一个定量的三维层析成像评估肠道巨噬细胞浸润的小鼠结肠炎模型使用 F4/80-specific 荧光介导的断层扫描。

视频: Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)

Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 provide information about the burden of disease. However, despite multiple validation studies of CAC3-5, and C-IMT2,6, these modalities do not accurately assess plaque characteristics7,8, and the composition and inflammatory state of the plaque determine its stability and, therefore, the risk of clinical events9-13.
[18F]-2-fluoro-2-deoxy-D-glucose (FDG) imaging using positron-emission tomography (PET)/computed tomography (CT) has been extensively studied in oncologic metabolism14,15. Studies using animal models and immunohistochemistry in humans show that FDG-PET/CT is exquisitely sensitive for detecting macrophage activity16, an important source of cellular inflammation in vessel walls. More recently, we17,18 and others have shown that FDG-PET/CT enables highly precise, novel measurements of inflammatory activity of activity of atherosclerotic plaques in large and medium-sized arteries9,16,19,20. FDG-PET/CT studies have many advantages over other imaging modalities: 1) high contrast resolution; 2) quantification of plaque volume and metabolic activity allowing for multi-modal atherosclerotic plaque quantification; 3) dynamic, real-time, in vivo imaging; 4) minimal operator dependence. Finally, vascular inflammation detected by FDG-PET/CT has been shown to predict cardiovascular (CV) events independent of traditional risk factors21,22 and is also highly associated with overall burden of atherosclerosis23. Plaque activity by FDG-PET/CT is modulated by known beneficial CV interventions such as short term (12 week) statin therapy24 as well as longer term therapeutic lifestyle changes (16 months)25.
The current methodology for quantification of FDG uptake in atherosclerotic plaque involves measurement of the standardized uptake value (SUV) of an artery of interest and of the venous blood pool in order to calculate a target to background ratio (TBR), which is calculated by dividing the arterial SUV by the venous blood pool SUV. This method has shown to represent a stable, reproducible phenotype over time, has a high sensitivity for detection of vascular inflammation, and also has high inter-and intra-reader reliability26. Here we present our methodology for patient preparation, image acquisition, and quantification of atherosclerotic plaque activity and vascular inflammation using SUV, TBR, and a global parameter called the metabolic volumetric product (MVP). These approaches may be applied to assess vascular inflammation in various study samples of interest in a consistent fashion as we have shown in several prior publications.9,20,27,28

Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography FDG-PET/CT

There is great need to identify atherosclerosis non-invasively, and here we demonstrate how FDG-PET/CT can be used to detect and quantify atherosclerotic plaque activity and vascular inflammation.

动脉粥样硬化斑块的活动和血管炎症的定量使用[18 F]，氟正电子发射断层扫描/电脑断层扫描（FDG-PET/CT）

Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1 and carotid intimal medial thickness (C-IMT)2 ...

科研

医学

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

在啮齿类动物的肠动力及胃肠壁的炎症分析功能评价一个标准化的模式肠道操作

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

鼠肾移植:移植排斥反应模型

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

的不可逆和可逆单侧输尿管梗阻小鼠模型

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

主动脉钙化和炎症血管平滑肌细胞和成像的钙化

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

人类肠道器官脂滴形成特征化和定量的荧光测定

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.