这项工作得到了Convocatoria Interfacultades的支持, 这些信息来自于Vicerrectoría de 门多萨市大学和美国国际开发署研究与创新奖学金计划。我们感谢布伊特拉戈和 Yeferzon 阿迪拉鱼的维护和援助。

下列协议由洛杉矶大学动物保育和使用委员会 (CICUAL) 批准。应严格遵守生物安全2级 (BSL-2) 准则, 以防止感染病原体 T. 虫. 
 注意: 动物护理和维护: 斑马鱼, 一个基因改造的斑马鱼的菌株 (斑马鱼 ) 是在本议定书中使用, 由于其宝贵的光学透明度在所有发展阶段。鱼纵使用最佳的护理条件为种类 14 , 在 14 h light - 10 h 黑暗的周期 , 在 28 和 # 177 ; 1 和 # 176 ; C , 在 pH ( 7 . 0 - 7 . 4 ) 控制体循环水系统。动物每天喂食两次, 活盐水虾 ( 卤虫藻 ), 富含饲养食物。所有的协议都由 CICUAL 在洛斯安大学 (C. FUA_14-017) 批准. 
 1. 准备卵水 <ol> <li> 在反渗透 (RO) 或去离子水中准备0.6 克/l 水族馆盐, 并在溶液中加入0.01 毫克/升亚甲基蓝. 
	注: 测量的电导率应为400-500 和 #181; S/厘米和 pH 值在 7.2-7.9。为了降低 pH 值, 通气了几个小时的卵水. </li>
</ol> 2。Tricaine 库存解决方案的制备 <ol> <li> 在97.9 毫升蒸馏水 (ddH 2 O) 中溶解400毫克 Tricaine (MS-222), 以准备 0.4% Tricaine 的库存解决方案。粉体完全溶解后, 将 ph 值调整为 7.0, 使用2.1 毫升1M 三 (ph 9.0), 并冷藏解决方案. </li>
</ol> 3。1.0% 低熔点琼脂糖的制备 <ol> <li> 将低熔点琼脂糖溶解在卵水中, 最终浓度为1.0%。在微波炉或热板上加热混合物, 连续搅拌直到琼脂糖溶液出现均匀. </li>
	<li> 将等分存储在4和 #176; C 不超过一周. </li>
</ol> 4。交配试验和胚胎采集 <ol> <li> 在注射前三天设置交配使用健康对雄性和雌性鱼在养殖罐。丰富的玻璃大理石和人造植物提高产卵。交配应该在下午建立, 并在一夜之间离开. </li>
	<li> 第二天早上, 通过过滤器将水箱排出, 然后用卵水冲洗鸡蛋, 收集产卵的卵。通过倒置过滤器, 将卵水通过过滤器倒入培养皿中, 除去所有的卵。为了保持胚胎健康, 用转移塑料吸管清除任何残骸和死胚来清洁他们的水. </li>
	<li> 将胚胎移植到恒温温度为28.5 和 #176 的孵化场; C 允许根据斑马鱼的分期标准开发胚胎 15 。每天检查两次胚胎, 并丢弃 inviable 卵以保持离合器的健康. </li>
</ol> 5。胚 Dechorionation 注意: 如果胚胎在注射时未孵出, 则此过程是必需的。在这个过程中, #34; 幼虫和 #34; 是动物从他们的绒毛膜从 48 h 岗位受精 (hpf) 向前. 
<ol> <li> 在实验当天, 将包含健康胚胎的培养皿置于解剖立体下. </li>
	<li> 用两个锋利的镊子抓住绒毛膜的两端 (#5), 轻轻地撕开并拉开绒毛膜。大约有5-6 只幼虫一次注射, 通常3-4 个被成像. </li>
	<li> 使用转移吸管将 chorions 从水中取出. </li>
</ol> 6。准备注塑材料 <ol> <li> 使用薄壁玻璃毛细管和微拉拔器设备准备1.0 毫米针, 其设置如下: 2 轻重量 + 1 重重量, 2 mm 顶拉力 + 5 mm 底拉, 75.9 和 #176; c (步骤 1) + 78.2 和 #176; c (步骤 2)。在一条建模粘土上的培养皿中储存针。理想的针应该有一个狭窄的尖端, 因为 T. 虫 测量大约 20 x 1-3 和 #181; m 并且将容易地通过针。尖端的长度可以变化: 一个较长的尖端是有用的, 以达到更深的结构, 但较短的小费将更刚性, 使注入更容易为用户. 
	注: 针尖大小取决于所使用的温度: 2 步的较高温度会导致更长更细的尖端. </li>
	<li> 在 ddH 中准备1.5% 琼脂糖溶液 2 O 并将其倒入10厘米的培养皿中。覆盖大约1.0 厘米的深度. </li>
	<li> 在琼脂糖上放置一个预制的显微注射模, 并使其固化. </li>
	<li> 将预制的显微注射模取出, 并在4和 #176 中添加鸡蛋水储存。这可以防止琼脂糖干燥. </li>
	<li> 将鸡蛋水添加到 tricaine 库存溶液中, 最终浓度为150毫克/l 存储在4和 #176; C. </li>
</ol> 7。寄生虫生长的细胞培养 <ol> 使用 CRL-1718 描述的方法在人类星形瘤细胞系中保存寄生虫, 桑布拉诺 et al. 16 </li> <li> 在 T25 培养烧瓶 (表面积 = 25 cm 2 ) 中启动人类细胞的培养, 密度为 2 x 10 5 或 4 x 10 5 细胞, 在4毫升的 RPMI-1640 培养基中有10% 的胎儿小牛血清 (FCS), 补充具有2毫米 l-谷氨酰胺, 4.5 克/升葡萄糖, 10 毫米 HEPES, 1 毫米丙酮酸, 1% 青霉素-链霉素 (称为和 #34; 完整的媒体和 #34;)。在 5% CO 2 环境中孵育37和 #176 的星形细胞瘤培养物. </li>
	<li> 一旦单层是汇合的, 分离的细胞使用2毫升的0.25% 的胰蛋白酶-EDTA 通过孵化3分钟, 在37和 #176; c. 视觉检查细胞脱离, 并阻止胰蛋白酶溶液使用3毫升的 RPMI-1640 10% ml 在15毫升管. </li>
	<li> 将电池悬浮液离心5分钟, 1350 x g. </li>
	<li> 在15毫升的管中用 DMEM 介质清洗产生的细胞颗粒, 在22和 #176 上离心5分钟, 1350 x g。
	<li> 在 Neubauer 室中手动计数单元格, 并在40X 放大倍数的光学显微镜下检查其可行性. </li>
	<li> 在4毫升的完整介质中轻轻悬浮细胞颗粒, 并用它来创建新的细胞培养使用 2 x 10 5 每 T25 培养瓶的细胞. </li>
</ol> 8. 虫 区域性和标记 <ol> <li> 寄生生物是最初从受感染的人身上获得的一种菌株, 其特征为 T. 虫 DA 菌株 (MHOM/CO/01/DA), 基因型离散类型单位 (DTU) 特克斯和凯科斯群岛 16 。在3或4天的培养后, 从人类星形细胞瘤培养上清液中收集移动的寄生虫, 并离心5分钟, 在 1350 x g. 丢弃介质. 
	注: 完全培养基中的寄生虫可用于 T25 培养瓶中星形胶质细胞瘤 re-culture 1:1 的比值. </li>
	<li> 在10毫升无菌1x 磷酸盐缓冲盐水 (PBS) 中用0.1% 个 FCS 和离心机轻轻地 re-suspend 5 分钟, 在 1350 x g 上. </li>
	<li> 在 PBS 1 毫升中丢弃上清和 re-suspend。以10和 #181; 我数自由寄生虫在 Neubauer 室. </li>
	<li> 采取其余的悬浮寄生虫 (990 和 #181; l), 并添加1和 #181; 羧基乙酸 Succinimi 酯的 ldyl 酯 (CFSE), 最终浓度5.0 和 #181; m. 室温下孵育10分钟. 
	注: CFSE 5 毫米的库存应 aliquoted 并保持在-20 和 #176; C. </li>
	<li> 颗粒的寄生虫 (1350 x g, 5 分钟)。通过弹管和洗涤10毫升的 1x PBS-0.1%fcs 后, 随后旋转 (1350 x g, 5 分钟), 重建寄生虫颗粒. </li>
	<li> 丢弃上清, 并在 1X PBS 中轻轻 re-suspend 标记的寄生虫颗粒, 适当的体积, 以获得最终浓度约10-20 寄生虫/nL 注射. </li>
	<li> 使用小容量 (#8764; 10 和 #181; L, 大约 1 x 10 4 -2 x 10 4 寄生虫) trypomastigotes 评估生存力和标记。倒置荧光显微镜可用于寄生虫的直接可视化。在透射光模式下, 检查寄生虫是否移动。在荧光模式下, 使用荧光素异硫氰酸酯 (FITC) 过滤器来评估寄生虫标记. </li>
</ol> 9。注射斑马鱼幼虫 <ol> <li> 寄生虫载入 <ol> <li> 从悬浮 (100 和 #181; l) 中寄生寄生虫, 用10和 #181 将它们装入密封的玻璃针中; l microloader 吸管尖端. </li>
		<li> 将玻璃针插入微的针架中, 使用磁性支架, 然后将其放在立体的旁边. </li>
		<li> 使用立体下的细钳, 切下针头, 创建一个钝开放端, 并测量滴大小, 以获得 1 nL 的注入量 (这相当于直径 0.12-0. 13 毫米时, 在矿物油中测量 17 )。较长的尖端是首选, 使针可以到达结构深处的鱼, 而不损害组织。在本协议中, 使用钝针, 但也可以使用斜面针. </li>
	</ol> </li> <li> 安装 <ol> <li> 麻醉 48 hpf 的幼虫, 150 毫克/升 tricaine。检查他们不回应触摸, 但确保心脏跳动. </li>
		<li> 将幼虫放在微注射琼脂糖模具中的横向位置. </li>
		<li> 将针座放在立体的旁边, 调整微, 使针的尖端位于视野的中心。将培养皿中的步骤 6.2-到 6.4, 使幼虫蛋黄接近针尖。检查幼虫的状况, 确保心脏继续跳动. </li>
		<li> 设置微量值, 如下所示: 注射压力每英寸9.6 磅 (psi); 保持压力:20 psi; 100 ms 范围的浇注; 周期值 1.9 (对应于10.9 毫秒)。射入的容量应该在 1-3 nL 之间, 与大约10-20 寄生生物或 nl. </li>
	</ol> </li> </ol> 10。注射寄生虫 <ol> <li> 在维的导管中将鱼插入蛋黄的前半部分。这一步骤应实行, 以确保动物生存后注射。成功地注射5-6 幼虫大约需要10分钟. </li>
	<li> 作为一个控制, 注入1-2 个相同的车辆体积 (1x PBS) 的幼虫. </li>
	<li> 立即将幼虫转移到新鲜的卵水中. </li>
</ol> 11。注射幼虫的 LSFM 安装 <ol> <li> 将带有寄生虫的幼虫转移到一个空的培养皿中, 用塑料吸管和吸水纸小心地除去周围的水分。立即添加100和 #181; 预热1.0% 低熔点琼脂糖 (参阅步骤3琼脂糖制剂) 覆盖幼虫。确保琼脂糖不超过40和 #176; C. </li>
	<li> 在 1.0 mm 玻璃毛细管内插入一根直线, 并将其用作柱塞, 以垂直位置吸收幼虫。确保在幼虫上面留下少量的琼脂糖。等到琼脂糖凝固后才露出幼虫 (这将需要几分钟)。如有必要, 将多余的琼脂糖放在幼虫下面, 并将其切断. </li>
	<li> 将毛细管放在显微镜标本夹中, 并把含有幼虫的末端推开, 直到它从毛细血管中游离出来。试样室应充满 tricaine 溶液 (150 毫克/升), 温度设置在28和 #176; C. </li>
	<li> 使用 XYZ-微系统将幼虫置于检测目标的瞳孔前面。对于垂直轴的旋转, 请使用旋转阶段。幼虫的定位应在透射光模式下进行, 以明确识别幼体的结构. </li>
</ol> 12。注射寄生虫的 LSFM 成像 <ol> <li> 更改为荧光模式并调整光照强度以及曝光时间以减少光并优化时间分辨率。对于这些特定的实验, 我们使用的功率为 2.8-3.0 兆瓦的样品, 并暴露了每帧 200 ms 使用相机与6.45 和 #181; m 像素和〜70% 量子效率的检测波长。这些设置应导致在大约5帧/秒内充分曝光图像. 确保使用尽可能高的数字光圈的目标. </li>
	<li> 开始对感兴趣区域 (ROI) 进行视频获取。在我们的例子中, 寄生虫被观察附着在瓣膜上, 并在心脏区域自由移动。它是建议采取一个单一的飞机一段时间的视频, 或使用微或压电振系统的焦点不同的飞机, 而寄生虫移动. 
	注意: 此过程适用于2小时的短时间捕获。对于长期采集, 应使用替代方法 20 来安装幼虫. </li>
</ol> 13。获取的数据的图像处理和分析 注意: 图像处理是在具有 2.90 GHz 处理器、8.00 gb 内存和具有 1.00 gb 内存的显卡的个人计算机上执行的. 
<ol> <li> 在选择的图像处理软件中打开获取的数据集。开放软件斐济推荐用于 LSFM 数据处理和分析 21 . </li>
	<li> 在图像分析软件中, 调整亮度和对比度级别以增强图像. 
	注意: 在这种情况下没有使用固定的参数, 但选择和 #34; 自动和 #34; 选项可用于获得初始增强. </li>
	<li> 选择 ROI. 
	注意: 对于追踪个别寄生虫, 斐济有手动和自动跟踪插件可用. </li>
</ol> 14。正在恢复映像幼虫 <ol> <li> 使用细钳和毛发循环工具从琼脂糖中小心翼翼地取出幼虫。将鱼放回新鲜的卵水中, 在15分钟后进行恢复, 并在28、#177、0.5、#176; C. </li>
	<li> 或者, 继续使用过量的 tricaine 来安乐影像幼虫。然后, 在次氯酸钠 (6.15% 次氯酸) 溶液中引入幼虫, 以杀灭寄生虫5分钟。根据机构和 #39 的标准协议进行处理. </li>
</ol>

<ol>
	<li>Bern, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagas's+Disease.">Chagas's Disease.</a> New England Journal of Medicine. 373 (5), 456-466 (2015).</li><li>Sosa Estani, S., Segura, E. L., Ruiz, A. M., Velazquez, E., Porcel, B. M., Yampotis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+chemotherapy+with+benznidazole+in+children+in+the+indeterminate+phase+of+Chagas&#8217;+disease.">Efficacy of chemotherapy with benznidazole in children in the indeterminate phase of Chagas&#8217; disease.</a> The American Journal of Tropical Medicine and Hygiene. 59 (4), 526-529 (1998).</li><li>Rosas, F., Roa, N., Cucunuba, Z. M., Cuellar, A., Gonzalez, J. M., Puerta, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagasic+Cardiomyopathy.">Chagasic Cardiomyopathy.</a> Cardiomyopathies - From Basic Research to Clinical Management. , (2012).</li><li>Finkelsztein, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altering+the+motility+of+Trypanosoma+cruzi+with+rabbit+polyclonal+anti-peptide+antibodies+reduces+infection+to+susceptible+mammalian+cells.">Altering the motility of Trypanosoma cruzi with rabbit polyclonal anti-peptide antibodies reduces infection to susceptible mammalian cells.</a> Experimental Parasitology. 150, 36-43 (2015).</li><li>Martins, R. M., Covarrubias, C., Rojas, R. G., Silber, A. M., Yoshida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+L-Proline+and+ATP+Production+by+Trypanosoma+cruzi+Metacyclic+Forms+as+Requirements+for+Host+Cell+Invasion.">Use of L-Proline and ATP Production by Trypanosoma cruzi Metacyclic Forms as Requirements for Host Cell Invasion.</a> Infection and Immunity. 77 (7), 3023-3032 (2009).</li><li>Engstler, 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=Hydrodynamic+Flow-Mediated+Protein+Sorting+on+the+Cell+Surface+of+Trypanosomes.">Hydrodynamic Flow-Mediated Protein Sorting on the Cell Surface of Trypanosomes.</a> Cell. 131 (3), 505-515 (2007).</li><li>Uppaluri, 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=Impact+of+Microscopic+Motility+on+the+Swimming+Behavior+of+Parasites:+Straighter+Trypanosomes+are+More+Directional.">Impact of Microscopic Motility on the Swimming Behavior of Parasites: Straighter Trypanosomes are More Directional.</a> PLoS Computational Biology. 7 (6), e1002058 (2011).</li><li>Heddergott, N., 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=Trypanosome+Motion+Represents+an+Adaptation+to+the+Crowded+Environment+of+the+Vertebrate+Bloodstream.">Trypanosome Motion Represents an Adaptation to the Crowded Environment of the Vertebrate Bloodstream.</a> PLoS Pathogens. 8 (11), e1003023 (2012).</li><li>Gratacap, R. L., Wheeler, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+zebrafish+for+intravital+study+of+eukaryotic+pathogen&#8211;host+interactions.">Utilization of zebrafish for intravital study of eukaryotic pathogen&#8211;host interactions.</a> Developmental &amp; Comparative Immunology. 46 (1), 108-115 (2014).</li><li>Meijer, A. H., Spaink, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-Pathogen+Interactions+Made+Transparent+with+the+Zebrafish+Model.">Host-Pathogen Interactions Made Transparent with the Zebrafish Model.</a> Current Drug Targets. 12 (7), 1000-1017 (2011).</li><li>White, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+Adult+Zebrafish+as+a+Tool+for+In+Vivo+Transplantation+Analysis.">Transparent Adult Zebrafish as a Tool for In Vivo Transplantation Analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li><li>Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slicing+embryos+gently+with+laser+light+sheets.">Slicing embryos gently with laser light sheets.</a> BioEssays. 34 (5), 406-411 (2012).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203 (3), 253-310 (1995).</li><li>Vargas-Zambrano, J. C., Lasso, P., Cuellar, A., Puerta, C. J., Gonz&#225;lez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+astrocytoma+cell+line+is+highly+susceptible+to+infection+with+Trypanosoma+cruzi.">A human astrocytoma cell line is highly susceptible to infection with Trypanosoma cruzi.</a> Mem&#243;rias do Instituto Oswaldo Cruz. 108 (2), 212-219 (2013).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> JoVE (Journal of Visualized Experiments). (25), e1115-e1115 (2009).</li><li>Lorenzo, 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=Live+cell+division+dynamics+monitoring+in+3D+large+spheroid+tumor+models+using+light+sheet+microscopy.">Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy.</a> Cell Division. 6, 22 (2011).</li><li>Edelstein, A. D., Tsuchida, M. A., Amodaj, N., Pinkard, H., Vale, R. D., Stuurman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+methods+of+microscope+control+using+&#956;Manager+software.">Advanced methods of microscope control using &#956;Manager software.</a> Journal of Biological Methods. 1 (2), e10 (2014).</li><li>Weber, M., Mickoleit, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+Mounting+for+Long-term+Light+Sheet+Microscopy+of+Zebrafish.">Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish.</a> Journal of Visualized Experiments. (84), (2014).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Ng, A. N. 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=Formation+of+the+digestive+system+in+zebrafish:+III.+Intestinal+epithelium+morphogenesis.">Formation of the digestive system in zebrafish: III. Intestinal epithelium morphogenesis.</a> Developmental Biology. 286 (1), 114-135 (2005).</li><li>Higuchi, M. D. 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=Correlation+between+Trypanosoma+cruzi+parasitism+and+myocardial+inflammatory+infiltrate+in+human+chronic+chagasic+myocarditis:+Light+microscopy+and+immunohistochemical+findings.">Correlation between Trypanosoma cruzi parasitism and myocardial inflammatory infiltrate in human chronic chagasic myocarditis: Light microscopy and immunohistochemical findings.</a> Cardiovascular Pathology: The Official Journal of the Society for Cardiovascular Pathology. 2 (2), 101-106 (1993).</li><li>Vago, A. R., Silva, D. M., Adad, S. J., Correa-Oliveira, R., d'Avila Reis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+Chagas+disease:+presence+of+parasite+DNA+in+the+oesophagus+of+patients+without+megaoesophagus.">Chronic Chagas disease: presence of parasite DNA in the oesophagus of patients without megaoesophagus.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 97 (3), 308-309 (2003).</li><li>Wiegertjes, G. F., Forlenza, M., Joerink, M., Scharsack, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite+infections+revisited.">Parasite infections revisited.</a> Developmental &amp; Comparative Immunology. 29 (9), 749-758 (2005).</li><li>De Souza, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+cell+biology+of+Trypanosoma+cruzi.">Basic cell biology of Trypanosoma cruzi.</a> Current Pharmaceutical Design. 8 (4), 269-285 (2002).</li><li>Langousis, G., Hill, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+and+more:+the+flagellum+of+Trypanosoma+brucei.">Motility and more: the flagellum of Trypanosoma brucei.</a> Nature Reviews. Microbiology. 12 (7), 505-518 (2014).</li><li>Johnson, C. 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=Cellular+response+to+Trypanosoma+cruzi+infection+induces+secretion+of+defensin+&#945;-1,+which+damages+the+flagellum,+neutralizes+trypanosome+motility,+and+inhibits+infection.">Cellular response to Trypanosoma cruzi infection induces secretion of defensin &#945;-1, which damages the flagellum, neutralizes trypanosome motility, and inhibits infection.</a> Infection and Immunity. 81 (11), 4139-4148 (2013).</li><li>Magalh&#227;es, L. M. D., Viana, A., Chiari, E., Galv&#227;o, L. M. C., Gollob, K. J., Dutra, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Activation+of+Human+Monocytes+and+Lymphocytes+by+Distinct+Strains+of+Trypanosoma+cruzi.">Differential Activation of Human Monocytes and Lymphocytes by Distinct Strains of Trypanosoma cruzi.</a> PLoS neglected tropical diseases. 9 (7), e0003816 (2015).</li></ol>

作者没有什么可透露的。

本研究着重介绍了斑马鱼作为一种动物模型来研究病原体行为的优点在体内。特别是, 本研究提出了一种在其自然环境中对病原体虫进行可视化的方法: 血循环。鱼类的循环系统环境与哺乳动物相似, trypanosomatids 已经进化出了旅行的机制, 躲避免疫系统, 并在该环境中附加到感染细胞的25。本协议提供了一个关于在人类细胞系中培养T. 虫的最佳过程的描述, 并随后对荧光标记的鞭毛形式进行隔离。它然后显示适当的设置为成功地注射寄生虫入透明斑马鱼为以后装载和形象化使用 LSFM。最后, 本协议为有效和有效的在体内成像的寄生虫的位置和流通使用 LSFM。
锥的鞭毛从它的后部区域涌现, 从细胞身体流动, 并且在有机体的前面自由垂悬在26。T. 虫通过在身体前方挥舞鞭毛来推动自身, 这波动了寄生虫的整个身体。鞭毛运动不仅是不可缺少的寄生虫运动, 如在T. 布27 (非洲锥虫病的致病剂), 但它也被用于细胞感染, 正如已经在T. 虫5 中演示的那样. ,28。虽然斑马鱼不是T. 虫的天然宿主, 但使用此处所述的协议, 可以在发展中的心血管循环系统中研究寄生虫的运动。此外, 还有锥种感染科类的斑马鱼, 如t. carassii和t. borreli25.这些寄生虫种类可用于实时研究这些 trypanosomatids 的运动和附着机制;这样的研究可以洞察哺乳动物细胞感染过程。
在这项研究中, 注入运动的T. 虫寄生虫被观察到通过接种鱼的心血管循环, 移动与不透明的红细胞, 并坚持在心血管系统壁的结构。我们使用了一个国产 LSFM 与10X 无色长工作距离空气目标 (17.6 毫米) 为照明臂与数字孔径0.25。一个40X 的物镜水浸泡物镜的数值孔径为 0.8, 工作距离为3.5 毫米, 用于检测臂。检测目标被浸入样品室, 而光照目标则在腔室外。如洛伦佐et al.中所描述的, 室内的一个端口用片和光学胶水密封, 使照明光束进入腔室。18为了照明, 我们在 488 nm 使用了50兆瓦的 DPSS 激光器, 其功率由声光可调谐滤波器调制。检测路径采用与绿色荧光蛋白 (GFP) 或 FITC 相容的过滤器。配有毛细管取样器 (理想情况下具有自动旋转) 的光板显微镜和样品室的温度控制应该适合这个应用。如有必要, 应根据制造商的说明书或用户的实验室标准协议对显微镜进行校准和标定。在本协议中, 我们使用 SPIM 软件19控制了显微镜。
重要的是要注意, 在斑马鱼的循环, 幼虫寄生附着是有效的。在大是大非的静脉, 寄生虫仍然连着长达几分钟;在心脏, 他们举行了阀门和墙壁, 摆动与心脏收缩。进一步的研究仍然是为了阐明T. 虫是否与流向血液流动方向的流动红细胞相互作用。以前的体外研究表明, 固体结构 (如: 即, 血细胞) 的存在, 或增加液体的粘度以模拟血液的体外, 对寄生虫的运动和速度有显著的影响。9。
有许多问题关于感染的过程中的T. 虫在人类无逃逸吞噬细胞后, 最初受感染的细胞类型29。例如, 他们如何到达他们的目标器官？什么是倾向于首选器官的机制, 如心脏、消化系统和中枢神经系统？有趣的是, 在这项研究中, 寄生虫最初是在心脏成像的, 因为它是寄生生物密度最高的部位。然而, CFSE 信号随后在开发的肚腑积累了7天后注射。虽然鱼类和哺乳动物的解剖是不同的, 这项研究的结果表明了一种取向的形式, 因为它被观察到, 寄生虫表现出倾向于已知的首选靶器官, 尽管个体的差异。这项研究的一个重要限制是有关实验中使用的温度。斑马鱼幼虫在整个过程中应保持在 28 °C 左右。虽然这种温度可能类似于向量寄主 (Triatominae 亚科的昆虫), 但它与组成最终寄主的 warm-blooded 哺乳动物 (大约37° c) 是完全不同的。T. 虫已知在两个主机上都有鞭毛的生存形式;然而, 重要的是要牢记, 这一因素可能会影响动物的行为在体内。
虽然 fish´s 自适应免疫系统是不成熟的, 直到4周后受精, 先天免疫系统是活跃的早期开发10。早在48或 96 hpf, 吞噬细胞被发现吞噬标记锥 (数据没有显示)。这就限制了寄生虫可视化的时间窗口。然而, 如果一项研究的重点是评估 fish´s 免疫反应, 注射在后期阶段可能会被推荐。此外, 将寄生虫注入带有标记的巨噬细胞或免疫系统的其他单元的转基因鱼系, 可有助于研究寄生虫附着和可能的吞机制。重要的是要注意, 如果寄生虫被标记为 CFSE, 转基因细胞标签不应该是 GFP, 并在黄色或红色的光谱结束的标志是必需的。
为了评估寄生虫运动的详细方向, 在3维 (3D) 中跟踪它们的轨迹可能是有用的。对于3D 的可视化和重建过程, 需要一个高速系统。在该协议中使用的设备, 只有在一个平面上可视化寄生虫。在这种情况下, 我们优先考虑在寄生虫运动期间保持焦平面的稳定性, 并在一个平面上记录它的轨迹。
本文提出的方法为进一步研究心血管循环中的寄生虫行为铺平了道路。总之, 在斑马鱼幼虫体内成像活体荧光寄生虫的基本步骤是:(i) 使用早期孵化的胚胎 (24-48 hpf) 或幼虫, 或 72-96 hpf 之间的动物, 无色素沉着, 使其透明且易于注射;(ii) 在注射后应尽快将影像幼虫清除, 以避免吞噬细胞的寄生虫;(iii) 将 LSFM 集中在感兴趣的地点 (例如, 心包区) 并保持焦点。这个新的程序允许在一个环境中的 trypomastigotes 的可视化与它的自然感染生态位, 第一次提供的可能性, 研究T. 虫在一个活生生的有机体。

恰加斯病是由原生动物寄生虫引起的T. 虫.全世界大约有6至700万人感染了T. 虫。这种疾病主要在拉丁美洲传播, 但在美国、加拿大和许多欧洲以及一些西太平洋国家都有报道, 主要是由于受感染的个人的迁徙1。恰加斯在很大程度上是通过与 Triatominae 亚科的 hematophagic 昆虫的粪便接触而传染给人类的, 俗称 "接吻虫"。但是, T. 虫也可以通过输血、从母亲到儿童的垂直转移或摄入受寄生虫感染的食物 (2) 进行传输。急性阶段的感染主要是无症状或组成症状和持续时间从6到8周, 在此之后, 免疫系统的参与控制寄生虫负荷, 但没有完全消除感染3。多数个体然后进入慢性无症状的阶段;然而, 近30% 的患者发展为症状性慢性期, 其中心脏系统和较少频繁消化系统和神经系统被危及4。这种情况对疾病的治疗和控制提出了挑战, 因为没有疫苗可供使用, 而且只有两种有效药物用于恰加斯: 唑和呋。这两种治疗方法都需要长时间的管理, 并且可能有严重的副作用2。
提高对T. 虫行为在体内的行为的理解是确定寄生迁移、细胞附着和宿主内部入侵的关键;缺乏在体内模型限制了新的治疗方法的发展。体外对T. 虫感染的研究表明, trypomastigotes 的运动对宿主细胞膜和随后的细胞侵袭5具有重要作用。trypomastigotes 中的能量消耗在与易感细胞系共培养中被证明可以减少细胞侵入6。有趣的是, 在 trypanosomatids, 鞭毛运动也被定性为一种规避寄生虫特异抗体的机制7。
鞭毛运动已被广泛研究在体外在锥布, 一个密切相关的寄生虫, 导致非洲锥虫病8。体外对这些锥的运动的研究表明, 模拟的血液或体液的条件, 包括粘度和存在的障碍代表血细胞, 是重要的寄生虫向前运动9.到目前为止, 还无法将寄生虫在血液中的运动在体内进行可视化。
斑马鱼幼虫是研究宿主-病原体相互作用的一个强有力的模型在体内。与其他已建立的恰加斯病的脊椎动物模型相比, 它们体积小, 价格低廉, 而且相对容易饲养。斑马鱼的先天和适应性免疫系统类似于人类, 但他们的适应性免疫系统开始发展4天后受精 (dpf), 并没有成熟的另一个几个星期10。在早期的发展过程中, 当只有巨噬细胞存在时, 有一个很大的窗口来研究寄生虫的行为, 而没有立即的免疫干扰10。然而, 利用斑马鱼幼虫作为研究病原体行为的脊椎动物模型的最大优势在于它们的光学透明度, 使它们适于显微镜检查和成像11。此外, 有多种工具来操纵鱼类遗传学。例如, "精灵" 菌株是斑马鱼的变种, 没有色素沉着, 使动物完全透明和有用的可视化的个别器官和 real-time 跟踪注入细胞12。
利用共焦或荧光显微镜观察活斑马鱼快速移动寄生虫的纵向观测的一个关键限制在于, 在高采集速度和大穿透深度下, 成像质量和低光的风险。光表荧光 micsroscopy (LSFM) 是一种新兴的成像技术, 克服这些限制, 允许这些观察。利用一个目标检测荧光和第二个正交光照目标, 只照亮检测目标的焦面, 就可以在共焦显微镜下获得高分辨光学截面, 但显着减少光, 甚至关于荧光显微镜13。这里使用的 LSFM 技术称为单平面照明显微镜 (SPIM), 其中一层薄薄的光照亮了斑马鱼幼虫中的一张飞机。
该方法的目的是建立斑马鱼幼虫作为一个可行的感染模型, 以了解T. 虫运动和相关行为在体内。为了做到这一点, 我们注射了透明的斑马鱼幼虫与荧光标记 trypomastigotes, 细胞形式负责感染人类, 并确定了运动的T. 虫在心血管循环的斑马鱼使用 LSFM。

<table border="0" cellpadding="0" cellspacing="0" width="653">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">0.5-10 &#956;L Micropipette</td>
			<td style="width:164px;">Fisherbrand</td>
			<td style="width:104px;">21-377-815</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Agarose RA</td>
			<td style="width:164px;">Amresco</td>
			<td style="width:104px;">N605</td>
			<td>Regular</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Agarose SFR</td>
			<td style="width:164px;">Amresco</td>
			<td style="width:104px;">J234</td>
			<td>Low Melting point</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Aquarium salt</td>
			<td style="width:164px;">Instant ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Count chamber</td>
			<td style="width:164px;">Boeco</td>
			<td>Neubauer&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture flasks</td>
			<td style="width:164px;">Corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Centrifuge</td>
			<td style="width:164px;">Sorvall</td>
			<td style="width:104px;">Legend RT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFSE</td>
			<td>ThermoFisher</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Detection objective</td>
			<td style="width:164px;">Nikon 40x 0.8NA&#160;</td>
			<td style="width:104px;">40x CFI APO NIR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM medium</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:231px;">Dumont #5 fine forceps</td>
			<td style="width:164px;">World precision Instruments</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:104px;">A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fetal calf serum (FCS)</td>
			<td style="width:164px;">Eurobio</td>
			<td>CVFSVF00-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Filter</td>
			<td style="width:164px;">Chroma</td>
			<td style="width:104px;">ET-525/50M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass capillaries for embryo mounting</td>
			<td style="width:164px;">Vitrez Medical</td>
			<td>160215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass capillaries for pulling needles</td>
			<td style="width:164px;">World precision instruments</td>
			<td>TW100-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td style="width:164px;">Gibco</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">HEPES</td>
			<td style="width:164px;">Gibco&#160;</td>
			<td>156300-80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Incubator</td>
			<td style="width:164px;">Thermo Corporation</td>
			<td>Revco</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Larval microinjection mold</td>
			<td>Adaptive Science Tools</td>
			<td>I-34</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Laser</td>
			<td style="width:164px;">Crystalaser</td>
			<td style="width:104px;">DL488-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td style="width:164px;">Gibco</td>
			<td>250300-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene blue</td>
			<td>Albor Qu&#237;micos</td>
			<td>12223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator&#160;</td>
			<td style="width:164px;">Narishige</td>
			<td>MN-153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator system</td>
			<td style="width:164px;">Sutter Instrument</td>
			<td>MP-200</td>
			<td>For LSFM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette puller device&#160;</td>
			<td>Narishige</td>
			<td style="width:104px;">PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope&#160;</td>
			<td>Olympus</td>
			<td>CX31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope (inverted)</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multipurpose microscope</td>
			<td style="width:164px;">Nikon</td>
			<td style="width:104px;">AZ100M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neubauer counting chamber</td>
			<td style="width:164px;">Boeco Germany</td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-streptomycin</td>
			<td style="width:164px;">Gibco&#160;</td>
			<td>15140-163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish 94x16</td>
			<td>Greiner bio-one</td>
			<td>633181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Plastic pasteur pipette</td>
			<td>Fisherbrand</td>
			<td>11577722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Rotation stage</td>
			<td>Newport</td>
			<td>CONEX-PR50CC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI-1640 medium</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:104px;">R4130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereoscope</td>
			<td style="width:164px;">Nikon</td>
			<td>C-LEDS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tricaine (MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>886-86-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">TRIS</td>
			<td style="width:164px;">Amresco</td>
			<td style="width:104px;">M151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:164px;">Gibco</td>
			<td>R-001-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tubes 15 ml</td>
			<td style="width:164px;">Corning</td>
			<td>05-527-90</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of larval zebrafish as an animal model to investigate trypanosoma cruzi motility in vivo

恰加斯病是由锥虫引起的寄生虫感染, 其运动不仅对局部化很重要, 而且对细胞的结合和侵袭也是如此。当前动物模型为研究T. 虫允许有限的观察寄生生物在体内,代表对了解寄生虫行为的挑战在最初的阶段在人的传染。这种原生动物在载体和哺乳动物宿主中都有一个鞭毛阶段, 但是没有研究描述它的运动在体内。该项目的目的是建立一个活脊椎动物斑马鱼模型, 以评估T. 虫运动的血管系统。透明斑马鱼幼虫注射荧光标记 trypomastigotes 和观察使用光片荧光显微镜 (LSFM), 一种无创的方法, 可视化活生物体高光学分辨率。由于这种技术相对于共焦或荧光显微镜的光风险较低, 寄生虫可以被可视化较长时间。T. 虫寄生虫在不同血管和蛋黄的活斑马鱼循环系统中观察到。他们也可以看到连接到卵黄囊壁和房室瓣, 尽管强烈的力量与心脏收缩有关。LSFM 的T. 虫-接种斑马鱼幼虫是一种有价值的方法, 可用于可视化循环寄生虫和评价其取向, 迁移模式, 并在动态环境中的活动动物心血管系统的运动。

最佳注射条件:
斑马鱼幼虫被注射在 24, 48, 72, 96, 和 120 hpf, 在不同的解剖部位, 他们的生存每天检查了5天。经过5天注射后, 注射在 24 hpf 的胚胎有 6.25% (2/32) 存活, 而 95% (38/40) 的幼虫注射在 48 hpf 存活。作为一种控制, 幼虫被注射 1x PBS 作为交通工具。在注射和注射寄生虫的幼虫之间, 生存率没有差异, 这表明没有寄生依赖性影响鱼类存活率 (p = 0.08)。在 72-120 hpf 之间注射的幼虫在恒定的注射量下有可比的存活率到 48 hpf 被注射的幼虫。对于这里提出的所有程序, 48 hpf 幼虫使用由于其易于操作, 并有发达的器官和容易穿透皮肤没有明显的损害后注射。
在 48 hpf 注射的幼虫在心包间隙、尾肌、后脑室、耳囊、脊索和维管中注入卵黄囊。在不同解剖部位注射的幼虫存活率无差异。然而, 最快和最容易注射的区域是位于卵黄囊前部的维导管 (图 1,电影 1)。在该网站注射, 使更高的体积, 更低的风险, 对重要的结构的伤害。另外, 在 24-72 hpf 之间, 这个区域是一个最佳的站点直接地访问开发的血管和心脏11。
使用 LSFM 的寄生虫可视化:
在将T. 虫注入维导管后8-10 分钟内, 斑马鱼幼虫使用 LSFM 由于其 CFSE 荧光信号和幼虫的光学透明度而确定了寄生虫。接种后, 观察到的寄生虫要么附着在循环系统周围的墙壁上, 要么沿着血液流向 (图 2,图 3)。当一个寄生虫附着在心脏结构上, 如房室瓣膜, 它会随着心脏的收缩而振荡, 这表明寄生虫的黏附分子机制在我们的脊椎动物模型中可能是有效的 (电影 2, 电影3, 补充电影 1)。T. 虫还附着在幼虫卵黄囊的壁上 (图 2,电影 2), 该结构稍后将被吸收并成为斑马鱼肠道的一部分22。这可能是类似的发生在慢性疾病阶段在感染的人, 那里的寄生虫是在心肌细胞和消化系统神经系统的23,24。当不附着时, 寄生虫通过血液流动与红细胞 (图 3,电影 4) 相同的方向漂移。寄生虫可以观察到不同大小的鱼的船只, 但更丰富的心包间隙和邻近的蛋黄区域, 包含血流 (图 2,图 3,补充电影 2)。
在10分钟的注射后, 更难发现单一形式的寄生虫, 由于他们的分布沿血管, 并无法迅速筛选不同的解剖部位的鱼由于有限的视野的 LSFM (在40X 倍放大).在 24 h 后注入 (hpi) 后, CFSE 信号开始在发育中的小肠附近的区域中积聚 (补充图 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56238/56238fig1.jpg"> 
图 1:最佳注入站点(a) 幼虫 48 hpf 的图像, 在维 (黄色箭头) 的导管上使用常规立体显示最佳注射部位。(B) 放大的框视图, 显示维 (黄色箭头) 的管道。刻度栏 = 200 µm (a), 50 µm (B)。<a href="//ecsource.jove.com/files/ftp_upload/56238/56238fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56238/56238fig2.jpg"> 
图 2:LSFM 48 hpf 幼虫中的静态寄生虫的图像.T. 虫寄生生物 (黄色箭头) 保持在蛋黄囊的壁上, 在整个时移序列 (7.2 s、17.2 s 和 27.2 s), 大约约〜15分钟寄生虫注射。在至少三十年代的收购期内, 观察到寄生虫的位置没有变化. a, 心房;v, 心室。缩放栏 = 50 µm.<a href="//ecsource.jove.com/files/ftp_upload/56238/56238fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56238/56238fig3.jpg"> 
图 3:在心包间隙中使用 LSFM 的寄生虫的轨迹.当在心包间隙 (PS) 中漂流时, 可以跟踪T. 虫寄生生物, 跟随血液流动的方向 (显示在红色的轨道上) 大约在寄生虫注射后15分钟。缩放栏 = 50 µm.<a href="//ecsource.jove.com/files/ftp_upload/56238/56238fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Movie 1" src="/files/ftp_upload/56238/56238mov1.jpg"> 
影片 1: 蛋黄的血液循环谷在幼虫 48 hpf.电影的幼虫 48 hpf 显示血液循环山谷或管道维使用定期立体。在视频中, 不同的区域会聚焦在整个导管的红色血液细胞中。黄色箭头显示最佳注入位置。影片被记录了大约10-15 分钟在寄生虫注射以后。<a href="//ecsource.jove.com/files/ftp_upload/56238/Movie_1.mp4" target="_blank">请单击此处下载视频.</a>
<img alt="Movie 2" src="/files/ftp_upload/56238/56238mov2.jpg"> 
电影 2: T. 虫寄生虫附着在蛋黄囊壁上.LSFM 的一个幼虫 48 hpf 的电影显示, T. 虫寄生虫在寄生虫注射后10-15 分钟仍附着在卵黄囊中。在至少三十年代的收购期内, 观察到寄生虫的位置没有变化. a, 心房;v, 心室。<a href="//ecsource.jove.com/files/ftp_upload/56238/Movie_2.mp4" target="_blank">请单击此处下载视频.</a>
<img alt="Movie 3" src="/files/ftp_upload/56238/56238mov3.jpg"> 
电影 3: T. 虫寄生虫附着在心脏的壁上.LSFM 电影的幼虫 48 hpf 显示, T. 虫寄生虫 remain 黏附在心血管壁, 尽管强的心脏收缩大约10-15 分钟在寄生虫射入以后。红细胞可以被观察为黑色圆形阴影。<a href="//ecsource.jove.com/files/ftp_upload/56238/Movie_3.mp4" target="_blank">请单击此处下载视频.</a>
<img alt="Movie 4" src="/files/ftp_upload/56238/56238mov4.jpg"> 
电影 4: 在心包间隙中移动的寄生虫.LSFM 电影的幼虫 48 hpf 显示T. 虫寄生虫漂浮在心包间隙。两个寄生虫可以追溯到不同的时间点 (id 1, 在红圈, 和 id 2 在黄色圆圈), 遵循类似的轨迹。这部电影在寄生虫注射后记录了大约10-15 分钟。<a href="//ecsource.jove.com/files/ftp_upload/56238/Movie_4.mp4" target="_blank">请单击此处下载视频.</a>
补充图 1: 蛋黄中 CFSE 荧光信号的积累.立体 48 hpf 的野生幼虫在维的导管上注射的图像。CFSE 荧光信号逐渐积累在蛋黄, 经过两天后注射 (48 hpi)。缩放栏 = 500 µm.<a href="//ecsource.jove.com/files/ftp_upload/56238/Supplemental_figure_1.tif" target="_blank">请单击此处下载该图.</a>
补充电影 1: 附着在循环系统的墙壁和阀门上的寄生虫.立体时间序列图像的野生型幼虫注入 48 hpf。图像以0.2 秒的间隔拍摄, 捕获T. 虫寄生生物在房室瓣膜中与心肌收缩同步运动。影片被记录了大约30分钟在寄生虫注射以后。<a href="//ecsource.jove.com/files/ftp_upload/56238/Supplemental_movie_1.mp4" target="_blank">请单击此处下载视频.</a>
补充影片 2: 移动和黏附的寄生虫在脑室空间和蛋黄.LSFM 电影的幼虫 48 hpf 显示T. 虫寄生虫漂流或附着在心包间隙或蛋黄。在前5秒观察到透射光的观点。荧光观察从 5.2-25.8 s。这部电影在寄生虫注射后记录了大约10-15 分钟。<a href="//ecsource.jove.com/files/ftp_upload/56238/Supplemental_movie_2.mp4" target="_blank">请单击此处下载视频.</a>

Watch this Scientific Journal Video about Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo at JoVE.com

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

在本协议中, 荧光标记为T. 虫被注射到透明的斑马鱼幼虫中, 并使用光片荧光显微镜观察到体内的寄生虫运动。

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

establishment-larval-zebrafish-as-an-animal-model-to-investigate

Research

JoVE Journal

Developmental Biology

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

10.6K 次观看.  Universidad de los Andes. 在本协议中, 荧光标记为T. 虫被注射到透明的斑马鱼幼虫中, 并使用光片荧光显微镜观察到体内的寄生虫运动。

视频: Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

科研

发育生物学

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

建立有限元模型，研究斑马鱼下颌生物力学

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

原位卵巢移植术对雌性小鼠卵巢衰老的影响及寿命及健康范围研究

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For functional measurements of visual performance, electroretinography (ERG) is an ideal non-invasive method, which has been well established in higher vertebrate species. This approach is increasingly being used for examining the visual function in zebrafish, including during the early developmental larval stages. However, the most commonly used recording electrode for larval zebrafish ERG to date is the glass micropipette electrode, which requires specialized equipment for its manufacture, presenting a challenge for laboratories with limited resources. Here, we present a larval zebrafish ERG protocol using a cone-shaped sponge-tip electrode. The novel electrode is easier to manufacture and handle, more economical, and less likely to damage the larval eye than the glass micropipette. Like previously published ERG methods, the current protocol can assess outer retinal function through photoreceptor and bipolar cell responses, the a- and b-wave, respectively. The protocol can clearly illustrate the refinement of visual function throughout the early development of zebrafish larvae, supporting the utility, sensitivity, and reliability of the novel electrode. The simplified electrode is particularly useful when establishing a new ERG system or modifying existing small-animal ERG apparatus for zebrafish measurement, aiding researchers in the visual neurosciences to use the zebrafish model organism.

Here, we present a protocol that simplifies the measurement of light evoked electroretinogram responses from larval zebrafish. A novel cone-shaped sponge-tip electrode can help to make the study of visual development in larval zebrafish using the electroretinogram ERG easier to achieve with reliable outcomes and lower cost.

一种新型锥形海绵尖电极在幼虫斑马鱼中的电录记录

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

用于测试 Vivo 中巨噬菌体招募的异位化学表达模型

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

为梭状体开发拉瓦尔斑马鱼感染模型

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...