Name: real time in vivo tracking of thymocytes in the anterior chamber of the eye by laser scanning microscopy
Uploaded: 2018-10-02T13:30:00
Description: Watch this Scientific Journal Video about Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy at JoVE.com

这项工作得到了 NIH 赠款 R56DK084321 (ac)、R01DK084321 (ac)、R01DK111538 (ac)、R01DK113093 (ac) 和 R21ES025673 (ac) 以及 BEST/2015/043 赠款 (Consellería Educació、文化宫 i 电子竞技、加泰罗尼亚自治区、巴伦西亚、西班牙) (《雇佣条例》) 的支持。作者感谢派出的团队在瓦伦西亚天主教 Mártir, 瓦伦西亚, 西班牙和中心 de Investigación 圣多美和普林西比马萨, 瓦伦西亚, 西班牙的帮助下, 视频拍摄和编辑。

迈阿密大学的机构动物保育和使用委员会 (IACUC) 根据 IACUC 指南批准了所有的实验。
1. 新生儿 Thymi 的分离和修整
<ol><li>用热处理或其他方法制备所有试剂和仪器, 确保无菌条件。</li>
	<li>为了减少污染, 执行所有的手术程序在层流罩下。</li>
	<li>在 euthanizing 捐赠小鼠之前, 用无菌 prechilled 1x 磷酸盐缓冲盐水 (PBS, pH 值 7.4) 填充60毫米无菌皿, 并将其放在冰上。它将用于冲洗和储存...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+studies+in+living+animals+using+multiphoton+microscopy.">Functional studies in living animals using multiphoton microscopy.</a> ILAR Journal. 49, 66-77 (2008).</li><li>Caetano, S. S., Teixeira, T., Tadokoro, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+the+mouse+thymus+using+2-photon+Microscopy.">Intravital imaging of the mouse thymus using 2-photon Microscopy.</a> Journal of Visualized Experiments. (59), e3504 (2012).</li><li>Li, J., Iwanami, N., Hoa, V. Q., Furutani-Seiki, M., Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+intravital+imaging+of+thymocyte+dynamics+in+medaka.">Noninvasive intravital imaging of thymocyte dynamics in medaka.</a> Journal of Immunology. 179 (3), 1605-1615 (2007).</li><li>Adeghate, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-graft+circulation+and+vascular+morphology+in+pancreatic+tissue+transplants+in+rats.">Host-graft circulation and vascular morphology in pancreatic tissue transplants in rats.</a> Anatomical Record. 251, 448-459 (1998).</li><li>Adeghate, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+tissue+grafts+are+reinnervated+by+neuro-peptidergic+and+cholinergic+nerves+within+five+days+of+transplantation.">Pancreatic tissue grafts are reinnervated by neuro-peptidergic and cholinergic nerves within five days of transplantation.</a> Transplant Immunology. 10 (1), 73-80 (2002).</li><li>Speier, S., Nyqvist, D., K&#246;hler, M., Caicedo, A., Leibiger, I. 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由于 T 细胞成熟过程对个体免疫能力4的重要性以及前体细胞动力学对胸腺2、3所产生的成熟 T 细胞的推定影响, 已经投入了大量的努力。开发替代传统的固定组织快照方法。
虽然组织切片和其他外植体在再生组织结构上明显优于单分子膜或胸腺14、15的总器官培养, 但由?...

胸腺 t 细胞分化和 t 细胞亚群选择是发展和维持细胞介导免疫在脊椎动物1的关键过程。这一过程涉及复杂有序的活动序列, 包括从血液中招募祖细胞, 增殖和迁移, 膜蛋白的差异表达, 以及大规模程序性细胞死亡的子集选择。其结果是, 成熟的 T 细胞的释放反应到丰富的外国抗原, 同时显示最小的反应, 自肽, 最终殖民的外围淋巴器官的个体2,3。αβTCR 的异常胸腺细胞选择导致自身免疫性疾病或免疫失衡4 , 主要来源于在阴性或阳性前体选择过程中的缺陷。
胸腺胸腺的定向迁移是 T 细胞成熟的所有阶段的内在特性, 它被设想为一系列同时或连续的多重刺激, 包括趋化因子、粘合剂和脱粘细胞外基质 (ECM)。蛋白质相互作用3,5。对固定组织的研究为胸腺微环境5、6中胸腺细胞迁移线索的表达模式提供了关键信息, 而活体研究揭示了两种常见的胸腺细胞在两个组织学不同区域的迁移行为: 皮质缓慢随机运动, 髓质7,8,9,10,11,12,13. 移徙率增加与胸腺阳性选择13相关, 阴性选择与爬行行为有关, 这一假说表明, 通过胸腺进行的旅程的动力学决定适当的胸腺细胞的成熟。尽管它们的相关性, 胸腺细胞间质细胞相互作用的拓扑结构和胸腺组织运动的动力学在器官微环境期间在 T 体细胞成熟仍然是未定义的。
迄今进行的大多数活体研究包括胎儿或 reaggregate 胸腺器官培养14、15、组织切片或完整的胸腺叶外植体, 在那里胸腺细胞运动通过双光子激光扫描可视化。显微术 (TPLSM)8, 活体成像技术, 限制最大工作距离和成像深度1毫米根据组织检查16。与依赖于延长潜伏期形成3维结构的艰苦的胸腺器官培养相比, 胸腺切片技术和完整的胸腺叶方法允许控制引入预先标记的特定子集胸腺细胞进入一个本土的组织建筑环境。然而, 由于这些模型中没有血流, 它们显然仅限于研究胸腺沉降祖 (TSPs) 对胸腺实质的招募过程或成熟 T 细胞胸腺 egression 的动力学。
在活体模型中研究胸腺 T 细胞成熟生理学的小鼠包括移植的片段或整个器官裂片放置在肾囊17或 intradermally18。虽然这些选择显示了他们的效用, 以询问组织的系统性功能植入, 胸腺移植的位置在动物深处或覆盖层的不透明组织限制其使用在体内检查植入物TPLSM。
由于角膜的透明度, 眼睛的前腔提供了一个容易接近的空间, 直接监测任何接枝组织。的优势, 虹膜形成的室的基础是丰富的血管和自主神经的结局, 使移植的快速重建和神经19,20。凯塞多博士在过去21成功地利用这个解剖空间进行胰岛的维持和纵向研究。在这里, 我们表明, 这一策略不仅是研究胸腺细胞在原生器官结构中的动力学的有效方法, 而且是唯一允许将体内纵向记录扩展到祖招募研究和成熟的 T 细胞 egression 的步骤在小鼠。

<table>
	<tbody>
		<tr>
			<td>Isofluorane vaporizer w/isofluorane</td>
			<td>Kent Scientific Corp</td>
			<td>VetFlo-1215</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scope w/light source</td>
			<td>Zeiss</td>
			<td>Stemi 305</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine dissection forceps</td>
			<td>WPI</td>
			<td>500455</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium dissection forceps</td>
			<td>WPI</td>
			<td>501252</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved tip fine dissection forceps</td>
			<td>WPI</td>
			<td>15917</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas scissors</td>
			<td>WPI</td>
			<td>503371</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>WPI</td>
			<td>503243</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>WPI</td>
			<td>500353</td>
			<td></td>
		</tr>
		<tr>
			<td>40 mm 18G needles</td>
			<td>BD</td>
			<td>304622</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable transfer pipette</td>
			<td>Thermofisher</td>
			<td>201C</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat pad and heat lamp</td>
			<td>Kent Scientific Corp</td>
			<td>Infrarred</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>VWR</td>
			<td>83,813,360</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm sterile dish</td>
			<td>SIGMA</td>
			<td>CLS430166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 1x PBS pH(7,4)</td>
			<td>Thermofisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile wipes</td>
			<td>Kimberly-Clark</td>
			<td>LD004</td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs for pain management</td>
			<td>Sigma-Aldrich</td>
			<td>A3035-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution or Viscotears</td>
			<td>Novartis</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ FLIII</td>
			<td></td>
		</tr>
		<tr>
			<td>Head-holding adapter</td>
			<td>Narishige</td>
			<td>SG-4N-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas mask</td>
			<td>Narishige</td>
			<td>GM-4_S</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SP5 II</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Telstar</td>
			<td>BIO IIA</td>
			<td></td>
		</tr>
	</tbody>
</table>

real time in vivo tracking of thymocytes in the anterior chamber of the eye by laser scanning microscopy

所提出的方法的目的是首次显示, 新生儿 thymi 移植到等基因系成年小鼠的前眼室,在体内纵向实时监测 thymocytes´动态在血管内胸腺段。在移植后, 激光扫描显微镜 (LSM) 通过角膜允许在体内无创重复成像的细胞分辨率水平。重要的是, 该方法增加了以前的活体 T 细胞成熟成像模型的可能性, 连续祖细胞招募和成熟的 T 细胞出口记录在同一动物。该系统的额外优点是移植区的透明度, 允许对植入组织进行宏观的快速监测, 以及植入物的可移植性, 除了系统治疗外, 还可以进行局部化处理。主要的限制是组织的体积, 适合在减少的空间的眼睛室, 要求裂片修剪。通过解剖胸腺裂片, 在先前显示为成熟 T 细胞生产功能的模式中, 器官完整性最大化。该技术可能适合于询问与胸腺功能相关的医学相关问题的环境, 包括自身免疫、免疫机能丧失和中枢耐受;仍然机械化的过程。对诱导胸腺细胞迁移、分化和选择的机制进行精细的解剖, 将会导致新的治疗策略以发展 T 淋巴细胞为目标。

从 B6 中分离出新生小鼠胸腺。Cg-Tg (CAG-DsRed * MST) 1 纳吉/雅小鼠如本议定书所述 (步骤 1.1-1.9)。在这些转基因小鼠中, 鸡β肌动蛋白启动子引导红荧光蛋白质变体 DsRed 的表达。MST 的影响下, 巨细胞病毒 (CMV) 立即早期增强促进跟踪植入物。
为防止组织排斥, 等基因系个体的基因型: C57BL/6-Tg (不列颠哥伦比亚荧光蛋白) 30 scha...

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Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

激光扫描显微术实时跟踪眼球前室胸腺细胞

该协议的目的是通过激光扫描显微镜在小鼠眼前腔的胸腺植入物中显示胸腺细胞的纵向活体实时跟踪。角膜的透明度和移植的血管化允许持续记录祖细胞的招募和成熟的 T 细胞出口。

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

该方法有助于回答免疫学领域有关自身免疫、免疫缺陷、中央耐受性和胸腺介入机制的关键问题。该技术的主要优点是，它允许在体内纵向记录祖主招募和成熟的T细胞出口血管化和移植小鼠胸腺段。虽然这种方法可以提供对胸腺发育和功能的洞察，也可以应用于其他组织，如胰岛或肾球蛋白。要分离胸腺，请将鼠标放在无菌吸水纸巾上，放在层流罩的后侧直立位置，然后用 70% 乙醇喷洒和擦拭小鼠腹部。在下腹部做一个表面的V形切口，以暴露胸腔，并使用一对直10厘米解剖剪刀，使一个0.5至1厘米的切口在胸部沿腹中线。折叠胸部两侧的皮肤，露出胸腔，并通过隔膜和肋骨笼再进行两个更深的 0.5 至 1 厘米的侧切口，以进入前胸腔的优越介质。胸腺应该出现在心脏的上方两个苍白的叶。将弯曲的钳子尖放在胸腺下，垂直拉取完整的器官，防止带一对直钳的肋骨的折叠背面。然后将分离的胸腺淹没在冰上含有冷无菌PBS的无菌60毫升盘中，并使用手术刀切割结缔地峡以分离胸腺叶。移植前，标记和称重每只受助小鼠，将第一只动物置于侧卧位置，一只眼睛直接暴露在解剖显微镜的透镜下。使用Vannas剪刀，切除一个孤立的胸腺叶宽达一毫米的曲折图案，以确保每个段包含一些胸腺皮层和梅杜拉组织。在植入之前修剪胸腺至关重要，因为大块可能不能正确植入。接下来，从手术区域角膜底部开始，使用40毫米18贝针尖将小切口切成外部角膜层，以便可以引入一双解剖剪刀的尖端。使用剪刀直接在角膜底部周围进行 5 到 10 毫米的侧翼切口，同时使用扁平钳子牢牢抓住角膜，以防止重新密封。用平端钳抓住切角膜上皮，保持角膜的开口，同时通过开口插入胸腺部分。重要的是快速插入胸腺片通过角膜开口，以防止自发重新密封之前，组织被引入。在此步骤中要小心保持组织完整性。轻轻地按压眼睛表面，将引入的组织段滑到与瞳孔的横向位置，以保持眼睛的功能。并使用平钳按角膜开口的两侧，相互牢固地对对方三到五秒钟，促进自封。如果要在两只眼睛上进行移植，请将鼠标转向相反的侧侧进行第二次植入，正如刚才所证明的。手术完成后，将鼠标返回到用热灯预热的空笼中，并监控直至完全保持。在适当的实验时间点，将麻醉接受小鼠放在固定阶段显微镜平台上，放在热垫上的侧侧卧位置，一只眼睛朝上。并将鼠标头插入立体税头支架中。调整旋钮以将鼠标头置于侧侧位置，以便将显微镜目标直接接触到持有胸腺移植物的眼睛，并拉回眼睑，同时将眼睛放在角膜边缘，用一对钳子尖覆盖在连接到 UST-2 固体万维关节上的聚乙烯管上。在将5X显微镜目标放在小鼠眼睛上并定位显微镜场中的胸腺之前，在角膜和镜片之间加入几滴无菌盐水或人工泪液。然后切换到具有长工作距离的更高分辨率水浸浸目标，并在显微镜软件中选择采集模式。启动谐振扫描仪模式并选择 XYZT 成像模式。打开气量激光器，将功率调整为 30%，以进行荧光激发。选择适当的激发激光线，并使用反射检测同时对适当的发射波长设置交流光束分射器控制，以检测反散射器并描绘组织结构。收集所选波长的发射，并设置 512 x 512 像素分辨率。然后单击"实时"开始成像，并相应地调整增益级别。专注于胸腺植入物的顶部，并选择开始定义Z堆栈的开始。然后专注于植入胸腺中的最后一个平面，该平面可以可视化并选择结束，使用五微米的 Z 步长定义 Z 堆栈的末尾。将每个 Z 堆栈的采集时间间隔设置为 1.5 到 2 秒，然后选择连续映像的采集至停止选项。然后单击"开始"初始化录制。在这里，显示胸腺植入物同一区域的亮场和荧光图像，可对组织生长和进化和延续进行双宏观随从。亮场图像还有助于植入组织的血管化可视化，从而能够对胸腺生理学对血液供应的依赖性进行独特的研究。该模型允许可视化细胞从血液洪流向植入胸腺的迁移，跟踪进入胸腺植入物的GFP阳性祖细胞之间的接触，在分化和选择过程中使用RFP标记的驻留胸腺上皮和频闪细胞，以及监测从胸腺进入外周血管的入口免疫细胞。在尝试此程序时，重要的是要记住尽量减少组织暴露，以获得最佳的移植，并限制眼睛的切口大小，允许通过角膜进行无伤人片段切除，同时减少愈合期间的纤维化。按照这个程序，其他方法，如局部控制治疗和其他组织切口到反边眼，以便回答有关与感染有关的免疫功能障碍，进化决定因素和移植耐受机制的其他问题。该技术开发后，为免疫学领域的研究人员探索T细胞发育铺平了道路，包括祖体招募、胸腺-眼睛动力学和体内成熟的T细胞出口步骤。

Research

JoVE Journal

Biology

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

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科研

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