免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像
Summary
Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.
Abstract
肝脏炎症作为对损伤的响应是，涉及白细胞的不同的亚型，包括单核细胞，嗜中性粒细胞，T细胞亚群，B细胞，自然杀伤（NK）和NKT细胞的浸润高度动态的过程。肝脏用于监测免疫细胞迁移的活体显微镜是特别具有挑战性的，由于有关样品制备和固定，光学分辨率和动物长期存活的高要求。然而，炎症过程以及细胞相互作用研究的动力学能提供更好的理解炎性肝病的发生，发展和回归的关键信息。因此，建立了一个高度敏感的和可靠的方法来研究的迁移和不同的免疫细胞在小鼠肝脏在长时间内（约6小时），通过活体双光子激光扫描显微镜（TPLSM）细胞 – 细胞相互作用中有重症监护组合监控。
ENT“>所提供的方法包括：一个轻柔的制备和与器官的极小扰动肝脏的稳定固定;使用多色多光子显微镜，几乎没有光漂白或光毒性效果在长达6小时的时间段长期活体成像，允许跟踪具体的白细胞子集;而由于大量监测小鼠重要参数和流通，温度和气体交换的稳定稳定的成像条件。调查在肝脏炎症淋巴细胞迁移CXCR6.gfp敲入小鼠下基线条件和后诱导四氯化碳腹腔注射（多个）（CCL 4）的急性和慢性肝损伤进行活体肝成像。
CXCR6是表达在淋巴细胞趋化因子受体，主要对天然杀伤T（NKT） – ，天然杀伤（NK） – 和T淋巴细胞，如CD4 + T细胞亚群，而且粘膜associated不变（MAIT）T细胞1。继允许在肝损伤的详细洞察他们的行为改变，因此他们的病情恶化可能参与的迁徙模式CXCR6.gfp +免疫细胞和定位。
Introduction
细胞和在整个器官的细胞功能或甚至整个生物体的可视化受到了极大的兴趣超过50年，包括本体2的几乎所有的部分。因此，一些早期的研究已经采用的肝3,4的活体成像。但是，有几个局限存在最新关于长期稳定的高分辨率的肝组织的成像。
由于在与膜片和胃肠道5紧密接触肝脏的解剖位置，因为微观活体成像的最常见的问题是移动由于呼吸和，在较小程度上，蠕动肠道6。相较于其他实体器官，肝脏手术是特别具有挑战性。由于密集的微血管结构，手术操作可导致大量出血病灶，居民受损的微循环7，也激活我mmune细胞如Kupffer细胞8。因此，该组织的机械固定如别处公布6,9-可能妨碍与活体显微镜成像。
在一个健康的肝脏，总血量的10-15％驻留在肝脉管系统中，并且该机关收到整体心输出10的25％左右，使该器官极易受到在循环变化（ 例如 ，血压波动）。因此，由于例如 ，剪切应力，位移，伤害由过度的组织处理或集中式循环中断的肝血流量将导致人工改变在白细胞迁移行为，受损的肝的氧合，因此进一步的肝损伤，影响肝的免疫反应，以及作为器官保存和动物的整体使用寿命。
早期的微观研究是基于活体萤光英里croscopy，但一些技术限制，如光漂白和低渗透深度限制使用这种技术的长期的肝脏成像4,11,12。在20世纪90年代多光子显微术的发展，光漂白或穿透深度的限制，主要是解决了，因为这种新的方法，在技术上是能够在根据实际生活情况13-15几乎所有的器官进行成像研究。然而，对于肝脏成像主剩余的挑战是：呼吸运动，肝组织的自体荧光，确保为几个小时，16长时间不变的血流量在肝窦，特别是稳定的成像。
尽管一些研究涉及的功能和各种白细胞的迁移在肝脏17， 如 NKT细胞18-20，T细胞21,22，肝巨噬细胞23,24或25的中性粒细胞，长期的多光子米icroscopy成像尚未成功建立，任务更有挑战性的动物的急性或慢性肝病，由于现有的损害，因此，较高的敏感性，以进一步的损害26。然而，监测在实时肝白细胞的迁移行为和细胞功能允许在其特定的作用，在肝脏内稳态和疾病27新的见解。
趋化因子受体CXCR6上表达几种淋巴细胞亚群，包括自然杀伤（NK）细胞，NKT细胞和一些T细胞群18,28。在小鼠中之前的研究已经表明，CXCR6和其同源配体CXCL16可能动态平衡期间控制NKT细胞的巡逻肝血窦。因此，使用CXCR6.gfp小鼠（携带一敲，在绿色荧光蛋白[GFP]在CXCR6基因）被描述为调查淋巴细胞迁移的各种器官如脑29并且还肝脏18,20，示出了在炎症增加CXCR6.gfp细胞浸润。
在这项研究中所提供的方法有可能在一段时间稳定的条件下长时间遵循这些进程。活体多光子基于程序允许成像，这是与动物的器官极小扰动高度重复性;长期生存的动物被大量监控后严密控制呼吸和循环的优化;高度灵活，也容易采取其他实质器官如肾脏或脾脏。
Protocol
Representative Results
Discussion
我们研究的目的是开发一个高度标准化的，稳定的和可重复的方法肝脏活体成像TPLSM。一般活体成像给以下归巢和发展，动态平衡和疾病的不同白细胞群体的互动有价值的见解在现实生活条件的细胞行为。但是，肝脏的几分有挑战性解剖位置，由于其呼吸道和肠道蠕动运动直接被传递到肝以及它们的高脆性相对于其他固体器官施加用于稳定长期活体成像几个挑战。近年来，许多实验研究小鼠揭示?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors thank the Central Animal facility of the University Hospital Aachen for technical support. This work was supported by the German Research Foundation (DFG Ta434/2-1, DFG SFB/TRR 57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. This work was further supported by the Core Facility ”Two-Photon Imaging”, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.
Materials
| Anesthetics | |||
| Buprenorphine | Essex Pharma | 997.00.00 | Analgeticum, 0.1 mg/kg |
| Fentanyl | Rotex Medica | charge: 30819 | |
| Fluovac anesthesia system | Harvard Apparatus | 34-1030 | |
| Glucose 5% | Braun | ||
| ISOFLO (Isoflurane Vapor) vaporiser | Eickemeyer | 4802885 | |
| Isoflurane | Forene Abbott | B 506 | |
| Isotonic (0.9%) NaCl solution | DeltaSelect GmbH | PZN 00765145 | |
| Ketamin 10% | ceva | Charge: 36217/09 | |
| Xylazin 2% | medistar | Charge: 04-03-9338/23 | |
| Consumable supplies | |||
| 20ml Syringe | BD Plastipak | ||
| 250ml Erlenmeyer flask | Schott Duran | 21 226 36 | |
| 25mL Beaker 2x | Schott Duran | 50-1150 | |
| 2ml syringe | BD Plastipak | ||
| 4-0 Vicryl suture | Ethicon | V7980 | |
| Agarose | commercially available | ||
| Bepanthen Eye and Nose ointment | Bayer Vital GmbH | 6029009.00.00 | |
| Change-A-Tip Deluxe High-Temp Cautery Kit | Fine Science Tools Inc. | 18010-00 | |
| Cotton Gauze swabs | Fuhrmann GmbH | 32014 | |
| Cover Slip 24x50mm | ROTH | 1871 | |
| Durapore silk tape | 3M | 1538-1 | |
| Feather disposable scalpel | Feather | 02.001.30.011 | |
| Fine Bore Polythene Tubing 0,58mm ID | Smiths medical | 800/100/200 | |
| Histoacryl | Braun | 1050052 | 5x 0,5ml |
| Leukoplast | BSN Medical Inc. | ||
| Microscope Slides | ROTH | 1879 | |
| Poly-Alcohol Haut…farblos Antisepticum | Antiseptica GmbH | 72PAH200 | |
| Sterican needle 18 G x 1 | B. Braun | 304622 | |
| Sterican needle 27 3/4 G x 1 | B. Braun | 4657705 | |
| Tissue paper | commercially available | ||
| Surgical Instruments | |||
| Amalgam burnisher 3PL | Gatz | 0110? | |
| Blair retractors (4 pronged (blunt)) x2 | Storz&Klein | S-01134 | |
| Dumont No.7 forceps | Fine Science Tools Inc. | 91197-00 | |
| Graefe forceps curved x1 | Fine Science Tools Inc. | 11151-10 | |
| Graefe forceps straight x2 | Fine Science Tools Inc. | 11050-10 | |
| Heidemann spatula HD2 | Stoma | 2030.00 | |
| Needle holder Mathieu | Fine Science Tools Inc. | 12010-14 | |
| Scissor | Fine Science Tools Inc. | 14074-11 | |
| Semken forceps | Fine Science Tools Inc. | 11008-13 | |
| Small surgical scissors curved | Fine Science Tools Inc. | 14029-10 | |
| Small surgical scissors straight | Fine Science Tools Inc. | 14028-10 | |
| Standard pattern forceps | Fine Science Tools Inc. | 11000-12 | |
| Vannas spring scissors | Fine Science Tools Inc. | 15000-08 | |
| Equipment | |||
| ECG Trigger Unit | Rapid Biomedical | 3000003686 | |
| MICROCAPSTAR End-Tidal Carbon Dioxide Analyzer | AD Instruments | ||
| Minivent Typ 845 | Harvard Apparatus | 73-0043 | |
| Multiphoton microscope Trimscope I | LaVision | ||
| Perfusor Compact | B. Braun | ||
| PowerLab 8/30 8 channel recorder | AD Instruments | PL3508 | |
| Temperature controlled heating pad | Sygonix | 26857617 | |
| Temperature sensor | comercially available | ||
| Temperature controlled System for Microscopes -Cube&Box | Life Imaging Services |
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