用于研究小鼠视网膜循环中流动动力学的红细胞和白细胞的荧光染料标记
1National Healthcare Group Eye Institute,Tan Tock Seng Hospital, 2Singapore Eye Research Institute (SERI),Singapore National Eye Center, 3School of Material Science and Engineering,Nanyang Technological University, 4Department of Ophthalmology, Yong Loo Lin School of Medicine, National University Health Systems,National University of Singapore, 5Ophthalmology Academic Clinical Research Program,DUKE-NUS Graduate Medical School
Summary
眼睛循环中标记血细胞的活细胞成像可以提供关于糖尿病视网膜病变和年龄相关性黄斑变性的炎症和缺血的信息。描述了标记血细胞并在视网膜循环中成像标记细胞的方案。
Abstract
视网膜和脉络膜血流动力学可以提供各种眼部疾病如青光眼，糖尿病性视网膜病变，年龄相关性黄斑变性（AMD）和其他眼部炎症病症的病理生理学和后遗症的见解。它也可能有助于监测眼睛的治疗反应。血细胞的适当标记加上标记细胞的活细胞成像，允许调查视网膜和脉络膜循环中的流动动力学。在这里，我们分别描述1.5％靛青绿（ICG）和1％荧光素钠标记小鼠红细胞和白细胞的标准化方案。应用扫描激光检眼镜（SLO）可视化C57BL / 6J小鼠（野生型）视网膜循环中的标记细胞。两种方法都在小鼠视网膜循环中显示了不同的荧光标记细胞。这些标记方法可以在各种眼部疾病中具有更广泛的应用楷模。
Introduction
了解视网膜和脉络膜循环中血液细胞的流动动力学对于了解潜在的威胁眼睛的眼部疾病和其他眼部炎症状况的发病机制至关重要。然而，涉及荧光染料与血浆蛋白结合的常规血管造影技术不提供关于红细胞或白细胞1的动力学的任何信息。红细胞视网膜流动力学对于研究视网膜中的代谢有效循环和白细胞流动动力学是重要的，用于了解各种炎性疾病中的细胞迁移，识别，粘附和破坏2 。用于鉴定和表征各种细胞类型的几种荧光分子3 。血细胞的血液动力学可以用适当的荧光染色来测量适用的成像技术4 。
眼内疾病如年龄相关性黄斑变性（AMD）和糖尿病性视网膜病变（DR）中炎症反应的存在涉及淋巴细胞在患病区域5,6的积累。跟踪组织中的免疫细胞可以帮助了解涉及疾病发病机制的复杂事件。放射性同位素如51 Cr和125 I在早期研究中被用作细胞示踪剂。这些染料是有毒的并影响细胞活力。虽然放射性标记物3 H和14 C对细胞的毒性较小，但由于其发射能量较低，因此难以在系统7,8中检测到信号。引入了许多荧光染料以克服与w相关的潜在问题使用荧光显微镜和流式细胞术，体外放射性标记和轨道淋巴细胞迁移9,10 。 Hoechst 33342和噻唑橙是DNA结合荧光染料，用于在体内追踪淋巴细胞。 Hoechst 33342结合DNA中富含AT的区域，是膜可渗透的，保留荧光信号2 – 4天，耐猝灭9,10 。 Hoechst 33342和噻唑橙的缺点分别是抑制淋巴细胞增殖11和短半衰期9 。
钙黄绿素AM，荧光素二乙酸酯（FDA），2'，7'-双 – （2-羧乙基）-5-（和-6） – 羧基荧光素，乙酰氧甲基酯（BCECF-AM），5-（和-6）羧基荧光素二乙酸酯（CFDA）和5-（和-6） – 羧基荧光素二乙酸酯乙酰氧甲酯（CFDA-AM）是用于淋巴细胞迁移研究的细胞质荧光染料。然而，FDA，CFDA和CFDA-AM在细胞中的保留率较低9 。 BCECF-AM降低增殖反应并影响趋化性和超氧化物的生成9,12 。钙黄绿素AM是一种荧光染料，可用于短期体内淋巴细胞迁移研究。它发出强烈的荧光信号，不干扰大多数细胞功能，并保留荧光信号长达3天12,13 。荧光素异硫氰酸酯（FITC）和羧基荧光素二乙酸酯琥珀酰亚胺酯（CFDA-SE）是共价偶联荧光染料，用于淋巴细胞迁移研究。 FITC对细胞活力没有影响，与B淋巴细胞的亲和力比T淋巴细胞14,15更强</suP>。 CFDA-SE标记的淋巴细胞可以在体内追踪超过8周和最多8个细胞分裂9,16 。 C18 DiI（1,1'-二十八烷基-3,3,3'，3'-四甲基吲哚羰花青高氯酸盐），DiO（3,3'-二十八烷基羰基花青高氯酸盐），Paul Karl Horan（PKH）2，PKH3和PKH26是膜 – 插入用于标记白细胞和红细胞的荧光亲脂性碳菁染料。 C18 Dil和DiO在掺入细胞膜时表现出较高的信号，并且相对无毒12,17 。 PKH2，PKH3和PKH26标记的细胞表现出良好的荧光信号保留，毒性较小18,19,20,21,22。然而，PKH2下调CD62L表达并减少细胞活力23 。
大多数上述研究已经被执行用于跟踪淋巴细胞迁移和淋巴管中的增殖并研究非眼部循环中标记的红细胞。使用标记技术研究眼部循环中的血细胞的研究很少。扫描激光检眼镜（SLO）的应用在通过眼底血管造影技术研究体内视网膜和脉络膜循环中的标记细胞方面具有很大的优势。有几种荧光染料，例如ICG，吖啶橙，FITC，荧光素钠和CFDA，用于研究SLO 25,26,27,28,29,30,30在视网膜循环中的白细胞。class =“xref”> 31，32，33，34。吖啶橙26,27的光毒性和致癌性，FITC与细胞活性的干扰以及血管内造影剂对视网膜和脉络膜血管分辨率的要求限制了其在体内动物实验中的应用29 。荧光素钠和ICG无毒，经食品和药物管理局批准，可安全用于人类测试32,35 。大多数流动动力学研究与白细胞或红细胞的标记及其在视网膜和脉络膜血管中的可视化有关36,37,38,39 </s了>。在这里，我们描述了红细胞ICG标记的标准化方案，白细胞的荧光素钠标记，以及使用SLO跟踪小鼠视网膜循环中的可视化标记细胞。
Protocol
本研究中使用的动物方案由新加坡SingHealth的机构动物护理和使用委员会批准，并符合视力和眼科研究协会（ARVO）关于在眼睛和视力中使用动物的声明的指导原则研究。 荧光染料对红细胞和白细胞的标记 试剂的制备 通过将3mg的ICG溶解在1800μL无菌蒸馏水中来制备ICG（1.5mg / mL）。加入200μL10倍磷酸缓冲盐水（PBS）。 通过向6 mL无菌蒸馏水中加入4 mL 1x PBS制备40％1…
Representative Results
用1.5％ICG标记的红细胞在C57BL / 6J小鼠（野生型）的视网膜循环中显现。 1.5％ICG标记的红细胞的1％和5％血细胞比容在视网膜循环中是可区分的。然而，用1％红细胞比容为1.5％的ICG标记的红细胞，可以更清楚地观察到各个标记细胞的可视化（ 图1 ）。在5％的血细胞比容中，由于视网膜血管中的标记细胞数量很多，不可能标记个体细胞。在两个条件下?…
Discussion
研究视网膜和脉络膜循环中的血液动力学对于了解许多眼部疾病的病理生理学至关重要。视网膜循环中的血流动力学可以通过傅立叶域光学相干断层扫描（FD-OCT），激光散斑图（LSFG）和视网膜血氧饱和度检测来研究。尽管这些方法使用不同的方法来研究视网膜循环40,41,42,43,44,45中的总血流量，但是它们具有不能研究各种细胞类型的流动动力学的限制。视网膜和脉络膜组织的营养物质和氧气供应以?…
Declarações
The authors have nothing to disclose.
Acknowledgements
该研究项目由新加坡国立医学研究理事会（NMRC）提供的新研究者资助。该小组希望承认2012年11月至2014年10月在德国大学学院眼科研究所（IoO）向国立医学研究委员会（NMRC）海外研究培训奖学金提供的研究培训。大志志Agrawal博士获得了Shima博士实验室的细胞标记和活体成像的概念和技能。因此，团队希望在David Shima教授，Kenith Meissner教授，Peter Lundh博士和Daiju Iwata博士的培训研究中感谢监督和指导。
Materials
| Cardiogreen polymethine dye (Indocyanine green) | Sigma Aldrich | 12633-50MG | |
| Fluorescein 100 mg/mL | Novartis | U1705A/H-1330292 | |
| 10X Phosphate-buffered saline (PBS) Ultra Pure Grade | 1st BASE | BUF-2040-10X1L | |
| Bovine serum albumin | Sigma Aldrich | A7906-100G | |
| Microtainer tubes with K2E (K2EDTA) – EDTA concentration – 1.8 mg/mL of blood | BD, USA | REF 365974 | |
| Histopaque 1077 solution | Sigma Aldrich | 10771 | |
| Centrifuge 5810 R | Eppendorf | 05-413-401 | |
| Microcentrifuge tubes 2mL | Axygen | MCT-200-C-S | |
| Vortex mixer | Insta BioAnalytik pte. ltd | FINE VORTEX | |
| Shaker incubator | Lab Tech | ||
| Ceva Ketamine injection (Ketamine hydrochloride 100mg/mL) | Ceva | KETALAB03 | |
| ILIUM XYLAZIL-20 (Xylazine hydrochloride 20mg/Ml) | Troy Laboratories PTY. Limited | LI0605 | |
| 1% Mydriacyl 15 mL (Tropicamide 1%) | Alcon Laboratories, Inc. USA | NDC 0998-0355-15 | |
| 2.5% Mydfrin 5 mL (Phenylephrine hydrochloride 2.5%) | Alcon Laboratories, Inc. USA | NDC 0998-0342-05 | |
| Terumo syringe with needle 1cc/mL Tuberculin | Terumo (Philippenes) Corporation, Philippines | SS-01T2613 | |
| Vidisic Gel 10G | Dr. Gerhard Mann, Chem.-Pharm, Fabrik Gmbh, Berlin, Germany | ||
| Alcohol swabs | Assure medical disposables | 7M-004-L-01 | |
| Confocal laser scanning angiography system (Heidelberg Retina Angiograph 2) | Heidelberg Engineering, GmbH, Heidelberg, Germany | ||
| Hiedelberg Spectralis Viewing Module software, v4.0 | Heidelberg Engineering, GmbH, Heidelberg, Germany | ||
| Fluorescent microscope | ZEISS | Model: axio imager z1 |
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Agrawal, R., Balne, P. K., Tun, S. B. B., Sia Wey, Y., Khandelwal, N., Barathi, V. A. Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation. J. Vis. Exp. (125), e55495, doi:10.3791/55495 (2017).