重复采血车在成年斑马鱼验血
Summary
Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.
Abstract
重复采血是实验室动物进行的最普遍的技术之一。但是，还没有建立一个非致命性协议的采血从斑马鱼。以前的方法采血从斑马鱼是致命的，如外侧切口，斩首和尾巴切除。因此，我们已经开发了一种新的“重复”的血液采集方法，以及在座的详细协议，概述了此过程。这种方法是微创并导致非常低死亡率（2.3％），用于斑马鱼，从而使来自同一个体的重复采血。血液取样的最大体积是依赖于体重的鱼。间隔重复采集血样的体积应每周体重或≤1％，每2周，这是由血液中的血红蛋白的测定评价≤0.4％。此外，血红蛋白，空腹血糖，血浆甘油三酯（TG）和总碳在男性和女性的成年斑马鱼holesterol水平进行了测量。我们也应用这个方法来研究糖代谢饮食诱导的肥胖的失调。这种血液采集方法将允许许多应用，包括葡萄糖和脂质代谢和血液学研究，这将增加使用斑马鱼作为人类疾病模型有机体。
Introduction
斑马鱼正日益普及人类疾病的一种有价值的模式，因为他们的器官和遗传学类似于人类1,2。在发育生物学的领域中，许多研究已经证明，斑马鱼和人显示标记相似造血3，止血4,5，和髓6。成年斑马鱼也用于研究免疫学7，神经变性8和肥胖相关疾病9，因为此模式生物股与破坏在人类疾病共同通路。肥胖和肥胖相关疾病（糖尿病，脂肪肝和酒精性脂肪性肝炎和动脉粥样硬化），斑马鱼血糖和血脂水平已经在几个转基因和饮食诱导的肥胖模型10月13日被彻底调查。
从单个动物反复采血会减少动物的使用和DECrease个体差异。然而，重复的样品采集是因为它们具有相对小的血容量和缺乏方便船舶在小动物，如斑马鱼在技术上困难。已经开发了几种方法用于从斑马鱼一次性采血，虽然这些方法有其自身的缺陷，包括致命性，相关的组织损伤和有限的血容量。例如，1-5微升血液可以从约0.3厘米的横向切口，在背主动脉5的区域收获长度。斩首用剪刀通过肩带切割可收集5-10微升血液10。另一种方便的采血方法是尾切除14。心脏穿刺是用于从相同的鱼重复采血一个潜在的替代方法，但非常小的量，得到（约50 NL）与此过程限制的分析，可以是perfor的数量配有11。因此，新的协议需要启用反复的非致命的血液采样，这将是一个重要的进步所必需的这种微生物是一个标准的模式生物的人类疾病。这种技术将允许用于测试药理反应，发现分子生物标记物的诊断，确定预后，以及各种疾病，如代谢疾病，退行性疾病和多种类型的恶性肿瘤的监测。
因此，我们开发了获得血液从斑马鱼连续15微创方法。在这里，我们演示程序可视化，并提供该技术的详细协议。使用这种方法，基于各种参数，包括血红蛋白，空腹血糖，并在健康成年斑马鱼的血脂正常值进行了评价。此外，我们还评估了此方法是否适用于需要通过米系列样本的研究期间过度喂食实验onitoring中血糖水平的瞬时变化。
Protocol
Representative Results
Discussion
我们在座的详细协议，用于连续获得血液从成年斑马鱼。这种方法是简单的进行，我们使用它在实验室，每天。此采血方法是基于插入玻璃毛细管针进入斑马鱼的背主动脉。在此过程中，关键是要小心不烧蚀脊柱，因为它是标准用于搜索背主动脉。降低颈椎损伤会提高患者的生存率。虽然这种技术是简单，易于掌握，有可以保证高的成功率和存活率的最佳做法。一个熟练的研究员将需要1-2分钟进?…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.
Materials
| Glass capillaries with filament | Narishige | GD-1 | 1.0-mm-outer-diameter. |
| Needle puller | Narishige | PC-10 | To produce the needls |
| Heparin | Wako Pure Chemical Industries | 081-00136 | For heparinization |
| Aspirator tube assembly | Drummond | 2-040-000 | For blood collection |
| Bulb dispenser | Drummond | 1-000-9000 | For blood collection |
| 2-phenoxyethanol | Wako Pure Chemical Industries | 163-12075 | For anesthetizing the fish |
| DRI-CHEM3500V | Fujifilm | – | For hemoglobin measurement |
| DRI-CHEM Slides | Fujifilm | Hb-WII | For hemoglobin measurement |
| Glutest Neo Super | Sanwa Kagaku Kenkyusho | – | For bood glucose measurement |
| Wako L-type TG kit | Wako Pure Chemical Industries | 464-44201 | For TG measurement |
| Wako L-type CHO kit | Wako Pure Chemical Industries | 460-44301 | For total cholesterol measurement |
| Parafilm M | Alcan Packaging | PM996 | To expel the blood on |
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