斑马鱼高血糖的急性和慢性模型：评估高血糖对神经发生的影响和放射性标记的分子的生物分布的方法
Summary
这项工作描述了在斑马鱼中建立急性和慢性高血糖模型的方法。目的是调查高血糖对生理过程的影响，如组成型和损伤诱导的神经发生。这项工作还突出了使用斑马鱼跟踪放射性标记的分子（这里，[ 18 F] -FDG）使用PET / CT。
Abstract
高血糖是导致心血管和脑功能障碍的主要健康问题。例如，它与中风后增加的神经系统问题相关，并且显示损害神经源性过程。有趣的是，成年斑马鱼已经成为模拟高血糖/糖尿病并调查组成型和再生神经发生的相关和有用的模型。这项工作提供了开发高血糖斑马鱼模型的方法，以探讨在稳态和脑修复条件下高血糖对脑细胞增殖的影响。使用腹膜内注射D-葡萄糖（2.5g / kg体重）到成年斑马鱼中建立急性高血糖。通过将成人斑马鱼浸入含有水的D-葡萄糖（111mM）中14天来诱导慢性高血糖。对于这些不同的方法描述血糖水平测量。调查高血糖对组成型a的影响的方法通过描述端脑的机械损伤，解剖脑，石蜡包埋和切片切片，并进行免疫组织化学程序，进行再生神经发生。最后，还描述了使用斑马鱼作为研究放射性标记分子的生物分布的相关模型的方法（这里，[ 18 F] -FDG）使用PET / CT。
Introduction
高血糖定义为血糖过高。尽管可以反映急性压力的情况，但高血糖也是常常导致糖尿病诊断，糖尿病，胰岛素分泌和/或抵抗的慢性障碍的病症。 2016年全球糖尿病患者人数达到4.22亿，每年有150万人死于该病，成为重大的健康问题1 。事实上，不受控制的糖尿病导致影响心血管系统，肾脏和周围和中枢神经系统的几种生理学障碍。
有趣的是，急性和慢性高血糖症可能会改变认知功能，并有助于痴呆和抑郁症2,3,4,5,6。另外，病人入院高血糖与缺血性卒中7,8,9,10,11之间的功能，神经和生存结果较差有关。还显示高血糖/糖尿病影响成人神经发生，一种导致新神经元产生的过程，通过影响神经干细胞活性和神经元分化，迁移和存活2,12 。
与哺乳动物相比，硬骨鱼如斑马鱼在整个大脑中显示出强烈的神经源性活动，并且在成年期13,14,15,16期间表现出突出的脑修复能力。值得注意的是，由于neu的持续存在，这种能力是可能的ral干/祖细胞，包括放射状胶质细胞和神经母细胞17,18,19 。此外，斑马鱼近来已成为研究代谢紊乱的典范，包括肥胖和高血糖/糖尿病20,21,22 。
虽然斑马鱼是一个公认的高血糖和神经发生模型，但很少有研究调查了高血糖对脑内稳态和认知功能的影响12,23。为了确定高血糖对组成型和损伤诱导的脑细胞增殖的影响，通过腹膜内注射D-葡萄糖产生急性高血糖模型。此外，通过将鱼浸入补充水的水中再现慢性高血糖模型D-葡萄糖12 。斑马鱼在研究中表现出很多优势。它们在开发的第一阶段便宜，易于提高和透明，并且其基因组已被测序。在这项工作的背景下，他们还展示了几个额外的优点：（1）他们与人类共享类似的生理过程，使其成为生物医学研究的关键工具; （2）鉴于其广泛而强的神经源性活性，它们可以快速调查高血糖对脑内稳态和神经发生的影响;和（3）它们是替代模型，允许减少研究中使用的哺乳动物的数量。最后，斑马鱼可用作测试放射性标记分子和潜在治疗剂使用PET / CT的生物分布的模型。
以下程序的总体目标是视觉记录如何建立斑马鱼急性和慢性高血糖模型，使用zeb在高血糖条件下评估脑重塑，并使用PET / CT监测放射性标记的分子（这里为[ 18 F] -FDG）。
Protocol
将成年野生型斑马鱼（ Danio rerio ）保持在标准光周期（14/10h光/暗）和温度（28℃）条件下。所有实验均按照法国和欧洲共同体动物研究指南（86/609 / EEC和2010/63 / EU）进行，并经当地伦理委员会批准进行动物实验。 1.建立斑马鱼急性高血糖模型通过将400mg三卡因粉末溶解在97.9mL水和2.1mL 1M Tris / HCl缓冲液（pH9）中来制备三卡因（MS-222）的储备溶液。将pH调节至7，并…
Representative Results
使用本文所述的方法，在成年斑马鱼上进行腹膜内注射D-葡萄糖（2.5g / kg体重），并且在注射后1.5小时导致血糖水平的显着增加（ 图1A ）。注射后24小时，D-葡萄糖和PBS注射的鱼12之间的血糖水平相似。对于慢性治疗，将斑马鱼浸入D-葡萄糖水（111mM）中，并在其14天治疗结束时变成高血糖（ 图1B ），如前所述<su…
Discussion
这项工作描述了在斑马鱼中建立急性和慢性高血糖模型的各种方法。这些程序的主要优点是：（1）允许减少用于研究的哺乳动物数量，（2）易于建立和快速实施，（3）它们是经济的。因此，这些模型允许调查高血糖对大量动物的影响，以研究其对不同生理过程的影响，包括动脉粥样硬化血栓形成，心血管功能障碍，视网膜病变，血脑屏障渗漏以及组成型和再生性神经发生。这项工作描述了如何?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们非常感谢LaRéunion大学的杜蕾斯大学（DUN）编辑视频（特别是Jean-FrançoisFévrier，Eric Esnault和Sylvain Ducasse），Lynda-Rose Mottagan为配音，Mary Osborne-Pellegrin进行校对讲话和CYROI平台。这项工作得到了LaRéunion大学（BonusQualitéRecherche，Dispositifs incitatifs），ConseilRégionalde LaRéunion，欧盟（CPER / FEDER）和Philancia协会的资助。 ACD是LaRéunion大学（Contrat博士）德国国立教育学院获得奖学金的获得者。
Materials
| 1mL Luer-Lok Syringe | BD, USA | 309628 | |
| 4',6'-diamidino-2-phenylindole (DAPI) | Sigma-Aldrich, Germany | D8417 | |
| 7 mL bijou container plain lab | Dutscher, France | 080171 | |
| D-glucose | Sigma-Aldrich, Germany | 67021 | |
| Digital camera | Life Sciences, Japan | Hamamatsu ORCA-ER | |
| Disposable base molds | Simport, Canada | M475-2 | |
| Donkey anti-rabbit Alexa fluor 488 | Life Technologies, USA | A21206 | |
| Embedding center | Thermo Scientific, USA | Shandon Histocentre 3 | |
| Fluorescence microscope | Nikon, Japan | Eclipse 80i | |
| Fluorodeoxyglucose (18F-FDG) | Cyclotron, France | ||
| Glucometer test strip | LifeScan, France | One-Touch 143 Ultra | |
| Goat anti-mouse Alexa fluor 594 | Life Technologies, USA | A11005 | |
| In-Vivo Imaging System | TriFoil Imaging, Canada | Triumph Trimodality | |
| Microtome | Thermo Scientific, USA | Microm HM 355 S | |
| Monoclonal mouse anti-PCNA | DAKO, USA | clone PC10 | |
| Paraformaldehyde (PFA) | Sigma-Aldrich, Germany | P6148-500G | |
| Polyclonal rabbit anti-GFAP | DAKO, USA | Z033429 | |
| Slide drying bench | Electrothermal, USA | MH6616 | |
| Sodium chloride | Sigma-Aldrich, Germany | S9888 | |
| Sodium citrate trisodium salt dehydrate | Prolabo, France | 27833.294 | |
| Sterile needle | BD Microlance 3 | 30 G 1/2 ; 0.3 mm x 13 mm | |
| Student Dumont #5 Forceps | Fine Science Tools | 91150-20 | |
| Student surgical scissors | Fine Science Tools | 91400-14 | |
| Superfros Plus Gold Slides | Thermo Scientific, USA | FT4981GLPLUS | |
| Surgical microscope | Leica, France | M320-F12 | |
| Tissue embedding cassettes | Simport, Canada | M490-10 | |
| Tissue embedding medium | LeicaBiosystems, USA | 39602004 | |
| Toluene | Sigma-Aldrich, Germany | 244511 | |
| Tricaine MS-222 | Sigma-Aldrich, Germany | A5040 | |
| Triton X100 | Sigma-Aldrich, Germany | X100-500 mL | |
| Vectashield medium | Vector Laboratories, USA | H-1000 | |
| Xylene | Sigma-Aldrich, Germany | 534056 | |
| Fish Strain | AB | ||
| Saline phosphate buffer (10X PBS) pH 7.4 (for 1 liter) | For preparing 10X PBS, add the following salts and complete to 1 liter with distilled water | ||
| Potassium chloride (MM : 74.55 g/mol): 2.00 g | Sigma-Aldrich, Germany | 746436 | |
| Potassium phosphate monobasic (MM: 136,09 g/mol): 2.40g | Sigma-Aldrich, Germany | 795488 | |
| Sodium chloride (MM : 58.44 g/mol): 80.00 g | Sigma-Aldrich, Germany | S9888 | |
| Sodium phosphate dibasic (MM: 141,96 g): 14,40 g | Sigma-Aldrich, Germany | 795410 |
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Citazione di questo articolo
Dorsemans, A., Lefebvre d’Hellencourt, C., Ait-Arsa, I., Jestin, E., Meilhac, O., Diotel, N. Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules. J. Vis. Exp. (124), e55203, doi:10.3791/55203 (2017).