交替浸入葡萄糖中，在斑马鱼中产生长期血糖超血症
Summary
此协议非侵入性地诱发斑马鱼高血糖长达 8 周。利用这一协议，可以深入研究高血糖的不利影响。
Abstract
斑马鱼（Danio rerio）是研究慢性高血糖影响的优秀模型，这是 II 型糖尿病 （T2DM） 的标志。这种替代浸入协议是一种非侵入性的、循步骤的方法，可诱导高血糖长达八周。成年斑马鱼交替接触糖（葡萄糖）和水，每次24小时。斑马鱼开始用1%的葡萄糖溶液治疗2周，然后用2%的溶液治疗2周，最后在剩下的4周内进行3%的溶液治疗。与水处理（压力）和曼尼托处理（渗透）对照相比，葡萄糖处理斑马鱼的血糖水平显著较高。经过葡萄糖处理的斑马鱼的血糖水平是对照组的3倍，这表明在4周和8周后可以达到高血糖。持续性高血糖与胶质纤维酸性蛋白 （GFAP） 增加、心网膜核因子 Kappa B （NF-kB） 水平增加、生理反应下降以及认知缺陷有关，表明此协议可用于模拟疾病并发症。
Introduction
斑马鱼（丹尼奥·雷里奥）正在迅速成为一个广泛使用的动物模型，以研究疾病和认知1。基因操纵的易受干扰和胚胎透明性在早期发育阶段，使他们成为研究人类疾病的主要候选者与已知的遗传基础。例如，斑马鱼已经被用来研究霍尔特-奥拉姆综合征，心肌病，球状细胞性肾脏疾病，肌肉萎缩症和糖尿病（DM）等疾病1。此外，斑马鱼模型是理想的，因为该物种的体积小，维护方便，和高粪便2，3。
斑马鱼胰腺在解剖学上和功能上都与哺乳动物胰腺4相似。因此，斑马鱼具有体型大、肥大和内分泌结构相似的独特特征，因此斑马鱼成为研究DM相关并发症的合适人选。在斑马鱼中，有两种实验方法用于诱导DM特有的长时间高血糖：葡萄糖的流入（建模2型）和胰岛素分泌的停止（建模1型）5、6。在实验中，为了阻止胰岛素分泌，胰腺β细胞可以通过链球菌素（STZ）或Aloxan注射进行化学破坏。STZ已成功地用于啮齿动物和斑马鱼，导致并发症与视网膜病变7，8，9，认知障碍10，和肢体再生11。然而，在斑马鱼中，β细胞在治疗后再生，导致STZ的”助推器注射”成为维持糖尿病状况所必需的。或者，斑马鱼的胰腺可以去除6。这些都是高度侵入性的程序，由于多次注射，和广泛的恢复时间。
相反，高血糖可以通过接触外源性葡萄糖来非侵入性地诱发。在此协议中，鱼被淹没在高度浓缩的葡萄糖溶液中24小时5，13或连续2周14，15，16。外源性葡萄糖通过摄入和/或穿过刺，导致血糖水平升高而被跨皮吸走。由于这种非侵入性技术不直接操纵胰岛素水平，因此不能声称诱导2型DM。然而，它可以用来检查高血糖引起的并发症，这是2型DM的主要症状之一。
最近，斑马鱼突变pdx1-/-通过操纵胰腺和十二指肠家谱1基因，一种与人类2型DM的遗传原因相关的基因而开发的。利用这种突变体，研究人员已经能够复制胰腺发育中断，高血糖，并研究高血糖引起的糖尿病视网膜病变17，18。
本文描述了一种使用交替浸入协议的非侵入性高血糖诱导方法。此协议维持高血糖条件长达 8 周，并观察到随后的并发症。简言之，成年斑马鱼被放置在糖溶液中24小时，然后放入水溶液中24小时。与持续浸入外部葡萄糖溶液相反，糖和水之间的交替天模拟糖尿病中血糖的上升和下降。此外，交替葡萄糖协议还允许高血糖在较长时间内诱发，因为斑马鱼无法补偿高外部葡萄糖条件。作为原则的证明，我们提供的数据表明，使用此协议诱导的血糖过高会改变视网膜化学和生理学。
Protocol
所有程序都得到了美国大学动物护理和使用机构委员会的批准。 1. 准备解决方案罐 获得六个水箱，每个实验组两个（葡萄糖、曼尼托和水）。给两个储罐中的一个贴上”储罐”（它将容纳鱼）的标签，并给另一个”溶液罐”贴上标签（它将容纳溶液）。注：曼尼托治疗组为渗透控制，水处理组为处理/应力控制。重要的是要保持坦克，航空公司/航空石，盖子和清洁用品?…
Representative Results
使用此协议（图 1），经过 4 周和 8 周的治疗（图 2A），血糖值显著升高，高血糖的定义是水处理组和曼尼托治疗组的控制平均值的 3 倍。水处理控制每天进出水，提供压力/处理控制。曼尼托在体外葡萄糖研究19，20中充当渗透控制，因为它是一种6碳糖，如葡萄糖，但不被细胞占用。为了与这些研究和斑马鱼…
Discussion
糖尿病是一个全国性的问题。研究表明，到2030年，估计有4亿人将患有某种形式的糖尿病。在啮齿动物模型中，使用基因操作研究2型DM。在大鼠中，扎克糖尿病脂肪大鼠（ZDF）和大须卡长埃文斯德岛脂肪大鼠（OLETF）提供了更多关于2型DM10的影响的信息。此外，高脂肪饮食已用于啮齿动物诱发高血糖。这反映了本文中提出的非侵入性程序。使用我们的非侵入性协议，我们可以诱发?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢 VPC、CJR 和 MCP 为本协议的制定。EMM 获得美国大学文理学院研究生支持的资助，以开展此项研究。这项工作还得到了美国大学梅隆学院奖的支持，并通过美国大学艺术与科学学院（VPC）提供资金。
Materials
| Airline Tubing | petsmart | 5291863 | This can be used in the tank to circulate air |
| Airpump | petsmart | 5094984 | This can be used in the tank to circulate air |
| Airstones | petsmart | 5149683 | This can be used in the tank to circulate air |
| D-glucose | Sigma | G8270-5KG | |
| D-mannitol | Acros Organics | AC125340050 | |
| Freestyle Lite Meter | Amazon | B01LMOMLTU | |
| Freestyle Lite Strips | Amazon | B074ZN3H2Z | |
| Net | petsmart | 5175115 | |
| Tanks | Amazon | B0002APZO4 |
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Citar este artigo
McCarthy, E., Rowe, C. J., Crowley-Perry, M., Connaughton, V. P. Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish. J. Vis. Exp. (171), e61935, doi:10.3791/61935 (2021).