Name: acute and chronic models of hyperglycemia in zebrafish: a method to assess the impact of hyperglycemia on neurogenesis and the biodistribution of radiolabeled molecules
Uploaded: 2017-06-26T12:30:00.000+00:00
Duration: 13 min 35 s
Description: Watch this Scientific Journal Video about Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled

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。

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<ol><li>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，并将其等分储存在-20°C。 </li><li>通过将5mL三卡因（储备溶液）加入到100mL水中（最终三氯氰酸盐浓度:0.02％），为成年斑马鱼准备麻醉剂。 </li><li>将一个斑马鱼（3至6个月）从其坦克转移到麻醉剂，直至停止移动。 </li><li>回覆使用切割移液管移动鱼，并在吸收性纸巾上快速干燥。 </li><li>称重鱼</li><li>准备溶解在1X PBS中的D-葡萄糖注射器，注射50μLD-葡萄糖（2.5g / kg体重）。 注意:例如，0.6克鱼将接受50μL的3％D-葡萄糖/ PBS溶液。 </li><li>将鱼放在背部，将注射器的针头插入腹腔。 </li><li>缓慢注射D-葡萄糖/ PBS溶液，然后将鱼放回水中。 </li><li>检查鱼，直到它完全恢复。将其返回其罐，直到达到进行血糖测量所需的时间。 注意:注射后1.5小时测量血糖。 </li></ol> 2.建立斑马鱼慢性高血糖模型<ol><li>准备一个2升的清水鱼缸。 </li><li>将40g D-葡萄糖溶解在2L鱼水中，以得到最终D-葡萄糖浓度111 mM。 </li><li>在含D-葡萄糖的水中浸入5至7个成年斑马鱼。 </li><li>每2天更换D-葡萄糖鱼水，以避免细菌或其他微生物的生长。 </li><li>处理14天后，将鱼短暂置于淡水中，以便在进行血糖值测量之前除去身体外的D-葡萄糖。 </li></ol> 3.测量斑马鱼血糖水平<ol><li>用冰盖上鱼，以便快速安乐死。 注意:不要使用过量的三卡因，因为这会引起血糖水平的大幅变化。不要将鱼留在冰中太长时间，因为这会导致血液凝结。 </li><li>用吸收性纸巾擦拭鱼，以除去所有的水，并在血糖测量期间避免血液稀释。 </li><li>使用解剖镊子取下眼睛，等待眼睛充满血液。 </li><li>将测试条放在血糖仪上，将条带插入眼睛腔。 </li><li>测量血糖水平。 </li></ol> 4.分析高血糖后脑细胞增殖<ol><li> 解决方案和缓冲:要求和准备 <ol><li>通过向900mL蒸馏水（dH 2 O）中加入100毫升10倍的PBS制备1升1x PBS并混合。 </li><li>用0.2％洗涤剂（PBS-T;参见材料表）制备1x PBS。 注意:为了制备1L PBS-T，加入2mL洗涤剂（参见材料表 ）至1L PBS。 </li><li>准备乙醇系列（100％x2; 95％; 85％; 70％; 50％和30％）和0.85％NaCl。 </li><li>制备封闭缓冲液:含有0.5％至1％奶粉的PBS-T。 注意:为了制备200 mL封闭缓冲液，在200 mL PBST中加入2 g奶粉。 </li><li>准备抗原检索缓冲液，柠檬酸钠。 注意:为了制备1升柠檬酸钠缓冲液，加入2.94克柠檬酸三钠钠盐脱水至1升dH 2 0.将pH调至6，然后再填充至1 L. </li><li>制备4％多聚甲醛-PBS缓冲液，加入4g多聚甲醛至100mL的1x PBS。在58-60℃搅拌下加热，直到其完全溶解。 </li></ol></li><li> 免疫组织化学样品制备:固定和脱水 <ol><li>在测量血糖水平后，通过切割鳃后面的头部将头部与身体分开。 注意:或者，可以将大脑直接提取和冷冻用于其他实验，如mRNA提取。 </li><li>在4℃溶解于PBS（PFA-PBS）的4％多聚甲醛中将头固定过夜。 </li><li>第二天，在PBS中简单地洗头。 </li><li>用显微镜小心解剖脑部。用PBS冲洗固定头，使用针头将头部固定在解剖显微镜下。用镊子去除眼睛和头顶部。 Carefully提取大脑并将其置于1x PBS中。 </li><li>用1x PBS连续洗涤30分钟，0.85％NaCl 30分钟，70％EtOH / 0.85％NaCl（v / v）15分钟，70％EtOH 15分钟（两次），85％EtOH 20 min，95％EtOH 20分钟，100％EtOH 20分钟（两次）。在最终的100％乙醇溶液中孵育过夜。 注意:在石蜡包埋之前，脱水的脑可以在4℃的100％EtOH中保留数月。 </li></ol></li><li> 免疫组织化学样品制备:石蜡包埋的组织制备 <ol><li>将大脑放在一个小玻璃烧杯中。 注意:不要使用塑料容器，因为甲苯会损坏一些塑料。 </li><li>取出乙醇并用甲苯代替，因为大脑必须被甲苯完全回收。执行两个30分钟的甲苯浴。 </li><li>取出甲苯并将大脑置于嵌入式盒中。将封闭的盒子放在熔化的石蜡系列烧杯中58-60℃（每个石蜡烧杯中30分钟）。打开加温夹层钳。在石蜡浴的末端，取出盒子并将一些液体石蜡倒入模具中。 </li><li>将熔化的石蜡倒入模具中，将脑部放入内部。使用加温夹层钳定向大脑，使用大脑的前后方向作为指导。让石蜡在包埋机的冷却部分上硬化。 </li><li>由于技术原因，将大脑沿着前后轴定位。在解开石蜡块之后，将其固化并固定在盒子上，使熔融的石蜡以正确的方向进行横向切片。 </li><li>将石蜡块插入切片机的手臂。切割50μm厚的部分，直到达到脑样本水平。修剪石蜡块以获得一个空中飞行器，并将切片厚度调整为7μm。使用油漆刷收集石蜡带并将其放在黑色的pap上呃。每3至4节轻轻切割一次。 </li><li>将一个滑块放在暖气板上，并用dH 2 O水将其盖住。 </li><li>将切割的丝带轻轻放在水面上。当石蜡带充分展开/展开时，用吸收性纸巾去除水分。最后删除药液。从载玻片上取出水，让它们在30-40℃左右的温暖板上干燥至少3小时。 </li></ol></li><li> 免疫组化方法 <ol><li>通过将三分之一的二甲苯容器放置在每个容器中7分钟来除去石蜡。 </li><li>将载玻片放入两个100％乙醇的容器中，每次2分钟，然后加入95％EtOH，85％EtOH，70％EtOH和30％EtOH的浴，每次30秒，将部分加水。最后，简单地将幻灯片放在dH 2 O中，并在PBS中两次，每次5分钟。 </li><li>通过在微波炉中的柠檬酸盐缓冲液中孵育切片进行抗原恢复（在500W下2分钟）直到缓冲区开始沸腾。让幻灯片在室温下恢复15分钟。 </li><li>在PBST中洗涤三次，每次5分钟，并在含有1％牛奶的PBST中封闭切片45分钟。用阻断缓冲液（ 即 PBST，1％牛奶）稀释的初级抗体（ 例如增殖细胞核抗原（PCNA）用于增殖细胞染色; 1/100）孵育过夜。 </li><li>在PBST中洗涤3次，每次5分钟，并用封闭缓冲液和DAPI（1/500）稀释的合适的二次抗体（ 例如山羊抗小鼠Alexa Fluor 488; 1/200）孵育切片90分钟。 </li><li>在PBST中洗涤三次，每次5分钟，并用荧光安装介质安装载玻片（参见材料表）。 </li><li>使用落射荧光和/或共焦显微镜分析染色。 </li><li>定量至少三种不同的感兴趣区域的至少三个连续的脑切片上的脑细胞增殖imals。 注意:或者，如前所述，可以在琼脂糖中进行脑嵌入进行vibratome切片和自由浮动免疫组织化学。 </li></ol></li><li> 研究高血糖对脑修复机制的影响 注意:或者，急性和慢性高血糖症的脑修复调查可以在端脑刺伤伤后进行，如前所述， 24,25,26 。简述: <ol><li>麻醉成人斑马鱼与三叉戟。 </li><li>将鱼放在解剖显微镜下面。 </li><li>用一只手握住鱼。 </li><li>另一方面，将30 G注射器通过颅骨垂直插入右端脑半球的内侧区域。 </li><li>将鱼放回鲜鱼水，D-葡萄糖补充水（111mM），或控制鱼水。允许鱼在伤后7天生存。 注意:有关其余步骤，请参阅步骤4。 </li></ol></li></ol> 5.通过PET / CT在斑马鱼:氟脱氧葡萄糖（[18F] -FDG）中成像放射性标记的分子的生物分布来分析葡萄糖代谢<ol><li>准备一个含有20 MBq [18 F] -FDG盐水溶液的50μL注射器。 </li><li>将注射器放在辐射防护屏幕的后面。 </li><li>麻醉三叉戟的成年斑马鱼</li><li>将鱼放在防辐射屏幕的后面。 </li><li>将[18F] -FDG注入腹膜腔。用小纸巾擦拭注射部位，以防止可能从鱼肚泄漏出来的剩余[18F] -FDG。 </li><li>使用放射性同位素校准器来测量注射器和组织上包含的剩余活性，以计算精确的注射剂量。 </li><li>把鱼放在一个ew，用三叶草浸泡的小片吸收性组织轻轻地包裹起来。将吸收性组织的鱼放在PET / CT成像仪的床上。 </li><li>将床插入PET / CT成像系统并开始采集。 </li><li>继续进行PET / CT采集。 </li><li>在程序结束时，将鱼放回少量鲜鱼水中。 注意:或者，在[18F] -FDG注射后，鱼可以放回少量淡水10分钟至1小时以促进[18F] -FDG的扩散，然后麻醉鱼再次用于PET / CT成像。 </li></ol>

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(90), e51753 (2014).</li><li>Diotel, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+estradiol+in+adult+neurogenesis+and+brain+repair+in+zebrafish.">Effects of estradiol in adult neurogenesis and brain repair in zebrafish.</a> Horm Behav. 63 (2), 193-207 (2013).</li><li>Rodriguez Viales, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+helix-loop-helix+protein+id1+controls+stem+cell+proliferation+during+regenerative+neurogenesis+in+the+adult+zebrafish+telencephalon.">The helix-loop-helix protein id1 controls stem cell proliferation during regenerative neurogenesis in the adult zebrafish telencephalon.</a> Stem Cells. 33 (3), 892-903 (2015).</li><li>Kaslin, J., Ganz, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+neurogenesis+and+regeneration+in+the+non-mammalian+vertebrate+brain.">Proliferation, neurogenesis and regeneration in the non-mammalian vertebrate brain.</a> Philos Trans R Soc Lond B Biol Sci. 363 (1489), 101-122 (2008).</li><li>Kroehne, V., Freudenreich, D., Hans, S., Kaslin, J., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+adult+zebrafish+brain+from+neurogenic+radial+glia-type+progenitors.">Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors.</a> Development. 138 (22), 4831-4841 (2011).</li><li>Alunni, A., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+view+of+regenerative+neurogenesis+in+vertebrates.">A comparative view of regenerative neurogenesis in vertebrates.</a> Development. 143 (5), 741-753 (2016).</li><li>M&#228;rz, M., Schmidt, R., Rastegar, S., Str&#228;hle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+response+following+stab+injury+in+the+adult+zebrafish+telencephalon.">Regenerative response following stab injury in the adult zebrafish telencephalon.</a> Dev Dyn. 240 (9), 2221-2231 (2011).</li><li>Wullimann, M., Rupp, B., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroanatomy of the zebrafish brain: A topological atlas. , 1-144 (1996).</li><li>Pellegrini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+aromatase-positive+radial+glial+cells+as+progenitor+cells+in+the+ventricular+layer+of+the+forebrain+in+zebrafish.">Identification of aromatase-positive radial glial cells as progenitor cells in the ventricular layer of the forebrain in zebrafish.</a> J Comp Neurol. 501 (1), 150-167 (2007).</li><li>Zupanc, G. K., Hinsch, K., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+migration,+neuronal+differentiation,+and+long-term+survival+of+new+cells+in+the+adult+zebrafish+brain.">Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain.</a> J Comp Neurol. 488 (3), 290-319 (2005).</li><li>Sarras, M. P., Intine, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Zebrafish+as+a+Disease+Model+Provides+a+Unique+Window+For+Understanding+the+Molecular+Basis+of+Diabetic+Metabolic+Memory.">Use of Zebrafish as a Disease Model Provides a Unique Window For Understanding the Molecular Basis of Diabetic Metabolic Memory.</a> iConcept Press. , 2611-2619 (2013).</li><li>Connaughton, V. P., Baker, C., Fonde, L., Gerardi, E., Slack, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternate+Immersion+in+an+External+Glucose+Solution+Differentially+Affects+Blood+Sugar+Values+in+Older+Versus+Younger+Zebrafish+Adults.">Alternate Immersion in an External Glucose Solution Differentially Affects Blood Sugar Values in Older Versus Younger Zebrafish Adults.</a> Zebrafish. , (2016).</li><li>Prasad, S., Sajja, R. K., Naik, P., Cucullo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+Mellitus+and+Blood-Brain+Barrier+Dysfunction:+An+Overview.">Diabetes Mellitus and Blood-Brain Barrier Dysfunction: An Overview.</a> J Pharmacovigil. 2 (2), 125 (2014).</li></ol>

没有披露潜在的利益冲突。

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进...

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）。

<table><tbody><tr><td>1mL Luer-Lok Syringe</td><td>BD, USA</td><td>309628</td><td></td></tr><tr><td>4&#039;,6&#039;-diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich, Germany</td><td>D8417</td><td></td></tr><tr><td>7 mL bijou container plain lab</td><td>Dutscher, France</td><td>080171</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich, Germany</td><td>67021</td><td></td></tr><tr><td>Digital camera</td><td>Life Sciences, Japan</td><td>Hamamatsu ORCA-ER</td><td></td></tr><tr><td>Disposable base molds&amp;nbsp;</td><td>Simport, Canada</td><td>M475-2</td><td></td></tr><tr><td>Donkey anti-rabbit Alexa fluor 488</td><td>Life Technologies, USA</td><td>A21206</td><td></td></tr><tr><td>Embedding center</td><td>Thermo Scientific, USA</td><td>Shandon Histocentre 3</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon, Japan</td><td>Eclipse 80i</td><td></td></tr><tr><td>Fluorodeoxyglucose (&lt;sup&gt;18&lt;/sup&gt;F-FDG)</td><td>Cyclotron, France</td><td></td><td></td></tr><tr><td>Glucometer test strip</td><td>LifeScan, France</td><td>One-Touch 143 Ultra</td><td></td></tr><tr><td>Goat anti-mouse Alexa fluor 594</td><td>Life Technologies, USA</td><td>A11005</td><td></td></tr><tr><td>&lt;em&gt;In-Vivo&lt;/em&gt; Imaging System</td><td>TriFoil Imaging, Canada</td><td>Triumph Trimodality&amp;nbsp;</td><td></td></tr><tr><td>Microtome</td><td>Thermo Scientific, USA</td><td>Microm HM 355 S</td><td></td></tr><tr><td>Monoclonal mouse anti-PCNA</td><td>DAKO, USA</td><td>clone PC10</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich, Germany</td><td>P6148-500G</td><td></td></tr><tr><td>Polyclonal rabbit anti-GFAP</td><td>DAKO, USA</td><td>Z033429</td><td></td></tr><tr><td>Slide drying bench</td><td>Electrothermal, USA</td><td>MH6616</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich, Germany</td><td>S9888</td><td></td></tr><tr><td>Sodium citrate trisodium salt dehydrate&amp;nbsp;</td><td>Prolabo, France</td><td>27833.294</td><td></td></tr><tr><td>Sterile needle</td><td>BD Microlance 3</td><td>30 G 1/2 ; 0.3 mm&amp;times; 13 mm</td><td></td></tr><tr><td>Student Dumont #5 Forceps</td><td>Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Student surgical scissors</td><td>Fine Science Tools</td><td>91400-14</td><td></td></tr><tr><td>Superfros Plus Gold Slides</td><td>Thermo Scientific, USA</td><td>FT4981GLPLUS</td><td></td></tr><tr><td>Surgical microscope</td><td>Leica, France</td><td>M320-F12</td><td></td></tr><tr><td>Tissue embedding cassettes</td><td>Simport, Canada</td><td>M490-10</td><td></td></tr><tr><td>Tissue embedding medium</td><td>LeicaBiosystems, USA</td><td>39602004</td><td></td></tr><tr><td>Toluene</td><td>Sigma-Aldrich, Germany</td><td>244511</td><td></td></tr><tr><td>Tricaine MS-222</td><td>Sigma-Aldrich, Germany</td><td>A5040</td><td></td></tr><tr><td>Triton X100</td><td>Sigma-Aldrich, Germany</td><td>X100-500 mL</td><td></td></tr><tr><td>Vectashield medium&amp;nbsp;</td><td>Vector Laboratories, USA</td><td>H-1000</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich, Germany</td><td>534056</td><td></td></tr><tr><td>Fish Strain</td><td>AB</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Saline phosphate buffer (10X PBS) pH 7.4 (for 1 liter)&lt;/strong&gt;</td><td></td><td></td><td>For preparing 10X PBS, add the following&amp;nbsp; salts and complete to 1 liter with distilled water</td></tr><tr><td>&lt;em&gt;Potassium chloride (MM : 74.55 g/mol): 2.00 g&lt;/em&gt;</td><td>&lt;em&gt;Sigma-Aldrich, Germany&lt;/em&gt;</td><td>&lt;em&gt;746436&lt;/em&gt;</td><td></td></tr><tr><td>&lt;em&gt;Potassium phosphate monobasic (MM: 136,09 g/mol): 2.40g&lt;/em&gt;</td><td>&lt;em&gt;Sigma-Aldrich, Germany&lt;/em&gt;</td><td>&lt;em&gt;795488&lt;/em&gt;</td><td></td></tr><tr><td>&lt;em&gt;Sodium chloride (MM : 58.44 g/mol): 80.00 g&amp;nbsp;&lt;/em&gt;</td><td>&lt;em&gt;Sigma-Aldrich, Germany&lt;/em&gt;</td><td>&lt;em&gt;S9888&lt;/em&gt;</td><td></td></tr><tr><td>&lt;em&gt;Sodium phosphate dibasic (MM: 141,96 g): 14,40 g&lt;/em&gt;</td><td>&lt;em&gt;Sigma-Aldrich, Germany&lt;/em&gt;</td><td>&lt;em&gt;795410&lt;/em&gt;</td><td></td></tr></tbody></table>

acute and chronic models of hyperglycemia in zebrafish: a method to assess the impact of hyperglycemia on neurogenesis and the biodistribution of radiolabeled molecules

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。

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...

Watch this Scientific Journal Video about Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

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。

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

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

acute-chronic-models-hyperglycemia-zebrafish-method-to-assess-impact

Research

JoVE Journal

Developmental Biology

13.3K 次观看.  Université de La Réunion, INSERM. 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。 

视频: Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

斑马鱼模型的糖尿病和代谢记忆

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

科研

医学

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

斑马鱼神经母细胞瘤转移模型

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

癌症研究

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

以斑马鱼为模型系统的三室选择行为任务

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

行为学

Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo &#946;-Cell Function

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in &#946;-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, &#946;-cells experience an influx of Ca2+. Thus, imaging the glucose-stimulated Ca2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying &#946;-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca2+ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how &#946;-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate &#946;-cell activity in vivo. Here, a detailed protocol for Ca2+ imaging and glucose stimulation to investigate &#946;-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ dynamics in &#946;-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect &#946;-cell function.

<meta charset="utf8"></head><body>在活斑马鱼中同时进行钙成像和葡萄糖刺激以研究体内β细胞功能

Here, the study presents a protocol for calcium imaging and glucose stimulation of the pancreatic &#946;-cells of the zebrafish in vivo.

在活斑马鱼中同时进行钙成像和葡萄糖刺激以研究体内β细胞功能

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in ...

发育生物学

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

生物学

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

神经科学

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

通过鱼眼研究糖尿病: 成人 tg (fli: EGFP) 斑马鱼视网膜血管的显微解剖、可视化和分析

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This alternate immersion protocol is a noninvasive, step-wise method of inducing hyperglycemia for up to eight weeks. Adult zebrafish are alternately exposed to sugar (glucose) and water for 24 hours each. The zebrafish begin treatment in a 1% glucose solution for 2 weeks, then a 2% solution for 2 weeks, and finally a 3% solution for the remaining 4 weeks. Compared to water-treated (stress) and mannitol-treated (osmotic) controls, glucose-treated zebrafish have significantly higher blood sugar levels. The glucose-treated zebrafish show blood sugar levels of 3-times that of controls, suggesting that after both four and eight weeks hyperglycemia can be achieved. Sustained hyperglycemia was associated with increased Glial Fibrillary Acidic Protein (GFAP) and increased nuclear factor Kappa B (NF-kB)&#160;levels in retina and decreased physiological responses, as well as cognitive deficits suggesting this protocol can be used to model disease complications.

This protocol noninvasively induces hyperglycemia in zebrafish for up to 8 weeks. Using this protocol, an in-depth study of the adverse effects of hyperglycemia can be made.

交替浸入葡萄糖中，在斑马鱼中产生长期血糖超血症

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This ...

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

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此视频中的章节

斑马鱼模型的糖尿病和代谢记忆

斑马鱼神经母细胞瘤转移模型

以斑马鱼为模型系统的三室选择行为任务

在活斑马鱼中同时进行钙成像和葡萄糖刺激以研究体内β细胞功能

在活斑马鱼中同时进行钙成像和葡萄糖刺激以研究体内β细胞功能

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

通过鱼眼研究糖尿病: 成人 tg (fli: EGFP) 斑马鱼视网膜血管的显微解剖、可视化和分析

交替浸入葡萄糖中，在斑马鱼中产生长期血糖超血症