这项工作得到了 DZNE 和亥姆霍兹协会 (VH-NG-1021)、》、TU 德累斯顿 (FZ-111、043_261518) 和 DFG (KI1524/6) (酸) 的支持;由莱布尼茨协会 (SAW-2011-IPF-2) 和 BMBF (BioLithoMorphie 03Z2E512) (Y.Z.)。我们还要感谢乌莉霍夫曼肽合成, 南 Asokan, Prayag Murawala, 和艾利在拍摄过程中帮助田中。

本议定书是欧盟准则 (2010/63) 和欧洲生物和医学鱼类模型协会 (EuFishBioMed) 在卡尔斯鲁厄中国科学院技术 (KIT) 中建议的标准程序。所有在这里描述的方法都得到了道德委员会的批准 (Landesdirektion 德累斯顿; 文档编号 TVV-52/2015). 
 1. 制备 #946; 42 肽 <ol> <li> 合成多肽 (参见 表 1 ) 使用标准的 9-芴 (甲氧羰基) 化学与 2-(1 h-benzotriazol-1-基)-11, 33-tetramethyluronoiumhexafluorphosphate (HBTU) 作为自动固相多肽合成器的偶联试剂 52 。合成的规模是100和 #181; 摩尔. </li>
	<li> 将 n-芴甲氧羰基保护树脂的500毫克的自动多肽合成器加载为固相 (负载容量为0.2 摩尔/克)。装载被溶化的 n-芴甲氧羰基被保护的氨基酸在集中 0.5 M 在需要的容量和为各自合成器计算. 
	注: 进行了计算, 使每块 n-芴甲氧羰基被保护的氨基酸的偶联度为每一个积木的5倍, 超过了树脂的 47 . </li>
	<li> 溶解 dimethlyformamide (DMF) 中所需的多肽合成试剂。例如, 准备活化剂, HBTU, 在浓度为0.48 米, 45% 伏/v n-啉 (NMM) (基地), 和5% 伏/v 醋酸酐 (上限混合物) 上限的 non-reacted 氨基. </li>
	<li> 将所有的肽从树脂中通过连续混合的固体支持使用搅拌器在一个新准备的裂解混合物组成的乙酸酸 (TFA): Triisopropylsilane (TIS): 水: 糖 (数码) 在 90 (v/v): 5 (v/v): 2。5(五/五): 2.5 (m/五), 4 h. 为100和 #181 的合成规模使用10毫升; 摩尔. </li>
	<li> 通过将裂解混合物加入100毫升 ice-cold 二乙醚, 沉淀所裂解产物。通过含有聚四氟乙烯 (PTFE) 过滤器的过滤单元, 其孔径大小为0.45 和 #181; m 和20毫升 ice-cold 乙醚洗涤. </li>
	<li> 从滤纸中收集过滤后的肽, 在5毫升蒸馏水中溶解100毫克沉淀肽: 乙腈在 1:1. </li>
	<li> 通过反相高压液相色谱法 (hplc) 对 semi-preparative 的多孔聚苯乙烯苯柱尺寸10和 #181; m. 
	<ol> <li> 预热该列并保持在50和 #176; C 使用柱式加热装置。收集所有主要分数使用自动分数收集器, 应用梯度从5% 到100% 溶剂 B 超过25分钟, 在4毫升/分钟. 
		注: 溶剂 a 是 0.1% TFA 在水和溶剂 B 是 0.1% TFA 在乙腈 52 . </li>
		<li> 监测色谱在 220 nm, 收集适当的峰值, 并使用 LC-MS 进行分析. </li>
	</ol> </li> <li> 通过分析 C18 柱 (珠粒大小1.7 和 #181; m) 通过检测, 通过对紫外线探测器的分析, 在 220 nm 上通过解析反相超高压液相色谱 (UPLC) 来确认纯度。用质谱法确认多肽产品. 
	注: UPLC 是耦合到一个电喷雾电离质谱 (ESI-MS) 和串联四极探测器. </li>
	<li> 通过将0.052 毫巴的真空度应用到蓬松的粉末上, 将所需肽的正确分数 Lyophilize 在圆底烧瓶中。将真空泵耦合到-78 和 #176 的冷冻装置上; c.-80 和 #176; c, 无限期存储. </li>
	<li> 使用冻干肽在乙腈混合物中制备1毫米的库存溶液: DMF: 分析级水在 1:1: 1 用于实验。此解决方案可以存储至少6月, 因为该解决方案不聚合多肽. </li>
</ol> 2。喷射混合物的制备 <ol> <li> 准备磷酸盐缓冲盐水 (PBS) (137 毫米氯化钠, 2.7 毫米氯化钾, 10 mm Na 2 HPO 4 , 2 mm, 2 PO 4 , pH 7.4) 用于稀释冻干肽. </li>
	<li> 将该肽分解为20和 #181 的最终浓度; M 在 PBS 中。准备这个解决方案新鲜。混合好, 并在冰上储存, 直到注射。不要超过30分钟, 以防止在解决方案中聚合. </li>
</ol> 3。麻醉 <ol> <li> 准备麻醉剂的储存液, 0.1% 乙基氨基 methanesulphonate (MESAB), 从循环系统中的常规鱼水中。准备 anaesthetization 溶液, 最终浓度为 0.0025% (v/v). </li>
	<li> 将所需数量的鱼从其罐中移到一个带有5升系统水的运输容器中. </li>
	<li> 半填充一个塑料培养皿 (90 毫米) 与40毫升麻醉剂。用这个盘子做注射. </li>
	<li> 将鱼放入麻醉剂中, 直到盖运动停止. </li>
</ol> 4。脑室微注射 <ol> <li> 注射装置的制备 <ol> <li> 使用针牵引器制备玻璃注射毛细管, 其参数如下: 加热循环, 537; 牵引循环, 250; 速度1.5 s;时间, 80 毫秒 </li> <li> 将压力源上的压力设置为 25 psi. </li>
		<li> 将微量参数设置为: 按住压力 20 psi; 弹出压力 10 psi; 周期值 2.5; 浇注值100毫秒 </li>
		<li> 将玻璃毛细管与注射液一起加载。将玻璃毛细管插入微注射支架。将注塑角度调整为45和 #176;. 
		注意: 可以找到更详细的协议, 如参考资料 27 , 46 中所述。
	</ol> </li> <li> 将一条鱼放入一个充满 anaesthetization 解决方案的新培养皿中 (如步骤3.3 中所示). </li>
	<li> 拿着钳子的鱼和东方注射. </li>
	<li> 用30克针的尖端在光学顶上产生一个狭缝, 两个侧面板相遇。不要将笔尖插入脑组织, 这会导致出血和损伤。有关其他详细信息, 请参阅参考 27 、 45 、 46 。
	<li> 将玻璃毛细管插入狭缝中. 
	<ol> <li> 仅使用针头的尖端, 并且不穿透头骨超过1毫米。保持鱼和插入玻璃毛细管的尖端通过切口部位. </li>
		<li> 将玻璃毛细管的尖端朝向端的45和 #176; 角度。注入1和 #181; 我的解决方案。液体在注射后立即散开. </li>
	</ol> </li> </ol> 5。恢复 <ol> <li> 将鱼放回传输容器中, 直到它恢复为止。将容器与定期循环的鱼水连接, 以确保最佳的水质. 
	注意: 恢复通常需要1分钟。如果需要更长的时间, 必须将鱼放在麻醉剂中较短 (这需要由实验者进行优化). </li>
</ol > 6。组织准备和切片 <ol> <li> 在牺牲鱼之前等待一段希望的时间. 
	注意: 这取决于实验问题。淀粉样蛋白的沉积可以早在注射后1天就能看到. </li>
	<li> 用适当的方法牺牲鱼根据道德规则 ( 例如 , 对待鱼与 0.1 M MESAB). </li>
	<li> 将顶上的颅骨切开, 用尖头 cep 在背侧, 用手术刀解剖胸肋后方的头部. </li>
	<li> 在4和 #176 一夜之间使用2% 甲醛 (粉煤灰) 固定磁头; c. 使用3毫升的粉煤灰每头在一个塑料管与螺丝盖. 
	注意: 在处理粉煤灰时, 应佩戴适当的个人防护设备. </li>
	<li> 对于冻和脱, 洗头三次在0.1M 磷酸盐缓冲 (pH 值 7.4), 然后转移到 20% sucrose/20%ethylenediaminetetraacetic (EDTA) 溶液和孵化过夜在4和 #176; C. </li>
	<li> 将组织嵌入到切片树脂中, 将 7.5% gelatin/20%sucrose 溶液中的头冻结在干冰 (大约3-5 分钟以冻结块) 的塑料组织学模子中。将示例存储在-80 和 #176; C 或继续下一步 (cryosectioning). </li>
	<li> 将磁头分为12和 #181; m 厚 cryosections 使用低温描述的 28 , 42 。将 cryosections 直接转移到玻片上, 并存放在-20 和 #176; C 用于长期使用. </li>
</ol> 7。免疫组织化学染色与显微术 注意: 在湿化室中执行所有孵化步骤。所有的洗涤步骤为10分钟. 
<ol> <li> 在室温下将部分干燥30分钟。解冻后, 清洗两次在 pbs 和一次在 PBSTx (pbs 与 0.03% Triton-X-100). </li>
	<li> 用主抗体 (抗 A 和 #946; 42 抗体; 1:500 稀释在 PBSTx) 在4和 #176; C 隔夜. 和 #160; 第二天, 在 PBS 洗一次, 两次在 PBSTx. </li>
	<li> 在室温下, 用二次抗体 (荧光耦合检测, 1:500 稀释) 和 DAPI (1 和 #956; g/毫升) 在 PBSTx 中孵育剖面, 2 小时. </li>
	<li> 在 PBSTx 中洗三次。在最后的洗涤之后, 用片和 #160 安装幻灯片; 使用100和 #956; L 70% 甘油. </li>
	<li> 使用共聚焦显微镜获取荧光图像 (例如, 20X PL 0.7 目标具有488励磁范围和标准绿色滤镜; 图 2 ) 39 . </li> </ol>

作者没有透露

淀粉样肽可以被修改, 包括序列变化或各种标签。例如, 可以生成一个被炒的淀粉样肽, 多肽可以在肽末端的 N 端标上荧光标记, 或用载体肽39标记。同样, 在这个协议中, 载体肽是细胞穿透肽 TR, 因为它的有效运输货物深入到脑组织39。此外, 我们的方法允许注射和分析各种多肽, 可能导致有毒的聚合50,51。因此, 我们的系统提供了一个多用途的方法, 手动注射有毒蛋白质到斑马鱼的大脑, 并分析这种毒性的细胞效应。
淀粉样肽的聚合速度很快, 因此, 必须将其储存在水-DMF-乙腈溶液中, 并在注射前0.5 小时与 PBS 混合。如果溶液中有较大的聚合体, 注射液应在注射前只准备10分钟。注入效率是产生一致结果的重要参数。请参阅我们以前的朱庇特纸46 , 以执行良好且一致的注入。如果注入后未出现聚合, 则必须优化注入方法。
我们的方法是有限的, 根据大小的分子被注射。我们以前表明, 质粒或短的寡聚物可以有效地被采取由心室细胞45,46, 然而, 为高效率的交付入深脑组织, 大分子 (例如, 抗体, 大蛋白质) 不能使用。此外, 亲油性分子可能不易穿透深层组织, 因为这些分子将被隔离的前几层心室细胞。
小鼠模型的神经是耗时和维护这些模型是相当昂贵的。我们的注射方法是一种快速模型的毒性淀粉样沉积, 和神经。此外, 与哺乳动物模型相比, 斑马鱼具有优越的再生能力, 研究重点是脊椎动物的大脑如何能够在神经后的再生反应, 肯定会受益于快速检测系统斑马.
合成肽的质量及其纯度对实验的成功具有重要意义。为了保证这一质量, 必须用液相色谱和质谱对合成的多肽进行彻底的表征。此外, 还建议了39所描述的循环二和聚合研究。
注入鱼脑是一个关键的步骤, 以确保一致的聚集和病理结果。如果需要, 可以进行一项纵向研究, 分析淀粉样肽的聚集和间隙动力学。我们使用的淀粉样肽结合到载体肽, 以确保平等的分布和渗透到脑组织。不耦合的淀粉样肽也给出了类似的结果, 但分布和聚合的时间轴是不同的39。实验者可以根据目标选择所需的淀粉样肽的版本。
有很大的潜力, 我们的方法与其他操作研究结合使用。CVMI 淀粉样肽可以与药物治疗或其他化合物的注射结合, 以测试各种分子在疾病条件下的协同作用。最终, 我们的快速和新颖的模型也可以用于 small-scale 药物筛选方法, 在一个简单的实验室设置。
我们的手动显微注射方法可以有效地用于向成年斑马鱼脑中注入淀粉样肽, 以模拟淀粉样蛋白沉积。在大脑中, 淀粉样蛋白的沉积会引起细胞毒作用, 并导致 AD 病理, 从而显示急性神经退行性疾病。该方法的未来前景将是利用它在一个急性退行性模型研究神经病的斑马鱼脑和再生反应。这样的理解将为设计新的治疗策略提供一个重要的平台。

AD 是一种慢性渐进性疾病, 其特点是大脑皮层神经元和突触的丢失1,2,3,4,5。经典的病理标志的广告是淀粉样肽的沉积和形成的纤维缠结 (NFTs)6。老年斑块, 也称为淀粉样斑块, 是由淀粉样β (a) 肽组成的β褶结构在脑实质5。Aβ42在 AD 患者中的积累在疾病进展中具有早期和关键性的作用。AD 触发一连串的事件导致突触功能障碍, 可塑性受损, 神经元丢失7,8,9,10。
硬斑马鱼的成年大脑是研究干细胞可塑性调控的优良模型 11,12,13,141516、17、18、19、20以及中枢神经系统中的各种疾病 (包括 AD21、22、23 ,24。由于大量可用的实验方法 19,20,25,26,27,28,2930,31, 这些研究是翔实和可行的。斑马鱼可以补充中枢神经系统13,15,32,33,34,35,36,37, 38, 部分使用在神经元丢失后激活的分子程序19,39,40,4142,43, 44。因此, 在斑马鱼中建立一个神经退行性疾病模型可以帮助解决脊椎动物大脑再生能力和干细胞生物学方面的新问题。
最近, 我们通过注射合成 Aβ42肽 (表 1)39在成年斑马鱼脑中建立了淀粉样毒性模型。这种注射引起了神经表型的人脑病理学 (例如, 细胞死亡, 胶质活化, 突触变性, 和记忆缺陷), 表明斑马鱼可用于诱导神经在斑马鱼脑中, Aβ42肽可通过免疫组化染色检测, 并可识别成年斑马鱼中枢神经系统再生的分子机制39。在本协议中, 我们演示了使用脑室注射液 (CVMI) 方法27,39,45,46 , 将合成的淀粉样肽注射到斑马鱼脑中。模拟淀粉样蛋白沉积 (图 1)。CVMI 提供了一种新的方式提供的多肽, 其聚合后注射作为β板结构和施加毒性。这些肽分布在整个大脑中, 以整个 rostro 尾轴的心室面积为目标45。此外, 该方法还可以分析 NSPCs 在成年斑马鱼脑内淀粉样蛋白包裹体中的形态和分子反应。这些研究将为我们提供一个成功的哺乳动物大脑修复的洞察力。我们的方法可以用来理解的必要分子机制, 成功的再生反应后, AD 样症状, 以诱导补充的丢失神经元和功能恢复。

<table>
	<tbody>
		<tr>
			<td>Fmoc-protected amino acids</td>
			<td>IRIS Biotech GmbH (Marktredwitz, Germany)</td>
			<td></td>
			<td>Fmoc-based amino acids for solid phase peptide synthesis (SPPS)</td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)</td>
			<td>IRIS Biotech GmbH (Marktredwitz, Germany)</td>
			<td>RL-1030</td>
			<td>Activator</td>
		</tr>
		<tr>
			<td>Oxyma</td>
			<td>IRIS Biotech GmbH (Marktredwitz, Germany)</td>
			<td>RL-1180</td>
			<td>Racemization supressor</td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine</td>
			<td>IRIS Biotech GmbH (Marktredwitz, Germany)</td>
			<td>SOL-003</td>
			<td>Base</td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>IRIS Biotech GmbH (Marktredwitz, Germany)</td>
			<td>SOL-004</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>N-Methylmorpholine</td>
			<td>Thermo Fisher (Kandel) GmbH, Germany</td>
			<td>A12158</td>
			<td>Base</td>
		</tr>
		<tr>
			<td>1-Hydroxybenzotriazole hydrate (HOBT)</td>
			<td>Sigma-Aldrich Co. LLC. (St. Louis, MO, USA)</td>
			<td>157260 ALDRICH</td>
			<td>Activator</td>
		</tr>
		<tr>
			<td>Piperidine</td>
			<td>MERCK KGaA (Darmstadt, Germany)</td>
			<td>822299</td>
			<td>Fmoc deprotection reagent</td>
		</tr>
		<tr>
			<td>Dichlormethane (DCM)</td>
			<td>MERCK KGaA (Darmstadt, Germany)</td>
			<td>106050</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Formic acid (FA)</td>
			<td>MERCK KGaA (Darmstadt, Germany)</td>
			<td>100264</td>
			<td>Buffer component for HPLC</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>MERCK KGaA (Darmstadt, Germany)</td>
			<td>808260</td>
			<td>Clevage Mixture reagent</td>
		</tr>
		<tr>
			<td>Triisopropylsilane(TIS)</td>
			<td>MERCK KGaA (Darmstadt, Germany)</td>
			<td>233781 ALDRICH</td>
			<td>Clevage Mixture reagent</td>
		</tr>
		<tr>
			<td>Acetonitrile (for UPLC/LCMS)</td>
			<td>Sigma-Aldrich Laborchemikalien GmbH</td>
			<td>34967-1L</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Acetonitrile (for HPLC)</td>
			<td>VWR International Ltd, England</td>
			<td>83639.320</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Diethylether</td>
			<td>VWR International Ltd, England</td>
			<td>23811.326</td>
			<td>Solvent for peptide precipitation</td>
		</tr>
		<tr>
			<td>Dithiotritol (DTT)</td>
			<td>VWR International Ltd, England</td>
			<td>0281-25G</td>
			<td>Clevage Mixture reagent</td>
		</tr>
		<tr>
			<td>TentaGel S RAM Fmoc rink amide resin</td>
			<td>Rapp Polymere GmbH (Tuebingen, Germany)</td>
			<td>S30023</td>
			<td>Solid phase for SPPS</td>
		</tr>
		<tr>
			<td>Peptide synthesis 5 ml syringes with included filters</td>
			<td>Intavis AG (Cologne, Germany)</td>
			<td>34.274</td>
			<td>Reaction tube for SPPS and for clevage from the Solid Phase</td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene (PTFE) filter</td>
			<td>Sartorius Stedtim (Aubagne, France)</td>
			<td>11806-50-N</td>
			<td>Filteration of precipitated peptides</td>
		</tr>
		<tr>
			<td>Polyvinylidenefluoride (PVDF) syringe filter</td>
			<td>Carl Roth GmbH + Co. KG Karlsruhe</td>
			<td>KC78.1</td>
			<td>Pre-filteration for HPLC</td>
		</tr>
		<tr>
			<td>Peptide Synthesizer</td>
			<td>Intavis, Cologne, Germany</td>
			<td>ResPep SL</td>
			<td>Automated solid-phase peptide synthesizer</td>
		</tr>
		<tr>
			<td>Water Alliance HPLC</td>
			<td>Waters, Milford Massachusetts, USA</td>
			<td>Waters 2998, Waters e2695</td>
			<td>Semi-preparative reverse-phase high pressure liquid chromatography (HPLC)</td>
		</tr>
		<tr>
			<td>PolymerX, bead size 10&#956;m, 250x10 mm</td>
			<td>Phenomenex Ltd. Germany</td>
			<td>00G-4328-N0</td>
			<td>Porous polystyrene divinylbenzene HPLC column</td>
		</tr>
		<tr>
			<td>Milli-Q Advantage A10, with a Milli-Q filter</td>
			<td>EMD Millipore Corporation, Billerica, MA, USA</td>
			<td>LCPAK0001</td>
			<td>Water purification system</td>
		</tr>
		<tr>
			<td>Filtration Unit</td>
			<td>Sartorius Stedtim (Aubagne, France)</td>
			<td>16307</td>
			<td>Filtration unit for peptide precipitation</td>
		</tr>
		<tr>
			<td>UPLC Aquity with UV Detector</td>
			<td>Waters, Milford Massachusetts, USA</td>
			<td>M09UPA 664M</td>
			<td>Analytical reverse phase ultra HPLC for LC-MS</td>
		</tr>
		<tr>
			<td>ACQUITY UPLC BEH C18, bead size 1.7 &#956;m, 50x2.1 mm</td>
			<td>Waters, Milford Massachusetts, USA</td>
			<td>186002350</td>
			<td>Analytical C18 column</td>
		</tr>
		<tr>
			<td>ACQUITY TQ Detector</td>
			<td>Waters, Milford Massachusetts, USA</td>
			<td>QBB908</td>
			<td>Electrospray ionization mass spectrometry (ESI-MS)</td>
		</tr>
		<tr>
			<td>CHRIST ALPHA 2-4 LD plus + vacuubrand RZ6</td>
			<td>Martin Christ Gefriertrocknungsanlagen GmbH, Germany</td>
			<td>16706, 101542</td>
			<td>Lyophilizer with vaccum pump</td>
		</tr>
		<tr>
			<td>Paradigm plate reader</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MESAB (ethyl-m-aminobenzoate methanesulphonate)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sarstedt</td>
			<td>821.472</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Life Technologies, GIBCO</td>
			<td>10010-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Becton-Dickinson</td>
			<td>305178</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus, Leica, Zeiss</td>
			<td>Varies with the manufacturer</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr>
			<td>Gillies Dissecting Forceps</td>
			<td>World Precision Instruments</td>
			<td>501265</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass injection capillaries</td>
			<td>World Precision Instruments</td>
			<td>TWF10</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoNozzle</td>
			<td>World Precision Instruments</td>
			<td>5430-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic PicoPump</td>
			<td>World Precision Instruments</td>
			<td>SYS-PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring illuminator; Ring Light Guide</td>
			<td>Parkland Scientific</td>
			<td>ILL-RLG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling amyloid-&#946;42 toxicity and neurodegeneration in adult zebrafish brain

阿尔茨海默氏病 (AD) 是一种衰弱的神经退行性疾病, 其中有毒 amyloid-β42 (Aβ42) 肽的积累导致突触变性、炎症、神经元死亡和学习缺陷。由于神经干/祖细胞 (NSPCs) 的增殖能力受损, 神经元发生减少, 在 AD 的情况下, 人类无法再生丢失的神经细胞。因此, 有效的再生疗法也应提高 NSPCs 的增殖和神经源能力. 斑马鱼 (斑马鱼) 是一种再生有机体, 我们可以学习基本的分子程序, 我们可以设计处理 AD 的治疗方法。因此, 在斑马鱼中生成类似广告的模型是必要的。利用我们的方法, 我们可以引入 Aβ42肽的合成衍生物, 将组织穿透能力纳入成年斑马鱼脑, 并分析疾病病理和再生反应。比现有的方法或动物模型的优势是, 斑马鱼可以教我们如何一个脊椎动物的大脑可以自然再生, 从而帮助我们更好地治疗人类神经退行性疾病的目标内生 NSPCs。因此, 在成年斑马鱼脑中建立的淀粉样毒性模型可能为神经科学和临床医学领域的研究开辟新的途径。此外, 这种方法的简单执行, 允许 cost-effective 和有效的实验评估。这份手稿描述了 Aβ42肽的合成和注射到斑马鱼脑。

采用高效液相色谱法纯化了合成的多肽, 并利用质谱法对纯化的淀粉样β肽进行了表征。将 HPLC 柱加热至50° c, 以改善 a 肽的分离, 并收集所有的分数。为了识别正确的合成肽, 对所有的分数进行了质谱分析。UPLC 色谱显示了化合物的纯度。在 UPLC 上产生一个峰值的 HPLC 分数 (即, 所需的淀粉样β肽的正确质量比) 进一步处理实验 (图 2)。
在形成的聚合体中的多肽的功能可以使用各种光谱方法来评估39,48,49, 并通过在 PBS 室温下孵化多肽 1 h, 这将产量合计。在这种情况下, 应在溶液中看到沉淀物和大团聚体。
在脑室显微注射后, 多肽在大脑中聚集, 形成淀粉样蛋白沉积 (图 3)。这些聚集物主要被视为细胞内的证言, 但也在血管周围。这种聚合是在组织中成功积累淀粉样肽的迹象。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56014/56014fig1.jpg"> 
图 1: 多肽合成、纯化、注射和分析的示意图表示法.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56014/56014fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56014/56014fig2.jpg"> 
图 2: 本地 amyloid-β42肽的特性.原 amyloid-β42肽 (A) 和 CPP 标记的 amyloid-β42肽 (TR-Aβ42) 的质谱表征;B). (C) 在超高压液相色谱 (UPLC) 上纯化、原生和 TR-Aβ42肽的色谱。X 轴表示荷比率 (m/z) (A、 B) 和洗脱时间 (以分钟为单位) (C)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56014/56014fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56014/56014fig3.jpg"> 
图 3: 成人斑马鱼脑部淀粉样蛋白沉积的免疫组织化学染色1天后注射.Aβ42是绿色的, 原子核是蓝色的。检测绿色簇表示有效的 Aβ42聚合。缩放条形图 = 100 µm。此图已从引用39中进行了修改。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>肽</td>
			<td colspan="4">肽序列</td>
			<td>兆瓦 (克/摩尔)</td>
		</tr>
		<tr>
			<td>Aβ42</td>
			<td colspan="4">DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA</td>
			<td>4514。1</td>
		</tr>
		<tr>
			<td>TR-Aβ42</td>
			<td colspan="4">GWTLNSAGYLLGKINLKALAALAKKILDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA</td>
			<td>5919。8</td>
		</tr>
	</tbody>
</table>
表 1: Aβ42和 TR-Aβ42肽序列。

Watch this Scientific Journal Video about Modeling Amyloid-ÃŽÂ²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain at JoVE.com

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

本协议描述了单体 amyloid-β42肽在成年斑马鱼中产生淀粉样毒性的合成、表征和注射, 以建立阿尔茨海默病模型, 随后进行组织学分析和检测聚合.

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 ...

modeling-amyloid-42-toxicity-neurodegeneration-adult-zebrafish

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.1K 次观看.  German Centre for Neurodegenerative Diseases (DZNE) Dresden within Helmholtz Association. 本协议描述了单体 amyloid-β42肽在成年斑马鱼中产生淀粉样毒性的合成、表征和注射, 以建立阿尔茨海默病模型, 随后进行组织学分析和检测聚合.

视频: Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

在 C 在体内激光干切断线虫

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

科研

神经科学

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

与神经退化有关的成人 果 蝇的可视化突触变性

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

研究钝力损伤对成年斑马鱼影响的可扩展模型

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

穿梭盒测定作为使用成人斑马鱼进行学习和记忆研究认知评估的关联学习工具

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

摘要

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此视频中的章节

在 C 在体内激光干切断线虫

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

在完整的成年斑马鱼大脑电生理记录

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

成人脑切片长期抑郁症诱导评估

与神经退化有关的成人果蝇的可视化突触变性

研究钝力损伤对成年斑马鱼影响的可扩展模型

穿梭盒测定作为使用成人斑马鱼进行学习和记忆研究认知评估的关联学习工具

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

摘要

探索更多视频

此视频中的章节

在 C 在体内激光干切断线虫

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

在完整的成年斑马鱼大脑电生理记录

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

成人脑切片长期抑郁症诱导评估

与神经退化有关的成人 果 蝇的可视化突触变性

研究钝力损伤对成年斑马鱼影响的可扩展模型

穿梭盒测定作为使用成人斑马鱼进行学习和记忆研究认知评估的关联学习工具

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

与神经退化有关的成人果蝇的可视化突触变性