Name: a drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system
Uploaded: 2018-05-05T14:30:00
Description: Watch this Scientific Journal Video about A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System at JoVE.com

我们感谢杰西卡 Goldshteyn 的技术支持。宋实验室的工作由 NIH 赠款 R00NS088211 和智力和发展残疾研究中心 (IDDRC) 新项目发展奖资助。

1. 培养板和瓶的制备
<ol><li>葡萄汁琼脂板的研制<ol>
<li>加入10克琼脂粉, 200 毫升葡萄汁, 192 毫升 ddH2O 成烧杯和微波约4-5 分钟, 搅拌间歇, 直到琼脂完全溶解。</li>
		<li>在通风罩, 冷却的解决方案约60°c。加入4.2 毫升95% 乙醇和4.0 毫升的冰乙酸。将解决方案的总容量调整为400毫升, ddH2o 混合良好。</li>
		<li>对于每35毫米板, 增加约2-3 毫升的解决方案。制作大约120-150 片, 总共400毫升葡?...

<ol>
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在设置苍蝇十字架时, 使用的雌性和雄性的数量会因基因型和特定实验所需的幼虫数量而异。对于果蝇, 典型的十字架使用10女性和5男性。收集窗口可能会缩小, 这取决于需要的幼虫年龄的准确性。例如, 一个2小时的收集期将产生更均匀的种群幼虫。在这种情况下, 使用20或更多的处女雌性来建立十字架将有助于产生足够的鸡蛋。第一天的产量通常是稀缺的, 所以在设置房间后两天或更多的时间收集?...

在中枢神经系统受损后, 轴突无法再生, 可能会导致患者的永久性残疾, 并在神经退行性疾病中起到不可逆转的神经功能缺损的作用1,2 ,3,4,5。中枢神经系统的环境, 以及神经元的固有生长能力, 决定了神经轴突是否能够在创伤后再生。少突胶质、星形胶质和成纤维细胞源的细胞外因素已被证明阻碍神经元生长4,6,7, 8, 但这些分子的消除仅允许5的有限发芽。固有的再生信号可能影响再生成功5,9和代表潜在的治疗目标, 但这些过程仍然没有明确定义的分子水平。增加的营养因子信号或消除内源性制动器, 如 Pten 磷酸酶10, 可能导致轴突再生在某些情况下。发现单独有效的不同方法的组合也只提供有限的总体恢复到目前为止11,12,13,14。因此, 迫切需要确定有针对性治疗的其他途径。除了轴突再生的开始, 轴突是否和如何重新导线到正确的目标, 改革突触特异性, 并实现功能恢复是重要的悬而未决的问题。
总而言之, 目前对轴突再生机械的理解仍然是非常零碎的。问题的一部分是实时研究哺乳动物轴突再生的技术难点, 这种方法成本高昂, 耗时, 对进行大规模的基因筛有挑战性。另一方面,果蝇已被证明是研究复杂生物学问题的极其强大的系统。果蝇在定义基因和信号通路方面发挥了重要作用, 在人类中具有显著的保守性, 并且通过广泛的分子遗传学工具, 已经成为研究人类状况的成功模型, 如神经退行性疾病。可用于操作基因功能15。特别是, 果蝇被认为是发现涉及神经损伤和再生的基因的理想工具15,16。开发了几种飞行神经损伤模型, 包括成人头或幼虫腹侧神经线 (vnc) 刺针、幼虫 vnc 或神经粉碎、幼虫神经元激光切断、嗅受体神经元摘除、脑外植体损伤、和周围神经损伤由翼遣散15,17,18,19,20,21,22,23。扣人心弦, 最近利用果蝇损伤模型的工作提高了我们对神经系统用于神经损伤反应的细胞和基因通路的理解, 其中一些已被证明是在哺乳动物中保存的24 ,25。再次, 这强调了这个模型有机体的效用, 以确定新的机制, 神经修复。
这里描述的是一个双光子激光为基础的果蝇幼虫感官神经元损伤模型。在2003年的26中, 首次使用双光子激光在斑马鱼的体内切断轴突。同年, 第一个激光 dendriotomy 是在果蝇中使用脉冲氮气激光器27进行的。不久之后, 几个线虫实验室使用飞秒激光器建立轴突再生的模型28。在 2007年, 吴和同事们比较并报道了不同类型的激光器所诱导的激光损伤在C. 线虫中的差异29。在 2010年, 激光切断后的轴突再生首次显示在果蝇30中。在这一广泛的激光损伤文献的基础上, 我们开发了一种使用双光子激光器的苍蝇神经损伤模型, 它允许对靶点的损伤进行精确的诱导, 对邻近组织的微扰, 提供相对清洁的系统研究了单细胞分辨率 neuroregeneration 的内在和外在性质。具体来说, 我们已经建立了一套损伤方法的树突状树枝状 (da) 感官神经元的周围神经系统 (三七) 和中枢神经系统。Da 神经元可以分为四个不同的类, 主要由它们的枝晶分支复杂性区分: I 类到 IV31。我们发表的研究表明, 大神经元再生类似于表型和分子水平的哺乳动物损伤模型: 大神经元显示类特定的再生特性, 与 IV 类, 而不是类 I 或 III 大神经元表现再生在PNS;IV. 级神经元轴突在周围有强劲的再生, 但其再生潜能在中枢神经系统明显减少, 从而类似哺乳动物的背根神经节神经元;通过 Pten 删除或 Akt 过度表达增强 mTOR 活性, 增强了中枢神经系统中的轴突再生19。利用这一损伤模型, 我们一直在执行基因筛查, 并确定 RNA 处理酶 Rtca 作为轴突再生的进化保守抑制因子, 将轴突损伤与细胞应力和 RNA 修饰联系起来20.
在所提出的范式中, 损伤是通过激光切断/dendriotomy 的幼虫类 IV 或 III 大神经元, 分别标记为ppk-CD4-tdGFP或19-12-Gal4, UAS-CD4-tdGFP, repo-Gal80。在产卵 (h AEL) 后, 在2个nd到3个rd龄幼虫的 48-72 小时内进行损伤。对于切断, 病变是针对轴突的部分 20-50 µm 远离细胞体, 为中枢神经系统切断到一个面积20µm 直径在合生面交界的 VNC, 并 dendriotomy 到主要树枝状分支点。同一神经元在损伤后 8-24 小时 (ai) 被成像, 以确认完全的横断, 并在 48-72 h AI 评估再生。通过时间推移共焦成像, 可以随时监测在体内的单个轴突/树突受损的退化和再生。

<table border="0" cellpadding="0" cellspacing="0" width="919">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:444px;">Diethyl ether, ACS reagent, anhydrous</td>
			<td style="width:155px;">Acros Organics</td>
			<td style="width:153px;">AC615080010</td>
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			<td height="84" style="height:84px;width:444px;">Halocarbon 27 Oil</td>
			<td style="width:155px;">Genesee Scientific</td>
			<td style="width:153px;">59-133</td>
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			<td height="21" style="height:21px;">Phosphate buffered saline (PBS), 20x Concentrate, pH 7.5, supplier # E703-1L</td>
			<td style="width:155px;">VWR</td>
			<td>97062-948&#160;</td>
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			<td height="21" style="height:21px;">Agar powder, Alfa Aesar, 500GM</td>
			<td style="width:155px;">VWR</td>
			<td>AAA10752-36</td>
			<td></td>
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			<td height="21" style="height:21px;">Grape juice</td>
			<td>Welch&#8217;s</td>
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			<td height="21" style="height:21px;width:444px;">Ethanol 95% (Reagent Alcohol 95%)</td>
			<td style="width:155px;">VWR</td>
			<td style="width:153px;">64-17-5</td>
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			<td height="42" style="height:42px;width:444px;">Acetic acid</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:153px;">A6283</td>
			<td></td>
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			<td height="42" style="height:42px;width:444px;">Propionic Acid</td>
			<td style="width:155px;">J.T.Baker</td>
			<td style="width:153px;">U33007</td>
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			<td height="63" style="height:63px;width:444px;">Cover Glasses: Rectangles</td>
			<td style="width:155px;">Fisher Scientific</td>
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			<td>50 mm X 22 mm</td>
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			<td height="21" style="height:21px;width:444px;">Zeiss LSM 880 laser scanning microscope</td>
			<td style="width:155px;">Zeiss</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Zen software</td>
			<td style="width:155px;">Zeiss</td>
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			<td height="42" style="height:42px;width:444px;">Chameleon Ultra II</td>
			<td style="width:155px;">Coherent</td>
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</table>

a drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system

受损神经元的再生能力控制了神经系统损伤后的 neuroregeneration 和功能恢复。在过去的几十年中, 发现了影响轴突再生的各种内在和外在抑制因素。然而, 简单地去除这些抑制暗示是不够的成功再生, 表明存在的额外的管理机制。"果蝇" 是一种具有进化保守基因和信号通路的脊椎动物, 包括人类. 结合双光子激光切断/dendriotomy 的果蝇强大的遗传工具箱, 我们这里描述了果蝇感觉神经元-树突状树枝状 (da) 神经元损伤模型作为系统筛选的一个平台, 为新的再生调节器。简要地, 这个范例包括 a) 幼虫的准备, b) 使用双光子激光, c) 现场共聚焦成像后损伤和 d) 数据分析对枝晶 (s) 或轴突的损伤诱导。我们的模型能够在周围和中枢神经系统中使单个标记神经元、轴突和良好定义的神经元亚型的树突发生高度重复性损伤。

大脑神经元显示周围和中枢神经系统之间的差异再生潜能, 以及类特异性。这为筛查轴突再生所需的新因素提供了独特的机会 (使用 iv 类三七总损伤), 以及抑制再生 (iv. 类中枢神经系统损伤和 III. 类三七总损伤)。
在三七中的轴突再生
以 III. 类和 IV. 级大神经元的再生特性为例进行了?...

Watch this Scientific Journal Video about A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System at JoVE.com

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

在这里, 我们提出了一个协议使用果蝇感觉神经元-树突状树枝状 (da) 神经元损伤模型, 结合在体内活成像, 双光子激光切断/dendriotomy, 和强大的苍蝇遗传工具箱, 作为neuroregeneration 的潜在促进剂和抑制剂的筛选平台。

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

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A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

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