Name: a drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system
Uploaded: 2018-05-05T14:30:00.000+00:00
Duration: 9 min 55 s
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毫升葡萄汁琼脂溶液。</li>
		<li>等待10分钟冷却的盘子和固化琼脂溶液。将葡萄汁琼脂盘包装在自密封自封袋中, 贮存于4°c 内, 供日后使用。</li>
	</ol>
</li>
	<li>果蝇培养瓶的制备<ol>
<li>使用刀片在果蝇培养瓶的一堵墙上打一个1.5 厘米的侧长三角形孔, 并用 2-2. 5 厘米的棉花直径球来填充孔以供通风。</li>
		<li>用葡萄汁琼脂盘塞住瓶子, 补充约0.5 厘米的酵母膏3 。</li>
	</ol>
</li>
</ol>2. 收集果蝇幼虫
<ol><li>建立成人苍蝇的十字架<ol>
<li>放置10处女女性和5男性成年苍蝇一起在一个文化瓶插入一个葡萄汁琼脂板。</li>
		<li>将瓶子底部放在 25°c, 这样苍蝇就会在葡萄汁琼脂盘子上产卵。每天至少用酵母膏换一次盘子。在一个2小时的收集期内, 它允许收获一个均匀发育阶段的幼虫, 首先从20多名处女雌性和10只雄性2.1.1。</li>
		<li>在25°c 的60毫米培养皿中用湿纸巾培养盘子, 例如浸泡在0.5% 丙酸溶液中。排列组织, 使其不堵塞氧气流。 注: 丙酸溶液用于保持盘中的湿度, 避免霉菌的生长。</li>
	</ol>
</li>
	<li>收获特定年龄的幼虫<ol>
<li>用一对镊子将所需阶段的幼虫转移, 例如, 2nd到 3rd龄幼虫48-72 小时后产卵 (AEL), 到一个新的葡萄汁琼脂板没有酵母糊。</li>
		<li>通过让它们在新盘子上爬行来去除幼虫皮肤上的酵母, 以防止切断和成像对酵母的潜在干扰。或者, 通过在 PBS 的一碟中洗涤并在纸巾上简单的烘干, 彻底清洁幼虫。</li>
	</ol>
</li>
</ol>3. 双光子损伤与共焦成像
<ol><li>显微镜安装 注: 采用双光子激光共聚焦激光扫描显微镜进行了实验, 但其它具有同等设置的系统也就足够了。采用双光子激光 (930 nm) 进行损伤处理, 采用氩激光 (488 nm) 对 GFP 进行共焦成像。<ol>
<li>在每个会话开始时, 打开双光子激光器和/或共聚焦激光器, 以及显微镜。打开映像软件。</li>
		<li>对于双光子损伤, 为 930 nm (1950 兆瓦) 的双光子激光成像 GFP 设置以下参数。<ol>
<li>选择行扫描模式。把针孔打开将激光强度提高到 ~ 20% (390 兆瓦)。</li>
			<li>选择 512 x 512 作为帧扫描。使用最大扫描速度 (通常与像素停留时间在0.77 µs)。确保平均数字为 1, 位深度为8位。</li>
			<li>将增益设置为 ~ 750, 偏移量设为0。</li>
			<li>将此预置实验协议保存为2P GFP 930 消融, 以便在以后的实验中易于重用。</li>
		</ol>
</li>
		<li>为共焦成像, 设置以下参数的成像 GFP 与氩激光在 488 nm:<ol>
<li>选择购置选项卡, 然后单击Z 堆栈。</li>
			<li>在 "激光" 下, 打开 488 nm 氩激光器的电源。</li>
			<li>转到通道, 选择488毫米激光, 并将激光功率提高到5-10%。对于针孔, 使用1-2 通风单元 (AU)。调整增益为650。</li>
			<li>在捕获模式中, 选择 1024 x 1024 作为帧扫描, 使用最大扫描速度、平均数字2和位深度8位。</li>
			<li>将此预设置的实验协议保存为GFP 映像。</li>
		</ol>
</li>
	</ol></li>
	<li>乙基醚与安装的幼虫麻醉<ol>
<li>在通风罩中, 在15厘米的塑料培养皿中放置一个60毫米的玻璃盘子。折叠并放置一张纸巾在玻璃盘子的底部, 然后放置一个葡萄汁琼脂板上的组织。将二乙基醚加入到玻璃盘中, 放到纸巾被浸泡的地方, 盘子里还有一层液体醚。时刻保持盖子。</li>
		<li>用一滴卤化碳27油在中心准备一张玻璃滑梯。在幻灯片的四个角上添加4个真空润滑脂, 以后支持盖玻片。</li>
		<li>使用镊子捡起一个干净的幼虫, 把它放在60毫米的玻璃盘子的琼脂盘。用盖子盖上玻璃盘子, 等到幼虫停止移动。对于三七总损伤/成像, 一旦它的尾巴停止抽搐, 就取出幼虫。对于中枢神经系统, 等待, 直到整个幼虫变得静止, 特别是头段。 注意: 以太接触的时间是至关重要的。请参阅讨论。</li>
		<li>小心地拾起被麻醉的幼虫并且安置它头直立入卤化碳油下落在幻灯片。在幻灯片的顶部添加一个盖玻片。使用温和的压力向下推盖玻片, 直到它触及幼虫(图 1A)。</li>
		<li>调整幼虫的位置通过轻轻地推动盖玻片朝左或右滚动幼虫, 以便神经元/轴突/枝晶的兴趣是在顶部和最接近显微镜透镜。</li>
		<li>对于三七总损伤, 安装幼虫背侧向上, 使两个气管可见。然后将幼虫30度向左滚动, 以损伤 III. 级神经元轴突 (图 1B 和 1C), 90 度损伤 iv. 级神经元轴突 (图 1B 和 1E), 或30度损伤 iv. 大神经元树突 (图 2A)。</li>
		<li>对于中枢神经系统损伤, 将幼虫置于完全腹侧向上 (图 3A), 使感兴趣区域与 z 平面中的显微镜透镜最接近。</li>
	</ol></li>
	<li>双光子激光损伤<ol>
<li>将幻灯片与幼虫置于显微镜下, 并将其固定在舞台上的滑动架上。使用 10X (0.3 NA) 目的寻找幼虫。</li>
		<li>添加1滴目标油到盖玻片, 切换到 40X (1.3 NA) 目标, 调整焦点。</li>
		<li>切换到扫描模式并重用实验协议2P GFP 930 消融。确保针孔一直开着。 注意: 配置需要根据单个系统进行优化。</li>
		<li>启动Live模式以定位感兴趣的区域 (ROI), 并微调设置以使用适当的缩放来实现良好的图像质量。 注意: 这一步的目的是发现神经元/轴突/枝晶损伤, 而不是采取最佳质量的图像。因此, 使用足够的最小设置来可视化目标区域, 以避免过度曝光或漂白。</li>
		<li>停止实时扫描, 这样裁剪按钮将变为可用。让静止的图像作为路线图。选择裁剪函数并调整扫描窗口以将焦点放在要受伤害的目标上。</li>
		<li>减少投资回报率, 使之成为潜在伤害部位的大小。例如, 只要覆盖轴突或枝晶的宽度, 以确保伤害的准确性和减少对邻近组织的损害。如果需要, 在裁剪前放大 ROI, 从而更精确地伤害。</li>
		<li>打开一个新的图像窗口。降低扫描速度, 提高激光强度。根据在Live模式中扫描的组织荧光信号, 确定激光强度的增加。</li>
		<li>通常, 设置双光子激光强度从25% 开始为三七总伤害和50-100% 的 VNC 伤害。为保证轴突损伤, 确保激光强度为480兆瓦, 像素停留时间为8.19 微秒。对于 VNC 轴突损伤, 应确保激光强度和像素停留时间分别为965-1930 兆瓦和8.19-32.77 微秒。</li>
		<li>启动连续扫描。使光标悬停在连续按钮上。只要观察到荧光的急剧增加, 就能密切注视图像并停止扫描。 注: 荧光尖峰的出现是由于损伤部位的自动荧光引起的。</li>
		<li>通过重用设置切换回实时模式。通过调整焦点来找到感兴趣的区域。 注意: 一个很好的迹象表明, 成功的伤害是出现一个小火山口, 环形结构, 或局部碎片的权利在受伤地点。</li>
		<li>移动到下一个神经元并从步骤3.3.5 重复, 以伤害单个动物中的多个神经元。或重复步骤 3.3.5, 同时逐步增加功率和/或降低扫描速度, 如果最初的伤害是不够的。 注: 在激光功率过高的情况下, 在损伤后的实时扫描图像中会显示一个大面积的损坏区域。太多的伤害可能导致幼虫的死亡。</li>
		<li>通过仔细去除盖玻片并将受损的幼虫转移到新的盘子上, 以酵母膏来恢复幼虫。用镊子将琼脂盘上的几个洞穴沟上;或者, 在盘子里做一个琼脂岛, 而不是用整个盘子, 以减少幼虫爬出盘子的可能性。</li>
		<li>把盘子放在60毫米的培养皿中, 用湿纸巾 (浸泡0.5% 丙酸溶液), 在室温或25°c 时进行培养。 注意: 与25°c 相比, 幼虫在室温下将保持在幼虫阶段大约额外的一天 (22°C)。</li>
	</ol></li>
	<li>损伤后共聚焦成像<ol>
<li>在所需的时间点上想象受伤的幼虫, 用同样的麻醉和安装步骤 3.2, 然后用共焦激光进行成像, 准备幼虫。 注: 图像的幼虫在24小时后受伤 (AI) 确认轴突损伤和 48 h ai (IV 类大神经元) 或 72 h ai (III 类 da 神经元), 以评估再生。</li>
		<li>使用10X 目标定位幼虫, 然后切换到 25X (0.8 NA) 目标。重用实验性协议GFP 映像。</li>
		<li>单击实时按钮, 找到以前受伤的同一神经元。</li>
		<li>设置实时扫描窗口中的第一个和最后一个 Z 位置。按 "停止", 然后单击开始实验获取 Z 堆栈图像。 注意: 要确保在捕获图像时包括一个规范化点 (轴突汇合点), 以便可以量化再生 (图 1D, 1F)--在 "数据分析" 部分将进一步讨论这一点。</li>
		<li>切换到图像处理, 选择刚刚拍摄的图像, 并生成最大强度投影。同时保存 z 堆栈和最大强度投影图像。</li>
	</ol></li>
</ol>4. 数据分析
<ol><li>使用图像软件或 ImageJ 对图像进行处理和量化。</li>
	<li>三七中轴突再生的量化研究<ol>
<li>计算再生百分比, 它指的是损伤的所有轴突中再生轴突的百分比。regrows 轴突再生, 只要它超出了受伤部位。</li>
		<li>测量再生长度, 这是轴突长度的增加。如果用 ImageJ 进行量化, 请使用分段行工具跟踪再生轴突, 并在分析下拉菜单中使用度量以获取表示再生轴突的行的长度。</li>
		<li>计算再生指数, 即规范化轴突长度的增加。 注: 轴突长度由细胞体与轴突汇合点 (神龙公司)-轴突长度/神龙公司(图 1D和1F) 之间的距离正常化。这个值有助于解释任何由于幼虫结垢而引起的轴突生长。正值表示再生, 0的值表示不再生, 负值表示撤回。</li>
	</ol>
</li>
	<li>枝晶再生的量化<ol>
<li>计算再生率, 这是在所有那些显示明显的枝晶再生的细胞中 da 神经元的百分比。 注: 如果新的树突从收回的树枝状茎和损伤部位外再生, 枝晶的再生是阳性的。损伤部位是由地标决定的, 在某些情况下, 由于残余损伤诱发的自体荧光, 容易看到。</li>
		<li>计算分枝点的增加, 它统计损伤后新的树枝状分支点的添加。</li>
		<li>计算总枝晶长度的增加, 这是损伤后添加的所有新树突的累积长度。</li>
	</ol>
</li>
	<li>中枢神经系统轴突再生的量化研究 注: 如果病灶部位在第一次成像时显示变性 (图 3B), 则将包括在再生长度和速率的分析中。<ol>
<li>测量再生长度, 这是再生轴突的长度。 注意: 单个病变部位的再生轴突是在损伤前从原轴突路线开始的。</li>
		<li>计算规范化再生长度, 它将再生长度正常化为合生面段的长度, 即图3A 和3B 中 commissures ("Y") 之间的纵向距离. 注意: 这个值有助于纠正幼虫大小差异的影响。</li>
		<li>计算再生率, 它是损伤中所有段中再生段的百分比, 以反映特定基因型的再生能力。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Yakura, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Recovery following spinal cord injury. , (1996).</li><li>Harel, N. Y., Strittmatter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+regenerating+axons+recapitulate+developmental+guidance+during+recovery+from+spinal+cord+injury?.">Can regenerating axons recapitulate developmental guidance during recovery from spinal cord injury?.</a> Nature reviews. Neuroscience. 7, 603-616 (2006).</li><li>Jurewicz, A., Matysiak, M., Raine, C. S., Selmaj, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+Nogo-A,+an+inhibitor+of+axonal+regeneration,+as+a+biomarker+for+multiple+sclerosis.">Soluble Nogo-A, an inhibitor of axonal regeneration, as a biomarker for multiple sclerosis.</a> Neurology. 68, 283-287 (2007).</li><li>Yiu, G., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+inhibition+of+CNS+axon+regeneration.">Glial inhibition of CNS axon regeneration.</a> Nat Rev Neurosci. 7, 617-627 (2006).</li><li>Sun, F., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+intrinsic+barriers+for+axon+regeneration+in+the+adult+CNS.">Neuronal intrinsic barriers for axon regeneration in the adult CNS.</a> Curr Opin Neurobiol. , (2010).</li><li>Liu, B. P., Cafferty, W. B., Budel, S. O., Strittmatter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+regulators+of+axonal+growth+in+the+adult+central+nervous+system.">Extracellular regulators of axonal growth in the adult central nervous system.</a> Philos Trans R Soc Lond B Biol Sci. 361, 1593-1610 (2006).</li><li>Liu, K., Tedeschi, A., Park, K. K., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+intrinsic+mechanisms+of+axon+regeneration.">Neuronal intrinsic mechanisms of axon regeneration.</a> Annu Rev Neurosci. 34, 131-152 (2011).</li><li>Schwab, M. E., Strittmatter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nogo+limits+neural+plasticity+and+recovery+from+injury.">Nogo limits neural plasticity and recovery from injury.</a> Curr Opin Neurobiol. 27, 53-60 (2014).</li><li>He, Z., Jin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+Control+of+Axon+Regeneration.">Intrinsic Control of Axon Regeneration.</a> Neuron. 90, 437-451 (2016).</li><li>Park, K. K., 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=Promoting+axon+regeneration+in+the+adult+CNS+by+modulation+of+the+PTEN/mTOR+pathway.">Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway.</a> Science. 322, 963-966 (2008).</li><li>Geoffroy, C. G., Hilton, B. J., Tetzlaff, W., Zheng, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+Age-Dependent+Decline+in+Axon+Regeneration+in+the+Adult+Mammalian+Central+Nervous+System.">Evidence for an Age-Dependent Decline in Axon Regeneration in the Adult Mammalian Central Nervous System.</a> Cell Rep. 15, 238-246 (2016).</li><li>Geoffroy, C. G., 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+PTEN+and+Nogo+Codeletion+on+Corticospinal+Axon+Sprouting+and+Regeneration+in+Mice.">Effects of PTEN and Nogo Codeletion on Corticospinal Axon Sprouting and Regeneration in Mice.</a> J Neurosci. 35, 6413-6428 (2015).</li><li>Jin, D., 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=Restoration+of+skilled+locomotion+by+sprouting+corticospinal+axons+induced+by+co-deletion+of+PTEN+and+SOCS3.">Restoration of skilled locomotion by sprouting corticospinal axons induced by co-deletion of PTEN and SOCS3.</a> Nat Commun. 6, 8074 (2015).</li><li>Wang, X., 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=Axonal+regeneration+induced+by+blockade+of+glial+inhibitors+coupled+with+activation+of+intrinsic+neuronal+growth+pathways.">Axonal regeneration induced by blockade of glial inhibitors coupled with activation of intrinsic neuronal growth pathways.</a> Exp Neurol. 237, 55-69 (2012).</li><li>Fang, Y., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+degeneration+and+regeneration:+insights+from+Drosophila+models+of+nerve+injury.">Axon degeneration and regeneration: insights from Drosophila models of nerve injury.</a> Annual review of cell and developmental biology. 28, 575-597 (2012).</li><li>Venken, K. J., Simpson, J. H., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+genes+and+cells+in+the+nervous+system+of+the+fruit+fly.">Genetic manipulation of genes and cells in the nervous system of the fruit fly.</a> Neuron. 72, 202-230 (2011).</li><li>Leyssen, M., 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=Amyloid+precursor+protein+promotes+post-developmental+neurite+arborization+in+the+Drosophila+brain.">Amyloid precursor protein promotes post-developmental neurite arborization in the Drosophila brain.</a> The EMBO journal. 24, 2944-2955 (2005).</li><li>MacDonald, J. M., 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+Drosophila+cell+corpse+engulfment+receptor+Draper+mediates+glial+clearance+of+severed+axons.">The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons.</a> Neuron. 50, 869-881 (2006).</li><li>Song, Y., 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=Regeneration+of+Drosophila+sensory+neuron+axons+and+dendrites+is+regulated+by+the+Akt+pathway+involving+Pten+and+microRNA+bantam.">Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam.</a> Genes Dev. 26, 1612-1625 (2012).</li><li>Song, Y., 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=Regulation+of+axon+regeneration+by+the+RNA+repair+and+splicing+pathway.">Regulation of axon regeneration by the RNA repair and splicing pathway.</a> Nat Neurosci. 18, 817-825 (2015).</li><li>Kato, K., Forero, M. G., Fenton, J. C., Hidalgo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+regenerative+response+to+central+nervous+system+injury+is+enabled+by+pros-notch+and+pros-NFkappaB+feedback.">The glial regenerative response to central nervous system injury is enabled by pros-notch and pros-NFkappaB feedback.</a> PLoS Biol. 9, e1001133 (2011).</li><li>Fang, Y., Soares, L., Teng, X., Geary, M., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Drosophila+model+of+nerve+injury+reveals+an+essential+role+of+Nmnat+in+maintaining+axonal+integrity.">A novel Drosophila model of nerve injury reveals an essential role of Nmnat in maintaining axonal integrity.</a> Curr Biol. 22, 590-595 (2012).</li><li>Xiong, X., 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=Protein+turnover+of+the+Wallenda/DLK+kinase+regulates+a+retrograde+response+to+axonal+injury.">Protein turnover of the Wallenda/DLK kinase regulates a retrograde response to axonal injury.</a> J Cell Biol. 191, 211-223 (2010).</li><li>Brace, E. J., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+axon+regeneration+in+Drosophila.">Models of axon regeneration in Drosophila.</a> Exp Neurol. 287, 310-317 (2017).</li><li>Hao, Y., Collins, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+mechanisms+for+axon+regeneration:+insights+from+injured+axons+in+Drosophila.">Intrinsic mechanisms for axon regeneration: insights from injured axons in Drosophila.</a> Curr Opin Genet Dev. 44, 84-91 (2017).</li><li>Galbraith, J. A., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+damage+in+thick+specimens+by+multiphoton+excitation.">Controlled damage in thick specimens by multiphoton excitation.</a> Mol Biol Cell. 14, 1808-1817 (2003).</li><li>Sugimura, K., 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=Distinct+developmental+modes+and+lesion-induced+reactions+of+dendrites+of+two+classes+of+Drosophila+sensory+neurons.">Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.</a> J Neurosci. 23, 3752-3760 (2003).</li><li>Yanik, M. F., 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=Neurosurgery:+functional+regeneration+after+laser+axotomy.">Neurosurgery: functional regeneration after laser axotomy.</a> Nature. 432, 822 (2004).</li><li>Wu, Z., 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=Caenorhabditis+elegans+neuronal+regeneration+is+influenced+by+life+stage,+ephrin+signaling,+and+synaptic+branching.">Caenorhabditis elegans neuronal regeneration is influenced by life stage, ephrin signaling, and synaptic branching.</a> Proc Natl Acad Sci U S A. 104, 15132-15137 (2007).</li><li>Stone, M. C., Nguyen, M. M., Tao, J., Allender, D. L., Rolls, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+up-regulation+of+microtubule+dynamics+and+polarity+reversal+during+regeneration+of+an+axon+from+a+dendrite.">Global up-regulation of microtubule dynamics and polarity reversal during regeneration of an axon from a dendrite.</a> Mol Biol Cell. 21, 767-777 (2010).</li><li>Grueber, W. B., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tiling+of+the+Drosophila+epidermis+by+multidendritic+sensory+neurons.">Tiling of the Drosophila epidermis by multidendritic sensory neurons.</a> Development. 129, 2867-2878 (2002).</li><li>Kang, H., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+axon+regeneration+and+muscle+reinnervation+in+young+adult+and+aged+animals.">Motor axon regeneration and muscle reinnervation in young adult and aged animals.</a> J Neurosci. 33, 19480-19491 (2013).</li><li>Duan, X., 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=Subtype-specific+regeneration+of+retinal+ganglion+cells+following+axotomy:+effects+of+osteopontin+and+mTOR+signaling.">Subtype-specific regeneration of retinal ganglion cells following axotomy: effects of osteopontin and mTOR signaling.</a> Neuron. 85, 1244-1256 (2015).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22, 451-461 (1999).</li><li>Grueber, W. B., 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=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Buss, A., 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=NG2+and+phosphacan+are+present+in+the+astroglial+scar+after+human+traumatic+spinal+cord+injury.">NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury.</a> BMC Neurol. 9, 32 (2009).</li><li>McKeon, R. J., Jurynec, M. J., Buck, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chondroitin+sulfate+proteoglycans+neurocan+and+phosphacan+are+expressed+by+reactive+astrocytes+in+the+chronic+CNS+glial+scar.">The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar.</a> J Neurosci. 19, 10778-10788 (1999).</li><li>Raabe, I., Vogel, S. K., Peychl, J., Tolic-Norrelykke, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+nanosurgery+and+cell+enucleation+using+a+picosecond+laser.">Intracellular nanosurgery and cell enucleation using a picosecond laser.</a> J Microsc. 234, 1-8 (2009).</li><li>Hutson, M. S., Ma, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+and+cavitation+dynamics+during+pulsed+laser+microsurgery+in+vivo.">Plasma and cavitation dynamics during pulsed laser microsurgery in vivo.</a> Phys Rev Lett. 99, 158104 (2007).</li><li>Venugopalan, V., Guerra, A., Nahen, K., Vogel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laser-induced+plasma+formation+in+pulsed+cellular+microsurgery+and+micromanipulation.">Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation.</a> Phys Rev Lett. 88, 078103 (2002).</li><li>Bourgeois, F., Ben-Yakar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtosecond+laser+nanoaxotomy+properties+and+their+effect+on+axonal+recovery+in+C.+elegans.">Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans.</a> Opt Express. 16, 5963 (2008).</li><li>O'Brien, G. S., 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=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. , (2009).</li><li>Tsai, P. S., 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=Plasma-mediated+ablation:+an+optical+tool+for+submicrometer+surgery+on+neuronal+and+vascular+systems.">Plasma-mediated ablation: an optical tool for submicrometer surgery on neuronal and vascular systems.</a> Curr Opin Biotechnol. 20, 90-99 (2009).</li><li>Chung, S. H., Clark, D. A., Gabel, C. V., Mazur, E., Samuel, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+AFD+neuron+in+C.+elegans+thermotaxis+analyzed+using+femtosecond+laser+ablation.">The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation.</a> BMC Neurosci. 7, 30 (2006).</li><li>Williams, W., Nix, P., Bastiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+a+low-budget+laser+axotomy+system+to+study+axon+regeneration+in+C.+elegans.">Constructing a low-budget laser axotomy system to study axon regeneration in C. elegans.</a> J Vis Exp. , (2011).</li></ol>

作者没有什么可透露的。

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盘子。在进一步培养鸡蛋的盘子时, 建议在0.5% 丙酸溶液中浸泡60毫米培养皿中的组织, 而不是水, 这不仅有助于保持盘子里的湿度, 还能避免霉菌的生长。
在设置幼虫麻醉和安装装置时, 一定要使用玻璃器皿, 因为乙醚会通过塑料熔化。玻璃碟被放置在一个15厘米的塑料培养皿, 以防泄漏。纸巾有助于在盘子里保存乙醚, 这样幼虫就能被以太蒸气有效地击出。建议使用琥珀滴瓶子储存整除数醚, 并在水滴中加入乙醚。补充醚, 并始终保持一层的液体醚在底部的菜, 以最佳效果。
醚暴露的时间是至关重要的: 麻醉下会导致幼虫在成像过程中恢复, 图像不稳;麻醉过量, 或直接接触乙醚液体, 将导致致死。为了最大限度地生存和成功率, 我们的经验法则如下: 对于三七总损伤/成像, 一旦它的尾巴停止抽搐, 立即取出幼虫;对于中枢神经系统, 等待, 直到整个幼虫变得静止, 特别是头段。即使是轻微的运动也会干扰 VNC 轴突的损伤和成像。年长的幼虫往往要花更长的时间才能敲开。它通常需要少于2分钟的幼虫小于72小时 AEL 和2-5 分钟的幼虫比 72 h AEL。
在设置双光子激光的损伤强度时, 该值由 "活" 扫描得到的组织荧光信号决定。在步骤3.3 中, "连续" 扫描引入了损伤。损伤诱发荧光的增加是由于损伤部位的自体荧光作用, 是突起的一个很好的指标。通常, 它需要1-10 秒才能看到荧光尖峰。一旦荧光升高, 控制扫描时间是至关重要的。对于三七总损伤, 必须立即停止扫描。长期暴露会扩大损伤部位, 损害邻近组织。但是, 这确实提供了调整扫描时间和操作伤害严重性的机会。一个成功的伤害应该有一个小于3-4 微米的病变大小直径。对于 vnc 轴突损伤, vnc 轴突的嵌入比大脑神经轴突/树突, 在皮肤下方更深, 因此需要更高的激光强度。我们通常会让扫描继续几秒钟。这是为了确保整个轴突束被切断。如果病灶部位的半径超过合生面束宽度的一半, 此类损伤部位被算作不成功。这种幼虫的存活率很低, 不会被纳入分析中。
对于轴突再生, 再生能力在幼虫阶段是相似的。但对于单枝晶切后的枝晶再生, 在 72 h AEL19后, 再生电位减少。因此, 轴突损伤通常表现为 48 h-72 h AEL 和枝晶损伤 48h AEL, 并评估再生在 120h AEL。幼虫 24h AEL 也可以使用, 但他们需要更仔细的处理, 鉴于其较小的大小。幼虫蛹后120小时 AEL, 使成像更具挑战性。因此, 我们的端点通常是 120 h AEL。
退化和再生之间的相互关系是什么？对于三七总损伤, 在 24 h AI, 通常的远端轴突/枝晶在小波已经完成沃勒变性, 而再生尚未开始在这个时间点。因此, 我们认为, 在野生幼虫中, 断突起的变性只对再生有很有限的影响, 如果是的话。对于 VNC 损伤, 8 h ai 的轴突已经开始退化, 在 24 h ai, 轴突再生被观察, 而轴突碎片仍然可以发现周围的伤害地点。碎片似乎并没有阻止再生。然而, 在某些情况下, 退化和再生之间可能存在重叠甚至串扰。事实上, 据报道, 在老龄小鼠, 周围神经损伤后的碎片清除比年轻动物慢。伴随地, 神经肌肉结的慢神经被观察, 可能归因于更多障碍再生轴突遇见在老动物。然而, 令人惊讶的是, 年老动物的轴突在不面临碎片32的情况下快速再生和 reinnervate 神经肌肉结点。这表明, 促进碎片清除可能是促进再生的一个潜在战略。
与其他 neuroregeneration 模型相比, 蝇感官神经元损伤模型具有独特的优越性。老鼠模型通常需要数周到几个月的时间来执行, 不适合进行大规模的基因筛;线虫只有一个原始的中枢神经系统, 它可能不会密切重述哺乳动物中枢神经再生障碍;与哺乳动物不同, 斑马鱼中枢神经系统的轴突再生强劲。苍蝇的快速生命周期、飞行遗传学的多样性、飞行感官神经元的轴突/树突的可及性和定型模式, 以及飞行感官神经元的特征再生特性--亚型特异性再生在中枢神经系统中的三七和有限的再生-使果蝇da 神经元成为研究 neuroregeneration 的一个有吸引力的模型。此外, 最近的研究从粉碎小鼠视网膜神经节细胞 (RGCs) 也表明, 神经元亚型具有明显的再生能力;一些研资局子类型可以再生, 而在看似均匀的神经丛中的其他子种不能再生33。这项重要的发现表明, 应利用神经元型特定的策略来促进再生和功能恢复, 并强烈认为轴突损伤的诱导和后续的再生分析应在神经元子类型特定的方式。此外, 这种再生类型特异性的细胞和分子决定因素主要仍然是未知的19,33。因此, 大神经元损伤模型为解决这些问题提供了理想的范例。
与轴突再生相比, 聚焦于枝晶再生的研究更为稀缺。枝晶损伤确实发生, 如外伤性脑损伤, 中风, 和多种形式的变性, 但几乎没有什么是已知的树突的能力修复和改革神经连接。da 神经元损伤模型再次提供了一个高度可访问的系统, 显示定型模式, 类特异性和时间调节19, 以探索这个方向。
还值得一提的是, 虽然有可能损害的标签单轴突在期票, 在中枢神经系统中的伤害是执行的方式导致损伤的一束轴突。如果需要, MARCM34或 FLP 克隆35方法可用于标记 CNS 中的单个轴突。另外, 当胶质细胞同时标记为 mRFP (Repo-Gal4, 无人 mRFP) 时, 在病变部位特别观察到胶质瘤的堆积过程。此外, Ptp99A 的表达, 同源软骨素多糖 (CSPG) phosphacan/PTPRZ1 的苍蝇, 在病变部位进行了调节。Ptp99A 与胶质细胞本地化并包围损伤部位, 形成类似于哺乳动物的星形胶质疤痕报告的环状结构19, 36, 37.总之, 大神经元损伤模型, 当结合其他细胞类型的标记, 如胶质细胞或免疫细胞, 将允许体内实时监测受损神经元与周围的多细胞相互作用环境。
虽然这种蝇幼虫感官神经元损伤模型为我们提供了机会找到潜在的 neuroregeneration 监管者在期票和中枢神经系统, 它仍然有几个限制。第一, 现阶段的吞吐量并不高。通常, 5-6 基因型可以在一周内被一个人筛查。它需要进行优化, 以执行一个无偏见的屏幕。第二, 由于乙醚对幼虫的麻醉可以从几分钟持续到不超过二十分钟, 因此对长期成像并不理想。因此, 选择具有代表性的轴突退化和再生的特定时间点进行成像。第三, 尽管该协议正在引入一种精确损伤轴突的方法, 但不能完全排除对周围组织造成损伤的可能性。在 VNC 中, 这些组织可以是胶质细胞、轴突和其他神经元的树突。为了尽量减少这一潜在的警告, 我们尽量减少伤害部位, 尽可能应用最低的激光功率, 并同时与控制和实验组执行相同的程序。第四, 在建立中枢神经系统损伤范式的过程中, 对不同的损伤部位进行了测试, 包括接近轴突终点的部位以及我们选择使用的轴突进入 VNC 的入口点。进入点被发现更难伤害, 因为他们在组织更深, 更有可能移动。因此, 由于这些实际的原因, 我们选择了目前的方法, 这是更一致的, 更好的控制, 并有良好的大规模实验。如上文所述, 一个潜在的问题是可能伤害邻近组织, 如突触后的部分和胶质细胞。另一方面, 损伤周围组织对轴突退化和再生的影响是一个突出的问题。例如, 胶质疤痕如何影响轴突再生。这也是为什么我们的损伤模型可能与哺乳动物的损伤模型紧密相似的另一个原因, 在这种模式下, 病灶周围的轴突和组织通常同时受伤。
激光损伤后的时间推移显微镜是研究轴突/枝晶再生的敏感方法。然而, 这一分析的主要关注是感知成本, 从 < $ 1万的低端固态脉冲激光器, $ 25-100K 为飞秒激光器到 > $ 10万的双光子激光器。对于用于切断29、38、39、40、41、42、43、的不同激光系统, 有几个好的讨论44,45. 综上所述, 常规激光器是最佳的切割轴突在大约30-50 µm 从表面。与飞秒激光器相比, 纳米和皮秒激光器的间接损伤会更严重, 特别是当目标区域的深度增加45时。对于 VNC 中的轴突损伤, 其深度通常约为50-100 µm, 这对于减少组织损伤至关重要。在这种情况下, 双光子激光器是理想的, 它把激光功率聚焦到焦平面上, 减少副组织损伤而不损害组织穿透。总之, 双光子系统成本高昂, 但提供了最佳的精确度和组织保存。然而, 如果只有切断的信号轴突是目标, 传统的脉冲激光器可能是一个更实惠的选择。

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><tbody><tr><td>Diethyl ether, ACS reagent, anhydrous</td><td>Acros Organics</td><td>AC615080010</td><td></td></tr><tr><td>Halocarbon 27 Oil</td><td>Genesee Scientific</td><td>59-133</td><td></td></tr><tr><td>Phosphate buffered saline (PBS), 20x Concentrate, pH 7.5, supplier # E703-1L</td><td>VWR</td><td>97062-948&amp;nbsp;</td><td></td></tr><tr><td>Agar powder, Alfa Aesar, 500GM</td><td>VWR</td><td>AAA10752-36</td><td></td></tr><tr><td>Grape juice</td><td>Welch&amp;rsquo;s</td><td></td><td></td></tr><tr><td>Ethanol 95% (Reagent Alcohol 95%)</td><td>VWR</td><td>64-17-5</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>A6283</td><td></td></tr><tr><td>Propionic Acid</td><td>J.T.Baker</td><td>U33007</td><td></td></tr><tr><td>Cover Glasses: Rectangles</td><td>Fisher Scientific</td><td>12-544-D</td><td>50 mm X 22 mm</td></tr><tr><td>Zeiss LSM 880 laser scanning microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Zen software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Chameleon Ultra II</td><td>Coherent</td><td></td><td></td></tr></tbody></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描述。这些神经元位于每一个身体段的双边。在同一幼虫中, 多神经元可能受伤;一般情况下, 腹部部分右侧3-4 神经元 A7-A2。第三类和 IV 类 da 神经元可以由19-12-Gal4、UAS-CD4tdGFP、repo-Gal80和ppk-CD4tdGFP分别可视化。麻醉和芒 48-72 h AEL 幼虫, 如所述, 调整幼虫的位置, 使感兴趣的 da 神经元朝上 (图 1A和1B)。我们通常会伤害 III 类 ddaF 和 IV. ada 神经元 (图 1C和1E)。执行切断和恢复幼虫如所述。在受伤后不久 (AI), 幼虫将从手术中恢复, 并表现正常的运动。这里的生存率通常超过80%。丢弃死亡或生病的幼虫。重新装上所描述的余数并评估退化。在 24 h AI, 远端轴突应该已经完成退化在这个时间点19, 轴突茎将容易地可见 (图 1D和1F)。重新映像的幼虫为 IV. 级神经元的 48 h ai 或 72 h ai, 用于评估再生。我们通常的目的是评估至少20个受伤的神经元每个实验条件。在 (野生型) 动物中, 而70% 的被切断的 IV 级神经元会在损伤部位 (图 1F) 中再生, 第三类大神经元无法再生, 这是由生长锥停滞 (图 1D) 证明的。
枝晶再生
我们通常在 IV 级大神经元 ddaC 上执行 dendriotomy (图 2A)。如原理图所示, 损伤是针对主树枝状点。根据经验, 当受伤在48小时 AEL, 50% 的 ddaC 神经元再生他们的树突 (图 2B)。在余下的 50%, 相邻的树突侵入和覆盖空置空间。此外, 如果在后期发育阶段受伤, 这些神经元的再生潜能就会减少。
中枢神经系统的轴突再生
对于 VNC 轴突损伤, 幼虫的存活率变化很大, 取决于损伤的年龄。根据经验, 48-72 h AEL 的幼虫在不同的试验阶段中存活率最高 (> 60%)。小于48小时 AEL 的幼虫在受伤后生存不良, 而在72小时以上的 AEL, 在 VNC 中很难引入损伤。此外, 在后合生面段引起的损伤比前部更容易诱发, 因为这些后段的 VNC 更接近腹面, 从而更容易被激光 (图3A) 所吸引。
为了在中枢神经系统损伤 IV. 大神经元轴突, 装上前面描述的幼虫 (图 3A)。在显微镜下, 找到构成 VNC (图 3A) 一部分的轴突丛的阶梯状结构, 并按照所述执行切断。变性被证实在8小时 ai 和再生被评估在24和 72 h ai。如图 3B所示, 8 h ai 的轴突已经开始退化, 并且在 24 h ai 上观察到轴突再生, 而在损伤部位周围仍然可以找到轴突碎片。在 VNC 中, 小波轴突显示有限的再生, 无法重新连接损伤产生的间隙 (图 3B)。为了量化损伤后轴突的再生能力, 测量受伤后再生的长度, 并将合生面段的长度 (Y in 图3A) 用于规范化 (图3C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57557/57557fig1.jpg"> 图 1: 大脑神经轴突再生在外围显示类特异性。(A 和 B)显示幼虫位置的示意图。(C)第三类 da 神经元的示意图。(D)第三类 da 神经元 ddaF 的轴突, 标记为19-12-Gal4、UAS-CD4-tdGFP、repo-Gal80/+, 无法再生。(E) IV 类 da 神经元示意图。(F) IV 类 da 神经元 v ' ada 的轴突, 标记为ppk-CD4-tdGFP/+, 在病灶部位以外再生。(D 和 F)红线表示轴突长度, 而绿色虚线标志着细胞体与轴突汇合点 (神龙公司) 之间的距离。蓝点标志着轴突汇合点。缩放条 = 20 µm.<a href="/files/ftp_upload/57557/57557fig1large.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/57557/57557fig2.jpg"> 图 2: Da 神经元枝晶再生。(A) IV 类 da 神经元的说明性表示。(B)在 IV. da 神经元 ddaC 中的枝晶再生的说明性表示, 由ppk-CD4-tdGFP/+标记。激光消融是针对初级分枝点, 并进行48小时 AEL。在24小时的突起损伤横断被证实, 并且在 72 h ai 再生是定量的。ddaC 神经元的树突表现出大量的再生, 新的树枝状树枝从断茎处发芽, 以平铺空置的空间。值得注意的是, 新的终端分支不断添加到未受伤的树突在这个发育阶段。缩放条 = 20 µm.<a href="/files/ftp_upload/57557/57557fig2large.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/57557/57557fig3.jpg"> 图 3: VNC 中的大神经元轴突再生。(a)一个果蝇幼虫安装在幻灯片上并在显微镜下成像的示意图。在ppk-CD4tdGFP/+幼虫中可视化的 VNC 中的 IV 类 da 神经元轴突。放大图像和示意图中显示两个候选合生面段。他们每人有两个受伤地点 (红色圆圈)。(B) 8、24和 72 h 在受伤后 (AI) 中的一个受伤片段的共焦图像。红线描绘了再生轴突。(C)再生轴突的测量和规范化。缩放条 = 20 µm.<a href="/files/ftp_upload/57557/57557fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

用于研究周围和中枢神经系统神经再生的果蝇体内损伤模型医学|视频

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

Human Biology

Nervous System

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9.7K 次观看.  The Children's Hospital of Philadelphia. 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。

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

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

科研

神经科学

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

生物工程

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

发育生物学

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

一个简单的神经元机械损伤方法学研究<em&gt;果蝇</em&gt;运动神经元变性

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三星果蝇中枢神经系统活动的电生理记录

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

刺激和分析 果蝇 中枢脑中的成人神经发生

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

Two-Photon Laser-Induced Neural Injury: A Method to Observe Axon Degeneration and Regeneration in Drosophila Larvae

Source: Li, D., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/57557/" target="_blank">A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System</a>. J. Vis. Exp. (2018).
This video describes two-photon laser ablation of axons in a Drosophila melanogaster larva and an example protocol to induce injury and image the injury site.

双光子激光诱导的神经损伤:一种观察果蝇幼虫轴突变性和再生的方法

Source: Li, D., et al. A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System. J. Vis. Exp. 
...

JoVE Encyclopedia of Experiments

生物学

黑腹果蝇（果蝇）

幼虫

Inducing Targeted Neuronal Injury Using a High-Power Laser in a Drosophila Larva

Source: Li, D., Li, F., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57557">A Drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system.</a> J. Vis. Exp. (2018)
This video demonstrates a method for inducing targeted neuronal injury in Drosophila larvae using a high-power laser. After positioning the larva under a microscope, a low-power laser scans for fluorescent neurons, and then the laser power is increased to induce localized damage. The injury is confirmed by increased fluorescence and a crater at the injury site.

Source: Li, D., Li, F., et al. A Drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system. J. Vis. Exp. ...

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

摘要

探索更多视频

此视频中的章节

单果蝇 Ommatidium解剖和影像

使用微流体芯片实时成像和损伤研究的响应

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

调查中枢神经系统修复中受伤范式

一个简单的神经元机械损伤方法学研究

第三星果蝇中枢神经系统活动的电生理记录

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

刺激和分析果蝇中枢脑中的成人神经发生

双光子激光诱导的神经损伤:一种观察果蝇幼虫轴突变性和再生的方法

Inducing Targeted Neuronal Injury Using a High-Power Laser in a Drosophila Larva

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

摘要

探索更多视频

此视频中的章节

单果蝇 Ommatidium解剖和影像

使用微流体芯片实时成像和损伤研究的响应

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

调查中枢神经系统修复中受伤范式

一个简单的神经元机械损伤方法学研究

第三星果蝇中枢神经系统活动的电生理记录

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

刺激和分析 果蝇 中枢脑中的成人神经发生

双光子激光诱导的神经损伤:一种观察果蝇幼虫轴突变性和再生的方法

Inducing Targeted Neuronal Injury Using a High-Power Laser in a Drosophila Larva

刺激和分析果蝇中枢脑中的成人神经发生