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>

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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?...

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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar powder, Alfa Aesar, 500GM</td>
			<td style="width:155px;">VWR</td>
			<td>AAA10752-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Grape juice</td>
			<td>Welch&#8217;s</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:444px;">Cover Glasses: Rectangles</td>
			<td style="width:155px;">Fisher Scientific</td>
			<td style="width:153px;">12-544-D</td>
			<td>50 mm X 22 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Zeiss LSM 880 laser scanning microscope</td>
			<td style="width:155px;">Zeiss</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Zen software</td>
			<td style="width:155px;">Zeiss</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:444px;">Chameleon Ultra II</td>
			<td style="width:155px;">Coherent</td>
			<td style="width:153px;"></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?...

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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JoVE Journal

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Biology

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

Human Biology

Nervous System

SCIENTISTS IN ACTION

9.6K 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

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

科研

医学

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

在的鼠标Aortocaval瘘的技术方面

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

缺氧缺糖再给氧作为<em&gt;在体外</em&gt;缺血再灌注损伤模型的研究血脑屏障功能障碍

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

重复震荡颅脑损伤模型小鼠

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导:"电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

跟踪超副磁性氧化铁标记的美感血干细胞使用MRI后，创伤性脑损伤Murine模型内伤分娩

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...