Name: personalized needles for microinjections in the rodent brain
Uploaded: 2018-01-24T12:30:00.000+00:00
Duration: 10 min 6 s
Description: Watch this Scientific Journal Video about Personalized Needles for Microinjections in the Rodent Brain at JoVE.com

这项工作得到欧洲共同体的赠款支持 [FP7-PEOPLE-2011-IAPP 项目 285827 (EPIXCHANGE)]。

所有实验方案均由费拉拉大学动物实验伦理委员会和意大利卫生部批准。到达 (动物研究: 报告在活体实验11) 指南被跟随了。
1. 石英针的制备
<ol><li>清洁和消毒石英毛细管 (见材料表), 将它们放在蒸馏水中5分钟, 乙醇99% 的5分钟, 乙醚中的5分钟。</li>
	<li>把毛细血管放在引擎盖下至少1小时, 让它们完全干燥。</li>
	<li>根据不同的需要, 利用激光拉拔器的参数准备针头, 允许生产足够长、足够薄的针。 注: 我们使用激光拉拔器 (请参阅材料表)。使用两个线程序来拉动。第一条线使用大功率, 最大的扫描长度 (对应于花丝值), 没有拉力。这些参量的组合导致最大的部分毛细管的熔化以最低的延长的速度, 允许对长的针的塑造以大内外部大小和减少的小腿。这样, 针保持了高的机械阻力和宽的内径, 使干细胞或病毒颗粒通过不堵塞。该程序线的速度和延迟应停止毛细管延长之前, 尖端变得太小, 长, 缺少所需的机械和形状的特点。一旦石英冷却下来 (5 秒), 牵引程序的第二行自动执行。功率、灯丝和拉力的参数可用于切割尖端长度最短的刀尖和陡柄, 避免两针的分离和尖端断裂。通常, 用我们的设备我们使用以下: (a) 线 1 = 力量 990, 灯丝 5, 速度 130, 延迟 110, 拉扯 0;(b) 线 2 = 功率 970, 灯丝 3, 速度 0, 延迟 70, 拉0。</li>
	<li>把针放在以前用乙醇70% 清洗过的培养皿中。<ol>
<li>用塑料石蜡膜覆盖这道菜。</li>
	</ol>
</li>
	<li>在微解剖上切开毛细血管<ol>
<li>在启动时, 让激光运行一个技术的过程, 以验证电源和能量交付。然后设置切削参数。 注: 我们使用微解剖 (参见材料表) 和以下参数: 激光功率脉冲能量 (63 µJ), 激光束直径20% 最大孔径 (最大孔径: 1.0 µm), 切割速度10% 的最大速度 (最大速度:30 µm/秒)。</li>
		<li>将针固定在显微镜台上, 并将其尖端放置在电脑屏幕的中心。</li>
		<li>在菜单栏上, 点击铅笔图标, 并用它来为激光切割针画一个记号。 注: 切割应在大约45°的角度与针面有关。</li>
		<li>在侧栏功能中, 选择包含 "开始剪切" 按钮的标志, 以允许激光切割先前标记的点。</li>
		<li>如果需要, 通过重复 1.5. 2-3 中描述的程序来减少侧孔 (s);不需要重新定位针 (图 1)。</li>
	</ol>
</li>
	<li>切后清洗。<ol>
<li>通过塑料管将针头连接到空泵。</li>
		<li>打开泵并手动设置正向流速 (3 µL/分钟)。</li>
		<li>先抽出99% 乙醇, 然后蒸馏水去除由于切割而造成的杂质。</li>
		<li>再次吸取99% 乙醇以缓解干燥。 注: 每个步骤应至少持续3分钟。</li>
	</ol>
</li>
	<li>将针储存在适当的培养皿中直到手术当天。</li>
	<li>可重新使用针头;在实验结束时, 用漂白剂和乙醇清除溶液和可能的组织残渣。要进行更彻底的清洗, 把针头放在一个微的储藏罐中, 把针尖向下指向, 用蒸馏水填满瓶子, 直到贴上盖子, 煮沸1.5 分钟, 除去任何残留的纸巾, 然后用乙醇清洗。</li>
</ol>2. 程序
<ol><li>按照泵用户手册提供的说明, 选择 "设置" 菜单, 根据注射注射器的直径/体积来校准泵, 并设置0.3 µL/分钟的流速。</li>
	<li>装满10毫升的注射器 (装有钝针 (如, 汉密尔顿针, 30 克) 和一个短的聚四氟乙烯管) 与无菌的水, 并用它来填补注射注射器 (经过非常谨慎地删除了活塞) 和注射手动微注射泵 (基质金属蛋白酶) 组装件套件。 注: 该金属蛋白酶试剂盒用于在立体定向框架中安全地安装针 (图 2)。该套件包括: 聚四氟乙烯油管, 口管耦合器, 吸管支架和管不同外径的垫圈。</li>
	<li>将针头连接到金属蛋白酶试剂盒。<ol>
<li>通过10毫升的注射器, 通过系统冲洗更多的水, 并检查水是否从针头中出来。 注: 请务必通过重复灌装步骤来清除系统内的气泡。</li>
	</ol></li>
	<li>从注射注射器上拔下10毫升注射器, 同时缓慢地推动活塞, 以便在注射注射器的顶端留下一滴水。在注射注射器中插入活塞。</li>
	<li>将微注射注射器连接到泵上, 并设定流速在0.3 µL/分钟。 注意: 等待, 直到有一个滴从针尖的泄漏, 然后让它再去3分钟。</li>
	<li>设置0.1 µL/分钟的流量, 并开始手术程序。在进行手术时, 泵应该正在进行。 注意: 在本文所报道的实验中, 我们使用了300克 (3 月龄) 的雄性大大鼠。然而, 该程序适用于其他物种, 特别是小鼠。</li>
	<li>使用当地法规批准的方法诱导全身麻醉。 注: 我们使用的混合腹腔注射 (ip) 85 毫克/千克氯胺酮和8毫克/千克嗪20-50 分钟的麻醉。重要的是, 尾巴和/或脚趾捏是用来确保动物完全镇静。麻醉的水平和基本的生命体征是评估前手术开始, 并监测每10分钟执行手术。动物体温在手术前后保持恒定, 使用水再循环加热垫。动物被监测, 直到完全恢复 (例如, 他们是直立和流动)。此外, 动物接受镇痛药 (曲马多, 5-7 毫克/千克的 ip) 后手术每24小时三天。<ol>
<li>剃掉动物的头部, 并充分准备手术部位进行无菌手术。执行一个两阶段的擦洗与碘为基础的解决方案 (控告), 其次是70% 乙醇。此外, 为了保持无菌, 包裹在外科手术部位与手术的褶皱 (在视频已被省略为示范目的)。</li>
	</ol></li>
	<li>将大鼠定位在水循环加热毯上的立体定向框中。为提供额外的止痛, 在手术现场应用0.5% 利多卡因溶液 (不超过7毫克/千克)。在2% 戊二醛加7.05% 苯酚 (Sporodicin 或 Cidex) 浸入, 用无菌水或盐水冲洗后, 用微珠消毒器或液体冷消毒设备预先消毒所有手术器械。即使在程序之间, 也必须进行消毒。用一把消毒的手术刀在头骨上做一个纵向的切割。要温柔, 避免过度出血。用平的刮刀打开头皮, 并在皮瓣上应用手术夹, 以保持它的开放性。轻轻地 , 分离骨膜 , 露出 bregma 和头骨的区域 , 通过它 , 针头将入。</li>
	<li>将手臂安装在立体定向的框架上 (同时流动是持续的), 并将针头的尖端精确地移到 bregma 上, 注意不要折断它的顶端。注释前后和译侧坐标的 bregma, 并转换为所需的坐标。在显微镜的引导下, 降低针头, 直到它的尖端几乎触及头骨, 在提高了一个小的立体定位臂后, 标记钻孔点。</li>
	<li>钻头 (钻头直径: 0.8 mm; 钻头转速: 1000 rpm) 头骨在标记点上。在钻探过程中, 必须注意不要穿透硬脑膜。在钻孔过程中, 滴无菌盐水, 以避免骨损伤和坏死。</li>
	<li>切一小块塑料石蜡薄膜, 并把它放在头骨上。使用10µL 吸管, 使一滴溶液注入塑料石蜡薄膜。停止泵, 轻微地取回泵的推动器, 并通过轻轻地拉动微注射注射器的活塞, 在毛细管中创建一个小气泡。</li>
	<li>向下的立体定向手臂, 直到针头的尖端到达下降。抽取 (非常缓慢) 的注射注射器活塞, 以避免任何气泡形成的样品。更换泵的推动器与活塞接触, 并打开泵的流量为0.3 µL/分钟. 等待, 直到形成一个下降 (几分钟), 然后继续下一步。 注: 在本例实验中, 人工脑脊液 (aCSF) 注入纹状体 (AP: + 1.5, 毫升: ±2.7;DV:-5.0) 和海马背 (AP:-3.5, ML: ±2.1;DV:-3.5;在毫米从硬脑膜), 2 µL/现场。为了便于两种注射方法的比较, 每个脑区都在一个半球和一个26克钝尖或一个 30 g 锥尖端不锈钢针中注入了一个石英针。</li>
	<li>降低流量到0.1 µL/分钟, 尼克硬脑膜与不锈钢锥尖针和缓慢降低立体定向手臂的背腹轴, 进入脑组织。当达到所需的位置时, 向下增加0.1 毫米, 停止泵, 提高立体定向手臂0.1 毫米, 并重新启动泵在流到0.3 µL/分钟。 注意: 在降低针头的同时保持缓慢流动的原因是为了确保一个正压, 避免了针内组织小碎片的穿透。这样的事件会妨碍小费, 损害解决方案的交付。由于流速是非常缓慢的, 而降低针和降低的速度是大约十年代/毫米, 这意味着数量的解决方案, 保持沿针轨道是微不足道的相比, 那些注射在目标站点。在使用带有侧孔的针头时, 可以避免这种预防措施。</li>
	<li>启动定时器, 并等待所需的时间取决于所需的体积注入 (例如, 6 分钟四十年代, 如果注射2µL)。在注射时监控气泡运动。最后, 停止泵, 等待5分钟, 然后慢慢地取回针。当石英针完全拔出时, 再启动泵, 检查针尖处是否形成一滴。</li>
	<li>用骨蜡封住颅骨表面的开口, 取出止血夹, 缝合头皮, 用抗生素霜 (如, 孢) 将伤口周围的区域涂上, 并将动物带回笼中。 注意: 按照机构伦理委员会批准的安乐死协议, 对于本文所述的实验, 动物被深度麻醉, 用43毫克/千克氯胺酮和7毫克/千克嗪, 并被斩首的 ip 注射。大脑被移除, 后缀在福尔马林中的 48 h 和石蜡嵌入。冠状部分 (8 µm) 的大脑被切片使用切片。</li>
</ol>

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作者没有什么可透露的。

本文中描述的技术满足了简介中概述的需要, 用于优化为各种目的而执行的 microinjections12。这里被描述的针减少损伤到极小值, 根本上不可检测的水平;在与玻璃针 (容易弯曲) 的差异, 石英针是刚性的, 确保一个可靠的命中的理想的大脑区域, 即使在深坐标。此外, 侧孔确保了解决方案的交付, 即使在插入大脑组织的尖端孔被阻塞。
限制和替代方法。由于熔点高, 石英毛细管不能在传统的拉拔器上拉动,即, 它们需要使用昂贵的设备。玻璃毛细管在避免对组织的损伤方面是同样好的, 但对于深层结构和更脆弱 (即, 在设计方面不那么灵活) 注射的可靠性较差。然而, 如果目标是注射在肤浅的结构, 如皮质或背部海马, 并没有要求多孔, 将增加针头折断的风险, 玻璃针肯定代表一个有效的和廉价的替代品。
石英和玻璃针的限制是, 他们不允许多次注射在不同的时间点, 如不锈钢针耦合引导套管。因此, 钢针仍然是这些应用的最佳选择, 建议避免相对较大的 (26 克) 和/或钝边针。
协议中的关键步骤。总的来说, 上述的协议是非常简单的, 不应该很难使用的任何研究员在 microinjections 的经验。一旦针头可用, 常规的预防措施, 用于麻醉, 手术, 缓慢插入和去除针, 恒定和缓慢的输液率微调节, 将确保良好和重现性的结果。
优势和进一步发展。如上所述, 石英针的关键优势是在最小的机械损伤和个性化的针设计的可能性。我们准备了多个孔和不同直径的针, 可以适应特定的实验需要。例如, 可能需要不同的直径来注射病毒载体和细胞相比, 药物或毒素;多孔可能是有用的, 以确保在较大的范围内注入大量的解决方案, 可能会成为特别有用的几何困难的大脑区域。这项技术的巨大通用性, 也可以利用, 以优化传播的病毒载体的基因治疗或细胞。事实上, 我们使用这些针头来注射病毒载体或细胞。
其他优势包括重新使用针头的可能性。为此, 可以用乙醇和漂白剂清洗针头, 去除溶液和可能的组织残渣。尽管生产成本相对较高, 但单针的重复使用可以使程序具有成本效益。

Microinjections 已经使用了很长一段时间的交付药理活性化合物, 以调节神经元活动在特定的脑区。此外, 它们还被用于在特定的神经元群附近注射毒素, 模拟某些疾病的神经退行性事件, 例如 6-羟基多巴胺在体多巴胺系统中模拟帕金森病1,2或免疫 192 IgG 皂素损害胆碱能系统3。最近, 微注射程序已被用来提供病毒载体或细胞移植的基因或细胞治疗实验性脑疾病4,5。
在这些研究中使用的经典类型的针是由不锈钢制成的。虽然容易和实用, 钢针有许多问题6: 它们相对较大, 可能会导致组织损伤, 与血脑屏障的渗漏和星形胶质细胞的活化有关;此外, 它们可能产生脑组织的取心, 进入针头制造障碍, 甚至完全避免所需溶液的流动。最近, 从毛细管中制备的玻璃针ad hoc已在使用中引入78。这些不造成重大组织损伤或星形胶质细胞活化, 但相对地灵活和可能弯曲, 当介绍在深部结构时, 减少定位的准确性 (个人观察)。
因此, 有必要减少尽可能多的损害 (特别是在进行实验, 以愈合损害), 同时提高准确性和重现性 (即, 确保所有解决方案交付和确保正确的本地化)。此外, 最好使用不同的针头设计, 以确保在不同几何形状的脑区内注射溶液的最佳分布。在本文中, 我们描述了一个协议, 准备和使用石英针 microinjections 在啮齿动物的大脑。由于高熔点, 石英毛细管不能在一个传统的拉拔器, 因此, 没有被用于产生针过去。石英, 然而, 提供一些重要好处在玻璃, 特别是高刚性和断裂抵抗9。由于其硬度, 石英针是理想的适合注射到腹侧脑区。由于他们的高抗破坏, 他们可以建模, 包括多个孔, 获得的设计, 可能证明最有效的, 甚至当目标的大脑区域复杂的几何形状10。

<table><tbody><tr><td>Quartz capillaries</td><td>Sutter Instruments, Novato, CA USA</td><td>Q100-50-10</td><td>Without filament</td></tr><tr><td>Puller</td><td>Sutter</td><td>P2000</td><td></td></tr><tr><td>Micropipette storage jar</td><td>World Precision Instruments (WPI), Sarasota, FL, USA</td><td>E210</td><td></td></tr><tr><td>Laser microdissector</td><td>Leica Microsystems, Wetzlar, Germany</td><td>LMD6500</td><td></td></tr><tr><td>Hamilton syringe</td><td>Hamilton ILS Innovative Labor Systeme GmbH, St&amp;Yuml;tzerbach, Germany</td><td>19138-U</td><td></td></tr><tr><td>Microinfusion pump</td><td>Univentor, Zejtun, Malta</td><td>Model 864</td><td></td></tr><tr><td>Manual microinjection pump kit</td><td>WPI</td><td>Item#: MMP-KIT</td><td>Kit allowing for micropipettes to be securely mounted to the stereotactic frame</td></tr><tr><td>Precision Drill</td><td>Proxxon</td><td>28510 MicroMot 50/E</td><td>Ball bearing drive shaft with variable speed</td></tr><tr><td>Artficial Cerebral Spinal Fluid</td><td>Tocris</td><td>3525</td><td></td></tr><tr><td>Needles 26 G Blunt and 30 G Bevel</td><td>Hamilton</td><td>26 G Blunt: 19138-U&lt;br/&gt; 30 G Bevel: 20757</td><td></td></tr><tr><td>Microtome</td><td>Leica, Germany</td><td>LEICA RM212RT</td><td></td></tr></tbody></table>

personalized needles for microinjections in the rodent brain

Microinjections 已经用于在特定的脑区内运送药物或毒素很长一段时间, 最近, 他们被用来提供基因或细胞治疗产品。不幸的是, 目前的显微注射技术使用钢或玻璃针是次优的多种原因: 特别是, 钢针可能会导致组织损伤, 玻璃针可能弯曲时, 深入到大脑, 失去了目标地区。在本文中, 我们描述了一个协议, 准备和使用石英针, 结合了一些有用的功能。这些针头不会产生可检测的组织损伤, 而且非常僵硬, 即使在使用深层坐标时, 也能确保在所需的脑区内可靠的分娩。此外, 通过制作所需直径的多个孔, 可以个性化针的设计。多个孔便于在较大的区域内注入大量的溶液, 而大孔则便于注入细胞。此外, 这些石英针可以清洗和重新使用, 这样的做法成为成本效益。

我们比较了在大鼠背侧海马和纹状体的直接显微注射引起的损伤 (60 µM 外径尖端; 1 20 µM 直径侧孔; C 型,图 1) 与两个经典的, 26 g 钝端和 30 g 斜角边缘比较不锈钢针。为了达到这个目的, 我们在右侧和左背海马和纹状体中分别注射了2µL aCSF 的石英和钢针, 在注射后48小时, 用苏木精-曙红 (he) 染色对组织损伤进行了评价。我们选择了一个早期的时间点后注射, 以更好地理解急性机械损伤产生的不同的针头 (及时, 组织修复机制可能掩盖最初的损害)7。
按照上述协议, 石英针没有产生任何可检测的损伤和一个勉强检测到的针迹, 而通过26克钢钝端针注射的脑区显示出明显的损坏 (图 3)。30克斜角 (30 °) 不锈钢针诱导了一个更有限, 但清晰可检测的脑损伤在纹状体 (图 4B) 和背部海马 (图 5)。为了证明注射 aCSF 是同样成功的两针, 在一组单独的实验中, 我们注入2µL 的台盼蓝 (图 4A)。重要的是, 所有回收的石英针都完好无损,即, 我们从来没有观察到任何由于插入演习造成的损害。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55751/55751fig1.jpg"> 图 1: 石英针尖的显微图像, 显示用于注入流体的尖端和孔的不同直径.低放大率图片 (A) 和更高的放大倍数 (B) 和 (C)。针可以根据大脑区域的解剖、扩散目标和溶液的粘度 (如B) 和 (C) 所示, 以不同的方式 (不同长度、孔数和孔尺寸) 进行准备。注意用低功率激光切割法获得的孔和尖端的平滑切割。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55751/55751fig1large.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/55751/55751fig2.jpg"> 图 2: 设置 microinjections.图片显示的工具包用于确保石英吸管 (左) 和不锈钢针 (右) 的立体定向微。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55751/55751fig2large.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/55751/55751fig3.jpg"> 图 3: 使用石英针显示较少损伤的冠状截面.在纹状体水平上的大鼠大脑冠状段 (8 µm 厚度), 通过注射2µL aCSF 的石英针, 如图 1C (左侧) 和26克钝端不锈钢针 (钻机ht 侧)。在石英针侧面的皮质上产生了一些损伤, 这是由于不适当地深钻颅骨造成的。这些图像, 从两个不同的动物, 是代表所有5动物包括在实验 (规模酒吧 = 500 µm)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55751/55751fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55751/55751fig4.jpg"> 图 4: 传播和组织损伤.(a) 用石英针 (左) 与30克斜边不锈钢针 (右) 相比, 在纹状体中注入的台盼蓝分布。(B) 大鼠脑纹状体的冠状段, 染色与他显示了一个石英针与30克斜边不锈钢针 (刻度条 = 500 µm) 的比较。在框 (左, 右), 更高的放大倍数的损害 (规模酒吧 = 200 µm, 10X)。这些图像是5动物的代表, 包括在实验中。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55751/55751fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55751/55751fig5.jpg"> 图 5: 石英针和26克斜角边缘不锈钢针在海马背层引起的损伤.这里所示的两个例子是包括在实验中的5只动物的代表 (刻度条 = 400 µm)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55751/55751fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Personalized Needles for Microinjections in the Rodent Brain at JoVE.com

Personalized Needles for Microinjections in the Rodent Brain

我们在这里描述了一种使用石英针的啮齿动物脑内注射的协议。这些针头不会产生可检测的组织损伤, 并确保即使在深部区域也能可靠地提供。此外, 他们可以适应研究需要的个性化设计, 可以重新使用。

啮齿动物脑 Microinjections 的个性化针

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

personalized-needles-for-microinjections-in-the-rodent-brain

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

11.2K 次观看.  University of Ferrara. 我们在这里描述了一种使用石英针的啮齿动物脑内注射的协议。这些针头不会产生可检测的组织损伤, 并确保即使在深部区域也能可靠地提供。此外, 他们可以适应研究需要的个性化设计, 可以重新使用。

视频: Personalized Needles for Microinjections in the Rodent Brain

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

科研

神经科学

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

定向脑深部结构与配送药品，病毒载体，或细胞移植Microinjections

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

啮齿动物脑显微注射学习动机行为的分子基板

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

行为学

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Syringe-injectable Mesh Electronics for Stable Chronic Rodent Electrophysiology

Implantable brain electrophysiology probes are valuable tools in neuroscience due to their ability to record neural activity with high spatiotemporal resolution from shallow and deep brain regions. Their use has been hindered, however, by mechanical and structural mismatches between the probes and brain tissue that commonly lead to micromotion and gliosis with resulting signal instability in chronic recording experiments. In contrast, following the implantation of ultraflexible mesh electronics via syringe injection, the mesh probes form a seamless, gliosis-free interface with the surrounding brain tissue that enables stable tracking of individual neurons on at least a year timescale. This protocol details the key steps in a typical mouse neural recording experiment using syringe-injectable mesh electronics, including the fabrication of mesh electronics in a standard photolithography-based process possible at many universities, loading mesh electronics into standard capillary needles, stereotaxic injection in vivo, connection of the mesh input/output to standard instrumentation interfaces, restrained or freely moving recording sessions, and histological sectioning of brain tissue containing mesh electronics. Representative neural recordings and histology data are presented. Investigators familiar with this protocol will have the knowledge necessary to incorporate mesh electronics into their own experiments and take advantage of the unique opportunities afforded by long-term stable neural interfacing, such as studies of aging processes, brain development, and the pathogenesis of brain disease.

Mesh electronics probes seamlessly integrate and provide stable, long-term, single-neuron level recording within the brain. This protocol uses mesh electronics for in vivo experiments, involving the fabrication of mesh electronics, loading into needles, stereotaxic injection, input/output interfacing, recording experiments, and histology of tissue containing mesh probes.

注射器-注射用网状电子技术稳定的慢性啮齿动物电生理学

Implantable brain electrophysiology probes are valuable tools in neuroscience due to their ability to record neural activity with high spatiotemporal ...

生物工程

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

使用机器人微注射操作脑片中单神经干细胞和神经元

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递:对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

使用徒手方法对小鼠进行脑室内注射

Free-Hand Intracerebroventricular Injections in Mice

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. An intracerebroventricular (ICV) injection allows the widespread delivery of the experimental agent throughout the brain (particularly in the structures near the ventricles). Here, methods for making free-hand ICV injections in adult mice are described. By using visual and tactile landmarks on the heads of mice, injections into the lateral ventricles can be made rapidly and reliably. The injections are made with a glass syringe held in the experimenter&#39;s hand and placed at approximate distances from the landmarks. Thus, this technique does not require a stereotaxic frame. Furthermore, this technique requires only brief isoflurane anesthesia, which permits the subsequent assessment of mouse behavior and/or physiology in awake, freely behaving mice. Free-hand ICV injection is a powerful tool for the efficient delivery of experimental agents into the brains of living mice and can be combined with other techniques such as frequent blood sampling, neural circuit manipulation, or in vivo recording to investigate neuroendocrine processes.

作者聚焦：生殖神经控制的见解、创新和未来途径 

Here, a simple and rapid approach for performing intracerebroventricular injections in mice using a free-hand approach (that is, without a stereotaxic device) is described.

小鼠徒手脑室内注射

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. ...

啮齿动物脑 Microinjections 的个性化针

摘要

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此视频中的章节

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

定向脑深部结构与配送药品，病毒载体，或细胞移植Microinjections

啮齿动物脑显微注射学习动机行为的分子基板

同时电生理记录和抑制剂在啮齿动物大脑微注射

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