Name: convection enhanced delivery of optogenetic adeno-associated viral vector to the cortex of rhesus macaque under guidance of online mri images
Uploaded: 2019-05-23T13:00:00
Description: Watch this Scientific Journal Video about Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images at JoVE.com

这项工作得到了美国心脏协会博士后奖学金(AY)、国防高级研究计划局(DARPA)重组和可塑性的支持,以加速损伤康复(REPAIR;N66001-10-C-2010),R01。NS073940,由UCSF神经科学成像中心。这项工作还得到了国家卫生研究院尤尼斯·肯尼迪·希弗国家儿童健康与人类发展研究所、华盛顿国家灵长类研究中心(WaNPCR,P51 OD010425)以及神经技术中心(CNT,国家科学基础工程研究中心,由格兰特EEC-1028725)。我们感谢卡米洛·迪亚兹-博蒂亚、蒂姆·汉森、维克托·哈拉齐亚、丹尼尔·西尔弗史密斯、卡伦·麦克劳德、朱莉安娜·米兰尼和布莱克利·安德鲁斯在实验中的帮助,以及南天、何继伟、彼得·莱多克霍维奇、米歇尔·马哈比兹和托尼·豪恩的技术支持。

所有程序均经加州大学旧金山机构动物护理和使用委员会 (IACUC) 批准,并符合《实验室动物护理和使用指南》。以下手术分别使用两只8岁、7岁的成年雄性河河猴,体重分别为17.5公斤和16.5公斤(猴子G和猴子J)。
注:对所有外科手术使用标准无菌技术。
1. 基线成像
<ol><li>在异胶下对动物进行隔离和插管,维持一般麻醉(浓度根据需要变化在0至5%之间,取决于呼吸?...

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本文概述了一种可行、高效的技术,通过MR引导的CED在NHP原发性躯体感觉和运动皮层中实现大规模光遗传学表达。与NHP大脑中传统的病毒输注方法相比,MR引导的CED的使用具有显著的优势。其中一个优势是能够在需要输注的大面积区域获得表达。例如,在常规方法中,在直径为 2-3 mm 的区域1、2、5、17 中多次注入 1-2 μL 的载体...

光遗传学是一个强大的工具,允许操作神经活动和研究大脑中的网络连接。在非人类灵长类动物(NHPs)中实施这种技术有可能增强我们对灵长类动物大脑中大规模神经计算、认知和行为的理解。虽然光遗传学近年来在NHP中已经成功实施了1、2、3、4、5、6、7,这是一个挑战,研究人员面对的是这些动物在大脑大区域实现高水平的表达。在这里,我们提供一种高效和简化的方法,以实现高浓度的光遗传学表达在大脑皮层的大片区域在猴。这项技术具有巨大的潜力,可以结合最先进的记录8、9和光学刺激10技术,提高这些动物目前的光遗传学研究。
对流增强传递(CED)是一种既定的方法,将药理剂和其他大分子(包括病毒载体)输送到中枢神经系统11、12、13。传统的输液方法涉及分布在大脑小区域的多个低容量输注,而CED可以以更少的输注实现更广泛、更均匀的代理分配。输注过程中压力驱动的散装流体流动(对流)允许在使用CED传播病毒载体时更广泛地均匀地分布目标组织。在最近的研究中,我们使用磁共振(MR)引导的CED演示了大面积原马达(M1)和躯体感觉(S1)皮质9和丘拉丘14的转导和后续光遗传学表达。
在这里,我们概述了使用CED实现光遗传学表达在大型皮质区域,只需几个皮质注射。

<table>
	<tbody>
		<tr>
			<td>0.2 mL High Pressure IV Tubing</td>
			<td>Smiths Medical Inc., Dublin, OH, USA</td>
			<td>533640</td>
			<td></td>
		</tr>
		<tr>
			<td>0.32 mm ID, 0.43 mm OD Silica Tubing</td>
			<td>Polymicro Technologies</td>
			<td>1068150027</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 mm ID, 0.76 mm OD Silica Tubing</td>
			<td>Polymicro Technologies</td>
			<td>1068150625</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV2.5-CamKII-C1V1-EYFP</td>
			<td>Penn Vector Core, University of Pennsylvania</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ABS plastic</td>
			<td>Stratasys, MN, USA</td>
			<td>ABSplus-P430</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimicrobial incise drape</td>
			<td>3M</td>
			<td>6650EZ</td>
			<td>Ioban Drape</td>
		</tr>
		<tr>
			<td>Dental Acrylic</td>
			<td>Henry Schein, Inc.</td>
			<td>1013117</td>
			<td>Acrylic Bonding Agent</td>
		</tr>
		<tr>
			<td>Elevators</td>
			<td>VWR International, LLC.</td>
			<td>10196-564</td>
			<td>Langenbeck Elevator, Wide Tip</td>
		</tr>
		<tr>
			<td>Fine suture</td>
			<td>McKesson Medical-Surgical Inc.</td>
			<td>1034505</td>
			<td></td>
		</tr>
		<tr>
			<td>Gadoteridol</td>
			<td>Prohance, Bracco Diagnostics, Princeton, NJ</td>
			<td>0270-1111-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser for light stimulation</td>
			<td>Omicron-Laserage, Germany</td>
			<td>PhoxX 488-60</td>
			<td></td>
		</tr>
		<tr>
			<td>MR compatible 3cc syringe</td>
			<td>Harvard apparatus, Holliston, MA, USA</td>
			<td>59-8377</td>
			<td></td>
		</tr>
		<tr>
			<td>MR Imaging Software</td>
			<td>Pixmeo</td>
			<td>OsiriX MD 10.0</td>
			<td></td>
		</tr>
		<tr>
			<td>MR-Compatible Pump</td>
			<td>Harvard apparatus, Holliston, MA, USA</td>
			<td>Harvard PHD 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>MR-compatible stereotaxic frame</td>
			<td>KOPF</td>
			<td>1430M MRI</td>
			<td></td>
		</tr>
		<tr>
			<td>Perifix Clamp Style Catheter Connector</td>
			<td>B-Braun, Bethlehem, PA, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Screws</td>
			<td>Plastics 1</td>
			<td>0-80 x 1/8N</td>
			<td>Nylon screws</td>
		</tr>
		<tr>
			<td>Titanium screws</td>
			<td>Crist Instrument Co., Inc.</td>
			<td>6-YCX-0312</td>
			<td>Self-tapping bone screws</td>
		</tr>
		<tr>
			<td>Trephine</td>
			<td>GerMedUSA Inc,</td>
			<td>SKU:GV70-42</td>
			<td></td>
		</tr>
		<tr>
			<td>uPrinter SE 3D printer</td>
			<td>Stratasys, MN, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin E Capsule</td>
			<td>Pure Encapsulations, LLC.</td>
			<td>DE1</td>
			<td></td>
		</tr>
		<tr>
			<td>Wet sterile absorbable gelatin</td>
			<td>Pfizer Inc.</td>
			<td>AZL0009034201</td>
			<td>Gelfoam</td>
		</tr>
	</tbody>
</table>

convection enhanced delivery of optogenetic adeno-associated viral vector to the cortex of rhesus macaque under guidance of online mri images

在非人类灵长类动物(NHP)光遗传学中,用病毒载体感染大型皮质区域通常是一项困难而耗时的任务。在这里,我们演示了使用磁共振 (MR) 引导对流增强交付 (CED) 光遗传学病毒载体到原发性躯体感觉 (S1) 和运动 (M1) 皮质的猴获得高效, 广泛皮质表达光敏性电子通道。在MR引导的CED下,将编码红移蛋白C1V1的Adeno相关病毒(AAV)载体注射到恒河猴皮层中,将这种蛋白注入黄色荧光蛋白(EYFP)。输液后三个月,显微荧光成像在M1和S1中确认两只猴子有大面积的光遗传学表达(>130 mm 2)。此外,我们还能够使用微电皮学阵列记录来自表达区域的可靠光唤起电生理反应。后来对记者进行组织学分析和免疫染色后发现,M1和S1中广泛而密集的光遗传学表达与表显荧光成像所指示的分布相对应。与传统技术相比,这种技术使我们能够在较短的时间内获得皮层大面积的表达,损害最小,并且可能是大型动物(如NHPs)的光遗传学病毒传播的最佳方法。这种方法显示了在动物模型中具有细胞型特异性的神经回路的网络级操作的巨大潜力。

在 MRI 指导下对流增强交付 (CED)
病毒载体的传播在CED输注期间在在线MR图像的指导下监测(图3A)。在这项研究中,两只猴子的S1和M1成为攻击目标(图3B)。雅兹丹-沙赫莫拉德等人于2016年9月9日对MR图像(图3C)进行了事后分析,证实了对大?...

Watch this Scientific Journal Video about Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images at JoVE.com

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

在这里,我们演示了磁共振 (MR) 引导的对流增强病毒载体到皮层 (CED) 到皮层作为一种有效和简化的方法,以实现光遗传学表达在大型皮质区域在猴脑。

在在线 MRI 图像的指导下,对流增强将视遗传阿德诺相关病毒载体输送到 Rhesus Macaque 的皮质

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

非人类灵长类动物的光遗传病毒载体对流增强传递，或CED，将使研究人员能够大规模地操纵神经活动，以了解复杂的神经计算和行为。通过 CED，与传统方法相比，我们可以在短时间内只需几次注射，就可以在灵长类大脑的大面积区域实现高光遗传学表达。磁共振或 MR 兼容腔室植入是在非人类灵长类手术中采用标准做法进行的，该过程在文本协议中进行了详细描述，并在本视频中进行了简要概述。按照文本协议中描述的动物解毒、定位和初始切口，创建圆形颅骨切除术，以覆盖使用颤管进行注射的预谋轨迹。在计划颅骨切除术的中心创建一个缩进，在头骨中足够深，使用骨管的可调中心点锚定颤量。中心完成后，将骨质利降低到头骨上，顺时针和逆时针旋转颤刺，同时施加向下压力，直到骨盖用钳子去除。解除文本协议中详述的 dura 后，将杜拉从中心切到颅骨切除术的边缘，然后继续沿着边缘使用精细的眼科剪刀。将圆柱形定制对流增强型输送 MR 兼容室安装到颅骨切除术顶部的头骨上，在输液过程中提供管管支撑，使室法兰的曲率与头骨的曲率保持良好。使用塑料螺钉和牙科丙烯酸或几个钛螺丝将植入物固定到头骨上。在植入 MR 兼容腔室并将管状注射网格插入腔室后不久，请用 0.9% 盐水填充栅格，通过 MR 解剖图像可视化注射位置。还要用湿的无菌可吸收凝胶泡沫填充腔腔，以保持大脑的水分。用无菌抗菌不育窗帘覆盖皮肤和气缸，以保持气瓶在运输和 MR 输液期间的无菌性。放置维生素 E 胶囊，以标记注射网格的顶部，进行阳性识别。当动物保持插管时，将气管管从麻醉回路中分离出来，重新连接到便携式 MR 兼容异氟烃的机器上。将动物运送到 MRI 扫描仪。获取 T1 图像以计算与喷射网格顶部和皮质表面之间的距离。维生素E胶囊在T1图像中清晰可见，应用作注射网格顶部的标记。获取 T2 图像，根据输液目标站点确定每个站点的最佳管内导轨。管网网格中充满了盐水，在 T2 图像中最好可见。使用 MR 成像软件，滚动扫描日冕和下垂平面，以找到输注的目标位置。在室温下解冻病毒载体数小时后，通过移液器或涡流混合将病毒载体与 MR 对比剂 gadoteridol 混合。将混合病毒加载到0.2毫升高压IV管中。使用高压 IV 管，将长延长线连接到 MR 兼容的三毫升注射器，并采用盐水素。将长延长管的另一端连接到装有病毒的 0.2 毫升 IV 管。然后，使用夹紧式导管连接器将具有 1 毫升阶梯尖的反流管连接到该组件的端端。最后将注射器放在 MR 兼容泵中。使用获得的基线解剖 MR 图像，选择到达目标输液部位所需的注射网格位置和插入深度。使用无菌胶带标记管上的插入深度。以每分钟一微升的速度开始输液，通过观察从管尖排出的液体，直观地验证输液管中的液体流动。通过注射网格手动将管插入到目标深度，同时保持输液管中的流量，因为这样可以防止穿透的组织在插入过程中堵塞管。现在获取快速闪存T1加权图像，用于在线监测病毒载体输液。注入大约 10 微升的载体后，使足够的病毒注入 MR 扫描仪中检测，获取 MR 图像以验证正确的管株放置，这从观察到的病毒传播中可见一斑。如果插入的管有的深度不正确，请相应地调整深度或慢慢移除管，然后重新尝试插入。通过在线 MR 图像的指导监控输液。将输液率提高至每分钟五微升，每分钟增加一微升，每分钟一微升。重要的是要慢慢增加输液率，不要超过每分钟五微升，因为高输液率可能会导致组织损伤。注入约40微升病毒载体后，开始将输液率降低每分钟1微升。注射约50微升后停止输液。将管在原位放置十分钟。慢慢从大脑取出管，然后移动到下一个位置。在动物运输前，在注射结束时用无菌窗帘盖住气缸，然后将动物运回手术室。对连续冠状体部分的组织学分析揭示了输液点周围的大规模光遗传学表达。此处显示的是基线日冕 MR 图像和在同一 MR 日冕切片输液后对比剂的传播。冠状组织部分显示的大致相同。过氧化物酶染色反映了黄色荧光蛋白或EYFP记者的表达。EYFP 表达区域之间，使用表面荧光和组织染色测量，观察到良好的对齐。其中包括从 MR 图像估计的矢量分布区域。白点表示注射部位，整个黑色区域表示颅骨切除术暴露的区域。此处显示的是染有抗 GFP 抗体的日冕部分的低放大图像，显示了躯体感觉皮层中 YFP 表达的中侧侧向。黑色箭头指示管杆轨道的位置。相邻组织以更大的放大率显示，以显示 YFP 正细胞的层层分布。YFP正细胞的密集填充区域主要位于第二层到三层和五层到六层。进一步放大揭示了第二层到三层、第五层和第六层中典型的金字塔细胞。在流量停止后，将管管留在原位 10 分钟非常重要。这将确保病毒已经渗透到周围的靶向神经组织中。CED 是一种有效的方法，可跨哺乳动物的大型大脑区域实现均匀的高表达水平。它有可能扩大我们对神经回路和连通性的理解。在CED进行病毒转导后，随后的光遗传学刺激实验可以与电记录和行为论文配对，揭示大脑中复杂的电路动力学。Yazdan-Shahmorad 及其同事在 2018 年曾采用此种技术，其中光遗传学神经调制用于诱导感觉运动皮层中的网络广泛连接变化。

Research

JoVE Journal

Neuroscience

Intracranial Injection of Adeno-associated Viral Vectors

颅内注射腺相关病毒载体

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

科研

神经科学

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Optogenetic Functional MRI

光遗传学功能磁共振成像

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice

在成年小鼠中的亚型腺相关病毒9（AAV9）载体递送

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

经颅磁刺激在线联合方法对经颅交流电刺激对原发性运动皮层的影响

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

腺相关病毒介导的脊髓基因神经元的转基因表达

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Automated Segmentation of Cortical Grey Matter from T1-Weighted MRI Images

t1 加权 mri 图像中皮质灰质的自动分割

Within neuroimaging research, a number of recent studies have discussed the impact of between-study differences in volumetric findings that are thought to ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

重组腺相关病毒在成年小鼠中的直接注射

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

通过对流增强的传递将抗体输送到毛里脑

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...