Name: personalized needles for microinjections in the rodent brain
Uploaded: 2018-01-24T12:30:00
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?...

<ol>
	<li>Kirik, D., Rosenblad, C., Bj&#246;rklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+behavioral+and+neurodegenerative+changes+following+partial+lesions+of+the+nigrostriatal+dopamine+system+induced+by+intrastriatal+6-hydroxydopamine+in+the+rat.">Characterization of behavioral and neurodegenerative changes following partial lesions of the nigrostriatal dopamine system induced by intrastriatal 6-hydroxydopamine in the rat.</a> Exp Neurol. 152 (2), 259-277 (1998).</li><li>Paolone, G., Brugnoli, A., Arcuri, A., Mercatelli, D., Morari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eltoprazine+prevents+levodopa-induced+dyskinesias+by+reducing+striatal+glutamate+and+direct+pathway+activity.">Eltoprazine prevents levodopa-induced dyskinesias by reducing striatal glutamate and direct pathway activity.</a> Mov Disord. 30 (13), 1728-1738 (2015).</li><li>Paolone, G., Lee, T. M., Sarter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+to+pay+attention:+attentional+performance+time-stamped+prefrontal+cholinergic+activation,+diurnality,+and+performance.">Time to pay attention: attentional performance time-stamped prefrontal cholinergic activation, diurnality, and performance.</a> J Neurosci. 32 (35), 12115-12128 (2012).</li><li>Shoichet, M. S., Winn, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+delivery+to+the+central+nervous+system.">Cell delivery to the central nervous system.</a> Adv Drug Deliv Rev. 42, 81-102 (2000).</li><li>Simonato, 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=Progress+in+gene+therapy+for+neurological+disorders.">Progress in gene therapy for neurological disorders.</a> Nature RevNeurol. 9, 277-291 (2013).</li><li>Bj&#246;rklund, H., Olson, L., Hahl, D., Schwarcz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-and+Long-Term+Consequences+of+Intracranial+Injections+of+the+Excitotoxin,+Quinolinic+Acid,+as+Evidenced+by+GFA+Immunohistochemistry+of+Astrocytes.">Short-and Long-Term Consequences of Intracranial Injections of the Excitotoxin, Quinolinic Acid, as Evidenced by GFA Immunohistochemistry of Astrocytes.</a> Brain Res. 317, 267-277 (1986).</li><li>Paradiso, 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=Localized+delivery+of+fibroblast+growth+factor-2+and+brain-derived+neurotrophic+factor+reduces+spontaneous+seizures+in+an+epilepsy+model.">Localized delivery of fibroblast growth factor-2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model.</a> Proc Natl Acad Sci U S A. 106 (17), 7191-7196 (2009).</li><li>Falcicchia, C., 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=Silencing+Status+Epilepticus-Induced+BDNF+Expression+with+Herpes+Simplex+Virus+Type-1+Based+Amplicon+Vectors.">Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.</a> PLoS One. 11 (3), 1-17 (2016).</li><li>Munoz, J. L., Coles, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quartz+micropipettes+for+intracellular+voltage+microelectrodes+and+ion-selective+microelectrodes.">Quartz micropipettes for intracellular voltage microelectrodes and ion-selective microelectrodes.</a> J Neurosci Meth. 22 (1), 57-64 (1987).</li><li>Kilkenny, C., Browne, W., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+research:+reporting+in+vivo+experiments:+the+ARRIVE+guidelines.">Animal research: reporting in vivo experiments: the ARRIVE guidelines.</a> J Cereb Blood Flow Metab. 31, 991-993 (2011).</li><li>Torres, E. M., Trigano, M., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+of+cell+therapies+to+the+clinic:+characteristics+of+cell+suspensions+in+large-diameter+injection+cannulae.">Translation of cell therapies to the clinic: characteristics of cell suspensions in large-diameter injection cannulae.</a> Cell Transplant. 24, 737-749 (2015).</li></ol>

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...

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&#376;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 
			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...

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 ...

Research

JoVE Journal

Neuroscience

Simultaneous fMRI and Electrophysiology in the Rodent Brain

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

神经科学

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

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 ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

从大鼠脑的解剖脑表面血管及相关脑膜和脉络丛的可视描述，

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping

罗切斯特大学医学中心的转化脑图:通过个性化大脑映射保护大脑

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...