Name: intrathecal delivery of antisense oligonucleotides in the rat central nervous system
Uploaded: 2019-10-29T14:00:00
Description: Watch this Scientific Journal Video about Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System at JoVE.com

我们要感谢Ionis制药公司提供本文中描述的ASO。

所有体内程序均根据生物基因机构动物使用和护理委员会（IACUC）批准的协议进行，这些规程遵循美国国家卫生研究院关于照料和使用实验室动物的指南。
1. 材料和仪器制备
<ol><li>准备特殊的导引管。<ol>
<li>使用带有切断轮（或锋利锯）的旋转工具切断 19 G 指针的两端，从而产生 ±1.5⁄2 厘米长的导管（图 1Aiii）。使用旋转工具的砂轮使两...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+CNS+barriers:+evolution,+differentiation,+and+modulation.">Dynamics of CNS barriers: evolution, differentiation, and modulation.</a> Cellular and Molecular Neurobiology. 25 (1), 5-23 (2005).</li><li>Greene, C., Campbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junction+modulation+of+the+blood+brain+barrier:+CNS+delivery+of+small+molecules.">Tight junction modulation of the blood brain barrier: CNS delivery of small molecules.</a> Tissue Barriers. 4 (1), e1138017 (2016).</li><li>Daneman, R., Engelhardt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+barriers+in+health+and+disease.">Brain barriers in health and disease.</a> Neurobiology of Disease. 107, 1-3 (2017).</li><li>Ballabh, P., Braun, A., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier:+an+overview:+structure,+regulation,+and+clinical+implications.">The blood-brain barrier: an overview: structure, regulation, and clinical implications.</a> Neurobiology of Disease. 16 (1), 1-13 (2004).</li><li>Cardoso, F. L., Brites, D., Brito, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+the+blood-brain+barrier:+molecular+anatomy+and+possible+investigation+approaches.">Looking at the blood-brain barrier: molecular anatomy and possible investigation approaches.</a> Brain Research Reviews. 64 (2), 328-363 (2010).</li><li>Larsen, J. M., Martin, D. R., Byrne, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+delivery+through+the+blood-brain+barrier.">Recent advances in delivery through the blood-brain barrier.</a> Current Topics in Medicinal Chemistry. 14 (9), 1148-1160 (2014).</li><li>Brinker, T., Stopa, E., Morrison, J., Klinge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+cerebrospinal+fluid+circulation.">A new look at cerebrospinal fluid circulation.</a> Fluids Barriers CNS. 11, 10 (2014).</li><li>Standifer, K. M., Chien, C. C., Wahlestedt, C., Brown, G. P., Pasternak, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+loss+of+delta+opioid+analgesia+and+binding+by+antisense+oligodeoxynucleotides+to+a+delta+opioid+receptor.">Selective loss of delta opioid analgesia and binding by antisense oligodeoxynucleotides to a delta opioid receptor.</a> Neuron. 12 (4), 805-810 (1994).</li><li>Wahlestedt, 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=Antisense+oligodeoxynucleotides+to+NMDA-R1+receptor+channel+protect+cortical+neurons+from+excitotoxicity+and+reduce+focal+ischaemic+infarctions.">Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischaemic infarctions.</a> Nature. 363 (6426), 260-263 (1993).</li><li>Wahlestedt, C., Pich, E. M., Koob, G. F., Yee, F., Heilig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+anxiety+and+neuropeptide+Y-Y1+receptors+by+antisense+oligodeoxynucleotides.">Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides.</a> Science. 259 (5094), 528-531 (1993).</li><li>Mazur, 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=Development+of+a+simple,+rapid,+and+robust+intrathecal+catheterization+method+in+the+rat.">Development of a simple, rapid, and robust intrathecal catheterization method in the rat.</a> Journal of Neuroscience Methods. 280, 36-46 (2017).</li><li>Wolf, D. 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=Dynamic+dual-isotope+molecular+imaging+elucidates+principles+for+optimizing+intrathecal+drug+delivery.">Dynamic dual-isotope molecular imaging elucidates principles for optimizing intrathecal drug delivery.</a> Journal of Clinical Investigation Insight. 1 (2), e85311 (2016).</li><li>Becker, L. 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=Therapeutic+reduction+of+ataxin-2+extends+lifespan+and+reduces+pathology+in+TDP-43+mice.">Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice.</a> Nature. 544 (7650), 367-371 (2017).</li><li>Zhang, X., Hamblin, M. H., Yin, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long+noncoding+RNA+Malat1:+Its+physiological+and+pathophysiological+functions.">The long noncoding RNA Malat1: Its physiological and pathophysiological functions.</a> RNA Biology. 14 (12), 1705-1714 (2017).</li><li>Crooke, S. T., Witztum, J. L., Bennett, C. F., Baker, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Targeted+Therapeutics.">RNA-Targeted Therapeutics.</a> Cell Metabolism. 27 (4), 714-739 (2018).</li><li>DeVos, S. L., Miller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+intraventricular+delivery+of+drugs+to+the+rodent+central+nervous+system.">Direct intraventricular delivery of drugs to the rodent central nervous system.</a> Journal of Visualized Experiments. (75), e50326 (2013).</li><li>McCampbell, 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=Antisense+oligonucleotides+extend+survival+and+reverse+decrement+in+muscle+response+in+ALS+models.">Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models.</a> Journal of Clinical Investigation. 128 (8), 3558-3567 (2018).</li><li>Schoch, K. M., Miller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+Oligonucleotides:+Translation+from+Mouse+Models+to+Human+Neurodegenerative+Diseases.">Antisense Oligonucleotides: Translation from Mouse Models to Human Neurodegenerative Diseases.</a> Neuron. 94 (6), 1056-1070 (2017).</li><li>Lane, R. 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=Translating+Antisense+Technology+into+a+Treatment+for+Huntington's+Disease.">Translating Antisense Technology into a Treatment for Huntington's Disease.</a> Methods in Molecular Biology. 1780, 497-523 (2018).</li><li>Wurster, C. D., Ludolph, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+oligonucleotides+in+neurological+disorders.">Antisense oligonucleotides in neurological disorders.</a> Therapeutic Advances in Neurological Disorders. 11, (2018).</li><li>Hach&#233;, 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=Intrathecal+Injections+in+Children+With+Spinal+Muscular+Atrophy:+Nusinersen+Clinical+Trial+Experience.">Intrathecal Injections in Children With Spinal Muscular Atrophy: Nusinersen Clinical Trial Experience.</a> Journal of Child Neurology. 31 (7), 899-906 (2016).</li><li>Goodkey, K., Aslesh, T., Maruyama, R., Yokota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nusinersen+in+the+Treatment+of+Spinal+Muscular+Atrophy.">Nusinersen in the Treatment of Spinal Muscular Atrophy.</a> Methods in Molecular Biology. 1828, 69-76 (2018).</li><li>Wurster, C. 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本文展示了一种将治疗剂直接输送到大鼠CNS的有力方法。从理论上讲，类似的技术也可以在小鼠身上进行，但由于体积较小，这种方法可能更具挑战性。因此，我们小组在小鼠中执行中枢注射（ICV）注射，用于中枢神经系统药物输送，通过不同的给药途径达到相同的目标。另一项研究16中已经描述了这种方法。
此处描述的方法的优点是，它不需要昂贵的设备...

中枢神经系统（CNS）的血管系统已经演变为平衡、控制分子交通、提供营养和清除废物的关键调节器。该系统也是抵御外部病原体攻击的第一道防线，这要归功于内皮细胞壁上紧密结的密集分布。这些紧密的交汇点构成血脑屏障 （BBB） 的一个方面。虽然BBB允许输送满足营养和能量需求所需的分子（如离子、葡萄糖），但它也有选择地限制病原体和有毒化学品1、2、3的通过。
具有讽刺意味的是，BBB相同的保护功能，限制病原体和有毒化学品的通过，也是我们能否在系统给生物体进行系统管理后，通过治疗轻松获得中枢神经系统的主要障碍2， 4，5.BBB的这一作用促使大量新的药物分销技术和方法的发展。
克服这个障碍的一个方法是将药物直接注射到脑脊液（CSF），这种液不断渗透大脑和脊髓7，8，9，10。在本文中，我们将描述一种通过将导管内端完全置于大鼠脊柱的cauda equina空间中，成功地将代理传递到腰间空间的方法。马祖尔等人曾在其他地方11日发表了对这一程序的描述。
该方案非常有效，通过定量聚合酶链反应（qPCR）分析靶基因敲除8，使反义寡核苷酸（ASO）输送到CNS的成功率超过90%。手术对动物造成最小的不适，因为100%的老鼠在手术中幸存下来，手术伤口周围肿胀最小，没有痛苦的迹象（例如，多动、脱水、盘旋、失去平衡、食物摄入量减少，以及脱水）在手术后观察。此处描述的方法的另一个优点是，它不需要昂贵的设备，也不需要任何特殊工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3M Steri-Drape Small Drape with Adhesive Aperture</td>
			<td>3M</td>
			<td></td>
			<td>1020</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Decon Laboratories, Inc</td>
			<td></td>
			<td>8416-160Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swab sticks</td>
			<td>Dynarex</td>
			<td></td>
			<td>NO 1204</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://punchout.fishersci.com/shop/products/bd-general-use-syringes-5/22124967?crossRef=BD%20302830#?keyword=BD+302830">BD General Use Syringes 1 mL Luer-Lok tip</a></td>
			<td>BD</td>
			<td>1ml TB Luer-Lok tip</td>
			<td>BD 302830</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic PE Tubing</td>
			<td>BD</td>
			<td>Polyethylene tubing PE50 Diameter 0.023 in</td>
			<td>BD 427400 (10ft, Fischer Scientific 22-204008) or 427401 (100ft, Fischer Scientific 14-170-12P)</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic PE Tubing</td>
			<td>BD</td>
			<td>Polyethylene tubing PE10 Diameter 0.011 in</td>
			<td>BD 427410 (10ft, Fischer Scientific 14-170-11B) or 4274011 (100ft, Fischer Scientific 14-170-12B)</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic PE Tubing Adapters</td>
			<td>BD</td>
			<td>23 gauge intramedic luer stub adaper</td>
			<td>BD 427565 or Fisher Scientific 14-826-19E</td>
			<td>120V 1.2A</td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Single-use Needles 30G</td>
			<td>BD</td>
			<td></td>
			<td>BD 305128</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine Sustained Release-lab</td>
			<td>ZooPharm</td>
			<td>Prescription required</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene oxide sterilizer</td>
			<td>Andersen Sterilizer INC.</td>
			<td>AN 74i, gas sterilizer</td>
			<td>AN 74i</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide cannula</td>
			<td>BD</td>
			<td>19G x 1 WT (1.1 mm x 25mm) needle</td>
			<td>BD 305186</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 100ul</td>
			<td>Hamilton company</td>
			<td>Hamilton syringe 100ul</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead Sterilizer</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>STERILIZER MODELNO FST 250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Dechra veterranery product</td>
			<td></td>
			<td>17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Pocket Pro Pet Trimmer</td>
			<td>Braintree Scientific</td>
			<td></td>
			<td>CLP-9931 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone scrub</td>
			<td>PDI</td>
			<td></td>
			<td>S48050</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Baxter</td>
			<td>Sodium Chloride 0.9% Intravenous Infusion BP 50ml</td>
			<td>FE1306G</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>disposable scalpel</td>
			<td>No. 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal heating pad</td>
			<td>K&#38;H Manufacturing</td>
			<td></td>
			<td>Model # 1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Stylet Wire</td>
			<td>McMaster-Carr</td>
			<td>1749T14</td>
			<td>LH-36233780</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery Towel drape</td>
			<td>Dynarex</td>
			<td></td>
			<td>4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors and forceps</td>
			<td>FST and Fisher Scientific</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Ethicon</td>
			<td></td>
			<td>4-0 or 5-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Tool to make the Guide cannular</td>
			<td>Grainger</td>
			<td>Rotary tool (Dremel)</td>
			<td>14H446 (Mfr: EZ456)</td>
			<td>1.5&#8221; diameter, Pk5</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>EZ lock cut off Wheel</td>
			<td>1PKX5 (Mfr: 3000-1/24)</td>
			<td>1.5&#8221;, Pk2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Grinding Wheel, Aluminum Oxide</td>
			<td>38EY44 (Mfr: EZ541GR)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>EZ lock Mandrel</td>
			<td>1PKX8 (Mfr: EZ402-01)</td>
			<td>1.5&#8221; diameter</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Diamond wheel floor Tile</td>
			<td>3DRN4 (Mfr: EZ545)</td>
			<td></td>
		</tr>
		<tr>
			<td>Alternative source for pre-made and sterilized materials for this procedure</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dosing catheter system</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide cannula</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-GCA</td>
			<td></td>
		</tr>
		<tr>
			<td>Internal Catheters</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-INC</td>
			<td></td>
		</tr>
		<tr>
			<td>Stylet Wire</td>
			<td>SAI Infusion Systems</td>
			<td></td>
			<td>RIDC-STY</td>
			<td></td>
		</tr>
	</tbody>
</table>

intrathecal delivery of antisense oligonucleotides in the rat central nervous system

血脑屏障 （BBB） 是防止潜在毒性或致病剂从血液进入中枢神经系统 （CNS） 的重要防御手段。然而，它的存在也大大降低了系统管理的治疗剂对CNS的可及性。克服这一难题的一种方法是将这些制剂直接注入脑脊液（CSF），从而绕过BBB。这可以通过植入导管进行连续输注，使用渗透泵或单核聚子输送。在本文中，我们将描述通过直接植入成年大鼠脊柱的cauda equina空间的导管输送CNS靶向反义寡核苷酸（ASOs）的手术方案。作为代表性的结果，我们展示了通过该导管系统单次博鲁斯ASO内注射在大鼠CNS不同区域击倒目标RNA的功效。该程序安全、有效，不需要昂贵的设备或手术工具。此处描述的技术也可以用于以其他方式提供药物。

使用此处描述的方法，我们给两组成年雌性大鼠（250–300克;n = 10/组）注射了单一磷酸缓冲盐水PBS或300μgASO，针对长非编码（林肯）RNA Malat1;在我们的实验室中，我们经常使用Malat1 ASO作为工具化合物，因为马拉特1在所有组织（包括大脑和脊髓）中随处可见，并且含量很高。Malat1 ASO 通过 RNaseH1 介导机制15工作，该机制可降解 RNA，导致敲?...

Watch this Scientific Journal Video about Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System at JoVE.com

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

在这里，我们描述了一种通过将导管植入脊柱的腰间空间来向大鼠中枢神经系统输送药物的方法。我们专注于反义寡核苷酸的输送，尽管这种方法也适合其他治疗方式的交付。

大鼠中枢神经系统中反义寡核苷酸的细胞内传递

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...

该协议允许将治疗药物直接输送到大鼠的中枢神经系统，以评估大鼠疾病模型中新疗法的药代动力学、药理动力学和疗效。此过程安全、有效，不需要昂贵的设备或手术工具。演示程序将是易晨和易罗，我的实验室的研究科学家。要准备专用导轨，请使用带切断轮的旋转工具切断 19 量表针的两端，以获得大约 1 个半到 2 厘米长的导轨管。然后使用旋转工具的砂轮使两端平滑。要准备导管总成，请切割一块8厘米长、直径为011英寸的PE10管，作为每种动物的导管。使用防乙醇的标记笔，从每根管子的一端做两厘米的标记。对于每种动物，将一条 11 厘米长的线材从聚四氟乙烯涂层不锈钢丝切割到 8 厘米 PE10 导管的流明中。要准备输送导管组件，请切割一块直径为 023 英寸的 5 至 10 厘米 PE50 导管，并将 23 量轨导管适配器插入导管的一端。然后插入一个 30 量表针，将轮毂切断到大约 0.5 厘米的 PE10 油管中，并将 PE10 油管连接到导管的另一端。要准备导管针组件，请将导管放在 23 表针的端上。在开始手术之前，在手术台上放置一个加热垫，用无菌窗帘盖住垫。将 50mL 锥形离心管放在片上，确认麻醉实验鼠对头趾捏缺乏反应。称量动物后，将背部从尾巴剃到胸椎，然后将剃光的老鼠放在易发位置的无菌纸上。将 50mL 管放在腹部下，使脊柱在腰椎区域弯曲，并涂抹在眼睛的眼科软膏。接下来，皮下注射每公斤1毫克持续释放的布丙诺啡，并清洁暴露的皮肤与顺序的波维酮和酒精磨砂。用芬斯特化的无菌透明片将动物拖下水。对于导管的放置和复合物的注射，识别剃光的骨盆上方的肌肉之间的两个自然坑，用一只手握住凹坑，用另一只手轻轻按压和感觉脊柱从胸腔到旋转体方向，以定位椎骨之间的第一个主要凹痕。在确定S1和L6椎骨之间的椎间空间后，稍微移动一点，以识别L5和L6椎骨之间的椎间空间。使用手术刀，使一个不超过两厘米长的切口在这个部位的皮肤和肌肉胶囊沿中线从旋转到骨道方向，使注射部位将在切口的中心。使用解剖剪刀解剖结缔组织，直到肌肉层可以可视化。在肌肉胶囊中做一厘米切口，立即横向到L6腰椎的后脊柱过程。将导管针组件定位在第六个腰椎前侧附近，沿第六椎骨的前部将组件推入椎间空间，使针的端部穿透脊柱管。使用钝钳定位L6腰椎的背脊过程，然后沿着第六椎骨的前部将针头推入无脊椎空间。沿着针头将导管管推入原位，取出针头，并将导管丝组件插入导管。将导管以大约 45 度角向脊柱管倾斜，迫使导管的端端进入管中约 0.3 厘米。从导管的内部下部取出约 2.5 厘米的线，将导管推进到脊柱管中，直到导管上的两厘米标记在肌肉下方可见。当导管就位时，拆下导管管，使导管和样式线保持原位。将导管推进到脊柱管中，直到两厘米的标记位于运河的入口处，并完全收回样式线。脑脊髓液可以可视化进入植入导管。然后通过 30 量表针端将输送导管组件连接到植入导管的端端。接下来，将60微升无菌盐水装入100微升注射器，将30微升试验化合物的宝条装入第二个注射器中。将盐水注射器连接到输送导管组件的管状适配器端，将 20 微升无菌盐水冲洗到内部空间。当所有盐水都冲洗后，用装瓶的注射器更换盐水注射器，并在 30 秒内将 30 微升测试化合物注入内侧空间。当所有螺栓已交付后，用另一个盐水加载的注射器更换装有纤维的注射器，然后用另外 40 微升盐水冲洗导管。输送第二卷盐水后，将输送导管组件从植入导管中分离出来，并使用一对非常热的热消毒解剖钳夹住导管，以无菌切割和热封植入导管。使用可吸收的单丝缝合线将热密封导管固定到结缔组织，然后使用不可吸收的缝合线关闭皮肤。然后使用纱布和盐水清洗皮肤上的任何血液，并放置大鼠在加热培养箱与监测，直到完全恢复。在这个具有代表性的实验中，在单一玻利莫核苷酸传递后收集的所有区域都获得了很好的击倒。然而，在脊髓的敲击百分比最高时，测量了某种程度的区域变异性。在成功将反义寡核苷酸或其他治疗药物注射到适当的模型中后，可以评估药物动力学、药效学和治疗效果。

Research

JoVE Journal

Neuroscience

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

全身和局部给药治疗的中枢神经系统疾病的啮齿动物模型

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

科研

神经科学

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

视神经横断：一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

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. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

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 ...