Name: delivery of antibodies into the murine brain via convection-enhanced delivery
Uploaded: 2019-07-18T13:00:00
Description: Watch this Scientific Journal Video about Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery at JoVE.com

这项工作得到了苏黎世大学(FK-15-057)、诺华医学生物学研究基金会(16C231)和瑞士癌症研究(KFS-3852-02-2016,KFS-4146-02-2017)对约翰内斯·沃姆·伯格和BRIDGE概念证明(20B1-1)的资助。#177300) 给琳达·舍尔哈默。

此处描述的所有方法均已获得瑞士州兽医局批准,许可证号为 ZH246/15。
1. 步进导管的准备
<ol><li>为导管步骤制备熔融石英管
	<ol>
<li>将内径为 0.1 mm、壁厚为 0.0325 mm 的熔融硅毛细管切割至 30 mm 的长度。</li>
		<li>使用微锻件检查管道有无裂纹和热抛光,以确保管开口表面光滑。</li>
	</ol></li>
	<li>内管固定在金属针中
	<ol>
<li>将 27 G 针头安装在 10 μL 注射器...

<ol>
	<li>Scherrmann, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+via+the+blood-brain+barrier.">Drug delivery via the blood-brain barrier.</a> Vascular Pharmacology. 38 (6), 349-354 (2002).</li><li>Barua, N. U., Gill, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+drug+delivery:+prospects+for+glioblastoma+treatment.">Convection-enhanced drug delivery: prospects for glioblastoma treatment.</a> CNS Oncology. 3 (5), 313-316 (2014).</li><li>Bobo, R. H., 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=Convection-enhanced+delivery+of+macromolecules+in+the+brain.">Convection-enhanced delivery of macromolecules in the brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (6), 2076-2080 (1994).</li><li>Morrison, P. F., Laske, D. W., Bobo, H., Oldfield, E. H., Dedrick, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-flow+microinfusion:+tissue+penetration+and+pharmacodynamics.">High-flow microinfusion: tissue penetration and pharmacodynamics.</a> American Journal of Physiology. 266 (1 Pt 2), R292-R305 (1994).</li><li>Zhou, Z., Singh, R., Souweidane, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-Enhanced+Delivery+for+diffuse+intrinsic+pontine+glioma+treatment.">Convection-Enhanced Delivery for diffuse intrinsic pontine glioma treatment.</a> Current Neuropharmacology. 15 (1), 116-128 (2017).</li><li>Barua, N. U., 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=Intrastriatal+convection-enhanced+delivery+results+in+widespread+perivascular+distribution+in+a+pre-clinical+model.">Intrastriatal convection-enhanced delivery results in widespread perivascular distribution in a pre-clinical model.</a> Fluids and Barriers of the CNS. 9 (1), 2 (2012).</li><li>Shoji, T., 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=Local+convection-enhanced+delivery+of+an+anti-CD40+agonistic+monoclonal+antibody+induces+antitumor+effects+in+mouse+glioma+models.">Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models.</a> Neuro-Oncology. 18 (8), 1120-1128 (2016).</li><li>Souweidane, M. 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=Convection-enhanced+delivery+for+diffuse+intrinsic+pontine+glioma:+a+single-centre,+dose-escalation,+phase+1+trial.">Convection-enhanced delivery for diffuse intrinsic pontine glioma: a single-centre, dose-escalation, phase 1 trial.</a> The Lancet Oncology. , (2018).</li><li>Zhang, X., 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=Targeting+immune+checkpoints+in+malignant+glioma.">Targeting immune checkpoints in malignant glioma.</a> Oncotarget. 8 (4), 7157-7174 (2017).</li><li>Barua, N. U., Gill, S. S., Love, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+drug+delivery+to+the+brain:+therapeutic+potential+and+neuropathological+considerations.">Convection-enhanced drug delivery to the brain: therapeutic potential and neuropathological considerations.</a> Brain Pathology. 24 (2), 117-127 (2014).</li><li>Mehta, A. M., Sonabend, A. M., Bruce, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-Enhanced+Delivery.">Convection-Enhanced Delivery.</a> Neurotherapeutics. 14 (2), 358-371 (2017).</li><li>Krauze, M. T., 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=Reflux-free+cannula+for+convection-enhanced+high-speed+delivery+of+therapeutic+agents.">Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents.</a> Journal of Neurosurgery. 103 (5), 923-929 (2005).</li><li>Nash, K. R., Gordon, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection+Enhanced+Delivery+of+Recombinant+Adeno-associated+Virus+into+the+Mouse+Brain.">Convection Enhanced Delivery of Recombinant Adeno-associated Virus into the Mouse Brain.</a> Methods in Molecular Biology. 1382, 285-295 (2016).</li><li>Ohlfest, J. R., 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=Combinatorial+antiangiogenic+gene+therapy+by+nonviral+gene+transfer+using+the+sleeping+beauty+transposon+causes+tumor+regression+and+improves+survival+in+mice+bearing+intracranial+human+glioblastoma.">Combinatorial antiangiogenic gene therapy by nonviral gene transfer using the sleeping beauty transposon causes tumor regression and improves survival in mice bearing intracranial human glioblastoma.</a> Molecular Therapy. 12 (5), 778-788 (2005).</li><li>Yin, D., Forsayeth, J., Bankiewicz, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+cannula+design+and+placement+for+convection-enhanced+delivery+in+rat+striatum.">Optimized cannula design and placement for convection-enhanced delivery in rat striatum.</a> Journal of Neuroscience Methods. 187 (1), 46-51 (2010).</li><li>Mamot, 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=Extensive+distribution+of+liposomes+in+rodent+brains+and+brain+tumors+following+convection-enhanced+delivery.">Extensive distribution of liposomes in rodent brains and brain tumors following convection-enhanced delivery.</a> Journal of Neuro-Oncology. 68 (1), 1-9 (2004).</li><li>Saito, R., 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=Tissue+affinity+of+the+infusate+affects+the+distribution+volume+during+convection-enhanced+delivery+into+rodent+brains:+implications+for+local+drug+delivery.">Tissue affinity of the infusate affects the distribution volume during convection-enhanced delivery into rodent brains: implications for local drug delivery.</a> Journal of Neuroscience Methods. 154 (1-2), 225-232 (2006).</li><li>Oh, S., 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=Improved+distribution+of+small+molecules+and+viral+vectors+in+the+murine+brain+using+a+hollow+fiber+catheter.">Improved distribution of small molecules and viral vectors in the murine brain using a hollow fiber catheter.</a> Journal of Neurosurgery. 107 (3), 568-577 (2007).</li><li>Barua, N. U., 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=A+novel+implantable+catheter+system+with+transcutaneous+port+for+intermittent+convection-enhanced+delivery+of+carboplatin+for+recurrent+glioblastoma.">A novel implantable catheter system with transcutaneous port for intermittent convection-enhanced delivery of carboplatin for recurrent glioblastoma.</a> Drug Delivery. 23 (1), 167-173 (2016).</li><li>Rosenbluth, K. H., 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=Design+of+an+in-dwelling+cannula+for+convection-enhanced+delivery.">Design of an in-dwelling cannula for convection-enhanced delivery.</a> Journal of Neuroscience Methods. 196 (1), 118-123 (2011).</li><li>Debinski, W., Tatter, 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=Convection-enhanced+delivery+for+the+treatment+of+brain+tumors.">Convection-enhanced delivery for the treatment of brain tumors.</a> Expert Review of Neurotherapeutics. 9 (10), 1519-1527 (2009).</li><li>MacKay, J. A., Deen, D. F., Szoka, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+in+brain+of+liposomes+after+convection+enhanced+delivery;+modulation+by+particle+charge,+particle+diameter,+and+presence+of+steric+coating.">Distribution in brain of liposomes after convection enhanced delivery; modulation by particle charge, particle diameter, and presence of steric coating.</a> Brain Research. 1035 (2), 139-153 (2005).</li><li>Chen, Z. J., 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=A+realistic+brain+tissue+phantom+for+intraparenchymal+infusion+studies.">A realistic brain tissue phantom for intraparenchymal infusion studies.</a> Journal of Neurosurgery. 101 (2), 314-322 (2004).</li><li>Sampson, J. H., 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=Poor+drug+distribution+as+a+possible+explanation+for+the+results+of+the+PRECISE+trial.">Poor drug distribution as a possible explanation for the results of the PRECISE trial.</a> Journal of Neurosurgery. 113 (2), 301-309 (2010).</li><li>Wick, W., Weller, 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=Trabedersen+to+target+transforming+growth+factor-beta:+when+the+journey+is+not+the+reward,+in+reference+to+Bogdahn+et+al.+(Neuro-Oncology+2011;13:132-142).">Trabedersen to target transforming growth factor-beta: when the journey is not the reward, in reference to Bogdahn et al. (Neuro-Oncology 2011;13:132-142).</a> Neuro-Oncology. 13 (5), 559-560 (2011).</li><li>Saito, R., Tominaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convection-enhanced+delivery+of+therapeutics+for+malignant+gliomas.">Convection-enhanced delivery of therapeutics for malignant gliomas.</a> Neurologia Medico-Chirurgica. 57 (1), 8-16 (2017).</li><li>Bedussi, 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=Clearance+from+the+mouse+brain+by+convection+of+interstitial+fluid+towards+the+ventricular+system.">Clearance from the mouse brain by convection of interstitial fluid towards the ventricular system.</a> Fluids Barriers CNS. 12, 23 (2015).</li><li>Noroxe, D. S., Poulsen, H. S., Lassen, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+glioblastoma:+a+systematic+review.">Hallmarks of glioblastoma: a systematic review.</a> ESMO Open. 1 (6), e000144 (2016).</li><li>Boucher, Y., Salehi, H., Witwer, B., Harsh, G. R. t., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+fluid+pressure+in+intracranial+tumours+in+patients+and+in+rodents.">Interstitial fluid pressure in intracranial tumours in patients and in rodents.</a> British Journal of Cancer. 75 (6), 829-836 (1997).</li><li>Glushakova, O. Y., 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=Prospective+clinical+biomarkers+of+caspase-mediated+apoptosis+associated+with+neuronal+and+neurovascular+damage+following+stroke+and+other+severe+brain+injuries:+Implications+for+chronic+neurodegeneration.">Prospective clinical biomarkers of caspase-mediated apoptosis associated with neuronal and neurovascular damage following stroke and other severe brain injuries: Implications for chronic neurodegeneration.</a> Brain Circulation. 3 (2), 87-108 (2017).</li><li>Vom Berg, J., 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=Inhibition+of+IL-12/IL-23+signaling+reduces+Alzheimer's+disease-like+pathology+and+cognitive+decline.">Inhibition of IL-12/IL-23 signaling reduces Alzheimer's disease-like pathology and cognitive decline.</a> Nature Medicine. 18 (12), 1812-1819 (2012).</li><li>Vom Berg, J., 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=Intratumoral+IL-12+combined+with+CTLA-4+blockade+elicits+T+cell-mediated+glioma+rejection.">Intratumoral IL-12 combined with CTLA-4 blockade elicits T cell-mediated glioma rejection.</a> Journal of Experimental Medicine. 210 (13), 2803-2811 (2013).</li><li>Kurdi, 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=Continuous+administration+of+the+mTORC1+inhibitor+everolimus+induces+tolerance+and+decreases+autophagy+in+mice.">Continuous administration of the mTORC1 inhibitor everolimus induces tolerance and decreases autophagy in mice.</a> British Journal of Pharmacology. 173 (23), 3359-3371 (2016).</li></ol>

对流增强的输送,或压力介导的药物输注到大脑,首次提出在1990年初3。这种方法有望以可控的方式灌注血脑屏障后面的大脑容量2。然而,到目前为止,只有少数临床试验已经使用这种方法,部分是因为CED在临床设置已被证明是技术要求24,25。导管设计和输液方案的最新发展似乎已经克服了这些技术困难8,19。</s...

血脑屏障 (BBB) 形成一个半渗透边界,将中枢神经系统 (CNS) 与血液循环分离。然而,在各种疾病的背景下,如脑肿瘤、阿尔茨海默氏病(AD)或帕金森病(PD)等疾病,达到CNS是必要的。这成为新疗法开发的重要,特别是如果被测试的药物表现出较差的BBB渗透性或其全身暴露可能导致危险的毒性1,2。一些临床使用的抗体显示这两个功能。解决这个问题的方法是直接在BBB后面提供治疗药物。
对流增强输送(CED)是一种神经外科技术,能够有效灌注大脑容量。这是通过在目标区域通过手术安装一个或多个导管来实现的。在药物应用过程中,导管开口处形成压力梯度,成为组织3、4中迷剂的驱动力。因此,决定灌注范围2、4、5的不是输液的持续时间,而不是扩散系数。与传统的基于扩散的脑内注射方法2、6相比,这提供了在更大的脑体积上均匀地输送的内福剂。同时,这种分娩方式具有较低的组织损伤风险2。因此,CED 可以安全有效地给常规化疗药物用于治疗中枢神经系统肿瘤,以及提供免疫调节剂或许多其他中枢神经系统疾病中的抗性和对抗性抗体2 ,7,8,9.CED目前正在测试帕金森病,阿尔茨海默氏病,以及高档胶质瘤2,7,8,10,11的疗法。
导管设计和注射方案是影响CED 10、12、13、14、15、16结果的最重要因素之一。此外,它要求特定的物理化学性质,包括中等大小的颗粒,一个电子电荷,和低组织亲和力10,17。 这些参数中的每一个都必须根据大脑区域的组织特征进行潜在的调整,以瞄准2,10,17。
在这里,我们描述了将抗体溶液的CED执行到小鼠的牛锥(纹状体)的方法。此外,该协议包括在实验室设置中制备阶梯导管,在体外测试和执行CED。
文献中有多种导管设计,不同形状的导管、所用材料和导管开口数 12、15、18、19、20 ,21,22.我们使用的阶梯导管由熔融的二氧化硅毛细管制成,从钝端金属针伸出1毫米。这种导管设计可以很容易地在研究实验室制造,并可重复提供良好的CED结果,当在体外测试与基因胶块与物理参数类似于脑气囊在体内23。
此外,我们实施斜坡方案,在体内提供5μL的芬福。在这样的协议中,注射率从0.2μL/min提高到最大0.8μL/min,从而最大限度地减少了导管内液倒流的机会以及组织损伤的风险16。使用该协议,我们在11分钟30秒内成功地在5μL的PBS中用高达20μg的抗体给小鼠。
该协议可以很容易地调整其他输注量或注射各种其他物质,如化疗,细胞因子,病毒颗粒或脂质体2,10,14,18 ,22.如果使用与磷酸盐缓冲盐水 (PBS) 或人工脑脊液 (aCSF) 抗体溶液相比具有巨大物理化学特性的富化液,建议采取额外的验证步骤。对于导管组件、验证和 CED,我们使用一个立体定向机器人描述所有步骤,该机器人的钻头和喷射单元安装在常规立体定向框架上。此过程也可以通过连接到可编程微输液泵的手动立体定向框架执行,该泵可驱动所述玻璃微注射器。

<table>
	<tbody>
		<tr>
			<td>10 &#956;L syringe</td>
			<td>Hamilton</td>
			<td>7635-01</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G blunt end needle</td>
			<td>Hamilton</td>
			<td>7762-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Promega</td>
			<td>V3121</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezol</td>
			<td>Janssen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>Braun</td>
			<td>1029754</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Indivior Schweiz AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Pfizer AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dental drill bits, steel, size ISO 009</td>
			<td>Hager &#38; Meisinger</td>
			<td>1RF009</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Reuss-Chemie AG</td>
			<td>179-VL03K-/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Helvepharm AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Dextran, 2000 kDa</td>
			<td>Sigma Aldrich</td>
			<td>FD2000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Flumazenil</td>
			<td>Labatec Pharma AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>High viscosity cyanoacrylate glue</td>
			<td>Migros</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine solution</td>
			<td>Mundipharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidin</td>
			<td>Orion Pharma AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Roche Pharma AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Bausch + Lomb</td>
			<td>Vitamin A Blache</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFischer Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal goat anti-rat IgG (H+L) antibody coupled with Alexa Fluor 647</td>
			<td>Jackson Immuno</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Braun</td>
			<td>BB518</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica tubing internal diameter 0.1 mm, wall thickness of 0.0325 mm</td>
			<td>Postnova</td>
			<td>Z-FSS-100165</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame for mice</td>
			<td>Stoelting</td>
			<td>51615</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic robot</td>
			<td>Neurostar</td>
			<td>Drill and Injection Robot</td>
			<td></td>
		</tr>
		<tr>
			<td>Succrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Topical tissue adhesive</td>
			<td>Zoetis</td>
			<td>GLUture</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>ThermoFischer Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Bichsel</td>
			<td>1000004</td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of antibodies into the murine brain via convection-enhanced delivery

对流增强输送 (CED) 是一种神经外科技术,利用导管系统能够有效灌注大脑容量。这种方法提供了一种安全分娩方法,通过血脑屏障(BBB),从而允许治疗与不良BBB渗透性或那些不需要全身接触,例如,由于毒性的治疗。CED 要求优化导管设计、注射协议和内福的特性。通过该协议,我们描述了如何执行含有高达20μg的抗体的溶液的CED到小鼠的牛精。它描述了步进导管的制备,在体外测试,并使用斜坡注射程序在小鼠中执行CED。该协议可以很容易地调整其他输注量,并可用于注射各种示踪剂或药理活性或非活性物质,包括化疗剂、细胞因子、病毒颗粒和脂质体。

该协议允许制备步进导管 (图 1),用于实验室环境中的 CED 程序。为了控制导管的泄漏、沿着针道回流和堵塞,我们建议将染料(例如锥形蓝色溶液)注射到腺苷酸块中。图 3描述了使用 CED 导管在 0.5 μL/分钟下注入 1 μL 后形成锥形蓝色云(图 3A)。在导管步骤的开头,看不到沿着针道的回流。此外,分散的云形成了一个所需的球形。这与使?...

Watch this Scientific Journal Video about Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery at JoVE.com

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

对流增强输送(CED)是一种通过直接灌注大量组织,使治疗药物有效输送到大脑的方法。该过程需要使用导管和优化的注射程序。该协议描述了一种将抗体送入小鼠大脑的CED方法。

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 ...

该协议可用于使用步进导管系统对小啮齿动物进行对流增强传递，用于可调节的目标大脑区域的可调均匀灌注。对流增强的输送的优点是，它允许物质在绕过血脑屏障时进入感兴趣区域，而几乎没有组织损伤或回流。这种技术主要用于将抗体等治疗性物质输送到大脑。因此，可用于针对许多神经系统疾病。演示这个程序将是米哈尔·贝芬格，一个来自我实验室的博士后。首先切割一块内径为 0.1 毫米、壁厚为 0.325 毫米的熔融石英毛细管，长度为 30 毫米。检查管材有无裂纹后，使用微叉加热抛光两端，以确保管开口表面光滑。接下来，将 27 量针安装到 10 微升注射器上，将注射器放在立体定向机器人中。使用机器人将注射器移动到坚硬的表面上，然后用针尖触摸表面。注意到此位置后，提升针头，使融合的石英毛细管放置在针头内，使 20 毫米的毛细管从针头伸出。使用移液器将两个微升高粘度氰丙烯酸酯粘合剂均匀地铺在毛细管上，从金属针开始，在毛细管下端上方完成十毫米。使用立体定向机器人降低金属针，直到针尖在参考表面上一毫米，以固定金属针中熔融的石英毛细管，形成从金属针尖一毫米步。去除金属针端形成的任何多余的胶水，以避免插入导管步骤。在显微镜下检查尖端，确认所有多余的胶水都已去除。然后等待 15 分钟，让胶水硬化，然后用导管从机器人上取出注射器。对于步进导管测试，将凝固的0.6%的阿加罗斯凝胶切成20微20毫米块，并手动填充步进导管注射器与10微升过滤的0.4%trypan蓝色溶液。使用立体定向机器人，以每分钟 0.2 微升的速度分配一微升染料，以评估导管在固定过程中步骤的密封性。尝试蓝色溶液应仅在导管尖端可见。用纸纸巾擦去染料，在立体定向机器人中放置一个阿加罗斯块。校准机器人说，导管的尖端被引用在阿加罗斯块的表面。根据具体的实验计划设置注射量的比例序列。要将溶液注入 murine caudate putamen 中，请将注射设置为 1 毫米正面和 3.5 毫米深度的布雷格玛位置的 1 毫米到 2 毫米的横向。当针头就位时，启动对流增强的输送程序，将五微升的 trypan 蓝色溶液注入阿加罗斯块。评估红油中锥形蓝云的形状以及导管区域的潜在泄漏。金属针尖上没有大回流应可见。注射后，将导管留在到位两分钟，然后以每分钟一毫米的速度收回针头，以确保液体适当分散到大脑中，并在去除过程中密封注射区。将新的 agarose 块放入机器人中，然后以每分钟 0.2 微升的速度开始第二次喷射一微升，以评估 agarose 内导管的堵塞情况。注射开始后，锥形蓝色应立即从导管尖端再次形成云。然后评估注射器中的剩余体积是否对应于 3 微升，因为任何变化可能表明液体通过导管安装或注射器柱塞泄漏。用于将抗体注射到鼠皮线状体中确认对麻醉小鼠的皮肤捏合缺乏反应，并用修剪器剃光头部。用浸在碘溶液中的棉签消毒皮肤。使用手术刀在眼睛水平上沿着颅中线精加工 10 毫米的皮肤切口。使用鼻夹和耳杆将鼠标固定在立体形图框架中，注意头骨表面水平且安全。将注射器放在立体定向机器人中，并在参考点上将钻头与导管尖端同步。使用钳子缩回皮肤，并在头骨表面进行局部化。使用钻头在软件中参考啤酒，将钻头移动到一毫米正面和两毫米横向从钻头位置。钻一个毛刺孔，注意不要损坏杜拉母体，然后将注射器移过毛刺孔。从注射器中分配 0.5 至一微升，以确保导管中没有气泡。如所证明，开始对流增强传递，观察头骨表面是否有来自注射点的任何液体回流痕迹。在输送和导管拆卸结束时，以每分钟 0.2 微升的速度启动喷射泵，以检查喷射过程中导管堵塞的证据。如果未出现堵塞，应立即观察到来自导管尖端的注射液滴。在这张图像中，使用对流增强导管以每分钟 0.5 微升的速度注入一微升染料后形成的锥形蓝色云，在导管步骤开始时看不到沿针道的回流。分散的云形成了所需的球形。然而，在这张照片中，使用钝端针，可以观察到显著的回流。值得注意的是，与传统注射相比，对流增强的输送能够以统一和更少的组织损伤方式将大量体积灌入穆林脑中。在这两种类型的输送中，抗体和二分位粒子在语料库上都有典型的分布特征。然而，注射抗体的分散性特征比在对流增强传递后观察到的高分子量二分量更分散，这体现了不同不解物之间分布的差异。由于导管在脑输液过程中可能会堵塞，因此在缓慢分配少量灌注液后立即检查导管非常重要。这个过程可以作为一种向大脑提供药理活性化合物的方法，随后应密切监测疾病症状和预期的副作用。该技术能够注入精确的大脑区域与治疗，包括抗体，为中枢神经系统靶向疗法的发展开辟了新的可能性。

Research

JoVE Journal

Neuroscience

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 ...

科研

神经科学

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

基因传递到产后大鼠脑非心室质粒注射和电穿孔

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Miniosmotic植入泵和交货道示踪研究在病理情况下脑重组

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

鼠标显微注水技术进行有针对性的物质传递到中枢神经系统<i&gt;通过</i&gt;的颈内动脉

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...