Name: direct intrathecal injection of recombinant adeno-associated viruses in adult mice
Uploaded: 2019-02-15T13:00:00
Description: Watch this Scientific Journal Video about Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice at JoVE.com

这项工作的资金来自河北省人力资源和社会保障部的一笔赠款 (CY201605) 和河北省自然科学基金的赠款 (h201206101), 我们非常感谢高光平博士, 他为河北省提供了 aav。这项研究。

在河北神经内科重点实验室动物设施中培育了 fvb/nj 小鼠。所有的老鼠实验都是经河北医科大学第二医院伦理委员会批准的, 并按照中华人民共和国科学技术部颁布的实验动物管理规定进行的。中国。
1. 20% 盐酸利多卡因库存溶液的制备
<ol><li>称重2克盐酸利多卡因。加入5–6毫升的无菌水, 轻轻旋涡。用无菌水将体积增加到10毫升。</li>
	<li>1.2 通过0.2 微米过滤器过滤库存溶液?...

<ol>
	<li>Murlidharan, G., Samulski, R. J., Asokan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+adeno-associated+viral+vectors+in+the+central+nervous+system.">Biology of adeno-associated viral vectors in the central nervous system.</a> Frontiers in Molecular Neuroscience. 7, 76 (2014).</li><li>Lentz, T. B., Gray, S. J., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+gene+delivery+to+the+central+nervous+system.">Viral vectors for gene delivery to the central nervous system.</a> Neurobiology Disease. 48 (2), 179-188 (2012).</li><li>Yang, 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=Global+CNS+transduction+of+adult+mice+by+intravenously+delivered+rAAVrh.8+and+rAAVrh.10+and+nonhuman+primates+by+rAAVrh.10.">Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10.</a> Molecular Therapy. 22 (7), 1299-1309 (2014).</li><li>Guo, 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=A+Single+Injection+of+Recombinant+Adeno-Associated+Virus+into+the+Lumbar+Cistern+Delivers+Transgene+Expression+Throughout+the+Whole+Spinal+Cord.">A Single Injection of Recombinant Adeno-Associated Virus into the Lumbar Cistern Delivers Transgene Expression Throughout the Whole Spinal Cord.</a> Molecular Neurobiology. 53 (5), 3235-3248 (2016).</li><li>Hastie, E., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+at+50:+a+golden+anniversary+of+discovery,+research,+and+gene+therapy+success--a+personal+perspective.">Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success--a personal perspective.</a> Human Gene Therapy. 26 (5), 257-265 (2015).</li><li>Hocquemiller, M., Giersch, L., Audrain, M., Parker, S., Cartier, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-Associated+Virus-Based+Gene+Therapy+for+CNS+Diseases.">Adeno-Associated Virus-Based Gene Therapy for CNS Diseases.</a> Human Gene Therapy. 27 (7), 478-496 (2016).</li><li>Tanguy, 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=Systemic+AAVrh10+provides+higher+transgene+expression+than+AAV9+in+the+brain+and+the+spinal+cord+of+neonatal+mice.">Systemic AAVrh10 provides higher transgene expression than AAV9 in the brain and the spinal cord of neonatal mice.</a> Frontiers in Molecular Neuroscience. 8, 36 (2015).</li><li>Federic, 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=Robust+spinal+motor+neuron+transduction+following+intrathecal+delivery+of+AAV9+in+pigs.">Robust spinal motor neuron transduction following intrathecal delivery of AAV9 in pigs.</a> Gene Therapy. 19, 852-859 (2012).</li><li>Ayers, J. I., 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=Widespread+and+efficient+transduction+of+spinal+cord+and+brain+following+neonatal+AAV+injection+and+potential+disease+modifying+effect+in+ALS+mice.">Widespread and efficient transduction of spinal cord and brain following neonatal AAV injection and potential disease modifying effect in ALS mice.</a> Molecular Therapy. 23 (1), 53-62 (2015).</li><li>Li, D., 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=Slow+Intrathecal+Injection+of+rAAVrh10+Enhances+its+Transduction+of+Spinal+Cord+and+Therapeutic+Efficacy+in+a+Mutant+SOD1+Model+of+ALS.">Slow Intrathecal Injection of rAAVrh10 Enhances its Transduction of Spinal Cord and Therapeutic Efficacy in a Mutant SOD1 Model of ALS.</a> Neuroscience. 365, 192-205 (2017).</li><li>Borel, F., 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+rAAVrh10+Mediated+SOD1+Silencing+in+Adult+SOD1(G93A)+Mice+and+Nonhuman+Primates.">Therapeutic rAAVrh10 Mediated SOD1 Silencing in Adult SOD1(G93A) Mice and Nonhuman Primates.</a> Human Gene Therapy. 27 (1), 19-31 (2016).</li><li>Fairbanks, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+delivery+of+analgesics+in+experimental+models+of+pain+and+analgesia.">Spinal delivery of analgesics in experimental models of pain and analgesia.</a> Advanced Drug Delivery Reviews. 55 (8), 1007-1041 (2003).</li><li>Hylden, J. L., Wilcox, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrathecal+morphine+in+mice:+a+new+technique.">Intrathecal morphine in mice: a new technique.</a> European Journal of Pharmacology. 67, 313-316 (1980).</li><li>Wang, 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=Widespread+spinal+cord+transduction+by+intrathecal+injection+of+rAAV+delivers+efficacious+RNAi+therapy+for+amyotrophic+lateral+sclerosis.">Widespread spinal cord transduction by intrathecal injection of rAAV delivers efficacious RNAi therapy for amyotrophic lateral sclerosis.</a> Human Molecular Genetics. 23 (3), 668-681 (2014).</li><li>Wang, 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=scAAV9-VEGF+prolongs+the+survival+of+transgenic+ALS+mice+by+promoting+activation+of+M2+microglia+and+PI3K/Akt+pathway.">scAAV9-VEGF prolongs the survival of transgenic ALS mice by promoting activation of M2 microglia and PI3K/Akt pathway.</a> Brain Research. 1648, 1-10 (2016).</li></ol>

从技术上讲, 在清醒小鼠的 it 注射过程中, 有几个关键步骤。首先, 在整个手术过程中, 正确的手势和对老鼠的坚定控制是成功分娩的先决条件。其次, 最困难的一点是用针尖感觉椎间间隙, 因为在伤害动物或弯曲针尖的情况下, 有必要不要在没有阻力的情况下插入太深或在强阻力下强行插入。第三, 尽管利多卡因短暂的瘫痪为 it 注射质量提供了一个客观的指标, 但还需要更多的实践来取得一致和成?...

aav 可以介导中枢神经系统转导中长期广泛的基因表达, 副作用不大, 因此已成为治疗包括肌萎缩侧索硬化症 (als) 在内的中枢神经系统疾病最有希望的载体之一。疾病 (hd)、阿尔茨海默病 (ad)、溶酶体储存疾病 (lsd)、高彻病 (gd) 和神经元类癫痫 (ncl)1。目前, 已有 100多个 aav 血清型从人类和动物中分离出来。其中, 至少有12种已用于临床前和临床试验, 包括最常用的基因载体, 如 aav1、2、4、5、6、8、9、raavrh.8 和 rAAVrh.8.101、2、3、4、 5,6。
由于受影响的中枢神经系统区域和细胞类型不同, 不同的中枢神经系统疾病需要不同的 aav 传递策略。aav 可以传递的中枢神经系统区域和细胞类型因血清类型和传递方法的不同而不同。例如, raavrh10 已被证明可以通过全身静脉注射 (iv) 传播主要的星形胶质细胞, 而当通过鞘内注射4,7时, 它传递神经元和胶质细胞。此外, 实质注射导致局部转导到注射部位附近, 而通过脑室内或鞘内注射进入脑脊液 (csf) 导致广泛的中枢神经系统转导8.研究还证明了 aav 提供的基因治疗的治疗能力神经退行性疾病 9,10,11。在影响中枢神经系统广泛领域的疾病中, 如 als, 与全身分娩方法 4,10 相比, 鞘内注射到脑脊液中的药物中注射已证明覆盖了大多数受疾病影响的地区,剂量较低。最近的研究还表明, 腰椎穿刺可用于在 als 小鼠模型中注射 aav, 从而避免了与椎板切除术和鞘内导管插入4相关的潜在损伤.
实验性直接腰椎穿刺于 1885年12,13首次用于向脊髓输送药物, 特别是麻醉剂, 用于镇痛和麻醉。在本报告中, 我们说明了腰椎穿刺 it 注射方法的成人小鼠的援助1% 利多卡因盐酸利多卡因, 一种局部胺衍生麻醉剂, 在注射溶液中评估和监测注射质量。成功注射的特点是脂质引起的短暂性麻痹, 而失败的注射并没有显示这种行为。我们将瞬态弱点的水平分为五个等级之一, 以帮助预测注射效率。最后, 我们表明, raavrh10 转导水平可以通过瘫痪等级来预测。因此, 这种鞘内 aav 传递方法可用于增强 aava 介导的基因传递, 用于中枢神经系统疾病的实验治疗。

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	<tbody>
		<tr height="32">
			<td height="32" style="height:32px;width:284px;">FVB/NJ mice</td>
			<td style="width:219px;">Charles River Laboratories China</td>
			<td style="width:199px;"></td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Lidocaine hydrochloride monohydrate</td>
			<td>HEOWNS</td>
			<td>73-78-9</td>
			<td></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;">AAV</td>
			<td style="width:417px;">Viral Vector Core of the Gene Therapy Center at University of Massachusetts Medical School</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">25&#181;L&#160; Hamilton syringe/27-30g needle</td>
			<td>GASTIGHT</td>
			<td>1702</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">O.C.T compond</td>
			<td>SAKURA</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">H 2O 2</td>
			<td>SHUI HUAN PAI</td>
			<td>170401</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Goat serum</td>
			<td>Solarbio</td>
			<td>S9070</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Triton X-100</td>
			<td>LIFE SCIENCES</td>
			<td>T8200</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Rabbit anti-GFP</td>
			<td>Life tech</td>
			<td>G10362</td>
			<td>1:333 dilution</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">The second antibody (goat-anti rabbit)</td>
			<td>Jackson Immuno Research</td>
			<td>111-005-144</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">VECTASTAIN ABC REAGENT</td>
			<td>Vector Lab</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">ImmPACT DAB Peroxidase Substrate Kit</td>
			<td>Vector Lab</td>
			<td>SK-4105</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Mounting medium for fluorescence with DAPI</td>
			<td>Vectorshield</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:284px;">NaCl</td>
			<td>Yong Da Chemical</td>
			<td style="width:199px;"></td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:284px;">NaH2PO4&#183;2H2O</td>
			<td>Yong Da Chemical</td>
			<td style="width:199px;"></td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:284px;">Na2HPO4&#183;12H2O</td>
			<td>Yong Da Chemical</td>
			<td style="width:199px;"></td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Paraformaldehyde</td>
			<td>Yong Da Chemical</td>
			<td>307699</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Adhesion Microscope Slides</td>
			<td>CITOGLAS</td>
			<td>17083</td>
			<td>25*75 mm</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">SUPER-SLIP MICRO-GLAS</td>
			<td>Electro Microscopy Siences</td>
			<td>72236-60</td>
			<td>24*60 mm</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">15 ml Centrifuge tube</td>
			<td>CORNING</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">96 well cell culture cluster</td>
			<td>Coster</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">24 well cell culture cluster</td>
			<td>Coster</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">70% Ethanol</td>
			<td>WEN ZHI</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Gauze</td>
			<td>Wei AN</td>
			<td>05171112</td>
			<td>8cm*10cm*12cm</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">1mL syringe</td>
			<td>Hong Da</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:284px;">Microtubes</td>
			<td>Plasmed</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Micropipet&#160;</td>
			<td>eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Peppet tips</td>
			<td>Rainin</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Centirifuge</td>
			<td>eppendorf</td>
			<td>5427R</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Regerator</td>
			<td>Haier</td>
			<td>BCD-539WT</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Filter</td>
			<td>MILLEX GP</td>
			<td>R4PA42342</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:284px;">Pump</td>
			<td style="width:219px;">LongerPump</td>
			<td style="width:199px;">BT-100-2J/YZ1515X</td>
			<td style="width:131px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Microscope</td>
			<td>Olympus</td>
			<td>BX53</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:284px;">Freezing-microtome</td>
			<td style="width:219px;">Leica</td>
			<td style="width:199px;">CM1520</td>
			<td style="width:131px;"></td>
		</tr>
	</tbody>
</table>

direct intrathecal injection of recombinant adeno-associated viruses in adult mice

腺相关病毒 (aav) 的鞘内注射由于其安全性强、无创性和良好的转导效果, 在中枢神经系统基因治疗中引起了人们的极大兴趣。先前的研究已经证明了 aav 提供的基因治疗的治疗能力神经退行性疾病的 it 管理。然而, 据报道, 由于小动物信息技术管理的技术限制, 不可预测的故障率很高。在这里, 我们建立了一个评分系统, 以表明成功的程度, 腰椎穿刺在小动物通过添加1% 盐酸利多卡因注射溶液。进一步表明, 注入后瞬态弱点的程度可以预测 aav 的传导效率。因此, 该 it 注射方法可用于优化影响中枢神经系统广大区域的中枢神经系统疾病小鼠模型的治疗试验。

由于鞘内注射质量的不同, 在1% 利多卡因盐酸1% 的利多卡因注射 aav 溶液后, 小鼠表现出不同程度的瞬态虚弱。根据我们建立的半定量5级评分系统, 我们测试了不同程度的脂质诱导肢体无力小鼠 aav 的传导模式 (评分 0, n = 2; 得分 1, n = 1; 得分 4, n = 4; 评分 5, n = 3)。egfp 脊髓免疫染色显示小鼠腰椎脊髓无传导或很少转导得分为 0, 小鼠的转导略有增强, 得分为4或5的小鼠的转导性?...

Watch this Scientific Journal Video about Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice at JoVE.com

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

在这里, 我们提出了一个直接鞘内注射技术使用1% 利多因盐酸在病毒溶液中, 以确保有效的腺病毒传递给小动物, 并建立评分系统, 以预测在中央的传导效率神经系统根据利多卡因引起的短暂虚弱程度。

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, ...

这种方法有助于在清醒的小动物中有效传递与腺相关的病毒转基因，用于实验性治疗中枢神经系统疾病，如ALS。这种方法的主要优点是，病毒溶液包括1%利多基盐酸盐，在注射成功后诱导暂时性瘫痪。虽然此方法为判断内部自动化情况的成功率提供了视觉指标，但足够的实践对于提高交付的成功率至关重要。在开始手术之前，将 27 量针连接到 25 微升汉密尔顿注射器上，然后将针头的斜端与注射器上的体积刻度对齐。然后小心地将8微升的4×10到病毒溶液的第10个基因组拷贝放入注射器中，注意避免气泡。接下来，将一只清醒的30至70天12至30克的老鼠放在生物安全罩易发位置的床上，用无菌纱布覆盖上半身，使老鼠平静下来，避免被咬伤。用拇指一侧紧紧抓住鼠标的骨盆腰带，另一侧用食指和中指，用拇指和食指将皮紧绷在双边骨盆腰带之间。然后剃掉双边骨盆腰带之间的毛皮，用碘基磨砂和70%乙醇对裸露的皮肤进行消毒。对于腺相关病毒溶液的直接内部传递，在双边骨盆腰围之间的中线上对椎间空间进行点心，并使用指甲压入皮肤，以指示L5至L6椎间空间。轻轻旋转尾巴底部，露出脊柱的中线，并调整针斜向动物头部。将鼠标牢固固定到位，沿脊柱中线对齐针头，然后轻轻地垂直地将针头插入缩进中心，使注射器处于中央下垂平面中。当针头与骨骼接触时，慢慢将角度减小至约 30 度，然后将针头滑入椎间空间。突然的尾部轻拂是成功进入硬膜内空间的标志。当针头进入椎间空间时，尖端会感觉被牢牢地夹住。注入向量溶液，并在硬膜内空间内保持针头一分钟。然后缓慢地旋转地提取针头，以尽量减少泄漏。立即对小鼠四肢的短暂无力进行评分，以评估注射质量，并返回小鼠的笼子，从瘫痪中恢复。在适当的实验终点点，将注射小鼠的四肢和头部固定到泡沫盒盖上的易发位置，并使用剪刀将皮肤从头部剥离到囊中。将头骨夹在眼睛之间，沿着头骨的中线和小脑上方的水平线切割，并打开两侧的头骨。使用钳子取出腹骨，并使用眼科剪刀双边打开脊柱管。切开两侧的肋骨，小心地取下椎骨的上部。使用弯曲的钳子抬起大脑，切断头骨基的神经。然后小心提取整个大脑和脊髓，将组织修复为4%的甲醛24小时。在固定期结束时，在4摄氏度下，在30%蔗糖溶液中冷冻保护大脑、颈椎和腰椎，然后嵌入最佳切割温度化合物中，然后捕捉到液氮中的冷冻。然后使用低温计温器获取每个组织25微米厚的部分，将冷冻部分储存在0.01摩尔PBS中，在4摄氏度时。对于组织的免疫组织化学分析，用1%过氧化氢预处理自由浮动部分10分钟，然后用PBS清洗10分钟。接下来在PBS中孵育含有5%血清和0.3%非离子洗涤剂的阻断溶液中的样品1小时，随后在4摄氏度下用适当的原抗体进行过夜标记。第二天，用3个10分钟的PBS加Tween洗涤部分，并在最后一次洗涤后，用适当的相应的生物基化二次抗体孵育幻灯片，持续1小时。在孵育结束时，在新鲜 PBST 中清洗部分 3 次，正如刚刚演示的，用无限生物素过氧化物酶复合物孵育样品 40 分钟，然后用适当的色度剂染色。将标记部分安装到玻璃显微镜幻灯片上，并在安装部分完全干燥后将幻灯片浸泡在无水乙醇中 5 分钟。接下来，将幻灯片浸泡在二甲苯中 10 分钟，然后用适当的安装介质密封。然后用配备充电耦合装置的光学显微镜以 100、200 和 400 倍的放大倍率对幻灯片进行成像。eGFP对颈椎和腰椎组织部分进行免疫损伤，发现弱分为0的小鼠腰椎脊髓很少或没有转导，弱分为1的小鼠的转导率略有增强，弱分为4或5的小鼠的转导率则略有增强。显示不同程度瞬态肢体弱点的小鼠脊髓组织部分的GGFP染色强度的定量表明，病毒溶液注射后虚弱的严重程度与脊髓转导的程度密切相关。在大脑中，在嗅球、多侧前额皮质、凹陷陀螺和海马、小脑皮质和脑干边缘区域（包括面部核、胆管丛和阴性上皮细胞）的CA3区域检测到强大的eGFP信号。前角的运动神经元在脊髓的不同水平上也强烈转导。此外，在皮层中，检测到包括金字塔细胞在内的GP正神经元以及各种胶质细胞类型，包括微胶质、星形细胞和橄榄细胞。这项技术使神经学领域的研究人员能够探索小觉醒动物直接在内分娩对中枢神经系统疾病的治疗效果。

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

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. ...

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 ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial Direct Current Stimulation tDCS in Mice

经颅直流电刺激 (tDCS) 在小鼠中的作用

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

与腺相关病毒的向量的生产、纯化和质量控制

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

双直接注射血液到西斯特纳麦格作为苏巴拉奇内德出血的模型

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

使用腺相关病毒在小鼠中瞄准科菲林操纵的大脑皮层区域的电皮质记录

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

DREADDs Technology and Automated Blood Sampling

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Author Spotlight: Automated Infusion and Blood Sampling for Precise Hormonal Analysis in Conscious Mice

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of ...

Intracerebroventricular Injection in Mice

Intracerebroventricular Injection in Mice (Video) | JoVE 

小鼠脑室内注射

A Comprehensive Protocol for Delivering Intrathecal Injections in Neonatal Mice

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Author Spotlight: Intrathecal Injection &#8211; An Efficient and Reliable Delivery Method to Test the Efficacy of Gene Editing in Neonatal Mouse Brains

新生小鼠鞘内注射用于基因组编辑和药物递送

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...