Name: subpial adeno-associated virus 9 (aav9) vector delivery in adult mice
Uploaded: 2017-07-13T12:30:00
Description: Watch this Scientific Journal Video about Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice at JoVE.com

Este estudo foi apoiado pela bolsa SANPORC e ​​ALSA Foundation (Martin Marsala); Programa Nacional de Sustentabilidade, número de projeto LO1609 (Ministério da Educação, Juventude e Desporto); E RVO: 67985904 (Stefan Juhas e Jana Juhasova).

Esses estudos foram realizados sob um protocolo aprovado pelo Comitê Institucional de Cuidados e Uso Animal da Universidade da Califórnia, em San Diego, e estavam em conformidade com a Associação para Avaliação de diretrizes de Cuidados com animais de laboratório para uso em animais. Todos os estudos foram realizados de forma a minimizar o tamanho do grupo e o sofrimento dos animais. 1. Preparação geral de animais e cirurgiões <ol><li> Antes de iniciar o procedimento cir?...

<ol>
	<li>Foust, K. 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=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat Biotechnol. 27 (1), 59-65 (2009).</li><li>Duque, 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=Intravenous+administration+of+self-complementary+AAV9+enables+transgene+delivery+to+adult+motor+neurons.">Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.</a> Mol Ther. 17 (7), 1187-1196 (2009).</li><li>Gray, S. 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=Preclinical+differences+of+intravascular+AAV9+delivery+to+neurons+and+glia:+a+comparative+study+of+adult+mice+and+nonhuman+primates.">Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates.</a> Mol Ther. 19 (6), 1058-1069 (2011).</li><li>Meyer, K., 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=Improving+single+injection+CSF+delivery+of+AAV9-mediated+gene+therapy+for+SMA:+a+dose-response+study+in+mice+and+nonhuman+primates.">Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose-response study in mice and nonhuman primates.</a> Mol Ther. 23 (3), 477-487 (2015).</li><li>Foust, K. 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=Therapeutic+AAV9-mediated+suppression+of+mutant+SOD1+slows+disease+progression+and+extends+survival+in+models+of+inherited+ALS.">Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS.</a> Mol Ther. 21 (12), 2148-2159 (2013).</li><li>Passini, M. 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=Translational+fidelity+of+intrathecal+delivery+of+self-complementary+AAV9-survival+motor+neuron+1+for+spinal+muscular+atrophy.">Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy.</a> Hum Gene Ther. 25 (7), 619-630 (2014).</li><li>Bell, P., 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=Motor+neuron+transduction+after+intracisternal+delivery+of+AAV9+in+a+cynomolgus+macaque.">Motor neuron transduction after intracisternal delivery of AAV9 in a cynomolgus macaque.</a> Hum Gene Ther Methods. 26 (2), 43-44 (2015).</li><li>Miyanohara, 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=Potent+spinal+parenchymal+AAV9-mediated+gene+delivery+by+subpial+injection+in+adult+rats+and+pigs.">Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.</a> Mol Ther Methods Clin Dev. 3, 16046 (2016).</li><li>Xu, Q., 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=In+vivo+gene+knockdown+in+rat+dorsal+root+ganglia+mediated+by+self-complementary+adeno-associated+virus+serotype+5+following+intrathecal+delivery.">In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery.</a> PLoS One. 7 (3), 32581 (2012).</li><li>Xiao, X., Li, 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=Production+of+high-titer+recombinant+adeno-associated+virus+vectors+in+the+absence+of+helper+adenovirus.">Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.</a> J Virol. 72 (3), 2224-2232 (1998).</li></ol>

O presente estudo descreve uma técnica de entrega de vector subpial (AAV9) em camundongos adultos. Conforme demonstrado no vídeo acompanhante, esta abordagem e técnica podem ser efetivamente utilizadas, desde que os instrumentos necessários e a agulha pia penetrante e a agulha de injeção subpial sejam devidamente fabricadas, de acordo com as especificações estabelecidas e testadas. Variáveis ​​técnicas críticas na realização de uma injeção subpial consistente e seg...

O uso de vetores de AAV para tratar uma variedade de distúrbios neurodegenerativos da medula espinhal e do SNC está se tornando uma plataforma bem aceita para efetivamente aumentar ou silenciar a expressão dos genes de interesse. Uma das principais limitações para a utilização mais efetiva desta tecnologia para o tratamento de distúrbios do SNC / medula espinhal é a capacidade limitada de fornecer vetores AAV para o cérebro profundo ou parênquima da medula espinhal em mamíferos adultos. Foi demonstrado, por exemplo, que a distribuição sistêmica de AAV9 em roedores adultos, gatos ou primatas não humanos é apenas moderadamente eficaz para induzir a expressão do transgene nos neurônios do cérebro e da medula espinhal 1 , 2 , 3 . A administração intratecal mais efetiva de vetores AAV9 também mostrou que leva a apenas expressão limitada de transgenes em conjuntos de neurônios anatomicamente definidos. Mais especificamente, tem sido demôniosQue o fornecimento de AAV9 intracelular cisterno ou lumbo-sacral em primatas, porcos ou roedores não humanos leva a um alto nível de expressão do transgene nos motoneurônios α da espinha e neurônios ganglionares da raça dorsal segmentar. No entanto, observa-se uma expressão mínima ou nenhuma em interneurônios espinhais ou axônios ascendentes ou descendentes na matéria branca 4 , 5 , 6 , 7 . Coletivamente, esses dados mostram que existe uma barreira biológico-anatômica altamente efetiva, o que impede a difusão do AAV entregue por via intratéfica no parênquima espinhal mais profundo. Em um estudo anterior, utilizando ratos e porcos adultos, desenvolveu-se uma nova técnica de entrega de vetor subpial 8 . Ao usar essa abordagem, a expressão de transgene altamente potente e multi-segmentar foi demonstrada após uma entrega de AAV9 subpial de bolus único. A expressão GFP intensa foi consistentemente vistaNos neurônios, células gliais e axônios descendentes / ascendentes através dos segmentos espinhais injetados. Este estudo demonstrou pela primeira vez que a pia mater representa a barreira principal que limita a difusão efetiva de AAV9 no parênquima espinhal do espaço intratecal. Embora esta técnica previamente desenvolvida e o dispositivo de injeção subpial sejam relativamente fáceis de usar em roedores grandes (como ratos) ou porcos adultos, o sistema não é adequado para uso em animais pequenos, como ratos adultos. Devido ao elevado número de modelos de ratos transgênicos disponíveis de uma variedade de distúrbios neurodegenerativos, existe uma necessidade clara de desenvolvimento de uma técnica efetiva de parto espinhal parenquimatoso em camundongos. A disponibilidade de tal técnica permitiria o estudo do efeito de silenciamento de genes específicos ( por exemplo, usando shRNA) ou upregulation usando células não específicas ( por exemplo, citomegalovírus-CMV ou Ubiquitin) ou específicas de células ( por exemplo, sinapsina ou glial Ácido fibrilarProteína (GFAP)) durante o desenvolvimento pós-natal inicial ou em condições doentes. Conseqüentemente, no presente estudo, desenvolvemos e validamos um sistema de entrega de vetor subpial em miniatura que pode ser efetivamente usado em camundongos adultos. Da mesma forma, como em estudos anteriores de ratos e porcos, este trabalho demonstra uma expressão de transgênese potente ao longo do parênquima espinhal após uma entrega de AAV9 subpial de bolus único em camundongos. A simplicidade desta abordagem, a tolerabilidade muito boa dos ratos injetados à entrega de AAV9 subpial e a alta potência da expressão do transgene no parênquima espinal sugerem que esta técnica pode efetivamente ser implementada em qualquer configuração de laboratório e utilizada em experimentos visando a expressão do gene espinhal.

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			<td height="21" style="height:21px;width:255px;">C57BL/6J Mice</td>
			<td style="width:163px;">Jackson Labs</td>
			<td align="right" style="width:199px;">664</td>
			<td style="width:192px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Lab Standard Stereotaxic for Mice</td>
			<td style="width:163px;">Harvard Apparatus</td>
			<td style="width:199px;">72-9568</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Mouse Spinal Adaptor</td>
			<td style="width:163px;">Harvard Apparatus</td>
			<td style="width:199px;">72-4811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XYZ Manipulator</td>
			<td style="width:163px;">Stoelting</td>
			<td align="right" style="width:199px;">51604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Manual Infusion Pump</td>
			<td style="width:163px;">Stoelting</td>
			<td align="right" style="width:199px;">51218</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:255px;">34G Beveled Nanofill Needle</td>
			<td style="width:163px;">World Precision Instruments</td>
			<td style="width:199px;">NF34BV-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">36G Blunt Nanofill needle</td>
			<td style="width:163px;">World Precision Instruments</td>
			<td style="width:199px;">NF-36BL-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:255px;">Fluriso, Isoflurane</td>
			<td style="width:163px;">MWI Veterinary Supply</td>
			<td align="right" style="width:199px;">502017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Chlorhexidine Solution</td>
			<td style="width:163px;">MWI Veterinary Supply</td>
			<td align="right" style="width:199px;">501027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">20G Stainless Steel Needle</td>
			<td style="width:163px;">Becton-Dickinson</td>
			<td align="right" style="width:199px;">305175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">23G Stainless Steel Needle</td>
			<td style="width:163px;">Becton-Dickinson</td>
			<td align="right" style="width:199px;">305145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">30G Stainless Steel Needle</td>
			<td style="width:163px;">Becton-Dickinson</td>
			<td align="right" style="width:199px;">305128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Cotton Tipped Applicator</td>
			<td style="width:163px;">MWI Veterinary Supply</td>
			<td align="right" style="width:199px;">27426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glass Capillary Beveller&#160;</td>
			<td style="width:163px;">Narishige International</td>
			<td style="width:199px;">SM-25B</td>
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			<td height="21" style="height:21px;">Slide Microscope Superfrost</td>
			<td style="width:163px;">Leica Microsystems</td>
			<td style="width:199px;">M80</td>
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			<td height="21" style="height:21px;">50&#956;l Microsyringe&#160;</td>
			<td style="width:163px;">Hamilton</td>
			<td align="right" style="width:199px;">81242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BD Intramedic PE-20 Tubing</td>
			<td style="width:163px;">Becton, Dickinson</td>
			<td align="right" style="width:199px;">427406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BD Intramedic PE-10 Tubing</td>
			<td style="width:163px;">Becton, Dickinson</td>
			<td align="right" style="width:199px;">427401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 monofilament suture</td>
			<td style="width:163px;">VetOne</td>
			<td style="width:199px;">V1D397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Capillary Beveller&#160;</td>
			<td>Narishige</td>
			<td>Pipet Micro Grinder EG-40&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">5 min Epoxy (Epoxy Clear)</td>
			<td style="width:163px;">Devcon</td>
			<td align="right" style="width:199px;">14310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Euthanasia Solution</td>
			<td style="width:163px;">MWI Veterinary Supply</td>
			<td align="right" style="width:199px;">11168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin Inj 1000U/mL</td>
			<td style="width:163px;">MWI Veterinary Supply</td>
			<td align="right" style="width:199px;">54254</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td align="right" style="width:199px;">158127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:199px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Anti NeuN Antibody</td>
			<td style="width:163px;">EMD-Millipore</td>
			<td style="width:199px;">ABN78</td>
			<td>Primary Rabbit Polyclonal Antibody, 1:1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Anti-Choline Acetyltransferase (CHAT) Antibody</td>
			<td style="width:163px;">EMD-Millipore</td>
			<td style="width:199px;">AB144P</td>
			<td>Primary Goat Polyclonal Antibody, 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Anti GFP Antibody</td>
			<td style="width:163px;">Aves Labs</td>
			<td style="width:199px;">GFP-1020</td>
			<td>Primary Chicken Polyclonal Antibody, 1:1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594</td>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<td style="width:199px;">A21207</td>
			<td>Secondary Antibody, 1:1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">&#160;Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 680</td>
			<td style="width:163px;">ThermoFisher Scientific</td>
			<td style="width:199px;">A10043</td>
			<td>Secondary Antibody, 1:1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Donkey anti-Chicken IgY Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:163px;">Jackson Immunoresearch Labs</td>
			<td style="width:199px;">703-545-155</td>
			<td>Secondary Antibody, 1:1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Donkey Anti-Goat IgG H&#38;L (Alexa Fluor 647</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:199px;">ab150131</td>
			<td>Secondary Antibody, 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Slide Microscope Superfrost</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:199px;">12-550-143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ProLong Gold Antifade Mountant</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:199px;">P36930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Epifluorescence Microscope</td>
			<td style="width:163px;">Zeiss</td>
			<td style="width:199px;">Zeiss AxioImager M2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fluorescence Confocal Microscope</td>
			<td style="width:163px;">Olympus</td>
			<td style="width:199px;">Olympus FV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Dextran</td>
			<td style="width:163px;">Polysciences, Inc</td>
			<td align="right" style="width:199px;">19411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAV9-UBC-GFP</td>
			<td colspan="2">UCSD Viral Vector Core Laboratory</td>
			<td></td>
		</tr>
	</tbody>
</table>

subpial adeno-associated virus 9 (aav9) vector delivery in adult mice

O desenvolvimento bem-sucedido de uma técnica de entrega do vetor de adeno-associado subpial 9 (AAV9) em ratos adultos e porcos foi relatado anteriormente. Utilizando cateteres de polietileno colocados subpialmente (PE-10 ou PE-5) para a entrega de AAV9, demonstrou-se uma expressão potente do transgene através do parênquima espinhal (substância branca e cinzenta) em segmentos espinhais injetados subpialmente. Devido à ampla gama de modelos de ratos transgênicos de doenças neurodegenerativas, há um forte desejo pelo desenvolvimento de uma técnica de entrega de vetor segmentado do sistema nervoso central potente (SNC) em camundongos adultos. Consequentemente, o presente estudo descreve o desenvolvimento de um dispositivo de entrega de vetor subpial da coluna vertebral e técnica para permitir a administração segura e eficaz de AAV9 espinhal em ratos C57BL / 6J adultos. Nos ratinhos imobilizados e anestesiados, o pia mater (nível cervical 1 e segmentar 1-2) foi inciso com uma agulha afiada de 34 G usando um manipulador XYZ. Um segundo XYZ maO nipulador foi então usado para avançar uma agulha 36G brusca no espaço subpial lombar e / ou cervical. O vetor AAV9 (3-5 μL; 1,2 x 10 13 cópias do genoma (gc)) que codificam a proteína fluorescente verde (GFP) foi então injetado subpialmente. Após as injeções, a função neurológica (motor e sensorial) foi avaliada periodicamente, e os animais foram fixados por perfusão 14 dias após a administração de AAV9 com paraformaldeído a 4%. A análise das seções da corda espinal horizontal ou transversal mostrou expressão do transgene em toda a medula espinhal, tanto em matéria cinza quanto em substância branca. Além disso, observou-se uma intensa expressão de GFP mediada por retrogradabilidade nos axônios motores descendentes e nos neurônios no córtex motor, no núcleo e na formatio reticular. Não foi observada nenhuma disfunção neurológica em nenhum animal. Esses dados mostram que a técnica de entrega do vetor subpial pode ser usada com sucesso em camundongos adultos, sem causar lesão da medula espinhal relacionada ao procedimento, e está associada a expressões de transgênese altamente potentesDurante toda a neuraxis da coluna vertebral.

<html> Expressão Transgene Potente em Subpialmente Segmentos Injetados com AAV9: A análise da expressão de transgene (GFP) nas secções da medula espinhal aos 14 dias após a administração de AAV9 apresentou expressão de GFP dependente de AAV9 ao longo do parênquima espinhal. Primeiro, duas injeções bilaterais de 3 μL de AAV9-UBI-GFP injetadas no espaço subpial lombar superior foram associadas à infecção quase completa da matéria branca e cinzenta e...

Watch this Scientific Journal Video about Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice at JoVE.com

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

O objetivo do presente estudo foi desenvolver e validar a potência e a segurança da entrega de genes mediada pelo vírus adeno-associado à coluna vertebral (AAV9) usando uma nova técnica de entrega de genes subpial em camundongos adultos.

Vedação subjugada Adeno-associada do vírus 9 (AAV9) em ratos adultos

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

The overall goal of this subpial AAV9 delivery technique is to achieve a potent spinal parenchymal transgene expression throughout the entire length of spinal chord in adult mice. This method can facilitate studies targeted to manipulate spinal gene expression and help to understand the mechanism of several spinal neurodegenerative disorders including amyotrophic lateral sclerosis, spinal traumatic injury, or chronic pain. The main advantage of this technique is that highly potent transgene expression is achieved through the spinal gray and white matter in multiple spinal segments, which are adjacent to subpial AAV9 injected segment.Visual demonstration of this method is critical because the dura-opening step and placement of subpial needle are difficult to learn without visual demonstration. Demonstrating the procedure will be Takahiro Tadokoro, a post-doctorate fellow in our laboratory. Begin by properly anesthetizing the animal for surgery, isoflurane is used here.Ensure the absence of a paw pinch response before proceeding, then cover the eyes with ophthalmic ointment. Next, shave the back of the animal with clippers and clean the skin with 2%chlorhexidine. If lumbar subpial injections are to be performed, cut the skin overlying the T8 to L1 vertebrae with a scalpel and then use scissors to detach the paravertebral muscle from the T10 to T12 spinal vertebrae.Mount the animal into a standard stereotaxic frame, using mouse spinal clamps. Next, use a dental drill to shave both sides of the lamina of the T10 to T12 vertebrae until cracks appear then use forceps to remove cracked bone fragments and expose the dorsal surface of the lumbar spinal chord. The dura is now visible, use a 30 gauge stainless steel needle to make a one to two millimeter incision into the dura.It is important to carefully cut open the dura to avoid injury to the underlying pia membrane. Now, grasp the edge of the cut dura with forceps and extend the dura opening for up to five millimeters. If cervical subpial injections are to be performed, immobilize the head of the mouse by placing the head into a stereotaxic frame using ear bars and use scissors to make a 1.5 to two centimeter incision in the dorsal neck skin to expose the C1 and C2 segments and occipital bone.Then, shave a portion of exposed occipital bone using a dental drill and remove the bone fragments using forceps. Then, use a 23 gauge stainless steel needle and forceps to remove the atlanto occipital membrane of the cisterna magna. Use cotton swabs to clean the incision site of any tissue and bone debris, then cut open the dura, making a two to three millimeter incision.Mount the 34 gauge pia-penetrating needle and 36 gauge injection needle on to the Z arms of two separate X, Y, Z manipulators using glass capillary holders. Next, use a 50 microliter microsyringe connected with PE10 or PE20 tubing to load AAV9-UBI-GFP virus. After the virus is loaded, connect the end of the tubing to the injection needle.Next, while looking through a surgical dissecting scope set to eight to ten X magnification, use the X arm to lower the pia-penetrating needle into the pia to a depth of one millimeter. Keep the angle of the penetrating needle at five to 10 degrees relative to the tissue surface. After opening the pia, use the X arm to remove the pia penetrating needle from the subpial space.Note the penetrated site using a landmark such as a blood vessel. Move the X, Y, and Z arms of the second manipulator to position the tip of the AAV9 virus loaded injection needle into the pia penetrated site. Then, after inserting the tip of the injection needle about 0.5 millimeters in to the subpial space, use the Z arm to lift the pia about 0.3 millimeters, then use the X arm to continue advancing the needle horizontally into the subpial space.Advance the needle until it enters the subpial space at a depth of about two to three millimeters, then use the 50 microliter microsyringe to inject the AAV9-UBI-GFP virus into the subpial space. Remove the injection needle from the subpial space after the AAV9-UBI-GFP injection is complete. Close the muscle and skin using foromonofilament suture and surgical clips.Allow the animal to recover on a heating pad before housing in a clean cage. Two bilateral injections of AAV9-UBI-GFP were delivered into the upper lumbar subpial space and the animals were profusion fixed 14 days after AAV9 delivery. Intense GFP expression is seen in the gray matter located within the dotted area and white matter outside the dotted area.This extends from the lumbar to the upper thoracic segments in animals injected with three plus three microliters of AAV9. Animals injected with a lower volume of AAV9 have less intense expression of GFP. The following images depict potent retrograde and anterograde AAV9-UBI-GFP mediated GFP expression in brain motor and sensory centers.This low-power image clearly shows the presence of intense GFP positivity in the cervical spinal cord, medulla oblongata, cerebellum, and motor cortex. This high-power image taken from a sagittal brain section shows the presence of GFP florescence in neurons in the reticular formation, nucleus ruber, and axons of the spinocerebellar tract. This lower-power image taken from a coronal brain section demonstrates the presence of GFP florescence in pyramidal neurons in the motor cortex and in the terminals of the spinothalamic tract in areas of the reticular thalamic nuclei.Finally, this high-power image demonstrates an intense GFP expression in pyramidal neurons in the motor cortex. Once mastered, this technique can be completed in 30 to 40 minute. The use of this technique by researchers in the field of neuroscience will be an effective tool to study the role of specific genes in the evolution of spinal degenerative disorders.It will also help to explore potential treatment efficacy by suppressing mutated genes known to be linked with progressive spinal neuronal degeneration as in the familiar form of amyotrophic lateral sclerosis.

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Research

JoVE Journal

Neuroscience

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

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Injeção intracraniana da adeno-associado vetores virais

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

Neurociência

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Métodos e dicas para administração intravenosa de vírus Adeno-associado aos ratos e avaliação do sistema nervoso Central transdução

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Visualização e imagem ao vivo do Oligodendrocyte organelas em fatias Organotypic cérebro usando microscopia Confocal e vírus Adeno-associado

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

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

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Expressão do Transgene de mediada por vírus adeno-associado em geneticamente definidos neurônios da medula espinhal

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

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

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

Injeção intratecal de vírus Adeno-associado recombinantes em ratos adultos

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

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

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Produção, purificação e controle de qualidade para vetores de baseados em vírus Adeno-associado

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

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

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

A convecção aumentou a entrega do vetor viral adeno-associado Optogenetic ao córtice de Rhesus macaque a orientação de imagens em linha de MRI

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

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

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

Registro eletrocorticográfico de áreas do córtex cerebral manipulado usando um vírus associado ao Adeno visando cofilina em camundongos

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

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Imagem de lapso de tempo da arborização neuronal usando rotulagem de vírus associado adeno esparsa de populações de células de retina geneticamente direcionadas

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Entrega de punção lombar de AAV9 em ratos juvenis para terapia gênica do SNC

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

Autor em destaque: Avaliando a eficácia da terapia gênica intratecal em ratos juvenis

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

Entrega de vetor intratecal em ratos juvenis por injeção de cisterna lombar

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...