O estudo foi financiado (concedido a F-Q. Z) pelo NIH (R01NS064288, R01NS085176, R01GM111514, R01EY027347), a Fundação de Neilsen H. Craig e a Fundação de BrightFocus.

Todas as experiências em animais foram realizadas em conformidade com o protocolo animal aprovado pelo Comitê de uso e Johns Hopkins institucional Cuidado Animal.
1. materiais e reagentes
<ol><li>
Animais
	<ol>
<li>Use ratos CF1 fêmeas seis semanas de idade, pesando 30 – 35 g para os experimentos. Nota: Os ratos foram alojados em grupo (5 ratos por gaiola) em gaiolas esterilizados individualmente ventilados com 1/4" milho espiga da cama e 2" quadrado nestlets para o assentamento. As gaiolas foram mantidas em um ciclo controlado de claro-escuro de 12-h e a temperatura foi entre 19,4 ° C e 25 ° C. Os ratos foram alimentados com dieta de roedor global e sistema de fornecimento de água automática.</li>
	</ol>
</li>
	<li>
Instrumentos
	<ol>
<li>Use os seguintes instrumentos cirúrgicos para realizar eficientemente a cirurgia: pele de seringas descartáveis (27 G, 1,0 mL) por injeção intraperitoneal, tosquiadeira, microscópio estéreo dissecação, microcirurgia pinça, tesoura microcirurgia e pequeno osso ruginas e esterilizadas não tecida esponjas. Nota: Todos os instrumentos e os materiais são autoclavados antes da cirurgia ou pré-esterilizados pelos produtores. Durante a cirurgia, os instrumentos são pulverizados com 75% de álcool para manter a esterilização.</li>
	</ol>
</li>
	<li>
Soluções anestésicas
	<ol>
<li>Use solução anestésica #1 para induzir a anestesia e usar anestesia solução #2 antes da injeção de DRG para manter a anestesia.<ol>
<li>Para tornar a solução anestésica #1, diluir ketamina e xilazina em solução salina estéril em uma concentração final de 10 mg/mL e 1,2 mg/mL.</li>
			<li>Para tornar a solução anestésica #2, dilua avertin em solução salina estéril em uma concentração final de 20 mg/mL.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Plasmídeo de DNA e RNA Oligos
	<ol>
<li>Prepare os plasmideos utilizando o kit comercial, seguindo o protocolo do fabricante, em conformidade. Dilua o plasmídeo DNAs em água desionizada estéril na concentração de 2,0 µ g / µ l.</li>
		<li>Adicionar a solução de corante Fast Green para atingir uma concentração final de 0,005-0,01% (v/v) para melhor visualização do contorno DRG durante a injeção.</li>
		<li>Dissolva os oligos siRNA ou microRNA nos correspondentes buffers fornecidos pelos fabricantes para atingir uma concentração final de 50 µM.</li>
	</ol>
</li>
	<li>
Injeção de micro pipeta
	<ol>
<li>Puxe o vidro capilar usando o extrator micropipeta e corte a ponta da pipeta vidro capilar puxada com uma tesoura de microcirurgia para gerar uma abertura com um diâmetro externo aproximado de 50 µm. Em seguida esterilize a pipeta de vidro sob luz UV sobre uma bancada limpa por 20-30 min aproximadamente.</li>
		<li>Monte a pipeta de vidro esterilizado no suporte da pipeta conectada a uma microinjeção intracelular dispensar o sistema. Defina os parâmetros do sistema de microinjeção a 30 psi de pressão e 8 ms de duração.</li>
	</ol>
</li>
	<li>
Electroporation sistema
	<ol>
<li>Conectar os eletrodos ao sistema de eletroporação de onda quadrada e definir os seguintes parâmetros: pulsos de 15 ms em 35 V com intervalos de ms 950 para eletroporação na vivo .</li>
	</ol>
</li>
	<li>
Perfusão e solução de fixação
	<ol>
<li>Dissolva o pó de paraformaldeído (PFA) em PBS 1x em uma concentração de 4% (p/v). Armazenar a solução PFA em 4 ° C.</li>
	</ol>
</li>
</ol>2. experimentais procedimentos
<ol><li>
Exposição cirúrgica da L4 e L5 DRGs
	<ol>
<li>Anestesia o mouse usando uma injeção intraperitoneal de solução anestésica #1 preparado anteriormente (com cetamina 100mg/kg de peso corporal e xilazina 12 mg/kg de peso corporal).</li>
		<li>Raspe a área cirúrgica com o aparador de pelos. Nota: Uma vez que haverá outra cirurgia para o esmagamento de esquerda nervo ciático, a pele da região posterior da coxa esquerda pode ser raspada simultaneamente.</li>
		<li>Coloque o mouse sobre o cobertor aquecido (35,0 ° C) e monitorar a temperatura retal com uma sonda de temperatura.</li>
		<li>Para testar a anestesia, apertar os dedos do pé e cauda de rato e observar as respostas comportamentais.</li>
		<li>Tape os quatro membros do mouse sobre o quadro de avisos.</li>
		<li>Limpe a área cirúrgica com solução Iodofor e, em seguida, com 75% de álcool para remover o Iodofor antes de cortar a pele.</li>
		<li>Aplica a pomada oftálmica nos olhos para evitar ressecamento enquanto sob anestesia.</li>
		<li>Antes da incisão, marque ambos os lados das cristas ilíacas com uma caneta fina. Desenha uma linha conectando dois pontos das cristas ilíacas, a fim de facilitar a identificar as posições dos DRGs L5.</li>
		<li>Faça uma incisão de 3 cm ao longo da linha mediana da região lombar com microtesoura. Além disso, desanexar musculatura paraespinhal, tais como o multiﬁdus musculus e o musculus longissimus lombar, do L3 para processos espinhosos de S1 e expor as articulações faceta de L4-5 e L5-6.</li>
		<li>Use um micro-rongeur para remover as articulações faceta de L4-5 e L5-6. Além disso, remova o arco neural esquerdo de L4 e L5 para expor o lado dorsal os DRGs.</li>
	</ol>
</li>
	<li>
Injeção de DRG
	<ol>
<li>Injetar a solução anestésica #2 (com avertin 200 mg/kg de peso corporal) intraperitonealmente para manter a anestesia antes da injeção de DRG.</li>
		<li>Carrega o plasmídeo de DNA ou RNA oligos (1 µ l por DRG) dentro da pipeta capilar de vidro.</li>
		<li>Insira a ponta da pipeta capilar de vidro cuidadosamente no RDG.</li>
		<li>Gradualmente, injetar 1,0 solução µ l de plasmídeos de DNA ou RNA oligos em DRG usando a microinjeção intracelular dispensar sistemas (30 psi, 8 ms). Nota: A duração da injeção deve durar nada menos do que 5 minutos.</li>
	</ol>
</li>
	<li>
Electroporation
	<ol>
<li>Largue o PBS nas pontas dos eletrodos.</li>
		<li>Limpe o sangramento com gaze de algodão esterilizado de quadrados.</li>
		<li>Beliscar o alvo DRG com os eléctrodos suavemente e aplicar 5 pulsos quadrado elétricos com o sistema de eletroporação.</li>
		<li>Feche as camadas de músculo e pele respectivamente com suturas de nylon 5-0.</li>
		<li>Coloque o mouse sobre um cobertor aquecido (35,0 ° C) sob muita atenção até que completamente recuperou a consciência suficiente para manter a prostração esternal. Retorne o rato da gaiola em casa depois que recuperou totalmente da anestesia. Nota: Demora cerca de 60 min para o mouse para se recuperar completamente da anestesia do cobertor de 37 ° C. Dissolva um ibuprofeno comprimido (200 mg) em 1.000 ml de água (0,2 mg/ml) para a alimentação diária para aliviar a dor pós-cirúrgica.</li>
	</ol>
</li>
	<li>
Esmagamento do nervo ciático
	<ol>
<li>Dois ou três dias (dependendo do projeto experimental) após o electroporation DRG, anestesia o mouse intraperitonealmente com solução anestésica #2 (com avertin 400 mg/kg de peso corporal).</li>
		<li>Tape os quatro membros do mouse sobre o quadro de avisos.</li>
		<li>Faça uma incisão de 1 cm 0,5 cm para o lado esquerdo ao longo da linha mediana. Os músculos, como o músculo glúteo máximo e piriforme, cortado longitudinalmente. Expor o segmento do nervo ciático entre o forame isquiático maior e o entalhe sciatic.</li>
		<li>Esmagamento do nervo com fórceps de microcirurgia para 12 s e marca o local da queda com uma sutura de epineural nylon 10-0. Faça um nó na membrana dural para marcar o local da queda.</li>
		<li>Feche as camadas de músculo e pele com sutura de nylon 5-0.</li>
		<li>Coloque o mouse sobre um cobertor aquecido (35,0 ° C) com muita atenção até que completamente recuperou a consciência suficiente para manter a prostração esternal. Retorne o rato da gaiola em casa depois que recuperou totalmente da anestesia. Nota: Demora cerca de 60 min para o mouse para se recuperar completamente da anestesia do cobertor de 37 ° C. Dissolva um ibuprofeno comprimido (200 mg) em 1.000 ml de água (0,2 mg/ml) para a alimentação diária para aliviar a dor pós-cirúrgica.</li>
	</ol>
</li>
	<li>
Perfusão de mouse, DRG e colheita de nervo ciático
	<ol>
<li>Dois ou três dias após o esmagamento do nervo ciático (dependendo do design experimental), anestesiar o mouse intraperitonealmente com solução anestésica #2.</li>
		<li>Perfundir o rato transcardially com PBS (pH 7,4) seguido por gelada paraformaldeído 4% (PFA) (pH 7,5) em PBS.</li>
		<li>Após a perfusão, separe cuidadosamente os DRGs juntamente com as raízes nervosas e nervo ciático antes do joelho com microtesoura e pinça-micro sob o microscópio de dissecação. Coloque o nervo ciático diretamente em 4% PFA overnight a 4 ° C.</li>
	</ol>
</li>
	<li>
Imaging e medindo os axônios sensoriais fluorescência-etiquetada
	<ol>
<li>Tire o tecido anexado e membrana no nervo ciático fixo com micro tesouras e micropinça cuidadosamente sob o microscópio de dissecação. Em seguida, trocar o 4% PFA com PBS e lavar o nervo três vezes.</li>
		<li>Coloque o nervo ciático em um slide e mantê-lo reto. Adicione 80 µ l de solução antidesgaste em torno do nervo e, em seguida, deitar uma lamela nela. Achate o tecido todo montado com pressão.</li>
		<li>Coloque os tecidos de ﬂattened sobre o microscópio invertido epiﬂuorescent, equipado com um acessório para o mosaico de aquisição e processamento de imagem. Nota: Os comprimentos de excitação e emissão são 488 nm e 509 nm. Também é possível tomar várias imagens sobrepostas manualmente e costurar as imagens juntos usando o ImageJ com o plug-in de mosaico.</li>
		<li>Quando se mede o comprimento dos axônios regenerados, rastrear todos os identiﬁable ﬂuorescently-rotulado axônios no nervo ciático do local da queda (marcado com a sutura epineural de 10-0) para as extremidades do axônio distal.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Saijilafu, , 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=PI3K-GSK3+signalling+regulates+mammalian+axon+regeneration+by+inducing+the+expression+of+Smad1.">PI3K-GSK3 signalling regulates mammalian axon regeneration by inducing the expression of Smad1.</a> Nature Communications. 4, 2690 (2013).</li><li>Zhang, B. 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=Akt-independent+GSK3+inactivation+downstream+of+PI3K+signaling+regulates+mammalian+axon+regeneration.">Akt-independent GSK3 inactivation downstream of PI3K signaling regulates mammalian axon regeneration.</a> Biochemical and Biophysical Research Communications. 443 (2), 743-748 (2014).</li><li>Jiang, J. 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=MicroRNA-26a+supports+mammalian+axon+regeneration+in+vivo+by+suppressing+GSK3beta+expression.">MicroRNA-26a supports mammalian axon regeneration in vivo by suppressing GSK3beta expression.</a> Cell Death &amp; Disease. 6, 1865 (2015).</li><li>Krames, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+dorsal+root+ganglion+in+the+development+of+neuropathic+pain.">The role of the dorsal root ganglion in the development of neuropathic pain.</a> Pain Medicine. 15 (10), 1669-1685 (2014).</li><li>Salimzadeh, L., Jaberipour, M., Hosseini, A., Ghaderi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-viral+transfection+methods+optimized+for+gene+delivery+to+a+lung+cancer+cell+line.">Non-viral transfection methods optimized for gene delivery to a lung cancer cell line.</a> Avicenna Journal of Medical Biotechnology. 5 (2), 68-77 (2013).</li><li>Keeler, A. M., ElMallah, M. K., Flotte, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+2017:+Progress+and+Future+Directions.">Gene Therapy 2017: Progress and Future Directions.</a> Clinical and Translational Science. 10 (4), 242-248 (2017).</li><li>Neumann, E., Schaeferridder, M., Wang, Y., Hofschneider, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-Transfer+into+Mouse+Lyoma+Cells+by+Electroporation+in+High+Electric-Fields.">Gene-Transfer into Mouse Lyoma Cells by Electroporation in High Electric-Fields.</a> The EMBO Journal. 1 (7), 841-845 (1982).</li><li>Liu, C. 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=MicroRNA-138+and+SIRT1+form+a+mutual+negative+feedback+loop+to+regulate+mammalian+axon+regeneration.">MicroRNA-138 and SIRT1 form a mutual negative feedback loop to regulate mammalian axon regeneration.</a> Genes &amp; Development. 27 (13), 1473-1483 (2013).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240 (1), 237-246 (2001).</li><li>Saijilafu, E. M., Hur, F. Q., Zhou, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+axon+regeneration+via+in+vivo+electroporation+of+adult+mouse+sensory+neurons.">Genetic dissection of axon regeneration via in vivo electroporation of adult mouse sensory neurons.</a> Nature Communications. 2, 543 (2011).</li><li>Pan, 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=Shrinkage-mediated+imaging+of+entire+organs+and+organisms+using+uDISCO.">Shrinkage-mediated imaging of entire organs and organisms using uDISCO.</a> Nature Methods. 13, 859 (2016).</li><li>Rao, R. D., Bagaria, V. B., Cooley, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterolateral+intertransverse+lumbar+fusion+in+a+mouse+model:+surgical+anatomy+and+operative+technique.">Posterolateral intertransverse lumbar fusion in a mouse model: surgical anatomy and operative technique.</a> Spine Journal. 7 (1), 61-67 (2007).</li><li>Dommisse, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood+supply+of+the+spinal+cord.+A+critical+vascular+zone+in+spinal+surgery.">The blood supply of the spinal cord. A critical vascular zone in spinal surgery.</a> The Journal of Bone and Joint Surgery. British Volume. 56 (2), 225-235 (1974).</li><li>Sorensen, D. 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Os autores não têm nada para divulgar.

Várias etapas cirúrgicas requerem especial atenção. A L4 e L5 DRGs (localização de somas), que dominam o nervo ciático, precisam ser corretamente identificado e injetado com construções de gene. Caso contrário, o GFP-rotulagem estarão ausente em axônios do nervo ciático. As cristas ilíacas podem ser vistas como útil pontos anatômicos para localizar a L4 e L5 DRGs. Na maioria dos ratos, o aspecto comum entre as vértebras L5 e L6 é próxima de cristas ilíacas12. Alternativamente, ...

Lesões no sistema nervoso causaram por trauma neural ou várias doenças neurodegenerativas geralmente resultam em defeitos nas funções motoras, sensoriais e cognitivas. Recentemente, muito esforço tem sido dedicado para restabelecimento de potência regenerativa nos neurônios adultos para restaurar o functionsof fisiológicas ferido neurônios1,2,3. Neurônios sensoriais em DRG são um aglomerado de células nervosas que transmitem diferentes estímulos sensoriais, tais como dor, temperatura, toque ou postura do corpo, para o cérebro. Cada um desses neurônios é pseudo unipolar e contém um único axônio que se bifurca com um ramo, estendendo-se em direção a periferia e o outro ramo indo em direção a medula espinhal4. Os neurônios sensoriais adultos DRGs estão entre alguns neurônios de mamíferos maduros conhecidos para regenerar seus axônios ativamente após lesão. Daí, lesões de axônios sensoriais têm sido empregadas extensivamente como um modelo fundamental para estudar os mecanismos de regeneração axonal em vivo.
Na vivo técnicas transfeccao de gene, que são geralmente menos demorado para configurar e mais flexível do que usando animais transgénicos, jogar papéis essenciais em estudar as funções dos genes e sinalização de caminhos no sistema nervoso. As principais técnicas podem ser classificadas em duas abordagens: instrumento-vírus baseados e5. Viral-baseado na vivo entrega gene nos neurônios adultos pode fornecer precisa spatiotemporal manipulação do gene expressão6. No entanto, processos de trabalho intensivos estão envolvidos em métodos baseados em viral, tais como a produção e purificação de partículas virais, contendo o gene desejado. Além disso, muitos vetores virais poderiam ativar o sistema imunológico do hospedeiro, que pode interferir com a aquisição de dados, análise de dados e possivelmente induzir em erro a interpretação dos resultados experimentais. Eletroporação, uma abordagem típica do transfection baseado no instrumento, utiliza um pulso elétrico para aumentar a permeabilidade da célula e as membranas nucleares, transitoriamente, o que favorece o afluxo de vetores de gene ou pequeno RNA oligos do espaço fora das células7 . Em vitro eletroporação é amplamente reconhecida como uma estratégia transitória, mas altamente eficiente para manipular a expressão de genes alvo em muitos tipos de células. Embora na vivo electroporation leva apenas a expressão do gene transiente com baixa eficiência transfecting comparada com vetores virais, tem várias vantagens sobre abordagens virais. Por exemplo, pode ser aplicado a quase todos os tecidos e células de7,8,9. Além disso, qualquer codificação de genes de interesse ou pequenos oligos RNA (por exemplo, siRNAs, microRNAs) contra certas transcrições de plasmídeos podem ser injetados diretamente no tecido alvo e então eletricamente pulsados, que tornar o processo menos trabalho - e tempo - consumindo. Além disso, transfecting múltiplos plasmídeos e RNA oligos simultaneamente com eletroporação único é possível.
Nós temos estabelecido uma abordagem electroporation in vivo , para manipular a expressão gênica em neurônios sensoriais do rato adulto e aplicado com sucesso e validado tal abordagem em numerosos estudos pioneiros1,2,3 ,8,10. Aqui, apresentamos um protocolo detalhado para facilitar o uso desta abordagem para futuros estudos de regeneração do axônio dos mamíferos.

<table border="0" cellpadding="0" cellspacing="0" style="width:1039px;" width="1037">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:335px;">ECM 830 Square wave electroporation system</td>
			<td style="width:232px;">BTX Harvard Apparatus</td>
			<td style="width:121px;">45-0052</td>
			<td style="width:351px;">For in vivo electroporation</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:335px;">Tweezertrodes electrodes</td>
			<td style="width:232px;">BTX Harvard Apparatus</td>
			<td style="width:121px;">45-0524</td>
			<td>For in vivo electroporation, 1 mm flat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Picospritzer III</td>
			<td style="width:232px;">Parker Instrumentation</td>
			<td style="width:121px;">1096</td>
			<td>Intracellular Microinjection Dispense Systems</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass Capillary Puller</td>
			<td style="width:232px;">NARISHIGE</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments, Inc.</td>
			<td style="width:121px;">1902328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Stereo Dissection Microscope</td>
			<td style="width:232px;">Leica</td>
			<td style="width:121px;">M80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microsurgery Rongeur</td>
			<td style="width:232px;">F.S.T</td>
			<td style="width:121px;">16221-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microsurgery Forceps</td>
			<td style="width:232px;">FST by DUMONT, Switzerland</td>
			<td style="width:121px;">11255-20</td>
			<td>Only for sciatic nerve crush</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Glass Capillary</td>
			<td style="width:232px;">World Precision Instruments, Inc.</td>
			<td style="width:121px;">TW100-4</td>
			<td>10 cm, standard wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Tape</td>
			<td style="width:232px;">Fisherbrand</td>
			<td style="width:121px;">15-901-30</td>
			<td>For fixing the mouse on the corkboard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2, 2, 2-Tribromoethanol (Avertin)</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">T48402</td>
			<td>Avertin stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-methyl-2-butanol</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">152463</td>
			<td>Avertin stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">siRNA Fluorescent Universal Negative Control #1</td>
			<td style="width:232px;">Sigma-Aldrich</td>
			<td style="width:121px;">SIC003</td>
			<td>Non-target siRNA with fluorescence</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:335px;">microRNA Mimic Transfection Control with Dy547</td>
			<td style="width:232px;">Dharmacon</td>
			<td style="width:121px;">CP-004500-01-05</td>
			<td>Non-target microRNA with fluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Plasmids preparation kit</td>
			<td style="width:232px;">Invitrogen Purelink</td>
			<td style="width:121px;">K210016</td>
			<td>GFP-coding plasmid preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fast Green Dye</td>
			<td style="width:232px;">Millipore-Sigma</td>
			<td style="width:121px;">F7252</td>
			<td>For better visualization of the DRG outline during injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Ketamine</td>
			<td style="width:232px;">Putney, Inc</td>
			<td style="width:121px;">NDC 26637-731-51</td>
			<td>Anesthesia induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Xylazine</td>
			<td style="width:232px;">AnaSed</td>
			<td style="width:121px;">NDC 59399-110-20</td>
			<td>Anesthesia induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Acetaminophen</td>
			<td style="width:232px;">McNeil Consumer Healthcare</td>
			<td style="width:121px;">NDC 50580-449-36</td>
			<td>Post-surgical pain relief</td>
		</tr>
	</tbody>
</table>

investigating mammalian axon regeneration: in vivo electroporation of adult mouse dorsal root ganglion

Eletroporação é uma abordagem de transfecção do gene não-virais essenciais para introduzir DNA plasmídeos ou pequenas moléculas de RNA em células. Um neurônio sensorial no gânglio da raiz dorsal (DRGs) estende-se a um único axônio com dois ramos. Um ramo vai para o periférico nervo (ramo periférico) e o outro ramo entra a medula espinhal através da raiz dorsal (agência central). Após a lesão neural, o ramo periférico regenera robustamente Considerando que a agência central não se regenera. Devido a alta capacidade regenerativa, regeneração do axônio sensorial foi amplamente utilizada como um sistema modelo para estudar a regeneração do axônio mamíferos no sistema nervoso periférico (SNP) e sistema nervoso central (SNC). Aqui, descrevemos um protocolo de abordagem estabelecida anteriormente para manipular a expressão gênica em neurônios sensoriais maduros na vivo via eletroporação. Baseado na transfecção com plasmídeos ou pequeno RNA oligos (siRNAs ou microRNAs), a abordagem permite ambas as experiências de perda e ganho-de-função estudar os papéis de genes de interesse ou microRNAs no Regulamento do axônio regeneração em vivo. Além disso, a manipulação do gene expressão na vivo pode ser controlada tanto espacial e temporalmente dentro de um curso de tempo relativamente curto. Este modelo sistema fornece uma única ferramenta para investigar o mecanismo molecular pelo qual a regeneração do axônio mamíferos é regulamentado na vivo.

Para quantificar a citotoxicidade do protocolo atual e para validar que a taxa de transfecção de na vivo DRG eletroporação é alta o suficiente, nós injetamos e electroporated marcadas fluorescentemente microRNA ou siRNA em L4 e L5 DRGs. Os DRGs desanexados foram processados através de cryo-seccionamento e imuno-histoquímica (Figura 1A-B). Ao estimar a taxa de sobrevivência de células após a injeção e eletroporação, os ...

Watch this Scientific Journal Video about Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion at JoVE.com

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Eletroporação é uma abordagem eficaz para entregar genes de interesse em células. Aplicando-se esta abordagem na vivo sobre os neurônios do gânglio de raiz dorsal do rato adulto (DRG), descrevemos um modelo para estudo de regeneração do axônio em vivo.

Investigação regeneração do axônio mamíferos: In Vivo Electroporation do gânglio de raiz Dorsal do rato adulto

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

investigating-mammalian-axon-regeneration-vivo-electroporation-adult

Research

JoVE Journal

Neuroscience

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

8.1K visualizações.  Johns Hopkins University School of Medicine. Eletroporação é uma abordagem eficaz para entregar genes de interesse em células. Aplicando-se esta abordagem na vivo sobre os neurônios do gânglio de raiz dorsal do rato adulto (DRG), descrevemos um modelo para estudo de regeneração do axônio em vivo.

Vídeo: Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfecção de células de camundongo por Ganglionares da Retina In vivo Eletroporação

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Neurociência

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Em Eletroporação vivo de Retina do rato em desenvolvimento

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Estudo genético da regeneração do axônio com cultivadas Adulto neurônios do gânglio da raiz dorsal

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Medindo Spinal pré-sináptica Inibição em Ratos por raiz dorsal Gravação Potencial<em&gt; In Vivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

In Vitro Modelagem de Cancerous Neural Invasion: o gânglio Modelo de raiz dorsal

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Uma abordagem para reforçar Alinhamento e mielinização do gânglio de raiz dorsal neurônios

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Dorsal Root Injection gânglio da raiz dorsal e lesão por esmagamento como um modelo para Sensorial Axon Regeneração

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomy para acesso de gânglio lombar Dorsal raiz e injeção em suínos

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Análise de células do sistema imunológico em único nervos ciático e gânglio da raiz Dorsal de um único rato usando citometria de fluxo

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Uso da gravação in vivo da único-fibra e do gânglio dorsal intacto da raiz com nervo sciatic Unido para examinar o mecanismo da falha da condução

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...