This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology to M.I. (21229004 and 25221304).

O uso de ratos para a investigação estava em conformidade com as orientações estabelecidas pelo Comitê da Universidade de Tóquio Animal Welfare. 1.Preparation de DNA de plasmídeo <ol><li> Gerar os plasmídeos de ADN para electroporação in vivo por subclonagem do ADNc ou sequência de shRNA em um plasmídeo de expressão para células de mamífero 12. Usar um citomegalovírus precoce imediato intensificadoras e de frango β-actina promotor de fusão (CAG) plasmídeo conduzido pelo promotor 13 porque permite a expressão forte e estável. Para a expressão de shRNAs sob o controlo de um promotor CAG, usar um sistema de cassete shRNA baseada mir30 para subclonagem a 14 shRNA. </li><li> Purifica-se DNA de plasmídeo com um kit Maxi-prep de acordo com as instruções do fabricante, e ressuspender o ADN com solução salina tamponada com HEPES (NaCl 140 mM, 0,75 mM de Na 2 HPO 4, 25 mM de HEPES, pH 7,40). Ajustar a concentração de ADN para ≥4 ug / uL. </li><li&gt; Preparar a solução de DNA de plasmídeo a uma concentração de 4 mg / mL, e adicionar uma quantidade mínima de corante verde rápido (concentração final de 0,01%) para marcar o local da injecção. Quando é necessária a electroporação simultânea de vários plasmídeos, ajustar a concentração total da solução de DNA de plasmídeo a 4 mg / mL. Nota: A composição óptima dos DNAs de plasmídeo deve ser determinado de acordo com a eficiência de transfecção de cada plasmídeo. </li></ol> 2. A esterilização de instrumentos cirúrgicos e Saline <ol><li> instrumentos cirúrgicos autoclave e a solução de NaCl a 0,9%. </li></ol> 3. Preparação da micropipeta de vidro <ol><li> Puxe pipetas de vidro usando um extrator pipeta. Cortar a ponta da pipeta para um diâmetro de 30-50 ^ m. Utilize os seguintes parâmetros: calor, 600; Velocidade, 50; Tempo, 75. </li></ol> 4. Cirurgia animal, Injecção de ADN e electroporação Nota: Um excessoFace a esta etapa é descrita na Figura 1. Embora o procedimento de crias de ratos são descritos aqui, o método também é aplicável a animais mais maduros, utilizando o mesmo procedimento. <ol><li> Anestesiar ratos com isoflurano na caixa de indução até que o animal torna-se imóvel, ajustando o fluxo de oxigénio para 0,4 L / min e a concentração de isoflurano a 4% (vol / vol). Execute toe beliscar para confirmar anesthetization adequada. </li><li> Colocar o rato sobre o aquecedor pré-aquecido sob um microscópio binocular e manter a anestesia por administração de isoflurano continuamente através da máscara facial. Ajustar o fluxo de oxigénio para 0,2 L / min e a concentração de isoflurano a 2% (vol / vol). Use colírios para evitar a secura dos olhos, se os olhos do animal estão abertas. </li><li> Fixar as pernas com fita cirúrgica. </li><li> Limpar a pele na parte posterior da coxa com povidona-iodo, e fazer uma incisão com um bisturi. Nota: Raspar áreas cirúrgicas se as áreas cirúrgicas são cobertas com har. </li><li> Expor o nervo ciático, criando uma abertura entre os músculo quadríceps femoral e músculo bíceps femoral com agulhas de costura. </li><li> Molhar o nervo com uma solução de NaCl a 0,9%. Absorver o excesso de água com papel sem fiapos. </li><li> Inserir a base de uma micropipeta de vidro na tubagem flexível, e preencher a quantidade adequada de solução de ADN (pelo menos um microlitro) na micropipeta, aspirando suavemente. </li><li> Levantar o nervo exposto puxando suavemente o lado distal do nervo usando uma agulha. Nota: Não aplicar tensão no nervo para minimizar o stress mecânico. </li><li> Inserir a micropipeta de vidro no local distai do nervo, e injectar a solução de DNA através da aplicação de pressão (isto é, por sopro para dentro da extremidade aberta do tubo flexível). Injectar a solução de DNA até o nervo aparece verde (máximo de 1 ml). Porque inserção frequente da micropipeta pode danificar o nervo, não insira o micropipeta mais de duas vezes. </li><li> Coloque uma TWEezer tipo eletrodo de platina cerca de 1-2 mm do nervo. Preencher a lacuna entre o eletrodo e o nervo com uma solução de NaCl a 0,9%. Nota: Não segure o nervo com o eletrodo para evitar estresse mecânico sobre o nervo. </li><li> Aplicar impulsos eléctricos para o local da injecção usando um electroporador com o eléctrodo. Após o primeiro conjunto de pulso, inverter o eletrodo e aplicar outro conjunto de pulso. Utilize os seguintes parâmetros: tensão, 50 V; duração do pulso, 5 ms; intervalo de pulso, a 100 ms; número de pulsos, 4 vezes. </li><li> Limpar o local de electroporação com uma solução de NaCl a 0,9%. </li><li> Repita os passos 4,4-4,11 sobre o nervo ciático contralateral. </li></ol> 5. Pós-eletroporação <ol><li> Fechar as incisões com cola de cianoacrilato. </li><li> Após a secagem da cola, limpar a ferida com povidona-iodo. </li><li> Solte o filhote de cachorro da máscara facial. Aqueça o filhote em um mais quente, pelo menos durante uma hora, a fim de permitir que ele se recuperar totalmente da anestesia. Do não deixar o cachorro sem supervisão até que tenha recuperado a consciência suficiente. </li><li> Após a recuperação da anestesia, devolver o cachorro para o rato mãe. Não devolva o filhote até que esteja totalmente recuperado. </li></ol> 6. Pós-cirurgia <ol><li> Casa os filhotes de ratos na gaiola até a realização dos experimentos 11 (ver exemplos na Figura 3). Administrar carprofeno (5 mg / kg; ip), uma droga não-esteróide anti-inflamatório, ou buprenorfina (0,1 mg / kg, sc), um analgésico opióide, se necessário. Nota: Se o filhote de rato não cresce bem ou inflamação é observado ao redor do local da cirurgia, excluir o animal a partir dos experimentos. </li></ol>

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The authors declare that they have no competing financial interests.

In this paper, we describe a simple and efficient method that allows in vivo gene transfer to myelinating SCs in the rat sciatic nerve using electroporation. This method allows highly selective gene expression in myelinating SCs by simply applying electric pulses to the plasmid DNA-injected sciatic nerve. Because the molecular mechanisms of myelination and demyelination in the peripheral nervous system remain unclear, the present in vivo electroporation method will be a powerful tool to clarify the roles of multiple genes of interest in living animals.
A critical requirement of this method is to keep damage to the nerve during surgery to a minimal level. Should surgical damage cause excessive inflammation, the sciatic nerve may degenerate. To avoid this, one must conduct surgery with extreme care, so as to not damage the blood vessels around the nerve. Mechanical stress to the nerve during the surgery can also be a cause of nerve damage. To minimize mechanical stress, lifting the exposed nerve should be done as gently as possible, and the tweezer-type electrode should be placed close to the nerve without contact. Furthermore, electrical pulses that are too strong can cause undesirable large leg movement, which leads to mechanical stress, or can burn the nerve. If significant damages are observed in the nerves, we recommend reducing the electrical pulse intensities or placing the electrode further away from the nerve.
In our present protocol, CAG promoter-driven plasmids were used as expression vectors. CAG promoter-driven plasmids allow high levels of gene expression in myelinating SCs in vivo. We also have tried a CMV promoter, another widely used universal promoter for mammalian gene expression, but expression of the gene product was very weak. This is consistent with previous results, in which electroporation-mediated transfection was conducted in the embryonic brain20. Therefore, we recommend using CAG promoter-driven plasmids for the in vivo electroporation method.
Because axonal signaling is a key factor in myelination/demyelination21, gene modification in neurons is also important. However, delivery of transgenes using our in vivo electroporation method is limited to SCs. It has been reported that gene delivery into sciatic nerve axons can be achieved when in vivo electroporation is applied to dorsal root ganglion (DRG) neurons in adult rats22. This suggests that delivery of plasmid DNA to the cell body is likely to be critical for in vivo transfection of peripheral axons. Thus, to examine the involvement of axonal molecules in myelination/demyelination, researchers should use neuron-specific genetic methods such as genetically modified animals, neuron-specific viral vectors, or in vivo electroporation to DRG neurons.
Compared with current methods, such as the generation of genetically modified animal lines23 and delivery of transgenes by viral vectors4-6, gene modification of SCs by in vivo electroporation is simpler. This method only requires several days for plasmid DNA construction and one day for electroporation surgery. Plasmid DNA construction does not require a biohazard room that is usually essential for viral vector handling. In addition, one of the advantages of the electroporation method is the capacity for simultaneous expression of multiple gene products using a simple protocol. Our novel technique will be useful for analyzing the interaction of a variety of signaling molecules involved in myelination and demyelination. In particular, by permitting the cotransfection of a number of different intracellular fluorescent probes, our method should be a powerful tool for investigating intracellular signaling dynamics in SCs using live imaging experiments.

The rapid transmission of sensory and motor information in the peripheral nervous system is permitted by the myelin sheath, which is formed by myelinating Schwann cells (SCs)1. Insulation of axons by the myelin sheath enables saltatory conduction, which increases the speed of nerve impulses. In disorders in which the development or maintenance of the myelin sheath is impaired, nerve conduction speed is reduced. This results in neuropathy involving motor and sensory dysfunction. Although there are many studies on the molecular mechanisms of myelination and demyelination in the peripheral nervous system, the roles of the numerous proteins involved in these processes remain unclear.
To study the molecular mechanisms of SC myelination/demyelination in vivo, genetic approaches have been used to modify gene expression in animals. A powerful approach is the use of knockout/knockin or transgenic animals. However, the generation of these animals is expensive and time consuming. For SC-specific gene manipulation, crossing floxed strains with Cre mice or other conditional gene expression methods are necessary. This again is laborious and time intensive. In recent years, a cutting-edge genetic technology, the CRISPR-Cas9 system, has made the generation of genetically modified mice much quicker (about 4 weeks)2,3, but this method is hindered by target sequence limitations, and suffers from off-target effects. As an alternative method, viral vector-mediated gene transfer is a faster and easier method of achieving gene transfer into SCs in vivo4-6. Indeed, the generation of viral vectors is less expensive, and takes a shorter time (within a few weeks), and gene manipulation of SCs can be achieved by simply injecting engineered viral vectors, such as adenoviral vectors, adeno-associated viral (AAV) vectors, and lentiviral vectors, into the sciatic nerve. Because these viral vectors have different characteristics, users have to choose the one best suited for their purpose. Adenoviral vectors infect axons and SCs in both young and mature sciatic nerves. In particular, adenoviral vectors have higher selectively for non-myelinating SCs than myelinating SCs. Adenoviruses can cause immune responses, and accordingly, immunodeficient strains should be used5. AAV vectors are currently the most widely used viral vectors, and allow in vivo gene transfer with lower toxicity7. AAV can transduce both axons and SCs by direct injection into the nerve fibers8,9. However, AAV-mediated protein expression usually requires 3 weeks or longer to reach maximum levels7,9. Therefore, it is difficult to analyze myelination, which actively progresses during the two week postnatal period. Lentiviral vectors have higher selectively for myelinating SCs than non-myelinating SCs, and do not have toxic effects on sciatic nerves. However, lentiviral vectors do not infect SCs in more mature nerves5, and therefore are unsuitable for analyzing events such as the demyelination process.
Electroporation is another faster and easier approach to achieve in vivo gene transfer. It has been reported that in vivo transfection of SCs can be achieved when electroporation is applied to transected rat sciatic nerves10. However, because this method requires nerve transection for gene delivery, the application is limited to the analysis of the damaged nerves. Here, we describe an alternative method that allows the delivery of transgenes into myelinating SCs in intact rat sciatic nerves using electroporation11. This method requires plasmid construction, which can usually be completed within a week. Then, by simply delivering electric pulses to the site on the sciatic nerve where the plasmid DNA was injected, highly selective transfection of myelinating SCs can be achieved in neonatal as well as in more mature animals. By electroporating multiple plasmids, simultaneous expression of a variety of genes can be easily achieved. The ability to simultaneous express multiple molecules, such as signaling proteins, short-hairpin RNAs (shRNAs) and functional probes, is crucial for investigating complex processes such as myelination and demyelination. The novel in vivo electroporation method described in this paper will be a powerful tool, allowing researchers to analyze the function of a multitude of molecules and their interactions in myelinating SCs.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genopure Plasmid Maxi Kit</td>
			<td style="width:71px;">Roche</td>
			<td style="width:119px;">03 143 422 001</td>
			<td style="width:167px;">Plasmid DNA purification kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green CFC</td>
			<td>WAKO</td>
			<td>069-00032</td>
			<td>Dye for DNA injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GC 150T-10</td>
			<td>HARVARD APPARATUS</td>
			<td>30-0062</td>
			<td>Glass capillary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suction tubing</td>
			<td>Drummond</td>
			<td>05-2000-00</td>
			<td>Suction tubing for micro injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MODEL P-97</td>
			<td colspan="2">SUTTER INSTRUMENT CO.</td>
			<td>Micropipette puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CUY21 Single Cell</td>
			<td>BEX</td>
			<td>Electroporator CUY21 Single Cell</td>
			<td>Pulse generator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electric warmer</td>
			<td>KODEN</td>
			<td style="width:119px;">CAH-6A</td>
			<td>Warmer during the surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isofluolane</td>
			<td style="width:71px;">Mylan</td>
			<td style="width:119px;">1119701G1076</td>
			<td>Anesthetic</td>
		</tr>
	</tbody>
</table>

in vivo gene transfer to schwann cells in the rodent sciatic nerve by electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

Um exemplo de um nervo ciático transfectada com proteína fluorescente vermelha (RFP) -expressing plasmídeo é mostrada na Figura 2A. As células que mostram morfologia bipolar, uma característica de SCs, foram escassamente transfectadas com RFP. Nenhuma fluorescência RFP foi detectado em axónios. Normalmente encontramos ~ 100 SCs transfectadas em cada nervo. Esta eficiência de transfecção parece semelhante à in vivo SC eficiência de infecção utilizando vectores lentivirais 4. Experimentos imunocoloração mostrou que a maioria (~ 96%) as células RFP-positivo em P7 co-marcadas durante S100, um marcador SC (Figura 2B), e 91% de células RFP-positivo em P14 co-marcadas durante MBP, um marcador mielinizantes SC (Figura 2C), o que sugere que a transferência genética através de electroporação é altamente selectivo para myelinating SCs. A introdução de genes em múltiplas SCs <em&gt; in vivo será extremamente útil para investigar os mecanismos de mielinização / desmielinização. Uma grande vantagem do método de electroporação in vivo descrito aqui é a capacidade de transferência de genes múltiplos com um procedimento simples. A Figura 2D mostra uma imagem representativa de um nervo ciático transfectadas com uma mistura de GFP e os plasmídeos que expressam RFP utilizando electroporação em vivo. Cerca de 97% das SC foram GFP e RFP duplo positivo, sugerindo que a entrega altamente eficiente de genes múltiplos pode ser conseguido simplesmente electroporating misturas de vários plasmídeos. Em roedores, a mielinização inicia em torno do nascimento, aumentando dramaticamente durante as primeiras duas semanas após o parto, e depois diminui gradualmente. Assim, manipulando geneticamente SCs durante essas janelas de tempo de desenvolvimento, os mecanismos subjacentes a estas diferentes fases de mielinização pode ser esclarecida. lentivirais são uma boa ferramenta para umnalyzing mielinização, especialmente como eles têm uma toxicidade mínima, mas lentivírus única infectar neonatal nervo ciático 5,6. Em comparação, a transferência de genes mediada por electroporação funciona bem quando a transfecção é realizada na P3 (Figura 2E, topo) ou em P14 (Figura 2E, parte inferior). As aplicações do novo método em electroporação in vivo são descritos aqui. A Figura 3A mostra que as imagens ao microscópio óptico de GFP-expressando myelinating SCs em várias fases do desenvolvimento (P7, P14, P21 e P31). Por análise microscópica de luz, alterações nos parâmetros morfológicos, tais como comprimento e diâmetro, pode ser avaliado. Note-se que estes parâmetros têm valores semelhantes em comparação com ratos intactos 15,16 nervos periféricos, o que sugere que os nervos electroporadas desenvolver, sem efeitos prejudiciais significativos. A Figura 3B mostra uma imagem microscópica de electrões de LacZ-expressing myelinating SCs. Neste caso, LacZ foi utilizada como um marcador de expressão. β-galactosidase coloração utilizando Bluo-gal, um substrato insolúvel em etanol, possibilita a análise da estrutura de mielina de SCs transfectadas por microscopia eletrônica 11,17. Nestas experiências, o papel de moléculas de sinalização pode ser examinado por silenciamento ou aumentando a sua expressão, permitindo assim a análise da perda de função ou efeitos de ganho de função. Para além da análise de tecido fixo, in vivo, transferência de genes mediada por electroporação pode ser também aplicado a viver experiências de imagiologia. Por exemplo, a Figura 3C mostra uma mielinizantes SC co-expressando G-GECO1.1 18, um indicador verde fluorescente citosólica de Ca2 +, e R-GECO1mt 19, um indicador vermelho fluorescente Ca2 + mitocondrial. Ao expressar estes indicadores, identificamos uma via de sinalização que controla citosólica e mitocondrial de Ca2 + concentrações em myelinating SCs<html> . Assim, o presente método pode ser usado para estudar uma variedade de mecanismos de sinalização, especialmente quando as sondas fluorescentes geneticamente codificados estão disponíveis para detectar os sinais de interesse. <img alt="figura 1" src="/files/ftp_upload/54567/54567fig1.jpg" /> Figura 1:. Esquemática do i n vivo A electroporação Método Em primeiro lugar, o nervo ciático do rato anestesiado é exposta. Em segundo lugar, o ADN de plasmídeo é injectado no nervo ciático. Terceiro, os impulsos eléctricos são entregues no local da injecção, através do eléctrodo de uma pinça em forma de. Finalmente, a ferida é fechada com cola. Este procedimento pode ser repetido sobre o nervo contralateral. <a href="https://www.jove.com/files/ftp_upload/54567/54567fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> &quot;&gt; <img alt="Figura 2" src="/files/ftp_upload/54567/54567fig2.jpg" /> Figura 2: Resultados representativos sobre nervos ciáticos transfectadas (A) uma imagem representativa de um nervo ciático transfectadas.. O nervo foi transfectada com o plasmídeo que expressa RFP no P3, e fixado em P7. (B) uma imagem representativa de uma célula transfectada-RFP em P7 mostrando uma co-localização com S100, um marcador SC. (C) uma imagem representativa de um nervo ciático transfectada-RFP no P14 mostrando uma co-localização com MBP, um marcador mielinizantes SC. (D) A imagem representativa de um nervo ciático cotransfectadas com GFP e plasmídeos que expressam RFP. SCs transfectadas expressaram simultaneamente GFP e RFP. (E) Uma imagem de myelinating SCs no P31 transfectadas em P3, quando mielinização começa (em cima), e uma imagem de SCs myelinating no P31 transfectadas a P14, quando a maioria das grandes axônios se tornar mielinizado (parte inferior), sugerindo que a transfec ç ã o das SC myelinating pode ser conseguida não só em nervos neonatal, mas também em nervos mais maduros. Barras de escala = 200 uM (A); 50 mm (BE). Este valor foi modificado a partir de nossa publicação anterior 11. <a href="https://www.jove.com/files/ftp_upload/54567/54567fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54567/54567fig3.jpg" /> Figura 3: Aplicação de electroporação in vivo (a) uma análise microscópica de luz do desenvolvimento de myelinating SCs.. Os nervos ciáticos foram electroporadas com o plasmídeo que expressa GFP em P3, e foram fixados em vários estágios de desenvolvimento (P7, P14, P21 e P31). Imagens representativas de CT-GFP positivo estão apresentados à esquerda. A duração média e o diâmetro estão resumidos como média ± EPM (n = 30-47 de 3 nervos) no lado direito. O comprimento e diâmetro do myelinating SCs aumenta à medida que prossegue o desenvolvimento. (B) uma imagem de microscopia electrónica de um nervo ciático transfectadas com um plasmídeo que codifica para LacZ. Um transf SC (asterisco branco, à esquerda) foi finamente marcado com precipitados do produto da reacção de β-galactosidase. (C) Uma imagem de um SC cotransfectadas com G-GECO1.1, um indicador verde fluorescente citosólica de Ca2 +, e R-GECO1mt, um indicador vermelho fluorescente mitocondrial de Ca2 +. As regiões dentro dos retângulos pontilhadas brancas são mostrados ampliada nos painéis à direita. Barras de escala = 50 uM (A e C); 1 | iM (B). Este valor foi modificado a partir de nossa publicação anterior 11. <a href="https://www.jove.com/files/ftp_upload/54567/54567fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation at JoVE.com

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Em Gene Vivo Transferência de células de Schwann no nervo ciático de roedores por Eletroporação

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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Neuroscience

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

8.8K visualizações.  The University of Tokyo. Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Vídeo: In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

Isolamento axoplasma de nervo ciático do rato

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

Neurociência

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Mouse No Utero Eletroporação: Transfecção Gênica Controlled Spatiotemporal

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Transferência de genes para a orelha do camundongo Desenvolvimento Inner por<em&gt; In Vivo</em&gt; Eletroporação

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Entrega intraventricular direta de medicamentos para o Sistema Nervoso Central Rodent

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

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Transplante de células olfativas Ensheathing para avaliar a recuperação funcional após a lesão do nervo periférico

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Em Medidas Vivo eletrofisiológicos nos nervos ciático Rato

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultâneo<em&gt; Ex vivo</em&gt; Teste Funcional de Dois retinas por<em&gt; In vivo</em&gt; Sistema Eletrorretinograma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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 ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Análise Cinética 3D para a Avaliação Funcional no modelo de rat da lesão ciática de esmagamento nervoso

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Em Gene Vivo Transferência de células de Schwann no nervo ciático de roedores por Eletroporação

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