This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

NOTA: Todos os procedimentos e manejo de animais respeitadas as diretrizes de ética do NIH e requisitos protocolo aprovado do Cuidado e Uso Comitê animal Institucional da Universidade da Califórnia, em San Francisco. 1. Preparar o animal para a cirurgia <ol><li> Realizar procedimentos cirúrgicos em um espaço dedicado limpo. Autoclave todos os instrumentos cirúrgicos, esterilizar com um esterilizador de esferas de vidro antes da cirurgia. NOTA: Neste protocolo, utilize dia pós-natal (10-12 P10-12) camundongos FVB. Diferentes idades e linhagens de camundongos podem ser usados ​​para atender às necessidades de um projeto específico. Ratos com idade superior a P40 pode ser um desafio, porque o osso bolha fica mais difícil. </li><li> Anestesiar os ratos com injecção intraperitoneal de uma mistura de cloridrato de cetamina (100 mg / kg), hidrocloreto de xilazina (10 mg / kg), e acepromazina (2 mg / kg). Comece a preparação cirúrgica só depois que o animal já não responde a estímulos dolorosos, como toe pitada. Se necessary, administrar uma dose de reforço (um quinto da dose original) do cocktail de anestésico para restaurar o plano anestésico originais. Nota: Tenha cuidado nesta etapa porque a maioria da mortalidade observada nesta idade é causada por overdose de anestésico. </li><li> Cubra os olhos do animal com uma pomada oftálmica de proteção para manter os olhos úmidos durante a anestesia, suprimindo piscar reflexo do animal. </li><li> Posicione o mouse com o pescoço estendido sobre uma almofada de aquecimento durante todo o processo até que o mouse é totalmente acordado, para evitar pós-anestésica hipotermia. </li><li> Raspar a região pós-auricular esquerdo com um clipper e desinfectar com álcool 70% e iodopovidona antes de manipulação cirúrgica. </li></ol> 2. O procedimento cirúrgico e Vector Injection <ol><li> Use uma abordagem pós-auricular para expor a bula timpânica. Inciso a tecido subcutâneo com uma tesoura pequena para expor o músculo pós-auricular. Depois de recolher o tecido adiposo para poe lado posterior da incisão, para separar os músculos do lado direito e esquerdo perpendicularmente à incisão para expor o osso temporal. Certifique-se de que esta incisão é tempo suficiente para trabalhar confortavelmente com visualização adequada. </li><li> Perfurar a bula timpânica com uma agulha de 25 G e expandir o buraco, se necessário com uma pinça por descascar o osso de volta para permitir o acesso ao giro basal da cóclea, e, em seguida, ampliar o suficiente para visualizar a artéria estapediana ea membrana da janela redonda (VM) . </li><li> Perfurar o VM suavemente no centro com uma pipeta de borosilicato capilar. Observe efluxo fluido através da VM neste ponto; isso é normal. Aguarde até que o efluxo estabilizou (o fluido effluxed da cóclea é seca com um papel de filtro estéril). NOTA: Tome cuidado ao segurar e fazer avançar a agulha de vidro para que a perfuração RWM é tão pequeno quanto possível. Dependendo do microscópio utilizado, segurar as pipetas utilizando um micromanipulador ou à mão, utilizando uma pipetatitular. </li><li> Prepare as pipetas para injecção utilizando uma pipeta extrator da mesma forma que as pipetas de fixação de membranas são preparadas. Certifique-se de que o diâmetro da ponta é grande (cerca de 15 m de diâmetro), de modo que a pipetagem do vírus dentro e para fora é feito facilmente. </li><li> Elaborar 1-2 l de AAV1-VGLUT3, AAV1-GFP ou AAV2-GFP em uma pipeta de injeção. Depois do efluxo é estabilizada (5 a 10 min), microinject este volume fixo na rampa timpânica através do mesmo orifício feito previamente RWM. </li><li> Selar o nicho RW rapidamente depois de retirar a pipeta com uma pequena porção de músculo e fixe-o com uma pequena gota de cola de tecido colocado no músculo para evitar fugas da janela redonda. NOTA: Selar este furo muito bem após a agulha é removida com uma pequena camada de músculo para impedir qualquer fuga de perilinfa a cóclea-este passo é crítico para preservar audição. Falha para selar completamente o VM irá resultar em perda de audição ao longo do tempo. </li><li> Após a injeção, cobrir oburaco na bula auditivo com o tecido adiposo, os músculos retornar pós-auricular e tecido adiposo para a sua posição normal e suturar a ferida em camadas com uma sutura absorvível 6-0 crómico ou menor. </li><li> Desinfectar a ferida com iodopovidona. </li></ol> 3. Cuidados Pós-Operatórios <ol><li> Coloque os ratos em uma gaiola quente e não deixá-los abandonados até que sejam totalmente recuperado depois movê-los de volta com a mãe. Recomenda-se a remover o macho da gaiola antes de colocar os filhotes de volta com a mãe. </li><li> Administrar Carprofeno subcutânea (2 mg / kg) para analgesia pós-operatória e a cada 24 horas depois durante 3 dias, para gerir a inflamação e dor. Monitorar os animais diariamente para sinais de sofrimento, perda de peso anormal, dor ou infecção. Geralmente pelo 3º dia após a cirurgia todos os ratos devem estar agindo normalmente. Se quaisquer sinais de angústia ou doença aparecer em um rato após o 3º dia, considerar a eutanásia de tele rato conforme diretrizes institucionais. </li></ol> 4. Avaliação da função coclear Após Parto Viral Usando Auditivo de Tronco Encefálico (ABR) Recordings NOTA: O tronco encefálico (ABR) é um potencial auditivo extraído da atividade elétrica em curso no cérebro e gravado através de eletrodos colocados sob o couro cabeludo evocado. O animal é estimulado com som. A gravação resultante consiste de cinco ondas que reflectem a actividade eléctrica dos pontos sucessivos da via auditiva nos primeiros 10 ms após o início do estímulo auditivo. <ol><li> Anestesiar ratos por injeção intraperitoneal de uma mistura de cloridrato de cetamina e cloridrato de xilazina, conforme descrito no ponto 1.2 do presente protocolo, excepto sem acepromazina. </li><li> Posicione o mouse sobre uma almofada de aquecimento durante todo o teste de audição até que o mouse é totalmente acordado, para evitar pós-anestésica hipotermia. NOTA: Useos seguintes estímulos sonoros nesta experiência: clique (5 ms de duração, 31 de taxa de apresentação Hz). </li><li> Coloque eletrodos de agulha subdérmicos no vértice, abaixo do pavilhão auricular esquerdo (de referência), e abaixo da orelha contralateral (terra) e começar a gravação ABRs como descrito anteriormente em uma câmara à prova de som 27,28. Grave ondas ABR em 5 intervalos de nível de pressão sonora dB para baixo de amplitude máxima. </li><li> Determinar os limiares ABR postoperatively logo aos quatro dias após o parto viral NOTA: Threshold é definido como o nível mais baixo de estímulo no qual a resposta picos de ondas I-V foram claramente e repetidamente presente na inspeção visual. </li><li> Medida da onda I analisar a atividade do nervo coclear. O nível mais baixo estímulo que produz uma forma de onda ABR detectável é definido como o limiar. </li></ol> 5. Coclear Transgene Expression Protein, através de imunofluorescência <ol><li> Anestesiar ratos com uma sobredosagem de intraperitoneal injeção de uma mistura de cloridrato de cetamina e cloridrato de xilazina, conforme descrito no ponto 1.2 do presente protocolo, excepto sem acepromazina. </li><li> Decapitar a cabeça somente após o animal já não responde a estímulos dolorosos, como toe pitada. </li><li> Dissecar o cochleae fora e processo para imunofluorescência todo o monte, como descrito 26. Um protocolo detalhado do conjunto de montagem coclear imunofluorescência utilizando anticorpos anti-VGLUT3 7a e anticorpos anti-miosina é descrito em Akil et al., 2013 29. </li><li> Para a etiquetagem de GFP, incubar as cocleares inteiras montagens durante a noite a 4 ° C com um anticorpo de coelho anti-GFP em 1: 250 de diluição. </li><li> Lavar as cócleas duas vezes durante 10 min com PBS e depois incubar durante 2 horas em IgG de cabra anti-coelho conjugado com Cy2 diluído 1: 4000 em PBS. </li><li> Lavar as cócleas com PBS duas vezes durante 10 minutos e incubar com DAPI durante 15 min. </li><li> Monte o cochleae em lâminas de vidro e observar under um microscópio com imunofluorescência confocal. </li></ol>

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+cochlear+transgene+expression+mediated+by+recombinant+adeno-associated+virus.">Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus.</a> Gene Ther. 5 (2), 277-281 (1998).</li><li>Raphael, Y., Frisancho, J. C., Roessler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+into+guinea+pig+cochlear+cells+in+vivo.">Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.</a> Neurosci. Lett. 207 (2), 137-141 (1996).</li><li>Weiss, M. A., Frisancho, J. C., Roessler, B. J., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+mediated+gene+transfer+in+the+cochlea.">Viral mediated gene transfer in the cochlea.</a> Int. J. Dev. Neurosci. 15 (4=5), 577-583 (1997).</li><li>Pettis, R. M., Han, J. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracochlear+infusion+of+recombinant+adeno+associated+virus:+Analysis+of+its+dissemination+to+near+and+distant+tissues.">Intracochlear infusion of recombinant adeno associated virus: Analysis of its dissemination to near and distant tissues.</a> Assoc. Res. Otolaryngol. Abstr. 21, 673 (1998).</li><li>Konish, i. M., Kawamoto, K., Izumikawa, M., Kuriyama, H., Yamashita, T. <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+guinea+pig+cochlea+using+adeno-associated+virus+vectors.">Gene transfer into guinea pig cochlea using adeno-associated virus vectors.</a> J. Gene Med. 10 (6), 610-618 (2008).</li><li>Kaplitt, M. G., 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=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nature Genetics. 8 (2), 148-154 (1994).</li></ol>

No conflicts of interest declared.

Neste trabalho, nós descrevemos em pormenor uma técnica que pode ser utilizada para terapia génica coclear, com o objectivo de restaurar ou resgatar função auditiva normal, que está comprometida por um defeito genético. Como é tipicamente atraumática, esta abordagem é seguro para a transferência de genes coclear ou outras terapias moleculares potenciais 30. Outras abordagens para a terapia coclear foram descritas, incluindo uma abordagem ventral 24, cocleostomias 31,32 e saco ...

A terapia genética tem sido sugerida como um potencial tratamento para perda auditiva genética, mas o sucesso nesta área permaneceu uma incógnita 1. Até o momento, as metodologias de forma viral mediadas predominaram devido a capacidade teórica para atingir tipos específicos de células dentro da cóclea relativamente inacessível. Ambos os adenovírus (AV) e vírus adeno-associado (AAV) têm sido utilizadas para a entrega de genes coclear. AAV são vantajosos na cóclea por um número de razões. São vírus deficientes na replicação eficiente e pode transferir moléculas transgénicos para diferentes tipos de células incluindo neurónios, um alvo importante para um certo número de causas de perda de audição. Entrada de AAV na célula é mediada por receptores específicos 2; assim, a escolha de um serotipo particular, deve ser compatível com os tipos de células a serem transduzidas. AAV pode eficazmente transfectar células ciliadas 3 e incorporar no genoma hospedeiro, resultando em estabilidade, expressão de longo prazo do traproteína nsgenic e mudança fenotípica na célula 4. Embora não seja necessariamente vantajoso para as aplicações de curto prazo, tais como a regeneração de células de cabelo, expressão de longo prazo é muito importante para o salvamento estável de defeitos genéticos. Porque os AAV não estão associados com qualquer doença humana ou infecção e não demonstram 5,6,7 ototoxicidade, que é um candidato ideal para utilização na terapia de gene para formas herdadas de perda auditiva 8. Transferência de material genético exógeno para o ouvido interno de mamíferos utilizando vetores virais tem sido estudada ao longo da última década e está emergindo como uma técnica promissora para o tratamento de ambas as formas genéticas e adquiridas de perda auditiva 9. A cóclea é potencialmente um alvo ideal para terapia génica, por várias razões: 1) o seu volume pequeno necessita de uma quantidade limitada do vírus necessária; 2) seu relativo isolamento de outros efeitos limites de sistemas de órgãos secundários; e 3) suas câmaras cheias de líquido facilitar viralentrega ao longo do labirinto 10, 11,12,13,14, 15. Modelos de ratos com surdez congênita permitir o uso de vários métodos de estudo para monitorar o desenvolvimento do ouvido interno de uma forma sistemática, replicável. Enquanto o pequeno tamanho do rato cochleae não apresenta alguma dificuldade cirúrgica, o mouse funciona como um modelo extremamente importante no estudo de surdez genética, com várias vantagens em relação a outras espécies experimentais 16. Modelos de ratos permitir a avaliação de um conjunto de características por meio de análise de ligação genética, a coleta de observações morfológicas detalhadas, e simulação de cenários patogénicos; como tal, eles são bons candidatos para a terapia génica mediada por vírus. Estudos genéticos extensas em ratos, combinados com os avanços tecnológicos tornaram possível gerar camundongos geneticamente modificados de forma reproduzíveis em laboratório 17,18, 19, 20,21. Furthermore, existem inúmeros modelos para ambas adquiridas e herdou auditivos fenótipos perda em ratos, permitindo testes rigorosos neste modelo animal 22, 23,24. Assim, corrigindo audiência usando terapia genética mediada por vírus em um modelo de rato é um primeiro passo adequado na busca de uma cura para a doença humana. Nós já mostraram que camundongos transgênicos que faltam glutamato vesicular transporter 3 (VGLUT3) nascem surdos devido à falta de liberação de glutamato na fita sinapse IHC 25. Porque esta mutação não leva a uma degeneração primária das células sensoriais ciliadas, estes ratinhos mutantes são potencialmente um excelente modelo em que para testar a terapia genética coclear para a perda auditiva congênita. Até à data, foram descritos um número de técnicas de entrega virais para terapia genética coclear, incluindo membrana da janela redonda difusão, injecção membrana da janela redonda, e entrega através de um cocleostomias. Existem potenteial vantagens e desvantagens de cada uma destas abordagens 9. Aqui nós relatamos um método cirúrgico para entrega gene viral mediada para o ouvido interno VGLUT3 KO rato através da membrana da janela redonda (VM). O método de injeção RWM pós-auricular é minimamente invasivo, com excelente preservação auditiva, e é relativamente rápido. Como já publicado anteriormente, num esforço para restaurar a audição neste modelo de ratinho, um vector que transporta o gene AAV1 VGLUT3 (AAV1-VGLUT3) foi introduzido na cóclea destes ratinhos surdos, dia pós-natal 12 (P! @), Resultando em a restauração da audição 26. Ouvindo nos ratos VGLUT3 KO foi verificada por audiometria de tronco encefálico (ABR), enquanto que a expressão da proteína transgene foi verificada através de imunofluorescência (IF). Esta metodologia demonstra, portanto, que a terapia génica mediada por vírus pode corrigir um defeito genético que de outra forma seria resulta em surdez.

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			<td>&#160;Tucker-Davis Technologies</td>
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surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

A terapia genética, usada para conseguir a recuperação funcional de surdez neurossensorial, promete conceder uma melhor compreensão dos mecanismos moleculares e genéticas subjacentes que contribuem para a perda de audição. Introdução de vectores para o ouvido interno deve ser feito de uma maneira que largamente distribui o agente de toda a cóclea ao minimizar lesão das estruturas existentes. Este artigo descreve a abordagem cirúrgica pós-auricular que pode ser utilizado para a terapia da cóclea de rato usando molecular, farmacológica, e entrega viral para ratinhos 10 dias pós-natais e mais velhos através da membrana da janela redonda (VM). Esta abordagem cirúrgica permite a entrega rápida e directa na escala timpânica, minimizando a perda de sangue e evitar a mortalidade animal. Esta técnica envolve negligenciável ou nenhum dano às estruturas essenciais do ouvido interno e médio, bem como os músculos do pescoço, enquanto preservando totalmente a audição. Para demonstrar a eficácia da técnica cirúrgica, a glutam vesicularComeram transportador 3 nocaute (VGLUT3 KO) será utilizado como um exemplo de um modelo de ratinho de surdez congénita que recupera auditiva após a entrega de VGLUT3 para o ouvido interno utilizando um vírus adeno-associado (AAV-1).

Para verificar as características técnicas e utilidade da abordagem pós-auricular para a terapia molecular coclear, AAV1-VGLUT3, AAV1-GFP e AAV2-GFP foram entregues em camundongos P10-12 ouvido interno através da VM. Esta abordagem demonstra a expressão do transgene de sucesso dentro das células ciliadas internas (IHC) (VGLUT3 Figura 1 e Figura 2 GFP e GFP Figura 3A), células ciliadas externas (OHC) (GFP Figura 2) e células de suporte (GFP <stro...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Método cirúrgico para Virally Mediated Gene entrega à orelha de rato Inner através da janela redonda Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Research

JoVE Journal

Neuroscience

15.1K visualizações.  University of California, San Francisco. The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane. 

Vídeo: Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Microscopia intravital da Microcirculação cérebro de camundongo usando uma janela fechada Cranial

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neurociência

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Eletrofisiologia II: Fabricação Eletrodo de Sucção Extracelular

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolando-LacZ expressar Células de tecidos de camundongos Inner Ear usando Citometria de Fluxo

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

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 ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

A polido e reforçado Janela afinado crânio para longo prazo de imagens do cérebro de camundongo

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Gravações patch clamp em células ciliadas da orelha interna Isolado de Zebrafish

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Imagiologia pHluorin-tagged Inserção receptor para a membrana plasmática em primários de rato cultivadas Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

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

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizando metodologias combinadas para definir o papel do Plasma Membrane entrega durante Axon Ramificação e Neuronal morfogênese

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

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

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

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

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Técnica do mouse Microcirurgia de infusão para distribuição de substâncias alvejado no SNC<i&gt; via</i&gt; Artéria carótida interna

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

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Implante de janela de laminectomia e medula espinhal no mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...