Name: neonatal murine cochlear explant technique as an in vitro screening tool in hearing research
Uploaded: 2017-06-08T15:00:00.000+00:00
Duration: 8 min 30 s
Description: Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

Este trabalho foi apoiado pelos subsídios do Instituto Nacional de Surdez e Distúrbios da Comunicação R01DC015824 (KMS) e T32DC00038 (suporte de SD), o Departamento de Defesa concede W81XWH-15-1-0472 (KMS), a Fundação Bertarelli (KMS), o Nancy Sayles Day Foundation (KMS) e o Lauer Tinnitus Research Center (KMS). Agradecemos a Jessica E. Sagers, BA por comentários perspicazes sobre o manuscrito.

O protocolo do estudo foi aprovado pelo Comitê Institucional de Cuidados e Uso Animal (IACUC) do Massachusetts Eye and Ear. Os experimentos foram realizados de acordo com o Código de Ética da Associação Médica Mundial. 1. Preparando a dissecação <ol><li> Preparando a mesa cirúrgica <ol><li> Use etanol a 70% para desinfectar a mesa cirúrgica. </li><li> Coloque duas pastilhas não preparadas ao lado do microscópio. </li><li> Prepare a bandeja do instrumento, incluindo tesouras de operação esterilizadas por calor, uma alça de bisturí, uma microcarga e fórceps (duas em cada # 4 e # 55). Coloque a bandeja do instrumento e uma placa de Petri com fundo de parede de paredes claras de 50 mm sobre uma almofada não-estéril. </li><li> Obter uma lâmina de bisturi # 15 estéril; Uma bolsa ou recipiente de plástico selável (para descartar as carcaças de acordo com as diretrizes institucionais); Gelo em um balde; E solução de sal equilibrada Hank, estéril e fria (HBSS). Coloque esses itens noOutra almofada não-esterilizada. </li><li> Transfira o HBSS para um tubo de laboratório plástico de 50 mL e coloque-o sobre gelo. </li></ol></li><li> Preparando a cultura <ol><li> Prepare todos os materiais em uma capa de fluxo laminar. Para cada quatro espécimes, coloque quatro lâminas de cobertura de vidro redondas de 10 mm em autoclavagem nas quatro incrustações da placa de Petri de 35 mm de 4 poços. </li><li> Para cada 4 espécimes, misture um volume total de 300 μL que consiste no seguinte em um tubo de microcentrífuga: 10 μL de adesivo de tecido, 285 μL de NaHCO 3 e 5 μL de NaOH. </li><li> Coloque 45 μL da solução resultante em cada folha de cobertura e deixe-a lá durante pelo menos 20 minutos à TA; Esta solução pode ser deixada no lamínula durante várias horas, mas não pode ser deixada O / N. </li><li> Coloque uma ou duas placas de petri de 4 mm de 4 mm no interior de uma placa de Petri de 10 cm para criar duas barreiras contra a contaminação. </li><li> Preparar meio de cultura contendo 98% de Meio de Eagle Modificado por Dulbecco (DMEM), 1% de N-2 e 1% de ampicilina (65 μL por poço em um tubo de (micro) centrífuga). Aquecer a solução até 25 ° C antes da utilização. </li></ol></li></ol> 2. Dissecção Extrica Cochlear Murina <ol><li> Decapitação do mouse neonatal <ol><li> Obtenha uma placa de petri de plástico de 60 mm e pulverize 70% de etanol na sua tampa de modo que a superfície esteja molhada. </li><li> Despeje o HBSS frio do tubo de laboratório de plástico na parte inferior do plástico de 60 mm e a placa de Petri de fundo transparente com 50 mm de parede transparente. Armazene o tubo de laboratório de plástico e ambos os pratos de Petri no gelo para manter o tecido frio sempre que não estiver sendo dissecado. </li><li> Coloque um rato neonatal P3-P5-envelhecido no gelo em um dedo de corte de uma luva de látex e aguarde até que a hipotermia induz perda de consciência (aproximadamente 1-3 min). </li><li> Decapite o mouse rapidamente com tesoura em funcionamento e descarte o corpo na bolsa de plástico selável. Coloque a cabeça de recém-nascido em70% de etanol na tampa da placa PETRI de plástico de 60 mm. </li></ol></li><li> Dispersão macroscópica do osso temporal <ol><li> Remova a pele do crânio fazendo um corte superficial da parte anterior à posterior (diretamente na parte superior da sutura sagital) usando a lâmina do bisturi. </li><li> Corte os dois canais auditivos externos com a lâmina do bisturi e dobre a pele anteriormente para expor todo o crânio. NOTA: Embora não seja necessário nenhum microscópio de dissecação para esta etapa, ele pode ser usado para uma melhor visualização. </li><li> Abra o crânio ao longo da sutura sagital usando a lâmina # 15 do bisturi. Certifique-se de cortar o crânio movendo suavemente a lâmina para cima e para baixo de anterior para posterior para evitar a compressão das cócleas. NOTA: O crânio não deve ser ossificado nesta idade e deve cortar facilmente. </li><li> Remova e descarte o focinho fazendo um corte vertical diretamente posterior às órbitas usando o bisturi # 15lâmina. NOTA: A parte posterior do crânio ainda deve conter o cérebro e as cócleas. </li><li> Descarte o prosencéfalo, cerebelo e tronco cerebral através de dissecção sem corte com fórceps # 4. </li></ol></li><li> Extração microscópica das cócleas <ol><li> Ajuste o microscópio (ampliação 6.5X) e a fonte de luz colocando a pinça sob o escopo e otimizando a iluminação e o foco. </li><li> Coloque as duas metades do crânio na placa de petri de plástico de 60 mm, preenchida com HBSS frio. </li><li> Exponha completamente a cóclea / e, identificável como adjacente à artéria estapedial circundante (uma artéria tortuosa dentro do osso temporal) e separe sem rodeios o órgão do osso temporal usando fórceps # 4. </li><li> Transfira a cóclea para a placa de Petri com fundo de vidro de 50 mm, de paredes claras e posicione o prato sob o microscópio. </li><li> Coloque a placa de petri de plástico de 60 mm com a outra metade do crânio no gelo. </Li&gt; <li> Remova a cóclea do sistema vestibular e dissecar cuidadosamente a cápsula ocula coclear (tipicamente cartilaginosa a esta idade) com fórceps # 4. Use pinças # 55 para todas as etapas adicionais. </li></ol></li><li> Etapas finais do processamento de tecidos <ol><li> Continue com o processamento do tecido da orelha interna separando cuidadosamente o ligamento espiral aderente ao órgão de Corti do resto da cóclea e o modiolus usando a microcadeia ou duas fórceps. <ol><li> Corte na área mais escura e translúcida na fenda entre o ligamento espiral e a região das células ciliadas e proceda à remoção subseqüente do ligamento espiral, agarrando-o no aspecto mais basal e relaxando suavemente enquanto se move apicalmente. </li></ol></li><li> Posicione a amostra para obter uma visão longitudinal e sagital da cóclea e corte a cóclea em duas a três seções usando a microcâmara, classificando essas seções como apical, (meio) e bVoltas do Asal. Remova o tecido superior e inferior à camada real de células ciliadas e neurites, a fim de obter uma peça de explante coclear que pode ser colocada horizontalmente na placa de Petri. NOTA: Um tamanho apropriado para a adesão estável ao prato de cultura é aproximadamente metade de uma volta coclear. </li><li> Remova a membrana tectorial, uma camada translúcida muito fina, imediatamente superior ao órgão de Corti, descascando suavemente com fórceps. </li><li> Remova a membrana de Reissner, imediatamente superior às SGNs, tocando-a com fórceps em ambos os lados e descascando-a. NOTA: Remova todas as áreas de células de cabelo danificadas localizadas lateralmente, cortando-as com a microcadeira. </li></ol></li></ol> 3. Transferência de espécimes para as placas de cultura <ol><li> Remova os 45 μL de solução adesiva de tecido das quatro folhas de cobertura. Lavar cada deslizamento de cobertura com 50 μL de H2O estéril. Aspirar a água. </li><li> Pegue a pipeta de 1 mL. Molhe a ponta da pipeta com aproximadamente 120 μL de DMEM para evitar que a amostra adira ao interior da ponta. </li><li> Transfira os espécimes individualmente usando a pipeta de 1 mL. Pipetar um máximo de 70 μL da placa de Petri com fundo de vidro, pequeno, de paredes claras e com base em HBSS e colocar a amostra juntamente com o líquido em uma das 4 incrustações (preparadas com folhas de cobertura) na placa Petri de 4 poços . </li><li> Verifique sob o microscópio (aumento de 50X) que a amostra está na orientação correta. Certifique-se de que a área das células ciliadas a partir da qual a membrana tectorial foi removida fica voltada para cima. Certifique-se de que a membrana basilar, juntamente com a parte basal reduzida do modiolus, esteja voltada para baixo e adere ao lamínula. <ol><li> Se o espécime for orientado de cabeça para baixo após a inspeção, deposite o HBSS que permanece na ponta da pipeta na embutição e reposicione a peça até que esteja corretamente orientada. NÃOTE: isto impedirá que células ciliadas flutuem em meio de cultura sem suporte estrutural da lamínula, o que pode levar à degeneração. </li></ol></li><li> Aspirar excesso de HBSS usando a pipeta de 1 mL. Aguarde 15 s. </li><li> Pipetar 60 μL do meio de cultura quente preparado (98% de DMEM, 1% de N-2 e 1% de ampicilina) diretamente sobre a amostra. Tenha cuidado para não tocar na amostra com a pipeta. Substitua as tampas interna e externa do prato de Petri e incube O / N a 37 ° C. </li><li> Coloque todas as soluções de reserva em seus locais apropriados, descarte carcaças e resíduos residuais, descontaminar a mesa cirúrgica de acordo com diretrizes institucionais ( por exemplo, usando etanol ou hipoclorito), limpe e autoclave os instrumentos e descarte os objetos afiados no recipiente apropriado . </li></ol> 4. Adicionando meios culturais finais e substâncias de interesse NOTA: Os explantes cocleares estarão prontos para uso 10-16 h depois. <ol><li> Prepare um mixtuRe 97% de DMEM, 1% de soro bovino fetal (FBS), 1% de N-2 e 1% de ampicilina (65 μL por poço em um tubo de microcentrífuga, aquecer a solução até 25 ° C antes da utilização). </li><li> Adicione outras substâncias de interesse para as soluções para os respectivos grupos de tratamento. Se forem adicionadas substâncias fluorescentes ( p. Ex. Vírus da proteína fluorescente verde (GFP), em uma publicação recente, uma concentração de 10 10 GC de Vírus Adeno-Associados (AAVs) foi utilizada durante 48 h em 50 μL) 18 , proteja de luz. </li><li> Transfira as placas de Petri contendo explantes cocleares da incubadora e coloque-as sob o capô de fluxo laminar. </li><li> Aspirar o meio de cultura antigo (sem FBS) com a pipeta das inlays. </li><li> Adicionar 60 μL por poço do meio de cultura quente (tratamento) preparado (97% de DMEM, 1% de FBS, 1% de N-2, 1% de ampicilina e as substâncias de interesse). </li><li> Coloque a placa Petri de volta à incubadora (37 ° C). </li><li> Ver rPor exemplo, no microscópio para identificar sinais de contaminação, migração celular ou desprendimento do explante coclear e realizar manchas após um máximo de 7 dias. </li></ol> 5. Imunofluorescência - Dia 1 <ol><li> Preparar a solução de bloqueio (94% de solução salina tamponada com fosfato (PBS), 5% de soro normal de cabra ou cavalo (NGS / NHS, dependendo dos anticorpos secundários) e 1% de surfactante não iónico (ver Tabela de Materiais )) e estoque Solução de anticorpo (98,6% de PBS, 1% de NHS e 0,4% de surfactante não iónico com os respectivos anticorpos). NOTA: Os anticorpos incluem o coelho anti-Myo7A a uma concentração de 1: 400+ (miosina 7A, para células ciliadas internas e externas) e anti-TuJ1 1: 200+ (β-tubulina, para SGNs) OU mouse (IgG1) anti -CtBP2 1: 200+ (proteína de ligação c-terminal, para presinapse), mouse (IgG2a) anti-PSD95 1: 50+ (densidade pós-sináptica, pós-sincronismo) e frango anti-NF-H 1: 1000+ (neurofilamento, Para SGNs e fibras nervosas). &quot;+&quot; MeaNs &quot;ou superior&quot;. </li><li> Aspirar o meio de cultura usando uma pipeta. Tenha cuidado para não tocar na amostra. </li><li> Sob o capô de fluxo laminar, enxágue os explantes cocleares duas vezes com PBS estéril, colocando os espécimes em um agitador durante 5 minutos após cada lavagem. </li><li> Corrigir o tecido em paraformaldeído a 4% (PFA - CUIDADO) por 20 minutos no agitador (sob o capô). Aspirar o PFA. </li><li> Aplique 60 μL de PBS nos explantes cocleares e coloque a placa de Petri em um agitador durante 5 min. Aspirar o PBS. Repita 3x. </li><li> Coloque os explantes cocleares na solução de bloqueio preparada (94% de PBS, 5% de NHS e 1% de surfactante não iónico) durante 30 min no agitador. </li><li> Coloque os explantes cocleares em uma solução de anticorpo de estoque preparada (98,6% de PBS, 1% de NHS e 0,4% de surfactante não iónico) com os respectivos anticorpos primários (vortex antes do uso) O / N em um contador na RT. NOTA: Enrole as placas de Petri com toalhas de papel úmidas, incluídas em plástico (ou papel alumínio, se a solução adicionadaIon contém materiais fluorescentes, como vírus que expressam GFP) para manter a louça úmida e para evitar a evaporação da solução de anticorpo O / N. </li></ol> 6. Imunofluorescência - Dia 2 <ol><li> Prepare a mancha de 4 &#39;, 6-diamidino-2-fenilindole (DAPI) (300 nM) e use a solução de anticorpo de reserva para obter as misturas de anticorpos secundários corretas, complementares aos anticorpos primários no passo 5.1. (Por exemplo , cabra anti-coelho 488 1: 200+ e cabra anti-rato 568 1: 200+ OU cabra anti-rato (IgG1) 568 1: 1000+, cabra anti-rato (IgG2a) 488 1: 500+, E cabra anti-frango 647 1: 200+ OU Phalloidin 568 1: 200+ (para células ciliadas internas e externas)). </li><li> Aspirar a solução primária de anticorpos. </li><li> Aplique 60 μL de PBS nos explantes cocleares e coloque a placa de Petri em um agitador durante 5 min. Aspirar o PBS. Repita 3x. </li><li> Colocar os explantes cocleares na solução preparada de anticorpo em estoque (98,6% de PBS, 1% de NHS e 0,4% de surfactante não iónico) com resAnticorpos secundários pectivos (vortex antes do uso) durante 1,5 h em um contador e protegem da luz. </li><li> Aspirar a solução secundária de anticorpos e enxaguar os explantes cocleares com uma mancha DAPI (colocando em um agitador durante 1 min e depois aspirar). </li><li> Aplique 60 μL de PBS nos explantes cocleares e coloque a placa de Petri em um agitador durante 5 min. Aspirar o PBS. Repita 3x. </li><li> Use fórceps para remover lamínulas com espécimes da placa de 4 poços, mergulhe-os em um recipiente repleto de H 2 O destilado e retire os lamíninhos colocando-os verticalmente em contato com toalhas de papel. </li><li> Coloque uma gota de meio de montagem antifade no slide do microscópio e inverta as lamelas para mergulhar o tecido voltado para baixo no meio. </li><li> Selar a lamínula usando esmalte de asa transparente cobriu circunferencialmente a borda (sem esmagar a amostra sob o vidro). Deixe as lâminas deitado na área coberta longe da luz por 15 a 20 minutos até a secagem e a temperaturaColocá-los em uma caixa ou examinar sob o microscópio confocal. NOTA: A coloração fluorescente é melhor conservada armazenando as lâminas a 4 ou -20 ° C. </li></ol> 7. Imagens confocais <ol><li> Obtenha uma visão geral e imagens ampliadas para o órgão da região de Corti, incluindo neurites e SGNs. NOTA: Configurações padrão para o processo de imagem: Formato de 1.024 x 1.024 pixels, velocidade de 400 Hz, média de trama de 3, 20% de intensidade geral do laser e, geralmente, lente de 20X e 63X com imersão em óleo (potencialmente combinada com zoom digital 2X). As configurações de canal para o protocolo de coloração DAPI, Myo7A e TuJ1 são descritas acima: 405 5% DAPI, &quot;Smart Gain&quot; 766.8V, &quot;Smart Offset&quot; 0.0% - 488 15% isotiocianato de fluoresceína (FITC), &quot;Smart Gain&quot; 622.2V , &quot;Smart Offset&quot; 0,0% - 561 13% de isotiocianato de tetrametilrhomadina (TRITC), &quot;Smart Gain&quot; 562,4V, &quot;Smart Offset&quot; 0,0%. </li><li> Mesclar as imagens em uma pilha z usando oSoftware para obter uma projeção do eixo z. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+Factors+from+Human+Vestibular+Schwannomas+Can+Cause+Cochlear+Damage.">Secreted Factors from Human Vestibular Schwannomas Can Cause Cochlear Damage.</a> Sci Rep. 5, 18599 (2015).</li><li>Soares, V. 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=Extracellular+vesicles+derived+from+human+vestibular+schwannomas+associated+with+poor+hearing+damage+cochlear+cells.">Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells.</a> Neuro Oncol. , (2016).</li><li>Tong, M., Brugeaud, A., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerated+synapses+between+postnatal+hair+cells+and+auditory+neurons.">Regenerated synapses between postnatal hair cells and auditory neurons.</a> J Assoc Res Otolaryngol. 14 (3), 321-329 (2013).</li><li>Yuan, 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=Ouabain-induced+cochlear+nerve+degeneration:+synaptic+loss+and+plasticity+in+a+mouse+model+of+auditory+neuropathy.">Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy.</a> J Assoc Res Otolaryngol. 15 (1), 31-43 (2014).</li><li>Fernandez, K. A., Jeffers, P. W., Lall, K., Liberman, M. C., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+after+noise+exposure:+acceleration+of+cochlear+synaptopathy+in+&amp;#34;recovered&amp;#34;+ears.">Aging after noise exposure: acceleration of cochlear synaptopathy in &amp;#34;recovered&amp;#34; ears.</a> J Neurosci. 35 (19), 7509-7520 (2015).</li><li>Barclay, M., Constable, R., James, N. R., Thorne, P. R., Montgomery, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+sensory+stimulation+alters+the+molecular+make-up+of+glutamatergic+hair+cell+synapses+in+the+developing+cochlea.">Reduced sensory stimulation alters the molecular make-up of glutamatergic hair cell synapses in the developing cochlea.</a> Neuroscience. , 50-62 (2016).</li><li>Zinn, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Silico+Reconstruction+of+the+Viral+Evolutionary+Lineage+Yields+a+Potent+Gene+Therapy+Vector.">In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector.</a> Cell Rep. 12 (6), 1056-1068 (2015).</li><li>Wu, Z., Yang, H., Colosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+on+AAV+vector+packaging.">Effect of genome size on AAV vector packaging.</a> Mol Ther. 18 (1), 80-86 (2010).</li><li>Shu, 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=Identification+of+Adeno-Associated+Viral+Vectors+That+Target+Neonatal+and+Adult+Mammalian+Inner+Ear+Cell+Subtypes.">Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes.</a> Hum Gene Ther. , (2016).</li><li>Kao, S. Y., Soares, V. Y., Kristiansen, A. G., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRAIL-DR5+pathway+promotes+sensorineural+degeneration+in+the+inner+ear.">Activation of TRAIL-DR5 pathway promotes sensorineural degeneration in the inner ear.</a> Aging Cell. 15 (2), 301-308 (2016).</li></ol>

Os autores não têm nada a revelar.

Os pesquisadores devem aperfeiçoar a técnica de dissecação antes de realizar experiências envolvendo explantes cocleares. As células do cabelo são comumente danificadas durante as dissecações realizadas no início da curva de aprendizado e um momento particularmente problemático para sua integridade é a remoção da membrana tectorial, que requer mãos firmes, ferramentas adequadas e experiência. Para economizar tempo e recursos, um controle visual deve ser realizado sob o microscópio de dissecação e áre...

Perda auditiva sensorial (SNHL) reflete dano ao ouvido interno ou via auditiva ascendente. Embora a perda de audição seja o déficit sensorial mais comum em humanos 1 , as terapias curativas ainda não existem 2 . Embora os implantes de tronco encefálicos cocleares ou auditivos possam restaurar algum grau de audição para pacientes com SNHL grave a profunda, a audiência fornecida por esses dispositivos ainda é muito diferente da audiência &quot;natural&quot;, especialmente durante as tentativas de entender a fala em ruído ou para ouvir música. Embora a degeneração de células ciliadas tenha sido considerada a principal conseqüência de eventos auditivos traumáticos ( por exemplo, exposição ao ruído alto), há evidências crescentes de que as sinapses que transportam informações de células ciliadas para o nervo auditivo são pelo menos tão vulneráveis ​​a trauma acústico 3 , 4 , 5 </sup<html> &gt; , 6 . Uma vez que os limiares audiométricos humanos, o padrão ouro atual para a avaliação da função auditiva, não prevêem danos celulares específicos na orelha interna, são necessárias ferramentas mais refinadas para detectar a degeneração celular o mais rápido possível e para iniciar um tratamento adequado 7 . Os tratamentos farmacêuticos promissores para a perda auditiva são freqüentemente testados em culturas celulares homogêneas in vitro , mas esses sistemas não modelam com precisão o microambiente coclear. Sabe-se que as células cocleares secretam fatores tróficos que influenciam outros tipos de células dentro da cóclea 8 , 9 , um processo in vivo crucial que se perde quando o órgão de Corti 10 , 11 ou Neurônios de Ganglion Espiral (SGNs) 12 são cultivados isoladamente ou quando Marcadores moleculares são analisados13. No entanto, estudos in vivo que podem ser necessários para a validação de dados in vitro para estabelecer novos tratamentos personalizados para a perda de audição na busca de &quot;medicina de precisão&quot; requerem recursos e tempo significativos. Isto é especialmente relevante quando se considera Quanto esforço é necessário para aperfeiçoar e realizar injeções de membrana de orelha média ou arredondada com testes auditivos e a subsequente dissecção de montagens inteiras cocleares. O rastreio eficiente de compostos promissores em culturas orgotípicas ex vivo conhecidas como explantes cocleares fornece uma alternativa econômica e confiável 14 , 15 , 16 , 17 . Este artigo detalha um protocolo para gerar, manter e avaliar explantes cocleares tratados. As aplicações específicas para este modelo são enfatizadas, incluindo seu uso na seleçãoDe compostos potencialmente terapêuticos e a avaliação comparativa de vetores virais para terapia genética. Uma abordagem de explante ex vivo permite aos pesquisadores visualizar os efeitos de um determinado tratamento em diferentes populações celulares in situ , facilitando a identificação de mecanismos específicos do tipo celular e o aprimoramento subseqüente de terapias específicas. No geral, esta técnica fornece um modelo para estudar a cóclea ex vivo, ao mesmo tempo em que preserva a conversação vital entre os tipos de células muito diferentes que coexistem dentro da cóclea.

<table><tbody><tr><td>Ampicillin, Sodium Salt</td><td>Invitrogen</td><td>11593-027</td><td></td></tr><tr><td>anti-CtBP2 antibody, mouse(IgG1)</td><td>BD Transduction Laboratories</td><td>612044</td><td></td></tr><tr><td>anti-Myo7A antibody, rabbit</td><td>Proteus Biosciences</td><td>25-6790</td><td></td></tr><tr><td>anti-NF-H antibody, chicken</td><td>EMD Millipore</td><td>AB5539</td><td></td></tr><tr><td>anti-PSD95 antibody, mouse(IgG2a)</td><td>Antibodies Inc.</td><td>75-028</td><td></td></tr><tr><td>anti-TuJ1 antibody, mouse</td><td>BioLegend</td><td>801202</td><td></td></tr><tr><td>Cell-Tak Cell and Tissue Adhesive, 5 mg</td><td>Corning</td><td>354241</td><td></td></tr><tr><td>CELLSTAR 15 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile</td><td>Greiner Bio-One</td><td>188161</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 100 x 20 mm</td><td>Greiner Bio-One</td><td>664160</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 35 x 10 mm, 4 inner rings</td><td>Greiner Bio-One</td><td>627170</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 60 x 15 mm</td><td>Greiner Bio-One</td><td>628160</td><td></td></tr><tr><td>CELLSTAR 50 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>Clear Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Clear Wall Glass Bottom Dishes (Glass 40mm), PELCO&amp;reg;, Sleeve/20, 50 x 7 mm</td><td>Ted Pella Inc.</td><td>14027-20</td><td></td></tr><tr><td>Coverslips, Round, Glass, 10 mm diameter, Thickness #1, 0.13 - 0.16mm</td><td>Ted Pella Inc.</td><td>260368</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Distilled water, 500 mL</td><td>Thermo Fisher Scientific</td><td>15230-162&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM), high glucose, pyruvate, no glutamine, 500 mL</td><td>Thermo Fisher Scientific</td><td>10313-039</td><td></td></tr><tr><td>Dumont #4 Forceps</td><td>Fine Science Tools</td><td>11241-30</td><td></td></tr><tr><td>Dumont #55 Forceps (Dumostar)</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Ethyl alcohol (EtOH), Pure, 200 proof, anhydrous, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>459836-1L</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), qualified, USDA-approved regions, 500 mL</td><td>Thermo Fisher Scientific</td><td>10437-028&amp;nbsp;</td><td>Aliquot in 50 mL tubes and store in -20&amp;deg;C freezer</td></tr><tr><td>goat anti-chicken-647 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21469</td><td></td></tr><tr><td>goat anti-mouse(IgG)-568 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-11004</td><td></td></tr><tr><td>goat anti-mouse(IgG1)-568 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21124</td><td></td></tr><tr><td>goat anti-mouse(IgG2a)-488 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21131</td><td></td></tr><tr><td>goat anti-rabbit-488 secondary antibody</td><td>Thermo Fisher Scientific</td><td>R37116</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, sterile, EmbryoMax Ultra Pure Water, 500 mL</td><td>EMD Millipore</td><td>TMS-006-B</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS), calcium, magnesium, no phenol red, 500 mL</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Instrument Tray with Lid Stainless Steel</td><td>Mountainside Medical</td><td>TechMed4255</td><td></td></tr><tr><td>Micro (dissecting) knife &amp;ndash; angled 30&amp;deg;</td><td>Fine Science Tools</td><td>10056-12</td><td></td></tr><tr><td>Microscope slides, VistaVision, color-coded, 75 x 25 mm (3 x 1&quot;), 1 mm thick, white, pack of 72</td><td>VWR</td><td>16004-382</td><td></td></tr><tr><td>N-2 Supplement (100X), 5 mL</td><td>Thermo Fisher Scientific</td><td>17502-048</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;, Sodium Bicarbonate 7.5% solution, 100 mL</td><td>Thermo Fisher Scientific</td><td>25080-094</td><td></td></tr><tr><td>NaOH, sodium hydroxide solution, 1 L</td><td>Thermo Fisher Scientific</td><td>SS266-1</td><td></td></tr><tr><td>Normal Horse Serum (NHS)</td><td>Invitrogen</td><td>16050130</td><td></td></tr><tr><td>Operating scissors</td><td>Roboz Surgical Instruments Co.</td><td>RS-6806</td><td></td></tr><tr><td>Paraformaldehyde (PFA), Reagent Grade, Crystalline</td><td>Sigma-Aldrich</td><td>P6148</td><td>Prior to use: Establish Standard Operating Procedures based on protocols available online</td></tr><tr><td>PBS, pH 7.4, 500 mL</td><td>Thermo Fisher Scientific</td><td>10010-023&amp;nbsp;</td><td>Autoclave prior to use</td></tr><tr><td>Phalloidin, Alexa Fluor 568&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A12380</td><td></td></tr><tr><td>Prep Pad, Non Sterile&amp;nbsp;</td><td>Medline</td><td>05136CS</td><td></td></tr><tr><td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 mL</td><td>Eppendorf</td><td>022363719</td><td>Autoclave prior to use</td></tr><tr><td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 mL</td><td>Eppendorf</td><td>022363204</td><td>Autoclave prior to use</td></tr><tr><td>Scalpel Blades - #15</td><td>Fine Science Tools</td><td>10015-00</td><td></td></tr><tr><td>Scalpel Handle - #4</td><td>Fine Science Tools</td><td>10004-13</td><td></td></tr><tr><td>Stemi 2000-C Stereo Microscope</td><td>Zeiss&amp;nbsp;</td><td>000000-1106-133</td><td></td></tr><tr><td>TCS SP5 confocal microscope</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>Triton-X (non-ionic surfactant)</td><td>Integra</td><td>T756.30.30</td><td></td></tr><tr><td>VectaShield antifade mounting medium for fluorescence</td><td>Vector Laboratories, Inc.</td><td>H-1000</td><td></td></tr><tr><td>Zipper Bag, Reclosable, 4 x 6&#039;&#039; - 2 mm thick</td><td>Zipline</td><td>0609132541599</td><td></td></tr></tbody></table>

neonatal murine cochlear explant technique as an in vitro screening tool in hearing research

Embora tenha havido avanços notáveis ​​na pesquisa auditiva nas últimas décadas, ainda não há cura para a Perda auditiva sensorial (SNHL), uma condição que tipicamente envolve danos ou perda das delicadas estruturas mechanosensoriais da orelha interna. Ensaios sofisticados in vitro e ex vivo surgiram nos últimos anos, permitindo a triagem de um número crescente de compostos potencialmente terapêuticos, minimizando os recursos e acelerando os esforços para desenvolver curas para SNHL. Embora as culturas homogêneas de certos tipos de células continuem a desempenhar um papel importante na pesquisa atual, muitos cientistas agora contam com culturas organotípicas mais complexas de orelhas internas murinas, também conhecidas como explantes cocleares. A preservação de estruturas celulares organizadas dentro da orelha interna facilita a avaliação in situ de vários componentes da infra-estrutura coclear, incluindo células ciliadas internas e externas, neurônios ganglionares espirais, neurItes, e células de apoio. Aqui apresentamos a preparação, a cultura, o tratamento e a imunocoloração de explantes cocleares murinos neonatais. A preparação cuidadosa desses explantes facilita a identificação de mecanismos que contribuem para o SNHL e constitui uma ferramenta valiosa para a comunidade de pesquisa auditiva.

Enquanto muitos protocolos se concentram no órgão dos explantes de Corti, essa técnica tenta preservar a anatomia de toda a curva coclear, incluindo os SGNs. Isso dá aos pesquisadores a oportunidade de analisar os efeitos de um determinado tratamento sobre neurites e somata de SGNs, além do órgão de Corti. Realizar uma dissecção que preserva parte do modiolus, como descrito aqui, é mais tecnicamente desafiador do que explantar o órgão de Corti sozinho. No entanto, a área de ...

Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

Técnica de explante coclear murino neonatal como ferramenta de triagem in vitro em pesquisa auditiva | Medicina | Vídeo

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

O objetivo deste protocolo é demonstrar a preparação, cultura, tratamento e imunocoloração de explantes cocleares murinos neonatais. A técnica pode ser utilizada como ferramenta de seleção in vitro na pesquisa auditiva.

Técnica de Explante Coclear Murino Neonatal como Ferramenta de Triagem In Vitro na Pesquisa Auditiva

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

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Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

Human Biology

Sensory Systems

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17.5K visualizações.  Massachusetts Eye and Ear.  O objetivo deste protocolo é demonstrar a preparação, cultura, tratamento e imunocoloração de explantes cocleares murinos neonatais. A técnica pode ser utilizada como ferramenta de seleção in vitro na pesquisa auditiva. 

Vídeo: Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura da embrionárias mouse cocleares explantes e Gene Transferência de Eletroporação

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Biologia do Desenvolvimento

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

Making Sense of Listening: A bateria de testes IMAP

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

Medicina

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

A estratégia para identificar mutações de novo em Distúrbios comuns, tais como autismo e esquizofrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primária e Eletroporação Plasmid do Órgão de Corti murino.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biologia

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Longo prazo Time Lapse Imagem do mouse cocleares explantes

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Neurociência

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

A abordagem do rato Round-janela para ototóxicos Delivery Agent: A rápida e técnica confiável para induzir a degeneração das células da cóclea

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Preparação da superfície coclear no mouse adulto

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

Imagens de células vivas in vitro para explorar a migração celular em direção ao órgão de Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

Explantes Cocleares Neonatais Totais como Modelo In vitro

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

Cultivo in vitro eficiente de células ciliadas cocleares de camundongos

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. 
After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 &#176;C, and gently suspended in culture medium using a 200 &#181;L pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached &gt;90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

Autor em destaque: Avanços no cultivo de células ciliadas de camundongos para pesquisa auditiva 

Herein, we present a detailed protocol for isolating and culturing primary cochlear hair cells from mice. Initially, the organ of Corti was dissected from neonatal (aged 3-5 days) murine cochleae under a microscope. Subsequently, cells were enzymatically digested into a single-cell suspension and identified using immunofluorescence after several days in culture.

Isolamento e Cultura de Células Ciliadas Cocleares Primárias de Camundongos Neonatais

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for ...