PS tem um Canada Research Chair em Biologia Celular da retina e do Instituto de Pesquisa Alcon New Investigator Award. Este trabalho foi financiado por subvenções dos Institutos Canadenses de Pesquisa em Saúde (221.478), a Canadian Diabetes Association (OG-3-11-3329-PS), Ciências Naturais e do Conselho de Pesquisa do Canadá Engenharia (418637) e A Fundação Fighting Blindness Canadá. O suporte também foi fornecido pelo Réseau de Recherche en Santé de la Visão du Québec.

Declaração de Ética: Todos experimentação animal adere as diretrizes de cuidados de animais estabelecidas pela Associação de Pesquisa em Visão e Oftalmologia (ARVO) Declaração para o uso de animais em Ophthalmic and Vision Research e pelo Conselho Canadense de Cuidado Animal. 1. Oxigênio Induzida Retinopatia (OIR) <ol><li> Registar a data de nascimento de filhotes de rato como P0. </li><li> Registre todos os pesos dos animais aquando da entrada em O 2 para garantir uma faixa de peso adequado. Nota: Para C57BL / 6 ratos em P17, o peso corporal deve variar entre 5 e 7,5 g de NV máxima 32. A fim de manter a consistência do ambiente, é recomendado o uso da mesma ninhada de controlo (para ratinhos geneticamente modificados, bem como ratinhos que receberam tratamentos experimentais). Ao avaliar os efeitos de um vetor viral, deve-se considerar o tropismo do vírus e dar tempo suficiente para a plena expressão de transgenes por vírus entregues. Rapidamente expressar viralOs vectores, tais como os lentivírus 3 ª geração 24, 25, 33 são recomendados. </li><li> Filhotes lugar do mouse no P7 (C57BL / 6 ou desejado tensão) e um CD1 fomentar mãe em uma câmara de oxigênio fixado em 75% O 2 por 5 dias 27. Umidade e temperatura ambiental foram mantidas constantes durante toda a exposição O 2. Nota: As instalações de investigação equipados com uma fonte central de O 2 são ideais e limitar a substituição pesado de O vazio 2 tanques. Se estiver trabalhando com camundongos transgênicos ou nocaute, é importante para garantir que o controle e os ratos experimental são adquiridos do mesmo fornecedor para limitar desvios genéticos dentro das mesmas cepas 30, 29. </li><li> Em P12, remover ratinhos a partir da câmara de oxigénio e retornar os animais ao ambiente de O 2. </li></ol> 2. Injeção intravítrea para entrega de Compostos para o Inner Retina (When avaliar os efeitos de um composto farmacológico ou proteína recombinante) <ol><li> No P14, anestesiar ratos com 2% de isoflurano em oxigênio 2 L / min (ou proteção animal aprovado pelo comitê de anestésico de escolha). A fim de verificar a eficácia da anestesia, sequencialmente beliscar a cauda, ​​pé de trás e da orelha com uma pinça. </li><li> Posicione o mouse sobre o ventre. </li><li> Usando uma seringa esterilizada de 10 mL equipado com uma agulha de vidro puxado chanfrada, realizar uma injecção de um volume máximo de 1 ml de solução contendo o composto a ser investigada ou veículo (solução salina fisiológica) no limbo posterior do olho, com um 45 ° ângulo de evitar a lente. Nota: A-agulha de vidro puxado está ligado à seringa utilizando uma gota de epoxi-resina. </li><li> Aplique uma gota de pomada oftálmica lubrificante (de preferência com antibiótico) com um cotonete para o olho do mouse. </li><li> Retorne o mouse de volta para a jaula com a promoção mãe. Ratos são, então, cuidadosamente monitorizados até recoberto e totalmente ambulatorial. </li></ol> 3. Avaliação da perfusão navio e função de barreira (Integridade) por Angiofluoresceinografia <ol><li> Anestesiar ratos com isoflurano a 2% em oxigênio 2 L / min (ou proteção animal aprovado pelo comitê de anestésico de escolha). A fim de verificar a eficácia da anestesia, sequencialmente beliscar a cauda, ​​pé de trás e da orelha com uma pinça. Nota: Esta é normalmente realizada no P17 quando a regeneração é avaliada. Também levar a cabo a análise em P19 e P21 para determinar se a integridade vascular é preservada ao longo do tempo. </li><li> Uma vez anestesiados, pesar o mouse. </li><li> Faça uma incisão na linha média do abdome com uma tesoura de dissecação. Nota: Os instrumentos de dissecação devem ser regularmente verificados e afiada. </li><li> Cortar costelas lateralmente e levantar a caixa torácica com o auxílio de uma pinça. Nota: É necessário cortar lateralmente como possível, para evitar danos no coração. </li><li> Após a remoção peripHeral tecido do coração, prender a aorta descendente com pinça hemostática. </li><li> Lentamente injetar fluoresceína-dextrano para o ventrículo esquerdo usando uma agulha G 25. Nota: Se a função de barreira vascular é investigado, 70 kDa fluoresceína-dextrano é empregado como ele vai vazar para fora dos vasos quando a integridade dos vasos é comprometida. Se o investigador quer fundido vasos sanguíneos, 2 MDa fluoresceína-dextrano é usado. As etapas críticas: 1) Para garantir uma repartição homogênea, centrífuga fluoresceína-dextrano e injetar o sobrenadante, 2) a fim de evitar a constrição dos vasos, injetar solução de fluoresceína-dextrano aquecido, 3) O seu tempo de circulação não deve excesso de 4 min. </li><li> Decapitar ratos 2 min após a injeção com uma tesoura de operação. </li></ol> 4. Enucleação e Eye Fixation Nota: Ao avaliar as taxas de regeneração vascular, primeiro coletar retinas no P12 e, adicionalmente, no P14 e P17. Aumentar o número de ponto de tempo amostrados para determinação mais precisa das taxas de revascularização 24. <ol><li> Incline a cabeça do rato e colocá-lo de lado. </li><li> Retire a pele e as pálpebras cobrem os olhos com uma tesoura de dissecação. </li><li> Coloque uma pinça curva abaixo do olho e puxe-o até que o nervo óptico é cortada. </li><li> Vire a cabeça do rato para seu outro lado e realizar os mesmos passos (passos 4.2 e 4.3). </li><li> Para garantir uma melhor penetração do fixador, perfurar um buraco na câmara anterior do olho usando 30 G agulha. </li><li> Transferir os olhos para um tubo contendo 4% de paraformaldeído (PFA) e fixar durante 1 hora à temperatura ambiente. </li><li> Remover e lavar os olhos PFA 4 vezes com uma solução de PBS gelado. </li></ol> 5. Dissecção da retina <ol><li> Coloque rato olhos em uma placa de Petri contendo PBS frio e realizar dissecção das retinas sob microscópio estereoscópico. </li><li> Retire a gordura / tecido extra em torno do olho com micro-dissecção scissors. </li><li> Corte a córnea com uma tesoura micro-dissecção. </li><li> O uso de dois pares de pinças, minuciosamente descascar a esclera distância a partir da periferia em direcção ao nervo óptico e descartar. </li><li> Aperte a lente (bola esbranquiçada abaixo da córnea) com uma pinça e extraí-lo do copo olho. Utilizar um par de pinças, como um apoio, e a outra para agarrar e levantar cuidadosamente e remover a lente. </li><li> Separar os vasos hialóide da face interna da retina utilizando escovas pequenas (tamanho 0) e uma pinça. </li><li> Retirar feixes de vasos hialóide ligados ao disco óptico usando uma pinça. </li><li> Transferência dissecados retinas de dois tubos ml de microcentrífuga contendo PBS e colocar em gelo antes de se iniciar o processo de coloração. </li></ol> 6. Retinal Vascular Coloração <ol><li> Incubar as retinas dissecados durante a noite com agitação suave a 4 ° C em uma solução de fluorescência acoplado a isolectina B4 (rodamina-lectina ou outra) em PBS contendo 1 mM de CaCl2 </sub&gt; (uma diluição de uma solução B4 2 mg / ml isolectina é recomendado 1:100). Durante o procedimento de coloração inteira, cobrir os tubos com folha de alumínio ou uma película opaca para proteger da luz. </li><li> No dia seguinte, remover a solução de corante e lavar retinas 3x em PBS durante 10 min à temperatura ambiente. </li></ol> 7. Preparação de retina Flatmounts <ol><li> Retinas, do lado fotorreceptor para baixo de transferência, sobre uma lâmina de microscópio e fazer quatro incisões radiais equidistantes profundas utilizando um bisturi cirúrgico para dividir a retina em quatro quadrantes de igual tamanho. Durante as incisões, a cinta da retina, com uma escova, de modo que ele não se mova. </li><li> Usando duas escovas embebidas em PBS, alise cuidadosamente a quadrantes fotorreceptor side-down e mergulhe a retina em meio de montagem para evitar a foto-branqueamento. Em seguida, coloque cuidadosamente uma lamela sobre a superfície da retina montado sem a aplicação de pressão e garantir que as bolhas de ar não se acumulam sob a lamínula. &lt;/ Li&gt; </ol> 8. Imagem e Quantificação de Vasoobliteration (VO) e neovascularização (NV), como descrito anteriormente 31 <ol><li> Tire fotos de retinas inteiras montado com um microscópio de epi-fluorescência com uma ampliação de 10x. </li><li> Abra a imagem da retina em software de edição de fotos, unir e medir a área total da retina, ea área avascular. Área pode ser expressa em pixels. </li><li> Determinar a extensão de VO, dividindo o número de pixels na área avascular pelo número de pixels na área total da retina. </li><li> Determinar a extensão da NV-se dividindo o número de pixels de NV pelo número de pixels na área total da retina, tal como descrito 31. </li></ol>

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G., Jiang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis,+angiogenesis,+and+MRI+indices+of+functional+recovery+from+stroke.">Neurogenesis, angiogenesis, and MRI indices of functional recovery from stroke.</a> Stroke. 38, 827-831 (2007).</li><li>Li, L., 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=Angiogenesis+and+improved+cerebral+blood+flow+in+the+ischemic+boundary+area+detected+by+MRI+after+administration+of+sildenafil+to+rats+with+embolic+stroke.">Angiogenesis and improved cerebral blood flow in the ischemic boundary area detected by MRI after administration of sildenafil to rats with embolic stroke.</a> Brain Res. 1132, 185-192 (2007).</li><li>Robinson, R., Barathi, V. A., Chaurasia, S. S., Wong, T. Y., Kern, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+animal+models+of+diabetic+retinopathy:+from+molecular+approaches+to+mice+and+higher+mammals.">Update on animal models of diabetic retinopathy: from molecular approaches to mice and higher mammals.</a> Dis Model Mech. 5, 444-456 (2012).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat Genet. 37, 1181-1186 (2005).</li><li>Jenuth, J. P., Peterson, A. C., Shoubridge, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+selection+for+different+mtDNA+genotypes+in+heteroplasmic+mice.">Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.</a> Nat Genet. 16, 93-95 (1997).</li><li>Mattapallil, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rd8+mutation+of+the+Crb1+gene+is+present+in+vendor+lines+of+C57BL/6N+mice+and+embryonic+stem+cells,+and+confounds+ocular+induced+mutant+phenotypes.">The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.</a> Investigative ophthalmolog., &amp; visual science. 53, 2921-2927 (2012).</li></ol>

Os autores não têm nada a revelar.

Qual é a maneira mais eficaz de estimular o crescimento de novos vasos saudáveis ​​no tecido nervoso isquêmico? É terapeuticamente válida para interferir e acelerar ocorrem naturalmente rebrota vascular? Em patologias neuro-isquêmica, como retinopatias isquêmicas ou acidente vascular cerebral, degeneração vascular está associado à redução da função neuronal 35-38. Daí para combater lesão precoce, restabelecendo micro-circulação regional durante o segmento de imediato / precoce da doenç...

Ao longo do desenvolvimento do SNC, nervos, células do sistema imunológico e vasos sanguíneos estabelecer redes notavelmente acoplados para garantir adequada perfusão tecidual e permitir a transmissão de informações sensoriais 1-5. O colapso dos sistemas vascular resulta na oxigenação dos tecidos ea oferta insuficiente metabólica comprometida e é cada vez mais reconhecida como um importante contribuinte para a patogênese de doenças neurodegenerativas 6. Abandono vascular e a deterioração da unidade neurovascular dentro do cérebro, por exemplo, está associado com a demência vascular, lesões vasculares da matéria branca do cérebro 7 e doença de Alzheimer com estenose de arteríolas e vasos de pequeno calibre 8. Além disso, a função de barreira vascular prejudicada é pensado para contribuir esclerose múltipla 9 e esclerose lateral amiotrófica 10. De relevância direta para o modelo de retina descrito neste protocolo, cegandodoenças tais como a retinopatia diabética 11 e retinopatia da prematuridade 12, 13 são caracterizados por uma fase de degeneração vascular precoce. O estresse isquêmico que se seguiu na retina neurovascular desencadeia uma segunda fase de neovascularização excessiva e patológica que provavelmente se origina como uma resposta compensatória ao oxigênio restabelecer o fornecimento de energia e 14-16. Uma estratégia atractiva para superar o stress isquémico que é central para a progressão da doença é o de restaurar redes vasculares funcionais especificamente nas zonas isquêmicas do sistema neuro-retina (Figuras 2 e 3). Provocar uma resposta angiogênico controlado pode vir transversalmente como um contra-senso para uma condição em que os tratamentos anti-angiogênicos, tais como anti-VEGF são considerados tratamentos adaptados. No entanto, a evidência para a validade desta abordagem é montagem. Por exemplo, aumentando a rebrota vascular &quot;fisiológico-like&quot; em ischemretinopatias ic foi elegantemente demonstrado através da introdução de células precursoras endoteliais 17, a inibição da Müller VEGF expresso de células na supressão induzida de outros fatores angiogênicos 18, injeção de células progenitoras mielóides 19, a inibição da NADPH oxidase a apoptose induzida por 20, aumentando dietético ω-3 fatty poliinsaturados ingestão de ácido 21, o tratamento com um fragmento do terminal carboxilo do triptofano tRNA 22, e a administração directa de VEGF ou FGF-2, para a protecção de células gliais 23. Além disso, foi demonstrado que o modulando pistas de orientação neuronais clássicos, tais como netrinas Semaforinas ou em retinopatias isquêmicas acelera a regeneração vascular de vasos saudáveis ​​dentro da retina e, consequentemente, reduz a angiogénese patológica 24, 25. De relevância clínica direta, vários dos estudos realizados em animais acima mencionados fornecem evidências de que a promoção re vasculargeração durante a fase isquêmica precoce de retinopatias pode reduzir significativamente risco de vista pré-retinal neovascularização 19, 23, 24, 26, provavelmente através da redução da carga isquêmica. Elaboração de estratégias terapêuticas que estimulam a regeneração dos vasos funcionais continua a ser um desafio significativo para os biólogos vasculares. Aqui nós descrevemos um sistema experimental que utiliza o modelo do rato de retinopatia induzida por oxigênio (OIR), para explorar estratégias para modular a rebrota vascular dentro da retina. Desenvolvido por Smith et al., Em 1994, 27, este modelo serve como um proxy para retinopatias proliferativas humanos e consiste em expor filhotes P7 rato para 75% O 2 até P12 e, posteriormente, re-introduzir os filhotes para a sala ambiente O 2-tensão (Figura 1). Este paradigma vagamente imita um cenário onde um bebê prematuro é ventiladocom o O 2. A exposição de filhotes de rato à hiperóxia provoca degeneração dos capilares da retina e microcirculação, e produz uma área reprodutível de vaso-obliteração (VO) normalmente avaliada em saída de O 2 no P12, embora a área VO máxima é atingida aos 48 hr (P9) após exposição ao O 2 28. No ratinho, as zonas avasculares VO regenerar-se espontaneamente ao longo da semana seguinte re-introdução de ar ambiente e, eventualmente, as zonas de VO são completamente re-vascularização (figura 2). Re-introdução de ar ambiente, de ratinhos submetidos a OIR também provoca neovascularização pré-retinal (NV) (máximo a P17), que é tipicamente avaliada para determinar a eficácia de paradigmas de tratamento anti-angiogénicos. Na sua forma mais pura, o modelo OIR fornece uma ferramenta altamente reprodutível e quantificável para avaliar a degeneração vascular induzida por oxigénio e determinar a extensão da neovascularização destrutiva pré-retiniana 29-31. <p class = &quot;jove_content&quot;&gt; Vários paradigmas de tratamento exploratório que modulam a regeneração vascular CNS pode ser investigado usando o modelo OIR incluindo o uso de compostos farmacológicos, terapia genética, deleção do gene e muito mais. A propensão de uma determinada abordagem para influenciar rebrota vascular é avaliada passo a passo na janela entre P12 (VO máxima após saída da hiperóxia) e P17 (NV máxima). A avaliação dos resultados do tratamento na NV patológico pode ser rapidamente e facilmente determinada em paralelo e foi exaustivamente descrito por Stahl e colegas 30, 31. Aqui nós fornecemos um procedimento simples passo-a-passo para investigar a modulação da revascularização fisiológica dentro da retina neural por compostos farmacológicos, terapêuticos potenciais, vetores virais ou para estudar a influência dos genes candidatos em camundongos transgênicos ou nocaute.

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			<td height="21" style="height:21px;width:639px;">C57Bl/6 mice ((Other strains may be used; angiogenic response varies from one strain to the other)</td>
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			<td style="width:181px;">Zeiss</td>
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assessment of vascular regeneration in the cns using the mouse retina

A retina roedor é, talvez, o sistema de mamífero mais acessível que a investigar interacção neurovascular dentro do sistema nervoso central (SNC). É cada vez mais reconhecido que várias doenças neurodegenerativas, como a doença de Alzheimer, esclerose múltipla e esclerose lateral amiotrófica apresentam elementos de comprometimento vascular. Além disso, as causas mais importantes de cegueira em populações pediátricas e de trabalho idade (retinopatia da prematuridade ea retinopatia diabética, respectivamente) são caracterizadas por degeneração vascular e insuficiência de rebrota vascular fisiológico. O objetivo deste documento técnico é fornecer um protocolo detalhado para estudar a regeneração vascular CNS na retina. O método pode ser utilizado para elucidar os mecanismos moleculares que conduzem à falha de crescimento vascular após lesão isquémica. Além disso, possíveis modalidades terapêuticas para acelerar e restaurar plexos vasculares saudáveis ​​podem ser exploradas. Achados obtained utilizando a abordagem descrita pode fornecer meios terapêuticos para retinopatias isquêmicas, tais como diabetes ou que de prematuridade e possivelmente beneficiar outros distúrbios vasculares do SNC.

O modelo OIR é amplamente utilizada para estudar a degeneração vascular induzida por oxigênio e neovascularização patológica induzida por isquemia na retina e tem sido fundamental para o desenvolvimento de tratamentos atualmente empregados anti-angiogênicos para doenças oculares 27, 29, 30. Os resultados obtidos com este modelo pode ser vagamente extrapolados para retinopatias isquêmicas, como a retinopatia diabética proliferativa e retinopatia da prematuridade 30. <p class="jove_con...

Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

A retina de roedores tem sido reconhecida como uma janela de acesso para o cérebro. Neste documento técnico que proporcionam um protocolo que utiliza o modelo de ratinho de retinopatia induzida por oxigénio para estudar os mecanismos que levam à insuficiência de regeneração vascular dentro do sistema nervoso central após a lesão isquémica. O sistema descrito também pode ser aproveitada para explorar estratégias para promover o crescimento de vasos sanguíneos funcionais dentro da retina e do SNC.

Assessment of Vascular regeneração no SNC Usando o rato Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

assessment-of-vascular-regeneration-in-the-cns-using-the-mouse-retina

Research

JoVE Journal

Neuroscience

14.3K visualizações.  McGill University.  A retina de roedores tem sido reconhecida como uma janela de acesso para o cérebro. Neste documento técnico que proporcionam um protocolo que utiliza o modelo de ratinho de retinopatia induzida por oxigénio para estudar os mecanismos que levam à insuficiência de regeneração vascular dentro do sistema nervoso central após a lesão isquémica. O sistema descrito também pode ser aproveitada para explorar estratégias para promover o crescimento de vasos sanguíneos...

Vídeo: Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Quantificar a atividade de Cis Elements-Regulação na retina do rato por Eletroporação Remoção

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

Neurociência

In vivo Electroporation of Developing Mouse Retina

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

In vivo Electroporation of Developing Mouse Retina

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

Em Eletroporação vivo de Retina do rato em desenvolvimento

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

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Rápido Micro-iontoforese de glutamato e GABA: uma ferramenta útil para investigar Integração Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

A córnea microbolsa Ensaio: Um modelo de angiogênese no mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagem Ca<sup&gt; 2+</sup&gt; Dynamics nos terminais Cone fotorreceptores Axon do mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

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 ...

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

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

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

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

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

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

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

O poder do intervalo de Interstimulus para a avaliação do processamento Temporal em roedores

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

Fatias hipocampais horizontais do cérebro do rato

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Imagem transpupilar de dois fótons in vivo da retina do rato

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...