Os autores agradecem Voltaire Gungab de assistência com a preparação do manuscrito. Este trabalho foi apoiado em parte por concessões: o National Institutes of Health (T32 GM008440 para EJ Walker, R01 NS27713 a WL Young, P01 NS44155 a WL Young e H. Su) e R21 NS070153 para H. Su, eo norte-americano Heart Association (AHA 10GRNT3130004 para H. Su).

1. Limpeza de sangue de Vascularização <ol><li> Anestesiar rato por injeção intraperitoneal de ketamina 100 mg / kg de xilazina com 10 mg / kg diluído em solução salina 0,25 ml. </li><li> Uma vez que a anestesia foi confirmada por nenhuma resposta a uma pitada de pata, use a tesoura para fazer uma incisão no peito com uma tesoura, logo abaixo do apêndice xifóide, corte através do diafragma, e retira-se entre o segmento dorsal e ventral das costelas para expor cavidade torácica. Expor o coração, dobrando-se esterno e parede torácica adjacente e seguro com pinça hemostática. Abra o saco pericárdico com pinças para expor o coração. </li><li> Ligue bomba de perfusão (100mmHg de pressão) conectado a quente PBS acrescido de heparina (5 unidades / ml) solução em banho de água quente a 37 ° C e os tubos com uma agulha de calibre 22 sem corte na saída. </li><li> Inserir a agulha para o ventrículo esquerdo, perto do ápice do coração. Imediatamente cortado do átrio direito para permitir saída arterial sistêmica e continue lavando até que o sangue is retirado de circulação. </li></ol> 2. Perfusão de agente de elenco no Sistema Vascular <ol><li> Prepare Microfil (Flow Tech, Inc. Carver, MA) Solução de fundição por protocolo do fabricante: Misturar 5 ml de diluente MV com 4 ml de composto MV filtrada. Adicionar 450μl (5%) de catalisador (MV agente de cura). Tempo de solução de trabalho é de 20 minutos, mas mistura imediatamente antes do uso para assegurar viscosidade adequada. </li><li> Retirar fundição solução de agente para uma seringa de 10 ml, juntar uma agulha de calibre 20 e blunt a seringa e agulha preencher com a solução. </li><li> Inserir a agulha para o ventrículo esquerdo através do mesmo buraco utilizado para a lavagem do sangue. Agulha segura no lugar com pinça hemostática no coração. </li><li> Injectar a solução lentamente agente de elenco para o ventrículo esquerdo em cerca de 3 ml / min. Excesso de pressão pode levar ao incorreto de fluxo de agente de elenco ou ruptura navio potencial. </li><li> Olhe para os sinais de perfusão de sucesso, incluindo: visualização do elencoing agente no intestino e fígado vasculatura, descoloração membro distal, e nariz e descoloração da língua. Reajuste agulha se necessário para alcançar a perfusão adequada. </li></ol> 3. Coleta e Processamento de Tecido <ol><li> Remover cérebro e colocar em paraformaldeído a 4% durante a noite a 4 ° C. </li><li> Desidratar cérebro, colocando de paraformaldeído diretamente em EtOH cada vez mais concentrados em temperatura ambiente: 25% EtOH, 1 dia, 50% EtOH, 1 dia, 75% EtOH, 1 dia, 95% EtOH, 2 dias, e 100% EtOH, 2 dias . </li><li> Esclarecer o tecido cerebral ao redor dos vasos sanguíneos por imersão do cérebro perfundidos em salicilato de metila por 2 dias à temperatura ambiente. </li><li> Vascularização do cérebro unsectioned todo esclarecidas podem ser visualizados com o cérebro imerso em salicilato de metila sob um microscópio de dissecação (Fig. 1) (Leica MZFL microscópio III, Leica Microsystems, Bannockburn, IL) ou microscópio de campo claro (Fig. 2) (Leica DM / LS, Leica Microsystems). </li><li> Reclarificationpode ser feito se o tecido não está completamente esclarecido. De metil salicilato de transferência do cérebro, para EtOH 95% para 2 dias, 100% EtOH por 2 dias, e salicilato de metila por 2 dias. </li></ol> 4. Micro-CT imagem ou de Preparação de aplicação de tecidos-histológica <ol><li> O cérebro pode ser fotografada usando micro-CT logo após esclarecimento ou após a re-hidratação (Fig. 4). </li><li> Para preparar o tecido cerebral para análise histológica, reidratar o cérebro através de EtOH diluída serial: 100% EtOH, 1 dia, 95% EtOH, 1 dia, 75% EtOH, 1 dia, 50% EtOH, 2 dias, 25% EtOH, 2 dias ; loja e na PBS. O cérebro pode então ser embebidos em parafina usando um procedimento padrão. </li></ol> 5. Resultados representante Após vazamento vascular, o cerebrovasculature pode ser visto macroscopicamente na superfície do cérebro (Fig.1a). A árvore vascular no interior do parênquima podem ser visualizados seguinte esclarecimento (Figura 1B). A estrutura detalhada vascularpodem ser visualizados e analisados ​​sob um microscópio de campo claro (Fig. 2), dissecando escopo (Fig. 3), e com micro-tomografia computadorizada (Fig. 4). Usando a microscopia de campo claro, a vasculatura podem ser vistos em diferentes planos focais, sem seccionamento do cérebro (Fig. 2A), bem como microvasos individuais em maior ampliação (Fig. 2B). De acordo com um escopo de dissecação, pode-se ver toda a estrutura vascular em um plano focal, permitindo melhor visualização da estrutura tridimensional do cerebrovasculature (Fig. 3). Imagens tiradas em um microscópio de dissecação pode mostrar a morfologia de grandes vasos e vascularização do cérebro inteiro, mas falta os detalhes mostrados em fotos tiradas com a microscopia de campo claro (Fig. 2B). Esta técnica permitiu-nos detectar embarcações irregulares no nosso modelo experimental do rato BAVM adulto 2. A Figura 5 mostra um exemplo de as mesmas embarcações irregulares detectadas pelo micro-tomografia computadorizada (Fig. 5A) e microscópio de campo claro (Fig. 5B). Juntos, esses dados mostram que podemos analisar tele mesma amostra com múltiplas ferramentas de imagem após a fundição vascular. <img alt="Figura 1" src="/files/ftp_upload/2958/2958fig1.jpg" /> Figura 1. Cérebro de camundongo inteiro seguinte elenco vascular. Antes (A) e após (B) Clarificação por salicilato de metila solvente. Vasos podem ser claramente visualizados na superfície do cérebro (A) e no parênquima procedimento seguinte esclarecimento (B). <img alt="Figura 2" src="/files/ftp_upload/2958/2958fig2.jpg" /> Figura 2. Brightfield imagens de microscópio do cérebro vascular casted esclareceu. Cérebro pode ser trabalhada em vários planos e níveis de ampliação diferentes. Escala Bar 200μm (A) e 50 ìm (B). <img alt="Figura 3" src="/files/ftp_upload/2958/2958fig3.jpg" /> Figura 3. Dissecando imagem do microscópio em 1x mostrando todo adulto cerebrovasculature cérebro de camundongo a partir de uma visão dorsal. &lt;img alt = &quot;Figura 4&quot; src = &quot;files/ftp_upload/2958/2958fig4.jpg /&quot; /&gt; Figura 4. Micro-CT digitalização de imagens do cérebro do rato inteiro mesmo adulto mostrado na Figura 3 a partir de uma visão dorsal. <img alt="Figura 5" src="/files/ftp_upload/2958/2958fig5.jpg" /> Figura 5. Aplicação do método de fundição vascular para detectar uma malformação arteriovenosa cerebral. A) Micro-CT scan do cérebro vascular casted mostra região de alargada, irregular AVM-como vasos no hemisfério direito do ponto de vista ventral. Imagem ampliada da região (caixa branca) é mostrado à direita. B) O mesmo embarcações irregulares visualizados sob microscópio de campo claro com ampliação de 50x. Barra de escala 50 mm.

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Procedimentos experimentais para a utilização de animais de laboratório foram aprovados pelo Institutional Animal Care e do Comitê Use da Universidade da Califórnia, San Francisco (UCSF).

Nós relatamos aqui um método para fundição de cerebrovasculature rato adulto que pode ser trabalhada com diversas modalidades. Aplicação deste método para a BAVM modelo de doença permite a análise 3D dos vasos irregular. Potenciais futuros estudos incluem quantificação de micro-TC, a análise histológica do tecido, e diagnóstico de progressão da doença vascular. Problemas comuns encontrados e Sugestões Perfusão completa da vasculatura cerebral nem sempre é alcançado. Ter pressão suficiente para alcançar os vasos sanguíneos sem ruptura distal do átrio esquerdo ou aorta é um desafio. Para superar isso, aplicar uma pressão consistente de aproximadamente 100mmHg. Também, ajustar a posição da agulha para assegurar a vazão adequada de fundição agente para a aorta. É importante que as artérias carótidas não são restritas para que o agente de elenco pode atingir os vasos do cérebro. Procedimentos críticos que se correlacionam com pequenos vasos melhorou porfusão são: (1) remoção adequada do sangue dos vasos; (2) filtração do agente de elenco, e (3) a colocação da agulha durante a injeção do casting. Cuidados devem ser tomados para evitar a injecção de fundição agente para o átrio esquerdo eo pulmão. Outros investigadores com sucesso perfundidos mouse de 3 e 4 cerebrovasculature rato sem o uso de procedimentos adicionais. Análise manual dos vasos perfundidos Microfil contracorados com hemotoxylin mostrou 93% dos navios de perfusão lumen 5. Sempre descartar ou completamente limpa qualquer ferramenta que entra em contato com o agente de elenco como ele irá polimerizar rapidamente, e ferramentas não podem ser usados ​​para diversos assuntos. Clarificação do tecido ao redor do mouse cérebro pode ser um desafio. Após imersão em salicilato de metila, o tecido ainda pode aparecer opaco, tornando difícil de imagem navio. Assegurar a desidratação adequada e esclarecimento deve resolver este problema. Colocação de tecido cerebral em um roqueiro durante álcool e metil salicylate tratamento pode melhorar a clarificação dos tecidos. Desvantagens da técnica As limitações dessa técnica incluem: (1) é necessário para injetar a solução de agente de elenco imediatamente após a mistura-lo como ele vai engrossar rapidamente após o catalisador é adicionado; (2) o agente de elenco enche vasos de condutância grande facilidade, mas não confiável encher vasos menores, tais como arteríolas (vasos de resistência) e capilares (vasos nutritivos) e (3) não é o melhor contraste radiopaco para micro-TC, embora alguns grupos preferem Microfil para micro-TC 1,6. No entanto, seguindo as sugestões propostas anteriormente, incluindo a colocação de agulha apropriada, a posição do navio e pressão de perfusão, casting completa de navios menores podem ser alcançados. A adaptabilidade a várias ferramentas de análise faz deste agente um valioso instrumento para analisar a estrutura cerebral, incluindo a circulação arterial e venosa, com váriasperspectivas. Aplicações da Técnica O benefício deste método é seu potencial de aplicação ampla. O elenco preenche todos os navios, incluindo artérias, veias, capilares e de todos os tecidos dentro do corpo, por isso as estruturas vasculares em vários órgãos de várias espécies podem ser analisados. Anteriormente, outros grupos têm utilizado este perfusão para grandes e pequenos 7 8 navios em humanos, macacos, 9 de ovelhas 10, ratos 4,11,12 e mouse 3,13. Vamos agora mostrar evidências do uso desta técnica na análise da estrutura de microvasos no cérebro do rato, em estados sãos e doentes 2. Além disso, existem soluções Microfil com viscosidades diferentes disponíveis para perfundir vasos de diferentes tamanhos e cores diferentes para melhorar a visualização dos vasos em vários órgãos. Por exemplo, o vermelho pode ser facilmente visualizado no parênquima cerebral pálido e amarelo contrasta bem com avermelho escuro miocárdio. Além disso, como o material é radiopaco, vasculatura podem ser visualizados com micro-tomografia computadorizada para obter uma imagem 3D giratório. O tecido também pode ser usado para cortes em parafina e análise histológica depois de ser fotografada. Conclusão Demonstramos aqui um protocolo para fazer um molde vascular dos vasos sanguíneos do cérebro do rato adulto, que podem ser adaptados usando uma variedade de técnicas de análise para estudar a estrutura morfológica do cerebrovasculature. Nós também fornecemos evidências para a aplicação deste método para um modelo do estado de doença BAVM, representado por alargada, as embarcações de forma irregular que o sangue de shunt arterial para as circulações venosa. Há uma variedade de métodos de fundição disponíveis vascular. O protocolo simples que oferecemos aqui para um elenco vascular pode ser usado como uma ferramenta para examinar a vascularização do cérebro do rato adulto usando diversas modalidades de imagem.

<table border="1"><tbody><tr><td> Nome </td><td> Companhia </td><td> Número de catálogo </td><td> Notas </td></tr><tr><td> Microfil </td><td> FlowTech, Inc. </td><td> MV130 </td><td> 4 meses a vida de prateleira </td></tr><tr><td> Salicilato de metila </td><td> Fisher Scientific </td><td> O3695-500 </td><td> para esclarecer o tecido </td></tr></tbody></table>

cerebrovascular casting of the adult mouse for 3d imaging and morphological analysis

Vasculares imagem é crucial para o diagnóstico clínico e tratamento das doenças cerebrovasculares, como malformações arteriovenosas cerebrais (BAVMs). Modelos de animais são necessários para estudar as terapias etiopatogenia e potencial de doenças cerebrovasculares. Imagem da vasculatura em animais de grande porte é relativamente fácil. Entretanto, os métodos de imagem navio desenvolvimento de modelos murinos doença cérebro é desejável devido ao custo e disponibilidade de linhas geneticamente modificados mouse. Imagem da árvore vascular cerebral murina é um desafio. Em humanos e animais maiores, o padrão-ouro para avaliar a angioarquitetura no macrovascular (condutância) nível é x-ray cateter baseado em contraste angiografia, um método não é adequado para pequenos roedores. Neste artigo, apresentamos um método de fundição cerebrovascular que produz um esqueleto duráveis ​​de todo o leito vascular, incluindo artérias, veias e capilares que podem ser analisados ​​usando d muitosmodalidades IFERENTES. Fundição completo dos microvasos da cerebrovasculature mouse pode ser difícil, no entanto, estes desafios são abordadas neste protocolo passo a passo. Por meio de perfusão intracardíaca do material de fundição vascular, todos os vasos do corpo são fundidos. O cérebro pode então ser removido e clarificado com o salicilato de metila solvente orgânico. Três dimensional imagem dos vasos sanguíneos do cérebro pode ser visualizado de forma simples e barata com qualquer microscópio de campo claro convencional ou microscópio de dissecação. O cerebrovasculature casted também pode ser trabalhada e quantificados utilizando micro-tomografia computadorizada (micro-CT) 1. Além disso, depois de ser fotografada, o cérebro fundido pode ser incluído em parafina para análise histológica. O benefício deste método de fundição vascular, em comparação com outras técnicas é a sua ampla adaptação a diversas ferramentas analíticas, incluindo brightfield análise microscópica, tomografia computadorizada, devido à características radiopacoapanágio do material, bem como a análise histológica e imuno-histoquímico. Este uso eficiente de tecido pode salvar utilização de animais e reduzir os custos. Recentemente, demonstrou aplicação deste método para visualizar os vasos sanguíneos irregulares em um modelo do rato de BAVM adulto em um nível microscópico 2, e fornecer imagens adicionais dos vasos malformados fotografada por micro-tomografia computadorizada. Embora este método tem desvantagens e não pode ser ideal para todos os tipos de análises, é uma técnica simples e prática que pode ser facilmente aprendido e amplamente aplicado para fundição vascular dos vasos sanguíneos em todo o corpo.

Watch this Scientific Journal Video about Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis at JoVE.com

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Neste artigo, apresentamos uma técnica simples e prática para a fundição cerebrovascular que é fácil de executar e pode ser utilizado para a imagem da árvore vascular do cérebro do rato adulto.

Fundição vascular cerebral do rato adulto for Imaging 3D e Análise morfológica

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

cerebrovascular-casting-adult-mouse-for-3d-imaging-morphological

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JoVE Journal

Neuroscience

21.4K visualizações.  University of California, San Francisco. Neste artigo, apresentamos uma técnica simples e prática para a fundição cerebrovascular que é fácil de executar e pode ser utilizado para a imagem da árvore vascular do cérebro do rato adulto.

Vídeo: Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiplas mouse-Imagem por Ressonância Magnética neuroanatômicos

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Neurociência

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Lapso de tempo de imagem de migração neuroblastos em fatias agudas do cérebro anterior Rato Adulto

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualização e manipulação genética de dendritos e espinhas no córtex cerebral e hipocampo do rato usando<em&gt; In utero</em&gt; Eletroporação

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Os métodos para a modulação e Análise de NF-kB-dependente Adulto Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

Padrões morfológicos do potencial evocado cortical Visual específico do estímulo

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Avaliação morfológica e funcional de sinapses de fita em regiões de frequência específica da cóclea do rato

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and indocyanine green are used to enable the observation of blood flow and vessel perfusion status, respectively. The aim of this study is to apply FVA to verify real-time blood flow, vessel perfusion status and occlusion of aneurysms after induction of sidewall aneurysms in rabbits and rats, as well as to validate the procedure in these species.
Twenty sidewall aneurysms were created in 10 rabbits by suturing a decellularized arterial vessel pouch on the carotid artery of a donor rabbit. In addition, 48 microsurgical sidewall aneurysms were created in 48 rats. During follow-up at one month after creation, the parent artery/aneurysm complex was dissected and FVA was performed using an intravenous fluorescein (10%, 1 mL) injection via an ear vein catheterization in rabbits and a femoral vein catherization in rats. Aneurysms were then harvested, and patency was evaluated macroscopically.
Macroscopically, 14 out of 16 aneurysms in rabbits indicated no residual parent artery perfusion with totally occluded luminae, however 11 (79%) were detected by FVA. Four aneurysms were excluded due to technical problems. In rats, residual aneurysm perfusion was macroscopically observed in 25 out of 48 cases. Of the 23 without macroscopic evidence of perfusion, FVA confirmed the incidence of 22 aneurysms (96%). There were no adverse events associated with FVA. Fluorescein is easily applicable and no special equipment is needed. It is a safe and extremely effective method for evaluating parent artery integrity and aneurysm patency/residual perfusion in an experimental setting with rabbits and rats. FVA using fluorescein as a contrast agent appears to be effective in controlling patency of aneurysms and the underlying vessel and can even be adapted to bypass surgery.

We present a protocol to efficiently evaluate aneurysm perfusion and vessel patency of sidewall aneurysm in rats and rabbits, using fluorescein-based fluorescence video angiography (FVA). With a positive predictive value of 92.6%, it is a simple but very effective and economical method with no special equipment required.

Angiografia por fluorescência para avaliação da perfusão de aneurisma e patência da artéria parental em modelos de aneurisma de ratos e coelhos

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and ...

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

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

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

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

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

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 ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Imagem e Análise do Transporte de Neurofilamento em Nervo Tibial de Camundongo Excised

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...