Este trabalho foi financiado pelos Institutos Canadenses de Pesquisa em Saúde (CIHR), a Família Townsend, e Jack Brown e Família Alzheimer Research Foundation (para WAS). WS é o titular da Cátedra de Pesquisa do Canadá na Doença de Alzheimer. PTTL é suportado pelas Ciências Naturais e Conselheiro Pesquisa de Engenharia e Michael Smith Fundação de Saúde de Bolsas de Investigação.

1. Fixação de cérebros de camundongos e cryosectioning <ol><li> Camundongos transgênicos são sacrificados com gás de dióxido de carbono, seguido por decapitação eo cérebro extraído. </li><li> O cérebro é dissecada no sentido longitudinal no centro. Metade do cérebro será flash congelado em gelo seco para análise bioquímica. O outro será submerso em paraformaldeído a 4% (PFA) para pelo menos 48 horas a 4 ° C. </li><li> Após a fixação em PFA 4% para pelo menos 48 horas, o cérebro hemi é transferido diretamente para a solução de sacarose 30% e colocou a 4 ° C. No ponto, o cérebro hemi deve ser flutuante na solução de sacarose. Quando o cérebro hemi afundou para o fundo, normalmente exigindo cerca de 48 horas, ele está pronto para ser incorporado em outubro para cryosectioning. </li><li> Antes cryosectioning, monte uma camada fina de outubro na maçaneta e deixe congelar a -20 ° C. Remova o cerebelo do resto do cérebro hemi e coloque sobre a camada de outubro congelados. Cobrir imediatamente o cérebro hemi com outubro e deixe lentamente congelar a -20 ° C. Uma vez que a incorporação de outubro o cérebro se tornou opaco, ele está pronto para ser seccionado. </li><li> Definir a espessura de cada seção para 30 mm. Coletar cada fatia em um recipiente com solução de D&#39;Olomos. </li></ol> 2. Procedimento de imuno-histoquímica para placas neuríticas (free seções flutuante) <ol><li> Tratar cada seção em 88% de ácido fórmico por 15 minutos. Após o tratamento ácido fórmico, a seção vai desativar temporariamente opaco e pode aparecer a murchar. </li><li> Retire cada seção em um prato e lave com Tx-PBS (0,3% Triton X-100 em PBS) por 5 X 3 min, agitando suavemente. </li><li> Peróxido de bloco endógena bloqueio 0,5% H 2 O 2 em Tx-PBS, RT 30 min, agitando suavemente. </li><li> Lavar a lâmina com Tx-PBS por 10 min X 3, agitando suavemente. </li><li> Seção do bloco com 5% de leite desnatado sem gordura dissolvida em Tx-PBS em temperatura ambiente por uma hora, agitando suavemente. </li><li> Incubar seções em anticorpo primário (biotinilado 4G8, 1:500-1:2000, Signet) diluído em PBS-Tx contendo 5% de leite a 4 ° C durante a noite, agitando suavemente. </li><li> Lavar a lâmina com Tx-PBS por 10 min X 3, agitando suavemente. </li><li> Prepare protocolo ABC fabricante da solução seguinte (Elite ABC, Vector Laboratories). Incubar a mistura de seções ABC por 30 min, agitando suavemente. </li><li> Lavar a lâmina com Tx-PBS por 10 min X 3, agitando suavemente. </li><li> Prepare 3,3 tetrahidrocloreto &#39;diaminobenzidina (DAB) (Vector Laboratories) protocolo do fabricante seguinte. Incubar seções em DAB mistura de 5-10 min. Neste ponto, você vai observar uma cor acastanhada desenvolver em cada seção. </li><li> Lavar a lâmina com Tx-PBS por 10 min X 3, agitando suavemente. </li><li> Seções de livre flutuação do cérebro são montados em gelatina slides revestido, para ajudar na adesão do tecido. Seções são então ar seco seguido por limpeza com uma série de xileno e montados em Entellen. </li><li> Placas são visualizadas em microscopia de luz em aumento de 40X e são quantificados avaliando a contagem de placa médio por fatia para cada rato. </li></ol> 3. Thioflavin S coloração procedimento para a detecção de placas neuríticas <ol><li> Sacrifício do mouse e procedimento de extração do cérebro são, tal como indicado nos pontos 1.1 a 1.5. </li><li> Monte 4-5 seções cérebro hemi numa lâmina de vidro e deixar completamente o ar seco antes da coloração. </li><li> Lavagem das lâminas com etanol 70% por 1 minuto. </li><li> Lavagem das lâminas com etanol 80% por 1 minuto. </li><li> Incubar as lâminas em solução filtrada (filtro de 0,2 mm) thioflavin S (1% em 80% de etanol) por 15 minutos. (Solução Thioflavin S precisa ser filtrada antes de cada utilização). Nota: proteger thioflavin S da luz e proteger lâminas coradas da luz. </li><li> Lavagem das lâminas com etanol 80% por 1 minuto. </li><li> Lavagem das lâminas com 70% de etanol minutos 1. </li><li> Lavar com água destilada duas vezes. </li><li> Slides montagem em solução aquosa meios de montagem e permitir que as lâminas secar no escuro por pelo menos duas horas, seguido por lamínula de vedação com unhas polonês claro. Cortes corados deve ser armazenado no escuro a 4 ° C. </li><li> A fluorescência verde placas manchada poderia ser visualizado com microscropy fluorescente. Analisar slides dentro de uma semana porque a coloração vai desaparecer com o tempo. </li></ol> 4. Resultados representante Em nosso estudo recente sobre a eficácia do medicamento anti-epiléptico e ácido valpróico estabilizador de humor (VPA) para inibir a formação de placas neuríticas, foi utilizada a imunocoloração acima descritos e thioflavin S coloração procedimentos para identificar placas neuríticas em ratos AD modelo (3). A Figura 1 representa um típico 9 meses de idade APP23 camundongo transgênico, corados com anti-Aâ usando 4G8 biotinilado e detectados através do método ABC. Placas neuríticas são claramente identificados com o anticorpo e são indicados pelas setas brancas. Usando coloração thioflavin S, detectamos deposição de placas neuríticas 2 meses de idade APP23xPS transgenic ratos (Figura 2). Obrigado Thioflavin S placas neuríticas Aâ contendo parece verde sob microscopia de fluorescência (indicado por setas brancas). Nós mostramos que o tratamento com VPA, a formação de placas neuríticas é significativamente reduzido (3). Em outro estudo, estudamos a hipóxia ligação molecular subjacente e patogênese AD. Mais uma vez, usando tanto a coloração imuno-histoquímica anti-4G8 Aâ e coloração thioflavin S, descobrimos que a hipóxia facilitou a formação de Aâ contendo placas neuríticas (4). <img src="/files/ftp_upload/2831/2831fig1.jpg" alt="Figura 1"/> Figura 1. Análise imunohistoquímica da formação de placas neuríticas em camundongos transgênicos AD. APP23 ratos na idade de nove meses foram sacrificados e foram dissecados, fixo, e seccionados. Placas neuríticas no hipocampo foram detectados através de um anticorpo anti-4G8 Aâ e foram desenvolvidos utilizando os métodos ABC e DAB. As placas foram visualizadas por microscopia de luz com 40X. Setas brancas apontam para placas neuríticas Aâ. <img src="/files/ftp_upload/2831/2831fig2.jpg" alt="Figura 2"/> Figura 2. Coloração Thioflavin S de placas neuríticas em ratos AD modelado. APP23xPS45 dupla camundongos transgênicos às 8 semanas de idade foram sacrificados e foram dissecados, fixo, e seccionados. Pragas neuríticas no hipocampo estão foram confirmados pela coloração fluorescente thioflavin S e visualizados por microscopia em aumento de 40X. Setas brancas indicam placas neuríticas manchada thioflavin S.

<ol>
	<li>Glenner, G. G., Wong, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+initial+report+of+the+purification+and+characterization+of+a+novel+cerebrovascular+amyloid+protein.">Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein.</a> Biochem Biophys Res Commun. 120, 885-890 (1984).</li><li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Qing, H., He, G., Ly, P. T., Fox, C. J., Staufenbiel, M., Cai, F., Zhang, Z., Wei, S., Sun, X., Chen, C. H., Zhou, W., Wang, K., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+acid+inhibits+Abeta+production,+neuritic+plaque+formation,+and+behavioral+deficits+in+Alzheimer's+disease+mouse+models.">Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.</a> J Exp Med. 205, 2781-2789 (2008).</li><li>Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., Staufenbiel, M., Huang, L. E., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+facilitates+Alzheimer's+disease+pathogenesis+by+up-regulating+BACE1+gene+expression.">Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression.</a> Proc Natl Acad Sci U S A. 103, 18727-18732 (2006).</li></ol>

Todos os experimentos foram conduzidos de acordo com o The University of British Columbia Animal Care e da comissão Use e diretrizes CIHR.

Imunohistoquímica utilizando o anticorpo biotinilado rotulados manchas 4G8 placas neuríticas na AD modelado camundongos com especificidade. O resultado coloração permite a quantificação e comparação da carga de placa bacteriana entre os diferentes grupos de tratamento. Existem vários passos cruciais que poderiam afetar o resultado. Porque o cérebro hemi não são perfundidos antes da extração do crânio, o cuidado deve ser usado durante o processo de extração, a fim de evitar danos para o cérebro hemi. Além disso, uma vez que o cérebro é hemi passivamente perfundidos, recomendamos incubando-o em PFA 4% para pelo menos 48 horas a 4 ° C antes da imersão em solução de sacarose a 30%. Alternativamente, a perfusão transcardial poderia ser feito antes de extrair o cérebro. Se o cérebro é fixada corretamente, ele deve ter uma textura emborrachada. No caso em que o cérebro não é fixa adequadamente, as seções serão rasgões na solução Olomos D&#39;ou durante os procedimentos de coloração. O anticorpo monoclonal é 4G8 reativa para resíduos de aminoácidos 17-24 de Aâ e reconheceu o epítopo na seqüência do núcleo (VFFAE). Desde 4G8 é derivado de espécies mouse, ele tende a dar uma coloração mais fundo. Assim, optou-se incubar as seções na diluição indicada com considerações para alcançar um verdadeiro sinal, minimizando coloração de fundo. Além de imunocoloração para a detecção de placas neuríticas usando o anticorpo 4G8, um método thioflavin S coloração também é usado para identificar placas. Thioflavin S é uma mistura homogênea de corante que resulta de metilação de dehydrothiotoluidine com ácido sulfônico. Thioflavin S não se liga seletivamente conteúdo folha beta de proteínas, tais como aquelas em oligômeros amilóide. Após a ligação, thioflavin sofre uma mudança de característica azul do seu espectro de emissão. Por outro lado vinculativo thioflavin S com as formas monoméricas não vai provocar uma mudança de azul e não podia ser detectado com microscópio de fluorescência. Coloração Thioflavin S oferece uma alternativa rápida para triagem de amilóide como a intensidade de fluorescência permite boa visualização de pequenas quantidades de depósitos de amilóide. No entanto, uma desvantagem sobre coloração thioflavin S é a falta de especificidade. Muitos componentes do tecido, incluindo contendo folhas beta extensa, como fibrinoids, hialina, queratina, etc, têm uma afinidade bastante para este corante.

<table border="1"><tbody><tr><th> Nome do reagente e equipamentos </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Paraformaldeído </td><td> Sigma-Aldrich </td><td> P6148-1KG </td></tr><tr><td> Polyvinylpyrrolidene </td><td> Sigma-Aldrich </td><td> PVP40-100G </td></tr><tr><td> Sacarose </td><td> Fisher Scientific </td><td> S5-3 </td></tr><tr><td> Etilenoglicol </td><td> Sigma-Aldrich </td><td> 324558-2L </td></tr><tr><td> Tissue-Tek Compound outubro </td><td> Sakura </td><td> 4583 </td></tr><tr><td> 88% de ácido fórmico </td><td> Fisher Scientific </td><td> A118P-500 </td></tr><tr><td> Triton X-100 </td><td> Biomedicals ICN </td><td> 807426 </td></tr><tr><td> H2O2 </td><td> Sigma-Aldrich </td><td> H-1009 </td></tr><tr><td> Biotina rotuladas 4G8 Antibody </td><td> Cedarlane </td><td> SIG-39240-500 </td></tr><tr><td> Elite ABC kit </td><td> Vetor </td><td> PK-6100 </td></tr><tr><td> Imm PACTO DAB </td><td> Vetor </td><td> SK-4105 </td></tr><tr><td> Xileno </td><td> Fisher Scientific </td><td> X4-4 </td></tr><tr><td> Entellan </td><td> EM ciência </td><td> 65037-71 </td></tr><tr><td> Superfrost * Plus Lâminas de microscopia </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> Criostato </td><td> Leica </td><td> CM-3050-S </td></tr><tr><td> MX35 Premier micrótomo blade </td><td> Thermo Scientific </td><td> 3052835 </td></tr><tr><td> Thioflavin S </td><td> Sigma-Aldrich </td><td> T1892-25G </td></tr></tbody></table>

detection of neuritic plaques in alzheimer's disease mouse model

Doença de Alzheimer (DA) é a mais comum desordem neurodegenerativa levando à demência. Formação de placas neuríticas é uma das características patológicas da doença de Alzheimer. O componente central de placas neuríticas é uma pequena proteína filamentosa proteína chamada amilóide β (Aâ) 1, que é derivada da clivagem proteolítica seqüencial da proteína precursora beta-amilóide (APP) por β-secretase e γ-secretase. A hipótese amilóide implica que Aγ contendo placas como o mecanismo subjacente tóxicos em patologia 2. A análise post-mortem da presença de placas neuríticas confirma o diagnóstico da DA. Para promover nossa compreensão da neurobiologia Aγ na patogênese da AD, várias linhagens de camundongos expressando AD relacionados com mutações nos genes APP humanos foram gerados. Dependendo da gravidade da doença, esses ratos desenvolvem placas neuríticas em diferentes idades. Esses camundongos servem como ferramentas de valor inestimável para o estudo da patogênese e desenvolvimento de drogas que podem afetar o caminho de processamento APP e formação de placas neuríticas. Neste protocolo, empregamos um método imuno-histoquímico para detecção específica de placas neuríticas em camundongos modelo AD. Vamos especificamente discutir a preparação de extrair o cérebro meia, fixação paraformaldeído, cryosectioning, e dois métodos para detectar placas neurótico em camundongos transgênicos AD: detecção imuno-histoquímica usando o método ABC e DAB e detecção fluorescente usando o método thiofalvin S coloração.

Watch this Scientific Journal Video about Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model at JoVE.com

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

Uma das características patológicas da doença de Alzheimer é a formação de amilóide β proteína placas neuríticas positivo. Neste protocolo, descrevemos dois métodos para detectar placas neuríticas em camundongos transgênicos modelo AD: detecção imunohistoquímica utilizando o método ABC e DAB e detecção fluorescente usando o método thioflavin S coloração.

Detecção de placas neuríticas no rato da doença de Alzheimer Modelo

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

detection-of-neuritic-plaques-in-alzheimer-s-disease-mouse-model

Research

JoVE Journal

Neuroscience

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

36.4K visualizações.  The University of British Columbia. Uma das características patológicas da doença de Alzheimer é a formação de amilóide β proteína placas neuríticas positivo. Neste protocolo, descrevemos dois métodos para detectar placas neuríticas em camundongos transgênicos modelo AD: detecção imunohistoquímica utilizando o método ABC e DAB e detecção fluorescente usando o método thioflavin S coloração.

Vídeo: Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Preparação de Oligoméricas β-amilóide 1-42 Indução e de Impairment plasticidade sináptica em fatias do hipocampo

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Neurociência

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &amp;#946;-Protein, the Causative Agent of Alzheimer&#039;s Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Seleção de Aptamers para proteína β-amilóide, o agente causador da doença de Alzheimer

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

Morris Teste Maze Água para déficits de aprendizagem e memória em camundongos Alzheimer Disease Modelo

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

Detecção de hipóxia microrregional no Cortex Cerebral mouse por imagem de dois fótons de fluorescência endógena NADH

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Infusão estereotáxica de oligomica beta-amilóide no ratinho Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer&#039;s Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

Análise de interação psicofisiológicos generalizada (PPI) de memória relacionados com a conectividade em indivíduos em risco genético para a doença de Alzheimer

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Utilizando biossensores baseados em Enzyme medir Tonic e Phasic glutamato na doença de Alzheimer rato Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

Neurocomportamentais avaliações em um modelo do rato de lesão cerebral hipóxica-isquêmica Neonatal

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Rotulagem e imagem de placas amilóides no tecido cerebral usando a curcumina polifenóis naturais

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

Estimativa estereológica do comprimento da fibra cholinergica no Núcleo Basalis de Meynert do Cérebro do Rato

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...