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'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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

36.5K 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

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

Estabelecimento de um mímico valioso de doença de Alzheimer em modelo Animal de rato por injeção Intracerebroventricular de proteína Beta amiloide compostos

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

Comportamento

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

A técnica de coleta de líquido cefalorraquidiano Serial da Cisterna Magna em rato

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Biologia

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 ...

Neurociência

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

Detecção de doença associada α-sinucleína por ELISA reforçada no cérebro de ratinhos transgénicos que sobre-expressam A53T Humano Mutante α-sinucleína

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Medicina

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Injeção intracerebroventricular de peptídeos amilóide-beta em ratos normais para induzir Acutely déficits cognitivos Alzheimer-like

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

Correlativa Luz e Microscopia Eletrônica para estudar interações Microglial com placas de beta-amilóide

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

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.

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 ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

3D quantitativa<em&gt; In Silico</em&gt; Modelagem (q3DISM) de beta-amilóide cerebral fagocitose em modelos de roedores da doença de Alzheimer

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Avaliação de Alternância Espontânea, Reconhecimento de Objetos Novos e Clasping de Membros em Modelos de Rato Transgênico de Neuropatologia Amilóide-β e Tau

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Detecção de placas neuríticas no rato da doença de Alzheimer Modelo

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