Name: quantitative 3d in silico modeling (q3dism) of cerebral amyloid-beta phagocytosis in rodent models of alzheimer's disease
Uploaded: 2016-12-26T13:00:00.000+00:00
Duration: 9 min 33 s
Description: Watch this Scientific Journal Video about Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease at JoVE.com

M-V.G-S. is supported by a BrightFocus Foundation Alzheimer's Disease Research Fellowship Award (A2015309F) and an Alzheimer's Association, California Southland Chapter Young Investigator Award. T.M.W. is supported by an ARCS Foundation and John Douglas French Alzheimer's Foundation Maggie McKnight Russell-JDFAF Memorial Postdoctoral Fellowship. This work was supported by the National Institute on Neurologic Disorders and Stroke (1R01NS076794-01, to T.T.), an Alzheimer's Association Zenith Fellows Award (ZEN-10-174633, to T.T.), and an American Federation of Aging Research/Ellison Medical Foundation Julie Martin Mid-Career Award in Aging Research (M11472, to T.T.). We are grateful for startup funds from the Zilkha Neurogenetic Institute, which helped to make this work possible. &#8195;

Declaração de ética em pesquisa: Todos os experimentos envolvendo animais detalhados aqui foram aprovados pela University of Southern California Institutional Animal Care e Use Committee (IACUC) e realizado em estrita conformidade com os Institutos Nacionais de diretrizes de saúde e recomendações da Associação de Avaliação e Acreditação do Laboratório animal Care International. 1. Rodent isolamento Cérebro e Preparação para imunocoloração DIA 1: <ol><li> Coloque ratos TgF344-AD com idade (14 meses de idade) ou APP ratos / PS1 (12 meses de idade), sob anestesia isoflurano profunda contínua (4%). Avaliar a profundidade da anestesia por pitada dedo do pé e ausência de reflexo de retirada. </li><li> Cortar ambos os lados da caixa torácica e levante para expor o coração. Inserir uma agulha G 23 para o ventrículo esquerdo do coração e fazer uma pequena incisão no átrio direito. Continuar a exsanguinação por perfusão transcardial com soro fisiológico tamponado com fosfato arrefecido com gelo(PBS) utilizando uma bomba peristáltica (30 mL para ratos; 150-200 mL para ratos). </li><li> Fazer uma incisão na linha média na pele caudal e mover a pele e músculo de lado. Cortar através da parte superior do crânio, ao longo da linha média e entre os olhos. Retirar as placas ósseas e isolar todo o cérebro do crânio. </li><li> Coloque o cérebro em uma matriz de roedores cérebro coronal e cortá-lo em quartos. Incubar quartos posteriores S / N (16 h) no fixador de paraformaldeído (PFA a 4% em PBS) a 4 ° C. Lavar 3x em PBS, e depois transferir para 70% de etanol. Cuidado: PFA é tóxico e deve ser tratado sob um capuz químico com equipamento de protecção individual adequado. </li></ol> DIA 2: <ol start="5"><li> Coloque trimestres cerebrais em cassetes de embebimento e progressivamente desidratar o tecido em banhos de etanol 1 h sucessivamente mais concentrada (70%, 80%, 95%, e 100% x3). </li><li> Limpar o etanol a partir do tecido com três banhos sucessivos de xileno a 100% (1 h cada). Atenção: O xileno é tóxico e should ser tratadas sob um capuz químico com equipamento de protecção individual adequado. </li><li> Incorporar tecidos em blocos de parafina depois de dois banhos de fundição de cera de parafina (56-58 ° C, 90 min cada). </li></ol> DIA 3: <ol start="8"><li> Cortar 10 seções mm de espessura de cérebros embebidos em parafina usando um micrótomo. secções mergulhar em um banho de água (50 ° C durante 1 min) e aplicam-se a lâminas de microscópio. Deixar as lâminas secar S / N, garantindo adesão do tecido à lâmina. </li></ol> 2. A imunocoloração Nota: Diferentes combinações de anticorpos pode ser utilizada para o procedimento de coloração descrito a seguir. Misturas de anticorpos compatíveis com tecido cerebral de ratos e murganhos são listados na Tabela 1. DIA 4: <ol><li> Desparafinar secções cerebrais usando 2x 100% banhos de xileno (12 min cada). </li><li> Rehidratar secções do cérebro em banhos sucessivos de etanol - 100% durante 10 min, 95% durante 5 min, 80% durante 10 min, e, finalmente, 70% durante 15 min - followed por lavagens PBS 3x [5 min à temperatura ambiente, com agitação luz]. Enquanto isso, o calor solução de recuperação de antígenos a 95-97 ° C em uma placa quente com agitação barra magnética. </li><li> Incubar secções do cérebro em solução de recuperação de antigénio a 95-97 ° C durante 30 min. Em seguida, lavar 3x em PBS (5 min à temperatura ambiente, com agitação leve). </li><li> Rapidamente lâminas secas usando limpadores tarefa delicada para evitar a secagem do tecido, e desenhar uma barreira hidrofóbica ao redor da área do tecido com uma caneta barreira hidrofóbica. Encher o tecido região cercada com tampão de bloqueio [PBS contendo 0,3% de Triton X-100 e 10% de soro normal burro (NDS)], e incubar à temperatura ambiente durante 1 h numa câmara humidificada. </li><li> Substituir tampão com anticorpo primário Iba1 (diluído em tampão de bloqueio) a rotular fagócitos mononucleares, e incubar O / N a 4 ° C numa câmara humidif içada de bloqueio. Para hosts de anticorpos e diluições de trabalho, consulte a Tabela 1 e a Tabela de Materiais. </li></ol> DIA 5: <ol start="6"><li> Rinseanticorpo primário com banhos 3x PBS (5 min à temperatura ambiente com agitação leve). Incubar com anticorpo fluorescente secundário (conjugado com um fluoróforo de emissão 594 nm) durante 1 h (em tampão à TA no escuro bloqueio) seguido de banhos 3x com PBS (5 min à temperatura ambiente com agitação luz). Neste momento, manter secções no escuro para evitar branqueamento sinal fluorescente. </li></ol> Dia 6 - 7: <ol start="7"><li> Repita os passos 2.5 e 2.6 com CD68 (cérebros de ratos) ou anticorpos LAMP1 (tecido mouse) e anticorpos secundários apropriados (juntamente com um fluoróforo emissão de 488 nm) para rotular phagolysosomes. </li></ol> DIA 7-8: <ol start="8"><li> Repita os passos 2.5 e 2.6 com OC (tecido de rato) ou anticorpos 4G8 (cérebro do rato) e anticorpos secundários apropriados (acoplado a um fluoróforo 647 nm) para rotular depósitos de Ap. NOTA: Como alternativa, o anticorpo 6E10 pode ser utilizado com sucesso tanto em tecidos do rato e rato. Para combinações de anticorpos adequado, consulte a Tabela de Materiais </strong&gt;. </li><li> Permitir seções completamente O seco / N à temperatura ambiente no escuro. Em seguida, cobrir amostras com uma lamela selada por meios de montagem de fluorescência contendo DAPI. </li></ol> 3. Aquisição de grande Z-stack confocal conjuntos de dados Nota: Este protocolo requer um rastreio por laser microscópio confocal totalmente automatizado equipado com uma objectiva 60X e 405 nm, 488 nm, 594 nm e 647 nm lasers. Todo o equipamento é controlado por computador pelo software de imagem e controle laser. Antes de iniciar o protocolo de imagem, ligue o computador, lâmpada de epifluorescência, microscópio, lasers e câmera. DIA 9: <ol><li> Escolha a objetiva do microscópio 60X. Adicione o óleo de imersão para a lente, e colocar a amostra no suporte de microscópio estágio slide. Elevar o objetivo até que o óleo faz contato com o slide. Ajuste o plano focal para localizar placas amilóides no hipocampo ou córtex cerebral usando iluminação de epifluorescência através das oculares. </li><li> Adquirir im confocalidades de fagócitos mononucleares ativadas circundantes depósitos de amilóide no hipocampo ou córtex de roedores por microscopia confocal (60X ampliação, passos z-stack: 0,25 mm &lt;z &lt;0,40 mm, número de etapas de 25 &lt;n &lt;35). </li></ol> 4. q3DISM Nota: A fim de produzir resultados significativos, sugerimos analisar um mínimo de 3 imagens por animal / região de interesse. Para cada imagem, a abundância de células a analisar pode variar dependendo de paradigmas experimentais. Nos resultados representativos apresentados neste relatório, foram analisadas 3 células / condição (por exemplo, fagócitos mononucleares distantes ou associados com placas; ver Figuras 1C - D e 2C - D). DIA 10: <ol><li> Analisar conjuntos de dados confocal com co-localização de processamento de imagem 3D científico e análise de software (Coloc) módulo de proximidade espacial de Iba1 / CD68 (tecido de rato) ou Iba1 / LAMP1 (tecido mouse) coloração em todos os z-aviões simultaneamente.Criar Iba + / CD68 + ou canais Iba + / LAMP1 + co-localização que correspondem a phagolysosomes dentro de fagócitos mononucleares ativadas. <ol><li> Escolha TRITC para o canal A (correspondente a coloração Iba1 acoplada com 594 nm fluoróforo) e FITC para o canal B (que corresponde a CD68 ou coloração LAMP1 acoplada com 488 nm fluoróforo). No lado direito da janela do software para &#39;verificação modo,&#39; selecionar &#39;limite&#39; e por &#39;intensidades COLOC,&#39; selecionar &#39;canais de origem&#39;. </li><li> Clique em &quot;Editar&quot; para selecionar &quot;cor Coloc &#39;no lado direito da janela do software. </li><li> Para cada canal de forma independente, ajustar os limites para incluir coloração específica e não incluem sinais de fundo / não-específicos. Uma vez ajustado, não altere os limites entre as imagens para garantir uma análise imparcial. Os voxels colocalized (pixels de todos os z-stacks) será exibido na cor selecionada na etapa 4.1.2 em todas as z-stacks SIMultaneously. </li><li> Clique em &#39;construir canal Coloc&#39;. O canal de co-localização criado aparecerá na janela de ajuste de display. </li><li> Clique no canal Coloc para abrir estatísticas de canal. A &quot;% de volume de material / A acima limiar colocalized &#39;% representa o sinal de Iba1 (voxels correspondentes ao fluoróforo 594 nm) co-localizada com LAMP1 ou sinal de CD68 (voxels correspondentes ao fluoróforo 488 nm). Mais simplesmente, este é o volume ocupado pelos monócitos fagolisossomas (ver Figuras 1C e 2C). NOTA: &#39;% do volume de material / B acima do limiar colocalized&#39; corresponde a% de sinal LAMP1 ou CD68 colocalized com Iba1. Isto deve ser próximo de 100%, como fagolisossomas são estruturas intracelulares. Os valores são baseados na proporção de sinal de todos os z-stacks acima do limiar colocalized. </li></ol></li><li> Usando o módulo Coloc, analisar o canal Coloc criado na etapa 4.1 para proximidade espacial com OC (ti ratossue) ou 4G8 (tecido mouse) sinais de Ap. Isto permite a quantificação de Ap encapsulado dentro de fagolisossomas. <ol><li> Seleccionar o canal Um conjunto de dados Coloc (correspondente ao Iba1 / CD68 ou Iba1 / LAMP1 Coloc canal criado no passo 4.1) e Cy5 para o canal B (que corresponde a coloração ou OC 4G8 acoplada com 647 nm fluoróforo). </li><li> Construir um canal Coloc como descrito nos passos 4.1.2 a 4.1.5. </li><li> Clique no canal Coloc para abrir estatísticas de canal. A &quot;% de volume de material / A acima limiar colocalized &#39;representa a% de Iba1 / ou LAMP1 Iba1 / CD68 (voxels correspondente ao canal Coloc construído no passo 4.1.5.) Co-localizada com CO ou sinal 4G8 (voxels correspondente ao 647 fluoróforo nm). Este é o volume ocupado por fagolisossï¿½ico Ap (ver Figuras 1D e 2D). NOTA: &#39;% do volume de material / B acima do limiar colocalized&#39; corresponde a% de sinal de Ap total de colocalized com phagolysosomes.Isto pode ser usado para avaliar a fracção do total dos depósitos de Ap encapsulados dentro de fagolisossomas (não mostrado nos presentes resultados representativos). </li></ol></li><li> Use o módulo superar para reconstruir as pilhas de imagens confocal e gerar modelos 3D de Ap encapsulados dentro phagolysosomes monócitos. <ol><li> Na janela &quot;ajuste display &#39;, selecione TRITC (para mostrar coloração única Iba1). Na janela &quot;Propriedades de volume&quot;, clique em &quot;adicionar nova superfície&quot;. </li><li> No passo &#39;1/5 algoritmo&#39;, em &#39;Configurações&#39;, selecione &#39;superfície&#39;. Além disso, em &#39;cor&#39;, selecione o tipo de cor na paleta ou RGB e ajustar a transparência (vermelho é de transparência de 60% nas Figuras 1B e 2B). a caixa de seleção &quot;selecionar a região de interesse&quot;. Clique ao lado. </li><li> No Passo &#39;2/5 região de interesse, &quot;desenhar uma janela em torno da célula de interesse, ajustando X, Y e Z coordenadas (ver caixas brancas nas Figuras 1A </strong&gt; e 2A). Clique ao lado. </li><li> No Passo &#39;3/5 canal de origem &quot;, selecione o canal de origem TRITC. Marque a caixa &quot;suave&quot;, e definir o nível de detalhe área de superfície a 0,4 mm. Para &quot;limiar&quot;, selecione intensidade absoluta. Clique ao lado. </li><li> Na Etapa &#39;4/5 limiar,&#39; ajustar o limiar de forma que o volume criado sobrepõe-se perfeitamente com o sinal de canal TRITC. Clique ao lado. </li><li> No Passo &#39;5/5 classificar superfícies &quot;, na seção&quot; tipo de filtro &quot;, selecionar objetos dependendo do seu tamanho para ser incluído ou excluído dos volumes a serem criados. Clique em Concluir, executar todas as etapas de criação, e sair do assistente. </li><li> Repita os passos 4.3.1 a 4.3.6 para criar uma superfície 3D para o canal FITC (LAMP1 + ou CD68 + fagolisossomas). </li><li> Repita os passos 4.3.1 a 4.3.6 para criar uma superfície 3D para o canal Cy5 (depósitos 4G8 + Ap OC + ou). </li></ol></li></ol>

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Chem. 285 (47), 36945-36957 (2010).</li><li>Blurton-Jones, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+improve+cognition+via+BDNF+in+a+transgenic+model+of+Alzheimer+disease.">Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease.</a> Proc Natl Acad Sci U S A. 106 (32), 13594-13599 (2009).</li><li>Stalder, M., Deller, T., Staufenbiel, M., Jucker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-Reconstruction+of+microglia+and+amyloid+in+APP23+transgenic+mice:+no+evidence+of+intracellular+amyloid.">3D-Reconstruction of microglia and amyloid in APP23 transgenic mice: no evidence of intracellular amyloid.</a> Neurobiol Aging. 22 (3), 427-434 (2001).</li><li>Leinenga, G., G&#246;tz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+ultrasound+removes+amyloid-&#946;+and+restores+memory+in+an+Alzheimer's+disease+mouse+model.">Scanning ultrasound removes amyloid-&#946; and restores memory in an Alzheimer's disease mouse model.</a> Sci Transl Med. 7 (278), 33 (2015).</li><li>Liarski, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+distance+mapping+identifies+functional+T+follicular+helper+cells+in+inflamed+human+renal+tissue.">Cell distance mapping identifies functional T follicular helper cells in inflamed human renal tissue.</a> Sci Transl Med. 6 (230), 46 (2014).</li><li>Nichols, L., Pike, V. W., Cai, L., Innis, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+in+vivo+quantitation+of+beta-amyloid:+an+exemplary+biomarker+for+Alzheimer's+disease.">Imaging and in vivo quantitation of beta-amyloid: an exemplary biomarker for Alzheimer's disease.</a> Biol Psychiatry. 59 (10), 940-947 (2006).</li><li>Skovronsky, D. M., Zhang, B., Kung, M. P., Kung, H. F., Trojanowski, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+of+amyloid+plaques+in+a+mouse+model+of+Alzheimer's+disease.">In vivo detection of amyloid plaques in a mouse model of Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 97 (13), 7609-7614 (2000).</li><li>Lian, H., Litvinchuk, A., Chiang, A. C. -. A., Aithmitti, N., Jankowsky, J. L., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-Microglia+Cross+Talk+through+Complement+Activation+Modulates+Amyloid+Pathology+in+Mouse+Models+of+Alzheimer's+Disease.">Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease.</a> J Neurosci. 36 (2), 577-589 (2016).</li><li>Novotny, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+Synthetic+A&#946;+to+In+Vivo+Active+Seeds+and+Amyloid+Plaque+Formation+in+a+Hippocampal+Slice+Culture+Model.">Conversion of Synthetic A&#946; to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.</a> J Neurosci. 36 (18), 5084-5093 (2016).</li><li>Tartaro, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+fluorescence-based+in+vivo+phagocytosis+assay+to+measure+mononuclear+phagocyte+system+function+in+the+rat.">Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.</a> J Immunotoxicol. 12 (3), 239-246 (2015).</li></ol>

Os autores não têm nada para revelar.

O protocolo que descrevemos neste relatório para a verdadeira quantificação 3D de Ap fagocitose in vivo pelos fagócitos mononucleares depende de rotulagem específica dos compartimentos celulares e subcelulares, bem como depósitos de Ap. Especificamente, usamos Iba1 (ionizada-se ligar à molécula de cálcio Adaptador 1), uma proteína que está envolvida em membrana ruffling e fagocitose por activação de célula 18, 19, para corar os fagócitos mononucleares cerebrais. E...

Doença de Alzheimer (AD), a demência relacionada com a idade mais comum 1, caracteriza-se pela acumulação de amilóide cerebral-β (Ap) como placas &quot;senis&quot; beta-amilóide, neuroinflamaï¿½o baixo nível crónica, taupatia, perda neuronal, e perturbação cognitiva dois . No AD cérebros de pacientes, neuroinflamação é destinada pelos astrócitos e fagócitos mononucleares reativa (referido como microglia, embora as suas centrais vs. origem periférica permanece obscuro) em torno depósitos Ap 3. Como as sentinelas imunes inatas do SNC, microglia são posicionados centralmente para limpar Ap cérebro. No entanto, o recrutamento da microglia para placas de Ap é acompanhada por muito pouco, se algum, fagocitose Ap 4,5. Uma hipótese é que microglia são inicialmente neuroprotetor por phagocytozing pequenas montagens de Ap. No entanto, eventualmente estas células se tornam neurotóxico como carga Ap esmagadora e / ou d funcional relacionada com a idadeecline, provoca microglia em um fenótipo pró-inflamatório disfuncional, contribuindo para a neurotoxicidade e declínio cognitivo 6. Estudo de associação genômica ampla Recentes (GWAS) identificaram um conjunto de alelos de risco AD pertencentes ao núcleo vias imunitárias inatas 7 que modulam a fagocitose 8-11. Consequentemente, a resposta imune a deposição de amilóide cerebral tornou-se uma grande área de interesse, tanto em termos de compreender a etiologia AD e para o desenvolvimento de novas abordagens terapêuticas 12-14. No entanto, existe uma necessidade vital para a metodologia para avaliar a fagocitose Ap in vivo. Para atender a essa necessidade não satisfeita, temos desenvolvido 3D quantitativa na modelagem silico (q3DISM) para permitir uma verdadeira quantificação 3D da cerebral fagocitose Ap por fagócitos mononucleares em modelos de roedores de doença de Alzheimer-like. Limitado apenas pelo grau em que a doença recapitular, os modelos animais têmcomprovada de valor inestimável para a compreensão AD pathoetiology e para avaliar terapêutica experimental. Devido ao facto de que as mutações nos genes presenilina (PS) e da Proteína Precursora Amilóide (APP) independentemente causar AD autossómica dominante, estes transgenes mutantes têm sido extensivamente usados ​​para gerar modelos de roedores transgénicos. Camundongos transgênicos APP / PS1 coexpressï¿½ simultaneamente &quot;sueco&quot; mutante APP humana (swe APP) e Δ exon 9 mutante presenilina 1 humano (PS1ΔE9) presente com amiloidose cerebral acelerada e neuroinflamação 15,16. Além disso, geramos ratos bi-transgénicos co-injectado com APP swe e construções PS1ΔE9 (linha TgF344-AD, em um fundo Fischer 344). Ao contrário de modelos de ratos transgênicos de amiloidose cerebral, ratos TgF344-AD desenvolver amilóide cerebral que antecede tauopathy, perda apoptótica de neurônios, e comprometimento comportamental 17. Neste relatório, nós descrevemos um protocolo para immunostaining microglia, phagolysosomes e depósitos de Ap em seções do cérebro de ratinhos APP / PS1 e ratos TgF344-AD, e aquisição de imagens confocal grandes z-dimensional. Nós detalhe na geração e análise dos verdadeiros reconstruções 3D a partir de conjuntos de dados confocal permitindo a quantificação da captação de Ap em phagolysosomes microgliais silico. Mais amplamente, a metodologia que nós detalhes aqui pode ser usado para quantificar virtualmente qualquer forma de fagocitose in vivo.

<table><tbody><tr><td>Isoflurane</td><td>Abbott</td><td>NDC 0044-5260-05</td><td></td></tr><tr><td>Dissecting scissors</td><td>VWR</td><td>82027-582</td><td></td></tr><tr><td>Dissecting scissors Blunt tip</td><td>VWR</td><td>82027-588</td><td></td></tr><tr><td>Tweezers</td><td>VWR</td><td>94024-408</td><td></td></tr><tr><td>23 G needle</td><td>VWR</td><td>BD305145</td><td></td></tr><tr><td>peristaltic pump FH10</td><td>Thermo Scientific</td><td>72-310-010</td><td></td></tr><tr><td>PBS 10x</td><td>Bioland Scientific</td><td>PBS01-02</td><td>Phosphate-buffered Saline; Working concentration 1x</td></tr><tr><td>Adult Mouse Brain Matrix, Coronal slices, Stainless Steel 1 mm&amp;nbsp;</td><td>Kent Scientific</td><td>RBMS-200C</td><td></td></tr><tr><td>Adult Rat Brain Matrix, Coronal slices, Stainless Steel 1 mm&amp;nbsp;</td><td>Kent Scientific</td><td>RBMS-305C&amp;nbsp;</td><td></td></tr><tr><td>32% Paraformaldehyde aqueous solution (PFA)</td><td>EMS</td><td>15714-S</td><td>Caution: Toxic. Working concentration: 4% in PBS</td></tr><tr><td>Ethanol</td><td>VWR</td><td>89125-188</td><td>Various concentrations, see protocol</td></tr><tr><td>Tissue-Tek Uni-cassettes Sakura</td><td>VWR</td><td>25608-774</td><td></td></tr><tr><td>Embedding and Infiltration Paraffin</td><td>VWR</td><td>15147-839</td><td></td></tr><tr><td>Microtome Leica RM2125</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Disposable Microtome Blades&amp;nbsp;</td><td>VWR</td><td>25608-964</td><td></td></tr><tr><td>Water bath Leica HI 1210</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Micro slide Superfrost plus</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>534056-4X4L</td><td>Caution: Toxic&amp;nbsp;</td></tr><tr><td>Target Retrieval Solution 10x</td><td>DAKO</td><td>S1699</td><td>Working concentration 1x</td></tr><tr><td>KimWipes</td><td>VWR</td><td>21905-026</td><td></td></tr><tr><td>Hydrophobic PAP pen</td><td>VWR</td><td>95025-252</td><td></td></tr><tr><td>Triton X-100</td><td>VWR</td><td>97062-208</td><td></td></tr><tr><td>Normal Donkey Serum (NDS)</td><td>Jackson Immuno</td><td>017-000-121</td><td></td></tr><tr><td>Coverslips</td><td>VWR</td><td>48393081</td><td></td></tr><tr><td>Prolong Gold antifade reagent with DAPI</td><td>Life Technologies</td><td>P36935</td><td></td></tr><tr><td>Glass Slide Rack</td><td>VWR</td><td>100492-942</td><td></td></tr><tr><td>Iba1 antibody (polyclonal, rabbit)</td><td>Wako</td><td>019-19741&amp;nbsp;</td><td>Working concentration 1:200</td></tr><tr><td>Iba1 antibody (polyclonal, goat)</td><td>LifeSpan Bioscience</td><td>LS-B2645</td><td>Working concentration 1:200</td></tr><tr><td>rat CD68 [KP1] antibody (monoclonal, mouse)</td><td>Abcam</td><td>ab955</td><td>Working concentration 1:200</td></tr><tr><td>mouse CD68 [FA-11] antibody (monoclonal, rat)</td><td>Abcam</td><td>ab53444</td><td>Working concentration 1:200</td></tr><tr><td>mouse CD107a (LAMP1) antibody (monoclonal, rat)</td><td>Affymetrix</td><td>14-1071</td><td>Working concentration 1:100</td></tr><tr><td>Beta-Amyloid, 17 - 24 (4G8) antibody (monoclonal, mouse)</td><td>Covance</td><td>SIG-39220</td><td>Working concentration 1:200</td></tr><tr><td>Beta-Amyloid, 1 - 16 (6E10) antibody (monoclonal, mouse)</td><td>Covance</td><td>SIG-39320</td><td>Working concentration 1:200</td></tr><tr><td>OC antibody (polyclonal, rabbit)</td><td>Gifted by D. H. Cribbs and C. G. Glabe (UC Irvine)</td><td>Working concentration 1:200</td><td></td></tr><tr><td>Alexa Fluor 488 mouse secondary antibody</td><td>Invitrogen</td><td>A-11001</td><td>Working concentration 1:1,000</td></tr><tr><td>Alexa Fluor 488 rat secondary antibody</td><td>Invitrogen</td><td>A-11006</td><td>Working concentration 1:1,000</td></tr><tr><td>Alexa Fluor 594 rabbit secondary antibody</td><td>Invitrogen</td><td>A-11037</td><td>Working concentration 1:1,000</td></tr><tr><td>Alexa Fluor 594 goat secondary antibody</td><td>Invitrogen</td><td>A-11080</td><td>Working concentration 1:1,000</td></tr><tr><td>Alexa Fluor 647 mouse secondary antibody</td><td>Invitrogen</td><td>A-21235</td><td>Working concentration 1:1,000</td></tr><tr><td>Alexa Fluor 647 rabbit secondary antibody</td><td>Invitrogen</td><td>A-21443</td><td>Working concentration 1:1,000</td></tr><tr><td>Immersion oil</td><td>Nikon&amp;nbsp;</td><td></td><td></td></tr><tr><td>A1 Confocal microscope</td><td>Nikon&amp;nbsp;</td><td></td><td></td></tr><tr><td>NIS Elements Advanced Research software</td><td>Nikon&amp;nbsp;</td><td></td><td></td></tr><tr><td>Imaris:Bitplane software version 7.6</td><td>Bitplane</td><td></td><td>&quot;coloc&quot; and &quot;supass&quot; modules are used.&amp;nbsp;Alternatively, the open-source freeware&amp;nbsp;ImageJ can be used for colocalization&amp;nbsp;analysis of confocal z-stacks datasets.</td></tr></tbody></table>

quantitative 3d in silico modeling (q3dism) of cerebral amyloid-beta phagocytosis in rodent models of alzheimer's disease

Neuroinflamação é agora reconhecido como um importante fator etiológico em doenças neurodegenerativas. fagócitos mononucleares são células imunitárias inatas responsáveis ​​pela fagocitose e remoção de escombros e detritos. Estas células incluem macrófagos residentes do SNC conhecidos como microglia e fagócitos mononucleares infiltrantes a partir da periferia. A microscopia de luz tem sido geralmente usado para visualizar a fagocitose em roedores ou espécimes do cérebro humano. No entanto, os métodos qualitativos não forneceram provas definitivas da fagocitose in vivo. Aqui, nós descrevemos 3D quantitativa na modelagem de silício (q3DISM), um método robusto para a verdadeira permitindo a quantificação de 3D-amilóide β (Ap) a fagocitose por fagócitos mononucleares em modelos de roedores da doença de Alzheimer (AD). O método envolve a fluorescência visualizando Ap encapsulado dentro phagolysosomes em seções roedor cerebrais. Grandes conjuntos de dados confocal z-dimensional são então reconstrução 3D para quantificação de A &amp;# 946; colocalized espacialmente no interior do fagolisossoma. Nós de demonstrar a aplicação bem sucedida de q3DISM de ratinho e de rato cérebros, mas esta metodologia pode ser estendida para virtualmente qualquer modo fagocitária em qualquer tecido.

Utilizando a metodologia multi-fase para q3DISM detalhado acima, que são capazes de quantificar a absorção de Ap em fagolisossomas monócitos nos cérebros de murganhos APP / PS1 (Figura 1) e ratos TgF344-AD (Figura 2). Portanto, a metodologia q3DISM permitiu a análise de fagócitos mononucleares em ratinhos e ratos modelos da doença de Alzheimer. Curiosamente, o volume ocupado pelo CD68 + fagolisossomas é aumentado significativamente em...

Watch this Scientific Journal Video about Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease at JoVE.com

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

Desenvolvemos uma metodologia para 3D quantitativa na modelagem silico (q3DISM) de amilóide cerebral-β (Aâ) fagocitose por fagócitos mononucleares em modelos de roedores da doença de Alzheimer. Este método pode ser generalizado para a quantificação de praticamente qualquer evento fagocítica 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 ...

quantitative-3d-silico-modeling-q3dism-cerebral-amyloid-beta

Research

JoVE Journal

Medicine

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

7.9K visualizações.  University of Southern California (USC).  Desenvolvemos uma metodologia para 3D quantitativa na modelagem silico (q3DISM) de amilóide cerebral-β (Aâ) fagocitose por fagócitos mononucleares em modelos de roedores da doença de Alzheimer. Este método pode ser generalizado para a quantificação de praticamente qualquer evento fagocítica in vivo. 

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

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

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 ...

Neurociência

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

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 ...

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, ...

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 ...

Medicina

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 ...

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral A&#946; plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the A&#946; load in brain tissues derived from AD transgenic mice. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of A&#946; load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on A&#946;-positive area quantification, and show a strong correlation between the A&#946;-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread A&#946; deposition.

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

Quantificação total versus sub-regional da carga amilóide-beta nas seções cerebrais do rato

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of ...

Fabrication of Amyloid-&#946;-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic amyloid-&#946; (A&#946;) fragments, eventually leading to the classical features of the disease: amyloid plaques, neurofibrillary tangles and synaptic and neuronal loss. The lack of relevant non-transgenic preclinical models reflective of disease progression is one of the main factors hindering the discovery of effective drug treatments. To this end, we have developed a protocol for the fabrication of alginate microbeads containing amyloid-secreting cells useful for the study of the effects of chronic A&#946; production.
Chinese hamster ovary cells previously transfected with a human APP gene, secreting A&#946; (i.e., 7PA2 cells), were used in this study. A three-dimensional (3D) in vitro model for the sustained release of A&#946; was fabricated by encapsulation of 7PA2 cells in alginate. The process was optimized to target a bead diameter of 500-600 &#956;m for further in vivo studies. Optimization of 7PA2 cell encapsulation in alginate was performed altering fabrication parameters, e.g., alginate concentration, gel flow rate, electrostatic potential, head vibration frequency, gelling solution. Levels of secreted A&#946; were analyzed over time and compared between alginate beads and standard cell culture methods (up to 96 h).
A concentration of 1.5 x 106 7PA2 cells/mL and an alginate concentration of 2% (w/v) buffered with HEPES and subsequent gelation in 0.5 M calcium chloride for 5 min were found to fabricate the most stable microbeads. Fabricated microbeads were 1) of uniform size, 2) with an average diameter of 550 &#956;m, 3) containing about 100-150 cells per microbead and 4) able to secrete A&#946;.
In conclusion, our optimized method for the production of stable alginate microbeads containing amyloid-producing 7PA2 cells might enable the modeling of important aspects of AD both in vitro and in vivo.

This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-&#946; over time and can be used to study the effects of secreted amyloid-&#946; in in vitro and in vivo models.

Fabricação de microgrânulos de alginato de secreção de amiloide-β para uso na modelagem da doença de Alzheimer

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic ...

Biologia

Establishing an Alzheimer's Disease Rat Model Through an Amyloid-Beta Peptide Injection

Source: Xiaoguang, W., et al. <a href="https://app.jove.com/v/56157">Establishment of a Valuable Mimic of Alzheimer&#39;s Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein.</a> J. Vis. Exp. (2018).
This video demonstrates the procedure for injecting amyloid-beta peptides, aluminum chloride, and a neuroinflammatory growth factor, which collectively aids in toxic amyloid plaque formation in the hippocampus region. This causes memory impairment in the rat, characteristic of Alzheimer&#39;s disease.

Estabelecendo um modelo de rato com doença de Alzheimer por meio de uma injeção de peptídeo beta-amilóide

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

Direct Stereotaxic Brain Infusion of Amyloid-Beta Proteins for an Alzheimer's Disease Model

Source: Jean, Y. Y. et al., <a href="https://app.jove.com/v/52805">Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus.</a> J. Vis. Exp. (2015)
This video demonstrates the precise stereotaxic infusion of amyloid beta into the dentate gyrus to establish a mouse model of Alzheimer&#39;s disease. Accurate coordinate determination and controlled delivery of amyloid beta proteins result in neuronal degradation, disrupting memory processing and contributing to Alzheimer&#39;s disease development.

3D quantitativa

Resumo

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