Stefano Fumagalli é um companheiro da Fundação Monzino.

1. Imunofluorescência O protocolo seguinte é realizada em crio-secções cerebrais coronais, obtidos a partir de ratinhos transcardially perfundidos (20 ml de PBS, 0,1 mol / litro, pH 7,4, seguido por 50 ml de paraformaldeído 4% gelada em PBS). Após a perfusão, os cérebros foram cuidadosamente removidos e transferidos para 30% de sacarose em PBS a 4 ° C durante a noite para crioprotecção. Os cérebros são então rapidamente congelados por imersão em isopentano a - 45 ° C durante 3 minutos antes de ser selado em frascos e armazenada a -70 ° C até à sua utilização. Criocortes cerebrais coronais (20 um) são cortados em série e submetido a protocolos de imunofluorescência 11, 6. Todos os reagentes e amostras à temperatura ambiente antes do uso. Por favor note que as diluições de trabalho apresentados de anticorpos e soro resultou de estudos realizados, a fim de obter o melhor desempenho. Quando diferentes anticorpos e soro são utilizados, o protocolo deve ser validado. <pclass = &quot;jove_content&quot;&gt; Note que, porque os anticorpos primários, tanto anti-CD11b e anti-CD68 são feitos em ratos, para evitar sinal de cruz por anti-rato Alexa 546, foi utilizado um alta diluição de anti-CD11b seguido de amplificação do sinal fluorescente com kit TSA (Cy5 tiramida). Criámos a diluição de trabalho ideal para anti-CD11b realizando o protocolo descrito, exceto para a etapa 1.17 e usando pelo menos sete diluições diferentes de anti-CD11b como relatado na tabela 1. Como resultado, 1:30.000 foi escolhido ser o único diluição realização: 1) sinal visível com Cy5 no comprimento de onda de excitação 646 nm, 2) nenhum sinal com Alexa 546 no comprimento de onda de excitação de 532 nm. Desta forma, Alexa 546 sinal fluorescente está associada selectivamente com CD68. As diluições óptimas de anti-CD11b e anti-CD68 podem variar dependendo do tipo de tecido e deve ser definida antes do início do protocolo de co-marcação. <ol><li> Lavar criocortes cerebrais duas vezes com PBS. </li><li> Emcriocortes Cubate por 5 min em PBS contendo 1% de H 2 O 2 (etapa necessária uma vez que a amplificação de fluorescência precisa de incubação com peroxidase de rábano, com estreptavidina-HRP, ver passo 1.12). </li><li> Criocortes Lavar 2x com PBS. </li><li> Incubar durante 60 min criocortes em PBS contendo 10% NGS e 0,3% de Triton. </li><li> Incubar criocortes a 4 ° C durante a noite em PBS contendo rato primária Ab anti-CD11b [1:30.000], 10% NGS e 0,3% de Triton. </li><li> Lavar 2x criocortes (5 min) com PBS. </li><li> Incubar durante 60 min criocortes em PBS contendo secundário biotinilado anti-rato Ab [1:200] e 1% de NGS. </li><li> Criocortes Lavar 2x com PBS. </li><li> Lavar criocortes com TNT (Tris-HCl, NaCl, Tween: TRIS 0,1 M-HCl, pH 7,4, 0,15 M de NaCl, 0,05% de Tween 20). </li><li> Incubar criocortes durante 1,5 horas em TNB (tampão Tris-HCl-NaCl-Blocking: TRIS 0,1 M-HCl, pH 7,4, 0,15 M NaCl, 0,5% de reagente de bloqueio a partir de kit adequado (ver tabela de reagentes)). </li><li> Wash crioseções 3x vezes com TNT. </li><li> Incubar criocortes em TNB contendo estreptavidina-HRP [1:100]. </li><li> Criosecções Lavar 3x com TNT. </li><li> Incubar criosecções por 8 minutos em Amplificação diluente contendo Cyanine 5 tiramida [1:300]. </li><li> Criocortes Lavar 3x com PBS. </li><li> Incubar durante 60 min criocortes em PBS contendo 10% NGS e 0,1% de Triton. </li><li> Incubar criocortes a 4 ° C durante a noite em PBS contendo primário Rato Ab anti-CD68 [1:200], 3% de NGS e 0,3% de Triton. </li><li> Criocortes Lavar 3x com PBS. </li><li> Incubar criocortes durante 60 min em PBS contendo fluorconjugated secundário Ab Alexa 546 anti-rato [1:500] e 1% de NGS. </li><li> Criocortes Lavar 3x com PBS. </li><li> Incubar criocortes durante 10 min em PBS contendo Hoechst 1 ug / ml. </li><li> Criocortes Lavar 3x com PBS. </li><li> Monte criosecções em Prolong Gold. </li></ol> 2. Aquisição de imagens tridimensionais por microscopia confocal <p class = &quot;jove_content&quot;&gt; O microscópio utilizado foi um microscópio IX81 equipado com uma unidade de digitalização FV500 confocal com 3 linhas de laser: Ar-Kr (488nm), He-Ne vermelho (646nm), e verde He-Ne (532nm) e um diodo de UV. <ol><li> Selecione os lasers de excitação, dependendo dos comprimentos de onda dos corantes fluorescentes a ser animado (He-Ne vermelho para Cy5, CD11b, He-Ne verde para Alexa546, CD68 eo diodo UV para a Hoechst, núcleos). </li><li> Escolha a melhor combinação espelho dicróico para a coleta de sinal luminoso. </li><li> Configure a resolução da imagem em um mínimo de 800 x 600 pixels. </li><li> Identificar uma área de interesse, utilizando epi-fluorescência e aumentar progressivamente a ampliação para a objetiva de 40X. </li><li> Mudar para Laser Scanning Microscopy (LSM) modalidade. </li><li> Execute varreduras repetitivas para ajustar o fotomultiplicador (PMT) eo ganho para cada canal. Lasers podem ser ativados individualmente para facilitar a configuração. Manter ganho tão baixa quanto possível, para evitar sinais não específicos indesejados. </li><li> Com repetitive verificação em execução, mover o foco de controlo para definir extremos superior e inferior do eixo z (comprimento total do eixo z = 10 mm). </li><li> Pare de varredura repetitivo. </li><li> Definir tamanho do passo. Ele deve ser o mais próximo possível ao tamanho do pixel (0,225 mm) para se obter uma proporção de 1:1 de tamanho padrão ao longo do eixo z. </li><li> Activar filtro de Kalman, pelo menos, duas vezes. </li><li> Ative o modo de varredura seqüencial para evitar efeitos sangram-through. </li><li> Vá a meio caminho ao longo do eixo z e executar uma varredura seqüencial xy. Confira o set-up da PMT e ganho (boa relação sinal / ruído). Se não for satisfatória, repita o passo 2.6. </li><li> Execute aquisição xyz. </li><li> Exportar dados como arquivo multitiff. Cada arquivo multitiff normalmente contém 3 canais de cores (azul, verde e vermelho) e 44 planos focais. </li></ol> 3. Ver tridimensional de Aquisições confocal e Rendering tridimensional Fazer upload de arquivos para o software multitiff Imaris e processá-los da seguinte forma: <ol><li>Software Open. </li><li> Selecione a vista superar. </li><li> Faça o upload do arquivo multitiff. </li><li> Selecione a cor desejada para cada canal. </li><li> Remover o ruído de fundo, aumentando o valor mínimo no painel de exibição de ajuste. Ajuste cada canal individualmente. </li><li> Vá para a vista de seção. Mover ao longo do eixo z procurando co-localização (pixels amarelos). </li><li> Clique sobre uma área de amarelo para ver se a co-localização está presente ao longo do eixo z (ou seja, pertencem a um objecto sólido). Projecções do eixo Z são visíveis na parte direita e na parte inferior da figura. </li><li> Tome um instantâneo. </li><li> Volte para superar vista. </li><li> Colheita 3D para isolar uma célula ou um grupo de células. </li><li> Selecione um canal no painel de ajuste de exibição. </li><li> Selecione o superam / ​​superfícies algoritmo construtor. Etapas algoritmo: <ol><li> Selecione canal. </li><li> Definir limite (use o mesmo valor mínimo em tele exibir o painel de ajuste). </li><li> Definir redimensionamento. </li><li> Definir suavização (melhor = 0,200). </li><li> Definir aparência de cor. </li><li> Fim algoritmo. </li></ol></li><li> Repita o passo 3,12 para cada canal. </li><li> Desmarque canais de fluorescência no painel de ajuste de exibição e selecione todas as três superfícies. </li><li> Mova o volume para encontrar a melhor vista. </li><li> Tome um instantâneo. </li></ol>

<ol>
	<li>Davalos, D., 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=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nat. Neurosci. 8, 752-758 (2005).</li><li>Yenari, M. A., Kauppinen, T. M., Swanson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+in+stroke:+therapeutic+targets.">Microglial activation in stroke: therapeutic targets.</a> Neurotherapeutics. 7, 378-391 (2010).</li><li>Iadecola, C., Anrather, 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+immunology+of+stroke:+from+mechanisms+to+translation.">The immunology of stroke: from mechanisms to translation.</a> Nat. Med. 17, 796-808 (2011).</li><li>Jin, R., Yang, G., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+in+ischemic+stroke:+role+of+inflammatory+cells.">Inflammatory mechanisms in ischemic stroke: role of inflammatory cells.</a> J. Leukoc. Biol. 87, 779-789 (2010).</li><li>Schilling, M., Besselmann, M., Muller, M., Strecker, J. K., Ringelstein, E. B., Kiefer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+phagocytic+activity+of+resident+microglia+over+hematogenous+macrophages+following+transient+focal+cerebral+ischemia:+an+investigation+using+green+fluorescent+protein+transgenic+bone+marrow+chimeric+mice.">Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice.</a> Exp. Neurol. 196, 290-297 (2005).</li><li>Perego, C., Fumagalli, S., De Simoni, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+pattern+of+expression+and+colocalization+of+microglia/macrophage+phenotype+markers+following+brain+ischemic+injury+in+mice.">Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice.</a> Journal of Neuroinflammation. 8, 174-193 (2011).</li><li>Zanier, E. 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=Human+umbilical+cord+blood+mesenchymal+stem+cells+protect+mice+brain+after+trauma.">Human umbilical cord blood mesenchymal stem cells protect mice brain after trauma.</a> Crit. CareMed. 39 (11), 2501-2510 (2011).</li><li>Capone, C., 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=Neurosphere+derived+cells+exert+a+neuroprotective+action+by+changing+the+ischemic+microenvironment.">Neurosphere derived cells exert a neuroprotective action by changing the ischemic microenvironment.</a> PLoS ONE. 2, e373 (2007).</li><li>Bhatia, S., 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=Rapid+host+defense+against+Aspergillus+fumigatus+involves+alveolar+macrophages+with+a+predominance+ofalternatively+activated+phenotype.">Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance ofalternatively activated phenotype.</a> PLoS One. 6, e15943 (2011).</li><li>Raes, G., Noel, W., Beschin, A., Brys, L., de Baetselier, P., Hassanzadeh, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FIZZ1+and+Ym+as+tools+to+discriminate+between+differentially+activated+macrophages.">FIZZ1 and Ym as tools to discriminate between differentially activated macrophages.</a> Dev. Immunol. 9, 151-159 (2002).</li><li>Gesuete, 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=Recombinant+C1+inhibitor+in+brain+ischemic+injury.">Recombinant C1 inhibitor in brain ischemic injury.</a> Ann. Neurol. 66, 332-342 (2009).</li><li>Sica, A., Mantovani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+plasticity+and+polarization:+in+vivo+veritas.">Macrophages plasticity and polarization: in vivo veritas.</a> J. Clin. Invest. 122 (3), 787-795 (2012).</li></ol>

Autores têm nada a revelar.

Apresentamos aqui protocolos baseados em imunofluorescência, seguido por análise confocal tridimensional como uma abordagem poderosa para investigar a localização e co-expressão de marcadores do fenótipo M / M para a área isquêmica (para uma análise mais detalhada, ver ref 6). Este método combina a coloração específica de marcador relevante de activação M / M e um imagem confocal tridimensional. O ajuste fino de anticorpos no soro, e fluorconjugated diluições de trabalho permite melhor sinal-...

Após lesão cerebral aguda, microglia são rapidamente ativadas e passam por morfológica dramática e fenotípica muda 1-3. Esta resposta intrínseca está associada ao recrutamento de macrófagos nascido de sangue que migram para o parênquima cerebral 4,5 feridos. O papel da microglia e macrófagos que não são antigenicamente distintos (doravante referido como M / M) em lesão cerebral ainda é debatido. Um número crescente de estudos indicam que, à semelhança do que descrito para macrófagos periféricos, microglia e cérebro recrutados macrófagos pode assumir diferentes fenótipos cujos extremos correspondem ao clássico tóxico pró-inflamatória (M1) ou de protecção (M2) fenótipo anti-inflamatório. Os diferentes estados de activação, incluindo a secreção de factores pro-ou anti-inflamatórios, a libertação de moléculas neurotróficas e actividade lisossomal são caracterizadas por um padrão específico de marcadores fenotípicos, cuja expressão depende da temporaisevolução do ambiente circundante. A caracterização destes fenótipos M / M no cérebro feridos ainda é escasso. Nós usamos um modelo murino bem estabelecida de pMCAo para analisar a expressão M / M e evolução após o AVC. Protocolos de imunofluorescência base aqui apresentados visam a obtenção de uma visão sobre o aparecimento de marcadores específicos M / M fenótipo, sua localização e co-expressão celular na área isquêmica. Nós investigamos algumas moléculas associadas a diferentes estado de ativação ou fenótipo, ou seja, CD11b, um marcador de superfície expressa por leucócitos e um marcador amplamente utilizado de M / M ativação / recrutamento 6-8, CD68 um marcador de lisossomos 6,7 e Ym1 uma secreção proteína expressa pelo alternativamente ativados (M2) e macrófagos associados a recuperação e restauração da função 9-10. Quando dois marcadores são expressos pela mesma célula, mas em diferentes compartimentos subcelulares, colocalização si só pode não ser tanto informative. Neste caso, a análise de co-expressão pode ser realizada utilizando único plano e vista por representações tridimensionais. Nós aqui descrevemos um protocolo para obter uma análise do marcador co-expressão completa tridimensional.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD11b</td>
			<td>Kindly provided by Dr. A. Doni, Istituto Clinico Humanitas, Milan, Italy</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rat Anti-mouse CD68 </td>
			<td>AbD Serotec</td>
			<td>MCA 1957</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rabbit Anti-mouse Ym1 </td>
			<td>Stem Cell Technologies</td>
			<td>1404</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hoechst 33342</td>
			<td>Life technologies</td>
			<td>H21492</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse Anti-Neural Nuclei (NeuN)</td>
			<td>CHEMICON</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biotinilated Goat Anti-Rat antibody</td>
			<td>Jackson Immuno Research</td>
			<td>112-065-143</td>
			<td></td>
		 </tr>
		 <tr>
			<td>TSA Cyanine 5 System </td>
			<td>Perkin Elmer</td>
			<td>NEL705A001KT</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Prolong Gold </td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rat alexa 546</td>
			<td>Invitrogen</td>
			<td>A-11081</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse Alexa 488</td>
			<td>Invitrogen</td>
			<td>A-21121</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti-rabbit Alexa 594</td>
			<td>Invitrogen</td>
			<td>A-11037</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Normal Goat Serum</td>
			<td>Vectors Laboratories</td>
			<td>S-1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tritin X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma</td>
			<td>P4417-100</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Cryostat CM1850</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Olympus IX81 confocal microscope </td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>AnalySIS software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaris software 5.0</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Photoshop cs2</td>
			<td>Adobe Systems</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software packages GraphPad Prism version 4.0</td>
			<td>GraphPad Software Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

three-dimensional confocal analysis of microglia/macrophage markers of polarization in experimental brain injury

Depois de acidente vascular cerebral microglia / macrófagos (M / M) sofrer ativação rápida, com alterações morfológicas e fenotípicas dramáticas que incluem expressão de antígenos de superfície novas e produção de mediadores que se formam e mantêm a resposta inflamatória. Algumas evidências indicam que M / M são células altamente plástico que pode assumir anti-inflamatória (M2) de activação pró-inflamatória (M1) ou alternativo clássico após lesão cerebral aguda. No entanto, uma caracterização completa de M / M expressão do marcador fenótipo, sua co-localização e evolução temporal no cérebro ferido ainda está desaparecida. Protocolos de coloração por imunofluorescência especificamente marcadores relevantes de activação M / M pode ser realizada no cérebro isquémico. Apresentamos aqui os protocolos baseados em imunofluorescência, seguido por análise confocal tridimensional como uma abordagem poderosa para investigar o padrão de localização e de co-expressão de marcadores fenotípicos M / M, comoCD11b, CD68, Ym1, no modelo de rato de isquemia focal induzida por oclusão permanente da artéria cerebral média (pMCAO). Análise bidimensional da área manchada revela que cada marcador é associado a uma morfologia M / M definido e tem uma dada localização na lesão isquémica. Padrões de M / M fenótipo marcador co-expressão pode ser avaliada por imagem confocal tridimensional na área isquémica. As imagens podem ser adquiridos ao longo de um volume definido (10 m z-eixo e um tamanho de 0,23 mM passo, o que corresponde a um volume de 180 x 135 x 10 mm) com um modo de varrimento sequencial para minimizar os efeitos de passagem de purga e de evitar a sobreposição de comprimento de onda. As imagens são, então, processado para obter representações tridimensionais, por meio de software Imaris. Visão sólida de três representações tridimensionais permite a definição de expressão do marcador em grupos de células. Nós mostramos que M / M têm a capacidade de se diferenciar para uma multiplicidade de fenótipos, dependendo da localização do local da lesão e time após a lesão.

Um exemplo dos resultados obtidos quando os protocolos de rotulagem e aquisições confocal são realizadas na região isquémica é ilustrado nas Figuras 1A e 1B. Uma vista bidimensional de imagens adquiridas mostra que, em vinte e quatro horas após a isquemia (D), o marcador CD68 lisossomal (verde) é expresso em células hipertróficas amebóides CD11b (vermelho) presentes no núcleo isquémico. Na zona de fronteira células positivas (B) CD11b exib...

Watch this Scientific Journal Video about Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury at JoVE.com

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

Uma maneira de ganhar novos insights sobre a complexidade da resposta inflamatória do cérebro é apresentado. Descrevemos protocolos baseados em imunofluorescência, seguido por análise confocal tridimensional para investigar o padrão de co-expressão de marcadores fenotípicos microglia / macrófagos em um modelo de rato de isquemia focal.

Tridimensional Confocal análise de marcadores Microglia / macrófagos de polarização em Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

three-dimensional-confocal-analysis-microgliamacrophage-markers

Research

JoVE Journal

Neuroscience

11.5K visualizações.  IRCCS - Istituto di Ricerche Farmacologiche Mario Negri.  Uma maneira de ganhar novos insights sobre a complexidade da resposta inflamatória do cérebro é apresentado. Descrevemos protocolos baseados em imunofluorescência, seguido por análise confocal tridimensional para investigar o padrão de co-expressão de marcadores fenotípicos microglia / macrófagos em um modelo de rato de isquemia focal. 

Vídeo: Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

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

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

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

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

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

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

Neurociência

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

Protocolo para tridimensional confocal morfométrica Análise de astrócitos

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

Imagem tridimensional e análise de mitocôndrias dentro as fibras nervosas intraepidérmicas humana

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Redes neuronais modificadas por micro-tecidos tridimensionais com inspiração anatômica para a reconstrução, modulação e modelagem do sistema nervoso

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

Análise de marcha automatizados em ratos com lesão crônica de constrição

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

Imagem tridimensional em grande escala de organização celular no neocórtex Mouse

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Crônica implantação de matriz Electrocorticographic todo-cortical no sagui comum

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Modelagem e análise tridimensional da forma de estruturas do cérebro

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

Geração organoide do nervo motor tridimensional

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Modelo de Lesão Cerebral Traumática Leve em Camundongos com Controle Eletromagnético

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...