Esta pesquisa foi apoiada pelo INSERM, da Universidade de Aix-Marseille e um prêmio de Desenvolvimento de Carreira de HFSPO obtido por CL Forestier.

Declaração de ética: Todos os estudos com animais foram realizados de acordo com as diretrizes e protocolos que foram aprovados pelo Comitê de Cuidado e Uso da Université Aix-Marseille, França animal Institucional. Feminino camundongos C57BL / 6 a 8 - 10 semanas de idade foram comercialmente obtida e manipulada de acordo com as regras do Décret N ° 8 ° 87-848, 19 de outubro, 1987, Paris. Todos os experimentos usando L. donovani parasitas LD1S foram conduzidos de acordo com as normas de biossegurança da legislação francesa e da União Europeia. 1. Rato infecção com L. donovani promastigota Parasitas <ol><li> Prepara-se o meio de cultura para manutenção in vitro de L. donovani parasitas promastigotas, LD1S (MHOM / SD / 62 / 1S-CL2D). <ol><li> Use meio M199. Suplemento de meio M199 com 10% de soro fetal de vitela (inactivado pelo calor a 56 ° C durante 30 min), 25 mM de HEPES pH 6,9, 12 mM de NaHCO 3, 1 mM de glutamina, RPMI 16040 mistura de vitaminas, 10 mM de ácido fólico, 100 mM de adenosina, hemina 7,6 mM, 50 U / ml de penicilina e 50 mg / ml de estreptomicina. </li></ol></li><li> Cultura LD1S parasitas em 10 ml de meio M199 completo a 26 ° C num balão ventilado 25cm 2. </li><li> Usando sucessivos passos de centrifugação diferencial, preparar uma população de parasitas promastigotas que são enriquecidos em formas metacíclicas. <ol><li> Colher um mid-log cultura parasita fase. Conte com uma hemocytomer. Ressuspender 5 x 10 5 parasitas / ml em 10 ml de meio M199 completo. Crescer a cultura parasita em condições anaeróbias até atingirem a fase estacionária tardia (em cerca de 5-6 dias). </li><li> Girar a cultura LD1S a 1.200 xg durante 10 min a 26 ° C para sedimentar os parasitas não metacíclicos, recuperar o sobrenadante e girá-lo de novo a 2500 xg a 26 ° C durante 10 min. Nota: Este sedimento final é altamente enriquecido em parasitas promastigotas metacíclicos. </li><li> Ressuspender o sedimento em 100 ul de PBS. Retirar uma pequena alíquota para fixação com 25% de glutaraldeído 1/100 v: v e contar parasitas utilizando um hemocitómetro. </li></ol></li><li> Dilui-se a população de parasitas enriquecido-metacíclica a 1 x 10 7/100 ul em PBS. Injectar o parasita suspensão na veia da cauda de um rato anestesiado (veja abaixo), utilizando uma seringa de 1 ml com uma agulha de 30 G. Para os animais de controlo, injectar 100 ul de PBS na veia da cauda. </li></ol> 2. Procedimentos Cirúrgicos <ol><li> Desinfectar o espaço de trabalho com um spray de desinfectante e biocida todos os instrumentos cirúrgicos com etanol a 70% durante 30 min. </li><li> Prepara-se uma solução de anestésico, de acordo com o peso do animal, que contém 125 mg / kg de cetamina e 12,5 mg / kg de xilazina diluído em PBS. </li><li> Pesa-se o rato e intraperitoneal injectar a dose apropriada de anestésico usando uma seringa de 1 ml com uma agulha G 30. </li><li> Mantenha o mouse quente e verifique se o mouse é completamente anestesiaTized por beliscar a almofada do pé antes de prosseguir. Re-injetar uma meia dose da solução anestésica no rato a cada 60 min. </li><li> Raspar o abdome do rato e limpe-o com Vetedine. Fazer uma pequena incisão na pele e musculatura sob a caixa torácica, no lado esquerdo do abdómen. </li><li> Coloque um pedaço de gaze úmida no abdômen logo abaixo da área aberta. Expor o fígado e coloque-o sobre a gaze. Coloque outro pedaço de gaze úmida sobre o fígado. </li><li> Inserir um 24 x 60 mm2 a 150 um de espessura lamela, para uma estrutura de metal, sobre o fígado para visualização sob um microscópio ligado-cianoacrilato. </li><li> Proteger os olhos do rato com uma gaze molhada. </li><li> Após a sessão de imagiologia, o animal anestesiado eutanásia por deslocação cervical. </li></ol> 3. Microscopia intravital Imagem da Arquitetura fígado <ol><li> Realizar os experimentos de microscopia intravital em um microscópio confocal invertido equipado com pelocâmara, uma objectiva de imersão 63X glicerol óleo apochromatic (NA 1.4), argônio (488 nm) e HeNe (543 nm, 633 nm) lasers e um diodo azul (405 nm) hermostatic controlada. </li><li> Posicione o mouse no palco do microscópio com a lamela cobrindo o rosto fígado para baixo no objetivo. Ajustar a temperatura da câmara para 29 ° C. Ajuste o foco com o autofluorescência fígado para permitir a visualização dos sinusóides. </li><li> Para visualizar a vascularização, preparar uma solução de 500 ug de BSA-Alexa 647 diluído em 100 ul de PBS, para cada ratinho. Injectar esta solução por via intravenosa na veia da cauda do ratinho anestesiado utilizando uma seringa de 1 ml com uma agulha de 30 G. </li><li> Para visualizar os núcleos de células do fígado, preparar uma solução de Hoechst 33342 em PBS. Injectar esta solução a 8 mg / kg de rato intraperitonealmente utilizando uma seringa de 1 ml com uma agulha G 30. </li><li> Use o modo seqüencial normal, a objectiva de imersão 63X eo scanner a 400 Hz para a imagem da hepáticaos núcleos das células e a vasculatura hepática. Ligue a 405 nm diodo azul para Hoechst eo laser de excitação 633 nm para BSA-Alexa 647 excitação. Ligue o laser nm Argon 488 e definir uma banda grande aquisição para visualizar a autofluorescência fígado. </li></ol> 4. Microscopia intravital Imagem do Fígado para medição do fluxo sanguíneo velocidade <ol><li> Abra o software LAS do microscópio (LAS-AF espectador Versão 3.1.0 Build 8587). Selecione o modo de scanner de ressonância ao abrir o software microscópio. </li><li> Clique na guia de configuração para definir o hardware. Clique em laser e selecione o laser HeNe 633. Clique em Configurações e selecione 12 bits de resolução. </li><li> Clique no menu de aquisição. Na janela de configurações via o feixe, selecione o 63X objetiva e configurar a potência do laser 633 a 100%. Ative o ganho de sinal e desligar parâmetros definidos selecionando Alexa-633 a partir do menu drop-down PM1-3. </li><li> Na guia adquirem, selecione a aquisição &#39;XYT&#39;modo. Definir a largura em formato de 1024 x 1024. Selecione a velocidade a 8.000 Hz. Selecione o pinhole de 3 unidades Airy. Selecione um fator de zoom de 3. Não selecione o modo bidirecional. </li></ol> 5. Análise Quantitativa de RBC Velocidade Usando as Imagens XYT (Método 1) <ol><li> Clique no ícone &#39;Live&#39; para visualizar o fluxo de sangue em um modo de viver. Escolha da área de interesse e seleccionar um vaso sanguíneo específico para análise. Defina o vaso de interesse para a posição horizontal, ajustando o &quot;X&quot;, &quot;Y&quot; e digitalização de rotação de campo coordenadas no painel de controle USB. </li><li> Pare o modo &#39;Live&#39; uma vez que o vaso de interesse está definido. Mudar a largura do conjunto formato para 1024 x 256 no modo de aquisição janela, linha média = 1, quadro médio = 1 configuração, selecione &quot;empilha&quot; 150. Clique em &#39;start&#39; para adquirir imagens. </li><li> Para a análise quantitativa da velocidade RBC usando as imagens &quot;XYT&quot; abrir o software ImageJ. Selecione a &#39;File&#39;guia em ImageJ, clique em &quot;Abrir&quot; e escolha o arquivo de interesse. </li><li> Vá para o menu &quot;plugin&quot; em ImageJ, selecione LOCI e Bio-importar formatos de opções. Nesta janela, selecione a pilha visualizar parâmetros como &#39;hyperstack&#39;. Na opção ordem da pilha, selecione a opção: &quot;xyzct&quot;. Nota: Isso abre uma janela com toda a série de aquisições. </li><li> Clique em &#39;Plugins&#39; no menu e selecione a opção ImageJ MTrackJ. Clique no ícone &quot;Adicionar&quot; na pequena janela e clique no RBC para acompanhar. Clique no mesmo RBC em cada seguinte quadro e clique no ícone «medida» para medir a velocidade. Nota: O arquivo gerado pode ser salvo no formato .exl. </li><li> Acompanhe um mínimo de cinco hemácias por navio e analisar um mínimo de três navios individuais do mesmo tamanho. </li></ol> 6. Análise Quantitativa de RBC Velocity usando a linha de xt Imagens (Método 2) <ol><li> Clique em &#39;Live&#39; para visualizar o sangue fbaixo no modo ao vivo. Selecione um vaso sanguíneo específico para análise. Definir o vaso de interesse para a posição horizontal. Pare o modo &#39;Live&#39; uma vez que o vaso de interesse está definido. </li><li> Selecione o modo de digitalização &quot;xt&quot; e defina o lúmen central do navio selecionado ao longo da linha de varredura. Set largura em formato 1.024 x 512, velocidade = 8.000Hz, linha média = 32, o tempo = 512. Clique em &#39;start&#39; e adquirir as imagens de linha xt. </li><li> Gerar raias de análise quantitativa de velocidade RBC abrindo o arquivo .lif ea imagem linha xt de interesse abertura. Em software LAS-AF, selecione &quot;experimentos&quot;, abra o .lif e selecione a imagem. Nota: Ele vai abrir automaticamente um quimógrafo. </li><li> Medir o tempo (t, obtido no eixo Y) necessária para um traço de partículas (que mostra a reflexão escuro) para percorrer uma determinada distância (d, obtido no eixo X) nesta quimógrafo. Selecione a ferramenta &quot;desenhar linha&quot; e desenhar uma linha horizontal para a raia. Note-se a distância emum e o tempo em segundos gerados em resultado da aba na parte inferior da imagem. </li><li> Calcular a velocidade como V = distância / tempo usando os valores obtidos a partir da quimógrafo. </li><li> Quantificar um mínimo de cinco partículas raias imagem xt e por um mínimo de três navios individuais do mesmo tamanho. </li></ol>

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The authors have nothing to disclose.

O desenvolvimento recente de microscopia intravital do fígado do rato abre novas possibilidades para a investigação da resposta fisiológica à infecção in vivo e em tempo real, 5,9,10. Fluxo sanguíneo de órgãos é um parâmetro fisiológico crítico que é frequentemente alterados em muitas doenças. No entanto, o estado da hemodinâmica hepáticas, sob condições fisiológicas e infecciosas continua a ser uma área pouco explorado. Neste estudo, os métodos baseados em IVM, que foram previ...

Hemodinâmica específicos de órgãos são características fisiológicas importantes de qualquer órgão de mamíferos. Anormalidades no fluxo sanguíneo pode ser a consequência de inflamação e um sinal de disfunção de órgãos 1. Assim, o fluxo de sangue organização, estrutura e função de parâmetros aparecem como críticos para análise sob condições fisiológicas e patológicas. As técnicas que têm sido normalmente utilizadas para a análise de fluxo sanguíneo em um órgão específico contêm várias limitações, incluindo o limite de resolução da técnica em si (por exemplo, o Doppler do fluxo de sangue), a capacidade para a medição de fluxo sanguíneo absoluto única (volume de sangue por unidade que serve um órgão) (por exemplo, a tomografia de coerência óptica) ea medição de mudanças médias em velocidade em uma população grande e heterogêneo de vasos sangüíneos 2,3. Sistema circulatório do fígado liga diferentes subtipos de vasos que são heterogêneos em seu tamanho, estrutura e função. Dentroesta tecnologia de imagem estudo, microscopia intravital (IVM) é aplicado para avaliar a hemodinâmica do fígado in vivo, em tempo real, com elevada resolução e em paralelo para descobrir as características dos vasos sanguíneos individuais que formam o órgão hepático. O recente desenvolvimento desta técnica de imagem óptico potente permite ao pesquisador para coletar dados dinâmicos em animais vivendo em uma resolução espacial e temporal alta. Ao permitir a visualização direta e acompanhamento dos processos biológicos específicos e rápidos in vivo em tempo real, IVM fornece uma oportunidade única para que o pesquisador vasos sanguíneos imagem individual e medir e quantificar a velocidade de glóbulos vermelhos de solteiro (RBC) dentro de um especificamente selecionados embarcação hepática. Neste estudo, nós implementamos a técnica de IVM no fígado do rato para investigar a influência da infecção do mouse pelo parasita Leishmania hepatotrópico sobre a hemodinâmica do fígado. L. donovanié o agente responsável pela leishmaniose visceral, uma doença grave caracterizada por respostas inflamatórias agudas-em-uma patologia crónica e que está presente em vários órgãos, incluindo o baço e o fígado. Num modelo de rato experimental de leishmaniose visceral, a infecção do fígado é auto-resolução enquanto que a infecção do baço é progressiva 4. Estes resultados de infecção por Leishmania em relação aos órgãos individuais são ainda não completamente compreendidas. Investigação de hemodinâmica do fígado e baço em condições patológicas vai lançar uma nova luz sobre as interacções parasita-hospedeiro e patogênese da doença. Nosso sistema modelo experimental é baseado em expor e imagiologia do fígado de um rato anestesiado que receberam injeção intravenosa de corantes fluorescentes específicos para a rotulagem do intravasculature hepática. O fígado é um órgão favorável para microscopia intra-vital. Depois de realizar uma pequena incision no abdômen, o fígado é suavemente exteriorizada e colocada sobre uma gaze molhada, em seguida, em uma lamela com o objetivo de reduzir quaisquer artefatos de movimento devido à pulsação e respiração. O fígado é então colocado dentro da visão de uma lente do microscópio. Em comparação com o baço e nó de linfa, que exigem a utilização de dois fotões para estudos de microscopia de IVM, a vantagem de o fígado reside na sua arquitectura homogénea 3D / anatomia, que permite o uso de um microscópio confocal convencional, com uma profundidade de penetração máxima de aproximadamente 50 mm, para microscopia intravital imagiologia 5-8. Este estudo descreve dois métodos de imagem independentes para a medição quantitativa da RBC velocidade e velocidade do fluxo sanguíneo nos vasos sanguíneos individuais. No primeiro método, o fluxo de sangue no fígado é adquirida utilizando um modo de bi-dimensional xy ao longo do tempo. Os dados XYT resultantes são analisados ​​usando o plugin MtrackJ no software livre ImageJ, que permite o rastreamento de individual hemácias ao longo do tempo. No segundo método, um único vaso sanguíneo é seleccionado e a sua correspondente fluxo de sangue é analisada utilizando o modo de varrimento de linha rápida aquisição do microscópio confocal de varredura a laser. O vaso de interesse é digitalizada de alta frequência ao longo do seu eixo central através de uma linha axial. A velocidade do fluxo de sangue é então quantificada com base na diferença de contraste entre eritrócitos não marcados escuras e plasma marcado por fluorescência. As intensidades de fluorescência de glóbulos vermelhos e plasma adquiridos ao longo da linha de varrimento são representados graficamente contra o tempo para obter estrias, os ângulos dos quais são proporcionais às velocidades de um RBC indivíduo. O objetivo deste artigo é fornecer um método simples e reprodutível para a imagem latente e medir a velocidade do fluxo de sangue dentro dos vasos sanguíneos individuais do fígado e disponibilizar as ferramentas básicas para a realização bem-sucedida da cirurgia rato, IVM e análises quantitativas da velocidade de glóbulos vermelhos individuais. Tsua abordagem permitirá que os investigadores para ganhar novos insights sobre velocidade do sangue em condições patológicas.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-Alexa 647</td>
			<td>lifetechnologies</td>
			<td>A34785</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-FITC 500 mol wt</td>
			<td>SIGMA</td>
			<td>46947</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>PanPharma</td>
			<td>20434</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>KP07KEU</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetedine</td>
			<td>Pharma Animal</td>
			<td>6869029</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate liquid</td>
			<td>Cyanolit</td>
			<td>5833300005</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip frame: Membrane slide for microdissection part N&#176;: 5013</td>
			<td>Molecular machines</td>
			<td>50103</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip DiaPath 24x60 ep: 1.6 mm</td>
			<td>DiaPath</td>
			<td>61061</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica&#160;</td>
			<td>TCS-SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS-AF viewer&#160;</td>
			<td>Leica software</td>
			<td>Version 3.1.0 buid 8587</td>
			<td></td>
		</tr>
	</tbody>
</table>

intravital microscopy imaging of the liver following leishmania infection: an assessment of hepatic hemodynamics

Microscopia intravital (IVM) é uma técnica de imageamento óptico poderoso que tornou possível a visualização, monitoramento e quantificação de vários eventos biológicos em tempo real e em animais vivos. Esta tecnologia tem avançado muito a nossa compreensão de processos fisiológicos e fenômenos mediada por patógenos em órgãos específicos. Neste estudo, IVM é aplicada para o fígado do rato e protocolos foram concebidos para a imagem in vivo, o sistema circulatório do fígado e medir glóbulos vermelhos do sangue (RBC) velocidade em vasos hepáticos individuais. Para visualizar os diferentes subtipos dos vasos que caracterizam o órgão hepático e realizar medições da velocidade do fluxo sanguíneo, ratinhos C57BL / 6 são injectados por via intravenosa com um reagente fluorescente de plasma que rotula a vasculatura associada a fígado. IVM permite in vivo, em tempo real, a medição da velocidade de RBC em um vaso de interesse específica. Estabelecer esta metodologia tornará possívelinvestigar a hemodinâmica do fígado, em condições fisiológicas e patológicas. Em última análise, esta metodologia baseada na imagem será importante para o estudo da influência de L. infecção donovani na hemodinâmica hepáticas. Este método pode ser aplicado a outros modelos infecciosas e órgãos de rato e pode ainda ser alargado para ensaios pré-clínicos do efeito de uma droga sobre a inflamação através da quantificação do seu efeito sobre o fluxo sanguíneo.

A organização arquitectónico específico dos sinusóides do fígado pode ser visualizado com base na propriedade autofluorescente deste órgão (Figura 1, painel B e C, verde), a injecção intraperitoneal de Hoechst para a rotulagem dos núcleos de hepatócitos (Figura 1B, azul) e a injecção intravenosa de BSA fluorescente para a coloração do sistema circulatório hepática (Figura 1C, vermelho). O fígado é composto por vários subtipos dos vasos diferentes co...

Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Microscopia intravital Imagem do Fígado seguinte<em&gt; Leishmania</em&gt; Infecção: Uma Avaliação de hepáticas Hemodinâmica

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

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JoVE Journal

Immunology and Infection

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

11.4K visualizações.  University of Aix-Marseille. This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Vídeo: Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

In vivo de imagens de parasitas Leishmania transgênicas em um host ao vivo

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Imunologia e Infecção

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Microscopia intravital do linfonodo inguinal

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagem intravital da Thymus rato usando 2-Photon Microscopia

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Microscopia intravital do Baço: Análise Quantitativa da Mobilidade Parasite e do fluxo sanguíneo

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imagem intravital do Linfonodo Popliteal Rato

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Altamente Resolvido Microscopia intravital Striped-iluminação dos centros germinativos

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Um fluxo de Adesão Ensaio para o Estudo de Recrutamento de leucócitos para hepáticas humanas senoidais endotélio sob condições de estresse de cisalhamento

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Long Term Intravital Multiphoton Microscopia de imagem de células imunes no saudáveis ​​e doentes do fígado por meio CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Imagem de Intravital dois fotões de leucócitos durante a resposta imune em fígado de rato lipopolissacarídeo-tratados

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Interação de célula de imagem latente na Mucosa traqueal durante a infecção de vírus de Influenza usando microscopia Intravital dois fotões

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...