Este trabalho foi financiado pelo R01 AI32556 (NIAID, National Institute of Health). Este trabalho também foi apoiado em parte pela Louisiana Vaccine Center e Louisiana do Sul Instituto de Pesquisas de Doenças Infecciosas patrocinado pelo Conselho de Regentes Louisiana.

1. Inoculação vaginal com Candida albicans <ol><li> Três dias antes da inoculação, enquanto prender o animal para expor o abdomen, injetar 100 ml de óleo de gergelim contendo 0,1-0,5 mg de β-estradiol por via subcutânea na parte inferior do abdômen. Antecedência a agulha cerca de 5 a 10 mm lateral à pele para minimizar a perda no local da injeção. A administração subcutânea de estrogênio no abdômen inferior é ideal neste modelo devido à proximidade com o trato genital. Doses eficazes podem variar de acordo com linhagens de camundongos, idades ou derivados de estrógeno. Em estudos anteriores usando CBA-J (H-2 κ), C3H/HeN (H-2 κ), C57BL / 6 (H-2 b), Balb / c (H-2 d), DBA / 2 (H- 2 d), SJL (H-2 s) ratos em 6-8 semanas de idade, 0,1 mL mg/100 foi encontrado eficaz evidenciado pelo espessamento da parede vaginal, diminuição do muco vaginal eaumento da descamação das células epiteliais. Camundongos tratados com estrógeno acima nesta concentração exibem a colonização vaginal consistentes com Candida. Para inoculação em camundongos, de outras cepas e idades, um estudo piloto é recomendado para garantir a eficácia do estrogênio nas condições modificadas e aumentar a dose de estrogênio, se necessário. A solução de estrogênio deve ser preparado de cada vez no dia da injeção. Para garantir a solubilidade total de estrogênio no óleo de gergelim, misturar a solução com um vortex e calor de forma intermitente a 37 ° C. Repetir a injecção semanal durante todo o período do estudo. </li><li> Para preparar o inóculo, adicione um loopful de C. albicans blastoconidia de uma preparação subcultura recentes Sabouraud-dextrose agar (SDA) em 10 ml de caldo de Phytone-peptona suplementado com glicose 0,1%. Incubar a cultura caldo de fase estacionária por 18 horas a 25 ° C em banho-maria a tremer. </li><li> Após a incubação, recolher ªe cultura caldo em um tubo cônico de 15 ml e centrifugar a 800 xg por 5 min. Lave o pellet duas vezes com PBS estéril. </li><li> Enumerar blastoconidia viável em um hemocitômetro por exclusão corante azul de tripano. Ajustar a concentração celular de 2,5 x 10 6 ml / (ou a uma concentração de inóculo desejado) em PBS estéril. </li><li> Para estabilizar o mouse, mantenha a base da cauda com dois dedos e levante o quadril para cima de modo que a abertura vaginal faces em sua direção (Figura 1A). O ideal é que o rato é colocado sobre uma superfície plana ralado (por exemplo, topo de gaiola) para que o mouse pode proporcionar resistência contra a restrição da cauda. </li><li> Pipetar 20 mL (ou um volume desejado para não exceder 20 l) da suspensão de inóculo, inserindo a ponta da pipeta cerca de 5 mm de profundidade para dentro do lúmen vaginal (Figura 1B). Concluir esta etapa mais rápida e suavemente possível para minimizar o sofrimento do mouse. </li></ol> 2. Lavagens vaginais <ol><li> Eutanásia seguinte (ou anestesia), ho velho rato para baixo pela base da cauda com dois dedos de modo que a abertura vaginal fica exposta. </li><li> A lavagem do lúmen vaginal através da introdução de 100 ul de PBS estéril com aspiração repetida e agitação com uma ponteira. A ponta da pipeta pode ficar entupido com as células. Se isso ocorrer, dispensar as células e continuar obstruindo lavaging com PBS restante na vagina. Recolher o lavado em um tubo de microcentrífuga. </li><li> Em alternativa, lavagem vaginal pode ser realizado em ratos anestesiados com anestesia inalatória de isoflurano. Para isso, expor os ratos para vaporizado isoflurano até que estejam completamente sedados (~ 30 seg.). Segure os ratos para baixo pela base da cauda e gentilmente lavagem do lúmen vaginal utilizando 50 mL de PBS estéril. Certifique-se evitar a agitação dura com uma ponteira para minimizar o trauma da vagina durante este procedimento. Camundongos sedado deve se recuperar da anestesia dentro de 30 segundos de exposição ao ar ambiente. Isofluranopode ser vaporizado utilizando um vaporizador de isoflurano e O 2 (preferencial) ou um sistema de queda norma fechada câmara de anestésico sem que o sistema vaporizador (requer acompanhamento de perto do animal, enquanto sedado para evitar problemas respiratórios). Lavagens vaginais em ratos anestesiados deve ser o método de escolha para amostras de lavagem longitudinal sobre os ratos mesmo. Lavaging consecutivas não influencia a avaliação da carga de fungos ao longo do tempo 41. </li><li> Para wet-mount preparações, a transferência de 10 ml do lavado numa lâmina de vidro e observar com ampliação de 1000x 400 por microscopia de luz. Além disso, as frações celulares do lavado pode ser manchado para examinar células e morfologias nuclear. Para as preparações de esfregaço, de transferência de 10 ml do lavado numa lâmina de vidro e gentilmente se espalhar usando a parede exterior de uma ponta da pipeta. Preservar as amostras de esfregaço com fixador CytoPrep e mancha pela técnica de Papanicolaou padrão (exame de Papanicolaou). Observe a 400 × ampliação por microscopia de luz. </li></ol> 3. Quantificação da carga fúngica vaginal <ol><li> Em uma placa de 96 poços de fundo redondo, transferir o lavado em um poço da linha superior e 180 mL de PBS estéril nas seguintes 5 poços dessa coluna (para baixo da placa). </li><li> Faça 01:10 diluições em série de lavado vaginal, transferindo a 20 mL do fluido para o bem próxima na coluna. Misturar bem por aspiração repetida antes de cada transferência. Diluições em série de até 12 amostras de lavagem (uma linha horizontal completa) podem ser realizadas simultaneamente, utilizando uma pipeta de 12 canais. </li><li> Começando com a menor diluição, transferir 10 ml da amostra em Sabouraud-dextrose agar (SDA). Chapeamento de até 36 amostras podem ser realizadas em uma placa usando SDA preparada em pratos de petri com grade quadrada e uma pipeta multicanal ajustável espaçamento. </li><li> Enumerar unidades formadoras de colônia (UFC), após incubação a 34 ° C fou 48 h. </li></ol> 4. Extração de tecido vaginal <ol><li> Seguindo o procedimento vaginal lavagem, coloque o mouse sacrificados em suas costas e saturar a área da virilha com etanol 70%. Usando um par de fórceps, levante o orifício urinário para cima de modo que a abertura vaginal fica exposta. </li><li> Inserir um par de fórceps curvado para dentro do lúmen vaginal e localizar o colo do útero. Mantendo uma pegada firme com o fórceps, extrair o colo do útero através da cavidade vaginal (Figura 2A-C). </li><li> Vagina da Excise na base da abertura vaginal e, em seguida, remover o colo da vagina com uma tesoura cirúrgica. Tenha em mente que o tecido vaginal é involuído (interior epitélio lado da vagina é exposta para fora). O tecido pode ser invertida lateralmente para manter a orientação original da vagina ou aberto em uma folha, fazendo uma incisão lateral. Excisadas tecidos vaginais pode ser usado para: 1) colagem após a extração de linfócitosnase digestão (~ 1 × 10 4 mouse /) 40, 2) das células epiteliais isolamento digestão dispase seguinte (~ 5 do mouse × 10 4 /) 28, 3) preparações congeladas ou parafina para análise histológica 25. </li></ol> 5. Excisão do nó de linfa lombar <ol><li> Seguindo o procedimento vaginal lavagem, coloque o mouse sacrificados em suas costas e saturar o abdômen com etanol 70%. Faça uma incisão lateral a partir da parte inferior do abdome para o tórax e expor os órgãos internos. Usando um par de fórceps em ambas as mãos, mover os intestinos para cima de modo que os vasos sanguíneos centrais tornam-se visíveis. </li><li> Localizar a veia cava inferior e aorta abdominal. Normalmente, um par de gânglios linfáticos lombares podem ser identificadas adjacentes à aorta abdominal, localizado a meio caminho entre a origem das artérias ilíacas comuns renal e 39. Estes gânglios linfáticos podem ser visualmente diferenciado de tecido adiposo pela textura elásticae são mais leves e mais opaca na cor em relação ao tecido gorduroso (Figura 3). Estes linfonodos são visivelmente maior nos animais infectados em comparação com animais não inoculadas. </li><li> Especiais de consumo os gânglios linfáticos, colocando micropinças sob o nó e, em seguida, puxar para cima com cuidado para separar tecido circundante. </li></ol> 6. Isolamento de células linfóides em uma única célula suspensões <ol><li> Transferência dos gânglios linfáticos em uma tela de arame de malha estéril (aproximadamente 3 x 3 cm 2 de tamanho) colocado dentro de um prato de vidro estéril petri contendo ~ 10 ml de solução de Hanks salina balanceada (HBSS) (Figura 4). </li><li> Com a placa de Petri ligeiramente inclinado, pressione os linfonodos contra a tela com uma cabeça de êmbolo da seringa. Certifique-se de quebrar todos os linfonodos para que o conteúdo celular de nós passar pela tela enquanto os componentes não-celulares dos gânglios (ou seja, membranas, estroma, tecido adiposo) permanecem na tela. </li><li> Usando o êmbolo e seringa mesmo, aspirado tele HBSS contendo células. Lavar a tela com HBSS ~ 5 ml e recolher o líquido restante em um tubo de 15 ml. </li><li> Centrifugar a 800 xg por 10 min. Aspirar qualquer depósitos de gordura na parte superior do líquido com uma pipeta antes de descartar o líquido. Lave o pellet celular três vezes com HBSS. Ressuspender o sedimento em 1 ml de HBSS e enumerar células viáveis ​​por exclusão corante azul de tripano. </li></ol> 7. Resultados representativos: As frações celulares do lavado vaginal de&gt; 4 dias de camundongos inoculados tipicamente consistem de Candida, células epiteliais e infiltração celular (Figura 5). Por wet-mount microscopia, Candida podem ser identificados visualmente pela presença de hifas, bem como levedura (Figura 5A). Preparados esfregaços de lavado vaginal pode ser manchado pela técnica de Papanicolaou para examinar células epiteliais e leucócitos infiltrantes, das quais as principais células são neutrófilos identificado pelo tri-nuclelobos ar (Figura 5B). Neutrófilos muito poucos, se houver, são detectados em ratos não inoculadas (Figura 5C) 41. Um exemplo de carga fúngica vaginal é mostrado na Figura 6. Lavado vaginal coletadas em momentos específicos são cultivadas por CFU enumeração (Figura 6A). Colonização vaginal / infecção por Candida persiste por semanas em estrogênio tratados com camundongos inoculados (Figura 6B), enquanto Candida não consegue estabelecer a colonização vaginal não-tratadas com estrogênio camundongos inoculados (Figura 6C). Camundongos tratados com estrógeno não inoculadas permanecem negativos para Candida ao longo do tempo (dados não mostrados). Além disso, lavagens vaginais pode ser realizada uma única vez em ratos separados em cada momento ou longitudinalmente em camundongos mesmo sob anestesia. Os linfonodos lombares são os principais linfonodos de drenagem do trato genital e do site mais relevante para avaliar a resposta imune sistêmica a um desafio vaginal. Note que esses nódulos linfáticos podem tornar-se ampliado em camundongos inoculados, enquanto eles normalmente aparecem muito pequenas não inoculado em camundongos. Leucócitos recuperações celular normalmente variam de 8 × 10 do mouse 5 / não inoculadas a 5 × 10 do mouse 6 / inoculados. Além de gânglios linfáticos lombares, linfonodos inguinais, poplíteos e mesentérica também pode ser usado. <img alt="Figura 1" src="/files/ftp_upload/3382/3382fig1.jpg" /> Figura 1. Inoculação vaginal com Candida. A) Um rato restringido para inoculação. O rato é colocado em uma gaiola de arame e inserção realizada pela base da cauda, ​​ligeiramente para cima para levantar as pernas e expor a abertura vaginal. O quadril do mouse pode ser estabilizado com a mesma mão ao tentar resistir à contenção da cauda. B) Introdução do inóculo para o lúmen vaginal. A ponteira é gentilmente inserido cerca de 5 mm de profundidade para dentro do lúmen vaginal. O inóculo de suspensão é então depositado. Figura 2. Extração de tecido vaginal. AB Extraction) do colo do útero. O colo do útero está localizado com uma pinça curva e exposto para fora através da cavidade vaginal. Uma vez fora da cavidade vaginal, o colo do útero é ainda mais puxado para fora, para expor completamente a vagina. C) Extração da vagina. A vagina é retirado da vulva com uma tesoura. Uma vez desligada, remova o colo da vagina. <img alt="Figura 3" src="/files/ftp_upload/3382/3382fig3.jpg" /> Figura 3. Identificação dos linfonodos lombares. A localização dos gânglios linfáticos lombares entre os órgãos circunvizinhos / vasos sanguíneos nas imediações da pelve é indicado. A aorta abdominal. B, bexiga urinária. C, artéria ilíaca comum. I, intestinos. L, o fígado. R, reto. S, baço. U, Útero. <img alt="Figura 4" src="/files/ftp_upload/3382/3382fig4.jpg" /> Figura 4. Os linfonodos lombares colocado em uma tela de malha de arame. Os linfonodos são agrupados na tela colocada numa placa de Petri com HBSS. Os linfonodos são pressionados contra a tela com um êmbolo da seringa para obter células linfóides em uma única célula suspensões. <img alt="Figura 5" src="/files/ftp_upload/3382/3382fig5.jpg" /> Figura 5. Frações celulares do lavado vaginal de camundongos inoculados. Preparações A) Wet-mount e B) colpocitológico vaginal amostras coletadas lavagem quatro dias após a inoculação e C) de ratos não inoculadas. Imagens são mostradas em 1000 × (A) ou 400 × (B, C) de ampliação. A inserção na B mostra a morfologia nuclear de neutrófilos vaginal em 1000 ×. Candida levedura (Y) e hifas (H), células epiteliais (CE) e neutrohils (N) são indicados. <img alt="Figura 6" src="/files/ftp_upload/3382/3382fig6.jpg" /> Figura 6. Detectíon de carga fúngica vaginal. A) colônias Representante C. albicans cultivadas em uma placa de SDA. Amostras puras (N) lavagem de seis diferentes camundongos inoculados (linha superior) foram diluídas em série e cultivadas para a enumeração UFC. B) Quantificação da carga fúngica vaginal de estrogênio-tratados e C) não-estrogênio camundongos tratados. Ufc/100 mL do lavado de camundongos inoculados foi avaliada em pontos de tempo indicado.

<ol>
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Immun. 70, 7081-7081 (2002).</li><li>Pietrella, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+beta-glucan-conjugate+vaccine+and+anti-beta-glucan+antibodies+are+effective+against+murine+vaginal+candidiasis+as+assessed+by+a+novel+in+vivo+imaging+technique.">A beta-glucan-conjugate vaccine and anti-beta-glucan antibodies are effective against murine vaginal candidiasis as assessed by a novel in vivo imaging technique.</a> Vaccine. 28 (7), 1717-1717 (2010).</li><li>Redondo-Lopez, V., Cook, R. N., Sobel, J. 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Gynecol. 178 (2), 203-203 (1998).</li><li>Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+humoral+immune+responses+against+primary+and+repeat+Neisseria+gonorrhoeae+genital+tract+infections+of+17&#946;-estradiol-treated+mice.">Local and humoral immune responses against primary and repeat Neisseria gonorrhoeae genital tract infections of 17&#946;-estradiol-treated mice.</a> Vaccine. 26, 5741-5741 (2008).</li><li>Taylor, B. N. <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+virulence+of+Candida+albicans+isolates+causing+mucosal+infections+in+people+infected+with+the+human+immunodeficiency+virus.">In vivo virulence of Candida albicans isolates causing mucosal infections in people infected with the human immunodeficiency virus.</a> J. Infect. Dis. 182, 955-955 (2000).</li><li>Taylor, B. N., Saavedra, M., Fidel, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+Th1/Th2+cytokine+production+during+experimental+vaginal+candidiasis.">Local Th1/Th2 cytokine production during experimental vaginal candidiasis.</a> Med. Mycol. 38, 419-419 (2000).</li><li>Tirabassi, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mucosal+vaccination+approach+for+herpes+simplex+virus+type+2.">A mucosal vaccination approach for herpes simplex virus type 2.</a> Vaccine. 29 (5), 1090-1090 (2011).</li><li>Broeck, W. V. a. n. d. e. n., Derore, A., Simoens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+nomenclature+of+murine+lymph+nodes:+descriptive+and+nomenclatory+standardization+in+BALB/cAnNCrl+mice.">Anatomy and nomenclature of murine lymph nodes: descriptive and nomenclatory standardization in BALB/cAnNCrl mice.</a> J. Immunol. Methods. 312 (1-2), 12-12 (2006).</li><li>Wormley, F. L., Chaiban, J., Fidel, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecule+and+lymphocyte+activation+marker+expression+during+experimental+vaginal+candidiasis.">Cell adhesion molecule and lymphocyte activation marker expression during experimental vaginal candidiasis.</a> J. Immunol. Methods. 69, 5072-5072 (2001).</li><li>Yano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell-derived+S100+calcium-binding+proteins+as+key+mediators+in+the+hallmark+acute+neutrophil+response+during+Candida+vaginitis.">Epithelial cell-derived S100 calcium-binding proteins as key mediators in the hallmark acute neutrophil response during Candida vaginitis.</a> Infect. Immun. 78 (12), 5126-5126 (2010).</li></ol>

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Um modelo experimental do rato de Candida vaginite foi estabelecida e usada historicamente para as últimas décadas para estudar a resposta do hospedeiro mucosa a Candida, bem como para testar terapias antifúngicas 3,4,11,13,16,17,19,21,24, 25,37. Os protocolos aqui apresentados incorporam métodos eficientes e menos trabalhoso, e parecem ser um dos sistemas de modelo mais otimizada de Candida vaginite descritas até o momento. Estas técnicas permitem a quantificação rápida da...

<table border="1"><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Feminino camundongos CBA / J </td><td> Charles River Laboratories </td><td> 01C38 </td><td> 5-6 semanas de idade </td></tr><tr><td> Candida albicans (3153A) </td><td> Colecção Nacional de fungos patogênicos, UK </td><td> NCPF3153 </td><td></td></tr><tr><td> Óleo de gergelim </td><td> Sigma-Aldrich </td><td> S3547 </td><td> Não precisa ser pré-esterilizadas antes do uso </td></tr><tr><td> Β-estradiol 17-valerato </td><td> Sigma-Aldrich </td><td> E1631 </td><td> 0.1-0.5mg em óleo de gergelim </td></tr><tr><td> Phytone peptona </td><td> Becton Dickinson </td><td> 211906 </td><td> Suplemento com glicose 0,1% </td></tr><tr><td> Solução de azul de Trypan </td><td> Sigma-Aldrich </td><td> T8154 </td><td></td></tr><tr><td> Ágar Sabouraud dextrose </td><td> Becton Dickinson </td><td> 211584 </td><td></td></tr><tr><td> Colagenase tipo IV </td><td> Sigma-Aldrich </td><td> C5138 </td><td> 0,25% </td></tr><tr><td> Dispase </td><td> Invitrogen </td><td> 17105-041 </td><td> 1,7 U / ml </td></tr><tr><td> Malha de telas de arame </td><td> TWP </td><td> 060X060S0065W36T </td><td> No. 60 mesh, aço inoxidável </td></tr><tr><td> Solução de Hanks salina balanceada </td><td> Invitrogen </td><td> 24020-117 </td><td></td></tr><tr><td> CytoPrep fixador </td><td> Fisher Scientific </td><td> 12-570-10 </td><td> Preserva esfregaço </td></tr><tr><td> Papanicolaou mancha EA-65 </td><td> Merck Química </td><td> 7054X-85 </td><td></td></tr><tr><td> Papanicolaou mancha OG-6 </td><td> Merck Química </td><td> 7052X-85 </td><td></td></tr><tr><td> Hematoxilina de Harris Alum </td><td> Merck Química </td><td> 638A-85 </td><td></td></tr><tr><td> Isoflurano </td><td> Baxter Healthcare </td><td> NDC 10019-773-60 </td><td> Usado com vaporizador de isoflurano ou em um sistema fechado queda anestésico câmara </td></tr></table>

protocols for vaginal inoculation and sample collection in the experimental mouse model of candida vaginitis

Vulvovaginal candidíase (VVC), causada por espécies de Candida, é uma infecção fúngica do trato genital feminino, que afeta aproximadamente 75% das mulheres saudáveis ​​durante seus anos reprodutivos 18,32-34. Os fatores predisponentes incluem uso de antibióticos, diabetes descontrolado e perturbação nos níveis de hormônios reprodutivos devido à gravidez, contraceptivos orais ou terapias de reposição hormonal 33,34. VVC recorrente (RVVC), definida como três ou mais episódios por ano, afeta a 5 separada a 8% das mulheres sem fatores predisponentes 33. Um modelo experimental de rato VVC foi estabelecida e utilizado para estudar a patogênese e resposta do hospedeiro mucosa a Candida 3,4,11,16,17,19,21,25,37. Este modelo também foi empregado para testar potenciais terapias antifúngicas in vivo 13,24. O modelo exige que os animais sejam mantidos em estado de pseudoestrus para optimal Candida colonização / infecção 6,14,23. Sob tais condições, animais inoculados terá carga fúngica vaginal detectável por semanas a meses. Estudos anteriores mostram um paralelo extremamente elevada entre o modelo animal e infecção humana em relação a propriedades imunológicas e fisiológicas 3,16,21. Diferenças, entretanto, incluem a falta de Candida como flora vaginal normal e um pH neutro vaginal nos ratos. Aqui, nós demonstramos uma série de métodos-chave no modelo de mouse que vaginite incluem inoculação vaginal, rápida coleta de amostras vaginal, avaliação da carga fúngica vaginal, e as preparações para a extração do tecido celular / isolamento. Isto é seguido por resultados representativos para constituintes do lavado vaginal, carga fúngica, e drenagem linfática produz leucócitos nó. Com o uso de anestésicos, as amostras de lavagem podem ser coletados em momentos diversos na mesma camundongos para avaliação longitudinal dainfecção / colonização. Além disso, este modelo não necessita de agentes imunossupressores para iniciar a infecção, permitindo estudos imunológicos em condições host definido. Finalmente, o modelo e cada técnica introduzida aqui poderia potencialmente dar origem a utilização das metodologias para examinar outras doenças infecciosas do trato genital feminino (bacterianas, parasitárias, virais) e respectivas defesas do hospedeiro local ou sistêmica.

Watch this Scientific Journal Video about Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis at JoVE.com

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Principais técnicas a serem utilizados na avaliação de<em&gt; Candida</em&gt; Vaginite em um modelo animal experimental são descritos. Os métodos permite uma recolha rápida de amostras vaginais e linfócitos de linfonodos drenantes lombar. Estas técnicas podem dar origem a modelos de ratos de outras doenças na parte inferior do trato genital feminino.

Protocolos para inoculação vaginal e coleta de amostra no Modelo Experimental de Rato Candida Vaginite

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

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Immunology and Infection

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

32.7K visualizações.  Louisiana State University Health Sciences Center. Principais técnicas a serem utilizados na avaliação de Candida Vaginite em um modelo animal experimental são descritos. Os métodos permite uma recolha rápida de amostras vaginais e linfócitos de linfonodos drenantes lombar. Estas técnicas podem dar origem a modelos de ratos de outras doenças na parte inferior do trato genital feminino.

Vídeo: Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Metástase experimental e CTL Adoptive Transferência Modelo rato Imunoterapia

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Imunologia e Infecção

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Preparação de rato Imunogénio Pituitária para a indução de hipofisite Auto-Imune Experimental

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Um modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonização Vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizando os Efeitos de escarro no desenvolvimento de biofilmes usando um modelo Chambered lamela

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Estabelecimento do modelo humanizado duplo do rato de TK-NOG para a patogénese HIV-associada do fígado

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

Divulgando a coleta de Hemolinfas e a inoculação de blastósporos de Metarhizium em carrapatos de Rhipicephalus MICROPLUS para estudos de patologia de invertebrados

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

Modelo de inoculação do footpad do mouse para estudar respostas neuroinflamatórias induzidas pelo viral

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Uveíte Autoimune Experimental: Um Modelo de Rato Inflamatório Intraocular

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

Um Modelo Murino de Fibrose Hepática Crônica para o Estudo da Atresia Biliar

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Procedure for Swine Burn Wounding, Burn Wound Imaging, Wound Inoculation, and Biopsy Collection

To begin the procedure inside the properly arranged surgery room, place the anesthetized pig in the sternal recumbency position, and ensure all the connected lines and tubes are secured. Remove any residual hairs from the shaved area designated for wounding using hair removal cream. After sterilizing the wound sites, mark the area using a sterile burn template and a surgical skin marker.Set the burner by adjusting the set point on the control unit to 150 degrees celsius. The low set point to 145 degrees Celsius and the high set point to 155 degrees Celsius. Using a heated stainless steel blocks connected to the burn device, create a full thickness burn wound by placing the blocks on the skin for 60 seconds.Take a digital photo of the whole back of the pig, including a placard with the pig's identification time point and date. Then separately take images for each wound along with a ruler and a placard displaying the pig ID, wound ID and time point. To assess the wound microvascular perfusion in real time using laser speckle imaging or LSI, image all the wounds in a single recording using a LASCA blood perfusion imager.To measure trans epidermal water loss or TEWL, place a clean probe cover over the TEWL probe tip, which will be in contact with the wound tissue. Measure each wound five times, first in the center, followed by each corner. Then export all the readings to a spreadsheet.Perform a harmonic ultrasound or HUSD mapping by scanning the wound with an ultrasound probe from the midline or vertebral column, starting from normal skin and moving towards the lateral side of the pig over the wounds until the normal skin is again reached. Follow the scanning pattern for each wound in both B mode and tissue elastography mode. Change the annotation after each wound is imaged in both B mode and elasto mode, which is two recordings per wound.Once all the imaging is complete, cover the burn wounds individually with transparent film dressings or the test dressing. Then place a larger transparent film dressing over the entire wound area. Apply a second layer of roll gauze loosely around the entire trunk of the pig to absorb any fluid exuding from the wounds.Cover the gauze loosely with a layer of flexible elastic bandage. Cover the elastic bandage with a final layer of four inch elastic tape. After discontinuing the anesthetic gas upon completion of the bandaging, allow the pig to remain on oxygen for at least five minutes.To inoculate the burn wounds on day three post burn, disperse the inoculum across the surface of the exposed wounds using a pipette, and spread it evenly using a disposable spreader. To collect the tissue biopsies for analysis following imaging, infiltrate the area around the wound with 0.5%bupivacaine. Using a disposable scalpel with a size 10 blade, cut a 3-4 millimeter wide strip from one edge of the wound to the other, keeping small margins of normal skin on both sides.Cut a 6 millimeter punch biopsy from the wound either from the wound bed or wound edge. Achieve hemostasis after the samples are collected by gently pressing the wound with a sterile gauze. Cover the wound with a non-adherent dressing and bandage it as demonstrated previously.Analysis of the biopsy sample by hematoxylin and Eosin staining showed distortion and necrosis of all the histological skin layers. The established biofilm was further validated at day seven post inoculation by colony forming unit. Scan the electron microscopy imaging, and immunofluorescence staining.The burn wounds showed a thick biogenic membrane on the wound surface by day seven post inoculation, confirming the infection and establishment of biofilm. The wound area started to shrink by day 14, but incomplete healing to about 25%of the original wound size was observed by day 56 indicating wound chronicity. Wound chronicity and impaired wound healing were further confirmed by the TEWL showing high trans epidermal water loss at all measured time points.This was also confirmed by the suppression of the tight junctional proteins, ZO-1 and 2, and the impaired restoration of skin barrier function.