Name: inducing meningococcal meningitis serogroup c in mice via intracisternal delivery
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Os estudos foram apoiados em parte pelo PRIN 2012 [número de subvenção 2012WJSX8K]: "Modelos de interação host-micróbio em infecções mucosas: desenvolvimento de novas estratégias terapêuticas" e pela PRIN 2017 [2017SFBFER]: "Uma abordagem integrada para enfrentar a interação entre adaptação, condições estressantes e resistência antimicrobiana de patógenos desafiadores".

Este protocolo foi conduzido para minimizar o sofrimento dos animais e reduzir o número de camundongos de acordo com a Diretiva do Conselho das Comunidades Europeias de 24 de novembro de 1986 (86/609/CEE). Experimentos in vivo relatados neste estudo foram aprovados pelo Ethical Animal Care and Use Committee (Prot. número 2, 14 de dezembro de 2012) e pelo Ministério da Saúde italiano (Prot. número 0000094-A-03/01/2013). Todos os procedimentos devem ser realizados dentro do Gabinete de Biossegurança 2 (BSC2) em uma s...

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Neste estudo, descrevemos um protocolo experimental para induzir meningite meningocócica em camundongos adultos por i.cist. inoculação de bactérias meningocócicas. A nosso conhecimento, nenhum outro modelo da meningite meningococcal foi desenvolvido nos ratos do laboratório contaminados pelo i.cist. rota; no passado, desta forma tem sido explorada para fornecer modelos de meningite meningocócica em ambos os ratos31 e coelho32. É sabido que a maior taxa de doença me...

Neisseria meningitidis é um Gram negativo β-proteobacterium restrito ao hospedeiro humano, bem conhecido por ser uma das causas mais comuns de meningite e sepse na população humana em todo o mundo. Coloniza o trato respiratório superior (nariz e garganta) de portadores saudáveis e assintomáticos (2-30% da população), mas a bactéria às vezes evita várias defesas imunes do hospedeiro e se espalha da corrente sanguínea para o cérebro, causando um local descontrolado inflamação, conhecida como meningite meningocócica. Uma combinação de fatores do anfitrião e bacterianos parece contribuir à transição do commensal ao comportamento invasor1.
N. meningitidis é especializada exclusivamente em colonização humana e infecção. Tem uma escala estreita do anfitrião e, conseqüentemente, limitou estudos in vivo do pathogenesis devido à falta dos modelos animais apropriados que reproduzem a doença meningocócica humana. Como resultado, levou a lacunas fundamentais na compreensão em relação à patogênese da septicemia e meningite causada pelo meningococcus. Nas últimas décadas, o desenvolvimento de muitos sistemas in vitro permitiu a identificação de vários fatores de virulência meningocócica2,3,4. Embora esses estudos valiosos fornecessem insights importantes para entender o papel desses fatores para uma infecção meningocócica bem-sucedida, esses modelos não permitiram a avaliação das consequências das interações bacterianas com o humoral e celular sistema imunológico e ainda menos com todo o tecido. In vivo modelos animais de infecção são de grande relevância, bem como para a avaliação do grau de proteção conferida por formulações vacinais. Como patógeno humano-trópico, meningococcus possuem determinantes apropriados necessários para infecções bem-sucedidas, como estruturas superficiais (ou seja, pili tipo IV e proteínas de opacidade) e sistemas de captação de ferro para receptores humanos e proteínas de transporte (ou seja, transferrina e lactoferrina)5,6,7 para aderir adequadamente, sobreviver e invadir o hospedeiro humano. Finalmente, as habilidades de variação genética do patógeno para fugir e/ou bloquear a resposta imune humana contribuem ainda mais para o tropotismo de espécies altas8,9. Portanto, a ausência de fatores hospedeiros específicos, envolvidos na interação, pode bloquear etapas do ciclo de vida do patógeno, estabelecendo dificuldades significativas no desenvolvimento de pequenos modelos animais resumindo o ciclo de vida meningocócica.
Ao longo das últimas décadas, várias abordagens foram desenvolvidas para melhorar a nossa compreensão do ciclo infeccioso meningocócico. Infecções de dois modelos animais, camundongos e camundongos, intraperitoneally (i.p.) ou intranasally (i.n.), foram desenvolvidas para reproduzir a doença meningocócica10,11,12,13, 14 , 15,16,17. O rato de laboratório é provavelmente um dos animais mais versáteis para induzir a infecção meningocócica experimental.
No entanto, a forma de infecção do i.p. leva ao desenvolvimento de sepse grave, embora não imite a via natural da infecção, enquanto a via de infecção do i.n. foi útil para avaliar a patogênese meningocócica, mesmo que possa induzir infecção pulmonar antes da sepse10,11,12,13,14,15,16,17.
O modelo de mouse i.p. foi fundamental para avaliar a proteção contra o desafio meningocócico10,11,12. O modelo do rato da colonização meningocócica baseada na rota i.n. da infecção foi desenvolvido com ratos infantis, porque são mais suscetíveis aos meningococci, para reproduzir uma infecção invasora que imita o curso da doença meningocócica nos seres humanos 13,14,15,16,17. Além disso, para promover a replicação meningocócica no hospedeiro murine, um número crescente de estratégias técnicas também foram aplicadas, incluindo a administração do ferro para os animais para melhorar a infecção, o uso de alto inóculobacteriano , cepa bacteriana passagem por camundongos, bem como o emprego de animais infantis ou imunocomprometidos hospeda10,13,15,18,19. A expressão de fatores humanos específicos como CD4620 outransferrin21 aumentou a susceptibilidade dos camundongos para esta bactéria humano-trópico; o emprego do modelo de xenoenxerto de pele humana de infecção também tem sido útil para avaliar a capacidade de adesão de meningococos ao endotelio humano22,23. Coletivamente, o recente desenvolvimento de camundongos transgênicos humanizados melhorou a compreensão da patogênese meningocócica e suas interações hospedeiras.
Anteriormente, desenvolvemos um modelo de urina de meningite meningocócica, onde a inoculação de bactérias foi realizada na cisterna magna de camundongos adultos com bactérias de passagem de ratos24. Parâmetros clínicos e a taxa de sobrevida de camundongos infectados demonstraram o estabelecimento de meningite com características comparáveis às observadas no hospedeiro humano, bem como as análises microbiológicas e histológicas do cérebro. Destes camundongos infectados, as bactérias foram, também, recuperados de sangue, fígado e baço, e cargas bacterianas de órgãos periféricos correlacionados com a dose infecciosa. Em particular, este modelo foi empregado para avaliar a virulência de uma cepa mutante isógênica com defeito no transportador L-glutamato GltT24. Recentemente, usando nosso modelo do rato da meningite meningococcal baseada em i.cist. rota com cepa sorogrupo C 93/42862,24 e um mutante isogênico defeituoso na codificação de genes cssA para UDP-N-acetilglucosamina 2-epimerase25, analisamos o papel do ácido siálico exposto no estabelecimento de doença em camundongos.
Neste protocolo, descrevemos um método simples para induzir a meningite meningocócica experimental com base no i.cist. rota de infecção em camundongos adultos Balb/c. Este método é particularmente útil para a caracterização da infecção meningocócica em um hospedeiro de urina, bem como para a avaliação da virulência entre cepas de referência do tipo selvagem e mutantes isogênicos. A via intra-cisterna da infecção garante a entrega completa dos meningococos diretamente na cisterna magna, que por sua vez facilita a replicação bacteriana no líquido cefalorraquidiano (CSF) e induz meningite com características que imitam aqueles presente emseres humanos2,24,25,26.

<table>
	<tbody>
		<tr>
			<td>1,8 Skirted Cryovial With external thread</td>
			<td>Starlab</td>
			<td>E3090-6222</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>30 x 115mm</td>
		</tr>
		<tr>
			<td>Adson Forceps</td>
			<td>F.S.T.</td>
			<td>11006-12</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Alarm-Thermometer</td>
			<td>TESTO</td>
			<td>9000530</td>
			<td></td>
		</tr>
		<tr>
			<td>BactoTM Proteose Peptone</td>
			<td>BD</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Micro Fine syringe</td>
			<td>BD</td>
			<td>320837</td>
			<td>U-100 Insulin</td>
		</tr>
		<tr>
			<td>BD Plastipak syringe 1ml 25GA 5/8in</td>
			<td>BD</td>
			<td>300014</td>
			<td>05x16mm</td>
		</tr>
		<tr>
			<td>BD Plastipak syringe 5ml</td>
			<td>BD</td>
			<td>308062</td>
			<td>07 x 30mm</td>
		</tr>
		<tr>
			<td>BIOHAZARD AURA B VERTICAL LAMINAR FLOW CABINET</td>
			<td>Bio Air s.c.r.l.</td>
			<td>Aura B3</td>
			<td></td>
		</tr>
		<tr>
			<td>BioPhotometer</td>
			<td>Eppendorf</td>
			<td>Model #6131</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle D</td>
			<td>Tecniplast</td>
			<td>D</td>
			<td>Graduated up to:400ml, Total Volume 450ml, 72x72x122mm</td>
		</tr>
		<tr>
			<td>C150 CO2 Incubator</td>
			<td>Binder</td>
			<td>9040-0078</td>
			<td></td>
		</tr>
		<tr>
			<td>Cage Body Eurostandard Type II</td>
			<td>Tecniplast</td>
			<td>1264C</td>
			<td>267x207x140mm, Floor area 370cm2</td>
		</tr>
		<tr>
			<td>Cell Culture Petri Dish With Lid</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td>Working Volume: 5mL</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Microcentrifuge 5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvetta semi-micro L. Form</td>
			<td>Kartell S.p.A.</td>
			<td>01938-00</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Potassium hydrogen phosphate trihydrate</td>
			<td>Carlo erba</td>
			<td>471767</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium hydrogen phosphate anhydrous ACS-for analysis</td>
			<td>Carlo Erba</td>
			<td>480141</td>
			<td>g1000</td>
		</tr>
		<tr>
			<td>Diete Standard Certificate</td>
			<td>Mucedola s.r.l.</td>
			<td>4RF21</td>
			<td>Food pellet for animal</td>
		</tr>
		<tr>
			<td>Dumont Hp Tweezers 5 Stainless Steel</td>
			<td>F.S.T. by DUMONT</td>
			<td>AGT5034</td>
			<td>0,10 x 0,06 mm tip</td>
		</tr>
		<tr>
			<td>Electronic Balance</td>
			<td>Gibertini</td>
			<td>EU-C1200</td>
			<td>Max 1200g, d=0,01g, T=-1200g</td>
		</tr>
		<tr>
			<td>Eppendorf Microcentrifuge tube safe-lock</td>
			<td>Eppendorf</td>
			<td>T3545-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythromycin</td>
			<td>Sigma-Aldrich</td>
			<td>E-6376</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors</td>
			<td>F.S.T.</td>
			<td>14084-08</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Filter Top (mini- Isolator), H-Temp with lock clamps</td>
			<td>Tecniplast</td>
			<td>1264C400SUC</td>
			<td></td>
		</tr>
		<tr>
			<td>GC agar base</td>
			<td>OXOID</td>
			<td>CM0367</td>
			<td></td>
		</tr>
		<tr>
			<td>Gillies Forceps 1 x2 teeth</td>
			<td>F.S.T.</td>
			<td>11028-15</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Glicerin RPE</td>
			<td>Carlo Erba</td>
			<td>453752</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T.</td>
			<td>11052-10</td>
			<td>Serrated Tip Width: 0.8mm</td>
		</tr>
		<tr>
			<td>Inner lid</td>
			<td>Tecniplast</td>
			<td>1264C116</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron dextran solution</td>
			<td>Sigma-Aldrich</td>
			<td>D8517-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological Safety Cabinet BH-EN and BHG Class II</td>
			<td>Faster</td>
			<td>BH-EN 2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5ml&#160;</td>
			<td>BRAND</td>
			<td>PP780751</td>
			<td>screw cap PP, grad</td>
		</tr>
		<tr>
			<td>Mouse Handling Forceps</td>
			<td>F.S.T.</td>
			<td>11035-20</td>
			<td>Serrated rubber; Gripping surface:15 x 20 mm</td>
		</tr>
		<tr>
			<td>Mucotit-F2000</td>
			<td>MERZ</td>
			<td>61846</td>
			<td>2000ml</td>
		</tr>
		<tr>
			<td>Natural Latex Gloves</td>
			<td>Medica</td>
			<td>M101</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Classic C24 Incubator Shaker</td>
			<td>PBI international</td>
			<td>C-24 Classic Benchtop Incubator Shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri PS Dishes</td>
			<td>VWR</td>
			<td>391-0453</td>
			<td>90X14.2MM</td>
		</tr>
		<tr>
			<td>Pipetman Classic P20</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td>2-20microL</td>
		</tr>
		<tr>
			<td>Pipetman Classic P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td>20-200microL</td>
		</tr>
		<tr>
			<td>Pipetman Classim P1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td>200-1000microL</td>
		</tr>
		<tr>
			<td>Polyvitox</td>
			<td>OXOID</td>
			<td>SR0090A</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>J.T. Baker Chemicals B.V.</td>
			<td>0208</td>
			<td>250g</td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Phosphate</td>
			<td>J.T. Baker Chemicals B.V.</td>
			<td>0240</td>
			<td>1Kg</td>
		</tr>
		<tr>
			<td>PS Disposible forceps</td>
			<td>VWR</td>
			<td>232-0191</td>
			<td></td>
		</tr>
		<tr>
			<td>Removable Divider</td>
			<td>Tecniplast</td>
			<td>1264C812</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polypropylene Tubes</td>
			<td>Falcon</td>
			<td>352063</td>
			<td>5ml</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>MOLEKULA</td>
			<td>41272436</td>
			<td></td>
		</tr>
		<tr>
			<td>SS retainer and Polyester FilterSheet</td>
			<td>Tecniplast</td>
			<td>1264C</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Pattern Forceps</td>
			<td>F.S.T.</td>
			<td>11000-12</td>
			<td>Stainless</td>
		</tr>
		<tr>
			<td>Stevens Tenotomy Scissors</td>
			<td>F.S.T.</td>
			<td>14066-11</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Surgical Scissor - ToughCut</td>
			<td>F.S.T.</td>
			<td>14130-17</td>
			<td>Stainless</td>
		</tr>
		<tr>
			<td>Touch N Tuff disposible nitrile gloves</td>
			<td>Ansell</td>
			<td>92-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Low Temperature (ULT) Freezer</td>
			<td>Haier</td>
			<td>DW-86L288</td>
			<td>Volume= 288L</td>
		</tr>
		<tr>
			<td>Wagner Scissors</td>
			<td>F.S.T.</td>
			<td>14070-12</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

inducing meningococcal meningitis serogroup c in mice via intracisternal delivery

Neisseria meningitidis (meningococcus) é um microorganismo de alcance estreito, reconhecido mundialmente como a principal causa de meningite bacteriana. Meningococcus é um colonizador transitório da nasofaringe humana de aproximadamente 10% do sujeito saudável. Em circunstâncias particulares, adquire uma capacidade invasiva de penetrar na barreira da mucosa e invade a corrente sanguínea causando septicemia. No último caso, a sepse fulminante poderia surgir mesmo sem o consequente desenvolvimento da meningite. Por outro lado, as bactérias poderiam se multiplicar mal na corrente sanguínea, atravessar a barreira do cérebro sanguíneo, alcançar o sistema nervoso central, levando à meningite fulminante. Os modelos de urina de meningite bacteriana representam uma ferramenta útil para investigar as interações hospedógeno-patógeno e analisar os mecanismos pathogenéticos responsáveis por esta doença letal. Embora, vários sistemas de modelos experimentais tenham sido avaliados nas últimas décadas, nenhum deles foi capaz de reproduzir os eventos patológicos característicos da doença meningocócica. Neste protocolo experimental, descrevemos um procedimento detalhado para a indução da meningite meningocócica em um modelo de camundongo baseado na inoculação intracisternal de bactérias. Os sinais peculiares da meningite humana foram registrados no hospedeiro murina por meio da avaliação de parâmetros clínicos (por exemplo, temperatura, peso corporal), avaliação da taxa de sobrevida, análise microbiológica e exame histológico de lesão cerebral. Ao usar inóculo intracisternal (i.cist.), meningococos entrega completa diretamente em cisterna magna, levando a uma replicação meningocócica muito eficiente no tecido cerebral. Um aumento de 1.000 vezes da contagem viável de bactérias é observado em cerca de 18 h. Além disso, meningococos também são encontrados no baço, e fígado de camundongos infectados, sugerindo que o fígado pode representar um órgão-alvo para a replicação meningocócica.

Sobrevivência de camundongos infectados com N. meningitidis tipo selvagem e cepas mutantes isogênicos. As cepas Neisseria meningitidis utilizadas nestes resultados representativos são a estirpe de referência serogrupo C 93/4286 (ET-37) e o seu mutante isogênico 93/4286ΩcssA obtido por inativação insercional do gene cssA, codificação para o UDP-N-acetilglucosamina 2-epimerase, que mapeia na síntese cápsula locus25. Para avaliar o g...

Watch this Scientific Journal Video about Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery at JoVE.com

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Aqui, descrevemos um método para induzir a meningite meningocócica através de uma via intracisternal de infecção em camundongos adultos. Apresentamos um protocolo passo a passo de infecção meningocócica desde a preparação do inóculo para a infecção intracisternal; registre então a sobrevivência animal e avalie as cargas bacterianas em tecidos do murine.

Indução meningocócica meningite sorogrupo C em camundongos via parto intracisternal

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

Neisseria meningitidis é uma das causas mais comuns de sepse e meningite entre a população humana em todo o mundo, e a bactéria é restrita ao hospedeiro humano. Neste protocolo experimental, descreveremos o procedimento personalizado para a indução de meningite meningocócica em um modelo de camundongo baseado na inoculação intracisternal de bactérias. Este modelo murino reproduziu os eventos patológicos característicos da meningite meningocócica como uma doença humana grave.Assim, representa uma ferramenta útil para avaliar a interação hospedeiro-patógeno e analisar o mecanismo patogenético responsável por esta doença letal. O modelo de infecção poderia ser útil não apenas para avaliar a estratégia terapêutica inovadora para evitar a replicação de bactérias diretamente no CSF, mas também para analisar a eficácia de uma possível imunoterapia passiva contra patógenos humanos. A técnica requer a fase de treinamento para garantir o sucesso não só do inoculum intracisternal, mas mais em geral a indução da doença.Os procedimentos serão Roberta Colicchio e Chiara Pagliuca como pesquisadoras, e Elena Scaglione como doutoranda, todas pelo meu laboratório. Para começar, escolha uma única colônia de uma cultura fresca de meningite Neisseria na placa de ágar gonocócica suplementada com suplemento de 1%polibitox e inoculada em 10 mililitros gc caldo. Todos os procedimentos que envolvam o uso de meningococci devem ser feitos sob um armário de cubos de biosegurança de fluxo laminar.Cresça a bactéria a 37 graus Celsius em uma incubadora de shake orbital a 220 RPM e continue verificando a densidade óptica da cultura em diferentes pontos de tempo com um espectrofotômetro. Cresça a cultura até chegar a OD600 de 0,7 correspondente à fase exponencial inicial. Uma vez obtida essa densidade óptica, faça estoques congelados adicionando 10% de glicerol e distribuindo um mililitro da cultura nos frascos de criogeno.Armazene os frascos a 80 graus Celsius até o uso. Antes da infecção, descongele bactérias congeladas à temperatura ambiente, em seguida, transfira para um tubo de centrífuga em centrífuga por 15 minutos a 1500xG Remova o supernascimento e suspenda novamente em um mililitro de caldo GC fresco contendo cinco miligramas por quilograma de dextran de ferro. Antes de iniciar o experimento, pese os animais e avalie sua temperatura corporal.Raspe o animal do pescoço para baixo e desinfete o abdômen com 70% de etanol. Aproximadamente 2 horas antes da infecção, usando uma agulha calibre 25, injete dextran de ferro intraperitonealmente no quadrante inferior direito do abdômen. Após anestesiar o animal, verifique a profundidade da anestesia confirmando a ausência de resposta à dor ao beliscar o dedo do pé.Depois de colocar o rato no gabinete de fluxo laminar posicione-o em decúbito severo e estique cuidadosamente os membros e a coluna cervical para manter a coluna vertebral em uma posição reta. Para manter uma suspensão bacteriana consistente, misture-a suavemente antes de carregá-la em uma seringa com agulha de calibre 30 de oito milímetros. Desinfete a cabeça com 70% de etanol.Coloque o animal em recumbência lateral, puxe as orelhas para fora do caminho e flexione o pescoço moderadamente. Certifique-se de que a linha média do pescoço e da cabeça estão em posição perfeitamente paralela à parte superior da mesa. Em seguida, toque nas asas atlas, certificando-se de que elas se sobrepõem, e sinta o recuo natural na linha média onde a agulha é mais provável de entrar no orifício occipital.Encha uma seringa com uma agulha calibre 30 de 8 milímetros com a dose bacteriana sub-letal estabelecida de meningococci ou dextran de ferro complementado GC Caldo como controle em um volume total de 10 microliters. Use a agulha para identificar o ponto de injeção e, em seguida, injete o conteúdo da seringa na cisterna magna dos camundongos através de um orifício de rebarba occipital. Descarte a seringa e a agulha com segurança após a injeção.Coloque o animal na gaiola sob um armário de fluxo laminar. Aguarde o despertar e a recuperação completa do movimento e prossiga para a sobrevivência animal nos animais infectados, conforme descrito no protocolo. Depois de anestesiar o rato, desinfete o baú com 70% de etanol.Usando uma agulha calibre 25, retire de 600 a 700 microlitadores de sangue por punção cardíaca da cavidade torácica. Colete o sangue em um tubo contendo 3,8% de citrato de sódio e armazene a 80 graus Celsius para a avaliação posterior da contagem de células bacterianas viáveis. Coloque o rato eutanizado na posição supina.Usando tesouras e fórceps cortou a pele ao longo do plano sagital do corpo. Com pinos, conserte a pele com a pele nas laterais do corpo. Use uma tesoura afiada para cortar a membrana peritoneal.Em seguida, com tesouras e fórceps descartáveis, extirpar os órgãos, como baço e fígado, e colocar cada um em uma placa de Petri estéril separada com um mililitro de Caldo GC suplementado com 10% de glicerol. Usando um êmbolo de uma seringa de cinco milímetros, homogeneize os órgãos mecanicamente à temperatura ambiente por cerca de dois a três minutos até que a suspensão unicelular seja formada e transfira-a para um frasco criogênico escurdente. Coloque o tubo imediatamente no gelo seco.Faça placas de ágar GC com antibióticos e seque-as antes de usar a 37 graus Celsius por duas a três horas. Prepare 10 diluições seriais completas das amostras em Caldo GC de cada tecido homogeneizado. Emplaque-os em placas de ágar GC e incubar durante a noite a 37 graus Celsius com 5% de dióxido de carbono.Com 70% de etanol, desinfete a cabeça de um rato eutanizado. Usando uma pequena tesoura cirúrgica e uma fórceps de aço de ponta fina, ressecção da pele e da pele da cabeça removida, em seguida, prossiga em direção ao topo do crânio, a fim de ver claramente as suturas e orientar a abertura do crânio. Para abrir o crânio, insira a ponta da tesoura minúscula através do foramen magnum.Corte em direção ao centro do crânio e através da linha média do osso parietal para o lado oposto do crânio e corte suavemente ao longo do membro lateral da sutura lambdoid. Usando fórceps finos, começando pelo canto parietal posterior, levante o crânio e puxe-o diagonalmente para cima para descobrir o cérebro, certificando-se de que o tecido cerebral não está preso a nenhum osso da cabeça quando o crânio é levantado. Use fórceps descartáveis para remover qualquer tecido conjuntivo entre o crânio e o cérebro e não permita que o tecido cerebral seque.Use fórceps descartáveis para colocar imediatamente o cérebro em uma placa de Petri com um mililitro de caldo GC suplementado com 10% de glicerol. Use o êmbolo de uma seringa de cinco mililitros para homogeneizar o cérebro mecanicamente à temperatura ambiente por cerca de dois a três minutos até que uma suspensão unicelular seja formada. Transfira a suspensão para um tubo estéril de 2 mililitros e armazene a 80 graus Celsius para a avaliação posterior da contagem de células bacterianas viáveis.Foram avaliadas a sobrevivência de camundongos infectados com 93/4286 cepas de Neisseria meningitidis defeituosas do tipo selvagem ou cssA defeituosas. A dose letal mediana para a cepa do tipo selvagem correspondeu ao desafio meningocócica de 10 a 4ª UFC. Considerando que a taxa de mortalidade de 10 a 5ª UFC foi igual a 83,4% e 100% com 10 à 5ª dose da UFC.Para obter a dose letal mediana para a cepa mutante defeituosa cssA, foi necessária uma dose de 10 a 8ª UFC e uma quantidade 1000 dobras maior do que a cepa do tipo selvagem. Após a injeção, houve um rápido aumento de bactérias do tipo selvagem no tecido cerebral atingindo o pico em torno de 24 horas após a infecção. No cérebro de camundongos infectados com a cepa mutante, as contagens viáveis caíram progressivamente ao longo do tempo.Além disso, 33,3% dos camundongos infectados mutantes apresentaram folga bacteriana do local da infecção, ao contrário de animais infectados do tipo selvagem. 48 horas após a infecção, o mutante defeituoso do CSSA foi completamente limpo em baço e fígado, enquanto os animais infectados com a cepa selvagem apresentaram uma infecção persistente. O método experimental descrito poderia ser útil para analisar danos cerebrais associados a esta doença letal, por sua avaliação patológica, como imunohistoquímica, imunofluorescência e análise de sangramento cerebral.

inducing-meningococcal-meningitis-serogroup-c-mice-via-intracisternal

Research

JoVE Journal

Immunology and Infection

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Ratos Transnuclear com pré-definidos T Especificidades receptor da célula contra a Toxoplasma gondii Obtidos Via TNCS

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Imunologia e Infecção

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Inferência genotípica do HIV-1 Tropismo Usando de base populacional de Seqüenciamento V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

Um método para Etiquetagem Vascularização no embrião do rato

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Transmissão Oral de<em&gt; Listeria monocytogenes</em&gt; Em camundongos através de ingestão de alimentos contaminados

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia cultura do Sistema Nervoso Central Neonatal e Adulto

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Indução de Murino inflamação intestinal por transferência adotiva de Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; Células T em imunodeficientes Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

A<em&gt; In Vitro</em&gt; Caseum Ensaio de Ligação ao que prevê a penetração da droga na tuberculose Lesões

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Intracraniana optou rota de infecção para investigar papéis de biofilmes de Streptococcus suis em meningite em um modelo de infecção do rato

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

PCR quantitativo do bacteriófago T7 de Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

Estudos de probiótico em camundongos neonatais usando Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...