Os autores gostariam de acusar o recebimento da Pediatric Orthopaedic Society of North America Biomet Spine Grant e do National Institutes of Health Clinical and Translational Science Institute KL2 Grant, e do HH Lee Surgical Research Grant como principais fontes de financiamento para esses experimentos.

Todos os animais foram manuseados em estrita conformidade com as boas práticas animais, conforme definido nas regulamentações federais, conforme estabelecido na Lei de Bem-Estar Animal (AWA), no Guia para o Cuidado e Uso de Animais de Laboratório de 1996, na Política PHS para o Cuidado e Uso Humano de Animais de Laboratório, bem como nas políticas e procedimentos da instituição, conforme estabelecido no Manual de Treinamento de Cuidados e Uso de Animais, e todo o trabalho com animais foi aprovado pelo Comitê de Pesquisa Animal (ARC) da Universidade da Califórnia em Los Angeles.
1. Escolha da cepa bioluminescente de S. aureus
<ol><li>Utilizar a cepa bioluminescente de S. aureus Xen36 como inóculo de interesse. NOTA: Esta cepa foi derivada da cepa parental S. aureus ATCC-49525, que é um isolado clínico de um paciente séptico. S. aureus O Xen36 utiliza exclusivamente um operon ABCDE lux, que é otimizado e integrado ao plasmídeo nativo do hospedeiro. 19 Como resultado, a cepa Xen36 é capaz de produzir uma luz bioluminescente azul-esverdeada com um comprimento de onda de pico de emissão de 490 nm. Este sinal de emissão só é produzido por organismos bacterianos vivos metabolicamente ativos.</li>
</ol>2. Preparação de S. aureus para inoculação
<ol><li>Adicionar 200 μg/mL de canamicina ao Caldo Luria mais ágar 1,5% para isolar S. aureus Xen36 de potenciais contaminantes, utilizando o gene de resistência à canamicina ligado ao operon lux 19.</li>
	<li>Streak S. aureus Xen36 bactérias em placas de ágar de soja tríptica (caldo de soja tríptico [TSB] mais 1,5% de ágar) e incubar a 37 °C por 12-16 h.</li>
	<li>Isolar colônias únicas de S. aureus Xen36 e cultivar individualmente em TSB por 12-16 h a 37 °C em incubadora de agitação (200 rpm).</li>
	<li>Diluir a cultura resultante na proporção de 1:50.</li>
	<li>Cultura por mais 2 h a 37 °C para isolamento de bactérias da fase midlogarítmica.</li>
	<li>Pellet, ressuspender e lavar bactérias em solução salina tamponada com fosfato (PBS) três vezes.</li>
	<li>Medir a absorbância a 600 nm. O OD600 ideal está entre 0,700 e 0,750, o que corresponde a 4x105 UFC/mL. Realizar diluições seriadas para obter o inóculo bacteriano desejado (1 x 103 UFC/2 μL). NOTA: A concentração ideal de Xen36 para o estabelecimento de uma infecção crônica foi encontrada para ser 1 x 103 UFC. Doses mais baixas de bactérias foram eliminadas pelo sistema imunológico do hospedeiro e doses mais altas causaram a quebra da ferida. A quebra da ferida não diferencia entre uma infecção profunda do implante e uma infecção superficial da ferida e, portanto, é evitada neste modelo (Figura 1)20.</li>
</ol>3. Ratos
<ol><li>Use camundongos selvagens C57BL/6J machos de 12 semanas de idade.</li>
	<li>Ratos domésticos em gaiolas com um máximo de 4 de cada vez.</li>
	<li>Mantenha a água sempre disponível. Mantenha um ciclo claro/escuro de 12 horas e não realize experimentação durante a fase escura do ciclo.</li>
	<li>Use ração livre de alfafa para alimentação devido à interferência potencial com a sinalização fluorescente.</li>
	<li>Peça à equipe de pesquisa ou veterinária que avalie os camundongos diariamente para garantir o bem-estar dos animais durante todo o experimento.</li>
</ol>4. Procedimentos cirúrgicos em camundongos
<ol><li>Induzir anestesia colocando camundongos em uma câmara de isoflurano (2%) por aproximadamente 5 minutos. Confirme a profundidade apropriada da anestesia monitorando as respirações para que permaneçam rítmicas e mais lentas do que quando acordadas e não mudem em resposta a estímulos nocivos (por exemplo, manipulação cirúrgica, pinça dos dedos).</li>
	<li>Transfira camundongos anestesiados para uma estação de preparação e remova pelos do sacro para a coluna torácica superior com cortadores de roedores.</li>
	<li>Limpar e esterilizar a pele com lavagens triplas de solução alternada de betadina e álcool isopropílico.</li>
	<li>Transferir camundongos anestesiados e esterilizados em decúbito ventral para leito cirúrgico estéril mantendo anestesia com administração inalatória de isoflurano (2%) via cone nasal.</li>
	<li>Flexionar ao máximo os quadris e identificar a posição do joelho ao nível da coluna vertebral para aproximar o corpo vertebral lombar 4.</li>
	<li>Fazer uma incisão longitudinal de 2 cm através da pele com bisturi cirúrgico de 15 lâminas.</li>
	<li>Palpar os processos espinhosos para confirmar a linha média e continuar a incisão até o osso.</li>
	<li>Dissecar subperiostealmente no lado direito do processo espinhoso de L4, estendendo-se lateralmente ao processo transverso.</li>
	<li>Passar uma sutura absorvível trançada tamanho 5-0 cefálica e caudada ao corpo de L4 através da fáscia e deixar aberta, em preparação para futuro fechamento.</li>
	<li>Usando uma agulha espinhal de 25 G, resma o processo espinhoso de L4 usando uma agulha espinhal de 25 G e insira um implante de aço inoxidável de grau "L" de 0,1 mm de diâmetro e 1 cm de comprimento ao longo da lâmina com o braço longo posicionando cefálica.</li>
	<li>Inocular o implante com 1 x 103 UFC/2 μL bioluminescente S. aureus Xen36, tendo o cuidado de garantir que toda a solução entre em contato com o implante.</li>
	<li>Aguarde aproximadamente 10 segundos antes de amarrar a sutura absorvível previamente passada após a inoculação para garantir a contenção do inóculo no implante.</li>
	<li>Feche a pele de forma corrida com sutura absorvível.</li>
	<li>Administrar medicamentos para a dor através de injeção subcutânea de buprenorfina (0,1 mg/kg) imediatamente após a paragem e, em seguida, a cada 12 horas durante 3 dias subsequentes.</li>
	<li>Recupere ratos em uma almofada de aquecimento e monitore para retornar à atividade normal.</li>
	<li>Obter radiografias pós-operatórias para confirmar o posicionamento adequado do implante.</li>
</ol>5. Imagem longitudinal de bioluminescência in vivo para medir a carga bacteriana
<ol><li>Anestesiar camundongos com isoflurano inalatório (2%). Confirme a profundidade apropriada da anestesia monitorando as respirações para que permaneçam rítmicas e mais lentas do que quando acordadas e não mudem em resposta a estímulos nocivos (por exemplo, manipulação cirúrgica, pinça dos dedos).</li>
	<li>Remova os pelos do sacro para a coluna torácica superior com cortadores de roedores.</li>
	<li>Carregue camundongos no campo de visão da plataforma de imagem bioluminescente para realizar imagens de bioluminescência in vivo (BLI)19.</li>
	<li>Capture o sinal bioluminescente durante um tempo de aquisição de 5 min. Utilize configurações de binning grandes com um campo de visão de 15 cm (B).</li>
	<li>Repita as etapas 5.1-5.4 nos dias 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28 e 35 do pós-operatório (ou outros dias com base no planejamento experimental específico) para monitorar a carga bacteriana.</li>
	<li>Apresente dados BLI por meio de escala de cores e sobreposição em uma fotografia em tons de cinza. Isolar uma região ovoide padrão de interesse (ROI) usando o software BLI para quantificar o BLI em fluxo total (fótons por segundo) ou fluxo máximo médio (fótons/segundo/centímetro2/esteradiano).</li>
</ol>6. Quantificar bactérias aderentes aos implantes e tecidos circundantes
<ol><li>Eutanásia de camundongos no POD 35 ou em uma data pós-operatória alternativa de escolha com exposição ao dióxido de carbono de acordo com as diretrizes da AVMA. Confirmar eutanásia com luxação cervical secundária.</li>
	<li>Esterilizar a pele dorsal de acordo com o Passo 4.3 e posicionar o camundongo em decúbito ventral em um campo cirúrgico estéril.</li>
	<li>Incisar bruscamente a incisão anterior com bisturi cirúrgico de 15 lâminas.</li>
	<li>Use tesoura estéril para dissecar bruscamente o processo espinhoso de L4 e identificar o implante cirúrgico.</li>
	<li>Use um driver de agulha para torcer e remover suavemente o implante de sua posição no processo espinhoso L4.</li>
	<li>Com pinça e tesoura estéreis, retirar aproximadamente 0,1 g de osso espinhoso e tecido mole imediatamente ao redor do implante cirúrgico e colocar 1 mL de PBS em rinotubo cônico pequeno com 4 esferas homogeneizadoras afiadas.</li>
	<li>Registrar o peso dos tecidos moles pesando o tubo cônico antes e depois da colheita.</li>
	<li>Colocar o implante em 0,5 mL de Tween-80 a 0,3% em TSB e sonicar por 15 min.</li>
	<li>Vórtice a suspensão resultante do implante por 2 min e cultura durante a noite por 12-16 h.</li>
	<li>Homogeneizar os tecidos moles e os processos espinhosos previamente colocados em 1 ml de PBS ao redor do implante com homogeneizador.</li>
	<li>Vórtice a suspensão de tecidos moles resultante por 5 min e cultura durante a noite por 12-16 h.</li>
	<li>Após a cultura noturna, conte as UFC do implante e dos tecidos circundantes, respectivamente. Valor expresso como UFC total/g colhido para tecido mole e UFC/mL para implante sonicado.</li>
</ol>

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Os autores não têm conflitos de interesse a declarar.

Infecções relacionadas ao implante na coluna vertebral pressagiam maus resultados para os pacientes 1,2,3,4,5. Ao contrário de muitas outras áreas do corpo, o hardware infectado na coluna frequentemente não pode ser removido devido ao risco de instabilidade e comprometimento neurológico. Esse desafio único no cenário de bactérias do biofilme resistent...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated from:
Benjamin V. Kelley1 
Stephen D. Zoller1 
Danielle Greig1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles
to:
Benjamin V. Kelley1 
Christopher Hamad1 
Stephen D. Zoller1 
Danielle Greig1 
Zeinab Mamouei1 
Rene Chun1 
Kellyn Hori1 
Nicolas Cevallos1 
Chad Ishmael1 
Peter Hsiue1 
Rishi Trikha1 
Troy Sekimura2 
Brandon Gettleman3 
Autreen Golzar2 
Adrian Lin2 
Thomas Olson2 
Ameen Chaudry2 
Michael M. Le2 
Anthony A. Scaduto1 
Kevin P. Francis1 
Nicholas M. Bernthal1 
1Department of Orthopaedic Surgery, University of California Los Angeles 
2David Geffen School of Medicine, University of California Los Angeles 
3University of South Carolina School of Medicine, University of South Carolina

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60560">In vivo Mouse Model of Spinal Implant Infection</a>. The Authors section was updated.

Erratum: In vivo Mouse Model of Spinal Implant Infection

O objetivo deste método é descrever um novo modelo de infecção de implante espinhal (LIP) em camundongos. Esse modelo foi projetado para fornecer uma ferramenta barata e precisa para avaliar de forma flexível o efeito de variáveis do hospedeiro, patógeno e/ou implante in vivo. Testar potenciais estratégias terapêuticas e de tratamento para infecções de implantes espinhais neste modelo visa orientar o desenvolvimento de pesquisas antes da aplicação em modelos animais maiores e ensaios clínicos.
A infecção relacionada ao implante após cirurgia de coluna é uma complicação devastadora e, infelizmente, ocorre em aproximadamente 3% a 8% dos pacientes submetidos à cirurgia eletiva da colunavertebral 1,2,3,4,5 e em até 65% dos pacientes submetidos à cirurgia multinível ou de revisão6. O tratamento de infecções de implantes espinhais geralmente requer várias hospitalizações, múltiplas cirurgias e antibioticoterapia prolongada. As SIIs pressagiam resultados ruins para os pacientes, incluindo comprometimento neurológico, incapacidade e aumento do risco de mortalidade. O manejo da LIF é extremamente caro, custando mais de US$ 900.000 por paciente7.
Staphylococcus aureus é o patógeno virulento mais comum da LIF8,9,10,11. As bactérias podem semear o hardware diretamente durante a cirurgia, através da ferida durante o período pós-operatório, ou mais tarde via disseminação hematogênica. Na presença de implantes metálicos, o S. aureus forma biofilme que protege as bactérias da antibioticoterapia e das células imunes. Embora a remoção de hardware infectado possa ajudar efetivamente a erradicar uma infecção, isso frequentemente não é viável na coluna vertebral sem causar desestabilização e risco de comprometimento neurológico12.
Na ausência de explantio de hardware infectado, novas abordagens são necessárias para prevenir, detectar e tratar SII. Historicamente, há modelos animais limitados de SII para avaliar eficientemente a segurança e eficácia de novas terapias. Modelos animais anteriores de LIF requerem grande número de animais e coleta de pontos de dados que requerem eutanásia, incluindo contagem de colônias, histologia e cultura13,14,15. Na falta de monitoramento longitudinal in vivo, esses modelos fornecem apenas um ponto de dados por animal e, portanto, são caros e ineficientes.
Trabalhos anteriores estudando um modelo de infecção por artroplastia de joelho em camundongos estabeleceram o valor e a acurácia da imagem óptica in vivo não invasiva para monitorar longitudinalmente a carga de infecção16. A detecção de bioluminescência permite que a carga bacteriana seja quantificada ao longo de um curso longitudinal de tempo em um único animal de forma humana, precisa e eficiente. Além disso, estudos prévios demonstraram alta correlação entre bioluminescência in vivo e UFCs aderentes a implantes17. A capacidade de rastrear a infecção ao longo do tempo, levou a uma compreensão mais sutil da infecção relacionada ao implante. Além disso, monitorar a infecção longitudinal dessa forma permitiu avaliar com precisão a efetividade da antibioticoterapia e de novos antimicrobianos16,17,18.
Aproveitando essas ferramentas, desenvolvemos e validamos um modelo de infecção pós-operatória de implante espinhal. No método apresentado, utilizamos um inóculo de S. aureus Xen36 bioluminescente para estabelecer um modelo in vivo de SII em camundongos para monitorar longitudinalmente a carga bacteriana16,17,18. Este novo modelo fornece uma ferramenta valiosa para testar eficientemente potenciais estratégias de detecção, prevenção e tratamento para SII antes de sua aplicação em modelos animais maiores e ensaios clínicos.

<table>
	<tbody>
		<tr>
			<td>Analytical Balance ME104</td>
			<td>Mettler Toledo</td>
			<td>30029067</td>
			<td>120 g capacity, 0.1 mg readability, backlit LCD, internal adjustment, metal base</td>
		</tr>
		<tr>
			<td>BD Bacto Tryptic Soy Broth</td>
			<td>Becton Dickinson (BD)</td>
			<td>BD 211825</td>
			<td>BD Bacto Tryptic Soy Broth (Soybean-Casein Digest Medium)</td>
		</tr>
		<tr>
			<td>Biomate 3S UV-VIS Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>840-208300</td>
			<td>Spectrophotometer; Thermo Scientific; BioMate 3S; Six-position cell holder; Spectral bandwidth: 1.8nm; Long-life xenon lamp; Store up to 40 test methods; 16L x 13W x 9 in. H; 19 lb.; 100/240V US line cord</td>
		</tr>
		<tr>
			<td>Bioshield 720+ swinging bucket rotor</td>
			<td>Thermo Scientific</td>
			<td>75003183</td>
			<td>Rotor, Swinging bucket; Thermo Scientific; BIOShield 720 high speed; Capacity: 4 x 180mL (0.72L); Angle: 90 deg. ; Max. speed/RCF: 6300rpm/7188 x g; Max. radius: 16.2cm</td>
		</tr>
		<tr>
			<td>Branson Ultrasonics 2510R-MTH (Sonicator)</td>
			<td>Branson Ultrasonics</td>
			<td>CPX952217R</td>
			<td>*similar model, our model is discontinued* Branson Ultrasonics MH Series Heated Ultrasonic Cleaning Bath, 120V, 0.75 gal</td>
		</tr>
		<tr>
			<td>Bullet Blender Storm Homogenizer</td>
			<td>Next Advance</td>
			<td>BBY24M</td>
			<td>The Bullet Blender Storm is the most powerful member of the Bullet Blender family. Homogenize up to 24 of your toughest samples (mouse femur, skin, cartilage, tumor, etc.) in just minutes. Air cooling&#8482; minimizes sample heat up. Uses 1.5ml screw-cap RINO&#174; tubes or snap-cap Eppendorf&#174; Safe-lock&#8482; tubes.</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>Electron Microscopy Sciences</td>
			<td>66118-10</td>
			<td>The Germinator 500 is designed to decontaminate metal micro-dissecting instruments only. It is to be 
			used exclusively for research purposes. The Germinator 500 should not be used as a substitute for 
			traditional methods of terminal sterilization. Effective sterilization cannot be assured due to lack of routine 
			sterilization-efficacy monitoring methods for glass bead sterilization. The Germinator 500 has been 
			designed and built to pass the Validation of Dry Sterilizer Spore Suspension Test: USP XXIII, Part 1211.</td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026282</td>
			<td>Single 150L</td>
		</tr>
		<tr>
			<td>IVIS Lumina X5 Imaging System</td>
			<td>Perkin Elmer</td>
			<td>CLS148590</td>
			<td>The IVIS Lumina X5 high-throughput 2D optical imaging system combines high-sensitivity bioluminescence and fluorescence with high-resolution x-ray into a compact system that fits on your benchtop. With an expanded 5 mouse field of view for 2D optical imaging plus our unique line of accessories to accelerate setup and labeling, it has never been easier or faster to get robust data&#8212;and answers&#8212;on anatomical and molecular aspects of disease.</td>
		</tr>
		<tr>
			<td>MAXQ 4450 Digtial Incubating Bench Shaker</td>
			<td>Thermo Scientific</td>
			<td>SHKE4450</td>
			<td>Shaker, Incubated; Thermo Scientific; Digital; MaxQ 4450; Speed 15 to 500rpm +/-1rpm; 5 deg. C above ambient to 80 deg. C; 120V 50/60Hz</td>
		</tr>
		<tr>
			<td>PBS, Phosphate Buffered Saline</td>
			<td>Fisher Bioreagents</td>
			<td>BP24384</td>
			<td>PBS, Phosphate Buffered Saline, 1X Solution, pH 7.4</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21 Centrifuge, Ventilated</td>
			<td>Thermo Scientific</td>
			<td>75002436</td>
			<td>24 x 1.5/2.0mL rotor with ClickSeal biocontainment lid</td>
		</tr>
		<tr>
			<td>SORVALL LEGEND X1R 120V Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004261</td>
			<td>Centrifuge, Benchtop; Thermo Scientific; Sorvall Legend X1R (Refrigerated), 1L capacity; Max. Speed/RCF 15,200rpm/25,830 x g; CFC-free cooling -10C to +40C; 120V 60Hz</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus - Xen36</td>
			<td>Perkin Elmer</td>
			<td>119243</td>
			<td>Staphylococcus aureus - Xen36 bioluminescent pathogenic bacteria for in vivo and in vitro drug discovery. This product was derived from a parental strain from the American Type Culture Collection, used under license. Staph. aureus-Xen36 possesses a stable copy of the Photorhabdus luminescens lux operon on the native plasmid.</td>
		</tr>
		<tr>
			<td>TUTTNAUER AUTOCLAVE 2540E 120V</td>
			<td>Heidolph Tuttnauer</td>
			<td>23210401</td>
			<td>Sterilizer, Benchtop; Heidolph; Tuttnauer; Model 2540E; Self-contained design with refillable reservoir controls water purity for sterilization; 120V 50/60Hz; 1400w. With electronic controls</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Fisher Bioreagents</td>
			<td>BP338-500</td>
			<td>Tween 80, Fisher BioReagents, Non-ionic detergent for selective protein extraction</td>
		</tr>
		<tr>
			<td>Vortex mixer VX-200</td>
			<td>Labnet Internation</td>
			<td>S0200</td>
			<td>120V touch or continuous mixer, 230V: 0 - 2,850 rpm,120V: 0 - 3,400 rpm</td>
		</tr>
		<tr>
			<td>0.9% Sodium Chloride</td>
			<td>Pfizer Injectables/Hospira</td>
			<td>00409-4888-10</td>
			<td>0.9% Sodium Chloride Injection, USP</td>
		</tr>
	</tbody>
</table>

in vivo mouse model of spinal implant infection

As infecções por implantes na coluna vertebral pressagiam resultados ruins, pois o diagnóstico é desafiador e a erradicação cirúrgica está em desacordo com a estabilidade mecânica da coluna vertebral. O objetivo deste método é descrever um novo modelo de camundongo de infecção por implante espinhal (SII) que foi criado para fornecer uma ferramenta in vivo barata, rápida e precisa para testar potenciais estratégias terapêuticas e de tratamento para infecções de implantes espinhais.
Neste método, apresentamos um modelo de cirurgia espinhal por abordagem posterior, no qual um fio k de aço inoxidável é transfixado no processo espinhoso L4 de camundongos selvagens C57BL/6J com 12 semanas de idade e inoculado com 1 x 103 UFC de uma cepa bioluminescente da bactéria Staphylococcus aureus Xen36. Os camundongos são então fotografados longitudinalmente para bioluminescência in vivo nos dias de pós-operatório 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28 e 35. Sinais de imagem de bioluminescência (BLI) de um campo de visão padronizado são quantificados para medir a carga bacteriana in vivo.
Para quantificar as bactérias aderidas aos implantes e ao tecido peri-implantar, camundongos são eutanasiados e o implante e o tecido mole circundante são colhidos. As bactérias são destacadas do implante por sonicação, cultivadas durante a noite e, em seguida, as unidades formadoras de colônias (UFCs) são contadas. Os resultados obtidos a partir deste método incluem contagens bacterianas longitudinais medidas pela bioluminescência de S. aureus in vivo (fluxo máximo médio) e contagens de UFC após eutanásia.
Enquanto modelos animais anteriores de infecção instrumentada da coluna vertebral envolveram análise invasiva ex vivo de tecido, o modelo de SII em camundongos apresentado neste artigo aproveita imagens ópticas in vivo não invasivas e em tempo real de bactérias bioluminescentes para substituir o estudo de tecido estático. As aplicações do modelo são amplas e podem incluir a utilização de cepas bacterianas bioluminescentes alternativas, a incorporação de outros tipos de camundongos geneticamente modificados para estudar contemporaneamente a resposta imune do hospedeiro e avaliar as atuais ou investigar novas modalidades diagnósticas e terapêuticas, como antibióticos ou revestimentos de implantes.

O procedimento aqui apresentado foi utilizado para avaliar a eficácia de esquemas de antibióticos em um modelo in vivo de SII em camundongos. Especificamente, a eficácia da antibioticoterapia combinada vancomicina e rifampicina foi comparada à vancomicina em monoterapia e controles infectados não tratados.
Antes da cirurgia, os camundongos foram randomizados para terapia combinada, monoterapia ou controle infectado. Uma análise de poder estatístico foi realizada para calcular o tamanho ...

Watch this Scientific Journal Video about In Vivo Mouse Model of Spinal Implant Infection at JoVE.com

In Vivo Mouse Model of Spinal Implant Infection

O protocolo descreve um novo modelo in vivo de infecção de implante espinhal em camundongos, onde um implante de fio k de aço inoxidável é infectado com Staphylococcus aureus Xen36 bioluminescente. A carga bacteriana é monitorada longitudinalmente com imagens bioluminescentes e confirmada com contagens de unidades formadoras de colônias após a eutanásia.

Modelo in vivo de infecção de implante espinhal em camundongos

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

in-vivo-mouse-model-of-spinal-implant-infection

Research

JoVE Journal

Medicine

2.2K visualizações.  University of California Los Angeles. O protocolo descreve um novo modelo in vivo de infecção de implante espinhal em camundongos, onde um implante de fio k de aço inoxidável é infectado com Staphylococcus aureus Xen36 bioluminescente. A carga bacteriana é monitorada longitudinalmente com imagens bioluminescentes e confirmada com contagens de unidades formadoras de colônias após a eutanásia.

Vídeo: In Vivo Mouse Model of Spinal Implant Infection

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex Infecção Vivo de tecido vivo com Vírus Oncolíticos

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Medicina

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Avaliação longitudinal do rato Perda Hind Limb óssea após lesão medular usando Novel, In vivo Metodologia,

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Testes clínicos e Remoção da Medula Espinhal em um modelo do rato para a Esclerose Lateral Amiotrófica (ELA)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

Um modelo de contusão grave de lesão medular em ratos

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Combinado<em&gt; In vivo</em&gt; Optical e μCT imagem para monitorar infecções, inflamações, and Bone Anatomy em uma infecção Orthopaedic Implant em Ratos

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Controlado Modelo Impact Cortical para lesão cerebral traumática

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetria Intrauterine medir Rato contrátil Pressão<em&gt; In Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

A Neonatal do rato Spinal Cord Injury Compression Modelo

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

De células-tronco Baseado Imunidade Engineered contra infecção por HIV no modelo humanizado mouse

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...