Los autores desean agradecer la recepción de la subvención de la columna vertebral Biomet de la Sociedad Ortopédica Pediátrica de América del Norte y la subvención KL2 del Instituto de Ciencias Clínicas y Traslacionales de los Institutos Nacionales de Salud, y la subvención de investigación quirúrgica HH Lee como las principales fuentes de financiación para estos experimentos.

Todos los animales fueron manejados en estricta conformidad con las buenas prácticas animales según se define en las regulaciones federales según lo establecido en la Ley de Bienestar Animal (AWA), la Guía de 1996 para el Cuidado y Uso de Animales de Laboratorio, la Política de PHS para el Cuidado y Uso Humanitario de Animales de Laboratorio, así como las políticas y procedimientos de la institución según lo establecido en el Manual de Capacitación para el Cuidado y Uso de Animales. y todo el trabajo con animales fue aprobado por el Comité de Investigación Animal (ARC) del Canciller de la Universidad de California en Los Ángeles.
1. Elección de la cepa bioluminiscente de S. aureus
<ol><li>Utilice la cepa bioluminiscente de S. aureus Xen36 como inóculo de interés. NOTA: Esta cepa se derivó de la cepa parental S. aureus ATCC-49525, que es un aislado clínico de un paciente séptico. S. aureus Xen36 utiliza de forma única un operón ABCDE lux, que está optimizado e integrado en el plásmido nativo del huésped. 19 Como resultado, la cepa Xen36 es capaz de producir una luz bioluminiscente azul-verde con una emisión de longitud de onda máxima de 490 nm. Esta señal de emisión solo es producida por organismos bacterianos vivos metabólicamente activos.</li>
</ol>2. Preparación de S. Aureus para inoculación
<ol><li>Añadir 200 μg/mL de kanamicina al caldo Luria más agar al 1,5% para aislar S. aureus Xen36 de posibles contaminantes, utilizando el gen de resistencia a la kanamicina unido al operón lux 19.</li>
	<li>Escarbar la bacteria S. aureus Xen36 en placas de agar tríptico de soja (caldo de soja tríptico [TSB] más agar al 1,5%) e incubar a 37 °C durante 12-16 h.</li>
	<li>Aislar colonias individuales de S. aureus Xen36 y cultivar individualmente en TSB durante 12-16 h a 37 °C en una incubadora agitadora (200 rpm).</li>
	<li>Diluir el cultivo resultante en una proporción de 1:50.</li>
	<li>Cultivo durante 2 h adicionales a 37 °C para aislar bacterias de fase logarítmica media.</li>
	<li>Gellet, resuspenda y lave las bacterias en solución salina tamponada con fosfato (PBS) tres veces.</li>
	<li>Mida la absorbancia a 600 nm. El OD600 ideal está entre 0,700 y 0,750, lo que corresponde a 4x105 UFC/mL. Realice diluciones seriadas para lograr el inóculo bacteriano deseado (1 x 103 UFC/2 μL). NOTA: Se encontró que la concentración óptima de Xen36 para el establecimiento de una infección crónica era de 1 x 103 UFC. El sistema inmunitario del huésped eliminó una dosis más baja de bacterias y una dosis más alta provocó la ruptura de la herida. La ruptura de la herida no diferencia entre una infección profunda del implante y una infección superficial de la herida y, por lo tanto, se evita en este modelo (Figura 1)20.</li>
</ol>3. Ratones
<ol><li>Utilice ratones machos C57BL/6J de 12 semanas de edad.</li>
	<li>Ratones domésticos en jaulas con un máximo de 4 a la vez.</li>
	<li>Mantenga agua disponible en todo momento. Mantenga un ciclo de luz/oscuridad de 12 horas y no realice experimentos durante la fase oscura del ciclo.</li>
	<li>Utilice comida libre de alfalfa para la alimentación debido a la posible interferencia con la señalización fluorescente.</li>
	<li>Haga que el personal de investigación o veterinario evalúe a los ratones diariamente para garantizar el bienestar de los animales durante todo el experimento.</li>
</ol>4. Procedimientos quirúrgicos con ratones
<ol><li>Inducir la anestesia colocando ratones en una cámara de isoflurano (2%) durante aproximadamente 5 minutos. Confirme la profundidad adecuada de la anestesia monitoreando las respiraciones para que permanezcan rítmicas y más lentas que cuando están despiertos y no cambien en respuesta a estímulos nocivos (p. ej., manipulación quirúrgica, pellizco de los dedos de los pies).</li>
	<li>Transfiera a los ratones anestesiados a una estación de preparación y elimine el vello del sacro a la parte superior de la columna torácica con una maquinilla para roedores.</li>
	<li>Limpiar y esterilizar la piel con lavados triples de solución alterna de betadine y alcohol isopropílico.</li>
	<li>Transferir ratones anestesiados y esterilizados en decúbito prono a una cama quirúrgica estéril manteniendo la anestesia con la administración de isoflurano inhalado (2%) a través del cono nasal.</li>
	<li>Flexionar al máximo las caderas e identificar la posición de la rodilla a nivel de la columna vertebral para aproximarse al cuerpo vertebral lumbar 4.</li>
	<li>Haga una incisión longitudinal de 2 cm a través de la piel con un bisturí quirúrgico de 15 hojas.</li>
	<li>Palpe las apófisis espinosas para confirmar la línea media y continúe la incisión hasta el hueso.</li>
	<li>Diseccionar subperiósticamente en el lado derecho de la apófisis espinosa L4, extendiéndose lateralmente hasta la apófisis transversa.</li>
	<li>Pasar una sutura trenzada reabsorbible tamaño 5-0 cefálica y caudad al cuerpo L4 a través de la fascia y dejar abierta, en preparación para un futuro cierre.</li>
	<li>Con una aguja espinal de 25 G, escariar la apófisis espinosa de L4 con una aguja espinal de 25 G e insertar un implante de acero inoxidable de grado quirúrgico en forma de "L" de 0,1 mm de diámetro y 1 cm de largo a lo largo de la lámina con el brazo largo que yace cefálico.</li>
	<li>Inocular el implante con 1 x 103 UFC/2 μL de S. aureus Xen36 bioluminiscente, teniendo cuidado de que toda la solución entre en contacto con el implante.</li>
	<li>Espere aproximadamente 10 segundos antes de atar la sutura absorbible previamente pasada después de la inoculación para asegurar la contención del inóculo en el implante.</li>
	<li>Cierre la piel en forma de corrido con sutura absorbible.</li>
	<li>Administrar analgésicos mediante inyección subcutánea de buprenorfina (0,1 mg/kg) inmediatamente por vía posoperatoria y luego cada 12 horas durante 3 días a partir de entonces.</li>
	<li>Recupere los ratones en una almohadilla térmica y controle el regreso a la actividad normal.</li>
	<li>Obtener radiografías postoperatorias para confirmar la colocación adecuada del implante.</li>
</ol>5. Imágenes longitudinales de bioluminiscencia in vivo para medir la carga bacteriana
<ol><li>Anestesiar ratones con isoflurano inhalado (2%). Confirme la profundidad adecuada de la anestesia monitoreando las respiraciones para que permanezcan rítmicas y más lentas que cuando están despiertos y no cambien en respuesta a estímulos nocivos (p. ej., manipulación quirúrgica, pellizco de los dedos de los pies).</li>
	<li>Retire el vello desde el sacro hasta la parte superior de la columna torácica con una maquinilla para roedores.</li>
	<li>Cargue ratones en el campo de visión de la plataforma de imágenes bioluminiscentes para realizar imágenes de bioluminiscencia (BLI) in vivo19.</li>
	<li>Captura la señal bioluminiscente durante un tiempo de adquisición de 5 minutos. Utilice configuraciones de agrupación grandes con un campo de visión de 15 cm (B).</li>
	<li>Repita los pasos 5.1-5.4 en los días postoperatorios 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28 y 35 (u otros días basados en un diseño experimental específico) para controlar la carga bacteriana.</li>
	<li>Presente los datos BLI a través de una escala de colores y superponga en una fotografía en escala de grises. Aísle una región de interés ovoide (ROI) estándar utilizando el software BLI para cuantificar BLI en flujo total (fotones por segundo) o flujo máximo medio (fotones/segundo/centímetro2/esteradiano).</li>
</ol>6. Cuantificar las bacterias adheridas a los implantes y al tejido circundante
<ol><li>Sacrificar ratones en POD 35 o en una fecha postoperatoria alternativa de elección con exposición al dióxido de carbono de acuerdo con las Directrices AVMA. Confirmar la eutanasia con luxación cervical secundaria.</li>
	<li>Esterilizar la piel dorsal de acuerdo con el paso 4.3 y colocar al ratón boca abajo en un campo quirúrgico estéril.</li>
	<li>Haga una incisión aguda en la incisión anterior con un bisturí quirúrgico de 15 hojas.</li>
	<li>Utilice unas tijeras estériles para diseccionar sin rodeos la apófisis espinosa L4 e identificar el implante quirúrgico.</li>
	<li>Utilice un destornillador de agujas para girar suavemente y retirar el implante de su posición en la apófisis espinosa L4.</li>
	<li>Con pinzas y tijeras estériles, extraiga aproximadamente 0,1 g de hueso de proceso espinoso y tejido blando que rodea inmediatamente el implante quirúrgico y colóquelo en 1 ml de PBS en un pequeño tubo cónico de rinoceronte con 4 perlas homogeneizadoras afiladas.</li>
	<li>Registre el peso de los tejidos blandos pesando el tubo cónico antes y después de la cosecha.</li>
	<li>Colocar el implante en 0,5 mL de Tween-80 al 0,3% en TSB y sonicar durante 15 min.</li>
	<li>Agitar la suspensión del implante resultante durante 2 min y cultivar durante la noche durante 12-16 h.</li>
	<li>Homogeneizar los tejidos blandos y las apófisis espinosas previamente colocadas en 1 ml de PBS rodeando el implante mediante homogeneizador.</li>
	<li>Agitar la suspensión de tejidos blandos resultante durante 5 min y cultivar durante la noche durante 12-16 h.</li>
	<li>Después del cultivo durante la noche, cuente las UFC del implante y de los tejidos circundantes, respectivamente. Exprese el valor como UFC/g totales recolectadas para tejidos blandos y UFC/ml para implantes sonicados.</li>
</ol>

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Los autores no tienen conflictos de intereses que revelar.

Las infecciones relacionadas con implantes en la columna vertebral presagian malos resultados para los pacientes 1,2,3,4,5. A diferencia de muchas otras áreas del cuerpo, el hardware infectado en la columna vertebral con frecuencia no se puede eliminar debido al riesgo de inestabilidad y compromiso neurológico. Este desafío único en el entorno de bacterias...

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

El propósito de este método es describir un nuevo modelo murino de infección por implante espinal (SII). Este modelo fue diseñado para proporcionar una herramienta económica y precisa para evaluar de manera flexible el efecto de las variables del huésped, el patógeno y/o el implante in vivo. La prueba de posibles terapias y estrategias de tratamiento para las infecciones de implantes espinales en este modelo tiene como objetivo guiar el desarrollo de la investigación antes de su aplicación en modelos animales más grandes y ensayos clínicos.
La infección relacionada con los implantes después de la cirugía de columna vertebral es una complicación devastadora y, por desgracia, ocurre en aproximadamente el 3-8% de los pacientes que se someten a cirugía electiva de columnavertebral 1,2,3,4,5 y hasta el 65% de los pacientes que se someten a cirugía multinivel o de revisión 6. El tratamiento de las infecciones de los implantes espinales a menudo requiere múltiples hospitalizaciones, múltiples cirugías y terapia antibiótica prolongada. Los SII presagian malos resultados para los pacientes, como el compromiso neurológico, la discapacidad y un mayor riesgo de mortalidad. El manejo del SII es extremadamente costoso, con un costo de más de $900,000 por paciente7.
Staphylococcus aureus es el patógeno virulento más común del SII 8,9,10,11. Las bacterias pueden sembrar el hardware directamente durante la cirugía, a través de la herida durante el período postoperatorio o más tarde a través de la diseminación hematógena. En presencia de implantes metálicos, S. aureus forma una biopelícula que protege a las bacterias de la terapia antibiótica y de las células inmunitarias. Si bien la eliminación del hardware infectado puede ayudar a erradicar eficazmente una infección, esto con frecuencia no es factible en la columna vertebral sin causar desestabilización y riesgo de compromiso neurológico12.
En ausencia de la explantación de hardware infectado, se necesitan enfoques novedosos para prevenir, detectar y tratar el SII. Históricamente, ha habido modelos animales limitados de SII para evaluar de manera eficiente la seguridad y eficacia de las nuevas terapias. Los modelos animales anteriores de SII requieren un gran número de animales y la recolección de puntos de datos que requieren eutanasia, incluido el recuento de colonias, la histología y el cultivo13,14,15. Al carecer de un seguimiento longitudinal in vivo, estos modelos solo proporcionan un punto de datos por animal y, por lo tanto, son caros e ineficientes.
Trabajos anteriores que estudiaron un modelo de ratón de infección por artroplastia de rodilla establecieron el valor y la precisión de las imágenes ópticas in vivo no invasivas para monitorizar longitudinalmente la carga de infección16. La detección de la bioluminiscencia permite cuantificar la carga bacteriana a lo largo de un curso de tiempo longitudinal en un solo animal de forma humana, precisa y eficiente. Además, estudios previos han demostrado una alta correlación entre la bioluminiscencia in vivo y las UFC adheridas a implantes17. La capacidad de rastrear la infección a lo largo del tiempo ha llevado a una comprensión más matizada de la infección relacionada con los implantes. Además, el monitoreo de la infección longitudinal de esta manera, ha permitido evaluar con precisión la efectividad de la terapia antibiótica y de los nuevos antimicrobianos16,17,18.
Aprovechando estas herramientas, desarrollamos y validamos un modelo de infección postoperatoria de implantes espinales. En el método presentado, utilizamos un inóculo de S. aureus Xen36 bioluminiscente para establecer un modelo murino in vivo de SII para monitorear longitudinalmente la carga bacteriana16,17,18. Este novedoso modelo proporciona una herramienta valiosa para probar de manera eficiente las posibles estrategias de detección, prevención y tratamiento del SII antes de su aplicación en modelos animales más grandes y ensayos 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

Las infecciones de los implantes de columna vertebral presagian malos resultados, ya que el diagnóstico es difícil y la erradicación quirúrgica está en desacuerdo con la estabilidad mecánica de la columna. El propósito de este método es describir un nuevo modelo de ratón de infección por implante espinal (SII) que se creó para proporcionar una herramienta in vivo económica, rápida y precisa para probar posibles terapias y estrategias de tratamiento para las infecciones de implantes espinales.
En este método, presentamos un modelo de cirugía espinal de abordaje posterior en el que se transfija un alambre k de acero inoxidable en la apófisis espinosa L4 de ratones de tipo salvaje C57BL/6J de 12 semanas de edad y se inocula con 1 x 103 UFC de una cepa bioluminiscente de la bacteria Staphylococcus aureus Xen36. A continuación, se obtienen imágenes longitudinales de la bioluminiscencia de los ratones in vivo en los días postoperatorios 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28 y 35. Las señales de imágenes de bioluminiscencia (BLI) de un campo de visión estandarizado se cuantifican para medir la carga bacteriana in vivo.
Para cuantificar las bacterias que se adhieren a los implantes y al tejido periimplantario, se sacrifica a los ratones y se extrae el implante y el tejido blando circundante. Las bacterias se desprenden del implante mediante sonicación, se cultivan durante la noche y luego se cuentan las unidades formadoras de colonias (UFC). Los resultados obtenidos de este método incluyen recuentos bacterianos longitudinales medidos por bioluminiscencia in vivo de S. aureus (flujo máximo medio) y recuentos de UFC después de la eutanasia.
Si bien los modelos animales anteriores de infección instrumentada de la columna vertebral han implicado un análisis invasivo de tejido ex vivo, el modelo de ratón de SII presentado en este artículo aprovecha las imágenes ópticas in vivo no invasivas y en tiempo real de bacterias bioluminiscentes para reemplazar el estudio de tejido estático. Las aplicaciones del modelo son amplias y pueden incluir la utilización de cepas bacterianas bioluminiscentes alternativas, la incorporación de otros tipos de ratones modificados genéticamente para estudiar simultáneamente la respuesta inmunitaria del huésped y la evaluación de las modalidades diagnósticas y terapéuticas actuales o la investigación de nuevas modalidades, como antibióticos o recubrimientos de implantes.

El procedimiento que aquí se presenta se utilizó para evaluar la eficacia de los regímenes antibióticos en un modelo de SII en ratones in vivo. Específicamente, se comparó la eficacia de la terapia combinada con antibióticos con vancomicina y rifampicina con la monoterapia con vancomicina y los controles infectados no tratados.
Antes de la cirugía, los ratones fueron aleatorizados a terapia combinada, monoterapia o control infectado. Se realizó un análisis estadístico de potencia pa...

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

El protocolo describe un nuevo modelo de ratón in vivo de infección por implante espinal en el que un implante de alambre k de acero inoxidable está infectado con Staphylococcus aureus Xen36 bioluminiscente. La carga bacteriana se monitoriza longitudinalmente con imágenes bioluminiscentes y se confirma con recuentos de unidades formadoras de colonias después de la eutanasia.

Modelo de ratón in vivo de infección por implante espinal

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 vistas.  University of California Los Angeles. El protocolo describe un nuevo modelo de ratón in vivo de infección por implante espinal en el que un implante de alambre k de acero inoxidable está infectado con Staphylococcus aureus Xen36 bioluminiscente. La carga bacteriana se monitoriza longitudinalmente con imágenes bioluminiscentes y se confirma con recuentos de unidades formadoras de colonias después de la eutanasia.

Video: 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 infección in vivo del tejido vivo con virus oncolíticos

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

Investigación

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.

Evaluación longitudinal de ratón posteriores pérdida de masa ósea del miembro después de una lesión de la médula espinal utilizando Novel, En vivo Metodología

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.

Los ensayos clínicos y de eliminación de la médula espinal en un modelo murino de esclerosis 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.

Un modelo Contusión de la lesión de la médula espinal severa en ratas

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; En vivo</em&gt; Óptica y μCT imágenes para vigilar la infección, inflamación y Hueso anatomía en una infección de implantes ortopédicos en ratones

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 de Impacto cortical de la lesión 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.

Telemetría intrauterina para medir la presión del ratón contráctil<em&gt; En 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.

Un ratón neonatal lesión de médula espinal de compresión 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.

-Base de células madre Inmunidad Diseñado frente a la infección por VIH en el modelo de ratón humanizado

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.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

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 ...