The authors are grateful for the support from the Center for Predicative Medicine Animal Models (Carol Vanover, Ashley Biller and Jennifer Kraenzle) and Microbiology (Daniel Cramer and Julie Sotsky) Core Facilities. This work was supported by funding from the NIH (HHSN272201000033I to M.B.L and J.M.W.).

NOTA: Todos los procedimientos descritos aquí fueron revisados ​​y aprobados por la Universidad de Louisville Comité Institucional de Bioseguridad (protocolo # 13-056) e Institucional Cuidado de Animales y el empleo Comisión (protocolo # 13-064). 1. Preparación del Tinte <ol><li> Diluir 0,1% (w / v) azul brillante de Coomassie en PBS y esterilizar filtro utilizando un filtro de jeringa de 0,45 mM. </li></ol> 2. Preparación de Pseudomonas aeruginosa Cultura <ol><li> 15 horas antes de la instilación, se inoculan 3 ml de caldo de cultivo con una sola colonia bacteriana. </li><li> Crece la cultura hr 15 a 37 ° C en un agitador (200 rpm). </li><li> Centrifugar 1 ml de cultivo en un tubo de microcentrífuga de 1,5 ml 12.000 xg durante 30 seg. </li><li> Retire el medio y volver a suspender el sedimento en 1 ml de PBS. </li><li> Diluir una alícuota de la suspensión bacteriana Stock 1:10 en PBS y medir el OD 600 de la suspensión bacteriana diluida para determinar elconcentración bacteriana. </li><li> Diluir la suspensión bacteriana de valores en PBS a la concentración deseada de inóculo bacteriano, utilizando un volumen de entrega de 50 l para la inoculación IMIT. </li></ol> 3. IMIT instilación <ol><li> Colocar un grupo de ratones en la cámara de inducción de anestesia con isoflurano y anestesiar utilizando un 2 - mezcla de isoflurano / oxígeno del 3%. </li><li> En el inicio de la sedación inicial, pescuezo del ratón, sostiene el ratón en posición vertical, y administrar 10 l de una solución de lidocaína al 2% en aguja de sonda a la parte posterior de la garganta y permitir que la solución fluya hacia abajo para la epiglotis. Devuelva el ratón a la cámara de la anestesia. <ol><li> Deje un mínimo de 5 minutos para permitir la lidocaína a surtir efecto completo como anestésico local. </li></ol></li><li> Cuando los ratones han alcanzado el nivel deseado de sedación (tasa de ~ 60 bpm de respiración), reducir el isoflurano al 2% para mantener la sedación. </li><li> Precarga tinte o inóculo bacteriano en un 250 l hermética a los gasesen forma de jeringa de precisión con una aguja roma largo 22 G. <ol><li> En primer lugar la elaboración de 150 l de aire, medida por el émbolo de teflón de la jeringa. A continuación, la elaboración de 50 l de inóculo haciendo avanzar el émbolo de teflón de la marca de 150 l hasta la marca de 200 l en el cuerpo de la jeringa. NOTA: Cuando la muestra es expulsada en un ratón intubado, la suspensión 50 l será entregado primero, seguido por un cojín de aire 150 l que distribuirá el inóculo en todo el pulmón. </li></ol></li><li> Retire un ratón desde la cámara de inducción y poner en posición supina sobre una plataforma de intubación. Asegure el ratón a la plataforma enganchando sus incisivos con una junta tórica adjunta a la tira de velcro, y asegurar el velcro a la plataforma. Levante el ratón a una inclinación de 45 °. </li><li> El uso de un aplicador de micro algodón, retraer la lengua con un movimiento de balanceo, utilizando la mano dominante. </li><li> Con la mano no dominante, utilice un ajuste otoscopio operando con un espéculos intubación tanto maintain retracción de la lengua y visualizar la glotis. </li><li> Con la mano dominante, utilice un alambre de guía roscada a través de un catéter de 20 G de intubar el ratón, asentar el catéter a una profundidad de 10 mm en la tráquea de ratón (catéter está en forma de un manguito de silicio con 10 mm de catéter expuesto). Retire el otoscopio / espéculos. </li><li> Confirmar que el ratón ha sido intubado correctamente por asegurar el catéter con la mano no dominante mientras se coloca brevemente una longitud conectado Luer de 1/16 &quot;tubo transparente que contiene un colorante de color. NOTA: El tinte migrar rápidamente hacia atrás y adelante en respuesta a la respiración. </li><li> No continúe con los pasos siguientes, si la confirmación de la intubación no se ha establecido en este punto. Si la intubación intentado realizar falló, restablecer el alambre y catéter guía para un intento adicional en la intubación. NOTA: Es aconsejable intentar más de dos intubaciones de un ratón en una sesión sin causar traumatismo en el ratón. </li><li> Continuarasegurar el catéter con la mano no dominante, mientras que la inserción de la / aguja roma jeringa de precisión que contiene el amortiguador de suspensión líquido / aire. </li><li> Dispensar el líquido / aire directamente en el pulmón en un solo movimiento fluido y retire inmediatamente la aguja / catéter del ratón. </li><li> Devuelva el ratón a una jaula y permitir la recuperación de la anestesia. </li></ol> 4. Caracterización de las IMT Entrega <ol><li> Después de la instilación IMIT, la eutanasia el ratón por asfixia de CO 2 en un momento apropiado después de la inoculación. </li><li> Asegure el ratón sacrificados en un tablero de disección y tomar el pecho y el abdomen con EtOH al 70% utilizando una jeringa. </li><li> Si la evaluación de la distribución de un agente de imagen en todo el pulmón, eliminar los pulmones de los animales utilizando una técnica estéril y mostrar los pulmones en su caso para la imagen. NOTA:. Los pulmones pueden ser preparados por técnicas de tinción histológicas adicionales a través de la fijación o la criopreservación apropiado &lt;/ Li&gt; <li> Si la evaluación de la carga bacteriana del tejido pulmonar infectado, eliminar los pulmones del animal utilizando una técnica estéril. Coloque los pulmones en un estéril, previamente pesado 1 oz bolsa de muestra. Pesar y registrar el peso de la bolsa de muestra + pulmones. <ol><li> Añadir 1 ml de PBS 1x estéril a cada bolsa de tejidos + muestra. Vuelva a sellar la bolsa de muestra. </li><li> Homogeneizar los tejidos por una suave rodando 25 ml pipeta serológica sobre la bolsa de muestra + tejido. </li><li> Generar diluciones seriadas del homogeneizado de pulmón en PBS estéril y placa en placa de agar (LB, o en su caso de las especies de bacterias en estudio): <ol><li> Llevar a cabo una dilución en serie de seis veces en una placa de fondo en U 96 por pipeta multicanal a continuación, la placa muestras por triplicado por multicanal en la placa de agar. </li></ol></li><li> Incubar las placas de agar durante la noche a 37 ° C y enumerar unidades formadoras de colonias al día siguiente. </li></ol></li></ol>

<ol>
	<li>Reznik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy,+physiology,+and+function+of+the+upper+respiratory+tract.">Comparative anatomy, physiology, and function of the upper respiratory tract.</a> Environ Health Perspect. 85, 171-176 (1990).</li><li>Hoyt, R. F. J., Hawkins, J. V., St. Clair, M. B., Kennet, M. B., Fox, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2.">Chapter 2.</a> The Mouse in Biomedical Research, Volume 2, Second Edition: Diseases (American College of Laboratory Animal Medicine). 2, 23-90 (2007).</li><li>Visweswaraiah, A., Novotny, L. A., Hjemdahl-Monsen, E. J., Bakaletz, L. O., Thanavala, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+tissue+distribution+of+marker+dye+following+intranasal+delivery+in+mice+and+chinchillas:+a+multifactorial+analysis+of+parameters+affecting+nasal+retention.">Tracking the tissue distribution of marker dye following intranasal delivery in mice and chinchillas: a multifactorial analysis of parameters affecting nasal retention.</a> Vaccine. 20, 3209-3220 (2002).</li><li>Eyles, J. E., Spiers, I. D., Williamson, E. D., Alpar, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution+of+radioactivity+following+intranasal+administration+of+radioactive+microspheres.">Tissue distribution of radioactivity following intranasal administration of radioactive microspheres.</a> J Pharm Pharmacol. 53, 601-607 (2001).</li><li>Southam, D. S., Dolovich, M., O'Byrne, P. M., Inman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+intranasal+instillations+in+mice:+effects+of+volume,+time,+body+position,+and+anesthesia.">Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia.</a> Am J Physiol Lung Cell Mol Physiol. 282, 833-839 (2002).</li><li>Miller, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+murine+intranasal+dosing+efficiency+using+luminescent+Francisella+tularensis:+effect+of+instillation+volume+and+form+of+anesthesia.">Visualization of murine intranasal dosing efficiency using luminescent Francisella tularensis: effect of instillation volume and form of anesthesia.</a> PLoS ONE. 7, (2012).</li><li>Lathem, W. W., Crosby, S. D., Miller, V. L., Goldman, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+primary+pneumonic+plague:+a+mouse+model+of+infection,+pathology,+and+bacterial+transcriptional+activity.">Progression of primary pneumonic plague: a mouse model of infection, pathology, and bacterial transcriptional activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 17786-17791 (2005).</li><li>Warawa, J. M., Long, D., Rosenke, R., Gardner, D., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+diagnostic+imaging+to+characterize+altered+respiratory+tract+colonization+by+the+Burkholderia+pseudomallei+capsule+mutant.">Bioluminescent diagnostic imaging to characterize altered respiratory tract colonization by the Burkholderia pseudomallei capsule mutant.</a> Front Microbiol. 2, 133 (2011).</li><li>Gutierrez, M., Pfeffer, T. L., Warawa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+3+Secretion+System+cluster+3+is+a+critical+virulence+determinant+for+lung-specific+melioidosis.">Type 3 Secretion System cluster 3 is a critical virulence determinant for lung-specific melioidosis.</a> Submitted. , (2014).</li><li>Fodah, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+Klebsiella+pneumoniae+comparative+genetic+analyses+with+virulence+profiles+in+a+murine+respiratory+disease+model.">Correlation of Klebsiella pneumoniae comparative genetic analyses with virulence profiles in a murine respiratory disease model.</a> PLoS ONE. In revision, (2014).</li><li>Soto-Montenegro, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+airway+distribution+of+transnasal+solutions+in+mice+by+PET/CT+imaging.">Assessment of airway distribution of transnasal solutions in mice by PET/CT imaging.</a> Mol Imaging Biol. 11, 263-268 (2009).</li></ol>

The authors have nothing to disclose.

IMIT instillation offers key improvements to existing respiratory disease models in the ability to reproducibly instill reagents directly into the lung. It is a rapid approach which is ideally situated for a team of two researchers, one of which manages the logistics of anesthesia and caging, and the other who performs the IMIT technique. Large studies may be conducted using IMIT with an average time commitment of 2 - 3 min per mouse. Because the approach makes use of isoflurane as an anesthetic, mice recover rapidly from the anesthesia, reducing the husbandry time of monitoring animals through recovery.
The most technically challenging aspect of the IMIT method is the initial step of intubating mice. Individuals learning to perform IMIT are able to focus on this first step of catheter placement and ensuring that intubation has been achieved through the visual confirmation of dye movement. The benefit of the approach is that lung-specific instillation is guaranteed through use of the confirmation of intubation, which increases the confidence of both the new researcher as well as the expert attempting to intubate a difficult animal. The key elements of optimizing the likelihood of a successful intubation are: i) achieving a deep sedation to allow sufficient working time, ii) correct placement of the specula in the mouth to allow good visualization of the epiglottis, iii) good depth placement of the specula so that the tongue remains retracted throughout the procedure, and iv) use of the tilting platform to support the researcher&#8217;s hands so that the procedure is conducted relaxed and with a steady approach.
One of the limitations of the IMIT procedure is related to frequency of IMIT instillation events. Due to the potential trauma associated with a missed intubation, it is not recommended that more than two intubation attempts be conducted in a single session (up to two misses). IMIT has an excellent potential in its ability to be used to deliver therapeutics into the murine lung, however therapeutic regimens which make use of very frequent delivery of reagent into the lung may not be suitable for IMIT. It may be possible that IMIT could be used daily to deliver reagents into a murine lung without causing significant trauma, but only when conducted by a highly skilled researcher, as the majority of trauma associated with intubation is thought to be associated with a missed intubation event. Such high-frequency IMIT should be discussed with local veterinarians and IACUC.
An additional potential limitation of IMIT is the size of the mouse which is being intubated. The IMIT procedure described above was developed using mice of approximately 17 - 22 g, where a 20 G catheter was found to be a suitable size for the trachea of mice in this size range. Larger catheters have been successfully used in older mice; the initial development of IMIT made use of an 18 G catheter in BALB/c mice which are &#62;20 g. Importantly, if alternate catheter sizes are used, blunt needles should be sourced which fit the lumen of the catheter and are trimmed to a length that extends just 1mm beyond the catheter tip. Intubation of mice smaller than 17 g may be possible but is not recommended due to the expertise required, and would require use of smaller catheters and specula than are described above.
We have used IMIT for the delivery of several respiratory pathogens in addition to P. aeruginosa, including B. pseudomallei9 and Klebsiella pneumoniae10. The IMIT model has made important advances to our studies of B. pseudomallei respiratory disease, having identified that intranasal inoculation causes an early, URT-related morbidity of mice rather than the systemic disease endpoint observed in human disease9. B. pseudomallei is a Tier 1 select agent of biodefense impact, and as such, respiratory disease models are being developed for aerosol exposure which models a potential biodefense related route of entry for weaponized pathogens. Because current aerosol models result in infection of both the URT and LRT, the same potential early morbidity phenotypes we have identified for the intranasal model of B. pseudomallei respiratory disease may apply to the aerosol model. A future adaptation of the IMIT model could be an intubation-mediated aerosol delivery (IMAD), in which mice are intubated for target aerosol delivery only into the lung. Mechanical ventilators are currently available to maintain isoflurane anesthesia, which could be adapted to deliver an aerosolized, rather than liquid based, pathogen challenge.
IMIT was developed initially as an approach to optimize the delivery of bacteria to the lung, but also has application for the delivery of other reagents into the mouse lung. As discussed above, intranasal delivery of compounds into mice results in a low efficiency, highly variable delivery of reagents into the target organ of the lung. Intranasal delivery of Positron Emission Tomography (PET) imaging reagents to the murine lung yielded a 40% delivery efficiency11, whereas we have demonstrated that IMIT offers an excellent alternative to other lung delivery approaches with its &#62;98% delivery efficacy and multilobar distribution. This improvement in targeted delivery to the lung has the potential to increase the reproducibility of therapeutic delivery for treatment of pulmonary disease. IMIT could similarly offer benefits to studies of: i) the impact of environmental pulmonary irritants, ii) lung cancer phenotypic studies, iii) lung-specific siRNA knock-down.

Los ratones se han utilizado para modelar numerosas manifestaciones de enfermedades humanas, incluyendo una gran variedad de enfermedades respiratorias. Modelos de enfermedades sustitutos son a menudo incapaces de recapitular todos los aspectos de una enfermedad modelado, por lo general debido a la fisiológica importante o diferencias inmunológicas en los dos modelos de acogida. Por lo tanto, un objetivo de la mejora de los sistemas de modelo es el desarrollo de métodos que permitan a los sustitutos reflejan más de cerca un proceso de enfermedad o de acogida que la respuesta observada en el sistema host original. Hay varias diferencias fisiológicas clave entre ratones y seres humanos en el mecanismo por el que se inspiran aire. Se incluyen en estas diferencias son diferencias significativas en el tamaño radiométricos entre la URT y LRT. Se ha estimado que los ratones que poseen&gt; 100 veces el área de superficie URT relativa a los seres humanos, normalizado contra el total de la capacidad pulmonar 1,2. Por lo tanto, los cornetes nasales del ratón permiten más extensa de filtrado de aire inspirado para facilitar una mayor tasa de breathing, que puede tener un impacto significativo en los estudios de neumonía si la infección de la cavidad nasal juega un papel importante en la progresión de la enfermedad. Se han empleado varios enfoques diferentes para infundir bacterias en los pulmones de ratones para estudiar la enfermedad respiratoria similar a la humana. El más común de estos enfoques es la inoculación intranasal, en el que se aplica una suspensión líquida en uno o ambos orificios nasales de un ratón. Aunque es relativamente sencillo, advertencias, como el volumen de la instilación y el tipo de anestesia que se utiliza puede afectar la eficiencia de la instilación en la LRT a través de la inoculación intranasal 3-5. Específicamente, Miller et al. han mostrado que la instilación intranasal de Francisella tularensis en los volúmenes de menos de 50 l no dio lugar a la instilación de las bacterias en la LRT 6. Observaron además mejor instilación LRT al utilizar isoflurano inhalado en lugar de inyectar ketamina / xilacina para la anestesia. Sin embargo, nuestra experience con Yersinia pestis inoculación intranasal indica la inoculación más consistente se puede lograr usando ketamina / xilacina en comparación con isoflurano (MBL, datos no publicados). Estas diferencias podrían atribuirse a patógeno utilizado o a la variación en los procedimientos de laboratorio, pero importante destacar la variabilidad potencial en esta técnica. Además, los pulmones cosechadas poco después de la instilación intranasal muestran que un porcentaje relativamente bajo del inóculo bacteriano inicial alcanza el pulmón (en el caso de Y. pestis, sólo el 10% se recuperaron 1 hr después de la instilación 7), lo que sugiere que un gran número de bacterias podría ser retenido en la URT (o se ingiere en el tracto gastrointestinal). En ciertos modelos de enfermedad, esta deposición significativa de bacterias en la mucosa URT puede confundir nuestra comprensión de la evolución de la enfermedad si el organismo es capaz de colonizar la cavidad nasal murino de manera incompatible con la enfermedad humana. Por ejemplo, el uso in vivo &lt;/ Em&gt; de imágenes, se ha observado que Burkholderia pseudomallei, que no colonizan la URT humano, causa una infección oportunista abrumadora de la cavidad nasal murino cuando se entrega por el método de la instilación intranasal 8. Otros métodos para inculcar bacterias en los pulmones de ratones también se han empleado en investigación de enfermedades infecciosas. Sin embargo, en comparación con la instilación intranasal estos métodos tienden a requerir más conocimientos técnicos y / o un equipo costoso, sin eliminar el potencial para la iniciación de la infección en múltiples sitios (por ejemplo, aerosol [URT y LRT]; transoral [tracto digestivo y LRT]; y intratraqueal quirúrgica [LRT y corriente de la sangre]). Dadas las complicaciones potenciales que podrían estar asociados con los sitios secundarios de la infección, hemos tratado de desarrollar un enfoque intratraqueal que no pasa por la URT y entrega patógeno directamente en los pulmones de ratones anestesiados, pero también limita inoculados inadvertidade iones en el torrente sanguíneo o en el tracto GI. Hacia este fin, intratraqueal (IMIT) instilación intubación mediada fue desarrollado como un procedimiento no quirúrgico que garantiza LRT instilación de inóculo mediante la inclusión de un paso intermedio para verificar la colocación del catéter adecuado antes de la instilación. Este método se describe usando el tinte de la instilación de demostrar visualmente amplia distribución del inóculo en todo el pulmón, y P. aeruginosa instilación para demostrar la entrega altamente eficaz (&gt; 98% del inóculo) de este método para el pulmón. Es importante destacar que, mientras que originalmente desarrollado para la entrega bacteriana, IMIT también ofrece una herramienta eficaz para: i) la instilación de diferentes moléculas para el estudio de otros modelos de enfermedades respiratorias, ii) la entrega terapéutico-pulmonar específica, y iii) los estudios de función pulmonar básica, incluyendo dirigido siRNA entrega al pulmón.

<table border="0" cellpadding="0" cellspacing="0" width="1035">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Name of Material/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:447px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Rodent, Tilting WorkStand</td>
			<td style="width:160px;">Hallowell EMC</td>
			<td style="width:119px;">000A3467</td>
			<td>Base should be detached when working in a BSC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Operating Otoscope Head</td>
			<td style="width:160px;">Welch Allyn</td>
			<td style="width:119px;">21700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Otoscope 3.5 V Li Battery</td>
			<td style="width:160px;">Welch Allyn</td>
			<td style="width:119px;">71900</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:311px;">Mouse Intubation Specula short, Autoclaveable</td>
			<td style="width:160px;">Hallowell EMC</td>
			<td style="width:119px;">200A3589S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Incisor Loops</td>
			<td style="width:160px;">Hallowell EMC</td>
			<td style="width:119px;">210A3490A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Cotton fine tip applicator</td>
			<td style="width:160px;">Puritan</td>
			<td style="width:119px;">871-PC DBL&#160;</td>
			<td>Used for tongue retraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">I.V. Catheter, 20G</td>
			<td style="width:160px;">Exel Int</td>
			<td style="width:119px;">26741</td>
			<td>Optional: fit a silicon sleeve with 10mm exposed catheter surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Gas tight syringe, 250ul</td>
			<td style="width:160px;">Hamilton</td>
			<td style="width:119px;">81120</td>
			<td>Used for delivery of liquid inoculum by IMIT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Blunt Needle, 22G</td>
			<td style="width:160px;">Hamilton</td>
			<td style="width:119px;">91022</td>
			<td>Trim to length to protrude 1mm from 20G catheter</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Guide wire (Fiber optic wire, 0.5mm)</td>
			<td style="width:160px;">TheFiberOpticStore.com</td>
			<td style="width:119px;">FOF .50</td>
			<td>Cut to 6&#34; length: used as guide wire for intubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Tuberculin syringe, 1ml</td>
			<td style="width:160px;">Becton Dickinson</td>
			<td style="width:119px;">309659</td>
			<td>Assemble with fiber optic wire as guide wire</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Brilliant Blue R (Coomassie)</td>
			<td style="width:160px;">Sigma</td>
			<td style="width:119px;">B0149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Tygon tubing, 1/16&#34;</td>
			<td style="width:160px;">Saint Gobain</td>
			<td style="width:119px;">ALC00002&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Male luer 1/16&#34; barb</td>
			<td style="width:160px;">Cole Parmer</td>
			<td style="width:119px;">45503-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Female luer 1/16&#34; barb</td>
			<td style="width:160px;">Cole Parmer</td>
			<td style="width:119px;">45500-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Lidocaine, USP</td>
			<td style="width:160px;">Spectrum</td>
			<td style="width:119px;">LI102</td>
			<td>pH lidocaine into solution at 2%(w/v) pH7.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Sample bag, 1oz</td>
			<td style="width:160px;">Whirl-Pak</td>
			<td style="width:119px;">B01067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">U-bottom 96 well plate, sterile</td>
			<td style="width:160px;">Greiner</td>
			<td style="width:119px;">650161</td>
			<td></td>
		</tr>
	</tbody>
</table>

intubation-mediated intratracheal (imit) instillation: a noninvasive, lung-specific delivery system

Estudios de enfermedades respiratorias típicamente implican el uso de modelos murinos como sistemas sustitutos. Sin embargo, hay diferencias fisiológicas significativas entre el murino y los sistemas respiratorios humanos, especialmente en sus vías respiratorias superiores (URT). En algunos modelos, estas diferencias en la cavidad nasal murino pueden tener un impacto significativo sobre la progresión de la enfermedad y la presentación en el tracto respiratorio inferior (LRT) cuando se utilizan técnicas de instilación intranasal, limitando potencialmente la utilidad del modelo de ratón para estudiar estas enfermedades. Por estas razones, sería ventajoso desarrollar una técnica para inculcar bacterias directamente en los pulmones del ratón con el fin de estudiar la enfermedad LRT en ausencia de afectación de la URT. Hemos denominado este mediada por la intubación intratraqueal técnica de entrega (IMIT) la instilación específica del pulmón. Esta técnica no invasiva minimiza el potencial para la instilación en el torrente sanguíneo, lo cual puede ocurrir durante traditi más invasivaonal enfoques quirúrgicos intratraqueales infección, y limita la posibilidad de entrega tracto digestivo incidental. IMIT es un proceso de dos pasos en el que los ratones son intubados primero, con un paso intermedio para asegurar la colocación correcta del catéter en la tráquea, seguido por la inserción de una aguja roma en el catéter para mediar la entrega directa de las bacterias en el pulmón. Este enfoque facilita una eficacia&gt; 98% de la ejecución en los pulmones con una excelente distribución de reactivo de todo el pulmón. Por lo tanto, IMIT representa un nuevo enfoque para estudiar la enfermedad LRT y suministro terapéutico directamente en el pulmón, la mejora de la capacidad de utilizar los ratones como sustitutos para estudiar la enfermedad respiratoria humana. Además, la exactitud y reproducibilidad de este sistema de administración también hace que sea susceptible de Normas de Buenas Prácticas de Laboratorio (GLP), así como la entrega de una amplia gama de reactivos que requieren la entrega de alta eficiencia en el pulmón.

To visualize the distribution of instilled material via the IMIT method, 50 &#181;l of 0.1% Coomassie Brilliant Blue dye was instilled into the lungs of an anesthetized mouse. The mouse was immediately euthanized and the lungs were removed by sterile necropsy. Figure 1 shows that the dye was delivered to all lobes of the lung.
To determine the amount of bacteria delivered to the lungs via the IMIT method, three groups of mice (n = 3) were instilled with three different concentrations of P. aeruginosa (1.21 x 108, 1.21x107, and 1.21 x 106 colony forming units [CFU] per 50 &#181;l). Immediately after IMIT instillation, mice were euthanized, lungs removed, and bacterial numbers were enumerated and compared to the inoculums (Figure 2). Delivery of the inoculum is highly efficient via this method, with &#62;98% of the inoculum recovered from the lungs of instilled animals. Furthermore, IMIT instillation was highly reproducible regardless of the concentration of the inoculum (R2 = 0.9951).
<img alt="Figure 1" src="/files/ftp_upload/52261/52261fig1highres.jpg" /> 
Figure 1: IMIT instillation distributes inoculum throughout the lungs. Lungs from mice instilled with 50 &#181;l of 0.1% Coomassie Brilliant Blue show blue dye distributed in all lobes.
<img alt="Figure 2" src="/files/ftp_upload/52261/52261fig2highres.jpg" /> 
Figure 2: IMIT instillation of bacteria into lungs. Mice were instilled with P. aeruginosa and the number of bacteria instilled into the lungs (Log10 CFU &#8211; recovered) were compared to the estimated inoculum (Log10 CFU &#8211; delivered). Each circle represents the CFU/lung of an individual mouse (n = 3 for each bacterial dose).

Watch this Scientific Journal Video about Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System at JoVE.com

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

La intubación mediada intratraqueal (IMIT) instilación: A, Sistema de Entrega de pulmón-específica no invasiva

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

intubation-mediated-intratracheal-imit-instillation-noninvasive-lung

Research

JoVE Journal

Medicine

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

24.0K vistas.  University of Louisville Medical School. Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Video: Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Un nuevo enfoque quirúrgico para la administración intratraqueal de agentes bioactivos en un modelo de ratón fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Investigación

Medicina

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Intraoperatoria detección de endometriosis sutil: un nuevo paradigma para la detección y tratamiento del dolor pélvico asociado con la pérdida de la integridad Peritoneal

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

No Invasiva Intratraqueal Intubación para Estudiar la Patología y Fisiología de pulmón de ratón

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

El implante de gel de fibrina en pulmón de ratón para el Estudio de la angiogénesis pulmón específico

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

Instilación y Fijación Métodos Útiles en pulmón de ratón Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

Un método multimodal Imaging- y basados ​​en la estimulación de evaluar relacionados con la conectividad cerebral excitabilidad en pacientes con epilepsia

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Retroductal submandibular Glándula instilación y con localización fraccionado irradiación en un modelo de rata de hipofunción salival

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

Un modelo de derrame Pleural en ratas por la instilación intratraqueal de poliacrilato/Nanosílice

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Inducir lesión pulmonar aguda en ratones por instilación directa de lipopolisacárido intratraal

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Desarrollo de un Modelo Experimental No Invasivo, Asistido por Láser de Pérdida Celular Endotelial Corneal

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...