Los autores agradecen a Dan Firl, Chris Corbett y Jesse Denson, PhD. La financiación fue proporcionada por el NIH NINDS R01 NS060765 a SR, el P30 Programa Piloto de Cobre a LJ y el Programa de Salud Infantil Firma de LJ en la Universidad de Nuevo México.

Atención Institucional y el empleo Comités en el Hospital de Niños de Boston, tanto y la Universidad de Nuevo Centro de Ciencias de Salud de Nuevo México aprobó todos los procedimientos experimentales. NOTA: Antes de comenzar el procedimiento, el sello, esterilizar y autoclave todos los instrumentos quirúrgicos y paños quirúrgicos. Además, preparar medicamentos post-operatorios en viales estériles incluyendo 0,125% bipivucaine y 0,1 mg / kg de buprenorfina. También hay que preparar la solución de lipopolisacárido (LPS) de forma estéril: 0,04 mg / ml de LPS (0111: B4) en solución salina estéril que contiene diluir colorante azul de Evan. 1. Anestesia <ol><li> Inducir la anestesia en un día embrionario 18 (E18) de ratas Sprague Dawley embarazada con una mezcla de 3% de isoflurano equilibrado 70% de nitrógeno y 30% de oxígeno. </li><li> Retire la rata de la cámara de inducción y coloque la supina rata en quirúrgico envuelto manta de agua que circula a 37 ° C. Traslado anestesia para cono de la nariz y reducir isofluranivel ne a 2%. </li><li> Aplique suavemente pomada oftálmica a cada ojo para prevenir la sequedad de la córnea. Durante el procedimiento de monitorear continuamente la temperatura, la tasa de respiración y la frecuencia cardiaca del animal. Fisiología materna debe permanecer estable durante todo el procedimiento. </li></ol> 2. Preparación quirúrgica y Scrub <ol><li> Usando pequeñas tijeras de animales eliminan todo el pelo en la región abdominal inferior. Afeitarse en un patrón rectangular con cuidado para evitar mellar los pezones o la generación de erupción de afeitar que puede ser irritante para el futuro de la enfermería de los cachorros nacidos vivos. </li><li> Preparar la piel abdominal por la alternancia de aplicación de povidona yodada y el 70% de etanol frote con hisopos de algodón estériles. Repita el matorral de tal manera que la povidona yodada y etanol 70% son cada aplicado 3x de forma alterna. Deje que se seque. </li><li> Confirme la profundidad de la anestesia a través de ausencia de reflejo toe-pinch. En ausencia de reflejo y estímulos de dolor, reducir el nivel de isoflurano al 1%. </li><li> El uso de toallas quirúrgicos estériles,cuelgue el animal. Tenga cuidado de colocar las cortinas en un ángulo apropiado de tal manera que maximizan la cantidad de fluido de irrigación que absorben mientras que no obstruye el flujo de sangre a los cuernos uterinos. </li></ol> 3. La laparotomía abdominal <ol><li> Con un bisturí hacer una incisión en la línea media de 3 cm en la piel abdominal preparada. Sin rodeos diseccionar la capa de piel de la fascia abdominal con tijeras. Usando fórceps y tijeras quirúrgicas, elevar la capa fascial abdominal y hacer una incisión de la línea alba avascular para obtener acceso a la cavidad peritoneal. </li><li> Coloque gasa quirúrgica en el exterior de la incisión y humedecer con solución salina estéril. Con unas pinzas romas y presión externa sobre el abdomen, retire con cuidado los cuernos uterinos de la cavidad peritoneal y organizar en la gasa humedecida. </li><li> Evitar con cuidado el enredo y el contacto con los intestinos. Organizar fetos utilizando pinzas contactando sólo el tejido muscular en entre sacos amnióticos individuales.Exponer y aislar a las 4 arterias uterinas mediante disección roma. NOTA: Se debe tener cuidado para diseccionar las arterias uterinas. Tejido circundante y los vasos mismos son extremadamente delicada. El daño a los vasos maternos puede causar sangrado, y en casos severos, muerte fetal y materna. </li></ol> 4. Colocación de los clips de aneurismas <ol><li> Coloque una G clip de aneurisma de la rata 30 en cada arteria uterina. Asegurar la cesación del flujo de sangre, incluyendo proximal y pulsos distales, y oscurecimiento de los vasos uterinos incluyendo placentas individuales. Cubra los cuernos a la vista y campo quirúrgico entero con una gasa y regar con solución salina estéril. Tenga cuidado de mantener el terreno húmedo con riego aproximadamente cada 10 min. </li><li> Después de 60 minutos, retire la gasa y el riego del campo. Asegúrese de que los cuernos uterinos y los vasos se humedecen de manera adecuada para la eliminación de clip de éxito. Retire con cuidado cada clip de aneurisma utilizando fórceps. Tenga cuidado de no causar traumatismos en el recipiente, y maintain la integridad del tejido durante la extracción. </li><li> Completamente regar los cuernos uterinos y el campo, teniendo cuidado de eliminar cualquier hilo callejeros de gasa de las bolsas amnióticas. </li></ol> 5. La inyección de lipopolisacárido para amniótico Sacs <ol><li> En la base de cada saco amniótico individual, justo anterior a la placa de la placenta, inyecte 100 l de LPS (4 mg / saco) con colorante azul diluido de Evan en el líquido amniótico. El uso de fórceps romos para estabilizar y girar cada saco amniótico para una posición óptima para la inyección. Colorante azul de Diluir Evan es un agente de contraste que es útil para confirmar la colocación correcta de la jeringa e inyección. NOTA: Utilice sólo un ultra-fino jeringa 0,3 ml de insulina con el adjunto 8 mm 31 G de agujas para las inyecciones intra-amniótica. El uso de grandes agujas de calibre dará lugar a la pérdida de líquido amniótico crónica, muerte fetal y reabsorción del embarazo. Pequeñas cantidades de fuga de líquido amniótico tras la retirada de la jeringa pueden ser mitiglización creada por presión directa en el saco amniótico. Algunos fetos de rata pueden tolerar un grado de oligohidramnios. Sin embargo, la pérdida de líquido amniótico aguda de la punción con grandes agujas de calibre, o punción accidental que resulta en pérdida de líquido crónica, los resultados en la pérdida fetal y en los casos graves, pérdida de embarazos vecinos. </li><li> Riegue el útero cuernos 3x con solución salina estéril. </li></ol> 6. Cierre de la laparotomía <ol><li> Con unas pinzas, devuelva cuidadosamente los cuernos uterinos de la cavidad peritoneal. Garantizar un espacio adecuado entre los sacos amnióticos y la incisión en la línea media, volver a aproximar los bordes de la capa musculofascial utilizando una sutura de seda 3-0 en funcionamiento. Sea consciente de la colocación de los sacos amnióticos mientras se cierra la incisión muscular. Tenga cuidado de no suturar hacia oa través de un saco. </li><li> Re-aproximar la capa de piel, utilizando una sutura de seda 3-0 en funcionamiento, el cierre de la capa de la piel. NOTA: La laparotomía debe ser cerrado en dos capas de suturas continuas que permitanpara la piel y la expansión muscular con el aumento de la gestación. Suturas continuas permiten la tensión de la herida uniformemente distribuida. Suturas interrumpidas son menos deseables como múltiples nudos son irritantes y pueden ser fácilmente masticadas por la rata tras recuperarse de la anestesia. Grapas quirúrgicas no son deseados. Las colas de los nudos quirúrgicos se deben cortar muy corto (&lt;3 mm). </li><li> Se inyecta 1 ml de 0,125% por vía subcutánea bupivacaína bordes alrededor de la herida utilizando una aguja de 26 G. Administrar una dosis de 0,1 mg / kg de buprenorfina por vía subcutánea en la nuca del cuello. </li><li> Apague el isoflurano y toalla rata seca como sea necesario. Colocar en jaula limpia y seguimiento de la recuperación de la anestesia. Asegurar la rata no se convierta en hipotermia. </li></ol> 7. Recuperación postoperatoria y Cuidado <ol><li> Supervisar la rata cada 8 a 12 horas durante 72 horas y luego todos los días hasta las crías nacen (aproximadamente E22 o E23). Administrar dosis adicionales de q8-12 buprenorfina hr / 72 hr o prn según lo dictado por el IACUC. </li><li> Monitorear las ratas para detectar signos de dolor, malestar, sangrado vaginal o sangrado de la zona quirúrgica. Inspeccione las suturas y la incisión y tener cuidado de asegurarse rata no está masticando o retirar sus suturas prematuramente. Aunque excepcionalmente raro, las ratas que comprometen sus suturas están en riesgo de dehiscencia de la herida. NOTA: De vez en cuando las ratas pueden ingerir cantidades excesivas de ropa de cama o artículos no alimentarios, llamado pica, como un efecto secundario de la administración de buprenorfina. Aunque es muy poco común, las ratas deben ser monitorizados para la pica y el potencial de obstrucción intestinal posterior. </li></ol> 8. Procesamiento de tejidos y cryosectioning <ol><li> Para prepararse para hematoxilina y eosina (H &amp; E) tinción del cerebro postnatal, retirar las crías de su jaula en postnatal día 2 (P2). Con unas tijeras quirúrgicas decapitan crías de ratas y suavemente retire el cerebro del cráneo. </li><li> Caída de corrección cerebro en un tubo cónico de 15 ml que contiene 7 ml de 4% de paraformaldehído en tampón fosfato salino(PBS). Coloque cerebro a 4 ° C y fijar durante 72 horas. </li><li> Después de 72 horas, transferir cerebros para una solución estéril que contiene 30% PBS sacarosa (w / v) y volver a 4 ° C. NOTA: Una vez que el cerebro caen en la solución de sacarosa que están listos para ser seccionado en un criostato. </li><li> Rápidamente congelar cerebros y montar en una maja criostato para la adquisición de secciones coronales congeladas. Cortar 20 micras secciones coronales congeladas y montar en las diapositivas. Asegurar secciones se recogen en serie. </li><li> Permitir que las diapositivas se sequen a temperatura ambiente durante la noche. Tienda desliza a -20 ° C. </li></ol> 9. hematoxilina y eosina tinción <ol><li> Tome diapositivas montadas secciones congeladas y caliente a temperatura ambiente. NOTA: Preparar todas las soluciones fresco. </li><li> Colocar los portaobjetos en una diapositiva conjunto más caliente a 50 ° C durante 2 horas. </li><li> Transferencia desliza a un estante de tinción. Dip desliza 10x en agua desionizada doblemente destilada (ddH2O). </li><li> Incubar los portaobjetos en 100% hematoxilina durante 5 min. Timí en hematoxilina se puede optimizar dependiendo del grado de coloración púrpura. </li><li> Diapositivas Dip 4x en grifo H2O, y dejar reposar en la limpia del grifo H2O durante 1 min. </li><li> Dip desliza 15x en alcohol ácido (250 ml 70% de etanol + 1 ml de ácido clorhídrico concentrado). </li><li> Diapositivas Dip 4x en grifo H2O, y dejar reposar en la limpia del grifo H2O durante 1 min. </li><li> Incubar en 1% de carbonato de litio durante 2 min. </li><li> Diapositivas Dip 4x en grifo H2O, y dejar reposar en la limpia del grifo H2O durante 1 min. </li><li> Incubar en etanol 95% durante 1 min. </li><li> Diapositivas Dip 7x en el 100% Eosina. Número de caídas en Eosina puede ser modificado en función del grado de coloración rosada. </li><li> Diapositivas Dip 5x en etanol al 95%. </li><li> 5x diapositivas inmersión en un nuevo cambio de etanol al 95%. </li><li> Incubar los portaobjetos en etanol al 100% durante 1 min. </li><li> Incubar los portaobjetos en un nuevo cambio de etanol 100% durante 1 min. </li><li> Incubar los portaobjetos en 100% xileno durante 15 min. NOTA: Los pasos de xileno ycubrición debe ocurrir en una campana de humos. </li><li> Incubar los portaobjetos en cambios frescas de xileno durante 15 min. </li><li> Cubreobjetos con Permount y dejar secar en campana de humos. </li><li> Imagen desliza con un microscopio óptico. </li></ol>

<ol>
	<li>Blencowe, H., 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=Preterm+birth+associated+neurodevelopmental+impairment+estimates+at+regional+and+global+levels+for+2010.">Preterm birth associated neurodevelopmental impairment estimates at regional and global levels for 2010.</a> Pediatr Res. 74, 17-34 (2013).</li><li>Mwaniki, M. K., Atieno, M., Lawn, J. E., Newton, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+neurodevelopmental+outcomes+after+intrauterine+and+neonatal+insults+a+systematic+review.">Long term neurodevelopmental outcomes after intrauterine and neonatal insults a systematic review.</a> Lancet. 379, 445-452 (2012).</li><li>Dammann, O., Leviton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+or+sustained+systemic+inflammation+and+the+preterm+brain.">Intermittent or sustained systemic inflammation and the preterm brain.</a> Pediatr Res. 75, 376-380 (2014).</li><li>Leviton, 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=Microbiologic+and+histologic+characteristics+of+the+extremely+preterm+infant's+placenta+predict+white+matter+damage+and+later+cerebral+palsy.">Microbiologic and histologic characteristics of the extremely preterm infant's placenta predict white matter damage and later cerebral palsy.</a> the ELGAN study Pediatr Res. 67, 95-101 (2010).</li><li>Redline, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+responses+in+the+placenta+and+umbilical+cord.">Inflammatory responses in the placenta and umbilical cord.</a> Semin Fetal Neonatal Med. 11, 296-301 (2006).</li><li>Lee, J., 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=Chronic+chorioamnionitis+is+the+most+common+placental+lesion+in+late+preterm+birth.">Chronic chorioamnionitis is the most common placental lesion in late preterm birth.</a> Placenta. 34, 681-689 (2013).</li><li>Lee, S. M., 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=Acute+histologic+chorioamnionitis+is+a+risk+factor+for+adverse+neonatal+outcome+in+late+preterm+birth+after+preterm+premature+rupture+of+membranes.">Acute histologic chorioamnionitis is a risk factor for adverse neonatal outcome in late preterm birth after preterm premature rupture of membranes.</a> PloS one. 8, e79941 (2013).</li><li>Pappas, 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=Chorioamnionitis+and+early+childhood+outcomes+among+extremely+low+gestational+age+neonates.">Chorioamnionitis and early childhood outcomes among extremely low gestational age neonates.</a> JAMA Peds. 168, 137-147 (2014).</li><li>Gaillard, R., Arends, L. R., Steegers, E. A., Hofman, A., Jaddoe, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+and+third+trimester+placental+hemodynamics+and+the+risks+of+pregnancy+complications+the+Generation+R+Study.">Second and third trimester placental hemodynamics and the risks of pregnancy complications the Generation R Study.</a> Am J Epidemiol. 177, 743-754 (2013).</li><li>Trivedi, S., 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=Fetal+placental+inflammation+but+not+adrenal+activation+is+associated+with+extreme+preterm+delivery.">Fetal placental inflammation but not adrenal activation is associated with extreme preterm delivery.</a> Am J Obstet Gynecol. 206, 236 (2012).</li><li>Yanowitz, T. D., 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=Hemodynamic+disturbances+in+premature+infants+born+after+chorioamnionitis+association+with+cord+blood+cytokine+concentrations.">Hemodynamic disturbances in premature infants born after chorioamnionitis association with cord blood cytokine concentrations.</a> Pediatr Res. 51, 310-316 (2002).</li><li>Jantzie, L. L., Corbett, C. J., Firl, D. J., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+Erythropoietin+Mitigates+Impaired+Cerebral+Cortical+Development+Following+Subplate+Loss+from+Prenatal+Hypoxia+Ischemia.">Postnatal Erythropoietin Mitigates Impaired Cerebral Cortical Development Following Subplate Loss from Prenatal Hypoxia Ischemia.</a> Cereb Cortex. , (2014).</li><li>Jantzie, L. 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=Erythropoietin+attenuates+loss+of+potassium+chloride+co+transporters+following+prenatal+brain+injury.">Erythropoietin attenuates loss of potassium chloride co transporters following prenatal brain injury.</a> Mol Cell Neurosci. 61, 152-162 (2014).</li><li>Jantzie, L. L., Miller, R. H., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin+signaling+promotes+oligodendrocyte+development+following+prenatal+systemic+hypoxic+ischemic+brain+injury.">Erythropoietin signaling promotes oligodendrocyte development following prenatal systemic hypoxic ischemic brain injury.</a> Pediatric Res. 74, 658-667 (2013).</li><li>Mazur, M., Miller, R. H., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+erythropoietin+treatment+mitigates+neural+cell+loss+after+systemic+prenatal+hypoxic+ischemic+injury.">Postnatal erythropoietin treatment mitigates neural cell loss after systemic prenatal hypoxic ischemic injury.</a> J Neurosurg Peds. 6, 206-221 (2010).</li><li>Robinson, S., 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=Developmental+changes+induced+by+graded+prenatal+systemic+hypoxic+ischemic+insults+in+rats.">Developmental changes induced by graded prenatal systemic hypoxic ischemic insults in rats.</a> Neurobiol Dis. 18, 568-581 (2005).</li><li>Delcour, M., 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=Neuroanatomical+sensorimotor+and+cognitive+deficits+in+adult+rats+with+white+matter+injury+following+prenatal+ischemia.">Neuroanatomical sensorimotor and cognitive deficits in adult rats with white matter injury following prenatal ischemia.</a> Brain Pathol. 22, 1-16 (2012).</li><li>Delcour, M., 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=Impact+of+prenatal+ischemia+on+behavior+cognitive+abilities+and+neuroanatomy+in+adult+rats+with+white+matter+damage.">Impact of prenatal ischemia on behavior cognitive abilities and neuroanatomy in adult rats with white matter damage.</a> Behav Brain Res. 232, 233-244 (2012).</li><li>Delcour, M., 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=Mild+musculoskeletal+and+locomotor+alterations+in+adult+rats+with+white+matter+injury+following+prenatal+ischemia.">Mild musculoskeletal and locomotor alterations in adult rats with white matter injury following prenatal ischemia.</a> Intl J Devel Neurosci. 29, 593-607 (2011).</li><li>Jantzie, L. 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=Complex+pattern+of+interaction+between+in+utero+hypoxia+ischemia+and+intra+amniotic+inflammation+disrupts+brain+development+and+motor+function.">Complex pattern of interaction between in utero hypoxia ischemia and intra amniotic inflammation disrupts brain development and motor function.</a> J Neuroinflam. 11, 131 (2014).</li><li>Back, S., 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=Selective+vulnerability+of+late+oligodendrocyte+progenitors+to+hypoxia+ischemia.">Selective vulnerability of late oligodendrocyte progenitors to hypoxia ischemia.</a> J Neurosci. 22, 455-463 (2002).</li><li>Jantzie, L. 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=Developmental+Expression+of+N+Methyl+d+Aspartate+(NMDA)+Receptor+Subunits+in+Human+White+and+Gray+Matter+Potential+Mechanism+of+Increased+Vulnerability+in+the+Immature+Brain.">Developmental Expression of N Methyl d Aspartate (NMDA) Receptor Subunits in Human White and Gray Matter Potential Mechanism of Increased Vulnerability in the Immature Brain.</a> Cereb Cortex. 25, 482-495 (2015).</li><li>Jantzie, L. L., Robinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+Models+of+Encephalopathy+of+Prematurity.">Preclinical Models of Encephalopathy of Prematurity.</a> Devel Neurosci. , (2015).</li><li>Workman, A. D., Charvet, C. J., Clancy, B., Darlington, R. B., Finlay, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+transformations+of+neurodevelopmental+sequences+across+mammalian+species.">Modeling transformations of neurodevelopmental sequences across mammalian species.</a> J Neurosci. 33, 7368-7383 (2013).</li><li>Herlenius, E., Lagercrantz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+neurotransmitter+systems+during+critical+periods.">Development of neurotransmitter systems during critical periods.</a> Exper Neurol. 190, S8-S21 (2004).</li><li>Kelsom, C., Lu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+specification+of+GABAergic+cortical+interneurons.">Development and specification of GABAergic cortical interneurons.</a> Cell Biosci. 3, 19 (2013).</li><li>Kinney, H., Back, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+oligodendroglial+development+Relationship+to+periventricualr+leukomalacia.">Human oligodendroglial development Relationship to periventricualr leukomalacia.</a> Semin Pediatr Neuro. 5, 180-189 (1998).</li><li>Robinson, S., Li, Q., DeChant, A., Cohen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+loss+of+gamma+amino+butyric+acid+pathway+expression+after+human+perinatal+brain+injury.">Neonatal loss of gamma amino butyric acid pathway expression after human perinatal brain injury.</a> J Neurosurg Peds. 104, 396-408 (2006).</li><li>Xu, 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=Late+development+of+the+GABAergic+system+in+the+human+cerebral+cortex+and+white+matter.">Late development of the GABAergic system in the human cerebral cortex and white matter.</a> J Neuropathol Exp Neurol. 70, 841-858 (2011).</li><li>Segovia, K. N., 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=Arrested+oligodendrocyte+lineage+maturation+in+chronic+perinatal+white+matter+injury.">Arrested oligodendrocyte lineage maturation in chronic perinatal white matter injury.</a> Ann Neurol. 63, 520-530 (2008).</li><li>Robinson, S., Li, Q., Dechant, A., Cohen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+loss+of+gamma+aminobutyric+acid+pathway+expression+after+human+perinatal+brain+injury.">Neonatal loss of gamma aminobutyric acid pathway expression after human perinatal brain injury.</a> J Neurosurg. 104, 396-408 (2006).</li><li>Boksa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prenatal+infection+on+brain+development+and+behavior+a+review+of+findings+from+animal+models.">Effects of prenatal infection on brain development and behavior a review of findings from animal models.</a> Brain Behav Immun. 24, 881-897 (2010).</li><li>Burd, I., Brown, A., Gonzalez, J. M., Chai, J., Elovitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+term+chorioamnionitis+unraveling+causes+of+adverse+neurological+outcomes.">A mouse model of term chorioamnionitis unraveling causes of adverse neurological outcomes.</a> Repro Sci. 18, 900-907 (2011).</li><li>Bergeron, J. D., 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=White+matter+injury+and+autistic+like+behavior+predominantly+affecting+male+rat+offspring+exposed+to+group+B+streptococcal+maternal+inflammation.">White matter injury and autistic like behavior predominantly affecting male rat offspring exposed to group B streptococcal maternal inflammation.</a> Devel Neurosci. 35, 504-515 (2013).</li><li>Uchida, K., 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=Effects+of+Ureaplasma+parvum+lipoprotein+multiple+banded+antigen+on+pregnancy+outcome+in+mice.">Effects of Ureaplasma parvum lipoprotein multiple banded antigen on pregnancy outcome in mice.</a> J Reprod Immunol. 100, 118-127 .</li></ol>

Los autores no tienen nada que revelar.

Encefalopatía del prematuro es difícil de modelar en los animales debido a la compleja interacción de causas, evolución en el tiempo del desarrollo neurológico, complejidad de la formación de la red cerebral humana, mecanismos de lesión se superponen, y los diversos fenotipos de SNC insulta manifiesta en los recién nacidos prematuros humanos. EOP se asocia con vulnerabilidades específicas de tipo celular (es decir, los oligodendrocitos inmaduros) 21, así como diversos caminos regulados por ...

Con más del 12% de los bebés nacidos en los Estados Unidos antes de la edad de las 37 semanas de gestación estimada 1, lesión cerebral perinatal (PBI) de la prematuridad es una causa importante de discapacidad permanente. PBI de la prematuridad, la encefalopatía también denominado del prematuro (EOP), afecta a todo el sistema nervioso central (SNC). Lesión del SNC a menudo comienza en el útero, y se ve exacerbada por procesos prenatales incluyendo corioamnionitis y las complicaciones postnatales tales como la hipoxia y sepsis. PBI de insultos sistémicos altera el desarrollo neurológico y conduce a la parálisis cerebral, epilepsia, retraso cognitivo y numerosos trastornos neuropsiquiátricos que afecta a la regulación emocional, la memoria y la función ejecutiva 1,2. Aunque se ha avanzado mucho, una comprensión limitada sigue siendo cómo las consecuencias celulares y moleculares de la lesión del SNC desde el nacimiento prematuro se traducen en la multitud de secuelas neurológicas en los niños que nacen prematuros. Esta falta de conocimiento traserares diagnóstico en tiempo real de la gravedad de la lesión del SNC y la dosificación informado de las intervenciones emergentes. Además, las estrategias terapéuticas apropiadas a su edad para esta población de pacientes vulnerables siguen siendo difícil de alcanzar. Inflamación intrauterina es muy común en la prematuridad extrema e implica una compleja cascada inflamatoria fetal-materno-placentaria 3. La infección intrauterina suele ser subclínica. Hallazgos placentarios específicos consistentes con la inflamación aguda o corioamnionitis histológica, son los principales determinantes de la respuesta inflamatoria fetal y son coincidentes con lesión cerebral asociada con el parto prematuro 3-5. De hecho, la respuesta inflamatoria fetal tiene implicaciones clínicas distintas de resultados a largo plazo desde el nacimiento prematuro. Los bebés que son pequeños para la edad gestacional (PEG) o que experimentar la infección son excepcionalmente vulnerables a los déficit neurológico 3,4. La corioamnionitis es un diagnóstico típico patológica tras el parto prematuro <sup&gt; 6,7, y el examen histológico revela signos de inflamación en el 70% de las placentas de los bebés nacidos muy prematuros 4. Además, la corioamnionitis se asocia con deterioro cognitivo a los dos años de 8. La evidencia de hipoperfusión vascular materna en la placenta de los recién nacidos extremadamente prematuros también se asocia con parálisis cerebral en la infancia 9. El impacto sinérgico de defectos de perfusión corioamnionitis y la placenta está bien ilustrado por el notable alto riesgo de resultados neurológicos anormales en esta población de pacientes a los dos años de 10,11 años. Para imitar los defectos de perfusión placentaria sistémicos humanos y corioamnionitis asociados con la inflamación inducida por patógenos, hemos desarrollado un modelo de prenatal transitoria hipoxia-isquemia sistémica (Tshi) combinado con lipopolisacárido intra-amniótica (LPS) en ratas. Nuestro objetivo era adaptar nuestro modelo de Tshi solo en ratas 12-16 para incluir la inflamación intrauterina,para facilitar el modelado preclínica de lesión del SNC asociado con el parto prematuro. Tshi solo ha revelado pérdida persistente de células del linaje oligodendroglial, las neuronas corticales, el aumento de la muerte celular, y niveles elevados de citoquinas pro-inflamatorias, con intervalos de isquemia progresiva que conduce a un patrón graduada de la lesión consistente con lesión cerebral prenatal 16. Las modificaciones a los componentes isquémicos de este modelo han demostrado también déficits en la codificación de la memoria, y la memoria a corto a largo plazo y alteraciones musculoesqueléticas leves en ratas, ya que 17-19 de envejecen. De hecho, hemos demostrado previamente que la combinación de Tshi + LPS recapitula las características fisiopatológicas de la EOP, incluyendo los oligodendrocitos y la pérdida neuronal, lesión axonal, la inflamación celular y las anormalidades funcionales 20.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Saline Solution, 0.9%</td>
			<td >Sigma</td>
			<td >S8776</td>
			<td ></td>
		</tr>
		<tr >
			<td >LPS 011B4</td>
			<td>Sigma</td>
			<td>L2630</td>
			<td></td>
		</tr>
		<tr >
			<td >Evan&#39;s Blue Dye</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical gloves</td>
			<td>Biogel</td>
			<td>40870</td>
			<td></td>
		</tr>
		<tr >
			<td >OR Towels</td>
			<td>Cardinal Health</td>
			<td>287000-008</td>
			<td>Sterile</td>
		</tr>
		<tr >
			<td >PDI Alcohol Prep Pads</td>
			<td>Fisherbrand</td>
			<td>06-669-62</td>
			<td></td>
		</tr>
		<tr >
			<td >Mini Arco Rechargeable Clippers</td>
			<td>Kent Scientific Corp.</td>
			<td>CL8787</td>
			<td></td>
		</tr>
		<tr >
			<td >Betadine surgical scrub</td>
			<td>Purdue Products L.P.</td>
			<td>67618-151-17</td>
			<td></td>
		</tr>
		<tr >
			<td >Eye Lubricant</td>
			<td>Refresh Lacri Lube</td>
			<td>00023-0312</td>
			<td></td>
		</tr>
		<tr >
			<td >Blunt Forceps</td>
			<td>Roboz</td>
			<td>RS-8100</td>
			<td></td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td>Roboz</td>
			<td>RS-6808</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical Scissors</td>
			<td>Roboz</td>
			<td>RS-5880</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical Scissors</td>
			<td>F.S.T.</td>
			<td>14002-16</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td>1 ml</td>
		</tr>
		<tr >
			<td >Needle</td>
			<td>BD</td>
			<td>305122</td>
			<td>25G 5/8</td>
		</tr>
		<tr >
			<td >Needle</td>
			<td>BD</td>
			<td>305128</td>
			<td>30G 1</td>
		</tr>
		<tr >
			<td >Cotton-tipped Applicators</td>
			<td>Fisherbrand</td>
			<td>23-400-114</td>
			<td>Small, 6 inch sterile</td>
		</tr>
		<tr >
			<td >Cotton Gauze Sponge</td>
			<td>Fisherbrand</td>
			<td>22-362-178</td>
			<td></td>
		</tr>
		<tr >
			<td >Needle Holders</td>
			<td>Kent Scientific Corp.</td>
			<td>INS600109</td>
			<td>12.5 CM STR</td>
		</tr>
		<tr >
			<td >Vessel Clips</td>
			<td>Kent Scientific Corp.</td>
			<td>INS600120</td>
			<td>30G Pressure</td>
		</tr>
		<tr >
			<td >3-0 Perma Hand Silk Sutures</td>
			<td>Ethicon</td>
			<td>1684G</td>
			<td>Black braided, 3-0 (2 metric), 18&#34;, non-absorbable,&#160; PS-1 24mm needle, 3/8 circle</td>
		</tr>
		<tr >
			<td >Insulin Syringes</td>
			<td>BD</td>
			<td>328438</td>
			<td>0.3cc 3mm 31G</td>
		</tr>
		<tr >
			<td >Pentobarbital</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Buprenorphine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Bupivacaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Lithium Carbonate</td>
			<td>Acros Chemicals</td>
			<td>554-13-2</td>
			<td></td>
		</tr>
		<tr >
			<td >Superfrost Plus Microscope Slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr >
			<td >Hematoxylin</td>
			<td>Leica</td>
			<td>3801521</td>
			<td>Surgipath Gill II Hematoxylin</td>
		</tr>
		<tr >
			<td >Eosin</td>
			<td>Leica</td>
			<td>3801601</td>
			<td>Surgipath Eosin</td>
		</tr>
		<tr >
			<td >Xylenes</td>
			<td>Fisherbrand</td>
			<td>X3S-4</td>
			<td>Histological Grade</td>
		</tr>
		<tr >
			<td >Permount</td>
			<td>Fisherbrand</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr >
			<td >Coverglass</td>
			<td>Fisherbrand</td>
			<td>12-548-5P</td>
			<td>Fisher Finest Premium Coverglass</td>
		</tr>
	</tbody>
</table>

modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats

Encefalopatía del prematuro (EOP) es un término que abarca el sistema nervioso central (SNC) anormalidades asociadas con el parto prematuro. Para mejores objetivos de traslación antelación y descubrir nuevas estrategias terapéuticas para la lesión cerebral asociada con el parto prematuro, modelos preclínicos de EOP deben incluir mecanismos similares de lesión mundial prenatal observado en los seres humanos y la participación de múltiples componentes del sistema materno-placentaria-fetal. Idealmente, los modelos deben producir un espectro similar de déficit funcional en el animal maduro y recapitular los múltiples aspectos de la fisiopatología. Para imitar los defectos humanos sistémicas placentarios perfusión, hipoperfusión placentaria y / o corioamnionitis asociados con la inflamación inducida por patógenos en el parto prematuro temprano, hemos desarrollado un modelo de prenatal transitoria hipoxia-isquemia sistémica (Tshi) combinado con lipopolisacárido intra-amniótica (LPS). En embarazadas ratas Sprague Dawley, Tshi través de oclusión de la arteria uterina on día embrionario 18 (E18) induce un defecto hipoperfusión placentaria graduada asociada con el aumento de daño en el SNC en el feto. Cuando se combina con inyecciones de LPS intra-amniótico, la placenta se incrementa la inflamación y daño en el SNC se ve agravado con la materia, de la marcha y de imagen anomalías blancos asociados. Prenatal Tshi y Tshi + LPS insultos prenatales cumplen varios de los criterios de un modelo de EOP incluyendo recapitulando el insulto intrauterino, causando la pérdida de neuronas, oligodendrocitos y los axones, la pérdida de la placa de conexión, y los déficits funcionales en animales adultos que imitan a los observados en los niños nacidos extremadamente prematuro. Además, este modelo permite la disección de la inflamación inducida por los tipos de lesiones divergentes.

Siguiendo Tshi + LPS a E18, hematoxilina y eosina revela anomalías significativas histopatológicos en tanto la placenta (Figura 1) y en el cerebro (Figura 2). Placentas examinadas en E19 y E21 son groseramente edematosa con micro-hemorragia y necrosis en toda la decidua y laberinto. También se observa infiltrado inflamatorio importante y aumento de la vascularización. Los cerebros examinados en P2 revelan ventrículomegalia, así como la materia blanca y la pérdida de neuronas sobr...

Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modelado Encefalopatía del Prematuro El uso prenatal hipoxia-isquemia con lipopolisacárido intraamniótica en ratas

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

modeling-encephalopathy-prematurity-using-prenatal-hypoxia-ischemia

Research

JoVE Journal

Medicine

11.2K vistas.  University of New Mexico. Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Video: Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Modelado de accidente cerebrovascular en ratones - Oclusión arteria cerebral media con el modelo de filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Investigación

Medicina

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

De imagen no invasivas y Análisis de la isquemia cerebral en ratas vivas utilizando Tomografía por emisión de positrones con<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Un modelo del cochinillo de Neonatología encefalopatía hipóxico-isquémica

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

La evaluación de la encefalopatía espongiforme transmisible Especies Barreras con un<em&gt; In Vitro</em&gt; Prion Análisis de Conversión de proteínas

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Simultánea de imágenes PET / RM Durante cerebral del ratón hipoxia-isquemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Un accidente cerebrovascular trombótico Basado En transitoria cerebral por hipoxia-isquemia Modelo

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Circunscrita capsular del infarto de modelado mediante técnica photothrombotic

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Preparación De Andamios Renales Descelularizados En Ratas

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Análisis de muestras crudas y procesadas de rizoma de Cyperi utilizando cromatografía líquida-espectrometría de masas en tándem en ratas con dismenorrea primaria

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

Manipulación estandarizada de Tuina para la artrosis de rodilla en ratas

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Autor destacado: Entendiendo el mecanismo de la manipulación de la tuina en ratas 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Manipulación de Tuina para reducir la inflamación y la pérdida de cartílago en ratas con artrosis de rodilla

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...