We thank Katherine Clark for technical assistance. A.D.R.G. is funded in part by 5K01MH097957-03

Adultos (3-4 meses de edad) los ratones macho en una mixta C57BL / 6 antecedentes fueron utilizados en este protocolo. Los animales se mantuvieron en un 12 horas de luz / oscuridad ciclo, y se les permite el acceso libre a comida y agua. Todos los procedimientos realizados en este protocolo se realizaron de acuerdo a los protocolos aprobados por el Comité de Cuidado y Uso de Animales Institucional Universidad Drexel. 1. Preparación Área Quirúrgica <ol><li> Desinfectar mesa quirúrgica con etanol al 70%, y luego cubrir todo el banco quirúrgica con almohadillas absorbentes y organizar instrumentos quirúrgicos adyacente a estereotáxica. </li><li> Configurar equipos estereotáxica sin brazo manipulador. Organizar la almohadilla térmica en el estereotáxica y se puso a 37 ° C. Evite el sobrecalentamiento del animal mediante la colocación de un pequeño pedazo de toalla de papel o almohadilla quirúrgica entre el animal y la resistencia de calentamiento. </li><li> Con unas tijeras en autoclave, cortar pequeñas piezas de espuma de gel en autoclave en una placa Petri estéril que contiene solución salina al 0,9% estéril hasta ready para su uso. Nota: Mantener entorno de trabajo estéril utilizando instrumentos autoclave y suministros quirúrgicos estériles. Re-esterilizar instrumentos quirúrgicos durante el procedimiento o en medio de animales por inmersión en un esterilizador de cuentas de 10 a 15 segundos, según sea necesario. Mantener guantes limpios durante todo el procedimiento por el roce de las manos con etanol al 70%, según sea necesario, de desinfectar. </li></ol> 2. Prepping ratón de Cirugía <ol><li> Retire del ratón de jaula y pesar (g). </li><li> Coloque el ratón en la cámara de inducción isoflurano y oxígeno a establecer 2 L / min y vaporizador de isoflurano a 5 para inducir un plano quirúrgico de anestesia, aproximadamente 3-5 min. Vigile la respiración lenta e inmovilización. Compruebe que el ratón está completamente sedado utilizando el reflejo pizca dedo del pie. </li><li> Cuando el ratón está completamente sedado, coloque en el marco estereotáxico, asegure la nariz en el cono de la nariz, que se adjunta con la tubería al isoflurano. Inserte barras de oído en el canal auditivo y ajuste, asegurando la cabeza es estable. </li><li> Afeitarse la cabeza de oreja a oreja, y de entre los ojos de detrás de las orejas. </li><li> Esterilizar la piel con alternancia de toallitas de alcohol isopropílico y solución de yodo betadine, 3 veces cada una. </li><li> Aplicar lágrimas artificiales para los dos ojos para evitar que se sequen durante el procedimiento quirúrgico. </li></ol> 3. Procedimiento Quirúrgico <ol><li> Supervisar la profundidad de la anestesia pellizcando la punta o la cola. El ratón es en el plano quirúrgico apropiado cuando no hay respuesta, y la respiración es lenta y uniforme. </li><li> Hacer una incisión en la piel parasagital desde justo detrás de los ojos hasta casi entre las orejas en un solo movimiento firme utilizando una hoja de bisturí No. 11. Mueva la piel a un lado y el clip lado derecho con pinza hemostática. </li><li> Claro cráneo de la membrana que recubre utilizando el lado opaco de la N º 11 bisturí y punta de algodón aplicadores. Opcionalmente, limpie el cráneo con algodón aplicador con punta sumergida en solución salina al 0,9%. Deje que se seque por completo. </li><li> Nosotrosing una pequeña regla, marcar el borde anterior de la craneotomía en 1 mm de caudal a la sutura coronal, y el borde izquierdo de la craneotomía en 1 mm lateral a la sutura sagital (Figura 1), con un marcador permanente. A continuación, marque los bordes derecho e caudales del craneotomía en 4 mm de las suturas sagital y coronal, respectivamente (Figura 1). </li><li> Con una broca de 0,5 mm, comenzar a hacer craneotomía perforando lentamente siguiendo el esquema marcador permanente. Asegúrese de no romper el cráneo completo. Presione suavemente en la pieza aislada del hueso parietal con el número 5 pinzas, áreas de debilidad dará paso a la presión. Cuando el hueso adelgazado es suficientemente débil en todo el perímetro, la pieza ósea está listo para el retiro. NOTA: Si la dificultad experiencias investigador retirar el hueso en una sola pieza, esto sugiere que el hueso no se ha adelgazado lo suficiente durante la perforación. Considere la perforación del cráneo aún más en los animales posteriores a FACILITAretiro fácil te de la pieza ósea. </li><li> Usando una jeringa de 10 ml equipado con una aguja de 23 G, aplicar una pequeña cantidad de 0,9% de solución salina para empapar el hueso aislado y el área perforado. </li><li> Fije el brazo manipulador al equipo estereotáxica. Adjunte una nueva hoja de bisturí No. 11 en el soporte de la sonda con el lado afilado de la hoja hacia rostral. NOTA: Aunque los tejidos rostral y caudal experimentan los bordes afilados y romos de la hoja, respectivamente, el daño mecánico inducido por la lesión penetrante es comparable en toda la extensión de la lesión. Observamos diferencias apreciables en las principales características de gliosis reactiva incluyendo la regulación positiva de la expresión de GFAP o proliferación, entre las secciones rostral y caudal. </li><li> Mantener el brazo manipulador fuera del camino, levante con cuidado el hueso aislado utilizando 5/45 fórceps en ángulo. Inserte la punta de la pinza en el lado del hueso y ascensor aislado, el uso de apalancamiento de sacar el trozo de hueso dejado atrás en una full movimiento. NOTA: Tenga cuidado de no apuñalar el cerebro o perturbar la dura debajo del cráneo. </li><li> Tome un pequeño pedazo de la espuma de gel absorbible empapado y colocarlo sobre el cerebro al descubierto para evitar que se reseque y disfrutar de sangre que pueda estar presente. </li><li> Una vez que la espuma de gel está en su lugar, hacer pivotar el brazo manipulador en su lugar y ajustar la cuchilla hacia el centro de la craneotomía sobre la espuma de gel. Retire la espuma de gel y baje la hoja hasta que la punta toca la duramadre sin perforación de la duramadre. Marcos dorsal / ventral coordenadas utilizando la escala vernier en el brazo vertical de la estereotáxica. </li><li> Utilizando el brazo manipulador, baje lentamente la hoja exactamente 3 mm en el cerebro. Esto se logra mediante el uso de las marcas de escala vernier en el brazo manipulador. Permita que la cuchilla para permanecer en el lugar durante 5-10 seg. Mueva el brazo estereotáxica con rostral acoplamiento a la hoja a caudal tres veces lo que permite que la hoja alcance los límites rostral y caudal de la craneotomía antes de pasar al opposite final. NOTA: La duramadre no se elimina antes de insertar la cuchilla. En contraste con la duramadre de rata, que es de ~ 80 m de espesor 7, la duramadre ratón es considerablemente más delgada (sólo unas pocas capas de células de espesor) y no produce una resistencia apreciable a la hoja de bisturí durante la inserción. Utilice una nueva hoja de bisturí para cada ratón para asegurar que cada animal recibe una lesión consistente. </li><li> Lentamente levante el brazo estereotáxica, la eliminación de la hoja desde el cerebro. Después de la eliminación de la hoja, colocar inmediatamente otra pieza de espuma de gel en la superficie del cerebro para absorber cualquier exceso de sangre o fluido. </li><li> Mientras tanto, retire el brazo estereotáxica y disponer de la hoja de bisturí No. 11. Una vez que el sangrado se ha detenido, quitar la espuma de gel. </li><li> Cierre de la herida mediante sutura la piel con sutura no absorbible, tal como Ethilon o prolene. Las suturas deben ser retirados de 9-10 días después de la cirugía. </li><li> Devuelva el ratón a su jaula, y permitir que el ratón para recuperar lentamente en un heating almohadilla y el monitor para detectar cualquier signo de malestar. Recuperación de la anestesia inducida por isofluorano-ocurre típicamente dentro de 2-5 min después de la eliminación de la isofluorano. No dejar al animal sin vigilancia hasta que se haya recuperado recumbancy esternal. </li><li> Administrar 0,5-1 ml de lactato por vía subcutánea solución de Ringer para asegurar la hidratación. </li></ol> Cuidado 4. Post-quirúrgica <ol><li> Seguir de cerca los animales después de la operación hasta que la recuperación de la anestesia antes de regresar a la colonia. <ol><li> Para reducir el dolor y las molestias después de la operación, administrar 0,05-0,1 mg / kg de buprenorfina por inyección ip inmediatamente después del procedimiento. </li><li> Observar los animales durante 2-3 días después de la operación de signos graves de angustia como los movimientos restringidos, falta de aseo, o pérdida de peso. La eutanasia a los animales que demuestran cualquiera de estos síntomas de angustia y retirar del estudio. </li></ol></li><li> Para examinar la histopatología de los tejidos lesionados, la eutanasia de animales por stanperfusión intracardiaca dard. <ol><li> Brevemente, anestesiar los animales con una sobredosis de ketamina / xilazina, a continuación, intracardially perfundido con 15-20 ml de NaCl al 0,9%, o hasta que el hígado se borra de la sangre, seguido de 60 ml de paraformaldehído al 4% en la conclusión del experimento. </li></ol></li><li> Diseccionar los cerebros y post-corrección para 2-4 h en paraformaldehído al 4% antes de ser transferido a la solución de sacarosa al 30%. Sección cerebros en un criostato de 40 a 60 micras, y el proceso mediante procedimientos histológicos o inmunohistoquímicos estándar, o como se describe en Garcia 8,9. </li></ol>

<ol>
	<li>Hamby, M. E., Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+astrocytes+as+therapeutic+targets+for+CNS+disorders.">Reactive astrocytes as therapeutic targets for CNS disorders.</a> Neurotherapeutics. 7 (4), 494-506 (2010).</li><li>Bush, T. 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=Leukocyte+infiltration,+neuronal+degeneration,+and+neurite+outgrowth+after+ablation+of+scar-forming,+reactive+astrocytes+in+adult+transgenic+mice.">Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice.</a> Neuron. 23 (2), 297-308 (1999).</li><li>Faulkner, J. R., 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=Reactive+astrocytes+protect+tissue+and+preserve+function+after+spinal+cord+injury.">Reactive astrocytes protect tissue and preserve function after spinal cord injury.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 24 (9), 2143-2155 (2004).</li><li>Okada, 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=Conditional+ablation+of+Stat3+or+Socs3+discloses+a+dual+role+for+reactive+astrocytes+after+spinal+cord+injury.">Conditional ablation of Stat3 or Socs3 discloses a dual role for reactive astrocytes after spinal cord injury.</a> Nature Medicine. 12 (7), 829-834 (2006).</li><li>Voskuhl, R. R., 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=Reactive+Astrocytes+Form+Scar-Like+Perivascular+Barriers+to+Leukocytes+during+Adaptive+Immune+Inflammation+of+the+CNS.">Reactive Astrocytes Form Scar-Like Perivascular Barriers to Leukocytes during Adaptive Immune Inflammation of the CNS.</a> Journal of Neuroscience. 29 (37), 11511-11522 (2009).</li><li>Burda, J. E., Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+Gliosis+and+the+Multicellular+Response+to+CNS+Damage+and+Disease.">Reactive Gliosis and the Multicellular Response to CNS Damage and Disease.</a> Neuron. 81 (2), 229-248 (2014).</li><li>Maikos, J. T., Elias, R. A., Shreiber, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+dura+mater+from+the+rat+brain+and+spinal+cord.">Mechanical properties of dura mater from the rat brain and spinal cord.</a> Journal of neurotrauma. 25 (1), 38-51 (2008).</li><li>Garcia, A. D., Doan, N. B., Imura, T., Bush, T. G., Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP-expressing+progenitors+are+the+principal+source+of+constitutive+neurogenesis+in+adult+mouse+forebrain.">GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain.</a> Nat Neurosci. 7 (11), 1233-1241 (2004).</li><li>Garcia, A. D., Petrova, R., Eng, L., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+discrete+populations+of+astrocytes+in+the+adult+mouse+forebrain.">Sonic hedgehog regulates discrete populations of astrocytes in the adult mouse forebrain.</a> J Neurosci. 30 (41), 13597-13608 (2010).</li><li>Amat, J. A., Ishiguro, H., Nakamura, K., Norton, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+diversity+and+kinetics+of+proliferating+microglia+and+astrocytes+following+cortical+stab+wounds.">Phenotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds.</a> Glia. 16 (4), 368-382 (1996).</li><li>Hozumi, I., 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=Administration+of+prosaposin+ameliorates+spatial+learning+disturbance+and+reduces+cavity+formation+following+stab+wounds+in+rat+brain.">Administration of prosaposin ameliorates spatial learning disturbance and reduces cavity formation following stab wounds in rat brain.</a> Neuroscience letters. 267 (1), 73-76 (1999).</li><li>Ness, R., David, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptomeningeal+cells+modulate+the+neurite+growth+promoting+properties+of+astrocytes+in+vitro.">Leptomeningeal cells modulate the neurite growth promoting properties of astrocytes in vitro.</a> Glia. 19 (1), 47-57 (1997).</li><li>Byrnes, K. R., Fricke, S. T., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+differences+between+rats+and+mice+after+spinal+cord+injury.">Neuropathological differences between rats and mice after spinal cord injury.</a> Journal of magnetic resonance imaging : JMRI. 32 (4), 836-846 (2010).</li><li>Steward, O., 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=Genetic+approaches+to+neurotrauma+research:+opportunities+and+potential+pitfalls+of+murine+models.">Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models.</a> Experimental neurology. 157 (1), 19-42 (1999).</li><li>Davalos, 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=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nature neuroscience. 8 (6), 752-758 (2005).</li><li>Hanisch, U. -. K., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+active+sensor+and+versatile+effector+cells+in+the+normal+and+pathologic+brain.">Microglia: active sensor and versatile effector cells in the normal and pathologic brain.</a> Nature neuroscience. 10 (11), 1387-1394 (2007).</li><li>Neumann, H., Kotter, M. R., Franklin, R. 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=Debris+clearance+by+microglia:+an+essential+link+between+degeneration+and+regeneration.">Debris clearance by microglia: an essential link between degeneration and regeneration.</a> Brain. 132 (2), 288-295 (2008).</li><li>Nimmerjahn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+Microglial+Cells+Are+Highly+Dynamic+Surveillants+of+Brain+Parenchyma+in+Vivo.">Resting Microglial Cells Are Highly Dynamic Surveillants of Brain Parenchyma in Vivo.</a> Science. 308 (5726), 1314-1318 (2005).</li><li>Horn, K. P., Busch, S. A., Hawthorne, A. L., van Rooijen, N., Silver, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Another+Barrier+to+Regeneration+in+the+CNS:+Activated+Macrophages+Induce+Extensive+Retraction+of+Dystrophic+Axons+through+Direct+Physical+Interactions.">Another Barrier to Regeneration in the CNS: Activated Macrophages Induce Extensive Retraction of Dystrophic Axons through Direct Physical Interactions.</a> Journal of Neuroscience. 28 (38), 9330-9341 (2008).</li><li>Ip, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+Cells+Contribute+to+Myelin+Degeneration+and+Axonopathic+Changes+in+Mice+Overexpressing+Proteolipid+Protein+in+Oligodendrocytes.">Immune Cells Contribute to Myelin Degeneration and Axonopathic Changes in Mice Overexpressing Proteolipid Protein in Oligodendrocytes.</a> Journal of Neuroscience. 26 (31), 8206-8216 (2006).</li><li>Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+systemic+inflammation+to+chronic+neurodegeneration.">Contribution of systemic inflammation to chronic neurodegeneration.</a> Acta neuropathologica. 120 (3), 277-286 (2010).</li><li>McKeon, R. J., Jurynec, M. J., Buck, 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=The+chondroitin+sulfate+proteoglycans+neurocan+and+phosphacan+are+expressed+by+reactive+astrocytes+in+the+chronic+CNS+glial+scar.">The chondroitin sulfate proteoglycans neurocan and phosphacan are expressed by reactive astrocytes in the chronic CNS glial scar.</a> Journal of Neuroscience. 19 (24), 10778-10788 (1999).</li><li>Silver, J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+beyond+the+glial+scar.">Regeneration beyond the glial scar.</a> Nature reviews. Neuroscience. 5 (2), 146-156 (2004).</li><li>Zamanian, J. 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=Genomic+analysis+of+reactive+astrogliosis.">Genomic analysis of reactive astrogliosis.</a> J Neurosci. 32 (18), 6391-6410 (2012).</li><li>Buffo, 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=Origin+and+progeny+of+reactive+gliosis:+A+source+of+multipotent+cells+in+the+injured+brain.">Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (9), 3581-3586 (2008).</li><li>Sirko, 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=Reactive+glia+in+the+injured+brain+acquire+stem+cell+properties+in+response+to+sonic+hedgehog.+[corrected].">Reactive glia in the injured brain acquire stem cell properties in response to sonic hedgehog. [corrected].</a> Cell stem cell. 12 (4), 426-439 (2013).</li></ol>

The authors have nothing to disclose.

Es fundamental que el cráneo o dura subyacente no se dañen durante la perforación. Utilice una ligera presión durante la perforación para asegurar el cráneo no está pinchado. Además, se debe tener cuidado al levantar la pieza del cráneo para asegurar la duramadre no se levanta con el hueso. La lesión de arma blanca del cerebro anterior describe aquí los modelos de una lesión penetrante en el SNC. Aunque menos clínicamente traducible que los modelos de TBI como FPI o CCI, el mode...

A major challenge for developing successful therapies to treat the injured CNS is an incomplete understanding of the complex multicellular events that are triggered by the trauma. Reactive astrocytes are gaining increasing recognition as a promising target for novel therapies1. Though historically regarded as hostile to neural repair, reactive astrocytes are now recognized as critical components of a complex, multicellular neuroprotective response that includes attenuation of inflammatory processes and limiting secondary damage and neurodegeneration2-6. Although the neuropathological characteristics of reactive gliosis have long been well defined, the cellular and molecular mechanisms regulating reactive gliosis, and the diverse array of downstream consequences remain poorly understood. Understanding the mechanisms that drive reactive gliosis, as well as the subsequent cellular and molecular events, is an important step towards developing strategies aimed at promoting the neuroprotective properties of reactive gliosis, while attenuating the detrimental effects. 
Here we demonstrate a method to induce severe reactive astrogliosis in the forebrain of adult mice using a stab injury. In contrast to other traumatic brain injury (TBI) models, such as controlled cortical impact (CCI) or fluid percussion injury (FPI), which require specialized equipment to produce an injury, the forebrain stab requires only a stereotaxic device to stabilize the head and a No. 11 scalpel blade. Thus the forebrain stab lesion model is more broadly accessible to a wide range of laboratories that do not have access to the specialized devices necessary for creating an FPI or CCI injury. The method described here enables investigators to reliably and reproducibly trigger a robust gliosis response to investigate subsequent cellular and molecular events. Once recovered from surgery, animals that have received a forebrain stab injury can survive for prolonged periods without the need for specialized care and can be returned to the colony for acute, intermediate, or chronic studies. Though less clinically translatable than FPI or CCI models of TBI, a forebrain lesion produced by a stab injury serves as a simple yet useful experimental model to investigate basic biological mechanisms underlying reactive gliosis and other neuropathological events following trauma to the CNS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stereotax</td>
			<td>Harvard Apparatus</td>
			<td>726049</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed micro drill</td>
			<td>Harvard Apparatus</td>
			<td>724950</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel scalpel blade, #11</td>
			<td>MedVet</td>
			<td>JOR581S</td>
			<td></td>
		</tr>
		<tr>
			<td>5/45 angled forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam sponge 12cmx7mm</td>
			<td>Fisher</td>
			<td>NC9841478</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies and other reagents</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td>Dilution (brightfield) 
			Dilution (fluorescence)</td>
		</tr>
		<tr>
			<td>Rb anti-GFAP</td>
			<td>DAKO</td>
			<td>&#160;Z033429-2</td>
			<td>1:20k 
			1:1k</td>
		</tr>
		<tr>
			<td>Shp anti-BrdU</td>
			<td>Abcam</td>
			<td>ab1893</td>
			<td>1:2k 
			1:500</td>
		</tr>
		<tr>
			<td>Biotinylated goat anti-rabbit</td>
			<td>Vector Laboratories</td>
			<td>BA-1000&#160;</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Biotinylated rabbit anti-sheep</td>
			<td>Vector Laboratories</td>
			<td>BA-6000</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexafluor 488 goat anti-rabbit</td>
			<td>Life Technologies</td>
			<td>A-11008</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexafluor 568 donkey anti-sheep</td>
			<td>Life Technologies</td>
			<td>A-21099</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>DAPI Nucleic Acid Stain</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Cresyl Violet Acetate</td>
			<td>Sigma Aldrich</td>
			<td>C5042-10G</td>
			<td>1%</td>
		</tr>
	</tbody>
</table>

triggering reactive gliosis in vivo by a forebrain stab injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Porque los animales sometidos a este procedimiento no requieren períodos de supervivencia especializados cuidados postoperatorios, cortos o de tiempo a largo plazo se incorporan fácilmente en el estudio, en función de la necesidad de investigar la patología aguda o crónica después de una lesión. Principales características de gliosis reactiva, como la regulación positiva de GFAP y la hipertrofia del soma, se pueden observar ya en 2-3 días después de la lesión. La fase pico de la proliferación de astrocitos ...

Watch this Scientific Journal Video about Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury at JoVE.com

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Disparo reactiva Gliosis<em&gt; En Vivo</em&gt; Por un Prosencéfalo Lesiones Puñalada

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

triggering-reactive-gliosis-in-vivo-by-a-forebrain-stab-injury

Research

JoVE Journal

Medicine

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

10.4K vistas.  Drexel University. This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods. 

Video: Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Un In vivo Modelo de roedores de la contracción inducida por la lesión y la monitorización no invasiva de la recuperación

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Investigación

Medicina

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Implantación de un manguito de la carótida para provocar esfuerzo cortante aterosclerosis inducida en ratones

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo MRI de 19F para el seguimiento de la célula

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

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

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

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

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

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

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Detección de Anastasis en Vivo por CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

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

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

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

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

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

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

La traqueostomía abierta ácido gástrico aspiración modelo murino de lesión pulmonar aguda Resultados en máxima no letal aguda lesión pulmonar

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Reproducible lesión arterial Denudación por pinzamiento aórtico abdominal infrarrenal en un modelo murino

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

Terapia regenerativa por autoinjerto Suprachoroidal célula en Degeneración Macular seca relacionada con la edad: informe preliminar en Vivo

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

Un modelo de lesión de Drosophila en Vivo para el estudio de Neurorregeneración en el periférico y sistema nervioso Central

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...