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>

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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><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 12 cm x 7 mm</td><td>Fisher</td><td>NC9841478</td><td></td></tr><tr><td>Rb anti-GFAP</td><td>DAKO</td><td>&amp;nbsp;Z033429-2</td><td>Dilution - 1:20,000 (bright-field); 1:1,000 (fluorescence)</td></tr><tr><td>Shp anti-BrdU</td><td>Abcam</td><td>ab1893</td><td>Dilution - 1:20,000 (bright-field); 1:500 (fluorescence)</td></tr><tr><td>Biotinylated goat anti-rabbit</td><td>Vector Laboratories</td><td>BA-1000&amp;nbsp;</td><td>Dilution - 1:400 (bright-field)</td></tr><tr><td>Biotinylated rabbit anti-sheep</td><td>Vector Laboratories</td><td>BA-6000</td><td>Dilution - 1:400 (bright-field)</td></tr><tr><td>Alexafluor 488 goat anti-rabbit</td><td>Life Technologies</td><td>A-11008</td><td>Dilution - 1:400 (bright-field)</td></tr><tr><td>Alexafluor 568 donkey anti-sheep</td><td>Life Technologies</td><td>A-21099</td><td>Dilution - 1:1,000 (fluorescence)</td></tr><tr><td>DAPI Nucleic Acid Stain</td><td>Life Technologies</td><td>D3571</td><td>Dilution - 1:1,000 (fluorescence)</td></tr><tr><td>Cresyl Violet Acetate</td><td>Sigma Aldrich</td><td>C5042-10G</td><td>Dilution - 1% (bright-field)</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 Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

Un paradigma de Lesiones de investigar reparación del sistema nervioso central en<em&gt; Drosophila</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Investigación

Neurociencias

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Trauma cerebral aguda en ratones seguido de Longitudinal dos fotones de imágenes

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Medicina

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Puñalada de Lesiones de la herida del pez cebra Telencéfalo adultos: un método para investigar Vertebrados Cerebro neurogénesis y Regeneración

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelado astrocitoma Patogénesis<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Uso cortical astrocitos o células troncales nerviosas de ratones condicionales, Genéticamente Manipulados

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

Modelo de lesión por puñalada del Tectum óptico adulto utilizando pez cebra y medaka para el análisis comparativo de la capacidad regenerativa

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Biología del Desarrollo

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 ...

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

Estimulación y análisis de la neurogénesis adulta en el cerebro central de Drosophila

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Modelo electromagnético controlado de cabeza cerrada de lesión cerebral traumática leve en ratones

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

Imágenes intravitales de la expresión de proteínas fluorescentes en ratones con una lesión cerebral traumática de cráneo cerrado y ventana craneal utilizando un microscopio de dos fotones

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...

Stab-Wound Mouse Model for Studying Hemorrhage and Inflammation in Traumatic Brain Injury

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including concussions, brain contusions, hematomas, and skull fractures. To replicate these different causes, various TBI mouse models have been developed using distinct protocols. Physical brain injury leads to both primary and secondary brain injuries, which exacerbate neuronal loss. Primary injury occurs immediately after the damage, often due to hemorrhage, and subsequently triggers secondary injuries, including inflammation around the lesion. Developing a TBI model suitable for assessing hemorrhage extension and inflammatory severity is therefore crucial. This protocol introduces a method for mimicking penetrating brain injury, referred to as the stab-wound TBI mouse model, to study mechanisms of hemorrhage, inflammation, and neuronal loss associated with TBI pathology. This model is created by puncturing the skull and brain with needles and is simple to execute without the need for specialized experimental equipment. Additionally, the minor injury inflicted on the mouse cerebral cortex using a needle does not affect the animal's behavior post-surgery. This feature allows researchers to study the localized effects of brain injury without concerns about broader behavioral consequences. Sample data from stab-wounded mouse cerebral cortices demonstrate the model's effectiveness in assessing blood leakage into the parenchyma, glial activation, and inflammatory cytokine production. Furthermore, this protocol facilitates the evaluation of blood coagulants and anti-inflammatory compounds, aiding in the development of therapeutic agents for TBI.

Simplified traumatic brain injury (TBI) models have facilitated the development of therapeutic approaches. This protocol outlines the creation of a stab-wound mouse cortex using needles, enabling the analysis of hemorrhage and inflammation. The stab-wound TBI mouse model offers the advantage of being performed without requiring specialized equipment.

Modelo de ratón con herida de puñalada para estudiar la hemorragia y la inflamación en lesiones cerebrales traumáticas

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including ...

Disparo reactiva Gliosis

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Un paradigma de Lesiones de investigar reparación del sistema nervioso central en