Name: triggering reactive gliosis in vivo by a forebrain stab injury
Uploaded: 2015-06-29T13:00:00.000+00:00
Duration: 7 min 46 s
Description: Watch this Scientific Journal Video about Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury at JoVE.com

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

Adulto (3-4 meses de idade) ratos machos em um C57BL / 6 fundo misto foram utilizados neste protocolo. Os animais foram mantidos em um ciclo claro / escuro de 12 horas de luz, e permitiu o livre acesso a comida e água. Todos os procedimentos realizados neste protocolo foram realizados de acordo com protocolos aprovados pela Comissão de Drexel University Institutional Animal Care e Use. 1. Preparar Área Cirúrgica <ol><li> Desinfetar mesa cirúrgica com etanol 70%, em seguida, cobrir toda a bancada cirúrgica com absorventes higiênicos e providenciar instrumentos cirúrgicos adjacente ao estereotáxica. </li><li> Configurar equipamento estereotáxico sem braço manipulador. Organizar a almofada de aquecimento na estereotáxico e ajustado para 37 ° C. Evitar o sobreaquecimento do animal, colocando um pequeno pedaço de papel toalha ou almofada cirúrgica entre o animal ea almofada de aquecimento. </li><li> Com uma tesoura autoclavados, cortar pequenos pedaços de gelfoam autoclavado em uma placa de Petri estéril contendo solução salina 0,9% estéril até ready para uso. Nota: Manter ambiente de trabalho estéril, utilizando instrumentos esterilizados e suprimentos cirúrgicos estéreis. Re-esterilizar instrumentos cirúrgicos durante o procedimento ou entre animais por meio de imersão em um esterilizador talão de 10-15 segundos, conforme necessário. Manter luvas limpas durante todo o procedimento, esfregando as mãos com álcool a 70%, conforme necessário, de desinfectar. </li></ol> 2. Prepping mouse para Cirurgia <ol><li> Remover rato de gaiola e pesar (g). </li><li> Colocar o mouse na câmara de indução isoflurano e definir oxigênio a 2 L / min e vaporizador isoflurano a 5 para induzir um plano cirúrgico de anestesia, cerca de 3-5 min. Monitore a respiração desacelerou e imobilização. Verifique se o mouse está totalmente sedado utilizando o reflexo toe pitada. </li><li> Quando o mouse está totalmente sedado, coloque no quadro estereotáxico, prenda o nariz no cone do nariz, que está ligado com a tubulação para o isoflurano. Insira barras de ouvido no canal do ouvido e aperte, garantindo a cabeça é estável. </li><li> Raspar a cabeça de orelha a orelha, e de entre os olhos para atrás das orelhas. </li><li> Esterilizar a pele com toalhetes alternados de álcool isopropílico e uma solução de betadine iodo, 3 vezes cada um. </li><li> Aplicar lágrimas artificiais para ambos os olhos para evitar que sequem durante o procedimento cirúrgico. </li></ol> 3. Procedimento Cirúrgico <ol><li> Monitore a profundidade da anestesia por beliscar a ponta ou cauda. O rato está localizado no plano cirúrgico apropriado quando não houver nenhuma resposta, e a respiração é lenta e uniforme. </li><li> Faça uma incisão na pele parasagittal de apenas atrás dos olhos para quase entre as orelhas em um único movimento, firme, usando uma lâmina de bisturi n ° 11. Mova pele de lado e clip lado direito com pinça hemostática. </li><li> Limpar crânio de membrana sobreposta usando o lado maçante do No. 11 bisturi e algodão derrubou aplicadores. Opcionalmente, limpe o crânio com algodão aplicador derrubado mergulhados em solução salina a 0,9%. Deixar secar completamente. </li><li> Nósção uma pequena régua, marcar a borda anterior da craniotomia a 1 mm caudal para a sutura coronária, e a borda esquerda da craniotomia em 1 mm lateral à sutura sagital (Figura 1), com um marcador permanente. Em seguida, marque a direita e fronteiras caudal da craniotomia a 4 mm das suturas sagital e coronal, respectivamente (Figura 1). </li><li> Usando uma broca de 0,5 mm, começar a fazer a craniotomia por perfuração lentamente seguindo o contorno marcador permanente. Tenha cuidado para não romper o crânio completamente. Pressione suavemente a parte isolada de osso parietal com No. 5 fórceps, áreas de fraqueza dará lugar à pressão. Quando o osso diluído é suficientemente fraca ao longo do perímetro, a peça óssea está pronta para remoção. NOTA: Se a dificuldade experiências investigador remoção do osso em uma única peça, isso sugere que o osso não estava suficientemente diluído durante a perfuração. Considere a perfuração do crânio ainda mais em animais subsequentes para Facilitate fácil remoção da peça óssea. </li><li> Usando uma seringa de 10 ml, equipado com uma agulha G 23, aplicam-se uma pequena quantidade de 0,9% de solução salina para absorver o osso isolado e área perfurada. </li><li> Prenda o braço manipulador ao equipamento estereotáxico. Anexar uma nova lâmina de bisturi n ° 11 para o titular da sonda com o lado afiado da lâmina virada para rostralmente. Nota: Embora os tecidos rostral e caudal experimentar as arestas vivas e sem corte da lâmina, respectivamente, a danos mecânicos induzida pela lesão penetrante é comparável em toda a extensão da lesão. Observamos há diferenças consideráveis ​​das principais características da gliose reativa incluindo upregulation de expressão ou a proliferação GFAP, entre as seções rostral e caudal. </li><li> Mantendo o braço manipulador fora do caminho, levante cuidadosamente o osso isolado usando 5/45 fórceps anguladas. Insira a ponta da pinça para o lado do osso isolada e elevador, usando alavanca para retirar o pedaço de osso deixado para trás em um full movimento. NOTA: Tenha cuidado para não apunhalar o cérebro ou perturbar a dura por baixo do crânio. </li><li> Pegue um pequeno pedaço da espuma gel absorvível embebida e coloque sobre o cérebro descoberto para evitar que ela seque e absorva todo o sangue que possa estar presente. </li><li> Uma vez que a espuma de gel está no lugar, balançar o braço manipulador no lugar e ajustar lâmina para o centro da craniotomia sobre a espuma gel. Remover a espuma de gel e reduzir a lâmina até que a ponta toca a dura-máter sem a perfuração da dura-máter. Mark dorsal / ventral coordenadas utilizando a escala vernier no braço vertical da estereotáxico. </li><li> Usando o braço manipulador, abaixe lentamente a lâmina precisamente três milímetros no cérebro. Isto é conseguido usando as marcas de escala vernier do braço manipulador. Permitir lâmina para ficar no lugar de 5-10 seg. Mova o braço estereotáxico com acessório lâmina rostral para caudal três vezes, permitindo que a lâmina para alcançar os limites rostral e caudal da craniotomia antes de passar para o opposite fim. NOTA: A dura-máter não é removido antes de inserir a lâmina. Em contraste com a dura-máter de rato, que é ~ 80 um de espessura 7, a dura-máter do rato é consideravelmente mais finas (apenas algumas camadas celulares de espessura) e não produz uma resistência apreciável para a lâmina de bisturi durante a inserção. Usar uma nova lâmina de bisturi para cada rato para assegurar que cada animal recebe uma lesão consistente. </li><li> Lentamente levantar o braço estereotáxico, retirando a lâmina do cérebro. Após a remoção da lâmina, coloque imediatamente um outro pedaço de gelfoam na superfície do cérebro para absorver qualquer excesso de sangue ou fluido. </li><li> Enquanto isso, retirar o braço estereotáxico e dispor da lâmina de bisturi n ° 11. Uma vez que o sangramento parou, remova o gelfoam. </li><li> Fechar a ferida por sutura da pele com sutura não absorvível, tal como ethilon ou prolene. As suturas devem ser removidos 9-10 dias após a cirurgia. </li><li> Devolver o mouse para sua gaiola, e permitir que o mouse para se recuperar lentamente em um heating pad eo monitor para detectar quaisquer sinais de angústia. A recuperação da anestesia induzida por isoflurano tipicamente ocorre dentro de 2-5 minutos após a remoção do isofluorano. Não deixe o animal sem supervisão até que ele recuperou recumbancy esternal. </li><li> Administrar 0,5-1 ml de solução de Ringer lactato subcutaneamente para garantir a hidratação. </li></ol> 4. Cuidados pós-cirúrgica <ol><li> Acompanhar de perto os animais no pós-operatório até a recuperação da anestesia, antes de voltar para a colônia. <ol><li> Para reduzir a dor e desconforto pós-operatório, administrar 0,05-0,1 mg / kg de buprenorfina por injecção ip imediatamente após o procedimento. </li><li> Observe os animais por 2-3 dias após a cirurgia para sinais severos de angústia, como movimentos restritos, a falta de preparação, ou perda de peso. Euthanize animais que demonstram algum destes sinais de sofrimento e retire do estudo. </li></ol></li><li> Para examinar histopatológico de tecidos lesados, eutanásia animais por stanperfusão intracardíaca dard. <ol><li> Resumidamente, anestesiar os animais com uma overdose de cetamina / xilazina e, em seguida perfundidos intracardial com 15-20 mL de NaCl a 0,9%, ou até que o fígado é limpa de sangue, seguido por 60 ml de paraformaldeído a 4% no final da experiência. </li></ol></li><li> Dissecar cérebros e pós-correção para 2-4 h em paraformaldeído a 4% antes da transferência para solução de sacarose 30%. Secção cérebros num crióstato a 40-60 uM, e processo por procedimentos histológicos ou imuno-histoquímicos convencionais, ou como descritos em Garcia 8,9. </li></ol>

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The authors have nothing to disclose.

É fundamental que o crânio ou dura subjacente não sejam danificados durante a perfuração. Use uma leve pressão durante a perfuração para garantir o crânio não é perfurada. Além disso, deve ser tomado cuidado ao levantar o pedaço do crânio para garantir a dura não é levantado com o osso. A lesão forebrain facada descrito aqui modelos de uma lesão penetrante no SNC. Embora menos clinicamente traduzível do que os modelos de TBI como FPI ou ICC, o modelo de lesão forebrain fa...

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 os animais submetidos a esse procedimento não requerem períodos de sobrevivência especializados cuidados pós-operatórios, curtos ou de tempo de longo prazo são facilmente incorporados no estudo, dependendo da necessidade de investigar patologia aguda ou crônica após a lesão. Principais recursos de gliose reativa, como regulação alta de GFAP e hipertrofia de soma, pode ser observado logo em 2-3 dias após a lesão. A fase de pico da proliferação de astrócitos reativos é durante os dias 3-5 após les...

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 Reactive Gliose<em&gt; In Vivo</em&gt; Por um Stab Lesão Encéfalo anterior

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

10.5K visualizações.  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. 

Vídeo: 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.

Um Paradigma para Investigar Lesão de reparação do sistema nervoso central em<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. ...

Neurociência

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.

Aguda Cérebro Trauma em camundongos seguiu por Longitudinal dois fótons de imagem

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.

Stab ferida ferimento do peixe-zebra adulto Telencéfalo: Um método para investigar Vertebrados Cérebro Neurogênese e Regeneração

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.

Modelagem astrocitoma Patogênese<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Usando Cortical astrócitos ou células-tronco neurais de condicional, Geneticamente Modificadas Ratos

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 Lesão por Arma Branca do Adulto Óptico Utilizando Zebrafish e Medaka para Análise Comparativa da Capacidade Regenerativa

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

Biologia do Desenvolvimento

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.

Um modelo de lesão in vivo em Drosophila para estudar a neuroregeneração do sistema nervoso periférico e 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.

Estimulando e analisando neurogênese adulta no cérebro 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 de Lesão Cerebral Traumática Leve em Camundongos com Controle Eletromagnético

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.

Imagem Intravital da Expressão de Proteínas Fluorescentes em Camundongos com Traumatismo Cranioencefálico de Crânio Fechado e Janela Craniana Usando um Microscópio de Dois Fótons

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 camundongo com ferida por faca para estudar hemorragia e inflamação em lesão cerebral traumática

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

Disparo Reactive Gliose

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