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

Erwachsene (3-4 Monate alt) männlichen Mäusen auf eine gemischte C57BL / 6 Hintergrund wurden in diesem Protokoll verwendet. Die Tiere wurden auf einem 12 Stunden Licht / Dunkel-Zyklus gehalten und freien Zugang zu Nahrung und Wasser. Alle Verfahren in diesem Protokoll durchgeführt wurden nach Protokollen, die von der Drexel University Institutional Animal Care und Verwenden Committee genehmigt durchgeführt. 1. Vorbereitung OP-Bereich <ol><li> Desinfizieren Sie OP-Tisch mit 70% Ethanol, dann decken die gesamte chirurgische Bank mit absorbierenden Kissen und vereinbaren chirurgische Instrumente neben stereotaktischen. </li><li> Up stereotaktischen Gerät ohne Manipulatorarmes gesetzt. Ordnen Sie das Heizkissen auf der stereotaktischen und auf 37 ° C eingestellt. Vermeiden Sie eine Überhitzung des Tieres, indem Sie ein kleines Stück Papiertuch oder einem chirurgischen Polster zwischen dem Tier und dem Heizkissen. </li><li> Verwendung autoklaviert Schere, geschnitten kleine Stücke autoklaviert Gelschaum in eine sterile Petrischale gegeben, die 0,9% steriler Kochsalzlösung, bis ready zum Einsatz. Hinweis: Halten Sie sterile Arbeitsumgebung durch die Verwendung autoklaviert Instrumente und sterile OP-Bedarf. Erneute Sterilisation chirurgischer Instrumente während des Eingriffs oder zwischen Tieren durch Eintauchen in eine Perle Sterilisator für 10-15 sec, je nach Bedarf. Pflegen Sie saubere Handschuhe während des gesamten Verfahrens durch Reiben Hände mit 70% Ethanol, bei Bedarf zu desinfizieren sein. </li></ol> 2. Prepping Maus für Chirurgie <ol><li> Entfernen Sie Maus von zu Hause aus Käfig und wiegen (g). </li><li> Bewegen Sie die Maus in Isofluran Induktionskammer und setzen Sauerstoff zu 2 l / min und Isofluran Verdampfer bis 5, eine chirurgische Ebene der Anästhesie, etwa 3-5 min zu induzieren. Monitor für verlangsamte Atmung und Immobilisierung. Überprüfen Sie, ob die Maus ist komplett mit der Zehe Prise Reflex sediert. </li><li> Bei der Maus vollständig sediert wird, legen Sie in stereotaktischen Rahmen, sichern Sie die Nase in der Nasenkegel, der mit Schlauch an den Isofluran angebracht ist. Stecken Ohr Bars in den Gehörgang ein und ziehen Sie, die Gewährleistung der Kopf ist stabil. </li><li> Rasur des Kopfes von Ohr zu Ohr, und zwischen den Augen, um hinter den Ohren. </li><li> Sterilisieren der Haut mit abwechselnden Wischtücher aus Isopropylalkohol und Betadin Jodlösung jeweils 3 mal. </li><li> Künstliche Tränen für beide Augen, um sie vor dem Austrocknen während des chirurgischen Verfahrens zu verhindern. </li></ol> 3. Chirurgische Verfahren <ol><li> Überwachung der Narkosetiefe durch Kneifen der Zehe oder Schwanz. Die Maus in die entsprechende chirurgische Ebene, wenn es keine Antwort gibt, und die Atmung ist langsam und gleichmäßig. </li><li> Machen Sie eine parasagittal Hautschnitt von gerade hinter den Augen, fast zwischen den Ohren in einer einzigen, festen Bewegung unter Verwendung eines Nr 11 Skalpellklinge. Bewegen Sie die Haut beiseite und Clip rechts mit Gefäßklemme. </li><li> Klar Schädel des darüberliegenden Membran mit der stumpfen Seite der No. 11 Skalpell und Wattestäbchen Applikatoren. Gegebenenfalls wischen Sie die Schädel mit Wattestäbchen in 0,9% Kochsalzlösung eingetaucht. Komplett trocknen lassen. </li><li> Unsing ein kleines Lineal, markieren Sie den vorderen Rand des Kraniotomie bei 1 mm kaudal der Kranznaht, und dem linken Rand des Kraniotomie bei 1 mm lateral der Pfeilnaht (Abbildung 1), mit einem wasserfesten Stift. Markieren das rechte und Schwanz Grenzen der Kraniotomie bei 4 mm von der sagittalen und koronalen Naht, bzw. (Abbildung 1). </li><li> Unter Verwendung einer 0,5 mm Bohrer, beginnen, Kraniotomie durch Bohren langsam nach dem Permanentmarker Umriss zu machen. Achten Sie darauf, durch den Schädel vollständig zu brechen. Drücken Sie vorsichtig auf den isolierten Stück Scheitelbein mit No. 5 Zangen, Schwachstellen werden bis zum Druck zu geben. Wenn die ausgedünnt Knochen ist im gesamten Umfang ausreichend schwach, ist die Knochenstück zum Ausbau bereit. HINWEIS: Wenn die Ermittler Erfahrungen Schwierigkeiten beim Entfernen der Knochen in einem Stück, bedeutet dies, dass Knochen nicht ausreichend beim Bohren ausgedünnt. Betrachten Sie weitere Bohren des Schädels in den Folge Tiere facilitate ein leichtes Entfernen des Knochenstück. </li><li> Mit einem 10-ml-Spritze mit einer 23 G-Nadel ausgestattet, eine kleine Menge von 0,9% Salzlösung, um das isolierte Knochen gebohrt und Umgebung genießen. </li><li> Befestigen Sie den Manipulatorarm zu der stereotaktischen Geräte. Befestigen Sie eine neue Nummer 11 Skalpellklinge auf die Sondenhalter mit der scharfen Seite der Klinge gegen rostral. HINWEIS: Obwohl die rostral und Schwanzgewebe erleben Sie die scharfen und stumpfen Kanten der Klinge bzw. ist die mechanische Beschädigung durch den durchdringenden Verletzungen induziert in der gesamten Ausdehnung der Läsion vergleichbar. Wir beobachten keine nennenswerten Unterschiede in der Hauptmerkmale der reaktiven Gliose einschließlich Hochregulation von GFAP-Expression oder Proliferation, zwischen rostralen und kaudalen Abschnitte. </li><li> Halten Sie den Manipulatorarm aus dem Weg, heben Sie die isolierten Knochen mit 5/45 abgewinkelt Pinzette. Legen Sie die Spitze der Zange in die Seite des isolierten Knochen und Lift, mit Hebelwirkung, um das Knochenstück hinter einem ful links abziehenl Bewegung. HINWEIS: Achten Sie darauf, um das Gehirn zu erstechen oder stören die Dura unter dem Schädel. </li><li> Nehmen Sie ein kleines Stück des getränkten resorbierbare Gel-Schaum und auf den freigelegten Gehirn, um sie vor dem Austrocknen zu verhindern und genießen Sie kein Blut, die vorhanden sein könnten. </li><li> Sobald das Gel Schaum vorhanden ist, schwingen die Manipulatorarm in Stelle und passen Klinge Zentrum Kraniotomie über das Gel-Schaum. Entfernen Sie die Gel-Schaum und senken Sie die Klinge, bis die Spitze der Dura berührt, ohne Punktion der Dura. Mark dorsal / ventral Koordinaten unter Verwendung des Nonius auf der vertikalen Arm der stereotaktischen. </li><li> Unter Verwendung des Manipulatorarmes, langsam senken die Klinge genau 3 mm in das Gehirn. Dies wird mit Hilfe der Noniusskala Markierungen auf dem Manipulatorarm erreicht. Lassen Sie Messer, um im Platz für 5-10 sec bleiben. Bewegen Sie den stereotaktischen Arm mit Blattansatz rostral bis dreimal so das Blatt, um die rostralen und kaudalen Grenzen der Kraniotomie zu erreichen, bevor er nach dem Einspruch kaudalte Ende. HINWEIS: Die Dura wird nicht entfernt, bevor die Klinge eingesetzt wird. Im Gegensatz zur Ratten-Dura, das ist ca. 80 um Dicke 7 ist die Maus dura wesentlich dünner (nur wenige Zellschichten dick) und keinen nennenswerten Widerstand auf die Skalpellklinge während des Einsetzens erzeugen. Verwenden ein neues Skalpellklinge für jede Maus, um sicherzustellen, dass jedes Tier eine konsistente Verletzung. </li><li> Langsam heben die stereotaktischen Arm, das Entfernen der Klinge aus dem Gehirn. Nach dem Entfernen der Klinge, sofort mit einem weiteren Stück Gelschaum auf der Hirnoberfläche zu tanken überschüssige Blut oder Flüssigkeit. </li><li> In der Zwischenzeit, entfernen Sie den stereotaktischen Arm und entsorgen Sie die No. 11 Skalpellklinge. Sobald Blutung aufgehört hat, entfernen Sie die Gelschaum. </li><li> Schließen Sie die Wunde durch Naht der Haut mit nicht-resorbierbares Nahtmaterial, wie zum Beispiel Ethilon oder Prolene. Nähte sollten nach der Operation entfernt werden 9-10 Tage. </li><li> Rückflug die Maus, um seinen eigenen Käfig, und lassen Sie die Maus, um langsam auf einem heatin erholeng-Pad und Monitor für jegliche Anzeichen von Leiden. Erholung von Isofluran-induzierten Anästhesie tritt normalerweise innerhalb von 2-5 min nach Entnahme aus dem Isofluran. Stellen Sie das Tier nicht unbeaufsichtigt lassen, bis es sternale Festliegen wiedererlangt. </li><li> Verabreichen 0,5-1 ml Ringer-Laktat-Lösung subkutan Hydratisierung sicherzustellen. </li></ol> 4. Post-OP-Pflege <ol><li> Tiere nach der Operation genau zu überwachen, bis aus der Narkose vor der Rückkehr in die Kolonie. <ol><li> Um Schmerzen und Beschwerden nach der Operation zu reduzieren, zu verwalten 0,05-0,1 mg / kg Buprenorphin durch ip-Injektion unmittelbar nach dem Eingriff. </li><li> Beobachten Sie Tiere für 2-3 Tage nach der Operation für schwere Anzeichen von Leiden, wie beschränkt Bewegungen, mangelnde Pflege oder Gewichtsverlust. Euthanize Tieren demonstriert jede dieser Anzeichen von Leiden und von der Studie zu entfernen. </li></ol></li><li> Um Histopathologie der verletzten Gewebe zu untersuchen, einschläfern Tiere durch standard intrakardiale Perfusion. <ol><li> Kurz anästhesiert Tiere mit einer Überdosis an Ketamin / Xylazin, dann intrakardial mit 15-20 ml 0,9% NaCl perfundiert, oder bis die Leber von dem Blut eliminiert, gefolgt von 60 ml einer 4% Paraformaldehyd bei Abschluss des Experiments. </li></ol></li><li> Sezieren Gehirne und post-fix für 2-4 Stunden in 4% Paraformaldehyd vor der Übertragung auf 30% Saccharose-Lösung. Abschnitt Gehirn auf einem Kryostaten bei 40-60 um und Prozess durch histologische Standard oder immunhistochemische Verfahren, oder wie in Garcia 8,9 beschrieben. </li></ol>

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The authors have nothing to disclose.

Es ist kritisch, daß der Schädel oder der zugrunde liegenden Dura nicht während des Bohrens beschädigt wird. Mit leichtem Druck während des Bohrens, um den Schädel zu gewährleisten wird nicht punktiert. Außerdem sollte darauf geachtet werden, beim Anheben der Schädelteil auf die Dura zu gewährleisten ist nicht mit dem Knochen abgehoben werden. Das Vorderhirn Stichverletzung hier beschriebenen Modelle eine durchdringende Verletzungen des ZNS. Obwohl weniger klinisch übersetzbar als...

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.

Weil Tiere unterziehen dieses Verfahren benötigen keine spezielle postoperative Pflege, kurz- oder langfristige Überlebenszeitperioden lassen sich leicht in die Studie aufgenommen, je nach Notwendigkeit, akute oder chronische Pathologie nach der Verletzung zu untersuchen. Hauptmerkmale der reaktiven Gliose, wie Hochregulation von GFAP und Hypertrophie von Soma, kann so früh wie 2-3 Tage nach der Verletzung beobachtet werden. Die Hochphase der Proliferation für reaktive Astrozyten ist während der Tage 3-5 nach der V...

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.

Auslösen Reactive Gliose<em&gt; In Vivo</em&gt; Durch eine Vorderhirn Stab Injury

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

10.5K Aufrufe.  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. 

Serie: 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.

Eine Verletzung Paradigm Central Nervous System Repair in Untersuchen<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. ...

Forschung

Neurowissenschaften

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.

Akute Brain Trauma in Mäusen Gefolgt durch Längs Zwei-Photonen-Imaging

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

Medizin

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.

Stichwunde Verletzung des Zebrafisch Erwachsene Telen: eine Methode, um Wirbel Gehirn Neurogenese und Regeneration Untersuchen

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.

Modellierung Astrozytom Pathogenese<em&gt; In-vitro-</em&gt; Und<em&gt; In Vivo</em&gt; Verwenden kortikale Astrozyten oder neuralen Stammzellen aus dem Bedingten, gentechnisch veränderte Mäuse

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.

Stichwundenverletzungsmodell des adulten optischen Tektums unter Verwendung von Zebrafisch und Medaka zur vergleichenden Analyse der Regenerationsfähigkeit

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

Entwicklungsbiologie

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.

Eine Drosophila In Vivo Verletzungen Modell für das Studium Neuroregeneration in den peripheren und zentralen Nervensystems

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.

Stimulation und Analyse der adulten Neurogenese im zentralen Gehirn von 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.

Elektromagnetisch gesteuertes Closed-Head-Modell für leichtes Schädel-Hirn-Trauma bei Mäusen

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.

Intravitale Bildgebung der fluoreszierenden Proteinexpression bei Mäusen mit Schädel-Hirn-Trauma und Schädelfenster mit einem Zwei-Photonen-Mikroskop

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.

Stichwunden-Mausmodell zur Untersuchung von Blutungen und Entzündungen bei traumatischen Hirnverletzungen

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

Auslösen Reactive Gliose

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Eine Verletzung Paradigm Central Nervous System Repair in Untersuchen