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

Adulti (3-4 mesi) topi maschi su un misto C57BL / 6 di fondo sono stati utilizzati in questo protocollo. Gli animali sono stati tenuti al 12 hr luce / buio ciclo, e consentito l&#39;accesso gratuito al cibo e all&#39;acqua. Tutte le procedure eseguite in questo protocollo sono stati condotti secondo protocolli approvati dalla University Istituzionale cura e l&#39;uso degli animali Comitato Drexel. 1. Preparazione Area Chirurgica <ol><li> Disinfettare tavolo operatorio con il 70% di etanolo, quindi coprire l&#39;intera panchina chirurgica con assorbenti e organizzare strumenti chirurgici adiacente stereotassica. </li><li> Impostare attrezzature stereotassico senza braccio manipolatore. Disporre il rilievo di riscaldamento sul stereotassica e impostato a 37 ° C. Evitare il surriscaldamento l&#39;animale mettendo un piccolo pezzo di carta assorbente o tampone chirurgica tra l&#39;animale e il rilievo di riscaldamento. </li><li> Utilizzando le forbici in autoclave, tagliare piccoli pezzi di Gelfoam autoclavato in una piastra di Petri sterile contenente soluzione fisiologica sterile allo 0,9% fino a ready all&#39;uso. Nota: Mantenere ambiente di lavoro sterile, utilizzando strumenti in autoclave e rifornimenti chirurgici sterili. Re-sterilizzare gli strumenti chirurgici durante la procedura o in tra gli animali mediante immersione in uno sterilizzatore tallone per 10-15 secondi, a seconda delle necessità. Mantenere guanti puliti durante l&#39;intera procedura sfregando le mani con il 70% di etanolo, se necessario, da disinfettare. </li></ol> 2. Prepping mouse per Chirurgia <ol><li> Rimuovere mouse dalla gabbia di casa e pesare (g). </li><li> Posizionare il mouse nella camera di induzione isoflurano e ossigeno impostata su 2 L / min e vaporizzatore isoflurano a 5 per indurre un piano chirurgico di anestesia, circa 3-5 minuti. Monitor per la respirazione rallentata e l&#39;immobilizzazione. Verificare che il mouse sia completamente sedato con la reflex pizzico piedi. </li><li> Quando il mouse è completamente sedato, metterli in telaio stereotassico, fissare il naso nel cono naso, che è collegato con un tubo alla isoflurano. Inserire barre di orecchio in canale uditivo e stringere, garantendo la testa è stabile. </li><li> Radere la testa da un orecchio all&#39;altro, e da mezzo agli occhi di dietro le orecchie. </li><li> Sterilizzare la pelle con alternanza di salviette di alcol isopropilico e soluzione betadine iodio, 3 volte ciascuno. </li><li> Applicare lacrime artificiali per entrambi gli occhi per evitare che si secchi durante la procedura chirurgica. </li></ol> 3. Procedura chirurgica <ol><li> Monitorare la profondità dell&#39;anestesia pizzicando la punta o la coda. Il topo è nel piano chirurgica appropriata quando non vi è alcuna risposta, e la respirazione è lenta e uniforme. </li><li> Effettuare una incisione cutanea parasagittale da appena dietro gli occhi per quasi tra le orecchie in un unico movimento ferma con una lama n ° 11 bisturi. Spostare la pelle da parte e la clip a destra con pinza emostatica. </li><li> Cancella cranio di membrana sovrastante con il lato opaco del bisturi n ° 11 e il cotone punta applicatori. Facoltativamente, pulire il cranio con cotone applicatore capovolto immerso in soluzione salina allo 0,9%. Lasciare asciugare completamente. </li><li> Noiing un piccolo righello, segnare il bordo anteriore del craniotomia a 1 millimetro caudale alla sutura coronale, e il bordo sinistro del craniotomia a 1 mm lateralmente alla sutura sagittale (figura 1), con un pennarello indelebile. Quindi contrassegnare il bordo destro e caudale della craniotomia a 4 mm dal suture sagittali e coronali, rispettivamente (Figura 1). </li><li> Utilizzando una punta 0,5 millimetri, cominciare a fare craniotomia perforando lentamente seguendo il marcatore contorno permanente. Fare attenzione a non sfondare il cranio completamente. Premere delicatamente sul pezzo isolato di osso parietale con n ° 5 pinze, aree di debolezza darà modo alla pressione. Quando l&#39;osso assottigliata è sufficientemente debole per tutto il perimetro, il pezzo di osso è pronto per la rimozione. NOTA: Se la difficoltà esperienze investigatore rimuovendo l&#39;osso in un unico pezzo, questo suggerisce che l&#39;osso non era sufficientemente diluito durante la perforazione. Si consideri la foratura ulteriormente il cranio nelle successive animali al facilitafacile rimozione te del pezzo dell&#39;osso. </li><li> Usando una siringa da 10 ml munito di ago G 23, applicare una piccola quantità di soluzione fisiologica 0,9% per assorbire l&#39;osso isolata e zona forata. </li><li> Fissare il braccio manipolatore all&#39;apparecchiatura stereotassica. Collegare un nuovo n ° 11 bisturi al titolare sonda con il lato tagliente della lama rivolta verso rostralmente. NOTA: Sebbene i tessuti rostrale e caudale sperimentano gli spigoli smussati e della lama, rispettivamente, il danno meccanico indotto dalla ferita penetrante è comparabile in tutta l&#39;estensione della lesione. Osserviamo differenze apprezzabili caratteristiche principali di gliosi reattiva tra cui upregulation di espressione GFAP o la proliferazione, tra le sezioni rostrali e caudale. </li><li> Tenendo il braccio manipolatore di mezzo, sollevare con cautela l&#39;osso isolato con 5/45 pinze ad angolo. Inserire la punta della pinza nella parte dell&#39;osso isolata e ascensore, utilizzando la leva per tirare fuori il pezzo di osso lasciato in uno fulil movimento l. NOTA: Fare attenzione a non colpire il cervello o turbare la dura sotto il cranio. </li><li> Prendete un piccolo pezzo di schiuma gel assorbibile imbevuto e posizionare sul cervello scoperto per evitare che si secchi e assorbire il sangue che potrebbero essere presenti. </li><li> Una volta che la schiuma di gel è a posto, oscillare il braccio manipolatore in posizione e regolare lama centro craniotomia sopra la schiuma gel. Rimuovere la schiuma gel e abbassare la lama fino a quando la punta tocca la dura, senza pungere la dura. Mark dorsale / ventrale coordinate utilizzando il nonio sul braccio verticale della stereotassico. </li><li> Utilizzando il braccio manipolatore, abbassare lentamente la lama precisamente da 3 mm nel cervello. Ciò si ottiene utilizzando le marcature nonio sul braccio manipolatore. Lasciare lama per rimanere sul posto per 5-10 secondi. Portare il braccio stereotassica con attacco lama rostrale a caudale tre volte permettendo la lama per raggiungere i confini rostrale e caudale della craniotomia prima di passare alla opposifine te. NOTA: La dura non viene rimosso prima di inserire la lama. In contrasto con la dura madre ratto, che è ~ 80 micron di spessore 7, la dura mouse è considerevolmente più sottile (spessore di pochi strati di cellule) e non produce una resistenza apprezzabile bisturi durante l&#39;inserimento. Utilizzare una nuova lama di bisturi per ogni mouse per garantire che ogni animale riceve una lesione coerente. </li><li> Lentamente sollevare il braccio stereotassico, rimuovere la lama dal cervello. Dopo la rimozione della lama, immediatamente posto un altro pezzo di Gelfoam sulla superficie del cervello per assorbire qualsiasi eccesso di sangue o fluido. </li><li> Nel frattempo, smontare il braccio stereotassica e smaltire la lama n ° 11 bisturi. Una volta emorragia si è fermata, rimuovere il Gelfoam. </li><li> Chiudere la ferita sutura la pelle con sutura non riassorbibile, come ad esempio Ethilon o prolene. Le suture devono essere rimossi 9-10 giorni dopo l&#39;intervento. </li><li> Ritorna il mouse per la sua gabbia di casa, e lasciare il mouse per recuperare lentamente su un heating pad e monitor per eventuali segni di sofferenza. Il risveglio dall&#39;anestesia isofluorane indotta si verifica in genere entro 2-5 minuti dopo la rimozione dal isofluorane. Non lasciare l&#39;animale incustodito fino a quando non ha riconquistato recumbancy sternale. </li><li> Somministrare 0,5-1 ml di soluzione di Ringer lattato per via sottocutanea per garantire idratazione. </li></ol> Cura 4. post-chirurgico <ol><li> Seguire da vicino gli animali dopo l&#39;intervento fino al recupero dall&#39;anestesia, prima di tornare alla colonia. <ol><li> Per ridurre il dolore e il disagio post-operatorio, amministrare 0,05-0,1 mg / kg buprenorfina per iniezione ip immediatamente dopo la procedura. </li><li> Osservare gli animali per 2-3 giorni dopo l&#39;intervento per gravi segni di sofferenza, come i movimenti limitati, la mancanza di cura, o la perdita di peso. Eutanasia animali che dimostrano uno qualsiasi di questi segni di sofferenza e rimuovere dallo studio. </li></ol></li><li> Per esaminare istopatologia dei tessuti lesi, eutanasia animali da stanDard perfusione intracardial. <ol><li> Brevemente, anestetizzare animali con un sovradosaggio di ketamina / xilazina, quindi intracardiaca perfuso con 15-20 ml di NaCl allo 0,9%, o fino a quando il fegato è liberata di sangue, seguiti da 60 ml di 4% paraformaldeide a conclusione dell&#39;esperimento. </li></ol></li><li> Sezionare cervelli e post-correzione per 2-4 ore in paraformaldeide al 4% prima del trasferimento a una soluzione di saccarosio al 30%. Cervelli sezione su un criostato a 40-60 micron, e il processo di procedure istologiche o immunoistochimiche standard, oppure come descritto in Garcia 8,9. </li></ol>

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The authors have nothing to disclose.

E &#39;fondamentale che il cranio o dura sottostante non vengano danneggiati durante la perforazione. Utilizzare una leggera pressione durante la perforazione al fine di garantire il cranio non è forato. Inoltre, si deve prestare attenzione mentre si solleva il pezzo del cranio per garantire la dura non è decollato con l&#39;osso. L&#39;infortunio prosencefalo pugnalata qui descritto modelli una ferita penetrante al SNC. Anche se meno clinicamente traducibile di modelli TBI come la FPI e C...

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.

Dato che gli animali sottoposti a questa procedura non richiedono periodi sopravvivenza specializzati cura post-operatorio, brevi o time-lungo termine sono facilmente incorporati nello studio, a seconda delle necessità di indagare patologie acute o croniche a seguito di infortunio. Caratteristiche principali di gliosi reattiva, come l&#39;up-regolazione di GFAP e l&#39;ipertrofia del soma, si può osservare come già 2-3 giorni dopo un trauma. La fase di picco della proliferazione di astrociti reattivi è nei giorni 3-...

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.

Attivazione reattiva Gliosi<em&gt; In Vivo</em&gt; Da un Forebrain 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

10.4K Views.  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 Infortunio per Indagare riparazione del sistema nervoso centrale in<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. ...

Ricerca

Neuroscienze

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.

Acuta cerebrale Trauma nei topi Seguito By longitudinale a due fotoni di imaging

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 Ferita Lesione del Zebrafish adulti Telencefalo: Un metodo per Indagare vertebrati cervello neurogenesi e rigenerazione

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.

Modellazione Astrocitoma Patogenesi<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Utilizzo corticale astrociti o cellule staminali neurali da condizionale, topi geneticamente ingegnerizzati

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.

Modello di lesione da coltellata del tectum ottico adulto utilizzando zebrafish e medaka per l'analisi comparativa della capacità rigenerativa

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

Biologia dello sviluppo

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 modello di ferita Drosophila In Vivo per lo studio della neurorigenerazione la periferica e del sistema nervoso centrale

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.

Stimolare e analizzare la neurogenesi adulta nel cervello centrale della 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.

Modello elettromagnetico controllato a testa chiusa di lieve lesione cerebrale traumatica nei topi

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.

Imaging intravitale dell'espressione di proteine fluorescenti nei topi con una lesione cerebrale traumatica a cranio chiuso e una finestra cranica utilizzando un microscopio a due fotoni

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.

Modello murino con ferita da taglio per lo studio dell'emorragia e dell'infiammazione nella lesione cerebrale traumatica

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

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