Gli autori ringraziano il Voltaire Gungab per l&#39;assistenza nella preparazione del manoscritto. Questo lavoro è stato sostenuto in parte da finanziamenti: il National Institutes of Health (T32 GM008440 di EJ Walker, R01 NS27713 di WL Young, P01 NS44155 di WL H. Young e Sop) e R21 NS070153 a H. Su, e l&#39;americano Heart Association (AHA 10GRNT3130004 a H. Sop).

1. Compensazione del sangue da vasi <ol><li> Anestetizzare topo mediante iniezione intraperitoneale di 100 mg / kg di ketamina con 10 mg / kg xylazina diluiti in soluzione salina 0,25 ml. </li><li> Una volta che l&#39;anestesia è stata confermata da alcuna risposta a un pizzico zampa, usare le forbici per fare un&#39;incisione sul petto con le forbici appena sotto il processo xifoideo, tagliare diaframma, e tagliare tra il segmento dorsale e ventrale della gabbia toracica per esporre cavità toracica. Esporre cuore piegando fino allo sterno e della parete toracica adiacente e sicuro con hemostat. Aprire il sacco pericardico con le pinzette per esporre il cuore. </li><li> Accendere la pompa perfusione (100 mmHg di pressione) collegato al caldo PBS addizionato con eparina (5 unità / ml), soluzione in bagno di acqua calda a 37 ° C e il tubo con un brusco 22 gauge sul deflusso. </li><li> Inserire l&#39;ago nel ventricolo sinistro, vicino all&#39;apice del cuore. Immediatamente taglio aperto l&#39;atrio destro per permettere deflusso arteriosa sistemica e continuare a sciacquare fino a sangue is ritirate dalla circolazione. </li></ol> 2. Perfusione di Casting Agent nel sistema vascolare <ol><li> Preparare Microfil (flusso Tech, Inc. Carver, MA), soluzione di fusione per protocollo del produttore: Mescolare 5 ml di diluente MV con 4 ml di composto MV filtrata. Aggiungere 450μl (5%) di catalizzatore (induritore MV). Tempo di soluzione di lavoro è di 20 minuti, ma miscelare al momento dell&#39;uso per garantire la corretta viscosità. </li><li> Prelevare fusione soluzione agente in una siringa da 10 ml, allegare un brusco ago di calibro 20 alla siringa e riempire l&#39;ago con soluzione. </li><li> Inserire l&#39;ago nel ventricolo sinistro attraverso il foro stesso usato per il lavaggio del sangue. Fissare l&#39;ago in posizione con pinza emostatica sul cuore. </li><li> Iniettare agente soluzione di colata lentamente nel ventricolo sinistro di circa 3 ml / min. Pressione in eccesso può portare a errate flusso di agente di casting o di rottura serbatoio potenziale. </li><li> Cercare i segni di perfusione di successo, tra cui: la visualizzazione del castcomunemente in commercio, vasi intestino e fegato, scolorimento distali degli arti, e il naso e scolorimento della lingua. Regolare di nuovo l&#39;ago, se necessario, per raggiungere la perfusione corretta. </li></ol> 3. Raccolta ed elaborazione del tessuto <ol><li> Togliere il cervello e mettere in paraformaldeide 4% durante la notte a 4 ° C. </li><li> Disidratare cervello mettendo da paraformaldeide direttamente in EtOH sempre più concentrata a temperatura ambiente: 25% EtOH, 1 giorno, 50% EtOH, 1 giorno, 75% EtOH, 1 giorno, 95% EtOH, 2 giorni e il 100% EtOH, 2 giorni . </li><li> Chiarire tessuto cerebrale intorno ai vasi sanguigni immergendo il cervello perfuso in salicilato di metile per 2 giorni a temperatura ambiente. </li><li> Vascolarizzazione di tutto il cervello unsectioned chiarito può essere ripreso con il cervello immerso in salicilato di metile sotto un microscopio da dissezione (Fig. 1) (MZFL microscopio Leica III, Leica Microsystems, Bannockburn, IL) o microscopio campo chiaro (Fig. 2) (Leica DM / LS, Leica Microsystems). </li><li> Reclarificationpuò essere fatto se il tessuto non è completamente eliminato. Dal cervello salicilato di metile, il trasferimento, al 95% EtOH per 2 giorni, 100% EtOH per 2 giorni, e salicilato di metile per 2 giorni. </li></ol> 4. Micro-CT Imaging o istologica Application-Preparazione del tessuto <ol><li> Il cervello può essere ripreso con micro-CT subito dopo chiarimenti o dopo il re-idratazione (Fig. 4). </li><li> Per preparare il tessuto cerebrale per l&#39;analisi istologica, reidratare il cervello attraverso seriale EtOH diluito: 100% EtOH, 1 giorno, 95% EtOH, 1 giorno, 75% EtOH, 1 giorno, 50% EtOH, 2 giorni, il 25% EtOH, 2 giorni ; e conservare in PBS. Il cervello può essere quindi inclusi in paraffina utilizzando una procedura standard. </li></ol> 5. Rappresentante Risultati A seguito di fusione vascolare, la cerebrovasculature può essere visto macroscopicamente sulla superficie del cervello (Fig.1a). L&#39;albero vascolare all&#39;interno del parenchima può essere visualizzata precisa quanto segue (Fig. 1B). La struttura dettagliata vascolarepuò essere ripreso e analizzato al microscopio campo chiaro (Fig. 2), dissezione portata (Fig. 3), e con micro-TAC (Fig. 4). Utilizzando il microscopio chiaro, il sistema vascolare può essere vista a diversi piani focali senza sezionare il cervello (Fig. 2A), così come microvasi individuali a maggiore ingrandimento (Fig. 2B). In un ambito di dissezione, si può vedere l&#39;intera struttura vascolare in un unico piano focale, permettendo una migliore visualizzazione della struttura tridimensionale della cerebrovasculature (Fig. 3). Le immagini scattate sotto un microscopio da dissezione può mostrare morfologia grande nave e vascolarizzazione intero cervello, ma mancano i dati riportati nelle immagini scattate con la microscopia brightfield (Fig. 2B). Questa tecnica ci ha permesso di individuare i pescherecci irregolari nel nostro modello sperimentale topo adulto BAVM 2. La Figura 5 mostra un esempio dei vasi stessi irregolari rilevate da micro-TAC (Fig. 5A) e microscopio a campo chiaro (Fig. 5B). Insieme, questi dati dimostrano che siamo in grado di analizzare tlo stesso campione con strumenti di imaging più dopo la fusione vascolare. <img alt="Figura 1" src="/files/ftp_upload/2958/2958fig1.jpg" /> Figura 1. Cervello di topo intero successivo getto vascolare. Prima (A) e dopo (B) chiarificazione con salicilato di metile solvente. Le navi possono essere chiaramente visualizzate sulla superficie del cervello (A) e nella procedura di chiarificazione parenchima seguente (B). <img alt="Figura 2" src="/files/ftp_upload/2958/2958fig2.jpg" /> Figura 2. Immagini al microscopio di Brightfield chiarito cerebrali vascolari fuso. Cervello può essere ripreso in diversi piani e livelli diversi di ingrandimento. Scale Bar 200μm (A) e 50 micron (B). <img alt="Figura 3" src="/files/ftp_upload/2958/2958fig3.jpg" /> Figura 3. Dissezione immagine al microscopio a 1x mostrando tutta topo adulto cerebrovasculature cervello da una vista dorsale. &lt;img alt = &quot;Figura 4&quot; src = &quot;/ files/ftp_upload/2958/2958fig4.jpg&quot; /&gt; Figura 4. Micro-CT scansione delle immagini del cervello stesso intero topo adulto mostrato in Figura 3 da una vista dorsale. <img alt="Figura 5" src="/files/ftp_upload/2958/2958fig5.jpg" /> Figura 5. Applicazione del metodo di fusione vascolare per rilevare una malformazione arterovenosa cerebrale. A) Micro-TAC del cervello fuso vascolare mostra regione allargata, irregolari AVM-come navi in ​​emisfero destro da una vista ventrale. Immagine ingrandita della regione (scatola bianca) viene mostrato sulla destra. B) Lo stesso vasi irregolari visualizzati al microscopio campo chiaro con ingrandimento 50x. Scala bar 50 micron.

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	<li>Bolland, B. J., Kanczler, J. M., Dunlop, D. G., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+in+vivo+muCT+evaluation+of+neovascularisation+in+tissue+engineered+bone+constructs.">Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs.</a> Bone. 43, 195-202 (2008).</li><li>Walker, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteriovenous+malformation+in+the+adult+mouse+brain+resembling+the+human+disease.">Arteriovenous malformation in the adult mouse brain resembling the human disease.</a> Ann. Neurol. , (2011).</li><li>Iqbal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+novel+mono-+and+multivalent+VHH-fragments+and+their+application+for+molecular+imaging+of+brain+tumours.">Kinetic analysis of novel mono- and multivalent VHH-fragments and their application for molecular imaging of brain tumours.</a> Br. J. Pharmacol. 160, 1016-1028 (2010).</li><li>Fukunaga, A., Kawase, T., Uchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+after+simultaneous+transplantation+with+neuro-epithelial+stem+cells+and+adjacent+mesenchymal+tissues+into+infarcted+rat.">Functional recovery after simultaneous transplantation with neuro-epithelial stem cells and adjacent mesenchymal tissues into infarcted rat.</a> 145, 473-480 (2003).</li><li>Chugh, B. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothesis:+vasa+vasorum+and+neovascularization+of+human+coronary+arteries.+A+possible+role+in+the+pathophysiology+of+atherosclerosis.">Hypothesis: vasa vasorum and neovascularization of human coronary arteries. A possible role in the pathophysiology of atherosclerosis.</a> N. Engl. J. Med. 310, 175-177 (1984).</li><li>Fossett, D. T., Caputy, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Operative Neurosurgical Anatomy. , (2002).</li><li>Reynolds, D. G., Brim, J., Sheehy, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+architecture+of+the+small+intestinal+mucosa+of+the+monkey+(Macaca+mulatta).">The vascular architecture of the small intestinal mucosa of the monkey (Macaca mulatta).</a> Anat. Rec. 159, 211-218 (1967).</li><li>Bernard, S., Luchtel, D. L., Polissar, N., Hlastala, M. P., Lakshminarayan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+size+of+bronchopulmonary+anastomoses+in+sheep+lung.">Structure and size of bronchopulmonary anastomoses in sheep lung.</a> Anat. Rec. A. Discov. Mol. Cell. Evol. Biol. 286, 804-813 (2005).</li><li>Brady, J. M., Cutright, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+of+measuring+blood+vessel+volume+in+bone+applied+to+the+mandible+and+humerus+of+the+rat.">A new technique of measuring blood vessel volume in bone applied to the mandible and humerus of the rat.</a> Anat. Rec. 170, 143-146 (1971).</li><li>Evan, A. P., Dail, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efferent+arterioles+in+the+cortex+of+the+rat+kidney.">Efferent arterioles in the cortex of the rat kidney.</a> Anat. Rec. 187, 135-145 (1977).</li><li>Murphy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Notch4+signaling+induces+hallmarks+of+brain+arteriovenous+malformations+in+mice.">Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 10901-10906 (2008).</li></ol>

Procedure sperimentali per l&#39;utilizzo di animali da laboratorio sono stati approvati dalla cura degli animali e del Comitato Istituzionale uso della University of California, San Francisco (UCSF).

Riportiamo qui un metodo per la fusione di cerebrovasculature topo adulto che può essere ripreso con diverse modalità. L&#39;applicazione di questo metodo al BAVM modello di malattia consente l&#39;analisi 3D dei vasi irregolari. Potenziali futuri studi includono la quantificazione dei micro-CT immagini, analisi istologica dei tessuti, e la diagnosi di progressione della malattia vascolare. Problemi comuni riscontrati e suggerimenti Perfusione completa del sistema vascolare del cervello non è sempre raggiunto. Avere una pressione sufficiente a raggiungere i vasi sanguigni distale senza rompersi l&#39;atrio sinistro e l&#39;aorta è una sfida. Per superare questo problema, applicare una pressione costante di circa 100 mmHg. Inoltre, regolare l&#39;ago grado di garantire deflusso appropriata di fusione agente nell&#39;aorta. E &#39;importante che le arterie carotidi non si limita così l&#39;agente di casting può raggiungere i vasi cerebrali. Procedure critiche che correlano con il miglioramento dei piccoli vasi perfusione sono: (1) adeguata compensazione del sangue dai vasi, (2) filtrazione del agente di casting, e (3) posizionamento dell&#39;ago durante l&#39;iniezione agente di casting. Si deve prestare attenzione per evitare l&#39;iniezione di casting agent in atrio sinistro e del polmone. Altri ricercatori sono riusciti a perfusi del mouse 3 e 4 ratto cerebrovasculature senza l&#39;utilizzo di ulteriori procedure. Analisi manuale di Microfil vasi perfusi di contrasto con hemotoxylin ha mostrato 93% nave perfusione lumen 5. Sempre scartare o completamente pulire qualsiasi tipo di strumento che viene a contatto con l&#39;agente di fusione in quanto dovrà polimerizzare rapidamente, e gli strumenti non possono essere utilizzati per soggetti multipli. Chiarimento del tessuto cerebrale circostante il mouse può essere impegnativo. A seguito di immersione in salicilato di metile, il tessuto può apparire opaca, rendendo difficile l&#39;imaging nave. Assicurare adeguata disidratazione e chiarificazione dovrebbe risolvere questo problema. Posizionamento tessuto cerebrale su un bilanciere durante l&#39;alcool metilico e di strattamento alicylate può migliorare chiarificazione dei tessuti. Inconvenienti della Tecnica I limiti di questa tecnica sono: (1) è necessario per iniettare la soluzione di agente di casting immediatamente dopo la miscelazione come sarà addensare rapidamente dopo il catalizzatore è aggiunto (2) l&#39;agente di casting riempie vasi di conduttanza di grandi dimensioni facilmente, ma non affidabile riempire navi più piccole, come le arteriole (vasi di resistenza) e capillari (vasi nutritivi) e (3) non è il miglior contrasto radiopaco per la micro-TC, anche se alcuni gruppi preferiscono Microfil per la micro-TC 1,6. Tuttavia, seguendo i suggerimenti proposti in precedenza, tra cui posizionamento dell&#39;ago, le posizioni nave e pressione di perfusione, fusione completa di navi più piccole può essere raggiunto. L&#39;adattabilità a vari strumenti di analisi rende questo agente di un prezioso strumento per analizzare la struttura cerebrovascolari, tra cui la circolazione arteriosa e venosa, con variprospettive. Le applicazioni della tecnica Il vantaggio di questo metodo è il suo potenziale applicazione ampio. Il cast riempie tutte le navi, comprese le arterie, vene e capillari di tutti i tessuti all&#39;interno del corpo, così le strutture vascolari in molti organi di diverse specie possono essere analizzati. In precedenza, altri gruppi hanno usato questa perfusione per grandi e piccoli 7 8 unità umani, scimmia 9, 10 pecore, ratto 4,11,12 e mouse 3,13. Mostriamo ora la prova di usare questa tecnica per analizzare la struttura dei microvasi nel cervello di topo, negli stati sani e malati 2. In aggiunta, ci sono soluzioni Microfil con diverse viscosità a disposizione di perfusione vasi di diverse dimensioni e colori diversi per migliorare la visualizzazione dei vasi in vari organi. Per esempio, il rosso può essere facilmente visibili nel parenchima cerebrale pallido e giallo contrasta bene conrosso scuro miocardio. Inoltre, poiché il materiale è radiopaco, vasi può essere ripreso con micro-TC per ottenere un&#39;immagine 3D ruotabile. Il tessuto può essere utilizzato anche per le sezioni di paraffina e l&#39;analisi istologica dopo essere stato fotografato. Conclusione Si dimostra qui un protocollo per fare un cast vascolari dei vasi sanguigni del cervello di topo adulto, che può essere adattato usando una varietà di tecniche di analisi per studiare la struttura morfologica del cerebrovasculature. Abbiamo anche fornito prove per l&#39;applicazione di questo metodo per un modello di stato di malattia BAVM, rappresentata dal allargata, i vasi di forma irregolare che il sangue arterioso al shunt da circolazione venosa. Ci sono una varietà di metodi di fusione vascolare disponibili. Il protocollo semplice forniamo qui per un cast vascolare può essere utilizzato come uno strumento per valutare la vascolarizzazione del cervello di topo adulto utilizzando varie modalità di imaging.

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Numero di catalogo </td><td> Note </td></tr><tr><td> Microfil </td><td> Flowtech, Inc. </td><td> MV130 </td><td> 4 mesi shelf life </td></tr><tr><td> Salicilato di metile </td><td> Fisher Scientific </td><td> O3695-500 </td><td> per chiarire i tessuti </td></tr></tbody></table>

cerebrovascular casting of the adult mouse for 3d imaging and morphological analysis

Di imaging vascolare è di fondamentale importanza nella diagnosi clinica e la gestione delle malattie cerebrovascolari, come malformazioni artero-venose cerebrali (BAVMs). Modelli animali sono necessari per lo studio delle terapie eziopatologia e le potenzialità di malattie cerebrovascolari. Imaging del sistema vascolare in animali di grandi dimensioni è relativamente facile. Tuttavia, lo sviluppo di metodi di imaging vaso di modelli murini della malattia del cervello è auspicabile a causa del costo e la disponibilità di organismi geneticamente modificati linee del mouse. Imaging del murino albero vascolare cerebrale è una sfida. Negli esseri umani e animali più grandi, il gold standard per la valutazione della angioarchitecture al macrovascolari (conduttanza) di livello è x-ray catetere contrasto a base di angiografia, un metodo non adatto per piccoli roditori. In questo articolo, presentiamo un metodo di fusione cerebrovascolari che produce uno scheletro durevole del letto vascolare intero, comprese le arterie, vene e capillari che possono essere analizzati utilizzando molti dmodalità ifferent. Fusione completa dei microvasi della cerebrovasculature mouse può essere difficile, tuttavia, queste sfide vengono affrontate in questo step-by-step del protocollo. Attraverso la perfusione intracardial del materiale di colata vascolare, tutte le navi del corpo sono fuso. Il cervello può quindi essere rimosso e chiarito utilizzando il salicilato di metile, solventi organici. Tre immagini tridimensionali dei vasi sanguigni del cervello possono essere visualizzati e getta con qualsiasi microscopio brightfield convenzionali o microscopio da dissezione. Il cerebrovasculature fuso può essere ripreso e quantificati utilizzando micro-tomografia computerizzata (micro-CT) 1. Inoltre, dopo essere stato fotografato, il cervello fuso possono essere inclusi in paraffina per l&#39;analisi istologica. Il vantaggio di questo metodo di fusione vascolare rispetto ad altre tecniche è il suo adattamento ampio per vari strumenti analitici, tra cui l&#39;analisi microscopica campo chiaro, la TC a causa delle caratteristiche radiopacoteristic del materiale, nonché l&#39;analisi istologica ed immunoistochimica. Questo uso efficiente di tessuto può salvare l&#39;utilizzo degli animali e ridurre i costi. Abbiamo recentemente dimostrato applicazione di questo metodo per visualizzare i vasi sanguigni irregolari in un modello murino di BAVM adulti a livello microscopico 2, e fornire altre immagini dei vasi malformati ripreso da micro-TC. Anche se questo metodo ha inconvenienti e non possono essere l&#39;ideale per tutti i tipi di analisi, si tratta di una semplice tecnica pratica che può essere facilmente appreso ed ampiamente applicata al casting vascolari dei vasi sanguigni in tutto il corpo.

Watch this Scientific Journal Video about Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis at JoVE.com

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

In questo articolo vi presentiamo una semplice tecnica pratica per la fusione cerebrovascolari che è facile da eseguire e possono essere utilizzati per l&#39;immagine dell&#39;albero vascolare del cervello di topo adulto.

Casting cerebrovascolare del topo adulto per l&#39;imaging 3D e analisi morfologica

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

cerebrovascular-casting-adult-mouse-for-3d-imaging-morphological

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JoVE Journal

Neuroscience

21.4K Views.  University of California, San Francisco. In questo articolo vi presentiamo una semplice tecnica pratica per la fusione cerebrovascolari che è facile da eseguire e possono essere utilizzati per l'immagine dell'albero vascolare del cervello di topo adulto.

Video: Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Multiple-topo Imaging neuroanatomici Risonanza Magnetica

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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Neuroscienze

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

Time-lapse imaging delle Migrazioni neuroblasti in fette acuta del prosencefalo topo adulto

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualizzazione e manipolazione genetica di dendriti e spine nella corteccia cerebrale e nell&#39;ippocampo mouse utilizzando<em&gt; In utero</em&gt; Elettroporazione

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Metodi per la modulazione e analisi di NF-kB-dipendente adulti neurogenesi

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

Modelli morfologici potenziali evocati di tempo evocati da corticali specifici dello stimolo

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Valutazione morfologica e funzionale delle sinapsi del nastro nelle specifiche regioni di frequenza della Coclea del topo

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and indocyanine green are used to enable the observation of blood flow and vessel perfusion status, respectively. The aim of this study is to apply FVA to verify real-time blood flow, vessel perfusion status and occlusion of aneurysms after induction of sidewall aneurysms in rabbits and rats, as well as to validate the procedure in these species.
Twenty sidewall aneurysms were created in 10 rabbits by suturing a decellularized arterial vessel pouch on the carotid artery of a donor rabbit. In addition, 48 microsurgical sidewall aneurysms were created in 48 rats. During follow-up at one month after creation, the parent artery/aneurysm complex was dissected and FVA was performed using an intravenous fluorescein (10%, 1 mL) injection via an ear vein catheterization in rabbits and a femoral vein catherization in rats. Aneurysms were then harvested, and patency was evaluated macroscopically.
Macroscopically, 14 out of 16 aneurysms in rabbits indicated no residual parent artery perfusion with totally occluded luminae, however 11 (79%) were detected by FVA. Four aneurysms were excluded due to technical problems. In rats, residual aneurysm perfusion was macroscopically observed in 25 out of 48 cases. Of the 23 without macroscopic evidence of perfusion, FVA confirmed the incidence of 22 aneurysms (96%). There were no adverse events associated with FVA. Fluorescein is easily applicable and no special equipment is needed. It is a safe and extremely effective method for evaluating parent artery integrity and aneurysm patency/residual perfusion in an experimental setting with rabbits and rats. FVA using fluorescein as a contrast agent appears to be effective in controlling patency of aneurysms and the underlying vessel and can even be adapted to bypass surgery.

We present a protocol to efficiently evaluate aneurysm perfusion and vessel patency of sidewall aneurysm in rats and rabbits, using fluorescein-based fluorescence video angiography (FVA). With a positive predictive value of 92.6%, it is a simple but very effective and economical method with no special equipment required.

Angiografia di fluorescenza per la valutazione dell'aneurisma perfusione e patriera arteriosa dei genitori nei modelli di aneurisma per ratti e conigli

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Analisi kinematica 3D per la valutazione funzionale nel modello di ratto di lesioni da schiacciamento del nervo sciatico

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Preparazione della superficie cocleare nel topo adulto

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Imaging e analisi del trasporto neurofilamento nel nervo tibiale del topo escresato

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...