PS detiene una Canada Research Chair in biologia delle cellule della retina e la Alcon Research Institute di New Investigator Award. Questo lavoro è stato sostenuto da sovvenzioni dal Canadian Institutes of Health Research (221.478), la Canadian Diabetes Association (OG-3-11-3329-PS), le scienze naturali e ingegneria Research Council del Canada (418.637) e The Foundation Fighting Blindness Canada. Il sostegno è stato fornito anche dal Reseau de Recherche en Santé de la Vision du Québec.

Dichiarazione etica: Tutto sperimentazione animale rispetta le linee guida per la cura degli animali, istituito dall&#39;Associazione per la Ricerca e la Visione e Ophthalmology (ARVO) Dichiarazione per l&#39;uso di animali in Oftalmica e Vision Research e del Consiglio canadese delle Animal Care. 1. Ossigeno indotta retinopatia (OIR) <ol><li> Data di nascita di cuccioli di topo come P0 Record. </li><li> Registrare tutti i pesi degli animali al momento dell&#39;entrata in O 2 al fine di garantire un adeguato intervallo di peso. Nota: Per C57BL / 6 topi a P17, il peso corporeo deve essere compresa tra 5 e 7,5 g per NV massimo 32. Al fine di mantenere la coerenza ambientale, si raccomanda di utilizzare cucciolata il controllo (per i topi geneticamente modificati, così come i topi ricevere trattamenti sperimentali). Nel valutare gli effetti di un vettore virale, si deve considerare tropismo del virus e consentire un tempo sufficiente per la piena espressione dei transgeni viralmente-consegnati. Rapidamente esprimere viralevettori quali 3 lentivirus terza generazione 24, 25, 33 sono raccomandati. </li><li> Cuccioli posto del mouse a P7 (C57BL / 6 o desiderato deformazione) e una CD1 promozione madre in una camera di ossigeno a 75% O 2 per 5 giorni 27. Umidità ambiente e la temperatura sono stati mantenuti costanti per tutto O 2 esposizione. Nota: Strutture di ricerca equipaggiati con una sorgente centrale di O 2 sono ideali e limitano la sostituzione ingombrante di O 2 serbatoi vuoti. Se si lavora con transgenici o knockout topi, è importante garantire che il controllo e topi sperimentali sono acquisiti dal medesimo fornitore di limitare derive genetiche entro gli stessi ceppi 30, 29. </li><li> A P12, togliere i topi dalla camera di ossigeno e tornare animali ambient O 2. </li></ol> 2. Iniezione intravitreale per la consegna dei composti al Retina interna (WhEffetti en Valutare di un composto farmacologico o proteina ricombinante) <ol><li> A P14, anestetizzare i topi con il 2% isoflurano in ossigeno 2 L / min (o di protezione degli animali comitato approvato anestetico di scelta). Al fine di verificare l&#39;efficacia dell&#39;anestesia, in sequenza pizzicare la coda, piede posteriore e l&#39;orecchio con una pinza. </li><li> Posizionare il mouse sulla pancia. </li><li> Usando una siringa sterile 10 microlitri con ago tirato vetro smussato, eseguire una iniezione di un volume massimo di 1 ml di soluzione contenente il composto in fase di studio o veicolo (soluzione fisiologica) al limbus posteriore dell&#39;occhio, con un 45 ° angolo evitando la lente. Nota: Il vetro-ago tirato è attaccato alla siringa con una goccia di resina epossidica. </li><li> Applicare una goccia di lubrificante pomata oftalmica (idealmente con antibiotico) con un tampone per l&#39;occhio del mouse. </li><li> Ritorno il mouse torna alla gabbia con la promozione madre. I topi sono poi accuratamente controllati fino a quando recoperto e completamente ambulatoriale. </li></ol> 3. Valutazione della nave Perfusione e Barrier Function (Integrity) di angiografia con fluoresceina <ol><li> Anestetizzare i topi con il 2% isoflurano in ossigeno 2 L / min (o di protezione degli animali comitato approvato anestetico di scelta). Al fine di verificare l&#39;efficacia dell&#39;anestesia, in sequenza pizzicare la coda, piede posteriore e l&#39;orecchio con una pinza. Nota: Questo è in genere eseguita a P17 quando viene valutata la rigenerazione. Carry out anche l&#39;analisi a P19 e P21 per determinare se l&#39;integrità vascolare è conservato nel tempo. </li><li> Una volta anestetizzato, pesare il mouse. </li><li> Effettuare una incisione addominale mediana con le forbici dissezione. Nota: gli strumenti per dissezione devono essere regolarmente controllati e affilate. </li><li> Tagliare costole lateralmente e sollevare la gabbia toracica con l&#39;ausilio di pinze. Nota: È necessario tagliare lateralmente come possibile per evitare danni al cuore. </li><li> Dopo la rimozione peripHeral tessuto dal cuore, bloccare il aorta discendente con una pinza emostatica. </li><li> Iniettare lentamente fluoresceina destrano al ventricolo sinistro utilizzando un ago G 25. Nota: Se la funzione di barriera vascolare è indagato, 70 kDa fluoresceina destrano è impiegato come sarà fuoriuscire delle navi quando integrità imbarcazione è compromessa. Se l&#39;investigatore vuole lanciare vasi sanguigni, viene utilizzato 2 MDa fluoresceina destrano. Passaggi critici: 1) Per garantire una ripartizione omogenea, centrifuga fluoresceina destrano e iniettare il surnatante, 2) Al fine di prevenire la vasocostrizione, iniettare soluzione di fluoresceina destrano riscaldato, 3) Il suo tempo di circolazione non dovrebbe eccesso di 4 min. </li><li> Decapitare topi 2 minuti dopo l&#39;iniezione con le forbici operativi. </li></ol> 4. Enucleazione e la fissazione degli occhi Nota: Nel valutare i tassi di rigenerazione vascolare, in primo luogo raccogliere retine in P12 e, in aggiunta a P14 e P17. Aumentare il numero di punto temporale campionatos per la determinazione più accurata dei tassi di rivascolarizzazione 24. <ol><li> Inclinare la testa del mouse e posizionarlo su un fianco. </li><li> Togliere la pelle e le palpebre che coprono gli occhi con le forbici dissezione. </li><li> Posizionare pinze curve sotto l&#39;occhio e tirare delicatamente fino al nervo ottico è reciso. </li><li> Girare la testa del topo sull&#39;altro lato ed eseguire gli stessi passi (passi 4.2 e 4.3). </li><li> Per garantire una migliore penetrazione del fissativo, perforare un foro nella camera anteriore dell&#39;occhio utilizzando 30 ago G. </li><li> Trasferire occhi in una provetta contenente 4% paraformaldeide (PFA) e fissare per 1 ora a temperatura ambiente. </li><li> Rimuovere e lavare gli occhi PFA 4 volte con una soluzione di PBS ghiacciato. </li></ol> 5. Dissezione della retina <ol><li> Mettere gli occhi di topo in una capsula di Petri contenente PBS freddo e di eseguire la dissezione delle retine sotto uno stereomicroscopio. </li><li> Rimuovere il grasso in / tessuto circostante l&#39;occhio con micro-dissezione scissors. </li><li> Tagliare la cornea con le forbici micro-dissezione. </li><li> Utilizzando due coppie di pinze, minuziosamente sbucciare la sclera lontano dalla periferia verso il nervo ottico e scartare. </li><li> Stringere la lente (sfera biancastra sotto la cornea) con una pinza ed estrarlo dalla tazza occhio. Utilizzare un paio di pinze come supporto, e l&#39;altra per afferrare e sollevare con attenzione e rimuovere la lente. </li><li> Staccare i vasi hyaloid dal lato interno della retina con piccoli pennelli (dimensione 0) e pinze. </li><li> Rimuovere fasci di navi hyaloid collegati al disco ottico utilizzando una pinza. </li><li> Trasferimento sezionato retine a 2 tubi ml microcentrifuga contenenti PBS e posto sul ghiaccio prima di avviare la procedura di colorazione. </li></ol> 6. Retina vascolare colorazione <ol><li> Incubare retine sezionato notte con delicata agitazione a 4 ° C in una soluzione di fluorescenza accoppiata isolectin B4 (rodamina-lectina o altro) in PBS contenente 1 mM CaCl 2 </sub&gt; (a 1:100 diluizione di una soluzione B4 2 mg / ml isolectin raccomandata). Durante l&#39;intera procedura di colorazione, tubi coprire con un foglio di alluminio o un foglio opaco al riparo dalla luce. </li><li> Il giorno seguente, rimuovere la soluzione colorante e lavare retine 3x in PBS per 10 minuti a temperatura ambiente. </li></ol> 7. Preparazione della retina Flatmounts <ol><li> Trasferire retine, fotorecettori rivolto verso il basso, su un vetrino da microscopio e fare quattro profonde incisioni radiali equidistanti con un bisturi chirurgico per dividere la retina in quattro quadranti di uguale dimensione. Durante le incisioni, rinforzare la retina con una spazzola in modo che non si muove. </li><li> Utilizzando due spazzole imbevute di PBS, appiattire con attenzione la quadranti fotorecettore lato verso il basso e immergere la retina in mezzo di montaggio per impedire fotometabolismo. Poi posizionare accuratamente un coprioggetto sulla superficie della retina montata senza applicare pressione e fare in modo che le bolle d&#39;aria non si accumulano sotto il vetrino. &lt;/ Li&gt; </ol> 8. Imaging e quantificazione dei Vasoobliteration (VO) e neovascolarizzazione (NV), come descritto in precedenza 31 <ol><li> Prendere immagini di retine intere montato con un microscopio a epifluorescenza con un ingrandimento di 10X. </li><li> Aprire l&#39;immagine retinica in software di editing fotografico, cucire insieme e misurare l&#39;area retinica totale, e la zona avascolare. Area può essere espressa in pixel. </li><li> Determinare misura di VO dividendo il numero di pixel nella zona avascolare per il numero di pixel nell&#39;area retinica totale. </li><li> Determinare misura di NV dividendo il numero di pixel di NV per il numero di pixel per la regione retinica totale come descritto 31. </li></ol>

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G., Jiang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis,+angiogenesis,+and+MRI+indices+of+functional+recovery+from+stroke.">Neurogenesis, angiogenesis, and MRI indices of functional recovery from stroke.</a> Stroke. 38, 827-831 (2007).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+improved+cerebral+blood+flow+in+the+ischemic+boundary+area+detected+by+MRI+after+administration+of+sildenafil+to+rats+with+embolic+stroke.">Angiogenesis and improved cerebral blood flow in the ischemic boundary area detected by MRI after administration of sildenafil to rats with embolic stroke.</a> Brain Res. 1132, 185-192 (2007).</li><li>Robinson, R., Barathi, V. A., Chaurasia, S. S., Wong, T. Y., Kern, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+animal+models+of+diabetic+retinopathy:+from+molecular+approaches+to+mice+and+higher+mammals.">Update on animal models of diabetic retinopathy: from molecular approaches to mice and higher mammals.</a> Dis Model Mech. 5, 444-456 (2012).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat Genet. 37, 1181-1186 (2005).</li><li>Jenuth, J. P., Peterson, A. C., Shoubridge, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+selection+for+different+mtDNA+genotypes+in+heteroplasmic+mice.">Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.</a> Nat Genet. 16, 93-95 (1997).</li><li>Mattapallil, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rd8+mutation+of+the+Crb1+gene+is+present+in+vendor+lines+of+C57BL/6N+mice+and+embryonic+stem+cells,+and+confounds+ocular+induced+mutant+phenotypes.">The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.</a> Investigative ophthalmolog., &amp; visual science. 53, 2921-2927 (2012).</li></ol>

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Qual è il modo più efficace per stimolare la crescita di nuovi vasi sani in tessuto nervoso ischemico? È terapeuticamente valida per interferire e accelerare naturalmente ricrescita vascolare? In patologie neuro-ischemica, quali retinopatie ischemiche o ictus, la degenerazione vascolare è associata a una ridotta funzione neuronale 35-38. Quindi, per contrastare il pregiudizio presto, ripristinando regionale micro-circolazione durante il segmento immediato / precoce della malattia può rivelarsi utile. In ...

Nel corso dello sviluppo del sistema nervoso centrale, nervi, cellule immunitarie e vasi sanguigni istituire reti notevolmente accoppiati per garantire un&#39;adeguata perfusione tissutale e consentire la trasmissione delle informazioni sensoriali 1-5. La ripartizione dei sistemi vascolari risultati dell&#39;ossigenazione tessutale insufficiente e fornitura metabolica compromesso ed è sempre più riconosciuto come un fattore importante per la patogenesi delle malattie neurodegenerative 6. Dropout vascolare e il deterioramento dell&#39;unità neurovascolare all&#39;interno del cervello, per esempio, è associato con demenza vascolare, lesioni vascolari della materia bianca del cervello 7 e il morbo di Alzheimer con stenosi delle arteriole e piccole imbarcazioni 8. Inoltre, alterata funzione barriera vascolare è pensato per contribuire sclerosi multipla 9 e sclerosi laterale amiotrofica 10. Di rilevanza diretta per il modello retinica descritto in questo protocollo, accecantemalattie come la retinopatia diabetica 11 e retinopatia della prematurità 12, 13 sono caratterizzati da una fase precoce degenerazione vascolare. Lo stress ischemico conseguente sulla retina neurovascolare innesca una seconda fase di eccessiva e patologica neovascolarizzazione che probabilmente nasce come risposta compensatoria a ripristinare ossigeno e fornitura di energia 14-16. Una strategia attraente per superare lo stress ischemico che è fondamentale per la progressione della malattia è ripristinare reti vascolari funzionali specificamente nelle zone ischemiche del neuro-retina (figure 2 e 3). Provocando una risposta angiogenica controllata può venire attraverso come contro-intuitivo per una condizione in cui i trattamenti anti-angiogenici come anti-VEGF sono considerati trattamenti adattati. Eppure, la prova della validità di questo approccio è il montaggio. Ad esempio, aumentando &quot;fisiologica-like&quot; ricrescita vascolare in ischemretinopatie ic è stato elegantemente dimostrata attraverso l&#39;introduzione di cellule precursori endoteliali 17, l&#39;inibizione del VEGF Müller-cell espresso down-regulation indotta da altri fattori angiogenici 18, iniezione di progenitori mieloidi 19, l&#39;inibizione della NADPH ossidasi apoptosi indotta 20, aumentando la dieta ω-3 acidi grassi polinsaturi assunzione di acido 21, il trattamento con un frammento carbossi-terminale del triptofano tRNA sintetasi 22, e somministrazione diretta di VEGF o FGF-2 per la protezione delle cellule gliali 23. Inoltre, abbiamo dimostrato che la modulazione classici spunti di orientamento neuronali quali Semaforine o netrins in retinopatie ischemiche accelera la rigenerazione vascolare dei vasi sani all&#39;interno della retina e di conseguenza riduce angiogenesi patologica 24, 25. Di rilevanza clinica diretta, molti degli studi sugli animali di cui sopra forniscono la prova che la promozione re vascolaregenerazione durante la prima fase ischemica delle retinopatie può ridurre significativamente vista minacciando neovascolarizzazione retinica pre-19, 23, 24, 26, probabilmente attraverso la riduzione degli oneri ischemica. L&#39;elaborazione di strategie terapeutiche che stimolano la rigenerazione dei vasi funzionali rimane una sfida significativa per biologi vascolari. Qui si descrive un sistema sperimentale che utilizza il modello murino di ossigeno indotta retinopatia (OIR) per esplorare le strategie per modulare la ricrescita vascolare all&#39;interno della retina. Sviluppato da Smith et al. Nel 1994 27, questo modello funge da proxy per retinopatie proliferative umane e consiste nell&#39;esporre cuccioli di topo P7 al 75% O 2 fino a P12 e successivamente re-introdurre i cuccioli di ambiente della stanza O 2-tensione (Figura 1). Questo paradigma imita vagamente uno scenario in cui un neonato prematuro è ventilatocon O 2. L&#39;esposizione dei cuccioli di topo all&#39;iperossia provoca la degenerazione dei capillari retinici e microcircolo, e produce una superficie riproducibile di vaso-obliterazione (VO) in genere valutato caso di uscita dal O 2 a P12, anche se zona VO massima è raggiunta a 48 ore (P9) dopo esposizione a O 2 28. Nel topo, le zone VO avascolari rigenerano spontaneamente nel corso della settimana successiva reintroduzione di aria ambiente ed eventualmente zone VO sono completamente ri-vascolarizzati (Figura 2). Reintroduzione di aria ambiente di topi sottoposti a OIR provoca anche neovascolarizzazione pre-retinica (NV) (massimo a P17) che è in genere valutata per determinare l&#39;efficacia di paradigmi di trattamento anti-angiogenici. Nella sua forma più pura, il modello OIR fornisce uno strumento altamente riproducibile e quantificabile per valutare la degenerazione vascolare indotta da ossigeno e determinare l&#39;entità del distruttiva neovascolarizzazione pre-retinica 29-31. <p class = &quot;jove_content&quot;&gt; Diversi paradigmi di trattamento esplorative che modulano la rigenerazione vascolare del SNC può essere studiato utilizzando il modello OIR compreso l&#39;uso di composti farmacologici, terapia genica, delezione genica e altro ancora. La propensione di un determinato approccio di influenzare la ricrescita vascolare è valutata graduale nella finestra tra P12 (VO massimo dopo l&#39;uscita da iperossia) e P17 (NV massima). Valutazione del risultato del trattamento su NV patologica può essere rapidamente e facilmente determinato in parallelo ed è stato accuratamente descritto da Stahl e colleghi 30, 31. Qui forniamo una semplice procedura step-by-step per studiare la modulazione di rivascolarizzazione fisiologico all&#39;interno della retina neurale da composti farmacologici, potenziali terapie, vettori virali o di studiare l&#39;influenza dei geni candidati in transgenici o knockout mice.

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			<td height="21" style="height:21px;width:639px;">C57Bl/6 mice ((Other strains may be used; angiogenic response varies from one strain to the other)</td>
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			<td height="38" style="height:38px;width:639px;">CD1 nursing mothers</td>
			<td style="width:181px;">Vendor of choice</td>
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			<td height="42" style="height:42px;width:639px;">Operating Scissors straight</td>
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			<td height="42" style="height:42px;width:639px;">Dissecting Scissors straight</td>
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			<td style="width:175px;">72-8483</td>
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			<td height="42" style="height:42px;width:639px;">Iris Forceps, curved, serrated</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">15915</td>
			<td></td>
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			<td height="21" style="height:21px;width:639px;">Brushes 362R size 0</td>
			<td style="width:181px;">Dynasty</td>
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			<td height="42" style="height:42px;width:639px;">Dumont Forceps #3; straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">500338</td>
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			<td height="21" style="height:21px;width:639px;">Surgical Blade, size 10</td>
			<td style="width:181px;">Bard-Parker</td>
			<td style="width:175px;">371110</td>
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			<td height="21" style="height:21px;width:639px;">Rhodamine Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td style="width:181px;">Vector Laboratories, Inc</td>
			<td style="width:175px;">RL-1102</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Microscope slides</td>
			<td style="width:181px;">VWR</td>
			<td style="width:175px;">16004-368</td>
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			<td height="42" style="height:42px;width:639px;">Fluoromount G</td>
			<td style="width:181px;">Electron Microscopy Sciences</td>
			<td style="width:175px;">17984-25</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;"></td>
			<td></td>
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			<td height="42" rowspan="2" style="height:42px;width:639px;">Leica MZ9.5 Stereomicroscope</td>
			<td rowspan="2" style="width:181px;">Leica</td>
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			<td height="21" style="height:21px;width:639px;">Fluorescein isothicyanate-dextran, 70000</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:175px;">46945</td>
			<td></td>
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assessment of vascular regeneration in the cns using the mouse retina

La retina roditore è forse il sistema mammiferi più accessibile in cui indagare interazione neurovascolare all&#39;interno del sistema nervoso centrale (CNS). È sempre più riconosciuto che diverse malattie neurodegenerative come l&#39;Alzheimer, la sclerosi multipla e la sclerosi laterale amiotrofica presentano elementi di compromissione vascolare. Inoltre, le cause più importanti di cecità nella popolazione in età pediatrica e di lavoro (retinopatia della prematurità e la retinopatia diabetica, rispettivamente) sono caratterizzate da degenerazione vascolare e il fallimento di fisiologica ricrescita vascolare. Lo scopo di questo documento tecnico è quello di realizzare un protocollo dettagliato per studiare la rigenerazione vascolare CNS nella retina. Il metodo può essere impiegato per chiarire i meccanismi molecolari che portano al fallimento di crescita vascolare dopo la lesione ischemica. Inoltre, i potenziali modalità terapeutiche per accelerare e ripristinare plessi vascolari sani possono essere esplorati. Giudizio richiederlod utilizzando l&#39;approccio descritto può prevedere vie terapeutiche per retinopatie ischemiche come quella del diabete o prematurità ed eventualmente beneficiare di altri disturbi vascolari del sistema nervoso centrale.

Il modello OIR è ampiamente utilizzato per studiare indotta da ossigeno degenerazione vascolare e neovascolarizzazione patologica indotta da ischemia nella retina ed è stato fondamentale nello sviluppo di attualmente impiegati trattamenti antiangiogenici per le malattie oculari 27, 29, 30. I risultati ottenuti con questo modello possono essere liberamente estrapolati per retinopatie ischemiche come la retinopatia diabetica proliferativa e la retinopatia della prematurità 30. <p class="jove_c...

Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

La retina roditore è da tempo riconosciuta come finestra accessibile al cervello. In questa nota tecnica fornendo un protocollo che utilizza il modello murino di retinopatia indotta da ossigeno per studiare i meccanismi che portano al fallimento di rigenerazione vascolare nel sistema nervoso centrale dopo lesione ischemica. Il sistema descritto può anche essere sfruttata per esplorare le strategie per promuovere la ricrescita dei vasi sanguigni funzionali all&#39;interno della retina e del SNC.

Assessment of Vascular rigenerazione nel SNC Utilizzo del Retina mouse

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

assessment-of-vascular-regeneration-in-the-cns-using-the-mouse-retina

Research

JoVE Journal

Neuroscience

14.3K Views.  McGill University.  La retina roditore è da tempo riconosciuta come finestra accessibile al cervello. In questa nota tecnica fornendo un protocollo che utilizza il modello murino di retinopatia indotta da ossigeno per studiare i meccanismi che portano al fallimento di rigenerazione vascolare nel sistema nervoso centrale dopo lesione ischemica. Il sistema descritto può anche essere sfruttata per esplorare le strategie per promuovere la ricrescita dei vasi sanguigni funzionali all'inter...

Video: Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Quantificare le attività di Cis-Regolamentazione Elementi nella retina di topo Elettroporazione Espianto

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

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Neuroscienze

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

Il corneale MICROPOCKET Assay: Un modello di angiogenesi nel Eye mouse

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imaging Ca<sup&gt; 2+</sup&gt; Dinamica a cono fotorecettore Axon Terminali della retina di topo

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Tecnica mouse Microchirurgia infusione per Targeted Sostanza consegna nel sistema nervoso centrale<i&gt; via</i&gt; Carotide interna

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Inquirenti rigenerazione dell'assone dei mammiferi: In Vivo elettroporazione del ganglio di radice dorsale del topo adulto

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Il potere di Interstimulus intervallo per la valutazione di elaborazione temporale in roditori

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

Fette ippocampali orizzontali del cervello del topo

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillare Due fotoni In vivo Imaging della retina del topo

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...