Gli autori sono grati per l&#39;assistenza della signora Sharon Spana nel monitoraggio dei roditori, il trattamento e la sperimentazione. Sostegno finanziario PhD è stato fornito da Università di Sydney e centro di ricerca australiano per l&#39;eccellenza in Vision.

Questo protocollo segue le linee guida per la cura degli animali di Università di Sydney. 1. Realizzazione di elettrodi <ol><li> Costruire l&#39;elettrodo positivo (quello che contattare la cornea) da un breve (5 cm) lunghezza del filo di platino 1-2 mm di diametro. Adatti in un loop di pochi mm di diametro. Collegare questo circuito ad un cavo tradizionale, abbastanza a lungo per raggiungere la fase di ingresso dell&#39;amplificatore (vedi Figura 4). </li><li> Costruire l&#39;elettrodo negativo (che andrà in bocca all&#39;animale) utilizzando un Ag / AgCl pellet 1-2 mm di diametro, anche collegata ad un cavo convenzione (vedi Figura 4). </li><li> Come un elettrodo di riferimento (che entrerà in groppa dell&#39;animale), utilizzare un ago ipodermico pulita (23 G), anche collegata ad un cavo di lunghezza appropriata (vedere Figura 4). </li><li> Idealmente, utilizzare cavi a tre lead forniti dai produttori di strumenti, per collegare i tre elettrodi (positiva U94; cornea, negativo → bocca, riferimento → groppa) all&#39;amplificatore. </li></ol> 2. Connessione e calibrazione di stimolo luminoso e ERG Set-up <ol><li> Creare (o individuare) un piccolo laboratorio di registrazione, che può essere fatta scuro. Dotare con uno o entrambi di una luce over-the-banco fatto rosso o una testa-spia rossa. </li><li> Utilizzare un metro lux per confermare che l&#39;illuminamento a luci rosse raggiungere l&#39;occhio del ratto durante l&#39;installazione non supera 1 lux. Nota: un filtro a densità neutra può essere utilizzato per ridurre la luminosità della lampada e la sorgente di luce della lampada deve specificamente emettere luce rossa. Adattamento scuro sarà compromessa se fonti di luce emettono inferiori (visibili) lunghezze d&#39;onda. </li><li> Sigillare tutta la luce parassita di entrare nel laboratorio di registrazione (questo spesso richiede la persistenza con nastro opaco) e preparare un filtro a densità neutra (questo può essere acquistato in fogli) grande abbastanza da contenere oltre, e così debole, qualsiasi schermo del computer si avrà nel laboratorio. Nota: luce diffusa ela luce di uno schermo sono sufficienti a pregiudicare adattamento scuro dell&#39;occhio ratto. </li><li> Collegare l&#39;amplificatore di hardware di acquisizione dati. Collegare i cavi, negativi e positivi di riferimento per l&#39;amplificatore. Assicurarsi che il computer e l&#39;unità di alimentazione Ganzfeld LED siano collegati a una presa di terra. Nota: Alcuni laboratori sono specializzati punti di terra, collegato ad una terra dell&#39;edificio; un tubo di acqua è un&#39;alternativa efficace. </li><li> Calibrare la sorgente luminosa a LED con un radiometro di ricerca di qualità. Fissare sensore dello strumento nella posizione in cui l&#39;occhio dell&#39;animale sarà situato durante un esperimento. </li><li> Programmare i LED Ganzfeld per eseguire un protocollo di ERG a tutto campo con aumenti passo-saggio in energia flash, durata del flash, flash ripetizione e tempo tra lampi, chiamato intervallo interstimuls (ISI), le impostazioni. Ad esempio, un protocollo a tutto campo si veda la tabella 1. Nota: Il full-campo ERG lampi passano da ripetitivi dim lampeggia per Blampeggia a destra in modo saggio passo. Il programma Twin Flash fa seguito al protocollo a tutto campo e consente l&#39;isolamento di coni e bastoncelli risposte. </li></ol> 3. Giorno Prima di ERG Sperimentazione <ol><li> Scuro adattare ratti Sprague-Dawley di 12 ore prima della registrazione. È conveniente fare questo in laboratorio di registrazione, una volta che la luce esterna è stata eliminata. </li></ol> 4. Giorno di ERG Sperimentazione <ol><li> Disporre per l&#39;animale sia dolcemente riscaldata durante la registrazione. Usiamo una piattaforma di metallo leggero costruito in modo che la testa dell&#39;animale può riposare al giusto punto all&#39;entrata al Ganzfeld. La piattaforma ha integrato tubo attraverso cui si pompa acqua preriscaldata a 40 ° C in un bagno d&#39;acqua. Nota: L&#39;esperienza dimostra che questo mantiene la temperatura interna dell&#39;animale a 37 ° C. </li><li> Pesare il ratto in condizioni di oscurità. Peso record e portare corretto ketamina (60 mg / kg) e xilazina (5 mg / kg) dose. Trattenere il ratto gently e iniettare anestetico per via intraperitoneale. </li><li> Nota momento dell&#39;iniezione. Una volta che l&#39;animale è incosciente (di solito entro 5 min) verificare la profondità dell&#39;anestesia pizzicando leggermente un rilievo del piede, per vedere se una risposta riflessa è presente. E &#39;meglio aspettare fino a quando questo riflesso è assente o debole, prima di procedere. </li><li> Applicare una sola goccia di atropina e un altro di proxmethacaine alla cornea. </li><li> Tagliare una lunghezza 10 centimetri di filo nero. Fare un cappio con un nodo semplice e scivolare l&#39;anello sopra l&#39;equatore dell&#39;occhio. Stringere leggermente; l&#39;effetto è quello di disegnare il bulbo oculare leggermente in avanti, con pressione minima. Ciò mantiene la cornea risulta dalle palpebre. </li><li> Applicare occhio carbomer scende verso la superficie della cornea. Garantire carbomer rimane sulla superficie corneale e non fuoriesca sulle palpebre o la faccia. </li><li> Posizionare assestamento assorbente in cima piattaforma riscaldato. </li><li> Posizionare topo sulla biancheria da letto, con la testa nel posto consigliato nell&#39;apertura del Ganzfeld. </li><li> Inserisci intsonda di temperatura ernal nel retto. Sonda di temperatura sicuro in posizione con nastro adesivo il cavo della sonda fino alla coda. </li><li> Inserire l&#39;elettrodo di riferimento (l&#39;ago 23 G) per via sottocutanea nella gamba posteriore, e la connessione all&#39;amplificatore. </li><li> Inserire l&#39;elettrodo negativo (il pellet Ag / AgCl) saldamente in bocca. Per evitare che questo scivolare fuori dalla bocca, apporre il cavo di collegamento di una superficie stabile. </li><li> Posizionare l&#39;elettrodo positivo sul centro della cornea. Utilizzando un micromanipolatore, assicurarsi che l&#39;elettrodo tocca la cornea delicatamente. </li><li> Controllare la temperatura del corpo è al 37,0-37,5 ° C. </li><li> Una volta che l&#39;animale è posizionato correttamente e gli elettrodi sono in atto, copre l&#39;intero setup (Ganzfeld e animale) con un materiale opaco (per preservare adattamento al buio). Usiamo un panno morbido nera. </li><li> Nel software di acquisizione fissato a una frequenza di campionamento 2 KHz con un tempo di raccolta di 100-1000 msec con 5 msec di prelevamento dei campioni pre-collezione. Impostare i filtri passa banda a 1-1,000Hz e assicura che il campionamento viene attivato per assaggiare il periodo di ~ 250 msec dopo un lampo. </li><li> Controllare la linea di base della registrazione. Dovrebbe essere esente da disturbi esterni, ma mostrare un po &#39;di rumore amplificatore e una oscillazione respiratoria. </li><li> Se la linea di base mostra rumori estranei, iniziare la risoluzione dei problemi. La maggior parte dei problemi sono legati allo scivolamento in posizione degli elettrodi, o incaglio. Utilizzare una gabbia di Faraday per garantire registrazioni siano privi di rumore estraneo. </li><li> Eseguire un lampo di prova, 0,4 log scot cd.sm -2. Una forma d&#39;onda ERG simile alla figura dovrebbe apparire 2A. Nelle nostre risposte tipiche di laboratorio per un 0,4 log scot cd.sm -2 Flash sono (a-wave: -474 ± 39 mV e b-wave: 1.512 ± 160 mV, n = 11). </li><li> Lasciare animale scuro riadattare per 10 min. E &#39;conveniente usare questi 10 minuti di ricontrollare la linea di base. </li><li> A seguito della conferma del segnale stabile avviare la registrazione. </li><li> Alla fine della sessione di registrazione, controllare che il corpo temperature è stata mantenuta. Rimuovere gli elettrodi. Riapplicare carbomer polimero cornee. Permettere agli animali di recuperare su un blocco di calore finché non è completamente mobile e attivo, prima di tornare a stabulazione degli animali. </li></ol> 5. Ischemia Remote <ol><li> Eseguire ischemia a distanza sia in roditori svegli o anestetizzati. </li><li> Se l&#39;animale è anestetizzato, adagiarlo su una piattaforma riscaldata (sopra) e infilare il bracciale sfigmomanometro sulla parte superiore della arti posteriori, chiara del ginocchio. </li><li> Se gli animali sono abituati ad essere manipolati, è possibile eseguire questa procedura senza anestesia; questo richiede due persone. Una persona trattiene l&#39;animale delicatamente e il secondo si applica il bracciale sfigmomanometro e gestisce il misuratore di pressione. </li><li> Per gli animali svegli, utilizzare un pezzo di asciugamano ~ 15 centimetri x 30-50 cm per avvolgere delicatamente l&#39;animale, con uno degli arti posteriori gratuito. Posare l&#39;animale sul dorso su (ad esempio) l&#39;avambraccio sinistro, con la testa infilata tra il braccio del titolare e il tronco, e luogoil bracciale come appena descritto. </li><li> Sgonfiare il bracciale e garantire la valvola dell&#39;aria viene chiusa. Pompare il bracciale a 160 mmHg in animali anestetizzati, e a 180 mmHg negli animali svegli. Ciò supera la pressione sistolica (di solito 140 mmHg e 160 mmHg, rispettivamente). </li><li> Mantenere queste pressioni, come richiesto, utilizzando la pompa a mano. </li><li> Trascorso il tempo previsto per ischemia (usiamo 2 periodi di 5 minuti separati da 5 min riperfusione), sgonfiare la pressione del bracciale svitando la valvola dell&#39;aria. </li><li> Confermare l&#39;effetto di ischemia remota con una sonda di temperatura della pelle attaccata alla zampa. La temperatura cutanea scende tipicamente 32-30 ° C, più di 5 minuti e recupera su riperfusione. </li></ol> 6. Danni Luce <ol><li> Assicurarsi che i ratti sono in un buio adattato durante la notte, prima che la procedura di danno chiaro. </li><li> Al momento opportuno a seguito di ischemia degli arti (nei nostri esperimenti senza indugio), ogni animale viene posto singolarmente in scatole di plexiglass, with acqua e cibo in contenitori di pavimento-based. Nota: il danno indotto dalla luce può essere intrapresa solo in animali albini. </li><li> Accendere una luce bianca pre-calibrato 1.000 lux a un tempo standard (di solito 09:00) e mantenere questa condizione per 24 ore. </li></ol> 7. ERG Estrazione dei dati e analisi <ol><li> Acquisire forme d&#39;onda medio della ERG. Se necessario, corretto per una linea di base non-zero, per sottrazione. </li><li> Misurare l&#39;ampiezza della a-onda (presentato a metà a intensità elevate stimolo), come la differenza di tensione tra la linea di base e la prima (&lt;30 msec latenza) trogolo (Figura 1). </li><li> Misurare l&#39;ampiezza dell&#39;onda b come la differenza di tensione tra il picco di una onda e il positivo delle seguenti onda, tipicamente si verificano in una latenza di 80-100 msec (Figura 1). </li><li> Isolare potenziali oscillatorie utilizzando una trasformata di Fourier per filtrare i dati 60-235 Hz, con una banda di transizione 90 Hz <sup&gt; 7. Se necessario il isolato potenziale segnale oscillatorio può essere sottratta dalla forma d&#39;onda non filtrato per confermare l&#39;identità di un trogolo onda. </li><li> Il tempo implicito (latenza) del tipo A e B onda picchi può anche essere una misura utile (Figura 1). Utilizzare i gemelli lampeggia per isolare la risposta canna. Sottrarre la risposta cono (flash 2) dalla risposta mista (flash 1) per isolare la risposta asta (Figura 2). </li><li> Normalizzare l&#39;intensità della luce individuale un onda e ampiezze b-onda (post-trattamento / post-trattamento-base) o media per i gruppi di trattamento. Curve Intensity-risposta tracciare le ampiezze di gruppo e l&#39;errore contro l&#39;energia del flash. </li></ol>

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A., Bush, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+relationship+of+the+scotopic+and+photopic+ERG+to+photoreceptor+cell+loss+in+light+damaged+rats.">Quantitative relationship of the scotopic and photopic ERG to photoreceptor cell loss in light damaged rats.</a> Experimental Eye Research. 70 (5), 693-705 (2000).</li><li>Machida, S., 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=P23H+rhodopsin+transgenic+rat:+Correlation+of+retinal+function+with+histopathology.">P23H rhodopsin transgenic rat: Correlation of retinal function with histopathology.</a> Investigative Ophthalmology &amp; Visual Science. 41 (10), 3200-3209 (2000).</li><li>Brandli, A., Stone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+Ischemia+Influences+the+Responsiveness+of+the+Retina.">Remote Ischemia Influences the Responsiveness of the Retina.</a> Observations in the Rat. Investigative Ophthalmology &amp; Visual Science. 55 (4), 2088-2096 (2014).</li><li>Maccarone, R., Di Marco, S., Bisti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saffron+supplement+maintains+morphology+and+function+after+exposure+to+damaging+light+in+mammalian+retina.">Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina.</a> Investigative Ophthalmology &amp; Visual Science. 49 (3), 1254-1261 (2008).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+abnormal+rod+photoreceptor+activity+with+the+a-wave+of+the+electroretinogram:+Applications+and+methods.">Assessing abnormal rod photoreceptor activity with the a-wave of the electroretinogram: Applications and methods.</a> Documenta Ophthalmologica. 92 (4), 253-267 (1996).</li><li>Robson, J. G., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rod-driven+a-wave+of+the+dark-adapted+mammalian+electroretinogram.">The rod-driven a-wave of the dark-adapted mammalian electroretinogram.</a> Progress in Retinal and Eye Research. 39, 1-22 (2014).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+COMPUTATIONAL+MODEL+OF+THE+AMPLITUDE+AND+IMPLICIT+TIME+OF+THE+B-WAVE+OF+THE+HUMAN+ERG.">A COMPUTATIONAL MODEL OF THE AMPLITUDE AND IMPLICIT TIME OF THE B-WAVE OF THE HUMAN ERG.</a> Visual Neuroscience. 8 (2), 107-126 (1992).</li><li>Wachtmeister, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+potentials+in+the+retina:+what+do+they+reveal.">Oscillatory potentials in the retina: what do they reveal.</a> Progress in Retinal and Eye Research. 17 (4), 485-521 (1998).</li></ol>

Jonathan Stone è il direttore del CSCM Pty Ltd

Il flash metodo di ERG adattata al buio sopra descritto è un metodo affidabile per valutare la funzione della retina nei ratti. Sia l&#39;una-wave e b-wave sono stati ridotti del danno chiaro. Remote precondizionamento ischemico mitigata riduzioni danni indotti luce del un-wave e b-wave. Questa conservazione della funzione retinica suggerisce che a distanza precondizionamento ischemico ha indotto neuroprotezione, simile ad altre forme di precondizionamento protettivi quali ipossia, ischemia e l&#39;esercizio 8-10....

L&#39;ERG è un potenziale elettrico generato dalla retina in risposta alla luce, e registrato dalla superficie corneale dell&#39;occhio. Quando le condizioni di registrazione vengono gestite accuratamente, l&#39;ERG può essere utilizzato in una varietà di modi per valutare la funzione retinica. Qui abbiamo descritto come registrare il &#39;Flash ERG&#39;, il potenziale generato quando la retina è esposto a un breve lampo, luminoso presentato in uno sfondo Ganzfeld. Il Ganzfeld disperde la luce in modo omogeneo e il lampo di luce raggiunge tutta la retina di circa uniforme. Se la retina è scuro adattato prima della registrazione, e il buio-adattamento è mantenuto come l&#39;animale è preparato per la registrazione, l&#39;ERG ha ottenuto è generato da due bastoncelli e coni. Il-scuro adattato Flash ERG ha una forma d&#39;onda caratteristica, che è stato analizzato in due modi. Innanzitutto, precoci e tardive componenti della forma d&#39;onda ERG sono stati distinti, e relativi alla sequenza di neuroneal attivazione nella retina. Il primo componente è una breve latenza andamento negativo potenziale, l&#39;a-onda (Figura 1). Questo è seguito da un potenziale positivo continuo, chiamato b-wave. La fase di salita del b-wave mostra oscillazioni, che sono considerate un componente separato (potenziali oscillatori o PO). L&#39;a-onda è considerato essere generato da fotorecettori, il b-wave dalle cellule dello strato nucleare interno e dei PO dalle cellule amacrine 1. Sulla base della forza dello stimolo, risposte a molto fioche lampeggia definito la risposta di soglia scotopica sono possibili. La risposta di soglia scotopica è inteso essere generato dalle cellule gangliari retiniche 2-4. In secondo luogo, il flash ERG può essere separata da adattamento alla luce, o da un protocollo due Flash descritto di seguito, in componenti di noccioli e cono guidato. In condizioni fotopiche, l&#39;a-onda non è rilevabile nei ratti, perché la popolazione cono è basso, ma PO e un b-wave sonochiaro 5. Nei primati, le cui retine avere popolazioni cono più elevati, sia Rod- e percorsi di cono generano un rilevabile un onda 6. Due misure utili spesso estratte dal flash ERG sono le ampiezze dei a- e b-onde, misurate come nella figura 1, con risposte in flash tipici mostrati in Figura 2. Quando la popolazione fotorecettore è ridotto, per esempio mediante esposizione a pericolosamente luminoso luce, tutti i componenti del ERG sono ridotti. Interventi neuroprotettivi, come ad esempio a distanza precondizionamento ischemico (RIP), possono essere convalidati dalla conservazione delle ampiezze delle a- e b-onde (Figura 3). In sintesi, l&#39;analisi della ERG consente confronti tra sano, leggero e della retina danneggiate neuroprotected.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PC computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab, 4 channel acquistion hardware</td>
			<td>AD Instruments</td>
			<td>PL 35044</td>
			<td>Acquistion of ERG</td>
		</tr>
		<tr>
			<td>Animal Bio Amp</td>
			<td>AD Instruments</td>
			<td>FE 136</td>
			<td>Amplifier for ERG</td>
		</tr>
		<tr>
			<td>Lab chart</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Signal collection software</td>
		</tr>
		<tr>
			<td>Ganzfield</td>
			<td>Photometric solutions</td>
			<td>FS-250A</td>
			<td>Light stimulus</td>
		</tr>
		<tr>
			<td>Ganzfield operating system</td>
			<td>Photometric solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Research Radiometer</td>
			<td>International light technologies</td>
			<td>ILT-1700</td>
			<td>calibrate light series</td>
		</tr>
		<tr>
			<td>Lux meter</td>
			<td></td>
			<td>LX-1010B&#160;</td>
			<td>check red light illumanation</td>
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		<tr>
			<td>Excel</td>
			<td>microsoft</td>
			<td></td>
			<td></td>
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			<td>Lead wires</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Connect postive, negative ground electrodes to amplifier</td>
		</tr>
		<tr>
			<td>Lead wires -aligator</td>
			<td>AD Instruments</td>
			<td></td>
			<td>ground ganzfield and acquistion hardware to computer</td>
		</tr>
		<tr>
			<td>Platinum wire 95%</td>
			<td>A&#38;E metals</td>
			<td></td>
			<td>postive electrode</td>
		</tr>
		<tr>
			<td>Mouth electrode Ag/AgCl Pellet</td>
			<td>SDR</td>
			<td>E205</td>
			<td>negative electode</td>
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			<td>26 gauge needle</td>
			<td>BD</td>
			<td></td>
			<td>ground electode</td>
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			<td>Water pump</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Tubing</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Homeothermic blanket system with flexible probe</td>
			<td>Harvard Appartus</td>
			<td>507222F</td>
			<td></td>
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		<tr>
			<td>Atropine 1% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical mydriasis</td>
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		<tr>
			<td>Proxmethycaine 0.5% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical anaesthetic</td>
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		<tr>
			<td>Visco tears eye drops</td>
			<td>Novartis</td>
			<td></td>
			<td>carbomer polymer</td>
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		<tr>
			<td>Thread</td>
			<td></td>
			<td></td>
			<td>retract eye lid</td>
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		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reusable adhesive</td>
			<td>Blu tac</td>
			<td></td>
			<td>Dim red headlamp. Affix electrodes</td>
		</tr>
		<tr>
			<td>Absorbent bedding</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td></td>
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			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamil - ketamine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>dissociative</td>
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		<tr>
			<td>Xylium - Xylazine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>muscle relaxant</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td></td>
			<td></td>
			<td></td>
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using the electroretinogram to assess function in the rodent retina and the protective effects of remote limb ischemic preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

Il protocollo può essere utilizzato per misurare la funzione visiva di roditore retina in vivo. L&#39;a-onda, una misura della funzione fotorecettori, e b-wave, una misura della funzione retina interna, vengono annotati in Figura 1. I asta dominato ERG segnale aumenta con la crescente stimolo luminoso, come mostrato nella figura 2A. L&#39;a-onda diventa chiaro a ~ 0,4 log scot cd.sm -2 e l&#39;ampiezza delle onde un aumenta fino a satura...

Watch this Scientific Journal Video about Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning at JoVE.com

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

Utilizzando il Elettroretinogramma per valutare la funzione in roditori Retina e gli effetti protettivi del Remote Limb ischemico precondizionamento

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

using-electroretinogram-to-assess-function-rodent-retina-protective

Research

JoVE Journal

Neuroscience

16.1K Views.  University of Sydney. The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

Video: Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

Strategie per lo Studio delle Neuroprotezione dal freddo precondizionamento

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

Ricerca

Neuroscienze

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Preparazione del tessuto e immunocolorazione di fibre nervose che innervano il mouse sensoriali della pelle e ossa degli arti

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Consegna diretta intraventricolare di farmaci per il sistema nervoso centrale, Roditore

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

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). ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

Analisi elettroretinogramma della risposta visiva in Zebrafish larve

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Imaging Fibre serotoninergici nel midollo spinale mouse Uso del CHIAREZZA / Tecnica CUBIC

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In Vivo Gene Transfer di cellule di Schwann nel nervo sciatico roditori da elettroporazione

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Effetti del Neurotrauma indotta da Blast il roditore pressurizzato Middle arterie cerebrali

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

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 ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Combinare il comportamento e l'EEG per studiare gli effetti della meditazione di consapevolezza sulla memoria episodica

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...