Die Autoren sind dankbar für die Unterstützung von Frau Sharon Spana in Nagetier-Überwachung, Handhabung und Experimentieren. PhD finanzielle Unterstützung wurde von University of Sydney und Australian Research Centre for Excellence in Sicht zur Verfügung gestellt.

Dieses Protokoll folgt den Richtlinien der Tierpflege University of Sydney. 1. Herstellung Elektroden <ol><li> Konstruieren Sie die positive Elektrode (die eine, die die Hornhaut treten wird) von einem kurzen (5 cm) Länge der Platindraht 1-2 mm im Durchmesser. FaShion sie in einer Schleife wenige mm im Durchmesser. Verbinden Sie diese Schleife zu einer herkömmlichen Blei, lange genug, um die Eingangsstufe des Verstärkers zu erreichen (siehe Abbildung 4). </li><li> Konstruieren Sie die negative Elektrode mit einer Ag / AgCl-Pellet 1-2 mm Durchmesser, auch mit einem Kongress Leitung verbunden (die in das Maul des Tieres gehen wird) (siehe Abbildung 4). </li><li> Als eine Referenzelektrode (die in das Tier Rumpf gehen), mit einem sauberen Injektionsnadel (23 G), auch mit einer Leitung von geeigneter Länge verbunden (siehe Abbildung 4). </li><li> Idealerweise verwenden Drei-Blei-Kabel vom Gerätehersteller zur Verfügung gestellt, um die drei Elektroden anschließen (positive U94; Hornhaut, negativ → Mund, Referenz → Hinterteil) an den Verstärker. </li></ol> 2. Anschluss und Kalibrierung der Lichtreiz und ERG Set-up <ol><li> Erstellen (oder suchen) einen kleinen Aufnahme Labor, das dunkel gemacht werden können. Rüsten Sie sich mit einer oder beiden der Over-the-Bank hellrot gemacht oder einem roten Kopf-Lampe. </li><li> Verwenden Sie ein Luxmeter zu bestätigen, dass rotes Licht Beleuchtungsstärke erreicht Auge der Ratte während der Installation ist 1 Lux nicht übersteigen. Hinweis: Ein Neutraldichtefilter verwendet werden, um die Helligkeit der Lampe zu verringern und die Quelle der Lampenlicht müssen ausdrücklich rotes Licht emittieren. Dunkeladaption wird gefährdet, wenn Lichtquellen strahlen unteren (sichtbaren) Wellenlängen. </li><li> Abdichtung alle Streulicht in das Aufnahmelabor (dies oft erfordert Ausdauer mit Deckband) und erstellt einen Neutraldichtefilter (dies kann in Platten gekauft werden) groß genug, um zu passen, und so dunkel, jeder Computer-Bildschirm finden Sie in der haben Labor. Hinweis: Streulicht unddas Licht eines Bildschirms ausreichend sind, um Dunkeladaption des Auges der Ratte zu beeinträchtigen. </li><li> Schließen Sie den Verstärker an Datenerfassungshardware. Schließen Sie positive, negative und Referenzleitungen an den Verstärker. Sicherstellen, dass der Computer und die LED-Ganzfeld-Netzteil fest mit einer Massequelle verbunden ist. Hinweis: Einige Labors haben Erdungspunkten spezialisiert, zu einem Gebäude Masse verbunden ist; eine Wasserleitung ist eine effektive Alternative. </li><li> Kalibrieren Sie die LED-Lichtquelle mit einer Forschungs hochwertige Radiometer. Fix-Sensor des Messgeräts in der Position, an der das Auge des Tieres wird während eines Experiments befinden. </li><li> Programmieren Sie die Ganzfeld-LEDs, um eine Vollfeld-ERG-Protokoll mit stufenweisen Anstieg der Blitzenergie, Blitzdauer, Flash-Wiederholung und die Zeit zwischen den Blitzen führen, bezeichnet interstimuls Intervall (ISI), Einstellungen. Ein Beispiel für Vollfeld-Protokoll finden Sie in Tabelle 1. Hinweis: Die Vollfeld-ERG blinkt erhöhen aus sich wiederholenden dim Wallungen zu brechts blinkt in einer schrittweisen Art und Weise. Das Doppel Flash-Programm folgt auf die Vollfeld-Protokoll und ermöglicht Isolierung von Stäbchen und Zapfen Antworten. </li></ol> 3. Tag vor der ERG Experimentation <ol><li> Dunkle Sprague-Dawley Ratten anpassen 12 Stunden vor der Aufnahme. Es ist zweckmäßig, diese in der Aufzeichnung Labor tun, einmal Streulicht eliminiert wurde. </li></ol> 4. Tag der ERG Experimentation <ol><li> Vereinbaren Sie für das Tier sanft während der Aufnahme erwärmt werden. Wir verwenden eine Leichtmetall-Plattform gebaut, damit Kopf des Tieres kann an der richtigen Stelle an der Einreise in das Ganzfeld ruhen. Die Plattform verfügt über eingebaute Schläuche, durch die wir die Pumpe Wasser auf 40 ° C vorgewärmt in einem Wasserbad. Anmerkung: Die Erfahrung zeigt, dass dies hält die Kerntemperatur des Tieres auf 37 ° C. </li><li> Wiegen Sie die Ratte bei schlechten Lichtverhältnissen. Nehmen Sie Gewicht und machen richtig Ketamin (60 mg / kg) und Xylazin (5 mg / kg) Dosis. Restrain die Ratte gently und injizieren Narkose intraperitoneal. </li><li> Hinweis Zeitpunkt der Injektion. Sobald das Tier bewusstlos ist (in der Regel innerhalb von 5 min) zu überprüfen Narkosetiefe durch leichtes Kneifen einem Fuß-Pad, um zu sehen, wenn ein Reflexantwort vorhanden ist. Es ist am besten zu warten, bis dieser Reflex nicht vorhanden oder schwach ist, bevor Sie fortfahren. </li><li> Tragen Sie einen Tropfen Atropin und andere der proxmethacaine zu Hornhaut. </li><li> Schneiden Sie ein 10 cm Länge der schwarzen Faden. Machen Sie eine Schleife mit einem einfachen Knoten und schieben Sie die Schleife über dem Äquator des Auges. Leicht anziehen; Der Effekt wird auf den Augapfel leicht nach vorne zu ziehen, mit minimalem Druck. Dies hält die Hornhaut von den Augenlidern klar. </li><li> Bewerben Carbomer Augentropfen auf die Hornhautoberfläche. Stellen Sie sicher, Carbomer bleibt auf Hornhautoberfläche und nicht auf den Augenlidern oder das Gesicht zu verschütten. </li><li> Zeigen saugfähigen Einstreu auf der Oberseite des beheizten Plattform. </li><li> Position Ratte auf dem Bettzeug, mit dem Kopf in der empfohlene Ort in der Öffnung des Ganzfeld. </li><li> Int Inserternal Temperatursonde in das Rektum. Sichere Temperaturfühler in Position durch Abkleben Sondenkabel mit dem Schwanz. </li><li> Legen Sie die Referenzelektrode (die 23 G-Nadel) subkutan in die hintere Bein, und eine Verbindung zum Verstärker. </li><li> Zeigen die negative Elektrode (die Ag / AgCl-Pellet) sicher in den Mund. Um dies zu verhindern ein Herausrutschen des Mundes, befestigen Sie die Anschlussleitung auf eine stabile Oberfläche. </li><li> Positionieren Sie die positive Elektrode über der Mitte der Hornhaut. Mit einem Mikromanipulator, sicherzustellen, dass die Elektrode die Hornhaut sanft berührt. </li><li> Überprüfen Sie die Körpertemperatur bei 37,0 bis 37,5 ° C. </li><li> Sobald das Tier richtig positioniert ist und Elektroden in aufgeführt sind, drapieren den gesamten Setup (Ganzfeld und Tier) mit einem undurchsichtigen Material (zur Dunkeladaptation zu erhalten). Wir verwenden Sie ein weiches schwarzes Tuch. </li><li> In der Erfassungssoftware bei einer 2 kHz Abtastrate mit einer Erfassungszeit von 100-1000 ms mit 5 msec der Pre-Kollektion Probenahme eingestellt. Stellen Sie die Bandpassfilter, um 1-1000Hz und sicher, dass Probenahme ausgelöst wird, um den Zeitraum von ~ 250 ms nach einer Flash-kosten. </li><li> Überprüfen Sie die Aufnahme Grundlinie. Es sollte frei von Fremdgeräusche, aber zeigen einige Verstärkerrauschen und eine Atmungsschwingung. </li><li> Wenn die Grundlinie zeigt Nebengeräusche, beginnen die Fehlersuche. Die meisten Probleme sind ähnlich wie in Elektrodenposition oder Erdungs ​​Schlupf. Verwenden Sie einen Faraday-Käfig, um sicherzustellen, Aufnahmen sind frei von Nebengeräuschen. </li><li> Führen Sie einen Testblitz, 0,4 log scot cd.sm -2. Eine ERG Wellenform ähnlich zu 2A erscheinen soll. In unserem Labor typische Antworten für eine 0,4 log scot cd.sm -2 Blitz sind (a-Welle: -474 ± 39 &amp; mgr; V und b-Welle: 1512 ± 160 &amp; mgr; V, n = 11). </li><li> Erlauben Tier zu dunkel wieder Anpassung für 10 min. Es ist zweckmäßig, diese 10 Minuten zu verwenden, um die Basislinie erneut prüfen. </li><li> Nach der Bestätigung der stabilen Signal Aufzeichnung starten. </li><li> Am Ende der Aufnahme-Session, überprüfen Sie, dass Körper temperature wurde beibehalten. Elektroden entfernen. Wenden Carbomer Polymers an Hornhäuten. Lassen Sie das Tier auf einem Heizkissen erholen, bis er vollständig mobil und aktiv, vor der Rückkehr in Stallungen. </li></ol> 5. Fern Ischämie <ol><li> Führen Sie Fern Ischämie entweder wach oder anästhesiert Nagetiere. </li><li> Wird das Tier narkotisiert, legen Sie es auf eine beheizte Plattform (oben) und schieben Sie die Blutdruckmanschette über den oberen Teil der Hintergliedmaße, klare des Knies. </li><li> Wenn die Tiere auf der Handhabung verwendet wird, ist es möglich, dieses Verfahren ohne Betäubung durchzuführen; dies sind zwei Personen erforderlich. Eine Person hält den Tier sanft und die zweite gilt die Blutdruckmanschette und betreibt das Blutdruckmessgerät. </li><li> Für wach Tiere, verwenden Sie ein Stück Tuch ~ 15 cm x 30-50 cm vorsichtig wickeln Sie das Tier, mit einem Hinterlauf frei. Legen Sie das Tier auf den Rücken (sagen wir) den linken Unterarm, mit seinem Kopf zwischen dem Halter Arm und Oberkörper, und Ort verstecktdie Manschette wie gerade beschrieben. </li><li> Lassen Sie die Luft der Manschette und sicherzustellen, dass die Luftdruckventil geschlossen ist. Pumpen Sie die Manschette auf 160 mmHg in anästhesierten Tieren und bis 180 mmHg in wach Tiere. Dies übersteigt systolische Druck (in der Regel 140 mmHg und 160 mmHg beziehungsweise). </li><li> Pflegen Sie diesen Druck nach Bedarf, mit der Handpumpe. </li><li> Nach der geplanten Zeit für Ischämie (wir verwenden 2 Perioden von 5 Minuten mit dem 5 min Reperfusion getrennt), die Luft aus dem Manschettendruck durch Lösen der Druckluftventil. </li><li> Bestätigen Sie die Wirkung des Fern Ischämie mit einer Hauttemperatursonde in die Pfotenballen befestigt. Hauttemperatur fällt in der Regel 32 bis 30 ° C, während 5 min und stellt auf der Reperfusion. </li></ol> 6. Lichtschaden <ol><li> Stellen Sie sicher, dass Ratten in einem dunkeladaptierten über Nacht, bevor die Lichtschäden Verfahren. </li><li> Zum geeigneten Zeitpunkt folgenden Extremitäten-Ischämie (in unseren Versuchen ohne Verzögerung), wird jedes Tier einzeln in eine Plexiglas-Boxen, wi platziertth Wasser und Nahrung in Boden-basierten Container. Hinweis: Lichtschäden können nur in Albino-Tieren durchgeführt werden. </li><li> Anschalten einer vorkalibrierten 1.000 Lux weißes Licht bei einer Standardzeit (in der Regel 9.00), und dieser Zustand für 24 h aufrechtzuerhalten. </li></ol> 7. ERG Datenextraktion und -analyse <ol><li> Erwerben gemittelten Wellenformen der ERG. Falls erforderlich, für ein Nicht-Null-Grundlinie korrekt ist, durch Subtraktion. </li><li> Messen der Amplitude der A-Welle (in mittleren bis hohen Reizstärken dargestellt), da die Spannungsdifferenz zwischen der Basislinie und der ersten (&lt;30 ms Latenz) Wanne (Abbildung 1). </li><li> Messung der b-Wellenamplitude als die Spannungsdifferenz zwischen der Spitze des a-Welle und der positiven der folgenden Welle, typischerweise bei einer Latenzzeit von 80-100 ms (Figur 1) auftritt. </li><li> Isolieren oszillierenden Potentiale unter Verwendung einer Fourier-Transformation, um Daten von 60 bis 235 Hz zu filtern, mit einer 90 Hz Gangsband <sup&gt; 7. Bei Bedarf kann der isolierte Schwingungspotentialsignal kann dann von der ungefilterten Wellenform subtrahiert werden, um die Identität des a-Wellental zu bestätigen. </li><li> Die implizite Zeit (Latenz) der a- und b-Wellenberge können auch eine sinnvolle Maßnahme (Abbildung 1) ist. Verwenden Sie die Zwillings blinkt, um den Stab Reaktion zu isolieren. Subtrahieren Sie die Konus-Antwort (Flash-2) aus dem Mischantwort (Flash-1), um den Stab Reaktion zu isolieren (Abbildung 2). </li><li> Normalisieren einzelnen Lichtintensität einen Wellen und b-Wellenamplituden (Nachbehandlung / Nachbehandlung-Basislinie) oder gemittelt für den Behandlungsgruppen. Intensity-Wirkungs-Kurven zeichnen die Gruppe Amplituden und Fehler gegen Blitzenergie. </li></ol>

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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 ist der Direktor der CSCM Pty Ltd

Die oben beschriebene dunkeladaptierten Flash ERG Verfahren ist eine zuverlässige Methode zur Beurteilung Retinafunktion bei Ratten. Sowohl die A-Welle und b-Welle wurden durch Lichtschäden reduziert. Remote ischämischen Präkonditionierung gemildert Lichtschäden induzierte Reduktionen in der a-Welle und b-Welle. Diese Erhaltung der Netzhautfunktion legt nahe, dass Remote-ischämischen Präkonditionierung induziert hat Neuroprotektion, ähnlich andere Formen der Schutz Präkonditionierung wie Hypoxie, Ischämie und ...

Die ERG ist ein elektrisches Potential von der Netzhaut als Reaktion auf Licht erzeugt und von der Hornhautoberfläche des Auges aufgezeichnet. Wenn die Bedingungen der Aufnahme wird sorgfältig gesteuert, kann die ERG in einer Vielzahl von Möglichkeiten, um die Funktion der Retina zu bewerten verwendet werden. Hier haben wir beschrieben, wie man die &quot;Flash-ERG, die erzeugt werden, wenn die Netzhaut zu einer kurzen, hellen Blitz in einem Ganzfeld Hintergrund präsentiert ausgesetzt Potenzial aufzuzeichnen. Die Ganzfeld streut das Licht homogen und der Lichtblitz erreicht die gesamte Netzhaut etwa gleichmäßig. Wenn die Netzhaut vor der Aufnahme angepasst dunkel, und die dunkel Anpassung beibehalten wird, wie das Tier wird für die Aufnahme bereit, erhalten die ERG wird sowohl von Stäbchen und Zapfen-Photorezeptoren erzeugt. Die dunkeladaptierten Flash ERG eine charakteristische Wellenform, die in zwei Arten analysiert wurde. Erste, frühe und späte Komponenten des ERG-Wellenform werden unterschieden, und mit der Sequenz des Neurons bezogenenal Aktivierung in der Netzhaut. Die früheste Komponente ein kurzer Latenz negativ werdenden Potentials der a-Welle (1). Dies wird durch einen positiv verlaufenden Potential gefolgt, die so genannte b-Welle. Die steigende Phase der b-Welle zeigt Schwingungen, die als eine separate Komponente (Schwingungspotentiale oder OP) werden. Die a-Welle wird als von Photorezeptoren erzeugt werden, die b-Welle durch Zellen der inneren Körnerschicht und der OP durch Amakrinzellen 1. Auf der Grundlage der Reizstärke, Antworten auf sehr dunkel blinkt bezeichnet die skotopischen Schwellenreaktion möglich. Skotopischen Schwellenreaktion versteht man aus den retinalen Ganglienzellen 2-4 erzeugt werden. Zweitens kann der Flash-ERG durch Lichtanpassung zu trennen, oder durch eine zwei Flash-Protokoll weiter unten beschrieben, in Stab- und kegel angetriebenen Bestandteile. Unter photopischen Bedingungen ist der a-Welle nicht bei der Ratte nachweisbar sind, da der Konus Bevölkerung ist gering, aber OP und eine b-Welle sindklar 5. In Primaten, deren Netzhaut eine höhere Konus Populationen sowohl Stab- und kegelWege ein nachweisbares eine Wellen 6. Zwei nützliche Maßnahmen oft aus dem Flash-ERG extrahiert sind die Amplituden der A- und B-Wellen gemessen, wie in Figur 1, mit typischen Flash-Antworten in 2 gezeigt. Wenn die Fotorezeptorpopulation verringert wird, beispielsweise durch Einwirkung von schädigend hellen Licht, sind alle Komponenten des ERG reduziert. Neuroprotektive Eingriffe, wie Fern ischämischen Präkonditionierung (RIP), kann durch den Erhalt der Amplituden der A- und B-Wellen (3) überprüft werden. Zusammenfassend ist die Analyse der ERG ermöglicht Vergleiche zwischen gesund, leicht beschädigt und neuroprotected Netzhaut.

<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>
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			<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>
		</tr>
		<tr>
			<td>Excel</td>
			<td>microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>26 gauge needle</td>
			<td>BD</td>
			<td></td>
			<td>ground electode</td>
		</tr>
		<tr>
			<td>Water pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic blanket system with flexible probe</td>
			<td>Harvard Appartus</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine 1% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical mydriasis</td>
		</tr>
		<tr>
			<td>Proxmethycaine 0.5% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical anaesthetic</td>
		</tr>
		<tr>
			<td>Visco tears eye drops</td>
			<td>Novartis</td>
			<td></td>
			<td>carbomer polymer</td>
		</tr>
		<tr>
			<td>Thread</td>
			<td></td>
			<td></td>
			<td>retract eye lid</td>
		</tr>
		<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>
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			<td></td>
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			<td>Ketamil - ketamine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>dissociative</td>
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			<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>
		</tr>
	</tbody>
</table>

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.

Das Protokoll kann verwendet werden, um die Sehfunktion von Nagetier Retina in vivo zu messen. Die a-Welle, ein Maß für Photorezeptor-Funktion und die b-Welle, ein Maß für innere Retina-Funktion werden in 1 gekennzeichnet. Die stab dominiert ERG-Signal mit zunehmendem Lichtreiz, wie in 2A gezeigt. Die a-Welle wird bei ~ 0,4 log Scot cd.sm -2 und die Amplitude der A-Welle zu, bis Sättigung bei 2,5 log Scot cd.sm -2 (nicht d...

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.

Verwenden der Funktion zur Elektroretinogramm im Nager Retina und die schützende Wirkung des Fern Limb ischämischen Präkonditionierung bewerten

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 Aufrufe.  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.

Serie: 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.

Strategies for Study of Neuroprotektion von Cold-Vorkonditionierung

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

Forschung

Neurowissenschaften

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.

Tissue Vorbereitung und Immunfärbung von Maus sensorischen Nervenfasern innervieren Haut und Limb Bones

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.

Direkt Intraventrikuläre Abgabe von Arzneimitteln an die Nager 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 ...

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.

Beurteilung der Gefäß Regeneration im ZNS Verwenden der Maus Retina

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.

Elektroretinogramm Analyse der Sicht Antwort in Zebrafisch-Larven

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 Serotonerge Fasern im Rückenmark der Maus Mit der CLARITY / CUBIC Technik

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 - Gentransfer auf Schwann - Zellen in der Nagerischiasnerv durch Elektroporation

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.

Auswirkungen der Explosion-induzierte Neurotrauma auf Druck Nagetier mittlerer Hirnarterien

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.

3D Kinematische Analyse zur funktionellen Bewertung im Rattenmodell von Ischias-Nerven-Crush-Verletzungen

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.

Kombination von Verhalten und EEG, um die Auswirkungen von Achtsamkeitsmeditation auf das episodische Gedächtnis zu untersuchen

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