Les auteurs sont reconnaissants de l&#39;aide de Mme Sharon Spana dans la surveillance des rongeurs, la manipulation et l&#39;expérimentation. soutien financier de doctorat a été fournie par l&#39;Université de Sydney et du Centre australien de recherche pour l&#39;excellence dans la Vision.

Ce protocole suit les directives de protection des animaux de l&#39;Université de Sydney. 1. réaliser des électrodes <ol><li> Construire l&#39;électrode positive (celui qui prendra contact avec la cornée) d&#39;un court (5 cm) de longueur de fil de platine de 1-2 mm de diamètre. Façonner dans une boucle de quelques mm de diamètre. Connectez cette boucle à une connexion classique, assez long pour atteindre le stade de votre amplificateur d&#39;entrée (voir Figure 4). </li><li> Construire l&#39;électrode négative (qui ira dans la bouche de l&#39;animal) en utilisant un Ag / AgCl culot 1-2 mm de diamètre, également reliée à une avance de convention (voir Figure 4). </li><li> Comme une électrode de référence (qui ira dans la croupe de l&#39;animal), utiliser une aiguille hypodermique propre (23 G), également relié à un fil d&#39;une longueur appropriée (voir Figure 4). </li><li> Idéalement, utiliser des câbles de trois plomb fournies par les fabricants d&#39;instruments, pour relier les trois électrodes (U positif94; cornée, négative → bouche, référence → croupe) à l&#39;amplificateur. </li></ol> 2. Raccordement et étalonnage du stimulus lumineux et ERG Set-up <ol><li> Créez (ou localiser) un petit laboratoire d&#39;enregistrement, qui peut être fait sombre. Equipé d&#39;un ou des deux une lumière over-the-banc en rouge ou une lampe frontale rouge. </li><li> Utilisez un luxmètre pour confirmer que l&#39;éclairement de la lumière rouge atteindre l&#39;oeil du rat lors de l&#39;installation ne dépasse pas 1 lux. Remarque: Un filtre de densité neutre peut être utilisé pour réduire la luminosité de la lampe et la source de lumière de la lampe doit spécifiquement émettre de la lumière rouge. Adaptation à l&#39;obscurité sera compromise si les sources lumineuses émettent plus faibles longueurs d&#39;onde (visibles). </li><li> Sceller toute lumière parasite entrer dans le laboratoire d&#39;enregistrement (ce qui nécessite souvent la persistance avec du ruban adhésif opaque) et préparer un filtre de densité neutre (ce qui peut être acheté en feuilles) assez grand pour accueillir plus, et si faible, un écran d&#39;ordinateur que vous aurez dans le laboratoire. Note: La lumière parasite etla lumière d&#39;un écran sont suffisantes pour porter atteinte adaptation à l&#39;obscurité de l&#39;oeil de rat. </li><li> Branchez l&#39;amplificateur de matériel d&#39;acquisition de données. Branchez, fils négatifs et positifs référence à l&#39;amplificateur. Assurez-vous que l&#39;ordinateur et l&#39;unité d&#39;alimentation de Ganzfeld LED sont bien connectés à une source de masse. Remarque: Certains laboratoires sont spécialisés points de mise à la terre, relié à une masse d&#39;un bâtiment; une conduite d&#39;eau est une alternative efficace. </li><li> Calibrer la source de lumière LED avec un radiomètre la recherche de qualité. Fixer le capteur de l&#39;appareil de mesure dans la position à laquelle l&#39;oeil de l&#39;animal sera situé au cours d&#39;une expérience. </li><li> Programme des LED Ganzfeld Pour exécuter un plein champ protocole ERG avec des augmentations graduelles de l&#39;énergie flash, durée de flash, flash répétition et le temps entre les bouffées, appelé intervalle de interstimuls (ISI), les réglages. Pour un protocole exemple plein champ voir le tableau 1. Remarque: Le plein champ ERG clignote augmentent à partir répétitives clignote sombres à bdroite clignote dans un sage manière progressive. Le programme flash double suite au protocole de plein champ et permet d&#39;isoler des réponses des bâtonnets et des cônes. </li></ol> 3. Jour Avant ERG Expérimentation <ol><li> Adapter foncé rats Sprague-Dawley pendant 12 heures avant l&#39;enregistrement. Il est commode de le faire dans le laboratoire d&#39;enregistrement, une fois la lumière parasite a été éliminée. </li></ol> 4. Jour de l&#39;ERG Expérimentation <ol><li> Prendre des dispositions pour l&#39;animal d&#39;être chauffé doucement pendant l&#39;enregistrement. Nous utilisons une plate-forme de métal léger construit de telle sorte que la tête de l&#39;animal peut se reposer au point correcte à l&#39;entrée de la Ganzfeld. La plate-forme dispose d&#39;un tube intégré à travers lequel nous pomper l&#39;eau préchauffé à 40 ° C dans un bain d&#39;eau. Remarque: L&#39;expérience montre que ce qui maintient la température à coeur de l&#39;animal à 37 ° C. </li><li> Peser le rat dans des conditions sombres. Enregistrer le poids et maquillage kétamine correcte (60 mg / kg) et de xylazine (5 mg / kg) la dose. Retenez le rat gently et injecter un anesthésique par voie intrapéritonéale. </li><li> Remarque moment de l&#39;injection. Une fois que l&#39;animal est inconscient (généralement dans les 5 min) vérifier la profondeur de l&#39;anesthésie en pinçant légèrement un plot de pied, pour voir si une réponse réflexe est présent. Il est préférable d&#39;attendre jusqu&#39;à ce que ce réflexe est absente ou faible, avant de poursuivre. </li><li> Appliquer une seule goutte d&#39;atropine et un autre de proxmethacaine à la cornée. </li><li> Couper une longueur de 10 cm de fil noir. Faire une boucle avec un nœud simple et glisser la boucle sur l&#39;équateur de l&#39;œil. Serrer légèrement; l&#39;effet est d&#39;attirer l&#39;œil légèrement vers l&#39;avant, avec une pression minimale. Cela permet de maintenir la cornée claire des paupières. </li><li> Appliquer œil carbomère tombe à la surface de la cornée. Assurer carbomère reste sur la surface de la cornée et ne se renverse sur les paupières ou le visage. </li><li> Placez la literie absorbante sur le dessus de la plate-forme chauffée. </li><li> Position rat sur la literie, avec la tête à l&#39;endroit recommandé à l&#39;ouverture de la Ganzfeld. </li><li> Insérer internal sonde de température dans le rectum. Sonde de température sécurisé en position en collant la sonde cordon à la queue. </li><li> Insérez l&#39;électrode de référence (l&#39;aiguille 23 G) sous-cutanée dans la patte arrière, et se connecter à un amplificateur. </li><li> Placer l&#39;électrode négative (la pastille Ag / AgCl) en toute sécurité dans la bouche. Pour éviter ce glisser hors de la bouche, apposer le câble de raccordement sur une surface stable. </li><li> Placez l&#39;électrode positive sur le centre de la cornée. Utilisation d&#39;un micromanipulateur, veiller à ce que l&#39;électrode touche doucement la cornée. </li><li> Vérifiez la température du corps est à 37,0 à 37,5 ° C. </li><li> Une fois que l&#39;animal est correctement positionné et les électrodes sont en place, le drapé toute configuration (Ganzfeld et animale) avec un matériau opaque (pour préserver adaptation à l&#39;obscurité). Nous utilisons un chiffon doux noir. </li><li> Dans le logiciel d&#39;acquisition fixé à un taux d&#39;échantillonnage de 2 kHz avec un temps de collecte de 100-1000 msec avec 5 ms de pré-collecte échantillonnage. Réglez les filtres passe-bande à 1-1,000Hz et veiller à ce que l&#39;échantillonnage est déclenché pour goûter à la période de ~ 250 ms à la suite d&#39;un flash. </li><li> Vérifiez la ligne de base de l&#39;enregistrement. Il doit être exempt de bruits parasites, mais montrer un peu de bruit de l&#39;amplificateur et une oscillation respiratoire. </li><li> Si la ligne de base montre les bruits parasites, commencer le dépannage. La plupart des problèmes sont liés à des dérapages dans la position de l&#39;électrode, ou d&#39;échouement. Utilisez une cage de Faraday pour assurer des enregistrements sont exemptes de bruit parasite. </li><li> Exécuter un flash test, 0,4 log scot cd.sm -2. Une forme d&#39;onde ERG similaire à la figure 2A devrait apparaître. Dans notre laboratoire réponses typiques pour un 0,4 log scot cd.sm -2 Flash sont (a-onde: ± 39 mV -474 et b-ondes: 1512 ± 160 mV, n = 11). </li><li> Laisser animal sombre ré-adapter pendant 10 min. Il est commode d&#39;utiliser ces 10 min pour revérifier la ligne de base. </li><li> Suite à la confirmation de signal stable commencer l&#39;enregistrement. </li><li> À la fin de la session d&#39;enregistrement, vérifiez que le corps temperature été maintenue. Retirer électrodes. Appliquez de nouveau polymère carbomère cornées. Permettre à l&#39;animal de récupérer sur un pad thermique jusqu&#39;à ce qu&#39;il soit entièrement mobile et actif, avant de retourner au logement des animaux. </li></ol> 5. ischémie à distance <ol><li> Effectuer une ischémie à distance soit en rongeurs éveillés ou anesthésiés. </li><li> Si l&#39;animal est anesthésié, le poser sur une plate-forme chauffée (ci-dessus) et glisser le brassard de tensiomètre sur la partie supérieure du membre postérieur, claire du genou. </li><li> Si les animaux sont habitués à être manipulés, il est possible d&#39;effectuer cette procédure sans anesthésie; cela nécessite deux personnes. Une personne retient l&#39;animal doucement et le second applique le brassard de tensiomètre et exploite le tensiomètre. </li><li> Pour animaux éveillés, utiliser un morceau de serviette ~ 15 cm x 30-50 cm pour envelopper doucement l&#39;animal, avec l&#39;un des membres postérieurs libre. Couchez l&#39;animal sur le dos sur (par exemple) l&#39;avant-bras gauche, avec sa tête coincée entre le bras et le torse, et la place de titulairele brassard que nous venons de décrire. </li><li> Dégonfler le brassard et d&#39;assurer la soupape de pression d&#39;air est fermée. Pomper le brassard à 160 mmHg chez les animaux anesthésiés, et à 180 mm Hg chez les animaux éveillés. Cela dépasse la pression systolique (généralement 140 mmHg et 160 mmHg respectivement). </li><li> Maintenir ces pressions que nécessaire, à l&#39;aide de la pompe à main. </li><li> Après le temps prévu pour l&#39;ischémie (nous utilisons 2 périodes de 5 minutes séparées par 5 min de reperfusion), dégonfler la pression du brassard en desserrant le robinet de pression d&#39;air. </li><li> Confirmer l&#39;effet de l&#39;ischémie à distance avec une sonde de température de la peau attachée à la patte. la température de la peau tombe typiquement de 32 à 30 ° C, pendant 5 minutes et récupère sur reperfusion. </li></ol> 6. Light Damage <ol><li> Veiller à ce que les rats sont dans un avant adaptés à l&#39;obscurité la nuit, la procédure de légers dégâts. </li><li> Au moment opportun, suivant une ischémie des membres (dans nos expériences sans retard), chaque animal est placé seul dans une des boîtes en plexiglas, wie de l&#39;eau et de la nourriture dans des contenants à base de sol. Remarque: les dommages induits par la lumière ne peut être effectuée chez les animaux albinos. </li><li> Allumez une lumière blanche 1000 lux pré-calibré à un temps standard (généralement de 9 h) et maintenir cet état pendant 24 heures. </li></ol> 7. ERG Extraction et analyse des données <ol><li> Acquérir des formes d&#39;ondes moyennes de l&#39;ERG. Si nécessaire, pour corriger un niveau de référence différent de zéro, par soustraction. </li><li> Mesurer l&#39;amplitude de l&#39;onde-(présenté à moyen et à de fortes intensités de relance), comme la différence de tension entre la base et le premier (&lt;30 ms de latence) bac (Figure 1). </li><li> Mesurer l&#39;amplitude de l&#39;onde b que la différence de tension entre le pic de l&#39;a-onde et le positif de la vague suivante, survenant généralement à une latence de 80-100 ms (Figure 1). </li><li> Isoler les potentiels oscillatoires en utilisant une transformée de Fourier pour filtrer des données 60-235 Hz, avec une bande de transition 90 Hz <sup&gt; 7. Si nécessaire, le signal de potentiel oscillatoire isolé peut ensuite être soustraite de la forme d&#39;onde non filtrée pour confirmer l&#39;identité de l&#39;auge une onde. </li><li> Le temps implicite (latence) des A et B-ondes pics peut aussi être une mesure utile (Figure 1). Utilisez les bouffées de jumeaux pour isoler la réponse de la tige. Soustraire la réponse des cônes (flash 2) de la réponse mixte (flash 1) pour isoler la réponse de la tige (Figure 2). </li><li> Normaliser l&#39;intensité de la lumière individu une onde et des amplitudes b-ondes (post-traitement / post-traitement de base) ou en moyenne pour les groupes de traitement. Courbes intensité-réponse tracer les amplitudes de groupe et l&#39;erreur contre l&#39;énergie flash. </li></ol>

<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 est le directeur du CSCM Pty Ltd

Le flash méthode adaptée à l&#39;obscurité ERG décrit ci-dessus est une méthode fiable pour évaluer la fonction rétinienne chez les rats. Tant l&#39;un d&#39;onde et b-ondes ont été réduites par des dommages de la lumière. Distance préconditionnement ischémique atténué réductions induit des dommages-légers dans le un-ondes et b-ondes. Cette préservation de la fonction rétinienne suggère que le préconditionnement ischémique à distance a induit la neuroprotection, ressemblant à d&#39;autres formes...

L&#39;ERG est un potentiel électrique généré par la rétine en réponse à la lumière, et enregistré à partir de la surface de la cornée de l&#39;oeil. Lorsque les conditions d&#39;enregistrement sont gérés avec soin, l&#39;ERG peut être utilisé dans une variété de façons d&#39;évaluer la fonction rétinienne. Ici, nous avons décrit comment enregistrer l&#39;&#39;éclair ERG », le potentiel généré lorsque la rétine est exposée à un bref flash lumineux présenté dans un fond de Ganzfeld. Le Ganzfeld disperse la lumière de façon homogène et le flash de lumière atteint la rétine entière près uniformément. Si la rétine est sombre adapté avant l&#39;enregistrement, et l&#39;adaptation à l&#39;obscurité est maintenue tant que l&#39;animal est préparé pour l&#39;enregistrement, l&#39;ERG obtenue est générée par les deux photorécepteurs à bâtonnets et des cônes. La adaptés à l&#39;obscurité éclair ERG a une forme d&#39;onde caractéristique, qui a été analysée de deux manières. Tout d&#39;abord, précoces et tardives composants de la forme d&#39;onde ERG ont été distingués, et liée à la séquence de neuroneal activation de la rétine. Le premier composant est un court temps de latence devenant négative potentielle, l&#39;un d&#39;onde (Figure 1). Il est suivi par un potentiel positif en cours, appelé b-ondes. La phase de montée de l&#39;onde b montre des oscillations, qui sont considérés comme une composante distincte (des potentiels oscillatoires ou PO). L&#39;un d&#39;onde est considéré être généré par des photorécepteurs, la b-ondes par les cellules de la couche nucléaire interne, et les PO par les cellules amacrines 1. Sur la base de l&#39;intensité de stimulation, les réponses à éclairs très faibles dits la réponse scotopique de seuil sont possibles. La réponse du seuil scotopique est entendu être générés à partir des cellules ganglionnaires de la rétine 4.2. Deuxièmement, le flash ERG peut être séparé par adaptation à la lumière, ou par un protocole en deux flash décrit ci-dessous, en composants Rodgers et entraîné de cône. Dans des conditions photopiques, l&#39;un d&#39;onde est pas détectable chez les rats, parce que la population de cône est faible, mais les PO et un b-ondes sont5 clair. Chez les primates, dont les rétines ont des populations de cône supérieur, tant de bâtonnet et les voies de cône génèrent un détectable onde-6. Deux mesures utiles souvent extraites du flash ERG sont les amplitudes de la A et B-ondes, mesurées comme dans la figure 1, avec des réponses flash typiques de la figure 2. Lorsque la population de photorécepteur est réduite, par exemple par l&#39;exposition à dommageable lumineux la lumière, toutes les composantes de l&#39;ERG sont réduits. Interventions neuroprotecteurs, comme à distance préconditionnement ischémique (RIP), peuvent être validés par la préservation des amplitudes de la A et B-ondes (figure 3). En résumé, l&#39;analyse de l&#39;ERG permet des comparaisons entre la santé, la lumière et de la rétine endommagée 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>
		</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>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<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>
		</tr>
		<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>
		</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.

Le protocole peut être utilisé pour mesurer la fonction visuelle de la rétine rongeur in vivo. L&#39;un-ondes, une mesure de la fonction des photorécepteurs et le b-ondes, une mesure de la fonction de la rétine interne, sont annotés dans la figure 1. Les ERG augmentations de signaux de tige-dominé avec le stimulus lumineux croissante, comme le montre la figure 2A. L&#39;un d&#39;onde devient apparente à ~ 0,4 log scot cd.sm -2 et ...

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.

Utilisation de la Électrorétinogramme pour évaluer la fonction dans le Retina rongeurs et les effets protecteurs de la distance préconditionnement ischémique Limb

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

16.0K vues.  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.

Vidéo: 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.

Stratégies pour l&#39;étude de neuroprotection du froid préconditionnement

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

Recherche

Neurosciences

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.

Préparation des tissus et des fibres Immunocoloration souris nerveuses sensitives innervant la peau et les os du membre

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.

Livraison intraventriculaire directe des médicaments pour le système nerveux central rongeurs

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.

Évaluation de la régénération vasculaire dans le SNC Utilisation de la rétine de souris

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.

Électrorétinogramme Analyse de la réponse visuelle chez le poisson zèbre larves

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.

Imagerie fibres sérotoninergiques dans la moelle épinière de souris Utilisation du CLARITY / Technique 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 Transfert de gènes à des cellules de Schwann dans le nerf sciatique Rongeur par électroporation

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.

Effets de neurotraumatologie induite par le souffle sur les rongeurs sous pression du milieu des artères cérébrales

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.

Analyse cinématique 3D pour l’évaluation fonctionnelle dans le modèle de rat de la blessure d’écrasement de nerf sciatique

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.

Combiner le comportement et l’EEG pour étudier les effets de la méditation de pleine conscience sur la mémoire épisodique

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