Cette étude a été soutenue par l&#39;Institut de recherche en ophtalmologie de l&#39;Australie (ORIA). Nous remercions Prof. Algis Vingrys et le Dr Bang Bui, Université de Melbourne, pour d&#39;abord nous aider à développer la technique d&#39;enregistrement VEP.

Déclaration éthique: Toutes les procédures impliquant des animaux ont été effectuées en conformité avec le Code australien de pratiques pour le soin et l&#39;utilisation des animaux à des fins scientifiques et les lignes directrices de la Déclaration ARVO pour l&#39;utilisation d&#39;animaux dans ophtalmique et Vision Research, et ont été approuvés par le Comité d&#39;éthique animale de l&#39;Université de Macquarie. 1. VEP implantation d&#39;électrodes <ol><li> Anesthésier les animaux par une injection intraperitoneale de kétamine (75 mg / kg) et la médétomidine (0,5 mg / kg). Remarque: Après l&#39;induction de l&#39;anesthésie, observer les réflexes de retrait (test de pincement, réflexe cornéen et palpébrale, etc.) et leur absence comme une indication pour commencer la chirurgie. Surveiller continuellement les animaux tout au long de la chirurgie et administrer anesthésique supplémentaire (10% de la dose de kétamine initial par top-up) si les réflexes sont présents. Des rats adultes (&gt; 12 semaines) sont utilisés dans les expériences. </li><li> Raser la peau de la zone chirurgicale.Placez l&#39;animal sur un coussin chauffant (37 ° C) afin de maintenir la température du corps pendant l&#39;intervention chirurgicale. Préparer la peau grâce à l&#39;application topique de povidone-iode. Appliquer drapage chirurgical. Appliquer une pommade ophtalmique topique pour prévenir la sécheresse de la cornée sous anesthésie générale. Maintenir l&#39;asepsie en utilisant des instruments stériles. </li><li> Faire une incision longitudinale de la peau sur la ligne médiane de la peau de la tête. Désactivez le tissu conjonctif pour obtenir une bonne exposition du crâne. </li><li> Soigneusement, percer de petits trous de bavures manuellement à l&#39;aide d&#39;une perceuse à main micro à 7 mm derrière le bregma et 3 mm latéralement de la ligne médiane. </li><li> Implant à vis des électrodes à travers le crâne dans le cortex (zone 17), pénétrant dans le cortex à une profondeur d&#39;environ 0,5 mm. Implanter une électrode de vis de référence sur la ligne médiane 3 mm rostral au bregma. Appliquer ciment dentaire pour envelopper et fixer les vis (pas toujours nécessaire). </li><li> Suturer la peau de la tête, administrer une pommade antibiotique sur la peau et permettre aux animaux de recover de l&#39;anesthésie sur un coussin chauffant. Remarque: vous pouvez électrodes peuvent être laissés exposés, de sorte que la peau n&#39;a pas besoin d&#39;être rouvert dans chaque enregistrement. Immédiatement après la cessation de la chirurgie, mais avant la récupération anesthésique, administrer un médicament non-stéroïdiens anti-inflammatoire non stéroïdien (AINS) (si pas administré en pré-opératoire) ou un analgésique opioïde. Surveiller les animaux constamment jusqu&#39;à ce que le rétablissement complet de l&#39;anesthésie et entièrement ambulatoire. </li><li> Prévoyez au moins 1 semaine pour les animaux de récupérer de la chirurgie avant l&#39;enregistrement VEP. </li></ol> 2. Optic Nerve Injection <ol><li> Anesthésier l&#39;animal, préparer la peau et appliquer drapage comme ci-dessus (1.1 et 1.2). </li><li> Ajouter un 1- à 1,5 cm incision dans la peau au-dessus de l&#39;orbite de l&#39;oeil choisi de façon aléatoire. Ouvrez le tissu sous-cutané pour atteindre la cavité orbitaire en utilisant l&#39;iris ciseaux fins. Ouvrir la conjonctive et de la partie antérieure de la capsule de Tenon sous le microscope opératoire. </li><li> Rétracter le extraoculaireles muscles et les glandes lacrymales intraobital pour exposer environ 3 mm de longueur du nerf optique. Ouvrez la dure-mère et l&#39;arachnoïde matière couches autour du nerf optique en utilisant une lame longitudinalement ophtalmique. </li><li> Insérez la pipette en verre dans le nerf optique à une distance de 2 mm en arrière du globe. La micropipette en verre est fixée à une seringue Hamilton. </li><li> Injecter 1% lysolécithine (0,4 à 1,0 pi, avec 0,02% de bleu d&#39;Evan qui n&#39;a pas d&#39;effets sur la myélinisation) lentement dans le nerf environ sur une période de 30 sec. </li><li> Suturer l&#39;incision de la peau. Appliquer une pommade antibiotique pour prévenir l&#39;infection. Compagnons yeux peuvent être servis que des contrôles internes pour les enregistrements électrophysiologiques. </li><li> Placez les animaux sur un coussin chauffant pour récupérer de l&#39;anesthésie. </li></ol> 3. VEP Enregistrement <ol><li> Anesthésier l&#39;animal et de préparer la peau en tant que 1,1 et 1,2. Remarque: Une dose réduite d&#39;anesthésiques peut être utilisé pour reco électrophysiologiquerding (kétamine à 40 mg / kg de médétomidine et 0,25 mg / kg). </li><li> Placez le rat dans une pièce sombre et lui permettre d&#39;adapter à l&#39;obscurité pendant 5 - 30 min. Dans certains cas, les rats peuvent être respectivement O adapté foncé / N pour scotopique ou de lumière adapté pour les enregistrements photopique de VEP 8. </li><li> Maintenir la température du corps à 37 ± 0,5 ° C par le système de couverture homéothermes avec une sonde de thermomètre rectal. </li><li> Dilater les pupilles avec 1,0% tropicamide gouttes oculaires. Ouvrir la peau sur le crâne pour accéder à la pré-électrodes placés dans de vis situ. </li><li> Connectez la vis sur le cortex visuel de l&#39;oeil controlatéral stimulée et la vis de référence à l&#39;amplificateur. Insérez une électrode de l&#39;aiguille dans la queue que le sol. Mesurer et maintenir l&#39;impédance de l&#39;électrode en dessous de 5 kQ. </li><li> Placer un stimulateur mini-Ganzfeld directement sur ​​la peau autour des paupières pour assurer une isolation supérieure 7 de l&#39;oeil. L&#39;illumination du stimulateur doit être étalonné au préalablepar un photomètre. </li><li> Livrer la stimulation lumineuse à travers clignote 100 fois à une fréquence de 1 Hz, avec des réglages de filtre passe-bande basse et haute de 1 et 100 Hz, respectivement. Le taux d&#39;échantillonnage du signal est à 5 kHz. Remarque: Les signaux doivent être échantillonnés au moins à environ 250 à 300 Hz pour assurer que plus de deux échantillons sont prélevés au cours de chaque cycle. </li><li> Suturer la peau du dos et de garder les animaux sur un coussin chauffant pour récupérer de l&#39;anesthésie. L&#39;enregistrement peut être enregistrée de façon répétée sur des animaux individuels pour surveiller les changements fonctionnels sur une période de temps. </li><li> À la fin du point, administrer une injection surdose de pentobarbital de sodium (100 mg / kg, ip) pour euthanasier l&#39;animal. Confirmez l&#39;euthanasie par arrêt cardiaque, arrêt respiratoire et la diminution de la température du corps. </li></ol> 4. Préparation des tissus et histologie <ol><li> Retirer les nerfs optiques à partir d&#39;animaux euthanasiés au microscope et fixer le tissu dans 1% de paraformaldehyde O / N. </li> &lt;li&gt; Laver le tissu à fond avec une solution saline. Traiter le tissu dans un processeur automatique de tissu et l&#39;intégrer dans de la paraffine. sections coupées (5 - 10 um) à l&#39;aide d&#39;un microtome rotatif. Remarque: Pour l&#39;étude d&#39;immunohistochimie, fixer le tissu dans 1% de paraformaldehyde, laver avec une solution saline et incuber avec 30% de saccharose O / N. Incluez le tissu dans l&#39;OCT milieu d&#39;inclusion et de faire cryosections l&#39;aide d&#39;un cryostat. </li><li> Incuber les coupes dans 0,1% de solution de bleu rapide tels que Luxol (dans 95% d&#39;éthanol) O / N à 56 ° C. Différencier les sections de carbonate de lithium à 0,05% pendant 30 secondes et ensuite éthanol à 70% pendant 30 sec. Enfin, contre-colorer dans une solution de violet de crésyl à 0,1% pendant 30 secondes avant de monter les sections. Utilisez la coloration au bleu rapide pour identifier la myéline dans le nerf optique 5. </li></ol>

<ol>
	<li>Balcer, 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=Clinical+practice.+Optic+neuritis.">Clinical practice. Optic neuritis.</a> N Engl J Med. 354 (12), 1273-1280 (2006).</li><li>Lassmann, H., Waxman, S. 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> Multiple sclerosis as a neuronal disease. , 153-164 (2005).</li><li>Fahle, M., Bach, M., Heckenlively, J., Arden, 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> Principles and practice of clinical electrophysiology of vision. , 207-234 (2006).</li><li>Halliday, A. M., McDonald, W. I., Mushin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+visual+evoked+response+in+optic+neuritis.">Delayed visual evoked response in optic neuritis.</a> Lancet. 1, 982-985 (1972).</li><li>You, Y., Klistorner, A., Thie, J., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latency+delay+of+visual+evoked+potential+is+a+real+measurement+of+demyelination+in+a+rat+model+of+optic+neuritis.">Latency delay of visual evoked potential is a real measurement of demyelination in a rat model of optic neuritis.</a> Invest Ophthalmol Vis Sci. 52 (9), 6911-6918 (2011).</li><li>You, Y., Klistorner, A., Thie, J., Gupta, V. K., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+loss+in+a+rat+model+of+optic+neuritis+is+closely+correlated+with+visual+evoked+potential+amplitudes+using+electroencephalogram+based+scaling.">Axonal loss in a rat model of optic neuritis is closely correlated with visual evoked potential amplitudes using electroencephalogram based scaling.</a> Invest Ophthalmol Vis Sci. 53, 3662 (2012).</li><li>You, Y., Klistorner, A., Thie, J., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+reproducibility+of+VEP+recording+in+rats:+electrodes,+stimulus+source+and+peak+analysis.">Improving reproducibility of VEP recording in rats: electrodes, stimulus source and peak analysis.</a> Doc Ophthalmol. 123 (2), 109-119 (2011).</li><li>Heiduschka, P., Schraermeyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+visual+function+in+pigmented+and+albino+rats+by+electroretinography+and+visual+evoked+potentials.">Comparison of visual function in pigmented and albino rats by electroretinography and visual evoked potentials.</a> Graefes Arch Clin Exp Ophthalmol. 246 (11), 1559-1573 (2008).</li><li>You, Y., Thie, J., Klistorner, A., Gupta, V. K., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+visual+evoked+potentials+using+underlying+electroencephalogram+levels+improves+amplitude+reproducibility+in+rats.">Normalization of visual evoked potentials using underlying electroencephalogram levels improves amplitude reproducibility in rats.</a> Invest Ophthalmol Vis Sci. 53 (3), 1473-1478 (2012).</li><li>Levkovitch-Verbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+optic+nerve+diseases.">Animal models of optic nerve diseases.</a> Eye (Lond). 18 (11), 1066-1074 (2004).</li><li>Henry, K. R., Rhoades, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+albinism+and+drugs+to+the+visual+evoked+potential+of+the+mouse).">Relation of albinism and drugs to the visual evoked potential of the mouse).</a> J Comp Physiol Psychol. 92 (2), 271-279 (1978).</li><li>Murrell, J. C., Waters, D., Johnson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+effects+of+halothane,+isoflurane,+sevoflurane+and+desflurane+on+the+electroencephalogram+of+the+rat.">Comparative effects of halothane, isoflurane, sevoflurane and desflurane on the electroencephalogram of the rat.</a> Lab Anim. 42 (2), 161-170 (2008).</li><li>Makela, K., Hartikainen, K., Rorarius, M., Jantti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+F-VEP+during+isoflurane-induced+EEG+suppression.">Suppression of F-VEP during isoflurane-induced EEG suppression.</a> Electroencephalogr Clin Neurophysiol. 100 (3), 269-272 (1996).</li><li>Boyes, W. K., Padilla, S., Dyer, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+temperature-dependent+and+independent+actions+of+chlordimeform+on+visual+evoked+potentials+and+axonal+transport+in+optic+system+of+rat.">Body temperature-dependent and independent actions of chlordimeform on visual evoked potentials and axonal transport in optic system of rat.</a> Neuropharmacology. 24 (8), 743-749 (1985).</li><li>Hetzler, B. E., Boyes, W. K., Creason, J. P., Dyer, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependent+changes+in+visual+evoked+potentials+of+rats.">Temperature-dependent changes in visual evoked potentials of rats.</a> Electroencephalogr Clin Neurophysiol. 70 (2), 137-154 (1988).</li><li>Mitchell, 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+effects+of+lysolecithin+on+non-myelinated+axons+in+vitro.">The effects of lysolecithin on non-myelinated axons in vitro.</a> Acta Neuropathol. 58 (4), 243-248 (1982).</li><li>Meyer, R., 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=Acute+neuronal+apoptosis+in+a+rat+model+of+multiple+sclerosis.">Acute neuronal apoptosis in a rat model of multiple sclerosis.</a> J Neurosci. 21 (16), 6214-6220 (2001).</li><li>Lachapelle, F., 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=Failure+of+remyelination+in+the+nonhuman+primate+optic+nerve.">Failure of remyelination in the nonhuman primate optic nerve.</a> Brain Pathol. 15 (3), 198-207 (2005).</li></ol>

None of the authors have competing interests or conflicting interests.

The optic nerve is very susceptible to mechanical damage. Optic nerve crush injury over a duration of 1 s can lead to about 75% loss of RGC over a period of 2 weeks10. Therefore, extreme care is required while performing the surgical procedures. According to the authors&#8217; experience, it is much better to adapt a blunt dissection approach to expose and make way through the tissues around the optic nerve along the orientation of the nerve, rather than penetrating in a perpendicular orientation to the optic ...

Optic neuritis is one of the most common form of optic neuropathy, causing complete or partial loss of vision1. Histologically, it is featured by inflammatory demyelination, retinal ganglion cell axonal loss and varying degrees of remyelination in the optic nerve2. Optic neuritis is usually the manifest onset of multiple sclerosis. The visual evoked potential (VEP) is a non-invasive tool for investigating the function of the visual system. It reflects the post-retinal function from the retina to the primary visual cortex and is affected in many optic nerve disease conditions3. The VEP has been predominantly used in optic neuritis patients to assess the integrity of the visual pathway4. 
The latency of VEP, which reflects the velocity of signal conduction along the visual pathway, is considered to be an accurate measurement of the level of myelin associated changes in the optic nerve5; while the amplitude of VEP is believed to be closely correlated with axonal damage of the retinal ganglion cells (RGC)6. This hypothesis has been fairly well established using the rat model of lysolecithin-induced optic nerve demyelination5. 
Here, we explicate a comprehensive protocol of optic nerve microinjection technique in rodents, which can minimise the surgical manipulation-related damage to the nerve per se as well as to the adjacent tissues such as extraocular muscles and blood vessels. Also, the skull electrode implantation surgery has been described for VEP recording in animals7. The VEP recordings can be repeatedly carried out on animals over a period of time to assess demyelination/remyelination related changes as well as impact on axonal integrity in the optic nerve.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ketamine 100 mg/ml (Ketamil)</td>
			<td>Troy Laboratories</td>
			<td>AC 116</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine 1 mg/ml (Domitor)</td>
			<td>Pfizer</td>
			<td>sc-204073</td>
			<td></td>
		</tr>
		<tr>
			<td>Tropicamide 1.0% (Mydriacyl)</td>
			<td>Alcon</td>
			<td>sc-202371</td>
			<td></td>
		</tr>
		<tr>
			<td>Homoeothermic blanket system</td>
			<td>Harvard Apparatus</td>
			<td>NC9203819</td>
			<td></td>
		</tr>
		<tr>
			<td>Impedance meter&#160;</td>
			<td>Grass</td>
			<td>F-EZM5</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw electrodes&#160;</td>
			<td>Micro Fasteners</td>
			<td>M1.0&#215;3mm Csk Slot M/T 304 S/S</td>
			<td></td>
		</tr>
		<tr>
			<td>Subdermal needle electrodes&#160;</td>
			<td>Grass</td>
			<td>F-E3M-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid Repair&#160;</td>
			<td>DeguDent GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light-emitting diode&#160;</td>
			<td>Nichia</td>
			<td>NSPG300A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioamplifier</td>
			<td>CWE, Inc.</td>
			<td>BMA-400</td>
			<td></td>
		</tr>
		<tr>
			<td>CED system</td>
			<td>Cambridge Electronic Design, Ltd.</td>
			<td>Power1401</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe&#160;</td>
			<td>Hamilton</td>
			<td>87930</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysolecithin</td>
			<td>Sigma</td>
			<td>L4129</td>
			<td></td>
		</tr>
		<tr>
			<td>Evan&#8217;s blue&#160;</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td></td>
		</tr>
	</tbody>
</table>

visual evoked potential recording in a rat model of experimental optic nerve demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Traces de VEP intrasessions reproductibles sont présentés dans la figure 1 et un retard important dans la latence N1 peuvent être observés après l&#39;injection du nerf optique. Lésions du nerf optique partielles de démyélinisation peuvent être observées sur des coupes histologiques utilisant Luxol coloration au bleu rapide 5. La figure 2 montre une section représentative avec une petite lésion démyélinisée focal dans le centre du nerf optique. Notez que sectio...

Watch this Scientific Journal Video about Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination at JoVE.com

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Enregistrement Potentiel évoqué visuel dans un modèle de rat of Experimental Optic Nerve démyélinisation

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

visual-evoked-potential-recording-rat-model-experimental-optic-nerve

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12.1K vues.  Macquarie University. Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection. 

Vidéo: Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Section du nerf optique: un modèle de l&#39;apoptose neuronale des adultes dans le système nerveux central

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

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Neurosciences

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Méthodes de manipulations expérimentales après section du nerf optique dans le SNC des mammifères

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Un modèle Optic Nerve Crush blessures murins pour étudier la rétine de survie des cellules ganglionnaires

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Détermination des espèces d&#39;oxygène Potentiel membrane mitochondriale et réactive dans les neurones corticaux de rat en direct

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

Technique mini-invasive pour injection dans Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

Enregistrement simultané de Électrorétinographie et potentiels évoqués visuels chez des rats anesthésiés

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Résection partielle du nerf optique chez le rat : un modèle mis en place avec une nouvelle approche du dispositif d’évaluation secondaire dégénérescence des cellules ganglionnaires rétiniennes

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

Des enregistrements de potentiels évoqués visuels chez les souris en utilisant un multicanal Non invasif de la sec du cuir chevelu EEG capteur

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Modèle de rat de démyélinisation corticale cérébrale généralisée induite par une injection intracérébrale de cytokines pro-inflammatoires

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

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

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

Modèles morphologiques potentiels d'evoked Visual

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