Name: visual evoked potential recording in a rat model of experimental optic nerve demyelination
Uploaded: 2015-07-29T14:00:00.000+00:00
Duration: 6 min 49 s
Description: Watch this Scientific Journal Video about Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination at JoVE.com

Questo studio è stato sostenuto dal oftalmica Research Institute of Australia (ORIA). Ringraziamo il Prof. Algis Vingrys e il dottor Bang Bui, Università di Melbourne, per primo ci aiuta a sviluppare la tecnica di registrazione VEP.

Etica Dichiarazione: Tutte le procedure che coinvolgono gli animali sono state condotte in conformità con il Codice di condotta australiano per la cura e l&#39;uso di animali a scopi scientifici e gli orientamenti della Dichiarazione ARVO per l&#39;uso di animali in Oftalmica e Vision Research, e sono stati approvati dalla Animal Comitato Etico della Macquarie University. 1. VEP elettrodi impianto <ol><li> Anestetizzare l&#39;animale con una iniezione intraperitoneale di ketamina (75 mg / kg) e medetomidina (0,5 mg / kg). Nota: Dopo l&#39;induzione dell&#39;anestesia, osservare i riflessi di prelievo (test pizzico, riflessi cornea e della palpebra, ecc) e la loro assenza come indicazione per iniziare un intervento chirurgico. Monitorare continuamente gli animali in tutta la chirurgia e amministrare anestetico supplementare (10% della dose di ketamina iniziale per top-up) se sono presenti i riflessi. Ratti adulti (&gt; 12 settimane) sono usati negli esperimenti. </li><li> Radere la pelle della zona chirurgica.Posto l&#39;animale su un blocco di riscaldamento (37 ° C) per mantenere la temperatura corporea durante l&#39;intervento chirurgico. Preparare la pelle attraverso l&#39;applicazione topica di povidone-iodio. Applicare drappeggio chirurgica. Applicare attualità pomata oftalmica per prevenire la secchezza della cornea in anestesia generale. Mantenere asepsi utilizzando strumenti sterili. </li><li> Effettuare una incisione cutanea longitudinale sulla linea mediana della pelle testa. Eliminare il tessuto connettivo, per ottenere una buona esposizione del cranio. </li><li> Con cautela, praticare dei piccoli fori radica manualmente utilizzando un trapano micro mano a 7 mm dietro il bregma e 3 mm lateralmente alla linea mediana. </li><li> Elettrodi vite implantare attraverso il cranio nella corteccia (area 17), penetrando la corteccia ad una profondità di circa 0,5 mm. Impiantare un elettrodo di vite di riferimento sulla linea mediana 3 mm rostrale al bregma. Applicare cemento dentale di racchiudere e fissare le viti (non sempre necessario). </li><li> Suturare la pelle della testa, amministrare pomata antibiotica sulla pelle e consentire all&#39;animale di recover dall&#39;anestesia su un blocco di riscaldamento. Nota: In alternativa, gli elettrodi possono essere lasciati esposti, in modo che la pelle non deve essere riaperto in ogni registrazione. Immediatamente al momento della cessazione di un intervento chirurgico, ma prima del recupero anestetico, somministrare un farmaco non steroideo anti-infiammatori (FANS) (se non somministrato in fase preoperatoria) o un analgesico oppiaceo. Monitorare gli animali costantemente fino al pieno recupero da anestetico e completamente ambulatoriale. </li><li> Lasciare almeno 1 settimana per gli animali di recuperare da un intervento chirurgico prima della registrazione VEP. </li></ol> 2. Optic Nerve iniezione <ol><li> Anestetizzare l&#39;animale, preparare la pelle e applicare drappeggio come sopra (1.1 e 1.2). </li><li> Fare un 1 a 1,5 cm incisione nella pelle sopra l&#39;orbita di un occhio selezionata casualmente. Aprire il tessuto sottocutaneo per raggiungere la cavità orbitale con iris sottili forbici. Aprire la congiuntiva e la capsula di Tenon anteriore sotto il microscopio operatorio. </li><li> Ritrarre la extraocularemuscoli e ghiandole lacrimali intraobital per esporre circa 3 mm di lunghezza del nervo ottico. Aprire la dura madre e materia aracnoide strati attorno al nervo ottico longitudinalmente utilizzando una lama oftalmica. </li><li> Inserire la pipetta di vetro nel nervo ottico ad una distanza di 2 mm posteriormente al globo. La micropipetta di vetro è attaccato ad una siringa Hamilton. </li><li> Iniettare 1% lisolecitina (0,4-1,0 microlitri, con 0,02% di blu di Evan che non ha effetti sulla mielinizzazione) lentamente nel nervo circa per un periodo di 30 sec. </li><li> Suturare la incisione cutanea. Applicare una pomata antibiotica per prevenire l&#39;infezione. Occhi Compagni possono essere serviti come controlli interni per le registrazioni elettrofisiologiche. </li><li> Mettere gli animali su un blocco di riscaldamento di recuperare da anestesia. </li></ol> 3. VEP registrazione <ol><li> Anestetizzare l&#39;animale e preparare la pelle come 1.1 e 1.2. Nota: Lower dose di anestetici può essere utilizzato per reco elettrofisiologicarding (ketamina 40 mg / kg e medetomidina 0,25 mg / kg). </li><li> Mettere il topo in una stanza buia e permettono di adattarsi al buio per 5-30 min. In alcuni casi, i ratti possono rispettivamente essere scuro O adattato / N per scotopica o la luce adatta per le registrazioni fotopica VEP 8. </li><li> Mantenere la temperatura corporea a 37 ± 0,5 ° C dal sistema coperta homoeothermic con una sonda termometro rettale. </li><li> Dilatare le pupille con 1,0% tropicamide collirio. Aprire la pelle sopra il cranio per accedere al pre-posto in elettrodi vite situ. </li><li> Collegare la vite sulla corteccia visiva controlaterale dell&#39;occhio stimolata e la vite riferimento all&#39;amplificatore. Inserire un elettrodo ago nel coda come la terra. Misurare e mantenere l&#39;impedenza dell&#39;elettrodo sotto 5 kΩ. </li><li> Mettere uno stimolatore mini-Ganzfeld direttamente sulla pelle intorno alle palpebre per fornire l&#39;isolamento occhio superiore 7. L&#39;illuminazione dello stimolatore necessario calibrare anticipoda un fotometro. </li><li> Invia stimolazione luminosa attraverso lampi di luce 100 volte alla frequenza di 1 Hz, con impostazioni del filtro passa-banda bassa e alta di 1 e 100 Hz, rispettivamente. La frequenza di campionamento del segnale è a 5 kHz. Nota: I segnali devono essere campionati almeno a circa 250-300 Hz per garantire che più di due campioni vengono raccolti durante ogni ciclo. </li><li> Suturare la pelle posteriore e tenere gli animali su un blocco di riscaldamento di recuperare da anestesia. La registrazione può essere ripetutamente registrati su singoli animali per monitorare le variazioni funzionali per un periodo di tempo. </li><li> Al punto finale, somministrare una iniezione sovradosaggio di pentobarbitone sodio (100 mg / kg, ip) euthanize all&#39;animale. Conferma eutanasia arresto cardiaco, arresto respiratorio e diminuzione della temperatura corporea. </li></ol> 4. Preparazione del tessuto e Istologia <ol><li> Rimuovere i nervi ottici da animali eutanasia sotto il microscopio e fissare il tessuto in 1% paraformaldeide O / N. </li> &lt;li&gt; Lavare accuratamente i tessuti con soluzione fisiologica. Trattare il tessuto in un processatore automatico ed incorporare in paraffina. Sezioni tagliate (5-10 micron) utilizzando un microtomo rotativo. Nota: per studio immunoistochimica, fissare il tessuto in 1% paraformaldeide, lavare con soluzione fisiologica e incubare con il 30% di saccarosio O / N. Tessuto Incorpora in OCT mezzo di inclusione e fare criosezioni utilizzando un criostato. </li><li> Incubare sezioni 0,1% soluzione blu veloce come il Luxol (nel 95% di etanolo) O / N a 56 ° C. Differenziare le sezioni a 0,05% di carbonato di litio per 30 sec e poi 70% di etanolo per altri 30 sec. Infine, colorazione di contrasto in soluzione violetto cresolo 0,1% per 30 sec prima di montare sezioni. Utilizzare la veloce colorazione blu per identificare mielina nel nervo ottico 5. </li></ol>

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	<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 nerve. Also, the dura and arachnoid mater should be unwraped longitudinally to avoid any axotomy-like damage to the RGC axons. Evan&#8217;s blue is usually used to facilitate the microinjection, as presence of the dye makes the nerve light blue in colour at the injection site.
The reproducibility of VEP recording is critical for this experiment. Different types of anaesthetics and the depth of anaesthesia can affect the VEP waveforms11. We do not recommend using inhaled isoflurane anaesthesia for the VEP study, as isoflurane can lead to burst suppress on the electroencephalogram response12, probably due to GABAergic activity, resulting in more variable VEP traces13. Combination of ketamine/medetomidine anaesthesia provides stable anaesthesia that can quickly be reversed by administration of an alpha receptor antagonist such as atipamazole (2.5 mg/kg i.p). Body temperature can also have a significant impact on the speed of nerve conduction, especially in demyelinated nerves. Temperature-dependant changes in visual evoked potentials have been observed in rat VEPs and latencies were observed to be significantly longer as body temperature was lowered14,15. Hence, it is important to maintain a uniform body temperature using a homoeothermic blanket system during the VEP recordings.
Also, it is important to understand that the pathogenesis of lysolecithin-induced demyelination differs from that in optic neuritis or multiple sclerosis. Lysolecithin-induced demyelination results from the detergent like action of the toxin upon myelin lipids rather than immune-mediated inflammatory reaction, which is usually seen in the real disease scenarios16. If the study aims to investigate the mechanisms of demyelination/axonal damage in multiple sclerosis, an immune-mediated demyelination model, such as experimental autoimmune encephalomyelitis (EAE)17, should be considered. The advantage of using the toxin-induced demyelination model is that a focal lesion in the optic nerve can be produced and monitored longitudinally. In contrast to the brain lesions, the effects of demyelinated lesions on the optic nerve are more clinically apparent and measureable (lesion size can be accurately determined by VEPs or MRI imaging). Although remyelination in the optic nerve appeared to be not as pronounced as that observed previously in the case of spinal cord18, this optic nerve injection model, in conjunction with the VEP recording technique, is able to provide a superior tool to monitor the process of demyelination and remyelination in vivo, and to potentially evaluate new remyelinating therapies.

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><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&amp;nbsp;</td><td>Grass</td><td>F-EZM5</td><td></td></tr><tr><td>Screw electrodes&amp;nbsp;</td><td>Micro Fasteners</td><td>M1.0&amp;times;3mm Csk Slot M/T 304 S/S</td><td></td></tr><tr><td>Subdermal needle electrodes&amp;nbsp;</td><td>Grass</td><td>F-E3M-72</td><td></td></tr><tr><td>Rapid Repair&amp;nbsp;</td><td>DeguDent GmbH</td><td></td><td></td></tr><tr><td>Light-emitting diode&amp;nbsp;</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&amp;nbsp;</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&amp;rsquo;s blue&amp;nbsp;</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.

Riproducibili PEV tracce intra-sessione sono mostrati in Figura 1 e un significativo ritardo nella latenza N1 possono essere osservati dopo l&#39;iniezione nervo ottico. Lesioni del nervo ottico parziali di demielinizzazione può essere osservato su sezioni istologiche utilizzando Luxol veloce colorazione blu 5. La Figura 2 mostra una sezione rappresentativa con una piccola lesione demyelinated focale nel centro del nervo ottico. Si noti che la sezione trasversale non rappresenta il volume totale di lesione. L&#39;area demyelinated può essere misurata su ciascuna sezione consecutivi del nervo dedurre il volume della lesione utilizzando ricostruzione tridimensionale. Abbiamo dimostrato una forte correlazione tra il ritardo di latenza e il volume delle lesioni utilizzando questo modello in nostri studi precedenti e non c&#39;era VEP ritardo di latenza nei controlli saline a iniezione 5. Si ritiene che i componenti PEV primi seguenti l&#39;illuminazione flash sono più stabili 7 </sup&gt; E principalmente influenzato dalla eccitazione della corteccia visiva primaria tramite fibre retino-genicolate. Il nostro studio ha dimostrato che il ritardo N1 ha la più forte relazione lineare con demielinizzazione nel nervo ottico 5. Pertanto, si consiglia di N1 latenza dovrebbe essere utilizzato per l&#39;analisi dei dati e per il monitoraggio VEP longitudinale nel valutare l&#39;impatto delle terapie rimielinizzanti. L&#39;ampiezza di VEP, anche se più variabile rispetto alla latenza è più indicativo della funzione degli assoni del nervo ottico 6. Ridimensionamento basato Elettroencefalogramma-può essere presa in considerazione per l&#39;analisi di ampiezza 9. <img alt="Figura 1" src="/files/ftp_upload/52934/52934fig1.jpg" /> Figura 1. VEP ritardo dopo l&#39;iniezione del nervo ottico. Rappresentante VEP traccia da un topo individuale prima e 2 giorni dopo la microiniezione nervo ottico (0,8 ml lisolecitina). Registrazioni VEP sono state ripetute in ogni giorno (intra-sessione tracce sono spettacolon dello stesso colore) per dimostrare la riproducibilità di questo protocollo di registrazione VEP. (Barra della scala verticale: 10 mV; bar scala orizzontale: 10 msec). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52934/52934fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52934/52934fig2.jpg" /> Figura 2. Demielinizzazione nel nervo ottico. Rappresentante sezione del nervo ottico da un ratto dopo Lysolecithin microiniezione. Il componente mielina è macchiato blu utilizzando Luxol blu veloce. Una piccola lesione focale di demielinizzazione può essere visto nel centro della sezione. Zona demielinizzati può essere misurata su sezioni seriali longitudinali per stimare il volume delle lesioni in una scala tridimensionale. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52934/52934fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

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.

Registrazione potenziale evocato visivo in un modello murino di sperimentale del nervo ottico demielinizzazione

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 ...

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12.2K Views.  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. 

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

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Singola unità In vivo Registrazioni dal chiasma ottico di Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Ricerca

Neuroscienze

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

L&#39;impianto e la registrazione di Wireless Elettroretinogramma e potenziale visivo evocato ratto conscio

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

Comportamento

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Rat Modello di fotochimica-Induced posteriore Neuropatia ottica ischemica

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

Medicina

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.

Registrazione simultanea di elettroretinografia e Potenziali evocati visivi nei ratti anestetizzati

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

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.

Registrazioni di potenziali evocate visive nei topi utilizzando un multicanale Non invasiva a secco del cuoio capelluto EEG sensore

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 ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

In vivo Metodi per valutare la funzione e la struttura delle cellule gangliari retiniche e del nervo ottico in animali di grandi dimensioni

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...

Inducing Local Demyelination in the Optic Nerve Using Lysolecithin

Source: You, Y. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52934">Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination.</a> J. Vis. Exp. (2015)
This video demonstrates the procedure of surgically accessing the optic nerve in an anesthetized animal and precisely delivering lysolecithin to induce focal demyelination, creating neuropathological conditions for further study.

Induzione della demielinizzazione locale nel nervo ottico mediante lisolecitina

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

Preparing a Rat Model to Assess Visual Pathway Integrity Using an Electrophysiology Setup

Source: Nguyen, C. T. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54158">Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates the preparation of a rat model to study retinal health and visual pathway integrity. Visual evoked potential and electroretinography reference and active electrodes are connected to the rat, followed by aligning the platform with the rat in front of the Ganzfeld bowl for even retinal illumination and closing the Faraday cage to avoid external interference.

Preparazione di un modello di ratto per valutare l'integrità del percorso visivo utilizzando una configurazione di elettrofisiologia

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Neurophysiology

Electrophysiology Techniques

Electron Microscopic Analysis of Myelin Damage in a Rat Model of Optic Nerve Injury

Take fixed optic nerve sections from a control rat and a rat with optic nerve injury.
The control section exhibits axons with tightly packed myelin sheaths containing concentric plasma membrane layers. The injured section exhibits decompacted myelin with large gaps.
Incubate with an osmium-ferrocyanide complex that binds to lipids and enhances the myelin sheath&#39;s contrast.
Treat with thiocarbohydrazide to create additional binding sites for osmium.
Reapply osmium to further enhance lipid contrast.
Stain with heavy metals, which bind to macromolecules and enhance structural contrast.
Dehydrate using increasing alcohol concentrations and rinse with acetone for resin embedding.
Embed the tissue in resin, and coat the resin with gold to improve image quality.
Use serial block-face scanning electron microscopy to simultaneously slice and image. Focus the electron beam on the surface and detect the backscattered electrons to generate images.
Myelin sheaths appear dark, while spaces within damaged sheaths appear lighter, allowing differentiation between normal and damaged myelin.

Analisi al microscopio elettronico del danno mielinico in un modello di ratto di lesione del nervo ottico

Neuromuscular Disorders

Recording of Visual Evoked Potentials via the Skull Electrodes in a Rat Model

Source: You, Y., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52934">Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination.</a> J. Vis. Exp. (2015).
This video demonstrates the process of measuring visual evoked potentials or VEPs in a dark-adapted, anesthetized rat with pre-implanted skull electrodes, which record the electrical signals from the brain&#39;s visual cortex in response to light stimuli.

Registrazione dei potenziali evocati visivi tramite gli elettrodi del cranio in un modello di ratto

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. VEP ...