Brug af elektroretinogrammet at vurdere Funktion i gnaver Retina og beskyttende virkninger af fjernbetjening Limb iskæmisk prækonditionering
Summary
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.
Abstract
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 ‘flash’ 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.
Introduction
ERG er et elektrisk potentiale genereret af nethinden som reaktion på lys og registreres fra hornhindens overflade af øjet. Når betingelserne for optagelse styres omhyggeligt, kan ERG anvendes i en række forskellige måder at vurdere retinal funktion. Her beskrev vi, hvordan at registrere »flash ERG«, potentialet genereres, når nethinden udsættes for en kort, lyse flash præsenteret i en Ganzfeld baggrund. Den Ganzfeld spreder lyset homogent og lysglimt når hele nethinden ca. ensartet. Hvis nethinden er mørkt tilpasset før optagelse, og den mørke-tilpasning opretholdes som dyret er forberedt til optagelse, ERG opnåede genereres af både stang og kegle fotoreceptorer.
Den mørke-tilpassede flash ERG har en karakteristisk bølgeform, som er blevet analyseret på to måder. For det første har tidlige og sene komponenter af ERG bølgeform blevet adskilt og knyttet til sekvensen af neuronal aktivering i nethinden. Den tidligste komponent er en kort latenstid negativt gående potentiale, a-wave (figur 1). Dette efterfølges af en positivt gående potentiale, kaldet b-bølgen. Den stigende fase af b-bølgen viser svingninger, som betragtes som et separat element (oscillerende potentialer eller OP). A-bølge anses at blive genereret af fotoreceptorer, b-bølgen af celler i det indre nukleare lag og OP ved amacrine celler 1.
Baseret på stimulus styrke, reaktioner på blinker meget dim betegnet er muligt scotopic reaktionstærskel. Den scotopic tærskel respons forstås at blive genereret fra retinale ganglieceller 2-4. For det andet kan flash ERG være adskilt af lys tilpasning, eller af en to flash protokol beskrevet nedenfor, til stang- og kegle-drevne komponenter. Under fotopiske forhold, a-bølge ikke påvises i rotter, fordi keglen befolkning er lav, men OP og en b-bølge erklar 5. I primater, hvis nethinder har højere kegle populationer, både stang- og kegle- veje genererer et påviseligt en-bølge 6.
To nyttige foranstaltninger ofte udvundet fra flash-ERG er amplituderne af a- og b-bølgerne, målt som i figur 1, med typiske flash responser vist i figur 2. Når fotoreceptor befolkning er reduceret, for eksempel ved udsættelse for et skadeligt niveau lyse lys, er alle komponenter af ERG reduceret. Neuroprotektive interventioner, såsom remote iskæmisk konditionering (RIP), kan godkendes af bevarelsen af amplituder af a- og b-bølger (Figur 3). Sammenfattende analyse af ERG muliggør sammenligninger mellem sund, lys beskadiget og neuroprotected nethinden.
Protocol
Representative Results
Discussion
Den mørke-tilpassede flash ERG fremgangsmåde beskrevet ovenfor er en pålidelig metode til vurdering af nethindens funktion hos rotter. Både a-bølge og b-bølge blev reduceret med lys skader. Remote iskæmisk prækonditionering afbødes lysskade-inducerede reduktioner i a-bølge og b-bølge. Denne bevarelse af nethindens funktion antyder, at remote iskæmisk prækonditionering har fremkaldt neurobeskyttelse, der ligner andre former for beskyttende forkonditionering såsom hypoksi, iskæmi og motion 8-10. …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne er taknemmelige for den hjælp fra fru Sharon Spana i gnavere overvågning, håndtering og eksperimentering. Ph.d. finansieringsstøtte er leveret af University of Sydney og australske Forskningscenter for Excellence i Vision.
Materials
| PC computer | |||
| Powerlab, 4 channel acquistion hardware | AD Instruments | PL 35044 | Acquistion of ERG |
| Animal Bio Amp | AD Instruments | FE 136 | Amplifier for ERG |
| Lab chart | AD Instruments | Signal collection software | |
| Ganzfield | Photometric solutions | FS-250A | Light stimulus |
| Ganzfield operating system | Photometric solutions | ||
| Research Radiometer | International light technologies | ILT-1700 | calibrate light series |
| Lux meter | LX-1010B | check red light illumanation | |
| Excel | microsoft | ||
| Lead wires | AD Instruments | Connect postive, negative ground electrodes to amplifier | |
| Lead wires -aligator | AD Instruments | ground ganzfield and acquistion hardware to computer | |
| Platinum wire 95% | A&E metals | postive electrode | |
| Mouth electrode Ag/AgCl Pellet | SDR | E205 | negative electode |
| 26 gauge needle | BD | ground electode | |
| Water pump | |||
| Water bath | |||
| Tubing | |||
| Homeothermic blanket system with flexible probe | Harvard Appartus | 507222F | |
| Atropine 1% w/v | Bausch & Lomb | topical mydriasis | |
| Proxmethycaine 0.5% w/v | Bausch & Lomb | topical anaesthetic | |
| Visco tears eye drops | Novartis | carbomer polymer | |
| Thread | retract eye lid | ||
| Tweezers | |||
| Reusable adhesive | Blu tac | Dim red headlamp. Affix electrodes | |
| Absorbent bedding | |||
| Ketamil – ketamine 100 mg/ml – 50 ml | Troy Laboratories Pty Ltd | dissociative | |
| Xylium – Xylazine 100 mg/ml – 50 ml | Troy Laboratories Pty Ltd | muscle relaxant | |
| Scale |
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