Full-field Electroretinography in Healthy Rabbits According to ISCEV with Ganzfeld Stimulation
A.M.V. Safatle; A.P. Hvenegaard; D. Otsuki; E. Rodrigues; P.S.M. Barros
Electroretinogram (ERG) is an objective diagnostic method (Tzekov & Arden 1999), not invasive (Safatle et al. 2005) and capable to evaluate retinal function, making possible an early detection of lesions at retinal external layers, even earlier than the appearance of any clinical signs (Komaromy et al. 1998). This diagnostic method has been widely used in both veterinary and human medicine. In humans, ERG is used in many conditions for the diagnosis of retinotoxic effects of systemic drugs, hereditary diseases (Berezovsky et al. 2008) such as pigmentary retinitis; to detect and follow the proliferative and non proliferative retinopathy in diabetic patients and to assess retinal function in patients with opaque ocular means and in suddenly blindness (Ofri et al. 1993). The ERG is influenced by dark adaptation time, pupil size, stimulus intensity, electrodes, conscience level and other factors (Yanase et al. 1995). The exam is performed following the International Society for Clinical Electrophysiology of Vision (ISCEV) standards since 1989 (Marmor & Zrenner 1999). This guideline contains recommendations for calibration of the electrophysiological equipment and enabled clinics to compare their recordings worldwide (Brigell et al. 1998). The exam is constituted of five responses: rod response; maximal response in the dark-adapted eye; oscillatory potentials; light adapted cone response; and responses to a 30-Hz flickering stimulus. ERG parameters are measured from peak-to-peak amplitudes differences, between negative a-wave and positive b-wave, in microvolt; and implicit times, times from the stimulus presentation to a-and b-waves peaks, in milliseconds (ms). The exam takes 60 minutes, being the first thirty minutes spent for dark adaptation. In animals, where direct verbal communication is impossible, electrophysiological testing adds valuable information to the diagnosis of visual disorders (Rosolen et al. 2002). ERG exam has shown itself of great importance in small animals, although it can also be used in large (Komaromy et al. 2003) and exotic animals. In dogs, ERG is mostly utilized for preoperative evaluation in patients presenting cataracts; for the characterization of disturbances causing blindness, like glaucoma; achromatopsia (Rubin 1971, Hurn et al. 2003); retinal dysplasias; degenerative retinopathies (Safatle et al. 2005); optic nerve hypoplasia; sudden acquired retinal degeneration syndrome; neuronal ceroid lipofuscinoses (Narfstrom 2006); among the utilization of dogs as animal model in scientific researches (Petersen-Jones 2006). In cats ERG can be used for the diagnosis of retinal diseases such as hereditary retinal degeneration, non inflammatory retinopathy and central retinal degeneration caused by a lack of taurine in their diet (Sims 1999). In rabbits, ERG examination was already performed by Gjorloff et al. (2004) following a different protocol. As several pharmacological and toxicological tests are performed in rabbits, ERG can be useful in order to quantify the possible side effects in ophthalmologic experimental studies (Gjörloff et al. 2004). The purpose of this study was to evaluate and standardize the electroretinographic responses in healthy young New Zealand rabbits using monopolar ERG-jet electrode according to ISCEV protocol.
Materials and Methods
Seventeen healthy young New Zealand rabbits (average weight--2.2 kg) totalizing 34 eyes were studied. The rabbits were kept in a dark room for thirty minutes, prepared under dim red illumination, and anesthetized by intramuscular injection of solutions containing ketamine (50mg/ml) and xylazine (10mg/ml). Their pupils were dilated with eye drops of 1% tropicamide (Mydriacyl®--Alcon Labs. do Brasil Ltda., São Paulo, SP, Brazil) and 10% phenylephrine hydrochloride (Fenilefrina 10%®, Allergan Produtos farmacêuticos Ltda., São Paulo, SP, Brazil). The cornea was anaesthetized with 1% proparacaine HCl drops (Anestalcon®--proxymetacaine hydrochloride 0,5%, Alcon Labs. do Brasil Ltda., São Paulo, SP, Brazil). Monopolar disposable contact lenses ERG-jet electrodes (Universe SA, La Chaux-de-Fons, Switzerland) were put on both corneas with methylcellulose 2% (Metilcelulose 2%®--Ophthalmos Ind. Farmacêutica, São Paulo, SP, Brazil). After the region has been trimmed and cleaned, a reference electrode, previously filled with electrolytic gel was placed at the temporal cantus of the eyelid, while the ground electrode also filled with electrolytic gel, was placed at the left earlobe. Then, the head of the rabbits were placed in a Ganzfeld stimulator of the Veris system 5.1.12 (Electro-Diagnostic Imaging Inc., San Mateo, USA) and the ERG was simultaneously recorded from both eyes. The procedure was performed according to the standard protocol recommended by the ISCEV, recording five responses in the following order: a) a maximal intensity white light attenuated by 2.4 log neutral density filter to elicit a dark-adapted rod response, b) a maximal intensity white light to elicit a mixed dark-adapted rod and cone response, c) the maximal intensity white light with a 100-Hz low-cut and 1-KHz high-cut filter to elicit oscillatory potential (OP); and the maximal intensity white light on a 30 cd/m2 background presented, d) 1 Hz and e) 30 Hz to elicit cone responses (after 10 minutes light adaptation). Twenty responses were computer-averaged for each step, except for the 30-Hz flicker for which 50 responses were averaged. Peak-to-peak amplitudes and b-wave implicit times were measured for all responses, except OPs. To avoid diurnal variation in ERG responses, all exams were performed at the same time of the day (3 pm).
Statistical analysis included: mean, standard deviation, median, minimum and maximum values were determined for each parameter and was accomplished by using a computer program (Excel 2003, Microsoft Corporation, USA). Rod amplitudes varied from 82.3 μV to 194,6 μV (mean 125.3 μV, standard diversion--SD -27.7 and median of 121.8); bwave implicit time at rod responses varied from 28.5 ms to 57.5 ms (mean 37.5 ms, SD 5.8 and median 36.7); Maximal response amplitude varied from 108.6 to 255.8 μV (mean 164.2 μV, SD 35.2 and median 36,7); b-wave implicit time on maximal response varied: from 27.5 ms to 43.5 ms (mean 33.9 ms, SD 3.5 and median 33.2); Ratio b/a varied from 1.47 to 2.85 (mean 2,0, SD 0.3 and median 1.97); Oscillatory potential amplitudes varied from 15.4 μV to 46.3 μV (mean 25.4 μV, SD 7.5 and median 24); Cone amplitudes varied from 42 μV to 154.5 μV (mean 92.5 μV, SD 29.6 and median 84.7); Results from b-wave implicit time on cone response varied from 28.5 ms to 33.5 ms (mean 30.8 ms, SD 1.5 and median 30.5); Flicker amplitudes at 30 Hz varied from 23.6 μV to 91.5 μV (mean 43 μV, SD 14.5 and median 41); Implicit time on b-wave at flicker varied from 23 ms to 25.5 ms (mean 24 ms, SD 0.6 and median 24).
Discussion and Conclusions
Nowadays, rabbits are widely used in ophthalmic experimental studies of drugs and surgical methods. The retinal toxicity caused by several drugs and surgical proceedings in this specie could be evaluated by ERG exam. This study standardized retinal responses in healthy rabbits using ERG-jet electrodes according to ISCEV protocol. ERG examination in one or both eyes simultaneously depends of the apparatus and exam indication (Narfström 2006). In this study, both eyes were stimulated at the same time utilizing the Ganzfeld dome with Veris system. Studies showed difficulties on utilizing Ganzfeld dome in veterinary practice; besides the cost and its complicate installation, there is some difficulty in stimulate both eyes simultaneously, and in keeping animal heads inside the sphere (Maehara et al. 2005). Ganzfeld is really expensive, on the other hand, retina is uniformly stimulated and it performs bilateral responses when the whole head of the rabbit is kept inside the dome. A 38 cm of diameter Ganzfeld was utilized in this study and we did not observe any difficulty in keeping, gently, their heads inside the dome. Dark adaptation was either realized based on ISCEV standards taking 30 minutes and was achieved keeping rabbits inside covered travel carriers in a dark room. Dark adaptation procedure had special attention to avoid prolonged time, which would result in higher amplitudes at scotopic phase. The same 30 minutes were utilized by Maehara et al. in 2005. ERG exams were performed in a dark room, with an auxiliary red light to introduce electrodes correctly and track anesthesia. Anesthesia protocol was efficient to realize the exam. Responses vary with the chosen electrode. ERG-jet electrodes were utilized in this study to be easily bought and to be capable to register the responses properly. This electrode generated good quality registers and prevented eyelid closure during exam. Corneal abrasion was not observed after exams; as so, we believe that 2% methylcellulose and anesthetic eye drops were fundamental to avoid such complication. All animals came from the same breeder and were fed with the same food (pellets). The differences observed in the responses between animals were not caused by alimentary variations. Similarly to the responses obtained by Gjorloff et al. (2004) the ERG exams performed in rabbits, b-wave amplitudes varied between animals and the implicit time in 30Hz flicker was found to be a stable parameter either. The current study standardizes ERG examination in healthy rabbits using monopolar ERG-jet electrodes. Authors believe that this protocol will be useful in other studies to detect and/or to follow up retinal alterations in ophthalmic experimental researches.
1. Tzecov R, Arden GB. 1991. The electroretinogram in diabetic retinopathy. Surv Ophthalmol. 44:53-60.
2. Safatle AMV, Salomão S, Berezovsky A, Sacai P, Fantoni D, Yasbek K, Barros PSM. 2005. Retinal degeneration in a Pit Bull dog: electroretinographic findings. Arch Vet Sci.10:119-124.
3. Komaromy AM, Smith PJ, Brooks DE. 1998. Electroretinography in dogs and cats. Part I. Retinal morphology and physiology. Compend Contin Educ. 20:343-354.
4. Berezovsky A, Pereira JM, Salomão SR, Santos VR, Schor P. 2008. Validation of a new fiber electrode prototype for clinical electroretinography. Arq Bras Oftalmol. 71:316-320.
5. Ofri R, Dawson WW, Foli K, Gellat KN. 1993. Chronic ocular hypertension alters local retinal responsiveness. Br J Ophthalmol. 77:502-508.
6. Yanase J, Ogawa H, Ohtsuka H. 1995. Rod and cone components in the dog electroretinogram during dark adaptation. J Vet Med Sci. 57:877-881.
7. Marmor MF, Zrenner E. 1998. Standard for clinical electroretinography (1999 update). Doc Ophthalmol. 97:143-56.
8. Brigell M, Bach M, Barber C, Kawasaki K, Kooijman A. 1998. Guidelines for calibration of stimulus and recording parameters used in clinical electrophysiology of vision. Calibration Standard Committee of the International Society for Clinical Electrophysiology of Vision (ISCEV). Doc Ophthalmol. 95:1-14.
9. Rosolen SG, Rigaudiere F, Lachapelle P. 2002. A practical method to obtain reproducible binocular electroretinograms in dogs. Doc Ophthlamol. 105:93-103.
10. Komaromy AM, Andrew ST, Sapp Jr. HL, Brooks DE, Dawson WW. 2003. Flash electroretinography in standing horses using the DTLtm microfiber electrode. Vet Ophthalmol. 6:27-33.
11. Rubin LF. (1971). Clinical features of hemeralopia in adult Alaskan Malamute. J Vet Med Assoc. 158:1696-1698.
12. Hurn SD, Hardman C, Stanley RG. 2003. Day-blindness in three dogs: clinical and electroretinographic findings. Vet Ophthalmol. 6:127-130.
13. Narfström K. 2006. Electroretinographic testing in larger animals, p.923-933. In: Heckenlively JR, Arden GB. (2. Ed.) Principles and Practice of Clinical Electrophysiology of Vision, Cambridge, The Mit Press.
14. Petersen-Jones S, Tuntivanich N, Montiani-Ferreira F, Khan N. 2006. Electroretinograms of dog and chicken p, 911-921. In: Heckenlively JR, Arden GB. (2nd Ed.) Principles and Practice of Clinical Electrophysiology of Vision, Cambridge, The Mit Press.
15. Sims MH. 1999. Electrodiagnostic evaluation of vision, p.483-507. In: Gelatt KN. (3rd Ed.) Vet Ophthalmol. Philadelphia: Lippincott Williams e Wilkins.
16. Gjörloff K, Andréasson S, Ehinger B. 2004. Standardized full-field electroretinography in rabbits. Doc Ophthal 109:163-168.
17. Maehara S, Itoh N, Wakaiki S, Tsuzuki K, Seno T, Kushiro T, Yamashita K, Izumisawa Y, Kotani T. 2005. Electroretinography using contact lens electrode with built-in light source in dogs. J Vet Med Sci. 67:509-514.