Visual Neurophysiology Normalization of Visual Evoked Potentials Using Underlying Electroencephalogram Levels Improves Amplitude Reproducibility in Rats Yuyi You,1 Johnson Thie,1 Alexander Klistorner,1,2 Vivek K. Gupta,1 and Stuart L. Graham1,2 PURPOSE. The visual evoked potential (VEP) is a frequently used encephalogram (EEG)-based signals that form the family of noninvasive measurement of visual function. However, high- cortical evoked potentials (EPs).2 The ongoing cortical activity, amplitude variability has limited its potential for evaluating which has a major influence on sensory processing, should not axonal damage in both laboratory and clinical research. This be regarded as merely random background noise.3–5 In a neu- study was conducted to improve the reliability of VEP ampli- rophysiologically based mathematical model, Jansen et al.6 tude measurement in rats by using electroencephalogram demonstrated that the spontaneous EEG and the flash VEP are (EEG)-based signal correction. generated by some of the same neural structures. METHODS. VEPs of Sprague-Dawley rats were recorded on three It was suggested that the amplitude of the VEPs reflects the separate days within 2 weeks. The original VEP traces were number of functional optic nerve fibers. Our previous work re- normalized by EEG power spectrum, which was evaluated by vealed a positive correlation between retinal nerve fiber layer 7,8 Fourier transform. A comparison of intersession reproducibil- thickness and VEP amplitude in optic neuritis patients. We ity and intersubject variability was made between the original recently also demonstrated a strong linear relationship between and corrected signals. the axonal loss and amplitude decrease in experimental optic nerve demyelination.9 However, the relationship between axonal RESULTS. Corrected VEPs showed lower amplitude intersession damage and amplitude was weaker than that between demyeli- within-subject SD (Sw), coefficient of variation (CoV), and nation and latency delay, which may be due to the higher vari- repeatability (R ) than the original signals (P Ͻ 0.001). The 95 ability of the VEP amplitude.9 The VEP has been extensively used intraclass correlation coefficient (ICC) of the corrected traces in pharmacologic experiments and in assessing the neuroprotec- (0.90) was also better than the original potentials (0.82). For tive effects of compounds, as it provides a means of monitoring intersubject variability, the EEG-based normalization improved neural activity and sensory processing in vivo.10 The reproduc- the CoV from 44.64% to 30.26%. A linear correlation was ibility of VEP amplitude is critical if the parameter is used to observed between the EEG level and the VEP amplitude (r ϭ evaluate nerve functional change or neuronal apoptosis over time 0.71, P Ͻ 0.0001). in some optic nerve disease models.11 CONCLUSIONS. Underlying EEG signals should be considered in Rats are the most common species used in laboratory VEP measuring the VEP amplitude. In this study, a useful technique studies because of their accessibility and short growth cycle.12 was developed for VEP data processing that could also be used We have recently improved the reproducibility of VEP mea- for other cortical evoked potential recordings and for clinical surements in rats by using a modified electrode configuration, VEP interpretation in humans. (Invest Ophthalmol Vis Sci. a novel mini-Ganzfeld visual stimulator, and early-peak analy- 2012;53:1473–1478) DOI:10.1167/iovs.11-8797 sis.13 However, the variability of the amplitude was still rela- tively high, which may limit the use of the VEP in assessing he visual evoked potential (VEP) is a measurement in visual axonal damage. The variability of VEP amplitude has been Telectrophysiology that is frequently used as an objective attributed to many factors in both animals and humans, such as means of evaluating the function of the visual pathway. It is cortical anatomy,14 the conductivity of the electrodes and understood to be generated at the level of the striate cortex by underlying tissues,13 strain and age,15 stimulus design,13,16 and 1 combined activity of postsynaptic potentials. The VEP is an general level of cortical activity.17–19 Some factors (e.g., elec- analogous phenomenon of auditory evoked potential (AEP) trode configuration and stimulus intensity) are easy to rule out and somatosensory evoked potential (SEP), and all are electro- by using a standardized recording protocol. However, the ongoing cortical activity is very difficult to control during different recording sessions. As mentioned, the VEP is an EEG- 1 based signal, and we have reported the effect of EEG on From the Department of Ophthalmology, Australian School of 20 Advanced Medicine, Macquarie University, Sydney, New South Wales, multifocal VEP in humans. Australia; and the 2Save Sight Institute, Sydney University, Sydney, New In this study, we scaled the original VEP traces using the South Wales, Australia. ongoing EEG power spectrum level to minimize the effect of Supported by the Ophthalmic Research Institute of Australia brain activity and to improve the reliability of VEP amplitude (ORIA) and the National Multiple Sclerosis Society (NMSS). AK was measurement, as just described. We repeatedly performed the supported by the Sydney Foundation for Medical Research. VEP recording in rats by using a well-established protocol9,13 Submitted for publication October 14, 2011; accepted January 23, and analyzed the intersession reproducibility and intersubject 2012. variability of the original and corrected VEPs, respectively. The Disclosure: Y. You, None; J. Thie, None; A. Klistorner, None; V.K. Gupta, None; S.L. Graham, None purpose of this study was to explore a reliable data-processing Corresponding author: Yuyi You, Australian School of Advanced technique to improve VEP amplitude reproducibility for labo- Medicine, Macquarie University, F10A, Level 1, 2 Technology Pl, North ratory research in animal models. The technique could also be Ryde, Sydney, NSW 2109, Australia; [email protected]. considered for clinical VEP interpretation in humans. Investigative Ophthalmology & Visual Science, March 2012, Vol. 53, No. 3 Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc. 1473 Downloaded from tvst.arvojournals.org on 09/24/2021 1474 You et al. IOVS, March 2012, Vol. 53, No. 3 METHODS reference screw were connected to the amplifier using hook clips. A needle electrode (F-E3M-72; Grass, Warwick, RI) was inserted into the Animals tail as the ground. The electrode impedance was measured (F-EZM5 Impedance Meter; Grass) and maintained below 5 k⍀. Visual stimuli Twenty-five male Sprague-Dawley rats with a body weight of 300 to were generated by a mini-Ganzfeld stimulator (3 cd ⅐ s/m2), and the 350 g (10–12 weeks; Animal Research Centre, Perth, Western Austra- photic stimulation was delivered 100 times at a frequency of 1 Hz. The lia, Australia) were used. All animals were maintained in an air-condi- time of measurement was 200 ms, and the sampling rate was 5000 tioned room with controlled temperature (21 Ϯ 2 °C) and fixed daily samples/s. Responses were amplified 20,000 times and filtered by 12-hour light/dark cycles. All procedures involving animals were con- band-pass filter, with low and high cutoff frequencies of 1 and 100 Hz, ducted in accordance with the Australian Code of Practice for the Care respectively (BMA-400 Bioamplifier, CWE, Inc., Ardmore, PA). After and Use of Animals for Scientific Purposes and the guidelines of the the recording, the wound was closed and antibiotics administered. ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animals were anesthetized with an intraperitoneal in- EEG-Based Correction jection of ketamine (75 mg/kg) and medetomidine (0.5 mg/kg) for both surgery and electrophysiology recording. Fourier transform was used to quantify the amplitude of the underlying EEG during each recording session. The Fourier coefficients of the EEG VEP Recording sequence, x[n], were evaluated on the entire 100-second sequence. Let X[m] denote the Fourier coefficient, defined as Stainless steel skull screws (Micro Fasteners, Melbourne, Australia) were implanted through the skull into the visual cortex (7 mm behind the NϪ1 bregma and 3 mm lateral of the midline) as the positive electrodes. A 1 X͓m͔ ϭ ͸x͓n͔eϪ͓͑2␲imn͒/M͔, m ϭ 0,...,M Ϫ 1. reference screw electrode was placed on the midline, 3 mm rostral to the N bregma. Dental cement (Rapid Repair; DeguDent GmbH, Hanau, Ger- nϭ0 many) was used to fix the screws. The skin of the head was closed, and at least 1 week was allowed for the animals to recover from the surgery. The Fourier transform was performed by MatLab Function FFT with 13 VEP recording was performed on the same eye (randomly selected) M ϭ 2 (The MathWorks, Inc., Natick MA). The choice of M allows the on three separate days within 2 weeks. The animals were anesthetized, use of the Fast Fourier transform algorithm and ensures that the placed in a dark room, and allowed to adapt to darkness for 5 minutes frequency interval is at most 1 Hz/point. (The sampling frequency was (we have recently confirmed the adequacy of such a short adaptation 5000 samples per second.) time using our methodology).9,13 The body temperature was main- The amplitude of EEG was evaluated according to 30-Hz band tained at 37°C by a homoeothermic blanket system with a rectal power thermometer probe (Harvard Apparatus; Holliston, MA). The pupils b were dilated with 1.0% tropicamide eyedrops (Alcon Laboratories, Fort 1 Worth, TX). The skin over the skull was opened. The positive screw EEG ϭ ͸X2͓m͔ b Ϫ a ϩ 1 over the contralateral visual cortex of the stimulated eye and the mϭa FIGURE 1. (A) Raw EEG sequence (100 seconds). (B) Corresponding EEG power spectrum (Fast Fourier transform, 1–30 Hz). Downloaded from tvst.arvojournals.org on 09/24/2021 IOVS, March 2012, Vol.