December 2002
Volume 43, Issue 12
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Retina  |   December 2002
Effect of Body Temperature on Electroretinogram of Mice
Author Affiliations
  • Atsushi Mizota
    From the Department of Ophthalmology and Visual Science, Graduate School of Medicine, Chiba University, Japan.
  • Emiko Adachi-Usami
    From the Department of Ophthalmology and Visual Science, Graduate School of Medicine, Chiba University, Japan.
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 3754-3757. doi:
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      Atsushi Mizota, Emiko Adachi-Usami; Effect of Body Temperature on Electroretinogram of Mice. Invest. Ophthalmol. Vis. Sci. 2002;43(12):3754-3757.

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Abstract

purpose. To investigate the ERG alterations induced by changes in body temperature in mice.

methods. Three-week-old BALB/c mice were used. Rectal temperature was measured with a digital thermometer and taken as the body temperature. In experiment 1, the body temperature was kept at 33°C, and the ERGs elicited by a constant stimulus intensity were recorded every 5 minutes. In experiment 2, the body temperature was lowered in five steps from 38°C to 33°C, 28°C, 23°C, and 18°C. At each body temperature, ERGs elicited by different stimulus intensities were recorded.

results. In experiment 1, the mean amplitudes and implicit times of both the a- and b-waves did not change significantly. In experiment 2, the amplitude of both the a- and b-waves decreased significantly with a decrease in body temperature, and the implicit times of the a- and b-waves were prolonged with a decrease in body temperature.

conclusions. Body temperature greatly affects the amplitude and timing of the ERG. Great care must be taken to maintain as normal a body temperature as possible when using the ERG to evaluate the retina, especially in small animals such as mice.

The study of transgenic and selective gene knockout animal models has contributed significantly to medical research, and animal models of human diseases have been used to investigate the roles proteins and enzymes play in normal and abnormal developmental processes. The majority of these animal models are mice, and in ophthalmology, many transgenic and knockout mice have been studied to determine the mechanism of retinal and other ocular diseases. 
Electroretinography (ERG) is one of the most reliable methods for evaluating retinal function in mice, and several reports have reported ERG changes in gene-altered mice. 1 2 3 4 Although ERG recordings in animals and humans are relatively easy to perform, there are many factors that can alter the ERG. The body temperature has been shown to alter the ERG, and in small animals, such as the mouse, the body temperature can be easily influenced by the ambient temperature. 
Because of the increasing use of gene-altered mice, it became important to determine the extent and characteristics of the ERG alterations induced by changes in body temperature. To accomplish this, we recorded ERGs from mice whose body temperature was systematically altered. 
Materials and Methods
Three-week-old BALB/c mice (body weight: 12 g) were used. The animals were kept on a 12-hour light–dark schedule, and the ambient temperature in the animal quarters was 25°C to 30°C. The treatments and procedures performed on the mice were conducted with animals under anesthesia induced by intramuscular injection of ketamine (11 mg/kg), xylazine (14 mg/kg), and urethane (500 mg/kg). All the experiments in this study conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
The rectal temperature of the mouse was measured with a digital thermometer (TD-300; Shibaura Electric Co. Ltd., Tokyo, Japan) and was taken as the body temperature. With the animal under anesthesia, the body temperature was altered by changing the ambient temperature and was continuously monitored. 
For the ERG recording, a cotton wick electrode was placed on the cornea and was referred to an electrode placed subcutaneously on the nasal bone. The stimulus light for eliciting ERGs was obtained from a 150-W quartz halogen light bulb. The light was collected and focused onto a 3-mm-diameter fiber-optic bundle, and the tip of the other end of the bundle was placed 5 mm in front of the cornea of the mouse. The illuminance of the unattenuated stimulus on the surface of cornea was 140,000 lux. Neutral density filters (NDFs) were used to reduce the stimulus intensity. The duration of the stimulus of 20 ms was controlled by an electromagnetic shutter, and the stimulus frequency was 0.2 Hz. The responses were amplified with a preamplifier (MEG-2100; Nihon Kohden, Tokyo, Japan), and eight responses were averaged (DS-6411; Iwatsu, Tokyo, Japan). 
The a-wave amplitude was measured from the baseline to the trough of the a-wave, and b-wave amplitude was measured from the trough of the a-wave to the top of the b-wave. The a-wave implicit time was measured from the beginning of the stimulus to the trough of the a-wave, and the b-wave implicit time was measured from the beginning of the stimulus to the top of the b-wave. 
Experiment 1
For these experiments, the bandwidth of the preamplifier was set at 15 and 300 Hz, and the stimulus intensity was set at 1400 lux (2.0 log NDF). During the 30 minutes of dark-adaptation, the body temperature was changed to 33°C, and, in five mice, it was kept at 33°C. ERGs were recorded every 5 minutes for 90 minutes, and the recordings were stopped when the mice recovered from the anesthesia. 
Experiment 2
During 30 minutes of dark-adaptation, the body temperature was lowered in five steps from 38°C to 33°C, 28°C, 23°C, and 18°C. At each selected body temperature, ERGs were recorded. The body temperature was kept within ±1°C during the ERG recordings. The number of animals tested at each of the five temperatures was five, five, six, eight, and eight, respectively. 
Responses were recorded beginning with a 6.0 log NDF, and the stimulus intensity was increased by 0.5-log steps to the full-intensity stimulus with a 1-minute interval between one stimulus intensity and the next. The bandwidth of the preamplifier was set at 1 and 100 Hz, and the amplitudes of a- and b-waves were measured. From the stimulus intensity-response curves, we determined the maximal amplitude (V max). 
Results
Experiment 1
The ERGs recorded from a mouse in which body temperature was kept at approximately 33°C (range, 32.4–33.3°C) for 55 minutes are shown in Figure 1 . In this experiment, one mouse began to recover at approximately 60 minutes and another mouse at 75 minutes, and thus the number of animals was five recovering before 60 minutes, four between 65 and 75 minutes, and three after 80 minutes. The amplitudes and implicit times of both the a- and b-waves did not change significantly when the mouse was kept at a constant body temperature of 33°C (Fig. 1 ,Fig. 2)
Experiment 2
The ERGs recorded from a mouse at body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C are shown in Figure 3 . The increase in the mean amplitudes of the a- and b-waves with increase in stimulus intensity is shown in Figure 4 . The amplitude of the b-wave increased with increase in stimulus intensity until the stimulus intensity increased to 2.0 log NDF; then it began to decrease at all body temperatures (Fig. 3 ,Fig. 4b) . The amplitude of the a-wave could be measured at body temperatures higher than 28°C (Fig. 3) . The a-wave first appeared at a stimulus intensity of 4.0 log NDF, increased with increase in stimulus intensity until 2.0 log NDF, and then began to decrease (Fig. 3 ,Fig 4a)
The mean V max of the a-wave decreased with the decrease in body temperature (Fig. 5a) , and the difference between V max at 38°C and 33°C was statistically significant (P < 0.05). The V max of the b-wave also decreased with the decrease in body temperature (Fig. 5b) , but the difference between V max at 38°C and 33°C was not statistically significant. However, the differences between V max at 38°C and 28°C and lower were statistically significant (P < 0.005). 
The implicit times of the a- and b-waves increased with a decrease in body temperature (Fig. 3) . Retinal sensitivity improved with the increase in body temperature (Fig. 4)
Discussion
Many external factors, including body temperature, have been shown to alter the ERG. Hypothermia decreases general body metabolism, and this decreased metabolism leads to a general reduction in the rate of chemical reactions. The body temperature of small animals, such as the mouse, can be easily affected by the ambient temperature, and in our preliminary experiment, the body temperature of anesthetized mice kept at a room temperature of 25°C was decreased from approximately 38°C to less than 30°C within 30 minutes (the period of dark adaptation). 
Anesthesia also affects metabolism, and its influence on the ERG depends on the level of anesthesia. In our experiment 1, we continued to record ERGs until the mice were alert or began to move, and no significant changes were found in the amplitudes and implicit times of the a- and b-waves when the body temperature was maintained at 33°C. Therefore, we conclude that the depth of anesthesia used in these experiments did not affect the a- and b-waves. 
In experiment 2, the amplitude of both a- and b-waves decreased with higher stimulus intensities. We believe that this decrease of amplitude was caused by the light-adaptation with a stimulus frequency of 0.2 Hz. 5  
With a decrease in body temperature, the respiration and heart rates are reduced, and retinal hypoxemia can occur. Derwent and Linsenmeier 6 reported that the a-wave of the ERG in the cat retina is more resistant to severe hypoxemia than the b-wave, but in our mice, the amplitudes of both the a- and b-waves decreased concomitantly. Thus, we suggest that the changes in the ERG were not due to hypoxemia but were caused mainly by the decrease in body temperature. 
Armington and Adolph 7 reported that the amplitude of the a- and b-waves of the ERG of the isolated carp retina decrease with a decrease in retinal temperature but the oscillatory potentials persist. In eyecup preparations of turtles, Adolph 8 reported that the b-wave amplitude was strongly temperature dependent, but the amplitudes of the a-wave and slow PIII were less sensitive. He also found that the time course of all components of the ERG slowed markedly as the temperature decreased. In the all-cone retina of the iguana, Meneghini and Hamasaki 9 showed that the amplitudes of the a- and b-waves decreased as the body temperature decreased. This indicates that the cones are also sensitive to body temperature. 
Tazawa and Seaman 10 reported a slight decrease in the amplitude and prolongation of the implicit times of the a- and b-waves of isolated, perfused bovine eyes. Niemeyer 11 reported that the amplitude of the b-wave increases, whereas its latency and peak time decreases with increasing perfusate temperature in the range of 26°C to 39°C in perfused cat eyes. Horiguchi and Miyake 12 reported that cooling the vitreous cavity during closed vitrectomy in humans results in a markedly increased peak time and reduced amplitude with a 30-Hz flicker stimulus. The results in the isolated eyes of mammals and humans are similar to those in our in vivo experiments. 
Wolin et al. 13 reported a significant increase in the latency and a decrease in the amplitude of visual evoked potentials in cats and guinea pigs as the body temperature decreases. They concluded that the changes in the latency are due to the effect of cold on the photochemical and neurochemical reactions and that the reduced amplitudes may be a function of fewer receptors and/or nerve fibers of different calibers and myelination being excited at any given time. 
Lachapelle et al. 14 cooled the retinas of rabbits by wrapping plastic tubing around the eye and reported that the amplitude of the a-wave was reduced to 66.9% and the b-wave to 90.9% at a retinal temperature of approximately 18°C. The oscillatory potentials were most severely affected. The decrease in amplitude was more severe in our mice, which may be partly due to the general condition of the animals and partly to the difference in the blood supply between rabbits and mice. 
In summary, body temperature greatly affected the amplitude and timing of the ERG. These findings were made in normal mice, and whether a similar pattern is to be found in transgenic or gene-knockout mice must be determined. In any case, great care must be taken to maintain as normal a body temperature as possible when using the ERG to evaluated the retina, especially in small animals such as mice. 
 
Figure 1.
 
ERGs of a mouse with body temperature kept at approximately 33°C.
Figure 1.
 
ERGs of a mouse with body temperature kept at approximately 33°C.
Figure 2.
 
Mean (±SEM) amplitudes of the a- and b-waves of mice (n = 5) at a constant body temperature of approximately 33°C.
Figure 2.
 
Mean (±SEM) amplitudes of the a- and b-waves of mice (n = 5) at a constant body temperature of approximately 33°C.
Figure 3.
 
ERGs recorded from a mouse at body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C.
Figure 3.
 
ERGs recorded from a mouse at body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C.
Figure 4.
 
The relationship between the mean amplitude of the (a) a- and (b) b-waves and stimulus intensity at body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C (n = 8, 8, 6, 5, and 5, respectively).
Figure 4.
 
The relationship between the mean amplitude of the (a) a- and (b) b-waves and stimulus intensity at body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C (n = 8, 8, 6, 5, and 5, respectively).
Figure 5.
 
The relationship between body temperature and the mean (±SEM) V max of the (a) a- and (b) b-waves as a function of body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C (n = 8, 8, 6, 5, and 5, respectively).
Figure 5.
 
The relationship between body temperature and the mean (±SEM) V max of the (a) a- and (b) b-waves as a function of body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C (n = 8, 8, 6, 5, and 5, respectively).
The authors thank Duco I. Hamasaki (Bascom Palmer Eye Institute, Miami, Florida) for assistance and advice. 
Kobayashi A, Higashide T, Hamasaki D, et al. HRG4 (UNC119) mutation found in cone–rod dystrophy caused retinal degeneration in a transgenic model. Invest Ophthalmol Vis Sci. 2000;41:3268–3277. [PubMed]
Ripps H, Peachey NS, Xu X, et al. The rhodopsin cycle is preserved in IRBP “knockout” mice despite abnormalities in retinal structure and function. Vis Neurosci. 2000;40:97–105.
Salchow DJ, Gouras P, Doi K, et al. A point mutation (W70A) in the rod PDG-gamma gene desensitizing and delaying murine rod photoreceptors. Invest Ophthalmol Vis Sci. 1999;40:3262–3267. [PubMed]
Kameya S, Araki E, Katsuki M, et al. Dp260 disrupted mice revealed prolonged implicit time of b-wave in ERG and loss of accumulation of beta-dystroglycan in the outer plexiform layer of the retina. Hum Mol Genet. 1997;6:2195–2203. [CrossRef] [PubMed]
Kueng-Hitz N, Rol P, Niemeyer G. The electroretinogram (ERG) of the mouse: normal values, optimal stimulation and recording (in German). Klin Monatsbl Augenheilk. 1999;214:288–290. [CrossRef]
Derwent JK, Linsenmeier RA. Effects of hypoxemia on the a- and b-wave of the electroretinogram in cat. Invest Ophthalmol Vis Sci. 2000;41:3634–3642. [PubMed]
Armington JC, Adolph AR. Temperature effects on the electroretinogram of the isolated carp retina. Acta Ophthalmol. 1983;62:498–509.
Adolph AR. Temporal transfer and nonlinearity properties of turtle ERG: tuning by temperature, pharmacology, and light intensity. Vision Res. 1985;25:483–492. [CrossRef] [PubMed]
Meneghini KA, Hamasaki DI. The electroretinogram of the iguana and tokay gecko. Vision Res. 1967;7:243–251. [CrossRef] [PubMed]
Tazawa Y, Seaman AJ. The electroretinogram of the living extracorporeal bovine eye: the influence of anoxia and hypothermia. Invest Ophthalmol Vis Sci. 1972;11:691–698.
Niemeyer G. The function of the retina in the perfused eye. Doc Ophthalmol. 1975;39:53–116. [CrossRef] [PubMed]
Horiguchi M, Miyake Y. Effect of temperature on electroretinograph readings during closed vitrectomy in humans. Arch Ophthalmol. 1991;109:1127–1129. [CrossRef] [PubMed]
Wolin LR, Massopust LC, Jr, Meder J. Electroretinogram and cortical evoked potentials under hypothermia. Arch Ophthalmol. 1964;72:521–524. [CrossRef] [PubMed]
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Figure 1.
 
ERGs of a mouse with body temperature kept at approximately 33°C.
Figure 1.
 
ERGs of a mouse with body temperature kept at approximately 33°C.
Figure 2.
 
Mean (±SEM) amplitudes of the a- and b-waves of mice (n = 5) at a constant body temperature of approximately 33°C.
Figure 2.
 
Mean (±SEM) amplitudes of the a- and b-waves of mice (n = 5) at a constant body temperature of approximately 33°C.
Figure 3.
 
ERGs recorded from a mouse at body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C.
Figure 3.
 
ERGs recorded from a mouse at body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C.
Figure 4.
 
The relationship between the mean amplitude of the (a) a- and (b) b-waves and stimulus intensity at body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C (n = 8, 8, 6, 5, and 5, respectively).
Figure 4.
 
The relationship between the mean amplitude of the (a) a- and (b) b-waves and stimulus intensity at body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C (n = 8, 8, 6, 5, and 5, respectively).
Figure 5.
 
The relationship between body temperature and the mean (±SEM) V max of the (a) a- and (b) b-waves as a function of body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C (n = 8, 8, 6, 5, and 5, respectively).
Figure 5.
 
The relationship between body temperature and the mean (±SEM) V max of the (a) a- and (b) b-waves as a function of body temperatures of 18°C, 23°C, 28°C, 33°C, and 38°C (n = 8, 8, 6, 5, and 5, respectively).
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