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Erg Dark Adaptation in the Unanesthetized Rabbit
ERG D A R K A D A P T A T I O N IN T H E U N A N E S T H E T I Z E D R A B B I T G.
W.
CIBIS, M.D,
A N D H.
M.
BURIAN,
M.D
Iowa City, Iowa
The amplitude of the rabbit scotopic electroretinogram ( E R G ) is notoriously labile both in day-to-day variations in the same animal as well as in the wide range seen in normal controls. This seriously impairs the rabbit's usefulness as a model in long-term experiments. Part of the reason for this difficulty appears to lie in the traditional methods of obtaining and assessing adaptation preparatory to performing the ERG—specifically, in the amount of light pre-adaptation and the subsequent length of dark adaptation which the animal undergoes. Full dark adaptation is thought to have been reached when b-wave amplitude fails to increase materially with further time in the dark. It is the lack of firm knowledge regarding the length of time necessary for dark adaptation in the rabbit that prompted this investigation. The degree of exposure to light prior to dark adaptation has been shown to have a significant effect on the length of time necessary to reach full dark adaptation. ' Elenius measured the ERG amplitude in anesthetized rabbits after four hours in the dark without light pre-adaptation, (8000 Lux for 30, 10, three, and one minutes), and followed the subsequent increase in ERG amplitude until the value of 100%, total dark adaptation for that animal, was reached. He found that at one minute and three minutes of light pre-adaptation the animal took 90 to 100 minutes to return to the 100% level. With 30 minutes of light pre-adaptation this 1
2 3
3
From the Department of Ophthalmology and the Neurosensory Center, Paper 199, College of Medicine, University of Iowa, Iowa City. The Neurosensory Center is supported by Program-Project Grant NS03354 of the NINDS of the Department of Health, Education and Welfare, and this study was supported by Postdoctoral Research Fellowship 1F02-EY44718-01 from the National Eye Institute (Dr. Cibis). Reprint requests to Hermann M. Burian, M . D , Department of Ophthalmology, University of Iowa, Iowa City, Iowa 52240.
was stretched out to three to four hours, and with 10 minutes, to three hours. In long-term ERG studies it is of value not to have to use systemic anesthesia. However, the amount of light actually entering the eye of an unanesthetized animal during light pre-adaptation is difficult to control. Therefore, we wished to determine the actual dark adaptation time of nonanesthetized, non-light pre-adapted rabbits. EXPERIMENTAL
CONDITIONS
Animals used were six California females weighing between 4 and 5 kg. Each was placed in a restraining harness inside a copper-shielded room and silver grounding electrodes were clipped to each ear. A mechanical shutter was used, the motor of which came from a Grass model III-D encephalograph pen writer. This was activated by a Grass Model S-4 functional stimulator to provide an approximately square light stimulus 100 msec in duration twice per second. The shutter was located between the light source and one end of a %-inch random fiber optic which ran into the shielded room. The other end of the fiber optic was fitted flush into a %-inch hole in the center of a 12-mm corneal contact lens. With maximal pupillary dilatation, using 1% tropicamide (Mydriacyl) and the fiber optic-contact lens fitted directly onto the cornea, an artificial pupil 5.5 mm in diameter was created. The amount of stimulus light as measured at the corneal surface was about 80 foot lamberts. 4
The corneal contact electrode consisted of a 1.5-mm band of platinum fitted flush with the inner surface of the contact lens running around the artificial pupil. The indifferent electrode, also of platinum, was fitted onto the outside of the contact lens providing contact with the rabbit's palpebral conjunctiva. A separate pinhole in the lens was connected to tubing to provide low suction on the prin-
1113
AMERICAN JOURNAL OF OPHTHALMOLOGY
1114
240 220
A
/
am,
*_*
"
V
MAY,
1971
~
200 180
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: H'
....^:r-•r.r•«H^ ^• ' ,
r
, ï ,
^160 140 120 100 fe
80 60
_4...*-
i i —i i i i i
40 20 0
i • i , i • i • i 20
40
60
• i • i • i • i • i 80
100
120
140
TIME IN MINUTES Fig. 1 (Cibis and Burian). Plot of b-wave amplitude time in the dark for each of six rabbits. The experiments were done without light pré-adaptation or systemic anesthesia and show that there is a steady rise in b-wave amplitude which reaches a plateau of its dark adaptation curve. Each point on a curve represents an average of 20 consecutive ERGs.
ciple of the Henke's suction lens. The fiber optic was supported so as to avoid any drag or strain on the eye. Proparacaine H C l 0.5% (Ophthetic), was used as topical anesthesia. Insertion of the lens and all other preparations were done in normal room light. Lights were then turned out in the shielded room and stimulation and recording begun. Recording equipment consisted of an A C pre-amplifier Grass model P5-B. Voltage gain used was 28,000 x input. The pre-amplifier was connected to a Tectronix 565 oscilloscope for visual monitoring of the ERG and to a CAT biological digital computer of 5
average transients. The computer averaged 20 consecutive ERGs, writeout being on an X - Y plotter. The entire system was standardized prior to each test. RESULTS
Figure 1 shows the results of each of the six tests and depicts the height of the b-wave versus time in the dark for each animal. Of course, the maximum height of the b-wave is different for each animal. However, the time after the beginning of dark adaptation at which each animal's b-wave amplitude reaches a plateau is approximately the same. This occurs at 70 to 90 minutes. By two
VOL. 71, NO. S
ERG D A R K A D A P T A T I O N
IIIS
hours, it is obvious that each animal is well onto the plateau of its dark adaptation curve. All figures were obtained without the use of light pre-adaptation or systemic anesthesia. The latter, accounting for the lack of a perfectly smooth curve in all cases. Each point on a curve represents an average of 20 consecutive ERGs.
eyes of different animals because of eye movements, pupillary diameter and the like. Therefore, even if a dark adaptation curve were worked out for any given level of light pre-adaptation, there is no certain way of knowing whether the plateau has been reached. Since this is especially true for unanesthetized animals, as already pointed out, it would appear that for conditions such as DISCUSSION used in our experiments the most reliable Whenever experiments are contemplated procedure would be to dark adapt the animal in which the amplitude of the ERG compo- for two hours without any light pre-adaptanent waves are used as a criterion, it is es- tion and then run the ERG. We have done sential that a steady state be achieved in the this, and as expected, found that there was experimental animal to allow comparative no further significant rise in E R G amplifigures. One must therefore avoid taking tudes. ERGs during the period of dark adaptation SUMMARY when the amplitudes are still rising. This is generally attempted by standardized light Dark adaptation was tested by E R G ampre-adaptation so as to bring all eyes to the plitude in six unanesthetized rabbits who had same initial level of sensitivity. ' However, received no light pre-adaptation. It was in unanesthetized animals the exact amount found that the electrophysiologic dark adapof adapting light entering the eye is actually tation curve reached a plateau after approxidifficult to control because of eye movements mately 90 minutes. It is recommended that if and the like. It would appear, therefore, that E R G tests are run in unanesthetized rabbits, it might be preferable to employ a technique these be done without light pre-adaptation and in which it is guaranteed that the eye has taken after a two-hour dark adaptation pereached an approximate steady state with re- riod. gard to dark adaptation. Our results show that for rabbits taken out of room light this REFERENCES occurs at 70 to 90 minutes. 1. Spivey, B. E , and Pearlman, J. T . : Day-to6 7
It is of interest to note that our curves on day variations in the ERG of humans and rabbits. Am. J. Ophth. 55:1013,1963. rabbits taken directly from room light com2. Auerbach, E , and Burian, H. M. : Studies on pare very closely to those obtained by Elen- the photopic-scoptic relationships in human electroius on rabbits which had had one and three retinograms. Am. J. Ophth. 40:59,1955. 3. Elenius, V. : Recovery in the dark of the rabminutes of light pre-adaptation at 8000 Lux. bit's ERG. Acta Physiol. Scandinav. 44(Suppl.) : Nonetheless, we realize of course that the 150, 1958. 4. Pearlman, J. T.: Computer averaging techplateau of the dark adaptation curve is nique for routine ERG studies: A normative series reached at different times from the begin- in rabbits. In Burian, H. M , and Jacobson, J. H. ning of dark adaptation in accordance with (eds.) : Clinical Electroretinography. Oxford, Pergamon Press, 1964, p. 93. the history of the eye prior to the onset of 5. Henkes, H. E , and von Balen, A. : Techniques dark adaptation. It is, therefore, clearly in- of recording of the hitherto unrecordable ERG in adequate to proceed without light pre-adap- the human eye. Acta Fac. Med. Univ. Brunensis 4: tation and expect the ERGs obtained after 21,1955. 6. Müller-Limmroth, W. : Elektrophysiologie des five or 10 minutes to be comparable in differ- Gesichtssinns. Berlin, Springer-Verlag, 1959, p. 84. ent experimental subjects or in the same 7. Ronchi, L , and Ercoles, A. M. : On the variability of the course of electroretinographic response subject on successive examinations. 3
On the other hand, light pre-adaptation does not guarantee an equalization of the
during incoming adaptation to light Pubblicazioni del Istituto Nazionale Di Ottica, Serie II : N.
1131.
W.
CIBIS, M.D,
A N D H.
M.
BURIAN,
M.D
Iowa City, Iowa
The amplitude of the rabbit scotopic electroretinogram ( E R G ) is notoriously labile both in day-to-day variations in the same animal as well as in the wide range seen in normal controls. This seriously impairs the rabbit's usefulness as a model in long-term experiments. Part of the reason for this difficulty appears to lie in the traditional methods of obtaining and assessing adaptation preparatory to performing the ERG—specifically, in the amount of light pre-adaptation and the subsequent length of dark adaptation which the animal undergoes. Full dark adaptation is thought to have been reached when b-wave amplitude fails to increase materially with further time in the dark. It is the lack of firm knowledge regarding the length of time necessary for dark adaptation in the rabbit that prompted this investigation. The degree of exposure to light prior to dark adaptation has been shown to have a significant effect on the length of time necessary to reach full dark adaptation. ' Elenius measured the ERG amplitude in anesthetized rabbits after four hours in the dark without light pre-adaptation, (8000 Lux for 30, 10, three, and one minutes), and followed the subsequent increase in ERG amplitude until the value of 100%, total dark adaptation for that animal, was reached. He found that at one minute and three minutes of light pre-adaptation the animal took 90 to 100 minutes to return to the 100% level. With 30 minutes of light pre-adaptation this 1
2 3
3
From the Department of Ophthalmology and the Neurosensory Center, Paper 199, College of Medicine, University of Iowa, Iowa City. The Neurosensory Center is supported by Program-Project Grant NS03354 of the NINDS of the Department of Health, Education and Welfare, and this study was supported by Postdoctoral Research Fellowship 1F02-EY44718-01 from the National Eye Institute (Dr. Cibis). Reprint requests to Hermann M. Burian, M . D , Department of Ophthalmology, University of Iowa, Iowa City, Iowa 52240.
was stretched out to three to four hours, and with 10 minutes, to three hours. In long-term ERG studies it is of value not to have to use systemic anesthesia. However, the amount of light actually entering the eye of an unanesthetized animal during light pre-adaptation is difficult to control. Therefore, we wished to determine the actual dark adaptation time of nonanesthetized, non-light pre-adapted rabbits. EXPERIMENTAL
CONDITIONS
Animals used were six California females weighing between 4 and 5 kg. Each was placed in a restraining harness inside a copper-shielded room and silver grounding electrodes were clipped to each ear. A mechanical shutter was used, the motor of which came from a Grass model III-D encephalograph pen writer. This was activated by a Grass Model S-4 functional stimulator to provide an approximately square light stimulus 100 msec in duration twice per second. The shutter was located between the light source and one end of a %-inch random fiber optic which ran into the shielded room. The other end of the fiber optic was fitted flush into a %-inch hole in the center of a 12-mm corneal contact lens. With maximal pupillary dilatation, using 1% tropicamide (Mydriacyl) and the fiber optic-contact lens fitted directly onto the cornea, an artificial pupil 5.5 mm in diameter was created. The amount of stimulus light as measured at the corneal surface was about 80 foot lamberts. 4
The corneal contact electrode consisted of a 1.5-mm band of platinum fitted flush with the inner surface of the contact lens running around the artificial pupil. The indifferent electrode, also of platinum, was fitted onto the outside of the contact lens providing contact with the rabbit's palpebral conjunctiva. A separate pinhole in the lens was connected to tubing to provide low suction on the prin-
1113
AMERICAN JOURNAL OF OPHTHALMOLOGY
1114
240 220
A
/
am,
*_*
"
V
MAY,
1971
~
200 180
/ "
: H'
....^:r-•r.r•«H^ ^• ' ,
r
, ï ,
^160 140 120 100 fe
80 60
_4...*-
i i —i i i i i
40 20 0
i • i , i • i • i 20
40
60
• i • i • i • i • i 80
100
120
140
TIME IN MINUTES Fig. 1 (Cibis and Burian). Plot of b-wave amplitude time in the dark for each of six rabbits. The experiments were done without light pré-adaptation or systemic anesthesia and show that there is a steady rise in b-wave amplitude which reaches a plateau of its dark adaptation curve. Each point on a curve represents an average of 20 consecutive ERGs.
ciple of the Henke's suction lens. The fiber optic was supported so as to avoid any drag or strain on the eye. Proparacaine H C l 0.5% (Ophthetic), was used as topical anesthesia. Insertion of the lens and all other preparations were done in normal room light. Lights were then turned out in the shielded room and stimulation and recording begun. Recording equipment consisted of an A C pre-amplifier Grass model P5-B. Voltage gain used was 28,000 x input. The pre-amplifier was connected to a Tectronix 565 oscilloscope for visual monitoring of the ERG and to a CAT biological digital computer of 5
average transients. The computer averaged 20 consecutive ERGs, writeout being on an X - Y plotter. The entire system was standardized prior to each test. RESULTS
Figure 1 shows the results of each of the six tests and depicts the height of the b-wave versus time in the dark for each animal. Of course, the maximum height of the b-wave is different for each animal. However, the time after the beginning of dark adaptation at which each animal's b-wave amplitude reaches a plateau is approximately the same. This occurs at 70 to 90 minutes. By two
VOL. 71, NO. S
ERG D A R K A D A P T A T I O N
IIIS
hours, it is obvious that each animal is well onto the plateau of its dark adaptation curve. All figures were obtained without the use of light pre-adaptation or systemic anesthesia. The latter, accounting for the lack of a perfectly smooth curve in all cases. Each point on a curve represents an average of 20 consecutive ERGs.
eyes of different animals because of eye movements, pupillary diameter and the like. Therefore, even if a dark adaptation curve were worked out for any given level of light pre-adaptation, there is no certain way of knowing whether the plateau has been reached. Since this is especially true for unanesthetized animals, as already pointed out, it would appear that for conditions such as DISCUSSION used in our experiments the most reliable Whenever experiments are contemplated procedure would be to dark adapt the animal in which the amplitude of the ERG compo- for two hours without any light pre-adaptanent waves are used as a criterion, it is es- tion and then run the ERG. We have done sential that a steady state be achieved in the this, and as expected, found that there was experimental animal to allow comparative no further significant rise in E R G amplifigures. One must therefore avoid taking tudes. ERGs during the period of dark adaptation SUMMARY when the amplitudes are still rising. This is generally attempted by standardized light Dark adaptation was tested by E R G ampre-adaptation so as to bring all eyes to the plitude in six unanesthetized rabbits who had same initial level of sensitivity. ' However, received no light pre-adaptation. It was in unanesthetized animals the exact amount found that the electrophysiologic dark adapof adapting light entering the eye is actually tation curve reached a plateau after approxidifficult to control because of eye movements mately 90 minutes. It is recommended that if and the like. It would appear, therefore, that E R G tests are run in unanesthetized rabbits, it might be preferable to employ a technique these be done without light pre-adaptation and in which it is guaranteed that the eye has taken after a two-hour dark adaptation pereached an approximate steady state with re- riod. gard to dark adaptation. Our results show that for rabbits taken out of room light this REFERENCES occurs at 70 to 90 minutes. 1. Spivey, B. E , and Pearlman, J. T . : Day-to6 7
It is of interest to note that our curves on day variations in the ERG of humans and rabbits. Am. J. Ophth. 55:1013,1963. rabbits taken directly from room light com2. Auerbach, E , and Burian, H. M. : Studies on pare very closely to those obtained by Elen- the photopic-scoptic relationships in human electroius on rabbits which had had one and three retinograms. Am. J. Ophth. 40:59,1955. 3. Elenius, V. : Recovery in the dark of the rabminutes of light pre-adaptation at 8000 Lux. bit's ERG. Acta Physiol. Scandinav. 44(Suppl.) : Nonetheless, we realize of course that the 150, 1958. 4. Pearlman, J. T.: Computer averaging techplateau of the dark adaptation curve is nique for routine ERG studies: A normative series reached at different times from the begin- in rabbits. In Burian, H. M , and Jacobson, J. H. ning of dark adaptation in accordance with (eds.) : Clinical Electroretinography. Oxford, Pergamon Press, 1964, p. 93. the history of the eye prior to the onset of 5. Henkes, H. E , and von Balen, A. : Techniques dark adaptation. It is, therefore, clearly in- of recording of the hitherto unrecordable ERG in adequate to proceed without light pre-adap- the human eye. Acta Fac. Med. Univ. Brunensis 4: tation and expect the ERGs obtained after 21,1955. 6. Müller-Limmroth, W. : Elektrophysiologie des five or 10 minutes to be comparable in differ- Gesichtssinns. Berlin, Springer-Verlag, 1959, p. 84. ent experimental subjects or in the same 7. Ronchi, L , and Ercoles, A. M. : On the variability of the course of electroretinographic response subject on successive examinations. 3
On the other hand, light pre-adaptation does not guarantee an equalization of the
during incoming adaptation to light Pubblicazioni del Istituto Nazionale Di Ottica, Serie II : N.
1131.