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1 From the Departments of Biomedical Engineering, and 2 Neurobiology and Physiology, and the 3 Institute for Neuroscience, Northwestern University, Evanston, Illinois.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionAppendix 1References |
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METHODS. The ERG of dark-adapted, anesthetized cats was recorded between an Ag-AgCl electrode in the vitreous humor and a reference electrode near the eye. Responses to bright flashes of diffuse white light were recorded at 3-minute intervals during hypoxemic episodes lasting 15 minutes to 2 hours.
RESULTS. The cat a-wave was well described by the Lamb and Pugh a-wave model during normoxia and hypoxemia. During mild hypoxemia (PaO2 of 50–60 mm Hg), small changes in a-wave amplitude were detected but did not become greater during severe hypoxemia. The mean decrease in the a-wave amplitude during severe hypoxemia (PaO2 of 20–30 mm Hg) was 8.9% from the mean amplitude during air breathing. The effects of hypoxemia were more severe on the b-wave amplitude. The mean decrease in the b-wave was 35% at PaO2 of 20–30 mm Hg.
CONCLUSIONS. The a-wave is more resistant to severe hypoxemia than the b-wave. This implies that photoreceptor transduction works almost normally during hypoxemia and that failure of inner retinal PO2 regulation causes the decrease in the b-wave. Previously observed changes in the amplitudes of slow ERG components during hypoxemia may result from changes in the ionic environment, rather than a failure of photoreceptor energy metabolism.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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It is unclear why the outer retina is sensitive to hypoxemia, but the inner retina is resistant, because the inner retinal responses are dependent on signals from the photoreceptors. A key element missing from previous studies was direct evaluation of photoreceptor electrophysiology. For more than 60 years, it has been known that the leading edge of the a-wave is associated with photoreceptor activity.8 9 However, the a-wave could not be used in a quantitative way to reveal receptor response properties until Hood and Birch10 and Lamb and Pugh11 proposed a computational model of the rod response. Breton et al.12 demonstrated that the a-wave exhibits the same kinetics and amplification as the photocurrents of single primate rods. The Lamb and Pugh11 model fits the a-wave reasonably well, giving the fraction of circulating current (i.e., photocurrent) as a function of time in terms of an amplification constant, an effective delay time, and the number of photoisomerizations.
The purpose of this study was to examine the effects of hypoxemia on dark-adapted photoreceptors in the cat retina by recording the a-wave in response to bright flashes. The a-wave model parameters were analyzed to examine phototransduction during normoxia and hypoxemia. The a-wave was also compared with the b-wave to simultaneously evaluate the effects of hypoxemia on the outer and inner retina.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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Visual Stimulation
Bright flashes of diffuse white light, produced by a photoflash
(Model 283; Vivitar, Santa Monica, CA) were used to elicit the a- and
b-waves. Stimuli were reflected to the eye by a ganzfeld hemisphere 30
cm in diameter with an interior surface coated with reflective white
paint. Flash intensity was varied over 6 log units by means of neutral
density filters (Eastman Kodak, Rochester, NY) available in steps of
0.5 log units. At 0 log units, the intensity of the flash at the cornea
was 28,057 scotopic candelas (cd)-seconds per square meter (measured
with a photometer; model 40X; UDT, Hawthorne, CA) in photopic units and
converted to scotopic units by assuming the spectral distribution of
the xenon-filled flash tube).13
The conversion from the
intensity of the flash at the cornea to the number of
photoisomerizations per rod per flash (
), which was used in the
a-wave model, was performed according to Breton et al. (see Reference
12
, equation 13) with a few corrections for cat eye. The is a
product of Q, the retinal illuminance in scotopic
troland-seconds (scot td-sec) and K, the overall conversion
factor in photoisomerizations/rod per scot td-sec. The pupil area was
113 mm2, and retinal illuminance Q in
our study was therefore 3.17 x 106 scot
td-sec (or 12.2 log q/deg2). The conversion
factor K was adjusted for the cat eye by considering the
following three factors: 1) the smaller loss in media and tapetal
reflection, a difference of 0.24 log units from human14
;
2) the smaller posterior nodal distance in cat, differing 0.26 log
units from human14
; and 3) the smaller area of the rod
outer segment, assumed to have an average diameter of 1.3 µm in
cat.15
The value of K was then 27.13
photoisomerizations/rod per scot td-sec. Our maximum unattenuated
retinal illuminance corresponded to 7.93 log photoisomerizations per
rod per flash. To test for a cone contribution, responses were obtained
to short-wavelength flashes (Wratten 47B filter; Eastman Kodak,
Rochester, NY) and long-wavelength flashes (Wratten 26 filter).
Recording
The ERG was measured between a vitreal electrode and a reference
electrode behind the eye. The vitreal electrode was a chlorided silver
wire in a 0.5-mm glass tube. A 20-gauge needle was inserted in the eye
to hold the Ag-AgCl vitreal electrode in place. The reference electrode
was a chlorided silver plate, which was sewn into tissue near the eye.
Potentials were amplified (DC to 3 kHz, M4A Amplifier; WPI, Sarasota,
FL) and displayed on an oscilloscope. The responses were then digitized
at a rate of 5 kHz by an analog-to-digital board in a computer. The
flashes were triggered by the computer, and the data were digitized by
use of commercial software (Epic-XL; LKC, Gaithersburg, MD). After each
flash, the eye was allowed to dark adapt for at least 3 minutes. All
responses shown were to single flashes, not averages.
a-Wave Analysis
The responses to all flash energies were fitted to equation 1 based on the Lamb and Pugh model:11
 | (1) |
is the number of photoisomerizations
per rod, teff is a brief delay, and
A is an amplification constant. When applied to the a-waves,
F(t) is equivalent to the normalized a-wave from
flash onset to the peak of the a-wave:
 | (2) |
Double-Flash Experiments
Two photoflashes (Vivitar) were used to elicit two a-wave
responses to test recovery from a bright flash during hypoxemia (e.g.,
References 16, 17). Both stimulus flashes were reflected to the eye by
the ganzfeld. Flash intensity was varied by means of neutral-density
filters. A test flash (5.93 log photoisomerizations per rod per
flash) was given at time 0, and a probe flash followed at a
predetermined interstimulus interval (ISI). The probe flash (6.93 log
photoisomerizations per rod per flash) was used to produce amplitude
saturation of the a-wave. The photoflashes were triggered by computer
(LabTech Notebook software running within a Visual Basic program;
Microsoft, Redmond, WA). The ISI was controlled by the
computer. The ISI used during hypoxemia was determined in a preliminary
experiment in which the probe flash was given at varying times after
the test flash, usually ranging from 500 to 3000 msec. The responses
were digitized at a rate of 500 Hz by an analog-to-digital board. After
each pair of responses, the eye was allowed to dark adapt for 3
minutes.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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,
but at the higher intensities, A and
teff declined in
human11
12
and rat.18
In cats, A
also declined at higher intensities (Fig. 2)
.
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where R(I) is a-wave peak amplitude,
I is intensity, Rmax is the
maximum a-wave amplitude, and 
is half saturation. The Naka–Rushton
equation (n = 1) did not fit the peak amplitude data well,
although it fit when the a-wave was measured at a fixed time after the
flash (not shown). The average Hill equation parameter was 4 ± 0.3 log units attenuation for white light (for three cats), 2 ± 0.2 log units attenuation for blue light (for three cats), and 1 log
unit attenuation for red light (for one cat). The responses to white,
blue, and red light were separated from each other by a fixed amount
over the entire range, suggesting that there was no Purkinje shift and
no significant cone contribution. When a response to red light at 0 log
units attenuation was subtracted from a response to blue light at 1 log
unit attenuation, the subtracted response was virtually zero giving
more evidence that there was little or no cone intrusion. The inset to
Figure 3 shows the superimposed responses to blue (1 log unit) and red
(0 log units) lights. These data indicate that the responses recorded
were the rod ERG uninfluenced by a cone component.
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were not significantly different.
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14%) was observed a few minutes after the
onset of moderate hypoxemia, but the a-wave was then stable over time,
implying that 15-minute episodes of hypoxemia allowed a good estimate
of the a-wave during hypoxia and that the photoreceptor can function
almost normally during moderate hypoxemia for long periods. The b-wave
was also stable at this level of hypoxemia. This indicates that the
changes in the a- or b-wave are directly related to the
PaO2, and not the
duration of the hypoxemic episode.
The Lamb and Pugh Model in Hypoxemia
The Lamb and Pugh model11
was applied to determine
the values of A and teff
during hypoxemia. Table 2
shows the parameters of the Lamb and Pugh
model for five cats. At each level of hypoxemia, five responses to a
flash at a fixed attenuation (5.93 log photoisomerizations per rod per
flash) were compared with the control (normoxic) a-wave fits except in
cat 205 (three responses to 4.93 log photoisomerizations per rod per
flash) and cat 209 (five responses to 4.93 log photoisomerizations per
rod per flash). Both the normoxic and hypoxic a-waves were well
fitted by the Lamb and Pugh model. The overall amplitudes decreased
significantly as the percentage of oxygen decreased; however, the
change was small enough that the values of A and
teff were not different from the
normoxic a-wave fits. The rate of rise of the a-wave did not change
with lowering of the percentage of oxygen. The time to peak and the
shape of the a-wave also did not change during severe hypoxemia,
whereas the b-wave was essentially gone in some cats (Fig. 4A
, inset).
Double-Flash Experiments
To study the full time-course of the rod flash response, the
double-flash or paired-flash method was used to examine the recovery
kinetics of the rod16
17
during hypoxemia. Figure 6
(inset) shows the a-wave responses to the probe flash at various ISIs.
When the ISI was less than 1500 msec, almost no probe a-wave was
observed. For the hypoxemia experiments, an ISI of 2500 msec was used.
As in the hypoxemia experiments described earlier, 15-minute steps of
hypoxemia were used, but paired flashes rather than single flashes were
issued at 3-minute intervals.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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in the Breton et al.12
work was
6.08 log photoisomerizations per rod per flash, whereas in the present
work the maximum was 7.93 log photoisomerizations per rod per
flash. The amplification (A) is constant at low intensities, but at high intensities it declines because of saturation in the rate of rise of the photocurrent in human11 12 and rat.18 A similar result was obtained in cat (Fig. 2) . The maximum value of A was 5 to 10 seconds-2 in humans,12 whereas in rats the maximum was 2 seconds-2.18 The value of A for monkey was 4 to 7 seconds-2 (Pugh and Lamb19 estimated from data of Baylor et al.20 ). The maximum value of A in our cat data was approximately 0.7 seconds-2. Pugh and Lamb19 reported that A for mammalian rods at body temperature (37°) was 4 to 10 seconds-2 and that of amphibian rods at room temperature (22°) was 0.04 to 0.1 seconds-2. There could be several reasons for the difference between our value for A and that of Pugh and Lamb.19 First, when Pugh and Lamb19 estimated A for mammals (using experiments from other studies) they assumed teff to be 5 msec. Breton et al.12 and our calculations showed that teff was between 2 and 3 msec for both cat and human. The higher teff used by Pugh and Lamb19 could explain the higher A (see equation 1 ). Second, the value of A that we obtained represents the intact cat eye, whereas in the other mammalian work (monkey20 and cat21 ) Pugh and Lamb estimated the amplification constant from isolated retina. Finally, the conversion factor K was corrected for cat in this study, but it is not clear that the correction was made in the Pugh and Lamb19 summary. It is also possible that there may be some error in our conversion factor K, although we used the best values available.
Hypoxemia
Previous studies of hypoxic effects on the retina did not examine
the photoreceptor activity directly. The data presented here provide
the first information on the electrical activity of photoreceptors and
on phototransduction during graded hypoxia. The present work showed
that there was a small change, a decrease on average of approximately
9%, in the a-wave amplitude at
PaO2 between 50 and 60 mm
Hg. At this level there was no change in the b-wave. For
PaO2 between 20 and 30 mm
Hg, a large change in the b-wave amplitude was observed, but the a-wave
amplitude never decreased by more than 14.7%, indicating that the
a-wave is more resistant to hypoxemia than the b-wave. The Lamb and
Pugh model was applied to the hypoxic a-wave and even at very low
PaO2, the parameters were
not different from those in normoxia (Table 2)
except for a small
decrease in amax. The
intensity–response functions showed little change at any intensity.
The preservation of a-wave amplitude and the parameters of the Lamb and
Pugh model suggest that transduction works almost normally during
severe hypoxemia.
The recovery of the photoreceptor response after an intense flash was examined by using a double-flash technique. We hypothesized that, although hypoxemia had little effect on the flash response, it might impair the ability of the photoreceptor to recover from a flash. However, rather than being impaired, the probe a-wave amplitude was also resistant to hypoxemia.
There are a few concerns that should be addressed at this point. The first is that the b-wave amplitude may be overestimated in Figure 5 , because it often did not reach steady state at the most severe level of hypoxemia studied. However, the a-wave was not overestimated, because its amplitude was relatively stable even at low PaO2. Thus, the difference between the a- and b-waves was even greater than suggested by Figure 5 . It would be useful to know at what PO2 the a-wave deteriorates, but the cats could not tolerate more severe hypoxemia. Second, the a- and b-waves were stable over long periods (2 hours) during moderate hypoxemia, indicating that any changes in the amplitudes were due to the changes in oxygen level and not the duration of the hypoxemic episode. Third, it is possible that there is a regional failure of photoreceptors during hypoxemia, which may contribute to a decrease in the maximum amplitude. The amax may have decreased because some photoreceptors are affected more by hypoxemia than others or because each photoreceptor was affected a little by hypoxemia. Currently, there is no way to evaluate the regional loss of the photoreceptors. Fourth, there may be a concern that the a-wave appeared resistant to hypoxemia, because flashes nearly saturated its amplitude, but intensity–response data (Table 2) show that the effect of hypoxemia was small at all intensities. Finally, there was a concern that a reduction in the photoreceptor response might actually be more substantial than the a-wave indicates, because the a-wave change in hypoxemia might be obscured by a reduction in the b-wave. Rodieck22 proposed this as one explanation for failure to see a change in the a-wave during anoxia. This cannot explain the current results because in response to intense flashes the a-wave peaks well before the b-wave starts23 and there is no evidence that the b-wave obscures the a-wave peak. Instead, the results are consistent with those of Noell24 and Granit8 who found that even during anoxia there was relatively good preservation of the a-wave.
Possible Mechanism
Both the a- and b-waves were resistant to hypoxemia, but the
b-wave was more affected by low
PaO2. It can now be
suggested that when the b-wave decreases, it does so not because of a
failure of the photoreceptor signals that initiate it, but more likely
from a failure of the retinal circulation to maintain oxygenation of
the inner retina. It is possible that the failure of the b-wave does
not result directly from an effect of hypoxemia on Müller cells
or bipolar cells but from a failure of synaptic transmission in the
outer plexiform layer. There are currently no data that would allow us
to distinguish between these possibilities. In either case, the
decrease in the b-wave would be tied to changes in retinal circulation,
because the inner nuclear and outer plexiform layers both rely on
retinal circulation. Linsenmeier and Braun6
and
Enroth–Cugell et al.1
showed that the inner retinal
PO2 during normoxia was low (18 ± 12 mm Hg), but that it was well regulated during hypoxemia. Mean
inner retinal PO2 was significantly
affected only at PaO2
below 45 mm Hg, which corresponded with the point at which the b-wave
and ganglion cell sensitivity begin to break down in this and earlier
studies.1
4
25
Perhaps the most puzzling aspect of the current findings is that we know that photoreceptor oxidative metabolism decreases,6 and slow ERG components, which are dependent on the photoreceptors, change over a range of PaO2 when there is little change in the a-wave.3 Linsenmeier and Steinberg7 suggested that slowing of the Na+-K+ pump was responsible for changes in potassium activity during hypoxemia, which eventually led to changes in the slow ERG components. It now seems necessary to postulate that when oxidative metabolism is affected, the photoreceptors switch more to glycolysis for energy production. Linsenmeier and Braun6 calculated that the increase in adenosine triphosphate (ATP) production from glycolysis could potentially compensate for the decrease in oxidatively derived ATP production.
The present data may not require a fundamental alteration in our understanding of the mechanism of changes in the slow ERG components during hypoxemia. It is possible that the changes in [K+]o are due to changes in the mechanism of ATP production during hypoxemia, rather than the amount of ATP production, as was previously thought. Yamamoto and Steinberg26 showed that systemic hypoxia further acidifies the extracellular space outside rods in dark-adapted cat retina, a region that is already acidic in normoxia. Yamamoto et al.27 suggested that the acidity found outside rods is from rod glycolysis. This compensatory mechanism, an increase in ATP production by glycolysis that is needed to maintain rod function, may be responsible for keeping the a-wave unchanged when the slow ERG components are affected during hypoxemia. The slow ERG components may change because of acidosis accompanying hypoxemia, rather than because of hypoxemia per se. Respiratory acidosis alone is known to cause similar changes in the standing potential, c-wave, and light peak to those caused by hypoxemia.3 28 Perhaps the change in [K+]o during hypoxia occurs not because of a change in the pump rate but rather because of other changes that occur in the subretinal space during hypoxemia. One possibility is that there may be ionic redistribution that could occur because of the H+ change. The exact mechanism of ion redistribution is unknown.
A more likely possibility is that the compensation for the loss of oxidative metabolism is not quite complete, allowing a small change in [K+]o. The increase in [K+]o during hypoxemia may account for the small changes in the a-wave amplitude during mild hypoxemia. When the fractional changes in the photoreceptor light response in hypoxia (due to changes in K+) were calculated, there was a small change in rod voltage responses (for calculation, see Appendix). There was an approximately 5% change in the calculated membrane potential, which could account for the small changes (average of 3.4% ± 2.8%) in the a-wave amplitude during mild hypoxemia. The b-wave might not be altered by this small change in rod voltage responses, because the amplification at rod synapses is large, and the full b-wave can be produced with only a small rod hyperpolarization.
To test the idea that glycolysis compensates for the loss of oxidative metabolism, the a-wave was recorded during hypoglycemia, which will be discussed in a future article.
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Appendix 1 |
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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Let
in the dark. To a first approximation, rod membrane potential in the
dark can be expressed by the chord conductance equation29
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During strong illumination, all Na+ channels
close so z = 0, and the voltage difference between
light and dark is
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Assume that in the dark z does not change during hypoxia so
that the
factor drops out. Then,
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Acknowledgements |
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Footnotes |
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Submitted for publication December 28, 1999; revised May 22, 2000; accepted June 8, 2000.
Commercial relationships policy: N.
Corresponding author: Robert A. Linsenmeier, Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3107. r-linsenmeier{at}nwu.edu
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TopAbstractIntroductionMethodsResultsDiscussionAppendix 1References |
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) varied from 5 to
0 log units attenuation. An intensity series to red light (
) varied
from 5 to 0 log units attenuation. The Hill equation parameters for
white light were: Rmax = 729 µV,
n = 0.53, 
