(Investigative Ophthalmology and Visual Science. 2001;42:127-136.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
Characterization of Retinal Injury Using ERG Measures Obtained with both Conventional and Multifocal Methods in Chronic Ocular Hypertensive Primates
William A. Hare,
Hau Ton,
Guadalupe Ruiz,
Barbara Feldmann,
Mercy Wijono and
Elizabeth WoldeMussie
From the Department of Biological Sciences, Allergan Inc., Irvine, California.
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Abstract
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PURPOSE. To characterize, using both conventional and multifocal
electroretinogram (ERG) recordings as well as histologic measures,
retinal injury in the chronic ocular hypertensive primate model for
experimental glaucoma.
METHODS. Ocular hypertension was induced in the right eye of 7 cynomolgous
monkeys, Macaca fascicularis, using laser injury to the
aqueous outflow tissue at the anterior chamber angle. At 16 months
after IOP elevation, ERG recordings were made from both eyes of all
animals using both conventional and multifocal methods. After
electrophysiological recording, animals were killed and retinal samples
were radially sectioned for histologic analysis.
RESULTS. Histologic measures showed that ocular hypertensive injury was largely
or completely limited to a loss of retinal ganglion cells (RGCs). The
degree of RGC loss was similar in central and peripheral retina.
Amplitudes of conventional ERG responses were mostly unaffected in eyes
having severe loss of RGCs, a finding that is consistent with limited
injury to photoreceptors, bipolar cells, and amacrine cells. Peaks in
both the first- and second-order multifocal ERG responses were
attenuated in ocular hypertensive eyes, and amplitude of these peaks
was highly correlated with the density of surviving RGCs.
CONCLUSIONS. The results are consistent with a conclusion that both first- and
second-order components of the multifocal ERG response from the monkey
reflect a significant contribution from activity in RGCs and may
provide a useful measure for the clinical diagnosis and management of
glaucoma.
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Introduction
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The chronic ocular hypertensive (COHT) primate, originally
described by Gaasterland and Kupfer,1
has been studied in
a number of different laboratories as a model for glaucomatous injury
to the retina and optic nerve. In this model, chronic elevation of
intraocular pressure (IOP) results in retinopathy and neuropathy that
reflect an apparently selective loss of retinal ganglion cells (RGCs)
and their axons.2
3
This pattern of injury to the retina
and optic nerve, combined with the fact that the insult consists of an
elevation of IOP, has led many investigators to refer to this model as
"experimental glaucoma." Results of studies using electroretinogram
(ERG) recordings for functional characterization of retinal injury have
shown that pattern reversal,4
5
6
scotopic
negative,3
and photopic negative7
responses
provide a measure of RGC injury in this model, whereas flash a-wave,
b-wave, and oscillatory potential (OP) responses are relatively
unaffected by hypertensive injury.3
4
5
7
Multifocal ERG (mfERG) recordings from humans show evidence for
contributions from activity of inner retinal cells including
RGCs.8
9
10
11
12
Glaucoma has also been associated with changes
in the mfERG response,13
14
15
16
though a recent report showed
that measures of the local mfERG response were not well correlated with
local glaucomatous sensitivity losses.17
In rhesus
macaque, evidence for a contribution from RGCs to mfERG responses has
also been demonstrated.18
These findings support the
notion that mfERG recordings may have utility in the diagnosis and
management of glaucomatous injury to RGCs. We report here our results
from histologic measures of retinal injury as well as measures of ERG
responses obtained using both conventional and multifocal techniques in
the COHT primate. Some preliminary findings from this study have been
published previously in a brief report.19
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Methods
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Animal Subjects
Seven young adult female cynomolgous monkeys, Macaca
fascicularis, were used for this study. All experimental
procedures as well as animal care and handling adhered to the
guidelines outlined in the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research. All experimental procedures were also
reviewed and approved by an internal institutional review committee for
animal use.
Ocular Hypertension
IOP in the right eye of each animal was elevated using a
procedure similar to that originally described by Gaasterland and
Kupfer.1
Briefly, animals were anesthetized with an
intramuscular injection of ketamine (15 mg/kg) and topical application
of proparacaine (0.5%) in the right eye. Pupillary miosis was induced
with instillation of 2.0% pilocarpine. After placement of a goniolens,
energy from an argon laser (488 nm + 519 nm, model Novus 2000;
Coherent, Inc., Palo Alto, CA) was directed through the anterior
chamber to the trabecular meshwork. Individual burns were produced
using focused laser spots of 1-W power, 50-µm diameter, and
0.5-seconds duration. An initial treatment consisted of 30 to 40 burns
applied over the superior 180° of the chamber angle. Two weeks later,
the inferior 180° of the trabecular meshwork was similarly treated.
IOP was measured in both eyes of each animal at regular intervals under
light anesthesia (intramuscular ketamine, 5 mg/kg) using a
pneumotonometer (Digilab, Norwell, MA).
Electrophysiology
ERG recordings were made from both eyes of each animal at
approximately 16 months after induction of ocular hypertension. Animals
were anesthetized with an intramuscular injection of ketamine (10
mg/kg) in combination with acepromazine (0.5 mg/kg) before placement of
an endotracheal tube and intravenous catheter. Instillation of one drop
of 1% tropicamide maintained pupil diameter at approximately 6 mm
during the recording session. Animals were then positioned in a holder
that used soft pressure points and a bite bar to stabilize the head.
Neuromuscular block was induced with an intravenous bolus of
norcuronium bromide (0.06 mg/kg) and maintained for the duration of the
session with continuous infusion (0.04 mg/kg-hr.). Anesthesia was
maintained with periodic intramuscular injection of ketamine (10
mg/kg). Animals were mechanically ventilated with 100% oxygen. Heart
rate, rectal temperature, and expired
PCO2 were continuously
monitored.
Corneal voltage was recorded using a bipolar contact lens electrode
(Hansen Ophthalmic Laboratories, Iowa City, IA) configured such that
the cornea was active and the speculum was reference. The indifferent
electrode consisted of a subcutaneous needle located at the glabella.
Corneal voltage signals were amplified using a Grass model 12 amplifier
(Astro Med, West Warwick, RI) and digitized online at 2 kHz using a
model DAS 1200 converter (Keithley Metrabyte/Asyst, Taunton, MA).
Conventional ERG responses were elicited with stimuli of 10-µsec
duration and an intensity of 0.09 J/flash (intensity setting of
"1") generated by a Grass model PS33 xenon flash tube. The stimulus
was positioned at 10 cm from the cornea on the visual axis and
subtended approximately 50° of visual angle centered on the fovea as
shown in Figure 1A
. Ambient background room illumination was approximately 0.05
footcandles during recording. For flash and OP responses, stimuli were
delivered every 10 seconds. For flicker responses, 30-Hz stimulus
trains of 512-msec duration were delivered at 1-second intervals.
Averages of 10, 25, or 30 responses were used for analysis of flash,
OP, and flicker responses, respectively. OP responses were isolated
using analog bandpass filtering from 100 to 1000 Hz, whereas all other
conventional responses were bandpass filtered from 3 to 1000 Hz. Line
frequency noise was minimized with an analog 60-Hz notch filter.
For multifocal recordings, stimuli were generated on a 21-inch Radius
Intelicolor monitor (Radius, Inc., San Jose, CA) using VERIS 1
Scientific Software and video driver board (Electro Diagnostic Imaging,
Inc., San Mateo, CA) and consisted of an array of 61 hexagonal elements
of equal size as shown in Figure 1B . At the test distance of 30 cm, the
entire array subtended approximately 50° of visual angle, each
element having an angular subtense of approximately 5.5°. The
intensity of each element was temporally modulated in a stepwise
fashion at a frame rate of 67 Hz between 95 cd/m2
(white) and 5 cd/m2 (black) according to a binary
m-sequence.20
21
A steady background of 45
cd/m2 surrounded the stimulus field to minimize
contributions from light scatter. Responses to
215 stimulus frames were recorded
(m-sequence = 15), resulting in records of approximately 8-minutes
duration for each eye. The corneal voltage signal was digitized at
approximately 1000 Hz and bandpass filtered from 3 to 300 Hz in
conjunction with 60-Hz notch filtering.
Experimental Procedure for Electrophysiological
Recording
The same recording sequence was used for all animals: (1)
multifocal ERG OD, (2) conventional ERG OD, (3) multifocal ERG OS, and
(4) conventional ERG OS. During recording, the contralateral eye was
always occluded. After placement of the contact lens electrode,
retinoscopy was performed to determine the best spherical equivalent
lens power to make the retina optically conjugate to the stimulus
monitor. A lens of this power (typically +3 to +5 diopter) was then
placed at 1 cm anterior to the cornea. The stimulus monitor was then
positioned at 30 cm anterior to the cornea such that the estimated
visual axis projected to the center of the stimulus field. A series of
multifocal recordings of approximately 2-minutes duration (m
sequence = 13) was then used to adjust the stimulus position such
that the fovea projected to the center of the central stimulus element
and a clear amplitude maximum was elicited by the central stimulus
element for both first- and second-order responses. The precision of
this method for stimulus alignment was verified in several eyes by
optically projecting the fundus onto the stimulus monitor and noting
the location of the optic nerve head and macula. Stimulus alignment was
also verified for each recording by observing the location of the optic
nerve head projection (response minimum) in the first-order response
trace array. After stimulus alignment, a multifocal recording of 8
minutes duration was made. The stimulus monitor was then covered with a
light-tight shield, the corrective lens was removed, and the xenon
flash stimulator was positioned. After a 5-minute period of dark
adaptation, conventional recordings of the flash, OP, and flicker
response were made in that order.
Histology
Within several days after ERG recording, animals were deeply
anesthetized with intramuscular injection of a combination of ketamine
(15 mg/kg), xylazine (1 mg/kg), and acepromazine (0.2 mg/kg) and
transcardially perfused with heparinized saline (37°C) followed
by a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) in
phosphate buffer (pH 7.0–7.2, 37°C). Sutures were used to mark the
12 o’clock position on each eye before enucleation. After removal of
the anterior segment and vitreous, the retina/choroid was removed and
flat-mounted on a glass slide. Using a trephine and a template made
from transparent film, 3 x 3-mm samples of the retina/choroid
were obtained from eight regions, including one sample centered on the
fovea (PF), three perimacular samples (1–3), and four samples from the
far periphery (samples 4–7) as shown in Figure 2
. Tissue samples were then dehydrated and paraffin-embedded for
sectioning.
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Figure 2. Location of retinal sample regions for histologic analysis of ocular
hypertensive injury is shown here for a right (OD) eye. The
retina/choroid was flat-mounted on a glass slide (RGC layer up) and
samples were cut from eight locations with the orientation indicated.
Each sample region was 3 mm x 3 mm. The perifoveal sample was
centered on the fovea. Samples 1 to 3 were located on the horizontal
and vertical meridians from 3.5 to 6.5 mm from the fovea. Samples 4 to
7 were located on the oblique meridians from 8.5 to 11.5 mm from the
fovea. Sections were cut parallel to the heavy border as indicated for
each sample.
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For samples 1 to 7, radial sections of 7-µm thickness were cut as
indicated in Figure 2
. Six sections obtained at 50-µm intervals from
each sample were then stained with hematoxylin & eosin (H&E). In each
of these sample regions, the normal ganglion cell layer is comprised of
a single layer of cells, and ganglion cell counts were obtained by
manually counting and averaging cells in the ganglion cell layer of all
six sections. The perifoveal ganglion cell layer is six to eight cells
thick, and ganglion cell counts for the perifoveal sample (PF) were
obtained differently. Beginning at the inferior border, horizontally
oriented radial sections were made until the foveal pit was located.
Three sections were then selected from the region of highest ganglion
cell density between 0.5 and 0.7 mm from the center of the foveal pit.
Ganglion cell counts in these three sections were made using a
BIOQUANT imaging system (R&M Biometrics, Inc., Nashville, TN)
and stereology software.
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Results
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Ocular Hypertension
Mean IOP of all seven laser-treated (OD) eyes is summarized for
the duration of the study in Figure 3
. Following soon after the second laser treatment (inferior 180° of
chamber angle), there was a dramatic rise in IOP. The magnitude of this
initial elevation, as well as the pressure over the remainder of the
study, varied considerably among eyes, as indicated by the large SDs
from the group means. IOPs of some eyes were maintained at relatively
high levels, whereas in other eyes the IOP gradually dropped to some
lower level at a rate that also varied among those eyes. When mean
pressure elevation was expressed for each eye as the integral of IOP
elevation above baseline (
20 mm Hg) over the duration of the study,
there was a strong correlation between mean IOP elevation and
histologically measured loss of retinal ganglion cells (data not
shown). That is, higher mean pressures were associated with greater
loss of retinal ganglion cells. Pressure in the contralateral
(normotensive OS) eyes showed only the normal degree of variation over
the course of the study (data not shown). The relationship between the
level of IOP elevation and the degree of retinal ganglion cell loss is
beyond the scope of this report and will described in detail in a later
article.
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Figure 3. IOP history of all animals for the duration of the study. Each data
point represents the mean IOP for the laser-treated (OD) eyes of all
seven animals at that timepoint. Three baseline IOP measures were made
before the first laser treatment. The zero timepoint corresponds to the
IOP measure made 1 day before the second laser treatment. Note that the
IOP response to laser treatment varied considerably for individual eyes
as indicated by the large SDs (vertical bars) for mean
pressure.
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Ocular hypertension resulted in optic neuropathy that was most severe
in eyes with the highest mean IOP elevation. Fundus photographs from
one animal, obtained approximately 2 months before killing, are shown
in Figure 4
. Each panel is actually one of the two images from a stereo pair. The
IOP for the hypertensive (OD) eye of this animal remained relatively
high over the entire course of the study. The atrophic appearance of
the nerve head in the hypertensive eye is readily apparent from a
comparison with the normotensive (OS) eye.
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Figure 4. Fundus photographs of the hypertensive OD (A) and
normotensive OS (B) eyes from one animal. Each panel is one
frame of a stereo pair. The photographs were taken approximately 2
months before sacrifice (approximately 13 months after IOP elevation).
The pressure in the hypertensive eye of this animal remained relatively
high over the duration of the study. The atrophic appearance of the
hypertensive optic nerve head is readily apparent from a comparison
with the contralateral normotensive nerve head.
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Histology
PF sections from each eye of the same animal whose fundus
photographs are shown in Figure 4
are shown in Figure 5
. Comparison of the two sections shows that the RGC layer is
approximately six to seven cells thick in the normotensive eye but is
reduced to a single layer in the hypertensive eye. This is in contrast
to the appearance and thickness of the inner nuclear layer, outer
plexiform layer, and outer nuclear layer, which show no evidence of
injury in the hypertensive eye. Measures of inner and outer nuclear
layer thickness in sections from all sample regions of all eyes showed
no significant difference between normotensive and hypertensive eyes
(data not shown), evidence that supports the notion that hypertensive
injury is largely or completely limited to a loss of RGCs.
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Figure 5. Perifoveal H&E stained sections from the normotensive OS (A)
and hypertensive OD (B) eyes of the same animal whose fundus
photographs are shown in Figure 4
. Each of the sections was cut
orthogonal to the vertical meridian at 500 to 700 µm inferior to the
center of the foveal pit as described in the text (see also Fig. 3
).
The field of the micrographs is centered on the vertical meridian. Note
that the ganglion cell layer (GCL) in the normotensive eye is
approximately six to seven cells thick, whereas the hypertensive eye
has only a single sparsely populated row of cells. The thickness and
appearance of the inner nuclear layer (INL), outer plexiform layer
(OPL), and outer nuclear layer (ONL) are similar in the two eyes. The
calibration bar in (B) is equal to 100 µm and applies to
(A) and (B).
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Figure 6
summarizes results of ganglion cell counts from all seven hypertensive
eyes. For each sample region, ganglion cell counts from each
hypertensive eye were normalized with respect to counts obtained from
the same region in the contralateral eye. Mean normalized values for
all eyes are shown with SE bars. When compared with the perifoveal
sample region, no other region showed a significantly different
(P < 0.05) level of RGC loss.
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Figure 6. Summary of RGC counts from the eight retinal sample regions in all
seven animals. Counts of cells in the ganglion cell layer of each
sample region were made as described in the methods section. For each
animal, RGC counts in the hypertensive eye were normalized with respect
to counts obtained from the same sample region in the contralateral
normotensive eye (OD/OS). For each sample region, mean normalized
counts for all animals are shown with SE bars. Note that there is no
significant difference (P < 0.05) in the degree of
RGC loss/survival for any sample region.
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Conventional ERG Responses
Conventional ERG responses obtained from the normotensive eye of
one animal are illustrated in Figure 7
. For flash responses, amplitude of the a-wave and b-wave peak voltage
was measured (Fig. 7A)
. OP response amplitude was measured as the RMS
voltage over a window extending from 10 to 75 msec after the stimulus
(Fig. 7B) according to the equation:
 | (1) |
where VRMS is RMS voltage,
vi is voltage at each timepoint over the
range 10 to 75 msec, and n is number of voltage measures
(n = 130).
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Figure 7. Conventional ERG response measures. Traces represent averaged responses
recorded from a normotensive eye as described in the methods.
(A) Flash response. Stimulus delivered at 0 msec. Peak
amplitude of a-wave (a) and b-wave (b) measured as indicated. Trace is
average of 10 responses. (B) Oscillatory potential. Stimulus
delivered at 0 msec. Response amplitude measured as RMS voltage from 10
 75 msec after the stimulus. Trace is average of 25 responses.
(C) Flicker response. Stimulus consists of 30-Hz train of
512 msec duration beginning at 0 msec. Average peak-to-peak amplitude
of last three response cycles measured as indicated. Trace is average
of 30 responses.
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Amplitude of the 30-Hz flicker response was measured as the average
peak-to-peak voltage of the last three cycles in the response train
(Fig. 7C)
.
For each animal, amplitude measures from the hypertensive eye were
normalized with respect to measures obtained from the contralateral
normotensive eye (OD/OS). Results of previous recordings from normal
monkeys indicated that comparing responses from both eyes recorded
during the same recording sessions might yield less variability than
comparing responses obtained from the same eye during different
recording sessions (unpublished observations). These normalized
amplitudes are plotted for each response measure as a function of
normalized PF ganglion cell counts (OD/OS) in Figure 8
. In each panel, a single data point thus represents the amplitude
measure and ganglion cell number obtained from an individual animal.
Results from linear regression analysis of these plots show that a-wave
amplitude was not correlated with histologic measures of ganglion cell
loss/survival (Fig. 8A)
, whereas amplitude measures for the b-wave
(Fig. 8B) , OPs (Fig. 8C)
, and flicker (Fig. 8D)
responses showed a weak
correlation. That is, a reduction in the number of surviving ganglion
cells was associated with a reduction in b-wave, OP, and flicker
response amplitude. However, the slopes for the linear regression plots
were all less than 0.30, and eyes which lost most of their RGCs showed
only a modest reduction in ERG response amplitude. Similar results were
obtained when response amplitudes were plotted as a function of either
total RGC counts (sum of counts in all eight sample regions),
perimacular counts (sum of samples 1–3), or peripheral counts (sum of
samples 4–7; data not shown).
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Figure 8. Summary of correlation between conventional ERG response amplitude and
counts for the number of surviving cells in the perifoveal (PF) RGC
layer. Methods for measures of response amplitude and cell counts as
described in text. ERG response amplitude and cell counts for the
hypertensive eye are normalized, for each animal, with respect to
values obtained for the normotensive eye (OD/OS). In each panel, the
straight line represents the linear regression plot. (A)
Correlation of a-wave amplitude with RGC counts: regression slope = +0.07, r = 0.18. (B) Correlation of b-wave
amplitude with RGC counts: regression slope = +0.18,
r = 0.71. (C) Correlation of OP RMS voltage
with RGC counts: regression slope = +0.28, r =
0.90. (D) Correlation of flicker response amplitude with RGC
counts: regression slope = +0.28, r = 0.76. Note
that (C) has only six data points since OP responses were
not recorded from one of the animals with moderate RGC loss.
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Multifocal ERG Responses
Amplitude measures were made for both first- and second-order
multifocal ERG responses. Each of the 61 traces in Figure 9A
represents the local first-order retinal response from a normotensive
(OS) eye to the stimulus element in that location of the stimulus
field. The central seven highlighted traces correspond to responses
from the macular region of the retina (0° 8° eccentricity),
whereas the more peripheral traces correspond to responses from
perimacular retina (8° 25° eccentricity). Summation of traces
in either of these groupings yields the macular and perimacular
responses that are shown in Figure 9B
. Note that central and peripheral
response waveforms differ most markedly in the amplitude and kinetics
of the later peaks. The macular response consists of five transients:
an early negativity (N1) having a time-to-peak of approximately 17
msec, an early positivity (P1) having a time-to-peak of approximately
33 msec, a late negativity (N2) having a time-to-peak of approximately
50 msec, a late positivity (P2) having a time-to-peak of approximately
70 msec, and a late negativity (N3) having a time-to-peak of
approximately 95 msec. Amplitude measures for the five peaks of the
macular response were made as indicated. The first-order trace array
for the hypertensive (OD) eye from the same animal is plotted in Figure 9C
, whereas macular and perimacular responses from this eye are shown
in Figure 9D
.
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Figure 9. First order mfERG responses from both eyes of an animal with severe
hypertensive injury. Recording made as described in Methods.
(A) Normotensive (OS) eye. Trace array of 61 responses, each
of which represents the local retinal response corresponding to the
stimulus element at that location in the stimulus field. The central 7
traces (shaded) represent responses from macular retina
extending to approximately 8° retinal eccentricity. The surrounding
54 traces (unshaded) represent responses from perimacular
retina extending from approximately 8° 25° retinal
eccentricity. Calibration bars, 200 nV, 100 msec. Note that amplitude
of individual traces is expressed in units of volts since each trace
represents the response from retinal areas of the same size.
(B) Macular and perimacular responses obtained by summation
of either the central 7 or surrounding 54 response traces,
respectively, from (A). Amplitude measures for the 5 peaks
of the macular response were made as indicated. Calibration bars, 5 nV/deg2 and 25 msec. Note that macular and
perimacular response amplitude is expressed as response density
(volts/unit retinal area) since these responses reflect retinal
stimulus areas of different size. (C) Sixty-one response
array obtained from the hypertensive (OD) eye of the same animal whose
responses are shown in (A) and (B). Calibration
as for (A). (D) Macular (top trace)
and perimacular (bottom trace) responses obtained from
(C). Calibration as for (B).
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Second-order responses obtained from the same recording that generated
the responses of Figures 9A
and 9B
are shown in Figures 10A and 10B
. The macular response contains a prominent biphasic waveform:
an early positivity (P, time-to-peak of approximately 45 msec) and a
later negativity (N, time-to-peak of approximately 90 msec). This
biphasic component is relatively much smaller in the perimacular
response. A single peak-to-peak amplitude measure (PN) of the
second-order macular response was made as indicated. Second-order
responses from the hypertensive (OD) eye of the same animal are shown
in Figures 10C
and 10D
.
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Figure 10. Second order mfERG responses from the same recordings which produced
the first-order responses shown in Figure 9
. (A)
Normotensive (OS) eye. Sixty-one response trace array with responses
from macular (shaded) and perimacular (unshaded)
retina as indicated. Calibration bars, 200 nV, 100 msec. (B)
Macular and perimacular response obtained by summation of responses
from the macular and perimacular retina, respectively, in
(A). A single peak-to-peak measure of second-order macular
response amplitude was made as indicated. Calibration bars, 2.5
nV/deg2 and 25 msec. (C) Sixty-one
response array from hypertensive (OD) eye. Calibration as for
(A). (D) Macular (top trace) and
perimacular (bottom trace) responses obtained from
(C). Calibration as for (B).
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Responses from Figures 9B
and 9D
and 10B
and 10D
are replotted in
Figure 11
, where heavy traces represent responses from the normotensive (OS) eye
and light traces are from the hypertensive (OD) eye. Histologic
analysis showed that few ganglion cells remained in the hypertensive
eye of this animal (10% of the density measured in the normotensive
eye), whereas there was no apparent loss of cells in other retinal
layers. A comparison of first-order macular responses from the two eyes
shows that although the amplitude of N1, N1P1, and P1N2 is greater for
the hypertensive eye, the amplitude of N2P2 is smaller. Comparison PN
amplitudes for the second-order responses shows an even greater
relative reduction in the hypertensive eye. Hypertensive injury was
associated with relatively less effect on either first- or second-order
perimacular responses. Similar effects on macular mfERG responses were
seen in all hypertensive eyes that had severe RGC loss.
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Figure 11. Comparison of macular and perimacular responses from the hypertensive
(OD, light traces) and normotensive (OS, heavy
traces) eyes shown in Figures 9
and 10
. Histologic analysis
showed that normalized (OD/OS) perifoveal RGC density in the
hypertensive eye was 0.11. See text for description of responses. First
order response calibration bars, 5 nV/deg2 and 25 msec;
second-order calibration bars, 2.5 nV/deg2 and 25 msec.
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First- and second-order macular responses from Figure 11
are replotted
in Figure 12
,
trace A, whereas responses from both eyes of the other six animals are
plotted as traces B through G. The traces in this figure are ordered
from greatest (A) to lowest (G) RGC loss in the hypertensive eye
(expressed as normalized RGC density measures, OD/OS) with values equal
to A, 0.11; B, 0.15; C, 0.21; D, 0.69; E, 0.92; F, 0.96; and G, 1.41.
Note that, for animals with severe hypertensive injury (A through C),
later peaks of the first-order response (left column) are smaller in
the hypertensive eye, whereas, for all animals, amplitude of peaks N1
and N1P1 is greater in the hypertensive eye. Furthermore, amplitudes of
all first-order peaks are larger in hypertensive eyes that had little
or no RGC loss (E through G). This may be explained if recordings from
normotensive (OS) eyes produced generally smaller first-order
responses, whereas hypertensive injury is associated with a selective
attenuation of later response peaks. Inspection of second-order
response traces (right column) shows that, when compared with the
normotensive eye, PN amplitude is greatly reduced in eyes with severe
hypertensive RGC loss (A through C), whereas there is a tendency for PN
amplitude to be somewhat greater in hypertensive eyes with little or no
RGC loss (F, G). These observations are illustrated in Figure 13
, where normalized macular mfERG response amplitude is plotted as a
function of normalized perifoveal ganglion cell density for each
response measure in all animals. For the first-order response,
amplitude of the three late peaks (P1N2, N2P2, P2N3) is strongly
correlated with histologic measures of ganglion cell survival (Figs. 13C
13D
13E)
. Amplitude of these late peaks decreases with
decreasing numbers of surviving ganglion cells in the hypertensive
eyes. Amplitude of the early peaks (N1, N1P1) was unaffected in even
the most severely injured eyes (Figs. 13A
3B)
. Second-order response
amplitude (PN) is also strongly correlated with the number of surviving
ganglion cells (Fig. 13F)
. Similar results were obtained for
correlations with either total RGC counts (sum of all 8 retinal sample
regions), perimacular counts (sum of three perimacular regions), or
peripheral counts (sum of four peripheral regions; data not shown).
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Figure 12. Comparison of first-order (left column) and second-order
(right column) macular responses from normotensive (OS,
heavy traces) and hypertensive (OD, light
traces) eyes of all seven animals. RGC loss in hypertensive
eyes was expressed as normalized (OD/OS) histologic measures of
perifoveal RGC density and yielded values for each animal equal
to: A, 0.11; B, 0.15; C, 0.21; D, 0.69; E, 0.92; F, 0.96; G, 1.41.
Calibrations for all first-order responses, 5 nV/deg2 and
50 msec. Calibrations for all second-order responses, 5.0
nV/deg2 and 50 msec.
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Figure 13. Summary of the correlation between amplitude for both first- and
second-order mfERG responses and counts for the number of surviving
cells in the perifoveal (PF) RGC layer. Measures of response amplitude
and cell counts as described in Methods. In each panel, response
amplitude and RGC counts from the hypertensive eye are each normalized,
for each animal, with respect to values obtained from the contralateral
normotensive eye. Straight-line plots in
each panel represent results of linear regression analysis.
(A) First order peak N1: regression slope = -0.11,
r = 0.27. (B) First order peak N1P1:
regression slope = +0.11, r = 0.22. (C)
First order peak P1N2: regression slope = +0.77, r = 0.80. (D) First order peak N2P2: regression
slope = +1.04, r = 0.93. (E) First
order peak P2N3: regression slope = +0.76, r =
0.64. (F) Second order peak PN: regression slope =
+0.60, r = 0.92.
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Discussion
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|---|
Ocular Hypertensive Retinal Injury
Earlier studies of the chronic ocular hypertensive monkey model
have shown that retinal injury appears to be limited to a loss of
RGCs.2
3
Our own results from histologic analysis show
that, even in eyes that had the most severe ganglion cell loss, there
was no evidence for loss of any other retinal cell type. Furthermore,
amplitudes of conventional ERG responses were relatively unaffected by
hypertensive injury. Because these responses are believed to reflect
primarily the activity of retinal cell types other than
RGCs,22
23
this finding is consistent with a
conclusion that hypertensive injury has relatively little effect on the
function of photoreceptors, bipolar cells, or amacrine cells.
Our histologic analysis, however, could detect only gross
manifestations of cellular injury such as loss of somas and
neuropil. More subtle structural changes would presumably not
be detected, and functional integrity may be compromised in cells that
otherwise have a normal histologic appearance. It is also possible that
some functionally distinct subset of non-RGC retinal cell was lost in
hypertensive eyes but that this loss was not obvious because it makes
up such a small fraction of all cells in the retinal layer. In
addition, any loss of displaced amacrine cells, which comprise
approximately 10% to 15% of cells in the macular RGC
layer,24
would have been counted as lost RGCs in our
analysis. The combination of histologic and electrophysiological
results, however, are consistent with a conclusion that hypertensive
injury is at least largely limited to a loss of RGCs.
Results of ganglion cell counts from both central and peripheral retina
showed that the degree of ganglion cell loss in hypertensive eyes was
similar in all retinal regions. This finding is in agreement with
earlier reports of histologic studies in ocular hypertensive
monkeys3
25
and is also consistent with the fact that
macular mfERG response amplitude was similarly correlated with RGC
counts from either central or peripheral retina. It is possible that
regional differences in RGC loss might have been seen if the animals
had been killed at an earlier time. That is, RGC loss may have occurred
earlier or at a greater rate in some regions, but this difference was
not apparent at 16 months after IOP elevation. Also, our analysis did
not include a discrimination between morphologically distinct RGC
subtypes and thus does not permit any conclusion with regard to
regional subtype sensitivity to hypertensive injury. Furthermore, the
variability inherent in our method for quantifying RGC loss does not
allow us to exclude the presence of small regional differences.
Electrophysiological Measures of RGC Injury
Flash, OP, and 30-Hz flicker ERG responses are thought to reflect
primarily the activity of retinal cells other than
RGCs.22
23
26
27
28
29
These responses were chosen to provide a
functional measure of injury to non-RGC retinal cells. The weak
correlation that exists between amplitudes of these responses and
histologic measures of RGC injury may be taken as evidence for two very
different interpretations: (1) Ocular hypertension results in injury to
retinal cells other than RGCs. The extent of this injury is much less
than that seen for RGCs but is somewhat correlated with the degree of
RGC loss. (2) These ERG responses reflect a small but significant
contribution from RGC activity. A detailed discussion of these two
possibilities is beyond the scope of this article. It is sufficient to
note that the results of histologic analysis showed no evidence for
injury to any retinal cell type other than RGCs and that either of
these two interpretations for the conventional ERG results is
consistent with this conclusion.
The first-order mfERG responses obtained from normal eyes in this study
are similar in waveform and kinetics to those obtained from humans
under similar conditions.21
30
These responses, however,
are rather different in comparison to published responses obtained from
rhesus macaque,18
which are much more oscillatory in
nature. Although it is possible that this may reflect a species
difference, it is more likely the consequence of different recording
conditions. First, the rhesus recordings were made using the
contralateral cornea as the voltage reference. This method has been
reported to enhance contributions of an "optic nerve head
component" (ONHC), which gives the response a more oscillatory
appearance.9
Second, the responses in the present study
were made using analog 60-Hz notch filtering to remove line noise. A
comparison of responses recorded from a normal eye with (heavy traces)
and without (light traces) notch filtering is shown in Figure 14
. Note that notch filtering eliminates late oscillatory components in
the first-order macular response, whereas there is a moderate
attenuation of N1, N1P1, and P1N2. Notch filtering also attenuates an
early oscillation in the second-order response but has relatively less
effect on peak PN. Thus, it may be assumed that notch filtering had a
moderate effect to attenuate the amplitude of peaks that were measured
in both first- and second-order responses of the present study. The
attenuation of oscillatory components is more pronounced and, because
these components have a presumed origin in activity of inner retinal
cells,9
18
it is possible that inner retinal contributions
to the responses of the present study were reduced by notch filtering.
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Figure 14. First- (A) and second-order (B) macular
(top traces) and perimacular (bottom
traces) responses obtained from a normal eye either with
(heavy traces) or without (light traces) use of
the 60-Hz notch filter. Recording was first made with the notch filter
active and immediately followed by a recording with the notch filter
inactivated. Stimulus and recording conditions were, otherwise,
identical with those described in the Methods section. Calibrations, 10
nV/deg2 and 20 msec (A), and 5
nV/deg2 and 20 msec (B).
|
|
Ocular hypertensive injury was associated with a relative reduction in
amplitude for peaks in both the first- and second-order multifocal ERG
response (see Figs. 11
12
). These same response components are
prominent in normal macular retina where RGC density is high and
relatively smaller outside the macula where RGC density is low.
Amplitude of these response components is highly correlated with the
number (density) of surviving ganglion cells, with slopes for this
relationship ranging from approximately 0.6 to 1.0 (see Fig. 13
). Also,
RGC loss is the only obvious histologic consequence of hypertensive
retinal injury. Taken together, these observations are consistent with
a conclusion that activity in RGCs makes a contribution to both the
first- and second-order multifocal ERG responses in monkey.
For the conventional flash response, a-wave amplitude was uncorrelated
with RGC counts in hypertensive eyes, whereas the b-wave showed a very
weak correlation (see Fig. 8
). The early negative (N1) and positive
(N1P1) peaks of the first-order multifocal ERG response were also
uncorrelated with RGC counts. These results are consistent with a
previous report that concluded that the early negative and positive
peaks in the first-order mfERG response from humans appear to be driven
by activity in the same retinal cells that drive the flash a-wave and
b-wave.31
Although N1 and N1P1 amplitudes were not
correlated with RGC loss (or IOP history), there was a tendency for the
amplitude of these peaks to be relatively greater in the hypertensive
(OD) eye (see Figs. 12 13
). This observation may be a consequence of
the order for recording responses from the two eyes. Because OD was
always recorded first and OS was recorded approximately 45 minutes
later, the additional time under anesthesia might have resulted in a
generalized decrease in OS response amplitude that is similar for all
animals. For this reason, OS responses from Figure 12
were linearly
scaled by a factor that made peak N1 amplitude equal for both eyes.
Scaled OS responses have been replotted with the original OD responses
in Figure 15
. Note that, except for linear scaling of OS responses, this figure is
identical with Figure 12
. Also note that second-order OS responses have
been scaled by the same factor used for the first-order response. A
comparison of Figures 12
and 15
shows that scaling the OS response in
this manner makes OD and OS responses more similar in animals that had
little or no RGC loss and enhances the difference in animals having
severe RGC loss. When amplitude measures were made using scaled OS
responses for normalization and results replotted as in Figure 13
, the
slopes for linear regression were unchanged but the correlation
coefficients for P1N2, N2P2, P2N3, and PN increased (not shown).
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Figure 15. Responses from Figure 12
are replotted here. For each animal, the
first- and second-order response from the normotensive (OS,
heavy trace) eye has been linearly scaled by a factor
which makes peak N1 amplitude equal to that obtained from the
hypertensive (OD, light trace) eye. Responses to the
hypertensive eye are identical with those plotted in Figure 12
. OS
responses were linearly scaled by the following factors: A, 1.46; B,
1.44; C, 1.34; D, 1.47; E, 1.22; F, 1.34; G, 1.41. Calibrations are 5
nV/deg2 and 50 msec (first-order responses), and 5.0
nV/deg2 and 50 msec (second-order responses).
|
|
Multifocal ERG as a Measure for RGC Function
Perhaps the best evidence for an RGC-specific component in the
mfERG is found in nasotemporal response asymmetries, which have
properties consistent with their generation by impulse conduction in
axons of the retinal nerve fiber layer.8
9
This ONHC has
been shown to be blocked by both TTX and NMDA (agents that block
voltage-gated sodium channels and glutamatergic ligand-gated channels,
respectively) in monkey14
18
and has also been shown to be
reduced in both glaucoma patients and glaucoma suspects.13
Because TTX- and NMDA-sensitive mechanisms are relatively localized to
inner retinal cells, this provides further support for an inner retinal
origin of the ONHC. As discussed earlier, the recording conditions for
the present study likely minimized any ONHC contribution. Using
recording conditions similar to those used in the present study, we
have previously identified a small OHNC in responses from normal monkey
eyes (unpublished observations). We also found that it could be blocked
by intravitreal application of TTX but that TTX had a much greater
effect on other components of the first- and second-order response,
including the peaks that were most affected by RGC loss in this study.
It was recently reported15
that patients with glaucoma
show evidence for amplitude reduction in peaks of first- and
second-order macular responses, which are similar to the findings of
the present study. It has also been reported that branch retinal artery
occlusion results in relatively selective reduction of late components
in the first-order response and complete elimination of the
second-order response in that retinal region corresponding to ischemic
insult.11
Because branch artery occlusion is thought to
result in ischemic insult, which is relatively localized to inner
retinal elements, this finding is consistent with an inner retinal
origin for these response components. More recently, however, changes
in implicit times but not amplitude for peaks of the first-order
response were seen in patients with glaucoma,16
whereas,
in another study, measures of either first- or second-order responses
showed no correlation with visual sensitivity loss in subjects with
glaucoma.17
Early studies using the COHT monkey model of glaucoma showed that
pattern ERG (PERG) and pattern VEP (PVEP) amplitude were reduced in
eyes having normal conventional flash ERG responses,4
5
6
and similar results have been obtained in clinical glaucoma studies.
Results of the present study as well as results from other work with
animal models and human subjects are consistent with the notion that
mfERG responses contain significant contributions from inner retinal
cells including RGCs. The development of optimal stimulus and recording
conditions as well as effective strategies for response component
analysis can be expected to enhance the utility of the mfERG for
assessment of function in the visual pathways. Evaluation of these
methods in animal models is especially useful in this regard.
|
Acknowledgements
|
|---|
The authors are grateful for the expert assistance of James Burke,
who performed the laser treatment on all animals in this study, Don
Long, who provided assistance with animal preparation and anesthesia,
and also for many technical discussions with Erich Sutter.
|
Footnotes
|
|---|
Submitted for publication December 20, 1999; revised August 10, 2000; accepted September 20, 2000.
Commercial relationships policy: E.
Corresponding author: William A. Hare, Department of Biological Sciences, RD-2C, Allergan, Inc., 2525 Dupont Drive, Irvine, CA 92713. hare_william{at}allergan.com
|
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Top
Abstract
Introduction
