Download Survival of some photoreceptor cells in albino rats following

Investigative Ophthaltnology
January 1976
64 Reports
From the Laboratory of Vision Research,
National Eye Institute, and the Laboratory of
Biochemical Genetics, National Heart and Lung
Institute, National Institutes of Health, Bethesda,
Md. 20014. Submitted for publication Aug. 15,
1975.
REFERENCES
Fig. 3. Oscillogram of a Ca++-induced spike from
chick retinal pigment epithelium in culture. The
grid indicates 10 millivolts vertically and 200 milliseconds horizontally. Positively is upward.
1. Newsome, D. A., Fletcher, R. T., Robinson,
W. G., Jr., et al.: Effects of cyclic-AMP and
sephadex fractions of chick embryo extract on
cloned retinal pigmented epithelium in tissue
culture, J. Cell Biol. 61: 369, 1974
2. Nelson, P. G., Ruffner, B. W., and Nirenberg,
M.: Neuronal tumor cells with excitable membranes grown in vitro, Proc. Nat. Acad. Sci.
64: 1004, 1969.
3. Hudspeth, A. J., and Yee, A. G.: The intercellular junctional complexes of retinal pigment epithelia, INVEST. OPHTHAL. 12: 354,
+h
centration of Ca in the culture media less than
0.01 millimoles. Since the solution has to be applied near the cells in question, the local concentration required to induce these responses must
be higher than this. An order of magnitude estimate of this triggering concentration can be obtained by assuming that the pigment epithelium
cell membrane approximates a K+ electrode, an
assumption supported by the fact that K+ is very
effective in depolarizing these cells (Table I ) .
The depolarization produced by 10 microliters of
0.5 M KC1 was 36 ± 12 mv., which would be
produced by about a 20 to 40 millimole increase
in K+ outside the cell. The triggering concentration of Ca++ is probably near this range.
It would be interesting to know whether this
curious phenomenon induced by Ca++ plays any
role in the normal function of retinal pigment
epithelium. As far as we know spikes have only
been found in nerve and muscle and never in
any epithelial cell.
The hyperpolarizing wave that precedes the
spikes bears some resemblance to the c-wave of
the electroretinogram which is a hyperpolarizing
potential induced in intact retinal pigment epithelium by light impinging on the photoreceptors.4 Steinberg and Miller5 have suggested that
the light-induced Na+ conductance change in the
photoreceptors leads to a local reduction of extracellular K+ and hence to a hyperpolarization of
the retinal pigment epithelium. Some direct support for this hypothesis has recently been obtained.0 If Ca++ were released from the photoreceptors by light, as has also been suggested,7
the late hyperpolarization it produces on pigment
epithelial cells could also contribute to the c-wave
of the electroretinogram.
We would like to thank Drs. Fernando de Meto
and Dr. Marshall Nirenberg for their assistance in
this project.
1973.
4. Steinberg, R. H., Schmidt, R., and Brown,
K. T.: Intracellular responses to light from
cat pigment epithelium: origin of the electroretinogram c-wave, Nature (London) 227:
728, 1970.
5. Steinberg, R. H. and Miller, S.: Aspects of
electrolyte transport in frog pigment epithelium, Exp. Eye Res. 16: 365, 1973.
6. Oakley, B., and Green, D. G.: The ionic
basis of the c-wave of the electroretinogram,
ARVO meeting abstracts, 1975, p. 5.
7. Yoshikami, S.t and Hagins, W. A.: Light,
calcium, and the photocurrent of rods and
cones, Biophys. J. II. Abstract 1TM-E 16,
1971.
Survival of some photoreceptor cells in
albino rats following long-term exposure to continuous light. MATTHEW M.
LAVAIL.
Fischer albino rats, seven weeks of age, were
exposed to continuous light at 65 foot-candle
incident illuminance for up to 264 days. Other
Fischer rats, seven months of age, were exposed
to continuous light at 140 foot-candle incident
illuminance for up to 147 days. In all cases, a
small percentage of the photoreceptors survived.
The identification of the surviving cells as photoreceptors was made by light microscopy on the
basis of nuclear heterochromatin pattern and staining and by electron microscopy by the presence
of ribbon synapses and ciliary basal bodies with
ciliary filaments. No outer segment membranes
were observed. The percentage of cones progressively increased from the normal 1.5 per cent
to about 60 per cent with increasing exposure
time, indicating that cone cells are more resistant
than rods to destruction by constant light.
Continuous illumination causes photoreceptor
cell degeneration in normal albino rats. The rate
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Volume 15
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Reports 65
Table I. Number of photoreceptor cells* in sections of retinas from Fischer rats in different
lighting conditions
Lighting condition
CyL
—
—
3.4 months
Perfusion
CyL
CL
CL
Age into CL:
7 weeks
7 months
—
CL duration:
31 days
54 days
Age at fixation:
8 months
10.4 months
3.4 months
Type of fixation:
Immersion
Perfusion
Perfusion
Posterior retina:
Cones
4.4 ± 0.4
3.4 ± 0.4
1.7 ±0.3
2.3 ± 0.4
Rods
275.0 ± 4.2
228.3 ± 3.6
6.1 ±0.7
3.7 ±0.6
Per cent cones
1.6
1.5
21.8
38.3
Peripheral retina:
Cones
1.9 ±0.4
2.6 ± 0.4
2.1 ±0.3
1.1 ±0.2
Rods
161.1 ± 10.6
125.9 ±6.6
18.2 ± 2.4
14.3 ± 2.2
1.6
Per cent cones
1.5
10.3
7.1
'Mean ± S.E.M. per 180 ftm length of retina; counts based on fifteen 180 /im lengths, five consecutive lengths in each of
three sections, beginning in each section either about 400 /an from the optic disc and progressing peripherally (posterior retina) or at the ora serrata and progressing centrally (peripheral retina). CL, continuous light; CyL, cyclic light.
Table II. Number of photoreceptor cells* in sections of retinas from Fischer rats
Lighting condition
CL
Age into CL
CL duration
Age at fixation
Type of fixation
Cones
Rods
Per cent cones
7 weeks
178 days
7.6 months
Immersion
20.2 ± 2.0
15.6 ± 1.8
56.4
CL
7 weeks
264 days
10.4 months
Perfusion
14.2 ±2.0
8.2 ± 1.8
63.4
CL
7 months
70 days
9.3 months
Perfusion
42.2 ± 1.6
28.8 ± 1.1
59.4
CL
7 months
147 days
11.9 months
Perfusion
31.8 ±5.0
19.2 ± 1.7
62.4
"Mean ± S.E.M. per section of retina from optic disc to oraserata; counts based on five sections. CL, continuous light.
of the degeneration is influenced by the age 1 and
body temperature2 of the animal, the intensity of
illumination,3- 4 and the exposure time.2' 3< 5'7
Behavioral studies of albino rats whose retinas
were apparently devoid of photoreceptor cells due
to exposure to continuous light in the 20 to 70
foot-candle incident illuminance range indicate
that these animals are able to perform light-dark
and pattern discrimination as well, or almost as
well, as normal control animals.8'10
It had previously been thought that all photoreceptors degenerate in RCS rats with inherited
retinal dystrophy because none were recognizable
by conventional histology in animals of several
months of age. In a recent behavioral and cytological study, however, it was found that these
rats can discriminate light intensity at ages up
to 2 years, and, using cytological procedures that
intensify photoreceptor heterochromatin staining,
numerous surviving photoreceptor cells were observed.11 In view of these observations, it was
of interest to examine the retinas of albino rats
maintained in long-term continuous light with the
same cytological procedures as used for study
of the RCS rats.
Methods. One litter of nine rats, inbred de-
scendents of Cesarean-derived Fischer rats
(Charles River Breeding Laboratories, Inc., Wilmington, Mass.), were born and reared in a 12-hour
light-12-hour dark environment in cages illuminated
at less than 15 foot-candles and at a room temperature of 24 ± 1° C. The rats were maintained
in transparent polycarbonate cages with stainless
steel wire-bar covers with food (Charles River 18
RF pellets) and water provided ad libitum. The
bedding used was a No. 5 birch cube ("Bettachip," Northeastern Products Corporation, Warrensburg, N. Y.). At seven weeks of age, six of
the rats were transferred to a room with constant
fluorescent lighting with illuminance at cage level
of about 16 foot-candles. In addition, a lamp
containing two 15W fluorescent bulbs (ITT, Daylight No. F15T8/D) was positioned about 70 cm.
above the bottom of the cage. The lamp continuously provided an incident illuminance of
about 65 foot-candles at the floor of the cage. This
illuminance designation does not allow for calculation of the effective retinal illuminance, but
it is used to facilitate comparison with published
studies on constant light damage. The eyes were
taken from two rats after 54 days in constant
light, from two rats after 178 days, and from
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Investigative Ophthalmology
January 1976
66 Reports
two after 264 days. At each time eyes were taken
from one littermate control rat kept in cyclic light.
Throughout the light exposure the temperature in
the cage remained at 24 ± 1° C.
Three additional Fischer rats were exposed with
a somewhat higher illuminance level beginning at
seven months of age. Fluorescent lights were positioned about 40 cm. over a white, opaque, polyethylene cage with an in-cage feeder so that the
floor of the cage had a uniform incident illuminance of about 140 foot-candles. Eyes were
taken from one rat after 31 days in constant light,
from one after 70 days, and from one after 147
days.
Eyes were either enucleated and immersed in
fixative or dissected out after vascular perfusion
of the rats with fixative (Tables I and II). The
eyes were then postfixed in osmium tetroxide,
stained en bloc with uranyl acetate and embedded
in an Epon-Araldite mixture as described elsewhere.11- 12 The combined use of glutaraldehyde,
formaldehyde, and calcium ions in the initial fixative and the use of en bloc staining with uranyl
acetate result in an increased binding of toluidine
blue to photoreceptor nuclear heterochromatin.13
One to 1.5 Mm sections were cut such that the
retina from the optic disc to the ora serrata was
included in a single section, and selected areas
were examined by electron microscopy.
Results.
Rats in cyclic light. The retinas from the control
rats maintained in cyclic light were normal (Fig.
1, F). Most of the photoreceptor cells had small
nuclei, usually less than 5.5 i"m in diameter, with
one large, central clump of heterochromatin. These
features were typical of rod cells. About 1.5 per
cent of the photoreceptor cell nuclei contained
multiple small clumps of heterochromatin, were
larger than about 5.5 jum, were usually somewhat
ovoid in shape and were located in the outer onethird to one-half of the outer nuclear layer (Fig.
1, F and Table I ) . Cells with these features have
been described as cones in the rat retina (see
references in 11) and although they do not display
typical cone inner and outer segment structure,
they will be referred to as cones in this report to
distinguish them from the rods.
Rats placed into continuous illumination at seven
weeks of age. After 54 days in constant light the
outer nuclear layer in most regions of the retinas
was reduced to one incomplete row of photoreceptor nuclei (Fig. 1, A). The outer plexiform
layer essentially was missing in these areas, and
many of the photoreceptor cells appeared to be
displaced into the outermost part of the inner
nuclear layer. In the apparent absence of inner and
outer segments, the identification of the strongly
basophilic nuclei as those of photoreceptor cells
was supported by their arrangement as a row
continuous with an outer nuclear layer comprised
of one to two rows of nuclei in a small region of
peripheral retina (Fig. 1, B). The photoreceptor
cells in this region had remnants of at least some
inner segments and an underlying outer plexiform
layer. Degeneration and disappearance of photoreceptor cells was more extensive at the posterior
pole of the eye than at the periphery (Table I ) .
After 178 and 264 days in constant light, although most of the photoreceptor nuclei had
disappeared from the retinas (Table 11°), some
could be seen at the outermost aspect of the inner
nuclear layer either as one or two nuclei per field
(Fig. 1, C) or in clusters of several nuclei (Fig.
1, D). Occasionally, the nuclei were displaced into
the middle or inner part of the inner nuclear layer
(Fig. 1, E).
Fewer photoreceptor nuclei appeared to survive
in the retinas of rats exposed to constant light for
264 days than in those retinas exposed for 178
days (Table II). Other differences in the retinas
at these time points were more gliosis, greater
disruption of the inner layers, and increased
vascularization of the pigment epithelium from
the retinal capillaries in animals with the longer
exposure. These features have been described
previously14; the only exception was that no
anastomoses of retinal and choroidal vessels were
"Note that because so few surviving cells were present at
later intervals, cell counts are expressed as number per
retinal section in Table II rather than per 180 /im length
of retina as in Table I. The retinal length from optic
disc to ora serrata in a three-month-old Fischer rat is
approximately 5,000 /im, or 28-180 fim lengths. Thus,
the number of cells per section in Table II can be
divided by 28 to obtain an approximate number per 180
/im retinal length to compare with data in Table I.
Fig. 1. A through E. Light micrographs of retinas from Fischer rats after continuous light
exposure for different periods of time. A, 54 days of exposure; posterior retina. An incomplete
row of photoreceptor nuclei is present. B, 54 days of exposure; peripheral retina. One to two
rows of photoreceptor nuclei remain, as do some remnants of inner and outer segments.
C, 178 days of exposure. A few single photoreceptor nuclei are present, and the heterochromatin of a presumed rod cell nucleus appears contracted (arrow; cf. with Fig. 1, F). D,
264 days of exposure. A cluster of surviving photoreceptor nuclei is illustrated. E, 178 days of
exposure. A photoreceptor nucleus has been displaced or has migrated deep into the inner
nuclear layer (arrow). F, Outer nuclear layer of a 3.4-month-old Fischer rat reared in cyclic
light. Three cone nuclei (arrows) are present among the rod nuclei, c, capillary; pe, pigment
epithelium. 1 to 1.5 pm Epon-Araldite sections. Toluidine blue. All x960.
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Volume 15
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Reports 67
jr~-?«f»fc
B
\
Fig. 1. For legend, see opposite page.
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Investigative Ophthalmology
January 1976
68 Reports
observed in the present study. However, 1 to 1.5
Wn plastic sections do not lend themselves to
extensive serial sectioning which might be required
to observe such a feature.
Rods apparently respond more quickly than
cones to the damaging effects of continuous illumination because initially they were lost in higher
proportion. For example, after 54 days in constant light almost 98 per cent of the rods had
disappeared, but only about 60 per cent of the
cones were missing from the posterior retina. In
the peripheral retina almost 90 per cent of the
rods were missing, but few, if any, of the cones
had disappeared (Table I ) . This disproportionate
loss of rods resulted in the progressive increase in
the percentage of- cones, such that after 178 and
264 days of constant light exposure about 60 per
cent of the photoreceptors were classified as cones
(Table II).
The classification of cell types based on nuclear
morphology was relatively simple in the 54-day
retinas, but became more difficult at later intervals.
At the later intervals most of the cells became
ovoid instead of rounded, and the central clump of
heterochromatin of most rods appeared contracted
(Fig. 1, C). Some small nuclei counted as rods
may have been tangential sections through cone
nuclei. Thus, the percentage of cones in the 178and 264-day animals may be underestimated.
The retinas of animals exposed 178 and 264
days to continuous illumination were also examined
by electron microscopy, and features of the surviving photoreceptors at both intervals were
similar. In residual outer plexiform layers, photoreceptor synaptic terminals were observed which
were not in the plane of section with the parent
cell body (Fig. 2, A and B). In other cases, cells
with clumped nuclear heterochromatin were found
with synaptic ribbons in their perikaryal cytoplasm
(Fig. 2, C). In photoreceptor perikarya, conspicuous Golgi complexes, abundant ribosomes,
rough endoplasmic reticulum, and ciliary basal
bodies (Fig. 2, D), some with ciliary filaments
(Fig. 2, E ) , usually were found, but rod outer
segment membranes were never observed. Occasionally, small whorls of membranes were seen
between cells; they were not in obvious continuity
with any cell type and were probably a degenerative feature. Ribbon and conventional synapses
were present in the inner plexiform layer.
Rats placed into continuous illumination at seven
months of age. Photoreceptors degenerated slightly
faster when older rats were placed in constant
light of a somewhat higher illumination (Table I ) .
Other features of photoreceptor degeneration in
the older rats were similar to those of the younger
animals (Table II).
Discussion. It has been reported that all photoreceptor cells are missing from albino rat retinas
following continuous light exposure of 70 footcandle incident illuminance for 30 days7 or 36
foot-candle incident illuminance for 120 days.10
These retinas may have contained some surviving
photoreceptors, perhaps undetected with the
cytologic procedures that were employed, because
in the present study some photoreceptors survived
more than 8.5 months of continuous light at an
incident illuminance of 65 foot-candles (Table
II). The survival of these cells may have been
due to the age of the animals, since the rats were
seven weeks old at the start of light exposure.
Photoreceptors in seven-week-old rats are more
resistant to degeneration than are those in older
animals.1 The report of age-dependency,1 therefore, prompted the use of an even higher (140
foot-candle) illuminance level and a cage with
white reflective sides in an attempt to augment
the destruction of photoreceptor cells in sevenmonth-old rats. Under these conditions most of the
photoreceptor cells had disappeared after 31 days
of exposure (Table I), but about 50 cells per
section still remained after 147 days of exposure
(Table II). It is also possible that differences in
rat strains in the various studies might have influenced photoreceptor survival, although Noell
and co-workers2 found that four different albino
strains showed about the same susceptibility to the
damaging effect of light.
If some photoreceptor cells were, in fact,
present in the retinas of rats described in
behavioral studies on light-damaged retinas,8'10
thes6 surviving cells may have mediated the visually guided behavior. In the present study the
surviving cells were synaptically related to postsynaptic processes of the inner nuclear layer, and
the synaptic cytoarchitecture of the inner plexiform
layer appeared intact. After long-term constant
light exposure, rats showed progressive behavioral
performance deficits10 which might reflect either
the progressive loss of residual photoreceptor cells
as described in the present report or subtle
synaptic rearrangements in the visual system.15
Although it has been suggested that other
retinal cells may be responsible for the behavioral
response to light in the rats with light-damaged
retinas,9 the simplest hypothesis is that some
surviving photoreceptor cells mediate the behavioral responses. If so, the transduction mechanism remains unclear since outer segments are the
first part of the photoreceptor cells to be damaged
by light,5- G and the cells in the present study
snowed no outer segments. Possibly photoreceptive
molecules reside in the plasma membrane or other
parts of the surviving photoreceptor cells.
It is thought that the rat retina contains both
rods and cones (see references in 11). Cones in
albino rats appear to be more resistant than rods
to destruction by constant light. The reason for the
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Volume 15
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Reports 69
Fig. 2. Electron micrographs of retinas from Fischer rats after continuous light exposure for
either 178 or 264 days. A and B, photoreceptor synaptic terminals located in the residual outer
plexiform layer not in the plane of section with the parent cell bodies. Both x22J900. C, a surviving photoreceptor cell is illustrated with synaptic ribbons in its cytoplasm. x22,900. D, basal
bodies (arrows) are present in a surviving photoreceptor cell, p, pigment epithelial cell processes, x 17,540. E, A basal body in another photoreceptor cell has ciliary filaments extending
from it (arrow). x20,260.
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Investigative Ophthalmology
January 1976
70 Reports
difference in sensitivity between rods and cones
is not clear, but may depend upon a basic difference in the metabolism of the two cell types.
Thus, constant light joins a number of destructive
agents that affect rods earlier or to a greater extent
than cones, including iodoacetate,16 X-irradiation,1G
and the mutant genes responsible for inherited
retinal degeneration in the mouse (Carter and
LaVail, in preparation), inherited retinal dystrophy
in the rat (LaVail, in preparation), progressive
retinal atrophy in the Irish Setter17 and Norwegian
Elkhound (see references in 17), and retinitis
pigmentosa in man. is
The author wishes to thank Carolyn O. Gerhardt, Patricia Ann Ward, and Donna M. Colicchio for technical assistance.
From the Department of Neuropathology, Harvard Medical School and the Department of
Neuroscience, Children's Hospital Medical Center,
Boston. This investigation was supported in part
by United States Public Health Service Research
Grant EY-01202 and Career Development Award
EY-70871 from the National Eye Institute. Submitted for publication Aug. 18, 1975. Reprint
requests: Dr. Matthew LaVail, Department of
Neuroscience, Children's Hospital Medical Center,
300 Longwood Ave., Boston, Mass. 02115.
Key words: light-induced photoreceptor degeneration, rods and cones, albino rats.
REFERENCES
1. O'Steen, W. K., Anderson, K. V., and Shear,
C. R.: Photoreceptor degeneration in albino
10. Bennett, M. H., Dyer, R. F., and Dunn, J. D.:
Visual-deficit following long-term continuous
light exposure, Exp. Neurol. 38: 80, 1973.
11. LaVail, M. M., Sidman, M., Rausin, R., et al.:
Discrimination of light intensity by rats with
inherited retinal degeneration. A behavioral
and cytological study, Vision Res. 14: 693,
1974.
12. LaVail, M. M., and Battelle, B.-A.: Influence
of eye pigmentation and light deprivation on
inherited retinal dystrophy in the rat, Exp.
Eye Res. 21: 167, 1975.
13. Ladman, A. J.: Immersion fixation of dog
retina: factors affecting heterochromatin
stainability of photoreceptor nuclei, Anat. Rec.
175: 365,. 1973. (Abstr.)
14. O'Steen, W. K., Shear, C. R., and Anderson,
K. V.: Retinal damage after prolonged exposure to visible light. A light and electron
microscopic study, Am. J. Anat. 134: 5, 1972.
15. Fifkova, E.: Effect of light on the synaptic
organization of the inner plexiform layer of
the retina in albino rats, Experientia 29: 851,
1973.
16. Noell, W. K.: Aspects of experimental and
hereditary retinal degeneration, in: Biochemistry of the Retina, Graymore, C. N.,
editor. London, 1965, Academic Press, pp. 5172.
17. Aguirre, G. D., and Rubin, L. F.: Rod-cone
dysplasia (progressive retinal atrophy) in
Irish Setters, J. Amer. Vet. Med. Assoc. 166:
157, 1975.
18. Verhoeff, F. H.: Microscopic observations in
a case of retinitis pigmentosa, Arch. Ophthalmol. 5: 392, 1931.
rats: dependency on age, INVEST. OPHTHALMOL. 13: 334, 1974.
2. Noell, W. K., Walker, V. S., Kang, B. S.,
et al.: Retinal damage by light in rats, INVEST.
OPHTHALMOL. 5: 450,
1966.
3. Noell, W. K., and Albrecht, R.: Irreversible
effects of visible light on the retina: role of
vitamin A, Science 172: 76, 1971.
4. Weisse, I., Stotzer, H., and Seitz, R.: Ageand light-dependent changes in the rat eye,
Virchows Arch. Pathol. Anat. Histol. 362:
145, 1974.
5. Kuwabara, T., and Gorn, R. A.: Retinal
damage by visible light, Arch. Ophthalmol.
79: 69, 1968.
6. Grignolo, A., Orzalesi, N., Castellazzo, R.,
et al.: Retinal damage by visible light in
albino rats, Ophthalmologica 157: 43, 1969.
7. O'Steen, W. K., and Anderson, K. V.: Photically evoked responses in the visual system of
rats exposed to continuous light, Exp. Neurol.
30: 525, 1971.
8. Anderson, K. V., and O'Steen, W. K.: Blackwhite and pattern discrimination in rats without photoreceptors, Exp. Neurol. 34: 446,
1972.
9. Bennett, M. H., Dyer, R. F., and Dunn, J. D.:
Light-induced retinal degeneration: effect
upon light-dark discrimination, Exp. Neurol.
34: 434, 1972.
Effect of the ocular media on the main
wavelengths of argon laser emission.
O L E G P O M E R A N T Z E F F , H I R O S H I KANEKO,
R.
H.
J. W .
D O N O V A N , C.
L.
SCHEPENS, AND
MCMEEL.
The purpose of this study is threefold: (1)
spectrally selective scattering of light in the eye
media has been reported.1-2 We show that it
affects the argon green wavelength (514.5 nm.)
and the argon blue wavelength (488 nm.) differently during photocoagulation. (2) The presence
of yellow pigment has been shown3 to absorb
more strongly the argon blue than green. We show
that it enhances the difference in effect due to
scattering alone. (3) In the human eye, yellow
staining and scattering of the media are both
increased with age.2- 4 The influence of this
factor is noticeable when comparing the results of
photocoagulation in old and young patients.
Materials and methods. Eight owl monkeys
were selected for the study of scattering media
devoid of yellow pigment. Two macaques were
utilized for study of the effect of yellow pigment
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