No. 10
Reports
1433
Evidence for Rod Outer Segment Lipid Peroxidotion following
Constont Illuminotion of the Rot Retino
Rex D. Wiegond, Normo. M. Giusto, Laurence M. Rapp, and Robert E. Anderson
Constant illumination for three days (100-125 foot-candles)
caused degeneration of photoreceptor cells in the albino rat
retina and was accompanied by a reduction in the levels of
docosahexaenoic acid (22:6w3), the major polyunsaturated
fatty acid in rod outer segments (ROS). An increase in the
level of lipid conjugated dienes, a measure of lipid hydroperoxides, also was observed in ROS after 24-72 hours of
constant illumination. These data support the suggestion that
peroxidation of long-chain polyunsaturated fatty acids in ROS
may be a factor in light-induced retinal degeneration. Invest
Ophthalmol Vis Sci 24:1433-1435, 1983
The retina of the albino rat exposed to constant illumination at levels below the threshold for thermal
burns can be damaged irreversibly (reviewed in ref.
1). Initial ultrastructural changes are found in the rod
outer segments (ROS) of photoreceptor cells and consist of small, vesicular pertubations in the membranous
discs. Complete disorganization of the ROS rapidly
follows, and cell death may be observed as early as 12
hours after the onset of light. As would be expected,
these ultrastructural changes are accompanied by the
loss of retinal function, measured as a progressive decrease in the amplitude of the electroretinogram.
The biochemical etiologic factors of light-induced
retinal degeneration are not known, although it has
been suggested that peroxidation of rod outer segment
(ROS) membrane lipids may be involved.2'3 ROS
membranes are ideal substrates for peroxidative reactions, since the membrane phospholipids contain
the highest level of long-chain polyunsaturated fatty
acids of any membrane studied thus far.4 The major
polyunsaturate, docosahexaenoic acid (22:6<o3), is
especially susceptible to peroxidation.5
We recently have begun a series of experiments to
investigate the role of lipid peroxidation reactions in
light-induced retinal degenerations in the albino rat.3
In this paper, we present evidence for the specific loss
of 22:6co3 from ROS membranes during constant illumination and for the increased production of lipid
hydroperoxides.
Materials and Methods. Female albino rats (Sprague
Dawley) weighing between 225-275 grams were maintained in our vivarium under cyclic light (12 hours
light/12 hours dark) of about 10-15 foot-candles (ftc) at the front of the cage nearest the light. One week
prior to any experiment, the rats were placed in a
metabolic chamber (Freas Model 818, Precision Scientific, Chicago, IL) at 25°C under the same lighting
regime at 10-15 ft-c provided with a Vita-Lite fluorescent lamp of 40 watts (Duro-Test Corp., North
Bergen, NJ). The experiment was begun by exposing
the rats at the usual time of light onset to constant
illumination of either 10-15 ft-c or 100-125 ft-c. At
various times thereafter, the animals were sacrificed
and their retinas were removed for biochemical analysis. Retinal tissue also was examined for morphologic
changes typical of light damage. Dark-adapted animals
or animals exposed for 1 hour at 10-15 ft-c (L-l Hr)
served as controls. Experimental animals were exposed
for 1 day (H-l Dy) or 3 days (H-3 Dy) to 100-125 ftc of constant illumination. Dark-adapted retinas were
processed under dim red illumination until lipid extraction. Retinas from L-l Hr, H-l Dy, and H-3 Dy
animals were processed in dim room light.
ROS were isolated by discontinuous sucrose gradient
centrifugation. All solutions were saturated with argon
to minimize lipid oxidation during the preparation
and extraction procedures. Retinas (10 or less) were
homogenized gently in 6 ml of 1.175 g/ml sucrose
buffered with 10 mM Tris-acetate (pH 7.4) containing
70 mM NaCl, 2 mM MgCl 2 , and 0.1 mM EGTA.
Homogenization was achieved by 10 strokes of a Teflon
pestle #21G (TRI-R Instruments, Inc., Rockville
Centre, NY) in a glass homogenization tube #S35 (TRIR Instruments, Inc.). Clearance between pestle and
tube was 0.11-0.15 mm. The homogenate was transferred to a 17-ml cellulose nitrate centrifuge tube and
overlaid sequentially with 6 ml of 1.140 g/ml sucrose,
followed by 5 ml 1.115 g/ml sucrose. These latter two
sucrose solutions were buffered with 10 mM Tris-acetate (pH 7.4) and contained 0.2 mM MgCl2 and 0.1
mM EGTA. The sucrose density gradient tube was
spun at 82,000 X g for 2 hours at 4°C in a swinging
bucket centrifuge. The sucrose solution above 2 mm
of the 1.115/1.140 interfacial band was discarded and
the band carefully removed to a high speed centrifuge
tube, diluted with 10 volumes of 50 mM Tris-acetate
(pH 7.4) containing 5 mM MgCl2 and 0.1 mM EGTA
(wash buffer) and centrifuged at 27,000 X g for 30
minutes. The membranes were washed once again with
wash buffer and once with deionized water. The final
ROS pellet was transferred in a convenient aliquot of
water to a homogenizing tube and resuspended by
homogenization. Aliquots of the membrane suspension
were taken for Lowry protein quantitation and for
lipid-extraction.6
0146-0404/83/1000/1433/$0.95 © Association for Research in Vision and Ophthalmology
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / October 1983
1434
Table 1. Composition of the major fatty acids of
total phospholipids of rat rod outer segments
following constant illumination
Light status^
nmol/mg Protein^
Fatty
acids*
16:0
18:0
18:1
20:4«6
22:6a>3H
L-l Hr (4)\
302
581
85
108
889
± 60
±23
±22
± 13
± 86
H-l Dy (4)
266
576
66
86
841
± 31
± 42
± 10
±20
± 43
H-3 Dy (5)
328
560
113
114
654
± 30
± 28
± 24
± 19
± 100
* 16:0-palmitic acid; 18:0-stearic acid; 18:l-oleic acid; 20:4o>6-arachidonic
acid; 22:6u>3-docosahexaenoic acid.
t Refer to text for explanation of light status.
% Values in parenthesis are the number of membrane preparations from at
least 10 pooled retinas.
§ Values reported as means ± SD.
11 Statistical comparisons: L-l Hr vs. H-l Dy, not significant; L-l Hr and
H-l Dy vs. H-3 Dy, P < 0.005.
The lipid extract was evaporated under nitrogen at
room temperature and made to a known volume with
chloroform:methanol (19:1, v/v, saturated with H2O).
Aliquots were removed for lipid phosphorus assay as
well as for quantitation of the total phospholipid fatty
acids.6 A portion of the total lipid extract (2-3 ng
phosphorus) was applied to a silica gel-HR plate (5
X 20 cm) and developed in hexane:ethyl ethenglacial
acetic acid (60:40:1, by vol.). The region on the plate
containing the total phospholipids (the origin) was
scraped into a screw-capped tube. Methyl esters were
prepared with 14% BF3-methanol and their masses
quantitated by gas-liquid chromatography (GLC) using
heneicosanoic acid (21:0) as an internal standard.6
Conjugated dienes in ROS lipids were determined
spectrophotometrically.7 A portion of the lipid extract
was dried under argon and dissolved immediately in
an appropriate volume of argon-purged absolute
ethanol. The absorbance at 233 nm was determined
against a solvent blank on a Cary 219 recording spectrophotometer. Subsequently, phosphorus was determined on the lipid extract and the phospholipid concentration determined. The data are expressed as absorbance units/ml solvent/mg phospholipid.
Table 2. Lipid conjugated diene content of rat rod
outer segments following constant illumination
Group
Conjugated diene*
Control
Experimental
0.49 ± 0 . 1 1 (6)t
1.00 ± 0.41 (8)
P < 0.01
• Absorbance at X = 233 nm (1 cm optical path) per mg phospholipid
dissolved in 1 ml absolute ethanol.
t ROS were prepared from pooled retinas of individual rats. Values reported
as means ± SD for groups of six or eight animals.
Vol. 24
Results. The main effect of constant illumination
on the albino rat retina was the selective degeneration
of photoreceptor cells. Within a 24-hour exposure to
100-125 ft-c, structural changes were evident in the
photoreceptor inner and outer segments which showed
swollen mitochondria and vesiculated disk membranes,
respectively. Photoreceptor losses were not apparent
after a 24-hour exposure, although some nuclei were
pyknotic. Significant losses of photoreceptor nuclei
were evident by day 3.
Table 1 shows the levels of the major fatty acids of
the total phospholipids expressed as nmoles per mg
ROS protein. One of the chemical changes in lightinduced retinal degeneration was the reduction of the
long-chain polyunsaturated fatty acid, 22:6to3, after 3
days of constant illumination. Arachidonic acid
(20:4o>6), the other long-chain polyunsaturated fatty
acid, as well as the major saturated acids, palmitic and
stearic, were not affected. In addition, when the fatty
acids of the major phospholipid classes were examined
(data not shown), the level of 22:6o>3 was reduced at
3 days in each class by roughly 10% (absolute) compared with the L-l Hr or H-l Dy animals.
The level of lipid conjugated dienes in ROS lipids
is presented in Table 2. Control values include darkadapted and L-l Hr retinas; experimental values are
from H-l Dy and H-3 Dy retinas. Animals exposed
to constant illumination had twice the levels of conjugated dienes as control animals.
Discussion. The yield of ROS from light-damaged
animals was less than from controls. This is in part
from loss of ROS caused by the degeneration and in
part because the ROS membranes became more dense.
There was an increase in opsin-containing membranes
(determined by polyacrylamide gel disc electrophoresis)
at the 1.140/1.175 interface compared with controls.
Membranes from this interface were not studied further
because they are a heterogenous population derived
from many different cellular organelles. Thus, the ROS
membranes we analyzed may be considered "minimally damaged."
Three days of constant illumination reduced significantly the level of 22:6a>3, the major polyunsaturated fatty acid of ROS. None of the other fatty acids,
including the polyunsaturate arachidonic acid, were
changed significantly over the three-day time period.
Concomitant with the selective decrease in 22:6co3 was
an increase in the levels of lipid conjugated dienes.
The latter are formed during the oxidation of polyunsaturated fatty acids, and are an accepted measure
of lipid hydroperoxides.7 Both the loss of polyunsaturates and the increase in conjugated dienes provide
evidence for lipid peroxidation. This conclusion supports studies of Kagan et al8 which showed an increase
in lipid conjugated dienes in frog retinas exposed in
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No. 10
Reports
vitro to light for 30 minutes. Further studies by Kagan
et al2 demonstrated an increase in lipid peroxides in
the retinas of rats after 24 hours of constant highintensity illumination, measured spectrophotometrically as conjugated dienes. Interestingly, in this latter
study, animals deficient in vitamin E had greater
amounts of conjugated dienes than controls exposed
to the same levels of illumination. Decreases in vitamin
E in whole retinas of rats under constant illumination,
which preceded the loss of 22:6a>3, also have been
observed (C. D. Joel, personal communication).
Whether or not lipid peroxidation is the cause or
the consequence of light damage is difficult to determine. Clearly, peroxidation occurs at a higher level in
ROS of constant-light animals than in ROS of animals
maintained on a diurnal lighting cycle. Also, parallel
studies with frogs9 and rats (Rapp and Anderson, unpublished observations) injected intravitreally with
Fe+2, a potent pro-oxidant, suggest that lipid peroxidation may be a common mechanism for both lightand chemically induced photoreceptor degenerations.
There is a concomitant loss of 22:6a>3 and increase in
conjugated dienes in frog retinas following Fe+2 injection.9 In rats, both Fe+2 and constant light10 cause
the same regional pattern of degeneration with the
superior retina showing greater photoreceptor losses
than the inferior. These data indicate that light mediates
the peroxidation of ROS polyunsaturated fatty acids
and support the hypothesis that lipid peroxidation may
be involved in the cause of light-induced retinal degeneration.
Key words: retinal degeneration, photoreceptor membranes,
light damage, docosahexaenoic acid, lipid peroxidation, lipid
conjugated dienes
Acknowledgments. W e thank Maureen B. M a u d e for helpful support throughout the course of this work, a n d Janice
Cason for her patience in typing the manuscript.
From the Cullen Eye Institute, Baylor College of Medicine, Houston, Texas. Supported in part by grants from Retina Research Foundation (Houston, Texas), National Retinitis Pigmentosa Foundation,
Research to Prevent Blindness, Inc., and the National Eye Institute
1435
(NIH). Dr. Anderson is the recipient of a Dolly Green Special Scholar's
Award from Research to Prevent Blindness, Inc. Dr. Giusto is the
recipient of an International Travel Fellowship from Research to
Prevent Blindness, Inc. Norma M. Giusto's current address: Instituto
de Investigaciones Biochimicas, Universidad Nacional del Sur-Consejo Nacional de Investigaciones Cientificas y Tecnicas Avenida Alem
1253, 8000 Bahia Blanca, Argentina. Submitted for publication: November, 23, 1982. Reprint requests: Rex D. Wiegand, Cullen Eye
Institute, Baylor College of Medicine, Houston, TX 77030
References
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YP: Accumulation of lipid peroxidation products and depression
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to high-intensity light. Bull Exp Biol Med 91:144, 1981.
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albino rat rod outer segments following constant illumination.
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