Exp. Eye Res. (1985) 41. 67‘76
`
`CYAN EXHIBIT 1035
`
`Cataracts in the Royal College of Surgeons Rat: Evidence for
`Initiation by Lipid Peroxidation Products
`
`J. S. ZIGLER, JR. AND H. H. HESS
`
`Laboratory of Vision Research, National Eye Institute. National Institutes of Health,
`Bethesda, MD 20205, USA.
`
`(Received 25 September 1984 and accepted 6 March 1985, New York)
`
`The Royal College of Surgeons (RCS) rat has been extensively studied as a model system for
`inherited retinal degeneration. As in a number of human retina] degenerative diseases, posterior
`subcapsular cataracts (PSC) are associated with the retinal changes. It has been hypothesized
`recently that such cataracts may be initiated by toxic products generated by the peroxidation
`of polyunsaturated lipid components from degenerating photoreceptor outer segments. In the
`present study, the possibility that such a mechanism might be responsible for cataract initiation
`in the ROS rat has been investigated.
`The degeneration of the rod outer segments (ROS) occurs rapidly in these animals, beginning
`a few weeks after birth. Due to the failure of the retinal pigmented epithelium to phagocytize
`normally, ROS degeneration is accompanied by an accumulation of debris in the eye. During the
`brief period of maximal debris accumulation there is a marked increase in lipid peroxidation
`products in the vitreous. Cataract formation is correlated temporally with these events, becoming
`evident immediately following the time during which peroxidation products are present in the
`vitreous. In addition, the primary damage detected in the RCS lenses is an increase in the passive
`permeability of the lens membranes. Similar lens damage has been found in studies in which normal
`rat lenses were exposed to degenerating ROS in vitro. These findings are consistent with the
`hypothesis that cataracts in the ROS rat may be initiated by toxic lipid peroxidation products.
`Key words: Royal College of Surgeons (RCS) rat; retinal degeneration; posterior subcapsular
`cataract formation; lipid peroxidation;
`lens culture; rubidium ei’flux: microdissection: high-
`performance liquid chromatography (HPLC).
`
`1. Introduction
`
`The association between posterior subcapsular cataracts (PS0) and retinal degenerative
`diseases such as retinitis pigmentosa (Heckenlively, 1982) and gyrate atrophy
`(Kaiser-Kupfer, Kuwabara, Uga, Takki and Valle, 1983) has long been recognized;
`however, the mechanism accounting for this association has not been established.
`Although the cataracts have been considered by some to be intrinsic expressions of
`the genes responsible for retinal degeneration, the weight of the evidence supports the
`hypothesis that they are the result of secondary effects (Heckenlively, 1982). Berliner
`(1949) postulated that a ‘toxic substance ’ released from the degenerating retinal tissue
`was responsible for the initiation of cataract development. Recently there have been
`two studies reported which suggest that such a ‘toxic substance’ might be a product(s)
`generated by peroxidation of the polyunsaturated lipid released from degenerating
`photoreceptor outer segments (Goosey, Tuan and Garcia, 1984; Zigler, Bodaness, Gery
`and Kinoshita, 1983). In both studies lens damage was correlated with lipid
`peroxidation. This hypothesis is consistent with the facts that the retinal photoreceptor
`cells have very high rates of oxygen consumption and that the outer segments of these
`cells, which are the primary site of degeneration in these diseases, have extremely high
`
`Please address correspondence to J. Samuel Zigler, Jr, Bldg. 6, Rm. 235, National Institutes of Health,
`Bethesda, MD 20205, U.S.A.
`
`0014—4835/85/0100674— 10 $03,00/0
`
`© 1985 Academic Press Inc. (London) Limited
`
`

`

`68
`
`J..\‘.Zl(}l,ER AND H.H.HF}S-\‘
`
`levels of polyunsaturated lipid. Coupled with the marked tissue disruption occurring
`during the degenerative process, these factors would produce an ideal environment
`for lipid peroxidation.
`To investigate this hypothesis further the cataracts present in the Royal College
`of Surgeons (ROS) rat were studied. Although this strain was initially developed as
`a cataract model (Bourne, Campbell and Pyke, 1938), it has been studied primarily
`as a model of retinal degeneration. Only in 1982 was it reported by Hess, Newsome,
`Knapka and Westney that RCS rats fed the standard NIH rat diet all developed
`bilateral PSC at 7—8 weeks of age, as detected by slit lamp examination. Consistent
`with earlier work (LaVail. Sidman and Gerhardt, 1975), it was found that only 23 "a,
`of the rats had mature cataracts by 1 yr of age.
`The timing of the appearance of the P80 is consistent with the hypothesis that the
`lens changes may be initiated by toxic products released from degenerating rod outer
`segments (ROS). Dowling and Sidman (1962) have reported an elegant histologic
`analysis of the course of the retinal changes in these animals. The first observable
`damage is seen at about 12 days of age in the outer segments; by 18 days the outer
`segment layer is twice normal thickness and includes much lamellar outer segment
`debris (LaVail and Battelle, 1975). At about 4 weeks of age this layer reaches its
`maximal thickness and inner segments and photoreceptor nuclei begin to degenerate.
`By 40 days both the inner and outer segments are gone and the debris layer is
`narrowing and becoming less lamellar with a more homogeneous appearance. This
`layer of debris, which accumulates due to the inability of the retinal pigment
`epithelium to phagocytize normally (Herron. Riegel, Myers and Rubin, 1969; Mullen
`and LaVail, 1976), gradually disappears over a period of several months. Thus the
`initial appearance of cataractous changes in the lens (7—8 weeks) closely follows the
`period of maximal accumulation of outer segment debris. The possible relationship
`between these two events has been investigated by first determining whether lipid
`peroxidation is occurring in the ROS rat eye during degeneration of the ROS and,
`secondly, by comparing the condition of the lens in these animals with that of normal
`rat lenses exposed to lipid peroxidation products in Vitro.
`
`2. Materials and Methods
`
`All animals used in the present study were pink-eyed. tan-hooded dystrophic and control
`RCS rats (rdy/rdy, 11/1) and rdyfl p/p, respectively). All rats were fed the standard NIH-07
`natural ingredient rodent diet and were maintained in plastic cages under a 12-hr on/ 12—hr
`off light schedule.
`For preparation of vitreous, animals were killed and the eyes enucleated and frozen on
`crushed dry ice. Each eye was then bisected into anterior and posterior portions and the
`anterior portion discarded. While still frozen, the portion of lens remaining with the posterior
`part of the globe was removed and the vitreous was then carefully peeled from the outer layers
`of the eye. Any adherent retina was removed from the colorless vitreous under a dissecting
`microscope. Thiobarbituric acid assays were performed on isolated vitreous by the method
`of Buege and Aust (1978). For fluorescence studies, measured volumes of vitreous from ROS
`and congenic control animals were solubilized in sodium dodecyl sulfate (SDS) at a final
`concentration of 0-1 % SDS.
`Lens incubation studies were performed in TC-199 medium lacking phenol red and modified
`to increase buffering capacity as follows: to 160 ml TC-199 (Hanks salts) was added 26 ml
`H20, 108 mg glucose and 61-38 mg CaClz.2H20. After complete dissolution, 64 ml of a stock
`bicarbonate buffer (7-65 g NaHCOs, 0368 g KHCOa, 0-743 g Na0] and 0-085 g KCl pehi liter)
`was added to bring the total volume to 250 ml. Following addition of 30 mM fructose
`
`

`

`CATARACTS IN THE RCS RAT
`
`69
`
`(1-35 g/ 250 ml medium), the osmolarity of the medium was determined and NaCl added to
`bring the final osmolarity to 298;}- 2 mOsM. The medium was equilibrated with 95 "/5 air/5 0,-0
`CO2 by bubbling and was filter-sterilized following addition of 0-3 ml penicillin—streptomycin
`(Difco) per 100 ml. This is a modification of a medium previously developed specifically for
`lens organ culture (Kinoshita, Merola and Tung, 1968).
`lenses
`Rubidium efflux experiments were performed by incubating ROS and control
`individually for 16 hr in 2-0 ml medium containing tracer levels of 86RbCl. Following this
`period, half of the lenses in each group were removed and the uptake of radioactive label
`was determined as previously described (Zigler et al., 1983). The remaining lenses were rinsed
`in medium without label, transferred to fresh medium (20 ml) lacking 8“Rb, and incubated
`for 5 additional hr. Each lens was subsequently harvested and the content of 86Rh remaining
`in the lens was determined as above. Comparison of the label remaining in the lenses following
`the 5-hr leak—out period with that present after 16 hr uptake yielded the efflux data, which
`are reported for the dystrophic lenses as the percentage of the efliux from age—matched control
`lenses.
`.
`
`Lenses for microdissection were carefully removed from the globe by the posterior
`approach. The cataractous zones were clearly visible under a dissecting microscope. Such
`opaque areas and adjacent clear zones were dissected manually using a micro dissecting knife
`(Roboz Instrument Co. Inc., Washington, DC). Samples were homogenized in 0-25—0-5 ml
`0'05M Tris buffer. pH 7'1, containing 01 M KCl,
`1 mM EDTA and 1 mM dithiothreitol.
`Following centrifugation at 100000 g for 30 min, aliquots of the supernatants were applied
`to a 60 cm TSK-3000 column fitted to an LKB high-performance liquid chromatography
`(HPLC) system. The sample was eluted at 0-5 ml min‘1 and was monitored at 280 nm. Protein
`determinations on insoluble protein were performed in 0-5 % sodium dodecyl sulfate using
`the BCA protein assay (Pierce Chemical Company).
`
`3. Results
`
`To determine whether lipid peroxidation occurs in the RCS rat eye during outer
`segment degeneration, samples ofvitreous were carefully dissected from RCS dystrophic
`and congenic control animals of various ages and tested for the presence of lipid
`peroxidation products. Table I gives the results of thiobarbituric acid (TBA) assays
`on these preparations. There was a definite burst of TBA reactivity at about 4 weeks
`of age in the dystrophic eyes coincident with the time of maximal accumulation of
`debris from the ROS. The presence of the dialdehydes, with which TBA reacts, is of
`short duration, with the levels returning to the control range within several weeks.
`
`TBA reactivity in the vitreous*
`
`TABLE 1
`
`
`
`
`
` Animals Age 0.D.532
`
`RCS dystrophic
`
`2% weeks
`4 weeks
`8 weeks
`6 months
`
`0010
`0-057
`0008
`0003
`
`0006
`4 weeks
`ROS control
`
`6 months 0003
`
`* Each value represents a determination made on a sample of pooled vitreous taken from six to eight
`eyes. Similar results were obtained from two additional, separately prepared sets of vitreous samples.
`Comparison of the mean values (is.D.) for the 4-week dystrophic (00551-0013) and 4-week control
`(0-008i0'002) preparations from the three experiments by Student’s t—test revealed that they were
`significantly different (P < 005).
`
`

`

`70
`
`J.S.Z1GLERANI) H.H.HESS
`
`intensity
`Relmive
`
`500
`
`600
`
`J}:moo
`
`NO
`
`300
`
`400
`
`Wavelength (nm)
`
`FIG. 1. Corrected fluorescence emission and excitation curves for ROS dystrophic and control vitreous
`isolated from animals approximately 7 months of age. Emission curves were obtained with excitation
`at 350 nm. The excitation spectra, shown at the left, were obtained with emission set at 450 nm. In both
`pairs of curves the dystrophic vitreous is the upper spectrum.
`
`These highly reactive species would not be expected to persist for very long in the
`eye since they will react rapidly with any molecules containing amino groups.
`However, the products of the reaction of such aldehydes with amino groups are stable
`and give characteristic fluoresence emissions near 450 nm (Ex 2 350 nm) resulting
`from the iminopropene bonds formed (Chic and Tappel, 1969). Figure 1 demonstrates
`an increase in fluorescence emission in the 450 nm range in the vitreous of older RCS
`dystrophic rats relative to agevmatched congenic controls.
`Figure 2 is a photograph of the postcror surface of the lens from an RCS dystrophic
`rat at the age of about 8 weeks. The opacity is located at the posterior pole of the
`lens and has a ‘sugar-grain’ appearance. At this stage the opacity is immediately below
`the posterior capsule; however, in most of the lenses the opacity becomes ‘ internalized ’
`within several weeks (Hess, Newsome, Knapka and Westney, 1983), with normal-
`appearing transparent fiber cells being laid down external to the opacity. Figure 3 is
`a similar view of another RCS rat lens at about 9 weeks of age in which new lens fibers
`can be seen covering most of the lesion. Note the formation of the sutures directly
`over the opacity. Figure 4 shows a lens from a 14-month-old RCS rat in which the
`opacity can be seen from the side as a discrete are well inside the posterior capsule.
`The lens fibers both anterior and posterior to the opaque area are transparent.
`Since previous studies by the present authors had demonstrated that peroxidation
`products, particularly aldehydes, damaged the membranes of organ-cultured rat
`lenses making them abnormally permeable to “Rb, lenses were removed from RCS
`rats at different ages and their rubidium effiux rates were compared with those of
`age-matched congenic controls. Data are reported in Fig. 5 as percentage of control,
`with the 100% line representing the values for age-matched control lenses. The
`dystrophic animals first showed abnormally high efl‘lux rates at about 40 days, shortly
`after the time of greatest debris accumulation in the retina. The efflux rate continued
`to rise until about 60 days of age when it began to decrease, returning to normal levels
`by about 3 months of age. This fall in efiiux corresponds to the time at which the PSC
`becomes ‘internalized’. It was also observed that in some of the lenses the opacity
`did not become internalized. When those lenses were selected from animals of 3—4
`
`months of age, the rubidium efliux rates were found still to be elevated (Fig. 5).
`The nature of the PSC produced in the ROS rat, particularly its highly reproducible
`occurrence and its discrete localization, make it an ideal subject for study by the
`microdissection technique (Horwitz, Neuhaus and Dockstader, 1981). Such studies
`
`

`

`CATARACTS IN THE ROS RAT
`
`71
`
`
`
`FIG. 2‘ Posterior subcapsular cataract (PSC) in pink-eyed, tan-hooded RCS rat at 8 weeks of age,
`Photograph of posterior aspect of dissected lens immersed in saline, taken with Zeiss stereomicroscope
`SR with darkfield/brightfield stand and MC-63 photomicrographic camera system. Note the boundary
`of the initial lesion (arrow) and the ‘sugar-grain' appearance of some areas. (Microscope magnification
`in (a) x 32. (h) x 50.)
`
`

`

`72
`
`J‘ S. ZIGLER AND H. H. HESS
`
`
`
`F10.. 3, Early (9-week) stage of ' internalization " of the posterior subcapsular cataract (PSC). Note that
`new clear healthy lens fibers have appeared posterior to the opacity and are meeting to form sutures.
`Photographed as in Fig. 2. (Microscope magnification. X 50,)
`
`FIG. 4. Late (14—month) stage of 'internalizat-ion’ of posterior subcapsular cataract (PSC). The depth
`of the internalized arc of the opacity (arrow) is age-dependent. Photographed using a. Zeiss operating
`microscope with camera attchmcnt and darkfield background with synchronized flash from the side.
`
`

`

`CATARACTS IN THE RCS RAT
`
`73
`
`250
`
`
`
`
`
`Rubidiumefflux(“/0ofcontrol)
`
`ZOOl
`
`ISO
`
`IOO
`
` 50 .—-_L
`
`O
`
`25
`
`50
`
`1—.
`75
`
`IOO
`
`Age (days)
`
`FIG. 5. Rubidium efflux data from lenses of ROS dystrophic rats of varying age reported as the
`percentage of the efflux from agematehed congenic controls. Each point represents the mean value for
`a group of four to seven lenses. Points falling near the 100 % line Were not different from control values.
`The point at 105 days (I) represents a group of lenses from dystrophic rats in which the opacity did
`not become internalized. Error bars denote i 1 SD.
`
`
`(a)
`COI—
`
`A280nm
`
`r
`
`
`(b)
`
`0-01r
`
`
`
`
`
`0 IO
`
`I5
`
`30
`25
`20
`Elulion volume (ml)
`
`FIG. 6. Elution profiles from the TSK-3000 SW column for the water-soluble protein from (a) opaque
`and (b) clear zones of a lens from a 2-month-old ROS dystrophic rat. Details of the chromatography
`are given in Materials and Methods. Electrophoretic analysis revealed that a-crystallins eluted in the
`initial peak, the fl—crystallins eluted between 15 and 20 ml, and the ‘y—crystallins between 20 and 25 ml.
`Material eluting after 25 ml did not register on standard SDS electrophoretic gels. The approximate
`amount of protein applied was '75 fig for the opaque sample and 250 pg for the clear sample (Bio—Rad
`protein assay).
`
`have been initiated to determine the effects of cataract development in this model
`system on the lens soluble proteins. Figure 6 shows the elution patterns obtained from
`an HPLC gel filtration column for the water-soluble extracts from the cataractous area
`of an ROS lens and from an adjacent clear zone of the same lens. Clearly there was
`a marked decrease in all the soluble crystallins in the opaque tissue. It is unclear
`whether this loss resulted from protein degradation or insolubilization; however, as
`shown in Fig. 6, many of the samples of opaque areas showed increased amounts of
`
`

`

`74
`
`J. S. ZIGLER AND H. H. HESS
`
`apparent low molecular weight material which could represent protein degradation
`products. Furthermore, protein determinations on the water—insoluble fractions
`revealed little or no increase in insoluble protein in the opaque areas relative to
`adjacent clear zones. The absolute amount of insoluble protein (as a percentage of
`tissue wet weight) was less than 50.1,
`in all samples analysed.
`
`4. Discussion
`
`The initial damage to the lens in the ROS dystrophic rat is seen as the formation
`of a ‘sugar-grain’ posterior subcapsular opacity appearing at 7~8 weeks of age in all
`such animals fed a standard rodent diet. In most of the animals the PSC does not
`
`progress substantially, and after a few weeks can be seen separated from the capsule
`with normal-appearing transparent tissue between the opaque area and the capsule.
`This internalization of the lesion suggests the possibility that the toxic substance or
`condition which is responsible for initiating the formation of the opacity may be
`present for only a rather brief period of time (Hess et al., 1983). Since the appearance
`of this opacity closely follows the degeneration of the photoreceptor outer segments,
`it is reasonable to suspect that material toxic to the lens might be released during this
`degenerative process. We have discussed above the conditions which make degenerating
`outer segments ideal substrates for lipid peroxidation. Furthermore, previous studies
`by the present authors (Zigler et al._. 1983) and those of a number of other laboratories
`have demonstrated that peroxidation can occur in the retina both in vitro (Farnsworth
`and Dratz, 1976) and in vivo (e.g. Hiramitsu, Hasegawa, Hirata, Nishigki and Yagi,
`1976) under a variety of conditions. Indeed it has been reported that lipid peroxidation
`occurs in certain retinal degenerations (Anderson, Rapp and Wiegand, 1984). Thus the
`finding of apparent lipid peroxidation products (dialdehydes) in the vitreous of RCS
`rats during the time of outer segment degeneration is not surprising.
`It has been demonstrated previously (Zigler et- al., 1983) that normal rat lenses
`exposed in vitro to lipid peroxidation products are damaged, and that the primary
`damage is an increase in the passive permeability of the lens membranes which is
`largely attributable to the presence of toxic aldehyde products produced by the
`peroxidative process. The present study indicates that the membranes of the ROS
`cataracts also are abnormally permeable. The increased rubidium efflux levels appear
`initially at about 6 weeks of age. shortly before the opacity becomes evident by slit
`lamp examination. The lenses become progressively more leaky until about 10 weeks
`of age, at which point most of them begin to recover. returning to normal over a period
`of several weeks. The recovery correlates temporally with the ‘internalization’ of the
`opacity within the lens. This correlation is further substantiated by the fact that in
`those cases in which the opacity does not become internalized, the lens remains
`maximally leaky. Preliminary observations indicate that lenses in which the PSC fail
`to become internalized consistently develop mature cataracts (H. Hess. unpublished
`results). That light exposure may accelerate cataract maturation in the ROS rat is
`suggested by the finding of LaVail et al.
`(1975) that while 2‘ ‘33) of pink-eyed
`dystrophics develop mature cataracts by 9 months of age. only 3% of pigmented
`dystrophics have mature cataracts by the same age. despite an onset of P80 at 7—8
`weeks in both strains (Hess et al.. 1983). The present observation suggests that such
`a potentiating effect of light must occur during the early stages of lens damage.
`Evidence has been presented that lipid peroxidation occurs during degeneration of
`
`

`

`CATARACTS IN THE ROS RAT
`
`75
`
`the ROS in the ROS rat and that dialdehyde products of this process are present in
`the vitreous during the time of outer segment degeneration. It has also been shown
`that the ROS rat lens becomes abnormally permeable shortly after the time at which
`these peroxidation products are present. This finding correlates well with the previous
`report that normal rat lenses cultured in the presence of lipid peroxidation products
`become abnormally permeable. In most of the animals the lenses begin producing
`normal tissue again after the cessation of outer segment degeneration. These findings
`all are consistent with the hypothesis that the ROS cataract is initiated by toxic
`products of lipid peroxidation which are present in the eye in appreciable amounts
`only during the period of photoreceptor outer segment degeneration. Since posterior
`subcapsular cataract is a frequent secondary effect in a variety of human retina]
`degenerative diseases (Heckinlively, 1982), it is possible that peroxidation of unsatu—
`rated lipids from the retina may play a role in initiating these cataracts as well.
`
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`
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`
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`75
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`.I.S.ZIGI.EI{ AND H.H.HESH
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`LaVail, M. M.. Sidman. R. L. and Gerhardt, (I. (1975). Congenic strains of ROS rats with
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`(1976). Inherited retinal dystrophy: primary defect in
`pigment epithelium determined with experimental rat chimeras. Science 192. 799—801.
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`