Exp. Eye Res. (1982) :}5, 157-171

Studies on Experimental ly Induced Ret inal Degenerat ion . I. Effect of Lipid Peroxides on Electroret inographic Activity

in the Albino Rabbit

DONALD ARMSTRONG,* T A D A H I S A H I R A M I T S U , t J O H N G U T T E R I D G E , ~

AND SVEN E R I K N I L S S O N w

* Department of Ophthalmology, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A., and Department of Ophthalmology, University of

Linkoping, Sweden, tDepartment of Ophthalmology, School of Medicine, Fujita-Gakuen Univeristy, Toyoake-shi, Aichi-ken, Japan, $ National Institute for

Biological Standards and Control, London, England, w Department of Ophthalmology, University of Linkoping, Linkoping, Sweden

(Received 10 August 1981 and accepted 5 February 1982, New York)

Lipid hydroperoxides (LHP) have been synthesized and purified from linoleic, linolenic, arachidonic and docosahexaenoic acids, using soybean lipoxygenase and oxygen. Intravitreal injections into the eyes of mature, albino rabbits produced an early and then progressive decrease in the amplitude of a-, b- and c-waves of the ERG. Depending upon the amount and activity of the LHP preparation, ERG's were markedly decreased in amplitude ( > 50 ~o ) within 4 days following the injection and by 12 days, the activity from perioxide treated eyes was essentially non- recordable. In preliminary studies, these effects were less pronounced in adult pigmented rabbits of similar age, however, a younger pigmented rabbit was only slightly less susceptible to damage than the albino animals.

In other experiments, peroxidized native phospholipids, malonaldehyde, hydrogen peroxide and sodium iodate were also shown to be cytotoxic, but not all were as toxic as the LHP. In contrast, retinol, vitamin A acetate and retinoic acid had no effect upon ERG activity, nor did the parent fatty-acid compounds or the borate buffer in which they were injected.

These studies confirm previous reports where indirect produCtion of lipid peroxides caused retinal degeneration. The present report extends these observations to demostrate that when the retina and RPE are exposed to a sample of purified LHP, retinal function is altered in an irreversible way. We also demonstrate that a metabolic by-product (malonaldehyde) is likewise cytotoxic. However, the mechanisms by which the parent LHP and/or metabolites might act could be quite different.

This new animal model should prove useful in evaluating further the ultrastructural changes which are observed during peroxidative damage of the retina in vivo, as well as in evaluating the therapeutic approaches to these problems of retinal degeneration.

Key words : lipid peroxides; MDA ; vitamin A; intra-vitreal injection ; retinotoxicity; maturation ; ERG ; horseradish ; peroxidase ; melanin.

1. I n t r o d u c t i o n

Irradiation, oxygen (Hiramitsu, 1974; Yagi and Ohishi, 1976), light (Kuwabara and Gorn, 1968; Kagan, Shvedova, Novikov and Kozlov, 1973), and iron induced oxidation (Hiramitsu, Hasegawa, Hirata, Nighigaki and Yagi, 1976 ; Kagan, Shvedova, Novikov and Kozlov, 1975), are known to produce retinal damage. The resulting retinal degeneration may be due to free radical formation (Kagan et al., 1973) and lipid peroxidation (Hiramitsu et al., 1976; Delmelle, 1977). The latter term refers specifically to the autoxidation of lipids where primary lipid peroxides and secondary by-products (aldehydes) are formed (Gutteridge and Stocks, 1976).

Reprint requests to: Donald Armstrong, Ph.D., Department of Ophthalmology, Box J-284, J. Hillis Miller Health Center, University of Florida, Gainseville, FL 32610, U.S.A.

0014-4835/82/080157 + 15 $02.00/0 �9 1982 Academic Press Inc. (London) Limited

6-2

158 D. ARMSTRONG ET AL.

Lipid hydroperoxides (LHP) are increased in the rabbit retina after exposure to either high concentrations of oxygen (Hiramitsu et al., 1976), or to X-irradiation (Hiramitsu, Majima, Hasegawa and Hirata, 1974). Furthermore, in each instance, there is a decrease in amplitude of the electroretinogram (ERG), secondary aldehydes are formed, and there is a marked degeneration of the photoreceptor cell layers. I t has also been reported that following lipid peroxidation in the retina, visual pigments (Kawamura and Mizuno, 1970) and retinal oxygen consumption decrease (Hiramitsu, 1969). Because photoreceptor outer segments are rich in polyunsaturated fa t ty acids (Daemen, 1973) they are uniquely susceptible to lipid oxidation (Mead and Fulco, 1976; Katz, Stone, and Dratz, 1978). The present study was undertaken to (1) enzymatically prepare the peroxides of four polyunsaturated fat ty acids, (2) determine their electrophysiological effect on the neural retina and pigment epithelium, and (3) compare these changes to the effects produced by other compounds.

2. Materia ls and M e t h o d s

Preparation of lipid hydroperoxides The peroxides of linoleic (18:206), linolenic (18:3o~3), arachidonic (20:406), and docosa-

hexaenoic (22 : 603) acids were prepared by a modification of Egmond, Brunori and Fasells, 1976, using the enzyme, lipoxygenase (Fig. 1). The yield and purity of lipid peroxide produced by this method was found to be considerably greater than when prepared by the oxygen, ultraviolet-catalyzed technique (Ottlenghi, Bernheim, and Wilbur, 1955). Ten millimolar concentrations of each of the polyunsaturated fatty acids (PUFA) were prepared in 100 ml of 0"l M-sodium borate pH 9, then 10 mg of soybean lipoxygenase (Sigma Chemical Co.) was added and the mixture incubated in an oxygen saturated atmosphere at 4~ for 2 hr. After incubation, the lipid peroxides were extracted twice with a volume of diethyl ether equal to the reaction mixture. The ether extract was evaporated to dryness under nitrogen, resuspended in chloroform-methanol (2 : 1) and applied as a continuous band across the width of a silica gel F254 chromatographic plate, 1"5 cm from the bottom. Each preparation was subjected to one dimensional thin layer chromatography (chloroform, methanol, water 75 : 25:4"2) to isolate the lipid peroxide from the untreated PUFA. The silica gel plates were covered with a clean glass plate leaving 1 in exposed on the right and left sides of the underlying chromatographic plate. One exposed side was sprayed with concentrated sulphuric acid and charred to identify the free fatty acid while the other side was sprayed with Nu-Peroxy Spray (Supelco) to identify the pink peroxide derivative. After identification, the glass plate was removed, the area corresponding to the lipid peroxide on the underlying, unsprayed plate scraped off and resuspended in 5 ml of solvent (chloroform, methanol 2: 1). Using this technique, approximately 90 ~o conversion to the hydroperoxide was achieved. Subsequently this step was omitted and the preparation after incubation were evaporated directly, resuspended in buffer and sonicated prior to use. Phospholipid peroxides were prepared by purging a solution of phospholipids from bovine brain (Sigma Chemical Co.) continuously with oxygen under u.v. light for one week at 4~ The concentration of hydroperoxides was calculated from the absorbance at 234 nm using a molar absorption coefficient of 25 000 cm -1. Hydroperoxide standards were stored under nitrogen at 0~ until ready for use.

Preparation of malonaldehyde Malonaldehyde (MDA) was prepared by adding 2 g of Dowex 50W-X8 (H +) to 0"5 ml of

1,1,3,3-tetramethoxypropane (TMP) in 5 ml of metal-free (chelex-100 treated) water. The hydrolyzed TMP was incubated at 37 ~ for 30 min, and then 0"1 ml of the incubate chromatographed on a Sephadex G-10 column. Twelve 1-ml fractions were eluted with metal-ion free water and tested for thiobarbituric acid (TBA) reactivity at room temperature as follows: 0"2 ml of sample, 0"2 ml of 1% TBA, and 0"2 ml of 25 % HCI. A characteristic blue- red color developed rapidly within 2 min in fractions 2-4. These were pooled and constituted the MDA sample (Gutteridge, Heys and Lunec, 1977).

L I P I D P E R O X I D E R E T I N O T O X I C I T Y 159

i00 ml 0.i M sodiu~n borate, pH 9.0 i0 mg lipc~yganase, Sigma type 1

Polyunsaturated fatty acid to 10 ram concentration

Place into 125 ml Erleflmeyer flask

! Saturate with 02, cap flask

Incubate for 2 hours at 4~

! Extract with i00 ml diethyl ether (3x)

!

upper phase Lower phase (discard)

Evaporate to 1 ml with nitrogen

! Add 0.5 ml 0.i M sodium borate, pH 9

! Evaporate remaining et/her with nitrogen

! Sonicate with two 5 second bursts

in an ice hath

Lipid peroxide concentration calculated from the absorbance

at 234 nm using a molar extinction coefficient of 25,000 am -i

FIG. 1. Flow diagram of lipid peroxide preparation s.

Preparation of apoperoxidase and other enzymes Ten mg of horseradish peroxidase (Sigma, type II) was dissolved in 20 ml of methyl ethyl

ketone saturated with 0'05 N-HC1 (Mauk and Girotti , 1974). The heine group was extracted into the ketone phase. When the phases separated, the aqueous HC1 phase was removed, re-extracted four more times and the final ketone layer evaporated under nitrogen. The sample was next dialyzed overnight against 0"02 N-HC1 to restrict free heme from re- associating with the apoprotein, then dialyzed another 48 hr against 0"15 M-NaCI and finally, concentrated on an Amicon PM-10 membrane under pressure. The product showed no enzymatic act ivi ty using p-phenylenediamine and hydrogen peroxide as substrates in the conventional peroxidase assay system (Armstrong, Connole, Feeney and Berman, 1978b).

Horseradish peroxidase types VII, VI I I and IX, catalase (CAT) and superoxide dismutase (SOD) were all purchased from Sigma Chemical Co., were resuspended in normal saline and used directly.

Standardization of the electroretinogram (ERG) All animal experiments were carried out in Colorado, Norway and Sweden from 1977 to

1980, using New Zealand albino rabbits weighing 2-5-3"0 kg. In other preliminary studies, two pigmented adult rabbits of comparable size and one young 2- to 3-week-old pigmented rabbit were examined. No experiments were conducted in England. The rabbits were housed under standard laboratory conditions with 12 hr l ight-dark cycles.

160 D. A R M S T R O N G ET AL.

The E R G s were obtained using a LifeTech model 7101 recorder and a model 7305 st imulator. A zinc-zinc sulfate electrode with cot ton t ip was used as the ac t ive corneal electrode and the reference electrode a t tached subcutaneously over the forehead.

Tropicamide ophthalmic solution (1'0 %) was applied top ica l ly as a mydr ia t ic and the animals were allowed to dark adap t for l0 min while restrained in a box with only the head exposed. A few drops of 0"5 % proparacaine hydrochl0ride were applied to the eye and the eyelids reflected with a small Murdock eye speculum. The recording electrode was posi t ioned against the cornea using a micromanipula tor . Dur ing recordings, the eyes were rinsed frequent ly wi th normal saline to p reven t corneal drying. The recorder was set a t 40 # V / c m , chart speed was l0 m m / s e c and the storage t ime set t ing was 250 msec. The s t imula tor was set on manual flash (1/min) wi th an in tens i ty of 3, which is equal to 5'35 x 103 lumen-sec /cm. The l ight s t imulus from a Xenon helical flash bulb was rout inely posi t ioned 16 cm from the cornea.

For simultaneous, a-, b- and c-wave E R G recordings, animals were main ta ined under general anesthesia with a continuous i.v. infusion of 0-23 ml H y p n o r m R (Leo, Sweden) per kg body wt . /h r . Following di lat ion of the pupils and dark adaptat ion, 18:2 hydroperoxide was injected into one eye and a similar amoun t of 18:2 into the o ther eye. S tandard a- and b-wave E R G s were recorded as well as d.c. recordings using fiber optic s t imulat ion, suction contact lenses, matched calomel half-cell electrodes and low-drift d.c. amplifiers as previously repor ted (Nilsson and Skoog, 1975). This technique produced higher values for a- and b-wave ac t iv i ty than obta ined with the zinc electrode system.

Injection technique Prior to the injections, two drops of a 0-5 ~o proparacaine hydrochlor ide ophthalmic

solution were applied to the eye. I t was flushed with sterile saline and a 25-gauge x 3 /8 in needle used to evacuate a small amoun t of fluid from the anter ior chamber. A 0"l ml a l iquot (19-34 mg) of the appropr ia te solution was injected direct ly into the vi t reous approx imate ly 3-4 mm above the l imbus of the superior- temporal quadran t in an inferior-posterior direction. A 27-gauge needle was used for this purpose and was always inserted its entire length to insure consistency in present ing solutions a s tandard dis tance from the ret inal surface. Fol lowing injection, Neosporin ophthalmic solution was applied to re tard infections. These studies were of one to three weeks durat ion and infection was rarely encountered.

3. R e s u l t s

T h e E R G reco rd ings used in t he se e x p e r i m e n t s were r o u g h l y d i v i d e d i n t o t h r e e ser ies

a n d were p r o d u c e d b y t w o d i f f e ren t o p e r a t o r s o v e r a 3 -yea r p e r i o d o f t ime . To

i l l u s t r a t e r e p r o d u c i b i l i t y , t h e m e a n a m p l i t u d e a n d __ S.D. o f con t ro l , p r e - i n j e c t i o n

a - w a v e a c t i v i t y in t h e series w h i c h t o t a l l e d 75 a n i m a l s we re : ser ies 1 -- 5 7 _ 8 # V

(N -- 32); series 2 = 4 3 + l l (N = 14) a n d series 3 = 6 9 + 2 1 # v (N = 29). T h e corres-

TABLE I

Decrease* in ERG activity following injection of linoleic acid hydroperoxide

Days post-injection a-wave b-wave

1 (n = 25) 53'8+11"1 59"3_ 11'6 2 (n = 14) 59'3___8"2 62"8___9"4 3 (n = 10) 62"6+-- 11'3 64"6___6'0 4 (n = 4) 67"7+- 12"9 72"0___4"6 5 (n = 10) 76"2___9"8 71"4___6"2 6 (n = 11) 80"5+-11"3 73'1+-9'2

7-12 (n = 23) 98"6• 89-0___8"3

* Percentage below pre-injection value, _+ S.D.

L I P I D P E R O X I D E R E T I N O T O X I C I T Y 161

LINOLEIC ACID HYDROPEROXIDE LINOLEIC ACID

BEFORE

INJECTION % 1 Se•

b b

i \ ' \

a : 5 5 # v a : 7 0 . b : 9 0 ~ v b 9 0 ~

1 DAY b

AF TE R ,/~'= J"

m~ L a a

a ; 5 ~ v a : 5 5 p v b = S O ~ v b 9 5 ~ v

b

4 DAYB b ]~(

I N J E C T I O N a

a~ 20 juv a ~ 65pV b ~ 4 0 ~ v b= 9 5 u v

b

12 DAYB ~ .

AFTER b k

..... L a

i s e c a , O ~ V a~65pv b = O H V b : 9 5 p v

Fro. 2. The effect of linoleic acid (18:2) and its hydroperoxide on the albino rabbit E R G from series 1. Both compounds (19 rag) were dissolved in 0'1 M-sodium borate buffer prior to intravitreal injection. The E R G components are indicated and a downward deflection represents a negative potential.

ponding b-wave activities were: series 1 - - 9 0 + 1 5 # V ; series 2 - - 1 0 9 + 2 0 # V and series 3 = 105-t-21 FV.

The rabbits injected with 18:2 hydroperoxide showed a consistent, progressive and irreversible decrease in ERG activity (Table I). By 1 day post-injection, more than one-half of the a- and b-wave activity was lost. During subsequent days, both activities continued their decline until activity essentially ceased after 7 days of exposure. The adverse effect of LHP on b-wave activity seemed slightly greater for the first 4 days, however, the rate of decline shows relative to the decrease in a-wave activity thereafter. In a representative experiment, 19 mg oflinoleic acid was injected into one eye as the control preparation and a similar concentration of L H P was injected into the contralateral eye (Fig. 2). Since the average vo lume of vitreous from rabbits of this size is 3"5 ml, the concentration within the eye following injection is approximately 20 mM. At 1 day post-injection, the LHP had decreased the ERG to 55 % (a-wave) and 33 % (b-wave) of normal activity. Four days after injection, the a- and b-wave activities had decreased to 64 and 54 % respectively. Twelve days later, the ERG was non-recordable. In contrast, the parent P U F A compound had no effect on ERG activity during the same interval, except for a small (13 %) transient decrease

162 D. ARMSTRONG ET AL.

LINOLENIC ACID ARACHIDONIC ACID DOCOSAHEXANOIC ACID

HYDROPEROXIDE HYDROPEROXlDE HYDROPEROXlDE

B E F O R E

INJECTION ~I , f\, ~ ?~.,,,,r~

L_ ' i

,oo ~v ., i a a

sec

a : 5 o ~ v a= 70 ,uv a= 6 o , u v b= 70 juV b: 70 ,uV b, 75 p V

1 DAY

AFTER b

a a 1oo .=v

I ser a= 35 ,uV a= 3 5 p V a: 3 5 , u v b= 2 0 p V b= 40 ,uV b; 25 ,uV

4 0 A Y S

INJECTION / ~ '

~00 p v | a a a

L - - - 1 sec ~o Ec~ ,oo

a : 25 p v a : 25 p V a: 15 ~ v b = 30 pV b: 45 pV b; 3OpV

12 DAYS i=

A F T E R b b

, .JECT'ON - - - - - - ~ - - ~ - ~ ~ ~.z-.-----.~.,.--~ a ~, a

~ 0 0 ~v L a

I s e c

a: 5 pV a : 3 0 . u v a= ~ O p V b : l O ,uV b : 7O,UV b: 1 0 , u V

19 DAYS b AFTER ;~

iNJECTION ~ ~ .~..~ ,~,~Ap ~ b ~ . , ~

1 ~ e c

a : O p V a = 2 5 p V a , O u V b : O p V b = S O p V b : 0 p V

FIG. 3. The effects oflinolenic (18 : 3), arachidonic (20 : 4) and docosahexaenoic (22 : 6) acid hydroperoxides on the albino rabbit ERG from series 1. The concentrations used are described in the text and are similar to that used in Fig. 2.

of a-wave ac t iv i ty seen after the first day. In 30 other l inoleie acid control animals , a-and b-wave act ivit ies were 49__ l 0 and 95__ 23 FV at 2 days post- inject ion and 44_-t- 7 and 88 _-t- 19 FV at 2 weeks post- injection. Likewise, the sodium borate buffer was well tolerated by the rabbit retina and no change was noted in the E RG even several weeks after injection.

Similar results were obtained with the hydroperoxide derivat ives of l inolenic and docosahexaenoic acid at the concentrat ions used for l inoleic acid (Fig. 3). The former peroxides appeared to have somewhat less effect upon the a-wave than those shown in Fig. 2, but a more profound effect upon b-wave ac t iv i ty was observed. Thus at one day post- injection, the decrease in a-wave ac t iv i ty for 18:3 and 22:6 hydroperoxides was 30 and 42 %, whereas the decrease in b-wave ac t iv i ty was 71 and 67 % respectively.

L I P I D P E R O X I D E R E T I N O T O X I C I T Y 163

After four days, there were no further changes in the b-wave, but a-wave amplitude continued to fall with the 22 : 6 sample showing the greatest effect (75 ~ decline). At 12 days, only minimal ERG activity remained in four rabbits injected with 18:3 and 10 rabbits injected with 22:6 hydroperoxides. By 19 days after injection, activities were always nonrecordable.

Arachidonic acid hydroperoxide (19 mg) showed an early adverse effect through the first four days following injection. By 12 days, the ERG had stabilized and remained so thereafter. Similar results were obtained in two separate experiments. As noted in the 18:2 hydroperoxide experiment, none of the three PUFA employed here had any effect on ERG activity, at 2 weeks post-injection, the average deviation from pre-injection values was 3 _ 3 ~o for a-wave and 6 • 4 ~o for b-wave activity (n = 17).

When higher concentrations of each L H P were used (above 24 mg), dramatic ERG changes could be demonstrated within 2 hr of the injection.

":- / ..,,,~ ~..'

i FIG. 4. D.c. ERG's, from series 3, including the c-wave region from (1) a control eye 15 days after

injection with linoleic acid (left record) and (2) the corresponding experimental eye injected with 18:2 hydroperoxide 2 days (middle record) and 15 days (right record) post-injection. A 1 sec light stimulus is indicated below each recording. Amplitude calibration = 250/~V.

After injection of 18:2 hydroperoxide, the c-wave of the ERG followed the same reduction in amplitude as observed for the a- and b-waves (Fig. 4) during a 2-15 day post-injection period.

In separate single experiments, phospholipids from bovine brain (Sigma Chemical Co.) were peroxidized by the interaction of oxygen and u.v. light. When solutions of un-oxidized phospholipid and the oxidized derivative were injected in experiments comparable to the LHP studies, they showed no adverse effect on the ERG, whereas the oxidized product was as damaging as the L H P described above. Monomeric MDA synthesized and purified from TMP was very TBA reactive and showed extreme cytotoxicity. For example, when a single injection of 30 #g was used, the pre-injection a-wave value of 66/tV was reduced to 7 ttV in just one week. The b-wave was reduced from 132 to 68/tV during the same interval. Less reactive forms of MDA are produced when prepared using acid hydrolysis and when the sodium or bis-bisulfite salts are precursors. With this type of MDA, we observed no changes in ERG at 30#g concentrations, and even when the injected dose was increased to 2 • 10 -3 M, three weeks were required to achieve the same degree of damage as observed in one week with the more reactive form.

That the adverse effects we recorded were indeed specific for the peroxide group(s) is illustrated in Fig. 5. Prior to the injection of 18:2 hydroperoxide, 0"5 mg of horseradish peroxidase (Sigma, type IX, basic isoenzyme) was delivered into the vitreous of one rabbit eye. Thirty minutes later, identical solutions of the L H P were injected into both eyes and ERG recorded daily over the following seven days. At the end of one week, the LHP injected alone had destroyed all electrical activity. However, under the same experimental conditions, prior injection of the peroxidase

164 D. ARMSTRONG ET AL.

LINOLEIC ACID

HYDROPEROXlDE

0.5 mg, HRP &

LINOLEIC ACID

HYDROPEROXIDE

b

BEFORE

INJECTION a

lO0.~v

1 Sec

b

a

a: 40 a=35

b : 70 b = 100

b 1 DAY b

INJECTION | a "

100 juV L a

1 Sec a= 40 a=35

b = 35 b=63

b

3 DAYS ~ ' ~ , ~ b

INJECTION ~ a

100~v a,

1 Sec. a = 30 a=45

b= 0 b= 90

b B

7 DAYS b /~"~

AFTER _ _ ~ ~ ~'~ ~ ' ~ ' ~ INJECTION | a V

100,uV L a

I Sec. a=0 a=45

b =0 b=85

Fro. 5. The effect of Sigma type 9, basic horseradish peroxidase (HRP) on LHP damage to the albino rabbit from series l, Linoleic acid hydroperoxide (19 mg) was injected into both eyes. Experimental conditions are explained in the text.

had a marked protective effect. In the control experiment, heme was first extracted from horseradish peroxidase to inactivate enzymatic activity and the apoenzyme tested in a similar manner (Fig. 6B). No protective effect was observed after the active site was removed. Several other enzymes were subsequently tested in single experiments. Thus, neither of the two acidic horseradish peroxidase isoenzymes (Sigma type 7 and 8) we examined were protective (Figs 6C, D), nor was a similar concentration of superoxide dismutase (Fig. 6E). In another experiment, 0"5 mg of bovine catalase was injected prior to the L H P (Fig. 6F). After four days, there were

L I P I D P E R O X I D E R E T I N O T O X I C I T Y 165

A A B B

a

. C C D O

E E ~ F F b

Fro. 6. The non-protective effect of other anti-peroxidative enzymes on L H P damage to the albino rabbi t injected prior to 19 mg of linoleic acid hydroperoxide from series 2. The dose of each enzyme was 0-5 mg and the procedure identical to that described in Fig. 5. For each experiment (A through F), the recording to the left indicates the pre-injection value and the one to the right is 10 days post-injection. Figure A = LHP only; B = LHP and the apoenzyme of H R P ; C = L H P and type 7 acidic H R P ; D = L H P and type 8 acidic H R P ; E = L H P and superoxide dismutase; F = L H P and catalase. The control a-wave amplitudes from A through F are 33, 49, 50, 52, 51 and 45 pV and the b-waves are 93, 88, 91, 80, 85 and 90/tV respectively. Residual b-wave activity in E is indicated (b).

TABLE II

Effect of various compounds on ERG activity, at three weeks post-injection

Intravitreal ERG activity (~o of original activity) Compound tested concentration (M) a-wave b-wave

Untrea ted 100 100 Vitamin A acetate (10 -a) 100 100 Retinal (10 -a) 100 100 Retinoic Acid (10 -3) 100 100 Hydrogen peroxide (10 -4) 100 100 Hydrogen peroxide (10 -a) 90 100 Hydrogen peroxide (10-2) * 20 38 Sodium iodate (10 -7) 100 100 Sodium iodate (10 -s) 88 100 Sodium iodate (10 -~) 90 100 Sodium iodate (10-4)t 14 65 Sodium iodate (10-3)t 4 75

These data were produced during the series 3 experimental adult albino rabbits.

* 12 days, post-injection. t I day, post-injection.

period and represent single recordings from

166 D. ARMSTRONG ET AL.

no changes in a-wave activity, but the b-wave was decreased 43 To below the pre-injection value. However by 10 days, a- and b-wave activities were markedly decreased 67 and 75% respectively, indicating non-protection. Neither horseradish peroxidase, catalase or superoxide dismutase at 0"5 mg concentrations were toxic to the ret ina when injected alone.

In other experiments, v i tamin A acetate, retinal and retinoic acid were examined and found to have no effect on the ERG at concentrations similar to the L H P (Table 2). Likewise, hydrogen peroxide at 10 -4 and 10 -a M concentrations were essentially ineffective in damaging the ERG response. However, when raised to 10 -2 M levels, only 20 To of the a-wave and 38 % of the b-wave activi ty remained 12 days after exposure. On the other hand, at 10-4M and higher, sodium iodate, a known retinotoxic compound, rapidly inactivated the E R G in only one day.

The effect of L H P on retinal function in pigmented eyes has also been examined briefly. In three experiments, concentrations of lipid peroxides in the 19 mg range required considerably more time to produce adverse effects. Thus, in adult rabbits no a- or b-wave ERG changes were evident during the first 18 days after injection, but by 24 days, the a-wave had decreased 53 % and the b-wave was reduced by 41%. At 31 days, the a-wave was then 70 % and the b-wave, 65 % below normal. However, in one young rabbit two to three weeks of age, there was an initial decrease of approximately 80 % in both a- and b-wave activi ty after only seven days. By 16 days post-injection, the ERG's were decreased by more than 90 %.

4. D i s c u s s i o n

When long-chain, unsaturated lipids come in contact with atoms or molecules having unpaired electrons (free radicals), these highly reactive species readily extract electrons which result in the formation of a lipid free radical (Gutteridge, 1978). This radical then reacts spontaneously with oxygen, or in the presence of metal ions, yields a variety (alkoxy, peroxy) of lipid peroxides. Such linkages are unstable and rapidly break down, liberating MDA and other aldehyde fragments. In biological systems, MDA is capable of cross-linking the pr imary amine groups of numerous chemical compounds into Schiff-bases. The interaction of these various processes results in the formation of fluorescent lipopigments. The cytotoxic nature of products of free radical oxidation and reactions intitiated by them, is well known (Schauenstein, Esterbauer, and Zollner, 1977; Willson, 1979; Roubal, 1970; and Yau, 1979). Anti-oxidant deficient animals develop retinal dystrophies and accumulate increased amounts of lipopigments in the R P E (Robison, Kuwabara , and Bieri, 1979; Farnsworth and Dratz, 1976).

The present study has shown tha t the peroxide derivatives of four PUFA were extremely toxic to retinal function in the albino rabbit. The soybean lipoxygenase catalyzed reaction results in greater than 95 % conversion oflinoleic and linolenic acids to the 13-hydroperoxy isomer (Aoshima, 1978). In our study, this non-heme iron containing protein also catalyzed the oxidation of arachidonic and docosahexaenoic acids (strong absorbance at 234 nm), since they also contain the cis, cis-1, 4-pentadiene system necessary for the insertion of molecular oxygen (Pistorius, Axelrod, and" Palmer, 1976). Lipoxygenase in blood platelets has been reported to convert arachi- donic acid into a hydroperoxide (Nugteren, 1975).

To date more than 50 rabbit eyes have been injected with 18:2 hydroperoxide during these studies and in every instance, a progressive decline of the E R G was

LIPID PEROXIDE RETINOTOXICITY 167

observed within variable periods of time following exposure. Examples of this phenomenon are shown in Figs 2, 4 and 5. The rate of these changes is due to the amount of peroxide produced by each preparation of lipoxygenase. In each instance however, the ERG usually reaches non-recordable levels by 7-10 days and this activity never returns. Three other L H P have also been tested in five additional rabbit experiments and they too have shown similar and reproducible changes.

The system employed in this study did not elicit ERGs as high in amplitude as generally reported, however we have demonstrated that under our standardized conditions an adequate signal could be produced from un-injected eyes which allowed adequate comparisons to those made with the injected eyes. In those instances where compounds were toxic, the ERG showed a progressive decline and finally was extinguished. Other compounds showed no effect upon ERG activity. In one experiment where conditions were more optimal (Fig. 4; control a-wave -- 150 #V and b-wave -- 450/~V), the same toxic effect of L H P was observed.

During the first 24 hr following injections of LHP, a-wave activity was affected most and the largest decreases were due to 18:2 and 20:4 hydroperoxides. By four days, the degenerative effect of the 22 : 6 hydroperoxide appeared to be greater. In general, each of the b-wave ERGs were less affected at one day post-injection, but by four days the decreases were of the same magnitude as observed for the a-wave. At 12 days post-injection, all ERGs were markedly decreased except for the 20 : 4 hydroperoxide which showed an apparent recovery of b-wave activity. However, at concentrations above 19 mg, the ERG was completely and irreversibly extinguished by the 20:4 hydroperoxide. Since our main interest in this paper concentrated on the effects of 18 : 2 hydroperoxides, we did not pursue the rebound phenomena of 20 : 4 hydroperoxide. However, arachidonic acid is normally converted in vivo by lipoxygenase and/or cyclo-oxygenase to aperoxide before entering the pathway for prostaglandin synthesis (Porter, 1980) and it is conceivable that the effect we observed was in part due to presenting the retina with a precursor for prostaglandin synthesis which resulted in a dilution effect of the initial 20:4 hydroperoxide level. Prostaglandin E~ synthesis does occur to some extent in the rabbit retina (Kass and Holmberg, 1979). An alternative hypothesis might be that the 20:4 hydroperoxide was conjugated to glutathione to form leukotriene-C4 (Malik and Wong, 1981), which would also tend to reduce the initial concentration of peroxide. In previous studies, intravitreal injections of 20:4 hydroperoxides also had little effect on the rabbit ERG (Wallenstein and Bito, 1978).

The data presented in Figs 3 and 5 show that the peroxide group is most probably responsible for the toxic action of LHP. Thus, not only did horseradish peroxidase block the toxic effect of 18 : 2 hydroperoxide, but removal of the heme prosthetic group resulted in no protection at all. There are no reports of H R P acting on L H P substrates but leukocyte pero=(idase has recently been shown to degrade the lipid peroxides of 18:2 and 20:4 (Schwerer and Benheimer, 1978).

I t is of interest that catalase was not effective in de-toxifying the LHP. We have recently reported that catalase is only present in the canine retina, but not in the photoreceptor outer segments or in the pigment epithelium (Armstrong, Santangelo, and Connole, 1981~. Thus, it appears tha t peroxidase is the primary protective mechanism in the RPE where at least part of the c-wave orginates. However, the type of peroxidase present in vivo may be quite important, since only the major, basic isoenzyme of horseradish peroxidase (Fig. 5) was effective in protecting against LHP, whereas the acidic isoenzymes (Fig. 6D, E) were not. I t has been reported that cellular

168 D. ARMSTRONG ET AL.

uptake of the basic horseradish peroxidase is greater than the acidic isoenzyme (Davies, Rennke and Cotran, 1980) and one might suspect negatively charged enzymes to be more indicative of biological function. Previous studies have suggested tha t the R P E peroxidase may be composed of isoenzymes so knowledge of the exact isoenzyme composition will be important to determine (Armstrong, Connole, Feeney, and Berman, 1978b). Superoxide dismutase was also not effective in protecting against L H P damage, suggesting that superoxide radicals are not involved in the destructive process observed during L H P administration.

Other compounds were compared with the results produced by L H P and MDA. For example, sodium iodate showed ERG damage at concentrations lower (10 -4 M) than seen with LHP. This known R P E toxin also reduces and then abolishes c-wave activi ty while a- and b-wave activities are still retained for some t ime (Nilsson, Knave and Persson, 1977). Hydrogen peroxide was only effective in reducing E R G activi ty at levels (10 -2 M) greater than those used for LHP. This finding suggests tha t the rabbi t neural-retina is well protected enzymatically from hydrogen peroxide damage and in fact, contains both catalase and two peroxidases (Armstrong et al., 1981). Three vi tamin A derivatives were also tested and each were well tolerated by the retina, even at millimolar concentrations. I t would have indeed been surprising if these compounds caused significant problems for the retina since among other things, they normally exert an anti-lipoperoxidative action (Callari and Billitteri, 1976).

The gradual decline over one to two weeks of a- and b-wave E R G activities in these studies is proof tha t the observed changes are due to a progressive re t inopathy and not simply the result of retinal detachment. In the lat ter instance, c-wave act ivi ty is lost immediately after: detachment and this is followed by a slower decline of the a- and b-wave (Marmor, 1979). In the studies described in Fig. 4 we have shown tha t the c-wave is reduced to > 50 % of normal two days after injection and then continues to decline until the E R G is essentially extinct, 15 days later.

The possible protective effect of ocular melanin has been briefly examined. Because of its known action as a free radical scavenger (Cope, Sever and Polis, 1963) we were interested in comparing the effects of L H P on E R G act ivi ty between albino and pigmented rabbits. Individual differences in the rabbi t E R G have been noted as a function of age and degree of pigmentat ion (Reuter, 1974). Injections of 18 : 2 peroxide (19 mg) produced no changes in the E R G of pigmented rabbits until roughly 24 days, in contrast to the albino eye, where changes were evident at one day or less. Furthermore, the adult pigmented rabbi t never fully developed sufficient damage to totally extinguish the ERG. Besides its scavenging ability, an additional measure of protection afforded by melanin in pigmented eyes, could be provided from the peroxidase activity which is associated with melanin granules (Siakotos, Armstrong, Koppang and Connole, 1978; Okun, Edelstein, Patel and Donnellan, 1973 ; Armstrong et al., 1978b, 1981). Following phagocytosis, peroxidase could offer an initial protection in the R P E from any peroxides tha t were formed in the lipid rich ROS and secondarily, if free radical species were generated as a result of intracellular reactions from undegraded peroxides (Mason, Ingram, and Allen, 1960). In confirmation of our findings, a recent report has shown tha t the pigmented rabbi t eye is definitely more resistant to lipid peroxidation than the albino eye (Dontsov, Sakina and Ostrovski, 1980).

We also conducted a similar experiment using a young pigmented rabbit . Injections of L H P (19 rag) into the eye of a 2-3 week old animal resulted in a more accelerated, degenerative process than observed in the adult pigmented rabbit . Thus, by one week

LIPID PEROXIDE RETINOTOXICITY 169

post-injection, significant electrical act ivi ty was lost, and by two weeks the E R G was virtually eliminated. Retinal and R P E peroxidases show a maturat ional profile (Armstrong et al., 1978a) and this effect is known for other tissues where peroxidases are a prominent enzyme (Yamashina and Barka, 1974). R P E melanin, although present in utero, also increases in amount with age (Toda and Fitzpatrick, 1972). Therefore, it was anticipated tha t younger animals might have less protection against L H P because of the sub-optimal levels of peroxidase and melanin. In line with these maturat ional changes, it is fortuitous tha t the R P E in young animals is not overwhelmed by ROS material since ROS volume (Hollyfield and Rayborn, 1979}, number of phagosomes /RPE (Tamai and Chader, 1979), and deposition of fluorescent lipopigments (Feeney, 1978) all increase with age. Fortunately, increases in peroxidase and melanin apparent ly keep pace with these events.

Previous studies by others have shown tha t high intensity light (Kagan et al., 1973), addition of ferrous iron and ascorbate to the media of isolated frog retina in vitro (Schvedova, Sidorox, Novikov, Galushchenko and Kagen 1979), or experimentally induced ocular siderosis (Declereq, Meridith, and Rosenthal, 1977; Hiramitsu, Hasegawa, Hirata , Nishigaki, and Yagi, 1976b), result in retinal degeneration with an associated loss of ERG activity. Such manipulations induce lipid peroxidation with the formation of 234 nm absorbing compounds and other degradative products. Moreover, the non-enzymatic oxidation of lipids may yield different toxic species other than those produced by lipoxygenase. Nevertheless, retinotoxicity can indeed be demonstrated by the E R G technique.

We have carried the peroxide induced experiments further, by showing tha t the direct injection of a known, enzymatically produced L H P causes retinal degeneration. This type of model will be useful in testing the protective effects of certain antioxidative compounds as others have reported (Hiramitsu, Majima, Hasegawa and Hirata, 1974; Patterson, Sweasey, Roberts, and Patt ison, 1974), as well as the specific effects L H P have on retinal ultrastructure. In the lat ter context, histological evaluations of the present experiments will be reported separately to document the early and late pathology which is associated with E R G changes reported here.

A C K N O W L E D G E M E N T S

The authors acknowledge support provided for these studies by the National Eye Institute, Grant No. 02627, and in part by a grant from Fight for Sight, Inc., New York City, New York, the National Society for the Prevention of Blindness, an unrestricted departmental grant from Research to Prevent Blindness, Inc., the Swedish Medical Research Council (Project No. 12X-734), the Children's Brain Disease Foundation, and the Norwegian Agricultural Research Council. Portions of this work were performed at the University of Colorado Health Sciences Center and the National Veterinary Institute in Oslo, Norway. Dr Armstrong is the recipient of a Research Career Development Award, K04 EY 00130. We thank Per Persson and Cathryn Wilson for technical assistance with ERG recordings.

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