The Role of Tocopherol in Oxygen-Induced Retinopathy: Kitten Model

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The

Role

of Tocopherol

in Oxygen.lnduced

Retinopathy:

Kitten

Model

Dale L. Phelps, M.D., and Arthur L. Rosenbaum, M.D.

From the Departments of Pediatrics and Ophthalmology. University of California at Los Angeles School of

Medicine

ABSTRACT. The effect of tocopherol (vitalnin E) on oxygen-induced retinopathy was studied in 75 kittens following the development of a 12-point scoring systenl to quantitate the

degree of retinopathy seen at three weeks. The tocopherol was found to be beneficial when given daily from the day of birth (P < .0001) with oxygen exposures of two to three days (79% FU.) beginning on day 3. Caution is urged in applying these data to humans because (1) hepatosplenomegaly was noted in the treated animals, and (2) the kitten model of oxygen-induced retinopathy is not entirely satisfactory,

Pedi-atrics, 59:998-1005, 1977, RETROLENTAL FIBROPLASIA,

OXY-GEN TOXICITY, VITAMIN E, RETINOPATHY, KITTEN.

The increased survival of prematurely born infants coupled with the developmental scrutiny they receive has revealed a continued problem with retinopathy of the premature or retrolental fibroplasia (RLF).’ Since this disease was first clearly linked with oxygen in 1956,2 animal studies have led to significant gains in our under-standing of its pathophysiology36 while newer diagnostic techniques of indirect ophthalmoscop and fluorescein angiography have led to a greater

understanding of the incidence and natural course

of the disease in humans. The problem remains that, whereas arterial hyperoxemia is the primary cause of RLF, additional factors must account for

(

1) the persistence of the disease despite careful 02 monitoring, (2) the wide variability in the severity of the disease among infants receiving approximately equal amounts of oxygen, and (3) occasional cases of RLF in cyanotic infants or in infants receiving no oxygen therapy.’ To deter-mine these factors Johnson, Schaffer, and BoggsM

began to study the antioxidant, tocopherol (vitamin E), for a possible protective effect based on (1) the 1949 findings of Owens and Owens” that vitamin E is useful against RLF, (2) the known tocopherol-deficient state of human pre-mature infants,b0h1 and (3) the biochemical

func-tion of tocopherol. (Tocopherol is one of the naturally occurring antioxidants that protect cell membranes against peroxidation by free oxygen radicals.) Their preliminary results suggest that it

may be protective.

We were excited by these findings and elected to study the effectiveness of tocopherol in the recognized kitten model of RLF. The progress of retinal vascularization from birth to 3 weeks of age in the kitten closely corresponds to the human fetus from 6 months gestation to term.’ Also, extensive studies of hperoxia in the kitten’6 have shown it to be an excellent model of the acute proliferative changes seen in human RLF,

al-(Received November 24, 1976; revision accepted for publi-cation January 11, 1977.)

Presented before the Western Society for Pediatric Research, Carmel-by-the-Sea, California, February 6, 1976.

Supported by U.S. Public Health Service grant EY01507 and in part by CR5 grant 5 501 RR 05354 from the UCLA School of Medicine. Computing assistance was obtained from the health sciences computing facility, UCLA, supported by Special Resources grant RR-3 from the National Institutes of Health.

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2

Sacrifice

HYPEROXIA/RETINAL

RESPONSE CURVE

(logistic

regression)

points

omitted

for

regression

12

11

10

9

8 7 6 5 4

3

0

. .

#{149}.#{149}.

I I I I I I I I I I I

0 10 20 30 40 50 60

70

80 90 100 110

though not necessarily of the late cicatricial

changes, since the cat rarely develops retinal detachment. This limitation of the animal model has important implications for any conclusions to be drawn from this work.

MATERIALS AND METHODS

The kittens were studied in two phases: in the

first phase a quantitative scoring system was

developed for the retinopathy, and in the second the effect of tocopherol was tested. The basic

techniques and materials central to both phases will be described together.

The kittens were from an established breeding colony where mothers were fed a standard dry cat chow ad lib (Purina Cat Chow), supplemented with daily pet tuna or chicken during pregnancy and lactation. The pregnant queens were ob-served once daily and the date on which kittens were discovered was designated day 1. On day 3 (Fig. 1) the kittens to be exposed to hyperoxia were placed in a standard infant incubator (Model C-77, Air Shields) with normal air circulation, maximum humidity, and controlled temperature (30 C). Continuous flow oxygen was maintained at an FO of 79 ± 1% and checked at frequent

intervals with a Beckman Oxygen Analyzer which

was calibrated daily. The queens alternated the time spent with their kittens in 79% oxygen and

a:

0

C-) (#1

>-0

0

a:

Phase I

Hyperoxia

I

1---:-t-1

4” 1 2 3

I Birth age, weeks ->

Sacrifice

Phasell

ri

58

1 g

hrs

I

‘1’

Birth age,

weeks-FIG. 1. Time course and treatment in the two phases of the studs’.

their kittens living in room air in a control incubator, experiencing no more than 12 hours per day in high oxygen. No queens or kittens evidenced apparent pulmonary oxygen toxicity. All procedures were carried out through portholes with plastic sleeves to maintain the stable environment.

After the designated number of hours in oxygen the kittens were abruptly removed to room air

Hyperoxia

exposure

(hrs)

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Score

Category 0 1 2 3 4

*

Equal to control

*

Mildly retarded especially temporally Retarded growth all around Small circle of vessels only Major Vessel Pattern B

3 pairs of vessels, delicate pattern Piled on vessels peripherally linifora piled on vessels; basic pattern still discernable Heavily piled on vessels no basic 3 pairs visible Pen-arterlolar capillary free zone C Zone apparent around each arteriole to the periphery Patchy loss of clear zone

Uniforui loss of clear zone

Capillary tuft formation

D

None

1 or 2 large or small at the periphery

More than 3 scattered near the periphery

Small or large extending towards the disk

Extensive tuft formation with a mask of vessels over the disk

FIG. 3. The retinal scoring system. A value is judged in each of the four categories and the sum

taken as the final score.

and returned to a large cage where they were raised by their mothers to age 21 ± 1 days, at which time they were killed following a 4- to 16-hour fast. After barbiturate overdose, blood was collected with EDTA on ice, the plasma sepa-rated within four hours and stored at -20.0 C until determination of the tocopherol level by the method of Bieri.’2 Next, the aorta was catheter-ized, the external jugular veins were opened, and 100 cc of warmed saline was flushed through the vessels followed by a two- to five-minute flush with 50% Higgins india ink diluted with normal saline. (This technique was shown to D.L.P. by Arnall Patz, M.D., Johns Hopkins Medical School.) After a 30-minute waiting period in the head-down position, the eyes were enucleated, placed in 10% formalin (buffered with marble chips)’3 for a minimum of 48 hours. One or both

eyes from each kitten were then rinsed in running water overnight, and the retina was removed and mounted flat on a glass slide in Kaiser’s jelly’4 by making several radial cuts in the tissue and removing most of the vitreous.

Phase 1

Eight litters (37 kittens) were studied. At least one kitten from each litter served as a room air control, and the remaining 28 kittens were exposed to 79% oxygen from 21 to 1 10 hours. The

retinal flat mount prepared from the left eve of each kitten was examined stereoscopically. After

standardizing the scoring system (see Results

section), each retina was scored by both

investiga-tors independently and the average of these

results was used as the kitten’s score. A

dose-response relationship of the score to hours of hyperoxia was then estimated (Fig. 2).

Phase 2

From the estimated dose-response curve, four

exposure times, (47, 58, 68, 80 hours) were selected to produce minimal to severe retinop-athy to test the effect of tocopherol treatment. Four litters were assigned to each exposure tniie

(

75 kittens), and no one queen appeared twice in

any one exposure time. On day 1, each litter was separated equally into placebo and treatment

groups. Attempts were made to divide the two largest kittens between treatment and placebo, and then the next two largest, etc.; however, most litters had equally sized kittens and, therefore,

assignments were often made by color, balancing the number of black, striped, grey. white, calico, or carrot-colored kittens between tocopherol and placebo. Thirty-nine kittens received 50 mg of dl-alpha-tocopherol acetate 1M daily from day 1 imtil the day of death (9 in the 47-hour, 8 in the

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47-RESULTS

Statistical Methods

Fu;. 4. Exaniples of four retinas scored in categories A and B: \\‘(kitten 293): A 0, B 0; X (kitten 253): A = 1, B = 1; Y(kitten 530); A 2, B 2; Z(kitten 235): A 3, B 3.

hour, 7 in the 58-hour, 10 in the 68-hour, and 10 in the 80-hour group) received daily injections of an equal volume of a vehicle placebo (drug and placebo supplied by Hoffman-La Roche, Nutley, New Jersey); 50 mg represents a 1 log,1, increase over the approximate daily requirements of vi-tamin E for kittens, 5 mg.’5 The tocopherol acetate was in a vehicle of disodium edetate, Emulphor, glycerin, sodium acetate, acetic acid, and thimerosal. The placebo had the same chem-ical composition without the tocopherol. All were placed in oxygen on day 3, removed at the appropriate time, and killed at 3 weeks as described. In this phase, retinal flat mounts were prepared from both eyes and each kitten’s final score was an average of left and right scored by each investigator.

Statistical analysis was conducted by analysis of variance using the P value from the type II sums of squares unless otherwise indicated.

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FIG. 5. Examples of four retinas on higher magnification scored in categories C and D: W(kitten

111): C = 0, D 0; X(kitten 006): C 1, D 2; \‘(kitten 803): C 1. D :3: Z(kitten 231):

C = 2, D = 4.

As the severity of the retinopathy increases, the

maximal outreach of vessel growth at 3 weeks of

age decreases and the number of new and

exces-sive arterioles increases, first in the periphery and

(

in the worst cases) even from the disk. Figure 5

shows magnified

(

x

3) portions of retinas at the

advancing edge of vessel growth. These again

show increasing retinopathy where the loss of the

periarteriolar capillary free zone and the

increas-ing amount of neovascularization are readily

apparent. When the 37 retinas were scored on

this finalized system, there was excellent

intraob-server, as well as interobserver correlation. In the

few instances where the total score differed by

more than 2.0 points. masked rescoring nearly

always revealed a recognizable error in scoring by

one observer or the other, and the corrected score

was used.

Figure 6 shows the histologic features of the

retina in a normal and an 80-hour oxygen-exposed

retina. This clearly shows that a large portion of

the neovascularization is extraretinal as is

apparent through the stereoscopic microscope

used for scoring the flat mounts. The

oxygen-exposed retina is also thickened.

The oxygen response curve (Fig. 2) was

esti-mated by simple logistic regression. The two

indicated outlying data points were omitted as we

wished to predict future responses with this

curve. Essentially no retinopathy (that can be

identified 2#{189}weeks later) occurs with less than 32

hours of exposure. The severity then gradually

increases with the duration of exposure (with a

large variance) until reaching maximal values of

12 at 80 to 100 hours. The striking degree of

variation within and between litters began to be

apparent at this point. The two retinas that

represented the outlying points were reviewed

several times. There was no scoring error. The

retinopathy score of 3.5 in a kitten that received

no oxygen represented primarily

neovasculariza-tion that was unquestionably present in this

kit-ten (score = A(O.5) + B(O.25) + C(O) + D(2.75)

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FIG. 6. Photomicrograph of retinal cross sections showing normal architecture (Y) and the thickened retina with extraretinal neova.scularization (Z) as seen in an 80-hour oxygen exposure. Both micrographs are taken at the same magnification and the

arrow points out the poorly defined retinal surface in the abnormal section (hematoxlin and eosin).

Tocopherol Trial

Tocopherol therapy in doses of 50 mg/day was effective in reducing but not eliminating the oxygen-induced retinopathy (P < .0001). The variables that were significant in explaining either the A, B, C, or D subscores or the total score were the use of tocopherol (P < .0001), the number of hours spent in oxygen (P < .0001), and

the mother (P < .0009). None of the interactive terms was significant (Table I). This means that when we know the kitten’s mother, how much oxygen he received, and whether he was treated with tocopherol, we can explain 88% of the variance seen in the resulting scores. Craphically,

Figure 7 shows these data where each line

represents one litter with the average of the

TABLE I

TOCOPHEROL PROPHYLAXIS IN EXPERIMENTAL OXYGEN-INDUCED RETINOPATHY: ANALYSIS OF VARIANCE-PROBABILITY VALUES#{176}FOR VARIABLES RELATED TO RETINOPATHY SCORES

Score Mother No. of

Hours of

Oxygen

Tocopherol

or Placebo

(Hours 02)

X

(Tocopherol)

(Mother)

X

(Tocoplierol)

(Moth

X

(Hours er)

02)

A .000lt .0001 .0001 .630 .790 .642

B .0009 .0001 .0001 .706 .814 .518

C .0001 .0001 .0001 .418 .495 .958

D .0001 .0001 .0001 .101 .046 .098

Total .0001 .0001 .0001 .286 .284 .819

aType II Sums of Squares.

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l0

. Placebo from 1 litter

0 )(Tocopherol from 1 litter

.-o Values from a single litter

6

4

\

2

HOURS OF OXYGEN EXPOSURE

2 4 6 8 10 2

RETINOPATHY SCORE, O.S.

FIG. 8. Correlation between the scores from the left eye and right eye in each kitten.

L&J

0

C-)

>-=

I-0 z

FIG. 7. Effect of tocopherol treatment on oxygen-induced retinopathy.

placebo kits in that litter connected to the average of the tocopherol-treated kittens in the same litter. In 15 of 16 litters, there was improve-ment with tocopherol treatment. The differences between the placebos from one litter to the next

makes the “mother” effect obvious.

Of additional interest was the considerable variation between the scores of the left and right

eyes. Figure 8 shows the plot of these with the

line of identity; the variance around that line is considerable with some pairs differing by as much

as five points.

Plasma levels of tocopherol in the tocopherol-treated kittens were significantly elevated over the placebo kittens at 3 weeks of age, (12.3 ± 7.1

mg/100 ml, ± SD vs 0.8 ± 0.6 mg/100 ml,

P < .001, t test). The range among the treated kittens was large (3.7 to 31.5 mg/100 ml) although it did not overlap the range among the placebo kittens (0.1 to 3.2 mg/100 ml). No significant

effect on the score could be demonstrated for the tocopherol level when the kittens were killed (P = .08), nor the hematocrit or weight at that time. In the phase 2 study at the time they were killed, the kittens given placebos weighed 240 ± 40 gm (1, SD) (range, 177 to 339) and the tocopherol-treated kittens weighed 225 ± 63 gm (range, 115 to 400 gm).

a: 0

C-) “I

>-=

a-0

a:

DISCUSSION

These data demonstrate a clear beneficial effect of tocopherol on oxygen-induced retinop-athv in kittens, adding weight to the positive side

of the controversy over the potential efficacy of

tocopherol. In our study this model has proven to be much like the human disease with considerable variation between the left and right eyes, as well

as between animals from a different genetic

background. This discrepancy in scores among

animals exposed to identical oxygen environments

but differing genetic backgrounds may permit

study of this distressing phenomenon commonly

noted in humans as well. However, it must be

remenibered that the kitten as a model of the late,

or cicatricial, changes of RLF is not highly acceptable. Both Ashton et al.4 and Patz5 point out the failure of the cat to develop retinal detachment after severe oxygen stress as a serious drawback of this model. Additionally, in the

human disease, it has not been possible to clearly

correlate the severity of the acute proliferative

changes with the final cicatricial grade of the

disease.’7 If tocopherol is beneficial in the acute

proliferative stages, but not the cicatricial end grades of the disease, we have gained little.

Although tocopherol has been assumed to be a

nontoxic substance, LD() levels of approximately

500 mg/kg have been established in animals’8 and

(8)

treated kittens in this series. Awad and

Gilbreath’” have found evidence of hepatic

toxicity in rabbits treated with 100 times the

recommended daily allowance (for rabbits).

Toxicity levels are not known in human infants.

Implications

It seems Ol)ViOUs that we must ask ourselves

whether tocopherol treatment of the human

premature infant will help prevent some

blind-ness and some myopia, or will it, in some as yet

unknown way harm our infants as other

appar-ently beneficial drugs (for example, oxygen) have

in the past? Problems of drug absorption will be

significant, but clinical trials must be conducted

that are randomized, prospective, and

impecca-bly evaluated to answer these questions. One has

begun,5 and we hope more will begin soon.

SUMMARY

A quantitative retinopathy scoring system was developed for the kitten model of RLF where a

disturbing variation in retinopathy was noted

under identical environmental oxygen stress. This

system was used to test the effectiveness of

preventing this disease with tocopherol or

vitamin E. The drug was found to be beneficial and these data provide support for conducting

controlled human trials.

REFERENCES

1. James LS, Lanman JT: History of oxygen therapy and retrolental fibroplasia. Pediatrics 57(suppl): 591, 1976.

2. Kinsey yE, jacobtis JT, Hemphill FM: Retrolental fibroplasia: Cooperative study of retrolental fibro-plasia and the use of oxygen. Arch Ophthalmol

56:481, 1956.

3. Ashton N, \Vard B, Serpell C: Role of oxygen in the genesis of retrolental fibroplasia: A preliminary report. Br J Ophthalmol 37:513, 1953.

4. Ashton N, Ward B, Serpell C: Effect of oxygen on developing retinal vessels with particular reference to the problem of retrolental fibroplasia. Br J

Ophthalmol 38:397, 1954.

5. Patz A: Experimental studies. Am JOphthalmol 40:174, 1955.

6. Flower RW, Patz A: Oxygen studies in retrolental

fibroplasia: IX. The effects of elevated arterial oxygen tension on retinal vascular dynamics in the kitten. Arch Ophthalmol 85:197, 1971.

7. Flynn JT: Acute proliferative retrolental fibroplasia: Evolution of the lesion. Albrect von Graefes Arch Kim Ophthalmol 195:101, 1975.

8. Johnson L, Schaffer D, Boggs TR Jr: The premature infant, vitamin E deficiency and retrolental fibro-plasia. Ani J Clin Ntitr 27: 1 158, 1974.

9. Owens WC. Owens EU: Retrolental fibroplasia in premature infants: II. Studies on the prophylaxis of the disease: The use of alpha tocopheryl acetate. Am J Ophthalmol 32:1631, 1949.

10. Melhorn DK, Gross 5: Vitamin E-dependent anemia in the premature infant: I. Effects of large doses of medicinal iron. J Pediatr 79:569, 1971.

11. Ritchie JH, Fish NIB, McMasters V, Grossman M: Edema and hemolvtic aneniia in premature infants: A vitamin E deficiency syndrome. N Engl J Med 279:1185, 1968.

12. Bieri JG: Chromatography of tocopherols, in Marinetti CV (ed): Lipid Chrolnatographic Analysis. New

York, Marcel Dekker Inc, 1969, vol 2, pp 459-478.

13. Roth AM, Foos RY: A system for the macro examination of eyes in the laboratory. Am J Clin Pathol 59:674,

1973.

14. Mallory FB: Pathological Technique. New York, Hafner Publishing Co. 1938, p 1(X).

15. Scott PP: Minerals and vitamins in feline nutrition, in Graham-Jones 0 (ed): Canine and Feline

Nutri-tional Requirelnents. New York, Pergamon Press,

1965, pp 75-89.

16. Michaelson IC: The mode of developnent of the vascular system of the retina, with some

observa-tiOlls 011 its significance for certain retinal diseases.

Trans Ophthalniol Soc UK 68: 137, 1948.

17. Silverman WA, Blodi FC, Locke JC, et al: Incidence of retrolental fibroplasia 10 a New York nursery. Arch Ophthalmol 48:698, 1952.

18. Bauernfeind JC: Vitaniin E information brochure. Nutley, New Jersey, Hoffman-LaRoche Inc. 19. Awad AB, Gilbreath RL: Flypervitaminosis E in

athero-sclerotic rabbits. Nutr Reports Int 11:409, 1975.

ACKNOWLEDGMENT

The authors wish to express their appreciation to some of the many people who provided intellectual and sometimes

time-consuming support: Lila Aftergood, Ph.D.; Douglas Clarkson; Jean Cordon; Lois Johnson, M.D.; Sylvia Mansour; Charles Phelps, Ph.D.; William Silverman, M.D.; and Celia Stewart.

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1977;59;998

Pediatrics

Dale L. Phelps and Arthur L. Rosenbaum