`
`Retinal degeneration in monkeys induced by
`
`deficiencies of Vitamin E or A
`
`K. C. Hayes
`
`The effects of vitamin E or vitamin A deficiency on the retina were assessed in monkeys for
`as long as two and three-quarters years. A macular degeneration developed after two years
`in monkeys fed vitamin E-deficient diets. The lesion was characterized by focal, massive disrup-
`tion of photoreceptor outer segments attributed to lipid peroxidation of these lipoprotein
`structures containing highly unsaturated fatty acids. The focal nature of the lesion precluded
`any evidence of clinical blindness. Vitamin A deficiency was typical of that described by
`others, and was accompanied by xerophthalmia, keratomalacia, and clinically impaired vision.
`Anatomic evidence suggested that
`the structural disruption of photoreceptors was more
`advanced in cones and was most pronounced in the macula with a lesser involvement of the
`peripheral retina.
`
`Key words: vitamin A, vitamin E, retina, blindness, macular degeneration.
`
`Experimental vitamin E deficiency has
`
`elicited a multitude of pathologic entities
`in a variety of species. In terms of ocular
`pathology, cataract formation has been re-
`ported‘- ‘-’ but retinal degeneration has not
`been previously described. A group of dogs
`found deficient
`in vitamin E at necropsy
`was found to have advanced retinal de-
`
`its etiology and patho-
`generation,3 but
`genesis were unclear. Children afllicted
`with abetalipoproteinemia suffer from vis-
`ual
`impairment thought to be related to
`a failure in absorption of fat and the fat-
`soluble vitamins,‘ and the onset of neuronal
`
`From the Department of Nutrition, Harvard School
`of Public Health, Boston, Mass. 02115.
`This work was supported in part by United States
`Public Health Service Research Grants EY-00631
`and HL-10098, and the Fund for Research and
`Teaching, Department of Nuuition, Harvard
`School of Public Health.
`
`Submitted for publication Jan. 21, 1974.
`
`(Batten’s disease), a
`ceroid-lipofuscinosis
`syndrome resembling the ceroid accumula-
`tion of chronic vitamin E deficiency,“ is
`usually accompanied by impaired vision
`and progressive blindness of undetermined
`pathogenesis.“ These conditions and the ob-
`servation that vitamin E is concentrated in
`
`(OS)’
`the photoreceptor outer segments
`suggest that tocopherol may have a spe-
`cific function in the retina.
`
`The present study in primates provides
`details of a retinal degeneration in the
`macula of two species of monkeys follow-
`ing long-term deprivation of vitamin E.
`In addition,
`the lesion is compared with
`that produced by vitamin A deficiency in
`one of these species and difierences be-
`tween the deficiencies are discussed.
`
`Methods
`
`The complete description of the animals, diets,
`and procedures for the vitamin E study has been
`reported elsewherv:-3.3 Two species of monkeys ap-
`
`499
`
`
`
`500 Hayes
`
`Investigative Ophthalmology
`July 1974
`
`Table I. Composition of semi-purified
`diet low in vitamin A
`
`Grams per
`.100 grams
`Ingredients
`20.0
`Casein, vitamin-free
`29.6
`Dextrin
`10.0
`Sucrose
`10.0
`Cottonseed-soybean oil
`23.8
`Cellulose“
`4.0
`Salt mix’:
`2.2
`Vitamin mixt
`0.3
`Choline chloride
`0.1
`Vitamin D3 (1,250 IU per gram)
`°Alphacel, General Biochemicals, Chagrin Falls, Ohio.
`iflegsted, D. M., et al.: J. Biol. Chem. 138: ‘459, 1941.
`tThe vitamins added to 1,854 grams of dextrin were (in
`grams):
`a—tocopherol
`succinate, 10; ascorbic acid, 90;
`inositol, 10; menadione, 4.5- p-aminobenzoic acid, 10;
`nicotinic acid 9;
`riboflavin, E; pyridoxine~ HCl,_ 2;
`thia-
`mine-HC1
`i;
`calcium pantothenate
`6; biotin; 0.04;
`folic acid, 0.18; and cyanocobalamine, 0.005.
`
`proximately 14 months of age and born. and
`raised in our primate colony were used. These
`included 12 New World cebus (Cebus albifrons
`and apella) and 14 Old World cynomolgus (Macaca
`fascicularis) monkeys. Animals were fed- a. semi-
`purified diet containing either stripped safllower
`oil
`(tocopherol removed) or coconut oilpwith or
`without a vitamin E supplement for as "long as two
`and three-quarters years. Three deficient monkeys
`died unexpectedly whereas all others were killed
`at the end of the experimental period. Eyes were
`either perfused in situ via the left ventricle ‘of
`the heart using a 1 per cent formaldehyde:l.25
`per cent glutaraldehyde fixative buffered to pH 7.4
`with 0.1 M phosphate buffer, or
`the eye was
`removed from the anesthetized monkey and the
`posterior hemisphere immersed in 2 per .cent
`osmium tetroxide in 0.1 M phosphate buffer.
`Representative eyes were fixed in formalin and
`embedded in parafiin for routine hematoxylinand
`eosin (H & E) sections.
`~
`Six adult female cebus monkeys weighing 1,400
`to 1,700 grams were used for the study of vitamin
`A deficiency. They were fed an agar cake diet
`lacking in vitamin A (four monkeys) or the same
`diet with added vitamin A (two monkeys)
`(Table I)
`for as
`long as 21 months. Periodic
`serum samples were taken for vitamin A analysis.”
`Although the entire retina was available for light
`microscopy, only paramacular and mid-peripheral
`retina was examined by electron microscopy in the
`vitamin A-deficient monkeys.
`two anesthetized
`In both experiments one or
`monkeys were visually screened along with the
`control animals
`for
`their b-wave threshold in
`response to a
`full-field (Ganzfeld) white-light
`stimulus.” All
`the monkeys were housed under
`identical
`lighting and environmental conditions
`and were cared for by the same personnel. Cyclic
`
`light (12-hour periods) from overhead fluorescent
`lamps provided an approximate cage illumination
`of 8-foot candles.
`
`Vitamin E deficiency. The general pathophysiol-
`ogy of vitamin E deficiency in these monkeys5
`as well as the details of the anemia that devel-
`
`In sum-
`oped5 have been previously ylescribed.
`mary, deficient monkeys fed polyunsaturated saf-
`flower oil were most severely affected, developing
`severe hemolytic anemia, muscle degeneration,
`testicular degeneration, and marked ceroid ac-
`cumulation in several tissues. Retinal degeneration
`occurred in seven of
`the eleven monkeys
`fed
`either safllower oil or coconut oil without vitamin
`E. None of the vitamin E—supplemented monkeys
`developed retinal damage, and none of those fed
`coconut oil became anemic. Blindness was never
`
`apparent clinically. The full-field electroretinogram
`(ERG) b—wave threshold was considered normal
`in two deficient monkeys tested on two occasions,
`later found to have histologic degeneration of the
`macula. No fundus abnormality was detected with
`the indirect ophthalmoscope. There was no evi-
`dence of xerophthalmia and the monkeys appeared
`healthy and alert. The plasma vitamin E concen-
`tration was generally less
`than 100 fig/dl
`in
`advanced deficiency with both dietary oils. Con-
`trol values were in excess of 600 fig/dl.S
`The plasma vitamin A levels varied with the
`species and dietary treatment, being lower in the
`cebus than the cynomolgus monkeys and lowest
`in the vitamin E-deficient monkeys fed safliower
`oil.5 However,
`the most extensive degeneration
`was observed in a cebus monkey fed coconut oil
`for the longest period of time (33 months), and
`the vitamin A concentration was among the highest
`recorded for the cebus (20 [lg/dl).
`Light microscopy of the retina revealed a focal
`disruption of the photoreceptor OS layer that was
`restricted to the macula (Fig. 1).
`The minimal
`change detected by electron
`microscopy was vacuolization and disorientation
`of OS lamellae among isolated photoreceptors of
`‘the macula. Adjacent OS were often peculiarly
`spared in these areas of minimal damage (Fig. 2).
`This alteration most often involved the distal
`third. of the OS and included both rods and cones.
`Inner‘ segments of these photoreceptors appeared
`normal (Fig. 3). In more advanced degeneration
`the entire OS was markedly swollen and lamellae
`were increasingly disrupted, often broken into
`short
`tubules or empty vesicles (Figs. 4 and 5).
`Other tubules and vesicles were packed with an
`electron-dense, finely granular debris (Figs. 5 and
`6)
`similar
`to the material seen in liver mito-
`chondria of
`these monkeys5
`and thought
`to
`represent peroxidized lipid. Adjacent OS often
`remained intact
`(Fig. 4). In other areas of the
`macula the OS were totally disrupted and had
`formed dense clusters of
`lamellar debris with
`
`
`
`Volume 13
`Number 1
`
`Retinal degeneration in monkeys 501
`
`'
`
`‘_,s-.-.
`
`'.
`
`-v
`
`,
`
`
`Fig. l.'I'heperipherolretim(A){mmavitnminE-cleficientmonloey
`has intact inner (IS)
`and outer (05) segments of photoreoeptors that do not appear dilferent from control sec-
`tions, whereas those from the nncnla in the same monkey (B) are markedly disrupted (ar-
`rows). On the other hand, both peripheral retina (C) and macula (D) in the vitamin A-
`cleficient monkey have degenerated outer segments, the latter appearing much worse than the
`former. Thinning of the ONL has also occurred in the macula. ( X200).
`Fig. 2. Early stage of macular degeneration in vitamin E deficiency. An essentially normal
`rod outer segment (top) is adjacent to one undergoing degeneration (below). Disorientntion
`end vesioulafion of lomellae are apparent. (x27,550).
`
`whorls of myelin figures (Figs. 6 and 7). The
`pigment epithelial cells appeared to sustain phago-
`cytic activity, often being distended with lipo-
`fuscin-type lysosomes and larger aggregates of
`phagocytized OS debris (Figs. 7, 8, and 9). The
`inner layers of the retina were without visible
`change except for an occasional pyknotic nucleus in
`the outer nuclear layer (ONL).
`Vitamin A deficiency. Clinical manifestation of
`
`vihuninA deficienoyoecurredin three ofthefour
`depleted monkeys after 16, 19, and 20 months of
`study. Plasma vitamin A levels in the deficient
`monkeys were negligible after a year (< 5 Jag/dl,
`whereas control values were 15 to 20 pg/dl).
`After 12 to 15 months body weight loss become
`apparent. Xerophthalmia with reddening of the
`conjunctiva became evident as well. In the more
`advanced stage, lethargy, anorexia, and insnition
`
`
`
`Investigative Ophfiwllnology
`My 1974
`
`by its width and dense aggregation
`segments were degenerating. The C15 is
`of mitochondria. Intercellular junctions constituting the outer limiting membrane are visible
`(an-aw). Portions of outer segments are sectioned tangentially below. (x4.300).
`Fig. 4. Swelling of photoreceptor outer segments and vesiculatitm of lamellae were predomi-
`nant changes in moderately advanced stlgee of retinal degeneration in vitamin E deficiency.
`Portions of adjacent OS appear normal. Clusters of electron-dense particles (arrow) were
`daaracteristic of
`this degeneration. The pigment epithelium (PE) contains cigar-shaped
`gment granules and less dense lysosomal bodies presumed to represent an end-stage in the
`removal of the phagosornes (P). (X9500).
`
`
`
`vorume 13
`Number 7
`
`Retinal degeneration in. monkeys 503
`
`
`
`J 1
`1
`1
`«.1 C V ; " 1
`‘A
`
`
`Fig. 5. Many degenerating lemellee form tubules and vesicles packed with fine, granular,
`electron-dense particles (arrows) thought to represent peroxidized lipoprotein or lipofusoin
`formation in vitamin E deficiency. (x4'!,900).
`Fig. 6. Marked degeneration of outer segments is represented by swelling and disruption of
`outer segments, forrning whorls of lamellar debris (d) and myelin-like figures (My). Electron-
`dense particles fill tubules of degenerating lamellae (arrow). (X15300 ).
`
`
`
`were accompanied by advanced night blindness,
`evidenced by an inability to maneuver in a dimly
`lighted room. Xerosis and keratinization of focal
`areas of the cornea were associated with fotma-
`tion of corneal ulcers and the rapid development
`ofkeratomalacia (Fig. 10).
`The youngest and smallest. monkey was killed
`
`in an advanced state of deficiency after 16 months
`and one eye was taken for electron rnicroseopy
`(Figs. 12, 13, and 14) and the other for light
`microscopy (Fig. 1). Two of the other three
`deficient monkeys presented similar clinical condi-
`dons after 19 and 20 months. One of
`these
`monkeys, progressing from night blindness
`to
`
`
`
`r outer s
`
`mg. 7. Massive disruption of
`egments is apparent in the mac-uln of a
`. Formation of dense myelin-like flgures~topresents accumulation
`vitamin E-deficient monkey
`t epithelium contains masses of
`pha
`timed
`ter
`g-
`of extracellular debris. The pigmen
`(LOS) and in more typpciycal den‘: ph:;o-
`memlgbothlooselypaclcedinthebasal
`some: (arrows). Increased I
`granulee and cigar-shaped pigment granules are visible
`In the apical cytoplasm. ($1,700).
`
`impaired vision in a lighted room, was tested and
`found to have a nondetectable h-wave in the full-
`field ERG. One eye was enucleabed under anes-
`thesia and prepared for electron microscopy (Fig.
`15). All
`the monkeys were then refed com-
`mercial monkey daow. The monkey with the
`enudcated eye died two weeks later having devel-
`oped acute panophthalmitis of its remaining eye.
`
`regained their
`The other two ddicient
`appetite,
`activity, and body weight.
`Xerophtlmlmia and loeratomalacia disappeared in
`the monkey so alfected and vision appeared to be
`adequate, although slight corneal opacity remained
`after several months.
`
`Ugh! microscopy of major organs from the two
`vitamin A-deficient monkeys necropsied at 16 and
`
`
`
`Retinal degeneration in monkeys 505
`
`Fig. 8. Pigment epithelial cells from a vitamin E-deficient monkey contains distinctly dilferent
`stages of phagosome digestion (A, B, G, and D). The middle cell is packed with lysosomes
`and appears to be pulling away from Bruclfs membrane (arrow), presumably to migrate
`into the inner retina. (365,100).
`Fig. 9. Dilferent inclusions are observed in this pigment epithelial cell from a vitamin E-
`deficlent monkey. A large loosely packed portion of outer segment (A) is contrasted by a
`more densely packed phagosorne (B) and a lysosome (C). A cigar-shaped melanin granule
`is also visible. Mitaochondrié (M). (x13,8D0).
`
`revealed squnmmis metaplasia and
`21 months
`keratinization of the trachea, conjunctiva, excretory
`ducts in the parotid gland, and transitional epi-
`thelium of
`the renal pelvis
`(Fig. 11). Pyclo-
`nephritis was associated with the latter change in
`both monkeys.
`The one eye examined by light microscopy after
`16 montlu revealed a shortened, disrupted pattern
`
`among all photoreceptor OS in the mncnla and
`many in the paramacular retina. This degenera-
`tion was less apparent in the peripheral retina. The
`ONL was reduced in thickness and contained
`degenerating nuclei corresponding to the degree
`of OS degeneration (Fig. 1). The superonasal
`quadrant of
`the retina was nliected, but
`to a
`lesser degree. In the opposite eye only the mid-
`
`
`
`508 Hayes
`
`lnecrtigrrliue Ophthalmology
`Jlllv 1974
`
`Fig. 10. Classical kemcomalacin with exudative conjunctivitis was produced after 16 months
`on a vitamin A-free diet in this adult monkey.
`Fig. 11. Keratinizntion of the renal pelvis transitional epithelium provides histologic evi-
`dence of vitamin A deficiency. The inflammatory cells visible in the renal papilla were asso-
`ciated with pyelonephritis. (X450).
`
`examined by electron
`retina was
`peripheral
`revealing selective damage of
`the
`microscopy.
`cone 05. This 08 degeneration was characterized
`by vesiculntion, distention, disorientation, and loss
`of the Isunellar discs (Fig. 12). Adjacent rod 05
`were
`structurally intact or were nrlifactually
`distorted by cleits produced during in vivo perfu-
`
`sion fixation. In other cones, lamellae were inter-
`rupted by formation of vesicles and tubules con-
`taining irregularly granular dense particles within
`some of
`them (Fig. 13). The enuclented eye
`taken after 21 months from the second monkey
`with an
`extinguished b-wave
`revealed more
`advanced OS disruption with seemingly equal
`
`
`
`Valium 13
`Number '7
`
`Retinal degeneration in monkeys 507
`
`Fig. 12. A degenerating cone outer segment (CO8) is visible in this section from the mid-
`peripheral
`retina. of a vitamin A-deficient monkey. Numerous
`lamellse have developed
`vesiculation
`typical of this deficiency. Adjacent rod mater segments appear normal except
`for cleft: due to fixation artifact. (19,800).
`Fig. 13. Another cone outer segment in the mid-peripheral retina from the same vitamin
`A-deficient: monkey
`has lost most
`lamellse. The proximal portion contains vesicles packed
`with irregular granules. (x9,500).
`
`in electron
`and cones
`rods
`involvement of
`the paxamacular region
`microscopic sections of
`(Fig. 14). The pigment epithelium contained an
`extraordinary number of partially digested cyto-
`plasmic lipid droplets, but was otherwise nonnal
`(Fig. 15). Phagosomes were routinely observed.
`and melanin granules were located in their usual
`apical positmn. Panophthalmitis of the other eye
`
`in this monkey resulted in total dissolution of all
`retinal layers.
`
`Discus’sion
`
`These descriptions serve to emphasize
`that prolonged vitamin E deficiency in at
`least
`two species of monkeys can result
`
`
`
`508 Hayes
`
`Fig. 14. In more advanced vitamin A deficiency nod outer segments were also disrupted as
`evidenced by these tips hum three outer segments adjoining the pigment epithelium (PE)
`in the paramacular retina. 0:12.700).
`Fig. 15. Pigment epithelium from the same vitamin A-deficient monkey contains an ahnonnal
`number of lipid-laden Iysosomes. (x8,'lD0).
`
`
`
`in retinal damage having certain similarities
`and dissimilarities to those produced with
`insuificient vitamin A. On the one hand,
`protracted vitamin A depletion in adult
`monkeys produced classical signs of de-
`ficiency including xerophthalmia and kera-
`
`tomalacia. Rupture of ‘the cornea resulted
`in destructive panophthalmitis in one mon-
`key. These monkeys demonstrated clinical
`evidence of impaired vision prior to ad-
`vanced ophthalmitis and the ERG b-wave
`from one of them was undetectable. In
`
`
`
`Volume 13
`Number 7
`
`Retinal degeneration in monkeys
`
`509
`
`the first case light microscopy revealed
`relatively moderate to minor disruption of
`OS, primarily restricted to photoreceptors
`in the macula and paramacular
`retina.
`Electron microscopy suggested that
`the
`earliest OS damage, in the mid-peripheral
`retina examined, predominated in cones.
`Both rods and cones appeared damaged
`in the macula and in the surrounding retina
`of the more advanced lesion. Degeneration
`of the ONL and numerous lipid—laden lyso-
`somes
`in the pigment epithelium were
`the only other changes observed.
`On the other hand, vitamin E deficiency
`resulted in no signs of deficiency disease
`except the malaise and pallor accompanying
`severe anemia, and this only in monkeys
`fed the polyunsaturated fat. Retinal de-
`generation, however, occurred in animals
`fed both types of fat following an extra-
`ordinarily long depletion period. This de-
`generation was not manifest by clinical
`evidence of blindness or a change in b-
`wave threshold. Like vitamin A deficiency,
`the initial lesion was obsered in the photo-
`receptor OS. Unlike vitamin A deficiency
`it was restricted to the macula and more
`
`totally disrupted the affected OS. The in-
`tact retina surrounding the macula could
`explain the finding of a normal full-field
`ERG b-wave threshold.
`
`These differences reveal something of
`the pathogenesis of
`the lesions. From
`knowledge of vitamin A function in vision,
`it is predictable that depletion should re-
`sult in uniform loss of the visual pigments,
`rhodopsin and iodopsin,
`throughout
`the
`retina and thereby disrupt
`the electro-
`physical aspects of vision followed by struc-
`tural dismantling of the OS themselves.“
`It is interesting that peripheral cones and
`the cone-rich macula appeared to degener-
`ate first, a finding previously reported in
`monkeys.” Even in the rod-dominant reti-
`nas of vitamin A—deficient rats and guinea
`pigs, the central area of the retina is first
`to degenerate“) 1“ The observation that
`cone structure may be more critically af-
`fected by the lack of vitamin A than that
`of rods is surprising since cone visual pig-
`
`ments are synthesized more rapidly than
`those of rods,“ and cones have a much
`
`greater affinity for retinaldehyde.” Further-
`more, psychophysical testing in man sug-
`gests that the functional correlates of the
`two photoreceptors are equally sensitive
`to the night blindness of vitamin A de-
`pletion.“" ” Perhaps the structural integrity
`of the cone is more difficult
`to maintain
`due to its slower
`turnover rate.“ Cone
`function and structure have been found
`altered in the cone-dominant retina of the
`
`ground squirrel made vitamin A deficient.”
`The function of tocopherol in the retina
`is not known, but it has been isolated from
`bovine OS’ and might be required as a
`lipid antioxidant for the extraordinary con-
`centration of polyunsaturated fatty acids
`found in the OS."° This hypothesis is en-
`hanced by the vulnerability of the visual
`cell to x-irradiation, oxygen poisoning, and
`excessive light, all of which are manifest
`by disruptive oxidizing reactions in this
`delicately balanced
`structure.“ 22 One
`should also consider
`the possibility that
`vitamin E may protect retinaldehyde in
`the retina from peroxidative destruction;
`however,
`if true, the retinal degeneration
`and visual impairment measured by ERG
`might be expected to resemble vitamin
`A deficiency more closely.
`The susceptibility of the macula in vita-
`min E deficiency would again emphasize
`the peculiar character of this area, perhaps
`as a function of its cone density and the
`metabolic or structural correlates specific
`to this photoreceptor. It was not determined
`whether cone damage preceded or was
`more extensive than rod disruption in this
`deficiency, as both appeared equally in-
`volved. The focal, massive degeneration
`of retinal OS in vitamin E deficiency was
`in contrast to the less disruptive disman-
`tling and progressive shortening of OS in
`vitamin A deficiency. The remarkable ac-
`cumulation of lipofuscin pigment
`in the
`pigment epithelium of the vitamin E-de-
`ficient monkeys is
`identical
`to that pre-
`viously described in dogs3 and suggests
`that
`the pigment epithelium is capable
`
`
`
`510 Hayes
`
`Investigative Ophthalmology
`Iuly 1 974
`
`of extreme phagocytic activity and lyso-
`somal digestion in this deficiency state.
`Whether the retinal degeneration induced
`by tocopherol deficiency has a counterpart
`in any other species is unknown. It is note-
`worthy that a previous description of pro-
`longed vitamin E deficiency in dogs was
`associated with extensive retinal degenera-
`tion“ and that children with abeta1ipopro-
`teinemia have remarkably low circulating
`levels of tocopherol. The retinal degenera-
`tion found in these children can be in-
`
`fluenced by vitamin A.“ Whether vitamin
`E is
`limiting has not been determined.
`In any event,‘ further work is needed to
`explore the role of antioxidants in photo-
`receptor function and structure.
`
`Grateful acknowledgment is given Antonio Ruiz
`for his care of the animals. I am grateful to Tom
`Faherty, Barbara Burgess, and Claudia Starr for
`their diligent microscopy and photographic ex-
`pertise and to Arnold Rabin for his help with
`the electroretinographic measurements. Thanks are
`also due Ann Blanchard for preparation of
`the
`manuscript.
`
`REFERENCES
`
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`IN-
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