CYAN EXHIBIT 1026
`
`The Effect of Vitamin A Deficiency on the
`Fine Structure of the Retinal’
`
`JOHN E. DOWLINGL’ AND I. R. GIBBONS
`
`Biological Laboratories, Harvard University, Cambridge, Massachusetts
`
`Introduction
`
`FOR MANY YEARS it has been known that severe vitamin A deficiency
`causes histological degeneration in the retina (Tansley, 193.3; johnson,
`1939), and it has been suggested that this structural damage might be
`responsible for the long lasting or permanent effects of night blindness
`that persist after the deficiency has been relieved (johnson, 1943; VVald,
`1955). However, experiments to examine this point have been limited by
`the fact that Vitamin A has a function in general body metabolism in
`addition to its specific function in the retina. VVhen animals are main-
`tained on a diet completely free 0-f vitamin A, they lose Weight and die
`soon after the onset of retinal degeneration (Johnson, 1943; Dowling and
`Wald, 1958).
`
`Recently, we have found that vitamin A acid, when fed to rats on a
`vitamin A-free diet, keeps them healthy and growing normally, but does
`not support the visual cycle ( Dowling and VVald, 1960). Thus, rats main-
`tained on a Vitamin A—free diet, supplemented with Vitamin A acid grow
`normally, but gradually become -extremely night-blind.
`The result of one such experiment is shown in Fig. 1. After exhaus-
`tion of its stored Vitamin A, the rat given no supplement lost weight
`rapidly and died. The rat receiving vitamin A acid grew normally and
`appeared healthy throughout the experiment. Eleetroretinograms of this
`animal after 5 months are shown at the right of the figure compared
`with those of a normal rat. The deficient rat is highly night—blind: his
`visual threshold is 3.25 log units (about 1800 times) above normal.
`In-
`dependent measurements have shown that this rise of threshold cor-
`responds to the loss of 96 to 98% of the rhodopsin content of the eye.
`In such animals, therefore, we can study the effects of a dietary
`night blindness uncomplicated by other somatic symptoms.
`In this paper we shall describe the effects of this deficiency and its
`
`1 This investigation has been supported by grants from the National Science
`Foundation and United States Public Health Service.
`
`E. D. held a Post-Sophomore Fellowship
`2 Part of this work was done while
`of the Public Health Service, on leave from Harvard Medical School (1959-1960).
`
`35
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`86
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`JOHN E. DQWLINC A~ D J. H. C l BBONS
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`relief on the structure of the retina. Deta Ued correlati on of these histo(cid:173)
`logica l changes \Vith the simultaneolls changes in biochemical composi (cid:173)
`tion and ph)'siologiea l activit)' has been made else\Vhere ( Dowling and
`Walcl, 1960 ), ancl will be mentionecl onl)' hrieR)' here.
`
`350
`
`250
`
`E. R.G.
`v,ramlfl
`aod rat
`o
`lIJg
`3.25
`nreS>"'aI"'ti'--_-' __ _
`
`"f--z..- Rat Q/vt!n
`I4'ta.mitf A acid
`su.p~ ment
`
`o
`
`o
`
`25
`
`50
`
`75
`Days
`
`100
`on D iet
`
`125
`
`150
`
`F Ic. 1. Biological nctivity of vj tamin A acid.
`Litter mates were placed on vitamin A-clefi cien l diet. The animal recelvmg no
`supplement grew until vitamin A stores \Vere exhausted, thcn lost weight rapidly.
`and ùieel on the 57th clay of the experiment. The animal givcn vitamin A acid grew
`throughout the experiment ( 5 months) and rcmained in good condition. The picture
`of th is 11nimal \Vas taken at the end of the experiment, as \Vcre the clectrorctinograms
`shown at the right, compared with those of a nOlnlal animal. They show that titis
`rat L'\ highly nigbt-blind: it has a ViSlI;ll threshold 3.25 loglO unHs above normal,
`correspond ing to loss of 96 to 98% of the rhodopsin From the eye ( From Dowling
`and Wald, 1960).
`
`Experime ntal
`
`HEAIl L" C OF THE RATS
`
`Groups of albino, weanling rats \Vere raised on Standard D.S.P.
`vitami n A-test di ets supp1ementecl on\lI)' with 50 f(,g/ cla)' of vitamin A
`aciel, disso1vecl in vegetab le oil. As contro1s, li tter mates were raisecl
`
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`STHUCTURAL CHANCES OF VITAMIl'l" A D EFICIENCy
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`87
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`similarly on vitamin A-free di ets) supplemented with vitamin A alcohol.
`Th e animaIs were sacrinced nt times ranging from 2 to 10 months after
`having been placed on the diet.
`In certain cases, deficient rats were fed large doses of vitam in A ta
`permit us to examin e the course of recovery From the denciency.
`
`PHEPARATION Fon LIGHT AND ELEGrRON Nhenoscopy
`
`\Vithout regard ta conditions of light or dark adaptation, animaIs
`were anesthetized with Nembutal and the eyes enucleated. The cornea
`and the lens were removed and the whole back of the eye fixed for 1 hr
`in a 2% solution of osmium tetroxide, buffered ta pH 7.8 with Veron al
`acetate, and containing 45 mg per milliliter sucrase and 0.002 M calcium
`chloride. The specimens were dehydrated in graded acetone-water mix(cid:173)
`ture and embedded in Araldite epoxy resin (Glauert and Glauert, 1958 ).
`The whole back of the eye was embedded in an attempt ta retain the
`normal relationship of the retina ta the pigment epithelium. However,
`some separation usually did occur, and only in occasional areas was the
`normal approximation preserved.
`Thin sections were eut with a Porter-Blum microtome and stained
`with saturated uranyl acetate in 50% ethanol. They were examined in an
`ReA EMU-3D electron microscope operated at 100 kv.
`For light microscopy, thick (2-4 Il ) sections were eut from the same
`specimens with the same microtome. The sections were mounted on
`slides with Mayer's albumen, and stained for 6 ta 24 hr with 2% Giemsa
`blood stain. They were then washed briefly with ethanol, allowed ta dry
`in the air, and mounted in paraffin oil.
`
`Observations
`
`RETINAL DEGENERATION
`
`The changes observable with the light microscope during the course
`of degeneration are shawn in Fig. 2. The retina of a control animal that
`had been kept 10 months on a vitamin A-free diet supplemented with
`vitam in A appears entirely normal ( Fig. 2a).
`In rats on the diet supplemented with vitamin A acid, the first signs
`of degeneration are noted after about 2 months, when the outer seg(cid:173)
`ments of the visual œlls begin ta stain less intensely than the normal
`and present a somewhat fragil e and broken appearance ( Fig. 2b ). The
`inner ~egments and the nudei appear normal, along with the other
`retinal tissues.
`After 6 months on the diet ( Fig. 2c ), the visu al cell layer has de-
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`88
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`JOHN E. DOWLINC A ND 1. R. GIBBONS
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`teriorated considerahly: only fragments of the outer segments remain.
`and the nuclei and iuner segments are greatly reduced in l1lnnher. The
`inner segments that remain are' shorter and trucker than norm al. H ow(cid:173)
`ever, the other retinal cells-hipolar and ganglion cells- and the pigment
`epithelium-appear normal.
`After 10 months ( Fig. 2d), the visual cells have almost completely
`disappeared. Ouly one inegnlar row of visual cell nuclei remains, and
`no inner or outer segments can he distinguished. The rest of the retina
`
`FIG. 2. Retinal histology of rats raised on vitamin A-free diets and supplemented
`\Vith vitarnin A add.
`F IG. 2a. The retina from a control animal that had becn raised for 10 months
`on vitamin A-frce di et with vitamin A (alcohol) supplementation. The structure is
`entirely normal.
`F IG. 2b. The retina of an animal raised for 2 months on vitamin A-free diet sup(cid:173)
`plemented \Vith vitamin A acid. The primary change has occurred in the Quter seg(cid:173)
`ments which are disoriented and stain less intensely. The Test of the visual cell appears
`normal, as do the other layers of the retina.
`FIG. 2e. After 6 months, the outer sebrments have almost entircly clisappearecl.
`Ooly fragments and occasional large spherical structures remain. The inne!' segments
`and visual cell nuclei are reduced ta about half the normal number, and tlle inner
`segments have become squat and rowlded. The rcst of the retina and the pigment
`epithelium appear normal.
`FIG. 2d. Retina from an animal maintainecl for 10 months on the met \Vith
`vitamin A acid supplementation. The visual cells have disappeared, except for one
`irregular row of visual cell nuclci. The rod-bipolar synapse layer is thinner than
`nonnal. Other parts of the retina appear normal.
`F IC. 3. An electron mierograph of a longitudinal section of a normal rat rad
`from the retina shawn in Fig. 2a. A5 in other animaIs, tlle outer segment cOllsists of
`stacks of transverse dÜ;iks, enclosed within the ceU membrane. The disks are about
`150 À in over-all thicl'uess and 1.5 Jl in diamcter, and arc separated from one another
`by a space of about 100 À. The short tubules associated \Vith the incision appear
`here, in trausverse section, as sU1al1 cir.cles similar in diameter ta tlle thickness of
`the disk. Magnification: X 16,500.
`FIG. 4. Cross section of the proximal end of a nOInlal rad outer segment sho\Ving
`the circular shape of the disks, and the single incision. The incisions of adjacent disks
`are !ined up 50 that together they form a small channel running the length of the
`outer segment. Numerous, short tubular projections extcnd from the disk into the
`incision. Occasional tuhular projections can be seen also at the periphery of the disk,
`extending inta the space between Ùle disk and the cell membrane. A clustcr of singlet
`outer fibers fram the connecting cilium appears just within the cell membrane op(cid:173)
`posite the incision. In the upper left of the figure is a cross section of a connecting
`cilium bclonging ta another eell. Magnification : X 13,000.
`F IG. 5. A longitudinal section of a rad outer segment showing an early stage of
`degeneration. Stades of intact disks altemate WiÙl distended disks that have often
`pinched off ta form large vesicles and tubules. At this stage the rad main tains much
`of its normal cyündrical shape. Magnification: X 20,000.
`
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`STn UCTUHAL CHANCES OF VITAMu'l A DEFIC IEXCY
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`89
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`and the pigment epith eliurn still apperu- normal , except for the sug(cid:173)
`gestion of sorne thin lling of the layer of bipolar ceUs. The retina and the
`pigment epithelium adhere tightly ta one another in this condition, alld
`
`2c
`
`2d
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`90
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`j OH1\T E. DQWU NC AND L R. GIBBONS
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`show no tendency ta separate in the course of the preparation. ( Unlike
`normal or slightly cleficient eyes, in which some separation almost al",ays
`Occurs. )
`Before cons idering the changes in fi ne sh·ucture that occur during
`degeneration, w e must first mention certain features of the normal retina.
`Figure 3 shows a longitudinal section through part of a rad outer seg(cid:173)
`ment from the conh·ol retina shawn in Fig. 2a. As in other animaIs
`(Sjosh·and, 1953 ), the outer segment consists of a stack of Hattened
`transverse disks enclosed by the cell membrane. Each disk is itself com(cid:173)
`posed of an outer membrane enveloping a less densely stained internaI
`space.
`Figure 4, a cross section cnt near the proximal end of an outer seg(cid:173)
`ment, shows that the disks are approximately circular in face view, \Vith
`a single incision. The incisions of adjacent disks are ail stacked in line,
`sa that together they form a small channel rwming up the interior of the
`outer segment. Rather numerous, short tubules extend From the body of
`the disks into this interior channel (Fig. 4 ). When these tubules are cut
`in cross section, as frequently occurs in longitudinal sections of th e outer
`segment, they appear as sm ail cu·cles, of about the same diameter as the
`thickness of a disk (Fig. 3 ).
`In the course of vitamin A deficiency, this highly ordered structure
`of the outer segment gradually breaks clown. The degeneration begins
`with a marked swelling of the transverse disks, which pinch off ta form
`large vesicles and tubules ( Fig. 5). At this early stage, one frequently
`finds sm ail stacks of apparently normal disks separated by others that
`are swollen and have broken into vesicles.
`After a high proportion of Ùle disks has degenerated in this way,
`the outer segment begins ta lose its normal elongated, cylindrical shape,
`and may end by ra un ding up ta become almost spherica!. Figure 6 shows
`Ùle fin e sh·ucture of one such spl)ere. It cannat be confused ",ith the
`cross section of a normal outer segment, sinee the latter is ollly about
`1.5 fl in diameter, whereas such spherical remnants of rads are 3 ta 5 fl
`across. Most of the interior is filled with distended vesicl es and tubules,
`and only a very few intact disks remain. Il is not clear ta what extent
`
`FIG. 6. A Jate stage in the dcgeneration of au outer sc!,rment. The structure is
`almost entirely 6Hed with distended tubules and vesicles. With fcw inhlct disks re(cid:173)
`maining, the outer segment has assumed the characteristic sphericnl shape of il
`highly degenerate rod. Magnification : X 12,500.
`FIC. 7. An appnrcntly normal iuncr segment alongsidc a deteriorated, spherical
`outer segment, from an animal that had been on the diet for about 6 mOllths. The
`mitochondria, cytoplasmic membranes, and cytoplasmic granules a11
`look normal.
`Magnification: X 12,500.
`
`

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`STHUCTUHAL CHANGES OF VITAM LN A DEFI C ΠNCY
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`91
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`JOHN E. DOWLING AND I. R. CIBBONS
`
`such highly (Jjstorted outer segments retain contact with the inner seg(cid:173)
`ments. vVe have frequently observed them Iying loose in the space
`between the retina and pigment epithelium, but this may have been an
`artUaet of preparation.
`The change in shape of the inner segments and their gradual re(cid:173)
`duction in number have already been mentioned. Theu' fin e structure,
`however, appears to remain quite nonnal. The mitochondria, the cyto(cid:173)
`l'las mie granules, and the cytoplasmic membranes seem unaffected by the
`drastic changes going on in the outer segments (Fig. 7 ). We have some(cid:173)
`tÎlnes seen inner segments and degenerating outer segments in direct
`continuity with each other, without the ciliary constriction that normally
`intervenes. This would seem to imply the degeneration of the connecting
`cilium. However, in other cases, such as Fig. 7, the cilium is still present.
`As the deficiency progresses, the terminal processes of Müller's
`fibers become highly conspicuous in the spaces left between the remain(cid:173)
`ing inner segments (Fig. 9 ). In the normal retina these terminal proe(cid:173)
`esses are long eylindrieal structures, about 0.1 ft in diameter, which
`envelop the proximal portion of the inner segment, forming what has
`been called th.e "fiber basket" (Polyak, 1941 ). In electron micrographs
`they are difficult ta trace beyond the outer limiting membrane, but it is
`known From light microscopy that they are derived from glial cells deep
`in the retina (Polyak, 1941 ).
`In the most highly deficient animaIs we have examined, after 10
`months on the diet, almost ail the visual cells have disappeared. Figure 8
`shows a low magnification elecb'on micrograph of part of the retina
`from sueh an animal. The visual cell nuclei are reduced to one irregular
`row. Those that remain, however, still appear normal. Only small frag(cid:173)
`ments of inner and outer segments are visible, pressed against the proc(cid:173)
`esses of the pigment epithelium. O,n the right in the figure is a gap jn
`
`F IG. 8. A gcneral vicw of the retina, pigment epithelium ( PE), and choroid
`(Ch ), of an animal maintained on deficient diet, supplemented \Vith vitamin A acid
`for 10 months. Scattered fragments of inuer and outer segments are tightly pressed
`against the processes of the pigment epithcJium. Ooly one irregular row of visual
`cell l1udei ( RC N) remains. At the right of the micrograph is a gap in this row of
`nuclei, where the rod-bipolar synapse layer (SYN) extends to the pigment epithelium.
`Even at this stage, tbe remaining synapses and visual œ U nuclei appear as they
`usually do, although both are drasticaUy reduced in number. At the lower left is part
`of a bipo!.r cell nucleus (BeN). M.gnific.tion : X 3000.
`FIG, 9, An electron micrograph showing the base of the inner segments and a
`region of the external limiting membrane, in a 6-month deficient rat. Many of the
`visual ceUs have disappeared, leaving gaps between the remaining inner segments,
`The figure shows one such gap in which the terminal processcs of Müller's fibers are
`higbly conspicuous. Magnifica~ion : X 11,000.
`
`

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`$TRUC!'UHAL errANCES OF VITAMIN A DEFIClENCY
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`93
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`JOHN E. DOWLL.~C AND I. R. GIBBONS
`
`the row of rod nuclei. Here the rod-bipolar synapses (outer plexiform
`layer ) are in direct contact with the pigment epithelium. This synaptic
`layer looks as it usually does but is much reduced in thickness-pre(cid:173)
`sumably through the loss of afferents from the visual cells.
`
`RETINAL H ECOVEHY
`
`If the degeneration of the visual cells has not progressed too far,
`sh'llctural and physiological recovery can be indu ced by giving vitamin
`A ta the deficient animals.
`We have found that animals maintained on the deficient di et for
`about 6 months are the most suitable for observing regeneration. At this
`stage, there are very few intact outer segments left, and the number of
`visual cells has been reduced ta about one-half.
`In a typical experiment, three litter mates \Vere raised on a vitamin
`A-free diet for 60 months. The control animal was fed vitamin A
`throughout the experiment, while the other two animals were fed vitamin
`A acid. Th e recovery animal was fed a large dose of vitamin A ( 500 flg)
`and then periodically fed further vitamin A for 16 days.
`Sections of the retinas !rom these animals are shawn in Fig. 10. The
`conh'ol ( Fig. 10a ) has a normal retina. The deficient animal ( Fig. lOb )
`
`FIG. 10. Recovery From deficiency. TInce litter mates \Vere raised on vitumin
`A-free diets for 6 ~ months; one \Vas supplemented with vitamin A, the other two
`\Vith vitam in A acid. Sixteen days prior to Ù1C end of the experiment, the recovery
`animal \Vas fed a large dose of vitamin A.
`FIG. 10a. The retina From control animal maintained on vitamin A throl1ghout
`the experiment.
`FIG. l Ob. The retina From the deficient animal. Almost aIl of the outer segments
`have disappeHrcd along with about haH of the visual cell nuclei and inner segments.
`The inner segments that remHin are shortcl; and wider than normal.
`F IG. IOc. The retina from the recovery animal. New outer segments, normal in
`length and width, have regenerated. There has bcen no increase in number of visual
`ceIl nuclei or inner segments, and the inner segments have remained squat. The
`number of regenerated outer segments, thercfore, is only about half the normal
`Humber.
`FIG . 11. An carly stage in the regeneration of an outer segment. A cilium, ex(cid:173)
`tending from the inner segment at the bottom of the figure, has expanded at the
`distal end; a few vesicles are present in the structure; and the ciliary fibers extend
`much of its length. At the far distal end, arc small membranous saccules, oriented
`longitudinally with respect ta the rest of the cilium. Magnification: X 11 ,500.
`FIC. 12. A later stage of outer segment regeneration. Many more membranous
`saccules are present. The saccules are mostly oriented longituc1inally but, at the
`proximal end, appear to be assuming the normal transverse arrangement. Magnifica(cid:173)
`tion: X 11,500.
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`STHUCTUBAL CI-lANCES OF V JTA~Uj\" A DEF IC ΠNCy
`
`95
`
`shows typicaJ changes as described previously. The anima l fed vitam in
`A ( Fig. l Oc), however, has regenerated ne\\' outer segments that appear
`norm al. There has been no illcrease ù) th e l1umber of visual cells, sa that
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`96
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`JOH N E . DOWLINC Al'1D 1. R. GIBBONS
`
`only about one-haU the normal number of inner and outer segments are
`present. The inner segments have remained short.
`In arder ta discover the detailed process by which the outer seg(cid:173)
`ments regellerate, we have examined the fine structure of retinas from
`recovering animais, 3 ta 16 days after beginning ta feed vitamin A.
`After 3 days of recovely, one Bnds elongated cilia protruding from
`occasional inner segments. The ends of these cilia are enlarged, and
`contain many vesicles. At a later stage, the distal end of the cilium has
`broadened fmther, and contains a number of smail membranous saccules
`at Ùle clistal end (Fig. 11 ). Later, Ùle number of saccules has greaÙY
`increased, and they have begun ta assume the form of h'ansverse disks
`(Fig. 12 ). After 16 days of recovery, the outer segments "ppear essen(cid:173)
`tially normal, and no further regeneration is apparent. The fully regen(cid:173)
`erated outer segments have Ùle same dimensions as Ùle normal (about
`1.5 fl diameter and 15 fl long) .
`It is difficult ta deduce the dynamic sequence of events from static
`micrographs, and our observations on regenerating visual cells are not'
`sufficienÙy numerous for a detailed hypothesis conceming Ùle mechanism
`of Ùle process. vVe can, however, point out the resemblance between our
`micrographs of regenerating outer segments and the published micro(cid:173)
`graphs of other workers who have studied the normal emblyological de(cid:173)
`velopment of the visu al cells (De Robertis, 1956; Tokuyasu and Yamada,
`1959) . The process by which the outer segments regenerate after vitamin
`A deBciency is very similru' ta ilieir nmmal morphogenesis.
`
`PIGMENT E P ITHELIUM
`It has been known for many years that a very close relationship
`exists between Ùle outer segments of Ùle visu al cells and the pigment
`epithelium, and Ùlat tile retina is incapable of functioning without Ùlis
`association. Wald (1958) has recently discussed this subject.
`Many features of the fine structure of the pigment epithelium have
`been described by previous workers (Porter, 1957; Yamada et al., 1958).
`vVe have extended Ùlese observations, and our detailed findings will be
`reported later. For the present we will mention only one fea ture that is
`directly related ta our present subject. This concerns the disappearance
`of the myeloid bodies in vitamin A deficiency.
`The myeloid bodies OCCllI frequently in ilie pigmen t epithelinm
`cells of normal rats. Their over-all shape varies, but they bave a unuorm
`fine structure consisting of a number of flattened, more or less parallel
`saccules, which resemble the disks of the rad outer segment. Each
`myeloid body is bounded by a single densely staining membrane. No
`instances of continuity have been observed between the internai saccules
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`STRUCTURAL CHANGES OF VlTA1H.I."J A DEFICIENCY
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`97
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`and th e general sys tem of membranes in the cytoplasm, as described for
`the myeloid bod.ies of the frog pigment epithelium (Porter, 1957 ).
`In the pigment epithelium of vitamin A-deRcient rats, we have found
`no typical myeloid bodies. How they clisapperu' is not yet known. When
`the deSciency is relieved, the mye laid hodies quickly reappear, becoming
`conspicuous within 3 days after beginning ta feed vitam in A.
`
`Discussion
`Detai\ed meaSUl"ell1ents (Dowling and Wald, 1958, 1960) on the
`biochell1ical composition of the outer segments have shawn Ùlat the level
`of rhodopsin (vitamin A aldehyde-op sin campI ex ) begins ta fali early in
`vitamin A deficiency, declining ta 5 ta 10% of normal after 2 monÙls.
`The visual protein, opsin, declines more slowly, ahout 50% remaining
`after 2 months. No histological changes oecUl" during the early phase of
`deficiency, when the level of rhodopsin is falling. TItis was ta be ex(cid:173)
`pected, for the level of rhodopsin decreases also in normal light adapta(cid:173)
`tion. The anatomical de eneration of Ùle rod outer se ments a ears,
`rotein, a sin that OCCUl"S later
`rather, ta ar el the loss of the visual
`in deficiency. Opsin is a major cOll1ponent a
`t le outer segments [e.g.,
`14% of the dry weight in cattle, and 40% of the dry weight in frogs
`('Wald, 1955 ) ], sa that its loss may weil be direcÙY responsible for the
`breakdown of structure.
`lt is less clear why whole visu al cells shou!d die following Ùle loss of
`theu' outer segments. The celis begul ta disappear only late in the de(cid:173)
`Seiency, when the outer segments have almost gone. lt may be that the
`cells die as the result of lack of stimulation. However, Ùw possibility that
`vitamin Amay be neeessary for a further specifie role in the life of the
`visual celi must also be considered.
`It has been suggested that certain types of primary relina! de(cid:173)
`generalion, as UI 1'etinitis pigmentosa, might be the result of a highly
`localized vitamin A defici ency (Cogan, 1950), caused perhaps by a
`metabolic defect involving one of the enzymes of the visua! cycle. This
`hypothesis has been criticized on the grounds that the eHects of vitamin
`A deRciency occur throughout the animal, and fuat no one has shawn
`that deprivation of vitamin A can cause specific degeneration of visu al
`cells ( Noell, 1953) . The present results show that lack of vitamin A
`alone does cause the death of visual ceHs, and that animaIs maullained
`on the vitamin A-deRcient diet supplemented with vitamin A acid come
`ta mimic closely the condition observed m reti11,it'is p-ig11lentosa.
`B~th ÙIe lImer and outer segments characteristicaHy change Ùleu'
`shapes durmg the COUl"se of vitamin A deficiency and recovery. The mner
`
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`98
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`JOHN E. DOWLING AND I. fi. CrBBON$
`
`segments, normaUy forced ta maintain an elongated, cylindrical fo rm by
`the pressure of the surrounding inner segments, assume a m Ore globu lar
`form, without any change in fin e structure, wben the number of sur(cid:173)
`rounding inner segments decreases. Helief of the deficiency does not
`increase the number of the inner segments, and they remain squat and
`rou nded. The outer segments, on the other hand, lose their normal shape
`during deficiency because of th e breakd own of theu' internaI structure.
`This internaI struchlre is reformed after givin g vitamin A, and the seg(cid:173)
`ment then regains its nonnal shape and size.
`The disappearance of th e myeloid bodies during vitamin A de(cid:173)
`ficiency and their reappearance in recovery demonstrates that tbese
`structures, like the outer segments, are dependent on vitam in A for their
`maintenance and formation. This, and their morphological resemblance
`ta part of a rad outer segment, suggests a functional activity in the lue
`of the pigment epithelium cell .
`
`Summary
`Rats raised on a vitamin A-free diet and supplemented with vitamin
`A acid appear normal and grow well, but develop a high degree of night
`blindness. With time, the visual cells display anatomical changes and
`eventually disappear. The initial anatomieal disturbance seems limited
`ta the outer segments, and consists of a swelling and segmenting of the
`disks ta fonn large vesicles and tubules. Eventually these remnants of
`outer segments disappear completely. At a late stage of deficiency, the
`inner segments and visuaI cell nuclei c1ecrease in number; no obviollS
`changes in nne structure have been observed prior ta theu' loss.
`Provided that deficiency has not been allowed ta progress tao far,
`outer segments of the visual cells can .be regenerated by feeding vitamul
`A ta the de6cient animais. The development of the regenerating outer
`segments is very sirnilar to their normal morphogenesis. HO\vever, once
`a vi sual cell has been completely lost, it is not replaced.
`
`A CK..r\JOWLEDGl\'IENT
`
`1vVe should like to thank Professor George \ Vald fOT many helpful discussions
`cOllcern ing this study.
`
`REFERENCES
`
`( 1950 ) . Tralls. Am. Acad. Opht1wlmol. ancl Otola/'yngol. July-
`Cogan, D. C.
`August, p. 629.
`( 1956 ). J. Biopltys. 13iocltem. Cytol. 2, 319.
`De Hobertis, E.
`Dowlillg, J. E., and Wald, G.
`( 1958 ). Proc. Natl. Acad. Sei. U.S. 44, 648.
`Dmvling, J. E., and Wald, G.
`(1960 ). Proc. Na tl. Acad. Sei. U.S. 46, 587.
`( 19.58 ). J. 13i0l'hys. Biocltem . Cytol. 4, 191.
`Glauert, A. M., and Glauert, H. H.
`
`

`
`STBuc r URAL CI·IANGES OF VITAMIN A DEFIC IENC Y
`
`99
`
`]. Exl'/l. Zool. 81, 67.
`(1939).
`Johnson, ~I. L.
`(1943). A.M.A. Arch. Ol'ht/wlmol. 29, 793.
`Johnson. ~ I. L.
`(1953 ), USAF School of Aviation, Medicine l'roject No. 21-1201-
`Noell, \\'. K.
`0004 , Report No. l.
`l'olyak, S. L.
`( 1941 ) , "The Retinn," pp. 343-365. University of Chicago Press,
`Chicago, Illinois.
`Porter, K. R.
`( 1957 ), Harvey L ectures, Ser. 51, 175.
`]. Cellllio r Camp. Physiol. 42, 15.
`Sjostmnd, F. S.
`( 1953) .
`( 1933). Proc. Roy. Soc. B114, 79.
`Tansle,', 1:.
`] , Biophys. Biochem. Cytol. 6, 225.
`Toku yasu, K,. and Yê1lllada E.
`( 1959 ).
`( IfI55 ). Am . }. Ol'htholllloi. 40, 18.
`Wald, C.
`( 19.58 ). EXI'/l. CeU Reseorc" , SIII'I'I. 5, 389.
`Wald , C.
`Yamada, E., Toku yasu, K., and Iwaki, S.
`( 1958) .
`]. Electummic/'oscoP!J ( Chiba ),
`S, 42.
`
`DISCUSS iON
`
`}.IR. FRAN K M OYEU [Johns Hopkins University, Baltimore, Md.]: You have
`pointed out very well, Dr. Dowling, that there is a similarity between the kind of
`degencration that you observe in vHamin A de.6ciency and the fine structu re that
`Dr. De Robertis has observed in the C3H mouse. :Might it not be Ùlat Ùle two
`degenerations arc related in sorne \Vay? Dr. Pearse showed that there \Vas a high
`alcohol dehyd rogenase concentration in the pigmented epithelium normally. May it
`not be that the defect in the C3H mousc could be in an enzyme system, \Vhich sup(cid:173)
`plied the substrate for the formation of retinene, and that the failure of retinene to
`be formed may have something to do with the degeneration you observe? In tM\;
`respect, therefore, 1 wouid like ta ask if Dr. De Robertis has observed anything
`abnormal about the pigment epithclium in the C3H mouse or if Dr. Pearse has
`observed if there is an alcohol debydrogenase in the pigment epithelium of the C3H
`mouse.
`DR. DE HOUEHTIS [Buenos Aires, Argentinal: The process of degeneration in
`the C3H mouse looks rather diffemnt from that described by Dr. Dowling. There,
`the jnncr segments arc aŒected whereas in vitam in A deficiency the defect is mainly
`apparent in the outer segment. The process Dr. Dowling descri hed seems to have
`sorne resemblance ta that induced by iodoaceta te in which the outer segments arc
`first affected. One can sec in this proccss a crumbling of the membrane, production
`of vesicles exactly as seen in ad vanccd stages of vitamin A deflcieney.
`DR. DOWLING (Cambridge, Mass. l: Dr. De Robertis has pointed out that the
`changes he observes in the C3H mOllse secm to take place initially in the inncr
`segment, while the changes we observe in vitamin A dcSciency take place initially
`in the ou ter segments, indicating, l think, a fundamental difference in the two con(cid:173)
`ditions. l \\"ould point out, however, that in bath cases the entire visual ccli gradually
`degeneratcs and that the cventual rctinal histology in the two instances is quite
`sinlilar.
`l n both C.lses, the retinas may appcar remarkabl y normal except for the
`lack of visu al ccUs. 1 would further point out that a similar picture has been described
`for several other conditions, aud Dr. Cogan has made the suggestion that perh aps
`SOme hereditary night blindness diseases, such as reti nitis pigmentosa, ma)' be due
`to a highly localizcd vitamin A deSciency, perhaps caused by an enzyme defect.