Ultrastructure of the Pigment Epithelium in the Domestic Sheep

ULTRASTRUCTURE O F THE PIGMENT EPITHELIUM IN T H E DOMESTIC S H E E P A L P H O N S E L I :URE-DUPREE

Chicago, Illinois The functional interrelationship between the pigment epithelium and the visual cells has been recognized since 1879, when Kiihne 1 demonstrated that the pigment epi­ thelium was necessary for the regeneration of rhodopsin. There is considerable evidence that maintenance and integrity of the visual cells depend in part upon the pigment epithelium. 2 " 5 T h e fact that loss of vision results from retinal detachment—the visual layer being separated from the pigment epi­ thelium—and can be restored if contact is reestablished emphasizes such interrela­ tionship. Pharmacologic and electrophysiologic investigations have indicated that the pigment epithelium m a y regulate the move­ ment of ions and metabolites between the visual cells and the choroidal vessels.*' 7 A number of electron microscopic studies of vertebrate pigment epithelium have been made and possible roles for this structure have been suggested. 8 " 12 T h e only study on the pigment epithelium of the sheep was a light microscopic investigation of melanin formation during embryonic development. 13 This paper reports a study of the fine struc­ ture of the pigment epithelium of the sheep and its relationship with the choroid and re­ ceptor outer segments. MATERIAL AXD METHODS

The eyes were removed either from sheep under anesthesia or from heads tained from an abbatoir. T h e cornea, and lens were dissected away and the

the ob­ iris, eye

From the Eye Research Laboratories, The Uni­ versity of Chicago, and the Departments of Anat­ omy and Ophthalmology, Washington University School of Medicine, St. Louis, Missouri. This in­ vestigation was supported by USPHS Project Grant NB-03358 from the National Institute of Neurological Diseases and Blindness, and USPHS Research Grant B-621 and GMO-3784 from the In­ stitute of General Medical Sciences.

immersed in cold 3 % glutaraldehyde buf­ fered at p H 7.35 in 0.01 M cacodylate buf­ fer 14 containing 0.0015 M CaCU. After one hour the vitreous was removed and the eyes were transferred into fresh fixative. Fixa­ tion was continued for two hours. T h e retinas, along with the choroid and sclera, were dissected into small pieces and then postfixed overnight in cold in 0.1 M phos­ phate-buffered 1% osmium tetroxide. 1 5 T h e tissues were dehydrated through a graded series of ethanol, followed by treatment with propylene oxide, and then embedded in Epon 812. 1C Ultrathin sections were obtained with an L K B ultratome, using glass o r dia­ mond knives, and mounted on uncoated cop­ per grids. T h e sections were stained with a saturated aqueous or alcoholic solution of uranyl acetate 17 followed by lead hydrox­ ide. 18 T h e preparations were examined in an R C A E M U - 3 G electron microscope oper­ ated at 50 kv. Sections of lu, were also cut and examined under phase contrast prior to electron microscopic observation. OBSERVATIONS

T h e pigment epithelium consists of a sin­ gle layer of polygonal cells whose basal sur­ faces rest on a "basement membrane" or basal lamina. T h e plasma membrane of the basal surfaces is characterized by numerous infoldings. T h e imaginations are so exten­ sive that they form compartments within the cell. T h e infolded regions are devoid of cytoplasmic organelles except for a few ran­ domly distributed ribosomes ( R N P parti­ cles). T h e basement lamina appears as a lin­ ear density of amorphous or fibrillar mate­ rial roughly 600 A in thickness and closely conforming to the undulant contours of the basal portions of the cells (figs. 6 and 7 ) . T h e apical surface (vitreal side) of the pigment epithelium consists of long micro-

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Fig. 1 (Leure-duPree). Apical portion of pigment epithelial cells in horizontal section. Microvilli con­ taining pigment granules (arrows), cross sections of receptor outer segments (Os) and inclusion bodies (lb) are present. (X 16,500.) villous processes which interdigitate with the receptor outer segments (fig. 1 ) . Although very close contact is observed, no junctional complex or direct communication between

these two structures (as reported by Becher 1 9 ) was seen. T h e nuclei of the pigment epithelial cells, which are round, ovoid-shaped or irregularly

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Fig. 2 (Leure-duPree). Intermediate portion of pigment epithelium, showing abundance of smoothsurfaced endoplasmic reticulum (Sm), nucleus (N), nuclear pores (arrows), Golgi complex (G), roughsurfaced endoplasmis reticulum (Rs), pigment granules (pg) and mitochondria (m). Circle indicates intimate association of mitochondria with lateral plasma membranes (X 17,000.) contoured, occupy the center of the cell. T h e nuclear envelope has numerous fenestrations, 400-600 A in diameter (fig. 2) with a fairly regular pattern. T h e inner and outer membranes of the nuclear envelope are con­

tinuous with each other around the margin of each pore, and there is an absence of chromatin in this a r e a ; however, marginal condensation of chromatin is observed else­ where on the inner nuclear membrane. T h e

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rod-shaped, and their cristae often traverse their width. They are located in the in­ termediate portion of the cell, below the level of the junctional complexes, where they are interspersed between the tubular cisternae of the smooth-surfaced endoplasmic reticulum. However, they often exhibit a preferential orientation along the lateral margins of the cells and between the basal convoluted por­ tion and the nucleus (figs. 2 and 8). Mito­ chondria are extremely rare in the apical portion of the cell, and are never observed in the microvilli. Free aggregates of ribosomes ( R N P par­ ticles) are distributed in limited numbers throughout the cytoplasm, except in the api­ cal portion of the cell. The most conspicuous and characteristic components of the pigment epithelium are the pigment granules. They are confined to the intermediate and apical cytoplasm in­ The cytoplasmic matrix of the pigment cluding the microvilli. The granules are quite epithelial cell is characterized by a compact, variable in size and configuration. They ap­ elaborate network of tubular and vesicular pear as round, ellipsoidal, spherical, rod- or forms of smooth-surfaced endoplasmic retic­ cigar-shaped profiles, ranging from 1.0 to ulum. These constitute the bulk of the mem­ 1.5 microns. The melanin contained within branous elements in the cell. They ramify the granules is quite dense to both visible throughout the cytoplasm from a region ad­ light and electrons. The individual granule is jacent to the basal zone to the microvillous surrounded by a thin limiting membrane, projections, where they are less compact which is often in close association with the (figs. 1 and 2). Continuity between the smooth and rough-surfaced endoplasmic re­ smooth-surfaced reticulum and elements of ticulum (figs. 1, 3, 4, and 5). The internal structure of the pigment appears homoge­ the granular ER is frequently observed. The Golgi complex is usually located in neous, although a few granules have an in­ structure characteristic of the perinuclear region. The exact limits of ternal lamellated 22 melanosomes. Most of the mature granules the complex are often difficult to define in a are not uniformly opaque, the outer margins cytoplasm so rich in smooth-surfaced mem­ being somewhat less dense than the center of branes. The stacks of curved cisternae of the the granule, giving it a cortical and "nu­ Golgi complex are arranged concentrically to clear" substructure. one another. The outer cisternae are dilated but become progressively flattened. The cen­ Inclusion bodies are another prominent tral core of the cytoplasm circumscribed by feature of the pigment epithelium. These the Golgi complex contains small granules, structures form a rather heterogeneous and occasionally mitochondria are seen in group, varying in size, shape, and appear­ close relationship with this complex (fig. 2). ance ; morphologically they resemble the in­ Structurally, these mitochondria are simi­ clusion bodies described by Dowling and 9 lar to those seen in other epithelial cells. Gibbons in the pigment epithelium of the They are either cylindrical, spherical, or albino rat and the lamellated bodies observed

cytoplasmic surface of the nuclear envelope is studded with ribosomes, and communica­ tion between the perinuclear space and the cisternae of the rough-surfaced endoplasmic reticulum is frequently observed. The granu­ lar ER occurs in a segregated group of four to eight parallel stacked cisternae, studded with ribosomes on their outer surfaces (fig. 2). This preferred orientation is found in protein-secreting cells.20'21 The granular ER is confined to the intermediate portion of the epithelial cell and is never observed in the basal or apical portions. There is at times a close paralleling between the membranes of the rough-surfaced reticulum and the outer limiting membrane of some mitochondria. When a portion of the membrane of the granular ER is in intimate association with the outer mitochondrial membrane, the surface of the reticulum next to the mito­ chondria is agranular.

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Fig. 3 (Leure-duPree). 'Horizontal section of three adjacent pigment epithelial cells at the level of the intermediate junctional complex (Jc) and zonula occludens (Zo). Note the presence of associated filamen­ tous material (f). Pigment granules (pg) and inclusion bodies (lb) are represented (X 16,000). by Bairati and Orzalesi 1 1 in man. They are unlike the myeloid bodies observed in frog and turtle pigment epithelium. 10 ' 23 These organelles vary from 0.5 to 3 microns in diam­ eter, and as many as 20 can be observed in a single section through a pigment epithelial

cell. T h e bodies consist of granular matrix enclosed in a single membrane. I n some bod­ ies the matrix is amorphous while in others there are also lamellated circular membranes (figs. 3, 4, and 5 ) . Within the matrices of certain of the larger inclusion bodies are

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Figs. 4 and 5 (Leure-duPree). Higher magnification of the inclusion bodies (lb) seen in figure 3. The morphological heterogeneity of tliese bodies is clearly visible. Pigment granules (pg) and smooth-surfaced endoplasmic reticulum (SM) are also present (X32.000).

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concentric membranes regularly arranged while within others stacked membranes are found. Some inclusion bodies are entirely la­ mellar and have a structure similar to the outer segments of the photoreceptors (figs. 4 and 5 ) . T h e lateral membranes of adjacent epithe­ lial cells are attached to each other without interdigitations, by specialized intermediate junctional complexes similar to those de­ scribed by F a r q u h a r and Palade 2 4 in other epithelial cells. The junctional complexes are situated at the junction of the apical onethird of the cell with the basal two-thirds. A distinctive feature of these complexes is an extensive band of filamentous material of moderate density which is observed on the cytoplasmic aspects of the membranes of apposed cells. The filamentous nature of this material is quite discernible in sections par­ allel to the basement membrane. T h e fila­ ments run parallel to each other and to the lateral margins of the cells in which they are located (fig. 3 ) . There are no cytoplasmic organelles in the regions occupied by these filaments, which thus form a cortical zone in this region of the cells. The plasma mem­ brane is conspicuous within the junctional complexes, and the interval between cell membranes is often occupied by an extracel­ lular substance presumed to be a polysaccharide. Typical tight junctions (zonulae occludentes), which are characterized by contin­ uous fusion of the outer leaflets of the plasma membrane, are found at the apical surfaces of apposed epithelial cells. No cell-to-cell at­ tachments are observed at the basal portions of the cells.

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ment lamina is an inner collagen lamina, which consists of loosely arranged and ran­ domly oriented collagen fibers (figs. 6 and 7 ) , which may extend to the basement lam­ ina and the next layer (probably the elastic lamina). T h e presumed middle elastic lamina consists of bundles of parallel fibers showing no preferential orientation. Unlike the colla­ gen fibers, these fibers show no cross band­ ing or periodicity. Contiguous with this lam­ ina is a layer with structural features similar to the inner collagen layer. This outer colla­ gen fiber lamina often has "elastic fibers" in­ termingled with the collagen fibers. The col­ lagen fibers of both inner and outer collagen fiber laminae have a periodicity of 610 A. Peripheral to the outer collagen fiber lamina is the basement lamina of the endothelial cells of the choriocapillaries. T h e elastic and outer collagen fiber layers extend around the choriocapillaries and condense with the base­ ment lamina of the endothelial cells. I n areas where the choriocapillaries are absent, the outer collagen fiber layer blends with the collagenous network of the choroid. DISCUSSION

B R U C H ' S MEMBRANE

T h e role of the pigment epithelium in the physiology of vision and the transport and exchange of metabolities and fluid from the vascular compartment of the choroid to the photoreceptors has been discussed by various investigators. 9 " 11 ' 25 ' 26 T h e description of the pigment epithelium presented here provides further morphologic basis for understanding these functions. These observations are con­ sistent with the ideas that this structure is concerned with nutrition and metabolism and that transport of materials may be one of its functions.

Bruch's membrane is interposed between the pigment epithelium and the choroid. In those areas where the choriocapillaries are present, Bruch's membrane consists of five distinct laminae. T h e first of these, the "basement membrane" or basement lamina, is in closest apposition to the basal surfaces of the epithelial cells. External to the base­

T h e extensive infoldings of the basal plasma membranes and the numerous microvillous processes at the apical portions of the cells, which provide for an increase in surface area, are common features of epithelia engaged in transport. 27 " 29 However, two features which are usually associated with these membrane specializations are lacking

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Fig. 6 (Leure-dnPree). Basal portion of a pigment epithelial cell. The basal plasma membrane (double arrow) is markedly invaginated and free of cytoplasmic organelles, except for a few aggregates of RNP particles (asterisk). Bruch's membrane (B), which consists of the basement lamina of the epithelial cell (Epbl) ; inner (Ic) and outer (Oc) collagen layers; the elastic layer (E) and the basement lamina of the endothelial cell (Enbl), can be observed. Endothelial cell (En), capillary lumen (L), and red blood cell (Rbc) are present. (X 16,500.) in the pigment epithelium of the sheep : ( 1 ) there are no mitochondria in the region of the basal infoldings, and ( 2 ) the microvillous projections contain no cytoplasmic com­ ponents which would indicate the release or absorption of materials. The receptor outer segments are devoid of cytoplasmic orga­ nelles usually associated with synthetic and

energy-requiring processes and are in inti­ mate association with the pigment epithelium which seems to provide the fundamental pathway for the maintenance of nutrition and metabolism. This method of transport of metabolites from the choroid to the pig­ ment epithelium is comparable, to some ex­ tent, to that seen in the cornea, although the

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Fig. 7 (Leure-duPree). A high magnification micrograph of portions of the basal zone of the pigment epithelium and Bruch's membrane. Arrows point to the infoldings of the basal plasma membranes. The basal lamina of the pigment epithelium (Epbl), inner (Ic) and outer (Oc) collagen layers and the elastic layer (E) of Bruch's membrane are clearly visible. (x6O,000.)

layers of the cornea do not correspond in number and morphology to those of Bruch's membrane. F o r metabolites to reach the cor­ nea epithelium from the limbus tears and the aqueous humor, four layers must be tra­ versed: ( 1 ) the endothelial lining of the an­ terior chamber, ( 2 ) Descemet's membrane which represents a specialized basal lamina of the corneal endothelium, ( 3 ) the collagenous layer of the substantia propria, and ( 4 ) Bowman's membrane. Studies using fluorescein30 and electron-opaque markers 3 1 have clearly demonstrated that substances move freely across the cornea; however,

these studies have not been fully extended to the pigment epithelium. T h e elastic nature 7 ' 3 2 ' 3 3 of Bruch's mem­ brane suggests that one function may be to serve as a mechanical filter to dampen pul­ sating waves set up by the numerous blood vessels of the choroid. T h e collagenous and elastic layers and associated fluid of Bruch's membrane could be considered analogous to a mechanical filter of dashpots and springs, such as that proposed by Loewenstein and Skalak 34 for the Pacinian corpuscle. T h e fluid between the collagen and elastic layers constitutes the dashpot system. T h e elastic

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Fig. 8 (Leure-duPree). Intermediate and basal portions of a pigment epithelial cell. In the intermediate portion the nucleus (N), nucleous (n), rough-surfaced endoplasmic reticulum (ER), and mitochondria which are located between the nucleus and the basal convoluted portion of the cell are observed. The infoldings of the plasma membranes (double arrows), which contains ribosomes (R) are seen in the basal portion of the cell. The basal lamina of the pigment epithelium and the inner collagen layer (Ic) of Bruch's membrane are visible. (X20,000.)

and collagenous layers function as springs to bring Bruch's membrane back to its original shape after the pulse dissipates—the elastic layer being the main elastic component and the collagen supplying mainly tensile strength and support as well as a small amount of elasticity. This arrangement

would thus dampen the pulsation transmitted from the vascular compartment of the choroid and thereby reduce deformation or jar­ ring of the receptor outer segments. The prominent and highly structured in­ termediate junctions have been described in the pigment epithelium of the frog,10 turtle,23

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Fig. 9 (Leure-duPree). Diagrammatic representation of the pigment epithelium and the choriocapillaries of the sheep. mouse,8 monkey,35 and human.11'36 These specializations are probably supportive or cytoskeletal in that they increase tensile strength of adhesiveness and thereby insure resistance to mechanical stress. The intimate association between the mitochondria and the lateral plasma membranes, and their presence between the nucleous and the basal convoluted portion of the cell, may furnish the necessary energy for the transfer of ions in a preferred direction. The extensively developed agranular ER is a characteristic feature of the vertebrate pigment epithelium. Agranular ER is seen in similar configuration and abundance in cells

engaged in the biosynthesis of steroids, such as the cells of the foetal and adult adrenal cortex,3-38 the cells of the corpus luteum,39 and the interstitial cells of the testis.40 The smooth-surfaced reticulum has been impli­ cated in carbohydrate and lipid metabolism in the liver41 and there is evidence that it participates in certain phases of absorption and transport.42'43 The pigment epithelium is believed to pro­ vide storage of vitamin A during light-dark adaptation4'44*46 and also the enzymes neces­ sary to mediate esterification and isomerization of vitamin A to retinene (vitamin A aldehyde) for synthesis of the visual pig-

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Fig. 10 (Leure-duPree). Drawing representing, in three dimensions, the structure of the pigment epithelial cells, with the underlying Bruch's membrane and choriocapillaries.

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ments. Two of the enzymes involved in the rhodopsin cycle are present in the pigment epithelium: (1) an enzyme which catalyzes the esterincation of all-trans retinene to vita­ min A during light adaptation44 and (2) ret­ inene isomerase, which catalyzes the isomerization of all-trans retinene, since reti­ nene must be in the 11-cis configuration be­ fore it is able to combine with the protein opsin to form rhodopsin.45 Therefore, the smooth-surfaced ER may be involved in the transport of vitamin A back and forth be­ tween the pigment epithelium and the retina and in the resynthesis and interconversion of vitamin A to retinene. It is noteworthy that the embryonic development of the smooth-surfaced endoplasmic reticulum par­ allels that of the receptor outer segments.47 The biosynthetic function of the pigment epithelium is further emphasized morpholog­ ically by the interrelationship of the smooth ER with the granular ER and the mito­ chondria. The well-oriented cytoplasmic membrane system of the rough-surfaced endoplasmic reticulum, although not abundant, suggests that the pigment epithelium is engaged in protein synthesis. The granular ER is usually not concentrated in cells which synthesize but do not discharge protein, as compared to pro­ tein-secreting glandular cells. This unique organization of the rough-surfaced reticu­ lum, a prominent feature of the cell, has been reported only in pigment epithelium of man38'48 and monkey.49 There is no cytologic evidence of protein biosynthesis for export in this layer; therefore the stratified cisternae of the granular ER may synthesize some of the proteins for the visual cycle and for the metabolism of the pigment epithelium. Opsin, an essential requisite for the vis­ ual cycle, may be synthesized by the gran­ ular ER. The origin of this protein is un­ known ; however, the amount of visual pig­ ment synthesized depends upon its availability.3 The granular ER is frequently associated with the pigment granules. This positional

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relationship may have functional significance in the initiation of the synthesis of melanin. It has been suggested that in the biosyn­ thesis of melanin, the rough-surfaced ER is involved in the synthesis of pro-tyrosinase and tyrosinase in a manner not unlike that proposed by Caro and Palade20 for the syn­ thesis of zymogen granules in the exocrine pancreas.50 Tyrosinase, for example, would be synthesized in the ribosomes of the gran­ ular ER and then transferred by the intracisternal dilations to the Golgi complex, where it is incorporated or accumulated in vesicles. These vesicles increase in size, and the tyrosinase molecules become aligned in an ordered pattern on an acquired system of internal membranes. At this stage the pig­ ment granule is known as a premelanosome.51 Biochemical studies using differential centrifugation have shown that in vivo incorporation of labeled amino acid by soluble tyrosinase is localized in the microsomal fraction.52 Radioautographic stud­ ies have shown that tritiated dihydroxyphenylalanine ( D O P A ) , a labeled intermedi­ ate in the formation of melanin, was local­ ized first over the rough-surfaced endo­ plasmic reticulum.53 These studies provide evidence that the granular ER participates to some extent in the biosynthesis of melanin. Whether melanogenesis is a continuous pro­ cess in the pigment epithelium, as it is in the epidermis, is unknown. The pigment granules may function in the absorption of light which has traversed the neural retina and thereby prevent backscatter, particularly in species without a tapetum. This mechanism would aid in the refinement of the stimulus to the photoreceptors and thereby assure better visual acuity. To the author's knowledge there is no evi­ dence of phototropic displacement of pig­ ment granules in the mammalian pigment epithelium such as has been observed in fish, turtles, and Amphibia.10*47 The lysosomelike bodies observed in this study and those in the pigment epithelial cells of other vertebrates9'11'64 resemble

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strikingly the autophagic vacuoles of DeDuve55 and the cytolysomes of Novikoff and associates.56 The morphologic heteroge­ neity characteristic of these inclusion bodies may reflect various stages of degradation of sequestration.57 These structures are unlike the myeloid bodies found in the pigment epi­ thelial cells of fish, amphibia, reptiles, and birds by Porter and Yamada10 and by Yamada23 which, they suggested, may be photosensitive and may activate pigment mi­ gration in these cells. Feeney54 observed two types of myeloid bodies in the human pig­ ment epithelium. One type was continuous with the smooth-surfaced endoplasmic retic­ ulum, as observed in the frog10 and turtle.28 Such continuity was not observed in our in­ vestigation. Since many of the inclusion bod­ ies resemble the outer segment discs and membranes, it has been suggested that these bodies may be digested fragments broken from the outer segments, implying auto­ phagic or phagocytic activity or both.9'54 Evidence for phagocytosis is supported by the studies of Droz58 and Young59 who, using radioautographic techniques, observed a significant turnover of the outer segment material moving from the inner segment to the distal end of the outer segment. Although the inclusion bodies often re­ semble the receptor outer segments, the pres­ ence of rhodopsin in these bodies has not been reported. An interesting observation of Davis and Blasic60 was that antiserum pre­ pared against rhodopsin stained not only the receptor outer segments but also unidentified structures of the pigment epithelium. Stud­ ies to determine whether there is acid-phosphatase activity in the inclusion bodies and whether the pigment epithelium is auto­ phagic or phagocytic would help to deter­ mine the nature of these structures. SUMMARY

The foregoing observations indicate that the fine structure of the pigment epithelium of the domestic sheep is similar to that of other vertebrates. The extensive infoldings

MARCH, 1968

of the basal plasma membranes and the microvilli which provide increased surface area, suggest that the pigment epithelium is engaged in transport. Certain cytoplasmic organelles indicate specific functions. The smooth-surfaced endoplasmic reticulum, a characteristic feature of the pigment epithe­ lium, and the rough-surfaced endoplasmic reticulum and the Golgi complex all suggest a biosynthetic function. The inclusion bod­ ies or lysosomelike structures may be an ex­ pression of autophagic or phagocytic activity or both. Bruch's membrane, which is inter­ posed between the pigment epithelium and the choroid, may serve not only in a suppor­ tive capacity but also as a mechanical filter. 950 East 59th Street (60637) ACKNOWLEDGMENT

I wish to thank Professor Hewson Swift for his helpful discussion of this paper and for a critical reading of the manuscript. REFERENCES

1. Kiihne, W.: Chemische vorgange in der netzhaut. In Hermann, I. (ed.) : Handbuch der Physiologie. Leipzig, F. C. W. Vogel, 1879, ed. 3, pt. 1, p. 321. 2. Bliss, A. F.: Properties of pigment layer fac­ tor in the regeneration of rhodopsin. J. BioL Chem. 193 :52S, 1951. 3. Wald, G.: The photoreceptor process in vision. Am. J. Ophth. 40:, 1955. 4. Hubbard, R.: Retinene isomerase. J. Gen. Physiol. 39 :935, 1956. 5. Glocklin, V. C, and Potts, A. M.: The metab­ olism of retinal pigment cell epithelium. II. Respir­ ation and glycolysis. Invest. Ophth. 4:226, 1965. 6. Noell, W. K.: Studies on the electrophysiology and the metabolism of the retina. School of Avia­ tion Med., Report No. 1. Randolph Field, Texas, 1953. 7. Lasansky, A., and de Fisch, F. W.: Potential, current, and ionic fluxes across the isolated retinal pigment epithelium and choroid. J. Gen. Physiol. 49 :913, 1966. 8. Cohen, A. I.: The ultrastructure of the rods of the mouse retina. Am. J. Anat. 107:23, 1960. 9. Dowling, J. E., and Gibbons, I. R.: The fine structure of the pigment epithelium in the albino rat. J. Cell Biol. 14:459, 1962. 10. Porter, K. R., and Yamada, E.: Studies on the endoplasmic reticulum. V. Its form and differ­ entiation in the pigment epithelial cells of the frog retina. J. Biophys. Biochem. Cytol. 8:181, 1960. 11. Bairati, A. J., and Orzalesi, N : The ultrastructure of the pigment epithelium and the photo-

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receptor-pigment epithelium junction in the human retina. J. infrastructure Res. 9:484, 1963. 12. Berstein, M. H.: Functional architecture of pigment epithelium. In Smelser, George K. (ed.) : The Structure of the Eye. New York, Academic Press, 1961, p. 139. 13. Guttes, E.: Die herkunf t des augenpigmentes beim kaninchen-embryo. Z. fur Bellforsch u. Mikroskop. 39:168, 1953. 14. Sabatini, D. D., Bensch, K., and Barrnett, R. J.: The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17:19, 1963. 15. Millonig, G.: Advantages of phosphate buffer for osmium tetroxide solution in fixation. J. Ap­ plied Physics 32:1637,1961. 16. Luft, J. H.: Improvement in epoxy resin embedding method. J. Biophys. Biochem. Cytol. 9:409, 1961. 17. Watson, M. L.: Staining of tissue sections for electron microscopy with heavy metals. J. Bio­ phys. Biochem. Cytol. 4:475, 1958. 18. Millonig, G.: A modified procedure for lead staining of thin sections. J. Biophys. Biochem. Cytol. 17:736,1961. 19. Becher, H.: Intern. Kongr. Elektronenmikroskopie 4. Berlin, Verhandl. 2:425, 1960. 20. Caro, L. G., and Palade, G. E.: Protein syn­ thesis, storage, and discharge in the pancreatic exocrine cell. An autoradiographic study. J. Cell Biol. 20:473, 1964. 21. Brenner, R. M.: Fine structure of adrenocortical cells in adult male Rhesus monkeys. Am. J. Anat. 119:429, 1966. 22. Moyer, F . : Electron microscope observation on the origin, development, and genetic control of melanin granules in the mouse eye. In Smelser, George K. (ed.) : The Structure of the Eye. New York, Academic Press, 1961, p. 469. 23. Yamada, E.: The fine structure of the pig­ ment epithelium in the turtle eye. In Smelser, George K. (ed.) : The Structure of the Eye. New York, Academic Press, 1961, p. 73. 24. Farquhar, M. G., and Palade, G. E.: Junctional complexes in various epithelia. J. Cell Biol. 17 :375, 1963. 25. Lowry, O. H., Roberts, N. R., and Lewis, C.: The quantitative histochemistry of the retina. J. Biol. Chem. 220 :879,1956. 26. Cohen, A. I.: Vertebrate retinal cells and their organization. Biol. Rev. 38:427, 1963. 27. Pease, D. C.: Infolded basal plasma mem­ branes found in epithelia noted for water transport. J. Biophys. Biochem. Cytol. 2(Supp.) :203, 1956. 28. Homberg, A.: Ultrastructure of the ciliary epithelium. Arch. Ophth. 62:935, 1959. 29. Hamilton, D. W.: Microvillous cells in am­ pullae of the lizard inner ear. J. Morph. 116:339, 1965. 30. Maurice, D. M.: The movement of fluorescein and water in the cornea. Am. J. Ophth. 49:1011, 1960. 31. Kaye, G. I., and Pappas, G. D.: Studies on the cornea. I. The fine structure of the rabbit cor­

397

nea and the uptake and transport of colloidal parti­ cles by the cornea in vivo. J. Cell Biol. 12:457, 1962. 32. Lerche, W.: Elektronenmikroskopische beobachtungen an der Bruchschen Membran des menschlichen auges. Bericht d. Ophthalm. Ges. 65:384, 1963. 33. Leeson, T. S., and Leeson, C. R.: The bloodretina barrier: A study of the choriocapillaries and lamina elastics of the rat. In Uyeda, Ryozi (ed.) : Electron Microscopy. Vol. II. Tokyo, Maruzen Co., Ltd., p. 513. 34. Loewenstein, W. R., and Skalak, R.: Me­ chanical transmission in a pacinian corpuscle. An analysis and a theory. J. Physiol. 182:346, 1966. 35. Cohen, A. I.: The fine structure of the extrafoveal receptors of the Rhesus monkey. Exper. Eye Res. 1:128, 1961. 36. Fine, B. S.: Limiting membranes of sensory retina and pigment epithelium: an electron micro­ scopic study. Arch. Ophth. 66:847, 1961. 37. Ross, M. H., Pappas, G. D ; Lanman, J. T. and Lind, J.: Electron microscopic observation on the endoplasmic reticulum in the human fetal adre­ nal. J. Biophys. Biochem. Cytol. 4:659, 1958. 38. Enders, A. C, Schlafke, S., and Warren, R.: Cytology of the fetal zone of the adrenal gland of the armadillo. Anat. Rec. 154:807, 1966. 39. Enders, A. C.: Observations on the fine structure of lutein cells. J. Cell Biol. 12 :101, 1962. 40. Christensen, A. K.: The fine structure of testicular interstitial cells in guinea pigs. J. Cell Biol. 26:911, 1965. 41. Porter, K. R., and Bruni, C.: An electron mi­ croscope study of the early effects of 3-Me-DAB on rat liver cells. Cancer Res. 19:997, 1959. 42. Palay, S. L., and Karlin, L. J.: An electron microscopic study of the intestinal villus. II. The pathway of fat absorption. J. Biophys. Biochem. Cytol. 5:373,1959. 43. Strauss, E. W.: The absorption of fat by intestine of golden hamster in vitro. J. Cell Biol. 17:597,1963. 44. Dowling, J. E.: Chemistry of visual adapta­ tion in the rat. Nature 188:144,1960. 45. Hubbard, R., and Dowling, J. E.: Forma­ tion and utilization of 11-cis vitamin A by the eye tissues during light and dark adaptation. Nature 3:341, 1962. 46. Sidman, R. L., and Dowling, J. E.: Autoradi­ ographic localization of C"-vitamin A in dark and light-adapted rat eyes. Anat. Rec. 145:286, 1963. 47. Yamada, E.: Some observations on the fine structure of the human retina. Fukuoka Acta Medica 57:163,1966. 48. Yamada, E., and Ishikawa, T.: Some obser­ vations on the submicroscopic morphogenesis of the human retina. In Rohen, J. W. (ed.) : The Struc­ ture of the Eye. II Symposium. Stuttgart, Schattauer-Verlag, 1965, p. 5. 49. Keefe, J. R., Ordy, J. M., and Samorajski, T.: Prenatal development of the retina in a diurnal primate (Macaca mulatta). Anat. Rec. 154:759, 1966.

398

AMERICAN JOURNAL OF OPHTHALMOLOGY

MARCH, 1968

55. DeDuve, C.: From cytases to lysosomes. Fed. Proc. 23:1045, 1964. 56. Noyikoff, A. B., Essner, E , and Quintana, N.: Golgi apparatus and lysosome. Fed. Proc. 23 :1010, 1964. 57. Swift, H., and Hruban, Z.: Focal degrada­ tion as a biological process. Fed. Proc. 23:1026, 1964. 58. Droz, B.: Dynamic condition of proteins in the visual cells of rats and mice as shown by radioautography with labeled amino acids. Anat. Rec. 145:157, 1963. 59. Young, R. W.: The renewal of photoreceptor cell outer segments. J. Cell Biol. 33 :61, 1967. 60. Davis, D. P., and Blasie, J. K.: Immunofluorescent localization of rhodopsin in the retina of the frog. J. Histochem. Cytochem. 14:789, 1966.

50. Breathnach, A. S., and Wyllie, L. M.-A.: in­ frastructure of retinal pigment epithelium of the human fetus. J. Ultrastructure Res. 16:584, 1966. 51. Moyer, F. H.: Genetic variations in the fine structure and ontogeny of mouse melanin granules. Am. Zoologist 6:43, 1966. 52. Seiji, M., Shimao, K., Birbeck, M. S. C, and Fitzpatrick, T. B.: Subcellular localization of mela­ nin biosynthesis. Ann. N. Y. Acad. Sci. 100:497, 1963. 53. Zelickson, A. S., Hirsch, H. M., and Hartmann, J. F.: Localization of melanin synthesis. J. Invest. Derm. 45 :458, 1965. 54. Feeney, L. J., Grieshaber, J. A., and Hogan, M. J.: Studies in human ocular pigment. In Rohen, J. W. (ed.) : The Structure of the Eye. II Sympo­ sium. Stuttgart, Schattauer- Verlag, 1965, p. 105.

STRESS-INDUCED BIREFRINGENCE O F T H E CORNEA GERALD W.

NYQUIST,

M.S.

Detroit, Michigan

Since the discovery of birefringence of the cornea by David Brewster 1 in 1815, many researchers have used the phenomenon as a tool to aid in investigating the anatomic structure of the cornea. The birefringence of interest in anatomic studies is of intrinsic or form origin and is a function of the submicroscopic structure. In recent years, the possibility of using measurements of the temporary stress-in­ duced birefringence or photoelastic effect of the cornea for diagnosing eye diseases has been the motivating force of many re­ search efforts (Stanworth 2 and Zandman,3 among others). Hopefully, one would mea­ sure the magnitude of the birefringence in vivo, using an optical instrument known as a polariscope. Variation in corneal birefrin­ gence from that observed in the normal healthy eye would indicate an abnormality (such as glaucoma). From the Engineering Mechanics Department, Biomechanics Research Center, Wayne State Univer­ sity. This study was supported in part by U.S. Public Health Service grant NB-05641-02.

This paper describes a basic study of the stress-induced birefringence of the cornea conducted as part of a program aimed at de­ termining the change in birefringence in vivo caused by increased intraocular pres­ sure. Stress-induced birefringence properties as evidenced by uniaxial tensile tests on strips of corneal tissue are discussed. Pig corneas were used because of the availability of fresh specimens. PHOTOELASTICITY

Photoelasticity deals with the correlation between the optical property of temporary (stress-induced) birefringence and the inter­ nal stresses and strains of a material. Light impinging on certain materials is resolved into two components which travel through the structure at different velocities. The material exhibits two refractive indices and is said to be double-refracting, or birefringent. Birefringence is caused by the anisotropic structure of matter. If, optically, a material is more densely constructed in one direction