ACTA OPHT HAL MOL O G ICA VOL. 55 1977
Department o f Ophthalmology, University of Linkofling (Head: S . E . Nilsson), National Board of Occupational Safety and Health, Stockholm (Head: N . Lundgren), and Department of Clinical Neurophysiology Karolinska Hospital, Stockholm (Head: L. Wide'n),
CHANGES IN ULTRASTRUCTURE AND FUNCTION
OF THE SHEEP PIGMENT EPITHELIUM AND RETINA INDUCED BY SODIUM IODATE')
I. The Ultrastructure of the Normal Pigment Epithelium
of the Sheep BY
SVEN ERIK G. NILSSON, BENGT KNAVE and HANS E. PERSSON
The normal ultrastructure of the sheep pigment epithelial cells is described as a basis for the interpretation of toxic (sodium iodate) effects on these cells dealt with in two following papers. The morphological features of the different cell membranes and cell organelles, particularly the phagosomes and the lipid droplets, are discussed in relation to renewal of the photoreceptor outer segment, pigment epithelial and retinal metabolism, barrier mechanisms and electrical properties. Key words: electron microscopy - pigment epithelium
-
retina
-
sheep.
The ERG-waves arise as the result of an integration of several underlying processes in the neuroretina and the pigment epithelium. In one of our earlier investigations (Knave, Pex-sson & Nilsson 1974) sodium iodate was used with 1)
This investigation was supported by grants from the Swedish Medical Research Council (Projects No. 12X-734 and 04X-3119), the Magnus Bergvall Foundation for Scientific Research and from Karolinska Institutet.
Received March 18, 1977. 994
Ultrastructure of Sheep Pigment Epithelium
the intention to selectively block the activity of the pigment epithelium while studying the influence of barbiturate on the neuroretina of the sheep. Such a model can probably be used also in experiments concerning other drugs. For this purpose it would be desirable to know in more detail the nature of and the correlation of the changes in pigment epithelial and retinal ultrastructure and function induced by sodium iodate. In this connection particularly the early changes are of interest. Previous studies on the subject (Noel1 1953, 1954, 1963; Grignolo et al. 1966; Suyama 1965; Babel 1970) either do not include histology of the early stages or do not correlate morphological findings with electroretinography, however. For this reason an investigation was undertaken to map and to correlate mainly the early changes in ultrastructure and ERG of the sheep pigment epithelium and retina after administration of sodium iodate. For the necessary background as to the normal ultrastructure of the sheep neuroretina the reader is referred to earlier papers (Nilsson, Knave, Persson & Lunt 1973; Nilsson, Knave, Lunt & Persson 1973), but with respect to the pigment epithelium the studies already available (Leure-duPree 1968; Nilsson, Knave, Persson & Lunt 1973) needed to be completed with additional material. Thus this first paper in a series of three describes the normal ultrastructure of the sheep pigment epithelium. The other two papers will deal with the early and the delayed effects of sodium iodate, respectively (Nilsson, Knave & Persson 1977a,b).
Material and Methods The eyes from two light-adapted (for about 8 h) domestic sheep were fixed by perfusion via the common carotid arteries with 1 o/o glutaraldehyde in 0.1 M cacodylate buffer. Thereafter the eyes were enucleated. The anterior segment of each eye was removed and fixation was continued for three days by means of immersion in the same fixative. Then the eyes were cut in smaller pieces, and the sclera and most of the choroid were dissected away. At this stage such a dissection could be performed without giving rise to retinal detachment. 1 O / o osmium tetroxide in the same buffer was used for postfixation, and after dehydration in acetone the specimens were embedded in Vestopal W. Pieces of retina and the pigment epithelium taken from the extratapetal area of the posterior part of the eye were sectioned for light and electron microscopy. The thin sections were examined in a Philips 300 electron microscope.
Observations The nucleus of the pigment epithelial cell was generally oval and located in the basal part of the cell. It was often seen to contain a nucleolus (Fig. 1). 995
Sven Erik G . Nilsson,Bengt Knave and Hans E . Persson
The apical plasma membrane showed slender processes coming into close contact with the rod outer segments (Fig. 1). Attachment zones (junctional complexes) were present between the lateral membranes of adjacent cells, not far from the apical surface of the cell (Fig. 1). Melanin pigment granules of different sizes and shapes, mainly elongated, were abundant except close to the basal plasma membrane (Figs. 1 and 5). A moderate number of rod-shaped mitochondria were found in the basal half of the cell (Figs. 2 and 4). The Golgi complex, consisting of a number of flattened saccules and vesicles (Fig. 2), was in most cases seen in the vicinity of the nucleus. Rough-surfaced endoplasmic reticulum and free ribosomes were observed most frequently in the basal part of the cell (Figs. 3 and 4). Throughout the cytoplasmic compartment the very abundant tubular-shaped profiles of the smooth-surfaced endoplasmic reticulum were found (Fig. 5 ) . The basal plasma membrane was characterized by frequent infoldings (Fig. 4). The basement membrane between the pigment epithelial cells and the endothelial cells of the choroidal capillaries is seen in Figs. 3 and 4. In the apical part of the cell different kinds of granules and inclusion bodies were present. From a morphological point of view the evenly light grey and membrane-bound granules appear to contain lipids (Figs. 5-7). Thus they are called lipid-droplets. The larger bodies are so-called phagosomes, containing incorporated outer segment discs (Figs. 5-7). The phagosome (Ph 1) in Fig. 5 shows fairly well organized and to a great extent parallel membranes, whereas in Fig. 6 as well as in Fig. 7 a big phagosome (Ph 2) is seen to contain membranes in a concentric or an irregular arrangement. Other phagosomes (Ph 3) were found to have a content of small bodies of concentrically arranged membranes or of a homogeneous substance (Figs. 5 and 6). Occasionally also lipid droplets were observed within a phagosome (Ph 4) (Fig. 6). The possible origin and function of the lightly stained granules labelled X in Fig. 7 will be dealt with in the discussion.
Discussion The pigment epithelium described in the present investigation was taken from the extratapetal area of the fundus. It contained a large number of melanin pigment granules as a striking difference from the melanin-lacking pigment epithelium overlaying the tapetum (Nilsson, Knave, Persson & Lunt 1973). Many of the basic features of the melanin-containing pigment epithelium of the sheep were also described by Leure-duPree (1968). 996
Ultrastructure of Sheep Pigment Epithelium
Fig. I . Survey picture of a pigment epithelial cell (PE). N: nucleus, No: nucleous, M: melanin granule, A: apical plasma membrane with processes, Az: attachment zone, R: rod outer segment. x 14 500.
The participation in the turnover of the rod outer segments is an important feature of the pigment epithelial cells. It was shown by Nilsson (1964) that the outer segment disks in the young tadpole developed as invaginations of the plasma membrane at the base of the outer segment. Since basal membrane invaginations of the same kind were observed also in adult receptor outer segments, Nilsson (1964) proposed that new disks were formed in the same way also at the adult stage. In elegant autoradiographic studies by Young (1967) 99 7 Acta ophthal. 55, 6
64
Sven Erik G. Nilsson, Bengt Knave and Hans E . Persson
Fig. 2. Mitochondria (Mi) and a Golgi complex (G) are seen in the basal half of the cell. x 59 000.
and by Young & Droz (1968) this idea was proven to be correct. By the use of labelled amino acids it was found that the rod outer segment was continuously renewed from the base with a turnover-time of 9-10 days for mouse and rat and about 6 weeks for frog. It appears quite obvious that the same principle is valid also for the sheep. Young & Bok (1969) could demonstrate that the tips of the frog rod outer segments were repeatedly incorporated into the pigment epithelium, where
Figs. 3 and 4.
A basement membrane (BM) separates the pigment epithelial cell from the endothelial cell (E) of the chorioidal capillaries. The basal plasma membrane (B) shows numerous infoldings. In the cytoplasm rough-surfaced endoplasmic reticulum (RER), free ribosomes (FR) and mitochondria (Mi) are seen. x 27 500 and 34 000 resp.
998
Ultrastructure of Sheep Pigment Efiithelium
999 64*
Fig 5. A phagosome with faily well organized membranes (Ph 1) and a phagosome consisting of concentrically arranged membranes together with a homogeneous substance (Ph 3) are shown. L: lipid droplet, SER: profiles of smooth-surfaced endoplasmic reticulum, R: rod outer segment. ~ 4 2 0 0 0 . 1000
Ultrastructure of Sheep Pigment Epithelium
Figs 6 and 7. Phagosomes containing concentrically and irregularly arranged membranes (Ph 2), small bodies of concentrically organized membraneous material (Ph 3) or lipid droplets (Ph 4). L: lipid droplet, X: lightly stained granules. x 27 700 and 34 200 resp. 1001
Sven
Erik G . Nilsson, Bengt Knave and Hans E. Persson
so-called phagosomes are formed. Such inclusion bodies had been observed earlier (Dowling & Gibbons 1962; Bairati & Orzalesi 1963) but the exact nature of the structures was somewhat unclear. It appears that the rod outer segment tip (about 5-40 disks) can be separated from the major part of the outer segment either by protrusion of pigment epithelial processes, which latter then completely engulf the disks and withdraw them into the cell body of the pigment epithelial cell (Spitznas & Hogan 1970 (human)) or by shedding of the stack of disks primarily by infolding of the outer segment plasma membrane (Young 1971 (rhesus monkey)). Later it has been shown that also cone outer segments may be ensheathed by pigment epithelial processes (Steinberg & Wood 1974 (cat); Steinberg, Wood & Hogan 1977 (human)) and that also the tip of cone outer segments can be engulfed into the pigment epithelium by protrusion of pigment epithelial processes (Hogan, Wood & Steinberg 1974 ; Steinberg, Wood & Hogan 1977 (human)). It has recently been shown that shedding of outer segment disks is a cyclic daily process, where rods predominantly shed their disks at the onset of the light period (LaVeil 1976 (rat), Basinger, Hoffman & Matthes 1976 (frog)), and where shedding from cones occurs mainly at the beginning of the dark period (Young & O’Day 1977 (goldfish)). In the present investigation the animals were dark-adapted for about 8 h prior to the experiment. The phagosomes then undergo degradation through a series of stages (Ishikawa & Yamada 1970; Spitznas & Hogan 1970; Johnson 1975). The phagosomes labelled Ph 1-Ph 4 in Figs. 5-7 of the present investigation are considered to represent successive stages in such a degradation. The disintegration of the content seems to be accomplished by means of lytic enzymes, such as acid phosphatase (Ishikawa & Yamada 1970; Marshall 1970; Hollyfield & Ward 1974). There are also morphological evidence supporting the idea that these enzymes may originate in the Golgi compIex, and that the enzymes then are stored in lysosomal granules, which interact with the young phagosome (Ishikawa & Yamada 1970). The lightly stained granules (X) seen in Fig. 7 could possibly represent such lysosomes. However, it cannot be excluded that they are instead final stages of phagosomal degradation. The presence of cathepsin D (a proteolytic enzyme) in the pigment epithelium indicates that also this enzyme may take part in the digestion of the phagosomes (Hayasaka, Hara & Mizuno 1975). Certain hereditary retinal degenerations can be explained by a defect in the phagocytic mechanism described above (Bok & Hall 1969, 1971; Herron, Riegel, Myers & Rubin 1969; Herron, Riegel Brennan & Rubin 1974). The oil droplets of the frog pigment epithelium are known to concentrate vitamin A (the precursor of retinal) and fatty acids (Young & Bok 1970; Bibb &
1002
Ultrastructure
of
Sheep Pigment Epithelium
Young 1974). It seems most likely that the lipid droplets of the sheep pigment epithelium correspond to the frog oil droplets. It cannot be excluded that part of the lipid material is derived from outer segment degradation products. Besides the renewal of rod outer segments by means of membrane replacement, both rods and cones renew their membranes also by diffuse molecular replacement. This is true for proteins as well as for lipids. It appears that the pigment epithelium plays an important role in lipid metabolism of outer segment membranes (Bibb & Young 1974; Young 1974). The pigment epithelium is part of the “blood retina barrier”. Its plasma membranes possess certain passive and active (selective) transport mechanisms (Steinberg & Miller 1973; Miller & Steinberg 1977a,b). The extensive infoldings of the basal plasma membrane somewhat resemble those of the kindney tubular cells, where extensive transport is known to take place. Attachment zones (junctional complexes) connected the lateral membranes of adjacent cells. Close to the apical surface of the cells they are of the tight junction type (zonulae occludentes), and just basally to the tight junctions zonulae adherentes are present (Leure-duPree 1968). It was clearly shown by Peyman, Spitznas & Straatsma (1971) that diffusion of peroxidase was abruptly stopped at the zonulae occludentes in the intercellular spaces of the pigment epithelium. Thus, the tight junctions together with the selective membrane transport mechanisms can effectively block free flux between the choroidal capillaries and the neuroretina. The difference in transport and permeability properties between the apical and the basal membranes of the pigment epithelial cells gives rise to the trans-pigment epithelial potential (Steinberg & Miller 1973; Miller & Steinberg 1977a,b), which appears to be the major component of the standing potential of the eye (Noel1 1954; Heck & Papst 1957; Gouras 1969). Toxic agents that affect the properties of the basal and/or apical membranes are thus very likely to influence the trans-pigment epithelial potential and hence the standing potential of the eye. Furthermore, it is to be expected that the c-wave of the ERG, which is generated mainly across the apical membrane of the pigment epithelium (Steinberg, Schmidt & Brown 1970; Schmidt & Steinberg 1971; Steinberg & Miller 1973; Oakley & Green 1976), is also affected at the same time. Sodium iodate is a toxic agent of this kind, which will be shown in the following papers (Nilsson, Knave & Persson 1977a,b). In conclusion it can be said that the pigment epithelium is a most important barrier layer as well as a very metabolically active layer interposed between the receptor cells and the circulation, and upon which the receptor cells are highly dependent.
1003
Sven Erik G . Nilsson, Bengt Knave and Hans E. Persson
References Babel J. (1970) The retina. In: Bischoff A,, Ed. Ultrastructure of the Peripheral Nervous System and Sense Organs, pp. 339-441. G. Thieme Verlag, Stuttgart. Bairati A., Jr. & Orzalesi N. (1963) The ultrastructure of the pigment epithelium and of the photoreceptor pigment epithelium junction in the human retina. J . Ultrastruct. Res. 9, 484-496. Basinger S., Hoffman R. & Matthes M. (1976) Photoreceptor shedding is initiated by light in the frog retina. Science 194, 1074-1076. Bok D. & Hall M. 0. (1969) The etiology of retinal dystrophy in RCS rats. Invest. Ophthal. 8, 649-650. Bok D. & Hall M. 0. (1971) The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J . Cell Biol. 49, 664-682. Bibb C. & Young R. W. (1974) Renewal of fatty acids in the membranes of visual cell outer segments. J . Cell Biol. 61, 327-343. Dowling J. E. & Gibbons I. R. (1962) The fine structure of the pigment epithelium in the albino rat. J. Cell Biol. 14, 459-474. Gouras P. (1969) Clinical electro-oculography. In: Straatsma B. R., Ed. The Retina, pp. 565-581. University of California Press, Berkeley & Los Angeles. Grignolo A,, Orzalesi N. & Calabria G. A. (1966) Studies on the fine structure and the rhodopsin cycle of the rabbit retina in experimental degeneration induced by sodium iodate. E x p . Eye Res. 5, 86-97. Hayasaka S., Hara S. & Mizuno K. (1975) Distribution and some properties of cathepsin D in the retinal pigment epithelium. Exp. Eye Res. 21, 307-313. Heck J. & Papst W. (1957) Uber den Ursprung des comeo-retinalen Ruhepotentials. Bibl. ophthal. (Basel) 48, 96-107. Herron W. L., Riegel B. W., Myers 0. E. & Rubin M. L. (1969) Retinal dystrophy in the rat - A pigment epithelial disease. Invest. Ophthal. 8, 595-604. Herron W. L., Riegel B. W., Brennan E. & Rubin M. L. (1974) Retinal dystrophy in the pigmented rat. Invest. Ophthal. 13, 87-94. Hogan M. J., Wood I. & Steinberg R. H. (1974) Phagocytosis by pigment epithelium of human retinal cones. Nature (Lond.) 252, 305-307. Hollyfield J. G. & Wa r d A. (1974) Phagocytic activity of the pigmented retinal epithelium. 111. Interaction between lysosomes and ingested polystyrene spheres. Invest. Ophthal. 13, 1016-1023. Ishikawa T. & Yamada E. (1970) The degradation of the photoreceptor outer segment within the pigment epithelial cell of the rat retina. J . Electron Microscopy 19, 85-99. Johnson N. F. (1975) Phagocytosis in the normal and ischaemic retinal pigment epithelium of the rabbit. Ex@. Eye Res. 20, 97-107. Knave B., Persson H. E. & Nilsson S. E. G. (1974) The effect of barbiturate on retinal functions. 11. Effects on the c-wave of the electroretinogram and the standing potential of the sheep eye. Acta physiol. scand. 91, 180-186. LaVail M. M. (1976) Rod outer segment disk shedding in rat retina: relationship to cyclic lighting. Science 194, 1071-1073. Leure-duPree A. (1968) Ultrastructure of the pigment epithelium in the domestic sheep. Amer. J . Ophthal. 65, 383-398.
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Ultrastructure of Sheep Pigment Epithelium Marshall J. (1970) Acid phosphatase activity in the retinal pigment epithelium. Vision Res. 10, 821-824. Miller S. S. & Steinberg R. H. (1977a) Passive ionic properties of frog retinal pigment epithelium. In preparation for publication. Miller S. S. & Steinberg R. H. (197713) Active transport of ions across frog retinal pigment epithelium. In preparation for publication Nilsson S. E. G. (1964) Receptor cell outer segment development and ultrastructure of the disk membranes in the retina of the tadpole (Xana pipiens). /. Ultrastruct. Res. 11, 581-620. Nilsson S. E. G., Knave B., Lunt T. & Persson H. E. (1973) The morphology of the sheep retina. 11. The inner nuclear layer, the ganglion cells and the plexiform layers. Acta ophthal. (Kbh.) 51, 612-627. Nilsson S. E. G., Knave B. & Persson H. E. (1977a) Changes in ultrastructure and function of the sheep pigment epithelium and retina induced by sodium iodate. 11. Early effects. Acta ophthal. (Kbh.) 55, 1007-1026. Nilsson S. E. G., Knave B. & Persson H. E. (197713) Changes in ultrastructure and function of the sheep pigment epithelium and retina induced by sodium iodate. 111. Delayed effects. Acta ophthal. (Kbh.) 55, 1027-1043. Nilsson S. E. G., Knave B., Persson H. E. & Lunt T. (1973) The morphology of the sheep retina. I. The receptor cells and the pigment epithelium. Acta ophthal. (Kbh.) 51, 599-611. Noell W. K. (1953) Studies on the electrophysiology and the metabolism of the retina. Project report U . S. Air Force SAM Project No. 21-1201-0004, pp. 1-122. Randolph Field, Texas. Noell W. K. (1954) The origin of the electroretinogram. Amer. 1. Ophthal. 38, 78-93. Noell W . K. (1963) Cellular physiology of the retina. /. o p t . SOC.Amer. 53, 36-48. Oakley I1 B. & Green D. G. (1976) Correlation of light-induced changes in retinal extracellular potassium concentration with c-wave of the electroretinogram. /. Neurophysiol. 39, 1117-1 133. Peyman G. A., Spitznas M. & Straatsma B. R. (1971) Peroxidase diffusion in the normal and photocoagulated retina. Invest. Ophthal. 10, 181-189. Schmidt R. & Steinberg R. H. (1971) Rod-dependent intracellular responses to light recorded from the pigment epithelium of the cat retina. /. Physiol. (Lond.) 217, 71-91. Spitznas M. & Hogan M. J. (19iO) Outer segments of photoreceptors and the retinal pigment epithelium. Arch. Ophthal. (Chicago) 84, 810-819. Steinberg R. H. & Miller S. (1973) Aspects of electrolyte transport in frog pigment epithelium. Exp. Eye Res. 16, 365-372. Steinberg R. H., Schmidt R. & Brown K. T. (1970) Intracellular responses to light from cat pigment epithelium: origin of the electroretinogram c-wave. Nature (Lond.) 227, 728-730. Steinberg R. H. & Wood I. (1974) Pigment epithelial cell ensheathment of cone outer segments in the retina of the domestic cat. Proc. roy. SOC.Lond. B 187, 461-478. Steinberg R. H., Wood I. & Hogan M. J. (1977) Pigment epithelial ensheathment and phagocytosis of extrafoveal cones in human retina. Philosoph. Trans. R. SOC.Lond., B. 277, 459-474. Suyama T. (1965) Electron microscopic study on experimental retinal degeneration induced by sodium iodate injection. Acta SOC. ophthal. jab. 69, 440-460.
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Sven Erik G. Nilsson, Bengt Knave and Hans E. Persson Young R. W. (1967) The renewal of photoreceptor cell outer segments. /. Cell Biol. 33, 61-72. Young R. W. (1971) Shedding of discs from rod outer segments in the rhesus monkey. /. Ultrastruct. Res. 34, 190-203. Young R. W. (1974) Biogenesis and renewal of visual cell outer segment membranes. Ex#. Eye Res. IS, 215-223. Young R. W. & Bok D. (1969) Participation of the retinal pigment epithelium in the rod outer segment renewal process. /. Cell Biol. 42, 392-403. Young R. W. & Bok D. (1970) Autoradiographic studies on the metabolism of the retinal pigment epithelium. Invest. Ophthal. 9, 524-536. Young R. W. & Droz B. (1968) The renewal of protein in retinal rods and cones. /. Cell Biol. 39, 169-184. Young R. W. & O’Day W. T. (1977) Personal communication.
Authors’ addresses: Prof. Sven Erik G. Nilsson, Department of Ophthalmology, University of Linkoping, University Hospital, S-581 85 Linkoping, Sweden. Assoc. Prof. Bengt Knave, National Board of Occupational Safety and Health, S-100 26 Stockholm 34, Sweden. Assistant Prof. Hans E. Persson, Department of Clinical Neurophysiology, Karolinska Hospital, S-104 01 Stockholm 60, Sweden. Communications to Prof. Sven Erik Nilsson.
1006
Department o f Ophthalmology, University of Linkofling (Head: S . E . Nilsson), National Board of Occupational Safety and Health, Stockholm (Head: N . Lundgren), and Department of Clinical Neurophysiology Karolinska Hospital, Stockholm (Head: L. Wide'n),
CHANGES IN ULTRASTRUCTURE AND FUNCTION
OF THE SHEEP PIGMENT EPITHELIUM AND RETINA INDUCED BY SODIUM IODATE')
I. The Ultrastructure of the Normal Pigment Epithelium
of the Sheep BY
SVEN ERIK G. NILSSON, BENGT KNAVE and HANS E. PERSSON
The normal ultrastructure of the sheep pigment epithelial cells is described as a basis for the interpretation of toxic (sodium iodate) effects on these cells dealt with in two following papers. The morphological features of the different cell membranes and cell organelles, particularly the phagosomes and the lipid droplets, are discussed in relation to renewal of the photoreceptor outer segment, pigment epithelial and retinal metabolism, barrier mechanisms and electrical properties. Key words: electron microscopy - pigment epithelium
-
retina
-
sheep.
The ERG-waves arise as the result of an integration of several underlying processes in the neuroretina and the pigment epithelium. In one of our earlier investigations (Knave, Pex-sson & Nilsson 1974) sodium iodate was used with 1)
This investigation was supported by grants from the Swedish Medical Research Council (Projects No. 12X-734 and 04X-3119), the Magnus Bergvall Foundation for Scientific Research and from Karolinska Institutet.
Received March 18, 1977. 994
Ultrastructure of Sheep Pigment Epithelium
the intention to selectively block the activity of the pigment epithelium while studying the influence of barbiturate on the neuroretina of the sheep. Such a model can probably be used also in experiments concerning other drugs. For this purpose it would be desirable to know in more detail the nature of and the correlation of the changes in pigment epithelial and retinal ultrastructure and function induced by sodium iodate. In this connection particularly the early changes are of interest. Previous studies on the subject (Noel1 1953, 1954, 1963; Grignolo et al. 1966; Suyama 1965; Babel 1970) either do not include histology of the early stages or do not correlate morphological findings with electroretinography, however. For this reason an investigation was undertaken to map and to correlate mainly the early changes in ultrastructure and ERG of the sheep pigment epithelium and retina after administration of sodium iodate. For the necessary background as to the normal ultrastructure of the sheep neuroretina the reader is referred to earlier papers (Nilsson, Knave, Persson & Lunt 1973; Nilsson, Knave, Lunt & Persson 1973), but with respect to the pigment epithelium the studies already available (Leure-duPree 1968; Nilsson, Knave, Persson & Lunt 1973) needed to be completed with additional material. Thus this first paper in a series of three describes the normal ultrastructure of the sheep pigment epithelium. The other two papers will deal with the early and the delayed effects of sodium iodate, respectively (Nilsson, Knave & Persson 1977a,b).
Material and Methods The eyes from two light-adapted (for about 8 h) domestic sheep were fixed by perfusion via the common carotid arteries with 1 o/o glutaraldehyde in 0.1 M cacodylate buffer. Thereafter the eyes were enucleated. The anterior segment of each eye was removed and fixation was continued for three days by means of immersion in the same fixative. Then the eyes were cut in smaller pieces, and the sclera and most of the choroid were dissected away. At this stage such a dissection could be performed without giving rise to retinal detachment. 1 O / o osmium tetroxide in the same buffer was used for postfixation, and after dehydration in acetone the specimens were embedded in Vestopal W. Pieces of retina and the pigment epithelium taken from the extratapetal area of the posterior part of the eye were sectioned for light and electron microscopy. The thin sections were examined in a Philips 300 electron microscope.
Observations The nucleus of the pigment epithelial cell was generally oval and located in the basal part of the cell. It was often seen to contain a nucleolus (Fig. 1). 995
Sven Erik G . Nilsson,Bengt Knave and Hans E . Persson
The apical plasma membrane showed slender processes coming into close contact with the rod outer segments (Fig. 1). Attachment zones (junctional complexes) were present between the lateral membranes of adjacent cells, not far from the apical surface of the cell (Fig. 1). Melanin pigment granules of different sizes and shapes, mainly elongated, were abundant except close to the basal plasma membrane (Figs. 1 and 5). A moderate number of rod-shaped mitochondria were found in the basal half of the cell (Figs. 2 and 4). The Golgi complex, consisting of a number of flattened saccules and vesicles (Fig. 2), was in most cases seen in the vicinity of the nucleus. Rough-surfaced endoplasmic reticulum and free ribosomes were observed most frequently in the basal part of the cell (Figs. 3 and 4). Throughout the cytoplasmic compartment the very abundant tubular-shaped profiles of the smooth-surfaced endoplasmic reticulum were found (Fig. 5 ) . The basal plasma membrane was characterized by frequent infoldings (Fig. 4). The basement membrane between the pigment epithelial cells and the endothelial cells of the choroidal capillaries is seen in Figs. 3 and 4. In the apical part of the cell different kinds of granules and inclusion bodies were present. From a morphological point of view the evenly light grey and membrane-bound granules appear to contain lipids (Figs. 5-7). Thus they are called lipid-droplets. The larger bodies are so-called phagosomes, containing incorporated outer segment discs (Figs. 5-7). The phagosome (Ph 1) in Fig. 5 shows fairly well organized and to a great extent parallel membranes, whereas in Fig. 6 as well as in Fig. 7 a big phagosome (Ph 2) is seen to contain membranes in a concentric or an irregular arrangement. Other phagosomes (Ph 3) were found to have a content of small bodies of concentrically arranged membranes or of a homogeneous substance (Figs. 5 and 6). Occasionally also lipid droplets were observed within a phagosome (Ph 4) (Fig. 6). The possible origin and function of the lightly stained granules labelled X in Fig. 7 will be dealt with in the discussion.
Discussion The pigment epithelium described in the present investigation was taken from the extratapetal area of the fundus. It contained a large number of melanin pigment granules as a striking difference from the melanin-lacking pigment epithelium overlaying the tapetum (Nilsson, Knave, Persson & Lunt 1973). Many of the basic features of the melanin-containing pigment epithelium of the sheep were also described by Leure-duPree (1968). 996
Ultrastructure of Sheep Pigment Epithelium
Fig. I . Survey picture of a pigment epithelial cell (PE). N: nucleus, No: nucleous, M: melanin granule, A: apical plasma membrane with processes, Az: attachment zone, R: rod outer segment. x 14 500.
The participation in the turnover of the rod outer segments is an important feature of the pigment epithelial cells. It was shown by Nilsson (1964) that the outer segment disks in the young tadpole developed as invaginations of the plasma membrane at the base of the outer segment. Since basal membrane invaginations of the same kind were observed also in adult receptor outer segments, Nilsson (1964) proposed that new disks were formed in the same way also at the adult stage. In elegant autoradiographic studies by Young (1967) 99 7 Acta ophthal. 55, 6
64
Sven Erik G. Nilsson, Bengt Knave and Hans E . Persson
Fig. 2. Mitochondria (Mi) and a Golgi complex (G) are seen in the basal half of the cell. x 59 000.
and by Young & Droz (1968) this idea was proven to be correct. By the use of labelled amino acids it was found that the rod outer segment was continuously renewed from the base with a turnover-time of 9-10 days for mouse and rat and about 6 weeks for frog. It appears quite obvious that the same principle is valid also for the sheep. Young & Bok (1969) could demonstrate that the tips of the frog rod outer segments were repeatedly incorporated into the pigment epithelium, where
Figs. 3 and 4.
A basement membrane (BM) separates the pigment epithelial cell from the endothelial cell (E) of the chorioidal capillaries. The basal plasma membrane (B) shows numerous infoldings. In the cytoplasm rough-surfaced endoplasmic reticulum (RER), free ribosomes (FR) and mitochondria (Mi) are seen. x 27 500 and 34 000 resp.
998
Ultrastructure of Sheep Pigment Efiithelium
999 64*
Fig 5. A phagosome with faily well organized membranes (Ph 1) and a phagosome consisting of concentrically arranged membranes together with a homogeneous substance (Ph 3) are shown. L: lipid droplet, SER: profiles of smooth-surfaced endoplasmic reticulum, R: rod outer segment. ~ 4 2 0 0 0 . 1000
Ultrastructure of Sheep Pigment Epithelium
Figs 6 and 7. Phagosomes containing concentrically and irregularly arranged membranes (Ph 2), small bodies of concentrically organized membraneous material (Ph 3) or lipid droplets (Ph 4). L: lipid droplet, X: lightly stained granules. x 27 700 and 34 200 resp. 1001
Sven
Erik G . Nilsson, Bengt Knave and Hans E. Persson
so-called phagosomes are formed. Such inclusion bodies had been observed earlier (Dowling & Gibbons 1962; Bairati & Orzalesi 1963) but the exact nature of the structures was somewhat unclear. It appears that the rod outer segment tip (about 5-40 disks) can be separated from the major part of the outer segment either by protrusion of pigment epithelial processes, which latter then completely engulf the disks and withdraw them into the cell body of the pigment epithelial cell (Spitznas & Hogan 1970 (human)) or by shedding of the stack of disks primarily by infolding of the outer segment plasma membrane (Young 1971 (rhesus monkey)). Later it has been shown that also cone outer segments may be ensheathed by pigment epithelial processes (Steinberg & Wood 1974 (cat); Steinberg, Wood & Hogan 1977 (human)) and that also the tip of cone outer segments can be engulfed into the pigment epithelium by protrusion of pigment epithelial processes (Hogan, Wood & Steinberg 1974 ; Steinberg, Wood & Hogan 1977 (human)). It has recently been shown that shedding of outer segment disks is a cyclic daily process, where rods predominantly shed their disks at the onset of the light period (LaVeil 1976 (rat), Basinger, Hoffman & Matthes 1976 (frog)), and where shedding from cones occurs mainly at the beginning of the dark period (Young & O’Day 1977 (goldfish)). In the present investigation the animals were dark-adapted for about 8 h prior to the experiment. The phagosomes then undergo degradation through a series of stages (Ishikawa & Yamada 1970; Spitznas & Hogan 1970; Johnson 1975). The phagosomes labelled Ph 1-Ph 4 in Figs. 5-7 of the present investigation are considered to represent successive stages in such a degradation. The disintegration of the content seems to be accomplished by means of lytic enzymes, such as acid phosphatase (Ishikawa & Yamada 1970; Marshall 1970; Hollyfield & Ward 1974). There are also morphological evidence supporting the idea that these enzymes may originate in the Golgi compIex, and that the enzymes then are stored in lysosomal granules, which interact with the young phagosome (Ishikawa & Yamada 1970). The lightly stained granules (X) seen in Fig. 7 could possibly represent such lysosomes. However, it cannot be excluded that they are instead final stages of phagosomal degradation. The presence of cathepsin D (a proteolytic enzyme) in the pigment epithelium indicates that also this enzyme may take part in the digestion of the phagosomes (Hayasaka, Hara & Mizuno 1975). Certain hereditary retinal degenerations can be explained by a defect in the phagocytic mechanism described above (Bok & Hall 1969, 1971; Herron, Riegel, Myers & Rubin 1969; Herron, Riegel Brennan & Rubin 1974). The oil droplets of the frog pigment epithelium are known to concentrate vitamin A (the precursor of retinal) and fatty acids (Young & Bok 1970; Bibb &
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Ultrastructure
of
Sheep Pigment Epithelium
Young 1974). It seems most likely that the lipid droplets of the sheep pigment epithelium correspond to the frog oil droplets. It cannot be excluded that part of the lipid material is derived from outer segment degradation products. Besides the renewal of rod outer segments by means of membrane replacement, both rods and cones renew their membranes also by diffuse molecular replacement. This is true for proteins as well as for lipids. It appears that the pigment epithelium plays an important role in lipid metabolism of outer segment membranes (Bibb & Young 1974; Young 1974). The pigment epithelium is part of the “blood retina barrier”. Its plasma membranes possess certain passive and active (selective) transport mechanisms (Steinberg & Miller 1973; Miller & Steinberg 1977a,b). The extensive infoldings of the basal plasma membrane somewhat resemble those of the kindney tubular cells, where extensive transport is known to take place. Attachment zones (junctional complexes) connected the lateral membranes of adjacent cells. Close to the apical surface of the cells they are of the tight junction type (zonulae occludentes), and just basally to the tight junctions zonulae adherentes are present (Leure-duPree 1968). It was clearly shown by Peyman, Spitznas & Straatsma (1971) that diffusion of peroxidase was abruptly stopped at the zonulae occludentes in the intercellular spaces of the pigment epithelium. Thus, the tight junctions together with the selective membrane transport mechanisms can effectively block free flux between the choroidal capillaries and the neuroretina. The difference in transport and permeability properties between the apical and the basal membranes of the pigment epithelial cells gives rise to the trans-pigment epithelial potential (Steinberg & Miller 1973; Miller & Steinberg 1977a,b), which appears to be the major component of the standing potential of the eye (Noel1 1954; Heck & Papst 1957; Gouras 1969). Toxic agents that affect the properties of the basal and/or apical membranes are thus very likely to influence the trans-pigment epithelial potential and hence the standing potential of the eye. Furthermore, it is to be expected that the c-wave of the ERG, which is generated mainly across the apical membrane of the pigment epithelium (Steinberg, Schmidt & Brown 1970; Schmidt & Steinberg 1971; Steinberg & Miller 1973; Oakley & Green 1976), is also affected at the same time. Sodium iodate is a toxic agent of this kind, which will be shown in the following papers (Nilsson, Knave & Persson 1977a,b). In conclusion it can be said that the pigment epithelium is a most important barrier layer as well as a very metabolically active layer interposed between the receptor cells and the circulation, and upon which the receptor cells are highly dependent.
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References Babel J. (1970) The retina. In: Bischoff A,, Ed. Ultrastructure of the Peripheral Nervous System and Sense Organs, pp. 339-441. G. Thieme Verlag, Stuttgart. Bairati A., Jr. & Orzalesi N. (1963) The ultrastructure of the pigment epithelium and of the photoreceptor pigment epithelium junction in the human retina. J . Ultrastruct. Res. 9, 484-496. Basinger S., Hoffman R. & Matthes M. (1976) Photoreceptor shedding is initiated by light in the frog retina. Science 194, 1074-1076. Bok D. & Hall M. 0. (1969) The etiology of retinal dystrophy in RCS rats. Invest. Ophthal. 8, 649-650. Bok D. & Hall M. 0. (1971) The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J . Cell Biol. 49, 664-682. Bibb C. & Young R. W. (1974) Renewal of fatty acids in the membranes of visual cell outer segments. J . Cell Biol. 61, 327-343. Dowling J. E. & Gibbons I. R. (1962) The fine structure of the pigment epithelium in the albino rat. J. Cell Biol. 14, 459-474. Gouras P. (1969) Clinical electro-oculography. In: Straatsma B. R., Ed. The Retina, pp. 565-581. University of California Press, Berkeley & Los Angeles. Grignolo A,, Orzalesi N. & Calabria G. A. (1966) Studies on the fine structure and the rhodopsin cycle of the rabbit retina in experimental degeneration induced by sodium iodate. E x p . Eye Res. 5, 86-97. Hayasaka S., Hara S. & Mizuno K. (1975) Distribution and some properties of cathepsin D in the retinal pigment epithelium. Exp. Eye Res. 21, 307-313. Heck J. & Papst W. (1957) Uber den Ursprung des comeo-retinalen Ruhepotentials. Bibl. ophthal. (Basel) 48, 96-107. Herron W. L., Riegel B. W., Myers 0. E. & Rubin M. L. (1969) Retinal dystrophy in the rat - A pigment epithelial disease. Invest. Ophthal. 8, 595-604. Herron W. L., Riegel B. W., Brennan E. & Rubin M. L. (1974) Retinal dystrophy in the pigmented rat. Invest. Ophthal. 13, 87-94. Hogan M. J., Wood I. & Steinberg R. H. (1974) Phagocytosis by pigment epithelium of human retinal cones. Nature (Lond.) 252, 305-307. Hollyfield J. G. & Wa r d A. (1974) Phagocytic activity of the pigmented retinal epithelium. 111. Interaction between lysosomes and ingested polystyrene spheres. Invest. Ophthal. 13, 1016-1023. Ishikawa T. & Yamada E. (1970) The degradation of the photoreceptor outer segment within the pigment epithelial cell of the rat retina. J . Electron Microscopy 19, 85-99. Johnson N. F. (1975) Phagocytosis in the normal and ischaemic retinal pigment epithelium of the rabbit. Ex@. Eye Res. 20, 97-107. Knave B., Persson H. E. & Nilsson S. E. G. (1974) The effect of barbiturate on retinal functions. 11. Effects on the c-wave of the electroretinogram and the standing potential of the sheep eye. Acta physiol. scand. 91, 180-186. LaVail M. M. (1976) Rod outer segment disk shedding in rat retina: relationship to cyclic lighting. Science 194, 1071-1073. Leure-duPree A. (1968) Ultrastructure of the pigment epithelium in the domestic sheep. Amer. J . Ophthal. 65, 383-398.
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Ultrastructure of Sheep Pigment Epithelium Marshall J. (1970) Acid phosphatase activity in the retinal pigment epithelium. Vision Res. 10, 821-824. Miller S. S. & Steinberg R. H. (1977a) Passive ionic properties of frog retinal pigment epithelium. In preparation for publication. Miller S. S. & Steinberg R. H. (197713) Active transport of ions across frog retinal pigment epithelium. In preparation for publication Nilsson S. E. G. (1964) Receptor cell outer segment development and ultrastructure of the disk membranes in the retina of the tadpole (Xana pipiens). /. Ultrastruct. Res. 11, 581-620. Nilsson S. E. G., Knave B., Lunt T. & Persson H. E. (1973) The morphology of the sheep retina. 11. The inner nuclear layer, the ganglion cells and the plexiform layers. Acta ophthal. (Kbh.) 51, 612-627. Nilsson S. E. G., Knave B. & Persson H. E. (1977a) Changes in ultrastructure and function of the sheep pigment epithelium and retina induced by sodium iodate. 11. Early effects. Acta ophthal. (Kbh.) 55, 1007-1026. Nilsson S. E. G., Knave B. & Persson H. E. (197713) Changes in ultrastructure and function of the sheep pigment epithelium and retina induced by sodium iodate. 111. Delayed effects. Acta ophthal. (Kbh.) 55, 1027-1043. Nilsson S. E. G., Knave B., Persson H. E. & Lunt T. (1973) The morphology of the sheep retina. I. The receptor cells and the pigment epithelium. Acta ophthal. (Kbh.) 51, 599-611. Noell W. K. (1953) Studies on the electrophysiology and the metabolism of the retina. Project report U . S. Air Force SAM Project No. 21-1201-0004, pp. 1-122. Randolph Field, Texas. Noell W. K. (1954) The origin of the electroretinogram. Amer. 1. Ophthal. 38, 78-93. Noell W . K. (1963) Cellular physiology of the retina. /. o p t . SOC.Amer. 53, 36-48. Oakley I1 B. & Green D. G. (1976) Correlation of light-induced changes in retinal extracellular potassium concentration with c-wave of the electroretinogram. /. Neurophysiol. 39, 1117-1 133. Peyman G. A., Spitznas M. & Straatsma B. R. (1971) Peroxidase diffusion in the normal and photocoagulated retina. Invest. Ophthal. 10, 181-189. Schmidt R. & Steinberg R. H. (1971) Rod-dependent intracellular responses to light recorded from the pigment epithelium of the cat retina. /. Physiol. (Lond.) 217, 71-91. Spitznas M. & Hogan M. J. (19iO) Outer segments of photoreceptors and the retinal pigment epithelium. Arch. Ophthal. (Chicago) 84, 810-819. Steinberg R. H. & Miller S. (1973) Aspects of electrolyte transport in frog pigment epithelium. Exp. Eye Res. 16, 365-372. Steinberg R. H., Schmidt R. & Brown K. T. (1970) Intracellular responses to light from cat pigment epithelium: origin of the electroretinogram c-wave. Nature (Lond.) 227, 728-730. Steinberg R. H. & Wood I. (1974) Pigment epithelial cell ensheathment of cone outer segments in the retina of the domestic cat. Proc. roy. SOC.Lond. B 187, 461-478. Steinberg R. H., Wood I. & Hogan M. J. (1977) Pigment epithelial ensheathment and phagocytosis of extrafoveal cones in human retina. Philosoph. Trans. R. SOC.Lond., B. 277, 459-474. Suyama T. (1965) Electron microscopic study on experimental retinal degeneration induced by sodium iodate injection. Acta SOC. ophthal. jab. 69, 440-460.
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Sven Erik G. Nilsson, Bengt Knave and Hans E. Persson Young R. W. (1967) The renewal of photoreceptor cell outer segments. /. Cell Biol. 33, 61-72. Young R. W. (1971) Shedding of discs from rod outer segments in the rhesus monkey. /. Ultrastruct. Res. 34, 190-203. Young R. W. (1974) Biogenesis and renewal of visual cell outer segment membranes. Ex#. Eye Res. IS, 215-223. Young R. W. & Bok D. (1969) Participation of the retinal pigment epithelium in the rod outer segment renewal process. /. Cell Biol. 42, 392-403. Young R. W. & Bok D. (1970) Autoradiographic studies on the metabolism of the retinal pigment epithelium. Invest. Ophthal. 9, 524-536. Young R. W. & Droz B. (1968) The renewal of protein in retinal rods and cones. /. Cell Biol. 39, 169-184. Young R. W. & O’Day W. T. (1977) Personal communication.
Authors’ addresses: Prof. Sven Erik G. Nilsson, Department of Ophthalmology, University of Linkoping, University Hospital, S-581 85 Linkoping, Sweden. Assoc. Prof. Bengt Knave, National Board of Occupational Safety and Health, S-100 26 Stockholm 34, Sweden. Assistant Prof. Hans E. Persson, Department of Clinical Neurophysiology, Karolinska Hospital, S-104 01 Stockholm 60, Sweden. Communications to Prof. Sven Erik Nilsson.
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