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Feline retinal ultrastructural changes induced by a high cystine diet
NUTRITION RESEARCH, Vol. 10, pp. 1385-1400,1990 0271-5317/90 $3.00 + .00 Printed in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.
FELINE RETINAL ULTRASTRUCTURAL CHANGES INDUCED BY A HIGH CYSTINE DIET H. Imaki, Ph.D. and J.A. Sturman, Ph.D. Department of Developmental Biochemistry Institute for Basic Research in Developmental Disabilities 1050 Forest Hill Road, Staten Island, NY 10314, USA
ABSTRACT Cats fed a high cystine diet containing no taurine developed extreme signs of neurotoxicity and either died or had to be killed within 7 months. The presence of 0.05% taurine in the diet greatly ameliorated the neurotoxic effects of the cystine. Many developed retinal ultrastructural changes which were quite different from those previously described for cats on taurine-free diets. The most striking changes were reductions in the number of cells and extensive swelling of some remaining cells in the inner nuclear layer and in the ganglion cell layer. The most severely affected cells were amacrine cells, in contrast to photoreceptor cells in cats fed the taurine-free diet. The changes resemble those reported in other species caused by excitotoxic amino acids. Key words: Cat retina - Cystine neurotoxicity - Retinal ultrastructure - Taurine deficiency INTRODUCTION Cats are dependent on dietary source of tanrine, a sulfur-containing amino acid abundant in meat and animal tissues but poor in plants and vegetables. Earlier studies established that cats fed taurine-deficient diets develop a number of abnormalities including visual dysfunction, attributable to progressive degeneration of photoreceptor cells in the central retina, and disorganization of tapetum lucidum, a triangular sheet of light-reflecting cells behind the retina (1-14). Recently we demonstrated that weanling (8-week-old) kittens born to and nursed by taurine-deprived cats sustain similar degenerative changes both in the retina and tapetum (15-16) in addition to delayed, or abnormal, patterns of development in the cerebellum and visual cortex (17-18). Although cats have a limited biosynthetic capacity for taurine, they do have some activity of hepatic cysteinesulfinic acid decarboxylase, and can convert limited amounts of cyst(e)ine to taurine (19). We recently concluded a study in which a group of cats were fed with a synthetic diet enriched with cystine in order to test whether effects of taurine deficiency could be ameliorated by supplying an excess of its biosynthetic precursor. It has been reported that dietary cysteine (and methionine) supplementation in smaller amounts than used here was ineffective in restoring the reduced ERG response or delayed cone bwave implicit time in taurine-deprived cats (6,20), and that cysteine and some other sulfurcontaining amino acids administered orally or parenterally to young rodents were extremely neurotoxic (21-29). Here we report the light and electron microscopic observations made in the retinas of cats fed the cystine-rich diet with or without an adequate amount of taurine. Preliminary morphological findings and other biological and biochemical results obtained from the same set of cats have been reported previously (30). 1385
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MATERIALS AND METHODS Mature female domestic cats (1 - 3 years of ase) raised in the IBR colony and vaccinated against rhinotracheitis, panleukopenia, calicl virus [FVR-C-P (MLV) PitmanMoore, Washington Crossing, NJ] were fed ad libitum a completely defined, synthetic diet (taurine-free) containing 5% cystine with or without 0.05% taurine (BioServe, Frenchtown, NJ). The basal defined synthetic diet contained (grams/100g): casein (vitamin-free), 43.0; chicken fat, 20.0; dextrin, 13.5; sucrose, 13.5; salt mix (see below), 6.4; cellulose, 2.4; vitamin mix (see below), 0.6; L-cystine, 0.3; choline chloride, 0.3. The salt mix contained (in grams/kilogram): potassium phosphate dibasic, 328; calcium carbonate, 290; sodium chloride, 162; magnesium sulfate, 99; calcium phosphate dibasic, 73; magnesium oxide, 32; ferric citrate, 13; manganese sulfate, 1.22; zinc chloride, 0.91; cupric sulfate, 0.29; potassium iodide, 0.077; chromium acetate, 0.044; sodium fluoride, 0.023; sodium selenite, 0.0043. The vitamin mix contained (in grams-kilogram): dextrin, 857; inositol, 100; D,L-a-tocopheryl acetate (500 IU/g), 20; niacin amide, 8.0; calcium pantothenate, 5.0; retinyl acetate (500,000 IU/g), 5.0; riboflavin, 1.60; cholecalciferol (200,000 IU/g), 1.25; thiamin-HC1, 0.80; pyridoxine.HC1, 0.80; folic acid, 0.80; menadione, 0.10; biotin, 0.04; cyanocobalamin, 0.03. The cats were autopsied as soon as found dead, or killed by cardiac puncture under Nembutal anesthesia when they were moribund, or at the end of experimental period of 12 months. The right eye from each cat was fixed by perfusion by injecting 3% glutaraldehyde in 0.1 M Sorensen's phosphate buffer (pH 7.4), with a needle inserted in the opposite pole of equator for drainage. The posterior eye cup, cut radially into 12 sectors around the area centralis, was postfixed with 1% OsO a in 0.1 M Sorensen's buffer, dehydrated in an ascending series of acetone and embedded in .El?on 812. Light micrographs were taken from 0.75 urnthick sections stained with 1% toluidlne blue on a Zeiss Photomicroscope III using Kodak Panatomic X film. Electron micrographs were taken from thin sections, gray-silver in interference colors, mounted on copper grids and stained with uranyl acetate and lead citrate, in a Philips EM 300 operated at 80 kv, using Kodak EM film (4489). Micrographs taken from transverse sections otretinas from cats fed the diets described above were compared with those from cats fed the control diet which consisted of the same synthetic formula supplemented with 0.05% taurine but not with cystine. RESULTS All the 9 cats fed the 5% cystine, taurine-free diet developed fulminant neurological symptoms which included lethargy, rigidity, absence and epileptic seizures, and either died or were killed when they clearly appeared dying. The mean survival time of these cats was 5.2 • 2.0 months, with the shortest and the longest being 1.5 and 7 months, respectively, from the start of the diet. Of the 9 cats fed the 5% cystme plus 0.05% taurine diet, 4 died after showing minimal symptoms, such as slight lethargy and unsteady postures, with the mean survival time of 3.5 • 1.3 months, while the others outlived the experimental period without showing any obvious symptoms. None of the animals, while alive, exhibited apparent visual dysfunction as judged from their behavioral patterns. Light microscopic examination revealed small, yet noticeable, reductions in the number of ceils in the inner nuclear and ganglion cell layers of retinas from cats fed the diet containing 5% cystine, either with or without 0.05% taurine, in comparison to the retinas from cats fed the control diet (Figs. la, b and c). The overall thickness of such retinas, nevertheless, appeared unchanged or even increased, due largely to the massive swelling of the neuropil in the inner plexiform layer, and sometimes in the outer plexiform layer as well. In addition, many cells occurring in the inner part of the inner nuclear layer and some in the ganglion cell layer also were greatly swollen, while others appeared shrunken or compressed. Occasionally, the inner nuclear layer contained what appeared to be large spaces between cells. These changes usually were widespread and no appreciable difference in form or magnitude could be found between different areas of the same retina, although some variation existed between retinas from different cats. The number, structure and organization of photoreceptors, as well as cells forming retinal endothelium, epithelium or tapetum lucidum were comparable to the corresponding
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cells of cats fed the control diet. The retina from one of the cats fed the taurine-free diet containing 5% cystine showed a great reduction in the number of photoreceptor cells (completely absent from the superior-nasal sectors, and 1/3 to 1/2 o f the normal in the inferior-temporal sectors), and unusually swollen tapetal ceils in the mid-outer layers which surrounded more compact and denser cells of the inner tapetum. Cells in the inner retina of this cat exhibited relatively normal morphology, except for their somewhat undifferentiated disposition and lack of the typical layered arrangement (Fig. ld). As might be expected, the retinas from cats found dead exhibited massive swelling and generalized disorganization of nearly every structure as a result of post-mortem decomposition, with a notable exception of tapetal ceils which usually remained intact (Fig. le). By electron microscopy, the most severely affected cells in the retinas of cats fed the cystine-rich diets were identified as amacrine cells which occupied the inner half of the inner
FIG. 1 Light micrographs of cat retinas in transverse section through area centralis. Toluidine blue, x 400. a: Retina from a cat fed the basal synthetic diet containing 0.05% taurine (control); b: Retina from a cat fed the synthetic diet containing 0.05% taurine and 5% cystine; c: Retina from a cat fed the synthetic diet containing 5% cystine, but taurine-free; d: Retina from a different cat fed the same diet as c; e: Retina from a third cat fed the same diet as c. GCL: Ganglion cell layer; IPL: Inner plexiform layer; INL: Inner nuclear layer; OPL: Outer plexiform layer; ONL: Outer nuclear layer; IS and OS: Inner and outer segments of photoreceptors, respectively; RPE: Retinal pigmented epithelium; T: Tapetum lucidum.
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nuclear layer and a substantial portion of the ganglion cell layer as "displaced" amacrine cells. The normal amacrine cells observed in control cat retinas were characterized by relatively large, often deeply lobated, nuclei with homogeneous finely granular nucleoplasm, and relatively abundant perikarya rich in granular cisternae of endoplasmic reticulum (ER), rosettes of free ribonucleoprotein (RNP) particles, clusters of Golgi cisternae and mediumsize mitochondria, distributed evenly throughout the cytoplasm (Fig. 2a). The majority of amacrine cells found in the affected retina, in contrast, exhibited somewhat bloated nuclei with coarse clumps of chromatin in rather pale or bleached nucleoplasm, and grossly distended perikarya which contained greatly expanded spherical mitochondria, vacuolar cisternae of ER and Golgi, and small numbers of RNP particles, often clustered in the perinuclear region of otherwise sparse cytoplasm (Fig. 2b). In addition, varying numbers of large vacuoles containing membraneous or flocculent materials were found m peripheral reglon.s of cytoplasm. Both bipolar and horizontal cells of retinas with many degenerative amacrme cells usually showed similar but less pronounced ultrastructural changes. The nuclei of these cells, which in the normal retinas showed slightly undulating contours and small clumps of chromatin scattered in moderately dense nucleoplasm (Fig. 2a), displayed denser and coarser chromatin patterns and irregular surface indentations, often juxtaposed with greatly expanded mitochondria which generally are slightly larger than those in the amacrine cells, and irregularly shaped vacuoles similar in appearance to those found in degenerating amacrine cells (Fig. 2b). Muller cells, whose small cell bodies were located mid-level of the inner nuclear layer, displayed homogeneously electron-dense nuclear and toplasmic matrices in the retinas of cats fed 5% cystine (Fig. 2b) as those of the control cats Fig. 2a). The more angular profiles of these cells in the former appeared to be a result of excessive swelling and deformation of neighboring cells. The ganglion cells located in the ganglion cell layer of the control cat retinas were notable for their large nuclei and prominent nucleoli, and enormous cytoplasm containing extensive arrays of Golgi complexes, granular cisternae of ER and clusters of RNP particles. There were at least 2 morphological subtypes readily identified: the more common type was usually spherical in overall shape, and relatively electron-lucent, appearing more like amacrine cells than the second type, which exhibited complex surface contours and unusually high matrix densities in the nucleus and cytoplasm (Figs. 3a and 4a). Both subtypes of ganglion cells in the retinas of cats fed 5% cystine exhibited degenerative features, but the first type, which often exhibited heterochromatic nucleoplasm and loss of many cytoplasmic organelles, as noted in degenerating amacrine cells, seemed to be more severely affected than the second type, whose structural changes usually were limited to swollen mitochondria and dilated cisternae of ER and Golgi complexes (Figs. 3b and 4b).
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FIG.2, Electron micrographs of cat retinas in transverse section through the inner nuclear
layer, a: Retina trom a cat fed the control diet, showing fine structures of normal amacrine cells (Am) with relatively large, electron-lucent perikarya, horizontal cells (Ho) with rather large ovoid mitochondria and somewhat heterochromatic nuclei, bipolar cells (Bi) with denser nucleoplasm and scanty perinuclear cytoplasm and Muller cells (Mu) with homogeneously dense nuclear and cytoplasmic matrices. Vitreous is toward the top. b: Retina from a cat fed the diet contaimng 5% cystine but no taurine, showing degenerative profiles of amacrine (Am) horizontal (Ho) and bipolar (Bi) cells, with spherical, swollen mitochondria and vacuoles of varying size containing amorphous flocculent material or membraneous inclusions. Note the large expanse of amacrine cell cytoplasm devoid of organelles, while the neighboring Muller cell (Mu) remains compact and appears intact. Despite the irregular surface configuration and occasional intercellular spaces, the majority of cells in the inner nuclear layer and the ganglion cell layer of the affected retina appeared to maintain remarkably intact and intimate intercellular contacts with one another, comparable to those observed in normal retinas (Figs. 2a and b, 3a and b). Similarly close and normal intercellular contacts and junctional structures were observed between various neuronal processes in the inner plexiform layer as well as in the outer plexiform layer of
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FIG. 3
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FIG.3. Electron micrographs of cat retinas in transverse section through parts of ganglion cell
and inner plexiform layers, a: Retina from a cat fed the control diet, illustrating the fine structure of a normal ganglion cell (G1) with a large ovoid nucleus (n) and ample c~(toplasm filled with numerous granular cisternae of endoplasmic reticulum, free nbonucleoprotein particles and well-developed Golgi complexes, Muller cell processes (Mu) filled with dense skeins of filaments, and synaptic processes of neurons forming the inner plexiform layer (IPL) En: endothelial cell; Lu: Retinal vascular lumen; P: Pericyte. b: Retina from a cat fed the diet containing 5% cystine and 0.05% taurine, showing a degenerating ganglion cell consisted of an expanded nucleus with clumps of chromatin granules and edematous cytoplasm contaimng scattered clumps of vacuolar mitochondria and ER. The second ganglion cell (G2) contains clear vacuoles of varying size and shape in addition to swollen mitochondria, in the dense cytoplasm.
retinas from cats fed 5% cystine as in those fed the control diets (Figs. 3c and 4, 4a and b). However, virtually all postsynaptic processes found in the inner plexiform layer in the former retinas were extremely swollen and empty-looking, while presynaptic and Muller cell processes appeared unaffected, except for some mildly swollen mitochondria (Fig. 4d). The synaptic elements in the outer plexiform layer of these retinas usually appeared less affected than those in the inner plexiform layer, in some cases being indistinguishable from those in control retinas (Figs. 5a). Some retinas from cats fed the taurine-free diet containing 5% c3,stine exhibited generalized swelling of photoreceptor cytoplasm, including synaptic, axonal and somal elements (Fig. 5b) as well as inner and outer segments (Fig. 5c), whereas others had a relatively normal appearance (Fig. 5d). The retinal epithelial cells were unaffected by 5% cystine in the diet in most cats, but those in some cats fed the taurine-free variety tended to display slightly denser cytoplasmic matrix, possibly as a result of compression by an increased number of large vacuoles in such cells. Phagosomes, lysosomes and lipofuscin granules also were more frequently encountered in some of these cells (Fig. 5d). The tapetum of cats fed the diets containing 5% cystine exhibited normal fine structure except in one cat (on the taurine-free diet) which revealed unusually compact tapetal cells in the inner layers and swollen cells, containing some irregularly-spaced or loosely arranged tapetal rods, in the mid-outer layers (Figs. 6a and b). The retinal epithelial cells of this particular cat, which lacked photoreceptor cells entirely in some regions, were found to be extremely attenuated, and completely devoid of phagosomes or lipofuscin granules and, in some areas, apical microvilli as well (Fig. 6a). In nearby areas of the same retina were fine finger-like microvilli of the epithelial cells interdigitating with equally slender extensions of Muller cells, and giant lysosomes containing diverse inclusion bodies and occurring in the relatively thick and rich cytoplasm of an epithelial cell (Fig. 6c). Cells found vitread to the Muller cells in this retina appeared less differentiated and metabolically less active and were harder to identify even though none of these cells exhibited the obvious pathological features noted in the retina of the majority of cats fed the same diet (5% cystine, no taurine). Taurine concentrations in selected tissues from the animals used in the present study are summarized in Table 1 along with data obtained previously in corresponding tissues from similar groups of cats fed the same synthetic diet containing either 0 or 0.05% taurine, but no excess cystine, for comparison. For each pair of data sets, the taurine concentrations in tissues from taurine-supplemented cats were significantly greater than the same tissues from unsupplemented cats (with a single exception, lung o f animals receiving the 5% cystine supplement). Of note with re~ard to the ultrastructural results presented here was that retinal taurine concentration in cats supplemented with 0.05% and 5% cystine was significantly smaller than in cats supplemented with 0.05% taurine alone.
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FI6.4. Electron micrographs of cat retinas in transverse sections through the ganglion cell layer a: Retina from a cat fed the control diet, showing the normal ultrastructure of perinuclear region of a "dense" ganglion cell with a prominent nucleolus (no) occupying the middle of nucleus (n), which exhibit homogeneous nucleoplasm, and gently undulating nuclear envelope. The cytoplasm is occupied by prominent granular cisternae of ER, medium-size mitochondria, and well-developed Golgi complex, b: Corresponding area of a . . . . den se n ganghon cell from a cat fed the. diet contannng 5% cystme and 0.05% taurine. Note large intracellular spaces which may represent greatly dilated cisternae of ER or Golgi complex. The appearance of nucleus (n) including its centrally located nucleolus (no) is relatively unchanged. It
Electron micrographs of cat retinas in transverse section through the inner plexiform layer, c: Retina from a cat fed the control diet, showing complex arrangement of synaptic processes containing variable numbers of synaptic vesicles, neurofilaments and tubules, in addition to normal-looking mitochondria (m). d: Retina from a cat fed the diet containing 5% cystine and 0.05% taurine, showing greatly swollen and deformed postsynaptic processes in intimate contact with relatively intact presynaptic processes loaded with synaptic vesicles. Many mitochondria (m) of presynaptic process, however, appear swollen as those in the postsynaptic processes. DISCUSSION The results reported here werepart of an experiment initiated in an attempt to increase taurine biosynthesis in cats fed a taurine-free diet by supplying an excess 5%) of its natural metabolic precursor, cystine. Although there was no obvious mmediate effect of this diet and cats maintained their normal dietary intake, after several months the first cats started to exhibit acute neurotoxic symptoms. A major feature was the sudden onset and the rapid progression to a moribund state or death, usually within 48 hours. All nine cats fed the 5% cystine, taurine-free diet succumbed in this fashion, the longest survival time being 7 months from the start of the diet. This is the first reported incidence of neurotoxicity resultin~ from excess dietary cystine, although cats have not previously been used in such studies. It should be noted here that cats fed the same synthetic diet with or without 0.05% taurine, but not containing 5% cystine, did not show any overt neurotoxic symptoms, even after several years on such diets (12, Sturman, unpublished observations on cats fed these diets for more than 4 years). Several recent reports had examined the effect of feeding a diet containing 5% cystine to rats, and although this resulted in increased cholesterol levels and cholesterol biosynthesis, no adverse physical effects were noted (31-34). A recent report indicated that the addition of 1% cystine to the diet of rats fed 3.5% histidine resulted in hepatomegaly and a decreased liver cholesterol content (35). The morphological changes observed in the retinas of a majority of cats fed the cystine-rich diets were comparable to those described in the retinas of young rodents treated with glutamate or certain other acidic amino acids, collectively referred to as "excitotoxic", as their neurotoxic effects appear to be linked to their neuroexcitatory function (21). Earlier studies established that this type of retinal lesions were characterized by selective, yet randomized and widely disseminated destruction of the inner retina: the acute phase of response includes massive swelling of post-synaptic, or post-synaptic, or dendrosomatic, elements of neurons in the inner nuclear layer and ganglion cell layers, followed by extensive degeneration of intracellular organelles and nuclear pyknosis, as described in detail in the retinas of infant rodents treated with glutamate and other amino acids including L-cysteine (24-26,29,36). The delayed, or long-term, response involves reductions in the number of the same types of neurons
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FIG. 5. Electron micrographs of cat retinas in transverse section through photoreceptor and retinal epithelial cells, a: Synaptic region between photoreceptor cells and neurons of the inner retina from a cat fed the control diet, showing normal appearance of a pedicle (p) with typically large, but undisturbed, mitochondria and distinctive synaptic ribbons, several spherules containing smaller, normal mitochondria, and parts of cell body of inner-most rod cells (R). b: Synaptic region between photoreceptor cells and neurons of the inner retina from a cat fed the diet contaimng 5% cystine and no taurine, showing greatly expanded rod cell (R) cytoplasm including spherucles (s). c: Distal parts of rod photoreceptors of the same cat as b, showing slightly swollen mitochondria in the inner segments (is) and ruffled, wavy disc membranes in the outer segments (os). d: Distal portions of photoreceptors and retinal pigment epithelium (RPE) from a different cat fed the same diet as b (5% cystine, taun~ne-free), showing the fine structure of cone and rod outer segments (cos and ros) that are indistinguishable from those of cats fed the control diet. T: Tapetum. and of myelinated axons in the optic nerve, which are especially pronounced when experimental animals were treated early in development either with glutamate (37-40) or cysteine (27). Although there are indications that cysteine itself might be responsible for the cytotoxicity (41,42), cysteine administered systemically is believed to be converted locally to cysteinesulfinic acid or cysteic acid to act as a neuroexcitant, to account for its somewhat delayed, but more widespread pattern of pathological response, especially in the brain, where a neutral amino acid such as L-cysteine gains entry into several regions while acidic amino acids do not (21,22,25,26). The excess cystine ingested by the cats might be converted to similar acidic amino acids before it elicited structural damages observed in the retina which are characterized by both the acute and chronic patterns of response, i.e. massive swelling of some neurons and dendrites, and reductions in number of the same type of neurons, at the same time. This is most likely due to the fact that these cats were completely dependent on the cystine-enriched diet for prolonged periods, instead of a single or short-term exposure that animals in the other studies were subjected. It is possible that the differential susceptibility to the high cystine diets observed among different neurons in the cat retina may simply be due to differences in physiological and metabolic states of the cell: amacrine and ganglion cells in general might appear to sustain greater damage than bipolar and horizontal cells because they are associated with greater numbers of ER and mitochondria which are particularly vulnerable and respond by excessive swelling and disintegration. The reductions in the number of cells in the inner retina of these cats may be the result of such a differential susceptibility of individual neurons. Thus, the number of cells in the inner nuclear layer showed a relatively small decrease since amacrine cells comprise a small proportion of cells among other more numerous types, while the reduction in the number of cells in the ganglion cell layer was more obvious due to the fact that the latter layer is mostly composed of ganglion and amacrine cells. A greater reduction in amacrine cells, relative to ganglion cells, contributed to the overall reduction, as these "displaced" amacrme cells form some 80% of the total neuron population of the cat ganglion cell layer (43-44). These findings in cat retinas regarding the type and degree of neurons affected by excess cystine agree with the earlier observations by Cohen (38), who reported that number of amacrine and ganglion cells are more severely reduced than bipolar cells in the retina of mice treated with MSG. However, the appearance of surviving ganglion cells in cats fed a high-cystine diet were quite variable depending on the morphological subtypes and individual cells, contrary to the observation in the above mouse retinas in which they reportedly appeared "normal except for their smaller size". As repeatedly demonstrated in other animals treated with various excitotoxins postnatally, Muller cells and other non-neuronal cells in the retinas of cats fed the high cystine diet did not show any appreciable structural abnormalities. The photoreceptor cells of these cats (except one), likewise, were virtually indistinguishable from those found in cats fed the control diet. The retinal lesions induced by excessive dietary cystine, therefore, are
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FIG. 6 Electron micrographs of the central-temporal retina from a cat fed the taurinefree diet containing 5% cystine, a: Transverse section through the middleouter levels of retina and inner tapetum lucidum (T), showing parts of several neurons, in the upper half, a Muller cell (Mu) containing dense clumps of amorphous debris (*) and directly in contact with the retinal pigment epithelium (RPE). Photoreceptor cells are completely missing in this region. b: Transverse section through mid-outer levels of tapetum, showing several cells with loosely arranged tapetal rods, in comparison to those in normal tapetal cells, or those in the inner layers, as seen in a. The nucleus (n) is normal looking, c: Transverse section through retina - epithelial cell junction showing a dense aggregate (*) in a Muller cell cytoplasm, tightly interdigitating processes of Muller cell and microvilli of epithelial cell, which contains a large hagosome with heteromorphic inclusions and typically large mitochondria m).
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fundamentally different from those observed in the taurine-deprived cats and kittens studied previously. The retinas of some cats fed the taurine-free cystine-enriched diet,
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however, showed photoreceptors with subtle ultrastructural changes, such as slight loosening of disc membranes and ruffled plasma membranes, which were much less severe than the changes described in the retinas of cats fed a taurine-free diet without cystine. This ma X be due to the marginal increases in tissue taurine concentration, but it seems more hkely the result of the shorter feeding duration. The cause for the drastic reduction in the number of photoreceptor cells observed in one of the cats fed the same diet is not dear, but it may be due to genetic as well as nutritional factor as none of the other retinas examined here exhibited such extreme changes. Such features resemble those described in the retinas of certain strains of Abyssinian cats with inherited photoreceptor degeneration (45-47). The supplementation of the high cystine diet with adequate taurine seems to prevent certain degenerative changes in the photoreceptor cells but not neurons in the inner retinas of cats. Other neurotoxic effects of cystme and premature death observed in the cats studied here appeared to be significantly reduced or delayed by the inclusion of adequate taurine m the diet, supportin.g the suggestions that taurine plays a neuroprotective role against the excxtotoxac damages in mammalian systems either in viv0 or in vitro (48-55). TABLE 1 Concentration of taurine in selected tissues of cats fed the basal synthetic diet with or without additional suplements of taurine (0.05%) or cystine (5%)
Taurine alone a
None a
Cystine alone b
Taurine b + cystine
moles/g wet weight Retina Liver Heart Adrenal Kidney Lung Cerebellum Occipital lobe Spinal cord
42.8_+ 4.6 13.1_+ 2.2 12.6_+ 3.1 11.4 _+3.2 9.6_+ 1.0 7.8_+ 2.2 3.8 -+ 0.8 2.3+ 0.8 1.3_+ 0.4
15.0_+ 3.0 0.5_+ 0.3 1.4_+ 0.7 4.3 _+ 1.4 0.9_+ 0.1 2.1+ 0.6 0.6 _+ 0.2 0.4_+ 0.1 0.4_+ 0.2
17.7_+ 3.0 4.3_+ 0.6 4.7_+ 0.8 4.3 _+0.6 2.8-+ 0.4 4.3-+ 0.5 1.3 -+ 0.1 1.3-+ 0.1 0.5_+ 0.1
29.4_+ 3.7 10.1_+ 1.3 9.6+ 1.0 8.0 + 1.3 6.8_+ 1.9 5.8_+ 1.1 2.4 _+ 0.3 3.1-+ 0.2 1.2_+ 0.1
a Mean • SEM of 4 female cats fed the respective diet for 2 years (12). P < 0.01 for all values in these groups.. b Mean_* SEM of 9 female cats used in the present experiment. In case of retina, the left eye was used for taurine assay, and the right for morphological analysis (30). P < 0.05 for all values in these groups, except lung, which was not significantly different. REFERENCES 1. 2.
Scott PP, Greaves JP, Scott MG. Nutritional blindness in the cat. Exp. Eye Res. 1964; 3:357-64. Rubin LF, Lipton DE. Retinal degeneration in kittens. J. Am. Vet. Assoc. 1973; 162:467-9.
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Hayes KC, Carey RE, Schmidt SY. Retinal degeneration associated with taurine deficiency in the cat. Science. 1975; 188:949-51. Hayes KC, Rabin AR, Berson EL. An ultrastructural study of nutritionally induced and reversed retinal degeneration in cats. Am. J. Pathol. 1975; 78:505-24. Schmidt SY, Berson EL, Hayes KC. Retinal degeneration in cats fed casein. I. Taurine deficiency. Invest. Ophthalmol. 1976; 15:47-52. Schmidt SY, Berson EL, Watson G, Huang C. Retinal degeneration in cats fed casein. III. Taurine deficiency and ERG amplitudes. Invest. Ophthalmol. Vis. Sci. 1977; 16:673-8. Wen GY, Sturman JA, Wisniewski HM, Lidsky AA, Cornwell AC, Hayes KC. Tapetum disorganization in taurine-depleted cats. Invest. Ophthalmol. Vis. Sci. 1979; 18:1201-6. Barnett KC, Burger IH. Taurine deficiency retinopathy in the cat. J. Small Anim. Pract. 1980; 21:521-34. Sturman JA, Hayes KC. The biology of taurine in nutrition and development. Adv. Nutr. Res. 1980; 3:231-99. Sturman JA, Wen GY, Wisniewski HM, Hayes KC. Histochemical localization of zinc in the feline tapetum. Effect of taurine depletion. Histochemistry. 1981; 72:341-50. Sturman JA, Wen GY, Wisniewski HM, Niemann WH, Hayes KC. Taurine and tapetum structure. In Huxtable, RJ, Pasantes-Morales H, eds. Taurine in Nutrition and Neurology, Plenum Publishing Corp. 1982: 65-77. Sturman JA, Gargano AD, Messing JM, Imaki H. Feline maternal taurine deficiency: Effect on mother and offspring. J. Nutr. 1986; 116:655-67. Burger IH, Barnett KC. The taurine requirement of the adult cat. J. Small Anim. Pract. 1982; 23:533-7. Ricketts JD. Feline central retinal degeneration in the domestic cat. J. Small Anim. Pract. 1983; 24:221-7. Imaki H, Moretz R, Wisniewski H, Sturman J. Maternal taurine deficiency and development of retina and tapetum in kittens. Invest. Ophthal. Vis. Sci. 1985; 26(Suppl.): 65 (Abstr). Imaki H, Moretz RC, Wisniewski HM, Sturman JA. Feline maternal taurine deficiency: Effects on retina and tapetum of the offspring. Dev. Neurosci. 1986; 8:160-81. Sturman JA, Moretz R, French J, Wisniewski HM. Taurine deficiency in the developing cat: Persistence of the cerebellar external granule cell layer. J. Neurosci. Res. 1985; 13:405-16. Palackal T, Moretz R, Wisniewski H, Sturman J. Abnormal visual cortex development in the kitten associated with dietary taurine deprivation. J. Neurosci. Res. 1986; 15:223-39. Knopf K, Sturman JA, Armstrong M, Hayes KC. Taurine: An essential nutrient for the cat. J. Nutr. 1978; 108:773-78. Berson EL, Ha~,es KC, Rabin AR, Schmidt SY, Watson G. Retinal degeneration m cats fed casein. II. Supplementation with methionine, cysteine, or taurine. Invest. Ophthalmol. 1976; 15:52-8. Olney JW. Toxic effects of glutamate and related amino acids on the developing central nervous system. In Nyhan, WN ed. Heritable Disorders of Amino Acid Metabolism: Patterns o f Clinical Expression and Genetic Variation. John Wiley & Sons, NY, 1974: 501-512. Olney JW. Brain damage and oral intake of certain amino acids. Adv. Exp. Med. Biol. 1976; 69:497-506. Olney JW. The toxic effects of glutamate and related compounds in the retina and the brain. Retina 1982; 2:341-59. Olney JW, Ho OL. Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine. Nature 1970; 227:609-11. Olney JW, Ho OL, Rhee V. Cytotoxic effects of acidic and sulphur containing amino acids on the infant mouse central nervous system. Exp. Brain Res. 1971; 14:61-76.
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Olney JW, Ho OL, Rhee V, Schainker B. Cysteine-induced brain damage in infant and fetal rodents. Brain Res. 1972; 45:309-13. Pederson OO, Lund Karlsen R. The toxic effect of L-cysteine on the rat retina: A morphological and biochemical study. Invest. Ophthal. Vis. Sci. 1980; 19:886-92. Lund Karlsen R, Grofova I, Malthe-Sorenssen D, Fonnum F. Morphological changes in rat brain induced by L-cysteine injection in newborn animals. Brain Res. 1981; 208:167-80. Lund Karlsen RL, Pederson OO. A morphological study of the acute toxicity of L-cysteine on the retina of young rats. Exp. Eye Res. 1982; 34:65-9. Sturman JA, Messing JM, Gargano AD, Rerecich M, Imaki H, Rudelli R. Cystine neurotoxicity is increased by taurine deficiency. NeuroToxicology. 1989; 10:15-28. Rukaj A, Serougne C. Effect of excess dietary cystine on the biodynamics of cholesterol in the rat. Biochim. Biophys. Acta 1983; 753:1-5. Serougne C, Ferezou J, Rukaj A. Plasma and lipoprotein cholesterol in rats fed L-amino acid-supplementation diets. Ann. Nutr. Metab. 1983; 27:386-95. Serougne C, Ferezou J, Rukaj A. Effects of excess dietary L-cystine on the rat plasma lipoproteins. Ann. Nutr. Metab. 1984; 28:311-20. Serougne C, Ferezou J, Rukaj A. A new relationship between cholesterolemia and cholesterol synthesis determined in rats fed an excess of cystine. Biochim. Biophys. Acta 1987; 921:522-30. Ohmura E, Ishikawa T, Takagi M, Aoyama Y, Yoshida A. Effects of dietary methionine, taurine, cystine and glycine on liver and serum lipids in rats fed with histidine-excess diet. Agr. Biol. Chem. Tokyo 1988; 52:1027-32. Olne), JW. Glutamate-induced retinal degeneration in neonatal mice. Electron macroscopy of the acutely evolving lesion. J. Neuropath. Exp. Neurol. 1969; 28:455-74. Lucas DR, Newhouse JP. The toxic effect of sodium L-glutamate on the inner layers of the retina. A.M.A. Arch. Ophthalmol. 1957; 58:193-201. Cohen AI. An electron microscopic study of the modification by monosodium glutamate of the retinas of normal and "rodless" mice. Am. J. Anat. 1967; 120:319-55. Hansson HA. Ultrastructural studies on the long term effects of sodium glutamate on the rat retina. Virchows Arch. Abt. B. Zellpath. 1970; 6:1-11. Lund Karlsen R, Fonnum F. The toxic effects of sodium glutamate on rat retina: Changes in putative transmitters and their corresponding enzymes. J. Neurochem. 1976; 27:1437-41. Misra CH. Cysteine-induced effect on amino acids in neonatal rat brain. Experientia. 1982; 38:383-4. Keller HJ, Do KQ, Zollinger M, Winterhalter K, Cuenod M. Cysteine: Depolarizaton-induced release from rat brain in vitro. J. Neurochem. 1989; 52:1801-6. Hughes A, Wieniawa-Narkiewicz E. A newly identified population of presumptive microneurons in the cat retinal ganglion cell layer. Nature. 1980; 284:468-70. Wong ROL, Hughes A. The morphology, number, and distribution of a large population of confirmed displaced amacrine cells in the adult cat retina. J. Comp. Neurol. 1987; 255:159-77. Narfstrom K, Nilsson E. Progressive retinal atrophy in the Abyssinian cat: Electron microscopy. Invest. Ophthalmol. Vis. Sci. 1986; 27:1569-76. Narfstrom K, Nilsson SEG. Hereditary rod-cone degeneration in a strain of Abyssinian cats. Prog. Clin. Biol. Res. 1987; 247:349-68. Voaden MJ, Curtis R, Barnett KC, Leon A, Doshi M, Hussain AA. Dominant rod-cone dysplasia in the Abyssinian cat. Prog. Clin. Biol. Res. 1987; 247:36980. Sanberg PR,. Staines W, McGeer EG. Chronic taurine effects on various neurochemical indices in control and kainic acid-lesioned neostriatum. Brain Res. 1979; 161:367-70.
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Farieilo RG, Golden GT, Pisa M. Homotaurine (3-amino-propane-sulfonic acid;3-APS) protects from the convulsant and cytotoxic effect of systematically administered kainJc acid. Neurology. 1982; 32:241-5. Lehmann A, Hagberg H, Hamberger A. A role for taurine in the maintenance of homeostasis in the central nervous system during hyperexcitation. Neurosci. Lett. 1984; 52:341-6. Lehmann A, Hagberg H, Huxtable RJ, Sandberg M. Reduction of brain taurine: Effects on neurotoxic and metabolic actions of kainate. Neurochem. Internat. 1987; 10:265-74. CheSney RW. Taurine: Its biological role and clinical implications. Adv. Pediat. 1985; 32:1-42. French ED, Vezzani A, Whetsell WO Jr., Schwarcz R. Anti-excitotoxic actions of taurine in the rat hippocampus studied in vivo and in vitro. Adv. Exp. Med. Biol. 1986; 203:34~-'62. Trenkner E, Dykens JA. Taurine moderates kainate and kynurenine excitotoxicity in vitro. Soc. Neurosci. Abstr. 1986; 12:96. Pasantes-Morales, H, Arzate ME, Quesada O, Huxtable RJ. Hi~her susceptibility of taurine-deficient rats to seizures induced by 4-amino pynome. Neuropharmacol. 1987; 26:1721-5.
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Accepted for publication May 29, 1990.
FELINE RETINAL ULTRASTRUCTURAL CHANGES INDUCED BY A HIGH CYSTINE DIET H. Imaki, Ph.D. and J.A. Sturman, Ph.D. Department of Developmental Biochemistry Institute for Basic Research in Developmental Disabilities 1050 Forest Hill Road, Staten Island, NY 10314, USA
ABSTRACT Cats fed a high cystine diet containing no taurine developed extreme signs of neurotoxicity and either died or had to be killed within 7 months. The presence of 0.05% taurine in the diet greatly ameliorated the neurotoxic effects of the cystine. Many developed retinal ultrastructural changes which were quite different from those previously described for cats on taurine-free diets. The most striking changes were reductions in the number of cells and extensive swelling of some remaining cells in the inner nuclear layer and in the ganglion cell layer. The most severely affected cells were amacrine cells, in contrast to photoreceptor cells in cats fed the taurine-free diet. The changes resemble those reported in other species caused by excitotoxic amino acids. Key words: Cat retina - Cystine neurotoxicity - Retinal ultrastructure - Taurine deficiency INTRODUCTION Cats are dependent on dietary source of tanrine, a sulfur-containing amino acid abundant in meat and animal tissues but poor in plants and vegetables. Earlier studies established that cats fed taurine-deficient diets develop a number of abnormalities including visual dysfunction, attributable to progressive degeneration of photoreceptor cells in the central retina, and disorganization of tapetum lucidum, a triangular sheet of light-reflecting cells behind the retina (1-14). Recently we demonstrated that weanling (8-week-old) kittens born to and nursed by taurine-deprived cats sustain similar degenerative changes both in the retina and tapetum (15-16) in addition to delayed, or abnormal, patterns of development in the cerebellum and visual cortex (17-18). Although cats have a limited biosynthetic capacity for taurine, they do have some activity of hepatic cysteinesulfinic acid decarboxylase, and can convert limited amounts of cyst(e)ine to taurine (19). We recently concluded a study in which a group of cats were fed with a synthetic diet enriched with cystine in order to test whether effects of taurine deficiency could be ameliorated by supplying an excess of its biosynthetic precursor. It has been reported that dietary cysteine (and methionine) supplementation in smaller amounts than used here was ineffective in restoring the reduced ERG response or delayed cone bwave implicit time in taurine-deprived cats (6,20), and that cysteine and some other sulfurcontaining amino acids administered orally or parenterally to young rodents were extremely neurotoxic (21-29). Here we report the light and electron microscopic observations made in the retinas of cats fed the cystine-rich diet with or without an adequate amount of taurine. Preliminary morphological findings and other biological and biochemical results obtained from the same set of cats have been reported previously (30). 1385
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MATERIALS AND METHODS Mature female domestic cats (1 - 3 years of ase) raised in the IBR colony and vaccinated against rhinotracheitis, panleukopenia, calicl virus [FVR-C-P (MLV) PitmanMoore, Washington Crossing, NJ] were fed ad libitum a completely defined, synthetic diet (taurine-free) containing 5% cystine with or without 0.05% taurine (BioServe, Frenchtown, NJ). The basal defined synthetic diet contained (grams/100g): casein (vitamin-free), 43.0; chicken fat, 20.0; dextrin, 13.5; sucrose, 13.5; salt mix (see below), 6.4; cellulose, 2.4; vitamin mix (see below), 0.6; L-cystine, 0.3; choline chloride, 0.3. The salt mix contained (in grams/kilogram): potassium phosphate dibasic, 328; calcium carbonate, 290; sodium chloride, 162; magnesium sulfate, 99; calcium phosphate dibasic, 73; magnesium oxide, 32; ferric citrate, 13; manganese sulfate, 1.22; zinc chloride, 0.91; cupric sulfate, 0.29; potassium iodide, 0.077; chromium acetate, 0.044; sodium fluoride, 0.023; sodium selenite, 0.0043. The vitamin mix contained (in grams-kilogram): dextrin, 857; inositol, 100; D,L-a-tocopheryl acetate (500 IU/g), 20; niacin amide, 8.0; calcium pantothenate, 5.0; retinyl acetate (500,000 IU/g), 5.0; riboflavin, 1.60; cholecalciferol (200,000 IU/g), 1.25; thiamin-HC1, 0.80; pyridoxine.HC1, 0.80; folic acid, 0.80; menadione, 0.10; biotin, 0.04; cyanocobalamin, 0.03. The cats were autopsied as soon as found dead, or killed by cardiac puncture under Nembutal anesthesia when they were moribund, or at the end of experimental period of 12 months. The right eye from each cat was fixed by perfusion by injecting 3% glutaraldehyde in 0.1 M Sorensen's phosphate buffer (pH 7.4), with a needle inserted in the opposite pole of equator for drainage. The posterior eye cup, cut radially into 12 sectors around the area centralis, was postfixed with 1% OsO a in 0.1 M Sorensen's buffer, dehydrated in an ascending series of acetone and embedded in .El?on 812. Light micrographs were taken from 0.75 urnthick sections stained with 1% toluidlne blue on a Zeiss Photomicroscope III using Kodak Panatomic X film. Electron micrographs were taken from thin sections, gray-silver in interference colors, mounted on copper grids and stained with uranyl acetate and lead citrate, in a Philips EM 300 operated at 80 kv, using Kodak EM film (4489). Micrographs taken from transverse sections otretinas from cats fed the diets described above were compared with those from cats fed the control diet which consisted of the same synthetic formula supplemented with 0.05% taurine but not with cystine. RESULTS All the 9 cats fed the 5% cystine, taurine-free diet developed fulminant neurological symptoms which included lethargy, rigidity, absence and epileptic seizures, and either died or were killed when they clearly appeared dying. The mean survival time of these cats was 5.2 • 2.0 months, with the shortest and the longest being 1.5 and 7 months, respectively, from the start of the diet. Of the 9 cats fed the 5% cystme plus 0.05% taurine diet, 4 died after showing minimal symptoms, such as slight lethargy and unsteady postures, with the mean survival time of 3.5 • 1.3 months, while the others outlived the experimental period without showing any obvious symptoms. None of the animals, while alive, exhibited apparent visual dysfunction as judged from their behavioral patterns. Light microscopic examination revealed small, yet noticeable, reductions in the number of ceils in the inner nuclear and ganglion cell layers of retinas from cats fed the diet containing 5% cystine, either with or without 0.05% taurine, in comparison to the retinas from cats fed the control diet (Figs. la, b and c). The overall thickness of such retinas, nevertheless, appeared unchanged or even increased, due largely to the massive swelling of the neuropil in the inner plexiform layer, and sometimes in the outer plexiform layer as well. In addition, many cells occurring in the inner part of the inner nuclear layer and some in the ganglion cell layer also were greatly swollen, while others appeared shrunken or compressed. Occasionally, the inner nuclear layer contained what appeared to be large spaces between cells. These changes usually were widespread and no appreciable difference in form or magnitude could be found between different areas of the same retina, although some variation existed between retinas from different cats. The number, structure and organization of photoreceptors, as well as cells forming retinal endothelium, epithelium or tapetum lucidum were comparable to the corresponding
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cells of cats fed the control diet. The retina from one of the cats fed the taurine-free diet containing 5% cystine showed a great reduction in the number of photoreceptor cells (completely absent from the superior-nasal sectors, and 1/3 to 1/2 o f the normal in the inferior-temporal sectors), and unusually swollen tapetal ceils in the mid-outer layers which surrounded more compact and denser cells of the inner tapetum. Cells in the inner retina of this cat exhibited relatively normal morphology, except for their somewhat undifferentiated disposition and lack of the typical layered arrangement (Fig. ld). As might be expected, the retinas from cats found dead exhibited massive swelling and generalized disorganization of nearly every structure as a result of post-mortem decomposition, with a notable exception of tapetal ceils which usually remained intact (Fig. le). By electron microscopy, the most severely affected cells in the retinas of cats fed the cystine-rich diets were identified as amacrine cells which occupied the inner half of the inner
FIG. 1 Light micrographs of cat retinas in transverse section through area centralis. Toluidine blue, x 400. a: Retina from a cat fed the basal synthetic diet containing 0.05% taurine (control); b: Retina from a cat fed the synthetic diet containing 0.05% taurine and 5% cystine; c: Retina from a cat fed the synthetic diet containing 5% cystine, but taurine-free; d: Retina from a different cat fed the same diet as c; e: Retina from a third cat fed the same diet as c. GCL: Ganglion cell layer; IPL: Inner plexiform layer; INL: Inner nuclear layer; OPL: Outer plexiform layer; ONL: Outer nuclear layer; IS and OS: Inner and outer segments of photoreceptors, respectively; RPE: Retinal pigmented epithelium; T: Tapetum lucidum.
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nuclear layer and a substantial portion of the ganglion cell layer as "displaced" amacrine cells. The normal amacrine cells observed in control cat retinas were characterized by relatively large, often deeply lobated, nuclei with homogeneous finely granular nucleoplasm, and relatively abundant perikarya rich in granular cisternae of endoplasmic reticulum (ER), rosettes of free ribonucleoprotein (RNP) particles, clusters of Golgi cisternae and mediumsize mitochondria, distributed evenly throughout the cytoplasm (Fig. 2a). The majority of amacrine cells found in the affected retina, in contrast, exhibited somewhat bloated nuclei with coarse clumps of chromatin in rather pale or bleached nucleoplasm, and grossly distended perikarya which contained greatly expanded spherical mitochondria, vacuolar cisternae of ER and Golgi, and small numbers of RNP particles, often clustered in the perinuclear region of otherwise sparse cytoplasm (Fig. 2b). In addition, varying numbers of large vacuoles containing membraneous or flocculent materials were found m peripheral reglon.s of cytoplasm. Both bipolar and horizontal cells of retinas with many degenerative amacrme cells usually showed similar but less pronounced ultrastructural changes. The nuclei of these cells, which in the normal retinas showed slightly undulating contours and small clumps of chromatin scattered in moderately dense nucleoplasm (Fig. 2a), displayed denser and coarser chromatin patterns and irregular surface indentations, often juxtaposed with greatly expanded mitochondria which generally are slightly larger than those in the amacrine cells, and irregularly shaped vacuoles similar in appearance to those found in degenerating amacrine cells (Fig. 2b). Muller cells, whose small cell bodies were located mid-level of the inner nuclear layer, displayed homogeneously electron-dense nuclear and toplasmic matrices in the retinas of cats fed 5% cystine (Fig. 2b) as those of the control cats Fig. 2a). The more angular profiles of these cells in the former appeared to be a result of excessive swelling and deformation of neighboring cells. The ganglion cells located in the ganglion cell layer of the control cat retinas were notable for their large nuclei and prominent nucleoli, and enormous cytoplasm containing extensive arrays of Golgi complexes, granular cisternae of ER and clusters of RNP particles. There were at least 2 morphological subtypes readily identified: the more common type was usually spherical in overall shape, and relatively electron-lucent, appearing more like amacrine cells than the second type, which exhibited complex surface contours and unusually high matrix densities in the nucleus and cytoplasm (Figs. 3a and 4a). Both subtypes of ganglion cells in the retinas of cats fed 5% cystine exhibited degenerative features, but the first type, which often exhibited heterochromatic nucleoplasm and loss of many cytoplasmic organelles, as noted in degenerating amacrine cells, seemed to be more severely affected than the second type, whose structural changes usually were limited to swollen mitochondria and dilated cisternae of ER and Golgi complexes (Figs. 3b and 4b).
~y
FIG.2, Electron micrographs of cat retinas in transverse section through the inner nuclear
layer, a: Retina trom a cat fed the control diet, showing fine structures of normal amacrine cells (Am) with relatively large, electron-lucent perikarya, horizontal cells (Ho) with rather large ovoid mitochondria and somewhat heterochromatic nuclei, bipolar cells (Bi) with denser nucleoplasm and scanty perinuclear cytoplasm and Muller cells (Mu) with homogeneously dense nuclear and cytoplasmic matrices. Vitreous is toward the top. b: Retina from a cat fed the diet contaimng 5% cystine but no taurine, showing degenerative profiles of amacrine (Am) horizontal (Ho) and bipolar (Bi) cells, with spherical, swollen mitochondria and vacuoles of varying size containing amorphous flocculent material or membraneous inclusions. Note the large expanse of amacrine cell cytoplasm devoid of organelles, while the neighboring Muller cell (Mu) remains compact and appears intact. Despite the irregular surface configuration and occasional intercellular spaces, the majority of cells in the inner nuclear layer and the ganglion cell layer of the affected retina appeared to maintain remarkably intact and intimate intercellular contacts with one another, comparable to those observed in normal retinas (Figs. 2a and b, 3a and b). Similarly close and normal intercellular contacts and junctional structures were observed between various neuronal processes in the inner plexiform layer as well as in the outer plexiform layer of
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FIG.3. Electron micrographs of cat retinas in transverse section through parts of ganglion cell
and inner plexiform layers, a: Retina from a cat fed the control diet, illustrating the fine structure of a normal ganglion cell (G1) with a large ovoid nucleus (n) and ample c~(toplasm filled with numerous granular cisternae of endoplasmic reticulum, free nbonucleoprotein particles and well-developed Golgi complexes, Muller cell processes (Mu) filled with dense skeins of filaments, and synaptic processes of neurons forming the inner plexiform layer (IPL) En: endothelial cell; Lu: Retinal vascular lumen; P: Pericyte. b: Retina from a cat fed the diet containing 5% cystine and 0.05% taurine, showing a degenerating ganglion cell consisted of an expanded nucleus with clumps of chromatin granules and edematous cytoplasm contaimng scattered clumps of vacuolar mitochondria and ER. The second ganglion cell (G2) contains clear vacuoles of varying size and shape in addition to swollen mitochondria, in the dense cytoplasm.
retinas from cats fed 5% cystine as in those fed the control diets (Figs. 3c and 4, 4a and b). However, virtually all postsynaptic processes found in the inner plexiform layer in the former retinas were extremely swollen and empty-looking, while presynaptic and Muller cell processes appeared unaffected, except for some mildly swollen mitochondria (Fig. 4d). The synaptic elements in the outer plexiform layer of these retinas usually appeared less affected than those in the inner plexiform layer, in some cases being indistinguishable from those in control retinas (Figs. 5a). Some retinas from cats fed the taurine-free diet containing 5% c3,stine exhibited generalized swelling of photoreceptor cytoplasm, including synaptic, axonal and somal elements (Fig. 5b) as well as inner and outer segments (Fig. 5c), whereas others had a relatively normal appearance (Fig. 5d). The retinal epithelial cells were unaffected by 5% cystine in the diet in most cats, but those in some cats fed the taurine-free variety tended to display slightly denser cytoplasmic matrix, possibly as a result of compression by an increased number of large vacuoles in such cells. Phagosomes, lysosomes and lipofuscin granules also were more frequently encountered in some of these cells (Fig. 5d). The tapetum of cats fed the diets containing 5% cystine exhibited normal fine structure except in one cat (on the taurine-free diet) which revealed unusually compact tapetal cells in the inner layers and swollen cells, containing some irregularly-spaced or loosely arranged tapetal rods, in the mid-outer layers (Figs. 6a and b). The retinal epithelial cells of this particular cat, which lacked photoreceptor cells entirely in some regions, were found to be extremely attenuated, and completely devoid of phagosomes or lipofuscin granules and, in some areas, apical microvilli as well (Fig. 6a). In nearby areas of the same retina were fine finger-like microvilli of the epithelial cells interdigitating with equally slender extensions of Muller cells, and giant lysosomes containing diverse inclusion bodies and occurring in the relatively thick and rich cytoplasm of an epithelial cell (Fig. 6c). Cells found vitread to the Muller cells in this retina appeared less differentiated and metabolically less active and were harder to identify even though none of these cells exhibited the obvious pathological features noted in the retina of the majority of cats fed the same diet (5% cystine, no taurine). Taurine concentrations in selected tissues from the animals used in the present study are summarized in Table 1 along with data obtained previously in corresponding tissues from similar groups of cats fed the same synthetic diet containing either 0 or 0.05% taurine, but no excess cystine, for comparison. For each pair of data sets, the taurine concentrations in tissues from taurine-supplemented cats were significantly greater than the same tissues from unsupplemented cats (with a single exception, lung o f animals receiving the 5% cystine supplement). Of note with re~ard to the ultrastructural results presented here was that retinal taurine concentration in cats supplemented with 0.05% and 5% cystine was significantly smaller than in cats supplemented with 0.05% taurine alone.
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FI6.4. Electron micrographs of cat retinas in transverse sections through the ganglion cell layer a: Retina from a cat fed the control diet, showing the normal ultrastructure of perinuclear region of a "dense" ganglion cell with a prominent nucleolus (no) occupying the middle of nucleus (n), which exhibit homogeneous nucleoplasm, and gently undulating nuclear envelope. The cytoplasm is occupied by prominent granular cisternae of ER, medium-size mitochondria, and well-developed Golgi complex, b: Corresponding area of a . . . . den se n ganghon cell from a cat fed the. diet contannng 5% cystme and 0.05% taurine. Note large intracellular spaces which may represent greatly dilated cisternae of ER or Golgi complex. The appearance of nucleus (n) including its centrally located nucleolus (no) is relatively unchanged. It
Electron micrographs of cat retinas in transverse section through the inner plexiform layer, c: Retina from a cat fed the control diet, showing complex arrangement of synaptic processes containing variable numbers of synaptic vesicles, neurofilaments and tubules, in addition to normal-looking mitochondria (m). d: Retina from a cat fed the diet containing 5% cystine and 0.05% taurine, showing greatly swollen and deformed postsynaptic processes in intimate contact with relatively intact presynaptic processes loaded with synaptic vesicles. Many mitochondria (m) of presynaptic process, however, appear swollen as those in the postsynaptic processes. DISCUSSION The results reported here werepart of an experiment initiated in an attempt to increase taurine biosynthesis in cats fed a taurine-free diet by supplying an excess 5%) of its natural metabolic precursor, cystine. Although there was no obvious mmediate effect of this diet and cats maintained their normal dietary intake, after several months the first cats started to exhibit acute neurotoxic symptoms. A major feature was the sudden onset and the rapid progression to a moribund state or death, usually within 48 hours. All nine cats fed the 5% cystine, taurine-free diet succumbed in this fashion, the longest survival time being 7 months from the start of the diet. This is the first reported incidence of neurotoxicity resultin~ from excess dietary cystine, although cats have not previously been used in such studies. It should be noted here that cats fed the same synthetic diet with or without 0.05% taurine, but not containing 5% cystine, did not show any overt neurotoxic symptoms, even after several years on such diets (12, Sturman, unpublished observations on cats fed these diets for more than 4 years). Several recent reports had examined the effect of feeding a diet containing 5% cystine to rats, and although this resulted in increased cholesterol levels and cholesterol biosynthesis, no adverse physical effects were noted (31-34). A recent report indicated that the addition of 1% cystine to the diet of rats fed 3.5% histidine resulted in hepatomegaly and a decreased liver cholesterol content (35). The morphological changes observed in the retinas of a majority of cats fed the cystine-rich diets were comparable to those described in the retinas of young rodents treated with glutamate or certain other acidic amino acids, collectively referred to as "excitotoxic", as their neurotoxic effects appear to be linked to their neuroexcitatory function (21). Earlier studies established that this type of retinal lesions were characterized by selective, yet randomized and widely disseminated destruction of the inner retina: the acute phase of response includes massive swelling of post-synaptic, or post-synaptic, or dendrosomatic, elements of neurons in the inner nuclear layer and ganglion cell layers, followed by extensive degeneration of intracellular organelles and nuclear pyknosis, as described in detail in the retinas of infant rodents treated with glutamate and other amino acids including L-cysteine (24-26,29,36). The delayed, or long-term, response involves reductions in the number of the same types of neurons
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FIG. 5. Electron micrographs of cat retinas in transverse section through photoreceptor and retinal epithelial cells, a: Synaptic region between photoreceptor cells and neurons of the inner retina from a cat fed the control diet, showing normal appearance of a pedicle (p) with typically large, but undisturbed, mitochondria and distinctive synaptic ribbons, several spherules containing smaller, normal mitochondria, and parts of cell body of inner-most rod cells (R). b: Synaptic region between photoreceptor cells and neurons of the inner retina from a cat fed the diet contaimng 5% cystine and no taurine, showing greatly expanded rod cell (R) cytoplasm including spherucles (s). c: Distal parts of rod photoreceptors of the same cat as b, showing slightly swollen mitochondria in the inner segments (is) and ruffled, wavy disc membranes in the outer segments (os). d: Distal portions of photoreceptors and retinal pigment epithelium (RPE) from a different cat fed the same diet as b (5% cystine, taun~ne-free), showing the fine structure of cone and rod outer segments (cos and ros) that are indistinguishable from those of cats fed the control diet. T: Tapetum. and of myelinated axons in the optic nerve, which are especially pronounced when experimental animals were treated early in development either with glutamate (37-40) or cysteine (27). Although there are indications that cysteine itself might be responsible for the cytotoxicity (41,42), cysteine administered systemically is believed to be converted locally to cysteinesulfinic acid or cysteic acid to act as a neuroexcitant, to account for its somewhat delayed, but more widespread pattern of pathological response, especially in the brain, where a neutral amino acid such as L-cysteine gains entry into several regions while acidic amino acids do not (21,22,25,26). The excess cystine ingested by the cats might be converted to similar acidic amino acids before it elicited structural damages observed in the retina which are characterized by both the acute and chronic patterns of response, i.e. massive swelling of some neurons and dendrites, and reductions in number of the same type of neurons, at the same time. This is most likely due to the fact that these cats were completely dependent on the cystine-enriched diet for prolonged periods, instead of a single or short-term exposure that animals in the other studies were subjected. It is possible that the differential susceptibility to the high cystine diets observed among different neurons in the cat retina may simply be due to differences in physiological and metabolic states of the cell: amacrine and ganglion cells in general might appear to sustain greater damage than bipolar and horizontal cells because they are associated with greater numbers of ER and mitochondria which are particularly vulnerable and respond by excessive swelling and disintegration. The reductions in the number of cells in the inner retina of these cats may be the result of such a differential susceptibility of individual neurons. Thus, the number of cells in the inner nuclear layer showed a relatively small decrease since amacrine cells comprise a small proportion of cells among other more numerous types, while the reduction in the number of cells in the ganglion cell layer was more obvious due to the fact that the latter layer is mostly composed of ganglion and amacrine cells. A greater reduction in amacrine cells, relative to ganglion cells, contributed to the overall reduction, as these "displaced" amacrme cells form some 80% of the total neuron population of the cat ganglion cell layer (43-44). These findings in cat retinas regarding the type and degree of neurons affected by excess cystine agree with the earlier observations by Cohen (38), who reported that number of amacrine and ganglion cells are more severely reduced than bipolar cells in the retina of mice treated with MSG. However, the appearance of surviving ganglion cells in cats fed a high-cystine diet were quite variable depending on the morphological subtypes and individual cells, contrary to the observation in the above mouse retinas in which they reportedly appeared "normal except for their smaller size". As repeatedly demonstrated in other animals treated with various excitotoxins postnatally, Muller cells and other non-neuronal cells in the retinas of cats fed the high cystine diet did not show any appreciable structural abnormalities. The photoreceptor cells of these cats (except one), likewise, were virtually indistinguishable from those found in cats fed the control diet. The retinal lesions induced by excessive dietary cystine, therefore, are
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1396
FIG. 6 Electron micrographs of the central-temporal retina from a cat fed the taurinefree diet containing 5% cystine, a: Transverse section through the middleouter levels of retina and inner tapetum lucidum (T), showing parts of several neurons, in the upper half, a Muller cell (Mu) containing dense clumps of amorphous debris (*) and directly in contact with the retinal pigment epithelium (RPE). Photoreceptor cells are completely missing in this region. b: Transverse section through mid-outer levels of tapetum, showing several cells with loosely arranged tapetal rods, in comparison to those in normal tapetal cells, or those in the inner layers, as seen in a. The nucleus (n) is normal looking, c: Transverse section through retina - epithelial cell junction showing a dense aggregate (*) in a Muller cell cytoplasm, tightly interdigitating processes of Muller cell and microvilli of epithelial cell, which contains a large hagosome with heteromorphic inclusions and typically large mitochondria m).
~
fundamentally different from those observed in the taurine-deprived cats and kittens studied previously. The retinas of some cats fed the taurine-free cystine-enriched diet,
1397
CATS ON HIGH CYSTINE DIET
however, showed photoreceptors with subtle ultrastructural changes, such as slight loosening of disc membranes and ruffled plasma membranes, which were much less severe than the changes described in the retinas of cats fed a taurine-free diet without cystine. This ma X be due to the marginal increases in tissue taurine concentration, but it seems more hkely the result of the shorter feeding duration. The cause for the drastic reduction in the number of photoreceptor cells observed in one of the cats fed the same diet is not dear, but it may be due to genetic as well as nutritional factor as none of the other retinas examined here exhibited such extreme changes. Such features resemble those described in the retinas of certain strains of Abyssinian cats with inherited photoreceptor degeneration (45-47). The supplementation of the high cystine diet with adequate taurine seems to prevent certain degenerative changes in the photoreceptor cells but not neurons in the inner retinas of cats. Other neurotoxic effects of cystme and premature death observed in the cats studied here appeared to be significantly reduced or delayed by the inclusion of adequate taurine m the diet, supportin.g the suggestions that taurine plays a neuroprotective role against the excxtotoxac damages in mammalian systems either in viv0 or in vitro (48-55). TABLE 1 Concentration of taurine in selected tissues of cats fed the basal synthetic diet with or without additional suplements of taurine (0.05%) or cystine (5%)
Taurine alone a
None a
Cystine alone b
Taurine b + cystine
moles/g wet weight Retina Liver Heart Adrenal Kidney Lung Cerebellum Occipital lobe Spinal cord
42.8_+ 4.6 13.1_+ 2.2 12.6_+ 3.1 11.4 _+3.2 9.6_+ 1.0 7.8_+ 2.2 3.8 -+ 0.8 2.3+ 0.8 1.3_+ 0.4
15.0_+ 3.0 0.5_+ 0.3 1.4_+ 0.7 4.3 _+ 1.4 0.9_+ 0.1 2.1+ 0.6 0.6 _+ 0.2 0.4_+ 0.1 0.4_+ 0.2
17.7_+ 3.0 4.3_+ 0.6 4.7_+ 0.8 4.3 _+0.6 2.8-+ 0.4 4.3-+ 0.5 1.3 -+ 0.1 1.3-+ 0.1 0.5_+ 0.1
29.4_+ 3.7 10.1_+ 1.3 9.6+ 1.0 8.0 + 1.3 6.8_+ 1.9 5.8_+ 1.1 2.4 _+ 0.3 3.1-+ 0.2 1.2_+ 0.1
a Mean • SEM of 4 female cats fed the respective diet for 2 years (12). P < 0.01 for all values in these groups.. b Mean_* SEM of 9 female cats used in the present experiment. In case of retina, the left eye was used for taurine assay, and the right for morphological analysis (30). P < 0.05 for all values in these groups, except lung, which was not significantly different. REFERENCES 1. 2.
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50. 51. 52. 53. 54. 55.
Accepted for publication May 29, 1990.