Beneficial
Effect
of Intravenous
on Electroretinographic Chiaki
Shimada,
Disorder Shuichi
Kiyotsugu Research
and
Development
Center,
Fuso
Received
Tanaka,
Isaka,
November
Hasegawa,
Sano and Hiromasa
Industries,
12, 1991
Infusion
in Taurine
Michinori
Mitsuo
Pharmaceutical
Taurine
Ltd.,
Accepted
2-3-11, January
Deficient Sumio
Rats
Kuroda,
Araki
Morinomiya,
Joto-ku,
Osaka
536, Japan
22, 1992
ABSTRACT-We investigated the effect of intravenous taurine infusion on the electroretinogram (ERG) of taurine-deficient rats produced by treatment with guanidinoethyl sulfonate (GES), a taurine transport inhibitor. Mother rats were fed a taurine-free diet and given drinking water containing 1% GES from 2 weeks of gestation to weaning. The same feeding conditions were applied to male offspring after weaning. Both ERG measurement and continuous infusion of taurine at a dose of 10, 30 or 100 mg/animal/day were performed for 3 weeks from 7 to 10 weeks of age. GES-treatment reduced a and b-wave amplitudes to 50% of the control levels and also increased b-wave latencies. Intravenous infu sion of taurine improved these ERG abnormalities in a dose-dependent manner. Taurine concentrations in plasma, eyes and brain were also decreased by treatment with GES, and dose-dependent recovery was observed after infusion with taurine, although the concentrations of other amino acids were not affected by GES-treatment and infusion of taurine. Observations of morphological changes revealed that the retinal damage in GES-treated animals was decreased by taurine infusion. These results indi cate that the changes in ERG and retinal structure observed in taurine deficiency are improved by in travenous infusion of taurine. Keywords:
Taurine,
Electroretinogram,
Guanidinoethyl
Taurine, a sulfur-containing amino acid, has been re ported to distribute to many excitable tissues as a free amino acid (1). Retina is one of the sites that take up taurine with a high affinity (2-4). It has been reported that the release of taurine from a retinal preparation is induced by light stimulation (5), electrical stimulation to photoreceptor cells (6) or addition of K+ ions to the medium (7), and that taurine exhibits protective effects against the degeneration of photoreceptor cells caused by light stimulation (8), superoxide (9) or osmotic im balance (10). Therefore, taurine is thought to be re leased by certain light stimuli and to exhibit a protec tive role against photochemical damage of the photore ceptor membrane (11, 12). It has been well-documented that morphological de generation of the retina and concomitant disorder of the electroretinogram (ERG) or visual acuity are observed in cats and rhesus monkeys fed taurine-free diets during their infancy (13-17). In general, because of the well-developed ability of taurine synthesis in rats (18, 19), it is not possible to produce taurine-deficient
sulfonate,
Retina,
Taurine
deficient
rat
rats only by elimination of dietary taurine. However, treatment of rats with guanidinoethyl sulfonate (GES), a transport inhibitor of taurine, has been reported to elicit taurine depletion in many tissues including the retina (20-25), in which there is retinal degeneration, especially in the photoreceptor cells (24, 26, 27). Fur thermore, these taurine-depleted rats exhibit ERG abnor malities characterized by reduced amplitudes in the b and/or a-waves (28-33). Whereas it is obvious that these retinal disorders are elicited by GES in rats, it is yet not known if administration of taurine can normal ize the decreased tissue taurine levels as well as the ERG disorder in taurine-deficient animals. On the other hand, it has been reported that a slight ERG dis order and low plasma taurine levels in human infants treated with taurine-free amino acid solutions in long term parenteral nutrition are normalized by addition of taurine to the solution (34). In this study, we investigated whether or not taurine infusion in taurine deficient rats produced by long-term treatment with GES could improve the taurine deple
tion and structure.
concomitant
disorders
in ERG
and
retinal
MATERIALS AND METHODS Chemicals and animals GES used this study was synthesized by the method of Huxtable et al. (20). The drug was confirmed to be 99.9% pure by thin layer chromatography and con tained less than 0.01% taurine, which was determined with an amino acid analyzer (LP-8500, Hitachi). Female Sprague-Dawley rats at 2 weeks of gestation (Charles River Japan) were assigned to 3 feeding condi tion groups: 1) normal animals fed an ordinary labora tory chow (MF, Oriental Yeast) and tap water, 2) food control animals fed a casein-based diet and tap water, and 3) GES-treated animals fed a casein-based diet and tap water containing 1% GES. After weaning at 21 days of age, the same feeding conditions were applied to the male offspring. These rats had free access to food and water, and they were housed 5 per cage under a cycle of 12 hr light (300 lux)/ 12 hr dark at a room temperature of 23 ± 2°C and the humidity of 55 ± 5%. Measurement of ERG At 7 weeks of age, animals weighing 175 -277 g in each feeding condition were used for the first ERG re cordings. On the day of ERG recording, animals moved from the overnight dark room into the dark room with indirect red dim light were anesthetized with an i.p.-injection of 40 mg/kg sodium pentobarbital (Dai-Nippon Pharmaceuticals). They were fixed to a stereotaxic apparatus with a heating pad (Deltaphase, Braintree Scientific) which kept the body temperature constant at 37°C. The left pupil was dilated by 0.1% atropine solution (atropine sulfate, Sigma). A bipolar contact lens electrode (Kyoto Contact Lens) was attached to the left eye ball, and active and indifferent electrodes were placed on the cornea and around the sclera, respectively. A Xenon lamp unit was placed 50 cm above the left eye ball and connected to a photosti mulator (3G22, San-ei). Electrical potentials responded to 10 times photostimuli of a 10 joule intensity, 0.1 msec duration and 60 sec intervals were amplified using a bioelectric amplifier (1253A, San-ei) with a 0.1 sec time constant and a 1 kHz high cut filter. All wave forms were taken into an analog-to-digital converter (PCN-2198, Neolog) connected to a personal computer (PC-9801RX2, NEC). Waveforms acquired in each animal were averaged from 10 recordings with software for this purpose (Biosignal analyzer, Medical Research Equipment). Amplitudes and peak latencies of both a and b-waves were automatically measured from aver
aged waveforms and the ratio of b-wave amplitude and a-wave amplitude (b/a ratio) was calculated. Administration of taurine Just after the first ERG recordings, a sterilized sili cone tube with a 0.5-mm inner diameter (Dow Corn ing) was inserted into the right jugular vein for con tinuous infusion of taurine. The tube was covered by a spring coil and connected to a 50-m1 syringe through a cannula swivel which allowed the rat to move freely. Animals treated with GES were divided into 4 groups; one of the 4 groups was infused with saline, and the other groups were infused with taurine dissolved in saline at doses of 10, 30 and 100 mg/animal/day, re spectively, at the rate of 0.1 ml/hr with an infusion pump (STC-501; Terumo). Normal and food control animals were infused with saline at the same infusion rate. Continuous infusion of saline or taurine solution were made for 21 days. The ERG recordings described above were also performed at 7, 14 and 21 days after the start of infusion. Feeding conditions described above were continued for each animal during the infu sion. Any animal in which disconnection or occlusion of the infusion tube occurred was eliminated from the study, and 7 or 8 animals per group were finally used. Assay of amino acid After the last ERG recordings, arterial blood was withdrawn from the abdominal aorta with a heparinized syringe, and the plasma was obtained by centrifugation (3000 rpm, 10 min at 4°C). Eyes and the brain without cerebellum and olfactory bulb were isolated after perfu sion with ice-cold saline. Deproteinization was per formed by addition of an equal, 10-times or 5-times volume of ice-cold 5% sulfosalicylic acid solution to the plasma, eyes or brain, respectively. The eyes and brain were homogenized with an ultrasonic homogenizer (Physcotron, Nichi-on Irikakikai). Each deproteinized suspension was centrifuged (3000 rpm, 15 min at 4°C), and the supernatant was separated as a sample for quantitative assay of amino acids using an amino acid analyzer (LP-8500, Hitachi). Morphological evaluation To determine the morphological influence of taurine deficiency in the eyes, animal models like those used for the ERG recording were produced. After the infu sion of taurine or saline, 2 4 rats per group were per fused with 0.5 1/kg of 10 mM phosphate-buffered saline, and the tissues were fixed with perfusion of 1 l/kg of 0.1 M phosphate buffer containing 4% paraformalde hyde, 0.2% picric acid and 0.35% glutaraldehyde. The eyes were removed from the animals, and the eyecups
were isolated. They were postfixed for 2 days in the fixative without glutaraldehyde and immersed for 2 days in 0.1 M phosphate buffer containing 15% sucrose. For light microscopy, the paraffin sections were prepared according to the usual method and stained by hema toxylin and eosin (HE) staining solution. Statistical analysis All data represent the mean ± standard error of 7 or 8 observations. Statistical evaluation was performed by the unpaired Student's t-test or Welch's test.
Table
1.
Effect
of intravenous
infusion
of taurine
RESULTS Effect of i. v. infusion of taurine on the ERG of GES treated rats Changes in body weight during the infusion were not significantly different among the 6 groups (data not shown). Table 1 shows the amplitudes and latencies of both a and b-waves and the b/a ratio for amplitude in the ERG measured before and 7, 14 and 21 days after infusion of drugs in each group. There were no signifi cant differences in any ERG parameters between the normal and food control groups. Marked decrease in
on
ERG
parameters
in GES-treated
rats
amplitudes of both a and b-waves, which reduced to 50% of those of the normal or food control group, and increased latencies of b-wave were observed in GES treated animals before infusion of taurine. In GES-treat ed animals infused with taurine, as compared with the animals infused with saline (GES control), significant increases in amplitudes of both waves and a decrease in latencies of b-waves were observed at 14 and 21 days after infusion of taurine at doses of 30 and 100 mg/animal/day. A slight but not significant increase in amplitudes of both waves was observed at a dose of 10 mg/animal/day. The increases of amplitudes of both waves in animals treated with taurine were dose-de pendent at 21 days after infusion. Especially at a dose of 100 mg/animal/day, significant increases in ampli tudes of both waves were observed at 7 days after the infusion, and the b-wave latency at 14 days and ampli tude at 21 days recovered to the levels of the food con trol group. Significant differences in the b/a ratio be tween taurine-treated and food control groups were observed at 7 days, but not at 14 and 21 days after the
infusion. The average waveforms observed in the 6 groups are shown in Fig. 1. These waveforms reveal the ERG disorder in GES-treated animals (all waves for the GES control and waves before taurine infusion). Dose-dependent recovery of the ERG was observed in animals infused with taurine. Effect of i.v.-infusion of taurine on amino acid concen trations in plasma, eyes and brain of GES-treated rats Table 2 shows observed concentrations of amino acids in plasma, eyes and brain of each group at 21 days after infusion of saline or taurine. A marked de ficiency of taurine in each tissue of the GES control animals was observed, in which taurine in each tissue was reduced to 20 30% of that of the food control group. Infusion of taurine improved the taurine concen tration in a dose-dependent manner. There were signifi cant differences in taurine concentration between the GES control group and each taurine-treated group. Taurine infusion at doses of 30 and 100 mg/animal/day increased the plasma taurine level up to levels similar
Fig. 1. Typical waveforms of electroretinogram evoked by a 10-joule flush light stimuli in dark-adapted rats fed an ordi nary laboratory chow (normal) or a casein-based diet (food control) with tap water or a casein-based diet with tap water containing 1% GES before and after infusion of saline (GES control) or 3 doses of taurine. Arrows indicate the a-wave and b-wave peaks, respectively. First ERG recordings were performed just before infusion of drugs in rats at 7 weeks of age. All waveforms were obtained by averaging 10 ERG recordings.
Table
2.
Effect
of GES-treated
of intravenous
infusion
of
taurine
on
amino
acids
concentration
of plasma,
eyes
and
brain
rats
and higher, respectively, compared with that of the food control group. On the other hand, taurine concen trations in the eyes and brain of taurine-treated groups were still lower than that of the food control group. With respect to the concentrations of other amino acids, decreased glutamic acid levels in the eyes were observed in the GES-treated animals compared with that of the food control group. In addition, a significant decrease of y-aminobutyric acid (GABA) concentration in the eyes of animals treated with 100mg/animal/day of taurine was observed compared with that of the GES control group. Effect of i.v. infusion of taurine on morphological changes in retinas of GES-treated rats Figure 2 shows light micrographs of retinas from the food control, GES control and each dose of taurine treated animals. Both outer and inner segments of the photoreceptor cell layer of the GES control animals appear to be degenerated in density and show disorga nization of the cells compared with that of the food control in which the photoreceptor cell layer structure was well-organized. Large vacuoles and cell loss in the pigment epithelium were also observed in the GES con
trol animals. Taurine-treated animals revealed photore ceptor cell layer and pigment epithelium structures similar to those of the food control, except some degen eration of outer segments and the large vacuoles in the pigment epithelium at a dose of 10 mg/animal/day and small vacuoles in the pigment epithelium at a dose of 30 mg/animal/day were observed.
DISCUSSION Continuous and intravenous infusion of taurine to GES-treated rats markedly improved the ERG abnor malities and taurine deficiency in the plasma, eyes and brain in a dose-dependent manner. Similarly, structural abnormalities in the retina were also observed in GES treated rats, and dose-dependent improvements were observed in taurine-treated animals. Taurine concentra tions in the eyes of the GES-control, 10, 30 and 100 mg/animal/day of taurine infusion groups, were 21.5, 30.8, 45.0 and 65.5%, respectively, of the level of the food control group. Whereas the recovery of ERG and retinal structure in animals treated with the largest dose of taurine was almost complete and the plasma concen tration of taurine was higher (147.5%) than that of the
Fig. 2. Light micrographs of the retinas in rats of the food control, GES control and 10, 30 or 100 mg/animal/day of taurine infusion groups. Photoreceptor cell layers in the GES-control appear to be less dense and show disorganization of the cells. The large vacuoles are localized in the piment epithelium of the GES control. Taurine-treated animals markedly improved these changes at doses of 30 and 100 mg/animal/day. PE, pigment epithelium; OS, photoreceptor outer seg ments; IS, photoreceptor inner segments. ON, outer nuclear layer; OP, outer plexiform layer; IN, inner nuclear layer; IP, inner plexiform layer; GC, ganglion cell layer.
food eyes
control group, the concentration was still lower. Although there
of taurine in the is no systematic
study concerning the correlation between levels and either ERG or retinal structure, suggest that the taurine concentration
tissue taurine these results in the eyes
obtained here at the largest dose of taurine may be close to the critical level necessary to maintain both electrical potential normal level.
and
structure
of the
retina
at the
On the other hand, treatment with GES and/or taurine did not affect the concentration of other neuroactive amino acids determined except the concen trations of glutamic acid and GABA in the eyes. There is no evidence why the changes of these two amino acids levels have been induced. However, these changes are not considered to be related directly to the ERG abnormality and taurine deficiency, because the largest difference in the glutamic acid concentration was observed between the normal and food control groups, and the GABA concentration was not affected by GES
treatment. Thus, the ERG disorder observed in GES treated rats is probably not caused by a direct action of either GES or other amino acids. In addition, the be neficial effect of taurine obtained here suggests that an active transport system of external taurine to the retina should be recovered by intravenous infusion of taurine even in the presence of GES. Decreased amplitudes and increased latencies of b and/or a-wave in the ERG of GES-treated rats have already been reported in many papers (28-33). Cocker and Lake (32) have reported that ERG changes in duced by 4 weeks treatment with GES are reversed by long-term (55 days) cessation, but not by short-term (20 days) cessation of GES-treatment. We produced taurine deficient rats by long-term treatment with GES includ ing a premature period, and the measured ERG re sponded to photostimuli of a relatively high intensity. As a result, amplitudes of both a and b-waves were de creased to 50% of the control values. In spite of the se vere ERG disorder observed here compared with other
studies, intravenous infusion of taurine exerted a recov ery effect on ERG in GES-treated animals, which was almost complete after a 21-day infusion of the largest taurine dose. Our findings suggest that infusion of taurine can be utilized for improving retinal function even under conditions of severe taurine deficiency. In the clinical study of Geggel et al. (34), children re ceiving total parenteral nutrition without taurine exhi bited a low concentration of plasma taurine (47% of normal) and a slight ERG abnormality, which reversed after the addition of taurine in doses of 150 to 187.5 mg/kg/day for 12 to 14 weeks. These clinical doses were close to the dose of 30 mg/animal/day used here. It is not easy to compare the clinical findings of Geggel et al. with the findings of our study because of the use of GES-treatment to produce taurine deficiency and the difference in the properties of retinal photoreceptor cells between humans and rats, etc. However, when taurine infusion is applied to animals with mild taurine deficiency, taurine at doses lower than those used in our study may be efficacious. The mechanism of the beneficial effect of taurine on retinal function is still unclear. In this study, amplitudes of a and b-waves in GES-treated rats were decreased by almost same degree so that the b/a ratio was not affected by either treatment with GES or infusion of taurine. Cocker and Lake (32) have also suggested from their kinetic analysis of ERGs that ERG abnor malities in GES-treated rats are induced by a simple change in the resistive pathway common to both a and b-waves, that is not a transmission system but the re sponsiveness of photoreceptor cells. In agreement with our results, several morphological studies have also shown that taurine deficiency induced by GES leads to the degeneration of pigment epithelium and photore ceptor cells, mainly the rod outer segment (24, 26-30), which is accompanied by ERG abnormalities (28-30). Since it has been suggested that taurine transport to the retina is regulated by pigment epithelium (35) and it is well-known that photoreceptor cells have well-de veloped ability for renewal of the cell membrane (36), infusion of taurine, therefore, is thought to exert a be neficial effect on the regenerative function of photore ceptor cells by recovering the taurine concentration in the retina via the pigment epithelium. This considera tion would be supported by the findings that light de privation has reduced the severity of ERG abnormali ties in GES-treated rats (30, 33) and that taurine pro tects against light-induced disruption in the rod outer segment (8). It is not clear how taurine infused intravenously ex erts its beneficial effect on the physiological and mor phological changes in the retina of GES-treated ani
mals. In any event, the protective effect of taurine on retinal dysfunction observed in taurine deficiency may be expected in the case of long-term total parenteral nutrition containing taurine to human infants that lack enzymes for taurine synthesis (37). REFERENCES
1 Jacobsen, J.G. and Smith, L.H.: Biochemistry and physiology of taurine and taurine derivatives. Physiol. Rev. 48, 424-511 (1968) 2 Lake, N., Marshall, J. and Voaden, M.J.: High affinity up take sites for taurine in retina. Exp. Eye Res. 27, 713-718 (1978) 3 Schmidt, S.Y. and Berson, E.L.: Taurine uptake in isolated retinas of normal rats and rats with hereditary retinal degen eration. Exp. Eye Res. 27, 191-198 (1978) 4 Salceda, R.: High-affinity taurine uptake in developing re tina. Neurochem. Res. 5, 561-571 (1980) 5 Pasantes-Morales, H., Klethi, J., Urban, P.F. and Mandel, P.: Light stimulated release of 35S-taurine from chicken re tina. Brain Res. 51, 375-378 (1973) 6 Schulze, E. and Neuhoff, V.: Artificial release of 3H-taurine after electrical stimulation of retinae. Int. J. Neurosci. 21, 15-23 (1983) 7 Smith, L.F.P. and Pycock, C.J.: Potassium-stimulated release of radio-labeled taurine and glycine from the isolated rat re tina. J. Neurochem. 39, 653-658 (1982) 8 Pasantes-Morales, H., Ademe, R.M. and Quesada, 0.: Pro tective effect of taurine on the fight-induced disruption of iso lated frog rod outer segment. J. Neurosci. Res. 6, 337-348 (1981) Pasantes-Morales, H. and Cruz, C.: Protective effect of taurine and zinc on peroxidation-induced damage in photore ceptor outer segments. J. Neurosci. Res. 11, 303-311 (1984) 10 Lopez-Escalera, R., Moran, J. and Pasantes-Morales, H.: Taurine and nifedipine protect retinal rod outer segment structure altered by removal of divalent cations. J. Neurosci. Res. 19, 491 496 (1988) 11 Pasantes-Morales, H.: Taurine function in excitable tissues: the retina as a model. In Retinal Transmitters and Modula tors: Models for the Brain, Edited by Morgan, W.W., Vol. 2, 9
p. 33-62, CRC Press, Boca Raton (1987) Pasantes-Morales, H. and Cruz, C.: Taurine: a physiological stabilizer of photoreceptor membranes. In Taurine: Biological Actions and Clinical Perspectives, Edited by Oja, S.S., Ahtee, L. and Paasonen, M.K., p. 371-381, Alan R. Liss, New York (1985) 13 Schmidt, S.Y., Berson, E.L. and Hayes, K.C.: Retinal de 12
generation in cats fed casein: I. Taurine deficiency. Invest. Ophthalmol. 15, 45-52 (1976) 14 Berson, E.L., Hayes, K.C., Rabin, A.R., Schmidt, S.Y. and Watson, G.: Retinal degeneration in cats fed casein. II. Sup plementation with methionine, cysteine, or taurine. Invest. Ophthalmol. 15, 52-58 (1986) 15 Schmidt, S.Y., Berson, E.L., Watson, G. and Huang, C.: Retinal degeneration in cats fed casein. III. Taurine deficien cy and ERG amplitudes. Invest. Ophthalmol. Vis. Sci. 16, 673 678 (1977)
16 Imaki, H., Moretz, R., Wisniewski, M., Neuringer, M. and Sturman, J.: Retinal degeneration in 3-month-old rhesus monkey infants fed a taurine-free human infant formula. J. Neurosci. Res. 18, 602-614 (1987) 17 Neuringer, M., Imaki, H., Sturman, J., Moretz, R. and Wisniewski, M.: Abnormal visual acuity and retinal morphol ogy in rhesus monkey fed a taurine-free diet during the first three postnatal months. In Advances in Experimental Medi cine and Biology, Edited by Huxtable, R.J., Franconi, F. and Giotti, A., Vol. 217, p. 125-134, Plenum Press, New York (1987) 18 Jacobsen, J.G., Thomas, L.L. and Smith, L.H.: Properties and distribution of mammalian L-cysteine sulfinate carboxy lyases. Biochim. Biophys. Acta 85, 103-116 (1964) 19 Hardison, W.G.M., Wood, C.A. and Proffitt, J.H.: Quanti fication of taurine synthesis in the intact rat and cat liver. Proc. Soc. Exp. Biol. Med. 155, 55-58 (1977) 20 Huxtable, R.J., Laird, H.E. and Lippincott, S.: The trans
21 22
23
24
25
26
port of taurine in the heart and the rapid depletion of tissue taurine content by guanidinoethyl sulfonate. J. Pharmacol. Exp. Ther. 211, 465-471 (1979) Lake, N.: Depletion of retinal taurine by treatment with guanidinoethyl sulfonate. Life Sci. 29, 445-448 (1981) Huxtable, R.J.: Guanidinoethane sulfonate and the disposi tion of dietary taurine in the rat. J. Nutr. 112, 2293-2300 (1982) Marnela, K.-M., Kontro, P. and Oja, S.S.: Effects of pro longed guanidinoethanesulphonate administration on taurine and other amino acids in rat tissues. Med. Biol. 62, 239-244 (1984) Bonhaus, D.W., Pasantes-Morales, H. and Huxtable, R.J.: Actions of guanidinoethane sulfonate on taurine concentra tion, retinal morphology and seizure threshold in the neonatal rat. Neurochem. Int. 7, 263-270 (1985) Ejiri, K., Akahori, S., Kudo, K., Sekiba, K. and Ubuka, T.: Effect of guanidinoethyl sulfonate on taurine concentrations and fetal growth in pregnant rats. Biol. Neonate 51, 234-240 (1987) Pasantes-Morales, H., Quesada, A., Carabez, A. and Huxt able, R.J.: Effects of taurine transport antagonist, guanidino ethane sulfonate, and 9-alanine on the morphology of rat re tina. J. Neurosci. Res. 9, 135-143 (1983)
27
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34
35
36 37
Lake, N. and Malik, N.: Retinal morphology in rats treated with a-taurine transport antagonist. Exp. Eye Res. 443, 331 346 (1987) Hageman, G.S. and Schmidt, S.Y.: Taurine-deficient pig mented and albino rats: early retinal abnormalities and dif ferential rates of photoreceptor degeneration. In Degenera tive Retinal Disorders: Clinical and Experimental Laboratory Investigations, Edited by Hollyfield, J.G., LaVail, M.M. and Anderson, R.E., p. 497-515, Alan R. Liss, New York (1987) Rapp, L.M., Thum, L.A., Tarver, A.P. and Wiegand, R.D.: Retinal changes associated with taurine depletion in pig mented rats. In Degenerative Retinal Disorders: Clinical and Experimental Laboratory Investigations, Edited by Holly field, J.G., LaVail, M.M. and Anderson, R.E., p. 485-496, Alan R. Liss, New York (1987) Rapp, L.M., Thum, L.A. and Anderson, R.E.: Synergism between environmental lighting and taurine depletion in caus ing photoreceptor cell degeneration. Exp. Eye Res. 46, 229 238 (1988) Lake, N.: Electroretinographic deficits in rats treated with guanidinoethyl sulfonate, a depletor of taurine. Exp. Eye Res. 42, 87 91 (1986) Cocker, S.E. and Lake, N.: Electroretinographic alterations and their reversal in rats treated with guanidinoethyl sulfon ate, a taurine depletor. Exp. Eye Res. 45, 977-987 (1987) Quesada, O. and Pasantes-Morales, H.: Effect of light de privation on the ERG responses of taurine-deficient rats. Exp. Eye Res. 46, 13 20 (1988) Geggel, H.S., Ament, M.E., Heckenlively, J.R., Martin, D.A. and Kopple, J.D.: Nutritional requirement for taurine in patients receiving long-term parenteral nutrition. N. Engl. J. Med. 312, 142-146 (1985) Miller, S. and Steinberg, R.H.: Transport of taurine, L methionine and 3-O-methyl-D-glucose across frog retinal pig ment epithelium. Exp. Eye Res. 23, 177 189 (1976) Young, W.R.: The renewal of photoreceptor outer segments. J. Cell Biol. 33, 61 -72 (1967) Gaull, G.E., Sturman, J.A. and Raiha, N.C.R.: Develop ment of mammalian sulfur metabolism: Absence of cys tathionase in human fetal tissues. Pediatr. Res. 6, 538 547 (1972)
Effect
of Intravenous
on Electroretinographic Chiaki
Shimada,
Disorder Shuichi
Kiyotsugu Research
and
Development
Center,
Fuso
Received
Tanaka,
Isaka,
November
Hasegawa,
Sano and Hiromasa
Industries,
12, 1991
Infusion
in Taurine
Michinori
Mitsuo
Pharmaceutical
Taurine
Ltd.,
Accepted
2-3-11, January
Deficient Sumio
Rats
Kuroda,
Araki
Morinomiya,
Joto-ku,
Osaka
536, Japan
22, 1992
ABSTRACT-We investigated the effect of intravenous taurine infusion on the electroretinogram (ERG) of taurine-deficient rats produced by treatment with guanidinoethyl sulfonate (GES), a taurine transport inhibitor. Mother rats were fed a taurine-free diet and given drinking water containing 1% GES from 2 weeks of gestation to weaning. The same feeding conditions were applied to male offspring after weaning. Both ERG measurement and continuous infusion of taurine at a dose of 10, 30 or 100 mg/animal/day were performed for 3 weeks from 7 to 10 weeks of age. GES-treatment reduced a and b-wave amplitudes to 50% of the control levels and also increased b-wave latencies. Intravenous infu sion of taurine improved these ERG abnormalities in a dose-dependent manner. Taurine concentrations in plasma, eyes and brain were also decreased by treatment with GES, and dose-dependent recovery was observed after infusion with taurine, although the concentrations of other amino acids were not affected by GES-treatment and infusion of taurine. Observations of morphological changes revealed that the retinal damage in GES-treated animals was decreased by taurine infusion. These results indi cate that the changes in ERG and retinal structure observed in taurine deficiency are improved by in travenous infusion of taurine. Keywords:
Taurine,
Electroretinogram,
Guanidinoethyl
Taurine, a sulfur-containing amino acid, has been re ported to distribute to many excitable tissues as a free amino acid (1). Retina is one of the sites that take up taurine with a high affinity (2-4). It has been reported that the release of taurine from a retinal preparation is induced by light stimulation (5), electrical stimulation to photoreceptor cells (6) or addition of K+ ions to the medium (7), and that taurine exhibits protective effects against the degeneration of photoreceptor cells caused by light stimulation (8), superoxide (9) or osmotic im balance (10). Therefore, taurine is thought to be re leased by certain light stimuli and to exhibit a protec tive role against photochemical damage of the photore ceptor membrane (11, 12). It has been well-documented that morphological de generation of the retina and concomitant disorder of the electroretinogram (ERG) or visual acuity are observed in cats and rhesus monkeys fed taurine-free diets during their infancy (13-17). In general, because of the well-developed ability of taurine synthesis in rats (18, 19), it is not possible to produce taurine-deficient
sulfonate,
Retina,
Taurine
deficient
rat
rats only by elimination of dietary taurine. However, treatment of rats with guanidinoethyl sulfonate (GES), a transport inhibitor of taurine, has been reported to elicit taurine depletion in many tissues including the retina (20-25), in which there is retinal degeneration, especially in the photoreceptor cells (24, 26, 27). Fur thermore, these taurine-depleted rats exhibit ERG abnor malities characterized by reduced amplitudes in the b and/or a-waves (28-33). Whereas it is obvious that these retinal disorders are elicited by GES in rats, it is yet not known if administration of taurine can normal ize the decreased tissue taurine levels as well as the ERG disorder in taurine-deficient animals. On the other hand, it has been reported that a slight ERG dis order and low plasma taurine levels in human infants treated with taurine-free amino acid solutions in long term parenteral nutrition are normalized by addition of taurine to the solution (34). In this study, we investigated whether or not taurine infusion in taurine deficient rats produced by long-term treatment with GES could improve the taurine deple
tion and structure.
concomitant
disorders
in ERG
and
retinal
MATERIALS AND METHODS Chemicals and animals GES used this study was synthesized by the method of Huxtable et al. (20). The drug was confirmed to be 99.9% pure by thin layer chromatography and con tained less than 0.01% taurine, which was determined with an amino acid analyzer (LP-8500, Hitachi). Female Sprague-Dawley rats at 2 weeks of gestation (Charles River Japan) were assigned to 3 feeding condi tion groups: 1) normal animals fed an ordinary labora tory chow (MF, Oriental Yeast) and tap water, 2) food control animals fed a casein-based diet and tap water, and 3) GES-treated animals fed a casein-based diet and tap water containing 1% GES. After weaning at 21 days of age, the same feeding conditions were applied to the male offspring. These rats had free access to food and water, and they were housed 5 per cage under a cycle of 12 hr light (300 lux)/ 12 hr dark at a room temperature of 23 ± 2°C and the humidity of 55 ± 5%. Measurement of ERG At 7 weeks of age, animals weighing 175 -277 g in each feeding condition were used for the first ERG re cordings. On the day of ERG recording, animals moved from the overnight dark room into the dark room with indirect red dim light were anesthetized with an i.p.-injection of 40 mg/kg sodium pentobarbital (Dai-Nippon Pharmaceuticals). They were fixed to a stereotaxic apparatus with a heating pad (Deltaphase, Braintree Scientific) which kept the body temperature constant at 37°C. The left pupil was dilated by 0.1% atropine solution (atropine sulfate, Sigma). A bipolar contact lens electrode (Kyoto Contact Lens) was attached to the left eye ball, and active and indifferent electrodes were placed on the cornea and around the sclera, respectively. A Xenon lamp unit was placed 50 cm above the left eye ball and connected to a photosti mulator (3G22, San-ei). Electrical potentials responded to 10 times photostimuli of a 10 joule intensity, 0.1 msec duration and 60 sec intervals were amplified using a bioelectric amplifier (1253A, San-ei) with a 0.1 sec time constant and a 1 kHz high cut filter. All wave forms were taken into an analog-to-digital converter (PCN-2198, Neolog) connected to a personal computer (PC-9801RX2, NEC). Waveforms acquired in each animal were averaged from 10 recordings with software for this purpose (Biosignal analyzer, Medical Research Equipment). Amplitudes and peak latencies of both a and b-waves were automatically measured from aver
aged waveforms and the ratio of b-wave amplitude and a-wave amplitude (b/a ratio) was calculated. Administration of taurine Just after the first ERG recordings, a sterilized sili cone tube with a 0.5-mm inner diameter (Dow Corn ing) was inserted into the right jugular vein for con tinuous infusion of taurine. The tube was covered by a spring coil and connected to a 50-m1 syringe through a cannula swivel which allowed the rat to move freely. Animals treated with GES were divided into 4 groups; one of the 4 groups was infused with saline, and the other groups were infused with taurine dissolved in saline at doses of 10, 30 and 100 mg/animal/day, re spectively, at the rate of 0.1 ml/hr with an infusion pump (STC-501; Terumo). Normal and food control animals were infused with saline at the same infusion rate. Continuous infusion of saline or taurine solution were made for 21 days. The ERG recordings described above were also performed at 7, 14 and 21 days after the start of infusion. Feeding conditions described above were continued for each animal during the infu sion. Any animal in which disconnection or occlusion of the infusion tube occurred was eliminated from the study, and 7 or 8 animals per group were finally used. Assay of amino acid After the last ERG recordings, arterial blood was withdrawn from the abdominal aorta with a heparinized syringe, and the plasma was obtained by centrifugation (3000 rpm, 10 min at 4°C). Eyes and the brain without cerebellum and olfactory bulb were isolated after perfu sion with ice-cold saline. Deproteinization was per formed by addition of an equal, 10-times or 5-times volume of ice-cold 5% sulfosalicylic acid solution to the plasma, eyes or brain, respectively. The eyes and brain were homogenized with an ultrasonic homogenizer (Physcotron, Nichi-on Irikakikai). Each deproteinized suspension was centrifuged (3000 rpm, 15 min at 4°C), and the supernatant was separated as a sample for quantitative assay of amino acids using an amino acid analyzer (LP-8500, Hitachi). Morphological evaluation To determine the morphological influence of taurine deficiency in the eyes, animal models like those used for the ERG recording were produced. After the infu sion of taurine or saline, 2 4 rats per group were per fused with 0.5 1/kg of 10 mM phosphate-buffered saline, and the tissues were fixed with perfusion of 1 l/kg of 0.1 M phosphate buffer containing 4% paraformalde hyde, 0.2% picric acid and 0.35% glutaraldehyde. The eyes were removed from the animals, and the eyecups
were isolated. They were postfixed for 2 days in the fixative without glutaraldehyde and immersed for 2 days in 0.1 M phosphate buffer containing 15% sucrose. For light microscopy, the paraffin sections were prepared according to the usual method and stained by hema toxylin and eosin (HE) staining solution. Statistical analysis All data represent the mean ± standard error of 7 or 8 observations. Statistical evaluation was performed by the unpaired Student's t-test or Welch's test.
Table
1.
Effect
of intravenous
infusion
of taurine
RESULTS Effect of i. v. infusion of taurine on the ERG of GES treated rats Changes in body weight during the infusion were not significantly different among the 6 groups (data not shown). Table 1 shows the amplitudes and latencies of both a and b-waves and the b/a ratio for amplitude in the ERG measured before and 7, 14 and 21 days after infusion of drugs in each group. There were no signifi cant differences in any ERG parameters between the normal and food control groups. Marked decrease in
on
ERG
parameters
in GES-treated
rats
amplitudes of both a and b-waves, which reduced to 50% of those of the normal or food control group, and increased latencies of b-wave were observed in GES treated animals before infusion of taurine. In GES-treat ed animals infused with taurine, as compared with the animals infused with saline (GES control), significant increases in amplitudes of both waves and a decrease in latencies of b-waves were observed at 14 and 21 days after infusion of taurine at doses of 30 and 100 mg/animal/day. A slight but not significant increase in amplitudes of both waves was observed at a dose of 10 mg/animal/day. The increases of amplitudes of both waves in animals treated with taurine were dose-de pendent at 21 days after infusion. Especially at a dose of 100 mg/animal/day, significant increases in ampli tudes of both waves were observed at 7 days after the infusion, and the b-wave latency at 14 days and ampli tude at 21 days recovered to the levels of the food con trol group. Significant differences in the b/a ratio be tween taurine-treated and food control groups were observed at 7 days, but not at 14 and 21 days after the
infusion. The average waveforms observed in the 6 groups are shown in Fig. 1. These waveforms reveal the ERG disorder in GES-treated animals (all waves for the GES control and waves before taurine infusion). Dose-dependent recovery of the ERG was observed in animals infused with taurine. Effect of i.v.-infusion of taurine on amino acid concen trations in plasma, eyes and brain of GES-treated rats Table 2 shows observed concentrations of amino acids in plasma, eyes and brain of each group at 21 days after infusion of saline or taurine. A marked de ficiency of taurine in each tissue of the GES control animals was observed, in which taurine in each tissue was reduced to 20 30% of that of the food control group. Infusion of taurine improved the taurine concen tration in a dose-dependent manner. There were signifi cant differences in taurine concentration between the GES control group and each taurine-treated group. Taurine infusion at doses of 30 and 100 mg/animal/day increased the plasma taurine level up to levels similar
Fig. 1. Typical waveforms of electroretinogram evoked by a 10-joule flush light stimuli in dark-adapted rats fed an ordi nary laboratory chow (normal) or a casein-based diet (food control) with tap water or a casein-based diet with tap water containing 1% GES before and after infusion of saline (GES control) or 3 doses of taurine. Arrows indicate the a-wave and b-wave peaks, respectively. First ERG recordings were performed just before infusion of drugs in rats at 7 weeks of age. All waveforms were obtained by averaging 10 ERG recordings.
Table
2.
Effect
of GES-treated
of intravenous
infusion
of
taurine
on
amino
acids
concentration
of plasma,
eyes
and
brain
rats
and higher, respectively, compared with that of the food control group. On the other hand, taurine concen trations in the eyes and brain of taurine-treated groups were still lower than that of the food control group. With respect to the concentrations of other amino acids, decreased glutamic acid levels in the eyes were observed in the GES-treated animals compared with that of the food control group. In addition, a significant decrease of y-aminobutyric acid (GABA) concentration in the eyes of animals treated with 100mg/animal/day of taurine was observed compared with that of the GES control group. Effect of i.v. infusion of taurine on morphological changes in retinas of GES-treated rats Figure 2 shows light micrographs of retinas from the food control, GES control and each dose of taurine treated animals. Both outer and inner segments of the photoreceptor cell layer of the GES control animals appear to be degenerated in density and show disorga nization of the cells compared with that of the food control in which the photoreceptor cell layer structure was well-organized. Large vacuoles and cell loss in the pigment epithelium were also observed in the GES con
trol animals. Taurine-treated animals revealed photore ceptor cell layer and pigment epithelium structures similar to those of the food control, except some degen eration of outer segments and the large vacuoles in the pigment epithelium at a dose of 10 mg/animal/day and small vacuoles in the pigment epithelium at a dose of 30 mg/animal/day were observed.
DISCUSSION Continuous and intravenous infusion of taurine to GES-treated rats markedly improved the ERG abnor malities and taurine deficiency in the plasma, eyes and brain in a dose-dependent manner. Similarly, structural abnormalities in the retina were also observed in GES treated rats, and dose-dependent improvements were observed in taurine-treated animals. Taurine concentra tions in the eyes of the GES-control, 10, 30 and 100 mg/animal/day of taurine infusion groups, were 21.5, 30.8, 45.0 and 65.5%, respectively, of the level of the food control group. Whereas the recovery of ERG and retinal structure in animals treated with the largest dose of taurine was almost complete and the plasma concen tration of taurine was higher (147.5%) than that of the
Fig. 2. Light micrographs of the retinas in rats of the food control, GES control and 10, 30 or 100 mg/animal/day of taurine infusion groups. Photoreceptor cell layers in the GES-control appear to be less dense and show disorganization of the cells. The large vacuoles are localized in the piment epithelium of the GES control. Taurine-treated animals markedly improved these changes at doses of 30 and 100 mg/animal/day. PE, pigment epithelium; OS, photoreceptor outer seg ments; IS, photoreceptor inner segments. ON, outer nuclear layer; OP, outer plexiform layer; IN, inner nuclear layer; IP, inner plexiform layer; GC, ganglion cell layer.
food eyes
control group, the concentration was still lower. Although there
of taurine in the is no systematic
study concerning the correlation between levels and either ERG or retinal structure, suggest that the taurine concentration
tissue taurine these results in the eyes
obtained here at the largest dose of taurine may be close to the critical level necessary to maintain both electrical potential normal level.
and
structure
of the
retina
at the
On the other hand, treatment with GES and/or taurine did not affect the concentration of other neuroactive amino acids determined except the concen trations of glutamic acid and GABA in the eyes. There is no evidence why the changes of these two amino acids levels have been induced. However, these changes are not considered to be related directly to the ERG abnormality and taurine deficiency, because the largest difference in the glutamic acid concentration was observed between the normal and food control groups, and the GABA concentration was not affected by GES
treatment. Thus, the ERG disorder observed in GES treated rats is probably not caused by a direct action of either GES or other amino acids. In addition, the be neficial effect of taurine obtained here suggests that an active transport system of external taurine to the retina should be recovered by intravenous infusion of taurine even in the presence of GES. Decreased amplitudes and increased latencies of b and/or a-wave in the ERG of GES-treated rats have already been reported in many papers (28-33). Cocker and Lake (32) have reported that ERG changes in duced by 4 weeks treatment with GES are reversed by long-term (55 days) cessation, but not by short-term (20 days) cessation of GES-treatment. We produced taurine deficient rats by long-term treatment with GES includ ing a premature period, and the measured ERG re sponded to photostimuli of a relatively high intensity. As a result, amplitudes of both a and b-waves were de creased to 50% of the control values. In spite of the se vere ERG disorder observed here compared with other
studies, intravenous infusion of taurine exerted a recov ery effect on ERG in GES-treated animals, which was almost complete after a 21-day infusion of the largest taurine dose. Our findings suggest that infusion of taurine can be utilized for improving retinal function even under conditions of severe taurine deficiency. In the clinical study of Geggel et al. (34), children re ceiving total parenteral nutrition without taurine exhi bited a low concentration of plasma taurine (47% of normal) and a slight ERG abnormality, which reversed after the addition of taurine in doses of 150 to 187.5 mg/kg/day for 12 to 14 weeks. These clinical doses were close to the dose of 30 mg/animal/day used here. It is not easy to compare the clinical findings of Geggel et al. with the findings of our study because of the use of GES-treatment to produce taurine deficiency and the difference in the properties of retinal photoreceptor cells between humans and rats, etc. However, when taurine infusion is applied to animals with mild taurine deficiency, taurine at doses lower than those used in our study may be efficacious. The mechanism of the beneficial effect of taurine on retinal function is still unclear. In this study, amplitudes of a and b-waves in GES-treated rats were decreased by almost same degree so that the b/a ratio was not affected by either treatment with GES or infusion of taurine. Cocker and Lake (32) have also suggested from their kinetic analysis of ERGs that ERG abnor malities in GES-treated rats are induced by a simple change in the resistive pathway common to both a and b-waves, that is not a transmission system but the re sponsiveness of photoreceptor cells. In agreement with our results, several morphological studies have also shown that taurine deficiency induced by GES leads to the degeneration of pigment epithelium and photore ceptor cells, mainly the rod outer segment (24, 26-30), which is accompanied by ERG abnormalities (28-30). Since it has been suggested that taurine transport to the retina is regulated by pigment epithelium (35) and it is well-known that photoreceptor cells have well-de veloped ability for renewal of the cell membrane (36), infusion of taurine, therefore, is thought to exert a be neficial effect on the regenerative function of photore ceptor cells by recovering the taurine concentration in the retina via the pigment epithelium. This considera tion would be supported by the findings that light de privation has reduced the severity of ERG abnormali ties in GES-treated rats (30, 33) and that taurine pro tects against light-induced disruption in the rod outer segment (8). It is not clear how taurine infused intravenously ex erts its beneficial effect on the physiological and mor phological changes in the retina of GES-treated ani
mals. In any event, the protective effect of taurine on retinal dysfunction observed in taurine deficiency may be expected in the case of long-term total parenteral nutrition containing taurine to human infants that lack enzymes for taurine synthesis (37). REFERENCES
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