Exp. Eye Rrs. (1990) 50, 647-649

Confirmation ARITAKE Department

of Lens Hydration MIZUNOab,


by Raman




of a Biochemistry, b Ophthalmology and c Internal Medicine, The Jikei Medicine, Nishi- Shinbashi, Minato - ku, Tokyo 7 05, Japan

MORI” University



Lenshydration was monitoredby laserRamanspectroscopyin WBN/Kob rats which form spontaneous diabetesat about 1 yr of age and developcataractsapproximately 6 monthsafter the onsetof diabetes. Lenshydration in responseto chronic hyperglycemicstressin theserats appearedpredominantly in the cortical region while the hydration state in the nuclear region was fairly preserved.The 9-month treatment of similarlydiabeticrats with the aldosereductaseinhibitor Ponalrestatsufficientlysuppressed lenshydration in the cortical and nuclear regions. Key words: Raman spectroscopy:diabetic cataract: lens hydration ; WBN/Kob rat: spontaneous diabetes. 1. Introduction Laser Raman spectroscopy is a powerful non-destructive technique for probing cataractous or noncataractous lenses.By measuring the Raman spectrum of the lens, changes in the secondary structure and information about protein subgroups such as tryptophan, tyrosine, sulfhydryl and disulfide can be monitored in situ. Lens hydration is a major factor in many types of cataract formation, especially diabetic cataracts. It has been experimentally observed in galactosemic and diabetic animals that the accumulation of polyols in the lenses of these animals produces hyperosmotic effects which result in increases of lens water and subsequent lens opacification (Dvornik et al., 1973: Kinoshita, 1974). Polyols accumulate in the lens when excess aldose sugars such as galactose and glucose are reduced by aldose reductase. Inhibitors of aldose reductase have an anti-cataract effect on these sugar cataracts (Varma and Kinoshita, 19 76). It has been reported that measuring the intensity of the Raman band due to the hydroxyl stretching mode of lens water (3390 cm-‘) by laser Raman spectroscopy provides a useful marker for lens hydration in precataractous or cataractous lenses (Iriyama et al., 1982). With this technique, we have reported dramatic increasesin lens water in the cataractous lenses of streptozotocin-induced diabetic rats and observed the prevention of lens hydration by aldose reductase inhibitor (Mizuno et al., 1987; Nozawa, Yaginuma and Mizuno, 1988). It has been reported that the WBN/Kob-strain of male rats is a new diabetic animal model which forms spontaneous diabetes mellitus with age (Nakayama et al., 1985; Tsuchitani et al., 1985; Mori et al., 1988a). This strain is derived from a Wistar rat colony which originated at the Institute of Gerontology in Base1and was maintained at the Institute of Pathology at the University of Bonn (Kobori, Gedigk and Totovic, 19 7 7). Diabetesmellitus spontaneously develops in an inherited manner only in the aging male rats. The 0014-4~35/90/0~0~47+03


onset of diabetes in these males occurs after 9 months of age, and by 15 months of age 85 % of male rats develop diabetes. Lens opacities begin to appear 6 months after the onset of diabetes, and by 23 months of age nearly 100% of these rats develop totally opaque cataracts (Mori et al., 1988b). In this study we have investigated the diabetic lenses from these animals with laser Raman spectroscopy and monitored the effects of the aldose reductase inhibitor Ponalrestat. 2. Materials and Methods The WBN/Kob-strain of rats were supplied by the Shizuoka Laboratory Animal Center, Shizuoka, Japan. The animals were housed in a barrier maintained animal room and fed ad libitum standard rat chow with/without the aldose reductase inhibitor (ARI) Ponalrestat (ICI 128 436/MK-538 from the ICI Pharmaceutical Co., Ltd, U.K.) which was added to the rat chow at a concentration of O-046 ‘$&Eighty percent of male WBN/Kob rats developed diabetes by 14 months of age, and these diabetic male rats were selected and randomly placed into two groups, with one receiving the AR1 while the other served as a nontreated diabetic control. The rats were kept until they were 23 months old at which time all non-treated rats had developed total cataracts. Glucose levels in urine samplesfrom these rats were monitored biweekly with Test-Tape. Plasma glucose levels were determined -by the glucose-oxidase method. Average plasma glucose levels were 569 + 88 mg dl-’ for the non-treated group and 5 53 + 59 mg dl-’ for the AR&treated group, at the age of 23 months. There was no significant difference between the two groups. All eyes were enucleated under anesthesia with a lethal dose of pentobarbital. The lens was gently excised in HEPES buffered lens medium solution and transferred to the cuvette cell filled with HEPES buffered lens medium (Iwata and Takehana, 1980) where it was placed horizontally on the bottom of the 0 1990 AcademicPressLimited


cell. The incident laser beam was illuminated from the bottom and the scattered light was detected by the Raman spectrometer at 90” as previously described (Nozawa et al.. 1988). The Raman optical dissection technique for obtaining one-dimensional images of lens constituents along the horizontal axis (center to equator) was employed in this study. The Kaman band at 3390 cm~‘. assigned as the hydroxyl stretching mode. was used as a marker for lens hydration. ‘I’hc relative water content was estimated from the ratio of Raman intensities at 1559,,/f2935,where the band at 2 9 3 5 cm-’ represents the carbon-hydrogen stretching mode, mainly from lens proteins. The Raman spectra of clear or opaque lenses were obtained with a laser Raman spectrometer system consisting of a JASCO R 1100 double monochrometer, a Hamamatsu Photonics R 649 photomultiplier tube with cooling unit and a data processor. The excitation wavelength at 514.5 nm was provided by a NEC GLG 3200 argon-ion laser tube. 3. Results The cataractous lens from a 23-month-old WBN/ Kob rat is shown in Fig. 1(A). The lens opacity appears totally spread: however, the opacity at the center portion appears thinner than that of the cortex near the equator. In the early stagesof this diabetic cataract. the opacity appeared first in the cortex near the

FIG. 1. Lensesexcised from 23-month-old WBN/Kob diabeticrats. In (A) lensfrom a non-treated diabetic rat is illustrated, while (B) representsa lens from a diabetic rat treated with the aldosereductaseinhibitor Ponalrestat.



T w-e











1.2 0.9 mm








0.3 center

Fxc. 2. Relativewater content along the horizontal axis of rat lensesdetectedby the optical dissectionmethod using Ramanspectroscopywhere water content wasestimatedby the ratio of 13390/12935. The three groups examined were WBN/Kob rat lensesfrom non-treatedrats (m), WBN/Kob rat lensesfrom rats treated with Ponalrestat(a). and lenses from non-diabeticWistar rats (A). Eachpoint representsthe mean+ S.D.determinedfrom the centerof the nucleusto the equator on the horizontal axis (n = 4-6). equator and then spread superficially, while in the mid-stage the opacity appearedlocalized in the cortical portion. It might be suggestedthat the central nuclear region of the lens still maintained transparency. The Raman data by the optical dissection method confirmed that the nuclear region of the lensesfrom nontreated rats was only affected slightly by prolonged diabetic stress as reflected in lens hydration which appears only slightly increased (Fig. 2). The opaque cortical portion of the lens from non-treated rats showed increased hydration compared with normal. non-diabetic control rats (Fig. 2). This indicates that the lens fibres of the cortical region are hydrated to a greater extent than those in the nucleus in these lenses with prolonged exposure to hyperglycaemia. Figure l(B) shows the lens from a 23-month-old rat treated for 9 months with the aldose reductase inhibitor Ponalrestat. A small subcapsular opacity is visible in the posterior portion. but the transparency of the lens is fairly well preserved. The Raman analysis of lens hydration for the ARI-treated lenses indicated that the relative water content of diabetic lenses treated with Ponalrestat were reduced in all portions. especially the cortex, compared to lenses from nontreated diabetic rats. In the center of the nucleus, lens dehydration was almost completely preserved with AR1 treatment; however, the elevation of the lens water in response to prolonged hyperglycaemia was not fully suppressedin the cortical area. 4. Discussion Lens hydration is believed to be a good marker for lens opacification (Iriyama et al.. 1982). Lens hy-




dration has also been observed to be a good marker for investigating the pre-opacification stage of diabetic cataracts when vacuoles begin to form (Mizuno et al.. 1987). In human diabetic cataracts, opacities appear in various regions of the lens with posterior subcapsular or cortical opacities common in the early stages.In the cortical type of diabetic cataract. the opacities spread along lens fibres from the equator to the center. The lens cortex appears to be predominantly involved in human cataracts under long-term diabetic stress (Caird, Pirie and Ramsell, 1969). In streptozotocin-induced diabetes, the onset of diabetes is quite rapid and diabetic cataract formation appears to be relatively acute. Raman spectroscopy studies of these diabetic lenses revealed that lens hydration appeared first in the cortex and then in the nucleus (Mizuno et al., 1987: Nozawa et al.. 1988). Chronic diabetic stress on WBN/Kpb rat lenses resulted in the appearance of total cataracts [Fig. 1(A) ] and severe hydration in the cortical area. but the dehydration state in the nucleus was fairly well preserved (Fig. 2). These results suggested that the nuclear portion might still be transparent in such an opaque lens. It has been reported that many aldose reductase inhibitors can prevent the accumulation of polyols in the lens and suppress the formation of cataracts in galactosemic and diabetic animals (Dvornik et al., 1973 : Varma, Mikuni and Kinoshita, 1975 : Varma, Mizuno and Kinoshita 1977; Fukushi, Merola and Kinoshita, 1980). In streptozocin-induced diabetes, the formation of cataract and corresponding lens hydration monitored with Raman spectroscopy was completely suppressedby an aldosereductase inhibitor for 60 days after the onset of diabetes. After 60 days, all diabetic lenses became opaque and increased hydration in the nucleus region was detected by Raman spectroscopy. This acute onset of diabetes caused severe lens hydration both in the cortex and the nucleus (Mizuno et al., 1987: Nozawa et al., 1988). On the other hand, chronic hyperglycemic stress resulting from spontaneous diabetes in the WBN/Kob rat only produced severe hydration in the cortical region, and this was reduced in similar rats treated with the aldosereductase inhibitor Ponalrestat. The dehydration state in the nuclear portion was fairly well-preserved in these diabetic lenses,suggesting that the fibers in the nuclear region were not affected to the same extent

as those in the cortex

by the prolonged spon-

chronic hyperglycaemia stress resulting from taneous diabetes in the WBN/Kob rat.

Acknowledgment This work was supported in part by a grant from the Cooperative Kesearch Group from Jikel Ilniversity for 1988.

References Caird, F. I., Pirie. A. and Ramsell, T. G. (1969). Clinical aspect of cataract in diabetes. In Diabetes and the Eye. P. 12 7. Blackwell : Oxford. Dvornik, D.. Simard-Duquesne, N., Kraml, M.. Sestanj. K., Gabbay, K. H.. Kinoshita. J. H., Varma, S. D. and Merola, L. 0. (1973). Inhibition of aldose reductase in vivo. Science 182, 1146-7. Fukushi. S.. Merola, L. 0. and Kinoshita. 1. H. (1980). Altering the course of cataracts in diabetic rats. Irn~t. OphthalnloI. Vis. Sci. 19. 31 3-15. Iriyama. K.. Mizuno. A., Ozaki. Y.. Itoh. K. and Matsuzaki, H. I 1982). An application of laser Raman spectroscopy to the study of hereditary cataractous lens: on the Raman band for a diagnostic marker of cataractous signatures. (‘1Irr. E!jr Res. 2. 486-92. Iwata. S. and Takehana. M. (1980). Biochemical study of human cataractous lens (fourth report) Actn Sot. Ophthalrnol. jpn. 84. 2542-7. Kinoshita. J. H. (1974). Mechanism initiating cataract formation. Proctor Lecture. Invest. Ophthnlrnol. 1 3. i 13-24. Kobori, 0.. Gedigk. 0. and Totovic. V. ( 197,). Adenomatous changes and adenocarcinoma of glandular stomach in Wistar rats induced by N-methyl-N-nitro-N-nitrosoguanidine. An electron microscopic and histochemical study. Virchows. Arch. 373. 37-54. Mizuno. A., Nozawa. H.. Yaginuma. T.. Matsuzaki. H.. Ozaki, Y. and Iriyama. K. (1987). Effect of aldose reductase inhibitor on experimental diabetic cataract monitored by laser Raman spectroscopy. Esp. E!/r &s. 45. 185-6. Mori. Y.. Yokoyama, J.. Nishimura. M. and Ikeda. Y. (1988a). A new diabetic strain of rat with exocrine pancreatic insufficiency. In Frontiers in Dinbetic KPsrarch. Lessonfrotn Anitnal Dicrbetes II (Eds Shafrir. E. and Renold. A. E.). Pp. 324-6. John Libbey and Co.: London. Mori. Y.. Yokoyama. J.. Nishimura. M. and Ikeda. Y. (1988b). A new diabetic strain of rat with exocrine pancreatic insufficiency. In Diabetes Secondary to Pancreopathy (Eds Tiengo, A., Albert, K. G. M. M., Del Prato, S. and Vranic, M.). Elsevier: New York. Nakayama, K., Shichinoe, K., Kobayashi. K.. Naito. K..

Yasuhara, K. and Tobe. M. (1985). Spontaneous diabetic-like syndrome in WBN/Kob rats. Actn Dinbetol. Lat. 22. 335-42. Nozawa, H.. Yaginuma. T. and Mizuno, A. (1988). Raman spectroscopic study of the effect of aldose reductase inhibitor on experimental diabetic cataract. Acta Ser. Ophthalmol. jpn. 92, 194-201. Tsuchitani, M.. Saegusa. T.. Narama. I., Nishikawa. T. and Gonda. T. (1985). A new diabetic strain of rat (WBN/Kob).Laboratory Animals 19, 200-7. Varma. S. D. and Kinoshita, J. H. (1976). Inhibition of lens aldose reductase by flavonoids -their possible role in the preventionof diabeticcataracts.Biochem. Pharmacol. 25. 2505-l 3. Varma. S. D.. Mikuni, I. and Kinoshita, J. H. (1975). Flavonoids as inhibitors of lens aldose reductase. Science 188. 1215-17. Varma. S. D.. Mizuno. A. and Kinoshita, J. H. ( 1977). Diabetic cataracts and flavonoids. Science 195. 205-6.

Confirmation of lens hydration by Raman spectroscopy.

Lens hydration was monitored by laser Raman spectroscopy in WBN/Kob rats which form spontaneous diabetes at about 1 yr of age and develop cataracts ap...
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