galactose-induced cataract formation in guinea pigs: morphologic

Galactose-Induced Cataract Formation in Guinea Pigs:Morphologic Changes and Accumulation of Galactitol

Jasmina B. Mackic* Fred N. Ross-Cisneros* J. Gordon McComb,-\ Isaac Bekhor,%Martin H. Weiss,* Ram Kannan,% and Berislav V. Zlokovic*-f%

Purpose. To develop and characterize a new model of galactose-induced cataract formation inyoung, 3- to 4-week-old Hartley guinea pigs.

Methods. Experimental animals were fed 50% galactose in powdered guinea pig chow contain-ing 0.5 g ascorbate/kg diet. Control animals were fed normal powdered guinea pig chow (0.5 gascorbate/kg diet). Lenses from all animals were subjected to photo-slit-lamp examination,light microscopic analysis, and high-pressure liquid chromatography (HPLC) analysis of polyolcontent.

Results. Photo-slit-lamp examination indicated initial opacities in equatorial subcapsular re-gion between 3 and 5 days in all galactose-fed animals (20/20); opacities progressed toward theanterior pole when diet was extended to 14 days. Histologic analysis of the equatorial changesconfirmed progressive cataract formation consisting of small intrafibrillar vacuoles in thepreequatorial region (3 days), an increased number of enlarged and coalesced vacuoles (6days), and progressive tissue swellings with cellular disruption and signs of epithelial mul-tilayering (14 days). The anterior epithelium showed increased cell height and swelling after 3days of the galactose diet. HPLC analysis of lens tissue indicated progressive accumulation ofgalactitol, 18 mM after 3 days, which plateaued to about 30 mM between 6 and 14 days. Thelevel of myo-inositol dropped from a control value of 2.8 ± 0.7 mM to 1.5 ± 0.7 mM after 3days, and was nearly undetectable after 14 days of the galactose diet.

Conclusions. The current study suggests that the guinea pig model may serve as a valuable newtool to study sugar-induced cataract formation and to characterize the early morphologic andbiochemical events in cataractogenesis. Invest Ophthalmol Vis Sci. 1994; 35:804-810.

JCjxperimental models of the galactose-induced cata-ract have been used extensively to study the morpho-logic and biochemical changes in the lens during ca-taractogenesis. Lenticular changes in rats include vacu-olization and liquefaction of cortical fibers in theequatorial zone, multilayering of lens epithelium, lossof regular honeycomb-like structure,1 and increasedheight of the central lens epithelium.2 The accumula-

Fmm the * Department of Neurological Surgery and fDivision of Neurosurgery,Children's Hospital of IJOS Angeles, University of Southern California School ofMedicine, %Doheny Eye Institute, and ^Veterans Administration Outpatient Clinic,Los Angeles, California.Supported by the Hoover Foundation, the USC Faculty Research and InnovationFund, and National Institutes of Health grant EY09399.Submitted for publication July 9, 1993; revised October 4, 1993; accepted October22, 1993.Proprietary interest category: N.Reprint requests: Berislav V. Zlokovic, M.D., Ph.D., Department of NeurologicalSurgery, USC School of Medicine, 2025 Zonal Avenue, RMR 508, Los Angeles,CA 90033.

tion of galactitol, depletion of glutathione (GSH), cat-ion imbalance, decrease in the free amino acids pool,depletion of myo-inositol, and changes in phosphatemetabolism have all been demonstrated.3"5 It is be-lieved that the key event in the galactose-induced cata-ract is the activation of the polyol pathway, with con-version of galactose into galactitol by aldose reductase.Because the cellular membranes of the lens are imper-meable to galactitol, this leads to hyperosmotic cellswelling, which produces scattering of light and dimin-ished lens transparency.3"5

Guinea pigs, like rats, have a high aldose reductaseactivity in the lens,6 but, like humans, they rely on di-etary supplementation of ascorbic acid,7 making thisspecies suitable for the study of the role of ascorbicacid in sugar-induced cataractogenesis. Previous stud-ies in guinea pigs used different galactose diets in com-bination with low89 or normal10" ascorbic acid con-

804Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3Copyright © Association for Research in Vision and Ophthalmology

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Galactose Cataract in Guinea Pigs 805

tent to produce cataract. Findings from our labora-tory indicate that cataract formation in galactosemicguinea pigs is associated with altered lens GSH homeo-stasis.10" This study examines the time-course-re-lated morphologic changes and rate of galactitol accu-mulation in the lens of guinea pigs fed 50% galactosewith a normal content of ascorbate in the diet.

MATERIALS AND METHODS

All procedures used in these studies were approved bythe University of Southern California InstitutionalAnimal Care and Use Committee, and were in accor-dance with the NIH guidelines and the ARVO Resolu-tion on the Use of Animals in Research. Hartleyguinea pigs (Charles River Farms, Ballardville, MA) ofeither sex, 3 to 4 weeks old, and weighing 225 to 250 g,were fed a 50% galactose diet in powdered Teklad(Harlan-Teklad, Madison, WI) guinea pig laboratorychow containing 0.5 g/kg of ascorbic acid. Controlanimals were fed powdered guinea pig Teklad chowcontaining 0.5 g/kg of ascorbic acid. A slit-lamp bio-microscope retroillumination examination was per-formed before the start of the diet to exclude animalswith preexisting ocular lesions. Slit-lamp examinationswere performed under light ketamine anesthesia (15mg/kg body weight) using 1% tropicamide sterile oph-thalmic solution to dilate the pupils in control andexperimental animals every second day during the 14days of the diet. At 3, 6, and 14 days, both eyes in theexperimental and control groups were enucleated us-ing deep xylazine:ketamine (7:35 mg/kg body weight)anesthesia, before sacrifice. The lenses were preparedfor histologic and biochemical analyses as describedbelow.

Histologic Analysis

A 5- to 6-mm posterior scleral incision was made toremove the vitreous body, and the rest of the eye wasinitially immersed into the fixative (2.5% glutaralde-hyde, 4% sucrose in 0.05 M cacodylate buffer) for 24hours, followed by rinsing in 0.1 M cacodylate buffer,and then postfixing for several days in 10% formalde-hyde in neutral phosphate-buffered saline (PBS). Thelenses were sagittally cut in half and fixed in 4% glutar-aldehyde in cacodylate buffer for several days, fol-lowed by rinsing in 0.1 M cacodylate buffer overnight.Tissues were dehydrated through a series of alcohols,and embedded using a Historesin kit (Reichert-Jung,Heidelberg, Germany). Semithin (2-iim) sagittal sec-tions were cut with a glass knife and stained with he-matoxylin and eosin or toluidine-blue. Fibers in theequatorial region and portions of anterior and poste-rior lens cortex were sectioned longitudinally. Black-and-white photographs were taken using a Nikon (To-kyo, Japan) Microphot-FX. Computer-aided imaging

(Jandel-JAVA [San Francisco, CA] image analysis sys-tem) was used to determine the height of the centralanterior lens epithelium.

High-Pressure Liquid ChromatographyAnalysis of Polyols

A previously described high-pressure liquid chroma-tography (HPLC) method,5 based on a derivatizationprocedure in which a urethane bond is formed be-tween the phenylisocyanate and hydroxyl groups ofpolyols, was used to separate myo-inositol, galactitol,and sorbitol in lens tissue extracts. This required thatthe lens tissue be sonicated for 15 seconds in coldwater, containing glucose diethyl mercaptal as an in-ternal standard. Proteins in the lens tissue homoge-nates were precipitated with ethanol (70% of the finalvolume) and separated by centrifugation at 10,000g at4°C for 5 minutes. Aliquots (100 /A for controls and25 /x\ for galactosemic guinea pig lenses) were lyophi-lized, dissolved in 70 1̂ of pyridine, and derivatizedwith 20 /A phenylisocyanate (60 minutes at 55°C). Sam-ples were diluted with pyridine to a final volume of200 n\. Standards were prepared by lyophilizingaqueous solutions containing the internal standard.Separation and quantification were carried out on afully automatized HPLC system (Shimadzu, Columbia,MD) equipped with a variable-wavelength ultravioletdetector (SPD-6AV; set at 240 nm), an automaticsampler (SIL-6B), and an Axxi-Chrom (Cole Scien-tific, Moorepark, CA) stainless steel column (25 cm X4.6 mm I.D.). Aliquots of derivatized samples (20 fx\)were eluted with acetonitrile:ethanol:water (5:2:3,v:v:v) in the isocratic mode with a flow' rate of lm 1/min. The tissue polyol content was quantified by theuse of calibration curves.

SuppliesChemicals and reagents were obtained from the follow-ing suppliers: D-Galactose (Sigma Chemical Co., St.Louis, MO); ketamine (Ketaset; Aveco Co., Inc., FortDodge, IA); xylazine (AnaSed; Lloyd Laboratories,Shenandoah, IA); tropicamide (Mydriacyl; Alcon, Hu-macao, PR); glutaraldehyde and sodium cacodylate(Ted Pella, Redding, CA); formaldehyde in PBS(Banco, Fort Worth, TX); historesin-embedding kit(Reichert-Jung, Heidelberg, Germany); HPLC gradeacetonitrile, ethanol, water, pyridine, phenylisocyan-ate, galactitol, myo-inositol, sorbitol, and glucosediethyl mercaptal (Aldrich, Milwaukee, WI).

RESULTS

Morphologic Changes

All guinea pigs fed a 50% galactose with 0.5 g ascor-bate/kg diet had initial lenticular opacities in the equa-


806 Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3

to rial subcapsular regions after 3 days (20/20) asnoted on slit-lamp examination (data not shown).These opacities spread further into the anterior cortexthe longer the duration of the diet. At 6 days, theycovered one third to one half of the diameter of theanterior cortex, and after 14 days of galactose feeding,most of the anterior cortex was opaque. No changeswere observed in control animals examined at thesame time intervals, fed normal guinea pig chow with0.5 g ascorbate/kg diet.

Gross and light microscopic changes correlatedwell. Typical histologic changes are illustrated in Fig-

A

ACa

b

FIGURE l. Photomicrograph of a sagittal section of guineapig lens. Equatorial (A) and anterior (a) region in controllens. After 3 days of galactose feeding, the first small vacu-oles in the equatorial region (B) and increased height of thecells in the central anterior epithelium (b) can be seen. After6 days of galactose feeding, enlargement and coalescence ofvacuoles in the equatorial region (C) and increased cellheight in the central epithelium (c) are evident. Vacuolesindicated by small white arrows; large black arrows show co-alesced vacuoles. ACa = anterior capsule; Ep - epithelium.Historesin semithin (2-Mm) sections stained with toluidineblue; equatorial region X100, central epithelium X225.

FIGURE 2. Photomicrograph of a sagittal section of guineapig lens after 14 days of galactose feeding. Preequatorialregion showing fiber cell swelling, liquefaction, and accumu-lation of liquid under the anterior capsule, XI60 (A). Ante-rior epithelium, multilayering denoted with white arrows,XI25 (B). Large liquefaction under posterior capsule; fibersfacing liquid are swollen and irregular in shape and size(black arrowheads), X82 (C); Ca = capsule; Ep = epithelium;Li = liquid accumulated in the subcapsular region; ACa =anterior capsule; PCa = posterior capsule; f = swollenfibers. A and C, historesin sections, toluidine blue staining;B, paraffin section, H & E stain.

ures 1 and 2. The first changes occurred after 3 days ofgalactose feeding and consisted of intracellular vacu-oles in the secondary fibers of the bow in the equato-rial zone (Fig. IB) and increased height and roundednuclei of the central (anterior) epithelium (Fig. lb).The lenses in the control animals fed normal guineapig chow with 0.5 g ascorbate/kg diet exhibited nor-mal appearance of the equatorial zone (Fig. 1A) andelongated nuclei in the central lens epithelium (Fig.la). The average height of the central anterior epithe-lium was 9.2 ± 0.5 jum and 16.3 ± 0.6 /xm at 3 days forcontrol and galactose-fed guinea pigs, respectively.This difference in height, 77%, was significant {P< 0.05). After 6 days of galactose feeding, equatorialyacuolization had progressed both anteriorly and pos-



A

nm)

o

LU

BA

NC

DC

oCOCO

\ (0

yo-in

o

A A

orb

it

oCO

^ VT I""

(0

capi

oE

3 K

;e d

iet

5001

C3

A

B

14 21 28 35

28 35

Time (min)

FIGURE 3. Chromatograms of derivatized lens tissue extractsfrom control (A) and 14-day galactose-fed guinea pigs (B).Glucose diethyl mercaptal was added as an internal stan-dard.

teriorly, producing different sizes of vacuoles andcysts (Fig. 1C). The changes in the anterior epithelialregion were not advanced over those seen after 3 daysof galactose feeding (Fig. lc). After 14 days of treat-ment, the equatorial region showed a disorganized

bow area, large coalesced vacuoles and cysts, and li-quefied subcapsular areas (Fig. 2A). The elongated epi-thelial cells were markedly swollen and the nucleirounded. Extremely swollen fiber cells (three to fourtimes the diameter of corresponding cells in the con-trol lenses) were seen posterior to the lens bow. Fur-ther increases in height, more rounded nuclei, andmultilayering were noted in the anterior epithelium(Fig. 2B). Large liquefactions were present under theposterior capsule (Fig. 2C). Fibers in contact with thisarea were markedly swollen and very irregular in sizeand shape.

Polyol Content

In control animals fed 0.5 g ascorbate/kg diet, lenshomogenates contained myo-inositol and sorbitol onHPLC, whereas galactitol was undetectable (Fig. 3A).In contrast, the galactose-fed guinea pigs exhibited anaccumulation of galactitol and loss of myo-inositol, asshown in Figure 3B. Table 1 summarizes galactitol andmyo-inositol levels in the lenses of control and galac-tose-fed guinea pigs after different lengths of diet. Thelens galactitol content was 18 mM after 3 days on thegalactose diet, and plateaued to 30 mM at 6 and 14days. After 3 days of galactose feeding, the lens myo-inositol content decreased twofold in comparison tocontrols, and was nearly undetectable after 14 days.

DISCUSSION

The current study finds the guinea pig to be a suitablemodel for galactosemic cataract investigations, with anadditional advantage in that the guinea pig's dietaryrequirement for ascorbic acid is similar to that of hu-mans. Previous attempts to develop a galactose cata-ract in guinea pigs met with a variable degree of suc-cess, unless the animals were on a galactose diet withno ascorbic acid,8 or had a drastically reduced ascorbicacid content in the chow (< 0.04 g/kg diet),9 as re-viewed in Table 2. The current study, however, demon-strates that gross and light microscopic morphologicchanges associated with alterations in polyol metabo-

TABLE l. Galactiol and Myo-Inositol Content in the Lens of Normaland Galactose-Fed Guinea-Pigs

Myo-Inositolyunol/g Lens Wet WtTreatment

Galactitolfimol/g Lens Wet Wt

Control3-day galactose6-day galactose14-day galactose

N.D.17.6 ± 2.830.1 ± 9.728.0 ± 1.8

2.8 ±0.71.5 ±0.70.5 ±0.20.3 ±0.2

Results are expressed as the mean ± SE from 6 to 10 lenses; ND = nondetectable.


808 Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3

TABLE 2. Development of Structural Changes during Galactose-Induced Cataract Formationin the Guinea-Pig and Rat

Species

Guinea-pig225-250 g

Guinea-pig300 g

Guinea-pig

Rai 60-70

gRai 50-60

gRiu 1 50 g

Rai 90 -I 2 ( ) g

Rai 50 g

Diet

50% galaaosein chowcontaining0.5 g/kgascorbate

10% galactosecontainingnoascorbatein drinkingwater

0% galactosecontainingno

ascorbatein drinking

water10% galaciose

containing10 g/kgascorbatein drinkingwater

10% galaciosein drinkingwater and 1

gAgascorbatein chow

10% galactosein drinkingwater and<0.04 g/kgascorbatein chow

50% galactosein chow


30% galaciosein chow



IncreasedHeight ofthe

AnteriorEpithelium

3 clays(+77%)

1.5 days(+47%)

InitialEpithelialMultilayering

14 days

4 clays

6 clays

InitialVacuolesin theEquator

3 days

IncompleteRing ofVacuolesin theEquators

3-5 clays

9 days

No changes observed

LargeCoalescen tVacuolesand Cystsin theEquator

6 days

during study

No changes observed during study

2 days

2 days 2 days

6 clays

3 days

4 days

CompleteRing ofVacuolesin theEquator

10-12days

25 clays

14 days

0/22lenses(no

changes)

14 days10/22lenses

4 days

10 days

15 days

7 clays

Liquefactionof the Cortex

14 days

10 days

20 clays

15 days

10-12 days

Reference

Presentstudy,10

8

8

8

9

9

2

1

13

14

15

lism can be induced by feeding guinea pigs with 50%galactose in powdered chow with no lowering of ascor-bate in the diet. It is noteworthy that 0.5 g ascorbate/kg diet as used in the current study is not a low vitaminC diet, because the recommended nutrient allowanceof vitamin C for the growing guinea pig can be fulfilled

with normal intake of a diet containing 0.2 g ascor-bate/kg.12 In addition, it has been shown that a scor-butic diet per se does not result in cataract formationor any other morphologic lens changes in guinea pigs.8

Thus, the difference between the current and previousstudies in guinea pigs89 may be attributed mainly to



the fact that previous studies used a galactose diet wellbelow the 50% level of the current study, as, for exam-ple, only 10% galactose given in drinking water.8-9

The galactose-induced cataractogenesis in thecurrent model was highly reproducible, and corre-sponded well with the changes previously described inrats1213"15; however, the time course of cataract for-mation and magnitude of changes varied between thetwo species, as outlined in Table 2. Gross morphologicand histologic changes in guinea pig lenses after 3, 6,and 14 days of galactose feeding approximate stages la(day 2), Ib (day 4), and II (day 10), respectively, ofSippel's classic classification for young 50- to 60-g ratson a 50% galactose powdered diet.1 For example,abundant equatorial vacuoles found after 4 days inrats2 do not appear until after 6 days in guinea pigs(Fig. IB). The epithelial multilayering noted in ratsafter 6 days1 and 4 days2 was not observed before 14days in the guinea pig (Fig. 2C). A significant increasein the height of the anterior epithelium as describedafter 36 hours in rats2 was found in guinea pigs (Fig.lb) after 3 days of galactose feeding. A better correla-tion in structural changes was found between guineapigs in the current study and somewhat older, 100- to150-g rats fed 30% galactose.1314

This study provides evidence for the activation ofthe polyol pathway in the lens of galactose-fed guineapigs (Fig. 3). The necessary requirements for the acti-vation of the polyol pathway described for mammalianlenses, including high aldose reductase activity, highpyridine nucleotide content, and active hexose mono-phosphate shunt, also exist in the guinea pig lens.6 Thelens galactitol concentration in galactose-fed guineapigs reached a value of about 30 mM at 6 days, and wasno higher thereafter (Table 1). This level, however,was 2.7-fold lower than 80 mM value reported for 60-to 70-g rats,2 and 1.6 times lower than the galactitollevel of 49 mM in 200- to 250-g rats measured after 21days on a 30% galactose diet.5 The maximum lenticu-lar levels of galactitol in these rat studies appear to berelated to the age of animals and the percentage ofgalactose in the diet. Our study confirms that myo-ino-sitol depletion, described previously in rats,5 can alsobe seen in the lens of galactosemic guinea pigs (Table1). It has been suggested that the reduction of myo-inositol may be related to changes in cellular mem-brane transport, but the exact mechanism by whichaldose reductase inhibitors influence myo-inositol lev-els under these conditions and the relationship be-tween myo-inositol depletion and cataract formationare still not completely understood.

Our previous work substantiates that the biochem-istry of the normal guinea pig lens is similar to that ofother mammalian lenses with respect to metabolic

pathways for GSH synthesis and degradation.11 By us-ing the same galactose guinea pig model, we alsofound a significant decrease in GSH lens levels of 50%after 3 days and 70% after 14 days of treatment,11

although the ability of lens to synthesize GSH was notaffected by galactose feeding. The decrease of GSH inlenses of galactosemic guinea pigs was not as large asthat in 50- to 60-g rats, in which lens GSH decreasedby 80% to 90% after 1 to 2 days of galactose treat-ment.34

We conclude that galactose-induced cataracto-genesis in guinea pigs with a 50% galactose and 0.5g/kg ascorbic acid diet is slower and less severe than inrats. This might yield an important experimental ad-vantage in the detection of early morphologic, bio-chemical, and membrane transport changes duringcataractogenesis.10 The manipulation of galactosecontent in the diet, in combination with different vita-min C regimens, may allow acceleration or decelera-tion in the different phases of cataractogenesis, whichin turn may offer useful extension of this new model.

Key Words

guinea pig, galactose cataract, photo slit lamp, light micros-copy, polyols

A cknowledgmen ts

The authors thank Dr. Nalin Unakar and Dr. Tom Ogdenfor much helpful discussion.

References

1. Sippel TO. Changes in water, protein, and glutathionecontents of the lens in the course of galactose cataractdevelopment in rats. Invest Ophthalmol. 1966; 5:568-575.

2. Robison WG, Houlder N Jr, KinoshitaJ. The role oflens epithelium in sugar cataract formation. Exp EyeRes. 1990; 50:641-646.

3. Lou MF, Dickerson JE, Garadi JR, York BM. Glutathi-one depletion in the lens of galactosemic and diabeticrats. Exp Eye Res. 1988; 46:517-530.

4. Reddy VN, Schwass D, Chakaprani B, Lim CP. Bio-chemical changes associated with the developmentand reversal of galactose cataract. Exp Eye Res. 1976;23:483-493.

5. Miwa I, Kanbara M, Wakazono H, OkudaJ. Analysisof sorbitol, galactitol, and myo-inositol in lens and sci-atic nerve by high-performance liquid chromatogra-phy. Anal Biochem. 1988; 173:39-44.

6. Markus HB, Raduscha M, Haris H. Tissue distributionof mammalian aldose reductase and related enzymes.Biochem Med. 1983; 29:31-45.

7. Burns JJ. Overview of ascorbic acid metabolism. AnnNY AcadSci. 1975; 258:5-23.


810 Investigative Ophthalmology 8c Visual Science, March 1994, Vol. 35, No. 3

8. Kosegarten D, Maher TJ. Use of guinea-pigs as modelto study galactose-induced cataract formation. JPharm Sci. 1978; 67:1478-1479.

9. Sasaki H, Yokoyama T, Giblin FJ, Reddy VN. Ascor-bate deficiency increases the rate of galactose-inducedcataract in guinea-pigs: Study of a possible mecha-nism. Invest Ophthalmol Vis Sci. 1993; 34:915.

10. Zlokovic BV, Mackic JB, McComb JG, et al. Impairedblood to lens transport of glutathione in galactosemicguinea-pigs. Invest Ophthalmol Vis Sci. 1992; 33:1110.

11. Kannan R, Tang D, Mackic JB, Zlokovic BV, Fer-nandes-Checa JC. A simple technique to determineglutathione (GSH) levels and synthesis in ocular tis-sues as GSH-bimane adduct: Application to normaland galactosemic guinea-pigs. Exp Eye Res. 1993;56:45-50.

12. National Research Council (US) Subcommittee onLaboratory Animal Nutrition. Nutrient requirementsof the guinea pig. In: Nutrient Requirements of Labora-tory Animals. Washington, DC: National Academy ofSciences, 1978:65-66.

13. Cotlier E. Hypophysectomy effect on the lens epithe-lium mitosis and galactose cataract development inrats. Arch Ophthalmol. 1962; 67:476-482.

14. Kato K, Nakayama K, Ohta M, et al. Effects of novelhydantoin derivatives with aldose reductase inhibitingactivity in galactose-induced cataract in rats. Jpn JPharmacol. 1990; 54:355-364.

15. Unakar, NJ, Genyea C, Reddan JR, Reddy VN. Ultra-structural changes during the development and rever-sal of galactose cataracts. Exp Eye Res. 1978; 26:123-133.


galactose-induced cataract formation in guinea pigs: morphologic

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