A C T A O P H T H A L M O L O G I C A

61 (1983) 751-755

Departments of Ophthalmology (Head: L. Berggren) and Medical and Physiological Chemictly (Head: T. C. hurent)

University of Uppala, U@salu, Sweden

REDUCTION OF THE HYALURONATE CONCENTRATION IN RABBITAQUEOUS HUMOUR BY TOPICAL PREDNISOLONE

BY

U L U 6. G. LAUREN1

Rabbits were treated topically with prednisolone for 5 or 10 days and aqueous humour removed immediately after treatment as well as 10 and 20 days later. The hyaluronate concentration was significantly lower in the treated eyes compared to control eyes. It returned to the normal level after withdrawal of the drug. Repeated sampling of aqueous humour at intervals of 10 days did not influence the hyaluronate concentration in the control group.

Kq wwdr: corticosteroids - prednisolone - hyaluronic acid - aqueous humour - rabbit.

An effect of hyaluronidase on the out-flow resistance in the trabecular meshwork of experimental animals was already shown by Bardny and co-workers 30 years ago Barany & Woodin 1955). Furthermore, a connection between polysaccharides in the anterior segment and cortison-induced glaucoma has been suggested (see e.g. Francois 1978; Knepper et al. 1978). However, the effect of corticosteroids on the hyaluronate concentration of aqueous humour has not been investigated. A recently developed radioassay (Laurent & Tengblad 1980; Laurent 1981) has now been employed for such a study in rabbits.

Received on February 9th, 1983.

75 1

Laurent Steroid effect on hyaluronate in aqueous

Material and Methods

Twenty-four female rabbits (New Zealand White) weighing between 2.9 and 4.8 kg each were divided into groups of eight. One group was kept as a control group. In the second and third groups the right eye of each rabbit was treated topically for 5 and 10 days with 0.5% prednisolone acetate (one drop (= 28 f 1 pl) 4 times a day). The animals had a healthy appearance throughout the experiment.

Aqueous humour was collected 3 times: at 0, 10 and 20 days. Zero time for treated animals was immediately after treatment. The fluid (about 300 pl) was removed from the eyes during anaesthesia. The rabbits were given pentobarbital (30 mg/kg body weight) iv and tetracaine topically. The fluid was withdrawn through a puncture in the cornea with a 1 ml syringe provided with a 0.4 mm needle. The eyes were inspected repeatedly for adverse reactions. Slight injection and miosis could sometimes be seen after 8 h, but after 24 h all eyes appeared normal.

The aqueous humour samples were stored frozen until analyzed. The hyaluro- nate concentration was determined as described previously (Laurent & Tengblad 1980; Laurent 1981). Each sample was analyzed 3 times at different occasions, and the mean value was used in the calculations.

Resu I ts

The results are shown in Fig. 1 . The hyaluronate level in the control group was essentially constant throughout the experiment. The level in the treated (right) eyes was lower than in the controls immediately after treatment. The concentration increased in all treated eyes after withdrawal of the drug. A similar tendency was found in the left (untreated) eyes presumably due to a systemic effect of the drug.

An analysis of the values for the right eyes revealed that immediately after treatment there was a significant difference between the control group and the 2 treated groups combined (P < 0.02). There was no significant difference between the 2 treated groups. An analysis on the values for the left eyes did not show any significant difference between untreated and treated animals (P > 0.1). The increase in concentration in individual eyes between day 0 and day 10 was significant in the treated groups I1 and 111 (Pit < 0.01; Pi11 < 0.05). This analysis was based on right eye values, but the same tendency was found in the left eyes. At day 20 there were no significant differences between the 3 groups.

752

Laurent Steroid effect on hyaluronate in aqueous

PS

1L

1 2

1.0

00

nl I I1 111

T

0 10 20 0 10 20

Fig. I .

0 10 20 days

Sodium hyaluronate concentration in the aqueous humour of untreated rabbits ( I ) , rabbits treated for 5 days with topical prednisolone in the right eye (11) and rabbits treated similarly for 10 days (Ill) . Right eyes ( 0 ) ; left eyes (0). Each point represents the mean (+ SEM) of samples from 8 animals.

Discussion

The impetus to this investigation came during the screening of aqueous humour from human subjects (Laurent 1983). It was found that 2 patients, who had been treated with prednisolone, had low levels of hyaluronate (0.19 and 0.40 pg/g) compared with the normal ledel of 1.14 pg/g. Although these observations per se were no proof that corticosteroids have an effect on the hyaluronate concentration they called for an experimental verification.

The animal experiments verified that topical prednisolone lowers the level of hyaluronate in aqueous humour moderately but significantly. The level rises after withdrawal of the drug.

The experiment was planned with a control group, which received no treatment, rather than taking samples before treatment in the 2 groups receiving steroids. The possibility that paracenthesis might affect the hyaluronate production could not be excluded. In retrospect this does not seem to be the case with intervals of 10 days between samplings since the hyaluronate level stayed constant in the control group.

753 AcU ophthal. ti l , 5

4x

Laurent Steroid effect on hyaluronate in aqueous

The effect of corticosteroids on polysaccharide metabolism has been studied in tissue culture by several authors. It has been shown that steroids usually reduce the synthesis of hyaluronate (Castor 1962; Moscatelli & Rubin 1976; Saarni & Hopsu- Havu 1978; Mapleson & Buchwald 1981). The decrease in synthesis observed in tissue culture is of the same order of magnitude as the decrease in hyaluronate level in the aqueous humour observed in this investigation. Previous studies indicate that at least part of the hyaluronate in the aqueous humour may by synthesized in the anterior segment (Laurent & Granath 1983; Laurent & Fraser 1983) and rapidly removed from the anterior chamber by bulk flow (Laurent & Fraser 1983). The in vitro observations lend support to the assumption that prednisolone inhibits the biosynthesis in the eye. The concentration is, however, not only determined by biosynthesis but also by the rate of disappearance. It cannot be excluded that the hyaluronate concentration is reduced by an increased flow rate.

The present results contradict the assumption that cortison-induced glaucoma is due to an overproduction of hyaluronate in the anterior segment of the eye.

Acknowledgments

Technical assistance from Ms Kajsa Lilja is gratefully acknowledged. Dr Adam Taube has been of great help to the author in the statistical analyses. The project was supported by the Swedish Medical Research Council (03X-4) and Carmen and Bertil Regner’s fund.

References

Barany E H & Woodin A M (1955): Hyaluronic acid and hyaluronidase in the aqueous

Castor C W ( 1962): Adrenocorticoid suppression of mucopolysaccnaride formation in human

Francois J (1978): Corticosteroid glaucoma. Metabol Ophthalmol2: 3- 11. Knepper P A, Breen M, Weinstein H G & Blacik L J (1978): Intraocular pressure and

glycosaminoglycan distribution in the rabbit eye: effect of age and dexamethasone. Exp Eye Res 27: 567-575.

humour and the angle of the anterior chamber. Acta Physiol Scand 33: 257-290.

connective tissue cell cultures. J Lab Clin Med 60: 788-798.

Laurent U B G (1981): Hyaluronate in aqueous humour. Exp Eye Res 33: 147- 155. Laurent U B G (1983): Hyaluronate in human aqueous humor. Arch Ophthalmol 101 :

Laurent U B G & Fraser J R E (1983): Turnover of hyaluronate in the aqeuous humour and

Laurent U B G & Granath K A (1983): The molecular weight of hyaluronate in the aqueous

129- 130.

vitreous body of the rabbit. Exp Eye Res 36: 493-504.

humour and vitreous body of rabbit and cattle eyes. Exp Eye Res 36: 48 1-492.

754

Laurent Steroid effect on hyaluronate in aqueous

Laurent U B G & Tengblad A (1980): Determination of hyaluronate in biological samples by a specific radioassay technique. Anal Biochern 109: 386-394.

Mapleson J L & Buchwald M (1981): Effect of cycloheximide and dexamethasone phosphate on hyaluronic acid synthesis and secretion in cultured human skin fibroblasts. J Cell Physiol 109: 215-222.

Moscatelli D & Rubin H (1976): Hormonal control of hyaluronic acid production and relation

Saarni H & Hopsu-Havu V K (1978): The decrease of hyaluronate synthesis by anti- to nucleic acid and protein synthesis. J Cell Physiol91: 79-88.

inflammatory steroids in vitro. Br J Dermatol98: 445-449.

Author’s address: Ulla Laurent, Department of Ophthalmology, University Hospital, S-75 1 85 Uppsala, Sweden.

755 48*