Graefe's Arch Clin Exp Ophthalmol (1996) 234:258-263 © Springer-Verlag 1996

Michael Eichhorn Matthias Schreckenberger Ernst R. Tamm Elke Liitjen-Drecoll

Carbonic anhydrase activity is increased in retinal pigmented epithelium and choriocapillaris of RCS rats

Received: 30 January 1995 Revised version received: 6 June 1995 Accepted: 13 July 1995

M. Eichhorn • M. Schreckenberger E.R. Tamm • E. Ltitjen-Drecoll (~) Anatomisches Institut, Lehrstuhl II, Universit~it Erlangen-Ntirnberg, D-91054 Erlangen, Germany Tel. +49-9131-852865, Fax: +49-9131-852862

Abstract • Background: In Royal College of Surgeons (RCS) rats the retinal pigmented epithelium (RPE) exhibits defective phagocytosis of rod outer segments, causing degen- eration of the photoreceptor layer. It is not known whether another function of the RPE, ion and fluid transport, is also affected by the disease. One enzyme involved in modulation of RPE transport acti- vities is carbonic anhydrase (CA). To clarify whether changes in CA activity are correlated with the process of retinal degeneration, the localization of CA activity in RCS rat eyes was investigated. • Methods: Eyes of 12 RCS rats and 12 age-matched congenic controls of different ages were studied, using a modified histo- chemical method of Hansson for light and electron microscopy. • Results: Control eyes showed CA staining in corneal endothelium, both layers of ciliary epithelium, Mtiller cells, inner segments of

photoreceptors, and RPE cells. In RPE the apical membranes were most intensely stained. In RCS rats, changes in CA staining were seen only in the posterior segment of rats 6 and 7 months of age. Most of the RPE cells were more intensely stained than those of age-matched controls, especially due to in- creased CA activity in the basolat- eral membrane infoldings. Adjacent endothelial cells of the chorioca- pillaris and of retinal capillaries de- veloped staining for CA activity. • Conclusion: Changes in CA ac- tivity in the RPE and adjacent cap- illary endothelium, together with previously described changes in RPE morphology in RCS rats, indi- cate changes in ion and fluid trans- port across the RPE. Since the reti- na was already impaired when the increase in CA activity occurred, we hypothesize that the causative factor is not the genetic defect per se, but the destruction of the retina.

Introduction

It is well established that in Royal College of Surgeons (RCS) rats with autosomal recessive retinal dystrophy, the retinal pigmented epithelial (RPE) cells exhibit de- fective phagocytosis of shed rod outer segments, causing accumulation of debris in the subretinal space and subse- quent degeneration of the photoreceptor cell layer [1, 10, 16, 20]. It is not known, however, whether another im- portant function of the RPE, ion and fluid transport, is

also involved in this disease. In normal RPE cells, mito- chondria, apical microvilli, basolateral membrane in- foldings, and an intact blood-retina barrier at the level of the RPE tight junctions are regarded as important for regular ion and fluid transport. In 2-month-old RCS rats, however, reductions in basolateral membrane infoldings and apical microvilli have been observed in a consider- able number of RPE cells [2]. In addition, using peroxi- dase as a tracer, a breakdown of the blood-retina barrier has been shown at the RPE tight junctions [3]. Such mor-

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phological changes seem to point towards alterations in ion and fluid t ransport at this location. One enzyme which appears to be important in modulation of RPE t ransport activities is carbonic anhydrase (CA). In hu- man eyes, there is CA activity at the basolateral and, more intense, at the apical membranes of the RPE [11, 22]. Wolfensberger et al. [22] showed, by immunohisto- chemistry on cultured RPE cells, staining of the apical membranes for membrane-bound CA isozyme IV. Inhibi- tion of CA activity by acetazolamide had been reported to increase fluid movement f rom the retina across the RPE to the choroid [13-15, 17, 21] and seems to aug- ment the adherence of the retina to the RPE [13, 15].

To clarify whether changes in CA activity are corre- lated with the process of retinal degeneration, we investi- gated the localization of CA activity in eyes f rom RCS rats of different ages and their age-matched congenic controls.

Material and methods

Eyes from 12 RCS rats and 12 age-matched congenic controls, 6 weeks and 4, 6, and 7 months of age, were investigated. The ani- mals were anesthetized with 5 mg/ml thiopental i.p. and subse- quently perfused from the left ventricle with 200 ml plasma ex- pander (Expafusin, Pfrimmer, Erlangen, Germany) containing 10 IU heparin/ml, followed by 200 ml 4% formaldehyde in phos- phate-buffered saline (PBS, pH 7.4). After enucleation, the eyes were cut equatorially, the lens was gently removed, and the eyes post-fixed (6 h) in 4% formaldehyde for histochemistry and in 2.5% glutaraldehyde for ultrahistochemistry. The posterior eye segments were cut radially, beginning at the optic nerve head, into specimens containing the entire choroid and retina from center to periphery.

Carbonic anhydrase histochemistry

For histo- and ultrahistochemistry, specimens were embedded in water-soluble hydroxypropyl methacrylate (Historesin, Reichert and Jung, Heidelberg, Germany), or JB4, (Polysciences, Warring- ton, Pa., USA) according to RidderstrSle [18]. For the histochemi- cal demonstration of CA, 2-btm-thick sections were stained for 0.5-9 min according to the method of Hansson [9], modified by Ridderstrgde [18]. For ultrahistochemistry, a modification of the method was used [6]. In brief, 4-gm-thick methacrylate sections were stained histochemically, post-fixed in 1% osmium tetroxide, and subsequently embedded in Epon. Ultrathin sections parallel with the primary cutting plane were investigated with Zeiss elec- tron microscopes (EM 109 and 902) without counterstaining.

For control the CA inhibitor acetazolamide (10 .5 M) was added to the incubation medium.

For semiquantitative evaluation of changes in CA activity sec- tions through the posterior eye segments of three 6-month-old RCS rats and three 6-month-old controls were stained simultaneously, and first appearance of CA staining was evaluated after 30 s and 1, 3, and 5 min. The findings are listed in Table 1.

Table 1 Incubation time (min) necessary for first appearance of carbonic anhydrase staining in 6-month-old Royal College of Sur- geons (RCS) and control rats (RPE=retinal pigmented epithelium)

Tissue Control RCS

Choriocapillaris O 1 Apical RPE membranes l 0.5-1 Basal RPE membranes 3 0.5-1 Retinal Mtiller cells 1 0.5-1 Retinal capillaries adjacent O 1 to RPE cells

~=No staining

Results

Control rats

In the anterior eye segment, there was intense staining of the basolateral cell membranes of the corneal endotheli- um and weak staining of the cy toplasm of these cells. In the ciliary body, the nonpigmented (NPE) and pigmented (PE) epithel ium showed CA activity in the apical and, most intense, in the basolateral cell membranes (Fig. 1A). CA staining of the cytoplasm was observed in the PE, but not in the NPE (Fig. 1A). The fenestrated capil- lary endothelial cells adjacent to the ciliary epithelium did not stain. In the iris, both the pigmented epithel ium and the dilator muscle cells showed membrane-bound staining, whereas only the dilator muscle revealed addi- tional cytoplasmic staining.

In the posterior eye segment, the RPE cells showed staining of their apical and basolateral cell membranes. Staining appeared earlier in the apical cell membranes (after 0.5 min of incubation) than in the basolateral ones (after 3 min of incubation; Fig. 3A, Table 1). No staining of the endothelial cells of the choriocapillaris was found. In the retina, Mtiller cells and the inner segments of the photoreceptor cells were CA positive after 0.5 min of incubation (Table 1). This staining pattern was the same in 4-, 6-, and 7-month-old control rats.

RCS rats

In the anterior portion of the eye, no staining differences between RCS rats and control rats were found (Fig. 1B).

In the posterior eye segment of 6-week-old and 4- month-old RCS rats, staining was unchanged compared to the congenic control rats. In these animals nearly all photoreceptors were already degenerated (Fig. 2). In 6- month-old RCS rats, marked differences became appar- ent. In most RPE cells both the apical and basolateral cell membranes stained after 0.5 or 1 min of incubation (Table 1, Fig. 3B). In a few cells staining was first seen in the apical, in others first in the basolateral cell mem- branes. A few individual RPE cells were weakly stained

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Fig. 1A, B Light micrographs of sagittal sections through the ciliary body of the eye of a normal control rat and an RCS rat eye [6 months of age; his- tochemical staining for car- bonic anhydrase (CA), 6 min of incubation. 7(280]. A Both cell layers of the ciliary ep- ithelium as well as the iris ep- ithelium and the epithelial dilator muscle are stained. The fenestrated capillaries in the ciliary body stroma contain some stained erythrocytes, but the endothelium is unstained. B As described previously [19], in RCS rats the pars plana of the ciliary body is elongated (arrow), whereas the ciliary processes are shorter than in the controls. In the anterior eye segment staining for CA activity is the same as in the controls

Fig. 2 Light micrograph of a sagittal section through the retina of a 6-month-old RCS rat (Richardson's stain, ×280). Note that the photoreceptor layer is completely degenerated so that the inner nuclear layer (arrow) is in direct contact with the RPE

or even unstained (Fig. 3C). In marked contrast to control rats, endothelial cells of choroidal capillaries showed intense histochemical CA reaction (Fig. 3B, C). The staining was confined exclusively to those capillaries di- rectly adjacent to CA-posit ive RPE cells. In large capil- laries, only those endothelial cells facing the RPE were stained (Fig. 3C). Capillaries adjacent to unstained RPE

cells did not stain (Fig. 3C). Ultrahistochemical investi- gation conf i rmed that CA activity was strictly localized to the cell membrane of RPE cells. Basal infoldings and apical microvill i were present in CA-stained RPE cells, but to a lesser extent than in control eyes (Fig. 4). In addition, choroidal capillaries did not obviously differ f rom those of controls and showed numerous fenestra- tions (Fig. 4). In animals 6 and 7 months of age, capil- laries of the dystrophic retina were found to contact the RPE, which stained for CA activity. Staining was con- fined to capillaries within the RPE (Fig. 5A) or to those parts of the capi l lary wall adjacent to a RPE cell. Elec- t ron-microscopic investigation revealed that fenestra- tions were also observed in retinal capillaries that were in contact with the RPE and stained for CA activity (Fig. 5B).

All sections of both control and RCS rat eyes incubat- ed in the presence of acetazolamide in the medium re- mained completely unstained.

Discussion

In control rats, the histochemical demonstrat ion of CA activity was rather similar to what has been described in pr imate eyes [11]. The only difference was the absence of staining in fenestrated capillaries of ciliary body and choroid. In previous studies, we have demonstrated that in pr imates there is a significant correlation between fen- estrations and CA staining in capil lary endothelial cells of many organs, including the eye [7, 11, 12]. We as-

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Fig. 3 Light micrographs of sagittal sections through the retinal pigmented epithelium (RPE) of 7-month-old control rats (A) and RCS rats (B, C) stained histochemically for CA activity (?<800). A Control rat (incubation time 30 s). After 30 s of incubation only the apical microvilli are stained. B The RPE of the dystrophic RCS retina strongly reacts for CA. All cell membranes are intensely stained. In addition, the endothelium of the chorio- capillaries (arrowheads) adja- cent to the RPE is intensely stained. Arrow CA-positive Mtiller cells (incubation time 9 min). C At places where the RPE shows less intense or no staining of the cell membranes (short arrow'), the adjacent capillaries (asterisk) also re- main unstained. In CA-posi- tive capillaries of the choroid, only that side of the capillary wall facing the RPE cells is stained (arrowheads). Arrows CA-positive Maller cells (in- cubation time 9 min)

T

A

snmed that CA might be necessary for maintenance of capil lary fenestrations in these species [12]. However, based on the present finding this does not hold true, at least for the fenestrated capillaries in the rat eye.

In RCS rats, no changes in CA activity were observed in the anterior eye segment. The ciliary epithelial cells showed staining of their basolateral membranes , al- though a reduction of these infoldings has recently been demonstrated by electron microscopy in some individual RCS ciliary epithelial cells [19, 23]. Thus, either CA activity does not correlate with the ultrastructure of cil- iary epithelial cells in RCS rats, or the number of changed cells is too small to be detected by histochem- istry.

In contrast, m the posterior eye segment, there were pronounced changes in CA activity in RPE cells and ad- jacent vasculature. In the RPE we observed increase in CA staining in the basolateral cell membranes , so that both apical and basolateral membranes stained already after 30 s of incubation. The capil lary endothelial cells of the choriocapillaris stained intensely for CA, even if no obvious differences in number of fenestrations com- pared to age-matched controls were found. We therefore assume that in the RCS rat development of CA staining in the choriocapit laris is related to changes in functional morphology of the retina and RPE. In addition, changes in CA activity in RPE cells and adjacent capillaries were seen only in animals 6 months of age and older, when

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Fig. 4 Ultrahistochemical demonstration of CA activity in the RPE and adjacent choriocapillaris of a 7-month-old RCS rat (9 rain of incubation, × 14 000). A strong histochemical CA reac-

tion is seen in the basal cell membranes (arrows) of the RPE and in the endothelium of the adjacent capillaries. In the endothelial cells numerous fenestrations (arrowheads) are present

Fig. 5 A Light micrograph of a sagittal section through an RCS rat retina (7 month of age; incubation time 9 rain, X1600). Capillaries of the central retinal artery (asterisk) which contact the RPE are in- tensely stained. The pericytes adjacent to the thick basal membrane of the capillaries also take the stain (arrow). B Ultrahistochemical demonstra- tion of CA activity in the RPE and adjacent central retinal capillaries of an RCS rat (7 months old; 9 rain of incuba- tion, X8800). A typical capil- lary (C) of the central retinal artery with a thick basal mem- brane (asterisk) is surrounded by RPE cells. CA staining is seen at the membrane infold- ings (thin arrows) of the RPE cells and in the capillary en- dothelial cells (thick arrow). Note that the stained capillary endothelial cells are fenestrat- ed (arrowheads)

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v i r t u a l l y al l p h o t o r e c e p t o r ce l l s were lost. We the re fo re s uppose that changes in C A ac t iv i ty are not c aused by the gene t ic de fec t pe r se, but are r a the r due to des t ruc t ion o f the re t ina .

We do not know the func t iona l i m p l i c a t i o n s o f these changes . W h e t h e r they are r e s t r i c t ed to RCS rats or are mere ly p a r t o f a gene ra l p h e n o m e n o n due to r e t ina l de- genera t ive p r o c e s s e s o f o ther causes and in o the r spec ies is not known. S ince the changes o b s e r v e d in this s tudy c o n c e r n CA s ta in ing o f cel l m e m b r a n e s , it is t e m p t i n g to specu l a t e that an i nc rea se in C A ac t iv i ty is a c c o m p a n i e d by changes in f lu id t r a n s p o r t ac ross the RPE in advanced s tages o f re t ina l dys t rophy.

In pa t ien ts wi th re t in i t i s p i g m e n t o s a (RP), m a c u l a r e d e m a is a f requent c o m p l i c a t i o n in the late s tage o f the d isease . A c e t a z o l a m i d e , a C A inh ib i to r , has been shown

to resolve m a c u l a r e d e m a and to improve visua l a cu i t y in some pat ien ts wi th m a c u l a r e d e m a , i n c l u d i n g a n u m b e r o f pa t ien ts wi th RP [4, 5, 8]. This migh t ind ica te that in eyes o f RP pat ien ts , c ha nge s in C A ac t iv i ty o f R P E and c h o r i o c a p i l l a r i e s may c o n t r i b u t e to the d e v e l o p m e n t o f m a c u l a r edema . In RCS rats there were no s igns o f ede- ma, but in these an ima l s no m a c u l a wi th its s p e c i a l i z e d vascu la r supp ly exists . In addi t ion , the l i fe span o f the an ima l s may be too sho r t for d e v e l o p m e n t o f edema . C lea r ly fu r the r s tudies are n e c e s s a r y to c l a r i f y these ques t ions .

Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, Germany (Dre 124/3- 2) and the RP Foundation Fighting Blindness, Baltimore, Mary- land, USA

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