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(Investigative Ophthalmology and Visual Science. 2001;42:472-475.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Electrophysiological Effects of Corticosteroids on the Retinal Pigment Epithelium

Carl-Friedrich Arndt1,2, Anouar Sari1, Magali Ferre1, Eric Parrat1, Didier Courtas2, Jérôme De Seze3, Jean-Claude Hache2 and Régis Matran1

1 From the Département de Physiologie (Equipe d’Accueil 2689 & Institut Fédératif de Recherche 22), Université Lille Il; 2 Exploration Fonctionnelle de la Vision, CHR&U Lille; 3 Clinique Neurologique, CHR&U Lille, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. As corticosteroids appear to intervene in pathogenesis of central serous chorioretinopathy, ion transport changes within the retinal pigment epithelium (RPE) might be involved. Electrophysiological responses to corticosteroid administration were recorded in vivo and in vitro.

METHODS. Clinical study: The standing ocular potential was recorded during intravenous (IV) infusion of glucose 5% and glucose 5% + prednisolone 0.2% in 14 patients with relapsing multiple sclerosis. The results were compared with a control group receiving two successive identical glucose 5% infusions. In vitro study: Native tissue explants (RPE + choroid, porcine, and bovine) were placed in a Ussing-type chamber. After baseline determination of the transepithelial potential (PD), short circuit current (Isc) and transepithelial resistance (Rt), the effect of apical hydrocortisone (HC) 10-4 M was determined.

RESULTS. Clinical study: A significant rise of the standing potential was found after glucose infusion (P = 0.005), whereas no change was detected after IV glucose + prednisolone (P = 0.695). In vitro study: In the porcine RPE, the mean baseline PD and Isc were significantly reduced (both P = 0.012) after applying apical 10-4 HC. Rt was also significantly reduced (P = 0.01). The same type of response, observed in bovine RPE, was reduced in low chloride/low bicarbonate conditions.

CONCLUSIONS. Corticosteroids modified electrophysiological parameters representing RPE function in vivo. The existence of an RPE-specific effect was confirmed in vitro. Further work is required to link the observed ion transport changes to a reduction of apical, subretinal fluid absorption.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Retinal pigment epithelial (RPE) cells form a monolayer that regulates the transport of fluids, ions, and metabolites between the sensory retina and the vascular choroidal system. The RPE constitutes the outer part of the blood–retinal barrier. An impairment of this barrier, as it is observed in central serous chorioretinopathy, presents with subretinal fluid accumulation. As demonstrated by indocyanine green angiography, fluid accumulation is currently thought to be linked to vascular abnormalities within the choriocapillaris.1 2 However, it is probable that RPE dysfunction is equally associated, thus leading to localized breakdown of the outer blood–retinal barrier.

Currently, there is some evidence for pathophysiological links between corticosteroid treatment and the incidence of central serous chorioretinopathy.3 4 5 6 The existence of this hypothesized effect at the RPE level has never been demonstrated clearly. Fluid transport changes across an intact RPE are related to transepithelial ion flux, which necessarily induce measurable electrophysiological changes. The purpose of this study was to demonstrate the existence of RPE-related electrophysiological effects of corticosteroids in vivo and in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Clinical Study
Healthy volunteers were recruited among the staff of Lille University Hospital (Lille cedex, France). Patients with multiple sclerosis without any history of visual impairment were included in the study. The research followed the tenets of the Declaration of Helsinki; informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study. The patients were scheduled for treatment with systemic corticosteroids (1 g of prednisolone during 4 hours) for relapsing multiple sclerosis. A clinical ophthalmologic examination was performed on all patients, evaluating the best corrected visual acuity and oculomotricity; fundus examination by indirect ophthalmoscopy was also performed. Patients with ocular motility palsy, clinical evidence of optic neuritis (visual acuity < 20/25, optic disc pallor), or uveitis were not included. In all patients, the recordings were initiated between 1:30 and 2:15 PM. All patients had finished their meals by 12:30 PM at the latest.

In a preliminary study, the standing ocular potential was recorded in the group of 14 healthy controls. Skin electrodes were placed in accordance with ISCEV guidelines to perform electro-oculography.7 The subject was placed in Ganzfeld conditions at a luminance of 60 cd/m2 for 10 minutes. Then the recording procedure started. Every 2 minutes, during a 58-minute period, the standing potential of the eye was recorded as the subject performed target-triggered saccades. The analysis of the mean standing potential of the right eye revealed a slow oscillation response with a mean periodicity of 34 minutes (Fig. 1) . The first trough occurred at 12 minutes, the second at 46 minutes. The shift between the minimum (trough) to the maximum amplitude (peak) was determined. This shift was found to be statistically significant (Table 1) .



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  		Figure 1. Recording of the mean standing potential of the right eye in 14 healthy volunteers with no infusion.

 

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  		Table 1. Effect of Intravenous Prednisolone on the Standing Potential of the Eye

 
In the patients with multiple sclerosis, a similar procedure was used. However, the procedure was split in two recording sessions of 24 minutes each (phase 1 and 2), to allow the patient to take a rest for 10 minutes, remaining in the same light-adapted conditions. The first intravenous infusion (80 drops/min) was initiated at t = 11 minutes (phase 1) and the second at t = 45 minutes (phase 2). The duration of both infusions was 13 minutes.

The first group of consecutive patients was assigned to the prednisolone group. They first received (at t = 11 minutes) an intravenous infusion of glucose 5% and then (at t = 45 minutes) an intravenous infusion of glucose 5% and prednisolone 0.2%. It was estimated that the plasma level of 100 µM was obtained within 10 minutes.8 The second group of patients was assigned to the glucose group, in which only two successive infusions of glucose 5% were compared, and no prednisolone was added to the second infusion.

Results are shown as means ± SD. Only the recording of the right eye of each patient was analyzed. Comparisons of paired sets of data (trough to peak shift) within each group of subjects (phase 1 versus phase 2) were made using a Wilcoxon rank test. Comparisons of unpaired sets of data (trough-to-peak shift) between the different groups of subjects (phase 1: glucose versus prednisolone, phase 2: glucose versus prednisolone) were made using a Mann–Whitney U test. For both series of tests, P < 0.05 was considered significant.

In Vitro Tissue Study
The research followed the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Large white pigs were enucleated after premedication with intramuscular ketamine and general anesthesia with pentobarbital. Bovine eyes were provided by a local slaughterhouse. The eyes were transferred to phosphate-buffered saline (composition in mM/l: NaCl 140, KCl 4, Na2HPO4 0.5, KH2PO4 0.15 [pH 7.4]).

The eyes were opened posterior to the limbus (3 mm in porcine eyes, 6 mm in bovine eyes) with a surgical blade, then cut on the entire circumference with surgical scissors. The choroid was separated form the sclera by blunt dissection. The retina was then gently removed from the underlying RPE.

The native tissue (pigment epithelium + choroid) was placed between two half-Ussing–type chambers within 4 hours after enucleation. A 0.163 cm2 area of the tissue sample was exposed to 10 ml of solution on each side (apical side = retina and basolateral side = choroid) and gassed with 12% O2–5% CO2%–83% N2, as low oxygen increases longevity of RPE function.9 The experiments were conducted at 37°C. The short-circuit current (Isc) was monitored continuously using a DVC 1000 voltage clamp (WPI, Aston, UK), and the potential difference (PD) was measured every 2 minutes. Voltage-sensing electrodes and the current passing bridges consisted of 3 M KCl-agar; the reference electrodes were placed at the basolateral side. Transepithelial resistance (Rt) was determined by clamping the PD to +10 mV at 15-second intervals, recording the deflection of Isc, and applying Ohm’s law. The preparations were allowed to equilibrate. Stabilization of bioelectrical parameters was achieved within 30 to 40 minutes. Only tissue samples with an Rt > 80 {Omega} · cm2 were considered for analysis. Basal bioelectrical activity was monitored for 15 minutes before the addition of the reagents. Hydrocortisone (10-4) was dissolved in the KBR solution (vehicle) and added to the apical or the basolateral bath. This concentration was chosen in accordance with the estimated plasma and vitreous levels in the clinical part of this study.8 10 Changes in PD, Isc, and Rt were calculated as the variation between the values measured immediately before the addition of reagents and the values corresponding to the peak phase.

Krebs–Ringer bicarbonate buffer (KRB) had the following composition (in mM/l): 140 Na, 120 Cl, 5.2 K, 1.2 Mg, 1.2 Ca, 2.4 HPO4, 0.4 H2PO4, 25 HCO3, 5.5 glucose, and 1.0 glutathione (pH 7.3).

Cl-free solution was obtained by replacing NaCl and KCl by Na and K gluconate salts. In the bicarbonate-free solution, NaHCO3 was replaced with NaGluconate.

Results are expressed as means ± SD for each set of preparations. Comparisons of paired sets of data (PD, Isc, Rt, respectively before and after addition of HC) were made using a Wilcoxon rank test.

Comparisons of unpaired sets of data (PD, Isc, Rt) corresponding to different bath solutions (KRB versus Cl-free solution, Cl-free solution versus Cl-free solution + bumetanide, Cl-free solution versus Cl-free–HCO3-free solution) were made using a Mann–Whitney U test. For both series of tests, P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical Study
The first 14 consecutive patients were included in the prednisolone group. The evolution of the mean standing potential is shown in Figure 2 . Before IV infusion of glucose, the standing potential of the eye decreased. After starting the IV infusion of glucose (phase 1), it increased as it did in healthy volunteers with no infusion (Fig. 1) . The rise of the standing potential was found to be less pronounced after infusion of glucose + prednisolone (phase 2), compared with the phase 1 response (obtained with the glucose infusion). The phase 1 rise reached statistical significance at t = 20, 22, and 24 minutes. The phase 2 rise of the standing potential after infusion of glucose + prednisolone never reached statistical significance. Table 1 summarizes the effect of intravenous prednisolone on the standing potential of the eye and on the peak-to-trough shift.



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  		Figure 2. The effect of glucose versus glucose + prednisolone on the mean standing potential of the right eye of 14 multiple sclerosis patients (prednisolone group).

 
In the prednisolone group, comparing the peak-to-trough shift after glucose infusion (phase 1) with the peak-to-trough shift after glucose + prednisolone infusion (phase 2), the shift was found to be higher after glucose infusion in 12 out of 14 patients.

The following 14 patients were included in the glucose group. No significant difference of the standing potential of the eye could be found between the two recording sessions (Fig. 3) . The peak to trough shift was found to be statistically significant in both sessions (phase 1 and phase 2); it was slightly higher after the second glucose infusion, however this difference did not reach statistical significance (Table 1) .



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  		Figure 3. The effect of two successive glucose perfusions on the mean standing potential of the right eye of 14 multiple sclerosis patients (control group).

 
The observed difference of the trough-to-peak shift between the prednisolone group and the glucose group did not reach statistical significance, in either phase 1 or 2 of the recording procedure.

Bovine In Vitro Study
In the bovine RPE, Table 2 summarizes the effects of HC in KRB, in low chloride and low chloride-low bicarbonate solutions. Apical 10-4 HC reduced the transepithelial potential, the short circuit current, and the transepithelial resistance in the KRB bath. When using the low chloride solution, the HC-mediated reduction of PD, Isc, and Rt appeared to be inhibited, although not completely blocked. Adding apical bumetanide 10-4, a selective inhibitor of transepithelial chloride flux, to the low chloride bath, did not significantly modify the hydrocortisone response, when comparing it with the response observed in low chloride solutions without bumetanide. In a bicarbonate-free–low chloride bath, the response to hydrocortisone appeared to be completely blocked.


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  		Table 2. Effect of Hydrocortisone on Bioelectrical Parameters

 
Porcine In Vitro Study
In 8 of 20 porcine eyes, the basal electrophysiological recordings were stable, and Rt was sufficient to conduct the procedure. In eyes with low Rt, the previous blunt dissection separating the choroid from the sclera appeared to be difficult. Table 2 summarizes the effects of hydrocortisone on the electrical parameters of the porcine adult RPE. Apical hydrocortisone reduced the PD, Isc, and Rt significantly (Table 2) . Basolateral hydrocortisone alone produced no measurable change in PD, Isc, or Rt (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study are compatible with an inhibitory effect of corticosteroids on RPE fluid absorption in vivo and in vitro. However, it is crucial to stress the difference between the current experimental setup, which evaluated a short-term response, and the clinical presentation of central serous chorioretinopathy, which represents a chronic disease.

The electro-oculogram (EOG) is known to be one of the most appropriate ways of studying the function of human RPE,7 which compares the light response to the dark trough after dark adaptation. However, the light response is thought to be generated by the interaction of retina and RPE, which seems to be essentially linked to increased basolateral Cl conductance.11 Only the standing potential recorded at constant luminance conditions can be considered to be representative of RPE function. For this reason, the authors chose to develop an original protocol of EOG recording. It established that corticosteroids could induce changes of the standing potential when compared with controls. However, the hypothesized RPE-related origin of this EOG finding remained to be confirmed by in vitro recordings.

HC was found to modify the electrical parameters of the bovine RPE. A marked decrease of PD and Isc was observed after administration of HC on the apical side of the bovine RPE. No such response was observed on the basolateral side of the preparation. Certain transport mechanisms of the bovine RPE appear to be species-dependent, yet a similar response to HC in tapetum-free porcine RPE–choroid explants confirmed these findings in the present study, in conditions that might be functionally closer to human RPE. However, the experienced firm adherence between choroid and sclera made blunt dissection more difficult, thus probably altering the electrical properties of the tissue explant. This might explain the low potential difference obtained in the current porcine study, compared with bovine RPE, one of the most widely used animal models.

Physiological functions of corticosteroid hormones involve activation of intracellular receptors as well as poorly understood membrane receptors. HC is believed to diffuse freely across the cellular membrane to be linked to a cytosol receptor.12 In the past, some neurophysiological results suggested that corticosteroids could act nongenomically and specifically through their membrane receptor on a neuronal surface.13 The presence of a nuclear corticosteroid receptor has been recently demonstrated in the RPE.14 However, no membrane receptor for corticosteroids, which could explain the exclusive response to apical stimulation, has yet been identified.

Although the exact cellular mechanisms of HC in the RPE remain to be determined, the present study provides evidence for chloride/bicarbonate dependency of the response induced by HC. Both Cl- and HCO3- have previously been linked to transepithelial fluid transport in bovine RPE.15 However, there was no evidence for the implication of NaK2Cl cotransport in this chloride mediated response, as it remained unchanged by pretreatment with apical bumetanide.

In previously published data on bovine RPE, an increase of the PD has been linked to apical epinephrine stimulation, enhancing apical to basal chloride flux and transepithelial fluid transport, which were both inhibited by pretreatment with apical bumetanide.16 The effect of epinephrine is known to implicate intracellular Ca++ as a second messenger.17

The opposite effect has been associated with increased intracellular cyclic adenosine monophosphate (cAMP), inhibiting fluid absorption in bullfrog RPE18 and basolateral membrane chloride conductance in chick RPE.19 Intracellular cAMP might not be directly implicated as a second messenger in the response observed with HC in the present study, but there is some evidence for synergistic cellular effects between cAMP and corticosteroid mediated mechanisms20 that remain to be demonstrated in RPE cells.


   Acknowledgements
 
The authors thank Danièle Basset for collecting the data, Dorothea Arndt for her English corrections, Jean-Marie Jacquesson for his help in statistics, and Marie-Dominique Salomez for her kind assistance with Ussing chambers.


   Footnotes
 
Supported by a grant from the Programme Hospitalier de Recherche Clinique, Grant No. 98–1957 du Centre Hospitalier et Universitaire de Lille provided by the Direction de l’Hospitalisation et l’Organisation des Soins, France.

Submitted for publication April 7, 2000; revised September 7, 2000; accepted September 15, 2000.

Commercial relationships policy: N.

Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 1999.

Corresponding author: Carl-Friedrich Arndt, Exploration Fonctionnelle de la Vision, Hôpital Roger Salengro, 2 avenue Oscar Lambret, F-59037 Lille cedex, France. carndt{at}univ-lille2.fr


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Prünte, C, Flammer, J. (1996 Jan) Choroidal capillary and venous congestion in central serous chorioretinopathy Am J Ophthalmol 121,26-34[Medline][Order article via Infotrieve]
  2. Uyama, M, Matsunaga, H, Matsubara, T, et al (1999) Indocyanine green angiography and pathophysiology of multifocal posterior pigment epitheliopathy Retina 19,12-21[Medline][Order article via Infotrieve]
  3. Harada, T, Harada, K. (1985) Six cases of central serous choroidopathy induced by systemic corticosteroid therapy Doc Ophthalmol 60,37-44[Medline][Order article via Infotrieve]
  4. Bouzas, EA, Scott, MH, Mastorakos, G, et al (1993) Central serous chorioretinopathy in endogenous hypercortisolism Arch Ophthalmol 111,1229-1233[Abstract/Free Full Text]
  5. Polak, BC, Baarsma, GS, Snyers, B. (1995) Diffuse retinal pigment epitheliopathy complicating systemic corticosteroid treatment Br J Ophthalmol 79,922-925[Abstract/Free Full Text]
  6. Spraul, CW, Lang, GE, Lang, CK (1998) Retinal pigment epithelial changes associated with systemic corticosteroid treatment: report of cases and review of the literature Ophtalmologica 212,142-148
  7. Marmor, MF, Zrenner, E. (1993) Standard for clinical electro-oculography. International Society for Clinical Electrophysiology of Vision Arch Ophthalmol 111,601-604[Abstract/Free Full Text]
  8. Lawson, GJ (1985) Rapid method for the measurement of methylprednisolone and its hemisuccinate in plasma and urine following "pulse therapy" by high-performance liquid chromatography J Chromatogr 342,251-260[Medline][Order article via Infotrieve]
  9. Winkler, BS, Giblin, FJ (1983) Glutathione oxidation in retina: effects on biochemical and electrical activities Exp Eye Res 36,287-297[Medline][Order article via Infotrieve]
  10. Ziada, G, el-Haddad, S, Fatouh, M, et al (1985) Radionuclide study of the blood ocular barrier Eur J Drug Metab Pharmacokinet 10,325-328[Medline][Order article via Infotrieve]
  11. Gallemore, R, Steinberg, R. (1993) Light evoked modulation of the basolateral Cl conductance in chick retinal pigment epithelium: the light peak and the fast oscillation J Neurophysiol 70,1669-1680[Abstract/Free Full Text]
  12. Rousseau, GG, Higgins, SJ, Baxter, JD, et al (1975) Binding of glucocorticoid receptors to DNA J Biol Chem 250,6015-6021[Abstract/Free Full Text]
  13. Chen, YZ, Hua, SY, Wang, CA, et al (1991) An electrophysiological study on the membrane receptor-mediated action of glucocorticoids in mammalian neurons Neuroendocrinology 53 Suppl 1,25-30
  14. Schönfeld, CL (1998) Expression des nukleären Glukokortikoid-Rezeptors in humanem retinalen Pigmentepithel Ophthalmologe 956,400-403
  15. Kenyon, E, Joseph, DP, Miller, SS (1994) Evidence for electrogenic Cl-dependent Na-HCO3 cotransport at the apical membrane of bovine RPE [ARVO Abstract] Invest Ophthalmol Vis Sci 35(4),S1759Abstract nr 2336.
  16. Edelmann, JL, Miller, SS (1991) Epinephrine stimulates fluid absorption across bovine retinal pigment epithelium Invest Ophthalmol Vis Sci 32,3033-3040[Abstract/Free Full Text]
  17. Joseph, DP, Miller, SS (1992) Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium J Gen Physiol 99,263-290[Abstract/Free Full Text]
  18. Hughes, BA, Miller, SS, Machen, TE (1984) Effects of cyclic AMP on fluid absorption and ion transport across frog retinal pigment epithelium. Measurements in the open-circuit state J Gen Physiol 83,875-899[Abstract/Free Full Text]
  19. Kuntz, CA, Crook, RB, Dmitriev, A, Steinberg, RH (1994) Modification by cyclic adenosine monophosphate of basolateral membrane chloride conductance in chick retinal pigment epithelium Invest Ophthalmol Vis Sci 35,422-433[Abstract/Free Full Text]
  20. Penuelas, I, Encio, IJ, Lopez-Moratalla, N, Santiago, E. (1998) cAMP activates transcription of the human glucocorticoid receptor gene promoter J Steroid Biochem Mol Biol 67,89-94[Medline][Order article via Infotrieve]



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