The Glucocorticoid Triamcinolone Acetonide Inhibits Osmotic Swelling of Retinal Glial Cells via Stimulation of Endogenous Adenosine Signaling

The Glucocorticoid Triamcinolone Acetonide Inhibits Osmotic Swelling of Retinal Glial Cells via Stimulation of Endogenous Adenosine Signaling

The Glucocorticoid Triamcinolone Acetonide Inhibits Osmotic Swelling of Retinal Glial Cells via Stimulation of Endogenous Adenosine Signaling

The Glucocorticoid Triamcinolone Acetonide Inhibits Osmotic Swelling of Retinal Glial Cells via Stimulation of Endogenous Adenosine Signaling Ortrud Uckermann, Franziska Kutzera, Antje Wolf, Thomas Pannicke, Andreas Reichenbach, Peter Wiedemann, Sebastian Wolf, and Andreas Bringmann Paul Flechsig Institute of Brain Research (O.U., T.P., A.R.), Interdisziplinäres Zentrum für Klinische Forschung (O.U., A.W.), and Department of Ophthalmology and Eye Clinic (F.K., P.W., A.B.), University of Leipzig Medical Faculty, Leipzig, Germany; and Department for Ophthalmology, University Bern, Inselspital, Bern, Switzerland (S.W.) Received July 13, 2005; accepted August 31, 2005 ABSTRACT The glucocorticoid triamcinolone acetonide is clinically used for the treatment of macular edema.

However, the edema-resolv- ing mechanisms of triamcinolone are incompletely understood. Since cell swelling is a central cause of cytotoxic edema in the brain and retina, we determined the effects of triamcinolone acetonide on the swelling of retinal ganglion and Müller glial cells in acutely isolated retinas from rats and guinea pigs in situ. Triamcinolone acetonide (100 ␮M) had no effect on the swelling of ganglion cells that was evoked in isolated whole mounts of the guinea pig retina by acute application of glutamate (1 mM) or high K⫹ (50 mM). However, triamcinolone reversed the os- motic swelling of Müller glial cells in retinas of the rat that was observed under various experimental conditions: in retinas iso- lated at 3 days after transient retinal ischemia, in retinas of eyes with lipopolysaccharide-induced ocular inflammation, and in control retinas in the presence of Ba2⫹ (1 mM), H2O2 (200 ␮M), arachidonic acid (10 ␮M), or prostaglandin E2 (30 nM).

The inhibiting effect of triamcinolone on osmotic glial cell swelling was mediated by stimulation of transporter-mediated release of endogenous adenosine and subsequent A1 receptor activation, resulting in an elevation of the intracellular cAMP level and activation of the protein kinase A, and, finally, in an opening of extrusion pathways for K⫹ and Cl⫺ ions. The inhibitory effect on the cytotoxic swelling of glial cells may contribute to the fast edema-resolving effect of vitreal triamcinolone observed in hu- man patients.

Cystoid macular edema is an important complication of various ocular diseases such as diabetic retinopathy and retinal vein occlusion. In patients with uveitis or diabetic retinopathy, macular edema is the major cause of severe visual deterioration. Although the precise cause of cystoid macular edema is still uncertain, inflammatory and isch- emic-hypoxic conditions have been causally implicated in the development of retinal edema (Tso, 1982; Yanoff et al., 1984; Guex-Crosier, 1999; Marmor, 1999). Extracellular (vaso- genic) fluid accumulation, as a main causative factor of tissue swelling and cyst formation, occurs after breakdown of the blood-retina barriers (Gass et al., 1985) induced by vascular endothelial growth factor (VEGF) (Antcliff and Marshall, 1999; Marmor, 1999) and/or inflammatory mediators (Derev- janik et al., 2002).

Vascular leakage results in serum protein extravasation and osmotically driven water inflow into the retinal tissue. In addition to this vasogenic mechanism, wa- ter accumulation within retinal neurons and glial cells (re- sulting in cell swelling, i.e., cytotoxic or intracellular edema) may contribute to the edema in the ischemic and postisch- emic retina (Pannicke et al., 2004; Uckermann et al., 2004b). During diabetic retinopathy, the swelling of ganglion cell bodies and their processes precedes the loss of these cells and the gliosis of the inner retinal layers (Duke-Elder and Do- bree, 1967).

There are good arguments for a contribution of glial (Müller) cell swelling to the development of cystoid This work was supported by Interdisziplinäres Zentrum für Klinische For- schung at the Faculty of Medicine of the University of Leipzig Project C21 and by DFG Grants BR 1249/2-1 and GRK 1097/1.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.092353. ABBREVIATIONS: VEGF, vascular endothelial growth factor; LPS, lipopolysaccharide; PGE2, prostaglandin E2; LY341495, (2S)-2-amino-2- [(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; CSC, 8-(3-chlorostyryl) caffeine; CPA, N(6)-cyclopentyladenosine; MPEP, 2-methyl-6-(phenylethynyl)-piperidine; BAPTA/AM, bis-(o-aminophenoxy)ethane-N,N,N⬘,N⬘-tetra-acetic acid acetoxymethyl ester; pCPT, 8-(4-chlorophenylthio); IBMX, 3-isobutyl-1-methylxanthine; H-89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-iso- quinolinesulfonamide; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; NBTI, N-nitrobenzylthioinosine; ARL-67156, 6-N,N-diethyl-D-␤,␥- dibromomethylene ATP; AOPCP, adenosine-5⬘-O ␤ - methylene)-diphosphonate; DMSO, dimethyl sulfoxide.

0022-3565/05/3153-1036–1045$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 315, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 92353/3063312 JPET 315:1036–1045, 2005 Printed in U.S.A.

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macular edema, e.g., in cases without significant angio- graphic vascular leakage (Fine and Brucker, 1981; Yanoff et al., 1984). A similar situation has been described for the brain where the postischemic or traumatic edema is charac- terized by swelling of astroglial cells (Kimelberg, 1995). In the retina, dysregulated fluid-absorbing mechanisms nor- mally carried out by pigment epithelial and glial cells (Bring- mann et al., 2004) may contribute to edema formation, re- sulting in an ineffective redistribution of fluid from the retina into the blood.

The observation that clinically significant diabetic macular edema occurs only when (in addition to vascular leakage) also the active transport mechanisms of the blood-retinal barriers are dysfunctional (Mori et al., 2002) underlines the importance of disturbed fluid clearance in the development of retinal edema.

Anti-inflammatory corticosteroids are commonly used to treat inflammatory eye diseases, and among them, triamcin- olone acetonide (9␣-fluoro-16␣-hydroxyprednisolone) has the advantage to form crystals that represent local depots for the sustained release over relatively long periods of time. Intra- vitreal triamcinolone is used clinically for the rapid resolu- tion of macular edema (Ip et al., 2003; Massin et al., 2004). However, the mechanisms by which triamcinolone exerts its fast edema-resolving effect are incompletely understood. There is evidence that triamcinolone inhibits vasogenic edema and inflammation.

Triamcinolone decreases vascular leakage (Ando et al., 1994), reduces the secretion of VEGF by pigment epithelial cells during oxidative stress (Matsuda et al., 2005), down-regulates the expression of the VEGF gene in vascular smooth muscle cells (Nauck et al., 1998), and reduces the vitreal level of VEGF in patients with diabetic retinopathy (Brooks et al., 2004). Furthermore, triamcino- lone decreases the paracellular permeability of cultured ep- ithelial cells, down-regulates the inflammatory expression of endothelial adhesion molecules (Penfold et al., 2000), and inhibits leukocyte-endothelial interactions in the diabetic retina of the rat (Tamura et al., 2005).

However, it is not known whether triamcinolone, in addition to its inhibitory action on vasogenic edema, also has an effect on the devel- opment of cytotoxic (intracellular) edema. Inhibition of cyto- toxic edema may contribute to the rapid edema-resolving effect of triamcinolone observed in human patients. There- fore, we investigated whether triamcinolone inhibits the swelling of retinal neurons or glial cells. Neuronal cell swell- ing was evoked by application of glutamate or high K⫹ onto acutely isolated whole mounts of the guinea pig retina, which is known to rapidly induce swelling of retinal neurons by activation of ionotropic glutamate receptors, resulting in in- tracellular Na⫹ and Cl⫺ overload and water influx (Ucker- mann et al., 2004b).

Müller cell swelling was investigated in slices of the rat retina during exposure of a hypotonic solu- tion (a situation that resembles hypoxia-induced cytotoxic edema in the brain). We investigated the osmotic swelling of retinal glial (Müller) cells during various experimental con- ditions: in a rat model of transient retinal ischemia reperfu- sion, in a model of ocular inflammation induced by intravit- real injection of lipopolysaccharide (LPS), during acute application of the K⫹ channel blocker Ba2⫹ or of arachidonic acid and prostaglandin E2 (PGE2), respectively, and during oxidative stress induced by acute application of H2O2.

Materials and Methods Materials. Mitotracker Orange (chloromethyltetramethylro- samine) was purchased from Molecular Probes (Eugene, OR). Ket- amine was obtained from Ratiopharm (Ulm, Germany) and xylazine from BayerVital (Leverkusen, Germany). PGE2 was delivered from Calbiochem (San Diego, CA), and LY341495 was from Tocris Cook- son Inc. (Bristol, UK). Triamcinolone acetonide, acetazolamide, ara- chidonic acid, 4-bromophenacyl bromide, indomethacin, 8-cyclopen- tyl-1,3-dipropylxanthine (DPCPX), 8-(3-chlorostyryl) caffeine (CSC), N(6)-cyclopentyladenosine (CPA), 2-methyl-6-(phenylethynyl)-piper- idine (MPEP), bis-(o-aminophenoxy)ethane-N,N,N⬘,N⬘-tetra-acetic acid acetoxymethyl ester (BAPTA/AM), 8-(4-chlorophenylthio) (pCPT)-cAMP, forskolin, 3-isobutyl-1-methylxanthine (IBMX), 2,3- dideoxyadenosine, H-89, flufenamic acid, 5-nitro-2-(3-phenylpro- pylamino)benzoic acid (NPPB), N-nitrobenzylthioinosine (NBTI), ARL-67156, adenosine-5⬘-O ␤ - methylene)-diphosphonate (AOPCP), and all other substances used were purchased from Sigma Chemie (Deisenhofen, Germany).

Animal Models. All experiments were carried out in accordance with applicable German laws and with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Adult Long-Evans rats (250–350 g) were used to induce transient retinal ischemia in one eye of the animals; the other eye remained untreated and served as control. Anesthesia was induced with intramuscular ketamine (100 mg/kg) and xylazine (5 mg/kg). The anterior chamber of the treated eye was cannulated from the pars plana with a 27-gauge infusion needle, connected to a bag containing normal saline.

The intraocular pres- sure was increased to 160 mm Hg for 60 min by elevating the saline bag. Sham treatment (i.e., using the same procedures without ele- vation of the saline bag) does not induce alterations of the osmotic swelling characteristics of Müller glial cells when compared with control (Pannicke et al., 2004). Ocular inflammation alters the swell- ing characteristics of Müller glial cells in a similar fashion as isch- emia reperfusion (Pannicke et al., 2005). We used an animal model of uveoretinitis induced by intravitreal injection of LPS (Becker et al., 2000). During ketamine/xylazine anesthesia, LPS from Escherichia coli 055:B5 (0.5%; Sigma-Aldrich) dissolved in 2 ␮l of saline was injected into one eye of the animals.

At 3 days after transient retinal ischemia or at 7 days after intravitreal injection of LPS, the animals were killed by CO2, and the retinas were removed. Glial Cell Swelling. To determine volume changes of Müller glial cells evoked by hypotonic stress, the cross-sectional area of Müller cell somata in the inner nuclear layer of retinal slices was measured. Acutely isolated retinal slices (thickness, 1 mm) were placed in a perfusion chamber and loaded with the vital dye Mitotracker Orange (10 ␮M). It has been shown that Mitotracker Orange is taken up selectively by Müller glial cells, whereas neurons remain unstained (Uckermann et al., 2004a).

The stock solution of the dye was pre- pared in dimethyl sulfoxide (DMSO) and resolved in extracellular solution that contained 136 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 11 mM glucose, adjusted to pH 7.4 with Tris. The recording chamber was continuously perfused with extra- cellular solution; test substances were added by rapid changing of the perfusate. The hypotonic solution was made by adding distilled water. Ba2⫹ (1 mM) was added to the extracellular solution, and was preincubated for 10 min. H2O2, PGE2, and triamcinolone were ap- plied simultaneously with the hypotonic solution.

A stock solution of triamcinolone was made by using DMSO; vehicle alone had no effect on cell volume. Blocking substances were preincubated for 10 to 45 min. All experiments were performed at room temperature. The slices were examined by using a confocal laser scanning microscope LSM 510 Meta (Carl Zeiss GmbH, Jena, Germany). Mitotracker Orange was excited at 543 nm, and emission was recorded with a 560-nm long-pass filter. During the experiments, the Mitotracker Orange-stained somata in the inner nuclear layer were recorded at Triamcinolone Inhibits Glial Cell Swelling 1037 at ASPET Journals on February 7, 2015 jpet.aspetjournals.org Downloaded from

the focal plane of their largest extension. To assure that this focal plane was precisely recorded, the cell soma investigated was contin- uously refocused in the course of the experiments. Neuronal Cell Swelling. To investigate neuronal cell swelling, the nonvascularized guinea pig retina was used, which (in compar- ison with the retina of the rat) has the advantage that the cell bodies in the ganglion cell layer can be recorded without interference with blood vessels and astrocytes. The animals (550–650 g) were killed by CO2, and the retinas were removed. Pieces of retinal whole mounts (5 mm2 ) were explanted and mechanically fixed in a perfusion chamber, with their vitreal surface up.

The whole mounts were loaded with Mitotracker Orange resolved in extracellular solution. Elevation of the K⫹ concentration was generated by equimolar reduction of Na⫹ .

Images were taken at two focal planes: the ganglion cell and inner plexiform layers (Fig. 1A). Reflected light was acquired by using a 650-nm HeNe laser and a 340-nm long-pass filter. Data Analysis. To determine the extent of glial cell swelling, the cross-sectional area of Mitotracker Orange-stained cell bodies in the inner nuclear layer of retinal slices was measured manually using the image analysis software of the laser scanning microscope. The values are expressed in percentage of the control data measured before iso- or hypotonic challenge (100%). To determine the extent of neuronal cell swelling, two parameters were estimated as described previously (Uckermann et al., 2004b): the cross-sectional area of neuronal cell bodies in the ganglion cell layer, as well as the cross- sectional area of Müller cell fibers that pass through the inner plexiform layer; a decrease of the fiber area is caused by swelling of synapses structures located in this layer.

Data are expressed as means ⫾ S.E.M. Statistical analysis was made by using the Prism program (GraphPad Software Inc., San Diego, CA); significance was determined by Mann-Whitney U test or by analysis of variance followed by comparisons for multiple groups. Curve fits were made by using the Boltzmann equation.

Results Neuronal Cell Swelling. The swelling of neuronal cells was investigated in whole mounts of the guinea pig retina. It is known that acute application of glutamate (1 mM) to the whole mounts causes rapid swelling of neuronal cell bodies in the ganglion cell (by ⬃30%; P ⬍ 0.001) (Fig. 1B) and inner nuclear layers (not shown) (Uckermann et al., 2004b). Simul- taneously, the glial (Müller) cell fibers in the inner plexiform layer decrease their thickness (by ⬃30%; P ⬍ 0.001) (Fig. 1C), caused by swelling of synaptic structures. A similar neuronal cell swelling can be observed during application of a high-K⫹ (50 mM) solution to the acutely isolated retinal whole mounts, as well as in the ischemic guinea pig retina just after reperfusion (Uckermann et al., 2004b).

The high- K⫹ -evoked neuronal cell swelling is mediated by stimulation of endogenous glutamate release and subsequent activation of ionotropic glutamate receptors (Uckermann et al., 2004b). To investigate a possible effect of triamcinolone on neuro- nal cell swelling, the cross-sectional areas of neuronal cell bodies in the ganglion cell layer and of Müller cell fibers in the inner plexiform layer were recorded. Extracellular appli- cation of triamcinolone acetonide (100 ␮M) did not decrease the magnitude of neuronal cell swelling in the ganglion cell layer evoked either by glutamate (1 mM) or by high K⫹ (Fig.

1D). Likewise, the glutamate- or high-K⫹ -evoked shrinkage of the glial cell profiles in the inner plexiform layer was not altered by triamcinolone (Fig. 1E). The data suggest that acute application of triamcinolone does not inhibit neuronal cell swelling. Fig. 1. Acute application of glutamate or high-K⫹ evokes morphological alterations in whole-mount guinea pig retinas that are not inhibited by triamcinolone. The effects of glutamate (1 mM) and high (50 mM) K⫹ were determined after 10 and 5 min of exposure, respectively. Triamcinolone (100 ␮M) was applied either alone or simultaneously with glutamate or high K⫹ .

A, in an acutely isolated retinal slice, Mitotracker Orange (10 ␮M) selectively stained Müller glial cells (green). The morphological alterations were recorded in whole mounts at two focal planes. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NFL, nerve fiber layer; ONL, outer nuclear layer; OPL, outer plexiform layer. B, application of glutamate (1 mM) caused swelling of neuronal cell bodies in the GCL. The Müller cell end feet are green-stained, and the unstained neuronal cell bodies reflect light (red). Yellow-appearing elongated structures are nerve fiber bundles.

C, green-stained Müller cell profiles in the IPL decreased their thickness in response to glutamate; the synaptic structures reflect red light. Scale bars, 20 ␮m. D, mean cross-sectional area of the neuronal cell bodies in the GCL. E, mean cross-sectional area of the Müller cell profiles in the IPL. The bars represent values obtained in 20 to 103 cells. Significant differences versus control: ⴱ, P ⬍ 0.001.

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Glial Cell Swelling. As shown earlier (Pannicke et al., 2004), the somata of Müller glial cells in slices of 3-day postischemic retinas responded to acute hypotonic stress by an increase in their cross-sectional area by 10 to 20%, whereas cell somata in control retinas did not increase their size (Fig. 2A). A similar osmotic soma swelling was observed in retinal slices isolated at 7 days after intravitreal applica- tion of LPS (Fig. 2D) (Pannicke et al., 2005), as well as in control retinas when the K⫹ channels were blocked by extra- cellular Ba2⫹ ions (Fig.

3A). The osmotic swelling of the cells in diseased retinas has been shown to be caused by the down-regulation of distinct K⫹ channels, which impairs the extrusion of K⫹ ions under hypotonic conditions and can be mimicked by closure of K⫹ channels in control cells in the presence of the K⫹ channel blocker Ba2⫹ (Pannicke et al., 2004).

Although triamcinolone (100 ␮M) had no effect on the size of glial cell somata when the slices of 3-day postischemic rat retinas were perfused with an isotonic extracellular solution, it fully inhibited the soma swelling evoked by perfusion of a hypotonic solution (P ⬍ 0.001) (Fig. 2, A and B). In contrast, acetazolamide (100 ␮M), a carbonic anhydrase blocker that is used clinically to resolve macular edema, did not reverse the osmotic swelling of glial cell somata in postischemic retinas (Fig. 2C). Triamcinolone (100 ␮M) also inhibited the osmotic swelling of glial cell somata in slices obtained at 7 days after intravitreal application of LPS (Fig.

2D), as well as in control retinas in the presence of Ba2⫹ (1 mM) (Fig. 3B). The inhib- itory effect of triamcinolone on the Ba2⫹ -evoked hypotonic glial cell swelling was dose-dependent (Fig. 3C). The concen- tration estimated to evoke half-maximal inhibition was 1.3 ␮M. Vehicle (DMSO) alone had no effect (Fig. 3F). Oxidative stress is a major cause of retinal injury after ischemia (Osborne et al., 2004). To determine whether oxi- dative stress induces swelling of glial cell somata, slices of control rat retinas were perfused with H2O2. As shown in Fig. 3D, H2O2 did not induce swelling of glial cell somata at isotonic conditions.

However, hypotonic challenge evoked soma swelling in the presence (but not in the absence) of H2O2. The swelling-inducing effect of H2O2 was dose-depen- dent, with a half-maximal effect at 4 ␮M (Fig. 3D, inset). When triamcinolone (100 ␮M) was applied simultaneously with the hypotonic solution, the soma swelling evoked by H2O2 was fully inhibited (Fig. 3E).

It is known that arachidonic acid evokes vasogenic and cytotoxic brain edema (Chan et al., 1983) and swelling of cultured astrocytes (Staub et al., 1994). Metabolites of ara- chidonic acid such as prostaglandins (especially PGE2) are implicated in the development of retinal edema during ocular inflammation. To determine whether arachidonic acid or PGE2 may cause glial cell swelling, both substances were tested acutely in slices of control rat retinas. Arachidonic acid (Fig. 4A) and PGE2 (Fig. 4B) evoked swelling of glial cell somata when the substances were applied simultaneously with the hypotonic solution.

An involvement of endogenous arachidonic acid and PGE2 in the osmotic soma swelling in postischemic retinas is suggested by the blocking effects of the inhibitor of the phospholipase A2, 4-bromophenacyl bro- mide (Fig. 4, C and D), and of the cyclooxygenase inhibitor, indomethacin (Fig. 4, E and F), respectively. Triamcinolone inhibited the soma swelling evoked by arachidonic acid or PGE2 (Fig. 4, A and B).

Mechanism of the Inhibitory Effect of Triamcino- lone on Glial Swelling. It is known that adenosine (which is rapidly released within the retina upon ischemia; Roth et al., 1997) exerts a protective effect against ischemic injury in the retina by the activation of A1 receptors (Larsen and Osborne, 1996; Ghiardi et al., 1999). A swelling-inhibiting effect of adenosine may contribute to this protective effect in ischemic injury. To determine whether adenosine inhibits the osmotic swelling of glial cell somata, we applied adeno- sine simultaneously with the hypotonic solution to retinal slices.

Adenosine inhibited the osmotic swelling of glial cell somata in postischemic retinas (Fig. 5A) and in control reti- nas in the presence of extracellular Ba2⫹ (Fig. 5B) or arachi- donic acid (Fig. 5C). This effect of adenosine was mediated by activation of A1 receptors since a selective A1 receptor ago- nist, CPA, also inhibited the swelling, and since the effect of Fig. 2. Effect of triamcinolone on the osmotic soma swelling of Müller glial cells in slices from 3-day postischemic retinas and from endotoxin-treated eyes, respectively. Swelling was induced by changing the perfusate into a hypotonic solution (60% of control osmolarity).

A, hypotonic challenge resulted in soma swelling in the case of postischemic retinas, whereas Müller cells in control retinas did not increase the cross-sectional area of their somata. Application of triamcinolone (100 ␮M) simultaneously with the hypotonic solution fully inhibited the soma swelling. The insets show original records of two dye-filled somata in a postischemic retina, before (left) and during (right) exposure of hypotonic medium. Scale bar, 5 ␮m. B and C, mean cross-sectional area of Müller cell somata in slices of postischemic retinas, in dependence on the recording conditions.

D, mean soma area in retinal slices derived from eyes at 7 days after intravitreal injection of LPS. Triamcinolone (100 ␮M) or acetazolamide (100 ␮M) were applied simultaneously with the iso- or hypotonic solution, as indicated. The values were measured after 4-min perfusion with the iso- or hypotonic solution and are expressed as percentage of the control value measured before application of the test solution (100%). The bars represent values obtained in 4 to 19 cells. Significant differences versus control (100%): ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001. Significant blocking effects: †, P ⬍ 0.05 , P ⬍ 0.001.

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adenosine was blocked by the selective antagonist of A1 receptors, DPCPX (Fig. 5B). In contrast, the swelling-inhib- iting effect of adenosine was not modified by a selective A2a receptor antagonist, CSC (Fig. 5B). To determine whether triamcinolone inhibits the osmotic swelling of glial cell somata via stimulation of the release of endogenous adenosine and subsequent stimulation of aden- osine receptors, it was applied in the presence of antagonists of adenosine receptors and transporters. Theophylline, a blocker of A1 and A2 receptors, fully reversed the effect of triamcinolone (Fig. 6A).

To determine the subtype of adeno- sine receptors that mediates the swelling-inhibiting effect of triamcinolone, selective antagonists of A1 and A2a receptors, DPCPX and CSC, respectively, were tested. As shown in Fig. 6, B and C, DPCPX fully blocked the swelling-inhibiting effect of triamcinolone, whereas CSC had no effect. On the other hand, MPEP and LY341495, antagonists of subtype 5 of groups I and II metabotropic glutamate receptors, did not block the effect of triamcinolone (Fig. 6B). The data suggest that the effect of triamcinolone is mediated by a release of endogenous adenosine and subsequent activation of A1 re- ceptors.

Thus, we tried to determine whether this adenosine was released from intracellular compartments via stimula- tion of nucleoside transporters or generated by degradation Fig. 3. Triamcinolone inhibits the osmotic soma swelling of Müller glial cells in slices of control retinas. The swelling was induced by changing the perfusate into a hypotonic solution (60% of control osmolarity) in the presence of extracellular Ba2⫹ (1 mM) or H2O2. A, in the presence of Ba2⫹ , hypotonic challenge evoked a reversible swelling of Müller cell somata in control retinas. The mean cross-sectional area of glial cell somata was measured in dependence on the recording time.

B, triamcinolone (100 ␮M) inhibited the osmotic swelling of Müller cell somata in the presence of Ba2⫹ .

Mean cross-sectional area of the somata in dependence on the recording conditions. C, dose dependence of the inhibiting effect of triamcinolone on the Ba2⫹ -evoked osmotic soma swelling. The curve was fitted with the Boltzmann equation and a half-maximal inhibitory concentration of 1.3 ␮M. D, H2O2 evoked dose dependently soma swelling under hypotonic conditions. Inset, data points were fitted with the Boltzmann equation, with a half-maximal effect at 4 ␮M. E, triamcinolone (100 ␮M) inhibited the osmotic soma swelling in the presence of H2O2 (200 ␮M). F, vehicle (DMSO; 0.1%) had no effect on the soma swelling in the presence of Ba2⫹ (1 mM) or H2O2 (200 ␮M).

Triamcinolone was applied simultaneously with the iso- or hypotonic solutions. B to F, values were measured after 4-min perfusion of the iso- or hypotonic solution and are expressed as percentage of the control value measured before application of the solution (100%). The bars represent values obtained in 4 to 45 cells. Significant difference versus control (100%): ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001. Significant blocking effect: †, P ⬍ 0.05 , P ⬍ 0.01 , P ⬍ 0.001.

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of extracellular ATP. For this purpose, various blocking sub- stances were tested. The inhibitor of nucleoside transporters, NBTI, blocked the effect of triamcinolone (Fig. 6D). On the other hand, the ecto-ATPase inhibitor, ARL-67156, and the ectonucleotidase inhibitor, AOPCP, did not block the swell- ing-inhibiting effect of triamcinolone (Fig. 6E). These data suggest that the effect of triamcinolone was mediated by stimulation of transporter-mediated release of adenosine from intracellular compartments but not by extracellular formation of adenosine via degradation of ATP.

Tetrodotoxin failed to interfere with the effect of triamcinolone (Fig. 6F), suggesting that it is independent of the activation of retinal neurons.

Finally, we tried to identify the intracellular signaling mechanisms that inhibit swelling after A1 receptor stimula- tion. The swelling-inhibiting effects of triamcinolone and of adenosine remained unaltered in the presence of the mem- brane-permeable Ca2⫹ chelator BAPTA/AM (Fig. 7, A and B), suggesting that an intracellular Ca2⫹ response was not in- volved in the effects of both agents. On the other hand, application of a cAMP-enhancing cocktail that contained pCPT-cAMP (a membrane-permeable cAMP analog), forsko- lin (a direct activator of adenylyl cyclase), and IBMX (a phosphodiesterase inhibitor), reduced the osmotic glial soma swelling by approximately two-thirds in postischemic retinas (Fig.

7C) and in control retinas in the presence of Ba2⫹ (1 mM) (Fig. 7D). To explore whether the effects of triamcino- lone and adenosine were mediated by an activation of the adenylyl cyclase and a subsequent activation of protein ki- nase A, blockers of both enzymes were tested. 2,3-Dideoxy- Fig. 4. Arachidonic acid (AA) and PGE2 induce swelling of Müller cell somata at hypotonic conditions, which are inhibited by triamcinolone. A, triamcinolone (100 ␮M) inhibits the AA (10 ␮M)-evoked swelling in control retinas. B, triamcinolone (100 ␮M) decreases the soma swelling evoked by PGE2 (30 nM) in control retinas.

C, the inhibitor of the phospholipase A2, 4-bromophenacyl bromide (300 ␮M), reduces the osmotic soma swelling in postischemic retinas and in control retinas in the presence of Ba2⫹ (1 mM) (D). E, the cyclooxygenase inhibitor, indomethacin (10 ␮M), reversed the osmotic swelling in postischemic retinas and F, in control retinas in the presence of Ba2⫹ . The test substances were simultaneously applied with the hypotonic solution. The bars represent values obtained in 4 to 15 cells. Significant differences versus control (100%): ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001. Significant blocking effect , P ⬍ 0.01 , P ⬍ 0.001.

Fig. 5. Adenosine inhibits the osmotic swelling of Müller cell somata via activation of A1 receptors. Retinal slices were perfused with a hypotonic solution, and the relative soma size was measured before (100%) and after 4-min perfusion of the solution. A, soma size in control and postischemic retinas, in the absence and presence of adenosine (10 ␮M). B, soma size in control retinas in the presence of Ba2⫹ (1 mM). The swelling-inhibiting effect of adenosine (10 ␮M) was largely blocked by the selective antagonist of A1 receptors, DPCPX (100 nM), and remained unaltered in the presence of the A2a receptor antagonist CSC (200 nM).

The A1 receptor agonist CPA (100 nM) mimicked the swelling-inhibiting effect of adenosine. C, the osmotic soma swelling induced by AA (10 ␮M) was largely inhibited by adenosine (10 ␮M). The test substances were simultaneously applied with the hypotonic solution; blocking substances were preincubated for 10 min. The bars represent values obtained in 4 to 35 cells. Significant differences versus control (100%): ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001. Significant blocking effect , P ⬍ 0.01 , P ⬍ 0.001. Significant inhibition of the agonist effect: EEE, P ⬍ 0.001. Triamcinolone Inhibits Glial Cell Swelling 1041 at ASPET Journals on February 7, 2015 jpet.aspetjournals.org Downloaded from

adenosine, an inhibitor of the adenylyl cyclase, blocked the effect of triamcinolone (Fig. 7E). Likewise, an inhibitor of the protein kinase A, H-89, reduced the effects of triamcinolone (Fig. 7E) and adenosine (Fig. 7F) by approximately two thirds. Arachidonic acid has been shown to inhibit the efflux of osmolytes such as amino acids and Cl⫺ from cultured astro- cytes, an effect that may contribute to osmotic swelling (Sanchez-Olea et al., 1995). Thus, the inhibition of cell swell- ing may involve the opening of extrusion pathways for os- molytes. To determine whether adenosine inhibits osmotic swelling by opening of such extrusion pathways for K⫹ and/or Cl⫺ , the K⫹ channel blocker, quinine, and the Cl⫺ channel blockers, flufenamic acid and NPPB, were tested.

Indeed, these substances were found to block the effect of adenosine (Fig. 7G). Taken together, our data suggest that the inhibitory effect of triamcinolone on osmotic Müller cell swelling involves the release of endogenous adenosine and subsequent activation of A1 receptors. A1 receptor activation results in enhancement of the intracellular cAMP level and activation of the protein kinase A, which finally causes a compensatory efflux of K⫹ and Cl⫺ ions. Discussion Various ischemic and inflammatory ocular diseases are associated with the development of macular edema that may be caused by vascular leakage (vasogenic, extracellular edema) and/or by swelling of retinal cells (cytotoxic, intracel- lular edema).

It has been suggested that swelling of Müller glial cells contributes to macular edema (e.g., in cases with- out significant angiographic vascular leakage; Fine and Brucker, 1981; Yanoff et al., 1984) and that a dysregulation of retinal fluid absorption, normally carried out by pigment epithelial and Müller glial cells (Bringmann et al., 2004), is likely to be crucial for the formation of chronic edema (Mori et al., 2002). Since Müller cells mediate the water transport between the inner retinal tissue and the blood and vitreous (Bringmann et al., 2004), Müller cell swelling, when it occurs in vivo, may reflect such a dysregulated fluid clearance.

It has been shown that experimental ischemia reperfusion of the rat retina evokes a biphasic water accumulation in the retina; the retinal water content increases strongly during the ischemic episode and increases again slowly within days after reperfusion (Stefánsson et al., 1987). The edema that Fig. 6. The swelling-inhibiting effect of triamcinolone is mediated by endogenous release of adenosine and activation of A1 receptors. Osmotic swelling of Müller cell somata was induced by hypotonic challenge in slices of control retinas in the presence of Ba2⫹ (1 mM). A, the swelling-inhibiting effect of triamcinolone (100 ␮M) was blocked by the nonselective antagonist of adenosine receptors, theophylline (250 ␮M).

B, the effect of triamcinolone (100 ␮M) was blocked by DPCPX (100 nM) and remained unaltered in the presence of MPEP (50 ␮M) and LY341495 (100 ␮M). C, CSC (200 nM) did not block the swelling-inhibiting effect of triamcinolone (100 ␮M). D, NBTI (10 ␮M) reversed the swelling-inhibiting effect of triamcinolone (100 ␮M). E, ARL-67156 (50 ␮M) or AOPCP (250 ␮M) had no effects on the swelling-inhibiting action of triamcinolone (100 ␮M). F, the effect of triamcinolone (100 ␮M) remained unaltered in the presence of tetrodotoxin (TTX; 1 ␮M). The bars represent values obtained in 3 to 19 cells. Significant differences versus control (100%): ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001.

Significant blocking effects: †, P ⬍ 0.05 , P ⬍ 0.01 , P ⬍ 0.001. Significant inhibition of the agonist effects: E, P ⬍ 0.05.

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develops during the ischemic period is likely caused by neu- ronal cell swelling and is mediated, at least in part, by overstimulation of ionotropic glutamate receptors, which al- lows strong ion fluxes into retinal neurons, accompanied by water uptake (Uckermann et al., 2004b). The edema that develops after reperfusion may be caused, at least in part, by glial cell swelling (Pannicke et al., 2004). A similar pattern of neuronal and glial cell edema was described in the ischemic retina of the rabbit; during ischemic episodes, both plexiform layers and the cytoplasm of neuronal cells become edema- tous, whereas the Müller glial cells appear to be unaffected.

However, in the postischemic tissue, the Müller glial cells become edematous, whereas neural elements degenerate (Johnson, 1974).

Experimental ischemia reperfusion or endotoxin-evoked ocular inflammation cause a significant alteration of the osmotic swelling characteristics of Müller glial cells in the rat retina (Pannicke et al., 2004, 2005). During hypotonic stress, Müller cells in postischemic retinas (Fig. 2, A and B) or in retinas obtained from eyes displaying ocular inflammation (Fig. 2D) increase their soma volume, whereas cells in control retinas do not swell (Fig. 2A). The experimental model of acute hypotonic challenge used in the present study may be representative for pathological in vivo conditions. Hypotonic challenge mimics the osmotical conditions at the glio-vascu- lar and glio-vitreal interfaces during pathological states, such as ischemia, in which the retinal parenchyma becomes hyperosmotic in comparison with the blood and vitreous.

When the Müller glial cells take up excess osmolytes (e.g., K⫹ and Na⫹ /glutamate during periods of pathological neuronal hyperexcitation) and are impaired in their ability to redis- tribute these osmolytes into the blood and vitreous, an in- Fig. 7. The swelling-inhibiting effect of triamcinolone is largely mediated by activation of the adenylyl cyclase and protein kinase A and by opening of K⫹ and Cl⫺ channels. Osmotic swelling of Müller cell somata was induced by hypotonic challenge in slices of 3-day postischemic retinas or of control retinas in the presence of Ba2⫹ (1 mM). A, preincubation of the slices with BAPTA/AM (100 ␮M) for 30 min did not block the swelling-inhibiting effects of triamcinolone (100 ␮M) or of B, adenosine (10 ␮M).

C, application of a cAMP-enhancing cocktail that contained pCPT-cAMP (100 ␮M), forskolin (10 ␮M), and IBMX (100 ␮M) reduced the osmotic soma swelling in postischemic retinas as well as in control retinas (D). E, 2,3-dideoxyadenosine (20 ␮M) and H-89 (1 or 30 ␮M), respectively, reduced the swelling-inhibiting effect of triamcinolone (100 ␮M) in control retinas. F, H-89 (1 or 30 ␮M) reduced the effect of adenosine (10 ␮M). G, the swelling-inhibiting effect of adenosine (10 ␮M) was blocked by quinine (200 ␮M) and by the Cl⫺ channel blockers flufenamic acid (500 ␮M) and NPPB (100 ␮M), respectively.

The bars represent values obtained in 4 to 26 cells. Significant differences versus control (100%): ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001. Significant blocking effects: †, P ⬍ 0.05 , P ⬍ 0.01 , P ⬍ 0.001. Significant inhibition of the agonist effects: EE, P ⬍ 0.01; EEE, P ⬍ 0.001.

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creased intracellular osmolarity may constitute an osmotical driving force for water movement from the blood and vitreous into the Müller cells, resulting in cell swelling (Pannicke et al., 2004). However, the experimental model of hypotonic stress used in the present study may also reflect the condi- tions at the glio-synaptic interface since it is known that glial uptake of excess K⫹ causes a reduction of the extracellular osmolarity (Dietzel et al., 1989).

Ion fluxes from the extra- cellular space into the synapses through ionotropic receptors may contribute to the fall of extracellular osmolarity during neuronal activity. Therefore, experimental hypotonic stress may mimic the osmotic gradients during neuronal hyperex- citation that occurs during ischemic insults. The present results suggest that inflammatory mediators and/or oxidative stress (that is one factor causing neuronal degeneration during reperfusion; Osborne et al., 2004) medi- ate the alteration of the osmotic swelling characteristics of Müller glial cells. Müller cell swelling in the postischemic retina may be induced by inflammatory mediators, due to the activation of phospholipase A2 and cyclooxygenase by osmotic stress.

The mechanisms how arachidonic acid, PGE2, or H2O2 induce hypotonic glial cell swelling are unclear. It has been shown that arachidonic acid inhibits distinct outwardly di- rected K⫹ currents in isolated Müller cells (Bringmann et al., 1998), and it is conceivable (but remains to be proven) that arachidonic acid and PGE2 close plasma membrane path- ways that are involved in osmolyte extrusion out of the cells. A failure in osmolyte extrusion upon hypotonic challenge would cause water movement into the cells and, thus, cell swelling. The observation that blockade of K⫹ and Cl⫺ chan- nels inhibits the effect of adenosine (Fig.

7G) strongly sup- ports the view that a closure of ion release pathways is involved in the induction of osmotic swelling. It is known that arachidonic acid induces both vasogenic and cytotoxic edema in the brain (Chan et al., 1983; Wahl et al., 1993) and evokes swelling of cultured astrocytes (Staub et al., 1994), and it has been suggested that the swelling-inducing effect of arachi- donic acid is mediated by a direct inhibition of the efflux of osmolytes such as amino acids and Cl⫺ (Sanchez-Olea et al., 1995).

Corticosteroids such as triamcinolone are used clinically to resolve macular edema (Ip et al., 2003; Massin et al., 2004). The mechanisms of the triamcinolone effects are incom- pletely understood; present research is focused on the inhi- bition of vascular edema. It has been shown recently that triamcinolone induces a very rapid reduction of macular edema in patients with retinal vein occlusion and diabetic retinopathy, which is evident as early as 1 h after treatment (Miyamoto et al., 2005). However, the regulation of tight junctional and VEGF protein expression by triamcinolone is suggested to occur at the gene expression level that needs several hours.

Therefore, it has been suggested that triam- cinolone acts, at least additionally, via activation of signaling cascades (Miyamoto et al., 2005). Here, we present data supporting the idea that triamcinolone inhibits Müller cell swelling in situ via stimulation of endogenous adenosine signaling. A1 receptor stimulation after release of endoge- nous adenosine causes an activation of the adenylyl cyclase and of the protein kinase A that results in a stimulation of compensatory ion release by Müller glial cells. The lack of effects of inhibitors of the ecto-ATPase and the ectonucleoti- dase on the effect of triamcinolone (Fig.

6E) suggests that triamcinolone selectively stimulates the transporter-medi- ated release of adenosine from retinal cells, whereas it has no effect on the extracellular formation of adenosine via degra- dation of ATP. It is conceivable that triamcinolone stimulates the water clearance function of Müller cells in situ by the activation of ion secretion via Müller cell end feet into the blood and vitreous. Similar rapid effects of triamcinolone (within minutes) have been described for other cell systems, e.g., for the remodeling of the nuclear envelope structure in Xenopus oocytes (Shahin et al., 2005).

Our results suggest that stimulation of A1 receptors by adenosine results in an enhancement of the intracellular cAMP level. Although in most cell systems activation of A1 receptors causes a de- crease of the cAMP level, there are observations that (in dependence on the density of the receptors expressed by the cells) A1 receptors may couple also to stimulating G proteins, resulting in an enhancement of the intracellular cAMP level (Cordeaux et al., 2000). However, the inhibitory effect of cAMP elevation on cell swelling was only partial (Fig. 7, C and D). Similarly, blockade of the adenylyl cyclase or of the protein kinase A resulted in a partial inhibition of the effects of triamcinolone and adenosine (Fig.

7, E and F), suggesting that a second intracellular pathway may contribute to the signaling from A1 receptors to ion channels. In the present study, we used triamcinolone in a concen- tration of 100 ␮M (i.e., 43 ␮g/ml), unless stated otherwise. In human patients, a vitreal dosage of 1 mg/ml is used regularly (4 mg of triamcinolone in 4 ml of human vitreous volume), and peak concentrations between 2 and 7 ␮g/ml in aqueous humor samples were described to occur within days after a single 4-mg intravitreal injection of triamcinolone (Beer et al., 2003). We observed a half-maximal effect of triamcino- lone at 1.3 ␮M, i.e., 0.56 ␮g/ml (Fig.

3C), which is well in the range of concentration observed in human subjects. In summary, we show that triamcinolone does not inhibit glutamate- and high-K⫹ -evoked swelling of retinal neurons but inhibits the osmotic swelling of Müller glial cells during retinal ischemia reperfusion and inflammation. This inhibi- tory effect of triamcinolone on cytotoxic glial cell swelling may contribute to the fast edema-resolving effect of this substance observed in human patients. This view is sup- ported by the observation that triamcinolone also rapidly resolves cystoid macular edema in patients without signifi- cant angiographic vascular leakage.

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