- Email: [email protected]
Endothelin receptors in the detached retina of the pig
Neuroscience Letters 384 (2005) 72–75
Endothelin receptors in the detached retina of the pig Ianors Iandiev a , Susann Uhlmann b , Uta-Carolin Pietsch c , Bernd Biedermann a , Andreas Reichenbach a , Peter Wiedemann b , Andreas Bringmann b,∗ a
Paul Flechsig Institute of Clinical Brain Research, University of Leipzig Medical Faculty, D-04109 Leipzig, Germany b Department of Ophthalmology and Eye Clinic, University of Leipzig Medical Faculty, D-04103 Leipzig, Germany c Klinik f¨ ur An¨asthesie und Intensivmedizin, University of Leipzig Medical Faculty, D-04103 Leipzig, Germany Received 21 March 2005; received in revised form 8 April 2005; accepted 16 April 2005
Abstract Endothelin-1 (ET-1) is a potent vasoconstrictor that causes hypoperfusion of the neurosensory retina. We investigated immunohistochemically the expression of the receptors for ET-1, ETA and ETB , in control and locally detached retinas of the pig. Immunoreactivity for ETA was expressed in the innermost retinal layers and in the outer plexiform layer in control retinas, and was additionally strongly expressed by retinal blood vessels at 7 days after detachment of the sensory retina from the pigment epithelium. Immunoreactivity for ETB was expressed by the innermost retinal layers, by ganglion cell somata, and by M¨uller glial cells in the control tissue, and was not altered in its expression after detachment. The vascular expression of ETA may suggest a hypoperfusion of the retina after detachment. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Endothelin; Immunohistochemistry; Blood vessels; M¨uller cells; Retinal detachment; Pig
Detachment of the neurosensory retina from the pigment epithelium causes complex alterations and remodeling of the retinal tissue. In addition to the deconstruction of outer segments and photoreceptor cell death, there are morphological and biochemical alterations of the inner retinal neurons [5], as well as a fast activation of pigment epithelial and macroand microglial cells [6,20]. The activation of glial cells in the experimentally detached retina begins within minutes of detachment [6] and develops during the first hours and days after creation of the detachment [20]. The gliosis of M¨uller cells (the predominant macroglial cell in the retina) is characterized by the upregulation of the expression of immunoreactivity for the intermediate filaments vimentin and glial fibrillary acidic protein, by cellular hypertrophy, and by distinct physiological alterations such as upregulation of the responsiveness upon stimulation of distinct receptors [20]. A variety of growth and activating factors may induce glial cell activation in the injured retina. Upon retinal detachment, mechanical stress may activate glial cells, and in∗
Corresponding author. Tel.: +49 341 9721557; fax: +49 341 9721659. E-mail address: [email protected] (A. Bringmann).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.04.056
traretinally released growth factors and cytokines activate mitogen-activated protein kinases and transcription factors, as observed previously [6]. Other factors that were proposed to induce glial cell activation upon injury, include the endothelins that bind at endothelin (ET) A and B receptors. In the brain and spinal cord, activation of astrocytes is normally accompanied by upregulation of ETB receptors [7,21] while it is not known if this is also the case in the sensory retina. ET1 is a potent vasoconstrictor of retinal blood vessels [1,11], as well as a mitogen for retinal vascular smooth muscle cells and pericytes [8], and has been suggested to be implicated in the pathogenesis of important retinopathies such as glaucoma [10] and diabetic retinopathy [8]. Enhanced action or levels of ET-1 are associated with decreased blood flow in the retinas of diabetic animals. It has been shown that ET-1 promotes gliosis of optic nerve head astrocytes in vitro [13], and that experimental glaucoma or optic nerve transection increases the expression of ET-1 and ETB in optic nerve astrocytes [12,14]. By using autoradiography, it has been shown that ETA -like binding sites are expressed by blood vessels of the human retina while ETB -like binding sites are localized to neural and glial cells of the retina [9]. In other studies,
I. Iandiev et al. / Neuroscience Letters 384 (2005) 72–75
the presence of ETA and ETB receptors on vascular smooth muscle cells of retinal vessels, and of ETB receptors within the ganglion cell layer, has been shown [2,17]. In retinas of diabetic rats, an upregulation of endothelins, ETA and ETB has been described [3]. However, it is not known whether M¨uller glial cells in the sensory retina express receptors for endothelins, and whether the expression is altered during retinal injury. Therefore, we investigated the expression of ETA and ETB proteins in retinal slices derived from pig eyes. We immunohistochemically stained slices from control retinas, as well as from retinas that were detached from the pigment epithelium for 7 days. As a marker for glial cells, the slices were co-stained against vimentin. All experiments were carried out in accordance with applicable German laws and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Three young adult domestic pigs (17–22 kg; both sexes) were used. Twenty-four hours before and after surgery, the food intake of the animals was restricted with free access to water. Intravenous azaperon (15 mg/kg; Cilag-Janssen, Neuss, Germany), atropin (0.2 mg/kg; Braun, Melsungen, Germany) and ketamine (3 mg/kg; Ratiopharm, Ulm, Germany) were administered for premedication. The anesthesia was induced with thiopental (8 mg/kg i.v.; Trapanal, Byk Gulden, Konstanz, Germany) and maintained with isoflurane (Forene, Abbott, Wiesbaden, Germany). Ventilation was performed using the Julian respirator (Draegerwerk AG, Luebeck, Germany) with a Fi O2 of 40%. Rhegmatogenous detachment was created in one eye per animal; the other eye served as untreated control. The pupils of the eyes were dilated by topical tropicamide (1%; Ursapharm, Saarbr¨ucken, Germany) and phenylephrine hydrochloride (5%; Ankerpharm, Rudolstadt, Germany), and a lateral canthotomy was created. Hemostasis was achieved with wet-field cautery. After pars plana sclerotomy, a circumscript vitrectomy was performed in the area of the future detachment. Thin glass micropipettes attached to 250 l glass syringes (Hamilton, Reno, NV) were used to create a retinal detachment by subretinal injection of saline followed by 0.25% sodium hyaluronate (Healon; Pharmacia & Upjohn, D¨ubendorf, Switzerland) in saline. The retina below the optic nerve head was detached while the retina above the optic nerve head remained attached. After surgery, gentamicin (5 mg) and dexamethasone (0.5 mg) were injected subconjunctivally. The lateral cauthotomy was closed with 5–0 silk sutures, and atropine (1%) eye drops were instilled into the conjunctival sac. After a survival time of 7 days, the animals were anaesthetized as described, the eyes were excised, and the animals were killed by i.v. T61 (embutramid mebezonium iodide; 0.65 ml/kg body weight; Hoechst, Unterschleißheim, Germany). Isolated retinas were fixed in 4% paraformaldehyde for 2 h. After several washing steps in buffered saline, the tissue was embedded in saline containing 3% agarose (w/v), and 70-m thick slices were cut by using a vibratome. The slices were incubated in 5% normal goat serum plus 0.3% Triton X-100 in saline for 2 h and, subsequently, in primary
73
antibody overnight at 4 ◦ C. After washing in 1% bovine serum albumin in saline, the secondary antibodies were applied for 4 h at room temperature. The following antibodies were used: mouse anti-vimentin (1:500; V9 clone, SigmaAldrich, Taufkirchen, Germany), anti-ETA (1:100; Alomone Labs, Jerusalem, Israel), anti-ETB (1:100; Alomone), Cy3conjugated goat anti-rabbit IgG (1:400; Dianova, Hamburg, Germany), and Cy2-coupled goat anti-mouse IgG (1:400; Dianova). In control retinas, the immunoreactivity for vimentin was most strongly expressed in the innermost retinal layers (nerve fiber and ganglion cell layers) that reflects the prominant expression of vimentin by retinal astrocytes and endfeet of M¨uller cells (Fig. 1A and B). In addition, M¨uller cell fibers that pass through the inner retinal layers displayed faint staining for vimentin. In retinal tissue that was detached for 7 days, the vimentin labeling of M¨uller cells increased strongly when compared to control, with labeling of M¨uller cell fibers from the inner to the outer limiting membranes. In control retinas of the pig, the immunoreactivity for ETA was expressed in the innermost retinal layers and in the outer plexiform layer (Fig. 1A). At 7 days after detachment, immunoreactivity for ETA was strongly expressed in the blood vessels of the inner retina. In control tissue, immunoreactivity for ETB was expressed within the ganglion cell and nerve fiber layers, as well as by M¨uller cell fibers (Fig. 1B). In respect to M¨uller cells, co-staining of vimentin and ETB immunoreactivities was observed at the fibers that pass through the inner plexiform and nuclear layers, and through the outer retina. Additionally, the outer limiting membrane was ETB immunopositive. In addition to ganglion cell bodies that express immunoreactivity for ETB , it is likely that both the astrocytes, which are localized preferentially around the vessels in the ganglion cell/nerve fiber layers, and the M¨uller cell endfeet express immunoreactivity for ETB . In the detached retinas, no apparent alteration of the ETB immunostaining was observed. Endothelin-1 is a major regulator of the retinal blood flow that produces vasoconstriction of the retinal microvessels and subsequent decrease of retinal blood flow, predominantly by activation of ETA receptors [11]. In the present study, we found different expression of ETA and ETB immunoreactivities in the porcine retina: In control retinas, immunoreactivity for ETA was expressed in the innermost retinal layers (likely by retinal astrocytes) and in the outer plexiform layer, while immunoreactivity for ETB was expressed by the innermost retinal layers, by ganglion cell somata, and by M¨uller glial cells. Moreover, we found a different regulation of receptor protein expression upon retinal detachment: whereas the immunoreactivity for ETA was strongly upregulated in retinal blood vessels, the immunoreactivity for ETB was not altered in its expression after detachment. The functional significance of the vascular upregulation of ETA receptors upon detachment is unknown. It has been shown that intravitreal application of ET-1 produces vasoconstriction and retinal ischemia [19]. There is evidence
74
I. Iandiev et al. / Neuroscience Letters 384 (2005) 72–75
Fig. 1. Experimental detachment alters the expression of the immunoreactivity for ETA protein in the pig retina. The slices were derived from control retinas, and from retinas that were detached from the pigment epithelium for 7 days. (A) Co-immunostaining against ETA and vimentin. Arrowheads indicate large blood vessels in the innermost retinal layers. Arrows mark ETA -positive intraretinal blood vessels. (B) Immunoreactivities for ETB and vimentin. Arrows indicate vimentin-positive fibers in the inner plexiform layer that were stained also for ETB . Cell nuclei were counterstained using Hoechst 33258 (blue). Scale bars, 20 m. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OLM, outer limiting membrane; ONL, outer nuclear layer; and OPL, outer plexiform layer.
I. Iandiev et al. / Neuroscience Letters 384 (2005) 72–75
suggesting that retinal detachment is associated with disturbances of retinal circulation. The retinal blood flow rate is decreased in patients with retinal detachment compared to control [18]. Retinal circulation times of the detached areas are longer than those of the non-detached areas in patients with retinal detachment, and both are longer than those of normal subjects [16]. The extent of retinal detachment correlates with the decrease of ocular blood flow velocities [15]. It is conceivable that enhanced responsiveness of ETA receptors on vascular cells may induce ischemia–hypoxia in the inner retinal tissue that may contribute to the induction of morphological and biochemical alterations of the inner retinal neurons, and of glial cell activation, as observed in the detached inner retina [5]. In addition, stimulation of ETA receptors may contribute to the induction of proliferation of cells associated with the vasculature observed in experimental detachment [4]. We found no detachment-induced alteration in the expression of ETB immunoreactivity by M¨uller cells. Astrocytes in the brain and in the optic nerve strongly enhance the expression of ETB after lesion [14,21] and during glaucoma [12], respectively. The functional significance of the constitutive expression of ETB receptors in M¨uller cells of the pig retina remains to be established.
[10]
Acknowledgements
[15]
The authors thank Ute Weinbrecht for excellent technical support. This work was supported by grants from the S¨achsisches Ministerium f¨ur Wissenschaft und Kunst (SMWK; HWP program) and from the DFG (Br 1249/2-1).
[6]
[7]
[8]
[9]
[11]
[12]
[13]
[14]
[16]
[17]
References [1] S.E. Bursell, A.C. Clermont, B. Oren, G.L. King, The in vivo effect of endothelins on retinal circulation in nondiabetic and diabetic rats, Invest. Ophthalmol. Vis. Sci. 36 (1995) 596–607. [2] J.A. De Juan, F.J. Moya, A. Ripodas, R. Bernal, A. Fernandez-Cruz, R. Fernandez-Durango, Changes in the density and localisation of endothelin receptors in the early stages of rat diabetic retinopathy and the effect of insulin treatment, Diabetologia 43 (2000) 773–785. [3] D. Deng, T. Evans, K. Mukherjee, D. Downey, S. Chakrabarti, Diabetes-induced vascular dysfunction in the retina: role of endothelins, Diabetologia 42 (1999) 1228–1234. [4] S.K. Fisher, P.A. Erickson, G.P. Lewis, D.H. Anderson, Intraretinal proliferation induced by retinal detachment, Invest. Ophthalmol. Vis. Sci. 32 (1991) 1739–1748. [5] S.K. Fisher, G.P. Lewis, M¨uller cell and neuronal remodeling in retinal detachment and reattachment and their potential consequences
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for visual recovery: a review and reconsideration of recent data, Vision Res. 43 (2003) 887–897. S.F. Geller, G.P. Lewis, S.K. Fisher, FGFR1, signaling, and AP-1 expression after retinal detachment: reactive M¨uller and RPE cells, Invest. Ophthalmol. Vis. Sci. 42 (2001) 1363–1369. N. Ishikawa, M. Takemura, Y. Koyama, Y. Shigenaga, T. Okada, A. Baba, Endothelins promote the activation of astrocytes in rat neostriatum through ETB receptors, Eur. J. Neurosci. 9 (1997) 895–901. H.C. Lam, J.K. Lee, C.C. Lu, C.H. Chu, M.J. Chuang, M.C. Wang, Role of endothelin in diabetic retinopathy, Curr. Vasc. Pharmacol. 1 (2003) 243–250. M.W. MacCumber, S.A. D’Anna, Endothelin receptor-binding subtypes in the human retina and choroid, Arch. Ophthalmol. 112 (1994) 1231–1235. W. Noske, J. Hensen, M. Weiderholt, Endothelin-like immunoreactivity in aqueous humor of primary open-angle glaucoma and cataract patients, Graefes Arch. Clin. Exp. Ophthalmol. 235 (1997) 551–552. K. Polak, A. Luksch, B. Frank, K. Jandrasits, E. Polska, L. Schmetterer, Regulation of human retinal blood flow by endothelin-1, Exp. Eye Res. 76 (2003) 633–640. G. Prasanna, C. Hulet, D. Desai, R. Krishnamoorthy, S. Narayan, A.M. Brun, A.M. Suburo, T. Yorio, Effect of elevated intraocular pressure on endothelin-1 in a rat model of glaucoma, Pharmacol. Res. 51 (2005) 41–50. G. Prasanna, R. Krishnamoorthy, A.F. Clark, R.J. Wordinger, T. Yorio, Human optic nerve head astrocytes as a target for endothelin1, Invest. Ophthalmol. Vis. Sci. 43 (2002) 2704–2713. S.D. Rogers, E. Demaster, M. Catton, J.R. Ghilardi, L.A. Levin, J.E. Maggio, P.W. Mantyh, Expression of endothelin-B receptors by glia in vivo is increased after CNS injury in rats, rabbits, and humans, Exp. Neurol. 145 (1997) 180–195. M. Roldan Pallares, I.A. Ossama, E. Contreras, J. Hernandez, T. Ganado, C. Bravo, Ocular blood flow and characteristics of retinal detachment, Arch. Soc. Esp. Oftalmol. 76 (2001) 351–356. Y. Satoh, Retinal circulation in rhegmatogenous retinal detachment demonstrated by videofluorescence angiography and image analysis. I. The condition of retinal circulation before retinal detachment surgery, Nippon Ganka Gakkai Zasshi 93 (1989) 1002–1008. A.W. Stitt, U. Chakravarthy, T.A. Gardiner, D.B. Archer, Endothelinlike immunoreactivity and receptor binding in the choroid and retina, Curr. Eye Res. 15 (1996) 111–117. H. Tagawa, G.T. Feke, D.G. Goger, J.W. McMeel, H. Furukawa, Retinal blood flow changes in eyes with rhegmatogenous retinal detachment and scleral buckling procedures, Nippon Ganka Gakkai Zasshi 96 (1992) 259–264. K. Takei, T. Sato, T. Nonoyama, T. Miyauchi, K. Goto, S. Hommura, A new model of transient complete obstruction of retinal vessels induced by endothelin-1 injection into the posterior vitreous body in rabbits, Graefes Arch. Clin. Exp. Ophthalmol. 231 (1993) 476–481. S. Uhlmann, A. Bringmann, O. Uckermann, T. Pannicke, M. Weick, E. Ulbricht, I. Goczalik, A. Reichenbach, P. Wiedemann, M. Francke, Early glial cell reactivity in experimental retinal detachment: effect of suramin, Invest. Ophthalmol. Vis. Sci. 44 (2003) 4114–4122. U. Wilhelmsson, L. Li, M. Pekna, C.H. Berthold, S. Blom, C. Eliasson, O. Renner, E. Bushong, M. Ellisman, T.E. Morgan, M. Pekny, Absence of glial fibrillary acidic protein and vimentin prevents hypertrophy of astrocytic processes and improves post-traumatic regeneration, J. Neurosci. 24 (2004) 5016–5021.
Endothelin receptors in the detached retina of the pig Ianors Iandiev a , Susann Uhlmann b , Uta-Carolin Pietsch c , Bernd Biedermann a , Andreas Reichenbach a , Peter Wiedemann b , Andreas Bringmann b,∗ a
Paul Flechsig Institute of Clinical Brain Research, University of Leipzig Medical Faculty, D-04109 Leipzig, Germany b Department of Ophthalmology and Eye Clinic, University of Leipzig Medical Faculty, D-04103 Leipzig, Germany c Klinik f¨ ur An¨asthesie und Intensivmedizin, University of Leipzig Medical Faculty, D-04103 Leipzig, Germany Received 21 March 2005; received in revised form 8 April 2005; accepted 16 April 2005
Abstract Endothelin-1 (ET-1) is a potent vasoconstrictor that causes hypoperfusion of the neurosensory retina. We investigated immunohistochemically the expression of the receptors for ET-1, ETA and ETB , in control and locally detached retinas of the pig. Immunoreactivity for ETA was expressed in the innermost retinal layers and in the outer plexiform layer in control retinas, and was additionally strongly expressed by retinal blood vessels at 7 days after detachment of the sensory retina from the pigment epithelium. Immunoreactivity for ETB was expressed by the innermost retinal layers, by ganglion cell somata, and by M¨uller glial cells in the control tissue, and was not altered in its expression after detachment. The vascular expression of ETA may suggest a hypoperfusion of the retina after detachment. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Endothelin; Immunohistochemistry; Blood vessels; M¨uller cells; Retinal detachment; Pig
Detachment of the neurosensory retina from the pigment epithelium causes complex alterations and remodeling of the retinal tissue. In addition to the deconstruction of outer segments and photoreceptor cell death, there are morphological and biochemical alterations of the inner retinal neurons [5], as well as a fast activation of pigment epithelial and macroand microglial cells [6,20]. The activation of glial cells in the experimentally detached retina begins within minutes of detachment [6] and develops during the first hours and days after creation of the detachment [20]. The gliosis of M¨uller cells (the predominant macroglial cell in the retina) is characterized by the upregulation of the expression of immunoreactivity for the intermediate filaments vimentin and glial fibrillary acidic protein, by cellular hypertrophy, and by distinct physiological alterations such as upregulation of the responsiveness upon stimulation of distinct receptors [20]. A variety of growth and activating factors may induce glial cell activation in the injured retina. Upon retinal detachment, mechanical stress may activate glial cells, and in∗
Corresponding author. Tel.: +49 341 9721557; fax: +49 341 9721659. E-mail address: [email protected] (A. Bringmann).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.04.056
traretinally released growth factors and cytokines activate mitogen-activated protein kinases and transcription factors, as observed previously [6]. Other factors that were proposed to induce glial cell activation upon injury, include the endothelins that bind at endothelin (ET) A and B receptors. In the brain and spinal cord, activation of astrocytes is normally accompanied by upregulation of ETB receptors [7,21] while it is not known if this is also the case in the sensory retina. ET1 is a potent vasoconstrictor of retinal blood vessels [1,11], as well as a mitogen for retinal vascular smooth muscle cells and pericytes [8], and has been suggested to be implicated in the pathogenesis of important retinopathies such as glaucoma [10] and diabetic retinopathy [8]. Enhanced action or levels of ET-1 are associated with decreased blood flow in the retinas of diabetic animals. It has been shown that ET-1 promotes gliosis of optic nerve head astrocytes in vitro [13], and that experimental glaucoma or optic nerve transection increases the expression of ET-1 and ETB in optic nerve astrocytes [12,14]. By using autoradiography, it has been shown that ETA -like binding sites are expressed by blood vessels of the human retina while ETB -like binding sites are localized to neural and glial cells of the retina [9]. In other studies,
I. Iandiev et al. / Neuroscience Letters 384 (2005) 72–75
the presence of ETA and ETB receptors on vascular smooth muscle cells of retinal vessels, and of ETB receptors within the ganglion cell layer, has been shown [2,17]. In retinas of diabetic rats, an upregulation of endothelins, ETA and ETB has been described [3]. However, it is not known whether M¨uller glial cells in the sensory retina express receptors for endothelins, and whether the expression is altered during retinal injury. Therefore, we investigated the expression of ETA and ETB proteins in retinal slices derived from pig eyes. We immunohistochemically stained slices from control retinas, as well as from retinas that were detached from the pigment epithelium for 7 days. As a marker for glial cells, the slices were co-stained against vimentin. All experiments were carried out in accordance with applicable German laws and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Three young adult domestic pigs (17–22 kg; both sexes) were used. Twenty-four hours before and after surgery, the food intake of the animals was restricted with free access to water. Intravenous azaperon (15 mg/kg; Cilag-Janssen, Neuss, Germany), atropin (0.2 mg/kg; Braun, Melsungen, Germany) and ketamine (3 mg/kg; Ratiopharm, Ulm, Germany) were administered for premedication. The anesthesia was induced with thiopental (8 mg/kg i.v.; Trapanal, Byk Gulden, Konstanz, Germany) and maintained with isoflurane (Forene, Abbott, Wiesbaden, Germany). Ventilation was performed using the Julian respirator (Draegerwerk AG, Luebeck, Germany) with a Fi O2 of 40%. Rhegmatogenous detachment was created in one eye per animal; the other eye served as untreated control. The pupils of the eyes were dilated by topical tropicamide (1%; Ursapharm, Saarbr¨ucken, Germany) and phenylephrine hydrochloride (5%; Ankerpharm, Rudolstadt, Germany), and a lateral canthotomy was created. Hemostasis was achieved with wet-field cautery. After pars plana sclerotomy, a circumscript vitrectomy was performed in the area of the future detachment. Thin glass micropipettes attached to 250 l glass syringes (Hamilton, Reno, NV) were used to create a retinal detachment by subretinal injection of saline followed by 0.25% sodium hyaluronate (Healon; Pharmacia & Upjohn, D¨ubendorf, Switzerland) in saline. The retina below the optic nerve head was detached while the retina above the optic nerve head remained attached. After surgery, gentamicin (5 mg) and dexamethasone (0.5 mg) were injected subconjunctivally. The lateral cauthotomy was closed with 5–0 silk sutures, and atropine (1%) eye drops were instilled into the conjunctival sac. After a survival time of 7 days, the animals were anaesthetized as described, the eyes were excised, and the animals were killed by i.v. T61 (embutramid mebezonium iodide; 0.65 ml/kg body weight; Hoechst, Unterschleißheim, Germany). Isolated retinas were fixed in 4% paraformaldehyde for 2 h. After several washing steps in buffered saline, the tissue was embedded in saline containing 3% agarose (w/v), and 70-m thick slices were cut by using a vibratome. The slices were incubated in 5% normal goat serum plus 0.3% Triton X-100 in saline for 2 h and, subsequently, in primary
73
antibody overnight at 4 ◦ C. After washing in 1% bovine serum albumin in saline, the secondary antibodies were applied for 4 h at room temperature. The following antibodies were used: mouse anti-vimentin (1:500; V9 clone, SigmaAldrich, Taufkirchen, Germany), anti-ETA (1:100; Alomone Labs, Jerusalem, Israel), anti-ETB (1:100; Alomone), Cy3conjugated goat anti-rabbit IgG (1:400; Dianova, Hamburg, Germany), and Cy2-coupled goat anti-mouse IgG (1:400; Dianova). In control retinas, the immunoreactivity for vimentin was most strongly expressed in the innermost retinal layers (nerve fiber and ganglion cell layers) that reflects the prominant expression of vimentin by retinal astrocytes and endfeet of M¨uller cells (Fig. 1A and B). In addition, M¨uller cell fibers that pass through the inner retinal layers displayed faint staining for vimentin. In retinal tissue that was detached for 7 days, the vimentin labeling of M¨uller cells increased strongly when compared to control, with labeling of M¨uller cell fibers from the inner to the outer limiting membranes. In control retinas of the pig, the immunoreactivity for ETA was expressed in the innermost retinal layers and in the outer plexiform layer (Fig. 1A). At 7 days after detachment, immunoreactivity for ETA was strongly expressed in the blood vessels of the inner retina. In control tissue, immunoreactivity for ETB was expressed within the ganglion cell and nerve fiber layers, as well as by M¨uller cell fibers (Fig. 1B). In respect to M¨uller cells, co-staining of vimentin and ETB immunoreactivities was observed at the fibers that pass through the inner plexiform and nuclear layers, and through the outer retina. Additionally, the outer limiting membrane was ETB immunopositive. In addition to ganglion cell bodies that express immunoreactivity for ETB , it is likely that both the astrocytes, which are localized preferentially around the vessels in the ganglion cell/nerve fiber layers, and the M¨uller cell endfeet express immunoreactivity for ETB . In the detached retinas, no apparent alteration of the ETB immunostaining was observed. Endothelin-1 is a major regulator of the retinal blood flow that produces vasoconstriction of the retinal microvessels and subsequent decrease of retinal blood flow, predominantly by activation of ETA receptors [11]. In the present study, we found different expression of ETA and ETB immunoreactivities in the porcine retina: In control retinas, immunoreactivity for ETA was expressed in the innermost retinal layers (likely by retinal astrocytes) and in the outer plexiform layer, while immunoreactivity for ETB was expressed by the innermost retinal layers, by ganglion cell somata, and by M¨uller glial cells. Moreover, we found a different regulation of receptor protein expression upon retinal detachment: whereas the immunoreactivity for ETA was strongly upregulated in retinal blood vessels, the immunoreactivity for ETB was not altered in its expression after detachment. The functional significance of the vascular upregulation of ETA receptors upon detachment is unknown. It has been shown that intravitreal application of ET-1 produces vasoconstriction and retinal ischemia [19]. There is evidence
74
I. Iandiev et al. / Neuroscience Letters 384 (2005) 72–75
Fig. 1. Experimental detachment alters the expression of the immunoreactivity for ETA protein in the pig retina. The slices were derived from control retinas, and from retinas that were detached from the pigment epithelium for 7 days. (A) Co-immunostaining against ETA and vimentin. Arrowheads indicate large blood vessels in the innermost retinal layers. Arrows mark ETA -positive intraretinal blood vessels. (B) Immunoreactivities for ETB and vimentin. Arrows indicate vimentin-positive fibers in the inner plexiform layer that were stained also for ETB . Cell nuclei were counterstained using Hoechst 33258 (blue). Scale bars, 20 m. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; OLM, outer limiting membrane; ONL, outer nuclear layer; and OPL, outer plexiform layer.
I. Iandiev et al. / Neuroscience Letters 384 (2005) 72–75
suggesting that retinal detachment is associated with disturbances of retinal circulation. The retinal blood flow rate is decreased in patients with retinal detachment compared to control [18]. Retinal circulation times of the detached areas are longer than those of the non-detached areas in patients with retinal detachment, and both are longer than those of normal subjects [16]. The extent of retinal detachment correlates with the decrease of ocular blood flow velocities [15]. It is conceivable that enhanced responsiveness of ETA receptors on vascular cells may induce ischemia–hypoxia in the inner retinal tissue that may contribute to the induction of morphological and biochemical alterations of the inner retinal neurons, and of glial cell activation, as observed in the detached inner retina [5]. In addition, stimulation of ETA receptors may contribute to the induction of proliferation of cells associated with the vasculature observed in experimental detachment [4]. We found no detachment-induced alteration in the expression of ETB immunoreactivity by M¨uller cells. Astrocytes in the brain and in the optic nerve strongly enhance the expression of ETB after lesion [14,21] and during glaucoma [12], respectively. The functional significance of the constitutive expression of ETB receptors in M¨uller cells of the pig retina remains to be established.
[10]
Acknowledgements
[15]
The authors thank Ute Weinbrecht for excellent technical support. This work was supported by grants from the S¨achsisches Ministerium f¨ur Wissenschaft und Kunst (SMWK; HWP program) and from the DFG (Br 1249/2-1).
[6]
[7]
[8]
[9]
[11]
[12]
[13]
[14]
[16]
[17]
References [1] S.E. Bursell, A.C. Clermont, B. Oren, G.L. King, The in vivo effect of endothelins on retinal circulation in nondiabetic and diabetic rats, Invest. Ophthalmol. Vis. Sci. 36 (1995) 596–607. [2] J.A. De Juan, F.J. Moya, A. Ripodas, R. Bernal, A. Fernandez-Cruz, R. Fernandez-Durango, Changes in the density and localisation of endothelin receptors in the early stages of rat diabetic retinopathy and the effect of insulin treatment, Diabetologia 43 (2000) 773–785. [3] D. Deng, T. Evans, K. Mukherjee, D. Downey, S. Chakrabarti, Diabetes-induced vascular dysfunction in the retina: role of endothelins, Diabetologia 42 (1999) 1228–1234. [4] S.K. Fisher, P.A. Erickson, G.P. Lewis, D.H. Anderson, Intraretinal proliferation induced by retinal detachment, Invest. Ophthalmol. Vis. Sci. 32 (1991) 1739–1748. [5] S.K. Fisher, G.P. Lewis, M¨uller cell and neuronal remodeling in retinal detachment and reattachment and their potential consequences
[18]
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