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Keywords:

  • bipolar cell;
  • cell swelling;
  • glial;
  • ischemia;
  • nerve growth factor;
  • retina

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
Thumbnail image of graphical abstract

Osmotic swelling of neurons and glial cells contributes to the development of retinal edema and neurodegeneration. We show that nerve growth factor (NGF) inhibits the swelling of glial (Müller) and bipolar cells in rat retinal slices induced by barium-containing hypoosmotic solution. NGF also reduced Müller and bipolar cell swelling in the post-ischemic retina. On the other hand, NGF prevented the swelling of freshly isolated Müller cells, but not of isolated bipolar cells, suggesting that NGF induces a release of factors from Müller cells that inhibit bipolar cell swelling in retinal slices. The inhibitory effect of NGF on Müller cell swelling was mediated by activation of TrkA (the receptor tyrosine kinase A), but not p75NTR, and was prevented by blockers of metabotropic glutamate, P2Y1, adenosine A1, and fibroblast growth factor receptors. Basic fibroblast growth factor fully inhibited the swelling of freshly isolated Müller cells, but only partially the swelling of isolated bipolar cells. In addition, glial cell line-derived neurotrophic factor and transforming growth factor-β1, but not epidermal growth factor and platelet-derived growth factor, reduced the swelling of bipolar cells. Both Müller and bipolar cells displayed TrkA immunoreactivity, while Müller cells were also immunostained for p75NTR and NGF. The data suggest that the neuroprotective effect of NGF in the retina is in part mediated by prevention of the cytotoxic glial and bipolar cell swelling.

Cytotoxic cell swelling contributes to retinal neurodegeneration. Nerve growth factor (NGF) inhibits the osmotic swelling of glial cells by acting at TrkA, release of bFGF, and opening of K+ and Cl channels. The NGF-induced glial release of cytokines like bFGF inhibits the osmotic swelling of bipolar cells, suggesting that the neuroprotective effect of NGF is in part mediated by prevention of cytotoxic cell swelling.

Abbreviations used
bFGF

basic fibroblast growth factor

DPCPX

8-cyclopentyl-1,3-dipropylxanthine

EGF

epidermal growth factor

GDNF

glial cell line-derived neurotrophic factor

GFAP

glial fibrillary acidic protein

LY341495

(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid

MRS2179

N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate

NGF

nerve growth factor

NPPB

5-nitro-2-(3-phenylpropylamino)benzoic acid

p75NTR

p75 neurotrophin receptor

PD173074

N-[2-[[4-(diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N′-(1,1-dimethylethyl)urea

PDGF

platelet-derived growth factor

PKC

protein kinase C

TGF

transforming growth factor

VEGF

vascular endothelial growth factor

One major vision-threatening condition of ischemic-hypoxic and inflammatory retinal diseases such as diabetic retinopathy, uveitis, and atherosclerotic vascular disorders is the development of tissue edema. In addition to vascular hyperpermeability (vasogenic edema), water accumulation in neurons and glial cells resulting in cellular swelling (cytotoxic edema) may contribute to the development of edema and neurodegeneration in the retina (Bringmann et al. 2005). Water accumulation in retinal glial (Müller) cells was found in various animal models of retinal ischemia-hypoxia and diabetic retinopathy (Stepinac et al. 2005; Kaur et al. 2007; Kumar et al. 2013).

Neuronal activity in the retina is associated with a decrease in the extracellular fluid osmolarity (Dmitriev et al. 1999). Hypoosmolarity was shown to induce a swelling of bipolar cells that is mediated by a release of endogenous glutamate and activation of metabotropic glutamate receptors, resulting in sodium and water flux into the cells (Vogler et al. 2013). In contrast, Müller cells do not swell under hypoosmotic stress (Vogler et al. 2013). The different volume regulation of Müller and bipolar cells has led to the suggestion that retinal glial cells play a role in mediating the homeostasis of the extracellular space volume (Vogler et al. 2013). Müller cell volume regulation was shown to depend on the passive transmembrane potassium flux (Pannicke et al. 2004) and activation of an endogenous glutamatergic–purinergic signaling cascade (Uckermann et al. 2006; Wurm et al. 2008). Because various neuroprotective factors including vascular endothelial growth factor (VEGF), erythropoietin, and osteopontin activate this signaling cascade (Wurm et al. 2008; Krügel et al. 2010; Wahl et al. 2013), inhibition of cytotoxic Müller cell swelling might represent one neuroprotective mechanism.

Various growth factors, cytokines, and neurotrophins including basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor (GDNF) promote the survival of photoreceptors and retinal neurons such as ganglion and bipolar cells under pathological conditions (Sievers et al. 1987; Faktorovich et al. 1992; Mansour-Robaey et al. 1994; Wexler et al. 1998; Hauck et al. 2006). It has been shown that the simultaneous action of various trophic factors is required to effectively promote photoreceptor survival (Ogilvie et al. 2000), and that the survival of photoreceptors and neurons is, at least in part, indirectly supported via regulation of the trophic factor production in Müller glial cells (Wexler et al. 1998; Harada et al. 2002).

Nerve growth factor (NGF) controls neuronal survival in the retina via activation of high-affinity TrkA receptors, that transmit prosurvival signals, and low-affinity p75 neurotrophin receptors (p75NTR) that transmit antisurvival signals (Ali et al. 2008; Coassin et al. 2008; Lebrun-Julien et al. 2009; Bai et al. 2010). NGF acting at TrkA protects retinal neurons from excitotoxicity (Kokona et al. 2012). NGF delays retinal cell degeneration in animal models of inherited retinitis pigmentosa, diabetic retinopathy, glaucoma, retinal ischemia, retinal detachment, and optic nerve transection (Carmignoto et al. 1989; Siliprandi et al. 1993; Hammes et al. 1995; Lenzi et al. 2005; Sun et al. 2007; Colafrancesco et al. 2011). NGF protects neuronal and glial cells from oxidant-induced cell death (Sampath et al. 1994; Mattson et al. 1995; Giardino et al. 1998). However, activation of p75NTR was shown to have either pro- or antisurvival effects in the retina, in dependence on the experimental model studied (Wexler et al. 1998; Harada et al. 2000, 2002). NGF was suggested to decrease the production of bFGF by Müller cells resulting in increased photoreceptor apoptosis (Harada et al. 2000; Nakamura et al. 2005). In another study, activation of p75NTR was suggested to trigger release of bFGF from Müller cells, resulting in increased survival of bipolar cells (Wexler et al. 1998). Because water accumulation in retinal neurons and glial cells is a pathogenic factor involved in retinal degeneration under ischemic-hypoxic and inflammatory conditions, we tested whether NGF may influence the osmotic swelling of rat Müller glial and bipolar cells. We found that NGF prevents the osmotic swelling of Müller cells by inducing autocrine/paracrine FGF signaling, and indirectly the swelling of bipolar cells by inducing a release of cytokines from Müller cells.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Materials

Mitotracker Orange was obtained from Life Technologies (Darmstadt, Germany). Papain was from Roche Molecular Biochemicals (Mannheim, Germany). N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate and (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495) were from Tocris Cookson (Ellisville, MO, USA). Human recombinant bFGF, rat recombinant GDNF, TrkA receptor inhibitor (Calbiochem 648450), and TAT-conjugated Pep5 (506181) were obtained from Merck Millipore (Darmstadt, Germany). Rat recombinant epidermal growth factor, human recombinant platelet-derived growth factor, and human recombinant transforming growth factor-β1 (TGF-β1) were purchased from R&D Systems (Wiesbaden, Germany). Rat recombinant NGF-β (N2513) and all other agents used were obtained from Sigma-Aldrich (Taufkirchen, Germany), unless stated otherwise. The following antibodies were used: rabbit anti-NGF (1 : 100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-TrkA (1 : 100; Santa Cruz Biotechnology), rabbit anti-p75NTR (1 : 100; Promega, Mannheim, Germany), mouse anti-glutamine synthetase (1 : 200; Merck Millipore), mouse anti-glial fibrillary acidic protein (GFAP, 1 : 200; Sigma-Aldrich), mouse anti-protein kinase Cα (PKCα, 1 : 200; Santa Cruz Biotechnology), Cy3-coupled goat anti-rabbit IgG (1 : 200; Jackson Immuno Research, Newmarket, UK), and Cy2-coupled goat anti-mouse IgG (1 : 100; Jackson Immuno Research).

Animals and experimental retinal ischemia–reperfusion

All experiments were done in accordance with the European Communities Council Directive 86/609/EEC, and were approved by the local authorities (University of Leipzig Medical Faculty and Landesdirektion Leipzig). Adult Long-Evans rats (250–350 g; both sexes) were bred in the Medical-Experimental Center of the University of Leipzig Medical Faculty. Animals were killed with carbon dioxide. In four animals, retinal ischemia–reperfusion was induced in one eye by elevating the intraocular pressure to 160 mmHg for 60 min, as previously described (Pannicke et al. 2004). The contralateral eyes served as control. The animals were killed after 3 days.

Cell soma recording

Retinal slices and suspensions of dissociated retinal cells were prepared as described (Wurm et al. 2008). All experiments were performed at 20–23°C. Retinal slices or isolated cells were transferred to a custom-made perfusion chamber and kept submerged in extracellular solution. Slices or cells were loaded with the vital dye Mitotracker Orange (1 μM) for 3 min. Recordings were made with a confocal laser scanning microscope (LSM 510 Meta; Zeiss, Oberkochen, Germany) and an Achroplan 63×/0.9 water immersion objective (Zeiss). The pinhole was set at 151 μm; the thickness of the optical section was adjusted to 1 μm. Mitotracker Orange was excited at 543 nm with a HeNe laser (Zeiss), and emission was recorded with a 585 nm long-pass filter.

The recording chamber was continuously perfused with extracellular solution; test substances were applied by rapidly changing the perfusate. The extracellular solution consisted of (mM) 136 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 11 glucose, adjusted to pH 7.4 with Tris. The hypoosmotic solution (60% osmolarity) was made up by adding distilled water. Barium chloride (1 mM) was added to the hypoosmotic solution; the slices or cells were preincubated in barium-containing isoosmotic solution for 10 min. Blocking agents were preincubated for 15–45 min, and receptor agonists were applied within the hypoosmotic solution.

Immunostaining

Isolated retinas were fixed in 4% paraformaldehyde for 1 h. After washing in buffered saline, the tissues were embedded in saline containing 3% agarose (w/v), and 60-μm thick slices were cut with a vibratome. The slices were incubated in 5% normal goat serum plus 0.3% Triton X-100 and 1% dimethylsulfoxide in saline for 2 h at 20-23°C and, subsequently, in primary antibodies overnight at 4°C. After several washing steps with saline, the secondary antibodies were applied overnight at 4°C. No specific staining was found in negative control slices stained without primary antibodies (data not shown). Dissociated retinal cells were fixed in 4% paraformaldehyde for 10 min, incubated in 5% normal goat serum plus 0.03% Triton X-100 and 0.1% dimethylsulfoxide in saline for 2 h at 20-23°C and, subsequently, in primary antibodies overnight at 4°C. After washing with saline, the secondary antibodies were applied overnight at 4°C.

Data analysis

To determine the extent of cell soma swelling, the cross-sectional area of Mitotracker Orange-stained cell bodies was measured off-line using the Zeiss LSM Image Examiner version 3.2.0.70 (Zeiss). Data are presented as means ± SEM. Statistical analysis was made with Prism (Graphpad Software, San Diego, CA, USA). Significance was determined by one-way anova followed by Bonferroni's multiple comparison test and by Mann–Whitney U test, respectively, and was accepted at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Hypoosmotic swelling of Müller and bipolar cell somata

It was shown recently that Müller and bipolar cells differ in their cell volume regulation in response to osmotic stress (Vogler et al. 2013). We measured the cross-sectional area of Müller and bipolar cell somata recorded in Mitotracker Orange-loaded freshly isolated retinal slices (Fig. 1b and c) and cells (Fig. 2d). Müller and bipolar cells were identified in retinal slices and cell suspensions according to their characteristic morphologies (Figs 1b and 2d). The different cell morphologies and different localization of the somata within the inner nuclear layer was verified by immunohistochemical staining of Müller cells (glutamine synthetase) and rod bipolar cells (PKCα; Gabriel et al. 2001) in retinal slices (Fig. 1a). Superfusion of retinal slices from control animals with a hypoosmotic extracellular solution (60% osmolarity) for 4 min did not induce a significant alteration in the size of Müller cell somata (Fig. 1d) while bipolar cell somata swelled under these conditions (Fig. 1e). Similarly, somata of freshly isolated Müller cells did not change their size under hypoosmotic conditions (Fig. 2a) while somata of freshly isolated bipolar cells swelled significantly under these conditions (p < 0.05; Fig. 2b). As previously described (Pannicke et al. 2004), potassium channel-blocking barium ions induced a significant (p < 0.05) hypoosmotic swelling of Müller cell bodies (Figs 1d and 2a). Müller cells in slices of post-ischemic retinas displayed a hypoosmotic swelling of their somata in the absence of barium ions (Fig. 1f); the osmotic glial swelling after retinal ischemia–reperfusion was suggested to be caused by the down-regulation of Kir4.1, the major potassium channel of Müller cells (Pannicke et al. 2004). The data are in agreement with previous studies that showed that Müller cells, but not bipolar cells, possess endogenous volume-regulatory mechanisms that prevent cellular swelling under hypoosmotic conditions in the normal retina (Vogler et al. 2013), and that these mechanisms are impaired after retinal ischemia–reperfusion (Pannicke et al. 2004).

image

Figure 1. Nerve growth factor (NGF) inhibits the osmotic swelling of Müller cell and bipolar cell somata in slices of control and post-ischemic retinas. (a) Immunohistochemical staining of control retinal slices against the glial marker glutamine synthetase (GS) and the rod bipolar cell marker protein kinase Cα (PKCα). The focus is at the inner nuclear layer (INL). Arrow, Müller cell soma. Arrowhead, bipolar cell soma. Note that Müller cell somata are localized in the central part of the INL while bipolar cell somata are localized near the outer border of the INL. (b) Example of a freshly isolated retinal slice loaded with Mitotracker Orange. (c) Original records of cell somata obtained before (left) and during (right) superfusion of control retinal slices with a barium (1 mM)-containing hypoosmotic solution in the absence (left-hand side images) and presence (right-hand side images) of NGF (1 ng/mL). (d and e) Dose dependencies of the NGF effects on the osmotic swelling of Müller cell (d) and bipolar cell somata (e) in control retinal slices. The concentrations of NGF (ng/mL) are given in the bars. The cross-sectional area of cell somata was measured after a 4-min superfusion of the slices with an iso- or hypoosmotic solution (60% osmolarity), and are expressed in percent of the soma size recorded before beginning of the superfusion (100%). The effects of the hypoosmotic solution were determined in the absence and presence of barium chloride (1 mM). (f) Effects of NGF (1 ng/mL) on the osmotic swelling of Müller and bipolar cell somata in slices of 3-day-post-ischemic retinas. The slices were superfused with the hypoosmotic solution without barium chloride. Each bar represents values obtained in 3–34 cells. *p < 0.05 compared with hypoosmolarity (d) and isoosmolarity control (e), respectively. ●p < 0.05 compared with hypoosmolarity plus barium (d) and hypoosmolarity control (e), respectively. ○p < 0.05 compared with Müller cell somata in control retinas (f). #p < 0.05 compared with Müller cell and bipolar cell somata, respectively, in ischemic retinas in the absence of NGF (f). IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 5 μm.

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image

Figure 2. Effects of nerve growth factor (NGF) (1 ng/mL) on the osmotic soma swelling of Müller (a and c) and bipolar cells (b) freshly isolated from retinas of control animals. (c) The inhibitory effect of NGF on the osmotic soma swelling of isolated Müller cells was prevented in the presence of a TrkA inhibitor (500 nM) but not in the presence of a p75NTR inhibitor, TAT-conjugated Pep5 (TAT-Pep5; 1 μM). (d) Original records of Mitotracker Orange-loaded freshly isolated Müller (left) and bipolar cells (right). Filled arrowheads, cell soma. Unfilled arrowheads, Müller cell endfoot. Arrows, inner process of bipolar cells. Data were measured after a 4-min superfusion of the cells with an iso- or hypoosmotic solution, and are expressed in percent of the soma size recorded before beginning of the superfusion (100%). The effects of the hypoosmotic solution were recorded in the absence and presence of barium chloride (1 mM). Each bar represents values obtained in 7–20 cells. *p < 0.05 compared with hypoosmolarity (a) and isoosmolarity control (b), respectively. ●p < 0.05 compared with hypoosmolarity plus barium control (a and c). ○p < 0.05 compared with NGF control. Scale bars: 10 μm.

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Swelling inhibition by NGF

To reveal whether NGF affects the volume regulation of Müller and bipolar cells, retinal slices were superfused with a hypoosmotic solution containing barium chloride. NGF was simultaneously administered with the hypoosmotic solution. As shown in Fig. 1(d) and (e), the hypoosmotic swelling of Müller and bipolar cell somata in retinal slices from control animals was dose-dependently inhibited by NGF. NGF also reduced significantly (p < 0.05) the swelling of Müller and bipolar cell somata in slices of post-ischemic retinas (Fig. 1f). However, the inhibitory effect of NGF on the osmotic swelling of bipolar cell somata in slices of post-ischemic retinas (Fig. 1f) was significantly (p < 0.05) lower than the effect observed in slices of control retinas. On the other hand, while NGF fully prevented the osmotic soma swelling of freshly isolated Müller cells (Fig. 2a), it did not inhibit the soma swelling of isolated bipolar cells (Fig. 2b). The data suggest that Müller cells, but not bipolar cells, express NGF receptors that are coupled to cell volume-regulatory signaling mechanisms. The inhibitory effect of NGF on the swelling of bipolar cell somata in retinal slices might be an indirect effect, via NGF-induced stimulation of Müller cells which subsequently release factors that induce volume-regulatory signals in bipolar cells.

NGF binds with at least two classes of receptors, TrkA and p75NTR. To reveal the types of receptors that mediate the inhibitory effect of NGF on the osmotic swelling of Müller cell somata, we tested a TrkA receptor inhibitor and a p75NTR signaling inhibitor, TAT-conjugated Pep5. As shown in Fig. 2(c), the TrkA inhibitor fully prevented the inhibitory effect of NGF on the swelling of Müller cell somata, while the p75NTR inhibitor had no effect.

Dependence of the NGF effect on receptor transactivation

Rat Müller cells were shown to possess an endogenous signaling cascade that mediates the agonist-induced homeostasis of cellular volume under hypoosmotic conditions; this signaling cascade involves the autocrine/paracrine activation of glutamatergic and purinergic receptors (Uckermann et al. 2006; Wurm et al. 2008). The inhibitory effect of NGF on the swelling of Müller cell somata was abrogated in the presence of the selective antagonist of group II metabotropic glutamate receptors, LY341495 (Figure S1a). This suggests that NGF induces a release of endogenous glutamate, likely from Müller cells (Wurm et al. 2008), that subsequently activates metabotropic glutamate receptors on the cells. On the other hand, LY341495 inhibited the swelling of bipolar cells in retinal slices, but did not alter the inhibitory effect of NGF (Figure S1b). This finding is in agreement with a recent study that showed that the osmotic swelling of bipolar cell somata in rat retinal slices is mediated by induction of the release of endogenous glutamate and subsequent activation of metabotropic glutamate receptors (Vogler et al. 2013).

The inhibitory effects of NGF on the swelling of Müller and bipolar cell somata in retinal slices were fully abrogated in the presence of the selective antagonist of purinergic P2Y1 receptors, N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179), and the antagonist of adenosine A1 receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), respectively (Figure S1c and d). The data suggest that the effects of NGF on the swelling of Müller and bipolar cell somata are mediated by transactivation of P2Y1 and A1 receptors, likely via inducing a release of ATP and adenosine from Müller cells.

Dependence of the NGF effect on cytokine release

It was shown that exogenous glutamate, ATP, and adenosine inhibit the hypoosmotic swelling of Müller cell somata, but not of bipolar cell somata (Vogler et al. 2013). Therefore, it is unlikely that glutamate, ATP or adenosine released from Müller cells mediate the inhibitory effect of NGF on the swelling of bipolar cell somata in retinal slices (Fig. 1e). In order to test the assumption that the swelling-inhibitory effect of NGF on bipolar cells in retinal slices is an indirect effect mediated by other Müller cell-derived factors, we tested the FGF receptor tyrosine kinase inhibitor N-[2-[[4-(diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N′-(1,1-dimethylethyl)urea (PD173074) (Mohammadi et al. 1998). bFGF is a major neuroprotectant in the retina (Sievers et al. 1987; Faktorovich et al. 1992), and Müller cells are known to produce bFGF (Harada et al. 2000, 2002; Hauck et al. 2006). We found that the inhibitory effects of NGF on the osmotic swelling of Müller and bipolar cell somata in retinal slices were completely blocked by PD173074 (Fig. 3a and b). The data suggest that the effects of NGF are mediated by transactivation of FGF receptors. We found that exogenous bFGF fully blocked the osmotic soma swelling in isolated Müller cells (Fig. 3c), but inhibited the osmotic soma swelling in isolated bipolar cells by approximately 30% (Fig. 3d). This may suggest that, in addition to bFGF, further Müller cell-derived factors play a role in mediating the inhibitory effect of NGF on the osmotic swelling of bipolar cell somata in retinal slices.

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Figure 3. The inhibitory effect of nerve growth factor (NGF) on the osmotic swelling of Müller cell somata is mediated by the activation of fibroblast growth factor (FGF) signaling. The data were measured after a 4-min superfusion of retinal slices (a and b) and isolated cells (c–e) with a hypoosmotic solution containing barium chloride (1 mM), and are expressed in percent of the soma size recorded before beginning of the superfusion (100%). (a and b) The inhibitory effects of NGF (1 ng/mL) on the osmotic swelling of Müller (a) and bipolar cell somata (b) are prevented by the FGF receptor inhibitor N-[2-[[4-(diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N′-(1,1-dimethylethyl)urea (PD173074) (500 nM). (c and d) Effects of basic FGF (10 ng/mL) on the osmotic soma swelling in isolated Müller (c) and bipolar cells (d). In isolated Müller cells, the antagonist of adenosine A1 receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (100 nM), did not significantly block the swelling-inhibitory effect of basic FGF (c). (e) The inhibitory effect of adenosine (10 μM) on the osmotic swelling of Müller cell somata is prevented by PD173074 (500 nM). Each bar represents values obtained in 7–23 cells. ●p < 0.05 compared with hypoosmolarity plus barium control. ○p < 0.05 compared with NGF (a, b) and adenosine control (e).

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In order to determine further factors that inhibit the osmotic swelling of bipolar cell somata, we tested various cytokines. GDNF and TGF-β1 significantly (p < 0.05) reduced the osmotic swelling of bipolar cell somata in retinal slices while epidermal growth factor and platelet-derived growth factor had no effects (Fig. 4a). To distinguish whether GDNF and TGF-β1 directly inhibit the swelling of bipolar cell somata or indirectly via their effects in Müller cells, we tested the cytokines in freshly isolated bipolar cells. As shown in Fig. 4(b), both cytokines reduced significantly (p < 0.05) the osmotic soma swelling in isolated bipolar cells, albeit to degrees lower than that observed in retinal slices (Fig. 4a). Coadministration of GDNF and TGF-β1 resulted in a reduction in the soma swelling in isolated bipolar cells (Fig. 4b) to a degree similar to that observed in retinal slices (Fig. 4a). Coadministration of bFGF, GDNF, and TGF-β1 did not further decrease the extent of soma swelling in isolated bipolar cells (Fig. 4b).

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Figure 4. Cytokine-induced inhibition of the osmotic swelling of bipolar cell somata. The data were measured after a 4-min superfusion of retinal slices (a) and freshly isolated cells (b) with a hypoosmotic solution containing barium chloride (1 mM), and are expressed in percent of the soma size recorded before beginning of the superfusion (100%). (a) Effects of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), glial cell line-derived neurotrophic factor (GDNF), and transforming growth factor-β1 (TGF-β1) on the swelling of bipolar cell somata. (b) Effects of GDNF, TGF-β1, and of coadministrations of GDNF and TGF-β1, and of basic fibroblast growth factor (bFGF), GDNF, and TGF-β1, respectively, on the swelling of bipolar cell somata. Each cytokine was tested at 10 ng/mL. Each bar represents the values obtained in 10–45 cells. *p < 0.05 compared with hypoosmolarity plus barium control. ●p < 0.05 compared with TGF-β1.

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Glial bFGF release occurs downstream from A1 receptor activation

We found that the inhibitor of adenosine A1 receptors, DPCPX, fully blocked the inhibitory effect of NGF on the osmotic Müller cell swelling in retinal slices (Figure S1c). However, DPCPX blocked only partially the swelling-inhibitory effect of bFGF in isolated Müller cells (Fig. 3c). This suggests that the NGF-induced release of bFGF from Müller cells occurs downstream from autocrine/paracrine A1 receptor activation. This assumption is supported by the finding that the inhibitory effect of adenosine on the osmotic Müller cell swelling (Uckermann et al. 2006) is blocked in the presence of the FGF receptor inhibitor PD173074 (Fig. 3e).

Dependence of swelling inhibition on ion channel activation

We described previously that the agonist-mediated inhibition of osmotic Müller cell swelling is mediated by activation of potassium and chloride channels (Uckermann et al. 2006). We found that the potassium channel blocker clofilium and the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) blocked the inhibitory effects of NGF and bFGF on the osmotic soma swelling of isolated Müller cells (Fig. 5a). Both blockers also prevented the inhibitory effect of GDNF plus TGF-β1 on the osmotic soma swelling of isolated bipolar cells (Fig. 5b).

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Figure 5. Dependence of the swelling-inhibitory cytokine effects on ion channel activation. (a) In freshly isolated Müller cells, the effects of nerve growth factor (NGF) (10 ng/mL) and basic fibroblast growth factor (bFGF) (10 ng/mL) were abrogated in the presence of the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (100 μM) and the potassium channel blocker clofilium (10 μM), respectively. (b) Both blockers also abrogated the inhibitory effect of glial cell line-derived neurotrophic factor (GDNF) plus transforming growth factor-β1 (TGF-β1) (each at 10 ng/mL) on the osmotic soma swelling of freshly isolated bipolar cells. The data were measured after a 4-min superfusion of the cells with a hypoosmotic solution containing barium chloride (1 mM), and are expressed in percent of the soma size recorded before beginning of the superfusion (100%). Each bar represents values obtained in 7–22 cells. ●p < 0.05 compared with hypoosmolarity plus barium control. ○p < 0.05 compared with cytokine control.

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Retinal localization of NGF, TrkA, and p75NTR

To determine whether Müller and bipolar cells of the rat retina express NGF, TrkA, and p75NTR, we immunostained retinal slices and cells. The slices and cells were colabeled against glutamine synthetase (predominantly expressed by Müller cells), GFAP (mainly expressed by astrocytes), and PKCα (marker of rod bipolar cells; Gabriel et al. 2001). NGF immunoreactivity displayed a widespread distribution throughout the retinal tissue (Figure S2a). In addition to other structures, the somata of retinal ganglion cells and cell bodies in the inner nuclear layer were surrounded by NGF immunoreactivity (Figure S2a). Müller cells displayed immunoreactivity for NGF, as indicated by their labeled processes that traverse the outer nuclear layer (Figure S2a). GFAP-expressing astrocytes localized to the ganglion cell layer were not immunolabeled for NGF (Figure S2b). Isolated Müller cells displayed a punctate NGF immunolabeling at their whole cell bodies (Fig. 6a) while isolated bipolar cells did not display immunolabeling for NGF (Fig. 6b). The data are in agreement with a previous study that showed expression of NGF in rat retinal ganglion and Müller cells (Chakrabarti et al. 1990).

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Figure 6. Immunolocalization of nerve growth factor (NGF) (a and b), TrkA (c and d), and p75NTR (e and f) in freshly isolated Müller (a, c, and e) and bipolar cells (b, d, and f) of the rat retina. Müller cells were colabeled with an antibody against glutamine synthetase (GS). Bipolar cells were colabeled with an antibody against protein kinase Cα (PKCα). (a and e) Insets: Müller cell soma at higher magnification. (c) Insets: Soma (left) and endfoot (right) of Müller cells at higher magnification. Arrows, Müller cell endfeet. Arrowheads, Müller cell somata. Scale bars: 5 μm.

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Retinal slices displayed a widespread immunostaining for TrkA; among others, retinal ganglion cell and amacrine cell bodies were immunopositive for TrkA (Figure S3). Both isolated Müller and bipolar cells displayed a punctate TrkA labeling (Fig. 6c and d). The retinal cell suspensions contained also TrkA-positive, glutamine synthetase- and PKCα-negative cell bodies (data not shown), suggesting that further neuronal cell types, for example, amacrines (Figure S3), express TrkA. The data are in agreement with previous studies that showed a widespread distribution of TrkA in the rat retina (Coassin et al. 2008). Retinal slices were immunolabeled for p75NTR throughout all retinal layers (Figure S4). Among others, Müller cells displayed immunoreactivity for p75NTR, as indicated by the colabeling with glutamine synthetase in both plexiform layers and at the outer limiting membrane (Figure S4). Isolated Müller cells displayed p75NTR immunoreactivity at their whole cell bodies (Fig. 6e) while isolated bipolar cells were devoid of p75NTR immunolabeling (Fig. 6f). The data are in agreement with previous studies that showed localization of p75NTR in Müller cells but not bipolar cells in the rat retina (Hu et al. 1998; Wexler et al. 1998; Lebrun-Julien et al. 2009).

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Water accumulation in retinal neurons and glial cells resulting in cellular swelling contributes to the development of retinal edema and neuronal degeneration (Bringmann et al. 2005). Here, we examined whether NGF prevents the cytotoxic swelling of rat Müller and bipolar cells under hypoosmotic conditions, via activation of cell volume-regulatory mechanisms. We found that NGF inhibits the osmotic swelling of Müller and bipolar cells in control and post-ischemic retinal slices (Fig. 1d–f). On the other hand, NGF prevented the swelling of freshly isolated Müller cells (Fig. 2a) but not of isolated bipolar cells (Fig. 2b). The data may suggest that NGF induces a release of soluble factors from Müller cells that inhibit the swelling of bipolar cells in retinal slices. One such Müller cell-derived factor may represent bFGF, because the FGF receptor inhibitor PD173074 blocked the inhibitory effects of NGF on the swelling of Müller and bipolar cells in retinal slices (Fig. 3a and b). However, bFGF fully blocked the swelling of isolated Müller cells (Fig. 3c) and only in part the swelling of isolated bipolar cells (Fig. 3d). This suggests that further Müller cell-derived factors also mediate the inhibitory effect of NGF on bipolar cell swelling. We found that, in addition to the inhibitor of FGF receptors, blockers of P2Y1 and adenosine A1 receptors abrogated the inhibitory effect of NGF on the swelling of Müller and bipolar cells in retinal slices (Figure S1c and d). However, because agonists of P2Y1 (ATP) and A1 receptors (adenosine) do not block the hypoosmotic swelling of bipolar cells, as recently described (Vogler et al. 2013), it is unlikely that Müller cell-derived purinergic receptor agonists mediate the inhibitory effect of NGF on the bipolar cell swelling in retinal slices. PD173074 was described to block the tyrosine kinase activities of FGF and VEGF receptors (Mohammadi et al. 1998). It was shown that VEGF does not block the hypoosmotic swelling of bipolar cells (Vogler et al. 2013); thus, it is also unlikely that Müller cell-derived VEGF is involved in mediating the NGF-induced inhibition of bipolar cell swelling. In addition, glutamate does not block the osmotic swelling of bipolar cells (Vogler et al. 2013). Instead, glutamate is involved in mediating the osmotic swelling of bipolar cells, via activation of metabotropic glutamate receptors (Vogler et al. 2013); inhibition of these receptors prevented the swelling of bipolar cells (Figure S1b).

The kinds of the other factors that are released from Müller cells after stimulation with NGF and that inhibit the swelling of bipolar cells remain to be determined in future experiments. Two candidate molecules are GDNF and TGF-β1. We found that both cytokines reduced, when simultaneously administered to isolated cells, the extent of the bipolar cell swelling by two-third (Fig. 4b). However, because both cytokines alone reduced the swelling of isolated bipolar cells (Fig. 4b) to degrees lower than that observed in retinal slices (Fig. 4a), we assume that GDNF and TGF-β1 act by two mechanisms: via direct effects in bipolar cells and via indirect effects involving Müller cells. It was shown that Müller cells are a source of TGF-β (Ikeda et al. 1998) and that GDNF induces the expression of bFGF in Müller cells (Harada et al. 2003). However, because coadministration of bFGF, GDNF, and TGF-β1 did not fully inhibit the swelling of isolated bipolar cells (Fig. 4b), it is likely that further Müller cell-derived factors not investigated in the present study are also involved in mediating the effect of NGF on the swelling of bipolar cells. It can not be ruled out that various subtypes of bipolar cells express different kinds of cytokine receptors. In this case, the NGF-induced release of a variety of cytokines from Müller cells increases the efficiency of the glial control of bipolar cell volume.

The inhibitory effect of NGF on the osmotic swelling of Müller cells is mediated by transactivation of metabotropic glutamate, P2Y1, and adenosine A1 receptors (Figure S1a and c). These effects are likely mediated by the NGF-induced release of glutamate, ATP, and adenosine from Müller cells, as previously described for the swelling-inhibitory effects of other receptor ligands (Uckermann et al. 2006; Wurm et al. 2008; Krügel et al. 2010; Wahl et al. 2013). We found that the FGF receptor inhibitor PD173074 fully prevented the inhibitory effect of adenosine in isolated Müller cells (Fig. 3e), suggesting that the NGF-induced release of bFGF from Müller cells occurs downstream from autocrine/paracrine A1 receptor activation (Fig. 7). The data suggest that NGF induces activation of the previously described swelling-inhibitory glutamatergic–purinergic signaling cascade in Müller cells (Uckermann et al. 2006; Wurm et al. 2008). The final steps of the cascade may represent the adenosine-induced release of bFGF that subsequently activates FGF receptors, resulting in activation of potassium channels (likely two-pore domain channels; Skatchkov et al. 2006) and chloride channels (Fig. 5a). The ion flux compensates the osmotic gradient across the Müller cell membrane and thus prevents the cellular swelling. However, further experiments are required to determine more precisely the NGF-induced signaling mechanisms in Müller cells. Activation of potassium and chloride channels is also involved in mediating the inhibitory effects of GDNF and TGF-β1 on the osmotic swelling of bipolar cells (Fig. 5b).

image

Figure 7. Hypothetical scheme of the nerve growth factor (NGF)-induced signaling involved in the volume regulation of Müller glial and bipolar cells in the rat retina. NGF acting at TrkA activates a glutamatergic–purinergic signaling cascade in Müller cells. This cascade consists of the consecutive release of glutamate, ATP, and adenosine, and the activation of metabotropic glutamate receptors (mGluRs), P2Y1, and adenosine A1 receptors. Activation of A1 receptors induces a release of basic fibroblast growth factor (bFGF) from Müller cells that activates FGF receptors, resulting in the opening of potassium and chloride channels in the Müller cell membrane. The ion efflux compensates the transmembranous osmotic gradient and thus prevents the cellular swelling under hypoosmotic conditions. Müller cell-derived cytokines such as bFGF, glial cell line-derived neurotrophic factor (GDNF), and transforming growth factor-β (TGF-β), and possibly further cytokines, inhibit the hypoosmotic swelling of bipolar cells. While glutamate inhibits the osmotic swelling of Müller cells, it induces the osmotic swelling of bipolar cells. The glutamate-induced swelling of bipolar cells is prevented by Müller cell-derived cytokines. Different steps of the signaling cascade can be blocked by antagonists (left side), resulting in cellular swelling when the extracellular osmolarity decreases.

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The inhibitory effect of NGF on the osmotic Müller cell swelling was mediated by the activation of TrkA, but not p75NTR (Fig. 2c). Müller and bipolar cells display TrkA immunoreactivity (Fig. 6c and d). Because NGF inhibited the swelling of isolated Müller cells (Fig. 2a), but not of isolated bipolar cells (Fig. 2b), we assume that TrkA is coupled to cell volume-regulatory intracellular signaling mechanisms in Müller cells but not bipolar cells. The presence of TrkA-immunolabeled Müller cells (Fig. 6c) is in disagreement with a recent study that showed the absence of TrkA immunoreactivity in rat Müller cells (Kokona et al. 2012). However, the inhibitory effect of the TrkA inhibitor on the NGF effect in isolated Müller cells (Fig. 2c) suggests the presence of functional TrkA in Müller cells that is coupled to volume-regulatory signaling. The reasons for the different immunohistochemical data are unclear and may include, for example, the use of different rat strains and differences in the recognizability of plasma membrane stainings between retinal slices and isolated cells. Müller cells are also a source of NGF in the rat retina, as indicated by the punctate NGF labeling of isolated cells (Fig. 6a).

We found that the inhibitory effect of NGF on the osmotic swelling of bipolar cells in slices of post-ischemic retinas (Fig. 1f) was lower than that of the effect observed in slices of control retinas (Figs 1e and 3b). This may suggest a decreased efficiency of the NGF-induced glial control of the bipolar cell volume in the ischemic retina. The reasons for the decreased efficiency are unclear and may include alterations in the NGF-induced cytokine release from Müller cells and in the responsiveness of bipolar cells to glial-derived cytokines.

Dependent on specific conditions, NGF was shown to promote neuronal survival in the retina by activation of TrkA or to be neurotoxic via activation of p75NTR (Ali et al. 2008; Coassin et al. 2008; Lebrun-Julien et al. 2009; Bai et al. 2010). It has been suggested that NGF may represent a promising agent for the treatment of degenerative retinal diseases such as glaucoma and age-related macular degeneration (Lambiase et al. 2011). However, NGF may have also TrkA-dependent detrimental effects in the retina including promotion of retinal and choroidal neovascularization, and of Müller cell proliferation (Ikeda and Puro 1994; Steinle and Granger 2003; Liu et al. 2010). The present data suggest that the neuroprotective effect of NGF in the retina is in part mediated by preventing the cytotoxic swelling of glial and bipolar cells that contributes to neuronal degeneration in ischemic and inflammatory retinal diseases (Bringmann et al. 2005).

Acknowledgments and conflict of interest disclosure

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

This work was supported by grants from the Deutsche Forschungsgemeinschaft (GRK 1097/1, RE 849/16-1). T.B.G. was recipient of a graduate student fellowship from CNPq-Brazil. The authors have no conflicts of interest to disclose.

All experiments were conducted in compliance with the ARRIVE guidelines.

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  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12822-sup-0001-FigS1-S4.pdfapplication/PDF3981K

Figure S1. The inhibitory effects of NGF on the osmotic swelling of Müller and bipolar cell somata in retinal slices are mediated by receptor transactivation.

Figure S2. Immunolocalization of NGF in slices of the rat retina.

Figure S3. Immunolocalization of TrkA in slices of the rat retina.

Figure S4. Immunolocalization of p75NTR in slices of the rat retina.

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