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Published in final edited form as: Mol Immunol. 2011 Aug 6;48(0):2151–2158. doi: 10.1016/j.molimm.2011.07.012

Complement Mediated Apoptosis Leads to the Loss of Retinal Ganglion Cells in Animal Model of Glaucoma

Purushottam Jha 1, Himanshu Banda 1, Ruslana Tytarenko 1, PS Bora 1, NS Bora 1
PMCID: PMC3653641  NIHMSID: NIHMS465729  PMID: 21821293

Abstract

This study investigated the role of complement in the protection of retinal ganglion cells (RGCs) in chronic ocular hypertension model of glaucoma. Intraocular pressure (IOP) was elevated in the right eye of Lewis rats by laser photocoagulation (two treatments, 7 days apart) of episcleral and limbal veins. Left eye did not receive laser treatment and served as control. Animals were injected with cobra venom factor every fifth day starting day 7 after first laser, to deplete the complement system. Animals were sacrificed at six week post-laser. Levels of C3 split products and membrane attack complex (MAC) were elevated in the retina of eyes with increased IOP and complement depletion reduced the loss of Brn3a+ RGCs accompanied by decreased expression of GFAP and reduced MAC deposition. In complement depleted rats with increased IOP, reduced TUNEL+ cells in ganglion cell layer, and decreased levels of active caspase-8 and active caspase-9 was observed compared to PBS treated complement sufficient rats with increased IOP. Interestingly, complement depletion also resulted in reduction of calcium influx and levels of BAD in the retinal cells of the eyes with increased IOP. Together, our results provide evidence that complement mediated apoptosis plays a pivotal role in the loss of RGCs in chronic ocular hypertension model of glaucoma.

Keywords: Apoptosis, Glaucoma, Retinal ganglion cells, Complement system, Membrane attack complex, Inflammation

1. Introduction

Glaucoma, one of the leading causes of vision loss, is characterized by gradual loss of retinal ganglion cells (RGCs) and damage to the optic nerve. Elevated intraocular pressure (IOP) is one of the major risk factors for glaucoma and has been associated with the damage of RGCs and optic nerve observed in glaucomatous eyes (Rivera et al., 2008). Different mechanisms have been reported to be responsible for the loss of RGCs (Rivera et al., 2008). It is known that apoptosis leads to the degeneration of RGCs in glaucomatous eyes (Nickells, 2007). However the exact cellular and molecular events involved in apoptosis leading to progressive loss of RGCs in glaucoma are not well understood. There is growing evidence suggesting the role of immune system in the pathogenesis of glaucoma (Grus and Sun, 2008). Studies reported in the literature have shown the presence of complement system and complement activation products in the eyes with glaucoma (Kuehn et al., 2006, 2008; Tezel et al., 2010). However, the precise role of complement system in glaucoma is unknown. Additionally, the mechanisms by which the complement activation damages the RGCs have not yet been explored.

Complement system can be activated by three proteolytic cascades, namely the classical, the alternative and the lectin pathways. Although the initial signal for the activation of each cascade is different, all three pathways lead to the formation of membrane attack complex (MAC) (Ross, 1986; Muller-Eberhard, 1988, Bora et al., 2008; Jha et al., 2006, 2007). Activated complement system can lead to cell death by different mechanisms (Cole and Morgan, 2003). It is known that MAC can induce cell lysis and apoptosis that result in cell death (Cole and Morgan, 2003). In the current study, we used the model of chronic ocular hypertension in Lewis rats to investigate the role of complement in the loss of RGCs in glaucoma. We also explored the underlying mechanisms responsible for complement mediated loss of RGCs.

2. Materials and methods

2.1. Animals

Pathogen-free male Lewis rats (5–6 wk old) were obtained from Harlan Sprague Dawley (Indianapolis, IN). This study was approved by the Institutional Animal Care and Use Committee, University of Arkansas for Medical Sciences, Little Rock, AR. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

2.2. Induction of glaucoma

Glaucoma was induced in the right eye of each animal using laser photocoagulation as previously reported by others (Chiu et al., 2007; Li et al., 2006; Ji et al., 2004). Briefly, the limbal vein and the three episcleral veins were photocoagulated (power 1,000 mV; duration, 0.1 s) using an Argon laser. Approximately 80 laser spots around the limbal vein (except the nasal area) and 15 laser spots on each episcleral vein were applied. A second laser treatment at the same setting was applied 7 days later to maintain the increased IOP. IOP was measured with a Tonolab tonometer (Colonial Medical Supply, Franconia, NH) before the first laser treatment and at different time points after the first laser treatment.

2.3. Histology

Freshly enucleated rat eyes were fixed in 2.5% glutaraldehyde and embedded in epoxy. Semi-thin sections (1μm) of epoxy embedded tissue were cut on Ultracut E (Reichert-Jung, Wien, Austria) and stained with toluidine blue and basic fuchin (#14950, Electron Microscopy Sciences, Ft. Washington, PA). Sections were examined using a light microscope (Carl Zeiss Meditec, Inc., Thornwood, NY).

2.4. Immunohistochemistry

Freshly enucleated rat eyes were fixed in neutral buffered 10% formalin solution (Sigma-Aldrich, St. Louis, MO) for 24 hours at room temperature, dehydrated in ethanol through ascending series of ethanol concentrations and embedded in paraffin. The paraffin embedded tissue sections (4 μm) were immunostained for MAC, C3, Brn3a, GFAP and BAD. For MAC detection, polyclonal rabbit anti-rat C9 was used as the primary antibody. This antibody was kindly provided by Prof. B.P. Morgan (School of Medicine, Cardiff University, Cardiff, UK). For C3 detection, goat anti-rat C3 (MP biomedicals, Solon, OH) was used as primary antibodies and for detecting GFAP, CY-3 labeled mouse anti- GFAP (Abcam, Cambridge, MA) was used. Rabbit anti-Brn3a antibody (Abcam, Cambridge, MA) was used to detect Brn3a+ cells. Mouse anti-BAD and anti-BID (Abcam, Cambridge, MA) were used to detect BAD and BID. Control stains were performed with non-relevant antibodies (IgG whole molecule from rabbit serum) at concentrations similar to those of the primary antibodies. Additional controls consisted of staining by omission of the primary or secondary antibody. The sections were covered with mounting medium (ProLong Gold antifade reagent with DAPI; Invitrogen, Carlsbad, CA) and were examined under fluorescence microscope (Carl Zeiss Meditec Inc., Thornwood, NY). To obtain the total number of Brn3a+ RGCs in each image, the individual confocal images were analyzed using ImageJ 1.40g software (NIH, USA). The nuclei stained red and blue (Brn3a+, DAPI) in the RGC layer were manually marked and quantified for each image. The percentage of Brn3a+ cells was calculated as a ratio of the total number of cells (stained blue for DAPI) and the cells stained red and blue (Brn3a+, DAPI).

2.5. TUNEL

Terminal deoxynucleotidyl transferase (TdT)–mediated d-UTP-biotin nick end labeling (TUNEL) assay (Roche, Indianapolis, IN) was used to detect the apoptotic cells according to manufacturer's instructions. Negative control slides were treated identically, but the enzyme solution was omitted. The sections were covered by antifade reagent with DAPI (Invitrogen, Carlsbad, CA) and were examined by fluorescence microscope (Carl Zeiss Meditec Inc., Thornwood, NY).

2.6. Western blot analysis

Retinas harvested from all animal groups were pooled separately. Pooled tissue was homogenized and solubilized in ice-cold PBS containing protease inhibitors and total protein concentration was determined. After SDS–PAGE on 12% linear slab gel, separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were incubated with goat anti-rat C3 (MP Biomedicals, Solon, OH), or rabbit anti-rat C9 (kindly provided by Prof. B.P. Morgan, University of Wales College of Medicine, Cardiff, UK), or monoclonal anti-β-actin (mouse IgG1; Sigma–Aldrich). After washing and incubation with HRP-conjugated secondary antibody, blots were developed using the ECL Western blotting detection system “ECL Plus” (Amersham Biosciences, Piscataway, NJ).

2.7. Intracellular calcium staining

The eyes were removed and fixed for 1 h in 10% phosphate buffered formalin. The cornea and the lens were removed and the retina was carefully dissected from the eyecup. Retinal flat mounts were first stained with rabbit anti-rat C9 (1: 1000) and the respective CY3 labeled secondary antibody. In some cases the flat mounts were incubated for 30 minutes at 37°C with 10 μM Calcium Green-1 AM in DMSO (Molecular probe, Eugene, OR) and then washed. Tissue was flat-mounted, with the sclera side facing down, on a glass slide in antifade reagent with DAPI. Flat mounts were examined under a confocal microscope (Zeiss LSM510; Carl Zeiss Meditec, Inc., Thornwood, NY). The intracellular Ca2+ stained green whereas MAC stained red.

2.8. Statistical Analysis

All experiments were repeated three times with similar results. The data are expressed as the mean ± SD. Data were analyzed and compared using Student's t test, and differences were considered statistically significant with p < 0.05.

3. Results

3.1. Complement activation in eyes with elevated IOP

We first investigated if there was increased complement activation in the eyes of Lewis rats with elevated IOP by comparing the deposition of C3 split products and MAC in the retina harvested from the eyes with elevated IOP and normal IOP. Rat eyes received laser treatment on two occasions – at day 0 and at day 7 after first laser treatment to elevate the IOP as described previously (Chiu et al., 2007; Li et al., 2006; Ji et al., 2004). In control eyes that did not receive any laser treatment, normal IOP (15.3±1.21 mm Hg) was noted after second laser treatment (Fig. 1A). However, elevated IOP (39.94±2.7 mm Hg) was observed in the laser-treated eyes at this time point. IOP remained elevated till day 42 weeks post-laser treatment (Fig. 1A). Animals were sacrificed at 6 week after first laser treatment, eyes were harvested and retina was isolated for immunostaining and Western blot analysis. The immunofluorescent staining of rat eyes from rats with elevated IOP showed increased C3 (Fig. 1C) and MAC (Fig. 1E) deposition on the RGCs compared to the eyes with normal IOP (Fig. 1B and D respectively). Semi quantitative Western blot analysis demonstrated that iC3b (Fig. 1F; upper panel and G) as well as MAC (Fig. 1F; lower panel and H) levels were increased in the retina of animals with increased IOP compared to the retina from the eyes with normal IOP (Fig. 1F-H). Taken together our results demonstrated that there was increased complement activation in the retina of rats with increased IOP.

Fig. 1.

Fig. 1

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Complement activation in the retina of Lewis rats with increased IOP. (A) IOP of eyes treated with laser (■) and the control eyes not treated with laser (□) measured at different time points using Tonolab tonometer. (B-E) Immunofluorescent staining of C3 slit products (B and C) and MAC (D and E) in retina of rat with normal IOP (B and D) and elevated IOP (C and E) at week 6 post-laser. Objective magnification: 10X. (F) Western blot analysis for C3 split products (upper panel) and MAC (lower panel) in the retina of glaucomatous and control eyes at 6 weeks post laser. For the panel showing C3 split products (upper panel), lane 1 represents the molecular weight marker (Magic Marker XP, Invitrogen, Carlsbad, CA), lane 2 represents the control eyes not treated with laser (normal IOP) and lane 3 represents the eyes with elevated IOP at 6 weeks post-laser. For the lower panel showing MAC, lane 1 represents the control eyes not treated with laser (normal IOP) and lane 2 represents the eyes with elevated IOP at 6 weeks post-laser. Bands with strong and equal intensity of β-actin represent equal protein loading in each well. (G and H) Cumulative data for densitometric analysis of C3 (G) and MAC (H) Western blots from three separate experiments is shown. The intensity of protein bands was quantitated using an image analyzer, and the relative intensity was expressed as the ratio of the intensity of C3 (G) and MAC (H) bands to the intensity of β-actin bands. GCL: ganglion cell layer. *p<0.05.

3.2. Role of complement in the loss of RGCs

Lewis rats were divided in two groups and animals in each group received two laser treatments 7 days apart. The animals in the first group received CVF from Naja naja kaouthia (35 units/animal, i.p.; Quidel, San Diego, CA) immediately after the first laser treatment (i.e. at day 7 after first laser treatment) and then after every five days i.e. on days 12, 17, 22, 27, 32, 37, and 42 after first laser treatment. The second group received similar treatment with equal volume of PBS. Animals were sacrificed at 6 week after first laser and the eyes were processed for histology and immunostaining. The animals that received CVF treatment did not show any loss or thinning of RGC layer (Fig. 2A). In contrast histological analysis of H&E stained eyes demonstrated loss of RGCs (shown with arrows in Figure 2B) and thinning of RGC layer in the lasered animals treated with PBS (Fig. 2B). Immunofluorescent staining using anti-Brn3a antibody was used to confirm that CVF treatment protects the loss of RGCs. The paraffin sections from CVF treated and PBS treated laser-treated animals were stained for detection of Brn3a+ cells. Brn3a is used as a reliable marker to identify RGCs (Nadal-Nicolas et al., 2009). After immunostaining the Brn3a+ cells were counted using ImageJ software. Immunofluorescent staining demonstrated greater proportion of Brn3a+ cells in CVF treated lasered animals (41.5±6.3%; Fig. 2C and E) compared to PBS treated lasered animals (28.42 ± 2.97%; Fig. 2D and E). Taken together our results clearly demonstrated that complement depletion protects the RGCs in the laser-induced model of chronic ocular hypertension in Lewis rats. This protection is not due to reduction in IOP by CVF treatment because our results clearly show that CVF treatment does not reduce the IOP in lasered animals (Fig. 2F). No significant difference in IOP was observed between CVF treated lasered animals and PBS treated lasered animals (Fig. 2F).

Fig. 2.

Fig. 2

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Effect of complement on RGCs in the eyes with elevated IOP. (A and B). Histological analysis of the harvested eyes revealed no loss of RGCs or thinning of RGC layer in CVF treated (complement depleted) Lewis rats with increased IOP (A). The loss of RGCs and thinning of RGC layer (shown by arrow) was evident in PBS treated (complement sufficient) lasered-Lewis rats (B). Objective magnification: 20X. Immunofluorescent staining for Brn3a (green) in retina of lasered rats with increased IOP treated with CVF (C) and PBS (D). (E) The percentage of Brn3a positive (green fluorescence) cells in retina of lasered rats with increased IOP treated with CVF or PBS. (F) IOP of the eyes of lasered Lewis rats treated with CVF or PBS measured at 6 weeks post laser using Tonolab tonometer. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. *p<0.05.

The role of complement in the loss/damage of RGCs was further explored by immunofluorescent staining for MAC and GFAP. It is well known that MAC formation is a measure of complement activation (Bora et al., 2008; Jha et al., 2006, 2007) and GFAP expression increases in eyes with increased IOP (Woldemussie et al., 2004). The immunostaining of paraffin embedded sections demonstrated the presence of basal levels of GFAP (red fluorescence in Fig. 3A) and MAC (green fluorescence in Fig. 3A) in the RGC layer of naïve animals. Similar staining was observed in CVF treated lasered animals with increased IOP (Fig. 3B). In contrast an increased expression of GFAP was observed in the retina of eyes of rats with increased IOP that were treated with PBS (red fluorescence in Fig. 3C). Interestingly high deposition of MAC (green fluorescence in Fig. 3C) co-localized with the GFAP expression (yellow fluorescence in Fig. 3C) in these eyes. No fluorescence was detected in the negative controls that were stained with the secondary antibody alone (Fig. 3D). The decrease in MAC deposition in CVF-treated animals was confirmed by immunostaining of retinal flat mounts from Lewis rats with increased IOP treated with PBS (Fig. 3E) or CVF (Fig. 3F). Our results revealed that CVF treatment in the rats with increased IOP reduced MAC deposition (Fig. 3F) in the retina compared to the PBS treated animals with elevated IOP (Fig. 3E).

Fig. 3.

Fig. 3

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Immunofluorescent staining for GFAP (red) and MAC (green) in retina of naïve rats (A) and lasered rats with increased IOP treated with CVF (B) or PBS (C). Both GFAP and MAC staining decreased in the CVF-treated rats (B) compared to PBS (C) treated rats and was similar to that in naïve rats (A). No fluorescence was observed in negative control (D). (E and F) Retinal flat mounts were stained with rabbit antiserum to rat C9 to detect MAC. Intense red fluorescent staining was observed for MAC in PBS-treated lasered rats (E), whereas only weak MAC staining was observed in CVF-treated rats with increased IOP (F). Objective magnification: 40X. GCL: ganglion cell layer.

3.3. Mechanism of complement mediated loss of RGCs

We also explored the underlying mechanisms responsible for complement mediated loss of RGCs in rat eyes with elevated IOP. We investigated the effect of complement on apoptosis of RGCs as well as on calcium influx in RGCs.

3.3.1. Apoptosis

Lewis rats were divided in two groups and each animal received two laser treatments 7 days apart. The animals were treated with CVF or PBS as described above and the eyes were harvested at day 42 post-laser. Apoptosis of RGCs were investigated by TUNEL staining. TUNEL staining revealed the presence of several TUNEL positive apoptotic RGCs in the retina of PBS injected rats with increased IOP (Fig. 4A and C). However, only few apoptotic RGCs could be detected in the CVF-treated rats (Fig. 4B and C).

Fig. 4.

Fig. 4

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Effect of complement depletion on apoptosis of RGCs. (A and B) TUNEL staining of retina harvested from lasered rats with elevated IOP treated with PBS (A) or CVF (B). The apoptotic cells (green) are shown with arrows. (C) Cumulative results from three separate experiments showing the percentage of TUNEL positive (green fluorescence) cells in retina of lasered rats with increased IOP treated with CVF or PBS. Objective magnification : 63X; *p<0.05. (D-G) Western blot analysis of cleaved caspase-8 (D) and cleaved caspase-9 (E) in retina harvested from lasered rats with elevated IOP treated with PBS (lane 1) and CVF (lane 2). Bands with equal intensity of β-actin demonstrate equal protein loading in each well. Cumulative data for densitometric analysis of caspase-8 (F) and caspase-9 (G) Western blots from three separate experiments. The intensity of protein bands was quantitated using an image analyzer, and the relative intensity was expressed as the ratio of the intensity of caspase-8 (F) and caspase-9 (G) bands to the intensity of β-actin bands. GCL: ganglion cell layer; INL: inner nuclear layer.

Next we investigated if the presence and the absence of complement system affected the intrinsic and/or extrinsic pathways of apoptosis in the eyes of Lewis rats with increased IOP. Lewis rats were divided in two groups and each animal received two laser treatments 7 days apart. CVF or PBS treated animals were sacrificed at day 42 post-laser and the harvested eyes were processed for protein extraction. Protein samples were used for Western blot analysis for activated caspase-8 and activated caspase-9. Western blot analysis showed that CVF treatment blocked the activation of caspase-8 (Fig. 4D and F) and caspase-9 (Fig. 4E and G) in the eyes with elevated IOP. In contrast, increased levels of both activated caspase-8 (Fig. 4D and F) and activated caspase-9 (Fig. 4E and G) were detected in the eyes of PBS treated rats with increased IOP.

Collectively, these findings suggest that the apoptosis of RGCs in the retina of the eyes with elevated IOP requires the presence and the activation of the complement system.

3.3.2. Calcium influx

We hypothesized that MAC formation on the surface of RGCs due to complement activation induces apoptosis by increasing intracellular calcium levels. To test this hypothesis, animals with increased IOP treated with PBS (control) or CVF were sacrificed and eyes were enucleated. Retinal flat mounts were stained with calcium Green I and calcium influx was determined by confocal microcopy. In a separate set of experiments the retinal flat mounts were stained with calcium Green I and anti-rat C9 (MAC staining). High levels of intracellular calcium were noted in the animals treated with PBS (Fig. 5A). In contrast, low levels of calcium were observed in the retinal flat mounts of rats with increased IOP and treated with CVF (Fig. 5B). Interestingly, confocal analyses demonstrated increased MAC (red fluorescent in Fig. 5C) staining on the cell surface of the cells with increased intracellular calcium levels (green fluorescent in Fig. 5C). No fluorescence was observed in the absence of calcium Green I or anti-rat C9 (negative control; Fig. 5D). These results demonstrated that the calcium influx observed in the retinal cells of animals with increased IOP is complement-dependent.

Fig. 5.

Fig. 5

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Effect of complement depletion on calcium influx in RGCs. (A and B) Calcium green-1 staining (green fluorescence) in the flat mount of retina harvested from rats with increased IOP and treated with PBS (A) or CVF (B). (C) Calcium green-1 staining (green fluorescence) and immunofluorescent staining for MAC (red fluorescence) in the flat mount of retina harvested from rats with increased IOP. (D) The negative control flat mounts were not stained with calcium green or primary antibody for rat C-9. Objective magnification: 63X

3.3.3. Levels of BAD in RGCs

It has been reported that Ca2+ can induce apoptosis through calcineurin mediated modulation of Bcl-2 family member, Bcl-2 associated death domain (BAD) (Huang et al., 2005). Therefore we investigated the levels of BAD in RGCs of CVF and PBS-treated lasered rat eyes. Lewis rats were divided in two groups and each animal received two laser treatments 7 days apart. The animals were treated with CVF or PBS as described above and the eyes were harvested at day 42 post-laser. The protein levels of BAD in cytoplasm of cells in RGC layer were investigated by immunofluorescent staining. Immunofluorescent staining revealed increased cytoplasmic staining for BAD in cells present within the RGC layer of PBS injected lasered Lewis rats(green fluorescence in Fig. 6A) compared to CVF injected lasered Lewis rats (green fluorescence in Fig. 6B). Interestingly, the levels of BAD in RGC layer of CVF injected Lewis rats (Fig. 6B) were similar to those observed in naïve Lewis rats (Fig. 6C). No staining was observed in the negative controls (Fig. 6D) that were incubated with only the secondary antibody.

Fig. 6.

Fig. 6

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Effect of complement depletion on levels of BAD in RGCs. Paraffin-embedded sections prepared from the eyes harvested from lasered Lewis rats at 6 week post laser were stained with anti-BAD (A-C). Green fluorescence represents the staining for BAD. BAD staining decreased in the CVF treated rats (B) compared to PBS treated lasered rats (a). The levels of BAD in CVF treated rats were similar to those in naïve rats (C). Sections stained with secondary antibody only (negative control) did not show any stain (D). Objective magnification: 63X. GCL: ganglion cell layer.

Together our results clearly demonstrated that complement activation plays a critical role in the apoptosis of RGCs in glaucoma.

4. Discussion

Glaucoma characterized by elevated intraocular pressure is a chronic ocular disease and is a leading cause of blindness worldwide (Rivera et al., 2008). There is growing evidence suggesting the role of immune system in the pathogenesis of glaucoma (Grus and Sun, 2008; Grus et al., 2008). However, the underlying pathogenic mechanisms are not well understood. In the present study, we used an animal model of glaucoma to assess the role of complement system in this disease and investigated the underlying mechanisms. Using rat model of laser-induced chronic ocular hypertension we demonstrated that complement system plays an important role in the loss of RGCs in eyes with elevated IOP. Importantly, we observed that the complement depletion, even after established elevated IOP, reduces the loss of RGCs in the retina. Our results further demonstrate that complement mediated apoptosis leads to the loss of RGCs in the eyes with increased IOP.

Our findings reported here demonstrate that the levels of complement component C3, its activation products and MAC increased in the retina of rats with elevated IOP. Other investigators have also reported elevated levels of complement components and complement activation products such as C1q, C4a, C3 and MAC in both rat and human glaucomatous eyes (Kuehn et al., 2006; Miyahara et al., 2003; Ahmed et al., 2004). In the current study we used CVF to deplete the complement system. To confirm that repeated CVF injections deplete the complement system of the host, complement activity was checked on every seventh day post-laser using hemolytic assay as described previously by us (Jha et al., 2006). Complement activity (as measured by hemolytic assay) was barely detectable in rats that received repeated injections of CVF (data not shown). Thus, repeated CVF injections did not have any adverse effect on its ability to deplete the complement system of the host. Our results show that complement depletion using CVF protected the RGCs from cell death. The fact that in our study the complement was depleted after the second laser is significant since the rats already had elevated IOP for one week. Furthermore we have shown that complement depletion reduces the GFAP expression and MAC deposition in eyes with elevated IOP. GFAP is present in Müller cells and astrocytes in the normal retina. However if retina is damaged (after elevation of IOP), GFAP expression is elevated (Woldemussie et al., 2004). Reduction of GFAP staining after CVF treatment shows that complement depletion reduced the loss of RGCs. Histological analysis of eyes with elevated IOP, harvested from complement depleted and complement sufficient animals confirmed these findings. Complement system has been reported to play an important role in the destruction of RGCs (Kuehn et al., 2006, 2008; Tezel et al., 2010; Miyahara et al., 2003; Ahmed et al., 2004; Stasi et al., 2006; Stevens et al., 2007; Shields et al., 1976).

We have shown here that complement depletion reduces the apoptosis of RGCs in retina of rats with increased IOP. Furthermore, complement depletion inhibited the activation of both caspase-8 and caspase-9. Apoptosis has been reported to play a central role in the death of RGCs in glaucoma and both caspase-8 (extrinsic pathway) and caspase-9 (intrinsic pathway) have been reported to be activated in glaucomatous eyes (Qu et al., 2010). It is well established that MAC deposited on the cell membrane can induce apoptosis (Hughes et al., 2000; Sato et al., 1999). Therefore, it is likely that the apoptosis of the RGCs observed in glaucoma may be induced by increased MAC deposition. MAC deposition on the cell surface causes imbalance between the intracellular and extracellular ion concentrations (Carney et al., 1990). Results from our present study show that the intracellular Ca2+ concentration decreases dramatically in retina of complement depleted rats with elevated IOP compared to complement sufficient animals. Our results further show that in complement sufficient rats, increased MAC deposition directly correlated with high intracellular Ca2+ concentration. Increase in cytosolic-free Ca2+ can induces apoptosis through calcineurin-mediated dephosphorylation of proapoptotic molecules such as BAD leading to activation of caspases (Huang et al., 2005; Wang et al., 1999). Furthermore, it is known that the levels of many BH3-only proteins like BAD are increased under stress Lomonosova and Chinnadurai, 2008). Our results clearly demonstrated that the depletion of complement system resulted in the decrease in the levels of BAD which otherwise is increased in lasered Lewis rats with intact complement system. Therefore, based on our results we propose that the increased MAC formation in the eyes with elevated IOP increases Ca2+ influx in the RGCs. This causes apoptosis of RGCs in the glaucomatous eyes.

Taken together, our results demonstrated that increased MAC deposition (in the RGC layer) resulting from enhanced complement activation causes the apoptosis of RGCs in the glaucomatous eyes. Furthermore, complement depletion inhibits both extrinsic and intrinsic pathways of apoptosis and depletion of complement system blocks Ca2+ influx in the RGCs of glaucomatous eyes. Finally, results from the present study strongly indicate that inhibition of complement system is a potential strategy for preventing damage of RGCs in the eyes with glaucoma.

Highlights.

  • Increased MAC deposition resulting from enhanced complement activation causes the apoptosis of RGCs in the glaucomatous eyes.

  • Complement depletion inhibits both extrinsic and intrinsic pathways of apoptosis in rat eyes with elevated IOP.

  • Depletion of complement system blocks Ca2+ influx in the RGCs of glaucomatous eyes.

  • Inhibition of complement system is a potential strategy for preventing damage of RGCs in the eyes with glaucoma.

Acknowledgements

We thank Valeriy V. Lyzogubov MD, Ph.D., for help with histologic studies and the facilities in the University of Arkansas for Medical Sciences Digital and Confocal Microscopy Laboratory supported by Grant Number 2 P20 RR 16460 (PI: Larry Cornett, INBRE, Partnerships for Biomedical Research in Arkansas) and Grant Number 1 S10 RR 19395 (PI: Richard Kurten, “Zeiss LSM 510 META Confocal Microscope System“) from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), is acknowledged.

Support: This work was supported by grant from Jones Eye Institute, Little Rock, AR.

Footnotes

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Conflict of Interest: The authors declare no conflict of interest.

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