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Brilliant Blue G protects against photoreceptor injury in a murine endotoxin-induced uveitis model
Experimental Eye Research 177 (2018) 45–49
Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Brilliant Blue G protects against photoreceptor injury in a murine endotoxininduced uveitis model
T
Kenji Sakamoto∗, Miho Inukai, Asami Mori, Tsutomu Nakahara Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, 108-8641, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Retina Lipopolysaccharide P2X7 receptor Photoreceptor degeneration
We previously reported that P2X7 receptor antagonists prevented the retinal injury caused by N-methyl-D-aspartic acid. It has been reported that activation of P2X7 receptor is involved in the secretion of proinflammatory cytokines by macrophages, monocytes, and microglia. Although retinal inflammation is known to cause photoreceptor cell death, it is unclear whether a noncompetitive antagonist of P2X7 receptor can protect photoreceptor cells against inflammation-induced injury. We examined whether Brilliant Blue G (BBG), a potent noncompetitive antagonist of P2X7 receptor, had neuroprotective effects on photoreceptor cell injury in a murine endotoxin-induced uveitis (EIU) model. EIU was evoked by lipopolysaccharides (LPS; 10 mg/kg/day) administered intraperitoneally once a day for 4 days. BBG (50 mg/kg/day) and indomethacin (10 mg/kg) were also injected intraperitoneally just before LPS injection. BBG significantly prevented photoreceptor cell loss and reduction of the amplitudes of dark-adapted and light-adapted flush electroretinograms induced by LPS, whereas indomethacin did not show such protective effects. These results indicated that BBG is protective against photoreceptor cell injury in EIU in the mice in vivo, suggesting that P2X7 receptor antagonists may be good candidates for preventing photoreceptor degeneration induced by inflammation.
Inflammation has been considered to be a pathogenic factor in various retinal diseases. Thus, manipulation of inflammation may be a good approach for the treatment of retinal diseases. Upregulation of proinflammatory cytokines, such as vascular endothelial growth factor (Caldwell et al., 2003), interleukin-6 (Izumi-Nagai et al., 2007), and angiotensin II (Nagai et al., 2005), has been reported to cause diabetic retinopathy and age-related macular degeneration. The P2X7 receptor, a kind of purinergic P2 receptor, is a ligandgated ion channel that is stimulated by extracellular ATP and passes Na+ and Ca2+ (North, 2002; Egan and Khakh, 2004). It had been reported that relatively higher ATP levels, which are often observed in inflammatory or severely damaged tissues, are required to activate the P2X7 receptor. Activation of the P2X7 receptor in the brain has been reported to cause the release of proinflammatory cytokines from microglia and macrophages (Ferrari et al., 1997a, 1997b; Monif et al., 2010). It has been reported that P2X7 receptor activation is one of the causes of neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease, and acute injury of neural tissues, such as spinal cord injury and stroke (Peng et al., 2009; Skaper et al., 2010; Takenouchi et al., 2010). Also, in the retina, high ocular pressure
induced retinal ganglion cell (RGC) death has been reported to be mediated at least in part by P2X7 receptor activation (Resta et al., 2007; Zhang et al., 2005; Reigada et al., 2008). We recently reported that P2X7 receptor activation was involved in RGC loss by intravitreal Nmethyl-D aspartic acid (NMDA) (Sakamoto et al., 2015a). It has been reported that the P2X7 receptor is expressed not only in RGCs and amacrine cells (Brändle et al., 1998; Pannicke et al., 2000; Ishii et al., 2003), but also photoreceptor cells and retinal pigmented epithelial cells (RPE) in the retina (Notomi et al., 2011; Yang et al., 2011). Under inflammatory conditions, RPEs produce various cytokines. Autocrine/paracrine mechanisms of the cytokines may cause degeneration of the outer retina induced by nitric oxide, expression of major histocompatibility complex class II and adhesion molecules in the cell surface, and disruption of the outer blood-retinal barrier (Holtkamp et al., 2001; Sasaki et al., 2009). However, it has not been clarified whether P2X7 receptor antagonists are protective against photoreceptor cell injury under retinal inflammation. A murine endotoxin-induced uveitis (EIU) model has been used as a model of the inflammation of the outer retina, because EIU has been reported to upregulate various inflammatory cytokines and to cause photoreceptor cell death and impairment of visual function that was
∗ Corresponding author. Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 9-1 Shirokane 5-chome, Minato-ku, Tokyo, 108-8641, Japan. E-mail address: [email protected] (K. Sakamoto).
https://doi.org/10.1016/j.exer.2018.07.028 Received 24 May 2018; Received in revised form 11 July 2018; Accepted 27 July 2018 Available online 29 July 2018 0014-4835/ © 2018 Elsevier Ltd. All rights reserved.
Experimental Eye Research 177 (2018) 45–49
K. Sakamoto et al.
Fig. 1. Effect of brilliant blue G (BBG) and indomethacin (INDO) on reduction in the amplitude of electroretinogram (ERG) induced by lipopolysaccharides (LPS). (A) Representative scotopic ERG recorded 5 days after the first administration of LPS. (B) Data analyses of scotopic ERG a-wave and b-wave amplitudes 5 days after the first administration of LPS. Each datum is presented as the mean ± SEM of 5–8 independent experiments. *P < 0.05, vs. PBS group. (C) Representative photopic ERG recorded 5 days after the first injection of LPS. (D) Data analyses of photopic ERG b-wave amplitude 5 days after the first injection of LPS. Each datum is presented as the mean ± SEM of 4–6 independent experiments. *P < 0.05, versus PBS group. #P < 0.05, versus LPS group.
et al., 2015a). The dose of indomethacin was reported as ED50 on carrageenan rat paw edema with a single-dose administration (Cong et al., 2015). Full-field flush scotopic and photopic electroretinograms (ERG) were measured 5 days after the first administration of LPSs, according to our previous report (Sakamoto et al., 2015b) with a slight modification. Responses to white light flashes (1000 cd/m2, 3 ms in the dark-adapted animals; 11,000 cd/m2, 3 ms in the light-adapted animals) were measured for scotopic and photopic ERG. Histological evaluation was done according to our previous reports (Sakamoto et al., 2009, 2010a, 2010b, 2011, 2014). Animals were sacrificed with cervical dislocation 5 days after the first administration of LPS. The enucleated right eye was fixed with Davidson solution and embedded in paraffin. Five-micrometer-thick retinal horizontal sections were cut and stained with hematoxylin and eosin. The number of the cells in the outer nuclear layer (ONL) was counted. The procedure for immunohistochemistry using goat anti-P2X7 receptor antibody (1:100; Abcam, Cambridge, UK) was described previously (Sakamoto et al., 2015a). The enucleated left eye was fixed with 4% paraformaldehyde in
evaluated by electroretinogram (ERG) (Nagai et al., 2005; Kurihara et al., 2006; Ozawa et al., 2008; Sasaki et al., 2009). The current study aimed to clarify whether Brilliant Blue G (BBG), a potent non-competitive antagonist of P2X7 receptor, has neuroprotective effects on photoreceptor cell injury in the murine EIU model. The experimental procedures used in the present study conformed to the Regulations for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Kitasato University. Male ICR mice at 7–8 weeks of age were purchased from Japan SLC (Hamamatsu, Japan). The animal room was air-conditioned at 25 °C with a 12-h:12-h light-dark cycle. The mice used in the present study were fed and watered ad libitum. Lipopolysaccharides (LPS) from Escherichia coli (Nacalai Tesque, Kyoto, Japan) were dissolved in phosphate-buffered saline (PBS) and intraperitoneally administered at 10 mg/kg once a day for 4 days. BBG (50 mg/kg; Sigma, St. Louis, MO) and indomethacin (10 mg/kg; Sigma) were dissolved in saline and injected intraperitoneally just before LPS administration. The dose of BBG have previously been shown to antagonize the effect of BzATP, a P2X7 agonist in the retina (Sakamoto 46
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Fig. 2. Intraperitoneal lipopolysaccharides (LPS)-induced histological damage examined 5 days after the first injection of LPS. (A) Representative photomicrographs of the hematoxylin-eosin-stained retinal sections were shown. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, the outer nuclear layer. Scale bar = 25 μm. Original magnification is × 200. (B) The number of the cells in the outer nuclear layer (ONL) were counted. Each datum is presented as the mean ± SEM of 4–6 independent experiments. *P < 0.05, versus PBS group. #P < 0.05, versus LPS group. (C) Representative photomicrographs of the retinas stained with Alexa fluor 488-conjugated peanut agglutinin (PNA) were shown. Scale bar = 100 μm. Original magnification is × 200. (B) The number of the PNA-positive cells in 0.2 mm2 of the whole mount retina were counted. Each datum is presented as the mean ± S.E.M. of 6–8 independent experiments. *P < 0.05, versus PBS group. #P < 0.05, versus LPS group. (E) Immunohistochemical analysis using goat anti-P2X7 antibody in the retina. P2X7 receptors were expressed strongly in retinal pigment epithelium and relatively weakly in photoreceptor inner segment. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, the outer nuclear layer; IS, photoreceptor inner segment; OS, photoreceptor outer segment; RPE, retinal pigment epithelium. Scale bar = 25 μm. Original magnification, × 200.
LPSs-induced histological damage in the retina (Fig. 2A–D). LPS significantly shortened the thickness of ONL and decreased the number of the cells in ONL, indicating that retinal inflammation caused the loss of photoreceptor cells. In the whole-mount retina stained with Alexa fluor 488-conjugated PNA, LPS significantly decreased the number of the PNA-positive cells, suggesting that EIU caused the loss of cone photoreceptor cells. BBG, but not indomethacin, significantly prevented these retinal injuries induced by LPS, suggesting that blockade of P2X7 receptors was protective against histological injury caused by EIU. Indomethacin did not show such protective effects, suggesting that inflammatory prostaglandins did not play a central role in the histological damage of photoreceptor cells in the EIU model mice. P2X7 receptor activation has been reported to cause acute neuronal injury as well as chronic neurodegenerative diseases (Peng et al., 2009; Skaper et al., 2010; Takenouchi et al., 2010). The P2X7 receptor is expressed mostly in activated microglial cells and macrophages in the brain, and its activation elicits cytolysis (Collo et al., 1997; Rathbone et al., 1999). In the present study, we clarified whether the Iba1-positive cells, which were considered as microglial cells and macrophages, were found in the retina of the mice injected with LPS. We could not find the change in the distribution of the Iba1-positive cells by LPS injection (data not shown). In fact, a previous report showed that hematopoietic cell infiltration and glial cell activation were not apparent
0.1 M of PBS saline for 1 h at 4 °C. Whole mount retinas were prepared and stained with Alexa fluor 488-conjugated peanut agglutinin (PNA) to label cone photoreceptor cells. Images were taken using a confocal laser microscope (LSM710, Carl Zeiss, Oberkochen, Germany). The area in which the number of the PNA-positive cells was counted was 0.2 mm2. The number of PNA-positive cone photoreceptor cells was counted using image-processing software (Adobe Photoshop CS5, San Jose, CA) in the four areas (superior, nasal, inferior, and temporal) that were 500 mm away from the optic disc. Each datum was presented as the mean ± standard error of the mean (SEM) of 4–8 independent experiments. We used Dunnett's test for multiple comparisons. Differences were considered to be statistically significant if p < 0.05. First, we clarified the effects of BBG and indomethacin on LPSsinduced functional retinal impairment by ERG (Fig. 1). The amplitudes of scotopic a- and b-wave and photopic b-wave were significantly decreased by LPSs. BBG tended to preserve the amplitudes of scotopic aand b-waves, and significantly preserved that of photopic b-wave, suggesting that blockade of P2X7 receptors was protective against visual dysfunction caused by EIU. Indomethacin did not show such protective effects, suggesting that inflammatory prostaglandins did not play a major role in photoreceptor cell dysfunction in EIU. Second, we clarified the effects of BBG and indomethacin on the 47
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in the outer retina of the EIU model mice (Sasaki et al., 2009). These results suggest that P2X7 receptors expressed in microglial cells and macrophages are not involved in the damage of the outer retina in EIU model mice. P2X7 receptors have recently been reported to be involved in oxidative stress in the outer retina of Sod1 knockout mice (Carver et al., 2017). We investigated whether the 8-OHdG-positive cells, which were one of markers of the cells injured by oxidative stress, were found in the retina of the mice injected LPS. We could find few 8-OHdG-positive cells in the retina of EIU model mice (data not shown), suggesting that oxidative stress was not a major cause of the photoreceptor damage in EIU model mice. In the current study, we demonstrated that P2X7 receptors were expressed strongly in retinal pigment epithelium and relatively weakly in photoreceptor inner segments (Fig. 2E). Previous reports have shown that BzATP, a P2X7 agonist, induced photoreceptor cell death (Notomi et al., 2011) and death of the cultured RPE (Yang et al., 2011). We propose that P2X7 receptor activation in photoreceptor cells and RPE may cause photoreceptor cell damage in the EIU model mice. Although the source of the endogenous agonists of P2X7 receptors, such as ATP, in the EIU model remain unknown, BBG showed significant protective effects in the present study. These current results are consistent with those of the previous reports (Notomi et al., 2011; Yang et al., 2011). BBG has also been shown to have a neuroprotective effect on RGC damage induced by optic nerve crash (Kakurai et al., 2013) and intravitreal NMDA injection in vivo (Sakamoto et al., 2015a). In the present study, BBG evoked better functional protection in cone photoreceptors than in rod photoreceptors (Fig. 1), which is consistent with the protection of PNA-positive cells in the whole mount retinas. These results suggest that contribution of P2X7 receptors in the LPS-induced degeneration may be higher in cone photoreceptors than in rod photoreceptors. Unfortunately, we could not determine the difference in the expression of P2X7 receptors between rod and cone photoreceptors because the number of cone photoreceptors in a retinal horizontal section is too small to distinguish these two photoreceptor cell types. Further studies are clearly needed to clarify the underlying mechanism which causes better protective effects of BBG in cone photoreceptors. Bacterial infection in other tissues and sepsis rarely causes endogenous endophthalmitis (LaMattina, 2017). Although the patients are subjected to intravitreal and intravenous antibiotics and/or vitrectomy for treatment, their vision prognosis is often poor (LaMattina, 2017). The results in the present study suggest that BBG is a preventive drug for visual loss induced by endophthalmitis. Recently, BBG has been used in macular surgery for staining the inner limiting membrane to peel it (Almony et al., 2012). BBG has been reported to a better safety profiles than indocyanine green and trypan blue (Lüke et al., 2008; Baba et al., 2012). The cytoprotective effects found in the present and previous studies may reflect the superiority of BBG in inner limiting membrane peeling. In conclusion, we proposed that activation of P2X7 receptors at least in part cause injury of the outer retina in EIU model mice. The current study shows that blockade of P2X7 receptor is a good candidate for protection against outer retinal degeneration induced by inflammation and implies superiority of BBG in inner limiting membrane staining for macular surgery.
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Acknowledgments This work was supported by the Japan Society for the Promotion of Science KAKENHI (Scientific Research (C); Grant Number #24590329 (KS), #15K07971 (KS) and #18K06689 (KS)). References Almony, A., Nudleman, E., Shah, G.K., Blinder, K.J., Eliott, D.B., Mittra, R.A., Tewari, A.,
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Contents lists available at ScienceDirect
Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Brilliant Blue G protects against photoreceptor injury in a murine endotoxininduced uveitis model
T
Kenji Sakamoto∗, Miho Inukai, Asami Mori, Tsutomu Nakahara Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, 108-8641, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Retina Lipopolysaccharide P2X7 receptor Photoreceptor degeneration
We previously reported that P2X7 receptor antagonists prevented the retinal injury caused by N-methyl-D-aspartic acid. It has been reported that activation of P2X7 receptor is involved in the secretion of proinflammatory cytokines by macrophages, monocytes, and microglia. Although retinal inflammation is known to cause photoreceptor cell death, it is unclear whether a noncompetitive antagonist of P2X7 receptor can protect photoreceptor cells against inflammation-induced injury. We examined whether Brilliant Blue G (BBG), a potent noncompetitive antagonist of P2X7 receptor, had neuroprotective effects on photoreceptor cell injury in a murine endotoxin-induced uveitis (EIU) model. EIU was evoked by lipopolysaccharides (LPS; 10 mg/kg/day) administered intraperitoneally once a day for 4 days. BBG (50 mg/kg/day) and indomethacin (10 mg/kg) were also injected intraperitoneally just before LPS injection. BBG significantly prevented photoreceptor cell loss and reduction of the amplitudes of dark-adapted and light-adapted flush electroretinograms induced by LPS, whereas indomethacin did not show such protective effects. These results indicated that BBG is protective against photoreceptor cell injury in EIU in the mice in vivo, suggesting that P2X7 receptor antagonists may be good candidates for preventing photoreceptor degeneration induced by inflammation.
Inflammation has been considered to be a pathogenic factor in various retinal diseases. Thus, manipulation of inflammation may be a good approach for the treatment of retinal diseases. Upregulation of proinflammatory cytokines, such as vascular endothelial growth factor (Caldwell et al., 2003), interleukin-6 (Izumi-Nagai et al., 2007), and angiotensin II (Nagai et al., 2005), has been reported to cause diabetic retinopathy and age-related macular degeneration. The P2X7 receptor, a kind of purinergic P2 receptor, is a ligandgated ion channel that is stimulated by extracellular ATP and passes Na+ and Ca2+ (North, 2002; Egan and Khakh, 2004). It had been reported that relatively higher ATP levels, which are often observed in inflammatory or severely damaged tissues, are required to activate the P2X7 receptor. Activation of the P2X7 receptor in the brain has been reported to cause the release of proinflammatory cytokines from microglia and macrophages (Ferrari et al., 1997a, 1997b; Monif et al., 2010). It has been reported that P2X7 receptor activation is one of the causes of neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease, and acute injury of neural tissues, such as spinal cord injury and stroke (Peng et al., 2009; Skaper et al., 2010; Takenouchi et al., 2010). Also, in the retina, high ocular pressure
induced retinal ganglion cell (RGC) death has been reported to be mediated at least in part by P2X7 receptor activation (Resta et al., 2007; Zhang et al., 2005; Reigada et al., 2008). We recently reported that P2X7 receptor activation was involved in RGC loss by intravitreal Nmethyl-D aspartic acid (NMDA) (Sakamoto et al., 2015a). It has been reported that the P2X7 receptor is expressed not only in RGCs and amacrine cells (Brändle et al., 1998; Pannicke et al., 2000; Ishii et al., 2003), but also photoreceptor cells and retinal pigmented epithelial cells (RPE) in the retina (Notomi et al., 2011; Yang et al., 2011). Under inflammatory conditions, RPEs produce various cytokines. Autocrine/paracrine mechanisms of the cytokines may cause degeneration of the outer retina induced by nitric oxide, expression of major histocompatibility complex class II and adhesion molecules in the cell surface, and disruption of the outer blood-retinal barrier (Holtkamp et al., 2001; Sasaki et al., 2009). However, it has not been clarified whether P2X7 receptor antagonists are protective against photoreceptor cell injury under retinal inflammation. A murine endotoxin-induced uveitis (EIU) model has been used as a model of the inflammation of the outer retina, because EIU has been reported to upregulate various inflammatory cytokines and to cause photoreceptor cell death and impairment of visual function that was
∗ Corresponding author. Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 9-1 Shirokane 5-chome, Minato-ku, Tokyo, 108-8641, Japan. E-mail address: [email protected] (K. Sakamoto).
https://doi.org/10.1016/j.exer.2018.07.028 Received 24 May 2018; Received in revised form 11 July 2018; Accepted 27 July 2018 Available online 29 July 2018 0014-4835/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Effect of brilliant blue G (BBG) and indomethacin (INDO) on reduction in the amplitude of electroretinogram (ERG) induced by lipopolysaccharides (LPS). (A) Representative scotopic ERG recorded 5 days after the first administration of LPS. (B) Data analyses of scotopic ERG a-wave and b-wave amplitudes 5 days after the first administration of LPS. Each datum is presented as the mean ± SEM of 5–8 independent experiments. *P < 0.05, vs. PBS group. (C) Representative photopic ERG recorded 5 days after the first injection of LPS. (D) Data analyses of photopic ERG b-wave amplitude 5 days after the first injection of LPS. Each datum is presented as the mean ± SEM of 4–6 independent experiments. *P < 0.05, versus PBS group. #P < 0.05, versus LPS group.
et al., 2015a). The dose of indomethacin was reported as ED50 on carrageenan rat paw edema with a single-dose administration (Cong et al., 2015). Full-field flush scotopic and photopic electroretinograms (ERG) were measured 5 days after the first administration of LPSs, according to our previous report (Sakamoto et al., 2015b) with a slight modification. Responses to white light flashes (1000 cd/m2, 3 ms in the dark-adapted animals; 11,000 cd/m2, 3 ms in the light-adapted animals) were measured for scotopic and photopic ERG. Histological evaluation was done according to our previous reports (Sakamoto et al., 2009, 2010a, 2010b, 2011, 2014). Animals were sacrificed with cervical dislocation 5 days after the first administration of LPS. The enucleated right eye was fixed with Davidson solution and embedded in paraffin. Five-micrometer-thick retinal horizontal sections were cut and stained with hematoxylin and eosin. The number of the cells in the outer nuclear layer (ONL) was counted. The procedure for immunohistochemistry using goat anti-P2X7 receptor antibody (1:100; Abcam, Cambridge, UK) was described previously (Sakamoto et al., 2015a). The enucleated left eye was fixed with 4% paraformaldehyde in
evaluated by electroretinogram (ERG) (Nagai et al., 2005; Kurihara et al., 2006; Ozawa et al., 2008; Sasaki et al., 2009). The current study aimed to clarify whether Brilliant Blue G (BBG), a potent non-competitive antagonist of P2X7 receptor, has neuroprotective effects on photoreceptor cell injury in the murine EIU model. The experimental procedures used in the present study conformed to the Regulations for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Kitasato University. Male ICR mice at 7–8 weeks of age were purchased from Japan SLC (Hamamatsu, Japan). The animal room was air-conditioned at 25 °C with a 12-h:12-h light-dark cycle. The mice used in the present study were fed and watered ad libitum. Lipopolysaccharides (LPS) from Escherichia coli (Nacalai Tesque, Kyoto, Japan) were dissolved in phosphate-buffered saline (PBS) and intraperitoneally administered at 10 mg/kg once a day for 4 days. BBG (50 mg/kg; Sigma, St. Louis, MO) and indomethacin (10 mg/kg; Sigma) were dissolved in saline and injected intraperitoneally just before LPS administration. The dose of BBG have previously been shown to antagonize the effect of BzATP, a P2X7 agonist in the retina (Sakamoto 46
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Fig. 2. Intraperitoneal lipopolysaccharides (LPS)-induced histological damage examined 5 days after the first injection of LPS. (A) Representative photomicrographs of the hematoxylin-eosin-stained retinal sections were shown. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, the outer nuclear layer. Scale bar = 25 μm. Original magnification is × 200. (B) The number of the cells in the outer nuclear layer (ONL) were counted. Each datum is presented as the mean ± SEM of 4–6 independent experiments. *P < 0.05, versus PBS group. #P < 0.05, versus LPS group. (C) Representative photomicrographs of the retinas stained with Alexa fluor 488-conjugated peanut agglutinin (PNA) were shown. Scale bar = 100 μm. Original magnification is × 200. (B) The number of the PNA-positive cells in 0.2 mm2 of the whole mount retina were counted. Each datum is presented as the mean ± S.E.M. of 6–8 independent experiments. *P < 0.05, versus PBS group. #P < 0.05, versus LPS group. (E) Immunohistochemical analysis using goat anti-P2X7 antibody in the retina. P2X7 receptors were expressed strongly in retinal pigment epithelium and relatively weakly in photoreceptor inner segment. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, the outer nuclear layer; IS, photoreceptor inner segment; OS, photoreceptor outer segment; RPE, retinal pigment epithelium. Scale bar = 25 μm. Original magnification, × 200.
LPSs-induced histological damage in the retina (Fig. 2A–D). LPS significantly shortened the thickness of ONL and decreased the number of the cells in ONL, indicating that retinal inflammation caused the loss of photoreceptor cells. In the whole-mount retina stained with Alexa fluor 488-conjugated PNA, LPS significantly decreased the number of the PNA-positive cells, suggesting that EIU caused the loss of cone photoreceptor cells. BBG, but not indomethacin, significantly prevented these retinal injuries induced by LPS, suggesting that blockade of P2X7 receptors was protective against histological injury caused by EIU. Indomethacin did not show such protective effects, suggesting that inflammatory prostaglandins did not play a central role in the histological damage of photoreceptor cells in the EIU model mice. P2X7 receptor activation has been reported to cause acute neuronal injury as well as chronic neurodegenerative diseases (Peng et al., 2009; Skaper et al., 2010; Takenouchi et al., 2010). The P2X7 receptor is expressed mostly in activated microglial cells and macrophages in the brain, and its activation elicits cytolysis (Collo et al., 1997; Rathbone et al., 1999). In the present study, we clarified whether the Iba1-positive cells, which were considered as microglial cells and macrophages, were found in the retina of the mice injected with LPS. We could not find the change in the distribution of the Iba1-positive cells by LPS injection (data not shown). In fact, a previous report showed that hematopoietic cell infiltration and glial cell activation were not apparent
0.1 M of PBS saline for 1 h at 4 °C. Whole mount retinas were prepared and stained with Alexa fluor 488-conjugated peanut agglutinin (PNA) to label cone photoreceptor cells. Images were taken using a confocal laser microscope (LSM710, Carl Zeiss, Oberkochen, Germany). The area in which the number of the PNA-positive cells was counted was 0.2 mm2. The number of PNA-positive cone photoreceptor cells was counted using image-processing software (Adobe Photoshop CS5, San Jose, CA) in the four areas (superior, nasal, inferior, and temporal) that were 500 mm away from the optic disc. Each datum was presented as the mean ± standard error of the mean (SEM) of 4–8 independent experiments. We used Dunnett's test for multiple comparisons. Differences were considered to be statistically significant if p < 0.05. First, we clarified the effects of BBG and indomethacin on LPSsinduced functional retinal impairment by ERG (Fig. 1). The amplitudes of scotopic a- and b-wave and photopic b-wave were significantly decreased by LPSs. BBG tended to preserve the amplitudes of scotopic aand b-waves, and significantly preserved that of photopic b-wave, suggesting that blockade of P2X7 receptors was protective against visual dysfunction caused by EIU. Indomethacin did not show such protective effects, suggesting that inflammatory prostaglandins did not play a major role in photoreceptor cell dysfunction in EIU. Second, we clarified the effects of BBG and indomethacin on the 47
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in the outer retina of the EIU model mice (Sasaki et al., 2009). These results suggest that P2X7 receptors expressed in microglial cells and macrophages are not involved in the damage of the outer retina in EIU model mice. P2X7 receptors have recently been reported to be involved in oxidative stress in the outer retina of Sod1 knockout mice (Carver et al., 2017). We investigated whether the 8-OHdG-positive cells, which were one of markers of the cells injured by oxidative stress, were found in the retina of the mice injected LPS. We could find few 8-OHdG-positive cells in the retina of EIU model mice (data not shown), suggesting that oxidative stress was not a major cause of the photoreceptor damage in EIU model mice. In the current study, we demonstrated that P2X7 receptors were expressed strongly in retinal pigment epithelium and relatively weakly in photoreceptor inner segments (Fig. 2E). Previous reports have shown that BzATP, a P2X7 agonist, induced photoreceptor cell death (Notomi et al., 2011) and death of the cultured RPE (Yang et al., 2011). We propose that P2X7 receptor activation in photoreceptor cells and RPE may cause photoreceptor cell damage in the EIU model mice. Although the source of the endogenous agonists of P2X7 receptors, such as ATP, in the EIU model remain unknown, BBG showed significant protective effects in the present study. These current results are consistent with those of the previous reports (Notomi et al., 2011; Yang et al., 2011). BBG has also been shown to have a neuroprotective effect on RGC damage induced by optic nerve crash (Kakurai et al., 2013) and intravitreal NMDA injection in vivo (Sakamoto et al., 2015a). In the present study, BBG evoked better functional protection in cone photoreceptors than in rod photoreceptors (Fig. 1), which is consistent with the protection of PNA-positive cells in the whole mount retinas. These results suggest that contribution of P2X7 receptors in the LPS-induced degeneration may be higher in cone photoreceptors than in rod photoreceptors. Unfortunately, we could not determine the difference in the expression of P2X7 receptors between rod and cone photoreceptors because the number of cone photoreceptors in a retinal horizontal section is too small to distinguish these two photoreceptor cell types. Further studies are clearly needed to clarify the underlying mechanism which causes better protective effects of BBG in cone photoreceptors. Bacterial infection in other tissues and sepsis rarely causes endogenous endophthalmitis (LaMattina, 2017). Although the patients are subjected to intravitreal and intravenous antibiotics and/or vitrectomy for treatment, their vision prognosis is often poor (LaMattina, 2017). The results in the present study suggest that BBG is a preventive drug for visual loss induced by endophthalmitis. Recently, BBG has been used in macular surgery for staining the inner limiting membrane to peel it (Almony et al., 2012). BBG has been reported to a better safety profiles than indocyanine green and trypan blue (Lüke et al., 2008; Baba et al., 2012). The cytoprotective effects found in the present and previous studies may reflect the superiority of BBG in inner limiting membrane peeling. In conclusion, we proposed that activation of P2X7 receptors at least in part cause injury of the outer retina in EIU model mice. The current study shows that blockade of P2X7 receptor is a good candidate for protection against outer retinal degeneration induced by inflammation and implies superiority of BBG in inner limiting membrane staining for macular surgery.
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