June 2008
Volume 49, Issue 6
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Retina  |   June 2008
Neuroprotective Effects of Naloxone against Light-Induced Photoreceptor Degeneration through Inhibiting Retinal Microglial Activation
Author Affiliations
  • Ying-qin Ni
    From the Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, People’s Republic of China; and the
  • Ge-zhi Xu
    From the Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, People’s Republic of China; and the
  • Wen-zheng Hu
    Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana.
  • Le Shi
    From the Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, People’s Republic of China; and the
  • Yao-wu Qin
    From the Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, People’s Republic of China; and the
  • Cui-di Da
    From the Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, People’s Republic of China; and the
Investigative Ophthalmology & Visual Science June 2008, Vol.49, 2589-2598. doi:10.1167/iovs.07-1173
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      Ying-qin Ni, Ge-zhi Xu, Wen-zheng Hu, Le Shi, Yao-wu Qin, Cui-di Da; Neuroprotective Effects of Naloxone against Light-Induced Photoreceptor Degeneration through Inhibiting Retinal Microglial Activation. Invest. Ophthalmol. Vis. Sci. 2008;49(6):2589-2598. doi: 10.1167/iovs.07-1173.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine the role of microglial activation in light-induced photoreceptor degeneration and the neuroprotective effects of naloxone as a novel microglial inhibitor.

methods. Sprague–Dawley rats were exposed to intense blue light for 24 hours. Daily intraperitoneal injection of naloxone or PBS as a control was given 2 days before exposure to light and was continued for 2 weeks. Apoptotic cells were detected by the TUNEL assay, and anti-OX42 antibody was used to label retinal microglia. Western blot was applied to evaluate the retinal interleukin (IL)-1β protein levels. Retinal histologic examination and electroretinography (ERG) were also performed to evaluate the effects of naloxone on light-induced photoreceptor degeneration.

results. TUNEL-positive cells were noted in the outer nuclear layer (ONL) of the retina as early as 2 hours and peaked at 24 hours after exposure to light. OX42-positive microglia occurred in the ONL and subretinal space at 6 hours, peaked at 3 days, and changed morphologically from the resting ramified to the activated amoeboid. Expression of IL-1β protein was also significantly increased at 3 days. Compared with the control, the number of microglia in the outer retina was significantly decreased in the naloxone-treated group at 3 days, and the thickness of ONL and the amplitudes of dark-adapted a- and b-waves were also well preserved at 14 days.

conclusions. The activation and migration of microglia and the expression of neurotoxic factor (IL-1β) coincide with photoreceptor apoptosis, suggesting that activated microglia play a major role in light-induced photoreceptor degeneration. Inhibiting microglial activation by naloxone significantly reduces this degeneration.

Microglia are considered the major resident immune cells in the central nervous system (CNS). In the mature brain and under physiological conditions, resting microglia have a characteristic ramified morphologic appearance and serve the roles of immune surveillance and host defense. 1 2 3 Microglial cells are particularly sensitive to changes in their microenvironment and are quickly activated in response to various insults. 4 5 6 7 8 Activated microglia undergo dramatic morphologic changes from resting ramified to activated amoeboid and secrete a host of proinflammatory and neurotoxic factors, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, which contribute to neurodegeneration. 9 10 11 In contrast to a tremendous amount of research on the brain, little is known about the role of microglia in retinal photoreceptor degeneration. In photic injury, microglia migrate from the inner retinal layers to the outer nuclear layer (ONL) and the subretinal space. 12 13 Similar microglial migration was found in retinal dystrophic Royal College of Surgeons (RCS) rats. 14 15 16 Zhang et al. 17 demonstrated a significantly increased level of monocyte chemotactic protein (MCP)-3 preceding the infiltration of retinal microglia into the outer retina in photic injury. However, the relationship between microglial activation and photoreceptor apoptosis and the possible role of microglia in retinal photoreceptor degeneration remains unclear. 
The important roles of microglial activation in neurodegeneration and potentially in the pathogenesis of Parkinson disease have prompted brain researchers to speculate that the inhibition of microglial activation might be neuroprotective. Microglia-targeted pharmacotherapies, such as protein kinase C (PKC) inhibitors, 18 19 macrophage/microglia inhibiting factor (MIF), 20 21 and extracts of the medicinal herb Scutellaria baicalensis, 22 were reported to inhibit the activation of microglia and to promote neuronal survival in vivo. However, the inability of these drugs to penetrate the blood-brain barrier and the possible side effects may limit their long-term clinical use. Liu et al. 23 24 recently discovered that naloxone, a classic opioid receptor antagonist, was able to attenuate LPS-induced microglial activation and the degeneration of dopaminergic neurons in vitro and in vivo, suggesting that naloxone might also be neuroprotective in light-induced photoreceptor degeneration. Compared with other microglial inhibitors, naloxone has several advantages, including its ability to penetrate the CNS, and fewer side effects, presenting a new potential therapeutic option for light-induced photoreceptor degeneration. 25  
The purpose of the present study was to evaluate the possible role of microglial activation in light-induced photoreceptor apoptosis and to explore the use of naloxone as a novel microglial inhibitor for neuroprotection against photoreceptor degeneration. The study design was focused on the relationships among photoreceptor apoptosis, retinal microglial activation, infiltration into the outer retina, and upregulation of the proapoptotic factor IL-1β. The inhibitory effects of naloxone on the activation of microglia were also examined in an animal model of light-induced photoreceptor degeneration. 
Materials and Methods
Animals
All procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Adult male Sprague–Dawley rats, each weighing 225 to 250 g, were housed in a 12-hour light/12-hour dark cycle. Commercial rat diet and water were provided ad libitum. Animals were anesthetized by intramuscular injection of ketamine (120 mg/kg) and xylazine (6 mg/ kg) during the examination and were humanely killed by overdose of pentobarbitone at the end of the experiment. 
Light Exposure
It is well known that excessive exposure to light induces photoreceptor apoptosis in albino rats 26 27 28 and mice. 29 30 31 32 Therefore, these animals are used as a classic photoreceptor degeneration model. After 24-hour dark adaptation, 160 rats were randomly divided into several groups and placed separately in cages, allowing both eyes to be exposed to evenly distributed, bright blue light. The floor of each cage was illuminated by approximately 2500 lux. The temperature inside each cage was maintained at 24°C. Rats were placed under these conditions at the same time of day and were returned to their normal light/dark cycle after 24 hours of intense blue light exposure. 
Treatment with Naloxone
Naloxone (Sigma-Aldrich, St. Louis, MO) was freshly prepared at a concentration of 1 mg/mL with 0.1 M phosphate-buffered saline (PBS). Seventy-eight rats received daily intraperitoneal injections of naloxone at dosages of 0.25, 0.5, 0.75, or 1 mg/d, respectively, from 2 days before to 14 days after light exposure. Control rats received the same volume (1 mL) injection of 0.1 M PBS. Dosage levels were chosen according to a previous study by Liu et al., 23 which demonstrated a dose-dependent neuroprotective effect of naloxone in an animal model of Parkinson disease, with a maximum effect at 1 mg/d. 
Histopathology and Immunohistochemistry
Anesthetized rats were transcardially perfused with 200 mL of 0.9% saline, followed by 4% paraformaldehyde (PFA) solution in 0.1 M PBS (pH 7.4). The eyes were then enucleated and immersion fixed in 4% PFA for 1 hour, transferred to 10% neutral-buffered formalin overnight, and processed for routine paraffin-embedded sectioning using an automated tissue processor (Shandon Pathcenter; Thermo Shandon Inc., Pittsburgh, PA). The eyes were embedded sagittally, and 5-μm serial sections were cut with a rotary microtome (Microm HM 330; McBain Instruments, Chatsworth, CA) and were stained with hematoxylin and eosin. All eyes were cut vertically, and only sections through optic nerves were collected for subsequent studies. ONL thickness was measured at 18 locations of the retinal sections (superior quadrant, S1-S9; inferior quadrant, I1-I9; Fig. 1A ) starting from either side of the optic nerve, with each segment at 0.5 mm apart using an image analysis system (Qwin QG2–32; Leica Microsystems, Bensheim, Germany). 
For immunohistochemical studies of retinal sections, eyecups were fixed in 4% PFA for 2 hours after removal of the cornea and lens. The eyecups were cryoprotected in graded sucrose solutions (20%–30% in PBS) at 4°C, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Ted Pella, Inc., Redding, CA), and frozen in liquid nitrogen. Ten-micrometer sections were cut using a cryostat (Bright Instruments Ltd., Huntingdon, UK). Sections within approximately 1 mm of the optic nerve head were recovered onto gelatin-coated slides. Air-dried slides were incubated with blocking buffer (PBS containing 5% goat serum) at room temperature for 1 hour. After three washes with 0.01 M PBS, the sections were incubated with the primary antibody (mouse anti-OX42, 1:100 [Chemicon, Temecula, CA]; mouse anti-ED1, 1:100 [Serotec, Oxford, UK]) overnight at 4°C. The sections were then rinsed three times with 0.01 M PBS and incubated with the secondary antibody (anti-mouse IgG-CY3 conjugate, 1:200; Sigma-Aldrich) at 37°C for 45 minutes. For immunostaining of retinal whole mounts, retinas were dissected out after 2-hour fixation in 4% PFA. Retinas were permeabilized in 0.3% Triton X-100 in PBS for 1 hour and were washed with PBS three times. They were then incubated with primary antibody (mouse anti-OX42, 1:100; Chemicon) and were rinsed thoroughly with PBS before application of the secondary antibodies (anti-mouse IgG-CY3 conjugate, 1:200; Sigma-Aldrich). After three rinses with 0.01 M PBS, the sections or retinal whole mounts were mounted with antifade mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and examined under a confocal laser microscope (TCS SP2; Leica Microsystems). 
TUNEL Staining
TUNEL staining was performed with an in situ cell death detection kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. TUNEL-positive cells were counted in the entire area of each retinal section. Retinal histology and TUNEL staining were examined under a light microscope (DM IRB; Leica Microsystems) and analyzed with Leica software (Qwin QG2-32). 
Electroretinography
Electroretinograms (ERGs) were recorded 14 days after exposure to light (UTAS-E3000 Visual Electrodiagnose System; LKC Technologies, Gaithersburg, MD). Scotopic flash ERGs were recorded from dark-adapted rats by placing a golden-ring electrode in contact with the cornea and a reference electrode through the tongue. A grounding electrode was attached to the scruff of the neck. The pupils were dilated with 1% tropicamide and 2.5% phenylephrine, and the corneas were kept moist with the application of 1% carboxymethylcellulose as needed. All procedures were performed in dim red light, and the rats were kept warm during the entire procedure. Ten responses to a 2500 cd/m2 white light flash (10 μs, 0.1 Hz) from a Ganzfeld integrating sphere were amplified and averaged (1902 Signal Conditioner/1401 Laboratory Interface; CED, Cambridge, UK). The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave, and the a-wave was measured as the difference in amplitude between the recording at 5 ms and the trough of the negative deflection. Baseline recordings were taken at least 7 days before treatment. 
Western Blot Analysis
Retinas were homogenized in RIPA buffer containing 1% Triton X-100, 5% SDS, 5% deoxycholic acid, 0.5 M Tris HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 × 10−12 μg/ml aprotinin, 1 × 10−12 μg/ml leupeptin, 1 × 10−12 μg/ml pepstatin, 200 mM sodium orthovanadate, and 200 mM sodium fluoride. Tissue extracts were incubated on ice for 10 minutes and centrifuged at 10,000g for 25 minutes at 4°C. Total protein in retinal extracts was measured using a standard BCA assay (Pierce, Rockford, IL), and the protein concentration was determined by the Lowry method (Bio-Rad Life Science, Mississauga, ON, Canada). Retinal extracts were resuspended in 5× sample buffer (60 mM Tris HCl, pH 7.4, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, 0.1% bromophenol blue), boiled for 5 minutes, and resolved on a 12% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose membrane (Hybond-C; GE Healthcare, Little Chalfont, Buckinghamshire, UK), and blots were stained (Ponceau S; Sigma-Aldrich) to visualize the protein bands and to ensure equal protein loading and uniform transfer. The blots were then washed with Tween-Tris-buffered saline solution and were blocked with 5% nonfat dry milk in TBST buffer (20 mM Tris HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20) for 45 minutes. The blots were probed with the primary antibody against IL-1β (1:1000 dilution; Sigma-Aldrich) for 24 hours, followed by the horseradish-peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:10,000 dilution). Bound antibodies were detected using an enhanced chemiluminescence system (ECL; GE Healthcare) and x-ray film. Signal intensity was measured (ImageMasterRVDS; GE Healthcare), and the optical densities (mean ± SD) for each sample were obtained from three measurements on three separate blots. 
Data Analysis
Data on the mean ONL thickness of the entire retina and from each defined location across the retina, mean numbers of TUNEL-positive cells in the ONL, and OX42-positive cells in the outer retina from various groups were compared with one-way ANOVA or Student t-test. Amplitudes of dark-adapted ERG a- and b-waves were compared by two-sample Wilcoxon rank-sum test. 
Results
Light-Induced Photoreceptor Degeneration
Albino rats exposed to intense blue light showed swelling and disorganization of the inner and outer segments of the photoreceptors 24 hours after exposure to light, with some pyknotic nuclei scattered in the ONL and subretinal space (Fig. 1B) . Significant photoreceptor cell loss was seen in the experimental group 3 days after exposure to light, combined with progressive thinning of the ONL (56.8% reduction), compared with the normal rats (Fig. 1C ; 14.77 ± 1.21 μm vs. 34.21 ± 0.99 μm; P < 0.05). There was regional susceptibility, with the superior S3 region (as illustrated in Fig. 1A ) exhibiting the most severe damage. In this region, the photoreceptors almost completely disappeared with only one row of cells remained 14 days after photic injury (Fig. 1D)
The TUNEL assay, a common method for detecting the DNA fragmentation that results from apoptotic signaling cascades, was used to explore the process of cell death in our model. Positive staining was rarely seen in normal control retinas (Fig. 2A) ; however, TUNEL-positive cells were seen in the ONL as early as 2 hours after exposure to light and peaked at 1 day (Fig. 2B) , indicating remarkable apoptosis of photoreceptors from direct photic injury. The absolute number of TUNEL-positive cells gradually decreased at 3 days and 7 days (Figs. 2C 2D) , combined with significant thinning of the ONL. 
Activation and Migration of Retinal Microglia in Photic Injury
The OX42 antibody was used to identify retinal microglia. In normal retinal sections, a few OX42-positive microglia with thin cellular processes were seen in the ganglion cell layer (GCL) and the inner plexiform layer (IPL) but not in the ONL or subretinal space (Fig. 3A) . At 6 hours after exposure to light, a small but increased number of OX42-positive microglia appeared in the ONL and subretinal space (Fig. 3B) , and the number of OX42-positive cells increased rapidly and peaked at 3 days (Fig. 3C) . Immunofluorescent staining in retinal whole mounts clearly revealed that retinal microglia underwent dramatic morphologic changes from resting ramified (Fig. 3D)to activated amoeboid 3 days after exposure to light (Fig. 3E) . Coexistence of the two phenotypes was observed 7 days after exposure to light (Fig. 3F)
Additionally, the ED1 antibody was used to label possible macrophage/microglia infiltrating the outer retina. As shown in Figure 4B , only a few scattered ED1-positive cells were found in the ONL of the superior region 7 days after exposure to light. However, no such cells were observed in the normal retina (Fig. 4A)or in samples from 1 day and 3 days after exposure to light (data not shown). The number of ED1-positive cells (3.25 ± 2.22) in the ONL was significantly less than OX42-positive cells (28.08 ± 4.01) at 7 days (P < 0.01). The relatively late appearance, the position, and the amount of ED1-positive cells suggested that these cells might be infiltrating macrophages. 
Upregulation of IL-1β Protein after Photic Injury
As a proapoptotic and cytotoxic factor, IL-1β is known to be secreted by glial cells, especially activated microglia. 33 34 We hypothesized that an increase in endogenous levels of IL-1β after photic injury plays an important role in the pathogenesis of photoreceptor degeneration by exacerbating photoreceptor cell death. Therefore, we examined the levels of retinal IL-1β at various times after exposure to light. Compared with the controls, retinal IL-1β protein levels in the light-exposed group were elevated by approximately twofold at 2 hours after exposure to light (P < 0.01), increased continuously, and peaked at 3 days elevated by approximately 50-fold (P < 0.01). The time-dependent fashion of IL-1β upregulation was consistent with that of microglial activation and migration following the apoptotic peak of the photoreceptors (Fig. 5)
Neuroprotective Effects of Naloxone on Photoreceptor Degeneration
Similar to morphologic findings in the untreated control animals, the thickness of the ONL was markedly reduced in the PBS-treated group 14 days after photic injury (Figs. 6A 6C) . In contrast, treatment with 1 mg/d naloxone provided significant protection of photoreceptors against photic injury in the superior and the inferior quadrants of the retina, especially in the posterior area (Figs. 6B 6D 7) . The extent of restoration of ONL thickness by naloxone was dose related, with the maximum effect seen in the 1-mg/d group (Fig. 8) . Morphometric analysis showed that the naloxone-treated group (1 mg/d) had significantly higher numbers of normal-appearing photoreceptor nuclei than the PBS-treated group (70.73% vs. 39.91%; P < 0.01). On the other hand, the same naloxone treatment showed no influence on the thickness of the ONL in normal rats (data not shown). 
The effects of naloxone (1 mg/d) treatment on light-induced photoreceptor degeneration were further examined by electrophysiologic analyses. The a- and b-wave amplitudes were expressed as percentages of the normal baseline value. An approximately 90% loss of ERG a- and b-waves was observed in the model control and PBS-treated groups at 14 days after 24-hour exposure to 2500-lux blue light compared with the baseline value obtained in normal rats 7 days before light exposure. However, the naloxone-treated group showed significantly less loss of a- and b-wave amplitudes when compared with the PBS-treated group (P < 0.05 and P < 0.01, respectively) (Fig. 9)
Retinas of naloxone-treated (1 mg/d) and PBS-treated rats were also evaluated with TUNEL assay for photoreceptor cell apoptosis at 1, 3, and 7 days after exposure to light. As stated, abundant TUNEL-positive cells were seen in the ONL of PBS-treated retinas at 1 day and 3 days (Fig. 10A) , and treatment with naloxone (1 mg/d) showed significantly fewer TUNEL-positive cells at 3 and 7 days but not at 1 day after light exposure (Figs. 10B 10C) , suggesting that naloxone may further reduce photoreceptor cell apoptosis after initial direct light-induced photoreceptor damage. 
Effects of Naloxone on Light-Induced Retinal Microglia Activation and IL-1β Protein Upregulation
Three days after exposure to light, the peak time of microglial migration, there were fewer OX42-positive amoeboid cells in the outer retinas in the 1 mg/d naloxone-treated group (86.33 ± 12.59) than in the PBS-treated group (138.33 ± 11.57; P < 0.01; Fig. 11 ). No significant difference was observed at 1 day and 7 days after exposure to light (Fig. 11C)
Western blot analysis showed that naloxone dose dependently (0.25–1.0 mg/d) inhibited IL-1β production, with a maximum effect in the 1-mg/d treated group at 1 day and 3 days after exposure to light (Fig. 12 ; P < 0.01). 
Time-related and dose-dependent effects of naloxone on retinal microglial activation and IL-1β production suggested that the neuroprotective effects of naloxone on photoreceptor degeneration possibly occurred through inhibition of the activation of microglia and the release of the toxic factor IL-1β, which, in turn, further reduced photoreceptor cell death. 
Discussion
In the course of this study, we sought to determine whether retinal microglia became activated after exposure to intense blue light, contributing to the progression of photoreceptor apoptosis. We also evaluated the efficacy of naloxone, a kind of microglial inhibitor, in ameliorating light-induced photoreceptor degeneration. First, we showed that retinal microglia became activated and migrated to the outer retina after exposure to light. Second, the peak of microglial migration followed the peak of photoreceptor apoptosis, in parallel with the elevation of retinal IL-1β protein levels. Finally, naloxone treatment decreased the infiltration of retinal microglia into the outer retina, downregulated the expression of IL-1β, and inhibited apoptosis, leading to an overall neuroprotective effect against light-induced photoreceptor degeneration. 
Microglia are unique glial cells in the CNS and represent approximately 5% to 20% of the total glial cell population. 7 35 In the normal adult CNS, microglia are found in a highly ramified resting state but are actively and sensitively surveying their environment for signs of even the most subtle changes, acting as so-called sensors of pathologic events of the CNS. 1 2 3 4 36 37 In pathologic situations, microglia can quickly migrate to the site where injured neurons send out some signals and try to remove the dying neurons. 3 5 38 39 On the other hand, the activated microglia can also secrete some cytotoxic factors, amplifying the pathologic cascade. 40 41 42 43 Several researchers have found that microglia in the eye migrate to the outer retina in the photic injury model and in RCS rats, 12 13 14 15 16 suggesting the possible involvement of activated microglia in photoreceptor degeneration. Our study further demonstrated this time-related migration of microglia in response to photoreceptor apoptosis. Furthermore, immunostaining of retinal whole mounts with the OX42 antibody, which recognizes the complement type 3 receptor (CR3), clearly demonstrated microglial morphologic changes after photic injury from the resting state (a structure conducive to surveillance of the microenvironment) to cells with shortened, hypertrophied processes or amoeboid form (conducive to phagocytosis) combined with the upregulated expression of their constitutive marker CR3. 7 44 45  
Activated microglia are known to be able to secrete a variety of proapoptotic factors, including IL-1β, TNF-α, and reactive oxygen species. These factors may be cytotoxic to neurons or may amplify the cascade of microglial activation, causing eventual degeneration of neurons through mechanisms that are not completely understood. 10 46 47 48 49 50 In rd mice, Zeng et al. 51 observed activation of microglia and increased expression of chemokines and microglia-derived toxic factor (TNF-α). Our results demonstrated that intense light exposure led to upregulation of the proapoptotic cytokine IL-1β in a time-related manner, in parallel with microglial activation and migration after the initial photoreceptor apoptosis. The most likely source of IL-1β is the activated microglia; however, other retinal cells, such as Müller cells, may also release neurotoxic cytokines in response to injury. 52 On their release, these cytokines can aggravate the apoptotic progression of photoreceptors. 
Given that microglial activation and neurotoxic factor release underlie the progression of neurodegeneration, inhibiting microglial activation would be neuroprotective. In a number of neuronal systems, researchers have found that treatments that reduce microglial activation can increase neuronal cell survival, including neuroprotective effects of minocycline on photoreceptor degeneration. 20 36 53 54 55 Naloxone is a nonselective antagonist of the G-protein–linked classic opioid receptors, which are widely expressed on cells in the CNS. It has been reported that naloxone can reduce LPS-induced microglial activation and production of IL-1β and TNF-α in vitro, leading to the protection of cultured cortical neurons in the cortical mixed glia cultures and dopaminergic neurons in an acute model of Parkinson disease. 23 24 The neuroprotective effects of naloxone appear to be unrelated to the opioid system because this ineffective opioid receptor antagonist, (+)-naloxone, is also equally effective in the attenuation of LPS-induced nigral dopaminergic neurodegeneration. 23 24 56 57 Furthermore, the effects of naloxone isomers are not limited to LPS-induced neurodegeneration. The degeneration of cortical or mesencephalic neurons in neuron-glia cultures induced by treatment with Aβ (1–42) is also significantly reduced by naloxone. 58 The potential mechanism is attributed to the inhibition of the production of superoxide in Aβ (1–42)–activated microglia. 58  
In this regard, it was our interest to determine whether naloxone has potential neuroprotective effects on light-induced photoreceptor degeneration. Our results first showed that naloxone reduced the infiltration of microglia into the outer retina and the expression of proapoptotic factor IL-1β and that it remarkably ameliorated the loss of photoreceptors from intense light exposure. 
Of note, we could not completely rule out the possibility that naloxone treatment had a direct neuroprotective effect on photoreceptor degeneration, leading to the reduction of infiltrating microglia. A comparison of the number of the TUNEL-positive cells in the ONL between the naloxone-treated and PBS-treated animals demonstrated that naloxone reduced the number of TUNEL-positive cells at 3 days and 7 days but not at 1 day after exposure to light. This indicated that naloxone probably protected against further degeneration of photoreceptors at 3 to 7 days by inhibiting the activation of microglia and the production of neurotoxic IL-1β. 
Despite intensive studies, the histogenic origin of microglia and macrophages in the CNS is still a matter of debate. Unlike macroglia (astrocytes and Müller cells) and neurons, which originate from neuroectoderm, microglia share a common bone marrow origin with blood-borne monocytes. 2 59 60 61 Depending on the state of microglial activation, some markers—such as ED-1, -2, and -3, phosphotyrosine, and isolectin B4—can be used to distinguish resting microglia from macrophages. 15 62 However, no such markers can distinguish the activated microglia from macrophages. 63 In a study of photic injury, Moore and Thanos 36 used a retrolabeling technique to demonstrate that selectively labeled microglia from the inner retina migrated to the ONL. Using 5D4 antibody, Ng and Streilein 64 showed that the retina itself (the inner layer) was the predominant source of the 5D4-positive cells found in the subretinal space after photic injury, and most of those cells were microglia. However, several other studies suggest that blood-borne macrophages may be involved in retinal degeneration. 16 In our study, we found that some OX42-positive microglia extended their processes to the ONL, with the body still located in the OPL at the initial stage, and most of the infiltrated microglia first appeared along the inner part of the ONL, suggesting that they might have migrated from the inner retina. However, the possibility of invasion of circulating macrophages cannot be excluded because a few ED1-positive cells that could not be found at either 1 day or 3 days after exposure to light were observed in the superior region at 7 days. Therefore, we speculate that most of these migrating cells are retinal microglia, especially at early stages after photic injury, and the blood-borne macrophages may enter at later stages, after severe disruption of the blood-retina barrier. 
In conclusion, our results indicated that, in the photic injury model, the retinal microglia became activated and migrated to the outer retina along with the upregulation of proapoptotic factor IL-1β, suggesting its potentially important role in photoreceptor apoptosis. Inhibiting microglial activation by naloxone had significant neuroprotective effects on photoreceptor degeneration. 
 
Figure 1.
 
Photomicrographs of rat retinas in the light-induced photoreceptor degeneration model with hematoxylin-eosin staining. (A) Schematic diagram of the retinal vertical sections through the optic nerve. S1–S9 indicates nine measurements in the superior quadrant every 0.5 mm. I1–I9 indicates nine measurements in the inferior quadrant every 0.5 mm. All the photographs were taken in the S3 region, which was most susceptible to intense light exposure. (B) One day after exposure to light, showing disorganization of the outer segment of photoreceptors (black arrow). (C) Three days after exposure to light, showing remarkable thinning of the ONL (black arrow). (D) Fourteen days after exposure to light, showing only one to two rows of photoreceptors remaining (black arrow).
Figure 1.
 
Photomicrographs of rat retinas in the light-induced photoreceptor degeneration model with hematoxylin-eosin staining. (A) Schematic diagram of the retinal vertical sections through the optic nerve. S1–S9 indicates nine measurements in the superior quadrant every 0.5 mm. I1–I9 indicates nine measurements in the inferior quadrant every 0.5 mm. All the photographs were taken in the S3 region, which was most susceptible to intense light exposure. (B) One day after exposure to light, showing disorganization of the outer segment of photoreceptors (black arrow). (C) Three days after exposure to light, showing remarkable thinning of the ONL (black arrow). (D) Fourteen days after exposure to light, showing only one to two rows of photoreceptors remaining (black arrow).
Figure 2.
 
Representative examples of the photoreceptor cell death in the S3 region, as visualized with the TUNEL assay. (A) No TUNEL-positive cells were seen in the retinas of normal rats. (B) One day after exposure to light, there was a large number of TUNEL-positive cells in the ONL (black arrow). (C) TUNEL-positive cells were less abundant in the ONL at 3 days after exposure to light and dramatically decreased at 7 days (D). The thickness of the ONL was significantly reduced at 3 and 7 days after exposure to light.
Figure 2.
 
Representative examples of the photoreceptor cell death in the S3 region, as visualized with the TUNEL assay. (A) No TUNEL-positive cells were seen in the retinas of normal rats. (B) One day after exposure to light, there was a large number of TUNEL-positive cells in the ONL (black arrow). (C) TUNEL-positive cells were less abundant in the ONL at 3 days after exposure to light and dramatically decreased at 7 days (D). The thickness of the ONL was significantly reduced at 3 and 7 days after exposure to light.
Figure 3.
 
Immunolabeling of retinal microglia with the OX42 antibody. Arrowheads indicate the microglia in all panels. (A) In normal retinas, OX42-positive microglia were seen only in the GCL and IPL. (B) At 6 hours after exposure to light, a small number of microglia began to appear in the ONL and subretinal space. Microglia migrating to the outer retina increased at 1 day and peaked at 3 days after exposure to light. (C) Immunolabeling in the superior area of the retinal whole mounts showed that retinal microglia underwent significant morphologic changes from the normal resting ramified, with long, slim processes (D), to the activated amoeboid at 3 days after exposure to light (E). (F) The two phenotypes of the microglia were found to coexist at 7 days after exposure to light.
Figure 3.
 
Immunolabeling of retinal microglia with the OX42 antibody. Arrowheads indicate the microglia in all panels. (A) In normal retinas, OX42-positive microglia were seen only in the GCL and IPL. (B) At 6 hours after exposure to light, a small number of microglia began to appear in the ONL and subretinal space. Microglia migrating to the outer retina increased at 1 day and peaked at 3 days after exposure to light. (C) Immunolabeling in the superior area of the retinal whole mounts showed that retinal microglia underwent significant morphologic changes from the normal resting ramified, with long, slim processes (D), to the activated amoeboid at 3 days after exposure to light (E). (F) The two phenotypes of the microglia were found to coexist at 7 days after exposure to light.
Figure 4.
 
Immunolabeling of retinal macrophage/microglia with the ED1 antibody. (A) No ED1-positive cells were detected in normal retinas. (B) Only a few ED1-positive cells with oval shapes were found in the thinning ONL of the S2 region at 7 days after exposure to light.
Figure 4.
 
Immunolabeling of retinal macrophage/microglia with the ED1 antibody. (A) No ED1-positive cells were detected in normal retinas. (B) Only a few ED1-positive cells with oval shapes were found in the thinning ONL of the S2 region at 7 days after exposure to light.
Figure 5.
 
Changes in IL-1β protein expression (as measured by Western blot; three individual samples per time point) after intense light exposure. Retinal IL-1β protein expression began to increase at 6 hours after exposure to light and peaked at 3 days, which was concomitant with the peak of microglia activation and migration. β-actin was used as the loading control.
Figure 5.
 
Changes in IL-1β protein expression (as measured by Western blot; three individual samples per time point) after intense light exposure. Retinal IL-1β protein expression began to increase at 6 hours after exposure to light and peaked at 3 days, which was concomitant with the peak of microglia activation and migration. β-actin was used as the loading control.
Figure 6.
 
Photomicrographs of the retinas in the PBS-treated and naloxone-treated groups at 14 days after exposure to light. In the superior S3 region, the PBS-treated group (A) showed significant thinning of the ONL with only one row of cells remaining (arrow), whereas ONL thickness was better preserved in the naloxone-treated group (B). In the inferior I3 region, the ONL in the naloxone-treated group (D) also showed better preservation than that in the PBS-treated group (C).
Figure 6.
 
Photomicrographs of the retinas in the PBS-treated and naloxone-treated groups at 14 days after exposure to light. In the superior S3 region, the PBS-treated group (A) showed significant thinning of the ONL with only one row of cells remaining (arrow), whereas ONL thickness was better preserved in the naloxone-treated group (B). In the inferior I3 region, the ONL in the naloxone-treated group (D) also showed better preservation than that in the PBS-treated group (C).
Figure 7.
 
Measurements of the ONL thickness in the superior and inferior quadrants of the retina at 14 days after exposure to light. The photoreceptors were most severely damaged in the superior and posterior regions in the PBS-treated group. Naloxone treatment showed significant preservation of ONL thickness in both the superior and the inferior retina. Student’s t-test for two groups, (mean ± SD; n = 12). *P < 0.05; **P < 0.01.
Figure 7.
 
Measurements of the ONL thickness in the superior and inferior quadrants of the retina at 14 days after exposure to light. The photoreceptors were most severely damaged in the superior and posterior regions in the PBS-treated group. Naloxone treatment showed significant preservation of ONL thickness in both the superior and the inferior retina. Student’s t-test for two groups, (mean ± SD; n = 12). *P < 0.05; **P < 0.01.
Figure 8.
 
Neuroprotective effects of naloxone on light-induced photoreceptor degeneration 14 days after exposure to light were dose related. Mean ONL thickness in the three groups receiving naloxone injection (0.5, 0.75, 1 mg/d) was significantly increased compared with that in the PBS-treated group, respectively, and 1 mg/d showed the maximum effect. One-way ANOVA (mean ± SD; n = 6). **P < 0.01.
Figure 8.
 
Neuroprotective effects of naloxone on light-induced photoreceptor degeneration 14 days after exposure to light were dose related. Mean ONL thickness in the three groups receiving naloxone injection (0.5, 0.75, 1 mg/d) was significantly increased compared with that in the PBS-treated group, respectively, and 1 mg/d showed the maximum effect. One-way ANOVA (mean ± SD; n = 6). **P < 0.01.
Figure 9.
 
Representative dark-adapted photic ERGs recorded in (A) normal rats 7 days before light exposure (baseline), (B) normal rats as model control, (C) naloxone-treated group, and (D) PBS-treated group at 14 days after 24-hour light exposure. Both a- and b-waves were reduced in amplitude after exposure to intense light. However, the naloxone-treated group exhibited better preservation of the wave forms than did the control and PBS-treated groups. (E) Relative preservation of the a-wave and b-wave amplitudes against light-induced photoreceptor degeneration in the naloxone-treated group at 14 days after exposure to light. Baseline recordings were taken 7 days before light exposure. Amplitudes were expressed as the mean ratio between each record after treatment and its own baseline. Wilcoxon rank sum test for two groups. Median and interquartile range (n = 12). *P < 0.05; **P < 0.01.
Figure 9.
 
Representative dark-adapted photic ERGs recorded in (A) normal rats 7 days before light exposure (baseline), (B) normal rats as model control, (C) naloxone-treated group, and (D) PBS-treated group at 14 days after 24-hour light exposure. Both a- and b-waves were reduced in amplitude after exposure to intense light. However, the naloxone-treated group exhibited better preservation of the wave forms than did the control and PBS-treated groups. (E) Relative preservation of the a-wave and b-wave amplitudes against light-induced photoreceptor degeneration in the naloxone-treated group at 14 days after exposure to light. Baseline recordings were taken 7 days before light exposure. Amplitudes were expressed as the mean ratio between each record after treatment and its own baseline. Wilcoxon rank sum test for two groups. Median and interquartile range (n = 12). *P < 0.05; **P < 0.01.
Figure 10.
 
Effects of naloxone on light-induced photoreceptor death as measured by TUNEL assay. In the corresponding S3 region, TUNEL-positive cells were most abundant in the ONL in the PBS-treated group at 3 days after exposure to light (A) but were less abundant in the 1 mg/d naloxone-treated group (B). (C) Statistical analysis showed that the number of TUNEL-positive cells was significantly lower in the naloxone-treated than in the PBS-treated group at 3 days and 7 days after exposure to light. Student’s t-test for two groups. Mean ± SD (n = 6). **P < 0.01.
Figure 10.
 
Effects of naloxone on light-induced photoreceptor death as measured by TUNEL assay. In the corresponding S3 region, TUNEL-positive cells were most abundant in the ONL in the PBS-treated group at 3 days after exposure to light (A) but were less abundant in the 1 mg/d naloxone-treated group (B). (C) Statistical analysis showed that the number of TUNEL-positive cells was significantly lower in the naloxone-treated than in the PBS-treated group at 3 days and 7 days after exposure to light. Student’s t-test for two groups. Mean ± SD (n = 6). **P < 0.01.
Figure 11.
 
OX42 immunolabeling of retinal microglia at 3 days after exposure to light from the corresponding S3 region. (A) In the PBS-treated group, massive infiltration of amoeboid OX42-positive microglia were noted in the ONL and subretinal space (arrows). (B) However, fewer OX42-positive microglia were seen in the ONL of the naloxone-treated group (arrows). (C) Statistical analysis showed that significantly fewer microglial cells infiltrated the outer retina in the naloxone-treated group than in the PBS-treated at 3 days. No difference was found at 1 day and 7 days. Student’s t-test for two groups. Mean ± SD (n = 6). **P < 0.01.
Figure 11.
 
OX42 immunolabeling of retinal microglia at 3 days after exposure to light from the corresponding S3 region. (A) In the PBS-treated group, massive infiltration of amoeboid OX42-positive microglia were noted in the ONL and subretinal space (arrows). (B) However, fewer OX42-positive microglia were seen in the ONL of the naloxone-treated group (arrows). (C) Statistical analysis showed that significantly fewer microglial cells infiltrated the outer retina in the naloxone-treated group than in the PBS-treated at 3 days. No difference was found at 1 day and 7 days. Student’s t-test for two groups. Mean ± SD (n = 6). **P < 0.01.
Figure 12.
 
Retinal IL-1β protein expression (as measured by Western blot, three individual blots per dose) in the naloxone-treated and the PBS-treated groups at 1 day and 3 days after exposure to light, respectively. IL-1β protein expression was lower in the 0.5 mg/d and the 1.0 mg/d naloxone-treated groups than in the PBS-treated groups at both time points. Effects of naloxone on the downregulation of retinal IL-1β protein were dose dependent, with 1 mg/d showing the maximum effect. β-actin was used as the loading control. One-way ANOVA. Mean ± SD. *P < 0.05; **P < 0.01.
Figure 12.
 
Retinal IL-1β protein expression (as measured by Western blot, three individual blots per dose) in the naloxone-treated and the PBS-treated groups at 1 day and 3 days after exposure to light, respectively. IL-1β protein expression was lower in the 0.5 mg/d and the 1.0 mg/d naloxone-treated groups than in the PBS-treated groups at both time points. Effects of naloxone on the downregulation of retinal IL-1β protein were dose dependent, with 1 mg/d showing the maximum effect. β-actin was used as the loading control. One-way ANOVA. Mean ± SD. *P < 0.05; **P < 0.01.
The authors thank Pei-quan Zhao and Xiao-ling Gu for their critical reading and revision of the manuscript and Yi-min Tian for excellent technical assistance with Western blot analyses. 
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Figure 1.
 
Photomicrographs of rat retinas in the light-induced photoreceptor degeneration model with hematoxylin-eosin staining. (A) Schematic diagram of the retinal vertical sections through the optic nerve. S1–S9 indicates nine measurements in the superior quadrant every 0.5 mm. I1–I9 indicates nine measurements in the inferior quadrant every 0.5 mm. All the photographs were taken in the S3 region, which was most susceptible to intense light exposure. (B) One day after exposure to light, showing disorganization of the outer segment of photoreceptors (black arrow). (C) Three days after exposure to light, showing remarkable thinning of the ONL (black arrow). (D) Fourteen days after exposure to light, showing only one to two rows of photoreceptors remaining (black arrow).
Figure 1.
 
Photomicrographs of rat retinas in the light-induced photoreceptor degeneration model with hematoxylin-eosin staining. (A) Schematic diagram of the retinal vertical sections through the optic nerve. S1–S9 indicates nine measurements in the superior quadrant every 0.5 mm. I1–I9 indicates nine measurements in the inferior quadrant every 0.5 mm. All the photographs were taken in the S3 region, which was most susceptible to intense light exposure. (B) One day after exposure to light, showing disorganization of the outer segment of photoreceptors (black arrow). (C) Three days after exposure to light, showing remarkable thinning of the ONL (black arrow). (D) Fourteen days after exposure to light, showing only one to two rows of photoreceptors remaining (black arrow).
Figure 2.
 
Representative examples of the photoreceptor cell death in the S3 region, as visualized with the TUNEL assay. (A) No TUNEL-positive cells were seen in the retinas of normal rats. (B) One day after exposure to light, there was a large number of TUNEL-positive cells in the ONL (black arrow). (C) TUNEL-positive cells were less abundant in the ONL at 3 days after exposure to light and dramatically decreased at 7 days (D). The thickness of the ONL was significantly reduced at 3 and 7 days after exposure to light.
Figure 2.
 
Representative examples of the photoreceptor cell death in the S3 region, as visualized with the TUNEL assay. (A) No TUNEL-positive cells were seen in the retinas of normal rats. (B) One day after exposure to light, there was a large number of TUNEL-positive cells in the ONL (black arrow). (C) TUNEL-positive cells were less abundant in the ONL at 3 days after exposure to light and dramatically decreased at 7 days (D). The thickness of the ONL was significantly reduced at 3 and 7 days after exposure to light.
Figure 3.
 
Immunolabeling of retinal microglia with the OX42 antibody. Arrowheads indicate the microglia in all panels. (A) In normal retinas, OX42-positive microglia were seen only in the GCL and IPL. (B) At 6 hours after exposure to light, a small number of microglia began to appear in the ONL and subretinal space. Microglia migrating to the outer retina increased at 1 day and peaked at 3 days after exposure to light. (C) Immunolabeling in the superior area of the retinal whole mounts showed that retinal microglia underwent significant morphologic changes from the normal resting ramified, with long, slim processes (D), to the activated amoeboid at 3 days after exposure to light (E). (F) The two phenotypes of the microglia were found to coexist at 7 days after exposure to light.
Figure 3.
 
Immunolabeling of retinal microglia with the OX42 antibody. Arrowheads indicate the microglia in all panels. (A) In normal retinas, OX42-positive microglia were seen only in the GCL and IPL. (B) At 6 hours after exposure to light, a small number of microglia began to appear in the ONL and subretinal space. Microglia migrating to the outer retina increased at 1 day and peaked at 3 days after exposure to light. (C) Immunolabeling in the superior area of the retinal whole mounts showed that retinal microglia underwent significant morphologic changes from the normal resting ramified, with long, slim processes (D), to the activated amoeboid at 3 days after exposure to light (E). (F) The two phenotypes of the microglia were found to coexist at 7 days after exposure to light.
Figure 4.
 
Immunolabeling of retinal macrophage/microglia with the ED1 antibody. (A) No ED1-positive cells were detected in normal retinas. (B) Only a few ED1-positive cells with oval shapes were found in the thinning ONL of the S2 region at 7 days after exposure to light.
Figure 4.
 
Immunolabeling of retinal macrophage/microglia with the ED1 antibody. (A) No ED1-positive cells were detected in normal retinas. (B) Only a few ED1-positive cells with oval shapes were found in the thinning ONL of the S2 region at 7 days after exposure to light.
Figure 5.
 
Changes in IL-1β protein expression (as measured by Western blot; three individual samples per time point) after intense light exposure. Retinal IL-1β protein expression began to increase at 6 hours after exposure to light and peaked at 3 days, which was concomitant with the peak of microglia activation and migration. β-actin was used as the loading control.
Figure 5.
 
Changes in IL-1β protein expression (as measured by Western blot; three individual samples per time point) after intense light exposure. Retinal IL-1β protein expression began to increase at 6 hours after exposure to light and peaked at 3 days, which was concomitant with the peak of microglia activation and migration. β-actin was used as the loading control.
Figure 6.
 
Photomicrographs of the retinas in the PBS-treated and naloxone-treated groups at 14 days after exposure to light. In the superior S3 region, the PBS-treated group (A) showed significant thinning of the ONL with only one row of cells remaining (arrow), whereas ONL thickness was better preserved in the naloxone-treated group (B). In the inferior I3 region, the ONL in the naloxone-treated group (D) also showed better preservation than that in the PBS-treated group (C).
Figure 6.
 
Photomicrographs of the retinas in the PBS-treated and naloxone-treated groups at 14 days after exposure to light. In the superior S3 region, the PBS-treated group (A) showed significant thinning of the ONL with only one row of cells remaining (arrow), whereas ONL thickness was better preserved in the naloxone-treated group (B). In the inferior I3 region, the ONL in the naloxone-treated group (D) also showed better preservation than that in the PBS-treated group (C).
Figure 7.
 
Measurements of the ONL thickness in the superior and inferior quadrants of the retina at 14 days after exposure to light. The photoreceptors were most severely damaged in the superior and posterior regions in the PBS-treated group. Naloxone treatment showed significant preservation of ONL thickness in both the superior and the inferior retina. Student’s t-test for two groups, (mean ± SD; n = 12). *P < 0.05; **P < 0.01.
Figure 7.
 
Measurements of the ONL thickness in the superior and inferior quadrants of the retina at 14 days after exposure to light. The photoreceptors were most severely damaged in the superior and posterior regions in the PBS-treated group. Naloxone treatment showed significant preservation of ONL thickness in both the superior and the inferior retina. Student’s t-test for two groups, (mean ± SD; n = 12). *P < 0.05; **P < 0.01.
Figure 8.
 
Neuroprotective effects of naloxone on light-induced photoreceptor degeneration 14 days after exposure to light were dose related. Mean ONL thickness in the three groups receiving naloxone injection (0.5, 0.75, 1 mg/d) was significantly increased compared with that in the PBS-treated group, respectively, and 1 mg/d showed the maximum effect. One-way ANOVA (mean ± SD; n = 6). **P < 0.01.
Figure 8.
 
Neuroprotective effects of naloxone on light-induced photoreceptor degeneration 14 days after exposure to light were dose related. Mean ONL thickness in the three groups receiving naloxone injection (0.5, 0.75, 1 mg/d) was significantly increased compared with that in the PBS-treated group, respectively, and 1 mg/d showed the maximum effect. One-way ANOVA (mean ± SD; n = 6). **P < 0.01.
Figure 9.
 
Representative dark-adapted photic ERGs recorded in (A) normal rats 7 days before light exposure (baseline), (B) normal rats as model control, (C) naloxone-treated group, and (D) PBS-treated group at 14 days after 24-hour light exposure. Both a- and b-waves were reduced in amplitude after exposure to intense light. However, the naloxone-treated group exhibited better preservation of the wave forms than did the control and PBS-treated groups. (E) Relative preservation of the a-wave and b-wave amplitudes against light-induced photoreceptor degeneration in the naloxone-treated group at 14 days after exposure to light. Baseline recordings were taken 7 days before light exposure. Amplitudes were expressed as the mean ratio between each record after treatment and its own baseline. Wilcoxon rank sum test for two groups. Median and interquartile range (n = 12). *P < 0.05; **P < 0.01.
Figure 9.
 
Representative dark-adapted photic ERGs recorded in (A) normal rats 7 days before light exposure (baseline), (B) normal rats as model control, (C) naloxone-treated group, and (D) PBS-treated group at 14 days after 24-hour light exposure. Both a- and b-waves were reduced in amplitude after exposure to intense light. However, the naloxone-treated group exhibited better preservation of the wave forms than did the control and PBS-treated groups. (E) Relative preservation of the a-wave and b-wave amplitudes against light-induced photoreceptor degeneration in the naloxone-treated group at 14 days after exposure to light. Baseline recordings were taken 7 days before light exposure. Amplitudes were expressed as the mean ratio between each record after treatment and its own baseline. Wilcoxon rank sum test for two groups. Median and interquartile range (n = 12). *P < 0.05; **P < 0.01.
Figure 10.
 
Effects of naloxone on light-induced photoreceptor death as measured by TUNEL assay. In the corresponding S3 region, TUNEL-positive cells were most abundant in the ONL in the PBS-treated group at 3 days after exposure to light (A) but were less abundant in the 1 mg/d naloxone-treated group (B). (C) Statistical analysis showed that the number of TUNEL-positive cells was significantly lower in the naloxone-treated than in the PBS-treated group at 3 days and 7 days after exposure to light. Student’s t-test for two groups. Mean ± SD (n = 6). **P < 0.01.
Figure 10.
 
Effects of naloxone on light-induced photoreceptor death as measured by TUNEL assay. In the corresponding S3 region, TUNEL-positive cells were most abundant in the ONL in the PBS-treated group at 3 days after exposure to light (A) but were less abundant in the 1 mg/d naloxone-treated group (B). (C) Statistical analysis showed that the number of TUNEL-positive cells was significantly lower in the naloxone-treated than in the PBS-treated group at 3 days and 7 days after exposure to light. Student’s t-test for two groups. Mean ± SD (n = 6). **P < 0.01.
Figure 11.
 
OX42 immunolabeling of retinal microglia at 3 days after exposure to light from the corresponding S3 region. (A) In the PBS-treated group, massive infiltration of amoeboid OX42-positive microglia were noted in the ONL and subretinal space (arrows). (B) However, fewer OX42-positive microglia were seen in the ONL of the naloxone-treated group (arrows). (C) Statistical analysis showed that significantly fewer microglial cells infiltrated the outer retina in the naloxone-treated group than in the PBS-treated at 3 days. No difference was found at 1 day and 7 days. Student’s t-test for two groups. Mean ± SD (n = 6). **P < 0.01.
Figure 11.
 
OX42 immunolabeling of retinal microglia at 3 days after exposure to light from the corresponding S3 region. (A) In the PBS-treated group, massive infiltration of amoeboid OX42-positive microglia were noted in the ONL and subretinal space (arrows). (B) However, fewer OX42-positive microglia were seen in the ONL of the naloxone-treated group (arrows). (C) Statistical analysis showed that significantly fewer microglial cells infiltrated the outer retina in the naloxone-treated group than in the PBS-treated at 3 days. No difference was found at 1 day and 7 days. Student’s t-test for two groups. Mean ± SD (n = 6). **P < 0.01.
Figure 12.
 
Retinal IL-1β protein expression (as measured by Western blot, three individual blots per dose) in the naloxone-treated and the PBS-treated groups at 1 day and 3 days after exposure to light, respectively. IL-1β protein expression was lower in the 0.5 mg/d and the 1.0 mg/d naloxone-treated groups than in the PBS-treated groups at both time points. Effects of naloxone on the downregulation of retinal IL-1β protein were dose dependent, with 1 mg/d showing the maximum effect. β-actin was used as the loading control. One-way ANOVA. Mean ± SD. *P < 0.05; **P < 0.01.
Figure 12.
 
Retinal IL-1β protein expression (as measured by Western blot, three individual blots per dose) in the naloxone-treated and the PBS-treated groups at 1 day and 3 days after exposure to light, respectively. IL-1β protein expression was lower in the 0.5 mg/d and the 1.0 mg/d naloxone-treated groups than in the PBS-treated groups at both time points. Effects of naloxone on the downregulation of retinal IL-1β protein were dose dependent, with 1 mg/d showing the maximum effect. β-actin was used as the loading control. One-way ANOVA. Mean ± SD. *P < 0.05; **P < 0.01.
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