March 2016
Volume 57, Issue 3
Open Access
Glaucoma  |   March 2016
The Effect of A2A Receptor Antagonist on Microglial Activation in Experimental Glaucoma
Author Affiliations & Notes
  • Xiaohong Liu
    Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, Shanghai, China
  • Ping Huang
    Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, Shanghai, China
  • Jing Wang
    Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, Shanghai, China
  • Zijian Yang
    Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, Shanghai, China
  • Shouyue Huang
    Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, Shanghai, China
  • Xunda Luo
    Department of Ophthalmology, Scheie Eye Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States
    Department of Pathology and Laboratory Medicine, Temple University Hospital, Philadelphia, Pennsylvania, United States
  • Jin Qi
    Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, Shanghai, China
  • Xi Shen
    Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, Shanghai, China
  • Yisheng Zhong
    Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, Shanghai, China
  • Correspondence: Yisheng Zhong, Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, Shanghai, China 200025; yszhong68@126.com
  • Xi Shen, Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, Shanghai, China 200025; carl_shen2005@126.com
  • Footnotes
     PH and XL contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science March 2016, Vol.57, 776-786. doi:10.1167/iovs.15-18024
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      Xiaohong Liu, Ping Huang, Jing Wang, Zijian Yang, Shouyue Huang, Xunda Luo, Jin Qi, Xi Shen, Yisheng Zhong; The Effect of A2A Receptor Antagonist on Microglial Activation in Experimental Glaucoma. Invest. Ophthalmol. Vis. Sci. 2016;57(3):776-786. doi: 10.1167/iovs.15-18024.

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

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Abstract

Purpose: We investigated the effect of A2A receptor (A2AR) antagonist on microglial activation and retinal ganglion cell (RGC) survival under chronic ocular hypertension (COH), and explored the relationship between microglial activation and RGC survival by means of in vitro and in vivo experiments.

Methods: An animal model of COH was induced in one eye of male Sprague-Dawley (SD) rats by ligation of three episcleral veins. The survival of RGCs and the activation of microglia under COH without or with intravitreous injection of A2AR antagonist ZM241385 were assessed by fluorescent labeling, real time PCR and Western blot. ELISA was used to measure the secretion of inflammatory mediators by microglia when glutamate and/or ZM241385 was added into the culture system.

Results: Compared to the baseline, RGC density 2 weeks after COH induction decreased at the central (2436 ± 143 cells/mm2 pre- and 2130 ± 148 cells/mm2 post-COH induction) and peripheral (2219 ± 140 cells/mm2 pre- and 1953 ± 142 cells/mm2 post-COH induction) retina. The microglia changed their ramified morphology to an amoeboid form with increase in TNF-α and IL-1β expression after COH. These changes, however, were ameliorated with intravitreous ZM241385 (RGC density only dropped to 2287 ± 135 cells/mm2). The upregulation of those proinflammatory cytokines secreted by microglia in vitro under high concentration of glutamate was downregulated when ZM241385 was added into the culture system.

Conclusions: A2AR antagonist ZM241385 could reduce the activation of microglia and downregulate the proinflammatory cytokines expression under the conditions of COH and high concentration of glutamate, which may be one of the mechanisms that protected RGCs in experimental glaucoma.

Glaucoma is a neurodegenerative disease that results in progressive retinal ganglion cell (RGC) death and subsequent visual field defects if poorly managed. Although elevated IOP is an established risk factor for glaucoma,1 microglial responses are found in the glaucomatous eyes, suggesting a primary role of inflammation in glaucoma.27 
As a guardian of the central nervous system (CNS), microglial cells help to maintain CNS integrity.8 In physiological conditions, microglia adopt a characteristic ramified morphology. A “spider effect” of microglial morphologic change has been noted in which microglia transform from highly ramified cells to spherical cells during activation.9 In pathologic conditions, overactivated microglia take on an ameboid form and contribute to neuronal damage by releasing harmful substances, such as inflammatory cytokines, nitric oxide (NO), reactive oxygen species (ROS), and proteinases.1013 
Microglia-mediated neuroinflammation has been associated with a broad spectrum of neurodegenerative disorders in CNS. Mediators released from microglia are associated with neuron degeneration in Alzheimer's disease (AD) and Parkinson's disease (PD).1417 Reduction of microglia-derived mediators counteracts neurodegeneration in the animal model of PD.18 Increased levels of inflammatory mediators, such as TNF-α, IL-1β, IL-6, IL-9, IL-10, IL-12, and NO are found in the retina and aqueous humor of glaucoma patients and experimental glaucoma.1924 While basal levels of TNF-α are required for retinal neurotransmission and homeostasis, excessive soluble TNF-α is responsible for the RGC death in glaucoma.24 Microglia and astrocytes are the main sources of these inflammatory molecules in glaucoma.25,26 
Neurotransmitter receptors, such as adenosine receptors (ARs), adenosine triphosphate (ATP) receptors, glutamate receptors, gamma-aminobutyric acid (GABA) receptors, cholinergic receptors, adrenergic receptors, and dopamine receptors, are expressed on microglia.27 Neurotransmitter receptor–coupling modulates microglial-mediated neuroinflammation.27 Four subtypes of ARs have been identified so far, namely A1 receptor (A1R), A2AR, A2BR, and A3R.28 
As one of the key molecules in the neural network, A2AR has sophisticated interactions with dopamine receptors, A1R, glutamate receptors, and cannabinoid receptors, modulating a wide range of physiological and pathologic functions, including sleep, cognition, memory, motivation, and habit formation.29 A2AR modulates microglial proliferation, cyclooxygenase-2 (COX-2), and NO synthase-II expression, inflammatory mediator synthesis and release.30,31 Pharmacological inhibition of A2AR exerts profound neuroprotection in animal models of several cerebral disorders, especially in PD.3234 
Neuroinflammation is a common feature in CNS neurodegenerative disorders and retinal degenerative diseases.35 Previous studies have demonstrated that A2AR antagonists can modulate the microglial activation and neuroinflammation, which could result in neuroprotection in CNS neurodegenerative disease.35 A recent study shows that A2AR antagonists prevent neuroinflammation-induced death of RGCs caused by elevated hydrostatic pressure in vitro.36 The purpose of this study was to determine whether ZM241385, a selective A2AR antagonist, could protect RGCs in a rat model of glaucoma and to investigate its effect on microglial activation. As elevated glutamate levels have been implicated in glaucomatous degeneration,37,38 the effect of ZM241385 on microglia under high concentration of glutamate in vitro also was studied. 
Materials and Methods
Rat Glaucoma Model
A total of 89 adult male Sprague-Dawley (SD) rats (Slaccas, Shanghai, China) between 200 and 250 g in body weight were used. Rats were housed in a standard animal room with food and water provided ad libitum, a 12-hour light/dark cycle, and a constant room temperature of 22°C. For all surgeries and injections, the rats were anesthetized with intraperitoneal injection of xylazine (8 mg/kg) and ketamine (75 mg/kg), and topical 0.5% proparacaine (s.a. Alcon-Couvreur n.v., Puurs, Antwerp, Belgium). The use of animals in experiments was approved by the institutional review board of Ruijin Hospital, Shanghai, China. All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Chronic ocular hypertension (COH) was induced in the right eyes of rats by ligation of three episcleral veins.39 Briefly, two dorsal episcleral veins and one ventral episcleral vein were isolated from the surrounding tissues. Each vein was ligated with 10–0 nylon suture (Alcon Laboratories, Ft. Worth, TX, USA), and then severed without damaging the neighboring tissues. The left eyes of these animals were not used as control eyes. An additional group of animals with sham operation served as the control group (CTRL) instead. 
Intraocular pressure measurements were made under brief systemic anesthesia with isoflurane inhalation (2∼4%; Sigma-Aldrich Corp., St. Louis, MO, USA) to minimize the variation caused by stress and movement. Intraocular pressure was measured at preoperation and postoperation immediately, and weekly until the end of the experimental period. To avoid the effect of circadian rhythm, IOP was measured between 10 AM and 2 PM. Each IOP data point was an average of 6 consecutive measurements taken with a TonoLab Rebound Tonometer (Icare, Espoo, Finland), as described previously by others.40 
Rats with IOP elevation at least 1.3-fold above the baseline during the observation period were enrolled in the experimental glaucoma group. Intraocular pressure of rats with sham operation remained at approximately baseline. 
Drugs and Intravitreal Injection
The rat pupil was dilated with 0.5% tropicamide. One μL 50 μM ZM241385 dissolved in 10% dimethylsulfoxide (DMSO) was injected intravitreally, resulting in a concentration of approximately 1 μM in the vitreous, considering the adult rat vitreous volume to be approximately 56 μl.41 The intravitreal injection procedure was as described previously.42 Eyes that received only an injection of vehicle solution (10% DMSO) in the same manner served as vehicle controls (COH+vehicle group). Rat eyes with satisfactory IOP elevation (1.3-fold above baseline) accepted injections 1 hour after surgery and again 1 week later (COH+ZM group and COH+vehicle group). The vitreous cavity was taken as a drug storage owing to its avascularity and slow metabolism. The animals were killed 2 weeks after the surgery. 
Retrograde Labeling of RGCs and Quantification of RGCs
To determine RGC densities, retrograde labeling of RGCs was performed in 38 rats (6 in the COH4w group; 8 in each group of CTRL, COH2w, COH+vehicle, and COH+ZM) with fluorescent tracer 1,1--dioctadecyl--3,3,30,30--tet--ramethyl--indocarbocyanin perchlorate (DiI, Life Technologies, Carlsbad, CA, USA), as described previously.42 A 1.5 μL 4% solution of DiI in 10% DMSO in PBS was slowly injected into the superior colliculi (SC) bilaterally using a Hamilton syringe. 
Seven days after DiI injection, the eyes were enucleated with a part of nasal and superior rectus muscle retained for orientation. After being immersed in 4% paraformaldehyde (PFA) for 24 hours at 4°C, eyes were bisected at the corneoscleral junction. The retinae were isolated, flattened through four radial cuts, mounted on slides with the vitreal-side up and coverslipped with a fade-retardant mounting medium (Prolong Gold; Invitrogen, Carlsbad, CA, USA). Labeled RGCs were imaged using Zeiss microscopy (Carl Zeiss Imaging, Inc., Jena, Germany). 
Each retina was divided into four quadrants (nasal, temporal, superior, and inferior) and images were obtained at 2 different eccentricities of 2/6 (central) and 4/6 (peripheral) of the retinal radius in each quadrant (Fig. 1). Retinal ganglion cells were counted using QuantityOne software in duplicate by two independent masked investigators. The RGC densities in four quadrants were averaged in the central and peripheral retina, respectively. 
Figure 1
 
Calculation of RGC density. The circle on the left is a schematic of the retina. Each retina was divided into four quadrants (superior, inferior, nasal, and temporal) and images were obtained at 2 different eccentricities of 2/6 (central) and 4/6 (peripheral) of the retinal radius in each quadrant, as represented by 8 small squares on the circle.
Figure 1
 
Calculation of RGC density. The circle on the left is a schematic of the retina. Each retina was divided into four quadrants (superior, inferior, nasal, and temporal) and images were obtained at 2 different eccentricities of 2/6 (central) and 4/6 (peripheral) of the retinal radius in each quadrant, as represented by 8 small squares on the circle.
Immunohistochemistry
After the rats had been transcardially perfused with ice-cold saline and then 4% PFA, eyes were enucleated and fixed in 4% PFA at 4°C for 48 hours. The eyes then were bisected, and the lens and vitreous were removed. After being washed in PBS, the eyecups were immersed in 30% sucrose solution for 4 hours at 4°C before being frozen in optimum cutting temperature (OCT) compound (Sakura, Japan). Cryosection of 10 μm thickness were cut in an orientation parallel to the center of the pupil and through the optic nerve using a cryostat (model CM3050S, Leica,Wetzlar, Germany) and collected on Superfrost Plus glasses (Thermo Fisher Scientific, Waltham, MA, USA). 
Sections were washed in PBS for 5 minutes and incubated in blocking solution (PBS containing 5% donkey serum and 0.1% Triton X-100) for 2 hours. Primary antibody (goat anti-Iba-1 polyclonal antibody; Abcam, Cambridge, MA, USA) and secondary antibody (donkey anti-goat IgG-FITC; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were diluted in PBS containing 5% donkey serum (1:500 and 1:100, respectively). Primary antibody was allowed to react with the sections overnight at 4°C, and secondary antibody for 2 hours at room temperature. The sections then were coverslipped with the fade-retardant mounting medium and examined under Zeiss microscopy. 
Western Blotting
For protein extraction, retinae were immersed in RIPA Lysis Buffer (Sigma-Aldrich Corp.) with a cocktail of protease inhibitors (Complete Protease Inhibitor cocktail; Roche Applied Science, Penzberg, Germany) on ice. Retinae then were homogenized and centrifuged. Supernatant was collected and protein concentrations were quantified. 
Each retina served as an individual sample (n = 6 per group). Equal amounts of protein were separated on polyacrylamide gels and then electrotransferred onto a polyvinylidene fluoride membrane (PVDF; Merck Millipore, Billerica, MA, USA). After blocking for 2 hours in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% fat-free milk, membranes were incubated overnight at 4°C with primary antibodies (β-actin, Iba-1, TNF-α and IL-1β, Abcam) in TBST containing 3% BSA. The dilution of 1:5,000 was used for β-actin and 1:1,000 for the other antibodies. Membranes then were washed and incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 hour at room temperature. Antigen–antibodies complexes were visualized as bands by chemiluminescence reagent kit (Thermo Fisher Scientific). The densities of the bands on the membrane were scanned using a Bio-Rad Versadoc imaging system (Bio-Rad, Hercules, CA, USA) and analyzed with Image Pro Plus version 6.0 (Media Cybernetics, Silver Spring, MD, USA). Data are expressed as ratio of the protein of interest to β-actin of the identical sample. 
Quantitative Real-Time RT-PCR
Total RNA was isolated from rat retina using Trizol reagent (Invitrogen) following the manufacturer's instructions. The RNA then was converted to cDNA using the reverse transcriptase kit PrimeScript RT Master Mix (Takara Bio, Inc., Shiga, Japan). Primers were designed using Primer premier 5.0 software (Thermo Fisher Scientific). The primer pairs used were as follows: β-actin (forward) 5′-GCGCTCGTCGTCGACAACGG-3′, (reverse) 5′-GTGTGGTGCCAAATCTTCTCC-3′; TNF-α (forward) 5′-CTCCCAGAAAAGCAAGCA-3′, (reverse) 5′-CCTCTGCCAGTTCCACAAC-3′; and IL-1β (forward) 5′-AGCAGCTTTCGACAGTGAGG-3′, (reverse) 5′-CACACACTAGCAGGTCGTCA-3′. Quantitative real-time PCR was performed using the SYBR Premix Ex Taq II kit (Takara Bio, Inc.) in a total volume of 20 μL on a 7500 real-time PCR (Applied Biosystems, Foster City, CA, USA): 95°C for 30 seconds; 40 cycles of 95°C for 5 seconds, 60°C for 34 seconds, and 72°C for 30 seconds. Expression levels of each sample were detected in duplicates. β-Actin was used as a reference gene. Relative quantification of the gene expression was calculated using the 2−ΔΔCt method as described previously.43 
Primary Rat Retinal Microglia Cultures
Microglial cells were isolated from retinae of newborn rats (within 72 hours) as described by Tomas Deierborg,44 with minor modifications. Briefly, retinae were collected and washed with ice-cold D-HBSS and digested with 0.05% trypsin-EDTA (Invitrogen) containing DNase I (Sigma-Aldrich Corp.) 100 U/mL at 37°C for 10 minutes. The cells were collected by centrifugation, resuspended by adding Dulbecco's modified Eagle's medium (DMEM) + GlutaMAX medium containing 4.5 g/L glucose (Life Technologies) supplemented with 10% FBS and penicillin/streptomycin (100 U/mL and 0.1 mg/mL) and plated in 75 cm2 culture flasks at a density of 1 × 107 cells/ml. The cells were cultured in a humidified incubator at 37°C and 5% CO2 and the medium was changed after 48 hours, then changed once every 7 days. The supernatant from the mixed cell culture was preserved and labeled as M.C. supernatant, which contained cytokines from astrocytes and was able to promote the microglial survival. Two weeks later, microglial cells were harvested in culture medium by shaking the flasks at 100 rpm for 2 hours. The cells were resuspended in culture medium containing 10% M.C. supernatant and seeded. The cells were identified by immunocytochemical staining analysis for CD11b and Iba-1. The purity of the microglial cultures was determined by flow cytometric analysis. 
Flow Cytometry
Microglia were harvested by shaking. Cell viability determined by Trypan Dye was approximately 100%. After washes, the cells were incubated with an anti-rat CD11 b/c PE (eBioscience, Inc., San Diago, CA, USA) and an appropriate isotype control monoclonal antibodies (mAbs) at a 1:150 dilution for 30 minutes on ice in a dark place. Following washes, cells were fixed with 1% paraformaldehyde for 30 minutes. After washes again, approximately 10,000 cells were acquired for each sample by software Cellquest Pro using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cells were identified as microglia by gating on CD11 b/c positive events. 
Cell Treatments With Glutamate and ZM241385
Microglial cells collected from the culture flasks were resuspended and seeded at a density of 1 × 106 cells/mL in 24-well plates coated with poly-l-ornithine (Sigma-Aldrich Corp.). One day after seeding, the cells in one plate were incubated in the media containing different concentrations of glutamate (0, 0.1, 1, and 10 mM) for 24 hours and the supernatant was collected for preliminary ELISA analysis. The cells in other plates were incubated in the media with the indicated concentration of glutamate and 0.5 μM ZM241385 for 24 hours at 37°C and the media then was collected for ELISA analysis. 
ELISA Analysis
Conditioned media collected from microglia treated with glutamate and ZM241385 for 24 hours was assayed for soluble TNF-α and IL-1β using ELISA kit (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China) according to the manufacturer's instructions. Optical density was determined at 450 nm. 
Statistical Analysis
SPSS version 19.0 software was applied to perform statistical analyses. All results were expressed as mean ± SD. Retinal ganglion cell densities were ranked first. Then, all the data were analyzed using the 1-way ANOVA test, followed by least significant difference (LSD) test. P < 0.05 was considered statistically significant. Graphs were prepared with the aid of GraphPad Prism 6.04 (GraphPad Software, Inc., La Jolla, CA). 
Results
Change of RGC Density Induced by COH
In the control eyes (sham-operated rat), a higher RGC density (2436 ± 143 cells/mm2) was observed in the 2/6 retinal radius (central retina), while a lower density (2219 ± 140 cells/mm2) was in the 4/6 retinal radius (peripheral retina). Two weeks following the induction of COH, the RGC density in the central (2130 ± 148 cells/mm2) and peripheral (1953 ± 142 cells/mm2) retina were significantly lower than that of the control group (central, P = 0.002; peripheral, P = 002). The values dropped to 1999 ± 147 (center) and 1751 ± 135 (periphery) cells/mm2 after 4 weeks of COH (Fig. 2). 
Figure 2
 
Changes of RGC density induced by COH. Two and 4 weeks following the induction of COH, the RGC density in the central and peripheral retina were significantly lower than that of control group (*P < 0.05). The values of 4-week COH dropped significantly than that of 2-week COH (#P < 0.05). Scale bar: 100 μm. Whiskers: minimum to maximum.
Figure 2
 
Changes of RGC density induced by COH. Two and 4 weeks following the induction of COH, the RGC density in the central and peripheral retina were significantly lower than that of control group (*P < 0.05). The values of 4-week COH dropped significantly than that of 2-week COH (#P < 0.05). Scale bar: 100 μm. Whiskers: minimum to maximum.
Changes of Microglia and Inflammatory Mediators Induced by COH
Resting microglia free from COH were ramified with fine and long processes (white arrow in Fig. 3A CTRL). After the induction of COH, some retinal microglia changed their ramified morphology into a round or amoeboid morphologic profile, and the amount of microglia increased (Fig. 3A 1w--4w). Compared to the control group (sham-operated rat), the mRNA expression of TNF-α and IL-1β in retinae increased (Fig. 3B), and the protein expression of TNF-α, IL-1β, and Iba-1 in retinae also increased (Fig. 3C) at 2 and 4 weeks following the induction of COH. 
Figure 3
 
Changes of microglial morphology and inflammatory mediators induced by COH. (A) Changes of microglial morphology and number. After the induction of COH, the microglia changed their ramified morphology into an amoeboid or round morphologic profile (white arrows) and the amount of microglia increased. Scale bar: 50 μm. (B) The mRNA expression of TNF-α and IL-1β in retinae increased at 2 and 4 weeks following the induction of COH. (C) The protein expression of TNF-α, IL-1β, and Iba-1 in retinae increased at 2 and 4 weeks following the induction of COH. Each panel represents the average value. Error bar: SD (mean ± SD).
Figure 3
 
Changes of microglial morphology and inflammatory mediators induced by COH. (A) Changes of microglial morphology and number. After the induction of COH, the microglia changed their ramified morphology into an amoeboid or round morphologic profile (white arrows) and the amount of microglia increased. Scale bar: 50 μm. (B) The mRNA expression of TNF-α and IL-1β in retinae increased at 2 and 4 weeks following the induction of COH. (C) The protein expression of TNF-α, IL-1β, and Iba-1 in retinae increased at 2 and 4 weeks following the induction of COH. Each panel represents the average value. Error bar: SD (mean ± SD).
Changes of RGC Density by ZM241385 in the COH Rats
Retinal ganglion cells at the central retina were better preserved following intravitreal injection of ZM241385 in the COH rats (COH+ZM group) than that following vehicle injection (COH+ vehicle group; 2287 ± 135 vs. 2078 ± 151 cells/mm2; P = 0.004), while the RGC density at the peripheral retina had a trend of less decrease with ZM injection but not significant compared to the COH+ vehicle group (1989 ± 171 vs 1906 ± 164 cells/mm2, P = 0.464). Intravitreal injection of vehicle (COH+ vehicle group) had no effect on the RGCs counting compared to COH group at the central (P = 0.866) and peripheral (P = 0.839) retina (Fig. 4). 
Figure 4
 
Changes of RGC density at the central and peripheral retina induced by COH and ZM241385. *P < 0.05 represents comparison to the CTRL group; #P < 0.05 represents comparison to the COH+vehicle group. Scale bar: 100 μm. Whiskers: minimum to maximum.
Figure 4
 
Changes of RGC density at the central and peripheral retina induced by COH and ZM241385. *P < 0.05 represents comparison to the CTRL group; #P < 0.05 represents comparison to the COH+vehicle group. Scale bar: 100 μm. Whiskers: minimum to maximum.
Changes of Microglia and Inflammatory Mediators by ZM241385 in the COH Rats
In the COH rats, the amount of round or ameboid microglia in the retina decreased following intravitreal injection of ZM241385 (COH+ZM group) than that following the vehicle injection (COH+vehicle group) and that without any injection (COH group). The microglia also extended 1 to 2 thick and short processes after ZM241385 injection, but still less ramified than that in the CTRL group (Fig. 5A, white arrows pointed to microglia). The mRNA expressions of TNF-α and IL-1β in retina were downregulated by ZM241385 injection compared to the vehicle injection in the COH rats (n = 6, P = 0.024 and P = 0.000, respectively; Fig. 5B). Moreover, the protein expressions of TNF-α , IL-1β, and Iba-1 in retinae also were downregulated by ZM241385 injection compared to vehicle injection in the COH rats (n = 6, P = 0.000, 0.001 and 0.001, respectively; (Fig. 5C). Intravitreous vehicle injection in the COH rats showed no significant change on the microglial morphology or quantity and no significant effect on the expressions of TNF-α , IL-1β, and Iba-1 in retinae compared to the COH rats that did not accept any intravitreous injection (n = 6, P > 0.05; Fig. 5). 
Figure 5
 
Changes of microglial morphology and inflammatory mediators in the rat retinae with or without ZM241385 injection 2 weeks after the induction of COH. (A) The morphologic and quantity change of the microglia in different groups. In the COH rats, the amount of round or ameboid microglia in the retina decreased following intravitreal injection of ZM241385 (COH+ZM) than that following the vehicle injection (COH+vehicle) and that without any injection (COH). The microglia extended 1 to 2 thick and short processes in the group of COH+ZM, but still less ramified than that in the CTRL. White arrows pointed to microglia. Scale bar: 50 μm. (B) Changes of the mRNA expressions of TNF-α and IL-1β in retinae of the different groups. (C) Changes of the protein expressions of TNF-α, IL-1β, and Iba-1 in retinae of the different groups. *P < 0.05 represents comparison to CTRL group; #P < 0.05 represents comparison to COH+vehicle group. Each panel represents the average value. Error bar: mean ± SD.
Figure 5
 
Changes of microglial morphology and inflammatory mediators in the rat retinae with or without ZM241385 injection 2 weeks after the induction of COH. (A) The morphologic and quantity change of the microglia in different groups. In the COH rats, the amount of round or ameboid microglia in the retina decreased following intravitreal injection of ZM241385 (COH+ZM) than that following the vehicle injection (COH+vehicle) and that without any injection (COH). The microglia extended 1 to 2 thick and short processes in the group of COH+ZM, but still less ramified than that in the CTRL. White arrows pointed to microglia. Scale bar: 50 μm. (B) Changes of the mRNA expressions of TNF-α and IL-1β in retinae of the different groups. (C) Changes of the protein expressions of TNF-α, IL-1β, and Iba-1 in retinae of the different groups. *P < 0.05 represents comparison to CTRL group; #P < 0.05 represents comparison to COH+vehicle group. Each panel represents the average value. Error bar: mean ± SD.
Morphology and Identification of Cultured Microglia cells
Freshly isolated microglia cells were small round cells and weakly attached to the surface of the plate. Several days after separation seeding on poly-l-ornithine coated coverslips, a portion of microglia cells displayed a slightly ramified shape with sparse small processes (Figs. 6A–C). Microglia cells were fixed and immunostained by CD11b (Fig. 6B) or Iba-1 (Fig. 6C). The purity of isolated microglia population was approximately 95% determined by flow cytometric analysis (Fig. 6D) 
Figure 6
 
Morphology and identification of cultured microglia cells. (A) Isolated microglia cells were imaged under the inverted phase contrast microscope. (B) The microglia cells were immunostained by CD11b. (C) The microglia cells were immunostained by Iba-1. (D) The purity of cultured microglia cells was approximately 95.54% determined by flow cytometric analysis.
Figure 6
 
Morphology and identification of cultured microglia cells. (A) Isolated microglia cells were imaged under the inverted phase contrast microscope. (B) The microglia cells were immunostained by CD11b. (C) The microglia cells were immunostained by Iba-1. (D) The purity of cultured microglia cells was approximately 95.54% determined by flow cytometric analysis.
TNF-α and IL-1β Secretion of Cultured Microglia Cells After Treatment With Glutamate and ZM241385
Glutamate at the concentration of approximately 0.1 to 10 mM remarkably promoted the secretion of TNF-α and IL-1β compared to the control group (n = 6, each P = 0.000) and the maximum secretion was found in the group at the glutamate concentration of 1 mM (Glu1 group; Fig. 7). When 0.5 μM ZM241385 was added into the culture media containing 1 mM glutamate (Glu1+zm0.5 group), the secretion of TNF-α and IL-1β decreased compared to the Glu1 group (n = 6, P = 0.000 and P = 0.000); however, 0.5 μM ZM241385 alone had almost no effect on the secretion of TNF-α and IL-1β compared to control group (n = 6, P = 0.239 and P = 0.911, respectively). 
Figure 7
 
Tumor necrosis factor-α and IL-1β secretion of the cultured microglia cells after treatment with glutamate and ZM241385. *P < 0.05 represents comparison to ctrl group; #P < 0.05 represents comparison to Glu1 group. Ctrl, control. Glu0.1, Glu1, Glu10 represent the culture media containing 0.1, 1, and 10 mM glutamate, respectively, Glu1+zm0.5 represents the culture media containing 1 mM glutamate and 0.5 μM ZM241385; zm0.5 represents the culture media containing 0.5μM ZM241385. Each panel represents the average. Error bar: mean ± SD.
Figure 7
 
Tumor necrosis factor-α and IL-1β secretion of the cultured microglia cells after treatment with glutamate and ZM241385. *P < 0.05 represents comparison to ctrl group; #P < 0.05 represents comparison to Glu1 group. Ctrl, control. Glu0.1, Glu1, Glu10 represent the culture media containing 0.1, 1, and 10 mM glutamate, respectively, Glu1+zm0.5 represents the culture media containing 1 mM glutamate and 0.5 μM ZM241385; zm0.5 represents the culture media containing 0.5μM ZM241385. Each panel represents the average. Error bar: mean ± SD.
Discussion
Glaucoma typically is associated with IOP elevation, which remains the only treatable risk factor up to now and, therefore, remains the predominant independent variable in experimental animal models.45 It was reported that RGC apoptosis was observed at 1 week after episcleral vein ligation.39 Previous studies showed that approximately 13% RGCs were lost at 2 weeks after experimental glaucoma that induced by preventing the backflow of aqueous humor including episcleral vein ligation, injection of hypertonic saline into episcleral veins, and laser photocoagulation of the trabecular meshwork and/or episcleral veins, and so forth.4648 In the present study, we also found that RGC density decreased significantly in the central and peripheral retinae at 2 and 4 weeks after episcleral vein ligation, which demonstrated that the COH model was reliable. 
Resting microglia adopts a ramified morphology. Microglia maintains CNS integrity by constantly surveying the surrounding environment.9,49 They become less ramified and increase their soma size, culminating in an ameboid or spheroidal form in response to infection, injury, ischemia, and hypoxia.9,49 The same transformation and an increase in the number of ameboid microglia occurred in the rat retinae following the induction of COH in our study. We also found that the Iba-1 (ionized calcium binding adaptor molecule 1) expression, the upregulation of which served extensively as an indicator of microglia activation in the CNS,50 was significantly augmented in retinae at 2 and 4 weeks following the induction of COH, which confirmed the retinal microglia activation in the COH rats. 
Microglia activation results in their production of proinflammatory cytokines, such as IL-1, IL-6, and TNF-α.9 The release of these factors typically is intended to prevent further damage to CNS tissue, whereas they also may be toxic to neurons and other glia.51 Mounting evidence indicates that the chronic microglia activation may contribute to the secondary injury of neurons and may be the source of the chronic neuroinflammation.5254 A recent study identifies glia-derived soluble TNF-α as a major inducer of RGC death via activation of calcium-permeable AMPA receptors (CP-AMPAR), establishing a new link between neuroinflammation and RGC loss in glaucoma.24 In our study, RGC death following the induction of COH was in parallel with the sustained neuroinflammation, which also suggested a relation between them. 
Antagonist A2AR is a promising pharmacological agent for CNS degenerative diseases.17,55 The neuroinflammation regulation of A2AR antagonists may be one of the mechanisms of the neuroprotection effect.56,57 Some studies found that genetic blockade of A2AR could alleviate the ischemic brain damage by reducing the readouts of IL-1, IL-6, and IL-12 in the mice model of ischemic brain injury.56,57 As it comes to retina, Madiera et al.36 demonstrated that A2AR mediated RGC death, microglia activation, and cytokine production in organotypic cultures of retina exposed to elevated hydrostatic pressure. Likewise, we found a similar effect of A2AR antagonist in our study. We found that ZM241385 could provide neuroprotection for RGCs in the central retina following the induction of COH. Failure to protect the peripheral RGCs may be due to the longer axons facing more challenges,58 for example, the nutrients delivery blockage. Further investigation is needed on whether ZM241385 could protect the peripheral RGCs under COH if given longer observation period and more frequent pharmacologic interventions. 
Glutamate is an important neurotransmitter in CNS, and the elevation of extracellular glutamate is a key factor in retinal neurodegeneration occurring in glaucoma and other retinal pathologies characterized by ischemic events.59,60 The glutamate concentration inside the cells is at approximately 1 to 10mM.61 In normal conditions, glutamate concentration released into a synaptic cleft reaches to a high level of approximately 1 mM and remains momentarily.62 In excitotoxic states, the extracellular concentrations of glutamate may reach a millimolar range, causing degeneration of neurons through excessive stimulation of glutamate receptors.63 
The modulation of glutamate by A2AR antagonist also may be one of the mechanisms of the neuroprotection effect. Previous studies have shown that the neuroprotection of A2AR antagonists may be elicited by reducing presynaptic glutamate release and modulation of glutamate uptake.64,65 The ability of A2AR antagonists for reducing the excitotoxicity glutamate release had been confirmed in electrophysiological experiments with the corticostriatal slices by using ZM241385.66,67 
We found that high concentration of glutamate caused an increased expression of TNF-α and IL-1β in vitro. This result was consistent with previous study.68 However, this response was downregulated when ZM241385 was added into the culture system. The result seems in conflict with previous studies reporting that the activation of A2AR reduces microglia reactivity using primary retinal microglia cultures exposed to LPS or hypoxia.59,69 This may be due to the following factors: (1) the supernatant from mixed cell cultures was added into microglia culture system, which contained cytokines from astocytes and other cells, and (2) glutamate was used as the stimulator. It was pointed out that the A2AR effect on microglial cells in vitro is dependent on the extracellular glutamate concentration.70 The two points might affect the microglia status and the control by A2AR of microglia reactivity. In this case, we concluded that the neuroprotection of ZM241385 in vivo might be partially due to its downregulation of proinflammatory cytokines from microglia under high concentration of glutamate. 
Collectively, the data of the present study suggested that A2AR antagonist ZM241385 could downregulate the activation of microglia and proinflammatory cytokines under the conditions of COH and the high concentration of glutamate, which may be one of the mechanisms that protected RGCs in experimental glaucoma. 
Acknowledgments
Supported by the National Natural Science Foundation of China (No.81000373, No.81371014, and No.81470639) and Natural Science Foundation of Shanghai (14411968400). 
Disclosure: X. Liu, None; P. Huang, None; J. Wang, None; Z. Yang, None; S. Huang, None; X. Luo, None; J. Qi, None; X. Shen, None; Y. Zhong, None 
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Figure 1
 
Calculation of RGC density. The circle on the left is a schematic of the retina. Each retina was divided into four quadrants (superior, inferior, nasal, and temporal) and images were obtained at 2 different eccentricities of 2/6 (central) and 4/6 (peripheral) of the retinal radius in each quadrant, as represented by 8 small squares on the circle.
Figure 1
 
Calculation of RGC density. The circle on the left is a schematic of the retina. Each retina was divided into four quadrants (superior, inferior, nasal, and temporal) and images were obtained at 2 different eccentricities of 2/6 (central) and 4/6 (peripheral) of the retinal radius in each quadrant, as represented by 8 small squares on the circle.
Figure 2
 
Changes of RGC density induced by COH. Two and 4 weeks following the induction of COH, the RGC density in the central and peripheral retina were significantly lower than that of control group (*P < 0.05). The values of 4-week COH dropped significantly than that of 2-week COH (#P < 0.05). Scale bar: 100 μm. Whiskers: minimum to maximum.
Figure 2
 
Changes of RGC density induced by COH. Two and 4 weeks following the induction of COH, the RGC density in the central and peripheral retina were significantly lower than that of control group (*P < 0.05). The values of 4-week COH dropped significantly than that of 2-week COH (#P < 0.05). Scale bar: 100 μm. Whiskers: minimum to maximum.
Figure 3
 
Changes of microglial morphology and inflammatory mediators induced by COH. (A) Changes of microglial morphology and number. After the induction of COH, the microglia changed their ramified morphology into an amoeboid or round morphologic profile (white arrows) and the amount of microglia increased. Scale bar: 50 μm. (B) The mRNA expression of TNF-α and IL-1β in retinae increased at 2 and 4 weeks following the induction of COH. (C) The protein expression of TNF-α, IL-1β, and Iba-1 in retinae increased at 2 and 4 weeks following the induction of COH. Each panel represents the average value. Error bar: SD (mean ± SD).
Figure 3
 
Changes of microglial morphology and inflammatory mediators induced by COH. (A) Changes of microglial morphology and number. After the induction of COH, the microglia changed their ramified morphology into an amoeboid or round morphologic profile (white arrows) and the amount of microglia increased. Scale bar: 50 μm. (B) The mRNA expression of TNF-α and IL-1β in retinae increased at 2 and 4 weeks following the induction of COH. (C) The protein expression of TNF-α, IL-1β, and Iba-1 in retinae increased at 2 and 4 weeks following the induction of COH. Each panel represents the average value. Error bar: SD (mean ± SD).
Figure 4
 
Changes of RGC density at the central and peripheral retina induced by COH and ZM241385. *P < 0.05 represents comparison to the CTRL group; #P < 0.05 represents comparison to the COH+vehicle group. Scale bar: 100 μm. Whiskers: minimum to maximum.
Figure 4
 
Changes of RGC density at the central and peripheral retina induced by COH and ZM241385. *P < 0.05 represents comparison to the CTRL group; #P < 0.05 represents comparison to the COH+vehicle group. Scale bar: 100 μm. Whiskers: minimum to maximum.
Figure 5
 
Changes of microglial morphology and inflammatory mediators in the rat retinae with or without ZM241385 injection 2 weeks after the induction of COH. (A) The morphologic and quantity change of the microglia in different groups. In the COH rats, the amount of round or ameboid microglia in the retina decreased following intravitreal injection of ZM241385 (COH+ZM) than that following the vehicle injection (COH+vehicle) and that without any injection (COH). The microglia extended 1 to 2 thick and short processes in the group of COH+ZM, but still less ramified than that in the CTRL. White arrows pointed to microglia. Scale bar: 50 μm. (B) Changes of the mRNA expressions of TNF-α and IL-1β in retinae of the different groups. (C) Changes of the protein expressions of TNF-α, IL-1β, and Iba-1 in retinae of the different groups. *P < 0.05 represents comparison to CTRL group; #P < 0.05 represents comparison to COH+vehicle group. Each panel represents the average value. Error bar: mean ± SD.
Figure 5
 
Changes of microglial morphology and inflammatory mediators in the rat retinae with or without ZM241385 injection 2 weeks after the induction of COH. (A) The morphologic and quantity change of the microglia in different groups. In the COH rats, the amount of round or ameboid microglia in the retina decreased following intravitreal injection of ZM241385 (COH+ZM) than that following the vehicle injection (COH+vehicle) and that without any injection (COH). The microglia extended 1 to 2 thick and short processes in the group of COH+ZM, but still less ramified than that in the CTRL. White arrows pointed to microglia. Scale bar: 50 μm. (B) Changes of the mRNA expressions of TNF-α and IL-1β in retinae of the different groups. (C) Changes of the protein expressions of TNF-α, IL-1β, and Iba-1 in retinae of the different groups. *P < 0.05 represents comparison to CTRL group; #P < 0.05 represents comparison to COH+vehicle group. Each panel represents the average value. Error bar: mean ± SD.
Figure 6
 
Morphology and identification of cultured microglia cells. (A) Isolated microglia cells were imaged under the inverted phase contrast microscope. (B) The microglia cells were immunostained by CD11b. (C) The microglia cells were immunostained by Iba-1. (D) The purity of cultured microglia cells was approximately 95.54% determined by flow cytometric analysis.
Figure 6
 
Morphology and identification of cultured microglia cells. (A) Isolated microglia cells were imaged under the inverted phase contrast microscope. (B) The microglia cells were immunostained by CD11b. (C) The microglia cells were immunostained by Iba-1. (D) The purity of cultured microglia cells was approximately 95.54% determined by flow cytometric analysis.
Figure 7
 
Tumor necrosis factor-α and IL-1β secretion of the cultured microglia cells after treatment with glutamate and ZM241385. *P < 0.05 represents comparison to ctrl group; #P < 0.05 represents comparison to Glu1 group. Ctrl, control. Glu0.1, Glu1, Glu10 represent the culture media containing 0.1, 1, and 10 mM glutamate, respectively, Glu1+zm0.5 represents the culture media containing 1 mM glutamate and 0.5 μM ZM241385; zm0.5 represents the culture media containing 0.5μM ZM241385. Each panel represents the average. Error bar: mean ± SD.
Figure 7
 
Tumor necrosis factor-α and IL-1β secretion of the cultured microglia cells after treatment with glutamate and ZM241385. *P < 0.05 represents comparison to ctrl group; #P < 0.05 represents comparison to Glu1 group. Ctrl, control. Glu0.1, Glu1, Glu10 represent the culture media containing 0.1, 1, and 10 mM glutamate, respectively, Glu1+zm0.5 represents the culture media containing 1 mM glutamate and 0.5 μM ZM241385; zm0.5 represents the culture media containing 0.5μM ZM241385. Each panel represents the average. Error bar: mean ± SD.
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