Blockade of microglial adenosine A2A receptor suppresses elevated pressure‐induced inflammation, oxidative stress, and cell death in retinal cells

Abstract Glaucoma is a retinal degenerative disease characterized by the loss of retinal ganglion cells and damage of the optic nerve. Recently, we demonstrated that antagonists of adenosine A2A receptor (A2AR) control retinal inflammation and afford protection to rat retinal cells in glaucoma models. However, the precise contribution of microglia to retinal injury was not addressed, as well as the effect of A2AR blockade directly in microglia. Here we show that blocking microglial A2AR prevents microglial cell response to elevated pressure and it is sufficient to protect retinal cells from elevated pressure‐induced death. The A2AR antagonist SCH 58261 or the knockdown of A2AR expression with siRNA in microglial cells prevented the increase in microglia response to elevated hydrostatic pressure. Furthermore, in retinal neural cell cultures, the A2AR antagonist decreased microglia proliferation, as well as the expression and release of pro‐inflammatory mediators. Microglia ablation prevented neural cell death triggered by elevated pressure. The A2AR blockade recapitulated the effects of microglia depletion, suggesting that blocking A2AR in microglia is able to control neurodegeneration in glaucoma‐like conditions. Importantly, in human organotypic retinal cultures, A2AR blockade prevented the increase in reactive oxygen species and the morphological alterations in microglia triggered by elevated pressure. These findings place microglia as the main contributors for retinal cell death during elevated pressure and identify microglial A2AR as a therapeutic target to control retinal neuroinflammation and prevent neural apoptosis elicited by elevated pressure.

strategies that modulate microglial cell response to damage have been suggested to afford neuroprotection in glaucoma (Almasieh et al., 2012;Bosco et al., 2008;Langmann, 2007;. Adenosine is a neuromodulator in the central nervous system acting through the activation of four receptors, A 1 , A 2A , A 2B , and A 3 . We demonstrated that the blockade of adenosine A 2A receptor (A 2A R) confers neuroprotection to the retina in glaucoma models through the control of microglia responsiveness (Boia et al., 2017;Madeira, Elvas, et al., 2015;Santiago et al., 2014). However, the specific role of microglial cells was not addressed, as well as the impact of A 2A R blockade in microglial cells in the context of glaucoma. Since elevated intraocular pressure is considered the main risk factor of glaucoma, we focused on the contribution of microglia to the death of retinal neurons under elevated pressure to model glaucoma in vitro (Aires, Ambrósio, & Santiago, 2016). In addition, we also studied whether the pharmacological blockade or the genetic inactivation of A 2A R in microglia could attenuate the inflammatory profile of microglia, using a murine microglia cell line, primary retinal microglia cultures, rat retinal neural cell cultures, and human retinal tissue exposed to elevated pressure.

| Animals
All experiments using animals were approved by the Animal Welfare
Cells were cultured at 37 C in a humidified atmosphere of 5% CO 2 for 7 days. The primary retinal neural cell culture is composed of retinal neurons, Müller cells, astrocytes, and microglia.

| Primary retinal microglial cell cultures
Primary retinal microglial cell cultures were obtained from 3 to 4 days old Wistar rats, as described previously (Li, Qu, & Wang, 2015), with some modifications, as follows. The retinas were dissociated, and the cell suspension was plated in uncoated T75 flasks (corresponding to four retinas in each flask) and maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) with GlutaMAX™ (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 10% FBS (GIBCO, Invitrogen, Carlsbad, CA), 100 U/mL penicillin, and 100 μg/mL streptomycin (GIBCO, Invitrogen, Carlsbad, CA). The culture medium was replaced every week. After 2 weeks, the cultures were shaken for 1 hr at 110 g and microglial cells were collected from the supernatant.
Culture medium was added to the remaining adherent cells, and microglia were collected every 1-2 weeks, in a total of four collections.

| Human organotypic retinal cultures
Retinal pieces with similar sizes were cultured in transwell inserts with a 0.4 μm pore diameter (Millipore Bioscience Research Reagents, Billerica, MA) with the ganglion cell layer facing up in Neurobasal-A medium (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 2% FBS, 2% B27, 200 mM L-glutamine and antibiotics (Antibiotic-Antimycotic Solution, Sigma-Aldrich, St Louis, MO), as described previously (Niyadurupola, Sidaway, Osborne, Broadway, & Sanderson, 2011;Portugal et al., 2017). Immediately after dissection, retinal pieces were placed in a standard cell incubator with 5% CO 2 for 1 hr before beginning the experiment. From each donor, the three experimental conditions were prepared in duplicate, thus allowing an internal control per experiment.

| Cultures treatment
Cell cultures were incubated with 50 nM of the selective A 2A R antagonist SCH 58261 (Tocris Bioscience, Bristol, UK) 45 min before placing the cultures inside the pressure chamber.
In BV-2 microglial cells, the knockdown of A 2A R was accomplished by small interfering ribonucleic acid (siRNA). Cells were transfected with 12 pmol of siRNA against Adora2A (siRNA ID: s62046) or with nontargeting control sequences (Silencer ® Select negative control #1 siRNA; Ambion, Foster City, CA). Briefly, cells were plated in Opti-MEM ® (GIBCO, Invitrogen, Carlsbad, CA) at a density of 6 × 10 3 cells/cm 2 and then incubated with siRNA for 48 hr (medium replacement and reinforcement of siRNA at 24 hr). The transfections were performed using lipofectamine ® RNAiMAX reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
Microglial contribution to EHP-induced cell death was elucidated by depleting microglia from primary retinal neural cell cultures before exposure to EHP. Briefly, on the third day in culture, primary retinal neural cell cultures were incubated with 2% clodronate-loaded liposomes (Van Rooijen & Sanders, 1994) in fresh culture medium for 24 hr. Then, the cell culture medium was replaced by previously collected supernatant.
Cultures were submitted to elevated hydrostatic pressure (EHP; 70 mmHg above atmospheric pressure) for 4 hr or 24 hr, as previously described (Madeira, Elvas, et al., 2015;Sappington, Chan, & Calkins, 2006). Control cultures were kept at atmospheric pressure in a standard cell incubator.

| Western blot
Protein extracts were prepared in ice-cold radioimmunoprecipitation assay (RIPA) buffer with 1 mM of dithiothreitol (Sigma-Aldrich, St. Louis, MO) and complete mini protease inhibitor cocktail tablets (Roche, Sigma-Aldrich, St. Louis, MO). Western blot was performed as previously described . Membranes were probed with the antibodies indicated in Table 1. Blots were developed with ECL (Clarity™ from Bio-Rad, CA) and WesternBright Sirius from Advansta, Menlo Park, CA) or with ECF™ (GE Healthcare Amersham™, Little Chalfont, UK), in accordance with the manufacturer's instructions. Membranes were reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control.

| Morphometric analysis of microglia
Morphological alterations in microglia from primary retinal neural cultures were assessed as previously described (Kurpius, Wilson, Fuller, Hoffman, & Dailey, 2006). First, threshold was arbitrarily but uniformly applied to confocal images labeled with CD11b. Next, the particle measurement feature of ImageJ (http://rsb.info.nih.gov/ij/) was SCR_002074), as previously described (Tavares et al., 2017). From this analysis, the morphologic parameters assessed were a total number of processes, total length, last intersection radius, and Sholl analysis.

| Dihydroethidium staining
The production of reactive oxygen species (ROS) was assessed by dihydroethidium (DHE) (Invitrogen, Carlsbad, CA) staining (Reyes, Brennan, Shen, Baldwin, & Swanson, 2012). Culture medium was collected and the cells were incubated with 5 μM DHE prepared in fresh culture medium for 1 hr in the cell incubator. Then, the medium with DHE was replaced by the previous collected medium and the cultures were maintained for 4 hr in EHP or in control conditions. The cell cultures were rinsed with warm PBS and fixed.

| Griess reaction assay
The production of NO was assessed by the Griess reaction method.
The culture medium was collected and centrifuged to remove cell debris and then incubated (1:1) with the Griess reagent mix (1% sulfanilamide in 5% phosphoric acid with 0.1% N-1-naphtylenediamine) for 30 min, protected from light. The optical density was measured at 550 nm using a microplate reader (Synergy HT; Biotek, Winooski, VT).
The nitrite concentration was determined by comparison to a sodium nitrite standard curve.

| Cell proliferation assay
Cell proliferation was assessed using the Click-iT ® EdU cell prolifera-

| Phagocytosis assays
Phagocytosis was assayed with fluorescent latex beads (1 μm diameter) in BV-2 cells and primary microglia as we previously described (Madeira, Boia, et al., 2016), with minor modifications as follows. Cells were incubated with 0.025% beads for 60 min at 37 C. In the end, cells were fixed, and BV-2 cells were stained with phalloidin conjugated to tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC, 1:500; Sigma-Aldrich, St Louis, MO) or labeled with anti-CD11b in primary microglia using the antibodies described in Table 1. Nuclei were stained with DAPI (1:2,000). The phagocytic efficiency (Phago Eff.) was calculated with the following formula, as previously described (Madeira, Boia, et al., 2016;Pan et al., 2011).
xn represents the number of cells containing n beads (n = 1,2,3, ... up to a maximum of 6 points for more than 5 beads per cell).
In primary retinal microglia, phagocytosis was also evaluated with dead cells. Primary retinal neural cell cultures were exposed to UV light (200-280 nm) for 30 min and then cultured overnight. Dying/ dead cells were then labeled with 1 μg/mL of propidium iodide (PI) and washed twice with PBS. The number of PI + cells was counted and 5 × 10 4 cells/mL were added to microglia 1 hr before the end of the experiments. Microglial cells were washed, fixed, and then immunolabeled using the CD11b antibody (Table 1). Nuclei were stained with DAPI (1:2,000).

| Scratch wound assay
Confluent BV-2 cells, plated in six-well plates, were wounded with a sterile p200 pipet tip and washed to remove nonadherent cells. Cells were subsequently cultured for 4 hr in control or EHP conditions. Images (before, immediately after and 4 hr after the wound) were acquired with an inverted fluorescence microscope (Zeiss Axio HXP-120, Zeiss, Oberkochen, DE). The number of cells in the scratch before was subtracted to the number of cells in the scratch after

| Boyden chamber migration assay
Microglia migration was assessed in BV-2 cells and in primary microglia using the Boyden chamber migration assay as described (Siddiqui, Lively, Vincent, & Schlichter, 2012). BV-2 cells were kept in serumfree medium for 24 hr before beginning the experiment. Cells (BV-2 cells in 2% FBS or primary microglia in 10% FBS) were plated in transwell culture inserts with 8.0 μm pore diameter (Merck, Millipore, Billerica, MA) at a density of 3 × 10 4 cells/cm 2 and then cultured for 4 hr in control or EHP conditions. Inserts were washed with warm PBS and following fixation in 4% paraformaldehyde with 4% sucrose for 10 min the cells in upper side were removed with a cotton swab.
Nuclei were stained with DAPI (1:2,000) to allow cell counting. The membrane was removed and mounted with Glycergel in glass slides.
The preparations were observed in an inverted fluorescence microscope (Zeiss Axio HXP-120, Oberkochen, DE) and five random images per preparation were acquired (10× objective). The number of cells in the bottom side of the insert (the cells that migrated) was counted.

| Real-time quantitative polymerase chain reaction
The cells were washed with ice-cold RNAse-free PBS and total RNA was extracted using the TRIZOL reagent (Life Technologies, Carlsbad, CA). RNA concentration and purity were determined using  Portugal). To confirm nongenomic DNA synthesis a standard endpoint PCR for β-actin, using intron-spanning primers (Table 2), was performed with 2× MyTaq Red Mix (Bioline, London, UK), as previously described (Santiago et al., 2009). The cDNA samples were diluted 1:2 and kept at −20 C until further analysis.
The mRNA expression levels were quantified by quantitative polymerase chain reaction (qPCR) in a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA) using a mix of 2 μL of 1:2 cDNA, 200 nM primers (Table 2; Sigma-Aldrich,
Three housekeeping candidate genes were tested: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (ywhaz), hypoxanthine phosphoribosyltransferase 1 (hprt1), and TATA box binding protein (tbp). All samples were analyzed using NormFinder (Andersen, Jensen, & Orntoft, 2004), and the most stable gene among all samples and conditions was used as a housekeeping gene. In our conditions, ywhaz was the most stable gene and was used as a housekeeping gene.

| Enzyme-linked immunosorbent assay
The quantification of IL-1β and TNF in the culture medium was performed by enzyme-linked immunosorbent assay (ELISA), in accordance with the manufacturer's instructions (PeproTech, London, UK).
Culture supernatants were collected and kept at −80 C until further analysis. Readings of the optical density were collected for 2 hr in 5 min intervals at 405 nm with wavelength correction set at 650 nm, using a microplate reader (Synergy HT; Biotek, Winooski, VT).

| Statistical analysis
Results are presented as mean AE SEM. Shapiro-Wilk normality test was used to assess the normality of the data. Statistical analysis was performed using GraphPad Prism software version 6.01 for Windows    (Santiago et al., 2007;Santos-Carvalho, Elvas, Alvaro, Ambrósio, & Cavadas, 2013). In these cultures, all (100%) CD11b-immunoreactive microglial cells express A 2A R (Figure 1c). This observation was confirmed by the omission of the antibody anti-CD11b, in which cells immunoreactive to A 2A R have morphology consistent with microglia (Supporting Information Figure S1), faint labeling was also observed in other cells of the culture (Figure 1c). The exposure to EHP increased A 2A R immunoreactivity in CD11b + cells (Figure 1d).

| A 2A R pharmacological blockade or genetic deletion prevents EHP-elicited increase in microglia migration and phagocytic efficiency
Taking into consideration that EHP increased A 2A R density in microglia, we evaluated whether impeding A 2A R signaling could affect the motility of microglia. BV-2 microglial cells were exposed to EHP for  (d) The number of cells in the wound per field was counted, n = 3. In every experiment with siRNA A 2A R, the decrease in the protein levels was confirmed by Western blot. (e) Phagocytosis was assessed in BV-2 cells exposed to EHP for 24 hr using fluorescent beads. Representative images of BV-2 cells stained with phalloidin (red) with incorporated beads (green). Nuclei were counterstained with DAPI (blue). (f ) The phagocytic efficiency was calculated, n = 4-6. *p < 0.05, **p < 0.01, ****p < 0.0001, compared with control; #p < 0.05, ##p < 0.01, compared with EHP; Kruskal-Wallis test, followed by Dunn's multiple comparison test. Scale bar: 50 μm [Color figure can be viewed at wileyonlinelibrary.com] increase in microglia phagocytosis (Phago Eff. = 13.1 AE 3.6%; p < 0.05) ( Figure 3d). Microglia proliferation was evaluated with EdU ( Figure 3e and f ), an analog of thymidine that intercalates in the DNA during S phase, and, therefore, a nuclear marker of cell division (Salic & Mitchison, 2008 Figure 4a and b). In order to assess if the increase in microglial cell number was due to cell proliferation, we performed a proliferation assay (Figure 4a and c) The antagonist of A 2A R prevents EHP-induced changes in microglia morphology and cell response. Primary retinal neural cell cultures were pretreated with 50 nM SCH 58261 following exposure to EHP for 24 hr. (a) Microglial cells were labeled using anti-CD11b (red) and cell proliferation was measured by counting the number of EdU + cells (green). Nuclei were counterstained with DAPI (blue). (b) The number of CD11b + cells per field was counted, n = 6-7. (c) The number of microglial cells proliferating (EdU + CD11b + cells) was counted and the results are expressed as the ratio EdU + CD11b + /CD11b + , n = 6-8. (d) The circularity index was determined using ImageJ, n = 6-8. The mRNA expression levels of CD11b (e), TSPO (f ) and MHC II (g) were assessed by qPCR and the results are presented as fold change of the control, n = 4-6. **p < 0. We also assessed the circularity index (CI) of microglial cells, which can be used as a measurement of the microglia morphology ( Figure 4d). Microglia shifted from a more ramified morphology in control conditions (CI = 0.19 AE 0.01) toward a more amoeboid morphology when exposed to EHP for 24 hr (CI = 0.41 AE 0.05; p < 0.01).

| Blockade of A 2A R prevents oxidative stress and the release of pro-inflammatory mediators triggered by EHP
The impact of EHP on oxidative stress of retinal neural cells was assessed by evaluating DHE staining following exposure to EHP for 4 hr (Figure 5a). The exposure to EHP significantly increased the num- Overall, this group of results suggests that EHP triggers neural cell death mediated by TNF and IL-1β and that the A 2A R antagonist is able to prevent this effect probably by decreasing the levels of these proinflammatory cytokines. In order to further confirm this hypothesis, we co-incubated retinal neural cells with anti-TNF, anti-IL-1β, and SCH 58261 prior the exposure to EHP and determined cell death. The neutralization of the actions of TNF and IL-1β decreased the number of TUNEL + cells induced by EHP, which was not significantly different when the cells were co-incubated with SCH 58261 (Supporting Information Figure S2). This lack of additive effects indicates that the protective effects of A 2A R antagonist in EHP conditions occur by the modulation of these pro-inflammatory cytokines. Interestingly, when each antibody against the pro-inflammatory mediators has incubated alone the effect on the reduction of cell death was not so pronounced (Supporting Information Figure S2).  Figure 6d).

| A 2A R blockade prevents EHP-induced cell death and engulfment of dead cells by microglia
The expression of triggering receptor expressed on myeloid cells 2 (Trem2) has been associated with phagocytosis of apoptotic neuronal cells (Hsieh et al., 2009;Kawabori et al., 2015). The mRNA levels of Trem2 increased by 2.2 AE 0.5-fold in cultured retinal cells exposed to EHP (p < 0.001; Figure 6e), when compared with the control. The incubation with SCH 58261 decreased the expression of Trem2

| Microglia depletion prevents EHP-induced cell death in primary retinal neural cell cultures
In order to assess the role of microglia in neural cell death in EHP conditions, microglia were depleted from the cultures with clodronateloaded liposomes (Kumamaru et al., 2012). The incubation with clodronate liposomes for 24 hr was able to eliminate microglia, as observed by the absence of CD11b + cells (Figure 7a). The depletion of microglia clearly decreased the number of TUNEL + cells (120.2 AE 17.5% of the control; p < 0.05) when compared with cultures exposed 24 hr to EHP (284.9 AE 41.1% of the control; p < 0.01). In addition, when the cultures were incubated with 25 nM PLX3397, an inhibitor of colony stimulating factor 1 receptor (Elmore et al., 2014), thus decreasing the number of microglial cells under EHP to 41.9 AE 2.2% of the control, the EHPinduced cell death was prevented (Supporting Information Figure S3).
These results strongly suggest that microglia contribute to the neural cell loss under EHP conditions.
In order to establish whether microglial cells are the source of TNF and IL-1β under EHP conditions, the concentration of these cytokines was measured in supernatants of microglia-depleted cultures IL-1β and exposed to EHP for 24 hr. Cell death was assessed with TUNEL assay. The number of TUNEL + cells (green) was counted (g). Nuclei were stained with DAPI (blue). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with control; #p < 0.05, ##p < 0.01, ###p < 0.001; compared with EHP, Kruskal-Wallis test, followed by Dunn's multiple comparison test (b, c, e, g) or one-way ANOVA followed by Sidak's multiple comparisons test (d). Scale bar: 50 μm [Color figure can be viewed at wileyonlinelibrary.com] (Supporting Information Figure S4). The levels of TNF were maintained elevated in the clodronate-treated cultures exposed to EHP (Supporting Information Figure S4A), while the levels of IL-1β decreased to control levels (Supporting Information Figure S4B).
These results indicate that in these cultures when exposed to EHP, microglia release IL-1β but not TNF. was performed by Sholl analysis (Figure 8f and g). Human microglia exposed to EHP presented a reduction in branching complexity as a function of distance from the nucleus compared with control, which was slightly reestablished by the treatment with SCH 58261. The maximum intersection radius (extracted from the Sholl analysis), which provides an estimation of the size of the "territory" occupied by microglia, revealed a 1.6 decrease in microglia last intersection, indicating that human microglia exposed to EHP are less complex. Treatment with SCH 58261 prevented the decrease in microglia arborization, as shown by the increase in the maximum intersection radius (from 30.8 AE 2.2 μm in EHP to 43.1 AE 2.9 μm in EHP with SCH 58261; p < 0.05).
We then addressed if elevated pressure increased oxidative stress in human organotypic cultures and if A 2A R blockade was able to prevent this effect. The production of ROS was determined by DHE staining (Figure 8h and i). Exposure to EHP for 4 hr significantly

| DISCUSSION
In the present study, we showed that microglial cells act as main players in retinal cell degeneration triggered by elevated pressure and unveiled the protective properties of microglial A 2A R blockade. Moreover, this is the first study reporting that the A 2A R selective antagonist prevents human retinal microglial cell response to elevated pressure.
Previous reports from us and others show that A 2A R is expressed in the retina, including in microglia (Huang et al., 2014;Liou et al., 2008;Madeira, Elvas, et al., 2015;Vindeirinho, Costa, Correia, Cavadas, & Santos, 2013;Zhong, Yang, Huang, & Luo, 2013). The expression of A 2A R has been demonstrated to be upregulated in brain chronic noxious conditions (Cunha, 2005;Vindeirinho et al., 2013;Wittendorp, Boddeke, & Biber, 2004). Elevated intraocular pressure is the main risk factor of glaucoma (Almasieh et al., 2012). In this work, cultures have been exposed to EHP in order to mimic elevated intraocular pressure in vitro (Aires et al., 2016).  2016; Madeira, Elvas, et al., 2015). One of the mechanisms that may explain the protective properties of A 2A R antagonists is the control of microglia-mediated neuroinflammation (Cunha, 2005;Gomes et al., 2011). There is a controversy on the effects mediated by A 2A R in pathological conditions, since A 2A R activation in peripheral immune cells is anti-inflammatory (Hasko & Cronstein, 2013;Hasko & Pacher, 2008;Sitkovsky & Ohta, 2005), and in chronic conditions of the cen- Evidence shows that upon a noxious stimulus microglia become The number of Iba-1 + cells per field was counted, n = 5. Microglia morphologic features were assessed from 3D reconstructed images (c). The total number of processes (d), total length (e), Sholl analysis (f ), and last intersection radius (g) were analyzed from four independent donors in a total of 21-28 cells analyzed per condition. Results represent the average morphologic features from the total number of cells analyzed. ROS production was assessed by DHE staining (h). Nuclei were stained with DAPI (blue). (i) The number of DHE + cells (red) was counted n = 6-7. *p < 0.05, ***p < 0.001, ****p < 0.0001, compared with control; #p < 0.05, ##p < 0.01, compared with EHP; Kruskal-Wallis, followed by Dunn's multiple comparison test (b, d, and f ) or one-way ANOVA followed by Sidak's multiple comparisons test (e, g, and i). Scale bar: 50 μm [Color figure can be viewed at wileyonlinelibrary.com] responsive and migrate toward the site of injury (Lourbopoulos, Erturk, & Hellal, 2015). ATP (Davalos et al., 2005;Dou et al., 2012;Gyoneva, Orr, & Traynelis, 2009;Imura et al., 2013), adenosine (Orr, Orr, Li, Gross, &NO (Scheiblich et al., 2014) are key mediators for microglia mobilization. In this study, we found that the pharmacological blockade or genetic ablation of A 2A R decreased microglia migration elicited by elevated pressure. Elevated pressure triggers an increase in extracellular levels of ATP and adenosine (Madeira, Elvas, et al., 2015;Rodrigues-Neves et al., 2018), suggesting that ATP-derived adenosine signals through A 2A R that is upregulated in elevated pressure conditions, mediating microglia migration.
Adenosine through activation of the A 2A R modulates microglia process retraction, inducing the amoeboid morphology characteristic of motile responsive microglial cells (Gyoneva et al., 2014;Koizumi, Ohsawa, Inoue, & Kohsaka, 2013;Orr et al., 2009  . In this work, hampering the activity mediated by A 2A R was sufficient to prevent the effects of elevated pressure in morphology and motility, probably by directly hindering microglia process retraction and therefore decreasing cell motility. Another important function of microglia is the clearance of cell debris, dying and dead cells (Kettenmann, Kirchhoff, & Verkhratsky, 2013;Wolf, Boddeke, & Kettenmann, 2017). In chronic degenerative diseases, microglia become phagocytes with substantial deleterious effects for neurons or glial cells (Brown & Neher, 2014;Fu, Shen, Xu, Luo, & Tang, 2014;Napoli & Neumann, 2009 (George et al., 2015;Gomes et al., 2013). In primary retinal neural cell cultures, proliferation was not exclusive to microglia. Astrocytes and Müller cells also play important roles in the inflammatory response, and pro-inflammatory mediators increase their proliferation (de Hoz et al., 2016;Dyer & Cepko, 2000;Farina, Aloisi, & Meinl, 2007). Therefore, since these cells are also present in the culture, we cannot rule out the possibility that these cells are also proliferating (Bejarano-Escobar, Sanchez-Calderon, Otero-Arenas, Martin-Partido, & Francisco-Morcillo, 2017).
Retinal neuronal progenitor cells have also been identified in rat retinal cell cultures (Alvaro et al., 2008) and may also be proliferating. The with a response of microglia to elevated pressure. Also, A 2A R antagonist prevented this increase, suggesting that microglial A 2A R controls neuroinflammation, as has been suggested previously (Boia et al., 2017;. The mechanisms underlying microglia alterations in glaucomatous optic neuropathy are not clarified yet. The increase in ROS has been described as an early event in the pathophysiology of neurodegenerative diseases, including glaucoma, perpetuating the activation of microglia and the release of pro-inflammatory cytokines (Block & Hong, 2005;Block, Zecca, & Hong, 2007;Clausen et al., 2008;Liu & Hong, 2003;Lull & Block, 2010;Smith, Das, Ray, & Banik, 2012;Ye et al., 2016). IL-1β and TNF are pro-inflammatory cytokines produced by microglia that are involved in retinal neurodegeneration Tezel & Wax, 2000;Wang et al., 2016;Yuan & Neufeld, 2001). Elevated pressure elicited a pro-inflammatory environment, characterized by increased production of ROS, and the release of NO, IL-1β, and TNF. In brain glial mixed cultures, the activation of A 2A R increases ROS production by microglial cells (Saura et al., 2005).
Contrarily, the blockade of A 2A R prevented the formation of a proinflammatory environment, decreasing the release of proinflammatory mediators and reducing oxidative stress. Taking into consideration that similar results are observed in the four experimental models (microglia cell line, primary retinal microglia cultures, rat retinal neural cultures, and human tissue cultures), one can hypothesize that the effects are mediated by the A 2A R present in microglia.
The neutralization experiments using anti-IL-1β and anti-TNF antibodies further corroborate the causal role of inflammation to retinal cell death. In our previous works (Madeira, Boia, et al., 2016;Madeira, Elvas, et al., 2015), we could not identify the cells releasing these cytokines, since other cells may participate in the inflammatory response (Chong & Martin, 2015;Soto & Howell, 2014;Wang, Cioffi, Cull, Dong, & Fortune, 2002;Yuan & Neufeld, 2000). In this work, we clearly demonstrate that microglia orchestrate an inflammatory response that is absolutely critical to damage retinal neurons. Interestingly, in the primary retinal neural cultures, we were able to identify microglia as the source of IL-1β, but not TNF. In these cultures, TNF may be released by astrocytes and Müller cells, in the inflammatory response (Vargas & Di Polo, 2016;Yuan & Neufeld, 2000). This might be particular to the experimental conditions, where several cell types are present, since primary retinal microglia express TNF, when exposed to elevated pressure (Madeira, Boia, et al., 2016), despite at lower levels when compared with the expression of IL-1β.
Elevated pressure increased neural cell death, and under these circumstances, apoptotic microglial cells were rarely observed.
Although we cannot discard a direct impact of elevated pressure on neuronal function, since elevated pressure induces apoptosis in neuronal cell lines (Agar, Li, Agarwal, Coroneo, & Hill, 2006;Agar, Yip, Hill, & Coroneo, 2000), our findings show that elevated pressure changes microglia phenotype and these cells mount a pro-inflammatory response that culminates in neuronal apoptosis. The essential role of microglia for neural apoptosis was further confirmed with the clodronate liposomes experiments. Under elevated pressure conditions, we detected microglia with engulfed dead/dying cells, as already observed in other disease models (Ferrer-Martin et al., 2014;Jones et al., 1997;Petersen & Dailey, 2004). Microglia clearance of dead/ dying cells is part of a mounted strategy to control the neuronal damage (Neumann, Kotter, & Franklin, 2009). Indeed, elevated pressure increases the phagocytosis by BV-2 cells, without increasing BV-2 cell death (data not shown). Taken together, these results indicate that the blockade of A 2A R halts microglial cell response, thus inhibiting retinal cell death and reducing phagocytosis.
The potential anti-inflammatory and protective effects of the A 2A R antagonist were evaluated in human organotypic retinal cultures. SCH 58261 is also a potent and selective antagonist for human A 2A R (Jones et al., 1997;Ongini, Dionisotti, Gessi, Irenius, & Fredholm, 1999) and it is suitable for in vitro pharmacological studies (Yang et al., 2007). In this model, we found that A 2A R blockade by SCH 58261 was able to prevent microglia morphological alterations and ROS production triggered by elevated pressure, further reinforcing its role in the control of retinal neuroinflammation.
In summary, our results demonstrate that the blockade of microglial A 2A R affords protection to retinal cells through the control of microglia response to damage, thus identifying microglial cells as major contributors for retinal cell death induced by elevated pressure.
In addition, we also demonstrate that the A 2A R antagonist prevents microglial cell response in the human retina. The A 2A R emerges as an attractive target to manage retinal neuroinflammation in glaucoma.