The immortalized murine BV-2 microglia cell line was cultured in Roswell Park Memorial Institute (RPMI) medium, supplemented with 10% fetal bovine serum (FBS) and 1% of antibiotics (penicillin 100 U/ml, streptomycin 100 μg/ml) and maintained at 37°C under a humidified atmosphere with 5% CO₂. For experiments, cells were cultured in RPMI with 2% FBS and 1% antibiotics at a density of 6 × 10³ cells/cm² in 6-well-plates or at density of 1 × 10⁴ cells/cm² in 12-well-plates.

Cells were exposed to elevated hydrostatic pressure (EHP, 70 mmHg above atmospheric pressure) for 4 or 24 h, as previously described (Madeira et al., 2016). Control cultures were kept in a standard incubator at atmospheric pressure.

The extracellular levels of ATP were measured with a luciferin-luciferase assay kit, as previously described (Cunha et al., 2000; Madeira et al., 2015b). Briefly, cell culture supernatants were collected and stored at −80°C until used. Cells were collected and protein concentration was determined by the bicinchoninic acid (BCA) method (Pierce Biotechnology). Supernatants were quickly defrosted and incubated with ATP assay mix (Sigma-Aldrich) in an opaque 96-well plate. ATP levels were measured using a VICTOR multilabel plate reader (Perkin Elmer). The ATP concentration in each sample was determined by interpolation with a standard curve obtained from an ATP stock solution and was normalized to the amount of protein. Results are presented as percentage of control.

The extracellular levels of adenosine were quantified using high-performance liquid chromatography (HPLC), as previously described (Vindeirinho et al., 2013). Briefly, cell supernatants were analyzed in Beckman-System Gold HPLC apparatus, with a computer controlled 126 Binary Pump Model using a 166 Variable UV detector (detection at 254 nm) and a Lichrospher 100 RP-18 (5 mm) column from Merck. An isocratic elution with 10 mM phosphate buffer (NaH₂PO₄; pH 6.0) and 14% methanol was performed with a flow rate of 1.5 ml/min. Adenosine was quantified by considering the retention time and absorption spectra, and then comparing with the standard curve. Results are presented as percentage of control.

BV-2 microglial cells were washed with warm reaction buffer (in mM: 2 MgCl₂, 125 NaCl, 1 KCl, 10 glucose, 10 HEPES; pH 7.4), and then incubated with 2 mM adenosine monophosphate (AMP; Sigma-Aldrich) for 40 min at 37°C. The medium was collected to quantify the free inorganic phosphate, as a measurement of AMP degradation with the Malachite Green Phosphate Assay kit, following the instructions provided by the manufacturer (Cayman Chemicals). Inorganic free phosphate was detected by spectrophotometry (620–630 nm). Results are presented as fold-change of control.

BV-2 cells were washed twice with ice-cold phosphate-buffered saline (PBS, in mM: 137 NaCl, 2.7 KCl, 10 Na₂HPO₄, 1.8 KH₂PO₄, pH 7.4) and lysed in 50 mM Tris-HCl (pH 7.2), supplemented with complete mini protease inhibitor cocktail tablets (Roche). The enzymatic activity of ADA was measured following the instructions provided by the manufacturer (Diazyme). Results were normalized to the amount of protein quantified by the BCA method, and are presented as percentage of control.

BV-2 cells were washed twice with ice-cold PBS at 4°C. Cells were lysed in radioimmunoprecipitation assay buffer [RIPA, in mM: 50 Tris HCl, pH 7.4; 150 NaCl; 5 EDTA; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate (SDS)] supplemented with complete mini protease inhibitor cocktail tablets and 1 mM of dithiothreitol (DTT) at 4°C. Samples were denatured by adding 6x concentrated sample buffer (0.5 M Tris, 30% glycerol, 10% SDS, 0.6 M DTT, 0.012% bromophenol blue) and heating for 5 min at 95°C. When blotting for CD39, cells were lysed in non-reduced conditions.

Samples were separated in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% milk and then were incubated with the antibodies indicated in Table 1. Immunoreactive bands were visualized using Enhanced Chemi-Fluorescence system (ECF; GE Healthcare) on a Storm device (Molecular Dynamics, GE Healthcare) or with Enhance Chemiluminescence system (ECL; Bio-Rad) on a Versadoc (Bio-Rad). Digital quantification of band intensity was performed using Quantity One software (Bio-Rad). The membranes were reprobed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to control for similar amounts of protein.

Cells were washed with ice-cold PBS and fixed using 4% of paraformaldehyde (PFA) with 4% sucrose for 10 min at room temperature (RT). After fixation, the cells were washed with PBS and permeabilized during 5 min with 1% Triton X-100 in PBS. Then, cells were blocked with 3% of bovine serum albumin (BSA; NZYtech) and 0.2% Tween-20 in PBS for 1 h at RT to prevent the non-specific binding. Afterwards, cells were incubated with the primary antibody diluted in the blocking solution for 90 min at RT (Table 2). After washing, the cells were incubated with the secondary antibody in the same solution for 1 h at RT in the dark. Then, cells were rinsed with PBS. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI 1:2,000; Molecular Probes, Life Technologies) and F-actin was stained with phalloidin conjugated to Tetramethylrhodamine B isothiocyanate (TRITC, 1:500; Sigma-Aldrich). Upon rising with PBS, the coverslips were mounted in glycergel mounting medium and observed with a fluorescence microscope (Axio Observer.Z1), using a LD Plan-Neofluar 40x/0.6 Korr Ph2 M27 objective.

Representative images were acquired with a confocal microscope (LSM 710, Zeiss) on an Axio Observer.Z1 microscope using Plan-Apochromat 63x/1.40 Oil Dic M27 objective.

BV-2 cells were cultured in serum-free medium for 24 h before the experiment. Then, cells were plated in transwell cell culture inserts (8.0 μm pore diameter; Merck Millipore) in RPMI with 2% FBS and 1% antibiotics. Cells were incubated with apyrase (30 U/mL) and ADA (1 U/mL) followed by exposure to EHP for 4 h. At the end of the experiment, cells were washed with warm PBS and the cells in upper side of the insert were removed with a cotton swab. After fixation with 4% PFA with 4% sucrose during 10 min, the nuclei were stained with DAPI (1:2,000). The membranes were mounted in glass slides with glycergel mounting medium, and the preparations were observed with a fluorescence microscope (Axio Observer.Z1) using a N-Achroplan 5x/0.15 M27 objective. Five random images per sample were acquired. The number of cells in the bottom side of the insert (the cells that migrated) was counted and the results were expressed as percentage of control.

BV-2 cells were plated in RPMI with 2% FBS and 1% antibiotics and exposed to EHP for 24 h. Cells were incubated with apyrase (30 U/mL) and ADA (1 U/mL) 2 h before the end of the experiment. One hour before the end of the experiment, BV-2 cells were incubated with 0.025% fluorescent beads at 37°C. Then, cells were washed with warm PBS and fixed with 4% PFA with 4% sucrose. BV-2 cells were stained with TRITC-conjugated phalloidin (1:500; Sigma-Aldrich) and nuclei were stained with DAPI (1:2,000). Cells were observed with a confocal microscope (LSM 710, Zeiss) using a Plan-Apochromat 20x/0.8 M27 objective and five random fields were acquired from each condition. The total number of cells in each field, the number of cells with incorporated beads and the number of fluorescent beads phagocytized by each cell were counted. The phagocytic efficiency was calculated:

where xn represents the number of cells containing n microspheres (n = 1, 2, 3 … up to a maximum of 6 points for more than 5 microspheres ingested per each cell).

Results are presented as mean ± SEM. The number of independent experiments is indicated in each bar. Statistical analysis was performed using GraphPad Prism Version 6 (GraphPad Software). The normality of the data was assessed with Shapiro-Wilk test. Data was analyzed using the non-parametric Kruskall-Wallis test, followed by Dunn's multiple comparison test. Differences were considered significant for p < 0.05.

BV-2 cells were exposed to elevated pressure for 4 and 24 h and the levels of ATP (Figure 1A) and adenosine (Figure 1B) were quantified in cell culture medium supernatants. The exposure of microglia to EHP for 4 and 24 h increased the extracellular levels of ATP to 233.1 ± 49.9% (p < 0.01) and 187.9 ± 33.4% of control, respectively, and the adenosine levels to 124.1 ± 9.6% and 131.9 ± 9.6% of the control (p < 0.05), respectively.

Adenosine can be formed through the hydrolysis of adenine nucleotides [ATP, adenosine di-phosphate (ADP) and AMP] by a cascade of ectonucleotidases, including CD39 and CD73 that are expressed in several cell types, including microglia (Haskó et al., 2005).

Here, we addressed whether EHP could affect the expression of CD39 as well as AMP catabolism, both involved in adenosine formation through ATP hydrolysis. CD73 was not detected in BV-2 cells either by qPCR or Western blot (data not shown). The protein levels of CD39 significantly increased in BV-2 cells exposed to EHP for 4 and 24 h (147.3 ± 23.1% and 128.6 ± 11.0% of the control, respectively; p < 0.05; Figure 2A), which is in agreement with the previous proposal that CD39 might be a potential indicator of increased extracellular levels of ATP in retina cells (Lu et al., 2007). However, the dephosphorylation of AMP into adenosine was not altered in BV-2 cells exposed to 4 h EHP (1.0 ± 1 fold-change; Figure 2B).

Adenosine can be removed from the extracellular space by degradation into inosine mediated by ADA, while the intracellular the removal of adenosine also occurs by the conversion into AMP mediated by adenosine kinase (Yegutkin, 2008).

The exposure of microglial cells to EHP for 4 and 24 h did not affect the protein levels of ADA and ADK (Figures 3A,C). Nevertheless, the activity of ADA (Figure 3B) significantly decreased in microglia exposed to EHP for 24 h to 67.9 ± 10.2% of the control (p < 0.05).

Extracellular adenosine levels are typically regulated by nucleoside transporters or metabolism (Latini and Pedata, 2001). Hence, we also evaluated the impact of EHP exposure on the expression of nucleoside transporters ENT1, ENT2, and CNT2 in BV-2 cells.

ENT1 was not detected by Western blot (data not shown). The protein levels of ENT2 in BV-2 microglia exposed to EHP for 4 h were similar to the control. However, exposure to EHP for 24 h significantly decreased the protein levels of ENT2 in microglia (46.5 ± 9.6% of the control; p < 0.05; Figure 4A). The protein levels of CNT2 were not altered in BV-2 cells exposed to EHP (83.8 ± 13.5% and 105.2 ± 10.0% of the control, for 4 and 24 h, respectively; Figure 4B).

The actions of adenosine are dependent on the activation of the adenosine receptors. We evaluated the impact of EHP on the expression of A₁R and A₃R receptors in BV-2 microglial cells.

The exposure of BV-2 cells to EHP, for 4 and 24 h, decreased the protein levels of A₁R to 64.5 ± 15.6% and 45.9 ± 4.2% (p < 0.05) of the control, respectively, as assessed by Western blot (Figure 5A). As we previously reported (Lopes et al., 2003), Western blot for A₃R yields three bands but we quantified the more intense 44 kDa that corresponds to the expected molecular weight of A₃R. This analysis revealed that the protein levels of A₃R (Figure 5B) did not change in microglial cells upon exposure to EHP for 4 and 24 h (97.4 ± 17.2% and 124.0 ± 27.8% of the control, respectively).

The effects of EHP on the immunoreactivity of A₁R and A₃R were also assessed by immunocytochemistry in microglia (Figures 5C,D). In microglial cells exposed to EHP, A₁R immunoreactivity decreased when compared with the control, whereas the A₃R immunoreactivity was similar to the control, supporting the results obtained by Western blotting.

A feature of reactive microglia is their ability to migrate toward the site of injury, where they release pro-inflammatory mediators and phagocyte cell debris (Karlstetter et al., 2010). Although the role of the purinergic system in microglia chemotaxis is well established (Honda et al., 2001; Davalos et al., 2005; Wu et al., 2007), the contribution of ATP and adenosine to the motility of microglia in conditions of elevated pressure it still unknown. Therefore, we evaluated whether EHP could affect microglia migration and if ATP and adenosine affected this process (Figure 6). When microglial cells were exposed to EHP for 4 h, the number of microglia that migrated to the bottom side of the transwell significantly increased to 220.5 ± 22.8% of the control (p < 0.05). The treatment of BV-2 cells with 30 U/ml apyrase, to remove the extracellular ATP, prevented the effect of EHP in microglia migration (98.0 ± 10.4% of the control; p < 0.01 compared with EHP). BV-2 cells were also pre-treated with 1 U/ml ADA, to remove the extracellular adenosine. The removal of extracellular adenosine also prevented the migration of microglial cells to the bottom side of the membrane elicited by EHP (115.5 ± 19.2% of the control, p < 0.01 compared with EHP). This strict dependency on extracellular ATP and adenosine for the increased migration of microglia upon EHP is also an important control to exclude the possibility that migration in EHP conditions might result from cells that were just pushed through the pores.

Increased phagocytic efficiency is a feature of reactive microglia (Kettenmann et al., 2011). The contribution of adenosine and ATP to the phagocytic efficiency of microglia upon exposure to EHP was assessed by incubating microglia with fluorescent microbeads (Figure 7). The phagocytic efficiency of microglia exposed to EHP increased by nearly 200% when compared with control cells (from 13 ± 2% in control conditions to 25 ± 3% under EHP; p < 0.01). Incubation with apyrase (30 U/ml) and ADA (1 U/ml) was unable to decrease the phagocytic efficiency in cells under EHP (23 ± 2% and 19 ± 3%, respectively).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.