Introduction
Multiple sclerosis (MS) is a progressive demyelinating inflammatory disease of the central nervous system (CNS). In MS, as well as in its well-characterized animal model, experimental autoimmune encephalomyelitis (EAE), peripheral autoreactive T-cells specific for myelin antigens infiltrate the CNS and initiate an inflammatory reaction resulting in dysfunction of the blood–brain barrier (BBB), demyelination and neurodegenerative changes (Lassmann
2018). Local inflammation is fueled by T cell-derived cytokines which further activate both microglia and astroglia. Excessive activation of glial cells has deleterious consequences in the form of the release of signaling molecules involved in inflammation and neurodegeneration such as cytokines, reactive oxygen/nitrogen species and glutamate (Dheen et al.
2007).
One of the functions of microglia is to receive signals sent by injured cells and to react by removing cellular remnants in the process of phagocytosis (Monif et al.
2010). It has been reported that CNS-resident microglia are inert (Amadio et al.
2017) or less active (Barnett and Prineas
2004) during the initial stage of MS/EAE and participate rather in later phases of the disease, contributing to the release of proinflammatory cytokines and the removal of myelin debris within plaques. In MS patients, positive correlation was found between activation of microglia and destruction of myelin sheaths (Lassmann et al.
2007). Increased reactivity of microglia was also identified in EAE mice in the symptomatic phase correlating with inflammatory infiltration of parenchyma (Ayers et al.
2004). Without fail, activation of microglia accompanies development of both MS and EAE. Moreover, regulation of microglia activity may influence the outcome of the disease. Inhibition of macrophages/microglia at the onset of EAE symptoms (i.e., day 7 post immunization) was found to significantly decrease the progression of neurological deficits (Bhasin et al.
2007). It does not mean, however, that activated microglia only exert an adverse proinflammatory impact. Phagocytosis of myelin debris in MS lesions, expression of anti-inflammatory and tissue repair factors by activated microglia are essential processes to promote remyelination (Luo et al.
2017; Napoli and Neumann
2010). As it has been reported, functions of activated microglia are complex and the final effect depends on the timing and the form (proinflammatory M1/anti-inflammatory M2) of activation (Gao and Tsirka
2011).
Extracellular ATP is a strong signaling molecule in the CNS responsible for intercellular communication (Cotrina et al.
2000; Inoue et al.
2007) and acts as a natural agonist of an array of ionotropic (P2X) and metabotropic (P2Y) purinergic receptors. Among the P2X type of ATP-gated ion channels, the P2X7 receptor is widely expressed in brain cells (Sperlagh et al.
2006) and plays a substantial role in numerous brain pathologies, including MS (Sperlagh and Illes
2014).
Purinergic signaling, particularly P2X7R-mediated signaling, plays a pivotal role in activation and proliferation of microglia as shown in cultures of hippocampus tissue (Monif et al.
2009). Numerous reports have also showed that over-expression/over-activation of this receptor underlies a microglia-induced inflammatory reaction which is associated with the release of inflammatory and bioactive substances (Suzuki et al.
2004; Inoue
2002). Released inflammatory mediators drive a self-propagating cycle via an autocrine mechanism which further promotes neuroinflammation and neurodegeneration (Monif et al.
2010). P2X7R-induced depolarization and associated K
+ efflux (Riedel et al.
2007) leads to activation of a protein complex known as the inflammasome, via a caspase 1-dependent mechanism. The activated inflammasome causes proteolytic cleavage of the inactive form of IL-1β (pre-IL-1β, 30-35 kDa) and secretion of a mature form of interleukin IL-1β (18 kDa) (Mariathasan et al.
2006) which initiates the inflammatory cascade (Mingam et al.
2008). Evidence also exists that P2X7R is involved in the release of IL-6 (Solini et al.
1999). Thus, this receptor significantly contributes to the inflammatory process.
In spinal cords of MS patients, P2X7 was found to be upregulated in plaques formed around blood vessels mainly in activated microglial cells/macrophages (Amadio et al.
2017). Moreover, P2X7R-deficient mice were found to be more resistant to EAE than wild-type mice exhibiting reduced neuroinflammation and axonal damage (Sharp et al.
2008).
With the knowledge that activated microglia-dependent inflammation is implicated in the pathogenesis of MS, we focused on the response of this pool of glial cells during the course of EAE. We have addressed the question of whether microglia are activated in the pre-onset phase of EAE, and release proinflammatory cytokines and whether this activation is P2X7R dependent. We started the analysis at the preclinical stage (day 2–4 p.i.), well before the first neurological deficits appeared, and concentrated on obtaining evidence of activation of microglia and protein expression of cytokines such as interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor (TNF-α). The potential role of the P2X7R purinergic receptor in inducing activation of microglia in brains of immunized rats was verified using Brilliant Blue G (BBG), a selective antagonist of P2X7R.
Materials and methods
Animal model of EAE
Female Lewis rats weighing 190–200 g and sourced from the Animal House at the Mossakowski Medical Research Centre of the Polish Academy of Sciences (Warsaw, Poland) were used throughout the experiments. Spinal cords for rat immunization were isolated from guinea pigs obtained from Charles River Laboratories International Inc., Germany. Experimental procedures involving animals were performed in accordance with EU Directive 2010/63/EU and approved by the local Experimental Animal Care and Use Committee (Approval no. 48/2011). The number of Ethics Committee approval (48/2011) consists of the decision number (48) and the year of decision (2011).
EAE was induced according to our standard protocol. Briefly, rats were immunized with inoculum containing spinal cord of guinea pig homogenized in PBS and emulsified in Freund’s complete adjuvant with 2 mg/mL of
Mycobacterium tuberculosis (H37Ra) (Difco, Detroit, MI, USA). A single intradermal injection of 100 μL of inoculum was administered into each footpad of animals. A selective antagonist of P2X7R, Brilliant blue G (BBG), was dissolved in saline solution and administered daily to EAE rats in a dose of 50 mg/kg b.w. starting from day 0 until day 6 post immunization via a catheter implanted into the internal jugular vein. Appropriate control groups were also used. The vehicle control group received saline instead of BBG. The dose of the antagonist was selected based on the literature (Carmo et al.
2014; Geraghty et al.
2017) and our own preliminary studies.
The condition of the animals was monitored daily. Disease progression was assessed based on the developing neurological deficits scored as described previously (Grygorowicz et al.
2016) using the following scale: 0- no symptoms; 1- limp tail; 2- hind limb weakness; 3- hind limb paralysis; 4- ascending paralysis; and 5- moribund (Kerschensteiner et al.
2004). Animals were sacrificed at different time-points of the disease: in the asymptomatic (4 d.p.i.) and symptomatic phases (12 d.p.i.). To analyze the temporal profile of cytokine proteins, EAE rats were sacrificed at different time-points (2, 4, 6, 8 days) during the asymptomatic phase and in the symptomatic phase (12 d.p.i.). Neither EAE nor EAE + BBG animals were housed longer, until recovery phase of the disease.
After decapitation and rapid preparation, the brains were washed in 50 mM phosphate buffer, pH 7.4, frozen in liquid nitrogen and stored at − 80° C.
Western blot analysis
To prepare homogenates, the forebrains were homogenized in 50 mM phosphate buffer, pH 7.4 containing 10 mM EGTA, 10 mM EDTA, 0.1 mM PMSF and 100 mM NaCl in the presence of a protease inhibitor cocktail (1 μg/mL leupeptin, 0.1 μg/mL pepstatin and 1 μg/mL aprotinin). The protein concentration in homogenates was measured according to the method of Lowry et al. (
1951) using bovine albumin as a standard.
Samples of 20–40 μg of protein/lane were subjected to SDS-PAGE in 10% acrylamide mini-gels, transferred further onto nitrocellulose membranes (Hybond-ECL, Amersham, UK) and examined for the expression of proinflammatory cytokines. Blots were incubated with primary antibody: anti-IL-1β (1:500, R&D System, MN, USA), anti-IL-6 (1:250; R&D System, MN, USA), anti-TNF-α (1:500, R&D System, MN, USA). Monoclonal anti-actin antibody (specific towards α, β, γ forms of the actin) was used as internal standard (1:1000; MP Biomedicals, Warsaw, Poland). Thereafter, the respective secondary anti-goat or anti-mouse HRP-conjugated antibody (Sigma Aldrich, Inc., St. Louis, MO, USA) was applied at a dilution of 1:10,000. Bands were visualized using the ECL kit and quantified by densitometric analysis using ImageScanner III (GE Healthcare) and the ImageQuant TL v2005 program.
Immunohistochemical procedure and microscopic analysis
Animals (four per group) were anesthetized with a lethal dose of Narcotan–Halothanum (Zentiva, Prague, Czech Republic) and perfused through the heart with phosphate-buffered saline (PBS) at pH 7.4 and subsequently with 250 mL of ice-cold fixative (4% paraformaldehyde; Sigma-Aldrich, Inc., St. Louis, MO, USA; in PBS). After post-fixation in the same fixative for 1.5 h, brains were cryoprotected overnight in 10% sucrose in PBS, followed by 20% sucrose for 2 days and 30% sucrose for 4–5 days. Thereafter, frozen tissue was cut into 40-µm sections. The sections were collected free-floating in PBS, pH 7.4 with 0.1% sodium azide and then stored at − 20 °C in antifreeze medium (30% sucrose, 60% glycol ethylene, 0.05 M PBS buffer, pH 7.4).
Immunostaining was performed using primary anti-Iba-1 (1:500; Abcam, Cambridge, GB) and anti-P2X7R antibodies (1:200; Alomone Labs, Jerusalem, Israel), and further with secondary antibody conjugated with Alexa Fluor (1:200; Invitrogen Corp., Carlsbad, CA, USA). To control immunostaining specificity, the primary antibody was omitted from the incubation mixture. Brain sections were mounted on silane slides, air-dried and coverslipped under Vectashield Mounting Medium (Vector). Images were obtained using a confocal laser scanning microscope (Zeiss LSM 510) and processed using the Zeiss LSM 510 software package v. 3.2. Mean fluorescence intensity on micrographs was measured on the whole image area using ZEN Black Edition 2012 software.
Statistical analysis
The results are expressed as the mean ± SD from n experiments. Evaluation of significant differences among groups was performed using one-way analysis of variance (ANOVA) followed by the post hoc Dunnett’s test. p < 0.05 was considered significant.