Background
Multiple Sclerosis (MS) is a chronic autoimmune, inflammatory and degenerative disease of the central nervous system (CNS) that causes significant disability. Current drugs improve the course of the disease but with limited efficacy, serious side effects and inconvenient routes of administration. For these reasons, there is a need to develop more efficacious drugs (targeting inflammation and also neurodegeneration) that are safer (avoiding life-threatening adverse events, fatal infections or cancer), have non-serious adverse events that impair quality of life (e.g., flu-like symptoms), can be administered orally and have a good profile for eventual combination therapy.
Microglial cells, the resident macrophage populations in the CNS, sustain and propagate inflammation within the CNS through antigen and/or cytokine/chemokine secretion, which are important effectors of the demyelination and neurodegeneration described in MS [
1]. Perivascular microglia act as antigen-presenting cells to myelin-specific T cells and promote the CNS-confined inflammatory process. Once the process is initiated, parenchymal microglial cells are activated and elicit myelin damage and neurodegeneration by secreting pro-inflammatory and neurotoxic factors such as tumor necrosis factor alpha (TNF-α), prostaglandins, interleukin-6 (IL-6), nitric oxide (NO) or reactive oxygen species (ROS) [
2,
3]. Thus, microglial cells are a potential therapeutic target in inflammatory CNS disorders such as MS.
Potassium (K
+) channel modulation is widely pursued as novel pharmaceutical strategy for the treatment of neurological disorders and autoimmune diseases [
4]. In MS, activation on T cells depends on K
+ channel and selective targeting of two-pore domain K
+ channels (K
2P5.1), voltage-gated K
+ channel K
V1.3 and calcium-activated K
+ channel IKCa1 have been proposed for the treatment of CNS inflammation and degeneration [
5‐
7]. ATP-sensitive K
+ (K
ATP) channels are large hetero-octameric complexes consisting of four pore-forming inward-rectifying K
+ subunits (Kir6.x) and four regulatory sulfonylurea receptor (SURx) subunits [
8]. They are considered metabolic sensors that couple cellular energy metabolism to membrane excitability by regulating potassium flux. These channels act as energy sensors of ATP production and are believed to regulate various physiological functions, such as muscle contraction and insulin secretion, by coupling cell metabolism to membrane potential [
9‐
11]. K
ATP channels are also present at the mitochondrial inner membrane (mito-K
ATP) and they participate in the regulation of mitochondrial volume and membrane potential. Furthermore, their activity is related to electronic transport, metabolic energy, ROS production and mitochondrial welfare [
12,
13]. K
ATP channels are found in a range of tissues and they are also widely expressed in various brain regions, where they couple electrical activity of the neuron to its metabolic state, and modulate neuronal excitability in different physiological and pathological conditions [
14‐
16].
We previously reported that activated microglia in a rat model of neurodegeneration and in postmortem samples of patients with Alzheimer's disease (AD) strongly expressed K
ATP channel SUR components similar to those in neurons and pancreatic beta-cells [
17]. In this context, controlling the extent of microglial activation and neuroinflammation may offer prospective clinical therapeutic benefits for inflammation-related neurodegenerative disorders. Other authors have documented that pharmacological activation of K
ATP channels can exert neuroprotective and anti-inflammatory effects on the brain against ischemia, trauma and neurotoxicants [
18‐
21]. Therefore, the expression of K
ATP channels by activated microglia indicates that K
ATP channel openers (KCOs), such as diazoxide, could be used as therapeutic agents to treat inflammatory and neurodegenerative diseases like MS.
Diazoxide (7-chloro-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide) is a well-known small molecule that activates K
ATP channels in the smooth muscle of blood vessels and pancreatic beta-cells by increasing membrane permeability to potassium ions. It is structurally related to the thiazide diuretics, but does not possess any discernible diuretic activity. Its binding site is located on other regions of the SUR protein than the site for other KCOs and binding with similar affinities to SUR1 and SUR2B [
22]. Diazoxide-induced hyperpolarization of cell membranes prevents calcium entry via voltage-gated Ca
2+ channels (VGCCs), resulting in vasorelaxation and the inhibition of insulin secretion [
23,
24]. As a consequence, diazoxide increases the concentration of plasma glucose and produces a fall in blood pressure by a vasodilator effect on the arterioles and a reduction in peripheral resistance. Due to these actions, diazoxide has been approved and used since the 1970s for treating malignant hypertension and hypoglycemia in different European countries, the United States and Canada [
25,
26].
Others authors found that diazoxide-mediated cytoprotection is independent of the conductance of the mito-K
ATP channel inhibiting succinate oxidation and succinate dehydrogenase activity [
27]. These data implicate a direct mitochondrial respiratory inhibition-triggered ROS signaling mechanism in the protection of tissues by diazoxide [
28].
The aims of the present study were to: (a) analyze the expression of KATP channels on microglial cells and whether its pharmacological activation by diazoxide modulates the release of inflammatory mediators, and (b) study the effects of diazoxide oral administration on myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced experimental autoimmune encephalomyelitis (EAE), a murine model of MS.
Methods
Primary cell culture and cell line
The mouse microglial cell line BV-2 was purchased at the Istituto Nazionale per la Ricerca sul Cancro (IST, Genova, Italy), while primary glial cultures were obtained from 2- to 4-day old C57BL/6J mice as described previously by Saura
et al. [
29].
Mice
Female C57BL/6J mice, 8 to 10 weeks of age, were purchased from Charles River (Sulzfeld, Germany) and maintained on a 12:12 h light:dark cycle, with standard chow and water freely available. Animals were handled according to European legislation (86/609/EEC) and all manipulations were performed in accordance with European legislation (86/609/EEC). All efforts were made to minimize the number of animals and their suffering during the experiments, and procedures were approved by the Ethics Committee of the University of Barcelona under the supervision of the Generalitat of Catalunya, Spain.
Reagents
Diazoxide was purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions (50 mM) of diazoxide were prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich). Solutions for cell treatment were prepared by diluting stock solutions in culture media immediately before being added to the cells (DMSO concentration: 0.5%). Solutions for animal treatment were prepared by diluting stock solution in water every day of the treatment (DMSO concentration: 0.3%).
Cell culture and treatment
For primary mixed glial cultures, cells were seeded at a density of 4 × 10
5 cells/mL and cultured in Dulbecco's modified Eagle medium-F-12 nutrient mixture supplemented with 10% heat-inactivated fetal bovine serum (FBS), 0.1% penicillin-streptomycin and 0.5 μg/mL amphotericin B (Fungizone
®) (all from Gibco Invitrogen, Paisley, Scotland, UK). Cells were maintained at 37°C in a 5% CO
2 humidified atmosphere. Medium was replaced every 7 days. After 19 to 21 days
in vitro (DIV), microglia were isolated as described by Saura and collaborators [
29]. Cultures obtained following this method contained > 98% of microglia. The following day, mixed glial and microglial cultures were treated with different concentrations of diazoxide 30 min before stimulation with lipopolysaccharide (LPS) (
E. coli serotype 026:B6) 100 ng/mL and recombinant mouse interferon gamma (IFNγ) (both from Sigma-Aldrich, St. Louis, MO, USA) 10 pg/mL. As control, unstimulated cells and unstimulated cells pretreated with highest diazoxide concentration (100 μM) were used. Both contained the same final concentration of vehicle as the compound-containing wells.
BV-2 cells were cultured in RPMI-1640 medium (Gibco Invitrogen, Paisley, Scotland, UK) supplemented with 10% FBS and 0.1% penicillin-streptomycin. Cells were maintained at 37°C in a 5% CO2 humidified atmosphere. BV-2 cells were seeded at a density of 5 × 104 cells/mL. The following day, cells were treated with diazoxide 30 min before stimulation with LPS 100 ng/mL and IFN-γ 50 pg/mL. Control wells contained the same final concentration of vehicle as the compound-containing wells.
Culture supernatants of BV-2 and primary cells were collected 24 h after LPS/IFN-γ stimulation and stored at -20°C until assayed for nitrites, TNF-α and IL-6 content. Cell viability after treatment was determined by the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction method.
Nitrite, TNF-α and IL-6 quantification
Nitrite levels were quantified by the Griess reaction. Briefly, 50 μL of culture medium was mixed in a 96-well plate with 25 μL of Griess reagent A (sulfanilamide) and 25 μL of reagent B (N-1- naphthyl ethylene -diamine). After color development (10 min at 23 to 25°C), samples were measured at 540 nm on a microplate reader (BioTek ELX800, BioTek Instruments Inc., Vermont, USA). Nitrite concentration was determined from a sodium nitrite standard curve. The amount of TNF-α and IL-6 released into the culture medium was determined using an Enzyme-linked immunosorbent assay (ELISA) kit specific for mouse TNF-α (Murine TNF-α ELISA Development Kit, Peprotech, Rocky Hill, NJ, USA) and for mouse IL-6 (Mouse IL-6 Ready-SET-Go!®, eBioscience, San Diego, CA, USA) according to the manufacturer's instructions.
Immunofluorescence cell staining
BV-2 cells were activated with LPS/IFN-γ for 24 h, as described above. Then, cells were fixed with cold methanol (-20°C) for 5 minutes. Cultures were blocked in phosphate buffered saline (PBS) solution containing 10% donkey serum (Sigma-Aldrich, St. Louis, MO, USA) and 1% bovine serum albumin (BSA) (VWR International Ltd, UK) for 20 minutes. Cells were then incubated with primary antibodies anti-Kir6.1 and anti-Kir6.2 (1:300 dilution, Alomone, Jerusalem, Israel), anti-CD11b (1:500 dilution, Serotec, Oxford, England, UK) at 4°C overnight, followed by secondary antibodies Alexa®488 and 596 (1:500, Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h in blocking solution. Slides were mounted in ProLong Gold antifade medium (Molecular Probes, Invitrogen, Eugene, OR, USA) and images were acquired by SP1 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany), located at the Institut de Biologia Molecular de Barcelona, Microscopy Unit, Parc Científic de Barcelona, Barcelona, Spain.
Phagocytosis assay
The phagocytic ability of microglia was determined by the uptake of 2-μm red fluorescent microspheres (Molecular Probes, Invitrogen, Eugene, OR, USA) by BV-2 cells. Cells were treated with diazoxide 100 μM and activated with LPS/IFN-γ, as described above, and then incubated with microspheres at a concentration of 0.01% for 30 min in the dark at 37°C and 5% CO2. Cells were rinsed twice in PBS solution, pelleted at 1,000 g for 5 min and resuspended in 300 μL PBS. Cells were kept on ice and analyzed by flow cytometry. The single-cell fluorescent population was selected on a forward-side scatter scattergram using an Epics XL flow cytometer (Coulter Corporation, Miami, Florida) located at Technical and Scientific Center-University of Barcelona, Parc Científic Barcelona, Barcelona, Spain.
Some samples were fixed with 3% paraformaldehyde solution and stained using FITC conjugated anti-α-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA) and Hoechst 34580 (Molecular Probes, Invitrogen, Eugene, OR, USA) nuclear staining for image acquisition.
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction method
MTT reduction assay was used as an indicator of cell viability. MTT (Sigma-Aldrich, St. Louis, MO, USA) was added to a well at a final concentration of 0.5 mg/mL. After MTT incubation at 37°C, DMSO was added and cells were gently resuspended. Absorbances at 560 and 620 nm were recorded with a microplate reader (BioTek ELX800, BioTek Instruments Inc., Vermont, USA).
Isolation of total protein
For spinal cord total protein extraction, tissue (100 mg) was placed into a 1.5-mL microtube on ice containing 500 μL ice-cold RIPA extraction buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with complete protease inhibitor cocktail tablets (Roche Diagnostics, Basel, Switzerland). The sample was homogenized with a pipette tip on ice for 30 min. The homogenate was centrifuged at 6000 g for 15 min at 4°C. The supernatant was separated and stored at -80°C until use. For isolation of total proteins from cell cultures, after a cold PBS wash, total proteins were recovered in 100 μL per well of RIPA buffer supplemented with complete protease inhibitor cocktail tablets. The samples were sonicated and stored at -80°C. Protein amount was determined by the Lowry assay (Total Protein Kit micro-Lowry, Sigma-Aldrich, St. Louis, MO, USA).
Western blot
30 to 40 μg of proteins from denatured (100°C for 5 min) total extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis together with a molecular weight marker (Full Range Rainbow Molecular Weight Marker, Amersham, Buckinghamshire, UK), and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After washing in Tris-buffered saline (TBS: 20 mM Tris, 0.15 M NaCl, pH 7.5) for 5 min, dipping in methanol for 10 s and drying in air, the membranes were incubated with the following primary antibodies overnight at 4°C: polyclonal rabbit anti-Kir 6.1 or polyclonal rabbit anti-Kir 6.2 (both 1:500, Alomone, Jerusalem, Israel), polyclonal rabbit anti-inducible nitric oxide synthase (iNOS) (1:200, Millipore, Bedford, MA, USA), polyclonal rabbit anti-cyclooxygenase-2 (1:2000, Santa Cruz Biotechnology, St. Cruz, CA, USA) and monoclonal mouse anti-β-actin (1:50000, Sigma-Aldrich, St. Louis, MO, USA) diluted in immunoblot buffer (TBS containing 0.05% Tween-20 and 5% non-fat dry milk). The membranes were then washed twice in 0.05% Tween-20 in TBS for 15 s and incubated with the following horseradish peroxidase (HRP)-labeled secondary antibodies for 1 h at 23 to 25°C: donkey anti-rabbit (1:5000, Amersham, Buckinghamshire, UK) or goat anti-mouse (1:5000, Santa Cruz Biotechnology, St. Cruz, CA, USA). After extensive washes in 0.05% Tween-20 in TBS, they were incubated in ECL-Plus (Amersham, Buckinghamshire, UK) for 5 min. Membranes were then exposed to the camera and the pixel intensities of the immunoreactive bands were quantified using the percentage adjusted volume feature of Quantity One 5.6.4 software (Bio-Rad Laboratories, Hercules, CA, USA). Data are expressed as the ratio of the band intensity of the protein of interest to the loading control protein band (β-actin).
EAE induction and treatment
EAE was induced by immunization with > 95% pure synthetic MOG35-55 peptide (rat MOG35-55, MEVGWYRSPFSRVVHLYRNGK; EspiKem Srl, Florence, Italy). Mice were injected subcutaneously at one side of the flank with 100 μL solution containing 150 μg of rat MOG in complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA) and 5 mg/mL Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA). Mice also received intraperitoneal injections of 150 ng pertussis toxin (Sigma-Aldrich, St. Louis, MO, USA) in 100 μL PBS immediately after MOG injection and 48 h later. Mice were scored daily for signs of EAE on a scale of 0 to 6 using the following criteria: 0, no clinical signs; 1, distal limp tail; 1.5, complete limp tail; 2, mild paraparesis of the hind limbs, unsteady gait and impairment of righting reflex; 3, moderate paraparesis, partial hind limb paralysis, voluntary movements still possible and ataxia; 4, paraplegia and forelimb weakness; 5, tetraparesis; 6, moribund state. When clinical signs were intermediate between two grades of the disease, 0.5 was added to the lower score. To study the effects of the drug, two different administration protocols were performed: in the first one, treatment began on the first day of EAE induction whereas the second one started when the EAE clinical score was ≥ 1 (appearance of clinical signs). The MOG-immunized mice were administered either 0.8 mg/kg diazoxide (treated group) or diluent (0.3% DMSO in water, vehicle group) for 30 or 15 days by oral gavage, respectively.
Blood glucose measurements
Blood glucose measurements were performed using an Accu-Chek® Aviva glucometer (Roche Diagnostics, Basel, Switzerland). Blood samples were obtained from a small incision made at the distal part of the mice tail. Blood glucose concentrations higher than 176 mg/dL were considered hyperglycemic, according to animal welfare guidelines.
Histological and immunohistochemical analysis
To analyze the efficacy of diazoxide during the chronic effector phase of EAE, histological spinal cord analysis was performed in animals treated from the appearance of the first clinical signs. At the end of treatment, animals were anesthetized, transcardially perfused with 0.01 M PBS, followed by 4% paraformaldehyde solution. Spinal cords were then collected and post-fixed in fresh fixative solution for 4 h. For cryoprotection, they were placed in 30% sucrose for 24 h. Tissue was frozen in isopentane and dry ice and stored at -80°C. Coronal sections (20 μm) at the cervical, thoracic and lumbar levels were obtained in HM550 Cryostat (Thermo Scientific, Waltham, MA, USA) at -22°C and deposited onto poly-L-lysine-coated microscope slides.
Hematoxylin and eosin (H&E), Luxol fast blue (LFB), Nissl and Bielschowsky silver staining were used for histological studies.
For immunohistochemical studies, sections were first blocked in PBS (0.5% Triton) containing 10% goat serum (Sigma-Aldrich, St. Louis, MO, USA) for 2 h. The sections were then incubated with primary antibodies at 4°C overnight, followed by secondary antibodies for 2 h in blocking solution. The following antibodies were used: anti-Kir6.1 and anti-Kir6.2 (1:150 dilution, Alomone, Jerusalem, Israel), anti-CD11b and anti-CD3 (1:400 and 1:300 respectively, Serotec, Oxford, England, UK), anti-glial fibrillary acidic protein (GFAP) (1:2000, Dako, Glostrup, Denmark), anti-CD20 (1:300, Santa Cruz Biotechnology, St. Cruz, CA, USA) and anti-Neuronal nuclei (NeuN) (1:500, Millipore, Bedford, MA, USA). The secondary antibodies used were Alexa®488 and 596 (from 1:2000 to 1:1000, Molecular Probes, Invitrogen, Eugene, OR, USA). To assess the number of cells, the nuclear stain Hoechst 34580 (2 μg/mL; Molecular Probes, Invitrogen, Eugene, OR, USA) was added prior to final washes after secondary antibody addition. Sections were mounted using ProLong Gold antifade medium (Molecular Probes, Invitrogen, Eugene, OR, USA). As absolute controls, non-immunized healthy mice were also analyzed.
Quantification of histology and immunohistochemistry
Images were captured using both wide field microsope Leica AF7000 (Leica Microsystems GmbH, Wetzlar, Germany) located at the Institut de Biologia Molecular de Barcelona, Microscopy Unit, Parc Científic de Barcelona, and SP1 confocal microscope. The analyses were carried out on three randomly selected sections of cervical, thoracic and lumbar spinal cord per animal (n = 4 to 8 animals/group) to assess demyelination, number of inflammatory/infiltration lesions, reactive microglial-macrophage areas, astrocytic reactivity and number of infiltrating cells. To assess axonal loss area and for neuronal counting, the thoracic region (n = 6 to 8 animals/group) was used.
The resulting area and cell measurements were quantified using ImageJ software analysis (National Institute of Health, USA). For astrocytic reactivity, after defining the threshold for background correction, the integrated density of GFAP labeling was measured. The integrated density is the area above the threshold for the mean density minus the background. All analyses were performed blind with respect to the experimental groups.
Statistical Analysis
Data are expressed as the mean ± SEM unless specified. Statistical analysis of cell treatments was carried out using one-way ANOVA followed by Newman-Keuls post test when three or more experimental groups were compared. Data on the effect of EAE treatment on clinical signs, histological and immunohistochemical analysis were analyzed by Student's t-test or Mann-Whitney test for nonparametric data. Values of p < 0.05 were considered statistically significant.
Discussion
K
ATP channels are well known as linkers between cell metabolism and membrane potential. This activity has been classically described in pancreatic beta-cells, where an increase in plasma glucose promotes a calcium-dependent release of insulin due to the closing of K
ATP channels as a result of glycolysis-mediated increases in cytoplasmic ATP levels. K
ATP channels have also been described in the mitochondria, located on the inner membrane of these organelles where they play a crucial role in the maintenance of mitochondrial homeostasis and the proton gradient involved in the respiratory chain [
30].
Besides pancreatic beta-cells, physiologically functional K
ATP channels have been described in numerous cell types such as myocytes, neurons, astrocytes and oligodendrocytes [
31‐
33]. In recent years, the expression of these channels in microglial cells has also been reported [
17,
20]. Whereas Zhou and colls. only asserted the presence of SUR2 and Kir6.1 in microglial mitochondria
in vitro, Ramonet and colls. demonstrated the expression of SUR1 as well as SUR2 in microglia
in vivo. In the present study, we found
in vitro and
in vivo inmunoreactivity for Kir6.1 in microglia and also a clear positive signal for Kir6.2. Moreover, using a specific fluorescence antibody for the K
ATP channel Kir6.X subunits and for the microglial cell membrane marker CD11b, we found that K
ATP channels were not restricted only to the mitochondria of BV-2 microglial cells. Our hypothesis is that microglial cells present functional K
ATP channels at both mitochondrial and cytoplasmic membranes. Further studies are needed to analyze their functional cellular localizations in order to understand how compounds that regulate the activity of this channel affect microglial behavior.
In this way, compounds that can regulate ionic influx in microglia could represent a novel therapeutic approach for the treatment of CNS pathologies associated with microglial-mediated neuroinflammation, including EAE. In the present study, we demonstrated that diazoxide inhibited microglial inflammatory activity
in vitro, coincidently with other authors [
20,
34]. Diazoxide treatment partially inhibited the inflammatory pattern induced by LPS/IFN-γ in microglial cells, inducing a decrease in NO production that could be because of the decreased expression of iNOS detected. We also observed a decrease of two major inflammatory cytokines IL-6 and TNF-α release. These pro-inflammatory agents have been shown to mediate the neurotoxic effects of reactive glial cells in vitro, and the inhibition of their production has been shown to protect against the neurotoxicity induced by reactive glial cells [
35,
36]. For example, expression of inducible iNOS is abundant in EAE and at the edges of MS lesions and NO is one of the main effectors of demyelination [
37,
38]. Microglial IL-6 secretion during EAE has been directly associated to neuronal damage [
39] and leukocyte activation within the CNS [
40]. TNF-α increases severity of EAE, chronic macrophage/microglial reactivity, and demyelination [
41] and its inhibition prevents clinical disease despite activated T cell infiltration to the central nervous system [
42] and promotes axon preservation and remyelination [
43].The absence of any significant effect of diazoxide on COX-2 expression could be explained by the presence of different contributors in the final regulation of COX-2, TNF-α, IL-6 and iNOS genes under inflammatory stimuli [
44,
45]. Furthermore, our results showed that diazoxide had no effect on microglial phagocytosis
in vitro. Since the clearance of debris by microglia is a primordial step for the reparative process in the spinal cord following an autoimmune attack [
46,
47], the maintenance of a phagocytic microglial phenotype with suppressed inflammatory behavior could be an interesting feature in demyelinating diseases. Because activated microglia (and macrophages) could exert a neuroprotective role and promote remyelination [
48,
49], modulation of microglia behavior would be more interesting than a total inhibition of their activation for the treatment of these diseases.
KCOs can decrease rotenone-induced mitochondrial depolarization and p38/c-Jun N-terminal kinase activation in microglia [
20] by acting at the mito-K
ATP channel level but the mechanisms involved with cytoplasmic membrane K
ATP channels, which include changes in membrane potential and calcium influx, are yet to be elucidated. Recent studies have shown that the inhibition of N-type voltage-gated calcium channels reduced the severity of EAE neurological symptoms and decreased demyelination and infiltration areas [
50,
51]. The authors indicated microglia/macrophages as the principal effectors of this improvement, demonstrating that inhibition of these voltage-gated calcium channels regulates microglial activation.
Although the action of KCOs on microglia would be sufficient to explain the improvements observed in EAE mice after diazoxide treatment, the presence of functional K
ATP channels in other glial cells and neurons could explain additional positive CNS effects induced by KCOs, especially diazoxide. In astrocytes, diazoxide exerts a neuroprotective effect by different mechanisms, including the facilitation of glutamate uptake [
52] and amelioration of mitochondrial and connexin 43 dysfunction [
53]. We also observed a decrease in nitrite production and inflammatory cytokines release in primary cultures that included both astrocytes and microglia and a decrease of GFAP reactivity in the gray matter of diazoxide treated EAE mice. In oligodendrocytes, diazoxide has been reported to stimulate oligodendrocyte precursor cell proliferation in a calcium-dependent manner as well as promoting myelination
in vivo and preventing hypoxia-induced periventricular white matter injury [
33]. In neurons, the positive actions of diazoxide on cell survival after cytotoxic and hypoxic/ischemic insult have been well described [
19,
54‐
57]. Moreover, a recent study in a triple transgenic mouse model of AD has demonstrated the beneficial effect of diazoxide on the improvement in cognitive tasks, reduction of anxiety, decrease in the accumulation of amyloid-beta oligomers and hyperphosphorylation of tau proteins [
58]. Diazoxide may also exerts neuroprotective effects independently of K
+ channel activation by decreasing neuronal excitability and activation of
N-methyl-
D-aspartate (NMDA) receptors [
18] or by increasing currents trough α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [
59]. The possibility of diazoxide binding to other ion channels composed by SUR subunits despite of K
ATP channels [
60,
61] should not to be discarded and would need future research.
Taken together, these results reinforce our findings and could explain the differences observed between diazoxide-treated and untreated EAE mice, which included improvements in the neurological score, axon preservation and neuronal survival in addition to a decrease in glial reactivity and myelin loss.
Diazoxide-treated animals showed a decrease in disease severity a few days after the first clinical signs were observed, corresponding to the acute inflammatory phase of the disease [
62]. Interestingly, we did not observe any changes in the number of infiltrating lymphocytes in the spinal cord of diazoxide-treated EAE mice when compared to vehicle-treated ones. Furthermore, the appearance of EAE signs was not prevented by diazoxide pretreatment, suggesting that oral treatment with diazoxide has no effect on the first steps of the pathology that include auto-antigen recognition, adaptive immune response and lymphocyte [
63,
64]. However, the effect of diazoxide on the immune system should be further explored, including direct actions of the compound on lymphocytes and peripheral macrophage populations as well as the distribution of leukocyte subpopulations during the course of EAE. Diazoxide could diminish autoimmune attacks on white matter by inhibiting microglial cells, without altering the initial immune response and infiltration regulating the pro-inflammatory environment and intercellular interactions.
Competing interests
NV, JFEP, PM, APZ, MJR, NM and MP have applied for a PCT application "Diazoxide for use in the treatment of a central nervous system (CNS) autoimmune demyelinating disease" (application number PCT/EP2011/050049).
JG declares no competing interests.
Authors' contributions
NV, JFEP and MP designed the study; NV, JFEP, PM, APZ and JG performed it; NV and JFEP analyzed the data; and NV, JFEP and MP wrote the manuscript. MJR and NM participated in the design of the study and helped draft the manuscript. All authors have read and approved the final version of the manuscript.