The Endocannabinoid System Is Dysregulated in Multiple Sclerosis and in Experimental Autoimmune Encephalomyelitis

Diego Centonze; Monica Bari; Silvia Rossi; Chiara Prosperetti; Roberto Furlan; Filomena Fezza; Valentina De Chiara; Luca Battistini; Giorgio Bernardi; Sergio Bernardini; Gianvito Martino; Mauro Maccarrone

Disclosures

Brain. 2007;130(10):2543-2553. 

In This Article

Subjects and Methods

The study was approved by the Ethics Committee of the University of Rome Tor Vergata.

MS Patients

We collected peripheral blood and/or CSF from 26 patients suffering from relapsing-remitting MS (17 females and 9 males, aged 19-43 years), who were admitted to the neurological clinic of the University of Rome Tor Vergata for a relapse of the disease.These patients were suffering from a new symptom or a recrudescence of an old one, since at least two days. At the moment of blood and/or CSF withdrawal, patients were free of corticosteroids and MS-specific immunoactive therapies. The diagnosis of relapsing-remitting MS was established by clinical, laboratory and MRI parameters,and matched published criteria (McDonald et al., 2001). In all instances, patients underwent detection of oligoclonal banding in the CSF, and brain and/or spinal cord MRI. Expanded Disability Status Scale (EDSS) scores were between 0.5 and 3.

As controls, we used peripheral blood from 25 age- and gender-matched healthy volunteers (15 females and 10 males, aged 22-41 years), and CSF from 11 subjects (7 females and 4 males, aged 22-47 years) without inflammatory or degenerative diseases of the central or peripheral nervous system. These subjects underwent lumbar puncture because of clinical suspect of acuteperipheral neuropathy, meningitis or subarachnoidal haemorrhage.All the subjects gave their written informed consent to the study.

MRI Acquisition and Analysis

Three Tesla MRI examination during the disease relapse consisted of dual-echo proton density, FLAIR, T2-weighted spin-echo images and pre-contrast and post-contrast T1-weighted spin-echo images.All images were acquired in the axial orientation with 3-mm thick contiguous slices. T2 lesion volume was determined by manual tracing and the number of gadolinium (0.2 ml/kg e.v.)-enhancing lesions was counted by a neuroradiologist who was unaware of the patient's clinical details.

Correlation with endocannabinoid levels was estimated by regression analysis in Prism 3.0 (Graphpad Software Inc., San Diego, CA)and was inspected visually using scatter plots to determine linearity.

EAE Mice

As described (Pluchino et al., 2003), chronic, relapsing EAE was induced in C57BL/6 mice by subcutaneous immunization with 300 μl of 200 μg MOG(35-55) (Multiple Peptide System) in incomplete Freund's adjuvant containing 8 mg/ml Mycobacterium tuberculosis (strain H37Ra; Difco). Pertuss is toxin (Sigma)(500 ng) was injected on the day of the immunization and again 2 days later. Body weight and clinical score (0 = healthy; 1= limp tail; 2 = ataxia and/or paresis of hindlimbs; 3 = paralysis of hindlimbs and/or paresis of forelimbs; 4 = tetraparalysis;5 = moribund or death) were recorded daily.

Mice receiving or not vehicle instead of MOG were employed as controls. Data from these two control groups gave comparable results, so that they were pooled together. All efforts were made to minimize animal suffering and to reduce the number of mice used, in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC).

Analysis of the Endocannabinoid System

All human and mouse specimens were sent blind to the biochemistry laboratory. For the evaluation of endogenous levels of AEA or 2-AG, lipids were extracted from human CSF or lymphocytes, or from mouse striatal slices, and the organic phase was dried under nitrogen (Maccarrone et al., 2005). Dry pellet was resuspended in 20 μl of methanol, and was processed and analyzed by high performance liquid chromatography with fluorimetric detection(Wang et al., 2001), as reported (Maccarrone et al., 2005).In the case of human lymphocytes, blood from 9 control and 9MS donors (25 ml each) was pooled in three aliquots (3 donors/aliquot),that were used to extract and quantify AEA.

The hydrolysis of [3H]AEA by fatty acid amide hydrolase (E.C.3.5.1.4; FAAH) was assayed in cell or brain homogenates (10μg/test), incubated at pH 9.0 with 5 μM [3H]AEA.The release of [3H]arachidonic acid from [3H]AEA was evaluated through reversed phase-high performance liquid chromatography(RP-HPLC), as already reported (Maccarrone et al., 2003). FAAH activity was expressed as pmol [3H]arachidonic acid released per min per mg protein. FAAH expression was determined in lymphocyte extracts by enzyme-linked immunosorbent assay (ELISA), using anti-FAAH polyclonal antibodies (1:250) and GAR-AP (diluted1:2000), as reported (Maccarrone et al., 2003). Wells were coated with cell extracts (20 μg/well), and the absorbance values at 405 nm of unknown samples were within the linearity range of calibration curves drawn with different amounts of extracts(in the range 0-40 μg/well).

The synthesis of AEA through the activity of N-acyl-phosphatidylethanolamines(NAPE)-hydrolysing phospholipase D (E.C. 3.1.4.4; NAPE-PLD)was assayed in cell or brain homogenates (100 μg/test),using 100 μM [3H]NArPE as reported (Fezza et al., 2005).NAPE-PLD activity was expressed as pmol [3H]AEA released per min per mg protein.

For cannabinoid receptor studies, membrane fractions were prepared from human lymphocytes or mouse brain areas as reported (Maccarrone et al., 2005), and were stored at -80°C for no longer than 1 week. Membrane fractions were used in rapid filtration assays with the synthetic cannabinoid [3H]CP55.940 (400 pM), as described (Gubellini et al., 2002; Maccarrone et al., 2005). The concentration of 400 pM [3H]CP55.940 was chosen on the basis of previous data obtained in similar experimental conditions to avoid saturating effects (Gubellini et al., 2002). In all binding experiments, non-specific binding was determined in the presence of 1 µM ‘cold' agonist (Maccarrone et al., 2005).

Chemicals used in all biochemical assays were of the purest analytical grade. Anandamide (N-arachidonoylethanolamine, AEA), and 5-(1,1'-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)cyclohexyl]-phenol (CP55.940) were purchased from Sigma Chemical Co. (St Louis, MO, USA). N-arachidonoyl-phosphatidyl-ethanolamine (NArPE) was synthesized from arachidonic acid and phosphatidylethanolamine, as reported (Fezza et al., 2005). [3H]AEA (205 Ci/mmol), and [3H]CP55.940 (126 Ci/mmol) were from Perkin-Elmer Life Sciences, Inc. (Boston, MA, USA). [3H]NArPE (200 Ci/mmol) was from ARC (St Louis, MO, USA). Anti-FAAH polyclonal antibodies were prepared by Primm S.r.l. (Milan, Italy) as reported (Maccarrone et al., 2003). Goat anti-rabbit antibodies conjugated to alkaline phosphatase (GAR-AP) were from Bio-Rad (Hercules, CA, USA). N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-3-pyrazole carboxamide (SR141716A) and N-[(1S)-endo-1,3,3-trimethy-1-bicyclo [2.2.1]-heptan-2-yl]5-(4-choro-3-methyl-phenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide (SR144528) were a kind gift of Sanofi-Aventis Recherche (Montpellier, France). Biochemical data reported in this paper are the means ± SD of at least three independent experiments, each performed in duplicate. Statistical analysis was performed by the non-parametric Mann-Whitney U test, elaborating experimental data by means of the InStat 3 program (GraphPAD Software for Science, San Diego, CA).

Electrophysiology on EAE Mice

Corticostriatal coronal slices (200 µm) were prepared from tissue blocks of the mouse brain with the use of a vibratome (Centonze et al., 2005). A single slice was then transferred to a recording chamber and submerged in a continuously flowing artificial cerebrospinal fluid (ACSF) (33°C, 2-3 ml/min) gassed with 95% O2- 5% CO2. The composition of the control solution was (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 glucose, 25 NaHCO3.

In both control and EAE brains, the striatum could be readily identified under low power magnification, whereas individual neurons were visualized in situ using a differential interference contrast (Nomarski) optical system. This employed an Olympus BX50WI (Japan) non-inverted microscope with ×40 water immersion objective combined with an infra-red filter, a monochrome CCD camera (COHU 4912), and a PC compatible system for analysis of images and contrast enhancement (WinVision 2000, Delta Sistemi, Italy). Recording pipettes were advanced towards individual striatal cells in the slice under positive pressure and, on contact, tight GΩ seals were made by applying negative pressure. The membrane patch was then ruptured by suction and membrane current and potential monitored using an Axopatch 1D patch clamp amplifier (Axon Instruments, Foster City, CA, USA). Whole-cell access resistances measured in voltage clamp were in the range of 5-20 MΩ.

Whole-cell patch clamp recordings were made with borosilicate glass pipettes (1.8 mm o.d.; 3-5 MΩ), in voltage-clamp mode, at the holding potential (HP) of -80 mV. To study evoked glutamate-mediated excitatory post-synaptic currents (EPSCs), the recording pipettes were filled with internal solution of the following composition (mM): K+-gluconate (125), NaCl (10), CaCl2, (1.0), MgCl2 (2.0), 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA; 0.5), N-(2-hydroxyethyl)-piperazine-N-s-ethanesulfonic acid (HEPES; 19), guanosine triphosphate (GTP; 0.3), Mg-adenosine triphosphate (Mg-ATP; 1.0), adjusted to pH 7.3 with KOH. Bicuculline (10 µM) was added to the perfusing solution to block GABAA-mediated transmission. Conversely, to detect evoked GABAA-mediated inhibitory post-synaptic currents (IPSCs), intraelectrode solution had the following composition (mM): CsCl (110), K+-gluconate (30), ethylene glycol-bis (ß-aminoethyl ether)-N,N,N',N'-tetra-acetic acid (EGTA; 1.1), HEPES (10), CaCl2 (0.1), Mg-ATP (4), Na-GTP (0.3). MK-801 (30 µM) and CNQX (10 µM) were added to the external solution to block, respectively, NMDA and non-NMDA glutamate receptors. For synaptic stimulation, bipolar electrodes were placed on corticostriatal fibres to study EPSCs, and within the striatum to elicit IPSCs. EPSCs and IPSCs were induced at the frequency of 0.1 Hz, while pulse interval was 50 ms (for EPSCs) or 70 ms (for IPSCs) for the experiments on PPR. The electrophysiological traces were stored on P-CLAMP 9 (Axon Instruments) and analyzed off-line on a personal computer.

Three to seven cells per animal were recorded. For each type of experiment at least four distinct animals were employed. Throughout the text ‘n' refers to the number of cells, unless otherwise specified. For data presented as the mean ± SEM, statistical analysis was performed using a paired or unpaired Student's t-test or Wilcoxon's test. The significance level was established at P < 0.05.

Drugs were applied by dissolving them to the desired final concentration in the bathing ACSF. Drugs were: CNQX (10 µM), HU210 (0.3, 1 µM), MK-801 (30 µM), (from Tocris Cookson, Bristol, UK); bicuculline (10 µM) (from Sigma-RBI, St Louis, USA); SR141716A (1 µM) (from Sanofi-Aventis Recherche, Montpellier, France).

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