The experiments were carried out in accordance with the protocol for animal care approved by the European Communities Council Directive (2010/63/EU) with permission of the State Veterinary and Food Administration of the Slovak Republic (4451/14–221 and 4247/15–221) under the supervision of the ethical council of the Institute of Neurobiology BMC SAS. Every effort was made to minimise animal suffering and reduce the number of animals used. Adult male albino Wistar rats (bred at a certified vivarium of the Institute of Neurobiology BMC SAS originating from Velaz, Czech Republic) weighing 330–350 g were maintained on a 12 h light/dark cycle and given food and water ad libitum. Food was withdrawn one day before surgery.

Experimental Design

Animals were randomly divided into 4 groups. Groups A and B were pre-treated with remote ischaemic conditioning (RIPC, tolerant), while C and D underwent the same procedure without a hind limb tourniquet (non-RIPC, non-tolerant). One hour later, glutamate was injected into the right ventricle with (A, C) or without (B, D) the addition of the EAAT activity inhibitor Evans Blue. The samples of blood and CSF were collected before the procedure (control, C) and 5, 10, 30 and 60 minutes after glutamate administration (Fig. 1).

Fig. 1

Experimental design. Groups A and B- animals pre-treated with remote ischaemic conditioning (RIPC, tolerant); C and D - animals underwent the same procedure as A and B without a hind limb tourniquet (non-RIPC, non-tolerant). One hour later, glutamate (Glut.) was injected into the right ventricle with (A, C) or without (B, D) the addition of the EAAT activity inhibitor Evans Blue. The samples of blood and CSF were collected before the procedure (control, C) and 5, 10, 30 and 60 min after glutamate administration

Intraventricular Administration of Glutamate

Post-ischaemic glutamate release was imitated by a single dose of glutamate injected into the right lateral ventricle. Animals were anaesthetised with 4% isoflurane in the anaesthetic cage and during surgery were maintained with 1.5% isoflurane. The animal was fixed in the stereotaxic apparatus and the hole was drilled in the skull 1.49 mm right and 0.95 mm posterior to bregma. A single dose of 20 mM glutamate (L-glutamic acid, Sigma-Aldrich) (10 µl) was administered through the hole to the right lateral ventricle by needle (0.95 mm posterior and 1.49 mm lateral to the bregma, 3.6 mm deep). According to Cserr and Berman (1978) [12], the volume of rat CSF is 250 µl, so the injected 20 mM glutamate (or saline as a placebo, 10 µl) in the ventricle was diluted 25-fold to a concentration 800 µM. EAAT activity was inhibited by the intraventricular co-administration of glutamate doses and Evans Blue in a final concentration of 220nM. Evans Blue (EB) is a potent competitive inhibitor of vesicular glutamate uptake. EB is a structural analog of glutamate that shows severalfold higher affinity to the glutamate transporter- while Evans Blue is efficient for competitive inhibition of glutamate transport with a value of inhibitor constant Ki in the nanomolar scale, the Km value of the glutamate uptake has been determined to be millimolar [13, 14].

RIPC Treatment and Blood/Liquor Samples Collection

Right hind-limb ischaemia was induced by placing an elastic rubber band tourniquet on the proximal part of the limb for 5 min, followed by a 5-min period of reperfusion according to [15] (tolerant, RIPC). The procedure was performed under light isoflurane anaesthesia (0.5%) in three cycles 1 h before blood sampling. Non-tolerant (non-RIPC) animals underwent the same procedure without the tourniquet application. The samples of blood/liquor were collected in rated intervals (see experimental design) from the jugular vein or cisterna magna respectively, via the polyethylene tubing (BTPE10, Instech, USA) applied before the glutamate application.

Determination of Blood Glutamate and Glutathione

First, samples of blood/liquor were deproteinised by adding ice-cold 1M perchloric acid (PCA, 1:1 v/v, 10 min on ice) and centrifuged (12,000 rpm, 10 min, 4°C). The supernatant was collected and stored at -80°C for subsequent analysis. In liquor samples of the group where Evans Blue inhibition was performed, BSA in final concentration 0.5 mg/ml was applied for 10 min before deproteinisation to bind the rest of the dye.

The glutamate concentration of samples was measured by a modified enzymatic-fluorometric method [5] based on [16]. The detection of NADH fluorescence resulting from the reaction of glutamate in precipitated samples and NAD+ catalysed by glutamate dehydrogenase was used to determine glutamate content. The glutamate concentration is directly proportional to the concentration of NADH in a reaction. Briefly, 10 µl of supernatant was pipetted into a black 96-well plate, and 190 µl of reaction buffer (0.25 M hydrazine hydrate/0.3 M glycine buffer, pH 8.6) containing 200 nM NAD+ and 15 U of glutamate dehydrogenase was added. After 30 min of incubation at RT, the fluorescence intensity of the final product (NADH) was read on a Synergy™ 2 Multi-Mode Microplate Reader (BioTek) at 460 nm with an excitation wavelength of 360 nm. The concentration of glutamate in all samples was recalculated per litre of total blood (µmol/l of blood) and CSF (µmol/l of CSF). To determine glutamate changes in liquor/blood 10 min after its intravenous application (rapid phase, Fig. 5), the value was expressed as a percentage and the value in the 5th min was set as 100%. All chemicals used for glutamate concentration measurements were purchased from Fluka.

Total glutathione content in the samples of whole blood was measured using the colorimetric method from [17] with minor modifications. Samples of precipitated blood were incubated with 10 mM Ellman’s reagent in 500 mM TRIS buffer (pH 8.2) with 10 mM EDTA (10 min, RT). After incubation, the absorbance at 415 nm was measured, and the total glutathione concentration was expressed per litre of blood. Glutathione (scale from 0 to 15 nM) was used as a calibrator for the assay.

Determination of Oxidative StressComet Assay

The comet assay was carried out under alkaline conditions as reported by [18], with minor modifications to evaluate DNA strand breaks in peripheral lymphocytes. Microscope slides were pre-coated with 1% normal-melting-point agarose in PBS (pH 7.4) and allowed to dry on a flat surface at room temperature (RT). A lymphocyte suspension was mixed with 1% low-melting-point agarose in PBS (pH 7.4), spread over agarose pre-coated slides, covered by coverslips and allowed to dry in a refrigerator for 20 min. When the agarose gel solidified, the coverslips were removed and the slides were immersed in lysis solution (2.5 mol/L NaCl, 100 mmol/L Na2EDTA, 10 mmol/L Tris, 1% Triton X-100, 10% DMSO, pH 10) for 1 h at 4°C. The slides were transferred to alkaline electrophoresis buffer (5 mol/L NaOH, 200 mmol/L Na2EDTA, pH 13) for 20 min at 4°C and then electrophoresis was carried out at 25V for 25 min at 4°C. After electrophoresis, the slides were neutralised in a neutralisation buffer solution (400 mmol/L Tris, pH 7.5) for 15 min at 4°C. After air-drying, DNA was visualised using SYBR Green and images were captured using a fluorescence microscope (Olympus BX51, ex.f.485, em.f.520 nm) equipped with a camera (Olympus DP50). Images were analysed using the Comet ScoreTM v1.5 image analysis system (TriTek Corp., USA). DNA damage was assessed by the parameter “% DNA in tail” (100% of cell fluorescence intensity minus intensity of the head % DNA).

Superoxide Dismutase Antioxidant Defence

Superoxide dismutase (SOD) activity was measured by a modification of the protocol described by [19]. The standard assay substrate mixture contained 1 M xanthine (Sigma), 0.1 M EDTA, 5.6 x 10− 2 M NBT (p-nitrotetrazolium blue grade III, Sigma-Aldrich, Steiheim, Germany) and 1 M BSA (bovine serum albumin, Fluka) in 0.1 M sodium phosphate (pH 7.8). The assay uses xanthine-xanthine oxidase as a superoxide generator to prevent NBT reduction by superoxide. The absorbance of NBT reduced to blue formazan by superoxide at 560 nm at room temperature was measured. SOD in the reaction prevents the synthesis of blue formazan. In blood cells, one unit of SOD activity was defined as the amount that reduced the absorbance change by 50%. The SOD activity was expressed as kU per milliliter of blood (kU/ml of blood).

Statistical Analysis

Data were analysed and plotted using GraphPad Prism (Graph-Pad Software, San Diego, California, USA). Statistical analysis was performed using one-way and two-way ANOVA, followed by the Dunnett post hoc test. The criterion for statistical significance was p < 0.05.