Inhibition of Vesicular Glutamate Transporters (VGLUTs) with Chicago Sky Blue 6B Before Focal Cerebral Ischemia Offers Neuroprotection

Animals and Experimental Design

All experiments were performed on male Sprague‒Dawley rats (280–320 g, Charles Rivers). Animals were randomly allocated into the following experimental and timepoint groups: the SHAM 12 h, MCAO 12 h, CSB6B SHAM 12 h, CSB6B MCAO 12 h, SHAM 24 h, MCAO 24 h, CSB6B SHAM 24 h, CSB6B MCAO 24 h, SHAM 3 d, MCAO 3 d, CSB6B SHAM 3 d, CSB6B MCAO 3 d. Each group had an n of 6–8 animals. Timepoints 12 h, 24 h, and 3 d refer to the time lapse between the onset of reperfusion and animal decapitation and tissue collection. The animals were maintained on a normal day-night cycle at 22 ± 2 °C with free access to food and water. The experimental protocols were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and were approved by the First Local Ethical Committee at Jagiellonian University in Krakow (permit no: 11/2017). All studies involving animals are reported according to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines, including the procedure for blinding the investigators to the identities of the animals at each point of the experiment. CSB6B or artificial cerebrospinal fluid as a control was administered to rats 2 h before MCAO. CSB6B (1 mg/mL, dissolved in artificial cerebrospinal fluid) was slowly injected into the contralateral ventricle (i.c.v.) (anteroposterior (AP): 0.0 mm; mediolateral (ML): 1.6 mm; dorsoventral (DV): 4.0 mm, all coordinates according to the bregma) at a volume of 5 μL (0.5 μL/min) using a stereotaxic frame. The dosage was based on our preliminary studies and available literature [29]. The efficacy of administration was confirmed during brain tissue collection.

Focal Cerebral Ischemia Model

MCAO was elicited according to the method of Longa et al. as previously described [30]. All surgical procedures were carried out under a stereoscopic microscope (Leica, A60F; Germany), and body temperature was maintained at a physiological level using a heating blanket (Homeothermic Blanket System; Harvard Apparatus). Arterial occlusion was confirmed using the laser speckle contrast analysis system (PeriCam HR PSI, Perimed, Sweden), and a 70% blood flow reduction was considered to indicate a successful procedure. The rats were anesthetized with 5% isoflurane for induction and 2.5% isoflurane for maintenance. After exposure of the left external carotid artery (ECA), the internal carotid artery (ICA), and the common carotid artery (CCA), all branches of the ECA were coagulated. The ECA, ICA, and CCA were temporarily secured with microvascular clips. A silicone-coated filament (Doccol, USA) was introduced into the lumen of the ECA and advanced until the blood flow decreased. The clip was removed from the CCA, and the wound was secured with silk sutures. The occlusion was maintained for 90 min. Afterward, the wound was reopened, the filament removed to restore blood flow, and the wound closed. The sham operation was carried out as described above without insertion of the filament. The mortality rate for MCAO was 4% for 12 h timepoint, 6.2% for 24 h timepoint, and 9.35% for 3 d timepoint. The success rate of the optimal MCA occlusion was 86.4%.

Neurological Deficit

Neurological deficits were assessed at 12 h, 24 h, and 3 d after reperfusion. For each assessed group, n = 8 animals. The 10-point grading system of Philips et al. (2000) was used [31], as previously described [32]. The following neurological symptoms were assessed and scored: 4 points were given when animals pushed in a contralateral direction did not show any resistance, 3 points were given when animals circled in a contralateral direction, if animal demonstrated contralateral shoulder adduction, 2 points were added, and 1 point was given if contralateral forelimb flexion was observed. Zero indicates a lack of neurological deficit, whereas 10 points was the maximal neurological deficit.

TTC Staining and Measurement of the Infarct Volume

To determine the infarct volume, separate experimental groups were prepared (for each group, n = 6). Twenty-four hours after MCAO, animals were decapitated, and their brains were immediately removed and cut using a brain matrix (Harvard Apparatus, USA). Coronal Sects. (2 mm thick) were stained with a 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC) dye (Sigma Aldrich, USA) in 0.01 M phosphate-buffered saline (Sigma Aldrich, USA) at 37 °C for 10 min in the dark. Next, sections were fixed in 10% phosphate-buffered formalin (Sigma Aldrich, USA) for 30 min at 4 °C. The stained and fixed brain slices were photographed using a surgical microscope equipped with a camera (Leica S9D with Flexacam C3, Leica, Germany) by an investigator blinded to the subject identity. The infarct volume was determined using NIH ImageJ software (National Institutes of Health, version 8.0) by the same investigator. The infarct volume was calculated as a sum of each outlined white area multiplied by the thickness of the brain section and was expressed in mm3.

Microdialysis of the Motor Cortex

Guide cannulas were implanted 24 h prior to microdialysis. The animals were anesthetized with 2.5% isoflurane and then stereotaxically implanted with guide cannulas (MAB 4; AgnTho’s, Sweden) aimed at the frontal cortex (anteroposterior (AP): − 0.48 mm; mediolateral (ML): + 2.0 mm; dorsoventral (DV): − 1.2 mm) according to the atlas of Paxinos and Watson (2007). The guide cannulas were affixed to the skulls with acrylic dental cement and cranial screws. Obturators were placed in the cannulas until the microdialysis probes were inserted. Next, the obturators were removed from the guide cannulas, and microdialysis probes (MAB 4, membrane with a molecular weight 6-kDa cut off, 2-mm length, 0.24-mm outer diameter, AgnTho’s AB, Sweden) were inserted into the guide cannulas. To wash inserted probes, brain structures were perfused for 2 h with artificial cerebrospinal fluid (aCSF) (in mM: 147 NaCl, 4.0 KCl, 1.0 MgCl2, 2.2 CaCl2, pH 7.4) at a constant flow rate (2 μL/min). After this period, samples were collected every 30 min as follows: two baseline samples, three samples during MCAO, and two samples during the reperfusion period. At 24 h and at 3 d after reperfusion, the microdialysis procedure on freely-moving animals was repeated, maintaining 2 h washing period of the probe, followed by three samples collection every 30 min. All samples were immediately frozen and stored at − 80 °C until the LC‒MS assay. For each experimental group, n = 8 animals. After collecting the last sample at 3 d after reperfusion, the localization of guide cannula in the motor cortex was verified.

LC‒MS Analysis of Glu

The chromatographic separation was performed on a Waters ACQUITY UPLC® H-Class system (Waters Corporation, Milford, MA, USA) comprised of a quaternary pump, column oven, autosampler (with 100 µL of ANSI-384 well), and photodiode array detector. The MS/MS instrument was a Xevo TQ-S® mass spectrometer operating in positive electrospray ionization mode, and two multiple reaction monitoring (MRM) transitions were monitored per component. Optimization of the MS/MS parameters was performed by infusion of 1 µg/mL standard solutions of each analyte and IS into the Xevo TQ-S® mass spectrometer at a flow rate of 10 µL/min, in combination with the mobile phase (50% A/50% B, flow rate: 0.2 mL/min) using the IntelliStart Fluidics system.

The aCSF samples were analyzed in gradient mode with a ZIC®-HILIC column (5 µm, 200 Å, 150 × 21.2 mm; Merck, Darmstadt, Germany). The temperature of the column thermostat was set at 40 °C. The flow rate of the mobile phase was 0.3 mL/min. The mobile phase consisted of water with the addition of 0.02 M formic acid (solvent A) and acetonitrile with the addition of 0.02 M formic acid (solvent B). The initial gradient of solvent A was 50% (for 0.06 min), then was decreased to 3% (for 0.6 min), maintained at 3% (for 1.2 min), and finally increased to 50% (for last 2.8 min.). Pairs of ions were monitored in the assay using the following values of m/z: 148.1/84.1 for Glu and 153.1/89.1 for Glu-C13 (Internal Standard). The results were analyzed using MassLynx software V4.2 (Waters, Milford, MA, USA). Levels of Glu were calculated using the calibration standard curves, which were constructed by linear regression analysis of peak area versus concentration.

The ion source parameters were as follows: ion spray voltage (IS): 5400 V; nebulizer gas (gas 1): 30 psi; turbo gas (gas 2): 20 psi; temperature of the heated nebulizer (TEM): 550 °C; curtain gas (CUR): 30 psi. Nitrogen (99.9%) from Peak NM20ZA was used as the curtain and collision gas. The quantitation analysis was performed using the MRM mode and tandem LC/MS.

Western Blot

The tissues of the frontal cortex and dorsal striatum were homogenized in 2% SDS containing 1 mM PMSF, 1 mM Na2VO4, 20 mM NaF, and a mixture of phosphatase-proteinase inhibitors (Sigma Aldrich) using Ultra-Turrax and ultrasonic homogenizers. After denaturation at 95 °C for 10 min, insoluble debris was removed by centrifugation at 10,000 × g for 10 min at 4 °C. The protein concentration in the supernatants was determined using a BCA protein assay kit (Thermo Scientific, USA). After setting a proper protein concentration, the solutions were mixed with loading buffer (containing 10% 2-mercaptoethanol) at a ratio of 1:1 and heated for 10 min at 95 °C. Samples were loaded on gradient 8–16% SDS polyacrylamide gels (Criterion, TGX-Stain-free gel, Bio-Rad) at a total protein concentration of 30 µg/10 µL, and electrophoresis (200 V, 45 min) was performed. Next, proteins were semidry transferred (TurboBlot, Bio-Rad) to PVDF membranes, and the total protein content on the membrane was visualized with stain-free procedure. Next, membranes were blocked in a 5% solution of albumin. Membranes were incubated overnight at 4 °C with primary antibodies at the appropriate concentration. After an overnight incubation, the membranes were washed in TBST and then in 1% albumin solution and incubated with the respective secondary antibodies conjugated with peroxidase in 1% albumin solution for 1 h at RT. After washing, the membranes were developed using the ECL method (Western Bright Quantum, Advansta Inc., USA). The chemiluminescence of the membranes was imaged with a G-Box Imaging System (Syngene, USA), and the protein expression was analyzed with Gene Tools software (Syngene, USA) and expressed relative to the total protein content in the sample. Each experimental group consisted of n = 6 animals.

RT‒qPCR

The rats were decapitated 12 h, 24 h, or 3 d after reperfusion or sham operation. The brains were removed, and the ipsilateral brain structures were isolated and immersed in fixRNA solution (EURx, Poland) for 24 h at 4 °C to preserve RNA. Next, total RNA was extracted using TRIzol reagent and purified with a microcolumn system according to the manufacturer’s protocol. The concentration and purity of the RNA were determined by measuring the A260/A280 ratio with a Nanoquant plate (Tecan, Austria). cDNA was synthesized using 2 µg of total RNA and a Smart First Strand cDNA Synthesis Kit (EURx, Poland) according to the manufacturer’s protocol. The cDNA was stored at − 80 °C until use. RT‒qPCR amplification was performed on a CFX Connect system (Bio-Rad, USA) using appropriate TaqMan Gene Expression Assays (Applied Biosystems, USA), 200 ng of template cDNA and Probe qPCR Master Mix (EURx, Poland) according to the manufacturer’s instructions. The thermal cycling conditions were as follows: 95 °C for 15 min (initial denaturation) followed by 35 cycles of 94 °C for 15 s (denaturation), 55 °C for 30 s (annealing), and 72 °C for 30 s (extension). Reactions for each sample were performed in triplicate. The efficiency of the PCR for each TaqMan probe was verified. The fold change in the expression of each gene was calculated using the ΔCt method using three housekeeping genes, Ppia, Ywhaz, and Hprt, as we described recently [33]. Next, these values were used for further statistical analysis. The TaqMan probes used for RT‒qPCR are listed in Table 1. Each experimental group consisted of n = 6 animals.

Table 1 Characteristics of TaqMan™ probesTissue Fixation for Immunofluorescent Double Staining

Groups of animals (n = 6 animals per group) used for immunofluorescent staining were subjected to intracardiac 4% paraformaldehyde (PFA) perfusion 24 h after the surgical procedure. First, the animals were deeply anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg). Then, the rats were transcardially perfused with 250 mL of NaCl solution (0.9%; 32 °C) until all the remaining blood was removed. Next, animals were perfused with 500 mL of 4% PFA in a 0.1 M PBS. After perfusion, the brains were removed and kept in the same 4% PFA solution overnight. Afterward, the brains were transferred to 10% sucrose solution containing 0.1% sodium azide in 0.1 M PBS and stored for 24 h. Next, the brains were transferred to 20% and then to 30% sucrose solution with 0.1% sodium azide in 0.1 M PBS and stored until they sank. The brains were then cut into 20 μm coronal sections using an automatic cryotome (Leica CM 1860). The sections were mounted on SuperFrost (Thermo, USA) microscopic slides and stored at − 20 °C until staining and analysis.

Immunofluorescent Double Staining

Brain sections were placed on microscopic slides and stored at − 20 °C until staining. A double immunostaining procedure was performed to visualize the colocalization of either VGLUT1 or VGLUT2 proteins with the neuronal marker MAP2; thus, appropriate mixtures of the two antibodies were used for tissue staining. The antibodies used in this method are listed in Table 2. First, slides were immersed in 70% ethanol for a few seconds and air dried for 3 min. Slides were washed twice in 0.01 M PBS for 10 min. Next, the slides were washed once in 0.3% Triton X-100 in 0.01 M PBS for 10 min in order to permeabilize. Nonspecific binding was blocked by incubating slides in 10% goat normal serum (GNS) in 0.3% Triton X-100 in 0.01 M PBS for 1 h. Next, tissues were incubated in primary antibody solutions overnight at 4 °C in humidity chambers. Primary and secondary antibody solutions were prepared by in 2% GNS in 0.3% Triton X-100 in 0.01 M PBS. The following day, the slides were washed twice in 0.3% Triton X-100 in 0.01 M PBS and then twice in 2% GNS in 0.3% Triton X-100 in 0.01 M PBS. Each of these washes was for 10 min. Tissues were incubated in secondary antibody solution for an hour in the dark. Next, the slides were washed four times in 0.01 M PBS for 10 min. All washing and incubation steps were performed using a rocking shaker. Slides were dried with a paper towel, covered in mounting medium (Vectashield Vibrance with DAPI, Vectorlabs) and coverslipped. Slides were stored at 4 °C until visualization. Images were obtained using a Leica Stellaris 8 WLL DLS confocal microscope and Leica LAS X software. Each scan of the particular staining of the particular brain structure was performed using the same settings of the microscope, including the same pinhole (0.9 AU), the same power of lasers and detector, the same laser frequency (300 Hz) as well as the same characteristics of the z-stack and similar part of the brain structure located on the border between the core of ischemia, and the periinfarct zone. The resolutions of gathered scans were 2048 × 2048 pixels. The fluorescence intensity was analyzed in LAS X software selectively for each focal plane, before stitching the z-stack (35 focal planes). The fluorescence intensity analysis refers to the total surface of brain slice visible in a scan of a singular focal plane, corrected by the fluorescence of the background. Results are expressed as a mean fluorescence intensity in mean fluorescence intensity (MFI); for each experimental group, n = 6 animals; for each animal brain slices were scanned at 3–4 positions per brain structure (cortex or dorsal striatum), each position is a z-stack consisting 35 scans of focal planes.

Table 2 The list of antibodies used in immunofluorescent double staining protocolStatistical Analyses

All data are expressed as the mean ± standard error of the mean (SEM). The infarction volume and protein and mRNA expression data were analyzed using one-way ANOVA. If statistical significance was found after ANOVA, Sidak’s post hoc test was conducted to test the comparisons between experimental groups. Microdialysis results were analyzed by two-way repeated-measures ANOVA with the Sidak post hoc test. A Mann‒Whitney U test was used for the analysis of neurological deficits. Each statistical analysis compared the following groups: treatment alone, treatment + MCAO, sham, and MCAO. A calculated p value < 0.05 was considered statistically significant. Calculations were performed using GraphPad Prism software ver. 8.2.1 (GraphPad Software, USA).

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