Animals, animal models, treatment
Female, six-week-old C57BL/6J mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France). The NOD/ShiLtJ mice (Female, six-week-old) originate from the internal breeding facility (ZETT) at the University of Düsseldorf. EAE mouse model.
Mice were immunized with 200 μg of myelin oligodendrocyte glycoprotein fragment 35–55 (MOG35–55), purchased from BIOTREND emulsified in 200 μl of complete Freund’s adjuvant (CFA), supplemented with 800 μg of heat-killed Mycobacterium tuberculosis (MT) H37Ra, both purchased from BD Difco (injected subcutaneous, distributed over four spots on the hind and front flank) and additional intraperitoneal injections of 200 ng of pertussis toxin (PTX) from Sigma-Aldrich on days 0 and 2 post immunization (p.i.). The sham control group (sham) also received PTX and CFA, but no MOG35–55 peptide. The substances used for treatment, their concentration, mode of action, treatment interval and treatment start are described in Table 1. The used concentrations for each substance were adapted from recent dose finding studies found in the literature investigating the optimal dose.
The experimental protocol was reviewed and approved by the “State Office for Nature, Environment and Consumer Protection of North Rhine-Westphalia, Germany “Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (LANUV) under approval number Az. 81-02.04.2019.A063.
The in vitro concentrations of 2 µM and 5 µM flecainide used for treating primary mouse brain microvascular endothelial cells (pMBMECs) were selected as part of a dose-finding approach based on previously published studies17,18. These concentrations were chosen to approximate pharmacologically relevant levels corresponding to the systemic in vivo dosage of 30 mg/kg administered subcutaneously in our EAE model, while allowing the assessment of dose-dependent effects on endothelial gene expression and barrier function.
OCT measurements
Periodic OCT measurements were conducted using the Spectralis® HRA+OCT device (Heidelberg Engineering, Germany) with several adaptations for rodents, as previously described19. Segmentation of retinal volume scans was performed using the Heidelberg Eye Explorer software, with manual control for segmentation errors. The volume of the parapapillary region was assessed using the ETDRS grid, excluding the center with the disc, as published previously20. Inner retinal layer (IRL) thickness was examined at defined intervals after irradiation and compared with baseline measurements (IRL: NFL, GCL, and IPL layer).
For the measurements, the animals were anesthetized using isoflurane (Vaporizer from Harvard Apparatus Anesthetic Vaporizors; Isofluran from Piramal critical care). Specifically, induction was carried out at 3.5% isoflurane, followed by maintenance at 2% isoflurane, with a flow rate of 0.6 L/minute of oxygen. The gas was transferred through nose cones to the mice.
Optomotor response (OMR) measurements
OMR measurements were performed periodically, exclusively in C57BL/6J mice, using the OptoMotry® device from Cerebral Mechanics, in parallel with OCT measurements. Spatial frequency was monitored as a parameter for visual function. The spatial frequency threshold was determined by randomly changing the spatial frequency to identify the threshold at which the mouse could track the grids, as previously described20,21.
Histology (optic nerves, retinal cross sections and retinal whole mounts)
Mice were euthanized using 100 mg/kg Ketamine und 20 mg Xylazin intraperitoneal (i.p). (in 250 µl NaCl 0,9%) followed by cardiac perfusion using phosphate-buffered saline (Gibco, Carlsbad, USA).
The optic nerves were then isolated and fixated in 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany) overnight. After fixation, the optic nerves were subjected to a sucrose gradient for dehydration and subsequently embedded in O.C.T. compound (Sakura™ Finetek, Alphen aan den Rijn, Netherlands). Longitudinal sections of five micrometers were cut and prepared for fluorescence staining. Longitudinal sections of the optic nerves were used for quantifying T-Lymphocytes (CD3 (Clone 17A2), 1:400, Biolegend), assessing the state of myelination (MBP (Clone 12), Merck Millipore, 1:500), evaluating microglial activation (Iba1 (Clone GT10312), 1:500, Merck) using Leica HyD detector attached to a Leica DMi8 confocal microscope (63 × objective lens magnification). Astrocytic activation (GFAP (Clone 173,004), 1:1000 Synaptic System) was assessed using retinal cross sections. Cy3 goat anti-mouse (1:500 Millipore), Cy3 goat anti-rat (1:500, Millipore) and Cy3 goat anti-rabbit (1:500, Invitrogen) were used as secondary antibodies. The numbers of cells stained with CD3 and Iba1 were analyzed using ImageJ software, applied by blinded raters, and expressed as a ratio to DAPI staining. The overall signal for MBP (positive total area in the red channel) was analyzed using ImageJ software. Cell counts for CD3 and Iba1, as well as the positive area for MBP and GFAP, were assessed in the whole field across 6–9 images per sample. The mean values were calculated and utilized for analysis.
RGC count was calculated by a semi-automated count of Brn3a+cells on retinal flat mounts. Briefly, retinae were stained with Brn3a (1:200, Santa Cruz Biotechnology, cat# sc-31984) antibody and flat-mounted on glass slides. Each retina was then divided into four quadrants (three areas per quadrant: central, mid-periphery, and far-periphery). For each eye, Brn3a+cell count was summed up from all 6–12 areas imaged.
Evan’s Blue Dye assay
Evan’s Blue Dye (EBD) is a diazo dye that binds to serum albumin, creating a large molecular complex that normally does not cross the intact BBB. However, in pathological conditions leading to increased BBB permeability, the EBD-albumin complex can cross the BBB and accumulate in brain tissue, thus providing a measurable indication of barrier disruption. At the pre-determined time points of 18 days post-immunization, the mice were injected intravenously with 4% Evan’s Blue Dye in saline at a dosage of 4 ml/kg body weight. The dye was allowed to circulate for 3 h to ensure systemic distribution. After the circulation period, the mice were anesthetized and transcardially perfused with phosphate-buffered saline (PBS) to remove intravascular dye. The brains and spinal cords were then carefully extracted, weighed, and homogenized in N,N-dimethylformamide to extract the dye from the tissue. The samples were then centrifuged, and the supernatants were collected for spectrophotometric analysis. Quantification of the EBD was performed using a TECAN spectroscopic device. The absorption of the extracted solutions was measured at 565 nm, a wavelength at which EBD exhibits a distinct peak. EBD concentration was calculated from the absorbance values using a standard curve generated with known concentrations of EBD. The amount of EBD in the brain tissue was then expressed as µg of EBD per g of brain tissue. All data were collected and analyzed using appropriate statistical methods.
Flow cytometry analysis
Flow cytometry was used to examine the lymphocyte subpopulations in spinal cord and spleen of the EAE mice at specified time points. Spinal cords were carefully harvested and mechanically and enzymatically (Collagenase, DNAse) dissociated. Lymphocytes were then isolated from the suspension using a LymphoprepTM-gradient (Stemcell technologies). Spleen was collected and mechanically dissociated by passing it through a 70 µm cell strainer. Red blood cells were lysed with ACK Lysis Buffer and suspension was again passed through a 70 µm cell strainer.
Lymphocytes from both organs were centrifuged and resuspended in FACS buffer, containing 2 mM EDTA and 2% fetal calf serum. Cells were then stained with fluorochrome-conjugated monoclonal antibodies (Tables 2, and 3, respectively) in the dark 4 °C for 30 min. The cells were then washed with FACS buffer and prepared for analysis. Cells were analyzed on a CytoFLEX S (Beckman Coulter) and data was interpreted using Kaluza Analysis Software (Beckman Coulter). Lymphocytes were identified based on forward and side scatter properties, with specific populations determined by surface marker expression (representative gating strategy is stated in Fig. S1). Data are reported as the total cell counts (via flowrate check) of each cell type within the total lymphocyte population.
Isolation of pMBMECs
pMBMECs were isolated according to different protocols for performing the quantitative PCR or the permeability assay. In both procedures, the pMBMECs were never passaged between isolation and experiment. Flecainide treatment was applied for 24 h prior to qPCR analyses and for 7 days prior to Western blot analyses, starting after pMBMECs had reached confluency.
Isolation pMBMECs for quantitative PCR and western blot
We began by euthanizing six- to eight-week-old female C57BL/6 mice. After euthanasia, the brains were carefully extracted and immediately transferred onto sterile filter paper to facilitate the removal of the meninges. Following this step, the brain tissue was homogenized to a uniform consistency and subsequently processed according to the protocol of the Adult Brain Dissociation Kit (Miltenyi Biotec), which is optimized for effective dissociation of murine brain tissue. To isolate microvascular endothelial cells, the resulting single-cell suspension was subjected to magnetic-activated cell sorting (MACS). First, CD45+ immune cells were labeled using CD45 MicroBeads and removed by magnetic separation via LS columns and the MACS Separator. The flow-through, containing CD45− cells, was then incubated with CD31 MicroBeads to isolate CD31+ endothelial cells. These CD45−CD31+ cells were collected and plated in 12-well plates for culture. Prior to seeding, the plates were coated overnight at 4 °C with a coating solution consisting of 500 µl dH2O, 400 µl collagen, and 100 µl fibronectin (200 µl per well), to enhance cell adhesion. The isolated endothelial cells were seeded in 1.5 ml of MBMEC medium per well. For the initial two days, cells were cultured in MBMEC medium supplemented with 0.1% pyromycin (10 µl pyromycin per 10 ml medium) to select for the desired cell population. Thereafter, the medium was replaced with pyromycin-free MBMEC medium. The MBMEC culture medium consisted of 40 ml DMEM high glucose, 10 ml fetal calf serum (FCS), 25 µl basic fibroblast growth factor (bFGF) and 50 µl heparin.
Isolation of pMBMECs for permeability assay
Cells were isolated by the method of Coisne et al.22 as follows: for each preparation cortices from six- to ten-weeks old gender matched mice were isolated and meninges were removed. Preparations were pooled and homogenized in Hank’s balanced salt solution containing 0.1% bovine serum albumin. The homogenate was mixed with 30% dextran and centrifuged at 3000g for 25 min at 10 °C. The pellet containing the vascular fraction was collected. Centrifugation and pellet harvesting was repeated once. The collected vascular fraction was then filtered through a 60 μm nylon mesh. The capillary-enriched filtrate was digested in DNase I (10 mg/mL), TLCK (0.147 mg/mL), and collagenase/dispase (2 mg/mL) for 30 min at 37 °C. The digestion was stopped by an excess of wash buffer and filtered through a 20 μm nylon mesh. The crude cell preparation of pMBMECs were cultured for 48 h in the presence of 4 µg/ml puromycin, which allowed selective growth of pMBMECs only.
Western blot analysis of protein expression in pMBMECs
pMBMECs were cultured to confluence as described in “Isolation pMBMECs for quantitative PCR and western blot” section, then treated for 7 days with flecainide (2 µM or 5 µM) or vehicle. After treatment, cells were washed with PBS and lysed in NP-40 buffer (150 mM NaCl, 50 mM Tris/HCl, 1% NP-40, pH 8.0) on ice for 20 min and then scraped off using a pipette tip. Lysates were cleared by centrifugation (12,000 × g, 10 min, 4 °C), and protein concentration was determined using the BCassay kit (Interchim, France). Equal protein amounts (25 µg) were mixed with 1 × Laemmli buffer, denatured at 95 °C for 5 min, separated by SDS-PAGE, and transferred onto 0.2 µm nitrocellulose membranes. Membranes were blocked for 10 min at room temperature with EveryBlot buffer (Bio-Rad, USA), then incubated overnight at 4 °C with the following primary antibodies: anti-Actin (Invitrogen, PA1-183, 1:4000), anti-PECAM1 (Antibodies Online, ABIN669006, 1:1000), anti-β-Integrin (ABIN739029, 1:1000), anti-JAM3 (ABIN1386406, 1:1000), and anti-JAM2 (ABIN3187667, 1:1000). Following three washes with PBS containing 0.05% Tween-20, membranes were incubated for 1.5 h on a shaker at room temperature with IRDye-labeled secondary antibodies: Goat anti-Rabbit 680RD (LI-COR, 926-68071) and Goat anti-Mouse 800CW (LI-COR, 926-32210). Protein bands were visualized using the ChemiDoc system (Bio-Rad) and quantified with ImageLab software. Expression levels were normalized to β-actin and analyzed using GraphPad Prism.
Quantitative PCR analysis
For our qPCR analysis, we utilized the QuantStudio 3 from Thermo Fisher Scientific, employing SYBR Green as our detection chemistry. After preparing and loading our samples, we conducted the qPCR run analyzing the genes listed in Table 4 (in different experimental approaches). The machine’s sophisticated technology measured DNA quantity using the fluorescence of the SYBR Green. Post-run, we processed and interpreted the data using the QuantStudio Design and Analysis Software from Thermo Fisher Scientific, enabling us to calculate relative gene expression levels using the ΔΔCt and ΔCt, respectively.
The treatment groups in the in vitro treatment experiments included cells exposed to 2 µM or 5 µM flecainide or PBS vehicle control 24 h prior to cell harvesting. The in vitro concentrations were used based on previous experiments with flecainide found in literature17,18.
Permeability assay
Permeability assays were performed in triplicates as reported by Coisne et al.22, with minor adaptations: pMBMECs were grown on Matrigel-coated Transwell® filter inserts (0.4 μm pore size, 6.5 mm diameter; article number 662640, Greiner Bio-One Vacuette Schweiz GmbH, St. Gallen, Switzerland) for 6 to 8 days. Alexa Fluor 680-dextran (3 kDa, 10 μg/ml; LuBioScience, Luzerne, Switzerland) was used as permeability tracer. Diffused dextran was quantified using the Odyssey Imaging System (LI-COR, Bad Homburg, Germany) and the clearance value (Pe, in cm/min) of the pMBMECs calculated as reported by Coisne et al. 2005. Flecainide treatment at 5 µM was for 24 h and IL-1β treatment at 20 ng/ml was for 16 h. The in vitro concentrations of flecainide were used based on previous experiments found in literature17,18. After the experiment, each filter was examined for confluent growth of pMBMECs by staining with phalloidin-rhodamine and subsequent fluorescence microscopy. The inflammatory state of pMBMECs after IL-1β stimulation was monitored in parallel samples by staining with the homemade rat anti-mouse ICAM-1 monoclonal antibody 29G1 followed by a secondary donkey anti-rat Cy5 antibody (Jackson ImmunoResearch, Milan Analytica AG, Rheinfelden, Switzerland).
Isolation of splenocytes for proliferation assay and qPCR analysis
Immediately following euthanasia, spleens were aseptically removed and placed into wash buffer composed of DMEM supplemented with fetal calf serum and antibiotics. For tissue dissociation, each spleen was transferred onto a pre-wetted 40 µm cell strainer positioned on a conical tube. The spleen was mechanically dissociated by gently pressing it through the strainer using the plunger of a syringe. The cell strainer was then rinsed with additional wash buffer to collect all cells. The resulting single-cell suspension was centrifuged at 4 °C. After removal of the supernatant, the cell pellet was resuspended in pre-warmed ACK lysis buffer to lyse erythrocytes. The suspension was incubated at room temperature, and erythrocyte lysis was subsequently stopped by adding wash buffer, followed by a second centrifugation step at 4 °C. The final cell pellet was resuspended in wash buffer and passed through a freshly rinsed 40 µm cell strainer into a new conical tube to ensure maximal recovery of splenocytes.
Proliferation assay using CFSE and Ki-67
To investigate the effects of flecainide on lymphocyte proliferation, we performed a combined assay using CFSE (carboxyfluorescein succinimidyl ester) labeling and intracellular Ki-67 staining. Splenocytes were isolated as described in “Isolation of splenocytes for proliferation assay and qPCR analysis” section. Prior to culture, cells were labeled with CFSE by incubation in PBS supplemented with fetal calf serum and the dye at 37 °C in the dark. The labeling reaction was quenched by the addition of cold wash medium, followed by incubation on ice. After two washing steps, cells were resuspended in murine T cell medium composed of IMDM supplemented with fetal calf serum, β-mercaptoethanol, L-glutamine, and antibiotics. T cell activation was achieved by seeding the CFSE-labeled splenocytes into anti-CD3-coated 96-well plates. Cells were treated with either vehicle (containing 0,005% DMSO), 2 µM flecainide, or 5 µM flecainide and incubated for five days at 37 °C in a humidified CO2 incubator. On day three, half of the culture medium was carefully replaced with fresh treatment medium. At the end of the incubation period, proliferation was assessed by flow cytometric analysis of CFSE dilution and intracellular Ki-67 expression. For Ki-67 staining, cells were fixed, permeabilized, and stained with a fluorochrome-conjugated anti-Ki-67 antibody according to the manufacturer’s protocol. Data were acquired and analyzed within defined immune cell subsets.
Statistics and data interpretation
Statistical analysis was performed using Prism (version 9, Graphpad Software, Inc.) and IBM SPSS Statistics (version 20, IBM Corporation, USA). Total and percent changes of the acquired retinal parameters (OCT and OMR) were analyzed using generalized estimation equation (GEE) models, accounting for within-subject inter-eye correlations, to test for differences between the two groups. For non-paired data, group means analyses were compared using a one-way ANOVA with the Dunnett´s post hoc test, utilizing one optic nerve per animal for the histological investigations. The thickness of the inner retinal layers, ranging from the inner limiting membrane to the bottom of the inner plexiform layer, the total retinal thickness and outer retinal layers were assessed in volume scans around the optic disc as the primary OCT-based outcome parameters. The spatial frequency served as a functional primary readout. The other histology-derived parameters were assessed as secondary outcome criteria. The severity of EAE symptoms, extending from minor hind limb weakness to complete paralysis, was assessed through a standardized scoring system (ranging from 0 to 5), providing a quantitative outcome measure of neuroinflammation and neurodegeneration. The EAE scores served as an integral part of the study, illustrating the clinical manifestation and progression of the disease, offering a real-time evaluation of the neurological impairment. QPCR was deployed for the precise quantification of targeted gene expression changes. The relative changes in gene expression levels were examined as one of the primary outcome measures, offering key insights into the molecular mechanisms. The permeability of the BBB was further evaluated by using the Evans Blue assay. This approach involved the systemic administration of Evans Blue dye, the penetration of which into the brain tissue served as a direct indicator of BBB disruption. The quantified extent of dye penetration, extracted and measured spectrophotometrically, offered a primary outcome measure of BBB integrity. The data derived from the Evans Blue assay yielded essential insights into the degree and timing of BBB permeability changes in response to neuroinflammation and following various therapeutic interventions.