Brain tissue metabolic profiling

Tissue samples from the peri-ischemic area and from the contralateral side, collected at 1 dpi as described above, were weighed and 80% methanol was added (v/v H²O, LC-MS Ultra CHROMASOLV, Fluka) in a ratio of 350 μl solvent/100 mg tissue.

Extraction was facilitated by breaking the tissue with Teflon coated plastic staff followed with water sonication for 10 min (BRANSON GWB 2200). Samples were then vortexed and centrifuged (Eppendorf centrifuge 5804 R, 13000 rpm, 5 min at 4°C). Supernatant was collected and filtered to HPLC bottles through Acrodisc CR 4 mm (0.45 μm) filters. Quality control samples were made by taking 5 μl of solution from each sample. The samples were analyzed using a UHPLC-qTOF-MS system (Agilent Technologies, Waldbronn, Karlsruhe, Germany) that consisted of a 1290 LC system, a Jetstream electrospray ionization (ESI) source, and a 6540 UHD accurate-mass qTOF spectrometry as described in Puurunen et al. 2016[305]. Two different chromatographic techniques were used, i.e. reversed phase (RP) and hydrophilic interaction (hilic) chromatography and acquired data in both positive (+) and negative (−) polarity. The sample tray was kept at 4°C during the analysis.

MassHunter Acquisition B.04.00 (Agilent Technologies) software was used for data acquisition. The quality control samples were injected in the beginning of the analysis as well as every 12th injection. The analysis order of samples was randomized. Mass spectrometry data processing was performed using MassHunter Profinder B.06.00

(Agilent Technologies, USA). The batch recursive feature extraction function was used to extract ion to molecular features exhibiting isotopic peaks, dimers, and common adducts. Final alignment and quality control of peak spectra were done manually. The data were transferred as compound exchange format files into the Mass Profiler Professional (MPP) software (version 13, Agilent Technologies) for statistical analysis. MS-DIAL ver. 2.52[306] and MassHunter Qualitative Analysis B.07.00 (Agilent Technologies, USA) softwares were used for metabolite identification against MSMS spectra found in public and in-house standard libraries.

4.2 IN VITRO MODELS 4.2.1 Primary murine cultures

4.2.1.1 Cortical neurons

Primary cortical neuron cultures were prepared from C57BL/6J E15 embryos as described in Malm et al., 2015[307]. Briefly, cortices were dissected and the tissue was dissociated with trypsin for 15 min at 37°C (0.0125%, Sigma-Aldrich, St. Louis, USA).

Neurons were seeded in complete Neurobasal medium containing 2% B27 supplement, 500 μM L-glutamine and 10 μg/ml gentamycin (all reagents ThermoFisher Scientific, Waltham, USA), at a density of 12.5×10⁴ cells/well on 48-well or 1.8×10⁶ cells/well on 6-well plate format coated with poly-D-lysine (Sigma-Aldrich, St. Louis, USA). On 5 DIV after seeding 50% of the media was changed and the cultures were used for experiments on 7 DIV.

4.2.1.2 Microglia

Primary microglial cultures were prepared from C57BL/6J neonatal mice on postnatal day 0–3 as described elsewhere[307]. Briefly, mice were sacrificed by decapitation and their brains dissected. Tissue was mechanically dissociated and incubated in DMEM/F-12 supplemented with 1% penicillin/streptomycin and 0.05%

Trypsin-EDTA (all ThermoFisher Scientific, Waltham, MA, USA). Trypsin was inactivated with complete media DMEM/F-12 containing 10% iFBS, 1%

penicillin/streptomycin (all ThermoFisher Scientific, Waltham, MA, USA) and the tissue homogenized. Cells were seeded on 15 cm dishes and left at 37°C, 5% CO² for three weeks. Thereafter, the astrocyte layer from mixed glial culture was trypsinized, the remaining microglia collected and plated on 48-well or 6-well plate format at the density of 12.5×10⁴ cells/well and 1×10⁶ cells/well, respectively.

4.2.1.3 Primary cortical neurons and BV2 microglia co-cultures

Primary cortical neuron cultures were prepared as described above. For HX600 study, BV2 cells transfected with negative control siRNA (ThermoFisher Scientific, Waltham, MA, USA) or NURR1 siRNA (Silencer select siRNA for NR4A2, ThermoFisher Scientific, Waltham, MA, USA) were used. On 5 DIV BV2 cells were seeded on the top of neurons at 1:5 ratio (25×10³ BV2 cells per 125×10³ neurons) in complete Neurobasal medium and left for 2 h to allow attachment. Thereafter, the experiments were performed.

4.2.2 Neuro2a (N2a) cell line

The mouse neuroblastoma 2a cell line (N2a) was maintained on standard 10 cm cell culture dishes in complete DMEM (1X) with GlutaMAX-I containing D-glucose (25 mM) and sodium pyruvate (1 mM) supplemented with 10% iFBS and 1%

penicillin/streptomycin (all ThermoFisher Scientific, Waltham, MA, USA). Cells were passaged by gentle trituration at 80% confluency and seeded at 1:14 ml of cell suspension to fresh medium ratio. Passages 2-10 were used for experiments. The cell line was negatively tested for mycoplasma with MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).

4.2.3 BV2 microglial cell line

The mouse microglial BV2 cell line was maintained on standard 10 cm cell culture dishes in complete RPMI-1640 medium (Sigma-Aldrich, St. Louis, USA) supplemented with 1% GlutaMAX, 10% heat inactivated FBS and 5 μg/ml gentamycin (all reagents ThermoFisher Scientific, Waltham, USA). Cells were passaged by gentle trituration at 80% confluency and seeded at 1:9 ml of cell suspension to fresh medium ratio. Passages 2-10 were used for experiments. The cell line was negatively tested for mycoplasma with MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).

4.2.4 RAW 264.7 macrophages

Murine RAW 264.7 macrophages were maintained on standard 10 cm cell culture dishes in complete DMEM (1X) with GlutaMAX-I containing D-glucose (25 mM) and sodium pyruvate (1 mM) supplemented with 10% iFBS and 1%

penicillin/streptomycin (all ThermoFisher Scientific, Waltham, MA, USA). Cells were passaged by gentle trituration at 80% confluency and seeded at 1:9 ml of cell suspension to fresh medium ratio. Passages 2-7 were used for experiments.

4.2.5 In vitro experiments

4.2.5.1 Glutamate excitotoxicity assay

On 5 DIV after seeding 50% of the media was changed and the cultures were used for experiments on 6 DIV. Cells were treated with 400 μM L-glutamic acid (Sigma-Aldrich, St. Louis, USA) for 24 h prior to measurement of cell viability by the MTT assay or RNA isolation.

4.2.5.2 Hypoxia and oxygen/glucose deprivation with reoxygenation For OGD/R exposures, 24 h after plating N2a cells media was replaced with DMEM depleted from glucose and sodium pyruvate (ThermoFisher Scientific, Waltham, USA). Cells were then placed into the 37^oC hypoxia chamber with 1% O2 and 5% CO2

(ProOx C21, Biospherix Ltd, Parish, NY, USA) for 2 or 3 h, then the media was changed for complete DMEM and cells were returned to standard conditions for 24 h reoxygenation. The control plates were treated similarly, but instead maintained in complete DMEM media in a standard CO2 incubator.

4.2.5.3 Cytokine, endotoxin and drug treatments

Primary microglia were exposed to 20 ng/ml IFN-γ (Sigma-Aldrich, St. Louis, MO, USA) for 24 h followed by 10 ng/ml LPS (Sigma Aldrich, St. Louis, MO, USA) for another 24 h (IFN-γ/LPS treatment referred as M1), in combination with or without 20 ng/ml IL-13 (ThermoFisher Scientific, Waltham, MA, USA). In HX600 study, microglia were pretreated with 1 μM HX600 for 24 h, after which the cells were exposed to 50 ng/ml LPS (Sigma Aldrich, St. Louis, MO, USA) for 3 h, while keeping the concentration of HX600 unaltered. BV2 cells were treated with 50 ng/ml LPS or 20 ng/ml IL-4 for 24 h.

On 6 DIV primary cortical neurons were treated with 100 nM or 1 μM HX600 in the presence of 500 μM glutamate (Sigma-Aldrich, St. Louis, USA) for 24 h. Alternatively, on 6 DIV neurons were pre-exposed to HX600 for 24 h and followed by treatment with glutamate (500 μM) for another 24 h, without changing the HX600 concentration. In the miRNA study, neurons were treated with 400 μM glutamate for 24 h prior to measurement of cell viability by the MTT assay or RNA isolation.

Primary cortical neuron and BV2 co-cultures were pretreated with 1 μM HX600 for 6 h, after which they were exposed to 100 ng/ml LPS (#L2630, serotype O111:B4, Sigma Aldrich, St. Louis, MO, USA) and 30 ng/ml IFN-γ (Sigma-Aldrich, St. Louis, MO, USA) for 48 h. Concentration of HX600 was kept constant during LPS/IFN-γ exposure.

Mouse neuroblastoma N2a cells were seeded together with RAW 264.7 macrophages in ratio 1:1 at a density of total 200×10³ cells/well on 12-well plate format, in DMEM

supplemented with 10% iFBS and 1% P/S (all reagents ThermoFisher Scientific, Waltham, MA, USA). 24 h after plating, co-cultures were treated for 24 h with vehicle PBS or 25 ng/ml LPS (Sigma Aldrich, St. Louis, MO, USA) with or without 20 ng/ml IL-13 (ThermoFisher Scientific, Waltham, MA, USA). Then, co-cultures were exposed for another 24 h to vehicle or 100 ng/ml LPS and 25 ng/ml IFN-γ (Sigma-Aldrich, St.

Louis, MO, USA) with or without 20 ng/ml IL-13. Thereafter, the cells were collected, spun down and prepared for FACS analyses, and the media was saved for NO release measurements.

4.2.5.4 Co-culture neuroinflammation assay

Primary cortical neuron-BV2 co-cultures were prepared as described above. After treatment as described above, cells were fixed with 4% PFA, washed with PBS and neuronal viability was evaluated as previously described[231]. Briefly, the co-cultures were stained with MAP2 peroxidase labelling and then the ABTS peroxidase substrate kit (Vector) was used according to manufacturer’s instruction. After 30 min incubation in the dark the plate was gently agitated and 150 μl of the supernatant was transferred to a 96 well plate. The absorbance was measured with microplate reader Victor 2.0 (Perkin Elmer, MA, USA) at 405 nm and the results were calculated as relative absorbances compared to control wells. Alternatively, the co-cultures were stained with MAP2 antibody (Sigma-Aldrich, St. Louis, MO, USA) followed by incubation with Alexa-488 conjugated secondary antibody. The extent of MAP2 immunoreactivity indicative of neuronal viability was imaged under fluorescent microscope and quantified using ImagePro software (Media Cybernetics Inc., Rockville, MD, USA).

4.2.5.5 Lentiviral transductions

N2a or BV2 cells were seeded at a density of 12×10⁴ cells/well on 12-well plate format and lentiviral vector transduction was performed 24 h later. LV1-GFP (control) or LV1-miR-669c constructs were added into the fresh cell culture media at MOI 30.

Transduction was performed for 24 h, media were exchanged and transduced cells visualized under a fluorescent microscope (Carl Zeiss AG, Jena, Germany) by virtue of GFP expression. GFP positive cells were sorted with BD FACSAria III to establish a cell line stably expressing GFP-control or GFP-miR-699c.

4.2.5.6 miRNA mimics and siRNA transfections

For miRNA pulldown assay, biotinylated mmu-miR-669c-3p and biotinylated control cel-miR-39-3p (both miRCURY LNA microRNA mimics, Premium, Biotin, Exiqon) were transfected. For HX600 study, BV2 cells were transfected with negative control siRNA (ThermoFisher Scientific, Waltham, MA, USA) or NURR1 siRNA (Silencer select siRNA for Nr4a2, ThermoFisher Scientific, Waltham, MA, USA).

Transfections were performed with Viromer Blue transfection reagent (Lipocalyx GmbH, Halle, Germany) in Opti-MEM media (ThermoFisher Scientific, Waltham, USA) for 4 h, after which medium was changed to complete DMEM or complete RPMI-1640 for 20 h. For all transfections, 50 nM oligonucleotide concentrations were used. Transfection efficiency with siRNA was assessed by qPCR for the gene of interest.

4.2.5.7 miRNA pulldown

N2a or BV2 cell lines were seeded at a density of 3×10⁶ cells/dish or 2×10⁶ cells/dish on 10 cm dishes in complete DMEM or complete RPMI-1640 medium, respectively.

RNA pulldown was performed with modifications as described elsewhere[308].

After transfection with biotinylated miRNA mimics, N2a and BV2 cells were exposed to 2 h of OGD and then returned to standard conditions for 24 h reoxygenation.

Thereafter cells were collected and the pulldown experiment was performed. RNA was extracted via magnetic Dynabeads MyOne Streptavidin C1 (ThermoFisher Scientific, Waltham, USA) using mirVana miRNA Isolation Kit. RNA was reverse transcribed and cDNA templates were used for qPCR reactions with selected TaqMan gene expression assays. The results were normalized to control lysate values and then to fold changes calculated against control miRNA.

4.2.6 Outcome analyses

4.2.6.1 MTT reduction and LDH release assays

The MTT reduction assay was performed as previously described[309]. Briefly, following removal of media, MTT stock was added to new culture media at 1.2 mM final concentration. Cells were incubated with MTT containing media for 2 h (N2a) of 4 h (primary microglia) at 37°C in a CO² incubator until visible purple formazan crystals were formed. Following removal of the media, crystals were dissolved in DMSO. 100 μl aliquots were transferred to 96-well plates and absorbances were read at 585 nm with Wallac Victor2 1420 microplate reader (PerkinElmer Inc, Waltham, MA, USA). Results were calculated as percentages of relative absorbances compared to control wells.

LDH release assay was performed according to manufacturer’s instructions. Briefly, media were transferred to a 96-well plate and mixed with reaction mixture. After a 30 min incubation at RT, reactions were stopped by adding stop solution. Absorbance at 490 nm and 680 nm was measured using a microplate reader (PerkinElmer Inc, Waltham, MA, USA) to determine LDH activity. The results are presented as percentage of LDH release in relation to LDH positive control.

4.2.6.2 Real-time quantitative polymerase chain reaction (qPCR) 4.2.6.2.1 qPCR for mRNA and miRNA levels

Total RNA from cell culture lysates was isolated with the mirVana miRNA Isolation Kit according to manufacturer’s instruction (ThermoFisher Scientific, Waltham, USA). The subsequent isolation steps and qPCR reactions were as described in sections 4.1.6.4.1 and 4.1.6.4.2.

4.2.6.3 Cytometric bead array (CBA)

The cytometric bead array Mouse Inflammation kit (BD Biosciences, Franklin Lakes, NJ) was used to analyze IL-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70 cytokine concentrations in cell culture supernatants according to manufacturer’s instructions.

Data was acquired using FACSCalibur (BD Biosciences, San Jose, USA) or CytoFLEX S (Beckman Coulter, Indianapolis, USA) and analyzed by FCAP Array software (Soft Flow Inc., St. Louis Park, MN).

4.2.6.4 Immunocytochemistry (ICC)

Primary cortical neurons and BV2 co-cultures were stained with MAP2 antibody (Sigma-Aldrich, St. Louis, MO, USA) followed by incubation with Alexa Fluor 488-conjugated secondary antibody. The extent of MAP2 immunoreactivity indicative of neuronal viability was imaged under a fluorescent microscope and quantified using ImagePro software (Media Cybernetics Inc., Rockville, MD, USA).

4.2.6.5 Flow cytometry analysis of N2a cell death in co-culture with RAW 264.7 murine macrophages

Cells were incubated with a 1:200 dilution of CD11b-Alexa Fluor 647 (BD Biosciences, San Jose, CA, USA) for 30 min in the dark at 4°C, then washed with HBSS containing 3% iFBS (ThermoFisher Scientific, Waltham, MA, USA), resuspended in HBSS with 3% iFBS and counterstained with propidium iodide (PI) at a concentration of 2.5

g/ml (Sigma Aldrich, St. Louis, MO, USA). To assess the percentage of dead N2a cells, Alexa Fluor 647 and PI double staining was analyzed with CytoFLEX S (Beckman Coulter, Indianapolis, IN, USA). Dead N2a cells were detected as Alexa Fluor 647 (CD11b) negative and PI positive (UL quadrant).

4.2.6.6 Nitric oxide release measurements

NO production was indirectly assessed as described previously[310] by detection of nitrites in media samples obtained from N2a-RAW 264.7 co-cultures. A standard curve was prepared using 0-100 μM sodium nitrite (Sigma Aldrich, St. Louis, MO, USA) in cell culture media. All samples in triplicates were transferred in 50 μl volume to a 96-well plate and 50 μl of Griess reagent was added per well. The absorbance was then measured with a microplate reader Victor 2.0 (Perkin Elmer, MA, USA) at 544 nm and the nitrite concentration was calculated.

4.3 STATISTICAL ANALYSES AND EXCLUSION CRITERIA Prior to experiments, animals were randomized to treatment groups using GraphPad QuickCalcs software. All data collected from the study were analyzed blinded to the treatment groups. The statistical analyses were performed with GraphPad Prism (GraphPad Software Inc, La Jolla, CA, USA) using statistical tests indicated in the figure legends to compare means of interest assuming homoscedasticity and normality of variables. Statistically significant outliers as calculated by Grubb's tests using GraphPad Prism software were excluded from the datasets. Based on predetermined exclusion criteria, animals with unsuccessful ischemia induction or visible hemorrhages in MRI images were excluded from analyses. Data is reported as mean ± SEM unless otherwise stated. P-values <0.05 were considered as statistically significant.

5 RESULTS

5.1 miR-669c OVEREXPRESSION IS PROTECTIVE IN A MOUSE ISCHEMIC STROKE MODEL BY INCREASING M2A ALTERNATIVE MICROGLIAL POLARIZATION, AND BY TARGETING MYD88 (I) Study (I) was carried out to investigate how a member of microRNA cluster C2MC, miR-669c, is implicated in cerebral infarction pathology and to evaluate the potential role of this miRNA in regulation of ischemic stroke-induced neuroinflammatory events. In order to elucidate that we used two cerebral ischemia models, pMCAo and tMCAo, in combination with several analytical methods including MRI, behavioral testing with composite neuroscore, as well as immunohistochemistry, ex vivo cell morphology analyses and qPCR. In order to validate miR-669c-3p relevant gene targets under inflammation and hypoxia, we used an in vitro miRNA pulldown assay.

5.1.1 miR-669c-3p expression is elevated upon excitotoxic or ischemic neuronal injury

To assess whether OGD/R, glutamate exposure in vitro or ischemic stroke in vivo modulate the expression of miR-669c-3p, N2a cells were exposed to OGD for 1, 2 or 3 h followed by reoxygenation for 24 h. OGD/R, mimicking transient ischemic stroke, increased the expression of miR-669c-3p (Fig. 1A: +102% for 1 h, +118% for 2 h and +98.5% for 3 h comparing to control cells, p≤0.0001). Excitotoxic exposure with 400 μM glutamate resulted in significant induction of miR-669c-3p levels (Fig. 1B: +82%

comparing to vehicle-exposed cells, p=0.009). In order to evaluate if miR-669c overexpression is directly neuroprotective in vitro, N2a cells transduced with lentiviral vectors were subjected to OGD for 2 hours followed by 24 hours of reoxygenation. Lentiviral-mediated overexpression of miR-669c did not prevent OGD/R-induced N2a cells death (Fig. 1C). Mice were subjected to either pMCAo or tMCAo to induce ischemic stroke. pMCAo increased miR-669c-3p expression in the peri-ischemic (PI) area both at 1 (Fig. 1D: +61.5%, p=0.0443) and 3 dpi (Fig. 1E: +143%, p<0.0001), compared to contralateral cortex, whereas the levels of miR-669c-3p in tMCAo were only increased at 3 dpi (Fig. 1G: +180%, p=0.0101). On the contrary, the levels of the other arm of this miRNA hairpin precursor, miR-669c-5p, were not changed (p=0.8385) between PI and contralateral cortex at 3 dpi in pMCAo model (data not shown).

5.1.2 miR-669c overexpression influences inflammatory responses in BV2 cells by enhancing their M2a polarization

To evaluate the impact of miR-669c on microglial function, we first measured levels of miR-669c-3p in LPS- and IL-4-exposed BV2 cells or LPS- and IFN-/LPS-exposed primary murine microglia. While both LPS and IL-4 did not alter

miR-669c-3p expression in BV2 or primary microglia (Fig. 2A), LPS/IFN- challenge increased the expression of miR-669c-3p in primary microglia (Fig. 2B: +102% for LPS/IFN-

challenged cells comparing to vehicle, p=0.0003). Next, we evaluated whether primary astrocytes express similar amounts of miR-669c-3p than primary microglia and found that miR-669c-3p is significantly more prevalent in primary microglia (Fig.

2C: +310% for primary microglia comparing to primary astrocytes, p=0.0032). We confirmed miR-669c-3p overexpression in LV-miR-699c-transduced BV2s (Fig. 2D:

+56.5% for LV-miR-669c BV2s in comparison to cells transduced with control vector, p=0.0001). To investigate how miR-669c influences inflammation-related gene expression and cytokine production we challenged with LPS BV2 cells transduced with lentivirus to overexpress miR-699c. miR-669c overexpression under proinflammatory conditions decreased expression of Iba1 (Fig. 2E: -25% for LV-miR-669c BV2s comparing to control cells, p=0.011), fractalkine receptor CX3CR1 (Fig. 2F:

-67%, p=0.0002), and proinflammatory markers MMP9 (Fig. 2G: -68%, p=0.0001), TNF-α (Fig. 2H: -69%, p<0.0001), IL-6 (Fig. 2I: -84%, p<0.0001), IL-1β (Fig. 2J: -74%, p<0.0001) and CCL2 (Fig. 2K: -72%, p<0.0001), in comparison to GFP transduced control cells. Interestingly, miR-669c overexpression resulted in a significant increase of the M2a-type, alternative microglia markers Arg1 (Fig. 2L: +250% for LV-miR-669c BV2s comparing to control cells, p=0.0014), Chil3/Ym1 (Fig. 2M: +511%, p<0.0001) and PPAR-γ (Fig. 2N: +94%, p=0.0071). In contrast, the expression levels of M2c-type immunosuppressive cytokines IL-10 (Fig. 2O: -40% for LV-miR-669c BV2s comparing to control cells, p=0.0059) and TGF-β (Fig. 2P: -23%, p=0.0143) were reduced in LV1-miR-669c-transduced BV2 cells.

To determine the impact of miR-669c on microglial cytokine secretion, miR-669c transduced BV2 cells were treated with LPS and levels of cytokines measured in conditioned media. miR-669c overexpressing cells showed a decreased proinflammatory response to LPS challenge, since production of TNFα (Fig. 3A, -33.5% comparing to transduced with control vector, LPS-treated cells, p<0.0001), IL-6 (Fig. 3B: -57%, p<0.0001) and IL-12p70 (Fig. 3C:-85%, p=0.0019) was significantly alleviated compared to LPS-exposed control cells. In contrast, levels of MCP-1 were increased (Fig. 3D: +13%, p=0.0009) and levels of IL-10 (Fig. 3E) remained unaltered.

5.1.3 miR-669c overexpression confers protection in in vivo tMCAo

Next, we tested whether the lentivirus-driven miR-669c overexpression is neuroprotective in vivo in tMCAo in mice. miR-669c lentiviral vector and LV1-GFP control vector were injected into the caudate putamen of C57BL/6J mice. The animals were subjected to tMCAo 3 weeks after injections and the infarction volume was measured using MRI. The LV1-miR-669c injected mice had significantly smaller ischemic lesions both at 1 (-22.5% smaller comparing to control animals, p=0.0489) and 3 dpi (-26%, p=0.0044) (Fig. 4A-F). Concomitantly, the LV1-miR-669c injected mice exhibited improved sensorimotor functions as evaluated by the composite neuroscore testing at both of these time points (Fig. 4G: -30% decrease of deficits in comparison to control, LV1-GFP-injected stroke animals, p=0.0427 and 4H: -47%, p=0.0014).

5.1.4 Injected LV1-miR-669c induces alternative microglial polarization and changes microglial morphology in the ischemic brain

To assess the impact of miR-669c overexpression on ischemia-induced microglial activation, the brains of stroked mice were evaluated by IHC staining for typical microglial/macrophage marker Iba1[311] and the alternative activation marker Arg1[312]. As compared to LV1-miR-669c shams, ischemic stroke increased total Iba1 immunoreactivity (p=0.0355) in LV1-miR-669c tMCAo animals (data not shown).

There was no change in total microglial Iba1 between miR-669c overexpressing animals and the control group at 3 dpi (Fig. 5A-C, p=0.5429). However, we found that Arg1 staining was significantly stronger in LV1-miR-669c tMCAo animals at 3 dpi, compared to control-injected mice (Fig. 5F-J: +363%, p=0.0044). Arg1 is a M2 specific marker contributing to extracellular matrix remodeling and tissue repair[313]. We also quantified CD45, a leukocyte marker highly expressed in infiltrating myeloid cells and to lower extent in resting microglia, while upregulated in reactive microglial

There was no change in total microglial Iba1 between miR-669c overexpressing animals and the control group at 3 dpi (Fig. 5A-C, p=0.5429). However, we found that Arg1 staining was significantly stronger in LV1-miR-669c tMCAo animals at 3 dpi, compared to control-injected mice (Fig. 5F-J: +363%, p=0.0044). Arg1 is a M2 specific marker contributing to extracellular matrix remodeling and tissue repair[313]. We also quantified CD45, a leukocyte marker highly expressed in infiltrating myeloid cells and to lower extent in resting microglia, while upregulated in reactive microglial

In document Modulation of neuroimmune responses as potential treatment strategies in ischemic stroke (sivua 60-0)