Sulfosuccinimidyl oleate sodium is neuroprotective and alleviates stroke-induced neuroinflammation

The mouse BV2 microglial cell line was kindly provided by professor Rosario Donato (University of Perugia, Italy). Cells were maintained in a humidified chamber with 5% CO2 and 37 °C in RPMI-1620 medium (Invitrogen, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen) and 0.6 μg/ml gentamicin (Sigma-Aldrich, MO, USA). Cells were seeded at a density of 1.5 × 10⁵ cells/ml for both protein and RNA extraction. Culture media was replaced by serum-free RPMI media 24 h after seeding. On the next day, the cells were stimulated with 100 ng/ml lipopolysaccharide (LPS, Sigma-Aldrich) and 5 ng/ml interferon-γ (IFN-γ, Peprotech, NJ, USA) and co-treated with SSO (TRC, Toronto, Canada) for a further 24 h.

Primary neuronal cultures were prepared as described previously [15]. Briefly, cortical tissue from E15 C57BL/6J embryos was dissected out after carefully removing the meninges. The dissected cortices were transferred to Krebs buffer (0.126 M NaCl, 2.5 mM KCl, 25 mM NaHCO₃, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 2.5 mM CaCl₂, pH 7.4) containing 0.025% trypsin and incubated at 37 °C for 15 min. Then, the reaction was stopped by using trypsin inhibitor containing DNase (Sigma-Aldrich). Neurons were dissociated, counted, and plated in poly-l-lysine (Sigma-Aldrich)-coated 48-well plates at a density of 1.25 × 10⁵ cells/well in neurobasal media (Gibco, NY, USA) supplemented with 2% B27 (Thermo Fisher Scientific, MA, USA), 500 μM L-glutamine (Thermo Fisher Scientific), and 1% penicillin-streptomycin (P/S, Thermo Fisher Scientific). Fifty percent of the media was changed 5 days after seeding. At day 6, the neurons were exposed to 50 μM of SSO for 2 h and then 400 μM of glutamate in the presence of 50 μM SSO for 24 h.

Primary microglia cultures were prepared as described previously [15]. Briefly, P0-P2 C57BL/6J mouse brains were dissected and rinsed in phosphate buffer solution (PBS) containing 1 g/l glucose. Brains were mechanically dissociated and digested using 0.5% trypsin-EDTA for 20 min at 37 °C. The tissue homogenate was suspended in DMEM/F12 (Gibco) containing 10% heat-inactivated FBS and 1% P/S (Thermo Fisher Scientific) and plated on 150-mm cell culture dishes for 3 weeks. After reaching confluency, astrocytes were removed using 0.25% trypsin in Hank’s Balanced Salt Solution (HBSS, Gibco) in 1:4 dilution in serum-free DMEM/F12. After careful washes with PBS, the microglia attached on the bottom of the plates were isolated by using 0.25% trypsin in PBS. Fresh culture medium containing FBS was added to neutralize the effect of trypsin. Cells were then centrifuged and plated for co-culture studies.

For primary neuron-BV2 co-culture, cortical neurons were plated onto a 48-well plate at a density of 1.25 × 10⁵ cells/well in neurobasal media as described previously. At day 5, BV2 cells were plated on top of neurons at a density of 1:5 (25,000 BV2 cells for 125,000 neurons) for 2 h. Cells were pre-treated with SSO for 2 h and then exposed to 100 ng/ml LPS and 5 ng/ml IFNγ for 48 h. The co-cultures were rinsed with PBS, fixed with 4% PFA for 20 min, and washed again with PBS. Neuronal viability in the co-cultures was evaluated as described previously [16]. The co-cultures were stained with microtubule-associated protein 2 (MAP-2) peroxidase labeling, and the ABTS peroxidase substrate kit (Vector) was used to develop the color for the analysis according to the manufacturer’s instructions. The green-colored product was formed after incubation for 30 min with ABTS kit in the dark. One hundred fifty microliter of the substrate solution was transferred to a 96-well plate, and the absorbance was measured using a microtiter plate reader Victor 2.0 (Perkin Elmer, MA, USA) at 405 nm. For primary neuron-microglia co-culture, cortical neurons were similarly plated onto 48-well plates at a density of 1.25 × 10⁵ cells/well. At day 5, primary microglia were plated on top of neurons in neurobasal media at a density of 1:2 (62,500 microglia for 125,000 neurons). On the next day, the cells were pre-treated with SSO for 2 h and then exposed to 100 ng/ml LPS and 30 ng/ml IFN-γ for 48 h. The cells were then rinsed with PBS, fixed with 4% PFA, and the neuronal viability assessed by ABTS peroxidase substrate kit as described above.

Nitric oxide (NO) production was assessed in the culture media by using Griess assay 24 h after LPS/IFNγ stimulation and SSO treatment. Fifty microliter of culture supernatant was incubated with an equal volume of Griess reagent for 10 min at room temperature (RT), and the optical density was measured at 544 nm using Victor 2.0 plate reader.

Cell viability was measured using resazurin assay 24 h after exposure. Briefly, cells were incubated with 10 μM resazurin (Sigma-Aldrich) diluted in culture media for 2 h at 37 °C. The absorbance was then quantified at 485 nm using Victor 2.0 plate reader.

Culture supernatants obtained 24 h after SSO treatment were used to determine the levels of interleukin-6 (IL-6), IL-10, monocyte chemoattractant protein 1 (MCP-1), TNF-α, IFN-γ, and IL-12p70 using a mouse anti-inflammatory cytometric bead array (CBA) kit (BD Biosciences, CA, USA). After staining, the samples were run on a FACS Calibur flow cytometer (BD Biosciences). The results were analyzed using FCAP array software (Soft Flow Hungary Ltd., Pecs, Hungary).

Twenty-four hours after SSO treatment, BV2 cells were washed with PBS and collected in SDS sample buffer (0.0625 M TRIS-HCl, 2.3% SDS, 5% β-mercaptoethanol, 10% glycerol, bromophenol blue). For analyzing the nuclear localization of NFκB, BV2 cells were treated as described above and subcellular fractions were generated with Cell Fractionation kit (Cell Signaling) according to the manufacturer’s protocol. Ten micrograms of protein were loaded and run on 10% SDS-PAGE gels. The proteins were transferred onto PVDF-hybond membranes for 1 h at 4 °C. The membranes were then blocked in 5% milk in 0.2% PBS-tween (PBST) for 30 min. Primary antibodies for total cell lysates (nitric oxide synthase 2 (NOS2), 1:2000 dilution, EMD Millipore, MA, USA; COX-2, 1:1000 dilution, BD Biosciences; heme oxygenase-1 (HO-1), 1:1000 dilution, ENZO Lifesciences, NY, USA; P-p38, 1:1000 dilution, Cell Signaling Technology, MA, USA) and for nuclear fractions (NFκB p65, 1:1000 dilution, Santa Cruz, TX, USA) in 5% BSA in PBS and 0.02% NaN3 were incubated with the membranes overnight at 4 °C. After washing with PBST, appropriate secondary antibodies were incubated for 2 h at RT diluted at 1:2000 in a blocking solution (anti-mouse ECL-HRP; GE healthcare life science, Uppsala, Sweden, or anti-rabbit IgG HRP Conjugate, Bio-Rad, CA, USA). The membranes were washed again with PBST, and the protein bands were visualized using an ECL-plus kit (Thermo Fisher Scientific) under Storm 860 scanner (Molecular dynamics, Caesarea, Israel). β-actin (1:2000 dilution, Sigma-Aldrich) and histone-3 (H3) (1:1000 dilution, Abcam, Cambridge, UK) antibodies were used as loading controls for total cell lysates and nuclear fractions, respectively. Their immunoreactivities were detected with Cy3-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, PA, USA) and scanned with G:BOX imaging system (Syngene, Cambridge, UK). Densitometric analysis was performed using ImageQuant TL software (GE healthcare life science).

The relative expression levels of IL-6, NOS2, nuclear factor erythroid 2-related factor-2 (Nrf2), and HO-1were measured in BV2 cells exposed to LPS/IFNγ and SSO using quantitative PCR (qPCR). Total RNAs were isolated using the RNeasy mini kit (Qiagen, CA, USA) according to the manufacturer’s instructions. The concentration and purity of RNA samples were determined using a NanoDrop 2000 (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized by using 500 ng of total RNA, Maxima Reverse Transcriptase, dNTP, and random hexamer primers (Life Technologies, CA, USA). The final concentration of cDNA was 2.5 ng/μl. qPCR was run using the StepOne Plus Real-Time PCR system (Applied Bioscience, CA, USA). Gene expression analysis was carried out using comparative C_t method (△△C_T) where the threshold cycle of the target genes was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ribosomal RNA internal housekeeping gene controls (△C_T). The mRNA expression is presented as a fold change.

A total of 21 4-month-old male BALB/cABom mice (Taconic, Köln, Germany) obtained by breeding in-house were used in this study. The mice were housed under a 12-h light/dark cycle and allowed for food and water ad libitum. All the animal experiments were approved by the National Animal Experiment Board of Finland and followed the Council of Europe Legislation and Regulation for Animal Protection. The animals were randomized and divided into two groups, SSO treated (n = 10) and control (n = 11) using GraphPad QuickCalcs software (GraphPad Software, Inc. La Jolla, CA, USA).

All animals underwent distal permanent occlusion of the middle cerebral artery (MCA) (pMCAo) as described previously [17]. Briefly, mice were anesthetized with 5% isoflurane for induction and 2% isoflurane for maintenance (70% N₂O/30% O₂). The temperature of the mice was maintained at 36-5 ± 0.5 °C using a thermostatically controlled heating blanket connected to a rectal probe (PanLab, Harvard Apparatus, Barcelona, Spain). The temporalis muscle was retracted to expose the skull in between the ear and the eye, and a small hole of approximately 1 mm was drilled at the site of the MCA. The dura was carefully removed to expose the MCA. The artery was then gently lifted up and cauterized using a thermocoagulator (Bovie Medical Corporation, Clearwater, FL, USA). After the procedure, the muscle was lifted back and the skin wound was sutured. The animals were then placed back to their home cages. SSO was emulsified in 0.5% methyl cellulose and administered once by single oral gavage at the dose of 50 mg/kg immediately after the surgery, when the mice had retained their consciousness. The administration routes for SSO in vivo have been described previously [18, 19]. In addition, we performed a dose-response study and found that SSO at 50 mg/kg was suitable to see a beneficial effect after stroke. There was no mortality in this study.

Infarct size was imaged by magnetic resonance imaging (MRI) at 3 days post-injury using a vertical 9.4T Oxford NMR 400 magnet (Oxford Instrument PLC, Abingdon, UK). Mice were anesthetized and maintained at 1.5% isoflurane during the imaging. A quadrature volume radiofrequency coil was used for transmission and reception. Multislice T2-weighted images (repetition time 3000 ms, echo time 40 ms, matrix size 128 × 256, field of view 19.2 × 19.2 mm², slice thickness 0.8 mm, and number of slices 12) were taken with double spin-echo sequences with adiabatic refocusing pulses. Lesion volume was outlined manually on T2-weighted images using an in-house software (Aedes) under MATLAB (Math-works, Natick, MA, USA) environment. The total lesion volume and the volume of the left and right hemispheres were calculated by multiplying the number of pixels with the pixel size and slice thickness. The relative percentage of the infarction volume was calculated as described previously: infarction volume = (volume of left hemisphere − (volume of right hemisphere − measured infarct volume))/volume of left hemisphere [20]. The analysis was performed blinded to the study groups.

All mice were sacrificed immediately after MRI imaging at 3 days post-injury. The mice were anesthetized using 250 mg/kg of avertin and transcardially perfused with heparinized saline (2500 IU/l). The brains were removed and post-fixed in 4% paraformaldehyde (PFA) for 24 h followed by cryoprotection in 30% sucrose for 48 h. The brains were snap frozen in liquid nitrogen and cut into 20-μm-thick serial coronal sections at the start of the lesion using a cryostat (Leica Microsystems GmbH, Wetzlar, Germany).

For the detection of microglia, astrocytes, COX-2, and HO-1 immunoreactivity, a set of six consecutive sections at 400 μM intervals starting from the beginning of the lesion were used. The sections were incubated against primary antibody ionized calcium binding adapter molecule 1(Iba-1, 1:250 dilution, Wako Chemical GmbH, Neuss, Germany), CD45 (1:100 dilution, BioRad, Hercules, CA, USA), NeuN (1:200 dilution, EMD Millipore, Temecula, CA, USA), glial fibrillary acidic protein (GFAP, 1:500 dilution; DAKO; Glostrup, Denmark or 1:200 dilution; EMD Millipore), COX-2 (1:500 dilution; Cayman Chemicals, Ann Arbor, MI, USA), and HO-1 (1:1000, ENZO) overnight at RT. The sections were then incubated with biotinylated secondary antibody (1:200 dilution, Vector, Burlingame, CA, USA) for 2 h and then avidin-biotin complex reagent (Vector) according to the manufacturer’s instructions. Alternatively, Alexa 568-conjugated secondary antibody (1:200 dilution; Invitrogen, Eugene, OR, USA) was used. Immunoreactivities were visualized by the development with nickel-enhanced 3, 3′-diaminodenzidine.

Immunoreactivities in the peri-ischemic area adjacent to the infarct border were imaged at ×10 magnification using AX70 microscope (Olympus Corporation, Tokyo, Japan) with an attached digital camera (Color View 12 of F-view, Soft Imaging System, Munster, Germany) and a computer running analysis software (Soft imaging system). The intensity of the immunoreactivities was quantified from an area of 718 × 532 μm adjacent to the infarct border using ImagePro Software (Media Cybernetics, Silver Spring, MD, USA) blinded to the study groups. A total of seven to ten mice per group were analyzed.

Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., CA, USA) by running one-way ANOVA with Tukey’s post hoc test or student t test where appropriate. Statistical outliers were excluded from the data set after performing Grubb’s test using GraphPad Prism QuickCalcs. All the data are expressed as mean ± SEM and P < 0.05 was used for the determination of statistical significance.

The ability of SSO to alter cell viability was first assessed with naïve BV2 cells or BV2 cells stimulated with 100 ng/ml LPS and 5 ng/ml IFNγ. Two concentrations of SSO (20 μM and 50 μM) were used. SSO alone did not alter the cellular viability as measured by the resazurin assay (Fig. 1a). Exposure to 100 ng/ml LPS + 5 ng/ml IFNγ modestly, yet significantly reduced the viability of the BV2 cells. Co-treatment with SSO prevented the LPS + IFNγ-induced reduction in the cell viability (Fig. 1a). In addition, LPS + IFNγ exposure induced a massive NO production in BV2 cells which was blocked by co-treatment with SSO at both concentrations (Fig. 1b).

We next assessed whether SSO is directly neuroprotective in cultured primary neurons exposed to glutamate-mediated excitotoxicity. Primary neurons were pre-treated with 50 μM SSO for 2 h followed by the exposure to 400 μM glutamate and 50 μM SSO for 24 h. SSO alone was not toxic to the neurons, yet it was unable to prevent glutamate-induced neuronal death (Fig. 5a). Since SSO was not able to rescue neuronal viability in pure neuronal cultures but has anti-inflammatory properties, we assessed whether SSO can prevent inflammation-induced neuronal death using primary neuron-BV2 co-cultures. The co-cultures were pre-treated with two concentrations of SSO (20 μM and 50 μM) followed by the exposure to 100 ng/ml LPS and 5 ng/ml IFNγ. Measurement of neuronal viability using peroxidase-ABTS kit showed that SSO alone was not causing any loss of MAP-2 immunoreactivity. Moreover, SSO dose-dependently prevented the LPS/IFNγ-induced neuronal death (Fig. 5b). To confirm these findings on primary microglia, we exposed a primary neuron-primary microglia co-culture model to 20 μM SSO, and similarly, 2-h SSO pre-treatment significantly prevented LPS + IFNγ-induced neuron death (Fig. 5c).

Based on our in vitro data, we then tested the therapeutic effect of SSO in a mouse model of pMCAo. We chose the dose of 50 mg/kg of SSO, which caused no adverse effect to the mice. The mice underwent MRI imaging at 3 days post-injury. Quantification of the lesion volumes revealed that orally administered SSO significantly reduced the cortical ischemic infarct size compared to vehicle-treated controls (Fig. 6).

Ischemia-induced brain microgliosis was analyzed by immunohistochemical staining against Iba-1. As expected, we detected a significant upregulation of microgliosis in the peri-ischemic area of both the SSO-treated and control mice when compared to the corresponding area in the contralateral side at 3 days post-stroke (Fig. 7a). However, the extent of Iba-1 immunoreactivity in the peri-ischemic area of SSO-treated mice was significantly reduced compared to their vehicle-treated counterparts (Fig. 7a). Ischemia induced significant upregulation of GFAP immunoreactivity in the peri-ischemic area compared to the contralateral side, yet SSO failed to reduce stroke-induced increased GFAP immunoreactivity (Fig. 7b).

Due to the ability of SSO to reduce the expression of COX-2 in vitro, we analyzed the extent of COX-2 immunoreactivity in the peri-ischemic area of the stroked animals at 3 days post-stroke. pMCAo induced a significant upregulation in COX-2 immunoreactivity in the peri-ischemic area (Fig. 8a). SSO-treated animals showed a reduced extent of COX-2 expression compared to vehicle-treated controls (Fig. 8a). To evaluate the cell types expressing COX-2, we carried out immunohistological double stainings with COX-2 and microglial/macrophage marker CD45, neuronal marker NeuN, and astrocytic marker GFAP. The double stainings revealed that COX-2 immunoreactivity was mainly localized in microglia/macrophages and neurons but not in astrocytes (Fig. 8f). To evaluate the impact of SSO to induce antioxidant response, we carried out a staining against HO-1 and detected a significant upregulation in HO-1 in the peri-ischemic area in the SSO-treated animals when compared to vehicle-treated controls (Fig. 8g). Double stainings of HO-1 with CD45, NeuN, and GFAP revealed colocalization similar to COX-2, HO-1 was mainly expressed in microglia/macrophages and neurons, but not in astrocytes (Fig. 8l).