Expression of sphingosine kinase 1 in amoeboid microglial cells in the corpus callosum of postnatal rats

Wistar rats aged 1, 3, 5, 7, 14 and 21 days were used for immunofluoresence studies (n = 3, at each age). The rats were anesthetized with Nembutal (sodium pentobarbital, 60 mg/kg) and perfused transcardially with Ringer's solution followed by 2% paraformaldehyde in 0.1 M phosphate buffer. Following perfusion, the brain was removed, post-fixed in the same fixative and cryoprotected in 30% sucrose for 24 h. Frozen sections at 30 μm were cut coronally through the forebrain with a cryostat (Model CM 3050; Leica Instruments GmbH, NUBLOCH, Germany) and mounted onto gelatin-coated slides and stored at -20°C until use.

Fifty four 5-day-old rats were divided into 6 groups. In each group (n = 9), 3 rats were used for immunofluoresence staining and 6 for reverse transcription polymerase chain reactions (RT-PCR) and western blotting. In the experimental groups, the rats were given an intraperitoneal (i.p.) injection of 100 μL lipopolysaccharide (LPS) (1 mg/kg; Cat. No. L2654, Sigma-Aldrich, MO, USA). Three rats each were sacrificed at 30 min, 1 h and 6 h after LPS injection; 9 rats receiving an equal volume of saline served as matching controls. In another experimental group receiving DMS and LPS treatments, the rats were given an i.p. injection of 100 μl DMS (100 μM, Cat. No.310500, Calbiochem, USA) followed by LPS (3 mg/kg) injection 15 min later. For immunostaining, the brain was removed and sectioned, and the tissue sections stored with the same procedure as described above. For western blotting and quantitative RT-PCR analysis, the brain was exposed and the cerebral hemispheres dissected under deep anaesthesia. Fresh tissue samples of the corpus callosum above the lateral ventricles were carefully dissected and kept in liquid nitrogen until further use.

Forty five rats, aged 5 days, were divided into 5 groups (n = 9 each). Twenty seven rats were placed in a chamber filled with a gas mixture of 5% oxygen and 95% nitrogen (MCO18M; Sanyo Biomedical Electrical Co Ltd, Tokyo, Japan) for 2 h. They were allowed to recover in air and at room temperature for 15 min, 1 h and 6 h before being sacrificed. In another experimental group, the rats received DMS pretreatment followed by hypoxia exposure 15 min later. Nine rats were given firstly with an i.p. injection of 100 μl DMS (100 μM, Cat. No.310500, Calbiochem, USA), followed by an acute hypoxic exposure in an incubator for 2 h. Nine rats kept outside of the chamber were used as normal control. In each group, 3 rats were used for immunofluorescence staining, with the remaining 6 rats used for RT-PCR and western blotting analysis. Tissue sections and fresh corpus callosum were removed for different methods as described above.

Tissue sections were rinsed in phosphate-buffered saline (PBS) and then incubated in 5% normal goat serum diluted in PBS for 1 h at room temperature. Following removal of serum, tissue sections were incubated overnight with a mixture of primary antibodies containing OX-42 (1:300, mouse monoclonal IgG, Cat. No. CBL1512, Chemicon, USA) and anti-SphK1 (1:100, rabbit polyclonal, Cat. No.sc-48825IgG, Santa Cruz, USA) antibodies at room temperature. Some sections were incubated with preimmune rabbit IgG (Cat. No. 17-615, Millipore, USA) as isotypic controls. Sections from LPS or DMS/LPS treated samples were also incubated with OX-42 along with either anti-tumor necrosis factor-α (TNF-α), anti-interleukin-1β (IL-1β) (TNFα, rabbit polyclonal, Cat. No. AB1837P; IL-1β, rabbit polyclonal, Cat. No. AB1832P, Chemicon, USA) or anti-NFκB (1:200, rabbit polyclonal, sc-109, Santa Cruz) antibodies. On the next day, the sections were washed in PBS-Triton X 100 (TX). They were incubated with a mixture of secondary antibody: FITC-conjugated goat anti-rabbit IgG (1:200, Chemicon, USA) and Cy3-conjugated goat anti-mouse IgG (1:200, Chemicon, USA) for 1 h. After washing in PBS-TX, sections were stained with 4', 6-diamidino-2-phenylindole (DAPI, 1 μg/mL) for 2 min at room temperature. They were then rinsed in PBS-TX and mounted with a fluorescent mounting medium (DAKO Cytomation, Glostrup, Denmark). Colocalization was examined under a confocal microscope (LSM FV1000; Olympus Company Pte. Ltd., Tokyo, Japan).

Proteins were extracted from the frozen corpus callosum of saline-, LPS- and DMS+LPS-injected rats using a protein extraction kit (Pierce Biotechnology Inc, IL, USA). The concentration of protein was measured using bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA) as a standard. Samples of supernatant containing 50 μg protein were heated at 95°C for 4 min and separated in 10% sodium dodecyl sulfate-polyacrylamide gels. The proteins were transferred to immobilon polyvinylidene difluoride transfer membranes using a semi-dry electrophoretic transfer cell (Bio-Rad, CA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The membranes were incubated with the primary antibody anti-Sphk1 (1:1000, rabbit polyclonal IgG, Cat. No.sc-48825, Santa Cruz, USA) at 4°C overnight with shaking. After thorough washing in Tris buffered saline with Tween-20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, goat anti-rabbit IgG, Cat. No. GF9501119, Pierce, USA) for 1 h at room temperature. The immunoproducts were detected using a chemiluminescence detection system according to the manufacturer's instructions (Supersignal West Pico Horseradish Peroxidase Detection Kit, Pierce Biotechnology, IL, USA) and developed on a film. The membranes were washed and stripped with Western Blot Stripping Buffer (Prod #21059, Thermo, USA). They were then incubated with primary antibody anti-β-actin (1:10,000, mouse monoclonal, Sigma-Aldrich, MO, USA) at 4°C overnight with shaking. After membranes were incubated with HRP-conjugated secondary antibody (1:5000, goat anti-mouse IgG, Cat. No. GE9542222, Pierce, USA), immunoproducts of β-actin were developed on a film. The optical density of each protein band was quantified by a scanning densitometer and Quantity One Software, version 4.4.1 (Bio-Rad, CA, USA). Each lane of protein band density was normalized with corresponding β-actin density.

Total RNA was extracted from frozen corpus callosum or primary culture microglia using RNeasy Mini kit (Qiagen, Valencia, CA, USA) and its concentration was quantified with a biophotometer (Eppendorf, Westbury, NY, USA). Reverse transcription was carried out as follows: A mixture of 2 μg total RNA and 2 μl Oligo (dT) was heated at 70 °C for 5 min, rapidly cooled down on ice and mixed with reaction mixture containing 5 μl M-MLV RT 5X Buffer (Cat. No M531A, Promega, USA), 0.5 μl deoxyribonucleotide triphosphate (dNTP, 25 μM, Cat No. U1240, Promega, USA), 0.7 μl RNAse inhibitor (2500U, RNasin^® Cat. No N211A, Promega, USA) and 1 μl M-MLV reverse transcriptase (10000u; Cat. No M170A, Promega, USA). The reaction was made up to 25 μl with RNA-free water and then incubated at 42 °C for 1 h and at 70 °C for 10 min. cDNA was synthesized and stored at -80°C until its use. RT-PCR was carried out on a LightCycler instrument using QuantiTect SYBR Green PCR Kit (Cat. No. 204141, QIAGEN, USA) according to the manufacturer's instructions. For RT-PCR, reaction mixture containing 5 μl QuantiTect SYBR Green PCR Master Mix, 0.5 μl of 10 μM forward primer, 0.5 μl of 10 μM reverse primer, cDNA template and RNAse-free water was prepared and transferred into capillary glass tubes. PCR was performed under the following conditions: denaturation at 95 °C for 15 min, followed by 45 cycles of denaturation at 94 °C for 15 s, annealing at 61 °C for 22 s, and extension at 72 °C for 30 s.

Expression of target genes was measured in triplicate and was normalized to β- actin, an internal control. The RT-PCR products were subjected to 2% agarose gel electrophoresis, stained with 1mg/mL ethidium bromide, and photographed using ChemiGenius2 image analyzer (SYNGENE, Cambridge, UK). Forward and reverse primer sequences for each gene and their corresponding amplicon sizes are provided in Table 1. The predicted size and single band confirmed the amplicons and that the respective primers were specific. Fold change of target genes' expression was calculated according to the formula: fold change = 2^{-[[Ct (control) gene X-Ct (control) actin]-[Ct (activated) gene X-Ct (activated) actin]]} [28].

Mixed glial cells were prepared from the cerebrum in neonatal rats aged 3 days and cultured in a flask containing Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), 10 ml/L antibiotic-anti-mycotic (Cat. No. A5955, Sigma, USA), 0.1 mM nonessential amino acid (Cat. No. 11140-050, Invitrogen, USA) and 1 ml/L insulin (Cat. No. I-0516, Sigma, USA). The complete medium was replaced at 24 h and then every 3-4 days. Microglial cells were isolated at 10 days with 0.25% trypsin including 1 mM ethylene diamine tetra acetic acid (EDTA) for 10-20 min at 37 °C[29]. With a complete detachment of upper cell layer, microglial cells remained attached to the bottom of the flask and DMEM containing 10% FBS was added for trypsin inactivation. Microglial cells were cultured in complete medium overnight for their identification. The purity of microglial cells was confirmed by OX-42 labeling.

Microglial cells were plated on poly-L-lysine coated coverslips and cultured in complete medium overnight. The complete medium was then replaced by basic medium and LPS (1 μg/mL) was added into the basic medium for 1 h with or without pretreatment of DMS (15 μM, 15 min). Cells cultured in the basic medium served as the control. The cells were then rinsed with PBS and fixed with 4% paraformaldehyde in phosphate buffer for 30 min at room temperature. Following incubation in normal goat serum, all coverslips with adherent cells were incubated in a mixture of antibodies containing anti-SphK1 (1:100, Santa Cruz, USA) and OX-42 (1:200, Chemicon, USA) at 4°C overnight. The coverslips were washed and incubated with Cy3-conjugated goat anti-rabbit IgG (1:100, Chemicon, USA) and FITC-conjugated goat anti-mouse IgG (1:100, Chemicon, USA) for 1 h at room temperature. Cell nuclei were counterstained with DAPI (1 μg/mL) for 2 min. The coverslips were then washed and mounted with a fluorescent mounting medium (DAKO Cytomation, Glostrup, Denmark). All images were captured under a confocal microscope (LSM FV1000; Olympus Company Pte. Ltd., Tokyo, Japan).

Immunofluorescence staining of SphK1, IL-1β, TNF-α and NFκB in microglial cells was digitally captured by using a confocal microscope, and the average intensity of immunofluorescence in microglial cells was estimated using a software system (Fluoview, FV1000). Background (nonspecific extracellular signal) was optimized. These settings, used to optimize image quality, were then applied as standards for the processing of all cell images. Between seven and ten intact microglial cells (showing cellular and nuclear integrity) from each slide were selected and specified by drawing outlines, and their images were acquired in a single channel (showing single colour). The mean value of the intensity in the specified region was measured and normalized with the intensity value obtained from isotypic control sections. Fold changes were calculated and presented in a histogram. The total number of microglial cells on each slide was manually counted in a specified unit area. The percentages of SphK1-, IL-1β-, TNF-α-, and NFκB-positive cells were also calculated by counting the number of positive cells in OX-42-stained microglia.

AMC in the corpus callosum at different age groups was found to show moderate but definite expression of SphK1 protein as revealed by immunofluorescence staining (Figure 1Aa-Dc, Figure 2B, C). The identification of SphK1-immunoreactive AMC was confirmed by their colocalization with OX42 immunofluorescence. Sections incubated with rabbit IgG as the isotypic control for anti-SphK1 did not show any immunoreactivity, confirming the specificity of SphK1 immunoreactivity (Figure 2Aa-c). The intensity of SphK1 expression in AMC was most intense in corpus callosum of 1- to 5-day-old rats compared to that of those aged 7 d (Figure 1Da-c, Figure 2C). In rats aged 14 d (Figure 1Ea-c, Figure 2C) and 21 d (data not shown), in which microglia had assumed a ramified shape, SphK1 immunofluorescence was undetectable. Quantitative analysis revealed that the number of SphK1-positive AMC was more numerous in corpus callosum of 1-5 d rats than that of 7-14 d rats (Figure 2B).

In 5-d-old rats injected with LPS, the incidence of SphK1-immunopositive AMC as well as SphK1 immunoreactivity in corpus callosum was increased compared to that of corresponding control rats with saline injection (Figure 3Aa-Dc, Figure 4A, B). The number of SphK1-positive AMC (co-expressing OX-42) was found to be significantly increased at 1 h compared with that at 30 min or 6 h after LPS injection and that of controls (Figure 3Ba-c, Ca-c, Da-c, Figure 4A). The intensity of SphK1 immunofluorescence was found to be increased significantly at 30 min to 1 h compared with that 6 h after LPS injection and that of controls (Figure 4B). Further, mRNA expression of SphK1 (Figure 4C) was detected in primary microglial cells in vitro and its expression was induced significantly in cells exposed to LPS for 1 h (Figure 4D).

In rats subjected to hypoxic exposure, SphK1 immunoexpression in AMC was drastically increased (Figure 5A-D, Figure 6A). Virtually all OX-42-positive AMC exhibited more intense SphK1 immunofluorescence 1 h after hypoxia, compared with that in control rats (Figure 5Ca-c, Figure 6A). At 6 h after hypoxia, SphK1 immunofluorescence in AMC appeared to be diminished, although OX-42 immunofluorescence in AMC remained intense and the cells were hypertrophic (Figure 5Da-c).

As mentioned previously, LPS upregulated SphK1 mRNA and protein expression in AMC of corpus callosum (Figure 7). However, injection of DMS prior to LPS evidently suppressed LPS-induced SphK1 expression in AMC as manifested by its reduced immunofluorescence staining and mRNA expression (Figure 7A-D, Figure 8A-C). A similar phenomenon was observed in microglial cell cultures treated with LPS and DMS + LPS when compared with corresponding controls (Figure 9A-D, Figure 10A, B). The suppression of SphK1 by DMS pretreatment was corroborated by western blot analysis (Figure 10C,D) and RT-PCR (Figure 8A). DMS alone had no significant effect on the expression of SphK1 (Figure 7B, 9C) and this was confirmed by RT-PCR (Figure 8A). LPS-induced TNF-α immunofluorescence intensity (Figure 11C, Figure 12A,B) was found to be reduced in AMC after DMS pretreatment and there was no marked change in LPS-induced IL-1β (Figure 13C, Figure 14A,B) immunofluorescence intensity in those cells. However, real time RT-PCR analysis showed that LPS-induced TNF-α and IL-1β mRNA expression was suppressed in primary culture microglia treated with DMS prior to LPS (Figure 12C and Figure 14C). On the other hand, the intensity of NFκB immunoexpression in AMC found in corpus callosum was significantly increased at 1 h after LPS injection, and NFκB immunofluorescence colocalized with OX-42 (Figure 15Aa-Cc, Figure 16B). Moreover, nuclear translocation of NFκB appeared to be more evident in those cells (Figure 15Bb, Figure 16A). Pretreatment of rats with DMS reduced the incidence of OX-42-labeled, NFκB-immunopositive AMC and NFκB nuclear translocation (Figure 15C). In vitro analysis using primary cultures of microglia also showed that LPS treatment induced NFκB translocation from cytoplasm to the nucleus (Figure 17Ba-Bc). This translocation of NFκB was reversed in activated cells pretreated with DMS (Figure 17Da-Dc).