Sphingosine Kinase 1 Deficiency Exacerbates LPS-Induced Neuroinflammation

<div><p>The pathogenesis of inflammation in the central nervous system (CNS), which contributes to numerous neurodegenerative diseases and results in encephalopathy and neuroinflammation, is poorly understood. Sphingolipid metabolism plays a crucial role in maintaining cellular processes in the CNS, and thus mediates the various pathological consequences of inflammation. For a better understanding of the role of sphingosine kinase activation during neuroinflammation, we developed a bacterial lipopolysaccharide (LPS)-induced brain injury model. The onset of the inflammatory response was observed beginning 4 hours after intracerebral injection of LPS into the lateral ventricles of the brain. A comparison of established neuroinflammatory parameters such as white matter rarefactions, development of cytotoxic edema, astrogliosis, loss of oligodendrocytes, and major cytokines levels in wild type and knockout mice suggested that the neuroinflammatory response in SphK1−/− mice was significantly upregulated. At 6 hours after intracerebroventricular injection of LPS in SphK1−/− mice, the immunoreactivity of the microglia markers and astrocyte marker glial fibrillary acidic protein (GFAP) were significantly increased, while the oligodendrocyte marker O4 was decreased compared to WT mice. Furthermore, western blotting data showed increased levels of GFAP. These results suggest that SphK1 activation is involved in the regulation of LPS induced brain injury.</p> <h3>Research Highlights</h3><p>• Lipopolysaccharide (LPS) intracerebral injection induces severe neuroinflammation. • Sphingosine kinase 1 deletion worsens the effect of the LPS. • Overexpression of SphK1 might be a potential new treatment approach to neuroinflammation.</p> </div

Analysis of cortex area for oligodendrocyte expression.

<p>(<b>A</b>) Piriform cortex area of the analysis is depicted. Interaural: 4.39; bregma: 0.74. (<b>B</b>) Loss of oligodendrocytes (arrows) after intracerebroventricular injection of LPS 1 mg/kg in wild type and SphK1−/− mice. Nuclei were stained with propidium iodide. 40× magnifications are shown. (<b>C</b>) Number of oligodendrocytes was calculated per taken area of piriform cortex and analyzed statistically. The significant loss of the oligodendrocytes was determined in wild type group after LPS 1 mg/kg was injected intracerebrally (*p<0.0001) and between wild type saline and SphK1−/saline groups (**p<0.001).</p

(A) Immunohistochemistry of the frozen sections of the brains.

<p>GFAP specific antibody were used as a marker of reactive astrocytes (arrows), propidium iodide was used as a marker of all cells in the studied lateral septal complex area of the animals’ brain. SphK1−/− mice after LPS induction expressed the most of reactive astrocytes. Magnifications of 40× are shown. (<b>B</b>) Reactive astrocytes were quantified and statistically analyzed using two-way ANOVA. Significant results were reached in SphK1−/− groups after treatment with LPS 1 mg/kg (*p = 0.0150). SphK1−/− LPS 1 mg/kg injected animals expressed the highest number of the reactive astrocytes.</p

(A) Western blot analysis of the GFAP protein expressed in the total brain extract.

<p>(<b>B</b>) Statistical analysis of the integrated densities of the bands of the western blot. Significance was reached in wild type group after LPS induction (*p<0.0001) and in saline injected group between wild type and SphK1−/− animals (**p<0.001).</p

Hematoxylin and eosin staining of the frozen sections of the SphK1−/− type mice.

<p>(<b>A</b>) Wild type mice underwent the intracerebral injection of either saline or LPS 1 mg/kg for 6 hours. Frozen sections of the brains were prepared and stained with hematoxylin and eosin. LPS injected mice exhibit enlarged ventricles (stars) and white matter rarefactions (arrows), two of the neuroinflammation marker. Magnification of 4×, 10× and 20× are shown. (<b>B</b>) Hematoxylin and eosin staining of the frozen sections of the SphK1−/− type mice. Enlarged ventricles (stars) and white matter rarefactions (arrows) were observed in control SphK1−/− animals, these neuroinflammatory parameters were increased after LPS injection. Magnification of 4×, 10× and 20× are shown. (<b>C</b>) Two-way ANOVA statistical analysis of the increase of the sizes of the lateral ventricles. Injection of LPS 1 mg/kg into lateral ventricle of the brain significantly (p<0.0001) affected development of the edema in SphK1−/− group. When Bonferroni’s multiple comparison was performed, statistical significance was reached between similarly treated, but genetically different groups, such as wild type LPS 1 mg/kg vs. SphK1−/− LPS 1 mg/kg (*p<0.0001); wild type saline vs. SphK1−/− saline (*p<0.0001). (<b>D</b>) Statistical analysis of the loss of the white matter in the brains of the animals intracerebroventicularly injected with saline or LPS 1 mg/kg. Statistical significance was reached after the injection of LPS 1 mg/kg in wild type and SphK1−/− mice (*p<0.0001). SphK1 deletion had a significant effect (**p<0.0001) in the development of leukoaraiosis.</p

Immunohistochemistry using anti- CD 68 and ferritin light chain antibodies.

<p>(<b>A</b>) Two-way ANOVA analysis revealed significant difference (*p = 0.0451) in anti-CD68 (FITC) staining after LPS 1 mg/kg intracerebroventricular injection in wild type and SphK1−/− mice. (<b>B</b>) Bonferroni’s multiple comparison of the anti-ferritin light chain staining (TRITC) determined significance between wild type LPS 1 mg/kg vs. SphK1−/− LPS 1 mg/kg (**p = 0.0181) and in mutant group after LPS injury (*p = 0.0123).</p

Immunohistochemistry of brain sections after intracerebroventricular LPS injection.

<p>(<b>A</b>). Lateral septal complex area of the brain was chosen for the analysis based on <i>in situ</i> hybridization database. Interaural: 4.39 mm: bregma: 0.74 mm. (<b>B</b>). Immunohistochemistry of the frozen sections of the brains of wild type mice that received intracerebroventricular injection of saline or LPS 1 mg/kg. Reactive microglia was visualized by double staining with anti-CD68 (FITC – thick arrows) and anti-ferritin light chain (TRITC – arrow heads) antibody. DAPI stain was used to detect nuclei of all cells. Magnifications of 40× are shown. (<b>C</b>). Anti-CD68 (FITC), anti-ferritin light chain (TRITC) positive staining and DAPI staining of the frozen brain sections of SphK1−/− experimental animals. Magnifications of 40× are shown.</p