RESUMEN
BACKGROUND: Cranial irradiation is a common therapy for the treatment of brain tumors, but unfortunately patients suffer from side effects, particularly cognitive impairment, caused by neurodegenerative and neuroinflammatory mechanisms. Finding a therapeutic agent protecting hippocampal neurons would be beneficial. Fingolimod (FTY720), a sphingosine-1-phosphate receptor modulator approved for multiple sclerosis, is an immunosuppressant and known to enhance proliferation and differentiation of neuronal precursor cells (NPCs). OBJECTIVES: To investigate whether pre-treatment with FTY720 protects NPCs in vitro and in vivo from irradiation-induced damage. METHODS: Neuronal precursor cells were isolated from E13 C57BL/6 wildtype mice, treated at day 0 of differentiation with FTY720 and irradiated on day 6 with 1 Gy. NPCs were analyzed for markers of cell death (PI, caspase-3), proliferation (Ki67), and differentiation (DCX, ßIII-tubulin). Adult C57BL/6 wildtype mice were treated with FTY720 (1 mg/kg) and received a single dose of 6 Gy cranial irradiation at day 7. Using immunohistochemistry, we analyzed DCX and BrdU as markers of neurogenesis and Iba1, GFAP, and CD3 to visualize inflammation in the dentate gyrus (DG) and the subventricular zone (SVZ). B6(Cg)-Tyrc-2J/J DCX-luc reporter mice were used for bioluminescence imaging to evaluate the effect of FTY720 on neurogenesis in the DG and the spinal cord of naïve mice. RESULTS: FTY720 protected NPCs against irradiation induced cell death in vitro. Treatment with FTY720 dose-dependently reduced the number of PI+ cells 24 and 96 h after irradiation without effecting proliferation or neuronal differentiation. In vivo treatment resulted in a significant survival of DCX+ neurons in the DG and the SVZ 4 weeks after irradiation as well as a slight increase of proliferating cells. FTY720 inhibited microglia activation 24 h after X-ray exposure in the DG, while astrocyte activation was unaffected and no lymphocyte infiltrations were found. In naïve mice, FTY720 treatment for 4 weeks had no effect on neurogenesis. CONCLUSION: FTY720 treatment of NPCs prior to X-ray exposure and of mice prior to cranial irradiation is neuroprotective. No effects on neurogenesis were found.
RESUMEN
Bioluminescence imaging is a sensitive approach for longitudinal neuroimaging. Transgenic mice expressing luciferase under the promoter of doublecortin (DCX-luc), a specific marker of neuronal progenitor cells (NPC), allow monitoring of neurogenesis in living mice. Since the extent and time course of neurogenesis during autoimmune brain inflammation are controversial, we investigated neurogenesis in MOG-peptide induced experimental allergic encephalomyelitis (EAE) using DCX-luc reporter mice. We observed a marked, 2- to 4-fold increase of the bioluminescence signal intensity 10 days after EAE induction and a gradual decline 1-2 weeks thereafter. In contrast, immunostaining for DCX revealed no differences between EAE and control mice 2 and 4 weeks after immunization in zones of adult murine neurogenesis such as the dentate gyrus. Ex vivo bioluminescence imaging showed similar luciferase expression in brain homogenates of EAE and control animals. Apart from complete immunization including MOG-peptide also incomplete immunization with complete Freund´s adjuvant and pertussis toxin resulted in a rapid increase of the in vivo bioluminescence signal. Blood-brain barrier (BBB) leakage was demonstrated 10 days after both complete and incomplete immunization and might explain the increased bioluminescence signal in vivo. We conclude, that acute autoimmune inflammation in EAE does not alter neurogenesis, at least at the stage of DCX-expressing NPC. Effects of immunization on the BBB integrity must be considered when luciferase is used as a reporter within the CNS during the active stage of EAE. Models with stable CNS-restricted luciferase expression could serve as technically convenient way to evaluate BBB integrity in a longitudinal manner.