Fig 1.
In vivo bioluminescence imaging of neurogenesis in mice with EAE and after incomplete immunization.
(A) Experimental autoimmune encephalomyelitis (EAE) was induced in doublecortin (DCX)-luc reporter mice by immunization with 200 μg MOG35–55 peptide emulsified in CFA+PTX (n = 7). Control mice received incomplete immunization (CFA PTX; n = 5) or PBS (n = 6). The clinical course of EAE was followed for 28 days. (B and C) Bioluminescence of the brain was recorded from minute 5 to 20 after i.p. injection of 150 mg/kg D-Luciferin. The maximum photon flux integrated over 59 seconds is shown. (B) illustrates representative examples, (C) temporal changes of the average bioluminescence signal at 4 time points. Mean + SEM is shown. *p<0.05, **p<0.01 (ANOVA). (D and E) Neurogenesis in the dentate gyrus (DG) 28 days after immunization. DCX positive cells in the DG were counted and presented as the total number of positive cells per hemisphere (mean ± SEM; statistical analysis by ANOVA).
Fig 2.
DCX-expression and ex vivo bioluminescence remain constant despite increased in vivo bioluminescence after EAE induction.
(A) In vivo bioluminescence imaging of the brain recorded from minute 5 to 20 after i.p. injection of 150 mg/kg D-Luciferin in DCX-luc mice with antigen-specific (200 μg MOG35–55+CFA+PTX, n = 6) or incomplete (CFA only, n = 4; PTX only, n = 4) immunization and in the PBS control group (n = 5). The maximum photon flux integrated over 59 seconds is shown. Only mice with antigen-specific immunization developed clinical signs of EAE (not shown). Mice were sacrificed after 14 days and brains were used for ex vivo detection of luciferase activity. (B) Brain hemispheres were homogenized and luciferase activity measured by addition of excess D-Luciferin and ATP. (C and D) DCX-luc mice were immunized as detailed above (PBS, n = 6; CFA+PTX+MOG, n = 6) and perfused after 14 days at the peak of EAE. Sagittal brain sections were immunostained for DCX (shown in green) and analyzed in the dentate gyrus (DG), subventricular zone (SVZ), and rostral migratory stream (RMS). (C) A representative example is shown (PBS-treated). (D) DCX positive cells in the DG were counted and presented as the total number of positive cells per hemisphere. DCX positive cells in the SVZ and the RMS are shown as the total area (pixel2) of positive cells per hemisphere. Results are presented as mean ± SEM per group. **p<0.01 (ANOVA in A, B; Mann Whitney U test in D).
Fig 3.
Disruption of the blood-brain-barrier after antigen-specific and incomplete immunization.
(A) At day 10 after antigen-specific and incomplete immunization (CFA+PTX+MOG, n = 3; CFA+PTX, n = 4; PBS n = 4) mice were intravenously injected with 4% Evans blue dye (EBD) and sacrificed after 20 minutes of EBD circulation. Fluorescence microscopy revealed an increased blood-brain barrier (BBB) permeability in both immunized groups, most prominent between the DG and the thalamus, in the choroid plexus and around small vessels (insets). (B) Measurement of extracted EBD from brain parenchyma, expressed as μg per g tissue confirms leakage of the BBB after immunization. Results are presented as a mean ± SEM per group. *p<0.05 (ANOVA).