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Analysis of neurogenesis during experimental autoimmune encephalomyelitis reveals pitfalls of bioluminescence imaging.

Ayzenberg I, Schlevogt S, Metzdorf J, Stahlke S, Pedreitturia X, Hunfeld A, Couillard-Despres S, Kleiter I - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, St. Josef-Hospital, Ruhr-University, Bochum, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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).
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pone.0118550.g002: 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).

Mentions: To validate that the steep rise in bioluminescence 10–14 days after immunization reflected increased expression of the DCX-reporter in the CNS and to determine which compound (CFA or PTX) is critical, we did a second EAE experiment, followed by measurement of luciferase activity in brain tissue homogenates at the peak of disease. Six days after antigen-specific immunization, bioluminescence signal intensity increased 2-fold and reached 4-fold compared to baseline between days 10 and 13 (group differences, day 3: p = 0.304, day 6: p = 0.007, day 10: p = 0.006, day 13: 0.006) (Fig. 2A). In contrast to mice with complete (CFA+PTX+MOG) and incomplete (CFA+PTX) immunization (see Fig. 1), no changes of bioluminescence signal intensity were observed in unimmunized mice and mice injected with CFA or PTX alone. Measurement of ex vivo luciferase activity in brain homogenates at day 14 post immunization revealed no significant differences between the experimental groups (photons/s/cm2/sr, mean ± SD: PBS 9.8x106 ± 1.1x106, CFA 8.7x106 ± 0.7x106, PTX 8.6x106 ± 0.9x106, CFA+PTX+MOG 10.2x106 ± 0.7x106, p = 0.074) (Fig. 2B). In line with these results, immunostaining for DCX-positive cells in all major neurogenic niches of the murine CNS failed to show differences between mice with antigen-specific immunization and controls (cells/hemisphere, mean ± SD: DG: PBS 10438 ± 560, CFA+PTX+MOG 10128 ± 1219, p = 0.675; pixel2/hemisphere, mean ± SD: SVZ: PBS 404175 ± 54789, CFA+PTX+MOG 444851 ± 89574, p = 0.771; RMS: PBS 919802 ± 342667, CFA+PTX+MOG 921790 ± 257355, p = 0.875) (Fig. 2C, D).


Analysis of neurogenesis during experimental autoimmune encephalomyelitis reveals pitfalls of bioluminescence imaging.

Ayzenberg I, Schlevogt S, Metzdorf J, Stahlke S, Pedreitturia X, Hunfeld A, Couillard-Despres S, Kleiter I - PLoS ONE (2015)

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).
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pone.0118550.g002: 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).
Mentions: To validate that the steep rise in bioluminescence 10–14 days after immunization reflected increased expression of the DCX-reporter in the CNS and to determine which compound (CFA or PTX) is critical, we did a second EAE experiment, followed by measurement of luciferase activity in brain tissue homogenates at the peak of disease. Six days after antigen-specific immunization, bioluminescence signal intensity increased 2-fold and reached 4-fold compared to baseline between days 10 and 13 (group differences, day 3: p = 0.304, day 6: p = 0.007, day 10: p = 0.006, day 13: 0.006) (Fig. 2A). In contrast to mice with complete (CFA+PTX+MOG) and incomplete (CFA+PTX) immunization (see Fig. 1), no changes of bioluminescence signal intensity were observed in unimmunized mice and mice injected with CFA or PTX alone. Measurement of ex vivo luciferase activity in brain homogenates at day 14 post immunization revealed no significant differences between the experimental groups (photons/s/cm2/sr, mean ± SD: PBS 9.8x106 ± 1.1x106, CFA 8.7x106 ± 0.7x106, PTX 8.6x106 ± 0.9x106, CFA+PTX+MOG 10.2x106 ± 0.7x106, p = 0.074) (Fig. 2B). In line with these results, immunostaining for DCX-positive cells in all major neurogenic niches of the murine CNS failed to show differences between mice with antigen-specific immunization and controls (cells/hemisphere, mean ± SD: DG: PBS 10438 ± 560, CFA+PTX+MOG 10128 ± 1219, p = 0.675; pixel2/hemisphere, mean ± SD: SVZ: PBS 404175 ± 54789, CFA+PTX+MOG 444851 ± 89574, p = 0.771; RMS: PBS 919802 ± 342667, CFA+PTX+MOG 921790 ± 257355, p = 0.875) (Fig. 2C, D).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, St. Josef-Hospital, Ruhr-University, Bochum, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus