A Novel Ex Vivo Model to Study Therapeutic Treatments for Myelin Repair following Ischemic Damage
Abstract
:1. Introduction
2. Results
2.1. Slice Culture Viability Following Photothrombotic Ischemia
2.2. NG2 Glia within the Border Zone of Ischemic Lesion
2.3. Promotion of Oligodendroglial Differentiation within the Border Zone upon Parbendazole Exposition
2.4. Characterization of Cells Invading the Scratch on Myelination Co-Culture
2.5. Rescue of Oligodendroglial Differentiation Competence by Means of Parbendazole Treatment Following Chemical Ischemia after 3 d In Vitro
3. Discussion
4. Materials and Methods
4.1. Ethics Statements for Animal Experiments
4.2. Mouse Line
4.3. Oligodendroglial Cell Culture
4.4. Myelinating Co-Cultures
4.5. Scratch Assay of Myelinating Co-Cultures and Stimulation
4.6. Tamoxifen Administration
4.7. Induction of Cerebral Photothrombosis (cPT)
4.8. Preparation of Organotypic Coronal Slice Culture (OCSCs)
4.9. Slice Culture Conditions and Stimulation
4.10. TTC Staining
4.11. Fixation
4.12. Embedding of Slices for Frozen Sections
4.13. Immunocytochemistry (ICC)
4.14. Immunohistochemistry
4.15. Data Analysis
4.16. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
2-DG | 2-deoxy-D-glucose |
BCAS1 | Breast carcinoma amplified sequence 1 |
BDNF | Brain derived growth factor |
BSA | Bovine serum albumin |
CC1 | Monoclonal antibody anti-adenomatous polyposis coli (APC) clone |
CNS | Central nervous system |
cPT | Cerebral photothrombosis |
DAPI | 4′,6-diamidin-2-phenylindol |
DMSO | Dimethylsulfoxid |
DOAJ | Directory of open access journals |
DIV | Days in vitro |
FBS | Fetal bovine serum |
GFAP | Glial fibrillary acidic protein |
HBSS | Hank’s balanced salt solution |
ICC | Immunocytochemistry |
IHC | Immunohistochemistry |
IRB | Institutional Review Board |
LD | Linear dichroism |
MBP | Myelin basic protein |
MDPI | Multidisciplinary Digital Publishing Institute |
NaN3 | sodium azide |
NDS | Normal donkey serum |
NG2 | Neural/glial antigen 2 |
NGF | Nerf growth factor |
NGS | Normal goat serum |
Olig2 | Oligodendrocyte transcription factor 2 |
OL | Oligodendrocyte |
OPC | Oligodendroglial precursor cell |
OCSC | Organotypic coronal slice culture |
PDGFR-α | Platelet-derived growth factor receptor alpha |
PDL | Poly-D-lysine |
PFA | Paraformaldehyde |
PBS | Phosphate buffered saline |
ROI | Region of interest |
RT | Room temperature |
SD | Standard deviation |
SEM | Standard error of the mean |
SFM | Serum-free medium |
SSM | Serum-supplemented medium |
SOX10 | SRY (sex determining region Y) -Box Transcription Factor 10 |
T3 | Tri-iodo-thyronine |
tdT+ | td-Tomato positive cells |
TLA | Three letter acronym |
TTC | 2,3,5-Triphenyltetrazolium chloride |
wt | Wild type |
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Medium | Composition | Supplier |
---|---|---|
Serum-free medium (SFM) | Neurobasal medium 2% B27 supplement 1% N2 supplement 1% L-Glutamine (×100) 0.5% Glucose 1% Antibiotic/antimycotic (×100) | Gibco Cat# 21103-049 Gibco Cat# 17504-044 Gibco Cat#17502-048 Gibco Sigma-Aldrich, Cat# SLBF1738V Sigma-Aldrich, Cat# A5955 |
Serum-supplemented medium (SSM) | Neurobasal medium 10% Fetal bovine serum (FBS) 0.5% Glucose 1% Antibiotic/antimycotic (×100) | Gibco Cat# 21103-049 Roth, Cat# 8076.2 Sigma-Aldrich, Cat# SLBF1738V Sigma-Aldrich, Cat# A5955 |
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Werner, L.; Gliem, M.; Rychlik, N.; Pavic, G.; Reiche, L.; Kirchhoff, F.; Silva Oliveira Junior, M.; Gruchot, J.; Meuth, S.G.; Küry, P.; et al. A Novel Ex Vivo Model to Study Therapeutic Treatments for Myelin Repair following Ischemic Damage. Int. J. Mol. Sci. 2023, 24, 10972. https://doi.org/10.3390/ijms241310972
Werner L, Gliem M, Rychlik N, Pavic G, Reiche L, Kirchhoff F, Silva Oliveira Junior M, Gruchot J, Meuth SG, Küry P, et al. A Novel Ex Vivo Model to Study Therapeutic Treatments for Myelin Repair following Ischemic Damage. International Journal of Molecular Sciences. 2023; 24(13):10972. https://doi.org/10.3390/ijms241310972
Chicago/Turabian StyleWerner, Luisa, Michael Gliem, Nicole Rychlik, Goran Pavic, Laura Reiche, Frank Kirchhoff, Markley Silva Oliveira Junior, Joel Gruchot, Sven G. Meuth, Patrick Küry, and et al. 2023. "A Novel Ex Vivo Model to Study Therapeutic Treatments for Myelin Repair following Ischemic Damage" International Journal of Molecular Sciences 24, no. 13: 10972. https://doi.org/10.3390/ijms241310972