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Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Published on: February 16, 2018
Microglia-Dependent and Independent Brain Cytoprotective Effects of Mycophenolate Mofetil During Neuronal Damage
Joshua Kleine1, Urszula Hohmann1, Tim Hohmann1
1Department of Anatomy and Cell Biology, Medical Faculty, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany.
Abstract:
Acute lesions of the central nervous system often lead to permanent limiting deficits. In addition to the initial primary damage, accompanying neuroinflammation is responsible for progression of damage. Mycophenolate mofetil (MMF) as a selective inhibitor of inosine 5-monophosphate dehydrogenase (IMPDH) was shown to modulate the inflammatory response and promote neuronal survival when applied in specific time windows after neuronal injury. The application of brain cytoprotective therapeutics early after neuronal damage is a fundamental requirement for a successful immunomodulation approach. This study was designed to evaluate whether MMF can still mediate brain cytoprotection when applied in predefined short time intervals following CNS injury. Furthermore, the role of microglia and changes in IMPDH2 protein expression were assessed. Organotypic hippocampal slice cultures (OHSC) were used as an in vitro model and excitotoxically lesioned with N-methyl-aspartate (NMDA). Clodronate (Clo) was used to deplete microglia and analyze MMF mediated microglia independent effects. The temporal expression of IMPDH2 was studied in primary glial cell cultures treated with lipopolysaccharide (LPS). In excitotoxically lesioned OHSC a significant brain cytoprotective effect was observed between 8 and 36 h but not within 8 and 24 h after the NMDA damage. MMF mediated effects were mainly microglia dependent at 24, 36, 48 h after injury. However, further targets like astrocytes seem to be involved in protective effects 72 h post-injury. IMPDH2 expression was detected in primary microglia and astrocyte cell cultures. Our data indicate that MMF treatment in OHSC should still be started no later than 8-12 h after injury and should continue at least until 36 h post-injury. Microglia seem to be an essential mediator of the observed brain cytoprotective effects. However, a microglia-independent effect was also found, indicating involvement of astrocytes.
Insights
Mycophenolate mofetil (MMF) shows brain cytoprotective effects in central nervous system (CNS) injury models when initiated early. Its benefits are largely microglia-dependent but may involve astrocytes, highlighting therapeutic timing for neuroinflammation.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Acute central nervous system (CNS) injuries cause lasting deficits, with neuroinflammation exacerbating damage.
- Mycophenolate mofetil (MMF), an inosine 5-monophosphate dehydrogenase (IMPDH) inhibitor, modulates inflammation and promotes neuronal survival.
- Early intervention with cytoprotective therapies is crucial for effective immunomodulation after CNS injury.
Purpose of the Study:
- To determine if MMF provides brain cytoprotection when administered within specific short timeframes after CNS injury.
- To investigate the role of microglia in MMF-mediated effects and assess changes in IMPDH2 protein expression.
- To evaluate MMF's efficacy in an in vitro model of excitotoxic CNS injury.
Main Methods:
- Organotypic hippocampal slice cultures (OHSC) were subjected to excitotoxic injury using N-methyl-aspartate (NMMA).
- Clodronate (Clo) was used to deplete microglia, enabling analysis of MMF's microglia-independent effects.
- Temporal IMPDH2 expression was studied in primary glial cells stimulated with lipopolysaccharide (LPS).
Main Results:
- Significant brain cytoprotection was observed in NMDA-lesioned OHSC between 8 and 36 hours post-injury, but not between 8 and 24 hours.
- MMF's protective effects were primarily microglia-dependent at 24, 36, and 48 hours post-injury.
- IMPDH2 expression was detected in microglia and astrocytes; astrocytes may contribute to MMF's protective effects at 72 hours post-injury.
Conclusions:
- MMF treatment for CNS injury should commence within 8-12 hours and continue until at least 36 hours post-injury for optimal cytoprotection.
- Microglia are essential mediators of MMF's brain-protective effects in this model.
- A microglia-independent component of MMF's action suggests involvement of other cell types, such as astrocytes.
