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Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke
Published on: February 4, 2014
Mitogen-activated protein kinases support survival of activated microglia that mediate thrombin-induced striatal
Masatoshi Ohnishi1, Hiroshi Katsuki, Yasuhiko Izumi
1Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Abstract:
Intracerebral hemorrhage-associated tissue damage is triggered by blood-derived serine proteases such as thrombin. In addition, our previous studies have suggested that mitogen-activated protein (MAP) kinases contribute to intracerebral hemorrhage- and thrombin-induced striatal tissue damage in vivo. Here we addressed the mechanisms of MAP kinase involvement in thrombin cytotoxicity in rat corticostriatal slice culture, focusing on striatal tissue damage. Thrombin induced apoptotic nuclear condensation and fragmentation in striatal cells, which was suppressed by DEVD-CHO, a caspase-3 inhibitor. DEVD-CHO also prevented shrinkage of the striatal tissue induced by thrombin. Phagocytotic activity may be involved in tissue deterioration, because a phagocytosis inhibitor (cytochalasin D) and an inhibitor of phagocytosis of apoptotic cells (O-phospho-L-serine) suppressed shrinkage of the striatal tissue. OX42 immunostaining revealed that apoptosis-like microglial cell death was induced only when thrombin treatment was combined with application of inhibitors of MAP kinase/extracellular signal-regulated kinase kinase (PD98059), p38 MAP kinase (SB203580), or c-Jun N-terminal kinase (SP600125). Thrombin-induced increase in the number of microglia was also prevented by these inhibitors of MAP kinase pathways. We also found that thrombin-induced production of tumor necrosis factor (TNF)-alpha was inhibited by PD98059, SB203580, and SP600125. Finally, thrombin-induced neuronal apoptosis and shrinkage of the striatal tissue were significantly inhibited by anti-TNF-alpha neutralizing antibody. These results suggest that MAP kinases contribute to thrombin-induced striatal damage by supporting survival of activated microglia, which induce neuron death by producing TNF-alpha and cause tissue shrinkage by phagocytosing apoptotic cells.
Insights
Mitogen-activated protein (MAP) kinases promote brain tissue damage after intracerebral hemorrhage by supporting microglia survival. Activated microglia produce tumor necrosis factor-alpha, leading to neuron death and tissue shrinkage.
Area of Science:
- Neuroscience
- Cell Biology
- Pathology
Background:
- Intracerebral hemorrhage causes tissue damage via thrombin.
- Mitogen-activated protein (MAP) kinases are implicated in thrombin-induced brain injury.
Purpose of the Study:
- To investigate the role of MAP kinases in thrombin-induced damage in rat corticostriatal slice cultures.
- To elucidate the mechanisms of MAP kinase involvement in thrombin cytotoxicity.
Main Methods:
- Used rat corticostriatal slice cultures.
- Applied thrombin and inhibitors of caspases, phagocytosis, and MAP kinase pathways (PD98059, SB203580, SP600125).
- Assessed apoptosis, microglial activity, and tumor necrosis factor-alpha (TNF-α) production.
Main Results:
- Thrombin induced striatal cell apoptosis and tissue shrinkage, inhibited by caspase-3 inhibitor.
- MAP kinase inhibitors induced microglial apoptosis and prevented thrombin-induced microglial proliferation.
- MAP kinase inhibitors reduced thrombin-induced TNF-α production, which was neutralized by an anti-TNF-α antibody.
Conclusions:
- MAP kinases support activated microglia survival, contributing to thrombin-induced striatal damage.
- Microglia induce neuronal apoptosis and tissue shrinkage via TNF-α production and phagocytosis.
- Targeting MAP kinases or TNF-α may offer therapeutic strategies for intracerebral hemorrhage.
