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Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase.

ABSTRACT: In this study, microglial migration and phagocytosis were examined in mouse organotypic hippocampal slice cultures, which were treated with N-methyl-D-aspartate (NMDA) to selectively injure neuronal cells. Microglial cells were visualized by the expression of enhanced green fluorescent protein. Daily observation revealed microglial accumulation in the pyramidal cell layer, which peaked 5 to 6 days after NMDA treatment. Time-lapse imaging showed that microglia migrated to the pyramidal cell layer from adjacent and/or remote areas. There was no difference in the number of proliferating microglia between control and NMDA-treated slices in both the pyramidal cell layer and stratum radiatum, suggesting that microglial accumulation in the injured areas is mainly due to microglial migration, not to proliferation. Time-lapse imaging also showed that the injured neurons, which were visualized by propidium iodide (PI), disappeared just after being surrounded by microglia. Daily observation revealed that the intensity of PI fluorescence gradually attenuated, and this attenuation was suppressed by pretreatment with clodronate, a microglia toxin. These findings suggest that accumulating microglia phagocytosed injured neurons, and that PI fluorescence could be a useful indicator for microglial phagocytosis. Using this advantage to examine microglial phagocytosis in living slice cultures, we investigated the involvements of mitogen-activated protein (MAP) kinases in microglial accumulation and phagocytosis. p38 MAP kinase inhibitor SB203580, but not MAP kinase/extracellular signal-regulated kinase inhibitor PD98059 or c-Jun N-terminal kinase inhibitor SP600125, suppressed the attenuation of PI fluorescence. On the other hand, microglial accumulation in the injured areas was not inhibited by any of these inhibitors. These data suggest that p38 MAP kinase plays an important role in microglial phagocytosis of injured neurons.

SUBMITTER: Katayama T

PROVIDER: S-EPMC3398896 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase.

Katayama Takahiro T Kobayashi Hayato H Okamura Toshiyuki T Yamasaki-Katayama Yuko Y Kibayashi Tatsuya T Kimura Hiroshi H Ohsawa Keiko K Kohsaka Shinichi S Minami Masabumi M

PloS one 20120717 7

In this study, microglial migration and phagocytosis were examined in mouse organotypic hippocampal slice cultures, which were treated with N-methyl-D-aspartate (NMDA) to selectively injure neuronal cells. Microglial cells were visualized by the expression of enhanced green fluorescent protein. Daily observation revealed microglial accumulation in the pyramidal cell layer, which peaked 5 to 6 days after NMDA treatment. Time-lapse imaging showed that microglia migrated to the pyramidal cell layer ...[more]

PMID: 22815830

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Project description:BackgroundPyrethroids, such as bifenthrin (BF), are among the most widely used class of insecticides that pose serious risks to human and wildlife health. Pyrethroids are proposed to affect astrocytic functions and to cause neuron injury in the central nervous system (CNS). Microglia are key cells involved in innate immune responses in the CNS, and microglia activation has been linked to inflammation and neurotoxicity. However, little information is known about the effects of BF-induced toxicity in primary microglial cells as well as in organotypic hippocampal slice cultures (OHSCs).MethodsOxidative stress and inflammatory responses induced by BF were evaluated in primary microglial cells and OHSCs incubated with different concentrations of BF (1-20 μM) for 4 and 24 h. mRNA and protein synthesis of cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), nuclear erythroid-2 like factor-2 (Nrf-2), and microsomal prostaglandin synthase-1 (mPGES-1) was also studied by qPCR and Western blot. Cell viability was analyzed by MTT-tetrazolio (MTT) and lactate dehydrogenase (LDH) assays. Neurotoxicity in OHSCs was analyzed by propidium iodide (PI) staining and confocal microscopy.ResultsExposure of microglial cells to BF for 24 h resulted in a dose-dependent reduction in the number of viable cells. At sub-cytotoxic concentrations, BF increased reactive oxygen species (ROS), TNF-alpha synthesis, and prostaglandin E2 (PGE2) production, at both 4- and 24-h time points, respectively. Furthermore, BF incubation decreased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and increased lipid peroxidation, protein oxidation, and H2O2 formation. In addition, BF significantly induced protein synthesis and mRNA expression of oxidative and inflammatory mediators after 4 and 24 h, including Nrf-2, COX-2, mPGES-1, and nuclear factor kappaB (NF-kappaB). A 24-h exposure of OHSCs to BF also increased neuronal death compared to untreated controls. Furthermore, depletion of microglia from OHSCs potently enhanced neuronal death induced by BF.ConclusionsOverall, BF exhibited cytotoxic effects in primary microglial cells, accompanied by the induction of various inflammatory and oxidative stress markers including the Nrf-2/COX-2/mPGES-1/NF-kappaB pathways. Moreover, the study provided evidence that BF induced neuronal death in OHSCs and suggests that microglia exert a protective function against BF toxicity.

Project description:Neurogenesis in the adult hippocampus has become an intensively investigated research topic, as it is essential for proper hippocampal function and considered to bear therapeutic potential for the replacement of pathologically lost neurons. On the other hand, neurogenesis itself is frequently affected by CNS insults. To identify processes leading to the disturbance of neurogenesis, we made use of organotypic hippocampal slice cultures (OHSC), which, for unknown reasons, lose their neurogenic potential during cultivation. In the present study, we show by BrdU/Prox1 double-immunostaining that the generation of new granule cells drops by 90% during the first week of cultivation. Monitoring neurogenesis dynamically in OHSC from POMC-eGFP mice, in which immature granule cells are endogenously labeled, revealed a gradual decay of the eGFP signal, reaching 10% of initial values within 7 days of cultivation. Accordingly, reverse transcription quantitative polymerase chain reaction analysis showed the downregulation of the neurogenesis-related genes doublecortin and Hes5, a crucial target of the stem cell-maintaining Notch signaling pathway. In parallel, we demonstrate a strong and long-lasting activation of astrocytes and microglial cells, both, morphologically and on the level of gene expression. Enhancement of astroglial activation by treating OHSC with ciliary neurotrophic factor accelerated the loss of neurogenesis, whereas treatment with indomethacin or an antagonist of the purinergic P2Y12 receptor exhibited potent protective effects on the neurogenic outcome. Therefore, we conclude that OHSC rapidly lose their neurogenic capacity due to persistent inflammatory processes taking place after the slice preparation. As inflammation is also considered to affect neurogenesis in many CNS pathologies, OHSC appear as a useful tool to study this interplay and its molecular basis. Furthermore, we propose that modification of glial activation might bear the therapeutic potential of enabling neurogenesis under neuropathological conditions.

Project description:Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.

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Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase.

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Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase.

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