ABSTRACT
Treatment of excitotoxically injured organotypic hippocampal slice cultures (OHSC) with clodronate is known to result in the inhibition of microglial activation. We hypothesized that this is due to direct effects of clodronate on microglial cells, and investigated microglial proliferation in OHSC, and cytokine and NO secretion in isolated microglial cells. N-methyl-D-aspartate (NMDA) lesioning of OHSC resulted in a massive increase in the number of proliferating, bromo-desoxy-uridine (BrdU)-labeled cells that was reduced to control levels after treatment with clodronate (0.1, 1, 10 microg/ml). Triple-labeling revealed that clodronate abrogated the proliferation of both glial fibrillary acidic protein (GFAP)-labeled astrocytes and Griffonia simplicifolia isolectin B4 (IB4)-labeled microglial cells. Furthermore, isolated microglial cells were treated with clodronate after stimulation with lipopolysaccharide (LPS) or macrophage colony stimulating factor (M-CSF). Clodronate (0.01, 0.1, 1 microg/ml) significantly down-regulated the LPS-stimulated microglial secretion of tumor necrosis factor (TNF)-alpha, Interleukin (IL)-1beta and NO, but not of IL-6. In contrast, clodronate significantly reduced the microglial IL-6-release induced by M-CSF, indicating different intracellular pathways. The number and morphology of isolated microglial cells did not change significantly after treatment with clodronate. In summary, the number of proliferating microglial cells and astrocytes after excitotoxic injury is reduced to control levels after treatment with clodronate. Furthermore, clodronate inhibits microglial secretion of proinflammatory cytokines and NO. Clodronate could therefore prove to be a useful tool in the investigation of interactions between damaged neurons and microglial cells.
Subject(s)
Clodronic Acid/pharmacology , Cytokines/antagonists & inhibitors , Gliosis/drug therapy , Hippocampus/drug effects , Microglia/drug effects , Nitric Oxide/antagonists & inhibitors , Animals , Animals, Newborn , Antimetabolites/pharmacology , Astrocytes/drug effects , Astrocytes/metabolism , Cell Count , Cell Division/drug effects , Cell Division/physiology , Cytokines/metabolism , Down-Regulation/drug effects , Down-Regulation/physiology , Gliosis/chemically induced , Gliosis/pathology , Hippocampus/metabolism , Hippocampus/pathology , In Vitro Techniques , Inflammation Mediators/pharmacology , Interleukin-1/antagonists & inhibitors , Interleukin-1/metabolism , Microglia/metabolism , N-Methylaspartate , Neurotoxins , Nitric Oxide/metabolism , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolismABSTRACT
In this study we investigated whether treatment with the immunosuppressant mycophenolate mofetil (MMF) has beneficial effects on neuronal damage after excitotoxic injury. Organotypic hippocampal slice culture (OHSC), lesioned by the application of N-methyl-d-aspartate (NMDA) after 6 days in vitro, showed an improved preservation of the hippocampal cytoarchitecture after continuous treatment with MMF for 3 further days (10 or 100 micro g/mL). Treatment with NMDA and MMF (100 microg/mL) reduced the number of damaged propidium iodide (PI)+ neurons by 50.7% and the number of microglial cells by 52%. Continuous treatment of lesioned OHSCs with MMF for 3 days almost abrogated the glial proliferative response, reflected by the 91.5% reduction in the number of bromo-desoxy-uridine (BrdU)-labelled microglial cells and astrocytes. Microglial cells in MMF-treated OHSCs contained fragmented nuclei, indicating apoptotic cell death, an effect which was also found in isolated microglial cells treated with MMF. The beneficial effect of MMF on neuronal survival apparently does not reflect a direct antiexcitotoxic effect, as short-term treatment of OHSCs with NMDA and MMF for 4 h did not reduce the number of PI+ neurons. In conclusion, MMF inhibits proliferation and activation of microglia and astrocytes and protects neurons after excitotoxic injury.
