The inflammatory cytokines interleukin-1β and tumor necrosis factor-α (TNF-α) have been identified as mediators of several forms of neurodegeneration in the brain. However, they can produce either deleterious or beneficial effects on neuronal function. We investigated the effects of these cytokines on neuronal death caused by exposure of mouse organotypic hippocampal slice cultures to toxic concentrations of AMPA. Either potentiation of excitotoxicity or neuroprotection was observed, depending on the concentration of the cytokines and the timing of exposure. A relatively high concentration of mouse recombinant TNF-α (10 ng/ml) enhanced excitotoxicity when the cultures were simultaneously exposed to AMPA and to this cytokine. Decreasing the concentration of TNF-α to 1 ng/ml resulted in neuroprotection against AMPA-induced neuronal death independently on the application protocol. By using TNF-α receptor (TNFR) knock-out mice, we demonstrated that the potentiation of AMPA-induced toxicity by TNF-α involves TNF receptor-1, whereas the neuroprotective effect is mediated by TNF receptor-2. AMPA exposure was associated with activation and proliferation of microglia as assessed by macrophage antigen-1 and bromodeoxyuridine immunohistochemistry, suggesting a functional recruitment of cytokine-producing cells at sites of neurodegeneration. Together, these findings are relevant for understanding the role of proinflammatory cytokines and microglia activation in acute and chronic excitotoxic conditions.
Glutamate and NPY have been implicated in hippocampal neuropathology in temporal lobe epilepsy. Thus, we investigated the involvement of NPY receptors in mediating neuroprotection against excitotoxic insults in organotypic cultures of rat hippocampal slices. Exposure of hippocampal slice cultures to 2 microM AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate) induced neuronal degeneration, monitored by propidium iodide uptake, of granule cells and CA1 pyramidal cells. For dentate granule cells, selective activation of Y1, Y2, or Y5 receptors with 1 microM [Leu31,Pro34]NPY, 300 nM NPY13-36 or 1 microM 500 nM NPY(19-23)-(Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34)-PP, respectively, had a neuroprotective effect against AMPA, whereas only the activation of Y2 receptors was effective for CA1 pyramidal cells. When the slice cultures were exposed to 6 microM kainate, the CA3 pyramidal cells displayed significant degeneration, and in this case the activation of Y1, Y2, and Y5 receptors was neuroprotective. For the kainic acid-induced degeneration of CA1 pyramidal cells, it was again found that only the Y2 receptor activation was effective. Based on the present findings, it was concluded that Y1, Y2, and Y5 receptors effectively can modify glutamate receptor-mediated neurodegeneration in the hippocampus.
Receptor agonist/antagonist mediated modulation of the excitotoxic effect of AMPA was studied in organotypic hippocampal slice cultures. Treatment of developing cultures for 2 weeks with a subtoxic dose of 2 μM kainate reduced the toxicity of 3 μM AMPA, applied for 48 h with 24 h of recovery, as measured by cellular uptake of the fluorescent dye propidium iodide. In contrast long-term treatment with 0.3μM of the AMPA/KA antagonist NBQX increased the susceptibility of the cultures to an even lower dose of 2 μM AMPA. The modulatory effects of long-term application of low doses of kainate and NBQX, have implications for the development and use of related drugs that aim to protect against glutamate receptor-mediated disturbances.
Abstract The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA). Exposure of 2‐week‐old slice cultures, derived from 7‐day‐old C57BL/6 mice, to 8 µ m AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y 2 receptor agonist [NPY(13‐36), 300 n m ]. This effect was sensitive to the presence of the selective Y 2 receptor antagonist (BIIE0246, 1 µ m ), but was not affected by addition of TrkB‐Fc or by a neutralizing antibody against brain‐derived neurotrophic factor (BDNF). Moreover, addition of a Y 1 receptor antagonist (BIBP3226, 1 µ m ) or a NPY‐neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 µ m AMPA for 24 h, displayed, as measured by an enzyme‐linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up‐regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF‐immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA‐receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y 1 and Y 2 ), which can be affected by BDNF released by microglia and neurons.
Abstract We characterized organotypic ventral mesencephalic (VM) cultures derived from embryonic day 12 (E12) mice (CBL57/bL6) in terms of number of dopaminergic neurons, cell soma size and dopamine production in relation to time in vitro and tested the effects of 1‐methyl‐4‐phenylpyridinium (MPP + ) and glial derived neurotrophic factor (GDNF) to validate this novel culture model. Dopamine production and dopaminergic neuron soma size increased dramatically with time in vitro , whereas the number of dopamine neurons declined by approximately 30% between week 1 and week 2, which was further reduced after week 4. GDNF treatment (100 ng/mL) increased dopaminergic neuron soma size (up to 43%) and DOPAC production (approximately three‐fold), but not the number of dopamine neurons in control cultures. One‐week‐old cultures were more vulnerable to MPP + , than three‐week‐old cultures. The EC 50 for dopamine depletion after 2 days exposure and 15 days of recovery were 0.6 and 7 µm, respectively. Both pre‐treatment and post‐treatment with GDNF are important to obtain maximal protection against MPP + toxicity. In one‐week‐old cultures (5 µm MPP + , 2 days) GDNF provided potent neuroprotection with dopamine contents reaching control levels and number of tyrosine hydroxylase (TH) + cells up to 80% of control, but in three‐week‐old cultures (10 µm MPP + , 2 days) the protective potential of GDNF was markedly reduced. Long recovery periods after MPP + exposure are required to distinguish between reversible or irreversible toxic and/or trophic effects.
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