Abstract
BackgroundImmunosuppressants such as mycophenolate mofetil (MMF) have the capacity to inhibit microglial and astrocytic activation and to reduce the extent of cell death after neuronal injury. This study was designed to determine the effective neuroprotective time frame in which MMF elicits its beneficial effects, by analyzing glial cell proliferation, migration, and apoptosis.MethodsUsing organotypic hippocampal slice cultures (OHSCs), temporal dynamics of proliferation and apoptosis after N-methyl-D-aspartate (NMDA)-mediated excitotoxicity were analyzed by quantitative morphometry of Ki-67 or cleaved caspase-3 immunoreactive glial cells. Treatment on NMDA-lesioned OHSCs with mycophenolate mofetil (MMF)100 μg/mL was started at different time points after injury or performed within specific time frames, and the numbers of propidium iodide (PI)+ degenerating neurons and isolectin (I)B4+ microglial cells were determined. Pre-treatment with guanosine 100 μmol/l was performed to counteract MMF-induced effects. The effects of MMF on reactive astrocytic scar formation were investigated in the scratch-wound model of astrocyte monolayers.ResultsExcitotoxic lesion induction led to significant increases in glial proliferation rates between 12 and 36 hours after injury and to increased levels of apoptotic cells between 24 and 72 hours after injury. MMF treatment significantly reduced glial proliferation rates without affecting apoptosis. Continuous MMF treatment potently reduced the extent of neuronal cell demise when started within the first 12 hours after injury. A crucial time-frame of significant neuroprotection was identified between 12 and 36 hours after injury. Pre-treatment with the neuroprotective nucleoside guanosine reversed MMF-induced antiproliferative effects on glial cells. In the scratch-wound model, gap closure was reached within 48 hours in controls, and was potently inhibited by MMF.ConclusionsOur data indicate that immunosuppression by MMF significantly attenuates the extent of neuronal cell death when administered within a crucial time frame after injury. Moreover, long-lasting immunosuppression, as required after solid-organ transplantation, does not seem to be necessary. Targeting inosine 5-monophosphate dehydrogenase, the rate-limiting enzyme of purine synthesis, is an effective strategy to modulate the temporal dynamics of proliferation and migration of microglia and astrocytes, and thus to reduce the extent of secondary neuronal damage and scar formation.
Highlights
Immunosuppressants such as mycophenolate mofetil (MMF) have the capacity to inhibit microglial and astrocytic activation and to reduce the extent of cell death after neuronal injury
Temporal dynamics of proliferation and apoptosis after acute excitotoxic injury To investigate temporal patterns of glial cell proliferation and apoptosis, indices of Ki-67 and cleaved caspase3 immunoreactive astrocytes and microglial cells were assessed at different proximate time points after the onset of acute excitotoxic lesion
Apoptosis indices of control Organotypic hippocampal slice culture (OHSC) were markedly low: only 2.2% microglial cells and 0.5% astrocytes were immunoreactive for cleaved caspase-3 (Figure 1F,G)
Summary
Immunosuppressants such as mycophenolate mofetil (MMF) have the capacity to inhibit microglial and astrocytic activation and to reduce the extent of cell death after neuronal injury. Within the scope of this phenomenon, highly complex and dynamic neuron–glia interactions lead to the activation, proliferation, and recruitment of astrocytes, microglial cells, and blood-borne immune cells. Inflammatory cascades carried by direct cell–cell interactions via contact-dependent communication (such as Ephrin receptors or repulsive guidance molecules) determine the mode and dimension of glial activation, glutamate sensitivity or axonal regrowth [5,6,7,8]. Neuroinflammatory responses in the aftermath of acute neuropathologies may have detrimental effects, suggesting that an inhibition of glial activation by means of immunosuppression may be beneficial for neuronal survival and recovery [15,16,17]
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