After ischemic stroke, the loss of blood supply to the affected region of the brain leads to a series of pathological events known as ischemic cascades, that include excitotoxicity and microglia/macrophage over-activation resulting in damaging inflammatory responses. Studies from our research group suggest that the viable neurons in the ischemic penumbra (location receiving less profound damage, as compare to ischemic core) produce and release a potent anti-inflammatory cytokine, interleukin(IL) -4. We propose that this neuronal response to sublethal ischemia is designed to guide surrounding microglia/macrophages to a reparative and anti-inflammatory (M2) phenotype.
The research included in this study is designed to establish the mechanisms that underlie neuronal production and secretion of IL-4 under ischemic injury. To investigate this process we used primary rat cortical neurons in culture and a well-validated in vitro ischemic injury model that is based on transient oxygen- and glucose- deprivation, (OGD). In this model, only longer durations of OGD result in neuronal death. We discover that short-duration, sublethal OGD, as determined by LDH release assay, more LDH release correspond to greater damage) induces neuronal IL-4 production at the gene (RT-qPCR) and protein (ELISA) level. Furthermore, we show that mild excitotoxic stress produced by N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation (process that normally occurs in the cerebral ischemia) triggers IL-4 production and release by neurons in the primary neuron culture. We also implicated calcineurin and nuclear factor of activated T cell (NFAT) as potential players in the transcriptional regulation of the IL-4 synthesis in neurons. Finally, using neuron conditioned medium transfer to microglia, we find that neuronal IL-4 is capable of polarizing microglia toward a restorative, anti-inflammatory, and phagocytic phenotype.
For the first time, this study demonstrates that the ischemia-evoked NMDAR activation, through calcineurin/NFAT pathway induces IL-4 production by neurons, and that this neuron-secreted IL-4 is capable of regulating microglia phenotype change. This cross-talk between neurons and microglia could represent a therapeutic target for cerebral ischemia
