A Dynamic Balance between Neuronal Death and Clearance in an in Vitro Model of Acute Brain Injury

In in vitro models of acute brain injury, neuronal death may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. Neurons undergoing programmed cell death are in this queue, and are the most visible and frequently quantified measure of neuronal death after injury. However, the size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for 2 weeks. Altering phagocytosis rates (e.g., by changing the number of microglia) dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for stains that label dying neurons. Canonically neuroprotective interventions, such as seizure blockade, and neurotoxic maneuvers, such as perinatal ethanol exposure, were mediated by effects on microglial activity and the membrane permeability of neurons undergoing programmed cell death. These canonically neuroprotective and neurotoxic interventions had either no or opposing effects on healthy surviving neurons identified by the ongoing expression of transgenic fluorescent proteins.