Alcohol has well known effects on brain development and function, but its effects specifically on microglial cells (MG) are much less well understood. Here we will utilize in vitro and in vivo mouse brain tissue preparations to study the effects of ethanol on MG activation, motility, migration to injured neurons, and phagocytic clearance of dead cells. We expect that alcohol- induced neuronal injury and apoptotic cell death can be recapitulated in organotypic brain slice cultures derived from neonatal mice, thereby establishing an experimentally tractable in vitro tissue model for studies of glial responses to alcohol-induced neurodegeneration. We hypothesize that alcohol induces brain resident MG to activate rapidly, prior to neuronal cell death, and that MG activation and recruitment to injured neurons via fractalkine signaling promotes neuronal survival and limits secondary neuronal injury. We will test aspects of this hypothesis, first, by assessing the timing of MG activation (changes in MG morphology and gene expression) relative to alcohol-induced initiation of neuronal apoptosis (cleaved caspase-3 labeling) and subsequent cell death (Sytox/Propidium iodide labeling) in mouse hippocampal or cortical slice cultures. To determine whether MG activation precedes, follows, or indeed requires apoptotic neuronal death, we will examine MG activation in slices from mice lacking BAX, an apoptosis regulator protein. Next, we will test whether fractalkine signaling regulates MG activation and recruitment to alcohol-injured neurons in slices from fractalkine receptor null mice (CX3CR1GFP/GFP). Finally, we will use transcranial multiphoton imaging in live, alcohol- treated GFP reporter mice (CX3CR1GFP/+) to examine whether alcohol affects MG basal motility or mobilization to injured neurons in developing or adult cortical brain tissues in vivo. These exploratory studies will yield the first direct observations and analysis of glial cell behaviors in live, intact brain tissues during and following conditions of high blood alcohol and withdrawal. Results from these studies will lay the groundwork for future studies aimed at elucidating the consequences of MG activation for neuroprotection or neurotoxicity. This information will help generate new ideas on the consequences of alcohol for normal brain function as well as for impaired immune surveillance and response to injury in the CNS.
Alcohol-related neuronal injury in humans is an increasing health concern and a growing burden on the economy of our society. Alcohol exposure during critical periods of neuronal development induces damage to or death of neurons, and the response of nearby glial cells, including microglial cells, may promote the survival of injured neurons or exacerbate the injury. The data generated here will help identify responses of microglia to alcohol and alcohol-induced neuronal injury, and thus help guide therapeutic strategies after alcohol-induced brain injury in developing and adult humans.
