Date of Degree
Access restricted until 01/31/2019
PhD (Doctor of Philosophy)
Michael E. Dailey
Fetal alcohol exposure is the most common known cause of preventable mental retardation, yet we know little about how microglia respond to, or are affected by, alcohol in vivo. Using an acute (single day) model of moderate (3 g/kg) to severe (5 g/kg) alcohol exposure in postnatal day (P) 7 or P8 mice we have found that alcohol-induced cortical neuroapoptosis is closely correlated in space and time with the appearance of activated microglia near dead cells. Microglia found in close proximity to dying neurons selectively engulfed those that were in later stages of apoptosis. Remarkably, most dead cells were cleared and microglia began to deactivate within 1-2 days of the initial insult. Coincident with microglial activation and deactivation, in the 5 g/kg alcohol model, there was a transient but substantial increase in pro-inflammatory factor (PIFs) expression. Work in BAX-null mice demonstrated that microglial activation and PIF expression were linked to BAX-dependent neuroapoptosis. As such, the level of microglial activation scaled with alcohol-induced cell death. Therefore, acute alcohol exposure in the developing cortex causes transient microglial activation and mobilization, promoting clearance of dead cells and tissue recovery. Moreover, cortical microglia show a remarkable capacity to rapidly deactivate following even severe neurodegenerative insults in the developing brain.
Given that alcohol exposure on either P7 or P8 induced comparable levels of neuroapoptosis and microglial activation, we hypothesized that alcohol exposure on two sequential days (P7 and P8) would exacerbate neuroapoptosis and extend microglial activation. Instead, we found that the period of neuroapoptosis and microglial activation was similar after one day of alcohol exposure on P7 or after two days of exposure on P7 and P8. This was true for both the moderate and severe alcohol paradigms. Potentially, the low levels of cell death produced by the second day of injection may be due to neuroprotective mechanisms elicited by the first day of alcohol injection. In support of this idea, a preliminary microarray analysis of cortical gene expression 12 and 24 h after 5 g/kg alcohol exposure shows a decrease in expression of several pro-apoptotic factors and an increase in the expression of pro-survival factors, including neurotrophins. Of particular interest, BDNF, which has previously been shown to inhibit alcohol-induced neuroapoptosis, showed an eight-fold increase in expression at 24 h following 5 g/kg alcohol exposure and in situ hybridization showed strong BDNF expression near cortical regions with high levels of cell death. Future studies will be needed to extend the analysis of microglial activation states in this two-day injection model and to further explore the possibility that BDNF expression by microglia enacts neuroprotective mechanisms against a second insult.
Finally, work in cell culture has suggested that chronic alcohol exposure may potentiate or inhibit microglial phagocytosis of dead cells. These studies raise the possibility that alcohol may directly affect microglial mobility which is important for their surveillance and synaptic remodeling functions. Therefore, we measured the effect of increasing doses of alcohol (0, 0.25, 0.5 and 1%) on microglial migration, branch motility, and morphology in dissociated BV-2 cell cultures and in acutely isolated neonatal (P5-6) brain slices. The results indicate that alcohol dose-dependently inhibits microglial migration and ruffling in cell culture, but in brain slices even high alcohol concentrations (0.5%) only reduce microglial branch motility by ~2%. When combined with our evidence for efficient microglial phagocytic clearance of dead cells in the neonatal cortex, these data suggest that while there is a measurable effect of acute alcohol exposure on microglial mobility, it does not impede microglia from performing their surveillance and phagocytic functions in vivo.
Apoptosis, Development, Fetal Alcohol Spectrum Disorders, Migration, Phagocytosis, Protective Mechanism
xii, 134 pages
Includes bibliographical references (pages 122-134).
Copyright © 2014 Katelin Eloyce Ahlers
Available for download on Thursday, January 31, 2019