ASTROCYTE AND NEURON INTERACTIONS FOLLOWING LASER INDUCED LYSIS

Astrocytes are important immune responders in the central nervous system (CNS). Astrocytes are involved with the progression of many different neurological diseases such as Amyotrophic Lateral Sclerosis (ALS) and Alzheimer's disease. It is known that defects in the astrocytes ability to phagocytose can lead to the progression of some neurodegenerative diseases, however the underlying mechanizes are not well understood. The purpose of this study is to investigate the interactions of astrocytes with other astrocytes and with neurons after inducing damage using laser irradiation. Two different laser systems were used including an 800nm Ti:Sapphire femtosecond pulsed laser and a 532 nm Nd:YVO₄ diode pumped nanosecond laser. In combination with a Zeiss axiovert inverted microscope and a CO2 controlled stage incubator, primary E12 cortical cells were observed via time lapse imaging. Utilizing the computer program Robolase we were able to visualize the interactions in real time, control the exposure of the laser, and target a specific area of the cell. This allowed for damage from the laser to be controlled, allowing us to damage a single cell and leave the neighboring cells unharmed. Cellular interaction were characterized by the formation of processes between cells and by subsequent phagocytosis. Phagocytosis appears to be dependent on the type of connection present between the cells prior to laser exposure. Cells were categorized into two major categories: (1) direct contact with either other astrocytes or neurons (connected), or (2) those that had no direct contact with other astrocytes or neuron (non-connected). The connections can be by individual (or multiple) filopodia, or by a larger amount of shared plasma membrane in clusters of cells. Connected cells had a higher chance of phagocytosing the laser-damage (lysed) cell. Furthermore, the connected cells were more likely to phagocytose the cell if they were in a group as compared to those that had a thin filopodial connection. Sixty-five percent of the connected cells reacted to the lysed cell by phagocytosing the cellular remnants (debris) while only 22% of the non-connected cells reacted in the same form. Astrocytes behaved similarly to laser-induced lysis of neurons. Lysis of neurons was induced by targeting two different cellular regions: the cell body and the axon. Similar to the astrocyte-astrocyte interaction, connected cells had a higher chance of phagocytosing the axon than non-connected cells. In conclusion, this study suggests that astrocytes play a key role in phagocytosis of damaged nervous system cells (astrocytes and neurons). Parameters that can affect their ability to phagocytose include the amount of shared membrane between cells, as well as the location in the cell of laser-induced lysis.