ÉTUDE IN VITRO DE LA SURVIE DE CELLULES NEURONALES APRÈS AXOTOMIE PAR LASER.

CAROLYNE DION; D. KUNIK; T. OZAKI; L.A. LEVIN; S. COSTANTINO

Montreal QC Canada

Jornada; 22e Journée de la recherche en ophtalmologie de l'Université de Montréal; 2010

Introduction:  Irreversible blindness is most frequently caused by the death of retinal neurons. The most common cause of blindness worldwide is glaucoma, a disease of the optic nerve where the retinal ganglion cell axons are injured. Understanding how neurons die, including the transduction of the cell death process, is crucial in leading to cures for what is otherwise permanent visual loss. In addition, understandin neuronal and axonal function on a single cell basis is essential for the evaluation of neuroprotective and neuroregenerative strategies. In this context, we have combined microscopy and ultrafast laser transsection to provide a platform for studying intracellular processes of cell death and regeneration after axotomy at a single cell scale. Here we present the capabilities of this system and preliminary results on the effects of axotomy. Methods: The system combines laser nanosurgery capabilities and fluorescence imaging by coupling a picosecond laser beam to an inverted microscope. The 7 ps 1064 nm laser pulses are generated at a repetition rate of 76 MHz and 100 nJ per pulse. The laser intensity at the sample is adjusted using a half wave plate and a polarizer. The axial location of the focal volume is fixed using a two-lens telescope and the laser exposure is controlled by an electromechanical shutter. The surgery beam fills the back aperture of an 60x objective yielding a 1 µm laser spot size at the sample. The microscope camera, motorized stages, and shutter are controlled by a custom-written computer program that allows nanosurgery and time-lapse imaging of multiple cells. Results: Axonal injuries were performed on differentiated RGC-5 cells in culture. Significant morphological changes were observed within 2 hours after axotomy. Rapid swelling was followed by reduction of soma volume body and retraction of the injured axon. Conclusion: We have developed an optical system and demonstrated it suitability for the study of cell death process after axotomy at the single cell scale. Our future work will focus on using specific dyes to track the induction of apoptosis after axotomy.