INVESTIGADORES
KUNIK Dario
congresos y reuniones científicas
Título:
25. NEURONAL SURVIVAL STUDY AFTER LASER AXOTOMY
Autor/es:
DARÍO KUNIK; C. DION; T. OZAKI; L.A. LEVIN; S. COSTANTINO
Lugar:
Montreal QC Canada
Reunión:
Jornada; 17e JOURNÉE DE LA RECHERCHE de l'HÔPITAL MAISONNEUVE-ROSEMONT; 2010
Resumen:
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, understanding 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 its 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.