THE VIABILITY OF CONFOCAL LASER SCANNING MICROSCOPY OF RABBIT NASAL SEPTAL CART

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Identification of proliferating chondrocytes along the periphery of laser ablation sites in irradiated cartilage has led to interest in studying the use of laser heating alone to stimulate chondrocyte growth. However,excessive heat produced by a laser can also cause chondrocyte necrosis and apoptosis. The objective of this study was to evaluate acute injury to cartilage following irradiation by an Nd:YAG (¿f=1.32¿m) laser in intact ex-vivo tissue specimens. Rabbit nasal septal cartilage was irradiated using an Nd:YAG laser using pulse durations (4, 6, and 8 seconds) and power (4,6, and 8 watts) settings previously determined to produce cell division. Immediately after laser irradiation, the extent of thermal injury to the cartilage samples was evaluated using a Live/Dead¿ cell viability assay combined with confocal microscopy. Thermal injury was assessed with respect to distribution of live and dead cells surrounding the laser spot where regeneration was previously observed. The cell viability assay identified necrotic tissue within and immediately around the laser spot. Moving away from the center of the laser spot, a mixed population of necrotic and live chondrocytes was observed. As expected, a correlation between irradiation time, power and degree of injury was found. The results of this experiment will be used determine the threshold required to produce regeneration while minimizing thermal injury.