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

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在照射的软骨中，沿着激光消融部位周围的增殖的软骨细胞的鉴定引起了人们对单独使用激光加热来刺激软骨细胞生长的研究兴趣。然而，激光产生的过高热量也会导致软骨细胞坏死和凋亡。本研究的目的是在完整的体外组织标本中评价激光照射后对软骨的急性损伤。用脉冲持续时间(4、6和8秒)和功率(4、6和8瓦)设置的脉冲照射兔鼻中隔软骨，用来产生细胞分裂。激光照射后即刻，用活/死细胞活性测定结合共聚焦显微镜评价软骨样本的热损伤程度。根据先前观察到再生的激光光斑周围活细胞和死亡细胞的分布来评估热损伤。细胞存活率测定确定了激光光斑内及其周围的坏死组织。远离激光光斑中心，可观察到混合的坏死性和活生性软骨细胞群。正如预期的那样，照射时间、功率和损伤程度之间存在相关性。这项实验的结果将被用来确定在将热损伤降至最低的同时产生再生所需的阈值。

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.