DEVELOPMENT OF INTRAVITAL LASER MICROSCOPE FOR VISUALIZATION OF MICROVASCULAR PO_2 DISTRIBUTION

为了阐明氧在骨骼肌中的传输和氧在骨骼肌中的分布，我们设计了一种基于氧依赖猝灭磷光技术的活体激光显微镜。将pd -卟啉溶液注射到全身血液中，利用光电倍增管捕获其在直径为15 μm的组织区域内被N_2/染料脉冲激光激发的磷光。体外和体内氧张力（pO_2）测量结果表明，该方法无创、准确、反应时间快。将该方法应用于大鼠肌微循环pO_2的测定。在按分支顺序分类的几个区域进行了小动脉和静脉pO_2测量。不同阶动脉的平均pO_2随着分支的增加而降低（A1: 72.4, A2: 51.8, A3: 44.1 mmHg），而静脉pO_2与血管阶无关（V1: 27.5, V2: 28.6, V3: 32.0 mmHg）。邻近不同阶小动脉间质pO_2明显低于血管内pO2 （I1: 47.8, I2: 35.8, I3: 24.4 mmHg）。综上所述，尽管目前还需要更多的组织中pO_2值的区域数据，但本研究表明，激光荧光猝灭显微镜可以用来阐明O_2在微血管中的运输和O_2在组织中的分布。如果详细阐明组织损伤的可能性，则使用激光激发磷光探针可以照亮较小的组织区域（<10μm）。此外，我们发现不同级别的小动脉的pO_2水平明显降低，血液和组织界面的pO_2梯度较大。这些发现表明毛细血管不是向周围组织供应氧气的唯一来源。

To clarify the oxygen transport across the microvessed and oxygen distribution in the skeletal muscle, we have newly designed an intravital laser microscope with oxygen-dependent quenching of phosphorescence technique. Pd-porphyrin solution was injected into the systemic blood, and its phosphorescence excited by N_2/dye pulse laser in the tissue area of 15 μm in diameter was captured by a photomultiplier. In vitro and in vivo oxygen tension (pO_2) measurements revealed the efficiency of this method because of its non-invasiveness, accuracy, and rapid response time. This technique was applied to pO_2 measurements in the rat cremaster muscle microcirculation. The arteriolar and venular pO_2 measurements were performed at the several regions classified by branching order. The mean arteriolar pO_2 of different orders decreased according to increase the branching (A1 : 72.4, A2 : 51.8 and A3 : 44.1 mmHg), however venular pO_2 were independent on vessel orders (V1 : 27.5, V2 : 28.6 and V3 : 32.0 mmHg). Interstitial pO_2 adjacent to arterioles of different orders were significantly lower than those of intravascular pO2 (I1 : 47.8, I2 : 35.8 and I3 : 24.4 mmHg). In conclusion, the present study has shown that the phosphorescence quenching laser microscope can be used to clarify the transport of O_2 across microvessels and the distribution of O_2 in tissues, although more regional data on pO_2 values in tissues are required in the current. The use of lasers to excite phosphorescent probes enables a smaller area of tissue to be illuminated (<10μm), if the potential for tissue damage is clarified in detail. In addition, we found a large reduction in pO_2 levels of different order arterioles as well as a large pO_2 gradient at the interface between blood and tissue. These findings suggest that capillaries are not the sole source of oxygen supply to surrounding tissue.