DEVELOPMENT OF INTRAVITAL LASER MICROSCOPE FOR VISUALIZATION OF MICROVASCULAR PO_2 DISTRIBUTION

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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.