Intra-operative identification of malignant glioma using real-time fluorescence spectroscopic analysis and double staining method

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The first purpose of this study is to establish the glioma identification method of PDD using spectroscopic analysis and double staining method. Our second purpose is to develop the next generation PDD agent, which has the excellent tumor specificity. Our third purpose is to achieve the basic studies on the ALA-PDT.To analyze the fluorescence spectrum using fiber-optic spectroscopic tumor sensor, we could identify the tumor specific spectrum. The time resolution of spectroscopic identification is very short, so that this sensing method can be adapted for the sensor type surgical robot system. Next, we developed the new fluorescent agent (FLS-HAS) to conjugate the FLS and human albumin. The FLS-HAS has the excellent tumor specificity compared with 5-ALA. We found that the EPR (electron spin resonance method) could be applicable to the direct detection of ROSs (reactive oxygen species) in the living cell during PDT. The major ROSs of ALA-PDT was singlet oxygen. As for the mechanism of cell death after ALA-PDT, we found that the increases in the activity of both caspase-3 and -9, a decrease in MMP (mitochondrial membrane potential), and a marked increase in cytochrome c in the cytosolic fl-action were found after cells were subjected to PDT. These results indicate that a dysfunction of MMP is followed by mitochondrial cytochrome c release, which triggers apoptosis through a mitochondrial pathway.