Yasuhisa Fujibayashi

UNLABELLED: An ideal hypoxia imaging agent should have high membrane permeability for easy access to intracellular mitochondria and low redox potential to confer stability in normal tissue, but it should be able to be reduced by mitochondria with abnormally high electron concentrations in hypoxic cells. In this context, nitroimidazole residues are not considered to be essential. In this study, Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM), a 62Cu-bisthiosemicarbazone complex, with high membrane permeability and low redox potential, was evaluated as a possible hypoxia imaging agent, using electron spin resonance spectrometry and the Langendorff isolated perfused rat heart model as well as rat heart left anterior descending occlusion model. METHODS: Nonradioactive Cu-ATSM was incubated with rat mitochondria, after which reduction of Cu(II) to Cu(I) was measured with electron spin resonance. As a model of hypoxic mitochondria, rotenone (Complex I inhibitor)-treated mitochondria were used. RESULTS: In this study, Cu-ATSM was reduced by hypoxic but not by normal mitochondria. CONCLUSION: Thus, retention of 62Cu-ATSM was studied serially in perfused rat hearts under conditions of normoxia (95% O2 + 5% CO2), hypoxia (95% N2 + 5% CO2) and reoxygenation (95% O2 + 5% CO2). In normoxia and reoxygenation, 62Cu-ATSM injected as a single bolus showed low retention (23.77% and 22.80%, respectively) 15 min after injection, but retention was increased markedly under hypoxic conditions (81.10%). Also, in the in vivo left anterior descending occluded rat heart model, 62Cu-ATSM retention was inversely correlated with accumulation of 201Tl, a relative myocardial blood flow marker.

UNLABELLED: We have evaluated Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM), an effective marker for the delineation of hypoxic but viable tissue, in vitro in the EMT6 carcinoma cell line under varying degrees of hypoxia and compared it with the flow tracer 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM) and the hypoxic tracer 18F-fluoromisonidazole (MISO). We have also compared the uptake of Cu-ATSM and Cu-PTSM in vivo and ex vivo in a murine animal model bearing the EMT6 tumor. METHODS: Uptake of 64Cu-ATSM, 64Cu-PTSM and 18F-MISO in vitro into EMT6 cells was investigated at the dissolved oxygen concentrations of 0, 1 x 10(3), 5 x 10(3), 5 x 10(4) and 2 x 10(5) ppm. Biodistribution performed at 1, 5, 10, 20 and 40 min compared 64Cu-ATSM with 64Cu-PTSM in BALB/c mice bearing EMT6 tumors. To determine long-term retention of 64Cu-ATSM, biodistribution was also performed at 1, 2 and 4 h. Ex vivo autoradiography of tumor slices after co-injection of 60Cu-PTSM (60Cu, T1/2 = 23.7 min) and 64Cu-ATSM (64Cu, t1/2 = 12.7 h) into the same animal was performed. RESULTS: After 1 h, 64Cu-ATSM was taken up by EMT6 cells: 90% at 0 ppm, 77% at 1 x 10(3) ppm, 38% at 5 x 10(3) ppm, 35% at 5 x 10(4) ppm and 31% at 2 x 10(5) ppm. 18F-MISO also showed oxygen concentration dependent uptake, but with lower percentages than 64Cu-ATSM. 64Cu-PTSM showed 83%-85% uptake into the cells after 1 h, independent of oxygen concentration. Biodistribution data of 64Cu-ATSM and 64Cu-PTSM showed optimal tumor uptake after 5 and 10 min, respectively (0.76% injected dose (ID)/organ for 64Cu-ATSM and 1.11%ID/organ for 64Cu-PTSM). Ex vivo imaging experiments showed 60Cu-PTSM uniform throughout the EMT6 tumor, but heterogeneous uptake of 64Cu-ATSM, indicative of selective trapping of 64Cu-ATSM into the hypoxic tumor cells. CONCLUSION: Cu-ATSM exhibits selectivity for hypoxic tumor tissue both in vivo and in vitro and may provide a successful diagnostic modality for the detection of tumor ischemia.

Eye contact provides a communicative link between humans, prompting joint attention. As spontaneous brain activity might have an important role in the coordination of neuronal processing within the brain, their inter-subject synchronization might occur during eye contact. To test this, we conducted simultaneous functional MRI in pairs of adults. Eye contact was maintained at baseline while the subjects engaged in real-time gaze exchange in a joint attention task. Averted gaze activated the bilateral occipital pole extending to the right posterior superior temporal sulcus, the dorso-medial prefrontal cortex, and the bilateral inferior frontal gyrus. Following a partner's gaze toward an object activated the left intraparietal sulcus. After all the task-related effects were modeled out, inter-individual correlation analysis of residual time-courses was performed. Paired subjects showed more prominent correlations than non-paired subjects in the right inferior frontal gyrus, suggesting that this region is involved in sharing intention during eye contact that provides the context for joint attention.