[Distribution of electroencephalograph power density in patients with severe obstructive sleep apnea during different sleep stages].

Objective: To investigate the variation of electroencephalograph(EEG) power density during different sleep stages in OSA for understanding of the mechanisms underlying the brain dysfunction in OSA as well as its earlier diagnosis and treatment. Methods: Sixteen-channel EEGs from OSA patients and normal controls in stage wake, sleep stage 1, sleep stage 2, sleep stage 3 and rapid eye movement stage were analyzed by time-frequency analysis method. The EEG power density in different frequency bands (including δ, θ, α, σ, ß and γ) was respectively compared between the 2 groups. The correlation between the variation in the EEG power and primary indices of polysomnography was further analyzed. Results: The EEG power density in δ band in stage wake [OSA: (0.82±0.13) µV(2)/Hz, Control: (0.66±0.02) µV(2)/Hz, t=4.309, P<0.05], stage 1 [OSA: (1.28±0.07) µV(2)/Hz, Control: (0.92±0.04) µV(2)/Hz, t=-3.369, P<0.05] and stage 3 [OSA: (2.74±0.22) µV(2)/Hz, Control: (2.04±0.07) µV(2)/Hz, t=-2.669, P<0.05] was significantly higher in OSA, compared with that in the control. Statistical analysis showed that the EEG power density was significantly higher in frontal and central regions in stage wake [frontal: OSA: (0.90±0.02) µV(2)/Hz, Control: (0.66±0.02) µV(2)/Hz, t=8.539, P<0.01; central: OSA: (1.15±0.06) µV(2)/Hz, Control: (0.72±0.02) µV(2)/Hz, t=6.669, P<0.01] and stage 1 [frontal: OSA: (1.23±0.03) µV(2)/Hz, Control: (0.99±0.03) µV(2)/Hz, t=5.983, P<0.01; central: OSA: (1.52±0.05) µV(2)/Hz, Control: (1.14±0.04) µV(2)/Hz, t=5.714, P<0.01], as well as central region in stage 3 [OSA: (3.24±0.17) µV(2)/Hz, Control: (2.71±0.08) µV(2)/Hz, t=2.707, P<0.05]. The correlation analysis showed that the power density in central region in stage 1 and stage 3 was positively correlated with arousal index (r=0.877 in stage 1, 0.656 in stage 3), implying that sleep fragmentation was closely related to the variation of EEG power density during nocturnal sleep in OSA. Conclusions: The feature stages for OSA are stage wake, stage 1 and stage 3. The EEG power density in OSA (δ band) was significantly higher than that in the control. The EEG power density in OSA and the control shows differences in frontal and central regions in stage wake and stage 1, as well as in central region in stage 3. The results indicate that low-frequency EEG power density giving priority to frontal area and central area has improved in severe OSA, which may be related to the neurologic deficits in corresponding brain areas.

Thawed human hepatocytes in primary culture.

In drug metabolism studies, isolated and cultured human hepatocytes provide a useful model for overcoming the difficulty of extrapolating from animal data. In vitro studies with human hepatocytes are scarce because of the lack of livers and suitable methods of storage. After developing a new method for cryopreservation of human hepatocytes, we evaluated the effects of deep freezing storage on their viability, morphology, and functional and toxicological capabilities in classical culture conditions. Freshly isolated human hepatocytes were cryopreserved in medium containing 10% Me2SO and 20% fetal calf serum, using a Nicool ST20 programmable freezer (-1.9 degrees C/min for 18 min and -30 degrees C/min for 4 min). Cells were stored in liquid nitrogen. Viability of thawed human hepatocytes was 50-65% as assessed by erythrosin exclusion test prior to purification on a Percoll density gradient. Morphological criteria showed that thawed human hepatocytes require an adaptation period to the medium after seeding. Functional assessments showed that human hepatocytes which survive freezing and thawing preserve their protein synthesis capabilities and are able to secrete a specific protein, anionic peptidic fraction, which is involved in the hepatic uptake of bile-destined cholesterol. We then studied Midazolam biotransformation to test metabolic functions, and erythromycin toxicity by Neutral Red test (cell viability) and 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide test (cell metabolism). All of these experiments indicated that thawed human hepatocytes should be used 38 h after seeding for optimum recovery of their functions: membrane integrity, protein synthesis, and stabilization of drug metabolism enzymes.

Freshly isolated or cryopreserved human hepatocytes in primary culture: Influence of drug metabolism on hepatotoxicity.

The use of human hepatocytes in drug metabolism studies overcomes the difficulty in extrapolating from animal data. As drug metabolites are often involved in pharmacological activity and toxicity, it is essential to determine these compounds early in the development of a drug. We previously demonstrated that mitoxantrone metabolism exhibits a great interspecies variability: the two main metabolites in humans being dicarboxylic acid (the major metabolite) and monocarboxylic acid. In this paper we analyse their respective cytotoxicities with two endpoint tests: neutral red uptake (cell viability) and MTT reduction (cell metabolism) after a 24- or 48-hr exposure. Results demonstrated less or no toxicity of mono- and dicarboxylic acid derivatives compared with mitoxantrone. In vitro studies using human hepatocytes are scarce because of the limited availability of human livers. After developing a new method for cryopreservation of human hepatocytes, we evaluated them before and after cryopreservation using erythromycin as a test molecule. The cell viability test was more reproducible than the cell metabolism test after 38 hr of cell seeding.