Real-Time Dynamics of Ca2+, Caspase-3/7, and Morphological Changes in Retinal Ganglion Cell Apoptosis under Elevated Pressure

Quantitative information on the dynamics of multiple molecular processes in individual live cells under controlled stress is central to the understanding of the cell behavior of interest and the establishment of reliable models. Here, the dynamics of the apoptosis regulator intracellular Ca2+, apoptosis effector caspase-3/7, and morphological changes, as well as temporal correlation between them at the single cell level, are examined in retinal gangling cell line (differentiated RGC-5 cells) undergoing apoptosis at elevated hydrostatic pressure using a custom-designed imaging platform that allows long-term real-time simultaneous imaging of morphological and molecular-level physiological changes in large numbers of live cells (beyond the field-of-view of typical microscopy) under controlled hydrostatic pressure. This examination revealed intracellular Ca2+ elevation with transient single or multiple peaks of less than 0.5 hour duration appearing at the early stages (typically less than 5 hours after the onset of 100 mmHg pressure) followed by gradual caspase-3/7 activation at late stages (typically later than 5 hours). The data reveal a strong temporal correlation between the Ca2+ peak occurrence and morphological changes of neurite retraction and cell body shrinkage. This suggests that Ca2+ elevation, through its impact on ion channel activity and water efflux, is likely responsible for the onset of apoptotic morphological changes. Moreover, the data show a significant cell-to-cell variation in the onset of caspase-3/7 activation, an inevitable consequence of the stochastic nature of the underlying biochemical reactions not captured by conventional assays based on population-averaged cellular responses. This real-time imaging study provides, for the first time, statistically significant data on simultaneous multiple molecular level changes to enable refinements and testing of models of the dynamics of mitochondria-mediated apoptosis. Further, the platform developed and the approach has direct significance to the study of a variety of signaling pathway phenomena

Long-term effects of a Palaeolithic-type diet in obese postmenopausal women: a 2-year randomized trial

BACKGROUND/OBJECTIVES: Short-term studies have suggested beneficial effects of a Palaeolithic-type diet (PD) on body weight and metabolic balance. We now report long-term effects in obese postmenopausal women of a PD on anthropometric measurements and metabolic balance, in comparison with a diet according to the Nordic Nutrition Recommendations (NNR). SUBJECTS/METHODS: Seventy obese postmenopausal women (mean age 60 years, body mass index 33 kg/m(2)) were assigned to an ad libitum PD or NNR diet in a 2-year randomized controlled trial. The primary outcome was change in fat mass as measured by dual energy X-ray absorptiometry. RESULTS: Both groups significantly decreased total fat mass at 6 months (−6.5 and −2.6 kg) and 24 months (−4.6 and −2.9 kg), with a more pronounced fat loss in the PD group at 6 months (P<0.001), but not at 24 months (P=0.095). Waist circumference and sagittal diameter also decreased in both groups, with a more pronounced decrease in the PD group at 6 months (−11.1 vs. −5.8 cm, P=0.001 and −3.7 vs. −2.0 cm, P<0.001, respectively). Triglyceride levels decreased significantly more at 6 and 24 months in the PD group versus the NNR group (P<0.001 and P=0.004). Nitrogen excretion did not differ between groups. CONCLUSIONS: A PD has greater beneficial effects versus an NNR diet regarding fat mass, abdominal obesity and triglyceride levels in obese postmenopausal women; effects not fully sustained for anthropometric measurements at 24 months. Adherence to protein intake was poor in the PD group. The long-term consequences of these changes remain to be studied