The paper presents results of the estimation of bioenergetic parameters in acute slices of brain rat by measuring mitochondrial pool and rate of NADH production using fluorescence imaging. NAD+ and NADH play crucial roles in mitochondrial energy metabolism due to their participation in the tricarboxylic acid cycle and in electron transport chain. That is why it is essential to know variability of its content in cells. In the study we used an approach based on the registration of auto-fluorescence of NADH. An experimental setup was designed and assembled for the imaging of the acute brain slices but also capable of measuring brain cell cultures. BDL-SMN-375 laser (Becker&Hickl, Germany) was used to excite fluorescence at the wavelength of 375 nm. The parameters of NADH content in both acute brain slices and cell cultures of midbrain and cortex were estimated. The obtained results can be of interest for better understanding of development of neurodegenerative diseases of mitochondrial disfunction origin.
NADH is one of the central signalling molecules that serve as a substrate for many vital processes, in particular, is a donor for the electron transport chain in mitochondria. At the same time, mitochondrial activity and the intensity of metabolic processes in the tissues of different brain regions drastically differ. This work aimed to compare the NAD(P)H production rate in the tissues of various brain regions (cortex, cerebellum, hippocampus, brainstem) assessed by the parameters of dynamics of its autofluorescence. We studied acute slices of the rat brain of the corresponding regions. The intensity of NAD(P)H autofluorescence and its rate of production was determined to assess the activity of mitochondria. Whereafter the slices were analysed by histological examination. It has been observed that mitochondrial activity in hippocampal tissues is significantly higher than in other brain regions, which may be associated with more complex cognitive functions of the hippocampus in mammals. The results of this research can help explain the selectivity of hippocampal lesions during ischemic injuries and neurodegenerative diseases.
The overall aim of this study was verification of the possibility to register the change of NADH fluorescence in live tissue by a portable diagnostical laser system with fibre optical probe output and excitation by compact semiconductor UV light source. The measurements were conducted in fresh brain tissue slices of Wistar rat pups. The fluorescence measurements were conducted simultaneously at intervals of 5 s by the microscopic system with excitation at 360 nm and registering of the emitted fluorescence light at 455 nm and by the tested diagnostical system equipped with the fibre optical probe with excitation at 365 nm and registration of the fluorescence spectrum by the inbuilt spectroscopic subsystem. To modulate the mitochondrial function in the living cells, in the chamber sequentially were added 1 μM FCCP and 1 mM NaCN. The comparisons between the curves registered by the methods allowed us to find well agreement between the microscopic measurements and measurements by the fibre optical probe. The obtained results prove that the tested diagnostic system is capable of sensing the changes in brain metabolic activity associated with the NADH content alterations within the physiological range.
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