A comparative analysis of bioenergetic parameters of brain cells in acute slices and cell culture using fluorescence microscopy
NAD+ and NADH play crucial roles in mitochondrial energy metabolism due to their participation in the tricarboxylic acid cycle and electron transfer chain. Сomplex I oxidizes NADH on the inner side of the membrane in mitochondria but NAD+ reduction occurs in the matrix as a result of citrate cycle and β-oxidation. NAD+ and NADH are needed to maintain a balance between the production of active oxygen forms and their neutralization, energy homeostasis and metabolism. It should be noted that they also participate in death, aging of brain cells and tissue damage in various brain diseases.
Therefore, it is possible to estimate bioenergetic parameters of brain cells by measuring mitochondrial pool, redox index and rate of formation of NADH/ NAD(P)H with using fluorescence microscopy. This method is based on the registration of auto-fluorescence of endogenous fluorophore – NADH/ NAD(P)H. The experimental unit with BDL-SMN-375 laser source (Becker&Hickl, Germany) was assembled for measurement in order to excite fluorescence at 375 nm wavelength that registers mitochondrial NADH/ NAD(P)H content in cells of both acute slices and brain cell culture.
Experiments were performed on brain acute slices of 12-week-old Wistar rats and cell culture of midbrain and cortex. The brain was located in chilled Hanks and cut by hand after euthanasia by dislocation of the cervical spine. Later the acute slice was placed in an excavated slide with 150 µl HBSS. In turn, 500 µl HBSS was added to a Petri dish with the primary culture in it. After that FCCP (4 µM) and NaCN (5 mM) were used to determine the upper and lower values of the mitochondrial NADH pool. The basic level of endogenous fluorescence as well as its changes after addition of each reagent were recorded. Each stage of measurement lasted for 3 minutes with the interval of 2 s between frames.
It should be noted that FCCP is a protonophore that transports protons. After its addition cell respiration was maximal but NADH fluorescence level was minimal due to complete oxidation of NADH with formation of non-fluorescent NAD in mitochondria. NaCN blocked complex IV of the respiratory chain and the oxidation of NADH. Therefore, the level of fluorescence was recorded at the maximum level.
Data were obtained for cell cultures. The mitochondrial rate of NADH formation was 27,32±8,79 a.u. in the cortex, in the midbrain it was 15,16±3,6 a.u. The mitochondrial NADH pool was 100±12 units in the cortex, in the midbrain it was 49±7 units. The mitochondrial redox index was 45±11% in the cortex, in the midbrain it was 56±16%. Data were also obtained for acute slices. The mitochondrial rate of NADH formation was 2,45±0,7 a.u. in the cortex, in the midbrain it was 2,66±0,52 a.u. The mitochondrial NADH pool was 87±6 units in the cortex, in the midbrain it was 113±6 units. The mitochondrial redox index was 54±4% in the cortex, in the midbrain it was 66±8%.
Thus, the experimental unit with BDL-SMN-375 laser source (Becker&Hickl, Germany) is able to register mitochondrial pool, redox index and rate of formation of NADH/ NAD(P)H by fluorescence microscopy in cells of both acute brain slices and cell culture. It allows to estimate metabolic activity and mitochondrion work. In this way, bioenergetic parameters of brain cells can be analyzed that may be used in preventing neurodegenerative diseases.
This study was supported by the grant from the Russian Federation Government no. 075-15-2019-1877.
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Dolgikh Angelina (firstname.lastname@example.org)
Orel State University
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