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NADH growth rate evaluation in different rat brain regions by fluorescence spectroscopy

Alexander Palalov, Orel State University, Russian; Pavel Gorlin, Orel State University, Russia; Evgeniya Seryogina, Orel State University, Russia; Alexander Alekseev, Orel State University, Russia; Evgeny Zherebtsov, Orel State University, Russia, University of Oulu, Finland; Andrey Abramov, Orel State University, Russia, UCL Queen Square Institute of Neurology, London

Abstract

NADH is an essential signaling molecule that serves as a substrate for a plenty of vital processes. Particularly, NADH is an electron donor in mitochondria, where cellular respiration and ATP synthesis occur. Dysfunction of any element of cellular respiration metabolic pathway results in decreasing of ATP production, different degenerative changes and cell death. This phenomenon can be observed in the majority of pathological conditions but makes great difference in the diseases of central nervous system. Based on the literature, neurons and glial cells have low resistance to hypoxia and die rapidly with the mitochondrial dysfunction. Mitochondrial impairment also plays a meaningful role in neurodegenerative diseases like Alzheimer’s disease. It should be mentioned that cells of different areas of central nervous system have various intensity of metabolic rates. There is also a certain hierarchical lesion of brain regions during hypoxia, which necessitates a differentiated assessment of the cellular respiration intensity in the central nervous system. Evaluating the rate of key metabolites synthesis (NADH) one can consider the safety of cellular respiration. It is common to measure NAD(P)H in vitro by fluorimetric method due to its high sensitivity (the method is based on NAD(P)H ability to emit fluorescence when excited by a light beam with a wavelength of 375 nm, giving a maximum at 480 nm). Thus, the aim of the paper is to evaluate changes in the NAD(P)H fluorescence intensity in tissues of various brain regions.
Acute brain slices of male Wistar rats aged 10 to 12 weeks (n = 2) of the cortex, cerebellum, hippocampus and brainstem were studied. We performed 6 tissue slices from each analyzed brain area. Quantify mitochondrial forms of NADH an experimental setup with a laser source M365FP1 was assembled to excite NADH fluorescence at a wavelength of 375 nm. A series of measurements of the basal fluorescence level, fluorescence intensity after adding FCCP and after adding NaCN for 3 minutes each was performed. The interval between frames was 2 sec. To control the viability of tissues and the degree of morphological changes the analyzed samples were fixed in 10% buffered formalin solution and subjected to histological examination after the experiment. According to the data obtained, the hippocampus has a higher rate of NAD(P)H formation than other parts of the brain. Statistically significant differences were received for the hippocampus with the cerebellum (p <= 0.01) and brainstem (p <= 0.001). Morphological examination shows moderate hypoxic changes, confirming the data obtained with fluorescence spectroscopy.
Thus, the research demonstrates a higher rate of NAD(P)H formation in the hippocampal tissue, which reflects the high intensity of mitochondrial metabolism and supposes a good ability to restore the initial level of bioenergetic parameters in cells. Vice versa, during hypoxia the hippocampus has a higher risk of disruption of the bioenergetics and as a consequence the occurrence of neurodegenerative changes.
This study was supported by the grant of the Russian Federation Government no. 075-15-2019-1877.

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Alexander Palalov (d.alexanderpalalov@yandex.ru)
Orel State University
Russia

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