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9B-O-6
BIOMEDICAL PHOTONICS
FLIM imaging of pathological liver during regeneration
I.D. Shchechkin12, S.A. Rodimova12, N.V. Bobrov13, , D.P. Krylov12, D.S. Kozlov12, V.V. Elagin1, M.M. Karabut1, A.M. Mozherov1, V.E. Zagainov13, E.V. Zagaynova12, D.S. Kuznetsova12
1 - Privolzhsky research medical university, 603005, Nizhny Novgorod, Minin and Pozharsky sq. 10/1
2 - Lobachevsky Nizhny Novgorod National Research State University, 603022, Nizhny Novgorod, Gagarina 23 3 - The Volga District Medical Centre of Federal Medical and Biological Agency, 603000, Nizhny Novgorod, Ilinskaya st. 14 e-mail: daria.s.kuznetsova@gmail.com
Surgical liver resection remains the most effective treatment of liver tumors [1]. However, in the presence of hepatic pathologies, the regenerative potential of the liver is significantly reduced [2]. Standard clinical methods do not allow predicting the function of the liver remnant. Modern label-free methods of multiphoton microscopy with fluorescence lifetime imaging microscopy (FLIM) and second harmonic generation (SHG) expand the possibilities of studying the structural and functional state of liver tissue at the cellular level [3]. Thus, the search for criteria for intraoperative express assessment of the regenerative potential of the liver in the presence of liver pathologies remains an urgent task.
A series of experiments were carried out on Wistar rats. We induced toxic liver fibrosis by CCl4 injections and steatosis by 60% high-fat diet, the regenerative process was induced by 70% partial hepatectomy. Using multiphoton microscopy, we analysed the structure of the liver tissue on 3rd and 7th day after surgery. Using FLIM, we determined the fluorescence lifetime contributions of the free and bound forms of NADH and NADPH. Morphological analysis and a standard biochemical blood test were performed as controls.
As a result, we revealed the features of the structural and functional state at different stages of liver regeneration with steatosis and fibrosis. In case of steatosis, we identified zones with a reduced NADH autofluorescence intensity, corresponding to lipid infiltration or fibrosis. We also showed a decrease in the contributions of the bound form of NADH and NADPH already in the early stages of steatosis. During regeneration with the presence of steatosis, there was no sharp increase in the contributions of the bound form of NADH and NADPH on the 3rd day after hepatectomy, which we previously found during normal regeneration. This may be due to mitochondrial dysfunction of hepatocytes. In case of fibrosis, we also identified zones with a reduced signal of NADH autofluorescence intensity, which corresponded to fibrosis. There was sharp a decrease in the contributions of the bound form of NADH and NADPH in the early stages of pathology, followed by an increase in these parameters in the later stages. Such changes are associated with mitochondrial dysfunction in the early stages and the progression of compensatory processes in the later stages of pathology.
The work was supported by the Grant from the Russian Science Foundation №19-15-00263 (metabolic imaging, analysis of FLIM data), and by the Grant from the Russian Science Foundation №22-25-00098 (morphological analy-
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