CC BY
2The 30th International Conference on Advanced Laser Technologies B-P-2
ALT'23
FLIM reveals criteria for toxic liver damage in tissue slices
M.M. Karabut1, D.P. Krylov12, S.A. Rodimova1, I.D. Shchechkin12, D.S. Kozlov12, A.M. Mozherov1, 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
e-mail: [email protected]
Abuse with hepatotoxic agents is a major cause of acute liver failure. The search for new criteria indicating the acute or chronic pathological processes is still a challenging issue that requires the selection of effective tools and a research models. 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 [1]. Multiphoton microscopy with second hamonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) are modern label-free methods of optical biomedical imaging for assessing the metabolic state of hepatocytes, therefore reflecting the functional state of the liver tissue. Using precision-cut liver slices model (PCLSs) allows to preserve of the key intercellular interactions and cellular components of pathological changes caused by most widely used hepatotoxic agents - acetaminophen (APAP), carbon tetrachloride (CCl4), and ethanol.
Vibrating microtome 7000 cm3-2 was used to obtain liver slices using the protocol of Pearen et al. [2], and were placed in separate wells of a 12-well plate with a standard CO2-conditioned form of DMEM supplemented with 0.1 ^m of dexamethasone and 10% FBS, and incubated at 37 °C on orbital shaker (90 rpm). To induce APAP toxic damage, the liver slices were placed for 3 h in a 10 mM solution of APAP diluted in DMEM. To induce ethanol toxic damage, liver slices were placed for 3 h in 25 mM ethanol diluted in DMEM. For the CCl4 model the liver slices were incubated for 3 h with 2 mL standard culture medium, and a piece of filter paper soaked in 10 ^L of CC14 was attached to the lid of the 12-well plates. As a control, we used liver slices cultivated in DMEM without toxins. Monitoring were performed after 3 h, 24 h and 48 h of incubation. Using FLIM, we analyzed the metabolic state of hepatocytes based on fluorescence lifetime contributions of the free and bound forms of NADH and NADPH.
We have determined characteristic optical criteria for toxic liver damage, and these turn out to be specific for each toxic agent, reflecting the underlying pathological mechanisms of toxicity. Using multiphoton microscopy, we identified liver cells with both high and low NAD(P)H autofluorescence intensity, indicating cell damage. Interestingly, APAP-induced toxic damage was characterized by an increase in the contribution of the bound form of NAD(P)H, while exposure to ethanol and CCl4 showed a significant decrease in the contribution of the bound form of NAD(P)H, which reflects differences in the mechanisms of damage by each toxic agent. The results obtained are consistent with standard methods of molecular and morphological analysis. Thus, our approach, based on optical biomedical imaging, is effective for intravital monitoring of the state of liver tissue in the case of toxic damage or even in cases of acute liver injury.
The work was supported by the Grant from the Russian Science Foundation №22-25-00098.
[1] M. S. Roberts, Y. Dancik, T.W. Prow, et al., Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy. Eur J Pharm Biopharm, 77(3), 469-488, (2011).
[2] Pearen M. A., Lim H. K., Gratte F. D. et al. Murine precision-cut liver slices as an ex vivo model of liver biology. JoVE, (157), e60992, (2020).
