CC BY
16B-O-5
BIOMEDICAL PHOTONICS
Contrast-free FLIM diagnostics of the quality of pancreas islet of
Langerhans
A. Kashina1, P. Ermakova1, I. Kornilova1, A. Bogomolova1, A. Kashirina1, N. Naraliev1, D. Kuchin2,
E. Zagaynova u, V. Zagainov 14
1-Privolzhsky Research Medical University, Nizhny Novgorod, Russia 2- Nizhny Novgorod Regional Clinical Hospital named after N.A. Semashko».
3- Lobachevsky State University of Nizhny Novgorod, Russia 4-Nizhny Novgorod Regional Clinical Oncological Dispensary bardina-polina@mail.ru
Insulin-deficient conditions such as type 1 and type 2 diabetes mellitus, pancreatogenic diabetes are a major global public health problem. p cell stress and dysfunction precede progressive loss of P cell mass in both type 1 diabetes (T1D) and type 2 diabetes (T2D). p cell dysfunction is at least in part due to remodeled glucose metabolism as a component of a conserved pro-survival signaling program. In healthy p cells, as glucose concentrations increase, there is a proportionate increase in adenosine triphosphate (ATP) generation from oxidative phosphorylation (OxPhos) that, in turn, acts on membrane channels, electrically coupling oxidative phosphorylation to insulin secretion. It has been suggested that there is increased glycolysis in stressed P-cells, which is largely unrelated to oxidative phosphorylation in both T1DM and T2DM, consistent with such a high survival-inducing Warburg level characteristic of cancer cells.
Most studies of cell metabolism are based on methods such as PCR, immunocytochemistry, transcriptomic studies, which, on the one hand, do not give a complete picture of the islets in native tissue, and, on the other hand, are quite contradictory. Metabolic FLIM represents a powerful tool that may potentially provide a diagnostic tool for treatment aimed at restoring islet cell function in diabetes. To bring FLIM closer to clinical reality, new FLIM criteria are needed to identify islet quality and metabolism in various insulin-deficient conditions.
In this study based on FLIM and intracellular metabolite NAD(P)H was developed non-invasive and label-free method of the quality assessment of isolated islet cells and pancreatic islets in tissue. For fluorescence lifetimes (t1, t2) and fluorescence lifetimes contributions free and bound forms of NAD(P)H (a1, a2) analysis we used LSM 880 (Carl Zeiss, Germany), equipped with short-pulse femtosecond Ti:Sa laser Mai Tai HP with a pulse repetition rate of 80 MHz, duration of 140±20 fsec (Spectra-Physics, USA) and FLIM system for time resolved microscopy (Becker&Hickle GmbH, Germany).
Using the FLIM approach we checked FLIM opportunity to assess the islet cells quality in the pathological (T1D, T2D/chronic pancreatitis) and normal pancreas. We found that FLIM parameters (a1 and a1/a2) of islet cells in the pathological and normal pancreas were different. In particular, we demonstrated more glycolytic phenotype islet cells in pancreas with T1D and T2D/chronic pancreatitis. Metabolic FLIM imaging was also applied to assess the isolated islet cells metabolism and viability. The isolated islets were characterized by typical for NAD(P)H fluorescence lifetimes which indicates the viability and metabolically active status of the islets after their isolation.
Non-contrast FLIM diagnostics can be used both to obtain new FLIM criteria for the identification of islet cells quality and metabolism and to develop a rapid technique for islets viability analysis in order to help for islets transplantation in clinic.
This work has been financially supported by the Ministry of Health of the Russian Federation (state assignment № АААА-А20-120022590096-6).
ALT'22
