Liver regeneration has been studied for many decades, and the mechanisms underlying regeneration of normal liver following resection are well described. However, no less relevant is the study of mechanisms that disrupt the process of liver regeneration. First of all, a violation of liver regeneration can occur in the presence of concomitant hepatic pathology, which is a key factor reducing the liver’s regenerative potential. Understanding these mechanisms could enable the rational targeting of specific therapies to either reduce the factors inhibiting regeneration or to directly stimulate liver regeneration. This review describes the known mechanisms of normal liver regeneration and factors that reduce its regenerative potential, primarily at the level of hepatocyte metabolism, in the presence of concomitant hepatic pathology. We also briefly discuss promising strategies for stimulating liver regeneration and those concerning methods for assessing the regenerative potential of the liver, especially intraoperatively.

Although fluorescence lifetime imaging microscopy (FLIM) has been extensively applied to study cellular metabolism in the liver, there is neither an established approach to analyze the data, nor have appropriate protocols been developed to maintain the optical metabolic characteristics in the ex vivo liver tissue sample. Here, we show that a tri-exponential decay fitting model for the fluorescence signal from nicotinamide adenine dinucleotide (NAD(P)H) and the use of ex vivo samples allows the most appropriate processing of the FLIM data. Moreover, we determine the medium that maintains the initial metabolic state of hepatocytes (liver cells), most effectively. Our results should be particularly relevant for the interrogation of liver samples, not only in laboratory research, but also in clinical settings in the future.

Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.

To reduce the risk of post-hepatectomy liver failure in patients with hepatic pathologies, it is necessary to develop an approach to express the intraoperative assessment of the liver’s regenerative potential. Traditional clinical methods do not enable the prediction of the function of the liver remnant. Modern label-free bioimaging, using multiphoton microscopy in combination with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), can both expand the possibilities for diagnosing liver pathologies and for assessing the regenerative potential of the liver. Using multiphoton and SHG microscopy, we assessed the structural state of liver tissue at different stages of induced steatosis and fibrosis before and after 70% partial hepatectomy in rats. Using FLIM, we also performed a detailed analysis of the metabolic state of the hepatocytes. We were able to determine criteria that can reveal a lack of regenerative potential in violated liver, such as the presence of zones with reduced NAD(P)H autofluorescence signals. Furthermore, for a liver with pathology, there was an absence of the jump in the fluorescence lifetime contributions of the bound form of NADH and NADPH the 3rd day after hepatectomy that is characteristic of normal liver regeneration. Such results are associated with decreased intensity of oxidative phosphorylation and of biosynthetic processes in pathological liver, which is the reason for the impaired liver recovery. This modern approach offers an effective tool that can be successfully translated into the clinic for express, intraoperative assessment of the regenerative potential of the pathological liver of a patient.

We studied the structural and functional state of liver tissue during the regeneration process using multiphoton microscopy in combination with fluorescence lifetime imaging (FLIM) and second-harmonic generation microscopy, supplementing our findings with a mass spectrometry analysis. The regeneration process was induced by 30% or 70% partial hepatectomy (PH) of rats. The multiphoton microscopy and FLIM were used to evaluate the structure of the liver tissue and to assess the metabolic state of the hepatocytes, respectively. As a result, there was a statistically significant increase in the contribution of the bound forms of NAD(P)H during liver regeneration, indicating a metabolic shift towards oxidative phosphorylation and an increase in the intensity of biosynthesis processes in hepatocytes. Time-of-flight secondary ion mass spectrometry was used to study changes in the lipid and amino acid composition of liver tissue during regeneration. The approach used in the present work is effective for analyzing the dynamics of changes in the structural and functional state of liver tissue during the regeneration process. These results can be useful for the further development of criteria for the assessment of the regenerative potential of the liver and for predicting the dynamics of its recovery after the resection.

The great challenge in liver surgery is of pathological changes to the liver that may lead to a reduction of regenerative potential in diseased liver tissue. The main methods to assess both liver quality and the pathological changes include ultrasound, positron emission tomography (PET), magnetic resonance imaging (MRI), computed tomography (CT), biochemical blood analysis, morphological and immunohistochemical analysis of biopsy specimens, and clearance tests using indocyanine, as well as various radioactive isotopes.^{1 – 3} Ultrasound, PET, MRI, and CT are quite informative, but they only provide information about the liver tissue at the level of the whole organ, not allowing resolution at a cellular level. Biochemical blood analysis is among the simplest methods for studying liver function.⁴ Most of the indices are not organ-specific and may not reflect the true functional state of the liver. Moreover, these figures in most cases do not change in focal liver diseases since there are no pathological disorders in the healthy liver parenchyma. The morphological and immunohistochemical analysis of biopsy specimens requires additional staining with both standard histological dyes and specific, expensive antibodies to detect particular markers.⁵ There is also the likelihood of nonspecific background staining, which may give false results. Clearance tests are based on an assessment of the rate of removal of various substances from biological fluids or body tissues, and can characterize the absorption and excretory function of the liver.¹ However, they do not provide information about the processes occurring in the liver tissues at the level of individual hepatocytes. Thus, these conventional techniques are insufficient to describe precisely the internal structure, heterogeneous cell populations, and the dynamics of the biological processes of the diseased liver during surgery. There is a need for a rapid and safe method for the successful diagnosis of liver disease in order to plan surgery and to avoid postoperative liver failure.