## 4 Discussion  
Our study of ALF after ELR demonstrated that all the animals survived in the experimental groups (_iHSC_, _aHSC_, and _total RNA_), while the _control_ group had a 50% mortality rate by Day 7. Therefore, the ELR model was adequate and appropriate for the assessment. In the experimental groups, cell lysis markers (ALT, AST, ALP, and total bilirubin) first elevated and then decreased strongly compared to the _control_ group, reaching normal ranges by Day 10-14. Moreover, the groups of _aHSC_ and _total RNA_ had a more rapid recovery rate than _iHSC_. The _control_ group reached complete recovery biochemical markers and liver mass only up to Day 20. For albumin levels, the _control_ group kept the same dynamics with no recovery achieved by Day 14 (Figure 4D). In the groups of _iHSC_, _aHSC_, and _total RNA_, the albumin levels decreased less than those in the _control_ group. Generally, no fatal outcomes in animals at _iHSC_, _aHSC_, and _total RNA_ (Figure 3A) together with a more efficient biochemical recovery (Figure 4A-E) reveal a successful liver regeneration. Figure 6: **Morphometrics for mono- and binuclear hepatocytes by 48 h after extended liver resection. ** Median values are provided with 25 th and 75 th percentiles: (**A**)—cell area, \(\upmu\)m\({}^{2}\); (**B**)—nucleus area, \(\upmu\)m\({}^{2}\); (**C**)—cytoplasm area, \(\upmu\)m\({}^{2}\); (**D**)—relative number of binuclear cells, %; (**E**)—total number of hepatocytes per 50,000 \(\upmu\)m\({}^{2}\). **—**the difference is significant compared with the control group; \(p<0.05\). Cytoplasm vacuolization on the histological slides of the liver can be a feature of cell autophagy [37], while a more pronounced and earlier vacuolization in the groups of _aHSC_ and _total RNA_ may be due to a more pronounced ability of apoptotic cells and their components to stimulate adaptive changes [38]. Interestingly, no histological signs for types of cellular death other than apoptosis and autophagy were observed, and no irreversible apoptosis was found at all. The mitotic activity of hepatocytes showed differences between the groups. The experimental groups exhibited an increased mitotic activity compared to controls (Figure 4F). We observed the highest rate of MI for the _aHSC_ group by 24 h, by 48 h in the _total RNA_ group, and by 72 h for the _iHSC_ group. The values of these MI peaks differed. For instance, the _total RNA_ group provided the highest MI, while the _aHSC_ group saw the most rapid increase. This finding may be explained by the immediate arrival of signal molecules from apoptotic cells to the liver [16]. Proliferation after ELR is, however, simultaneous with cell death, either reversible or irreversible. We found that most hepatocytes expressed elevated levels of caspase-9, a reversible apoptosis marker, in two days after ELR, i.e., at the same time with higher vacuolization. The data are consistent with reports about autophagy and reversible apoptosis involved in reparative regeneration [37; 38; 39]. Such an adaptation covers both metabolic and cellular mechanisms [40; 41]. Extended liver resection changes the proliferative potential of hepatocytes, as we already demonstrated previously [42]. The current study develops this idea, demonstrating iHSCs, aHSCs, and total RNA to be adequate tools for autophagy and reversible apoptosis induction together with cell proliferation in the liver, and that is consistent with the common concepts of liver regeneration [43; 44]. Interestingly, there was no increase in the absolute number of cells or binuclear cells per field of view in the experimental groups. Considering a higher rate of Ki-67 expression, we interpret this as, first of all, a hallmark for a strong apoptosis with mitigating cell number increase and, further, a strong role of hypertrophic regeneration and not only a proliferative one in response to HSC-derived stimuli. The total RNA of HSCs, which is shown as the most appropriate method in the current study, has not been widely studied before in liver regeneration.