**3.1 RIPC in experimental models**

*Liver Disease and Surgery*

hospitalization [55].

*2.2.2 IPC in orthotopic liver transplantation*

transcripts, which are associated with hepatic damage [58].

**3. Remote ischemic preconditioning**

in young patients requiring a prolonged period of inflow occlusion and in the presence of steatosis [44, 47]. Other clinical trials also suggest that IPC (10 min ischemia/10 min reperfusion) provides both better intraoperative hemodynamic stability and anti-ischemic effects compared with intermittent clamping [48, 49]. Regarding the molecular basis of IPC (10 min ischemia/10 min reperfusion) in clinical PH, its beneficial effects have been shown to be linked to the down-regulation of potentially cytotoxic functions of PMNLs elicited by the Pringle Maneuver [50]. In addition, IPC (10 min ischemia/15 min reperfusion) increased the generation of adenosine and attenuated the degradation of purines in patients undergoing PH. Moreover, IPC appeared to attenuate apoptotic response of the liver remnant after resection [51]. Other clinical trial revealed that IPC (10 min ischemia/10 min reperfusion) stimulated the expression of the IL-1-RA, inducible nitric oxide synthase (iNOS), and Bcl-2 which decreased the inflammatory response and abrogated liver I/R injury [52]. Interestingly, since the ischemic period and pathophysiology are similar in partial hepatectomy and living donor liver transplantation, IPC could reduce damage and improve liver regeneration failure, a relevant risk factor in living donor liver transplantation [34]. Moreover, IPC could be implemented as an appropriate surgical strategy for the use of suboptimal livers, such as steatotic ones, in the clinical practice. Different results indicate that in patients with liver cirrhosis, IPC (5 min ischemia/5 min reperfusion) has been a suitable method to decrease liver I/R injury [53, 54]. Recently, the protective mechanism of IPC in patients with liver cirrhosis subjected to PH has been associated with changes in MAPK pathways [54]. In contrast, IPC applied for 15 min followed by 5 min reperfusion did not improve liver tolerance to I/R injury after PH in patients with liver cirrhosis [55]. In fact, RIPC did not induce changes in the postoperative levels of transaminases, bilirubin, and albumin nor reduced the morbidity and mortality rates and the duration of

Clinical trials in liver transplantation report different results on the effects of IPC

RIPC is a surgical technique by which preconditioning of one organ or vascular bed provides protection to distant organs or vascular beds during a sustained period of ischaemia (**Figure 1**). Few experimental and clinical studies, most of them from

the last years, have addressed the effects of RIPC in livers submitted to I/R.

against hepatic I/R injury. An IPC of 10 min ischemia/10 min reperfusion before liver transplantation reduced inflammatory response, improved ischemia tolerance, and decreased early graft function [56]. However, although the application of IPC (10 min ischemia/15 min reperfusion) reduced hepatocellular necrosis, it showed no clinical benefits [57]. In the largest prospective randomized trial of 10 min period IPC in liver transplantation from cadaveric donors, I/R injury was greater when IPC was applied [45], and it was called the "IPC paradox." This was in accordance with the results obtained in experimental model of liver transplantation from cadaveric donors indicating that brain death abrogates the benefits of IP on post-operative outcomes [41, 42]. In fact, a microarray analysis in a randomized trial of 10 min IPC in deceased donor liver transplantation identified alteration of the expression of different antioxidant, immunological, lipid biosynthesis, cell development and growth

**108**

### *3.1.1 RIPC in warm ischemia without liver resection*

When RIPC is applied in the hind limb, it reduced hepatic warm I/R injury of mice, rats, and rabbits. RIPC (5–10 min ischemia/5–10 min reperfusion) has been shown to improve hepatic oxygenation and microcirculation and to reduce hepatic acidosis and damage [59, 60]. RIPC (4 min ischemia/4 min reperfusion) induced eNOS activation, leading to NO production to preserve sinusoidal structure and blood flow [61]. In addition, RIPC (5 min ischemia/5 min reperfusion) regulated the expressions of iNOS and eNOS and the expressions of miR-34a, miR-122, and miR-27b injury related miRs in fatty livers, thus attenuating I/R injury [62, 63]. RIPC (10 min ischemia/10 min reperfusion) also induced the up-regulation of HO-1, induced autophagy, and then reduced the damaged mitochondria to inhibit apoptosis and eventually protect hepatic cells from I/R injury [64, 65]. Moreover, RIPC (5 min ischemia/5 min reperfusion) reduced neutrophil activation and adhesion and TNF-α [66]. Controversial results have been described in a rat model in which RIPC protocol included 3 cycles of 10 min ischemia interspersed with 10 min of reperfusion periods [67]. Regarding the hemodynamic and microcirculatory alterations, RIPC protocol had beneficial effect; however, the histopathological findings were paradox [67, 68]. In addition to RIPC in the hind limb, when RIPC (5 min ischemia/5 min reperfusion) is applied in kidney, it has also been shown to protect liver against I/R injury, improving blood flow, histology, and redox-state [69]. **Figure 2** shows some of the protective mechanisms of RIPC in the hepatic I/R injury.

#### *3.1.2 RIPC in liver resections*

A recent study in mice showed that RIPC (3 cycles of 5 min of ischemia each followed by 5 min of reperfusion) applied in the right femoral vascular bundle did not affect regeneration after 70%-PH [70]. However, of clinical interest, the same protocol of RIPC improved liver weight gain and hepatocyte mitoses after 90%-PH [70].

#### *3.1.3 RIPC in orthotopic liver transplantation*

In an experimental model of OLT, RIPC based on 4 cycles of 5 min of ischemia and 5 min of reperfusion was applied on the infrarenal aorta. The results suggested that RIPC might confer potent protection against the detrimental effects of I/R injury including apoptosis and inflammation [71]. In addition, authors suggest that HO-1 overexpression could play an orchestrating role in RIPC (5 min ischemia/5 min reperfusion)-mediated organ protection [71]. In addition, a recent study showed that the same protocol of RIPC also exhibits protective effects, as indicated by increased portal venous flow and microcirculation, as well as decreased AST and ALT levels and a reduced Suzuki score in a model of OLT [72]. Authors suggest that the RIPC inhibited the macrophage migration inhibitory factor (MIF), which resulted in the modulation of further downstream pro-survival mechanisms (iNOS, RISK-, SAFE-pathways), protecting graft injury [72].

#### **3.2 RIPC in clinical trials**

Only three studies dated in 2017 and 2018 have addressed the effects of RIPC in the clinical liver surgery.

#### *3.2.1 RIPC in liver resections*

In major HP, RIPC was shown to reduce liver I/R injury as indicated by a reduction in post-operative transaminases and increased ICG clearance [73]. To induce RIPC, a tourniquet was inflated to induce 10 min of ischemia and then deflated for 10 min to reperfuse the leg. This was repeated twice prior to commencing the operation. RIPC has potential to reduce liver injury following PH [73]. In addition, other clinical trial where RIPC was induced by three cycles of 5 min of ischemia of right upper limb followed by 5 min of reperfusion showed hepatic cytoprotective effects assessed by cholinesterase and bilirubin levels during liver resection [74]. Authors suggest that a shorter protocol of RIPC is safe and of equal effect, although the mechanisms of this effect must be investigated in future studies [74].

### *3.2.2 RIPC in orthotopic liver transplantation*

The first trial to investigate the feasibility of RIPC in liver transplant recipients was addressing by Robertson et al. [75]. The trial involved randomization of adult recipients undergoing deceased donor liver transplantation. To induce RIPC, a tourniquet was inflated for 5 min and then deflated for 5 min to reperfuse the leg. This was repeated twice and completed prior to the transplant procedure. Authors demonstrated that RIPC is feasible, acceptable to patients and safe in this group of patients but clinical benefits within the first 3 months post transplantation were not detected [75]. Authors suggest that 5 min cycles are insufficient to create localized ischemia in the limb [75].
