Effects of Ischemic Preconditioning on Regenerative Capacity of Hepatocyte in the Ischemically Damaged Rat Livers

doi:10.1016/j.jss.2004.11.028

Journal of Surgical Research

Volume 125, Issue 1, 1 May 2005, Pages 42-48

https://doi.org/10.1016/j.jss.2004.11.028 Get rights and content

Background

Liver regeneration after partial hepatectomy is regulated by several factors that activate or inhibit hepatocyte proliferation. A short period of ischemia-reperfusion (IR), called ischemic preconditioning (IPC), protects the liver against subsequent sustained ischemic insults. The present study investigated the effects of IPC on liver regeneration after partial hepatectomy under IR in rats.

Materials and methods

Male Wistar rats were subjected to 45 min of total hepatic ischemia, and 70% hepatectomy was performed just before reperfusion. Animals were pre-treated with either IPC (10/15 min) (IPC + PHx group) or not (ischemia + PHx). The survival rate, serum transaminases, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 levels, hepatocyte proliferation and histological change of the remnant liver were measured in both groups and compared with non-ischemic controls subjected to 70% hepatectomy alone (PHx group).

Results

The survival rate was significantly better in the IPC + PHx group than in the ischemia + PHx group. Furthermore, IPC reduced liver injury determined by liver histology and serum transaminases. There was an early rise in serum TNF-α and IL-6 levels in the ischemia + PHx group. Compared with non-ischemic controls, IPC significantly decreased TNF-α, but not IL-6 during the late (24 and 48 h) phases of reperfusion. Rats subjected to 70% hepatectomy and 45 min of hepatic ischemia showed significantly reduced hepatocyte proliferation (mitotic index, proliferating cell nuclear antigen, and relative liver weight) when compared with animals subjected to hepatectomy alone. However, hepatocyte proliferation was markedly increased in rats pretreatment with IPC when compared with ischemic controls.

Conclusion

These results suggest that ischemic pre-conditioning ameliorates the hepatic injury associated with ischemia-reperfusion and has a stimulatory effect on liver cell regeneration that may make it valuable as a hepatoprotective modality. Il-6 appears to be key mediator in promoting regeneration after combined ischemia and hepatic resection.

Introduction

Hepatic resection is considered today to be the most important and efficient curative treatment for hepatic malignancies. Clinically, hepatic resection is usually performed under the total or partial exclusion of the blood inflow to the liver to control bleeding during parenchymal dissection. Ischemia-reperfusion (IR) injury is inevitable in this setting of liver surgery and it remains a serious problem. It is known that liver regeneration is disturbed after hepatectomy under IR in rats [1]. Many cytokines are up-regulated during acute liver injury, including tumor necrosis factor-α (TNF-α), interleukins 1 and 6 (IL-1, IL-6), hepatocyte growth factor (HGF), macrophage inflammatory protein-2 (MIP-2), stem cell factor (SCF), and many others [2, 3, 4]. Although many of these molecules contribute to hepatic inflammation via direct effects, effects on the vascular endothelium, and/or neutrophil recruitment and activation, they have also been shown to be involved in hepatic repair and regeneration [5, 6, 7]. Studies in the partial hepatectomy model have shown that initiation of the regenerative response depends on early activation of TNF-α and IL-6 responsive transcription factors [8, 9, 10]. Different strategies have been reported to confer a state of protection against IR injury. Ischemic preconditioning (IPC) is a process in which a transient period of ischemia before an extended ischemic insult leads to hepatic protection. After preconditioning, liver demonstrate enhanced hepatic function, improved hepatic tissue flows, and reduced cellular disturbances [11, 12]. Although the beneficial effects of IPC on the liver include improvement of survival, reduction of the extent of liver necrosis, inhibition of apoptosis, and stabilization of ATP after IR have been described, the effects of IPC on hepatic regeneration are not well established. In the present study, we hypothesized that IPC would have unfavorable effects on liver regeneration after hepatectomy under inflow occlusion. To achieve our hypothesis, we examined the effects of IPC on survival rate, injury, and regeneration of the liver after partial hepatectomy under IR. In addition, the concentration of TNF-α and IL-6 in the serum were measured.

Section snippets

Materials and methods

The experimental protocols were conducted with the approval of the Animal Research Committee at Gazi University, Ankara. All animals were maintained in accordance with the recommendations of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Results

In non-ischemic controls (PHx group), all rats survived for 7 days after partial hepatectomy. Among the ischemia + PHx group, four rats died of acute liver failure within 24 h and one died on the third day after the surgical procedure. IPC significantly improved the 7-day survival rate to 90% when compared with that in the ischemia + PHx group (P = 0.04). Animal survival rates are shown in the Table 1.

Serum AST and ALT levels of all groups rapidly increased after partial hepatectomy, reaching

Discussion

Major tissue loss of the liver associated with concomitant periods of ischemia is common in trauma, transplantation, and hepatic surgery. For example, transient occlusion of the portal vein and hepatic artery (Pringle maneuver) is routinely performed during liver resection to minimize intra-operative blood loss. The effects of warm ischemia on liver regeneration have been studied in several models combining both ischemia and major hepatectomy. The IR procedure has been shown to significantly

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Cited by (44)

Effects of ischemic preconditioning in a pig model of large-for-size liver transplantation
2015, Clinics
In most cases of pediatric liver transplantation, the clinical scenario of large-for-size transplants can lead to hepatic dysfunction and a decreased blood supply to the liver graft. The objective of the present experimental investigation was to evaluate the effects of ischemic preconditioning on this clinical entity.
Eighteen pigs were divided into three groups and underwent liver transplantation: a control group, in which the weights of the donors were similar to those of the recipients, a large-for-size group, and a large-for-size + ischemic preconditioning group. Blood samples were collected from the recipients to evaluate the pH and the sodium, potassium, aspartate aminotransferase and alanine aminotransferase levels. In addition, hepatic tissue was sampled from the recipients for histological evaluation, immunohistochemical analyses to detect hepatocyte apoptosis and proliferation and molecular analyses to evaluate the gene expression of Bax (pro-apoptotic), Bcl-XL (anti-apoptotic), c-Fos and c-Jun (immediate-early genes), ischemia-reperfusion-related inflammatory cytokines (IL-1, TNF-alpha and IL-6, which is also a stimulator of hepatocyte regeneration), intracellular adhesion molecule, endothelial nitric oxide synthase (a mediator of the protective effect of ischemic preconditioning) and TGF-beta (a pro-fibrogenic cytokine).
All animals developed acidosis. At 1 hour and 3 hours after reperfusion, the animals in the large-for-size and large-for-size + ischemic preconditioning groups had decreased serum levels of Na and increased serum levels of K and aspartate aminotransferase compared with the control group. The molecular analysis revealed higher expression of the Bax, TNF-alpha, I-CAM and TGF-beta genes in the large-for-size group compared with the control and large-for-size + ischemic preconditioning groups. Ischemic preconditioning was responsible for an increase in c-Fos, IL-1, IL-6 and e-NOS gene expression.
Ischemia-reperfusion injury in this model of large-for-size liver transplantation could be partially attenuated by ischemic preconditioning.
Effect of ischemic preconditioning and postconditioning on liver regeneration in prepubertal rats
2014, Transplantation Proceedings
Citation Excerpt :
The small number of studies [27,28] that link ischemic postconditioning to liver regeneration under the conditions used in this study make comparisons difficult. In our study, we used well-established parameters [8,22,23] in the evaluation of liver regeneration and observed greater regeneration in ischemic postconditioning than preconditioning. Yang et al [27] related regeneration after ischemic postconditioning with activation of TNF-α/IL-6/STAT-3 and the cyclinD1-Cdk4 complex, which enhances the proliferation of hepatocytes.
Liver regeneration has great importance for transplantation, especially in children; however, it has not been studied sufficiently in development animals. Ischemia-reperfusion injury is a problem, and strategies such as ischemic preconditioning and postconditioning are not well defined regarding regeneration.
This study sought to evaluate liver regeneration with modulation by ischemic preconditioning and postconditioning in prepubertal rats subjected to total ischemia and reperfusion.
Thirty-five 5-week-old female Wistar rats were divided into groups of 7 animals each: control group (SHAM), 70% hepatectomy (HEP), total ischemia 30 minutes before hepatectomy (IR), ischemic preconditioning 10/10 minutes before ischemia (PRE), and two 30/30-second ischemic postconditioning cycles after ischemia and hepatectomy (POS). All animals were subjected to 24-hour reperfusion. Aspartate aminotransferase and alanine aminotransferase activity were measured to evaluate liver damage, and histological analysis, proliferating cell nuclear antigen (PCNA) and regenerated mass liver were used to evaluate liver regeneration. Statistical analyses were performed using ANOVA and Kruskal-Wallis test.
Alanine aminotransferase and aspartate aminotransferase levels were significantly lower in conditioned groups than in the IR group. Regarding mitotic index, IR > control group and HEP (P < .05), PRE and POS were not significantly different from IR, and POS > HEP (P < .05). PCNA analysis showed that IR > HEP (P < .01), PRE < IR (P < .01), and no significant differences were observed between POS and IR groups. No significant differences in regenerated mass liver were observed between conditioned groups and HEP.
Ischemic postconditioning prevented ischemic injury, promoted greater liver regeneration, and should be further investigated as an alternative better than ischemic preconditioning.
Remote ischemic perconditioning protects the liver from ischemiaereperfusion injury
2013, Journal of Surgical Research
Citation Excerpt :
Likewise, significantly attenuated TNF-α levels were observed after 6 h of reperfusion; nevertheless, in the 24-h reperfusion group, the serum cytokine levels were remarkably reduced without any noticeable differences between the experimental groups. Bedirli et al. [27] reported similar serum TNF-α expression characteristics in a rat model of hepatectomy and ischemic preconditioning in 2005. Besides proinflammatory cytokine expression, reperfusion-associated sinusoidal endothelial injury, imbalance between vasoconstrictive and vasodilatative effects, as well as excessive oxidative stress, and consequential free radical production are also represented as key factors in the development of microcirculatory impairment and no-reflow phenomenon [28,29].
Ischemia–reperfusion (IR)–induced injury is a frequent sequel of major liver resections. IR injury after prolonged surgical interventions could be the source of increased risk of postoperative morbidity and mortality. Hepatoprotective effects of this new feasible method called remote ischemic perconditioning (RIPER) were investigated in our rat model of IR injury.
Male Wistar rats underwent ischemia for 60 min on two-thirds of their livers, followed by 1, 6, and 24 h of reperfusion (n = 72, 8 per group). During liver ischemia, but before reperfusion, rats in the treated groups received four cycles of brief infrarenal aortic clamping as perconditioning. Liver microcirculation was monitored by laser Doppler flowmeter parallel with mean arterial pressure measurements. Liver tissue injury and redox homeostasis were investigated. Furthermore, serum tumor necrosis factor alpha (TNF-α) levels were measured.
In the RIPER group, compared with the IR group, serum transaminase levels were significantly lower after each reperfusion period (alanine aminotransferase: 1 h, P < 0.001; 6 h, P < 0.05; 24 h, P < 0.01 and aspartate aminotransferase: 1 h, P < 0.001; 6 h, P < 0.05; 24 h, P < 0.05). Reperfusion microcirculatory parameters significantly improved in the perconditioned group compared with those in the IR group (reperfusion area: P = 0.005; maximal plateau: P = 0.0002). Regarding TNF-α levels, significant differences were detected between the two IR injured groups (RIPER versus IR: 1 h, 34.3 ± 12.8 pg/mL versus 205.7 ± 60.9 pg/mL, P < 0.001; 6 h, 60.6 ± 11.7 pg/mL versus 110.4 ± 21.6 pg/mL, P < 0.05). Results of the histologic assessment and redox state measurements also showed favorable changes.
Our team firstly reported the protective effects of RIPER on liver morphology, redox homeostasis, and microcirculation and proposed the changes of TNF-α expression.
Remote ischemic preconditioning promotes early liver cell proliferation in a rat model of small-for-size liver transplantation
2013, Journal of Surgical Research
The size of the liver donor graft is a major concern in living donor liver transplantation. Rapid regeneration is essential for the survival of these grafts. The purpose of this study was to investigate the effect of remote ischemic preconditioning (RIPC) on liver regeneration in a rat small-for-size liver transplantation model.
We established rat models of small-for-size liver transplantation (30%) in the presence or absence (control) of remote ischemic preconditioning. We observed liver mass regeneration, serum alanine aminotransferase, hepatic pathologic alterations, flow cytometry, and Ki-67 antigen immunohistochemistry. In addition, using Western blotting and reverse-transcriptase–polymerase chain reaction, we assessed the activation of cell cycle progression as well as tumor necrosis factor-α and interleukin-6 expression.
Compared with the control group, serum alanine aminotransferase activity was significantly lower and histopathology changes were significantly attenuated in the RIPC group. Remote ischemic preconditioning induced a high level of interleukin-6 mRNA in small grafts, but suppressed the expression of tumor necrosis factor-α. The proliferation index, indicated by the S-phase and G2/M-phase ratio [(S+G2/M)/(G0/G1+S+G2/M)], was signiﬁcantly increased in the RIPC group at 24 h (58.25% ± 0.506% versus 53.405% ± 1.25%; P = .007). Meanwhile, cell cycle progression and regeneration (Ki-67) were initiated early in liver grafts treated with RIPC.
These results suggest that RIPC can protect liver cells against ischemia reperfusion injury in the small grafts and enhance liver regeneration. Interleukin-6 may be a critical mediator in the stimulatory effect on liver cell regeneration, which may make RIPC valuable as a hepatoprotective modality.
Ischemic preconditioning enhances hepatocyte proliferation in the early phase after ischemia under hemi-hepatectomy in rats
2012, Hepatobiliary and Pancreatic Diseases International
Ischemia/reperfusion (I/R) injury is an important barrier to liver surgery and transplantation because it impairs remnant liver/reduced-size-graft regeneration. Ischemic preconditioning (IPC), as an effective measure to overcome I/R injury, has been shown to enhance the regenerative capacity of hepatocytes. However, investigations have always focused on regeneration in the late phase after reperfusion. This study aimed to investigate whether IPC enhances hepatocyte proliferation in the early phase after reperfusion and possible underlying mechanisms.
A total of 90 rats were divided into three groups: hemi-hepatectomy alone (PHx group), 60 minutes of ischemia plus hemi-hepatectomy (I/R group), and a cycle of 10 minutes of alternating I/R prior to 60 minutes of ischemia plus hemi-hepatectomy (IPC group). Each group was divided into five subgroups sacrificed after 0.5, 2, 6, 12 or 24 hours (n=6/subgroup). Subsequently, serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-alpha (TNF-a), and interleukin-6 (IL-6) were measured; caspase-3 and proliferating cell nuclear antigen (PCNA) proteins were also determined by Western blotting. Furthermore, PCNA was detected by immunohistochemistry to identify the expression site.
Serum ALT and AST levels after 2–24 hours of reperfusion in the PHx and IPC groups were remarkably decreased compared to the I/R group, and the serum TNF-α was relatively lower. A significant increase of serum IL-6 levels was found in the PHx and IPC groups compared with the I/R group at each time point. Furthermore, PCNA expression was remarkably increased in the IPC group after 6–12 hours of reperfusion, and in the earlier 0.5 and 6 hours time points after reperfusion have shown the massive PCNA-positive hepatocytes. At the same time, the expression of liver p-JNK was higher in the IPC group in the early phase after reperfusion than that of the I/R group and its expression was consistent with the PCNA.
IPC can initiate hepatocyte proliferation in the early phase after ischemia under hemi-hepatectomy, and may be associated with p-JNK expression and triggered by TNF-α/IL-6 signals.
Activation of p38 MAPK participates in brain ischemic tolerance induced by limb ischemic preconditioning by up-regulating HSP 70
2010, Experimental Neurology
This study investigates whether activation of p38 MAPK by the up-regulation of HSP 70 participates in the induction of brain ischemic tolerance by limb ischemic preconditioning (LIP). Western blot and immunohistochemical assays indicated that p38 MAPK activation occurred earlier than HSP 70 induction in the CA1 region of the hippocampus after LIP. P-p38 MAPK expression was up-regulated at 6 h and reached its peak 12 h after LIP, while HSP 70 expression was not significantly increased until 1 day and peaked 2 days after LIP. Neuropathological evaluation by thionin staining showed that quercetin (4 ml/kg, 50 mg/kg, intraperitoneal injection), an inhibitor of HSP 70, blocked the protective effect of LIP against delayed neuronal death that is normally induced by lethal brain ischemic insult, indicating that HSP 70 participates in the induction of brain ischemic tolerance by LIP. Furthermore, SB 203580, an inhibitor of HSP 70, inhibited HSP 70 activation in the CA1 region of the hippocampus induced by LIP either with or without the presence of subsequent brain ischemic insult. Based on the above results, it can be concluded that activation of p38 MAPK participates in the brain ischemic tolerance induced by LIP at least partly by the up-regulation of HSP 70 expression.

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GastrointestinalEffects of Ischemic Preconditioning on Regenerative Capacity of Hepatocyte in the Ischemically Damaged Rat Livers

Background

Materials and methods

Results

Conclusion

Introduction

Section snippets

Materials and methods

Results

Discussion

J. Surg. Res.

J. Surg. Res.

Am. J. Transplant.

Surgery

Surgery

Transplant. Proc.

Hepatology

Hepatology

Gastroenterology

J. Biol. Chem.

J. Biol. Chem.

Ischemia impairs liver regeneration following major tissue loss in the rodent

Hepatology

Neutrophils, cytokines, and adhesion molecules in hepatic ischemia and reperfusion injury

J. Am. Coll. Surg.

Beneficial effect of deletion variant of hepatocyte growth factor for impaired hepatic regeneration in the ischemically damaged liver

World J. Surg.

Gastrointestinal
Effects of Ischemic Preconditioning on Regenerative Capacity of Hepatocyte in the Ischemically Damaged Rat Livers