Yoritaka Nakano

Role of platelets on liver regeneration after 90% hepatectomy in mice

BACKGROUND/AIMS Mortality after 90% partial hepatectomy in mice was associated with severe acute liver failure. Recently, we revealed that platelets have a strong promotional effect on hepatic regeneration. In the present study, we investigated the effect of thrombocytosis on liver regeneration after 90% hepatectomy in mice. METHODS For thrombocytosis induction PEG-rHuMGDF was injected 5 days before operation. Hepatectomy, sparing only the caudate lobe, was performed in normal and thrombocytotic BALB/c mice. Survival rate, platelet number, liver weight/body weight ratio, proliferating cell nuclear antigen, serum parameters, signal transduction and overexpressed genes were examined. RESULTS Platelet number was significantly higher in thrombocytotic group. All mice in normal group died within 30 h after hepatectomy. Survival rate in thrombocytotic group was 6/11 at 30 h and 3/11 one week after hepatectomy. Activation of Akt and STAT3 signaling pathways in thrombocytotic group was observed earlier and recognized to be stronger compared to normal group. Cell cycle, signaling pathways, metabolism and transport genes were significantly overexpressed in thrombocytotic group up to 24h after hepatectomy. CONCLUSIONS Under the thrombocytotic condition, liver regeneration occurred even in 90% hepatectomized mice. Platelets contribute to cell cycle progression and metabolic pathways in addition to preventing acute liver failure.

Platelets contribute to the reduction of liver fibrosis in mice

Background and Aim:  Several recent studies have reported that liver cirrhosis (LC) can be ameliorated, but few adequate strategies are available against liver fibrosis. Although LC clinically shows thrombocytopenia and hypersplenism, the correlation with liver fibrosis and platelets remains unclear. The aim of the present study was to investigate the effect of platelets on liver fibrosis in mouse models.

Platelet Administration Via the Portal Vein Promotes Liver Regeneration in Rats After 70% Hepatectomy

Objective:This study examines the applicability of platelet infusion therapy for liver regeneration in vivo. Background:We recently reported that platelets accumulate in the liver immediately after extended hepatectomy and promote residual liver regeneration. Liver regeneration depends on the number of accumulated platelets in the sinusoids. Methods:Male Sprague-Dawley rats underwent 70% hepatectomy and were then assigned to groups that were infused with 1 mL of either platelet-rich plasma (PRP; 1 × 109 platelets/mL) in normal saline (NS) or NS via the portal vein. We then analyzed liver regeneration and the signaling pathways that are related to liver regeneration and function. The dynamics of platelets infused via the portal vein were visualized before and after hepatectomy. Results:The liver/body weight ratio after 70% hepatectomy was significantly higher and the Ki-67 labeling index was higher in the PRP, than in the NS group. The Akt pathway was activated earlier in the PRP, than in the NS group with concurrent ERK1/2 pathway activation, but this was prolonged in the PRP group. Many more platelets infused via the portal vein accumulated in the sinusoid after 70% hepatectomy, and serum liver function tests and histological findings revealed that portal infusion did not cause liver damage. Conclusions:Platelets infused via the portal vein promoted liver regeneration after 70% hepatectomy in rats without liver damage. These findings indicate that PRP administration could be a useful part of liver regeneration therapy.

Single Administration of Thrombopoietin Prevents Progression of Liver Fibrosis and Promotes Liver Regeneration After Partial Hepatectomy in Cirrhotic Rats

Objective:To evaluate the effect of thrombopoietin on liver regeneration after hepatectomy and antifibrosis under conditions of liver cirrhosis in rats. Summary Background Data:We revealed that platelets induced by thrombopoietin administration promote liver regeneration after hepatectomy in the normal liver. Methods:Seventy percent hepatectomy was carried out in rats, which were subsequently divided into 4 groups: (1) normal group without any treatment, (2) liver cirrhosis (LC) group, (3) combined thrombopoietin and liver cirrhosis (LC+TPO) group, and (4) combined thrombopoietin, antiplatelet serum and liver cirrhosis (LC+TPO+APS) group. Growth kinetics in the liver regeneration and growth factors were analyzed. Liver fibrotic area and activation of hepatic stellate cells were also investigated. Results:In LC group, liver regeneration was significantly delayed compared with normal group 24 hours after hepatectomy. On the other hand, liver regeneration of LC+TPO group increased significantly compared with LC group, to a level that was the same as that recorded in normal group. In LC group, liver fibrotic area before hepatectomy was significantly higher compared with the normal group. Liver fibrosis of LC+TPO group was significantly reduced compared with LC group. The antifibrotic and liver regeneration promoting effects of LC+TPO group were inhibited by antiplatelet serum in LC+TPO+APS group. Conclusion:The administration of thrombopoietin reduces liver fibrosis and stimulates regeneration after hepatectomy through increment and accumulation of platelets in the cirrhotic liver. This could be a potentially useful treatment for liver cirrhosis.

Platelet dynamics in the early phase of postischemic liver in vivo.

BACKGROUND In liver surgery, ischemia/reperfusion injury occasionally leads to liver failure by activating Kupffer cells (KCs) and leukocytes. However, few reports have demonstrated a relationship between KCs and platelets in vivo. This study investigated the relationship between these cells using intravital microscopy. MATERIALS AND METHODS Male Wistar rats were divided into two groups: (1) KC+ group, receiving 1 mL saline; and (2) KC- group, intravenously injected with liposome-encapsulated dichloromethylene disphosphonate for elimination of KCs. At 48 h after administration, 20 min of total normothermic hepatic ischemia was induced. Rhodamine-6G-labeled platelets and sinusoidal alterations were monitored using intravital microscopy up to 120 min after reperfusion. P-selectin, accumulated leukocytes and morphological damage, and alanine aminotransferase were evaluated. RESULTS In the KC+ group, numbers of adherent platelets increased significantly within 30 min after reperfusion. Endothelial cells of sinusoids in which KCs were mainly located were destroyed and the sinusoids were significantly constricted after reperfusion. Conversely, in the KC- group, adherent platelets in sinusoids were suppressed, and sinusoidal perfusion, endothelial cell damage and serum alanine aminotransferase levels were significantly improved. P-selectin on sinusoidal endothelial cells was not observed up to 120 min after reperfusion in either group. CONCLUSIONS Adherent platelets appear to reflect activation of KCs and lead to leukocyte accumulation, resulting in sinusoidal perfusion disturbance and liver failure. Evaluation of adherent platelets in the microcirculation offers an important marker of hepatic injury.

Platelets prevent acute liver damage after extended hepatectomy in pigs

Background/PurposePlatelets develop tissue repair and promote liver regeneration. We investigated whether platelets prevented acute liver damage after extended hepatectomy in pigs.MethodsThrombocytosis was induced by the following two methods; afterwards 80% hepatectomy was performed in pigs. In the first method, the pigs received administration of thrombopoietin [TPO (+) group], and they were compared with a control group [TPO (−) group]. In the second method, the pigs received a splenectomy [Sp (+) group], and theywere compared with another control group [Sp (−) group]. Platelet counts, biochemical examination of blood, and histopathological findings of the residual liver were examined.ResultsSerum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and total bilirubin (T-Bil) levels were significantly decreased in the thrombocytotic groups compared with the control groups in the early period after hepatectomy. In the histopathological findings, hemorrhagic necrosis with a bile plug was observed in the control groups, but this phenomenon was not observed in the thrombocytotic groups. On transmission electron microscopy, the sinusoidal endothelial lining was destroyed and detached into the sinusoidal space with enlargement of Disse’s spaces in the thrombocytotic groups, but these findings were not observed in the control groups.ConclusionAn increased number of platelets prevents acute liver damage after extended hepatectomy.

Platelet adhesion in the sinusoid caused hepatic injury by neutrophils after hepatic ischemia reperfusion

Liver ischemia-reperfusion (I/R) injury is one of the most serious complications of hepatic surgery. In I/R, activated Kupffer cells cause platelet adhesion to sinusoidal endothelium as well as neutrophils and cause liver dysfunction. The aim of this study was to evaluate platelet dynamics in the hepatic microcirculation after I/R by intravital microscopy (IVM) and to clarify the relationship between platelet adhesion and neutrophil activation. Male Sprague-Dawley (SD) rats were divided into two groups: the control (administration of saline) group and the sivelestat group in which neutrophil activation was suppressed by sivelestat before I/R. The number of adherent platelets in sinusoid was observed up to 120 minutes after I/R by IVM. Samples of liver tissue and blood were taken for examination of histological findings, liver enzymes and inflammatory cytokines. The number of adherent platelets was significantly increased after I/R in both groups. Compared with the control group, the number of adherent platelets significantly decreased after hepatic I/R in the sivelestat group. Moreover, sivelestat improved changes of histological findings and elevation of liver enzymes. However, there was no significant difference in inflammatory cytokines of TNF-α, IL-1β or IL-6. Platelet adhesion in the sinusoid is associated with liver dysfunction after I/R as well as neutrophils. Activated neutrophils induce platelet adhesion in the sinusoid of the liver.

Interaction between Kupffer cells and platelets in the early period of hepatic ischemia–reperfusion injury—An in vivo study

BACKGROUND Hepatic ischemia-reperfusion (I/R) leads to activation of Kupffer cells (KCs). The activated KCs cause platelet and leukocyte adhesion to the sinusoidal endothelium. Previously, we reported that platelet-endothelium interactions occur earlier than leukocyte responses. The aim of this study was to evaluate the interaction between platelets and KCs in the hepatic microcirculation after I/R. MATERIALS AND METHODS Sprague-Dawley rats were divided into three groups: the no-ischemia group (control group; n = 6); the 20-min ischemia group (I/R group; n = 6); and the 20-min ischemia + anti-rat platelet serum group (APS group; n = 6). KCs were labeled using the liposome entrapment method. The number of adherent platelets was observed for up to 120 min after reperfusion by intravital microscopy. To investigate the effects of platelets on I/R injury, rats were injected intravenously with rabbit APS for platelet depletion. RESULTS In the I/R group, the number of adherent platelets increased significantly after I/R. More than 50% of the adherent platelets adhered to KCs. Electron microscopy indicated that the platelets attached to the KCs after hepatic ischemia. The histologic findings indicated liver damage and apoptosis of hepatocytes in zone 1. In the I/R group, but not in the control and APS groups, serum ALT increased immediately after reperfusion. CONCLUSIONS We succeeded in visualizing the dynamics of both KCs and platelets in the hepatic sinusoids. Liver ischemia induced the adhesion of platelets to KCs in the early period, which could play a key role in reperfusion injury of the liver.

Prevention of leukocyte activation by the neutrophil elastase inhibitor, sivelestat, in the hepatic microcirculation after ischemia-reperfusion.

BACKGROUND Liver ischemia-reperfusion (I/R) injury is one of the most serious complications of hepatic surgery. However, no effective treatment is yet clinically available. Although neutrophil elastase inhibitor (NEI) has been used clinically in acute lung injury, the effect of NEI on leukocyte dynamics in the liver microcirculation after hepatic I/R remained unclear. The purpose of this study was to use intravital microscopy (IVM) to evaluate the effect of NEI on leukocyte dynamics in the liver microcirculation after hepatic I/R. METHODS Hepatic ischemia was induced in male Sprague-Dawley (SD) rats. Sivelestat, a specific NEI, or normal saline (NS) was given as a continuous intravenous infusion before ischemia. The number of adherent leukocytes and the disturbances of sinusoidal perfusion in hepatic microcirculation were observed up to 120 min after reperfusion. Samples of liver tissue and blood were taken for histological examination and measurement of liver enzymes and tissue malondialdehyde (MDA). RESULTS Compared with NS, sivelestat significantly decreased the number of adherent leukocytes and prevented perfusion disturbance. In addition, sivelestat obviously improved liver injury as assessed by histological findings and liver enzymes, and prevented the increase of MDA. CONCLUSIONS Administration of sivelestat before ischemia effectively suppressed the activation of leukocytes and lipid peroxide, and it consequently prevented hepatic I/R injury.

Autonomic Regulation of Liver Regeneration After Partial Hepatectomy in Mice

BACKGROUND/AIMS The autonomic vagus nerve is thought to play an essential role in liver regeneration since hepatic vagotomy delays hepatic DNA synthesis. However, how the parasympathetic vagus nerve is involved in liver regeneration remains obscure. Kupffer cells are located in liver sinusoids adjacent to hepatocytes and might regulate liver regeneration by releasing interleukin-6 (IL-6). The present study examines the role of the vagus nerve and how Kupffer cells are involved in parasympathetic nerve-mediated liver regeneration in mice. METHODS We performed surgical vagotomy of the hepatic branch and then partial hepatectomy (PH); some mice received acetylcholine (ACh) agonist/antagonist before PH. We then evaluated liver regeneration and signal transducer and activator of transcription-3 (STAT3) activation. We also investigated whether ACh stimulates IL-6 release from Kupffer cells. RESULTS Surgical vagotomy impaired liver regeneration. STAT3, which is activated by IL-6 after hepatectomy and plays a pivotal role in liver regeneration, was less activated in vagotomized mice after PH. Post-PH STAT3 activation was recovered by administering vagotomized mice with an ACh agonist. Furthermore, ACh stimulated IL-6 release in Kupffer cells in vitro. CONCLUSION The parasympathetic system (vagus nerve) contributes to liver regeneration after hepatectomy by stimulating IL-6 release from Kupffer cells followed by STAT3 activation in hepatocytes.