This study was presented at the 93rd Annual Meeting of the Swiss Surgical Society, where it was awarded the 2006 Research Prize.
Potential conflict of interest: Nothing to report.
Hepatic ischemia and reperfusion (I/R) leads to the formation of leukocyte–platelet aggregates. Upon activation, platelets generate reactive oxygen species and release proapoptotic and proinflammatory mediators as well as growth factors. In cold hepatic ischemia, adhesion of platelets to endothelial cells mediates sinusoidal endothelial cell apoptosis. Furthermore, platelet-derived serotonin mediates liver regeneration. We hypothesized that platelets may contribute to reperfusion injury and repair after normothermic hepatic ischemia. The aim of this study was to assess the impact of platelets in normothermic hepatic I/R injury using models of impaired platelet function and immune thrombocytopenia. Inhibition of platelet function in mice was achieved via clopidogrel feeding. Immune thrombocytopenia was induced via intraperitoneal injection of anti-CD41 antibody. Platelet-derived serotonin was investigated using mice lacking tryptophan hydroxylase 1. Mice were subjected to 60 minutes of partial hepatic ischemia and various time points of reperfusion. Hepatic injury was determined via AST and histological analysis of the necrotic area as well as leukocyte infiltration. Liver regeneration was determined via proliferating cell nuclear antigen and Ki67 immunohistochemistry. Neither inhibition of platelet function nor platelet depletion led to a reduction of I/R injury. Liver regeneration and repair were significantly impaired in platelet-depleted animals. Mice lacking peripheral serotonin were deficient in hepatocyte proliferation, but otherwise displayed normal tissue remodeling. Conclusion: Platelets have no direct impact on the pathogenesis of normothermic I/R injury. However, they mediate tissue repair and liver regeneration. Furthermore, platelet-derived serotonin is a mediator of hepatocyte proliferation in the postischemic liver, but has no impact on tissue remodeling. (HEPATOLOGY 2007;45:369–376.)
Platelets are specialized blood constituents that play pivotal roles in physiological and pathological processes, including hemostasis, inflammation, host defense, and wound healing. Upon activation, platelets release inflammatory mediators, growth factors, and proteases that can either enhance or limit tissue injury depending on the specific context. For example, inhibition of thrombocyte activity reduces ischemic tissue injury in the heart,1 lung,2 and pancreas.3
In the liver, platelets and leukocytes synergistically trigger sinusoidal endothelial cell apoptosis upon reperfusion of cold preserved organs.4–9 During transplantation, the liver is subjected to a combination of hypothermic (cold) ischemia, intentionally applied to protect the liver graft, and normothermic (warm) ischemia.10, 11 Apart from the transplantation setting, warm ischemia also occurs during liver resection or in shock. In contrast to cold ischemia, warm ischemia is tolerated only for a short period and rapidly leads to hepatocellular injury and tissue necrosis.12, 13
Little is known about the impact of platelets on normothermic ischemia/reperfusion (I/R) injury of the liver. Mice deficient for P-selectin, an adhesion molecule critical to the postischemic platelet–endothelial cell interaction,14 display reduced platelet and neutrophil sequestration and a better survival following warm ischemia.15 In addition, the inhibition of platelet adhesion by the administration of anti-fibrinogen antibody decreases short-term liver injury after ischemia.16 However, these experimental approaches also inhibit leukocyte-mediated effects; thus, the specific contribution of platelets to warm I/R injury of the liver is still unknown. Ischemic liver injury also induces tissue repair, a process that includes inflammation, tissue resorption, and remodeling, as well as hepatocyte proliferation. Interestingly, the initiation of hepatocellular proliferation and liver regeneration have recently been shown to involve the release of platelet-derived serotonin.17
Consequently, platelets could exert opposing effects by mediating ischemic liver injury on the one hand and by facilitating the ensuing tissue repair process on the other hand. Therefore, using an established protocol of pharmacological platelet inhibition and a new model of platelet depletion, we evaluated the role of platelets in normothermic ischemia and reperfusion injury of the liver. We further investigated how platelets influence postischemic inflammation, and whether they play a critical role in the repair process following warm hepatic ischemia. Finally, we assessed the impact of platelet-derived serotonin in mediating tissue repair following an ischemic insult.
Experiments were performed on 8-week-old to 12-week-old male wild-type (Harlan, the Netherlands) and tryptophan hydroxylase 1 (Tph1)−/− mice (kindly provided by M. Bader18), both with a C57BL/6 background. Animals were kept in the animal facility of the University Hospital of Zurich with access to standard chow and water ad libitum. All procedures were approved by the Veterinary Office of the Canton of Zurich and were performed in accordance with institutional animal care guidelines.
Inhibition of Platelet Function.
Inhibition of platelet adhesion and aggregation with clopidogrel (Sanofi-Synthelabo-Recherche, Toulouse, France; 5 mg/kg/day) was performed as described.17 Clopidogrel or equal volumes of saline (controls) were administered via daily gavage starting 5 days before ischemia and continued until harvesting.
Immune Thrombocytopenia Model.
Thrombocytopenia was induced in C57BL/6 mice via intraperitoneal injection of 1 mg/kg anti-CD41 (glycoprotein GPIIb, clone MWReg 30, BD Biosciences, Franklin Lakes, NJ) diluted in 200 μl phosphate-buffered saline. This strategy leads to a reduction of platelets below 10% of their initial value (see Supplementary Figure). Control mice were treated via intraperitoneal injection of an equivalent amount of nonimmune IgG2 (BD Biosciences, Franklin Lakes, NJ). Whole blood samples (20 μl) were collected from the tail vein before injection and 24, 48, 72, and 96 hours after injection and diluted in 20 μl EDTA (12 mM). Blood cell counts were assessed using a Coulter AcT diff counter (Beckman Coulter, Nyon, Switzerland). Sustained platelet depletion (7 d) was achieved via additional daily injection of 0.5 mg/kg anti-CD41 starting on the first postoperative day.
Partial Hepatic Ischemia.
A model of 70% hepatic ischemia was used in all experiments as described.19 Group size was n = 5 unless otherwise indicated. Mice were anesthetized with inhalation of isoflurane/O2 (Halocarbon Laboratories, River Edge, NJ), and a constant gas mixture was maintained with a vaporizer system (Provet, Basel, Switzerland). After midline laparotomy, the liver was freed from its ligaments. Subsequently all structures of the portal triad to the left and median hepatic lobes were occluded for 60 minutes with a microvascular clamp (Aesculap, San Francisco, CA). In this model, mesenteric congestion is prevented by allowing intestinal blood flow through the right and caudate lobes. Reperfusion was initiated via removal of the clamp.
Serum AST Levels.
Blood samples were obtained from the inferior vena cava after different periods of reperfusion and were immediately centrifuged at 2,000g for 6 minutes. AST levels were measured using a serum multiple biochemical analyzer (Ektachem DTSCII; Johnson & Johnson Inc., Rochester, NY).
Liver tissue was immersion-fixed in 4% buffered formaldehyde embedded in paraffin, sectioned, and stained with hematoxylin-eosin using standard histological techniques. In addition, slides were immunostained for myeloperoxidase (polyclonal rabbit antibody; NeoMarkers, Fremont, CA), proliferating cell nuclear antigen (PCNA) (polyclonal rabbit antibody; clone PC-10, InnoGenex, San Ramon, CA), and Ki-67 (monoclonal rabbit clone SP6; NeoMarkers) using the Ventana Discovery automated staining system with the DAB Map kit (Ventana, Tucson, AZ). All immunostains were counterstained with hematoxylin. Polymorphonuclear leukocyte (PMN) infiltration was scored semiquantitatively on a scale of 1 (none) to 4 (severe). PCNA-positive and Ki-67–positive cells were counted in 10 randomly selected high-power fields (×400) per slide. All histologic analyses were performed by 2 investigators blinded with respect to the experimental group.
Quantitative Real-Time PCR.
Total RNA was extracted from 50 mg of liver tissue using TRIzol reagent (Invitrogen, Paisley, Scotland, UK) following the manufacturer's instructions. Five micrograms of RNA was reverse-transcribed using the ThermoScript RT-PCR System (Invitrogen, Basel, Switzerland) kit yielding the complementary DNA template. Quantitative real-time PCR amplification and data analysis were performed using an ABI Prism 7000 Sequence Detector System (PE Applied Biosystems, Rotkreuz, Switzerland). TaqMan gene expression assays (PE Applied Biosystems, Rotkreuz, Switzerland) for IL-1β (Mm00434228_m1), IL-6 (Mm 00446190_m1), Cxcl2 (Mm 00436450_m1), and tumor necrosis factor-α (TNF-α) (Mm 00443258_m1) were used to quantify mRNA expression of the respective genes. Messenger RNA expression levels of each sample were normalized to 18S RNA (TaqMan ribosomal RNA control reagents, PE Applied Biosystems). The results shown represent fold induction after ischemia versus baseline levels in control mice before ischemia.
TNF-α and IL-6 levels in serum were determined by ELISA (DuoSet mouse TNF-α and IL-6; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Data are expressed as the mean ± SD. Continuous variables were compared with the Student t test, ordinal variables with the Mann-Whitney U test. A P value of 0.05 was considered significant. Data analysis was performed with SPSS 12.0.1 for Windows (SPSS Inc., Chicago, IL).
Does Depletion or Functional Inhibition of Platelets Reduce Normothermic Hepatic I/R Injury?
To test whether platelets mediate normothermic hepatic I/R injury, platelets were depleted with anti-CD41 antibody or were functionally inhibited with clopidogrel (starting 5 d before ischemia) before 1 hour of ischemia. Liver injury was determined by AST release (Fig. 1A,C) after 1, 4, 24, and 48 hours of reperfusion as well as histological quantification of liver necrosis (Fig. 1B,D) after 4 and 24 hours of reperfusion. No difference in AST release or histological liver injury was detected between control animals and either strategy of platelet inhibition. These findings suggest that platelets do not contribute directly to ischemic liver injury.
Does Depletion or Functional Inhibition of Platelets Reduce Postischemic Leukocyte Infiltration?
Platelets are known to interact with leukocytes and may therefore play a role in the inflammatory reaction following hepatic ischemia. To assess this interaction, we quantified neutrophils in a semiquantitative fashion using myeloperoxidase immunostaining 24 hours after reperfusion. Whereas PMN infiltration was not affected by clopidogrel treatment (Fig. 2A), platelet-depleted animals showed significantly less leukocyte infiltration of the postischemic liver (Fig. 2B). A representative microphotograph is shown in Fig. 2C.
Does Depletion or Functional Inhibition of Platelets Reduce Postischemic Expression of Inflammatory Mediators?
Attraction of neutrophils to the site of ischemic tissue injury is mediated by the release of cytokines and chemokines. TNF-α, IL-6, and IL-1β are major proinflammatory cytokines in the liver, while the chemokine Cxcl2 (also known as MIP-2) is a strong chemoattractant for neutrophils, which is involved in ischemic liver injury.20, 21 The presence of transcripts coding for these proinflammatory molecules was analyzed via real-time PCR. Whereas TNF-α and IL-1β transcripts (Fig. 4A,C) were only slightly reduced, expression levels of IL-6 and Cxcl2 (Fig. 4B,D) were significantly lower in platelet-depleted animals. Consistent with reduced neutrophil infiltration, platelet depletion caused a marked reduction in postischemic TNF-α and IL-6 release (Fig. 5), as determined via ELISA. Importantly, inhibition of platelet aggregation by clopidogrel did not affect the expression of proinflammatory mediators (Fig. 3), in accordance with the lack of effect on PMN infiltration (Fig. 2A). Taken together, these data suggest that platelets are required for the secretion of proinflammatory cytokines, which in turn are crucial for neutrophil infiltration.
Does Platelet Depletion Reduce Postischemic Liver Repair and Regeneration?
Inflammation is crucial for the containment and consecutive resorption of damaged tissue. In view of reduced inflammation and neutrophil infiltration, we asked whether platelet depletion would also delay tissue repair in the postischemic liver. To this end, we quantified residual liver necrosis 7 days after ischemic insult (Fig. 6). Whereas control animals displayed minimal residual necrosis (3% of liver tissue), platelet-depleted animals still showed more than 10% necrosis, indicating a delayed resolution.
Restitution of hepatic parenchyma after an ischemic insult requires a combination of tissue resorption and remodeling as well as hepatocellular proliferation. Because platelets are critical mediators of liver regeneration after hepatectomy,17 we hypothesized that they would also play a role in the restitution of hepatic parenchyma following ischemia. Quantification of hepatocyte regeneration by PCNA and Ki67 immunostaining revealed a significant reduction of hepatocellular proliferation in platelet-depleted animals only at 48 hours, pointing to an impaired rather than delayed liver regeneration (Fig. 7).
Does Platelet-Derived Serotonin Mediate Postischemic Liver Regeneration and Tissue Repair?
Platelets mediate liver regeneration through the release of serotonin.17 The finding that platelets are involved in postischemic liver repair and regeneration prompted us to investigate the role of platelet-derived serotonin using Tph1−/− mice. These animals have a disrupted gene for Tph1, rendering them devoid of serotonin outside of the central nervous system.18 Initially, we tested whether a lack of serotonin results in a reduction of I/R injury. As for platelet depletion, serotonin deficiency did not decrease tissue injury (data not shown). We then analyzed neutrophil infiltration and the expression of proinflammatory markers as well as the resolution of necrotic tissue. In contrast to platelet-depleted animals, serotonin deficiency did not affect the sequel of inflammation and tissue resorption; this is reflected by an equal amount of remnant necrosis 7 days after the initial ischemic insult (Fig. 8A). Because serotonin has mitogenic properties, we further analyzed the regenerative activity of hepatocytes and found that the lack of serotonin significantly reduced hepatocyte proliferation (Fig. 8B,C). This further supports a role for serotonin in liver regeneration, while the presence of other platelet-derived factors is instrumental for tissue resorption and remodeling.
This study was designed to assess the role of platelets in normothermic I/R injury of the liver. Neither abrogation of platelet aggregation nor platelet depletion reduced postischemic tissue injury. Instead, postischemic inflammation, as well as liver regeneration and consequently tissue repair, were strikingly impaired. In particular, platelet-derived serotonin mediates hepatocyte proliferation, which is an integral component of postischemic tissue repair.
Research on the role of platelets in normothermic I/R injury has been limited by the absence of a suitable model. Although various protocols targeting platelets have been available, thrombocytes either remained physically present,22 or leukocytes were concomitantly affected.14, 23 It was only recently that a pure model of platelet-depletion using a monoclonal antibody against a platelet epitope was described.17 Here, we established a model of immune thrombocytopenia using a commercially available antibody directed against the platelet-associated epitope GPIIb. Intraperitoneal administration of this antibody selectively reduced platelet counts by more than 90%, whereas leukocyte and erythrocyte counts remained unaffected (Supplementary Figure), indicating a specific thrombocytopenia. This approach may also be instrumental in investigating platelet function in organs other than the liver. We also employed mice lacking Tph1, which is the rate-limiting enzyme for the synthesis of peripheral serotonin. Using this model, we recently demonstrated platelet-derived serotonin to mediate hepatocyte proliferation after partial hepatectomy.17
We found that I/R injury as determined by AST levels and liver tissue necrosis was not affected by the absence or impaired function of platelets. This result stands in contrast with previous studies.15, 16 One of these studies used anti-fibrinogen antibodies to inhibit platelet–endothelial cell interactions in a model of 90 minutes of ischemia and assessed reperfusion injury only up to 60 minutes.16 Tissue necrosis and repair do not manifest themselves during 1 hour of reperfusion and thus cannot be conclusively assessed at this time point. The short reperfusion time might have been chosen because of the severe ischemic insult, because 90 minutes of hepatic ischemia lead to subtotal tissue necrosis. In pilot experiments, we assessed an ischemic period of 90 minutes, which was associated with high mortality in the platelet-depleted group (data not shown). Furthermore, as the authors suggested, the beneficial effects seen after functional platelet inhibition could be attributed to reduced leukocyte sequestration, because activated platelets have been reported to modulate leukocyte adherence through secretion of chemotactic agents as well as surface expression of adhesion molecules.24 Indeed, this mechanism is supported by our finding of decreased postischemic PMN infiltration in platelet-depleted animals, whereas the mere inhibition of platelet aggregation did not have such an effect.
Another study, which showed reduced postischemic liver injury in mice deficient in P-selectin,15 must also be interpreted with caution, because these animals display reduced endothelial–leukocyte adhesion in addition to their platelet phenotype. Our results imply that the observed protection is related to an inhibition of leukocyte infiltration rather than a specific platelet-mediated effect. Taken together, the presence or activation of platelets does not contribute to tissue injury and necrosis following hepatic ischemia.
In our study, platelet depletion reduced the hepatic expression and release of proinflammatory mediators as well as the recruitment of neutrophils. This effect could be attributed to a disturbed interaction of platelets and neutrophils at the endothelial level. However, in the setting of inhibited platelet aggregation, neutrophil infiltration remained unaffected. This hints against a predominant role of secondary capture of neutrophils by platelets in this setting. These results suggest that these blood constituents play an important role in chemoattraction to the postischemic liver. Although the reduced expression and release of these mediators in platelet-depleted animals can already be found at 4 hours of reperfusion, this partial attenuation of the inflammatory reaction is apparently insufficient to reduce the ensuing injury. Because a depletion of neutrophils has been convincingly shown to protect the liver from reperfusion injury,25 our data point to the possibility that the amount of neutrophils infiltrating the postischemic liver independently of platelet function are sufficient to exert their adverse effects, resulting in extensive liver damage. This interpretation is also supported by the finding that IL-1 receptor–deficient mice display reduced neutrophil recruitment without effects on the extent of liver injury.21
Recovery from liver damage has been termed the liver wound healing response.26 This requires distinct mechanisms such as inflammation, tissue resorption, and remodeling, as well as hepatocyte proliferation. We have recently shown platelet-derived serotonin to mediate liver regeneration in a mouse model of partial hepatectomy.17 Consistent with these findings, both thrombopenic animals and mice lacking peripheral serotonin exhibited reduced hepatocyte proliferation 48 hours after the initial ischemic insult. Thus, the effect on liver regeneration in platelet-depleted mice could also be ascribed to the diminished expression of TNF-α or IL-6, both of which have been identified as key mediators of regeneration.27–29 However, in our experiments, the expression levels of TNF-α and IL-6 did not differ in postischemic livers of Tph1−/− and wild-type mice. Yet there was a significant reduction in regeneration in these animals. This observation points to two independent pathways that act synergistically on hepatocyte proliferation.
The role of TNF-α and IL-6 in liver regeneration has been extensively discussed.27–29 They appear to initiate the hepatocyte cell cycle entry into the G1 phase. These studies were predominantly performed in models of partial hepatectomy, where the loss of liver mass is instantaneous, triggering a synchronous regenerative response in the remnant liver. In contrast, after ischemia and reperfusion the injury is focal and less synchronized, leading to a regenerative activity mainly at the site of damage. This may explain the only partial induction of hepatocyte proliferation. In the serotonin-dependent model, the postischemic regenerative response was more severely impaired, suggesting that serotonin is required to secure hepatocyte proliferation.
Although hepatic inflammation is detrimental, it is also a part of the normal wound healing process. Platelets contain an array of preformed peptide mediators and growth factors, including platelet factor-4, platelet-derived growth factor, vascular endothelial growth factor, and many others. These mediators are rapidly released upon tissue injury and play critical roles in the induction of inflammation and tissue repair. Vascular endothelial growth factor promotes migration of endothelial cells, thus initiating angiogenesis at the tissue repair stage.30 In turn, platelet factor-4 and platelet-derived growth factor are chemotactic for neutrophils, monocytes, and fibroblasts,31, 32 all of which are active players in the resolution of liver injury. A reduction of neutrophil recruitment has indeed been reported to impair wound closure in the skin.33 Consistent with these findings, the reduced neutrophil infiltration in platelet-depleted animals was associated with a prolonged persistence of necrotic tissue. This effect was caused by the absence of platelets and platelet-derived factors, resulting in a reduced expression of proinflammatory molecules in the liver.
The fact that mice lacking peripheral serotonin displayed reduced hepatocellular proliferation in spite of normal injury, inflammation, and resolution of necrotic tissue suggests that other platelet-derived factors are involved at different steps during postischemic tissue repair and regeneration.
In conclusion, our results convincingly acquit platelets of causing postischemic liver injury, but highlight for the first time their importance in different steps of postischemic tissue repair through the modulation of inflammation and the release of serotonin. Taken together, these findings point to a novel role of platelets in hepatic wound healing.
We thank Udo Ungethüm and Marion Bawohl for technical assistance. We are also grateful to Bernhard Odermatt for help in establishing the immune thrombocytopenia model.