Liver Wound Research Articles

Divergent Transforming Growth Factor-β Signaling in Hepatic Stellate Cells after Liver Injury: Functional Effects on ECE-1 Regulation

In liver wound healing, transforming growth factor-beta (TGF-beta) plays a critical role in stellate cell activation as well as signaling cascades in the fibrogenic response to injury. We postulate that the TGF-beta-dependent downstream signaling pathway may vary according to the mechanism of stellate cell activation; this study was undertaken to ascertain whether the downstream signaling pathways mediated by TGF-beta vary in different liver injury models. We measured Smad3 and MAP kinase activation after isolating stellate cells from rat livers injured by either bile duct ligation (BDL) or repeated carbon tetrachloride (CCl(4)) administration. Phospho-Smad3 was dramatically up-regulated in stellate cells after CCl(4) injury, but not after BDL-induced injury. TGF-beta signaling in stellate cells activated after BDL was mediated prominently through ERK activation, whereas activation induced by CCl(4) injury or culture led to a cross-signaling mechanism involving both Smad3 and p38. The divergent Smad signaling pathways observed appeared to be attributable to the differential regulation of the early growth response gene-1 (Egr-1), an apparent negative transcriptional factor for Smad3 in our system. In addition, inhibition of ERK activation in stellate cells from BDL-injured liver led to a decrease in expression of endothelin-converting enzyme-1, a critical regulator of endothelin-1. We speculate that TGF-beta signaling proceeds through differential signaling pathways depending on the mechanism of liver injury that leads to stellate cell activation.

A simple hemostasis model for the quantitative evaluation of hydrogel-based local hemostatic biomaterials on tissue surface

Several hemostat hydrogels are clinically used, and some other agents are studied for safer, more facile, and more efficient hemostasis. In the present paper, we proposed a novel method to evaluate local hemostat hydrogel on tissue surface. The procedure consisted of the following steps: (step 1) a mouse was fixed on a cork board, and its abdomen was incised; (step 2) serous fluid was carefully removed because it affected the estimation of the weight gained by the filter paper, and parafilm and preweighted filter paper were placed beneath the liver (parafilm prevented the filter paper's absorption of gradually oozing serous fluid); (step 3) the cork board was tilted and maintained at an angle of about 45° so that the bleeding would more easily flow from the liver toward the filter paper; and (step 4) the bleeding lasted for 3 min. In this step, a hemostat was applied to the liver wound immediately after the liver was pricked with a needle. We found that (1) a careful removal of serous fluid prior to a bleeding and (2) a quantitative determination of the amount of excess aqueous solution that oozed out from a hemostat were important to a rigorous evaluation of hemostat efficacy. We successfully evaluated the efficacy of a fibrin-based hemostat hydrogel by using our method. The method proposed in the present study enabled the quantitative, accurate, and easy evaluation of the efficacy of local hemostatic hydrogel which acts as tissue-adhesive agent on biointerfaces.

Wound healing and local neuroendocrine regulation in the injured liver

The hepatic wound-healing response is a complex process involving many different cell types and factors. It leads to the formation of excessive matrix and a fibrotic scar, which ultimately disrupts proper functioning of the liver and establishes cirrhosis. Activated hepatic myofibroblasts, which are derived from cells such as hepatic stellate cells (HSCs), play a key role in this process. Upon chronic liver injury, there is an upregulation in the local neuroendocrine system and it has recently been demonstrated that activated HSCs express specific receptors and respond to different components of this system. Neuroendocrine factors and their receptors participate in a complex network that modulates liver inflammation and wound healing, and controls the development and progression of liver fibrosis. The first part of this review provides an overview of the molecular mechanisms governing hepatic wound healing. In the second section, we explore important components of the hepatic neuroendocrine system and their recently highlighted roles in HSC biology and hepatic fibrogenesis. We discuss the therapeutic interventions that are being developed for use in antifibrotic therapy.

Clinical Usefulness of Biliary Scope for Pringle’s Maneuver in Laparoscopic Hepatectomy

D T m a s o r o uring the last few years, there have been significant deelopments in the area of laparoscopic surgery. Previous tudies reported a successful laparoscopic hepatectomy for epatocellular carcinoma (HCC). The major problem in aparoscopic surgery for parenchymal organs, such as the iver, is successfully performing hemostasis from the reected stump. In particular, HCC usually occurs from a irrhotic liver, and this liver condition often leads to bleedng problems. In addition, the blood loss is a prognostic actor of postoperative morbidity and mortality after heatic resection. For these reasons, hemostasis is important n laparoscopic liver surgery, especially for hepatic alignancies. In 1908, Pringle recommended compression of the heatic hilar vessels to control bleeding from liver wounds. is report that vascular control could enable a major heatic resection without excessive blood loss resulted in a rogressive decrease in mortality and morbidity associated ith liver resection. Since that report, this technique has een widely used in liver surgery to reduce intraoperative lood loss. But this procedure cannot be easily applied for laparoscopic hepatectomy because of the narrow field of ision under laparoscopy. This report describes a new techique using a biliary scope to easily perform Pringle’s maeuver during a laparoscopic hepatectomy.

Preclinical safety of recombinant human thrombin

Recombinant human thrombin (rhThrombin) is being developed as an alternative to thrombin products purified from pooled human or bovine plasma, which are currently marketed for topical hemostasis. Preclinical studies of rhThrombin were conducted prior to its evaluation as a topical adjunct to surgical hemostasis in clinical trials. No overt clinical pathology or signs were observed in cynomolgus monkeys following implantation of a gelatin sponge containing either rhThrombin or bovine thrombin to a surgical liver wound, and similar gross and microscopic wound healing characteristics were observed over an eight-week recovery period with either compound. Repeated subcutaneous injections of rhThrombin or bovine thrombin to cynomolgus monkeys produced no treatment-related effects. Whereas no monkeys demonstrated anti-rhThrombin antibody seroconversion, specific anti-bovine antibodies were detected in all tested monkeys exposed to bovine thrombin. Addition of rhThrombin or bovine thrombin to mouse fibroblast cells resulted in expected detachment and shape change. Topical application of rhThrombin to rabbits did not cause irritation to the eye, normal skin, or abraded skin. These studies showed that topical, subcutaneous, or implanted rhThrombin was minimally immunogenic, safe, and well tolerated in nonclinical models, and supported the clinical evaluation of rhThrombin in a variety of surgical settings.

The Role of Integrin-linked Kinase in Liver Wound Healing

Liver wound healing is an integrated process in which hepatic stellate cells play a major role. We hypothesized that the cellextracellular signaling protein integrin-linked kinase (ILK) is important in transducing signals from the extracellular matrix to stellate cells and thus plays a critical role in stellate cell activation and fibrogenesis during liver injury. Liver injury and subsequent stellate cell activation led to a 3-fold increase in ILK expression and increased kinase activity. Overexpression of ILK in isolated stellate cells led to enhanced motility and adhesion as well as increases in smooth muscle alpha-actin and type I collagen mRNA expression. The effects of ILK on stellate cell phenotypes were phosphatidylinositol 3-kinase-dependent. Forced expression of ILK in vivo led to increases in type I collagen, smooth muscle alpha-actin, transforming growth factor-beta, and extra domain A (EDA) fibronectin mRNAs (by 3.2-, 3.5-, 2.5-, and 2.2-fold, respectively; n = 8, p < 0.05 for each versus the control), whereas inhibition of ILK in vivo led to significant reductions in these mRNAs. Morphometric analysis revealed that ILK overexpression led to a 31.4% increase in liver collagen content (n = 8, p < 0.05 versus the control); in contrast ILK knockdown in vivo led to a significant reduction in fibrogenesis. We conclude that ILK plays an important pathophysiological role in vivo in liver wound healing.

Prostaglandins of the E-series inhibit connective tissue proliferation in the liver wound of the rat

The present study was undertaken to relate wound healing of an internal organ to prostaglandins of the E and F series. A small liver wound was induced by a galvanic cauter via the abdominal route under general anesthesia and prostaglandin E1, E2 and F2alpha were injected twice daily at a dose of 250 microg/kg. Proliferation of the connective tissue in the liver wound was estimated morphometrically 6 days after liver wound infliction. Levels of prostaglandins E2 and F2alpha were measured in the liver wound as well as in normal liver tissue from adjacent lobes using radioimmunoassay. The results show that exogenous prostaglandins of the E-series suppress connective tissue proliferation. Three minutes after the last prostaglandin E2 injection, high prostaglandin concentrations were measured both in the liver wound and in the liver tissue of the adjacent lobe. Prostaglandin F2alpha injections had no effect on wound healing. We believe that the rat thermic liver wound model can be used for different studies on wound healing mechanisms and that prostaglandins of the E-series are involved in wound healing in the specific time period studied.

Modulation of fibronectin gene expression in inflammatory mononuclear phagocytes of rat liver after acute liver injury

Background/Aims : In acute liver injury, fibronectin (FN) is deposited at the site of hepatocellular necrosis. We have previously shown that liver inflammatory mononuclear phagocytes (MNP), in contrast to quiescent hepatic macrophages, synthesize abundant amounts of FN. We now analyzed effects of agents known to influence macrophage functions to better understand liver damage and repair. Methods : Acute rat liver injury was induced by CCl 4. Liver cellular FN ( cFN) expression was analyzed by in situ-hybridization. Liver MNP were isolated and characterized immunocytochemically. Protein synthesis was studied by biosynthetic labeling, immunoprecipitation, and SDS-PAGE. RNA was analyzed by Northern Blotting. Results : cFN gene expression was localized by in situ-hybridization and immunohistochemistry within the pericentral inflammatory infiltrate. Treatment of inflammatory MNP with dexamethasone, or interferon-gamma, or lipopolysaccharide induced a dose-dependent decrease in cFN gene expression, whereas transforming growth factor-β increased cFN gene expression. Conclusions : 1. Inflammatory MNP express cFN. 2. Downregulation of cFN expression by dexamethasone in inflammatory MNP may explain delayed wound healing after corticosteroid therapy. Interferon-γ and lipopolysaccharide could also delay the repair process in the liver. Transforming growth factor-β may promote liver wound healing after acute liver injury by increasing local cFN synthesis in inflammatory mononuclear phagocytes of the inflammatory infiltrate.

Damage control: a tactical alternative for the management of exanguinating trauma patients

Despite the advances in the treatment of exanguinating patients, hemorrhage remains as the leading cause of early deaths. A great deal of attention has been given to "damage control" as a therapeutic alternative in this scenario. To appraise the definition, indications, operative techniques and results of damage control for the treatment of exanguinating trauma patients. Bibliographic review. Damage control introduces the concept of breaking the vicious cycle of metabolic acidosis, hypothermia and coagulopathy which results from hemorrhagic shock. Thus, the operation has to be interrupted before this irreversible stage, even if the injured organs were not given the definitive treatment at this moment. So, damage control involves three steps: an abbreviated operation, a recovering period in the intensive care unit, and the reoperation for the definitive treatment. At the abbreviated operation, operative techniques as stapling intestinal injuries or packing liver wounds are applied, allowing rapid control of the bleeding and spillage. In the intensive care unit, the patient is warmed, oxygen delivery and consumption are restored and coagulation factors administered. As soon as the hemodynamic stability, ideal body temperature and coagulation status are reached, the definitive operation is carried out. Damage control is a helpful option if correctly used. However, there are also severe complications that can occur. Therefore, it should be employed only in centers that could provide optimum resources. Damage control is an important tactical alternative for the treatment of exanguinating trauma patients.

Chemokines in liver inflammation and fibrosis.

Chemokines may be involved in the tissue response to injury regulating the influx of leukocytes, and modulating a number of other critical biologic actions, including angiogenesis, neoplastic growth, myo-fibroblast activation, and the response to viral infections. In the liver, up-regulated expression of different members of the chemokine system may be induced by almost all types of injury, and there is often a clear relation between the chemokine pattern activated by different types of injury and the predominant subclasses of leukocytes which infiltrate the liver. Neutralization of specific chemokines by passive immunization or the use of animals deficient in specific chemokines or chemokine receptors has indicated a causal relation between up-regulation of chemokines and leukocyte infiltration. Inflammation is part of the liver wound healing response, that in chronic conditions leads to the development of fibrosis and cirrhosis. Hepatic stellate cells, which play a leading role in the development of fibrosis following their transition to myofibroblasts, express different chemokines. Chemokine expression by stellate cells is regulated by soluble mediators, in particular pro-inflammatory cytokines, as well as growth factors, proteases, and products of oxidative stress. In addition, stellate cells also respond to chemokines with biologic actions relevant for tissue repair, such as cell migration or induction of other chemokines. These data indicate that chemokines in the liver may modulate the progression of liver fibrosis through actions on hepatic stellate cells.

Chemokines in liver inflammation and fibrosis

Chemokines may be involved in the tissue response to injury regulating the influx of leukocytes, and modulating a number of other critical biologic actions, including angiogenesis, neoplastic growth, myo-fibroblast activation, and the response to viral infections. In the liver, up-regulated expression of different members of the chemokine system may be induced by almost all types of injury, and there is often a clear relation between the chemokine pattern activated by different types of injury and the predominant subclasses of leukocytes which infiltrate the liver. Neutralization of specific chemokines by passive immunization or the use of animals deficient in specific chemokines or chemokine receptors has indicated a causal relation between up-regulation of chemokines and leukocyte infiltration. Inflammation is part of the liver wound healing response, that in chronic conditions leads to the development of fibrosis and cirrhosis. Hepatic stellate cells, which play a leading role in the development of fibrosis following their transition to myofibroblasts, express different chemokines. Chemokine expression by stellate cells is regulated by soluble mediators, in particular pro-inflammatory cytokines, as well as growth factors, proteases, and products of oxidative stress. In addition, stellate cells also respond to chemokines with biologic actions relevant for tissue repair, such as cell migration or induction of other chemokines. These data indicate that chemokines in the liver may modulate the progression of liver fibrosis through actions on hepatic stellate cells.

Influence of intraperitoneal phospholipid dosage on adhesion formation and wound healing at different intervals after surgery.

Adjuvant therapy is needed to prevent adhesion formation as a major cause of postoperative morbidity and mortality. The efficacy of phospholipids (PLs) has been proven; however, information on dosage and drug safety are still outstanding. Forty-eight Chinchilla rabbits underwent median laparotomy, abrasion of the peritoneum, jejunal anastomosis, and an electrocautery incision of the liver. The operation was completed by intraperitoneal administration of PLs in two different concentrations (30 mg/kg and 70 mg/kg body weight). In the control group, the abdomen was closed without additional treatment. Adhesion area, anastomotic bursting pressure, tensile strength of the midline incision, and healing of the liver wound were assessed on days 3, 5, 7, and 10, respectively. The mean areas of adhesions in the control group were slightly larger than in the PL groups after 3 days and 5 days. On day 7 and day 10, both PL groups presented with significantly smaller adhesion areas (P<0.05). In all groups, we measured equal anastomotic bursting pressures on the 3rd, 5th, and 10th postoperative days. After 7 days, the mean value of the PL 70-mg group (17.2 kPa) was significantly lower than in the other groups (control 22.1 kPa, PL 30 mg 20.7 kPa; P<0.05). The tensile strengths of the laparotomy wound measured after intervals of 5 days and 7 days were not statistically different. On day 3, it was reduced after 30 mg PL but enhanced after 70 mg PL, whereas 10 days after surgery the strength increased with the PL dosage. The inflammatory reparative response to hepatic injury, jejunal anastomosis, and midline incision was not affected by PLs as assessed by histological analysis. These results prove the efficacy of PLs in adhesion prevention in two concentrations. The findings reveal an unimpeded healing of anastomoses, laparotomy wounds, and liver incisions at different periods after surgery.

Lipoic Acid Prevents Suppression of Connective Tissue Proliferation in the Rat Liver Induced by n–3 PUFAs

As previously shown, dietary n–3 polyunsaturated fatty acids (n–3 PUFAs) suppress connective tissue proliferation in the rat liver wound concurrent with an elevated level of lipid peroxidation. The present study was undertaken to investigate the influence of α-lipoic acid (LA), a natural anti-oxidant, on these effects of n–3 PUFAs. Rats were fed with a commercial pellet diet (control group) or with diets enriched with 10% of sunflower oil (n–6 group) or 10% of fish oil (n–3 group) for 8 weeks followed by addition of LA to the same diets for 10 days. Then a liver thermic wound was induced and the administration of LA was continued for 6 days. The proliferation of the connective tissue, the level of lipid peroxidation and their peroxidizability and the content of prostaglandins E₂ and F_2α were measured in the liver wounds. LA prevented the suppression of connective tissue proliferation in the healing wound induced by n–3 PUFAs, avoided the increase in peroxidation of lipids, reduced peroxidizability of lipids and modulated the decrease in PGE₂ and PGF_2α. The results indicate that dietary LA may prevent the suppression of liver wound healing induced by n–3 PUFAs.

Stellate Cell Activation in Alcoholic Fibrosi—An Overview

There has been remarkable progress in our understanding of how chronic alcohol ingestion may lead to hepatic injury and scarring, or fibrosis. Hepatic fibrosis represents the liver's wound healing response and is characterized by accumulation of interstitial matrix, or scar. Fibrosis in the liver results from the activation of stellate cells, or resident mesenchymal cells. Stellate cell activation is a dramatic phenotype transition whose net effect is the replacement of normal liver matrix by scar. Features of stellate cell activation include increased cell accumulation from proliferation and directed migration, increased matrix production, enhanced contractility, accelerated degradation of the normal liver matrix, release of profibrogenic cytokines, and loss of cellular vitamin A. Alcohol may enhance fibrogenesis through stimulation of stellate cells by hypoxia, generation of lipid peroxides from damaged hepatocytes, production of acetaldehyde that may have direct fibrogenic activity, and through activation of Kupffer cells or resident macrophages. Unanswered questions remain to be studied, but the clarification of underlying mechanisms of fibrosis portends continued progress in our ability to treat alcoholic liver fibrosis.

Alterações morfológicas do fígado após secção hepática parcial e omentoplastia

A omentoplastia é um método útil para o tratamento de traumas hepáticos graves. Apesar dos bons resultados dessa tática operatória para controlar a hemorragia, seus efeitos sobre a arquitetura hepática ainda não são bem conhecidos. Dessa forma, o presente trabalho teve como objetivo verificar a influência da omentoplastia em trauma experimental do fígado. Este estudo foi realizado em dez ratos submetidos a secção parcial do lobo hepático direito. Um segmento de omento maior foi introduzido dentro da ferida hepática, que foi suturada com fio de categute simples 5-0. Os animais foram seguidos durante sete (n=5) ou 21 (n=5) dias. Decorridos esses períodos, os aspectos macro e microscópicos do fígado foram avaliados. Todos os ratos suportaram a operação e sobreviveram durante o período estudado neste experimento. Os principais achados foram, no sétimo dia, necrose e abscessos do fígado distal à secção hepática. No 21° dia, a parte distal do fígado tornara-se fibrótica. Concluindo, a omentoplastia é de grande ajuda para controlar sangramentos hepáticos intensos decorrentes de trauma; porém, em ratos, a parte distal de uma ferida profunda muda suas características morfológicas.

Inhibition of tumor implantation at sites of trauma by plasminogen activators

The authors report on the influence of plasminogen activators (PA) on implantation of TA3Ha mammary tumor cells in the healing hepatic wounds of syngeneic strain A mice. Intravenously injected TA3Ha cells, although they rarely metastasize to the liver, formed tumors in the hepatic wounds of a significant percent (42%, P less than 0.0001) of mice. The frequency of tumor formation declined as the interval between surgery and tumor cell inoculation was increased. Furthermore, preexposure of cells to fibrinogen, fibronectin, laminin, or peptides containing the arginine-glycine-aspartic acid-serine residues dramatically reduced the frequency of tumor formation in the hepatic wounds. These results indicate that TA3Ha cells interact with fibrinogen-related proteins in the wound to aid their attachment and growth. Because these proteins are susceptible to digestion by plasmin, PA were used in this study to examine whether administration of these drugs to the mice would modulate tumor formation in the liver wounds. Among the PA tested, human plasmin B-chain-streptokinase complex (B-SK) and recombinant tissue plasminogen activator (t-PA) inhibited tumor implantation in a dose-related manner. Administration of 900 units (U) of B-SK or 3300 U of t-PA per mouse reduced the frequency of tumor formation from 42% to 0% (P = 0.02) and 11% (P = 0.02), respectively. The B-SK was complexed with p-nitrophenyl-p-guanidinobenzoate; it did not activate the plasminogen or inhibit tumor formation in the hepatic wounds. Although urokinase activated the plasminogen, it did not inhibit tumor implantation in the hepatic wound. Heparin, an anticoagulant that prevents conversion of fibrinogen to fibrin without being fibrinolytic, had no influence on tumor formation in the hepatic wounds. The PA can generate plasmin that digests the cell attachment proteins in wounds and consequently inhibits tumor cell attachment.

Efficacy of Liver Wound Healing by Secondary Intent

Nonoperative treatment of liver injury raises questions about liver wound healing (LWH) when the edges are not approximated by primary intent. The efficacy of LWH was studied in 18 dogs and 18 pigs with a total of 108 standard 6-cm injuries. The 36 injuries in six dogs and six pigs were compressed for hemostasis and then allowed to heal by second intent. LWH in these animals was compared to 36 liver wounds in six dogs and six pigs following primary closure (hepatorrhaphy) and 36 liver wounds after omental buttress plus hepatorrhaphy in six dogs and six pigs. Average wound breaking strength (WBS) of liver wounds was studied at 3 weeks (54 wounds) and 6 weeks (54 wounds) with a Chatillon tensiometer and microscopic analysis. The WBS of liver wounds was also compared to normal uninjured liver WBS. The porcine WBS at 3 weeks after healing by second intent (0.31 kg/cm2) was similar to the WBS at 3 weeks after healing by hepatorrhaphy (0.30 kg/cm2) and omental buttress (0.25 kg/cm2). Porcine WBS at 6 weeks after healing by second intent was significantly greater than WBS at 6 weeks after hepatorrhaphy or omental buttress. The canine WBS at 3 weeks after healing by second intent exceeded WBS at 3 weeks after hepatorrhaphy or omental buttress. Canine WBS at 6 weeks after healing by second intent exceeded WBS at 6 weeks after omental buttress and was similar to WBS at 6 weeks after hepatorrhaphy. WBS in all groups paralleled the extent of fibrosis seen on microscopic analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemostasis of Solid Viscus Trauma by Intraparenchymal Injection of Fibrin Glue

Fibrin glue (FG) is an effective hemostatic agent applied topically to the spleen. In this study, FG was found to be an effective hemostatic agent when applied topically in standardized wounds of the canine liver, spleen, and pancreas. It was markedly more effective, however, when injected intraparenchymally. In a case of severe blunt trauma in a patient with acute alcoholic hepatitis, intraparenchymal FG was lifesaving. Fibrin glue is a useful adjunct in the management of trauma to all the abdominal solid viscera. Intraparenchymal injection is the preferred mode of FG application.

Hepatic Trauma

Routine drainage of liver wounds created by trauma has recently been challenged, prompting a prospective, randomized trial of drainage via a Penrose dam versus no drain in patients having emergency laparotomy for abdominal trauma. We excluded cases in which definite bile leak was noted at operation. Of 167 patients studied, six had obligatory drainage because of obvious bile leak. Among the remaining 161 patients, there was no significant difference as to demographics, mode of injury, volume of blood lost or used for resuscitation, incidence and severity of shock, number and types of associated injuries, or magnitude of liver wound between the 78 allocated to drainage and the 83 left without a drain. Resultant mortality, duration of hospitalization, incidence of wound and/or intra-abdominal infection, and likelihood of subsequent bile fistula were not different. Such data support the routine use of a drain only if bile leakage from the liver wound is found at laparotomy. Without obvious bile leak, drainage of a specific liver injury does not appear to be necessary.

Untersuchungen zur differenzierung der wirkung von laserstrahlen am herzmuskel und an der Leber durch Kombination von histochemischem succinatdehydrogenase-nachweis und acridinorange-fluorochromierung (MS-AO-technik)

In laser induced wounds of myocardium and liver of rats, 1 reversible zone and 3 irreversible zones of damage can be separated in dependence of time until 24 h by a combined enzyme-histochemical and fluorescence microscopical technique. Besides these zones, another carbonized zone is also detectable. A mainly reversible zone of contraction can be seen for a short time in the myocardium. The results indicate different types of manifestation of laser and ischemic induced tissue necroses.