Mesenchymal Stem Cells for Liver Regeneration in Liver Failure: From Experimental Models to Clinical Trials.

The liver centralizes the systemic metabolism and thus controls and modulates the functions of the central and peripheral nervous systems, the immune system, and the endocrine system. In addition, the liver intervenes between the splanchnic and systemic venous circulation, determining an abdominal portal circulatory system. The liver displays a powerful regenerative potential that rebuilds the parenchyma after an injury. This regenerative mission is mainly carried out by resident liver cells. However, in many cases this regenerative capacity is insufficient and organ failure occurs. In normal livers, if the size of the liver is at least 30% of the original volume, hepatectomy can be performed safely. In cirrhotic livers, the threshold is 50% based on current practice and available data. Typically, portal vein embolization of the part of the liver that is going to be resected is employed to allow liver regeneration in two-stage liver resection after portal vein occlusion (PVO). However, hepatic resection often cannot be performed due to advanced disease progression or because it is not indicated in patients with cirrhosis. In such cases, liver transplantation is the only treatment possibility, and the need for transplantation is the common outcome of progressive liver disease. It is the only effective treatment and has high survival rates of 83% after the first year. However, donated organs are becoming less available, and mortality and the waiting lists have increased, leading to the initiation of living donor liver transplantations. This type of transplant has overall complications of 38%. In order to improve the treatment of hepatic injury, much research has been devoted to stem cells, in particular mesenchymal stem cells (MSCs), to promote liver regeneration. In this review, we will focus on the advances made using MSCs in animal models, human patients, ongoing clinical trials, and new strategies using 3D organoids.

Prehepatic portal hypertension worsens the enterohepatic redox balance in thioacetamide-cirrhotic rats.

BACKGROUND: Oxidative stress has been reported as a key pathogenic factor in many human liver diseases and in experimental models of cirrhosis related to hepatotoxin administration. The aim of this study was to verify the hypothesis that prehepatic portal hypertension aggravates the enterohepatic redox imbalance in thioacetamide-cirrhotic rats. MATERIALS AND METHODS: Wistar male rats were used: Control (n=9); rats with prehepatic portal hypertension by triple partial portal vein ligation (TPVL; n=9); thioacetamide-cirrhotic rats (TAA; n=9) and TPVL-rats associated to TAA administration (TPVL+TAA; n=9). Three months after the operation, portal pressure (PP), mesenteric venous vasculopathy (MVV) and portosystemic collateral circulation were studied. Liver and ileal levels of malondialdehyde (MDA), as a lipid peroxidation marker, and catalase (CAT), glutathione peroxidase (GSH-Px), glutathione transferase (GSH-t) and cytosolic and mitochondrial superoxide dismutases (cSOD and mSOD), as antioxidative enzymatic mechanisms, were measured. RESULTS: Liver and ileal MDA increased in all the experimental groups, although the higher increase occurred in the ileum of rats with portal hypertension. CAT levels decreased in the liver and the ileum in the three experimental groups. The decrease in liver and ileal GSH-Px and GSH-t was greater in rats with portal hypertension, alone or associated with TAA. mSOD activation was demonstrated in the liver when portal hypertension was added to TAA. On the contrary, this compensatory response was not activated in the ileum, where mSOD was significantly decreased. CONCLUSION: Prehepatic portal hypertension by triple partial portal vein ligation impaired the enterohepatic antioxidative activity and aggravated the intestinal oxidative stress in thioacetamide-cirrhotic rats.

The inflammatory response recapitulates phylogeny through trophic mechanisms to the injured tissue.

The post-traumatic local acute inflammatory response is described as a succession of three functional phases of possible trophic significance: 1. Nervous or immediate (ischemia-reperfusion); 2. Immune or intermediate (infiltration by inflammatory and bacterial cells) and 3. Endocrine or late (angiogenesis with regeneration and/or cicatrization). Each of these phases emphasizes the trophic role of the mechanisms in the damaged tissue. Hence, the nervous phase is predominated by nutrition by diffusion; in the immune phase trophism is mediated by inflammatory cells and bacteria and, finally, in the endocrine phase, the blood circulation and oxidative metabolism play the most significant nutritive role. Since these trophic mechanisms are of increasing complexity, progressing from anoxia to total specialization in the use of oxygen to obtain usable energy, it could be speculated that they represent the successive reappearance of the stages that take place during the evolution of life on Earth, from ancient times without oxygen. In this sense, the inflammatory response could recapitulate phylogeny through the successive expression of pathophysiologic mechanisms that have a trophic meaning to the injured tissue.

Evaluation of two experimental models of hepatic encephalopathy in rats.

The serious neuropsychological repercussions of hepatic encephalopathy have led to the creation of several experimental models in order to better understand the pathogenesis of the disease. In the present investigation, two possible causes of hepatic encephalopathy, cholestasis and portal hypertension, were chosen to study the behavioral impairments caused by the disease using an object recognition task. This working memory test is based on a paradigm of spontaneous delayed non-matching to sample and was performed 60 days after surgery. Male Wistar rats (225-250 g) were divided into three groups: two experimental groups, microsurgical cholestasis (N = 20) and extrahepatic portal hypertension (N = 20), and a control group (N = 20). A mild alteration of the recognition memory occurred in rats with cholestasis compared to control rats and portal hypertensive rats. The latter group showed the poorest performance on the basis of the behavioral indexes tested. In particular, only the control group spent significantly more time exploring novel objects compared to familiar ones (P < 0.001). In addition, the portal hypertension group spent the shortest time exploring both the novel and familiar objects (P < 0.001). These results suggest that the existence of portosystemic collateral circulation per se may be responsible for subclinical encephalopathy.

Progressive hepatocytic fatty infiltration in rats with prehepatic portal hypertension.

BACKGROUND/AIMS: Homogenous evolution, with a narrow range of portal hypertension, degree of portosystemic shunt and hepatic atrophy has been described in the experimental model of prehepatic portal hypertension in the rat. However the great differences observed in the rats' liver weight could be attributed to a pathological alteration of the liver. Based on this, we performed an evolutive histological study of the liver. This study shows the existence of a progressive hepatocytic fatty infiltration. METHODOLOGY: Male Wistar rats with portal hypertension induced by triple stenosing ligation of the portal vein at 1 month (group II, n=4) and at 1 year (group IV, n=10) of postoperative evolution were used. The portal pressure, body, liver and splenic weights, types of collateral circulation and degree of mesenteric venous congestion were studied. The intracytoplasmatic lipid microvacuoles were quantified in hepatocytes with an image analyzer (software MIP/CID, Spain). The results were compared with those obtained in control rats with the same evolutive periods (Groups I and III). RESULTS: The hepatic fatty infiltration in Group II (TPVS 1 month) (30.12+/-53.92 micron2) is similar to that presented by Group III (Control 1 year) (16.52+/-45.20 micron2), while there is an increase (p<0.001) in Group IV (triple portal vein stenosis 1 year) (182.03+/-371.42 micron2) in relation to the other groups studied. The progressive hepatic fatty infiltration in triple portal vein stenosis rats is associated with a decrease of portal pressure and of the incidence of liver hepatic atrophy, portosystemic collateral circulation and mesenteric venous congestion. CONCLUSIONS: TPVS produces progressive hepatocytic fatty infiltration in the rat so that this prehepatic portal hypertension experimental model could also be considered as a hepatic steatosis model.

Posttraumatic inflammation is a complex response based on the pathological expression of the nervous, immune, and endocrine functional systems.

The successive phases that make up both the local and systemic posttraumatic acute inflammatory response could represent the expression of three concatenated pathological or "primitive" functional systems with trophic properties: the nervous, immune, and endocrine ones. The nervous functional system would play an important role in the phenomenon of ischemia-reperfusion, which would be represented by nutrition by diffusion that is either anaerobic (ischemia) or with defective use of oxygen (reperfusion) and, thus, with a limited energy requirement. The immune functional system would be represented by the infiltration of the tissues by inflammatory cells and bacteria, which would become mediators in providing nutrition to the injured tissues. Although the use of oxygen would still be defective, hypermetabolism and fever would occur. In these inflammatory response phases, the lymphatic is the most important circulation. The endocrine functional system would be the most specialized and would have high energy requirements because it would be represented by the blood capillary-mediated nutrition. Highly specialized epithelial cells would already possess a perfected oxidative metabolism. The successive expression of these three functional systems during embryonic development and also during the evolutionary development of our species could explain why the inflammatory response is a ubiquitous mechanism that is common to multiple diseases, because it is an integrator of the ontogeny and phylogeny.

Microsurgical extrahepatic cholestasis in the rat: a long-term study.

An experimental model of microsurgical cholestasis is studied as an alternative to the most frequently used surgical techniques, based on the section of the common bile duct. This microsurgical technique consists of the resection of the extrahepatic biliary tract, that is, of the common bile duct in continuity with the bile ducts that drain the four lobes of the rat liver. At 30 days of evolution, rats with microsurgical cholestasis do not develop biliary pseudocysts or intraperitoneal hilar hepatopulmonary abscesses and show an increase (p < 0.001) in total bilirubin (9.50 +/- 1.50 mg/dL vs. 1.60 +/- 0.35 mg/dL), bile acids (225 +/- 87 micromol/L vs. 12.5 +/- 14.50 micromol/L), gamma-glutamyltranspeptidase (375 +/- 143 U/L vs. 8 +/- 11 U/L), and alkaline phosphatase (73 +/- 25 U/L vs. 23 +/- 4 U/L) levels. The histological study shows fibrosis with biliary proliferation. The microsurgical cholestasis technique is a valid alternative to other techniques and can be an adequate experimental model for the study of etiopathogenic mechanisms of obstructive jaundice and especially to study extrahepatic biliary atresia.

Chronic portal hypertension in the rat by triple-portal stenosing ligation.

A surgical technique based on the development of a triple stenosing ligation is used to worsen the complications inherent to the prehepatic chronic portal hypertension. The results have been compared with those obtained in rats with a single-portal stenosing ligation. An increase (p <.05) in the body, liver, spleen, and kidney weights as well as a decrease (p <.001) in the testes weight to body weight ratio were produced in both groups of animals. In addition, the variability in the obtained weights, particularly in the liver weight, stands out. The incidence of portosystemic and portohepatic collateral circulation and of the mesenteric venous vasculopathy increases in the animals with triple-portal stenosing ligation. The new proposed technique is a valid alternative to the classic one that used single portal stenosing ligation.

Portal hypertension: return to fetal life to re-attempt differentiation?

We speculate on the final meaning of the alterations that characterize portal hypertensive enteropathy. The similarity of these alterations with certain morphofunctional characteristics of prenatal splanchnic development makes it possible to hypothesize that the dedifferentiation with return to early stages of development could constitute a portal hypertension induced pathogenic mechanism.

Neuro-immune-endocrine functional system and vascular pathology.

A new interpretation of the response to injury by the nervous, immune and endocrine system is proposed, in order to integrate biochemical knowledge into the respective clinical areas. The discovery that the signaling molecules of the classical nervous, immune and endocrine systems, that is, the neurotransmitters, cytokines and hormones, respectively, are expressed and perceived by the three systems, has enabled us to establish a functional concept of these systems. The hypothetical integration of different pathological processes in a functional response made up by three phases, the immediate or nervous, intermediate or immune and late or endocrine ones, makes it possible to consider that all of them represent different forms of expression of a functional response whose meaning is always the same, that is, inflammation. If the functions that characterize each one of these three phases represent the activity of the nervous, immune and endocrine systems, the biochemical knowledge could be integrated into the functional meaning of each system.

Behaviour of nucleolar organizer regions in the different Wistar rat liver lobes.

The area and number of silver-nucleolar organizer regions (Ag-NORs) in the hepatic lobes were determined in 3 male Wistar rats. There was a statistically significant increase in the percentage of Ag-NORs per nucleus in the right lateral and caudate lobe in relation to the left lateral and middle lobes. The area and number of Ag-NORs are greater in the caudate and right lateral lobes in relation to the left lateral and middle lobes. Since the Ag-NOR is a parameter which indicates hepatocytic protein synthesis, the different activity which corresponds to each lobe of the rat's liver makes it possible to assume that there is a functional heterogeneity which should be considered in the study of the hepatic regeneration according to the type of partial hepatectomy carried out.

Altered proteinogram in short term portal vein stenosed rats.

The electrophoretic pattern of serum proteins has been studied in short-term prehepatic portal hypertensive rats since atrophy is produced in the liver, which is the main origin of most of these proteins, during this postoperative period. After 28 days of evolution, rats (n = 9) with triple stenosing ligated portal vein showed hypoalbuminemia, hypo-alpha-globulinemia, hyper-alpha2-globulinemia and hyper-gamma-globulinemia, the albumin/globulin ratio decreased with respect to the control animals (n = 8). These alterations are associated with hepatic atrophy, portosystemic and portohepatic (44.4%) collateral circulation. The proteinogram alterations found in rats with short-term prehepatic portal hypertension suggest that hepatic failure exists in spite of potential portohepatic revascularization which is frequently originated by the development of portohepatic collateral circulation.

Auxiliary liver transplantation in the pig without portal blood: a study of a new technique for application to the heterotopic hepatic xenotransplant.

An auxiliary liver transplantation surgical technique in the pig, in which the graft only receives blood flow via the hepatic artery since an end-to-side portacaval shunt is performed in the donor animal prior to the transplantation, is described. The functional impairment of the receptor liver was done by a ligature of the choledoch, provoking death by sepsis in six of the transplanted animals at 8.16 +/- 6.61 days post transplantation. The auxiliary liver never suffered atrophy and prevented jaundice. It can be concluded that this simple surgical technique could be applied to the heterotopic xenotransplantation when the complications inherent in the immunological aggression are eliminated.

Changes in serum insulin levels during orthotopic liver transplantation in the pig.

Insulin and glucose levels were measured in 13 orthotopic liver transplantations in pigs. After portal revascularization a significant increase in the glucose levels takes place (p less than 0.01). When the infrahepatic vena cava clamp is removed, a significant increase in glucose and insulin concentrations (p less than 0.05) takes place. After the end of the operation the insulin levels decrease significantly with respect to initial values (p less than 0.05).

A 70% hepatectomy in the rat using a microsurgical technique.

An experimental model of 70% hepatectomy in the rat using a microsurgical technique is described. An operative microscope (Carl Zeiss 10x2) dissection made it possible to identify the hilar anatomical variations and then to avoid unexpected damage to the arterio-portal and biliary branches belonging to the remaining hepatic parenchyma. The use of this microsurgical technique allows for a more homogenous experimental model because it avoids the functional impairment of the remaining liver.

Washout of the pig liver with Haemaccel after hypothermic preservation.

In hepatic preservation by simple perfusion and hypothermic storage, a portal and hepatic washout before revascularization would avoid receptor hyperkaliema. In this report we study the effectiveness of this washout with Haemaccel at room temperature. Large-White pigs were used and eight livers were perfused "in situ" via the portal wein with Hartmann's solution containing 10,000 IU of heparin at 4 degrees C, and afterwards, via portal and arterial routes with C2 solution at 4 degrees C. After a cold ischemia time of less than 31/2 hours a liver washout via the portal vein and hepatic artery with Haemaccel before portal revascularization was done. The high concentrations of glucose, K+, GOT, GPT and LDH in the effluents obtained during the washout are attributed to Haemaccel hyperosmolarity. A portal and arterial hepatic washout associated with free drainage of the first 50-100 ml of portal venous blood after hepatic portal revascularization through the infrahepatic inferior vena cava (IH-IVC), prevents hyperkaliemia from occurring after a portal and arterial revascularization in the orthotopic liver transplant (OLT) in pigs.

Portal and arterial washout after hypothermic preservation of the pig liver: prevention of hyperkalaemia after revascularization.

In an orthotopic liver transplantation (OLT), portal revascularization may produce acidosis and hyperkalaemia due to loss of intracellular acid metabolites and K+ during hypothermic preservation. To verify the effectiveness of portal and arterial washout in preventing hypokalaemia after liver preservation, an OLT was done in 18 large-white pigs. The donor livers were perfused in situ via the portal vein with Hartmann's solution containing 1.000 IU of heparin at 4 degrees C. Afterwards, a cold Collins C2 solution was perfused either in vitro (group A) or in situ (group B). The cold ischemia time in both groups was less than 3 1/2 h. Before doing the portal revascularization of the donor livers, a washout via the portal vein and hepatic artery with saline serum was performed. The concentration of K+, glucose, GOT and LDH in effluents obtained through infrahepatic inferior vena cava were significantly lower in group B than in group A. Simple washout of the livers prior to revascularization prevented hyperkalaemia in both groups.

[Critical Care Surgery Unit in general and digestive system surgery]. / Unidad Quirúrgica de Cuidados Críticos en cirugía general y del aparato digestivo.

In a 5-bed Critical Care Surgery Unit (UQCC) where surgeons are in charge of care, 126 patients admitted over 5 consecutive months were divided into 4 groups according to the treatment given: Group I (n = 49): elective gastrointestinal surgery; Group II (n = 52): emergency gastrointestinal surgery; Group III (n = 15): non-gastrointestinal surgery; Group IV (n = 10): medical treatment. The mean stay of all patients in the Surgical Critical Care Unit was 6 +/- 7.7 days and the global mortality was 7.9% (n = 10). The mean age of the patients who died was 71.5 +/- 7.3 years and 70% of the deaths corresponded to Group II. Forty percent of the patients who died had systemic candidiasis. Among the factors implicated in the mortality acute gastrointestinal pathology per se, the coexistence of chronic systemic disease and advanced age were prominent. We discuss the need for typifying the role of Surgical Critical Care Units in General and Gastrointestinal Surgery.

Hepatic cytochrome oxidase in rats with microsurgical cholestasis or portocaval shunt.

AIMS: portocaval shunt and extrahepatic cholestasis are experimental models of chronic hepatic insufficiency of different etiology and histological characteristics, and which probably also differ in the mechanism of impairment of oxidative metabolism. To test this hypothesis we measured hepatic cytochrome oxidase. METHODS: cytochrome oxidase was assayed with a histochemical technique in three groups of Wistar rats: A (n = 8) control; B (n = 8) microsurgical extrahepatic cholestasis; and C (n = 8) end-to-side portocaval shunt. RESULTS: cytochrome oxidase activity was lowest in group B, both in the left middle (p = 0.00019) and in the inferior caudate (p = 0.00014) hepatic lobes, and was highest in group C in both hepatic lobes, especially in the left middle lobe (p = 0.0029). CONCLUSION: the decrease in cytochrome oxidase activity in the liver of rats with extrahepatic cholestasis and the increase in animals subjected to portal flow deprivation demonstrate the different nature of the impairment in hepatic oxidative metabolism in these two pathological conditions.

Cytochrome oxidase activity in splanchnic organs of portal hypertensive rats.

OBJECTIVE: Portal hypertension is characterized by hyperdynamic splanchnic circulation associated with the development of portosystemic portal collateral circulation. Since blood flow regulation mechanisms in the splanchnic organs can be metabolic, its metabolic capacity has been studied using the mitochondrial enzyme cytochrome C oxidase as histochemical marker. METHOD: Cytochrome oxidase was quantified with a histochemical technique in the liver, pancreas and small bowel of Wistar rats in the control group (n = 8) and in rats with portal hypertension by triple stenosing ligation of the portal vein (n = 9) at 28 days of evolution. RESULTS: All rats with portal hypertension develop portosystemic collateral circulation. In these animals, cytochrome oxidase activity increases (p < 0.01) in the liver (left lateral lobe, periportal zone: 91.81 +/- 5.18 vs. 86.03 +/- 2.82) exocrine pancreas (125.6 +/- 7.25 vs 117.57 +/- 6.43; p < 0.05) as well as in the mucosa (crypts) and duodenum serosa, jejunum and ileum while it decreases in the pericentral zone of the hepatic acinus and intestinal villi. CONCLUSION: Cytochrome oxidase is considered an endogenous marker of local tissular metabolic capacity, so that its increased activity in the small bowel mucosa, crypts, exocrine pancreas and visceral peritoneum may be a metabolic factor that induces splanchnic hyperdynamic circulation in short-term portal hypertensive rats.