Amiodarone has widespread applications in the treatment of supraventricular and ventricular tachyarrythmias. Long-term administration of amiodarone is well known to induce a full spectrum of hepatic manifestations from benign increases in aminotransferases levels to fatal hepatitis and cirrhosis .
Sustained liver injury causes the development of fibrosis, with subsequent portal hypertension, liver failure, and hepatocellular cancer. For patients with end-stage fibrosis (cirrhosis) or nonmetastatic cancer, liver transplantation is the only treatment option .
Currently, liver transplantation is the only effective treatment, but it is associated with several problems, including a shortage of donors, operative damage, the risk of immune rejection, and high costs. Furthermore, liver transplantation induces several long-term side effects, such as chronic renal failure, post-transplantation lymphoproliferative disorder, and cardiovascular complications. The emerging field of stem cell therapy may aid in improving the treatment of liver diseases .
Mesenchymal stem cells (MSCs) reside in most adult tissues, where they contribute to normal tissue turnover and repair. In addition to their direct capacity to differentiate into multiple cell lineages, for example osteoblasts, chondrocytes, tenocytes, and myocytes, undifferentiated cells with unlimited self-renewal capacity can be found in most organs and tissues of adult organisms . MSCs have been isolated from a number of organs or tissues including adipose tissue, muscle, and umbilical cord. MSCs have been considered as one of the most promising ‘off-the-shelf’ cell therapeutics . Human mesenchymal stem cells (hMSCs) have been commonly used in cell replacement therapy . It has been postulated that hMSCs represent a more economical and easily processed source of MSCs .
The present study aimed to determine the possible effect of MSC therapy on liver tissue damage using amiodarone-induced liver injury as a model in albino rats.
Materials and methods
The study was carried out at the animal house of Faculty of Medicine, Cairo University, according to the guidelines for the care and use of laboratory animals.
Amiodarone (cordarone) was used as ampoules 150mg/3ml dissolved in Tween 80 (Sanofi Corporation, Paris, France).
Twenty-three male albino rats weighing 150–200g were included in the study. They were housed in a temperature-controlled and light-controlled room (12-h light/dark cycle), with free access to food and water. They were divided into three groups:
Group I (control group): This group included six rats, three in each experimental group. Each rat was given 0.5ml Tween 80 (solvent of amiodarone) (Sanofi Corporation) daily orally for 2 weeks.
Group II (amiodarone group): This group included 10 rats that received 5.4mg amiodarone dissolved in Tween 80 daily orally for 2 weeks .
1. Three animals from this group were sacrificed to confirm liver injury.
2. The remaining seven animals received amiodarone for 2 weeks and were held for 4 weeks without therapy .
A stock solution of the drug was prepared weekly and kept at 4°C. The required dose for each rat was introduced into the mouth using a syringe with a metal tube instead of the needle.
Group III (stem cell therapy group): This group included seven rats that received amiodarone for 2 weeks and were then injected with 0.5ml cultured and labeled stem cells suspended in phosphate-buffered saline in the tail vein . The rats were sacrificed 4 weeks following stem cell therapy (the total time of sacrifice was after 6 weeks) . Stem cells were isolated from cord blood .
Cord blood collection was performed at the Gynaecology Department, Faculty of Medicine, Cairo University. Stem cell culture, labeling, and phenotyping were performed at the Hematology Unit, New Kasr El Aini Hospital, Cairo University.
Cord blood collection : The storage and transport temperature was 15–22°C, the transport time was 8–24h, the sample volume was 65–250ml, and none of the samples showed signs of coagulation or hemolysis.
Mononuclear cell fraction isolation and propagation : The mononuclear cell fraction was isolated by loading 30ml of whole blood onto 10ml of Ficoll density media (Healthcare Bio-Sciences,,Princeton, New Jersey, USA) in 50ml polypropylene tubes, which were centrifuged for 30min at room temperature. The interphase was collected after aspirating and discarding the supernatant. The interphase was washed with 20ml PBS and centrifuged at room temperature. The supernatant was aspirated and the cells were washed with PBS a second time. The cells were resuspended in the isolation media and transferred to culture dishes. The isolation medium was low-glucose DMEM (Dulbecco's modified Eagles medium; Cambrex Bio-Science, Walkersville, United States) supplemented with small amounts of dexamethazone (10−7 mol/l) (Sigma-Aldrich, Taufkirchen,Germany), penicillin (100IU/ml) (Invitrogen, Carlsbad, California, USA), streptomycin (0.1mg/ml) (Invitrogen), and ultraglutamine (2mmol/l) (Cambrex Bio-Science). Incubation was carried out at 38.5°C in a humidified atmosphere of 5% CO2.
Culture : The isolation media were replaced after overnight incubation (12–18h) in order to remove nonadherent cells. The media were replaced every 3 days until MSC colonies were observed. The cultures were inspected daily for the formation of adherent spindle-shaped fibroblastoid cell colonies. Subculturing was carried out by chemical detachment using 0.04% trypsin. Later, when cell numbers were sufficient, expansion was carried out in 25 or 75cm2 tissue culture flasks.
Labeling : MSCs were labeled by incubation with a ferumoxide injectable solution (25μg Fe/ml, Feridex; Berlex Laboratories, Montville, New Jersey, USA) in the culture medium for 24h, with 375ng/ml poly L lysine added 1h before cell incubation. Labeling was assessed histologically using Prussian blue. Feridex-labeled hMSCs were washed in PBS, trypsinized, washed, and resuspended in 0.01mol/l PBS at a concentration of 1×1 000 000 cells/ml.
Cell viability analysis: Cell viability was carried out using the trypan blue dye exclusion test. This method is based on the principle that viable cells do not take up certain dyes, whereas dead cells do.
Flow cytometry : Flow cytometric analyses were carried out on a Fluorescence Activated Cell Sorter (FACS) flow cytometer (Coulter Epics Elite, Miami, Florida, USA). hMSC were trypsinized and washed twice with PBS. A total number of 1×105 hMSC were used for each run. To evaluate the hMSC marker profile, cells were incubated in 100μl of PBS with 3μl of CD44-FITC for 20min at room temperature. The antibody concentration was 0.1mg/ml. Cells were washed twice with PBS and finally diluted in 200μl of PBS. The expression of the surface marker was assessed by the determination of the mean fluorescence of CD44 (a MSC marker). The percentage of cells positive for CD44 was determined by subtracting the percentage of cells stained nonspecifically with isotype control antibodies.
The animals were sacrificed using a lethal dose of ether. Liver specimens were removed and fixed in 10% formol saline for 24h. Paraffin blocks were prepared and 5μm thick sections were subjected to the following stains:
(1) H&E .
(2) Masson's trichrome stain .
Liver sections were stained with Prussian blue stain  for assessment of iron oxide-labeled mesenchymal therapeutic stem cells in the liver tissue.
CD44 immunostaining 
The marker for human MSCs (CD44) was mouse monoclonal antibodies (ab6124). The sections were pretreated using heat-mediated antigen retrieval with sodium citrate buffer for 20min and then incubated with CD44, 5μg/ml, at room temperature in a moist chamber for 15 min. DAB was used as the chromogen. The section was then counterstained with Mayer's hematoxylin. The reaction is membranous. Human kidney carcinoma sections were used as positive control specimens. However, one of the liver sections was used as a negative control by excluding the step of application of the primary antibody.
α-Fetoprotein immunostaining 
α-Fetoprotein (AFP) is a major plasma protein in the fetus, where it is produced by the yolk sac and the liver. Mouse monoclonal antibody (IW-MA1001) was used. The sections were pretreated using heat-mediated antigen retrieval with sodium citrate buffer for 20min and then incubated with 0.5ml AFP antibody for 60min at room temperature. DAB was used as the chromogen. The section was then counterstained with Mayer's hematoxylin. The reaction is nuclear. Human liver sections were used as positive control specimens. However, one of the liver sections was used as a negative control by excluding the step of application of the primary antibody.
Using the Leica Qwin 500 LTD software image analysis computer system, Cambridge, UK. In Masson's trichrome-stained sections, the area percent of collagen fibers was measured using color detect and the binary mode. The measurements were performed in 10 high-power fields in the control and the experimental groups using a magnification of ×200.
Assessment of the optical density of the AFP-positive cells was performed in AFP-immunostained sections using the interactive measurements menu. The measurements were performed in 10 high-power fields in the control and the experimental groups using a magnification of ×400.
Quantitative data were summarized as means and SDs and compared using one-way analysis of variance. Any significant analysis of variance was followed by a Bonferroni post-hoc test to detect which pairs of groups caused the significant difference. P-values less than 0.01 were considered statistically significant. Calculations were carried out on SPSS 9.0 software,IBM,United state. .
H&E-stained sections of the liver of the control group showed hepatic lobules, each consisting of a central vein surrounded by radiating hepatocytes separated by blood sinusoids. The hepatocytes showed an acidophilic cytoplasm and vesicular nuclei (Fig. 1). Between the hepatic lobules, there were portal tracts, each containing a portal vein, a hepatic artery, and a bile duct (Fig. 2).
In the amiodarone-treated group, some hepatic lobules showed congested dilated blood sinusoids (Figs 3 and 4) as well as pink homogenous materials in between the hepatocytes (Fig. 3). The hepatocytes showed variable degrees of effects. For instance, some hepatocyte nuclei showed margination of chromatin materials against the nuclear membrane, resulting in a crescent-shaped appearance (Figs 3 and 4). In addition, the anaphase stage of mitosis was detected in one hepatocyte nucleus (Figs 4 and 8). Some other hepatocytes had dark nuclei and a strong acidophilic cytoplasm (Figs 4 and 5), whereas others had dark nuclei (Fig. 5). Moreover, ballooned hepatocytes containing dark nuclei and a vacuolated cytoplasm were also observed (Fig. 6). Some portal areas showed intense mononuclear cellular infiltration including fibrocytes (Figs 7 and 8) and a congested dilated portal vein (Fig. 7). Eosinophilic Mallory bodies were also observed (Fig. 8).
In the stem cell therapy group, most of the hepatic lobules showed regularly arranged hepatocytes with an acidophilic cytoplasm and vesicular nuclei around a congested central vein. In addition, dilated blood sinusoids were observed (Fig. 9). Some of the hepatocytes surrounding the central veins showed dark nuclei. Moreover, the blood sinusoids were congested (Fig. 10).
Masson's trichrome-stained sections
In the control group, minimal collagen fibers were observed around the portal tract and the central vein (Fig. 11). In the amiodarone group, extensive collagen fibers existed around some central veins and extended between hepatocytes (Fig. 12) in and around some portal tracts (Fig. 13). In the stem cell therapy group, minimal amount of collagen fibers was detected around the central vein (Fig. 14) and in the portal tract (Fig. 15).
Prussian blue-stained sections
Sections in the liver of control rats showed negative staining with Prussian blue around the central vein (Fig. 16). The stem cell therapy group showed multiple Prussian blue-positive cells around the portal vein (Fig. 17).
Sections in the liver of control rats showed negative immunostaining with CD44 among hepatocytes (Fig. 18). The stem cell therapy group showed CD44-positive cells among the hepatocytes (Fig. 19).
Sections in the liver of control rats showed positive immunostaining with AFP in the hepatocytes (Fig. 20). In the amiodarone group, an apparent reduction in positive immunostaining was detected in the hepatocytes (Fig. 21). The stem cell therapy group showed multiple-positive AFP-immunostained cells (Fig. 22).
The mean area % of collagen fibers was significantly increased in the amiodarone group compared with the control and the stem cell therapy groups (Table 1 and Fig. 23). The mean optical density of AFP-positive immunostaining cells was significantly increased in the stem cell therapy group compared with the amiodarone group (Table 2 and Fig. 24).
The current study showed a modulating effect of cord blood stem cell therapy on amiodarone-induced liver injury in albino rats. This was indicated by histological, histochemical, immunohistochemical, and morphometric studies.
In the present study, the administration of amiodarone resulted in congested and dilated blood sinusoids in some hepatic lobules. This is in agreement with the observations of other investigators who have reported that hepatic sinusoidal dilatation is characterized by widening of hepatic capillaries . Sinusoidal dilatation was common in portal hypertension, which included various types of architectural alterations including perisinusoidal fibrosis or incomplete septal cirrhosis. These architectural alterations were related to xenobiotics, which act by inducing portal or sinusoidal obstruction. Other authors attributed the occurrence of sinusoidal dilatation to the activation of perisinusoidal cells that had contractile properties .
In the present study, in group II, some hepatocytes nuclei showed margination of chromatin materials against the nuclear membrane, resulting in a crescent-shaped appearance, whereas others showed dark nuclei. In addition, some hepatocytes showed dark nuclei with a homogenous strong acidophilic cytoplasm. These degenerative changes were confirmed by other investigators, who defined apoptosis as a specific morphological feature of cell death, and characterized by membrane blebbing, shrinkage of cells, chromatin condensation, and nuclear fragmentation . Others have reported that amiodarone, to varying degrees, could produce apoptosis and necrosis of the hepatocytes .
In addition, the anaphase stage of mitosis was detected in some hepatocyte nuclei. Some authors have reported the mitotic figures as proliferation indices .
In the current study, the administration of amiodarone resulted in intense mononuclear cellular infiltration; pink homogenous materials (fluid exudate) were detected between the hepatocytes and eosinophilic Mallory bodies. This was in agreement with the results of other investigators, who observed polymorph-nuclear infiltrates and Mallory hyaline degenerated bodies after the administration of amiodarone . Other authors have reported that amiodarone steatohepatitis is characterized by prominent Mallory hyaline and neutrophilic satellitosis  and that amiodarone-induced liver toxicity was related to accumulation of drug.
In the present study, some hepatocytes in group II appeared ballooned with a vacuolated cytoplasm and dark nuclei. This has been confirmed by other investigators , who called it a ‘foamy’ appearance of hepatocytes. In addition, they reported that the administration of amiodarone was associated with a different type of lipid accumulation called ‘phospholipidosis’, characterized by the accumulation of drug in the lysosomes and Kupffer cells.
In the present work, in the stem cell therapy group, there was a marked improvement in the hepatocytes. Still, some of the hepatocytes showed dark nuclei. This was in agreement with the results of other investigators, who showed that hMSCs could ameliorate hepatic injury and could represent a potential therapy for liver injury . They reported that hMSCs could inhibit apoptosis and facilitate proliferation. In addition, other authors have reported that stem cells are considered a ‘reservoir’ of potential cells at various stages of development used for the restoration and regeneration of damaged tissues and organs. Under proper conditions, stem cells may differentiate into specialized tissues and organs. They are self-sustaining and self-replicating for a long duration of time. These unique features make them a promising tool for studies on therapy for chronic liver disease. They could be differentiated into hepatocyte-like cells and also generated functional hepatocytes .
In the present study, in the portal area, cellular infiltration including multiple fibrocytes was detected in the amiodarone-treated group. This was concomitant with the presence of extensive collagen fibers around some central veins and portal tracts. The latter findings were confirmed by an increased area % of collagen fibers compared with the control and stem cell therapy groups. This has been confirmed by other researchers, who found portal and pericellular sinusoidal fibrosis and lymphocytic infiltrates with polymorphonuclear leukocytes after the administration of amiodarone . Others have reported that amiodarone increases mitochondrial formation of reactive oxygen species. Reactive oxygen species with lipid peroxidation products may cause mitochondrial dysfunction, which can lead to apoptosis and necrosis, activating a cascade leading to fibrosis .
Liver fibrosis occurs in response to drug abuse. Liver fibrosis is reversible, whereas cirrhosis, the end-stage result of fibrosis, is irreversible. Thus, efforts focus primarily on events that lead to the early accumulation of a fibrotic scar with the aim of identifying therapeutic targets to slow its progression .
In the present work, in group III, less cellular infiltration was observed. Reduced fibrosis was confirmed by minimal collagen fibers around the central veins and some portal tracts. This was in agreement with the results of other researchers, who showed that human umbilical cord mesenchymal stem cells (CBMSCs) could improve liver fibrosis . Other authors have shown that reduced fibrosis most likely relied on bioactive factors or cytokines released from the stem cells. These cytokines trigger liver regeneration, reduction in the activation of hepatic stellate cells, and enhancement in liver cell repair, and effectively cure liver fibrosis . Other investigators have reported that transplanted stem cells produce albumin, hepatocyte growth factor, and metalloproteinase in chemically injured livers to decrease liver collagen. Thus, stem cells may be useful for the treatment of liver fibrosis .
In the present work, the stem cell therapy group showed Prussian blue-positive cells around the portal vein. In addition, CD44-positive cells were observed among the hepatocytes. This was confirmed by other authors, who observed infused (CBMSCs) in the perivascular region of the liver . Other investigators have concluded that CBMSCs transplantation could significantly improve the survival of rats with liver injury. The underlying mechanisms involved might include transdifferentiation into hepatocyte-like cells and targeted migration of these cells to liver lesion sites .
In the present study, in the control group, the AFP immune reaction was positive in the hepatocytes, whereas in the amiodarone group, the reaction was weak positive. The stem cell therapy group showed multiple-positive AFP cells. The latter findings were confirmed by an increased optical density of AFP-positive cells in the stem cell therapy group compared with the amiodarone group. This was in agreement with the results obtained by other workers, who concluded that under certain conditions, these AFP-positive cells were products of liver cells differentiated from CBMSCs . Other investigators have reported that this differentiation occurred through the interaction of stem cells with damaged liver cells .
It can be concluded that cord blood MSC therapy can induce definite amelioration of the morphological changes associated with amiodarone-induced liver injury, provided therapy is initiated early in the development of the injury.
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Conflicts of interest
There are no conflicts of interest.
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