Fine structure and function of kupffer cells

Kupffer cells are macrophages that are attached to the luminal surface or inserted in the endothelial lining of hepatic sinusoids. In this site, Kupffer cells play a key role in host defense by removing foreign, toxic and infective substances from the portal blood and by releasing beneficial mediators. Under some conditions, toxic and vasoactive substances also are released from Kupffer cells which are thought to play a role in a variety of liver diseases. Many of these activities may be modulated by the levels of gut derived endotoxin normally present in the portal blood. The ultrastructural aspects of Kupffer cell structure function in situ are best studied using perfused-fixed livers. In fixed livers, transmission and scanning electron microscopy reveal Kupffer cells during health to be irregular in shape with their exposed surfaces presenting numerous microvilli, filopodia, and lamellopodia. Long filopodia penetrate endothelial fenestrae to secure Kupffer cells to the sinusoid lining. Specific membrane invaginations known as worm-like bodies or vermiform processes are seen in the cytoplasm of Kupffer cells as are numerous endocytotic vesicles and lysosomes which vary in density, shape and size. Sometimes, annulate lamellae connected to the rough endoplasmic reticulum also are found. The principal endocytic mechanisms of Kupffer cells are phagocytosis of particulates and cells, and bristle-coated micropinocytosis for fluid-phase endocytosis of smaller substances. Many of these events are mediated by specific receptors. In some species, Kupffer cells can be distinguished from other sinusoidal lining cells and monocytes by specific cytoplasmic staining or monoclonal antibodies. Kupffer cells have been shown to be of monocytic origin as well as having the capacity for self-replication.     

Fine structure of hepatic sinusoids and sinusoidal cells in disease

Liver sinusoids are special capillaries that are limited by fenestrated endothelial cells, without a genuine basement membrane, surrounded by perisinusoidal cells storing vitamin A, and harbouring Kupffer cells and pit cells, resident macrophages, and large granular lymphocytes, respectively. Each nonparenchymal cell and parenchymal cell of the liver interacts with all others and with the extracellular matrix. Therefore, the functional ability of each cell is constantly being modified by the metabolic activity of the others. Human liver biopsies (132), needle or surgical, perfusion-fixed with glutaraldehyde and processed for transmission electron microscopy (TEM), and occasionally for scanning electron microscopy (SEM), were examined. The study included liver diseases (such as alcoholic liver diseases, benign and malignant liver tumors, cholestasis of various origins, fulminant hepatitis, acute rejection after orthotopic liver transplantation, Budd-Chiari syndrome), as well as general or extrahepatic diseases (such as diabetes, hemochromatosis, hypervitaminosis A, various hematological disorders), and normal controls. Ultrastructural abnormalities are described and illustrated under two different headings: (1) elementary lesions of sinusoidal cells (endothelial, Kupffer, perisinusoidal and pit cells), nonsinusoidal cells (in the space of Disse and/or in the lumen), the extracellular matrix; and (2) the major pathological entities including perisinusoidal fibrosis, capillarization of sinusoids, sinusoidal dilatation, and peliosis. In the discussion, an overview of the major abnormalities reported in the literature is presented, and some specific questions regarding (1) perisinusoidal fibrosis in liver with normal histology, (2) the overload of perisinusoidal cells with lipids in non-hypervitaminosis A intoxication and (3) the etiological relationship of sinusoidal dilatation, peliosis, perisinusoidal fibrosis, or sinusoidal tumors with drugs and toxic compounds are discussed. In the event that lesions are not specific to any diagnosis, the knowledge of the ultrastructure of sinusoids is extremely useful from the perspective of the liver as an ecosystem.     

Use of multiphoton microscopy to diagnose liver cancer and lung metastasis in an orthotopic rat model

Liver or lung biopsy for suspicious lesions has several disadvantages such as bleeding, bile leak or pneumothorax, needle track seeding, and time-consuming histopathological procedure. The ability to directly observe cellular and subcellular details and then perform "optical biopsy" is a major goal in the development of new interventional techniques. Multiphoton microscopy (MPM) enables real-time noninvasive visualization of tissue architecture and cell morphology in live tissue. We performed a study to evaluate whether MPMcan make real-time optical diagnosis for liver cancer and lung metastasis using an orthotopic rat model with Morris hepatoma. We found that real-time high-resolution MPMimaging could clearly show tissue architecture and cell morphology. In the normal liver tissue, MPMimaging clearly revealed the blood-filled sinusoids and cords of hepatocytes. In the cancerous tissue, MPMimaging clearly illustrated that cancer cells displayed marked cellular and nuclear pleomorphism. MPMimages were comparable to golden standard hematoxylin-eosin staining images. Moreover, MPMimaging had deep penetration with the capability of optical sectioning. In short, MPMcan make real-time optical diagnosis for liver cancer and lung metastasis. This study provides the groundwork for further using multiphoton endoscopy to perform real-time noninvasive "optical biopsy" for liver cancer and lung metastasis in the near future.     

Tracing DiO-labelled tumour cells in liver sections by confocal laser scanning microscopy

Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO‐labelled colon carcinoma cells (CC531s) in 100 µm thick liver sections. In vitro and in vivo experiments revealed that DiO‐labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO‐labelled CC531s cells could be traced all over the tissue depth and anti‐metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine‐phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO‐labelled cells in tissue sections.     

Cytotoxic effects of permethrin on mouse liver and spleen cells

This study analyzed the histopathological and histochemical effects of different dosages of permethrin on liver and spleen cells of mice, in order to evaluate the toxic potential of this substance and the possible impairments that this chemical causes in different tissues of nontarget organisms (laboratorial conditions). The results showed that permethrin caused severe alterations in the liver cells, reducing the size of the nuclei and causing hydropic degeneration of the hepatocytes, in addition to stimulating the proliferation of Kupffer cells, altered the amount of proteins, polysaccharides, lipids, and vacuoles in the cytoplasm of the hepatocytes and congested the hepatic capillaries. As for the spleen of the treated mice, no alterations were observed in the morphology in relation to the control group, what would suggest that the spleen would continue performing its functions, without suffering morphological alterations even in the presence of the toxic agent.     

Imaging amoeboid cancer cell motility in vivo

The availability of multi-photon intravital microscopy has recently allowed researchers to start to visualise the dynamic behaviour of cancer cells in vivo. This imaging has revealed that many cancer cells ranging from carcinoma to melanoma move in an amoeboid manner in order to invade surrounding tissue and escape from the primary tumour. This mode on cell motility is extremely rapid and does not require the activity of proteases to degrade the extra-cellular matrix (ECM). This review details the techniques that are available to study cell motility in vivo and discusses the current knowledge about the mechanisms of amoeboid cell motility.     

Ultrastructure of in situ perfusion-fixed avian liver, with special reference to structure of the sinusoids

Broiler chicken and laying hen livers were fixed using a simple technique of in situ puncture perfusion of cacodylate-buffered fixative, which allowed characterisation of the fine structure of hepatic parenchyma, hepatocytes, bile ductules, and, in particular, the sinusoidal cells including endothelial, Kupffer, and Ito cells. Sinusoidal endothelial cells with their bulging perinuclear cytoplasm, evident in both transmission and scanning electron micrographs, were easily distinguishable from Kupffer cells, which possessed numerous pseudopodia. Bile ductular epithelium and hepatocytes of the laying hens contained large amounts of lipid. The ultrastructural characteristics of intercalated cells (putative extra-sinusoidal macrophages of chicken liver) are described and their possible role as precursors of Kupffer cells is discussed.     

Noninvasive 3D vital imaging and characterization of notochordal cells of the intervertebral disc by femtosecond near-infrared two-photon laser scanning microscopy and spatial-volume rendering

The central region of the intervertebral disc (IVD) in infant humans is made and maintained by notochordal cells (NCs). These cells disappear during maturation to be replaced by mature chondrocyte-like cells. NCs are completely different morphologically from the mature chondrocyte-like IVD cells and have complex and essential functions but little is known about them. Recently, two-photon laser scanning microscopy (TPLSM) using near-infrared (NIR) femtosecond pulsed lasers has emerged as a promising noninvasive optical technique for observing unfixed living 3D biological specimens in situ and in vitro. Several lines of evidence suggest that compared with conventional laser scanning confocal microscopy (LSCM), femtosecond NIR laser-based TPLSM has any number of advantages including 3D resolution without a spatial filter (confocal pinhole), minimal photobleaching, and photodamage above and below the focal plane, and importantly, greater depth penetration. We have thus taken advantage of these unique features of femtosecond laser-based TPLSM for vital 3D imaging in conjunction with advanced spatial-volume rendering modalities to compare morphologies of NCs/clusters from pig caudal discs with chondrocyte-like IVD cells from bovine caudal discs, both in ex vivo tissue and when isolated and grown in vitro within 3D alginate scaffolds. Our results provide evidence that (a) ex vivo notochordal tissue consists of areas with NC clusters, and those dominated by tubular structures of low cell density (b) within 3D in vitro scaffolds the morphology of NC is heterogeneous and the cells contain distinct cytoplasmic vacuole-like structures occasionally including acidic subinclusions (c) a quantitative determination based on 3D spatial and volumetric-rendering reveals an average NC diameter of 22.05 microm (range 11.96-46.63 microm) and NC volume of 9701 microm(3) (2041-36427 microm(3)) whereas chondrocyte-like cells have a mean volume of 3279 microm(3) and diameter of 12.20 microm. Taken together, this study demonstrates that femtosecond TPLSM has unique advantages over other conventional histological and in particular LSCM for high resolution noninvasive vital characterization of notochordal and chondrocyte-like cells of IVD over extended depths beyond 300-500 microm.     

Action of the chemical agent fipronil (active ingredient of acaricide Frontline®) on the liver of mice: an ultrastructural analysis

Fipronil, active ingredient of the acaricide Frontiline®, is a phenyl‐pyrazolic derivative, and its efficacy in the elimination of several plagues, even in low concentrations, has already been demonstrated; however, its effect on nontarget organisms has not been thoroughly explained. In this sense, the objective of this study was to evaluate the effects of different dosages of fipronil on the liver of mice in artificial conditions. Results showed that the animals exposed to fipronil present significant ultrastrucutural changes in hepatic cells with evident cellular and cytoplasm disorganization in hepatocytes characterized by an increase in the number of organelles, mainly mitochondria and rough endoplasmic reticulum, organelles that, in the case of the exposed animals, were probably responsible for the enzymes' synthesis that have the function of inactivating the toxic metabolites. A fat accumulation in the hepatocytes' cytoplasm (steatosis) was observed, in addition to extended vacuolated areas, mainly in regions next to the cell nucleus. Alterations observed in the nuclei of the hepatocytes pointed out cell death processes. Moreover, Kupffer cells increased in number (hyperplasia) suggesting an increase in the phagocytic activity of the liver in the exposed animals. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.     

Ultrastructural alterations in liver of mice exposed chronically and transgenerationally to aqueous extract of betel nut: Implications in betel nut‐induced carcinogenesis

The aqueous extract of betel nut (AEBN) induces the formation of preneoplastic nodules in the liver of Swiss Albino mice and leads to increased predisposition to cancer when administered transgenerationally. The aim of this investigation was to elucidate the alterations in ultrastructure of subcellular organelles in the liver nodules using transmission electron microscopy and to determine whether these alterations have implications in AEBN‐induced carcinogenesis. Male and female Swiss Albino mice were exposed to AEBN chronically and transgenerationally at a dose of 2 mg/mL in drinking water for 24 weeks. Extensive polymorphism was noted in nuclear shape and heterochromatin organization. Heterochromatin aggregation and marginalization were observed in the nuclei of chronically exposed mice, whereas transgenerationally exposed mice exhibited dispersion or loss of heterochromatin. The nuclear envelope was disrupted, and the nucleoli were enlarged in chronically exposed mice, whereas in transgenerationally exposed mice the nucleoli were reduced in size or totally absent. The cisternae of the rough endoplasmic reticulum were dilated and disrupted, and a large number of autophagic vesicles were observed in both chronically and transgenerationally exposed mice. Atypical mitochondria that underwent extensive cristolysis and progressively declined in size and number from the chronically exposed mice to the different generations of transgenerationally exposed mice were also observed. Thus, exposure to AEBN resulted in severe loss of ultrastructural integrity of cells in the liver nodules, and the progressive loss of mitochondrial function appeared to play a significant role in increasing the predisposition to cancer of mice exposed transgenerationally to AEBN. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.     

Morphological and molecular pathology of CCL4‐induced hepatic fibrosis in connexin43‐deficient mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl₄)‐induced hepatic fibrosis in mice. We administered CCl₄ to both Cx43‐deficient (Cx43^+/?) and wild‐type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real‐time PCR. We demonstrated that Cx43^+/? mice developed excessive liver fibrosis compared with wild‐type mice after CCl₄‐induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43^+/? mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43^+/? mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl₄‐treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Phagocytosis of apoptotic cells by liver: a morphological study

The present review deals with the morphological features of the removal of apoptotic cells by liver. The engulfment of cells undergoing apoptosis can be considered a specialized form of phagocytosis, playing a major role in the general tissue homeostasis in physiological and pathological conditions. In fact, defects of phagocytosis of apoptotic cells might have deleterious consequences for neighboring healthy cells, i.e., pathogenesis of inflammatory disease or dysregulation of the immune system. Phagocytosis of apoptotic cells by liver is a complex phenomenon, involving multiple molecular mechanisms of recognition (i.e., lectin-like receptors and receptors for externalized phosphatydilserine) of both parenchymal (hepatocytes) and nonparenchymal (Kupffer and endothelial cells) liver cells, often operating in cooperation. The data discussed in the present review are drawn from studies of phagocytosis of apoptotic cells in the liver, carried out with in vivo and in situ adhesion experiments as well as in vitro assays. Our results indicate that the three main liver cell types (hepatocytes, Kupffer, and endothelial cells) are able to recognize and internalize apoptotic cells by means of specific receptors (galactose and mannose-specific receptor; receptor for phosphatydilserine) and by cytoskeletal reorganization that favors the engulfment of the apoptotic cells. The "flags" for the identification of apoptotic cells by the liver are modifications of the surface of dead cells, i.e., sugar residues and phosphatydilserine exposition. Vitronectin receptor is not involved in such a recognition. The adhesions between modified cell surfaces of apoptotic cells and phagocytes generate cytoplasmatic signaling pathways that drive apoptotic cells to their final fate within the phagocytes (i.e., lysosomal digestion).     

Histochemical evidence of osteoclastic degradation of extracellular matrix in osteolytic metastasis originating from human lung small carcinoma (SBC-5) cells

The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc.     

SPAG9 promotes prostate cancer growth and metastasis

Sperm-associated antigen 9 (SPAG9) expression is increased in prostate tissues of prostate cancer patients. This experimental study aimed to investigate the role of SPAG9 in bone metastasis of prostate cancer. Immunohistochemical analysis showed that SPAG9 staining was positive in 81.67% of 240 cases of prostatic carcinoma but only in 6.67% of 120 cases of benign prostate hyperplasia. Strong PAG9 staining was positively correlated with Gleason score and bone metastasis in 240 prostate cancer patients (p < 0.05), but not with the age or serum prostatespecific antigen level (p > 0.05). PC-3 cells were transfected with shRNA against SPAG9, and CCK-8 assay in triplicate showed that PC-3 cell viability was inhibited by SPAG9 knockdown. In addition, transwell assay in triplicate showed that PC-3 cell invasion was inhibited by SPAG9 knockdown. Furthermore, total 2 × 106 PC-3 cells were injected subcutaneously into the right flank of nude mice which were randomly divided into three groups (N = 8) and treated by intratumoral injection of SPAG9 shRNA, control shRNA or PBS, respectively. SPAG9 shRNA inhibited the growth, invasion and angiogenesis while promoted apoptosis of xenografted PC-3 cells. SPAG9 knockdown led to the upregulation of E-cadherin and the downregulation of MMP2 and vimentin in xenografted tumors. In conclusion, this is the first study to provide evidence that SPAG9 promotes bone metastasis of prostate cancer, and SPAG9 is a promising target to prevent or treat bone metastasis of prostate cancer.     

Associations between CD133, CK19 and G2/M in cirrhotic HCV (genotype-4) patients with or without accompanying tumor

Hepatitis C virus (HCV)-cirrhotic patients have the highest threat of developing hepatocellular carcinoma (HCC) and may be at risk of extra hepatic cancer. The present study was designed to investigate CD133 and CK19 in HCV (genotype-4)-cirrhotic patients with/without HCC or extra hepatic cancer, to assess the degree of their correlation with cell cycle abnormalities and finally to assess the role of their combination as diagnostic tool for discrimination of cirrhotic patients with HCC from those with extra hepatic cancer. The study included 77 HCV-cirrhotic patients and 20 healthy non-disease control group. Patients were categorized histo-pathologically into: 24 have only liver cirrhosis, 26 with HCC, and 27 patients with extra hepatic cancer. Cell cycle abnormalities, CD133 and CK19 were determined by flow cytometry technique. CD133 and CK19 showed marked elevation in HCC and extra hepatic cancer compared with liver cirrhosis and control subjects (p<0.0001). Positive associations were noted between CK19, CD133 and G2/M. They were gradually increased with progression from liver cirrhosis to HCC. Combination of the three showed the best AUC (0.978) and accuracy (92.5%) for discrimination of HCC from extra hepatic cancer. Combined CD133 with G2/M and CK19 comprises an excellent diagnostic panel for discrimination of HCV-cirrhotic patients with HCC from those with extra hepatic cancer.     

Limited utility of acetoxymethyl (AM)-based intracellular delivery systems, in vivo: interference by extracellular esterases

The use of acetoxymethyl (AM) groups to deliver and trap exogenous optical probes inside cells is an established tool in cell biology/physiology, however, these probes have not been used extensively in vivo. In this study, the use of the acetoxymethyl delivery system for optical probes was evaluated, in vivo. Initial studies revealed very little trapped probe in intact tissues even when near saturating levels of probe were injected in living animals. We tested the hypothesis that extracellular esterases rapidly cleave the acetoxymethyl groups preventing the probes from entering cells, in vivo. The rates of hydrolysis of 11 acetoxymethyl probes in diluted porcine plasma revealed an essentially first order high rate dye cleavage with half times on the order of minutes or less. Studies on mice and rabbits revealed rates 10‐ to 2‐fold higher, respectively. These plasma studies suggested that the acetoxymethyl probes were being cleaved before having a chance to enter cells in tissues in vivo. This was confirmed using intravital 2‐photon excitation microscopy in muscle tissue where several acetoxymethyl probes were found to rapidly cleave in the vascular space during infusion and not be trapped in the muscle cells. Studies with succinimidyl esters that should quickly bind to proteins on cleavage also failed to enter cells, in vivo, consistent with the notion that the cleavage was occurring in the extracellular space. These data suggest that the high level of plasma and extracellular esterase activity render the classical acetoxymethyl probes ineffective for monitoring intracellular events, in vivo. Different approaches to trapping exogenous probes will need to be explored for physiological studies using intravital microscopy.     

A method to isolate human limbal basal cells enriched for a subset of epithelial cells with a large nucleus/cytoplasm ratio expressing high levels of p63

The objectives were to develop method of isolating viable human limbal basal cells in order to enrich a subset of small cells with a large Nucleus/Cytoplasm (N/C) ratio expressing high levels of p63, nuclear protein. Limbal tissues were treated with trypsin for 50 min at 37 degrees C in an orbital shaker at 100 rpm with epithelial side down followed by additional 5 min with epithelial side up and then with Dispase II to obtain various epithelial fractions. Isolated cell fractions were assessed for colony forming efficiency and DeltaNp63alpha, connexin (Cx43) mRNA levels. Cytospin smears were double-immunostained for p63 and any one of the stem cell (SC) related markers and analyzed using a laser scanning confocal microscope and advanced image analysis software (Leica Confocal software, 2.61 build 1537 version) for quantification of fluorescence intensity. The isolated limbal basal cells were highly positive for DeltaNp63alpha mRNA but expressing low Cx43 mRNA. They gave rise to higher number of large colonies with compact morphology in contrast to the limbal suprabasal/superficial (LS/S) colonies. Furthermore, a subset with a large N/C ratio expressing high levels of p63 was observed, as much as 25% among the limbal basal cell fraction, in contrast to only about 4% in the total limbal epithelial cells. Such cells were positive for K5 and negative for Ki67, Cx43, and 14-3-3s and were absent in the LS/S fraction. These results collectively substantiate our method of isolation of limbal basal layer cells containing an enriched population of cells with SC phenotype.     

Amphibia Kupffer cells

Amphibia Kupffer cells (i.e., liver resident macrophages) show many common characteristics when compared with Mammalia Kupffer cells: filopodia, microvillous-like structures, lamellipodia, fuzzy coat, coated vesicles, bristled vacuoles, nonspecific esterase activity, and pinocytotic and phagocytic activity are present both in Amphibia and Mammalia Kupffer cells. On the other hand, some differences are present between Kupffer cells of both zoological classes: phagocytosed red cells and their derivatives, iron-protein complexes, and lipofuscin bodies are normally present in Amphibia Kupffer cells, but absent in the same cells of healthy mammals. Worm-like structures are not seen in Amphibia and endogenous peroxidase activity is very weak in these animals compared with Mammalia. The most important difference lies in the ability of Amphibia Kupffer cells to produce melanins: in fact the tyrosinase gene is expressed, "melanosome centers" are present, and dopa oxidase activity is demonstrable.     

Micro‐CT measurements of tumoral vessels supplied by portal circulation in hepatic colorectal metastasis mouse model

The purpose of this study was to elucidate the micro CT findings of tumoral vessels supplied by portal circulation during establishment of hepatic metastasis of colorectal cancer in a mouse model. Hepatic metastases were induced in 15 BALB/c mice through the injection of murine colonic adenocarcinoma tumor cells into the mesenteric vein. Micro‐CT imaging of the tumoral vessels was obtained to clarify the microvascular architecture. We evaluated the sinusoidal structure, diameter of the tumoral vessels (DTV) and blood vessel density (BVD) according to tumor sizes ranging from 201 to 3,000 µm in diameter. A total of 116 tumors were observed on day 15 after cell injection. The mean diameter of a normal hepatic sinusoid was 11.7 ± 2.0 µm on micro CT. The DTV supplied by the portal vein of tumors measuring 1,001–1,500 µm in diameter was greater than that of tumors 200–1,000 µm in diameter. The mean BVD from the portal vein gradually decrease according to size of tumor from 201 to 3,000 µm in diameter (r² = ?0.584, P < 0.01). The characteristics of tumoral vessels supplied by portal circulation during establishment of hepatic colorectal metastases were well visualized with micro‐CT imaging. Microsc. Res. Tech. 77:415–421, 2014. © 2014 Wiley Periodicals, Inc.     

Sinusoidal endothelial cells of the liver: Fine structure and function in relation to age

Liver endothelial cells form a continuous lining of the liver capillaries, or sinusoids, separating parenchymal cells and fat-storing cells from sinusoidal blood. Liver sinusoidal endothelial cells differ in fine structure from endothelial cells lining larger blood vessels and from other capillary endothelia in that they lack a distinct basement membrane and also contain open pores, or fenestrae, in the thin cytoplasmic projections which constitute the sinusoidal wall. This distinctive morphology supports the protective role played by liver endothelium, the cells forming a general barrier against pathogenic agents and serving as a selective sieve for substances passing from the blood to parenchymal and fat-storing cells, and vice versa. Sinusoidal endothelial cells, furthermore, significantly participate in the metabolic and clearance functions of the liver. They have been shown to be involved in the endocytosis and metabolism of a wide range of macromolecules, including glycoproteins, lipoproteins, extracellular matrix components, and inert colloids, establishing endothelial cells as a vital link in the complex network of cellular interactions and cooperation in the liver. Fine structural studies in combination with the development of cell isolation and culture techniques from both experimental animal and human liver have greatly contributed to the elucidation of these endothelial cell functions. Morphological and biochemical investigations have both revealed little changes with age except for an accumulation of iron ferritin and a decrease in the activities of glucose-6-phosphatase, Mg-ATPase, and in glucagon-stimulated adenylcyclase. Future studies are likely to disclose more fully the role of sinusoidal endothelial cells in the regulation of liver hemodynamics, in liver metabolism and blood clearance, in the maintenance of hepatic structure, in the pathogenesis of various liver diseases, and in the aging process in the liver.