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).     

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.     

Ultrastructural characterization of isolated rat Kupffer cells by transmission X-ray microscopy

The ultrastructure of primary cultured rat Kupffer cells was studied using transmission X-ray microscopy as well as transmission electron microscopy. X-ray microscopical images of intact, hydrated Kupffer cells demonstrated structures such as cell nucleus separated by a nuclear membrane and filaments concentrated in the perinuclear area. Within the cytoplasm, a number of vacuoles were visible; some of these were crescent-shaped vacuoles that were half X-ray lucent, half X-ray dense; others were uniformly dense. The number of crescent-shaped vacuoles was predominant. After phagocytosis of haematite particles, enlarged vacuoles containing the ingested material were visible within the cytoplasm of Kupffer cells while crescent-shaped vacuoles were no longer detectable. Densitometric analysis of the two types of vacuole revealed that the X-ray absorption of the uniform vacuole was approximately half that of the dense part of the crescent-shaped vacuoles. This observation led to speculation on the existence of only one type of vacuole in the cytoplasm of Kupffer cells. The different morphological aspects — crescent-shaped versus uniform vacuoles — might be due to different three-dimensional orientation with respect to the image plane. Using transmission electron microscopy, the morphology of vacuoles differed more widely in diameter, density and shape. Two main types of vacuole were identified: electron-lucent and electron-dense. Based on the observation of only one type of vacuole by transmission X-ray microscopy, the different morphological aspects of vacuoles obtained by transmission electron microscopy could be explained by imaging several different sections of a crescent-shaped vacuole. From the present data it can be concluded that transmission X-ray microscopy is a versatile technique that reveals the ultrastructure of intact, unsectioned biological specimens in their aqueous environment, thereby allowing a more comprehensive interpretation of data obtained by transmission electron microscopy.     

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.     

A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.     

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.     

Organization of microtubules in cochlear hair cells

The organization of microtubules in hair cells of the guinea-pig cochlea has been investigated using transmission electron microscopy and correlated with the location of tubulin-associated immunofluorescence in surface preparations of the organ of Corti. Results from both techniques reveal consistent distributions of microtubules in inner and outer hair cells. In the inner hair cells, microtubules are most concentrated in the apex. Reconstruction from serial sections shows three main groups: firstly, in channels through the cuticular plate and in a discontinuous belt around its upper perimeter; secondly, forming a ring inside a rim extending down from the lower perimeter of the plate; and thirdly, in a meshwork underlying the main body of the plate. In the cell body, microtubules line the inner face of the subsurface cistern and extend longitudinally through a tubulo-vesicular track between the apex and base. In outer hair cells, the pattern of microtubules associated with the cuticular plate is similar, although there are fewer present than in inner hair cells. In outer hair cells from the apex of the cochlea, microtubules occur around an infracuticular protrusion of cuticular plate material. In the cell body, many more microtubules occur in the region below the nucleus compared with inner hair cells. The possible functions of microtubules in hair cells are discussed by comparison with those found in other systems. These include morphogenesis and maintenance of cell shape; intracellular transport, e.g., of neurotransmitter vesicles; providing a possible substrate for motility; mechanical support of structures associated with sensory transduction.     

Imaging molecular structure and physiological function of gap junctions and hemijunctions by multimodal atomic force microscopy

Gap junctions are specialized plasma membrane structures that join neighboring cells via specialized intercellular ion channels (hemichannels) and provide a direct pathway for cell-cell communication. They presumably mediate regulation of growth, transmission of developmental signals, coordination of muscle contraction, and maintenance of metabolic homeostasis. Hemichannels are also present in the non-junctional regions of the cell plasma membrane and they provide a direct pathway for communication between the cytoplasm and the extracellular region. Recent studies suggest that gap junctional communication is much more complex than previously anticipated, in terms of both its structure as well as its activity. While the mechanism of gap junction activity is being studied extensively, their quaternary structure, assembly, and conformational changes underlying gating of their activity as well as their physiological role are poorly understood because, due to their complex structure, these junctions are less amenable to existing techniques for high-resolution three-dimensional structure-function analyses. Atomic Force Microscopy (AFM) images molecular structure of biological specimens in an aqueous environment, allows on-line perturbations, and can be coupled with electrophysiological, biochemical, and other microscopic techniques. The present review examines the potential of AFM application for the study of the molecular structure of hydrated, native gap junctions and hemijunctions as well as their physiological functions. Special attention is paid to new, complementary, or provocative findings from AFM studies of both vertebrate and invertebrate gap junctions and hemijunctions.     

Immune system involvement in the regulation of ovarian function and augmentation of cancer

Increasing evidence indicates a role for the immune system and mesenchymal-epithelial interactions in the regulation of ovarian function. Cytokines produced by mesenchymal cells can stimulate development and regression of ovarian structures. We report here that mesenchymal cells releasing surface molecules among epithelial cells--namely vascular pericytes and monocyte-derived cells (MDC)--and intraepithelial T lymphocytes are associated with oogenesis and formation of new primary follicles in both fetal and adult human ovaries. These activated mesenchymal cells interact with the ovarian surface epithelium, which appears to be a source of secondary germ cells and granulosa cells. Activated pericytes and MDC are also associated with stimulation of thecal development during selection of growing secondary follicles from the cohort of primary follicles. However, survival of the dominant follicle during mid-follicular phase selection is associated with a lack of activity of mesenchymal cells and retardation of thecal development, since immature granulosa cells lacking aromatase are unable to resist high levels of thecal androgens. Once the selected follicle matures (late follicular phase), it shows enhanced activity of thecal mesenchymal cells and advanced thecal development. Corpus luteum (CL) development is accompanied by a high activity of vascular pericytes and MDC. In mature CL and CL of pregnancy, luteal MDC and pericytes show a stable (inactive) state. Regression of the CL is associated with regression of pericytes, transformation of MDC into dendritic cells, infiltration by T lymphocytes, and binding of immunoglobulin G to the luteal cells. The immunoglobulin M (IgM) binds to young but not mature luteal cells. In the CL of pregnancy, IgM binds to luteal vessels, but not to luteal cells. Regressing CL shows IgM binding to both luteal cells and vessels. In ovarian cancers, highly activated MDC and sometimes activated pericytes (poorly differentiated carcinomas) are present. IgM binding is similar to that seen in the CL of pregnancy. These data indicate that vascular pericytes, MDC, T cells, and immunoglobulins may play an important role in the regulation of ovarian physiology and contribute to the augmentation of ovarian cancer growth.     

An investigation of cell adhesion and growth on micro/nano-scale structured surface—Self-assembled micro particles as a scaffold

A controlled cellular behavior (adhesion, migration and growth) on a scaffold is a technical issue on development of cultured cell applications such as biosensor and tissue engineering. The present paper describes fundamental experiments about effects of some micro/nano-scale structures on cell adhesion and growth. Micro/nano-scale structures of scaffold are fabricated by both top-down and bottom-up processes using some unique materials. For fine spherical particles, they are self-assembled on a substrate. The micro/nano-structured particles provide non-angular and regularly-arranged surface asperity with two-dimensional opal structure, which is also permeable to a culture solution because of fine gaps among assembled particles. Some polymers are formed into an array of micro-ridges with rectangular cross-section by nano-imprint process. Most cells are selectively adhered on micro-structured silica particles, while a flat surface has low affinity for the cells. The present study intends to explain a preferential surface for cell adhesion and growth in terms of geometry and biochemical property of micro-structure.     

Dislocation structures in fatigued polycrystalline stainless steel

The dislocation structures produced in polycrystalline Fe-25Cr as a result of push-pull fatigue are described with reference to the Schmid factors of the active slip systems in different grains. The activity of the structures is discussed in relation to an etch which is indicative of localized shear activity. The ladder and labyrinth structures seen are compared with those which form in f.c.c. material.     

Study of the characteristics of forced homogeneous turbulence using band-pass Fourier filtering

Simulations of forced homogeneous isotropic turbulence with resolutions of 128³ and 256³ using the Lattice Boltzmann method are carried out. The multi-scale vortical structures are identified using the band-pass Fourier cutoff filtering. Three fields at each simulation are extracted and their characteristics are studied. The vortical structures are visualized using the Q-identification method. A new lattice segmentation scheme to identify the central axes of the vortical structures is introduced. The central points of each vortex are identified and they are connected using the direction cosines technique. Results show that the Q-spectrum of the fine scale field survives at low and high wave-numbers. However, the large and intermediate Q-spectra survives till wave-numbers less than or equal to twice the used velocity cutoff wave-numbers. It is found that the extracted central axes clearly resemble the corresponding vortical structures at each scale. Using the central axes scheme, the radii and lengths of the vortical structures at each scale are determined and compared. It is also found that the radii of the identified vortical structures at each scale in both simulations are of the order of several times the Kolmogorov microscales.     

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.     

Diversity of the vomeronasal system in mammals: the singularities of the sheep model

The enormous morphological diversity and heterogeneity of the vomeronasal system (VNS) in mammals--as well as its complete absence in some cases--complicates the extrapolation of data from one species to another, making any physiological and functional conclusions valid for the whole Mammalian Class difficult and risky to draw. Some highly-evolved macrosmatic mammals, like sheep, have been previously used in interesting behavioral studies concerning the main and accessory olfactory systems. However, in this species, certain crucial morphological peculiarities have not until now been considered. Following histological, histochemical and immunohistochemical procedures, we have studied the vomeronasal organ (VNO) and the accessory olfactory bulb (AOB) of adult sheep. We have determined: (1) that all structures which classically define the VNO in mammals are present and well developed, providing the morphological basis for functional activity. (2) that, conversely, there is only a scant population of scattered mitral/tufted cells. One morphological consequence of both details is that the strata of the AOB in adult sheep are not as sharply defined as in other species; moreover, the small number of the mitral/tufted cells in the AOB may imply that the VNS of adult sheep is not capable of functioning in the way a well-developed VNS does in other species. (3) the zone to zone projection from the apical and basal sensory epithelium of the VNO to the anterior and posterior part of the AOB, respectively, typical in rodents, lagomorphs and marsupials, is not present in adult sheep.     

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.     

Mechanisms of actin stress fibre assembly

Stress fibres are contractile acto-myosin structures found from many types of non-muscle cells, where they are involved in adhesion, motility and morphogenesis. Stress fibres typically display a periodic alpha-actinin-myosin II pattern and are thus suggested to resemble the sarcomeric actin filament structures of muscle cells. Mammalian cells contain three categories of stress fibres: ventral stress fibres that are attached to focal adhesions at both ends, dorsal stress fibres that are attached to focal adhesions typically at one end and transverse arcs that are curved acto-myosin bundles, which do not directly attach to focal adhesions. In this review, we discuss the definition of stress fibres, organization of actin filaments and other components within these contractile structures, and the mechanisms of stress fibre assembly.     

Cilia in the head of hornets: form and function

In the head of the Oriental hornet, beneath the cuticle, there are plaques of hair cells. These are distributed throughout the upper front part of the head; to wit: in the region of the vertex (i.e., around and behind the ocelli), in the genae around and behind the compound eyes (the ommatidia), and in the region of the forehead or frons. These hair cells are arranged with their thin whip-like part (i.e., cilia) directed outward and morphologically fall into three distinct groups: type (a) thin elongated cilia connected to each other alongside by side-links; type (b) thin elongated cilia of which two or more interconnect at their distal ends via a delicate nerve fiber bearing a knob at its center; and type (c) shorter and thicker cilia that roughly resemble a triangular thorn and are also interconnected by a thin thread, which, however, bears a ball rather then a knob at its center. The knob in the one case and the ball in the other vary in their diameter, but in both instances the interconnecting elements, be they nerve fibers or threads, are seemingly multidirectional. Beneath the frons, in the region of the coronal suture, the hair cells (cilial plaques) are inwardly directed and bear a large trachea at their center. Presumably, the "weighted" cilial cells that are directed toward the exterior of the body aid the hornet in navigation and gravity determination whereas the inwardly directed ciliary cells may possibly serve in acoustic communication. Another element worthy of mention within the hair cells are yellow granules (yg). These yg's originate from the whip-like portion of the ciliary cells that are distributed beneath the frons plate, and also in the yellow stripes of the gastral cuticle. Conceivably, these yellow granules, in both cases, may play a role in the absorption and storage of solar energy. In summary, ciliary structures are involved in the hornet in gravity sensing, in acoustical communication and in light sensing, i.e., with some similarity with what happens in vertebrates in the inner ear and in the photoreceptor.     

Extremely thin layer plastification for focused‐ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells

Since the recent boost in the usage of electron microscopy in life‐science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused‐ion beam scanning electron microscopy (FIB‐SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold‐labelled breast cancer SKBR3 cells was to visualise gold‐labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 m² cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back‐scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB‐SEM. Cross‐sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.     

Unique features of the basal cells of human prostate epithelium

The prostate gland is globally composed of epithelium and stroma. The epithelium plays an important role in the development of both benign and malignant disorders while the stroma is involved in benign prostatic hyperplasia. While the prostatic epithelium of the majority of laboratory animals is well recognized as a pseudostratified columnar, the classification of the human prostatic epithelium is controversial. Moreover, the role of the basal cells of the human prostatic epithelium is still uncertain. These cells have been described as undifferentiated cells, precursors of luminal cells, reserve and myoepithelial cells. The objective of the present study was to assess the similarities and/or differences between the epithelium of the human prostate and that of other laboratory animals and thus derive information about the potential functions of basal cells in the human prostate. In the human, basal cells form a continuous layer of cells resting on the basement membrane and upon which rests a layer of luminal cells. This results in a stratified columnar epithelium of two layers of cells, unlike the sporadic appearance of basal cells observed in other species where it results in a pseudostratified epithelium. In addition, the ratio of basal to luminal cells in the human is about 1:1, while the average ratio in the other animal species examined is about 1:7. Furthermore, the gap junctional proteins connexin 26 and 43, are present between basal and luminal cells in the human, thus suggesting that these cells communicate directly with each other. In addition, the ultrastructure of the human basal cells shows morphological evidence of differentiated but not of undifferentiated cells. Moreover, the presence of junction-like structures between adjacent basal cells suggests that these cells form a blood-prostate barrier. In this way, basal cells could prevent substances derived from the blood from directly coming in contact with the luminal cells. Human basal cells could thus regulate functions of the luminal cells by being part of a two-cell mechanism somewhat analogous to thecal and granulosa cells in the ovary.     

Intravital dual-colored visualization of colorectal liver metastasis in living mice using two photon laser scanning microscopy

A major challenge of cancer biology is to visualize the dynamics of the metastatic process in secondary organs at high optical resolution in vivo real-time. Here, we presented intravital, dual-colored imaging of liver metastasis formation from a single cancer cell to metastatic colonies in the living liver of living mice using two photon laser scanning microscopy (TPLSM). Red fluorescent protein expressing murine (SL4) or human (HT29) colorectal cancer cell lines were inoculated to the spleen of green fluorescent protein expressing mice. Intravital TPLSM was performed by exteriorizing and fixing the liver lobe of living mice. This was repeated several times for the long-term imaging of the same mouse. Viable cancer cells in the living liver of living mice were visualized intravitally at a magnification of over 600×. Single cancer cells were arrested within hepatic sinusoids 2 h after injection. Platelet aggregation surrounding a cancer cell was observed, indicating a phenomenon of tumor-cell induced platelet aggregation. Cancer cells were extravasated from hepatic sinusoids to the space of Disse. Protrusions of Kupffer cells surrounding a cancer cell were observed, indicating that Kupffer cells appear to phagocytose cancer cells. SL4 cells formed liver metastatic colonies with extensive stromal reaction. Liver metastases by HT29 cells were observed as a cluster of micrometastatic nodules. High-resolution, dual-colored, real-time visualization of cancer metastasis using intravital TLPSM can help to understand spatiotemporal tumor-host interactions during metastatic processes in the living organs of living animals.