Occurrence of fenestral diaphragms and knots in renal glomerular endothelia of diabetic mutant MafA−/−MafK+ mice as revealed in embedment‐free transmission electron microscopy

Using the advantages (high contrast and transparency and efficient 3D viewing) of embedment‐free section transmission electron microscopy (TEM), the occurrence of numerous fenestral diaphragms was clearly shown in 3D en‐face viewing of the renal glomerular capillary endothelium of severe overt diabetes mellitus mice, which were generally MafA‐deficient and simultaneously MafK‐overexpressed specifically in pancreatic β‐cells. This presents another example of nephritis‐induced diaphragmed fenestrae in the renal glomerular endothelium. In addition, knot‐/umbilicus‐like structures discrete from and larger than the central knots of regular diaphragms of fenestrated endothelium were clearly demonstrated to occur randomly in the renal glomerular endothelial fenestrae of mutant mice and wild ones. The knot‐structures were revealed to be protrusions of underlining basement lamina in conventional TEM by section‐tilting observation. Microsc. Res. Tech. 78:207–212, 2015. © 2015 Wiley Periodicals, Inc.     

Structure and function of endothelial caveolae

Caveolae are spherical invaginations of the plasma membrane and associated vesicles that are found at high surface densities in most cells, endothelia included. Their structural framework has been shown to consist of oligomerized caveolin molecules interacting with cholesterol and sphingolipids. Caveolae have been involved in many cellular functions such as endocytosis, signal transduction, mechano-transduction, potocytosis, and cholesterol trafficking. Some confusion still persists in the field with respect to the relationship between caveolae and the lipid rafts, which have been involved in many of the above functions. In addition to all these, endothelial caveolae have been involved in capillary permeability by their participation in the process of transcytosis. This short review will focus on their structure and components, methods used to determine these components, and the role of caveolae in the transendothelial exchanges between blood plasma and the interstitial fluid.     

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.     

Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro

Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.
AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (−15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).     

Identification of fenestrated capillary segments in microvascular corrosion casts of the rat exocrine pancreas

The capillary bed of the rat exocrine pancreas was studied by scanning and transmission electron microscopy of corrosion casts and tissue sections. Two types of capillaries were distinguished in corrosion casts. First, there were straight capillaries of relatively constant width (mean diameter 4.79±0.87 μm), which were characterized by numerous circular constrictions on their surface. About 37% of the capillaries belonged to this type. Second, there were undulating capillaries which showed smooth surfaced eccentric dilatations defined by similar surface constrictions. The bulging areas measured 8.43 ± 1.33 μm, the constrictions next to the bulges figured for 6.45 ± 1.53 μm. About 63% of the capillaries belonged to the second type. Two types of capillaries were also identified in tissue sections. First, there were capillaries with continuous endothelial lining (26% of capillary profiles; mean diameter 5.48 ± 1.67 μm); 27% of their endothelial lining was provided with underlying pericytes. Second, there were capillaries with fenestrated endothelium (64% of capillary profiles; mean diameter 6.16 ± 1.75 μm); 12% of their endothelial lining was accompanied by pericytes. According to frequency and dimension of these two types of capillaries, we conclude that bulged and undulating capillary casts correspond to fenestrated capillaries and straight capillary casts of constant width correspond to nonfenestrated capillaries. The frequency of crests on the surface of capillary casts correlates with the different frequency of pericyte processes on fenestrated and nonfenestrated capillaries. It is concluded that pericyte processes beneath the endothelium hold resistance against luminal pressure. Bulging areas of capillary casts correlate with fenestrated areas of endothelial lining, that is, areas which are not reinforced by pericyte processes.     

Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey

The modern exploration of endothelial cell biology is a largely interdisciplinary exercise. Cell biological, physiological, and more recently molecular biology approaches were used to study the pathways and the organelles involved in transcytosis of macromolecules in endothelial cell (EC). Here we discuss mainly the cell biological findings that revealed that EC have the attributes to fulfill the transport function. They are polarized cells, heterogeneous, and, thus, structurally and functionally adapted to the vascular bed in which they reside. The structural heterogeneity involves the number and distribution of plasmalemmal vesicles (caveolae), their generated channels, and the organization of intercellular junctions. The closely related functional heterogeneity comprises the degree of permeability for plasma molecules that vary as a function of organ. The EC are endowed with the cellular machinery to perform (1) endocytosis, that is to take up plasma proteins and the molecules they carry to be used for themselves (cholesterol-carrying low density lipoproteins, fatty acid carrying albumin, iron carrying transferrin, etc.), and (2) transcytosis, which implies to transport plasma proteins to the subjacent cells and tissues. The possible pathways for transport of molecules are transcellular, via caveolae and channels, and paracellular via intercellular junctions. Most of the results obtained, so far, indicate that transcytosis of albumin, low-density lipoproteins, metaloproteases, and insulin, is performed by cargo-vesicles and their generated channels. The paracellular pathway can be used for water and ions; in postcapillary venules, at the level of which approximately 30% of junctions are open to a space of 6 nm, small molecules may take this route. Recent data obtained by molecular biology techniques revealed that caveolae are endowed with the molecular machinery for fusion/fission, docking, and movement across cells. Moreover, the various and numerous molecules that have been detected in the caveolae membrane and the different functions assumed by this differentiated microdomain strengthen the postulate that there are at least two or more types of vesicles molecularly tailored for the local physiological requirements.     

Lymphatic vessels in the oral cavity: different structures for the same function.

A study using a light and transmission electron microscope was performed on some structural characteristics of the lymphatic capillaries in different regions of the human oral cavity. The lymphatic capillaries of dental pulp, masticatory mucosa (gingiva and peri-implant mucosa) and lining mucosa (cheek) were examined. Our attention was focused on the morphologic characteristics of the endothelial wall in the lymphatic capillaries. In particular, the connections between endothelial cells were investigated. In the lymphatic capillaries of the dental pulp, the endothelial wall was always very complex. It frequently presented protrusions of the endothelial cells that overlapped and formed intercellular channels. These channels were thus contained by the vessel endothelial wall with their extremities opening out towards the surrounding interstitium and the vessel lumen. The endothelial wall of the lymphatic capillaries of the cheek was very smooth and thin without complex intercellular junctions. The endothelial cells were joined by end-to-end junctions and open junctions were frequently observed. Intercellular channels were also found in the endothelial wall of lymphatic capillaries of the gingiva and the peri-implant mucosa. The presence of numerous clefts represented by the open junctions in the lymphatics of the cheek and the existence of complex intercellular adhesions with the formation of intercellular channels in the endothelial wall of the lymphatic capillaries of the dental pulp and gingiva induce us to believe that these may play a role in the various mechanisms used by lymphatic capillaries to absorb interstitial fluids. These mechanisms are based on the different morpho-functional characteristics of the surrounding tissue.     

Hydrodynamic flow in the cytoplasm of plant cells

Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007) shows that active transport of organelles gives rise to a drag in the cytosol, setting up a hydrodynamic flow, which contributes to a fast distribution of proteins and nutrients in plant cells. Here, we show experimentally that actively transported organelles produce hydrodynamic flow that significantly contributes to the movement of the molecules in the cytosol. We have used fluorescence recovery after photobleaching and show that in tobacco Bright Yellow 2 (BY-2) suspension cells constitutively expressing cytoplasmic green fluorescent protein (GFP), free GFP molecules move faster in cells with active transport of organelles than in cells where this transport has been inhibited with the general myosin inhibitor BDM (2,3-butanedione monoxime). Furthermore, we show that the direction of the GFP movement in the cells with active transport is the same as that of the organelle movement and that the speed of the GFP in the cytosol is proportional to the speed of the organelle movement. In large BY-2 cells with fast cytoplasmic streaming, a GFP molecule reaches the other side of the cell approximately in the similar time frame (about 16 s) as in small BY-2 cells that have slow cytoplasmic streaming. With this, we suggest that hydrodynamic flow is important for efficient transport of cytosolic molecules in large cells. Hydrodynamic flow might also contribute to the movement of larger structures than molecules in the cytoplasm. We show that synthetic lipid (DOPG) vesicles and 'stealth' vesicles with PEG phospholipids moved in the cytoplasm.     

Secretory pathways in animal cells: With emphasis on pancreatic acinar cells

Studies over the past three decades have clearly established the existence of at least two distinct pathways for the intracellular transport and release of secretory proteins by animal cells. These have been identified as the regulated and constitutive pathways. Many observations have indicated that in certain cells, such as those of the exocrine pancreas and parotid glands at least, these pathways coexist in the same cells. Although the general scheme of protein transport within these pathways is well established, many fundamental aspects of intracellular transport remain to be unraveled. How are proteins transported through the endoplasmic reticulum? How are the transitional vesicles formed and what are the underlying mechanisms involved in their fusion with the cis-Golgi cisterna? Even the general mode of transfer through the Golgi stack is debated: Is there a diffusion through the stack by flow through intercisternal tubules and openings or is there a vesicle transfer system where membrane quanta hop from one cisterna to the other? What is the fate of secretory proteins in the trans-Golgi area and by what mechanisms is a fraction of newly synthesized molecules of a given secretory protein released spontaneously while the majority of such nascent molecules are diverted into a secretory granule compartment? In this review, we have examined these and other aspects of intracellular transport of secretory proteins using pancreatic acinar cells as our reference model and we present some evidence to support the existence of a paragranular pathway of secretion associated with secretory granule maturation.     

Ultrastructural studies define soluble macromolecular,particulate, and cellular transendothelial cell pathways in venules,lymphatic vessels,and tumor-associated microvessels in man and animals

We present de novo studies and review published efforts from our laboratory, spanning 12 years (from 1988 to 2000), where we have used ultrastructural approaches to study the functional anatomy of the microvasculature in man and animals in health and disease. These efforts have defined a new endothelial cell organelle, termed the vesiculo-vacuolar organelle (VVO), which participates in the regulated transendothelial cell passage of soluble macromolecules. The studies defining this organelle utilized ultrathin serial sections, three-dimensional computer-assisted reconstructions, and ultrastructural electron-dense tracers to establish luminal to abluminal transendothelial cell continuity of VVOs. Commonality of VVOs and caveolae is suggested by the ultrastructural anatomy of individual units of VVOs and caveolae, the presence of caveolin in both structures, and a mathematical analysis of morphometric data, all of which suggest that VVOs form from fusions of individual size units equivalent to vesicles of caveolar size. Ultrastructural studies have localized potent permeability factors and their specific receptors to VVOs in in vivo tumor and allergic inflammation models. Regulation of permeability through VVOs has been quantified and shown to be increased in tumor microvessels and in control vessels exposed to potent permeability-inducing mediators. The transendothelial cell passage of particulate macromolecules occurs by vacuolar transport in tumor vessels; in permeability factor-exposed control vessels, colloidal carbon traversed endothelial cells via the development of pores that did not communicate with or disrupt intercellular junctions by gap formation. Serial section and computer-assisted reconstructions established these findings and suggested the possible development of transendothelial cell pores from VVOs. Serial sectioning and computer-assisted three-dimensional reconstructions of ultrastructural samples of an acute inflammation model revealed a transendothelial cell traffic route for motile neutrophils and platelets in the absence of classical ultrastructural criteria for regulated secretion from either cell.     

Hormone regulation of endothelial apoptosis and proliferation in vessel regression and angiogenesis

Apoptosis and endothelial proliferation represent two adverse events which take place during vessel regression and angiogenesis, respectively. Apoptosis, an intrinsically activated programmed cell death, regulates cell elimination during vessel regression. In contrast, angiogenesis involves endothelial cell proliferation, migration, and vascular formation. Several molecules, including growth factors and cytokines, produced by endothelial cells and by other cells within the vicinity of the capillary network, regulate apoptosis and angiogenesis. Hormones and endocrine peptides acting via specific receptors located on the endothelial and perivascular stromal cells also have been found to be involved in the regulation of these two major antagonistic processes. The need for a better understanding of the mechanisms involved in hormone regulation of endothelial cell during apoptosis and angiogenesis is of great importance. The accumulating knowledge of hormone regulation may contribute to the introduction of new therapeutic strategies targeting the endothelial cells.     

Flow near the meniscus of a pressure-driven water slug in microchannels

Micro-PIV system with a high speed CCD camera is used to measure the flow field near the advancing meniscus of a water slug in microchannels. Image shifting technique combined with meniscus detecting technique is proposed to measure the relative velocity of the liquid near the meniscus in a moving reference frame. The proposed method is applied to an advancing front of a slug in microchannels with rectangular cross section. In the case of hydrophilic channel, strong flow from the center to the side wall along the meniscus occurs, while in the case of the hydrophobic channel, the fluid flows in the opposite direction. Further, the velocity near the side wall is higher than the center region velocity, exhibiting the characteristics of a strong shear-driven flow. This phenomenon is explained to be due to the existence of small gaps between the slug and the channel wall at each capillary corner so that the gas flows through the gaps inducing high shear on the slug surface. Simulation of the shape of a static droplet inside a cubic cell obtained by using the Surface Evolver program is supportive of the existence of the gap at the rectangular capillary corners. The flow fields in the circular capillary, in which no such gap exists, are also measured. The results show that a similar flow pattern to that of the hydrophilic rectangular capillary (i.e., center-to-wall flow) is always exhibited regardless of the wettability of the channel wall, which is also indicative of the validity of the above-mentioned assertion.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Diversity in unity: the biochemical composition of the endothelial cell surface varies between the vascular beds

The vascular endothelium represents a population of squamous epithelial cells characterized by a particular histological localization (intima of blood vessels) and by several physiological functions such as the transport of substances between blood and tissues, the modulation of the vascular tone, the control of blood coagulation and that of the leukocyte extravasation. In spite of all these elements in common and of an identical embryonic origin, endothelial cells show definite morphological and physiological variations that divide them into types and subtypes, each specifically associated to various categories of organs. Even within the vasculature of the same organ, there are clear segmental (arterial/capillary/venous) differentiations of the endothelial cells. While the morphological and physiological differences between endothelial cells are well documented, there are very few data on the biochemistry underlying this heterogeneity. This work presents several data suggesting that, at present, the domain is ripe for a comprehensive analysis of this biochemical diversity, at least in what concerns the luminal aspect of the endothelial plasmalemma, a compartment of crucial importance in the biology and pathology of the cardiovascular system.     

Choroid plexus: target for polypeptides and site of their synthesis

Choroid plexus (CP) is an important target organ for polypeptides. The fenestrated phenotype of choroidal endothelium facilitates the penetration of blood-borne polypeptides across the capillary walls. Thus, both circulating and cerebrospinal fluid (CSF)-borne polypeptides can reach their receptors on choroidal epithelium. Several polypeptides have been demonstrated to regulate CSF formation by controlling blood flow to choroid plexus and/or the activity of ion transport in choroidal epithelium. However, many ligand-receptor interactions occurring in the CP are not involved in the regulation of fluid secretion. Increasing evidence suggests that the choroidal epithelium plays an important role in hormonal signaling via a receptor-mediated transport into the brain (e.g., leptin) and helps to clear certain CSF-borne polypeptides (e.g., soluble amyloid beta-protein). Thus, impaired choroidal transport or insufficient clearance of polypeptides may contribute to pathogenesis of systemic or central nervous system (CNS) disorders, such as obesity or Alzheimer's disease. CP epithelium is not only a target but is also a source of neuropeptides, growth factors, and cytokines in the CNS. These polypeptides following their release into the CSF may exert distal, endocrine-like effects on target cells in the brain due to bulk flow of this fluid. Distinct temporal patterns of choroidal expression of several polypeptides are observed during brain development and in various CNS disorders, including traumatic brain injury and ischemia. Therefore, it is proposed that the CP plays an integral role not only in normal brain functioning, but also in the recovery from the injury. This review attempts to critically analyze the available data to support the above hypothesis.     

Suspension state promotes extravasation of breast tumor cells by increasing integrin β1 expression

Mechanical microenvironment can strongly affect the metastatic efficiency of circulating tumor cells. However, the effect of suspension state on their extravasation and the mechanisms involved are still unclear. To explore the influence of suspension state on extravasation (including adhesion, spreading and transendothelial migration) of breast tumor cells and its relevant molecular mechanism, MDA-MB-231 cells were cultured on poly (2-hydroxyethyl methacrylate) coated 6-well plates to minic the suspension state. Suspension state promoted adhesion, spreading and transendothelial migration of MDA-MB-231 cells to EAhy926 endothelial cells (ECs) monolayer under both the static condition and 0.5 dyne/cm² flow shear stress (FSS). The number of cells adhered to ECs monolayer reached 2.15 (static condition, 3 d) and 1.67 (FSS, 3 d) times, and the number of migration reached 10.60 times, respectively, as compared to that in adhesion state. Moreover, MDA-MB-231 cells knockdown of integrin β1 provoked poor adhesion and transendothelial migration, as compared with MDA-MB-231 cells. But it made no difference in cell spreading. Our results implied the increasing expression of integrin β1 induced by suspension culture promoted the adhesion and transendothelial migration of MDA-MB-231 cells, but had no significant influence on their spreading.     

mAb 5-1-6 nephropathy and nephrin

It is well established that the glomerular capillary wall consists of three layers: endothelial cell, glomerular basement membrane, and the slit diaphragm bridging foot processes of glomerular epithelial cell. Which structure in the glomerular capillary wall represents the primary filter for retaining plasma proteins is not clearly elucidated. An anti-slit diaphragm monoclonal antibody (mAb) 5-1-6 causes massive proteinuria in rats by single intravenous injection, which clearly indicates that the slit diaphragm plays a critical role for maintaining the barrier function of the glomerular capillary wall. Recently, we concluded that mAb 5-1-6 recognized a rat homolog of nephrin, a gene product of NPHS1. The expression of nephrin decreased in puromycin aminonucleoside nephropathy and adriamycin nephropathy as well as mAb 5-1-6-induced nephropathy, which suggested that nephrin was involved in the development of proteinuria in these proteinuric states. In mAb 5-1-6 nephropathy, the slit diaphragm was maintained morphologically normal, although nephrin expression dramatically decreased. The finding suggested that nephrin was not a sole component of the slit diaphragm. To better understand the structure of the slit diaphragm, it is particularly important to identify other components that build up the structure of the slit diaphragm together with nephrin.     

Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: ultrastructure and uptake of tracers.

Mammalian olfactory neurons possess a well-developed system of endocytic vesicles, endosomes, and lysosomes in their dendrites and perikarya. Vomeronasal neurons are similar and also contain much perikaryal agranular endoplasmic reticulum (AER). Olfactory supporting cells contain endocytic vesicles and endosomes associated closely with abundant fenestrated AER, and vesicles and numerous large dense vacuoles are present basally. Vomeronasal supporting cells have little AER, and few dense vacuoles occur in their bases. In olfactory neurons, ultrastructural tracers (0.08% horseradish peroxidase, thorium dioxide, ferritin) are endocytosed by olfactory receptor endings and transported to the cell body, where their movement is halted in lysosomes. Higher concentrations (1%) of horseradish peroxidase penetrate olfactory receptor plasma membranes and intercellular junctions. In olfactory supporting cells, endocytosed tracers pass through endosomes to accumulate in dense basal vacuoles. These observations indicate that olfactory sensory membranes are rapidly cycled and that endocytosed materials are trapped within the epithelium. It is proposed that in the olfactory epithelium, endocytosis presents redundant odorants to the enzymes of the supporting cell AER to prevent their accumulation, whereas in the vomeronasal epithelium the receptor cells carry out this activity.     

Involvement of the choroid plexus in central nervous system inflammation

During inflammatory conditions in the central nervous system (CNS), immune cells immigrate into the CNS and can be detected in the CNS parenchyma and in the cerebrospinal fluid (CSF). The most comprehensively investigated model for CNS inflammation is experimental autoimmune encephalomyelitis (EAE), which is considered the prototype model for the human disease multiple sclerosis (MS). In EAE autoagressive CD4(+), T cells gain access to the CNS and initiate the molecular and cellular events leading to edema, inflammation, and demyelination in the CNS. The endothelial blood-brain barrier (BBB) has been considered the obvious place of entry for the circulating immune cells into the CNS. A role of the choroid plexus in the pathogenesis of EAE or MS, i.e., as an alternative entry site for circulating lymphocytes directly into the CSF, has not been seriously considered before. However, during EAE, we observed massive ultrastructural changes within the choroid plexus, which are different from changes observed during hypoxia. Using immunohistochemistry and in situ hybridization, we observed expression of VCAM-1 and ICAM-1 in the choroid plexus and demonstrated their upregulation and also de novo expression of MAdCAM-1 during EAE. Ultrastructural studies revealed polar localization of ICAM-1, VCAM-1, and MAdCAM-1 on the apical surface of choroid plexus epithelial cells and their complete absence on the fenestrated endothelial cells within the choroid plexus parenchyme. Furthermore, ICAM-1, VCAM-1, and MAdCAM-1 expressed in choroid plexus epithelium mediated binding of lymphocytes via their known ligands. In vitro, choroid plexus epithelial cells can be induced to express ICAM-1, VCAM-1, MAdCAM-1, and, additionally, MHC class I and II molecules on their surface. Taken together, our observations imply a previously unappreciated function of the choroid plexus in the immunosurveillance of the CNS.     

Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells

Critical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS-dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole-mount cells for TEM and AFM.