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.

Claudin-1, claudin-2 and claudin-11 are present in tight junctions of choroid plexus epithelium of the mouse.

The choroid plexus epithelium forms the blood-cerebrospinal fluid (CSF) barrier and is responsible for the secretion of the CSF from the blood. The morphological correlate of the blood-CSF barrier are the tight junctions of choroid plexus epithelium. By freeze-fracture electron microscopy it has been demonstrated that choroid plexus epithelial tight junctions form parallel strands resembling those of Sertoli cells building the blood-testis barrier and those of the myelin sheaths of oligodendrocytes. As the oligodendrocyte specific protein/claudin-11 has been shown to be the central mediator of parallel-array tight junctions in Sertoli cells and myelin sheaths in mice, we asked whether claudin-11 is present in the tight junctions of choroid plexus epithelial cells of the mouse. Here, we present the first direct evidence that claudin-11 besides claudin-1 and -2, occludin and the zonula occludens protein ZO-1 is present in choroid plexus epithelial tight junctions. During inflammation in the central nervous system such as experimental autoimmune encephalomyelitis, the molecular composition of choroid plexus epithelial tight junctions does not change considerably. Their unique molecular composition, with claudin-11 accompanied by claudin-1 and claudin-2 points to a unique regulatory mechanism of the blood-CSF-barrier function.

The superposition eye of Cloeon dipterum: the organization of the lamina ganglionaris.

The lamina ganglionaris of the superposition eye of Cloeon dipterum is composed of separate optic cartridges arranged in a hexagonal pattern. Each optic cartridge consists of one central, radially branched monopolar cell (Li) surrounded by a crown of seven retinula cell terminals and two more unilaterally branched monopolar cells (La1/La2) situated close together outside the cartridge. Projections to neighbouring cartridges have not been observed. In most cases, synaptic contacts could be seen between a presynaptic retinula cell and more than two other postsynaptic profiles, which belong to monopolar cells or sometimes to glial cells. Seven retinula cell fibers of one ommatidium pass in a bundle through the basement membrane, run into their respective cartridges without changing orientation and terminate at approximately equal levels in the lamina. Long visual fibers with endings in the medulla are not visible in the superposition eye lamina, but are present in the lateral apposition eye. The relationship between the behaviour of the animal, optic mechanisms of the superposition eye and the structure of the lamina is discussed.

The organization of the lamina ganglionaris of the hemipteran insects, Notonecta glauca, Corixa punctata and Gerris lacustris.

Neuronal elements, i.e. first and second order neurons, of the first optic ganglion of three waterbugs, N. glauca, C. punctata and G. lacustris, are analyzed on the basis of light- and electron microscopy. Eight retinula cell axons, leaving each ommatidium, disperse to different cartridges as they enter the laminar outer plexiform layer. Such a pattern of divergence is one of the conditions for neuronal superposition; it is observed for all three species of waterbugs. The manner in which the receptors of a single bundle of ommatidia split of within the lamina, whereby information from receptors up to three or five horizontal rows away can converge upon the same cartridge, differs among the species. Six of the eight axons of retinula cells R1--6, the short visual fibers end at different levels within the bilayered lamina, whereas the central pair of retinula cells R7/8, the long visual fibers, run directly through the lamina to a corresponding unit of the medulla. Four types of monopolar cells L1-L4 are classified; their branching patterns seem to be correlated to the splitting and termination of retinula cell axons. The topographical relationship and synaptic organization between retinula cell terminals and monopolar cells in the two laminar layers are identified by examination of serial ultrathin sections of single Golgi-stained neurons. An attempt is made to correlate some anatomical findings, especially the neuronal superposition, to results from physiological investigations on the hemipteran retina.

Nonselective nerve fibre damage in peripheral nerves after experimental thermocoagulation.

In this study the morphological effects of local heat application by controlled thermocoagulation to the sciatic nerve of rabbits are investigated. It concentrates on the question of a possible selective elimination of nerve fibres depending on their calibers. Temperatures of 50 degrees C, 55 degrees C, 60 degrees C, 70 degrees C and 90 degrees C were applied for 45 seconds. The nerves were examined histologically after 2-3 minutes, 7 and 12 days and 5 weeks. The light and electronmicrographs show no differential vulnerability of small myelinated or unmyelinated fibres as proposed by Sweet and Wepsic [20]. At a temperature of 50 degrees C no fibre is damaged whereas at 60 degrees C the fibre damage extends over nearly the total cut surface of the nerve. Even at the critical temperature of 55 degrees C affecting only part of the cross-section no predilection of any fibre caliber was observed. In summary thermocoagulation seems not to be able to cause selective fibre damage.

Familial lysosomal storage disease with generalized vacuolization and sialic aciduria. Sporadic Salla disease.

Two eight- and sixteen-year-old children with severe progressive neurologic disease revealed an ultrastructural finding of lysosomal vacuolization in mesenchymal or parenchymal cells of different organ biopsies (skin, muscle, nerve and liver), which may be very suggestive of mucolipidosis. However, in our patients biochemical tests available for these diseases yielded negative results, except for increased excretion of free sialic acid in urine and sialic acid storage in cultured fibroblasts. The clinical picture and the ultrastructural and biochemical findings were compatible with Salla disease, a rare lysosomal storage disease originally observed in Finland.

Proliferation of smooth muscle cells in the inner and outer layers of the tunica media of arteries: an in vitro study.

During the development of atherosclerotic and fibromuscular proliferates/lesions, smooth muscle cells (SMC) in the media, particularly near the lumen, are activated to migrate into the intima, where they continue to proliferate to form an intimal thickening. It is to date unclear whether SMCs situated adjacent to the adventitia possess a lower capacity to proliferate because they are a special subpopulation of medial SMCs or because the adventitia excerts an inhibitory effect. We have, therefore, developed an in vitro system whereby we have attempted to clear up this uncertainty. The following observations were made from the in vitro experiments: Media-explants from rabbit aorta were laid on a polycarbonate filter with pores 5 microns in diameter. The SMCs migrated through the pores and formed a fibromuscular proliferate on the other side of the filter. Endothelial cells were seeded on one side of the filter before media-explants were laid on the other side of the filter. The confluent endothelium inhibited migration of SMCs through the filter pores. Media-explants were placed between two polycarbonate filters (pores 5 microns diameter). In this "sandwich" arrangement SMCs migrated through both filters, i.e., in both directions. The quantity of migrating and proliferating cells through both filters was almost identical. This suggests that there is no difference in the migratory and proliferative capacity of SMCs in the inner and outer layers in the media of arteries. To investigate the influence of the adventitia on medial SMCs, media-explants were placed between a lower (5 microns) and an upper (0.2 micron) filter. On the 0.2 micron filter adventitia-explants were laid above the media-explants. The 0.2 micron filter prevented migration of SMCs from the media-explant into the adventitia and migration of fibroblasts from the adventitia into the media. Interestingly, the adventitial tissue inhibited proliferation of SMCs at the abluminal and migration and proliferation at the luminal side of the media-explant; the number of cells migrating through the 5 microns pores at the luminal side was diminished, suggesting that the adventitial tissue has an antiproliferative influence on SMCs. Moreover, it was found that in media-explants near the filter with adventitia, the medial SMCs were in a better preserved condition than at the de-endothelialised luminal side. As a control, cultures consisting of media-explants were incubated without filters (i.e., explant organ cultures). The proliferates in the concavity (luminal side) exhibited a pattern of proliferating SMCs different from that of the cells at the abluminal convexity.(ABSTRACT TRUNCATED AT 400 WORDS)

Fibromuscular proliferates induced in vitro using a trans-filter culture system.

A trans-filter co-culture system of vascular smooth muscle cells (SMC) and endothelial cells (EC) is compared with a trans-filter culture of SMC without EC. Explants from the tunica media of rabbit aorta are cultured on one side of a polycarbonate filter with a defined pore size (5 microns). Smooth muscle cells grow out from the media explants and migrate through the filter pores to the other side of the filter, where they proliferate. After 14 days in trans-filter culture, a multilayer resembling "fibromuscular plaques" seen in vivo is formed on the opposite side of the filter. Moreover, the proliferation rate of SMC that have migrated through the filter pores is demonstrated to be similar to that of SMC in intimal lesions induced by balloon catheter injury. In the trans-filter co-culture, a monolayer of endothelial cells (porcine, bovine, or human) is cultivated on one side of the filter. Media explants are placed on the other side. This arrangement mimics the in vivo situation of an arterial vessel wall with endothelium and media separated by a porous lamina. In this co-culture system, the few smooth muscle cells that migrate into the subendothelial space do not form a cell multilayer. A confluent endothelial cell layer is capable of inhibiting the formation of a proliferate in this co-culture system.

Induction of cell-rich and lipid-rich plaques in a transfilter coculture system with human vascular cells.

Cell-to-cell interactions are mainly involved in the control of the proliferation, migration, differentiation and function of different cell types in a wide range of tissues. In the arterial vessel wall, human arterial endothelial cells (haEC) and smooth muscle cells (haSMC) coexist in close contact with each other. In atherogenesis, haSMC can migrate from the media to the subintimal space to form fibromuscular and atheromatous plaques. In the present study, a transfilter coculture system is described, in which the interface between haSMC and confluent or proliferative haEC can be studied in detail. Cells were cocultured on the opposite sides of a porous filter which separates both cell types like the internal elastic lamina in vivo. In cocultures containing proliferative haEC, haSMC growth was significantly stimulated (33.4 +/- 5.7 cells/section, p < 0.05) compared to haSMC monocultures (22.9 +/- 2.5 cells/section) and cocultures containing confluent haEC (15.6 +/- 2.9 cells/section). If confluent haEC were injured mechanically, haSMC growth increased highly significantly (71.3 +/- 16.8 cells/section, p < 0.001). Thus, cell-rich proliferates containing 5-7 layers of haSMC embedded in extracellular matrix were formed after 14 days. On the other hand, after haSMC migration to the endothelial side had occurred, the addition of LDL and monocytes to cocultures with arterial media explants and haEC resulted in the formation of lipid-rich, low-cellular structures. After 28 days, characteristic in vitro plaque growth was induced; the plaque contained a lipid core with predominantly necrotic cells, extracellular lipid accumulations, atypically shaped lipid-loaded haSMC and macrophages, similar to in vivo foam cells, as well as an increased amount of extracellular matrix (collagen I, III and IV). These areas were surrounded by typical fibromuscular caps consisting of smooth muscle alpha-actin-positive haSMC. Finally, the formation of capillaries by haEC could also be observed within these structures.

Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation.

Tie-1 and Tie-2 define a new class of receptor tyrosine kinases that are specifically expressed in developing vascular endothelial cells. To study the functions of Tie-1 and Tie-2 during vascular endothelial cell growth and differentiation in vivo, targeted mutations of the genes in mice were introduced by homologous recombination. Embryos deficient in Tie-1 failed to establish structural integrity of vascular endothelial cells, resulting in oedema and subsequently localized haemorrhage. However, analyses of embryos deficient in Tie-2 showed that it is important in angiogenesis, particularly for vascular network formation in endothelial cells. This result contrasts with previous reports on Tie-2 function in vasculogenesis and/or endothelial cell survival. Our in vivo analyses indicate that the structurally related receptor tyrosine kinases Tie-1 and Tie-2 have important but distinct roles in the formation of blood vessels.