The aim of the study was to characterize the interendothelial junctions in tumor microvessels of five cases of human glioblastoma multiforme. In addition to morphological analysis, tumors were screened for the expression of junctional proteins, such as occludin, claudin-1, ZO-1 and catenins. The expression of the tight junction protein claudin-1 was lost in the majority of tumor microvessels, whereas claudin-5 and occludin were significantly down-regulated only in hyperplastic vessels. As shown by freeze-fracture analysis, under the conditions of tumor growth tight junction particles of endothelial cells were almost exclusively associated with the exocytoplasmic fracture face, providing evidence for a switch of the particles from the protoplasmic to the external leaflet of the endothelial membrane. These results suggest a relationship between claudin-1 suppression and the alteration of tight junction morphology, which is likely to correlate with the increase of endothelial permeability. Underlining the undifferentiated state of tumor microvessels, plakoglobin, a crucial protein for mature endothelial junctions, was not detectable in most microvessels, whereas beta-catenin was abundantly labeled. In this context, it is of particular interest that the majority of microvascular pericytes were negative for alpha-smooth muscle actin, which is a marker of differentiated pericytes, although pericytes were frequently found in electron micrographs. In conclusion, the data suggest that the increase in microvascular permeability in human gliomas, contributing to the clinically severe symptoms of brain edema, is a result of a dysregulation of junctional proteins.

The quality of the blood-brain barrier (BBB), represented mainly by endothelial tight junctions (TJ), is now believed to be dependent on the brain microenvironment and influenced by the basal lamina of the microvessels. In the highly vascularized glioblastoma multiforme (GBM), a dramatic increase in the permeability of blood vessels is observed but the nature of basal lamina involvement remains to be determined. Agrin, a heparan sulfate proteoglycan, is a component of the basal lamina of BBB microvessels, and growing evidence suggests that it may be important for the maintenance of the BBB. In the present study, we provide first evidence that agrin is absent from basal lamina of tumor vessels if the TJ molecules occludin, claudin-5 and claudin-1 were lacking in the endothelial cells. If agrin was expressed, occludin was always localized at the TJ, claudin-5 was frequently detected, whereas claudin-1 was absent from almost all vessels. Furthermore, despite a high variability of vascular phenotypes, the loss of agrin strongly correlated with the expression of tenascin, an extracellular matrix molecule which has been described previously to be absent in mature non-pathological brain tissue and to accumulate in the basal lamina of tumor vessels. These results support the view that in human GBM, BBB breakdown is reflected by the changes of the molecular compositions of both the endothelial TJ and the basal lamina.

In contrast to adult mammals, adult teleost fish regularly generate new neurons and glial cells in many brain regions. A previous quantitative mapping of the proliferation zones in the brain of adult Apteronotus leptorhynchus (Teleostei, Gymnotiformes) has shown that 75% of all mitotically active cells are situated in the cerebellum (Zupanc and Horschke [1995] J. Comp. Neurol. 353:213-233). By employing the thymidine analogue 5-bromo-2'-deoxyuridine, we have, in the present study, investigated the postembryonic development of this brain region in detail. In the corpus cerebelli and the valvula cerebelli, the vast majority of newborn cells originate in the respective molecular layers. Within the first few days of their life, these cells migrate toward specific target areas, namely, the respective granule cell layers. In the caudal part of the cerebellum, the granule cell layer of the eminentia granularis pars medialis displays the highest mitotic activity. From there, the cells migrate through the adjacent molecular layer to the granule cell layer of the eminentia granularis pars posterior. Combination of retrograde-tracing techniques with immunohistochemistry for 5-bromo-2'-deoxyuridine showed that at least a portion of the newly generated cells develop into granule neurons. Many of the newly generated cells survive for long periods of time. A large fraction of these cells is added to the population of already existing cells, thus resulting in a permanent growth of the target areas and their associated structures.

Human gingival keratinocytes (HGKs) were studied by means of freeze-fracture technique, conventional electron microscopy and the transepithelial electrical resistance for the investigation of intercellular contacts. For the purpose of comparison, MDCK cells and HaCat cells were also included. An unexpected finding was the presence of tight junctions in the HGKs. In vivo the tight junctions, which were of low complexity and P-face-associated, were co-distributed with desmosomes; in one case, the strands ran directly through desmosomal plaques. Where tight junctions and desmosomes occurred together, no gap junctions were seen. In contrast, where no tight junctions were present, gap junctions and desmosomes were co-localized. However, the unfavourable fracture planes through the tissue did not allow a clearcut allocation of gap junction/tight junction occurrence to certain strata. In vitro, HGKs also expressed tight junctions which formed networks of low complexity and high P-face association. Whereas desmosomes were highly expressed, gap junctions were not observed in cultured keratinocytes. Transepithelial electrical resistances (TEER) of cultured HGKs were higher than the values in low resistance-MDCK cells and HaCat cells but considerably lower than the values in high resistance MDCK cells, supporting the fundamental correlation between tight junction morphology and TEER. The results with this cell culture model of the human gingiva provide some valuable information about in vitro differentation and concommittent changes in cellular contacts of human gingival keratinocytes.

In contrast to adult mammals, adult teleost fish regularly generate new neurons and glial cells in many brain regions. A previous quantitative mapping of the proliferation zones in the brain of adult Apteronotus leptorhynchus (Teleostei, Gymnotiformes) has shown that 75% of all mitotically active cells are situated in the cerebellum (Zupanc and Horschke [1995] J. Comp. Neurol. 353:213-233). By employing the thymidine analogue 5-bromo-2'-deoxyuridine, we have, in the present study, investigated the postembryonic development of this brain region in detail. In the corpus cerebelli and the valvula cerebelli, the vast majority of newborn cells originate in the respective molecular layers. Within the first few days of their life, these cells migrate toward specific target areas, namely, the respective granule cell layers. In the caudal part of the cerebellum, the granule cell layer of the eminentia granularis pars medialis displays the highest mitotic activity. From there, the cells migrate through the adjacent molecular layer to the granule cell layer of the eminentia granularis pars posterior. Combination of retrograde-tracing techniques with immunohistochemistry for 5-bromo-2'-deoxyuridine showed that at least a portion of the newly generated cells develop into granule neurons. Many of the newly generated cells survive for long periods of time. A large fraction of these cells is added to the population of already existing cells, thus resulting in a permanent growth of the target areas and their associated structures.