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At least 505 records · Page 28Linked to original sources

PubMed1994

Mossy fiber growth and synaptogenesis in rat hippocampal slices in vitro.

Hippocampal slices from early postnatal rat were used to study mossy fiber (MF) growth and synaptogenesis. The ability of MFs to form new giant synapses within isolated tissue slices was established by a series of experiments involving synapsin I immunohistochemistry, electron microscopy, and whole-cell recordings. When hippocampal slices from immature rats were cultured for up to 2 weeks, the distribution of giant MF terminals was similar to that found in vivo. Using a lesioning procedure, we determined that MFs in slices extend and form appropriate synaptic connections with normal target CA3 pyramidal cells. MF terminals were dispersed more widely than normal within the CA3 pyramidal layer after a lesion, but electron microscopy indicated that synaptic junctions were still primarily associated with pyramidal cell dendrites and not the somata. Establishment of functional synaptic input in vitro was confirmed by whole-cell recordings of MF-driven excitatory postsynaptic currents (50 pA to 1 nA) in pyramidal cells. The results establish for the first time that an MF projection with appropriate and functional synaptic connections can be formed de novo and not just maintained in excised hippocampal slices. The cellular dynamics underlying MF growth and synaptogenesis were examined directly by time-lapse confocal imaging of fibers selectively stained with a fluorescent membrane dye (Dil or DiO). MFs growing deep within isolated tissue slices were tipped by small (5-10 microns), active growth cones that advanced at variable rates (5-25 microns/hr). Furthermore, dynamic filopodial structures were seen at small varicosities along the length of developing MFs, which may identify nascent en passant synaptic contacts. The hippocampal slice preparations are shown to support normal development of MF connections and allow for direct visualization of the cellular dynamics of synapse formation in a mammalian CNS tissue environment.

Animals↗

PubMed1994

Dynamic behaviors of growth cones extending in the corpus callosum of living cortical brain slices observed with video microscopy.

During development, axons of the mammalian corpus callosum must navigate across the midline to establish connections with corresponding targets in the contralateral cerebral cortex. To gain insight into how growth cones of callosal axons respond to putative guidance cues along this CNS pathway, we have used time-lapse video microscopy to observe dynamic behaviors of individual callosal growth cones extending in living brain slices from neonatal hamster sensorimotor cortex. Crystals of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) were inserted into the cortex in vivo to label small populations of callosal axons and their growth cones. Subsequently, 400 microns brain slices that included the injection site, the corpus callosum, and the target cortex were placed in culture and viewed under low-light-level conditions with a silicon-intensified target (SIT) camera. Time-lapse video observations revealed striking differences in growth cone behaviors in different regions of the callosal pathway. In the tract, which is defined as the region of the callosal pathway from the injection site to the corresponding target cortex, growth cones advanced rapidly, displaying continual lamellipodial shape changes and filopodial exploration. Forward advance was sometimes interrupted by brief pauses or retraction. Growth cones in the target cortex had almost uniform compact shapes that were consistently smaller than those in the tract. In cortex, axons adhered to straight radial trajectories and their growth cones extended at only half the speed of those in the tract. Growth cones in subtarget regions of the callosum beneath cortical targets displayed complex behaviors characterized by long pauses, extension of transitory branches, and repeated cycles of collapse, withdrawal, and resurgence. Video observations suggested that extension of axons into cortical targets could occur by interstitial branching from callosal axons rather than by turning behaviors of the primary growth cones. These results suggest the existence of guidance cues distinct for each of these callosal regions that elicit characteristic growth cone behaviors.

Animals↗

PubMed1994

Thalamocortical axons extend along a chondroitin sulfate proteoglycan-enriched pathway coincident with the neocortical subplate and distinct from the efferent path.

The distinct axonal tracts of the mature nervous system are defined during development by sets of substrate-bound and diffusible molecular signals that promote or restrict axonal elongation. In the adult cerebral cortex, efferent and afferent axons are segregated within the white matter. To define the relationship of growing efferent and afferent axons in the developing murine cortex to chondroitin sulfate proteoglycans (CSPGs) in the pericellular and extracellular matrix, we used the fluorescent tracer Dil to determine axonal trajectories and immunolabeling to disclose the distribution of CSPGs. Axons of neurons in the preplate are the first to leave the cortex; they arise in the CSPG-rich preplate and extend obliquely across it to enter the CSPG-poor intermediate zone. Slightly later, axons of cortical plate neurons extend directly across the CSPG-rich subplate, and then turn abruptly to run in the upper intermediate zone. In contrast, once afferent axons from the thalamus reach the developing cortical wall, their intracortical trajectory is centered on the CSPG-rich subplate, above the path taken by efferent axons. Our findings demonstrate a molecular difference between the adjacent but distinct efferent and afferent pathways in developing neocortex. Early efferents cross the subplate and follow a pathway that contains very little CSPG, while afferents preferentially travel more superficially within the CSPG-rich subplate. Thus, CSPGs and associated extracellular matrix (ECM) components in the preplate/subplate do not form a barrier to axonal initiation or outgrowth in the neocortex as they may in other locations. Instead, their distribution suggests a role in defining discrete axonal pathways during early cortical development.

Animals↗

PubMed1994

Independent control of dendritic and axonal form in the developing lateral geniculate nucleus.

To identify mechanisms that regulate neuronal form in the mammalian CNS, we have examined dendritic development in the lateral geniculate nucleus (LGN) during the period of segregation of retinal ganglion cell axons. The tracer Dil was used to label retrogradely LGN neurons that send their axons to primary visual cortex at different ages between embryonic day 36 (E36) and E60 in the cat. LGN neurons grow extensively during this period, in concert with the progressive restriction of ganglion cell axons from the two eyes to their appropriate eye-specific layers. At E36 neurons have simple bipolar morphology; by E60 all have acquired complex multipolar dendritic trees. During this period, soma size increases by 190% and total dendritic length increases 240%. Dendritic complexity, as measured by dendritic branch points, also increases. As dendrites grow, the number of spines increases, but their density remains constant at 0.015/micron throughout this period. Since it is known that blockade of action potential activity significantly alters the branching pattern and extent of retinal ganglion cell axonal arbors within the LGN, we also investigated whether the dendritic development of the postsynaptic LGN neurons is similarly susceptible. Following 2 weeks of the intracranial minipump infusion of TTX between E42 and E56, the morphology of LGN neurons was examined. Surprisingly in view of the striking effect of the treatment on the morphology of retinal ganglion cell axons, dendritic growth and development were essentially normal. However, the density of dendritic spines increased almost threefold, suggesting that this specific feature of dendritic morphology is highly regulated by action potential activity. These observations indicate that normally during this period of development, the previously described changes that occur in the morphology of the presynaptic inputs to LGN neurons are accompanied by a progressive growth of post-synaptic dendrites. Because the intracranial TTX infusions have almost certainly blocked all sodium action potentials, our results suggest that the basic dendritic framework of LGN neurons can be achieved even in the absence of this form of neural activity. Moreover, since the same treatment causes a profound change in the morphology of the presynaptic axons, at least some aspects of axonal and dendritic form must be controlled independently during this prenatal period of development.

Animals↗

PubMed1993

Organized growth of thalamocortical axons from the deep tier of terminations into layer IV of developing mouse barrel cortex.

The thalamocortical projection to rodent somatosensory ("barrell") cortex is highly ordered in both the radial and the tangential dimensions. During a brief period of postnatal development, thalamocortical axons establish two tiers of terminations, in the deep layers and in layer IV, and form whisker-specific clusters within layer IV; however, little is known about the cues that guide them to their appropriate radial and tangential positions. To gain insight into potential mechanisms underlying this process, we studied the development of thalamocortical termination patterns in mouse barrel cortex at high spatial resolution. Developing thalamocortical axons were labeled in fixed slices with the lipophilic carbocyanine dye Dil and imaged with a laser scanning confocal microscope. On the day of birth (postnatal day 0, P0) axons coursed through layers VI and V, with little or no branching. By P2 the lower tier of terminations, at the border of layers VI and V, was clearly identifiable. Below this tier axons coursed obliquely or tangentially, forming a dense meshwork of intersecting fibers, but with no apparent branching. By P4 the upper tier of terminations, in layer IV, was clearly recognizable, and consisted of periodic, dense clusters of terminal arborizations. In marked contrast to the oblique and apparently disorderly course followed by axons in layer VI and lower layer V, axons in upper layer V heading toward the upper tier were organized in loose bundles running radially, suggesting that axons destined to terminate in a particular layer IV barrel had already reached their appropriate tangential coordinates within the lower tier. Thus, the pattern of thalamocortical terminations in layer IV seems to be projected from the deep tier of terminations, and does not develop from an initially profuse arborization pattern through pruning of inappropriate branches.

Animals↗

PubMed1994

Olfactory glomeruli in the zebrafish form an invariant pattern and are identifiable across animals.

Glomeruli are anatomical and possibly functional modules in the vertebrate olfactory bulb. We investigated the spatial arrangement of glomeruli in the olfactory bulbs of adult zebrafish (Brachydanio rerio). A solution of the lipophilic tracer Dil was injected into the nasal cavities. Axons of sensory neurons projecting from the olfactory epithelium into the bulb were traced anterogradely, thus labeling the whole population of glomeruli. The glomerular distribution was analyzed in detail by confocal laser-scanning microscopy. We find that a typical olfactory bulb contains a small number of about 80 glomeruli that have a stereotyped configuration in all animals investigated. All glomeruli exhibit bilateral symmetry. Twenty-two single glomeruli could be identified from animal to animal by their characteristic position and morphology. The remaining glomeruli either are embedded in glomerular plexus and therefore cannot be delineated reliably, or belong to a densely clustered subpopulation of on average 49 glomeruli in the dorsal olfactory bulb. No sexually dimorphic glomeruli were identified. To test whether glomerular constancy is specific for the zebrafish, we performed similar tracing experiments in the goldfish and found several indications for a similar invariance of glomeruli in this species. The remarkable stereotypy of this pattern is reminiscent of the insect olfactory system and has been demonstrated here for the first time in a vertebrate. It will now be possible to examine whether these identifiable glomeruli are functionally specialized in terms of odor processing. If so, zebrafish may emerge as a tractable model system for studies on olfactory coding.

Animals↗

PubMed1993

Cultivation and characterization of micro- and macrovascular endothelial cells from the human heart.

In order to investigate processes, such as atherosclerosis and inflammation in vitro, it is necessary to obtain viable and pure endothelial cell cultures from human hearts. To this end, endothelial cells were isolated and cultured from the micro- and macrovasculature of human hearts obtained during heart transplantation. Isolation of capillaries after enzymatic digestion of heart muscle provided a source of microvascular endothelial cells. Contaminating non-endothelial cells were removed by a new technique: paramagnetic beads linked to the lectin ulex europaeus I (UEA-I) were used to select endothelial cells. The resulting cultures contained less than 2% of non-endothelial cells, as judged from immunological staining and fluorescence-activated cell sorting. Both types of endothelial cell displayed typical endothelial properties. They were all positive for factor VIII-related antigen and expressed the endothelial-specific adhesion molecules, CD31 and E-selectin (ELAM-1), after stimulation with cytokines. In addition, they could be labelled with Dil-Ac-LDL, contained angiotensin converting enzyme activity and secreted tissue plasminogen activator, thus demonstrating that typical endothelial functions were preserved in culture.

Capillaries↗

PubMed1993

Four different classes of inhibitors of receptor-mediated endocytosis decrease tumor necrosis factor-induced gene expression in human endothelial cells.

We have investigated the relationship between receptor-mediated endocytosis of TNF and TNF-induced gene expression in cultured human endothelial cells. Exposure of cells to hypertonicity, to cytoplasmic acidification, to treatment with phenylarsine oxide, or to treatment with primary amines such as putrescine or dansylcadaverine each inhibited receptor-mediated endocytosis by 30 to 75%, as measured by uptake of acetylated Dil-low density lipoprotein or of 125I-TNF. All four treatments also inhibited TNF-induced surface expression of ELAM-1 by 50 to 100%. Among these four treatments, only hypertonicity inhibited pinocytosis, as measured by uptake of fluorescein-BSA, and only phenylarsine oxide irreversibly inhibited protein synthesis, as measured by [35S]methionine incorporation. Notably, acidification or treatment with primary amines selectively inhibited the response to TNF, compared with the response to PMA, a drug that induces ELAM-1 through a pathway that bypasses surface receptors. Primary amines, which can be used for sustained periods under physiologic culture conditions without causing toxicity, were investigated further. Pretreatment of endothelial cells with 10 mM putrescine or 100 microM dansylcadaverine also inhibited TNF induction of ICAM-1 expression and VCAM-1 expression. Primary amines also inhibited IL-1-induced increases in ELAM-1, ICAM-1, and VCAM-1 measured 4 to 6 h after treatment and inhibited IFN-beta- and IFN-gamma-mediated induction of class I MHC molecules and IFN-gamma-mediated induction of class II MHC molecules measured 72 h after treatment with cytokine. Levels of mRNA encoding cytokine-inducible molecules were also selectively reduced by primary amines. A constitutively expressed surface molecule, gp96, was not affected in the same cells. These data are consistent with a role for receptor-mediated endocytosis in TNF-mediated gene induction and suggest a new potential target for anti-inflammatory therapy.

Arsenicals↗

PubMed1993

Membrane properties of ameboid microglial cells in the corpus callosum slice from early postnatal mice.

Microglial cells in culture are distinct from neurons, macroglial cells, and macrophages of tissues other than brain with respect to their membrane current pattern. To assess these cells in the intact tissue, we have applied the patch-clamp technique to study membrane currents in microglial cells from acute, whole brain slices of 6-9-d-old mice in an area of microglial cell invasion, the cingulum. As strategies to identify microglial cells prior to or after recording, we used binding and incorporation of Dil-acetylated low-density lipoproteins, binding of fluorescein isothiocyanate-coupled IgG via microglial Fc-receptors, and ultrastructural characterization. As observed previously for cultured microglial cells, depolarizing voltage steps activate only minute if any membrane currents, while hyperpolarizing voltage steps induced large inward currents. These currents exhibited properties of the inwardly rectifying K+ channel in that the reversal potential depended on the transmembrane K+ gradient, inactivation time constants decreased with hyperpolarization, and the current was blocked by tetraethylammonium (50 mM). This study represents the first attempt to assess microglial cells in situ using electrophysiological methods. It opens the possibility to address questions related to the function of microglial cells in the intact CNS.

4-Aminopyridine↗

PubMed1993

The effects of low density lipoprotein on calcium transients in isolated rabbit cardiomyocytes.

The purpose of this study was to examine the effects of low density lipoprotein (LDL) on Ca2+ transients of isolated rabbit cardiomyocytes. Incubation of cardiomyocytes with > or = 1 mg of LDL cholesterol/ml of perfusion medium induced a slow (> or = 30 min) but significant increase (2-fold) in the cellular Ca2+ transient. The time course for the effect was similar to that observed for the accumulation of cholesterol in the cells. Using Dil- labeled LDL as a fluorescent marker for LDL interaction with the cardiomyocytes, it was concluded that LDL interacted via a receptor-mediated event, but probably this was not the primary mechanism whereby the lipid entered the cell. LDL-treated cells were resistant to the depressant actions for ryanodine, nicardipine, and dichlorobenzamil on the cellular Ca2+ transient. Lowering the extracellular Ca2+ concentration removed the stimulatory effect of LDL on the Ca2+ transient. It is concluded that LDL can induce an increase in the magnitude of the Ca2+ transient in isolated cardiomyocytes. This is a relatively slow process. The mechanism appears to involve a stimulation of a transsarcolemmal Ca2+ transport pathway. These findings have important implications for cardiac contractile function in hypercholesterolemic and drug-treated hypercholesterolemic subjects.

Amiloride↗

PubMed1996

Retroviral misexpression of engrailed genes in the chick optic tectum perturbs the topographic targeting of retinal axons.

We have investigated the role of the homeodomain transcription factor genes En-1 and En-2, homologs of the Drosophila segment polarity gene engrailed, in regulating the development of the retinotopic map in the chick optic tectum. The En proteins are distributed in a gradient along the rostral-caudal axis of the developing tectum, with highest amounts found caudally. Previous evidence suggests that En-1 and En-2 may regulate the polarity of the rostral-caudal axis of the tectum and the subsequent topographic mapping of retinal axons. We have tested this hypothesis by using a recombinant replication-competent retrovirus to overexpress the En-1 or En-2 genes in the developing tectum. Anterograde labeling with the axon tracer Dil was used to analyze the topographic mapping of retinal axons after the time that the retinotectal projection is normally topographically organized. Overexpression of either En-1 or En-2 perturbed the topographic targeting of retinal axons. In En-infected tecta, nasal retinal axons form an abnormally diffuse projection with numerous aberrant axons, branches, and arbors found at topographically incorrect locations, colocalized with domains of viral infection. In contrast, temporal axons did not form a diffuse projection or discrete aberrant arbors; however, many temporal axons were stunted and ended aberrantly rostral to their appropriate TZ, or in other cases either did not enter the tectum or formed a dense termination at its extreme rostral edge. These findings indicate that En-1 and En-2 are involved in regulating the development of the retinotopic map in the tectum. Furthermore, they support the hypothesis that En genes regulate the polarity of the rostral-caudal axis of the tectum, most likely by controlling the expression of retinal axon guidance molecules.

Animals↗

PubMed1996

Rapid characterization of disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene by overexpression in COS cells.

To characterize disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene, COS cells are transfected with the mutant gene in an EBV-based expression vector and characterized by flow cytometry. Using antibodies against the LDL-receptor the amount of receptor protein on the cell surface is quantitated. The receptor activity is measured by incubating the cells with fluorescence labeled LDL (Dil-labelled LDL) at 37 degrees C and 4 degrees C. The transfected cells stained with anti-LDL-R antibodies can also be analysed by immunofluorescence microscopy allowing the study of the intracellular location of variants of the receptor. To evaluate these methods, we are analyzing four previously well-characterized LDL-R mutations, belonging to each of the classes 2 to 5. Preliminary data show that mutant genes belonging to class 3 and 4A give rise to receptor protein on the cell surface, but impaired LDL uptake, while mutant receptors belonging to class 2A and 5 can only be detected intracellularly. Expression of the class 2A mutation results in an ER staining pattern, whereas the class 5 mutation gives rise to an intracellular staining compatible with localization in the endosomal/lysosomal compartments. We conclude that this system is useful for a rapid functional analysis of newly discovered mutations in the LDL-R gene.

Cell Line↗

PubMed1996

Dynamics of target recognition by interstitial axon branching along developing cortical axons.

Corticospinal axons innervate their midbrain, hindbrain, and spinal targets by extending collateral branches interstitially along their length. To establish that the axon shaft rather than the axonal growth cone is responsible for target recognition in this system, and to characterize the dynamics of interstitial branch formation, we have studied this process in an in vivo-like setting using slice cultures from neonatal mice containing the entire pathway of corticospinal axons. Corticospinal axons labeled with the dye 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (or Dil) were imaged using time-lapse video microscopy of their pathway overlying the basilar pons, their major hindbrain target. The axon shaft millimeters behind the growth cone exhibits several dynamic behaviors, including the de novo formation of varicosities and filopodia-like extensions, and a behavior that we term "pulsation," which is characterized by a variable thickening and thining of short segments of the axon. An individual axon can have multiple sites of branching activity, with many of the branches being transient. These dynamic behaviors occur along the portion of the axon shaft overlying the basilar pons, but not just caudal to it. Once the collaterals extend into the pontine neuropil, they branch further in the neuropil, while the parent axon becomes quiescent. Thus, the branching activity is spatially restricted to specific portions of the axon, as well as temporally restricted to a relatively brief time window. These findings provide definitive evidence that collateral branches form de novo along corticospinal axons and establish that the process of target recognition in this system is a property of the axon shaft rather than the leading growth cone.

Animals↗

PubMed1996

Development of an identified spinal commissural interneuron population in an amniote: neurons of the avian Hofmann nuclei.

The commissural interneurons of the Hofmann nuclei (HN) of the avian spinal cord (The axonal projections of the Hofmann nuclei in the spinal cord of the late stage chicken embryo, Anat Embryol (Berl), A.L. Eide, 1996, Vol 193, pp 543-557) provide a unique opportunity to describe the development of an identified spinal commissural axon projection and its terminal collaterals in an amniote vertebrate. Here, we use the lipophilic tracer Dil to label these and other commissural projections anterogradely and retrogradely from the time the HN neurons are born. [3H]thymidine birthdating shows that the final mitoses of HN neurons occur at stages 21-24 [developmental day (d) 4]. By direct comparison, this follows the generation of motoneurons and of large, dorsally located commissural interneurons. The first HN neurons reach the ventrolateral margin of the spinal cord by d6 by a radial migration through the ventral horn. Radial migration occurs after the extension of HN axons across the midline. Thus, HN neurons are determined to be commissural interneurons before attaining their definitive locations. The HN neurons subsequently aggregate into segmentally iterated clusters at the ventrolateral margin of the spinal cord by d8. Also by d8 their logitudinal axons attain mature extent in the ventral funiculus of the contralateral side and begin to sprout collaterals. The collaterals are directed predominantly toward the medial aspect of the ventral horn at all stages, forming by d12 a dense thicket of terminals that thins out over several segments to each side of the HN of origin. The initial direction of collateral outgrowth is largely appropriate for the mature termination pattern of the HN. Terminal arbors, however, are less focused at early developmental stages than at later stages.

Animals↗

PubMed1996

Dynamics of cell migration from the lateral ganglionic eminence in the rat.

From previous developmental studies, it has been proposed that the neurons of the ventrolateral cortex, including the primary olfactory cortex, differentiate from progenitor cells in the lateral ganglionic eminence. The objective of the present study was to test this hypothesis. The cells first generated in the forebrain of the rat migrate to the surface of the telencephalic vesicle by embryonic day (E) 12. Using [3H]thymidine, we found that most of these cells contributed to the formation of the deep layer III of the primary olfactory cortex. To study the migratory routes of these cells, we made localized injections of the carbocyanine fluorescent tracers Dil and DiA into various parts of the lateral ganglionic eminence in living embryos at E12-E14 and subsequently maintained the embryos in a culture device for 17-48 hr. After fixation, most migrating cells were located at the surface of the telencephalic vesicle, whereas others were seen coursing tangentially into the preplate. Injections made at E13 and in fixed tissue at E15 showed that migrating cells follow radial glial fibers extending from the ventricular zone of the lateral ganglionic eminence to the ventrolateral surface of the telencephalic vesicle. The spatial distribution of radial glial fibers was studied in Golgi preparations, and these observations provided further evidence of the existence of long glial fibers extending from the ventricular zone of the lateral ganglionic eminence to the ventrolateral cortex. We conclude that cells of the primary olfactory cortex derive from the lateral ganglionic eminence and that some early generated cells migrating from the lateral ganglionic eminence transgress the cortico-striatal boundary entering the preplate of the neocortical primordium.

Animals↗

PubMedDate not supplied

Establishment of an immortalized Copenhagen rat bone marrow endothelial cell line.

Bone marrow endothelial cells are critical mediators in the processes of cell trafficking as well as cancer metastasis, however few established models exist. An immortal cell line of Copenhagen rat bone marrow endothelium was established after infection of primary cultured cells with Adenovirus-12 SV40 hybrid virus and designated YPBE-1. The established cell line has continued to proliferate more than 70 population doublings and has not undergone "crisis". It stains positively for SV40 T-antigen in its nuclei by immunohistochemistry and grows in a monolayer with a cobblestone appearance. It demonstrates Dil-Ac-LDL uptake as an endothelial marker. YPBE-1 does not express Integrin beta 3 or endothelin, but does express Integrin alpha 6 beta 1 on the plasma membrane and demonstrates tube formation in Matrigel. This cell line of rat bone marrow endothelial origin should be useful for studying mechanisms of bone metastasis and cell trafficking.

Animals↗

PubMed1996

A dynamic model of the blood-brain barrier "in vitro".

Cell culture models have been widely used for screening of neurotoxicants and represent a viable alternative to direct in vivo experiments. We have developed a dynamic in vitro blood-brain barrier model designed to allow for extensive toxicological, pharmacological and physiological testing. Induction of blood-brain barrier properties in a tri-dimensional hollow fiber culturing apparatus was investigated by co-culturing a bovine aortic endothelial cell line (or rat brain endothelial cells) with rat brain astrocytes (or C6 rat glioma cells) under pulsatile flow conditions to mimic intraluminal blood flow. Cell growth was monitored over time by measuring glucose consumption and lactate production: these experiments confirmed that the hollow fiber cell culturing systems can maintain viable cells in culture for extended (> 1 month) periods of time. Cells were visually inspected after culturing and dissociation from the hollow fiber cartridge and identified as endothelial (by fluorescent Dil-Ac-LDL uptake) or glial (by GFAP immunoreactivity). Blood-brain barrier properties were tested by intraluminal injection of horse-radish peroxidase (HRP, mol. weight 44,000), glucose (m.w. 180) or potassium. Either procedure demonstrated that aortic cells co-cultured with astrocytes (or C6 cells) developed a selective barrier with an estimated electrical resistance of 2,900 omega/cm2. The electrophysiological and morphological properties of BAEC were also affected by the co-culturing process, suggesting that astrocytes induced CNS properties in these cells. These results demonstrate that the hollow fiber cell co-culturing system may be used as a dynamic model of the mammalian blood-brain barrier.

Animals↗

PubMedDate not supplied

Establishment of immortalized Copenhagen rat prostate endothelial cell lines.

Since the introduction of culture methods for umbilical vein endothelial cells, many successful attempts for culturing endothelial cells have been reported. The successful establishment of immortalized cell lines of organ specific endothelium, however, has been rare. Primary cultured endothelial cells isolated from the Copenhagen rat prostate were immortalized by infection with an Adenovirus-12 SV40 hybrid virus. Two immortal cell lines of Copenhagen rat prostate endothelium were established and designated YPEN-1 and YPEN-2. The established cell lines have continued to proliferate more than 80 population doublings and have not undergone senescence. They stain positively for but are nonproducers of SV4O0 T-antigen and grow in a monolayer with a cobblestone appearance. They demonstrate Dil-Ac-LDL uptake as an endothelial marker. YPEN-1 and YPEN-2 cells exhibit positive staining for endothelin and MRC OX-43 and express Integrin a6 beta 1 and Integrin beta 3 on their plasma membrane and demonstrate tube formation in Matrigel. Doubling times of YPEN-1 and YPEN-2 are 26 hours and 21 hours, respectively. Genetically, YPEN-1 and YPEN-2 are both diploid. These cell lines of rat prostate endothelial origin should be useful for studying angiogenesis and its inhibition.

Animals↗