Biomedical subjects

I Bechmann

Publications and source records attributed to I Bechmann.

25 records · Page 2Linked to original sources

PubMed1999

FasL (CD95L, Apo1L) is expressed in the normal rat and human brain: evidence for the existence of an immunological brain barrier.

Despite the mechanical blood-brain barrier, activated T-cells can cross brain vessels. Thus, the CNS is routinely surveyed by immune competent cells; yet the healthy brain is not a target of antigen-specific immune reactions. Therefore, mechanisms must exist to prevent brain-antigen-specific T-cells from inducing immune responses. Data indicate that activated T-cells entering the CNS may undergo apoptosis rather than leaving the brain to induce immune responses. Applying RT-PCR, Western-blots, and immunocytochemistry, we have demonstrated expression of the apoptosis-inducing protein Fas ligand on astrocytes and neurons of apparently normal rat and human brains. FasL-positive astrocytes were often situated in close vicinity to cerebral blood vessels in vivo and induced apoptosis of Fas expressing Jurkat cells in vitro. We propose that similar to other immune privileged organs FasL-induced apoptosis of activated T-cells in the brain protects the tissue from self damaging immune attacks by forming an immunological brain barrier.

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PubMed1998

Tracing of the entorhinal-hippocampal pathway in vitro.

In vitro tract tracing allowing for continuous observation of the perforant path is a crucial prerequisite for experimental studies on the entorhinal-hippocampal interaction in an organotypic slice culture containing the entorhinal cortex, the perforant path, and the dentate gyrus (OEHSC). We prepared horizontal slices of the temporal entorhinal-hippocampal region of the rat on a vibratome, and the perforant path axons were traced by application of the fluorescent tracer Mini Ruby on the entorhinal cortex. After 2 days in vitro (div), the perforant path became visible in most cultures. Entorhinal neurons and single perforant fibers could be followed to the outer molecular layers of the dentate gyrus by in vitro fluorescence microscopy and it was possible to monitor the perforant path directly over a period of 25 div. Moreover, ultrastructural analysis proved the existence of traced perforant path boutons forming synapses with spines and dendritic shafts in the outer molecular layers of the dentate gyrus. Transsection of the prelabelled perforant path in vitro resulted in anterograde degeneration and subsequent phagocytosis of axonal material by activated microglial cells in the zone of denervation. In conclusion, in vitro tracing demonstrates the maintenance of the entorhinal-hippocampal pathway in OEHSCs and permits monitoring of dynamic changes in the prelabeled perforant path after various lesion paradigms, e.g., transsection or neurotoxin treatment. This approach permits further studies on the efficacy of neuroprotectants, cytokines, and growth factors in the treatment of lesion-induced neuronal degeneration.

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PubMed1997

Heterogeneity in the neuropeptide Y-containing neurons of the rat arcuate nucleus: GABAergic and non-GABAergic subpopulations.

Neuropeptide Y, produced in the arcuate nucleus of the hypothalamus, plays a key role in the central regulation of anterior pituitary and appetitive functions. The pleiotropic nature of neuropeptide Y in these mechanisms indicates the existence of heterogeneity in the hypothalamic neuronal population producing neuropeptide Y. In this study, we report the coexistence of neuropeptide Y and the amino acid transmitter, gamma-aminobutyric acid (GABA), in neuronal perikarya of the arcuate nucleus. Fluorescent double immunolabeling for neuropeptide Y and glutamic acid decarboxylase was carried out on vibratome sections collected through the hypothalamic arcuate nuclei of animals that were pretreated with colchicine. It was found that about one third of the neuropeptide Y-producing arcuate nucleus perikarya co-expressed glutamic acid decarboxylase. This population of neuropeptide Y-containing GABAergic neurons were distributed longitudinally within the arcuate nucleus located predominantly in its dorsomedial aspects. These results show that there are at least two distinct populations of neuropeptide Y-producing neurons in the arcuate nucleus: a subset of neuropeptide Y and GABA-co-producing neurons located in the dorsomedial arcuate nucleus and a subset of non-GABAergic neuropeptide Y cells located in the ventral arcuate nucleus. This heterogeneity in the neuropeptide Y-producing perikarya of the hypothalamus may help explain adverse neuroendocrine and behavioral effects of arcuate nucleus neuropeptide Y.

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PubMed1997

Adhesion molecule expression on phagocytic microglial cells following anterograde degeneration of perforant path axons.

Entorhinal cortex lesion (ECL) leads to anterograde degeneration of perforant path axons and is known to induce a rapid and intense reaction of astrocytes and microglial cells in the deafferented dentate gyrus. Phagocytosis of degenerating axons involves the establishment and maintenance of cell-matrix and cell-cell interactions by activated glial cells. It was thus our aim to investigate whether the process of axon phagocytosis is accompanied by the expression of adhesion molecules on activated microglial cells or reactive astrocytes, as such molecules mediate bot cell-matrix and cell-cell interactions. We found that the integrin adhesion molecules leukocyte function antigen-1 (LFA-1), very late antigen-4 (VLA-4), and the ligand for LFA-1, intercellular adhesion molecule-1 (ICAM-1), were expressed on microglial cells accumulating in the outer molecular layer of the deafferented dentate gyrus. This upregulation of adhesion molecule expression on microglial cells showing morphological criteria of activation occurred rapidly following ECL, reached its peak at 3 days post lesion (dpl), and gradually returned to control levels after 9 dpl. Astrocytes were never labeled by antibodies directed against these adhesion molecules. Prelabeling of the perforant path with a fluorescent tracer and subsequent ECL led to phagocytosis of fluorescent-labeled axonal debris by cells that were located in the outer molecular layer and showed typical microglial morphology. Double-fluorescence labeling demonstrated that microglial cells engaged in the phagocytosis of axonal debris expressed LFA-1, VLA-4, and the LFA-1-ligand ICAM-1. In conclusion, our results demonstrate that anterograde degeneration of perforant path axons results in adhesion molecule expression on activated microglial cells engaged in axon phagocytosis. The expression of such molecules could represent a mechanism that retains activated microglia in areas of axonal degeneration and perhaps enables the interaction of microglial cells with each other or with other immunocompetent cells.

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PubMed1997

Astrocytes and microglial cells incorporate degenerating fibers following entorhinal lesion: a light, confocal, and electron microscopical study using a phagocytosis-dependent labeling technique.

Entorhinal lesion leads to anterograde degeneration of perforant path fibers in their main termination zone in the outer molecular layers of the dentate gyrus. Concomitantly, astrocytes become hypertrophic, and microglial cells alter their phenotype, suggesting participation in anterograde degeneration. This study analyzes the involvement of these lesion-induced activated glial cells in the process of phagocytosis of degenerated axonal debris. We established a phagocytosis-dependent labeling technique that allows for direct and simultaneous visualization of both labeled incorporated axonal debris and incorporating glial cells. Stereotaxic application of small crystals of the biotin- and rhodamine-conjugated dextran amine Mini Ruby (MR) into the entorhinal cortex led to strong and stable axonal staining of perforant path axons. Following entorhinal lesion, labeled terminals and fibers condensed and formed small granules. Incorporation of these rhodamine-fluorescent granules resulted in a phagocytosis-dependent cell labeling. During the first 3 days, we were able to identify these cells as microglia by using double-fluorescence and confocal microscopy. The first unequivocally double-labeled astrocytes were found 6 days post lesion (dpl). Whereas in all stages a subpopulation of microglial cells remained devoid of MR-labeled granules, all astrocytes in the middle molecular layer were double-labeled after long survival times (20 dpl). On the ultrastructural level, labeled granules appeared to be perforant path axons containing the tracer. Both terminals and myelinated fibers could be seen inside the cytoplasm of microglial cells and astrocytes. Thus, anterograde degeneration is a sufficient stimulus to induce axon incorporation by both astrocytes and a subpopulation of microglial cells.

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PubMed1997

Expression of synaptophysin in sprouting neurons after entorhinal lesion in the rat.

Expression of the synaptic vesicle protein synaptophysin was studied in lesion-induced sprouting neurons of the contralateral entorhinal cortex and in the contralateral dentate gyrus using immunocytochemistry at the light- and electron-microscopic level. Perikaryal immunoreactivity for synaptophysin was found between 8 and 10 days postlesion. Light microscopy revealed that synaptophysin immunostaining was present in almost all neurons of layers II and III of the contralateral medial entorhinal cortex. These neurons give rise to the sprouting, crossed temporodentate pathway. In addition, some hilar neurons of the contralateral dentate gyrus, which are the parent cells of sprouting commissural fibers, were immunostained for synaptophysin. Transient immunostaining for synaptophysin was observed within cell bodies and dendrites. Additionally, the cell bodies were outlined by immunoreactive puncta, identified by electron microscopy as nerve terminals. Our results revealed that sprouting neurons express the major synaptic vesicle protein synaptophysin during reactive synaptogenesis in a pattern that reflects biosynthesis and sorting of this protein as seen in developing neurons during synapse formation.

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PubMed1997

Identification of phagocytic glial cells after lesion-induced anterograde degeneration using double-fluorescence labeling: combination of axonal tracing and lectin or immunostaining.

Retrograde and anterograde degeneration have been reported to be sufficient stimuli to activate glial cells, which, in turn, are involved in phagocytosis of degenerating material. Here we describe a double-fluorescence technique which allows for direct and simultaneous visualization of both labeled incorporated axonal debris and incorporating glial cells in the course of anterograde degeneration. Stereotaxic application of small crystals of biotinylated and tetramethylrhodamine (TRITC)-conjugated dextran amine Mini Ruby into the medial entorhinal cortex resulted in a stable rhodamine fluorescence confined to fibers and terminals in the middle molecular layer of the dentate gyrus, the stratum lacunosum-moleculare, and the crossed temporo-hippocampal pathway. Subsequent stereotaxic lesion of the entorhinal cortex induced transformation of rhodamine-fluorescent fibers and terminals into small granules. Incorporation of these granules by microglial cells [labeled by fluorescein isothiocyanate (FITC)-coupled Bandeiraea simplicifolia isolectin B4] or astrocytes (labeled by FITC-coupled glial fibrillary acidic protein antibodies) resulted in phagocytosis-dependent labeling of these non-neuronal cells, which could be identified by double-fluorescence microscopy. Electron microscopical analysis revealed that, following lesion, the tracer remained confined to entorhinal axons which were found to be incorporated by glial cells. Our data show that TRITC- and biotin-conjugated dextran amines are versatile tracers leading to Phaseolus vulgaris leucoagglutinin-like axonal staining. Lesion-induced phagocytosis of anterogradely degenerating axons by immunocytochemically identified glial cells can be directly observed by this technique on the light and electron microscopical levels.

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