Karl Meller

Scanning electron microscopic studies on the development of the chick retina

SummaryFixed retinae of chick embryos and chicks of the first week after hatching were fractured and examined with the scanning electron microscope. The matrix cells of the retina proliferate up to the beginning of the second week. The migrating cells are oriented in cell cords. This columnar organization prevails up to the development of the plexiform layers formed as a consequence of the outgrowth of the dendritic and axonal cell processes.Special attention was paid to the differentiation of the ganglion, bipolar and receptor cells, and the radial fibers (Müller cells).Two main morphological patterns are significant for the organization of the retina during neurogenesis:a)the cell to cell contacts of migrating cells andb)the spatial arrangement of Müller cells which could provide guidelines for migration of neuronal elements.

Human cornea and sclera studied by atomic force microscopy

Abstract.The purpose of this study was to investigate the ultrastructure of the extracellular matrix of human cornea and sclera by using the atomic force microscope (AFM). Specimens of human cornea (n=16) and sclera (n=10) were obtained from a cornea bank or from enucleated eyes (n=1; clinical and histopathological diagnosis: choroidal melanoma) and fixed in Karnovsky solution. The AFM resolved individual collagen fibrils in corneal and scleral tissue. Scleral collagen fibrils had a diameter ranging from 118.3 to 1268.0 nm and showed clear banding with a mean axial D-periodicity of 77.02 nm. The mean gap depth between the two overlaps was larger in the sclera than in the cornea. The diameter of corneal collagen fibrils ranged from 48.0 to 113.0 nm. In contrast to the sclera, the corneal collagen fibrils did not exhibit clear banding as their surface pattern. Closely attached fibrils with a beaded to globular structure were predominant in the cornea. The mean axial D-periodicity of the corneal collagen fibrils was 68.50 nm. In both tissues, the AFM resolved structures resembling cross-bridges between adjacent fibrils. The corneal collagen fibrils showed fibrillar properties that were different from those of the sclera, and that therefore might be essential for the spatial organization responsible for the optical quality of the cornea.

In vivo and in vitro formation of the junctional complex in choroid epithelium

SummaryThe Junctional complex of choroid epithelial cells was studied during in vivo formation, disaggregation after trypsin treatment, and in vitro reaggregation. The in vivo formation begins with the occurrence of amorphous patches of particles followed by the formation of small particulate rows and polygonal-ordered particle assemblies. Further arrangement of the zonula occludens continues with the confluence of particles and smooth contoured ridges. At the 9th day stage a fully developed zonula occludens has developed. In a subsequent step nexus become integrated within the tight junction formation. Disaggregation after trypsination results in fragmentation of the zonulae occludentes. Parts of the disassembling aggregates become incorporated in vacuoles indicating an endocytotic mode of “digestion”. The in vitro reconstruction of the zonula occludens proceeds from remnants of the former zonula occludens. On the 3rd to 4th day of cultivation mature tight junctions are visible. In vitro integrations of nexus were observed during a later phase. On the 7th day, cultivated choroid epithelial cells reveal well differentiated Junctional complexes consisting of continuous zonulae occludentes and integrated gap junctions.

Cyclic morphological changes of glial cells in long-term cultures of rat brain

SummaryBrain cells from embryonic rats were dissociated with trypsin, cultivated under constant conditions in Falcon flasks, and studied for periods of one year or more. Antisera against glial fibrillary acidic protein (GFA) and myelin basic protein (MBP) were used to identify glial cell types. For scanning electron microscopical (SEM) observation an embedding method in resin was developed that allows good preservation of the fine ultrastructural features of the cultivated cells and precise characterization of the cell types. Under our culture conditions, after four subcultures and 8–10 weeks of cultivation, the following cell types can be distinguished. (a) Flat epitheloid cells. From an immunocytological point of view these cells form a heterogeneous population composed of GFA- and MBP-positive and negative cells. They are the precursors of the following cell types. (b) Astroglial cells. SEM observations show a characteristic network of radially orientated prolongations. 92% of these cells are GFA-positive. (c) Oligodendroglial cells with characteristic dichotomously dividing branches. Secondary and tertiary branches end in flat amoeboid prolongations. These cells are MBP-positive.After approximately six weeks the most prominent cells are the flat epitheloid cells. The astroglial cells originate continuously from the epitheloid cells during the whole cultivation time. The formation of oligodendroglial cells, on the other hand, takes place at relatively precise intervals of time (approximately every 20–30 days) over the entire cultivation period (of more than one year).

Die Feinstruktur der Neuro-, Glio- und Ependymoblasten von Hühnerembryonen in der Gewebekultur

Summary1.The ultrastructure of embryonic cells of the central nervous system has been studied electronmicroscopically in tissue cultures of the telencephalon, spinal cord and retina of the chick.2.The cytoplasmic ultrastructure of neuroblasts and their differentiation to neurons is characterised particularly, by a special arrangement of the endoplasmic reticulum, leading to the formation of Nissl bodies. The synaptic contacts begin to differentiate by the formation of an electrondense material on the contiguous membranes. After incubating a 7 days old embryo for 3 days this process is already detectable. The number of synaptic vesicles increases with the increase in glucose concentration (up to 600 mg%) in the culture medium.3.Two types of ependymal cells have been observed in the spinal cord. Type one has a well-developed endoplasmic reticulum and a few cilia. Type two is distinguished by a filamentous cytoplasm and sparingly developed endoplasmic reticulum. The ependymal cells form rosettes in the telencephalon and in the retina. In the telencephalon the rosette-cells develop into mature ependymal cells, whereas in the retina they develop into cells resembling receptors, without, however, forming a regular outer segment.4.Criteria have been proposed for the identification of glioblasts in tissue culture. As a rule the nucleus appears strongly lobulated. The granular endoplasmic reticulum is only moderately developed. The cytoplasm of glioblasts is filled with characteristic filaments.

Impact of vegf on astrocytes: Analysis of gap junctional intercellular communication, proliferation, and motility

The purpose of the present study was to investigate the effects of vascular endothelial growth factor (VEGF) on gap junctional intercellular communication (GJIC), cell proliferation, and cell dynamics in primary astrocytes. VEGF is known as a dimeric polypeptide that potentially binds to two receptors, VEGFR‐1 and VEGFR‐2, however many effects are mediated by VEGFR‐2, for example, actin polymerization, forced cell migration, angiogenesis, and cell proliferation. Recently it has been shown that in case of hypoxia, ischemia or injury VEGF is upregulated to stimulate angiogenesis and cell proliferation. Besides this, VEGF reveals a potent therapeutical target for averting tumor vascularization, emerging in bevacizumab, the first humanized anti‐VEGF‐A antibody for treating recurrent Glioblastoma multiforme. To expand our knowledge about VEGF effects in glial cells, we cultivated rat astrocytes in medium containing VEGF for 1 and 2 days. To investigate the effects of VEGF on GJIC, we microinjected neurobiotin into a single cell and monitored dye‐spreading into adjacent cells. These experiments showed that VEGF significantly enhances astrocytic GJIC compared with controls. Cell proliferation measured by BrdU‐labeling also revealed a significant increase of astrocytic mitose rates subsequent to 1 day of VEGF exposure, whereas longer VEGF treatment for 2 days did not have additive effects. To study cell‐dynamics of astrocytes subsequent to VEGF treatment, we additionally transfected astrocytes with LifeAct‐RFP. Live‐cell imaging and quantitative analysis of these cells with aid of confocal laser scanning microscopy revealed higher process movement of VEGF‐treated astrocytes. In conclusion, VEGF strongly affects cell proliferation, GJIC, and motility in astrocytes.

Axoplasmic transport of horseradish peroxidase in single neurons of the dorsal root ganglion studied in vitro by microinjection

SummaryThe dependence of anterograde axoplasmic transport on cytoskeletal components was investigated using microinjection of horseradish peroxidase (HRP) into the somata of chick dorsal root ganglion cells in vitro. Microinjected HRP was transported anterogradely in the neurites and their branches; this transport was disturbed by colchicine in a drug-dependent and time-dependent manner. Cytochalasin B, a drug that depolymerizes actin, did not inhibit the transport of HRP, despite the formation of local swellings in neurites. The microinjection of polyclonal antibodies directed against tubulin and monoclonal antibodies (mAbs) against 200-kDa neurofilaments disturbed the axoplasmic transport of co-injected HRP, which then exhibited an irregular and discontinuous distribution in the axonal branches. The transport of HRP became discontinuous after the injection of anti-tubulin antibodies and led to the formation of globular deposits of HRP. Polyclonal antibodies against actin and mAbs to 160-kDa and 68-kDa neurofilaments seemed to have no effect on the axoplasmic transport of co-injected HRP. Microinjection of antibodies against tubulin induced formation of perinuclear bundles consisting of cytoskeletal components. The transport of HRP thus appears to be regulated by an intact microtubular system and cross-linker components (200-kDa neurofilaments) of the cytoskeleton. Actin and most intermediate filament proteins do not seem to play an essential role in the transport of HRP.

Neuronal migration during the early development of the cerebral cortex

SummaryFixed cerebral vesicles of mouse foetuses were fractured and examined with the scanning electron microscope. This method provides a study of the three dimensional developmental features of the pseudostratified columnar epithelium up to the formation of the early cortex plate.Matrix cells are a cell population of homogeneous shape, however, mitotic cells are easily identified by their spherical form. The external surface of the brain is formed by the closely packed end feet of these cells covered by a basal membrane. The formation of the cortical plate is the result of a continuous cell migration in columnar arrangement towards the pia. Glioependymal cells extend along the whole brain wall and most likely provide guidance for the migrating cell cords. The formation of the so-called migratory zone is a consequence of the growth of the basal and the horizontal prolongations of emigrating cells. The significance of the cell to cell contacts for the neuronal migration processes is discussed.

Ultrastructural aspects of the choroid plexus epithelium as revealed by the rapid-freezing and deep-etching techniques

SummaryBy use of rapid freezing with a nitrogen-cooled propane jet, differentiating epithelial cells of the choroid plexus can be studied without chemical and embedding procedures. The cytological features of the developing choroidal cells were investigated in chick embryos and three-week-old-chicks. After cryofixation, the material was fractured, deep-etched and rotary shadowed. Fractures of the Golgi apparatus reveal the particulate structure of the saccule membranes and show that the intracisternal surfaces are covered by particles which protrude into the intracisternal space. The inner membranes of mitochondrial cristae are characteristically covered by stalked particles; the intracristal space is collapsed. The endoplasmic reticulum possesses globular particles that occupy the width of the membrane, as well as granules and filament-like structures in its cisternae. The adherent ribosomes demonstrated by deep-etching consist of aggregated subunits. New aspects of the structure of the nuclear envelope and the pore complex are described. The circumference of the pore complex is formed by intramembranous aggregated particles in an octagonal symmetrical arrangement. The structure of the cilia and the ciliary necklace can be studied in their three-dimensional aspects, e.g., the structure of doublets and their relation to the cytomembrane. The junctional complexes of choroid epithelial cells differentiated both nexus and zonula occludens simultaneously. Tight junctions consist of isolated arrays of particles, which do not form fibrils in cryofixed material. Gap junctions are not always hexagonally arranged, are devoid of a crystalline form and occasionally show the presence of pits in the protoplasmic face.

The reaggregation of neurons and their satellite cells in cultures of trypsin-dissociated spinal ganglia

SummaryDorsal root spinal ganglia of chick embryos were trypsinized and the cellular components isolated. Suspensions of these cells reaggregate. During this process satellite glial cells and neurons established intimate contacts. The reconstructed glial envelopes around the neurons are morphologically similar to the ones formed in the intact animal during development. The formation of the characteristic glial-neuronal relation is an example of specific cell recognition.