Peter Eggli

A new approach for cryofixation by high‐pressure freezing

A newly designed high‐pressure freezing machine for cryofixation was established and tested (Leica EMPACT), based on ideas originally proposed by Moor & Riehle in 1968. The new machine, essentially an improved version of our prototype, pressurizes the sample to 2000 bar in a small container (using methylcyclohexane as hydraulic fluid) and at the same time cools the outer surface of the container with a jet of liquid nitrogen. The advantage of this approach is that the machine uses little liquid nitrogen and can be built small and light. The machine is able to vitrify and freeze well a variety of specimens, for example, plant leaves, yeast cells, liver or nerve tissue (more samples are shown at: http://www.ana.unibe.ch/empact). Cooling efficiency is the same as in the traditional machines that use liquid nitrogen to pressurize and simultaneously cool the sample.

A rapid microbiopsy system to improve the preservation of biological samples prior to high-pressure freezing

A microbiopsy system for fast excision and transfer of biological specimens from donor to high‐pressure freezer was developed. With a modified, commercially available, Promag 1.2 biopsy gun, tissue samples can be excised with a size small enough (0.6 mm × 1.2 mm × 0.3 mm) to be easily transferred into a newly designed specimen platelet. A self‐made transfer unit allows fast transfer of the specimen from the needle into the specimen platelet. The platelet is then fixed in a commercially available specimen holder of a high‐pressure freezing machine (EM PACT, Leica Microsystems, Vienna, Austria) and frozen therein. The time required by a well‐instructed (but not experienced) person to execute all steps is in the range of half a minute. This period is considered short enough to maintain the excised tissue pieces close to their native state. We show that a range of animal tissues (liver, brain, kidney and muscle) are well preserved. To prove the quality of freezing achieved with the system, we show vitrified ivy leaves high‐pressure frozen in the new specimen platelet.

Ultrastructural localization of hyaluronan in myelin sheaths of the rat central and rat and human peripheral nervous systems using hyaluronan-binding protein-gold and link protein-gold

Neural tissue of central (rat spinal cord) and peripheral origin (rat sciatic nerve, nerve fascicles of rat skin and iris and of human conjunctiva) was processed by osmium tetroxide/microwave fixation and embedded in epoxy resin. Hyaluronan-binding proteins and link proteins coupled to 15-20-nm gold particles were used as markers in a one-step post-embedding procedure for identifying hyaluronan (hyaluronic acid) at the ultrastructural level. All myelin sheaths in both rat and human material were found to be intensely labelled. The specificity of the hyaluronan-binding probes was demonstrated by the total loss of labelling following treatment of sections with hyaluronidase or by preincubating either the probes with hyaluronan oligosaccharides or the sections with unlabelled hyaluronan-binding protein. The identified hyaluronan appears to be located extracellularly, but is precise role here remains to be elucidated.

Mesoderm-derived cells proliferate in the embryonic central nervous system: confocal microscopy and three-dimensional visualization

Abstract In the chick and quail embryo, two cell populations migrate into the neural tube from the surrounding mesodermal tissues during the fourth day of incubation: individual cells which represent macrophages, and endothelial cells which remain continuous with the extraneural vessels. We report here on the proliferative capacity of these mesoderm-derived cells. A double-immunofluorescence protocol for two monoclonal antibodies of subtype IgG1, the endothelial cell/macrophage marker QH1, and the S-phase marker bromodeoxyuridine, was developed. With confocal laser scanning microscopy of thick microtome sections, labeling indices of intraneural individual QH1-positive cells (12%) and of endothelial cells (10%) were determined. In contrast, the labeling index of extraneural endothelial cells was 25%. With three-dimensional visualization of confocal data, the variable morphology of macrophages was shown. Our results indicate that: (1) proliferative activity of intraneural capillary endothelial cells is less than expected and that it is absent from sprouts; (2) both spheroidal and ramified macrophages proliferate inside the neural tissues; and (3) ramified macrophages frequently make contact with capillary endothelial cells. We conclude that most embryonic microglia may be derived from the early invasive QH1+ macrophages.

Topographic variations in rat brain oligodendrocyte morphology elucidated by injection of Lucifer Yellow in fixed tissue slices.

SummaryVisualisation of oligodendrocytes by fluorochrome labelling in fresh tissue is a relatively recent innovation, but its widespread applicability in comparative analyses between different regions of the brain has been hampered by the limited survival time of excised preparations. We here applied the technique of impaling and injecting these cells with Lucifer Yellow in fixed tissue slices. Using confocal laser scanning microscopy, we reconstructed the three-dimensional forms of oligodendrocytes derived from the optic nerve, corpus callosum, cerebellum and spinal cord of young adult rats. Differences in shape and size of the cell body, in the number of internodal segments supplied by a single cell, as well in their spatial orientation, and in the thickness of the myelinated fibre, were observed between the four white matter tracts analysed.

Embryonic central nervous system angiogenesis does not involve blood‐borne endothelial progenitors

We asked, whether, in the blood of avian embryos, endothelial precursor cells circulate that actually contribute to the growing vascular system in and around the central nervous system (CNS). We compared the morphology and distribution of QH1‐positive cells after transplantation of quail paraxial mesoderm, after blood transfusion, in quail‐chick parabiosis, or after quail bone‐marrow transplantation. After head mesoderm transplantation from quail to chick, we observed sprouting endothelial cells (ECs), capillary tube formation, and chimeric endothelial lining of large arteries in the host brain. These QH1‐positive quail cells showed EC morphologies that demonstrated three different aspects of CNS angiogenesis: invasion by means of filopodia, clonal proliferation and tube formation, and integration into preexisting EC layers. After blood transfusion or in chick‐quail parabiosis, blood‐borne QH1+ cells were found in the lumen of but not integrated into the wall of the host vascular system. Neither were QH1+ cells observed in the capillary walls of parabiotic chick chorioallantoic membranes. In both cases, the quail cells showed typical macrophage morphology. In chicks that had received quail bone marrow transplants onto their chorioallantoic membranes, QH1+ cells with macrophage, but not EC shape were occasionally seen near the inoculation site. We conclude that (1) blood‐borne cells do not become ECs or directly contribute to angiogenesis inside, or in vascular plexuses around the CNS during embryonic development; (2) blood‐borne cells do not contribute to the intraneural macrophage population of the embryonic CNS. J. Comp. Neurol. 436:263–274, 2001.

Actin accumulation in pseudopods or in the tail of polarized walker carcinosarcoma cells quantitatively correlates with local folding of the cell surface membrane.

We determined the actin distribution and the relationship between actin and the cell surface membrane in polarized Walker carcinosarcoma cells showing lamellipodia or blebs at the front in order to get a better insight into actins role in shape changes and cell locomotion. Using two different techniques, we found that actin is mainly present as a submembraneous layer. The actin concentration detectable in the cytoplasm was about 16X lower. F-actin staining was increased mainly at the contracted tail and to a lesser extent in lamellipodia. However, there is also accumulation of the cell surface membrane at these sites. The quantitative analysis of electron micrographs showed that the apparent accumulation of F-actin at the tail and in the leading lamellipodia was, on the average, fully explained by increased membrane folding. The cell membrane as well as the cortical actin may fold and unfold during shape changes and polarized cells have reserves of plasma membrane as well as of cortical actin at the tail. In addition, the cells may show spots where the surface membrane was dissociated from the cortical actin layer. Polarized cells showed no increase in actin within the blebs or at the basis of lamellipodia. In this respect, the distribution of polymerized actin was different from other currently studied locomoting metazoan cells. So far, the data are difficult to reconcile with models, postulating that polymerized actin within the protrusions is the direct force driving the membrane forward.

Growth characteristics of central and peripheral bovine corneal epithelial cells in vitro.

Primary cultures of pure bovine corneal epithelial sheets, isolated from either central or peripheral areas of the cornea and grown on an extracellular matrix, exhibited major differences in relation to their respective growth characteristics and morphology. After 15 days in culture, cells of peripheral origin covered a 40% greater area and were 2.75 times more numerous than those of central origin. Most peripheral cells were small with a polygonal morphology, whereas central cells varied considerably in both size and shape, although areas consisting of large cells were regularly observed. Differences in the rates of proliferation between central and peripheral corneal epithelial cells were maintained throughout the first and second subcultures. However, the growth rates were considerably lower in second passage cultures of both central and peripheral cells when compared with those of first passage. The growth characteristics of primary cultures of pure epithelial sheets were confirmed by further studies on corneal buttons in culture. Our results in vitro strongly support the concept of a slow but steady physiological movement of increasingly differentiated cells from the periphery of the cornea towards the centre, resulting in a constant renewal of epithelial cells in vivo.

Three-dimensional analysis of DNA replication foci: a comparative study on species and cell type in situ

Abstract Chromatin morphology of interphase nuclei in most cell lines of quail (Coturnix coturnix japonica) and chick (Gallus gallus domesticus) embryos shows typical interspecies differences. This intrinsic marker has been used in quail/chick chimerisation experiments, where also differences between cell types were noted. We asked whether similar differences between species and between cell types could be observed in S phase nuclei in situ. In this report, we used bromodeoxyuridine (BrdU) pulse labelling and anti-BrdU immunofluorescence to detect DNA replication foci in the nuclei of identified cells. In the central nervous system of 5- to 7-day-old quail and chick embryos, mesoderm-derived cells with strikingly different morphology and topographical distribution were studied: endothelial, i.e. polarised cells forming continuous tubes, and macrophages, i.e. non-polarised, ameboid or ramified individual cells. Using confocal microscopy, replication foci in the nuclei were assessed quantitatively and three-dimensional visualisations were produced. We consistently observed that: (1) chick, but never quail, nuclei displayed completely confluent replication sites, independent of cell type, and (2) macrophages, but not endothelial cells, had distinct perinucleolar replication sites, independent of species. We thus demonstrate a new relationship between cell type and spatial arrangement of DNA replication sites, and conclude that interspecies differences of chromatin distribution are conserved throughout S phase. Our results strongly recommend that work done on nuclear structure in vitro should not be extrapolated without reservation to cells in vivo.

Rat brain oligodendrocytes do not interact selectively with axons expressing different calcium-binding proteins

A single oligodendrocyte may endow ten to twenty vicinal axons with internodal segments, but its radial domain is neither exclusive of processes from other like cells nor are all nerve fibres within this zone myelinated. Whether oligodendrocytes are able to discriminate between axons on the basis of chemical or electrophysiological differences, or whether the tactic response is random, has yet to be established. In order to shed some light on this process, we investigated the ensheathment, by single oligodendrocytes, of axons distinguished on the basis of their calcium‐binding protein complexion. Rat brain oligodendrocytes were visualized either with the Rip‐antibody or by intracellular injection of Lucifer Yellow; subclasses of axons were immunolabelled with antibodies against one of the two calcium‐binding proteins parvalbumin or calretinin. Individual oligodendrocytes did not exhibit exclusivity with respect to their preferment for axons containing calcium‐binding proteins, associations with both non‐immunoreactive, as well as with parvalbumin‐ or calretinin‐positive ones, being encountered.