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

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.

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

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.

Plastic hollow waveguides: properties and possibilities as a flexible radiation delivery system for CO2-laser radiation.

BACKGROUND AND OBJECTIVES: One significant inconvenience of the CO2 laser is the lack of flexible fibers essential for endoscopic applications. The goal of this study is to test the feasibility of hollow waveguides in view of a practical use in medicine. STUDY DESIGN/MATERIALS AND METHODS: Various types of plastic hollow waveguides for flexible delivery of CO2 laser radiation were examined. The transmission losses, divergence angle, damage threshold, and input and output beam profiles were determined. The interaction process between radiation transmitted through these guides with soft as well as hard tissues was studied. RESULTS: Plastic hollow waveguides can transmit high power (up to 50 W) with low losses (straight guide 1 dB/m) even through bendings. The divergence angle is < 13 degrees. Cutting quality and extent of thermal damage are comparable to incisions performed with a free laser beam. CONCLUSION: The results of this study show good cutting quality and durability of these flexible plastic hollow waveguides, which render possible to deliver CO2 radiation in the power range needed for most surgical applications with affordable transmission losses. Plastic hollow waveguides are, therefore, a real alternative to replace the mirror arms.