In bandicoots, implantation takes place 9.5 days after mating and birth occurs 3 days later. It is therefore of interest to examine the lungs of newborn marsupials to ascertain the stage of lung development with respect to other organs and other mammals in view of the short period of time allowed for maturation of lung tissue. The newborn bandicoot lung is composed of a primitive system of branching airways which terminate in large blind sacs; alveolar ducts or alveoli are not present. The terminating sacs, which are delineated by thick connective-tissue septae, have a highly vascularised internal lining. Thin squamous cells and numerous type-II "surfactant"-secreting cells constitute the air interface of the sac. Corticosteroids influence the development of the eutherian fetal lung by accelerating pulmonary maturation and increasing the synthesis of surfactant. Since recent evidence indicates that the adrenal of the bandicoot is active at, or before birth, it is possible that this organ also plays an important role in preparing the lungs of the marsupial for extra-uterine life in the pouch.
Current methods of morphological analysis do not permit detailed imaging of individual myelinated fibres over substantial lengths without disruption of neighbouring, potentially significant, cellular and extracellular relationships. We report a new method which overcomes this limitation by combining aldehyde-induced fluorescence with confocal microscopy. Myelin fluorescence was intense relative to that from other tissue components, enabling individual myelinated nerve fibres to be traced for distances of many millimeters in whole PNS nerve trunks. Image obtained with a Bio-Rad MRC-600 confocal laser scanning microscope clearly displayed features of PNS and CNS myelinated fibres including nodes of Ranvier; fibre diameter; sheath thickness and contour; branch points at nodes; as well as (in the PNS) Schmidt-Lanterman incisures and the position of Schwann cell nuclei. Direct comparisons using the same specimens (whole nerve trunks; also teased fibres) showed confocal imaging to be markedly superior to conventional fluorescence microscopy in terms of contrast, apparent resolution and resistance to photobleaching. Development of the fluorophore was examined systemically in sciatic nerves of young adult rats. In separate experiments, animals were perfused systemically using (1) 5% glutaraldehyde; (2) Karnovsky's solution; (3) 4% paraformaldehyde; buffered with either 0.1 M sodium phosphate or sodium cacodylate (pH 7.4). The concentration of glutaraldehyde in the fixative solution was the principal determinant of fluorescence intensity. Confocal imaging was achieved immediately following perfusion with 5% glutaraldehyde or Karnovsky's. Fluorescence intensity increased markedly during overnight storage in these fixatives and continued to increase during subsequent storage in buffer alone. The fluorophore was stable and resistant to fading during storage (15 months at least), enabling data collection over extended periods. To demonstrate application of the method in neuropathology, individual fibres in transected sciatic nerve trunks were traced through multiple successive internodes: Classical features of Wallerian degeneration (axonal swelling and debris; ovoid formation and incisure changes; variation among fibres in the extent of degeneration) were displayed. The method is compatible with subsequent ultrastructural examination and will complement existing methods of investigation of myelinated fibre anatomy and pathology, particularly where preservation of 3-dimensional relationships or elucidation of spatial gradients are required.
In the newborn southern elephant seal the pineal gland is very large, and both pineal and plasma melatonin concentration is elevated. The pineal gland was investigated during the first 24 h, and up to 20 days of age, in elephant seal pups. A primary aim of this investigation was to determine whether there are obvious ultrastructural characteristics of pinealocytes that are exhibiting extraordinarily high levels of activity. Blood and pineal glands were collected from thirty seven pups of known age which were sampled at random from early September to early November (1985) at Macquarie Island. The pineal gland is large (mean weight, 4.71 +/- 0.35 gm, range 1-9.3 gm) and actively secreting melatonin at birth. Melatonin concentrations were extremely variable, yet very high in pups during the first 24 h post-partum. Mean melatonin plasma concentration for pups 0-24 h was 17632.8 +/- 5723.8 pmol/l (4090.8 +/- 1327.9 pg/ml), ranging from 126 pmol/l (29 pg/ml) to 297000 pmol/l (68904 pg/ml). Electron microscopic examination did not reveal any marked changes in pinealocyte ultrastructure suggestive of increased secretory activity during this period. The large and extremely active pineal gland in newborn southern elephant seal suggests that it is actively involved in thermoregulation.
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