Alpha-particle irradiation-induced change in bronchopulmonary macrophage morphology, in vitro.

Bronchopulmonary macrophages, isolated from canine lungs by saline lavage and grown in tissue culture for short periods, were acutely irradiated with a range of doses of either Americium-241 alpha particles (0.03-48 Gy) or 250 keV x-rays (0.5-24 Gy). Following a 24-hour reincubation and "expression" period, cells were examined for radiation-induced changes in overall viability, as well as in cell morphology and ultrastructure. Results indicated that neither quality of radiation had much effect on cell viability over dose ranges examined, but substantial changes in cell volume, surface topography, and cytoplasmic features were noted, especially in the alpha-particle-irradiated specimens. Results support the concept that the limiting plasma membrane of the targeted macrophage is a sensitive subcellular target for ionizing radiation, especially high-linear-energy-transfer heavy particles.

Developmental and radiobiologic characteristics of canine multinucleated, osteoclast-like cells generated in vitro from canine bone marrow.

We report here our initial observations on the growth and morphology, and developmental radiosensitivity of giant, multinucleated, osteoclast-like cells (MN-OS) generated through in vitro cultivation of hematopoietic progenitor-enriched canine bone marrow samples. Maximum cell densities of 5.5 x 10(3) to 6.5 x 10(3) MN-OS per cm2 of growth area were achieved following 10 to 14 days of culture at 37 degrees C. Acute gamma irradiation of the initial marrow inocula resulted in significant, dose-dependent perturbations of MN-OS formation, growth, and development. Attempts to estimate radiosensitivity of MN-OS progenitors from canine marrow yielded a range of Do values from a low of 212 cGy measured at six days of culture to higher values of 405 to 542 cGy following 10 to 22 days of culture. At the intermediate times of culture (10 to 14 days), the radiation-induced responses were clearly biphasic, reflecting either (a) the presence of multiple subpopulations of MN-OS progenitors with varying degrees of radiosensitivity or (b) the inherent biphasic nature of MN-OS development involving early progenitor cell proliferation followed by maturation and subsequent fusion. Morphologically, MN-OS generated from irradiated marrow inocula appeared only marginally altered, with alterations expressed largely in a biphasic, dose-dependent fashion in terms of smaller cell size, reduced number of nuclei, increased expression of both surface microprojections, and a unique set of crystalloid cytoplasmic inclusions. Functionally, MN-OS appeared to be impaired by irradiation of marrow progenitors, as evidenced by failure to initiate resorptive attachments to devitalized bone spicules in vitro.

SEM of canine chromosomes: normal structure and the effects of whole-body irradiation.

Canine chromosomes are not only numerous (38 autosomal pairs), but they are small (compared to human chromosomes) and morphologically similar as well. Analysis of the canine karyotype by light microscopy (LM) of banded chromosomes is, thus, difficult, and the literature on the canine karyotype is scanty. In this study, we describe examination of chromosomes from normal and chronically irradiated dogs with the scanning electron microscope (SEM). Metaphase chromosomes from bone marrow aspirates were Giemsa-banded with either 0.025% trypsin alone or 0.1% trypsin preceded by 10% H2O2 and prepared for SEM. Examination of chromosomes from normal dogs revealed cylindrical chromosome profiles with well-defined chromatids and centromeres. The chromosome arms were consistently marked by periodic grooves that had complementary structures on sister chromatids and may represent the trypsin-sensitive chromatic regions. The quality of the preservation varied from preparation to preparation and depended on the concentration and time of trypsin treatment. Chromosomes from irradiated dogs revealed translocations, deletions, and gaps. We conclude that SEM produces images superior to LM images of canine chromosomes; SEM images can be used not only to identify individual chromosomes, but also to identify genetic lesions in the chromosomes of chronically irradiated dogs. We further conclude that the two Giemsa-banding protocols used in the present study produced variable results, although 0.025% trypsin alone appeared to give better and more consistent results than 0.1% trypsin preceded by 10% H2O2.

The renal cortical lymphatic system in the rat, hamster, and rabbit.

Rat, hamster, and rabbit renal cortical lymphatics were examined by light and electron microscopy. Rat and hamster kidneys possessed both intra- and interlobular lymphatics that were structurally similar at the light microscopic level. Ultrastructural examination of the hamster lymphatic endothelium, however, revealed an unusual arrangement of cytoplasmic extensions not seen in the other two species. The intralobular lymphatics were related primarily to tubules, afferent arterioles, and renal corpuscles and were consistent with lymph formation from both plasma filtrate and tubular reabsorbate. Interlobular lymphatics were seen in connective tissue associated with the interlobular blood vessels. Rabbit cortex contained only interlobular lymphatics. Cross-sectional area, maximum diameter, volume density, and profile density were determined by stereological measurements using a computer-based image analyzer. The morphological data from the rat were used, in combination with published values for lymph flow, to calculate the rate of lymph formation per unit area of endothelium in lymphatics of the renal cortex. Among kidneys fixed by retrograde perfusion, the cortical lymphatic system was most extensive in maximum diameter, volume density, and profile density. It was smallest in the rabbit and intermediate in the rat. Lower volume and profile density were found for rat kidneys fixed by the dripping technique. It was concluded that: tubular reabsorbate probably contributes to renal lymph in the rat and hamster, but not in the rabbit; significant differences exist in the extent of the renal lymphatic systems among the three species, with the hamster kidney having the richest network and the rabbit the poorest; the method of fixation influences the measured size and density of renal cortical lymphatics; and the estimated rate of lymph formation in the kidney of the rat is roughly comparable to that in the dog.

Pattern and distribution of intrahepatic lymph vessels in the rat.

The pattern and distribution of intrahepatic lymph vessels were examined by light and electron microscopy in rat livers fixed by perfusion through the portal vein. Lymph vessels were found in the connective tissue of the larger portal canals, where they coursed in close association with branches of the hepatic artery. The smallest portal canals contained no lymphatics. Of the portal canals that lacked a lymphatic, over 50% also lacked an arterial component. Direct connections between the lymphatic lumen and the spaces of Disse or Mall were not observed but lymphatics were found close to Mall's space, separated by only a sparse connective tissue space containing a few collagen fibrils. Lymphatics were neither seen within the parenchyma, nor associated with intercalated (sublobular) veins. Cross-sectional area (223.2 +/- 48.7 micron2 SEM), maximum diameter (20.5 +/- 2.0 microns), volume density (0.00098 +/- 0.00046 micron3/micron3) and profile density (1.8 +/- 0.3 lymphatics per 1 mm2) of hepatic lymph vessels were determined by stereological measurement by a computer-based image analyzer. These data were used to estimate the rate of lymph formation in the liver. It was concluded that 1) initial lymphatics probably originate in the portal canals; 2) the concept that fluid in the space of Disse can be regarded as the principal source of fluid-forming hepatic lymph is questioned, since initial lymphatics appear to be separated from the space of Disse by hepatocytes and the space of Mall; and 3) the rate of lymph formation in the liver of the rat is approximately 0.06-0.08 microliter/min/cm2 of lymphatic endothelium.

An ultrastructural study of transport pathways across rat hepatic lymph vessels.

Hepatic lymph vessels in the rat were examined by qualitative and quantitative analyses in order to obtain data pertinent to the mechanism of lymph formation. The ultrastructually visible transport pathways across these vessels appeared to be by way of intracytoplasmic vesicles (89.6 micron mean diameter) and normal channels (22.6 micron wide) between endothelial cells. Three types of intercellular contacts were seen, end-to-end, overlapping, and interdigitating. Only one open junction (greater than 30 nm) was seen in 226 contacts examined. Specialized junctional complexes, either fasciae occludentes or fasciae adherentes, were seen in 65% of the contacts. Approximately one-third of the contacts had a dilatation along part of their length separating the opposing endothelial cells. Vesicles occupied 3.5% of the endothelial cytoplasmic volume and were distributed as follows: 40% opening onto or touching the luminal membrane, 34% without visible connection to either surface, 23% opening onto or touching the abluminal membrane, and less than 3% associated with membranes forming intercellular contacts. It was concluded that the mechanism of lymph formation in the liver is similar to that in the kidney and different from that in the dermis or diaphragm.