Edwin S. Boatman

A morphometric and morphological study of the lungs of rabbits after unilateral pneumonectomy

Boatman, E. S. (1977).Thorax, 32, 406-417. A morphometric and morphological study of the lungs of rabbits after unilateral pneumonectomy. A study was made to determine the changes occurring in the contralateral lung of adult rabbits as a result of left pneumonectomy. Stereological procedures for light microscopy and for electron microscopy were used to quantitate the changes. Lungs from control rabbits, ranging in body weight from 1·0 to 9·0 kg, were also analysed. Resin corrosion casts of left and right lungs were prepared. Left pneumonectomy causes the volume of the right lung to increase to the total paired lung volume in about four weeks. Total surface area, while increasing directly with lung volume in control rabbits, increased as two-thirds of the lung volume after pneumonectomy. In some right lungs there appeared to be an increase in the number of alveoli. Mean linear intercepts for control lungs were fairly constant, but the volume density of alveolar ducts increased with age. In pneumonectomised animals, mean air-space diameter of the right lung increased by about 25% and the volume density of alveolar ducts by 22-33%. The volume density of alveolar wall tissue decreased by about 30%. The average number of alveoli/cm3 for whole lungs was 2·31±0·46×106, whereas for right lungs eight weeks or more after pneumonectomy it was significantly less (1·70±0·38×106). Morphometry by electron microscopy revealed slight changes in the components of the alveolar septum and a reduction in the thickness of the air-blood barrier. The overall effect of left pneumonectomy was mainly one of a compensatory increase in the volume of the right lung with dilatation of alveoli and of alveolar ducts.

Morphology, morphometry and electron microscopy of HeLa cells infected with bovine Mycoplasma.

SummaryThe host-parasite relationship of HeLa M cells artificially infected with a bovine species of Mycoplasma was studied by light microscopy, transmission electron microscopy and scanning electron microscopy. The use of morphometry to quantitate some of the findings was explored. The parasites were seen in locations extracellular to the cell surface. The detection of small numbers of organisms by light microscopy was well demonstrated by use of the fluorescent antibody technique. Scanning electron microscopy proved to be an excellent method for revealing the surface details of cell-parasite morphology. Ultra-thin sections showed that the parasites are aligned mostly parallel to the plasma membrane of the host cell but separated by a gap of 10 nm. Morphometry indicated an average of 69 organisms per cell surface occupying 1.7% of the surface area. An increase of 26% in diameter of the HeLa cells, possibly as a result of infection, was observed.

The influence of protein malnutrition on ileal permeability to macromolecules in the rat

Ileal absorption of intact protein was assessed in groups of control and protein-deficient rats during a 5-month period of dietary protein restriction. At monthly intervals following diet initiation, 3 animals from both the control and deficient groups were administered horseradish peroxidase via ligated ileal loops either 15, 45 or 90 minutes before loop excision and processing for light and electron microscopy. Morphological evaluations revealed no change in pinocytotic activity during the 5 months of progressive protein malnutrition. After 3 months, however, partial deterioration of mucosal structure was observed with apparent separation of apical intercellular junctions and movement of peroxidase molecules directly between cells. These observations in the ileum are compared to previous findings in the jejunum. It is suggested that ileal and jejunal absorptive cells respond uniquely to protein deprivation but that eventual junctional deterioration occurs in both regions with potential detrimental consequences for the host.

Photographic Mapping of a Tissue Surface to Locate Fields for Electron Microscopy; Mouse Lung

Precise sampling from whole lobes of mouse lungs fixed in the inflated state and embedded in epoxy resin can be not only feasible but also efficient. A 1 μm section is cut from an embedded lobe with a rotary microtome and a steel knife. This section is stained and photographed, and from it a 35 × enlarged print is prepared. A grid of transparent plastic scored with 35 mm squares, lettered vertically and numbered horizontally, is superimposed over the photograph. The area chosen for electron microscopy thus becomes identifiable by a letter-number designation obtained from the grid. This area is then located by light microscopy on a 2 mm slice taken from the block from which the 1 μm section was cut, by use of oblique illumination and the calibrated mechanical stage of the light microscope. A block of 1.3 mm diameter is removed for electron microscopy from the tissue by a rotatable circular spring-loaded punch screwed into the objective turret of the microscope. The removed cylinder is mounted on a metal st...

A role for interstitial matrix in tissue tension of alveolar wall

Single alveolar walls subjected to length-tension studies in saline and Bicine (0.2 M) undergo a progressive decay in tissue tension (TTD). We have examined the effect of different solutions on this TTD and looked for corresponding changes in the ultrastructure. Lung parenchyma was dissected to single alveolar walls (30 X 30 X 150 microns) in phosphate-buffered saline (0.15 M). Transferred to a length-tension bath, the tissue was immersed in Bicine, saline, fortified Hanks solution, 0.25% Alcian blue in saline, or a sodium dodecyl sulfate solution, for variable periods. Cycled through a given extension with peak force measured over time, these same tissues were fixed in buffered glutaraldehyde/tannic acid and processed for electron microscopy. Single alveolar walls immersed in saline or Bicine showed a progressive TTD. Vacuoles or spaces appeared in the interstitium which with cellular disorganization progressed with the TTD. Seen within 0.3 h, the changes were well advanced at 0.6 h. In sodium dodecyl sulfate (70 mM), however, there was no TTD and structurally there was no interstitium, with only basement membranes and fibrous proteins remaining. In fortified Hanks solution or 0.25% Alcian blue the interstitial matrix, cell morphology and tissue tension were well preserved for 1 h. This study suggest that leaching of the interstitial matrix occurs in saline or Bicine, and an intact matrix is essential for the preservation of tissue tension.

Mycoplasmatales virus, MV-M1: Discovery inAcholeplasma modicum and preliminary characterization

A new virus, Mycoplasmatales virus-modicum 1 (MV-M1), was recovered from spontaneous plaques in lawns ofAcholeplasma modicum. Strain “mod” produced plaques onA. modicum strains but not on strains ofAcholeplasma laidlawii. Only MV-L3 of the three knownA. laidlawii viruses (MV-L1, MV-L2, and MV-L3) produced plaques onA. modicum. The MV-M1 virus was serologically distinct from the threeA. laidlawii viruses; filterable at 0.1 μm; partially sensitive to heat and Nonidet P-40; and chloroform labile. Spherical particles ranging from 105 to 160 nm were observed in electron micrographs of negatively stained preparations.

MORPHOLOGY AND ULTRASTRUCTURE OF MYCOPLASMAS

Investigations on the morphology and ultrastructure of the mycoplasmas during the last four years, although not extensive in terms of species examined, have shown great promise in some areas. Of the 90 or so publications during this period, about 30 were concerned with various physical, biochemical, and morphological aspects of the limiting membrane of Acholeplasma laidlawii. Of the remainder, M . gallisepticum? M. hominis,6 and M . pneumoniaelo were observed more frequently than others. Thus, many species still remain to be adequately characterized both morphologically and ultrastructurally . By “morphology” is meant the three-dimensional shape of the cells, and ultrastructure, as a generalization, refers to the external or internal structure of cells frozen or chemically fixed at a point in time and observed by microscopes capable of a resolution between 20 nm and about 0.4 nm. Fixation, resolution, and observation (Lee, selection of fields) comprise the foundation upon which statements about ultrastructure are made. What, then, is the present state of the art? Within the last year there has been a workshop on the Mycoplasmatales’ (to be published January, 1973, J. Infect. Dis. supplement), a monograph “The Biology of Mycoplasmas,”2 and a number of reviews, two on mycoplasmas in general,8.* and one on plant-associated mycoplasmas.6 Thus I shall speak mainly of recent observations and work indicating trends for the future. Morphologically, mycoplasmas are, at least in some phases of growth, a distinctly heterogeneous group of organisms. An example of this variability of shape is shown in FIGURE 1. Ideally, in spite of their small size, the growth of mycoplasmas should be followed by light microscopy. Interest in the use of the light microscope for the observation of growing cultures of Mycoplusma has been stimulated by the outstanding work of BredtE with the use of both cinematography and phase-contrast systems. In one such study, ten strains of M. hominis were observed. As growth proceeded, three basic morphological forms were seen: (1) coccoidal cells 0.3-0.8 pm diameter, (2) diploforms, and (3) filamentous forms with filaments 0.3-0.4 pm thick and up to more than 40 p m long. However, strains that had been subcultured a number of times developed mainly large coccoidal cells. Cinematography of single cells observed continuously for up to ten hours showed replication occurring by binary fission, by fragmentation of filaments and rings, and by a budding process. Under these conditions the generation time by binary fission was two to three hours. Under rather different conditions, Hubbard and Kite’ observed the growth of M. pneumoniue starting with filtered cultures containing a predominance of coccoidal cells. Generally within two to five days, filamentous forms were numerous, and at seven to ten days of growth small colonies were recognizable. Later stages (21-28 days) showed large colonies composed of coccoidal forms, but in these larger colonies only the edge of the colony could be resolved free of superimposed forms. Thus filamentous forms are still very much

Effects of lobar pulmonary blood flow on the evolution of oleic acid lung injury in dogs

We studied the effects of interruption of the pulmonary blood flow to the left diaphragmatic lung lobe on the evolution of canine oleic acid lung injury. We compared the morphology and edema of the left diaphragmatic lobe, whose pulmonary artery was ligated immediately after oleic acid injury, with the right diaphragmatic lobe, in which the blood supply was intact. The injury plus ligation resulted in hypoxemia and pulmonary hypertension with PaO2 falling from 98 +/- 4 to 72 +/- 21 torr (P less than 0.05) and pulmonary artery pressure increasing from 11 +/- 3 to 18 +/- 4 mm Hg (P less than 0.05). Animals were sacrificed 48 hr following the injury. Morphological examination of right and left lobes showed no consistent differences although wet/dry ratios indicated significantly greater edema (P less than 0.01) for the right diaphragmatic lobes (7.66 +/- 1.23) than the left (6.80 +/- 0.59). Both right and left lobes were substantially more edematous than our laboratory normal value of 4.74 +/- 0.54 (P less than 0.001 for both). We conclude that interruption of pulmonary arterial blood flow protects against edema formation in oleic acid injury but does not alter the morphologic evolution of the injury.

Ureaplasma Urealyticum Upper Urinary Tract Infection: Persistence and Pathogenicity in a Canine Model

Ureaplasma urealyticum is an opportunistic pathogen, commonly isolated from the lower urogenital tract. Although U. urealyticum has been cultured from the upper urinary tracts of patients with interstitial renal diseases and struvite renal calculi, the precise role of ureaplasmas in upper tract diseases is unknown. To evaluate their potential significance in the etiology of renal diseases, we studied survival, multiplication, and pathogenicity of U. urealyticum in canine kidneys with experimentally induced hydronephrosis. After inoculation of the prototypic serovar (Type 8, strain 960) of U. urealyticum, seral urine specimens were obtained using a subcutaneously positioned nephrostomy catheter. Although U. urealyticum survived for less than 48 hours in canine urine in vitro, organisms persisted in the obstructed upper urinary tract for at least 21 days. Urinary pH of infected renal units increased to 7.5 to 8.5, a most unfavorable range for U. urealyticum in culture. Renal parenchyma had higher concentrations of ureaplasmas (8.7 X 10(3) to 9.5 X 10(4) CFU/gm.) than either renal pelvis tissue (1.0 X 10(3) CFU/gm.) or urine (6.5 X 10(3) CFU/ml.). Histologic studies demonstrated progressive interstitial inflammation in infected kidneys but similar changes were not apparent in obstructed, uninfected kidneys. The obstructed upper urinary tract appears to provide favorable conditions for ureaplasmas which may cause progressive interstitial inflammation in the absence of other pathogens.

Digitizing and Quantitation

The aim of quantitative morphology as specifically restricted here to the electron microscopy of microorganisms, is to estimate in an as unbiased manner as possible, the volume, area, number, and size of cells or components of cells derived from the analysis of two-dimensional images of sectioned material by the use of relatively simple to moderately complex techniques and equipment.