Evaluation of lung diffusing capacity by physiological and morphometric techniques

Determinations of pulmonary diffusing capacity for CO (DLCO) by physiological and morphometric techniques have resulted in substantially different values for both DLCO and its major components. To evaluate the differences in these methods of measurement of DLCO, measurements were made under controlled conditions on isolated perfused dog lungs. Multiple gas-rebreathing techniques were used to measure DLCO, the membrane component of the diffusing capacity for CO (DmCO), and pulmonary capillary blood volume (Vc) in both anesthetized dogs and after isolation and perfusion of their lungs. The isolated perfused lungs were than perfusion fixed for morphometric analysis of the components of DLCO. The values obtained morphometrically for Vc were similar to those measured by physiological techniques. Perfusion fixation did not substantially alter the morphometric estimate of DmCO when compared with previous values obtained on inflation fixed lungs. However, the morphometric estimate of DmCO was over 10 times higher than that estimated physiologically. Analysis of the potential errors in the techniques suggests that the correct value for DmCO is substantially higher than that commonly estimated by use of physiological techniques and that the explanation for the difference is due to a number of factors that can influence the binding of CO to hemoglobin under in vivo conditions. The net effect of these factors can be represented by an unknown in each component of the Roughton-Forster relationship so that 1/DL = 1/(U1.Dm) + 1/(U2.theta Vc), where theta is the binding rate for CO to hemoglobin. Because the magnitudes of the unknown terms (U1 and U2) in the Roughton-Forster relationship are likely to be large, this relationship cannot be reliably used to determine Dm and Vc.     

Increase of melanogenesis by retinoic acid: an ultrastructural and morphometric study

Melanin is a dark pigment protecting the skin against UV radiation in some organisms. Studies on invasion and metastasis using retinoic acid as inhibitor agent are well known, but its role in melanin production (melanogenesis), especially at ultrastructural level and using morphometry were not well studied. In the present study, we analyzed the effects of retinoic acid on the melanosomes in B16F10 melanoma cells. These organelles were identified and quantified using routine electron microscopy and the specific HMB45 antibody. Other approaches such as immunofluorescence, and flow cytometry were also used. Our results indicated that retinoic acid increased the melanogenesis process in B16F10 melanoma cells. Furthermore, this work also provided evidence that this substance interferes at the subcellular level altering the numerical density of melanosomes, as well as the relative volume of the nucleus and nucleolus. In addition, the cells displayed altered morphology and an increase in the percentage of the relative volume of melanosomes, mainly the stages II-III and IV, leading to melanin formation. Furthermore, a decrease in the cells number after retinoic acid treatment was also observed.     

Comparative ultrastructural morphometric studies of micromyeloblastoid and lymphoblastoid paramyeloblasts.

One hundred lymphoblastoid and one hundred micromyeloblastoid paramyeloblasts, isolated from peripheral blood of untreated leukaemia patients, were studied by electron microscopic morphometry. Considerable differences are to be found between the micromyeloblastoid and lymphoblastoid paramyeloblasts as regards the size of the nucleolar "apparatus", both in the absolute average values and in the index showing the ratio of the nucleolous of the remaining nuclear surface (4.76% for myeloblastoid and 9.96% for lymphoblastoid paramyeloblasts). The central heterochromatin (scattered), which is discussed to be essential for the detection of an early prophase, was found in 3% of the myeloblastoid cells and in 14% of lymphoblastoid ones. The effect of cytostatic therapy is discussed by taking these data into consideration.     

A morphometric ultrastructural study of the nucleus of cerebellar granule cells

A quantitative approach to the nuclear ultrastructure of cerebellar granule cells is described here. The study was made using conventional electron microscopy from cerebellar cortices of adult rats by means of a semiautomatic image analyzer. The basic observation is that the nuclei of mature granule cells constitute a homogeneous population in terms of morphometric and stereologic data; in fact, the volume density of condensed chromatin within the nuclei remains practically constant in all nuclear sections. These results seem to indicate the existence of a cell-specific nuclear morphometric phenotype which might be considered as an effective criterion for the typification of this cellular lineage.     

Development of germ cell transplants: morphometric and ultrastructural studies.

Mouse-to-mouse transplants were studied at 10 min, 9 h, 24 h, 1 week, 1 month, 2 months, and 3 months post-transplantation. Data from a previous light microscope study were confirmed and extended using morphometric and ultrastructural techniques. As soon as 10 min after introduction of the germ cells from one mouse into the tubule lumen of a recipient mouse they developed relationships with small Sertoli cell processes. The extent of this surface-to-surface relationship increased in animals sacrificed up to 1 week post-transplantation. Most transplanted germ cells retained the characteristics of the donor germ cells after they had been isolated and pelleted. Nearly all transplanted cells eventually underwent phagocytosis by the recipient Sertoli cells. The presence of small apparent clones of germ cells after 1 week of transplantation indicated that some germ cells may divide and survive for short periods within the epithelium. No discernible qualitative subcellular changes in the host Sertoli cell accompanying the development of transplant spermatogenesis were noted. Macrophages were present in the region of the boundary tissue between myoid cells and appeared to increase in number in the peritubular tissue of transplanted testes. Images suggest that they migrated into the tubule to gain entrance to the lumen and there take on the form of activated macrophages. Some macrophages phagocytose sperm at 2 months and 3 months post-transplantation. A testis weight increase previously demonstrate to occur at 24 h post-introduction of germ cells was found to be due to an increase in the volume of the tubular lumen. The increase of lumen size at 24 h was not related to the volume of the injected material. It is suggested that the presence of injected cells, likely germ cells, in the tubule lumen stimulated increased secretion by the Sertoli cell.     

Ageing adipokinetic cells in Locusta migratoria: an ultrastructural morphometric study

Summary A morphometric study was made of the ultrastructure of adipokinetic cells in resting adults of Locusta migratoria at 3, 23, and 43 days after imaginal ecdysis. The nucleus, rough endoplasmic reticulum, and Golgi apparatus enlarge with age, which indicates that the synthesis and packaging of secretory substances increases during ageing. The size of the storage compartment, consisting of secretory and ergastoplasmic granules, does not increase earlier than 23–43 days after imaginal ecdysis. The lysosomal compartment markedly enlarges between 3 and 23 days; later on, the growth of this compartment, especially of autophagosomes, is less prominent. This suggests that lysosomal destruction initially compensates for the production of new secretory granules, assuming that exocytosis of secretory granules by adipokinetic cells is insignificant in resting locusts. Afterwards, lysosomal destruction may no longer be sufficient to prevent over-production of secretory granules, as is suggested by the increase in the number of these granules between 23 and 43 days. This coincides with the appearance of a considerable number of large ergastoplasmic granules, which represent a spatially more efficient form of storage of secretory material than the much smaller secretory granules. The increase with age in the amount of secretory products indicates that the biosynthetic activity of the adipokinetic cells is not (finely) tuned to their releasing activity.     

Changes in histological differentiation of human tumors transplanted to athymic nude mice: a morphometric study

Differing conclusions have been reached regarding the phenotypic stability of human tumors transplanted to athymic nude mice. Since previous conflicting studies of tumor histology have been largely subjective, quantitative methodology was applied to an analysis of 13 human adenocarcinoma tumor lines that were originally derived directly from surgical specimens. Glandular differentiation was quantitated, both in the original human tumor (OHT) and in a minimum of 6 serial passages of the nude mouse-grown tumors (MGT), by means of point counting. A significant change in differentiation was noted in 12 lines, with 9 showing a decrease. Variance from the OHT was most commonly noted in the initial MGT, but additional changes were also noted in 8 lines during subsequent passages. Most of the lines also showed increased necrosis in the MGTs. Since histological differentiation and necrosis are related to tumor aggressiveness, it would appear that the predominant tendency was to evolve toward a more malignant phenotype. These changes may mimic those seen in human tumor metastases.     

Use of lymphocytic DNA by the urothelium of bladder tumors

In 35 rats tumors of the urinary bladder were induced by nitrosomethylurea. A rubber loop was established on the neck of the urinary bladder and tightened for 1.5 h. This allowed one to completely isolate the tumor from the circulation. Shortly after the tightening of the loop 3H-thymidine was injected intraperitoneally. The rats were sacrificed at different times after the loop was removed. The tumors were examined by histoautoradiography. Lymphocytes were the first labeled cells that appeared in the stroma of the tumors 9 h after operation. Following 36 hours the radioisotope was detectable in the epitheliocytes. That meant that the label was reutilized by the tumor urothelium. It was shown that the only donor of the isotope could be a live labeled lymphocyte transmitting its own DNA in the course of a direct contact with the tumor cell. That was a manifestation of the trophic function of lymphocytes. It is concluded that lymphocytes support the proliferative activity of the tumorous tissue.     

Grading of urinary bladder tumors by automated microscopic image analysis

Studies were undertaken on biopsies from urinary papillomas and urothelial carcinomas of different grades (G1 to G3) along with normal urinary bladder mucosa from a total of 220 patients. They were performed by use of automated microscopic image analysis (system Robotron A 6471, software AMBA/R). As a result, the tumor grading was improved and lead to the separation of both papillomas and G1-carcinomas with high proliferative activity. In addition, it was possible to divide the G2-carcinomas into 2 groups which probably show a different biological behaviour.     

The floral nectaries of Hibiscus rosa-sinensis in. A morphometric and ultrastructural approach

The secretory hairs of Hibiscus rosa-sinensis nectaries have been studied ultrastructurally, particularly at stages before, during and after secretion. Morphometric cytology revealed considerable changes in the volume of endoplasmic reticulum (ER) and the vacuoles. At least three different forms of ER have been noticed, cisternal, tubular and vesicular. The intermediate cells of the hairs are ultrastructurally and morphometrically similar to the tip cell, suggesting their probable involvement both in a symplastic prenectar transport via the plasmodesmata and in nectar release into an extracellular space provided by the lateral cell walls. Nectar would then be apoplastically moving towards the tip cell, where it is forcibly expelled to the outside via transient pores of the cuticle.
Ultrastructural evidence indicates that ER is the cell compartment principally involved both in prenectar transport and nectar elimination. In the hair cells it provides a "secretory reticulum" mediating between prenectar accumulation and nectar release. Vacuoles are voluminous in the basal cells, and in all cell types of old nectaries. The results are discussed in relation with other plant glands, especially with the closely related Abutilon nectaries.     

Elastase-like enzymes in human neutrophils localized by ultrastructural cytochemistry

The thiol ester N-t-Boc-L-alanine-p-nitrothiophenyl ester (Boc-Ala-SNp) was synthesized and applied as an ultrastructural cytochemical substrate for intracellular elastase-like enzymes. Mature human neutrophils incubated with Boc-Ala-SNp and gold ions generate an electron-dense reaction product, gold p-nitrothiophenolate, which is found in the nuclear membrane, Golgi complex, endoplasmic reticulum, mitochondria, and granules of these cells. Enzyme activity against Boc- Ala-SNp is also observed in developing monkey bone marrow neutrophils and in other blood cells. The intracellular neutrophil enzyme activity is elastase-like because it is characterized by a slightly alkaline pH optimum and is inactivated by exposure of the cells to general and specific active site inhibitors of neutrophil elastase. This substrate appears to have important potential for use in ultrastructural studies of intracellular elastase-like enzymes.     

A comparative ultrastructural and morphometric study of six species of the diatom genus Stephanodiscus

The cellular composition of six species of Stephanodiscus commonin the Laurentian Great Lakes was compared using quantitativeultrastructural and morphometric techniques. The species studiedspan a considerable portion of the range of size and obviousmorphological differences known within the genus. Results showconsiderable differences between species in the amount of totalcell volume occupied by frustule, cytoplasm, vacuole and chloroplast.Other organelles, such as mitochondria, Golgi bodies and thenucleus occupy similar fractions of the total cytoplasmic massin all species studied. The main within-species difference isfound in the amount of storage products present, which appearsto be related to cell condition. The techniques utilized areapplicable to populations in mixed assemblages from the fieldand can be used to measure the cellular composition of suchpopulations with a high degree of accuracy and precision. ^*Contribution No. 406 of the Great Lakes Research Division,The University of Michigan.