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Computer-assisted morphometry using video-mixed microscopic images and computer graphics.

A microcomputer system has been developed for collection and analysis of morphometric data from video images of histological sections. Microscopic fields of view are visualized on a video monitor and are overlaid with computer-generated graphics by means of a video mixer. Planimetric data are entered by drawing on a digitizing tablet while observing the drawn image superimposed on the tissue. Programs are available to calculate various geometric parameters, count objects in a field, perform descriptive statistics, and measure volume and surface area of a solid from a series of cross sections. A program for point-counting stereology presents single points for scoring within a user-defined reference area. The potential applications of interactive graphics in morphometry are discussed.

Cell Nucleus

Morphometry of the human pulmonary acinus.

The geometry and morphometry of intraacinar airways in human lungs were studied on silicone rubber casts from two adult lungs. We defined acini as the complex of alveolated airways distal to the terminal bronchioles--that is, beginning with the first-order respiratory or transitional bronchiole. The morphological properties of pulmonary acini are described. The acinar volume averages 187 mm3 (SD +/- 79 mm3). Intraacinar airways branch dichotomously over about 9 generations (range 6-12). The internal airway diameter falls from 500 micron to 270 micron between acinar generations 0 and 10, whereas the outer diameter (including the sleeve of alveoli) remains constant at 700 micron. Towards the periphery the size of alveoli increases and clusters of alveoli become more numerous. The longitudinal path length of acinar airways (defined as the distance along the ducts from the transitional bronchiole to the alveolar sacs) averages 8.8 mm (+/- 1.4 mm). The morphometric data collected in this study are used to construct an idealized model of human acinar airways that can be related to existing models of the human bronchial tree.

Adult

In vivo morphometry and functional morphology of brown adipose tissue by magnetic resonance imaging.

Brown adipose tissue (BAT) is the main effector of nonshivering thermogenesis and diet-induced thermogenesis in mammals. Assessment of the magnitude and perturbations of BAT deposits in the intact, living body would be of much relevance for quantitative studies of BAT functions, but such studies have been impossible to date. In this paper it is shown that magnetic resonance imaging (MRI) morphometry can provide the means for accurate, repeated determinations of the volume of BAT deposits in a living animal; moreover, tissue modifications due to acclimation at different ambient temperatures are revealed in vivo by MRI, which correlates with histology and ultrastructure. Furthermore, MRI differentiates areas of BAT responsive to acute adrenergic stimulation, thereby giving information on the thermogenetically active tissue in the intact animal. Therefore, MRI represents a reliable tool for correlative morphological and functional studies of BAT in the living animal.

Adipose Tissue, Brown

A comparative study by morphometry of the microvasculature in normal and rheumatoid synovium.

Vascularity is said to be increased (subjectively) in rheumatoid arthritis, yet synovial fluid is hypoxic and acidotic. Morphometry by image analysis was used to quantify vascularity in normal and rheumatoid synovium from the knee joint. Capillaries were distributed more deeply in the rheumatoid synovium (93.3 microns, compared with 32.5 microns in normal synovium) and were significantly less densely arranged (80.2/mm2 rheumatoid, 241.5/mm2 normal). The blood volume fraction fell from 2.9% in normal knee synovium to 1.2% in rheumatoid synovium. These results imply that there is impairment of O2 transfer, which contributes to the intraarticular hypoxia, in rheumatoid arthritis.

Adult

Topography and morphometry of capillaries in the rat subfornical organ.

A comprehensive stereological analysis was performed to define capillary dimensions in individual subregions of the subfornical organ in Long-Evans, homozygous Brattleboro, and Sprague-Dawley rats. Capillary density, volume fraction, length, surface area, and diameter were assessed in four regions in the sagittal plane (rostral, "transitional," central, and caudal) and two zones in the coronal plane (dorsal and ventromedial). The ventromedial zones in the central and caudal regions correspond to areas of dense perikarya and neuropil containing neural afferent inputs to the subfornical organ (e.g., putative fiber terminals for angiotensin II), whereas the dorsal zones of these regions are apparently the predominant sites of perikarya having efferent projections directed outside of the organ. The morphometric analysis revealed heterogeneous capillary density across subregions of the subfornical organ (range of 132 to 931 capillaries/mm2 in the three rat groups). Capillaries in the ventromedial zones of the central and caudal regions had significantly greater density, volume fraction, and surface area, but smaller diameters, than those in the adjacent dorsal zones and more rostral regions. Across all subregions within the dorsal zone, there was generally a consistent morphometric pattern in the three rat groups. No differences in capillary dimensions in any part of the subfornical organ were found between the Long-Evans and Brattleboro rats. A qualitative electron microscopic investigation of endothelial cells in each subregion of the subfornical organ in Long-Evans rats revealed at least three types of capillary oriented according to region: in the rostral region were capillaries having no endothelial fenestrations or pericapillary spaces, and few vesicles, in the "transitional" region between the rostral and central regions, capillaries having no endothelial fenestrations, substantial numbers of vesicles, and narrow but perceptible pericapillary spaces were found, and in the central and caudal regions, capillaries having abundant endothelial fenestrations and vesicles, expansive pericapillary labyrinths, and relatively thin walls were present. These findings from light microscopic morphometry and electron microscopy in rats indicate a heterogeneity of capillary organization that shows topographical correspondence to the cytology and putative functions of the subfornical organ.

Animals

Bile canalicular morphometry in arteriohepatic dysplasia.

The diagnosis of arteriohepatic dysplasia may be difficult, particularly in very young patients with no family history, and the pathogenesis of the disorder remains obscure. It has been reported that the typical ultrastructural changes of cholestasis are scant in this condition and proposed that there is a failure of the hepatocytes to secrete bile into the canaliculi. In an attempt to expand on these observations and to test the value of ultrastructure as a differential diagnostic test for this condition, we chose two parameters felt to be associated with cholestasis that could be reliably and relatively simply determined by ultrastructural morphometry: canalicular dilatation and loss of canalicular microvilli. The values for these parameters were compared in arteriohepatic dysplasia with those in other infantile cholestatic conditions and with those in noncholestatic controls. The results of these morphometric studies corroborated the previous observation that canalicular dilatation in arteriohepatic dysplasia is minimal. There was, however, only a marginal difference in canalicular area between patients with arteriohepatic dysplasia and those with extrahepatic biliary atresia. There was significant overlap of values for canalicular area among all groups. Surprisingly, the studies showed no real loss of canalicular microvilli in any of the cholestatic conditions. The findings suggest that, at least for these two parameters, caution should be exercised in using qualitative electron microscopic evaluation as a diagnostic test for arteriohepatic dysplasia.

Bile Canaliculi

A computer program for morphometry of circular and elliptical profiles on the microscope.

We describe a short computer program, which is written in Pascal language, to measure the diameter of circular and elliptical profiles in sections. Coordinate pairs on the microscope stage, corresponding to two or three points of a profile, are input to obtain its diameter. The program enables one to take measurements directly on the microscope, thereby reducing photographic work and caliper measurement. While the program is largely designed for electron microscopy, it also may be useful for light microscopy morphometry.

Animals

Computer-assisted morphometry: point, intersection, and profile counting and three-dimensional reconstruction.

The use of computers in morphometry can involve 1) automated image analysis, semiautomated image analysis and point, intersection, intercept and profile counts of two-dimensional images on tissue sections with mathematical extrapolation to the third dimension, 2) direct measurement of volumes, surfaces, lengths, and curvature using x,y,z coordinates of serial sectioned images, or 3) stereologic techniques and serial sections which is a combination of 1 and 2 above. Automated and semiautomated image analysis are generally restricted to specimens that are characterized by differential contrast such as interalveolar septa in the lung or histochemically stained mucous granules in pulmonary epithelium. Point, intersection, and profile counts using hand-held, notebook PCs, portable PCs, or standard PCs and MS-DOS-based application programs are extremely efficient, precise, affordable, and convenient methods of quantitating average values of a population. When morphometric measurements of individual structures are required, computer-assisted three-dimensional reconstruction using x,y,z coordinates of the surface outline from serial sections is a tedious yet precise method. We describe a computer program that efficiently estimates mean caliper diameter, volume, and surface area with less than five percent error with five sections per structure. We also describe a program that does digital image subtraction on serial sections, superimposes digitally generated test systems on biological images, and accumulates point, intersection, and profile counts using a Macintosh II series computer.

Animals

Differences between early- and late-maturing genotypes of the platyfish (Xiphophorus maculatus) in the morphometry of their immunoreactive luteinizing hormone releasing hormone-containing cells: a developmental study.

Immunoreactive luteinizing hormone releasing hormone (ir-LHRH) containing perikarya, brain nuclei, and pituitary cells were studied during the postnatal development of male sibling platyfish (Xiphophorus maculatus) genetically determined to reach puberty at two different ages using immunocytochemical, cytological, and morphometric methods. Ir-LHRH-containing perikarya first appear in the nucleus olfactoretinalis (NOR) at 5 weeks of age in early maturers and at 11 weeks in late maturers at a maximum number which is similar in both genotypes and remains constant into adulthood. The dimensions of the NOR and its perikarya increase up to the initiation of sexual maturation (gonopodial stage 2) in both genotypes. In the nucleus preopticus periventricularis (NPP), ir-perikarya appear at stage 2 and are maximum in number at maturity (stage 6) when early maturers have 50% more than late maturers. Measurements for the NPP increase between 1 week and stage 6 in both genotypes. In the nucleus lateralis tuberis (NLT), ir-perikarya appear soon after stage 2 in early maturers but are never seen in late maturers. Late maturers also have fewer ir-LHRH containing pituitary cells than early maturers. In both genotypes, measurements for the NLT increase to stage 2 and then decrease to stage 6. During sexual development there are differences between early- and late-maturing genotypes in the morphometry of their LHRH-containing brain centers. The timing of sexual development creates significant differences in the cytological and cytometric characteristics of the three ir-LHRH-containing brain nuclei in fish of the same age but different genotype. Our results also show that for both genotypes there is a positive correlation between the total number of ir-LHRH brain neurons and the total number of ir-pituitary cells and both are lower for late maturers at every age and stage.

Animals

Fiber type morphometry and capillary geometry in free, vascularized muscle transfers.

In order to establish the relationship among intraoperative ischemia time, the recovery of contractile function, muscle fiber morphometry, and capillary geometry following free muscle transfer, 15 male dogs underwent unilateral, orthotopic free transfer of the left gracilis muscle with microneurovascular anastomosis. Intraoperative ischemia time varied from 0 to 4 hours. After a recovery period averaging 14 months, isometric contractile function was measured in the transfers and the contralateral, control muscles. Muscle fiber type composition, fiber size, and capillary geometry were quantified from histochemical sections. Transferred and control muscles had similar type I fiber percentages, type I fiber sizes, and capillary densities. Type II fiber sizes were reduced in transfers relative to controls. There was no significant effect of intraoperative ischemia on any morphometric variable. Although contractile function was generally reduced after free, vascularized transfer, there was only a weak correlation between the recovery of tetanic tension and type I fiber percentage (r = 0.53, P less than 0.05). It is concluded that free, vascularized muscle transfer results in minimal disturbance of muscle fiber type composition, fiber size, or capillary supply and that the diminished contractile function observed in this setting is still largely unexplained.

Analysis of Variance

Morphometry of bladder carcinoma: I. The automatic delineation of urothelial nuclei in tissue sections using an IBAS II image array processor.

Semi-automatic methods of morphometry require the boundaries of the objects under study to be drawn by hand. This is slow, tedious and does not guarantee accuracy. Complete automation, with recognition, detection and verification by computer is not yet feasible. Automatic detection of nuclear boundaries with observer verification provides a means of obtaining large amounts of data for analysis, quickly and with similar accuracy to manual tracing.

Cell Nucleus

Morphometry of gastric carcinoma: its association with patient survival, tumour stage, and DNA ploidy.

Morphometric image analysis of nuclear features was performed on tissue from 46 patients who had had curative resections for gastric cancer. Clinical, pathological, flow cytometric, and follow-up data were available for these patients, which were drawn from a larger, previously reported series. The morphometric data were compared with patient survival, clinico-pathological status, and DNA ploidy. Univariate survival analysis revealed that morphometric parameters were not significantly related to survival, but examination of clinico-pathological data showed lymph node involvement, involvement of the resection margin, and lymphatic invasion to be significantly associated (P < 0.01) with patient prognosis. Multivariate survival analysis using the Cox model found only lymph node and resection margin involvement to be independently related to survival. Comparison of morphometric results with the clinico-pathological parameters showed various features, relating to nuclear size, and its variation to be significantly associated (P < 0.01) with the presence of lymphatic invasion, resection margin involvement, and tumour pattern (intestinal/diffuse). A comparison of morphometry with flow cytometric analysis in these cases showed that nuclear size was not significantly related to either DNA aneuploidy or the DNA proliferative index.

Aged

Morphometry of right ventricular papillary muscle in rat during development and regression of hypoxia-induced hypertension.

Morphometric analyses of the right ventricular papillary muscle, as well as measurements of right ventricular pressure and weight, were carried out in the rat during the development and recovery of hypoxic pulmonary hypertension. Animals were divided into hypoxic and normobaric control groups. The hypoxic rats were placed in hypobaric chambers for 1, 2, and 3 wks; and after 3 wks exposure, subgroups of hypoxic rats were allowed to recover in normoxia for 1 to 9 wks. Hematocrit (HCT) and right ventricular systolic pressure (RVSP) were measured prior to sacrifice. The heart was perfused, and the right ventricle (RV) was separated from the left ventricle and septum (LV+S) and weighed. The papillary muscles were dissected and processed for ultrastructural morphometry. Results showed that HCT, RVSP, and RV weight increased in the rats during the hypoxic exposure and then gradually returned to control levels after 3 to 4 wks of normobaric recovery. The papillary muscle of the hypoxic rats showed increased volume density of interstitium, increased diameter and cross sectional area of the cardiac myocytes, reduced volume density of mitochondria, and reduced mitochondria to myofilament ratio. During normoxic recovery, these morphometric indices returned toward control values at various periods of time ranging from less than 3 wks to 9 wks. The results indicate that the adaptive ultrastructural changes of the papillary muscle in RV hypertrophy paralleled the RVSP changes, and also demonstrate the reversibility of these changes in ambient oxygen.

Animals

Morphometry of the corpus callosum in normal aging and Alzheimer's disease.

Changes of the human corpus callosum in normal aging and Alzheimer's disease were analysed by means of morphometry. A standardized computerized evaluation program was implemented allowing objective, quantitative and reproducible data. The various parts of the corpus callosum showed a different pattern of changes in normal aging as compared to Alzheimer's disease. In conclusion, in normal aging affects mostly the front-temporal interhemispheric fiber systems, whereas in Alzheimer's disease the parietotemporal commissural fibers are altered.

Aging

[Contribution to the morphometry of coronary arteriosclerosis (author's transl)].

In 94 cases with and without coronary arteriosclerosis or various cardiac diseases the coronary arteries were pressure fixed, filled with a mixture of barium sulfate and gelatine for coronary angiography and dissected in 1 cm long segments for morphometry. On consecutive cross sections of the three main branches of the coronary arteries the absolute area of lumen, intima and media as well as of the thickness of the intima and media were measured. From the numerous data of the coronary arteries different indices on coronary arteriosclerosis and by correlation to the heart weight also on coronary insufficiency had been calculated. Using these quantitative data age dependent intimal changes could be distinguished convincingly from cases with uncomplicated coronary arteriosclerosis and cases with hypertension or infarcts. Thus in forensic pathology comparative evaluations can be made in regard to the extent, severity and significance of acute and chronic coronary insufficiency.

Acute Disease

Characterization of an oxygen-tolerant cell line derived from Chinese hamster ovary. Antioxygenic enzyme levels and ultrastructural morphometry of peroxisomes and mitochondria.

To study the cellular defense mechanism against oxygen toxicity, an oxygen-tolerant cell line from Chinese hamster ovary (CHO) was obtained by multistep adaptation to increased O2 levels. The hyperoxia-adapted (HA) cells were able to proliferate under an atmosphere of 99% O2/1% CO2, an O2 tension lethal to the parental (control) cells. When grown under normoxic conditions (20% O2/1% CO2/79% N2) the cells remained tolerant for at least 8 weeks, suggesting a genetic basis for the oxygen tolerance. Compared to the parental cells, the HA cells were irregularly shaped, had larger mitochondria, contained more lipid droplets and showed a reduced growth rate. Ultrastructural morphometry revealed a 1.8-fold (p less than 0.001) increase of the mitochondrial volume fraction in the HA cells, resulting from an increase in both number and average volume of the mitochondria. The volume fraction of peroxisomes was increased over two-fold in the HA cells, as appeared from a approximately 1.9-fold (p less than 0.001) increase in number and a 1.2-fold (p less than 0.025) increase in size. There was no evidence for ultrastructural damage in the HA cells. Specific activities of antioxygenic enzymes were considerably higher in the HA cells compared to controls: CuZn-superoxide dismutase, X 2.5; Mn-superoxide dismutase, X 2.1; catalase, X 4.0; glutathione peroxidase, X 1.9. Oxygen tolerance in CHO cells is therefore associated with increased levels of antioxygenic enzymes, confirming the proposed important role of these enzymes in the defense against oxygen toxicity.

Adaptation, Physiological

Ultrastructural morphometry of parathyroid cells in rats of different ages.

Parathyroid glands of newborn to 1-year-old male Sprague-Dawley rats were fixed by perfusion or by immersion, and prepared for electron microscopy. Parathyroid glands fixed by immersion exhibited parenchymal cells with variable ultrastructure, indicating that these cells were in different stages of the proposed secretory cycle in parathyroid cells. In contrast, parathyroid cells of glands fixed by perfusion were uniform in ultrastructure, suggesting that all cells were in the same stage of secretory activity. Parathyroid glands of 3-, 4-, 6-, 8- and 10-week-old rats also were fixed by perfusion and analysed by electron-microscopic morphometry. These data demonstrated an increase in cell volume and in surface area of the membrane compartments concerned with parathyroid hormone secretion: these changes were not related to variations in serum calcium concentration. Both the qualitative observations and the quantitative data do not favour the idea of a secretory cycle in rat parathyroid cells.

Aging

Effects of TRH and somatostatin on releases of prolactin and growth hormone in vitro by the pituitary of Poecilia latipinna. II. Electron-microscopic morphometry using automatic image analysis.

Pituitary glands from a teleost fish were incubated in the presence of the synthetic hypophysiotropic peptides, thyrotrophin-releasing hormone and somatostatin, in two media of different osmotic pressure. The effects on prolactin and growth hormone cells were detected by electron-microscopic morphometry with the aid of an image analyser. Thyrotrophin-releasing hormone caused changes in prolactin cell ultrastructure consistent with stimulated hormone release and, in the low osmotic pressure medium, appeared to increase synthetic activity. There was no effect on growth hormone cells. After somatostatin treatment, both synthesis and release in prolactin cells appeared to be inhibited, and there was an obvious inhibition of synthesis and release in growth hormone cells. The response of both cell types to somatostatin did not appear to be dependent on the osmotic pressure of the medium.

Animals