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R P Bolender

Publications and source records attributed to R P Bolender.

18 recordsLinked to original sources
PubMed1992

Biological stereology: history, present state, future directions.

The development of a quantitative structural platform for experimental biology--extending across a hierarchy of sizes ranging from molecules to organisms--has been punctuated by a series of major achievements over the last 30 years. Stereology, a form of quantitative morphology, has contributed handsomely to this success. A personal view is presented highlighting key events in the development of biological stereology. We also examine stereology with a view toward future developments in biology and speculate how stereology might contribute to the new biological infrastructure currently being built with computers.

Animals↗
PubMed1992

Quantitative morphology for biologists and computer scientists: I. Computer-aided tutorial for biological stereology (version 1.0).

This paper describes a computer-aided tutorial for biological stereology. Stereology, a type of quantitative morphology, includes a collection of statistical methods that quantify the structural compartments that can be viewed in sections with light and electron microscopy. These methods provide volume, surface, length, shape, and number data, and help define the quantitative relationships among the structural compartments of biological hierarchies. Hierarchies, which connect structural data ranging in size from molecules to organs, serve as a central core to which the data of biological databases can be linked. The tutorial focuses on two objectives. It provides the user primarily interested in using quantitative morphology databases with background information, and offers a set of state-of-the-art tools to researchers wishing to use these methods in the laboratory. The main topics of the tutorial include: introduction to quantitative morphology, symbols/terms, data types, sampling, hierarchies, data interpretation, and utilities. The tutorial runs under the MS-DOS operating system and requires at least an IBM PC AT (or compatible), a color monitor (EGA, VGA), 540 KB of RAM, and 3 MB of hard disk space.

Animals↗
PubMed1992

Computer assisted data collection for stereology: rationale and description of point counting stereology (PCS) software.

The paper describes microcomputer software for point counting stereology. Stereology includes a collection of statistical methods that quantify the images of light and transmission electron microscopy. The methods use test grids placed over images to collect raw data, which includes counts of points, intersections, transections, and profiles. In turn, the counts are included in stereological equations that give estimates of compartmental volumes, surfaces, lengths, or numbers. These parameters describe the composition of a structure in three-dimensional space. The PCS (point counting stereology) System Software III serves as a data collection, storage, and management tool. Users set up point counting protocols without programming, enter data by pressing predefined function (MS-DOS) or alphabetic keys (UNIX), store data in files, select files for analysis, and calculate results as stereological densities. The latest version of the PCS software includes a new user interface and is designed as a research "front end" that can feed data either into the calculation tools of a stereology tutorial (Bolender, 1992, this issue) or into the analysis routines of quantitative morphology databases (Bolender and Bluhm, 1992).

Animals↗
PubMed1990

Counting cells with stereology: random versus serial sectioning.

Counts of cells and nuclei from sections provide information central to studying structural changes in cells, tissues, and organs. This study considers some of the practical problems associated with counting cells with the newer random and serial sectioning methods of stereology and tests the hypothesis that similar cell counts can be obtained with both random and serial sectioning methods. Using irregularly shaped nuclei from alveolar cells of the goat lung, we compared cell counts derived from random (electron microscopic) and serial sectioning (light microscopic) methods. The results showed that both sectioning methods gave similar cell counts (10(7)/cm3 of parenchyma) for type 1 epithelial cells (5.0 vs. 5.0; P=1.0), type 2 epithelial cells (8.6 vs. 9.8; P= 0.42) and interstitial cells (34.6 vs. 33.4; P=0.64), provided that corrections were introduced for section-related biases and that the nuclei of the random sectioning method were corrected for shape. We found counting biases of 5%-7% for nuclear shape and 16% for section compression. These observations support the hypothesis that similar cell counts can be obtained with random and serial sectioning, even when nuclei have irregular shapes.

Animals↗
PubMed1990

Heparin modulates the composition of the extracellular matrix domain surrounding arterial smooth muscle cells.

Heparin and related molecules influence vascular wall structure by their ability to inhibit smooth muscle cell (smc) proliferation and migration. However, little is known as to whether heparin has an effect on the extracellular matrix. In the present study, the effect of heparin on the content and regional distribution of elastin, collagen, and proteoglycans (PGs) in blood vessels following experimental injury was determined. Two groups of rats were subjected to left common carotid balloon injury and were infused with either 0.9% saline or heparin in a saline solution, for 2 weeks. Using a new morphometric method of analysis, the authors determined changes in volumes of elastin, collagen, and PGs contained within an 'extracellular matrix domain (ECM domain),' the average envelope of connective tissue surrounding each smc. Heparin treatment inhibited intimal thickening and decreased the elastin content in the ECM domain in the upper and lower arterial intima. Collagen also was found to be significantly decreased 5.0-fold and 7.6-fold in the ECM domains of upper and lower intima, respectively, of heparin-treated animals. The decrease in both elastin and collagen was balanced by a significant increase in amorphous and filamentous electron-dense material. Heparin also caused a significant 1.8-fold and 1.9-fold increase in the PG content in the ECM domain in the upper and lower intima, respectively. Immunohistochemical analysis, using antibodies to elastin and PG subclasses, supported the morphometric observations. This study has shown that heparin administered in vivo can alter the accumulation and distribution of each of the major vascular ECM components in a specific and differential manner.

Animals↗
PubMed1988

Numbers of synapses in laminae I-IV of the rat dorsal horn.

The present study determines numerical densities (NVsyn) and total numbers of synaptic discs in laminae I-IV of the rat S2 dorsal horn. Previous methods for NVsyn have the advantage of being relatively simple, but these assume that the discs are round, flat, and of uniform size. In our material, serial reconstructions indicate that these assumptions are not met. Accordingly we use a stereological method that is not as dependent on these assumptions. This method is to divide the surface density of the discs by the mean surface area of a disc (NVsyn = SVsyn/Ssyn). We refer to this as a reconstruction method because synaptic discs are reconstructed from serial sections. We also calculate numerical densities by several previously used standard methods, and the findings are similar but not identical. We find that numerical density and total synaptic numbers are smallest in lamina I, and densities and total numbers are not significantly different when lamina II is compared to laminae III and IV. Thus the intense labeling of terminals with certain compounds that characterize lamina I and II does not imply an increase in total synaptic numbers or in synaptic density. In addition there is a general increase in synaptic densities and numbers as one proceeds from lamina I to lamina IV. Another point is that the numerical density of synapses in the dorsal horn is approximately that of the cerebral cortex. These data will serve as a basis from which to judge the effects of denervations and other manipulations that purportedly change synaptic numbers.

Animals↗
PubMed1986

Counting parenchymal cells in the goat lung with serial section reconstruction and stereology.

Information about numbers of cells is needed to interpret cellular and tissue responses to injury. As a first step towards identifying changes in cell number and structure in injured lungs, this study reports the frequencies of 4 parenchymal cell types in the normal goat lung. Cells were counted using serial section reconstruction and light microscopy. Relative cell frequencies were (mean +/- standard error): type I cells (5.47 +/- 0.52%), type II cells 10.74 +/- 1.17%), capillary endothelial cells (47.50 +/- 1.42%), and interstitial cells (37.14 +/- 3.00%). These cell counts coming from serial section reconstructions allowed us to estimate mean nuclear diameters of the 4 cell types without assumptions for size, shape, and frequency distribution, and they were also used to evaluate counts of nuclear profiles taken from light and electron micrographs. When the same mean nuclear diameter was used, the counts of nuclear profiles from both light and electron microscopy gave similar stereological estimates for cell numbers, provided section compression was corrected. We conclude that lung parenchymal cells can be counted using light microscopy and serial section reconstruction and that this information provides a standard for evaluating stereological estimates of cell numbers.

Animals↗
PubMed1985

PCS System I: point counting stereology programs for cell biology.

PCS System I (PCS) is a set of four software modules designed to simplify the application of stereology to problems in cell biology. It is written in BASIC for the Tektronix 4052A microcomputer (Beaverton, OR). A Counting Module collects raw data counts in either a Density Mode (points, intersections, transections, profiles) or a Boundary Mode (intersections with complete nuclear profiles). This information is stored on tape or disk data files and can also be printed. Three analysis modules use data files created with the Counting Module. The Density Module uses Density Mode data files to calculate volume, surface, length, and numerical densities. The B Numerical Density Module uses both Boundary Mode and Density Mode data to calculate the means for the boundary, diameter, and surface area of a nuclear compartment. The mean nuclear surface area is then used with the nuclear surface density to estimate the nuclear numerical density, which, in turn, is used to calculate surface areas of membrane compartments in average cells and in 10(6) cells. The Format Module reformats raw data files for analysis with Tektronix statistical software.

Biometry↗
PubMed1979

Surface area ratios. I. A stereological method for estimating average cell changes in membrane surface areas.

The study (i) describes a method for estimating relative changes in membrane surface areas as they occur in stereological "average cells," and (ii) considers the effect of the controls on these estimates. The results indicate that changes in five membranes compartments of pancreatic exocrine cells--produced by a secretagogue (carbomylcholine chloride)--were detected similarly when related to either an average cell surface (surface area ratio method) or to an average cell volume (method of Loud, '68). Changes, however, detected with surface densities, which relate these membrane compartments to 1 cm3 of exocrine cell cytoplasm or pancreas, were notably different from the first two estimates. This inconsistency could be explained by the fact that the surface densities were influenced not only by membrane changes within the exocrine cells, but also by changes in the number of these cells filling the cm3 of reference volume. Relating the data to an average cell reference--instead of 1 cm3--improved the accuracy of the estimates for changes in membrane surface areas by as much as several fold; the choice of controls had a similar several-fold effect on the results.

Animals↗
PubMed1979

Surface area ratios. II. A stereological method for estimating changes in average cell volume and frequency.

The study describes a sterological method for detecting relative changes in cells--with respect to their average volumes and to their frequencies within one cm3 of exocrine cells. The method is based on surface area ratios (Bolender, '79) and can detect changes comparable to those obtained with the numerical density approaches (Loud, '68; Weibel et al., '69; Bolender, '74; Williams, '77). The new method requires only intersection counts--the same ones that are used for surface density estimates--and, as a result, avoids the difficult and often problematic procedures of the numerical densities. In analyzing the cellular changes in vitro, the choice of either a zero or timed control was found to exert a major influence on the results. Timed controls were required to "isolate" the experimentally induced changes from those produced by the incubation medium.

Animals↗
PubMed1979

Changes in membrane surface areas in mouse parietal cells in relation to high levels of acid secretion.

Levels of gastric acid secretion which may be maximal for the mouse were recorded following treatment with histamine and carbachol. A 30-fold increase over control levels was obtained in perfused animals, corresponding to a fourfold increase over highest levels recorded previously for stimulated mice. Stereological methods were used to estimate surface areas of membrane compartments of parietal cells in control and stimulated animals. Estimates of relative changes in membrane surface areas using a surface ratio method in this case substantiated changes detected by calculating surface densities. Main changes in membrane compartments of parietal cells from animals showing maximal acid secretion were a fourfold increase in free (luminal) surface, a 50% increase approximately in lateral and basal membrane, and a 90% reduction approximately in the tubulovesicular membrane compartment. Following withdrawal of secretagogues, acid secretion usually returned to control levels within 3 hours, but complete reconstitution of the tubulovesicular compartment was not seen within any survival period up to 5 hours. Reappearance of tubulovesicular elements first occurred shortly after the peak of a secretory response in focal cytoplasmic areas containing spherical and indented coated vesicles, and also numerous concentric membrane profiles not previously described in parietal cells. The way in which movement of membrane from the tubulovesicular compartment to the free surface occurs is not yet clear. However, reconstitution of the tubulovesicular compartment during a fall in acid secretion appears to involve movement of membrane from the free surface through coated vesicles, and their progression through indented forms and concentric membrane profiles to vesicles of the tubulovesicular compartment.

Animals↗
PubMed1978

Intergrated stereological and biochemical studies of hepatocytic membranes. I. Membrane recoveries in subcellular fractions.

Previous attempts to relate the structure and function of hepatocytic membranes have compared biochemical data of fractions to morphological data derived from either intact tissue or fractions. The effects of the original homogenization aside, biochemical recoveries comparing membrane marker enzymes of the homogenate to subsequent fractions suggest a general conservation of activity. A sterological study was undertaken to estimate membrane surface areas in the intact tissue, homogenate, and fractions of the same livers and then to test the comparability of these data with membrane marker enzymes by calculating both morphological and biochemical recoveries. The sterological data were corrected for errors due to section thickness and compression. The average total membrane sufrace area per 1 g of liver was 9.3 m2 in the intact tissue (T), 7.8 m2 in the homogenate (H), and 7.4 m2 in the fractions (F); recoveries for the membrane surface areas thus averaged 96% for the (F/H) and 81% for the (F/T) comparisons. In homogenate and fractions, the differentiability of membranes by morphological criteria was limited to rough- and smooth- surfaced membranes, as well as outer and inner mitochondrial membranes. The recoveries of rough-surfaced membranes were 101% for F/H and 92% for F/T; those of smooth-surface membranes were 89% for F/H and 107% for F/T. For mitochondrial membranes, a recovery of 100% for F/H was obtained, whereas it amounted to only 54% for F/T. With respect to F/H, the membrane recoveries compare well with the marker enzyme recoveries obtained biochemically. The extension of recovery calculations to the intact tissue (F/T) revealed satisfactory conservation of the procedures of homogenization and fractionation; it indicates, however, that a shift of a substantial part of mitochondrial membranes to the pool of unidentifiable smooth membranes may occur on homogenization.

Animals↗
PubMed1978

Integrated stereological and biochemical studies on hepatocytic membranes. III. Relative surface of endoplasmic reticulum membranes in microsomal fractions estimated on freeze-fracture preparations.

New methods are required for identifying membranes in subcellular fractions with respect to their origin, if such preparations are to be evaluated morphometrically. One method is freeze-fracturing which reveals intramembrane particles whose size, pattern, and numerical density differ for various membrane types. The question is examined whether the differences in numerical particle density per square micrometer of membrane (alpha) can be used to differentiate membrane vesicles found in microsomal fractions from liver cells with respect to their origin in the hepatocytes. It is found that the range of alpha for the protoplasmic face (PF) of endoplasmic reticulum (ER) membrane (1,900 less than alpha less than 3,250) is intermediate between those for plasma and mitochondrial membranes. Since PF(ER) should appear in the outer leaflet of microsomal vesicles, alpha was estimated on concave profiles of freeze-fracture preparations; the numerical frequency distribution of vesicles with respect to alpha was trimodal, with a major peak around 2,900/micrometer2 and 66% of the vesicles in the range determined for PF(ER). Using a new stereological method, it was calculated that 63% of the membrane surface in these microsomal fractions was of ER origin by this criterion. On the same preparations, an attempt was made to label the ER-derived membranes cytochemically for glucose-6-phosphatase. A line intersection count revealed 62% of the membrane surface to be of ER origin on the basis of marker enzyme labeling. These findings indicate a smaller part of ER membranes in microsomal fractions than would be predicted from biochemical data (77%). The possible reasons for such discrepancies are discussed; shifts in particle densities due to the preparation procedure could lead to an underestimate by freeze-fracturing, whereas the prediction from biochemical data could be overestimates if marker enzymes were not homogeneously distributed.

Animals↗
PubMed1977

Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study.

When biochemical studies on the liver are interpreted, the cells of the sinusoidal area frequently receive little attention because, compared to hepatocytes, their contribution to subcellular fractions is assumed insignificant. A systematic stereological analysis of liver parenchyma was therefore performed in order to determine the distribution of organelles and membranes between hepatocytic and nonhepatocytic cells, namely endothelial, Kupffer, and fat-storing cells. The livers were fixed by vascular perfusion and the data were corrected for systematic errors dur to section thickness and compression. The extracellular space compartment includes the lumina of sinusoids (10.6%), the space of Disse (4.9%), and the bile canaliculi (0.4%). Hepatocytes constitute 78% of parenchymal volume; the nonhepatocytes account for 6.3% and consist of 2.8% endothelial cells, 2.1% Kupffer cells, and 1.4% fat-storing cells. The nonhepatocytes contribute 55% of the volume of lipid droplets in the liver, 43% of the lysosomes, and 1.2% of the mitochondria. Although the nonhepatocytes account for only 8% of the total surface area of parenchymal membranes, they contain 26.5% of all the plasma membranes, 32.4% of the lysosomal membranes, 15.1% of the Golgi apparatus 6.4% of the endoplasmic reticulum, and 2.4% of the mitochondrial membranes. The data demonstrate the extent to which nonhepatocytic organelles can potentially contaminate subcellular fractions used for biochemical studies. Particularly important for the interpretation of studies on lysosomes, plasma membrane, and Golgi apparatus is the finding that an appreciable part of these organelles may be derived from cell types other than hepatocytes.

Adipose Tissue↗
PubMed1976

Stereological method for estimating relative membrane surface area in freeze-fracture preparations of subcellular fractions.

Microsomal fractions from liver cells are a mixture of vesicles derived from a number of different cellular membranes. These can be differentiated on freeze-fracture preparations by their characteristic density of intramembranous particles. A stereological method is developed which allows the estimation of the relative membrane surface of the various membrane types by using freeze-fracture preparations. The sample is restricted to concave profiles without cast shadow. The numerical frequency distribution of vesicles with respect to particle density is determined. The estimation of relative surface area must consider the effects on sampling of (a) variable size distributions of vesicles and (b) loss of small profiles. Correction coefficients are derived which allow a differential vesicle count to be transformed into an estimate of relative membrane surface.

Animals↗