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R P Bolender

Publications and source records attributed to R P Bolender.

At least 19 recordsLinked to original sources
PubMed1996

Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure.

The effects of long-term subclinical exposure to methylmercury on the number of neurons, oligodendrocytes, astrocytes, microglia, endothelial cells and pericytes within the thalamus from the left side of the brain of the monkey Macaca fascicularis has been determined by use of the Optical Volume Fractionator stereological method. The accumulated burden of inorganic mercury (IHg) within these same cell types has been determined by autometallographic methods. Four groups of monkeys were exposed to methylmercury (MeHg; 50 micrograms Hg/kg body weight/day) by mouth for 6 months, 12 months, 18 months, or 12 months followed by 6 months without exposure (clearance group). Neurons, oligodendrocytes, endothelia, and pericytes did not show a significant change in cell number for any exposure group. Astrocyte cell number exhibited a significant decline for both the 6 month and clearance exposure groups. The microglia, in contrast, showed a significant increase in the 18 month and clearance exposure groups. Results from mercury speciation and quantification analysis of contralateral matched samples from the thalamus of the right side of the brain from these same monkeys indicated that MeHg concentrations plateaued at around 12 months exposure, whereas the inorganic levels, presumably derived from demethylation of MeHg, continued to increase throughout all exposure durations. Autometallographic determination of the distribution of IHg by cell type indicates that both the astrocytes and microglia contain substantially elevated IHg deposits relative to all other cell types. The data suggest that the inorganic mercury present in the brains, accumulating after long-term subclinical methyl mercury exposure, may be a proximate toxic form of mercury responsible for the changes within the astrocyte and microglial populations.

Animals↗
PubMed1995

Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.

Allantois↗
PubMed1994

Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure.

The number of neurons, astrocytes, reactive glia, oligodendrocytes, endothelia, and pericytes in the cortex of the calcarine sulcus of adult female Macaca fascicularis following long-term subclinical exposure to methyl mercury (MeHg) and mercuric chloride (inorganic mercury; IHg) has been estimated by use of the optical volume fractionator stereology technique. Four groups of monkeys were exposed to MeHg (50 micrograms Hg/kg body wt/day) by mouth for 6, 12, 18, and 12 months followed by 6 months without exposure (clearance group). A fifth group of monkeys was administered IHg (as HgCl2; 200 micrograms Hg/kg body wt/day) by constant rate intravenous infusion via an indwelling catheter for 3 months. Reactive glia showed a significant increase in number for every treatment group, increasing 72% in the 6-month, 152% in the 12-month, and 120% in the 18-month MeHg exposed groups, and the number of reactive glia in the clearance group remained elevated (89%). The IHg exposed group showed a 165% increase in the number of reactive glia. The IHg exposed group and the clearance group had low levels of MeHg present within the tissue; however, the level of IHg was elevated in both groups. These results suggest that the IHg may be responsible for the increase in reactive glia. All other cell types, including the neurons, showed no significant change in number at the prescribed exposure level and durations. The identities of the reactive glial cells and the implications for the long-term function and survivability of the neurons due to changes in the glial population following subclinical long-term exposure to mercury are discussed.

Analysis of Variance↗
PubMed1994

Morphometric approaches for evaluating pulmonary toxicity in mammals: implications for risk assessment.

Recent advances in quantitative morphology provide all the tools necessary to obtain structural information in the lung that can be quantified and interpreted in the three-dimensional world of toxicology. Structural hierarchies of conducting airways and parenchyma of the lung provide: (1) numbers of cells per airway, lobe, or lung; (2) surface areas of cells, airways, and alveoli; (3) length of airways and vessels; and (4) volumes of cells, alveoli, airways, vessels, and individual lobes or the entire lung. Unbiased sampling of these subcompartments of the lung requires fractionation of lobes or individual airways. Individual airways of proximal and distal generations are obtained by airway microdissection along one axial pathway and comparisons made between airway generations. Vertical sections of selected airways are used to sample epithelium and interstitium. Using this unbiased approach of quantitative morphology, we have shown that inhalation of low ambient concentrations of ozone ([O3]0.15 ppm) near or at the United States National Ambient Air Quality Standard (NAAQS) (0.12 ppm O3) induces significant alterations in bronchiolar epithelium and interstitium in nonhuman primates but not rats. The alterations do not appear to be concentration- or time-dependent, thereby bringing into question the current NAAQS that may be at or above the threshold for distal airway injury in primates. Unbiased morphometric methods are critical in a quantitative evaluation of toxicological injury of mammalian tracheobronchial airways.

Air Pollutants↗
PubMed1993

Software for quantitative immunogold and in situ hybridization.

A well-deserved criticism of stereology is that it is often too difficult to understand and use. Nevertheless, it is rapidly becoming one of the most effective ways of collecting and interpreting structural data in experimental biology. Recent breakthroughs in theory have produced a remarkable set of tools that can be used to engineer new methods. The dilemma remains, however, in that some of these new methods continue to be too difficult to understand and use. One solution to this growing problem of technology transfer might be to simplify the methods with computer software. To test this idea, programs were written for quantitative immunogold and in situ hybridization. Simulators were used to develop and test new experimental designs, which, in turn, were translated into step-by-step laboratory tool kits. This paper shows how these tool kits can turn two-dimensional section data into estimates for the number of labeled molecules in three-dimensional organelles, cells, tissues, and organs. The results indicate that software can identify the key data of an experiment and reduce the computational requirements of a new stereological method to entering constants and variables into data entry forms.

Animals↗
PubMed1993

Software for counting cells and estimating structural volumes with the optical disector and fractionator.

We describe MS-DOS software for the optical volume fractionator (OVF), a stereological method combining the principles of the optical disector (Gundersen et al.: Acta Pathol. Microbiol. Immunol. Scand., 96:857-881, 1988) and fractionator (Gundersen: J. Microsc., 143:3-45, 1986). The OVF program estimates the volume of a fixed and embedded structure, the numerical density of cells, and the total number of cells in a structure. The hardware requirements include a PC computer (386 or 486 with VGA graphics) and a conventional light microscope fitted with a rotating stage, extension tube, and length gauge. The software includes an introduction, tutorial, simulator, laboratory tool kit, and report generator. The tool kit improves the efficiency of gathering stereological data with light microscopy and offers a convenient link between the data of light and electron microscopy. A novel algorithm, based on fractionator sampling, gives the volume of a fixed and embedded structure from the same set of sections used for cell counting. A laboratory example illustrates the operation of the software.

Algorithms↗
PubMed1993

Lung morphometry: a new generation of tools and experiments for organ, tissue, cell, and molecular biology.

Today all structural information of the lung can be quantified and interpreted in the three-dimensional space of real-world biology. Remarkable achievements in the theory and practice of biological stereology are creating a new generation of data suitable for constructing structural hierarchies. Such hierarchies serve to organize and link biological data, thereby providing a framework on which to build new information systems. In this review, we describe the new tools of quantitative morphology and show how they can be used to design new experiments for lung research.

Animals↗
PubMed1993

Estimation of tissue mRNAs by in situ hybridization.

We describe a new stereological method for analyzing data derived from the in situ hybridization procedure. This method should prove important, since data summarization in terms of grains per anatomic area by sampling of tissue sections may lead to faulty interpretations. Using computer simulation of measurements taken from a two- and a three-dimensional perspective, we show how the detection of molecular changes can be influenced by multiple structural events. Cell volume, the volume of the structure, and the number of labeled cells in an anatomic structure are all important parameters that must be assessed to obtain accurate results. We then outline in detail a simple and efficient method for estimation of the number of mRNA molecules in cells and in the total structure. By estimation of the abundance of mRNA molecules in three dimensions, we can make better inferences concerning gene expression in a structural context.

Animals↗
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↗
PubMed1983

Methods for decreasing the statistical variance of stereological estimates.

Three methods are described for decreasing the statistical variance of stereological estimates. Method 1 uses profile boundaries and surface densities of nuclear membranes, measured in thin sections, to estimate the mean diameter, surface area, and numerical density of spherical and nonspherical nuclei. For the guinea pig pancreas (number(m) = 4), the standard deviations (s.d.) as a percent of the mean for the estimates of the diameters of the exocrine, duct, and endothelial cell nuclei were 1.5%, 3.3% and 1.4%. The estimate for the mean diameter of exocrine nuclei (6.4 +/- 0.1 micron) was based on a spherical model, whereas the estimates for the diameters of the nonspherical (and nonconvex) nuclei of the duct (6.4 +/- 0.2 micron) and endothelial (6.7 +/- 0.1 micron) cells were calculated from the numerical density of the exocrine cells and the relative frequencies of the three cell types (determined from serial reconstructions). In an average cubic centimeter, there were 6.17 X 10(8) +/- 0.32 X 10(8) (s.d. 5.1% of mean) exocrine cells, 1.64 X 10(8) +/- 0.18 X 10(8) (10.9%) duct cells, and 0.803 X 10(8) +/- 0.13 X 10(8) (16.6%) endothelial cells. In contrast to method 1, conventional stereological approaches were found to have standard deviations two- to eightfold larger. Method 2 uses a mean nuclear surface area and a ratio of surface densities to estimate the surface area of a membrane compartment in an average cell. A s.d. equal to 6.5% of the mean was found for the surface area of the outer mitochondrial membrane in an average exocrine cell (672 +/- 43.6 micron 2), which represented an almost fourfold reduction in the s.d. compared with an earlier estimate (Bolender, 1974). Method 3 relates the surface area of a membrane compartment to a standard number of cells. Referenced to 10(6) cells, for example, the surface area of the inner nuclear membrane of endothelial cells had a s.d. equal to 2.9% of the mean, whereas the surface density of the same membrane compartment-referenced to a cm3 of cells-had a s.d. at 19.1% of the mean. In this case, method 3 produced almost a sevenfold reduction in the standard deviation. Similar results were found for exocrine and duct cells. The results of the study indicate that the standard deviation of a stereological estimate can be reduced to a minimum by using a mean nuclear profile boundary to generate an estimate for a nuclear numerical density, which, in turn, can be combined with a surface density to obtain average cell information.

Animals↗