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Biomedical subjects

H J Gundersen

Publications and source records attributed to H J Gundersen.

At least 19 recordsLinked to original sources
PubMed2001

Total regional and global number of synapses in the human brain neocortex.

An estimator of the total number of synapses in neocortex of human autopsy brains based on unbiased stereological principles is described. Each randomly chosen cerebral hemisphere was stratified into the four major neocortical regions. Uniform sampling with a varying sampling fraction in each region of neocortex was performed. The total volume of each neocortical region was estimated using point counting according to Cavalieri's principle. The ethanolic phosphotungstic acid staining technique was modified for synapses in human autopsy brains. The numerical density of synapses in each neocortical region studied was estimated using the disector at the electron microscopical level. The total number of neocortical synapses in each region was estimated as the product of the total volume of neocortex and the numerical density of synapses. The influence of the postmortem fixation delay on the number of synapses was investigated in five large mammals (one dog, one cow, and three pigs), the brains of which were kept under conditions similar to those under which human corpses are normally kept. The apparent decrease of 3.9% in the numerical density of synapses in the large mammals following a 2-day fixation delay was not significant. The average total number of synapses in the neocortex of five young male brains was 164 x 10(12) (CV = 0.17). An analysis of the precision of the estimate of the total number of synapses in neocortex indicates that blocks represent both the major source of variation and the largest workload. Using eight blocks per brain the imprecision of the estimate is, however, only 66% of the total variance.

Adult↗
PubMed2001

A precise and efficient stereological method for determining murine lung metastasis volumes.

We have developed a computer-assisted stereological method based on unbiased principles for estimating metastasis volumes in mouse lungs. We evaluated this method using the transplantable Lewis lung carcinoma. Twenty-one days after subcutaneous inoculation of 10(6) Lewis lung cells into C57BL/6J mice, the mice had primary tumors with an average volume of 2300 mm(3). After perfusion fixation, the lungs were removed, embedded in OCT compound, snap-frozen, and processed for stereology. The metastasis volumes were estimated by application of the Cavalieri principle after evaluation of single sections from several evenly distributed tissue levels. The metastasis volume in a group of nine mice varied between 0.01 and 14.4 mm(3), with an average of 6.1 mm(3). The coefficient of variation was 0.9. The coefficient of error of the volume estimation was determined in five cases and varied from 0.08 to 0.23. Thus, the variation on the metastasis volumes that is achieved by this method contributes very little, 2.5%, to the total variance within the group of mice. In conclusion, we have developed an efficient and unbiased method to determine the metastasis burden in mouse lungs.

Animals↗
PubMed2001

Estimating the number of complex particles using the ConnEulor principle.

An unbiased counting rule for the number of topologically simple objects of any shape, size and distribution in 3D space is a pertinent problem in stereology. Combining the disector principle with the object's 3D Euler number makes possible number estimation, which until now has been obtainable only by exhaustive serial sections. The disector is a set of two sections where the object's profiles in one section are compared with its profiles on the neighbouring section, and the number of new 2D topological events is recorded. In a disector of known volume the sum of topological events is a direct estimate of the disector contribution to the total Euler number, which forms the basis for an ultimate number estimator in 3D, the ConnEulor. The method is illustrated by an electron microscopic study of the number of mitochondria in the exocrine cells of the pancreas.

Animals↗
PubMed2001

Tissue shrinkage and unbiased stereological estimation of particle number and size.

This paper is a review of the stereological problems related to the unbiased estimation of particle number and size when tissue deformation is present. The deformation may occur during the histological processing of the tissue. It is especially noted that the widely used optical disector may be biased by dimensional changes in the z-axis, i.e. the direction perpendicular to the section plane. This is often the case when frozen sections or vibratome sections are used for the stereological measurements. The present paper introduces new estimators to be used in optical fractionator and optical disector designs; the first is, as usual, the simplest and most robust. Finally, it is stated that when tissue deformation only occurs in the z-direction, unbiased estimation of particle size with several estimators is possible.

Brain↗
PubMed2001

Size of neocortical neurons in control subjects and in Alzheimer's disease.

The aim of the present study was to estimate mean neuronal volume and absolute size distributions of the neocortical neurons in brains from controls and AD patients using stereological methods based on unbiased principles to determine whether changes in absolute cell size are part of the neuropathological pattern of Alzheimer's disease. The neocortex of 8 patients with Alzheimer's disease (AD), mean age 81.1 (68-94) y was compared with 9 nondemented controls, mean age 80.9 (65-101) y. The brains came from Johns Hopkins University Hospital (JHUH) in Baltimore, USA, the Netherlands Brain Bank (NBB), and from a large brain repository in Denmark. The rotator method was used to obtain an estimate of cell volumes providing absolute size distributions of the volume of both cell perikaryon and cell nuclei. The geometric mean volume of cell nuclei in neocortical neurons was 328 microm3 (interindividual CV = 0.15) in the Alzheimer group compared with 277 microm3 (interindividual CV = 0.17) in controls which was a statistically significant increase (P = 0.049). The perikaryal volume was 1117 microm3 in the Alzheimer group compared with 999 microm3 in controls which was a nonsignificant difference (P = 0.20). There was a highly significant correlation between the nuclear and perikaryal volumes in all individuals. The average slope of the regression lines was significantly higher in the Alzheimer patients than in the controls, illustrating that nuclear hypertrophy was more pronounced in the largest neurons.

Aged↗
PubMed2000

Remodeling of hippocampal synapses after hippocampus-dependent associative learning.

The aim of this study was to determine whether hippocampus-dependent associative learning involves changes in the number and/or structure of hippocampal synapses. A behavioral paradigm of trace eyeblink conditioning was used. Young adult rabbits were given daily 80 trial sessions to a criterion of 80% conditioned responses in a session. During each trial, the conditioned (tone) and unconditioned (corneal airpuff) stimuli were presented with a stimulus-free or trace interval of 500 msec. Control rabbits were pseudoconditioned by equal numbers of random presentations of the same stimuli. Brain tissue was taken for morphological analyses 24 hours after the last session. Synapses were examined in the stratum radiatum of hippocampal subfield CA1. Unbiased stereological methods were used to obtain estimates of the total number of synapses in this layer as well as the area of the postsynaptic density. The data showed that the total numbers of all synaptic contacts and various morphological subtypes of synapses did not change in conditioned animals. The area of the postsynaptic density, however, was significantly increased after conditioning in axospinous nonperforated synapses. This structural alteration may reflect an addition of signal transduction proteins (such as receptors and ion channels) and the transformation of postsynaptically silent synapses into functional ones. The findings of the present study indicate that cellular mechanisms of hippocampus-dependent associative learning include the remodeling of existing hippocampal synapses. Further studies examining various time points along the learning curve are necessary to clarify the issue of whether these mechanisms also involve the formation of additional synaptic contacts.

Animals↗
PubMed2000

Non-uniform systematic sampling in stereology.

Non-uniform systematic sampling designs in stereology are studied. Various methods of constructing non-uniform systematic sampling points from prior knowledge of the measurement function are presented. As an example, we consider area estimation from lengths of linear intercepts. The efficiency of two area estimators, based on non-uniform sampling of parallel lines, is compared to that of the classical 2D Cavalieri estimator, based on uniform sampling, in a sample of planar profiles from transverse sections of 41 small myelinated axons. The comparison is based on simulations. It is concluded that for profiles of this type one of the non-uniform sampling schemes is more efficient than the traditional uniform sampling scheme. Other examples where non-uniform systematic sampling may be used are in area estimation from lines emanating from a fixed point, area estimation from concentric circles or spirals and curve length estimation from sweeping lines. It is shown that proportional-to-size sampling is a special case of non-uniform systematic sampling. Finally, the effect of noise in the observations is discussed.

Axons↗
PubMed2000

Quantitation of regional cerebral blood flow corrected for partial volume effect using O-15 water and PET: I. Theory, error analysis, and stereologic comparison.

Limited spatial resolution of positron emission tomography (PET) can cause significant underestimation in the observed regional radioactivity concentration (so-called partial volume effect or PVE) resulting in systematic errors in estimating quantitative physiologic parameters. The authors have formulated four mathematical models that describe the dynamic behavior of a freely diffusible tracer (H215O) in a region of interest (ROI) incorporating estimates of regional tissue flow that are independent of PVE. The current study was intended to evaluate the feasibility of these models and to establish a methodology to accurately quantify regional cerebral blood flow (CBF) corrected for PVE in cortical gray matter regions. Five monkeys were studied with PET after IV H2(15)O two times (n = 3) or three times (n = 2) in a row. Two ROIs were drawn on structural magnetic resonance imaging (MRI) scans and projected onto the PET images in which regional CBF values and the water perfusable tissue fraction for the cortical gray matter tissue (hence the volume of gray matter) were estimated. After the PET study, the animals were killed and stereologic analysis was performed to assess the gray matter mass in the corresponding ROIs. Reproducibility of the estimated parameters and sensitivity to various error sources were also evaluated. All models tested in the current study yielded PVE-corrected regional CBF values (approximately 0.8 mL x min(-1) x g(-1) for models with a term for gray matter tissue and 0.5 mL x min(-1) x g(-1) for models with a term for a mixture of gray matter and white matter tissues). These values were greater than those obtained from ROIs tracing the gray matter cortex using conventional H2(15)O autoradiography (approximately 0.40 mL x min(-1) x g(-1)). Among the four models, configurations that included two parallel tissue compartments demonstrated better results with regards to the agreement of tissue time-activity curve and the Akaike's Information Criteria. Error sensitivity analysis suggested the model that fits three parameters of the gray matter CBF, the gray matter fraction, and the white matter fraction with fixed white matter CBF as the most reliable and suitable for estimating the gray matter CBF. Reproducibility with this model was 11% for estimating the gray matter CBF. The volume of gray matter tissue can also be estimated using this model and was significantly correlated with the results from the stereologic analysis. However, values were significantly smaller compared with those measured by stereologic analysis by 40%, which can not be explained by the methodologic errors. In conclusion, the partial volume correction was essential in quantitation of regional CBF. The method presented in this article provided the PVE-corrected regional CBF in the cortical gray matter tissue. This study also suggests that further studies are required before using MRI derived anatomic information for PVE correction in PET.

Animals↗
PubMed1999

How accurate are measurements on MRI? A study on multiple sclerosis using reliable 3D stereological methods.

Unbiased stereological principles for quantifying plaque volume and number in multiple sclerosis (MS) are described, and practical problems with their implementation are discussed. Plaque volume was estimated using stereological methods on 3 mm brain magnetic resonance imaging (MRI). Volume estimates made from thick sections are biased. For convex particles as plaques, bias can be corrected by excluding the slice of maximal area from the estimate. Comparing the corrected with the uncorrected results, bias varied between 15 and 200%, depending on object size. By analogy, for the estimates of plaque, bias varied between 15 and 90%. The use of 1 mm slices reduces bias to a value close to zero and should be preferred when precise plaque measurements are required. An unbiased three-dimensional counting rule-the disector-was used to estimate plaque number. The coefficient of error of the estimates was calculated. Stereological methods applied to MRI provide efficient and reliable estimates of MS plaque load.

Adult↗
PubMed1999

The efficiency of systematic sampling in stereology--reconsidered.

In the present paper, we summarize and further develop recent reseach in the estimation of the variance of sterelogical estimators based on systematic sampling. In particular, it is emphasized that the relevant estimation procedure depends on the sampling density. The validity of the variance estimation is examined in a collection of data sets, obtained by systematic sampling. Practical recommendations are also provided in a separate section.

Analysis of Variance↗
PubMed1999

Pronounced loss of cell nuclei and anisotropic deformation of thick sections.

The local deformation and variations in section thickness are studied in 100-microm thick vibratome sections of well-fixed human brain tissue. During processing, including drying on glass slides, the section thickness is reduced to less than half, but close to the edges there is less shrinkage of the section thickness. Close to both surfaces there is a pronounced reduction in the number of neuronal nucleoli. At the scale of the original section, the upper 15 microm and the lower 10 microm are depleted. The loss is most pronounced at the upper surface, which is unprotected during processing. In the central 70% of the section height, where one would ordinarily use an optical disector for sampling, there is no indication of non-uniform shrinkage. The simplest explanation for the observed loss of nucleoli is that all cells opened by the knife may lose their nuclei across an unprotected section surface. The observations do not generalize to other tissues and other preparation techniques, but illustrate the magnitude of some of the problems for uniform sampling and unbiased estimation in very thick sections. The uniform optical disector sampling of nucleoli in thick sections, as opposed to that of cell nuclei, raises a special problem, which is discussed briefly.

Adult↗
PubMed1997

Neocortical neuron number in humans: effect of sex and age.

Modern stereological methods provide precise and reliable estimates of the number of neurons in specific regions of the brain. We decided to estimate the total number of neocortical neurons in the normal human brain and to analyze it with respect to the major macro- and microscopical structural components, to study the internal relationships of these components, and to quantitate the influence of important physiological variables on brain structure. The 94 brains reported represent a consecutive collection of brains from the general Danish population. The average numbers of neocortical neurons were 19 billion in female brains and 23 billion in male brains, a 16% difference. In our study, which covered the age range from 20 years to 90 years, approximately 10% of all neocortical neurons are lost over the life span in both sexes. Sex and age were the main determinants of the total number of neurons in the human neocortex, whereas body size, per se, had no influence on neuron number. Some of the data presented have been analyzed by using new mathematical designs. An equation predicting the total neocortical neuron number in any individual in which sex and age are known is provided.

Adult↗
PubMed1997

The optical rotator.

The optical rotator is an unbiased, local stereological principle for estimation of cell volume and cell surface area in thick, transparent slabs. The underlying principle was first described in 1993 by Kiêu & Jensen (J. Microsc. 170, 45-51) who also derived an estimator of length. In this study we further discuss the methods derived from this principle and present two new local volume estimators. The optical rotator benefits from information obtained in all three dimensions in thick sections but avoids over-/ underprojection problems at the extremes of the cell. Using computer-assisted microscopes the extra measurements demand minimal extra effort and make this estimator even more efficient when it comes to estimation of individual cell size than many of the previous local estimators. We demonstrate the principle of the optical rotator in an example (the cells in the dorsal root ganglion of the rat), evaluate its efficiency and compare it with other unbiased, local stereological principles available for estimation of cell volume and surface area.

Animals↗
PubMed1997

Three-dimensional estimation of the glandular volume, and of the number and volume of epithelial cells in two glands from the antral mucosa of five healthy volunteers.

Specimens of antral mucosa were taken from five healthy volunteers and processed for microscopic evaluation. Consecutive 50-micron-thick sections were cut to estimate the volume of two antral glands. Two glands in each section were followed throughout by projecting their profiles from two microscopes mounted in parallel. The glandular volume was estimated using Cavalieri's principle. The mean volume of epithelial cells was estimated by systematic random sampling of the sections with an optical disector of known sample volume. The total number of cells per gland was calculated after determination of the volume of the glands and the mean volume of the epithelial cells. The mean volume of the antral glands was 13.6 +/- 1.67.10(6) microns3, whilst the mean volume of the epithelial cells was 1256 +/- 240 microns3. The total number of epithelial cells per gland was 11216 +/- 1104. In conclusion, using stereological methods, the total number of cells as well as the cell volume and the volume of antral glands can be determined in routine biopsy specimens of human stomach provided that the total depth of the mucosa is present in the section.

Adult↗
PubMed1996

Unbiased stereological estimation of the total number of synapses in a brain region.

Modern stereological methods have been used to make unbiased estimates of the total number of synapses in the striatum radiatum of the hippocampal CA1 region of five rabbits. The approach used involved a two stage analysis and is generally applicable to all parts of the nervous system. During the first stage of the analysis, the reference volume was estimated by point counting, at the light microscope level, according to the Cavalieri principle. During the second stage, the numerical density of synapses was estimated with dissectors at the electron microscopic level. The total number of synapses was calculated as the product of the numerical density and the volume of the region. The sampling with points and dissectors was carried out in all three dimensions of the entire CA1 region in a manner that ensured that all parts of the region and all synapses within it had equal probabilities of being sampled. An analysis of the precision of the estimate of total synapse number has been performed in terms of the variances of volume and synaptic numerical density at different levels of sampling, i.e. at the level of points, sections, individual animals and group of animals. Detailed descriptions of the procedures used to estimate the total number of synapses, evaluate the precision of the estimates, and optimize the sampling scheme are provided.

Analysis of Variance↗
PubMed1996

If you assume, you can make an ass out of u and me': a decade of the disector for stereological counting of particles in 3D space.

The year 1984 was a watershed in stereology. It saw the introduction of highly efficient and unbiased design-based methods for counting the number of arbitrary objects in 3-dimensional (3D) space using 2D sectional images. The only requirement is that the objects be unambiguously identifiable on parallel sections or successive focal planes. The move away from the ¿assumption-based' and ¿model-based' methods applied previously has been a major scientific advance. It has led to the resolution of several problems in different biomedical areas. The basic principle which makes possible 3D counting from sections is the disector. Here, we review the disector principle and consider its impact on the counting and sizing of biological particles. From now on, there can be no excuse for applying the biased counting methods of yesteryear. Their continued use, despite the availability of unbiased alternatives, should be seen as paying homage to History rather than advancing Science.

Animals↗
PubMed1995

Age and sex do not affect the volume, cell numbers, or cell size of the suprachiasmatic nucleus of the rat: an unbiased stereological study.

The circadian rhythms displayed by numerous biological functions are known to be sex specific and affected by aging. It has not been settled yet whether the sex- and age-related characteristics of circadian rhythms derive from changes in the anatomy of the suprachiasmatic nucleus. To shed light on these issues, we applied unbiased stereological techniques to estimate the volume of the suprachiasmatic nucleus as well as the total number of its cells and the mean volume of their somata and nuclei in progressively older groups of male and female Wistar rats (aged 1, 6, 12, 18, 24, and 30 months). The volume of the nucleus was estimated with the Cavalieri principle on serial sections. The total numbers of neurons and astrocytes were estimated by applying the optical fractionator, and the mean somatic and nuclear volumes of cells were estimated by using isotropic, uniform random sections and the nucleator method. On average, the volume of the suprachiasmatic nucleus was 0.044 mm3, and the total number of neurons and astrocytes was 17,400. Cells of the dorsomedial and ventrolateral components of the nucleus, which are morphologically different, have identical mean perikaryal and nuclear volumes, which we estimated to be 750 microns3 and 400 microns3, respectively. We further demonstrated that, at all ages analysed, the volume of the suprachiasmatic nucleus, the total cell number, and the mean somatic and nuclear volumes of its cells are affected neither by the age nor by the sex of the animal, regardless of the presence of sex- and age-related variations in circadian rhythms. However, the possibility that females may display changes in the volume of the suprachiasmatic nucleus at older ages cannot be ruled out. No effect of aging was observed in the total number of neurons or in the total number of astrocytes.

Age Factors↗