Design-based Stereological Methods Research Articles

Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy.

Stereological methods for estimating the 3D particle size and density from 2D projections are essential to many research fields. These methods are, however, prone to errors arising from undetected particle profiles due to sectioning and limited resolution, known as 'lost caps'. A potential solution developed by Keiding, Jensen, and Ranek in 1972, which we refer to as the Keiding model, accounts for lost caps by quantifying the smallest detectable profile in terms of its limiting 'cap angle' (ϕ), a size-independent measure of a particle's distance from the section surface. However, this simple solution has not been widely adopted nor tested. Rather, model-independent design-based stereological methods, which do not explicitly account for lost caps, have come to the fore. Here, we provide the first experimental validation of the Keiding model by comparing the size and density of particles estimated from 2D projections with direct measurement from 3D EM reconstructions of the same tissue. We applied the Keiding model to estimate the size and density of somata, nuclei and vesicles in the cerebellum of mice and rats, where high packing density can be problematic for design-based methods. Our analysis reveals a Gaussian distribution for ϕ rather than a single value. Nevertheless, curve fits of the Keiding model to the 2D diameter distribution accurately estimate the mean ϕ and 3D diameter distribution. While systematic testing using simulations revealed an upper limit to determining ϕ, our analysis shows that estimated ϕ can be used to determine the 3D particle density from the 2D density under a wide range of conditions, and this method is potentially more accurate than minimum-size-based lost-cap corrections and disector methods. Our results show the Keiding model provides an efficient means of accurately estimating the size and density of particles from 2D projections even under conditions of a high density.

Morphometric study of the testis in sheep embryos using unbiased design-based stereology.

Sheep have been used as translational models of human postnatal testicular development. However, the morphometric features of the normal developing testis in sheep embryos have not been previously investigated using stereology. The objective of the present work was to establish normal quantitative parameters for fetal testicular tissue components in sheep, using unbiased design-based stereological methods. Twenty-four sheep embryos were divided into four gestational age groups (9-11, 12-14, 15-17 and 18-20weeks of gestation) on the basis of the embryos' crown-rump length. Isotropic, systematic uniform random sections of the left testes were obtained by employing the orientator method. Testicular total volume, the absolute and proportional volumes occupied by the seminiferous tubules and interstitial tissue, as well as the seminiferous tubule length, were estimated using the point-counting system and the unbiased counting frame principle. All the parameters, with the exception of the interstitial tissue's fractional volume, gradually increased along with gestational age, with the maximum increase especially seen in the late fetal stages. The proportional volume of the interstitial tissue, on the other hand, showed a decreasing trend along with increasing gestational age. The absolute volume of the testes, of the seminiferous tubules and of the interstitial tissue, and the length of the seminiferous tubules showed a significant (p<0.05) positive linear correlation with gestational age. Several similarities were observed with human testicular embryogenesis. The stereological data emerging from the present study might prove useful as basic contribution to the fields of andrology and embryology and stimulate further research in these areas.

Stereologic Investigation of Mastoid Air Cell Geometry: Volume, Surface Area, and Anisotropy.

To assess mastoid geometry using computed tomography (CT) scans and design-based stereological methods. The anatomical organization of the mastoid air cell system (MACS) remains debated. Geometrical parameters have previously been determined by automated image-analysis algorithms. Stereology is an alternative approach, which has previously been applied to estimate the volume of the MACS, but has not previously been used to estimate surface area or study anisotropy. Twenty-three clinical CT scans of aerated temporal bones obtained from various ENT patients were studied. The structural orientation and anisotropy of the MACS was investigated by test-grid rotation and rose plots. Volume, surface area, and surface area-to-volume ratio were estimated with design-based stereology. Anisotropy of the mastoid air cells was demonstrated by a significant difference in surface area estimates between the axial and coronal planes (p = 0.0065). Rose plots illustrated variances in surface area estimates with different grid rotations, and a minimum value in the craniocaudal direction was shown. Sampling in the axial plane provided the least variance due to anisotropy. The mean (±SD) volume and surface area estimates were 5.71 ± 2.98 cm and 117 cm ± 60 cm, respectively. A large biological variation was noted. The mean (±SD) surface-to-volume ratio was 20.6 ± 2.8 cm. The stereological technique proved to be a robust method for volume and surface area estimation in clinical CT scans. The mastoid air cells constitute an anisotropic cell-system that seems to have a predominant orientation in the craniocaudal direction.

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on mice myocardial morphology

ABSTRACT: Studies have demonstrated sympathetic cardiac denervation in the MPTP mouse model. MPTP toxicity causes sympathetic nerve damage and depletion of heart norepinephrine. Previous evaluations of impairments in heart innervation have been based on imaging, electrophysiological and biochemical methods. However, these studies lacked information that can be obtained from morphoquantitative analyses. Thus, this study aimed to apply a design-based stereological method for evaluating the morphoquantitative alterations of myocardium following treatment with the neurotoxin MPTP in the C57/BL mouse. Our results showed that MPTP reduced the number of cardiomyocytes in the left ventricle.

Global Trends in Application of Stereology as a Quantitative Tool in Biomedical Research.

Stereology is a quantitative and comparative method that utilizes planes, lines, and points for the estimation of three-dimensional parameters in morphological studies. It primarily focuses on geometrical features of objects such as number, density, length, area, and volume. A scientometric study was conducted to analyze global research trends in application of stereology in biomedical research. Stereology has gained wide application resulting into design-based stereological methods. Data for this study were retrieved from the SCOPUS database. At least 5,732 publications employing stereology as analytical tool were produced in a period of 50 years between 1966 and 2016. Half (2,858; 49.87%) of these publications were produced in the last 12 years from 2005 to 2016. The relative growth rate (RGR) of publications decreased from 1967 (0.69) to 2016 (0.03) whereas the doubling time (DT) increased from 1.00 to 20.56 in the same period. A great majority (5,332; 93.02%) of the publications retrieved from SCOPUS were journal articles in various biomedical fields. The Journal of Microscopy tops the list of journals with at least 205 articles. The most productive country was USA with at least 1663 (23.10%) publications and Aarhus Universitet tops the list of institutions with at least 306 publications. J.R. Nyengaard was the most prolific author who contributed at least 125 publications. The highly cited article had a total of 2,054 citations with an average of over 82 citations per year. Given the growing importance of stereology in biomedical research, it is necessary to promote its application among scholars.

The quantification of testicular cells during the postnatal development in two Caviomorph rodents: the guinea pig (Cavia porcellus) and the cutia (Dasyprocta agouti).

The germinative, Sertoli and Leydig cells of two caviomorph rodents (Cavia porcellus and Dasyprocta agouti) were counted as well as the estimation of the total volume of the testis and the total volume of seminiferous tubules and interstitium in prepubertal, pubertal and adult animals. The number of spermatogonia, spermatocytes and spermatids cells increased during the pubertal phase in both rodents, notably the spermatid cells. The spermatocyte and spermatid slightly decreased in the adult of both rodents, but the increment in spermatogonia cells number was seen, mainly in cutias. The number of Sertoli cells increased in pubertal rodents, but in the adult the number reduced. Substantial number of Leydig cells was counted in pubertal and adult guinea pigs. In cutias, the number of Leydig cells increased in pubertal phase and decline in adults. The design-based stereological method has proven to be unbiased and reliable to be applied in reproduction studies.

Stereological analysis of size and density of hepatocytes in the porcine liver.

The porcine liver is frequently used as a large animal model for verification of surgical techniques, as well as experimental therapies. Often, a histological evaluation is required that include measurements of the size, nuclearity or density of hepatocytes. Our aims were to assess the mean number-weighted volume of hepatocytes, the numerical density of hepatocytes, and the fraction of binuclear hepatocytes (BnHEP) in the porcine liver, and compare the distribution of these parameters among hepatic lobes and macroscopic regions of interest (ROIs) with different positions related to the liver vasculature. Using disector and nucleator as design-based stereological methods, the morphometry of hepatocytes was quantified in seven healthy piglets. The samples were obtained from all six hepatic lobes and three ROIs (peripheral, paracaval and paraportal) within each lobe. Histological sections (thickness 16μm) of formalin-fixed paraffin-embedded material were stained with the periodic acid-Schiff reaction to indicate the cell outlines and were assessed in a series of 3-μm-thick optical sections. The mean number-weighted volume of mononuclear hepatocytes (MnHEP) in all samples was 3670±805μm3 (mean±SD). The mean number-weighted volume of BnHEP was 7050±2550μm3 . The fraction of BnHEP was 4±2%. The numerical density of all hepatocytes was 146997±15738 cellsmm-3 of liver parenchyma. The porcine hepatic lobes contained hepatocytes of a comparable size, nuclearity and density. No significant differences were identified between the lobes. The peripheral ROIs of the hepatic lobes contained the largest MnHEP with the smallest numerical density. The distribution of a larger MnHEP was correlated with a larger volume of BnHEP and a smaller numerical density of all hepatocytes. Practical recommendations for designing studies that involve stereological evaluations of the size, nuclearity and density of hepatocytes in porcine liver are provided.

Neocortical Development in Brain of Young Children-A Stereological Study.

The early postnatal development of neuron and glia numbers is poorly documented in human brain. Therefore we estimated using design-based stereological methods the regional volumes of neocortex and the numbers of neocortical neurons and glial cells for 10 children (4 girls and 6 boys), ranging from neonate to 3 years of age. The 10 infants had a mean of 20.7 × 109 neocortical neurons (range 18.0-24.8 × 109) estimated with a coefficient of variation (CV) = 0.11; this range is similar to adult neuron numbers. The glia populations were 10.5 × 109 oligodendrocytes (range 5.0-16.0 × 109; CV = 0.40); 5.3 × 109 astrocytes (range 2.7-8.3 × 109, CV = 0.39); and 0.32 × 109 microglia (range 0.15-0.43 × 109, CV = 0.31). Thus, the estimated mean composite number of neocortical neuron and glial cells was 36.8 × 109 (range26.8-48.3 × 109, CV = 0.21), of which approximately one-half were glial cells. There was a significant linear increase in oligodendrocyte and astrocyte numbers during the first 3 years of life, but no change in the total number of neurons. This is in line with our expectation that the total number of neocortical neurons is already determined in mid-fetal life.

First and second order stereology of hyaline cartilage: Application on mice femoral cartilage

Stereological techniques could be considered in research on cartilage to obtain quantitative data. The present study aimed to explain application of the first- and second-order stereological methods on articular cartilage of mice and the methods applied on the mice exposed to cadmium (Cd).The distal femoral articular cartilage of BALB/c mice (control and Cd-treated) was removed. Then, volume and surface area of the cartilage and number of chondrocytes were estimated using Cavalieri and optical dissector techniques on isotropic uniform random sections. Pair-correlation function [g(r)] and cross-correlation function were calculated to express the spatial arrangement of chondrocytes–chondrocytes and chondrocytes–matrix (chondrocyte clustering/dispersing), respectively.The mean±standard deviation of the cartilage volume, surface area, and thickness were 1.4±0.1mm3, 26.2±5.4mm2, and 52.8±6.7μm, respectively. Besides, the mean number of chondrocytes was 680±200 (×103). The cartilage volume, cartilage surface area, and number of chondrocytes were respectively reduced by 25%, 27%, and 27% in the Cd-treated mice in comparison to the control animals (p<0.03). Estimates of g(r) for the cells and matrix against the dipole distances, r, have been plotted. This plot showed that the chondrocytes and the matrix were neither dispersed nor clustered in the two study groups.Application of design-based stereological methods and also evaluation of spatial arrangement of the cartilage components carried potential advantages for investigating the cartilage in different joint conditions. Chondrocyte clustering/dispersing and cellularity can be evaluated in cartilage assessment in normal or abnormal situations.

Absolute number of parvicellular and magnocellular neurons in the red nucleus of the rat midbrain: a stereological study.

The absolute number of parvicellular and magnocellular neurons in the red nucleus was estimated using design-based stereological counting methods and systematic random sampling techniques. Six young adult male rats, and a complete set of serial 40-μm glycolmethacrylate sections for each rat, were used to quantify neuronal numbers. After a random start, a systematic subset (i.e. every third) of the serial sections was used to estimate the total volume of the red nucleus using Cavalieri's method. The same set of sampled sections was used to estimate the number of neurons in a known subvolume (i.e. the numerical density Nv ) by the optical disector method. Multiplication of the total volume by Nv yielded the absolute number of neurons. It was found that the right red nucleus consisted, on average, of 8400 parvicellular neurons (with a coefficient of variation of 0.16) and 7000 magnocellular neurons (0.12). These total neuronal numbers provide important data for the transfer of information through these nuclei and for species comparisons.

Elderly Female Rhesus Macaques Preserve Lung Alveoli With Estrogen/Progesterone Therapy.

The aging lung is associated with increased susceptibility to chronic inflammatory diseases such as chronic obstructive pulmonary disease where females have been reported to be more susceptible than males. The changes in reproductive hormones due to aging may directly or indirectly affect lung structure and function and little is known on the mechanism of these changes. Twenty female rhesus macaques were divided into four groups. Ovariectomy (OVX) was performed on eight animals with three receiving estrogen/progesterone therapy (HRT) and five animals given implants containing vehicle. The remaining 12 animals represented control groups of ages 10-14 years (n = 6) and ages 20-24 (n = 6). A design-based stereological method was employed to estimate the number of alveoli in the right middle lung lobe along with hormone analysis for possible correlation. A significant decrease was found in the number of alveoli in the vehicle OVX animals compared to intact younger adult females (P < 0.001). A significant increase in alveoli between OVX vehicle animals and those on HRT was also found (P < 0.0001). There was difference in the number of alveoli between younger adult animals and animals on HRT. The loss of ovaries and hormones had a significant effect on alveolar lung morphology. This result mimics what is seen in the aging process and could contribute to gender differences reported in the elderly. Hormone replacement, as reported here, could possibly slow the loss of alveoli due to the aging process or aid in alveolar regeneration through direct or indirect mechanisms. Anat Rec, 299:973-978, 2016. © 2016 Wiley Periodicals, Inc.

Stereological assessment of normal Persian squirrels (Sciurus anomalus) kidney.

The functions of the mammalian kidney are closely related to its structure. This suggests that renal function can be completely characterized by accurate knowledge of its quantitative morphological features. The aim of this study was to investigate the histomorphometric features of the kidney using design-based and unbiased stereological methods in the Persian squirrel (Sciurus anomalus), which is the only representative of the Sciuridae family in the Middle East. The left kidneys of five animals were examined. Total volume of the kidney, cortex, and medulla were determined to be 960.75±87.4, 754.31±77.09 and 206.1±16.89mm3, respectively. The glomerular number was 32844.03±1069.19, and the total glomerular volume was estimated to be 36.7±1.45mm3. The volume and length of the proximal convoluted tubule were estimated at 585.67±60.7mm3 and 328.8±14.8m, respectively, with both values being greater than those reported in the rat kidney. The volume and length of the distal convoluted tubule were calculated at 122.34±7.38mm3 and 234.4±17.45m, respectively, which are also greater than those reported in the rat kidney. Despite the comparable body weight, the total number and mean individual volume of glomeruli in the Persian squirrel kidney were greater than those in the rat kidney. Overall, the stereological variables of the kidneys elucidated in this study are exclusive to the Persian squirrel. Our findings, together with future renal physiological data, will contribute to a better understanding of the renal structure-function relationship in the Persian squirrel.

Neocortical Neuronal Loss in Patients with Multiple System Atrophy: A Stereological Study.

To determine the extent of neocortical involvement in multiple system atrophy (MSA), we used design-based stereological methods to estimate the total numbers of neurons, oligodendrocytes, astrocytes, and microglia in the frontal, parietal, temporal, and occipital cortex of brains from 11 patients with MSA and 11 age- and gender-matched control subjects. The stereological data were supported by cell marker expression analyses in tissue samples from the prefrontal cortex. We found significantly fewer neurons in the frontal and parietal cortex of MSA brains compared with control brains. Significantly more astrocytes and microglia were observed in the frontal, parietal, and temporal cortex of MSA brains, whereas no change in the total number of oligodendrocytes was seen in any of the neocortical regions. There were significantly fewer neurons in the frontal cortex of MSA patients with impaired executive function than in patients with normal executive function. Our results indicate that the involvement of the neocortex in MSA is far more widespread and substantial than previously thought. In addition, our results suggest that the increasingly recognized cognitive impairment in MSA may be related to neuronal loss in the frontal cortex.

Physical activity ameliorates neuron loss and memory deficits in Tg4-42 mice

N-truncated Aβ4–42 is one of the most abundant Aβ peptide species in the brain of sporadic Alzheimerʼs disease (AD) patients. We have recently developed a transgenic mouse model (Tg4–42) expressing Aβ4–42 without mutations under the control of the neuron-specific Thy1-promotor. These mice develop age-dependent deficits in spatial reference memory accompanied by robust CA1 pyramidal neuron loss. Using an enriched environment paradigm, the impact of enhanced physical activity on hippocampal neuron loss and behavioral deficits was assessed. In this study, Tg4–42 mice were housed under standard conditions or in enriched cages equipped with various objects and running wheels. The number of CA1 pyramidal and dentate gyrus (DG) granule cell layer neurons was assessed using design-based stereological methods. In addition, spatial reference memory using the Morris Water Maze task, as well as neurogenesis in the subgranular zone (SGZ) was analyzed. In addition to CA1 neuron loss, homozygous Tg4–42 at 6 months showed a ~ 45% decrease in SGZ neurogenesis, accompanied by a ~ 20% neuron loss in DG. Housing under enrichment conditions maintained both neurogenesis and granule cell neuron loss, while CA1 neuron loss and spatial reference memory deficits were partially rescued. Amelioration of CA1 neuron loss and working memory deficits could be also shown in 12-month-old hemizygous Tg4–42 animals. We provide evidence that enhanced physical activity due to environmental enrichment leads to an amelioration of neuronal loss and behavioral deficits in a transgenic mouse model reflecting the sporadic form of AD. This might indicate the relevance of increased physical activity as a potential strategy in the prevention of dementia.

Subchondral bone turnover, but not bone volume, is increased in early stage osteoarthritic lesions in the human hip joint

The pathogenesis of osteoarthritis (OA) is not fully understood, but bone changes are suggested to be important. Bone turnover and bone volume (BV) in human hip OA were investigated in relation to the overlying cartilage degeneration using design-based stereological estimators. Femoral heads were obtained from 25 end-stage OA patients and 24 controls (CTL). Design-based stereological methods were used for sampling and quantification to obtain absolute estimates of volume and surface in the central trabecular and the subarticular bone region. The subarticular bone was further subdivided into regions according to the OARSI-score of the overlying articular cartilage in which erosion and osteoid surfaces were estimated. In the subarticular region, bone volume (BV/TV) was 15.0% higher in OA patients compared to CTL; The fraction of erosive (ES/BS) and osteoid surfaces (OS/BS) were 56.2% and 72.8% higher in OA compared to CTL. In subarticular regions with none to mild cartilage degeneration (OARSI grade 0-2), ES/BS and OS/BS were 48.6% and 59.9% higher in OA compared to CTL, whereas BV/TV did not differ between OA and CTL. In human end-stage hip OA, BV and bone turnover correlate with the degree of local cartilage degeneration. Subarticular bone sclerosis was only present in regions corresponding to end-stage OA. However, in regions with only none to mild cartilage degeneration the underlying bone had significantly higher turnover in OA patients compared to the control group, suggesting that high bone turnover may contribute to the early pathogenesis of OA.

Counting glomeruli and podocytes: rationale and methodologies.

There is currently much interest in the numbers of both glomeruli and podocytes. This interest stems from a greater understanding of the effects of suboptimal fetal events on nephron endowment, the associations between low nephron number and chronic cardiovascular and kidney disease in adults, and the emergence of the podocyte depletion hypothesis. Obtaining accurate and precise estimates of glomerular and podocyte number has proven surprisingly difficult. When whole kidneys or large tissue samples are available, design-based stereological methods are considered gold standard because they are based on principles that negate systematic bias. However, these methods are often tedious and time consuming, and oftentimes inapplicable when dealing with small samples such as biopsies. Therefore, novel methods suitable for small tissue samples, and innovative approaches to facilitate high throughput measurements, such as MRI to estimate glomerular number and flow cytometry to estimate podocyte number, have recently been described. This review describes current gold-standard methods for estimating glomerular and podocyte number, as well as methods developed in the past 3 years. We are now better placed than ever before to accurately and precisely estimate glomerular and podocyte number, and examine relationships between these measurements and kidney health and disease.

Discrepancies in quantitative assessment of normal and regenerated peripheral nerve fibers between light and electron microscopy.

Quantitative estimation of myelinated nerve fiber number, together with fiber size parameters, is one of the most important tools for nerve regeneration research. In this study we used a design-based stereological method to evaluate the regenerative process in two experimental paradigms: crush injury and autograft repair. Samples were embedded in resin and morphometric counting and measurements were performed using both light and electron microscopes. Results show a significant difference in myelinated fiber number estimation between light and electron microscopes, especially after autograft repair; light microscope significantly underestimates the number of fibers because of the large number of very small axons that can be detected only in electron microscope. The analysis of the size parameters also shows a higher number of small fibers in electron microscopic analysis, especially in regenerated nerves. This comparative study shows that the integration of data obtained in light microscope with those obtained in electron microscope is necessary in revealing very small myelinated fibers that cannot be detected otherwise. Moreover, the difference in the estimation of total number of myelinated fibers between light and electron microscopes must be considered in data analysis to ensure accurate interpretation of the results.

Development of the Cell Population in the Brain White Matter of Young Children.

While brain gray matter is primarily associated with sensorimotor processing and cognition, white matter modulates the distribution of action potentials, coordinates communication between different brain regions, and acts as a relay for input/output signals. Previous studies have described morphological changes in gray and white matter during childhood and adolescence, which are consistent with cellular genesis and maturation, but corresponding events in infants are poorly documented. In the present study, we estimated the total number of cells (neurons, oligodendrocytes, astrocytes, and microglia) in the cerebral white matter of 9 infants aged 0-33 months, using design-based stereological methods to obtain quantitative data about brain development. There were linear increases with age in the numbers of oligodendrocytes (7-28 billion) and astrocytes (1.5-6.7 billion) during the first 3 years of life, thus attaining two-thirds of the corresponding numbers in adults. The numbers of neurons (0.7 billion) and microglia (0.2 billion) in the white matter did not increase during the first 3 years of life, but showed large biological variation.

Morpho-functional characteristics of rat fetal thyroid gland are affected by prenatal dexamethasone exposure

Thyroid hormones (TH) and glucocorticoids strongly contribute to the maturation of fetal tissues in the preparation for extrauterine life. Influence of maternal dexamethasone (Dx) administration on thyroid glands morpho-functional characteristics of near term rat fetuses was investigated applying unbiased stereology. On the 16th day of pregnancy dams received 1.0mg/Dx/kg/b.w., followed by 0.5mg/Dx/kg/b.w. on the 17th and 18th days of gestation. The control females received the same volume of saline. The volume of fetal thyroid was estimated using Cavalieri’s principle; the physical/fractionator design was applied for the determination of absolute number of follicular cells in mitosis and immunohistochemically labeled C cells; C cell volume was measured using the planar rotator. The functional activity of thyroid tissue was provided from thyroglobulin (Tg) and thyroperoxidase (TPO) immunohistochemical staining. Applying these design-based modern stereological methods it was shown that Dx treatment of gravid females led to a significant decrease of fetal thyroid gland volume in 19- and 21-day-old fetuses, due to decreased proliferation of follicular cells. The Tg and TPO immunohistochemistry demonstrated that intensive TH production starts and continues during the examined period in control and Dx-exposed fetuses. Under the influence of Dx the absolute number of C cells was lower in both groups of near term fetuses, although unchanged relation between the two populations of endocrine cells, follicular and C cells suggesting that structural relationships within the gland are preserved. In conclusion maternal glucocorticoid administration at the thyroid gland level exerts growth-inhibitory and maturational promoting effects in near term rat fetuses.

Estimating glomerular number: Why we do it and how

There is currently much interest in determining the number of glomeruli, and thereby nephrons, in the kidney. Researchers have been trying to count glomeruli since the 19th century and currently four general approaches are available: (i) acid maceration; (ii) counting glomerular profiles in histological sections; (iii) model-based stereology; and (iv) design-based stereology. Although design-based stereological methods are generally considered the gold-standard method, all current methods have limitations. A new approach using magnetic resonance imaging has recently been described and may ultimately enable glomerular imaging and quantification in vivo. This report considers the advantages and disadvantages of current methods for counting glomeruli and describes the new magnetic resonance approach. In addition, a method for counting glomeruli in developing kidneys is described.