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Design-based stereology is the gold standard for obtaining unbiased quantitative morphological data on volume, surface area, and length, as well as the number of tissues, cells or organelles. In cardiac research, the introduction of a stereological method to unbiasedly estimate the number of cardiomyocytes has considerably increased the use of stereology. Since its original description, various modifications to this method have been described. A particular field in which this method has been employed is the normal developmental life cycle of cardiomyocytes after birth, and particularly the question of when, during postnatal development, cardiomyocytes lose their capacity to divide and proliferate, and thus their inherent regenerative ability. This field is directly related to a second major application of stereology in recent years, addressing the question of what consequences intrauterine growth restriction has on the development of the heart, particularly of cardiomyocytes. Advances have also been made regarding the quantification of nerve fibers and collagen deposition as measures of heart innervation and fibrosis. In the present review article, we highlight the methodological progress made in the last 20 years and demonstrate how stereology has helped to gain insight into the process of normal cardiac development, and how it is affected by intrauterine growth restriction. [ABSTRACT FROM AUTHOR]

Obesity is a risk factor of cardiopulmonary disorders including left and right ventricular dysfunction and pulmonary hypertension (PH), and PH is associated with right ventricular (RV) hypertrophy and failure. Here, we tested the hypothesis that alterations of the RV capillary network under PH induced by chronic hypoxia are aggravated by alimentary obesity, thereby representing a predisposition for subsequent RV dysfunction. Male, 6-week-old C57BL/6N mice were assigned to one of the following groups: control diet (CD), CD/hypoxia (CD-Hyp), high-fat diet (HFD), HFD/hypoxia (HFD-Hyp). Mice were fed CD or HFD for 30 weeks, CD-Hyp and HFD-Hyp mice were exposed to normobaric hypoxia (13 % O 2) during the last 3 weeks of the experiments. Hearts were prepared for light and electron microscopy and right atria and RVs were analyzed by design-based stereology. HFD and hypoxia independently increased RV and cardiomyocyte volume. These changes were further enhanced in HFD-Hyp. The ratio between RV and body weights was similar in CD and HFD but enhanced in both hypoxia groups to a similar extent. The total length of capillaries was elevated in proportion with the RV hypertrophy, thus the area of myocardium supplied by an average capillary was similar in all groups. Similarly, the thickness of the capillary endothelium was not altered by HFD or hypoxia. In conclusion, in experimental PH capillaries of the RV myocardium showed similar adaptations in lean and obese mice. Thus, under chronic hypoxic conditions, obesity had no adverse effect on the capillarization of the right ventricle. • Obesity is associated with pulmonary hypertension (PH). Information about the additive role of obesity in PH is rare. • We utilized a clinically relevant mouse model of alimentary, pre-diabetic obesity and experimental PH. • Chronic hypoxia induced hypertrophy of the right ventricle and microvascular alterations irrespective of body weight. • Thus, microvascular adaptations of the right ventricle are not aggravated by obesity. [ABSTRACT FROM AUTHOR]

Alterations of the pulmonary vasculature are an important feature of human lung diseases such as chronic obstructive pulmonary disease, pulmonary hypertension, and bronchopulmonary dysplasia. Experimental studies to investigate the pathogenesis or a therapeutic intervention in animal models of these diseases often require robust, meaningful, and efficient morphometric data that allow for appropriate statistical testing. The gold standard for obtaining such data is design-based stereology. However, certain morphological characteristics of the pulmonary vasculature make the implementation of stereological methods challenging. For example, the alveolar capillary network functions according to the sheet flow principle, thus making unbiased length estimations impossible and requiring other strategies to obtain mechanistic morphometric data. Another example is the location of pathological changes along the branches of the vascular tree. For developmental defects like in bronchopulmonary dysplasia or for pulmonary hypertension, it is important to know whether certain segments of the vascular tree are preferentially altered. This cannot be overcome by traditional stereological methods but requires the combination of a three-dimensional data set and stereology. The present review aims at highlighting the great potential while discussing the major challenges (such as time consumption and data volume) of this combined approach. We hope to raise interest in the potential of this approach and thus stimulate solutions to overcome the existing challenges. [ABSTRACT FROM AUTHOR]

Left ventricular (LV) hypertrophy in response to hypertension and increased afterload frequently progresses to heart failure. It is under debate whether the loss of cardiomyocytes contributes to this transition. To address this question, female C57 BL/6 wild-type mice were subjected to transverse aortic constriction ( TAC) and developed compensated LV hypertrophy after 1 week, which progressed to heart failure characterized by reduced ejection fraction and pulmonary congestion 4 weeks post- TAC. Quantitative, design-based stereology methods were used to estimate number and mean volume of LV cardiomyocytes. DNA strand breaks were visualized using the TUNEL method 6 weeks post- TAC to quantify the number of apoptotic cell nuclei. The volume of the LV myocardium as well as the cardiomyocyte mean volume increased progressively after TAC. In contrast, the number of LV cardiomyocytes remained constant 1 and 4 weeks post- TAC in comparison to sham-operated mice. Moreover, there was no significant difference in the number of cardiomyocyte nuclei stained for DNA strand breaks at 6 weeks post- TAC. It was concluded that the loss of cardiomyocytes is not required for the transition from compensated hypertrophy to heart failure induced by TAC in the female murine heart. [ABSTRACT FROM AUTHOR]

Design-based stereology is the gold standard of morphometry in lung research. Here, we analyze the current use of morphometric and stereological methods in lung research and provide an overview on recent methodological developments and biological observations made by the use of stereology. Based on this analysis we hope to provide useful recommendations for a good stereological practice to further the use of advanced and unbiased stereological methods. [ABSTRACT FROM AUTHOR]

Changes in body mass due to varying amounts of calorie intake occur frequently with obesity and anorexia/cachexia being at opposite sides of the scale. Here, we tested whether a high-fat diet or calorie restriction ( CR) decreases the number of cardiac myocytes and affects their volume. Ten 6-8-week-old mice were randomly assigned to a normal (control group, n = 5) or high-fat diet (obesity group, n = 5) for 28 weeks. Ten 8-week-old mice were randomly assigned to a normal (control group, n = 5) or CR diet (CR group, n = 5) for 7 days. The left ventricles of the hearts were prepared for light and electron microscopy, and analysed by design-based stereology. In CR, neither the number of cardiac myocytes, the relationship between one- and multinucleate myocytes nor their mean volume were significantly different between the groups. In contrast, in the obese mice we observed a significant increase in cell size combined with a lower number of cardiomyocytes ( P < 0.05 in the one-sided U-test) and an increase in the mean number of nuclei per myocyte. The mean volume of myofibrils and mitochondria per cardiac myocyte reflected the hypertrophic and hypotrophic remodelling in obesity and CR, respectively, but were only significant in the obese mice, indicating a more profound effect of the obesity protocol than in the CR experiments. Taken together, our data indicate that long-lasting obesity is associated with a loss of cardiomyocytes of the left ventricle, but that short-term CR does not alter the number of cardiomyocytes. [ABSTRACT FROM AUTHOR]

Pulmonary lipofibroblasts are thought to be involved in lung development, regeneration, vitamin A storage, and surfactant synthesis. Most of the evidence for these important functions relies on mouse or rat studies. Therefore, the present study was designed to investigate the presence of lipofibroblasts in a variety of early postnatal and adult mammalian species (including humans) to evaluate the ability to generalize functions of this cell type for other species. For this purpose, lung samples from 14 adult mammalian species as well as from postnatal mice, rats, and humans were investigated using light and electron microscopic stereology to obtain the volume fraction and the total volume of lipid bodies. In adult animals, lipid bodies were observed only, but not in all rodents. In all other species, no lipofibroblasts were observed. In rodents, lipid body volume scaled with body mass with an exponent b 0.73 in the power law equation. Lipid bodies were not observed in postnatal human lungs but showed a characteristic postnatal increase in mice and rats and persisted at a lower level in the adult animals. Among 14 mammalian species, lipofibroblasts were only observed in rodents. The great increase in lipid body volume during early postnatal development of the mouse lung confirms the special role of lipofibroblasts during rodent lung development. It is evident that the cellular functions of pulmonary lipofibroblasts cannot be transferred easily from rodents to other species, in particular humans. [ABSTRACT FROM AUTHOR]

Summary: The vasculature of the heart is able to adapt to various physiological and pathological stimuli and its failure to do so is well-reflected by the great impact of ischaemic heart disease on personal morbidity and mortality and on the health care systems of industrial countries. Studies on physiological or genetic interventions as well as therapeutic angiogenesis rely on quantitative data to characterize the effects in a statistically robust way. The gold standard for obtaining quantitative morphological data is design-based stereology which allows the estimation of volume, surface area, length and number of blood vessels as well as their thickness, diameter or wall composition. Unfortunately, the use of stereological methods for this purpose is still rare. One of the reasons for this is the fact that the transfer of the theoretical foundations into laboratory practice requires a remarkable amount of considerations before touching the first piece of tissue. These considerations, however, are often based on already acquired experience and are usually not dealt with in stereological review articles. The present article therefore delineates the procedures for estimating the most important characteristics of the cardiac vasculature and highlights potential problems and their practical solutions. Worked examples are used to illustrate the methods and provide examples of the calculations. Hopefully, the considerations and examples contained herein will provide researchers in this field with the necessary equipment to add stereological methods to their study designs. [ABSTRACT FROM AUTHOR]

Design-based stereology provides efficient methods to obtain valuable quantitative information of the respiratory tract in various diseases. However, the choice of the most relevant parameters in a specific disease setting has to be deduced from the present pathobiological knowledge. Often it is difficult to express the pathological alterations by interpretable parameters in terms of volume, surface area, length, or number. In the second part of this companion review article, we analyze the present pathophysiological knowledge about acute lung injury, diffuse parenchymal lung diseases, emphysema, pulmonary hypertension, and asthma to come up with recommendations for the disease-specific application of stereological principles for obtaining relevant parameters. Worked examples with illustrative images are used to demonstrate the work flow, estimation procedure, and calculation and to facilitate the practical performance of equivalent analyses. [ABSTRACT FROM AUTHOR]

The growing awareness of the importance of accurate morphometry in lung research has recently motivated the publication of guidelines set forth by a combined task force of the American Thoracic Society and the European Respiratory Society (20). This official ATS/ERS Research Policy Statement provides general recommendations on which stereological methods are to be used in quantitative microscopy of the lung. However, to integrate stereology into a particular experimental study design, investigators are left with the problem of how to implement this in practice. Specifically, different animal models of human lung disease require the use of different stereological techniques and may determine the mode of lung fixation, tissue processing, preparation of sections, and other things. Therefore, the present companion articles were designed to allow a short practically oriented introduction into the concepts of design-based stereology (Part 1) and to provide recommendations for choosing the most appropriate methods to investigate a number of important disease models (Part 2). Worked examples with illustrative images will facilitate the practical performance of equivalent analyses. Study algorithms provide comprehensive surveys to ensure that no essential step gets lost during the multistage workflow. Thus, with this review, we hope to close the gap between theory and practice and enhance the use of stereological techniques in pulmonary research. [ABSTRACT FROM AUTHOR]

Structural and functional remodelling of cardiomyocytes, capillaries and cardiac innervation occurs in left ventricular hypertrophy ( LVH) and heart failure ( HF) in response to pressure-induced overload. However, the onset, time course and the extent of these morphological alterations remain controversial. In the present study, we tested the hypothesis that the progression from hypertrophy to HF is accompanied by changes in the innervation (hyper- or hypoinnervation). Left ventricles of wild-type murine hearts subjected to pressure overload-induced hypertrophy by transverse aortic constriction ( TAC) were investigated by morphometric and design-based stereological methods at 1 and 4 weeks after TAC and compared with sham-operated mice. Mice developed compensated LVH at 1 week and typical signs of HF, such as left ventricular dilation, reduced ejection fraction and increased relative lung weight at 4 weeks post- TAC. At the (sub-)cellular level, cardiomyocyte myofibrillar and mitochondrial volume increased progressively in response to mechanical overload. The total length of capillaries was not significantly increased after TAC, indicating a misrelationship between the cardiomyocyte and the capillary compartment. The myocardial innervation decreased already during the development of LVH and did not significantly decrease further during the progression to HF. In conclusion, our study suggests that early loss of myocardial innervation density and increased heterogeneity occur during pressure overload-induced hypertrophy, and that these changes appear to be independent of cardiomyocyte and capillary remodelling. [ABSTRACT FROM AUTHOR]

Quantitative information about the innervation is essential to analyze the structure-function relationships of organs. So far, there has been no unbiased stereological tool for this purpose. This study presents a new unbiased and efficient method to quantify the total length of axons in a given reference volume, illustrated on the left ventricle of the mouse heart. The method is based on the following steps: 1) estimation of the reference volume; 2) randomization of location and orientation using appropriate sampling techniques; 3) counting of nerve fiber profiles hit by a defined test area within an unbiased counting frame on paraffin sections stained immunohistochemically for protein gene product 9.5; 4) electron microscopic estimation of the mean number of axon profiles contained in one nerve fiber profile; 5) estimation of the degree of tissue shrinkage of specimens in paraffin; and 6) calculation of the total axon length within the reference volume, taking tissue shrinkage into account. In a set of five mouse hearts, the total length of axons ramifying between cardiomyocytes ranged between ∼50 and 100 m, with a mean of 75.98 m (SD 23.73). The time required for the microscopical analysis was ∼8 h/animal for an experienced observer. Using antibodies specific for different neuron subtypes and immunoelectron microscopy, this method is also suited to estimate the total axon length of neurons expressing different transmitters. This new and efficient method is particularly useful when structural remodeling takes place and is suspected to involve gain or loss of axons. [ABSTRACT FROM AUTHOR]