Surface-weighted star volume: concept and estimation

The estimation of ‘size’ for an interconnected nonconvex phase is an ongoing problem in materials science and biomedicine. Examples of this type of phase include the pore space in a porous rock such as sandstone or the marrow space found in cancellous bone. A reasonable definition of size for this type of phase is provided by the mean volume-weighted star volume. This is defined as the average volume of the phase of interest seen unobscured from a typical point within the phase. One practical advantage of this definition of size is that it is easy to estimate stereologically. In this paper we extend the concept of star volume to consider the mean surface-weighted star volume. The mean surface-weighted star volume is the volume of a continuous phase seen unobscured from a typical point of the interface. We expect that this parameter could be useful in biological problems involving mass transfer across an interface. The stereological estimation of mean surface-weighted star volume is described and the method is illustrated on piglet lung tissue.     

Somatostatin and Astroglial Involvement in the Human Limbic System in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the elderly. Progressive accumulation of insoluble isoforms of amyloid-β peptide (Aβ) and tau protein are the major neuropathologic hallmarks, and the loss of cholinergic pathways underlies cognitive deficits in patients. Recently, glial involvement has gained interest regarding its effect on preservation and impairment of brain integrity. The limbic system, including temporal lobe regions and the olfactory bulb, is particularly affected in the early stages. In the early 1980s, the reduced expression of the somatostatin neuropeptide was described in AD. However, over the last three decades, research on somatostatin in Alzheimer’s disease has been scarce in humans. Therefore, the aim of this study was to stereologically quantify the expression of somatostatin in the human hippocampus and olfactory bulb and analyze its spatial distribution with respect to that of Aβ and au neuropathologic proteins and astroglia. The results indicate that somatostatin-expressing cells are reduced by 50% in the hippocampus but are preserved in the olfactory bulb. Interestingly, the coexpression of somatostatin with the Aβ peptide is very common but not with the tau protein. Finally, the coexpression of somatostatin with astrocytes is rare, although their spatial distribution is very similar. Altogether, we can conclude that somatostatin expression is highly reduced in the human hippocampus, but not the olfactory bulb, and may play a role in Alzheimer’s disease pathogenesis.     

Statistical analysis of labelling patterns of mammary carcinoma cell nuclei on histological sections

It is of central interest for tumour biology to explore the mechanisms of tumour cell proliferation. In this study, methods of spatial statistics were used to study the spatial distribution of proliferating cells within tumour tissue quantitatively and objectively. Mammary cancer tissue was studied as an example. It was attempted to clarify whether cell division occurs entirely at random (random labelling), i.e. the process of division occurs at random, independently from the state of the neighbouring nuclei, or whether the spatial distribution of proliferation is more complex, e.g. in the form of actively proliferating clusters alternating with relatively silent zones. In the case of random labelling, the reduced second moment functions K(r) of the labelled and the unlabelled nuclei would be identical. The same would hold for the pair correlation functions g(r) . The alternative hypothesis is that the second‐order properties of the processes of the labelled and the unlabelled nuclei are different. Twenty cases of invasive ductal mammary carcinomas were studied. The nuclei of proliferating cells were stained immunohistochemically with the monoclonal antibody MIB‐1, which detects specifically the proliferation‐associated nuclear antigen Ki 67. The planar coordinates of the tumor cell nucleus profiles from two rectangular visual fields per case were recorded. For each visual field, the following investigations were performed: estimation of the explorative summary characteristics K(r) and g(r) , fitting of the parameters of a stationary Strauss hard‐core model to the observed point patterns, estimation of two distance‐dependent Simpson indices and Monte Carlo tests of all individual patterns on the null hypothesis of random labelling. Significant differences between the mean K‐functions and the mean g‐functions of the labelled and the unlabelled nuclei were found. Moreover, the mean interaction parameter γ of the stationary Strauss hard‐core model was significantly higher for the labelled nuclei than for the unlabelled nuclei. The estimates of the two distance‐dependent Simpson indices showed a tendency of points with the same label towards a positive spatial correlation. In the Monte Carlo tests, the null hypothesis of random labelling was rejected for the majority of the visual fields. These four lines of investigation led to the concordant conclusion that the labelling of mammary carcinoma nuclei by MIB‐1 is not simply random. The data suggest that the second‐order properties of the point process of the labelled nuclei are significantly different from those of the unlabelled nuclei. In particular, the process of the labelled nuclei shows a higher degree of clustering (increased strength of interaction) than the process of the unlabelled points.     

Automated identification of neurons in 3D confocal datasets from zebrafish brainstem

Many kinds of neuroscience data are being acquired regarding the dynamic behaviour and phenotypic diversity of nerve cells. But as the size, complexity and numbers of 3D neuroanatomical datasets grow ever larger, the need for automated detection and analysis of individual neurons takes on greater importance. We describe here a method that detects and identifies neurons within confocal image stacks acquired from the zebrafish brainstem. The first step is to create a template that incorporates the location of all known neurons within a population – in this case the population of reticulospinal cells. Once created, the template is used in conjunction with a sequence of algorithms to determine the 3D location and identity of all fluorescent neurons in each confocal dataset. After an image registration step, neurons are segmented within the confocal image stack and subsequently localized to specific locations within the brainstem template – in many instances identifying neurons as specific, individual reticulospinal cells. This image-processing sequence is fully automated except for the initial selection of three registration points on a maximum projection image. In analysing confocal image stacks that ranged considerably in image quality, we found that this method correctly identified on average ∼80% of the neurons (if we assume that manual detection by experts constitutes 'ground truth'). Because this identification can be generated approximately 100 times faster than manual identification, it offers a considerable time savings for the investigation of zebrafish reticulospinal neurons. In addition to its cell identification function, this protocol might also be integrated with stereological techniques to enhance quantification of neurons in larger databases. Our focus has been on zebrafish brainstem systems, but the methods described should be applicable to diverse neural architectures including retina, hippocampus and cerebral cortex.     

Quantitative Evaluation of Cellular Uptake and Trafficking of Plain and Polyethylene Glycol‐Coated Gold Nanoparticles

This study addresses the cellular uptake and intracellular trafficking of 15‐nm gold nanoparticles (NPs), either plain (i.e., stabilized with citrate) or coated with polyethylene glycol (PEG), exposed to human alveolar epithelial cells (A549) at the air–liquid interface for 1, 4, and 24 h. Quantitative analysis by stereology on transmission electron microscopy images reveals a significant, nonrandom intracellular distribution for both NP types. No particles are observed in the nucleus, mitochondria, endoplasmic reticulum, or golgi. The cytosol is not a preferred cellular compartment for both NP types, although significantly more PEG‐coated than citrate‐stabilized NPs are present there. The preferred particle localizations are vesicles of different sizes (<150, 150–1000, >1000 nm). This is observed for both NP types and indicates a predominant uptake by endocytosis. Subsequent inhibition of caveolin‐ and clathrin‐mediated endocytosis by methyl‐β‐cyclodextrin (MβCD) results in a significant reduction of intracellular NPs. The inhibition, however, is more pronounced for PEG‐coated than citrate‐stabilized NPs. The latter are mostly found in larger vesicles; therefore, they are potentially taken up by macropinocytosis, which is not inhibited by MβCD. With prolonged exposure times, both NPs are preferentially localized in larger‐sized intracellular vesicles such as lysosomes, thus indicating intracellular particle trafficking. This quantitative evaluation reveals that NP surface coatings modulate endocytotic uptake pathways and cellular NP trafficking. Other nonendocytotic entry mechanisms are found to be involved as well, as indicated by localization of a minority of PEG‐coated NPs in the cytosol.     

基于体视学的SAP混凝土气孔结构分析与研究

为了研究混凝土在添加高吸水性树脂后其内部气孔结构的变化规律,以及内部气孔结构的变化对混凝土试样抗压强度的影响,基于定量体视学的电子显微图像分析方法,对添加SAP混凝土的内部气孔结构特征展开了相关试验研究.采用干拌的方法拌和SAP混凝土,对不同配合比和不同SAP掺量的混凝土进行分批试验,测定出各组试样的气孔结构参数和抗压强度等,以深层次的剖析SAP对混凝土强度和内部气孔结构的影响.研究结果表明:混凝土内部气孔的平均孔径与间距系数随着SAP含量和水灰比的增加而增大,而混凝土抗压强度则呈现出相反的变化趋势.在配合比相同的情况下,小粒径SAP颗粒对混凝土气孔率的提高效果更为明显,而大粒径SAP颗粒对混凝土气孔平均孔径的提高效果更为明显;水灰比对气孔平均间距系数影响显著,而SAP粒径对混凝土内部气孔平均间距系数的影响不明显.     

Maternal dexamethasone treatment reduces ovarian follicle number in neonatal rat offspring

Elevated glucocorticoid levels in the gravid female circulation affect a number of endocrine functions in the fetuses and neonates. The aim of this study was to examine the effects of maternal dexamethasone (Dx) administration during late pregnancy on the ovaries of neonatal offspring. On the 16th day of pregnancy, experimental dams received subcutaneously 1.0 mg Dx/kg b.w., followed by 0.5 mg Dx/kg b.w./day on the 17th and 18th days of gestation. The control gravid females received the same volume of saline vehicle. Left ovaries from 5‐day‐old female pups were stereologically analyzed. The ovary volumes were estimated using Cavalieri's principle. The number of healthy and atretic primordial and primary follicles was estimated using a fractionator–physical disector method. The number of secondary follicles was determined by exact counts of every fourth section encompassing whole cross‐sections of the ovary. The ovary volume was significantly decreased (by 44.4%; P < 0.05) in the group of female pups from Dx‐treated mothers comparing to the controls. The numbers of healthy primordial and atretic follicles were 38.8% (P < 0.05) and 50.9% (P < 0.05), respectively, reduced in the ovaries of pups from the Dx‐treated mothers, when compared with the control values. There were 53.4% (P < 0.05) fewer healthy primary and 41.8% (P < 0.05) fewer healthy secondary follicles as well. The numbers of atretic primary and secondary follicles were reduced by 60.0% (P < 0.05) and 61.7% (P < 0.05), respectively. It can be concluded that fetal exposure to glucocorticoids decreased the pool of non‐growing follicles in the neonatal ovary, whereas the processes of folliculogenesis and atresia remained unaffected.     

Improved estimation of the pair correlation function of random sets

The texture of binary spatial structures can be characterized by second-order methods of spatial statistics. The pair correlation function, which describes the structure in terms of spatial correlation as a function of distance, is of central importance in this context. Conventionally, the pair correlation function of stationary and isotropic random sets is estimated as the ratio of the covariance to the square of volume fraction of the phase of interest. In the present paper, an improved estimator of the pair correlation function is presented, where the covariance is divided by the square of a distance-adapted estimator of volume fraction. The new estimator is explained mathematically and applied to simulated images of the Boolean model and to microscopic images from neoplastic and non-neoplastic human glandular tissues. It leads to a considerable reduction of bias and variance of estimated pair correlation functions, in particular for large distances.     

Monitoring age-related changes of collagen content and vascularity in ganglia using unbiased stereological methods

We describe for the first time application of unbiased stereological techniques to estimate total volume and volume fractions of interest in individual dorsal root ganglia (DRG). Volume estimates were obtained using a two-stage sampling design. Sections were systematically sampled following a random start, from DRG which were embedded in methacrylate and exhaustively sectioned. We further examined the efficiency of point counting irregular volume fractions housed in a regular reference volume. We found that the precision of volume estimates was relatively unaffected by exhaustive sampling, and that the magnitude of error was, in large part, determined by object shape.