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Sampling designs dictated by stereology have proven very useful in recent years to estimate in situ the total number of deposited particles, or of macrophages, in different lung compartments at the light microscopical level. The sampling methods are based on parallel slabs which are subsequently subsampled by disectors. The resulting number estimators are unbiased irrespective of tissue shrinkage or swelling, and they are readily applicable in other contexts (notably in neuroscience). Several variants of the design are available, however, and, although they all yield the same number estimates, their precision, and the mathematical prediction of it, vary among the different estimators and are subjected to theoretical improvement. The present paper constitutes a detailed survey of the latest advances, and it illustrates methods and formulae alike by way of a real example stemming from an earlier study on particle retention and clearance.

A new stereological method is proposed which combines vertical slice projections with the fractionator to estimate the total capillary length in a skeletal muscle. The method was demonstrated on the soleus muscle of a Wistar rat. The implementation required capillary highlighting, tissue sampling, and data acquisition in the form of intersection counts between capillary projections and cycloid test lines. The capillaries were demonstrated using vascular perfusion (with gelatine) of the hind leg of the rat. The sampling procedure followed the fractionator design, namely a multistage systematic sampling design with a known sampling fraction at each stage. To make the design unbiased, vertical slices were used; for efficiency, the vertical axis was chosen parallel to the main axis of the muscle. As prescribed to avoid bias, the cycloid test lines were superimposed on the slice projections, viewed under the light microscope, with their minor axes normal to the vertical axis. The estimation precision was compared for different sampling and subsampling fractions. The proposed method was globally highly efficient, unbiased, and easy to implement.

With the advent of many new tools over the last five years, stereology has become simpler, assumption-free, and more efficient but, at the same time, new terms and concepts have proliferated, which risk overwhelming potential users. The present review is intended to meet the urgent need for a structured classification and evaluation of the newest stereological methods. Being fairly comprehensive, the exposition is necessarily succinct: the reader is referred to selected references for the necessary details and examples.

Gamma-glutamyltranspeptidase-positive hepatocyte foci were produced in female rats given a single dose of diethylnitrosamine neonatally after birth and, after weaning, a diet containing phenobarbitone for 30 wk. The nucleator method, a new stereological approach, provided an efficient, unbiased estimate of mean cell volume in focal lesions and extrafocal areas. It also provided an unbiased sample of cells to estimate hepatocyte nuclear volume and the percentage of binucleated cells. The results showed an increase in the mean volume of mononucleated cells--from 4,700 micron3 in extrafocal areas to 12,700 micron2 in foci--and of binucleated cells--from 6,900 micron3 to 25,000 micron3. This demonstrated the hypertrophic effect of the carcinogenic treatment in focal lesions. A striking reduction in the proportion of binucleated cells was also observed in the preneoplastic lesions. Nuclear volume measurements from mononucleated and binucleated hepatocytes were used to assess ploidy. An apparent increase in nuclear ploidy, with no change in cellular ploidy, was noted in focal tissue when compared with nonfocal tissue. This appeared to be caused by an increase in mononucleated tetraploid cells and a reduction in binucleated cells with two diploid nuclei, indicating an altered mitotic mechanism in focal lesions. The significance of these changes in cell volume, apparent ploidy levels and binuclearity in preneoplastic foci is discussed in relation to the hepatocarcinogenic process.

Population sizing from still aerial pictures is of wide applicability in ecological and social sciences. The problem is long standing because current automatic detection and counting algorithms are known to fail in most cases, and exhaustive manual counting is tedious, slow, difficult to verify and unfeasible for large populations. An alternative is to multiply population density with some reference area but, unfortunately, sampling details, handling of edge effects, etc., are seldom described. For the first time we address the problem using principles of geometric sampling. These principles are old and solid, but largely unknown outside the areas of three dimensional microscopy and stereology. Here we adapt them to estimate the size of any population of individuals lying on an essentially planar area, e.g. people, animals, trees on a savanna, etc. The proposed design is unbiased irrespective of population size, pattern, perspective artifacts, etc. The implementation is very simple—it is based on the random superimposition of coarse quadrat grids. Also, an objective error assessment is often lacking. For the latter purpose the quadrat counts are often assumed to be independent. We demonstrate that this approach can perform very poorly, and we propose (and check via Monte Carlo resampling) a new theoretical error prediction formula. As far as efficiency, counting about 50 (100) individuals in 20 quadrats, can yield relative standard errors of about 8% (5%) in typical cases. This fact effectively breaks the barrier hitherto imposed by the current lack of automatic face detection algorithms, because semiautomatic sampling and manual counting becomes an attractive option.

The physiological role of 5-hydroxytryptamine 6 receptors in the central nervous system has not yet been elucidated. The high affinity of various psychotropic drugs for 5-hydroxytryptamine 6 receptors has led to the suggestion that this receptor type may be a novel target in neuropsychiatry. We have found that continuous intracerebroventricular administration of a 5-hydroxytryptamine 6 receptor antisense oligonucleotide, but not of a missense oligonucleotide, produced an anxiogenic-like response in rats using two different models of anxiety, the social interaction test and the elevated plus-maze. Neither oligonucleotide treatment modified locomotor activity, rectal temperature or food intake, suggesting a low or null neurotoxicity. The effectiveness of the treatment with the designed antisense oligonucleotide to block the synthesis of the protein encoded by the target mRNA was assessed by immunolabelling 5-hydroxytryptamine 6 receptors in the nucleus accumbens, where this receptor is highly expressed, using previously characterized specific antibodies. The density of the immunostaining was quantified by means of an unbiased three-dimensional stereologic procedure, which revealed a significant reduction (−25%) in the number of immunolabelled neuronal elements. These results suggest that, in addition to other 5-hydroxytryptamine receptor subtypes, 5-hydroxytryptamine 6 receptors in the nucleus accumbens may participate in anxiety-related neurobiological mechanisms.

Slow and fast twitch fibres were stereologically analysed in the morphologically defined and most strongly developed ventral and ventrolateral region of the external urethral sphincter (M. urethralis) using five sexually intact anestrous and five ovariectomized nulliparous beagles. The estimated mean total cross-sectional area of the investigated part of the muscle consisted of 4.2 mm2 (15.1%) type I fibre, 10.0 mm2 (32.1%) type II fibre, and 16.3 mm2 (52.8%) connective tissue in the control group. The corresponding absolute mean value of type I fibres (3.7 mm2/13.5%) was statistically lower in ovariectomized animals. No significant difference between groups was observed in the relative number of transverse profiles of type I and II fibres; type I fibres comprised 23.8% and type II 76.2% of all muscle fibres in the sexually intact group, but 21.8% and 78.2% in the ovariectomy group, respectively. The ovariectomized dogs exhibited a statistically significant lower type I and II fibre number and a concomitant slightly larger mean single profile area (diameter) of fibre type II compared with the control animals. The significantly reduced number and decreased total cross-sectional area of the fatigue-resistant type I fibres in ovariectomized dogs suggest a predominant weakening of the fibre type I portion of the M. urethralis as consequence of ovariectomy. The effect could be mediated by sex hormonal factors and may contribute to the development of postspaying urinary incontinence in female dogs.

The aim of this study was to develop an in vitro model to estimate the clearance of pulmonary administered cyclosporine A (CsA). To do this we estimated the volume of CsA particles phagocytosed by alveolar macrophages (AM) lavaged from hamsters. AM were cultured with CsA particles at two doses of particles (0.1 mg or 0.5 mg) and at three incubation times (1 h, 6 h or 24 h). The AM were also incubated with or without latex particles. After incubation, AM were processed for light and electron microscopy and the mean volume of phagocytosed particles was estimated stereologically from micrographs of the cells. Here, however, the CsA particles were dissolved during the embedding process and only their negative images (vacuoles) could be detected. An indirect method was therefore developed. The volume of cytoplasmic vacuoles (called 'background' vacuoles) was estimated in control macrophages (without particles or with latex particles and subtracted from the total volume of vacuoles in macrophages incubated with CsA, which gave the volume of phagocytosed CsA. The volume of the 'background' vacuoles remained constant in all study conditions. At a dose of 0.1 mg CsA the volume phagocytosed per macrophage was 13.83 microns3 at 1 h, 8.43 microns3 at 6 h and 4.50 microns3 at 24 h. At a dose of 0.5 mg CsA, the volume phagocytosed varied from 26.59 microns3 at 1 h, to 4.13 microns3 at 6 h and 49.10 microns3 at 24 h. These results show no statistically significant dependence on time for either dose, and a statistically significant dose effect only at 24 h. With latex particles, the phagocytosed volume increased significantly with time and dose and was significantly higher than for CsA particles. This study showed that CsA particles are phagocytosed by AM from hamsters but to a lesser extent than latex particles. This difference could be correlated with physical properties, i.e. a difference between particle size and shape and/or chemical properties, latex particles being inert and CsA particles being peptidic. Moreover, different surface receptors on AM could be involved in the process of phagocytosis of CsA and latex particles.

Macrophages play a central role in the defence of the respiratory tract against deposited particles. In addition to the well-studied alveolar macrophages, airway macrophages have been recognized as an important clearance factor. Bronchoalveolar lavage (BAL) has been used for functional and morphological investigations of macrophages in vitro, assuming that all macrophages are removed with equal probability from the lung surface. Airway macrophages have been found in close contact with the epithelial cells. These macrophages may not be easily removed by lavage, and they might constitute a functionally different macrophage population. We have tested the hypothesis that there exists a population of macrophages in the conducting airways that resists removal by lavage. We lavaged the lungs of four hamsters and fixed the lungs, thereafter, by intravascular perfusion. The number of macrophages in the intrapulmonary conducting airways was estimated with an unbiased stereological technique, the fractionator, and compared to the number of macrophages in the airways of four hamsters whose lungs had not been lavaged prior to fixation. This in situ study revealed that, in hamster lungs, 42% of the airway macrophages were not removed by BAL and that about 5% of all macrophages in the BAL fluid were airway macrophages. Additionally, ultrastructural alterations of the airway epithelium were found. It is concluded that there exists a population of airway macrophages that resists lavage. This is an aspect which has to be considered in studies performed with macrophages obtained by BAL, since they could represent a functionally different macrophage population.

The perfusion-fixed urethrae of 5 sexually intact nulliparous and 5 ovario-hysterectomized beagles were analyzed stereologically. Different urethral tissue components were estimated stereologically in each of the three proximal urethral quarters and compared between the two groups of dogs. Considerable individual variability within all groups was observed. In all the three urethral quarters considered, average smooth muscle and connective tissue volumes were generally lower in spayed than in intact animals, both in absolute and relative terms. However, only the difference between the mean relative volumes of urethral quarter I was statistically significant. In contrast, mean absolute and relative volumes of the vascular plexus were generally greater in the ovariohysterectomized dogs as compared to the animals of the other group – deviations in statistical relevance concerning the first urethral quarter were noted. Mean epithelial and striated muscle volumes did not vary essentially between the spayed and intact group of dogs. Despite the fact that all findings were not always statistically meaningful, our research nevertheless demonstrates that ovariohysterectomy decreases smooth muscle mass and connective tissue; it also elevates vascular plexus volumes in the urethra of spayed animals. Moreover, our results imply a general weakening of the urethral wall as a sequel to ovariohysterectomy, and this, in turn, could be seen to affect the closure mechanism of the urethra.

The M. urethralis was morphologically investigated in ten medium-sized female dogs of different breeds and age, as well as histochemically and stereologically analysed in a homogeneous group of five female beagles. Macroscopically, the muscle was essentially confined to the distal third and most strongly developed in the fourth quarter of the urethra. Here, it surrounded the urethra transversely at the ventral and lateral aspects, passing with its caudal fibres dorsally onto the vagina. The muscle fibres were assembled in groups of different sizes and usually separated by thick connective tissue septae. Based on the myofibrillar actomyosin ATPase (mATPase) reaction, type I and two main subtype II fibres could be differentiated. Type II fibres were, however, indistinguishable by their metabolic enzyme activities since both subclasses displayed oxidative-glycolytic properties. The subtype II fibres containing the more acid-labile mATPase activity were classified as IIA, whereas the other main subtype was designated IIS (subtype) and considered as peculiar to the dog. In addition, the investigation revealed a rare fibre type exhibiting the histochemical profile of IIC fibres. There was no evidence of classical glycolytic IIB fibres. The M. urethralis was composed of 24% type I and 76% type II fibres with an average diameter of 34.9 and 28.5 microns, respectively. Connective tissue constituted 52.8% of the total muscle volume. Due to the predominance of fast twitch II fibres, the urethral muscle is not designed to maintain a high tone over long periods of time. Its primary role is to function rapidly and intermittently guaranteeing urinary continence during stress situations when other continence factors are overburdened.

The number and functions of macrophages in the lungs are crucial factors for prevention and development of lung disease caused by inhaled particles. To examine whether airway macrophages are attracted to the site of particle deposition and what proportion of these macrophages is involved in phagocytosis, aerosols of 6-microns polystyrene particles (PSP) were inhaled by Syrian Golden hamsters under controlled conditions through an inhalation tubule and their lungs were fixed by intravascular perfusion within 20 min (PSP-1, PSP-1a), 40 min (PSP-2), and 24 h (PSP-3) after the beginning of the inhalation. The number and the phagocytic activity of airway macrophages were studied in situ with a fractionator, a stereologic method, on light microscopic sections. No significant increase in macrophage number was detected for the groups PSP-1 and PSP-1a. The increase for group PSP-2 was, however, between 2- and 3-fold, whereas for group PSP-3 the increase was between 1.5- and 2.5-fold with respect to control animals, which had inhaled ambient air through an intubation tubule (C-2) and whose lungs had been fixed after 40 min. There were no significant differences among the four groups with respect to the proportion of airway macrophages that had phagocytized polystyrene microspheres. Twelve to fifteen percent of the macrophages were found to be involved in phagocytosis. In the case of the mean number of particles per phagocytizing macrophage, there was a significant decrease for the PSP-3 group with respect to the pool of the three groups PSP-1, PSP-1a, and PSP-2 taken together. These studies demonstrate (1) that airway macrophages are rapidly recruited to the sites of particle deposition and (2) that only a small proportion of very active macrophages contributes to the clearance of particles, suggesting a great potential of airway macrophages to interact with many more particles than the hamsters were exposed to in this study.

A practical methodology is proposed for the stereological analysis of lung and other organs using recently developed unbiased procedures. This study concentrates on the unbiased estimation of lung volume using Cavalieri's principle compared with the fluid displacement method and measurement of pleural surface area using vertical sections. Furthermore, the proposed design, in addition to the sampling of extensive slices for the initial steps, also allows sampling of vertical sections for light and electron-microscopical stereology. The procedures are described in detail by reference to biological data from the right lungs of four rabbits. We found excellent agreement between estimates of lung volume measured with Cavalieri's principle and those measured by fluid displacement. Pertinent details of the statistical analysis of the sources of variation (namely biological, systematic sectioning and point-counting variation) are given in an appendix.

Biological mineralization kis a cell-mediated process which is believed to be triggered by a "nucleating agent." Various matrix structures, such as matrix vesicles, collagen fibrils and macromolecules, have been claimed to be the source of this substance, since these components have been found by transmission electron microscopy (TEM) of thin sections to be associated with early mineral crystals. Systematic image analysis of the relationships revealed in electron micrographs between specific matrix components and early mineral deposits has shown that unequivocal image interpretation is not possible. This is due principally to the problems posed by overprojection and truncation phenomena, since the structures being analyzed lie within the same dimensional range as thin section thickness. Various examples are illustrated and discussed. The site at which mineral crystals are initially laid down thus cannot be identified with any matrix structure using thin section TEM. Possible technical approaches to resolve this problem of image analysis are discussed.

The administration of nicotine (1 and 4 mg/kg/day, subcutaneously for up to 6 weeks) to male Sprague-Dawley rats first induced a time-and dose-dependent increase in catecholamine (CA) synthesis. This was followed by an increase in endogenous CA levels and in total volume and number of chromaffin cells, as measured by stereological methods on serially sectioned adrenal glands. Thus, continued stimulation of the sympathoadrenal system generated an increase in biosynthetic enzyme activity, and subsequently adrenal medullary hypertrophy and hyperplasia developed as an adaptive reaction. The proposed model is useful for quantifying both biochemically and morphometrically early adrenal changes long before irreversible pathologic alterations are manifested.

The molecular structure of mitochondria and their inner membrane has been studied using a combined approach of stereology and biochemistry. The amount of mitochondrial structures (volume, number, surface area of inner membrane) in a purified preparation of mitochondria from rat liver was estimated by stereological procedures. In the same preparation, the oxidative activity of the respiratory chain with different substrates and the concentration of the redox complexes were measured by biochemical means. By relating the stereological and biochemical data, it was estimated that the individual mitochondrion isolated from rat liver has a volume of 0.27 micron 3, an inner membrane area of 6.5 micron 2, and contains between 2,600 (complex I) and 15,600 (aa3) redox complexes which produce an electron flow of over 100,000 electrons per second with pyruvate as substrate. The individual redox complexes and the H+-ATPase together occur at a density of approximately 7,500/micron 2 and occupy approximately 40% of the inner membrane area. From the respective densities it was concluded that the mean nearest distance between reaction partners is small enough (70-200 A) to cause the formation of micro-aggregates. The meaning of these results for the mechanism of mitochondrial energy transduction is discussed.

Certain assumptions on the movement and signalling of a species, as well as on the dimensionality of the space, give rise to a variety of “spatial models”, and yield some conclusions relative to the gregarious behaviour of that species and the adjustment of the sex ratio in bisexual populations. In the former context, Hamilton's (1971) arguments are affected. In the latter, Kalmus & Smith's (1960) treatment is discussed and elaborated to include pertinent new factors. This is done in a quantitative manner by applying a number of occupancy models. It appears that the sex ratio which maximizes the expected number of fertilized females will be biased towards the mobile sex, subject to the relative fecundity of the males. It is argued that the optimal sex ratio constitutes a socially advantageous but individually disadvantageous character, and may survive under certain conditions.

SUMMARY ‘Vertical’ sections are plane sections longitudinal to a fixed (but arbitrary) axial direction. Examples are sections of a cylinder parallel to the central axis; and sections of a flat slab normal to the plane of the slab. Vertical sections of any object can be generated by placing the object on a table and taking sections perpendicular to the plane of the table. The standard methods of stereology assume isotropic random sections, and are not applicable to this kind of biased sampling. However, by using specially designed test systems, one can obtain an unbiased estimate of surface area. General principles of stereology for vertical sections are outlined. No assumptions are necessary about the shape or orientation distribution of the structure. Vertical section stereology is valid on the same terms as standard stereological methods for isotropic random sections. The range of structural quantities that can be estimated from vertical sections includes Vv, Nv, Sv and the volume-weighted mean particle volume vv, but not Lv. There is complete freedom to choose the vertical axis direction, which makes the sampling procedure simple and ‘natural’. Practical sampling procedures for implementation of the ideas are described, and illustrated by examples.

The longitudinal growth of bone depends on the activities of individual chondrocytes of the growth plate. Each chondrocyte remains in a fixed location throughout its life, and there accomplishes all of its functions. Although a cell may perform several or all of its activities simultaneously, one of these will usually predominate during a particular phase of its life. The two most prominent stages are those of cellular proliferation and hypertrophy (including the mineralization of matrix) before the resorption of tissue during vascular invasion. By applying recently developed stereological procedures and improved methods for the fixation of cartilage, we compared cellular shape modulation, various ultrastructural parameters (surface areas or volumes of endoplasmic reticulum, Golgi membranes, and mitochondria), the production of matrix, and cellular turnover for proliferating and hypertrophic chondrocytes within the proximal tibial growth plate of the rat. By the late hypertrophic stage, fourfold and tenfold increases in the mean cellular height and volume, respectively, and a threefold increase in the mean volume of the matrix per cell were achieved. The high metabolic activity of hypertrophic cells was reflected by a twofold to fivefold increase in the mean cellular surface area of rough endoplasmic reticulum, the Golgi membranes, and the mean cellular mitochondrial volume. Rates of longitudinal growth were determined by fluorochrome labeling and incident-light fluorescence microscopy. Using these values and the stereological estimators describing cellular height, the rates of cellular turnover were calculated. The rapid progression of the vascular invasion front was found to eliminate, for each column of cells, one chondrocyte every three hours; that is, eight cells a day. The maintenance of a steady-state structure for growth-plate cartilage in rats in a steady state of growth thus necessitates efficient compensation for these losses, which is achieved by a high rate of cellular proliferation and rapid hypertrophy.

SUMMARY The purpose of the present paper is twofold. Firstly, to compare the accuracy per unit cost achieved by two different methods of subsampling micrographs from sections of a material for stereology when the sections cannot be analysed as a whole at the required magnification. Secondly, to illustrate, by means of real data, the application of some of the methods and formulae proposed in the companion paper (Cruz-Orive & Weibel, 1981) for estimating ratios at the electron microscopic level. The final estimates of a same ratio obtained by either subsampling method (namely systematic (SQ) and systematic area-weighted quadrats (SAWQ)) agreed in the mean and they were about equally precise. The former fact indicates that the new SAWQ method is at least as reliable as the SQ method as far as bias is concerned. The latter result is a consequence of the well-known fact that subsampling is relatively unimportant in two-stage sampling. Yet, SAWQ subsampling enjoys definite practical advantages over other subsampling methods in certain situations.

An evaluation is made of the relative efficiency (precision of the final estimate per unit time of measurement on a given set of sections) of different methods for planar analysis aimed at estimating aggregate, overall stereological parameters (such as VV, SV). The methods tested are point-counting with different densities of test points (4 less than or equal to PT less than 900 per picture), semiautomatic computer image analysis with MOP and automatic image analysis with Quantimet, for obtaining VV and SV estimates. One biological sample as well as three synthetic model structures with known coefficients of variation between sections are used. The standard error of an estimate is mainly determined by the coefficient of variation between sampling units (= sections in the present paper) so that measuring each sample unit with a very high precision is not necessary. Automatic image analysis and point-counting with a 100-point grid were the most efficient methods for reducing the relative standard errors of the VV and SV estimates to equivalent levels in the synthetic models. Using a 64-point grid was as precise, and about 11 times faster than using a tracing device for obtaining the estimate of VV in the biological sample.

SUMMARY A serious problem in stereology is to ensure a consistent definition of reference spaces at different levels of magnification whenever the boundaries of such reference spaces are either fuzzy or non-existent, and hence they have to be defined artificially. (It is well known that inconsistent definitions of the reference space leads to unknown amounts of bias in stereological results.) In this paper a new application is found for the laser microbeam system used in microdissection, whereby the required boundaries can be easily and neatly traced (in fact, cut) directly onto uncovered sections for light microscopy. The dangers of bias inherent from inconsistencies of definition are thereby eliminated completely, and alternative, very expensive procedures requiring direct marking of paper prints with a pen are no longer necessary.

SUMMARY Two related problems are explored. Firstly, a single opaque solid Ω1 of arbitrary size and shape, containing an arbitrarily shaped phase Ω2, is considered. The problem is to estimate the minimum number of systematic sections m, necessary to estimate the volume ratio v = V(Ω2)/V(Ω1) with a coefficient of error of at most γ0 with a probability 1 - α. Secondly, we consider a population of such specimens. The second problem is to estimate the optimum number n of specimens to be sampled and the number m of systematic sections per specimen in order to estimate the mean volume ratio of the population with a relative error of at most ∍0 with a probability 1 - α. General guidelines for solving the two problems are presented. Practical results applicable to two populations of mouse and guinea-pig lymph nodes, exhibiting a wide variation in size and shape, are obtained.

A modified version of the fractionator was used to estimate the total number of polystyrene microspheres retained in the airways of hamster lungs at two different time points after inhalation. A systematic three-stage subsampling procedure with known sampling fractions was adopted. First, each lung was cut into slices, from which primary disectors were sampled systematically with a known sampling fraction. From each primary disector, smaller sub-disectors were subsampled, and the corresponding sampling fraction was estimated by point counting. Finally, a few particles were counted at the microscopic level in the sub-disectors, and the final estimate of total particle number (which is unbiased irrespective of any tissue deformations) was easily computed as a product of the counted number times the reciprocal of the successive sampling fractions. The error variance of each estimate was assessed from the data using a new estimator. An average of 6% of the deposited particles were retained on the epithelial surface of the intrapulmonary conducting airways shortly after the inhalation, from which at least one-third was already phagocytosed by macrophages. After 24 h, an average of 87% of the particles retained shortly after the inhalation had been cleared. The proportion of particles ingested by macrophages had increased to at least 87% in three out of four animals studied.

The accurate estimation of stereological parameters defined on anisotropic structures is a long-standing problem. In this paper we seek to estimate the capillary length density JV in skeletal muscle tissue. A well-known model for directional anisotropy in space, namely the 'spherical normal' or 'Fisher axial distribution' model, is found to fit the relevant data satisfactorily. Based on this model, a short-cut estimation method is proposed and illustrated with a numerical example. This method essentially consists in taking the ratio of mean capillary profile counts, as obtained from transversal and longitudinal sections of the muscle tissue, and making use of a table or a graph given in the paper to estimate JV. The conditions under which the methods are applicable and practicable are discussed in detail. Apart from an accurate estimation of JV, an important feature of our method is the possibility of quantifying the degree of anisotropy by a coefficient K (called the concentration parameter of the Fisher axial distribution), which enjoys both a biological significance and a sound statistical basis.

The quality of morphometric data obtained by stereological methods depends on the sampling scheme adopted. In the light of recent advances in sampling theory for stereology a sampling strategy has been developed which differs significantly from that used in previous studies. It operates with a cascade of four sampling levels and adopts the principle of A-weighted sampling, a method that should eliminate bias and possibly yield smaller variances. This method is described and discussed in detail. When compared with the old simpler sampling approach it is found that the new method (1) is at least as efficient and (2) that the estimates of morphometric parameters obtained by this method are similar to the data generated by the old method.

Perfusion-fixed urethrae of five sexually intact nulliparous female beagles in anestrus, between 2 and 3 years of age, were morphologically and morphometrically investigated. Absolute and relative volumes of different tissue components were stereologically estimated per urethral quarter. A discrete smooth muscle sphincter could neither be identified in the bladder neck nor in the urethra. However, the circularly arranged smooth muscle fibers in the proximal half of the urethra were regarded as being capable of sphincteric function. The connective tissue, composed of collagenous and longitudinally orientated numerous elastic fibers, accounted for the most predominant constituent. It comprised 77.3 to 78.2% of the total volume in the proximal three urethral quarters. The vascular plexus was most prominently developed in the proximal two urethral quarters, and its relative and absolute volumes significantly exceeded those of the third quarter. The discovered direct connections between small arteries and the sinusoids of the vascular plexus emphasize its importance as a major contributory component to urinary continence. The striated musculus urethralis was essentially confined to the distal half of the urethra and most strongly developed in the fourth quarter, where it surrounded the urethra transversely at its ventral and lateral aspects. The caudal muscle fibers continued dorsally to the vagina. A transitional epithelium bounded the urethral lumen. Its superficial cell layer progressively flattened towards the external urethral orifice, changing into a stratified squamous lining as also seen in the vestibulum. Recorded pressures, or pressure changes, could neither be correlated with volumes nor with morphology of particular tissue elements in the different urethral quarters.

Lung disease is the major cause of death in cystic fibrosis (CF), but the effect of gene mutation on the morphology of the main structural compartments of the lung is poorly understood. We show here, to our knowledge for the first time, a quantitative comparison of the fine pulmonary structures of cftr mutant versus non-cf mice. Pertinent volumes and surface areas were estimated in 10 homozygous cftrm1HGU mutants and 11 non-cf littermates by unbiased stereology at the light microscopic level. Our data did not reveal any statistical differences between group means for any of the 9 parameters considered. In other words, our data do not supply any significant evidence that the lack of the Cftr gene is accompanied by any developmental abnormalities in the lung, at least as far as the parameters studied are concerned.