Downstream effects of polypathology on neurodegeneration of medial temporal lobe subregions.

The medial temporal lobe (MTL) is a nidus for neurodegenerative pathologies and therefore an important region in which to study polypathology. We investigated associations between neurodegenerative pathologies and the thickness of different MTL subregions measured using high-resolution post-mortem MRI. Tau, TAR DNA-binding protein 43 (TDP-43), amyloid-ß and α-synuclein pathology were rated on a scale of 0 (absent)-3 (severe) in the hippocampus and entorhinal cortex (ERC) of 58 individuals with and without neurodegenerative diseases (median age 75.0 years, 60.3% male). Thickness measurements in ERC, Brodmann Area (BA) 35 and 36, parahippocampal cortex, subiculum, cornu ammonis (CA)1 and the stratum radiatum lacunosum moleculare (SRLM) were derived from 0.2 × 0.2 × 0.2 mm3 post-mortem MRI scans of excised MTL specimens from the contralateral hemisphere using a semi-automated approach. Spearman's rank correlations were performed between neurodegenerative pathologies and thickness, correcting for age, sex and hemisphere, including all four proteinopathies in the model. We found significant associations of (1) TDP-43 with thickness in all subregions (r = - 0.27 to r = - 0.46), and (2) tau with BA35 (r = - 0.31) and SRLM thickness (r = - 0.33). In amyloid-ß and TDP-43 negative cases, we found strong significant associations of tau with ERC (r = - 0.40), BA35 (r = - 0.55), subiculum (r = - 0.42) and CA1 thickness (r = - 0.47). This unique dataset shows widespread MTL atrophy in relation to TDP-43 pathology and atrophy in regions affected early in Braak stageing and tau pathology. Moreover, the strong association of tau with thickness in early Braak regions in the absence of amyloid-ß suggests a role of Primary Age-Related Tauopathy in neurodegeneration.

Anatomical segmentation of the human medial prefrontal cortex.

The medial prefrontal areas 32, 24, 14, and 25 (mPFC) form part of the limbic memory system, but little is known about their functional specialization in humans. To add anatomical precision to structural and functional magnetic resonance imaging (MRI) data, we aimed to identify these mPFC subareas in histological preparations of human brain tissue, determine sulci most consistently related with mPFC areal boundaries, and use these sulci to delineate mPFC areas in MRIs. To achieve this, we obtained three-dimensional MRI data from 11 ex vivo hemispheres and processed them for cyto- and myelo-architectonic analysis. The architectonic boundaries of mPFC areas were identified in histology and cortical surface length and volumes were measured. Unfolded maps of histologically determined boundaries were generated to identify the association of mPFC areal boundaries with sulci across cases. This analysis showed that cingulate and superior rostral were the sulci most consistently related to mPFC areal boundaries. Based on presence/absence and anastomosis between such sulci, 6 sulci patterns in the 11 hemispheres were found. A further analysis of 102 hemispheres of in vivo MRI scans (N = 51 males, mean ± SD 24.1 ± 3.1 years of age) showed similar sulci patterns, which allowed us to delineate the mFPC areas in them. The volumes of mPFC areas across histological, ex vivo and in vivo MRI delineations were comparable and probabilistic maps generated from the MRIs of the102 hemispheres. Probabilistic maps of mPFC areas were registered to MNI space and are available for regional analysis of functional magnetic resonance imaging data.

Stereology of isolated objects with the invariator.

The invariator is a new stereological design to generate motion invariant test lines in three dimensions on an isotropic plane through a fixed point. The theory has been published recently. The purpose of this paper is to illustrate the application of the invariator on a group of rat brains to estimate brain volume and external surface area. Each brain was first split into its two hemispheres and then embedded into a ball filled with agar following a configuration named the antithetic isector, with the idea of reducing the error variance. After rolling the ball at random it was scanned by magnetic resonance imaging into a stack of parallel systematic sections: this is the isotropic Cavalieri design which, combined with the antithetic isector idea, proves to be very accurate. The invariator used only an equatorial section of the ball, and in the present case the coefficient of error of the volume and surface area estimators of an individual brain was about 30%. As it is design unbiased, the invariator may prove its strength mainly to estimate population means.

Convergence of unimodal and polymodal sensory input to the entorhinal cortex in the fascicularis monkey.

The hippocampal formation is a key structure in memory formation and consolidation. The hippocampus receives information from different cortical and subcortical sources. Cortical information is mostly funneled to the hippocampus through the entorhinal cortex (EC) in a bi-directional way that ultimately ends in the cortex. Retrograde tracing studies in the nonhuman primate indicate that more than two-thirds of the cortical afferents to the EC come from polymodal sensory association areas. Although some evidence for the projection from visual unimodal cortex to the EC exists, inputs from other visual and auditory unimodal association areas, and the possibility of their convergence with polymodal input in the EC remains largely undisclosed. We studied 10 Macaca fascicularis monkeys in which cortical deposits of the anterograde tracer biotinylated dextran-amine were made into different portions of visual and auditory unimodal association cortices in the temporal lobe, and in polymodal association cortex at the upper bank of the superior temporal sulcus. Visual and auditory unimodal as well as polymodal cortical areas projected to the EC. Both visual unimodal and polymodal association cortices presented dense projections, while those from unimodal auditory association cortex were more patchy and less dense. In all instances, the projection distributed in both the superficial and deep layers of the EC. However, while polymodal cortex projected to all layers (including layer I), visual unimodal cortex did not project to layer I, and auditory unimodal cortex projected less densely, scattered through all layers. Topographically, convergence from the three cortical areas studied can be observed in the lateral rostral and lateral caudal subfields. The present study suggests that unimodal and polymodal association cortical inputs converge in the lateral EC, thereby providing the possibility for the integration of complex stimuli for internal representations in declarative memory elaboration.

Topographical and laminar distribution of cortical input to the monkey entorhinal cortex.

Hippocampal formation plays a prominent role in episodic memory formation and consolidation. It is likely that episodic memory representations are constructed from cortical information that is mostly funnelled through the entorhinal cortex to the hippocampus. The entorhinal cortex returns processed information to the neocortex. Retrograde tracing studies have shown that neocortical afferents to the entorhinal cortex originate almost exclusively in polymodal association cortical areas. However, the use of retrograde studies does not address the question of the laminar and topographical distribution of cortical projections within the entorhinal cortex. We examined material from 60 Macaca fascicularis monkeys in which cortical deposits of either (3)H-amino acids or biotinylated dextran-amine as anterograde tracers were made into different cortical areas (the frontal, cingulate, temporal and parietal cortices). The various cortical inputs to the entorhinal cortex present a heterogeneous topographical distribution. Some projections terminate throughout the entorhinal cortex (afferents from medial area 13 and posterior parahippocampal cortex), while others have more limited termination, with emphasis either rostrally (lateral orbitofrontal cortex, agranular insular cortex, anterior cingulate cortex, perirhinal cortex, unimodal visual association cortex), intermediate (upper bank of the superior temporal sulcus, unimodal auditory association cortex) or caudally (parietal and retrosplenial cortices). Many of these inputs overlap, particularly within the rostrolateral portion of the entorhinal cortex. Some projections were directed mainly to superficial layers (I-III) while others were heavier to deep layers (V-VI) although areas of dense projections typically spanned all layers. A primary report will provide a detailed analysis of the regional and laminar organization of these projections. Here we provide a general overview of these projections in relation to the known neuroanatomy of the entorhinal cortex.

Reciprocal connections between olfactory structures and the cortex of the rostral superior temporal sulcus in the Macaca fascicularis monkey.

Convergence of sensory modalities in the nonhuman primate cerebral cortex is still poorly understood. We present an anatomical tracing study in which polysensory association cortex located at the fundus and upper bank of the rostral superior temporal sulcus presents reciprocal connections with primary olfactory structures. At the same time, projections from this polysensory area reach multiple primary olfactory centres. Retrograde (Fast Blue) and anterograde (biotinylated dextran-amine and 3H-amino acids) tracers were injected into primary olfactory structures and rostral superior temporal sulcus. Retrograde tracers restricted to the anterior olfactory nucleus resulted in labelled neurons in the rostral portion of the upper bank and fundus of superior temporal sulcus. Injections of biotinylated dextran-amine at the fundus and upper bank of the superior temporal sulcus confirmed this projection by labelling axons in the dorsal and lateral portions of the anterior olfactory nucleus, as well as piriform, periamygdaloid and entorhinal cortices. Retrograde tracer injections at the rostral superior temporal sulcus resulted in neuronal labelling in the anterior olfactory nucleus, piriform, periamygdaloid and entorhinal cortices, thus providing confirmation of the reciprocity between primary olfactory structures and the cortex at the rostral superior temporal sulcus. The reciprocal connections between the rostral part of superior temporal sulcus and primary olfactory structures represent a convergence for olfactory and other sensory modalities at the cortex of the rostral temporal lobe.

Self-directed learning in the Gross Anatomy medical curriculum

The Medical School of the University of Castilla-La Mancha (UCLM) in Albacete is the most recentSchool of Medicine approved in Spain. The Institutionwas launched in the academic year 1998-99 with the specific aim of implementing educational innovations in the medical curriculum. The ultimate goal is to provide future doctors with the competences and skills for medicalpractice among the people of the region of Castilla-La Mancha, and Spanish society in general, by providing the means for easy integration into the job market of our society. The medical curriculum at UCLM, as in any other medical school in the country, is six years long and is divided into a basic sciences part (first to third years) and clinical sciences part (fourth to sixth years). Theteaching method of the UCLM Medical School departs from most Medical Schools in Spain by incorporating the most recent educational trends and technological advances, lead and directed by a Medical Education Unit. The UCLM Medical School organizes its medical curriculum accordingto two different, but not mutually exclusive, educational approaches: 1. self-directed learning, organized in modules of objectives (basic sciences), and 2. problem-based learning (PBL, for the clinical sciences). The ultimate goal of the curriculum is an integration of basic and clinical disciplines, both among courses in each year of the medical curriculum and among the different years of the degree at both the preclinical and clinical levels. Likewise, maximal interaction between Faculty and students is strongly encouraged, and indeedfacilitated by restricting the number of new studentsper year to a maximum number of 80, divided into four groups of 20 students (basic sciences), and 6 students in the clinical sciences. Gross Anatomy courses are given in the first and second years. During the first year, the locomotor system is presented as a 10-credit course(one credit equals 10 hours of teaching activity). During the second year, Anatomy and Embryology are integrated as a single course, along with Physiology and Histology, comprising 70 credits altogether. In both instances, the contents are organized into modules of objectives two tothree weeks long. Each module is divided into five phases. Phase 1 includes an introduction to the objectives and its resources (books, anatomicalCD programs, and other educational material), in order to help the student to accomplish the objectives. Phase 2 is a self-learning period, followed by Phase 3, in which the students expound on and discuss the contents related tothe objectives. Phase 4 is another period for self-learning and tutorials, while Phase 5 is the evaluation of individual or several thematically related modules.In Gross Anatomy, practical courses are interwoven in the modules in phases 2 and 4. In addition, this past year we have introduced 4 lectures per year in which the students attend to more general and clinical aspects of severalmodules of objectives. It is important to point out that in addition to the regular practical hours and learning periods, students carry out two gross anatomical dissections per year with the help of handouts and other reference material, after which they present a written report that is a percentage of the final score. Throughout the program, both the autonomy and interests of the students are emphasized. Here, preliminary theoretical and practical results will be discussed

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Postnatal development of calcium-binding proteins immunoreactivity (parvalbumin, calbindin, calretinin) in the human entorhinal cortex.

The entorhinal cortex is an essential component in the organization of the human hippocampal formation related to cortical activity. It transfers, neocortical information (ultimately distributed to the dentate gyrus and hippocampus) and receives most of the hippocampal output directed to neocortex. At birth, the human entorhinal cortex presents similar layer organization as in adults, although layer II (cell islands) and upper layer III have a protracted maturation. The presence of interneurons expressing calcium-binding proteins (parvalbumin, calbindin-D28K (calbindin) and calretinin) is well documented in the adult human entorhinal cortex. In many of them the calcium binding is co-localized with GABA. Parvalbumin-immunoreactive cells and fibers were virtually absent at birth, their presence increasing gradually in deep layer III, mostly in the lateral and caudal portions of the entorhinal cortex from the 5th month onwards. Calbindin immunoreactive cells and fibers were present at birth, mainly in layers II and upper III; mostly at rostral and lateral portions of the entorhinal cortex, increasing in number and extending to deep layers from the 5th month onwards. Calretinin immunoreactivity was present at birth, homogeneously distributed over layers I, II and upper V, throughout the entorhinal cortex. A substantial increase in the number of calretinin neurons in layer V was observed at the 5th month. The postnatal development of parvalbumin, calbindin and calretinin may have an important role in the functional maturation of the entorhinal cortex through the control of hippocampal, cortical and subcortical information.

Stereological study of normal and apoptotic cell populations in the adrenal gland in calves.

Morphometrical features of the adrenal gland in calves were assessed using stereological methods for normal and apoptotic cells. Adrenal glands were obtained from calves (n = 16) after they were slaughtered in a commercial abattoir. Quantitative data of nuclear size showed the highest values in the noradrenaline-storing region for all parameters, whereas the nuclei of the zona fasciculata were significantly larger than those of the other cortical regions. Data of nuclear size and shape of apoptotic cells reflected their morphometrical features; as expected, a notable decrease in the nuclear size and a more irregular shape were found in apoptosis with respect to the normal nuclei in all adrenal regions. The volume fraction data supplied the percentage of the adrenal gland occupied by each histological zone, highlighting the zona fasciculata as being the most extensive one. A significant increase in V(V)(nucl/tis), S(V)(nucl/tis), and Nv(nucl/tis) was observed in cortical regions with respect to the medullar regions. A comparison between normal and apoptotic nuclei indicated a highly significant decrease in V(V)(nucl/tis), S(V)(nucl/tis), and Nv(nucl/tis) for the apoptotic nuclei in all the adrenal zones. The apoptotic nuclei figures found in all the adrenal regions suggest that a process of cell renovation occurred in all the zones, although the higher number of apoptosis in zona reticularis is in agreement with previous results that have pointed out this zone as being a cell senescence region. The results of discriminant analyses gave the parameter required and the percentage of correct classifications for discriminating between normal nuclei, between apoptotic nuclei and between normal and apoptotic nuclei.

Morphological and quantitative study of the Leydig cells of pigs fed with anabolic doses of clenbuterol.

The effects of clenbuterol administered at anabolic doses on the testicular interstitium were studied in 30 pigs allocated to three experimental groups. The diet of two groups was supplemented with clenbuterol (Clb) (1 ppm), but whereas in the Clb+ group the treatment was given until slaughter (treatment period: 3 months), in the Clb- group the clenbuterol was withdrawn 2 weeks before slaughter (treatment period: 2-5 months); in the control group, the pigs were fed without clenbuterol. For histological procedures, a fractional sampling scheme was applied and routine techniques for light and transmission electron microscopy were used. The results of subjective morphology and morphometrics showed slight differences between the treated and the control groups. Conversely, the stereological results identified a prominent hyperplasia of the Leydig cells and ultrastructural analysis of these cells revealed a conspicuous increase in the organelles related to testosterone production, suggesting a functional activation of the interstitial cells in response to the clenbuterol treatment.

Stereological study of the adrenal gland of calves treated with clenbuterol.

Stereological methods were used to investigate the morphometric variations in the adrenal cell population of calves dosed orally with clenbuterol at an anabolic dose of 20 microg/kg bodyweight per day for 12 weeks. In the treated group the size of the nuclei increased with respect to the control group, and the increases were significant for the maximum linear parameters in all the regions except the zona fasciculata. The largest increases were observed in the epinephrine-producing cells. The quantitative stereological data showed highly significant increases in the nuclear volume fraction, the nuclear surface density and the numerical density in the zona fasciculata of the treated group.

Nuclear volume estimates in prostatic intraepithelial neoplasia.

OBJECTIVE: Prostatic intraepithelial neoplasia (PIN), the most likely precursor of prostatic adenocarcinoma, is divided into two grades, low and high. Pathologists may encounter difficulties in applying these criteria in daily practice. In view of the clinical significance of high grade PIN as strong predictor of carcinoma, the separation of low and high grade PIN plays an important role in patient management. The aim of the present study was to evaluate three-dimensional nuclear size estimation in normal prostatic glands, low and high grade PIN, and prostatic adenocarcinoma as an element in their classification. STUDY DESIGN: We studied 31 formalin-fixed, paraffin-embedded, whole-mounted radical prostastectomy specimens that contained foci of normal prostatic glands, low and high grade PIN, and prostatic adenocarcinoma. Hematoxylin-eosin-stained sections were selected for the stereologic estimation of volume-weighted mean nuclear volume by the "point-sampled intercepts" method. On each focus, an average of six fields of vision were systematically chosen. RESULTS: The quantitative results indicate a significant increase in nuclear volume from normal prostatic glands (mean, 209.0 micron 3; SD, 64.6 micron 3) to low grade PIN, high grade PIN and prostatic adenocarcinoma with increments of 49%, 88% and 109%, respectively (F = 29.1, P < .001). Two-group comparisons (Duncan procedure) showed differences between low and high grade PIN and prostatic adenocarcinoma (P < .01). The difference between high grade PIN and prostatic adenocarcinoma was not significant. CONCLUSION: Three-dimensional estimates of nuclear size discriminate low and high grade PIN. Lack of stereologic differences between high grade PIN and prostatic adenocarcinoma further supports high grade PIN as a precursor of prostatic adenocarcinoma.

Nuclear volume estimates in prostatic atypical adenomatous hyperplasia.

OBJECTIVE: To investigate nuclear volume estimates by the point-sampled intercepts method in atypical adenomatous hyperplasia (AAH) as compared with nodular hyperplasia and well-differentiated prostatic adenocarcinoma. STUDY DESIGN: The study group consisted of 27 formalin-fixed, paraffin-embedded, whole-mounted radical prostatectomy specimens that contained foci of nodular hyperplasia, atypical adenomatous hyperplasia and well-differentiated adenocarcinoma (Gleason pattern 1 and 2). Representative sections were selected for stereologic estimation of volume-weighted mean nuclear volume by the point-sampled intercepts method. On each focus, an average of five fields of vision were systematically chosen. RESULTS: The quantitative results indicate an increase in nuclear volume from nodular hyperplasia (209 +/- 65 micron 3) to AAH (237 +/- 85 micron 3) and prostate adenocarcinoma (436 +/- 106 micron 3). Significant differences were found (F = 39.0, P < .001) with two group comparisons (Scheffe's procedure) between prostate cancer and AAH (P < .001) or nodular hyperplasia (P < .001). The difference between AAH and benign hyperplasia was not signifcant. CONCLUSION: The results indicate that three-dimensional estimates of the nuclear size discriminate AAH and nodular hyperplasia from well-differentiated prostate adenocarcinoma. These findings suggest that AAH is probably a histologic variant of benign prostatic hyperplasia the exact relationship of which to prostatic adenocarcinoma remains to be determined.

Application of the fractionator and vertical slices to estimate total capillary length in skeletal muscle.

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.

Unbiased stereological estimation of the number and volume of nuclei and nuclear size variability in invasive ductal breast carcinomas.

The application of design-based stereological methods for estimating nuclear features quantitatively in invasive ductal breast cancer is described. Nuclear number, size and size variability are explored in relation to the tumour grade and patient prognosis. The study includes an examination of the efficiency in estimating different nuclear volumes, and two different estimators of the nuclear size variability are contrasted. Forty-two invasive ductal breast carcinomas diagnosed and graded by two pathologists were used. Both 5-microns and 25-microns-thick sections were obtained from paraffin blocks for stereological study. More undifferentiated tumours show significantly larger nuclei than low-grade tumours. The estimates based on the disector method demonstrate a decrease in the number of tumour cell nuclei per unit volume of tissue from grades 1 to 2 and especially from grades 2 to 3. The univariate survival analysis shows a high prognostic value of the nuclear volume estimates. The study shows that an efficient sampling procedure was performed, particularly when estimating volume-weighted mean nuclear volume using the point-sampled intercepts method. This method is more efficient than estimation of the number-weighted mean nuclear volume using the selector method; however, the latter provides paired estimates of volume- and number-weighted mean nuclear volume, as well as an estimate of the coefficient of variation of nuclear volume in the number distribution of the same cells.

Objective differential classification of thyroid lesions by nuclear quantitative assessment.

Quantitative nuclear parameters estimated by morphometric and stereological methods in combination with discriminant analysis were used in order to evaluate the diagnostic efficiency of thyroid lesions. This study includes 55 patients with thyroid pathology. Samples of follicular adenomas, follicular carcinomas, and papillary carcinomas were examined by image analysis to obtain size and form nuclear parameters. Stepwise discriminant analyses were performed. There was an increase in nuclear size from follicular adenomas to follicular carcinomas, and a greater increase from follicular carcinomas to papillary carcinomas. The increase was more significant when the three-dimensional estimates of the volume-weighted mean nuclear volume were assessed. No significant differences between follicular adenomas and follicular carcinomas were found with respect to the nuclear form factors, however, a significant increase in nuclear elongation and irregularity was registered between follicular and papillary tumors (p < 0.01). The overall accuracy rate of discrimination was 75% when the three lesions were included in the analysis, with an efficiency of 85% for papillary carcinoma samples. These percentages increased when two lesion discrimination was performed. The worst discrimination (69% of efficiency) was found between follicular adenomas and follicular carcinomas.

Quantitative histopathologic discrimination of rheumatic disorders using nuclear measurements of synoviocytes.

OBJECTIVE: To use several nuclear quantitative estimators of synoviocytes and advanced statistical tests to discriminate between rheumatic disorders with the purpose of providing an objective histopathologic differentiation and to illustrate the difficulty of establishing the implications of the vague diagnosis of chronic nonspecific synovitis. STUDY DESIGN: Synovial histologic material from 48 patients, including a control group and those diagnosed with osteoarthritis, rheumatoid arthritis and chronic nonspecific synovitis, was analyzed. Both morphometric and stereologic nuclear measurements were assessed in each case, and several stepwise discriminant analyses were performed to obtain linear discriminant functions. RESULTS: Differences in the nuclear size parameters were found between rheumatoid arthritis and the normal and osteoarthritis groups and between normal and chronic nonspecific synovitis. The volume-weighted mean nuclear volume was the most significant parameter. No differences were demonstrated between osteoarthritis and normal and chronic nonspecific synovitis or between rheumatoid arthritis and chronic nonspecific synovitis except for nuclear volume. Using stepwise discriminant analysis, 94% of the cases were correctly classified when differentiating the normal, osteoarthritis and rheumatoid arthritis groups. The overall accuracy of diagnosis decreased to 83% when chronic nonspecific synovitis samples were included. The misclassified samples were related mainly to normal cases and to osteoarthritis and chronic nonspecific synovitis cases. CONCLUSION: This study emphasized that nuclear quantitative features of synoviocytes may be useful in differentiating rheumatic disorders objectively, especially in combination with discriminant analysis. Thus, nuclear changes in the synovium in chronic nonspecific synovitis show a closer similarity to normal and osteoarthritis than to rheumatoid arthritis.

Grading and prognosis of infiltrating ductal breast carcinoma by mean nuclear volume estimates.

OBJECTIVE: Quantitative evaluation of nuclear size in breast cancer is performed with the aim of investigating whether the data obtained are related to subjective grading and prognosis. STUDY DESIGN: Unbiased stereologic estimates of the volume-weighted mean nuclear volume (nuclear vv) in paraffin-embedded tissue from 44 specimens of infiltrating ductal breast carcinoma were obtained by the point-sampled intercepts method. RESULTS: Histologically graded cases showed an overlap in nuclear vv estimates, but significant differences were demonstrated in which averaged values increased from grade 1 to 3 breast carcinomas. Nuclear vv estimates in grade 3 breast carcinoma (mean, 495.4 microns 3; SD, 101.7 microns 3) were significantly larger than those in grades 1 (mean, 327.8 microns 3; SD, 90.9 microns 3) and 2 (mean, 409.9 microns 3; SD, 72.6 microns 3) (P < .01). Minor statistically significant differences (P < .05) were found between grades 1 and 2. Estimates of nuclear vv based on systematic sampling within the specimen showed high efficiency, with > 80% of the total observed variance contributing to biologic differences between individual specimens. Univariate prognostic study showed that clinical stage, and not histologic grade, was of prognostic value in the cases studied. Similarly, nuclear vv estimates were associated with disease recurrence and survival, with a cutoff point of 425 microns 3. CONCLUSION: Based on the cases investigated, shape-independent nuclear vv estimates may be an adjuvant tool in the grading and prognostic evaluation of infiltrating ductal breast carcinoma.

Application of recent stereological tools for unbiased three-dimensional estimation of number and size of nuclei in renal cell carcinoma samples.

New stereological methods have appeared in recent years that allow an unbiased and efficient estimation of quantitative characteristics of three-dimensional biological structures. The aim of this study is to apply the new stereological tools on renal tissue and investigate the changes in the number and size of nuclei in renal cell carcinomas. The simple and easy use of the disector, point-sampled intercepts and selector methods on kidney biopsies is demonstrated. Using these approaches, the mean number of nuclei per unit reference volume: NV(nucl/tis), the volume-weighted mean nuclear volume: nu V(nucl) and the number-weighted mean nuclear volume: nu N(nucl) were estimated in histological sections from 20 renal cell carcinomas. The study was performed on 10 grade 2 and 10 grade 3 renal cell carcinomas previously diagnosed by the pathologist on the basis of nuclear size increase, irregularity and nucleolar prominence. Basically, serial optical kidney sections from a 25-millimicrons thick slice were used for stereological study. The size estimations of the individual particles nu V(nucl) and nu N(nucl) obtained by the 'point-sampled intercepts' and 'selector' methods, respectively, turned out to be the most adequate parameters employed in discriminating between renal carcinoma grades (P less than or = 0.001).

Estimation of capillary length density in skeletal muscle by unbiased stereological methods: I. Use of vertical slices of known thickness.

BACKGROUND: The unbiased estimation of the capillary length density in skeletal muscle tissue Lv(cap/mus) has been performed in this study applying a new stereological methodology based on the use of vertical slices and the intersections of cycloid test curves with capillaries in a three-dimensional space defined by systematically chosen fields of vision and the thickness of the sections. METHODS: The following simple requirements must be fulfilled: selection of a fixed vertical axis in skeletal muscle, adequate systematic muscle sampling, obtention of vertical slices of constant and known thickness but indifferent in magnitude, superposition of a cycloid test system with the minor axis of cycloid curves positioned perpendicularly to the vertical axis, and counting the intersections between cycloid curves and capillaries. In our study, the vertical axis was defined as that which is parallel to the natural, major axis of the muscle where fibres and capillaries are arranged parallel to this axis. The muscle sampling was performed using the fractionator method, and 25 microns thick sections were chosen. RESULTS: The application of the equation for estimation of Lv(cap/mus) permits determination of an average of 1,480 mm of capillaries per mm3 of muscle tissue, knowing the number of intersections, section thickness, and the points hitting the muscle with a known ratio between cycloid test curve length to a test point. CONCLUSIONS: The estimation of Lv(cap/mus) is efficient, unbiasedly obtained, and no assumptions on the degree of capillary anisotropy are required.