A Metric Fractionator for Estimation of Total Cell Number in Paraffin-Embedded Flat or Hollow Organs.

The physical fractionator is a convenient and practical solution for estimation of total cell number in a regulatory toxicology setting because it is insensitive to shrinkage allowing for paraffin processing/embedding and does not require measurement of the reference or organ volume. The principle involves sampling a known fraction of an organ in one or more steps and counting the total number of cells present in the final sample, physical disector section pairs. The total cell number in the organ is estimated by multiplying the cell count in the final fraction by the inverse of the sampling fraction(s). The key feature of the design is that tissue shrinkage due to paraffin processing occurs before the organ is uniformly sampled. Another requirement is that thermal expansion or contraction is avoided during the preparation of disector sections from the individual embedded subsamples, which ensures that the disector sections represent a known constant fraction. This vertical physical fractionator with subsampling is a simple and fast estimator to obtain precise and robust estimates of total cell number in large flat or hollow organs that do not prolong routine necropsy procedures. It is compatible with paraffin processing, avoids exhaustive sectioning, and allows for the collection of routine histopathology sections.

Bone-to-implant contact after maxillary sinus floor augmentation with Bio-Oss and autogenous bone in different ratios in mini pigs.

OBJECTIVES: The objective was to test the hypotheses: (i) no differences in bone-to-implant contact formation, and (ii) no differences between the use of autogenous mandibular or iliac bone grafts, when autogenous bone, Bio-Oss mixed with autogenous bone, or Bio-Oss is used as graft for the maxillary sinus floor augmentation. MATERIAL AND METHODS: Bilateral sinus floor augmentation was performed in 40 mini pigs with: (A) 100% autogenous bone, (B) 75% autogenous bone and 25% Bio-Oss, (C) 50% autogenous bone and 50% Bio-Oss, (D) 25% autogenous bone and 75% Bio-Oss, or (E) 100% Bio-Oss. Autogenous bone was harvested from the iliac crest or the mandible and the graft composition was selected at random and placed concomitant with the implant placement. The animals were euthanized 12 weeks after surgery. Bone-to-implant contact was estimated by stereological methods and summarized as median percentage with 95% confidence interval (CI). Bone-to-implant contact formation was evaluated by fluorochrome labelling and assessed by median odds ratios (OR) with 95% (CI). RESULTS: Median bone-to-implant contact was: (A) 42.9% (95% CI: 32.1-54.5%), (B) 37.8% (95% CI: 27.1-49.9%), (C) 43.9% (95% CI: 32.6-55.9%), (D) 30.2% (95% CI: 21.6-40.3%), and (E) 13.9% (95% CI: 11.4-16.9%). Bone-to-implant contact was significantly higher for A, B, C, D as compared to E (P < 0.0001). Bone-to-implant contact was not significantly influenced by the ratio of Bio-Oss and autogenous bone (P = 0.19) or the origin of the autogenous bone (P = 0.72). Fluorochrome labelling revealed extensive variation in bone-to-implant contact formation over time. The labelling at weeks 2-3 was significantly increased with A compared to E (OR = 8.1 CI: 5.0-13.1, P < 0.0001), whereas E showed a significantly increased labelling at weeks 8-9 compared to A (OR = 0.5 CI: 0.3-0.7, P = 0.0028). CONCLUSIONS: The hypothesis of no differences in bone-to-implant contact between the various treatment modalities was rejected since the bone-to-implant contact was significantly increased with autogenous bone or Bio-Oss mixed with autogenous bone as compared to Bio-Oss. Early bone-to-implant contact formation was more advanced with autogenous bone. No differences between the use of mandibular or iliac bone grafts were observed since the bone-to-implant contact was not significantly influenced by the origin of the bone graft.

Volumetric changes of the graft after maxillary sinus floor augmentation with Bio-Oss and autogenous bone in different ratios: a radiographic study in minipigs.

OBJECTIVE: The objective of the present study was to learn about the volumetric changes of the graft after maxillary sinus floor augmentation with Bio-Oss and autogenous bone from the iliac crest or the mandible in different ratios in minipigs. MATERIAL AND METHODS: Bilateral maxillary sinus floor augmentation was performed in 40 minipigs with: (A) 100% autogenous bone, (B) 75% autogenous bone and 25% Bio-Oss, (C) 50% autogenous bone and 50% Bio-Oss, (D) 25% autogenous bone and 75% Bio-Oss, and (E) 100% Bio-Oss. The autogenous bone graft was harvested from the iliac crest or the mandible and the graft composition was selected at random and placed concomitant with implant placement. Computed tomographies of the maxillary sinuses were obtained preoperatively, immediately postoperatively, and at euthanasia after 12 weeks. The volumetric changes of the graft were estimated using the Cavalieri principle and expressed as mean percentage with a 95% confidence interval (CI). RESULTS: The mean volume of the graft was reduced by (A) 65% (95% CI: 60-70%), (B) 38% (95% CI: 35-41%), (C) 23% (95% CI: 21-25%), (D) 16% (95% CI: 12-21%), and (E) 6% (95% CI: 4-8%). The volumetric reduction was significantly influenced by the ratio of Bio-Oss and autogenous bone (P<0.001), but not by the origin of the autogenous bone graft (P=0.2). CONCLUSIONS: The volume of autogenous bone grafts from the iliac crest and the mandible is reduced significantly after maxillary sinus floor augmentation in minipigs. The graft volume is better preserved after the addition of Bio-Oss and the volumetric reduction is significantly influenced by the ratio of Bio-Oss and autogenous bone. However, further studies are needed addressing the amount of new bone formation and bone-to-implant contact before the final conclusion can be made about the optimal ratio of Bio-Oss and autogenous bone.

Continuing education course #3: current practices and future trends in neuropathology assessment for developmental neurotoxicity testing.

The continuing education course on Developmental Neurotoxicity Testing (DNT) was designed to communicate current practices for DNT neuropathology, describe promising innovations in quantitative analysis and noninvasive imaging, and facilitate a discussion among experienced neuropathologists and regulatory scientists regarding suitable DNT practices. Conventional DNT neuropathology endpoints are qualitative histopathology and morphometric endpoints of particularly vulnerable sites (e.g., cerebral, cerebellar, or hippocampal thickness). Novel imaging and stereology measurements hold promise for automated analysis of factors that cannot be effectively examined in routinely processed specimens (e.g., cell numbers, fiber tract integrity). The panel recommended that dedicated DNT neuropathology data sets be acquired on a minimum of 8 sections (for qualitative assessment) or 3 sections (for quantitative linear and stereological analyses) using a small battery of stains to examine neurons and myelin. Where guidelines permit discretion, immersion fixation is acceptable for younger animals (postnatal day 22 or earlier), and peripheral nerves may be embedded in paraffin. Frequent concerns regarding DNT data sets include false-negative outcomes due to processing difficulties (e.g., lack of concordance among sections from different animals) and insensitive analytical endpoints (e.g., qualitative evaluation) as well as false-positive results arising from overinterpretation or misreading by inexperienced pathologists.

Design-based stereology: introduction to basic concepts and practical approaches for estimation of cell number.

In regulatory toxicology studies, qualitative histopathological evaluation is the reference standard for assessment of test article-related morphological changes. In certain cases, quantitative analysis may be required to detect more subtle morphological changes, such as small changes in cell number. When the detection of subtle test article-related morphological changes is critical to the decision-making process, sensitive quantitative methods are needed. Design-based stereology provides the tools for obtaining accurate, precise quantitative structural data from tissue sections. These tools have the sensitivity necessary to detect small changes by combining statistical sampling principles with geometric analysis of the tissue microstructure. It differs from other morphometric methods based on tissue section analysis by providing estimates that are statistically valid, truly three-dimensional, and referent to the entire organ. Further, because the precision of the stereological analysis procedure can be predicted, studies can be designed and powered to detect subtle, potentially toxicologically significant changes. Although stereological methods have not been widely applied in toxicologic pathology, recent advances have made it feasible to implement these methods in a regulatory toxicology setting, particularly methods for estimation of total cell number.

Design-based stereological analysis of the lung parenchymal architecture and alveolar type II cells in surfactant protein A and D double deficient mice.

Alveolar epithelial type II cells synthesize and secrete surfactant. The surfactant-associated proteins A and D (SP-A and SP-D), members of the collectin protein family, participate in pulmonary immune defense, modulation of inflammation, and surfactant metabolism. Both proteins are known to have overlapping as well as distinct functions. The present study provides a design-based stereological analysis of adult mice deficient in both SP-A and SP-D (A(-)D(-)) with special emphasis on parameters characterizing alveolar architecture and surfactant-producing type II cells. Compared to wild-type, A(-)D(-) mice have fewer and larger alveoli, an increase in the number and size of type II cells, as well as more numerous and larger alveolar macrophages. More surfactant-storing lamellar bodies are seen in type II cells, leading to a threefold increase in the total volume of lamellar bodies per lung, but the mean volume of a single lamellar body remains constant. These results demonstrate that chronic deficiency of SP-A and SP-D in mice leads to parenchymal remodeling, type II cell hyperplasia and hypertrophy, and disturbed intracellular surfactant metabolism. The design-based stereological approach presented here provides a framework for the quantitative lung structure analysis in gene-manipulated mice as well as in human lung disease.

Aging of the human cerebellum: a stereological study.

Cerebella from 19 normal Caucasian males, ages 19-84 years, were studied using stereological methods. Cerebellum was divided into four different regions: the anterior and posterior lobe, the vermis, and the flocculonodular lobe. Total volume of the cerebellar cortex and white matter, cerebellar surface area, total Purkinje and granule cell number, and the distribution of the volumes of the Purkinje cells and their nuclei were estimated in all four regions. The global white matter was reduced by 26% with age; the mean volume of the Purkinje cell body was decreased by 33% with no decrease in the volume of the Purkinje cell nuclei. A tendency towards a 16% total cerebellar volume loss was seen without a concomitant neuron loss. No global Purkinje or granule cell loss was detected with age, total Purkinje cell number being 28 x 10(6) (coefficient of variation, CV = 0.16) and total granule cell number 109 x 10(9) (CV = 0.17). However, a significant change was observed with age in the anterior lobe, where a selective 40% loss of both Purkinje and granule cells was found. Furthermore, a 30% loss of volume, mostly due to a cortical volume loss, was recorded in the anterior lobe, which is predominantly involved in motor control.

Aging and the human neocortex.

Neurostereology has been applied to quantitative anatomical study of the human brain. Such studies have included the total neocortical number of neurons and glial cells, the estimated size distribution of neocortical neurons, the total myelinated fiber length in the brain white matter, the total number of synapses in the neocortex, and the effect of normal aging on these structural elements. The difference in total number of neurons was found to be less than 10% over the age range from 20 to 90 years, while the glial cell number in six elderly individuals, mean age 89.2 years, showed an average number of 36 billion glial cells, which was not statistically significantly different from the 39 billion glial cells in the neocortex of six young individuals with a mean age of 26.2 years. The total myelinated fiber length varied from 150,000 to 180,000 km in young individuals and showed a large reduction as a function of age. The total number of synapses in the human neocortex is approximately 0.15 x 10(15) (0.15 quadrillion). Although the effect of aging is seen in all estimated structural elements, the effect of sex is actually higher. The functional relevance of these differences in neuron numbers in both age and gender is not known.

Assessment of air space size characteristics by intercept (chord) measurement: an accurate and efficient stereological approach.

The mean linear intercept (chord) length (L(m)) is a useful parameter of peripheral lung structure as it describes the mean free distance in the air spaces. It is often misinterpreted as a measure of "alveolar size," and its estimation is fraught with a number of pitfalls. We present two methods for the accurate estimation of L(m): 1) the indirect method, which derives L(m) from the volume-to-surface ratio of air spaces estimated by point counting methods, and 2) the direct method, which uses a set of random intercepts and calculates L(m) from their frequency distribution, for which we introduce a new and accurate method. Both methods are efficient and, with proper precautions, unbiased. The meaning of L(m) is assessed in two different examples. In a physiological study, the effect of different inflation levels is studied, showing that L(m) critically depends on lung inflation. In an experimental study on emphysema-like changes in a genetic mouse model, the effect of heterogeneity of air space size is assessed; these results are obtained partly because of differences in lung volume due to altered recoil in the emphysematous lungs. In conclusion, although L(m) is not a robust parameter of internal lung structure because it crucially depends on lung volume, it is still a valid measure for which accurate and efficient methods are available that yield additional parameters such as size distribution or alveolar surface area.

Stereological estimates of alveolar number and size and capillary length and surface area in mice lungs.

The major function of the lung is gas exchange and depends on alveolar and capillary parameters such as surface area and volume. The number of alveoli may report on the nature of structural changes in lung parenchyma during development, illness or changing environmental factors. We therefore developed an efficient and easily applicable stereological design for estimating and monitoring these structural parameters in the mouse lung. The estimation of volume fractions of different lung compartments has been carried out by point counting. A combination of cycloid grids superimposed on vertical sections was used to estimate the capillary surface area with isotropic test lines. Capillary length could be measured using the harmonic mean of the surface weighted diameter. The Euler characteristic applied in the physical fractionator with varying but known sampling fractions (Horovitz-Thompson estimator) enabled us to estimate alveolar number. In adult mice lungs, we obtained total values for alveolar number of 2.31 x 10(6) alveoli in a pair of lungs, alveolar surface area of 82.2 cm(2), capillary surface area of 124 cm(2), and capillary length of 1.13 km. All values are corrected for tissue shrinkage. With this study we present a highly efficient combination of several design-based stereological tools for the unbiased estimation of alveolar number and volume as well as length, surface area, and diameter of capillaries in the mice lung. Anat Rec, 292:113-122, 2009. (c) 2008 Wiley-Liss, Inc.

An empirical analysis of the precision of estimating the numbers of neurons and glia in human neocortex using a fractionator-design with sub-sampling.

Improving histomorphometric analysis of the human neocortex by combining stereological cell counting with immunohistochemical visualisation of specific neuronal and glial cell populations is a methodological challenge. To enable standardized immunohistochemical staining, the amount of brain tissue to be stained and analysed by cell counting was efficiently reduced using a fractionator protocol involving several steps of sub-sampling. Since no mathematical or statistical tools exist to predict the variance originating from repeated sampling in complex structures like the human neocortex, the variance at each level of sampling was determined empirically. The methodology was tested in three brains analysing the contribution of the multi-step sampling procedure to the precision on the estimated total numbers of immunohistochemically defined NeuN expressing (NeuN(+)) neurons and CD45(+) microglia. The results showed that it was possible, but not straight forward, to combine immunohistochemistry and the optical fractionator for estimation of specific subpopulations of brain cells in human neocortex.

The application of stereological methods for estimating structural parameters in the human heart.

This study describes and exemplifies generally applicable design-based stereological methods for obtaining quantitative estimates of the numbers and sizes of capillaries, cardiomyocytes, and cardiomyocyte nuclei in immersion-fixed human left ventricles (N = 6). The design-based stereological methods are valid in all cardiac investigations onto quantifying changes in structure and function as seen under various conditions such as during development, aging, hypertrophy, and following ischemia/reperfusion. The applied principles of unbiased stereology were as follows: 1) uniform random sampling was taken at all levels, also in respect to orientations, for estimates of length and mean sizes. 2) All global structural quantities were estimated as total quantity = density x volume of the left ventricle. As an example, the left ventricle contains 1.5 x 10(9) capillaries with a total length of just below 200 km. 3) Stereological methods were used for estimating the volume density, surface area density, and length density of capillaries and cardiomyocytes. The numerical density of cardiomyocyte nuclei and capillaries was estimated, using the optical and physical disector, respectively. 4) In all local quantities, "size" was estimated either directly, using unbiased estimators to obtain the average individual size and size distribution parameters, or indirectly, using the relationship that: average size = total quantity/total number. In the six hearts constituting this study, we observed the anticipated correlation between left ventricular volume and global estimates such as total number of capillaries. There were no correlation between local quantities and total left ventricular volume (e.g., average star volume of individual cardiomyocytes).

Stereological methods for estimating the myelin sheaths of the myelinated fibers in white matter.

In the present study, efficient and unbiased stereological techniques to investigate the myelin sheaths of the myelinated fibers in rat white matter were established. In the present design, four tissue blocks were obtained from the entire white matter of rat brain in a uniform, random fashion. Isotropic, uniform random (IUR) sections were ensured by the use of the isector technique. One section with the thickness of 60 nm was cut from the center of each epon block. Eight to 10 fields of vision were randomly photographed under a transmission electron microscope. The total length of the myelinated fibers and the total volume of the myelin sheaths in the white matter were the products of the length density, volume density, and the volume of the white matter obtained with the Cavalieri principle. The mean areas of the myelinated fibers profiles and myelin sheaths were estimated with the point counting technique. The inner and outer perimeters of the myelin sheaths were estimated by the use of a line grid, and the thickness of the myelin sheaths was estimated by direct orthogonal measurements in uniform, random locations. The described methods will provide very useful tools for future quantitative studies of changes in the myelin sheaths of white matter in various experimental conditions and in various neurodegenerative diseases.

Pyramidal neuron number in layer 3 of primary auditory cortex of subjects with schizophrenia.

Individuals with schizophrenia demonstrate impairments of sensory processing within primary auditory cortex. We have previously identified lower densities of dendritic spines and axon boutons, and smaller mean pyramidal neuron somal volume, in layer 3 of the primary auditory cortex in subjects with schizophrenia, all of which might reflect fewer layer 3 pyramidal neurons in schizophrenia. To examine this hypothesis, we developed a robust stereological method based upon unbiased principles for estimation of total volume and pyramidal neuron numbers for each layer of a cortical area. Our method generates both a systematic, uniformly random set of mapping sections as well as a set of randomly rotated sections cut orthogonal to the pial surface, within the region of interest. We applied our approach in twelve subjects with schizophrenia, each matched to a normal comparison subject. Primary auditory cortex volume was assessed using Cavalieri's method. The relative and absolute volume of each cortical layer and, within layer 3, the number and density of pyramidal neurons were estimated using our novel approach. Subject groups did not differ in regional volume, layer volumes, or pyramidal neuron number, although pyramidal neuron density was significantly greater in subjects with schizophrenia. These findings suggest that previously observed lower densities of dendritic spines and axon boutons reflect fewer numbers per neuron, and contribute to greater neuronal density via a reduced neuropil. Our approach represents a powerful new method for stereologic estimation of features of interest within individual layers of cerebral cortex, with applications beyond the current study.

The postnatal development of cerebellar Purkinje cells in the Göttingen minipig estimated with a new stereological sampling technique--the vertical bar fractionator.

The postnatal development of total number and perikaryon volume of cerebellar Purkinje cells was estimated in the Göttingen minipig cerebellar cortex using a new stereological approach, the vertical bar fractionator. Data were obtained from the brains of five neonate and five adult female Göttingen minipigs. The total number of Purkinje cells ranged from 1.83 x 10(6) in the neonate to 2.82 x 10(6) in the adult Göttingen minipig. The number-weighted mean perikaryon volume of Purkinje cells increased concurrently from around 6,800 microm(3) in the neonate to 17,600 microm(3) in the adult. The study demonstrates that a pronounced postnatal neurogenesis in Purkinje cell number and perikaryon volume is part of the growth and development of the cerebellum in the Göttingen minipig. The Purkinje cells of the Göttingen minipig were found to be substantially large compared with human and represents the largest cells described hitherto from mammalian cerebella. The vertical fractionator is a new sampling technique, which allows the combination of a fractionator design on vertical bar sections excluding exhaustive sampling and bias from artificial edges. By design, the sections are perfect stereological vertical sections and provide the basis for unbiased estimates of total number of structural entities in the brain, including surface area, fibre length and particle volume.

Assessment of in vivo MR imaging compared to physical sections in vitro--a quantitative study of brain volumes using stereology.

The object of the present study was to compare stereological estimates of brain volumes obtained in vivo by magnetic resonance imaging (MRI) to corresponding volumes from physical sections in vitro. Brains of ten domestic pigs were imaged using a 3-T scanner. The volumes of different brain compartments were obtained from MR images by two observers and from physical sections using the Cavalieri estimator in combination with point counting. Paired t tests revealed no significant differences between the two methods for any of the five compartments considered, except for the basal gray compartment. However, although intraobserver difference of MRI estimates was acceptable, the interobserver difference was not. A statistical highly significant difference of 11-41% was observed between observers for volume estimates of all compartments considered. The study demonstrates that quantitative MRI is susceptible to observer dependent interpretation of images.

Autogenous bone graft and ePTFE membrane in the treatment of peri-implantitis. II. Stereologic and histologic observations in cynomolgus monkeys.

The purpose of the present study was to evaluate the effect of autogenous bone graft particles and expanded polytetrafluoroethylene (ePTFE) membrane in the treatment of peri-implantitis with stereologic and histologic methods. Clinical and radiographic findings are reported elsewhere. Experimental peri-implantitis with a bone loss of 4-6 mm was established during 14-22 months around 64 implants with a titanium plasma-sprayed (TPS) surface in eight cynomolgus monkeys (Macaca fascicularis). The defects were treated with autogenous bone+membrane (B+M), autogenous bone (B), membrane (M), or a conventional flap procedure (control) (C). The animals were killed 6 months after surgery. Healthy peri-implant tissue was established irrespective of the applied treatment procedure. However, the amount of bone (autogenous bone graft particles and regenerated bone) and re-osseointegration were significantly higher in defects treated with B+M as compared with the three other treatment modalities. A mean bone-to-implant contact of 45% was estimated within defects treated with B+M. The corresponding values for the B, M, and C groups were 22, 21, and 14%. The present study therefore demonstrates that autogenous bone graft particles covered by an ePTFE membrane is a useful surgical treatment procedure of experimental peri-implantitis around implants with a TPS surface in cynomolgus monkeys. Obviously, there is a background for long-term evaluation in humans.

The number of alveoli in the human lung.

The number of alveoli is a key structural determinant of lung architecture. A design-based stereologic approach was used for the direct and unbiased estimation of alveolar number in the human lung. The principle is based on two-dimensional topology in three-dimensional space and is free of assumptions on the shape, size, or spatial orientation of alveoli. Alveolar number is estimated by counting their openings at the level of the free septal edges, where they form a two-dimensional network. Mathematically, the Euler number of this network is estimated using physical disectors at a light microscopic level. In six adult human lungs, the mean alveolar number was 480 million (range: 274-790 million; coefficient of variation: 37%). Alveolar number was closely related to total lung volume, with larger lungs having considerably more alveoli. The mean size of a single alveolus was rather constant with 4.2 x 10(6) microm3 (range: 3.3-4.8 x 10(6) microm3; coefficient of variation: 10%), irrespective of the lung size. One cubic millimeter lung parenchyma would then contain around 170 alveoli. The method proved to be very efficient and easy to apply in practice. Future applications will show this approach to be an important addition to design-based stereologic methods for the quantitative analysis of lung structure.

Detection of circulating tumor lysate-reactive CD4+ T cells in melanoma patients.

PURPOSE: We wanted to study whether an allogeneic melanoma lysate would be a feasible stimulatory antigen source for detection of a peripheral CD4+ T-cell immune response in patients with medically untreated malignant melanoma. The lysate was produced from a melanoma cell line (FM3.29) which expresses high amounts of melanoma antigens. METHODS: Fresh peripheral blood was incubated with and without lysate for 6 h in the presence of anti-CD28/anti-CD49d MoAb (for costimulation). After flow cytometric estimation of the frequency of CD69+/IFN-gamma+ cells in the CD4+ population, the response to lysate was calculated as the difference between the number of activated IFN-gamma-producing CD4+ cells in the lysate-stimulated and the nonstimulated sample. RESULTS: An immune response to lysate was observed in blood samples from 11 of 15 patients (73%) with metastatic melanoma. A weak response was found in 1 of 4 patients radically operated for localized disease, whereas no responders were seen among 7 healthy donors. The fraction of circulating lysate-activated T cells ranged from 0.0037% to 0.080% of the CD4+ population. A negative result of the assay was found occasionally, especially in donors with high background levels of spontaneous IFN-gamma production, indicating an inhibitory effect of the lysate. CONCLUSIONS: This method for detection of a peripheral T-cell immune response in melanoma patients has several advantages for clinical use. The tumor lysate preparations may contain large numbers of stimulating antigens (known, as well as unknown) and are easily prepared and handled. Potentially, the assay might be useful as a diagnostic tool, a marker of residual or recurrent disease, a prognostic factor, or a predictor or monitor of the effect of antineoplastic therapy including immune-modulating therapy.