Volume Of Cell Nuclei Research Articles

Microtopography-Induced Nuclear Deformation Triggers Chromatin Reorganization and Cytoskeleton Remodeling.

Cells can adapt to diverse topographical substrates through contact guidance, which regulates the cellular and nuclear morphologies and functions. How adaptive deformation of the cell body and nucleus coordinates to protect genetic material within mechanical microenvironments remains poorly understood. In this study, we engineered micrometer-level narrow-spacing micropillars to mimic constricted extracellular topographies in vivo, enabling us to explore variances in the nuclear architecture, cytoskeleton distribution, and chromatin conformation. The results showed that the area and volume of cell nuclei were distinctly smaller on micropillar topography. Actin and vimentin densely encapsulated the micropillars surrounding the nucleus, effectively segregating it from the micropillars. Additionally, nucleo-cytoskeleton lamin A/C exhibited a polarized distribution at the protrusion of the deformed nuclei. Notably, the degree of heterochromatin was altered in response to significant nuclear deformation, leading to a downregulation trend in H3K9me3 expression. These findings suggest that mechanical constraints imposed by microtopography profoundly influence cell behaviors, providing insights into disease diagnosis and therapeutic interventions in vivo.

Novel air-liquid interface culture model to investigate stiffness-dependent behaviors of alveolar epithelial cells

Pulmonary alveoli are functional units in gas exchange in the lung, and their dysfunctions in lung diseases such as interstitial pneumonia are accompanied by fibrotic changes in structure, elevating the stiffness of extracellular matrix components. The present study aimed to test the hypothesis that such changes in alveoli stiffness induce functional alteration of epithelial cell functions, exacerbating lung diseases. For this, we have developed a novel method of culturing alveolar epithelial cells on polyacrylamide gel with different elastic modulus at an air-liquid interface. It was demonstrated that A549 cells on soft gels, mimicking the modulus of a healthy lung, upregulated mRNA expression and protein synthesis of surfactant protein C (SFTPC). By contrast, the cells on stiff gels, mimicking the modulus of the fibrotic lung, exhibited upregulation of SFTPC gene expression but not at the protein level. Cell morphology, as well as cell nucleus volume, were also different between the two types of gels.

ANALYSIS OF THE VOLUME OF BONE MARROW CELL NUCLEI FOR ASSESSMENT OF CYTOSTATIC MYELOSUPPRESSION AND ITS PREVENTION BY ACTIVATED CARBON

Bone marrow suppression (myelosuppression) is a frequent complication of chemotherapy, and the need for monitoring and managing this side effect is still in great demand. Aim: to study the changes in the volume of nuclei in bone marrow cells of chemotherapy drug-treated laboratory animals with or without enterosorption by activated carbon (AC). Object and methods: both flow cytometry and confocal microscopy were used to analyze acridine orange (AO)-stained bone marrow samples of intact, doxorubicin (DOX)-, and DOX+AC-treated rats. Confocal Z-series that represent sequential scans of cell nuclei directed from top to bottom at 0.5-μm step size were acquired at 40× magnification using argon laser (488 nm) for excitation of AO. Green fluorescence emitted by DNA-bound AO was detected through a 505–530 nm band-pass filter, allowing distinct visualization of nuclei and their boundaries. Z-series were further processed and analyzed with ImageJ software to quantify the values of nuclear volumes. Results: there were apparent differences between the nuclear volumes in the bone marrow samples of intact, DOX-treated, and DOX+AC-treated rats. A significant increase of the volume of nuclei in DOX+AC-treated rats, compared with those in DOX-treated (1.42-fold) and intact rats (1.14-fold), is likely due to an active DNA replication, suggesting an ongoing recovery of the pool of nucleated cells. Notably, in these three groups, the populations of bone marrow nucleated cells, as estimated by flow cytometry, correlated well with the aforementioned values of nuclear volumes. However, the volume of nuclei may not necessarily correlate with the height of Z-series representing the thickness of nuclei, providing a clue that can help to delineate the role of nuclear deformability. Conclusion: analysis of the volume of bone marrow cell nuclei proposed in this work is important in terms of obtaining supplementary information in the study of the course of induced myelosuppression and of the ways of its prevention.

Hematological adaptations in diploid and triploid Salvelinus fontinalis and diploid Oncorhynchus mykiss (Salmonidae, Teleostei) in response to long-term exposure to elevated temperature

Hematology is a simple but reliable method to determine the influence of environmental stressors on the physiology and performance of teleost fish. Understanding the detailed impacts of elevated temperature on the hematology of fish can help us to evaluate fish responses to climate change. Therefore, cellular and biochemical changes in peripheral blood were investigated in diploid (2n) and triploid (3n) brook trout and in 2n rainbow trout exposed to 20 °C for 32 days in comparison to fish acclimated to 9 °C. Additionally, survival and growth rates and the relative mRNA expression of heat-shock proteins were recorded. All brook trout and rainbow trout survived exposure to 20 °C. At 20 °C, growth was decreased in 2n rainbow trout, increased in 2n brook trout and similar to growth at 9 °C in 3n brook trout. The erythrocyte cell volume, nucleus volume, cell surface, and nucleocytoplasmatic ratio were significantly lower at 20 °C than at 9 °C in all investigated species/ploidy levels. In contrast, the erythrocyte surface-to-volume ratio was not affected by temperature. In 2n and 3n brook trout, erythrocyte concentration and blood hemoglobin content were increased at 20 °C; in 2n rainbow trout, there were no differences in comparison to 9 °C. Exposure of 2n and 3n brook trout and 2n rainbow trout to 20 °C had no negative effect on cellular and molecular immune components in blood. Serum diagnostic enzymes and metabolites indicated neither organ inflammation nor disease nor disturbance in energy metabolism or nutrition status in fish exposed to 20 °C. In addition, the mRNA expression of Hsp70 and Hsp90 relative to the 28S ribosomal protein S32 did not differ between 9 °C and 20 °C.

A simple pipeline for cell cycle kinetic studies in the root apical meristem.

Root system architecture ultimately depends on precise signaling between different cells and tissues in the root apical meristem (RAM) and integration with environmental cues. This study describes a simple pipeline to simultaneously determine cellular parameters, nucleus geometry, and cell cycle kinetics in the RAM. The method uses marker-free techniques for nucleus and cell boundary detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining for DNA replication quantification. Based on this approach, we characterized differences in cell volume, nucleus volume, and nucleus shape across different domains of the Arabidopsis RAM. We found that DNA replication patterns were cell layer and region dependent. G2 phase duration, which varied from 3.5 h in the pericycle to more than 4.5 h in the epidermis, was found to be associated with some features of nucleus geometry. Endocycle duration was determined as the time required to achieve 100% EdU-positive cells in the elongation zone and, as such, it was estimated to be in the region of 5 h for the epidermis and cortex. This experimental pipeline could be used to precisely map cell cycle duration in the RAM of mutants and in response to environmental stress in several plant species without the need for introgressing molecular cell cycle markers.

EFFECT OF IMUNOFAN INFLUENCE ON THE STRUCTURE OF THE TESTES, HORMONAL AND CYTOKINE PROFILE OF IMMATURE EXPERIMENTAL ANIMALS

The aim: It was the establishing the features of changes in the structure of the testes of experimental animals, as well as immunological, hormonal and cytokine parameters of blood plasma during stimulation. Materials and methods: The study was carried out on 60 white male immature rats. Imunofan was used at a dosage of 50 μg. The organs were weighed, the relative mass was calculated, and the linear dimensions were determined. The morphometriv parameters of the epitheliospermatogenic layer were measured. The number of supporting cells and interstitial endocrinocytes was counted, as well as the volume of cell nuclei. The level of reproductive hormones in the plasma and the concentration of cytokines were determined. Results: The results obtained indicate the development of readaptation processes in the testes after the use of the Imunofan against the background of environmental immunosuppression. The ability of the drug to stimulate the production of cytokines and hormones normalizes the function of immunocompetent cells, which is manifested in the stabilization of the immune homeostasis of the testes. Conclusions: In response to the immunostimulating effect of Imunofan, a pronounced reaction is observed on the part of the testes of immature animals, which is due to the sensitivity of morphogenetic processes in the organ to external influences and the formation of mechanisms of their regulation, characteristic of this period of ontogenesis.

Three-dimensional virtual histology of the human hippocampus based on phase-contrast computed tomography

We have studied the three-dimensional (3D) cytoarchitecture of the human hippocampus in neuropathologically healthy and Alzheimer's disease (AD) individuals, based on phase-contrast X-ray computed tomography of postmortem human tissue punch biopsies. In view of recent findings suggesting a nuclear origin of AD, we target in particular the nuclear structure of the dentate gyrus (DG) granule cells. Tissue samples of 20 individuals were scanned and evaluated using a highly automated approach of measurement and analysis, combining multiscale recordings, optimized phase retrieval, segmentation by machine learning, representation of structural properties in a feature space, and classification based on the theory of optimal transport. Accordingly, we find that the prototypical transformation between a structure representing healthy granule cells and the pathological state involves a decrease in the volume of granule cell nuclei, as well as an increase in the electron density and its spatial heterogeneity. The latter can be explained by a higher ratio of heterochromatin to euchromatin. Similarly, many other structural properties can be derived from the data, reflecting both the natural polydispersity of the hippocampal cytoarchitecture between different individuals in the physiological context and the structural effects associated with AD pathology.

Cellular correlates of gray matter volume changes in magnetic resonance morphometry identified by two-photon microscopy

Magnetic resonance imaging (MRI) of the brain combined with voxel-based morphometry (VBM) revealed changes in gray matter volume (GMV) in various disorders. However, the cellular basis of GMV changes has remained largely unclear. We correlated changes in GMV with cellular metrics by imaging mice with MRI and two-photon in vivo microscopy at three time points within 12 weeks, taking advantage of age-dependent changes in brain structure. Imaging fluorescent cell nuclei allowed inferences on (i) physical tissue volume as determined from reference spaces outlined by nuclei, (ii) cell density, (iii) the extent of cell clustering, and (iv) the volume of cell nuclei. Our data indicate that physical tissue volume alterations only account for 13.0% of the variance in GMV change. However, when including comprehensive measurements of nucleus volume and cell density, 35.6% of the GMV variance could be explained, highlighting the influence of distinct cellular mechanisms on VBM results.

Quantifying Nanoscale Viscosity and Structures of Living Cells Nucleus from Mobility Measurements.

Understanding the mobility of nano-objects in the eukaryotic cell nucleus, at multiple length-scales, is essential for dissecting nuclear structure-function relationships both in space and in time. Here, we demonstrate, using single-molecule fluorescent correlation spectroscopies, that motion of inert probes (proteins, polymers, or nanoparticles) with diameters ranging from 2.6 to 150 nm is mostly unobstructed in a nucleus. Supported by the analysis of electron tomography images, these results advocate the ∼150 nm-wide interchromosomal channels filled with the aqueous diluted protein solution. The nucleus is percolated by these channels to allow various cargos to migrate freely at the nanoscale. We determined the volume of interchromosomal channels in the HeLa cell nucleus to 237 ± 61 fL, which constitutes 34% of the cell nucleus volume. The volume fraction of mobile proteins in channels equals 16% ± 4%, and the concentration is 1 mM.

Abstract 1631: Molecular and clinical associations of histopathological image features

Abstract Haematoxylin and eosin stained (H&E) histopathological images are widely used as a clinical routine for many cancers for diagnosis, but their interpretation is laborious and has a subjective element. Recent advances in computer vision using deep neural network offers an unprecedented opportunity to quantify patterns on H&E images typically used to classify tumour and normal. It is however possible to associate these traits with molecular alterations to obtain a precise understanding of the molecular underpinnings of histological changes. Here we demonstrate wide-spread associations of quantitative image traits with underlying genetic mutations including TP53, whole genome duplications (WDG) and molecular expression profiles ranging from cell morphology to T-cell infiltration. These traits are then on par with molecular data to predict patient survival. We use Inception-V4, a deep neural network, to extract 1536 image features from 9,754 tumour or normal H&E images of 28 cancers from TCGA (split into 7.9 million tiles of 512 by 512 pixels). Using these features, tissue-specific tumour/normal classification accuracy was high with an average of 0.95, ranging from 0.99 for Thyroid Carcinoma to 0.64 for Head and Neck Squamous Cell Carcinoma. We then used these features to predict molecular alterations using regression models. Out of 104 driver gene mutations, 30 showed an association with histology in at least one cancer (AUC > 0.5). Interestingly, TP53 the most frequent mutated gene in cancer, was found in measurably distinct histology in 10 of 28 cancers. WGD were associated with histology in 24 out of 27 cancers (AUC > 0.5), where 4 cancers showed an AUC as high as 0.8. As expected, presence of WGD also correlated with increased cell nucleus volume. For gene expression, 7.7% showed a positive correlation with image features (FDR < 0.1); 2.7% showed a correlation above 0.6. Interestingly, we detected significant associations for genes indicative of immune cell infiltration. Finally, we used image and clinical data to predict patient overall survival. We obtained a C-index range in [0.64, 1] for 25 cancers, which were comparable to models used expression and clinical data ([0.61, 1]). For 13 cancers, models with image data outperformed those with expression data with an improvement of C-index from 0.05% for Kidney Chromophope to 20% for Esophageal Adenocarcinoma. In this study, we used an established deep learning architecture to quantify tumour histology. These analyses showed a high accuracy of classifying tumour/normal images. We obtained a high accuracy in predicting molecular alterations with image features, indicating that these alterations shape the resulting tumour histology. We showed that image features can be used to predict clinical outcomes for cancer patients with a comparable to sometime higher accuracy compared to expression data, offering an easy and inexpensive method to complement the patients’ prognosis. Citation Format: yu fu, moritz gerstung. Molecular and clinical associations of histopathological image features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1631.

SIMULATION OF EARLY RADIATION-INDUCED DNA DAMAGE ON DIFFERENT TYPES OF CELL NUCLEI.

This work presents a comparison of simulated early radiation-induced DNA damage represented by yields of double-strand breaks (DSB) in three different human cell nuclei geometries representing fibroblasts, lymphocytes and endothelial cells for protons and alpha particles of different energies and for different irradiation configurations. Each cell nucleus model includes a multi-scale description of the DNA target from the molecular level to the whole human genome representation (6 Gbp) in the G0/G1 phase of the cell cycle and was generated with the DnaFabric software. The three nuclei differ in shape, volume, and therefore DNA density. A calculation chain based on Geant4-DNA that takes into account the physical, physico-chemical and chemical stages was used to simulate the irradiation of the different cell nuclei. Results show an increase of DSB/primary/μm with an increase of DNA density and an increase of DSB/Gy/Gbp with an increase of the cell nucleus volume which indicates that the cell nucleus shape and size have an impact on early DNA damage, which may play a role in latter effects.

Spirulina Platensis Affects Factors Involved in Spermatogenesis and Increases Ghrelin Receptors in Testis Tissue of Rats Fed a High-Fat Diet.

Ghrelin is a peptide hormone which plays important role in maintaining growth hormone release and energy homeostasis in vertebrates. Spirulina platensis (SP) has antioxidant and hypolipidemic effects due to its ingredients. In this study we aimed to investigate the effects of SP on the testicular structure and relation between ghrelin and testosterone in the testis of rats fed a high fat diet (HFD). Sixty four young adult male rats were used and divided to 8 equal groups. Experimental groups received addition of 10% cholesterol (CHL), 43% hydrogenated vegetable oil (HVO) and 3% SP alone or in combination to basal diet while the control group received only basal diet. Serum ghrelin and testosterone levels were measured with ELISA. Receptors for ghrelin and androgen were detected with immunohistochemistry. For histomorphometric investigation, tubulus seminiferus, intertubular area, tubulus seminiferus lumen, Leydig cell nucleus, Sertoli cell nucleus, germ cell nucleus, spermatocyte nucleus and elongated spermatid volume densities were determined stereologically. Serum ghrelin level was increased especially in HVO and CHL combination group compared to the control while serum ghrelin levels were close to control levels in SP-received groups. Ghrelin receptor level was increased in tubulus seminiferus with HVO+CHL administration but this effect was, however, limited in HVO+CHL and SP challenged groups. HVO+CHL administration caused a significant decrease in Leydig cell nucleus volume density, as well as in all SP-received groups, compared to the control. Significantly increased spermatocyte nucleus volume density in cholesterol-receiving groups was decreased to control level with SP alone and its combinations.

Histological parameters of the adrenal cortex after testosterone application in a rat model of the andropause.

Histological analysis of the adrenal cortex, after testosterone application in a rat model of the andropause, was the main subject of the present study. Middle-aged Wistar rats were divided into sham-operated (SO; n=8), orchidectomized (Orx; n=8) and testosterone treated orchidectomized (Orx+T; n=8) groups. Testosterone propionate (5 mg/kg b.w. /day) was administered for three weeks, while SO and Orx groups received the vehicle alone. Histological objectives were achieved using stereology, histochemistry and steroid receptor immunostaining. The concentrations of testosterone, aldosterone, corticosterone and DHEA were determined by immunoassays. Expectedly, increased (p<0.05) serum concentration of testosterone was observed in Orx+T group. The volume of ZG cells and nuclei increased in Orx+T animals by 50% and 25% (p<0.05) respectively, but the serum concentrations of aldosterone decreased (p<0.05) by 60%, all compared to the same parameters in Orx group. The immunostaining for androgen receptors (ARs) suggested their cytoplasmic localization in ZG cells of Orx+T rats. Volume of the ZF cell nuclei in Orx+T group decreased (p<0.05) by 17%, which was followed by the significant (p<0.05) fall in corticosterone production and secretion, all in comparison with Orx animals. Also, nuclear immunolocalization of ARs of high optical density was observed through the ZF of Orx+T group. In Orx+T rats volume of ZR cells and nuclei, and circulating DHEA concentration increased (p<0.05) by 68%, 22% and about 6.6 times respectively, compared to Orx animals. Besides the extra-receptor actions in adrenal cortex, testosterone supposedly affects some steroidogenesis-related gene expression, as indicated by centripetal rise in the number of nuclear ARs.

Phase-based x-ray scattering--a possible method to detect cancer cells in a very early stage.

This theoretical work contains a detailed investigation of the potential and sensitivity of phase-based x-ray scattering for cancer detection in biopsies if cancer is in a very early stage of development. Cancer cells in their early stage of development differ from healthy ones mainly due to their faster growing cell nuclei and the enlargement of their densities. This growth is accompanied by an altered nucleus-plasma relation for the benefit of the cell nuclei, that changes the physical properties especially the index of refraction of the cell and the one of the cell nuclei. Interaction of radiation with matter is known to be highly sensitive to small changes of the index of refraction of matter; therefore a detection of such changes of volume and density of cell nuclei by means of high angular resolved phase-based scattering of x rays might provide a technique to distinguish malignant cells from healthy ones if the cell-cell nucleus system is considered as a coherent phase shifting object. Then one can observe from a thin biopsy which represents a monolayer of cells (no multiple scattering) that phase-based x-ray scattering curves from healthy cells differ from those of cancer cells in their early stage of development. Detailed calculations of x-ray scattering patterns from healthy and cancer cell nuclei yield graphs and numbers with which one can distinguish healthy cells from cancer ones, taking into account that both kinds of cells occur in a tissue within a range of size and density. One important result is the role and the influence of the (lateral) coherence width of the radiation on the scattering curves and the sensitivity of phase-based scattering for cancer detection. A major result is that a larger coherence width yields a larger sensitivity for cancer detection. Further import results are calculated limits for critical sizes and densities of cell nuclei in order to attribute the investigated tissue to be healthy or diseased. With this proposed method it should be in principle possible to detect cancer cells in apparently healthy tissues in biopsies and/or in samples of the far border region of abscised or excised tissues. Thus this method could support established methods in diagnostics of cancer-suspicious samples.

Scaling the primate lateral geniculate nucleus: Niche and neurodevelopment in the regulation of magnocellular and parvocellular cell number and nucleus volume

New stereological assessments of lateral geniculate nucleus (LGN) neuron numbers and volumes in five New World primates (Cebus apella, Saguinus midas niger, Alouatta caraya, Aotus azarae, and Callicebus moloch) and compiled LGN volumes for an additional 26 mammals were analyzed for a better understanding of visual system evolution. Both the magnocellular (M)- and the parvocellular (P)-cell populations scale allometrically with brain volume in primates, P cells with a significantly higher slope such that, for every increase in M neuron number, P neuron numbers more than double (ln scale; y = 0.89x + 2.42R(2) = 0.664). In diurnal primates, the ratio of P to M cells was slightly but significantly higher than in nocturnal primates. For all mammals, including primates, LGN volume was unrelated to nocturnal or diurnal niche but showed marked differences in slope and intercept depending on taxonomic group. The allometric scaling of M and P cells can be related to the order of neurogenesis, with late-generated P cells increasing with positive allometry compared with the earlier-generated M cells. This developmental regularity links relative foveal representation to relative isocortex enlargement, which is also generated late. The small increase in the P/M cell ratio in diurnal primates may result from increased developmental neuron loss in the M-cell population as it competes for limited termination zones in primary visual cortex.

Quantitative Analysis of Adrenal Cortical Histological Alterations After Application of Medroxyprogesterone Acetate

Results. Gravimetric and variance analysis demonstrated the most remarkable decrease of the adrenal gland weight after the application of 75.0 mg/kg/bw MPA (from 35.66 ± 11.78 to 13.41 ± 8.41 mg (p<0.001). On the other hand, morphometric analysis demonstrated that adrenal cortex volume was the most significantly reduced from 11.30 ± 3.53 in the control group to 2.04 ± 0.90 mm3 in the group treated with 15.0 mg/kg bw MPA (p<0.0001). Adrenal cortex thickness was reduced from 846.06 ± 6.48 to 349.55 ± 4.84 μm after the application of 75.0 mg/kg bw (p < 0.001). After the application of the same dose the glomerular zone thickness was decreased from 48.02 ± 0.44 to 41.40 ± 0.43 μm (p < 0.001), the fascicular zone thickness from 480.72 ± 3.43 to 194.32 ± 2.80 μm (p <0.001) and the reticular zone thicknes reduced from 317.47 ± 4.80 to 113.85 ± 2.28 μm (p < 0.001). The volume of glomerular zone cell nuclei decreased most remarkably from 94.79 ± 0.96 μm3 after the application of 75.0 mg/kg bw to 60.12 ± 0.88 μm3 (p < 0.001). The volume of fascicular zone cell nuceli decreased from 166.30 ± 1.53 to 52.47 ± 0.89 μm3 (p < 0.001), while the volume of reticular zone cell nuceli reduced from 95.61 ± 1.03 to 47.68 ± 0.92 μm3 (p < 0.001). The analysis by means of χ2 test showed that the appearance of tissue necrotic changes in adrenal cortex had a statistical significance only after the application of the highest dose of MPA (p < 0.012).

The Nun Study

It is common to find substantial Alzheimer disease (AD) lesions, i.e., neuritic beta-amyloid plaques and neurofibrillary tangles, in the autopsied brains of elderly subjects with normal cognition assessed shortly before death. We have termed this status asymptomatic AD (ASYMAD). We assessed the morphologic substrate of ASYMAD compared to mild cognitive impairment (MCI) in subjects from the Nun Study. In addition, possible correlations between linguistic abilities in early life and the presence of AD pathology with and without clinical manifestations in late life were considered. Design-based stereology was used to measure the volumes of neuronal cell bodies, nuclei, and nucleoli in the CA1 region of hippocampus (CA1). Four groups of subjects were compared: ASYMAD (n = 10), MCI (n = 5), AD (n = 10), and age-matched controls (n = 13). Linguistic ability assessed in early life was compared among all groups. A significant hypertrophy of the cell bodies (+44.9%), nuclei (+59.7%), and nucleoli (+80.2%) in the CA1 neurons was found in ASYMAD compared with MCI. Similar differences were observed with controls. Furthermore, significant higher idea density scores in early life were observed in controls and ASYMAD group compared to MCI and AD groups. 1) Neuronal hypertrophy may constitute an early cellular response to Alzheimer disease (AD) pathology or reflect compensatory mechanisms that prevent cognitive impairment despite substantial AD lesions; 2) higher idea density scores in early life are associated with intact cognition in late life despite the presence of AD lesions.

Neuronal Hypertrophy in Asymptomatic Alzheimer Disease

The pathologic changes of Alzheimer disease (AD) evolve very gradually over decades before the disease becomes clinically manifest. Thus, it is not uncommon to find substantial numbers of Abeta plaques and neurofibrillary tangles in autopsy brains of older subjects with documented normal cognition, a state that we define as asymptomatic AD (ASYMAD). The goal of this study is to understand the morphometric substrate of ASYMAD subjects compared with mild cognitive impairment and definite AD cases. We used designed-based stereology to measure the volumes of neuronal cell bodies, nuclei, and nucleoli in 4 cerebral regions: anterior cingulate gyrus, posterior cingulate gyrus, primary visual cortex, and CA1 of hippocampus. We examined and compared autopsy brains from 4 groups (n = 15 each) of participants in the Baltimore Longitudinal Study of Aging: ASYMAD, mild cognitive impairment, AD, and age-matched controls. We found significant hypertrophy of the neuronal cell bodies, nuclei, and nucleoli of CA1 of hippocampus and anterior cingulate gyrus neurons in ASYMAD subjects compared with control and mild cognitive impairment cases. In the posterior cingulate gyrus and primary visual cortex, the hypertrophy was limited to the nuclei and nucleoli. The hypertrophy of cortical neurons and their nuclei and nucleoli in ASYMAD may represent an early reaction to the presence of neurotoxic Abeta or tau, or a compensatory mechanism that prevents the progression of the disease into dementia.

Filtering, reconstruction, and measurement of the geometry of nuclei from hippocampal neurons based on confocal microscopy data

The cell nucleus is often considered a spherical structure. However, the visualization of proteins associated with the nuclear envelope in rat hippocampal neurons indicates that the geometry of nuclei is far more complex. The shape of cell nuclei is likely to influence the nucleo-cytoplasmic exchange of macromolecules and ions, in particular calcium, a key regulator of neuronal gene expression. We developed a tool to retrieve the 3-D view of cell nuclei from laser scanning confocal microscopy data. By applying an inertia-based filter, based on a special structure detection mechanism, the signal-to-noise ratio of the image is enhanced, the signal is smoothed, gaps in the membrane are closed, while at the same time the geometric properties, such as diameters of the membrane, are preserved. After segmentation of the image data, the microscopy data are sufficiently processed to extract surface information of the membrane by creating an isosurface with a marching tetrahedra algorithm combined with a modified Dijkstra graph-search algorithm. All methods are tested on artificial data, as well as on real data, which are recorded with a laser scanning confocal microscope. Significant advantages of the inertia-based filter can be observed when comparing it to other state of the art nonlinear diffusion filters. An additional program is written to calculate surface and volume of cell nuclei. These results represent the first step toward establishing a geometry-based model of the-dynamics of cytoplasmic and nuclear calcium.

236. Effects of long term recombinant rat follicle-stimulating hormone replacement on the restoration of spermatogenesis after chronic suppression of gonadotrophins in adult rats

Follicle stimulating hormone (FSH) in short-term rat studies supports spermatogenesis at multiple levels, notably spermatogonial development. The role of FSH in supporting full spermatogenesis in rats is still in question as long-term studies have not been possible due the development of neutralising antibodies to heterologous FSH preparations. This study sought to assess the effects of a homologous recombinant rat FSH (rr-FSH) preparation on the long-term restoration of spermatogenesis. Adult rats were GnRH-immunised (GnRH-im) for 12 weeks then, administered an anti-androgen; flutamide (flut), alone or together with rr-FSH (8µg/rat/daily) for 56 days (1 spermatogenic cycle). Germ and Sertoli cell numbers were quantified using an optical disector stereological method. Testis weight, serum FSH and inhibin B and Sertoli cell nuclear volume were significantly reduced to 15%, 13%, 25% and 57% of controls respectively, following GnRH-im+flut treatment. GnRH-im+flut treatment reduced A/I spermatogonial, type B spermatogonial+preleptotene, leptotene+zygotene and early pachytene spermatocyte numbers to 28%, 68%, 50% and 19% (P &lt; 0.001) of controls respectively, with later germ cells rarely observed. After FSH treatment, no significant affect on testis weight, serum FSH and inhibin B or Sertoli cell number were observed. However, rr-FSH treatment significantly increased numbers of A/I spermatogonia, leptotene+zygotene and early pachytene spermatocytes from 28 = &gt;42%, 50 = &gt;69% and 19 = &gt;27% of controls, respectively, while no differences were observed in later germ cell types. rr-FSH also increased (P &lt; 0.05) the volume of Sertoli cell nuclei from 57 = &gt;66% of control. In conclusion, FSH is unable to support full rat spermatogenesis; however, FSH can partially support germ cells notably spermatogonia through to early pachytene spermatocytes, despite the absence of androgenic support.