Number Of Argyrophilic Nucleolar Organizer Regions Research Articles

A CNN-based approach for joint segmentation and quantification of nuclei and NORs in AgNOR-stained images

Background and ObjectiveOral cancer is the sixth most common kind of human cancer. Brush cytology for counting Argyrophilic Nucleolar Organizer Regions (AgNORs) can help early mouth cancer detection, lowering patient mortality. However, the manual counting of AgNORs still in use today is time-consuming, labor-intensive, and error-prone. The goal of our work is to address these shortcomings by proposing a convolutional neural network (CNN) based method to automatically segment individual nuclei and AgNORs in microscope slide images and count the number of AgNORs within each nucleus. MethodsWe systematically defined, trained and tested 102 CNNs in the search for a high-performing solution. This included the evaluation of 51 network architectures combining 17 encoders with 3 decoders and 2 loss functions. These CNNs were trained and evaluated on a new AgNOR-stained image dataset of epithelial cells from oral mucosa containing 1,171 images from 48 patients, with ground truth annotated by specialists. The annotations were greatly facilitated by a semi-automatic procedure developed in our project. Overlapping nuclei, which tend to hide AgNORs, thus affecting their true count, were discarded using an automatic solution also developed in our project. Besides the evaluation on the test dataset, the robustness of the best performing model was evaluated against the results produced by a group of human experts on a second dataset. ResultsThe best performing CNN model on the test dataset consisted of a DenseNet-169 + LinkNet with Focal Loss (DenseNet-169 as encoder and LinkNet as decoder). It obtained a Dice score of 0.90 and intersection over union (IoU) of 0.84. The counting of nuclei and AgNORs achieved precision and recall of 0.94 and 0.90 for nuclei, and 0.82 and 0.74 for AgNORs, respectively. Our solution achieved a performance similar to human experts on a set of 291 images from 6 new patients, obtaining Intraclass Correlation Coefficient (ICC) of 0.91 for nuclei and 0.81 for AgNORs with 95% confidence intervals of [0.89, 0.93] and [0.77, 0.84], respectively, and p-values < 0.001, confirming its statistical significance. Our AgNOR-stained image dataset is the most diverse publicly available AgNOR-stained image dataset in terms of number of patients and the first for oral cells. ConclusionsCNN-based joint segmentation and quantification of nuclei and NORs in AgNOR-stained images achieves expert-like performance levels, while being orders of magnitude faster than the later. Our solution demonstrated this by showing strong agreement with the results produced by a group of specialists, highlighting its potential to accelerate diagnostic workflows. Our trained model, code, and dataset are available and can stimulate new research in early oral cancer detection.

Evaluation of the Cytological Changes of the Oral Mucosa Among Smokers in Al Madinah Al Munawara Using Argyrophilic Nucleolar Organizer Region (AgNOR) Counts and Papanicolaus Stain.

Objective To evaluate the cytological changes of the oral mucosa among smokers using Argyrophilic nucleolar organizer region (AgNOR) counts and Papanicolaou (Pap) staining. Methodology The oral mucosal exfoliate smears of 500 individuals (200 nonsmokers and 300 smokers) aged between 18 and 80 years were prepared in Al Madinah. TheAgNOR count and Pap stain were used to generate a cytogenic smear to assess the presence of cytological changes suggestive of atypia, inflammation, dysplasia, keratinization, and proliferative activity of epithelial cells. Results Smokers have a considerably higher number of AgNORs per nucleus than nonsmokers (1.99 3.53 vs. 0.42 1.22). There were inflammatory changes in 127 (42.3%) of the cases and 40 (20%) of the controls. Multinucleated cells and atypia were found in 33 (11%) and 14 (4.5%) of the casesbut not in the controls. The results indicate higher proliferative activity in smoking patients compared to nonsmoking patients, even in the absence of clinical lesions. Conclusion To detect the effects of smoking on the oral mucosa, Pap staining alone is insufficient. Combining Pap staining with the AgNOR technique produces the desired results.

A Comparison of Proliferative Capacity of Reticular and Erosive Variants of Oral Lichen Planus by Argyrophilic Nucleolar Organizer Regions Method.

Oral lichen planus (OLP), an immune mediated disorder, has been recognized since 1869 and is presented as any one of the six variants. Reticular and erosive are the most frequently encountered. Its proliferative capacity can give some information regarding its progression. We adopted the argyrophilic nucleolar organizer regions (AgNORs) method because of its simplicity to use and dependable results. We evaluated AgNORs in basal, suprabasal, and squamous cell layers. We also compared these three layers within two variants, reticular, and erosive. Thirty clinically diagnosed patients of OLP were included in the study. Reticular and erosive variants were included in our study. This was followed by hematoxylin and eosin staining and later by the AgNOR method. The mean number of AgNORs per nucleus was calculated. Thirteen males and 17 females were the gender distribution. Twenty-three (76.67%) had reticular pattern and seven (23.33%) had erosive pattern. The basal cell layer had the highest mean AgNOR compared to suprabasal and squamous layers. Even among, erosive and reticular variants, the former had higher mean AgNOR counts. Our results suggest that the inflammatory infiltrate close to the epithelial cells can alter the proliferation index for the pattern of protein synthesis of these cells. Moreover, the high proliferative index in OLP can be related to a specific immunologic response. We conclude that AgNOR can be used as a proliferative marker in earlier lesions to detect the severity.

Analysis of Argyrophilic Nucleolar Organizer Regions (AgNORs) in Acute Leukemia in Adults.

The evaluation of argyrophilic nucleolar organizer regions (AgNORs) uses a simple method used in research into neoplasm. Bone marrow aspirates from 70 patients with acute leukemia underwent morphological, immunophenotypic, and genetic assessment and were stained with silver nitrate. In leukemic cells, the mean AgNORs number, mean AgNORs area, and mean AgNOR-area-to-nucleus-area ratio were calculated in patients with acute myeloid leukemia (AML), patients with acute lymphoblastic leukemia (ALL), and selected risk groups. A higher value of all measured AgNOR parameters was observed in patients with AML compared to the ALL group. In AML patients, a higher mean AgNOR area was found in the ELN3 cytogenetic group compared to the ELN2 cytogenetic group. A higher value of the mean AgNOR count was observed in patients with white blood cells (WBCs) > 12 × 109/L than in the group with WBCs ≤ 12 × 109/L, as well as in patients with >20% blasts in peripheral blood (PB) than in patients with ≤20% blasts in PB. In the ALL group, a higher mean AgNOR-area-to-nucleus-area ratio was found in group with the presence of Philadelphia chromosome Ph(+) than without the Philadelphia chromosome Ph(−). AgNOR parameter analysis is a valuable method for differentiation of AML and ALL in adults.

Effect of 50-Hz Magnetic Fields on Argyrophilic Nucleolar Organizer Regions in Bone Marrow Cells

Background: Exposure to an increasing amount of extremely low-frequency magnetic fields (MFs) causes some adverse effects. Considering a direct association between the sizes and numbers of argyrophilic nucleolar organizer regions (AgNORs) and cell proliferation, this study was conducted to evaluate the effect of 50-Hz MFs on the AgNORs of bone marrow (BM) cells. Methods: Eighty male rats were randomly divided into four exposed groups and one control group. The exposed groups received 50-Hz MFs with magnetic flux densities of 1, 100, 500, and 2000 µT two hours a day for two months. BM cells were aspirated from sacrificed rats’ femoral bones, smeared on glass slides, and then stained with silver nitrate for AgNORs. The area (AA), length (AL), and number (AN) of AgNORs were calculated by a microscope equipped with a camera and Scion Image software in 100 BM cells of each rat. The mean of AA, AL, and AN was computed for each group. Results: AA, AL, and AN significantly reduced in the 1 µT group compared with 2000 µT and control groups. Eventually, there was a nonlinear relationship between the effect of 50-Hz MFs and magnetic flux densities. Conclusion: Overall, 50-Hz MFs with a magnetic flux density of 1 µT reduced AgNORs in BM cells. However, 100, 500, and 2000 µT did not affect AgNORs. Therefore, 50-Hz MFs with low density may suppress BM cell proliferation.

Detection of antitumoral effects of quercetin through protein synthesis associated with argyrophilic nucleolar-regulating region

Quercetin is a natural flavonoid with potential anticancer properties without significant cytotoxicity in normal tissues. However, the effects of quercetin on Ehrlich ascites carcinoma (EAC) have not yet been clarified. The study aimed to show the antitumoral effect of quercetin through argyrophilic nucleolar organizer region (AgNOR) protein synthesis in mice carrying EAC. Thirty mice were used in the experiment (negative control (n = 6), tumor control (n = 8), quercetin 50 mg/kg (n = 8), and quercetin 100 mg/kg (n = 8) intravenously). The animals were euthanized on the 14th day, and the solid tumors were removed. Then, the total AgNOR area/nuclear area (TAA/NA) and average AgNOR number were calculated for each mice and the immunoreactivity of the factor VIII protein in tumor was evaluated. Tumor volumes and animal weights were statistically significant compared to the tumor control group (p < 0.05). Statistically significant differences were observed between the groups in terms of TAA/NA ratio (p < 0.05). Factor VIII expressions decreased in quercetin groups compared to control tumor tissue (p < 0.05). The current study showed that quercetin has an important function against cancer development.

Argyrophilic nucleolar organizer regions in oral mucosa cells of crack and cocaine users: a cross-sectional study.

The objective of this study was to analyze and compare the presence of argyrophilic nucleolar organizer regions (AgNORs) in exfoliated cells obtained from the buccal mucosa among crack and cocaine users and non-users by the AgNOR staining technique. Seventy-three males ≥ 18years were categorized into two groups: 38 crack and/or cocaine users and 35 non-user volunteers. They were interviewed and responded a questionnaire regarding general health and drug addiction. Exfoliative cytology specimens were collected from the clinically intact buccal mucosa, and cytological preparations were fixed and stained by AgNOR technique. The mean number of AgNORs (p = 0.02) and the percentage of epithelial cells with more than 3 (p = 0.01) and 4 (p = 0.04) AgNORs/nucleus were significantly higher in the non-user group. In conclusion, the frequency and diversity of substances present in the drugs-cocaine, crack, and alcohol-consumed by the volunteers of this study may have influenced the number of AgNORs and the response to damage and consequent effect on protein synthesis.

Use of argyrophilic nucleolar–organizer region-associated protein synthesis in skeletal muscle cells for prediction of chronic carbon monoxide exposure

This study aims to evaluate possible effects of chronic carbon monoxide (CO) exposure on argyrophilic nucleolar–organizer region (AgNOR)-associated protein synthesis in striated muscle cells. Eighteen rats were randomly divided into three groups. Rats in group II and group III were given CO gas (1000 and 3000 ppm, respectively) for 30 min a day for 7 d. Mean AgNOR number per nucleus and ratio of total AgNOR area to nuclear area (TAA/NA) were determined. Both TAA/NA ratio and mean AgNOR number provided information about the existence or absence of exposure. TAA/NA ratio was also an indicator of the level of exposure.

Identification of AgNORs and cytopathological changes in oral lichen planus lesions

ObjectiveTo evaluate cytopathological changes in epithelial cells of the oral mucosa of patients with oral lichen planus (OLP) compared with patients without OLP. Subjects and methodsSwabs were collected from the oral mucosa of 20 patients with OLP (case group) and 20 patients without OLP (control group) using liquid-based cytology. After Papanicolaou staining, the smears were characterized based on Papanicolaou classification and degree of maturation. Nuclear area (NA) measurements, cytoplasmic area (CA) measurements, and the NA/CA ratio were determined from 50 epithelial cells per slide. For quantification of argyrophilic nucleolar organizer regions (AgNORs), the smears were stained with silver nitrate, and the number of AgNORs was counted in 100 cells. ResultsIn both groups, there was a predominance of Papanicolaou Class I nucleated cells in the superficial layer. The average values of NA (p>0.05) and CA (p=0.000) were greater in the case group (NA=521.6, CA=22,750.3) compared with the control group (NA=518.9, CA=18,348.0). The NA/CA ratio was 0.025 for the case group and 0.031 for the control group (p=0.004). There was no significant difference between the mean AgNORs values of both groups (p>0.05). ConclusionThe oral mucosa of patients with OLP exhibited significant cytomorphometric changes. However, there was no evidence of malignancy.

Fractal analysis and the diagnostic usefulness of silver staining nucleolar organizer regions in prostate adenocarcinoma.

Pathological diagnosis of prostate adenocarcinoma often requires complementary methods. On prostate biopsy tissue from 39 patients including benign nodular hyperplasia (BNH), atypical adenomatous hyperplasia (AAH), and adenocarcinomas, we have performed combined histochemical-immunohistochemical stainings for argyrophilic nucleolar organizer regions (AgNORs) and glandular basal cells. After ascertaining the pathology, we have analyzed the number, roundness, area, and fractal dimension of individual AgNORs or of their skeleton-filtered maps. We have optimized here for the first time a combination of AgNOR morphological denominators that would reflect best the differences between these pathologies. The analysis of AgNORs' roundness, averaged from large composite images, revealed clear-cut lower values in adenocarcinomas compared to benign and atypical lesions but with no differences between different Gleason scores. Fractal dimension (FD) of AgNOR silhouettes not only revealed significant lower values for global cancer images compared to AAH and BNH images, but was also able to differentiate between Gleason pattern 2 and Gleason patterns 3–5 adenocarcinomas. Plotting the frequency distribution of the FDs for different pathologies showed clear differences between all Gleason patterns and BNH. Together with existing morphological classifiers, AgNOR analysis might contribute to a faster and more reliable machine-assisted screening of prostatic adenocarcinoma, as an essential aid for pathologists.

Effects of the interaction between hydroxyapatite nanoparticles and hepatoma cells

To gain a better understanding of the anticancer effects of hydroxyapatite (HAP) nanoparticles in vivo and in vitro, the effects of the interaction of HAP nanoparticles with hepatoma cells were explored. HAP nanoparticles were prepared by homogeneous precipitation and characterized by laser particle analysis and transmission electron microscopy (TEM). HAP nanoparticles were observed to be uniformly distributed, with rod-like shapes and diameters in the range of 42.1–87.1 nm. Overnight attached, suspended, and proliferating Bel-7402 cells were incubated with HAP nanoparticles. Inverted microscopy observation revealed that HAP nanoparticles with a cell membrane showed good adsorption. TEM demonstrated that HAP nanoparticles were present on the surface of cells, continuously taken up by cells through endocytosis, and transported in vesicles close to the nucleus. Fluorescence microscopy showed that the concentrations of intracellular Ca2+ labeled with Fluo-3 calcium fluorescent probe were significantly enhanced. In addition, inverted microscopy observation revealed that suspended cells treated with HAP nanoparticles did not adhere to the culture bottle, resulting in cell death. After the overnight attached cells were treated with HAP nanoparticles for 96 h with increasing doses of HAP nanoparticles, inverted microscopy observation revealed that cell proliferation was slowed and cell-cell adhesion was weakened. Feulgen staining and image analysis indicated that the nuclear DNA content of the cells was markedly reduced, and argyrophilic nucleolar organizer region (AgNOR) staining and image analysis indicated that the number of AgNORs was significantly decreased. Therefore, hepatoma cells brought about the adsorption, uptake, transport and degradation of HAP nanoparticles. In addition, HAP nanoparticles affected hepatoma cells with regard to cell-cell adhesion, cell and extracellular matrix adhesion, and DNA and protein synthesis; thus inhibiting cell proliferation. This understanding of the effects of interaction between HAP nanoparticles and hepatoma cells is useful for further study of the anticancer mechanisms of HAP nanoparticles.

The AgNORs count in predicting long-term survival in serous ovarian cancer

IntroductionThe value of argyrophilic nucleolar organizer regions (AgNORs) to predict survival in patients with ovarian cancer has not been clearly explained yet. The aim of study was to assess the value of analysis of the mean number of AgNORs per nucleus (mAgNOR) and mean percentage of nuclei with five or more AgNORs per nucleus (pAgNOR) in the prediction of disease-free survival (DFS) and overall survival (OS) in patients with serous ovarian cancer.Material and methodsThe study examined 52 patients treated for serous ovarian cancer with a follow-up period of 2-143 months. After silver staining paraffin specimens from primary surgery, mAgNOR and pAgNOR in cancer cells were counted and analyzed. Age, grading, radicality of surgery and FIGO staging were analyzed as covariates.ResultsMean mAgNOR equaled 4.4 ±0.9 and pAgNOR equaled 42.2 ±20.8%. Both mAgNOR and pAgNOR were the lowest in G1 tumors. The mAgNOR and pAgNOR were lower in stage I than stage IV cancers. The DFS and OS rates were respectively 15.4% and 21.2%. In univariate analysis FIGO staging, grading, and pAgNOR were associated with worse prognosis, while radicality of surgery remained a significant protective factor in terms of DFS. Higher FIGO staging and older age worsened OS. In multivariate analysis FIGO staging remained significantly associated with both DFS (HR 1.98; 95% CI 1.05-3.71) and OS (HR 1.76; 95% CI 1.00-3.10), while age affected OS rates (HR 1.78; 95% CI 1.04-2.95).ConclusionsmAgNOR and pAgNOR are useful markers of cellular kinetics. Prospective studies in larger populations are needed to confirm these results in terms of AgNORs’ effects on survival.

Prognostic Value of Microvessel Density in Tumor and Peritumoral Area as Evaluated by CD31 Protein Expression and Argyrophilic Nucleolar Organizer Region Count in Endothelial Cells in Uterine Leiomyosarcoma

The objective of this study was to investigate the prognostic value of microvessel density (MVD) in uterine leiomyosarcoma (LMS) and peritumoral area (PA) as evaluated by CD31 expression and argyrophilic nucleolar organizer region (AgNOR) count in endothelial cells. Tissue specimens from 66 patients with uterine LMS were examined. There were no significant differences in the mean MVD between tumor itself and the PA (P = 0.9); moreover, the MVD in the PA often exceeded that in the tumor. No correlation or significant differences were also found in the MVD between different grades of malignancy of LMS (r = 0.1; P = 0.07). The number of AgNORs in tumor endothelial cells was significantly higher in tumor vessels than in the peritumoral area (P < 0.005) and increased with the tumor grade. Analysis of the prognostic value of MVD in uterine LMS and PA showed that the density of tumor vessels was not an independent criterion, while the MVD in the PA affected 10-year survival to a significantly greater extent (χ 2 = 27.5; P = 0.0003). The number of AgNORs also had an important effect on survival of LMS patients: when the threshold of 11.6 granules was exceeded, prognosis was significantly more unfavorable than that prior to exceeding the threshold.

Significance of AgNOR analysis and the expression of Ki-67 in the precursor lesions of the breast carcinoma

Objective To study the diagnostic and differential diagnostic significance of argyrophilic nucleolar organizer region (AgNOR) and cellular proliferation index ( PlKi-67 ) in the precursor lesions of the breast carcinoma. Methods Image quantitative analysis of AgNOR and immunostains of Ki-67 in tissue microarray sections from 76 biopsies of the precursor lesions of the breast carcinoma. Results There were some differences in granulae sizes and shapes of AgNOR among the precursor lesions of the breast carcinoma. The number of AgNORs per nucleu, total area of AgNORs, mean area of a single AgNOR, PIKi-67 were increasing from ADH to high-grade DCIS, and there was statistical difference between ADH, low-grade DCIS,respectively and high-grade DCIS. The number of AgNORs per nucleu was statistically different between ADH and low-grade DCIS. Conclusion AgNOR and Ki-67 could be regarded as assistant indicators in the diagnosis and differential diagnosis of the precursor lesions of the breast carcinoma. Key words: Breast neoplasia; Atypical ductal hyperplasia; Ductal carcinoma in situ; Argyrophilic nucleolar organizer regions; Ki-67; Tissue microarray

Inhibition of migration but stimulation of proliferation of human retinal pigment epithelial cells cultured with uniform vesicles of silicone oil

It has been hypothesized that emulsification of silicone oil might stimulate preretinal membrane formation and proliferative vitreoretinopathy. This study aimed to test the effects of vesicles of silicone oil (VSO), which were prepared by a novel membrane emulsification technique, on the migration and proliferation of human retinal pigment epithelial (hRPE) cells in vitro. VSO were produced by extrusion of the oil through a Supor membrane (Pall Life Sciences, Port Washington, NY, USA) with 0.45 microm pore size under a pressure of 0.1 MPa. hRPE cells were incubated with stained 25% VSO for 24 h to assess whether hRPE cells could phagocytize VSO. A wound-healing model was used by denuding cells from a glass slide to assess the migration of hRPE cells. The cells were then incubated with 25%, 50% and 100% VSO with or without serum for up to 72 h. The number of cells that had entered the denuded area was counted under a microscope. To assess the proliferative activity, the cells were incubated with 25%, 50% and 100% VSO, with or without serum, for 24 h. A total of 100 nuclei were examined for each slide, and the numbers of stained argyrophilic nucleolar organizer regions (AgNORs) in the nuclei were measured. The mean vesicle diameter of VSO prepared was 4.25 +/- 0.77 microm. The stained 25% VSO were phagocytized by hRPE cells. After the cells cultured with serum-free Dulbecco's Modified Eagle Medium (DMEM) were incubated with VSO, the numbers of migratory cells at higher concentrations (50% and 100%) of VSO were significantly decreased (48.9 +/- 5.37 and 10.6 +/- 3.03 respectively) compared to controls (69.9 +/- 9.88; P < 0.01). After the cells cultured with serum-free DMEM were incubated with VSO, the number of AgNORs in nucleus at 100% VSO was significantly increased compared to controls (3.1 +/- 0.72 vs 1.6 +/- 0.6; P < 0.01). The result indicated that VSO inhibited the migration but stimulated the proliferation of hRPE cells.

Argyrophilic nucleolar organizer regions in neoplastic and non-neoplastic hepatocytes bearing Mallory bodies.

The proliferative activity of Mallory bodies (MB)-positive hepatocytes (neoplastic and non-neoplastic) was examined by counting the argyrophilic nucleolar organizer regions (AgNORs). Among 19 cases of hepatocellular carcinoma, the mean number of AgNORs was lower in the MB-positive carcinoma cells than in the negative ones in nine cases, higher in six, and there was no difference in four. In non-neoplastic cases (seven cases of advanced primary biliary cirrhosis and seven cases of alcoholic or nutritional liver injury), the mean number of AgNORs was lower in the MB-positive hepatocytes than that in the negative ones in eight cases, and approximately equal in number in six cases. These findings imply that MB formation does not directly represent the level of proliferative activity of hepatocytes, regardless of whether they are malignant or not.

Argyrophilic nucleolar organizer regions in inflammatory, premalignant, and malignant oral lesions: A quantitative and qualitative assessment

Argyrophilic nucleolar organizer regions (AgNORs) have found widespread application in the past, especially in tumor histopathology. This study was undertaken to evaluate the significance of various AgNOR parameters and to assess their role in differentiating hyperplastic, premalignant, and malignant lesions. The study sample consisted of archival biopsy specimens of ten squamous cell carcinomas, ten premalignant lesions, and five inflammatory lesions. Two biopsies from normal mucosa acted as control. AgNORs were assessed both quantitatively and qualitatively. The data were analyzed using Student's independent t-test, one-way analysis of variance (ANOVA), and multiple range test (Tukey-HSD). Quantitatively significant difference existed in the number of AgNORs between the normal mucosa, inflammatory lesions, and carcinomas, but the premalignant lesions failed to differ significantly from the normal mucosa. The number of AgNORs was found to be related to epithelial proliferation. Qualitatively, in terms of size, shape, and pattern of distribution, the normal mucosa and inflammatory lesion were alike, but the premalignant and malignant lesions differed significantly from the normal, with a marked degree of AgNOR pleomorphism being observed in carcinomas. AgNOR quantity is strictly proportional to the proliferative activity of the cell and does not necessarily indicate malignancy. It is the qualitative characteristics of AgNOR that help to differentiate hyperplastic, premalignant, and malignant lesions.

Morphometry and digital AgNOR analysis in cytological imprints of benign, borderline and malignant serous ovarian tumours

The aim of the study was to determine values of a quantitative morphometry analysis of nuclear characteristics and argyrophilic nucleolar organizer regions (AgNORs) in differential cytodiagnosis of benign, atypically proliferating (borderline) and malignant serous ovarian tumours. Cytological imprints of benign (n = 20), borderline (n = 19) and malignant (n = 20) ovarian serous tumours were analysed. A computerized, digital analysis was used to determine morphometric nuclear features, the number and characteristics of single AgNORs, cluster AgNORs, total AgNOR and AgNOR area/nucleus (relative area) ratio. According to their size AgNORs were classified in three categories. A one-way variance analysis and post hoc test (Scheffé) were used for statistical analysis. The morphometric nuclear analysis showed that benign, borderline and malignant serous ovarian tumours are statistically different (P < 0.001) according to the area and outline, the values being highest in malignant tumours and lowest in the borderline group. Digital analysis of AgNORs in benign, borderline and malignant groups showed that the total AgNOR number increases with progression of the lesion (meaning tumour malignancy) significantly (P < 0.001) between benign and malignant as well as between borderline and malignant serous ovarian tumours (P < 0.001). The progression of the lesion malignancy was accompanied by a significant (P < 0.001) progressive increase of the total and relative AgNOR area per nucleus. The AgNOR size increases from benign to malignant tumours and a statistically significant difference (P < 0.001) was observed in all three groups regarding small and large AgNORs. Combining different markers of morphometric nuclear characteristics and AgNOR values could improve differential cytodiagnosis of benign, borderline and malignant serous ovarian tumours.

Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer regions in squamous cell carcinoma: relationships with tumor stage and grade, and apoptosis

The aim of this study was to investigate whether levels of Fas and Fas-ligand (Fas-L) expression in oral and oropharyngeal squamous cell carcinomas (SCCs) are associated with tumor stage and grade, and to assess whether parameters related to argyrophilic nucleolar organizer regions (AgNORs) correlate with apoptosis in SCC cells and adjacent normal epithelium. Fifty-two specimens taken from the tumoral and neighboring normal tissues of 26 patients with oral and oropharyngeal SCC were analyzed for Fas/Fas-L expression, and 24 specimens from 12 patients for AgNOR parameters. Seventeen (65%) of the tumors were Fas and/or Fas-L-positive (by immunohistochemistry). A significant positive correlation was found between Fas/Fas-L expression and clinical tumor stage ( P < 0.01). Mean AgNOR number per nucleus, AgNOR size and the percentage area of each nucleus occupied by AgNORs (percent of nuclear area) were significantly increased in the SCC cells (4.49 ± 1.28, 4.48 ± 1.42, 5.56 ± 1.22, respectively) when compared with the control neighboring squamous epithelial cells (2.58 ± 0.61, 1.64 ± 0.59 and 4.35 ± 0.62%, respectively) ( P < 0.01). A significant positive correlation was found between the AgNOR parameters and Fas/Fas-L expression as apoptotic markers in the tumoral cells of SCC ( P < 0.05). There was also a significant positive correlation between the AgNOR parameters and the grading of tumors ( P < 0.05). In conclusion, AgNOR count was a strong proliferation marker in patients with SCC, and Fas and Fas-L staining was useful in tumor grading.

A stereological study of the tuberomammillary nucleus in the Wistar rat: Morphological and functional comparison among subgroups E1‐E2‐E3

AbstractIn the present study, we have calculated the total neuronal number and volume of the tuberomammillary nucleus and of each of its subgroups E1, E2 and E3 in the Wistar rat. This estimation was done using stereological methods that can produce accurate quantifications. Neuronal number was estimated by applying the optical fractionator and the total volume of the nucleus by Cavalieri's method. Simultaneously, we quantified the argyrophilic nucleolar organizer regions (Ag‐NORs) of the neurons from these subgroups. The number and area of the Ag‐NORs stained with the silver nitrate technique, reflect the protein synthesis activity of these neurons.Our results show that the tuberomammillary nucleus has a unilateral total neuronal number of 2,460 neurons and a unilateral volume of 0.08756 mm3. We also find statistically significant differences on a morphological level among the different subgroups of the tuberomammillary nucleus. Hence, E2 has the largest number of neurons followed by E3 whereas E1 is the subgroup with the smallest neuronal density. There were no significant differences in the functional parameters: area of neuronal nucleus and mean area and number of Ag‐NOR, among the groups.