AgNOR Parameters Research Articles

Electromagnetic Field Effect on Bone Marrow Stem Cells Differentiation and Nucleoli AgNOR

Purpose: Numerous studies have described the effect of Electromagnetic Fields (EMFs) in the promotion of Bone Marrow Stem Cell (BMSC) differentiation. We aimed to investigate the influence of frequency (10 and 100 Hz) and different pulse shapes (sine, rectangular, and triangular) of EMF on rats' BMSCs.
 Materials and Methods: The BMSCs in 6 groups were exposed to EMF for 1 h/ 7 days. The BMSCs viability was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The cresyl violet labeled the Nissl bodies, and the silver nitrate staining was done to evaluate the BMSCs nucleoli AgNORs.
 Results: The MTT test verified that EMF and pulse shape did not affect cell viability. In Nissl bodies staining most of the large neurons were related to the rectangular 10 Hz EMF group. The majority of the differentiated BMSCs were astrocytes, microglia, and oligodendrocyte in the triangular 100 Hz EMF group. Although the silver nitrate staining confirmed the effect of 10 Hz EMF, pulse shape alteration did not affect AgNOR parameters. In conclusion, we presented a low-magnetic flux density EMF (400 µT) to assess the responses of BMSCs nuclei.
 Conclusion: The findings showed that BMSCs differentiation was frequency-dependent. Further investigations are recommended for recognizing the function of EMF on BMSCs.

Komparativna evaluacija parametara argirofilnih regiona jedarnog organizatora kod benignih i malignih tumora dojke

Aim. The aim of the paper was to evaluate the AgNOR parameters for the discrimination of benign from malignant breast tumors via a new approach - the total AgNOR area/nuclear area (TAA/NA). Material and methods. Three groups, consisting of control (n = 14), benign (n = 18) and malignant (n = 28) participants were included in the study. The AgNOR staining technique was performed and both mean AgNOR number and TAA/NA ratio were evaluated. Results. While the differences between the control and patient groups were statistically significant for AgNOR number (p < 0.001), it was not significant between the malignant group and the benign group for mean AgNOR number (p > 0.05). For the ratio of TAA/NA, the differences between the control and benign group (p < 0.001), control and malignant group (p < 0.001), and malignant and benign patient groups were significant. (p < 0.05). Conclusion. We consider that the evaluation of the TAA/NA rate, when compared with the AgNOR number, can be more sensitive and useful tool for distinguishing benign from the malignant breast lesions.

The Comparasion of Some AgNOR Parameters of Purkinje Cells in The Laying and Broiler Chicks Exposed to Daily Cyclic Lighting During The Embryonic Period

Aydınlatma, modern tavuk yetiştiriciliğinde, üreme, büyüme, vücut sıcaklığı, yem tüketimi ve sindirim olayları da dahil olmak üzere birçok fizyolojik süreci etkileyen ve metabolik olaylarda belirleyici rol oynayan önemli unsurlardan biridir. Aydınlatmaya bağlı olarak oluşan değişiklikler, sinir sistemi ve endokrin sistemin görev aldığı fizyolojik olaylar sonucu meydana gelmektedir. Purkinje hücreleri, merkezi sinir sisteminin en iri hücrelerindendir. Bu hücreler, tavuk embriyosunun ilk günlerinde beyincik korteksine göç edip, kendilerine ait hücre tabakasını meydana getirmektedirler. DNA’nın rRNA sentezleyen genlerini içeren ve nukleolusu oluşturan bölgeleri, nukleolus düzenleyici bölgeler (NOR) olarak adlandırılmaktadır. NOR’ların gümüşleme metoduyla (AgNOR) aktif olarak transkripsiyon yapan bölgelerinin boyanması sırasında rRNA bölgeleri de boyanarak, ışık mikroskobunda küçük, koyu benekler halinde görünmektedirler. Bu çalışmada; etlik ve yumurtacı damızlık yumurtalarına kuluçkada uygulanan günlük döngüsel aydınlatmanın (16 saat aydınlık/8 saat karanlık) beyincikte Purkinje hücrelerinde hem histolojik (luxol fast blue & cresyl violet) hem de bu hücrelerin nukleus çap ve alanları ile NOR alanları (AgNOR gümüş boyama yöntemi ve istatistiksel analiz) üzerine etkisi incelenerek ileride yapılacak olan benzeri çalışmalara temel veriler sağlanması amaçlanmıştır. Kuluçkada aydınlatmanın kontrol grubuna göre etlik civcivlerde Purkinje hücrelerinde nukleus alanı ile NOR alanını, yumurtacılarda ise nukleus çapını artırdığı saptanmıştır. Sonuç olarak, kuluçkada aydınlatmanın etlik civcivlerde Purkinje hücrelerinde protein sentezini artırdığı dolayısı ile sitoplazmalarındaki protein miktarında artışa bağlı olarak nukleus alanının genişlediği yargısına varılmıştır.

Evaluation of genotoxic and cytologic effects of environmental stressin wheat species with different ploidy levels

Drought and salinity induce biochemical, physiological, morphological, and genetic responses in plants. To understand the damage to different wheat species caused by these stresses, we investigated root-shoot growth, cell division, chromosomal aberrations, and nucleolus organizer regions (AgNORs) in root tip cells. Inhibition of root and shoot growth were observed in diploid, tetraploid, and hexaploid wheat genotypes after salt and drought stresses. Both stresses had adverse effects on dividing cells in root tips of all wheat species and caused a decrease in mitotic index values. Salt and drought lead to the induction of chromosome aberrations and repression of interphase AgNOR parameters. Both treatments produced different mitotic abnormalities including chromosomal stickiness, c-mitosis, and micronuclei formation, which indicated their action on the mitotic spindle. Reduction in AgNOR parameters showed that salt and drought may decrease the level of ribosomal biogenesis and reduce the length of the cell cycle. Because of the toxicity of Na ions, salinity inhibited all parameters at a higher degree than drought in all Triticum species. Cytotoxic effects of salinity and drought were mainly observed in meristematic cells in all wheat species. This comprehensive analysis may provide valuable information for understanding the effects of salt and drought stresses on cytogenetics of wheat species.

Decreased ribosomal DNA transcription in dorsal raphe nucleus neurons differentiates between suicidal and non-suicidal death.

An involvement of the central serotonergic system has been implicated in the pathogenesis of suicide. The dorsal raphe nucleus (DRN) is the main source of serotonergic innervation of forebrain limbic structures disturbed in suicidal behaviour. The study was carried out on paraffin-embedded brainstem blocks containing the DRN obtained from 27 suicide completers (predominantly violent) with unknown psychiatric diagnosis and 30 non-suicidal controls. The transcriptional activity of ribosomal DNA (rDNA) in DRN neurons as a surrogate marker of protein biosynthesis was evaluated by the AgNOR silver staining method. Significant decreases in AgNOR parameters suggestive of attenuated rDNA activity were found in the cumulative analysis of all DRN subnuclei in suicide victims versus controls (U test P values < 0.00001). Our findings suggest that the decreased activity of rDNA transcription in DRN neurons plays an important role in suicide pathogenesis. The method accuracy represented by the area under receiver operating characteristic curve (>80 %) suggests a diagnostic value of the observed effect. However, the possible application of the method in forensic differentiation diagnostics between suicidal and non-suicidal death needs further research.Electronic supplementary materialThe online version of this article (doi:10.1007/s00406-015-0655-4) contains supplementary material, which is available to authorized users.

Higher AgNOR Expression in Metaplastic and Dysplastic Airway Epithelial Cells Predicts the Risk of Developing Lung Cancer in Women Chronically Exposed to Biomass Smoke.

We evaluated AgNOR expression in airway epithelial cells (AECs) as a risk factor of lung carcinogenesis in 228 nonsmoking women exposed to biomass fuel (BMF). A total of 185 age-matched women who cooked with cleaner fuel (liquefied petroleum gas [LPG]) were enrolled as study controls. Compared with controls, Papanicolaou-stained sputum samples showed 4 and 8 times higher prevalence of metaplasia and dysplasia, respectively, in AECs of BMF users. AgNOR staining showed significantly larger numbers of dots and larger size and percentage of AgNOR-occupied nuclear area in normal AECs of BMF users than in controls. Interestingly, AgNOR parameters increased dramatically when the cells were transformed from normalcy to metaplasia and dysplasia. Compared with LPG users, BMF users showed a marked rise in reactive oxygen species (ROS) generation and a depletion of superoxide dismutase (SOD), indicating oxidative stress. Indoor air of BMF-using households had 2-5 times more particulate pollutants (PM10 and PM2.5), 73% more nitrogen dioxide (NO2), and 4 times more particulate-laden benzo(a)pyrene [B(a)P], but no difference in sulfur dioxide was observed. A high-performance liquid chromatography (HPLC) study estimated a 6-fold rise in benzene metabolite trans, trans-muconic acid (t,t-MA) in urine of BMF users. After controlling confounding factors using multivariate logistic regression, positive associations were observed between cellular changes, AgNOR parameters, and PM10, PM2.5, NO2, B(a)P, and t,t-MA levels, especially the concentration of B(a)P. In conclusion, cumulative exposure to biomass smoke causes oxidative stress and enhances AgNOR expression in precancerous metaplastic and dysplastic AECs and appears to be a risk factor for developing lung cancer.

Postmortem-Assessed Impairment of Neuronal Activity in Depression: The Dominant Impact of Suicide

We report recent postmortem findings from the Magdeburg Brain Bank related to the evaluation of impaired activity of neuronal networks relevant for depression. Chronic changes in ribosomal DNA transcriptional activity have been revealed by the quantitative evaluation of silver-stained nucleolar organising regions (AgNORs). Abnormalities in AgNOR parameters have been found in neurons of the prefrontal limbic regions, the amygdala, the external globus pallidus, and the serotonergic dorsal raphe nucleus (DRN). The impact of the processes leading to suicide has been clearly accentuated in opposition to the weak influence of unipolar-bipolar dichotomy, which was only revealed in one area (the anterior cingulate cortex). The impact of suicide was most pronounced in the DRN. Our AgNOR studies show that the influence of psychotropic medication (antidepressants and typical neuroleptics, among others) is limited in the prevention of abnormal neuronal activity specific for suicide.

Minichromosome maintenance (MCM) and AgNOR proteins expression in desmoid tumours: a tissue microarray analysis.

In the present study, nuclear proliferative proteins: MCM2, MCM5, MCM7, Ki-67 and AgNORs expression was assessed in paraffin sections from sporadic desmoid tumours using a tissue microarray (TMA)-based immuno- and histochemistry, respectively. Nuclear expression of MCM7, where the percentage of positive cells was 0.87% (± 1.64) (range 0-5%), was found in 4/20 (20.0%) cases. In 32/32 (100%) of the examined desmoid cases no expression of nuclear proteins MCM2 and MCM5 was detected. Nuclear expression of Ki-67 was observed in 4/21 (19%) cases. Paraffin sections from 30 cases of desmoid tumours were silver-stained to visualize AgNORs. The following AgNOR parameters were calculated: mean AgNOR number per nucleus (N), mean AgNOR area per nucleus, mean AgNOR dot area per nucleus (A), and mean AgNOR content (C = N/A). In the investigated group the mean values of AgNOR parameters were the following number: 4.34 (± 0.11); area: 0.74 μm2 (± 0.19); dot area: 0.18 m2 (± 0.01), and AgNOR content: 23.73 (± 1.85). The mean AgNOR number per nucleus and mean AgNOR content in desmoid tumours were statistically significantly higher as compared to the controls (tonsil tissue) (p<0.001). This study observed low level of MCM7 and Ki-67 and lack of MCM2, MCM5 proteins expression which may explain commonly known low mitotic activity of desmoid tumour cells. The morphology of dots related to AgNORs (number, area) and their morphometric parameters point to elevated transcriptional activity of desmoid cells.

The impact of sodium chloride on root growth, cell division, and interphase silver-stained nucleolar organizer regions (AgNORs) in root tip cells of Allium cepa L.

To evaluate genetic damage, parameters including root growth, chromosomal aberrations, and interphase silver-stained nucleolar organizer regions (AgNORs) in root tip cells of Allium cepa L. were investigated after treatment with NaCl (40–160 mM). The results showed that NaCl caused a decrease in root growth. All concentrations of NaCl showed an inhibitory effect on dividing cells in root tips of A. cepa L. and caused a reduction in mitotic index values. Upon exposure to NaCl, roots exhibited various mitotic abnormalities, including c-mitosis, anaphase bridge, and chromosome stickiness. In addition, interphase cells with micronuclei, budding nuclei, and unequal-sized nuclei were observed. Moreover, total cell aberration increased with increasing NaCl concentration. For AgNOR parameters, the average number of AgNORs per nucleus decreased in roots treated at all NaCl concentrations. The singular AgNOR area and whole AgNOR area in the nucleus containing 1–3 AgNORs were inversely proportional to NaCl concentrations.

Demonstration of decreased activity of dorsal raphe nucleus neurons in depressed suicidal patients by the AgNOR staining method

Background Suicide and depression are closely related yet distinct phenomena. In both these phenomena, research has focused on central serotonergic system disturbances. The dorsal raphe nucleus (DRN) is the main source of serotonergic innervation of limbic structures crucial for the regulation of emotionally influenced behaviour. Methods The study was carried out on paraffin-embedded brains from 23 depressed patients (12 suicides and 11 non-suicides) and 26 matched controls without mental disorders. The karyometric parameters of DRN neurons were evaluated by the AgNOR silver staining method. Results The significant effect of suicide on the nuclear area was found in the cumulative analysis of all DRN subnuclei (ANOVA, P = 0.032). A decreased mean value of this parameter was observed in the suicides group versus controls ( t-test, P = 0.032). This effect was especially pronounced in the violent suicide victims ( t-test, P = 0.001), who also demonstrated a decreased AgNOR area versus controls ( t-test, P = 0.007). No significant effect of depression or polarity on AgNOR parameters was found. Limitations A major limitation of this study is relatively small case number. A further limitation is given by the lack of data on drug exposure across the whole life span. Conclusion Our findings suggest that hypoactivity of DRN neurons is a distinct phenomenon in depression, specific only for suicidal subgroup of depressed patients.

Computer-assisted analysis of cell proliferation markers in oral lesions

Abnormalities in any component of the cell cycle regulatory machine may result in oral cancer, and markers of cell proliferation have been used to determine the prognosis of tumor progression. The aim of this study was to determine whether silver-stained nucleolar organizer region (AgNOR) and Ki-67 measurements could improve the assessment of growth rates in oral lesions. Eighty-three oral biopsies were studied, 20 of which were classified as fibrous inflammatory hyperplasia (FIH), 40 as leukoplakia (LKP) and 23 as oral squamous cell carcinoma (OSCC). Within the LKP group, 22 out of 29 biopsies were diagnosed as non-dysplastic leukoplakia (LK) and 18 as dysplastic leukoplakia (DLK), presenting discrete, moderate and severe dysplasia. Ki-67 immunolabeling of the lesions increased steadily in the following order: FIH, DLK, LK and OSCC, indicating that Ki-67 is a good marker for predicting the proliferative fraction among benign, premalignant and malignant oral lesions. The median values of AgNOR parameters indicate that the morphometric index gives better results regarding the proliferative rate than the numerical one. A series of linear regressions between AgNOR parameters and Ki-67 showed positive associations. We conclude that a combination of Ki-67 and morphometric AgNOR analyses could be used as an aid in the determination of the proliferative status of oral epithelial cells in oral cancer.

Prognostic significance of DNA cytometry in combination with AgNOR investigation

Conclusion. While most results concerning DNA and nucleolar organizer region (AgNOR) parameters fit with previous studies, the percentage of aneuploidy looks like a promising prognostic parameter. The observed intratumoral heterogeneity could represent a possible source of conflicting and inconsistent results. Objectives. The aims of our study were to determine the prognostic relevance of different DNA and AgNOR parameters in laryngeal squamous cell carcinoma (SCC) and compare these findings with established prognostic factors including tumor stage and grade, as well as the detection of possible intratumoral heterogeneity. Materials and methods. Sections from 62 laryngeal SCCs were analyzed for DNA content, DNA index, S-phase, percentage of aneuploidy, and AgNOR. Of 62 samples, 31 morphologically similar tumor samples were analyzed for the same parameters in three different tumor areas defined as tumor center, invasive tumor margin, and transformation margin between tumor and normal-appearing mucosa. Results. Our study showed that DNA and AgNOR parameters correlated with T stage, lymph node involvement, and histologic grade regardless of tumor areas. Significant correlation was found between mean number of AgNOR per nucleus and percentage of aneuploidy. Clinical stage and percentage of aneuploidy correlated with survival (p<0.02). Heterogeneity DNA study revealed aneuploidy in central portions of 90% of tumors, while in margins aneuploidy was demonstrated in about half of the patients.

Proliferation in non-Hodgkin's lymphomas and its prognostic value related to staging parameters.

In malignant lymphomas, cell kinetics has shown to be related with histologic type as well as with the clinical behaviour. The aim of our study was to investigate the relevance of cell proliferation parameters on overall survival in non‐Hodgkin's lymphomas as well as their relationship with prognostic factors such as International Prognostic Index (IPI). We performed DNA‐flow‐cytometry (S‐phase fraction and detection of DNA‐aneuploidy) as well as cytologic examination and the AgNOR technique in material obtained by fine needle aspiration of lymph nodes at diagnosis. The majority of the patients were stage IV by Ann Arbor and intermediate risk by IPI (42/55). When analyzing all patients together, histologic type by the WHO classification, IPI and the presence of a DNA‐aneuploid clone could not separate well patients with a different survival. For all patients, univariate Cox analysis revealed S‐phase (SPF) and AgNOR parameters to be of prognostic value. In the multivariate analysis, however, only SPF remained in the final model. Yet, when stratifying for DNA‐ploidy, only the total number of AgNORs/nucleus was an independent parameter. Looking only at the DNA‐diploid cases, the AgNOR pattern remained the most important parameter, whereas for the DNA‐aneuploid cases this was true for SPF. When studying patients with B large cell lymphoma separately, only DNA‐ploidy was a prognostic factor. In summary, cell kinetic parameters reveal important prognostic information in NHL patients. Furthermore, DNA‐aneuploidy seems to interfere with the analysis of the AgNOR pattern.

Micronuclei, nuclear lesions and interphase silver-stained nucleolar organizer regions (AgNORs) as cyto-genotoxicity indicators in Oreochromis niloticus exposed to textile mill effluent

In this study, cyto-genotoxic effects of a textile mill effluent on fish Oreochromis niloticus were investigated using the micronucleus (MN) test and methods to analyze interphase silver-stained nucleolar organizer regions (AgNORs). Fishes were exposed to three different concentrations of textile mill effluent (5, 10 and 20% (v/v)) for 3, 6 and 9 days. Cyclophosphamide (2 mg/l) was used as a positive control. Micronucleus frequencies were examined in peripheral blood erythrocytes and gill cells. Nuclear abnormalities (NA) other than micronuclei such as binuclei, lobed nuclei, blebbed nuclei and notched nuclei were also evaluated in peripheral erythrocytes. Interphase AgNOR parameters were examined in epithelial cells obtained from the edge of caudal fins after 90 and 180 min of exposure. As a result, dose-dependent increases in the frequencies of micronuclei and other NA in erythrocytes were observed. MN frequencies in gill cells also significantly increased, while the interphase AgNOR parameters in fin cells decreased, as a result of textile effluent and cylophosphamide treatments.

The effect of desalivation on 4-nitroquinoline-1-oxide-induced tongue carcinogenesis: a morphometric study of nucleolar organizer regions.

Saliva is an important factor in the oral cavity and could be significant in protecting against carcinogens. In experimental models of carcinogenesis, saliva was shown to have a temporary protective effect against the carcinogens DMBA and 4-nitroquinoline-oxide (4NQO). Silver-stained nucleolar organizer regions (AgNORs) are considered markers for both proliferative capacity and prognosis. The purpose of the present study was to investigate the effect of desalivation on AgNOR parameters in lesions induced by the carcinogen 4NQO in a rat model, in order to trace early nuclear changes. The study group consisted of 120 male Wistar-derived rats. The experimental group (n=56) underwent surgical desalivation; the control group (n=56) underwent a sham operation, and both groups were administered a solution of 0.001% 4NQO in the drinking water. A normal group (n=8) did not receive surgery and drank tap water. Rats were sacrificed at 7, 14, 22, and 28 weeks. Formalin fixed, paraffin embedded sections of the tongue were silver-stained and AgNOR parameters were analyzed using computerized image analysis. In both desalivated and control groups, the nuclear area was significantly higher than the normal. This difference was already evident at 7 weeks. The mean AgNOR area was significantly higher in the desalivated group at week 7 and continued to increase over time. The mean AgNOR number was also significantly higher in the desalivated group at week 7. Differences between the desalivated and control groups diminished with time. These changes in proliferative activity, as expressed by AgNOR parameters, presented earlier changes in comparison to those observed in microscopic examination of the same slides. Results suggest that saliva in the oral cavity can delay malignanttransformation, but continued exposure to the carcinogen overrides this effect. AgNOR stain seems to be sensitive and allows for early identification of intranuclear changes.

Early progression stage of malignancy of uterine cervical dysplasia as revealed by immunohistochemical demonstration of increased DNA-instability.

The degree of DNA-instability as revealed by the immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (DNA-instability test) was used as a marker of malignancy. This was applied to mild dysplasia (42 cases), moderate dysplasia (43 cases), severe dysplasia (27 cases), squamous cell carcinoma in situ (CIS) (21 cases), invasive squamous cell carcinoma (SCC) (31 cases) and normal (7 cases) human uterine cervix. The expression of tumour suppressor gene p53 and oncogene bcl-2 was detected immunohistochemically. Proliferative activity was evaluated by PCNA immumohistochemistry and the quantitative analysis of the number, mean area, the largest area and maximum shape irregularities of AgNOR in a nucleus were performed for all these cases. The distribution of numeric chromosomal aberrations of chromosome 17 was also investigated in some of these cases. The results showed that 31 SCC (100%), 21 CIS (100%), 21 severe dysplasia (77.77%), 28 moderate dysplasia (65.11%), and 14 mild dysplasia (33.33%) were positively stained by the DNA-instability test diffusely or sporadically, indicating their malignancy. Reflecting the malignant character, these cases showed a remarkable increase in the PCNA-index with the loss of polarity of PCNA positive cell distribution and also an increase in number, mean and largest sizes and maximum shape irregularity of AgNOR dots. The mean chromosome index for chromosome 17, p53 and bcl-2 immunostaining positivity were also found to be significantly increased in moderate and severe dysplasia and in cancerous cases in comparison to normal and mild dysplasia cases. Moreover, the DNA-instability-test positive dysplasia cases showed statistically significant increased values of PCNA-index, AgNOR parameters, mean chromosome index, p53 and bcl-2 expression in comparison to those of DNA-instability-test negative dysplasia cases. In conclusion, some mild dysplasia (33.33%) and most of the moderate (65.11%) and severe dysplasia (77.77%) were regarded as malignant in nature, existing at an early stage of progression of malignancy.

Bax expression has prognostic significance that is enhanced when combined with AgNOR counts in glottic carcinomas.

Using nucleolar organizer regions (NORs) as a proliferative marker and Bax expression as a marker for apoptosis, we have studied the individual and combined prognostic significance of these markers. Successive sections of diagnostic, formalin-fixed and paraffin-embedded specimens from 69 patients with T1-4 tumours were stained with a rabbit anti-human Bax polyclonal antibody and silver nitrate for visualization of NORs (AgNORs). After classification for staining intensity and the percentage of Bax expression, a final score resulting in four classes of increasing Bax expression was obtained. AgNOR counts were expressed as mean counts (mAgNOR) and the percentage of tumour nuclei with more than one AgNOR (pAgNOR>1). Both AgNOR parameters were grouped in three classes with increasing values. Low Bax scores correlated significantly with poor prognosis (P = 0.0106). For mAgNOR and pAgNOR>1, high values correlated with poor prognosis (P = 0.0185 and P = 0.0003 respectively). A combined parameter, for which the Bax score was subtracted from the AgNOR scores, appeared to be statistically stronger than the individual parameters (P < 0.0001). Both Bax expression and AgNOR scores, and in particular the combination of these parameters, appear to be strong prognostic markers in glottic squamous cell carcinomas.

Relationship between AgNOR proteins, Ki-67 antigen, p53 immunophenotype and differentiation markers in archival breast carcinomas.

The present study investigated (i) the relationship between standardised morphometric AgNOR parameters (argyrophilic nucleolar organiser region-associated proteins) and MIB1 growth fraction, and (ii) their correlation with immunohistochemical p53, sex steroid receptor status and histopathological differentiation grade in serial paraffin sections from 39 breast carcinomas. Ten sections were double-stained for AgNOR/MIB1. AgNOR parameters correlated significantly with MIB1 growth fraction and p53 protein expression. Significant inverse correlation was found between proliferation markers and oestrogen/progesterone receptor status and histopathological grade. AgNOR expression was significantly higher in cycling (MIB1 positive) tumour cells, than in resting (MIB1 negative) ones, however with exceptions. We conclude, that standardised AgNOR parameters correlate with markers of increased malignant potential in breast carcinomas. However, AgNORs seem to reflect proliferation independent cellular and nucleolar activity of tumour cells, as well. We recommend the use of standardised AgNOR analysis for obtaining sound results in routine paraffin sections.

NOR activity in hippocampal areas during the postnatal development and ageing

The silver staining of the nucleolar organizer regions (Ag-NORs) was used in order to estimate the biosynthetic activity of three hippocampal areas (dentate gyrus, CA1 and CA3) during postnatal development and ageing. 32 Wistar rats were used and 4 groups were formed according to the age of the animals (14, 21, 90 days and 23 months). Several Ag-NOR parameters such as mean Ag-NOR area and the ratio between Ag-NOR and nuclear areas per neuronal cell were quantified using an image analysis system. High values of these parameters are associated with a high rate of rRNA transcription. In this way, the neural biosynthetic activity in all regions studied decreased as the older ages are reached. Differences between areas are shown with the dentate gyrus and CA1 areas decreasing faster. The different activity among these areas is discussed, taking into account the particular affect on these areas of some injuries and the ageing process. Our results support the hypothesis of NOR loss as a main cause of ageing as reported by other authors.

Standardized AgNOR analysis of the invasive tumour front in oral squamous cell carcinomas.

In the last decade, silver staining of nucleolar organizer region-associated proteins (AgNORs) has been widely used in tumour pathology both for diagnostic and for prognostic purposes. However, a reliable and reproducible assessment of these proteins on routinely processed archival tissues has only become possible since the recent introduction of standardized staining method and computer-aided morphometric analysis. In the present study, the AgNOR content at the invasive front of 80 squamous cell carcinomas of the floor of the mouth/tongue was investigated using this novel approach, with regard to prognosis and a variety of clinico-pathological parameters. All standardized AgNOR parameters [mean of AgNOR number, mean of AgNOR area, coefficients of variation (CV) of both AgNOR number and area] were statistically significantly associated with the clinical course. The strongest correlation was found for the AgNOR-area univariate analysis (P = 0.006). In multivariate analysis, the mean of AgNOR number could independently predict both overall (P = 0.01) and disease-free survival (P = 0.001). It is concluded that standardized staining and computer-aided analysis of AgNORs are prerequisites for an objective and reproducible AgNOR assessment, which has potential as a supplementary diagnostic and prognostic tool in oral cancer.