| Abstract|| |
Background: The periodontal lesions with cellular proliferation can be assessed by various methods. One of the most recent methods to determine the proliferative activity is silver-staining nucleolar organizer region (AgNOR) staining. The purpose of the present study was to evaluate, if AgNOR count can act as a proliferative marker and can aid in the diagnosis and prognosis of periodontal lesions.
Materials and Methods: For this study, subjects with healthy gingival status, non-neoplastic lesions, neoplastic lesions, and plaque-induced gingivitis were included. Following the provisional diagnosis of clinical entity, biopsies were taken from the respective selected sites for histopathological diagnosis. In plaque-induced gingivitis cases, a second biopsy was taken from the selected sites 3 weeks following scaling. After histological confirmation, one more section was prepared, which was subjected to AgNOR staining, and AgNOR numbers were counted by individual and cluster counts and statistically analyzed.
Results: Results showed the highest AgNOR count in neoplastic lesions. Non-neoplastic lesions showed a higher AgNOR count as compared to clinically healthy gingiva. Plaque-induced gingivitis showed a considerable reduction in AgNOR count after treatment.
Conclusion: Results of this study confirmed that AgNOR count reflects the cellular proliferation and has a limited diagnostic value. However, the prognostic value of AgNOR for periodontal lesions is dependable.
Keywords: AgNOR count, diagnostic, H and E staining, periodontal lesions, prognostic
|How to cite this article:|
Saluja M, Vandana K L. The diagnostic and prognostic implications of silver-binding nucleolar organizer regions in periodontal lesions. Indian J Dent Res 2008;19:36-41
The response of the gingival tissues to pathogenic factors may lead to reactive hyperplastic changes, which can be broadly segregated into inflammatory and neoplastic lesions. The clinical presentation of these lesions affecting periodontium may generate diagnostic challenges for the periodontist. Although most of the soft tissue lesions affecting the periodontium can be accurately diagnosed at the microscopic level with conventional laboratory procedures, sometimes histochemistry, immunochemistry, or immunofluorescence evaluations are needed to arrive at a precise diagnosis.
|How to cite this URL:|
Saluja M, Vandana K L. The diagnostic and prognostic implications of silver-binding nucleolar organizer regions in periodontal lesions. Indian J Dent Res [serial online] 2008 [cited 2013 May 20];19:36-41. Available from: http://www.ijdr.in/text.asp?2008/19/1/36/38930
Several methods have been used in the past for identification of proliferating cells in tissue sections such as the assessment of mitosis, use of DNA flow cytometry, autoradiographic methods, applications of DNA and RNA, and use of monoclonal antibodies to detect proliferation-related antigen,  Ki67,  proliferating cell nuclear antigen (PCNA), bromodeoxyuridine-labeling (Brdu), and silver-staining nucleolar organizer region (AgNOR) staining.  AgNORs have recently attracted much attention.
Nucleolar organizing regions (NORs are loops of ribosomal DNA, which occur in the nuclei of cells possessing the genes for synthesizing r-RNA. In the Homo sapiens , they are located on the short arm of five acrocentric chromosomes, i.e., chromosome numbers 13, 14, 15, 21, and 22. NOR staining represents actively transcribing NORs and the frequency of NORs per nucleus may prove useful as replicatory marker. It has been suggested that the numbers of AgNORs in nuclei may reflect their state of activation or indeed, degree of malignancy of the lesion involved.
AgNOR staining is also useful for studying normal proliferating cells, because it may be a quantitative marker of incipient cellular alterations before the histologic hallmarks appear.  This method has been considered to represent a new, additional diagnostic tool in tumor pathology. It has been employed to study the proliferative activity of various tumor cells including malignant lymphoma, sarcoma, breast cancer, and pleural malignancy.  It is also suggested to be of use in diagnosis of cutaneous tumors.  However, it has been reported that the difference in AgNOR numbers is not of diagnostic significance and cannot be used to distinguish various odontogenic cysts from one another, nor from unicystic ameloblastoma. 
One of the most important uses of AgNOR staining is that it acts as a reliable prognostic marker.  It was also stated that AgNOR count may provide information on breast cancer prognosis supplementary to that obtained from DNA flow cytometric analysis.  Studies of oral malignancies and submucous fibrosis have revealed high count in squamous cell carcinoma with the AgNOR count increasing further with poor differentiation of the neoplasm.  The lesions with higher counts were reported to exhibit poor prognosis.  The increased proliferative activity and poor prognosis of ossifying fibroma compared with peripheral ossifying fibroma was suggested through AgNORs expression. 
A few studies have investigated the proliferative activity of the periodontal tissues using the AgNORs method. The changes in AgNOR number and its distribution indicate the destruction and repairing stages of the experimentally induced marginal periodontitis in dogs.  The study of AgNOR in periodontics is a new found interest, as many periodontal lesions are proliferative in nature. The proliferative activity of long junctional epithelium using AgNOR count has been reported by Uno et al .  However, limited literature is available related to AgNORs count of periodontal tissues during health and disease. A first attempt is made in the present study to include neoplastic as well as non-neoplastic periodontal conditions to evaluate the diagnostic ability of AgNOR. The prognostic value of AgNOR count is also evaluated in plaque-induced gingivitis cases before and after treatment.
| Materials and Methods|| |
The review committee constituted by Rajiv Gandhi University of Health Sciences approved the protocol for human subjects. Fifty-four systemically healthy Indians (24 males, 30 females, age range 18-50 years) participated in this study. The study period extended from April 2004 to February 2006.
Subjects of various categories included individuals with clinically healthy gingival status (absence of bleeding on probing) who were subjected for minor esthetic surgical procedure such as crown lengthening; subjects with non-neoplastic lesions such as fibrous hyperplasia, peripheral ossifying fibroma, gingival fibromatosis, pyogenic granuloma, drug-induced enlargement, gingival polyp; and subjects with neoplastic lesions such as squamous cell carcinoma of alveolar mucosa for evaluation of diagnostic value of AgNOR. Subjects with plaque-induced gingivitis were also included in the study to evaluate the prognostic value of AgNOR. Exclusion criteria included subjects who had received any periodontal therapy in past 6 months, who had received any antibiotic therapy in past 1 month, smokers, pregnant women, and nursing mothers. After collecting information about this study such as the objectives, expected outcomes and the degree of discomfort that might occur, the subjects gave their informed consent.
The selected subjects were examined for their periodontal status. Following the provisional diagnosis, biopsies were taken from the respective selected sites for histopathological diagnosis. In plaque-induced gingivitis cases, a second biopsy was taken from the selected sites 3 weeks following scaling. The bleeding index (Aniamo and Bay, 1975) was recorded in plaque-induced gingivitis cases before and after treatment. The sections were stained with hematoxylin and eosin for histological confirmation. Following the confirmation, the other section was stained for NOR to examine the proliferative activity of periodontal lesion, and the counting was done using the Windows-based image analyzer software (Image Proplus version 184.108.40.206 for Windows by Media Cybernetics, USA). The slides were restained if required.
AgNOR staining was done by modified procedure of Smith and Crocker.  Sections of 5 µm thickness were cut from the routinely processed paraffin blocks and were dewaxed in two changes of xylene for 3-5 min. They were hydrated through descending grades of alcohol to distilled water. The AgNOR solution was prepared by mixing one part of solution A (2 g gelatin in 100 ml of 1% aqueous formic acid) with two parts of solution B (50% silver nitrate). This was immediately poured over the tissue sections and left for 40 min at room temperature in a dark place. The silver colloid was then washed off with distilled water and the sections dehydrated through graded alcohol, cleared in xylene, and mounted in a synthetic medium (DPX).
For counting, the images were first captured using three-chip CCD camera attached to trinocular research microscope (Olympus BX 51, Japan). The actual counting was done by using the Windows-based image analyzer software. For each case, 100 cells with clear field were selected using 100× objective and the AgNORs were visualized as black or brown dots. These images were captured and the stored images recalled on to the monitor and manual counting of AgNORs was done by two different methods, i.e., by considering the AgNOR as individual count and as cluster count. The individual count was done by counting the number of individually discernible and separable black dots in each nucleus  and, in addition, by counting the clearly discernible boundaries of black dots forming the cluster and the average for each case was computed. The cluster count was done by recording the aggregate AgNOR as one, when two or more dots were so closely aggregated within a nucleus that the precise number within the aggregate could not be counted, along with the individually discernible and separable black dots in each nucleus and the average for each case was computed. 
Descriptive data were presented as mean, SD and range values. Multiple group comparisons were made by one-way anova. Intragroup comparisons were made by Wilcoxon's signed rank test and intergroup comparisons by the Mann-Whitney test. For all the tests, a p-value of 0.05 or less was considered for statistical significance.
| Results|| |
A total of 13 patients with clinically healthy gingival status, 24 patients with non-neoplastic, 2 with neoplastic lesions, and 15 patients with plaque-induced gingivitis were included in this study.
AgNOR staining was performed for all the sections and AgNOR counts were compared by individual counting and cluster counting. Intragroup comparison of mean AgNOR count was done for gingivitis cases before scaling and 21 days after scaling by using Wilcoxon's signed rank test. Intergroup comparison was done between different groups using the Mann-Whitney test.
The mean AgNOR counts of different non-neoplastic and neoplastic lesions are shown in [Table - 1]. [Table - 2] presents the intergroup comparison of AgNOR count between controls, plaque-induced gingivitis and non-neoplastic lesions. [Table - 3] represents the intergroup comparison of AgNOR count between controls and fibrous hyperplasia. Intragroup comparison of AgNOR count and gingival bleeding index in plaque-induced gingivitis before and after treatment are presented in [Table - 4]. Comparison of AgNOR count by individual count and cluster count are presented in [Table - 5].
| Discussion|| |
Nucleolar organizer regions (NORs) are loops of DNA that contain ribosomal RNA genes. These genes are transcribed by RNA polymerase I and ultimately direct ribosome formation and protein synthesis. One of the proliferative marker which is focused is Argyrophilic nucleolar organizer region (AgNOR) staining in histopathological diagnosis. The amount of silver deposited in the nucleus in this cytochemical reaction is a reflection of the transcriptional activity of ribosomal genes.
Qualitative and quantitative changes in AgNORs may provide useful information about nucleolar activity in hyperplastic and neoplastic conditions. AgNOR staining is also useful for studying normal proliferating cells, because it may be a quantitative marker of incipient cellular alterations before the histological hallmarks appear. 
AgNOR count is more commonly used for diagnostic purpose as compared to prognostic determination. Crocker  demonstrated that malignant cells can be distinguished from corresponding benign or normal cells on the basis of a higher quantity of interphase AgNOR, thus representing a new additional diagnostic tool in tumor pathology. AgNOR counts would thus contribute to diagnosis if changes in these parameters are associated with the degree of malignant transformation.
Other than diagnostic aspect, AgNOR has been found to serve as a reliable prognostic marker too. It has been demonstrated that squamous cell carcinomas in poor prognostic group had higher pooled mean AgNOR than those in good prognostic group.  The diagnostic and prognostic aspect of AgNOR is dealt to a large extent in medical field and its applications in oral lesions, especially periodontal lesions, are scanty. Recently, Abe  demonstrated that changes in AgNOR number and its distribution indicate the destruction and repairing stages of the experimentally induced marginal periodontitis in dogs. However, limited literature is available, related to AgNOR count of periodontal tissues during health and disease.
The diagnostic value of AgNOR count was determined in various non-neoplastic and neoplastic lesions. The non-neoplastic lesions included lesions with reactive hyperplastic changes in response to various pathogenic factors, which were broadly segregated into three types of lesions, namely fibrous hyperplasia, pyogenic granuloma, and peripheral ossifying fibroma. Apart from this, drug-induced gingival enlargement, gingival fibromatosis, and gingival polyp were also included. The prognostic value of AgNOR was evaluated in plaque-induced gingivitis patients before and 21 days after scaling.
In this study, AgNOR staining was performed for all the sections and AgNOR dots were enumerated as it is considered to be a basic method, which is widely used.  Microscopically, the AgNORs were clearly recognized as variable-sized black dots with yellowish background in the nuclei, and the counting was done using two methods by individual and cluster counts. The individual count was done by modification of the method used by Smith and Crocker  and cluster count by the method proposed by Giri et al. 
Because the AgNOR count is basically associated with tumor pathology, wherein dysplasia is characteristically seen, the word non-neoplastic is preferred in this study to those lesions predominated with hyperplasia. The results of this study demonstrated AgNOR count of healthy gingival epithelium to be 2.01 ± 0.36 by individual count and 1.62 ± 0.27 by cluster count, which was similar to the mean AgNOR count of healthy junctional epithelium, i.e., 1.63 ± 0.23 as reported by Usuda et al . 
The intergroup comparison between non-neoplastic lesions and controls showed much lesser AgNOR count in controls as compared to cases by both individual and cluster counts and the difference was found to be highly significant. No studies have been done on pyogenic granuloma, drug-induced enlargement, and gingival polyp, whereas limited literature is available on AgNOR count of fibrous hyperplasia, peripheral ossifying fibroma, and gingival fibromatosis.
The mean AgNOR count of fibrous hyperplasia and peripheral ossifying fibroma was found to be much higher as compared to studies done by Fonseca and do Carmo  and Mesquita et al .,  respectively. The relatively higher mean AgNOR count in the present study could be attributed to modification of individual count method, wherein the overlapping AgNORs were not counted as single dots, which would have resulted in too low results for more active lesions. 
The AgNOR count of squamous cell carcinoma was highest in this study, in spite of only two cases, by both the counts. The AgNOR count was found to be similar to a study conducted by Fonseca and do Carmo  but higher as compared to Epivatianos.  According to these methods of AgNORs counting, the results support that the DNA content and the proliferative activity of neoplastic lesion is much higher as compared to normal and non-neoplastic lesions. Yue et al.  reported that neoplastic cells generally exhibit a rise in the synthesis of normal and abnormal products and thus frequently feature a significant rise in AgNOR material.
The comparison of AgNOR count in plaque-induced gingivitis before and after treatment showed definite reduction in AgNOR count after treatment, which was found to be highly significant by both individual and cluster counts, thus determining the prognostic value of AgNOR count. The significant reduction in bleeding index of plaque-induced gingivitis supported the effective treatment that would influence the cellular proliferative activity, which in turn decreases the AgNOR count in the post scaling period.
As there is considerable debate on which method is better, in the present study, counting was done by both the methods and compared. Individual count was found to be 1.2-1.4 times than that of cluster count, which was significant, as compared to a study that reported the individual count to be 2-3 times higher than that of cluster count.  Thus, it can be inferred that, although both the counting methods gave highly significant results, individual counting is more reliable as compared to cluster counting, as any of the stained NOR in the nucleolus is not ignored.
The possible limitation of this time-bound study is the inclusion of limited number of periodontal lesions especially gingival fibromatosis, drug-induced enlargement, and gingival polyp. However, the primary intention of the study was to evaluate if AgNOR count can serve as a proliferative marker in non-neoplastic lesions as a whole instead of individual type of lesion. However, further investigations are required to clarify this technique in large series of periodontal lesions.
| Conclusion|| |
The present study suggests that AgNOR count reflects the cellular proliferation of non-neoplastic lesions, such as fibrous hyperplasia, peripheral ossifying fibroma, gingival fibromatosis, pyogenic granuloma, drug-induced enlargement, and gingival polyp, which varies within each type of periodontal lesions, and hence has a limited diagnostic value, i.e., no specific AgNOR count can be designated to the type of periodontal lesion. The AgNOR count has a definite prognostic value as the reduction of AgNOR count in plaque-induced gingivitis cases reflects the decreased cellular proliferation due to effective treatment. Thus, it can be concluded that the prognostic value of AgNOR count in non-neoplastic periodontal lesions could serve as an additional tool similar to certain enzyme markers.
The use of AgNOR count as a prognostic marker for periodontal lesions requires study of AgNOR count in larger sample size in various types of proliferative periodontal lesions.
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K L Vandana
Department of Periodontics, College of Dental Sciences, Davangere, Karnataka
[Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5]