Argyrophilic nucleolar organizer regions (AgNORs) in odontogenic myxoma (OM) and ameloblastic fibroma (AF)

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Abstract: Ten cases of odontogenic myxoma (OM) and six cases of ameloblastic fibroma (AF) were subjected to comparative analysis by the AgNOR technique, in order to determine a possible difference in cell proliferation index between these lesions.
  Argyrophilicnucleolarorganizerregions(AgNORs)inmucosalepitheliumunderexperimentaldenturebasesinrats Tetsuya HaraTakashi SatoShingo MoriHajime ShiraiYukinori MaruoShogo Minagi Department of Removable Prosthodontics,Okayama University Dental School, Okayama, Japan Correspondence to: Tetsuya HaraDepartment of Removable Prosthodontics, OkayamaUniversity Dental School, 2-5-1, Shikata-cho,Okayama 700–8525, Japan Accepted for publication September 3, 1999 Copyright C Munksgaard 2000  J Oral Pathol Med .  ISSN 0904-2512Printed in Denmark . All rights reserved 33 Abstract:  The purpose of this study was to evaluate changesin the argyrophilic nucleolar organizer region (AgNOR) counts inmucosal epithelium induced by continuous or intermittent com-pressive pressure exerted through experimental denture basesand to examine the relationships between the AgNOR count,histopathological changes and the intensity of the pressureunder denture bases. Continuous or intermittent compressivepressure exerted through the denture bases was applied to thehard palate of the molar region in rats. A morphometric analysisof AgNORs was performed in denture-supporting tissue 3 daysand 1, 2, 4, 8, 12 and 20 weeks after the denture insertion.From the results of this study, it was found that non-pressurecontact of the denture bases with palatal tissues did notchange the AgNOR count. The AgNOR count was decreased by continuous or intermittent compressive pressure, and then re-covered to almost the same level as with the non-pressure con-tact at 20 weeks following a decrease of the pressure. TheAgNOR counts in the epithelium under the denture bases wererevealed to be related to the histopathological changes in thedenture-supporting tissues and the intensity of the pressureunder the denture bases. Key words:  AgNOR count; continuous compressive pressure;denture bases; epithelium; intermittent compressive pressure  J Oral Pathol Med 2000:  29:  33–8 Nucleolar organizer regions (NORs) are loops of ribosomal DNApresent in the nuclei of cells that possess ribosomal RNA (rRNA)genes (1). These rRNA are transcribed by RNA polymerase I andultimately direct protein synthesis (2). Transcriptionally activeNORs can be selectively stained by a silver colloid technique andvisualized as black dots (AgNORs) under an optical microscope (3,4). AgNORs have been proved to be a valuable marker of incipientcell alterations long before these can be detected in routine prepara-tions (5–7). Attempts have been made to distinguish between benignand malignant lesions, as well as between different degrees of ma-lignancy, on the basis of the AgNOR count per nucleus (8–11). Inoral mucosa, the AgNOR count has been used as a marker for deter-  Hara et al. mining diagnostic and prognostic values of normal, cancerous andprecancerous conditions (6, 7, 12).Pathological conditions of oral mucosa under denture bases,such as deformation of mucosa, decubital ulcer or denture stoma-titis, have been reported frequently (13–16). The cellular activity inthe mucosal epithelium under denture bases has been investigatedby histopathological and histochemical analysis (17–19). However,many factors could affect the histopathological changes in denture-supporting tissues (17, 20). The diversity of experimental conditionsmakes it difficult to establish conclusive findings on the cellularactivity in mucosal epithelium under denture bases. In order to cla-rify the causal relationship between denture bases and the histo-pathological changes arising in tissues under denture bases, wehave reported experimental studies utilizing well-classified and sim-plified factors related to the insertion of denture bases (21–26). Inthese reports, compression of epithelium, shortening of epithelialridges and their deformation were induced by a continuous com-pressive pressure (CCP) or intermittent compressive pressure (ICP),and shortened ridges were elongated following a decrease of thepressure (22, 23).The purpose of the present study was to evaluate the changesin the AgNOR counts in rat oral mucosal epithelium subjected ex-perimentally to CCP or ICP exerted through denture bases and toexamine the relationship between the AgNOR counts, histopatho-logical changes (21–23) and the intensity of these pressures underdenture bases (22, 23). Furthermore, we analyzed AgNOR counts inan attempt to contribute data to the interpretation of cellular alter-ations in mucosal epithelium subjected to the pressures exertedthrough denture bases. Material and methods Two hundred and eighty 15-week-old male Wistar rats (Japan SLCInc., Shizuoka, Japan) were used in this study. All the experimentswere conducted according to the animal experimental guidelinesapproved by the Animal Experiment Committee, Okayama Univer-sity Dental School. The animals were divided into eight groups (  n  35 for each group). Three types of experimental denture were con-structed to apply CCP (3 groups), ICP (3 groups) or non-pressurecontact (NPC, 1 group) to the hard palate of the molar region, re-spectively. The remaining group was kept untreated throughout theexperimental period [non-denture wearing (NDW) group].Each experimental denture consisted of a fixed metal frame-work part and a removable denture base, as described previously(21–23). The fixed metal framework was made of Ag-Au-Pd alloy, 34  J Oral Pathol Med  29:  33–8 and designed to be adherent to the maxillary molars of each individ-ual rat with dental adhesive (PANAVIA A EX, Kuraray Co., Ltd.,Osaka, Japan) (21).In the CCP group, the denture base was composed of heat-curedacrylic resin (Acron, GC Corp., Tokyo, Japan). Orthodontic tubeswere embedded in both the metal framework and denture base. Thedenture base was designed to be fixed rigidly to the metal frame-work by threading orthodontic wire through the orthodontic tubesin which the inner diameter was the same as the diameter of thethreading wire. A prescribed amount of CCP was exerted throughthis rigidly fixed denture base to the tissues under the denture base.The intensity of CCP applied to the mucosal surface was 1.5, 3.4 or4.9 kPa, determined by a method using loading equipment (23). Theforce necessary to transmit the prescribed amount of pressure wascalculated from the intended amount of the pressure and the areaof mucosal surface under the denture base, which was measured asthe area projected perpendicularly to the occlusal plane by using atwo-dimensional analyzing system (COSMOZONE 1SB, Nikon, To-kyo, Japan).In the ICP group, the removable denture base consisted of ametal (Ag-Au-Pd alloy) framework and heat-cured acrylic resin. Theremovable denture base was installed to cover the palatal mucosaof the molar region without any pressure unless occlusal force wasexerted by mastication. In mastication, the denture base was per-mitted to subside into the palatal mucosa by utilizing the differencein diameter between the threading orthodontic wire and the innerdiameter of the orthodontic tube that was embedded in the fixedmetal framework. The metal framework of the denture base con-tacted the fixed metal framework to regulate the subsidence of thedenture base, thus regulating the amount of pressure transmittedto the denture-supporting tissues. In this study, the experimentaldenture bases for these groups were designed to allow subsidenceof 13, 50 or 100  m m, which were shown to result in an ICP of 10.8,33.3 or 82.3 kPa, respectively (22). Each experimental group, there-fore, was subsequently designated as the 10.8 kPa ICP, 33.3 kPaICP or 82.3 kPa ICP group, respectively.In the NPC group, the denture base, which was composed of heat-cured acrylic resin, was constructed to make contact with thepalatal mucosa without any mechanical pressure (21).The denture bases were immersed in distilled water for 24 hprior to insertion in order to eliminate the possible effect of anyresidual monomer. For the denture-wearing groups (CCP, ICP andNPC groups), cleaning of the palate and removable denture baseswas carried out at 3- or 4-day intervals throughout the experimentalperiod. The palate was washed with a tap-water spray and thedenture base was cleaned by an ultrasonic cleaning apparatus(SUW-50D, Morita, Osaka, Japan) for 3 min using distilled water.  AgNORs in epithelium under denture bases All the intraoral procedures, except for cleaning the palate and thedenture base, were performed under anesthesia by intraperitonealinjection of sodium pentobarbital. The cleaning procedure was per-formed under inhalation anesthesia with diethyl ether. The ratswere given free access to water and food (MF, Oriental Yeast Co.,Ltd., Tokyo, Japan). Staining technique After observation periods of 3 days, 1, 2, 4, 8, 12 and 20 weeks, fiverats from each group were weighed and anesthetized by ether inhal-ation. The palatal mucosa stripped from the bone tissue was fixed in4% paraformaldehydeat 4 æ C for12 hand embedded inparaffin wax.Transverse sections were cut at 2  m m thickness from the paraffinblocks. AgNOR staining was performed as described by Lindner (4)with a slight modification. The outline of the modified method is asfollows. The AgNOR solution was prepared by dissolving gelatin in1 g/dl aqueous (distilled, deionized) formic acid at a concentration of 2 g/dl. This solution was mixed (1:2 volumes) with 50 g/dl aqueous(distilled, deionized) silver nitrate solution, to give a final workingsolution. This was immediately poured over the tissue sections andleft for 45 min at room temperature in the dark. The sections werewashed thoroughly in distilled water, immersed in 5% sodium thio-sulfate solution for 5 min, dehydrated in ascending ethanol concen-trations, cleared in xylene and mounted in balsam. Counting procedure and statistical analysis The observation site for counting the AgNORs was mucosal epithel-ium of the trough between the rugae in the middle part of the trans-verse section with the exception of the mucosal area covering thepalatal suture. AgNOR dots were counted in 100 randomly selectednuclei under a  ¿ 100 immersion objective to a total magnification of  ¿ 1000.ForAgNORenumeration,thestandardizedmethodbyCrock-er et al. (27) was followed: each AgNOR dot was counted as a unitwhen seen separately within the nucleoli. Undiscernible nucleolar Table 1.  Body weight (g) in each group Observation periodExperimental group 3 days 1 week 2 weeks 4 weeks 8 weeks 12 weeks 20 weeksNDW 423 ∫ 12 430 ∫ 14 440 ∫ 21 454 ∫ 15 486 ∫ 14 513 ∫ 19 550 ∫ 30NPC 400 ∫ 14 410 ∫ 14 429 ∫ 28 449 ∫ 32 473 ∫ 43 503 ∫ 33 541 ∫ 471.5 kPa CCP 410 ∫ 19 418 ∫ 18 434 ∫ 19 445 ∫ 20 476 ∫ 15 494 ∫ 16 544 ∫ 193.4 kPa CCP 407 ∫ 14 424 ∫ 15 432 ∫ 16 442 ∫ 20 467 ∫ 24 496 ∫ 26 530 ∫ 324.9 kPa CCP 408 ∫ 27 415 ∫ 15 425 ∫ 23 438 ∫ 31 452 ∫ 31 480 ∫ 32 524 ∫ 2710.8 kPa ICP 400 ∫ 16 403 ∫ 17 414 ∫ 22 431 ∫ 18 454 ∫ 43 478 ∫ 23 517 ∫ 3933.3 kPa ICP 396 ∫ 16 405 ∫ 15 419 ∫ 20 433 ∫ 17 451 ∫ 16 484 ∫ 33 513 ∫ 2582.3 kPa ICP 399 ∫ 16 409 ∫ 14 419 ∫ 20 441 ∫ 24 464 ∫ 18 489 ∫ 33 524 ∫ 26NDW: non-denture wearing; NPC: non-pressure contact; CCP: continuous compressive pressure; ICP: intermittent compressive pressure;  n  5. 35  J Oral Pathol Med  29:  33–8 Fig. 1.  Silver-stained black dots (AgNOR, arrows) are evident in the nucleiof epithelial cells. Arrowheads indicate basement membrane. (  ¿ 600) clusterswerecountedasasingledot.Oneexaminerwhowasunawareof the experimental conditions counted the number of AgNORs. ThemeannumberofAgNORspernucleus(AgNORcount)wascalculatedfor each rat, and a mean AgNOR count was calculated at each obser-vation period for each group. At each observation period, statisticaldifferences between the mean AgNOR counts of the NDW group andtheothergroups(CCP,ICPandNPCgroups)wereevaluatedusingtheMann-Whitney U-test. In order to clarify the effect of the CCP or ICPon the AgNOR count in the epithelium under the denture bases, stat-isticaldifferencesbetweenthemeanAgNORcountsoftheNPCgroupandboththeCCPandtheICPgroupswereevaluatedusingtheMann-Whitney U-test at each observation period.  P   values less than 0.05were considered to be significant. Results All animals increased body weight progressively. No significant dif-ference between body weight of each group was observed (Table 1).  Hara et al. Fig. 2.  Changes over time of AgNOR counts in mucosal epithelium. A)changes in CCP groups; B) changes in ICP groups. AgNOR counts in NDWand NPC groups are shown for comparison. Each dot stands for an AgNORcount for one subject animal, and each line shows the change in the meanAgNOR count for each group. (NDW: non-denture wearing; NPC: non-press-ure contact; CCP: continuous compressive pressure; ICP: intermittent com-pressive pressure.) Well-defined black silver-stained dots were observed in the nuclei(Fig. 1). The distributions of the AgNOR counts in each rat and themean AgNOR counts are shown in Fig. 2A & B. The results of thestatistical analysis are shown in Table 2. The mean AgNOR countsin both the NDW group and the NPC group ranged from 1.96 to 2.02 Table 2.  AgNOR count in each group and results of a statistical comparison of the mean AgNOR counts Observation periodExperimental group 3 days 1 week 2 weeks 4 weeks 8 weeks 12 weeks 20 weeksNDW 2.00 2.02 1.98 2.01 2.00 2.02 2.01NPC 1.99 2.02 1.96 1.99 1.99 1.98 1.971.5 kPa CCP 1.94 1.80 † * 1.81 † * 1.82 † * 1.84 † * 1.97 2.023.4 kPa CCP 1.82 † * 1.65 † * 1.66 † * 1.82 † * 1.83 † * 2.00 2.024.9 kPa CCP 1.77 † * 1.57 † * 1.60 † * 1.73 † * 1.88 † * 2.02 2.0210.8 kPa ICP 1.33 † * 1.45 † * 1.55 † * 1.70 † * 1.88 † * 1.96 1.9633.3 kPa ICP 1.24 † * 1.38 † * 1.48 † * 1.76 † * 1.82 † * 1.85 † * 1.9182.3 kPa ICP 1.03 † * 1.18 † * 1.21 † * 1.65 † * 1.80 † * 1.81 † * 1.89 † : vs NDW group; *: vs NPC group.NDW: non-denture wearing; NPC: non-pressure contact; CCP: continuous compressive pressure; ICP: intermittent compressive pressure. 36  J Oral Pathol Med  29:  33–8 throughout the experimental period, and there was no significantdifference between them.The AgNOR count in the 3.4 and 4.9 kPa CCP groups showedsignificantly smaller values at the 3-day stage than the NDW andthe NPC groups. The AgNOR count in the CCP groups at the 1-week stage was smaller than that at the previous stage, and theAgNOR count in the 1.5 kPa group showed a significant decrease(vs NDW and NPC groups). Although the AgNOR count in the 1.5kPa CCP group showed almost constant values from the 1- to 8-week stage, the count in the 3.4 and 4.9 kPa CCP groups graduallyincreased from the 2-week stage. The AgNOR counts in the CCPgroups were significantly smaller than those in the NDW and theNPC groups until the 8-week stage and recovered to almost thesame level as the NDW and the NPC groups after 12 weeks.In the ICP group, the AgNOR counts decreased significantlycompared with the value of the NDW and the NPC groups at the 3-day stage. The AgNOR count in the ICP groups gradually increasedover time and recovered to almost the same level as the NDW andthe NPC groups at 20 weeks. Discussion The results observed in this study were analyzed in relation to theresults in our previous studies, which were accomplished underidentical conditions and showed similar histopathological changes(21–23) associated with the same duration of mechanical pressureunder denture bases (22, 23). Under conditions identical to those of the NPC group in this study, slight blanching of epithelial ridgesunder the denture bases was observed from 2 to 12 weeks afterdenture insertion (21). The AgNOR count in the NPC group sug-gested that this slight morphological change in the epithelial ridgeswas not a consequence of the cellular alterations.The CCP and ICP groups in this study similarly showed com-  AgNORs in epithelium under denture bases pression of epithelium, and shortening and deformation of the epi-thelial ridges under the denture bases at an early stage (22, 23).In addition, these histopathological changes were seen to be mostremarkable at the 1-week stage in the CCP groups and in the 1- to2-week stages in the ICP groups. It is suggested that these histo-pathological changes may be closely related to the initial decreasein the AgNOR count induced by CCP or ICP. Moreover, the declinein the AgNOR count in the CCP and ICP groups was observedbefore histopathological changes could be detected. Therefore, mor-phometric evaluation of AgNORs revealed incipient cellular alter-ations that were not evident in routine preparations.Thereafter,thethicknessofepitheliumincreasedandtheepithelialridges noticeably blanched and elongated to almost their srcinallengthastheCCPandICPunderthedenturebasesdecreased(22,23).Elongation of the epithelial ridges to their srcinal length might berelated to the increase and recovery in the AgNOR counts followingthedecreaseintheCCPandICP.Therefore,theAgNORcountsinepi-thelialcellsunderthedenturebasesmayberelevanttoboththehisto-pathological changes in the tissues under the denture bases and theintensity of the CCP and ICP under the denture bases (22, 23).Turck (17) reported that mitosis in the epithelial cells under den-ture bases was more frequent than in normal epithelium when theouter layer of the epithelium was injured and showed a decrease orabsence of the keratin layer. In a preliminary (unpublished) study,we have shown that the AgNOR count in healing rat palatal mucosaafter experimental incision ranged from 2.69 to 3.39, and was appar-ently increased compared with that in the NDW group in this pres-ent study. It seems that the AgNOR count increases during therestoration period of the injured epithelium. The AgNOR count inthe CCP and ICP groups did not exceed the count in the NDWgroup throughout the observation period. This finding is consistentwith the histopathological finding that no injury was observed inthe mucosal epithelium under conditions identical to those of theCCP and ICP groups (22, 23).From the results of this study, the AgNOR counts in mucosalepithelium under denture bases were revealed to be relevant to thehistopathological changes in the tissues under the denture bases. Itis suggested that the AgNOR count in the mucosal epithelium underdenture bases could be used as an objective morphometric par-ameter of the tissue changes exerted by CCP and ICP through thedenture bases. References 1. Fawcett DW. A textbook of histology, 12th edn. New York: Chapmanand Hall, 1994: 1–56. 37  J Oral Pathol Med  29:  33–8 2. Leong, AS-Y, Gilham P. Silver staining of nucleolar organizer regionsin malignant melanoma and melanotic nevi.  Hum Pathol   1989;  20 : 257– 62.3. Ploton D, Menager M, Jeannesson P, Himber G, Pigeon F, Adnet JJ. Im-provement in the staining and in the visualization of the argyrophilicproteins of the nucleolar organizer region at the optical level.  Histochem J   1986;  18 : 5–14.4. Lindner LE. Improvements in the silver-staining technique for nucleolarorganizer regions (AgNOR).  J Histochem Cytochem  1993;  41 : 439–45.5. Schwint AE, Gomez E, Itoiz ME, Cabrini RL. 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