The Relationship between Proliferating Cell Nuclear Antigen Expression and Histomorphometrical Alterations in Cyclosporin A-Induced Gingival Overgrowth in Rats

Abstract

The aim of this study was to investigate the relationship between Proliferating Cell Nuclear Antigen (PCNA) expression and histomorphometrical alterations in cyclosporin A (CsA)-induced gingival overgrowth with or without microbial dental plaque accumulation. Forty male Wistar rats were equally divided into 4 groups; Group I (control); Group II (CsA); Group III (ligature); Group IV (ligature and CsA). After 8 weeks of experimental period, rats were subsequently decapitated and mandibular molars were dissected. Gingival overgrowth was determined by measuring depth of the gingival sulcus, then the mandible were decalcified and serial sections were obtained for histomorphometric and immunohistochemical analysis. Histomorphometric analysis included the measurement of epithelial thickness; immunohistochemical analysis included the assessment of PCNA expression in the oral and sulcular epithelium of buccal and lingual gingiva. Epithelial thickness and PCNA expression were significantly increased in buccal oral epithelium of Group II (p < 0.05) and in all regions in Group IV (p < 0.05) compared to control group. Also gingival overgrowth was more prominent in Group IV in comparison to Group II. These results indicate that CsA-induced gingival alterations are closely accociated with increased epithelial proliferative activity, and dental plaque accumulation seems not to be an essential but to be an aggrevating factor for the progression of the lesion.

Introduction

Cyclosporin A (CsA), a lipophilic, hydrophobic, cyclic endecapeptide, is used successfully as an immunosuppresive agent to prevent rejection of transplanted organs and to treat various autoimmune diseases (Laupacis et al., 1982). Various side effects associated with CsA treatment have been described; including nephrotoxicity, hepatotoxicity, neurotoxicity, hypertrichosis, and gingival overgrowth (Spolidorio et al., 2001). In dental literature, CsA-induced gingival overgrowth was first reported by Rateitschak-Plus et al. in 1983, but has been described in medical literature as a side effect of CsA therapy as early as 1979 (Calne et al., 1979). Although there are numerous studies about the cellular and molecular relations between CsA and gingival tissues, the etiology of CsA-induced gingival overgrowth still remains unclear.

Many risk factors have been suggested and investigated with both development and expression of CsA-induced gingival overgrowth (Seymour et al., 2000). These factors are individual factors such as age, gender, genetic predisposition (Seibel et al., 1989; Morisaki et al., 1993; Cebeci et al., 1996), pharmacologic factors such as drug dosage, serum concentration, treatment duration, combined therapy (Rostock et al., 1986; Fu et al., 1995; Morisaki et al., 1997) and local periodontal factors such as accumulation of microbial dental plaque and gingival inflammation (Tyldesley and Rotter, 1984; McGaw et al., 1987; Kitamura et al., 1990; Pernu et al., 1992; Fu et al., 1997). Although results of the studies about local periodontal factors are contradictory and inconclusive, it has only been confirmed that patients with CsA-induced gingival overgrowth have more microbial dental plaque than those without such overgrowth (Niimi et al., 1990). However, it is still not known, whether the dental plaque accumulation is essential for the initial lesion or it is simply a result of gingival overgrowth (Seymour et al., 2000).

Histopathologic features of the CsA-induced gingival overgrowth show great similarity with other drug induced gingival enlargements. The main microscopic characteristic of CsA-induced gingival overgrowth has been shown to be fibroepithelial hyperplasia (McGaw et al., 1987), the major components being fibroblasts, infiltrating inflammatory cells, and epithelial cells. There are many reports on fibroblasts (Bartold, 1987; McGaw and Porter, 1988) and infiltrating inflammatory cells (Deliliers et al., 1986; Friskopp et al., 1986) while epithelial cells in CsA-induced gingival overgrowth have been studied little. Also these limited studies have presented contradictory results; while keratinocyte growth factor which plays a prominent part in the proliferation and differentiation of epithelial cells has been found to be up-regulated (Das et al., 2001), in another study keratinocyte proliferation has been suggested to be decreased (Niimi et al., 1990). As far as we know, the effect of CsA alone or with dental plaque on epithelial cells in CsA-induced gingival overgrowth has not been fully understood yet.

In humans mitotic activity, as an indicator of cell proliferation, can be determined by counting the cells in the mitotic phase (Marwah et al., 1956). Subsequently certain nuclear antigens were identified in proliferating cells (Miyachi et al., 1978; Mathews et al., 1984). Proliferating cell nuclear antigen (PCNA) is one of these nuclear antigens which is defined as a 36kDa auxillary protein to DNA polimerase delta (Miyachi et al., 1978). The concentration of PCNA correlated directly with proliferative state of the cell which increased through G1 phase, peaked at the G1/S phase interface, decreased through G2 phase and reached to low levels during M phase and interphase (Kurki et al., 1986).

The aim of this study was to investigate the relationship between PCNA expression and histomorphometrical alterations in CsA-induced gingival overgrowth with or without microbial dental plaque accumulation.

Material and Methods

Experimental Design

Forty male Wistar rats (100 g) were randomly distributed into 4 groups of 10 animals each: Group I (control): 0.9% NaCl was injected daily; Group II (CsA): CsA (Sandimmun IV solution, Novartis, Sao Poulo, Brazil) was injected subcutaenously for 8 weeks with a daily dose of 10 mg/kg to provide plasma peak and trough levels of 1000 and 750 ng/ml, respectively (Wassef et al., 1985); Group III (ligature): After systemic anesthesia with intraperitoneal injection of 60 mg/kg ketamine-HCl, (Warner Lambert, Pfizer Inc., Istanbul, Turkey) 3.0 sterile silk ligatures were tyed on the necks of mandibular first molars which were kept in position to promote microbial dental plaque accumulation and inflammation during the experimental period (Keles et al., 2005) Group IV (ligature and CsA): 3.0 sterile silk ligatures were placed on the necks of mandibular first molars. Also, CsA was injected subcutaenously to the rats in this group for 8 weeks with a daily dose of 10 mg/kg to provide plasma peak and trough levels of 1000 and 750 ng/ml, respectively.

Rats were weighed daily and drug dosage adjusted accordingly. All rats were housed seperately in plastic cages and kept in a temperature-controlled room with a standard 12/12 h light-dark illumination cycle. All animal care and study protocols were in compliance with guidelines approved by the Animal Experiment Commitee with the assignment protocole CAM 10/57-07162001.

Macroscopic Analysis

Rats were decapitated after 8 weeks of the experimental period, the mandibles together with the surrounding gingiva and soft tissue were carefully removed, and soaked in neutral 10% formalin for 48 h. The probing pocket depth was measured from the top of the gingival margin to the buttom of gingival sulcus on the distobuccal and distolingual aspects of the mandibular first molars using a calibrated celluloid probe (kindly supplied by Dr. I. Morisaki, University of Osaka, Japan), approximately 250 μm in width with 50 μm bands of 5 different colours. The probe was inserted into the gingival sulcus with a light force of about 10 g under a stereo-microscope (Zeiss MC 80 DX, Jena, Germany). All measurements were performed by a single probe and by the same examiner in the manner of random and blind specimen sampling. The depth of the gingival sulcus and pseudo pocket was used as the degree of gingival overgrowth.

Histomorphometrical Analysis

After macroscopic observations, the mandibles were decalcified in 10% nitric acid for 48 h and 4 μm thick serial paraffin sections were cut in a bucco-lingual direction throughout the mandibular first molars. The sections were used for hematoxylin & eosin (H&E) and immunohistochemical staining. In the H&E-stained sections, gingival epithelium thickness was measured at 4 different regions; oral and sulcular epithelium of both buccal and lingual gingiva by a light microscope (Olympus BH2-Research Microscope, Tokyo, Japan) at a magnification of ×40. Measurements were made at the right angles to the outer epithelial surface from the latter up to the epithelium-connective tissue border in randomly selected 5 different areas of the oral and sulcular epithelium. The mean of the measurements was used for data analysis.

Tissue inflammation was evaluated in the same sections in both oral and sulcular epithelium of the buccal and lingual region by counting the inflammatory cells (macrophages and lymphocytes) in a 36 μm² area by a light microscope transferred to a monitor with a camera apparatus (Panasonic F10 CCD Camera, Objective ×3.3, Osaka, Japan) at a magnification of ×4165 (Keles et al., 2005).

Immunohistochemical Analysis

Sections were taken over poly-1-lysine coated lams, dried at room temperature, deparafinized, and washed with distilled water. PCNA was demonstrated using an improved biotin-streptavidin immunoperoxidase technique according to the manufacturer’s protocol (Universal Immunostaining Kit, DBS 1020 Serpentine Lane, # 111, Pleasanton, CA, 94566, USA). In order to reduce the non specific disclosing due to endogenous peroxidase, sections were washed twice with phosphate buffered solution (PBS, pH: 7.2), incubated in 3% H₂O₂ (10–15 min), and protein blocked (Goat DAKO, California, USA) (5 min) at room temperature. The sections were then incubated for 30 min at room temperature with primary monoclonal anti-PCNA antibody, (Mouse, monoclonal Clone: PC10, DAKO, CA, USA) washed 4 times with PBS, and stored in biotinylated secondary antibody (anti mouse&rabbit) for 20 min. After washing with PBS the slides were incubated for 20 min in streptavidin conjugated peroxidase solution. After washing 4 times with PBS, diaminobenzidine (DAB) was added for color development (3–5 min) and rinsed with distilled water. After washing for 2 min under tap water, the sections were lightly counterstained with Mayer’s hematoxylin (1 min) and washed in running water. They were stored 10 min in alcohol and 3 min in xylene and then mounted in synthetic resin. The section that was stained without incubation with primary monoclonal anti-PCNA antibody was used as negative control. The tonsilla palatina section was used as positive control.

In each section, more than 500 epithelial cells were counted in the basal layer of the epithelium with × 400 magnification. After determining the background staining in each section, every nucleus with brown color (darker than the background staining) was considered positive for PCNA regardless of staining intensity. PCNA-Proliferative index which was expressed as the percentage of PCNA positive cells per total number of nucleated epithelial cells counted (PCNA + %), was used as the indicator of proliferative activity (Zidar et al., 1996; Celenligil-Nazliel et al., 2000).

Statistical Analysis

Normalities of distributions were tested by using the Shapiro–Wilks procedure. In the comparison of the groups, in which the parameters were not in accordance with normal distribution, Kruskal–Wallis and Mann–Whitney U-non-parametric tests were used whereas in comparison of the groups having normal distribution, one-way analysis of variance (One-way ANOVA), and Post Hoc Tukey parametric tests were used. Spearman rank order correlation was used to test the relationship between epithelium thickness and epithelial cell proliferation. These analyses were performed using a statistical software package (SPSS 10.0 software package programme).

Results

Macroscopically, remarkable overgrowth was observed in the buccal gingiva of Group II, Group III, and in the buccal and lingual gingiva of Group IV compared to control Group I. According to our macroscopic observations, gingival overgrowth was more prominent in Group IV compared to Group II and III, and in the buccal aspect than the lingual aspect of the gingiva. (Figure 1A–D).

The average depth of the buccal and lingual gingival sulcus which was estimated by measuring the probing pocket depth of rat mandibles with a calibrated celluloid probe of rat mandibles in each group is shown in Table 1. These measurements confirmed the macroscopic observation that the overgrowth induced by CsA, as well as dental plaque accumulation, was more prominent in buccal than in lingual gingiva. Probing pocket depths in all experimental groups (Group II, III, IV) increased as compared to the control Group I.

Histomorphometric Findings

In the H&E-stained sections increased tissue dimensions especially increased epithelial thickness and elongated rete pegs were seen in the buccal gingiva of CsA-treated Group II and IV, more prominent in Group IV than in Group II. A well-organized tooth-gingiva interface was found in unligated Group I and II, whereas periodontal breakdown characterized by depressed soft tissue notch and alveolar bone loss was observed in the ligated Groups III and IV. (Figure 2A–D).

The linear measurements of epithelium thickness in the oral and sulcular epithelium of the buccal and lingual gingiva are shown in Table 2. Among all groups the highest epithelium thickness was found in the oral epithelium of buccal gingiva in Group IV (p < 0.05). There was a statistically significant increase in CsA-treated groups; in the buccal oral epithelium in Group II (p < 0.05) and in all regions in Group IV (p < 0.05) compared to the control Group I. No significant increase in any regions of Group III compared to control Group I was found (p > 0.05). While Group IV showed the highest epithelium thickness, there were no significant difference between Group II and III (p > 0.05).

Inflammatory cell infiltration was most prominent beneath the sulcular epithelium in all groups (p < 0.05). The number of the inflammatory cells was significantly higher in ligated Group III and IV compared to control group (p < 0.001) while, there was no significant difference between CsA-treated Group II and control Group I (p > 0.05) (Table 3).

Immunohistochemical Findings

PCNA+ stained cells were distributed heterogenously throughout the basal cell layer of all groups and were especially intense in the elongated rete pegs of CsA treated Group II and IV (Figure 3A–D). PCNA-Proliferative index (PCNA + %) was significantly higher in the buccal oral epithelium in Group II (p < 0.05), in the buccal sulcular epithelium in Group III (p < 0.05) compared to control group. Group IV showed the highest PCNA+ % in all regions of both buccal and lingual gingiva (p < 0.001) (Table 4).

When epithelial thickness and PCNA-proliferative index was compared in four different regions between all groups, no statistically significant correlation was found (p > 0.05).

Discussion

The rats are excellent models to study gingival overgrowth caused by CsA because they are small, inexpensive, easy to handle, and their response to CsA is more uniformly than humans (Kataoka et al., 2000; Spolidorio et al., 2002). In addition, variables such as genetic predisposition, age, gender, dose, and duration of the drug can be better standardized in rats (Nishikawa et al., 1996). In our study, all the rats responded positively and uniformly to CsA as subcutaneous injection of CsA (10 mg/kg) for 8 weeks resulted in both macroscopic and microscopic gingival overgrowth in all CsA-treated rats. It is important to note that besides CsA, dental plaque accumulation also alone caused gingival overgrowth.

Probing pocket depth of gingival sulcus has been measured by a calibrated probe where increase in probing pocket depth has been used as an indicator of drug-induced gingival overgrowth in experimental studies (Kitamura et al., 1990; Morisaki et al., 1993). Increase in probing pocket depth is also one of the indicators of periodontal breakdown due to periodontal diseases (Papapanou and Lindhe, 2003). In our study increased probing pocket depth in CsA-treated Group II was only related to drug-induced gingival overgrowth as there was no periodontal breakdown in this group of rats whereas, increased pocket depth in the ligated Group III was both related to the periodontal breakdown and inflammation-induced gingival overgrowth. However, while expecting the highest pocket depth in Group IV, the data showed that probing pocket depth in Group III was higher than that in Group IV. This might probably be related to the diminished periodontal breakdown due to inhibitory effect of CsA on immune system (Daley and Wysocki, 1984). In light of these observations we can suggest that probing pocket depth values not only show the degree of gingival overgrowth but also the progression of periodontal disease in our study.

Epithelial alterations, such as thickening of epithelium and elongated rete pegs appear to be characteristic features of CsA-induced gingival overgrowth (Deliliers et al., 1986; Rostock et al., 1986). Increased epithelial thickness has been described before (Niimi et al., 1990; Fu et al., 1997; Pilatti and Sampaio, 1997; Nurmenniemi et al., 2001; Spolidorio et al., 2001, 2002, 2003) likewise; our histometric data indicating increased epithelial thickness in CsA-treated Groups II and IV were in accordance with these reports. Although a synergistic effect of CsA treatment and ligature replacement was evident in macroscopic and microscopic findings, and the epithelial thickness (Table 2), a similar synergistic effect was not observed in the degree of gingival overgrowth based on probing depth measurements (Table 1). As mentioned above, the inhibitory effect of CsA on periodontal breakdown characterized by alveolar bone loss (Daley and Wysocki, 1984) might explain as why probing depth values do not correlate with the other findings.

It has been reported that increased epithelial thickness might be related to the influence of connective tissue on epithelial architecture and cytodifferentation (Spolidorio et al., 2002). Similarly, it has been suggested that epithelial alterations seen in CsA and nifedipine-induced gingival overgrowth are a local response related to epithelium-connective tissue alterations, instead of a response to these drugs (Spolidorio et al., 2003). In these studies it has not been investigated whether CsA might have a proliferative effect on gingival epithelium and increased epithelial thickness might be related to epithelial cell proliferation, or not.

It is known that the effect of CsA on growth of several epithelial cell types in different tissues is controversial. For example, CsA which is succesfully used for the treatment of psoriasis has an inhibitory effect on the proliferation of keratinocytes in this lesion (Wong et al., 1993). On the other hand, hypertrichosis, a well known side effect of CsA, is related to increased proliferative activity in hair follicles of keratinocytes (Wysocki and Daley, 1987). Our study shows that there was a statistically significant increase in the percentage of PCNA+ cells in buccal oral epithelium in Group II, in buccal sulcular epithelium in Group III, and in all epithelial regions in Group IV as compared to the control Group I. These results, the thickening of gingival epithelium, and increased proliferative activity in hair follicles of keratinocytes argue against an antiproliferative effect of CsA on epithelium. Several in vivo studies analyzed proliferative activity by showing the presence and localization of PCNA in gingival epithelial cells (Marwah et al., 1956; Celenligil-Nazliel et al., 2001). These clinical studies have analysed the effect of aging upon proliferative activity in human gingival epithelium and reported that proliferative activity has elevated in older subjects. Although the same method was used for the determination of proliferative activity, this is the first experimental report showing in vivo PCNA expression in epithelial cells, and analysing epithelial alterations in four different regions in CsA-induced gingival overgrowth.

Existing knowledge related to the effect of CsA on gingival epithelial cells in overgrown tissue is insufficent to provide unequivocal conclusions; proliferative activity has been reported as decreased (Niimi et al., 1990; Ayanoglou and Lesty, 1999) or increased (Das et al., 2001; Nurmenniemi et al., 2001). In an immunohistochemical study, it has been reported that DNA polymerase delta that is essential for cell proliferation, is detected only in few basal keratinocytes of CsA induced gingival overgrowth suggesting a decrease in keratinocyte proliferation (Niimi et al., 1990). A histomorphometric study has suggested that incompletely keratinized cells seen in CsA-incuced gingival overgrowth tissue reflect that increased oral epithelial thickness is not associated with epithelial cell proliferation (Ayanoglou and Lesty, 1999). On the other hand it has been found that in CsA-induced gingival overgrowth, expression of keratinocyte growth factor is up-regulated, which has been shown to play a prominent part in the proliferation and differentiation of epithelial cells (Das et al., 2001). In another clinical study, it has been shown that proliferative activity of keratinocytes increased in CsA and nifedipine induced gingival overgrowth (Nurmenniemi et al., 2001).

Based on the results presented in Table 4 the sulcular epithelium of both the buccal and lingual gingiva of ligature-placed Group III animals have higher proliferative activity than CsA-treated Group II animals which suggests that ligature-induced inflammation at these sites results in a greater increase in proliferative activity than that observed for animals treated with CsA alone. These results are consistent with reports in the literature demonstrating that the PCNA-proliferative index is significantly higher in inflamed gingiva compared to healthy gingiva (Celenligil-Nazliel et al., 2000). The results presented in Table 4 also suggest that compared to control Group I, both CsA and ligature-induced inflammation together contribute to the increased proliferative activity.

The results of studies supporting proliferative effect of CsA on keratinocytes in CsA-induced gingival overgrowth have suggested that drug-induced epithelial changes are a result of local gingival inflammation (Das et al., 2001; Nurmenniemi et al., 2001). Similarly it has been reported that the hyperplastic alterations of epithelium in gingival overgrowth may be relevant to ligature-induced inflammation rather than the drug alone (Fu et al., 1997). In contrast to above reports, our results clearly showed that proliferative activity increased in the buccal oral epithelium in CsA-treated Group II where number of the inflammatory cells was not different from that in the control group. This observation suggests that increased proliferative activity might be related to the proliferative effect of CsA on gingival epithelial cells rather than the effect of dental plaque and gingival inflammation. But this observation does not exclude the possible role of dental plaque on epithelial cell proliferation as proliferative activity also increased in the buccal sulcular epithelium of ligated Group III where number of the inflammatory cells was significantly higher than that of in the control group. The highest proliferative activity was found in all epithelial regions of ligature-placed and CsA-treated Group IV. In light of these findings, it is relevant to assume that the drug and microbial dental plaque accumulation might have independent effects on proliferative activity of gingiva. However, microbial dental plaque is not essential for the development of gingival overgrowth, but it is an aggregevating factor for the progression of the lesion.

In conclusion, this study suggests that epithelial alterations in CsA-induced gingival overgrowth is closely associated with increased proliferative activity, and PCNA immunolocalization can also be used as an index of the state of cell proliferation in drug-induced gingival overgrowth. This assumption does not exclude the possible role of other factors such as decrease in desquamation besides cell proliferation in the observed phenomenon as no significant correlation was found between epithelial thickness and epithelial cell proliferation. CsA-induced gingival overgrowth is a result of multi-causal intrinsic and extrinsic factors and further investigations are necessary for several issues which still await clarification.