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Keywords:

  • alveolar bone;
  • apoptosis;
  • cimetidine;
  • osteoclast;
  • osteoclastogenesis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Quantitative analyses
  6. Transmission electron microscopy (TEM)
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

It has been demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H1- and H2-receptors. Cimetidine is a H2-receptor antagonist used for treatment of gastric ulcers that seems to prevent bone resorption. In this study, a possible cimetidine interference was investigated in the number of alveolar bone osteoclasts. The incidence of osteoclast apoptosis and immunoexpression of RANKL (receptor activator of nuclear factor κB ligand) was also evaluated.

Adult male rats were treated with 100 mg kg−1 of cimetidine for 50 days (CimG); the sham group (SG) received saline. Maxillary fragments containing the first molars and alveolar bone were fixed, decalcified and embedded in paraffin. The sections were stained by H&E or submitted to tartrate-resistant acid phosphatase (TRAP) method. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) method and immunohistochemical reactions for detecting caspase-3 and RANKL were performed. The number of TRAP-positive osteoclasts, the frequency of apoptotic osteoclasts and the numerical density of RANKL-positive cells were obtained. Osteoclast death by apoptosis was confirmed by transmission electron microscopy (TEM). In CimG, TRAP-positive osteoclasts with TUNEL-positive nuclei and caspase-3-immunolabeled osteoclasts were found. A significant reduction in the number of TRAP-positive osteoclasts and a high frequency of apoptotic osteoclasts were observed in CimG. Under TEM, detached osteoclasts from the bone surface showed typical features of apoptosis. Moreover, a significant reduction in the numerical density of RANKL-positive cells was observed in CimG.

The significant reduction in the number of osteoclasts may be due to cimetidine-induced osteoclast apoptosis. However, RANKL immunoexpression reduction also suggests a possible interference of cimetidine treatment in the osteoclastogenesis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Quantitative analyses
  6. Transmission electron microscopy (TEM)
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Bone homeostasis is maintained by a coordinated and complex cascade of cellular and molecular processes that control the formation, activity and survival of bone cells (Sodek & Mckee, 2000). Several systemic and local factors play a role in the control of osteoblasts and osteoclasts, which in turn maintain the bone structural integrity (Phan et al. 2004). Among several osteoclast-activating factors, interleukins (ILs), such as IL-1, IL-3 and IL-6, tumoral necrosis factor alpha (TNF-α) and receptor activator of nuclear factor κB ligand (RANKL) have been demonstrated (Ikawa et al. 2007). Some factors, such as TNF-α, IL-1 and estrogen, play a role in the control of the osteoclasts life span (Glantschnig et al. 2003; Cruzoé-Souza et al. 2009).

It has been suggested that histamine, a biogenic amine, controls several cellular and molecular processes. The cellular action of histamine is mediated by four receptors – H1, H2, H3 and H4 – which are expressed by immunocompetent cells, neurons as well as bone marrow cells (Hill et al. 1997; Schneider et al. 2002). Some studies have demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H1- and H2-receptors. However, strong evidence suggests that H2-receptors seem to exert a more significant role in the histamine-induced osteoclast differentiation than H1-receptors (Dobigny & Saffar, 1997; Yamaura et al. 2003; Biosse-Duplan et al. 2009). Moreover, it has been demonstrated that a histamine intracellular receptor (Hic) mediates important cellular processes, including activity, division and apoptosis (Brandes et al. 1990; LaBella & Brandes, 2000).

There is evidence that histamine induces RANKL secretion by bone marrow cells, osteoblasts and mesenchymal cells (Deyama et al. 2002; Yamaura et al. 2003). Thus, RANKL binds to its receptor RANK, activating a signaling pathway that promotes osteoclast differentiation (Yasuda et al. 1999; Ikawa et al. 2007) and, consequently, bone resorption (Phan et al. 2004).

Cimetidine, a histamine H2-receptor antagonist used for the treatment of gastric ulcers (Alekseenko & Timoshin, 1999), seems to prevent bone loss. In mammals with induced inflammatory disease, such as rheumatoid arthritis and periodontitis, cimetidine was demonstrated to reduce bone resorption (Yamaura et al. 2003; Hasturk et al. 2006). The inhibitory effect of cimetidine on bone resorption has also been reported in estrogen-deprived female rats (Leclous et al. 2002, 2004; Lesclous et al. 2006). Under these conditions, TNF-α and IL-1 are increased in animals. As these factors play an important role in osteoclasts survival (Glantschnig et al. 2003), it has been suggested that cimetidine inhibits the production of these factors, thus interfering with bone resorption (Glantschnig et al. 2003; Hasturk et al. 2006).

Alveolar bone undergoes continuous remodeling due to tooth movements that occur, at least in part, as a consequence of the functional demand placed on it by the forces of mastication. Considering that alveolar bone turns over more rapidly than other parts of the skeleton (Garant, 2003), several osteoclasts are often present in the alveolar bone surface to promote resorption in specific sites and to accommodate tooth movement. However, alveolar bone resorption is focally located, allowing teeth maintenance in the alveolar socket (Nanci & Bosshardt, 2006). Therefore, the alveolar bone is a suitable in vivo model to investigate structural and molecular changes in osteoclasts (Cruzoé-Souza et al. 2009; Faloni et al. 2012).

In this study, we purposed to evaluate a possible interference of cimetidine in the number of alveolar bone osteoclasts, and to correlate this with the incidence of apoptosis and RANKL immunoexpression in treated rats for a prolonged period of time.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Quantitative analyses
  6. Transmission electron microscopy (TEM)
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Experimental procedures

In this study, the principles of animal care and experimental procedures were conducted following the national law on animal use. The research protocol was authorized by the Ethical Committee for Animal Research of the São Paulo State University, Brazil (Araraquara Dental School-UNESP).

Twelve male Holtzman rats (Rattus norvegicus albinus), weighing 250 ± 10 g, were maintained in plastic cages under a 12 : 12 h light–dark cycle at controlled temperature (23 ± 2 °C) and humidity (55 + 10%), with food and water provided ad libitum. The animals were distributed into two groups containing six animals each: the cimetidine group (CimG) and the sham group (SG). The animals from CimG received daily intraperitoneal injections of 100 mg of cimetidine (Hycimet®, Hypofarma, Brazil) per kg of body weight. This dosage was selected based on previous studies in which 125 mg kg day−1 of cimetidine was demonstrated to interfere with rat bone resorption (Dobigny & Saffar, 1997; Leclous et al. 2002, 2004; Lesclous et al. 2006). Thus, the cimetidine dose used in this study (100 mg kg−1; approximately six times higher than that prescribed for human) was used with the aim to produce effects in the alveolar bone of rodents, but not to mimic human pharmaceutical use. Considering that, in general, the treatment in humans is over a long period, the rats received cimetidine for 50 days.

The SG rats received the same dose of saline solution by the same route. At 24 h after the last injection, the CimG and SG rats were killed by an overdose of chloral hydrate (600 mg kg−1). The left and right maxillary fragments containing the first molars with surrounding alveolar bone were removed and immediately immersed in the fixative solutions.

Light microscopy

Fragments of maxilla were fixed for 48 h at room temperature in 4% formaldehyde (freshly prepared from paraformaldehyde) buffered at pH 7.2 with 0.1 m sodium phosphate. After decalcification for 60 days in 7% EDTA buffered at pH 7.2 with 0.1 m sodium phosphate buffer, the specimens were dehydrated and embedded in paraffin. The 6-μm sagittal sections were stained with hematoxylin and eosin (H&E) for morphological analysis; the tartrate-resistant acid phosphatase (TRAP) histochemical method was used as an osteoclast marker. Sections were also adhered to silanized slides for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method and immunohistochemical detection of RANKL and caspase-3.

TRAP

The TRAP method was carried out as described by Cerri et al. (2003). Deparaffinized sections were immersed in an incubation medium prepared from 50 mL of 0.2 mm sodium acetate buffer (pH 5.2) containing 70 mg of Fast Red Salt (Sigma Chemical Company, St Louis, MO, USA) and 50 mm sodium tartrate dihydrate; 8 mg of naphthol AS-BI substrate (Sigma Chemical Company) was dissolved in 500 μL of N-N-dimethylformamide (Sigma Chemical Company) and added to sodium acetate solution. This medium was filtered and the sections were incubated at 37 °C; after washings in distilled water the sections were counterstained with Carazzi's hematoxylin and mounted in an aqueous medium.

TUNEL method

The TUNEL method, for detection of DNA breaks (Gavrieli et al. 1992), was performed as previously described (Cerri et al. 2000) and according to the Apop-Tag Plus Kit (Chemicon International, Chemicula, CA, USA). Deparaffinized sections were washed in phosphate-buffered saline (PBS; 50 mm sodium phosphate, 200 mm NaCl) at pH 7.2 and treated with 20 μg mL−1 proteinase K (Sigma-Aldrich Chemie, Germany) for 15 min to expose the DNA strands. The sections were treated with 3% hydrogen peroxide and then immersed in equilibration buffer for 5 min. Subsequently the sections were incubated in solution containing terminal deoxynucleotidyl transferase (TdT), in a humid chamber at 37 °C, for 1 h. The sections were washed in PBS and incubated in the anti-digoxigenin peroxidase at room temperature, for 30 min. After washings in PBS, the reaction was revealed with 0.06% 3,3′-diaminobenzidine (DAB; Sigma-Aldrich), counterstained with hematoxylin and mounted in resin Permount®. Sections of involuting mammary gland provided by the manufacturer of the kit were used as positive controls. Negative controls were incubated in a TdT free-enzyme solution.

For demonstration of apoptosis in osteoclasts, some sections were submitted to the TUNEL method followed by the TRAP reaction, as described by Cerri et al. (2003). After washing in distilled water, the sections were counterstained with hematoxylin and mounted in glycerin medium.

Immunohistochemistry for cleaved caspase-3 detection

For antigen retrieval, deparaffinized sections were immersed in 10 mm sodium citrate buffer pH 6.0 and maintained at 90–94 °C, in a microwave oven for 30 min. After cooling at room temperature, the endogenous peroxidase was blocked with 3% hydrogen peroxide for 20 min. The slides were washed in 50 mm PBS at pH 7.2 and incubated with 2% normal horse serum (Vector Laboratories, Burlingame, CA, USA) at room temperature. Subsequently, the sections were incubated overnight at 4 °C with rabbit polyclonal primary antibody anti-cleaved caspase-3 (Chemicon, Millipore, Temecula, CA, USA) diluted 1 : 150 (Faloni et al. 2012). After washings in PBS, the sections were incubated for 30 min at room temperature with biotinylated anti-rabbit IgG (Vector Laboratories). Subsequently the sections were washed and incubated in Vectastain® Elite® ABC reagent (Vector Laboratories) for 30 min. Peroxidase activity was revealed by 0.06% DAB (Sigma-Aldrich Chemie) in PBS and the sections were counterstained with Carazzi's hematoxylin. For negative controls for immunohistochemical reaction, the primary antibody was replaced by non-immune serum. For positive controls, sections of involuting mammary gland were used.

RANKL immunohistochemistry

For antigen retrieval, the sections were immersed in 10 mm sodium citrate buffer, pH 6.0 and maintained at 70–75 °C, in a vapor cooker, for 30 min. After cooling at room temperature and inactivation of the endogenous peroxidase with hydrogen peroxide, the slides were washed in 50 mm PBS at pH 7.2 and incubated with 2% bovine serum albumin containing 1% Triton-X (Sigma-Aldrich Chemie) for 30 min, at room temperature. Subsequently, the sections were incubated overnight at 4 °C with goat primary anti-RANKL antibody (Santa Cruz Biotechnology, USA), diluted 1 : 100. After washings in PBS, the immunoreaction was detected by the Labeled StreptAvidin-Biotin system (LSAB-plus kit; DAKO, USA). Sections were incubated for 30 min at room temperature with multi-link solution containing biotinylated mouse/rabbit/goat antibodies, washed in PBS and subsequently the sections were incubated with streptavidin-peroxidase complex for 30 min at room temperature. The sections were washed, and the reaction was revealed by Betazoid DAB (Biocare Medical, USA); sections were counterstained with Carazzi's hematoxylin. For negative controls, the primary antibody was replaced by non-immune serum in the immunohistochemical reaction.

Quantitative analyses

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Quantitative analyses
  6. Transmission electron microscopy (TEM)
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Number of TRAP-positive osteoclasts per millimeter of alveolar bone surface

Three sagittal non-serial sections of the alveolar bone surface surrounding the first molar from each SG and CimG animal were used. The shortest distance between the sections was 60 μm. The linear surface of alveolar bone around the roots of the first molar was measured using a light microscope (Olympus, BX-51, Olympus, Tokyo, Japan) and an image analysis system (Image-Pro Express 6.0, Olympus), as previously described (Faloni et al. 2007; Cruzoé-Souza et al. 2009) at 4 × magnification. Thus, the entire alveolar bone surface of the first molar, from the mesial to the distal alveolar process, was computed (Fig. 1). Multinucleated TRAP-positive osteoclasts (containing two or more nuclei/cellular profile) in close juxtaposition to the alveolar bone surface were counted at 400 × magnification.

image

Figure 1. Light micrograph of a sagittal section of the first upper molar tooth submitted to the TRAP reaction and counterstained with hematoxylin. The alveolar bone (AB) surrounding the molar roots (R) exhibits TRAP-positive osteoclasts (arrows), as observed in high magnification (inset). The hatched line shows the surface of the alveolar bone (AB) from the mesial (M) to distal (D), which was measured; in this surface, the number of multinucleated TRAP-positive osteoclasts was counted. Furcation region of the periodontium (asterisks) is present between the roots (R). BM, bone marrow; D, dentine; E, enamel space; G, gingiva; P, dental pulp; PL, periodontal ligament. Scale bars: 200 μm; inset: 7 μm.

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Frequency of apoptotic osteoclasts

In each animal, three non-serial sagittal sections of the first molar were used. In these sections, 25 multinucleated osteoclasts apposed to the alveolar bone surface (from the mesial to the distal alveolar process) were captured, totalling 150 osteoclasts per group. The images were captured using a camera (DP-71, Olympus) attached to a light microscope (Olympus, BX-51), at 1750 × magnification. The number of osteoclasts exhibiting nuclei with typical cell death features was computed, and the frequency of apoptotic osteoclasts was obtained. These typical characteristics included nuclei with chromatin strongly stained by hematoxylin, and nuclei showing a half-moon and condensed peripheral chromatin (Gonçalves et al. 2008).

Numerical density of RANKL-immunolabeled cells

The number of RANKL-immunolabeled cells per mm2 of periodontium was obtained in the furcation region (Fig. 1). The furcation region, located between the roots of multi-radicular tooth (such as rat molars), was chosen due to its anatomical characteristics that facilitate the localization of this region in histological sections; thus, the periodontium furcation region is useful for establishing a standard area.

The number of RANKL-immunolabeled cells adjacent to the alveolar bone surface was counted at 1750 × using an image analysis system (Image Pro-Express 6.0, Olympus). For each representative animal from the SG and CimG, these cells were counted in a total standardized area (0.015 mm2 per animal) from the periodontal ligament of the furcation region. Thus, the number of RANKL-positive cells per mm2 was obtained for each rat.

Statistical analysis

The statistical analyses were performed using SigmaStat software version 3.2 (Jandel Scientific, Sausalito, CA, USA). The data were subjected to the unpaired Student's t-test for comparison between the groups (SG and CimG). The significance level was set as P ≤ 0.05.

Transmission electron microscopy (TEM)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Quantitative analyses
  6. Transmission electron microscopy (TEM)
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Specimens containing alveolar bone of the first molars were fixed for 16 h in a solution containing 4% glutaraldehyde and 4% formaldehyde buffered at pH 7.2 with 0.1 m sodium cacodylate. After decalcification for 60 days in a 7% solution of EDTA buffered at pH 7.2 in 0.1 m sodium cacodylate, the specimens were postfixed in cacodylate-buffered 1% osmium tetroxide at pH 7.2 for 1 h. Subsequently, the specimens were washed in distilled water and immersed in 2% aqueous uranyl acetate for 2 h. After washings, the specimens were dehydrated in graded concentrations of ethanol, treated with propylene oxide and then embedded in Araldite.

Semi-thin sections (600–800 nm) stained by 1% toluidine blue were examined in a light microscope, and suitable regions were carefully selected for trimming of the blocks. Ultrathin sections (70–85 nm) were collected onto grids and stained in alcoholic 2% uranyl acetate and in lead citrate solution, and examined in a Philips CM 100 TEM.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Quantitative analyses
  6. Transmission electron microscopy (TEM)
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Number of TRAP-positive osteoclasts in alveolar bone surface

The alveolar bone of maxilla of rats from both groups exhibited osteoclasts with conspicuous TRAP activity in their cytoplasm, which was stained in red (Figs 1a and 2a,b). However, few TRAP-positive osteoclasts were found in the bone surface of cimetidine-treated rats (Fig. 2b). The quantitative analysis revealed a statistically significant 42% reduction in the number of TRAP-positive osteoclasts per mm of bone surface in the CimG rats compared with SG (Table 1).

image

Figure 2. (a and b) Light micrographs showing portions of alveolar bone (AB) of sham (a) and cimetidine-treated rats (b) submitted to the TRAP reaction and counterstained with hematoxylin. Numerous TRAP-positive osteoclasts (OC) are juxtaposed to the alveolar bone surface in the CG (a). Note that in the CimG scarce TRAP-positive osteoclasts (OC) are adjacent to the bone surface. OB, osteoblasts; Ot, osteocytes; PL, periodontal ligament. Scale bars: 30 μm.

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Table 1. Number of TRAP-positive osteoclasts mm−1 of bone surface, frequency (%) of apoptotic osteoclasts and numerical density of RANKL-immunolabeled cells per square millimeter in rats from sham (SG) and cimetidine (CimG) groups.
 SGCimG
  1. Results are expressed as means ± SD (standard deviation).

  2. a

    P ≤ 0.01.

  3. b

    P ≤ 0.001.

  4. CimG, cimetidine group; OC, osteoclast; RANKL, receptor activator of nuclear factor κB ligand; SG, sham group; TRAP, tartrate-resistant acid phosphatase.

TRAP-positive OC mm−10.9 ± 0.20.5 ± 0.1a
Apoptotic OC (%)21.3 ± 5.438.0 ± 5.5b
RANKL-immunopositive cells mm−23089.6 ± 556.82027.5 ± 421.2a

Apoptosis in osteoclasts

Tartrate-resistant acid phosphatase-positive osteoclasts exhibiting shrunken cytoplasm and irregular nuclei with tortuous masses of condensed chromatin – strongly stained by hematoxylin – were found in the alveolar bone surface (Fig. 3a). When the sections were submitted to the TUNEL method, some multinucleated osteoclasts exhibited TUNEL-positive nuclei that were stained in yellow-brown (Fig. 3b). The sections submitted to the combined TUNEL/TRAP methods revealed TRAP-positive osteoclasts with TUNEL-positive nuclei (Fig. 3c,d). Caspase-3 immunolabeling was also detected in the cytoplasm of osteoclasts next to the alveolar bone surface; these osteoclasts exhibited condensed chromatin that was strongly stained by hematoxylin (Fig. 3e,f). Although images of osteoclasts exhibiting immunolabeling for caspase-3 and TUNEL-positivity were observed in the alveolar bone of rats from both groups, a significant increase in the number of apoptotic osteoclasts was verified in the cimetidine-treated rats (Table 1). Furthermore, numerous positive structures were observed in the involuting mammary gland sections, which were used as positive controls for the TUNEL method and caspase-3 immunohistochemistry. On the other hand, TUNEL-positive nuclei and caspase-3 immunolabeling were not detected in the negative control sections (data not shown).

image

Figure 3. (a–f) Light micrographs showing portions of alveolar bone (AB) of sham (c and e) and cimetidine-treated rats (a, b, d and f). (a) A TRAP-positive osteoclast (OC) adjacent to the bone surface exhibits irregular nuclei with peripheral chromatin strongly basophilic (arrows), typical of peripheral condensed chromatin. TRAP counterstained with hematoxylin. (b) Alveolar bone section submitted to the TUNEL method (brown-yellow color) and counterstained with hematoxylin. An osteoclast (OC) located in the resorption lacuna exhibits TUNEL-positive nuclei. (c and d) Portions of alveolar bone submitted to the TUNEL method (brown-yellow color) combined with TRAP reaction (red color). (c) Next to the bone surface a TRAP-positive osteoclast (OC) exhibits a TUNEL-positive nucleus (arrow). (d) The TRAP-positive osteoclast (OC) shows TUNEL-positive nuclei (arrows). (e and f) Portions of alveolar bone submitted to immunohistochemistry for caspase 3 detection (brown-yellow color) and counterstained with hematoxylin. Multinucleated osteoclasts (OC) adjacent to the bone surface exhibit immunopositive cytoplasm; these osteoclasts (OC) show nuclei (arrows) with peripheral condensed chromatin strongly stained by hematoxylin. Scale bars: 5 μm.

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The ultrastructural examination revealed multinucleated osteoclasts apposed to the alveolar bone surface in both groups. Some of them exhibited several vacuoles and vesicles near to the typical ruffled border (Fig. 4a,b). However, osteoclasts were also observed with irregular-shaped nuclei that exhibited conspicuous masses of condensed chromatin. Usually, these altered osteoclasts were detached from the alveolar bone surface. Several vacuolar and dense structures were observed throughout the cytoplasm. Some small bodies containing electron-dense material, similar to condensed chromatin, were also observed next to the bone surface. Usually, the ruffled border and clear zone were not observed in these altered osteoclasts (Fig. 5a,b).

image

Figure 4. (a and b) Electron micrographs of a portion of alveolar bone (AB) of a rat from SG. (a) A typical multinucleated and polarized osteoclast (OC) exhibiting ruffled border (RB) in close juxtaposition to bone surface is observed in the resorbing lacuna (arrows); adjacent to the ruffled border, several vacuoles (V) of varied sizes are present in the osteoclast cytoplasm. N, nuclei. Scale bar: 2.5 μm. (b) Outlined area in (a), shows the plasma membrane infoldings of the ruffled border (RB), which projects towards the bone surface (AB). Scale bar: 0.5 μm.

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image

Figure 5. (a and b) Electron micrographs of portions of alveolar bone (AB) of rats from CimG. (a) An osteoclast (OC1) next to the bone surface exhibits irregular nuclei (N) containing blocks of condensed chromatin (arrows); an irregular structure (S) showing irregular masses of condensed chromatin is seen in the osteoclast cytoplasm. Several vacuolar structures (V) are distributed by cytoplasm. Other osteoclast portion (OC2), showing nuclei with conspicuous masses of condensed chromatin (arrows), is adjacent to the bone surface. A small body containing electron-dense material (asterisks) is in close juxtaposition to the OC2. Scale bar: 1.5 μm. (b) A multinucleated osteoclast (OC) adjacent to the bone surface exhibits nuclei (N) with peripheral condensed chromatin (arrows); convoluted cytoplasm shows several vacuolar structures (V) and irregular/ovoid bodies with heterogeneous electron opacity (B). Scale bar: 1 μm.

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Numerical density of RANKL-immunolabeled cells

Periodontium sections for RANKL immunohistochemistry detection showed positive immunolabeling (brown-yellow color) in the cytoplasm of the SG and CimG osteoblasts and fibroblasts. However, an enhanced immunolabeling was verified in the periodontal ligament cells from SG when compared with CimG (Fig. 6a–f). In the SG, a strong immunoreaction was observed in the cytoplasm of large osteoblasts adjacent to the alveolar bone surface; moreover, RANKL-positive immunolabeling was also found in the cytoplasm of fibroblasts from the periodontal ligament (Fig. 6b,c). In contrast, osteoblasts and fibroblasts were weakly immunolabeled or negative for RANKL immunohistochemistry in CimG (Fig. 6d–f). No RANKL-positive cells were found in the negative control sections (data not shown).

image

Figure 6. (a–f) Light micrographs of portions of alveolar bone (AB) from sham (a–c) and cimetidine-treated (d–f) rats submitted to immunohistochemistry for detection of RANKL (brown-yellow color) and counterstained with hematoxylin. (a) An evident immunolabeling is observed in the furcation region of the periodontal ligament (PL). Osteoblasts (arrows) apposed to the bone surface and periodontal fibroblasts (Fb) exhibit positive immunoreaction. (b) Large osteoblasts (arrows) juxtaposed to the bone surface show strongly immunolabeled cytoplasm. (c) Fibroblasts (Fb) of the periodontal ligament (PL) exhibit conspicuous immunolabeling in their cytoplasm. (d–f) In the furcation region of the periodontal ligament (PL) cells exhibit RANKL immunolabeling. (e) Outlined area in (d), portion of alveolar bone, osteoblasts (arrows) exhibit immunolabeled cytoplasm. (f) Scarce immunolabeling is observed in fibroblasts (Fb) of the periodontal ligament (PL). Note an evident reduction in the RANKL immunolabeling in the periodontal ligament (PL) of the CimG (d–f) in comparison to SG (a–c). Scale bars: 35 μm (a and d); 12 μm (b, c, e and f).

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The quantitative analysis revealed a significant reduction in the numerical density of RANKL-positive cells in the furcation region of the periodontal ligament of the CimG rats in comparison to the SG (Table 1).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Quantitative analyses
  6. Transmission electron microscopy (TEM)
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Our findings clearly demonstrate that cimetidine, a histamine H2 antagonist, significantly reduces the number of osteoclasts in the alveolar bone and therefore may interfere with the bone resorption. The high incidence of apoptotic osteoclasts in the cimetidine-treated rats indicates that cell death may be, at least in part, responsible for the significant reduction in the number of osteoclasts and, probably, in bone resorption. In TRAP knockout mice, an increase in the thickness of the metaphyseal trabecular bone has been demonstrated (Blumer et al. 2012). Our results also showed that cimetidine inhibits RANKL immunoexpression and may consequently interfere with osteoclastogenesis. Therefore, our results strongly indicate that cimetidine interferes with osteoclasts survival and formation.

Studies have suggested that the histamine released during inflammation plays a role in the bone metabolism via the H2-receptor, stimulating the bone resorption (Crisp et al. 1986; Hall & Schaueblin, 1994; Nakamura et al. 1996; Dobigny & Saffar, 1997). Histamine induces the production of IL-1, IL-6, macrophage colony-stimulation factor and granulocyte macrophage colony-stimulating factor by hematopoietic cells (Mor et al. 1995) and stromal cells (Takamatsu & Nakano, 1998) via H2-receptors (Vannier & Dinarello, 1994; Mor et al. 1995; Jilka et al. 1998). These cytokines stimulate the osteoclast formation, differentiation and activity (Riggs, 2000; Phan et al. 2004). During tooth eruption, the concomitant increase in the mast cells and osteoclasts was also verified, suggesting a participation of mast cells in the bone resorption signaling (Cerri et al. 2010). In the present study, numerical and cellular changes observed in the osteoclasts indicate that cimetidine induces osteoclasts death resulting in a significant cell number decrease.

Evidence suggests that the treatment with cimetidine, a H2-receptor antagonist, prevents bone resorption in ovariectomized rats (Leclous et al. 2002, 2004; Lesclous et al. 2006) and rabbits with induced periodontal disease (Hasturk et al. 2006). Additionally, cimetidine prevents articular destruction in patients with rheumatoid arthritis (Yamaura et al. 2003). Thus, it has been suggested that cimetidine exerts a beneficial effect in preventing inflammation (Yamaura et al. 2003; Hasturk et al. 2006) and, consequently, has a role as an immunoregulatory agent (Sirois et al. 2000; Jutel et al. 2001). Considering that our findings revealed a significant reduction in the number of osteoclasts in the cimetidine-treated rats, it is possible that cimetidine might also interfere with bone homeostasis in the healthy periodontium.

Our findings revealed a significant reduction in the number of TRAP-positive osteoclasts on the alveolar bone surface in CimG rats. Some osteoclasts present on the alveolar bone surface showed nuclei with peripheral chromatin strongly stained by hematoxylin, a characteristic of condensed chromatin indicative of apoptosis. A higher frequency of osteoclasts with these morphological features was verified in the alveolar bone surface of CimG rats compared with SG. Additionally, TUNEL-positive nuclei were observed in some osteoclasts adjacent to the alveolar bone surface. The TUNEL method is widely used to identify cell death because it reveals DNA fragmentation, a characteristic indicative of apoptosis (Gavrieli et al. 1992; Cerri et al. 2003; Cerri, 2005; Faloni et al. 2007; Gonçalves et al. 2008). Moreover, strong immunolabeling for caspase-3 was also observed in these altered osteoclasts, which frequently exhibited nuclei with condensed chromatin. Normally, the caspases constitute a family of proteases that are involved in the complex cascade of molecular events that control cell death (Raff, 1998; Huppertz et al. 1999; Lockshin & Zakeri, 2004). Caspases contain a cysteine residue capable of cleaving an aspartic acid-containing motif on the next downstream caspase. Evidence suggests that caspase-3 is activated during osteoclasts apoptosis induced by tamoxifen (Wu et al. 2003, 2005) and estrogen (Faloni et al. 2012). Thus, morphological analysis taken together with the TUNEL reaction and caspase-3 immunohistochemistry are used to identify cell death by apoptosis (Caneguim et al. 2011; Faloni et al. 2012).

The ultrastructural images revealed that altered osteoclasts exhibited irregular nuclei containing conspicuous masses of condensed chromatin. Their cytoplasm exhibited several vacuoles and vesicles; moreover, the ruffled border and the clear zone were not observed in the altered osteoclasts. Usually, these osteoclasts were detached from the bone surface. These ultrastructural characteristics are typical of osteoclasts undergoing apoptosis, as described by other authors (Ito et al. 2001; Faloni et al. 2007, 2012; Cruzoé-Souza et al. 2009). Considering that the incidence of osteoclast apoptosis was significantly increased in the cimetidine-treated rats (CimG), it is conceivable to suggest that cimetidine may promote osteoclasts apoptosis in the alveolar bone. Although the mechanisms of cimetidine action on osteoclasts remains unclear, it has been suggested that the presence of an intracellular histamine receptor (Hic receptor) may perhaps modulate several cellular processes, such as apoptosis (Brandes et al. 1990).

Additionally, RANKL was immunohistochemically detected in the periodontium cells of CimG and SG rats. However, RANKL-immunolabeling pattern exhibited a clear difference between the CimG and SG groups; weak immunoreaction was verified in the cimetidine-treated rats in comparison to the sham-treated rats. Our quantitative analysis revealed a significant reduction in the number of RANKL-immunolabeled cells in the cimetidine-treated rats compared with the sham-treated rats. There is considerable evidence that, under certain circumstances, histamine regulates osteoclastogenesis to enhance RANKL expression in osteoblasts and stromal cells of the bone marrow (Deyama et al. 2002; Yamaura et al. 2003; Ikawa et al. 2007). In vitro studies have demonstrated that histamine stimulates binding of RANKL to RANK and, consequently, enhances osteoclasts maturation (Yasuda et al. 1999; Deyama et al. 2002; Ikawa et al. 2007). Adding 10 μm cimetidine to the rat bone marrow culture significantly inhibited the osteoclast formation (Ikawa et al. 2007). In the present study, the significant reduction of RANKL-immunolabeled cells in the CimG group indicates that cimetidine may inhibit RANKL immunoexpression, therefore reinforcing the idea that histamine participates in osteoclastogenesis.

For this study, the cimetidine dosage (100 mg kg−1) was not aimed to mimic human pharmaceutical use; therefore, the results cannot be directly correlated to human cimetidine use. Thus, further clinical studies are needed to determine the cimetidine effects in human alveolar bone.

Our results strongly indicate that cimetidine interferes in the rat alveolar bone homeostasis inhibiting bone resorption. The reduction in the RANKL immunoexpression was correlated to the increased frequency of apoptotic osteoclasts, suggesting that cimetidine inhibited osteoclastogenesis and promoted osteoclast death by apoptosis. Future studies are necessary to investigate whether the cimetidine acts on osteoclasts directly and/or indirectly.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Quantitative analyses
  6. Transmission electron microscopy (TEM)
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

The authors thank Mr Luis Antônio Potenza and Mr Pedro Sérgio Simões for technical support. This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - 2007/59374-6; 2010/03571-0; 2010/10391-9) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Quantitative analyses
  6. Transmission electron microscopy (TEM)
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References