Surg Radiol Anat (2006) 28: 157–162 DOI 10.1007/s00276-005-0065-9
O R I GI N A L A R T IC L E
G. Oktem Æ S. Uslu Æ S.H. Vatansever Æ H. Aktug M.E. Yurtseven Æ A. Uysal
Evaluation of the relationship between inducible nitric oxide synthase (iNOS) activity and effects of melatonin in experimental osteoporosis in the rat Received: 1 September 2005 / Accepted: 20 October 2005 / Published online: 15 December 2005 Springer-Verlag 2005
Abstract Inducible nitric oxide synthase (iNOS) plays a critical role in the pathogenesis of osteoporosis. iNOS generates nitric oxide (NO), a free radical contributing to the imbalance between bone formation and resorption caused by estrogen depletion. Melatonin is the major product of the pineal gland which is known to diminish iNOS expression and NO production significantly. The aim of this study was to determine the distribution of iNOS and the amount of apoptotic cells after melatonin treatment in ovariectomized rats. Since previous studies have shown that constitution of bone formation is primarily sustained in nucleus pulposus and epiphyseal cartilage, experiments were carried out on nucleus pulposus and epiphyseal cartilage; additional quantitation of osteoblasts and osteoclasts were evaluated on vertebral area as well. Vertebral sections of ovariectomized rats were obtained from formalin-fixed and parafin-embedded blocks. iNOS expression and quantitation of apoptotic cells in nucleus pulposus and epiphyseal cartilage were evaluated using indirect immunoperoxidase and TUNEL techniques, respectively. The number of osteoclasts and osteoblasts in trabecular bone was determined using histomorphometry. Ovariectomy increased iNOS expression and the number of apoptotic cells in nucleus pulposus and epiphyseal cartilage, whereas a 4-week treatment with melatonin (10 mg/kg/day) resulted in the reduction of both effects. These data indicate that there is strong influence of melatonin application on expression of iNOS, apoptosis, osteoclast and osteoblast numbers after ovariectomy. In
G. Oktem (&) Æ S. Uslu Æ H. Aktug Æ M.E. Yurtseven Æ A. Uysal Department of Histology and Embryology, School of Medicine, Ege University, Histoloji ve Embriyoloji A.D, TR-35100 Izmir, Turkey E-mail:
[email protected] Tel.: +90-232-390409136 S.H. Vatansever Department of Histology and Embryology, School of Medicine, Celal Bayar University, 35040 Manisa, Turkey
conclusion, melatonin besides its usual use as an antiaging hormone, may also be an effective hormone in treatment of bone changes in estrogen deficiency states Keywords Osteoporosis Æ Inducible nitric oxide synthase Æ Melatonin Æ Vertebra Æ Nucleus pulposus
Introduction Inducible nitric oxide synthase (iNOS) plays a considerable important role in various cell systems, one of which is the bone. The protective effect of estrogen in bone are mediated by the blocking of the synthesis of iNOS induced by a variety of stimuli in cell systems, thus the promoter induced by phorbol esters. Nitric oxide (NO) is produced constitutively by osteoblasts in vitro [8, 20] and stimulates their proliferation [22]. On the contrary, there is a strong role for iNOS in osteoclast activation after ovariectomy, through the regulation of the development of osteoclasts by osteogenic cells [25]. Estrogen depletion results in an increased iNOS synthesis in stromal cells and osteoblasts, leading to NO accumulation in the bone microenvironment. This, in turn, results in an increased formation of peroxynitrites and other inflammatory compounds [13]. Cuzzocrea et al. [4] have shown that iNOS is a key signaling molecule in determining the imbalance between bone resorption and bone formation caused by estrogen depletion in mice and have suggested that it is a potential target for therapy of postmenopausal osteoporosis in women. The mechanism of bone loss after ovariectomy remains unclear, but previous studies have indicated that depressed bone formation plays an important role [19]. The absence of iNOS becomes crucial when bone loss is induced by estrogen depletion, suggesting a predominant role of this enzyme in the osteoporosis consequent to deficient ovarian function. iNOS is indispensable for the development of osteoporosis consequent to systemic inflammation, and it has been proposed that the reduced bone formation mediated by
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iNOS is due to the dramatic increase in osteoblast apoptosis [15]. Melatonin (MT) (N-acetyl-5-methoxytryptamine), the major product of the pineal gland, plays a fundamental role in the neuroimmuno-endocrine system [2]. Current data suggest that MT treatment could reduce tissue damage as a result of free radicals in a number of models both in vivo or in vitro [10, 26]. MT treatment significantly abolished iNOS expression and NO production in murine macrophages [5]. Crespo et al. have demonstrated that MT is able to inhibit the expression of the iNOS mRNA levels induced by lipopolysaccharide in lung and liver of rats in vivo in a dose-dependent manner [3]. In the light of these observations, this study aims to investigate the effect of MT treatment on the iNOS immunoreactivity and apoptosis immunohistochemically, and on the number of bone cells histologically in the vertebra of bilateral ovariectomized rats. Knowing firstly that intervertebral disc consists of nucleus pulposus, annulus fibrosus and epiphyseal plate; secondly that ovariectomy-induced bone loss is due to an increase in bone resorption and degenerative changes; and thirdly that degenerative changes influence each intervertebral disc components in osteoporosis; the target tissue in this study has been selected as the nucleus pulposus and epiphyseal plate [9, 29].
Materials and methods Animals and experimental design Forty-five, 5-month-old female Wistar rats, weighing approximately 220–250g were used in this study. All experiments were approved by the Animal Ethics Committee at Ege University School of Medicine. Rats were housed as five rats per cage in 12:12 light/dark cycled rooms with the temperature at 24C and allowed free access to water and commercial pellet food ad libitum. Experiments were performed by the same surgeon using aseptic techniques and microsurgical dissection aided by a surgical loop (Heine Optotechnik 6X, West Germany). The animals were randomly divided into five groups as follows: Control group: consists of intact rats (n:5), ovariectomy (Ovx) group: consists of untreated, ovariectomized rats (n:10); ovariectomy + saline (Ovx + SF) group: consists of ovariectomized rats treated subcutaneously with 0.25 ml saline (n:10); ovariectomy + MT (Ovx + MT) group: consists of ovariectomized rats treated subcutaneously with MT (n:10); sham + MT group: consists of rats treated with MT after sham operation (n:10). MT treatment (10mg/k/day) was started 8weeks after the ovariectomy. MT (N-acetyl-5methoxytrptamine, SIGMA Chemical Co., USA) was injected subcutaneously in 0.25ml saline for 4weeks. At the end of 12weeks following operation, rats were sacrificed by vascular fixative perfusion under anesthesia. All surgical procedures were performed under
anesthesia with ketamine (60mg/kg, BAYER, United German Pharmaceutical Factories, Turkey) and sedation with xylasine (10mg/kg, Parke-Davis, Turkey).
Tissue processing and antibody application L4–L5 vertebras were removed and fixed in 10% neutral buffered formalin for 24h, and then decalcified in 10% formic acid at room temperature for 7days. Following fixation and decalcification, vertebrae specimens were dehydrated through graded ethanol, cleared in xylene, embedded in paraffin and cut on microtome (Leica MR 2145). Longitudinal sections of vertebras (3 lm thick) were picked up onto poly-L-lysine-coated glass slides. Some of the sections were stained with Haematoxylin– eosine and the rest were used for immunohistochemical analysis. For immunohistochemistry, after xylene for 30 min, sections soaking in a decreasing series of ethanol and washed with distilled water and phosphate-buffered saline (PBS), then treated with 2% trypsin in 50 mM Tris buffer (pH 7.5) at 37C for 15min and washed with PBS. Sections were delineated with a Dako pen (Dako, Denmark) and incubated in a solution of 3% H2O2 for 15min to inhibit endogenous peroxidase activity. The primary antibody specific for iNOS (Santa Cruz Biotechnology, USA; 1:100 dilution in PBS) was applied to the sections for 2 h. After biotinylated second antibody for 30min, washed and stained for 30min with streptavidin–peroxidase (HRP) enzyme conjugate and rinsed well with PBS. Color was developed with DAB (3,3diaminobenzidine tetrahydrochloride) as chromogen for 5min and counterstained with hematoxylin and mounted with glycerol vinyl alcohol aqueous-GVA (Zymed Laboratories, USA).
In situ identification of apoptosis using terminal dUTP nick end-labeling (TUNEL) This step was followed by the addition of apoptosis detection kit (deadend colorimetric terminal dUTP nick end-labeling (TUNEL) system, Promega, USA). Five micrometer sections were cut from the paraffin blocks of the samples. The sections were deparaffinized in xylene, rehydrated and incubated with 20 lg/ml proteinase K for 10min and rinsed in distilled water. Endogenous peroxidase activity was inhibited with 3% hydrogen peroxide. The sections were then incubated with equilibration buffer for 10–15s and TdT-enzyme in a humidified atmosphere at 37C for 60min. They were subsequently put into prewarmed working strength stop/wash buffer at room temperature for 10min and incubated with antistreptavidin–peroxidase for 45min. Each step was separated by careful washings in PBS. Staining was done with DAB and counterstaining was performed in Mayer’s hematoxylin.
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Evaluation of sections All sections were analyzed under a light microscope (Olympus BX40, Tokyo, Japan). Staining intensity was graded independently by two observers blinded to the experimental conditions. The selection of the area for the analysis was based on availability of sufficient area for immunohistochemical scoring. The degree of positive staining for iNOS was evaluated by scoring on a scale of 1–4 for intensity (I); i.e., none, mild, moderate and strong; and for distribution (D); i.e., none, focal, patchy and diffuse. Tissues with I · D less than or equal to 4 were considered weakly positive, and those with I · D greater than 4 were designated strongly positive. Results were also further simplified in positive and negative cases. For TUNEL; approximately 100 TUNEL positive and immunoreactive cells were counted in randomly chosen fields per case. The percentage of apoptotic cells stained brown was determined. Cells in areas with necrosis, poor morphology or in the margins of sections were not analyzed.
Table 1 Intensity of iNOS and dispersion of the apoptotic cells on nucleus pulposus and epiphyseal cartilage in control and study groups Intensity of iNOS
TUNEL
Nucleus
Pulposus
Epiphyseal
Cartilage
Control Ovx Ovx + MT Ovx + SF
(++) (+++) (++) (+++)
(+) (+++) (+) (+++)
Negative ( ) 77.66±1.22 20.28±0.8 76.98±1.25
Sham + MT
(+)
(++)
Negative ( ) 48.66±2.66 18.16±0.16 47.59±0.12 76.98 Negative ( )
Negative ( )
one-way analysis of variance, followed by post hoc analysis using Bonferroni test. Probability values less than 0.05 were considered to show a significant difference.
Results iNOS expression in intervertebral disc after estrogen depletion and melatonin treatment
Histomorphometrical analysis of osteoclast and osteoblast volume Blind histomorphometrical analysis was carried out by the semiautomatic digitizer (UTHSCSA Image Tool for Windows, version 1.28) after the images were transferred from a light microscope onto a computer. Osteoblast number per bone surface (Obl.N/BS; mm–1) and osteoclast number (Ocl.N/BS; mm–1) were measured on the haematoxylin–eosine-stained sections on vertebral area. Bone histomorphometry was performed by the quantification of a minimum of 5 adjacent fields at a magnification of ·100 in each section. L5 region studied was cancellous bone between 0.5mm distal to two ends of growth plate. All measurements were carried out as recommended previously [17].
The immunohistochemical profile of iNOS in nucleus pulposus and epiphyseal cartilage is shown in Table 1. In epiphyseal cartilage, iNOS immunoreactivity was mild in control and Ovx + MT groups (Fig. 1a), moderate in sham + MT group (Fig. 1b) and strong in Ovx and Ovx + SF groups (Fig. 1c). iNOS immunoreactivity was moderate in control and Ovx + MT groups (Fig. 2b) in nucleus pulposus, whereas it was strong in Ovx and Ovx + SF groups (Fig. 2c) and mild in sham + MT group (Fig. 2a). Immunoreactivity of iNOS was significantly changed after ovariectomy (P
