The pineal secretory product melatonin was recently shown to have free radical scavenging properties. Melatonin also activates several antioxidative enzymes including glutathione (GSH) peroxidase, modulates gene expression for several protective enzymes and reduces lipid peroxidation. Additionally, the antioxidant effects of melatonin are probably based on its stimulatory effect on the expression of superoxide dismutase, GSH peroxidase, GSH reductase and glucose-6-phosphate dehydrogenase and its inhibitory effect on nitric oxide synthase expression. On the other hand, the role of physiological levels of melatonin, which are known to decrease with age, in the prevention of this damage is not yet known. It regulates a wide variety of biological rhythms, including cardiac rhythms. Physiological and/or pharmacological doses of melatonin affect a variety of heart functions. These effects on the heart may be mediated directly and/or indirectly.

Pinealectomy-induced biochemical and behavioral changes have been the subject of many studies, but morphological changes and particularly cardiovascular changes have been less well studied. Since it was reported that pinealectomy may cause hypertension up to 60 days, we have used rats that were pinealectomized at 2 months before treatment with melatonin to eliminate any possible effect of pinealectomy-induced hypertension.

We have explored whether reduced melatonin levels in the circulation play a critical role in damage to the heart as a result of pinealectomy. Although the retina has a high capacity to synthesize melatonin, it does not seem to contribute significantly to plasma melatonin levels probably because of rapid catabolism in the retina. Night-time blood melatonin peaks are eliminated after pinealectomy. We also investigated the effects of melatonin on pinealectomy-induced changes of malondialdehyde (MDA), a major lipid peroxidation product, and GSH, which is a major protectant against lipid peroxidation. It has been proposed that antioxidants that maintain levels of GSH may restore cellular defense mechanisms against lipid peroxidation. Additionally, serum cholesterol levels and heart weight were determined. The present study was designed to investigate the effects of physiological and pharmacological levels of melatonin on rat hearts using pinealectomy and exogenous melatonin.

Female Wistar rats, weighing 150–200 g, were kept at a constant temperature (21±2°C) and humidity (60%) with a 12/12 h light/dark cycle. Rats were pinealectomized or not at 2 months before treatment with melatonin.

Pinealectomy was performed as described by. Rats were anesthetized with ketamine hydrochloride (75 mg/kg) and xylazine (8 mg/kg) which were administered intraperitoneally (i.p.) before operation. The entire procedure was completed within 15 min.Pinealectomy was confirmed by histological evaluation of the gland of each animal. Rats were divided into two groups: pinealectomized (n=12) and non-pinealectomized (n=6). Subsequently, pinealectomized rats were distributed over two groups: one group of rats were treated ip with vehicle, and one group with 4 mg/kg melatonin (Sigma, St. Louis, MO, USA) for 3 days. Non-pinealectomized rats were injected with vehicle only. At 24 h after the last treatment, rats were sacrificed, hearts and aortas were quickly removed and hearts were divided into two equal longitudinal parts. One of the parts was placed in a solution of 10% formaldehyde for routine histopathological examination using light microscopy. The other half of the hearts was placed in liquid nitrogen and stored at ?70°C until assayed for levels of MDA and GSH. Trunk blood was collected to determine the serum levels of cholesterol. Additionally, the weight of the hearts was determined. For these studies, melatonin was dissolved in ethanol and diluted in saline to give a final concentration of 5% ethanol.

All experiments were performed in accordance with the guidelines for animal research of the National Institute of Health and were approved by the Committee for Animal Research at Inonu University, Malatya, Turkey.

Heart tissue (200 mg) was homogenized in an ice-cold solution of KCl (150 mM) for the determination of MDA levels. MDA levels in homogenates were determined spectrophotometrically by measuring thiobarbituric acid-reactive substances. Levels of GSH were determined by spectrophotometry based on the use of Elman’s reagent. Results are expressed as nmol/g tissue. Serum levels of cholesterol were determined using an Olympus Autoanalyser (Olympus, Tokyo, Japan). Heart MDA and GSH levels were measured to compare our data with those in the literature and to assess the status of lipid peroxidation and antioxidant capacity in the animals in the three groups. Serum cholesterol levels were also found to be related to pinealectomy in animal experiments.

Samples of heart and aorta that had been fixed in 10% formaldehyde were routinely processed and paraffin embedded. Subsequently, 5 ?m-thick sections were stained by the following methods: hematoxylin and eosin (H&E), Van Gieson, Masson’s trichrome and PAS/Alcian blue at pH 2.5 (PAB).

Levels of MDA and GSH in the heart and serum cholesterol levels were analyzed by one-way ANOVA. Post hoc comparisons were performed using Tukey tests. The weight of hearts was analyzed by a Student’s t-test. Differences were considered significant when p<0.05. All results are expressed as mean±SEM.

Biochemical data with respect to tissue levels of MDA and GSH, and serum cholesterol levels are shown in Table 1. Heart MDA levels were significantly higher in the pinealectomized group than in the sham-operated group and the group of pinealectomized rats treated with melatonin; however, GSH levels were within the normal range, irrespective of the treatment. Serum cholesterol levels were higher in the pinealectomized group than in the sham-operated group and the group of pinealectomized rats treated with melatonin.

Hearts of pinealectomized rats were heavier than those of sham-operated rats, whereas hearts of pinealectomized rats that had been treated with melatonin had similar weights as the pinealectomized group.

Microscopical examination of the hearts and aortas revealed pathological effects of pinealectomy. First, we noticed myocardial fibrosis in both pinealectomized rats and pinealectomized rats treated with melatonin but not in the hearts of sham-operated rats. Fibrosis was detected optimally with Masson’s trichrome staining method. Second, myxomatous degeneration of heart valves was observed in pinealectomized rats and pinealectomized rats treated with melatonin, which showed similarities with the floppy mitral valve syndrome in humans, and this degeneration was best demonstrated with PAB staining. These changes may have occurred as follows: attenuation of the fibrosa layer of the valve was accompanied by focal thickening of the spongiosa layer and concomitant deposition of mucoid (myxomatous) material. Thickening of the mural endocardium of the left atrium was also observed. Coronary arteries were difficult to find, whereas aortas did not exhibit calcification or degeneration of elastic tissue. These morphological alterations were similar in the pinealectomized rats and pinealectomized rats treated with melatonin.