Stress-related disturbances of homeostasis and induction of the pathogenesis of various diseases are well established. Physical and emotional stress can be etiological factors underlying several diseases. Stress can induce inflammatory responses in various organs and when stress is chronic it may induce or aggravate chronic inflammatory diseases such as migraine, atopic dermatitis, urticaria, psoriasis, inflammatory bowel diseases and aseptic cystitis. The role of mast cells in inflammatory and allergic reactions including stress-induced inflammatory diseases is well documented. Mast cells are implicated in skin disorders such as atopic dermatitis, psoriasis and these diseases are either induced or worsened by stress. Skin of patients with atopic dermatitis contains significantly higher numbers of mast cells than intact skin. In psoriasis, the number of mast cells is significantly elevated in both lesioned skin and intact skin. Mast cells release inflammatory mediators upon activation, such as histamine, proteases, prostaglandins, leukotrienes and cytokines that have potent vasodilatory, and inflammatory properties. For example, histamine from mast cells increases vascular permeability upon activation and stimulates cutaneous sensory nerves. Stimulation of unmyelinated sensory nerves leads to release of neuropeptides such as substance P, which act on mast cells, blood vessels and leukocyte infiltration. It has been recently suggested that skin may have its own equivalent of the hypothalamic–pituitary–adrenal (HPA) axis because corticotropine-releasing hormone (CRH) and its receptors were shown to be present in the skin. Activated skin mast cells increase vascular permeability in rodents. It was also shown recently that acute stress induces local release of CRH in the skin, further implicating a local stress-induced HPA axis.

The pineal hormone melatonin has multiple functions in humans and its release is stimulated by beta-adrenergic receptors. Serum melatonin levels exhibit a circadian rhythm. Melatonin participates in the regulation of several physiological processes such as the seasonal biological rhythm, daily sleep induction, aging and modulation of immunobiological defence reactions. Furthermore, melatonin is highly lipophilic facilitating penetration of cell membranes and has been shown to have free radical scavenging properties. Pierrefiche et al. (1993) were the first to show that melatonin reduces lipid peroxidation (LP). Tan et al. (1993) has shown that melatonin scavenges the hydroxyl radical (OH), a radical that can initiate LP. Melatonin may play a role in the etiology and prevention of several dermatoses e.g. atopic eczema, psoriasis, and malignant melanoma (Fischer, 1999). Considering the fact that psychological stress induces or worsens various skin conditions, we investigated whether water avoidance stress (WAS) affects the occurence of mast cells in the skin and their degranulation and if so, whether melatonin can reduce mast cell activation.

Adult female Wistar albino rats weighing 200–250 g were housed individually in a light- and temperature-controlled room on a 12/12 h light/dark cycle, and fed a standard pellet lab chow. All experimental protocols were approved by the Animal Care and Use Committee of the Marmara University, School of Medicine, Istanbul, Turkey.

Rats were handled daily by the same investigator for 2 weeks before the start of the study and were then submitted to WAS as described previously. The procedure was as follows: rats were placed on a glass platform (8×6 cm) in the middle of a plastic container with a diameter of 90 cm and height of 50 cm that was filled with water of 25 °C to 1 cm below the level of the platform. Rats avoided the aversive stimulus (water) by staying on the platform for 2 h. All experimental procedures were performed between 8:00 and 10:00 to minimize the effect of circadian rhythms.

Melatonin treatment consisted of injections of 10 mg/kg melatonin (Sigma, St. Louis, MO, USA) ip before WAS induction to the animals. At the end of the experiments, rats were euthanized by decapitation.

Six groups of rats were treated as follows:

1. Control group (n=8): rats were placed on the platform in a container without water for 2 h and were injected with a saline solution.

2. Melatonin group (n=8): rats were injected with melatonin but were not exposed to WAS.

3. Acute stress (aWAS) group (n=8): rats were exposed to WAS for 2 h after injection of a saline solution.

4. Melatonin-treated acute stress (aWAS+mel) group (n=8): rats were exposed to WAS after an injection of melatonin.

5. Chronic stress (cWAS) group (n=8): rats were exposed to WAS for 2 h daily for 5 days after receiving each day an injection of a saline solution.

6. Melatonin-treated chronic stress (cWAS+mel) group (n=8): rats were exposed to WAS for 2 h daily for 5 days after receiving each day an injection of melatonin.

Pieces of skin from the back of approximately 2 cm2 (skin was taken from the same location in all rats) including the hypodermis were fixed in 10% formaldehyde and then routinely processed for paraffin embedding. Paraffin sections (5-?m thick) were stained with 1% toluidine blue (Merck, Darmstadt, Germany). Sections were examined at 400× magnification with a BH 2 photomicroscope (Olympus, Tokyo, Japan).

Sections of the skin were evaluated for mature granulated mast cells and degranulated mast cells. Ten randomly taken sections (the first, 11th, 21st, etc.) from each animal were selected and mast cells were counted without knowing to which treatment group the rat belonged. In each section, mast cells containing metachromatic granules were counted separately at 400× magnification in five consecutive dermal areas by one or more observer and the observers were blinded to the treatment of the animals. An eye-piece graticule (size 0.0785 mm2) was used in order to avoid overlap of areas to be counted. It had a vertical and a horizontal axis, so that the area was divided in four parts, which facilitated counting. Areas selected in each region were surveyed for mast cells and the mast cell density was then expressed as cell number per unit area. Data were analyzed by using one-way analysis of variance (ANOVA). Differences between groups were determined with the Dunnett’s multiple comparisons test and data were expressed as mean±standard error of the mean. Significance of differences was taken at the level p<0.05.

The results of the present study demonstrate that melatonin has a protective effect on acute and chronic psychological nontraumatic stress-induced numbers of mature mast cells in dermis of rats. WAS induces psychological stimuli and cWAS may be a model of “life stress” as its duration is 5 days. Recent clinical studies support the notion that chronic life stress predicts the outcome of symptom intensity in patients with functional bowel disease. Exposure for a few days to stress has been reported to worsen colitis in rats as well. Barrier dysfunction in the mucosa of the ileum and colon, morphological changes in epithelial cells, hyperplasia and activation of mast cells, infiltration of neutrophils and mononuclear cells, and increased myeloperoxidase activity are the results of induction of cWAS. Activation of mast cells has been shown in different stress models. Acute immobilization stress induces mast cell degranulation in dura and jejunum. Repetitive exposure to odors, Pavlovian conditioning and isolation stress conditions cause secretion of histamine from mucosal mast cells. We have previously observed that stress-induced inflammatory changes such as mast cell degranulation are mediated by capsaicin-sensitive sensory neurons in urinary bladder. Recently, it was reported that acute immobilization stress also induces mast cell degranulation in the skin, which may be linked to various inflammatory skin diseases. These authors also reported that capsaicin-sensitive sensory neurons are involved in stress-induced mast cell degranulation in skin.