The Effects of Melatonin on Open Wounds of Aged Mice Skin

  Melatonin is a hormone secreted by the pineal gland in the brain.¹ Researchers believe that the melatonin level may be related to aging as it has been shown that the plasma level of melatonin declines considerably with aging.² Melatonin is an antioxidant, a free radical scavenger^3,4 that stimulates the activity of several antioxidant enzymes.^5,6 Skin, the largest organ of the body, undergoes an aging process that presents multiple clinical manifestations and problems.^7,8 A majority of the skin changes associated with aging are due to decreased proliferative capacity that leads to cellular senescence and altered biosynthetic activity of skin derived cells, and from accumulation of oxygen species as a result of oxidative metabolism.^9,10 Therefore, aged skin heals more slowly than younger skin. The wound healing process consists of a sequence of events that ultimately leads to the re-establishment of tissue integrity and function.¹¹ Melatonin receptors are expressed in skin cells and have the potential to mediate phenotypic actions on cellular proliferation and differentiation. Melatonin may be a significant contributor to regulation of physical and functional integrity of the skin.¹² The effects of melatonin on the skin cells have been reported by researchers and there are different opinions about these effects. In the present study, the effects of melatonin on the healing process of open wounds in the skin of aged mice were evaluated by morphological and morphometric analysis.

Materials and Methods

  Forty 16-month-old male Suri mice that weighed 20 g–23 g were purchased from Iran Pasteur Institute and housed in a temperature controlled room at 23˚C ± 2˚C. The mice were maintained according to the guidelines and animal protocol approved by the research center of Tehran University of Medical Sciences. Forty mice were divided into a control group (n = 20) and an experimental group (n = 20). Under ether anesthesia, the hair on the back of the neck of both groups was shaved and a 2-cm long, full-thickness incision was made on each mouse’s neck and left unsutured. The experimental group was injected intraperitoneally (IP) with a single daily dose of 10-mg/kg melatonin in saline for 12 days. The control group received only saline for the same period of time. On days 9 and 12 after making the incision, 10 mice from each group were sacrificed (20 mice on day 9 and 20 on day 12). The skin around the area of the incision was carefully dissected and fixed in 10% formalin. The dissected skin tissues were dehydrated in graded concentration of alcohol and then cleared in xylene, infiltrated with paraffin, and finally embedded in paraffin. Paraffin blocks were cut into 5-µm thick pieces and stained with hematoxylin & eosin (H&E) to evaluate epithelization. Epithelial neoformation was scored 0–3. The mean diameter fibroblast nuclei were measured using a Leica DM LB image analyzer, Leica QWin software (Leica Microsystems, Buffalo Grove, IL), and an objective lens (x40 magnification). The major (a) and minor (b) axis’s mean profile diameters (√a.b) of longitudinally sectioned nuclear profiles were obtained.13 Van Gieson’s stain results were in a red color for collagen fibers and were scored 0–3 for collagen fiber deposition. For hydroxyproline measurement, tissue samples were hydrolyzed in 6N HCL (1 mL/10 mg tissue) at 107˚C for 16 hours and then neutralized with sodium hydroxide. Absorbance was measured at 560 nm with d-dimethylaminobenzaldehyde according to the method previously described.14   Statistical analysis was performed using SPSS software (IBM Corp., Somers, NY). The Mann-Whitney U test was applied to assess the significance of changes between control and experimental groups.

Results

  H&E stains of skin tissues 9 days after incision revealed that new epithelial cells had not yet completely bridged the epidermis; however, a single layer of epithelial cells had covered the dermis in the experimental group (Figure 1). The dermis consisted of fibroblasts with a significantly increased nuclei diameter in the experimental group compared to control group (P< 0.001; Table1). Histological analysis at 12 days post wounding demonstrated that epithelization in the control group was significantly less than melatonin-treated mice (P< 0.001; Table 1, Figure 1). Van Gieson’s stain showed an increase in collagen fibers 9 and 12 days after wound creation in the experimental group compared to control (P< 0.001). The organization of collagen fibers was irregular in the control group, while collagen fibers were oriented parallel to the epidermis in melatonin-treated mice (Figure 2). The tissue hydroxyproline levels on days 9 and 12 after wound creation in the control group were lower than experimental group (P< 0.001).

Discussion

  Structurally, in aged skin, the dermal cell population decreases, the epidermis becomes thinner and atrophic, and the biosynthetic capacity of fibroblasts is reduced15; therefore, wounds sustained in aged skin will heal more slowly than younger skin. In the present study, the histological effects of IP administration of melatonin on the wound healing process were evaluated. Recent research suggests that the skin contains melatonin receptors, which means that melatonin may have some role in the structure of skin cells, particularly keratinocytes and fibroblasts.16 Melatonin exerts positive effects on wound healing whether it is administered topically or systemically.17 Melatonin has a number of properties that could be beneficial to wound healing. The actions of melatonin, such as stimulation of fibroblasts in order to synthesize collagen fibers, and growth factors could alter the wound healing process¹⁸ since growth factors stimulate epithelial cell proliferation.¹⁹ A significant increase in the mean diameter of nuclei in fibroblasts was demonstrated in melatonin mice indicating that these cells were stimulated. According to the present results, epithelization on days 9 and 12 post wounding increased in the experimental group compared to control. Melatonin in mammals can regulate proliferation of epidermal keratinocytes.²⁰ The lack of melatonin causes prominent ultrastructural changes in rat skin, especially in the epidermis.21   Aging of the skin is associated with progressive changes in the architectural organization.22 The expression of different enzymes increases and this causes the degradation of the collagenous matrix.23 The Van Gieson’s stain showed that melatonin exerted positive effects on collagen synthesis. Collagen fibers have an important role in wound healing.18 Collagen is a protein that contains hydroxyproline. Hydroxyproline levels in the experimental group were significantly higher than control (P< 0.001) and shows that collagen fiber synthesis in melatonin-treated mice was higher than control. In 2009, Izykowska et al²³ evaluated the effects of melatonin in various doses added to culture medium 30 minutes before exposure of keratinocytes and fibroblasts to irradiation. Cell viability was evaluated and the protective role of melatonin on the skin cells was shown. Stimulation of cell proliferation by melatonin in a dose- and time-dependent manner could be due to enhanced antioxidative enzyme activity.²⁴ Antioxidants have been shown to promote the wound healing process.²⁵ According to the present findings, a statistically significant increase in epithelization was shown in mice treated with melatonin.

Conclusion

  The results of this study suggest that melatonin may be beneficial to the open wound healing process in aged mice.

Acknowledgements

  This research was supported by the vice-chancellor for research at Tehran University of Medical Sciences. The authors would like to thank Farzan Institute for Research and Technology for providing technical assistance.

References

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