**3. Effect of leptin on the wound healing of the skin**

energy expenditure by negative feedback at the hypothalamic nuclei [7]. Leptin is also known to exhibit a variety of physiological actions on lipid metabolism [8], hematopoiesis [9], thermogenesis [10], ovarian function [11], bone formation [12, 13], and angiogenesis [14, 15]. The leptin receptor (Ob-R) is expressed in various tissues including the hypothalamus [16, 17], adipose tissue [18], skeletal muscle [19], and hepatocytes [18, 20]. The multifunctionality of leptin and the wide distribution of its receptor suggest that leptin plays a variety of physiological roles not

The surface of the body is covered by skin to communicate with the external environment and to protect deeper tissues and organs by separating them from the external environment such as chemical, mechanical, and thermal stresses, infections, and dehydration [21, 22]. Normal dermal wound repair processes, such as inflammation, angiogenesis, contraction, deposition of extracellular matrix, granulation tissue formation, epithelialization, and remodeling, require various cellular and molecular signals [23]. In this biological process, skin fibroblasts interact with surrounding cells such as keratinocytes, inflammatory cells, and endothelial cells [21, 24]. Fibroblasts produce extracellular matrix, glycoproteins, adhesive molecules, and various cytokines [25, 26]. The lack of these signals may result in poor healing of wounds such

A certain study showed that skin wound healing delayed in leptin deficient *ob/ob* mice and that exogenously administered leptin restored this delayed wound healing by enhancing reepithelialization of the wound in these mice in diabetic condition [29]. Another some studies unveiled the effect of leptin on wound healing by demonstrating that leptin acted as an autocrine/paracrine regulator in the wounded site [30, 31]. These findings strongly suggest the possibility that leptin could be a potential medicine for promoting wound healing in skin. However, all previous studies refer to whole-body dosage administered intraperitoneally, and even when administered locally, the leptin must have been administered every day. So, we investigated whether local and single-dose administration of leptin exerted a promotive influence on the skin wound healing. Because, we thought that local and single administration of leptin could avoid the influence of its adverse effect such as metabolic disorders, hyper/ hypoglycemia caused by the fact that leptin is a multifunctional and potent systemic hormone, and could be advantageous for the lowering of patients' distress in some cases in its clinical

An immunohistochemical analysis of mouse skin using anti-leptin receptor antibody revealed that leptin receptor was expressed in prickle and granular cells of epidermis (**Figure**

only as a systemic hormone but also as a local growth factor.

26 Wound Healing - New insights into Ancient Challenges

**2. Localization of leptin receptor in mouse skin**

**1**). These findings showed that epidermal cells are target of leptin.

as diabetic ulcers [27, 28].

application.

To elucidate the effect of leptin on the wound healing of the skin, mouse skin chemical burn model was used. Eighteen 6-week-old male ICR mice were fed a normal diet and maintained under a 12-h light/12-h dark cycle. Chemical wounds were created on the back skin by applying two pieces (12 × 12 mm) of filter paper soaked with 20% sodium hypochlorite for 5 min. Wound formation was verified next day, and the wounds were covered with 15 g (12 × 12 × 1 mm) of MedGel (Med GEL Corp., Tokyo, Japan) containing 10 μL of 100 ng/mL leptin (R&D Systems, Minneapolis, USA) or phosphate-buffered saline (PBS) as a control. This hydrogel-contained leptin or PBS was attached to the chemical burn site and dressed. The size of the ulcer was measured on day 4 and 8 after burn formation, and the skin tissue around the wound was obtained for histological analysis. In consequence, at day 4, slightly enhanced re-epithelialization was observed in leptin-treated group, but no significant difference was noted between leptin-treated and control group. In contrast, at day 8, significantly enhanced re-epithelialization was observed in leptin-treated group (**Figure 2**). These experiments showed that the wound area decreased much faster in the leptin-treated group compared with the control group. These findings demonstrated that single and local administration of leptin using bioabsorbable hydrogel promoted the wound healing of skin.

Meanwhile, body weight (BW), and levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) or blood sugar (BS) were not affected through experiment period, showing that topically administered leptin had no systemic adverse effects (**Figure 3**). These findings certify that topically administered leptin is capable of promoting wound healing of the skin without any systemic adverse effects in this period. However, unfortunately, we could not elucidate whether local and single administration of leptin could avoid or not the influence of its side effect over a long period. This issue should be elucidated in the future investigation.

**Figure 2.** Effect of leptin on the wound healing of the mouse skin. (A) Histological findings of wound repair of skin at day 8 after initial wounding in leptin-treated group. (B) Histological findings of wound repair of skin at day 8 after initial wounding in control group. Spaces between arrow heads show ulcerative area without epithelium. Wound heal is significantly enhanced in leptin-treated group. (C) Skin wound healing at day 4 after wound creation. No significant difference in wound healing was noted between leptin-treated and control group. (D) Skin wound healing at day 8 after wound creation. Significantly enhanced re-epithelialization was observed in leptin-treated group. \*\*P < 0.01. H-E staining. Bars: 500 μm.

**Figure 3.** Changes in BW, AST, ALT, and BS. (A) BW, (B) BS, (C) AST, (D) ALT. None of these laboratory parameters were significantly affected by leptin application.
