A total of 24 hamsters (weight, 40–60 g; age, 8 weeks) were used, obtained from the Ürümqi Municipal Center for Disease Control and Prevention (Ürümqi, China). All animals were individually housed in stainless steel cages with a 12-h light-dark cycle and a controlled temperature. The hamsters received food and water ad libitum and were acclimatized for at least two weeks prior to thermal injury. All animal care and experimental procedures were approved by the Animal Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Ürümqi, China).

The body surface areas of the hamsters (equivalent to 0.0913 × weight^2/3) ranged between 106.8 and 139.9 cm² (19). The burn wound was circular, with a diameter of ~3 cm, accounting for 5–6.5% of their body surface area. The hamsters were anesthetized with ketamine (0.7 g/kg), and diazepam and atropine were used to maintain adequate anesthesia. Once anesthetized, the hair on the back of each hamster was removed to reveal a bare region with a diameter of ~3 cm. There are a variety of methods for producing deep second-degree burns in animal models (20–24); however, the majority of these methods are adapted to large animals and may be complex. The burn wound depth may be affected by the contact temperature, heat duration, burn area and pressure. Since hamsters are small in size, there is a risk of accidental mortality if the burn is not inflicted correctly. The end of a 50-ml syringe, with no plunger, was used to administer the burn. While the hamster was anesthetized, the end of the syringe was gently pressed onto the bare region on the back of the hamster to keep the hot water inside the syringe (Fig. 1A). Next, 20 ml water (75°C) was poured into the syringe, producing a circular burn wound with a diameter of ~3 cm. The durations of water contact with the back skin of the hamsters were 0, 6, 8, 10 and 12 sec, for the five groups of four hamsters. The wounds of all the hamsters were was hed with running water for 1 min and 1.5–1.8 ml isotonic saline was injected intraperitoneally for fluid resuscitation. The hamsters were insulated and received 700 mg/kg morphine when awake. The hamsters stopped experiencing any pain by day 2 after the burn was administered which was evident by their return to normal behavior. At 24 h after the burn, the wound tissues in the superficial layer of the deep fascia were removed, with the hamsters under ketamine anesthesia. The dissected tissues were immersed in 10% formaldehyde solution, with a volume ten times the volume of the tissues. The dissected tissues were divided into sections and stained with hematoxylin and eosin (H&E). The optimum duration of hot water contact for producing a deep second-degree burn was determined by H&E staining. According to the histologically determined durations of contact for deep second-degree burn, the wounds were created on the remaining four hamsters to observe wound healing durations.

Tissue samples were cut into slices for H&E staining. The sections were dewaxed with xylene for 15 min and soaked with 100% ethanol for 3 min, 95% ethanol for 3 min and 70% ethanol for 3 min. The sections were then was hed with water, air-dried, soaked with hematoxylin for 15 min and was hed with water a further three times. Subsequently, the sections were stained with eosin for 3 min and soaked with 95% ethanol I for 3 min, 95% ethanol II for 3 min, 100% ethanol I for 3 min and 100% ethanol II for 3 min. Once the ethanol had dried, the sections were soaked in xylene for 2 h and subsequently mounted using Permount^™ Mounting Medium purchased from Multi Sciences (Lianke) Biotech Co., Ltd. (Hangzhou, China).

Total RNA was extracted with TRIzol®(Invitrogen Life Technologies, Carlsbad, CA, USA), chloroform, isopropanol and 75% ethylene glycol, and stored in a refrigerator at −80°C. Reverse transcription was conducted by adding 1.5 µg sample RNA to a 20-µl reaction system that included 1 µl Oligo (dT) Primer, 1 µl reverse transcriptase and 4 µl buffer (Takara Biotechnology Co., Ltd., Dalian, China). The reaction mixture was incubated for 1 h at 37°C and then heated to 85°C for 5 min to terminate the reaction. For qPCR amplification, 5 µl reacted solution was extracted and added to a 20-µl reaction system that included 10 µl SYBR Premix Ex Taq, 0.25 µl target primer/0.25 µl β-actin primer (Takara Biotechnology Co., Ltd.) and 4.5 µl ddH₂O. The reaction was conducted on a Thermal Cycler Dice Real Time System (Takara Bio, Inc., Otsu, Japan). For the amplification of chymase, the following PCR procedure was used: 95°C for 3 min, 35 cycles of 95°C for 45 sec, 55°C for 45 sec and 72°C for 45 sec, and a final extension at 72°C for 5 min. For the amplification of β-actin, the following PCR procedure was used: 95°C for 4 min, 35 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 2 min, and a final extension at 72°C for 7 min. The PCR product was examined by dissociation curve analysis, and the relative quantification of gene expression was normalized against the internal standard, β-actin.

The primers were designed as follows: Chymase forward, 5-CTG AGA GGA TGC TTC TTC CTG C-3, and reverse, 5-AGA TCT TAT TGA TCC AGG GCC G-3; β-actin forward, 5-AAC TCC ATC ATG AAG TGT GA-3, and reverse, 5-ACT CCT GCT TGC TGA TCC AC-3.

Chymase activity in the burn tissue was determined using a radioimmunoassay (25). Burn tissue samples weighing 100 mg were repeatedly homogenized and added to 50 mM NaH₂PO₄ buffer (10 w/v, pH 7.4) for 15 min at 4°C. The samples were centrifuged at 3,000 × g for 20 sec at 4°C and the supernatants were discarded. The sediments were added to 50 mM NaH₂PO₄ buffer for ultrasonic homogenization (400 A, 5 sec/time with an interval of 10 sec, repeated three to five times) and the homogenized tissues were subsequently centrifuged at 30,000 × g for 20 min at 4°C. The homogenization and centrifugation steps were repeated three times and the supernatants, which contained chymase, were collected for each repeat. From each processed sample, 1,500 µl supernatant was collected and divided into three tubes (500 µl each) labeled A, B and C. For reaction system A, 6 ng AngI was added, while 6 ng AngI and 50 µM lisinopril were added to reaction system B and 6 ng AngI and 20 µM aprotinin were added to reaction system C. The tubes were placed in a water bath (37°C) for 15 min, and a 2.5-fold volume of precooled ethanol was added to terminate the reaction. The resulting concentrations of AngII in reaction systems A, B and C were determined using a radioimmunoassay kit (Beijing North Institute of Biological Technology, Beijing, China), according to the manufacturers instructions. The activity of angiotensin-converting enzyme (ACE) was calculated by subtracting the enzyme activity of reaction system B from that of A. The activity of other serine proteases was calculated by subtracting the enzyme activity of reaction system C from that of A. Finally, the activity of chymase was calculated by subtracting the ACE activity and the other serine protease activity from the enzyme activity of reaction system A. The enzyme activity required for the generation of 1 nmol AngII in 100 mg burn tissues per min was defined as 1 enzyme activity unit (U). The activity of chymase was expressed as U/mg.

SPSS software, version 16.0 (SPSS, Inc., Madison, WI, USA) was used for statistical analysis. The results are presented as the mean ± standard deviation. Analysis of variance and Dunnetts test were used to assess the differences between groups. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the degree of the burn wounds, hamsters were used as the animal model and H&E staining of the tissues was performed. All hamsters used in the study survived. The degree of paleness of the burnt skin varied at different time periods after the injury was inflicted. At 24 h after the burn was inflicted, the edge of the burn wound was red with swelling, while the wound itself exhibited swelling and was white in color, with no visible blisters (Fig. 1B). The burn contact durations analyzed were 0, 6, 8, 10 and 12 sec. Histological examination revealed that the epidermal cells were partially detached from the burn area in the 12-sec burn duration hamsters. H&E staining showed that the structure of the tissues was unclear and that blisters had formed on the skin. In addition, congestion had occurred in the dermal interstitial layer, collagen fibers had integrated into sheets and residual hair follicles were observed in the deep dermis. Subcutaneous vascular dilation, congestion and edema were evident; however, the tissue structure was not destroyed (Fig. 1C). Thus, it was determined that the damage caused by 12 sec hot water contact was equivalent to a deep second-degree burn. Deep second-degree burns were produced on the four hamsters that received hot water contact for 12 sec. There were no signs of infection during wound healing and re-epithelialization was complete after 18 days (Fig. 1D). These results indicated that a hamster model of deep second-degree burn was established by applying hot water contact for 12 sec.

A radioimmunoassay and qPCR were used to investigate the activity and mRNA expression levels of chymase in the burn tissues. Chymase activity was observed in all the post-burn stages and was significantly higher in the burnt tissue when compared with the normal skin tissue (day 0). However, there were no statistically significant differences in chymase activity amongst the various post-burn stages (P>0.05; Fig. 2A). At days 1, 3, 7 and 14 after burning, chymase mRNA expression levels were significantly higher when compared with those in the normal skin tissue (day 0; P<0.05). In addition, no statistically significant differences were observed amongst the chymase mRNA expression levels of the post-burn stages (P<0.05; Fig. 2B). These results demonstrated that chymase activity and mRNA expression levels were increased in the mast cells of the burned tissue and may be involved in the process of burn wound healing.