Experimental animals and the “comb” scald model
Adult male Sprague-Dawley rats weighing approximately (200- 250 g) were purchased from the Shanghai Slac Laboratory Animal Company (Shanghai, China). Adult male Sprague-Dawley rats (weighing approximately 200- 250 g) were purchased from the Shanghai Slac Laboratory Animal Company (Shanghai, China). The present study was performed according to protocols approved by the Committee on Animal Care and Use of the Second Affiliated Hospital Zhejiang University School of Medicine (2019-331), and strictly followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
The “comb” scald model was established according to previous reports[23,24]. Specifically, a customized rectangular brass comb (with a transverse area of approximately 20 mm×10 mm) was boiled in 100 °C water for 5 min and then applied to the shaved skin surface of the dorsum of rats for 20 s after anesthesia was induced (sodium pentobarbital, Sigma, St. Louis, MO, USA, 50 mg/kg, intraperitoneal), as shown in Fig. 1a. A row of four bands of full-thickness burns (20 mm×10 mm) was made with three interspaces of uninjured skin (20 mm×5 mm) to represent the zone of stasis. The burn wound area occupied approximately 4% of the total body surface area (TBSA). For the sham group, the brass comb was heated in 25 °C water and applied to the shaved dorsum of rats after anesthesia. During the operation, the breathing and heart rates of the burned rats were carefully monitored to ensure that all of the rats were under anesthesia and pain-free and were then allowed to recover from the anesthesia. Moreover, all rats were housed in individual cages and given 0.25 mg/kg buprenorphine via subcutaneous injection immediately and every 12 h after the burn injury for analgesia. In terms of pain-relieving intervention, pain and distress scales were used to assess pain in the burned rats immediately and every 6 h after recovery from anesthesia and informed medical treatment.
Research design and sample collection
Forty-eight animals were randomly assigned to 6 groups: the sham group (n = 8) and five burn groups (n = 8 per group) at different time points. The five burn groups were euthanized using an overdose of sodium pentobarbital 6, 12, 24, 48 or 72 h postburn, whereas the sham group was euthanized 72 h after pseudo-operation. One band of interspace skin surrounded by 2 mm of burned tissue on each side (20 mm×9 mm) was harvested from each burned rat and stored in 4% paraformaldehyde at 4 °C for subsequent histological and immunofluorescence analyses, and the remaining two bands of interspace skin (without burned tissue; 20 mm×5 mm) from each burned rat were stored at -80 °C for Western blot assays. The corresponding bands of unburned skin from each rat in the sham group were also collected as control samples. The general design of this study is shown in Fig. 1b.
Local blood flowgraphy
A PeriCam PSI system (Perimed, Sweden), which is based on laser speckle contrast analysis (LASCA) technology, was applied to evaluate the local blood flow changes in the burn wounds. Real-time images showing tissue blood perfusion were captured with the help of special PIMSoft software.
Histological preparation and pathologic examination
Cutaneous samples were biopsied and fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at a thickness of 3 μm using a rotary microtome (RM2245, Leica, Solms, Germany). After deparaffinization and rehydration, hematoxylin and eosin (HE) staining was performed for histological examinations, and the tissue slices were observed and imaged under a microscope (DM2500, Leica, Solms, Germany). Five visual fields (200×) from every slice were randomly selected for analysis.
Oxidative stress assessment
Burn wound tissue homogenates were analyzed with a thiobarbituric acid reactive species (TBARS) assay kit (KGT003–1, KeyGEN, Nanjing, China) to determine the MDA levels, which are expressed as nmol/mg protein. Tissue glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities in the zone of stasis were measured separately using commercial assay kits from KeyGEN Biotech (KGT010 and KGT00150, Nanjing, China) according to the manufacturer’s recommended protocol. The results are expressed as nmol/min/mg protein and U/mg protein. The absorbance values were measured using a microplate reader (Model 680 Microplate Reader, Bio-Rad, CA, USA).
A commercial cell-death detection kit purchased from Roche Diagnostics (Indianapolis, IN, USA) was used for TUNEL staining according to the manufacturer’s recommended protocol. The processed slices were observed under a microscope (DM2500, Leica, Solms, Germany), and representative images were captured. The index of apoptosis was determined and is presented as the percentage of apoptotic cells among all the cells counted in a double-blind manner. At least three visual fields per slide and five slides per group were evaluated by two investigators.
Transmission electron microscope-based detection of ultrastructure
Full-thickness skin tissues from the zone of stasis were dissected from the dorsum of sham-treated and scalded rats for electron microscopy. Briefly, the skin tissues were cut into pieces, immediately fixed in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.0) (National CHEM, China) at 4 °C overnight, postfixed in 1% osmium tetroxide (National CHEM, China) for 1-2 h, and dehydrated in a graded series of ethanol. Then, the specimens were placed in a 1:1 mixture of absolute acetone (National CHEM, China) and the final Spurr resin mixture (SPI-CHEM, West Chester, PA) for 1 h at room temperature, transferred to a 1:3 mixture of absolute acetone and the final resin mixture for 3 h and finally moved to a pure final Spurr resin mixture overnight. The prepared specimens were sectioned into 70-nm-thick sections with a microtome (EM UC7, Leica, Germany). The sections were sequentially stained with uranyl acetate (SPI-CHEM, West Chester, PA) and alkaline lead citrate (National CHEM, China) for 5 to 10 min, respectively. The stained tissue sections were examined initially at low magnification (1,5000×) to identify representative structures. The cells in the selected zone were then examined at high magnification (3,0000× or 50,000×) to reveal mitochondria. Briefly, digital images with scale bars were collected by TEM. Representative digital images with scale bars, which showed cellular mitochondria undergoing the process of mitophagy in the zone of stasis, were captured by transmission electron microscopy (H7650, Hitachi, Japan).
Prepared slices were washed in PBS for 10 min and then boiled in 0.01 mmol citrate buffer (pH = 6) for 10 min for antigen retrieval. After incubation with hydrogen peroxide for 10 min, 5% bovine serum albumin (BSA) was applied as the blocking solution for 20 min at room temperature. Without being washed, the sections were incubated with an anti-CC3 (1:200; #9664, Cell Signaling Technology, MA, USA), anti-p-Bad (1:200; sc-12969-R, Santa Cruz, CA, USA), anti-LC3 II (1:200; #4060, Cell Signaling Technology, MA, USA) or anti-TOMM20 (1:200; sc-12969-R, Santa Cruz, CA, USA) antibody overnight at 4 °C. After being rinsed with PBS, the sections were incubated with Alexa 488 (1:50; Abcam, China)- or Cy3 (1:50; BA1032, Boster, Wuhan, China)-labeled goat anti-rabbit secondary antibodies for 2 h at 37 °C in the dark. The sections were rinsed and then mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). All slices were observed and photographed under a fluorescence microscope (DM5500B, Leica, Solms, Germany).
Quantitative real‐time PCR (qRT‐PCR) detection
The expression level of HIF-1 in the zone of stasis was analysed via qRT-PCR. Briefly, total RNA was isolated from tissues with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and RNase-Free DNase I (Qiagen, Duesseldorf, Germany). The SuperScript First- Strand Synthesis System for reverse transcription PCR (Invitrogen, Carlsbad, CA, USA) was applied to synthesise cDNAs, and RNA and cDNA concentrations and purities were measured via BIO-RAD spectrophotometry (SmartSpecTM Plus, BIO-RAD, CA, USA). The primers were designed using Primer Premier 6.0 software, as a forward primer 5’-CGTGCCCCTACTATGTCGCTTT-3’ and a reverse primer 5’-GTCTTCTGCTCCATTCCATCCTGT-3’ and were synthesised by Shanghai Biological Engineering Co., Ltd. (Shanghai, China). PCR amplifications were conducted using the Power SYBR® Master Mix (Invitrogen, Carlsbad, CA, USA) in an iQTM 5 Real-time PCR system (BIO-RAD, CA, USA). Expression levels were assessed relative to that of GAPDH, as an internal standard, and the primer detail is shown as forward 5’-GAAGGTCGGTGTGAACGGATTTG-3’ and reverse 5’-CATGTAGACCATGTAGTTGAGG TCA-3’. Relative quantification of the target gene expression level was conducted using the 2−∆∆Ct method.
Western blot analysis
Skin samples were collected, frozen in an ultra-low freezer and cut into pieces, which were lysed with RIPA lysis buffer (AR0105, Boster, Wuhan, China) for 1 h on ice. The lysates were mixed with loading buffer and centrifuged at 14,000 x g for 10 min, and the protein samples were subjected to SDS-PAGE and then transferred onto nitrocellulose membranes via electrophoresis. In addition, aliquots of the samples were used to determine the protein concentration of each sample using a bicinchoninic acid (BCA) kit (KGPBCA, KeyGEN, Nanjing, China). Subsequently, the membranes were incubated in blocking buffer for 2 h and incubated overnight at 4 °C with the following primary antibodies: anti-cleaved caspase 3 (1:1000; #9664, Cell Signaling Technology, MA, USA), anti-cleaved caspase 9 (1:1000; #9507, Cell Signaling Technology, MA, USA), anti-p-Bad (1:2000; ab129192, Abcam, Cambridge, UK), anti-Bad (1:2000; ab62465, Abcam, Cambridge, UK), anti-Cytochrome C (1:1000, ab13575, Abcam, Cambridge, UK), anti-TOMM20 (1:500; ab56783, Abcam, Cambridge, UK), anti- SQSTM1 (1:200; ab56416, Abcam, Cambridge, UK ), anti-LC3II (1:1000; #4108, Cell Signaling Technology, MA, USA), anti-PARKIN (1:1000; ab179812, Abcam, Cambridge, UK), anti-BNIP3 (1:1000; ab109362, Abcam, Cambridge, UK), anti-BNIP3L (1:1000; ab109414, Abcam, Cambridge, UK), anti-FUNDC1 (1:200; ab173226, Abcam, Cambridge, UK), and anti-HIF-1 (1:10000; ab179483, Abcam, Cambridge, UK). GAPDH (1:10000; ab181602, Abcam, Cambridge, UK) was used as a control on the same membranes. After the application of secondary antibodies, the bands were detected with West Dura Extended Duration Substrate (Pierce, USA), and exposed X-ray films (Kodak, USA) were analyzed using Bandscan 5.0 software based on comparisons with GAPDH bands.
Under sodium pentobarbital anesthesia (50 mg/kg, intraperitoneal), mCherry-labeled adenoviruses (Advs, designed, produced and identified by Bioomin Technology Corp., Ltd., Shanghai, China), including Ad-negative control (NC) and Ad-shHIF-1, were injected subcutaneously into selected locations on the shaved dorsum of rats (nine locations) (Fig. 6A). Subsequent experiments were performed a minimum of 72 h after infection to allow sufficient time for gene silencing, and the effects of the adenoviruses were evaluated by a Bio-Real in vivo imaging system (IVIS spectrum, Perkin Elmer, MA, USA).
The data are presented as the means ± standard deviations (SDs). GraphPad Prism version 7 (San Diego, CA, USA) was used for the statistical analyses. Multiple comparisons were performed via one-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests. Unpaired t-tests were used for comparisons between two groups. P-values less than 0.05 were considered statistically significant.