Adult C57BL/6J mice (8-12 weeks, Shanghai SLAC Laboratory Animal Corporation, China) were used in this study. All animal experiments were conducted strictly followed the Guide for the Care and Use of Laboratory Animals of the China National Institutes of Health. All procedures involving mice were carried out with approval of The Tab of Animal Experimental Ethical Inspection of the Affiliated Lihuili Hospital of Ningbo University. All mice in this experiment were housed individually in standard experimental cages, with a 12-h light/dark cycle and free access to regular food and water before any experimental procedure. Thirty-six mice were randomly divided into the control and limonin groups (n = 18 each).
Reagents and antibodies
Limonin (C32H42O14, purity ≥98%) was purchased from Solarbio (Beijing, China). Diaminobenzidine (DAB) developer, pentobarbital sodium, and the hematoxylin and eosin (H&E) staining kit were provided by Solarbio (Beijing, China). Rabbit anti–cadherin 5 was purchased from Boster Biological Technology (A02632–2). Rabbit anti-GAPDH was acquired from Biogot Technology (AP0063). Rabbit anti–vascular endothelial growth factor (VEGF), anti–superoxide dismutase 1 (SOD1), anti–matrix metalloproteinase 9 (MMP9), anti-HO-1, and anti-CAPS3 were acquired from Proteintech (19003-1, 10269-1, 10375-2, 21327-1, and 19677-1). Rabbit anti–endothelial nitric oxide synthase (eNOS), anti–cytochrome c (CYC), and anti-Bax were purchased from Cell Signaling Technology (12994, 14796, and 32027). Horseradish peroxidase (HRP)–conjugated immunoglobulin G (IgG) secondary antibody was purchased from Santa Cruz Biotechnology. Fluorescein isothiocyanate (FITC)–conjugated IgG secondary antibody was obtained from Boyun Biotechnology, and the 4′,6-diamidino- 2-phenylindole (DAPI) solution was purchased from Beyotime Biotechnology. The Electrochemiluminescence (ECL) Plus Reagent Kit was obtained from PerkinElmer Life Sciences and the BCA kit was acquired from TermoFisher Scientifc.
Random-pattern skin flap model
Before surgery, mice were anesthetized by intraperitoneal injection of 1% (w/v) pentobarbital sodium 50 mg/kg. Then a caudal-based skin/panniculus carnosus ﬂap (size 1.5 × 4.5 cm2) was elevated on the mouse dorsum beneath the fascia using a mouse dorsal random ﬂap model as previously reported . After that, the right and left sacral arteries supporting the blood supply of this ﬂap were excised completely. Finally, the separated ﬂap was inset immediately into the donor bed and sutured using 5-0 silk. The random ﬂap area was divided into the proximal (area I), intermediate (area II), and distal (area III) zones, each with the same size. On day 7 post surgery, all mice were euthanized with an overdose of pentobarbital sodium. Six mice in each group were killed for Western blotting analysis, and six mice in each group were killed for immunohistochemistry (IHC) analysis, and H&E staining. Six mice in each group were used for the general evaluation of survival, the tissue edema assessment, and laser Doppler blood ﬂow imaging.
Limonin was dissolved in 1% dimethyl sulfoxide (DMSO)–saline solution to a concentration of 50 mg/ml. Each mouse in the limonin group received limonin (40 mg/kg per day) by intraperitoneal injection for 7 days after the surgery. Mice in the control group received equal volume of 1% DMSO–saline solution (vehicle control) for 7 days. All animals were euthanized by pentobarbital sodium (overdose), and skin samples were harvested.
General evaluation of flap survival
Macroscopic development and characteristics of texture, appearance, color, and hair condition of the ﬂaps were observed for 7 days after the surgery. At 3 and 7 days after the surgery, photographs of the skin ﬂap were acquired to estimate ﬂap viability. All photographs were measured using Image-Pro Plus imaging software (version 6.0; Media Cybernetics, Silver Spring, MD, USA) to calculate the survival area, and the percentage of viable area was measured as follows: the extent of viable area × 100%/total area.
Assessment of tissue edema
Tissue edema is a crucial indicator of necrosis in ischemic ﬂaps. Tissue edema was reﬂected according to the water content of the ﬂaps. On postoperative day 7, six ﬂap tissue samples from each group were weighed, dehydrated in an autoclave at 50°C, and weighed until weight remained stable for at least 2 days. The percentage of water was measured as [(wet weight − dry weight)/wet weight] × 100%.
Laser doppler blood flow imaging
The blood supply and vascular ﬂow to the ﬂaps were evaluated by laser Doppler blood ﬂow (LDBF) measurements . On postoperative day 7, a laser Doppler instrument (Moor Instruments, Axminster, UK) was introduced to scan the dorsal skin of mice in a safe environment under anesthesia. LDBF evaluation was performed according to the previous study . The entire dorsal skin of mice, including the ﬂap area, was scanned by laser Doppler. Laser Doppler blood ﬂow commonly provides deeper penetration, which enables reinforced visualization of small vessels under the tissue surface, which is suitable for angiogenesis assessment. Perfusion units were seen as indices of blood ﬂow. The blood ﬂow of the random-pattern skin ﬂaps was quantified by LDI Review software (version 6.1; Moor Instruments, Wilmington, DE, USA). Each mouse was measured three times and calculated the mean value.
Hematoxylin and eosin staining
On postoperative day 7, six skin samples (1 × 1 cm) of the central area in ﬂap area II were acquired and sampled after sacrifice. The specimens were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin for further transverse sectioning. Sections (5-mm thickness) were cut with a microtome and mounted on poly l-lysine–coated slides for H&E staining. Number of microvessels in per unit area (/mm2) was counted to assess the microvascular density (MVD) under a light microscope (×200 magnification; Olympus Corp., Tokyo, Japan).
Six sections of the central part of area II in each group were deparaffinized in xylene and then rehydrated in a graded ethanol series. After washing, 3% hydrogen peroxide solution was added to the sections to block endogenous peroxidase. Then, 10.2 mM sodium citrate buﬀer (pH 6.0) was used for antigen retrieval (20 min, 95°C). After blocking with 10% (w/v) bovine serum albumin for 1 hour, the sections were incubated with primary antibodies: CD34 (1:100), VEGF (1:200), cadherin 5 (1:200), CASP3 (1:200), and SOD1 (1:100) overnight at 4°C. After washing, the sections were further incubated with HRP-conjugated second antibody (1:1000), stained with a DAB detection kit, and counterstained using hematoxylin. Slides were imaged at 200× magnification using the DP2-TWAN image-acquisition system (Olympus). Integral absorbance of VEGF-, cadherin 5-, CASP3-, SOD1- and CD34-positive blood vessels was calculated using Image-Pro Plus software (Media Cybernetics). Six random fields in three random sections of each tissue sample was counted.
After the mice euthanasia, skin samples (0.5 × 0.5 cm) from the middle of area II ﬂaps (n = 6) in each group were harvested and stored at −80°C for western blotting analysis. After extracting the ﬂap tissues with lysis buﬀer, the protein concentration was measured using the BCA assay. An equal amount of 60 µg protein was separated by 10% (w/v) gel electrophoresis and transferred to polyvinylidene diﬂuoride membranes (Roche Applied Science, Indianapolis, IN, USA). After blocking with 5% (w/v) nonfat milk for 2 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight: VEGF (1:1000), MMP-9 (1:1000), cadherin 5 (1:1000), HO-1 (1:1000), eNOS (1:1000), SOD1 (1:1000), Bax (1:1000), CYC (1:1000), caspase 3 (1:1000), and GAPDH (1:1000). After washing, the membranes were incubated with HRP-conjugated IgG secondary antibody (1:5000) for 2 h at room temperature. The bands on the membranes were visualized using the ECL Plus Reagent Kit. Band intensity was quantified using Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA).
Statistical analyses were performed using GraphPad Prism (Version 8.0.1, CA, USA). Data were presented as mean ± standard errors of the means (SEM). Statistical differences in comparison to the control group were analyzed using unpaired student t-tests. Significance was considered with p value < 0.05.