2.1 Chemicals
ISL (CAS no. 5041-81-6, purity ≥ 98%) was purchased from Pufei De Bio Co.(Chengdu, China), its chemical structure is shown in Fig. 1. Dimethyl sulfoxide(DMSO), tricaine (ethyl 3-aminobenzoate methane- sulfonate) and hematoxylin and eosin (H&E) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trizols Reagent was purchased from Invitrogen (Carlsbad, CA, USA).
2.2 Animals and group assignments
Adult Sprague-Dawley (SD) female rats (weight 210–240 g) were purchased from the Experimental Animal Center of Suzhou University. Rats were housed in the single cage, fed with standard rat chow at an appropriate temperature (23–25 °C).
In total, 80 rats were randomly divided into ISL group (n = 40) and normal saline group (n = 40). All surgical procedures and protocols in this study were approved by the Animal Ethics Committee of Suzhou University. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals described by the National Research Council Drug Administration.
2.3 Operative technique
All rats were anesthetized with 2% pentobarbital sodium (40 mg/kg, intraperitoneal injection). After anesthesia, rats were fixed in the prone position on the sterile operating table, hair was removed using a pet electric shaver, and rats were deiodinated with alcohol after iodophor disinfection. The dorsal flap was designed according to Miyamoto [11]. To be specific, a trivascular perforator flap based on a perforating artery of deep iliac circumflex artery was design on the left dorsal of the rat, including two choke zones. In addition, the flap borders were as follows: medial border, the midline of the dorsum; the cranial border, subscapular angle; caudal border, the spina iliaca posterior superior, and the dorsal flap measuring 10 × 2.5 cm was designed. Firstly, the skin was cut along the medial border of the flap, and the subcutaneous tissue was separated just from the meat layer. The flap was gradually lifted to separate trivascular pedicles. The arteriathoracodorsalis perforator and the intercostal artery perforator were ligated. Finally, the arteriae circumflexa ilium profunda perforator was preserved and the flaps were completely separated, followed by identification of Choke I and II zone (Fig. 2) and corresponding marking on the epidermis using a marker pen (Fig. 2). After complete hemostasis, the flap sutured back into its original position with 4 − 0 silk. Wounds were post-operatively coated with chlortetracycline ointment to prevent infection. The flap was resected by the same surgeon to ensure the uniform thickness of each flap and to reduce the factors that might affect flap survival due to the different thickness of the flap.
2.4 Dosage and administration of drug
Rats in the experimental group were intraperitoneally injected with ISL solution at a dose of 100,200 µg/kg two days before operation, two hours before operation and two days after operation according to the pre-experiment results. Rats in the control group were intraperitoneally injected with the same volume of saline solution accordingly.
2.5 Gross observation of flap and calculation of flap survival rate
All flaps were grossly observed and recorded 1, 3, 5, and 7 days after operation, including the flap color, tissue elasticity, texture, skin and hair growth, infection, necrosis, and etc. After post-operative observation for seven days,ten rats were anesthetized and high-quality photographs of flap were obtained using a digital camera and imported into Image-Pro Plus v6.0 software to calculate the percentage of flap survival area. The flap survival rate in each group was calculated according to the following formula: flap survival rate = flap survival area / total flap area x100%. The standard of flap necrosis was as follows: the flap color was black, the tissue was hard, shrunk, dry necrosis, inelastic, and no blood outflow when cutting.
2.6 Perforator flap angiography
On the 7th day after operation, 10 rats were randomly selected from the control and ISL groups (n = 10 each, 20 in total), and subjected to gelatin-lead oxide angiography of the flap vessels. (1) Preparation of the perfusion solution: The preparation ratio of perfusion solution was: industrial gelatin 1 g, warm water 20 ml 40 ℃, lead oxide 20 g. First, the gelatin was dissolved in warm water for 3–4 h. After the gelatin was completely dissolved, lead oxide was added and stirred. Because the gelatin-lead oxide mixture was prone to solidify under low temperature, the entire procedure must be carried out in water bath at 40 ℃. The amount of perfusion depended on the size of rats, generally ranging from 40–50 ml / kg, not exceeding 50 ml / kg. (2) Perfusion process: rats were fixed in a supine position after anesthesia, and unilateral carotid artery was bluntly separated. Afterwards, No.24 indwelling catheter was inserted into the carotid artery, the catheter was ligated with silk thread fir fixation. The blood from was completely drained from the rat, rinsed with heparin saline. When the effluent was clear, the prepared gelatin-lead oxide mixture was slowly and uniformly injected into the carotid artery until the sclera and the distal limbs of the rat showed dot-like or patchy orange-red. Afterwards, rats were placed in a supine position in the refrigerator at 4 ℃ for about 24 h for gelatin solidification. (3) X-ray radiography: 24 hours after perfusion, the entire back skin of the rat was peeled off and photographed under X-ray camera (40kv, 50 mA and 100 ms exposure time). (4) Image analysis: The PACS system of our hospital was used to visualize and assess the blood vessels in Choke II zone of the flaps in rats from both groups.
2.7 Histological examination of microvessel density (MVD) in the Choke II zone of the flap
Seven days after operation, six rats from each group were anesthetized according to the previously described procedure. The flap tissue (2.0 cm × 0.5 cm in size) of Choke II zone was obtained, fixed in 40 g/L paraformaldehyde, routinely dehydrated, sliced into 5 µm-thick sections and subjected to HE staining. Afterwards, the staining results of the tissue sections were observed under the microscope. As a result, the cytoplasm was stained as pink by eosin, and the nuclei were stained as blue by hematoxylin. Firstly, the dense area of microvessels was found under 4 × 10 low magnification. Afterwards, five fields of view were randomly selected under 10 × 10 magnification microscope, and the number of blood vessels was counted manually for average to calculate the number of microvessels per unit area (number/mm2), which was used an indicator for MVD.
2.8 Immunohistochemistry (IHC) for VEGF expression in Choke II zone of the flap
The sections prepared in Sect. 2.7 (one for each rat) were routinely dewaxed, dehydrated, microwaved for antigen retrieval, incubated with 3% (volume fraction) peroxide at 37 ° C in water bath for 10 min to inactivate endogenous peroxidase, blocked with 5% (volume fraction) goat serum at 37 ° C for 30 min. The sections were subsequently incubated with anti-rat VEGF primary antibody (dilution 1:200) at 4 ° C overnight, reacted with HRP-conjugated goat anti-rat IgG secondary antibody at 37 ° C for 1 h, and incubated with HRP-conjugated streptomycin avidin at 37 ° C for 45 min. Afterwards, the sections were visualized by DAB, counterstained with hematoxylin, dehydrated with ethanol, transparented with xylene, sealed with neutral resin. The distribution of VEGF (brown yellow particles) in Choke II zone of the flap was observed under light microscope at 400 magnification and photographed under five randomly selected fields of view. Images were imported into Image Pro Plus 6.0 software to detect VEGF expression and the results were shown as integral absorbance values.
2.9 Western blot for VEGF expression in Choke II zone of the flap
Seven days after operation, total tissue protein was extracted from the Choke II zone of the flap from ten rats in each group. After adjusting protein concentration, equal amount protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to membranes and blocked with 50 g / L skim milk solution at room temperature for 2 h. The membranes were incubated with mouse anti-rat VEGF primary antibody (dilution 1:300), rabbit anti-mouse GAPDH primary antibody (dilution 1:500) overnight at 4 °C, followed by incubation with HRP-conjugated goat anti-mouse IgG secondary antibody (dilution 1:4000) or HRP-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:5000) at room temperature for 2 h. Afterwards, the membranes were visualized using chemiluminescence, and grayscale scanning analysis was performed using gel image analysis system. The band intensity of VEGF was quantified and presented relative to GADPH (internal control). This assay was performed in triplicate.