We established an external jugular vein bypass grafting model using rabbit carotid arteries and conducted vascular biomechanical experiments (pressurisation and stretching).
The establishment of rabbit animal model was carried out in the standardized animal medicine laboratory, and the animal experiment center of Hebei university Medical college was in charge of the rabbit feeding after the modelling. All experiments were carried out under general anesthesia in rabbits. Finally, the rabbits were killed by injecting air into the ear margin under general anesthesia condition. (the principle of euthanasia).
Our animal experiment scheme used in the research was reviewed and approved by the Animal Welfare and Ethical Committee of Hebei University(AWEC), and conformed to the principles of animal protection, animal welfare and ethics, as well as the relevant national regulations for experimental animal welfare ethics. Animal use permit: SYXK-2017-002. Approval Number: 2017011.
Male rabbits (n = 24; 3.0 ± 0.5 kg) were obtained from the Laboratory Animal Center of the Medical School, Hebei University and randomly divided into two groups: experimental and control (n = 12 per group). Rabbits had free access to water. Food was prohibited for 12 h before surgery.
Experimental instruments, equipment, and reagents
A myograph system was provided by Professor Huo Yunlong’s team, Beijing University College of Engineering. Other equipment included: a vertical oxygen cylinder (Huandong), rotameter (Zhenxing), thermostatic water bath (Taisite), small peristaltic pump (Shenchen), mercury sphygmomanometer (Yuyue), a computer connected to a stereomicroscope (Nikon) and camera (Canon). In addition chemicals for preparation of HEPES-PSS solution were from Damao Chemical Reagent Factory, α-actin monoclonal antibody was from US Dako, StreptAvidin-Biotin Complex (SABC) Staining Kits from Boster, experimental surgical instruments for small animals from Function Laboratory of the Medical School, Hebei University, and 5-0 prolene sutures and a medical micro needle holder were from Suzhou Xiehe Medical Equipment Factory.
Establishment of a rabbit venous arterialisation model (carotid artery, external jugular vein bypass grafting)
Rabbits in the experimental group received general anaesthesia and heparinisation (ethyl carbamate 4 ml/kg, 1% heparin 2 ml/kg) injected into a marginal ear vein, and were then fixed on a sterile operating table for small animals with the part of the neck requiring surgery fully exposed. Following skin preparation and standard disinfection, a 7–8 cm incision was made at the midpoint of the anterior region of the neck, and the subcutaneous tissue, fat layer, and muscle layer blunt dissected to expose 5–6 cm of the right carotid artery and the right external jugular vein. Next, lateral vascular anastomosis of the carotid artery and external jugular vein was conducted under a 2.5× portable magnifying glass, using 5-0 prolene sutures (taking 5 mm as the standard anastomotic length). The external jugular vein graft was ligated to the two stoma at both ends and the carotid artery ligated in the middle of the stoma with 6-0 sutures, which allowed the blood from the proximal artery to flow into the distal end via the vein conduit. After confirmation that blood flow was smooth and that there was no bleeding around the stoma, the surgical incision was closed, layer by layer. After regaining consciousness, rabbits were transferred to the Laboratory Animal Feeding Center of the Medical School, Hebei University. Three days after surgery, rabbits were administered penicillin injections, to prevent infection, and underwent small animal ultrasound, to monitor blood flow patency.
Collection of blood vessels
Four weeks after surgery, the arterialised venous conduit was excised and divided into three segments. Segment 1, a 5 mm vascular ring, was fixed in preservation fluid for haematoxylin and eosin (HE) staining（10% Formaldehyde Solution，at room temperature）. Segment 2, also 5 mm, was fixed and stored(preserve liquid as before), for immunohistochemical analysis. Finally, the third segment (approximately 3 cm long) was immediately stored in perseveration solution once excised for subsequent mechanical testing(0.9% Normal Saline). In the control group, 3 cm segments of carotid arteries and external jugular veins were excised from untreated rabbits, then HE staining, immunohistochemical analysis, and mechanical tests conducted in the same way as described for experimental animals.
The specimens were embedded in paraffin and sectioned into sections with a thickness of 5μm. The specimens were routinely dewaxed to water.Diluted with a-actin .(1:200 dilution USA Dako company )Conventional SABC staining was finished for monoclonal antibody PCNA (1:100 dilution USA Dako company), and hematoxylin restaining.Four high power fields of view for each specimen were randomly chosen for observation of the number of proliferating cell nuclear antigen (PCNA)-positive cells and corresponding total cells and the cell proliferation index calculated as follows:
Cell proliferation index = PCNA-positive cell number/total cell number × 100%
Blood vessel pressurisation and mechanical stretch testing
The myograph was configured with its two needles inserted into blood vessel fragments at each end and 4-0 sutures used for ligation. The position and distance of the needles were adjusted to maintain the vessel in the horizontal plane, with no stretching, overlapping, or twisting, and the air remaining in the system expelled using the principle of fluid dynamics. First, the active mechanical properties of the blood vessel were measured. Calcium and potassium ions can both cause vasoconstriction. Calcium ions combine with receptor proteins inside the cytosol, causing vasoconstriction and, when potassium ion concentration outside the cell increases, this may also cause vasoconstriction, due to the activity of voltage-sensitive calcium channels. Therefore, to measure their active vascular mechanical properties, vessels were bathed in HEPES-PSS solution containing a high concentration of potassium ions, while measurement of passive vascular mechanical properties was conducted using HEPES-PSS solution without calcium for stimulation, pressurisation, and mechanical stretching tests; detailed HEPES-PSS solution compositions are provided in Tables 1 and 2 [30–31].
Each blood vessel was fully immersed in high potassium HEPES-PSS solution, the pressure adjusted to 0 kPa (20 mmHg = 2.66 kPa), and the background set using the computer attached to the stereomicroscope and Canon camera. The initial length of the blood vessel (without stretching or relaxation) was first marked and recorded. Then, blood vessels were stretched to 1.3 times their initial length by adjusting the control arms of the two myograph needles and the rotameter applied to increase the pressure within the system to 23.94 kPa, followed by a decrease back to 0 kPa, while all the other experimental parameters were maintained. The pressurisation and depressurisation procedure was repeated 10 times, to minimise internal stress. Next, mechanical tests were initiated.
The internal pressure of the system was adjusted to 2.66 kPa using the oxygen cylinder, tube, and rotameter. Blood vessel diameters were measured 5 min after stabilisation (monitored by computer). After an initial data recording, the pressure was further raised to 5.32 kPa and the vessel diameter change noted. Then, pressure was raised stepwise by 2.55 kPa each time, and changes in the vessel recorded following each increase in pressure, to a maximum of 26.60 kPa, when a depressurisation procedure was initiated. Starting at 26.60 kPa, pressure was decreased stepwise by 2.66 kPa per step, with vessel diameter recorded after each step, until the internal pressure of the system returned to 0 kPa.
On completion of the 1.3-stretch-ratio pressurisation and stretch test, the internal system pressure reduced to 0 kPa and the blood vessel was allowed to rest for 15 min to fully release the remaining internal stress. Next, the same method was applied to stretch the blood vessel to 1.4 times its original length. Again, 10 rounds of the pressurisation and depressurisation procedure were conducted and data recorded, as described above. When measurements were complete, the chamber containing high-potassium HEPES-PSS was thoroughly cleaned before replacing the solution with calcium-free HEPES-PSS.
Blood vessels were completely rested in calcium-free HEPES-PSS solution and, when they were fully diastolic, the same method that was applied to measure active mechanical properties (described above) was used to evaluate passive mechanical properties. After all procedures were completed, a 3 mm long vascular ring was cut from the remaining blood vessel and incubated without manipulation in calcium-free HEPES-PSS solution for 30 min to eliminate internal stress. Subsequently, the thickness, size, and stretch angle after full extension of the vessel were determined.
1) HE staining outcomes and the immunohistochemical results from arterial and venous vessels. 2) Outer diameter changes in rabbit blood vessels in response to changes in pressure in the experimental and control groups.
All data were statistically analysed using SPSS 25.0 statistical software. Measurement data are presented as mean ± standard deviation. The significance of differences between groups were evaluated using the t-test, with a significance level of 0.05.