Graphical representation of the methods of the study is shown as Figure 1.
Animals
Adult SPF Long-Evans rats of both sexes, weighing 400-650 g and aging 5 months (± 1 month), were used in the experiment. Common carotid artery diameter ranged between 0.7-1.2 mm and the vessel was of sufficient length, without branches.
The rats were housed individually throughout the experiment and received a standard of care according to EU directive 2010/63/EU, including a 12/12 light schedule and free access to pelleted food (Sniff, Germany) and water.
The experiment was preceded by a pilot study focused on practicing surgical techniques and optimizing the methodology.
In the main study, the animals were randomly divided into two groups based on the type of suturing technique used. Group 1, consisting of 19 animals, underwent an anastomosis using the interrupted suture technique (Table 1). Group 2, consisting of 13 animals, underwent an anastomosis using the CRS technique (Table 2). Randomization via a coin toss determined which CCA was operated and which was used as a control.
Table 1
Interrupted suture technique group
| | First surgery | Second surgery |
| | Blood flow [ml/min] | | | Blood flow [ml/min] | |
Animal number | Gender | Intact left CCA | Intact right CCA | Anastomosis | Control | Anastomosis blood flow [%] | Suture time [min] | Blood loss [g] | Anastomosis | Control | Anastomosis blood flow [%] |
1 | M | 6.1 | 7.0 | 3.3 | 4.7 | 70.2 | 40 | 3.0 | 2.5 | 4.5 | 55.6 |
2 | M | 9.0 | 9.0 | 3.7 | 5.5 | 67.3 | 75 | 2.1 | 5.0 | 4.8 | 104.2 |
3 | F | 3.7 | 3.8 | 3.6 | 3.9 | 92.3 | 70 | 1.5 | 4.1 | 6.4 | 64.1 |
4 | F | 6.1 | 6.2 | 2.6 | 4.3 | 60.5 | 60 | 1.5 | 2.7 | 2.5 | 108.0 |
5 | M | 5.8 | 5.9 | 4.7 | 5.8 | 81.0 | 60 | 1.2 | 7.5 | 8.0 | 93.8 |
6 | M | 4.4 | 4.5 | 4.0 | 4.0 | 100.0 | 60 | 1.8 | 4.9 | 5.1 | 96.1 |
7 | M | 6.8 | 6.3 | 4.6 | 4.4 | 104.5 | 55 | 1.4 | 6.9 | 4.5 | 153.3 |
8 | M | 5.9 | 5.6 | 2.3 | 6.0 | 38.3 | 50 | 2.0 | 3.9 | 5.2 | 75.0 |
9 | F | 5.5 | 5.6 | 3.2 | 3.6 | 88.9 | 22 | 1.5 | 8.5 | 7.0 | 121.4 |
10 | F | 3.4 | 3.8 | 2.8 | 4.8 | 58.3 | 30 | 1.0 | 2.1 | 2.2 | 95.5 |
11 | F | 4.2 | 4.3 | 1.4 | 3.2 | 43.8 | 59 | 2.3 | 4.7 | 4.9 | 95.9 |
12 | F | 3.8 | 4.1 | 2.3 | 2.3 | 100.0 | 25 | 3.0 | 3.5 | 3.5 | 100.0 |
13 | F | 5.6 | 5.1 | 3.9 | 3.9 | 100.0 | 33 | 0.5 | 4.9 | 4.9 | 100.0 |
14 | F | 4.1 | 4.1 | 2.6 | 2.6 | 100.0 | 27 | 0.5 | 3.3 | 3.3 | 100.0 |
15 | F | 4.4 | 4.4 | 1.6 | 2.7 | 59.3 | 41 | 2.7 | 5.0 | 4.0 | 125.0 |
16 | F | 5.0 | 5.0 | 3.5 | 3.4 | 102.9 | 35 | 2.0 | 4.0 | 4.6 | 87.0 |
17 | F | 4.2 | 4.2 | 2.9 | 2.9 | 100.0 | 46 | 1.0 | 5.5 | 3.8 | 144.7 |
18 | M | 5.8 | 5.7 | 3.1 | 4.1 | 75.6 | 45 | 3.5 | 5.2 | 5.7 | 91.2 |
19 | F | 2.6 | 2.6 | 2.2 | 2.2 | 100.0 | 60 | 1.0 | 3.3 | 3.7 | 89.2 |
Table 2
Continuous running suture group
| | First surgery | Second surgery |
| | Blood flow [ml/min] | | | Blood flow [ml/min] | |
Animal number | Gender | Intact left CCA | Intact right CCA | Anastomosis | Control | Anastomosis blood flow [%] | Suture time [min] | Blood loss [g] | Anastomosis | Control | Anastomosis blood flow [%] |
1 | F | 4.1 | 4.2 | 3.3 | 2.8 | 117.9 | 22 | 1.0 | 2.6 | 2.3 | 113.0 |
2 | F | 4.7 | 4.7 | 2.8 | 3.7 | 75.7 | 40 | 1.0 | 3.3 | 3.5 | 94.3 |
3 | F | 2.8 | 2.8 | 2.9 | 3.0 | 96.7 | 35 | 0.5 | 4.8 | 4.8 | 100.0 |
4 | F | 5.0 | 4.8 | 2.8 | 2.7 | 103.7 | 20 | 4.0 | 6.4 | 5.9 | 108.5 |
5 | F | 3.9 | 3.9 | 3.2 | 3.2 | 100.0 | 25 | 0.5 | 3.1 | 3.1 | 100.0 |
6 | F | 4.7 | 4.7 | 3.1 | 6.1 | 50.8 | 30 | 0.5 | 5.7 | 4.2 | 135.7 |
7 | F | 5.7 | 5.4 | 2.6 | 5.1 | 51.0 | 25 | 1.0 | 3.3 | 5.8 | 56.9 |
8 | M | 4.9 | 4.9 | 4.6 | 4.8 | 95.8 | 35 | 0.0 | 3.2 | 3.5 | 91.4 |
9 | M | 5.5 | 5.5 | 5.3 | 6.0 | 88.3 | 23 | 0.5 | 6.1 | 7.0 | 87.1 |
10 | M | 3.6 | 3.6 | 3.4 | 4.0 | 85.0 | 25 | 0.0 | 4.6 | 3.9 | 117.9 |
11 | M | 3.2 | 3.2 | 2.8 | 2.9 | 96.6 | 35 | 2.0 | 4.3 | 4.7 | 91.5 |
12 | M | 5.3 | 5.2 | 2.7 | 3.6 | 75.0 | 30 | 1.0 | 6.2 | 5.3 | 117.0 |
13 | M | 5.2 | 5.3 | 2.8 | 3.7 | 75.7 | 35 | 1.0 | 3.7 | 4.5 | 82.2 |
At the beginning of the study, pilot experiments were conducted on the total of 24 animals to test various technical methodologies.
Only surviving animals with a patent anastomosis were included in the study.
Anaesthesia and analgesia
Surgery was performed under general anaesthesia. A freshly prepared anaesthetic mixture was in a syringe by mixing medetomidine (0.1 mg/kg; NarcoStart®, Produlab Pharma B.V., Netherlands), propofol (100 mg/kg; Propofol 2%, Fresenius Kabi, Germany), and nalbuphine (0.1 mg/kg; Nalbuphin Orpha, Orpha-Devel Handels und Vertriebs GmbH, Austria). Medetomidine and nalbuphine were diluted beforehand using sterile 0.9.% NaCl solution, in order to obtain working solutions of 0.1 mg/ml. The anaesthetized animal was placed on a tempered operating table and continually supplied with oxygen via a face mask. The pulse oximeter probe was attached to the hind paw or to the proximal part of the tail. Corneal reflexes, reactions to painful stimuli, pulse rate, oxygen saturation, depth and regularity of breathing were all continuously monitored. Animals were administered a reduced dose of anaesthetics (25% of the initial dose) every 40 minutes until the surgery was completed. The anaesthesia was then terminated by intramuscular application of atipamezole (0.5 mg/kg; NarcoStop®, Produlab Pharma B.V., Netherlands). The animals were then observed for the first 24 hours and received multimodal analgesia via tramadol (10 mg/kg) and carprofen (5 mg/kg) based on Zegre Cannon et al.[4] for the following 3 days. At the end of the second surgery (day 14), the animal was euthanized by an intracardial injection of potassium chloride under the general anaesthesia.
Experimental surgery
The entire surgery was performed using the surgical Zeiss OPMI CS NC-2 microscope (Zeiss Germany). Gentle microsurgical technique with high magnification was utilized.
In the supine position, a vertical straight-line midline incision over the neck was utilized to expose both carotid arteries in their maximal possible course (Figure 2). Structures were dissected along their anatomical margins to minimize bleeding and pain. Blood flow in both CCAs was measured with as little delay as possible.
In the first surgery the CCA blood flow was measured. Obtained values were recorded both in absolute number and in percentage of the blood flow of the contralateral intact CCA. The transit time flowmetry device – Transonic TS420 Flowmeter Module with the Transonic PR series TTF 1.5 mm probe (Transonic Systems Inc., USA), was used for blood flow measurement[5]. After the measurement, end-to-end anastomosis was performed. The artery chosen as surgical was clamped by a dual approximation clamp and cut transversally. After both artery stumps preparation, they were sutured together using Ethicon Ethilon 10-0 suture with a 3.8 mm needle.
In the interrupted suture group, Carrel’s triangulation technique was employed[6]. It was necessary to use 14 – 18 stitches depending on the size of the vessel to minimize the anastomosis leaking (Figure 3A). In the CRS group, two sutures were used, originating at the 3rd and 9th hour positions respectively. After suturing the back wall of the vessel, the end of the first suture was tied to the first knot of the second suture. The front wall anastomosis was completed with the second suture, which was tied to the first knot of the first suture (Figure 3B).
After finishing the anastomosis, clamps were removed. In a case of significant anastomosis bleeding, additional stitches were added. Based on the findings of the pilot study, the rats tolerated very little blood loss. Blood loss of more than 3 ml (circa 12% of animal’s blood volume) caused significant hemodynamic instability. Blood loss of more than 5 ml (circa 20% of animal’s blood volume) was shown to be lethal. Anastomosis leaks were the only significant causes of bleeding during the surgery.
After the clip removal, blood flow measurements were performed in the same manner as before the anastomosis execution. The wound was closed in two layers afterwards. Blood loss during the surgery was determined by the weight of used cottonoids.
A second surgery was performed 14 days later, in order to measure blood flow of both CCAs. Obtained values were recorded in the same manner as during the first surgery. The anastomosis was then excised for histological examination and the animal was euthanized by an intracardial injection of potassium chloride under the general anaesthesia.
Histological evaluation
For the histological examination, the samples were submerged in Tissue Freezing Medium (Leica, Germany) and frozen at 80°C. Afterwards, the samples were unfrozen and fixed by formalin, dehydrated and embedded in paraffin blocks. The blocks were cut to 5 µm thick histological sections at the site of anastomosis as well as a more remote site. The sections were stained with haematoxylin-eosin, Verhoeff’s haematoxylin and green trichrome to visualize connective tissue. Picrosirius red staining (Direct Red 80, Sigma Aldrich, Munich, Germany) was used to visualize type I and III collagen, using circularly polarized light (Figure 4). Orcein staining was used to visualize elastin. The sections were also processed immunohistochemically with anti-smooth a muscle actin antibody (dilution 1:1000, clone 1A4, Agilent Technologies, US at 4°C, overnight) to visualize the smooth muscle cells (SMC) and counterstained with Gill’s haematoxylin.
Data evaluation
Student’s t-test for paired samples was used to compare blood flow through intact carotid arteries at the beginning of the experiment. Normality test (Kolmogorov-Smirnov test) showed normal distribution in the interrupted suture group and abnormal distribution in the CRS group. Blood flow change between the first and the second surgery was analysed by t-test for the interrupted suture group and by Wilcoxon test in the CRS group. StatSoft STATISTICA software was used for statistical analysis.
Ethics Approval
The study was approved by the Animal Welfare Advisory Committee at the Faculty of Medicine in Pilsen and by the Ministry of Education, Youth and Sports of the Czech Republic
(approval ID: MSMT-10669/2016-3 and MSMT-33242/2018-5).
All experiments were performed in accordance with relevant guidelines and regulations.
The reporting in the manuscript follows the recommendations in the ARRIVE guidelines.