Comparison of the Microvascular Anastomosis Maturation in Continuous and Interrupted Suture Technique

Jiri Dostal Department of Neurosurgery, University Hospital and Faculty of Medicine in Pilsen, Charles University, Czech Republic Pavel Klein Biomedical Centre, Faculty of Medicine in Pilsen, Charles University, Czech Republic Tereza Blassova Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University, Czech Republic Vladimir Priban (  pribanv@fnplzen.cz ) Department of Neurosurgery, University Hospital and Faculty of Medicine in Pilsen, Charles University, Czech Republic


Introduction
Precise surgical technique is the key in creating a functional microvascular anastomosis. In order to achieve maximal possible blood ow, the anastomosis site must be able to expand over time. Mechanical factors leading to anastomosis maturation are arterial wall pulsatility and direct effect of blood pressure on the site of anastomosis. Use of interrupted sutures is the widely accepted standard technique. Presumed advantages are the ability of maturation and simple repair of technical errors. Usage of a continuous running suture (CRS) is less common. Presumed advantages are time e ciency [1] and bleeding reduction at the site of the anastomosis. Shorter surgical time is an important factor in the prevention of tissue ischemia [2]. However, it is unclear whether the continuous character of a running suture might negatively affect anastomosis elasticity, thus leading to restriction of maturation of the anastomosis. In our study, maturation of the microvascular anastomosis was monitored in a rat model. An end-to-end anastomosis of the rat common carotid artery (CCA) acts as a suitable laboratory microanastomosis model [3].
In order to quantitatively assess blood ow changes in time, transit time owmetry (TTF) measurement was used concurrently with histological examination of the arterial wall. The aim of this study was to determine whether both interrupted suture and CRS technique provide a suitable environment for microanastomosis maturation.
To authors' knowledge, no similar study using exact quantitative methods in measurement of delayed maturation of microanastomosis has been published.

Materials And Methods
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 su cient 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.
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.  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 re exes, 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 rst 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 magni cation 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 ow in both CCAs was measured with as little delay as possible.
In the rst surgery the CCA blood ow was measured. Obtained values were recorded both in absolute number and in percentage of the blood ow of the contralateral intact CCA. The transit time owmetry device -Transonic TS420 Flowmeter Module with the Transonic PR series TTF 1.5 mm probe (Transonic Systems Inc., USA), was used for blood ow 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 rst suture was tied to the rst knot of the second suture. The front wall anastomosis was completed with the second suture, which was tied to the rst knot of the rst suture ( Figure 3B).
After nishing the anastomosis, clamps were removed. In a case of signi cant anastomosis bleeding, additional stitches were added. Based on the ndings 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 signi cant 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 signi cant causes of bleeding during the surgery.
After the clip removal, blood ow 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. All experiments were performed in accordance with relevant guidelines and regulations.
The reporting in the manuscript follows the recommendations in the ARRIVE guidelines.

Results
The blood ow in both CCAs at the beginning of the experiment was not statistically different in all animals. The average blood ows in the right and left CCA were 4.844 ± 1.28 ml and 4.856 ± 1.26 ml respectively, and the hypothesis that ow rates are different was rejected (p =.004). The intact CCA was therefore used as a control for blood ow of the sutured CCA.
In the interrupted suture group, the anastomosis blood ow values immediately after completion of the suture ranged from 38.3-104.5% of the control CCA blood ow, with a median of 88.9% and mean average of 81.2%. The anastomosis blood ow was restricted (p =.002). Surgery time ranged from 22 to 70 minutes, with a median of 46 minutes and a mean average of 47 minutes. The length of surgery did not corelate with anastomosis ow restriction. After two weeks, the anastomosis blood ow ranged from 55.6-144.7% of the control CCA blood ow, with a median of 96.1%, and a mean average of 100.0%. The blood ow increase was present, but was statistically insigni cant (p=.073). Histological examination did not show any thrombosis within the artery lumen. The inner vessel diameter (ID) at the site of the anastomosis was approximately the same as the ID of the same vessel outside the anastomosis site. Histological ndings suggested that scar formation was complete in all cases ( Figure 4).
In the continuous running suture anastomosis group, the anastomosis blood ow values immediately after completion of the suture ranged from 50.8-117.9% of the control CCA blood ow, with a median of 88.3% and a mean average of 85.5%. The anastomosis blood ow was restricted (p =.025). Surgery time ranged from 22 to 40 minutes, with a median of 30 minutes and a mean average 29.2 minutes. The length of the surgery did not corelate with anastomosis ow restriction. After two weeks, the anastomosis blood ow ranged from 56.9-135.7% of the control CCA blood ow, with a median of 100.0% and mean average of 99.7%. The blood ow increase was statistically signi cant (p=.011). Histological examination did not show any thrombosis within the artery lumen. The inner vessel diameter (ID) at the site of the anastomosis was approximately the same as the ID of the same vessel outside the anastomosis site. Histological ndings suggested that scar formation was complete in all cases.
The amount of blood loss did not affect surgical time nor blood ow through the anastomosis.
In both groups, no vasospasms were noted during the surgeries. The time required to create a fully functional anastomosis was signi cantly shorter in the case of CRS, compared to the interrupted suture technique (p <.001), with an average 34,8% time reduction in CRS cases. The anastomosis maturation rate after two weeks was almost identical in both groups ( Figure 5).

Discussion
Brain revascularization surgeries, replantations in cases of limb loss resulting injuries or free ap procedures in reconstructive surgery all carry a risk of ischemic tissue damage. Time is therefore a limiting factor in the outcome of these surgeries [2]. which is supported by our study, as the patency rate after 2 weeks was 100% for both interrupted and CRS group. No maturation restriction in the CRS group was noted in our study, compared to the interrupted suture group. Both groups showed a similar blood ow increase pattern in the course of two weeks. CRS microanastomosis maturation is probably enabled by elasticity of the vessel wall and a su cient abundance of the suture length.
The main disadvantage of CRS is that management of error repairs is more di cult and time-consuming. This disadvantage can potentially negate the time saved using the CRS technique.
Cardiac output, blood viscosity and vessel size contribute to immediate blood ow through a vessel. In our study, both CCAs showed no difference in the blood ow before performing the anastomosis. The ability to compare blood ow through the intact CCA to the operated CCA with no time delay eliminates the need to consider circulatory parameters. Obtained anastomosis blood ow values were also expressed in percentage of the intact vessel blood ow value, thus direct comparison between immediate and delayed (after 2 weeks) anastomosis blood ow was possible.
Circulatory parameters during both of the surgeries can therefore be disregarded.
The scar at the site of the anastomosis was considered mature at the time of the second surgery, two weeks after performing the anastomosis.
Collagen types were histologically assessed in the anastomosis scar. Presence of type I collagen was consistent with nal stages of scar healing ( Figure 4), suggesting 14 days interval between the surgeries was su cient.
There was no intraluminal thrombosis in the anastomosis site. No specimens with low degree of maturation of the anastomosis after 14 days were associated with abnormal histological nding.
End-to-side microvascular anastomosis is usually used in neurosurgery when performing extra-intracranial bypass. End-to-side CCA (or ICA) to CCA microanastomosis carried high mortality in the pilot experiment preceding this study. In surviving animals, the need of transsection of the sternohyoid muscle needed for such anastomosis was too invasive and required high doses of analgesics. End-to-end microanastomosis was chosen for the experiment, because it provided the opportunity to perform delayed measurement and to precisely compare blood ow with the intact contralateral CCA. The authors are con dent, that the end-to-end anastomosis model provides su cient insight into the mechanical properties of microvascular anastomosis.

Limitations
The rat did tolerate only a very small blood loss. Any blood leakage from the anastomosis site had to be repaired by additional interrupted sutures.
In order to minimize the blood loss and to minimize the risk of restricting the blood ow of the anastomosis by these stitches, the vessel had to be clamped for the whole time of the repair. This resulted in long operating times and larger time variance.

Conclusions
In our study, both CRS and the interrupted suture technique allowed microanastomosis maturation in the course of two weeks. Maturation rate in both groups was comparable. The resultant time e ciency of CRS was signi cant and might potentially be important in a clinical setting.