vagus Cryosurgical Method for Nervus Vagus Ablation in Experimental Studies

: When studying the impact of nervous system on physiological and pathophysiological processes, a need arises to de- termine the contribution of each division of autonomic nervous system. For the nervus vagus ablation in cervical spine, chest and abdominal cavities the cryosurgical method has been designed to assess the effect of parasympathetic division on heart rate va- riability. There has been shown the possibility to implement the n. vagus ablation, as well as the arteria carotis cryodenervation using cryosurgical technique in the ‘carotid’ triangle area of the neck, and thereby to ‘switch off’ the vascular intima-located baroreceptors from a regulatory link. The use of endoscopic device and cryosurgical technique for vascular denervation was established to enable the aortic arch baroreceptor ablation while preserving an anatomical integrity of the vessel. Since the isolation procedure for the n. vagus abdominal branches in rat along the esophageal lateral edges is relatively accessible, it allows performing the cryodenervation of individual and both nerve branches along with esophagus.


Материалы и методы
Эксперименты на животных проводили в соответствии с Законом Украины «О защите жи-adaptation to constantly changing environmental conditions. It is known that the ANS is autonomous, consisting of sympathetic and parasympathetic branch, which participate in regulation of body's systems and have not only an antagonistic, but synergistic direction as well [7]. The parasympathetic division of ANS is represented by a pair of vagus nerves (n. vagus is the tenth cranial nerve pair), which are anatomically divided into the head, neck, thorax and abdomen compartments [13,14].
In order to determine the contribution of each ANS division into regulation of body's vital functions, the physiologists use different approaches such as: nerve blocking or stimulation [12]. The fi rst approach consists in the dissection of nerves and/or their branches. In the experiments, this procedure may be technically diffi cult and, in some cases, even impossible. There is, in particular, the nerve isolation from neurovascular bundle or arterial vessel denervation in order to 'switch off' the baroreceptors, located in vessel wall. When performing such a procedure in rat aorta, the signals coming to the ANS central parts are not totally blocked, that is explained by the branching of nerve fi bers coming from baroreceptors to the n. vagus main trunk [11]. A total blockade of signals from aortic baroreceptors is decisive when studying the mechanisms of heart rate regulation. Therefore, to study the heart rate variability (HRV) and to interpret correctly the frequency ranges of HRV spectral analysis, it is necessary to design the breakthrough technologies for baroreceptor 'switching off' [3].
In this aspect, one of the most effi cient ways is cryosurgical. In contrast to mechanical dissection of nerve fi bers, it destroys the tissue while preserving the elastic-collagen scaffold of organ (vessel, nerve) [1,5]. After vessel cryoablation, the intima cells, muscle and adventitious layers are completely destroyed [5]. Of note is a reversible character of post cryoablation lesions. The nerve tissues may restore within a few months [2,10].
The research aim was to design the denervation methods for n. vagus peripheral compartments using cryosurgical way.
The work was performed in 7-month-old Wistar male rats (n = 30), weighing 200-300 g under usual animal housing in the morning (from 9 to 12 a. m.).
The surgery was done with inhalation anesthesia. For additional anesthesia, Nalbuphine (Zdorovye, Ukraine) at a dose of 1 mg / kg was once administered, intramuscularly.
For n. vagus cryoablation we used the selfcontained dermatological cryogenic instrument KD-3 (B. Verkin Institute for Low Temperature Physics and Engineering of National Academy of Sciences of Ukraine, Kharkiv) [8,16] and the specially manufactured 1/3 open circle-shaped copper applicator with operating surface temperature of 120°C. Total time of manipulation was 60 s [5].
The surgeries in thoracic cavity were done using the endoscopic device, designed at the Department of Experimental Cryomedicine of the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine [4,6].
After animal sacrifi cing, the fragment of aortic endothelium at its arch level was morphologically analyzed. The semi-thin sections were obtained with UMTP-7 ultramicrotome (Elektron, Ukraine), stained with the methylene blue and basic fuchsinbased polychrome dye, then analysed with Granum R 4003 microscope (Granum, China), equipped with Panasonic WV-CP 470 digital video camera (Panasonic, Japan). The ultrathin sections were contrasted with a saturated aqueous solution of uranyl acetate and lead citrate solution according to Reynolds. To make the preparations, the aortic fragments after prefi xation and adventitia separation were impregnated with silver nitrate according to the standard method [9]. Aortic preparations were embedded into a glycerol-gelatin mixture and studied in transmitted light with light trinocular microscope Granum R 4003 (Granum) using DCM 510 camera (eTrek, China).
The n. vagus was isolated from the neurovascular bundle and its local cryoablation was carried-out ( Fig. 1B) without damaging the surrounding tissues. After careful hemostasis, the surgical wound was sutured in layers.
После криоденервации нервные волокна адвентициальной оболочки претерпели значитель-the signals along the glossopharyngeal nerve to the medulla oblongata, where the sympathetic ANS centers are situated.

Cryoablation technique for thoracic n. vagus.
When designing the technique for thoracic n. vagus cryoablation the aortic arch was cryodenervated to 'switch off' the aortic arch baroreceptors. The left vagus nerve, when entering the chest cavity, is known to be located on an anterior surface of the aortic arch, and the right one is found on an anterior surface of the initial section of the right subclavian artery [11].
After animal anesthetizing and fi xing to the operating table, a skin was incised under aseptic conditions in the area of the right and left II intercostal spaces. The pectoralis major and minor were separated apart.
The endoscopic method was used for precise verifi cation of the ascending aorta and aortic arch [4,6]. The 0° rhinoscope with 2.7 mm diameter (MGB, Germany) was inserted through a trocar (3.0 mm diameter) into the II intercostal space on the left. Two provisional W-shaped sutures were preliminarily laid with capturing the ribs II and III and suture fi xation. This technique helps to close wound rapidly and to prevent an open pneumothorax development after cryoablation completing, since the surgery was carried-out under spontaneous breathing with no apparatus for artifi cial lung ventilation.
After the clamp and trocar removing, the KD-3 cryoinstrument was introduced. A right-sided thoracotomy in the 2 nd intercostal space ensures an optimal access to aortic arch. Using a special tip as a cryoapplicator we can grasp an aortic arch and perform a total vessel cryoablation until the blood fl ow stops (Fig. 2B).
After cryoexposure and instrument removal the aortic site was thawed for 5-7 s and blood fl ow in aorta was restored. The rhinoscope was then removed from thoracic cavity. After hemostasis, the W-shaped sutures on both sides were tightened and the post-surgery wound was sutured in layers.
Light microscopy of semi-thin sections of the aorta in the norm showed the structure of all the layers in the studied vessel to be preserved. A sharp distinction by layers was seen (Fig. 3A).
An adventitial membrane was represented by collagen fi bers, fi broblasts and fat cells, as well as the nerve fi ber bundles, running along the vessel and crossing each other. No endothelial cells were detected in semi-thin sections. The inner elastic membrane was distinct, its connection with the muscle layer was preserved.
To verify the presence or absence of vessel endothelium after aorta cryoablation, the intima morphology was studied using the silver impregnation of histological preparations. This method enables determining the structure of cells and tissues, since they have different capability to retain or restore the silver salts.
After cryoablation, a structural organization of aortic endothelium, as the most cryolabile tissue, underwent signifi cant changes, entailing its desquamation. The preparations revealed the extensive sites of endothelium desquamation (Fig. 4B).
Previous fi ndings in arteria hepatica [5], as well as our own fi ndings showed the temperature of -120°С and 60 s exposure to provide the cryoablation of both vascular intima and adventitia.

Cryoablation technique for abdominal n. vagus.
For animal anesthesia and fi xation we used the described above techniques. After hair coat removal, an anterior abdominal wall was opened under aseptic conditions. An abdominal portion of esophagus and stomach were brought into surgical wound. The n. vagus branches were clearly determined on both sides of cardiac portion of the esophagus (Fig. 5A). Further, using cryoinstrument, the cryoablation of n. vagus anterior branch was performed (Fig. 5B).
After careful hemostasis, an anterior abdominal wall was sutured in layers and the skin sutures were laid to the wound. In a post-surgery period, no complications, changes in behavior and food intake, neither cases of post-surgery mortality were recorded.
This approach is crucial in studying the physiological mechanisms of heart rate variability, because of its prospects for surgeries, involving the heart cryodenervation, i. e. branches of sympathetic and parasympathetic nerves, as well as right atrial sinus node.

Conclusions
Cryosurgical method was established to enable the n. vagus ablation in various anatomical areas in experimental animals and allows excluding the impact of ANS parasympathetic branch on heart rate variability.
Using the proposed surgical approach to animal chest cavity by means of endoscopic device and a cryosurgical way for vascular denervation, the aortic arch baroreceptors may be destroyed while preserving the anatomical integrity of the vessel.
A relative accessibility of isolation of the n. vagus abdominal branches along the lateral edges of rat esophagus enables the cryodenervation of individual and both nerve branches along with esophagus.