Animals and study design
This pilot study was conducted in three phases. The first phase served to familiarise with the anatomy of the abdominal wall in pig cadavers and to practise the ultrasound (US) localisation of the needle in the appropriate interfascial plane between the IOM or its aponeurosis and the TAM. The cadavers of pigs (n = 3) deceased for reasons unrelated to infectious diseases and kept in the cold room for several days before being used in the study were obtained from the Veterinary Hygiene Service.
Next, the distribution of a methylene blue (MB) solution in a three-point TAP technique was examined in freshly euthanized cadavers (phase II, n = 3) within one hour after euthanasia. The abdominal organs were removed from cadavers by midline laparatomy based on the requirements of the primary study [18] and the abdominal wall was sutured. Then the TAP study was performed.
Finally, the distribution of MB solution was studied using a four-point TAP technique performed in anaesthetized pigs 15 minutes prior to euthanasia (phase III, n = 4). After that, the abdominal organs were removed from cadavers by midline laparotomy for the primary study. The abdominal wall was sutured before anatomical dissection was performed to assess the spread of MB solution.
Phases II and III were approved by the National Ethics Committee and National Veterinary Administration (approval number U34401-2/2021/5, approval date 3.3.2021) and conducted in accordance with European Directive 2010/63/EU, ARRIVE 2.0 guidelines [19], and PREPARE guidelines for planning animal research and testing [20]. The TAP study was designed in compliance with the 3Rs principle (Replacement, Reduction, Refinement) in addition to the primary study investigating the effects and safety of electroporation with bleomycin on liver vessels [18]. Based on the requirements of the primary study, TAP injections in phases II and III were performed after and 15 minutes before euthanasia, respectively, and MB spread was assessed after abdominal organ explantation.
Pigs (Sus scrofa domesticus), six female and one castrated male Landrace and Large White hybrids, aged 10 weeks, were procured from a local farm free of swine fever, Aujeszky's disease, porcine respiratory and reproductive syndrome, and salmonellosis. They were vaccinated against mycoplasmosis 4–6 weeks before purchase. During a two-week acclimatization period, they were housed in three groups (two pairs and one group of three pigs). After surgery (primary study), they were housed in separate indoor straw-bedded pens that enabled visual, auditory, and limited tactile contact. Seven days later, they were returned to the initial groups. They were exposed to a natural light-dark cycle with an ambient temperature of 19 to 21 ºC. Besides the straw bedding, various methods of environmental enrichment, such as plastic balls with openings for treats and hiding fruit and mealworms in the straw, were used. The pigs were fed a commercial pig diet twice daily, and water was provided ad libitum from nipple waterers. They were under continuous video surveillance and clinically examined three times daily until the end of experiment.
Each pig was anaesthetized twice for the purpose of the primary study [18]. They were first anaesthetized for a median laparatomy for resection and electroporation of the portal vein and adjacent tissues. Four weeks later, when the TAP study was carried out, they were anaesthetized again and euthanized with T61 (MSD, Intervet International B.V., Kenilworth, NJ, USA) 3 mL/10 kg intravenously.
TAP block technique
The TAP injections were performed by two anaesthetists (JS and AS) skilled in performing TAP blocks in dogs but not in pigs. The pigs were positioned in dorsal recumbency with the hindquarters turned slightly to the left or right. Injection sites were determined using anatomical landmarks and ultrasonography. An US device (M9 GI, Mindray, Nanshan, Shenzhen, P. R. China) with a linear 3.0–13.0 MHz transducer, always placed with the indicator oriented dorsally, was used. Sterile 0.9% NaCl solution (B Braun, Melsungen, Germany) was used to facilitate acoustic coupling. An 8 cm long 22-gauge echogenic needle (Stimuplex Ultra, B Braun, Melsungen, Germany) was advanced in-plane from the ventral to dorsal direction at an angle of 20–30º relative to the skin. A 1% MB solution (1% Metilensko modrilo, UKC, Slovenia) at a dose of 1 mg/kg was diluted with 5% glucose solution (B Braun, Melsungen, Germany) to the calculated volume and used as a dye. A total of 1.8 mL/kg (0.3 mL/kg per injection point) MB solution was used for the three-point TAP technique in phase II, and a total of 1.6 mL/kg (0.2 mL/kg per injection point) MB solution was used for the four-point TAP technique in phase III. When the tip of the needle was visualized in the interfascial plane, 1 mL of the MB solution was injected as part of the total amount available to confirm correct positioning before the entire dose was administered. If the positioning was incorrect, the needle was repositioned, and the procedure was repeated.
Three-point TAP technique: Injection points
First injection point: An orientation line between the caudal end of the xyphoid process and the most caudal end of the last rib’s body was drawn and divided into thirds. The US transducer was placed perpendicular to the spine between the second and the last third of the orientation line, just below the costal arch. The TAM and IOM aponeurosis were identified, and MB solution was injected between their fasciae.
Second injection point: A line perpendicular to the spine was drawn through the most caudal end of the last rib’s body. The US transducer was placed on this line at the halfway between the linea alba and the spine and slowly pushed dorsally to find the point where the aponeurosis of the TAM transforms into its muscle part. The MB solution was injected interfascially between the TAM and the IOM aponeurosis just before it transforms into its muscle part.
Third injection point: A line parallel to the spine was drawn through the ischial tuberosity. Two lines perpendicular to the spine were drawn, with the first passing through the most caudal end of the last rib’s body and the second cranial to the coxal tuberosity. The transducer was placed halfway between the intersection of perpendicular lines and a line parallel to the spine. The TAM and IOM were identified, and MB solution was injected in the interfascial plane, approximately at the same dorsoventral level as the second injection point.
Four-point TAP technique: Injection points
The locations of the injection points for the four-point TAP technique are shown in Fig. 1. For the first and second injection points, the orientation line was drawn between the caudal end of the xyphoid process and the most caudal end of the body of the last rib.
First injection point: The US transducer was placed perpendicular to the spine below the costal arch on a halfway of the orientation line. The rectus abdominis muscle (RAM) was identified ventrally and, with the transducer sliding dorsally towards the costal arch, the IOM aponeurosis and underlying TAM were identified. The MB solution was injected between the fasciae of the latter two structures (Fig. 2A and B).
Second injection point: The US transducer was placed perpendicular to the spine below the costal arch in a three-quarter orientation line from cranial to caudal direction. The MB solution was injected interfascially between the TAM and IOM aponeurosis (Fig. 2C and D).
The third (Fig. 3A and B) and fourth (Fig. 3C and D) injection points were the same as the second and the third injection points in phase II.
Anatomical dissection and evaluation of the spread of MB solution
All animals were dissected by a veterinary anatomist (JB) with few modifications of previous reports [15, 16]. A sagittal incision at both hemiabdomens was made through the skin medially to the teats because the ventral midline was previously incised and sutured for the purpose of the primary study. Further incisions were made perpendicular to the sagittal incision up to the dorsal midline between the xyphoid cartilage and the coxal tuberosity, removing the skin, the cutaneous trunci, and the latissimus dorsi muscles. Other layers, including the external oblique muscle (EOM), IOM, RAM, and TAM were dissected layer by layer. Special attention was given to the dissection of the IOM aponeurosis to reveal the layer where the ventral branches of the thoracic and lumbar nerves run. The RAM was dissected from the abdominal wall to better define the course of the caudal thoracic and the cranial lumbar nerves that run slightly caudoventrally and supply the abdominal wall muscles.
Thoracic nerves were identified according to the number of the ribs in each animal together with the position of the nerves relative to the last rib. The ventral branch of the last thoracic nerve, the costoabdominal nerve, was identified as the nerve running caudally to the last asternal rib. Other thoracic nerves were identified and named in a caudocranial direction as reported previously [15], including the second-last, third-last, fourth-last, etc. The first three lumbar nerves (L1, L2, and L3) were dissected and identified according to their cranio-caudal position [14].
Since the MB coloration of the tissue develops upon exposure to the air/oxygen [21], the evaluation of staining was performed 5 minutes after the removal of the IOM from the TAM. The MB staining of the nerves was evaluated through direct visualization by the anatomist and both anaesthetists. A positive nerve staining was defined as more than 1 cm of continuous staining of the nerve length [15, 16]. After evaluating the spread of MB solution in one hemiabdomen, the entire procedure was repeated on the other side. The number of ribs was determined at the end of the evaluation.
Statistical analysis
Binary variables (positive/negative) were used for nerve staining assessment. The average number of stained nerves and the proportion relative to all studied nerves were calculated. The weight of pigs in phase II and III is presented as mean ± standard deviation. Data were analysed using SPSS software (ver. 28, SPSS Inc., Il, USA).