Study design
All procedures were approved by Utrecht University’s Ethical Committee, as required under Dutch legislation (DEC number 2014.III.12.112) and performed at the Utrecht University Clinic for Companion Animals. Three research dogs were instrumented with the VAP system and the system was evaluated for a subsequent period of 5 months. After surgical implantation, the VAP system was used for intravenous infusion of liver cells intraoperatively and once daily for 2 days after surgery. The VAP system was also used for direct intraoperative measurement of portal venous pressure.
Dogs
Venous access port systems were implanted in three Beagle-Bedlington terrier crossbreeds with copper toxicosis due to COMMD1 deficiency [9]. Dog no. 1 was an intact male (5 years, 15.0 kg), dog no. 2 an intact male (8 months, 15.5 kg), and dog no. 3 an intact female (8 months, 10.6 kg).
Anaesthesia and monitoring
Pre-operatively, all dogs received cefazolin 20 mg/kg IV (Kefzol, Eurocept International B.V., Ankeveen, The Netherlands). The dogs were premedicated with glycopyrrolate 0.01 mg/kg IM (Robinul 0.2 mg/mL injection, Riemser Pharma GmbH, Greifswald, Deutschland) and methadone 0.5 mg/kg IV (Comfortan 10 mg/mL, Dechra, Bladel, The Netherlands). Propofol 1–4 mg/kg dosed to effect IV (Propofol 1%, MC Fresenius Kabi, Zeist, The Netherlands) was used for induction. After intubation, general anaesthesia was maintained with isoflurane dosed to effect in a mixture of oxygen and air 1:1 and fentanyl continuous rate infusion 10–20 μg/kg/h CRI (Fentanyl 0.05 mg/mL, Bipharma, Almere, The Netherlands).
Postoperatively, the dogs were admitted to the intensive care unit for analgesia and monitoring. A clinical examination including abdominal circumference and central venous pressure measurements were performed every 4 hrs for 3 days. Mean arterial pressure was monitored via an intra-arterial line for at least 24 h postoperatively. Venous blood analysis was performed pre-operatively, and 1 day, 2 days, 3 days, 1 week, 1 month, and 3 months post-operatively. Blood was sampled through the jugular catheter for the first three postoperative days and at later time points by venipuncture and was analysed for the following parameters: haematocrit, platelet count, activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen, total protein and albumin. Dogs received postoperative pain medication, initially consisting of fentanyl 3–5 μg/kg/h CRI (Fentanyl, Bipharma, Almere, The Netherlands) and ketamine 3–5 μg/kg/min CRI (Narketan, Vetoquinol, ‘s-Hertogenbosch, The Netherlands) to effect. After two to 3 days analgesia was continued for 3–5 days with tramadol 3–5 mg/kg four times daily per os (Tramadol HCl, Centrafarm, Etten-Leur, The Netherlands). As part of the liver cell transplantation protocol, dogs received cyclosporine 6.25 mg/kg two times daily per os (Sporimune, AST Farma, Oudewater, The Netherlands) from the day before surgery until 3 months post-operatively.
Surgery
First, the implantation of the VAP system was practiced on two fresh cadavers of female mixed breed dogs (28 kg), euthanized for non-related medical experiments (surplus material, Utrecht University 3R-policy). The surgical protocol was then applied to the three experimental dogs. All surgeries were performed by the same surgeons. A midline celiotomy was performed in dorsal recumbency and the falciform ligament was removed after cranial ligation. A left lateral hepatic lobectomy was performed as part of the liver cell transplantation study using double ligation of the large vessels near the hilus of the lobe with polydioxanone (PDS 0, Johnson & Johnson International, Amersfoort, The Netherlands) and additional electrocoagulation to seal the parenchyma peripheral to the ligated vessels.
The VAP system used was a PORT-A-CATH II POWER system (reference number: 21–4477-24, Smiths Medical Nederland B.V., Rosmalen, The Netherlands) that consisted of a polyurethane catheter with an outer diameter of 1.9 mm (5.8 Fr), a titanium connector and a single lumen portal made of polysulfone and titanium. The priming volume was provided by the manufacturer and manually confirmed pre-operatively. The volume amount of the catheter + portal = 0.7 + 0.3 ml = 1 ml. This is the total amount needed to fill a non-adjusted device. The VAP system was prepared and flushed with heparinized saline (50 IU/mL). A distal branch of the extrahepatic portal circulation (jejunal or splenic vein) was selected based on accessibility and size (Fig. 1). Two polyglecaprone 25 (Monocryl 4–0, Johnson & Johnson International, Amersfoort, The Netherlands) stay-sutures and a Rummel tourniquet (Fig. 1a, left) were placed around the vessel to fixate the vessel and to prevent bleeding. A stab incision was made between both sutures and elongated to approximately 5 mm over the length of the vein. With the aid of a vein hook (Fig. 1a, middle) the rounded tip of the VAP catheter was inserted into the vein and carefully fed towards the main stem of the extrahepatic portal vein, just one to two centimetres proximal to the hepatic entrance. The position was checked by palpation and the VAP catheter was fixed at the insertion site using a Chinese finger trap suture technique with polypropylene (Prolene 4–0, Johnson & Johnson, Amersfoort, The Netherlands) (Fig. 1a, right). The tourniquet was removed and the preplaced stay-sutures, one distal and one proximal to the catheter insertion, were tied. The VAP system was repeatedly flushed with heparinized saline (50 IU/mL).
The port of the VAP system was placed in a subcutaneous pocket lateral to the caudal part of the midline incision. A stab incision was made in the abdominal wall and the extravenous part of the catheter was inserted through the incision and connected to the portal outside the abdomen. The portal was fixated to the muscular fascia with three polypropylene (Prolene 2–0, Johnson & Johnson, Amersfoort, The Netherlands) interrupted sutures (Fig. 1b). The VAP system was flushed again to confirm patency and the subcutaneous pocket was closed with single interrupted polyglecaprone 25 sutures. A 19G gripper needle (reference number: 21–3280-24, Smiths Medical Nederland B.V., Rosmalen, The Netherlands) was placed percutaneously into the port to obtain venous access for the liver cell infusions and to enable portal venous pressure measurements (Fig. 1c). The system was flushed with saline to confirm patency. The abdomen was closed routinely.
Portal pressure measurements
We explored the possibility to measure the pressure in the portal vein directly and non-invasively via the VAP system. For this purpose, an extension tubing was connected to the gripper needle in the subcutaneous portal during surgery in two dogs. Venous pressure was measured by a water column method in centimetres H2O and electronically by a Merit Medical single transducer set (Gabarith® PMSET 1DT-XX 1rose) connected to a Datex-Ohmeda S5 Anaesthesia Monitor in millimetres Hg. In both cases, the level of the right atrium was used as zero reference.
Maintenance and patency checks of VAP
Patency of the VAP system was checked by flushing portal and catheter with saline once daily on the first 2 days after surgery before and after infusing cells through the VAP into the portal bloodstream. Images of the portal vein and the tip of the catheter were obtained by B-mode imaging and colour Doppler was applied to check for hepatopetal flow during infusion. The gripper needle was removed 5 days after surgery.
Subsequently, patency was checked by flushing the VAP system once a month. The skin overlying the portal was clipped, locally anesthetized with a combined lidocaine and prilocaine ointment EMLA Cream 5% (AstraZeneca, Den Haag, The Netherlands) and routinely disinfected. A 19G gripper needle (reference number: 21–3468-24, Smiths Medical Nederland B.V) was inserted percutaneously into the subcutaneous portal. Using a syringe, 5 ml of blood was withdrawn and the catheter was subsequently flushed with 10 ml of saline. Patency of the portal vein and the position of the intravenous catheter were also checked with abdominal ultrasonography. After each use of the VAP, a heparin lock was placed by infusion of 5 ml of heparinized saline (100 IU/ml).