This study was conducted from December 2011 until November 2012 and was approved by the animal experimentation committee of the Utrecht University, Utrecht, the Netherlands. All protocols on animals were according to the regulations of Good Laboratory Practice. The implantation study was approved by the Animal Experiments Commission (DEC) of the Utrecht University under DEC registration number 2011.II.06.112.
Animals
For this study, we used 18 female Dutch Landrace pigs (mean weight 31.0 kg ± 3.5). Preoperatively, the pigs had a normal diet and 1 daily dose of 100 mg calcium carbasalate from 6 days before surgery until sacrifice. For this study, we have chosen the porcine model as it is the most similar to the human conditions in weight, organ similarity, and vascular physiology. During the experiments, we encountered 5 terminal complications due to non-anastomosis–related factors, e.g., laryngospasm, which are known complications [
3,
4]. Pigs are difficult to intubate on the basis of a long narrow mouth and an angulation into the trachea. This difficulty increases the number of attempts needed to successfully intubate the animal, which results in the emergence of larynchospasm [
5]. Because no determinations on outcome measures could be made, these animals were excluded from our study.
Procedure
All procedures were performed by the first author (BdB). During the study, we performed surgery on 18 female Dutch Landrace pigs in six different survival groups: 4 h, 2 days, 1 week, 2 weeks, 3 weeks, and 6 months. In each animal, one bypass was made. Each bypass consisted of two separate end-to-side SEcl anastomoses, which were interconnected by a traditional hand sutured end-to-end anastomosis.
First, the pig was positioned in a supine position under general anesthesia with the neck in deflection. After a midline incision in the neck, the right common carotid artery (CCA) was harvested over approximately 10 cm. The vessel was then flushed using a heparin solution and divided into two equal parts (2 × 5 cm length). At one end of each 5-cm section, a SEcl was attached by guiding the donor through the ring and then folding the donor back over the ring (Fig.
1a,b). Subsequently, the SELANA catheter 2.0, a modified version of the standard ELANA laser catheter, was inserted. A fixation clip was then positioned over the donor and catheter, so a fixed entity was formed (Figs.
1b and
2a). Thereafter, with a 90° angle between the applier and the clip, the SEcl/donor complex was inserted in the recipient artery (Fig.
1c–e). The improved visibility over the SEsl technique and the insertion pins reduces the risk of mispositioning of the SEcl (Fig.
2b–d).
After correct positioning of the SEcl/donor complex on the recipient left CCA, we used 3 times 16.7 mJ to lase the arteriotomies (Fig.
1f).
After lasing and retrieval of the flap (Fig.
1 g,h), 5000 IE of heparin was administered in both anastomoses. When the flap was not retrieved together with the SELANA catheter 2.0, an escape procedure was performed to retrieve the flap. Two options were used. In one option, two temporary clips were placed proximal and distal to the SEcl anastomosis. Then, the SEcl was opened to allow retrieval of the flap by forceps. In the other option, we again ceased circulation by temporary clip, after which a longitudinal incision was made in the donor to access the anastomosis and manually retrieve the flap using forceps. Thereafter, the incision was closed by standing sutures.
Following the flap retrieval, both donor vessels were temporally clipped to prevent back flow from the CCA and were then end-to-end hand sutured with Prolene 8.0 (Ethicon Endo-Surgery (Europe) GmbH) to create an interposition jump-bypass. After flow measurement in the bypass, the left CCA was occluded between the anastomoses, so the anterior circulation was dependent on the bypass (Fig.
2f). The flow in the bypass was measured before wound closure.
Post-operatively, the animals were housed in a separate stable for one week to secure optimal wound healing. For 3 days the animals were scored on neurological function, and body temperature was measured. When no complications occurred within the first 7 days after surgery, the animals were housed in groups. Certified animal caretaker personnel performed monitoring of animal health and welfare according to our standardized and approved protocols.
Post-operative patency of the bypass was determined by angiography before termination in all animals, except in the 4-hour survival group in which we used the intra operative flow measurements to determine patency. In the animals in the 6-month survival group, an interim angiography was performed after three weeks of the initial bypass surgery. If the bypass was occluded, the animal was terminated.
Per survival group, one anastomosis was analyzed using electron microscopy and one by histology, distributed ad random, to determine endothelialization and remodeling. Hereto, the anastomoses were stored in formalin immediately after removal from the pig.
For histology, the anastomoses were embedded in plastic. After a 7-day fixation, coupes were made using a diamond saw and stained with hematoxylin and eosin.
Complications rates, flow values, endothelialization, and bypass patency were compared with historical series on laboratory animals [
6,
13].
Flow measurements
Intraoperative flow was measured using a single-vessel flow meter (Transonic Systems Inc.®, Ithaca, NY, USA). Before removing the right CCA, flow was determined in both CCAs. Subsequently, the flow was measured after removal of the right CCA and also when the bypass was patent. Before sacrifice, the flow in the bypass was assessed for the last time.