Surgical technique for developing a rabbit model of congenital diaphragmatic hernia and tracheal occlusion

Graphical abstract


Perioperative care
All animal procedures were carried out in strict accordance with the guidelines of the Canadian Council of Animal Care (CCAC) as approved by the Western University Animal Care Committee (AUP 2016-041). All efforts were made to minimize suffering as per the approved protocol and with veterinary oversight. Time dated pregnant New Zealand White rabbits (Charles River, Sherbrooke) arrived at 13 days gestation. Each month, a group of 5 does arrived at our facility and were individually housed with free access to water, food, and environmental enrichment (Fig. 1). Each doe underwent two surgeries, a CDH creation at 23 days gestation and a second surgery, the TO surgery 5 days later at 28 days gestation as previously described [15].
Pre-operatively, each doe received 0.12 mg/kg Buprenorphine SR SQ (conc. 3 mg/mL, Sigma-Aldrich Corporation, St. Louis, MO, USA) for analgesia, Penicillin G 300 000 IU IM (Pfizer Inc., New York, NY, USA) for the prevention of infection, and Depo-Provera 4.5 mg IM (Pfizer Inc.) to reduce the risk of spontaneous abortion. Anesthesia was induced with 5 mg/kg IM Ketamine (conc. 100 mg/mL), 0.15 mg/kg Dexmedetomidine IM (conc. 1 mg/mL, Clearsynth Labs Pvt. Ltd., Mumbai, India), 0.01 mg/ kg Glycopyrrolate SQ (conc. 0.2 mg/mL, Omega Laboratories Ltd., Montréal, QC, Canada), and maintained with 5% Isoflurane (Baxter Healthcare Corporation, Deerfield, IL, USA) via facemask. Each doe received a 4 cc/kg bolus of 0.9% NaCl SQ, followed by a maintenance rate of 5 mL/kg/hr. Lacrilube was applied to the eyes and a water circulating pad was used to maintain normothermia intraoperatively. Soft restraints were used on all 4 limbs to secure the doe. Vital signs were monitored and maintained within normal limits including heart rate 180-250, respiratory rate 30-60, and temperature 38-40 C.
Post-operatively, the does were placed under a red warming light and transferred to their housing cage once mobile. Does were closely monitored with daily weights, intake/output assessment, and incision checks. Meloxicam 0.2 mg/kg SQ (conc. 5 mg/mL, Boehringer Ingelheim Vetmedica Inc., St. Joseph, MO, USA) was administered every 24 h for 2 days for post-operative analgesia.

CDH creation
CDH creation was performed at 23 days of gestation in a total of 75 fetuses. The does were premedicated as described above. The abdominal fur was trimmed, and the abdomen was prepared with 2% chlorhexidine (Laboratoire Atlas Inc., Montréal, QC, Canada) and draped. A lower midline laparotomy incision was made and the uterus was exposed. The number of fetuses in each uterine horn was counted. Two fetuses were chosen from the most ovarian end of each uterine horn, for a total of 4 fetuses per doe (Fig. 2). Fetal position was determined by gentle palpation. A 1 cm hysterotomy was made on the anti-mesometrial side of the uterus with a #15-blade scalpel (Aspen Surgical Products Inc., Caledonia, MI, USA) (Fig. 3). A purse string suture was placed using a 6-0 Prolene 1 Fig. 2. Bicornuate Rabbit Uterus. The pregnant New Zealand rabbit uterus is bicornuate with a right and left uterine horn containing multiple fetuses. Fetuses located at the ovarian end were used for surgical experimentation. (Ethicon Inc., Somerville, NJ, USA). The left upper limb of the fetus was identified, exposed and retracted cephalad. A left-sided thoracotomy was made at the landmark between the lateral thoracic vessels using a 25-gauge needle (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and mosquito forceps (Fig. 3). The lung was retracted, exposing the fetal diaphragm. The diaphragm was grasped with mosquito forceps and a piece was cut with fine scissors, creating the diaphragmatic defect (Fig. 3). The chest wall was sutured with a 6-0 Prolene 1 (Ethicon Inc., Somerville, NJ, USA). The fetus was repositioned into the uterus and the purse string was tied, thus closing the hysterotomy. The laparotomy incision was closed with a 3-0 Vicryl 1 (Ethicon Inc., Somerville, NJ, USA) in a running fashion. The skin was closed in a running subcuticular fashion with a 4-0 Monocryl 1 (Ethicon Inc., Somerville, NJ, USA).
In the last set of 5 does, 10 sham CDH fetuses were generated. Surgical protocol was followed as previously described. Following CDH creation, two additional fetuses were chosen at the most ovarian end. A sham CDH was created by performing a left-sided thoracotomy without the creation of a diaphragmatic defect. We recommend that a maximum of 4 fetuses per doe undergo surgery for improved survival of fetuses. We found that adding sham fetuses and increasing the number of operative fetuses to 6 increased the operative time and surgical stress to the doe and fetuses, and resulted in a higher mortality rate.

Tracheal occlusion
TO was performed at 28 days gestation in a total of 17 fetuses. The does were pre-medicated as described above. Once anesthesia was induced, the abdomen was prepped and draped in a sterile fashion. The lower laparotomy incision was opened and fetuses were counted. Viability was noted. Half of the potentially viable CDH fetuses were chosen for TO. A hysterotomy was made and the fetal head was exposed. The snout was immediately covered with a saline soaked non-woven sponge to ensure no fetal breathing. A horizontal incision was made at the superior border of the thyroid gland. A fine snap was used to bluntly dissect down to the trachea. Once isolated, the trachea was double ligated with a 4-0 Vicryl 1 suture (Ethicon Inc., Somerville, NJ, USA) (Fig. 4). Fetal skin was sutured with 6-0 Prolene 1 (Ethicon Inc., Somerville, NJ, USA). The hysterotomy was closed with 6-0 Prolene 1 (Ethicon Inc., Somerville, NJ, USA) in a running locking fashion. The doe's fascia was sutured with 3-0 Vicryl 1 (Ethicon Inc., Somerville, NJ, USA) and the skin was sutured with 4-0 Monocryl 1 (Ethicon Inc., Somerville, NJ, USA). Post-operative treatment was as described above.

Euthanasia and organ retrieval
Fetal weights and lung tissue collection was performed at 31 days gestation. Each doe was euthanized individually in a room away from the remaining does. Each doe was sedated with 10-50 mg/kg Ketamine IM (conc. 100 mg/mL) and 2.5-10 mg/kg Xylazine IM (conc. 100 mg/mL, Bayer A hysterotomy has been made, the head exposed and neck extended. A small neck incision was used to isolate the trachea which was double ligated with sutures in order to perform the TO. Healthcare LLC, Shawnee, KS, USA), and euthanized with 100 mg/kg Pentobarbital Sodium IV (conc. 50 mg/mL, Oak Pharmaceuticals Inc., Lake Forest, IL, USA) administered through the ear vein. A midline laparotomy was made and the doe's heart was palpated to confirm the absence of cardiac activity. All fetuses were delivered (Fig. 5). The snouts were immediately covered with a saline soaked non-woven sponge and each fetus was decapitated to ensure no fetal breathing. All fetuses were weighed. A sternotomy and midline laparotomy were performed and the diaphragm was assessed for the presence or absence of diaphragmatic defect (Fig. 6). The neck was also incised in CDH + TO fetuses and the trachea was assessed for presence of ligation. One control unoperated fetus was chosen from each doe containing other surgical specimens and was selected based on its weight being the average of the litter. Total lung weight and right lung weight were measured for each fetus and left lung weight was calculated by subtraction (Fig. 5). Upper lung lobes were collected and stored in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and lower lung lobes were flash frozen in liquid nitrogen and stored in À80 C.

Method validation
This surgical technique of CDH creation resulted in lung hypoplasia which was reversed by tracheal occlusion [16]. This was recently validated in our research on the effects of tracheal occlusion on Wnt signaling in a rabbit model of CDH [16].