TITLE: Protocol for surgical implantation of chronic cecal cannula in pigs 
AUTHORS: Muriel Larauche, Karim Atmani, Yushan Wang, Zach Weitzner, Sandra Duarte-Vogel, Wentai Liu, Mulugeta Million, Justin Wagner

[DESCRIPTION]
Gastroduodenal and ileal cannulas in pigs are commonly used to evaluate food digestibility and nutrition research. However accessing the motility of the proximal colon for long term in conscious pigs remains a
challenge. In this protocol, we describe the surgical implantation of a cecal cannula in Yucatan minipigs,
together with details to design/3D-print a biocompatible cecal cannula that allows direct access to the cecum/proximal colon of pigs and the maintenance procedure. The cannula is very well tolerated by the animals for long-term use. The model enables not only a reliable longitudinal chronic colon function measurements (probes inserted through the cannula) to study gut motility in health and diseases but also for luminal sensing and stimulation using stationary or non-stationary wireless smart pills.







[STEPS]
SECTION: Surgical cannula implantation through laparotomy
2. Pre-operative diet regimen for intestine preparation

Five days prior to surgery, the animals were started on a low residue diet consisting of 2 cups of low residue food (Hill's Prescription Diet i/d Dry Dog Food or LabDiet Advanced 
Protocol 0043639 (594K)) plus one bottle of a liquid dietary supplement (Impact Advanced Recovery®, Nestlé Health Science or Enure Surgery Vanilla Flavor, Abbott Laboratories)  twice daily.  
  
  
  
  


Two days prior to the surgical procedure, 8 oz of Miralax (Polyethylene Glycol 3350, Bayer US) mixed in 32 oz of Gatorade or Pedialyte plus 2 pills of Dulcolax (Sanofi) was administered once a day for 2 days. Oral Gabapentin at 5 mg/kg and 20 mg of omeprazole were administered the day before surgery.
  
   
SECTION: Surgical cannula implantation through laparotomy
2.1. Surgery

Pigs were fasted for at least 12h prior to surgery with free access to water. 

Anesthesia was induced with Telazol (Tiletamine/zolazepam, 6 mg/kg, IM)  , followed by isoflurane delivery initially via face mask and later via endotracheal tube. Both ears were cleaned with 3 alternating scrubs of alcohol/chlorhexidine solution and IV catheters were inserted and secured into the lateral or medial veins, for administration of medications and fluids. 
  
  
  
 

For pre-operative analgesia animals received Meloxidyl (meloxicam, 0.3 mg/kg, IM), and
hydromorphone (0.1 mg/Kg, IV). 
  
  
  

Other medications administered pre-op include: Famotidine at 1 mg/Kg IV,  Cerenia (Maropitant citrate, Zoetis) at 1 mg/Kg IV, long acting Draxxin (tulathromycin, Zoetis)at 2.5 mg/Kg IM, long acting Excede (ceftiofur, Pfizer) at 5 mg/Kg IM, and Cefazolin at 30 mg/Kg IV.
  
  
  
  
  
 

Intra-operatively, a surgical plane of anesthesia was maintained with isoflurane (2-4%) delivered via endotracheal tube. The animals were  also mechanically ventilated with a tidal volume of 10-20 mL/Kg and a respiratory rate of 10-16 breaths per minute.   
   

Lactated Ringer’s solution was administered at 5-10 ml/kg/h IV throughout the surgery.  
  


In some experiments, prior to incision, 0.25% bupivacaine at  0.6 mL/Kg per site (2-4 sites)(Marcaine or bupivacaine hydrochloride, Henry Schein Animal Health) was injected in the transverse abdominalis plane using ultrasound to guide the regional block. Otherwise, lidocaine 2 mg/Kg was injected subcutaneously at the incision sites pre operatively followed by marcaine 2 mg/Kg at closure. 
  
  


During the surgical procedure, pigs were positioned on a heating pad (32°C) in supine position. The right medial thigh was also cleaned and the right femoral artery was catheterized to monitor blood pressure during the procedure. Heparinized saline was used to flush the line preventing clotting. 
  
  


The left and ventral abdomen side of the animals was scrubbed with Chlorhexidine using a pre-packed surgical brush. Hair in this area was clipped and the skin prepared aseptically alternating chlorhexidine solution and alcohol. Cannula exit was marked at the dorsal flank area with a sterile marker (between the last rib and the antero-dorsal region of the flexed stifle joint). 
  

The surgical site was draped  with  Ioban (Steri-Drape, 3M). Sterile drapes were placed on top of the Ioban-covered surface with an open small widow/surgical field. 
  

Autoclaved surgical tools were used for the procedure. Laparotomy was performed using a disposable scalpel and a monopolar electrocautery device.  A ~15 cm skin incision was made using a scalpel. Incision ran along the midline, distal from the navel. In males, the incision line around the navel and distal to it was ~2cm lateral from midline to avoid damage to the urethra. This was followed with dissection of, from superficial to deeper: subcutaneous fat and linea alba using cautery. The peritoneum was then elevated with forceps and incised sharply to access the peritoneal cavity. The peritoneal incision was widened with cautery, with care taken to avoid injury to underlying viscera.  

The cecum was located and a 5-cm purse-string suture (3-0 polydioxanone suture on SH needle, J316H, Ethicon) was placed approximately 5 cm proximal from the ileocecal junction. A longitudinal colotomy was created with cautery through the taenia coli within the purse-string suture. The base of the 3D-printed biocompatible T cannula was inserted into the cecum through this opening. The cannula were made of ABS-M30i material, biocompatible (ISO 10993 USP Class VI), measured 4.5 cm in width (base of the cannula inserted in the cecum), 4.8-5.2 cm in length (exteriorized length 1.8-2.1 cm), and had an internal diameter of 2 cm.  All together, with the plug, each cannula weighed between 24.3-25 g. The cannula was then secured (the base/flange in the cecum) by tightening the purse-string suture, around the body/barrel of the cannula.  
  
  

A cylinder of tissue at the marked cannula exit site on the flank (approximately 2 cm in diameter) was excised with cautery. The underlying tissues was gently split by blunt dissection. The fascia was divided with cautery and the flank muscle was dissected bluntly in a muscle-sparing fashion. The cecum was affixed to the abdominal wall in Stamm fashion with 4 circumferential sutures about the cannula site.  The cannula lumen was occluded with gauze to minimize contamination of the sterile surgical field and the body of the cannula was delivered through the abdominal wall. The cecum was confirmed to be free of kinks or twists. The sutures were then tied to complete the Stamm approximation. The gauze pack was removed and cannula was capped with a 3D printed cannula cap.  

The abdominal cavity was flushed with copious amount of warm saline and suction was used to removed excess fluid. The midline incision was closed in layers. The linea alba, was reapproximated with 0-looped PDS on CTX needle (PDP990G, Ethicon) and the skin was closed in a subcuticular fashion with absorbable suture 2-0 PDS on SH needle (7317H, Ethicon). 
  
  

The sutures skin incision  was then covered with skin glue (Vetbond Tissue Adhesive, 3M) and bandaged using Ioban and gauzes. Modified bandaging techniques were used for male pigs to avoid skin irritation due to urine.
 
  


Animals were continuously monitored post-surgery until fully awake and ambulatory. Postoperative pain medication was administered in addition to water and food without any postoperative fast.
SECTION: Cecal cannula care
3. The skin surrounding the exteriorization site was cleansed with a dilute Chlorhexidine solution (10% in saline) rinse twice daily as leakage of digesta around the site of exteriorization was occurring occasionally. 
   

The cannula sites and surrounding skin were then dried and coated generously with zinc oxide ointment (Desitin, 40% zinc oxide) once or twice daily throughout the study to protect skin from irritation caused by any leaked digesta.
  





SECTION: Animals
1. Four months old (18-22 kg) male, castrated at 7 days of age, and female Yucatan minipigs (S&S farms, Ramona, CA) , were singly housed in pens (either bedding or grate floor depending on housing availabilities - 2 pigs/pen, 42ft2) in an environmentally controlled room (lights on/off 6AM/6PM, 61-81ºF) under SPF conditions. 
  

All pigs were offered ad libitum access to diet (Laboratory Mini-Pig Grower Diet 5081, LabDiet) and filtered tap water. 
  

All husbandry practices and procedures conformed to the Guide for the Care and Use of Laboratory Animals (8th edition), the Animal Welfare Act and were reviewed and approved by the UCLA Institutional Animal Care and Use Committee.  
SECTION: Cannula care
5. The cecal cannula stayed fully capped when animals were not being tested.

The skin surrounding the exteriorization site was cleansed with chlorhexidine solution /saline rinse (10%) once to twice daily as leakage of digesta around the site of exteriorization was occurring occasionally. 

The cannula sites and surrounding skin were then coated generously with zinc oxide ointment (Desitin, 40% zinc oxide, Pfizer Animal Health, Inc.) once to twice daily throughout the study to protect skin from irritation caused by any leaked digesta. 
SECTION: Surgical procedures
4.1. Bupivacaine or lidocaine was used as local anesthetics and injected intradermally at the site of incision prior to starting the surgery. 

Bupivacaine 0.25%, 1.2 mL/Kg was injected in the transverse abdominalis plane using ultrasound to guide the local block. 

A disposable scalpel was used to make a 9-cm incision through the skin and first muscle layer. The incision was made 4 cm posterior and parallel to the last rib on the right side of the animal. The remaining muscle layers were spread by using manual manipulation and blunt dissection with hemostatic forceps. Scissors were used to make an opening in the peritoneum. 

The cecum and ileum were located, exteriorized, and isolated, and a 7-cm purse string suture (3-0 vicryl suture on SH needle) was inserted approximately 5 cm proximal from the cecocolic junction. The purse string suture consisted of two parallel lines of sutures, approximately 5 mm apart, made with one continuous suture. Each stitch was approximately 5 mm in length. 

A new disposable scalpel was used to make an incision between the pursestrings. The flange of the simple-T cannula was inserted into this opening. The cannula was secured in the cecum by tightening the pursestring suture. For added security, a second pursestring suture (3-0 vicryl suture on SH needle) was placed around the cannula. The cannula was exteriorized on the left side of the pig between the last two ribs, approximately 15 mm from the ends of the ribs. A circle of skin approximately 2 cm in diameter was excised, the underlying tissues were split by blunt dissection, and the peritoneum was cut with scissors. The cannula was pulled between the ribs and through this incision with the aid of Allis tissue forceps. 

To help prevent adhesions, sterile saline (approximately 500 ml) was poured into the body cavity and removed via suction. The peritoneum was sutured closed with 3-0 vicryl suture on SH needle. Individual muscle layers, including the subcutaneous muscle layer, were sutured together by using 2-0 PDS on SH needle, and the skin was closed in an intradermal pattern with absorbable suture (PDS II size 0 looped monofilament polydioxanone suture on CTX needle). The skin was then be covered by skin glue. 

The animal was continuously monitored postoperation until they are completely awake and ambulatory. Postoperative pain medication were administered in addition to water and food without any postoperative fast.
SECTION: Surgical procedures
4. Pigs were fasted for at least 12h prior to surgery with free access to water. 

For surgical level anesthesia, pigs were premedicated with midazolam (1 mg/kg), 
  
ketamine (15 mg/kg) ,
  
and meloxicam (0.3 mg/kg) injected intramuscularly. 
  

They were then intubated, connected to a respirator for ventilation (breathing rate maintained between 13-16 breaths/min), and maintained under general anesthesia with 1-3% inhaled isoflurane. 
  

Maintenance fluids (lactated ringers ) were administered at 10 ml/kg/h. 
  

During the surgical procedure, pigs were positioned on a heating pad (32°C) in supine position. A femoral heart line was placed. When needed, the heart line was flushed with sterile saline containing 0.5% heparin.
  
  
SECTION: Pre-surgery diet
3. Two days prior surgery, the animals were started on a low residue diet (Hills I/D diet) 
  plus a liquid supplement (Impact)
. The low residue diet was continued in the post-surgical recovery period (up to 10 days) to insure proper healing of the cecal cannula.

Two days prior the procedure, pigs received a colon cleanse consisting of 8 oz of miralax mixed in 32 oz of gatorade plus 2 pills of Dulcolax. The same was repeated the day before the surgical procedure.
2. 
SECTION: Training
2. During  the week of quarantine, pigs will be familiarized with the trainer.  Training will start one week after arrival and for 2-3 weeks or more  after the end of the quarantine. The chute is a metallic cart that has  adjustable sides to allow loose restraint. The chute will allow pigs to  move back and forth over short distances (20 cm) but does not allow  turning around. Training will start with short (30 min) stay in the  chute and will gradually increase over several days, up to a maximal 2h  30min stay. Habituation to the chute will be positively reinforced using  treats/food by the trainer.
SECTION: Surgical cannula implantation through laparotomy
2.2. Post-operative treatment

Post operative care was provided for 14 days as follows:
Hydromorphone 0.05 - 0.1 mg/kg IV every 4h - first overnight, then Buprenorphine 0.02 mg/kg IV/IM every 12 hours for the initial 48-72 hours post op or Ethiqa SR 0.2 mg/kg SC 4h after
the last administration of hydromorphone.
Meloxicam 0.3mg/kg SID (PO/or IM) x 7 days  
Excede 5mg/kg IM once 5 days after first injection 
Cerenia 2mg/kg SID PO x 1 day 
Omeprazole 20mg/day PO x 5 days
Famotidine 1 mg/kg SID x 3 days 
Gabapentin 5mg/kg BID PO; give 1 dose day of surgery, and continue x 10 days BID (12hrs apart)
Fecal output monitored daily
Cecal cannula assessed and cleaned daily
Surgical incisions assessed daily

   
  
  
  
  
  
  
  

The low residue diet regimen was continued throughout the post-surgical recovery period (up to 10 days) to insure proper healing of the cecal cannula.