May 02, 2024

Public workspaceProtocol for surgical implantation of chronic cecal cannula in pigs

DOI
dx.doi.org/10.17504/protocols.io.8epv5jwx5l1b/v1
Protocol for surgical implantation of chronic cecal cannula in pigs
  • 1University of California, Los Angeles;
  • 2UCLA David Geffen School of Medicine
Muriel Larauche
Open access
DOI: dx.doi.org/10.17504/protocols.io.8epv5jwx5l1b/v1
Protocol Citation: Muriel Larauche, Karim Atmani, Yushan Wang, Zach Weitzner, Sandra Duarte-Vogel, Wentai Liu, Mulugeta Million, Justin Wagner 2024. Protocol for surgical implantation of chronic cecal cannula in pigs . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jwx5l1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 26, 2023
Last Modified: May 03, 2024
Protocol Integer ID: 82529
Keywords: cecum, proximal colon, chronic implantation, cannulation, surgery
Funders Acknowledgement:
SPARC NIH
Grant ID: OT2OD024899
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
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.
Materials
  • Four months old (18-22 kg) male (castrated at 7 days of age) and female Yucatan minipigs (S&S farms, Ramona, CA)
  • Low residue food (Hill's Prescription Diet i/d Dry Dog Food, #2684397 or LabDiet Advanced Protocol 0043639 (594K))
  • Liquid dietary supplement (Impact Advanced Recovery‱, Nestlé Health Science or Ensure Surgery Vanilla flavor, Abbott Laboratories)
  • 8 oz of Miralax (Polyethylene Glycol 3350, Bayer US) mixed in 32 oz of Gatorade or Pedialyte
  • Dulcolax (Bisacodyl, 5 mg, Sanofi)
  • Oral gabapentin at 5 mg/Kg
  • 20 mg of omeprazole
  • Tiletamine and zolazepam at 6 mg/Kg, IM
  • Isoflurane (2-4%)
  • Alcohol/chlorhexidine solution
  • IV catheters
  • Meloxicam (0.3 mg/kg IM)
  • Hydromorphone 0.1 mg/Kg IV
  • Famotidine at 1 mg/Kg IV
  • Maropitant (Cerenia, Zoetis) at 1 mg/Kg IV
  • Buprenorphine 0.02mg/kg IV/IM
  • Ethiqa SR 0.2 mg/kg
  • Meloxicam 0.3mg/kg SID (PO/or IM)
  • Excede 5mg/kg IM
  • Cerenia 2mg/kg SID PO
  • Omeprazole 20mg/day PO
  • Long acting tulathromycin (Draxxin, Zoetis) at 2.5 mg/Kg IM
  • Long acting Ceftiofur (Excede, Pfizer) at 5 mg/Kg IM
  • Cefazolin at 30 mg/Kg IV
  • Lactated Ringer’s solution (USP, Preservative-Free, Baxter Henry Schein Animal Health Catalog #059380) at 5-10 ml/kg/h IV
  • 0.25% bupivacaine at 0.6 mL/Kg per site (2-4 sites) (Marcaine or bupivacaine hydrochloride, Henry Schein Animal Health)
  • Lidocaine 2 mg/Kg
  • Marcaine 2 mg/Kg
  • Heating pad (32°C)
  • Heparinized saline
  • Loban (Steri-Drape, 3M)
  • Autoclaved surgical tools
  • Disposable scalpel
  • Monopolar electrocautery device
  • 0-looped PDS on CTX needle (PDP990G, Ethicon)
  • Absorbable suture (2-0 PDS on SH needle (7317H, Ethicon)
  • Skin glue (Vetbond Tissue Adhesive, 3M Animal Care)
  • Chlorhexidine solution (Chlorhexidine 2% Solution, VetOne, Catalog #1054488)
  • Zinc oxide ointment (Desitin, 40% zinc oxide, Johnson & Johnson Pediatrics)
Protocol materials
ReagentVetbond Tissue Adhesive3M corporationCatalog #1469SB
Step 2.1
ReagentEthiqa XR (1.3 mg/ml)Fidelis PharmaceuticalsCatalog #NDC 86084-100-30
Step 2.2
ReagentImpact Advance RecoveryNestle Health ScienceCatalog #00043900943114
Step 2
ReagentHeparin Sodium Injection, USP, 10,000 unit/mLHenry Schein Animal HealthCatalog #067792
Step 4
ReagentYucatan minipigS&S Farms
Step 1
ReagentCereniaZoetis
In 2 steps
ReagentIoban 2 23x23 Sterile Surgical Drape Non-Fenestrated Henry Schein Animal HealthCatalog #7774142
In 2 steps
Reagent0-looped PDS on CTX needle EthiconCatalog #PDP990G
Step 2.1
ReagentLidocaine 2%VetOneCatalog #NDC 13985-222-04
Step 2.1
ReagentDraxxinZoetis
Step 2.1
ReagentSodium Chloride Injection, USP, Preservative-Free, 0.9%, BaxterHenry Schein Animal HealthCatalog #059382
In 2 steps
ReagentMarcaine Injection 0.5%Henry Schein Animal HealthCatalog #6312615
Step 2.1
ReagentDesitinJohnson & Johnson Pediatrics
Step 3
ReagentMiralax (Polyethylene Glycol 3350)Bayer
Step 2
ReagentEloxiJect (Meloxicam) Injection, 5mg/mLHenry Schein Animal HealthCatalog #049755
Step 4
ReagentLactated Ringers Injection, USP, Preservative-Free, BaxterHenry Schein Animal HealthCatalog #059380
In 2 steps
ReagentHeparin Sodium Injection USP. 1000U/mLPfizer (Hospira)Catalog #NDC 0409-2720-02
Step 2.1
ReagentDulcolax (Bisacodyl 5 mg)Sanofi
Step 2
ReagentEnsure Surgery Vanilla flavorAbbott
Step 2
ReagentKetamine HCl Injection, 200mg/20mL (10mg/mL), C3NHenry Schein Animal HealthCatalog #068317
Step 4
ReagentLaboratory Mini-Pig Grower Diet 5081LabDietCatalog #0001339
Step 1
ReagentChlorhexidine 2% solutionVetOneCatalog #11-09007
In 3 steps
ReagentTelazolZoetis
Step 2.1
ReagentExcedeZoetis
In 2 steps
Reagent3-0 polydioxanone suture on SH needleEthiconCatalog #J316H
Step 2.1
ReagentHydromorphone Hydrochloride Injection, USPTeva Pharmaceuticals USA, IncCatalog #NDC 0703-0113-03
In 2 steps
ReagentGabapentin (oral solution, 250 mg/5 ml)Amneal Pharmaceuticals LLCCatalog #NDC 65162-698-90
Step 2.2
ReagentIsoThesia (Isoflurane) SolutionHenry Schein Animal HealthCatalog #029405
In 3 steps
ReagentFamotidine USP 20 mgBluePoint LaboratoriesCatalog #NDC 68001-397-00
In 2 steps
ReagentMeloxidylZoetisCatalog #NDC 11695-6966-2
In 2 steps
ReagentCecal cannula ABS-M30i material, biocompatible Stratasys Direct Manufacturing
Step 2.1
ReagentPrilosec OTC (omeprazole 20 mg)Procter and GambleCatalog #NDC 37000-455-02
Step 2.2
ReagentMidazolam HCl Injection, 50mg/10mL (5mg/mL), C4Henry Schein Animal HealthCatalog #067695
Step 4
Reagenti/d Dry Dog FoodHill's Pet NutritionCatalog #2684397
In 2 steps
Reagent 2-0 PDS on SH needle EthiconCatalog #7317H
Step 2.1
ReagentCefazolin for Injection, USPPfizer (Hospira)
Step 2.1
ReagentLabDiet Advanced Protocol 0043639LabDietCatalog #594K
Step 2
Animals
Animals
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.
ReagentYucatan minipigS&S Farms

All pigs were offered ad libitum access to diet (Laboratory Mini-Pig Grower Diet 5081, LabDiet) and filtered tap water.
ReagentLaboratory Mini-Pig Grower Diet 5081LabDietCatalog #0001339

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.
Surgical cannula implantation through laparotomy
Surgical cannula implantation through laparotomy
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.
Reagenti/d Dry Dog FoodHill's Pet NutritionCatalog #2684397
ReagentLabDiet Advanced Protocol 0043639LabDietCatalog #594K
ReagentImpact Advance RecoveryNestle Health ScienceCatalog #00043900943114
ReagentEnsure Surgery Vanilla flavorAbbott


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.
ReagentMiralax (Polyethylene Glycol 3350)Bayer
ReagentDulcolax (Bisacodyl 5 mg)Sanofi
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.
ReagentTelazolZoetis
ReagentIsoThesia (Isoflurane) SolutionHenry Schein Animal HealthCatalog #029405
ReagentChlorhexidine 2% solutionVetOneCatalog #11-09007
For pre-operative analgesia animals received Meloxidyl (meloxicam, 0.3 mg/kg, IM), and hydromorphone (0.1 mg/Kg, IV).
ReagentMeloxidylZoetisCatalog #NDC 11695-6966-2
ReagentHydromorphone Hydrochloride Injection, USPTeva Pharmaceuticals USA, IncCatalog #NDC 0703-0113-03

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.
ReagentFamotidine USP 20 mgBluePoint LaboratoriesCatalog #NDC 68001-397-00
ReagentCereniaZoetis
ReagentExcedeZoetis
ReagentDraxxinZoetis
ReagentCefazolin for Injection, USPPfizer (Hospira)

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.   
ReagentIsoThesia (Isoflurane) SolutionHenry Schein Animal HealthCatalog #029405
Lactated Ringer’s solution was administered at 5-10 ml/kg/h IV throughout the surgery.
ReagentLactated Ringers Injection, USP, Preservative-Free, BaxterHenry Schein Animal HealthCatalog #059380


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.
ReagentMarcaine Injection 0.5%Henry Schein Animal HealthCatalog #6312615
ReagentLidocaine 2%VetOneCatalog #NDC 13985-222-04


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.
ReagentSodium Chloride Injection, USP, Preservative-Free, 0.9%, BaxterHenry Schein Animal HealthCatalog #059382
ReagentHeparin Sodium Injection USP. 1000U/mLPfizer (Hospira)Catalog #NDC 0409-2720-02


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).
ReagentChlorhexidine 2% solutionVetOneCatalog #11-09007
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.
ReagentIoban 2 23x23 Sterile Surgical Drape Non-Fenestrated Henry Schein Animal HealthCatalog #7774142
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. 
Reagent3-0 polydioxanone suture on SH needleEthiconCatalog #J316H
ReagentCecal cannula ABS-M30i material, biocompatible Stratasys Direct Manufacturing
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).
Reagent0-looped PDS on CTX needle EthiconCatalog #PDP990G
Reagent 2-0 PDS on SH needle EthiconCatalog #7317H
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.
ReagentVetbond Tissue Adhesive3M corporationCatalog #1469SB
ReagentIoban 2 23x23 Sterile Surgical Drape Non-Fenestrated Henry Schein Animal HealthCatalog #7774142


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.
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

ReagentHydromorphone Hydrochloride Injection, USPTeva Pharmaceuticals USA, IncCatalog #NDC 0703-0113-03
ReagentEthiqa XR (1.3 mg/ml)Fidelis PharmaceuticalsCatalog #NDC 86084-100-30
ReagentMeloxidylZoetisCatalog #NDC 11695-6966-2
ReagentExcedeZoetis
ReagentCereniaZoetis
ReagentPrilosec OTC (omeprazole 20 mg)Procter and GambleCatalog #NDC 37000-455-02
ReagentFamotidine USP 20 mgBluePoint LaboratoriesCatalog #NDC 68001-397-00
ReagentGabapentin (oral solution, 250 mg/5 ml)Amneal Pharmaceuticals LLCCatalog #NDC 65162-698-90
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.

Cecal cannula care
Cecal cannula care
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.
ReagentChlorhexidine 2% solutionVetOneCatalog #11-09007

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.
ReagentDesitinJohnson & Johnson Pediatrics