A Mouse Model of Intestinal Partial Obstruction

Podsumowanie

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Streszczenie

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFRα⁺, and neuronal cells during the development of intestinal obstruction.

Wprowadzenie

Intestinal obstructions are a partial or complete blockage in the small or large intestine which prevents digested food, fluids, and gas from moving through the intestines¹. Due to the obstruction, the blockage induces the intestinal walls to become thickened, narrowing the lumen². Intestinal obstruction can occur as a result of abdominal or pelvic surgeries that cause abdominal adhesion tissue formation or from GI disorders such as inflammatory bowel diseases (Crohn's disease), diverticulitis, hernias, volvulus, stricture, intussusception, constipation, fecal impaction, pseudo-obstruction, cancers and tumors^3,4,5. Intestinal obstructions in these cases often lead to the hypertrophy of the tunica muscularis of the intestine⁶.

PO of the lumen induces intestinal distention, and increases smooth muscle layer thickness around the obstruction in response to the need to continue functional peristalsis^{7,8,9,10,11,12,13}. Animal models of intestinal PO have been developed to study smooth muscle hypertrophy in mice⁷, rats¹⁰, guinea pigs¹¹, dogs¹², and cats¹³ that consistently develop similar hypertrophy within the intestinal muscle layers.

A mouse model of intestinal PO is the most cost effective way to generate and study intestinal obstructions in vivo. Small intestine obstructions are carried out in mice by using a silicone ring surgically placed surrounding the ileum. The PO mice showed an early increase in the number of cells (hyperplasia), and an increase in muscle layer thickness (hypertrophy) after PO surgery^8,15. SMC are the primary plastic cells that are growing within smooth muscle layers in response to the hypertrophic conditions¹⁴, but other cells such as ICC and PDGFRα⁺ cells that are closely associated with SMC, are also repopulated. We have previously reported that the PO mice develop hypertrophy in the small intestine, in which SMC are dedifferentiated into PDGFRα⁺ cells that are highly proliferative^7,15,16. Conversely, ICC are degenerated and lost within the hypertrophied smooth muscle layers during the development of intestinal obsruction⁷. Another major benefit of the PO model is its capacity to induce changes in the enteric nervous system and propagating neurogenic motor patterns. The major propagating neurogenic motor pattern in the mouse small bowel is the migrating motor complex (MMC), which is neurogenic and does not require ICC or electrical slow waves¹⁷. The PO model can provide clear insights into how MMCs and enteric nerves are remodeled by partial obstruction.

Here, we propose a murine protocol for intestinal PO surgery using a silicone ring. Mice receiving PO surgery reliably produce hypertrophy in the tunica muscularis of the small intestine. Within hypertrophic muscle, SMC, ICC, PDGFRα⁺, and neuronal cells are dramatically remodeled.

Protokół

The following protocol has been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Nevada-Reno (UNR) Animal Resources and complies with all institutional ethical guidelines regarding the use of research animals.

1. Animals.

1. Obtain mature (4-6 weeks old) C57BL/6 mice weighing between 20-30 g. House the colony of laboratory mice in a centralized animal facility at UNR Animal Resources.

2. Partial Obstruction Surgery

NOTE: Surgeries are performed in a room dedicated to surgical procedures. All surgical instruments are autoclaved prior to surgery. Sterile surgical gowns and gloves should be worn by all personnel in the surgical room at all times.

1. Preparation for the surgical mouse
   1. Check the anesthetic delivery system to ensure the supply of oxygen and isoflurane is adequate for procedure. Turn on the supply oxygen. Turn on the gas flowmeter and adjust it to 500-1000 mL/min. Place the animal in the induction chamber and seal the top.
   2. Turn on the isoflurane vaporizer to 5%, and monitor the animal until it becomes recumbent. Switch the anesthetic delivery system to the nose cone.
   3. Flush the induction chamber of any residual gas with oxygen, then turn off the induction chamber line while keeping the nose cone line open.
   4. Remove the animal from the chamber and carefully place ophthalmic ointment on the eyes of the animal.
   5. Place the nose cone on a preheated warm pad when gas continues to flow.
   6. Change the oxygen flow to 100-200 mL/min, with 2-3 % isoflurane. If the animal begins to move, gentlyrestrain the animal with the nose cone on until fully anesthetized again.
   7. Monitor the respiration and response to stimulation during the procedure and adjust the percentage of isoflurane (2-5 %) as needed. Animal anesthetization level is monitored by the lack of toe pinch reflex before surgery is performed.
   8. Inject pain medicine (buprenorphine, 1 µg/g of body weight) intraperitoneally away from the incision site.
   9. Apply hair removal lotion on the abdomen using a clean cotton swab. Let the lotion sit for 3-5 min on mouse, then remove the hair using gauze and cotton swabs. Repeat this step until all hair has been removed from the abdomen of the mouse.
   10. Clean the skin with 70 % ethanol using gauze and cotton swabs. Apply swabsticks or povidone-iodine solution to clean the abdomen.
2. Partial obstruction surgery
   1. Drape the surgical site using 25 x 50 cm sterile paper with a 2.5 x 2.5 cm opening in the middle for the surgery area. Secure the drape to the animal by placing sterile strips at the boundaries of the opening and skin.
   2. Make a ~3.0 cm abdominal incision longitudinally using a No. 15 blade scalpel, ensuring that only the skin is incised and avoiding cutting into the musculoperitoneal layer at this time.
   3. Using forceps and surgical scissors, carefully separate the skin from the musculoperitoneal layer without causing any incision to the musculoperitoneal layer. After the layers have been sufficiently separated (approximately 1 cm x 4 cm), identify the linea alba on the musculoperitoneal layer and cut ~2 cm along the linea alba to expose the intraperitoneal cavity using micro-forceps and scissors.
   4. Carefully locate and identify the cecum. Slowly and gently remove the cecum from the intraperitoneal cavity with micro-forceps, bringing the proximal colon and ileum with the cecum outside onto the sterile drape. Immediately moisten the intestinal tissue with 0.9 % sterile saline soaked gauze and keep exposed tissue moistened at all times while they are outside of the abdominal cavity.
   5. Locate and identify the mesentery between the ileum and proximal colon. Make an incision (~1 cm) parallel to, and just below, the ileum, in the mesentery and avoid cutting any vasculature.
   6. Take an autoclaved silicone ring (6 mm in length, 4 mm exterior diameter, 3.5 mm interior diameter). Cut longitudinally to open the tubing and open the ring with micro-forceps.
   7. Insert one end of the opened ring through the incision in the mesentery tissue. Return the ring to a completed ring shape by bringing one end into contact with another, with the ileum surrounded by the ring.
   8. Ensure that the silicone ring completely surrounds the ileum, close the ring with suture and carefully place the intestines back in the intraperitoneal cavity.
3. Surgery closure
   1. Perform a simple continuous suture on the musculoperitoneal layer along the linea alba to close the musculoperitoneal wound with an absorbable suture. After the suture is complete, clean any bleeding with 0.9 % sterile saline soaked gauze.
   2. To completely close the wound, with a separate nylon suture, perform a simple continuous suture on the skin.
   3. After both sutures have been completed, clean the wound with new swabstick or povidone-iodine.
   4. Intraperitoneally inject antibiotics (gentamicin, 150 μL per mouse based on 20-30 g body weight).
   5. After the completion of procedure, turn off the isoflurane vaporizer and allow the animal to breathe only flowing oxygen until it start gaining consciousness.
   6. Once the animal is awake, place the animal into a separate recovery area with thermal support until fully recovered.

3. Post-operation observation.

1. After the completion of surgery, move the animals to an incubator in a recovery room where temperature and humidity is regulated. Monitor the animals postoperatively every 15 min for the first hour then every 30 min for the second hour while the animals are in the incubator.
2. As soon as the prescribed observation is complete, move the animals to their own individual cage and monitor them daily for clinical indications of pain¹⁸, and to ensure that the surgical wound is healing properly without any signs of complications (dehiscence) present.

Wyniki

Partial obstruction (PO) was surgically induced in one month old mice by placing a silicone ring around the ileum close to the ileocecal sphincter. This ring created a partial blockage in the ileum. Sham operations (SO) were also performed without a ring on age/sex matched mice and these mice did not show any symptoms similar to those found in PO mice. Mice quickly recovered from PO surgery within a few hours. They showed no obvious behavioral changes or weakness within the first week, bu...

Dyskusje

We demonstrated that mice receiving the intestinal PO surgery consistently and reproducibly develop intestinal smooth muscle hypertrophy, which mimics human intestinal obstruction. Intestinal obstruction surgeries have been developed for different animals including mice⁷, rats¹⁰, guinea pigs¹¹, dog¹² and cats¹³. The mouse model of intestinal obstruction has time, cost, size, and phenotypic advantages ov...

Materiały

Name	Company	Catalog Number	Comments
Surgical drape	Medical and veterinary supplies	SMS40	40”X100 yards
Underpad, econ, pro plus	Medical and veterinary supplies	MSC281224	17x24”
Iris scissors	Braintree scientific, Inc	SC-i-130
Iris scissors	Vantage	V95-304
Dumont electronic & jeweler tweezers	Dumont	98-180-3
Braided absorbable suture	Covidien polysorb	SL-5687G	5-0, polyglactin
Nylon non-absorbable mono filament	AD surgical	S-N618R13	6-0, nylon
Surgical blade	Dynarex		No.15
Needle holder	Jacobson microvascular	36-1342TC	8.5 inch
Scalpel handle	Flinn scientific	AB1049
Microsurgical scissor	WPI	503305
Petrolatum ophthalmic ointment	Puralube VET		3.5 g
Fluriso (isoflurane)	Vetone	V1 502017	250 ml
Steri-strip reinforced skin closure	3M	R1547
Surgical gloves	Medline	MSG2270
Ear loop face mask	The safety zone	RS700
Avant gauze non-woven sponges	Caring	PRM25444
Surgical cup	Admiral  craft OYC-2	725-A42	2.5 oz
Swabstick	ChloraPrep	260103	2% w/v Chlorhexidine  Gluconate (CHG) and 70% v/v Isopropyl Alcohol (IPA)
Cotton tipped applicator	Puritan	806-WC
Buprenorphine	Zoo pharm	BZ8069317	1 mg/ml
Gentamycin sulfate	Vetone	G-6336-04	100 mg/ml
Fast acting gel cream remover	Veet	8111002
Syringe	AHS	AH01T2516	1 ml with needle
Silicon ring	VWR	60985-720	6 mm in length, 4 mm exterior diameter, 3.5 mm interior diameter
C57BL/6 mice	The Jackson Laboratory		4-6 weeks old