The research could not have been completed without the dedication and efforts of many persons at the Agriculture and Agri-Food Canada Research Centre, Lethbridge, Alberta, Canada. In that regard, we thank: Tara Shelton for pre- and post-operative care of animals, surgical preparation, and anesthesiology, analysis of samples, and assistance with video imaging; Randy Wilde and Estela Costa for pre- and post-operative care; Jenny Gusse for sample analysis; Byron Lee for the trocar image; and Brian Egland for sheep husbandry. We also thank Brent Selinger (Department of Biological Sciences, University of Lethbridge) for providing video equipment and advice, as well as other assistance (i.e. executive production), and the Curriculum Re-Development Centre, University of Lethbridge for videography expertise. This study was supported in part by grants from the Advanced Foods and Materials Network (G.D.I.), the Canada-Alberta Beef Industry Development Fund (G.D.I.), and Agriculture and Agri-Food Canada (G.D.I. and J.P.K.).

Experimental procedure:
<ol>
<li>All instruments are autoclaved; non-autoclaveable materials are sterilized in Germex cold sterilization solution for a minimum of 10 min.</li>
<li>Sheep are pre-medicated with acepromazine (0.05 mg/kg), glycopyrrolate (0.005 mg/kg), and butorphanol tartrate (0.2 mg/kg) IM, and left for 15-20 min.</li>
<li>The neck is shaved and the skin surgically scrubbed. Once the jugular vein is visualized, 0.1 ml of lidocaine is injected SC over the vein, the catheter is introduced into the vein and secured to the skin using livestock identification tag cement and 2" hospital tape. During surgery, the animals are given 10 ml/kg/hr of Plasmalyte 148 (by gravity flow). </li>
<li>Diazepam (0.2 mg/kg) is administered IV, and anesthesia is induced with thiopental sodium (10 mg/kg). Lidocaine endotracheal spray is applied to the vocal folds, the sheep is intubated, and maintained on isoflurane (1.5-2.5% in 100% O2 at a rate of 4 L/min) for the duration of the surgery.</li>
<li>The animal is placed in dorsal recumbency on a heated mat to help maintain body temperature during surgery. A stomach tube is inserted into the rumen to prevent excessive tympany. </li>
<li>The ventral abdomen is shaved and the skin is prepared for surgery with three Prepodyne surgical scrubs. The abdomen is then draped, ensuring that appropriate exposure of the surgical site (umbilicus to caudal abdomen) is maintained. </li>
<li>A 15-20 cm midline incision is made using an electroscapel, caudally from the umbilicus, and the ileocecal fold is located in the right lower quadrant of the abdomen. The caudal ileum and cecum are then exteriorized. Throughout the procedure, handling of the intestine is minimized and care is taken to ensure the exteriorized intestine is kept moist with warm (37-39&deg;C) sterile phosphate buffered saline (PBS; pH 7.2). </li>
<li>An 85 cm segment of ileum (i.e. the 'intestinal segment') is measured and then clamped with Doyen forceps (intestinal clamps) and large Kelly forceps at each end of the segment. Within the intestinal segment, 'loops' and 'interspaces' between the loops will be established. Doyen forceps are placed on the side of the ileum to be joined (anastomosis), and the Kelly forceps are placed on the intestinal segment side of the ileum.</li>
<li>The ileum is cut between each Doyen and Kelly forcep and the intestinal segment designated for loops is gently flushed twice with 60 ml of warm PBS to remove ingesta. </li>
<li>A cocktail of broad-spectrum antibiotics (200 mg enrofloxacin and 250 mg metronidazole in warm PBS, 60 ml total volume) is distributed throughout the intestinal segment and left for 30 min. </li>
<li>The proximal and distal cut ends of the ileum (excluding the intestinal segment) are joined to form a continuous and functional intestinal tract. First, the ileum is aligned by two stay sutures, placed at the mesenteric and antimesenteric borders of the intestine, respectively. Then, a simple continuous suture pattern (2-0 Vicryl) is completed on both sides of the intestine to join the intestine together. </li>
<li>Warm PBS (3-5 ml) is injected at the anastomosis site to check for leakage and suture failure. Then, the tip of Kelly forcep is pressed against the serosal surface of the intestine, close to the site of the anastomosis site, and advanced inwards at the anastomosis site to ensure the intestinal lumen is large and patent.</li>
<li>The antibiotic cocktail is removed from the intestinal segment. Each end of the segment is closed with a simple continuous suture pattern (absorbable suture; 2-0 Vicryl) followed by an inverting suture pattern using 2-0 Vicryl.</li>
<li>The 85 cm-long intestinal segment is partitioned into three 15 cm 'loops', two 15 cm-long 'interspaces' between the loops, and two 5 cm-long blunt-end compartments at the termini of the intestinal segment (Figure 1). The loops are created by placing a ligature around the intestine using 2-0 silk. It is noteworthy that major mesenteric vessels are not ligated during this procedure, as these are needed to supply the intestine with blood, maintaining intestinal viability. Also, the ligatures are left long, as they will be used to secure the catheters.</li>
<li>Catheters (i.e. silastic tubing) are individually identified using a permanent marker. A silicon ball (5 mm-diameter) encircling the catheter, is placed 4-5 cm from the catheter end to help prevent the catheter from sliding out of the intestine (Figure 1). After sterilization by autoclaving, a non-antibiotic ointment (Orbeseal) is aseptically inserted into the catheter end (3-5 mm); this ensures that the catheter remains patent (i.e. it does not become plugged by sloughed mucosa within the loop). </li>
<li>A small incision is placed at the cranial aspect of each of the three loops. Each hole is enlarged with a catheter introducer (i.e. 'vessel dilator'), and a 6-8 cm segment of catheter is inserted caudally into the loop, until the silcone ball passes into the lumen (Figure 1).</li>
<li>The intestinal wall is secured around the catheter with a purse string suture (2-0 Vicryl). The catheter is further secured to the intestine by tying the catheter with the ligature silk that delineated the cranial end of the loop.</li>
<li>Ampicillin (5 ml; 100 mg/ml in warm PBS) is injected into each loop and interspace, and the intestine is placed back in the abdominal cavity. Cefazolin (8 ml; 100 mg/ml in warm PBS) is injected into the peritoneal cavity through the abdominal incision. </li>
<li>A single small stab incision is made through the abdominal wall adjacent to the midline incision, and the catheters are exteriorized through this site.</li>
<li>The position and tension of the catheters is adjusted within the peritoneal cavity. Abdominal muscles are closed with a simple interrupted suture pattern (absorbable suture; Catgut #3), and skin is closed with an interrupted horizontal mattress pattern (non-absorbable suture; Supramid #1).</li>
<li>A curved hollow stainless steel tube (7 mm ID; 10 mm OD; 65 cm in length) is inserted under the skin near the abdominal incision, tunneled under the skin to an exit site just caudal to the neck and between the shoulders. The catheters are inserted into the tube and pushed forward for at least 50% of the tube length. The tube with the catheters is then pulled from the skin at the exit site, exposing the catheters.</li>
<li>The tension of the catheters is adjusted at the stab incision site, and the skin is closed with Supramid #1.</li>
<li>Individual catheters are identified and placed within a bandage "pouch" which is sutured to the skin with Supramid #1. The pouch protects the catheters from subsequent damage.</li>
<li>Post-operatively, sheep are injected IM with flunixin (s.i.d.) at an initial dose 2.2 mg/kg, followed by a maintenance dose of 1.1 mg/kg s.i.d. for 3 days. Sheep also are injected IM with Hemostam (3 ml) and enrofloxacin (2.5 mg/kg) s.i.d. for 3 and 5 days, respectively. Animals are maintained on intravenous Plasmalyte 148 with 5% dextrose solution, administered continuously with a peristaltic pump to ensure that flow (145 ml/hr) continues regardless of body position. They are allowed to drink ad libitum, but feed is restricted until normal rumen and bowel functions are restored. Food intake, water consumption, body temperature, passage of feces and urine, gut noises, abdominal discomfort, demeanor, and blood glucose concentrations are closely monitored (b.i.d.); when necessary, glucose concentrations in the intravenous fluids are adjusted to maintain blood glucose concentrations within the normal range. Blood chemistry and hematology (i.e. albumin, alkaline phosphatase, alanine aminotransferase, amylase, calcium, cholesterol, creatinine, glucose, inorganic phosphate, total bilirubin, total protein, urea, globulin, sodium, potassium, chloride, partial pressure oxygen, pH, partial pressure carbon dioxide, bicarbonate, and total carbon dioxide, red and white blood cell counts, hematocrit, hemoglobin, differential white blood cell count and platelet count) are monitored before and immediately after surgery, and every other day thereafter. Once normal bowel function commences, intravenous fluid therapy is terminated and animals are transferred to paddocks (i.e. 5-7 days post-surgery). Animals are maintained in individual pens (within sight of other animals), thereby reducing stress and the chance of sustaining damage to their catheter pouches (e.g. chewing by a penmate).</li>
<li>To inject treatments into individual loops, 5 ml of the treatment is injected into the catheter using a sterile syringe fitted with an 18 gauge, blunt-ended needle. To ensure the deposition of all of the treatment into the loop, the catheter is then injected with 3 ml of PBS.</li>
</ol>
Critical points:
Procedures of crucial importance to the success of this surgical procedure include: (1) verifying that the intestine is not twisted on itself before establishing the anastomosis; (2) ensuring the integrity and patency of the anastomosis; (3) adequately securing the catheter to the loop wall with the ligature delineating the cranial end of the loop, but ensuring that the attachment does not affect the patency of the catheter; (4) minimizing intestinal handling and ensuring that exteriorized intestine is kept moist; (5) ensuring the appropriate catheter tension; (6) ensuring that the integrity of the catheter is not affected during muscle and skin suturing; (7) preventing gorging by sheep post-surgery (i.e. during the period of post-operative ileus) which can cause ruminal impaction and impairment of rumen function; and (8) ensuring that animals receive sufficient intravenous fluid therapy for the first 3-5 days post-surgery, that blood glucose concentrations are carefully monitored, and glucose concentrations in the intravenous fluids are adjusted if necessary to prevent hypoglycemia. <img src="/files/ftp_upload/1301/1301fig1.jpg" alt="figure 1" /> Figure 1. A schematic representation of catheterized ileal loops. Note the three 15 cm-long catheterized 'loops', the two 15 cm-long non-catheterized 'interspaces' established between the loops, and the two 5 cm-long terminal segments, all established within an 85 cm-long 'intestinal segment' of a sheep ileum.

<ol>
	<li>Vidal, J. E., McClane, B. A., Saputo, J., Parker, J., Uzal, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Clostridium+perfringens+beta-toxin+on+the+rabbit+small+intestine+and+colon.">Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon.</a> Infect. Immun. 76, 4396-4404 (2008).</li><li>Santos, R. L., Zhang, S., Tsolis, R. M., Baumler, A. J., Adams, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+molecular+characterization+of+Salmonella+typhimurium+infection+in+neonatal+calves.">Morphologic and molecular characterization of Salmonella typhimurium infection in neonatal calves.</a> Vet. Pathol. 39, 200-215 (2002).</li><li>Uzzau, S., Leori, G. S., Petruzzi, V., Watson, P. R., Schianchi, G., Bacciu, D., Mazzarello, V., Wallis, T. S., Rubino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+enterica+serovar-host+specificity+does+not+correlate+with+the+magnitude+of+intestinal+invasion+in+sheep.">Salmonella enterica serovar-host specificity does not correlate with the magnitude of intestinal invasion in sheep.</a> Infect. Immun. 69, 3092-3099 (2001).</li><li>Gerdts, V., Uwiera, R. R. E., Mutwiri, G. K., Wilson, D. J., Bowersock, T., Kindane, A., Babiuk, L. A., Griebel, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+intestinal+'loops'+provide+an+in+vivo+model+to+analyse+multiple+mucosal+immune+responses.">Multiple intestinal 'loops' provide an in vivo model to analyse multiple mucosal immune responses.</a> J. Immunol. Methods. 256, 19-33 (2001).</li><li>Greibel, P. J., Davis, W. C., Reynolds, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+growth+and+differentiation+of+b+cells+isolated+from+follicles+from+ileal+Peyer's+patch+of+sheep.">An analysis of the growth and differentiation of b cells isolated from follicles from ileal Peyer's patch of sheep.</a> Immunology. 75, 601-607 (1992).</li><li>Motyka, G., Greibel, P. J., Reynolds, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agents+that+activate+protein+kinase+C+resue+sheep+ileal+Peyer's+patch+B+cells+from+apoptosis.">Agents that activate protein kinase C resue sheep ileal Peyer's patch B cells from apoptosis.</a> Eur. J. Immunol. 23, 1314-1321 (1993).</li></ol>

All procedures on experimental animals were approved by the Institutional Animal Care Committee (Animal Use Protocol #0609) and followed the guidelines set by the Canadian Council of Animal Care. 
We have nothing to disclose.

In this procedure, we were able to successfully establish catheters in intestinal loops established within the ileum of sheep. By adhering to stringent post-operative care, animals rapidly recovered from the procedure and resumed normal activity 5-7 days after surgery. We determined that the establishment of catheters within loops does not affect intestinal mucosal or immune function (data not shown) and animals can live for at least 8 wk after surgery. This procedure allows us to introduce multiple treatments into distinct immunological compartments over an extended interval, once animals have recovered from surgery, and to empirically measure the physiological and immunological effects of these treatments.
An important consideration for future experiments is the location of the intestinal segment within the small intestine. In this procedure we surgically created the intestinal segment within the &lsquo;immunologically functional ileum&rsquo; as often defined by ileal (continuous) Peyer&rsquo;s patches (5,6). This area can extend cranially up to 2 m beyond ileocecal fold. Although this region is considered anatomically as the jejunum, in the context of immunological function, it is ileum, highlighting the importance of considering function when describing intestinal regions.

<table border="1">
<tr><td>Category / Item	Number</td><td>Company</td><td>Catalogue</td></tr> 
<tr><td colspan="3">Surgeon supplies</td></tr>
<tr><td>Surgical gloves</td><td>WDDCa</td><td>103091</td></tr>
<tr><td>Surgical mask</td><td>WDDC</td><td>MAX53013</td></tr>
<tr><td>Surgical gown</td><td>CDMVb</td><td>3365</td></tr>
<tr><td>OB Apron</td><td>WDDC</td><td>900062</td></tr>
<tr><td>Surgical cap</td><td>WDDC</td><td>903271</td></tr>
<tr><td>EZ Scrub brush</td><td>WDDC</td><td>BDC377479</td></tr>

<tr><td colspan="3">Anesthesia and drugs</td></tr>
<tr><td>Anesthetic machine</td><td>Boyle</td><td>&#8212;</td></tr>
<tr><td>Sodasorb</td><td>WDDC</td><td>108638</td></tr>
<tr><td>Laryngoscope</td><td>WDDC</td><td>&#8212;</td></tr>
<tr><td>Laryngoscope blade (Miller blade #4)</td><td>WDDC</td><td>902667</td></tr>
<tr><td>Endotracheal tube (7.0 - 8.5 mm)</td><td>WDDC</td><td>&#8212;</td></tr>
<tr><td>Lidocaine endotracheal spray</td><td>WDDC</td><td>103364</td></tr>
<tr><td>Acepromazine</td><td>WDDC</td><td>AUS01020</td></tr>
<tr><td>Glycopyrrolate</td><td>WDDC</td><td>SAB2880</td></tr>
<tr><td>Butorphanol tartrate</td><td>WDDC</td><td>AYE304541</td></tr>
<tr><td>Diazepam</td><td>WDDC</td><td>sab790</td></tr>
<tr><td>Thiopental sodium</td><td>WDDC</td><td>103508</td></tr>
<tr><td>Epinephrine hydrochloride</td><td>WDDC</td><td>103856</td></tr>
<tr><td>Doxapram</td><td>Strathcona Pharmacy</td><td>&#8212;</td></tr>
<tr><td>Isoflurane</td><td>WDDC</td><td>100339</td></tr>
<tr><td>Hemostam</td><td>WDDC</td><td>103857</td></tr>
<tr><td>Flunixin</td><td>WDDC</td><td>sch140100</td></tr>
<tr><td>Enrofloxacin</td><td>WDDC</td><td>hll0049</td></tr>
<tr><td>Metronidazole</td><td>WDDC</td><td>101653</td></tr>
<tr><td>Ampicillin</td><td>WDDC</td><td>NOV67185-6</td></tr>
<tr><td>Cefazolin</td><td>WDDC</td><td>nov69397</td></tr>

<tr><td colspan="3">Surgical equipment and supplies</td></tr>		
<tr><td>Electroscapel</td><td>Macan</td><td>MV8</td></tr>
<tr><td>Heating mat</td><td>Kane</td><td>PHMD-48</td></tr>
<tr><td>Stomach tube (Nalgene tubing 5/16" ID)</td><td>Fisher Scientific</td><td>&#8212;</td></tr>
<tr><td>Needle driver</td><td>WDDC</td><td>108338</td></tr>
<tr><td>Addison brown forcep</td><td>WDDC</td><td>911075</td></tr>
<tr><td>Rat tooth forcep</td><td>CDMV</td><td>5275</td></tr>
<tr><td>Kelly forcep</td><td>WDDC</td><td>901133</td></tr>
<tr><td>Mosquito hemostat</td><td>WDDC</td><td>108471</td></tr>
<tr><td>Carmalt forcep</td><td>WDDC</td><td>901225</td></tr>
<tr><td>Scalpel handle</td><td>WDDC</td><td>900285</td></tr>
<tr><td>Scalpel blade #22</td><td>WDDC</td><td>900511</td></tr>
<tr><td>Metzenbaum scissor</td><td>WDDC</td><td>901166</td></tr>
<tr><td>Sharp-sharp scissor</td><td>WDDC</td><td>108264</td></tr>
<tr><td>Towel clamp</td><td>WDDC</td><td>901213</td></tr>
<tr><td>Allis tissue forcep</td><td>WDDC</td><td>901201</td></tr>
<tr><td>Doyen's forcep</td><td>WDDC</td><td>904226</td></tr>
<tr><td>Stainless steel bowl</td><td>WDDC</td><td>903601</td></tr>	
<tr><td>Stainless steel trocar (curved)</td><td>Local supplier</td><td>&#8212;</td></tr>
<tr><td>Catheter introducer</td><td>WDDC</td><td>11440</td></tr>
<tr><td>Surgical towel</td><td>CDMV</td><td>7972</td></tr>
<tr><td>Disposable drape</td><td>WDDC</td><td>900035</td></tr>
<tr><td>Steri drape</td><td>CDMV</td><td>2323</td></tr>
<tr><td>Drape (40 x 40 cm)</td><td>CDMV</td><td>8358</td></tr>
<tr><td>Surgical drape (7" x 11 &#188;" opening)</td><td>CDMV</td><td>2694</td></tr>
<tr><td>Vicryl (2-0)</td><td>WDDC</td><td>JJMJ333H</td></tr>
<tr><td>Silk 2-0</td><td>WDDC</td><td>105248</td></tr>
<tr><td>Catgut #3</td><td>WDDC</td><td>CSL547-2070</td></tr>
<tr><td>Supramid #1</td><td>WDDC</td><td>CSL547-3040</td></tr>
<tr><td>Needles (cutting and non-cutting)</td><td>WDDC</td><td>&#8212;</td></tr>

<tr><td colspan="3">Catheter</td></tr>
<tr><td>Silastic tubing (0.040" ID, 0.085" OD)</td><td>Fisher Scientific</td><td>11-189-15D</td></tr>
<tr><td>Orbeseal ointment</td><td>WDDC</td><td>106344</td></tr>
<tr><td>Clear silicone (732 multi-purpose sealant)</td><td>Local supplier</td><td>&#8212;</td></tr>
<tr><td>Blunt end needle</td><td>WDDC</td><td>905035</td></tr>
<tr><td>Marker (permanent industrial)</td><td>Local supplier</td><td>&#8212;</td></tr>
<tr><td>Catheter pouch (constructed of Elastoplast 7.5 cm)</td><td>WDDC</td><td>104094</td></tr>

<tr><td colspan="3">Bandages</td></tr>
<tr><td>Hospital tape (2")</td><td>WDDC</td><td>103842</td></tr>
<tr><td>Hospital tape (1")</td><td>WDDC</td><td>VLMRENF20</td></tr>
<tr><td>Gauze sponge 4x4</td><td>WDDC</td><td>107551</td></tr>
<tr><td>Gauze cling</td><td>WDDC</td><td>107633</td></tr>
<tr><td>Elastoplast 7.5cm</td><td>WDDC</td><td>104094</td></tr>
<tr><td>Elastoplast rippable bandage</td><td>WDDC</td><td>104093</td></tr>
		
<tr><td colspan="3">Needles and syringes</td></tr>		
<tr><td>Syringe (60 ml)</td><td>WDDC</td><td>104004</td></tr>
<tr><td>Syringe (10 ml)</td><td>WDDC</td><td>bdc301604</td></tr>
<tr><td>25G Needle</td><td>WDDC</td><td>BDC305122</td></tr>
<tr><td>20G Needle</td><td>WDDC</td><td>BDC305175</td></tr>
<tr><td>18G Needle</td><td>WDDC</td><td>903353</td></tr>

<tr><td colspan="3">Intravenous</td></tr> 
<tr><td>Intracath IV catheter (16G 8")</td><td>WDDC</td><td>bdc3831621</td></tr>
<tr><td>IV administration set (10 drop)</td><td>WDDC</td><td>101797</td></tr>
<tr><td>0.9% NaCl solution</td><td>WDDC</td><td>BAXJB1322P</td></tr>
<tr><td>Plasmalyte 148 solution</td><td>WDDC</td><td>BAXJB2534</td></tr>
<tr><td>Dextrose</td><td>WDDC</td><td>MCT60190</td></tr>
<tr><td>Peristaltic pump</td><td>Cole Parmer</td><td>7524-10</td></tr>
<tr><td>Livestock identification tag cement</td><td>Local supplier</td><td>&#8212;</td></tr>

<tr><td colspan="3">Sanatizers / disinfectants</td></tr>		
<tr><td>Germex solution</td><td>WDDC</td><td>mtc60270</td></tr>
<tr><td>Isopropyl alcohol</td><td>WDDC</td><td>104590</td></tr>
<tr><td>Stanhexidine cleanser</td><td>WDDC</td><td>109248</td></tr>
<tr><td>Prepodyne solution</td><td>WDDC</td><td>105710</td></tr>
<tr><td>Prepodyne scrub</td><td>WDDC</td><td>105711</td></tr>
</table>
aWestern Drug Distribution Center LTD, Edmonton, AB.
bThe Veterinary Medication Distribution Center Inc., St. Hyacinthe, QE.

catheterization of intestinal loops in ruminants

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (&lt;24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (&lt;6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period.

Watch this Scientific Journal Video about Catheterization of Intestinal Loops in Ruminants at JoVE.com

Catheterization of Intestinal Loops in Ruminants

We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

catheterization-of-intestinal-loops-in-ruminants

Nanyang Technological University

Research

JoVE Journal

Biology

13.6K Views.  University of Alberta. We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

Video: Catheterization of Intestinal Loops in Ruminants

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

A Mouse Model of Intestinal Partial Obstruction

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), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

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.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...