This work was supported by the Chair in Poultry Research (M. Boulianne) from the Faculty of Veterinary Medicine of the Universit&#233; de Montr&#233;al. Drs. Boulianne and Parent developed the model. Drs. Boulianne and Parent performed the surgeries. Dr. Burns assisted in developing the anesthesia protocol. Dr. Parent edited the video and wrote the manuscript. Drs Burns, Desrochers and Boulianne edited the manuscript.

All procedures with live animal use were authorized by the Ethical Committee of the Faculty of Veterinary Medicine, Universit&#233; de Montr&#233;al (C&#201;UA, &#39;Comit&#233; d&#39;&#233;thique de l&#39;utilisation des Animaux&#39;).
1. Considerations Before Surgery
<ol>
	<li>Select 10 weeks old specific pathogen free (SPF) leghorn chickens for the surgery. 
	NOTE: Their weight must be between 1.0 and 1.2 kg.</li>
	<li>Withdraw the feed 12 h prior to the procedures to empty the intestinal tract.</li>
	<li>Prior to surgery, place the chickens under general anesthesia following a protocol verified by an anesthesiologist. Premedicate each chicken 15 min prior to the surgery with an intramuscular (IM) injection of midazolam 1 mg/kg and butorphanol 4 mg/kg. Repeat, every 4 h, the injection of butorphanol 4 mg/kg IM or when the breathing pattern changes and frequency increases during anesthesia. For the general anesthesia, during all procedures, administer isoflurane at a concentration between 1.5 and 2.5% with an intratracheal tube. 
	NOTE: The use of premedication for sedation and analgesia is recommended as they&#160;will significantly improve the depth of anesthesia, reduce the quantity of anesthetics necessary during all procedures, as well as enable researchers to control pain levels.</li>
	<li>Monitor the anesthesia by recording body temperature, heart rate and electrocardiogram (ECG), respiratory rate and rhythm, oxygen saturation, expired carbon dioxide (CO2) and neurologic reflexes. 
	NOTE: A person must be assigned to monitor these parameters throughout all the procedures.</li>
	<li>Follow sterility principles throughout the surgical procedures. Ensure that the surgeon and the assistant surgeon dress with sterile gowns, gloves, a mask and protective goggles according to institutional preoperative protocols. Ensure that the surgeon scrubs the hands with a chlorhexidine gluconate impregnated sterile brush up to the elbow with emphasis on fingernails and each finger surface.</li>
	<li>Sterilize the surgical instruments with a sterilizer.</li>
</ol>
2. Surgical Site Preparation
<ol>
	<li>Firstly, manually remove the feathers from the abdomen with a gentle traction.</li>
	<li>Scrub the surgical site with a chlorhexidine gluconate detergent impregnated sterile brush. Gently scrub the skin from the center to the outside for a total contact time of 5 min without coming back to the center.</li>
	<li>Perform 3 alternative passages on the surgical site with a chlorhexidine gluconate solution and isopropyl alcohol using sterile gauzes. 
	NOTE: The passage must start from the middle of the surgical site to the borders to avoid contamination from the non-disinfected areas to the surgical site.</li>
	<li>Place the sterile surgical drape on the chicken.</li>
</ol>
3. Extraction of the Small Intestines from the Abdominal Cavity
<ol>
	<li>Incise the surgical drape with scissors to expose only the skin of the surgical site.</li>
	<li>Incise the skin by performing an &#39;&#39;L&#39;&#39; shape low-midline incision with a scalpel blade #3.
	<ol>
		<li>Start the first incision 1 cm caudal to the sternum and end it at 1 cm cranial to the cloaca.</li>
		<li>Perform the second incision perpendicular to the first incision. Start from the caudal end of the first incision and continue 5 cm to the left side of the abdomen by following the pelvis line. 
		NOTE: This opening will create a flap that will allow an easier extraction of the intestines.</li>
	</ol>
	</li>
	<li>With surgery scissors, cut the peritoneum and abdominal muscles with the same &#39;&#39;L&#39;&#39; shape pattern to open the abdominal cavity. 
	NOTE: This will expose the air sacs. Intestines are located underneath these structures. Air sacs must be humidified with gauzes saturated with sterile saline 0.85 % to avoid desiccation and possible rupture throughout the procedure.</li>
	<li>Insert a Snook spay hook in the abdominal cavity by following the left abdominal wall and work the way around the abdominal air sacs.</li>
	<li>Gently exteriorize the intestines and spread these out on the surgical field. 
	NOTE: Keep intestines humidified by frequently spraying sterile saline 0.9% throughout the procedure. Also, gauzes humidified with sterile saline can be placed on the intestines to avoid desiccation.</li>
	<li>Grab the intestines using a dry sterile gauze and gently pull out the small intestines by manual traction to expose the jejunum. 
	NOTE: Mesenteric vessels are fragile and excessive tension may rupture them which may lead to intestinal ischemic lesions and greatly compromise the outcome of the procedure.</li>
</ol>
4. Fabrication of Intestinal Loops
<ol>
	<li>Creation of loop no. 1.
	<ol>
		<li>Place a simple ligature with a polyglactin multifilament synthetic absorbable material in the proximal jejunum by avoiding the ligature of major mesenteric vessels.</li>
		<li>Place a distal ligature 2 cm away from the proximal ligature. 
		NOTE: Simple ligatures made with multifilament suture material are placed along the intestinal tract to create hermetic segments. A schema (Figure 1) describes the location of all ligatures. The loops consist of a proximal and distal ligature separated by 2 cm.</li>
	</ol>
	</li>
	<li>Creation of interloop no. 1.
	<ol>
		<li>Place a simple ligature 0.5 cm aborally to the distal ligature of loop no.1. 
		NOTE: Between successive loops, there are interloops of 1 cm, i.e. segments making a physical separation between the loops. To likely decrease the possible risk of cross-contamination from one loop to another, a simple ligature is placed in the middle of the interloop (0.5 cm from the distal and proximal ligature of 2 adjacent loops). This reduces the risk of a possible leaked contaminant from a loop to reach the ligature of an adjacent loop. These segments are not mandatory but will decrease the risk of cross-contamination.</li>
	</ol>
	</li>
	<li>Creation of subsequent loops and interloops.
	<ol>
		<li>Repeat steps 4.1 and 4.2 to obtain the number of desired loops. 
		NOTE: In this experiment, 9 loops and 8 interloops were created. Total length amounted to 26 cm. The number of loops can be adjusted depending on the study.</li>
	</ol>
	</li>
</ol>
5. Injection of Clostridium perfringens Strains Into the Loops
<ol>
	<li>Inject with a sterile syringe and needle 26 G the pathogen in loop no.1. Gently insert the needle on the antimesenteric side of the intestine at a 45&#176; angle. 
	NOTE: In this experiment, 0.2 mL of brain heart infusion (BHI) containing 1 x 106 CFU (colony forming units) of C. perfringens in the mid-log growth phase was injected in each loop. 5 different strains were injected in 5 different loops and the 4 remaining loops were injected with a negative control containing no bacteria (sterile BHI), alternating pathogens and sterile BHI loops.</li>
	<li>Repeat step 5.1 to inject all loops.</li>
	<li>Gently replace the intestinal tract in the dorsal area of the abdominal cavity, underneath the air sacs.</li>
</ol>
6. Closure of the Abdominal Cavity
<ol>
	<li>The suture of the peritoneum and abdominal muscles.
	<ol>
		<li>Suture the peritoneum and abdominal muscles along the pelvis (made in step 3.3) with a simple continuous suture pattern using a polyglactin multifilament synthetic absorbable material.</li>
		<li>Suture the peritoneum and abdominal muscles from the sternum to the cloaca (made in step 3.3. with a simple continuous suture pattern using a polyglactin multifilament synthetic absorbable material.</li>
	</ol>
	</li>
	<li>The suture of the skin.
	<ol>
		<li>Using a polyglactin multifilament synthetic material and a simple continuous suture pattern, close the incision of the skin along the pelvis made in step 3.2.</li>
		<li>Using the same multifilament ligature material and a simple continuous suture pattern, close the incision of the skin from the sternum to the cloaca made in step 3.2.</li>
	</ol>
	</li>
</ol>
7. Conclusion
<ol>
	<li>After the surgical procedures, keep the chicken under general anesthesia with proper use of analgesics to minimize animal pain during the infection time. Continue anesthetic monitoring to ensure an adequate anesthesia level.</li>
	<li>After the desired infection time, humanely euthanize the chicken by cervical dislocation under general anesthesia. 
	NOTE: For these procedures, the required time to induce microscopic lesions with C. perfringens pathogenic strains was 7 h.</li>
	<li>With a scalpel blade #3, cut a 0.5 cm to 1 cm long loop section and place it in 10% formalin for fixation overnight and further histopathological analysis.</li>
	<li>With the same scalpel blade, cut the remaining loop section and place it in a sterile microcentrifuge tube for the isolation of the C. perfringens strains in each loop further bacteriologic analysis for their genetic characterization.</li>
	<li>Repeat steps 7.2 and 7.3 for all loops by changing the scalpel blade between every loop.</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NetB,+a+new+toxin+that+is+associated+with+avian+necrotic+enteritis+caused+by+Clostridium+perfringens.">NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens.</a> PLoS Pathog. 4 (2), e26 (2008).</li><li>Timbermont, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+Clostridium+perfringens+isolates+determines+the+ability+to+induce+necrotic+enteritis+in+broilers.">Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers.</a> Comp Immunol Microbiol Infect Dis. 32 (6), 503-512 (2009).</li><li>Paradis, M. 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The authors have nothing to disclose.

Intestinal loops models have been described in numerous species to study host-pathogen interaction and pathogenesis of diseases caused by various intestinal pathogens, such as Clostridium perfringens, Clostridium difficile and Salmonella enterica7,9,13,14,15. It has also been used to analyze the mucosal immune response16, the efficacy of antibodies to neutralize bacterial toxins excreted by intestinal pathogens11 and to evaluate vaccine candidates to prevent intestinal diseases10. However, this model using numerous ligated loops in a single individual animal is poorly described in chickens and standardization of this technique is mandatory to obtain valid results as inadequate manipulations may lead to false results and misinterpretations. This manuscript shows the creation of 9 successive ligated loops located in the jejunum and ileum of the chicken gastro-intestinal tract. This study used Specific Pathogen Free (SPF) leghorn male chickens weighing between 1.0 and 1.2 kg. The maximum number of loops allowed by this breed at this weight was 9, since 26 cm of small intestines could be easily exteriorized from the coelomic cavity. It is not excluded that by using another breed and/or a heavier leghorn male chicken, the total number of loops created may be different, even higher than 9. Intestinal loops length could also be modified, but the 2-cm length was considered optimal to allow sample collection for histopathology and bacteriology while using the maximum capacity of the available intestinal length.
The intestinal ligated loop technique described in this article can be a valuable alternative to the current models available to study necrotic enteritis in chickens caused by Clostridium perfringens. According to the results described in a previous article12, the evaluation of 5 C. perfringens strains with 10 chickens was sufficient to differentiate highly virulent and commensal strains of C. perfringens. In comparison, other infection models using per os inoculation to reproduce necrotic enteritis with C. perfringens will require a large number of individuals to obtain the same results. In the literature, the number of birds needed to evaluate the virulence of C. perfringens strains ranges from 10 to 30 chickens per group17,18,19 to a hundred hosts per group20. In regard to the refinement principle for animal use in research, it would be advised to use the technique requiring the lowest number of hosts to obtain significant results. The intestinal ligated loop model described in this article is most likely the model requiring the lowest number of chickens to compare the virulence of C. perfringens strains.
Since cross-contamination by leakage from a ligature could be evaluated only after C. perfringens culture and characterization by pulsed field gel electrophoresis (PFGE) many days after the procedures, it was of the utmost importance to minimize this risk by including 1 cm interloops containing an interloop ligature mid-way between 2 adjacent loops. By taking these precautions, it was possible to avoid a possible cross-contamination. Indeed, while pathogenic strains caused microscopic necrotic lesions concordant with necrotic enteritis, there were no significant microscopic lesions in adjacent loops injected with non-pathogenic C. perfringens strains or the BHI culture medium negative control. This was indicative that different strains could be safely injected in adjacent intestinal loops of the same chicken.
One of the most challenging issues with this model was the exteriorization of the small intestines without compromising ventilation and intestinal vascularization. Birds have a different respiratory system than mammals. The diaphragm is absent, and ventilation is controlled by airsacs, thin-walled structures in the abdominal cavity. There are 7 of these in chickens and they are divided in the cervical (1), anterior thoracic (2), posterior thoracic (2) and abdominal (2) air sacs. These balloon-like structures are directly connected to the lungs and allow for the movement of air in the respiratory tract. Upon opening the abdominal cavity between the sternum and cloaca, abdominal airsacs are prominent and tend to be easily ruptured, especially if dry. For this reason, constant humidification with gauzes damped with sterile saline is important to ease proper ventilation and anesthesia during procedures. Also, it can be difficult to avoid small punctures in the abdominal airsacs while exteriorizing the intestines out of the abdominal cavity. It is important to monitor anesthetic parameters, such as respiratory rate and pattern, palpebral reflexes, heart rate and chicken&#39;s movements, to make sure the chicken stays under an adequate plane of anesthesia. Small air leakage is less likely to affect the quality of anesthesia, but this leakage must be minimized. It is important to hermetically close the peritoneum after the procedures. This will reestablish the internal air pressure and ventilation will return to normal after this step. After the closure of this structure, there should be no air leakage from the abdomen. This can be verified by spraying a small amount of sterile saline on the sutures and look for bubble formation indicating air coming from the abdominal cavity. If this happens, additional simple skin sutures can be placed at the air leakage site.
Another technically challenging aspect is related to the risk of ischemic lesions developing in the small intestine due to poorly executed surgical procedures. Ischemia happens when there is a restriction of blood supply to tissues, causing a shortage of oxygen and other molecules needed for cellular metabolism. In this case, the intestine will rapidly become macroscopically dark blue instead of pink-reddish and the intestinal mucosal cells will degenerate quickly. This will cause ischemic lesions unrelated to the injected pathogen and it will interfere with the histopathological interpretation. Two problematic situations can lead to ischemia. First, ligature of mesenteric vessels by inadvertence will cause this condition. For this reason, it is crucial to look carefully at the placement of the ligatures along the intestinal tract to avoid including the mesenteric vessels. Second, ischemia can happen if hemorrhages of the mesenteric vessels occur during the procedures. These structures are thin walled and fragile. Excessive tension can lead to their rupture and while manually exteriorizing the intestines, attention should be given to the tension on the mesenteric vessels to ensure their integrity during this step. Also, mesenteric vessels hemorrhage can occur if these are punctured with a surgical suture needle. The surgeon must look carefully at the location pierced through the mesentery with the needle. Bleeding can be stopped in both situations, but the coagulation process will stop or reduce significantly the blood flow to these intestinal segments, hence causing irreversible lesions and compromising the validity of the results.
This model is useful for the study of host-pathogen interactions and pathogenesis of intestinal diseases caused by infectious agents. The model proposed can be adapted depending on the study by changing the number of loops created or by modifying the length of each loop. Before implementing such changes, it is recommended to standardize the method to ensure significant results. Limitations of this technique are skill-oriented and only properly trained personnel should perform this surgery on live animals.

The use of animal models to study infectious diseases affecting humans and animals is a central tool in assessing virulence factors regulating the pathogenesis of a specific condition as well as to elaborate strategies to prevent or cure diseases1. Despite numerous advantages of using animal models to study various diseases, ethical concerns regarding this practice are arising. Researchers need to minimize animal use while obtaining significant and valid results. The concept of the 3 Rs principles (Replace, Reduce and Refine) was elaborated to ensure that welfare issues were addressed in such trials. In chicken necrotic enteritis studies, in vivo chicken models are used to investigate the etiology, prevention, and treatments of this condition2,3,4,5. Pathogenic strains of C. perfringens carrying specific virulence factors, such as the NetB toxin6, are administered to cause necrotic enteritis in chickens7. The replacement principle is, therefore, difficult to achieve in such case as these virulence factors may not be as critical in other animal species. Most models for necrotic enteritis in chickens use a combination of risk factors, such as coccidiosis and altered diet to induce necrotic enteritis followed by gavage of broth culture containing large numbers of C. perfringens2. These models will induce the disease in a large number of chickens, and mortality can be important in such trials8, thus raising concerns about the reduction and refinement principles in animal research.
Intestinal ligated loops models are a desirable alternative for the study of diseases induced by intestinal pathogens with respect to the principle reduction, and refinement. In these models, intestinal segments called &#39;loops&#39; are created by placing ligatures along the intestinal tract to form independent and hermetic compartments where pathogens can be injected alone9 or with other molecules, such as vaccine candidates10,11. The pathogens of interest are placed in close contact with the intestinal cells and after a few hours of infection time, intestinal samples can be recovered for further analysis. This allows the use of multiple treatment and control groups in the same animal. Statistical analysis can be performed with repeated measures models, which increases the power of discrimination between groups and reduces the number of necessary chickens compared to oral gavage trials. Also, surgical procedures and subsequent infection times are performed under continuous general anesthesia and analgesia, hence minimizing the animal pain. Closed loop ligations are an ideal template for reducing host numbers and creating a more humane system in animal research.
Intestinal ligated loops models are well described in various species, such as calves, rabbits and mice2,9, but poorly described in chickens7. For an optimal use of this surgical model, proper technique and execution are essential for the creation of ligated intestinal loops to avoid damages to the intestinal integrity. The goal of this manuscript is to describe a step by step method in the creation of multiple intestinal loops in a chicken model. This technique is limited by the surgeon&#39;s skill and experience, as accurate procedures are essential for the success of the project.

<table><tbody><tr><td>EZ-Scrub 747</td><td>Becton Dickinson and Company</td><td></td><td>4% chlorhexidine gluconate detergent impregnated sterile brush. No catalog number available. Web address: https://www.bd.com/en-us/offerings/capabilities/infection-prevention/surgical-hand-scrubs/ez-scrub-preoperative-surgical-scrub-brushes&amp;nbsp;</td></tr><tr><td>Isopropyl alcohol 70% USP 4 L</td><td>Commercial isopropyl alcohol</td><td>P016IP70</td><td>Web address: http://www.comalc.com/products/</td></tr><tr><td>Chlorexidine</td><td>Sigma-Aldrich</td><td>282227-1G</td><td>Chlorhexidine gluconate solution, must be diluted to 4%. Web address: http://www.sigmaaldrich.com/catalog/product/aldrich/282227?lang=fr&amp;amp;region=CA&amp;amp;gclid=EAIaIQ&lt;br/&gt;obChMI8vHr4_OY1wIV3rrACh2Z&lt;br/&gt;WQKuEAAYAiAAEgLKx_D_BwE</td></tr><tr><td>Surgical Drape Small (27 inch x 24 inch) with 3 x 6 inch Fenestration</td><td>Veterinary Specialty Products</td><td>32724</td><td>Web address: http://www.vetspecialtyproducts.com/index.cfm?fuseaction=ecommercecatalog.&lt;br/&gt;detail&amp;amp;productgroup_id=15</td></tr><tr><td>Scalpel blades #3</td><td>Swann-Morton</td><td>301</td><td>Web address: https://www.swann-morton.com/product/16.php</td></tr><tr><td>Sterile saline NaCl 0.9%</td><td>Sigma-Aldrich</td><td>S8776-100ML</td><td>Web address: http://www.sigmaaldrich.com/catalog/product/sigma/s8776?lang=fr&amp;amp;region=CA</td></tr><tr><td>Vicryl 3-0</td><td>Ethicon</td><td>D9003</td><td>Polyglactin multifilament absorbable suture material. Web address: http://www.ethicon.com/healthcare-professionals/products/wound-closure/absorbable-sutures/coated-vicryl-polyglactin-910-suture</td></tr><tr><td>Syringe 1 ml with 26G needle</td><td>Becton Dickinson and Company</td><td>329652</td><td>Web address: https://www.bd.com/en-us/offerings/capabilities/diabetes-care/insulin-syringes/bd-1-ml-conventional-insulin-syringes</td></tr><tr><td>Brain Heart Infusion</td><td>Sigma-Aldrich</td><td>1104930500</td><td>Web address: http://www.sigmaaldrich.com/catalog/product/mm/110493?lang=fr&amp;amp;region=CA&amp;amp;cm_sp=Insite-_-prodRecCold_xorders-_-prodRecCold2-1</td></tr><tr><td>Eppendorf tube</td><td>Sigma-Aldrich</td><td>T9661-500EA</td><td>Microcentrifuge tube. Web address: http://www.sigmaaldrich.com/catalog/product/sigma/t9661?lang=fr&amp;amp;region=CA&amp;amp;gclid=EAIaIQ&lt;br/&gt;obChMI-YizzfiY1wIVRkCGCh&lt;br/&gt;30SQjuEAAYAiAAEgLK5fD_BwE</td></tr><tr><td>Formalin solution, buffered neutral, 10%</td><td>Sigma-Aldrich</td><td>HT501128-4L</td><td>Ratio tissue : formalin of 1: 10 for adequate fixation. Web address: http://www.sigmaaldrich.com/catalog/product/sigma/ht501128?lang=fr&amp;amp;region=CA</td></tr><tr><td>&lt;em&gt;Clostridium perfringens&lt;/em&gt; strains</td><td>Universit&amp;eacute; de Montr&amp;eacute;al, Chaire en recherche avicole</td><td>N/A</td><td>Specific to each laboratory, available upon request to correspondant author</td></tr></tbody></table>

a ligated intestinal loop model in anesthetized specific pathogen free chickens to study clostridium perfringens virulence

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as coccidiosis and diet, with gavage or administration via the feed using Clostridium perfringens. In these models, the comparison of multiple C. perfringens strains for virulence studies requires a large number of hosts to obtain significant results. Mortality during the course of the study can be high depending on the experimental model, hence raising ethical concerns regarding animal welfare in research. The development of new infection models requiring fewer animals to study pathogenesis, yet providing statistically significant and valid results, is important in reducing animal use in research. Intestinal ligated loop models have been used to study clostridial infections in various species such as mice, rabbits and calves. Following surgical procedures to create ligated loop segments, C. perfringens strains are injected directly into the loops to establish a close contact between the bacteria and the intestinal mucosa. Samples of the small intestine and luminal contents are taken at the termination of the procedures after a few hours. Multiple bacterial strains can be inoculated in each animal, hence reducing the number of required subjects in the experiments. Also, procedures are performed under general anesthesia to reduce animal pain. In chickens, this model would be more appropriate than oral administration to compare C. perfringens strain pathogenicity because fewer animals are needed, no predisposing factors are required to induce the disease, and pain is controlled by analgesics. The intestinal ligated loop model is poorly described in chickens and standardization is essential for its optimal use. This manuscript provides all the necessary steps to create numerous intestinal ligated loops in chickens and brings information on the critical points to obtain valid results.

A schematic of the 9 intestinal loops and 8 interloops is shown in Figure 1. In this model, a total of 9 loops and 8 interloops are created with simple ligatures. A loop consists of proximal and distal ligatures spaced by 2 cm measured from the proximal ligature. Two adjacent loops are separated by an interloop of 1 cm. To decrease the possible risk of cross-contamination between loops by leakage from a ligature, an interloop ligature is placed mid-way from the distal ligature and proximal ligature of 2 adjacent loops. The number of loops can be adjusted depending on the requirements of the study.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57523/57523fig1.jpg" /> 
Figure 1: Schematic representation of ligated intestinal loops. <a href="/files/ftp_upload/57523/57523fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 2A (HPS stain) shows the microscopic appearance of an intestinal loop injected with a control sterile medium. The mucosal brush border is intact and there is no necrosis present in the section. Mild congestion may be present in the intestinal layers (mucosa, submucosa, muscularis, and serosa) of the segments. Figure 2B (HPS stain) shows mucosal necrosis 7 hours following the injection of a pathogenic strain of Clostridium perfringens recovered from a clinical case of necrotic enteritis on a chicken farm. Villi tips are eroded with necrotic enterocytes surrounding the villi tips. In the necrotic material surrounding the villi, clusters of large rod-shaped bacteria are observed (arrows). These bacteria were further identified as Gram-positive bacteria with a Twort&#39;s stain (not shown). This finding, combined with the isolation by bacteriology of large numbers of Clostridium perfringens, indicates the lesions were caused by this bacterium. Figure 2C (HPS stain) shows histological lesions of an ischemic loop, where necrosis is not related to the injection of pathogenic C. perfringens strains but the result of poorly executed procedures. This magnification shows severe coagulation necrosis that could be misinterpreted as lesions caused by Clostridium perfringens. However, no bacteria are observed in this section. Also, the blood vessels in the muscular layer are severely dilated by the accumulation of large numbers of degenerating erythrocytes (arrows). This is indicative of vascular congestion and in this case, the congestion was caused by ligated mesenteric blood vessels. Ischemic loops can be easily identified macroscopically during the surgical procedures; following severe vascular congestion, the serosa will be dark blue instead of its normal pink-reddish coloration.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57523/57523fig2.jpg" /> 
Figure 2: Histopathologic findings 7 h post-infection. (A) There are no necrotic lesions in the mucosa on histopathology following the injection of Brain Heart Infusion (BHI), a sterile culture medium. (B) In loops injected with a pathogenic strain of Clostridium perfringens, there is marked necrosis of the mucosa with numerous large Gram-positive rod-shaped bacteria covering the mucosa. (C) Ischemic lesions can resemble necrotic lesions caused by C. perfringens. <a href="/files/ftp_upload/57523/57523fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In this study, loops were injected with either a sterile control culture medium (BHI) or one of the five different strains of Clostridium perfringens recovered from various antibiotic free chicken flocks affected or not with necrotic enteritis12. Ten chickens were used in this study. Strains pathogenicity could be determined based on ability to repeatedly cause lesions compatible with necrotic enteritis to the mucosa. While strains recovered from clinical cases of necrotic enteritis showed the ability to cause mucosal necrosis 7 h post-infection, strains recovered from clinically healthy chicken farms did not induce mucosal necrosis and the mucosa was similar to the normal findings from the control loops injected with BHI12. By using numerous loops in the same animal, five C. perfringens strains and four control loops could be used in the same bird.

Watch this Scientific Journal Video about A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence at JoVE.com

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Here we present a protocol to surgically create &#39;intestinal ligated loops&#39; in chicken small intestines. This procedure allows for the comparison of multiple Clostridium perfringens strains&#39; virulence in situ in a single host. This method markedly decreases the number of chickens usually necessary for similar in vivo experiments.

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as ...

a-ligated-intestinal-loop-model-anesthetized-specific-pathogen-free

Nanyang Technological University

Research

JoVE Journal

Medicine

9.4K Views.  Université de Montréal. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van Chicken Necrotic Enteritis onderzoek. Zoals het vergelijken van de virulentie van specifieke Clostridium perfringens stammen die deze ziekte veroorzaken. Het belangrijkste voordeel van deze techniek is dat het opmerkelijk vermindert het aantal kippen meestal nodig voor soortgelijke ambivore experimenten.Hoewel deze methode inzicht kan geven in virulentiepotentieel van Clostridium perfringens stammen, ...

Video: A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Cecal Ligation Puncture Procedure

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the host. Currently, sepsis ranks among the top ten causes of mortality in the USA intensive care units 1. During sepsis there are two established haemodynamic phases that may overlap. The initial phase (hyperdynamic) is defined as a massive production of proinflammatory cytokines and reactive oxygen species by macrophages and neutrophils that affects vascular permeability (leading to hypotension), cardiac function and induces metabolic changes culminating in tissue necrosis and organ failure. Consequently, the most common cause of mortality is acute kidney injury. The second phase (hypodynamic) is an anti-inflammatory process involving altered monocyte antigen presentation, decreased lymphocyte proliferation and function and increased apoptosis. This state known as immunosuppression or immune depression sharply increases the risk of nocosomial infections and ultimately, death. The mechanisms of these pathophysiological processes are not well characterized. Because both phases of sepsis may cause irreversible and irreparable damage, it is essential to determine the immunological and physiological status of the patient. This is the main reason why many therapeutic drugs have failed. The same drug given at different stages of sepsis may be therapeutic or otherwise harmful or have no effect 2,3. To understand sepsis at various levels it is crucial to have a suitable and comprehensive animal model that reproduces the clinical course of the disease. It is important to characterize the pathophysiological mechanisms occurring during sepsis and control the model conditions for testing potential therapeutic agents. 
To study the etiology of human sepsis researchers have developed different animal models. The most widely used clinical model is cecal ligation and puncture (CLP). The CLP model consists of the perforation of the cecum allowing the release of fecal material into the peritoneal cavity to generate an exacerbated immune response induced by polymicrobial infection. This model fulfills the human condition that is clinically relevant. As in humans, mice that undergo CLP with fluid resuscitation show the first (early) hyperdynamic phase that in time progresses to the second (late) hypodynamic phase. In addition, the cytokine profile is similar to that seen in human sepsis where there is increased lymphocyte apoptosis (reviewed in 4,5). Due to the multiple and overlapping mechanisms involved in sepsis, researchers need a suitable sepsis model of controlled severity in order to obtain consistent and reproducible results.

The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Immunology and Infection

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.

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 ...

Biology

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein &#946;-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown ...

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...

Murine Model for Studying Postoperative Ileus through Intestinal Manipulation

Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

Mouse Ileal Loop Model: A Surgical Model to Study Intestinal Permeability in the Mouse

Source: Boerner, K., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62093">Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.</a> J. Vis. Exp. (2021)
This video demonstrates the generation of an ileal loop model to study intestinal permeability in a mouse model. This method provides a live model to understand the epithelial barrier function and consequent immune response.

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

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