Experimental Animal Models for Studying Intestinal Obstruction

Abstract

Understanding the pathophysiology of intestinal obstruction and exploring potential therapeutic interventions heavily relies on the utilization of experimental animal models. This book chapter provides a comprehensive overview of various animal models employed in the study of surgically induced intestinal obstruction. From rodents to large animals, a range of experimental setups and methodologies are discussed, each offering unique advantages and insights into the complexities of this condition. The chapter provides a guide for researchers aiming to investigate intestinal obstruction, reviewing all aspects of an experimental protocol. Ethical and regulatory regulations, anatomical and physiological differences among species, and surgically induced experimental intestinal obstruction are reviewed. The existing experimental animal models are evaluated regarding their reproducibility and efficacy, as many published models prove difficult to replicate in the laboratory or lack crucial information for researchers. A chapter of this nature would greatly benefit the research community and pave the way for future studies.

1. Introduction

Medicine has acknowledged intestinal obstruction (IO) as a clinical entity for thousands of years and has used experimental research ever since to bring light to our understanding regarding the symptoms, pathophysiology, and management. Ebers Papyrous was the first to present a definition in 1550 BC, and Hippocrates was the first to state a treatment for ileus {ειλεός/eileós}, meaning the squeezed or twisted bowel [1, 2]. Nowadays, IO is characterized by the level, the degree, and the possible cause of obstruction. Thus, it might be of intraluminal, extraluminal, or intramural cause, either partial or complete, and located anywhere across the gastrointestinal tract (GIT). The inability of the intestine to perform normal propulsion of its content, either liquid, solid, or gaseous, triggers adjusting mechanisms to overcome the site of obstruction, such as proximal distension of the intestinal lumen and distal collapse, inhibition of normal secretory and absorptive function and cease of normal gastrointestinal (GI) reflexes. Complete intestinal obstruction is a surgical emergency, whereas conservative management may be therapeutic for partial intestinal obstruction, particularly when located in the small bowel, with success rates reaching up to 90% [3]. Prompt treatment is crucial for prognosis in all types of IO as they pose a major cause of abdominal morbidity and mortality [3, 4, 5, 6]. The overall burden of IO has risen significantly in the last 30 years, from 56.91% in 1990 to 86.67% in 2019. There are geographic disparities of the age, type, sex, and pattern of IO due to the wide spectrum of pathologies hindered behind this clinical entity and the different socioeconomic, cultural, and dietary profiles, yet the overall rising trend establishes a significant public health issue [7]. Τhus, research on IO is an ongoing field of investigation.

Animal models have already had a major contribution to our understanding, regarding the clinical entity of IO. Animal models offer the unique ability of in vivo real-time study of complex physiological and pathological phenomena that are both challenging and ethically impossible to replicate in humans. In vivo research offers the ability to study the sequela of any IO on homeostasis and the function of other organs as well as evaluate the efficacy and safety of surgical and non-surgical treatments and drugs. Experimental animal models are also important mediators in the training of physicians to acquire and advance surgical skills and diagnostic modalities. On the other hand, the advent of medical and research ethics has led to a series of legal acts that protect animals’ rights and welfare and restrict the use of animal testing to cases involving the yet undefined pathophysiological aspects of intestinal obstructions and where viable and translatable alternatives to in vivo animal experimentation are unavailable.

In published literature, there is a large volume of experiments using one or multiple techniques of IO. All of these experiments are very heterogeneous as study protocols examine different aspects of IO and related preventive, therapeutic, or diagnostic insights. Unfortunately, most techniques are described briefly and are not accompanied by images of the procedure or comments regarding intraoperative difficulties or adverse events. For young researchers, all of these aspects might lead them to tough spots in the field. The main drawback, though, is the lack of comparative studies regarding the reliability and reproducibility of experimental intestinal obstruction techniques published by individual researchers.

Even though many aspects of the pathophysiology of IO have been clarified by previous experiments, there are still aspects of IO in healthy subjects or in combination with intestinal pathologies that require further research and perhaps experimental studies. The authors of this chapter encountered all the difficulties of designing a research protocol for experimental IO. Georgopoulos et al. [8] based on clinical observation, aimed to test the never-before-proven hypothesis that the expression or recurrence of Crohn’s disease in humans is related to narrow intestinal passings such as valves, strictures, or tight anastomoses. To prove this hypothesis, the authors set up an experimental research protocol using knock-in mice that combined partial intestinal obstruction and genetic predisposition in Crohn-like disease. Yet, none of the existing methods of partial IO could fulfill the criteria of a long-standing partial IO. The authors decided that a research protocol to define the most suitable model had to be preceded. After reviewing and replicating the existing methods of partial IO, Georgopoulos et al. [9] established a new, robust, reproducible, and refined model of partial IO, ultimately pioneering a novel triple suture technique. This research gave very interesting insights into Crohn’s disease, which has still many obscure pathophysiological pathways, showing evidence that this clinical observation may actually be accurate.

In the following chapter, we aim to provide researchers with a guide for proper protocol planning and decision-making regarding an experimental surgical intestinal obstruction study from scratch.

2. Choosing the most appropriate animal model

One of the parts that need thorough and in-depth study at the time of shaping a research protocol is the part of deciding the appropriate animal model for the translational research project at hand [10]. The aspects that need to be taken into consideration are summarized in Table 1.

1. Ethical and regulatory considerations

2. Availability of animals and housing facilities

3. Translational research suitability of animal species

4. Surgical and anesthetic considerations

5. Overall costs of the study

Table 1.

Crucial aspects to consider when choosing the appropriate animal model.

2.1 Ethical and regulatory considerations

Publications in esteemed journals, as early as the nineteenth century, show interest in the rights of experimental animals [11, 12]. Russell and Burch were the first to use the terms “replacement, reduction, and refinement” in 1959 [13], and a little later, the “3Rs principle” used by Smyth [14]. The 3Rs comprise a series of measures to be taken in order to perform animal experiments in medicine in a more humane way [15, 16].

The principle of the 3Rs is explained briefly in Table 2.

	Basic	Updated
Replacement	Avoiding or replacing the use of animals in areas where they otherwise would have been used.	Accelerating the development and use of predictive and robust models and tools, based on the latest science and technologies, to replace the use of animals in addressing important research questions.
Reduction	Minimizing the number of animals used is consistent with scientific aims.	Appropriately designed and analyzed animal experiments that are robust and reproducible and add to the knowledge base.
Refinement	Minimizing the pain, suffering, distress, or lasting harm that research animals might experience.	Advancing laboratory animal welfare by exploiting the latest in vivo technologies to minimize pain, suffering, and distress and improving understanding of the impact of welfare on scientific outcomes.

Table 2.

Definitions of 3Rs – basic and updated (by the National Centre for the Replacement, refinement, and reduction of animals in research – NC3Rs [13]).

These principles have been widely accepted and are used as a guide for experimental research worldwide, even though they are not implemented to the same degree in all countries and by all researchers [17]. Initiated by the National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs), a checklist of 20 items, the ARRIVE guidelines (Animal Research: Reporting of In Vivo experiments), was suggested in 2010 in order to help authors and journals identify and include the minimum information necessary to report in publications describing in vivo experiments [18]. This checklist includes information concerning the characteristics of animals used, details on housing and husbandry, details on drugs or surgical procedures applied and statistical and analytical methods used, including methods used to reduce subjective bias by the researchers [18]. The ultimate aim was to tackle the problem of serious omissions during experimental research and their potential scientific, ethical, and economic implications. Due to the limited effect on the transparency of reporting in animal research since their first publication in 2020, these guidelines were updated to further facilitate their use in practice [19]. Many national and international regulations and directives have encompassed these guidelines [20, 21], but still compliance with the 3Rs principle is not achieved to the desired extent [22].

2.2 Availability and housing facilities

Animals for research may be either purchased from licensed suppliers or provided from the in-house bred colonies of the research center. Depending on the content and the power analysis of the study, a certain number of animals of certain characteristics (age, sex, weight, etc.) will be needed throughout the study. The researcher should design a detailed timeline for the study, explaining the number of animals needed at certain times, the duration of their housing, the timing of each intervention (surgical, veterinarian, genotyping, breeding, etc.), and make all necessary arrangements to have the available animals throughout the study at the times needed [12, 23]. The researcher should beforehand make sure of the appropriate housing conditions accordingly to the animal species chosen and find the appropriate facility to house the study. Housing conditions such as cages, boxes, or even shelters and outdoor housing in larger animals, are important for the well-being of the animals. There are recommendations for the minimum space in the animal house for every animal species, so care should be shown to the availability of appropriate space for the number of animals being housed at the same time during the study. Special attention should be given to assure healthy environmental conditions, such as the circle of light and darkness, the noise, the temperature, and the humidity, and that adequate and proper – for the study and species – husbandry is readily available [12, 15].

2.3 Translational research suitability of animal species

Translational research is the process that leads from findings of basic science (ideas, insights, discoveries, etc.) to bedside medicine, such as novel treatments, prevention measures, and a better understanding of human disease. A basic step in deciding the appropriate animal model for a research protocol is choosing among the different animal species available for the one that has a higher potential translational impact. In other words, looking for an animal that has a greater genetic, anatomic, physiologic, pharmacologic, biochemical, etc., resemblance or comparability to human.

Both large and small animals have been used as subjects to study IO. Rodents and pigs have prevailed over dogs, monkeys, sheep, rabbits, and horses in experimental models of IO. Hatton et al. [24] reported that from 1966 to 2014, rodents consisted up to 90% of animal species in biomedical research, with rabbits, pigs, dogs, monkeys, and guinea pigs accounting for the rest 10%. The same trend toward rodents is underlined by Oliveira et al. [25], reporting that 73% of ischemia-reperfusion models utilize rodents as animal species, followed by pigs at 9%, dogs at 8%, and felines at 6%. Ewes’ models are mostly cited in the literature in the mid-twentieth century, an era when metabolic mechanisms hindered behind electrolyte changes and clinical outcomes were under the microscope, as well as research on the field of bowel response to trauma and pain [26, 27, 28]. Only 15% of sheep used in experimental research have served as models for gastrointestinal studies as they are herbivores with significant anatomic and physiologic differences compared to humans [10]. Non-human primates, dogs, cats, and Equidae have been used due to the better resemblance of their anatomy and physiology to humans, yet due to ethical concerns, they are protected by the “Animals Scientific Procedures Act,” and their use in experimental medicine is allowed only when other species are not eligible as animal models for a certain aim of study [29, 30]. Parameters guiding the proper animal species, in general, are dictated by the aim of the study and the ethical, legal, and regulatory requirements that protect animal welfare, which are not globally equal and are customized by each country’s constitution.

2.3.1 Small animals

Small animals such as rodents and rabbits, are readily available animals, easy to handle, and more cost-effective in terms of procurement, housing, and maintenance compared to larger animals (Figure 1). Laboratories with limited facilities have the space requirements to host small animals in large samples. Rodents are nocturnal animals which needs to be interpreted in their housing conditions. Rabbits newly introduced to a colony should undergo a mandatory quarantine period that allows the rabbits to adapt to their new environment. Providing rabbits between 12 and 14 hours of light ensures synchronization with the colony’s circadian biorhythm [31]. Genetic manipulation techniques are well established in small animals; thus, researchers have a wide range of modified animals to study specific genetic factors related to intestinal obstruction and explore potential therapeutic targets. Their short reproductive cycles allow for the generation of a large number of experimental subjects in a relatively short time. Gestation lasts approximately 19–21 days for mice with a litter size of 7–11 young, 21–23 days for rats with a litter size of 6–13 young, and approximately 30–32 days for rabbits with a litter size of 5 to 8 young [32]. This is particularly beneficial for studies requiring a significant sample size. On the other hand, the small size may restrict the feasibility of certain surgical procedures and utilizing imaging techniques, making it challenging to replicate complex clinical scenarios related to intestinal obstruction. Adult mice have an average weight of 20–40gr for males and 22–63gr for females. Rats are larger animals than mice, with an average adult weight of 267–500gr for males and 225–325gr for females, while rabbits weigh up to 3 kg at 5 months of age [31]; thus, surgical manipulation is more feasible in rats and rabbits than in mice. Small animals generally have shorter lifespans, limiting the duration of long-term studies and the ability to observe the chronic effects of intestinal obstruction. The average life span of mice is 12–36 months, and of rats is 26–40 months [32]. Gut-associated lymphoid tissue is distributed homologously in rodents and humans by means of quality and quantity [24]. Rodents’ genome is approximately 14% smaller than humans. These models offer several advantages, including ease of genetic manipulation, particularly in immunodeficient mouse models, cost-effectiveness, and the availability of well-established genetic tools. However, despite these benefits, it is important to acknowledge that there are significant differences between humans and rodents. These differences pose limitations in accurately simulating complex diseases and translating research findings to clinical practice.

Figure 1.

Main advantages and disadvantages of small animal models in experimental IO.

2.3.2 Large animals

Large animal models in experimental intestinal obstruction allow for a more realistic simulation of surgical procedures and render the use of surgical instruments more feasible. It is also more feasible to utilize advanced imaging techniques that provide valuable insights into the structural and functional changes associated with intestinal obstruction. Experimental scales are, in general, more comparable to humans in terms of intervention, clinical scenarios, and treatments; thus, they are also the preferred models in hands-on teaching seminars. Their longer lifespan enables researchers to extend observation periods regarding the progression of IO and its outcomes and the conduction of longitudinal studies. Pigs have a lifespan of about 27 years, and dogs of 10–13 years. Gestation in pigs lasts 114 days with a litter size of 12 piglets [33]. Porcine adulthood is reached by the first year of life. The first 6 months of life represent childhood and puberty, which lasts from 6 to 12 months of age [34]. Adulthood in dogs is reached approximately the second year of their life, and they are considered adolescents from 6 months to 24 months of age. Childhood is considered the period from birth till the sixth month of age. An important aspect is that large animal species’ gastrointestinal anatomy and physiology are widely similar to humans; thus, they are more representative models for investigating all types of effects of IO. The porcine genome is proven to be three times more homogenous to humans than the mouse genome, and the porcine immune function approximates humans by 80%, although there are variations in the porcine adaptive immune response [35]. The primary drawback of utilizing large animal models, besides ethical considerations, is the elevated expenditure associated with their maintenance and care. Another important limitation is the limited availability of genetically modified models. Larger animal species necessitate more extensive and specialized housing and surgical facilities, resulting in increased costs for feed, veterinary care, and surgical procedures (Figure 2). There is a scarcity of studies exploring the manipulation of the dog genome [36]. Genetic manipulation in pigs is not as developed as in rodents but has produced an ideal analog to human cystic fibrosis. Besides domestic pigs, there are 29 different lines of pigs, wild type and genetically engineered, available as experimental models [35].

Figure 2.

Main advantages and disadvantages of large animal models in experimental IO.

2.3.3 Interspecies anatomy and physiology

Anatomy and physiology are highly correlated to species evolution and vary depending on sex, age, diet, weight, and genome, not only among animal species but even between breeds. All these parameters are crucial when designing a study protocol and even more so when matching results to human conditions. Kararli et al. [37], Hatton et al. [24], and Zwart et al. [38] have published in-depth descriptions of the comparative anatomy and physiology of the human intestine and commonly used animal species.

Pigs and dogs are omnivore monogastric species with glandular-type stomachs like humans. Rodents are omnivorous monogastric species with both glandular and non-glandular stomachs separated by a bridge, the margot plicatus [38]. Their inability to vomit is attributed to this bridge in combination with their high metabolic rate; thus, no fasting is needed before anesthesia. Rabbits are herbivorous monogastric species with simple stomachs that lack specialized regions and are thin-walled (Figure 3). Rabbits, as rats, are also cecotrophic, meaning that they re-ingest fecal material. Rabbits, as rodents, are continuous feeders with the inability to vomit; thus, no fasting is required prior to anesthesia. Gastric transit time is 3-6 h, cecal material remains in the fundus of rabbits for 6-8 h, and normally, the stomach contains a mixture of food, hair, and fluid even after 24 hours of fasting [39]. Gastric emptying is incomplete in pigs, so they tend to retain food in the stomach even 24 hours later [38]. Cyclic gastric motility has the same phasic pattern and duration per phase in dogs as in humans and lasts approximately 2 hours [38].

Figure 3.

Interspecies gastric mucosal variations. (interpreted image by Kararli et al. [37]).

Humans and rabbits typically exhibit few organisms in the upper GI tract due to lower pH values in the fasting state, while other animals often harbor large numbers of bacteria in the stomach and upper intestine (Table 3). pH value variations along the GIT tract affect drug absorption and metabolism and need to be interpreted when translating research outcomes to human conditions (Table 4). Microflora exhibits lower heterogeneity in the lower intestine in all animals, including humans. While humans harbor a predominantly consistent population of Bacteroidetes and Firmicutes phyla in the GI tract, animals exhibit diverse gut microbiome compositions based on their dietary habits 85% of mouse gut microbiota are absent from human flora [40]. These microbiota variations play critical roles in nutrient absorption, fermentation processes, and disease modulation. Additionally, gut microbiota influence drug metabolism and absorption, with regional concentrations varying across species. Nonetheless, modified animal models, like germ-free rats, offer insights into drug stability and microbial interactions. Mouse models, sharing similarities with human microbiomes, are extensively utilized for host genetics and microbiome studies, facilitating extrapolation of data.

	Human	Mouse	Rat	Rabbit
Stomach	0–5	7–9	7–9	0–6
Proximal small intestine	0–5	7–9	6–8	0–5
Distal small intestine	6–7	7–8	7–8	6–7
Colon	7–10	8–9	8–9	8–9
Rectum/feces	10–11	9–10	9–10	9–10

Table 3.

Distribution of gut flora across different species in bacterial counts at different segments of the gastrointestinal tract quantified as the logarithm of viable organisms per gram of wet weight. Interpreted by Kararli et al. [37].

	Human	Pig	Dog	Mouse	Rat	Rabbit
Stomach; Fasted state	0.4–4		1.5 ± 0.04	4.04	3.9
Stomach fed state	1.5	4.5	3–5	2.98	3.2	1.5
Jejunum	6.6 ± 0.5	6.2	6.2	5.01	6.13	6.8
Ileum	7.5 ± 0.5	6.9–7.5	6.6–7.5	4.8–5.2	5.93	7.5–8
Colon	6.4 ± 0.6	6.8	6.5	5.02	6.23	7.2

Table 4.

Interspecies pH values of the GIT incorporated by Ηatton et al. [20].

The measurements of intestinal length in these studies refer to cadaveric observations, and as noted by the authors, measurements are longer than in vivo length. The jejunum is the longer part of the GIT in most animal species. Figure 4 shows the gross GIT anatomy of the most commonly used animal species, and Table 5 summarizes the differences in intestinal length between the human intestine and the most commonly used animal species. Porcine intestinal length is 20-fold longer than that of humans, approximately 23 m (range 15-24 m) [37], but if the size of intestinal length in meters is adjusted to body weight in kilograms, it results in a ratio of approximately 0.1 which correlates to the human ratio. The small intestine is twice the size of the human, approximately 14.5 m long, and the colon is 4.7 m. The caliber of the small intestine is around 3.5 cm, which is close to the human caliber and shares a similar finger-shaped architecture of the microvilli. The main differences between porcine models are the lack of an appendix, the enlarged cecum, the spiral configuration of the large bowel, and the inverted lymph node structure with lymphoid follicles present as meter-length strips [24, 35, 41]. The canine anatomy and physiology are more similar to humans than any other species. Canine intestinal length is half the size of the human intestine, approximately 4 m in total length [37]. Canine body length of 0.75 m correlates to 3.9 m of small intestine 3.9 m and 0.6 m of large intestine. The small intestine has longer and more slender villi, creating a capacious absorptive surface area with an estimated 34 microvilli per micrometer of villi and 23 villi per square millimeter of freeze-dried dog jejunum and ileum [24]. The diameter of the small intestine varies between different canine breeds. The cecum is proportionally equal to human cecum but coiled and appears as a diverticulum to the right of the ascending colon [24]. The main anatomical differences of canine models are the absence of an appendix, the absence of haustra on the large bowel, the shorter mesentery, which results in a relatively fixed position of the colon, and the absence of sigmoid colon.

Figure 4.

Differences in the macroscopic GI anatomy among species (incorporated image by Kararli et al. [37] and Ziegler et al. [40]).

	Human	Pig	Dog	Mouse	Rat	Rabbit
Average total intestine length (m)	7	23.5	4.82	0.5–0.55	1.2–1.7	5.82
Small intestine (m)	6.25	14.16	2.48	0.34	1	3.39
Cecum	0.15	0.23	0.08		0.04	0.44
Appendix	+	—	—	—	—	+
Colon(m)	1.5	4.27	0.6	0.09–0.142	0.26	1.23

Table 5.

Interspecies intestinal length interpreted by Hatton et al. [24], Kararli et al. [37], Zwart et al. [38], Ziegler et al. [40].

The total intestinal length of mice is approximately 0.5-055 m. The small intestine is approximately 0.34 m long and has a finger-shaped architecture of the microvilli, as seen in pigs and humans. The diameter of the small intestine ranges from 2.3 mm to 2.9 mm. The cecum is the most enlarged part, with a diameter of 4.9–5.4 mm, and has no appendix [42]. Their colon is approximately 0.09–0.14 m long with a diameter of 2.7–2.9 mm, lacks haustra, and it is not clearly divided into sections as the human colon [43]. Rats’ intestinal length is approximately 1.2–1.7 m. The small intestine is approximately 1–1.4 m with a diameter of 3–5 mm. The rat colon diameter is approximately 10 mm [37]. Microvilli has a tongue-shaped architecture with 65 microvilli per square micrometer of villi surface. Rats’ colon is approximately 0.22–0.26 m, lacks haustra, and its first part is structured like a cecum. The sigmoid colon is absent, and so is the gallbladder. Rabbits’ intestinal length is 5.8 m. The small intestine is approximately 2.70 m, and the large intestine is 1.95 m with a diameter of 0.3–0.5 mm. The jejunum is the dominant region of the small bowel and ends up with a well-defined appendix rich in lymphoid tissue [24]. The cecum in the rabbit is a well-defined, thin-walled, and coiled structure that can hold up to 40% of the GIT content, meaning it is 10 times more capacious than their stomach.

2.4 Surgical and anesthetic considerations

2.4.1 Surgical considerations

A series of surgical and anesthetic factors should be assessed by the research team (e.g., the surgeon, veterinarian, animal care staff, and the investigators) before concluding with the preferred experimental model. The presence of a multidisciplinary team in a surgical project seems to increase the likelihood of a successful outcome [44].

The surgical procedures are categorized as major or minor depending on the physical or physiological impairment or the presence of extensive tissue removal or other major alterations. Surgical procedures involving the intestine may be major if the effect is for example total intestinal obstruction. Animals recovering from major surgical procedures may require extra care for pain, discomfort, and complications, close postoperative monitoring to assure animal well-being, or euthanasia if pain or distress cannot be alleviated medically. Minor surgical procedures usually have minimal complications, and animals shortly return to normal function [15, 45].

The researcher should try to choose among surgical models that are reliable and reproducible. Many literature articles, as stated elsewhere in this chapter, fail to report all the needed information in order to be reproduced, and many seem never to have been replicated by other researchers. Reliable models are those that show results in correlation to the certain surgical effect inflicted. Reproducible models are those that have been replicated by other researchers with the same or comparative results [23].

Nevertheless, all surgeons to be involved in a study should be of adequate expertise for the certain procedure and agree to every detail relevant. The complexity and difficulty of the surgical procedure or intervention chosen should be relevant to the dexterity and expertise of the team. Surgical training in non-living models such as artificial simulators, animal tissues, animals euthanized for other reasons, and pilot studies may be required before commencing the experiment, in order to improve the available skills on a certain animal species, test the feasibility of the procedure and assure the consistency of the team [15, 23].

Surgical outcomes should be monitored continually and assessed by the research team for the protection of the well-being of the animals and in order to make appropriate changes in time for a successful outcome [15]. Other surgical details such as the use of aseptic technique, the preparation of the surgeon, and the sterility of instruments and materials should be answered, since they may create extra logistic concerns. The aseptic technique is widely accepted to reduce the likelihood of infection, though some small species such as the mouse or rat, show no complications with just clean surgical conditions [9, 15, 46].

2.4.2 Anesthetic considerations

Detailed guidelines and current best practices in the use of anesthetics and analgesics are outside the scope and purpose of this chapter. The researcher should seek this species-specific information in laboratory guides or veterinary directives according to the design, the available equipment, and team expertise. We are limited to a few general principles and tips that are potentially helpful for the researcher.

2.4.2.1 Analgesia

It is generally accepted that animals experience pain in a similar way to human. Pain in animals may affect the experimental results by delaying recovery, anorexia, altering their mental status, creating major endocrine responses, and more. Thus, scientific reasons, as well as ethical reasons, compel the use of analgesics in surgical research [12, 47, 48]. The regimens, the dosage, and the indications or contradictions depend on the animal and the surgical intervention and should be individualized in every experiment [12, 47, 48, 49]. Preemptive analgesia (preoperative and intraoperative analgesia) seems to be beneficial in optimizing the intraoperative and postoperative course of the animal and perhaps should be implied in experimental protocols with major surgical procedures [47, 49, 50, 51]. Post-operative analgesia should always be administered until at least the restoration of food and water intake and resuming of the normal behavior of the animal. Frequent monitoring of the animals’ behavior port-operatively will indicate the timing and length of treatment [50]. Even though simple analgesics usually do not interfere with the scientific findings and are widely used in research, special care should be given with regard to the effects of certain agents on certain tissues to avoid any unwanted confounding factors.

2.4.2.2 Anesthesia

The type of anesthetic agents used in an experimental study is usually guided by numerous factors such as the expected length of the surgical procedure, the available equipment of the operating facility, the expertise of the team, the presence of veterinarian, the animal species and first and foremost the accepted current practices in regard to animal welfare [47, 50]. The two main anesthetic regimen categories are injectable and inhalant. Injectable anesthetics can be administered through intramuscular, intraperitoneal, or intravenous injection. Inhalant anesthetics can be used by anesthetic chamber or face mask and maintained using a face mask (in smaller animals especially) or an endotracheal tube. Although inhaled anesthesia is considered safer than injectable, its use in literature is limited to larger animals due to lack of equipment or expertise of the researchers [47, 50, 52].

Intraoperative monitoring of anesthetic depth and vital signs, such as body temperature, cardiac rate, blood pressure, and respiratory rate, increases the likelihood of a successful surgical outcome. The recovering period post general anesthesia is of high risk for advent events that may be lethal. Continuous attention should be given until the effects of the anesthetic regimens have worn off, and the researchers should be vigilant for any supportive treatment to be given timely. During that time, it is perhaps the best time to administer the first dose of post-operative analgesia to benefit from the long-term effects of analgesia and less adverse events during recovery [50].

2.4.2.3 Euthanasia

The researchers should include in their study details of the euthanasia procedure, considering that practically all experimental animals will eventually be killed before their life span, with very few exceptions. The timing of killing may be scheduled beforehand by the design of the study, such as collection of tissues or reaching the end of their breeding life or stock use. Animals, though, should be killed if they are in severe pain or suffering without possibility of recovery (Article 8 of Council Directive no. 86/609/EEC). Killing an animal should be always carried out in a humane way [12, 20]. There are several ethically and scientifically acceptable methods of euthanasia available that are individualized for each species [47]. Two widely acceptable ways for most animal species are overdose of injectable anesthetic agents and overdose of anesthetic agents by inhalation with exposure to carbon dioxide [45, 48, 51, 53].

Before the study begins, humane endpoints should be defined. These refer to the earliest indication that an animal, during an experiment, is in severe pain, suffering, or distress, or there are signs of impending death. The study team should describe humane endpoints in meaningful terms, establish observation schedules for prompt recognition and make all arrangements for provision of measures to alleviate these symptoms or signs at sight. In case of irreversible situations, the animals shall be euthanized without delay [45, 50].

2.5 Overall cost of the study

The overall costs of the study are usually a definitive factor in an experimental study. In order to calculate a realistic budget for each study, it is crucial to have all the available information and costs from every aspect of it, which can be done after in-depth study and detailed design. Some important costs to take into consideration -where applicable- are the cost of purchasing the total number of animals, the cost of maintenance, housing, and husbandry per day and animal, the cost of drugs and surgical tools or materials, the cost of tests on the animal (blood, stool, urine, genetic, etc.), the cost for disposing of the animals according to international regulations and the cost of any special studies on the animals or the specimens (molecular, histologic, nuclear, radiologic, etc.) [23].

3. Types of experimental intestinal obstruction

All surgical procedures (Figure 5) must be preceded by proper housing, fasting, anesthesia, and prepping of the animals. Basic surgical skills are required in order to safely enter the abdominal cavity and handle the intra-abdominal tissues so as to prevent surgical complications and reduce perioperative mortality. Basic knowledge of instruments and tissue handling is also essential for researchers who aim to investigate an experimental IO.

Figure 5.

Experimental surgical intestinal obstruction models.

3.1 Mechanical intestinal obstruction

Mechanical IO refers to an anatomic barrier that might be either partial or complete. In complete IO, the propulsion of intestinal contents is totally halted beyond the site of obstruction, whereas in partial IO, some gaseous or liquid enteric content is able to pass distally. Mechanical IO is further divided into acute or chronic, depending on the time of onset of symptoms. The underlying pathology might be extrinsic such as adhesions, hernias, intra-abdominal masses, and volvulus, and intrinsic such as intestinal neoplasmatic lesions, inflammatory diseases, congenital malformations, duplications, or intraluminal, including intussusception, foreign bodies, hematomas, gallstones, and feces [3, 54, 55].

3.1.1 Complete mechanical obstruction

Ligation models: A simple ligation of the lumen at the desired level of obstruction has been a commonly cited technique since the beginning of the twentieth century [26, 27, 28, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73] and is probably the most feasible and cost-effective model to produce an acute complete intestinal obstruction. The desired intestinal loop is identified after entering the abdominal cavity, and the adjacent mesentery is carefully dissected so as to create a small mesenteric window but not to damage the mesentery itself or the mesenteric marginal vessels. A suture ligation is passed around the intestinal lumen and tied. Silk sutures are most commonly used, but any type of suture material is acceptable [59]. This technique is easy to perform even from researchers with basic surgical experience. The main drawback is that the pressure force of the ligating suture cannot be controlled, and neither is reported using measurable values. An overly tied ligation will crush the intestinal lumen and result in rapid necrosis. Enochsson et al. [74] reported a loose ligation around the intestinal lumen of rats, yet this is not an objectively measurable value. Many researchers note that they place the ligature in such a way so as not to strangulate the intestinal lumen. This is not yet proved by objectively measuring different levels of tightness.

Other methods used to generate an acute complete obstruction utilize clips, automatic mechanical suturing devices, rings of various materials such as silicone tourniquet catheters, GoreTex bands, cotton threads, umbilical tapes, and clamps placed around the intestinal lumen or in Ω shape [46, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87]. Transection of the bowel, either via open or minimally invasive approach, is also reported in order to induce an acute complete IO [88]. Correa-Martin et al. [85] utilized a forced laparoscopic suture at the ileum near the ileocecal valve of 10 large female pigs, reporting only one death due to bowel perforation. Berlin et al. [46] used a ligating clip to induce small bowel obstruction in 19 male rats, noting that tight apposition was avoided to prevent local necrosis without recording intraoperative adverse events. Zhang et al. [80] created an Ω shape clamp from a double flat iron-core binding wire of 3 mm width in order to produce a more graduate, stable, and reversible complete obstruction of the small intestine in rats. A total of 200 rats were used of which 60 were assigned to recovery groups after de-obstruction, reporting 10 deaths and minimal intestinal injury by the clamps. An obstructive device was developed by Fraser et al. [89, 90] by attaching a silicon rubber sheet at the balloon tip of a silicone rubber urethral catheter. The dimensions of the materials used to produce the device were adjusted to the predicted intestinal lumen of the three species they used: 5 monkeys, 7 dogs, and 20 rats. The device was wrapped around the intestinal lumen, the edges of the silicon sheet were sutured, and the free end of the catheter was fixated to the abdominal wall. As reported by the authors, they were able to predictably and progressively replicate both acute and chronic complete IO by changing the insufflation level of the balloon.

Strangulation models [78, 91, 92, 93, 94, 95, 96, 97, 98, 99]: By definition, strangulation refers to a disruption in the blood supply to the intestine. Vascular occlusion can be achieved either by ligating or clamping the vessels. A simple technique to strangulate the intestine is to ligate an intestinal loop with its adjacent mesentery by passing a simple ligating suture around its basis so as to create a “fan” from the loop and the mesentery. Another common technique is ligating separately an intestinal segment and its mesenteric feeding vessels or the main feeding vascular branch. This is called the segmental mesenteric vascular occlusion by Gonzalez et al. [100] while other authors report this as the closed-loop strangulation technique [101, 102, 103]. This model can be performed both in large animals and rodents, providing multiple treatment groups in a single animal as multiple loops can undergo varying durations of ischemia, with or without reperfusion, allowing for adjustment of the degree of injury. Oliveira et al. [25] in order to precisely locate the marginal artery branch, injected blue liquid dye in the jugular vein and carotid artery of a “pilot” rabbit before the experiment and managed to establish consistency of the occlusion site. Matsuo et al. [104] placed an acrylic ring around a balloon and a loop of the distal ileum of rats. By inflating the balloon, they managed to obstruct blood flow to the adjacent intra-ring loop, followed by deflation to reverse the strangulation. Fevang et al. [105] placed an infant blood pressure gasket around a loop of porcine ileum and increased the pressure until venous pressure reached 50 mmHg and managed to simulate strangulation. This technique is not applicable to small animals due to size restrictions. Manual rotation has been utilized by Doğan et al. [106] and Darien et al. [107] in order to simulate the volvulus of the small intestine in rats and the ascending colon in ponies, respectively. All of these models can be used to study ischemia and reperfusion intestinal injury by reversing the occlusion of the blood supply strangulation models are valuable to study clinical scenarios such as volvulus or incarcerated hernias.

Atresia models: Ligation of mesenteric vessels that perfuse a part of the small intestine of animal fetuses was the first described technique to induce congenital intestinal atresia based on the theory of an intrauterine vascular insult being the pathogenetic mechanism [108, 109, 110]. Baglaj et al. [111] used a microsurgical bipolar coagulator instead of ligation to produce the vascular ischemic event in chick embryos. Researches also utilize the intestinal loop ligation model, using fine sutures 9–0, 10–0, or 11–0 to ligate an intestinal segment, to surgically induce congenital IO in rat, rabbit, chick, and sheep fetuses [110, 112, 113, 114, 115, 116]. These techniques require advanced surgical skills, advanced equipment, and specialized laboratories to offer optimum care to mothers and offsprings. Certain pharmacological agents or toxins such as adriamycin [117] when administered to pregnant animals during critical stages of fetal development, disrupt normal intestinal development and induce atresia-like abnormalities in the offspring. Genetic engineering techniques have also been used to introduce specific mutations or alterations in key genes associated with intestinal development and homeostasis. This approach allows researchers to create animal models with genetic predispositions to intestinal atresia and study the underlying mechanisms involved [118].

3.1.2 Partial mechanical obstruction

Partial IO models exhibit great variation in literature regarding terminology. Partial or incomplete obstruction refers to difficulty passing intestinal content beyond a specific point, which could result from external pressure (such as from a tumor, aneurysm, and adhesion) or stenosis. Models to induce external pressure in order to induce partial IO are mainly modifications of the aforementioned techniques using rings of different materials, clips, and penetrating ligations placed on only a proportion of the enteric lumen [119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130]. Stenosis, on the other hand, specifically refers to a scenario where the intestinal lumen is reduced in diameter due to a lesion within the intestinal wall, characterized by localized narrowing, irregular muscularis, and thickened submucosa, which needs to be testified by histology. Partial IO might be the onset of a complete obstruction. Models of partial IO must justify a chronic obstruction and yield different degrees of incomplete obstruction or stenosis without crashing the intestinal lumen.

Intestinal rings [9, 76, 83, 123, 124, 126, 127, 128, 130, 131, 132, 133, 134, 135]: The intestinal ring technique is commonly employed in research, yet there is great heterogeneity regarding the size of rings and recoding of the reported ratio of obstruction. The main aspects of the technique are cutting a strip of available material (silicone, polyethylene, polyurethane, plastic film, and penrose) to form a band and pass it around the desired part of the intestine through a mesenteric window, followed by edge-to-edge suturing in order to form a closed circumferential structure around the intestinal lumen. In vitro replication prior to in vivo experimentation is advised, as it might be forceful to penetrate the edges of the strip, depending on the material used, thus causing inadvertent complications. The main drawback of this technique though is the lack of a standardized ratio of ring to intestinal diameter to produce a consistent type of partial IO. Morel et al. [122] described the use of multiple adjustable rings to an isolated loop of jejunum, without recording of ring sizes and material and concluded that maintaining ≥60% of the original diameter allows for normal flow of the content while a diameter of ≤30% results in bowel rupture.

Clips [9, 136]: The clip technique is easy to perform and requires basic surgical skills in order to enter the abdominal cavity and locate the desired intestinal loop. No handling of the mesentery is necessary. Besides its feasibility, this technique is not regularly reported in partial IO models. As reported by Georgopoulos et al. [9] it is not a somatometrically adjustable technique and it is difficult to create the same degree of stenosis and apply the exact closure force needed in order to create stenosis but to not crush the tissue. Another interesting note is the rejection of the clip at the time of autopsy, which might result in rise in the number of animals used and experimental time.

Ligation models: Ligation models can be divided into “tube ligation” and “simple ligation.” In tube ligation models, a tube is placed on the longitudinal axis of the desired portion of the intestine, and a ligation is passed around the tube and the intestine and tied. The tube is then removed, and the knot is left intact surrounding the intestine [9, 125, 137]. Different sizes and material of tubes have been reported in the literature. In a comparative study of partial IO by Yuan et al. [125], they concluded that the wide pipe group of their study was the most effective in replicating incomplete IO. This group used a pipe of 10 mm in length and 6 mm in width for “tube ligation” of rat ileum, yet animals were euthanized at 72 h post obstruction. Georgopoulos et al. compared experimental partial IO and reported mortality before 96 hours for all tube sizes, with only one animal surviving until the sixth postoperative day; thus, they concluded that it should be considered more of a complete obstruction model. “Simple ligation” [9] is penetrating the intestinal lumen on a proportion of its diameter. A non-absorbable suture is passed through the lumen, transmurally, on its antimesenteric border, and tied. This is also a technically not demanding technique as there is no dissection to the mesentery; it needs simple instruments and basic skills. Researchers must ensure obstruction of the enclosed tissue without crushing it as the knot is tied. Georgopoulos et al. [9] developed a triple suture technique, overcoming the vulnerability of the single partial ligation and achieving a long-term survival of animal models up to 4 weeks postoperatively. In the triple suture technique, three consecutive penetrating sutures were passed through the lumen, transmurally, on its antimesenteric border with a distance of approximately 1.5 mm between them.

Some authors have reported isolated partial IO models that are more challenging to replicate compared to the aforementioned techniques. Collins et al. [138] reported a technically challenging method on porcine models of creating a partial IO by combining a wide band of polytetrafluoroethylene placed around the intestine and folding the intestine telescopically on top of this band to create a valve mechanism. Besides being technically demanding, this technique also deems necessary at least 7 cm of intestine for its construction. Hiratsuka et al. [139] developed a porcine animal model of colonic stenosis using a silicon sheet (40 × 80 mm) onto which there were attached silicon rubbers at intervals of 5 mm. After locating the desired level of the colon by colonoscopy, they created a window at the mesocolon, passed the sheet circumferentially, and sutured its edges in order to produce the stenosis and observed the animals for 14-19 days. Putranto et al. [140] utilized three plastic cable ties placed at a 1 cm distance over an encircled standard plastic sheet covering the intestines and reported effective fibrosis in 24 h postoperatively when animals were euthanized. Lukas et al. [141] developed a model of surgically induced stricture to mimic CD anastomotic strictures. In this method, authors performed a modified Roux-en-Y side-to-side ileosigmoid anastomosis followed by at least four postoperative endoscopic injections of phenol/trinitrobenzenesulfonic acid solution at the anastomotic site; 15 of the 19 pigs used completed the 6 months follow up and only one adverse event of rectal prolapse is documented.

The main question raised in partial IO models is how long these animals survive with these techniques and how some of these partial obstruction models are different from chronic complete obstruction models, especially when the technique is about forming a ring-like structure around the intestinal lumen. Sun et al. [124, 142] commented that their 20 x 10 mm rectangular polyethylene ring placed on the jejunum of male rats to induce partial IO was looser than the 30 x 5 mm rectangular polyethylene ring on the ileum of male rats for complete IO, yet this conclusions lack proved consistency. Galvez et al. [121] utilized three techniques of experimental partial IO in rats: a longitudinal serosal approximation of the intestinal lumen estimated to cause 50% partial, a 5-mm-wide GoreTex ring around the colon estimated to cause an obstruction of approximately 35% of the intestinal lumen and a suture fixated to an avascular section of the mesentery before encircling the colon and tied up to a 15% partial IO. Of the three study groups, none of the gore-text ring subjects survived the cut-off of 6 weeks. Georgopoulos et al. [9], in an experimental comparative study of all techniques for partial IO, comment that all rings failed to meet the 4-week survival cut-off. Two weeks is considered the least time needed for histopathological characteristics of partial obstruction to develop [9, 75]. Thus, if a chronic partial intestinal obstruction such as stenosis is the aim of the study, the experimental model needs to establish a long-standing obstruction with survival rates longer than 2 weeks.

Adhesion models: Intraperitoneal adhesions can result in both complete and partial IO. As surgical instruments and materials are still evolving, experimental adhesion models are an ongoing field of research. All operative aspects may act as adhesiogenic factors by inducing a foreign-body type reaction. Mechanical and chemical intraoperative stimuli, such as types of gloves, types of gauze, moisture level of gauze, forceps, intraperitoneal anesthetic agents, or lavage, all induce inflammatory reactions to the intestinal wall and need to be carefully and in detail addressed in a protocol of experimental adhesive IO. Cecal abrasion alone or combined with other methods of intestinal manipulation is probably the most commonly cited method for postoperative intestinal adhesions, and it is accepted that the extent of adhesion formation is directly proportional to the extent of abrasion, even though quality and quantity are not standardized [23]. Simple laparotomy or laparoscopy with or without handling of the intestine, abrasion of cecum with or without abrasion of other organs, abrasion of the peritoneum, abrasion of the abdominal wall, intraperitoneal injection of various substances, drains, and modification of these experimental techniques by changing force, time, suturing materials, gloves, use of electrocautery, healing agents, meshes, inflammation agents and instrumentation have all been applied to replicate postoperative adhesive bowel obstruction [23, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153]. Authors have also published their experiments on how radiation, desiccation, thermal injury, bleeding, ischemia, endometriosis, cancer, pain, and peritoneal irritation affect the formation of adhesions [23, 154, 155, 156, 157, 158]. The main drawback of all of these techniques is the inability to standardize them in an objective countable method.

3.2 Functional intestinal obstruction

This type of IO refers to a wide spectrum of diseases characterized by intestinal transit alterations that mimic the signs and symptoms of mechanical IO as described in the introduction, yet there is no actual anatomic barrier. Functional IO represents an asynergia of the muscular and nervous intestinal plexus due to either a disruption of their homeostasis, genetic alterations, or because of a systematic disease. Also described as paralysis of the intestine or ileus, functional IO may affect both the small and the large intestine, whereas pseudo-obstruction is a functional obstruction affecting only the colon. Most common causes are postoperative ileus after any kind of surgery, intrabdominal infections, sepsis, decreased blood supply, pharmaceutical side effects, cystic fibrosis, neuromuscular diseases, and prolonged hospitalization [55, 159, 160]. Transgenic, knock-out and genetically modified animal models are available and commonly used to study functional IO. The surgical models of functional IO aim to induce postoperative ileus, intestinal ischemia, or intraperitoneal infections.

Postoperative ileus models: A common technique to replicate postoperative paralysis is laparotomy with the handling of the intestine by translocation, eventration, hand pressure, or exposure to room air for a period of time [161, 162, 163, 164, 165, 166, 167, 168, 169, 170]. This postoperative ileus model is cost-effective and not demanding by means of surgical skills, yet it is highly variable as pressure is not countable, and neither is the handling of the intestine. Authors utilize different time intervals of what they record as intestine manipulation (Table 6), and comparative studies do not exist regarding time intervals of tissue handling and means of manipulation. Gerring et al. [171], after exposure of the small intestine, used a dry swab to rub it vigorously for 10 minutes and then covered it with a dry towel for a further 30 minutes. Van Bree et al. [172] in order to eliminate pressure and handling variations, used a plexiglass onto which they rolled the intestine three to four times with the use of a cotton applicator and reported that they managed to reduce variability in outcomes; thus, they can reduce the number of animals used. Many authors record the intestine manipulation technique not as the study group but as the sham operation to compare the outcomes of intestinal motility, inflammation, management, and recovery compared to intervention groups.

Author	Method	Time (minutes)	Species	Adverse events	Sampling
Schwarz [161]	Eventration and placement on moist gauze, then light manipulation of the entire bowel with two moist cotton applicators	15’	Male rats	None	0 h, 24 h, 3d
Yong-Yu Li [162]	Eventration and manipulation with two moist cotton applicators	5’	Female mice	n/a	24 h
Morris [163]	Room air exposure	30′	Labrador dogs	n/a	1-22d
Vilz [164]	Eventration, placement on moist gauze, and manual evacuation by rolling two moist and sterile cotton applicators	n/a	Male mice	Caution for hemorrhage	24 hr
Hartmann [165]	Manipulation with two moist cotton applicators ±mechanical bowel preparation or selective decontamination	15 min	Mice	n/a	1 h, 3 h,9 h
The [166]	Exteriorization and manipulation with sterile, moist cotton applicators	5′	Female mice	n/a	24 hr
De Winter [167]	Evisceration on sterile gauze and manipulation by hand pressure	5′	Male rats	n/a	20 minutes
De Winter [168]	Evisceration on sterile gauze and manipulation (not defined)	5’	Male rats	n/a	20 minutes
Matsumoto [169]	Exposure and gentle manipulation with a sterile moistened cotton swab	3–5’	Male mice	n/a	n/a
Sun [170]	Evisceration on wet gauze and manipulation with a moist medical cotton swab three times	10′	Male mice	None	3 h, 24 h
Gerring [171]	Evisceration and vigorous rub with dry swab, then covered with dry towels	40’	ponies	none	3 h to 74 h
Van Bre [172]	Evisceration with wet gauze; Three times roll on Plexiglas platform with cotton applicator device Compression with moist cotton applicators	n/a	mice	n/a	24 h

Table 6.

Variations of commonly used techniques of experimental postoperative ileus.

Peritoneal irritation models: Functional ileus because of chemical or inflammatory origin is easily induced by injecting a substance intraperitoneally, subcutaneously, intragastrically, intravenously, intranasally, or inhaled. This is a feasible way to replicate both septic, systematic (such as diabetic), and pharmaceutical functional ileus, and there is a long-listed catalog of agents used depending on the aim of each protocol. Lipopolysaccharides, acetic acid, iodine, capsaicin, bacteria, viruses, chemotherapeutic agents, anesthetic drugs, and multiple other substances have been administered to animal models with subsequent study of the enteric transit time and pathophysiological reactions [173, 174, 175, 176, 177, 178, 179, 180, 181]. Some authors utilize the two-hit technique by sequential administration of two inflammatory stimuli. The cecal ligation and puncture technique is another commonly used technique to induce septic paralysis. The cecum is ligated directly beneath the ileocecal valve, generating an inflammatory response resulting in necrotic tissue. This step is followed by perforating the cecum, facilitating the leakage of fecal material into the typically sterile peritoneal cavity. As a consequence, animals exhibit classical symptoms of sepsis and typically succumb to the condition. [182]. These are mainly surgical models used to study the effect of sepsis, but the gut is also an affected organ in septic conditions and has been used to study septic ileus by Overhaus et al. and Köylüoğlu et al. [183, 184].

Ischemia-Reperfusion models: The intestine is one of the most vulnerable organs to ischemia-reperfusion injuries [185]. A common animal model of intestinal reperfusion injury is occlusion of the superior mesenteric artery (SMA) by atraumatic microvascular clamps or by ligation in rodents for varying time intervals. Heparin is often administered intravenously to prevent thrombus formation within the SMA, enabling the re-establishment of circulation once the clamps are removed. The outcome depends on the selected part of the intestine as perfusion differs along the GIT, with the jejunum, ileum, and large colon showing different levels of resistance [100]. SMA occlusion alone produces variable injury severity and results in high mortality rates. When SMA occlusion is combined with ligation of collateral arcades, a more consistent injury is induced with lower mortality levels. The low-flow ischemia model uses adjustable atraumatic clamps to lower SMA flow at 20% of baseline levels to replicate hypovolemic clinical scenarios and is reported mainly in cats. Segmental mesenteric vascular occlusion by clamping the local mesenteric vascular supply and cross-clamping the bowel is a strangulation model for intestinal reperfusion injury as described above. A more recent technique in porcine models involves embolization of the SMA using materials such as buthyl-2-cyanoacrylate or polyvinyl alcohol particles and gel foam. This approach allows for complete, irreversible ischemia without exposing or manipulating the abdominal contents, making it suitable for studies of acute mesenteric ischemia [100].

4. Conclusions

The basic principles of any IO experimental model are:

1. Selection of the proper animal species based on research objectives, availability, ethical considerations, and translational relevance to human physiology.

2. Adhere to ethical guidelines and regulations governing animal research, ensuring humane treatment of animal subjects and minimizing pain and distress associated with experimental procedures.

3. Plan a reproducible and consistent protocol for experimental intestinal obstruction.

4. Introduce a mechanical or functional obstruction in the intestine of the animal model.

5. Regularly monitor the animal model for signs of intestinal obstruction, including changes in behavior, food intake, abdominal distension, and bowel movements, and monitor for potential complications such as intestinal ischemia, perforation, and sepsis.

6. Conduct histological analysis of intestinal tissue samples to evaluate morphological changes associated with obstruction, such as mucosal damage, inflammation, and fibrosis, and perform molecular analyses to investigate gene expression patterns, signaling pathways, and molecular mechanisms involved in the development and progression of intestinal obstruction.

7. Consider conducting longitudinal studies to assess the progression of intestinal obstruction over time and evaluate the long-term effects on gastrointestinal function, mucosal integrity, and overall health.