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  1. Molecular imaging in living subjects: seeing fundamental biological processes in a new light
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  3. Two-photon Microscopy Protocols and Methods | Springer Nature Experiments

This chapter will describe a step-by-step protocol for using two-photon microscopy to track the colonization. This chapter will describe a step-by-step protocol for using two-photon microscopy to track the colonization of cancer cells to bone using frozen bone samples of xenograft mouse models. The tightly regulated permeance of the blood-brain barrier BBB greatly limits the range of therapeutic treatment options for central nervous system CNS diseases. The use of focused ultrasound FUS , in conjunction with circulating microbubbles,.

The use of focused ultrasound FUS , in conjunction with circulating microbubbles, is a unique approach whereby the transcranial application of acoustic energy, focused within targeted brain areas, can be used to induce a noninvasive, transient, and targeted increase in BBB permeability. This can provide an avenue for the delivery of therapeutic agents from the systemic circulation into the brain. The goal of this chapter is to outline each procedure, present options for experimental design, and highlight important considerations for the collection and interpretation of data.

A thorough understanding of the synaptic ultrastructure is necessary to bridge our current knowledge gap about the relationship between neuronal structure and function. Femtosecond-pulsed near-infrared laser was used to create fiducial marks around the dendrite of interest in aldehyde-fixed tissues. Thereafter, samples were subjected to en bloc staining with rOTO reduced osmium tetroxide-thiocarbohydrazide-osmium tetroxide , followed by lead aspartate and uranyl acetate to enhance tissue contrast. In order to mount a potent immune response, immune cells must move actively through tissues.

As an example, T-cell need to migrate within lymph nodes in order to scan the surface of many dendritic cells and recognize rare expressed antigens. The recent development of improved imaging approaches, such as two-photon microscopy, and the use of powerful mouse models have shed light on some of the mechanisms that regulate the migration of immune cells in many organs.

Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tissue samples. However, these studies lack a temporal dimension since samples have been fixed. In order to overcome some of these limitations, we describe, in this methodology chapter, an experimental system of fresh human adenoid slices to monitor the dynamics of resident T-lymphocytes that have been stained with directly-coupled fluorescent antibodies.

Combined with confocal fluorescent imaging, this preparation offers an effective approach to imaging immune cells in a three-dimensional 3D human lymphoid tissue environment. Laser scanning microscopy LSM is a technology that allows for direct observations of host-pathogen interactions during infection. In addition to high resolution and contrast,.

In addition to high resolution and contrast, these two technologies also provide high excitation penetrance in unsectioned samples. High penetrance allows for imaging of layers of tissue that are difficult to image with other more conventional microscopy approaches.

Thus, confocal and two-photon LSM open the possibility of observing infection in a three-dimensional context, where the natural architecture of a tissue is preserved. Few studies have used LSM technology to gain insights into Yersinia pestis pathogenesis in the mammalian host. The use of LSM in the plague field has an enormous potential for the discovery of the mechanisms that lie behind key aspects of pathogenesis such as colonization, dissemination, and tissue damage. This chapter provides guidance for the implementation of confocal or two-photon LSM to study Y.

This document provides specific instructions applied to imaging of Y. Two-photon calcium imaging became in recent years a very popular method for the functional analysis of neural cell populations on a single-cell level in anesthetized or awake behaving animals. Scientific insights about single-cell processing of.

Scientific insights about single-cell processing of sensory information but also analyses of higher cognitive functions in healthy or diseased states became thereby feasible. However, two-photon imaging is generally limited to depths of a few hundred micrometers when recording from densely labeled cell populations. Therefore, such recordings are often restricted to the superficial layers 1—3 of the mouse cortex, whereas the deep cell layers 4—6 are hardly accessible with standard two-photon imaging. Here, we provide a protocol for deep two-photon calcium imaging, which allows imaging of neuronal circuits with single-cell resolution in all cortical layers of the mouse primary cortex.

This technique can be readily applied to other species. The method includes a reduction of excitation light scattering by the use of a red-shifted calcium indicator and the minimization of background fluorescence by visually guided local application of the fluorescent dye. The technique is similar to previously published protocols for in vivo two-photon calcium imaging with synthetic calcium dyes Stosiek et al. Hence, only minor changes of a generic two-photon setup and some adaptations of the experimental procedures are required.

Our approach combines direct wavefront sensing of light from a guidestar formed by descanned fluorescence from Cy5. We achieve high signal-to-noise ratios in recordings of glutamate release from thalamocortical axons and calcium transients in spines of layer 5b basal dendrites during active tactile sensing. The cerebral vascular system services the constant demand for energy during neuronal activity in the brain.

Attempts to delineate the logic of neurovascular coupling have been greatly aided by the advent of two-photon laser scanning microscopy to. Attempts to delineate the logic of neurovascular coupling have been greatly aided by the advent of two-photon laser scanning microscopy to concurrently image blood flow and the activity of individual neurons and astrocytes involved in the control of the flow.

Here we review the procedures to generate optical access to the cortex for both rats and mice, determine the receptive fields of the exposed cortical areas, and use two-photon microscopy to accurately measure blood flow in individual cortical vessels concurrent with local cellular activity. We illustrate the techniques with acute recordings from rats and chronic recordings from mice. The recent introduction of multiphoton microscopy coupled with advances in optics, computer sciences, designer fluorophores, molecular labeling, and previously developed physiologic approaches have empowered investigators to quantitatively study the.

The recent introduction of multiphoton microscopy coupled with advances in optics, computer sciences, designer fluorophores, molecular labeling, and previously developed physiologic approaches have empowered investigators to quantitatively study the cell-specific dynamic events, such as endocytosis, within a functioning organ with subcellular resolution. This rapidly emerging field of investigation, with superior spatial and temporal resolution and high sensitivity, enables investigators to track molecules and determine their mode of cellular uptake, intracellular trafficking, and metabolism in a cell-specific fashion in complex heterogeneous organs such as the kidney with repeated determinations possible over a prolonged period of time.

This approach is enhanced by the ability to obtain and quantify volumetric data with using up to three different fluorophores simultaneously. We have utilized this intravital approach to understand and quantify kidney proximal tubule cell uptake and intracellular distribution and metabolism of fluorescently labeled molecules, including folic acid, gentamicin, and small interfering ribonucleic acid siRNA.

Limitations of this technique include tissue penetration, which is the major barrier to successful clinical utilization of this technology. However, its use in preclinical animal models offers new insight into physiologic processes and the pathophysiology and treatment of disease processes. The advent of two-photon microscopy has enabled us to visualize individual neurons in the intact brain. This technique, used in combination with whole-cell patch-clamp recordings, has facilitated targeted intracellular recording from particular. This technique, used in combination with whole-cell patch-clamp recordings, has facilitated targeted intracellular recording from particular neurons of interest.

This chapter provides a practical guide for implementing in vivo two-photon targeted patch-clamp recording and describes potential outcomes using the technique. Optical investigation of fast neuronal network dynamics in the intact neocortex—using appropriate activity-dependent indicators—requires single-cell resolution at large imaging depths and sufficient acquisition speed. These requirements are met by. These requirements are met by two-photon laser scanning microscopy, which has become one of the key methods for functional measurements of neuronal population activity in vivo, primarily in combination with calcium indicators.

In this chapter we focus on various advanced two-photon imaging techniques that were recently developed to improve scanning speed, to enable 3D sampling from large numbers of neurons, or to extend imaging towards measurements in freely behaving animals. In general, sampling speed and population size trade off against each other. Currently, about 1, neurons can be measured with good signal-to-noise ratio at 1—10 Hz or a few tens of neurons can be optically recorded at 1 kHz.

Measurements of local network activity have been used to either characterize spatial distributions of functional properties, such as orientation tuning in visual cortex, or reveal neuronal activation patterns on a fast time scale. We illustrate these new opportunities with examples from in vivo two-photon calcium imaging in mouse visual cortex. The chapter concludes with a discussion of the advantages and limitations of the various techniques and of future perspectives.

The direct observation of neuronal network dynamics in living animals no doubt will help to elucidate principles of operation of neocortical microcircuits. In this chapter I will give a brief general introduction to optical imaging and then discuss in more detail some of the methods specifically used for imaging cortical dynamics today.

Absorption and fluorescence microscopy can be used to form direct,. Absorption and fluorescence microscopy can be used to form direct, diffraction-limited images but standard methods are often only applicable to superficial layers of cortical tissue. Two-photon microscopy takes an intermediate role since the illumination pathway is diffraction-limited but the detection pathway is not.

Losses in the illumination path can be compensated using higher laser power. Since the detection pathway does not require image formation, the method can substantially increase the imaging depth. Understanding the role of scattering is important in this case since non-descanned detection can substantially enhance the imaging performance.

Finally, I will discuss some of the most widely used imaging methods that all rely on diffuse scattering such as diffuse optical tomography, laser speckle imaging, and intrinsic optical imaging. These purely scattering-based methods offer a much higher imaging depth, although at a substantially reduced spatial resolution. The present chapter describes how to apply optical neuroimaging to study brain lateralization in insects. It provides two complete protocols, one for in vivo imaging to obtain information on functional lateralization, and one on histochemical.

It provides two complete protocols, one for in vivo imaging to obtain information on functional lateralization, and one on histochemical techniques to study morphological asymmetries. Both sections start with the animal preparation, and illustrate the different possibilities for brain tissue labeling.

Then, imaging techniques are presented, concentrating on wide-field fluorescence microscopy, confocal, and two-photon laser scanning microscopy. After some remarks on the main methods for data analysis, studies on functional and morphological lateralization in insects are reviewed. Mammalian cortical neurons integrate sensory information that arrives through numerous synaptic inputs located on their dendrites.

Here we introduce an approach to identify sensory-evoked dendritic input sites in cortical neurons in vivo involving. Here we introduce an approach to identify sensory-evoked dendritic input sites in cortical neurons in vivo involving the use of two-photon calcium imaging combined with targeted whole-cell recordings.

We provide basic technical descriptions as well as experimental procedures of this method. First, we discuss various scanning modes for two-photon imaging of cortical neuron dendrites in vivo. Second, we focus on practical aspects of dye-loading by means of whole-cell recordings in vivo. Third, we provide a step-by-step experimental protocol and a data analysis algorithm for dendritic imaging in cortical neurons. We conclude that the combination of whole-cell recording and two-photon calcium imaging is a useful tool for the functional mapping of input sites in dendrites that can be readily applied to other cortical areas and layers, helping to reveal the fine organization of sensory information representation in the dendrites of mammalian cortical neurons in vivo.

This technique provides anatomically targeted, rapid and non-invasive staining of both neurones. We describe the protocols for staining cells in newborn, juvenile, adult and aged tissue; discuss critical steps and possible pitfalls and introduce the multicolor imaging approach for functional characterization of specific cell types. Finally, we illustrate the use of MCBL for two-photon calcium imaging of neurones and glia in the aged mouse cortex as well as the mouse cortex in an animal model of AD.

From brain slice to freely moving mouse, optical methods are being used to probe single neuron physiology and neural circuit function. Efforts in physics, engineering, and genetics have resulted in novel techniques that permit more refined optical. Efforts in physics, engineering, and genetics have resulted in novel techniques that permit more refined optical interrogation of brain function. The field of optogenetics has enabled neural activity to be driven by light, while optical read-out of neural activity has been improved in terms of speed, spatial resolution, and imaging depth.

Genetically encoded sensors and activators of neural activity can now be expressed in cell subtypes helping us to understand neural circuits with unprecedented detail and specificity. In this chapter, we discuss some of the currently available optical methods while highlighting their relative strengths and weaknesses. Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands.

This method is fast and is scalable for high-throughput screening of drugs targeting protein—protein interactions. In contrast to the immune dynamics in peripheral lymph nodes, the dynamics of immune response in PP. In contrast to the immune dynamics in peripheral lymph nodes, the dynamics of immune response in PP have not been extensively characterized in vivo by two-photon microscopy, mainly due to the PP location on the anti-mesenteric side of the small intestine and the associated peristaltic movement. Here, we describe an approach based on a custom-made spring-loaded platform to immobilize PPs and allow for two-photon microscopy imaging in vivo.

Two-photon intravital microscopy 2P-IVM is an advanced imaging technique that allows the visualization of dynamic cellular behavior deeply inside tissues and organs of living animals. Due to the deep tissue penetration, imaging of highly. Due to the deep tissue penetration, imaging of highly light-scattering tissue as the bone becomes feasible at subcellular resolution. To better understand the influence of blood flow on hematopoietic stem and progenitor cell HSPC homing to the bone marrow BM microvasculature of the calvarial bone, we analyzed blood flow dynamics and the influence of flow on the early homing behavior of HSPCs during their passage through BM microvessels.

Here, we describe a 2P-IVM approach for direct measurements of red blood cell RBC velocities in the BM microvasculature using repetitive centerline scans at the level of individual arterial vessels and sinusoidal capillaries to obtain a detailed flow profile map.

Furthermore, we explain the isolation and enrichment of HSPCs from long bones and the transplantation of these cells to study the early homing behavior of HSPCs in BM sinusoids at cellular resolution. This is achieved by high-resolution spatiotemporal imaging through a chronic cranial window using transgenic reporter mice. Nanoparticle-assisted localized optical stimulation NALOS of cultured neurons is an all-optical method that allows subcellular light stimulation to investigate localized signaling in neurons.

We demonstrate that this technique can be applied to cultured hippocampal neurons using commercially available bare AuNPs. This chapter describes how to apply two-photon neuroimaging to study the insect olfactory system in vivo. It provides a complete protocol for insect brain functional imaging, with some additional remarks on the acquisition of morphological.

It provides a complete protocol for insect brain functional imaging, with some additional remarks on the acquisition of morphological information from the living brain. We discuss the most important choices to make when buying or building a two-photon laser-scanning microscope. We illustrate different possibilities of animal preparation and brain tissue labeling for in vivo imaging.

Molecular imaging in living subjects: seeing fundamental biological processes in a new light

Finally, we give an overview of the main methods of image data processing and analysis, followed by a short description of pioneering applications of this imaging modality. Thus far, optical recording of neuronal activity in freely behaving animals has been limited to a thin axial range. We present a head-mounted miniaturized light-field microscope MiniLFM capable of capturing neuronal network activity within a volume. Here we present a protocol for analyses of axon regeneration and density in unsectioned adult mouse spinal cord.

This includes methods for injury and tracing of dorsal column sensory and corticospinal axons; clearing and staining of unsectioned. This includes methods for injury and tracing of dorsal column sensory and corticospinal axons; clearing and staining of unsectioned spinal cord; visualization of axon degeneration and regeneration in cleared and uncleared specimens using two-photon microscopy; and either manual or semi-automatic analysis of axon density and regeneration in 3D space using Imaris and ImageJ software.

This protocol can be used to elucidate the molecular and cellular mechanisms underlying nervous system degeneration and regeneration and to establish the therapeutic efficacy of candidate neuroregenerative treatments. In as such, this procedure is much easier to perform in the larger rat model as compared to mice. Furthermore, murine models have a considerably higher amount of genetically altered and highly conserved inbred strains to study intestinal inflammation when compared to rats, which are often composed of outbred stains Wistar and Sprague—Dawley rats and contain a less conserved genetic background [ 89 ].

Despite this, the rat is still a valuable animal model to study intestinal inflammation and it is conceivable that overtime, there could be an increase in the numbers of analytical tools and reagents available for rats, making rats an even more effective animal model to study intestinal inflammation in people. To exemplify this, Caenorhabditis elegans has been used to examine host-microbiome interactions in the intestine at the apical surface of epithelial cells [ 93 ]. In its natural habitat, C. Bacteria belonging to the Proteobacteria phylum act as commensal residents within the C. Notably, this intestinal model has been used to examine bacterial populations required to maintain intestinal homeostasis and to investigate mechanisms of epithelial defense [ 93 , 95 ].

The pathogenicity and intestinal injury caused by Listeria sp. As an example, Listeria monocytogenes induces intestinal epithelial changes in C. Similarities are also observed when comparing nematode and mammalian signalling pathways, protein secretion, and expression of transcription factors [ 98 ]. As examples, the C. Another advantage of using C. This attribute has been useful in measuring temporal changes involving intestinal cell integrity, and the subsequent progression of intestinal inflammation following challenge with pathogenic S.

Insects possess many of the same attributes as nematodes making them a valuable model to study intestinal function. Recently, the fruit fly Drosophila melanogaster has been used to study the mechanisms involved in intestinal function and disease. Specifically, the D. Drosophila can provide a highly applicable system to study mechanistic changes in the host genome.

The innate immune response of Drosophila is often associated with antimicrobial peptides AMPs and the reactive oxygen species ROS response produced by its epithelia, followed by immobilization of phagocytic haemocytes which engulf foreign materials [ ]. Presently, cell signalling pathways involving innate immune functions have been studied in the Drosophila intestine. Importantly, these events in the Drosophila intestinal epithelium are mechanistically similar to defences observed in human beings; furthermore, components of the epithelial architecture of the Drosophila intestinal epithelium are also similar to people [ , ].

Structurally, the epithelial monolayer and brush border, enterocytes, and crypts of Drosophila are also comparable to mammals [ ]. Caenorhabditis elegans and Drosophila can be used as effective invertebrate models for identifying early processes involved in the initiation and progression of innate aspects of intestinal inflammation [ ], cell signalling, epithelial barrier function, and the impact of bacterial populations on intestinal physiology [ 47 , 95 ]. A major limitation to the use of these invertebrate models is the lack of an adaptive immune response and some cellular processes that are present within the mammalian intestine [ ].

This is underscored by the induction of intestinal injury in the C. Furthermore and in general, the C. This observation is highlighted in a study that challenged C. This indicates that caution should be used when employing C. Finally, a potential limitation of insect models is that portions of the foregut and hindgut are lined with chitin. Certain holometabolous insects produce a peritrophic matrix that is functionally similar to the intestinal glycocalyx of mammals and serves to protect the gut from mechanical damage and also acts as a barrier against the invasion of microorganisms [ ].

Importantly, the peritrophic matrix is composed of microfibrils rich in chitin, a product that is not present in the mammalian intestine [ ]. Thus, insect models may not be appropriate for studying either the physiological functions of the intestinal glycocalyx or microbial interactions with the glycocalyx. Zebrafish Danio rerio have been used to model the human intestine for many years, and although this is a non-mammalian vertebrate model, it is a highly versatile model that provides researchers with the option to study both innate and adaptive immune responses [ 48 ].

Zebrafish are considered by many to be superior to invertebrate models as they have a larger repertoire of organs that exhibit pathologic changes. Similarly to C. The zebrafish intestine possesses similar cell types to mammals such as absorptive enterocytes, endocrine and goblet cells, a functional brush border with microvilli, and an epithelium that is continuously sloughed off into a luminal space and regenerated in a manner parallel to the murine and human intestine [ ]. Zebrafish do not have a defined stomach, therefore its strengths as a nutritional model are limited as most protein and fat absorption occurs in the lower intestine rather than in the small intestine [ ].

Despite this limitation, the zebrafish model has proven useful for studies of intestinal motility and peristaltic events through a mutation that leads to the loss of enteric neurons [ ]. The zebrafish model has been well established to study host-microorganism interactions and bacterially triggered immune responses [ ]. The establishment of a germ-free zebrafish model has enhanced its ability to be applied to microbiome research, and researchers have used germ-free fish to understand and compare the richness and abundance of microbial communities [ , ].

Significantly, the adaptive immune response in zebrafish develops to maturity in approximately 3weeks, and the use of zebrafish at 3-weeks of age or younger allows researchers to study innate responses without the interference of the adaptive immune response [ 48 ]. As zebrafish possess a functional innate and adaptive immune response, their use in combination with larger animal models can be very advantageous to elucidate the role of the intestinal microbiome on enteric disease. Applications of the zebrafish model to further understand the effects of acute and chronic inflammation on intestinal cells can be very useful, and it is expected that this model will become increasingly utilized once more biomolecules and techniques are developed.

The zebrafish intestine has the added advantage of being homologous to the human intestine structurally, and the ability to use direct live imaging to view the epithelial cells in real-time during infection significantly increases the effectiveness of this model. The pig is an excellent mammalian model to study the mechanisms involved in acute and chronic intestinal injury and inflammation, as the intestine is anatomically and functionally similar to the human intestine [ 49 ].

The anatomic structure of the pig gastrointestinal tract, in particular the stomach and small intestine, is analogous to the arrangement in human beings and differs only by the spiral orientation of the pig colon and the lack of an appendix [ ]. Despite this, primary intestinal functions such as nutrient and water absorption and microbial fermentation are still comparable to the human intestine [ , ].

Additionally, intestinal digestive enzymes, secretory proteins and the microbiome within the pig intestine are also comparable to people, facilitating the examination of the relationship between microbial communities, diet and intestinal health [ , ]. The pig model has also been used extensively to replicate the human microbiome in the pig intestine through fecal transplantation procedures [ ]. Several studies have also examined the pre-colonization of piglets with probiotic and avirulent bacterial strains common to the human microbiome, and concluded these strains were protective against subsequent infection with pathogenic bacteria [ , ].

Many of the immune cells and processes of the innate and adaptive immune system, namely populations of mucosal and intraepithelial B and T lymphocytes and the recognition of activators of innate immunity i. LPS and nucleic acids by macrophages, are also comparable to those in human intestinal immunity [ , ]. Although pigs are an excellent animal model to study intestinal inflammation of people, there are variations in the porcine adaptive immune response. In addition, expression of Th2 cytokine IL-4 is downregulated in pigs, as compared to human beings and mice, which show increased expression of IL-4 when induced [ ].

The use of pigs as a large animal model enables researchers to harvest large amounts of tissue, a distinct advantage when investigating intestinal inflammation. The size, slow growth rate and relatively slower reproductive rates of the pig however, are unfavourable qualities when studying intestinal inflammation relative to other animal models. These large animals also require large housing facilities and husbandry costs compared to smaller rodent and invertebrate models.

Collectively, the reproductive biology, increased costs of housing and the overall size of pigs can limit their use as an intestinal inflammation model in comparison to rodents, invertebrates and fish. Despite the drawbacks e. Non-human primates NHPs are considered the best animal model to study the mechanisms involved in acute and chronic inflammation, as there are irrefutable similarities to human intestinal physiology, function, immunology, and the intestinal microbiome.

Macaques and tamarins are most commonly used to study mechanisms involved in both the pathogenesis and treatment of intestinal disease [ ]. Interestingly, these NHPs often develop spontaneous colitis and subsequent colon cancer following extended periods of confined captivity, and thus are good models to examine the de novo generation of intestinal inflammation and neoplasia [ — ]. For instance, Gozalo et al. Although many of the cellular mechanisms involved in the development of spontaneous colitis remain unknown [ ], Ramesh et al.

Non-human primates are also used to investigate the effect of microorganisms on the development of intestinal inflammation, and have demonstrated that intestinal bacteria can influence the onset of disease [ ]. Importantly, the microbiome in NHPs, rodents, zebrafish, and human beings all possess similarities to one another, and imbalances made in the intestinal communities can lead to disease. Not only are the populations of the bacterial communities critical to maintaining homeostasis in the intestine, but species of Archaea within the intestine also contribute to the maintenance of a well-functioning microbiome.

Investigations have identified methane-producing Archaea species and sulphate-reducing bacteria SRB collectively produce metabolic by-products associated with poor colonocyte health and function [ 41 ]. Furthermore, the severity of disease increased with higher amounts of SRB. As these bacteria increased in number, the hydrogen sulfide concentration in the colon also increased [ ], suggesting a connection between methane-producing Archaea and SRB with intestinal health.

Further, Macaca sp. Finally, rhesus macaque populations have also shown that species-specific Helicobacter spp. The intestinal environment is in a constant state of flux and as such is either in a state of immunological quiescence or activation, and it has become clear that alterations of these states can impact brain function and the mental health of the host. As such, researchers are now using animal models to examine the effect of the intestine on mental health.

Non-human primates can be employed to investigate the relationship between the intestinal microbiome, the intestinal nervous system, and the impact on host well-being. The gut-brain axis is a functional link between the intestine, the autonomic nervous system, and the higher functions of the brain. Innervation of the autonomic nervous system has shown that perturbations in the intestinal microbiome influence brain function and behavior, and thus can result in changes in feeding behavior, anxiety-like behaviour, stress, depression, and pain perception [ 34 ].

Although mice have also been used to study certain aspects of the gut-brain axis [ ], NHPs likely are the best model to study neurological activity for human beings. Many NHPs characteristically develop deeper social bonds and display behaviours indicative of higher human-like intellect, giving them preference over the mouse and pig animal models for brain-related investigations. The use of NHPs appears to be the most representative animal model to simulate intestinal inflammation in people.

This model however, has considerable drawbacks. Most notably, the ethical use of highly intelligent animals closely related to human beings is controversial, and generates charged discussion between the scientific communities involved in animal research and the general public. Many people also believe NHPs such as the near extinct African Great Ape species should be banned from scientific research [ ].

NHPs also require specialized housing facilities with an extensive and expensive biosecurity infrastructure, as well as elaborate equipment for environmental enrichment [ ]. Moreover, several species of NHPs are large gorillas, chimpanzees and orangutans and can be potentially intractable and dangerous, requiring animal care staff and veterinarians with specialized training in animal husbandry, safety, and disease control.

Furthermore, NHPs may carry zoonotic organisms that are highly pathogenic and easily transferable to people. One of the most well-known, potentially fatal pathogens carried by NHPs is Cercopithecine herpesvirus B virus [ ], and although it is relatively innocuous in monkeys, people who are exposed to the virus through secretions from bites or scratches can develop a fatal form of viral myeloencephalitis [ ]. Sheep intestinal loop model. Campylobacter jejuni treated intestinal loops are markedly edematous, congested, and presented with numerous fibrino-hemorrhagic foci of mucosal necrosis.

A number of surgical models have been developed to study inflammation. Surgical models possess a multitude of advantages including the ability to manipulate physiological and microbiological processes within the intestine and circumvent some of the ethical issues encountered when working directly with human subjects. Also, surgical models deliver a number of logistical advantages including the ability to deliver and localize treatments, and to measure treatment effects in a highly prescribed manner. An example of a model that has been used to manipulate physiological and microbiological process is the cecectomy model in mice.

As the cecum is a major site of complex carbohydrate fermentation in mice, surgically removing the cecum significantly alters the microbial community structure and fermentation processes [ , ] and also affects colonization resistance. Surprisingly, this model has not been extensively used to study colonization resistance processes [ ], nor has it been applied to pigs or ruminants to date. Xenografts involve the transplantation of fetal intestinal segments from one species into a different recipient species.

The recipient mice are B- and T-lymphocyte deficient e. The transplant tissue is implanted under the skin of the back on recipient mice, and allowed to grow. Treatments are then injected directly into the lumen of the graft via a hypodermic needle. Transplantation of human, rat, and bovine intestinal xenografts has been successfully performed [ — ], and we recently and successfully transplanted porcine fetal intestines into SCID mice.

After transplantation of the xenograft, the species-specific integrity of the epithelium of the transplanted tissue is thought to be retained, however chimeric tissue can form within the submucosa and lamina propria [ ]. Xenograft models are considered to be axenic, but care must be taken to avoid the introduction of contaminant bacteria during the inoculation procedure. The intestinal xenograft model has been used to measure pathologic metrics incited by a variety of biotic incitants of inflammation including Clostridium difficile toxin A and B [ ], Cryptosporidium parvum [ — ], Entamoeba histolytica [ — ], Enterohemorrhagic E.

Metrics of inflammation in xenografts have focused on histopathologic changes, loss of barrier function, and differential expression of pro-inflammatory genes and proteins. Many studies using xenografts have utilized uninfected controls e. In porcine xenografts we noted that non-pathogenic E. In contrast, others have noted the pathogenic bacteria exacerbated the inflammatory response in xenografts relative to non-pathogenic bacterial controls [ , , ]. The potential of the xenograft model as a comparative pathogenicity model is currently uncertain.

Cannulation is a commonly applied method to allow researchers to temporally obtain samples from the gastro-intestinal tract of animals. For this strategy, a fistula is established into the target region of the gastrointestinal tract, and a cannula is inserted. Cannulation is commonly used to examine nutritional metrics in ruminants e. The model has also been applied to monogastric animals small intestine, cecum, colon including rabbits [ — ], dogs [ — ], pigs [ — ], and horses [ ].

The cannulation method has the advantage of allowing researchers to temporally sample mucosa, digesta or both. The salient limits of this method are the complexity of the surgical procedure, the restriction of sampling to prescribed regions of the intestine, and the inability of treatments to be localized.

Intestinal loops have the added advantage of mimicking normal intestinal physiologic, immunologic, and histopathologic responses. Importantly, treatments can be localized to a specific region of the intestine. Furthermore, treatments can be replicated within a single animal as part of the experimental design. Intestinal loop models can be divided into two basic types: recovery and non-recovery surgical procedures. For the non-recovery type, loops are established in animals under general anesthesia. Importantly, vascular and lymphatic functions are not disrupted by the procedure.

While the animal is under anesthesia, treatments are introduced into the loop lumen via injection. Although non-recovery models are much more commonly used than recovery loop models, they are limited to short-term measures and this is the primary limitation of the model. Non-recovery loop models have been established in a variety of animals including rats, rabbits, sheep, and cattle [ — ]. The intestine is then cut, and the intestinal segment designated for loops is flushed with broad-spectrum antibiotics or saline.

The non-intestinal segment side of the intestine is then rejoined to form a continuous and functioning intestinal tract. Animals are carefully monitored, and their recovery is uneventful. At the desired time, the animal is humanely euthanized, the compartmentalized intestinal segment is exposed, loops are removed, and samples are collected and processed Fig. The primary disadvantages of the recovery model are its technical complexity, need of surgical infrastructure, skill to successfully complete the surgical procedure, and requisite post-operative measures must be adhered including the administration of analgesics and antibiotics.

The single window of opportunity to administer treatments i. Furthermore, samples of both the intestinal mucosa and luminal contents e. For researchers studying bacteria and inflammatory processes, the administration of antibiotics and analgesics can directly affect the treatment itself and alter immune function, thereby confounding results. As a result, a catheterized loop model was developed in which long-term catheters were inserted into the loops [ ]. Notably, the establishment of catheters in loops allowed for the introduction of multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period.

Furthermore, observations from loop models that have been successfully established in sheep [ , ] and pig unpublished have suggested that there is no effect on intestinal function following establishment of the loop [ ]. A limitation of this model is that use of antibiotics does not eliminate microorganisms within the loops, and sloughed mucosa within the lumen of loops can interfere with the uniform distribution of administered treatments and sample collections.

All countries must adhere to standards for the ethical use of animals in research. Animal use is permissible only if the research promises to contribute to the understanding of fundamental biological principles, or to the development of knowledge that can reasonably be expected to benefit human beings or non-human animals. Animals should only be used if non-animal alternatives do not exist. In the study of inflammation, research must involve the use of animal models i.

Animals used in inflammation based research must be maintained in a manner that provides for their physical comfort and psychological well-being, and expert opinion must attest to the potential value of studies with animals before research commences. A hallmark of inflammation is pain, and thus degrees of pain or distress are concomitant in studies of inflammation.

The level of invasiveness and the procedures implemented to address this must be specified and evaluated in advance. Research studying inflammation commonly falls within invasiveness categories C i. However, in relatively rare instances, research may fall within invasiveness category E i. As pain must be minimized both in intensity and duration, research that is categorized as invasiveness category E will not be approved without strong justification.

The application of quantitative pain assessments is mandatory, and any animal observed to be experiencing severe and unrelenting pain or discomfort must be humanely euthanized i. As research on inflammation commonly involves the use of biological incitants of enteritis e. In this regard, all scientific activities conducted within signatory countries that involve pathogens must adhere to United Nations conventions on biosafety and biosecurity. Identifying the best animal model to study intestinal inflammation is an important consideration and requires a thorough understanding of the advantages and disadvantages of each model, as there are many factors to consider.

Animal models with comparable intestinal anatomy monogastric vs. Moreover, animal models that can be genetically engineered and have a similar genome to the human genome allow researchers to investigate specific genes related to intestinal disease. Most certainly, genetically modified mice have become instrumental to inflammation studies, due to their ability to display phenotypic traits definitive of specific gene manipulations.

Animal husbandry is also an important factor to consider, as the cost of the facilities and equipment can be prohibitive. Moreover, some animal species such as NHPs require enhanced training by animal care personnel and specialized veterinary care and service. The availability of biologic techniques and analytical tools necessary to study intestinal function and inflammation are also important factors to consider when choosing an appropriate animal model. Advantages and disadvantages of various animal models used to study intestinal inflammation based on the immune response elicited by biological and chemical incitants.

The induction and subsequent progression of intestinal inflammation is a complex, multifactorial interaction between the host and its environment. In particular, the physiological status of the host plays an important role in the onset and severity of disease, and as such, prior use of products such as antibiotics can contribute to the establishment of pathogenic transient bacteria in acute inflammation [ ].

Other factors such as age or genetic predisposition can also contribute to inflammatory disease development in chronic disease [ 18 ]. Although the onset of intestinal inflammation can occur spontaneously in various models [ , ], the use of either chemical or biological incitants can be effective in inducing a rapidly developing and robust response. The array of chemicals available to induce intestinal inflammation permits one to choose between acute and chronic intestinal responses, and some chemicals have the potential to incite both forms of inflammation.

Similarly, various bacterial agents can induce both acute and chronic forms of intestinal inflammation. Chemical and biological incitants of inflammation are necessary to simulate inflammation in appropriate models and often closely representative diseases may not be obtainable using a single model, necessitating the use of multiple models to accurately study the disease. Similar to choosing the best animal model to investigate specific aspects of intestinal inflammation, choosing the most effective chemicals and biological agents to incite inflammation must be carefully considered.

Acute mucosal and vascular injury in low concentrations, develops in UC-like symptoms. Dextran sulphate sodium is used either independently or in conjunction with other chemicals to induce inflammation. By adjusting the concentration and duration of DSS treatment, the mechanisms involved in both acute and chronic inflammation can be studied. In general, DSS incites inflammation by disrupting the epithelial barrier, causing vascular and mucosal injury through the exposure of the lamina propria to luminal contents and bacterial antigens [ ].

Furthermore, the factors involved in innate immunity are also affected by DSS treatment, as treatment with DSS changes the expression of MyD88, TLR4 and TLR9, and small changes in these mediators of innate immunity contribute to epithelial cell damage and subsequent intestinal inflammation [ 56 ]. Many factors affect the propensity of DSS to induce inflammation in different animal models. The bacterial population within the colon is a critical factor in modulating the severity of the tissue response caused by DSS treatment. The ability of DSS treatment to induce intestinal inflammation is also affected by the genetic background of the animal species.

In addition to mice, pigs have been used to examine DSS induced intestinal inflammation. Young et al. Other studies in pigs report increased lymphocyte infiltration in mucosal tissue as well as mucosal erosion and crypt destruction following DSS treatment, and that these tissue changes are similar to the intestinal lesions often present in people afflicted with IBD [ ]. Although DSS is an effective inducer of intestinal inflammation, there are potential drawbacks to its use in animal models.

Most notably, there can often be significant inter-animal variability in the severity of tissue injury. In many cases, marked inconsistency in the amount of mucosal damage and in particular epithelial cell necrosis is observed [ 56 , ]. Furthermore, the molecular composition and purity of the chemical product can vary between the product batches and chemical supplier, potentially leading to inaccurate concentrations and volumes of DSS administered to the test animals [ ].

Although the level of tissue injury can vary between treatment groups with DSS, it is still considered a model chemical incitant of intestinal injury and is commonly used to stimulate UC-like lesions in various animal models. The severity of inflammation can be enhanced by administering chemical incitants of inflammation in combination with another chemical inducer of inflammation. Furthermore, the administration of AOM with DSS is necessary to exacerbate the effects of DSS to induce the development of colorectal cancer [ ], an event that can on occasion occur in people with UC [ 2 ].

Thus, the combined use of both chemicals is ideal for investigating both inflammatory diseases of the intestine, as well as the pathophysiology of colorectal neoplasia. The administration of AOM alone has also been used to study mechanisms that induce cancer in the distal colon [ ]. Proposed mechanisms for the induction of inflammation and tumour formation by AOM include the upregulation of cyclooxygenases leading to the enhanced production of prostaglandin E 2 [ ], and the induction of pro-mutagenic epithelial changes caused by the O 6 methylation of guanine to induce tumour formation [ ].

Trinitrobenzene sulfonic acid is primarily used to establish acute intestinal inflammation in animal models, but can also be employed to induce chronic inflammation in rodents [ 79 , ], pigs [ ], rabbits [ 56 ], guinea pigs [ 52 ], and NHPs [ ]. To become chemically active, TNBS needs to be solubilized in ethanol, and this TNBS-ethanol mixture induces intestinal inflammation by altering host proteins through the formation of covalent bonds with trinitrophenyl haptens of TNBS [ ].

This process stimulates an immune-mediated inflammatory response. Moreover, ethanol also acts as an irritant that contributes to the damage of the epithelial barrier [ ]. Trinitrobenzene sulfonic acid can be used as an incitant of both acute and chronic inflammation. Research also suggests that a T-cell deficient mouse exhibit chronic enteric inflammation in the presence of IL, making this cytokine a determinative marker for chronic enteritis [ ].

Importantly, these observations are aligned with cytokine profiles in CD patients as these individuals display elevated levels of IL, IL and IL [ , ]. Rats supplemented with TNBS often lose weight, present with bloody diarrhea, and exhibit marked mucosal and transmural intestinal inflammation, similarly to people with IBD [ ]. Although intestinal inflammation has been established in rodents, swine, and NHPs using TNBS as the chemical incitant [ 52 , 56 ], evidence indicates that mice are the best models for investigating TNBS-ethanol induced colitis [ ].

When selecting the most appropriate mouse strain to examine TNBS induced tissue injury, the genetic background and phenotypic profile of the mouse are important factors to consider. Its use results in intestinal lesions associated with a predominant Th2 immune response. The tissue lesions manifested in mice following exposure to oxazolone are similar to UC-like lesions in people, with most lesions causing mucosal ulceration, submucosal edema, and tissue hemorrhaging [ ].

In mice, oxazolone administration has been attributed to body weight loss, diarrhea, ulcers, and loss of epithelial cells in the large intestine [ , ]. One of the advantages of using oxazolone to induce tissue injury is the rapid progression of tissue architecture alteration in comparison to other chemical agents [ ].

Indeed, the relatively fast induction of tissue damage makes oxazolone an ideal candidate to study UC-like disease in mice, as histological evidence shows an increase in IL-4, IL-5, and IL, cytokines that are indicative of a Th2 immune response [ ]. Although oxazolone is an effective inducer of acute inflammation, its effectiveness to induce chronic inflammation remains undetermined, as few investigations have examined its potential to cause long-term intestinal inflammation [ , ]. Chemical incitants induce tissue injury by initially disrupting the epithelial barrier, exposing the lamina propria to intestinal contents, and stimulating pro-inflammatory cytokine activity.

Dextran sulphate sodium, AOM, TNBS, and oxazolone cause tissue injury within the intestine, and have been especially effective in inducing injury within the distal colon [ ]. Each incitant has the ability to induce distinct tissue lesions accompanied by specific helper T-cell cytokine cascades during inflammation. Individually, these chemicals are all effective inducers of disease-specific injury. For instance, DSS is very useful as a chemical model for UC-like intestinal injury [ , ], whereas oxazolone provides the benefit of quick injury development and rapid tissue damage compared to the other three chemical agents [ ].

If developing chronic inflammation in the intestine is the main focus of study, then TNBS and DSS are the most appropriate chemicals to use. In summary, TNBS, DSS, AOM, and oxazolone are all useful chemicals to induce intestinal inflammation in animal models, and the best chemical agents to employ depends on the specific aspect of intestinal inflammation under investigation. Chemical incitants are the most common agents used to induce intestinal injury and inflammation, and are often considered the best methods to study the immune response associated in intestinal disease.

Chemicals agents are an inexpensive [ 47 ], quick, and effective method to cause inflammation, and these agents are valuable tools in the armamentarium for investigating the pathophysiology of intestinal inflammation. As an alternative to using chemical incitants, biological incitants have also been used to study common intestinal inflammatory diseases.

Biological incitants can be bacterial, viral, protozoal, or helminthic, and can be used to induce both acute and chronic inflammation. Herein we review the most commonly used biological agents to induce intestinal inflammation in animal models. The host intestinal tract contains a diverse community of bacteria totalling 10 13 —10 14 bacterial cells [ ], with species most often belonging to the Bacteroidetes , Firmicutes , Actinobacteria , Spirochetes , and Proteobacteria phyla [ , ]. Homeostatic interactions between the host and the resident microbiome occur in the intestine , and changes in bacterial species abundance can potentially lead to intestinal inflammation [ ].

It has been well investigated that the commensal bacteria are important in maintaining a healthy intestine by preventing the overgrowth of pathogenic microorganisms, and assisting in regulating and maintaining a quiescent intestinal immune system [ 41 ]. An uncontrolled immune response to commensal bacteria can lead to intestinal injury, and reports indicate that the development of aberrant immune responses can occur from increased exposure to the commensal bacteria [ 25 , ].

Moreover, modifications to the community structure of the intestinal microbiome can incite disease, often by the uncoordinated expression of pro-inflammatory cytokine profiles in concert with the simultaneous loss of anti-inflammatory signalling [ , ]. A well characterized model for studying acute inflammation involves using C. Infection with C.

Although C. Importantly, mice have been used to study the progression of tissue injury, and to identify the temporal relationship in cytokine expression by four different T helper CD4 T-cells subtypes Th1, Th17, Th2, and Treg [ ]. Citrobacter rodentium also serves as an alternative mouse model to study the virulence mechanisms related to EHEC and Enteropathogenic E.

Pathogenesis is associated with the presence of the locus of enterocyte effacement LEE pathogenicity island, which is responsible for the production of the Type 3 secretion system, Shiga toxins, Tir, intimin, and enterohemolysin [ ]. Citrobacter rodentium is also a LEE positive organism and utilizes attaching and effacing lesions to facilitate infection; however, it does not produce a Shiga toxin, meaning the tissue damage observed is not hemorrhagic , but displayed as transmissive colonic hyperplasia allowing the passage of immune cells into the lumen of the colon [ ].

A number of small and large animal models have been used to study EHEC and EPEC colonization and pathogenesis including mice, rats, rabbits, pigs, cows, dogs, baboons, and macaques [ ]. No single animal model can manifest lesions that are representative of EHEC-associated disease observed in people, as such using multiple animal models is a better strategy to understand intestinal inflammation in people caused by EHEC.

Helicobacter pylori are gram negative bacteria associated with the development of gastric ulcers in people. Helicobacter spp. Research groups are now investigating a link between H. Urease positive Helicobacter spp. The genetics of the animal model used has a significant effect on the pathophysiology of bacterial induced intestinal injury. Studies show that non- H. In such individuals, increased levels of Foxp3 and reduced intestinal inflammation have often been observed [ , ]. Experimental colitis leading to cancer has also been induced using H. Helicobacter hepaticus is often isolated in the livers and colons of infected mice [ ], and induces hepatitis, enteritis, typhlocolitis, and IBD-like tissue injury in many genetically modified mouse models [ 64 , ].

Challenge studies with H. As more information on the mechanisms involved in tissue injury caused by H. Investigations show S. A breach in the epithelium can also act as a conduit for prolonged mucosal translocation of the bacterium. Mice are relatively resistant to developing typhoid-like lesions following exposure to S.

This phenomenon suggests that disruptions in the intestinal microbiome can influence the effects of S. Further, pre-treatment with streptomycin in mice produces intestinal damage that includes epithelial crypt loss, mucosal erosion, and neutrophil infiltration that are similar to lesions observed in people with UC [ 72 , ]. These lesions can be accompanied by edema, disruption of the villus architecture, goblet cell depletion, and increased ICAM-1 expression [ ]. Murine lesions also show striking similarities to S. Finally, Salmonella spp. As well, Map has frequently been found in the environment of infected herds.

Individuals with CD who have been in close contact with or consumed milk from infected cattle also present with Map [ , ]. Research suggests Map is a useful associative model to investigate CD in people [ , , ]. Infections with Map have also been identified in captive rhesus macaques with chronic diarrhea and intestinal injury similar to lesions that are present in ruminants [ , ]. Collectively, these observations suggest Map is a good associative model used to study intestinal disease in people.

The above information suggests that C. Importantly, these models appear to represent intestinal changes observed in people with intestinal inflammatory disease. Similarly to other incitants of inflammation, these bacteria can be employed not only as primary inducers of inflammation, but also used in concert with other agents i. Helminths i. Nematodes i. The T. In addition, T. Interestingly, although helminths primarily produce a Th2 immune response in the mouse intestine, T. This event, however, fails to clear the parasite and facilitates the development of a chronic intestinal infection [ ], making T.

The severity of inflammation observed after exposure to helminths varies depending on the genetic background of the animal model. The progression of tissue injury and the type of immune response developed can be affected by the number of eggs administered. For example, T. Importantly, T. In conclusion, the T. Toxoplasma gondii has also been used in mouse models to promote intestinal inflammation. Toxoplasma gondii infection in susceptible mouse strains is able to produce a robust Th1 associated pro-inflammatory response in the small intestine [ ].

This suggests that T. Presently, there are few studies that conclusively demonstrate a direct link between viral infections and intestinal inflammation. Most of these studies show only an observational relationship between the induction of intestinal inflammation by viruses and the onset of IBS or IBD in people [ ]. Most studies examined coincidental associations between the presence of IBD in patients, the existence of viral pathogens and their remnants i. One example is the link between early onset childhood measles and the subsequent development of intestinal disease in which infants with previous history of measles-associated-pneumonia, diarrhea, and weight loss developed CD or UC later in life [ ].

Another example is the relationship between paramyxovirus and Epstein—Barr virus and the development of IBD, where either remnants of the virus or lymphocytes infected with viral particles, respectively, are present in the intestine [ 24 , ]. Although these studies do not conclusively prove viral infections induced intestinal inflammation in healthy people, it is possible that viruses can readily affect immunosuppressed individuals. Studies in immunocompromised NHP animal models and immunocompromised people with UC and CD demonstrate that intestinal inflammation and injury can be both induced [ ] and exacerbated [ 24 , , ] following exposure to viral pathogens.

Mouse models have also been used to facilitate acute colitis using cytomegalovirus to exacerbate DSS-induced colitis [ ]. From the information described, it appears that a causal association between viral infections and the induction of intestinal inflammation in immunocompetent individuals remains undetermined.

It suggests, however, that immunosuppressed individuals and animal models are more susceptible to the development of virally-induced intestinal inflammation. In as such, viruses may only be an effective tool to investigate the mechanisms involved in intestinal inflammation in immunodeficient animal models. Biological incitants offer the advantage of being able to study both acute and chronic inflammation using agents that naturally cause inflammation in human and non-human animal tissues.

A plethora of biological incitants exist that can be applied to examine inflammation; most of these agents have been investigated and are known to cause infection in the intestine of mammals. Bacterial incitants such as Salmonella spp. Most of these incitants are best used when examining the effects of acute inflammation, however helminth and protozoan models are better suited for chronic inflammatory studies. Long-term enteritis can be induced in susceptible murine models using low levels of T. The use of other agents can further enhance the effect of T. For instance, Stoicov et al.

The resulting lesions are associated with a prominent Th1 immune response and muted Th2 immune response that consequently develops into long-term injury to the upper gastrointestinal tract mucosa [ ]. Although Map and viral incitants have not been definitively associated with the onset of intestinal inflammation, the ability of these microorganisms to be either co-isolated with afflicted individuals or to exacerbate infection suggests a functional role for Map and viral agents in IBD immunity [ 24 , , ]. As the human intestine is occupied by many bacterial species that are critical to intestinal function and homeostasis, the use of models that can replicate this diverse and complex relationship yet allow alterations e.

Utilizing bacterial species that cause damage in both human and non-human animal intestines allows for comparable experimental conditions that can help in understanding dysbioses in relation to inflammatory bowel diseases. As the prevalence of intestinal inflammatory diseases continues to increase, it is becoming increasingly important to elucidate causes and possible mitigation strategies.

Intestinal disease can arise from a variety of factors, and the complex interactions between the host and the intestinal microbiome make determining the mechanisms involved in the induction and progression of disease challenging. Currently, a variety of animal models can be used to study the processes involved in intestinal inflammation, however, rodent models and in particular genetically engineered mice are the primary models used to study acute and chronic intestinal inflammation.

The ability to modify the genetic background in mice allows specific questions to be addressed, and importantly, the information from mice can be compared to other animal models and extrapolated to human beings. The generation of spontaneous, long-term intestinal inflammation can be a lengthy process, so the use of chemical and bacterial incitants to expedite the process is often necessary. Each incitant of inflammation has the inherent ability to develop specific manifestations of tissue injury as well as corresponding immune responses within various animal models, and as such, determining which agent chemical vs.

Furthermore, the association between the intestinal microbiome and the host adds another level of complexity to the pathobiology of intestinal inflammation. Together, these components facilitate our understanding into the mechanisms involved in the pathophysiology of intestinal disease and potentially set the foundation for the development of mitigation strategies that can treat intestinal inflammation in people. T follicular cells. Foxhead box protein 3. T-cell receptor. Canadian Council on Animal Care. APC gene, multiple intestinal neoplasia. Enterohemorrhagic Escherichia coli. Enteropathogenic Escherichia coli.

Adrenocorticotropic hormone. JAJ wrote the first draft of the manuscript. All authors read and approved the final manuscript. We thank the members of the Enteric Microbiology and Intestinal Health Research group at the University of Alberta and Lethbridge Research Centre for their research expertise, encouragement, and helpful discussions.

We also thank Estela Costa for her insights regarding the systemic effects of mucosal inflammation presented in Fig. This approval included the experiments involving the treatment of sheep intestines with C. Review Open Access. Animal models to study acute and chronic intestinal inflammation in mammals.

Uwiera 3 , G. Uwiera 2 Email author. Gut Pathogens 7 Abstract Acute and chronic inflammatory diseases of the intestine impart a significant and negative impact on the health and well-being of human and non-human mammalian animals. Although inflammatory diseases of the intestine are often referenced with regard to their localized and temporal inflammatory effects within the small or large intestine, uncontrolled inflammation of the intestine always imparts a systemic impact on the body [ 21 , 22 ] Fig.

Significantly, the etiology of both acute and chronic intestinal inflammatory disease is often enigmatic, [ 16 , 23 ] thereby compromising treatment choices and efficacy. Furthermore, chronic inflammatory diseases of the intestine such as IBD are often linked to prior acute inflammatory disease incited by viruses, bacteria, parasites [ 24 ], dysregulation of the intestinal immune response, or autoimmune disorders [ 25 ]. The appropriate use of animal models is essential to ascertain the etiology of intestinal inflammatory diseases, and is advantageous when elucidating the processes involved in the onset and progression of acute and chronic disease.

Effectively applied animal models are instrumental to the development and prevention of appropriate mitigation strategies. Understanding the limitations, benefits, differences and similarities between various animal models, and the chemical and biological methods that can be used to advance them is essential in the successful mechanistic understanding of disease. The immune system within the intestine is a complex system; combining coordinated responses between the innate and adaptive immune systems within the intestinal mucosa [ 26 — 28 ].

The innate and adaptive responses are composed of both cellular and non-cellular components Fig. Cellular components of innate immunity include macrophages, mast cells, neutrophils, eosinophils, natural killer NK cells, NK T-cells, and dendritic cells, which can engulf and eliminate harmful pathogens [ 31 ]. Macrophages, and in particular dendritic cells, also act as antigen presenting cells APC which engulf the recognized pathogens and present their antigens to components of the acquired immune system such as T-cells [ 32 ]. This process enables the two immune systems to operate in a coordinated manner.

Antibodies are the non-cellular components of the adaptive immune system produced by plasma B-cells and act to bind pathogens [ 33 ]. The pathogens are either neutralized by agglutination with antibodies, or are targeted for destruction by the following methods: 1 activation of the compliment system; 2 opsonisation to granulocytes; or 3 release of cytokine cascades for NK cells [ 30 ].

These function to create pores in the cell lipid bilayer and digest cellular material to promote apoptosis, respectively [ 28 ]. For example, the Th1 response is mostly associated with infections by intracellular pathogens i. Although the immune responses involved in intestinal inflammation are not the main theme of the current article, it is important to consider the components of the innate and adaptive immune response when examining the causes and manifestation of intestinal injury and inflammation.

Mice are the most commonly used animal model for intestinal studies, and genetically engineered mice are particularly important in studying intestinal inflammation [ 55 ]. There are many reviews that summarize and discuss the use of genetically modified murine models to study intestinal disease [ 56 — 58 ], and this section of the review will focus on the advantages and disadvantages of using rodent models.

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Their genetic lines can be modified to produce phenotypes that investigate specific aspects of intestinal inflammation associated with adaptive and innate immune responses, ranging from the activation of proteins involved in pathogen recognition to the activation of effector cells necessary to trigger both cell-mediated and humoral immune responses. There are other less frequently used animal models employed in intestinal inflammation and disease studies. Gnotobiotic juvenile beagles have been used to study colitis induced by C. More recent studies have used German Shepard dogs to study canine IBD, and attempt to make comparisons between cytokine expression in the dog intestine and alterations in gene expression observed in human patients with IBD [ , ].

Sheep as a ruminant model have also been used for intestinal investigations, but unlike monogastric species Homo sapiens , NHPs, rodents, and pigs , the majority of bacterial fermentation of carbohydrates tends to occur in the rumen and not in the large intestine [ ]. As such, ruminants are not ideal models to use for microbiological and nutritional studies of intestinal inflammation in people due to the importance of the rumen in ruminant nutrition; the rumen also harbors a microbial community that differs greatly from the large intestine of monogastric animals.

Most research involving nutrition in ruminants focusses on the rumen. The ruminant intestine remains an area less studied, although a few studies have utilized the fetal ovine intestine for inflammation-based research [ , ]. Some research has suggested that the presence of Mycobacterium avium subspecies paratuberculosis Map , a bacterium that causes intestinal disease in cattle, can be associated with people with CD [ ].

Although most studies provide contradicting evidence regarding the presence of Map in CD patients, some researchers have suggested that this cattle enteric pathogen can contribute to the onset of CD in human tissue, and vice versa [ , ]. The bovine animal model has also been used to study non-typhoid enteric infection induced by S. A few studies have used sheep as comparative models for human studies, using intestinal loops in neonatal sheep to study mucosal immune function [ ]. Although this model is good for studying the impact of pathogens on intestinal injury Fig.

The rabbit is another animal model that has been used to study colitis. Following muramyl dipeptide administration, mononuclear cell infiltration, lymphoid aggregation, and transmural inflammation were observed in the rabbit colon [ ]. Of late, preterm rabbit models have been used as a method to understand physiologic and biologic changes associated with intestinal dysfunction, neonatal necrotizing enterocolitis, and rectal-anal obstruction [ ].

Rabbits and guinea pigs have also been used to study intestinal lesions resulting from the administration of common chemical incitants to be discussed later in the review of intestinal inflammation [ 52 , 56 ]. Each animal model has an array of advantages and disadvantages to its use and therefore a comprehensive study examining multiple aspects of intestinal inflammation requires the use of two or more animal models. For instance, using an invertebrate model to study mechanisms involved in innate immunity in conjunction with a genetically engineered murine model could provide a broader understanding of the causes of intestinal inflammation with respect to both innate and adaptive immunity.

Alternatively, a mouse model can be used to determine the immunologic mechanisms of pathogen challenge on the intestine, and these observations paired with the effects of the pathogen on intestinal architecture and enterocyte function in the swine or NHP. In animal models of inflammation, chemicals are often used as fast, economic and effective strategies to induce intestinal tissue injury. The effectiveness of inducing tissue injury following treatment with chemical agents varies and depends on the molecular weight, concentration, manufacturer, and batch of the chemical [ ].

In addition, the species, gender [ 56 ], and the genetic background of the animal model being challenged influences the degree of tissue injury [ , ]. The method of administration also influences the induction and severity of disease, as some chemicals work well to induce inflammation after ingestion [ 56 ], while others function best when applied directly to the site of infection, such as the rectal administration of haptenating agents [ ].

Furthermore, microorganisms present in the intestine can interact with the chemical incitant and interfere with its ability to effectively incite tissue injury [ ]. In general, chemical incitants induce tissue damage that can effectively represent clinical cases of intestinal inflammation. Acknowledgements We thank the members of the Enteric Microbiology and Intestinal Health Research group at the University of Alberta and Lethbridge Research Centre for their research expertise, encouragement, and helpful discussions.

Two-photon Microscopy Protocols and Methods | Springer Nature Experiments

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What are the strengths and weaknesses of different models of collective bargaining?

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