The pig is very similar to humans in terms of anatomy, genetics and physiology. Choosing the right breed and age allows various surgical and non-surgical procedures typically used in human medicine, including catheterization, heart surgery, valve manipulation, endoscopy and broncho-alveolar lavages (1,2). These procedures are particularly difficult or impossible to perform in many animal models including rodents. In terms of genetics, the size and the composition of the porcine genome are comparable to those of humans. During the past half century, even though the germ-free (GF) pig has been increasingly recognized as a very valuable experimental animal model system in the investigation of pathogens (1), the number of functional GF pig facilities remains low, mainly because of the high costs and the technical complexity in establishing and maintaining the facility (2). Despite this financial difficulty, the advantages of the piglet’s models highlighted by these studies include physiological similarity to the human gut as well as to the pathological mechanisms of human diseases. Piglets, have also proven usefulness in the study of intestinal barrier function, surgical manipulation, and tissue intervention, as well as in biomaterial implantation and tissue transplantation (3). Available literature sources show that microbiota is necessary for the normal postnatal development of the structures of the gut wall. It has been suggested that pigs possess a “developmental window” in which the developing host–gut microbiota interactions are the easiest to manipulate and during which time the gut is the most susceptible to major disturbances (4). Therefore, the examination of GF animals before introduction of bacterial colonization, may provide us with better understanding of the communication that occurs between the host and its bacterial residents in the future.
Nowadays, the main focus of attention has been directed to study distribution of bacteria and their influence on development and maturation of piglet´s gut, but a smaller number of information is obtained from GF environment. Our study is focused on investigation of
The structure of the small intestine is very similar in humans and piglets, including macroscopic features such as the ratio of intestinal length
Gastrointestinal (GI) tract was removed from the sacrificed piglets immediately. Small intestine (jejunum) and large intestine (colon) 1–2 cm long bioptic samples were obtained and washed with cold saline and fixed in 4% paraformaldehyde. Histological sections (4–5 μm) were deparaffinized and rehydrated. The population of GCs present in the intestinal mucosa of jejunum and colon was detected using the PAS - reaction and Alcian blue histochemical staining method. For neutral mucosubstances and mucins detection the PAS-reaction modified according to McManus was used. Fresh-made 1% solution of PAS was prepared for the PAS-reaction. For further differentiation of cell nuclei, Mayer hematoxylin staining was performed. The goblet cells and glycocalyx were stained magenta by PAS-reaction, and cell nuclei were stained dark blue by Mayer’s hematoxylin (SIGMA-ALDRICH, Co). Alcian blue 8GX solution (pH 2.5) (Sigma–Aldrich, St. Louis, MO, USA) stains both sulfated and carboxylated acidic mucopolysaccharides and sulfated and carboxylated sialomucins (glycoproteins). Excessive amounts of non-sulfated acidic mucosubstances were visible in the cytoplasm of secretory GCs. Strongly acidic mucosubstances in the cytoplasm were stained blue, while nuclei were counterstained pink to red by nuclear red stain. Alcian blue/nuclear red stained tissues were acquired and the number of Alcian blue positive GCs was determined in 10 intestinal villi and corresponding intestinal crypts in each sample. All histochemically stained tissue sections were cover-slipped with Pertex (Histolab Products AB; Göteborg, Sweden).
All measurements were performed in order to ensure objectivity in blind conditions, by two observers (unaware of the experimental groups) for all experimental groups and methods, carrying out the measures of control and experimental samples of each segment of the gut under the same conditions. For the quantitative analyses of PAS positive and Alcian Blue positive GCs, we used five sections from both gut segments in all animals’ groups. All measurements were done using magnification 200x. For quantitative and qualitative analyses of histo chemical and histological methods for detection of mucin in GCs, light microscope OLYMPUS BX50 with a digital camera OLYMPUS SP350 (Olympus; Tokyo, Japan) and Quick PHOTO Industrial 2.3 image analyser software (Promicra; Prague, Czech Republic) were used. The statistical analysis was performed in GraphPad InStat ver. 3.10 for Windows (GraphPad Software Inc., San Diego, CA, USA). Quantitative evaluation of studied markers is expressed as mean ± SEM (standard error of the mean). The significance of the differences between experimental groups was analysed using one-way analysis of variance ANOVA test followed by a Tukey-Kramer multiple comparison test. The value of
Mucins possess potential binding sites for both commensal and pathogenic organisms and may perform a defensive role during establishment of the intestinal barrier. In this study, the effects on intestinal GCs mucin production in the gut of HC piglets and ECK piglets were examined. Numbers of GCs containing total acidic mucins in both, the jejunum and colon, differed significantly between HC and ECK piglets (Fig. 1).
In the epithelium of jejunum of ECK piglets, the epithelial lining exhibited a marked decrease in number of GCs which produced acidic mucins (***indicates the values differ significantly from the jejunum of HC piglets at
Number of PAS positive GCs (detection of neutral mucins in goblet cells) in the HC piglets was significantly different in comparison to the number of PAS positive cells in the ECK piglets (Fig. 3).
In the ECK piglets, jejunal GCs exhibited decrease in neutral mucins (**indicates the values differ significantly from the jejunum HC piglets at
Enteric infections with pathogenic bacteria play an important role in animal health with the initiation and perpetuation of diseases such as diarrheal disease caused by enterotoxigenic
Goblet cells are important cells of intestinal epithelial lining because they play important role in synthesis and secretion of mucus. Its major function is to protect the intestinal epithelium from damage caused by food and digestive secretions. The overlying mucus gel layer is the first line of defence that foreign bacteria and other pathogens encounter when they attempt to traverse the intestinal mucosa (12). However, simultaneously, mucin provides a desirable environment for proliferation of specific microflora due to its high carbohydrate content (13). Thus, the true chemical composition of mucus is essential for establishment of the intestinal barrier. Mucins can be classified into two broad categories: neutral and acidic depending on sugar type in the chains. These terms are derived from the chemical nature of the oligosaccharide sugar moieties (14).
Currently, small number of information is available describing the effects of bacterial colonization on the secretory histochemical pattern of intestinal mucins in piglets. Reference to numbers of GCs containing acidic mucins compared with neutral mucins in conventionally reared poultry has been reported (15). However, it has not been described in piglets from GF environment. Thus, the aim of the current study was to investigate the effects of bacterial colonization on mucin production in jejunal and colon´s GCs of piglets.
In the current study, we found that piglets infected with
This change in mucin profile in response to bacterial colonization suggests a potential role as a protective mechanism against pathogenic invasion of the intestinal mucosa during of gut mucosa development in piglets.
It can be concluded that colonization by
Therefore, using of piglets from GF environment as animal models may contributed to the acquisition of new knowledge to improve both animal and human health.
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