Bulgarian Journal of Veterinary Medicine (2011), 14 , N o 4, 201 −208
MORPHOLOGICAL AND HISTOCHEMICAL STUDY OF GUINEA PIG DUODENAL SUBMUCOSAL GLANDS
A. A. MOHAMMADPOUR Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
Summary
Mohammadpour, A. A., 2011. Morphological and histochemical study of guinea pig duode nal submucosal glands. Bulg. J. Vet. Med. , 14 , No 4, 201 −208.
The duodenum is largely responsible for the breakdown of food in the small intestine, using enzymes. Duodenal submucosal glands, which in general produce a mucous secretion, exist in all mammalian species. These glands are located in the submucosa of the proximal duodenum. The study aimed to demonstrate the morphological and histochemical properties of duodenum and duodenal submucosal glands in the small intestine of the guinea pig. The duodenum of 10 adult healthy animals constituted the material of the study. After dissecting them, three parts of duodenum (cranial, descending and ascending parts) were determined. For histological studies, after tissue preparation, duodenal tissue layers and duodenal submucosal glands in tunica submucosa were measured using the micrometre method. All parameters between the three parts of duodenum were analysed and compared using the ANOVA test. We concluded that duodenal wall thickness was variable in the three parts. It decreased from the cranial (1306.81±132.80 m) to the ascending part (1026.92±80.01 m) and in the cranial part was very distinctive. Duodenal or Brunner’s glands were composed of only mucous acini densely packed within the submucosa. The glands were well developed in the cranial part of duodenum. There was a significant difference in the thickness of duodenal submucosal glands in all parts of the duode num that decreased from the cranial to the ascending part (P<0.001) from 147.52±22.80 m to 38.25±12.30 m respectively. Histochemical examination revealed that the mucous glands reacted positively with Alcian blue pH 2.5 and Periodic Acid Schiff (PAS) stains. Key words : duodenal submucosal glands, duodenum, guinea pig, histochemistry
INTRODUCTION
Although the duodenum is a tiny fraction break down food, while bile aids the di of the small intestine, it is the site of most gestion and absorption of fats. The duo of the breakdown of the food passing denum is responsible for secreting hormo through it. The duodenum is lined with nes that trigger the pancreatic duct to re duodenal submucosal glands, which secre lease pancreatic juice and bile. In addi te an alkaline mucus that supports the in tion, the mucus secreted by duodenal sub testinal enzymes and aids in the absorp mucosal glands helps protecting the duo tion of nutrients. The pancreatic duct, denum from the acidity, making the duo which introduces bile and pancreatic juice denum much less sensitive than the rest of into the small intestine, is directly con the small intestine to the acidic chyme. nected to the descending duodenum. Pan Therefore, the duodenum protects the rest creatic juice contains enzymes that help of the small intestine by neutralizing the Morphological and histochemical study of guinea pig duodenal submucosal glands chyme to some extent before it passes into 1996; Gartner & Hiatt, 2003). Various the jejunum (Cunningham & Klein, 2007). studies dealing with the mucosubstance The general morphology of the duode histochemistry of duodenal submucosal num of some species was examined in glands, pyloric glands and goblet cells in a former studies. However, in few species large number of mammals show marked the distribution and morphology of the inter species and even within species va duodenal submucosal glands deviated riation (Poddar & Jacob, 1979). There are from the general pattern. In the South incomplete data about the duodenum and American opossum, as has also been re duodenal submucosal glands of guinea ported for Didelphis virginiana (Schuma pigs. cher & Krause, 1995), the duodenal sub The aim of this study was to examine mucosal glands were confined to a very the normal appearances of duodenum, to narrow region, immediately distal to the measure the inner diameter of duodenal pyloric sphincter. In comparison with the submucosal glands in the three parts of the other mammals examined, the secretory duodenum and to compare them each with units of duodenal submucosal glands in the other. Also, it aimed to determine the the raccoon were more dilated and lined histochemical properties of guinea pig with flatter cells. Similar observations re duodenal submucosal glands. garding the luminal size of duodenal sub mucosal glands in the mouse have been MATERIALS AND METHODS reported (Krause, 1981). The acini of duo denal submucosal glands in the raccoon were relatively dilated in comparison with Animals these of other mammals. In the duodenum, Ten healthy adult male guinea pigs ( Cavia goblet cells stained with Periodic Acid porcellus ), long haired breed, aged 8–10 Schiff (PAS) and Alcian blue at both pH months, weighing 500–550 g were used 1.0 and 2.5, while duodenal submucosal for the study. The animals were obtained glands stained only with PAS. The histol from the Razi institute in Mashhad, Iran ogy of the gastroduodenal junction of the and feed with regular food made of pel opossum differed from other mammals. In leted alfalfa grains. The animals were opossums, duodenal submucosal glands anaesthetised with sodium pentobarbital were concentrated in a very short portion and then euthanized by an overdose of the of the submucosa, extending from the same drug. distal part of the pyloric region of the stomach to the proximal duodenum (Schu Tissue samples macher et al., 2004). The small intestines, which were removed The existence of duodenal submucosal from the body of the animals by the im glands in the duodenum is uncontestable mersion method following the dissection (Botros et al. , 1990; Bloom & Fawcett, of the abdominal cavity were examined 1994; Burkitt et al. , 2000). However, the histochemically. Part of the tissue samples re remain doubts as to their exact location were first fixed in 10% neutral buffered along the full extent of the duodenal wall, formalin and then subjected to routine given that the existing opinions in the spe tissue processing for light microscopy. cialized literature are often incomplete or The resultant blocks were cut into sections difficult to interpret (Coutinho et al. , 5 m thick and stained with haematoxylin
202 BJVM, 14 , No 4 A. A. Mohammadpour and eosin for general histological exami nation. Periodic Acid Schiff (PAS) and Alcian blue (pH 2.5) were used for neutral TM and for acidic mucosubstance, respec DSG tively. In PAS staining technique, the sec tions were oxidized for 5 min in 1% aque ous periodic acid, washed under running tap water for 5 min, rinsed in distilled V water, and then treated with Schiff’s re agent for 15 min. Afterwards, the sections were washed with water for 10 min and counterstained with haematoxylin. Finally, the sections were dehydrated in alcohol, cleared in xylene, and mounted in a resin ous mountant. In Alcian blue method (pH Fig. 1. Optical photomicrograph of the cranial 2.5), sections were stained in freshly fil part of duodenal wall. Note the numerous tered 1% Alcian blue 8 GX in 3% acetic plicae circularis in this part, duodenal submu acid (pH 2.5) for 30 min and washed in cosal glands (DSG) are numerous, occupy the water. The sections were dried with fine entire duodenal submucosa and extend near to filter paper and were dehydrated in alco the base of villi (V). The tunica muscularis hol, cleared in xylene, and mounted in a (TM) was composed of thick inner and thin resinous mountant. outer layers. H&E staining, bar=100 m. Statistical analysis TM For histological studies, after tissue prepa ration, duodenal tissue layers and duode nal submucosal glands in tunica submu cosa were measured using the micrometre method. All parameters between the three parts of the duodenum were analysed and DSG compared using ANOVA test. V
RESULTS
In the guinea pig, duodenal submucosal Fig. 2. Optical photomicrograph of the cranial glands are compound, tubuloalveolar, part of duodenal wall. Note the duodenal composed only of mucous acini densely submucosal glands (DSG) located in tuinca packed within the submucosa. The cross submucosa. The glands are only mucous and section of each gland comprised six to ten tubuloalveolar. Tunica muscularis (TM) and cells around a narrow lumen and the cells Villi (V) are labelled. H&E staining, bar= 100 m. had a truncated pyramidal form. In these cells the nucleus was round, located near num (Fig. 1). The cranial part of duode the basement membrane, and with a dis num presented large quantities of duode tinct nucleolus. The glands were well de nal submucosal glands in all of the fields veloped in the cranial part of the duode examined. In this part, intestinal wall was
BJVM, 14 , No 4 203 Morphological and histochemical study of guinea pig duodenal submucosal glands thicker than descending and ascending parts. Plicae circularis were also well de veloped and detectable. The descending part also showed the presence of duodenal submucosal glands in the entire field exa mined, albeit in smaller quantities than in the cranial part (Fig. 2). In the ascending part, the number of duodenal submucosal glands in submucosa was variable and there was a minimal quantity of glands. In some of these sections, duodenal submucosal glands were few and poorly developed. In ascending part plicae circularis were not detected and the intestinal wall was thinner Fig. 4. Optical photomicrograph of the cranial than the other two parts (Fig. 3). part of duodenum. Secretory unit cells of duo denal submucosal glands (DSG) and goblet cells (arrow) reacted positively with Periodic acid shiff. Periodic acid shiff staining, bar= 100 m.
The inner diameter of duodenal sub mucosal glands in the three parts of the duodenum was different and larger in the DSG cranial part. There was a significant dif ference in inner diameters of duodenal submucosal glands in all parts of the duo denum, decreasing from the cranial (147.00±22.87 m) to the ascending part Fig. 3. Optical photomicrograph of the cranial part of duodenum. Secretory unit cells of duo (38.00±12.29 m). The thickness of tu denal submucosal glands (DSG) and goblet nica muscularis and total thickness of in cells (arrow) positive in Alcian blue (pH 2.5). testinal wall were reduced from the cranial Alcian blue staining, bar=100 m. to the ascending part (Table 1).
Table 1. Histometrical parameters of three parts of duodenum in guinea pig (mean ± SD; n=10)
Parameters ( m) Cranial part Descending part Ascending part Height of villi 785.00 ± 850.00 ± 828.84 ± 87.67 69.03 46.59 Inner diameter of duodenal submucosal 147.00 ± 58.00 ± 38.00 ± gland 22.87 A 14.73 B 12.29 B Thickness of tunica muscularis 293.18 ± 142.14 ± 105.00 ± 37.23 A 26.07 B 15.81 B Total thickness of intestinal wall 1306.81 ± 1039.50 ± 1026.92 ± 132.80 A 136.06 B 80.01 B Different letters within a row indicate difference, significant at P<0.001.
204 BJVM, 14 , No 4 A. A. Mohammadpour
a positive PAS reaction indicates the pres ence of neutral carbohydrates, while posi tive Alcian blue reactions at pH 2.5 indi cate the presence of acidic carboxylated residues, respectively. In this research, DSG goblet cells and duodenal submucosal glands were stained with PAS and Alcian blue at pH 2.5.
DISCUSSION
Since the duodenal submucosal glands Fig. 5. Optical photomicrograph of the des were discovered by Wepfer in 1679, stu cending part of duodenum. Duodenal submu dies in many animal species were con cosal glands (DSG) are present in tunica sub ducted to clarify the extent and the density mucosa; however, their numbers are fewer of their distribution, the types of cells than in the cranial part of duodenum. Secre forming the glands by light and electron tory unit cells of DSG and goblet cells (arrow) microscopy. However, there are only few reacted positively with Alcian blue (pH 2.5). histochemical studies on the composition Alcian blue staining, bar=100 m. of the secretion of this gland. In the pre sent study it was elucidated that the duo denal glands in the guinea pig were com posed only of mucous cells. The principal function of the glands is thought to be DSG protection of the duodenal mucosa against the erosive effects of the gastric juice by virtue of the mucoid nature of its secre tion, its alkalinity, and possibly by the buffering capacity of its bicarbonate con tent. No enzymatic activity involved in the digestive process has yet been found, al though in some species juices collected from the region of duodenal submucosal Fig. 6. Optical photomicrograph of the ascen glands contained a mucolytic enzyme. ding part of duodenum. Greatly reduced num Secretion of the glands is enhanced by bers of duodenal submucosal glands (DSG) humoral (crude secretion), nervous (para can be seen in the duodenal submucosa. The sympathetic), and mechanical stimuli (pre glands are only occasionally present in the duodenal submucosa. Secretory unit cells of sence of food or rubbing the mucosal sur DSG and goblet cells (arrow) reacted posi face). Although the similarities of the tively with Alcian blue (pH. 2.5). Alcian blue duodenal glands of various animals have staining, bar=100 m. been emphasized in the past, physiologi cal evidence of significant differences The results of the carbohydrate histo among species were reported in more re chemistry were shown on Fig. 3, 4, 5 and cent histochemical studies (Farkas et al. , 6. In classic carbohydrate histochemistry,
BJVM, 14 , No 4 205 Morphological and histochemical study of guinea pig duodenal submucosal glands
1989; Fuse et al. , 1990; Krause, 2000; (Krause, 2000). In horses, duodenal sub Morre et al. , 2000). mucosal glands occupy a very large area Cells, which compose the duodenal and extend approximately 6 m caudal to submucosal glands, vary with species. the pylorus. They are known to exist also These glands were reported to be com in the jejunum of pigs and large herbi posed of two types of cells, serous and vores (Verdiglione et al. , 2002). In rab mucous cells in rabbits and horses bits, the distribution of duodenal submu (Oduor Okelo, 1976; Pfeiffer & Dabarei cosal glands was determined to start from ner, 1992; Takehana et al. , 1989), while the pyloroduodenal junction and to extend they were composed of only mucous cells near the jejunum. In the pony (Takehana in the moose, elk, bison and white tailed et al. , 1989) mucous glands were reported deer (Krause, 1981; 2000). to be present along the duodenum, while Histological results in guinea pigs serous glands were determined to be lo showed that histological layers and rate of cated in the upper part of the duodenum duodenal submucosal glands secretion in within the region extending. the three parts of duodenum were vari Despite their similar morphological able. In cranial part of duodenum, the appearance in the H&E sections, the PAS submucosa had numerous spiral folds (the and Alcian blue (pH 1.0 and 2.5) staining plicae circulare), which increase the sur properties of duodenal submucosal glands face area for maximum absorption. Also, showed marked differences in our study. the number of duodenal submucosal In bison, deer, voles, and cotton tailed and glands was higher than those in the other domestic rabbits, they contain acidic sul parts and total wall thickness was higher. phated and carboxylated mucins, whereas We suggested that, in the cranial part of in humans, rhesus and Japanese ma duodenum, the acidity of gastric secretion caques, cats, raccoons, rats and opossums was higher compared to the other parts, they contain neutral mucins. This varia thus, more duodenal submucosal glands tion could not be attributed to either the are needed to neutralize the acidity of order or the diet of the mammals food. . In guinea pigs, duodenal submuco (Schumacher et al. , 2004). sal glands were observed in the three The general morphology of the duode parts, but their density decreased from the num of guinea pig examined in this study cranial to the ascending part. In most spe was in accordance with that described for cies, duodenal submucosal glands are dis mammals in general. In classic carbohy tributed in an area starting from the gas drate histochemistry, a positive PAS reac trointestinal junction and extending to tion indicates the presence of neutral car varying distances in the proximal small bohydrate, while positive Alcian blue re intestine (Alogninouwa et al. , 1996; Krau actions at pH 1.0 and 2.5 indicates the se, 2000; Takehana et al. , 2000; Verdi presence of acidic sulphated and acidic glione et al. , 2002), while in humans they carboxylated residues, respectively (Spi extend almost to the level of the papilla of cer & Schulte, 1992). In guinea pigs, his Vater (Treasure, 1978). In mammalian tochemistry of duodenal submucosal species and in eutherians, duodenal sub glands showed that secretory unit cells mucosal glands are located within the first reacted positively with Periodic acid shiff few millimetres of the proximal duode and Alcian blue (pH 2.5) stains. These num, just distal to the pyloric sphincter results indicated that the secretion of duo
206 BJVM, 14 , No 4 A. A. Mohammadpour denal glands in guinea pigs contained neu Bloom, W. & D. Fawcett, 1994. A Textbook th tral sugar and acid carbohydrate with of Histology, 12 edn, W. B. Saunders, sialic acid. Various studies dealing with Philadelphia. the mucosubstance histochemistry of duo Botros, K. G., S. L. el Hady & E. A. el denal submucosal glands in large numbers Mohandes, 1990. Prenatal development of of mammals show marked species varia the human Brunner’s glands. Anatomis- tion and variation even within the species cher Anzeiger , 171 , 23–30. Burkitt, H. G., G. Young. & J. W. Heath, (Poddar & Jacob, 1979). Therefore, de th pending on species, duodenal submucosal 2000. Weather’s Histologia Funcional, 4 edn, Guanabara Koogan, Rio de Janeiro. glands in guinea pig secreted neutral and acidic carboxylated mucin. In addition, Coutinho, H. B., T. I. Robalinho, V. B. the results showed that the goblet cells in Coutinho, A. M. Amorin, J. R. Almeida, J. T. Filho, E. Walker, G. King, H. F. Sewell guinea pig contained acidic carboxylated & D. Wakelin, 1996. Immunocytochemical and neutral mucins. These observations demonstration that human duodenal Brun suggest that the mucins secreted by goblet ner’s glands may participate in intestinal cells have been conserved with regard to defence. Journal of Anatomy , 189 , 193– their charge during the evolution of mam 197. mals (Ota et al. , 1998). In studies conduc Crescenzi, A., P. Barsotti, L. Anemona & V. ted using different techniques, depending Marinozzi, 1988. Carbohydrate histochem on species, duodenal submucosal glands istry of human Brunner’s glands. Histo- were reported to contain neutral or acidic chemistry , 90 , 47–49. mucin glycoproteins or the combination of Cunningham, J. G. & B. G. Klein, 2007. Text both types of mucin (Crescenzi et al. , book of Veterinary Physiology, 4 th edn, 1988; Takehana et al. , 1989; 1991a, 2000; Elsevier Health Sciences. Krause, 2000; Verdiglione et al. , 2002). Farkas, I. E. & G. Gero, 1989. The role of Generally, the duodenal glands are be Brunner's glands in the mucosal protection lieved to protect the duodenal mucosa of the proximal part of duodenum. Acta from the gastric hydrochloric acid. Physiologica Hungarica , 73 , 257–260. Fuse, Y., Y. Tsuchihashi, M. Takamasu, T. Kodama, S. Fujita & K. Kashima, 1990. ACKNOWLEDGEMENTS Thickness of Brunner's glands and its clinical significance in duodenal ulcer dis The author wishes to express his appreciation ease. Scandinavian Journal of Gastroen- to the Research Council of the Ferdowsi Uni terology , 25 , 165–172. versity of Mashhad for the financial support Gartner, L. P. & J. L. Hiatt, 2003. Tratado de (Research project no: 14654). Also, many Histologia Em Cores. 2 ed., Rio de Ja thanks to Mr. Gh. Pouradibi for his assistance. neiro, Guanabara Koogan, Krause, W. J., 1981. Morphological and histo REFERENCES chemical observations on the duodenal glands of eight wild ungulate species na Alogninouwa, T., K. C. Agba, E. Agossou & tive to North America. American Journal M. Kpodekon, 1996. Anatomical, histo of Anatomy , 162 , 167–181. logical and functional specifities of the di Krause, W. J., 2000. Brunner’s glands: A gestive tract in the male grasscutter ( Thry- structural, histochemical and pathological onomys swinderianus ). Anatomia, His- profile. Progress in Histochemistry and tologia, Embryologia , 25 , 15–21. Cytochemistry , 35 , 255–367.
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