NEUROBIOLOGY & PHYSIOLOGY Animal Cells and Systems, 2015 Vol. 19, No. 5, 289–294, http://dx.doi.org/10.1080/19768354.2015.1074105 Distribution of neuroendocrine cells in the small and large intestines of the mole-rats (Spalax leucodon) a a b Ali Bayrakdar , Mine Yaman * and Ramazan Ilgün a b Department of Histology and Embryology, Faculty of Veterinary Medicine, Fırat University, Elazığ, Turkey; Department of Anatomy, Faculty of Veterinary Medicine, Aksaray University, Aksaray, Turkey (Received 5 January 2015; received in revised form 21 April 2015; accepted 4 June 2015) In this research, we aimed to determine the regional distribution and relative frequency of immunoreactive (IR) endocrine cells of ghrelin, glucagon, somatostatin–14 (SOM–14), neuropeptide Y (NPY), calcitonin-gene-related peptide (CGRP), insulin and galanin in small and large intestines of mole-rats (Spalax leucodon) by using immunohistochemical staining methods. For this purpose, small and large intestine regions of six adult mole-rats were used as material. In the small and large intestines of mole-rats (S. leucodon), ghrelin, glucagon, SOM–14, NPY and CGRP were identified at various frequencies, but insulin and galanin were not detected in this study. Ghrelin-IR cells were only observed in the small intestine. In addition, the existence of positive staining in the cells was decreased while passing from duodenum to ileum. Glucagon-IR cells were mainly detected in the small intestine regions and colon. SOM–14-IR cells were detected throughout the small intestines with a lesser frequency. NPY-IR cells were also detected in the small and large intestines at various frequencies but not in cecum and rectum. CGRP-IR cells were scarce in duodenum and jejunum, but their numbers increased from ileum to colon. In conclusion, the present study revealed the existence and distributions of neuropeptide-secreting endocrine cells in small and large intestines of mole-rats (S. leucodon). Keywords: Mole-rats (Spalax leucodon); gastrointestinal tract; immunohistochemistry Introduction degree (Ku et al. 2002, 2003, 2004a, 2004b). However, as far as we know there is not any information concerning The rodents, which are the widest order of placental endocrine cells in gastrointestinal tracts of mole-rats apart mammals, comprise more than half of the mammals actu- from our report related to the existence of serotonin and ally known. The mole-rat is a representative of the Spalaci- some neuropeptides (Yaman et al. 2012). dae family, which constitutes a group of order of Rodentia The objective of this study was to clarify the regional (Kuru 1987; Demirsoy 1992). distribution and relative frequency of endocrine cells in Gastrointestinal endocrine cells, dispersed among the small and large intestines of mole-rats by immunohisto- epithelial cells of the gastrointestinal tract, together with chemical methods using specific antisera against ghrelin, the enteric nervous system play a vital role in the function- glucagon, somatostatin–14 (SOM–14), neuropeptide Y ing of the digestive system (Bell 1978). Several studies (NPY), calcitonin-gene-related peptide (CGRP), insulin have demonstrated the regional distributions and relative and galanin. frequencies of different endocrine cells in the gastrointesti- nal tract of various vertebrates, including several species and some of rodent species, and different kinds of these Materials and methods cells were described using immunohistochemical Animal and tissue samples methods. In the Rodentia, the localizations of endocrine cells in the gastrointestinal tract of the rat (Sakata et al. In the present study, six adult mole-rats, trapped by the 2002; Sarı et al. 2007), mice (Sandstrom & El-Salhy farmers, were used. After the mole-rats were anaesthetized 2000), gerbil (Lee et al. 2000), hairless mouse (Ku et al. with Pentothal (40 mg/kg, IP), tissue samples were taken 2002), C57BL/6 mouse (Ku et al. 2003), Balb/c mouse from the small and large intestines and were fixed in (Ku et al. 2004a), ddY mouse (Ku et al. 2004b), nude 10% neutral-buffered formalin. Tissues were then dehy- mice (Ku et al. 2006) and ddN mice (Lee et al. 2010) drated and embedded in paraffin. were demonstrated. Changes of regional distribution and relative frequency of some gastrointestinal endocrine Immunohistochemistry cells with aging were also reported in mice (Sandstrom & El-Salhy 2000) and the distribution and frequency of gas- Immunohistochemical staining was performed by the trointestinal endocrine cells in various mouse species has avidin–biotin–peroxidase complex (ABC) method by been shown with strain-dependent differences to a certain using the Ultravision Detection System Anti- Polyvalent, *Corresponding author. Email: firstname.lastname@example.org © 2015 Korean Society for Integrative Biology 290 A. Bayrakdar et al. Table 1. The details of the antibodies used in this study. Primer antibodies Dilution Trade name Cat. no. Ghrelin 1:1000 Santa Cruz Biotecnology, Inc. Germany sc-50297 Glucagon 1:200 Santa Cruz Biotecnology, Inc. Germany sc-13091 Som-14 1:1000 Chemicon (Millipore), Canada AB1976 NPY 1:200 Chemicon (Millipore), Canada AB1915 CGRP 1:1000 Chemicon (Millipore), Canada AB5920 Insulin 1:1000 Abcam ab63820 Galanin 1:500 Chemicon (Millipore), Canada AB5909 Note: All antisera were raised in rabbit (polyclonal). SOM–14, somatostatin–14; NPY, neuropeptide Y; CGRP, calcitonin-gene-related peptide. HRP/DAB kit (Thermo Scientific) according to the manu- location of the small and large intestines, different regional facturer’s protocols. In accordance with ABC methods, distributions and relative frequencies of these IR cells were dewaxed and re-hydrated sections were treated with 3% observed and these differences are given in Table 2. The H O in methanol for 10 minutes. In order to block unspe- regional distribution and relative frequency of intestinal 2 2 cific binding, incubation with normal goat serum (1.10) endocrine cells varied with the intestinal tract and some was performed. Sections were incubated with primary anti- unique distributional patterns were also found in the bodies for 16–20 hours at 4°C. The working dilutions and mole-rats (S. leucodon). the sources of the primary antibodies are listed in Table 1. Ghrelin-IR cells were localized in the whole small Negative control sections were performed by replacing the intestine at various frequencies except for large intestine. primary antibodies with PBS (phosphate-buffered saline). These cells were only observed in the villi and crypts of The sections were then incubated with biotinylated goat the mucosal layer. In the small intestine, a great majority anti-rabbit immunoglobulin G (sigma) followed by of ghrelin-IR cells were round-shaped closed-type cells. avidin–biotin–peroxidase complex (Dako) for 1 hour. A few ghrelin-IR cells were found in the duodenum, and Finally, sections were immersed in glucose oxidase–DAB they were situated within the epithelium of intestinal (diaminobenzidine)–nickel ammonium sulphate substrate crypts and villi (Figure 1(a) and (b)). In addition, rare (Shu et al. 1988) for 10 minutes and counterstained with ghrelin-IR cells were observed in the intestinal crypt hematoxylin. region of jejunum and ileum (Figure 1(c)). On the other The specificity of each immunohistochemical reaction hand, intensity of the staining was less in these regions was determined as recommended by Sternberger (1979), than the duodenum. including the replacement of specific antiserum by the Glucagon-IR cells were detected in the small and large same antiserum, which had been pre-incubated with its cor- intestines at various frequencies except for the cecum and responding antigen. Sections were examined with rectum. Moderate numbers of glucagon-IR cells were Olympus BX–51 microscope and photographs were found in the duodenum, although they were present at taken. The immunoreactive (IR) endocrine cells on each lesser frequencies in the jejunum and ileum. Great majority section were counted at 10 × 40 times magnification. The of glucagon-IR cells were oval and spindle-shaped closed- mean numbers of IR endocrine cells in each sample type cells. In the duodenum, closed-type cells were mostly obtained from small and large intestines were determined found in the epithelium of the villi, but were rarely detected by counting the IR endocrine cells in 10 randomly selected in the basal regions of crypt. However, a limited number of microscopic fields with 40 × magnification (quantification IR cells were located as open-type cells in villi (Figure 2(a) of IR cells numbers/microscopic field). The frequencies of and (b)). Only open-type cells were demonstrated in the IR cells were calculated as mean± standard deviation (SD) crypt regions of the jejunum (Figure 2(c)). In addition, of the total of 60 parts (10 fields in each animal) of each closed-type IR cells in the ileum resembled the IR cell local- intestinal region (Table 2). izations in the duodenum. In large intestines, glucagon-IR cells were detected in moderate numbers in the crypt epi- thelium of the colon. Open-type cells were mixed with closed-type cells in those regions and their shapes varied Results from triangular to oval (Figure 2(d)). Also, triangular- In this study, all five kinds of the IR-endocrine cells were shaped open-type cells were observed in basal crypts detected with the antisera against ghrelin, glucagon, regions (Figure 2(e)). SOM–14, NPY and CGRP in the small and large intestines SOM-14-IR cells were identified throughout the small of mole-rats (Spalax leucodon) for the first time. However, intestines with a rare frequency except for large intestines. insulin and galanin-IR cells were not identified in any The IR cells were located inside the epithelia of the crypts region of the small and large intestines. Based on the Animal Cells and Systems 291 Table 2. Regional distributions and relative frequencies of IR endocrine cells in the small and large intestines of the mole-rats (S. leucodon). Duodenum Jejunum Ileum Cecum Colon Rectum Ghrelin 2.1±0.94 0.7±0.46 0.6±0.49 –– – Glucagon 7.2±1.54 2.3±0.90 −2.5±1.20 – 2.8±1.47 −– Som-14 0.9±0.70 0.5±0.50 0.8±0.40 –– – NPY 3.1±1.22 0.3±0.46 0.5±0.50 – 6.9±1.51 – CGRP 0.9±0.54 0.7±0.46 0.8±0.60 – 2.7±1.1 – Insulin –– – – – – Galanin –– – – – – Note: Values are expressed as mean ± SD of 6 mole-rats (S. leucodon), of total 60 parts (ten field in each animal) of each the intestinal regions. but not in the villi. Open-type SOM-14-IR cells were exclusively located in the basal portion of the intestinal crypt regions changing in shape from round to spindle. Their cytoplasmic extensions appeared to be directed toward the crypt lumen (Figure 3(a)). Round-shaped closed-type cells were occasionally demonstrated in basal intestinal crypts. NPY-IR endocrine cells were situated along the whole intestine except for the cecum and rectum. In the small intestine, majority of NPY-IR endocrine cells were situated in the basal regions of the intestinal crypt. Most of these IR cells were round-shaped closed-type cells (Figure 3(b)). They were more numerous in the colon mucosa, particu- larly in surface epithelium and their shapes varied from elongated to triangular. Almost all of NPY-IR cells were open-type cells (Figure 3(c)). On the other hand, closed- type cells were round shaped and were also located in the crypt region. CGRP-IR cells were found both in small and large intestines at various frequencies, although none were found in cecum or rectum. The number of CGRP-IR cells was rare in the duodenum and jejunum, but their numbers increased from ileum to colon. In the duodenum, CGRP-IR cells were located in the basal region of the intes- tinal crypts. These IR cells were shuttle-shaped open-type cells and they had luminal contact via an apical cyto- plasmic process (Figure 3(d)). In the colon, they were situ- ated among the epithelial cells and in the basal crypt regions. Open-type cells were situated in the epithelium of the mucosal folds. These cells were elongated in shape. Only a few closed cells were detected in the basal crypt (Figure 3(e)). Insulin and galanin-IR cells were not detected in the present study. Discussion The localization of gastrointestinal endocrine cells in Figure 1. Microphotograph showing IR cells for ghrelin in the various vertebrates has been investigated using immunohis- small intestines of the mole-rats. (a) Closed-type IR-cells were tochemical methods (Sarı et al. 2007; Yaman et al. 2007). round shaped in the villus of duodenum. (b) Closed-type IR- The regional distribution patterns and relative frequencies cells were located in the intestinal crypts of duodenum. (c) Closed-type IR-cells were located in the intestinal crypts of ileum. of these cells show remarkable differences among animal 292 A. Bayrakdar et al. Figure 3. Microphotograph showing IR cells for SOM-14, NPY and CGRP in the small and large intestines of the mole-rats. (a) Open-type SOM-IR cells were located from round (arrowhead) to spindle (arrow) shaped in the intestinal crypt regions of duode- Figure 2. Microphotograph showing IR cells for glucagon in the num. (b) Closed-type NPY-IR cells were located in the intestinal small and large intestines of the mole-rats. (a–b) Closed- (arrow) crypts of duodenum. (c) Open-type NPY-IR cells were elongated and open (arrowhead)-type IR-cells were located in the villi of (arrowhead), shuttle (arrow) and triangular (big arrow) in shape in duodenum. (c) Open-type IR-cells were located in the crypts of colon. (d) Open-type CGRP-IR cells were located in the intestinal jejunum. (d–e) Triangular-shaped open-type IR-cells were crypts of duodenum. (e) Closed-type CGRP-IR cells were located located in basal crypt regions of colon. in the basal crypts of colon. Animal Cells and Systems 293 species and within parts of the gastrointestinal tracts. In (Falkmer & Van Noorden 1983). However, species-depen- addition, many studies have elucidated the regional distri- dent variations of the distributional patterns of these IR butions and relative frequencies of different endocrine cells have been reported. They were detected throughout cells in the gastrointestinal tracts of various vertebrates, the whole gastrointestinal tract of the rodent species including various species of rodents (Ku et al. 2004a, except for cecum and rectum (Ku et al. 2004b; Yaman 2004b; Yaman et al. 2007; Lee et al. 2010). The present et al. 2007; Lee et al. 2010), and they were detected through- study identified the existence and distributions of neuropep- out the whole gastrointestinal tract except for the colon and tide-secreting endocrine cells in the small and large intes- rectum of nude mice (Ku et al. 2006). Also, these IR-cells tines of mole-rats. In this study, ghrelin, glucagon, Som– were found in the whole gastrointestinal tract of the 14, NPY and CGRP were shown to exist in the intestines C57BL/6 mice except for the cecum, colon and the rectum of mole-rats for the first time. (Ku et al. 2003). In addition, SOM-IR cells in duodenum The distribution of ghrelin-IR cells in the gastrointesti- of NMRI mice were reported to decrease with age (Sand- nal tract was studied by using immunohistochemical tech- strom & El-Salhy 2000). In the rat, small frequencies of niques (Date et al. 2000; Sakata et al. 2002). In Rodents, these cells were detected in the small intestine (Sarı et al. ghrelin-IR cells were observed in all the regions of the gas- 2007). In the present study, SOM-14-IR cells were observed trointestinal tract. In duodenum, ileum, cecum and colon, throughout the whole intestinal tract except for the large ghrelin cells were scattered in the epithelia of crypts and intestines of mole-rats. These results correlated with those villi. The densities of ghrelin cells were dramatically of some rodent studies (Sandstrom & El-Salhy 2000;Ku decreased toward lower gastrointestinal tract regions et al. 2003; Sarı et al. 2007) but were quite different from (Sakata et al. 2002). Ghrelin-IR cells were also most those conducted on mice strains and porcupine (Ku et al. numerous in the stomach region, less abundant in the duo- 2004b, 2006; Yaman et al. 2007; Lee et al. 2010). denum, occasional in the jejunum, and rare in the ileum of Some researchers detected NPY-IR nerve fibers in the human gastrointestinal tract while absent in the large intes- intestinal tracts of rats (Sundler et al. 1983; Jackerott & tine (Gronberg et al. 2008). In this study, ghrelin-IR cells Larsson 1997). In these examinations performed on rats, were found only in small intestine crypts and villi of limited numbers of NPY-IR endocrine cells were observed mole-rats. In the small intestine, ghrelin-IR cells were in the small intestines. In the present study, NPY-IR cells decreased while passing from duodenum to ileum. The dis- were observed throughout the intestinal tract except for tributions and frequencies of these cells correlated with the the cecum and rectum, at higher frequencies in the colon. results of humans (Gronberg et al. 2008), but differed from The results of this study are similar to the results of the pre- those of other rodents (Sakata et al. 2002). These differ- vious study, except for the presence of these cells in the ences detected among various rodent species were con- colon (Jackerott & Larsson 1997; Matsuda et al. 2002). sidered as species-dependent characteristic distribution The distribution of CGRP-IR endocrine cells was pre- patterns. viously investigated in stomachs of rats (Kasacka 2009) Glucagon immunoreactivity has been demonstrated in and porcupines (Yaman et al. 2007). However, there are various mammals, and the distribution patterns of these no reports in the literature that describe CGRP-IR cells in cells in the gastrointestinal tract of mammals are con- the intestines of rodents with immunohistochemistry. In sidered to demonstrate species-dependent variations (Ku the present study, CGRP-IR endocrine cells were observed et al. 2003). Glucagon-IR cells were detected in stomachs in small and large intestines of mole-rats for the first time, and small intestines of Manchurian chipmunks (Lee et al. except for cecum or rectum. 1997). These cells were found throughout the gastrointes- The present study revealed the existence and distri- tinal tracts of musk shrews (Solcia et al. 1989) and butions of neuropeptide-secreting endocrine cells in the common tree shrews (Yamada et al. 1999), and appeared small and large intestines of mole-rats (S. leucodon). to be absent from the gastrointestinal tracts of gerbils Higher number of IR endocrine cell presence in the small (Lee et al. 2000). In the ddY mice (Ku et al. 2004b), gluca- intestine suggests primary roles of these segments of the gon-IR cells were detected in fundus, duodenum and gut in the control of food intake and thus the modulation jejunum. Especially, appearances of these IR cells were of the feeding behavior by strongly stimulating the also reported in large intestines in mice (Spangeus & El- smooth musculature of the gut and controlling the Salhy 1998). In the present study, glucagon-IR cells were mucous secretion of the glandular epithelium. This knowl- observed predominantly in duodenum and other intestinal edge allows us to design new strategies to implement the regions. The results of this study are compatible with the culture of this species. The general distribution patterns findings reported in the previous studies (Solcia et al. and relative frequencies of intestinal endocrine cells of 1989; Yamada et al. 1999). mole-rats were similar to those of certain rodent species. It is well known that somatostatin-IR cells have the However, some species-dependent unique distributions widest distribution throughout the gastrointestinal tract, and frequency characteristics of endocrine cells were also except for the large intestine, among all vertebrate species observed in the present study. The characteristic existence 294 A. Bayrakdar et al. Lee HS, Choi SH, Ku SK. 2010. 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Animal Cells and Systems
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Published: Sep 3, 2015
Keywords: Mole-rats ( Spalax leucodon ); gastrointestinal tract; immunohistochemistry