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Localization of the orexin system in the gastrointestinal tract of fallow deer
Acta Histochemica 114 (2012) 74–78
Contents lists available at ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
Localization of the orexin system in the gastrointestinal tract of fallow deer Cecilia Dall’Aglio a,∗ , Luisa Pascucci a , Francesca Mercati a , Cristiano Boiti b , Piero Ceccarelli a a
Sezione di Anatomia Veterinaria, Dipartimento di Scienze Biopatologiche Veterinarie ed Igiene delle Produzioni Animali ed Alimentari, Via S. Costanzo 4, 06100 Perugia, Italy Laboratorio di Biotecnologie Fisiologiche, Sezione di Fisiologia Veterinaria, Dipartimento di Scienze Biopatologiche Veterinarie ed Igiene delle Produzioni Animali ed Alimentari, Via S. Costanzo 4, 06100 Perugia, Italy b
a r t i c l e
i n f o
Article history: Received 18 January 2011 Received in revised form 11 February 2011 Accepted 13 February 2011
Keywords: Hypocretin Orexin cells Immunohistochemistry Neuroendocrine cells Gastrointestinal tract Deer
a b s t r a c t The aim of the present study was to investigate by immunohistochemistry the presence and distribution of the orexin system in the stomach and gut of fallow deer. Abundant orexin A-positive cells were localized in the middle and basal portions of the mucosal glands of the cardial and fundic regions of the stomach. In the same gastric areas, orexin B-positive cells were also found, mainly localized in the basal portion of glands. In the intestinal tract, orexin-containing cells were occasionally found in the duodenal epithelium and in the rectal intestinal glands. Immunoreactivity for orexin receptors, type 1 and 2 (OX1R and OX2R), was not detected in the same stomach regions. OX1R-immunopositivity was observed in the enteric neuron ganglia localized in the submucosal and muscular intestinal layers, while OX2R-immunopositivity was found close in contact with the cytoplasmic membrane of epithelial cells in the small intestine. © 2011 Elsevier GmbH. All rights reserved.
Introduction The orexin family is a complex system composed of two neuropeptides, orexin-A and orexin-B (OXA and OXB), and two cognate receptors, orexin type 1 and orexin type 2 (OX1R and OX2R). The two peptides derive from the same precursor, the pre-proorexin, through proteolytic cleavage and their sequences are at least 50% homologous. In particular, orexin-A is fully preserved in a large variety of mammals (Dyer et al., 1999; Sakurai et al., 1998; Shibahara et al., 1999; Smart and Jerman, 2002). The two receptors belong to the G-protein-coupled family and are 64% homologous (Sakurai et al., 1998). Recently, orexins have been studied extensively to comprehend their complex functional roles. In fact, since the first identification of OXA and OXB in rat hypothalamus (Sakurai et al., 1998), the presence of this system has been found in both the central and peripheral nervous systems as well as in several other different tissues and organs (Kukkonen et al., 2002; Heinonen et al., 2008). This widespread distribution leads to the supposition of new possible roles for the orexin system that extend beyond appetite control involving a large array of functions, depending on the tissue and/or organ where it is found. Orexins have been identified in humans (Nakabayashi et al., 2003; Erstróm et al., 2005; Heinonen et al., 2008), laboratory animals (Näslund et al., 2002; Sanchez de Miguel and Burrel, 2002)
and animals such as dogs, horses and ruminants (Zabielski, 2007; Dall’Aglio et al., 2008, 2009). They were found in peripheral tissues and, particularly, in endocrine cells and neurons of the enteric nervous system (ENS) of different portions of the gastrointestinal tract and in the pancreas and salivary glands (Dall’Aglio et al., 2010a,b). Many studies have emphasized the role of endocrine cells and ENS in the control of gut functions through the action of several neuropeptides, most of which can act as hormones and neuromediators, both in domestic (Ceccarelli et al., 1991, 1995) and wild mammals (Dall’Aglio et al., 1999). In this regard, the presence of the orexin system in the ENS may be interpreted as an additional mechanism of action through which the ENS can operate to control gut functions. As far as we know, data are lacking on the presence and distribution of the orexin system in the alimentary tract of wild animals. This information would be useful to analyse the gastrointestinal physiology of wild species which need to confront limited feed availability and which have different grazing habits. Therefore, the aim of this study was to evaluate, by means of immunohistochemistry, the localization of positive cells for each component of the orexin system in the gastrointestinal tract of fallow deer, a species which is expanding in the so called “marginal areas” that are not useful for traditional breeding of domestic species. Materials and methods
∗ Corresponding author. E-mail address: [email protected] (C. Dall’Aglio). 0065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2011.02.006
Samples were taken from a total of 5 adult fallow-deer, 3 males and 2 females, regularly slaughtered at the slaughterhouse of the
C. Dall’Aglio et al. / Acta Histochemica 114 (2012) 74–78
75
Table 1 Source and working dilutions of the reagents used. Antisera
Working dilutions
Sources
Normal goat serum Orexin-A Orexin-B Orexin type 1 receptor Orexin type2 receptor Goat anti-rabbit IgG, biotin conjugated Goat anti-mouse IgG, biotin conjugated ABC, Vector Elite Kit Diaminobenzidine (DAB)
1:10 1:100 1:100 1:100 1:100 1:200 1:200 1:100 As specified in the data sheet
S-1000, Vector Laboratories, Burlingame, CA, USA MAB763, R&D System Minneapolis, MN 55413, USA MAB734, R&D System Minneapolis, MN 55413, USA O 4514, Sigma, Missouri 63103, USA AB3094, Chemicon, Temecula, CA, USA 81-6140, Zymed, CA 94080, USA sc-2039, Santa Cruz Biotechnology PK-6100, Vector Laboratories, Burlingame, CA, USA SK-4100, Vector Laboratories, Burlingame, CA, USA
Cooperativa Ponte Parrano at Nocera Umbra, Perugia. In particular, specimens from the abomasum (cardial, fundic and pyloric regions), the duodenum and rectum were fixed by immersion in Bouin’s fluid at room temperature for 24 h. The tissue samples were then dehydrated through a graded series of ethanols, cleared in xylene, and embedded in paraffin. The immunohistochemical reaction was visualized on 5 m serial sections, mounted on poly-l-lysine coated glass slides, utilising the avidin–biotincomplex (ABC, Vector Elite Kit, Burlingame, CA, USA) and the 3,3 diaminobenzidine-4-HCl (DAB, Vector Laboratories) as the chromogen. To reduce variations in staining, tissue sections from each of the above described gastrointestinal portions were incubated together during each immunohistochemical procedure. In brief, dewaxed sections were microwaved for 15 min in 10 mM citric acid (pH 6.0) for antigen retrieval. All subsequent steps were carried out in a moist chamber at room temperature. To prevent non-specific binding of primary antibodies, after proper cooling, the sections were pre-incubated for 30 min with the normal serum (Table 1). Subsequently, serial sections were incubated overnight with one of the primary antibodies (Table 1): anti-OXA and anti-OXB mouse monoclonal antibodies and anti-OX1R and anti-OX2R rabbit polyclonal antibodies. The next day, after washing in phosphate-buffered saline (PBS), the sections were incubated for 30 min at room temperature with the corresponding secondary biotin-conjugated antibody (Table 1): a goat anti-mouse IgG for the two orexins and a goat anti-rabbit IgG for the two receptors. They were then processed for 30 min using the ABC kit. Subsequently, the tissue samples were repeatedly rinsed with PBS and developed with a chromogen solution. After several rinses in PBS, the sections were dehydrated and mounted in natural Canada balsam (BDH, Poole, Dorset, UK). Sections in which the primary antibodies were omitted or substituted with pre-immune gamma globulin were used as controls of non-specific staining. All tissue analyses were carried out on coded slides using a light microscope (Nikon Eclipse E800, Nikon Corporation, Tokyo, Japan) connected to a digital camera (Dxm 1200 Nikon digital camera). Images were processed using an image analysis system (Lucia, Laboratory Imaging Ltd.). The settings for image capture were standardized by subtracting the background signals obtained from the matched tissue sections which had not reacted with the primary antibodies and which were used as immunohistochemical controls. Variations in the intensity of immunolabeling for OXA and OXB were observed between different portions, possibly reflecting the expression of the corresponding antigens. However they were not quantified given the prevalently qualitative nature of the immunohistochemical technique in the tissue sections. Results The morphological studies together with immunohistochemical research revealed the presence of endocrine cells showing cyto-
plasmic positive reactions for orexin A and orexin B in the cardial and fundic regions of the abomasum. These cells showed morphological features typical of endocrine cells: they were oval or round shape and mainly of the closed type (Fig. 1a and b). OXA-positive cells were distributed in the basal and middle third of the tubular glands of the mucosa; their positivity appeared granular and prevalently localized around the nucleus (Fig. 1a). The OXB positive cells were localized exclusively in the basal third of glands; they were gathered, mainly of the closed type and contained many positive granules distributed throughout the cytoplasm (Fig. 1c). The number of OXA- and OXB-positive cells was particularly abundant in the cardial and fundic gastric regions, but in no case positive cells for orexins were visualized in the pyloric region. A few OXA and OXB positive cells were occasionally observed among the epithelial cells along the villi in the duodenum (Fig. 1d) and in the glands of the rectum (Fig. 1e). In the duodenum, such cells were elongated and spindle-shaped and, moreover, were prevalently of the open type, being in contact with the lumen of the gut via an apical cytoplasmic process (Fig. 1d). By immunohistochemistry, positive staining for OX1R was revealed in neurons of the ENS, which appeared localized in the submucosal (Fig. 1f) and muscular layers of the small and large intestine and were isolated or gathered in small or more voluminous groups. Positive staining for OX2R was localized exclusively in a few cells scattered among the epithelial cells in the villi of the duodenum (Fig. 1g). Immunoreaction characteristically seemed to be localized in the apical cytoplasmatic portion (Fig. 1h) in the tubular gland cells, as in the endocrine ones positive to OX2R. Staining was completely absent in the control sections where primary antibodies for orexins and cognate receptors were omitted (data not shown). Discussion Our immunohistochemical investigations of the orexin system showed the presence of different orexinic components in the gastrointestinal tract of fallow deer. The characteristic distribution in the digestive portions differs slightly from what was found previously in other animal species such as dogs and horses (Dall’Aglio et al., 2008, 2009). In fallow-deer, orexin A and orexin B-positive cells were numerous in the cardial and fundic gastric regions, but were completely absent in the pyloric region: their presence was observed again in the different areas of the intestinal tract though with a reduced frequency. Moreover, the orexins were localized only within the epithelial cells of glands of both abomasum and gut, whereas no staining was visualized in neurones and nerves in the submucosal and muscular layers. Orexins, being mainly distributed within the stomach and the intestinal glandular compartments of fallow-deer, confirm the results obtained in other animals such as dogs and horses (Dall’Aglio et al., 2008, 2009).
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Fig. 1. Orexin system immunohistochemical positivity in the gastrointestinal tract of fallow deer. (a and b) Many orexin-A positive cells in the basal and middle third of the fundic mucosa show an oval or round shape and a granular positivity localized prevalently around the nucleus. (c) Many orexin-B positive cells are seen in the basal third of fundic glands; they are typical round-shaped closed cells. (d and e) Some scattered orexin-B positive cells within the epithelial cells of the duodenum (d) and in the rectal intestinal glands (e) show an elongated or triangular shape. (f) Some orexin type 1 receptor-positive neurons in the duodenal sub-mucosal layer. (g and h) Orexin type 2 receptor-positivity localized within the epithelial cells (g) and in the intestinal glands (h); characteristic positive staining appeared localized near the apical cytoplasmic portions.
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Instead, the unsuccessful visualization of OXA in gastric neurons, though differing from what was observed in other animal species (Dall’Aglio et al., 2008; Nakabayashi et al., 2003), supports the results reported by Baumann et al. (2008) in men and mice and could reflect some species differences in the localization of the GI orexin system. However, the prevalent expression of OXA and OXB in cells of the abomasum suggests that, for the gastrointestinal tract, the two peptides are mainly produced in this anatomical region and are probably secreted into the bloodstream where OXA presence was documented in other animal species (Komaki et al., 2001). Thus they likely act in an endocrine way by controlling the brain–gut axis (Mendieta-Zerón et al., 2008) without any direct and/or indirect action within the stomach itself as evidenced by the different local distribution of their cognate receptors. In fact, neither OX1R nor OX2R were found in the gastric regions examined, but exclusively in the gut with a prevalent localization in the small intestine. OXR1 appeared distributed in an ubiquitarian way in the neurons of the enteric nervous system of each examined tract while OX2R appeared mainly localized in the intestinal glands of duodenum and in some scattered endocrine cells. The localization of the immunoreaction for OX2R is typical of the receptors linked to the cellular membrane, such as the G-protein coupled receptor, and is particularly evident in the intestinal glands where the positivity is limited to the apical portion of cytoplasm. The same pattern of distribution of the OX2R-immunoreaction was observed in the endocrine cells of intestinal villi, even if in this case positivity appeared more extended to the entire cytoplasm but with a much more evident concentration in the apical portion. Moreover, OX2R-positivity in the apical portion of epithelial intestinal cells may represent the organism’s response to orexin-like substances present in the gut lumen and reaching the surfaces of the glandular cells. Our results in fallow-deer, like those in humans, laboratory and domestic animals (Nakabayashi et al., 2003; Erstróm et al., 2005; Dall’Aglio et al., 2008, 2009), in peripheral tissues, particularly in endocrine cells, together with the aforementioned considerations, may extend the observation that the control of the gastrointestinal tract functions depends also on many substances, including orexins, in wild animals. These peptides, produced in a consistent way in specific gastric regions, probably have a hormonal endocrine action which may regulate not only gastrointestinal functions, such as gut motility, but also some central functions, such as appetite, thus controlling the brain–gut axis (Heinonen et al., 2008; Konturek et al., 2004; Kukkonen et al., 2002; Smart and Jerman, 2002). The present findings regarding the localization of the orexin system in the gastrointestinal apparatus of the fallow deer, in tune with what was observed in other species, underline even more the existence of a complex control mechanism of the gastrointestinal system that involves the orexin system, both at the central and peripheral level, with or without the collaboration of other peptides. This latter consideration, although requiring more thorough examination, is of great interest, because the existence of previous data concerning the distribution of endocrine cells (Ceccarelli et al., 1995), together with current information on the orexin system, would help us define a clear table concerning ENS and its functional connections with different neuropeptides.
Acknowledgments The authors wish to thank Mrs. G. Mancini for her excellent technical assistance.
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The authors gratefully acknowledge the revision of the English text by Patrick Raymer.
References Baumann CR, Clark EL, Pedersen NP, Hecht JL, Scammel TE. Do enteric neurons make hypocretin? Regul Peptides 2008;147:1–3. Ceccarelli P, Pedini V, Gargiulo AM. Endocrine cells in sheep fetuses. Small Ruminant Res 1991;6:85–93. Ceccarelli P, Pedini V, Gargiulo AM. Serotonin-containing cells in the horse gastrointestinal tract. Anat Histol Embryol 1995;24:97–9. Dall’Aglio C, Gargiulo AM, Pedini V, Ceccarelli P. Presence and distribution of neuroendocrine cells in the gastroenteropancreatic endocrine system of fallow-deer foetuses. Anat Histol Embryol 1999;28:331–6. Dall’Aglio C, Pascucci L, Mercati F, Giontella A, Pedini V, Scocco P, et al. Identification of orexin A- and orexin type 2 receptorpositive cells in the gastrointestinal tract of neonatal dogs. Eur J Histochem 2008;52:229–35. Dall’Aglio C, Pascucci L, Mercati F, Giontella A, Pedini V, Ceccarelli P. Immunohistochemical identification and localization of orexin A and orexin type 2 receptor in the horse gastrointestinal tract. Res Veter Sci 2009;86:189–93. Dall’Aglio C, Pedini V, Scocco P, Boiti C, Ceccarelli P. Immunohistochemical evidence of orexin-A in the pancreatic beta cells of domestic animals. Res Veter Sci 2010a;89:147–9. Dall’Aglio C, Pascucci L, Boiti C, Ceccarelli P. Immunohistochemical localization of the orexinic system in the sheep mandibular gland. In: Abstracts of the LXIV annual meeting of the Italian society for veterinary sciences; 2010b. p. 36. Dyer CJ, Touchette KJ, Carroll JA, Allee GL, Matteri LM. Cloning of porcine prepro-orexin cDNA and effects of an intramuscular injection of synthetic porcine orexin-B on feed intake in young pigs. Domest Anim Endocrinol 1999;16:145–8. Erstróm M, Gustafsson T, Finn A, Kirchgessner A, Grybäc P, Jacobsson H, et al. Inhibitory effect of exogenous orexin A on gastric emptying, plasma leptin, and the distribution of orexin and orexin receptors in the gut and pancreas in man. J Clin Endocr Metab 2005;90:2370–7. Heinonen MV, Purhonen AK, Mäkelä KA, Herzig KH. Functions of orexins in peripheral tissues. Acta Physiol 2008;192:471–85. Komaki G, Matsumoto Y, Nishikata H, Kawai K, Nozaki T, Takii M, et al. Orexin-A and leptin change inversely in fasting non-obese subjects. Eur J Endocrinol 2001;144:645–51. Konturek SJ, Konturek JW, Pawlik T, Brzozowski T. Brain–gut axis and its role in the control of food intake. J Physiol Pharmacol 2004;55:137–54. Kukkonen JP, Holmqvist T, Ammoun S, Åkerman KEO. Functions of the orexinergic/hypocretinergic system. Am J Physiol Cell Physiol 2002;283:1567–91. Mendieta-Zerón H, López M, Diéguez C. Gastrointestinal peptides controlling body weight homeostasis. Gen Comp Endocrinol 2008;155:481–95. Nakabayashi M, Suzuki T, Takahashi K, Totsune K, Muramatsu Y, Kaneko C, et al. Orexin-A expression in human peripheral tissues. Mol Cell Endocrinol 2003;205:43–50. Näslund E, Ehrström M, Ma J, Hellström PM, Kirchgessner AL. Localization and effects of orexin on fasting motility in the rat duodenum. Am J Physiol Gastrointest Liver Physiol 2002;282:470–9. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 1998;92:573–85.
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Contents lists available at ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
Localization of the orexin system in the gastrointestinal tract of fallow deer Cecilia Dall’Aglio a,∗ , Luisa Pascucci a , Francesca Mercati a , Cristiano Boiti b , Piero Ceccarelli a a
Sezione di Anatomia Veterinaria, Dipartimento di Scienze Biopatologiche Veterinarie ed Igiene delle Produzioni Animali ed Alimentari, Via S. Costanzo 4, 06100 Perugia, Italy Laboratorio di Biotecnologie Fisiologiche, Sezione di Fisiologia Veterinaria, Dipartimento di Scienze Biopatologiche Veterinarie ed Igiene delle Produzioni Animali ed Alimentari, Via S. Costanzo 4, 06100 Perugia, Italy b
a r t i c l e
i n f o
Article history: Received 18 January 2011 Received in revised form 11 February 2011 Accepted 13 February 2011
Keywords: Hypocretin Orexin cells Immunohistochemistry Neuroendocrine cells Gastrointestinal tract Deer
a b s t r a c t The aim of the present study was to investigate by immunohistochemistry the presence and distribution of the orexin system in the stomach and gut of fallow deer. Abundant orexin A-positive cells were localized in the middle and basal portions of the mucosal glands of the cardial and fundic regions of the stomach. In the same gastric areas, orexin B-positive cells were also found, mainly localized in the basal portion of glands. In the intestinal tract, orexin-containing cells were occasionally found in the duodenal epithelium and in the rectal intestinal glands. Immunoreactivity for orexin receptors, type 1 and 2 (OX1R and OX2R), was not detected in the same stomach regions. OX1R-immunopositivity was observed in the enteric neuron ganglia localized in the submucosal and muscular intestinal layers, while OX2R-immunopositivity was found close in contact with the cytoplasmic membrane of epithelial cells in the small intestine. © 2011 Elsevier GmbH. All rights reserved.
Introduction The orexin family is a complex system composed of two neuropeptides, orexin-A and orexin-B (OXA and OXB), and two cognate receptors, orexin type 1 and orexin type 2 (OX1R and OX2R). The two peptides derive from the same precursor, the pre-proorexin, through proteolytic cleavage and their sequences are at least 50% homologous. In particular, orexin-A is fully preserved in a large variety of mammals (Dyer et al., 1999; Sakurai et al., 1998; Shibahara et al., 1999; Smart and Jerman, 2002). The two receptors belong to the G-protein-coupled family and are 64% homologous (Sakurai et al., 1998). Recently, orexins have been studied extensively to comprehend their complex functional roles. In fact, since the first identification of OXA and OXB in rat hypothalamus (Sakurai et al., 1998), the presence of this system has been found in both the central and peripheral nervous systems as well as in several other different tissues and organs (Kukkonen et al., 2002; Heinonen et al., 2008). This widespread distribution leads to the supposition of new possible roles for the orexin system that extend beyond appetite control involving a large array of functions, depending on the tissue and/or organ where it is found. Orexins have been identified in humans (Nakabayashi et al., 2003; Erstróm et al., 2005; Heinonen et al., 2008), laboratory animals (Näslund et al., 2002; Sanchez de Miguel and Burrel, 2002)
and animals such as dogs, horses and ruminants (Zabielski, 2007; Dall’Aglio et al., 2008, 2009). They were found in peripheral tissues and, particularly, in endocrine cells and neurons of the enteric nervous system (ENS) of different portions of the gastrointestinal tract and in the pancreas and salivary glands (Dall’Aglio et al., 2010a,b). Many studies have emphasized the role of endocrine cells and ENS in the control of gut functions through the action of several neuropeptides, most of which can act as hormones and neuromediators, both in domestic (Ceccarelli et al., 1991, 1995) and wild mammals (Dall’Aglio et al., 1999). In this regard, the presence of the orexin system in the ENS may be interpreted as an additional mechanism of action through which the ENS can operate to control gut functions. As far as we know, data are lacking on the presence and distribution of the orexin system in the alimentary tract of wild animals. This information would be useful to analyse the gastrointestinal physiology of wild species which need to confront limited feed availability and which have different grazing habits. Therefore, the aim of this study was to evaluate, by means of immunohistochemistry, the localization of positive cells for each component of the orexin system in the gastrointestinal tract of fallow deer, a species which is expanding in the so called “marginal areas” that are not useful for traditional breeding of domestic species. Materials and methods
∗ Corresponding author. E-mail address: [email protected] (C. Dall’Aglio). 0065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2011.02.006
Samples were taken from a total of 5 adult fallow-deer, 3 males and 2 females, regularly slaughtered at the slaughterhouse of the
C. Dall’Aglio et al. / Acta Histochemica 114 (2012) 74–78
75
Table 1 Source and working dilutions of the reagents used. Antisera
Working dilutions
Sources
Normal goat serum Orexin-A Orexin-B Orexin type 1 receptor Orexin type2 receptor Goat anti-rabbit IgG, biotin conjugated Goat anti-mouse IgG, biotin conjugated ABC, Vector Elite Kit Diaminobenzidine (DAB)
1:10 1:100 1:100 1:100 1:100 1:200 1:200 1:100 As specified in the data sheet
S-1000, Vector Laboratories, Burlingame, CA, USA MAB763, R&D System Minneapolis, MN 55413, USA MAB734, R&D System Minneapolis, MN 55413, USA O 4514, Sigma, Missouri 63103, USA AB3094, Chemicon, Temecula, CA, USA 81-6140, Zymed, CA 94080, USA sc-2039, Santa Cruz Biotechnology PK-6100, Vector Laboratories, Burlingame, CA, USA SK-4100, Vector Laboratories, Burlingame, CA, USA
Cooperativa Ponte Parrano at Nocera Umbra, Perugia. In particular, specimens from the abomasum (cardial, fundic and pyloric regions), the duodenum and rectum were fixed by immersion in Bouin’s fluid at room temperature for 24 h. The tissue samples were then dehydrated through a graded series of ethanols, cleared in xylene, and embedded in paraffin. The immunohistochemical reaction was visualized on 5 m serial sections, mounted on poly-l-lysine coated glass slides, utilising the avidin–biotincomplex (ABC, Vector Elite Kit, Burlingame, CA, USA) and the 3,3 diaminobenzidine-4-HCl (DAB, Vector Laboratories) as the chromogen. To reduce variations in staining, tissue sections from each of the above described gastrointestinal portions were incubated together during each immunohistochemical procedure. In brief, dewaxed sections were microwaved for 15 min in 10 mM citric acid (pH 6.0) for antigen retrieval. All subsequent steps were carried out in a moist chamber at room temperature. To prevent non-specific binding of primary antibodies, after proper cooling, the sections were pre-incubated for 30 min with the normal serum (Table 1). Subsequently, serial sections were incubated overnight with one of the primary antibodies (Table 1): anti-OXA and anti-OXB mouse monoclonal antibodies and anti-OX1R and anti-OX2R rabbit polyclonal antibodies. The next day, after washing in phosphate-buffered saline (PBS), the sections were incubated for 30 min at room temperature with the corresponding secondary biotin-conjugated antibody (Table 1): a goat anti-mouse IgG for the two orexins and a goat anti-rabbit IgG for the two receptors. They were then processed for 30 min using the ABC kit. Subsequently, the tissue samples were repeatedly rinsed with PBS and developed with a chromogen solution. After several rinses in PBS, the sections were dehydrated and mounted in natural Canada balsam (BDH, Poole, Dorset, UK). Sections in which the primary antibodies were omitted or substituted with pre-immune gamma globulin were used as controls of non-specific staining. All tissue analyses were carried out on coded slides using a light microscope (Nikon Eclipse E800, Nikon Corporation, Tokyo, Japan) connected to a digital camera (Dxm 1200 Nikon digital camera). Images were processed using an image analysis system (Lucia, Laboratory Imaging Ltd.). The settings for image capture were standardized by subtracting the background signals obtained from the matched tissue sections which had not reacted with the primary antibodies and which were used as immunohistochemical controls. Variations in the intensity of immunolabeling for OXA and OXB were observed between different portions, possibly reflecting the expression of the corresponding antigens. However they were not quantified given the prevalently qualitative nature of the immunohistochemical technique in the tissue sections. Results The morphological studies together with immunohistochemical research revealed the presence of endocrine cells showing cyto-
plasmic positive reactions for orexin A and orexin B in the cardial and fundic regions of the abomasum. These cells showed morphological features typical of endocrine cells: they were oval or round shape and mainly of the closed type (Fig. 1a and b). OXA-positive cells were distributed in the basal and middle third of the tubular glands of the mucosa; their positivity appeared granular and prevalently localized around the nucleus (Fig. 1a). The OXB positive cells were localized exclusively in the basal third of glands; they were gathered, mainly of the closed type and contained many positive granules distributed throughout the cytoplasm (Fig. 1c). The number of OXA- and OXB-positive cells was particularly abundant in the cardial and fundic gastric regions, but in no case positive cells for orexins were visualized in the pyloric region. A few OXA and OXB positive cells were occasionally observed among the epithelial cells along the villi in the duodenum (Fig. 1d) and in the glands of the rectum (Fig. 1e). In the duodenum, such cells were elongated and spindle-shaped and, moreover, were prevalently of the open type, being in contact with the lumen of the gut via an apical cytoplasmic process (Fig. 1d). By immunohistochemistry, positive staining for OX1R was revealed in neurons of the ENS, which appeared localized in the submucosal (Fig. 1f) and muscular layers of the small and large intestine and were isolated or gathered in small or more voluminous groups. Positive staining for OX2R was localized exclusively in a few cells scattered among the epithelial cells in the villi of the duodenum (Fig. 1g). Immunoreaction characteristically seemed to be localized in the apical cytoplasmatic portion (Fig. 1h) in the tubular gland cells, as in the endocrine ones positive to OX2R. Staining was completely absent in the control sections where primary antibodies for orexins and cognate receptors were omitted (data not shown). Discussion Our immunohistochemical investigations of the orexin system showed the presence of different orexinic components in the gastrointestinal tract of fallow deer. The characteristic distribution in the digestive portions differs slightly from what was found previously in other animal species such as dogs and horses (Dall’Aglio et al., 2008, 2009). In fallow-deer, orexin A and orexin B-positive cells were numerous in the cardial and fundic gastric regions, but were completely absent in the pyloric region: their presence was observed again in the different areas of the intestinal tract though with a reduced frequency. Moreover, the orexins were localized only within the epithelial cells of glands of both abomasum and gut, whereas no staining was visualized in neurones and nerves in the submucosal and muscular layers. Orexins, being mainly distributed within the stomach and the intestinal glandular compartments of fallow-deer, confirm the results obtained in other animals such as dogs and horses (Dall’Aglio et al., 2008, 2009).
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Fig. 1. Orexin system immunohistochemical positivity in the gastrointestinal tract of fallow deer. (a and b) Many orexin-A positive cells in the basal and middle third of the fundic mucosa show an oval or round shape and a granular positivity localized prevalently around the nucleus. (c) Many orexin-B positive cells are seen in the basal third of fundic glands; they are typical round-shaped closed cells. (d and e) Some scattered orexin-B positive cells within the epithelial cells of the duodenum (d) and in the rectal intestinal glands (e) show an elongated or triangular shape. (f) Some orexin type 1 receptor-positive neurons in the duodenal sub-mucosal layer. (g and h) Orexin type 2 receptor-positivity localized within the epithelial cells (g) and in the intestinal glands (h); characteristic positive staining appeared localized near the apical cytoplasmic portions.
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Instead, the unsuccessful visualization of OXA in gastric neurons, though differing from what was observed in other animal species (Dall’Aglio et al., 2008; Nakabayashi et al., 2003), supports the results reported by Baumann et al. (2008) in men and mice and could reflect some species differences in the localization of the GI orexin system. However, the prevalent expression of OXA and OXB in cells of the abomasum suggests that, for the gastrointestinal tract, the two peptides are mainly produced in this anatomical region and are probably secreted into the bloodstream where OXA presence was documented in other animal species (Komaki et al., 2001). Thus they likely act in an endocrine way by controlling the brain–gut axis (Mendieta-Zerón et al., 2008) without any direct and/or indirect action within the stomach itself as evidenced by the different local distribution of their cognate receptors. In fact, neither OX1R nor OX2R were found in the gastric regions examined, but exclusively in the gut with a prevalent localization in the small intestine. OXR1 appeared distributed in an ubiquitarian way in the neurons of the enteric nervous system of each examined tract while OX2R appeared mainly localized in the intestinal glands of duodenum and in some scattered endocrine cells. The localization of the immunoreaction for OX2R is typical of the receptors linked to the cellular membrane, such as the G-protein coupled receptor, and is particularly evident in the intestinal glands where the positivity is limited to the apical portion of cytoplasm. The same pattern of distribution of the OX2R-immunoreaction was observed in the endocrine cells of intestinal villi, even if in this case positivity appeared more extended to the entire cytoplasm but with a much more evident concentration in the apical portion. Moreover, OX2R-positivity in the apical portion of epithelial intestinal cells may represent the organism’s response to orexin-like substances present in the gut lumen and reaching the surfaces of the glandular cells. Our results in fallow-deer, like those in humans, laboratory and domestic animals (Nakabayashi et al., 2003; Erstróm et al., 2005; Dall’Aglio et al., 2008, 2009), in peripheral tissues, particularly in endocrine cells, together with the aforementioned considerations, may extend the observation that the control of the gastrointestinal tract functions depends also on many substances, including orexins, in wild animals. These peptides, produced in a consistent way in specific gastric regions, probably have a hormonal endocrine action which may regulate not only gastrointestinal functions, such as gut motility, but also some central functions, such as appetite, thus controlling the brain–gut axis (Heinonen et al., 2008; Konturek et al., 2004; Kukkonen et al., 2002; Smart and Jerman, 2002). The present findings regarding the localization of the orexin system in the gastrointestinal apparatus of the fallow deer, in tune with what was observed in other species, underline even more the existence of a complex control mechanism of the gastrointestinal system that involves the orexin system, both at the central and peripheral level, with or without the collaboration of other peptides. This latter consideration, although requiring more thorough examination, is of great interest, because the existence of previous data concerning the distribution of endocrine cells (Ceccarelli et al., 1995), together with current information on the orexin system, would help us define a clear table concerning ENS and its functional connections with different neuropeptides.
Acknowledgments The authors wish to thank Mrs. G. Mancini for her excellent technical assistance.
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The authors gratefully acknowledge the revision of the English text by Patrick Raymer.
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