Immunohistochemical identification and localization of orexin A and orexin type 2 receptor in the horse gastrointestinal tract

Research in Veterinary Science 86 (2009) 189–193

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Immunohistochemical identification and localization of orexin A and orexin type 2 receptor in the horse gastrointestinal tract Cecilia Dall’Aglio a,*, Luisa Pascucci a, Francesca Mercati a, Andrea Giontella b, Vera Pedini a, Piero Ceccarelli a a

Dipartimento di Scienze Biopatologiche Veterinarie ed Igiene delle Produzioni Animali ed Alimentari, Sezione di Anatomia Veterinaria, Via San Costanzo 4, 06126 Perugia, Italy Dipartimento di Patologia, Diagnostica e Clinica Veterinaria, Sezione di Scienze Sperimentali e Biotecnologie Applicate, Facoltà di Medicina Veterinaria, Via San Costanzo 4, 06126 Perugia, Italy b

a r t i c l e

i n f o

Article history: Accepted 1 July 2008

Keywords: Hypocretin Immunohistochemistry Neuroendocrine cells Equine

a b s t r a c t The aim of the present study was to investigate the presence and the distribution of cells containing orexin A and orexin type 2 receptor in the horse stomach and gut, by means of immunohistochemical techniques. Orexin A was identified in the stomach fundic and pyloric regions and in the duodenum. In the same stomach regions, a large subset of orexin A-positive cells also showed orexin type 2 receptor-like immunoreactivity. Moreover, in the duodenum, many of them, seemed to store serotonin. Characteristically, enteric neurons or ganglia also displayed orexin A and, sometimes, orexin type 2 receptor immunoreaction. Orexin A and orexin type 2 receptor immunoreactivity was also found in the nerve fibers in the enteric submucosal layer. Our results, together with data present in the literature, could contribute to the understanding of complex mechanisms regulating the horse gut functionality that are depending very likely on the consequence of the co-operation of both a central and a peripheral control. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Orexins A and B (OXA and OXB) are two neuropeptides, recently identificated in the rat hypothalamus (Sakurai et al., 1998). Their name is from Greek (oreksis = appetite) and is justified by the observation that, when injected into the lateral ventricle, they caused an increase in food intake in nonfasted rats (Kukkonen et al., 2002): this is known as the orexinergic effect. They have also been called hypocretins, on the basis of their sequence homology with secretin and of the site of their first isolation, the hypothalamus. In fact, in the central nervous system, orexins are produced exclusively by a small group of neurons in the lateral hypothalamus, an area well known for being implicated in the control of appetite (Bernardis and Bellinger, 1996; Sakurai, 1999). Their effects are mediated by two receptors, orexin type 1 and orexin type 2 receptors (OX1R and OX2R), whose exact distribution in the brain, to date, has not been reported (Edwards et al., 1999). Recent investigations have shown that OXA and OXB sequences are at least 50% homologus and that the OXA sequence is fully preserved among a large variety of mammals; moreover, OXA’s bind-

* Corresponding author. Fax: +39 075 5857631. E-mail address: [email protected] (C. Dall’Aglio). 0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.07.001

ing affinity is the same for both receptor types (Smart and Jerman, 2002). It has been seen that orexin production is up-regulated by events like fasting and is down-regulated in obese mice, genetically deprived of the leptin protein (Ob/Ob) and/or of the leptin receptor (db/db). Moreover, orexin positive hypothalamic neurons also showed the presence of leptin receptor, thus increasing the evidence that leptin and, more generally, adipose tissue may influence their production (Horvath et al., 1999). A negative effect of leptin on orexin expression in the hypothalamus is probably exerted both, directly, via leptin receptors present on orexinergic cells and, indirectly, via leptin receptors present on other cells, forming clusters that are called ‘‘feedingcontrolling”, and that are related to orexinergic cells (Kukkonen et al., 2002; Sakurai et al., 1998). Recently, orexins have been identified in humans (Ehrstróm et al., 2005; Nakabayashi et al., 2003) and laboratory animals (Näslund et al., 2002; Sánchez de Miguel and Burrel, 2002) in peripheral tissues and, in particular, in endocrine cells and neurons (ENS) in different portions of the gastrointestinal tract. Many studies have emphasized the control of ENS on gut functions through the action of several neuropeptides, most of which can act as hormones and neuromediators. Data on the presence and distribution of orexin producing cells in the alimentary tract of large domestic animals are lacking.

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Therefore, with the aim of improving knowledges that would be useful also for clinicians, we studied the presence, the distribution and the quantification of orexin A- and orexin type 2 receptor-positive cells in the horse gastrointestinal tract, chosen for the anatomical complexity of its digestive tract and for the frequency of specific pathological events. The number of OXA and OX2R positive cells in each gastrointestinal tract studied was also evaluated. 2. Materials and methods In reference to what was previously mentioned regarding sequence homology among animal species, as well as their binding affinity to receptor types, we have started investigating the presence of orexin A and of the orexin type 2 receptor. For this study, samples were taken from a total of 10 horses, 5 males and 5 females, regularly slaughtered at the slaughterhouse of Ponte San Giovanni, at Perugia. In particular, specimens from the stomach (fundic and pyloric regions) and from the different portions of the small and large intestine were fixed by immersion in Bouin’s fluid at room temperature for 24 h. Then the tissue samples were dehydrated through a graded series of ethanols, cleared in xylene, and embedded in paraffin. The immunohistochemical reaction was visualized on 5 lm serial sections, mounted on poly-L-lysine coated glass slides, utilising the avidin–biotin-complex (ABC) and the 3,30 -diaminobenzidine-4-HCl (DAB) as the chromogen. To reduce variations in staining, tissue sections from each of the above-mentioned 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 a proper cooling the sections were pre-incubated for 30 min with the normal serum (Table 1). Subsequently, serial sections were incubated overnight with the primary antibodies (Table 1): anti-OXA 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 secondary biotin conjugated antibody (Table 1) and then processed for 30 min using the Vectastain 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 Canada Balsam Natural (BDH, Poole, Dorset, England). In a subsequent step, serial sections were stained with a set of primary antibodies: a monoclonal anti-serotonin antibody and a polyclonal goat anti-leptin receptor (ObR) antibody. Obviously, the secondary biotin conjugated antibody used was different in reference to the primary one: a goat anti-mouse IgG in the first case and a chicken anti-goat IgG in the second one. Sections in which the primary antibodies were omitted or substituted with pre-immune gamma globulin were used as control of unspecific staining.

The working dilutions and the sources of the antibodies are listed in Table 1. All tissue analyses and cell counts 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.). To count the orexin A- and orexin type 2 receptor-positive cells in the investigated gastrointestinal portions we randomly selected ten fields of 0.5 mm2 in some sections of the different portions and, in each field, the number of positive cells was assessed. The setting 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. The cells were considered positive only if cytoplasmic staining was present. Although we observed some changes among different portions in the intensity of immunolabeling for OXA and OX2R, which may reflect the expression of the corresponding antigens, they were not estimated given the prevalent qualitative nature of the immunohistochemical technique in the tissue sections. Statistical analysis was carried out by ‘‘R” software (R Development Core Team, 2007). Due to the reduced sample size, non-parametric tests were used: the Kruskal-Wallis’s test, followed by Wilkoxon’s test with Bonferroni’s correction, were used to compare OXA and OX2R in the four different portions, and Wilkoxon’s signed rank sum was used to compare OXA and OX2R in the same portion. 3. Results The immunohistochemical techniques revealed the presence of endocrine cells showing cytoplasmic positive reactions for orexin A and orexin type 2 receptor in the fundic and pyloric regions of the stomach and in the duodenum (Figs. 1 and 2). Both the number of OXA (P < 0.001) and OX2R cells (P < 0.001) were influenced by anatomical portion. In particular, the number of the OXA cells was less in the fundus than in the pylorus (P < 0.001); the difference between the pylorus and the duodenum was not significant. The OXA cells were not present in the large intestine. Also the OX2R cells were less in the fundus than in pylorus (P < 0.001), and these cells were absent both in the duodenum and in the large intestine. There was no difference in the number of OXA and OX2R cells in the pylorus, while in the fundus (P < 0.001) and in the duodenum (P < 0.001) the OX2R cell number was inferior to the number of the OXA cells . In the fundic region, the orexin A positive cells (10.5 ± 1.8) were gathered in groups in the basal third of the tubular glands and were mainly of the closed type, with an oval or round shape and contained many perinuclear granules. In the pyloric region (Fig. 1a), their immunolocalization was always in the tubular glands, principally in the basal third but with some cells also scattered in the glands middle third. They were of both the open type, with

Table 1 Source and working dilutions of the reagents used Antisera

Working dilutions

Sources

Orexin A Orexin type 2 receptor Serotonin Leptin receptor Goat anti rabbit IgG, biotin conjugated Goat anti-mouse IgG, biotin conjugated Chicken anti-goat IgG, biotin conjugated ABC, Vector Elite Kit

1:100 1:100 1:150 1:100 1:200 1:200 1:200 1:100

AB3098-Chemicon, Temecula, CA, USA AB3094-Chemicon, Temecula, CA, USA M0758-DAKO, Glostrup, Denmark sc-1834, Santa Cruz Biotechnology 81-6140-Zymed sc-2039, Santa Cruz Biotechnology sc-2984, Santa Cruz Biotechnology Vector Laboratories, Burlingame, CA, USA

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Fig. 1. Orexin A immunohistochemistry reaction in the horse gastrointestinal apparatus: (a) some OXA positive cells in the pyloric gland region (arrows), (b) a characteristic ‘‘open type” OXA cell (asterisk), among epithelial cells, with an apical process (arrow) reaching the lumen, and (c) a group of OXA positive neurons in the submucosa layer of the duodenum.

Fig. 2. Orexin type 2 receptor immunohistochemistry reaction in the horse gastrointestinal apparatus: (a) some OX2R positive cells in the basal third of the fundic region glands. The insert indicates a submucosal nervous fiber positive to the receptor (arrow), and (b) a group of neurons in the submucosal layer stained by OX2R.

a triangular or elongated shape, extending from the basal membrane to the gland lumen, and of the closed type (17 ± 1.8). In the duodenum, the orexin A positive cells (16 ± 1.2) were localized in the crypts and scattered among the epithelial cells along the villi and, moreover, they were prevalently of the open type, making contact with the lumen of the gut via an apical cytoplasmic process (Fig. 1b). On villi, in particular, they tended to be elongated and spindle shaped. Furthermore, some of these cells showed cytoplasmic processes that ran along the basement membrane and made contact with neighbouring cells. Immunohistochemical staining for serotonin seemed to evidence that a large subset of the orexin-containing cells in the small intestinal mucosa also contain this hormone (Fig. 3a and b): this last observation, together with the previously exposed morphological features, allowed us to consider these cells as being enterochromaffin (EC). The orexin type 2 receptor-positive cells showed the same morphological characteristics as those positive to orexin A; their number was considerably lower than the orexin A-cells and, in any case, they were not present in either duodenum or the large intes-

tine (Fig. 2a). In Graph 1 the variations in the number of OXA and OX2R positive cells in the different tract of gastrointestinal tract are clearly visualized. The immunohistochemical techniques revealed positivity for both the orexin A and the orexin type 2 receptor in the neurons and fibers of the enteric nervous system (Figs. 1c and 2b). They were localized in the submucosal and in the muscular layers all along the gastrointestinal tract examined, but with a more evident concentration in the intestine. Positive neurons appeared isolated or gathered in small or more voluminous groups among the connective tissue of the submucosal layer and, also in little groups, among the characteristic submucosal glands, in particular in the duodenum. Some interconnecting orexin A positive nerve fibers were also evident. Further immunohistochemical studies carried out to identify the orexin A positive neurons neurochemically seemed to evidence positivity to leptin receptor in some of them, both in the submucosal and muscular layers (Fig. 3c and d). Staining was completely absent in the control sections (data not shown).

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Fig. 3. (a) Some cells stained with anti-OXA antibody, in the duodenum; in the subsequent section (b), the same cells stained with the anti-serotonin antibody. (c) A group of neurons positive to the OXA, in the duodenal submucosa; in the subsequent section (d), some of these cells weakly stained with anti-ObR antibody.

20

***

18

cells/0.5mm2

16

***

14 12

orexin A

10

orexin type 2 receptor

8 6 4 2 0 pylorus

fundus

duod.

large int.

Graph 1. Mean number of endocrine cells positive to orexin A and orexin type 2 receptor antibodies, in the horse gastrointestinal tract.

4. Discussion Our immunohistochemical investigations enabled us to identify many endocrine cells positive for orexin A and orexin type 2 receptor in the alimentary tract mucosa of adult horses. Moreover, considerable immunoreactivity for both substances was evidenced in some neurons and in nervous fibers localized in the submucosal and muscular layers. The latter observation is in disagreement with a recent report that questions the presence of orexins in murine or human enteric neurons (Baumann et al., 2008). Nevertheless, the discrepant results between Baumann’s analysis and our immunohistochemistry findings in the horse gastrointestinal tract may be partly due to the difference of the antibody for orexin A used and/or the species reactivity (Nakabayashi et al., 2003).

Orexin A containing cells were numerous in the fundic and pyloric gastric regions, while their number remarkably decreased in the duodenum and completely disappeared in the large intestine. A large number of orexin type 2 receptor positive cells were evidenced in the gastric regions, in particular in the fundic one, while they completely disappeared in the gut. In the intestinal mucosa some of the orexin A positive cells also seemed to contain serotonin as previously shown in laboratory animals (Kirchgessner et al., 1992; Walsh, 1994). This observation led us to suppose that these substances are synergistically involved in the functional control of the small intestine also in horses. In particular, as it has been proposed in man (Ehrstróm et al., 2005), 5-HT, being released from entero-chromaffin cells by a luminal stimuli, could start the peristaltic reflex activating specific neurons while OXA, as has been demonstrated in vitro, could enhanced the peristaltic reflex (Kirchgessner and Liu, 1999). In any case, the presence of orexin A and type 2 receptor in entero-chromaffin cells seems to suggest that orexin A may be able to modulate serotonin release (Ehrstróm et al., 2005; Kirchgessner, 2002) and probably its own release in an autocrine fashion (Näslund et al., 2002). The presence of OXA and OX2R in the neurons of the horse gastrointestinal tract may indicate that orexin A has a direct and local action on intestinal motility (Heinonen et al., 2008; Kirchgessner, 2002). The documented localization of orexin A in the horse gastrointestinal tract may support efforts, already undertaken in man (Kukkonen et al., 2002), to distinguish a double control mechanism of the gastrointestinal system that expects an orexin activity that is both central and peripheral. In particular, the orexins central activity could affect the first phase of food ingestion and food transit through the alimentary canal via its specific innervation, while the orexin peripheral control, via the enteric neurons and/or endo-

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crine cells, could affect gut motility and exocrine secretion (Kukkonen et al., 2002). It is very likely that orexin control on food intake is exerted by both neuropeptides (OXA and OXB) (Ducroc et al., 2007), even if orexin A is thought to be more effective than orexin B. Indeed, the latter peptide, when administered intracerebroventricular, only occasionally stimulates food consumption and anyway with a shorter intervention than orexin A (Edwards et al., 1999). Our results in horses, like those in humans and laboratory animals, in combination with the aforementioned considerations, enhanced the observation that control of the gastrointestinal tract functions depends on the co-operation of many substances that act at the peripheral level, and probably at the central one as well. This peculiar condition is underlined by the apparent expression of two or more neuropeptides in the same cells of the ENS. However, even if the precise mechanism of action of orexins has not been clarified in most of the functional aspects of the gastrointestinal apparatus (Heinonen et al., 2008), in the last decade orexins have been the target of intense research that has produced many clues that may lead to explain the importance of orexins in appetite regulation, feeding behaviour and energy expenditure (Korczynski et al., 2006; Ducroc et al., 2007). Since the expression of orexins in the gastrointestinal apparatus is enhanced during fasting and it is during fasting that many effects of orexin stimulation are revealed (Komaki et al., 2001), one of the aspects that has been most studied is the link between feeding status, in particular leptin, and the orexins. Regarding this point, observations made during a sequence of experimental investigations on laboratory animals pointed out that a fasting condition induces a decline in the leptin plasma level and, thus, activation of orexin-positive neurons and an increase in orexin synthesis (Ehrstróm et al., 2005). These observations, supported by the well-documented presence of leptin receptor immunoreactivity in the same orexin A-positive neurons in both the central and enteric nervous systems of laboratory animals, suggested a relationship between orexin and leptin and led to the supposition of a functional response of these cells to the feeding status (Valassi et al.,2008; Näslund and Hellström, 2007). Our results, evidencing leptin receptor positivity in OXA positive neurons of the horse enteric nervous system, could be considered as confirmation of the latest data in laboratory animals and, therefore, could underline a possible link between orexin A and leptin. In conclusion, our results enable us to state that orexin A and orexin type 2 receptor are present in the horse gastrointestinal apparatus and that our data concerning the morphological characteristics and distribution of cells containing orexin A and orexin type 2 receptor are in agreement with those described in the literature regarding humans and laboratory animals. These personal considerations, together with bibliographic data related to experimental conditions in laboratory animals, allow us to suppose a peripheral intervention of orexin A in the control of the gastrointestinal apparatus also in horses and to hypothesize that a disorder in the ‘‘orexinergic system” could be also responsible for some pathological conditions (Rouet-Benzineb et al., 2004; Russo et al., 2008; Spinazzi et al., 2005). Acknowledgment The authors wish to thank Mrs. G. Mancini for her excellent technical assistance.

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