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Distribution and morphology of ghrelin-immunopositive cells in the cerebellum of the African ostrich
Tissue and Cell 44 (2012) 373–377
Contents lists available at SciVerse ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Distribution and morphology of ghrelin-immunopositive cells in the cerebellum of the African ostrich Jia-xiang Wang ∗ , Peng Li, Yue Zhou College of Animal Science, Yangtze University, Jingzhou 434103, PR China
a r t i c l e
i n f o
Article history: Received 29 July 2011 Received in revised form 18 June 2012 Accepted 18 June 2012 Available online 15 July 2012 Keywords: African ostrich Ghrelin Cerebellum Immunohistochemistry
a b s t r a c t Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, has been found in the cerebellum of many vertebrates and in the gastrointestinal tract of African ostrich chicks, but little is known about its distribution in the cerebellum of the African ostrich. In the present study, the distribution and morphological characteristics of ghrelin-producing cells in the cerebellum of the African ostrich were investigated using immunohistochemistry. The results indicate that the cerebellum is divided into two sections: the outer cerebellar cortex and the inner medulla of cerebellum. The cerebellar cortex comprises a molecular layer, a Purkinje cell layer and a granular layer; ghrelin-immunopositive (ghrelin-ip) cells were localized throughout the entire cerebellum, but sparsely in the medulla. The greatest number of ghrelin-ip cells was found in the stratum granulosum, and the density decreased gradually from the molecular layer to the Purkinje cell layer in the cerebellar cortex. The ghrelin-ip cells were fusiform or irregular polygons and their cytoplasm was stained intensely. These results clearly demonstrate the presence of ghrelin-ip cells in the cerebellum of the African ostrich. It is speculated that ghrelin may have a physiological function in the cerebellum. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Ghrelin is a brain-gut peptide that has been isolated as an endogenous ligand for the growth hormone secretagogue receptor in the rat stomach. This peptide comprises 28 amino acids, the third residue of which (serine, Ser3) is n-octanoylated; this side chain is essential to its biological activity in rats and humans (Kojima et al., 1999). In the chicken, ghrelin comprises 26 amino acids and Ser3 is conserved between the chicken and mammalian species, as is its acylation by either n-octanoic or n-decanoic acid (Kaiya et al., 2002). In the African ostrich, ghrelin comprises 28 amino acids and Ser3 could be n-octanoylated (Wang et al., 2011). Ghrelin is predominantly produced by X/A-like endocrine cells in the oxyntic mucosa of the stomach, which is the major source of circulating ghrelin (Date et al., 2000; Ariyasu et al., 2001; Dornonville et al., 2001). In mammals, ghrelin-immunopositive (ghrelin-ip) cells have been found in the intestine, pancreas, heart, liver, hypothalamus, cerebrum, cerebellum, thymus gland, hypothalamic arcuate nucleus, kidney and pituitary (Mondal et al., 2005; Kageyama et al., 2005; Kojima and Kangawa, 2005; Ghelardoni et al., 2006).
∗ Corresponding author at: Department of Anatomy, Histology and Embryology, College of Animal Science, Yangtze University, 1Nanhuan Road, Jingzhou, Hubei, PR China. Tel.: +86 716 8066 256; fax: +86 716 8066 256. E-mail address: [email protected] (J.-x. Wang). 0040-8166/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tice.2012.06.004
In the chicken, Saito et al. (2005) found ghrelin mRNA expression in the cerebellum in the chicken; ghrelin-ip cells have been also found in the stomach, intestines, pancreas, heart, liver, hypothalamus, cerebrum, cerebellum, thymus gland and bursa of Fabricius (Ye et al., 2010; Neglia et al., 2005; Ahmed and Harvey, 2002; Wei et al., 2010). However, in the African ostrich, they have examined only in the stomach and intestines (Wang et al., 2009). There have been no studies on the distribution of ghrelin in the cerebellum of the African ostrich. Therefore, in this study, the distribution, morphological characteristics and developmental changes of ghrelin-ip cells in the cerebellum were studied in detail using immunohistochemistry.
2. Materials and methods 2.1. Animals Four female African ostriches (age, 3 months; weight, 14.05 ± 2.12 kg) were used for this study. African ostrich (4 females) were obtained from a standard ostrich farm in Guangdong Province, China and were transported within 10 h to a battery house, where feed and water were made available ad libitum. All of the birds were maintained in a heated room with slatted plastic flooring and were fed a starter diet for postnatal days 7, which was formulated according to the specifications of the Elsenburg Ostrich Feed Database
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(Brand, 2000). All procedures were approved by the Animal Care and Welfare Committee of our Institute.
2.2. Tissue preparation The ostriches were deeply anesthetized with 10% urethane (Caoyang Secondary Chemical Plant, Shanghai, China) at a dose of 1 g/kg BW, and perfused, initially with 1000 mL of 0.85% normal saline (containing 0.075% sodium citrate) and thereafter with 1500 mL of 4% paraformaldehyde phosphate-buffered solution (0.1 mol/L, pH 7.4) at 4 ◦ C. Then the brains were removed and opened to be postfixed for more than 24 h with 4% paraformaldehyde. After immersion, the tissues were embedded in paraffin. Serial sections (5 m) were cut on a Leica microtome (Nussloch Gmbh, Germany), 2 suit sections were prepared; one suit was stained by haematoxylin and eosin (H&E) to observe the cytoarchitecture of cerebellum; the other was stained by immunohistochemistry (SABC) to observe the distribution, morphological characteristics of ghrelin-producing cells in the cerebellum of the African ostrich.
2.3. Immunohistochemistry Immunohistochemical detection of ghrelin cells using rabbit anti-ghrelin was carried out by the streptavidin-biotin-peroxidase complex (SABC) method. The production and specificity of the anti-human ghrelin serum used in this study were previously reported (Wang et al., 2009); it is established that this antiserum recognizes both N- and C-terminial of human ghrelin. Immunohistochemical staining was performed according to the following procedure. The sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol, then treated with 3% hydrogen peroxide (H2 O2 ) to block endogenous peroxidase for 10 min at room temperature. After rinsing with distilled water, the sections were incubated with a citrate buffer (pH 6.0) and placed in a microwave oven until the water boiled to fully expose the antigen. After rinsing with phosphate-buffered saline (PBS), the sections were incubated with 5% normal goat serum for 20 min. After removing superfluous liquid, the sections were incubated with rabbit anti-ghrelin serum (BA1619; Boster Corporation) diluted 1:100 in PBS for 12 h in a humid chamber at 4 ◦ C. After washing with PBS for 6 min, a second incubation with biotin-conjugated
Fig. 1. Microphotographs of histological observation of African Ostrich’s cerebellar (HE staining). (A) The cerebellum was divided into four layer: molecular layer, Purkinje cell layer, granular layer, medulla. (B) The cytoarchitecture of the molecular layer and Purkinje cell layer. (C) The cytoarchitecture of the Purkinje cell layer and granular layer. (D) The cytoarchitecture of the granular layer. (ML, Molecular layer; PL, Purkinje cell layer; GL,Granular layer; DL, Medulla; SC, stellate cells; BC, basket cells; KC, granulosa cells; GC, Golgi cells). (A–D)bar; 50 m.
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Fig. 2. Microphotographs of ghrelin-immunoposive cells in the cerebellar of African ostrich. (A) Ghrelin cells (arrows) were found in the molecular layer. (B) Ghrelin cells (arrows) were found in the Purkinje cell layer and granular layer. (C) Ghrelin cells (arrows) were found in the granular layer and medulla. (D) Microphotograph of absorption test in the cerebellum (ML, Molecular layer; PL, Purkinje cell layer; GL,Granular layer; DL, Medulla; →, Ghrelin-immunoposive cells). (A and B) bar; 20 m. (C and D) bar; 100 m.
anti-rabbit IgG serum (SA1022; Boster) was carried out for 20 min, and this was followed by further washing with PBS. Finally, the sections were incubated for 20 min with an SABC solution prepared according to the manufacturer’s instructions. After washing with PBS for 20 min, the sections were reacted in a diaminobenzidinetetrachloride kit (DAB kit, AR1022, Boster Corp) for 30 min to detect immunostaining. After washing with distilled water, the sections were dehydrated with a graded ethanol series, cleared in xylene, mounted with a coverslip, and viewed under a light microscope (BH-2; Olympus, Japan). All of the incubations were carried out in a humid chamber at room temperature. Control sections were prepared using the same method, omitting the primary antibody. To examine the specificity of rabbit anti-human ghrelin antiserum, the diluted antiserum (1:100) was incubated with human ghrelin (5 g/mL) at room temperature for 10 h, and mixtures were centrifuged at 12,000 rpm for 25 min at 4 ◦ C. The supernatant was used as the primary antiserum for absorption tests.
units were selected for experiments conducted in triplicate (30 measurements for each sample). After taking digital photographs under a light microscope with a digital camera (COOLPIX4500; Nikon, Japan), the number of ghrelin-ip cells in each section was counted using a computerized image analysis program, HMIAS2000 High-definition Chromatic Color Medical Science Figure Analysis Program (Qianping, Wuhan, China). The ghrelin-ip cells density was calculated as the number of ghrelin-ip cells per unit area.
2.4. Morphometric analysis
3. Results
The densities of ghrelin cells in various regions of the African ostrich cerebellar cortex were estimated. For each cerebellum tissue sample, 3 cross-sections were prepared after the samples had been stained with hematoxylin and eosin and SABC stain. Further, for each cerebellum cross-section, 10 intact, well-oriented
3.1. Cytoarchitecture of the cerebellum in the African ostrich
2.5. Statistical analyses Results are expressed as means ± standard errors on the mean (means ± S.E.). Statistical analysis was done using analysis of variance statistics software (SAS Institute, 2000) with Duncan’s multiple range test where appropriate. Differences of p < 0.05 were considered significant.
The cerebellum was divided into two sections: the outer cerebellar cortex and the inner medulla of the cerebellum (Fig. 1A). The cerebellar cortex comprised a molecular layer (ML), a Purkinje cell
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10
a
Ghrelin-ip cell Density (cells/mm2)
9 8 7 6 5
b c
4 3 2 1 0
A
B
C
Fig. 3. Histogram showing the densities of ghrelin-immunoposive cells (cells/mm2 ) in the cerebellar cortex of African ostrich. Ghrelin-immunoposive were present throughout the cerebellar cortex. (A–C) Different letters within the same column indicate significant differences among segments according to Duncan’s multiple range (p ≤ 0.05). (A) Molecular layer; (B) Purkinje cell layer; (C) Granular layer.
layer (PL) and a granular layer (GL). The ML, principally formed from stellate cells (SC) and basket cells (BC), was located in the shallow grooves of the cortex (Fig. 1B). The PL was situated beneath the molecular layer and comprised large, pear-shaped, round or oval Purkinje cells with a regular arrangement; nucleoli were obvious and well stained. Numerous rough dendrites extended from the apical portion of the cells to the molecular layer (Fig. 1C). The GL was the deepest, and formed from densely arranged granulosa cells (KC) and a small number of Golgi cells (GC, Fig. 1D). The cerebellar medial nucleus was fully developed and contained loosely arranged cells including a large number of neurons and mediumsized, light-colored cells. The nuclear perimeter was unapparent and it was difficult to distinguish between the nucleus cerebellaris medialis and the intercalated nucleus (Fig. 1A). The results of absorption tests using antiserum absorbed with 5 g/mL ghrelin showed complete disappearance of immunoreactivity in the cerebellum (Fig. 2D). 3.2. Distribution of ghrelin-immunopositive cells Ghrelin-ip cells were found in both the cortex and the medulla of the African ostrich cerebellum (Fig. 2A–C). They were sparse in the medulla (DL) and abundant in the cortex (Fig. 2A–C). In the molecular layer, the cytoplasm of the basket cells stained for ghrelin (Fig. 2A). In the PL, pear-shaped ghrelin-ip cells were observed; Purkinje cell soma and dendrites were well stained (Fig. 2B). In the GL, elliptic Golgi cells were immunopositive for ghrelin (Fig. 2B and C). 3.3. Morphometric analysis Morphometric analysis revealed that ghrelin-ip cells were localized preferentially in the stratum granulosum (p < 0.05) (Fig. 3); the cell density decreased gradually from the molecular layer to the Purkinje cell layer and the medulla, (p < 0.05) (Fig. 3). 4. Discussion 4.1. Cytoarchitecture of the cerebellum in the African ostrich The cytoarchitecture of the cortex in the African ostrich, which is divided into a molecular layer, a Purkinje cell layer and a granular
layer from outside to inside, is similar to that of silky fowl (Chen et al., 2005), pigeon and Sanhuang chicken (Luo et al., 2010). In the present study, the African ostrich cerebellar medial nucleus differed significantly from that of mammals. In Sanhuang chicken, the cross-section of the cerebellum is approximately round in shape, which is quite different from that of African ostrich. The nucleus cerebellaris medialis is fully developed in both Sanhuang chicken and pigeon, and in both birds it is difficult to distinguish between the nucleus cerebellaris medialis and the other two nuclei, their boundaries being not obvious (Luo et al., 2010). This is consistent with the findings in the African ostrich. In contrast, the silky fowl’s nucleus cerebellaris medialis is not fully developed and contains a small number of loosely arranged neurons, which is not consistent with the findings in the African ostrich. The cross-section of the cerebellum in silky fowl is approximately triangular, which differs from that of the African ostrich (Chen et al., 2005). The delamination of the African ostrich cerebellum is similar to that in the silky fowl. There are few neurons in the molecular layer. The Purkinje cell layer contains a large number of pyriform, round or elliptic Purkinje cells with a well stained nucleus. There are a large number of smaller nuclei packed tightly in the granular layer. The central nuclei of the silky fowl cerebellum are situated at both sides of the ventriculus cerebelli, the small number of nuclei differing from that in the African ostrich.
4.2. Distribution features of ghrelin-immunopositive cells In the chicken, ghrelin-ip neurons have been found in the cerebellar medial nucleus and the granular layer in the cerebellar cortex; they have not been observed in the molecular layer or Purkinje cell layer (Ye et al., 2010). In the present study, ghrelin-ip cells were detected in both the cortex and the medulla of the African ostrich cerebellum. In the goose, the largest numbers of immunoreactive neurons are in the Purkinje cell layer and the molecular layer, with a small number in the granular layer; no ghrelin-ip neurons or nerve fibers have been detected in the cerebellar medulla (Fang et al., 2008). In the present study, ghrelin-ip cells were found throughout the cerebellum, sparsely in the medulla with the greatest number in the stratum granulosum; the density decreased gradually from the molecular layer to the Purkinje cell layer in the cerebellar cortex of the African ostrich, unlike in the goose. Ghrelin stimulates GH release in vivo and in vitro in chicken (Ahmed and Harvey, 2002; Kaiya et al., 2002; Baudet and Harvey, 2003). ICV administration of ghrelin decreases food intake in neonatal chicks (Saito et al., 2002, 2005). Saito et al. (2005) revealed that the anorectic effect of ghrelin is mediated via corticotrophinreleasing factor (CRF): ICV injections of ghrelin increased plasma corticosterone levels in chicken and the increase in corticosterone and anorexia was attenuated by the co-administration of ghrelin and a CRF receptor antagonist, astressin. Ghrelin-ip neurons are distributed widely in the African ostrich cerebellum and with characteristic features, suggesting that ghrelin may result in diverse functions of ghrelin. The relationship between the distribution and functions of ghrelin-ip cells requires further research.
Acknowledgments We would like to thank Prof. Peng Keimei of Department of Anatomy, Histology and Embryology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University for his valuable comments on the experiments. This study was supported by the Department of Education of Hubei Province of China, no. Q20121214.
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References Ahmed, S., Harvey, S., 2002. Ghrelin: a hypotalamic GH-releasing factor in domestic fowl (Gallus domesticus). J. Endocrinol. 172, 117–125. Ariyasu, H., Takaya, K., Tagami, T., Ogawa, Y., Hosoda, K., Akamizu, T., Suda, M., Koh, T., Natsui, K., Toyooka, S., Shirakami, G., Usui, T., Shimatsu, A., Doi, K., Hosoda, H., Kojima, M., Kangawa, K., Nakao, K., 2001. Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J. Clin. Endocrinol. Metab. 86, 4753–4758. Baudet, M.L., Harvey, S., 2003. Ghrelin-induced GH secretion in domestic fowl in vivo and in vitro. J. Endocrinol. 179, 97–105. Brand, T.S., 2000. Elsenburg Ostrich Feed Databases. Elsenburg Agricultural Research Centre, Elsenburg, South Africa. Chen, W.Q., Peng, K.M., Liu, H.Z.H., Luo, G.Z.H., 2005. Anatomy and distribution of NPY immunoreactive neurons of the cerebellum in silky fowl. Prog. Vet. Med. 1 (26), 51–54 (in Chinese). Date, Y., Kojima, M., Hosoda, H., Sawaguchi, A., Mondal, M.S., Suganuma, T., Matsukura, S., Kangawa, K., Nakazato, M., 2000. Ghrelin, a novel growth hormone-releasing acylated peptides,is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141, 4255–4261. Dornonville, de la Cour, C., Bjorkqvist, M., Sandvik, A.K., Bakke, I., Zhao, C.M., Chen, D., Hakanson, R., 2001. A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control. Regul. Pept. 99, 141–150. Fang, F.G., Jiang, S.H.D., Li, M.Q., Zhang, L.X., Li, F.B., Ding, S.H.Q., 2008. Immunohistochemical localization of ghrelin in the cerebrum and cerebellum of adult Wanxi white goose. Chin. J. Vet. Sci. 28 (7), 809–811 (in Chinese). Ghelardoni, S., Carnicelli, V., Frascarelli, S., Ronca-Testoni, S., Zucchi, R., 2006. Ghrelin tissue distribution: comparison between gene and protein expression. J. Endocrinol. Invest. 29 (2), 115–121. Kageyama, H., Funahashi, H., Hirayama, M., Takenoya, F., Kita, T., Kato, S., Sakurai, J., Lee, E.Y., Inoue, S., Date, Y., Nakazato, M., Kangawa, K., Shioda, S., 2005. Morphological analysis of ghrelin and its receptor distribution in the rat pancreas. Regul. Pept. 126, 67–71. Kaiya, H., Van der Geyten, S., Kojima, M., Hosoda, H., Kitajima, Y., Matsumoto, M., Geelissen, S., Darras, V.M., Kangawa, K., 2002. Chicken ghrelin:
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purification, cDNA cloning, and biological activity. Endocrinology 143, 3454–3463. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Ghrelin is a growth hormone-releasing acylated peptide from stomach. Nature 402, 656–660. Kojima, M., Kangawa, K., 2005. Ghrelin: structure and function. Physiol. Rev. 85, 495–522. Luo, H.Q., Peng, K.M., Wang, J.X., Tang, L., Cheng, J.X., Zhang, G.Y., Song, H., Liu, H.Z.H., 2010. Distribution of GABA immunopositive cells in cerebellum of Sanhuang chicken. J. Huazhong Agric. Univ. 5 (26), 597–600 (in Chinese). Mondal, M.S., Date, Y., Yamaguchi, H., Toshinai, K., Tsuruta, T., Kangawa, K., Nakazato, M., 2005. Identification of ghrelin and its receptor in neurons of the rat arcuate nucleus. Regul. Pept. 126, 55–59. Neglia, S., Arcamone, N., Esposito, V., Gargiulo, G., Girolamo, P. de, 2005. Presence and distribution of ghrelinimmunopositive cells in the chicken gastrointestinal tract. Acta Histochem. 107, 3–9. Saito, E.S., Kaiya, H., Takagi, T., Yamasaki, I., Denbow, D.M., Kangawa, K., Furuse, M., 2002. Chicken ghrelin and growth hormone-releasing peptide-2 inhibit food intake of neonatal chicks. Eur. J. Pharmacol. 453, 75–79. Saito, E.S., Kaiya, H., Tachibanat, T., Tomonaga, S., Denbow, D.M., Kangawa, K., Furuse, M., 2005. Inhibitory effect of ghrelin on food intake is mediated by the corticotropin-releasing factor system in neonatal chicks. Regul. Pept. 125, 201–208. Wang, J.X., Peng, K.M., Liu, H.Z.H., Song, H., Chen, X., Liu, M., 2009. Distribution and developmental changes in ghrelin -immunopositive cells in the gastrointestinal tract of African ostrich chicks. Regul. Pept. 154, 97–101. Wang, J.X., Li, P., Peng, K.M., Jin, S.H.Z., 2011. cDNA cloning of ghrelin and ontogeny of ghrelin mRNA expression in the gastrointestinal tract of the African Ostrich chicks. Regul. Pept. 167, 50–55. Wei, F.M., Li, Y.G., Ye, Y.L., Zhang, Y., Ma, Y.J., Jiang, Q.Y., 2010. Localization and development of ghrelin-immunopositive cells in periphery organs of Broiler chickens. Acta Vet. Zootech. Sin. 41 (3), 3412–4346 (in Chinese). Ye, Y.L., Li, Y.G., Wei, F.M., Ma, Y.J., Zhang, Y., Jiang, Q.Y., 2010. Localization, distribution and development of ghrelin -immunopositive neurons and influence of fasting on the minbrain of Broiler chickens. Acta Vet. Zootech. Sin. 41 (9), 1177–1184 (in Chinese).
Contents lists available at SciVerse ScienceDirect
Tissue and Cell journal homepage: www.elsevier.com/locate/tice
Distribution and morphology of ghrelin-immunopositive cells in the cerebellum of the African ostrich Jia-xiang Wang ∗ , Peng Li, Yue Zhou College of Animal Science, Yangtze University, Jingzhou 434103, PR China
a r t i c l e
i n f o
Article history: Received 29 July 2011 Received in revised form 18 June 2012 Accepted 18 June 2012 Available online 15 July 2012 Keywords: African ostrich Ghrelin Cerebellum Immunohistochemistry
a b s t r a c t Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, has been found in the cerebellum of many vertebrates and in the gastrointestinal tract of African ostrich chicks, but little is known about its distribution in the cerebellum of the African ostrich. In the present study, the distribution and morphological characteristics of ghrelin-producing cells in the cerebellum of the African ostrich were investigated using immunohistochemistry. The results indicate that the cerebellum is divided into two sections: the outer cerebellar cortex and the inner medulla of cerebellum. The cerebellar cortex comprises a molecular layer, a Purkinje cell layer and a granular layer; ghrelin-immunopositive (ghrelin-ip) cells were localized throughout the entire cerebellum, but sparsely in the medulla. The greatest number of ghrelin-ip cells was found in the stratum granulosum, and the density decreased gradually from the molecular layer to the Purkinje cell layer in the cerebellar cortex. The ghrelin-ip cells were fusiform or irregular polygons and their cytoplasm was stained intensely. These results clearly demonstrate the presence of ghrelin-ip cells in the cerebellum of the African ostrich. It is speculated that ghrelin may have a physiological function in the cerebellum. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Ghrelin is a brain-gut peptide that has been isolated as an endogenous ligand for the growth hormone secretagogue receptor in the rat stomach. This peptide comprises 28 amino acids, the third residue of which (serine, Ser3) is n-octanoylated; this side chain is essential to its biological activity in rats and humans (Kojima et al., 1999). In the chicken, ghrelin comprises 26 amino acids and Ser3 is conserved between the chicken and mammalian species, as is its acylation by either n-octanoic or n-decanoic acid (Kaiya et al., 2002). In the African ostrich, ghrelin comprises 28 amino acids and Ser3 could be n-octanoylated (Wang et al., 2011). Ghrelin is predominantly produced by X/A-like endocrine cells in the oxyntic mucosa of the stomach, which is the major source of circulating ghrelin (Date et al., 2000; Ariyasu et al., 2001; Dornonville et al., 2001). In mammals, ghrelin-immunopositive (ghrelin-ip) cells have been found in the intestine, pancreas, heart, liver, hypothalamus, cerebrum, cerebellum, thymus gland, hypothalamic arcuate nucleus, kidney and pituitary (Mondal et al., 2005; Kageyama et al., 2005; Kojima and Kangawa, 2005; Ghelardoni et al., 2006).
∗ Corresponding author at: Department of Anatomy, Histology and Embryology, College of Animal Science, Yangtze University, 1Nanhuan Road, Jingzhou, Hubei, PR China. Tel.: +86 716 8066 256; fax: +86 716 8066 256. E-mail address: [email protected] (J.-x. Wang). 0040-8166/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tice.2012.06.004
In the chicken, Saito et al. (2005) found ghrelin mRNA expression in the cerebellum in the chicken; ghrelin-ip cells have been also found in the stomach, intestines, pancreas, heart, liver, hypothalamus, cerebrum, cerebellum, thymus gland and bursa of Fabricius (Ye et al., 2010; Neglia et al., 2005; Ahmed and Harvey, 2002; Wei et al., 2010). However, in the African ostrich, they have examined only in the stomach and intestines (Wang et al., 2009). There have been no studies on the distribution of ghrelin in the cerebellum of the African ostrich. Therefore, in this study, the distribution, morphological characteristics and developmental changes of ghrelin-ip cells in the cerebellum were studied in detail using immunohistochemistry.
2. Materials and methods 2.1. Animals Four female African ostriches (age, 3 months; weight, 14.05 ± 2.12 kg) were used for this study. African ostrich (4 females) were obtained from a standard ostrich farm in Guangdong Province, China and were transported within 10 h to a battery house, where feed and water were made available ad libitum. All of the birds were maintained in a heated room with slatted plastic flooring and were fed a starter diet for postnatal days 7, which was formulated according to the specifications of the Elsenburg Ostrich Feed Database
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(Brand, 2000). All procedures were approved by the Animal Care and Welfare Committee of our Institute.
2.2. Tissue preparation The ostriches were deeply anesthetized with 10% urethane (Caoyang Secondary Chemical Plant, Shanghai, China) at a dose of 1 g/kg BW, and perfused, initially with 1000 mL of 0.85% normal saline (containing 0.075% sodium citrate) and thereafter with 1500 mL of 4% paraformaldehyde phosphate-buffered solution (0.1 mol/L, pH 7.4) at 4 ◦ C. Then the brains were removed and opened to be postfixed for more than 24 h with 4% paraformaldehyde. After immersion, the tissues were embedded in paraffin. Serial sections (5 m) were cut on a Leica microtome (Nussloch Gmbh, Germany), 2 suit sections were prepared; one suit was stained by haematoxylin and eosin (H&E) to observe the cytoarchitecture of cerebellum; the other was stained by immunohistochemistry (SABC) to observe the distribution, morphological characteristics of ghrelin-producing cells in the cerebellum of the African ostrich.
2.3. Immunohistochemistry Immunohistochemical detection of ghrelin cells using rabbit anti-ghrelin was carried out by the streptavidin-biotin-peroxidase complex (SABC) method. The production and specificity of the anti-human ghrelin serum used in this study were previously reported (Wang et al., 2009); it is established that this antiserum recognizes both N- and C-terminial of human ghrelin. Immunohistochemical staining was performed according to the following procedure. The sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol, then treated with 3% hydrogen peroxide (H2 O2 ) to block endogenous peroxidase for 10 min at room temperature. After rinsing with distilled water, the sections were incubated with a citrate buffer (pH 6.0) and placed in a microwave oven until the water boiled to fully expose the antigen. After rinsing with phosphate-buffered saline (PBS), the sections were incubated with 5% normal goat serum for 20 min. After removing superfluous liquid, the sections were incubated with rabbit anti-ghrelin serum (BA1619; Boster Corporation) diluted 1:100 in PBS for 12 h in a humid chamber at 4 ◦ C. After washing with PBS for 6 min, a second incubation with biotin-conjugated
Fig. 1. Microphotographs of histological observation of African Ostrich’s cerebellar (HE staining). (A) The cerebellum was divided into four layer: molecular layer, Purkinje cell layer, granular layer, medulla. (B) The cytoarchitecture of the molecular layer and Purkinje cell layer. (C) The cytoarchitecture of the Purkinje cell layer and granular layer. (D) The cytoarchitecture of the granular layer. (ML, Molecular layer; PL, Purkinje cell layer; GL,Granular layer; DL, Medulla; SC, stellate cells; BC, basket cells; KC, granulosa cells; GC, Golgi cells). (A–D)bar; 50 m.
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Fig. 2. Microphotographs of ghrelin-immunoposive cells in the cerebellar of African ostrich. (A) Ghrelin cells (arrows) were found in the molecular layer. (B) Ghrelin cells (arrows) were found in the Purkinje cell layer and granular layer. (C) Ghrelin cells (arrows) were found in the granular layer and medulla. (D) Microphotograph of absorption test in the cerebellum (ML, Molecular layer; PL, Purkinje cell layer; GL,Granular layer; DL, Medulla; →, Ghrelin-immunoposive cells). (A and B) bar; 20 m. (C and D) bar; 100 m.
anti-rabbit IgG serum (SA1022; Boster) was carried out for 20 min, and this was followed by further washing with PBS. Finally, the sections were incubated for 20 min with an SABC solution prepared according to the manufacturer’s instructions. After washing with PBS for 20 min, the sections were reacted in a diaminobenzidinetetrachloride kit (DAB kit, AR1022, Boster Corp) for 30 min to detect immunostaining. After washing with distilled water, the sections were dehydrated with a graded ethanol series, cleared in xylene, mounted with a coverslip, and viewed under a light microscope (BH-2; Olympus, Japan). All of the incubations were carried out in a humid chamber at room temperature. Control sections were prepared using the same method, omitting the primary antibody. To examine the specificity of rabbit anti-human ghrelin antiserum, the diluted antiserum (1:100) was incubated with human ghrelin (5 g/mL) at room temperature for 10 h, and mixtures were centrifuged at 12,000 rpm for 25 min at 4 ◦ C. The supernatant was used as the primary antiserum for absorption tests.
units were selected for experiments conducted in triplicate (30 measurements for each sample). After taking digital photographs under a light microscope with a digital camera (COOLPIX4500; Nikon, Japan), the number of ghrelin-ip cells in each section was counted using a computerized image analysis program, HMIAS2000 High-definition Chromatic Color Medical Science Figure Analysis Program (Qianping, Wuhan, China). The ghrelin-ip cells density was calculated as the number of ghrelin-ip cells per unit area.
2.4. Morphometric analysis
3. Results
The densities of ghrelin cells in various regions of the African ostrich cerebellar cortex were estimated. For each cerebellum tissue sample, 3 cross-sections were prepared after the samples had been stained with hematoxylin and eosin and SABC stain. Further, for each cerebellum cross-section, 10 intact, well-oriented
3.1. Cytoarchitecture of the cerebellum in the African ostrich
2.5. Statistical analyses Results are expressed as means ± standard errors on the mean (means ± S.E.). Statistical analysis was done using analysis of variance statistics software (SAS Institute, 2000) with Duncan’s multiple range test where appropriate. Differences of p < 0.05 were considered significant.
The cerebellum was divided into two sections: the outer cerebellar cortex and the inner medulla of the cerebellum (Fig. 1A). The cerebellar cortex comprised a molecular layer (ML), a Purkinje cell
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10
a
Ghrelin-ip cell Density (cells/mm2)
9 8 7 6 5
b c
4 3 2 1 0
A
B
C
Fig. 3. Histogram showing the densities of ghrelin-immunoposive cells (cells/mm2 ) in the cerebellar cortex of African ostrich. Ghrelin-immunoposive were present throughout the cerebellar cortex. (A–C) Different letters within the same column indicate significant differences among segments according to Duncan’s multiple range (p ≤ 0.05). (A) Molecular layer; (B) Purkinje cell layer; (C) Granular layer.
layer (PL) and a granular layer (GL). The ML, principally formed from stellate cells (SC) and basket cells (BC), was located in the shallow grooves of the cortex (Fig. 1B). The PL was situated beneath the molecular layer and comprised large, pear-shaped, round or oval Purkinje cells with a regular arrangement; nucleoli were obvious and well stained. Numerous rough dendrites extended from the apical portion of the cells to the molecular layer (Fig. 1C). The GL was the deepest, and formed from densely arranged granulosa cells (KC) and a small number of Golgi cells (GC, Fig. 1D). The cerebellar medial nucleus was fully developed and contained loosely arranged cells including a large number of neurons and mediumsized, light-colored cells. The nuclear perimeter was unapparent and it was difficult to distinguish between the nucleus cerebellaris medialis and the intercalated nucleus (Fig. 1A). The results of absorption tests using antiserum absorbed with 5 g/mL ghrelin showed complete disappearance of immunoreactivity in the cerebellum (Fig. 2D). 3.2. Distribution of ghrelin-immunopositive cells Ghrelin-ip cells were found in both the cortex and the medulla of the African ostrich cerebellum (Fig. 2A–C). They were sparse in the medulla (DL) and abundant in the cortex (Fig. 2A–C). In the molecular layer, the cytoplasm of the basket cells stained for ghrelin (Fig. 2A). In the PL, pear-shaped ghrelin-ip cells were observed; Purkinje cell soma and dendrites were well stained (Fig. 2B). In the GL, elliptic Golgi cells were immunopositive for ghrelin (Fig. 2B and C). 3.3. Morphometric analysis Morphometric analysis revealed that ghrelin-ip cells were localized preferentially in the stratum granulosum (p < 0.05) (Fig. 3); the cell density decreased gradually from the molecular layer to the Purkinje cell layer and the medulla, (p < 0.05) (Fig. 3). 4. Discussion 4.1. Cytoarchitecture of the cerebellum in the African ostrich The cytoarchitecture of the cortex in the African ostrich, which is divided into a molecular layer, a Purkinje cell layer and a granular
layer from outside to inside, is similar to that of silky fowl (Chen et al., 2005), pigeon and Sanhuang chicken (Luo et al., 2010). In the present study, the African ostrich cerebellar medial nucleus differed significantly from that of mammals. In Sanhuang chicken, the cross-section of the cerebellum is approximately round in shape, which is quite different from that of African ostrich. The nucleus cerebellaris medialis is fully developed in both Sanhuang chicken and pigeon, and in both birds it is difficult to distinguish between the nucleus cerebellaris medialis and the other two nuclei, their boundaries being not obvious (Luo et al., 2010). This is consistent with the findings in the African ostrich. In contrast, the silky fowl’s nucleus cerebellaris medialis is not fully developed and contains a small number of loosely arranged neurons, which is not consistent with the findings in the African ostrich. The cross-section of the cerebellum in silky fowl is approximately triangular, which differs from that of the African ostrich (Chen et al., 2005). The delamination of the African ostrich cerebellum is similar to that in the silky fowl. There are few neurons in the molecular layer. The Purkinje cell layer contains a large number of pyriform, round or elliptic Purkinje cells with a well stained nucleus. There are a large number of smaller nuclei packed tightly in the granular layer. The central nuclei of the silky fowl cerebellum are situated at both sides of the ventriculus cerebelli, the small number of nuclei differing from that in the African ostrich.
4.2. Distribution features of ghrelin-immunopositive cells In the chicken, ghrelin-ip neurons have been found in the cerebellar medial nucleus and the granular layer in the cerebellar cortex; they have not been observed in the molecular layer or Purkinje cell layer (Ye et al., 2010). In the present study, ghrelin-ip cells were detected in both the cortex and the medulla of the African ostrich cerebellum. In the goose, the largest numbers of immunoreactive neurons are in the Purkinje cell layer and the molecular layer, with a small number in the granular layer; no ghrelin-ip neurons or nerve fibers have been detected in the cerebellar medulla (Fang et al., 2008). In the present study, ghrelin-ip cells were found throughout the cerebellum, sparsely in the medulla with the greatest number in the stratum granulosum; the density decreased gradually from the molecular layer to the Purkinje cell layer in the cerebellar cortex of the African ostrich, unlike in the goose. Ghrelin stimulates GH release in vivo and in vitro in chicken (Ahmed and Harvey, 2002; Kaiya et al., 2002; Baudet and Harvey, 2003). ICV administration of ghrelin decreases food intake in neonatal chicks (Saito et al., 2002, 2005). Saito et al. (2005) revealed that the anorectic effect of ghrelin is mediated via corticotrophinreleasing factor (CRF): ICV injections of ghrelin increased plasma corticosterone levels in chicken and the increase in corticosterone and anorexia was attenuated by the co-administration of ghrelin and a CRF receptor antagonist, astressin. Ghrelin-ip neurons are distributed widely in the African ostrich cerebellum and with characteristic features, suggesting that ghrelin may result in diverse functions of ghrelin. The relationship between the distribution and functions of ghrelin-ip cells requires further research.
Acknowledgments We would like to thank Prof. Peng Keimei of Department of Anatomy, Histology and Embryology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University for his valuable comments on the experiments. This study was supported by the Department of Education of Hubei Province of China, no. Q20121214.
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