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J. Vet. Sci. (2004),�5(3), 183–188

Changes of gastrointestinal argyrophil endocrine cells in the osteoporotic SD rats induced by ovariectomy

Sae-Kwang Ku1, Hyeung-Sik Lee2,*, Jae-Hyun Lee3

1Pharmacology & Toxicology Lab., Central Research Laboratories, Dong-Wha Pharm. Ind. Co., Anyang 430-017, Korea2Department of Herbal Biotechnology, Daegu Haany University, Daegu 712-715, Korea3Department of Histology, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Korea

The regional distributions and frequencies of argyrophilendocrine cells in gastrointestinal (GI) tract ofosteoporotic Sprague-Dawley rat induced by ovariectomywere studied using Grimelius silver stain. Theexperimental animals were divided into two groups, one isnon-ovariectomized group (Sham) and the other isovariectomized group (OVX). Samples were collectedfrom each part of GI tract (fundus, pylorus, duodenum,jejunum, ileum, cecum, colon and rectum) at 10th weekafter ovariectomy or sham operation. In this study,argyrophil cells were detected throughout the entire GItract with various frequencies regardless of ovariectomy.Most of these argyrophil cells in the mucosa of GI tractwere generally spherical or spindle in shape (open typecell) while cells showing round in shape (close type cell)were found occasionally in gastric and/or intestinal glandregions. The regional distributions of GI argyrophilendocrine cells in OVX were similar to those of Sham.However, significant decreases of argyrophil cells weredetected in OVX compared to those of Sham except forthe pylorus, jejunum and cecum. In pylorus and jejunum,argyrophil cells in OVX dramatically decreased comparedto those of Sham but significances were not recorded. Inaddition, argyrophil cells in cecum of OVX showed similarfrequency compared to that of Sham. The endocrine cellsare the anatomical units responsible for the production ofgut hormones that regulate gut motility and digestionincluding absorption, and a change in their density wouldreflect the change in the capacity of producing thesehormones and regulating gut motility and digestion.Ovariectomy induced severe quantitative changes of GIargyrophil endocrine cell density, and the abnormality indensity of GI endocrine cells may contribute to thedevelopment of gastrointestinal symptoms in osteoporosissuch as impairments of calcium and some lipids,

frequently encountered in patients with postmenopausalosteoporosis.

Key words: Ovariectomy, rat, argyrophil, endocrine, frequency

Introduction

Osteoporosis is caused by an imbalance between boneresorption and bone formation, which results in bone lossand fractures after mineral flux. The frequency of fracturessignificantly increases in osteoporosis, and hip fractures insenile patients are a very serious problem because it oftenlimits the patients quality of life. The postmenopausalosteoporosis model using ovariectomized rat is useful forevaluation of osteoporetic drugs, because several parametersclearly decrease by the ovariectomy within 4 weeks afteroperation [27]. In addition, the ovariectomized rat bone lossmodel is suitable for studying problems that are relevant topostmenopausal bone loss, because ovariectomy thatinduced bone loss in the rat and postmenopausal bone lossshare many similar characteristics including decreasedintestinal absorption of calcium [12].

Gastrointestinal (GI) endocrine cells dispersed in theepithelia and gastric glands of the digestive tract synthesizedvarious kinds of gastrointestinal hormones and played animportant role in the physiological functions of thealimentary tract [2]. Until now, the investigation ofgastrointestinal endocrine cells is considered to be animportant part of a phylogenic study [5] and the endocrinecells are regarded as the anatomical units responsible for theproduction of gut hormones, and a change in their densitywould reflect the change in the capacity of producing thesehormones [8]. Silver techniques have been regarded as ageneral method for detecting GI endocrine cells andGrimelius positive cells are classified as argyrophil cells[10,11].

The changes of distribution and frequency of GIargyrophil endocrine cells in some diseases are also well

*Corresponding authorTel: 82-53-819-1436; Fax: 82-53-819-1574E-mail: endohist@dhu.ac.kr

184 Sae-Kwang Ku et al.

demonstrated especially in some cancer status [9,22],gastritis including Helicobacter pylori [1,17] and some GItract disorder [7]. In addition, these GI argyrophil cells arealso changed after treatment of some drugs such asebrotidine [21] and omeprazole [4]. The distribution andfrequency of GI endocrine cells were varied with feedinghabits [23] and osteoporetic patients and/or animals showquite different feeding habits [25]. However, there was noreport dealing with the changes of GI argyrophil endocrinecells at osteoporotic status in spite of some clear disorder ofgastric absorption of calcium ion [13], lipids [15] and GIargyrophil endocrine cells showed somewhat differentfeeding habits, and osteoporosis induced by ovariectomy orpost-menopause is directly related to some endocrinesystem especially to estrogen [19,20].

The purpose of this study is to observe the changes ofregional distribution and frequency of GI argyrophilendocrine cells in a postmenopausal osteoporotic ratinduced by ovariectomy. In this study, each part of GI tract issampled at 10th week after ovariectomy or sham-operation.

Material and Methods

Experimental animalsTwenty Sprague-Dawley (SD) female rat (6-wk old upon

receipt, Charles River, Japan) were used afteracclimatization for 7 days. Animals were allocated 5 perpolycarbonate cage in a temperature (20-25oC) and humidity(30-35%) controlled room. Light: dark cycle was 12 hr:12 hr and feed (Samyang, Seoul, Korea) and water weresupplied free to access. Half rats were ovariectomized group(OVX) and remainders were sham-operated group (Sham).

Bilateral ovariectomyAll rats were anesthetized with Ketamine hydrochloride

(60 mg/2 ml/kg) and Xylazine hydrochloride (2.5 mg/2 ml/kg) combination and subjected to operation. Bilateralovariectomy was performed by removing both ovaries in theabdominal cavity, and sham operation (ovary identification)was performed in case of sham.

Histology and quantity analysesAfter phlebotomy, each region of GI tract, fundus,

pylorus, duodenum, jejunum, ileum, cecum, colon andrectum was collected from all experimental animals at 10thweek after ovariectomy and/or sham-operation after 18hrsfasting to GI empty. Collected samples fixed in Bouinssolution, then embedded in paraffin, sectioned (3~4 µm) andstained with hematoxylin-eosin stain for confirming normalarchitecture of each region of GI tract. For observing theregional distribution and frequency of argyrophil endocrinecells in each region of GI tract, Grimelius stain wasconducted [11].

Quantity analysisThe frequency of argyrophil cells was calculated using

automated image analysis (Soft Image System GmBH,Berlin Germany) under microscope (Carl Zeiss, Berlin,Germany) in the uniform area of GI mucosa among 1000parenchymal cells. Argyrophil cell numbers were calculatedas cell numbers/1000 parenchymal cells

Statistical analysisResults are expressed as the mean ± standard deviation.

Mann-Whitney U-Wilcoxon Rank Sum W test (M-W test)was used to analyze the significance of data with SPSS forWindows (Release 6.1.3, SPSS Inc., USA) and a p-value ofless than 0.05 was considered a significant difference.

Results

In this study, argyrophil endocrine cells were detectedthroughout the entire GI tract of rats in both OVX and Shamwith various frequencies. Most of these argyrophil cells inthe mucosa of GI tract were generally spherical or spindle inshape (open type), while occasionally round in shape (closetype cell) cells were also found in the gastric and intestinalgland regions. According to the location of the GI tract,different regional distributions and frequencies of argyrophilcells were observed. Argyrophil cells were mainly dispersedin the basal portions of gastric and intestinal mucosa ratherthan surface epithelia regions and they were morenumerously detected in the stomach and large intestinecompared to that of the small intestine regardless of sham(Fig 1a~l) and OVX (Fig 2a~j).

Quantity of argyrophil cells: Among 1000 parenchymalcells, argyrophil cells in sham were detected in the fundus,pylorus, duodenum, jejunum, ileum, cecum, colon andrectum with 291.70 ± 71.74, 68.70 ± 14.06, 21.80 ± 3.68,11.10 ± 2.47, 11.70 ± 2.11, 12.40 ± 3.24, 51.20 ± 12.33 and67.90 ± 15.60 cells, respectively. In OVX, argyrophil cellswere detected with 75.90 ± 19.26, 56.20 ± 9.98, 12.20 ±3.29, 9.10 ± 1.66, 9.10 ± 2.20, 12.10 ± 3.78, 21.10 ± 2.88and 10.30 ± 3.16 cells/1000 parenchymal cells in thefundus, pylorus, duodenum, jejunum, ileum, cecum, colonand rectum, respectively. In most regions of GI tract,argyrophil cells showed significant (p < 0.01 or p < 0.05)decrease in OVX compared to that of sham except for thepylorus, jejunum and cecum. In pylorus and jejunum,argyrophil cells in OVX dramatically decreased comparedto those of Sham but significances were not recorded. Inaddition, argyrophil cells in cecum of OVX showed similarfrequency compared to that of Sham (Fig 3).

Discussion

It is generally accepted that osteoporosis is metabolic and

Gastrointestinal argyrophil cells in ovariectomized osteoporotic rats 185

hormonal disorder that is clearly related to estrogen [19,20]and also osteoporetic patients and/or animals show quitedifferent feeding habits [25]. Silver techniques have beenregarded as a general method for detecting GI endocrinecells and Grimelius positive cells are classified as argyrophilcells [10,11]. The GI endocrine cells were generally dividedinto two types, one was round to spherical shaped close typecells which were located in the stomach regions, and theother was spherical to spindle shaped open type cells which

were situated in the intestinal regions. In addition, theendocrine cells are regarded as the anatomical unitsresponsible for the production of gut hormones, and achange in their density would reflect the change in thecapacity of producing these hormones [8].

In the present study, the changes of the argyrophilendocrine cells in the GI tract of SD rat after ovariectomywere observed by silver technique, the Grimelius method.The general distribution of the argyrophil cells in the GI tract

Fig. 1. Argyrophil endocrine cells in the GI tract of sham; Most of argyrophil cells were dispersed in the mucosa of the fundus (a),pylorus (b), duodenum (c and d), jejunum (e), ileum (f), cecum (g and h), colon (i and j) and rectum (k and l). a~c, e~g, i and k: 150; d,j and l: 300, Grimelius method.

186 Sae-Kwang Ku et al.

of OVX showed quite similar patterns compared to that ofsham. As results of ovariectomy, argyrophil cells significantly(p < 0.01 or p < 0.05) decreased throughout the entire GItract except for the pylorus, jejunum and cecum. In pylorusand jejunum, argyrophil cells in OVX dramaticallydecreased compared to those of Sham but significances werenot recorded. In addition, argyrophil cells in cecum of OVXshowed similar frequency compared to that of Sham. In thefundus and rectum, most dramatical changes were demonstrated.

It was generally accepted that the changes of argyrophilcells were clearly related to digestive status of animals. InHelicobacter pylori infection, hyperplasia of argyrophil cellswere demonstrated [17] and they also increased in patientswith ulcerative colitis and Crohns disease [7], atrophicgastritis [1], hypergastrinemia [3] and pernicious anemia[26]. It has been postulated that the changes in the GIendocrine cells are a selective process to meet the newdemands exerted by the dramatic decrease in intestinal

Fig. 2. Argyrophil endocrine cells in the GI tract of OVX; Most of argyrophil cells were dispersed in the mucosa of the fundus (a),pylorus (b), duodenum (c), jejunum (d), ileum (e), cecum (f and g), colon (h and i) and rectum (j). a~f, h and j: 150; g and i: 300,Grimelius method.

Gastrointestinal argyrophil cells in ovariectomized osteoporotic rats 187

absorption [6] and osteoporetic patients and/or experimentalanimals shows impairment of absorption of calcium ion[13,18] and increase of absorption of cholesterol and otherlipids [15]. Therefore, the decrease of GI argyrophilendocrine cells may be responsible for the malabsorption ofcalcium and lipids that occur in patients withpostmenopausal osteoporosis and these decrease ofendocrine cells are also detected with aging especially tocells that release the hormone regulating GI motility [16].

In conclusion, ovariectomy induced severe quantitativechanges of GI argyrophil endocrine cell density, and theabnormality in density of GI endocrine cells may contributeto the development of gastrointestinal symptoms inosteoporosis such as impairments of calcium and somelipids, frequently encountered in patients with postmenopausal

osteoporosis. However, the target or individual changes ofGI endocrine cells are not clear and the change ofargentaffin cells that are stained by other silver techniqueand generally used in GI endocrine researches [14] is alsounknown. Elucidation of the changes of individual GIendocrine cells using immunohistochemistry [24] and/orchange of argentaffin cells using another silver techniquewill provide mechanisms for understanding GI disorder thatoccurs in various disease. Further detailed studies withimmunohistochemical and another silver techniques will beneeded.

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Fig. 3. Number of argyrophil cells in GI tract and their changes after ovariectomy. *p < 0.01 compared to that of sham; **p < 0.05compared to that of sham. For each data point, n = 10 rats. Unit = argyrophil cells/1000 parenchymal cells.

188 Sae-Kwang Ku et al.

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