Radiol Oncol 1996; .30: 189-9.3. Gastric emptying in rats with gastroduodenal disease induced by N-methyl-N-nitro-N-nitrosoguanidine and alcohol Drazigost Pokorn1 and Anton Cerar2 ' Institute of Hygiene, 2 Institute of Pathology, Medical Faculty, University of Ljubljana, Slovenia The rale of gastric emptying was measured with a dye dilution technique in rats treated with N-methyl-N-nitro-N-nitrosoguanidine and alcohol. Gastric emptying was compared in rais with gastroduodenal inflammatory diseases and gastroduodenal neoplasms, and in those without gastroduodenal disease. Gastric emptying was found to be significantly increased following the intragastric injection of liquid meal in rats with gastroduodenal diseases as opposed to the control group of healthy rats. These findings suggest that an increased gastric emptying of liquids can be explained by abolition on the relaxation of the gastric wall in rats with gastroduodenal diseases. Key words: gastric emptying; dye dilution technique; stomach diseases-chemically induced; N-methyl-N-nitrosoguanidine Introduction There is an increasing tendency to incriminate abnormalities in gastric emptying in the pathogenesis of gastroduodenal disease. Many disorders are associated with delayed gastric emptying without evidence of a structural gastric outlet obstruction. '-4 Nevertheless, Nomiyama5 found, by use of the acetaminophen absorption method, that gastric emptying in patients with early gastric cancer was rather rapid, if compared with emptying in healthy subjects. These differences may be caused by differences in the methods of the gastric emptying measurement and in the test meal used.3- '' We supposed that liquids injected directly into the stomach by a sonde, would be emptied from a stomach with gastroduodenal disease more quickly than from a healthy stomach.7 Correspondence to: Prof. Dražigost Pokorn, PhD, MD, Institute of Hygiene, Medical Faculty, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia. UDC: 616.33-001.37:616.33-008. l We tested this hypothesis using the dye dilution test for gastric emptying"' 9 after treating Wistar rats with N-menthyl-N-nitro-N-nitrosoguanidine (MNNG) 1 and alcohol. Materials and methods Animals We used 70 male Wistar rats weighing 150-200 g. The animals were fed pelleted Knapka food, and maintained in macrolon cages at a constant temperature (22±2 °C) and relative humidity (60±5 %). All the animals were given MNNG (Fluka Chemie, Switzerland) at a concentration of 100 mg/litre10 in a drinking solution. We arbitrarily divided the experimental animals into two groups. One group had MNNG diluted in tap water, while the other, in a 12 % alcohol solution. The control group of 10 animals drank tap water. The experimental animals drank MNNG solution for 29 weeks; after that they drank tap water for an additional 29 weeks, at which time the experiment concluded. 190 Pokom D and Cerar A The experimental animals were observed daily and weighed once every 4 weeks. Autopsies were performed on ali except two animals. At the end of the 58 experimental weeks, the gastric emptying tests were performed. Gastric emptying studies The technique of Mangel and Noegel8" was used with modifications. Studies were performed on ali rats with the exception of the rats which spontaneously died during the experiment. During fasting the rats were maintained in wire-bottomed cages to avoid copraphagy. The experiments were performed in a room with the same environmental conditions (temperature, noise and humidity) as those in the breeding area. Ali experiments were done in the morning. The phenol red meal was prepared as follows: methyl cellulose was dissolved in water at about 80 °C and prepared in a final concentration of 1.5 %. The solution was stirred until dissolved, and phenol red (50 mg/100 ml) was then added to the stirring solution. Three ml of phenol red solution, maintained at 37 °C, was administered orally to the rats. The animals were then killed I O minutes (healthy rats) or 20 minutes (healthy rats and rats with gastroduode-nal diseases) after the ingestion of the phenol red meal by means of C02 inhalation. An incision was made for a middle laparotomy. The stomach was exposed and occluded at the pylorus and cardia. The stomach was then removed. cut along the greater curvature and washed out with 3 ml of 0.9 % saline. The gastric content was placed in 100 ml of O, 1 N NaOH with 0,9 % saline. Trichloracetic acid (0,5 ml) (20 % t/vol) was added to 5 ml of the mixture. This sample was centrifuged at 2,500 rpm for 30 min. The supernatant was removed and 4 ml of 0,5 N NaOH added. Samples were then read on a colour spectrophotometer (MA 9502) at 560 nm. The percentage of gastric remains was calculated as follows: % gastric remains = absorbtion value for stomach =- x 100 mean absorbtion value for test meal Values were reported as means ± SE. Statistical analysis was made using the Student's t test with a significance level criteria of 0,05. Morphologic evaluation The stomach and other visceral organs were examined macroscopically, fixed in a I 0 % neutral formalin and routinely processed for histopathological studies. Gastroduodenal lesions were classified his-topathologically into neoplastic (dysplasia, papilloma, squamous celi carcinoma, adenocarcinoma, sarcoma) and nonneoplastic (principally intlamma-tory) gastroduodenal diseases, following accepted histologic criteria.12 Statistical evaluation The significance of the percentage difference of the gastric content 20 minutes after the test-meal was evaluated by using the Student's t test. " Results Body weight gains of rats in the different MNNG-treated groups are shown in Figure 1. Figure l. Weight gain in rats during different dietary periods. One animal in the alcohol and two animals in the water group died from pneumonia and one animal in the alcohol group from an advanced gastric tumor. Autopsies were not done in two animals. As shown in Table 1, tumors were found in 23 rats (from a total of 60 rats). The incidence of gastroduodenal carcinoma was greater in the experimental group drinking the tap water with MNNG, in contrast to the group drinking alcohol with MNNG. The data on gastric emptying in rats with gastroduodenal diseases and those in healthy rats are summarised in Figure 2. The gastric emptying in rats with gastroduodenal lesions with or without gastric or duodenal tumors Gastric emptying in ru/s with gaslmduodenal disease 191 Table l. Effect of Various Test Meals on GastroLAL MCOIUM VAUJl CI ' ("( I'.',!,.!<>■(■ CC'JIU'l'i X wf/lüllf. M UK l>it I'.SI MiAl Figure 2. Distribution of the gastric content of the standard meal in the stomach of rats with and without gastroduodenal disease. No % 1 20.2 Chronic superficial duodenitis. 2 18.2 Fibrosis of fundic mucosa. Chronic superficial duodenitis. . 24.1 Chronic superficial duodenitis. 4 24.1 Chronic antral gastritis with focal fibrosis of mucosa an This includes lymphomas, carcinoma, Whipple's disease, and diseases that produce granulomas in the gastric wall. In our study, ali the animals with a prolonged gastric emptying with neoplasia had advanced carcinomas of the antrum and duodenum. On the other hand, the animals with shortened emptying generally had early gastric tumors. Gastric mucosal abnormalities can affect gastric emptying. Diseases of the gastric musculature, including the inflammatory and endocrine myopathias, muscular dystrophies, and infiltrative disorders, can result in significant gastroparesis.' Most patients with gastroparesis have a delay in the emptying of solid food, but the rate of liquid emptying is preserved. This observation suggests that the factors that regulate the fundic tone are preserved longer in most patients with gastroparesis.'7 The proximal stomach has two remarkable motor properties that allow it to carefully regulate intragastric pressure during gastric filling, namely: receptive relaxation and accommodation. The proximal stomach relaxes to receive the bolus of ingested food from the esophagus; hence the term receptive relaxation. Liquids entering the fin- Gastric emptying in rats with gastroduodenal disease 193 dus trigger vagally mediated initial receptive relaxation, so that the fundus plays a key role in the gastric emptying rate for liquids.IS The slight shortening of the time for gastric emptying could be accounted for as a result of the absence of the part of the receptive relaxation of the stomach when swallowing is eliminated. Accommodation to distension is a process whereby the stomach accepts increasing volumes without greatly increasing the intragastric pressure. In the present work, an increased gastric emptying (especially the initial emptying) of liquids can also be explained by the abolition of the vagally mediated receptive relaxation, and/or of the change in the fundic tone as the intragastric injection of the liquid test meal occurs. that raises the intraluminal pressure, elevates the pressure gradient between the stomach and duodenum, and allows more liquids into the duodenum.20 This abnormally rapid gastric emptying, compared with the emptying in healthy rats, may be caused by differences in the gastric tonus." In healthy group, the proximal stomach accommodated easily to distension. keeping the intragastric pressure low as the stomach filled. On the contrary in the group with gastroduodenal disease, larger increases in pressure occurred during gastric distension. The gastroduodenal diseases impaired the stomachs' accommodation and led to greater increases in intragastric pressure with gastric filling. Moreover, the greater increases in pressure in those animals with gastroduodenal diseases led to the more rapid gastric emptying of liquids for most of the rats studied. References 1. Chanduri TK, Fink S. Gastric emptying in human disease states. A111 J Gastroenterol 1991; 86: 533-8. 2. Griffith GH, Owen GM, hiset AP, Campbell H, Smith R. Gastric emptying in health and in gastroduodenal disease. Gastroenterology 1968; 51: 1-7. 3. Tatsuta M. Liski H, Okuda S. Gastric emptying in pa-lients with fundal gastritis and gastric cancer. Gut 1990; 31: 767-9. 4. Shih WJ. Domslated PA. Deland FH. Technetium - 99 111 triethelene tetramine polysterone resin gastric emptying studies in patients with various upper gastrointestinal diseases. Ciin Nucl Med 1985; 10: 494-7. 5. Nomiyama T. Gastric emptying in gastric cancer. In: Tsuneoka K, Miwa T, Sekiguchi T, eds. Gastroduode-nal motor disorder. Tokyo: Bunhodo, 1985: 178-81. 6. Minami H, Mc Collum RW. The physiology and pathophysiology of gastric emptying in humans. Gastroenterology 1984; 86: 1592-610. 7. Azpiroz F, Maalagelada JR. Gastric tone measured by an electronic barostat in health and postsurgical gast-roparesis. Gastroenterology 1987; 92: 934-43. 8. Scarpignato CT, Capovilla T, Beitaccini G. Action of caerulein on gastric emptying of the conscious rat. Arch Int Phamwcodyii 1980; 246: 286-94. 9. M;mgel AW, Koegl A. Effect of peptides on gastric emptying. Am J Physiol 1984; 246: (Gastrointest Liver Physiol 9): G342-G345. 10. Takalmshi M, Hasegawa R, Furukawa F, Toyoda K, Sato H, Hayeshi Y. Effect of ethanol, potasium metabisulfite, formaldehyde and hydrogen peroxide on gastric carcinogenesis in rats after initation with N-Me-thyl-N-Nitro-N-Nitrosoguanidine. Jpn J Cancer Res (Giiiiii) 1986; 77: 118-24. 11. Watanable H, Takahashi T, Okomoto T, Ogundigie PO, Ito A. Effect of Sodium Chloride and Ethanol on Stomach Tumorigenesis inACI Rats Treated with N-Methly-N-Nitro-N-Nitrosoguanidine; A Quantitative Morpho" metric Approach. Jpii J Cancer Res 1992; 83: 588-93. 12. Stinson SF, Schuller HM, Reznik G. Atlas of tumor pathology ofthe Fischer rat. Boca Raton, Florida: CRC Press, 1990'. 13. Petz B. Osnove sratisticke metode. Zagreb, 1970: 121-69. 14. Meerof JC, Schreiber DS, Trier JS et al. Abnormal gastric motor function in viral gastroenteritis. Ann liilem Med 1980; 92: 370-3. 15. MeddernJG, Horowitz M,Jamieson GG et al. Abnormalities of esophageal and gastiic emptying in progressive systemic sclerosis. Gastroenterology 1984; 87: 922-6. 16. Reddy AB, Wright RA, Wheeler GE et al. Non-obstructive gastroparesis in amyloidosis improved with metoclopramide. Arch Intern Med 1983; 143: 247-8. 17. Reynolds JL. Prokinetic agents: A key in the future of gastroenterology. Gastroenterol Clin North Am 1989; 18: 437-57. 18. Mistiaen W, Van Hee R, Block Pet al. Gastric emptying for solids in patients with duodenal ulcer before and after highly selective vagotomy. Dig Dis Sci 1990: 35: 310-6. 19. Hunt JN, Mac Donald I. The effect of intra-gastric instillation of meal on gastric function in man. J Physiol 1951; 113: 185-8. 20. Kelly KA. Gastric emptying of liquid and solids: roles of proximal and distal stomach. Am J Physiol 1989: 239: (Gastrointest Liver Physiol 2): G7I-G76.