A. A ..._, ‘m . , ‘ ,z: 2115': up \ ~.\ ‘ . ‘J‘TLI If» “ ‘f k. x' 4.; Lg" ; \. .. ., “NM,“ L--.9~\-.L -..E -.\ «<2 r“. 2 "..:.‘=. . .55) a ‘ ~. .' . Cu ‘.'. r... ’3 ¢. " u . , . -u " 13' '3'? 4"- ‘ 1. (1‘ "fl .g4v“:3i}"1), .. (xv . - w‘fi -3‘?~f«?‘.3): '" « \‘ . ‘ E c , ‘ , I ’4 ' a m2; ; ‘ ‘3‘ , g. .35. t nu. ., g. , ‘ .5555":- W’\ $ . '1). . 3‘1“ wk: 9%, In??? ' ‘ I! Date 0-7 639 UNIVERSILTY lllllllllllllllllllllllll\lllllHIllllllllllllllllll 3 1293 01018 8310 This is to certify that the thesis entitled CISPLATIN-lINDUCED PEPTIC ULCER, VAGOTOMY, ADRENAL AND CALCIUM MODULATION presented by AFSAR SOKHANSAN J has been accepted towards fulfillment of the requirements for .MASIEBLSAegree in WARTMENTAL B IOLOGI CAL SC I ENCE PROGRAM 6 L4 figogw/ Major professor JUNE 24, 1993 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State Unlverslty PLACE IN RETURN BOX to romovo this checkout from your rocord. TO AVOID FINES rotum on or bdoro duo duo. DATE DUE DATE DUE DATE DUE MSU loAn Nflnnotlw Action/Equal Opportunlty Inotltwon m M! ClSPLATIN-INDUCED PEPTIC ULCERS, VAGOTOMY, ADRENAL AND CALCIUM MODULATION BY AFSAR SOKHANSANJ A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Interdepartmental Biological Science Program 1993 ABSTRACT CISPLATlN-INDUCED PEPTIC ULCERS, VAGOTOMY, ADRENAL AND CALCIUM MODULATION BY AFSAR SOKHANSANJ Cisplatin (gfi-dichlorodiammineplatinum ll) treatment (9mg/kg) for 3 days causes the release of corticosterone from the zona fasciculata and zona reticularis of the adrenal gland and an increase in acid secretion from the parietal cells in the stomach, inducing ulcers. Cervical bilateral vagotomy has been used to control the ulceration. Morphological studies of the vagotomized adrenal gland show an increase in the number of the lipid droplets, reflecting a possible inhibition in the release of the hormones. Calcium is required for the release of acetylcholine from the neural endings of gastric smooth muscle; cisplatin inhibits this response by inducing hypocalcemia. Inhibition of the acetylcholine release leads to bloating of the stomach and possible induction of ulcers. Calcium injections (0.5 ml of 10% Calcium gluconate) before and during cisplatin treatment prevents stomach bloating and ulceration. This thesis is dedicated to my parents Mr. Ahmad Sokhansanj and Mrs. Badri-Jahan Ebrahimi-Nejad and my husband Farshid for their unlimited unconditional love and understanding. ACKNOWLEDGEMENTS I would like to thank my research advisor Dr. Surinder K. Aggarwal for his generous understanding and support through out my training, but more importantly I will consider myself to be truly blessed if I continue to meet people like him who have enriched so many aspects of my life. I would like to thank Dr. Thomas Corner for his advice and his willingness to meet with me and discuss my course work at all times. I also like to thank Dr. William Frantz for participating as a member of my committee. Special thanks go to Dr. James San Antonio for providing the 3H-acetylcholine incorporation and autoradiography experimental data as this work is being submitted as a joint paper for publication to the anti-cancer drugs. iv TABLE OF CONTENTS PAGE List of Figures and table. . . . . . . . . . . . .Vll Introduction. . 1 Material and Methods. . 3 Tissue Handling. . 4 Gastric Emptying. . 5 Statistical analysis . 5 3H-Choline Uptake Experiments. .5 Autoradiography. .6 Acetylcholinestrase Localization. .7 Results. . 9 Gastric Emptying. . 9 Acetylcholinestrase Localization. 1O 3H-Choline Uptake. 11 Autoradiography. 1 2 Ultrastructure Gastric Smooth Muscle And AxonalEndings. 12 Adrenal Morphology. . 12 Cis platin treatment. 14 Vagotomy. .14 Discussion. .55 References. . 63 Figure Table 1 . Figure 1 . Figure 2. Figure 3. Figure 4. Figure 5. LIST OF FIGURES AND TABLE Page Showing the experimental design. . . . . . . . . 16 Graph showing inhibiton of gastric emptying by 95% after(A) cisplatin treatment (9mg/kg) compared to (0) normal or cisplatin and vagotomy (.). The gastric emptying was not half as effected after cisplatin and vagotomy compared with cisplatinalone. ...............17 Light micrograph of a cross section through the cardiac portion of the stomach from a normal rat stained with hematoxylin. M, muscularis mucosa; C, circular muscle layer; L, longitudinal muscle layer. X520, Bar= 20pm. . . . . . . . . .19 Light micrograph of a cross section through the cardiac portion of the stomach from a rat after 3 days of cisplatin treatment (9mg/kg) showing small erosion in the mucosal layer (arrow). M, muscularis mucosa; C, circular muscle layer; L, longitudinal muscle layer. X520, Bar=20um. . . .21 Light micrograph of a cross section through the cardiac portion of the stomach of a rat after 5 days of cisplatin treatment (9mg/kg). Note the ulcer extending in to the deeper layers of mucosa (arrow). M, muscularis mucosa; C, circular muscle layer; L, longitudinal muscle layer. X520, Bar=20um. . . . . . . . . . . . . 23 Light micrograph of a cross section through the cardiac portion of the stomach of a rat after 8 days of cisplatin treatment (9mg/kg). Note the loose intensely staining blood cells in the mucosal layer (arrow). M, muscularis mucosa; VI Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. C, circular muscle layer; L, longitudinal muscle layer. The longitudinal muscle layer is not pictured in the field of view. X330, Bar=30um. . . . . . . .25 Light micrograph of a cross section (10pm) of the adrenal gland from a normal rat showing normal morphology of the gland after staining with hematoxylin. C, cortex; M, medulla; cap, capsule; ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis. Arrows point toward the normal capillaries in the gland. X192, Bar=0.5mm..................27 Light micrograph of a fresh frozen section of the adrenal gland from a normal rat, stained with Sudan black B showing the distribution of lipids in the zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR). Arrows point to the lipid granules. X1300, Bar=10um. . . . . . . . .29 Light micrograph of a fresh frozen section (10pm) of the corti-comedullary region of the adrenal gland from a normal rat stained with Sudan black B. C, cortex; M, medulla; c, capillaries. Arrows point to the lipids. X1300, Bar=10um. . . . . . . . . 31 Electron micrograph of a section through the cortical region of an adrenal gland from a normal rat showing the distribution of the lipid vesicles (arrows). Note the lamellae in one of the lipid vesicles (heavy arrows). N, nucleus. Arrowheads point toward mitochondria. X9540,bar=1um................33 Electron micrograph of a section through the medullary region of an adrenal gland from a normal rat showing the epinephrine (E) and norepinephrine granules (NE) containing cells. ,N, nucleus. Arrowheads point to the mitochondria. X8850,Bar=1pm................3S vii Figure 1 1 . Figure 12. Figure 1 3. Figure 14. Figure 1 5. Light micrograph of a fresh frozen section (10 pm) of the cortical region of an adrenal gland after 3 days of cisplatin treatment (9mg/kg) stained with Sudan black B. Note the intensely stained but less number of lipid globules in the various regions of the cortex compared with similar section from a normal gland (see Fig. 7). ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis. Arrows point to the lipid globules. X1300, Bar=10pm. . . .37 Light micrograph of a fresh frozen section 10pm) of the cotico-medullary region of the adrenal gland after 3 days of cisplatin treatment (9mg/kg) stained with Sudan black B. Cisplatin treatment induces complete absense of the lipids from cortico-medullary region. Note the distension of the capillaries so prominent in the medullary region. C, cortex; M, medulla; c, capillaries. Arrows point to the lipid droplets. X1300, Bar=10um. . . . . . . . . . . 39 Light micrograph showing the distension of the capillaries (arrows) in the zona glomerulosa (26) and zona fasciculata (ZF) of an adrenal gland after 3 days of cisplatin treatment (9mg/kg) stained with hematoxylin. X520, Bar=20um. . . . . . . . . .41 Light micrograph of a cross section (10pm) of the adrenal gland stained with hematoxylin. Cisplatin treatment (9mg/kg) induces significant telangiectasia in the capillaries (arrows ) of the cortex (C) and the medulla (M) just after 3 days. Note also the atrophy of the medulla. ZG, zona glomerulosa; ZF, zona fasciculata; ZR, reticularis;cap,capsule. X192, Bar=0.5mm. . . . 43 Electron micrograph of a section through the cortical region of an adrenal gland from a 3 day cisplatin treated (9mg/kg) rat. Note cisplatin treatment induces abnormal swelling of mitochondria (arrowheads) with large matrix and distruption of the inner membranes. N, nucleus. Arrows point to the lipids. X9540 Bar=1.5pm.............. viii . 4S. Figure 1 6. Figure 1 7. Figure 18. Figure 19 Electron micrograph of a section through the medullary region of an adrenal gland from a rat after 3 days of cisplatin treatment (9mg/kg). Note cisplatin treatment induces an abnormal swelling of mitochondria (arrowheads) and norepinephrine (NE) vesicles (arrows). N, nucleus. X21280 Bar=0.5 um. . . . . . . . . . 47 Light micrograph of a cross section of the adrenal gland from a vagotomized rat (3 days) showing hypertrophy of the medulla (M). Arrows point toward the distended capillaries in the zona reticularis. C, cortex; ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis. X192,Bar=0.5mm. . . . . . . . . . . . . . .49 Electron micrograph showing the cells of the cortical region in the vagotomized rat. Vagotomy (3 days) induces an increase in the number of the lipid droplets (arrows). N, nucleus. X9540, Bar=1pm. . .51 Electron micrograph of a section from the medullary region of an adrenal gland from a vagotomized rat (3 days). Vagotomy induces abnormal swelling of mitochondria (arrowheads) with distruption of internal organization and norepinephrine (NE) vesicles. N, nucleus. X21630, Bar=0.5pm. . . . . . . . . 53 INTRODUCTION Cisplatin (gigs-dichlorodiammineplatinum II), a potent broad spectrum anti-cancer agent, produces severe toxic side effects including kidney damage,1 nausea and vomiting?- loss of hearing,3 peripheral neuropathy,4 hypomagnesemia5 and hypocalcemia.5-7 The most severe dose-limiting side effects are kidney toxicity, nausea and vomiting. Kidney toxicity can be limited through slow infusion of the drug,3 hydration and diuresis of the patients,5 and use of special protective agents like WR27219: or by increased sodium chloride concentrations in the vehicle of administration.10 Nausea and vomiting can be so severe in some patients that the treatment has to be discontinued.11 However, this can be controlled to some extent through the use of anti-emetic drugs like metoclopramide12 or ondansteron.l3 Serotonin and serotonin 5HT3 seem to play an important role in the way chemotherapeutic agents produce nausea and emisis.14-16 The 5HT3 receptor antagonist ondansetron has been shown to control nausea and vomiting produced by cisplatin in both animals17 and patientsl‘lvlfi-19 and has been proven to be superior to metoclopramide.18-21 2 In the rat cisplatin causes undue distension of the stomachZZ'23 through the inhibition of gastric motility, leading to the development of ulcers. Adrenal steroidal factors and catecholamines have been implicated in the induction of ulcers in rats and can be blocked by adrenalectomy or bilateral vagotomy24 Vagotomy has been used as an alternative for the treatment of chronic ulcer patients7-5 and is an effective antiemetic therapy in cisplatin-induced nausea and vomiting.26 The vagus nerve also controls the acid secretion by parietal cells in the stomach.27 The present study is an effort to characterize cisplatin-induced changes in neuromuscular interactions of the stomach smooth muscle and in the adrenal gland (steroidal and catecholamines) and correlate these changes to the induction of peptic ulcers. MATERIALS AND METHODS Animals: Laboratory-bred male wistar rats [Crl:(Wl)BR] (Charles River Breeding Lab, Portage MI) weighing 200-300 g and approximately 3 months of age were used in these experiments. Animals were housed 4 per cage at 20t2°C on a 12:12 h light-dark cycle with free access to animal feed ( Allied Mills, Chicago, IL) and tap water ad libitum. Eight groups, 10 animals in each, were used in this experiment (Table 1). Animals in group 1 were injected with a single intraperitoneal dose of cisplatin (9mg/kg) dissolved in 0.85% saline. Control animals (group 2) received comparable saline injections. Cisplatin-treated animals in group 3 were denied access to food but were provided with free access to water. Yet another group of cisplatin-treated animals (group 4) was given daily intravenous injections of calcium (0.5 ml of 10% calcium gluconate) at 08:00 h on the day of cisplatin treatment and each day thereafter up to the day of euthanasia. Animals in group 5 were bilaterally vagotomized28 and after 24 hours were treated with cisplatin (9mg/kg) for 3 days. Animals in group 6 were sham operated to serve as controls. Animals in group 7 were bilaterally vagotomized. Animals in Group eight were sham 4 operated vagotomy. Animals were euthanatized and killed by decapitation on day 3, 5 and 8 post-treatment; The stomach and the adrenal glands were removed. The stomach was opened along the great curvature, emptied of its contents by flushing with 10 ml of 0.15% M NaCl and examined for evidence of ulceration. Tisu hnlin rce re: Stomach tissue was stretched in 4% buffered (0.05M cacodylate buffer pH 7.4) glutaraldehyde for 4 h before processing for light and electron microscopy, some of the tissue was used for frozen sections. Paraffin embedded and frozen sections (7-10 pm) were stained by Masson trichome, periodic acid-Schiff, Ehrlich hematoxylin and eosin, mercuric bromphenol blue and Sudan black B or oil red 0 methods}!9 Adrenal glands were cut into two halves. One half was prepared for freeze sectionining while the other half was fixed either in Bouin's fluid and processed for light microscopy or thin slices of adrenal were fixed in 4% buffered (0.05 M cacodylate pH 7.4) glutaraldehyde and 1% osmium tetroxide for 1-2 h at 4°C and embedded in araldite after proper dehydration . Thick (1pm) sections were stained with 1% solution of methylene blue in 1% borax for 60 S at 60°C. Thin sections (500-700A°) were stained with uranyl acetate and lead citrate. Stained sections were viewed under the Zeiss ultraphot Ill and Hitachi HU11E electron microscope operated at 75 KV respectively. Gastric Emptying: Gastric emptying was measured on day 3, 5 and 8 by the phenol red meal method.30 The meal consisted of a solution of 50 mg phenol red in 100 ml aqueous methylcellulose (1.5% W/v) given by oral intubation of hand-held conscious rats in a dose of 1.5 ml/rat. Gastric emptying was measured 3 h after the meal and phenol red content was determined according to the procedure described by Scarpignato.30 A group of 10 rats was killed immediately after the administration of the meal and the phenol red content of these animals served as a zero emptying point control group. Stomachs and the contents were homogenized in 0.1 M NaOH (100 ml). Proteins were precipitated using 20% trichloroacetic acid, alkalinized using 0.5 M NaOH and assayed using colorometry at 560 nm as a routine process. Gastric emptying was calculated for each rat as =[1-A/A’] x 100, where A represents absorption at 560 nm by gastric contents at 3 h after the meal and A’ represents absorption at zero emptying time. Statistical Analysis: Statistical analysis was performed using a one way analysis of variance with Student-Newsman-Keals follow-up test.32 EH-Cholinp Uptake Experiments: 6 Three days after cisplatin-treatment, 10 rats were injected intraperitoneally with choline chloride (Methyl-3H) (New England Nuclear, Boston, MA) at 6 uCi/Kg OR 60 uCi/Kg. Animals were sacrificed at 5 min, 3 h and 12 h intervals. Stomach was removed, emptied of its contents and divided into three parts by cutting along the line separating the cardiac from the pyloric region, and cutting 0.5 cm on both sides of the pyloric sphincter. Each piece was weighed, chopped into small pieces, and digested by adding 2.5 ml protosol (New England Nuclear, Boston, MA). After digestion for 48 h, 15 ml scintillation cocktail (5 g ppo, 100 g Naphthalene to 1 L Dioxane) was added. Sample radioactivity was measured using a Beckman scintillation counter. Data were statistically analyzed using student-Newsman-Keals test. A t r i r h : Small pieces 0me of tissue from the cardiac stomach were removed from the 3H-choline chloride injected animals and were fixed in 1% glutaraldehyde and 2% potassium pyroantimonate in 0.05% M cacodylate buffer (pH 7.4) for 12 h at 4°C. Tissues were post-fixed in 1% buffered osmium for 1 h, dehydrated and embedded in araldite. Sections (0.5 pm) were stained with 0.5% methylene blue before coating with Kodak NT B 2 emulsion kept at 43°C. Slides with emulsion coated stained sections were stored in black plastic slide boxes sealed with black electric tape for 6 weeks at 4°C. 7 After proper exposure, slides were developed in Microdol X (3 min), before examination using a Zeiss photo-microscope. For electron microscope autoradiography, thin sections (70-90 nm) were picked up on parlodion (4% solution in amyl acetate ) coated slides. Sections were coated with llford L4 emulsion (5 g in 20 ml distilled water). Thickness of the emulsion was standardized on a test slide so as to give purple diffraction using fluorescent light. Emulsion coated sections were developed after a 7 weeks exposure. A sharp knife was used to cut a circle (0.5 cm in diameter) around the sections. A drop of hydrofluoric acid (5%) was placed on the cut around the sections. The slide was slowly immersed in a dish of distilled water, resulting in circular section floating off onto the surface. Sections were picked up on copper grids and examined using a Hitachi HU11E electron microscope operated at 75. Apefllpholinpstgrgsg Lppalizatipn: Frozen sections (7 pm) of the pyloric sphincter and the stomach tissue were treated33 for the localization of cholinesterase activity. A brief fixation (3 min) in 4% buffered formaldehyde (0.05 M phosphate buffer pH 6.0) was employed before incubating sections in 2 mM acetylcholine iodide, 0.065 M Tris maleate buffer (pH 6.0), 5 mM sodium citrate, 3 mM copper sulfate, 1 ml distilled water, 0.5 mM potassium ferricynide, and 0.44 M surose. Controls included incubating sections in the incubation media without the substrate, acetylcholine iodide or in media containing physostigmine sulfate 8 (10‘4 M), or tetra mono isopropylpyrophosphoretetramide (iso—OMPA) (3X10—5 M)34 serving to inhibit acetylcholinestrase or pseudocholinestrase respectively. After a 45 min incubation sections were washed in 0.1 Tris Maleate buffer and mounted in glycerine jelly. The reaction product was visualized as reddish brown precipitate under the light microscope. For electron microscopy tissues were further treated with 1% osmium tetroxide and processed in a routine manner. RESULTS I m In : Cisplatin treatment induced gastric bloating mostly through inhibition of gastric emptying (Fig. 1). By day 3 the gastric emptying was down to about 33% of the normal and it further dropped down to about 20% of the total by day 5 when the weight of the stomach along with its contents increased by 5.5 fold of the normal stomach. Daily injections of calcium reversed this bloating process resulting in almost normal weights. The pH of the stomach contents as diluted with 10 ml of 0.85% saline solution was 4.85 in the normal rats and 2.6 by day 5 of post cisplatin treatment. Prominent peptic lesions could be detected in the cardiac portion of the stomach after 3 days of cisplatin treatment (Figs. 2-5). No such lesions were observed in the pyloric section of the stomach even in the 10 pm thick serial sections. The gastric lesions after cisplatin treatment varied in size from 15 pm to bleeding ulcers of a mm or more in diameter (Fig. 5). The number of these ulcers varied from about 7 to more than 40 between day 3 and day 5. Cisplatin-treated animals, denied any food, but allowed water ad libitum. did not show any gastric erosions of the mucosal 10 membranes. Vagotomized animals treated with cisplatin also did not show any gastric lesions even after day 8. The ulcers usually started as minute erosions (Fig. 3) in the mucosal layers after Day 3 of cisplatin treatment and extended into the deeper layers of mucosa (Fig. 4) leading to bleeding ulcers with raised margins. By day 5 of cisplatin treatment, intense granulation in the outermost layers of the mucosa was very prominent (Fig. 5) after hematoxylin and eosin staining such granulation was not observed in control rat stomach mucosal layers. This probably represents the increased gastrin/pepsin secretory activity due to cisplatin treatment. Apetylchplinpstgrage chalizgtipn: Acetylcholinesterase was histochemically localized in the gastric tissues from the normal and cisplatin-treated rats and the staining intensities and distributions were compared. The muscularis mucosa in the cardiac or the pyloric regions were negative whereas the circular and the longitudinal muscle layers were intensely positive only in localized places both in the normal and cisplatin treated tissues. These areas included mostly sections of nerve fibers, nerve fascicles and ganglia. There was no significant detectable difference observed between the normal and cisplatin treated animal tissues from the cardiac or the pyloric portions of the stomach. Similarly, the muscularis of the sphincter in the normal and cisplatin treated rats demonstrated no significant differences in staining intensities for acetylcholinesterase. Using 11 electron microscopical methods, acetylcholinesterase was demonstrated to be associated with the membranes of the axonal endings in smooth muscle in a uniform fashion. There was no difference in the distribution between the treated or untreated tissues. 3H-choline uptgkp: In order to determine if acetylcholine metabolism in the enteric nervous system was altered by cisplatin treatment, 3H-choline uptake in the stomach tissue of the normal rats was compared with that of cisplatin-treated rats. Radioactivity was measured in the cardiac, pyloric and pyloric sphincter regions of the stomach at 5 min, 3 h and 12 h after injections. In the cardiac region of the stomach the radioactivity was significantly higher in the cisplatin- treated animals than in controls at 5 min, 3 and 12 h. There was a significant increase in the radioactivity with time in both in the cisplatin-treated and nontreated rats. However, there was no significant difference in the 3H-choline incorporation in the pyloric region of the stomach between normal or cisplatin-treated tissues nor there was a significant change in the tissue radioactivity over time. In the pyloric sphincter 3H-choline incorporation in the cisplatin-treated rats was significantly higher compared to controls after 5 min and 12 h and this activity decreased over time in both the normal and cisplatin-treated animals. Thus when measured as a function of time following 3H-choline injection, the cardiac region 12 of the stomach demonstrated an increase in radioactivity with time whereas in sphincter tissue there was a decrease. WM Autoradiography was performed to determine the specificity of the 3H-choline uptake. Light and electron microscopic autoradiograms showed silver grains mostly localized over the cytoplasmic membranes of the smooth muscle cells and over the synaptic endings. Because of the large size of the silver grains in the case of the synaptic endings it was difficult to attribute these grains to neurotransmitter vesicles or mitochondria. Ultrastructpre gagtrip smooth mpsple and axpnpl pngingsz Two types of cells were easily recognized in the gastric smooth muscle tissue after cisplatin treatment for five days. There were cells with highly electron dense microfilaments and others that were quite electron lucent with swollen mitochondria. The axonal endings clearly demonstrate an increase in the synaptic vesicles after only three days of cisplatin treatment indicating a possible block in the release of the neurotransmitter. By day 5 these synaptic vesicles and the associated mitochondria break down leaving large axonal endings with no cellular orgenelles but membrane debri in them Adrnal In mrhl: 13 The adrenal glands consist of two regions, the cortex and the medulla. The cortical regions are divided in to 3 zones: zona glomerulosa, zona fasciculata and zona reticularis which occupy 15%, 70% and 7% of the total volume of the gland, respectively (Fig. 6). The normal morphology of the adrenal gland is well-known.35 Adrenal cortex is actively involved in the process of the steriodogenesis and the adrenal medulla is responsible for catecholamine synthesis.36 The zona glomerulosa of the adrenal cortex secrets mineralcorticoids principally aldosterone and the zona fasciculata produces glucocorticoids such as corticosterone. The Zona reticularis secrets the sex hormones like dehydroepiandrosterone. Lipid granules are abundant throughout the cortical region especially in the zona fasciculata and zona reticularis (Figs. 7,8). Lipid droplets are precursors of steroid hormones37 and have different sizes and contains a thin boundary membrane which seperates the lipids from the cytoplasm. Mitochondria in the adrenal cortical region are elongated (zona glomerulosa) , round (zona fasciculata) or ovoid (zona reticularis). Figure 9 shows an electron micrograph from zona fasciculata which contain round mitochondria with vesicular cristal arrangements. The outer nuclear membrane shows blebbing while the lipid droplets show lamellar concentrations which are probably parts of collapsed membranes of the lipid (heavy arrows) vesicles. Catecholamines (norpinephrine and epinephrine) are produced in the medullary region in two different cell types (Fig. 10). Norepinephrine vesicles 14 are more electron dense, smaller and irregular in shape compared to epinephrine vesicles, which are less electron dense, larger and their contents fill the vesicle. There is more variation in density in epinephrine vesicles than norepinephrine vesicles. There is a membrane surrounding both vesicles which is more evident around epinephrine vesicles. Sometimes the membrane of epinephrine vesicles is not apparent due to granules filling the space. Mitochondria are elongated or round and have tubular cristal arrangements (Fig. 10) . Cisplatin treatmpnt: Cisplatin treatment (9mg/kg) after 3 days induced a sharp decrease in the lipid contents of the cortex ( Fig. 11) compared to the normal (Fig. 7). Lipid vesicles are almost absent from the cortico- medullary region after cisplatin treatment (Fig. 12) compared to the normal (Fig. 8). Distension of the capillaries is very noticable after three days of cisplatin treatment (9mg/kg) in the cortical (Fig. 13- 14) and medullary regions (Fig. 12,14). Cisplatin causes an abnormal swelling of mitochondria with large matrix and inner membranes completely destroyed (Fig. 15). Medullary region is significantly atrophied after three days (Fig. 14) of cisplatin treatment with mitochondria being most effected as in the cortex and their number is also reduced. Norepinephrine containing granules show abnormal swelling (Fig. 16). 15 Vagotomy: After bilateral vagotomy there was observed a hypertrophy in the adrenal gland morphology (Fig. 17) with an increase in the lipid droplets of both the zona reticularis and the zona fasciculata cells (Fig. 18). Accumulation of these lipids after vagotomy is taken as an indication of steroid hormone precursor accumulation, in turn reflecting a depression in the functional activity of the zona fasciculata and reticularis. The medullary region is also hypertrophied (Fig. 17) within 3 days of vagotomy. In the medulla, norepinephrine vesicles are abnormally enlarged and some vesicles are almost empty of their contents. Mitochondria are swollen and are without any cristae ( Fig. 19). 16 op >Eouomm> 030L000 Emzm m 2 0.5.0 >Eouomm> n v m m 20.. 0c_.0m .mEo: 0....30 + 030.000 82.0. .2280 w v m m 3. .9.me8 500.006 + >Eouom0> m e m m 2.8 $32820 53.28 so. .6 .5 98 a8 + E .9358 5585 4 .V m m .803 :5 too; 9.. 05.00 .085: 0....30 c. E. .3853 55.3.0 m w m m 05.00 3.0% 83.0 3.. ".228 N v m m 05.00 .083: 2:03 :36 c. a. 60.388 53305 F £80 «an m was m 3:05800323... 230 mucmEumoCu wasp—m 3.: was...“ So u 6900p 3:05.090 05 9:265 ; 030... 17 Fig. 1. Graph showing inhibition of gastric emptying by more than 95% after (A) cisplatin treatment (9mg/kg) compared to (Q) normal or (.) cisplatin and vagotomy. The gastric emptying was half as affected after cisplatin and vagotomy compared with cisplatin alone. GASTRIC EMPTYINGI%I 20 ‘10 {‘4 18 \+ 1 3 ' 5 DAYS AFTER CISPLATIN TREATMENT l9 mg/kgl 19 Fig. 2. Light micrograph of a cross section through the cardiac portion of the stomach from a normal rat stained with hematoxylin. M, muscularis mucosa; C, circular muscle layer; L, longitudinal muscle layer. X 520, Bar=20um. 20 21 Fig. 3. Light micrograph of a cross section through the cardiac portion of the stomach after 3 days of cisplatin treatment (9mg/kg) showing small erosion in mucosal layer (arrow). M, muscularis mucosa; C, circular muscle layer; L, longitudinal muscle layer. X520, Bar=20um. 22 23 Fig. 4. Light micrograph of a cross section through the cardiac portion of the stomach of a rat after 5 days of cisplatin treatment (9mg/kg). Note the ulcer extending into deeper layers of mucosa (arrow). M, muscularis mucosa; C, circular muscle layer; L, longitudinal muscle layer. X520, Bar=20um. 24 25 Fig. 5. Light micrograph of a cross section through the cardiac portion of the stomach of a rat after 8 days of cisplatin treatment (9mg/kg). Note the loose intensely staining blood cells in the outer most mucosal layer (arrow). M, muscularis mucosa; C, circular muscle layer. The longitudinal muscle layer is not pictured in the field of view. X330, Bar=30um. 26 27 Fig. 6. Light micrograph of a cross section (10pm) of the adrenal gland from a normal rat showing normal morphology of the gland after staining with hematoxylin. C, cortex; M, medulla; cap, capsule; 1, zona glomerulosa; 2, zona fasciculata; 3, zona reticularis. Arrows point toward the normal capillaries in the gland. X192, Bar=0.5mm. 28 29 Fig. 7. Light micrograph of a fresh frozen section of the adrenal gland from a normal rat stained with Sudan black 8, showing the distribution of lipids in the zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR). Arrows point to the lipid granules. X1300, Bar=10pm. 30 9:. ,kzlwi... .. . L» ,, 1:. c m , . .i,.,..,.:,. H. 1 .I.» , 31 Fig. 8. Light micrograph of a fresh frozen section (10pm) of cortico- medullary region of the adrenal gland from a normal rat stained with Sudan black B. C, cortex; M, medulla; c, capillary. Arrows point to the lipid vessicles. X1300, Bar=10pm. 32 wag, a fir:- ‘ m an? _ flarew:;:.,. 7 n. 1...: 33 Fig. 9. Electron micrograph of a section through the cortical region of an adrenal gland from a normal rat showing the distribution of the lipid granules (arrows). Note the lamellae in one of the lipid vesicles (heavy arrows). N, nucleus. Arrowheads point toward mitochondria. X9540, Bar=1um. 34 35 Fig. 10. Electron micrograph of a section through the medullary region of an adrenal gland from a normal rat showing the epinephrine (E) and norepinephrine granule (NE) containing cells. N, nucleus. Arrowheads point to the mitochondria. X8850, Bar=1um. . I“? “It, . I: (a. d. i , b _ l. 6 I.- 36 37 Fig. 11. Light micrograph of a fresh frozen section (10 pm) of the cortical region of an adrenal gland after 3 days of cisplatin treatment (9mg/kg) stained with Sudan black B. Note the intensely stained but less number of lipid globules in various regions of the cortex compared with a similar section from a normal gland (see Fig. 7). ZG, zona glomerulosa, ZF, zona fasciculata, ZR, zona reticularis. Arrows point toward lipid droplets. X1300, Bar=10um. 38 39 Fig. 12. Light micrograph of a fresh frozen section (10pm) of the cortico-medullary region of the adrenal gland after 3 days of cisplatin treatment (9mg/kg) stained with Sudan black B. Cisplatin treatment induces complete absence of lipids from cortico- medullary region. Note the distension of capillaries (c) so prominent in the medullary region. C, cortex; M, medulla. X1300, Bar=10um. 40 41 Fig. 13. Light micrograph showing the distension of the capillaries (arrows) in the zona glomerulosa (2G) and zona fasciculata (ZF) of an adrenal gland after 3 days of cisplatin treatment (9mg/kg) stained with hematoxylin. X520, Bar=20um. 42 43 Fig. 14. Light micrograph of a cross section (10 pm) of an adrenal gland stained with hematoxylin. Cisplatin treatment (9mg/kg) induces significant telangiectasia in the capillaries (arrows) of the cortex (C) and the medulla (M) just after 3 days . Note also the atrophy of the medulla. ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis. X192, Bar=0.5mm. . I (W rt .. - . ‘ . ' . "\‘E ' , . f p n "v I, '. ‘ ' _ . . . v . _ - . . ‘ 4— I." » "' . 41 (71‘. ‘n'h 2:73:46??? . I. 5.! I I. .l‘; i .1 l l ;1 rig-A: i 3' as. I I I vi 5. -T (9;. T .‘l, -. i; '5» 45 Fig. 15. Electron micrograph of a section through the cortical region of an adrenal gland from a 3 day cisplatin treated (9mg/kg) rat. Note cisplatin treatment induces abnormal swelling of mitochondria (arrowheads) with large matrix and distruption of the inner membranes. N, nucleus. Arrows point to the lipid granules. X9540, Bar=1 .5um. 46 47 Fig. 16. Electron micrograph of a section through the medullary region of an adrenal gland after 3 days of cisplatin treatment (9mg/kg). Note cisplatin treatment induces an abnormal swelling of mitochondria (arrowheads) and norepinephrine (NE) vesicles (arrows). N, nucleus. X21280, Bar=0.5um. 48 49 Fig. 17. Light micrograph of a section of an adrenal gland from a vagotomized rat (3 days) showing hypertrophy of the medulla (M). Arrows point toward the distended capillaries in the zona reticularis . C, cortex, ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis. X192, Bar=0.5mm. 50 £11 a. . w...~ p~. 51 Fig. 18. Electron micrograph showing the cells of the cortical region in the vagotomized rat (3 days) induces an increase in the number of the lipid droplets (arrows). X9540, Bar=1um. 52 53 Fig. 19 Electron micrograph of a section from the medullary region of an adrenal gland from a vagotomized rat (3 days). Vagotomy induces abnormal swelling of (arrowheads) mitochondria (with distruption of internal organization) and norepinephrine (NE) vesicles. N, nucleus; X21630. Bar=0.5um. 54 55 DISCUSSION Various chemotherapeutic and noxious agents like aspirin, mercuric chloride and alcohol are known to induce gastric lesions leading to ulceration.24 In order to develope treatment for gastric ulcers, it is essential to learn the causes or mechanisms of induction of such ulcers. Different animal models have been developed through pyloric ligation, stress induction through restraint, cold and use of agents like alcohol, tobacco smoke, bile acids, corticosteroids, lysolectins and aspirin.38-39 All disrupt the continuity of the epithelial surface inducing increased shedding of the cells and possibly contributing to the development of the gastric mucosal erosions or ulcers- Various chemical agents affect different regions of the gastro-intestinal system. Some induce gastric lesions in the cardiac portions, others the pyloric region, and yet others the duodenal portion39 of the gastrointestinal system“. A variety of measures, mostly poorly understood, have been employed to protect the gastric mucosa in animal models.41 Adequate mucosal blood flow, normal levels of mucosal cells with Carbonic anhydrase, prostaglandins, sulfhydryl-containing drugs S6 (dimercaprol, cysteanine, dimercaptosuccinic acid), nonprotein thiols, natural amino acids (L-cysteine, methionine) and epidermal growth factor, are all responsible for protection of mucosal cells against erosive acid-peptic secretions.4~”--44 Therapeutic agents that stimulate mucus output seem to help in healing peptic ulcers.45 Cisplatin, a broad spectrum potent chemotherapeutic agent, causes gastric lesions in the cardiac portion of the stomach in Wistar rats within 3 days of its use at a chemotherapeutic dose of 9 mg/kg body weight; hemorrhagic gastric ulcers exist after day 5. Cisplatin treatment induces a release of corticosterones from the zona fasciculata and the zona reticularis while the glucocorticoids in the zona glomerulosa remain uneffected. Corticosterones have been shown to be essential in the induction of gastric lesions requiring the parasympathetic nervous system.24 Adrenalectomy completely prevents the inhibition of gastric emptying by sauvagine and CRF.46- 43 Cotricosterone administration, however, causes these peptides to recover their inhibitory activity thus demonstrating the important role of corticosterone in gastric emptying and induction of ulcers. Some chemical stressors like mercuric chloride have been shown to increase adrenal and plasma corticosterone levels in a dose and time dependent fashion49 therefore stimulate ulcer formation. In some cases, vagotomy is effective in controlling the release of corticosterone and mitigating the gastric ulceration. Subphragmatic vagotomy has been utilized as a method to control the gastric emptying and gastric lesions after various drug treatments, i.e., 57 Bombesine,50-51 Dermorphine52 or both in animalsSl'S4 and in clinical situations.55-56 It has been demonstrated that after vagotomy, the adrenal cortex shows hypertrophy and there is an accumulation of lipids which has been interpreted as a morphological sign of depressed activity.57 The medullary portion of the adrenal shows hypertrophy after vagotomy. There is an initial decrease in the number of secretory granules by Day 3 and subsequently, norepinephrine vesicles show a swelling with loss of of their contents. Various drugs are known to induce destructive, degenerative (senile nodular hypertrophy, telangiectasia, focal fatty deposits) changes in the adrenal gland.58 Cisplatin causes severe telangiectasia, loss of lipoidal vesicles from the zona fasciculata and the zona reticularis cells reflective of the depression of corticosteroids . In cisplatin treated rats, the medullary region of the adrenal undergoes an atrophy and also the vesicles in the medulla show a similar swelling and loss of contents (after cisplatin treatment of 3 days) as in vagotomized animals. The mitochondria of adrenal cells are most affected by cisplatin treatment. These are abnormally swollen with an enlarged matrix and their inner membranes are completely disrupted. Similar changes in the adrenal mitochondria have been described after treatment with the anticonvulsant, aminoglutethimide.59 Hydroxylated cisplatin uncouples oxidative phosohorylation in isolated mitochondria.60 Mitochondria seem to be the initial target for cisplatin and have been implicated in some toxicities, especially 58 nephrotoxicity where energy dependent membrane filtration is involved.6‘-63- Steroid hormones in the adrenal are synthesized by intimate contact between smooth endoplasmic reticulum, mitochondria and lipids which facilitates the exchange of enzymes and intermediate products in their synthesis.37 If the mitochondria are damaged then probably the synthesis of corticoids is also affected. Cisplatin is known to cause hypocalcemia and hypomagnesemia5-7 Calcium is required for the release of acetylcholine from the synaptic vesicles by their fusion with the axonal ending membrane.64 Hypocalcemia is probably the reason for the inhibition of acetylcholine release from the neural endings of the gastric smooth muscle and the pyloric sphincter, although the amount of acetylcholinesterase seems to be unaffected by cisplatin. Inhibition of acetylcholine release would lead to a chronic/spasmogenic effect on the pyloric sphincter thus inhibiting gastric emptying resulting in bloated stomachs. The starved, cisplatin-treated animals did not develop stomach bloating or ulcers. Bloating of the stomach in this case seems to be a requisite for ulceration. Bloating of the stomach has been associated with increased gastric acid and gastrin/pepsin secretion.65 Calcium is a necessary component in the process of gastric secretion66 and may influence parietal cell stimulus-secretion coupling.67-68 The common denominator for acetylcholine-induced gastric acid response is calcium, through enhanced calcium influx across the plasma membrane. Extracellular calcium seems to 59 control the secretory response to acetylcholine and pentagastrin which is decreased in its absence.68 Distension stimulus is known to involve some well recognized pathways including vagal reflexes, and intramural reflexes. In addition, there may be direct mechanical effects on the epithelium that enhance gastric acid secretion without the activation of nerves or known physiological ligands like gastrin , histamine, or acetylcholine.69-7O Acetylcholine, gastrin and histamine stimulate K+/H+ ATPase (parietal cells) to induce acid secretion. Acetylcholine exerts an acute and direct stimulative effect on the adrenocortical function by stimulating the production of 17- hydroxylated corticosteroids, while atropine blocks the acceleration of steroidogenesis by acetylcholine?"72 Acetylcholine transmits the nerve excitation and is a direct stimulant of the adrenal cortex in terms of hormone output. I Adrenalectomy, but not vagotomy, reverses the worsening effect of Ca2+ blockers on ethanol-induced gastric lesions in rats.48 Compounds with calcium blocking properties- verapmil, nifedipine and WY47037 have been used to reduce stress-induced ulceration.4s Ethanol induced ulceration or its aggravation by verapmil is antagonized by calcium gluconate.73 Again, adrenalectomy antagonizes ethanol lesion aggravation by nitrendipine or verapmil when it becomes gastroprotective, while dexamethasone restores the lesion-enhancing effects of both Ca2+ channel blockers. Ulcers have also been attributed to histamine and 60 5’-HT(5-hydroxytryptamine) released chiefly by stomach wall mast cell degranulation. Verapmil interferes with this degranulation and may be the way it acts as a protective agent.74 Stressful life events cause increased acid'secretion, ulceration or symptoms.75 Ulcers, if caused by gastric acid, may be prevented by antacids, anticholinergic agents or vagotomy.75'79 Yohimbine (alpha- blockers), propranolol, MJ 1999, alprenolol (beta-blockers) do show inhibition of ulceration and hemorrhage.80 Prostaglandins (PGE,PGE2) as well as certain methyl analogs of PGE2 inhibit gastric secretion in animals and humans,3l-83 prevent ulcer formation in animals and accelerate ulcer healing in humans.34-85 Most nonsteroidal anti-inflammatory compounds like aspirin, indomethacin, phenylbutazone, naproxene, ibuprofen can produce gastric damage in animals,35-87 and humans.33-90 Presence of acid plays a crucial role. Seratonin and seratonin 5-HT3 receptors also seem to play an important rule in the way chemotherapeutic agents produce nausea and vomiting.”-16 0f the antiemetic agents, ondansetron, a selective S-HT3 receptor antagonist has been proven to be effective in controlling nausea and vomiting in both laboratory animalsl7and patients.14,13-20.91 Release of large amount of 5-HT from enterochromaffin cells during the first 6 h of cisplatin treatment is thought to be a crucial factor in the initiation and maintenance of the vomiting reflex.l4.9-2 0ndansetron has proven to be superior as a S-HT3 receptor blocker to metaclopramide.” In our present studies, 61 accumulations of gastric acid through non-emptying of the stomach contents, or both, is hard to establish. We did not monitor the blood flow, but decresed blood flow has been implicated in the rse of gastric acid.93 Pharmacologically induced increases in mucosal circulation has been shown to prevent ulceration induced by bile salts and ' shock, thus the role of the circulation sweeping away excessive quantities of acid becomes very clear.94 Calcium supplements seem to alleviate gastric bloating and prevent gastric lesions.23 Calcium is involved in many diverse physiological processes. Calcium is responsible for initiating muscle contractions45v synthesis and secretion of transmitters, release of hormones95. increased enzyme activity and membrane permeability.96 Calcium (calcium gluconate or calcium Chloride) have been shown to ameliorate the nephrotoxicity and gastrointestinal toxicity due to cisplatin treatment.23 Calcium gluconate induces a transient rise in CaZ+ concentration in the glandular muscle layer and serum,73 while preventing any ulceration. Calcium probably is responsible for the release of acetylcholine from the vagus nerve fibers include relaxation of the pyloric sphincter and contraction of the gastric smooth muscles. In vitro fundal strips from cisplatin-treated rats have been demonstrated to be hypercontractile to acetylcholine and serotonin in calcium-free Tyrode solution, but contract normally in Tyrode solution with calcium , clearly demonstrating the important role of altered 62 calcium homeostasis in cisplatin treated smooth muscle contraction.97 So far it seems that every effort has been directed towards controlling the symptoms of various toxicities associated with cisplatin treatment23 and not the causes of these toxicities. Our studies clearly show that hypocalcemia and depletion of membrane associated calcium as being responsible for all the toxicities due to cisplatin treatment. It is important to note that simple calcium supplements given before cisplatin administration are used to control various toxicities, especially vomiting and nephrotoxicity. In conclusion, the induction of gastric lesion in cisplatin-treated rats seems to be due to hypocalcemia. This results in the inhibition of acetylcholine release, which in turn causes spasm of the pyloric sphincter. This effect on the pyloric sphincter causes bloating of the stomach by inhibiting gastric emptying, thereby producing excessive accumulation of gastric acid , gastrin and pepsin. These substances probably are responsible for the mucosal injury leading to ulceration. Calcium injections seem to elevate the serum calcium levels which in turn influence the release of acetylcholine. Acetylcholine reverses the pyloric sphincter stenosis and induces gastric smooth muscle contractility, stimulating gastric emptying and gastric blood flow. 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