Radiation and indomethacin prostaglandins, and motility ROBERT W. ANDREW J. Department of and Center for
effects on morphology, in dog jejunum
SUMMERS, CLYDE E. GLENN, FLATT, AND AHMED ELAHMADY Internal Medicine, Veterans Administration Medical Center, Digestive Diseases, University of Iowa College of Medicine, Iowa City, Iowa 52242
SUMMERS, ROBERT W., CLYDE E. GLENN, ANDREW J. FLATT, AND AHMED ELAHMADY. Radiation and indomethacin effects on morphology, prostaglandins, and motility in dog jejunum. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G145-GM, MC-Irradiation can have a profound effect on intestinal motor activity. Previous studies have suggested that prostaglandins may play some role in radiation-induced enteritis. The present study investigated the effects of abdominal Xirradiation with or without indomethacin treatment on jejunal myoelectric activity and prostaglandin synthesis by measuring the prostaglandin content of mesenteric arterial and venous plasma and in the intestinal lumen in dogs. After X-irradiation, venous concentrations and arteriovenous concentration differences of prostaglandin (PG)E,, PGF2,,, and 6-keto-PGF1,, increased markedly. The increased venous concentrations were in part attributable to increased mucosal and/or submucosal synthesis by inference from increased concentrations of these metabolites assayed from the jejunal lumen. Irradiation produced histological damage to the mucosa and submucosa and abnormalities in the migrating motor complex, jejunal slow waves, and a decrease in spike burst activity. Inhibition of prostaglandin synthesis by treating the animals with indomethacin reduced the severity of illness, the histological injury, and changes in myoelectric activity induced by irradiation. Such treatment should be evaluated further to treat patients exposed to large doses of irradiation. ionizing
radiation;
arachidonic
acid; intestinal
smooth muscle
IN ADDITION to its well-known effect on the intestinal mucosa, X-irradiation has been shown to cause profound changes in the functions of intestinal smooth muscle (9, 15, 24, 25, 31, 34). Our own recent study (33) demonstrated progressive inhibition of motor activity after radiation injury. The mechanisms involved in these changes are largely unknown, but they might involve arachidonic acid metabolites. Radiation increases prostaglandin synthesis in some organs; the tissues of the gastrointestinal tract have the capacity to synthesize prostaglandins from arachidonic acid, and their rate of synthesis could be increased by X-irradiation. Exogenous prostaglandins have been shown to affect both muscle function and neural activity in the gut (3, 4, 21, 26). Therefore, endogenously synthesized prostaglandins may be involved in the motility changes caused by irradiation. Two preliminary studies (6, 10) have suggested that prostaglandin synthesis is increased in the jejunum after irradiation. In both of these studies, however, prosta-
glandins were measured by a nonspecific bioassay. Inhibitors of prostaglandin synthesis have been reported to restore to normal the decreased intestinal transit time found in irradiated animals (6) and to relieve radiationinduced gastrointestinal symptoms in patients (19, 20). It is not established, however, whether prostaglandin metabolism in the intestine is altered by irradiation, whether irradiation effects on other tissues or organs alter prostaglandin concentrations in the blood and so affect intestinal function, or whether altering prostaglandin synthesis would reduce radiation effects on intestinal smooth muscle function. The present study was designed to explore the role of prostaglandins in canine jejunal muscle before and after irradiation. To determine the effect of irradiation on circulating prostaglandins, arterial and venous blood samples were collected from the vasculature of the intestine and analyzed for prostaglandins by radioimmunoassay (RIA). Luminal prostaglandins were measured to assessthe role of the mucosa and submucosa in prostaglandin metabolism after irradiation. Finally, another group of animals was treated with indomethacin before and after irradiation to determine whether reduced prostaglandin synthesis might reduce or prevent changes in histological morphology and motor function induced by X-irradiation. The results of these studies help to explain some of the abnormal smooth muscle motor functions that follow abdominal irradiation and elucidate the role of prostaglandins in this process. METHODS Preparation of animals. Mongrel female dogs weighing 18-25 kg were anesthetized with intravenous pentobarbital sodium (30 mg/kg) after the abdominal wall was shaved and prepped. Through a midline laparotomy incision, seven bipolar silver electrodes were sutured to the serosal surface of the jejunum at 3-cm intervals beginning 10 cm distal to the ligament of Treitz. A 16-Fr polyethylene Levin-type feeding tube was also sutured into the second portion of the duodenum for administration of water or formula if needed. Teflon-insulated 32-gauge stranded wire led from the electrodes to a 14-pin connector implanted in the animal’s abdominal wall. The proximal end of the feeding tube was tunneled through the abdominal wall muscle and subcutaneous tissue to exit along the lateral portion of the thoracic cage. Seven to
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G146
GI PROSTAGLANDINS
ten days were allowed for recovery before recordings of myoelectric activity were initiated. Irradiation procedure. Animals to be irradiated were anesthetized with intravenous pentobarbital and placed on an X-ray table. A GE Maxitron 250 X-ray machine was used to deliver the dose with 0.25 mm copper and 1 mm aluminum filters at 250 kVp, 30 mA, SSD 17 in. The thorax and pelvis were shielded with lead (thickness -0.5 cm), and the animal was exposed to one-half of the total dose lying on one side. Then the animal was turned and given the second half of the dose on the other side. Dogs were exposed to 1,250 rads as measured with a Victorean R meter. A preliminary investigation with thermoluminescent dosimetry chips placed in the jejunum of dog cadavers showed a dose of 938 cGy was delivered to the intestine by this procedure; chips placed under the shields received ~2.5 cGy absorbed dose. Six control dogs were anesthetized but not irradiated. Six dogs received irradiation as described above and six additional dogs received daily indomethacin 3 days before and 4 days after irradiation. Animals were given indomethacin (1 mg/kg iv) three times daily. Recording and analysis of myoelectric activity. Animals were fasted overnight for 18 h before recordings of myoelectric activity were made. The dogs were placed in a nylon-mesh sling and connected to a recorder using cables attached to the plug previously fixed in the lateral abdominal wall. The recorder was a Beckman R-611 physiological recorder (Beckman Instruments, Fullerton, CA) outfitted with “universal” 9853A couplers using a low-frequency cutoff of 5.3 Hz. The signals were routed to a rack of analog band-pass filters with corner frequencies of 10 and 30 Hz and from there to an MINC-11 computer (Digital Equipment, Maynard, MA) for sampling, storage, and analysis. Control recordings were made before irradiation or indomethacin treatment in all groups, daily in both groups that received irradiation, and just before irradiation in the group that received 3 days of indomethacin before irradiation. Recording periods consisted of 3 h of fasting activity followed by 1 h of fed activity, which was induced by infusing 120 ml of Cornpleat-B (Doyle Pharmaceutical, Minneapolis, MN) meat-based formula into the duodenal tube over 30 min. Modified electrical signals from the band-pass filter were digitized at an analog-to-digital conversion rate of 60 Hz and stored on hard disk during the experiment. Programs to detect spike bursts in all electrodes and to determine the number of bursts per minute (frequency), the duration (seconds), and length of propagation were employed to analyze the myoelectric data. Details of the methodology are provided in an earlier publication (33). Radioimmunoassay of prostaglandins. Studies were performed in the sham-irradiated dogs, the irradiated dogs, and the irradiated dogs treated with indomethacin at 96 h after irradiation. The animals were again anesthetized with intravenous pentobarbital, the abdomen was opened at the midline, and the jejunum was exteriorized and kept moist with physiological saline at 37OC. The six control dogs were treated in the same way. Two incisions, each -2 cm in length and 80 cm apart, were made in the midjejunum. A piece of plastic-coated wire was threaded into the lumen between these incisions, a
AFTER
X-RADIATION
dialysis bag (48 cm long) containing 90 ml of aqueous dialysis solution (5.6 g/l NaCl, 2.24 g/l KCl, 2.52 g/l NaHCO:J was connected to the wire, and the bag was pulled into the lumen (27). After 1 h, the bag was removed. Pre- and postintubation weighing showed negligible fluid loss from the bag. The dialysis fluid was frozen at -70°C until analyzed for prostaglandin (PG)Ez, PGF2,y, and 6-keto-PGF1,, content by RIA. [Note: 6-ketoPGF1,, is the inactive metabolite of prostacyclin, a very short-lived product of arachidonic acid metabolism.] Next the dogs were heparinized by intravenous injection of heparin sodium, and 9 ml of blood were collected in tubes containing 1 ml of Krebs solution and 100 mg indomethacin. Blood was collected from a mesenteric artery and a vein that supplied the irradiated intestine. Blood samples were kept on ice until they were centrifuged at 3,000 g. The plasma was separated from the cellular elements and stored at -70°C until the PGEz, PGFB,,, and 6-keto-PGF1,, content could be assayed by RIA (18). To verify the entry of luminal prostaglandins into the dialysis tubing, an open-ended dialysis bag was placed in a siliconized beaker containing 20 ml of dialysis buffer and 5 ml of buffer in the bag. Tritiated PGE2, PGFZ,,, and 6-keto-PGF,,, were then added to the beaker, which was kept at 37°C. Aliquots were withdrawn from the bag at 0, 15, 30, 45, 60, 90, and 120 min after addition of the standards and analyzed by RIA. After 60 min, all samples equilibrated at -90% of the total activity. Histology. Finally, a 4-cm segment of jejunum was removed from each dog, opened along the mesenteric border, stretched, pinned and fixed in buffered Formalin, and prepared for histological study. After these procedures, the animals were killed. Tissue was embedded in paraffin, sectioned, and prepared for staining with hematoxylin and eosin using standard methods. Slides from controls, irradiated, and irradiated plus indomethacintreated animals were coded and read blindly. Statistical analysis. Data were analyzed by the SAS general linear models procedure and the Wilcoxon rank sum test and the Kruskal-Wallis one-way analysis of variance (30). The nonparametric rank tests were used in preference to parametric tests, as scatter in the data gave large standard deviations and the data did not conform to a normal distribution. RESULTS
General observations. Animals were clinically unaffected by indomethacin treatment before irradiation. They ate normally and had normal bowel movements. On the second day after irradiation, animals not treated with indomethacin began to exhibit signs of irradiation sickness. They refused to eat; all had frequent emesis and began to develop bloody diarrhea. This became progressively more severe on days 3 and 4. The animals treated with indomethacin were not nearly as ill. They ate readily, and only two of six dogs vomited after eating, and they only exhibited mild diarrhea without gross blood. Unfortunately, these observations were only qualitative. Histology. The major histological changes from normal
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GI PROSTAGLANDINS
in irradiated animals were detected in the mucosa (Table 1). These consisted of vacuolization and atypia of the epithelial cells with a paucity of goblet cells. There was also moderate disruption of the villous architecture associated with superficial ulceration, loss of villi, crypt cell necrosis, and dilated glands (Fig. 1). Inflammation, predominantly mononuclear cell infiltrates and some segmented neutrophils and eosinophils, was present in the lamina propria and extended to a lesser degree into the submucosa near sites of severe mucosal damage. Changes in the muscularis propria and the myenteric plexus were minimal to absent except for some mild TABLE 1. Histological findings in controls, irradiation, and irradiated animals treated with indomethacin
Control (n = 6)
938 cGy (n = 6)
938 cGy + Indo (n = 6)
Mucosa Epithelial vacuolization 0 2.1 0.9 Epithelial atypia 0 2.6 1.1 Goblet cell (paucity) 0 3.1 1.1 Superficial ulceration 0 2.0 0.7 Crypt cell necrosis 0 1.9 0.6 Glandular dilatation 0 1.9 0.6 Villous architecture 0.2 1.9 1.4 disruption Inflammation 0 2.0 0.9 Submucosa Edema 0 1.8 0.6 Inflammation 0 0.3 0 Muscularis propria Inflammation 0 0.1 0 Vasodilatation 0 0.9 0 Myenteric plexus 0 0 0 inflammation/damage Total score 0.2 20.6 7.9 Grading of jejunal slides was done blindly according to the following scale: l+, mild, 2+, moderate; 3+, severe; if no change, animal was scored 0. Scores represent mean values from all of the dogs in the group. Individual scores for each finding were added and divided by number of animals in group analyzed. Using the Kruskal-Wallis oneway analysis of variance, total scores all differed from one another (P < 0.001). Indo, indomethacin treatment.
CONTROL
vasodilatation in the muscle layer. Histological abnormalities were more severe in the animals treated with irradiation alone. In only one animal treated with indomethacin, changes could not be distinguished from irradiated animals. The rest of the indomethacin-treated group consistently had only mild abnormalities (Table 0 Myoelectric actiuity. In the control fasting state, migrating myoelectric complexes (MMCs) were present in six of six animals in each of the groups (sham irradiation, irradiation only, and irradiation plus indomethacin). Four days after irradiation, none of the six dogs receiving no indomethacin exhibited MMCs during the 3-h recording period; however, they were present in all of the indomethacin-treated dogs. The absence of MMCs was statistically different from controls in the nonteated dogs (P < O.OOl), but the frequency per 3 h in the indomethacin-treated dogs (2.1 + 0.4) was not different from controls (2.5 ? 0.3). During the control period in all groups, slow waves were regular and demonstrated almost no variation in morphology (Fig. 2), but in the animals receiving irradiation alone, abnormalities in slow waves were noted. These consisted of variation in both morphology and irregularity in rhythm in all six dogs (P < 0.001). By morphology we mean the shape or configuration of the wave. The variability of the slow waves occurred in both the fasted and fed states, but was most clearly seen when spike bursts were absent, as in phase I. In three of six, uncoupling of slow waves was observed (P < 0.05). Changes were most severe on days 3 and 4 after exposure. In the indomethacin-treated group some abnormalities in slow waves during phase I of the MMC were observed in only 33% of the animals (P > 0.05), but the changes were much less severe and no evidence of uncoupling was recorded (Fig 2). Examples of recordings on day 4 after a meal are shown in Fig. 3. Before irradiation, spike bursts occurred irregularly but were evenly distributed. After irradiation, spike activity was reduced overall with occasional periods of intense activity. In irradiated dogs treated with indo-
IRRADIATION
FIG. 1. Representative histological sections from nonirradiated (middle) and with (ri&t ) indomethacin treatment.
G147
AFTER X-RADIATION
control dogs (left) and irradiated
IRRADIATION & INDOMETHACIN dogs without
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G148
GI
PROSTAGLANDINS
10 set
I-
C FI(G. 2. Recordings
from adjacent electrodes during phase I fromnonirradiated cant rol dogs (A), irradiated dogs without indomethacin treatment (H), and irradiated dogs treated with indomethacin (C). In A and C’, slow waves have uniform morphology and regular rhythm and are coupled. In H, morphology is variable, rhythm is irregular, and slow waves are not coupled.
methacin, spike activity was preserved, but there was a tendency for spike bursts to occur in clusters. The quantitative findings recorded 30 min after a meal are shown in Figs. 4-6. No significant changes in spike burst frequency, duration, or length of propagation were observed 1 day after irradiation, but at 4 days, all of these parameters were significantly reduced in the group treated with irradiation alone. However, there were no significant changes from control in any of the parameters in the group treated with indomethacin before or after irradiation. Arterial and venous plasma prostaglandin concentrations (Table 21. The venous plasma concentrations of all prostaglandins was greater than arterial concentrations in control animals, with venous plasma showing 1,060% more PGE2, 240% more PGFxCy,and 270% more 6-keto-
AFTER
X-RADIATION
PGFI,, than arterial plasma. Irradiation significantly increased the PGE, and PGF2,, content of arterial blood. This could have been due to increased prostaglandin synthesis by the irradiated blood vessels but probably reflects increased systemic concentrations. Venous blood showed a very large increase in PGE2, PGF2,?, and 6keto-PGF,,, after irradiation, although the concentrations varied considerably. The change in the postirradiation prostaglandin arteriovenous concentration differences increased significantly for all three prostaglandins. One pair of blood samples was inadvertently lost. Indomethacin effectively inhibited the rise in all three prostaglandins in arterial and venous blood so that the arteriovenous concentration differences were not statistically different from control values. Luminal prostaglandin concentration (Table 2). All three measured prostaglandins were found in the lumen of controls. The concentrations of all prostaglandins increased greatly (X0-fold) after irradiation. The percentage increase was greatest for PGE,. The increase in prostaglandins after irradiation was largely prevented by treating with indomethacin so that the concentrations were not significantly different from controls. DISCUSSION
The results confirm the capacity of the normal intestine to synthesize prostaglandin. Mesenteric venous concentrations of PGE2, PGFzCy,and 6-keto-PGF,,, were 3to IO-fold higher than arterial concentrations in control animals. Similar metabolites were found in this study and in previous investigations (5, 17, 28, 33) of prostaglandin synthesis in normal canine and other species, although relative levels were different. In the nonirradiated jejunum, luminal concentration of PGE2, PGF2,,, and 6-keto-PGF,,, were consistently higher than venous concentrations. This suggests that some synthesis of prostaglandins takes place in the mucosa or submucosa and is not merely “leaked” from the blood vessels. The marked increase in the arterial and venous con-
(-,,
FIG. 3. Typical recordings from fed animals representing each group. Four days after irradiation, spike burst activity is reduced overall, but some intense activity is present. In irradiated dogs treated with indomethacin, spike activity is similar to that seen in controls, with the exception of a tendency toward clustered activity.
CONTROL
IRRADIATION
IRRADIATION & INDOMETHACIN
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(:I
PROSTAGLANDINS
AFTER
G149
X-RADIATION 10
20 lz0 18 I
938 cGy
r m
938 cGy+lOmg/kg
INDOMETHACIN
938 cGy 938 cGy+l
Omg/kg
INDOMETHACIN
0
1
CONTROL
DAY
1
DAY 4
CONTROL
DAYS AFTER IRRADIATION HG. 4. Spike burst activity before and after irradiation exposure untreated dogs and in irradiated dogs with and without indomethacin treat merit. At dq -1, spike burst frequency was significantly reduced dogs not treated with indomethacin (*,I < 0.05).
in in
DAY
1
DAY 4
DAYS AFTER IRRADIATION HG. 6. Length of migration (in cm) was reduced in irradiated dogs not t,reated with indomet hacin (*II < 0.05). Mean length of migration was similarly less at 4 days in indomethacin-treated dogs, but variance was greater so that reduction was not statistically significant.
2. Mean plasma prostaglandin concentrations in mesenteric blood and jejunal lumen
TABLE am
938
cGy
938
cGy+l
Omg/kg
INDOMETHACIN
I’rost aglandin Arterial PGE, I’GFZ,, 6-keto-PGF,,, Venous PGE, PGF?,, 6-keto-PGF,,, A-V Difference PGE, CONTROL
DAYS
DAY
AFTER
1
DAY
4
PGF?,,
IRRADIATION
k-It;. 5. Spike burst duration in dogs not treated and treated indomet hacin before and after irradiation exposure. Spike burst t ion was significantly reduced at 4 days in dogs not treat,ed indomet hacin (*I’ < 0.05).
with durawith
centrations suggests that jejunal synthesis of prostaglandins is increased by irradiation. The elevated arterial plasma concentrations of prostaglandin after irradiation could be due in part to synthesis of prostaglandins at other sites. However, the increase in venous prostaglandin concentrations and the large arteriovenous concentration differences support increased local synthesis of prostaglandins in the jejunum. Decreased catabolism of prostaglandins is possible, but unlikely, because the rise in prostaglandins was prevented by treating with indomethacin, a known inhibitor of prostaglandin synthesis. Radiation has previously been shown to increase synthe-
6-keto-PGF,,, Jejunal lumen PGE, PGF?,, 6-keto-PGF,,,
Cant rol
Irradiation
(n = 6)
(n = 5)
9&l (S-29) 1:3t:3 (7-22) 198t23 (X38-297)
Irradiation + Indornet hack (n = 6)
44tfi” (:33-x3) 30*4* (25-40) :309t119 (99-762)
38t6 (16-54) 1853 (10-32) 83+19 (36-144)
l,828+1,105t (320-6,152)
93k 19 (46-147) t Y-t V+‘j (18-59) 5Z7k62 (380-l ,944)
(10%2,016) 8,76:3+4,005t ( 1,929-24,375)
204t 133 (40-867) 42t7 (24-68) 727+199 (203-1,:302)
85t18 (:38-161) 19t7 (O-45) 339t69 (220-614)
1,784+1,102* (280-6,098) 64%:358* (851,976) 8,454&4,012* ( 1,719-7,09S)
167t130 (:3-812) 24t8 (S-54) 644+198 (149-1,158)
428*7S (24%6:37) 4x3+40 (:38011,044) 161&21 (94-229)
16,589+6,009* (:3,012-40,250) 2,943a750* ( 1,508-6,238) 678t224* (2%1,699)
873t661 (8-4,150) x+22 (8-140) 25-t 10 (6-72)
677t360t
Values represent mean concentration in picograms per milliliter SE/( range). * P < 0.05 and t I’ < 0.01 compared with controls Wilcoxon rank sum test.
t hy
sis of prostaglandins in other tissues, including lung, spleen, kidney, and blood vessels or vascular endothelium (1, 11, 12, 21, 32). The relative contribution to the increased venous prostaglandins from the mucosa, submucosa, muscle layer,
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GE0
GI
PROSTAGLANDINS
or some combination remains unknown. Our study confirms the observation that irradiation most severely damages the mucosa, and the submucosa to a lesser degree acutely, and we observed all of the previously described histological abnormalities. Indomethacin treatment was protective in that radiation-induced changes in the epithelium, lamina propria, and submucosa were reduced by indomethacin treatment. The increase in prostaglandin synthesis was probably greatest in the mucosa, but alterations in prostaglandin metabolism also may have occurred in the submucosa as well as in the muscle layer. Luminal PGEg is also increased in active ulcerative colitis, reflecting increased PGE, synthesis in the inflamed mucosa (20). The epithelial injury was accompanied by a moderate inflammatory infiltrate. Therefore inflammatory cells, especially macrophages, in the lamina propria are a potential contributory source of the increased venous concentrations of the prostaglandins. Reduction in the number of tissue macrophages after indomethacin may have contributed to the lowering of prostaglandins after indomethacin. Exogenous prostaglandins placed in the lumen can affect intestinal function (29), but their effects may be reduced because of local degradation (2). The physiological role of each of the prostaglandins in intestinal smooth muscle function is still incompletely understood and based mostly on in vitro studies. The venous concentration of 6-keto-PGF,,, was the greatest of the three measured. Prostacyclin (PGI,), therefore, may be the most important metabolite in irradiated intestinal muscle, but it remains to be determined. Confirmation of its role in motility is difficult because of its rapid metabolism to the inactive 6-keto-PGF,,,. PGE, and PGFzty contract human, rat, and guinea pig ileal longitudinal muscle (33). In contrast, PGE, and PGI, relax canine and guinea pig circular small bowel muscle (28). PGEB and PGFzty also inhibit contraction of human, rat, and guinea pig ileal circular muscle (33). PGI, relaxes ileal longitudinal muscle from humans and opposes the actions of PGE, and PGFz,,. (3). The dominant effect of prostaglandins on human ileum may be increased contraction and tone of the longitudinal muscle and decreased contraction of the circular muscle (8). Indomethacin has opposite effects (7) From previous studies (9, 15, 24, 25, 31, 33, 34) it is known that radiation modifies intestinal smooth muscle function. It has been suspected that the decreased transit time that has been demonstrated in rats after irradiation (31) is due in part to reduced motor activity (24, 25). Spike burst activity, duration, and propagation were severely reduced in dogs by irradiation in the present studies. We would expect propulsion or resistance to flow to be reduced by less frequent and lower amplitude contractions that migrated over short lengths of intestine. The possible role of prostaglandins in these changes was first suggested by a reported rise in PGE, and PGFz,, levels in rat jejunum 7 days after 700 R whole body irradiation (21). Although the rise was not considered to be statistically significant because of scatter of the data, a 100% increase in content was reported. A second report also found an increase in prostaglandin-like material at 1 and 4 days postexposure and that increase was blocked
AFTER
X-RADIATION
by indomethacin (10). Both of these studies were inadequate because they used a nonspecific bioassay. The decreased transit time found by the same investigators after irradiation was, however, returned to normal levels by indomethacin treatment. Thus our study supports a role of prostaglandins in the reduced motor activity induced by irradiation. When prostaglandin synthesis was effectively inhibited by indomethacin, irradiation caused the animals to be less ill, the histological injury was reduced, and the changes in myoelectric activity were much less severe. Thus the prostaglandins appeared to have played an adverse role in the process of irradiation injury, and reduction in their production reduced morphological damage and abnormalities in physiological motor function. Pretreatment with indomethacin or aspirin has also been shown to reduce radiation injury to the opossum esophagus (22, 23). Furthermore, pre- and postradiation treatment with 16,16-dimethylprostaglandin E2 was associated with even more severe morphological injury than in control animals. Unfortunately, the effects of the nonsteroidal inflammatory drugs on radiation-induced motility changes were not reported. Salicylate therapy has also been reported to be beneficial symptomatic relief for radiation-induced enteritis in patients receiving pelvic radiotherapy (6, 19). The role of prostaglandins in irradiation injury remains unproven because of apparently contradictory data. Several investigators (13, 14, 35) have provided data to show that pretreatment with PGE, is radioprotective of the rodent intestine. It is difficult to reconcile the discrepant findings that prostaglandins and inhibitors of prostaglandin synthesis would both be radioprotective. Species variation is also possible, but seems an unlikely explanation. Different prostaglandins often exert differing biological actions. Although E, analogues may be radioprotective, other arachidonic acid metabolites may be inactive or may even increase radiation injury. Radiation undoubtedly induces a wide variety of effects in addition to increasing prostaglandin synthesis. Indomethacin, especially in doses as large as 3 mg kg-‘. 24 h-‘, is a nonselective inhibitor and could have inhibited synthesis of other arachidonic acid metabolites (leukotrienes), cytokines (interleukins), or other enhancers of inflammation. It is very possible that the early preirradiation administration of indomethacin reduced the migration of inflammatory cells into the submucosa by reducing production of chemotactic factors. Additional studies are required to clarify this issue. It is also important to recognize that the indomethacin was administered intravenously. When given orally, this agent may be ulcerogenic, especially in such high doses (16). When the drug is infused into the duodenum or intravenously it may not be as ulcerogenic because the drug has local injurious effects on the gastric mucosa partly by inhibiting the cyclooxygenase pathway of arachidonic acid metabolism. The results of this study support further evaluation of prostaglandin synthesis inhibitors in the treatment of radiation enteritis and the reduction of associated motility abnormalities. l
This study was supported by Department of Veterans Affairs.
medical
research
funds
from
the
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GI Address for reprint requests: Medicine, 1Jniv. of’ Iowa Hospitals Received
11 September
PROSTAGLANDINS
R. W. Summers, & Clinics, Iowa
1990; accepted
in final
form
AFTER
Dept. of Internal City, IA 52242. 25 February
1991.
REFERENCES 1. AI,I,EN, J. B., R. H. SAGEHMAN, ANI) M. J. STIJART. Irradiation decreased vascular prostacyclin formation with no concomitant effect on plat,elet thromboxane production. Lancet 2: 1193-l 198, 1981. 2. BENNETT, A., K. G. EI,EY, ANI) G. B. SCHOLES. Effect of prostaglandins E, and E, on intestinal motility in guinea pig and rat. Hr. J. Pharmacol. 34: 6:139-647, 1968. 3. BENNETT, A., K. G. EI,EY, AND G. B. SCHOLES. Effects of prostaglandins of human, guinea pig, and rat isolated small intestine. &-. J. Pharmacol. 34: 630-638, 1968. 4. BENNETT, A., C. N. HENSBY, G. J. SANCER, ANI> I. F. STANFORD. Metabolites of arachidonic acid found by human gastrointestinal tissues and their action on the muscle layers. Hr. J. Pharmacol. 74: 435-555, 1981. ANI) H. L. STOCKELY. Estimation 5. BENNETT, A., I. F. STANFORD, and charact,erization of prostaglandins in the human gastrointestinal tract. Hr. J. Pharmacol. 61: 579-586, 1977. A., S. SIERAKOWSKI, (J. MACKOWIAK, AND K. WIS6. BOROWSKA, NIEWSKI. A prostaglandin-like activity in small intestine and post irradiation gastrointestinal syndrome. Experientia Base1 35: 13681370, 1979. 7. BURAKOFE’, R., E. NASTOS, AND S. WON. Effects of PGF?,, and of indomethacin on rabbit small and large intestinal motility in vivo. Am. J. Ph.vsiol. 258 ((I‘astrointest. Liver Physiol. 21): G231-G237, 1990. 8. BURI,EIGH, D. E. The effects of indomethacin on the tone and spontaneous activity of the human small intestine in vitro. Arch. Int. Pharmacodyn. Ther. 225: 240-245, 1977. 9. CONARI), R. A. Some effects of ionizing radiation on the physiology of the gastrointestinal tract: a review. Radiat. Rex 5: 167-188, 1956. 10. EISEN, V., AND D. I. WALKER. Effect of ionizing radiation on prostaglandin-like activity in tissues. Hr. J. Pharmacol. 57: 527532, 1976. 11. EIBO, A., I. VLODAVSKY, E. HYAM, R. ATZMOM, ANI) Z. FUKS. The effect of radiation on prostacyclin (PGI,) production by cultured endothelial cells. Prostaglandins 25: 263-279, 1983. 12. HAHN, G. L., M. ,J. MENCONI, M. CAHII,L, AND P. POLGAR. The influence of gamma radiation on arachidonic acid release and prostacyclin synthesis. Prostaglandins 25: 783-791, 1983. 13. HANSON, W. R., ANI) K. DELAURENTIIS. Comparison of in vivo murine intestinal radiation by E-prostaglandins. Prostaglandins, Suppl. 33: 93-104, 1987. 14. HANSON, W. R., AND C. THOMAS. 16,16-dimethylprostaglandin E, increases survival of murine intestinal stem cells when given before photon radiation. Radiat. Rex 96: 393-398, 1983. 15. KAGNOFF, M. F. Motor activity in vitro of rat small intestine following whole body x-irradiation. Radiat. Rex 42: 565-576, 1970. 16. KENT, T. H., R. M. CARI~ELLI, AND F. W. STAMI,ER. Small intestinal ulcers and intestinal flora in rats given indomethacin. Am. J. Pathol. 54: 237-249, 1969. 17. LEDUC, L., AND P. NEEDLEMAN. Regional localization of prostacyclin and thromboxane synthesis in dog stomach and intestinal tract. J. Phamacol. Exp. Ther. 211: 181-188, 1979.
X-RADIATION
G151
18. LIFSHITZ, S., J. E. SAVAGE, K. A. TAYI,ER, H. H. TEWFICK, ANI) D. E. VAN ORIIEN. Plasma prostaglandin levels in radiationinduced enteritis. Int. J. Radiat. Oncol. Hiol. Ph-ys. 8: 275-277, 1982. A. T., AND V. DALLEY. Aspirin in radiation-induced 19. MENNIE, diarrhea. Law& 1: 1131, 1973. 20. ME:NNIE, A. T., V. M. DAUEY, L. C. DINSEEN, ANI) H. 0. COI,I,IER. Treatment of radiation-induced gastrointestinal distress with acetylsalicylate. Lancet 2: 442-443, 1975. 21. NAKAHATA, N., H. NAKANISHI, ANI) 1‘. SUZUKI. A possible negative feedback control of excitatory transmission via prostaglandins in canine small intestine. Hr. J. Pharmacol. 68: 393-398, 1980. 22. NORTHWAY, M. G., G. L. EASTWOOI), H. LIBSHITZ, M. S. FEI,DMAN, T. J. MAMEI,, AND R. ‘I’. SZWARC. Anti-inflammatory agents protect opossum esophagus during radiotherapy. Dig. Dis. Sci. 27: 923-928, 1982. 23. NORTHWAY, M. G., H. LIBSHIT~, M. B. M. OSBORNE, M. S. FELDMAN, T. J. MAMEL, ,J. H. WEST, ANI) I. A. SZWARC. Radiation esophagitis in the opossum: radioprotection with indomethacin. Gastroenterology 78: 883-892, 1980. 24. OTTERSON, M. F., S. K. SARNA, ANI) J. E. MOULI~ER. Effects of fractionated doses of ionizing radiation on small intestinal motor activity. C;a.stroenterology 95: 1249-1257, 1988. 25. QUASTEI,, M. R. Effect of whole body irradiation on spontaneous motility of rat isolated duodenum and its contractile response to acet,ylcholine and 5-hydroxytryptamine. Rr. J. Radiol. 41: 142-146, 1968. 26. RADOMIROV, R., V. PETKOV, ANI) S. YONEV. Effects of’prostaglandin F1,, on the contractile responses of guinea pig ileum to electrical stimulation. Experientia Base1 39: 754-755, 1983. D. S., G. E. SLADEN, L. ?J. F. YOULTEN. Rectal mucosal 27. RAMPTON, prostaglandin E, release and its relation to disease activity, electrical potential differences, and treatment in ulcerative colitis. (iut 21: 591-596, 1980. Prostaglandin synthesis by 28. SANDERS, H. M., ANI) T. E. NORTHUP. microsomes of circular and longitudinal gastrointestinal muscle. Am. J. Physiol. 245 (Cr’astrointest. Liver Physiol. 8): G442-G448, 1983. T. eJ., R. P. ROOD, M. Y. MAH, 29. SERNKA, ANI) C. H. TSENG. Antiabsorpt,ive effects of 16,16-dimethylprostaglandin E, in isolated rat colon. Prostaglandins 23: 411-426, 1982. Statistics for the Behavioral Sciences. 30. SIEGEL, S. Nonparametric New York: McGraw-Hill, 1956. 31. SPRUGEI,, W., M. SCHUBERT, P. METZNEGG, F. HEIM, G. HASI,, AND H. PAULY. Immediate X-irradiation induced contractions of the isolated guinea pig terminal ileum. The localization of the effects of drugs. Radiat. Environ. Hiophys. 14: 31-37, 1977. 32. STEEL, L. K., AND G. N. CATRAVAS. Radiation-induced changes in production of prostaglandins F?,,, E, and thromboxane B, in guinea pig parenchymal lung tissues. Znt. J. Radiat. Hiol. 42: 517-5:30, 1982. 33. SUMMERS, R. W., A. J. FI,ATT, M. ?J. PRIHODA, ANI) F. A. MITROS. Effect of irradiation on morphology and motility of canine small intestine. Dig. Dis. Sci. Z32: 1402-1410, 1987. 34. SUMMERS, R. W., T. H. KENT, ANI) ,J. W. OSBORNE. Effects of drugs, ileal obstruction and irradiation on rat gastrointestinal propulsion. Gastroenterology 59: 7:31-739, 1970. 35. THOMAS-DE I,A VEGA, J. E., B. F. BANNER, M. HIJBBARI), D. L. BOSTON, C. W. THOMAS, A. K. STRAUS, AND D. L. ROSMAN. Cytoprotective of prostaglandin E, in irradiated rat ileum. Surg. Gvnecol. Obstet. 158: Z-39-45. 1984.
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effects on morphology, in dog jejunum
SUMMERS, CLYDE E. GLENN, FLATT, AND AHMED ELAHMADY Internal Medicine, Veterans Administration Medical Center, Digestive Diseases, University of Iowa College of Medicine, Iowa City, Iowa 52242
SUMMERS, ROBERT W., CLYDE E. GLENN, ANDREW J. FLATT, AND AHMED ELAHMADY. Radiation and indomethacin effects on morphology, prostaglandins, and motility in dog jejunum. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G145-GM, MC-Irradiation can have a profound effect on intestinal motor activity. Previous studies have suggested that prostaglandins may play some role in radiation-induced enteritis. The present study investigated the effects of abdominal Xirradiation with or without indomethacin treatment on jejunal myoelectric activity and prostaglandin synthesis by measuring the prostaglandin content of mesenteric arterial and venous plasma and in the intestinal lumen in dogs. After X-irradiation, venous concentrations and arteriovenous concentration differences of prostaglandin (PG)E,, PGF2,,, and 6-keto-PGF1,, increased markedly. The increased venous concentrations were in part attributable to increased mucosal and/or submucosal synthesis by inference from increased concentrations of these metabolites assayed from the jejunal lumen. Irradiation produced histological damage to the mucosa and submucosa and abnormalities in the migrating motor complex, jejunal slow waves, and a decrease in spike burst activity. Inhibition of prostaglandin synthesis by treating the animals with indomethacin reduced the severity of illness, the histological injury, and changes in myoelectric activity induced by irradiation. Such treatment should be evaluated further to treat patients exposed to large doses of irradiation. ionizing
radiation;
arachidonic
acid; intestinal
smooth muscle
IN ADDITION to its well-known effect on the intestinal mucosa, X-irradiation has been shown to cause profound changes in the functions of intestinal smooth muscle (9, 15, 24, 25, 31, 34). Our own recent study (33) demonstrated progressive inhibition of motor activity after radiation injury. The mechanisms involved in these changes are largely unknown, but they might involve arachidonic acid metabolites. Radiation increases prostaglandin synthesis in some organs; the tissues of the gastrointestinal tract have the capacity to synthesize prostaglandins from arachidonic acid, and their rate of synthesis could be increased by X-irradiation. Exogenous prostaglandins have been shown to affect both muscle function and neural activity in the gut (3, 4, 21, 26). Therefore, endogenously synthesized prostaglandins may be involved in the motility changes caused by irradiation. Two preliminary studies (6, 10) have suggested that prostaglandin synthesis is increased in the jejunum after irradiation. In both of these studies, however, prosta-
glandins were measured by a nonspecific bioassay. Inhibitors of prostaglandin synthesis have been reported to restore to normal the decreased intestinal transit time found in irradiated animals (6) and to relieve radiationinduced gastrointestinal symptoms in patients (19, 20). It is not established, however, whether prostaglandin metabolism in the intestine is altered by irradiation, whether irradiation effects on other tissues or organs alter prostaglandin concentrations in the blood and so affect intestinal function, or whether altering prostaglandin synthesis would reduce radiation effects on intestinal smooth muscle function. The present study was designed to explore the role of prostaglandins in canine jejunal muscle before and after irradiation. To determine the effect of irradiation on circulating prostaglandins, arterial and venous blood samples were collected from the vasculature of the intestine and analyzed for prostaglandins by radioimmunoassay (RIA). Luminal prostaglandins were measured to assessthe role of the mucosa and submucosa in prostaglandin metabolism after irradiation. Finally, another group of animals was treated with indomethacin before and after irradiation to determine whether reduced prostaglandin synthesis might reduce or prevent changes in histological morphology and motor function induced by X-irradiation. The results of these studies help to explain some of the abnormal smooth muscle motor functions that follow abdominal irradiation and elucidate the role of prostaglandins in this process. METHODS Preparation of animals. Mongrel female dogs weighing 18-25 kg were anesthetized with intravenous pentobarbital sodium (30 mg/kg) after the abdominal wall was shaved and prepped. Through a midline laparotomy incision, seven bipolar silver electrodes were sutured to the serosal surface of the jejunum at 3-cm intervals beginning 10 cm distal to the ligament of Treitz. A 16-Fr polyethylene Levin-type feeding tube was also sutured into the second portion of the duodenum for administration of water or formula if needed. Teflon-insulated 32-gauge stranded wire led from the electrodes to a 14-pin connector implanted in the animal’s abdominal wall. The proximal end of the feeding tube was tunneled through the abdominal wall muscle and subcutaneous tissue to exit along the lateral portion of the thoracic cage. Seven to
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G146
GI PROSTAGLANDINS
ten days were allowed for recovery before recordings of myoelectric activity were initiated. Irradiation procedure. Animals to be irradiated were anesthetized with intravenous pentobarbital and placed on an X-ray table. A GE Maxitron 250 X-ray machine was used to deliver the dose with 0.25 mm copper and 1 mm aluminum filters at 250 kVp, 30 mA, SSD 17 in. The thorax and pelvis were shielded with lead (thickness -0.5 cm), and the animal was exposed to one-half of the total dose lying on one side. Then the animal was turned and given the second half of the dose on the other side. Dogs were exposed to 1,250 rads as measured with a Victorean R meter. A preliminary investigation with thermoluminescent dosimetry chips placed in the jejunum of dog cadavers showed a dose of 938 cGy was delivered to the intestine by this procedure; chips placed under the shields received ~2.5 cGy absorbed dose. Six control dogs were anesthetized but not irradiated. Six dogs received irradiation as described above and six additional dogs received daily indomethacin 3 days before and 4 days after irradiation. Animals were given indomethacin (1 mg/kg iv) three times daily. Recording and analysis of myoelectric activity. Animals were fasted overnight for 18 h before recordings of myoelectric activity were made. The dogs were placed in a nylon-mesh sling and connected to a recorder using cables attached to the plug previously fixed in the lateral abdominal wall. The recorder was a Beckman R-611 physiological recorder (Beckman Instruments, Fullerton, CA) outfitted with “universal” 9853A couplers using a low-frequency cutoff of 5.3 Hz. The signals were routed to a rack of analog band-pass filters with corner frequencies of 10 and 30 Hz and from there to an MINC-11 computer (Digital Equipment, Maynard, MA) for sampling, storage, and analysis. Control recordings were made before irradiation or indomethacin treatment in all groups, daily in both groups that received irradiation, and just before irradiation in the group that received 3 days of indomethacin before irradiation. Recording periods consisted of 3 h of fasting activity followed by 1 h of fed activity, which was induced by infusing 120 ml of Cornpleat-B (Doyle Pharmaceutical, Minneapolis, MN) meat-based formula into the duodenal tube over 30 min. Modified electrical signals from the band-pass filter were digitized at an analog-to-digital conversion rate of 60 Hz and stored on hard disk during the experiment. Programs to detect spike bursts in all electrodes and to determine the number of bursts per minute (frequency), the duration (seconds), and length of propagation were employed to analyze the myoelectric data. Details of the methodology are provided in an earlier publication (33). Radioimmunoassay of prostaglandins. Studies were performed in the sham-irradiated dogs, the irradiated dogs, and the irradiated dogs treated with indomethacin at 96 h after irradiation. The animals were again anesthetized with intravenous pentobarbital, the abdomen was opened at the midline, and the jejunum was exteriorized and kept moist with physiological saline at 37OC. The six control dogs were treated in the same way. Two incisions, each -2 cm in length and 80 cm apart, were made in the midjejunum. A piece of plastic-coated wire was threaded into the lumen between these incisions, a
AFTER
X-RADIATION
dialysis bag (48 cm long) containing 90 ml of aqueous dialysis solution (5.6 g/l NaCl, 2.24 g/l KCl, 2.52 g/l NaHCO:J was connected to the wire, and the bag was pulled into the lumen (27). After 1 h, the bag was removed. Pre- and postintubation weighing showed negligible fluid loss from the bag. The dialysis fluid was frozen at -70°C until analyzed for prostaglandin (PG)Ez, PGF2,y, and 6-keto-PGF1,, content by RIA. [Note: 6-ketoPGF1,, is the inactive metabolite of prostacyclin, a very short-lived product of arachidonic acid metabolism.] Next the dogs were heparinized by intravenous injection of heparin sodium, and 9 ml of blood were collected in tubes containing 1 ml of Krebs solution and 100 mg indomethacin. Blood was collected from a mesenteric artery and a vein that supplied the irradiated intestine. Blood samples were kept on ice until they were centrifuged at 3,000 g. The plasma was separated from the cellular elements and stored at -70°C until the PGEz, PGFB,,, and 6-keto-PGF1,, content could be assayed by RIA (18). To verify the entry of luminal prostaglandins into the dialysis tubing, an open-ended dialysis bag was placed in a siliconized beaker containing 20 ml of dialysis buffer and 5 ml of buffer in the bag. Tritiated PGE2, PGFZ,,, and 6-keto-PGF,,, were then added to the beaker, which was kept at 37°C. Aliquots were withdrawn from the bag at 0, 15, 30, 45, 60, 90, and 120 min after addition of the standards and analyzed by RIA. After 60 min, all samples equilibrated at -90% of the total activity. Histology. Finally, a 4-cm segment of jejunum was removed from each dog, opened along the mesenteric border, stretched, pinned and fixed in buffered Formalin, and prepared for histological study. After these procedures, the animals were killed. Tissue was embedded in paraffin, sectioned, and prepared for staining with hematoxylin and eosin using standard methods. Slides from controls, irradiated, and irradiated plus indomethacintreated animals were coded and read blindly. Statistical analysis. Data were analyzed by the SAS general linear models procedure and the Wilcoxon rank sum test and the Kruskal-Wallis one-way analysis of variance (30). The nonparametric rank tests were used in preference to parametric tests, as scatter in the data gave large standard deviations and the data did not conform to a normal distribution. RESULTS
General observations. Animals were clinically unaffected by indomethacin treatment before irradiation. They ate normally and had normal bowel movements. On the second day after irradiation, animals not treated with indomethacin began to exhibit signs of irradiation sickness. They refused to eat; all had frequent emesis and began to develop bloody diarrhea. This became progressively more severe on days 3 and 4. The animals treated with indomethacin were not nearly as ill. They ate readily, and only two of six dogs vomited after eating, and they only exhibited mild diarrhea without gross blood. Unfortunately, these observations were only qualitative. Histology. The major histological changes from normal
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GI PROSTAGLANDINS
in irradiated animals were detected in the mucosa (Table 1). These consisted of vacuolization and atypia of the epithelial cells with a paucity of goblet cells. There was also moderate disruption of the villous architecture associated with superficial ulceration, loss of villi, crypt cell necrosis, and dilated glands (Fig. 1). Inflammation, predominantly mononuclear cell infiltrates and some segmented neutrophils and eosinophils, was present in the lamina propria and extended to a lesser degree into the submucosa near sites of severe mucosal damage. Changes in the muscularis propria and the myenteric plexus were minimal to absent except for some mild TABLE 1. Histological findings in controls, irradiation, and irradiated animals treated with indomethacin
Control (n = 6)
938 cGy (n = 6)
938 cGy + Indo (n = 6)
Mucosa Epithelial vacuolization 0 2.1 0.9 Epithelial atypia 0 2.6 1.1 Goblet cell (paucity) 0 3.1 1.1 Superficial ulceration 0 2.0 0.7 Crypt cell necrosis 0 1.9 0.6 Glandular dilatation 0 1.9 0.6 Villous architecture 0.2 1.9 1.4 disruption Inflammation 0 2.0 0.9 Submucosa Edema 0 1.8 0.6 Inflammation 0 0.3 0 Muscularis propria Inflammation 0 0.1 0 Vasodilatation 0 0.9 0 Myenteric plexus 0 0 0 inflammation/damage Total score 0.2 20.6 7.9 Grading of jejunal slides was done blindly according to the following scale: l+, mild, 2+, moderate; 3+, severe; if no change, animal was scored 0. Scores represent mean values from all of the dogs in the group. Individual scores for each finding were added and divided by number of animals in group analyzed. Using the Kruskal-Wallis oneway analysis of variance, total scores all differed from one another (P < 0.001). Indo, indomethacin treatment.
CONTROL
vasodilatation in the muscle layer. Histological abnormalities were more severe in the animals treated with irradiation alone. In only one animal treated with indomethacin, changes could not be distinguished from irradiated animals. The rest of the indomethacin-treated group consistently had only mild abnormalities (Table 0 Myoelectric actiuity. In the control fasting state, migrating myoelectric complexes (MMCs) were present in six of six animals in each of the groups (sham irradiation, irradiation only, and irradiation plus indomethacin). Four days after irradiation, none of the six dogs receiving no indomethacin exhibited MMCs during the 3-h recording period; however, they were present in all of the indomethacin-treated dogs. The absence of MMCs was statistically different from controls in the nonteated dogs (P < O.OOl), but the frequency per 3 h in the indomethacin-treated dogs (2.1 + 0.4) was not different from controls (2.5 ? 0.3). During the control period in all groups, slow waves were regular and demonstrated almost no variation in morphology (Fig. 2), but in the animals receiving irradiation alone, abnormalities in slow waves were noted. These consisted of variation in both morphology and irregularity in rhythm in all six dogs (P < 0.001). By morphology we mean the shape or configuration of the wave. The variability of the slow waves occurred in both the fasted and fed states, but was most clearly seen when spike bursts were absent, as in phase I. In three of six, uncoupling of slow waves was observed (P < 0.05). Changes were most severe on days 3 and 4 after exposure. In the indomethacin-treated group some abnormalities in slow waves during phase I of the MMC were observed in only 33% of the animals (P > 0.05), but the changes were much less severe and no evidence of uncoupling was recorded (Fig 2). Examples of recordings on day 4 after a meal are shown in Fig. 3. Before irradiation, spike bursts occurred irregularly but were evenly distributed. After irradiation, spike activity was reduced overall with occasional periods of intense activity. In irradiated dogs treated with indo-
IRRADIATION
FIG. 1. Representative histological sections from nonirradiated (middle) and with (ri&t ) indomethacin treatment.
G147
AFTER X-RADIATION
control dogs (left) and irradiated
IRRADIATION & INDOMETHACIN dogs without
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G148
GI
PROSTAGLANDINS
10 set
I-
C FI(G. 2. Recordings
from adjacent electrodes during phase I fromnonirradiated cant rol dogs (A), irradiated dogs without indomethacin treatment (H), and irradiated dogs treated with indomethacin (C). In A and C’, slow waves have uniform morphology and regular rhythm and are coupled. In H, morphology is variable, rhythm is irregular, and slow waves are not coupled.
methacin, spike activity was preserved, but there was a tendency for spike bursts to occur in clusters. The quantitative findings recorded 30 min after a meal are shown in Figs. 4-6. No significant changes in spike burst frequency, duration, or length of propagation were observed 1 day after irradiation, but at 4 days, all of these parameters were significantly reduced in the group treated with irradiation alone. However, there were no significant changes from control in any of the parameters in the group treated with indomethacin before or after irradiation. Arterial and venous plasma prostaglandin concentrations (Table 21. The venous plasma concentrations of all prostaglandins was greater than arterial concentrations in control animals, with venous plasma showing 1,060% more PGE2, 240% more PGFxCy,and 270% more 6-keto-
AFTER
X-RADIATION
PGFI,, than arterial plasma. Irradiation significantly increased the PGE, and PGF2,, content of arterial blood. This could have been due to increased prostaglandin synthesis by the irradiated blood vessels but probably reflects increased systemic concentrations. Venous blood showed a very large increase in PGE2, PGF2,?, and 6keto-PGF,,, after irradiation, although the concentrations varied considerably. The change in the postirradiation prostaglandin arteriovenous concentration differences increased significantly for all three prostaglandins. One pair of blood samples was inadvertently lost. Indomethacin effectively inhibited the rise in all three prostaglandins in arterial and venous blood so that the arteriovenous concentration differences were not statistically different from control values. Luminal prostaglandin concentration (Table 2). All three measured prostaglandins were found in the lumen of controls. The concentrations of all prostaglandins increased greatly (X0-fold) after irradiation. The percentage increase was greatest for PGE,. The increase in prostaglandins after irradiation was largely prevented by treating with indomethacin so that the concentrations were not significantly different from controls. DISCUSSION
The results confirm the capacity of the normal intestine to synthesize prostaglandin. Mesenteric venous concentrations of PGE2, PGFzCy,and 6-keto-PGF,,, were 3to IO-fold higher than arterial concentrations in control animals. Similar metabolites were found in this study and in previous investigations (5, 17, 28, 33) of prostaglandin synthesis in normal canine and other species, although relative levels were different. In the nonirradiated jejunum, luminal concentration of PGE2, PGF2,,, and 6-keto-PGF,,, were consistently higher than venous concentrations. This suggests that some synthesis of prostaglandins takes place in the mucosa or submucosa and is not merely “leaked” from the blood vessels. The marked increase in the arterial and venous con-
(-,,
FIG. 3. Typical recordings from fed animals representing each group. Four days after irradiation, spike burst activity is reduced overall, but some intense activity is present. In irradiated dogs treated with indomethacin, spike activity is similar to that seen in controls, with the exception of a tendency toward clustered activity.
CONTROL
IRRADIATION
IRRADIATION & INDOMETHACIN
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(:I
PROSTAGLANDINS
AFTER
G149
X-RADIATION 10
20 lz0 18 I
938 cGy
r m
938 cGy+lOmg/kg
INDOMETHACIN
938 cGy 938 cGy+l
Omg/kg
INDOMETHACIN
0
1
CONTROL
DAY
1
DAY 4
CONTROL
DAYS AFTER IRRADIATION HG. 4. Spike burst activity before and after irradiation exposure untreated dogs and in irradiated dogs with and without indomethacin treat merit. At dq -1, spike burst frequency was significantly reduced dogs not treated with indomethacin (*,I < 0.05).
in in
DAY
1
DAY 4
DAYS AFTER IRRADIATION HG. 6. Length of migration (in cm) was reduced in irradiated dogs not t,reated with indomet hacin (*II < 0.05). Mean length of migration was similarly less at 4 days in indomethacin-treated dogs, but variance was greater so that reduction was not statistically significant.
2. Mean plasma prostaglandin concentrations in mesenteric blood and jejunal lumen
TABLE am
938
cGy
938
cGy+l
Omg/kg
INDOMETHACIN
I’rost aglandin Arterial PGE, I’GFZ,, 6-keto-PGF,,, Venous PGE, PGF?,, 6-keto-PGF,,, A-V Difference PGE, CONTROL
DAYS
DAY
AFTER
1
DAY
4
PGF?,,
IRRADIATION
k-It;. 5. Spike burst duration in dogs not treated and treated indomet hacin before and after irradiation exposure. Spike burst t ion was significantly reduced at 4 days in dogs not treat,ed indomet hacin (*I’ < 0.05).
with durawith
centrations suggests that jejunal synthesis of prostaglandins is increased by irradiation. The elevated arterial plasma concentrations of prostaglandin after irradiation could be due in part to synthesis of prostaglandins at other sites. However, the increase in venous prostaglandin concentrations and the large arteriovenous concentration differences support increased local synthesis of prostaglandins in the jejunum. Decreased catabolism of prostaglandins is possible, but unlikely, because the rise in prostaglandins was prevented by treating with indomethacin, a known inhibitor of prostaglandin synthesis. Radiation has previously been shown to increase synthe-
6-keto-PGF,,, Jejunal lumen PGE, PGF?,, 6-keto-PGF,,,
Cant rol
Irradiation
(n = 6)
(n = 5)
9&l (S-29) 1:3t:3 (7-22) 198t23 (X38-297)
Irradiation + Indornet hack (n = 6)
44tfi” (:33-x3) 30*4* (25-40) :309t119 (99-762)
38t6 (16-54) 1853 (10-32) 83+19 (36-144)
l,828+1,105t (320-6,152)
93k 19 (46-147) t Y-t V+‘j (18-59) 5Z7k62 (380-l ,944)
(10%2,016) 8,76:3+4,005t ( 1,929-24,375)
204t 133 (40-867) 42t7 (24-68) 727+199 (203-1,:302)
85t18 (:38-161) 19t7 (O-45) 339t69 (220-614)
1,784+1,102* (280-6,098) 64%:358* (851,976) 8,454&4,012* ( 1,719-7,09S)
167t130 (:3-812) 24t8 (S-54) 644+198 (149-1,158)
428*7S (24%6:37) 4x3+40 (:38011,044) 161&21 (94-229)
16,589+6,009* (:3,012-40,250) 2,943a750* ( 1,508-6,238) 678t224* (2%1,699)
873t661 (8-4,150) x+22 (8-140) 25-t 10 (6-72)
677t360t
Values represent mean concentration in picograms per milliliter SE/( range). * P < 0.05 and t I’ < 0.01 compared with controls Wilcoxon rank sum test.
t hy
sis of prostaglandins in other tissues, including lung, spleen, kidney, and blood vessels or vascular endothelium (1, 11, 12, 21, 32). The relative contribution to the increased venous prostaglandins from the mucosa, submucosa, muscle layer,
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GE0
GI
PROSTAGLANDINS
or some combination remains unknown. Our study confirms the observation that irradiation most severely damages the mucosa, and the submucosa to a lesser degree acutely, and we observed all of the previously described histological abnormalities. Indomethacin treatment was protective in that radiation-induced changes in the epithelium, lamina propria, and submucosa were reduced by indomethacin treatment. The increase in prostaglandin synthesis was probably greatest in the mucosa, but alterations in prostaglandin metabolism also may have occurred in the submucosa as well as in the muscle layer. Luminal PGEg is also increased in active ulcerative colitis, reflecting increased PGE, synthesis in the inflamed mucosa (20). The epithelial injury was accompanied by a moderate inflammatory infiltrate. Therefore inflammatory cells, especially macrophages, in the lamina propria are a potential contributory source of the increased venous concentrations of the prostaglandins. Reduction in the number of tissue macrophages after indomethacin may have contributed to the lowering of prostaglandins after indomethacin. Exogenous prostaglandins placed in the lumen can affect intestinal function (29), but their effects may be reduced because of local degradation (2). The physiological role of each of the prostaglandins in intestinal smooth muscle function is still incompletely understood and based mostly on in vitro studies. The venous concentration of 6-keto-PGF,,, was the greatest of the three measured. Prostacyclin (PGI,), therefore, may be the most important metabolite in irradiated intestinal muscle, but it remains to be determined. Confirmation of its role in motility is difficult because of its rapid metabolism to the inactive 6-keto-PGF,,,. PGE, and PGFzty contract human, rat, and guinea pig ileal longitudinal muscle (33). In contrast, PGE, and PGI, relax canine and guinea pig circular small bowel muscle (28). PGEB and PGFzty also inhibit contraction of human, rat, and guinea pig ileal circular muscle (33). PGI, relaxes ileal longitudinal muscle from humans and opposes the actions of PGE, and PGFz,,. (3). The dominant effect of prostaglandins on human ileum may be increased contraction and tone of the longitudinal muscle and decreased contraction of the circular muscle (8). Indomethacin has opposite effects (7) From previous studies (9, 15, 24, 25, 31, 33, 34) it is known that radiation modifies intestinal smooth muscle function. It has been suspected that the decreased transit time that has been demonstrated in rats after irradiation (31) is due in part to reduced motor activity (24, 25). Spike burst activity, duration, and propagation were severely reduced in dogs by irradiation in the present studies. We would expect propulsion or resistance to flow to be reduced by less frequent and lower amplitude contractions that migrated over short lengths of intestine. The possible role of prostaglandins in these changes was first suggested by a reported rise in PGE, and PGFz,, levels in rat jejunum 7 days after 700 R whole body irradiation (21). Although the rise was not considered to be statistically significant because of scatter of the data, a 100% increase in content was reported. A second report also found an increase in prostaglandin-like material at 1 and 4 days postexposure and that increase was blocked
AFTER
X-RADIATION
by indomethacin (10). Both of these studies were inadequate because they used a nonspecific bioassay. The decreased transit time found by the same investigators after irradiation was, however, returned to normal levels by indomethacin treatment. Thus our study supports a role of prostaglandins in the reduced motor activity induced by irradiation. When prostaglandin synthesis was effectively inhibited by indomethacin, irradiation caused the animals to be less ill, the histological injury was reduced, and the changes in myoelectric activity were much less severe. Thus the prostaglandins appeared to have played an adverse role in the process of irradiation injury, and reduction in their production reduced morphological damage and abnormalities in physiological motor function. Pretreatment with indomethacin or aspirin has also been shown to reduce radiation injury to the opossum esophagus (22, 23). Furthermore, pre- and postradiation treatment with 16,16-dimethylprostaglandin E2 was associated with even more severe morphological injury than in control animals. Unfortunately, the effects of the nonsteroidal inflammatory drugs on radiation-induced motility changes were not reported. Salicylate therapy has also been reported to be beneficial symptomatic relief for radiation-induced enteritis in patients receiving pelvic radiotherapy (6, 19). The role of prostaglandins in irradiation injury remains unproven because of apparently contradictory data. Several investigators (13, 14, 35) have provided data to show that pretreatment with PGE, is radioprotective of the rodent intestine. It is difficult to reconcile the discrepant findings that prostaglandins and inhibitors of prostaglandin synthesis would both be radioprotective. Species variation is also possible, but seems an unlikely explanation. Different prostaglandins often exert differing biological actions. Although E, analogues may be radioprotective, other arachidonic acid metabolites may be inactive or may even increase radiation injury. Radiation undoubtedly induces a wide variety of effects in addition to increasing prostaglandin synthesis. Indomethacin, especially in doses as large as 3 mg kg-‘. 24 h-‘, is a nonselective inhibitor and could have inhibited synthesis of other arachidonic acid metabolites (leukotrienes), cytokines (interleukins), or other enhancers of inflammation. It is very possible that the early preirradiation administration of indomethacin reduced the migration of inflammatory cells into the submucosa by reducing production of chemotactic factors. Additional studies are required to clarify this issue. It is also important to recognize that the indomethacin was administered intravenously. When given orally, this agent may be ulcerogenic, especially in such high doses (16). When the drug is infused into the duodenum or intravenously it may not be as ulcerogenic because the drug has local injurious effects on the gastric mucosa partly by inhibiting the cyclooxygenase pathway of arachidonic acid metabolism. The results of this study support further evaluation of prostaglandin synthesis inhibitors in the treatment of radiation enteritis and the reduction of associated motility abnormalities. l
This study was supported by Department of Veterans Affairs.
medical
research
funds
from
the
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GI Address for reprint requests: Medicine, 1Jniv. of’ Iowa Hospitals Received
11 September
PROSTAGLANDINS
R. W. Summers, & Clinics, Iowa
1990; accepted
in final
form
AFTER
Dept. of Internal City, IA 52242. 25 February
1991.
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