Effect of indomethacin on mesenteric circulation in mongrel dogs

Effect of Indomethacin on M e s e n t e r i c Circulation in Mongrel Dogs By Paul W. Cronen, Hirikati S. Nagaraj, Joseph S. Janik, Diller B. Groff, John C. Passmore, and Carl E. Hock Louisville, K e n t u c k y 9 Necrotizing enterocolitis has been attributed to the use of indomethacin (INDO) for medical closure of patent ductus arteriosus. To study the effect of I N D O on cardiac output and mesenteric circulation, I N D O was given by rectum (0.25 m g / k g , 0.5 m g / k g . 1.25 m g / k g - - 3 dogs in each group) and the control g r o u p received none; T h e cardiac output and organ blood flow were measured before and 1 hr after I N D O with radioactive microspheres using 4 isotopes (Cr =, Ni ss, Co sT, Sn'~). The blood flow to different parts of the G! tract was measured as percent of cardiac output using a gamma counter. Paired t test was used to calculate percent reduction in organ blood flow. During the experiment, there was no reduction in cardiac output in the entire group. Anesthesia had no effect on the control group, In the three INDO treated groups, percent reduction of mucosal blood flow of the stomach (63%, 3 2 % , 6 8 % , p < 0.01), mid ileum (19%, 59%, 5 7 % , p < 0.05) and terminal ileum (57%, 3 5 % , 5 4 % , p < 0.0151 w a s significant. A strong trend in reduction of organ blood flow was noted in other regions. There was no significant change due to different dosages of INDO. The area of ischemia in this dog model corresponds to clinical pathology noted in necrotizing enterocolitis. I N D E X W O R D S : Indomethacin; mesenteric circulation; necrotizing enterocolitis,

NDOMETHACIN (INDO), a prostaglandin

I synthetase inhibitor, has gained wide clinical usage for a variety of disease processes associated with prostaglandin imbalance. Recently, INDO has been used for pharmacologic closure of patient ductus arteriosus in infants with few complicationsJ While gastrointestinal side effects have been appreciated in adults, 2 only recently have bowel perforation and necrotizing enterocolitis been found in infants. In our NewFrom the Department of Surgery, Section of Pediatric Surgery, and the Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, By. Presented before the 30th Annual Meeting of the Surgical Section of the American Academy of Pediatrics, New Orleans, La, OctoberM-November 1, 1981. Address reprint requests to Hirikati S. Nagaraj, M.D., Department of Surgery, Ambulatory Care Building, University of Louisville, Louisville, Ky. 40292. 9 1982 by Grune & Stratton, Inc. 0022-3468/82/1705-0005501.00/0 474

born Intensive Care Unit we have reported gastrointestinal disease in 21 of 82 infants treated with INDO for patent ductus closure? Thirteen infants had necrotizing enterocolitis while 8 infants had focal bowel perforation of the gastrointestinal tract. These perforations occurred in the stomach, jejunum, and ileum primarily. The incidence rate of gastrointestinal side effects was higher in the group treated with INDO than in the group not treated with INDO. Prior laboratory studies4'5 have indicated that INDO may cause gastrointestinal perforation; however, no study has been available that examined the blood flow changes due to INDO. Intrigued by our clinical findings, we undertook a study to determine the actual effect that INDO had on mesenteric blood flow. MATERIALS AND METHODS Mongrel dogs (l 5 to 25 kg) were used as the experimental model. After the animals were anesthetized with Surital and Chlorolose (5 ml/kg), an endotracheal tube was inserted and mechanical ventilation begun with tidal volumes of 20 ml/kg bodyweight and a cycle rate of lO--15/min, An arterial catheter was then inserted into the right femoral artery and a central line placed into the left internal jugular vein by cutdown technique. Using pressure tracings, an angiography catheter was then placed into the left ventricle through the left internal carotid artery. Systemic and left ventricular pressures were then monitored. Arterial blood gases and hematocrit levels were obtained and appropriate ventilatory changes or bicarbonate infusions were given to correct any acid-base changes. A l-hr interval existed between anesthesia induction and blood flow determinations. Radioactive tagged microspheres, 15 • 3 ~ sizes were used to determine blood flow as described by Heymann et al. 6 Radioactive markers of Niobium 95, Chromium 57, Cobalt 51, and Tin 113 were employed. The microspheres were checked for appropriate size and absence of clumping by microscopy. Dosages used in the animals provide for 400 microspheres per tissue (6-8 g) specimen counted as recommended by Buckberg.7The microspheres (2 co) were then injected into the left ventricular catheter over 10 sec and the line flushed. A withdrawal pump was then attached to the femoral artery catheter and started at a constant rate of withdrawal of 14.8 ml/min prior to the microsphere injection. One minute collection was obtained starting with the sphere injection, which then served as a "reference organ." Following the baseline sphere injection, a rectal tube was inserted for the INDO administration. Four groups of three animals each were studied. One group received no INDO and Journal of Pediatric Surgery, Vo!. 17. No. 5 (October), 1982

EFFECTS OF INDO ON MESENTERIC CIRCULATION

475

served as a control group. The other groups received 0.25 mg/kg, 0.50 mg/kg and 1.25 mg/kg of INDO. An interval of 1 hr was used prior to the injection of a second different microsphere. Arterial blood gases were checked prior to the second microsphere injection and the pH balanced accordingly. After the second sphere injection the animal was sacrificed and specimens of the lung, liver, stomach, jejunum, mid-ileum, terminal ileum, and proximal and distal colon were obtained. The gastrointestinal segments were then separated into the mucosal and submucosal layers and the seromuscular segments. The tissue specimens were surfacedried, weighed, and placed in gamma counting containers. A Beckman gamma counter was used. Appropriate specimen heights, counting times, and crystal characteristics of our gamma counter were ascertained before the study. From the reference organ blood samples the cardiac output was calculated as follows: Cardiac output -

0.25 mg/Kg. DOSE 2.0

[ ] Pre - Indomethacin [ ] Post-Indomethacin

I.O IJ.

E3 0

g m

o

reference organ flow reference organ counts

STOMACH

JEJUNUM

TERM ILEUM

ASC. C O L O N SIGMOID

ORGAN

x Total Injection Counts Cardiac output -

MID ILEUM

Fig. 2. Blood flow changes in the mesenteric circulation of dogs following 0.25 mg/kg of INDO.

14.8 ml/min • total injected count reference blood samples count

Similarly, the specific organ blood flow was calculated by the same calculations listed. Results of these flows were computed as flow/g wt and listed as percent of cardiac output. The paired Student's t test was used for statistical analysis. RESULTS

N o s t a t i s t i c a l l y s i g n i f i c a n t c h a n g e in c a r d i a c o u t p u t o c c u r r e d in a n y o f t h e a n i m a l s , p H w a s maintained throughout the experiment and r a n g e d f r o m 7 . 3 6 t o 7 . 4 5 for t h e g r o u p . PO2s w e r e m a i n t a i n e d a b o v e 7 0 t o r r a t all t i m e s . N o observable hemodynamic effects occurred during

the experiments in any animal. Hematocrit values ranged from 34 to 35. The control group did not show any significant blood flow changes in any gastrointestinal organs, suggesting that anesthesia did not affect mesenteric blood flow (Fig. 1). A striking reduction in blood flow occurred in all mucosal and submucosal layers of the gastrointestinal tract of dogs who received INDO (Figs. 2-4). Seromuscular blood flow changes were not remarkable or statistically significant in all cases. A statistically significant

CONTROLS 3.0 A

[ ] Pre- Indomethacin [ ] Post-lndomethacin

E2.0

g i, E3 0 0 _1 m

1.0

0 Fig. 1 Blood flow changes in the mesenteric circulation of the control group of dogs without INDO.

STOMACH JEJUNUM

MID ILEUM

TERM ILEUM

ORGAN

ASC. COLON

SIGMOID

476

CRONEN ET AL.

0.5 mg/Kg. DOSE 3.0

1.25 mg/Kg. DOSE 2.0

[ ] Pre- Indomethacin

[ ] Pre - Indomethocin

[ ] Posf-lndomethacin

[ ] Post" Indomethacin

J

~1.o I,L o

MID STOMACH JEJUNUM ILEUM

0 0 .J r

TERM ASC. ILEUM COLON SIGMOID

ORGAN

Fig. 3. Blood flow changes in the mesenteric circulation of dogs following 0.5 m g / k g of INDO.

o

MID STOMACH JEJUNUM ILEUM

TERM ILEUM

ASC. COLON

SIGMOID

ORGAN

flow reduction was appreciated in the mucosal and submucosal layers of the stomach, midileum, and terminal ileum in all dosage groups (Table 1). A sharp reduction in blood flow occurred in the jejunum and proximal colon. No significant changes in blood flow occurred with INDO between the different dosage groups. Mean percent blood flow reduction was also reported in the INDO treated groups (Table 2). No actual perforations or mucosal ulcerations were noted in this acute animal model.

Fig. 4, Blood flow changes in the mesenteric circulation of dogs following 1.25 m g / k g of INDO.

ductus and mesenteric circulation. 8 In experimental situations prostaglandins have increased effects in premature infants. Kent 4 has shown that rats that have been orally fed INDO develop ulcerations along the mesenteric border, primarily in the distal small intestine. Multiple mechanisms may cause this phenomenon. Because of the enterohepatic circulation of 1NDO, the distal small intestine may have prolonged contact with INDO; this theory, however, would not explain the absence of ulcerations in the upper small bowel or their presence in the stomach or colon. The effects of a mucosal injury by INDO, coupled with bacterial invasion, have also been

DISCUSSION

As INDO has obvious vascular effects due to prostaglandin inhibition related to ductus arteriosus, it is reasonable to assume that mesenteric circulation is also affected. Prostaglandins E1 and E2 have a primary vasorelaxing effect on the

Table 1. Organ Blood Flow Before and A f t e r Indomethacin Organ Blood Flow as Percent of Cardiac Output 0.25 rng/kg

Organ Stomach Jejunum Mid ileum Terminal ileum Proximal colon

Control M S M S M

0.791 0.083 1.296 0.041 0.900

S M S M S

0.146 1.265 0.118 2.036 0.348

0.432 0.138 1.870 0,057 0.849 0.489 1.213 0,457 1.553 0.501

M = mucosa/submucosa; S = seromuscular. *p, statistically significant.

0.5 mg/kg

1.25 mg/kg

Pre

Post

Pre

Post

Pre

Post

0.816 0.331 0.648 0,187 0.621 0.368 0.893 0.459 1.337 0.175

0.302 0,158 0.518 0.142 0.503 0.348 0.384 0.172 0.727 0.136

0.707 0.168 0,922 0.383 1.266 2.812 0,625 1.054 2.225 0.430

0.484 0,128 0.685 0.093 0.522 0,184 0.408 0.179 1.627 0.090

1.312 0.059 1.227 0,150 0.845 0.134 0.636 0.225 2.106 0.098

0.423 0.069 0.437 0.095 0,367 0.155 0,293 0.177 1.25 0.091

p Value <0.01" <0.5 <0.1 <0.1 <0.05* <0.5 <0,15" <0.5 <0.1 <0.5

EFFECTS OF INDO ON MESENTERIC CIRCULATION

477

Table 2. Mean Percent Blood Flow Reduction of Different Organs After Indomethacin Mean Percent Reduction

Organ Mucosa/ Subrnucosa

0.25 mg/kg

0.5 mg/kg

1.25 mg/kg

p Value

Stomach Jejunum Mid Ileum Terminal Ileum Proximal colon

63 20 19 57 46

32 26 59 35 27

68 64 57 54 41

<0.01 * <0.1 <0.05" <0.15* <0.1

*p, statistically significant.

evaluated by Robert and A s a n o 9 in a study dealing with germ-free rats. Basically, no ulcerations occurred until m o n o c o n t a m i n a t i o n with bacteria occurred. In his other works, Robert l~ has suggested that prostaglandins have a cytoprotective effect on the intestinal mucosa a n d that an induced prostaglandin deficiency is the precipitating cause of these ulcerations. The actual changes in mesenteric blood flow following a d m i n i s t r a t i o n of I N D O have not been studied previously. Based on in vitro studies, 12'13 I N D O has been shown to have no m e a s u r a b l e effect on vascular smooth muscle by itself. However, isolated mesenteric artery segments show an increased response to epinephrine following I N D O t r e a t m e n t . N o w a k et al. 14 and W e n n m a Ion, 15 in h u m a n studies, showed a n increased mesenteric vascular resistance after large doses of I N D O were given intravenously. O u r study showed a significant decrease in the blood flow of the stomach, mid, and t e r m i n a l ileum. The effect of I N D O was much more profound in the muco-

sal and submucosal layers. From our data, we feel that I N D O m a y have a vascular effect that by itself or in c o m b i n a t i o n with its mucosal effect can cause gastrointestinal ulcerations. F u r t h e r more, the areas of decreased blood flow from our experimental studies are r e m a r k a b l y similar to those that have been observed in our clinical study group. 3 E x p e r i m e n t a l doses were on a m g / k g basis, which are similar to those used in our clinical experience, and were a d m i n i s t e r e d in a similar fashion. Rectal a d m i n i s t r a t i o n has been shown to give a d e q u a t e I N D O blood levels in relation to its vascular effects. 16 Bond 17 has docum e n t e d that after 1 hr of anesthesia, regional blood flows have r e t u r n e d to baseline, dispelling any question of whether anesthesia would change blood flow distribution. In s u m m a r y , we postulate first that I N D O m a y have vascular p h e n o m e n a on other organ beds, which accounts for the complications occurring after I N D O therapy for p a t e n t ductus arteriosus. O u r data suggest that a decrease in mesenteric blood flow occurs. Second, these areas of decreased flow correspond to areas of perforation a n d necrotizing enterocolitis as previously noted in our clinical study. Third, a dose response situation was not appreciated a m o n g our dosage groups because the action of I N D O was an e n z y m e inhibition process. Fourth, a strong trend in blood flow reduction was noted in other areas of the gastrointestinal tract. Determ i n a t i o n of the long t e r m effects of I N D O should be studied in chronic a n i m a l models.

REFERENCES

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systemic and pulmonary vascular resistance in man. Clin Sci Mol Med 54:141-145, 1978 16. Alpert BS, Lewins M J, Rowland DW, et al: Plasma indomethacin levels in preterm newborns with symptomatic patent ductus arteriosus. Clinical and echocardiographic

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assessments of response. Adv Prostaglandin Thromboxane Res 7:883-885, 1980 17. Bond JH, Prenthiss RA, Lavitt MD: Effect of anesthesia and laparotomy on blood flow to the stomach, small bowel, and colon of the dog. Surgery 87:313-318, 1981