The Effect of Live E. col i and Endotoxin on Mortal i ty F o l l o w i n g M a s s i v e Bowel Resect ion

By Karen West, Mark A. Malangoni, Thomas R. Weber, and Jay L. Grosfeld

Indianapol is, Indiana

�9 The distal small intestine and its mesentery contain abundant lymphoid tissues rich in B cells and IgM. An increased mortality due to sepsis has been observed after extensive distal bowel resection (80%) presumably related to excision of gut lym- phoid tissue and resultant impairment of humoral immunity. This study compares the effect of l ive E, coi l and endotoxin on mortality following massive distal bowel resect ion . O n e - h u n d r e d n ine ty Sprague-Dawley rats (200 g) underwent steri le midl ine laparotomy. Fifty animals had a sham opera- tion (CONTROL) and the remainder an 80% distal small bowel resection (DBR). At seven days after operation, rats were divided into four groups and received an intraperit0neal challenge with either 10 s organisms of l ive E. co l i o r 10 mg /kg of E. co l i endotoxin (ENDO) and were evaluated for morta l i ty at 72 hr. Mortality was 9% in controls + ENDO rats (2/25) (group I), 12% in CONTROL + E, co i l rats (3/25) (group II), 56% wi th DBR + ENDO (39/70) (group III) and 36% wi th DBR § E. co i l (25/70) (group IV). Mortality was increased in animals wi th DBR (groups III, IV) compared to controls regardless of the method of challenge (p < 0~05). Mor ta l i t y was significantly greater in DBR animals that received an ENDO challenge when compared to those given a challenge wi th l ive E. co i l (p < 0,05). These data indicate an increased mortality from both l ive E. co i l sepsis and /o r endotoxin challenge in animals with massive distal bowel resection. An LDlo of endotoxin had a greater influence on mortality than l ive E. c o i l These results confirm the observation that massive distal bowel resection increases the risk of morta l i t y related to gram negative sepsis probably due to decreased humoral immunity. Mor ta l i t y is adversely affected by release of endotoxin and its u l t imate effect on vital organs.

INDEX WORDS: Massive bowel resection; E. co i l sepsis.

From the Section of Pediatric Surgery, Department of Surgery, Indiana University School of Medicine and the James Whitcomb Riley Hospital for Children, Indianapolis, Ind.

Presented before the Twelfth Annual Meeting of the American Pediatric Surgical Association together with the British Association of Paediatric Surgeons, Tarpon Springs, Florida, April 2~May 2, 1981.

Address reprint reports to Jay L. Grosfeld, M.D., Surgeon-in-Chief, J.W. Riley Hospital for Children (K-21), 1100 W. Michigan Street, Indianapolis, Ind. 46223.

�9 1981 by Grune & Stratton, Inc. 0022-3468/81/1606~015501.00/0

T OTAL parenteral nutrition administered in the newborn period to babies requiring

massive bowel resection for midgut volvulus, intestinal atresia, and necrotizing enterocolitis has significantly increased their early survival. However, late mortali ty related to sepsis continues to be a clinical problem particularly when the distal small bowel and ileocecal valve are resected. It has been suggested that the extensive loss of the lymphoid-rich distal small bowel may account for the increased sepsis rate and mortality because of decreased humoral im- munity. This study evaluates this concept and compares the effect of live E. coli and E. coli endotoxin on mortality in animals undergoing extensive distal small bowel resection.

METHODS

A midline laparotomy was performed in 190 Spraguc- Dawley rats each weighing 200 g. The animals were then placed into four experimental groups: group I (n = 25) control and endotoxin; group lI (n - 25) control + E. col# group III (n = 70) distal bowel resection + endotoxin; group IV (n = 70) distal bowel resection and E. coli. The distal bowel resection (DBR) animals had an 80% distal resection with sacrifice of the ilieocecal valve and a one layer end- to-end anastomosis using interrupted 6-0 silk. Seven days postoperatively, intraperitoneal challenges with either live E. coli (1 x 10 organisms) or E. eoli endotoxin (10 mg/kg) (Sigma) were administered. Mortality was then evaluated at 72 hr. Statistical comparisons were made by R x C contin- gency tables utilizing • analysis.

RESULTS

Seventy-two hours after intraperitoneal chal- lenge, mortality was 9% (2/25) in group I; 12% (3/25) in group II; 56% (39/70) in group III; 36% in group IV (25/70). Mortality was increased in DBR animals regardless of the method of challenge (p < 0.05), and was signifi- cantly greater in resected animals receiving endotoxin when compared to the live E. coli challenge (p < 0.05). There was no significant differences in the death rates of the control animals.

DISCUSSION

Following massive distal bowel resection, sepsis is a frequent cause o f late morbidity and

846 Journal of Pediatric Surgery, Vol. 16, No, 6 (December), 1981

E. COLI AND ENDOTOXIN IN BOWEL RESECTION 847

mortality especially when the ileocecal valve is sacrificed. ~ This may be due in part to an increased transit time and malabsorption result- ing in protein calorie malnutrition and related immunosuppression. Reports by both Mavligit et aL E and Neiberger et al. 3 characterizing the lymphocytic subpopulations of the gut describe a predominance in the number of B cells and IgM in gut tissue in the mesenteric lymph nodes and the appendix, which is in contrast to peripheral blood, where T cells and IgG predominate. The number of mesenteric lymph nodes and Peyers patches increases significantly as the ileocecal valve is approached. This suggests that loss of these lymphoid rich areas may affect humoral immunity.

Endotoxin, a complex aggregate of lipopoly- saccharides and protein in the cell wall of gram negative bacteria, is released upon bacteriolysis. It affects both the humoral and cellular compo- nents of the host response by activating the complement cascade and coagulation mecha- nism as well as influencing the function of plate- lets, macrophages and endothelial cells. Investi- gators have determined that the complement system may be activated by two separate path- ways: (I) an antigen-antibody complex involv- ing IgM or IgG, and (2) naturally occurring polysaccharides. 4'5 Endotoxin produces tissue injury by initiation of coagulative changes through localized and generalized Shwartzman's reactions. 4'5 After initiating these changes, fibrin is deposited in a variety of tissues including lung, liver, heart, and spleen. 3

A previous report from our laboratory showed an increased mortality following endotoxin chal- lenge as a greater percentage of distal small bowel was resected. Massive small bowel resec- tions of 80% proximal, mid, and distal small bowel and 90% jejunoileal bypasses were performed in a large number of Sprague-Dawley rats. Twenty-four hours after an endotoxin chal- lenge, mortality was 9% in controls and 16% in the jejunoileal bypass animals. A mortality of 27% was noted in animals with proximal small bowel resection, 43% in those with the 80% mid small bowel resection, and 66% in those with distal small bowel resection. There was a signifi- cant increase in mortality when the distal bowel resection group was compared to all other groups studied. An assessment of T cell function was

done using skin allografts in each group. Recip- ient animals uniformly demonstrated skin graft necrosis in 7-10 days suggesting that cellular immunity was intact. Immunoglobulin evalua- tion of mesenteric lymphoid tissue demonstrated 53% B cells in distal small bowel and 63% B cells in the appendix. IgM was the predominant immunoglobulin in these same areas. The IgA levels in the lamina propria, where this immuno- globulin predominates, were not studied. Be- cause of the similar pattern of graft necrosis, it was concluded that mortality following an endo- toxin challenge was related to decreased hu- moral immunity. 1 Previous studies by Cooper et al. 6 revealed that excision of gut lymphoid tissue resulted in an antibody deficient syndrome with- out significant effect on the cell mediated system in the neonatal rabbit. Perey et al. further postu- lated that the gut lymphoid tissue was the mammalian equivalent of the avian bursa of Fabricus. 7

In this study, the effect of E. coli and endo- toxin were assessed in only those animals undergoing 80% distal small bowel resection. Animals with massive DBR had an increased mortality regardless of the method of challenge. However, there was a significant increase in the death rates of animals receiving endotoxin (at LD~0) when compared to those in whom a simi- larly lethal dose of live E. coli organisms was administered. This observation may be due to an increased specificity of end organ response to endotoxin.

Previous reports illustrate that bacterial endo- toxin has a profound effect on host systems In vitro and in vivo observations have showr, aa effect on coagulation, complement, endothelial cells, platelets, and macrophages. 4 Although endotoxin affects both the humoral and cellular immunity mediated systems, the results of this study suggest that a decrease in humoral com- ponents following extensive distal bowel re- section probably is responsible for the increased mortality in the endotoxin challenged animals. Moreover, an LD~o of endotoxin had a sig- nificantly greater influence on mortality than live E. coli. The increased late mortality due to sepsis in children with massive distal bowel re- section may reflect a decreased humoral immu- nity during the period of small bowel accommo- dation.

848 WEST ET AL.

REFERENCES

1. Malangoni MA, Cakmak O, Grosfeld JL: Adverse effects of endotoxin following massive distal bowel resection. J Pediatr Surg 14:708-712, 1979

2. Mavligit GM, Jubert A, Gutterman JU: Subpopulation of thymus dependent and thymus independent lymphocytes in human gut associated lymphoid tissue. Surg Gynecol Obstet 140:397400, 1975

3. Neiberger JB, Neiberger RG, Richardson JT: Distri- bution of T and B lymphocytes in lymphoid tissue of infants and children. Infect [mmun 14:110-121, 1976

4. Morrison DC, Ulevitch J: The effects of bacterial

endotoxin on last host mediation system. Am J Pathol 93:527-617, 1978

5. Mergenhagan SE, Synderman R: Significance of complement to the mechanism of actions of endotoxin. Curr Top Microbiol Immunol 50:37-77, 1969

6. Cooper MD, Perey DV, Gabrielsen AE: Production of an antibody deficient syndrome in rabbits by neonatal removal of organized intestinal lymphoid tissue. Int Arch Allerg 33:65-81, 1968

7. Percy DV, Cooper MD, Goode RA: The mammalian homologue of the avian bursa of Fabricius. Surgery 64:614- 621, 1968

D i s c u s s i o n

Marc I. Rowe (Miami): Did you characterize your bug? If you study different strains of E. coli, you find that they vary in susceptibility to host defense mechanisms. Some are killed only by immunoglobulins, others only by white blood cells. However, there are some E. coli strains that are not killed by either white blood cells or immunoglobins. As a result, when one discusses host defense deficiencies in E. coli septicemia, it is essential that you specify what the particular bacterial strain used is susceptible to.

Arnold Coran (Ann Arbor): This study attempts to explain the increased mortality after massive bowel resection as being due to decreased humoral immunity secondary to removal of lymphoid tissue in the distal small bowel. However, the decreased humoral immu- nity could well have been due to marked malnu- trition. Massive distal small bowel resection rapidly results in severe malnutrition, which is classically associated with both decreased humo- ral and cellular immunity. It might have been interesting to add another group of animals to this study who were starved and severely malnourished and then challenged with either live E. coli or endotoxin. Major operative trauma in the experimental animal significantly in- creases the organism's susceptibility to a septic challenge. I wonder whether or not the operative trauma itself rather than the distal small bowel resection was the significant factor in the increased susceptibility to the endotoxin and live E. coll. Finally, it is interesting to note that, in this group of animals, the endotoxin was more lethal than the live E. coli challenge. In our experience in producing septic shock in puppies,

live E. coli is a much more severe septic chal- lenge than endotoxin.

Eric Fonkalsrud (Los Angeles): I would like further clarification of the nutritional status of the experimental versus the control rats at the time of endotoxin or E. eoli challenge seven days postoperatively. Nutritional depletion has been shown to depress the immune response and make patients anergic to skin tests and make them particularly susceptible to infections and sepsis. Total parenteral nutrition may reverse the anergy and significantly lower the risk of infec- tion in depleted patients with intestinal disorders of various types. In your experimental model, can you rule out severe malnutrition as having as major an adverse effect as the loss of lymphoid tissue? Would it be possible to run total paren- teral nutrition in a group of rats in which you have removed large segments of ileum?

Peter Liebert (Philadelphia): I would like to ask whether the authors considered the use of passive immunity as a continuation of their experiments? Many of us, in the days before we knew about immunoglobulins, had a blind faith in the use of plasma in neonates who required major procedures and, especially, massive bowel resections. I wonder if the clinical impression from that experience can be substantiated in terms of experimental work?

Stephen Shochat (Stanford): I wonder whether a control group of animals in which the distal bowel was excluded but left in situ would help you get to the bottom of the nutrition versus the humoral immunity problems?

Karen West (Closure): Malnutrition per se was not assessed in this study, although we plan

E. COLI AND ENDOTOXIN IN BOWEL RESECTION 849

to reevaluate our data using TPN as a variable. A previous report by Dr. Malangoni (a coau- thor) showed that 90% jejunoileal bypass (which should result in a similar degree of malnutrition) did not make rats more susceptable to endotoxin challenge. Furthermore, as both E. coli and endotoxin challenged animals had the same amount of small bowel resected, they should be similarly nourished. In controls, the small bowel

was eviscerated for about 6 min of anesthesia time, the same as it took to do the bowel resec- tions. Dr. Rowe, we used a single batch of Sigma E. coli endotoxin for all of our experiments. The results obtained were similar to Dr. Malangoni 's study, which was reported about a year and a half ago although these were indeed different batches of endotoxin.