Functional innervation of the isolated bowel segment

Functional

Innervation

By Engin

Gtinel,

Ayse

of the Isolated Sahin,

Fatma Kon ya,

Purpose:The aim of this study is to investigate whether there might be an eventual change in the enteric nerve responses to electrical field stimulation (EFS) of the isolated bowel segment (IBS) created by omentoenteropexy. Methods: In the experimental group, an IBS was created in 10 rats using the omentum as the host organ by dividing of its mesentery 4 weeks later. In the control group, a jejunal loop of identical site and length to the IBS was studied in another 10 rats as normal jejunal segment. Longitudinal muscle strips were prepared from the IBSs (n = 20) and the normal jejunal segments (n = 20). The effects of atropine, tetrodotoxin, L-arginine, and L-nitroarginine methyl ester (L-NAME) on the responses to transmural EFS were examined in both the IBS and normal jejunal segment using the isometric tension recording technique. Results: Transmural EFS of the IBS strips tile responses. Atropine and tetrodotoxin

produced abolished

contracthe EFS-

REATION of an isolated bowel segment (IBS) has been attempted using a subcutaneous tunnel, the abdominal wall (myoenteropexy), and the liver (hepatoenteropexy) as host organs after division of the mesenteric vessels.1-3 Their functional studies proved that the motility and absorption capabilities of the IBS after mesenteric division remained norma1.4-6 In a previous study, we presented the technique for creation of an IBS in rats using omentum as the host organ when its mesentery was divided 4 weeks after omentoenteropexy. Viability of such an IBS was documented by histological observation at the level of the omentoenteropexy.7 The question remained as to whether it would be suitable for segmental esophageal substitution, bowel reconstruction, and by its lengthening procedure for surgical treatment of short bowel syndrome. The aim of this study is to investigate whether there might be an eventual change in the enteric nerve response to electrical field stimulation of the IBS, created by omentoenteropexy. Using the isometric tension recording technique, the functional innervation of the IBS was compared with that of the normal jejunum.

C

MATERIALS

AND

METHODS

Twenty Sprague-Dawley rats (180 to 240 g) were equally divided in to experimental and control groups, and anesthetized using intraperitoneal ketamine hydrochloride.

Journal

of Pediatric

Surgery,

Vol34,

No 3 (March),

1999:

pp 387-389

Caglayan,

and

Bowel ishak

Segment

Akllhoglu

Turkey

induced contractions of the IBS. Moreover, EFS-induced contractions were increased by the application of L-NAME, and were decreased by the application of L-arginine in the IBS. Mean percent change of IBS’s contraction was not found statistically different from mean percent change of normal jejunal segment’s contraction on the effects of L-NAME and L-arginine.

Conclusion: These results suggest that the IBS, created by omentoenteropexy, produced enteric nerve responses to EFS as seen as in normal jejunal segment. J Pediatr Surg 34:387-389. Copyright o 7999 by W.B. Saunders Company.

INDEX WORDS: Isolated functional innervation.

bowel

segment,

omentoenteropexy,

Creation of the IBS In the experimental group, the abdomen was entered by a midline incision. A lo-cm-long jejunal loop was isolated with the mesentery beginning 20 cm distal from the ligament of Treitz to create the IBS. The remaining bowel was reanastomosed to maintain bowel continuity. The omentoenteropexy was performed as described previously.7 A longitudinal seromuscular incision was made on the antimesenteric border on the IBS. The incision line was fixed to a pedicle of greater omentum and sutured with running 5-O Vicryl. Both IBS ends were sutured over, creating a closed loop in the abdominal cavity. The abdomen was closed in one layer using 3-O Prolene. After fasting for 24 hours, a standard rat diet was started. Four weeks after the first operation, the abdomen was reentered with the animal under general anesthesia. The mesentery of the IBS was ligated and divided in 10 rats. The IBS was then totaly free of its mesenteric blood supply. The abdomen was again closed in one layer using 3-O Prolene. All rats underwent reoperation 48 hours after division of the IBS mesentery. The IBS and the omental pedicule were resected en bloc. In the control group, the abdomen was entered by a midline incision, and a jejunal loop of identical site and length to the experimental group was resected in 10 rats in the first step of the procedure.

From the Departments of Pediatric Surgery and Pharmacology, Medical School of SelFuk University, Kenya, Turkey. Address reprint requests to Engin Giinel, MD, Assistant Professor of Pediatric Surgery, Babalzk mah. Vatan cad. Kartal Sitesi No:12/5 42040 Kenya, Turkey. Copyright o 1999 by WB. Saunders Company 0022-3468/99/3403-0003$03.00/O

387

GtiNEL

ET AL

Tissue Preparation The mucosa was removed from each specimen, and two longitudinal muscle strips, approximately 15 mm long and 3 mm wide, were prepared from each IBS and normal jejunal segment in the control group. These strips were mounted between a pair of parallel platinum electrodes and placed in 25-n& organ baths containing Krebs-Henseleit solution, aerated with 95% 0s and 5% COz, and maintained at 37°C. The composition of this physiological solution was (mmoUL): NaCl, 119; KCl, 4.7; MgS04, 1.5; KH2P04, 1.2; CaCl, 2.5; NaHCOs, 25; Glukoz, 11. The resting tension was adjusted to 1 g, and the tissues were allowed to equilibrate for 1 hour before the start of experiments. During the equilibration period, the bathing medium was replaced every 15 minutes.

Electrical Field Stimulation To obtain nerve-mediated contractions of jejunal strips, the tissues were subjected to EFS via parallel platinum electrodes by a dual impedance stimulator (Harvard). Stimulation lasted for 20 seconds at 100 V, with 0.5.millisecond pulse duration, at a frequency of 10 Hz. Mechanographic techniques were used to study the specimens in vitro. The contractions were recorded isometrically using an oscillograph (Harvard).

Drug Responses After control responses to EFS, the jejunal strips were incubated with a single concentration of atropine (1 X 10m6 mol/L, Sigma, St Louis, MO), L-arginine (1 X 10m4 mol/L, Sigma), L-NAME (1 X 10e5 moUL, Sigma), or tetrodotoxin (3 X IO-’ mol/L, Sigma) for 20 minutes, and EFS was repeated. EFS was repeated at 2minute intervals after at least three consistent contractions had been obtained. The Krebs-Henseleit solution always contained phentolamine (1 X 10m5 mol& Sigma) and propranolol(1 X 1 Od5 mol/L, Sigma) to minimize adrenergic activity in response to EFS. We observed separately the effects of atropine, L-arginine, L-NAME, and tetrodotoxin on the response to EFS using different preparations. All drugs were dissolved in distilled water and added to the organ bath in volume of 0.1 mL.

Statistical Analysis The effects of these agents on &he EFS-induced contractions were expressed as a percentage of the mean 2 SD of the three consecutive control responses before drug addition. For statistical analysis, unpaired Student’s t test was used. A P value of less than .05 was regarded as significant.

RESULTS

In the experimental group, EFS (100 V, 0.5 millisecond, 10 Hz) of the IBS strips produced reproducible contractile responsesasin the control group. It was thus uncertain whether responsesto EFS acted via the nerves or by a direct effect on the smoothmuscle.To clarify this point, the effects of EFS after blocking the nerves with tetrodotoxin were studied.Tetrodotoxin completely abolished the EFS responsesin both the IBS and normal jejunal segment. In the presence of 1 pmol/L atropine, EFS-induced contractions were abolishedin both the IBS and normal jejunal segment,which suggestedthe contractions to be mediatedby the cholinergic excitatory nerve.

A

L.ARG

Fig 1. The neurogenic responses Hz1 by the application of L-arginine segment [A) and the IBS (B) after cholinergic nerves. L-arg, L-arginine.

EFS

to EFS (100 V, 0.5 millisecond, 10 11 x IO-41 in the normal jejunal blockade of the adrenergic and

To determine whether the nonadrenergicnoncholinergic (NANC) inhibitory nerves were present in the IBS, the effects of L-NAME and L-arginine on EFS-induced contractionswere studied.EFS-induced contractionswere decreasedby the application of L-arginine (Fig 1). Mean percent change of IBS’s contraction (33.59 -t 2.96) also was not found statistically different from mean percent change of normal jejunal segment’s contraction (31.48 -t 2.48, P = .84) on the effect of L-arginine (Table 1). However, EFS-induced contractions were increased by the application of L-NAME, an NO synthaseinhibitor (Fig 2). Mean percent change of IBS’s contraction (22.06 2 3.34) was not found statistically different from meanpercentchangeof normaljejunal segment’scontraction (23.82 If: 3.84, P = .49) on the effect of L-NAME (Table 1). These results indicated that the enteric nerve responsesto EFS, mediatedby NANC inhibitory nerves, occured the same in both the IBS and normal jejunal segment. DISCUSSION

An IBS, that was preserved from normal bowel structures, can safely be created in the rat when its mesenterywasdivided 4 weeks after omentoenteropexy.7 We have proposedthat the IBS might be useful for bowel reconstruction and by its lengthening procedure for surgical treatment of short bowel syndrome becauseit offers a wider range of eventual clinical applications by virtue of its mobility.7 A technique for creation of an IBS in rats using omentum as the host organ was also presentedby Shoshany et alga9But they concluded that the appropriatetime for neovascularizationwas7 weeks.89 Viability and absorption capability of such a bowel had beendocumentedby experimental studieson the IBS, but motility studies of the IBS have not been documented yet.7,9,10 Table

1. Mean

Jejunum

Percent

Change

of Strips’

and the IBS by the Application

Contractions of L-NAME

NJS

L-NAME L-arginine Abbreviations:

23.82 31.48 NJS,

L-arginine

IBS

2 3.84 + 2.48

normal

of the Normal and

jejunal

22.06 33.59 segment;

s

t 3.34 k 2.96 S, significance.

P=

.49

P=

.84

FUNCTIONAL

*

EFS

INNERVATION

C.NAME

OF IBS

389

EFS

Fig 2. The neurogenic responses to EFS (100 V, 0.5 millisecond, 10 Hz) by the application of L-NAME (I x 1O-5) in the normal jejunal segment (A) and the IBS (B) after blockade of the adrenergic and cholinergic nerves. L-NAME, L-nitroarginine methyl ester.

In this study, we compared the contractility of the muscle strips of the IBS and control jejunum with or without influences by the agents mentioned above. Response of the prepared strips to transmural EFS were

examined in vitro using isometric tension recording technique. Responses to EFS after blockade of the adrenergic and cholinergic nerves in the IBS were found to be the same as the responses that were found in the normal jejunal segment, and the responses mediated by the NANC inhibitory nerves of the normal jejunum were also presented in the IBS specimens. Therefore, the neuroenteric structure of the IBS has not been interrupted during the neovascularization period from the omentum. Results of this study suggest that elimination of extrinsic innervation of an IBS by dividing its mesentery does not influence autonomy of myoelectrical changes. We propose that the IBS, created by omentoenteropexy, produced normal enteric nerve responses to EFS as seen in normal jejunal segment.

REFERENCES 1. Ienaga T, Kimura K, Hashimoto K, et al: Isolated bowel segment (Iowa model I): Technique and histological studies. J Pediatr Surg 25:902-904, 1990 2. Kimura K, Soper RT: Isolated bowel segment (model I). Creation by myoenteropexy. J Pediatr Surg 25:512-513,199O 3. Yamazato M, Kimura K, Yoshino H, et al: Isolated bowel segment (Iowa model II) created in the functioning bowel. .I Pediatr Surg 26:780-783, 1991 4. Jaume J, Kimura K, Ratner L, et al: Functional studies in an isolated bowel segment. Clin Res 35:797A, 1987 5. Yoshino H, Kimura K, Yamazato M, et al: Isolated bowel segment (Iowa model II): Absorption study of glucose and leucine. .I Pediatr Surg 26:1372-1375, 1991 6. Kimura K, Soper RT: A new bowel elongation technique for the

short-bowel syndrome using the isolated bowel segment Iowa Models. J Pediatr Surg 28:792-794, 1993 7. Gtinel E, Tavh L, Caglayan F, et al: Isolated bowel segment created by omentoenteropexy: Histologic findings. Pediatr Surg Int 12:364-366, 1997 8. Shoshany G, Cohen E, Mordohovich D, et al: The isolated bowel segment (IBS) created in animals by omentoenteropexy. J Pediatr Surg 29:1344-1351, 1994 9. Shoshany G, Mordohovich D, Lichting H, et al: Preserved viability of the isolated bowel segment, created by omentoenteropexy: A histological observation. J Pediatr Surg 30:1291-1293, 1995 10. Shoshany G, Diamond E, Mordechovitz D, et al: Jejunal mucosal function of the isolated bowel segment created by omentoenteropexy in dogs: A study by in situ hminal perfusion. J Pediatr Surg 30:402405,1995