大腸全摘後の下痢改善のための新しい手術術式の工夫(ileojejunal transpositionの消化管平滑筋運動に対する効果のin vivo及びin vitroでの検討)

大腸全摘後の下痢改善のための新しい手術術式の工

夫(ileojejunal transpositionの消化管平滑筋運動

に対する効果のin vivo及びin vitroでの検討)

著者

佐々木 巌

大腸全摘後の下痢改善のための新しい手術術式の工夫

(ileojejunaltrahspositionの消化管平滑筋運動に対する

･効果のinvivo及びinvitroでの検討)

(研究課題番号12557102)

平成1 2rl.4年度科学研究費補助金(基盤研究B2)

研究成果報告書

平成15年3月

研究代表者:佐々木巌

(東北大学大学院･医学系研究科･教授)

目 次

はじめに

研究組織

研究経費

研究発表

● 1 ● ● u ii 門間

研究成果

1 ･ Ileo-j'ejunal'transpo岳itionの胃排出と便性状に及ぼす

影響に関する結腸全摘犬を用いたinvivoの研究

(Effect of neo-jejunal･ transposition on g*C. emptying and定al

water contentindogs withtotalcolectomy)

2 ･ Ileo-jejunal'transpositionの回腸縦走平滑筋収縮に対

する影響に関するラットを用いたinvitroの研究

(EBTect of nee-jejunaltransposition onileal1｡ngitudinalsmo｡th

muscle contractile activityinratsinvitro) A

はじめに

大腸全摘･回腸征門吻合術は､潰癌性大腸炎､家族性大腸腺腫症に対する標準術

式であり､自然虻門を温存してこれらの疾患を根治出来る優れた術式である｡し

かしながら､大腸欠落による下痢が必発であるのが難点である｡多くの症例にお

いては時間経過と共に下痢は改善し､最終的には1日5-8回の排便回数にまで

減少するが､下痢が改善しないために日常生活に支障をきたしている症例も少な

くない｡下痢の改善薬剤も存在するが､大腸全摘後の難治性の下痢には無効であ

ることが多く､期待は出来ない｡

そこで､ ･本研究は､･外科的に大腸全摘後の難治性の下痢を改善する試みを動物で

行い､その消化管運動に対する効果について3年間にわたり検討した｡外科的手

術により消化管運動を抑制出来れば､それが下痢の治療に結びつく可能性がある｡

具体的には､ ileojejunalkmSPOSidon (町と大腸全摘をイヌで､ DT単独をラット

で行い､イヌにおいては胃排出と便中水分含有量･便の形態を､ラットでは回腸.

縦走平滑筋収縮を､検討した｡その結果､イヌではmを行うことにより胃排出

が遅延し､大腸全楠にmを併施した方が､大腸全摘単独群よりも便中水分含有

量が減少し便の形態も正常に近付くことが明かとなった｡また､ラットでは回腸:

縦走平滑筋収縮がDTにより抑制されること､ UTによってアセチルコリンに対す

る感受性が上昇することが判明した｡このような結果は､ mを臨床応用して行

く際の基礎的事実として重要である｡

今後もuTのを臨床応用出来るよう､検討を重ねて行く予定である｡

1

研究組織

研究代表者:佐々木巌 東北大学大学院･医学系研究科･教授

研究代表者:舟山裕士 東北大学･医学部附属病院･講師

研究代表者:福島浩平 東北大学大学院･医学系研究科･.･助手

研究代表者:柴田 近 東北大学･医学部附属病院･助手

研究経費

平成1 2年度10,302千円

平成13年度 2,100千円

平成1 4年度  600千円

合計   13,002千円

n

研究発表

Ueno T, Shibata C, Naito H, JinⅩし, Funayama Y, Fhkushlma K, Matsuno S, Sasaki I: Ileo-jejunaltran声POSition delays gasbic emptying and dareases fecalwater contentindogs withtotalcolectomy. Di島

目 次

#lG･ PARTI

Ileo-jejunaltranspositionの胃排出と便性状に及ぼす影響に関す

る緯腸全摘犬を用いたinvivoの研究

E触ct Qf neq･jejun山trwsposition on gastric emptyingand触1

water content in dogswith total colectomy

ABSTRA CT

INTRODU CTION

MATERIALS AND METHODS

Pt'epamtion of amimals

ExperimentalprotocoI

Data aLnalysis '. L

RESULTS

Gastric emptying I..

Gastrointestinalm'otility

Phsma concentrations of PYY

Fbcal water content.and stool form

Body weight and laboratory data

DIS CUSSION

A CENOⅥ牡一EDGMENT

REFEKNCES

TABLE

FIGURES

1 3 4 4 7 9 10 10 10 ll ll 12 12 17 18 22 24

#2#･ PART2

Ileo-jejunaltranspositionの回腸縦走平滑筋収縮に対する影響に

関するラットを用いた血vih:oの研究

Effect of止eo･jejunaltransposition on ileal longitudinalsmooth

musde contmctile activityinrats invitio

ABSTRACT

INTRODUCTION

●

MATERIALS ANDMETHODS

a

Preparation of amimds

Recording of contfatde activity

Experimental design

Data analysis

StatiStical comparisbns

Drugs

RESULTS

SpontaneousContractile activity

Response to bethanechol and noreplnePhrine

● Response to electrical丘eld stimulation

DIS CUSSION

A CKNOWLEDGMENT

REFEKNCES

TABLE

PART 1: EFFECT OF mEO･JEJUNAL TRANSPOSITION ON GASTRIC

EMPTYING AND FECAL WATER CONTENT IN DOGS WITH TOTAL

COLE CTOMY

ABSTRACT OFPART 1 I

Purpose: Theaim of the study was to investigate the effect of ileo-jejunaltransposition,

in which the distalilsum is interposed isoperistaltically into the proximal jejunum, on gastric emptying, gastrointe,stinalmotility,and fecalwater content in dogs with totalcolectomy･

Methods: D也gs were divided rinto three groups; _d9gS With intac.t colons (Control), total

colectomy alone (shamoperated group), or totalcolectomyand･ ileorjejunaltranspositiorll (UT･ A , ...

group). Thealimentary tract was reconstructed by ileai JIPOuCh-rectalanastomosis. Gastric

emptying was. measured by a validatedfre由6-drying method,and gastmintestinal mdtility''

was measured by strain gauge force transducers･ Plasmapeptide YY was measured by specific

radioimmunoassay･. Fecalwater content was measured in dogs withtotalcolectomy･

Results: Gastric emptying of solids in IJT group.was delayed later than120minut甲

after meal-ingestion compared to that in sham operated ･group. The duration of the digestive

state was prolonged in m group, only when compared to the control group･ Plasma peptide

YY was increased in IJT group compared to sham operated group･ Fecalwater content was decreased in IJT group compared to sham operated group･

Conclusions: Ileo-jejunal transposition delays gastric emptying of solidsand decreases

might be able to improve intractable watery diarrhea after totalcolectomy.

Key words: ileo-jejunaltransposition, peptide YY, gastric emptying, migrating motor

INTRODUCTION

severeand intractable diarrhea after totalor subtotalproctocolectomyperformed for

ulcerative colitis or fmi1ial adeno/matous polyposis interferes with patients, quality of lifel ･

This diarrhea largely ･arises because of the lack of colonic absorpt10n Of water in the stool･

watery diarrhea after these operations improves depending on the postoperative time, as the

small intestine adapts by increasing its capacityto absorb water2, changes in plasma

concentrations of selected peptide hormones3,and changes in gastmintestinal motility4･ we

studiedthe ,･effects of: ileo-jejumaltransposition (IJT) on release of peptide hormones･ morphology of. intestinalmucosa,血d gastmintestinalmotility in dogs with intact colons5･ 6･

because IJTmight be･ us的I to.improve watery diarrhea after totalcolectomy by enhancing

adaptation. IJT involves interposing one fourthof the length of the jejunoileum

isoperistaltically into the proximal-jejunum･ In dogs with intact colons･ IJT leads to marked

mucosalhypertrophyalong the whole small intestineand inhibition of gastric emptyingand

motility5-7･ These changes appear to be mediated by increases in plasma concentrations of

enteroglucagon and/or peptide YY (PYY) in response to contact of endocrine cells in the ileal

mucosa withundigested chyme. These findings suggest that ･IJTmight be effective in

improvlng diarrhea after totalcolectomy･ Study of dogs withtotalcolectomy is the next step

toward clinicalapplication of this operative procedure･ In dogs after totalcolectomy･ we

previously reported mucosalhypertrophy inthe small intestineandanincrease in plasma

enteroglucagon8 ; however, gastrointestinal motility, transit,and the character of the stool were

not studied.

Theaim of the present study was to investigate in dogswithtotalcolectomy,the effect

of IJT on gastric emptying, gastrointestinalmotility, plasma concentrations of PYY, water

content inthe stool,and consistency pfthe stool･ Our hypothesis was that gastric motilityand

emptying would be inhibited bythe increaseinplasma PYY,托Calwater content would be

decreased,and consistency of the ･.stool would improve, compared to dogs after total

colectomy butwithout Err.

A4ATEM ANDMETHODS

PreDarations of amimds: Procedur郎and subs印uentanimalCare were undertaken I

∝cording tothe Guide forthe Careand Use of Labwatory Animals (Tohoku University).All I

･surgiCalpreparati.ons wereperformedwithsterile techmique. Mongrel dogs weighing 12-17- kg ,

were used.All dogs wereanesthetized byanimitialintravenousinjection of 20 mg瓜g

thiopental(鮎vonar@; Tanabe■ Seiyaku C0., Osalq Japan)then maintained byaninhal血on.

of halothane (muothane⑳; Takeda Chemicals C0., Osaka)and oxygen.

Four mongrel dogs were used as controls for measurement of gastric emptying; a l･

silicone tube (SH No. 2; Create Medic Co., Yokohama, Japan) was positioned inthe flrSt

portion of the duodenumwiththetip atthe level of the main pancreatic duct. A catheter Crop

extension tube X2-50; Meditop Col, Tokyo, Japan) was positioned in the duodenallumen

withthetip 20 cm distaltothe main pancreatic duct. The proximalsilicone tube was tunneled subcutaneouslyand exteriorized through a stab wound betweenthe scapulae;the distaltube

was connected to a metal Cannula exteriorizedthrough a stab wound in theright

gastrointestinal motility. Eight strain gauge fわrce transducers (F-12IS, Star Medical lnc･,

Tokyo, Japan) were implanted onto the serosalsurface of the gastrointestinal tract at the

gastriCantrum 4cm proximal tothe gastroduodenal junction, the duodenum at the level of the

main pancreatic duct,and 6 sitesalong the jejunoileum 5, 30, 60, 150, 220, 235 cm血)m the

ligament of Treitz･ Lead wires fromthe transducer were tunneled subcutaneouslyand

exteriorized through a stab wound between the scapulae･ A silicone tube was positioned in the

superior vena cavavia the right extemaljugularvein for intravenous access･ The upper body

of the dog was covered with a canvas jacket to protectthe silicone tubeand wires from self- ∼

inflicted･ trauma･. Fourteen days after the surgery wereallowed as a recoveryperiod forthe

control dogs･ Dogs were.･fasted for the first 3 days after surgery･ and on each day dming this

･-period the dogs were given 1,000血L of Lactate Ringer solution intravenously (Lactec G⑳

Na: 130 HiEqA; Cl: 109 mEqm, K: 3 niEqn-, Calory: 200 kcalA; Otsuka PhamaceuticalCo･, Tokyo) contaiming I g of flomQXef sodium (Flumarin@, Shionogiand Col Ltd･, Osaka)･ The

dogs were fed solid 'food (CD-55α; Clea Japan, Inc･, Tokyo) at the rate of 20 g/kg once a day

after postoperative day 4･ This solid food contained 25g protein, 8g lipid･and 214kcalper 100

g･

Eight other dogs underwent totalcolectomy･ The teminalileumandthe rectum were

transected 5cm proximaltothe ileocecaljunctionand 5 cm proximal toperitonealreflection,

respectively,and the colon was excised. The dogs were divided into 2 groups (IJT groupand

sham･operated group) according tothe reconstruction method･ In the IJT group, the ileum was

transected 60 cm proximal to ･the ilealstump,and this ilealsegment was isoperistaltiCally ･

interposed intothe proximaljejunum 15cm distalto the ligament of Treitz. Hand-sewn

end-to-end jejuno-ileostomyand end-end-to-end ileo-JeJunOStOmy Wereperformed. The most distal

ileum after this procedure (60 cm proximal to the ileocecaljunction) was used to construct

ilealJ shaped pouch 10 cm long･ The ileum wasfolded 10 em proximal to the ilealstump,and

a･stapler (Linea Cutter⑳', Ethicon Japan, Tokyo) was inserted into the ileal1umtn via a small

stab wound in the tip of the pouchand fired twice to complete the ilealJ pouch･ Intestinal

continuity was restored by side-to-end ileal ∫ pouch-proctostomy (Figure 1). In仙e sham

operated group･the J-shaped ileal･ ･pouch was constructed using the most distalileum. Creation

ofanilealJIPOdch and reconstruction ofthealimentary tract were caLried out as in dogs ih

IJT group･ (Figure l･)･ The jejunum 15and 75 cm distal= to､the ligament of Treitz･ was transectedand immediately reanastomosed in the sham operated group to control forthe small

intestinalanastomoses at ithese sites in dogs withIJT･ Insertion of a silicone･ tube into the

superiorvena cava was performed as control dogs･ The upper body was covered witha canvas

jacket to protectthe silicone tube from self-innicted′ trauma. Twenty-eight days afterthe

surgery wereallowed as a recoveryperiod for dogs with totalcolectomy･Al1. dogs with

colectomy were fasted forthe first 5 days after surgery'and on each day dming thisperiod the

dogs ･were give･n I,000 mL of Amino Fluid solution intravenOuSly (Aminonuid@ Na: 35

niEqn･, Cl: 35 mEq凡, K: 20 mEq凡, Calory: 420 kcal凡; Otsuka PharmaceuticalCo.)

containing I, g offlomoxef sodium(Flumarin⑳). The dogs were fed solid food (CD-55α, 20

gnig) once a day after postoperative day 6･ A second operation wasperfomed on 8 dogs that

and 4 inthe shanoperated group. Silicone tubes as inthe control dogs were inserted for

measurement of gastric emptying. Other 8 dogs (4 forthe IJT groupand 4 forthe sham

operated group) underwent implan申tion of 8 strain gauge force transducers atthe same sites

as inthe control dogs. The postoperative care of the second operation wasthe same as forthe

co山rol dogs,and gastric emptyingand gastrointestinalmotility were measured 2 Weeks after

the second operation.

Experimenbl Protocol: Gastric emptying of･ solidand liquid was measured by a

previotkly validatedfreezeJrying method by Tanaka et･･alg･ Dogs were fastedfor 16 hours 1 before each expenmentand res血ned in a Pavlov sling ･duringthe experlmentS･ Amixture of

50 g･.solid fbd･,(ED1,･ 196･kcal)and lOOmiof 154 1mM NaCI solution contaiming 15%

polyethyle41eglycol (PEG, P-3640; Sigma ChemiCalCo., St. Louis, MO) was used as a test

meal･ This solid food contained 25･9 g protein, 8･2 'g lipid,and 7･6 g water per lOOg･

Fhenolsulfonphtalein(甘SP, Wako Pure ChemiCalIndustries, Ltd･, Osaka) solution (500 llg/mi,

pH 17.0) was infused at 3.8 Tnl/min throughthe proximalduodenaltube byaninfusion pump

(TE-151, Terumo Co., Tokyo) after warming to 370c by a magnetic stirrer (RCH-3, Eyela Co., Tokyo). The PSP was used to measure duodenalvolume. After a 30minute equilibration

period, 1.5 ml samples of duodenalcontents were collected throughthe distalduodenaltube

at 5-min intervals. Three consecutive samples were combined to produce a 151min sample;

gastric emptying at 6 hours after mealingestiOn was determined forthese 15-min intervalS･

After 2 more successive samples,all dogs were glVenthe test mealand nnished eatingwithin

5min. Concentration of PEGand触eze-dried absolute weight of each sample were. used as

the liquidand solid markers, respectively. The PEG concentration was calculated according to

the method of HydenlO. The duodenal sample (0.25 ml) was diluted to 5.0 ml wi血154mM

NaCI solution,then l･0mi10 % Ba,Cl2 , 2.0mi0.3 N Ba(OH)2and 2.0mi5 % ZnSO4 ･ 7H20

were added･ Thermixture was vortexedand filtered usingWhatman44 filter paper (Whatman

IntemationalLtd., Maidstone, England). The PEG concentration of each filtrate was

detemined at 650 nm by ･･a spectrophotometer (U- 1 100 spectrophotometer, Hitachi, Tokyo)

after adding 30 % trichloroacetic acid (TCA, Wako Ptlre ChemiCalIndustries, Ltd.). The same ･ filtrate was used to measurethe PSPI COnCentration by spectrophotometry at 560 nm after the

addition of 10 % ･NaOH. Onemiof each sample was completely driedina centrifugal

evaporator (vc-'360; Taitec Co., Saitama, Japan)and weighed:All these values of･PEGand

PSP concentrations,and absolute weight were applied to the appropriate formulas,and liquid and solid gastric emptying was me.asured simultaneouslyand separately. The gastric emptying

cuⅣe was血awm by subtracting･the weight of fわod emptied into the duodenum every 15 min ･

fromthe absolute weight of test meal(50 g) which was taken as lO0%.

Gastmintestinalmotility was recorded from the transducers toanamplifier (MS-08, Star

MedicalInc･),andthen to a computer program(Chart; AD Instrument, Victoria,I Australia)

throughMacLab (AD Instrument). Recording of gastrointestinal motility was started after 20

hour fasting. After 4 hour recording, Canned food (Vita One Crux; JapanPet Food Co., Tokyo,

25 gn(g) was given as a test meal･ This canned food contained lO% protein, 5% lipids,and

82% waterand had 85 Caloriesper lOOg.All dogs ate it within 5minutes. Recordings were

Blood samples were drawnfromthe silicone tube inthe supenor vena cava 5min before

and at 0, 5, 15, 30, 60, 90, 120, 180, 240min afterthe mealingestion during each experlment

to measure gastric emptying; /Samples were stored in chilled glass-tubes containing

ethylenediaminetetraacetic acid (m)and aprotinin･ Samples were centrifuged at 3000 rpm

for 15min,andthe plasma was separated･and stored at 400Cuntil assayed･ Plasma

concentrations ofpeptide YY (PYY) Were measured by a speciBc radioirrmoassay kit (RIK

7173; Peninsda Lab., hc., Sam Cados, CA).

Fd water content wits meaSufed 4 Weeks aftet.the imitialoperationindogswithtotal

colectomy･ :Feces samplesIWere COllectedfrom part of stool evacuated forthe丘rst･time after

9:00amfor consecutive 2 days. Within lminutes afterthe evacuation, Samples were weighed,

then driedwitha centrifugalevaporator (VC-360) for 12 hours･ The percentfeCalwater

content was calculated bythe ratio of dry w,eight to wet weight･ Photograph of feces was

taken to judge grade of stool･ forms. The grade of stool forms was determined according to a

previous reportll byaninvestigator who had no information aboutthis experiment･ Body

weight was measured beforeand 4 Weeks afterthe nrst operation,andpercent change was

calCulatedindogswith. totalcolectomy. Hemoglobinand･ totalprotein in･ plasma were

measured 4 Weeks afterthe伽st operation in dogswithtotalcolectomy･

Data atlalysis: Gastric emptying was measured twice in each dogandthe mean of two

studies was regarded as a representative value forthat dog･ Percent mealremamng inthe

stomach 60, 120, 180,and 300min after feeding was calculated to quantitate gastric emptying

of the liquidand solid, respectively.

We measured the duration of each phase of migrating motor complex (MMC). Phase I

is a period of quiescence,and irregular contractions occur during phase II･ Phasic contractions

with highamplitudeare regularly 9bserved during phase III. These phase Ill contractions

rmgrate from the stomach to the ileum;theperiod of the MMC cycles are loo-110min in

normaldogs12･ The duration of the digestive state, defined as the time from feeding to the first appearance of phase 班, was measured at the jejunum 5 cm distalto the ligament of Treitz.

Analysis of gastrointestinal motility wasperformed by visual inspection.

Values were expressed as meant Standard error of the mean(SEM),and p values less

than O･05 were regarded as signはcant.

RESULTS

GAStrlc emPtyin些: Tb were no differences in gastric emptying of liquids at 60, 120,

180,and 3001min after fbeding･ (Rgure 2A)･Alsothere were no differences inthe gastric

emptying curve of solids between cbntrolandthe shamoperated groups･ The gastric emptying

ctm of solids inthe-IJT group was simi1artothe other 2 groupsuntil 120min after fieding,

but was delayedand shifted totheright after120min (Rgure 2B). Thus,the percent of solids

remaining inthe stomach inthe IJT group was greaterthanthat in shamoperated group at 180

and 300min after feeding (Table 1), suggestingthat emptying of solid was delayed 120min

after feeding.

GaBtrOhtesdd JnOdJlty: We were able to detemine differences ill POStPraJldialaJld

hterdigestive pattems in control dogs;the MMC cycle was observed regularlyinthe stomach

触quency were induced by feeding;the MMC reappeared aftertime･ Postprandialand

interdigestive pattems were distinguishablein3 of 4 dogs in IJTand shamoperated groups

(Rgure 3), respectively; cyclic changes in motility similarto MMC were observed inthe

small intestine･ We, however, could not distinguish between posqmdialand interdigestive

pattems in I dog each inthe IJTand shamoperated groups･ The duration of the digestive state

was measurable, but measurement of the duratiop of each phase of the MMC was impossible

becapsethe change from phase I to rLand II to III wasunclearmost of the time. The duration

of the digesdve state inthe upperjejunuminthe IJT goup (ll.7土2.0 hours) was prolonged

compared to the･ controIT (5･4土0.名･hdurs, p<0.05) but was not different bm ･the sham

operated group (8;3 i 2.8 hours).

PbLStn OmαntmtiotLB OfPYY: No differences were observed amongthe 3 groups in

plasma PYY before feeding (Rgure･4)･Althoughplasma PYY increased postprandially inall

groups;the increaseinthe IJT group was greaterthaninthe remaiming 2 grcrups after 90

minutes (Rgure 3)･ Totalplasma PYY O1240minutes after feeding inthe IJT group (17.8土

3･6 ng-240min/ml) was greaterthanremaining 2 groups･ (Control; 9.6土2.5 ng-240min/ml,

sham; 5.0土0.9 ng-240min/mi).

Fecd y&血o仙t血t JLnd Stool fo-: Feed water content in IJT group (82二3土0.4%)

was decreased compared to shamoperated group (84.2土0.7%, p<0.05). Grade of stool forms

was 3 in 3 of 6 dogsinm group, while grade 3 was not observed in shamoperated group

Crable 2)･ Number of dogs whose grade of stool forms was 5 was land 4 in IJTand sham operated groups, respectively･ Overall grade in IJT group was signincantly betterthanthat in

Slmoperated group (p<0.05) Crd)le 2)･

Body wei申t and laboratory data: Body weight 4 weeks after IJT (79土5 %) was not

different血omthat inthe shamgroup (82土5 %). Hemoglobinand totalprotein inthe IJT

group (9.1土1.3 g/dland 5.1土0･1 g/dl, respectively) were not differentfromthose in sham

operated group (8.1土1.3 g/dland 5･0土0･3 g/dl, respectively), either･

DIS CUSSION.

various surgicalprocedures have been proposed to improve diarrhea after total colectomy･ Sdme surgeons have suggested constructing apantiperstaltic segment or creating a valve inthe I small intestine13, whereas others proposed constructing a jejunalJ-pouch14, tor

interposlnganileocecalValve151Althoughthese procedures were designed to improve

diarrhea by delaying small intestinaltransit, none of these procedures has ever been clinically

useful. Our method･IJT i岳differentfromthese previousaltemations because IJT intended to

improve diarrhea by changlngthe plasma peptidemiheu to enhance the small intestinal

function. Our hypothesisthat IJTmight improve diaxThea after totalcolectomy was derived

from our previous findings in dogs withintact colons; gastric emptyingand motility were

inhibitedand mucosal hypertrophy was observedalong the entire small intestine5･6･ Delay of

gastric emptyingand inhibition of gastric motilitywould letthe chyme contact with small intestinalmucosa for longertime, and enhanced small intestinalabsorption associated with

mucosalhypertrophy would occur･ These changesmight contribute toanimprovement of

postcolectomy diarrhea･ These changes after IJT were thought to be induced by the increase

exposed toundigested chyme would release these regulatory peptides･ IJTmightfunction as a

chronic model of the 'ilealbrake/ a phenomenonthat ilealperfusion with nutrients inhibits

gastric emptying, small intestinaltransit,and gastmintestinalmotilityl…･ The ilealbrake is

believed to be mediated by increase in plasma concentrations ofpeptides releasedfrom ･the ileum, including PYY, glucagon-likepeptide I (GLP-I),and enteroglucagon17･ 19･ 20･ we

soughtthe effect ･of IJT on gastric emptying, gastrointestinal motility,and fecalwater content

in dogs with totalcolectomy as a next step toward clinical･application of IJT･

･ we found that gastric emptying of solids was delayed later than120min after托eding･

and fecalwater content was decreased in dogs after colectom･y with IJT compared to dogs after totalcolectomy without Lrr･･ Delay of solid gastrid emptying was not considered to

suppress appetite or affect generalcondition, because the postoperative body weightand

laboratory data did not differ between the IJTand the sham operated groups･ Total plasma

pYy secretion was increased in dogs withIJT 90min after fecding･ Because PYY inhibits

gastric emptyingand motilityW･ 21, it is possiblethat this delay of 'solid emptying was related to

increased plasma PYY･ PYY isalso regarded as a most likely mediator of ilealbrake, because

fat-induced slowing Of small intestinaltransit was normalized withimmunoneutralization of pYY22,andthe delay of gastric emptying was related to increases in plasma PYY23･ showing

the inhibitory effect of intravenous administration of PYY on solid gastric emptying in

conscious dogs must be important to detemine the definitive role of PYY･ We･ however･

Consider that PYY mediates the inhibitory effect of IJT on gastric emptying･although itmight

not bethe sole factor. We could not detemine the role of GLP-I,another possible mediator of

ilealbrake19, in dogs with IJT because we did not measure plasma GLPll･

Fecalwater content in dogs after totalColectomy with IJT was reduced compared to

dogs after totalcolectomy without一IJT, suggesting that IJT improves watery diarrhea after

totalcolectomy. Severalreports correlatethe character of the stooland gastric emptying or

intestinaltransit: Gastric emptylng Was accelerated inthe diarrhea-typeof imitable bowel

syndrome24･ stoolformis associated with total oro-anal transit time25･ These results indicate

that delay or acceleration of･gastric emptying could contribute tothe changes of water content

inthe stool･ Becausethese reports･dealt withhumanwithout colectomy･ we cannot conclude that their data support correlation between gastric emptyingand fecalwater content in. dogs I I I after colectomy. The possibility, however, remains that delay 'of solid gastric emptying ･in ･

dogs with IJTmight be one of the mechanisms decreaslng fecalwater content･ We did not

measure small intestinaltransit whichmightalso contribute to the character of the stool

especially in dogs after totalcolectomy･ Differences of fecalwater content betwecnthe IJT

and the sham operated groups were only about 2%, which do not seem a big diuerence･

wenzl etalreportedthat fecalwater content of formed, semiformed, soft･ semiliquid,and

runny stools was about 72, 82, 86, 89,and 91%･ respectively26, suggesting that difference of

fecalwater content even in a couple of percent affects fecalconsistency･ In fact･ stool forms inspected visually in IJT group were betterthanthose in shamoperated group･ The duration of

the digestive state in the upper JeJunum, Which correlates to gastric emptying27･ was prolonged

in dogs after totalcolectomy with IJT compared with control dogs･ This phenomenon must

of each phase of the MMC,althoughglobaldigestiveand interdigestive motor pattem was

distinguishable. We considered that this fact must be associated with adhesion after subtotal

colectomy･ because this phenomen,on was observed not only in IJT butalso in shamgroups･

･ Weperformed experiments 6 weeks after colectomy, because thisperiod was

considered long enough for postoperative various changes to occur; increase in pl･asma PYY

and enteroglucagonand hypertrophy of the small intestinalmucosa were observed 4 and 12

weeks after ileojejunaltransposition6･ 28. The possibility, however, Cannot be denied that

different kind of adaptation would be observed laterthan6 weeks after colectomy. We

perfomed ilealJ pouch-proctostomy to reconstruct thealimentary tract but not a side-to-end

or end･to-end ileoproctosotmy. This procedure was chosen tomimic ilealJ ･p･ouch-anal

anastomosis in human. It is possible thatthe existence of ilealJ pouch changed the plasma

peptidemilieu in the ･intetdigesti've stateand resulted in vanishment of each phase of the

MMC in dogs wi也colectomy.

Asa sham operated ･group we prepared dogs with totalcolectomy and jejunal

transectionand reanastomosis but not dogs withtotalcolectomyalone. This was to control for

certain changes in gastric emptying in dogs with IJT LaSSOCiated withtransectionand reanastomosis in the upper JeJunum. Transectionand reanastomosis atthe proximaland distal

jejunum affects the small intestinaltransitand contractile activity within the jejunalsegment

in conscious dogs29･

In conclusion, gastric emptying of solid was delayedand fecalwater content was

reduced in dogs after totalcolectomy with IJT, compared withdogs after totalcolectomy

without IJT･ These findings suggest the possibilitythat surgicalalterations of Lrrmight

improve severeand intractable diarrhea after totalcolectomy,and delay of solid gastric

emptying was considered as one it.its mechanisms. Delay of solid gastric emptying in dogs

A CKNOWED GMENT

The authors wish to thank Dr. Michael G. Sarr, Division of General and

Gastroenterologic Surgery, Mayo Clinic, Rochester, Minnesota for correcting the English in

this article.

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small intestine after proctocolectomyand ilealpouch-analanastomosis･Ann Surg

1985;201:351-6.

2. Mibu R,･Itoh H, Nakayama F. Effect of totaland mucosalproctectomy on intestinal

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3.1Amstrong DN, Ballantyne GH, AdrianTE, Bilchik AJ, McMillen MA, Modlin IM･

Adaptive imerease in peptide YY and enterolglucagon a鮎r proctocolectomy and ileal

reservour construction. Dis Colon Rectum 199 1 ;34: I 19-25.

4. Soper NJ, Orkin BA, Kelly KA, Phillips SF, Brown ML. Gastrointestinaltransit after proctocolectomy withilealpouch-analanastomosis or ileostomy. J Surg Res 1989;46:3001 5.

5. Toda M, Sasaki I, Naito H, Funayama Y, Kamiyama Y, Suzuki Y, Tsuchiya T, Matsuno S･

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6. Ohtani N, Sasaki I, Naito H, Shibata C, Tsuchiya T, Matsuno S. Effects of ileo-jejunal transposition on.gastric emptying, gastrointestinal motility,and small intestinaltransit in dogs. ∫ Gastrointest Surg 1999;3:516-23･

7. Tsuchiya T, Ishizuka J, Sato K, Shimoda I, Rajaraman S, Uchida T, Townsend CM Jr, Thompson JC. Effect of ileo-jejunaltranSposition on the growth of the GI tractand

8. Tsuchiya T, Sasaki I, Naito H, Narui H, Funayama Y, Suzuki Y, Toda M. The infhence of ileo-jejunaltransposition on intestinalmucosalgrowthand gut hormones after total

colectomy (Japanese). Jpn J Surg 1986;87:87017.

9･ Tanaka T, Mizumoto A, Haga N, Itoh Z. A new method to measure gastric emptying ln

conscious dogs:.a validity study･and effects of EMl523and LINNA.AmJ Physiol

1997;272:G909-G9 15.

10) Hyden S. A ･turbidimetric method for the detemination 'of higher polyethyleneglycols in

biologiCalmaterialS. K LantbrHosgskAnnr 1955; 22:139-45.

1 I) Meretz HR, Beck CK, Dixon･W, Esquivel MA, H.ays RD, Shapiro MF･ Validation of new

measure of diamhea. Dig Dis Sci 1995; 40: 1873-1882

12)Itoh Z,Alzawa I, Takeuchi S, TakayanagiR. Diumalchanges in gastric motor activity in

conscious dogs. Am ∫ Dig Di島 1977;22:28-35.

1. Williams NS,King RF. The effect of a reversed ilealsegmentand artificialValve on

intestinaltransitand absorption followlng COlectomyand low ileorectalanastomosis in the

dogs･ Br ∫ Surg 1985;72:169-74.

14) Takahashi M, Williams JW, Kelly KA. Proctocolectomy with jejunalpouch-distalrectal

anastomosis:analtemative to ilealPouch construction. J Gastmintest Surg 1998;2:250-9･

15) Degen LP, von Flue MO, Collet A, Hamel C, Beghger C, Harder F. Ileocecal segment

transposition does notalter whole gut transit in humanS.Ann Surg 1997;226:746-5 I.

16) Spiller RC, TrotmanIF, Higgins BE, Ghatei MA, Grimble GK, Lee YC, Bloom SR,

Misiewicz JJ, Sin DBA. The ilealbrake-inhibition of jejunalmotility after ilealfat

perfusion in man. Gut 1984;25:365-74

17) Soper NJ, Chapman NJ, Kelly KA, Brown ML, Phillips SF, Go ⅥJ. The `ilealbrake'after

ilealpouch-analanastomosis. Gastroenterology 1990;98: I 1 1-6.

18)'Cherbut C, Aube AC, Blo山ere HM, Galmiche JP. Effects of short chain-fatty acids on

'gastmintestinal motility･ Scand J GastroenteroI Suppl 1997;222:58-61.

19) Wen'J, Phillips SF, Sarr MG,. Kost LJ, Holst JJ. PYYand GLP-1 contribute to fTeedback

inhibition from the canine ileumand colon. Am J Physiol 1995;269:G945-G952.

20) Spiller･ RC, Trotman IF, ･A血ian TE, Bloom SR, Misiewicz JJ, Silk DB. Further

I characterization of the ilealbrake reflex in man-effect･of･ilealinfusion of partialdigests of fat, protein,and starch on jejunalmotility and r'elease of neurotensin, enteroglucagon,

and peptide YY. Gut 1988;29: 1042-51

21). Ebert R･ Control of gastriC'emptying by regulatory･ peptides. Z GastroenteroI Verh

1998;23 : 165-70.

22) 'LinHC, Zhao XT, Wang L, Wong H. Fat-induced ilealbrake in the dog depends on

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Poggioli G, Morselli Labate AM, MonettiN, GozzettiG, Barbara L, Go Ⅵ一W.

Fat-induced ilealbrake in humanS: a dose-dependent phenomenon correlated tothe plasma

levels of peptide YY. Gastroenterology 1993; 105 :733-9.

24) Charles F, Phillips SF, CamilleriM, Thomforde GM･ Rapid gastric emptying in patients

25) Heaton KW, 0'Donnel LJ.AnOffice guide to whole-gut transittime. Patients'

recouection of也eir stool fbm. ∫ Clin Gastroenterol 1994; 19:28-30

26) WenzI HH, Fine KD, Schiller LR, FordtranJS. Deteminants of decreased fecal

Consistency ln Patients with diamhea. Gastroenterology 1995; 108: 1729-38.

27) Read NW,Al-Janabi MN, Edwards CA, Barber DC. Relationship between postprandial

motor activity in. the human ･ small intestine and gastrointestinal transit of fわod.

Gastroenterology 1984;86:721-7.

28) Toda M, Sasaki I, Naito FI, Funayama Y, Suzuki Y, ■Takahashi M, Matsuno S. Effect of

I ileo-jejunaltransposidon (IJT) on gastmintestinal hormonesand intestinalstructure in

dogs (Japanese). Jpn J Surg 1989;90: 1879-89.

29) Jolmson CP, Sarna SK, Cowles VE, Baytiyeh R, Zhu YR, Buchmann E, BonhamL, Roza AM, Adams MB･ Effects bf transectionand reanastomosis on postprandialjejunaltransit

and contractile activity. Surgery 1995;117:531-7.

Table 1: Percent remalnlng in也e stomach of solids

Time after feeding (min)

60        1 20        1 80        300

Contro1   83 ± 2     59 ± 1     39 ± 1    14 ± 2

m    79 ± 2     62 ± 5     48 ± 6*     30 ± 8*

-Sham    80 ± 1     56 ± 4      29 ± 5      5 ± 3

●

Table 2 : Fecal consistency ln each group

UT*      Sham

Stool forms Grade 1    0

Grade 2    0 Grade 3    3 Grade 4    2 Grade 5    1 Grade 6    0 0 0 0 1 4. 0

*pく0.05,00mpared to Sham (Mann-Whitney's U test)

FIGURES

SLJbtotal colectorny

Figure 1 : Operative preparations.

The colon was transected 5 cm proximal to theperitonealrefkction. The distalileum 60 cm

long was interposed isoperstaltical･ly into the proximal jejunum in dogs withIJT･ The

proximal jejunum 15and 75 cm distalto the ligament of Treitz was transectedand

reanastomosed in sham operated group･ Side-to-end ilealJ pouch-rectalanastomosis was

0  80 120 180糾0 30B360

Time･(min)

I) 80 12D lt)0 240 308 360

Time帥軸

Figure 2: Gastric emptying curves of liquid (A)and solid (B) in 3 groups.

Gastric emptying of liquid was not differentamong 3 groups (- △ - :Control,I

〇一: IJT group, -ロー: Shamoperated group). Gastric emptying curve of solid was

not different between control (-△-) and sham operated (-ロー) groups, Whereas

that in IJT group (- 0-) Shifted toward the right direction later than120min.

Ga豊trlc Antrum LP･一.一叫_-_ ‥ , NW, 【.._‥_.｡† Duod棚_虹〟°_山一__臥d血. _. L -A 10 cm _...■Jh』-.._〟,..,A..I_▲払.ー｣. ,JLL_一｣_▲▲Ju｣一._._ 30 cm ‥L､….,. ､JA,■-". ､ー‥ -‥ _...臥.J-_.一､,.州..__....…, 諏】博打EEコ璽蔓璽零璽璽E璽男璽璽璽璽璽ふよ璽璽璽::璽璽璽寧璽零 150 cm一一一●■▲._..A ‥ ､伽._ ､. A...I.∼q.一､-ふ.｡..肋.｡... 220 cm 235 cm ‥..I _‥-....__.._..____A.._I__∼,.._..A.A_._.-_

B GastdcAntrum

DLJOdenum __｡..__一■■__ _.-■■. ､...∼.‥_-...___一.-_I_.-■__叫.. LIOやm 一 一- 一---一三二-一一･一.-一一 ｣ 一一L -+一二-一･一一-90 cm -.････-.･一一一 60cm I- ･･1-二一JL 一･･････一-- ･･･--･Lr-小一 1郭qrtこナ=-て-･==一二二-=----亡二二一叫一丁- -JT--- -一一一ニ-:=-I 謝cm _... _.__一.._..._ゝ∴､ ､ ■_. ..一... 23S cm ...__… __一一_⊥ _i...1∴_..I. ∴∴._.__....､.…... The lnteryaI9. 60鵬m

Figure 3: Representative tracings showing gastmintestinal contractile activityin the

interdigestive state in IJT (A)and sham(B).

Postprandialand interdigestive pattems were distinguishable in 3 of 4 dogs in IJTand sham

operated groups, tespectively; cyclic changes in motility similarto MMC were observed in

buJhJ)AAd空昔ld

0

0    60    120   180   240

!edin, Tine(min)

Figure 4: Changes in plasma PYY after feeding in 3 groups･

No differencesamong groups were observed in plasma PYY before氏eding.Althoughplasma

PYY increased postprandially inal1 3 groups, increase in IJT group ( - ● -) was greater than

Control (--〇一一)and sham operated (--ローー) groups laterthan90minutes.

PART 2: EFFECT OF ILEO･JEJtJNAL TRANSPOSITION ON ILEAL

LONGITUDINAL SMOOTH MUSCLE CONTRACTmE ACTIVITY IN RATS IN

ABSTRACT OF PART 2

Aim: Theaim of the present study was to studythe effects of ileo-jejumaltransposition

(IJT) on ilealcontractile activityin vitro in rats. Methods: Male Sprague-Dawley rats were

divided into three groups; control, IJT,and sham･ In rats withIJT, the distalileum of

one-fourthof the length of the jejunoileum was isoperistaltically interposed into the proximal JeJunum. The jejunoileum was transectedandanastomosed at three sites in sham group･ Rats

were killed 17-20 Weeks postoperatively,and ilealsegment was removed･ IsomertriC

I contractile activity of the isolated ileallongitudinalmuscle was measured intissue chambers･

Results: Spontaneous contractile activity was decreased in IJT compared to control･ The

dose-response Curve tO CholinerglC agOnist bethanechol in IJT shifted tothe le氏compared to

other two groups,and the dose required to induce 200% response in IJT was less thancontrol

and sham (p<0.05), suggesting cholinergic hypersensitivity･ The dose-response curves to

adrenerglC agOnist noreplnePhrine were not different each other･ Electrical field

stimulation-induced inhibition of spontaneous activity was reversed by amitric oxide synthase inhibitor in

all groups. Conchsions: These results indicate that IJT induces cholinergic hypersensitivity

in rat ileallongitudinalsmooth muscle, which is likely to compensate decreased spontaneous

contractile activity.

INTRODUCTION

Ileo-jejunal transposition (UT) involves interposingthe distalileum isoperiStaltically into the proximd jejunum. We have done series of studies on the effects of IJT on the

function of the gastmintestinal tract,1-4 because this operative proceduremight be able to

improve severe diarrhea after restorative totalproctocolectomy for ulcerative colitis or

fmilial.adenomatous･ polyposis..In dogs with intact colons, ITT leads to marked mucosal

hypertfophy･along th色whole small intestine･1,2 wealso found in dogs withintact colons that

IJT delayed gastric emptying withinhibition of postprandialgastric contractions, but srnall

intestinalmotilityand transit was unchanged･3 In dogs with IJTand totalcolectomy, delayed

gastric emptying was observed, but･ measming small intestinalContractile activity was not

feasibleinthis model･4 These changes in IJT appearto be mediated by increases in plasma

concentrations of enteroglucagonand/or peptide YY (PYY) in response to coptact of

endocrine cells in ilealmucosa withundigested chyme･114

Previous studies on gastrointestinal motility reported that changes in small intestinal contractilitywere observed tin vitro in rats after small bowel transplantation which necessitates extrinsicand intrinsic denervation,although globalmotor pattems of small

intestine were notaltered in conscious dogs after in situ neuralisolation･518 Thus IJTmight

alter small intestinalContractility in vitro,althoughglobalmotor pattemsand transit were

unchanged after IJT in dogs withintact colons･ Theaim of the present study was to investigate the effect of IJT on ileallongitudinalcontractile activityin vitro in rats･

A4ATERIALS ANDMETⅡODS

Preparation ofanimals: ProceduresandanimalCare were perfomed according tothe guidelines of血e Animal Care and Use Committee of the Tohoku University.

Sprague-Dawley rats (SLC Co., Tokyo) weighing 250-300g were used inall experiments. Rats were

anesthetized by inhalation of halothane (Fluothane@: Takeda Chemicals C0., Osaka, Japan)

and intrapeitonealsodium pentobaribital(Nembutal⑳: Abbott, North Chicago, IL) at the dose

of lminig･ IJT in rats was performed similarly to procedures in dogs･3 Briefly, 15 cm long

ilealsegment (5 land '20 cm proximal to the ileocecalsphincter) was transectedand

isoperistaltically interposed intothe proximal jejunum 10 cm distaltothe ligament of Treitz

by an end-to-end jejunorileostomyandanend-to-end ileoIJeJunOStOmy. Thもremalnlhg ileum

was restored byanend-to-end ileo-ileostomy･Asa･ shamoperated group,･ JeJunOileum was

transectedand reanastomosed atthree sites; 5 cm distalto the ligament of Treitz,and 5and 20

cm proximal tothe ileocecalsphincter, respectively.All rats wereallowed fTree access to water and rat chow from the day of operation onward. Rats were studied 17-20 weeks after IJT (IJT;

N=6)and sham operation (sham; N=5). Rats withoutany surgiCalprocedure which survived

for 17120 Weeks after delivery to our institution served as controls (Control; N=8).

Rccording of contractile activity: A segment of the distalileum (10-15 cm proximalto

the ileocecalsphincter in controland sham,and 15-20 cm distal to the ligament of Treitz in

IJT) was removedand cutalong the mesenteric border in modified Krebs-Ringer bicarbonate

solution (concentrations in mM: NaCl 118.3, KC1 4.7, CaC12 2.5, MgSO4 1.2, KH2PO4 1.2,

NaHCO3 25･0, Calcium disodium edetate 0.26,andglucose l1. I). Eightfull-thickness muscle

bothends of the muscle strips･ The muscle strips were suspended vertically in 20-ml tissue

chambers filled with modified KrebsIRinger bicarbonate solution maintained at 37･50Cand

bubbled with95% 02and 5% C92･ The lower end of the muscle strip was connected to a

fixed plastic 'hook in the chamber, while the upper end was attached to a non-compliant force transducer,therebyallowlng measurement Of isometric force.

Experimental desigtI: After a 90- to 1201minequilibration period with replacement of

the buffer solution in the chamber every 30-40min, each strip was incrementally stretched at

15120min intervals to its optimal1ength (Lo), defined as the length beyond which山地er

stretching did not increase theamplitude of spontaneous activity･5 All subsequent experiments were. performed atthis Lo,and strips that did not show spontaneous activity were not used (lessthan5% of strips evaluated).

Two chambers (two muscle stripsper rat) were used fわr dose-response study to the

cholinerglC agOnist bethanecholand two other chambers for fbrthe adrenerglC agOnist horepinephrine. For the experiments withbethanechol, the lowest dose (lo一一 M) was

administered after taking 5min spontaneous activity as basalbaseline activity. The buffer was

washed 6min later,andthe next higher dose was glVen after a 5-6min equilibration time.

This procedure was repeated until the greatest dose of bethanechol (1014 M) had been given. After recording baseline spontaneous activity in two separate chambers, norepinephrine (10-8

- 10-1 M) was administered intothe chamber in a cumulative manner every 6min. Four other

chambers were used to assess the effect of electrical field stimulation (EPS) under

nonadrenergic, noncholinergiC (NANC) conditions produced by phentolamine (5 X IOl6 M),

propranolol (10-5 M),and atropine (1016 M). Square wave currents (amplitude = 30 V; pulse

width = 0.5 msec; stimulus duration; 10 seconds) were delivered every 4-5min,and the

responses to varying frequencies (1, 3, 5, 7, 10, 15, 20,and 30 Hz) were studied･ The chamber solution was washed after studies of EPS,and Noarginine monomethyl ester (L-NAME: 5 X

IO'3 M) as well as threeantagonists to produce NANC conditions was administered intothe

four chambers･ Similar･experiments with EPS were repeated 30min later to see the effects of

nitric oxide (NO) synthase inhibitor L-NAME on EPS-induced inhibition of ilealcontractile

activity.

Atth'e conclusion of the experiments, each tissue･was blottedand weighed･

Data analysis･: Integralof force generated was measured for･ 5min in eight chambers at

Lo as spontaneous basalactivity･ Responses to bethanecholand noreplnePhrine were

quantitated by measunng the integralof force for 5min immediately after drug administration･

The integralof force was measured by a computer program.･(MacLab, AD Instrument,

victoria, Australia)and standardizedperminper wet tissue weight of each muscle strip

(g/min-mg tissue). The dose-response curves for bethanecholand norepinephrine were

obtained by expresslng basalcontrol activity as lOO%･ To quantitatethese dose-response curves,the negative log of the equieffective concentration that caused a 200% (ED2..)and 50% (ED5｡) response was calculated fわr bethanechol and norepinephrine, respectively･

Spontaneous contractile activities beforeand 30min after admimistration of phentolamine,

propranolol,and atropine were compared to see the effect of NANC conditions on

generated duringthe 10-second stimulusand comparlng this value to the force of baseline

activity per 10 seconds deteminedfor each strip for a 5-min period immediately before

beginnlng EPS. Integralof force ,in response to each frequency was expressed as % when

baseline activity was taken as lOO･ Areas between the verticalline of lOO%andfrequency-response curves were calculated as 'areas of inhibition'to quantitate inhibitory effect of EFS･

All data were expressed as mean土Standard error of the mean(SEM)･

･ Statistical ･comparisons: Mann-Whitney test was used for comparison of 2 groups,and

paired Student's I-test was used to see if producing NANC conditionsaltered spontaneous

activity.

DrugS: Bethanechol chlorideand noreplnePhrine bitartarate were purchased from Sig.ma

ChemiCalCompany, St･ Louis, MO･ Atropine sulfate was kindly donated by Tanabe Seiyaku

Co･, tOsaka･ Phentolamineand propranolol chloride were purchased from Novartis Pharma Co,

Tokyo,and'Zeneca Yakuhin Col ,Tokyo, respectively･ RESULTS

Spontaneous contractile actiyity: Tracings of representative spontaneous activity in

the basalcondition in each group is shown in Figure l･ The integralofforce was decreased in

IJT group (0.16土0.03 g/min-mg tissue) compared to control (0･25土0･02 g/min-mg tissue,

p<0.05), but no statisticaldifference was noted between controland sham (0･21土0･04

g/min-mg tissue) or shamand IJTI

Response to bethamechol and noreplnePhrine: Bethanechol induced a dose-dependent

increase in contractile activityinall three groups (Figtue 2). The dose-response curve in IJT,

however, shifted to the leftcompared tothe other two groups due to tomic increase of the baseline above 10 5 M (Figures 2and 3A). ED200in IJT was significantly greater thancontrol and sham (p<0.05), signifying a lesser concentration of bethanechol to induce 200% response

of contractile activityandthus suggesting a hypersensitivityto bethanechol in IJT (Table l)I NoreplnePhrine dose-dependently inhibited spontaneous contractile activity ln a Similar

manner in three groups (Figure 4),and the dose-response Curves in IJTand sham were identicalto that in control (Figure 3B). ED50 Was not differentamong three groups (Table I)I

･ Respo鮎e tot electrical rleld stimulation: Admimistration of phentolamine, propranolol,

and atropine to produce NANC conditions did notalter spontaneous activity inal1 3 groups

(data not shown). E･FS induced inhibition of spontaneous. activity (Values less than100) at low

bequencies (2-10 Hz)and caused contractions at high frequencies (15-30 H21) in control rats

under basalConditions (Figures 5and 6A). This inhibition observed in lowfrequencies was

reversed inthe presence of L-NAME,andareas of inhibition were decreased in the presence of L-NAME compared to basalcondition (p<0.05, Figures 5and 6A)･ In IJTand sham,

frequency-response curves in basalcondition were simi1arto control,and the effect of L

NAME reversing inhibition at low鉦equencies wasalso noted in these 2 groups (Figures 6B

and6C).

DISCUSSION

We were interested in small intestinal smooth muscle contractility in vitro a氏er IJT,

because itmight bealtered even thoughIJT in dogs did not affect theglobalmotor pattems･

intestinalmotilityand transit in vivo after IJT. We foundthat after IJT basalspontaneous

activitywas reducedand sensitivityto cholinergic agonist bethanechol was increased･

Decreased basalContractile activity suggests the possibility thatthe small intestinaltransit

might be delayed, because mechanicalactivity often correlates with intestinaltransit･9 In dogs

with IJT postprandialgastric but not small intestinalmotility was inhibited,and this inhibition

was considered to be associated with increased plasma level of enteroglucagonand PYY.3 In

rats, IJT increased mucosalweightand DNA cohtent in the transposed ileum,and plasma

level of neurotensin.10 These results indicate that IJTincreases plasma level of peptides

mainly released h)m the ileal･hucosa･ Because those pepti.des inhibit gastrointestinal

contractility or delay transit,ll-13 d￠crease in spontaneous contractile activity i血the present

studymight be secondary to the increase in plasma level ofthosepeptides･

Hypersensitivityor supersensitivity is defined asthe phenomenon in which the dose of

a substance required to produce a given biologicalresponse is less thannormal,and thus the dose-response curve is shifted tothe left.14 sensitivity to acetylcholine is increased up to 100,000 times in skeletalmuscle after selective denervation of motor nerves, whereas increase range from 2- to 30-fold after transection of nerves directly innervating smooth muscle･14 our observation that muscle strips LTrom rats withIJT were 10112 times more sensitive to

bethanechol thanControl, estimatedfromthe difference in ED20.,fu1丘lls this criterion of

hypersensitivty. Previous studies reported extrinsic denervation-associated adrenergic hypersensitivityafter small bowel transplantation in rat ileumwith increased sensitivity to

norepinephrine 12 times higher thancontrol･5･6 This hypersensitivity was considered due to

increase of a血energlC reCeptOrS On the smoo血muscle but not on the enteric neⅣous

system･5･6 Thereare 5 subtypes (Ml-M5)for the muscarimic receptors.15 Bethanechol was

believed to stimulate M2 receptors when muscarinic receptors were classified only into Ml

and M2 in the past･16 M2and M3 receptorsare distributed in the rat ileum,andthe most

abundant receptor isthe M2 while M3 receptorsareminor･17 M2 receptors inhibit adenylate

cyclaseand M3 receptors stimulate phosphoinositide hydroly?is･17Asthere is no specific

agonist f♭r each musCadnic receptor subtype, bethanechol must stimulate both M2 and M3

receptors･ Osinskiand Bass reported active stress generation was increased in response to

bethanechol after extrinsicand myenteric denervation.18 Althoughwe cannot detemine the

mechanism of cholinergic hypersensitivityinduced by IJT (increase in cholinergic receptors

density, increase in the affinity of cholinergic receptors to bethanechol, Or a modification of

the receptor signaling), it is likely that cholinergic hypersensitivity was induced to

compensate d∝reased basal spontaneous contractility. I

EFS induced inhibition of ･contractile activity at low frequencies while it enhanced

contractions at highfrquencies･ Inhibition at lowfrequencies was reversed by a NO synthase

inhibitor L-NAME･ This observation was simi1arinal1 3 groups, suggesting that EFS-induced

inhibition at low frequencies is mediated via NO in normalratsandthat this inhibitory

nitrergic response was not modulated after IJTand shamoperation. These results in control

rats do not connict with those in previous studies in rat ileallongitudinalmuscle.6･19 Although

spontaneous activity was inhibited by NO in jejunallongitudinalmuscle, a NO synthase

by a NO synthase inhibitor, nor direct administration of NO into the tissue chamber did not

inhibit basalspontaneous activity in jejunaland ilealcircularsmooth muscle in rats.21･22 Thus,

the effect of NO or NO synthase inhibitor differs in each site even inthe same species. Results

inthe present study suggestthat the decrease in spontaneous after IJT must not be due to

changes of nitrergic neurons in the enteric nervous system･

血comclusion, IJT decreased spontaneous contractile activity and induced cholimergic

hypersensitivityin rat ileallongitudinalsmooth muscle in vitro･ Cholinergic hypersensitivity

was considered to.be induced to compensate decreased spontaneous contractile activity･

A CKNOnEDGEMENT

The authorsthank Michael G. Sarr, MD, Department of Surgery, Mayo ClimiC, Rochester, Minnesota for reviewlng this manuscript.

REFERENCES

l･ Tsuchiya T, Sasaki I, Naito H, Narui H, Funayama Y, Suzuki Y, Toda M･ The influence of ileo-jejunaltransposition on intestinalmucosalgrowthand gut homones after total colectomy (Japanese with English abstract). Jpn J Surg 1986;87:870-7.

2･ Toda M,･Sasaki I, Naito H, Funayama Y, Kamiyama Y, Suzuki Y, Tsuchiya T, Matsuno S･

Effect of ileo-jejunaltransposition on intestinalstructureandfunction in dogs. Biomedical

Res 1988;9, Supp1 3:157-62.

3･ Ohtani N, Sasaki I, Naito H, Shibata C, Tsuchiya T, Matsuno S. Effects of ileo-jejunal

transposition on gastric emptying, gastmintestinalmotility,and small intestinaltransit in

dogs･ J Gastmintest Surg 1999;3:516-23.

4･ Ueno T, Shibata C, Naito H, Jim XL, Funayama Y, Fukushima K, Matsuno S, Sasaki I･ Ileojejunaltransposition delays gastric emptyingand decreases fecalwater content in dogs

with total colectomy. Di島 Colon Rectum 2002;45 : 109-1 1 8.

5･ Shibata C, Balsiger BM,Anding WJ, Sarr MG: Adrenergic denervation hyper岳ensitivityin

ilealcircularsmooth muscle after small bowel transplantation in rats. Dig Dig Sci 1997;42:221 3-21.

6･ Ohtani N, Balsiger BM,Anding WJ, Duenes JA, Sarr MG･ Small bowel transplantation

induces adrenergic hypersensitivityin ileallongitudinalsmooth muscle in rats. J

Gastrointest Surg 2000;4:77-85.

7･ Murr MM, Miller VM, Sarr MG. Contractile properties of enteric smooth muscle after

smal1 bowel transplantation in rats.AmJ Surg. 1996;171:212-217;

8･ Zyromski NJ, Duenes JA, Kendrick ML, Libsch KD, Seiler R, Tanaka T, Sarr MG･ Differential adrenerglC response tO eXtrinsic denervation in canine longitudinaljejunaland

ilealsmooth muscle. J Gastmintest Surg 2002;6:4 I 8-425･

9. Siegle ML, Ehrlein HJ. Digestive motor patternsand transit of luminalcontents in canine

ileum. Am ∫ Physiol. 1988;254:G552-G559.

10･ Chu KUi Tsuchiya T, Ishizuka J, Uchida T, Townsend CM Jr, Thompson JC･ Trophic response of gutand pancreas after ileojejunaltransposition･ Ann Surg 1995;221 :249-256･

Ill Suzuki T, Nakaya M, Itoh Z, Tatemoto K, Mutt V･ Inhibition of interdigestive

℃ontractile activity in the stomach by peptide YY in Heidenhain pouch dogs･

Gastmenterology 1983;85: 1 14-121･

12. Shibata C, Naito H, Ueno T, Jim XL, Funayama Y, Fukushima K, Hashimoto A, Matsuno

S, Sasaki I: Effect ofglucagon,glucagonllikepeptide- 1 , -2,and glicentin on interdigestive

gastroduodenal motility in dogs with a vagally denervated gastric pouch･ ScanJ Gastroentero1 2001 ;36: 1049- 1055.

13. Schemann M, Ehrlein HJ. Effects of neurohormonal agents on jejunalcontraction spread and transit in the fed dog. Gastroenterology 1986;90: 1950-1955･

14. Flemmlng WW. Variable sensitivity of excitable cells: Possible mechanismsand

biologiCalsigmificance. Rev Neurosci ',2:43-90･

15･ Eglen RM, Hegde SS, Watson N･ Muscarinic receptor subtypes and smoo血muscle

function. PhamacoI Rev l996;48:53 I-565.

disorders. Dig Colon Rectum 1993;36:696-708.

17. Ehlert FJ, Ostrom RS, Sawyer GW. Subtypes of the muscarinic receptor in smooth muscle･

LifeSci 1997;61:17291740.

-I 8. Osinski MA, Bass P. -Increased active stress generation of denervated rat intestinalsmooth _ muscle: Functionalanalysis of muscarinic receptor population･ J PharmacoI Exp Ther

1994;268: 1368-1373.

19. Kanada A, Hata F, Suthamnatpong N, Maehara T, Ishii T, Takeuchi T, Yagasaki O･ Key roles of nitric oxideand cyclic GMP in nonadrenerglCand noneholinerglC inhibition in fat

ileum. Eur J Pharmacol 1992;216:287-292.

20･ Balsiger BM, Ohtani N,Anding WJ, Duenes JA, Sarr MG: Chronic extrinsic denervation

after small bowel transplantation in rat jejunum: Effectsand adaptation in nitrergiCand non-hitrergic neuromusCularinhibitory mechanisms･ Surgery･ 200 1 ; 1 29 :47 8-489 ･

21. Shibata C, Balsiger BM, Anding WJ, Duenes JA, MillFr VM, SaLT MG･ Functional

changes in nonadrenerglC, nOnCholinerglC inhibitory neurons in ilealcircularSmooth

muscle after small bowel transplantation in rats･ Dig Dis Sci 1998･,43:2446-2454･

22. Balsiger BM, Duenes JA, OhatmiN, Shibata C, Fa-gia G, Anding WJ, Sarr MG: ･Nitric

oxide (NO) pathways in circularmuscle of the rat jejunum beforeand after small bowel

transplantation (SBT). J Gastrointest Surg 2000;4:86-92･

Table l : ED20.for bethanecholand ED5. for noreplnePhrine in each group

Control IJT S ham ED200      4.6土0.2     5.6土0.3*     4.5土0.2

ED5｡     5.9土0.2     6.3'i 0.4     6.5土0.4 * p<0･05 compared to control and sham.

FIGURES

The lnteryals, 10 mh

0.5g

繊蜘鵬蜘細さ紬

Time lnteryaI9, 10 m套n

Tine Jnteryals, 10 niれ

Figure 1: Representative tracing of basal activity in control (A), IJT (B),and sham (C).

Spontaneous activity was decresaed in rats with IJT compared to the other 2 groups･

A

10･7 3XIO寸 10･6 3XIOJ6 1016 3XIOJ6 104 *  L   兼  ⊥  ♯  ⊥  ♯  ⊥  ♯

▲よこ｡.2｡

The JrlterY&Is, 1 0 nln

a.1u] 0.2g

______｣___._I.･..｣ ThTl● lrderyds, 1 0 rrIIn ___..ll.｣ C lq･7 3¥103 1.016 3¥10J6 19< 3X.10JS 1_OJ4

..嵐コ臥2g

璽璽璽竪琴艶零璽璽璽璽璽｣ ntery&Js, 1 0 rrlin

Figure 2: Representative tracing showingthe effect of.bethanechol in control (A), IJT (B),and

sham (C).

Bethanechol induced increase in contractility in a dose-dependent fashion inal1 3

groups･ Response to bethanechol in IJT group was greater than the other 2 groups at

the doses of 3X10-Sand 10-4 M because of tonic increase of baseline. Astreiks

伽棚紬訓Ⅷ

A 思毒性)8PJBB8q Controf 7   6   S   4

劫抑抑榊脚謝▲

一tT I

a) 萱旺P曇%

8   7   6   5   4

Dose of bethnechol tlo9kq Do事e Of nor叩Inephrhe trog叫

Figure 3: Dose-response curves to bethanechol (A)and norepinephrine (B).

Dose -response curve to bethanechol畠hifted o the left in IJT group compared to controland

sham (A)･ Dose-response cuⅣes to norepinephrine were not different each other (B).

LL I

Tfme JnteryaJs, 10 nh

J J ふ』  ｣  ｣ ｣,;;ここlここ_=;,_l=_こ_.;_=_=_._二i_;;1_,,=.=土､,.,､,.W,.Y.ⅦY.nム』

The lnteryals, 10 TTIbt

1D8 3Xllド1け7 3X1871tド3XID6 1DS 3X10S ID･J

I I 1 I I i 1 I 1

Figure 4: Representative tracing showing the effect of norepinephrine in control (A), IJT (B), and sham (C).

Noreplnephrine dose-dependently inhibited spontaneous contractile activity ln a Si血lar

EFS.7Hz

｢1

Time lntervaIs, 1 mtinule

EFS.7HZ

n

Time lntervals, 1 minute

Figure 5･'Representative tracing showing the effect of EPS in basalcondition (A)andwith

L-NAME (B).

EPS induced inhibition of spontaneous activity at low frequencies (2-10 Hz),and this

inhibition observed in lowfrequencies was reversed in the presence of L-NAME in control

rats under basal conditions.

婁-丘P喜l%

A

m且 E此 間L ¶山 E山 nH 亡可 ▲.一J 帥 軸 e 量 l こコBasaJ condition 串静W馳h L･触榊E 1 3 5 7 10 1520 30 F帽quOnCy tl軸 1 3 5 7 10 15全0 38 Froqu魯nCy糾め V塵遷虐tP!P事貞｢ tJOWPPEJb一事-層 e o 糞      ■1 0

Figure 6: Frequency-response to EFSand areas of inhibition in control (A), IJT (B),and sham

(C), in ゎasal condition and in the presence ofLNAME.

Inhibition of contractility was observed at low frequencies in basalconditions inall groups･ This inhibition was reversed by the pretreatment with L-NAME inall

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