Effect of Ischemia on the Canine Large Bowel: A Comparison with the Small Intestine 1

Transcription

1 JOURNAL OF SURGICAL RESEARCH 62, (1996) ARTICLE NO. 17 Effect of Ischemia on the Canine Large Bowel: A Comparison with the Small Intestine 1 IZUMI TAKEYOSHI, M.D., SHIMIN ZHANG, M.S., KENJIRO NAKAMURA, M.D., AKIRA IKOMA, M.D., YUE ZHU, M.D., THOMAS E. STARZL, M.D., PH.D., AND SATORU TODO, M.D.2 Pittsbrgh Transplantation Institte and the Department of Srgery, University of Pittsbrgh, Pittsbrgh, Pennsylvania Sbmitted for pblication September 12, 1995 Mcosal injry cased by ischemia and reperfsion has been well docmented with the small intestine, bt little is known abot the colon. In the present stdy, the effect of warm and cold ischemia on the canine colon was stdied and compared to that on the small intestine. After in sit flshing, the small intestine and the colon from six beagle dogs were removed and stored for.5, 1.5, and 3 hr at 37 C (warm ischemia) or for 1, 6, 12, 24, 36, and 48 hr at 4 C (cold ischemia). Electrophysiology, permeability, biochemistry, and histopathology of the specimens at each ischemic period and after reperfsion in the Ussing chamber were determined. Warm and cold ischemia indced dration-dependent sppression of electrophysiology in both organs, bt the colonic mcosa retained higher activity of absorptive enterocytes and cryptic cells than the small intestine. Only the colon showed increased permeability of FITC-conjgated Dextran from the mcosal srface to the sbmcosal layer after prolonged ischemia. Changes in adenine ncleotides and prine catabolites were not markedly different between the organs. Histopathologic abnormalities dring ischemia and after reperfsion were more serios with the small intestine than with the colon. Compared to warm ischemia, hypothermia lessened or delayed these morphofnctional derangements in both organs, which became niversally worsened after reperfsion. Colonic mcosa receives morphofnctional derangements from ischemia and reperfsion, bt the severity of the damage was mch less severe in the colon than in the small intestine Academic Press, Inc. INTRODUCTION Improvements in immnosppression, operative procedre, and posttransplant management have made clini- 1 Aided by Research Grants from the Veterans Administration and Project Grant DK from the National Instittes of Health, Bethesda, Maryland. 2 To whom correspondence shold be addressed at Pittsbrgh Transplantation Institte, 4C Falk Clinic, 361 Fifth Avene, Pittsbrgh, PA Fax: (412) cal intestinal transplantation feasible [1-3]. Since most patients that received intestinal transplantation lost most of their intestine and proximal colon (inclding the ileocecal valve), transplantation of the intestinal graft withot the colon often reslted in the development of postoperative high stomal otpt, dehydration, and malntrition. In several recent cases in or series, the colon has been inclded as a composite of intestinal grafts at transplantation to lessen these complications [4, 5]. Experimentally, it has been shown in rats that the inclsion of the ascending colon and the ileocecal valve significantly improves graft fnction [6, 7]. From the time of organ procrement to revasclarization, the intestinal graft is sbjected to varios degrees of tisse damage from cold and warm ischeinia. While the sensitivity of the small intestine to warm and cold ischeinia has been stdied extensively in terms of morphology and biocheinistry [8-12], only a few reports have examined the large bowel after warm ischeinia [13-17], The aim of this experiment is to evalate and compare the ssceptibility of the small and large intestine to warm and cold ischeinia sing the in vitro U ssing chamber techniqe, biocheinistry, and histopathology. MATERIALS AND METIIODS Animals. Six beagle dogs of both sexes, weighing 8-13 kg, were sed with approval ofthe University of Pittsbrgh Instittional Animal Care and Use Committee. The dogs were fasted from the evening prior to srgery. Under general anesthesia with endotracheal intbation and mechanical ventilation, the abdomen was opened throgh a midline incision. The lower abdominal aorta was dissected and cannlated with a 12-French trocar catheter (Sherwood Medical, St. Lois, MO). One liter of lactated Ringer's soltion containing 2 nits of heparin was infsed via the catheter to flsh the vasclar beds of the small bowel and the colon. After flshing, 2 cm of the terminal ilem (to 5 em proximal to the ileocecal valve) and 2 em of the colon (from 5 em distal to the ileocecal valve) were removed. The lmens of both segments were irrigated with 2 ml of the same soltion containing 3 mg/liter of neomycin slfate. Each segment was divided into two lo-cm pieces and preserved at either 37 C (warm ischemia) or 4 C (cold ischemia), immersed in the same soltion. At.5, 1.5, and 3 hr of warm ischemia, and at 1, 6, 12, 24, 36, and 48 hr of cold ischemia, tisse samples were collected for later analysis. At each time interval, a fll-thickness section of intestinal wall, 1 em in width, was sed for electrophysiologic and permeability $18. Copyright 1996 by Academic Press. Inc. Al! rights of reprodction in any form reserved.

2 ------~ ~--~ _. 42 JOURNAL OF SURGICAL RESEARCH: VOL. 62, NO.1, APRIL 1996 stdies and for histopathology. Mcosal tisse, 5 mg, was scraped by a glass microscope slide and then immediately frozen and stored in liqid nitrogen ntil biochemical analyses. Intestinal tisses taken prior to in sit vasclar flshing were sed as the control. Electrophysiologic stdy. Electrophysiologic measrements were performed sing essentially the same method as described by Madara and Kirkman [18]. The ilem and colon were rinsed with lactated Ringer's soltion to remove any remaining lminal contents. The mcosa and sbmcosa were separated from the seromsclar layer by blnt dissection. The separated sbmcosa and mcosa, containing no Peyer's patches, were monted on a 13.5-mm-diameter (1.43 cm 2 srface area) Ussing chamber (World Precision Instrments, Inc., Sarasota, FL). When monted in these chambers, the mcosal and sbmcosal membrane srfaces were perfsed with a recirclating, oxygenated (959, O 2,5% CO2) bffer soltion. The perfsion soltion, with a ph of 7.4, consisted of 12 NaCI, 4.7 KCI, 1.2 MgS4, 2.5 CaCh, 25 NaHCO", and 1.2 KH ZP 4 (in mm). The recirclating reservoirs were maintained at 37 C throghot the experiment. Using two different Us sing chambers, -two separate sets of electrophysiologic measrements were performed with individal samples. For baseline potential differential (pm, resistance (R), and shortcircit crrent (Isc) determinations, 1 mm D-glcose was added to the perfsion bffer soltion recirclating each cell of the chamber. After a 3-min eqilibration period, PD and Isc were directly measred every 5 min for 4 min. Resistance was calclated according to Ohm's law sing voltage deflection measrements of the transmcosal crrent (5 J1A). Potential difference, Isc, and R at 3 min of measrement were sed as representative vales of each sample. To determine electrophysiologic response to glcose stimlation, 1 mm mannitol was sbstitted for the glcose in the mcosal-side soltion, and then PD, R, and lsc were measred for the next 2 min. Mter completing the glcose stimlation experiment, 5 mm theophylline was added in the sbmcosal-side reservoir, and measrements were contined for another 2 min. Differences between the highest vales after stimlation and those before stimlation were determined. Permeability stdy. The permeability stdy was performed with the same chamber sed for baseline electrophysiologic measre ments. Immediately after 3 min of eqilibration, 5 mg of FITCconjgated Dextran (moleclar weight, 44; Sigma Chemical Co., St. Lois, MO) was added in the mcosal-side reservoir of the Ussing chamber. Before addition of the dye and after 4 min of measrements,.25 ml of soltion was collected from the sbmcosal-side reservoir. Soltion sampling prior to dye administration was done to eliminate the inflence of dye contamination to the recirclating system de to freqent ses and limited cleaning periods. The con- centration of florescent Dextran in each sample was measred at an excitation wavelength of 483 nm and at an emission wavelength of 517 nm sing a florescent spectrophotometer (RF5U, Shimadz, Japan). The permeability of the tisse sample was calclated from the difference in dye concentrations and expressed as ng/cm" ofmco sal srface areajmin. Biochemical ~tdy. Changes in tisse concentrations of adenine ncleotide (AN) and prine catabolites (PC) dring warm ischemia and cold ischemia were stdied sing high-performance liqid chromatography (HPLC) as described before [19]. Tisse samples were homogenized with a Polytron homogenizer (Brinkmann, Inc., Westbry, NY) in 6% perchloric acid containing.8 nmol/liter ethylenediaminetetraacetic acid. The homogenate was centrifged at 1,g and 4 C for 1 min. Mter centrifgation, the spernatant was extracted and injected to a Waters HPLC system (Waters Chromatography DivisionJMillipore Corporation, Milford, MAIModel 51 pmps, Model 484 absorbance modle and Model 717 WISP system). The system was eqipped with a Maxima 82 Chromatography work station (Waters), a reverse phase colmn [E. Merck, Darmstadt, Germany; LiChrospher 1 RP-18 (5 j.lm), 4 X 25 mm], and a precolmn (Waters; RCSS Gard-PAK). Concentrations of the AN and PC were monitored at 2fi4 nm (Waters 484, Tnable Absorbance Detectorl. Energy charge was calclated according to the formla by Atkinson [2]: (ATP + O.li ADP)/(AMP + ADP + ATP). Histopathologic stdy. Tisses, taken at the end of each ischemic period and 7 min after oxygenation in the Ussing chamber, were fixed with 1% bffered formalin, embedded in paraffin, ct into 5- j.lm sections, and then stained with hematoxilin and eosin. Histopathologic analyses were performed blindly by a single pathologist withot knowing the grops (warm or cold) or the ischemic periods. Modified Park's classification [8, 9] was sed to evalate the degree of tisse damage: () normal; (1) sbepithelial detachment at vills tip; (2) extended sbepithelial space; (3) epithelial lifting along vills side; (4) dendation of villi; (5) loss of vills tisse; (6) infarction of crypt layer; and (7) transmcosal infarction. Statistics. Vales were expressed as means::+: standard error of the mean. Using a statistical software package (Stat-View II, Abacs Concepts, Inc.), grop comparisons of electrophysiologic fnction and biochemistry were performed by variance analysis, and those of histopathologic changes were determined by a Wilcoxon rank sm test. RESULTS Warm Ischemia Electrophysiology. Warm ischemia indced dration-dependent sppression of electrophysiologic fnc- >- E # COLON 58 3 ;:;- E c: CD 2 c!! <I) ~1 'iii CI) II: 1 ~ --- COLON \ o Warm Ischemia (hors) FIG. 1. The baseline potential difference (left) and resistance (right) of the colon and the small intestine after warm ischemia (*P <.5 verss control; #p <.5 verss small bowel).

3 TAKEYOSHI ET AL.: ISCHEMIAJREPERFUSION INJURY i SO J Glcose sa ~4Iac ItIC Colon 8::ec i 1 8 J Warm Ischemia (hors) Theophylline.5 S8&ISC.Isc Colon disc talsc FIG. 2. Electrophysiologic response to glcose (left) or theophylline (right) stimlation of the colon and small bowel after warm ischemia. tion in both the small intestine and the colon to differing degrees. Potential difference, which was three times higher in the colon than in the intestine before indction of ischemia, was totally abolished in both organs by 1.5 hr (Fig. 1, left). Similarly, R of the colon markedly decreased at 1.5 hr, bt small intestinal R remained relatively high throghot the observation period (Fig. 1, right). Baseline Isc of both organs showed a redction similar to that of PD. However, colonic responses to glcose administration (Fig. 2, left) and theophylline administration (Fig. 2, right) at.5 hr of ischemia were similar to those of the control and were still detectable after 3 hr. In contrast, responses ofthe small bowel were barely detectable after only.5 hr of warm ischemia. The colon appears to retain more electrophysiologic activity than the small intestine. Permeability. Permeability of FITC-conjgated Dextran across mcosal specimens is shown in Fig. 3. Dring a 4-min perfsion of normal tisses,.65 ±.3 and.23 ±.2 ng/cm2/min offiorescent dye were fond to permeate the small intestine and the colon, respectively, acconting for.8 and.3% of the total amont of the dye injected into the mcosal-side reservoir. No changes in permeability were fond in ischemic small intestines, bt the colon developed a significant increase in permeability after 1.5 hr of warm ischemia (Fig. 3, left). Biochemistry. Levels of ATP and energy charge in mcosal tisses of the normal small intestine and normal colon were similar (Table 1). After the onset of warm ischemia, ATP and energy charge dropped significantly at.5 hr and were markedly depressed throghot the entire ischemic period. The rate of energy charge redction was slightly less in the small bowel compared to that of the colon. In contrast, tisse concentrations of AMP and xanthine (X) showed significant elevations in the small intestine by 3 hr, while the colon had greater concentrations of hypoxanthine (HX). Histopathology. Compared to the control, warm ischemia cased dration-dependent mcosal injry of eqal severity in both organs (Fig. 4A). Mter reperfsion, histological abnormalities were always agmented in both organs. Mcosal damage after.5 hr o Warm Ischemia (hors) o Cold Ischemia (hors) FIG. 3. Permeability of the colon and the small intestine after warm (left) or cold (right) ischemia of the colon and small bowel (*P <.5 verss control;.p <.5 verss small bowel).

4 44 JOURNAL OF SURGICAL RESEARCH: VOL. 62, NO.1, APRIL 1996 ' ~ o Co.).x- * * LC<o";<..;<OLC <OO>C<1C<1C<1<O oo""<c<icv:i~ OO~C'l?1""""I~1""""I ";<<O<OO>LC";< ~c<ioooocric<i C<1";< -x-.;.:- * -x- >> CO C':>LC OOtn t-r-i ~6C'i.,...itD~ <.....;<..;< ";<<oc<1c':>t-o cv:icv:i"":o"":ai.....;<c':> * -x- * -x ";<C<1>t-C':> C'\I O':lOC\l 1""""1""'" c<ioc<ic<ioocv:i t-oo><oo><o O>O>t-t-<O<O "":"":c<ioocri.....;<lc *.;<- * *-1:- ";<";<C':>OOLC LCC':>LCC<1 ""<c-ic<icv:i~c-i ':lr-io"""i'1""""it LCLCLCCOLCC':> ooaicri o>t:i... LCLC xt-c<1c':>lco>";< * * * COLCO";<COO>.,...iC'i~.,...itO~ 1""""ItOt-OO""'d'lC"l >>... <>..;< aiooai>t:i>t:icv:i... LC..;< ** OC<1<o<oC<1CO **-x C<1<OC':>";<";<O> ""<c-i~~"":"": t-c<1... C':>ti.G'lO)-.:::f4C\lT"""l cricv:icv:ioo>t:i>t:i 1""""I1""""I1""""Ic.DLC * x- x- -x- * <OCOO>O>O>C<1 OC<1C':>t-LCC<1 ooo...<c-ic-i..;<t-c<1<oc<1<o <oc':>olc... CO o""<c-ic-i""<~... C<1... * * * *' -:t *..;<LC<OO>O LCC<1tCtoooc-i~~ OCIr-1t-C\lr-l >... o.,...i.,...io..-ioo... 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C<1 -x-,~.x- -x- t- C\l en O>LCOt-C':>O...<>t:ic-iooo... t-""'c':> >C<1t-LCLC ""< r-< C<1 -x- *.:t * *-x- x- *"* * **OLC,,",<O OOC<1..;<... <o.,...i.,...ico..-i6... LOC"Ic,o.,..., OOOt-<OO> "":cv:icri C\lC\l '1""""1 et:ilo * * ~ -* * * -x- **OOOCr-I oo>";<or-<tc """;co..-ic4c1-:i,...i t-c<1c':> O<OOC<1tC<O "":c-icv:icv:iai 1""""IC\l1""""lC\lC\l 7C-*t-1""""IOJ-.::j'i * * * t-oc<1coc':><o tci..-ic4m..-i t-..;<c':>r-<,,",<<")<,...;r.o~~.,...ioo 1""""11""""1 M 1""""1...-! t-~l1""""i <OC':>C<1C<1C<1C<1 >t:i~oooo r-< tc""'o,,", ooot'-oolc\l < ischemia and reperfsion was significantly worse in the small intestine than in the colon, bt there was no important dhference after 1.6 hr. Cold Ischemia Electrophysiology. Cold preservation slowed the decrease in electrophysiologic fnctions by approximately 8 times in the small intestine and by approximately times in the colon. Potential difference, which was ndetectable in both organs after 1.6 hr of warm ischemia (Fig~. la), was ndetectable after 12 hr in the small bowel and at 24 to 36 hr in the colon (Fig. 6, left). Changes in R after cold ischemia showed a trend similar to those seen after warm ischemia (Fig. 6, right). Baseline Isc had a dration-dependent decline in both organs, bt response to the administration of glcose (Fig. 1o, left) and theophylline (Fig. 6, right) was more prominent with colonic tisses for p to 48 hr of cold ischemia. Ths, colonic mcosal cells retain more electrophysiologic activity dring cold ischemia. Permeability. Similar to warm ischemia (Fig. 3, left), cold ischemia did not case any change in mcosal permeability of the small intestine even after 48 hr of cold ischemia. Althogh significantly less than that of warm ischemia, there was an increase in colonic permeability after 36 hr of cold ischemia. Biochemistry. ATP and energy charge degradations were delayed nder cold ischemia in both organs (Table 1). Althogh ATP and energy charge decreased rapidly within 1 hr, the levels were mch higher for p to 48 hr than levels seen dring warm ischemia, being 1 to 2% of initial vales. The degradation of ATP dring cold ischemia compared to warm ischemia was reflected by a greater concentration of ADP and AMP, and a lesser accmlation of HX and X. Histopathology. Compared to the control, cold ischemia indced dration-dependent histologic damage in both organs. The damage was worse in the small bowel than in the colon at 12 hr of ischemia. Mter reperfsion, frther derangements of mcosal strctre developed in both organs. The severity of mcosal damage after reperfsion was greater in the small intestine than in the colon at 6 hr and at 12 hr. At 24 and 36 hr, histologic damage after ischemia and reperfsion was similar between the two organs, bt the colon was worse at 48 hr. DISCUSSION In the present stdy, we sed the in vitro Us sing chamber techniqe to simlate reoxygenation of enteric tisses after varios drations of warm and cold ischemia. Changes in electrophysiology, permeability, biochemistry, and histopathology before and after reoxygenation with asanginos perfsion flid were determined. Althogh this method cannot reprodce events similar to those which occr in the grafts after actal transplantation, particlarly de to the blood-

5 TAKEYOSHI ET AL.: ISCHEMIAIREPERFUSION INJURY 45 A Small Bowel Colon Pn- --.1on B Post.reperfUalon I * 6 6 CD CD CI CI as " III E 5 \ E as III 5 iii iii III 4 \ \ III 4 ;:) ;:) :i! 3 I :i! 3 ' -?:?: c 2 ii 2 CD > > CD ~ (/) 1.. ~ I:~II ' i Warm Ischemia (hors) B Sml!! Bowel Colon E !oPr_1an I 6 6 GJPotI-repet'ftikNI :... E 5 iii iii. ~ 4 ~ 4 ::I ::I (; (; ~ :E 2.~ 2 > ai ~ '. ai ~ ~... I Cold Ischemia (hors) FIG. 4. Histopathologic severity of the mcosal damage in the colon (left) and small intestine (right) at the end of warm ischemia (A) and cold ischemia (E). Horizontal bar indicates median score (*P <.5 verss control; #p <.5 verss colon). less perfsion system l21], it allows direct evalation of tisse damage cased by ischemia and reperfsion in a careflly controlled environment. Stdies on electrophysiologic fnction of intestinal and colonic mcosa showed an inverse relationship with the dration of ischemia. As ischemia progresses, electric activity, barrier fnction, vills absorptive fnction, and cryptic cell fnction are negatively affected. However, from the present experiment, electrophysiologic fnction of the colonic mcosa appears more resistant to ischemic inslts than small intestinal tisses. Colonic PD levels remained consistently higher than those of the small intestine ntil the activities were lost in both organs at 1.5 hr of warm ischemia and at 24 hr of cold ischemia. Similarly, Isc of the colon was higher than that of the small intestine throghot the ischemia period. More importantly, even when the electrophysiologic fnctions of absorptive enterocytes, determined by glcose stimlation, and cryptic cells, determined by theophylline stimlation, in the small intestine were abolished at longer periods of ischemia, colonic epithelial cells still responded to administration of both agents. An exceptional observation with or electrophysiologic measrements was the rather sstained small intestinal R even after prolonged ischemia. Althogh difficlt to explain, this may indicate better barrier fnction of the small intestine dring ischemia, or the inflence of a thicker sbmcosal layer of the small intestinal specimen. Barrier fnction of small intestinal and colonic mcosa was directly estimated by sing FITC-conjgated Dextran as a marker. Disrption of the mcosal barrier, or the movement of micro- and macromolecles from the lmen to the circlation across enterocytes, has been reported nder varios conditions [22]. Minimal bt similar movement of the marker sbstance throgh the enteric mcosa ofthe normal dog was demonstrated in this stdy, as described in the normal rat

6 46 JOURNAL OF SURGICAL RESEARCH: VOL. 62, NO.1, APRIL 1996 Cold Ischemia (hors) FIG. 5. The baseline potential difference (left) and resistance (right) ofthe colon and the small intestine after cold ischemia (*P <.5 verss control; 'P <.5 verss small bowel). intestine [23]. Pantzar et al., sing the same Us sing chamber techniqe, demonstrated that.3-.4% of the FITC-conjgated Dextran injected into the mcosal-side reservoir was recovered from the serosal-side soltion after 6 min perfsion. Permeability of the normal canine intestine was mch less than that of the normal rat. Permeability after perfsion of ischemic specimens showed differences between the organs. Althogh the small intestine had significant histologic mcosal damage, and the circlation-to-mcosa permeability has been demonstrated to increase with ischemia [1], no evident changes in the permeability were noted with or ischemic small intestines. In contrast, the colon developed increased permeability after 1.5 hr of warm ischemia and 36 hr of cold ischemia. This was an nexpected observation, becase the colon has generally been thoght less permeable to molecles than the small intestine [24], as seen with normal tisses in this stdy. Althogh the reslts with the permeability stdy and electrophysiologic measrement were similar, reasons for the difference between the two organs are nclear. At least, an artifact in preparing thinner colonic specimens can be exclded since all of the colonic mcosa with increased permeability had a standard error similar to that of the other specimens. Irreversible organ damage from prolonged ischemia has long been considered to be associated with the decrease of ATP and energy charge in the tisse. In addition, prine catabolites, particlarly HX, which accmlate as degradation prodcts of adenine ncleotides, are thoght to play an important role in reperfsion injry by prodcing speroxide radicals [1, 19]. Althogh the reversibility of mcosal damage was not determined, similar changes in adenine ncleotides 1 Glcose S81A1SC Isc 16 Colon ~blsc 7 Isc 14 t:i' ~ 6 E 12 ~5 ~ !!l.!!l Jheophylli ne S81A1SC Isc Colon Dblsc 12 Isc Cold Ischemia (hors) FIG. 6. Electrophysiologic response to glcose (left) or theophylline (right) stimlation of the colon and small bowel after cold ischemia.

7 TAKEYOSHI ET AL.: ISCHEMIAJREPERFUSION INJURY 47 and prine catabolites after warm and cold ischemia were obtained with the small intestine and the colon. Under warm ischemia, intestinal mcosa retained consistently higher ATP and energy charge than colonic mcosa, and the latter had greater hypoxanthine levels. These biochemical findings did not correlate with the more severe histologic mcosal damage seen in the small intestine dring ischemia and after reperfsion. Indeed, Canada et al. [25] indicated that xanthine was the only biochemical marker that correlated with the dration of ischemia, bt energy charge was of no vale in indicating the extent of injry in the small intestine. Hypothermic storage significantly retarded adenine ncleotide and prine catabolite changes. Becase ofthe architectral difference between the organs, it may not be appropriate to compare the degree of histopathologic damage to the mcosa, bt the small intestine developed extensive strctral abnormalities at earlier periods of warm and cold ischemia than the colon. This finding is consistent with the stdy reported by Robinson et al. [16], who compared fnctional and morphological changes in canine ilem and colon following in sit 2-hr stranglation ischemia. According to Robinson, immediately postischemia, both organs showed serios histologic damage, no amino acid and sgar transport, diminished Na+-K+-ATPase levels, and liberation of acid phosphatase into the circlation, bt the ilem was more sensitive to acte ischemia. The srgical procedre, transportation, dissection, and monting, followed by 7 min perfsion with an asanginos bffer soltion, cased no notorios inslts to the mcosal strctre, since normal specimens had no appreciable alterations except for one intestinal specimen. Mter reperfsion of the ischemic samples, the severity of mcosal damage was agmented in both organs. Reperfsion injry developed dring reoxygenation. Althogh reperfsion injry of organs is a wellestablished concept [1, 11], several stdies failed to demonstrate enhanced intestinal injry after in vivo warm ischemia and reperfsion [26, 27]. Recently, Park et al. [8, 9] confirmed the phenomenon histologically after warm and cold ischemia of the rat intestine. Or stdy is the first demonstration of intestinal reperfsion injry in vitro, particlarly with the colonic mcosa. Netrophils, and xanthine oxidase and hypoxanthine, have been considered factors involved in the reperfsion injry [1, 11], bt they had little effect in or investigations with cold ischemia becase we sed blood-free perfsion, and xanthine oxidase did not increase dring cold ischemia [28, 29]. Frther stdy is needed to clarify the case of intestinal reperfsion injry nder bloodless perfsion. 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