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Intestinal and Hemodynamic Impairment FollowingMesenteric Ischemia/Reperfusion1

Ashish Khanna, Ph.D.,*,2 Jon E. Rossman, M.S.,† Ho-Leung Fung, Ph.D.,* and Michael G. Caty, M.D.†

*Department of Pharmaceutics, University at Buffalo, Amherst, New York 14260; and †Department of Pediatric Surgery,Children’s Hospital of Buffalo, Buffalo, New York 14222

Journal of Surgical Research 99, 114–119 (2001)doi:10.1006/jsre.2001.6103, available online at http://www.idealibrary.com on

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Submitted for publication July 27,

Background. Clinical intestinal ischemia/reperfusion(I/R) injury results in local and systemic dysfunction. Arat model of transient mesenteric occlusion has beenused to study this phenomenon. However, a systematicanalysis of the rat model with respect to intestinal per-meability and hemodynamics has not been carried out.

Materials and methods. In anesthetized rats, the su-perior mesenteric artery was occluded for 60 min, fol-lowed by reperfusion for 4 h. Intestinal impairmentwas evaluated via histological examination and bymeasuring ex vivo apparent permeability coefficientsPapp) of mannitol (0.18 kDa), inulin (5 kDa), and dex-ran (70 kDa). Hemodynamic effects of intestinal I/Rere determined by monitoring mean arterial pres-

ure (MAP) and heart rate (HR) via a catheter placedn the femoral artery.

Results. The animal model was associated with in-reased ex vivo Papp for mannitol and inulin. Al-hough I/R injury was accompanied by significant his-ological disruption, there was no observablelteration in dextran permeability, suggesting thathe loss in normal barrier function was limited to low-olecular-weight compounds. Hemodynamic mea-

urements indicated that reperfusion induced a pre-ipitous and sustained fall in MAP. HR values fellharply following reperfusion but gradually increasednd eventually “overshot” to values greater than base-ine.

Conclusions. Our findings demonstrate the selectiveoss of barrier function of the small bowel following in-

1 This study was supported in part by a grant from the R. J.Stransky Foundation and the Women’s and Children’s Health Re-search Foundation of the Children’s Hospital of Buffalo.

2 To whom correspondence and reprint requests should be ad-

dressed at Department of Metabolism and Pharmacokinetics,Bristol–Myers Squibb Pharmaceutical Research Institute, MailstopHW 17-2.12, P.O. Box 5400, Princeton, NJ 08543. Fax: (609) 818-3675. E-mail: ashish.khanna@bms.com.

1140022-4804/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

0; published online May 14, 2001

estinal I/R. Furthermore, these results also illustratehe importance of selecting appropriate permeabilityarkers for the evaluation of intestinal damage. In light

f the significant hemodynamic disruption accompany-ng the animal model, our investigation also points to-ard the need for developing therapeutic strategies thatitigate the local and systemic effects of intestinal I/R

njury. © 2001 Academic Press

Key Words: small intestine; ischemia; reperfusion;superior mesenteric artery occlusion; permeability;histology; hemodynamics.

INTRODUCTION

Intestinal ischemia/reperfusion (I/R) injury isthought to be a causative mechanism for several gas-trointestinal diseases, such as necrotizing enterocolitis(NEC), mesenteric insufficiency in the elderly, and in-testinal dysfunction following bowel transplantation[1–3]. Reperfusion of ischemic tissue, although neces-sary for reparative mechanisms, has been shown toworsen acute ischemic injury via the release of reactiveoxygen species (ROS), e.g., superoxide [4, 5]. TheseROS initiate a cascade of events, including the activa-tion of neutrophils and the release of noxious stimulisuch as platelet activating factor (PAF) and histamine[6, 7]. Activated neutrophils infiltrate through intesti-nal epithelial and endothelial cells, causing mucosaland submucosal damage with the concomitant increasein bowel permeability [8]. This increase in permeabilityleads to a loss in the selective barrier function of thebowel, subsequently resulting in the translocation ofenteric bacterial products [1]. Furthermore, the releaseof neutrophils, bacterial products, and PAF leads to

significant distant pathophysiological effects, includ-ing hepatic and pulmonary dysfunction and systemichypotension [9–11]. The cumulative effect of these de-

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rangements and the paucity of suitable therapeuticoptions contribute to the significant mortality ratesassociated with these conditions [12].

To elucidate mechanisms associated with these dis-orders, it is important to develop animal models thatmimic some of the pathophysiology of clinical I/R.Hence, one goal of this work was to investigate theimpact of transient superior mesenteric artery occlu-sion on intestinal morphology, permeability, and sys-temic hemodynamics in the anesthetized rat. Further-more, depending on the size of the marker used fordetermination of intestinal permeability, several ex-perimental and clinical studies have reported conflict-ing results in conditions associated with intestinal in-flammation [13–15]. Therefore, another goal of thisstudy was to examine the effect of intestinal I/R on thepermeability of three solutes—mannitol, inulin, anddextran—that represent a wide range of molecularweights.

MATERIALS AND METHODS

Materials. Male Sprague–Dawley rats were obtained from Har-lan Sprague–Dawley, Inc. (Indianapolis, IN). The radiolabeled mark-ers were bought from American Radiolabeled Chemicals (St. Louis,MO). All other materials were purchased from Sigma Chemical Co.(St. Louis, MO).

Rat intestinal I/R model. All procedures used in the presentstudy have been approved by The State University of New York atBuffalo Animal Care Committee. The procedure used for inducingsuperior mesenteric artery (SMA) occlusion has been described by uspreviously [16]. Briefly, adult male Sprague–Dawley rats (225–275g) were fasted but had free access to water the night before theexperiment. Animals were anesthetized by the administration ofintramuscular ketamine (90 mg/kg) and xylazine (9 mg/kg). Follow-ing a midline laparotomy, the SMA was occluded for 60 min with anatraumatic vascular loop followed by reperfusion for a period of 4 h.Between surgical interventions, the incision was sutured and cov-ered with plastic wrap to minimize fluid losses. To maintain anadequate anesthetic plane, ketamine/xylazine was administered asnecessary and the rats were placed on heating pads at 37°C through-out the experiment. Time-matched animals, which had been sub-jected to midline laparotomy and dissection without SMA occlusion,served as controls. Following reperfusion, the entire small bowel washarvested and used for determining intestinal permeability and his-tology as described.

Intestinal histology. Sections for histological examination wereprocessed according to the method of O’Donnell et al. [17]. Briefly,

TABLE 1

Description of Histological Grading Schemea

Grade Histologic changes

0 Normal1 Subepithelial edema, partial separation of apical cells2 Epithelial cell slough from tips of villi3 Progression of slough to base of villi4 Partial mucosal necrosis of lamina propria

KHANNA ET AL.: LOCAL AND SYSTEMIC DISRU

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5 Total mucosal necrosis

a From O’Donnell et al. [17].

random distal segments were obtained from the remainder of theintestinal tissue and fixed in 10% buffered paraformaldehyde for 1week. They were then embedded in paraffin, cut, and stained withhematoxylin-eosin; histological assessment was carried out inblinded fashion by three investigators (A.K., J.E.R., and M.G.C.),with the mean of the observations being used for analysis. Thegrading scheme has been adapted from Oldham et al. [18] as modi-

ed from Chiu et al. [19]. Thus, injury was classified using a semi-uantitative grading system where a numerical score was assignedased on the type of mucosal and submucosal damage (Table 1).

Intestinal permeability. Ex vivo apparent permeability coeffi-ients (Papp) were determined for mannitol (molecular weight 582), inulin (molecular weight 5 5,000), and dextran (moleculareight 5 70,000). Harvested intestinal segments were flushed with

ce-cold Krebs’ bicarbonate buffer (KBR) (pH 7.4; in mM, NaCl 120,Cl 5.6, MgCl2 1.2, NaH2PO4 1.2, dextrose 10, NaHCO3 25, and

CaCl2 2.5, adjusted to 290 mOsm/L with NaCl) and placed on ance-cooled glass plate. A 15-cm segment of isolated tissue was thenannulated on both ends using PE-240 tubing, mounted in 10 ml ofBR at 37°C, and gassed with 95% O2 and 5% CO2 (Fig. 1). For each

intestinal segment, mucosal to serosal flux of 3H-mannitol (specificactivity 5 110 Ci/g) or 3H-inulin (210 mCi/g) was determined in thepresence of 14C-dextran (1.5 mCi/g) as the internal standard. Traceamounts of radiolabeled (“hot”) markers were used and adjusted tothe indicated final concentrations with nonradiolabeled (“cold”) com-pound. Hence, dextran (total 5 4.0 mM) along with mannitol (total 54.2 mM) or inulin (total 5 4.3 mM) was added to the mucosal side in1 ml of KBR. Serosal appearance of the radioisotopes was measuredby liquid scintillation counting (Tri-Carb Liquid Scintillation Ana-lyzer, Model 1900, Packard Instrument Company, Meriden, CT),with dual monitoring for 3H and 14C. Aliquots of 0.5 ml were takenfrom the serosal side every 15 min up to 90 min, with replenishmentof the sampled volume by fresh KBR. This dilution during samplingwas taken into account while calculating mucosal to serosal flux. Thesampling protocol was chosen to ensure maintenance of “sink” con-ditions throughout the experiment [20]. Mucosal to serosal flux wascalculated by normalizing the radioisotope appearance curve fortotal flux. At the end of 90 min, the intestinal segment was cut openalong the mesenteric border and its length was measured. Thebreadth of the segment was determined as the mean of five randommeasurements per segment. Surface area for each segment wascalculated as the product of length and mean breadth. Papps formannitol, inulin, and dextran were calculated as follows:

Papp 5DQ

DT 3 C0 3 SA 3 60 cm/s, (1)

FIG. 1. Experimental layout for in vitro intestinal permeabilitymeasurements.

115ONS IN MESENTERIC ISCHEMIA/REPERFUSION

here DQ/DT is flux, amount appearing in serosal side per unit timemol/min), C 0 is total concentration of marker initially added (mol/l), and SA is macro surface area of the intestinal segment (cm2).

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Measurement of mean arterial pressure and heart rate. A sepa-rate set of rats was anesthetized as described earlier and a polyeth-ylene catheter (PE-50) was inserted into the left femoral artery. Thecatheter was connected to the Cardiomax-II digital data acquisitionsystem (Columbus Instruments, Columbus, OH) via a Gould trans-ducer (Gould Statham Instruments, Inc., Cleveland, OH) for moni-toring hemodynamic parameters, viz., mean arterial pressure (MAP)and heart rate (HR). Periodically, the cannula was flushed withheparinized saline (100 ml) to maintain recording fidelity. After a0-min stabilization period, baseline values were measured in eachat by recording MAP and HR every 5 min over a 20-min period andalculated as the mean of the four observations. The animals werehen subjected to mesenteric ischemia followed by reperfusion asescribed earlier, with hemodynamic monitoring throughout the du-ation of the experiment. Data are expressed as the percentage ofhange from baseline values.Statistical analyses. Histology scores were obtained as the mean

alues reported from three independent blinded individuals and areherefore reported as mean 6 SEM. Similarly, baseline hemody-amic parameters are reported as mean 6 SEM since they werebtained by averaging the mean of four different observations perat. All other values are reported as mean 6 SD. Comparisonsetween groups were made by the Student’s t test at the P , 0.05evel.

RESULTS

Histological Damage Following I/R

Figure 2 shows a representative photomicrographof an intestinal section from a control animal. Therewas no evidence of epithelial disruption and the villiwere intact and clearly visible. Figure 3, obtainedfrom a representative rat subjected to intestinal I/R,demonstrates the presence of severe morphologicaldamage indicated by the appearance of epithelialsloughing, submucosal damage, and the progressionof injury toward the serosa. Tissue injury was quan-tified on the basis of our histological grading scheme

FIG. 2. Photomicrograph of representative intestinal tissue sectiprojections (villi) with intact epithelial cells are clearly visible.

116 JOURNAL OF SURGICAL RESE

(Table 1). Therefore, the I/R group was associatedwith a higher histology score compared to controlanimals, indicative of increased tissue damage

(2.03 6 0.24 vs 0.86 6 0.15 in controls, n 5 9 rats pergroup, P , 0.001).

Effect of I/R on Intestinal Permeability

Mucosal to serosal fluxes for mannitol, inulin, anddextran were calculated from the linear serosal radio-isotope appearance curves. Table 2 gives the calculatedapparent Papp of mannitol, inulin, and dextran. Thepermeabilities of mannitol and inulin were signifi-cantly higher in the I/R group (P , 0.05 vs control;Table 2), while dextran permeability remained unaf-fected. It is evident from Table 2 that the coefficient ofvariability associated with these values was very high(23.3 to 46.5%). However, when we divided Pappmannitol

or Pappinulin by the Pappdextran observed in the sameintestinal segment, the resultant relative permeabilitycoefficient (RPC) obtained exhibited lower variability(10.8 to 14.9%). Table 2 illustrates that animals in theI/R group were associated with higher RPC values formannitol and inulin in comparison to sham-surgeryanimals, indicating increased intestinal permeabilityin the disease group. In addition to reduced variabili-ties, the statistical significance of the difference betweendisease and control animals was also enhanced whenRPC was used as the indicator of permeability (Table 2).

Effect of Intestinal I/R on Hemodynamic Parameters

Similar baseline hemodynamic values in MAP(sham, 101 6 8 vs I/R, 102 6 9 mm Hg; P . 0.05) andHR (sham, 259 6 18 vs I/R, 254 6 14 beats/min; P .0.05) were observed in both groups. The time course ofMAP changes in animals subjected to I/R and respec-tive controls are depicted in Fig. 4. MAP in shamanimals was stable over the study period. However, in

from sham-surgery group (magnification 2503). Distinct finger-like

H: VOL. 99, NO. 1, JULY 2001

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I/R rats, mesenteric occlusion resulted in a pronounced(40%) but transient increase in MAP in comparison tosham-surgery animals (Fig. 4; P , 0.005). The eleva-

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tion in MAP returned to control values within 40 min ofischemia. Immediately following reperfusion, MAPdropped considerably and remained constant at ap-proximately 80% of control for the remainder of theobservation period (Fig. 4).

No significant change in HR was observed in sham-surgery animals during the study period (Fig. 5). ForI/R animals, HR values were also unaltered duringischemia. Reperfusion in I/R rats induced a precipitousdecrease in HR to approximately 60% of control (Fig.5). However, this immediate bradycardia was gradu-ally reversed during reperfusion and eventually “over-shot” the baseline values by about 20 mm Hg (P ,

.05 at t 5 280 and 300 min).

DISCUSSION

In the present study we have demonstrated thattransient mesenteric artery occlusion in rats is associ-ated with significant mucosal and submucosal damage.Histological evidence demonstrates extensive sheddingof epithelial cells from the villous surface, leading toexposure of the submucosa and basement membrane.Similar structural damage has been observed in clini-cal NEC [21]. These findings therefore support the

FIG. 3. Representative hematoxylin and eosin–stained intestinaith epithelial sloughing and progression of the injury toward the s

TAB

Effect of Intestinal I/R Injury on ex Vivo Peand Dextran in

Group Pappmannitol Pappdextran RPCmannitol

Sham 17.8 6 6.53 (36.7) 4.88 6 1.47 (30.1) 4.20 6 0.49 (11.7I/R 34.9 6 8.37* (24.0) 4.89 6 1.32 (27.0) 7.19 6 1.07** (14

27

KHANNA ET AL.: LOCAL AND SYSTEMIC DISRU

Note. Papp presented in cm/s (310 ). RPC denotes relative permeariation. n 5 3 rats per group for mannitol and n 5 6 rats/group for

* P , 0.05,** P , 0.001 vs control.

usefulness of the animal model to mimic morphologicalchanges associated with the disease.

To assess if structural damage was accompaniedby alterations in normal barrier function of the in-testine, we determined ex vivo permeabilities to

aracellular markers of different molecular weights.etermination of ex vivo apparent Papp may circum-ent problems such as systemic dilution of perme-ble markers and differences in gastrointestinalransit time that influence in vivo permeability mea-urements [22]. The data show that mesenteric I/Results in increased permeability to low-molecular-eight markers such as mannitol and inulin. Al-

hough the animal model was associated with severeucosal damage, this effect did not translate into

ltered dextran flux. These results are in agreementith studies in a porcine intestinal I/R model, where

he authors reported increased mannitol and inulinux but unaltered dextran (70 kDa) permeability22]. Increased permeability to mannitol and inulinay be due to mucosal and submucosal damage re-

ulting in greater exposure to the “leakier” cryptells at the base of the villi [15]. Permeability toextran may be unaltered since its diffusion is lim-

ction from the I/R group depicts significant morphological damage,sa (magnification 2503).

2

eability of Mannitol and Dextran or Inulinesthetized Rats

Pappinulin Pappdextran RPCinulin

8.73 6 3.74 (42.8) 5.08 6 2.36 (46.5) 1.76 6 0.19 (10.8)14.7 6 3.42* (23.3) 5.70 6 1.65 (29.9) 2.63 6 0.38** (14.4)

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ability coefficient. Number in parentheses indicates coefficient ofinulin. Data presented as means 6 SD.

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ited by the gap junctions between the crypt cellsrather than by exposure to them [15].

Normalizing the permeability of mannitol or inulinwith that of dextran results in calculation of the RPC,an index that is independent of fluid shifts and impre-cisions in measuring length and surface area of theintestinal segment. We have found that this approachreduces the variability accompanying ex vivo perme-ability coefficient measurements (Table 2). Normaliz-ing Papp with an internal standard has been shown toimprove prediction of in vivo peroral absorption from invitro intestinal permeability data [23]. Another reporthas used a similar approach to obtain better estimatesfor the in situ intestinal permeability of D-glucose inrats [24].

Our results also demonstrate that induction of intes-tinal ischemia results in a steep rise in MAP thatgradually diminishes, reaching preocclusion values bythe end of ischemia. Hayward and Lefer have proposedthat the abrupt rise in MAP induced by intestinalischemia may be mediated via a decrease in the barore-ceptor input to the medullary vasomotor center in re-sponse to reduced splanchnic perfusion [7]. The au-thors also reported that the return of MAP to controlvalues toward the end of ischemia may be due to thetransduction of fluid across the microcirculation [7].Reperfusion was accompanied by an abrupt and sus-tained decrease in MAP, indicating severe circulatoryshock. The precipitous decrease in MAP followingreperfusion has been shown to be primarily mediatedby the release of PAF from the postischemic intestine[7, 11, 25]. This is supported by evidence that the

FIG. 4. Mean arterial pressure measurements in I/R animals (F)nd corresponding sham-surgery animals (Œ). Data were recordedvery 20 min and are presented as mean 6 SD (n 5 5 rats in the I/Rroup and n 5 3 in the control group; *P , 0.05, **P , 0.005 vsime-matched control).

118 JOURNAL OF SURGICAL RESE

intravenous administration of PAF in control animalsinduces a 70% drop in MAP within 30 s of injection[26]. Furthermore, PAF antagonists have been shown

I

to prevent the circulatory collapse accompanyingreperfusion [11]. In both groups of animals studied, HRremained essentially unchanged during ischemia.Reperfusion resulted in an abrupt decrease in HR com-pared to sham-operated animals. However, HR gradu-ally rose to preocclusion values at the end of 2 h ofreperfusion and eventually resulted in an overshooteffect, with HR values significantly greater just beforetermination of the experiment than during preocclu-sion. While the initial bradycardia may be mediated byvagal stimulation or release of a myocardial depressantfactor, development of tachycardia may be a responseto decreased MAP or hypovolemia due to I/R [27].These results illustrate the complexity of hemody-namic regulation in I/R and indicate the need for fur-ther studies to delineate the mechanisms responsiblefor the cardiovascular response during I/R.

In conclusion, we have demonstrated altered bowelmorphology, permeability, and hemodynamics in a ratmodel of SMA occlusion. Loss of the normal barrierfunction of the small bowel results in increased perme-ability to mannitol and inulin. In addition, we haveshown that calculation of the RPC may reduce thevariability associated with permeability measure-ments, and we propose that this approach may beapplied to experimental studies in intestinal I/R andother gastrointestinal conditions, such as Crohn’s dis-ease and celiac sprue. Finally, we have described a ratmodel of intestinal I/R that appears to be useful forstudying mechanisms and therapeutic approaches inintestinal I/R injury. Our investigation also under-scores the importance of developing treatment modal-ities capable of alleviating local and systemic derange-ments following intestinal I/R.

FIG. 5. Heart rate measurements in I/R animals (F) and corre-

H: VOL. 99, NO. 1, JULY 2001

sponding sham-surgery animals (Œ). Data were measured every 20min in beats/min and are presented as mean 6 SD (n 5 5 rats in the/R group and n 5 3 in the control group; *P , 0.05, **P , 0.005

vs time-matched control).

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ACKNOWLEDGMENT

We thank Mr. David Soda, Department of Pharmaceutics, Univer-sity at Buffalo, for his excellent surgical assistance.

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