Reperfusion

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

Intestinal and Hemodynamic Impairment Following Mesenteric Ischemia/Reperfusion 1 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 Submitted for publication July 27, 2000; published online May 14, 2001

Background. Clinical intestinal ischemia/reperfusion (I/R) injury results in local and systemic dysfunction. A rat model of transient mesenteric occlusion has been used to study this phenomenon. However, a systematic analysis of the rat model with respect to intestinal permeability and hemodynamics has not been carried out. Materials and methods. In anesthetized rats, the superior mesenteric artery was occluded for 60 min, followed by reperfusion for 4 h. Intestinal impairment was evaluated via histological examination and by measuring ex vivo apparent permeability coefficients (Papp) of mannitol (0.18 kDa), inulin (5 kDa), and dextran (70 kDa). Hemodynamic effects of intestinal I/R were determined by monitoring mean arterial pressure (MAP) and heart rate (HR) via a catheter placed in the femoral artery. Results. The animal model was associated with increased ex vivo Papp for mannitol and inulin. Although I/R injury was accompanied by significant histological disruption, there was no observable alteration in dextran permeability, suggesting that the loss in normal barrier function was limited to lowmolecular-weight compounds. Hemodynamic measurements indicated that reperfusion induced a precipitous and sustained fall in MAP. HR values fell sharply following reperfusion but gradually increased and eventually “overshot” to values greater than baseline. Conclusions. Our findings demonstrate the selective loss of barrier function of the small bowel following in1

This study was supported in part by a grant from the R. J. Stransky Foundation and the Women’s and Children’s Health Research Foundation of the Children’s Hospital of Buffalo. 2 To whom correspondence and reprint requests should be addressed at Department of Metabolism and Pharmacokinetics, Bristol–Myers Squibb Pharmaceutical Research Institute, Mailstop HW 17-2.12, P.O. Box 5400, Princeton, NJ 08543. Fax: (609) 8183675. E-mail: [email protected].

0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

testinal I/R. Furthermore, these results also illustrate the importance of selecting appropriate permeability markers for the evaluation of intestinal damage. In light of the significant hemodynamic disruption accompanying the animal model, our investigation also points toward the need for developing therapeutic strategies that mitigate the local and systemic effects of intestinal I/R injury. © 2001 Academic Press Key Words: small intestine; ischemia; reperfusion; superior mesenteric artery occlusion; permeability; histology; hemodynamics. INTRODUCTION

Intestinal ischemia/reperfusion (I/R) injury is thought to be a causative mechanism for several gastrointestinal diseases, such as necrotizing enterocolitis (NEC), mesenteric insufficiency in the elderly, and intestinal dysfunction following bowel transplantation [1–3]. Reperfusion of ischemic tissue, although necessary for reparative mechanisms, has been shown to worsen acute ischemic injury via the release of reactive oxygen species (ROS), e.g., superoxide [4, 5]. These ROS initiate a cascade of events, including the activation of neutrophils and the release of noxious stimuli such as platelet activating factor (PAF) and histamine [6, 7]. Activated neutrophils infiltrate through intestinal epithelial and endothelial cells, causing mucosal and submucosal damage with the concomitant increase in bowel permeability [8]. This increase in permeability leads to a loss in the selective barrier function of the bowel, subsequently resulting in the translocation of enteric bacterial products [1]. Furthermore, the release of neutrophils, bacterial products, and PAF leads to significant distant pathophysiological effects, including hepatic and pulmonary dysfunction and systemic hypotension [9 –11]. The cumulative effect of these de-

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rangements and the paucity of suitable therapeutic options contribute to the significant mortality rates associated with these conditions [12]. To elucidate mechanisms associated with these disorders, it is important to develop animal models that mimic some of the pathophysiology of clinical I/R. Hence, one goal of this work was to investigate the impact of transient superior mesenteric artery occlusion on intestinal morphology, permeability, and systemic hemodynamics in the anesthetized rat. Furthermore, depending on the size of the marker used for determination of intestinal permeability, several experimental and clinical studies have reported conflicting results in conditions associated with intestinal inflammation [13–15]. Therefore, another goal of this study was to examine the effect of intestinal I/R on the permeability of three solutes—mannitol, inulin, and dextran—that represent a wide range of molecular weights. MATERIALS AND METHODS Materials. Male Sprague–Dawley rats were obtained from Harlan Sprague–Dawley, Inc. (Indianapolis, IN). The radiolabeled markers 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 present study have been approved by The State University of New York at Buffalo Animal Care Committee. The procedure used for inducing superior mesenteric artery (SMA) occlusion has been described by us previously [16]. Briefly, adult male Sprague–Dawley rats (225–275 g) were fasted but had free access to water the night before the experiment. Animals were anesthetized by the administration of intramuscular ketamine (90 mg/kg) and xylazine (9 mg/kg). Following a midline laparotomy, the SMA was occluded for 60 min with an atraumatic vascular loop followed by reperfusion for a period of 4 h. Between surgical interventions, the incision was sutured and covered with plastic wrap to minimize fluid losses. To maintain an adequate anesthetic plane, ketamine/xylazine was administered as necessary and the rats were placed on heating pads at 37°C throughout the experiment. Time-matched animals, which had been subjected to midline laparotomy and dissection without SMA occlusion, served as controls. Following reperfusion, the entire small bowel was harvested and used for determining intestinal permeability and histology as described. Intestinal histology. Sections for histological examination were processed according to the method of O’Donnell et al. [17]. Briefly,

TABLE 1 Description of Histological Grading Scheme a Grade

Histologic changes

0 1 2 3 4 5

Normal Subepithelial edema, partial separation of apical cells Epithelial cell slough from tips of villi Progression of slough to base of villi Partial mucosal necrosis of lamina propria Total mucosal necrosis

a

From O’Donnell et al. [17].

115

FIG. 1. Experimental layout for in vitro intestinal permeability measurements.

random distal segments were obtained from the remainder of the intestinal tissue and fixed in 10% buffered paraformaldehyde for 1 week. They were then embedded in paraffin, cut, and stained with hematoxylin-eosin; histological assessment was carried out in blinded fashion by three investigators (A.K., J.E.R., and M.G.C.), with the mean of the observations being used for analysis. The grading scheme has been adapted from Oldham et al. [18] as modified from Chiu et al. [19]. Thus, injury was classified using a semiquantitative grading system where a numerical score was assigned based on the type of mucosal and submucosal damage (Table 1). Intestinal permeability. Ex vivo apparent permeability coefficients (Papp) were determined for mannitol (molecular weight ⫽ 182), inulin (molecular weight ⫽ 5,000), and dextran (molecular weight ⫽ 70,000). Harvested intestinal segments were flushed with ice-cold Krebs’ bicarbonate buffer (KBR) (pH 7.4; in mM, NaCl 120, KCl 5.6, MgCl 2 1.2, NaH 2PO 4 1.2, dextrose 10, NaHCO 3 25, and CaCl 2 2.5, adjusted to 290 mOsm/L with NaCl) and placed on an ice-cooled glass plate. A 15-cm segment of isolated tissue was then cannulated on both ends using PE-240 tubing, mounted in 10 ml of KBR at 37°C, and gassed with 95% O 2 and 5% CO 2 (Fig. 1). For each intestinal segment, mucosal to serosal flux of 3H-mannitol (specific activity ⫽ 110 Ci/g) or 3H-inulin (210 mCi/g) was determined in the presence of 14C-dextran (1.5 mCi/g) as the internal standard. Trace amounts of radiolabeled (“hot”) markers were used and adjusted to the indicated final concentrations with nonradiolabeled (“cold”) compound. Hence, dextran (total ⫽ 4.0 ␮M) along with mannitol (total ⫽ 4.2 ␮M) or inulin (total ⫽ 4.3 ␮M) was added to the mucosal side in 1 ml of KBR. Serosal appearance of the radioisotopes was measured by liquid scintillation counting (Tri-Carb Liquid Scintillation Analyzer, Model 1900, Packard Instrument Company, Meriden, CT), with dual monitoring for 3H and 14C. Aliquots of 0.5 ml were taken from the serosal side every 15 min up to 90 min, with replenishment of the sampled volume by fresh KBR. This dilution during sampling was taken into account while calculating mucosal to serosal flux. The sampling protocol was chosen to ensure maintenance of “sink” conditions throughout the experiment [20]. Mucosal to serosal flux was calculated by normalizing the radioisotope appearance curve for total flux. At the end of 90 min, the intestinal segment was cut open along the mesenteric border and its length was measured. The breadth of the segment was determined as the mean of five random measurements per segment. Surface area for each segment was calculated as the product of length and mean breadth. Papps for mannitol, inulin, and dextran were calculated as follows:

Papp ⫽

⌬Q cm/s, ⌬T ⫻ C 0 ⫻ SA ⫻ 60

(1)

where ⌬Q/⌬T is flux, amount appearing in serosal side per unit time (mol/min), C 0 is total concentration of marker initially added (mol/ ml), and SA is macro surface area of the intestinal segment (cm 2).

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FIG. 2. Photomicrograph of representative intestinal tissue section from sham-surgery group (magnification 250⫻). Distinct finger-like projections (villi) with intact epithelial cells are clearly visible.

Measurement of mean arterial pressure and heart rate. A separate set of rats was anesthetized as described earlier and a polyethylene catheter (PE-50) was inserted into the left femoral artery. The catheter was connected to the Cardiomax-II digital data acquisition system (Columbus Instruments, Columbus, OH) via a Gould transducer (Gould Statham Instruments, Inc., Cleveland, OH) for monitoring hemodynamic parameters, viz., mean arterial pressure (MAP) and heart rate (HR). Periodically, the cannula was flushed with heparinized saline (100 ␮l) to maintain recording fidelity. After a 30-min stabilization period, baseline values were measured in each rat by recording MAP and HR every 5 min over a 20-min period and calculated as the mean of the four observations. The animals were then subjected to mesenteric ischemia followed by reperfusion as described earlier, with hemodynamic monitoring throughout the duration of the experiment. Data are expressed as the percentage of change from baseline values. Statistical analyses. Histology scores were obtained as the mean values reported from three independent blinded individuals and are therefore reported as mean ⫾ SEM. Similarly, baseline hemodynamic parameters are reported as mean ⫾ SEM since they were obtained by averaging the mean of four different observations per rat. All other values are reported as mean ⫾ SD. Comparisons between groups were made by the Student’s t test at the P ⬍ 0.05 level.

RESULTS

Histological Damage Following I/R Figure 2 shows a representative photomicrograph of an intestinal section from a control animal. There was no evidence of epithelial disruption and the villi were intact and clearly visible. Figure 3, obtained from a representative rat subjected to intestinal I/R, demonstrates the presence of severe morphological damage indicated by the appearance of epithelial sloughing, submucosal damage, and the progression of injury toward the serosa. Tissue injury was quantified on the basis of our histological grading scheme (Table 1). Therefore, the I/R group was associated with a higher histology score compared to control animals, indicative of increased tissue damage

(2.03 ⫾ 0.24 vs 0.86 ⫾ 0.15 in controls, n ⫽ 9 rats per group, P ⬍ 0.001). Effect of I/R on Intestinal Permeability Mucosal to serosal fluxes for mannitol, inulin, and dextran were calculated from the linear serosal radioisotope appearance curves. Table 2 gives the calculated apparent Papp of mannitol, inulin, and dextran. The permeabilities of mannitol and inulin were significantly higher in the I/R group (P ⬍ 0.05 vs control; Table 2), while dextran permeability remained unaffected. It is evident from Table 2 that the coefficient of variability associated with these values was very high (23.3 to 46.5%). However, when we divided Papp mannitol or Papp inulin by the Papp dextran observed in the same intestinal segment, the resultant relative permeability coefficient (RPC) obtained exhibited lower variability (10.8 to 14.9%). Table 2 illustrates that animals in the I/R group were associated with higher RPC values for mannitol and inulin in comparison to sham-surgery animals, indicating increased intestinal permeability in the disease group. In addition to reduced variabilities, the statistical significance of the difference between disease and control animals was also enhanced when RPC 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 ⫾ 8 vs I/R, 102 ⫾ 9 mm Hg; P ⬎ 0.05) and HR (sham, 259 ⫾ 18 vs I/R, 254 ⫾ 14 beats/min; P ⬎ 0.05) were observed in both groups. The time course of MAP changes in animals subjected to I/R and respective controls are depicted in Fig. 4. MAP in sham animals was stable over the study period. However, in I/R rats, mesenteric occlusion resulted in a pronounced (40%) but transient increase in MAP in comparison to sham-surgery animals (Fig. 4; P ⬍ 0.005). The eleva-

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FIG. 3. Representative hematoxylin and eosin–stained intestinal section from the I/R group depicts significant morphological damage, with epithelial sloughing and progression of the injury toward the serosa (magnification 250⫻).

tion in MAP returned to control values within 40 min of ischemia. Immediately following reperfusion, MAP dropped considerably and remained constant at approximately 80% of control for the remainder of the observation period (Fig. 4). No significant change in HR was observed in shamsurgery animals during the study period (Fig. 5). For I/R animals, HR values were also unaltered during ischemia. Reperfusion in I/R rats induced a precipitous decrease in HR to approximately 60% of control (Fig. 5). However, this immediate bradycardia was gradually reversed during reperfusion and eventually “overshot” the baseline values by about 20 mm Hg (P ⬍ 0.05 at t ⫽ 280 and 300 min). DISCUSSION

In the present study we have demonstrated that transient mesenteric artery occlusion in rats is associated with significant mucosal and submucosal damage. Histological evidence demonstrates extensive shedding of epithelial cells from the villous surface, leading to exposure of the submucosa and basement membrane. Similar structural damage has been observed in clinical NEC [21]. These findings therefore support the

usefulness of the animal model to mimic morphological changes associated with the disease. To assess if structural damage was accompanied by alterations in normal barrier function of the intestine, we determined ex vivo permeabilities to paracellular markers of different molecular weights. Determination of ex vivo apparent Papp may circumvent problems such as systemic dilution of permeable markers and differences in gastrointestinal transit time that influence in vivo permeability measurements [22]. The data show that mesenteric I/R results in increased permeability to low-molecularweight markers such as mannitol and inulin. Although the animal model was associated with severe mucosal damage, this effect did not translate into altered dextran flux. These results are in agreement with studies in a porcine intestinal I/R model, where the authors reported increased mannitol and inulin flux but unaltered dextran (70 kDa) permeability [22]. Increased permeability to mannitol and inulin may be due to mucosal and submucosal damage resulting in greater exposure to the “leakier” crypt cells at the base of the villi [15]. Permeability to dextran may be unaltered since its diffusion is lim-

TABLE 2 Effect of Intestinal I/R Injury on ex Vivo Permeability of Mannitol and Dextran or Inulin and Dextran in Anesthetized Rats Group

Papp mannitol

Papp dextran

RPC mannitol

Papp inulin

Papp dextran

RPC inulin

Sham I/R

17.8 ⫾ 6.53 (36.7) 34.9 ⫾ 8.37* (24.0)

4.88 ⫾ 1.47 (30.1) 4.89 ⫾ 1.32 (27.0)

4.20 ⫾ 0.49 (11.7) 7.19 ⫾ 1.07** (14.9)

8.73 ⫾ 3.74 (42.8) 14.7 ⫾ 3.42* (23.3)

5.08 ⫾ 2.36 (46.5) 5.70 ⫾ 1.65 (29.9)

1.76 ⫾ 0.19 (10.8) 2.63 ⫾ 0.38** (14.4)

Note. Papp presented in cm/s (⫻10 ⫺7). RPC denotes relative permeability coefficient. Number in parentheses indicates coefficient of variation. n ⫽ 3 rats per group for mannitol and n ⫽ 6 rats/group for inulin. Data presented as means ⫾ SD. * P ⬍ 0.05, ** P ⬍ 0.001 vs control.

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FIG. 4. Mean arterial pressure measurements in I/R animals (F) and corresponding sham-surgery animals (Œ). Data were recorded every 20 min and are presented as mean ⫾ SD (n ⫽ 5 rats in the I/R group and n ⫽ 3 in the control group; *P ⬍ 0.05, **P ⬍ 0.005 vs time-matched control).

ited by the gap junctions between the crypt cells rather than by exposure to them [15]. Normalizing the permeability of mannitol or inulin with that of dextran results in calculation of the RPC, an index that is independent of fluid shifts and imprecisions in measuring length and surface area of the intestinal segment. We have found that this approach reduces the variability accompanying ex vivo permeability coefficient measurements (Table 2). Normalizing Papp with an internal standard has been shown to improve prediction of in vivo peroral absorption from in vitro intestinal permeability data [23]. Another report has used a similar approach to obtain better estimates for the in situ intestinal permeability of D-glucose in rats [24]. Our results also demonstrate that induction of intestinal ischemia results in a steep rise in MAP that gradually diminishes, reaching preocclusion values by the end of ischemia. Hayward and Lefer have proposed that the abrupt rise in MAP induced by intestinal ischemia may be mediated via a decrease in the baroreceptor input to the medullary vasomotor center in response to reduced splanchnic perfusion [7]. The authors also reported that the return of MAP to control values toward the end of ischemia may be due to the transduction of fluid across the microcirculation [7]. Reperfusion was accompanied by an abrupt and sustained decrease in MAP, indicating severe circulatory shock. The precipitous decrease in MAP following reperfusion has been shown to be primarily mediated by the release of PAF from the postischemic intestine [7, 11, 25]. This is supported by evidence that the intravenous administration of PAF in control animals induces a 70% drop in MAP within 30 s of injection [26]. Furthermore, PAF antagonists have been shown

to prevent the circulatory collapse accompanying reperfusion [11]. In both groups of animals studied, HR remained essentially unchanged during ischemia. Reperfusion resulted in an abrupt decrease in HR compared to sham-operated animals. However, HR gradually rose to preocclusion values at the end of 2 h of reperfusion and eventually resulted in an overshoot effect, with HR values significantly greater just before termination of the experiment than during preocclusion. While the initial bradycardia may be mediated by vagal stimulation or release of a myocardial depressant factor, development of tachycardia may be a response to decreased MAP or hypovolemia due to I/R [27]. These results illustrate the complexity of hemodynamic regulation in I/R and indicate the need for further studies to delineate the mechanisms responsible for the cardiovascular response during I/R. In conclusion, we have demonstrated altered bowel morphology, permeability, and hemodynamics in a rat model of SMA occlusion. Loss of the normal barrier function of the small bowel results in increased permeability to mannitol and inulin. In addition, we have shown that calculation of the RPC may reduce the variability associated with permeability measurements, and we propose that this approach may be applied to experimental studies in intestinal I/R and other gastrointestinal conditions, such as Crohn’s disease and celiac sprue. Finally, we have described a rat model of intestinal I/R that appears to be useful for studying mechanisms and therapeutic approaches in intestinal I/R injury. Our investigation also underscores the importance of developing treatment modalities capable of alleviating local and systemic derangements following intestinal I/R.

FIG. 5. Heart rate measurements in I/R animals (F) and corresponding sham-surgery animals (Œ). Data were measured every 20 min in beats/min and are presented as mean ⫾ SD (n ⫽ 5 rats in the I/R group and n ⫽ 3 in the control group; *P ⬍ 0.05, **P ⬍ 0.005 vs time-matched control).

KHANNA ET AL.: LOCAL AND SYSTEMIC DISRUPTIONS IN MESENTERIC ISCHEMIA/REPERFUSION

ACKNOWLEDGMENT

14.

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

15.

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