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Epithelial Permeability Is Not Increased in Rats Following Small Bowel Resection
Journal of Surgical Research 97, 65–70 (2001) doi:10.1006/jsre.2001.6113, available online at http://www.idealibrary.com on
Epithelial Permeability Is Not Increased in Rats Following Small Bowel Resection 1 David P. O’Brien, M.D., Lindsey A. Nelson, M.D., Lawrence E. Stern, M.D., Jodi L. Williams, B.S., Christopher J. Kemp, B.S., Quan Wang, Ph.D.,* Patrick Tso, Ph.D.,† Christopher R. Erwin, Ph.D., Per-Olof Hasselgren, M.D.,* and Brad W. Warner, M.D. 2 Division of Pediatric Surgery, Children’s Hospital Medical Center, *Shriner’s Hospital for Children, and Department of Surgery and †Department of Pathology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039 Presented at the Annual Meeting of the Association for Academic Surgery, Tampa, Florida, November 2– 4, 2000; published online March 16, 2001
media. There were no permeability differences between SBR and Sham lymph-treated monolayers. Conclusion. The early adaptive response of the remnant intestine after SBR is associated with reduced permeability. These results suggest an alternative mechanism for the increased bacterial translocation that has been described following SBR. © 2001 Academic Press Key Words: epithelium; permeability; short bowel syndrome; intestinal adaptation.
Background. Increased intestinal permeability and translocation of bacteria and/or bacterial products may cause infection and liver dysfunction in patients with the short bowel syndrome. In previous studies, serum from mice undergoing small bowel resection (SBR) enhanced growth of cultured rat intestinal epithelial cells (RIEC-6), implicating a role for a serum factor(s) in the enterocyte response to SBR. These experiments tested the hypothesis that epithelial cell permeability is increased following SBR. Materials and methods. Male Sprague-Dawley rats underwent a 75% SBR or sham operation. Intestinal permeability in the remnant ileum was determined by Ussing chambers on Postoperative Day (POD) 3. Additionally, serum was collected on POD 1, 3, and 7 and mesenteric lymph was harvested on POD 3. Once confluent, RIEC-6 cells were incubated for 3 days in media supplemented with 10% fetal bovine serum (FBS; control), 1% FBS, 1% FBS plus 9% Sham serum, or 1% FBS plus 9% SBR serum or exposed to media with varied concentrations of SBR or Sham lymph. Monolayer permeability was determined by measuring the passage of dextran-rhodamine. Results. Intestinal permeability was reduced in rats undergoing SBR. Sham serum-treated monolayers demonstrated the greatest permeability. Incubation with SBR serum reduced permeability to near control
INTRODUCTION
Gut barrier failure with subsequent translocation of enteric bacteria and/or bacterial products has been proposed to be a leading cause of systemic infection, sepsis, and multisystem organ failure in multiple injury models including the short bowel syndrome (SBS) [1– 4]. In patients with SBS who receive parenteral nutrition, the central venous catheter is assumed to be the nidus of infection for most episodes of sepsis. However, there is evidence to suggest that sepsis in these patients may be derived from the gut as a result of increased intestinal permeability [5]. Following massive small bowel resection (SBR), the remnant intestine compensates for the loss of digestive and absorptive function through a process termed adaptation. The pathogenesis of the adaptive process is complex, but likely involves luminal nutrients, gastrointestinal secretions, growth factors, and circulating hormones [6]. The putative role of a humoral factor(s) in adaptation has been demonstrated using vascular parabiosis experiments [7]. This laboratory has been studying intestinal adaptation through the use of a unique hybrid experimental model in which serum from animals undergoing intestinal adaptation after
1
Supported by National Institutes of Health F32 DK09882 (LES), National Institutes of Health RO 1 DK53234 (B.W.W.), and a grant from the Children’s Hospital Campaign for Children Fund, Children’s Hospital Medical Center, Cincinnati, OH (B.W.W.). 2 To whom correspondence and reprint requests should be addressed at Division of Pediatric Surgery, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. Fax: (513) 636-7657. E-mail: [email protected].
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0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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SBR is added to the culture media of a fully differentiated rat intestinal epithelial cell line (RIEC-6). Initial studies utilizing this model have confirmed the existence of a circulating factor(s) that enhances intestinal cell proliferation [8]. In contrast, a similar model involving thermal injury revealed the presence of a serum factor(s) that inhibited intestinal epithelial cell proliferation and migration, altered permeability, and promoted apoptosis [9]. Such disparate results highlight the unique intestinal epithelial cell response to serum harvested after different stimuli. The effect of postresection intestinal adaptation on gut permeability is not currently known. To test the hypothesis that permeability is increased during intestinal adaptation, this study examined intestinal permeability after SBR in whole tissue with a Ussing chamber. Additionally, this study investigated permeability changes in epithelial cell monolayers following exposure to serum or lymph taken from adapting rats after massive small bowel resection. MATERIALS AND METHODS Animals. A protocol for this study was approved by the Children’s Hospital Research Foundation Institutional Animal Care and Use Committee (Children’s Hospital Medical Center, Cincinnati, OH). Male Sprague-Dawley rats (weight range 230 –259 g; The Harlan Laboratory, Indianapolis IN) were housed in groups of two at 21°C on 12-h day-night cycles (6 AM to 6 PM) for at least 5 days before experimentation in order to allow them to acclimate to their environment. The animals were given free access to standard rat chow. Cells. Rat intestinal epithelial cells (RIEC-6; American Type Culture Collection, Manassas, VA) that had undergone 13–18 passages were used for all experiments. Cells were incubated in 10% CO 2 and grown under the following media conditions: 95% Dulbecco’s modified Eagle’s medium (DMEM; 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 1.0 mM sodium pyruvate; American Type Culture Collection) supplemented with 0.1 unit/mL bovine insulin (GibcoBRL, Gaithersburg, MD), an antibiotic-antimycotic (100 units/mL penicillin G sodium, 100 g/ml streptomycin sulfate, and 0.25 g/mL amphotericin B; GibcoBRL), and 5% heat inactivated fetal bovine serum (FBS; GibcoBRL). Cell passage and plating were accomplished with a 5-min incubation in trypsin-EDTA (0.25% trypsin and 1 mM EDTA; GibcoBRL). Operative procedure. The rat model for a 75% proximal SBR and sham (transection) operation was performed as previously presented in detail [10]. Water was provided ad libitum for the first 24 h. Rats from each group subsequently were fed standard rodent diet. Serum and tissue collection. Under 2% isoflurane inhaled anesthesia, the midline sutures were removed and the bowel was retracted in order to expose the PV. Using aseptic technique, approximately 5 mL of PV blood was aspirated and placed into sterile collection tubes. Six centimeters (approximately 1 cm from the anastomosis) of ileum was excised and the luminal contents were gently expressed with cotton swabs. The proximal 1 cm was immediately fixed with 10% neutral buffered formalin and used for histology; the remaining 5 cm was weighed immediately used for Ussing chamber analysis. The rats then were euthanized with an intracardiac injection of xylazine. Blood from sham and SBR groups was pooled and the serum collected, aliquoted, and stored at ⫺80°C for future use. Lymph collection. Under 2% isoflurane inhaled anesthesia, the midline sutures were removed and the bowel was retracted in order to expose the mesenteric lymph duct, which was cannulated accord-
ing to the method of Bollman et al. [11]. Lymph was collected over a 5-h-period, pooled, and then stored at ⫺80°C until further use. Histology. Formalin-fixed samples of ileum were oriented and embedded in paraffin in order to provide cut sections parallel with the longitudinal axis of the bowel. Five-micrometer sections were mounted on poly-L-lysine-coated slides (Erie Scientific Products, Portsmouth, NH) and stained with hematoxylin/eosin. Measurements of the villus heights were performed using a light microscope with a video camera (Dage-MTI, Michigan City, IN) attached to an image capture card (National Instruments, Austin, TX) in a standard desktop computer with image analysis software (NIH Image, National Institutes of Health, Bethesda, MD). At least 20 villi were counted and averaged for each sample, and only villi with an intact central lymphatic channel were considered. Ussing chamber. The methodologies for the Ussing chamber experiments have been described previously [12]. Briefly, a 5-cm Peyer’s patch-free segment of ileum immediately distal to the anastomotic site was harvested. A 2-cm segment was opened along the mesenteric border, washed in ice-cold Krebs-Ringer solution (KBR; 114 mM NaCl, 1.65 mM Na 2HPO 4, 0.3 mM NaH 2PO 4, 5 mM KCl, 1.1 mM MgCl 2, 25 mM NaHCO 3, and 1.25 mM CaCl 2, pH 7.4), and mounted into the Ussing chamber with 0.3 cm 2 of exposed surface area. The mucosal and serosal surfaces were bathed independently in KBR. D-Glucose (10 mM) was present in the medium on the serosal side; a mannitol (10 mM) marker solution containing fluorescein isothiocyanate (FITC)-dextran (4.4 mg/mL) and horseradish peroxidase (HRP; 4 mg/mL) was substituted for D-glucose on the mucosal side. The KBR was oxygenated with 95% O 2 and 5% CO 2 in a gas-lift recirculation system and maintained at 37°C by waterjacketed reservoirs. The potential difference (PD) was measured with a voltage clamp (VCC-MC6, Physiologic Instruments, San Diego, CA) and calomel electrodes. Electrical continuity between the mucosal and the serosal media was maintained with 4% agar bridges. All experiments were performed under short-circuit conditions in which an external feedback circuit of the voltage clamp continuously maintained the PD at zero through the application of a current directly opposing the tissue current. The current was applied through Ag/AgCl electrodes that were in contact with the mucosal and serosal media via 4% agar bridges. The short-circuit current (I sc) magnitude was obtained directly from the digital readout of the voltage clamp. Membrane resistance was calculated using Ohm’s law after passing a 4-A current through the membrane and measuring the PD. The resistance measurements are expressed as (R; ohms ⫻ cm 2). Intestinal tissues were allowed to equilibrate under short circuit conditions for 30 min prior to experimentation. At 30-minute intervals, 0.22 mL was removed from the serosal side and replaced with an equal volume of KBR. All tissues were challenged with 1 mM theophylline at the end of the experiments as a measure of tissue viability. Tissues with less than a 10% increase in I sc were discarded. The tissues were visually inspected to ensure that they remained intact. Molecular probes. (FITC)-dextran (4.4 kDa) and HRP (40 kDa) probes were used to test permeability. Prior to experimentation, a 96-well plate (Maxisorp, Nunc, Denmark) was coated with a goat anti-HRP antibody (dilution 1:5000; Dulbecco’s phosphate buffered saline; Sigma, St. Louis, MO) and allowed to incubate for 2 h at room temperature. Serosal samples from the Ussing chamber were added to the plate in duplicate. The (FITC)-dextran concentration was determined by fluorescein detection (FL600 Microplate Fluorescence Reader, BIO-TEK Instruments, Inc., Winooski, VT). After an additional 2-h incubation at room temperature, the plate was washed three times with Tris-buffered saline containing 0.2% polyoxyethylenesorbitan (Sigma). TMB substrate (50 l; Dako, Carpenteria, CA) was added to each well and incubated for 15 min. The reaction was stopped with 0.4 N H 2SO 4 (50 L). The HRP concentration was determined by measuring the absorbance change from 550 to 450 nm on a spectrophotometer (ThermoMax, Molecular Devices, Menlo Park, CA).
O’BRIEN ET AL.: EPITHELIAL PERMEABILITY AFTER SBR
FIG. 1. Ileal wet weights from male Sprague-Dawley rats on Postoperative Days (POD) 1, 3, and 7 following 75% mid-SBR or sham operation. Results are in milligrams per centimeter and are reported as mean ⫾ SEM. N ⫽ 4 –7 per group. *P ⬍ 0.05, SBR versus Sham.
Cell monolayer formation. The RIEC-6 cells were seeded onto the apical chamber membrane of a Transwell cell culture system (pore size 3 m; Transwell, Corning, Corning, NY) at a density of 33,000 cells per well. Media in the apical and basal chambers were exchanged twice weekly. The cells were allowed to grow to confluence (approximately 14 –17 days), which was verified by impermeability to blue dextran (Sigma). Measurement of cell monolayer permeability. Upon confluence, the media in the apical and basal chambers were exchanged for DMEM supplemented with 1% FBS, 10% FBS, 1% FBS plus 9% sham PV serum (Sham serum), or 1% FBS plus 9% SBR PV serum (SBR serum). Alternatively, the monolayers were exposed to DMEM and 5% FBS with 0, 1, 5, and 10% sham or SBR mesenteric lymph. After a 72-h incubation, 1% dextran-rhodamine (Molecular Probes, Eugene, OR) were added to the apical chamber. After a 5-h incubation, the media in the basal chamber were removed and the amount of dextran-rhodamine present was determined spectrophotometrically at 570 nm. Permeability results are expressed as percentage of the amount of dextran-rhodamine that crossed the monolayer. Statistical analysis. Results are presented as mean values (⫾standard error of the mean). When experiments included only two groups, an unpaired Student’s t test was used. When the experiments included more than two groups, statistical differences were identified using a one-way analysis of variance (ANOVA) followed by the Student-Neuman-Keuls test. The SigmaStat statistical package (SPSS, Chicago, IL) was utilized for all statistical analyses. A P value of less than 0.05 was considered significant.
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FIG. 2. Villus heights from male Sprague-Dawley rats on POD 3 following mid-SBR or sham operation. Results are in micrometers and are reported as mean ⫾ SEM. N ⫽ 5 per group. *P ⬍ 0.05, SBR versus Sham.
tically significant, there was a trend toward decreased permeability to the larger probe HRP and increased membrane resistance (data not shown) in the intestine of the rats that had undergone a SBR (Fig. 3b). There
RESULTS
Survival in all groups was greater than 90%, and all rats were healthy and vigorous at the time of sacrifice. Intestinal adaptation in the SBR group was confirmed grossly by increased ileal wet weights (Fig. 1) and villus heights (Fig. 2). To assess intestinal barrier function in whole tissue, the extent of (FITC)-dextran and HRP passage across the remnant ileal tissue on POD 3 was determined by mounting isolated segments into the Ussing chamber. There was a significant decrease in permeability to (FITC)-dextran of the SBR group at all time points during the study period (Fig. 3a). Although not statis-
FIG. 3. Mucosal-to-serosal flux of (FITC)-dextran and horseradish peroxidase (HRP) across male Sprague-Dawley rat intestine on POD 3 following a 75% mid-SBR or sham operation. Serosal sampling was performed every 30 min over 120-min study period. (a) Serosal concentrations of (FITC)-dextran are in nanograms per milliliter. (b) Serosal concentrations of HRP are in picograms per milliliter. Results are reported as mean ⫾ SEM. N ⫽ 4 per group. *P ⬍ 0.05, SBR versus Sham.
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DISCUSSION
FIG. 4. RIEC-6 monolayers were analyzed for permeability to dextran-rhodamine following a 72-h exposure to culture media supplemented with 1% fetal bovine serum (FBS), 10% FBS, 1% FBS plus 9% Sham serum (Sham), and 1% FBS plus 9% SBR serum (SBR). Sham and SBR sera were harvested from the portal vein of male Sprague-Dawley rats on POD 1, 3, and 7 after undergoing a 75% mid-SBR or sham operation. Results are reported as mean ⫾ SEM. N ⫽ 5– 6 per group. *P ⬍ 0.05, SBR versus Sham and POD 1 SBR and Sham serum versus 10% FBS.
were no differences in PD and I sc, indicating similar degrees of tissue viability and electrogenic ion secretion between the two groups (data not shown). To determine whether these intestinal permeability changes are affected by a circulating factor(s), RIEC-6 monolayers were exposed to either serum or lymph from the Sham or SBR group for 72 h. Because parameters of adaptation in rats occur as early as POD 1, are dramatically increased by POD 3, and are complete by POD 7, serum was harvested at these time points. With respect to control media, monolayers treated with POD 1 serum taken after either sham operation or SBR demonstrated decreased permeability to the dextran-rhodamine (Fig. 4). The SBR serum-treated cells were significantly less permeable than Sham serum taken on POD 1 and 3. However, the permeability between cells exposed to SBR or Sham serum was no longer significantly different, and it was similar to that of the cells receiving control media by POD 7. Interestingly, monolayers that were deprived of FBS (DMEM with 1% FBS) demonstrated a significant decrease in permeability to dextran-rhodamine at all time points compared to all other treatments. Because of the potential for greater cytokine content, the effect of mesenteric lymph on epithelial cell permeability was also evaluated after SBR. There were no significant differences in permeability between monolayers treated with Sham or SBR lymph at 1, 5, or 10% concentration (Fig. 5). However, monolayers incubated with SBR and Sham lymph at a concentration of 10% demonstrated were more permeable when compared to monolayers treated with DMEM with 0 or 1% SBR or Sham lymph.
The purpose of this study was to determine the effects of massive small bowel resection and intestinal adaptation on intestinal epithelial permeability. The intact remnant ileum was significantly less permeable to (FITC)-dextran but not HRP after SBR. Furthermore, POD 1 and 3 serum from rats that underwent a SBR reduced the permeability of an RIEC-6 monolayer when compared to monolayers incubated with Sham serum. In contrast, lymph from the SBR group did not alter monolayer permeability to a greater or lesser extent than lymph from the Sham group. These results suggest that intestinal adaptation after massive SBR is associated with decreased, not increased, epithelial permeability during the early phase of intestinal adaptation. Further, it is likely that the permeability effects are mediated in part by a humoral factor(s). The results of a limited number of studies that have examined intestinal permeability and intestinal adaptation have been conflicting. Utilizing radiolabeled polyethylene glycol (PEG) and in vivo isolated intestinal loops in rats, Urban et al. was unable to detect differences in permeability at 2 and 4 weeks following a 50 or 70% proximal SBR and concluded that the adapted mucosa of the remnant ileum presented a formidable barrier [13]. Schulzke et al. measured PEG fluxes and epithelial resistance with an Ussing chamber in rats 8 weeks following a 70% proximal SBR [14]. In contrast, they found a 40% increase in PEG permeability in the SBR animals. Of note, they analyzed tight junction morphology by freeze-fracture electron microscopy, and no differences in strand number, meshwork depth, or depth of the total tight junction were noted between control and SBR animals. Al-
FIG. 5. RIEC-6 monolayers were analyzed for permeability to dextran-rhodamine following a 72-h exposure to culture media supplemented with 5% fetal bovine serum (FBS) and 0% (control media), 1, 5, and 10% mesenteric lymph harvested from male SpragueDawley rats on POD 3 after undergoing a 75% mid-SBR or sham operation. Results are reported as mean ⫾ SEM. N ⫽ 5– 6 per group. *P ⬍ 0.05, 10% SBR and Sham lymph versus control media.
O’BRIEN ET AL.: EPITHELIAL PERMEABILITY AFTER SBR
though neither of these studies corrected for changes in mucosal surface, Schulzke et al. did perform morphometric analysis on the intestinal villi and estiamted a villus height increase of 20% in the SBR rats. The authors suggested that this change might account for the increase in PEG fluxes in the bowel-resected animals [14]. In the current study, the resected animals demonstrated an overall reduction in permeability. Although the data could be flawed by the lack of correction to changes in surface area, any increase in surface are would further reduce the calculated permeability demonstrated in these animals. These disparate observations may be explained, in part, because of the varied time interval that these animals were studied following intestinal resection. Along these lines, Freeman et al. showed that upregulation of D-glucose transport in rats undergoing a 66% proximal small bowel resection did not occur for 6 weeks, suggesting that complete functional adaptation, including factors that affect intestinal permeability, may be delayed [15]. The experiments in this study were performed at relatively early time points during postresection adaptation. Future experiments using longer postoperative time intervals will be necessary to determine the effect of late adaptation on intestinal permeability. Because in vivo models of intestinal injury and permeability are confounded by the complexity and cellular heterogeneity of the gut, many investigators have utilized in vitro cell culture systems [16, 17]. Recent work with hemorrhagic shock [1], thermal injury [9], and endotoxin [3] models has demonstrated the importance of a factor(s) in the serum and/or lymph that affects epithelial barrier function. In the context of these experimental paradigms, confluent RIEC-6 cell monolayers exposed to serum from rats that underwent a SBR demonstrated reduced permeability when compared to serum from control animals. This novel finding suggests an alternative or opposing mechanism than that in hemorrhagic shock, thermal injury, and endotoxin exposure. In those injury models, serum from the animals increased cell monolayer permeability. Different responses to various noxious stimuli also have been observed with proliferation. Serum from burned animals reduces the proliferation of RIEC-6 [9], which stands in contradistinction to previous work in our laboratory in which proliferation of these cells was significantly enhanced following exposure to SBR serum [8]. In a study performed by Magnotti et al. cell monolayer permeability was increased after an incubation with mesenteric lymph harvested from rats during hemorrhagic shock [1]. They found that the increase in endothelial monolayer permeability was greater than that induced by portal vein serum. In contrast, this study did not demonstrate significant differences in the permeability changes caused by the SBR and Sham lymph.
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Increased translocation of bacteria to mesenteric lymph nodes and spleen has been previously demonstrated after intestinal resection in several animal models [18 –21]. It is likely that bacteria are able to translocate by mechanisms unrelated to permeability changes involving more complex bacterial-epithelial cell processing and immune function. Along these lines, respiratory epithelial cell apoptosis has been demonstrated to be an important response for preventing the systemic spread of administered Pseudomonas [22]. This laboratory has previously recognized increased enterocyte apoptosis during intestinal adaptation [23]. The relationship among enterocyte apoptosis, postresection adaptation, and bacterial translocation remains to be elucidated. REFERENCES 1.
Magnotti, L. J., Upperman, J. S., Xu, D. Z., Lu, Q., and Deitch, E. A. Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann. Surg. 228: 518, 1998.
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Williamson, R. C. Intestinal adaptation (second of two parts). Mechanisms of control. N. Engl. J Med. 298: 1444, 1978.
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Stern, L. E., Erwin, C. R., O’Brien, D. P., Huang, F. S., and Warner, B. W. Serum from mice after small bowel resection enhances epithelial cell growth. J. Pediatr. Surg., 2000.
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Varedi, M., Greeley, G. H., Jr., Herndon, D. N., and Englander, E. W. A thermal injury-induced circulating factor(s) compromises intestinal cell morphology, proliferation, and migration. Am. J. Physiol. 277: G175, 1999.
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Warner, B. W., Vander Kolk, W. E., Can, G., Helmrath, M. A., Shin, C. E., and Erwin, C. R. Epidermal growth factor receptor expression following small bowel resection. J. Surg. Res. 70: 171, 1997.
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Sheldon, R. J., Malarchik, M. E., Fox, D. A., Burks, T. F., and Porreca, F. Pharmacological characterization of neural mechanisms regulating mucosal ion transport in mouse jejunum. J. Pharmacol. Exp. Ther. 249: 572, 1989.
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JOURNAL OF SURGICAL RESEARCH: VOL. 97, NO. 1, MAY 1, 2001 after massive intestinal resection. Proc. Soc. Exp. Biol. Med. 172: 207, 1983. Schulzke, J. D., Fromm, M., Bentzel, C. J., Zeitz, M., Menge, H., and Riecken, E. O. Ion transport in the experimental short bowel syndrome of the rat. Gastroenterology 102: 497, 1992. Freeman, H. J., Ellis, S. T., Johnston, G. A., Kwan, W. C., and Quamme, G. A. Sodium-dependent D-glucose transport after proximal small intestinal resection in rat. Am. J. Physiol. 255: G292, 1988. Fink, M. P. Gastrointestinal mucosal injury in experimental models of shock, trauma, and sepsis. Crit. Care. Med. 19: 627, 1991. Cruz, N., Qi, L., Alvarez, X., Berg, R. D., and Deitch, E. A. The Caco-2 cell monolayer system as an in vitro model for studying bacterial-enterocyte interactions and bacterial translocation. J. Burn Care Rehabil. 15: 207, 1994. Aldazabal, P., Eizaguirre, I., Barrena, M. J., Garcia-Arenzana, J. M., Ariz, C., and Cuadrado, E. Bacterial translocation and T-lymphocyte populations in experimental short-bowel syndrome. Eur. J. Pediatr. Surg. 8: 247, 1998.
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Eizaguirre, I., Aldazabal, P., Barrena, M. J., Garcia-Arenzana, J. M., Alcorta, M., Ariz, C., Candelas, S., and Tovar, J. A. Bacterial translocation is favored by the preservation of the ileocecal valve in experimental short bowel with total parenteral nutrition. Eur. J. Pediatr. Surg. 9: 220, 1999.
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Grassme, H., Kirschnek, S., Riethmueller, J., Riehle, A., von Kurthy, G., Lang, F., Weller, M., and Gulbins, E. CD95/CD95 ligand interactions on epithelial cells in host defense to pseudomonas aeruginosa. Science 290: 527, 2000.
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Epithelial Permeability Is Not Increased in Rats Following Small Bowel Resection 1 David P. O’Brien, M.D., Lindsey A. Nelson, M.D., Lawrence E. Stern, M.D., Jodi L. Williams, B.S., Christopher J. Kemp, B.S., Quan Wang, Ph.D.,* Patrick Tso, Ph.D.,† Christopher R. Erwin, Ph.D., Per-Olof Hasselgren, M.D.,* and Brad W. Warner, M.D. 2 Division of Pediatric Surgery, Children’s Hospital Medical Center, *Shriner’s Hospital for Children, and Department of Surgery and †Department of Pathology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039 Presented at the Annual Meeting of the Association for Academic Surgery, Tampa, Florida, November 2– 4, 2000; published online March 16, 2001
media. There were no permeability differences between SBR and Sham lymph-treated monolayers. Conclusion. The early adaptive response of the remnant intestine after SBR is associated with reduced permeability. These results suggest an alternative mechanism for the increased bacterial translocation that has been described following SBR. © 2001 Academic Press Key Words: epithelium; permeability; short bowel syndrome; intestinal adaptation.
Background. Increased intestinal permeability and translocation of bacteria and/or bacterial products may cause infection and liver dysfunction in patients with the short bowel syndrome. In previous studies, serum from mice undergoing small bowel resection (SBR) enhanced growth of cultured rat intestinal epithelial cells (RIEC-6), implicating a role for a serum factor(s) in the enterocyte response to SBR. These experiments tested the hypothesis that epithelial cell permeability is increased following SBR. Materials and methods. Male Sprague-Dawley rats underwent a 75% SBR or sham operation. Intestinal permeability in the remnant ileum was determined by Ussing chambers on Postoperative Day (POD) 3. Additionally, serum was collected on POD 1, 3, and 7 and mesenteric lymph was harvested on POD 3. Once confluent, RIEC-6 cells were incubated for 3 days in media supplemented with 10% fetal bovine serum (FBS; control), 1% FBS, 1% FBS plus 9% Sham serum, or 1% FBS plus 9% SBR serum or exposed to media with varied concentrations of SBR or Sham lymph. Monolayer permeability was determined by measuring the passage of dextran-rhodamine. Results. Intestinal permeability was reduced in rats undergoing SBR. Sham serum-treated monolayers demonstrated the greatest permeability. Incubation with SBR serum reduced permeability to near control
INTRODUCTION
Gut barrier failure with subsequent translocation of enteric bacteria and/or bacterial products has been proposed to be a leading cause of systemic infection, sepsis, and multisystem organ failure in multiple injury models including the short bowel syndrome (SBS) [1– 4]. In patients with SBS who receive parenteral nutrition, the central venous catheter is assumed to be the nidus of infection for most episodes of sepsis. However, there is evidence to suggest that sepsis in these patients may be derived from the gut as a result of increased intestinal permeability [5]. Following massive small bowel resection (SBR), the remnant intestine compensates for the loss of digestive and absorptive function through a process termed adaptation. The pathogenesis of the adaptive process is complex, but likely involves luminal nutrients, gastrointestinal secretions, growth factors, and circulating hormones [6]. The putative role of a humoral factor(s) in adaptation has been demonstrated using vascular parabiosis experiments [7]. This laboratory has been studying intestinal adaptation through the use of a unique hybrid experimental model in which serum from animals undergoing intestinal adaptation after
1
Supported by National Institutes of Health F32 DK09882 (LES), National Institutes of Health RO 1 DK53234 (B.W.W.), and a grant from the Children’s Hospital Campaign for Children Fund, Children’s Hospital Medical Center, Cincinnati, OH (B.W.W.). 2 To whom correspondence and reprint requests should be addressed at Division of Pediatric Surgery, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. Fax: (513) 636-7657. E-mail: [email protected].
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0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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SBR is added to the culture media of a fully differentiated rat intestinal epithelial cell line (RIEC-6). Initial studies utilizing this model have confirmed the existence of a circulating factor(s) that enhances intestinal cell proliferation [8]. In contrast, a similar model involving thermal injury revealed the presence of a serum factor(s) that inhibited intestinal epithelial cell proliferation and migration, altered permeability, and promoted apoptosis [9]. Such disparate results highlight the unique intestinal epithelial cell response to serum harvested after different stimuli. The effect of postresection intestinal adaptation on gut permeability is not currently known. To test the hypothesis that permeability is increased during intestinal adaptation, this study examined intestinal permeability after SBR in whole tissue with a Ussing chamber. Additionally, this study investigated permeability changes in epithelial cell monolayers following exposure to serum or lymph taken from adapting rats after massive small bowel resection. MATERIALS AND METHODS Animals. A protocol for this study was approved by the Children’s Hospital Research Foundation Institutional Animal Care and Use Committee (Children’s Hospital Medical Center, Cincinnati, OH). Male Sprague-Dawley rats (weight range 230 –259 g; The Harlan Laboratory, Indianapolis IN) were housed in groups of two at 21°C on 12-h day-night cycles (6 AM to 6 PM) for at least 5 days before experimentation in order to allow them to acclimate to their environment. The animals were given free access to standard rat chow. Cells. Rat intestinal epithelial cells (RIEC-6; American Type Culture Collection, Manassas, VA) that had undergone 13–18 passages were used for all experiments. Cells were incubated in 10% CO 2 and grown under the following media conditions: 95% Dulbecco’s modified Eagle’s medium (DMEM; 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 1.0 mM sodium pyruvate; American Type Culture Collection) supplemented with 0.1 unit/mL bovine insulin (GibcoBRL, Gaithersburg, MD), an antibiotic-antimycotic (100 units/mL penicillin G sodium, 100 g/ml streptomycin sulfate, and 0.25 g/mL amphotericin B; GibcoBRL), and 5% heat inactivated fetal bovine serum (FBS; GibcoBRL). Cell passage and plating were accomplished with a 5-min incubation in trypsin-EDTA (0.25% trypsin and 1 mM EDTA; GibcoBRL). Operative procedure. The rat model for a 75% proximal SBR and sham (transection) operation was performed as previously presented in detail [10]. Water was provided ad libitum for the first 24 h. Rats from each group subsequently were fed standard rodent diet. Serum and tissue collection. Under 2% isoflurane inhaled anesthesia, the midline sutures were removed and the bowel was retracted in order to expose the PV. Using aseptic technique, approximately 5 mL of PV blood was aspirated and placed into sterile collection tubes. Six centimeters (approximately 1 cm from the anastomosis) of ileum was excised and the luminal contents were gently expressed with cotton swabs. The proximal 1 cm was immediately fixed with 10% neutral buffered formalin and used for histology; the remaining 5 cm was weighed immediately used for Ussing chamber analysis. The rats then were euthanized with an intracardiac injection of xylazine. Blood from sham and SBR groups was pooled and the serum collected, aliquoted, and stored at ⫺80°C for future use. Lymph collection. Under 2% isoflurane inhaled anesthesia, the midline sutures were removed and the bowel was retracted in order to expose the mesenteric lymph duct, which was cannulated accord-
ing to the method of Bollman et al. [11]. Lymph was collected over a 5-h-period, pooled, and then stored at ⫺80°C until further use. Histology. Formalin-fixed samples of ileum were oriented and embedded in paraffin in order to provide cut sections parallel with the longitudinal axis of the bowel. Five-micrometer sections were mounted on poly-L-lysine-coated slides (Erie Scientific Products, Portsmouth, NH) and stained with hematoxylin/eosin. Measurements of the villus heights were performed using a light microscope with a video camera (Dage-MTI, Michigan City, IN) attached to an image capture card (National Instruments, Austin, TX) in a standard desktop computer with image analysis software (NIH Image, National Institutes of Health, Bethesda, MD). At least 20 villi were counted and averaged for each sample, and only villi with an intact central lymphatic channel were considered. Ussing chamber. The methodologies for the Ussing chamber experiments have been described previously [12]. Briefly, a 5-cm Peyer’s patch-free segment of ileum immediately distal to the anastomotic site was harvested. A 2-cm segment was opened along the mesenteric border, washed in ice-cold Krebs-Ringer solution (KBR; 114 mM NaCl, 1.65 mM Na 2HPO 4, 0.3 mM NaH 2PO 4, 5 mM KCl, 1.1 mM MgCl 2, 25 mM NaHCO 3, and 1.25 mM CaCl 2, pH 7.4), and mounted into the Ussing chamber with 0.3 cm 2 of exposed surface area. The mucosal and serosal surfaces were bathed independently in KBR. D-Glucose (10 mM) was present in the medium on the serosal side; a mannitol (10 mM) marker solution containing fluorescein isothiocyanate (FITC)-dextran (4.4 mg/mL) and horseradish peroxidase (HRP; 4 mg/mL) was substituted for D-glucose on the mucosal side. The KBR was oxygenated with 95% O 2 and 5% CO 2 in a gas-lift recirculation system and maintained at 37°C by waterjacketed reservoirs. The potential difference (PD) was measured with a voltage clamp (VCC-MC6, Physiologic Instruments, San Diego, CA) and calomel electrodes. Electrical continuity between the mucosal and the serosal media was maintained with 4% agar bridges. All experiments were performed under short-circuit conditions in which an external feedback circuit of the voltage clamp continuously maintained the PD at zero through the application of a current directly opposing the tissue current. The current was applied through Ag/AgCl electrodes that were in contact with the mucosal and serosal media via 4% agar bridges. The short-circuit current (I sc) magnitude was obtained directly from the digital readout of the voltage clamp. Membrane resistance was calculated using Ohm’s law after passing a 4-A current through the membrane and measuring the PD. The resistance measurements are expressed as (R; ohms ⫻ cm 2). Intestinal tissues were allowed to equilibrate under short circuit conditions for 30 min prior to experimentation. At 30-minute intervals, 0.22 mL was removed from the serosal side and replaced with an equal volume of KBR. All tissues were challenged with 1 mM theophylline at the end of the experiments as a measure of tissue viability. Tissues with less than a 10% increase in I sc were discarded. The tissues were visually inspected to ensure that they remained intact. Molecular probes. (FITC)-dextran (4.4 kDa) and HRP (40 kDa) probes were used to test permeability. Prior to experimentation, a 96-well plate (Maxisorp, Nunc, Denmark) was coated with a goat anti-HRP antibody (dilution 1:5000; Dulbecco’s phosphate buffered saline; Sigma, St. Louis, MO) and allowed to incubate for 2 h at room temperature. Serosal samples from the Ussing chamber were added to the plate in duplicate. The (FITC)-dextran concentration was determined by fluorescein detection (FL600 Microplate Fluorescence Reader, BIO-TEK Instruments, Inc., Winooski, VT). After an additional 2-h incubation at room temperature, the plate was washed three times with Tris-buffered saline containing 0.2% polyoxyethylenesorbitan (Sigma). TMB substrate (50 l; Dako, Carpenteria, CA) was added to each well and incubated for 15 min. The reaction was stopped with 0.4 N H 2SO 4 (50 L). The HRP concentration was determined by measuring the absorbance change from 550 to 450 nm on a spectrophotometer (ThermoMax, Molecular Devices, Menlo Park, CA).
O’BRIEN ET AL.: EPITHELIAL PERMEABILITY AFTER SBR
FIG. 1. Ileal wet weights from male Sprague-Dawley rats on Postoperative Days (POD) 1, 3, and 7 following 75% mid-SBR or sham operation. Results are in milligrams per centimeter and are reported as mean ⫾ SEM. N ⫽ 4 –7 per group. *P ⬍ 0.05, SBR versus Sham.
Cell monolayer formation. The RIEC-6 cells were seeded onto the apical chamber membrane of a Transwell cell culture system (pore size 3 m; Transwell, Corning, Corning, NY) at a density of 33,000 cells per well. Media in the apical and basal chambers were exchanged twice weekly. The cells were allowed to grow to confluence (approximately 14 –17 days), which was verified by impermeability to blue dextran (Sigma). Measurement of cell monolayer permeability. Upon confluence, the media in the apical and basal chambers were exchanged for DMEM supplemented with 1% FBS, 10% FBS, 1% FBS plus 9% sham PV serum (Sham serum), or 1% FBS plus 9% SBR PV serum (SBR serum). Alternatively, the monolayers were exposed to DMEM and 5% FBS with 0, 1, 5, and 10% sham or SBR mesenteric lymph. After a 72-h incubation, 1% dextran-rhodamine (Molecular Probes, Eugene, OR) were added to the apical chamber. After a 5-h incubation, the media in the basal chamber were removed and the amount of dextran-rhodamine present was determined spectrophotometrically at 570 nm. Permeability results are expressed as percentage of the amount of dextran-rhodamine that crossed the monolayer. Statistical analysis. Results are presented as mean values (⫾standard error of the mean). When experiments included only two groups, an unpaired Student’s t test was used. When the experiments included more than two groups, statistical differences were identified using a one-way analysis of variance (ANOVA) followed by the Student-Neuman-Keuls test. The SigmaStat statistical package (SPSS, Chicago, IL) was utilized for all statistical analyses. A P value of less than 0.05 was considered significant.
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FIG. 2. Villus heights from male Sprague-Dawley rats on POD 3 following mid-SBR or sham operation. Results are in micrometers and are reported as mean ⫾ SEM. N ⫽ 5 per group. *P ⬍ 0.05, SBR versus Sham.
tically significant, there was a trend toward decreased permeability to the larger probe HRP and increased membrane resistance (data not shown) in the intestine of the rats that had undergone a SBR (Fig. 3b). There
RESULTS
Survival in all groups was greater than 90%, and all rats were healthy and vigorous at the time of sacrifice. Intestinal adaptation in the SBR group was confirmed grossly by increased ileal wet weights (Fig. 1) and villus heights (Fig. 2). To assess intestinal barrier function in whole tissue, the extent of (FITC)-dextran and HRP passage across the remnant ileal tissue on POD 3 was determined by mounting isolated segments into the Ussing chamber. There was a significant decrease in permeability to (FITC)-dextran of the SBR group at all time points during the study period (Fig. 3a). Although not statis-
FIG. 3. Mucosal-to-serosal flux of (FITC)-dextran and horseradish peroxidase (HRP) across male Sprague-Dawley rat intestine on POD 3 following a 75% mid-SBR or sham operation. Serosal sampling was performed every 30 min over 120-min study period. (a) Serosal concentrations of (FITC)-dextran are in nanograms per milliliter. (b) Serosal concentrations of HRP are in picograms per milliliter. Results are reported as mean ⫾ SEM. N ⫽ 4 per group. *P ⬍ 0.05, SBR versus Sham.
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DISCUSSION
FIG. 4. RIEC-6 monolayers were analyzed for permeability to dextran-rhodamine following a 72-h exposure to culture media supplemented with 1% fetal bovine serum (FBS), 10% FBS, 1% FBS plus 9% Sham serum (Sham), and 1% FBS plus 9% SBR serum (SBR). Sham and SBR sera were harvested from the portal vein of male Sprague-Dawley rats on POD 1, 3, and 7 after undergoing a 75% mid-SBR or sham operation. Results are reported as mean ⫾ SEM. N ⫽ 5– 6 per group. *P ⬍ 0.05, SBR versus Sham and POD 1 SBR and Sham serum versus 10% FBS.
were no differences in PD and I sc, indicating similar degrees of tissue viability and electrogenic ion secretion between the two groups (data not shown). To determine whether these intestinal permeability changes are affected by a circulating factor(s), RIEC-6 monolayers were exposed to either serum or lymph from the Sham or SBR group for 72 h. Because parameters of adaptation in rats occur as early as POD 1, are dramatically increased by POD 3, and are complete by POD 7, serum was harvested at these time points. With respect to control media, monolayers treated with POD 1 serum taken after either sham operation or SBR demonstrated decreased permeability to the dextran-rhodamine (Fig. 4). The SBR serum-treated cells were significantly less permeable than Sham serum taken on POD 1 and 3. However, the permeability between cells exposed to SBR or Sham serum was no longer significantly different, and it was similar to that of the cells receiving control media by POD 7. Interestingly, monolayers that were deprived of FBS (DMEM with 1% FBS) demonstrated a significant decrease in permeability to dextran-rhodamine at all time points compared to all other treatments. Because of the potential for greater cytokine content, the effect of mesenteric lymph on epithelial cell permeability was also evaluated after SBR. There were no significant differences in permeability between monolayers treated with Sham or SBR lymph at 1, 5, or 10% concentration (Fig. 5). However, monolayers incubated with SBR and Sham lymph at a concentration of 10% demonstrated were more permeable when compared to monolayers treated with DMEM with 0 or 1% SBR or Sham lymph.
The purpose of this study was to determine the effects of massive small bowel resection and intestinal adaptation on intestinal epithelial permeability. The intact remnant ileum was significantly less permeable to (FITC)-dextran but not HRP after SBR. Furthermore, POD 1 and 3 serum from rats that underwent a SBR reduced the permeability of an RIEC-6 monolayer when compared to monolayers incubated with Sham serum. In contrast, lymph from the SBR group did not alter monolayer permeability to a greater or lesser extent than lymph from the Sham group. These results suggest that intestinal adaptation after massive SBR is associated with decreased, not increased, epithelial permeability during the early phase of intestinal adaptation. Further, it is likely that the permeability effects are mediated in part by a humoral factor(s). The results of a limited number of studies that have examined intestinal permeability and intestinal adaptation have been conflicting. Utilizing radiolabeled polyethylene glycol (PEG) and in vivo isolated intestinal loops in rats, Urban et al. was unable to detect differences in permeability at 2 and 4 weeks following a 50 or 70% proximal SBR and concluded that the adapted mucosa of the remnant ileum presented a formidable barrier [13]. Schulzke et al. measured PEG fluxes and epithelial resistance with an Ussing chamber in rats 8 weeks following a 70% proximal SBR [14]. In contrast, they found a 40% increase in PEG permeability in the SBR animals. Of note, they analyzed tight junction morphology by freeze-fracture electron microscopy, and no differences in strand number, meshwork depth, or depth of the total tight junction were noted between control and SBR animals. Al-
FIG. 5. RIEC-6 monolayers were analyzed for permeability to dextran-rhodamine following a 72-h exposure to culture media supplemented with 5% fetal bovine serum (FBS) and 0% (control media), 1, 5, and 10% mesenteric lymph harvested from male SpragueDawley rats on POD 3 after undergoing a 75% mid-SBR or sham operation. Results are reported as mean ⫾ SEM. N ⫽ 5– 6 per group. *P ⬍ 0.05, 10% SBR and Sham lymph versus control media.
O’BRIEN ET AL.: EPITHELIAL PERMEABILITY AFTER SBR
though neither of these studies corrected for changes in mucosal surface, Schulzke et al. did perform morphometric analysis on the intestinal villi and estiamted a villus height increase of 20% in the SBR rats. The authors suggested that this change might account for the increase in PEG fluxes in the bowel-resected animals [14]. In the current study, the resected animals demonstrated an overall reduction in permeability. Although the data could be flawed by the lack of correction to changes in surface area, any increase in surface are would further reduce the calculated permeability demonstrated in these animals. These disparate observations may be explained, in part, because of the varied time interval that these animals were studied following intestinal resection. Along these lines, Freeman et al. showed that upregulation of D-glucose transport in rats undergoing a 66% proximal small bowel resection did not occur for 6 weeks, suggesting that complete functional adaptation, including factors that affect intestinal permeability, may be delayed [15]. The experiments in this study were performed at relatively early time points during postresection adaptation. Future experiments using longer postoperative time intervals will be necessary to determine the effect of late adaptation on intestinal permeability. Because in vivo models of intestinal injury and permeability are confounded by the complexity and cellular heterogeneity of the gut, many investigators have utilized in vitro cell culture systems [16, 17]. Recent work with hemorrhagic shock [1], thermal injury [9], and endotoxin [3] models has demonstrated the importance of a factor(s) in the serum and/or lymph that affects epithelial barrier function. In the context of these experimental paradigms, confluent RIEC-6 cell monolayers exposed to serum from rats that underwent a SBR demonstrated reduced permeability when compared to serum from control animals. This novel finding suggests an alternative or opposing mechanism than that in hemorrhagic shock, thermal injury, and endotoxin exposure. In those injury models, serum from the animals increased cell monolayer permeability. Different responses to various noxious stimuli also have been observed with proliferation. Serum from burned animals reduces the proliferation of RIEC-6 [9], which stands in contradistinction to previous work in our laboratory in which proliferation of these cells was significantly enhanced following exposure to SBR serum [8]. In a study performed by Magnotti et al. cell monolayer permeability was increased after an incubation with mesenteric lymph harvested from rats during hemorrhagic shock [1]. They found that the increase in endothelial monolayer permeability was greater than that induced by portal vein serum. In contrast, this study did not demonstrate significant differences in the permeability changes caused by the SBR and Sham lymph.
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Increased translocation of bacteria to mesenteric lymph nodes and spleen has been previously demonstrated after intestinal resection in several animal models [18 –21]. It is likely that bacteria are able to translocate by mechanisms unrelated to permeability changes involving more complex bacterial-epithelial cell processing and immune function. Along these lines, respiratory epithelial cell apoptosis has been demonstrated to be an important response for preventing the systemic spread of administered Pseudomonas [22]. This laboratory has previously recognized increased enterocyte apoptosis during intestinal adaptation [23]. The relationship among enterocyte apoptosis, postresection adaptation, and bacterial translocation remains to be elucidated. REFERENCES 1.
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