Increased transcellular permeability of rat small intestine after thermal injury

Bums (1992) 18, (2), 117-120

Prinled in Great Britain

117

Increased transcellular permeability intestine after thermal injury E. A. CarteF4,

of rat small

A. Gonnella4P6 and R. G. Tompkins5,’

‘Pediatric Gastrointestinal and Nutritional Laboratory, 2Massachusetts General Hospital, 3hriners Bums Institute, 4Department of Pediatrics and 5Department of Surgery, Harvard Medical School, Vhildrens Service, Boston Childrens Hospital and 7Surgical Services, Massachusetts General Hospital, Boston, USA

The pathway which results in a loss of intestinal bank function and kansepithelial tran5fer of macromolecules after cutaneous thermal injury is unknown. To determine the enhanced absorption pathway, transepithelial transport of horseradish peroxidase (HRP) was examined ultrastrucfurally afkr a thermal injury. Within 6 h after fhe injury, increased HRP uptake was seen in the portal and systemic blood with the maximal increase in uptake mmured at 18 h postinjury; permeability returned to normal by 72 h postinjuy. Morphologically, the itureased uptake was found to be franscellular through ultrastructurally normal intestinal absorptive cells. Occasional focal regions of enhanced HRP uptake were found and this enhanced uptake was attributed to focal intestinal epithelial disruptions. This increase in intestinal permeability represents a transient loss of infestinal barrier function and potenfially allows absorption of macromokcules such as endotoxin from the intestinal lumen into the portal circulation early afkr thermal injury.

Introduction Under normal conditions, adult small intestinal epithelium is largely impermeable to macromolecules and as such it provides a barrier to the absorption of potentially toxic materials including bacterial cell products (e.g. endotoxin and proteases) from the intestinal lumen. However, multiple studies suggest that this mucosal barrier may be altered and that macromolecules may be absorbed after thermal injury (Carter et al., 1987, 199Oa,b; Alexander et al., 1991; Epstein et al., 1991). Although the exact pathway for the enhanced absorption is unknown, preliminary studies suggest that mucosal integrity may be compromised. In thermal injury animal models (Mochizuki et al., 1984; Carter et al., 1986), a decreased small intestinal mucosal weight can be easily found after injury. In addition, Carter et al. (1986) have demonstrated biochemical alterations including a markedly diminished in vivo incorporation of thymidine and uridine into the small intestinal mucosa for a transient interval after these injuries; a decreased mucosal protein synthesis rate as measured by leucine incorporation; and diminished absorption of nutrients by the small intestine including calcium, glucose and leucine. These biochemical changes. are found as early as 6 h after the injuries and return to normal by 72 h postinjury. Although these changes can be readily demon0 1992 Butterworth-Heinemann 030%4179/92/020117-04

Ltd

strated, no morphological change in the small intestinal mucosa at the light microscopic level has been identified. In separate studies (Carter et al., 1988), because these functional changes could be a result of a diminished blood flow, small intestinal blood flow was measured by microsphere techniques and no significant decrease in intestinal blood flow was seen. Because the absorption of macromolecules such as endotoxin and proteases from the intestinal lumen may be important in the pathophysiological responses to injury, we studied transepithelial transfer of horseradish peroxidase ultrastructurally in the ligated loop model in rats.

Materials and methods Thermal injury model Female SpragueDawley rats (150-2OOg, Charles River Breeding Labs, Wilmington, MA, USA) were used in this study and were maintained in accordance with National Research Council Guidelines. Experimental protocols were approved by the Subcommittee on Animal Care, Committee on Research, Massachusetts General Hospital. There were between four and six animals in each of the sham and treated groups. A standardized thermal injury was performed on the rat according to the method described by Walker and Mason (1968) as modified by Carter et al. (1986, 1988). Briefly, after anaesthesia with intraperitoneal methohexitol (50 mg/kg body wt), the animal’s back was immersed in water at 100°C resulting in a full skin thickness scald injury involving 20 per cent of the body surface area. The animals were given an intraperitoneal injection of normal saline (50 ml/kg body wt) and placed in individual metal cages. Sham treatment (control) animals were handled exactly as the injured rats except for the scalding injury. For those studies continuing for more than 18 h after injury, the animals were given Purina Laboratory Chow (Purina, St Louis, MO, USA) and water ad libitum. Ligated loop in vivo At intervals (6, 18 and 72 h) after the thermal injury, general anaesthesia was induced with ether and segments (3-4 cm

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Bums (1992) Vol. 18iNo.2

Figure 1. Light microscopy of sham (A, C) and burned (B, D) intestine stained for peroxidase (& B) or with H & E (C, fij. ( x 381.)

long) of both proximal and distal (ileum) small intestine were ligated distally (one loop at each location per, animal); a loosely applied ligature was placed proximally, through which the horseradish peroxidase (HRP) solutions (lyophilized HRP (Sigma type VI, Sigma Chemical Co., St Louis, MO, USA)) diluted in phosphate-buffered saline (PBS) to a concentration of 10 pg/ml) were injected as previously described (Gonnella and Neutra, 1984; Weaver et al., IWO).The ligature was tightened around the needle as it was withdrawn providing a water-tight luminal compartment. In vivo ligated loops allow high concentrations of protein to be delivered to the mucosal surface allowing easier detection of the non-radiolabelled protein tracer to be seen at the electron microscopic level. Therefore, delivery of large amounts of protein to the small intestinal mucosa does not depend upon gastric emptying and peristalsis as seen with gavage feeding. For rinsing, a small cut at one end provided drainage while buffer solution was infused from the other end. After 60 min, portal blood samples were taken and mucosal tissue (six to ten samples) was surgically removed and infused with a solution consisting of 2 per cent fresh formaldehyde and 2.5per cent glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.4) at 22°C. Samples were immersed in fixative and sliced at 5 mm intervals. After 2-4 h of fixation, slices were rinsed in 0.1M cacodylate buffer at 4”C, postfixed in 1 per cent 0~0, (in the same buffer), rinsed in cacodylate buffer, dehydrated, and embedded in Epon-Araldite. Thin sections were stained for 2-3 minwith lead citrate and examined with a JEOL 100 CX electron microscope. For HRP histochemistry, tissues were fixed for 2-4 h, sliced at 100 pm, rinsed in 0.1 M sodium cacodylate buffer (pH 7.0) and 0.1M Tris buffer (pH 7.0). Mucosal slices were preincubated for 15 min (at 4°C in the dark) in a 0.05 per cent solution of 3-3’ diaminobenzidine tetrahydrochloride (Poly-

sciences Inc., Warrington, PA, USA) in Tris buffer (pH 7.0) and subsequently in the same solution containing 0.1per cent H,O,, rinsed, postfixed in OsO,, and routinely embedded for thin sectioning. In order to measure HRP activity in the portal blood, HRP activity was analysed in the supematant spectrophotometritally as previously described (Carter et al., logob). Statistical comparisons were made using Student’s two-tailed t-test for non-paired observations with equal variance and a level of significance of PC 0.05unless otherwise indicated.

Results The light microscopic picture of the sham and burned intestine is shown in Figure I. As can be seen, there was little gross difference in the peroxidase staining material at the microvillus membrane level between the control and the treated animals. The H&E sections also revealed little difference in the two groups. At the light microscopic level, there was only a faint indication of HRP staining material in the intestines of the sham-treated animals (Figure ZA). By contrast, in the electron microscope sections of the burned tissue, there was marked peroxidase staining material at the bush border level, in vesicles, and at the basal membrane level of the epithelial cell (FigureB). The tight junctions were the same in both the sham and burned animals, and appeared to be unaffected by either treatment. HRP was detected in the portal blood of the sham animals I h after the instillation of the HRP in the ligated loop, confirming previous reports (Warshaw et al., 1971; Walker and Isselbacher, 1974). HRF’-like activity was also detected in the portal blood of the burned rats. The activity was twice as high (PCO.01)as that seen in the control animals (450zt27 for the burned, 225f 29 for the sham; results expressed as nanomoles H,O, decomposed/h/loo ~1 of

Carter et al.: Transcelluhr intestinal permeability after bum injury

Figure 2. Electron micrographs of sham and burned rats. A, Absorptive cells from a ligated loop of an adult rat intestine (control) infused in vivo with HRP for 60min. HRP binds microvillar (M) and intermicrovillous (I) membranes, whereas apical vesicular compartments (V) and lateral membranes (LM) are virtually devoid of HRP. B, Absorptive cells from a ligated loop of intestine from a burned adult rat infused in vivo with HRP for 60 min. HRP is taken up into intracellular vesicular compartments (V) and is also present beyond the lateral membrane (arrow). ( x 59 375.)

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the ‘unstirred water layer’ (Westergaard and Diet&y, 1974; Lewis and Fordtran, 1975) and the glycocalyx mucous coat (Walker and Isselbacher, 1974). Once macromolecules have reached the intestinal epithelial cell membranes, absorption occurs either by transcellular or paracellular transport processes (Walker and Isselbacher, 1974). Transcellular transport occurs after endocytosis by the microvillus membrane, transcellular movement within vesicles, and reverse endocytosis (exocytosis) at the basolateral surfaces of the epithelial cell. Alternatively, paracellular transport occurs across tight junctions and diffusion within the intercellular spaces. For some macromolecules that have traversed the epithelial surface and have reached the basement membrane, an additional potential barrier is the epithelial basement membrane itself (Kingham et al., 1978). Since there are no demonstrable morphological alterations seen in the intestinal lining at the light microscopic level in these animals, subtle changes in any or all of these potential ‘barriers’ may be possible. Enhanced intestinal permeability to HRP in association with direct surgical trauma to the intestine has been demonstrated by Rhodes and Kamovsky (1971) as a widespread loss of intestinal barrier function in the small intestinal regions adjacent to the operative incision. The major defect in barrier function was a loss of integrity of the epithelial tight junctions, although increased transcellular transport accounted for a significant proportion of the HRP uptake. In our studies, the possibilities for the defect in the barrier function are also either an increased transcellular movement or loss of tight junction integrity with increased paracellular uptake. To determine the relative contributions of each of these routes will require further cytochemical localization studies using HRP.

Acknowledgements serum). This material demonstrated the same retention time as the HRP tracer instilled into the intestinal loop on a G-75

column

(Warshaw

et al., 1971; Walker

and Isselbacher,

1974).

Discussion This study demonstrates a transepithelial pathway for the enhancement of small intestinal macromolecular perrneability. In addition, enhanced intestinal permeability has been found in many conditions in humans using various permeability probes. Using the disaccharide probe, lactulose, and mannitol, Ziegler et al. (1988) demonstrated a general phenomenon of increased intestinal permeability in burned patients and O’Dwyer et al. (1988) reported similar findings in normal volunteers, following a single bolus of endotoxin. In other disorders in humans, enhanced small intestinal permeability has been measured using polyethylene glycols, EDTA, and other inert mono- and disaccharides with rheumatoid arthritis (Tagesson and Bengtsson, 1983), inflammatory bowel disease (Ewe et al., 1984; Peled et al., 1985; Gardner, 1988; Olaison et al., 1988; Murphy et al., 1989) and dermatological disorders such as eczema (Jackson et al., 1981; Pike et al., 1986). It is possible that the mechanism by which barrier function is altered in these disease and injury states is also operational after bum injury. An alteration in the rate-limiting component or ‘the barrier’ in the macromolecule movement from the intestinal lumen into the lamina propria will result in an increased absorption of macromolecules. The possible barriers include

This work was supported by the National Institutes of Health General Medical Sciences (GM 21700) and the Shriners Hospitals for Crippled Children.

References Alexander J. W., Boyce S. T., Babcock G. F. et al. (1991) The process of microbial translocation. Ann. Surg. 212, 496, Carter E. A., Harmatz P. R., Udall J. N. et al. (1987) Barrier defense function of the small intestine: effect of ethanol and acute burn trauma. tiv. Exp. Med. Biol. 216, 829. Carter E. A., Udall J. N., Kirkham S. E. et al. (1986) Thermal injury and gastrointestinal function I. Small intestinal nutrient absorption and DNA synthesis. 1. Burn Cure Rehabil. 7, 469. Carter E. A., Tompkins R. G., Yarmush M. L. et al. (1988) Redistribution of blood flow following thermal injury and hemorrhagic shock. _I.Appl. Physiol 65, 1782. Carter E. A., Hatz R. A., Yarmush M. L. et al. (1990a) Injuryinduced inhibition of small intestinal protein and nucleic acid synthesis. Gasfroenferology 98, 1445. Carter E. A., Tompkins R. G., Schiffrin E. J. et al. (199Ob) Cutaneous thermal injury alters macromolecular permeability of rat small intestine. Surgery 107, 335. Epstein M. D., Tchervenkov J. I., Alexander J. W. et al. (1991) Increased gut permeability following bum trauma. Arch. Surg. 126, 198. Ewe K., Wanitschke R. and Staritz M. (1984) Intestinal permeability studies in humans. In: Csaky T. Z. (ed.), Pharmacology of Intestinal Permeation II. New York: Springer-Verlag, p. 561.

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absorption of intact proteins. Ann. Rev. Nutr. 8,329. Gonnella P. A. and Neutra M. R. (1984) Membrane-bound and fluid phase macromolecules enter separate polysomal compartments in absorptive cells of suckling rat ileum. J. Cell Biol. 99, 909. Jackson P. G., Lessof M. H., Baker R. W. R. et al. (1981) Intestinal permeability in patients with eczema and food allergy. Lancet i, 1285. Kingham J. G. C., Baker J. H. and Loehry C. A. (1978) Autoradiographic study of the permeability characteristics of the small intestine. Gut 19,114. Lewis L. D. and Fordtran J. S. (1975) Effect of perfusion rate on absorption, surface area, unstirred water layer thickness, permeability, and intraluminal pressure in the rat ileum in viva. Gastroenferology 68, 1509. Mochizuki H., Trocki O., Dominioni L. et al. (1984) Mechanism of prevention of postbum hypermetabolism and catabolism by early enteral feeding. Ann. Surg. 200, 297. Murphy M. S., Eastham E. J., Nelson R. et al. (1989) Intestinal permeability in Crohn’s Disease. Arch. Dis. ChiM 64, 321. O’Dwyer S. T., Michie H. R., Ziegler T. E. et al. (1988) A single dose of endotoxin increases intestinal permeability in healthy humans. Arch. Surg. 123, 1459. Olaison G., Leandersson P., Sjodahl R. et al. (1988) Intestinal permeability to polyethyleneglycol 600 in Crohn’s disease. Peroperative determination in a defined segment of the small intestine. Gut 29, 196. Peled Y., Watz C. and Gilat T. (1985) Measurement of intestinal permeability using ‘ICr-EDTA. Am. ]. Gmfro&ferol. 80, 770. Pike M. G., Heddle R. J., Boulton P. et al. (1986) Increased permeability in atopic eczema. 1. Invest. Dewnafol. 86, 101.

Rhodes R. S. and Kamovsky M. J. (1971) Loss of macromolecular barrier function associated with surgical trauma to the intestine. Lab. Invest. 25, 220. Tagesson C. and Bengtsson A. (1983) Intestinal permeability to different sized polyethylene glycols in patients with rheumatoid arthritis. Stand. 1. Rheumafol. 12, 124. Walker W. A. and Isselbacher K. J. (1974) Uptake and transport of macromolecules by the intestine: possible role in clinical disorders. Gusfroenferology 6 7,53 1. Walker H. L. and Mason A. D. (1968) A standard animal bum. 1. Trauma 8, 1049. Warshaw A. L., Walker W. A., Cornell R. et al. (1971) Small intestinal permeability to macromolecules. Transmission of horseradish peroxidase into mesenteric lymph and portal blood. Lab. Invest. 25, 675. Weaver L. T., Gonnella P. A., Israel E. J. et al. (1990) Uptake and transport of epidermal growth factor by the small intestinal epithelium of the fetal rat. Gasfroenferology 98, 828. Westergaard H. and Dietschy J. M. (1974) Delineation of the dimensions and permeability characteristics of the two major diffusion barriers to passive mucosal uptake in the rabbit intestine. J C/in. Invest. 54, 718. Zeigler T. R., Smith R. J., O’Dwyer S. T. et al. (1988) Increased intestinal permeability associated with infection in burn patients. Arch. Stlrg. 123, 1313. Paper accepted

I7 October

1991.

Correspondence should be dressed to: Dr E. A. Carter, Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, Boston, MA 02114, USA.

James Laing Memorial Essay The British Burn Association has instituted a memorial essay to be awarded annually in memory of James Ellsworth Laing, Burn Surgeon, founder member of the British Burn Association and a former Editor of this Journal. There is a prize of up to E.500 for the winning essay. The subject for the eighth essay is: Who should lead the bum cllre team?’ I The essay should be confined to not more than 10 000 words and correspondingly less if up to 6 Figures and/or Tables are included. The substance of the essay should not already have been published since the winning essay will be published in this Journal. The essays will be assessed anonymously. All persons interested in the problems associated with burning injury are eligible to submit an essay. The deadline for submission of an essay (4 copies) is 31 December 1992. Completed essays and any queries should be sent to the Secretary of the British Burn Association: Dr J. N. Kearney, PhD, Yorkshire Regional Tissue Bank, Pinderfields General Hospital, Wakefield WFl4DG, West Yorkshire, U.K. The title and author(s) of the winning

essay will be announced

in April 1993.