Increased Mucosal Permeability After Intestinal Ischemia-Reperfusion Injury Is Mediated by Local Tissue Factors By Jacob
C. Langer
and Sarvjit
S. Sohal
Hamilton, Ontario 0 Subclinical intestinal ischemia-reperfusion injury (IRI) causes an increase in mucosal permeability and may represent an early event in the pathogenesis of necrotizing enterocolitis. The present study was undertaken to determine whether these changes are mediated by local or systemic factors. In 6-week-old weanling rats, the ileum was divided into two isolated loops with separate vascular supplies. The mesentery of the proximal loop was occluded for 30 minutes, following which the bowel was reperfused; permeability to “Cr EDTA was then assessed in the distal loop 30 minutes after reperfusion. In control groups, the distal loop was subjected to 30-minute IRI (“positive“ control) or IO-minute sham operation (“negative” control). Permeability in the distal loop was increased only with IRI to the distal bowel (15.4 f 3 counts/min/standard), and not with IRI to the proximal bowel (5.1 * 1) or with sham operation (6.5 + 2). To determine whether a mild “priming” injury might be necessary for systemic factors to have an effect, the distal loop was subjected to P-minute IRI and the proximal to 30.minute IRI or sham. Permeability was not increased in the distal loop in either of these groups (5.7 f 1 and 7.6 f 2, respectively). Thirty-minute IRI in the proximal loop did not increase permeability in the distal loop, with or without a priming injury. Only direct IRI in the distal loop resulted in a significant increase in permeability. We conclude that the permeability changes in this model are mediated through local tissue effects, rather than by systemic factors. Copyright o 1992 by W.B. Saunders Company INDEX WORDS: Necrotizing enterocolitis; sion injury; intestinal permeability.
ischemia-reperfu-
T
HE ETIOLOGY of necrotizing enterocolitis (NEC) is unclear, although intestinal &hernia, intraluminal substrate, and bacteria have all been implicated.’ We have suggested a unified theory in which multiple subclinical ischemic/hypoxic insults to the intestine may result in increased mucosal permeability to noxious intraluminal proteins and bacteria, leading to the characteristic inflammation seen in NEC. In previous studies in the rat designed to investigate the validity of this theory, we showed that subclinical ischemia-reperfusion injury (IRI) resulted in a significant increase in mucosal permeability to radiolabeled chromium ethylenediaminetetraacetic acid (%r EDTA). This phenomenon occurred in both weanling and newborn animals, with maximal permeability occurring 30 minutes after reperfusion.’ We are currently investigating mechanisms that may account for these changes in mucosal permeability. The present study was designed to determine whether the increase in permeability after IRI is mediated by local or systemic factors. JournalofPediarric
Surgery,
Vol27, No
3
(March), 1992: pp 329-332
MATERIALS
AND
METHODS
Animals Weanling male Sprague-Dawley (Charles River, Quebec) rats aged 6 weeks (weighing 150 to 180 g) were housed under standard conditions, fed Purina (Oakville, Ontario) rat chow, and given only water for 12 hours prior to all experiments. All studies were approved by the McMaster University Committee on Animal Experimentation.
Technique of Intestinal Ischemia Rats were anesthetized using intraperitoneal pentobarbital (45 mgikg). A catheter was inserted in the carotid artery for blood sampling and for infusion of fluid during the experiment. Through a midline laparotomy, the small intestine was divided 20 cm proximal to the ileocecal junction, creating two isolated ileal loops. The marginal vessels were also divided, resulting in complete separation of the vascular supply to both proximal and distal loops. The mesentery to either loop could then be occluded close to the bowel using a specially designed clip. On reperfusion, the bowel became pink and there was return of mesenteric pulsation. Anesthesia was maintained during the experiment by periodic intraperitoneal administration of pentobarbital (5 to 10 mgikg). Sham operation involved the same technique and exposure, without occlusion of the mesentery.
Measurement of Mucosal Permeability In each experiment, permeability was assessed in the distal loop of bowel, using a method adapted from Ramage et al.’ At the time of reperfusion, 1.0 mL of “Cr EDTA solution (0.065 mCi/mL in distilled water; McMaster Pharmacy Department) was injected into the isolated ileal loop. A I-mL sample of blood was taken 30 minutes after reperfusion, and the animal was killed. Blood samples were counted using a 1282 Compugamma counter (LKBWallac Corp, Turku, Finland). Results were expressed as counts per minute, and were standardized daily.
Experimental Protocol The first experiment was designed to examine whether a bloodborne (systemic) factor was released by the proximal loop in response to 30-minute IRI, which would result in an increase in permeability in the distal loop. The “negative*’ control group had sham operation alone. the “positive” control group had 30-minute
From the Intestinal Disease Research Unit and the Depanment of Surgery, McMaster University, Hamilton, Ontario. Supported in part by Medical Research Council of Canada Grant no. MA10878. Presented at the 22nd Annual Meeting of the American Pediatric Surgical Association, Lake Buena V&a, Florida, May IS-18,199l. Address reprint requests to Jacob C. Lunger, MD, Division of Pediatric Surgery, McMaster University Medical Centre, Rm 4E-2, 1200 Main St W, Hamilton, Ontario L8N 3Z.5 Canada. Copyright o I992 by W.B. Saunders Company 0022-3468l92/2703-OOll$O3.OOlO 329
330
LANGER AND SOHAL
IRI to the distal loop, and the experimental group had 30-minute IRI to the proximal loop. Permeability was measured in the distal loop in each case. The second experiment examined the possibility that a systemic factor might require a subclinical “priming” injury in order to have an effect on permeability. In both of these groups the distal loop was subjected to 2-minute IRI; in the control group the proximal loop was not occluded at all, and in the experimental group the proximal loop underwent 30-minute IRI. As in the previous experiment, permeability was measured in the distal loop in each case. Results in each group were compared with the negative control group using a Student’s t test, with a probability level of 0.05 considered significant. RESULTS
The results are summarized in Fig 1. As expected, 30-minute IRI of the distal loop (positive control) resulted in a significant increase in permeability to “Cr EDTA compared with the negative control group. Thirty-minute IRI of the proximal loop did not result in an increase in permeability in the distal loop. Two-minute IRI to the distal loop alone did not result in an increase in permeability, nor did 2-minute IRI to the distal loop combined with 30-minute IRI to the proximal loop. DISCUSSION
In other models, IRI involving skeletal muscle or intestine has been shown to result in a number of systemic effects, including hypotension, acute lung injury, and multiple system organ failure. These 20
1
lp
C. Langer
and Sarvjit
S. Sohal
Hamilton, Ontario 0 Subclinical intestinal ischemia-reperfusion injury (IRI) causes an increase in mucosal permeability and may represent an early event in the pathogenesis of necrotizing enterocolitis. The present study was undertaken to determine whether these changes are mediated by local or systemic factors. In 6-week-old weanling rats, the ileum was divided into two isolated loops with separate vascular supplies. The mesentery of the proximal loop was occluded for 30 minutes, following which the bowel was reperfused; permeability to “Cr EDTA was then assessed in the distal loop 30 minutes after reperfusion. In control groups, the distal loop was subjected to 30-minute IRI (“positive“ control) or IO-minute sham operation (“negative” control). Permeability in the distal loop was increased only with IRI to the distal bowel (15.4 f 3 counts/min/standard), and not with IRI to the proximal bowel (5.1 * 1) or with sham operation (6.5 + 2). To determine whether a mild “priming” injury might be necessary for systemic factors to have an effect, the distal loop was subjected to P-minute IRI and the proximal to 30.minute IRI or sham. Permeability was not increased in the distal loop in either of these groups (5.7 f 1 and 7.6 f 2, respectively). Thirty-minute IRI in the proximal loop did not increase permeability in the distal loop, with or without a priming injury. Only direct IRI in the distal loop resulted in a significant increase in permeability. We conclude that the permeability changes in this model are mediated through local tissue effects, rather than by systemic factors. Copyright o 1992 by W.B. Saunders Company INDEX WORDS: Necrotizing enterocolitis; sion injury; intestinal permeability.
ischemia-reperfu-
T
HE ETIOLOGY of necrotizing enterocolitis (NEC) is unclear, although intestinal &hernia, intraluminal substrate, and bacteria have all been implicated.’ We have suggested a unified theory in which multiple subclinical ischemic/hypoxic insults to the intestine may result in increased mucosal permeability to noxious intraluminal proteins and bacteria, leading to the characteristic inflammation seen in NEC. In previous studies in the rat designed to investigate the validity of this theory, we showed that subclinical ischemia-reperfusion injury (IRI) resulted in a significant increase in mucosal permeability to radiolabeled chromium ethylenediaminetetraacetic acid (%r EDTA). This phenomenon occurred in both weanling and newborn animals, with maximal permeability occurring 30 minutes after reperfusion.’ We are currently investigating mechanisms that may account for these changes in mucosal permeability. The present study was designed to determine whether the increase in permeability after IRI is mediated by local or systemic factors. JournalofPediarric
Surgery,
Vol27, No
3
(March), 1992: pp 329-332
MATERIALS
AND
METHODS
Animals Weanling male Sprague-Dawley (Charles River, Quebec) rats aged 6 weeks (weighing 150 to 180 g) were housed under standard conditions, fed Purina (Oakville, Ontario) rat chow, and given only water for 12 hours prior to all experiments. All studies were approved by the McMaster University Committee on Animal Experimentation.
Technique of Intestinal Ischemia Rats were anesthetized using intraperitoneal pentobarbital (45 mgikg). A catheter was inserted in the carotid artery for blood sampling and for infusion of fluid during the experiment. Through a midline laparotomy, the small intestine was divided 20 cm proximal to the ileocecal junction, creating two isolated ileal loops. The marginal vessels were also divided, resulting in complete separation of the vascular supply to both proximal and distal loops. The mesentery to either loop could then be occluded close to the bowel using a specially designed clip. On reperfusion, the bowel became pink and there was return of mesenteric pulsation. Anesthesia was maintained during the experiment by periodic intraperitoneal administration of pentobarbital (5 to 10 mgikg). Sham operation involved the same technique and exposure, without occlusion of the mesentery.
Measurement of Mucosal Permeability In each experiment, permeability was assessed in the distal loop of bowel, using a method adapted from Ramage et al.’ At the time of reperfusion, 1.0 mL of “Cr EDTA solution (0.065 mCi/mL in distilled water; McMaster Pharmacy Department) was injected into the isolated ileal loop. A I-mL sample of blood was taken 30 minutes after reperfusion, and the animal was killed. Blood samples were counted using a 1282 Compugamma counter (LKBWallac Corp, Turku, Finland). Results were expressed as counts per minute, and were standardized daily.
Experimental Protocol The first experiment was designed to examine whether a bloodborne (systemic) factor was released by the proximal loop in response to 30-minute IRI, which would result in an increase in permeability in the distal loop. The “negative*’ control group had sham operation alone. the “positive” control group had 30-minute
From the Intestinal Disease Research Unit and the Depanment of Surgery, McMaster University, Hamilton, Ontario. Supported in part by Medical Research Council of Canada Grant no. MA10878. Presented at the 22nd Annual Meeting of the American Pediatric Surgical Association, Lake Buena V&a, Florida, May IS-18,199l. Address reprint requests to Jacob C. Lunger, MD, Division of Pediatric Surgery, McMaster University Medical Centre, Rm 4E-2, 1200 Main St W, Hamilton, Ontario L8N 3Z.5 Canada. Copyright o I992 by W.B. Saunders Company 0022-3468l92/2703-OOll$O3.OOlO 329
330
LANGER AND SOHAL
IRI to the distal loop, and the experimental group had 30-minute IRI to the proximal loop. Permeability was measured in the distal loop in each case. The second experiment examined the possibility that a systemic factor might require a subclinical “priming” injury in order to have an effect on permeability. In both of these groups the distal loop was subjected to 2-minute IRI; in the control group the proximal loop was not occluded at all, and in the experimental group the proximal loop underwent 30-minute IRI. As in the previous experiment, permeability was measured in the distal loop in each case. Results in each group were compared with the negative control group using a Student’s t test, with a probability level of 0.05 considered significant. RESULTS
The results are summarized in Fig 1. As expected, 30-minute IRI of the distal loop (positive control) resulted in a significant increase in permeability to “Cr EDTA compared with the negative control group. Thirty-minute IRI of the proximal loop did not result in an increase in permeability in the distal loop. Two-minute IRI to the distal loop alone did not result in an increase in permeability, nor did 2-minute IRI to the distal loop combined with 30-minute IRI to the proximal loop. DISCUSSION
In other models, IRI involving skeletal muscle or intestine has been shown to result in a number of systemic effects, including hypotension, acute lung injury, and multiple system organ failure. These 20
1
lp
