Carbon monoxide inhalation protects rat intestinal grafts from ischemia/reperfusion injury

Abstract

Carbon monoxide (CO), a byproduct of heme catalysis by heme oxygenases, has been shown to exert anti-inflammatory effects. This study examines the cytoprotective efficacy of inhaled CO during intestinal cold ischemia/reperfusion injury associated with small intestinal transplantation. Orthotopic syngenic intestinal transplantation was performed in Lewis rats after 6 hours of cold preservation in University of Wisconsin solution. Three groups were examined: normal untreated controls, control intestinal transplant recipients kept in room air, and recipients exposed to CO (250 ppm) for 1 hour before and 24 hours after surgery. In air grafts, mRNA levels for interleukin-6, cyclooxygenase-2, intracellular adhesion molecule (ICAM-1), and inducible nitric oxide synthase rapidly increased after intestinal transplant. Histopathological analysis revealed severe mucosal erosion, villous congestion, and inflammatory infiltrates. CO effectively blocked an early up-regulation of these mediators, showed less severe histopathological changes, and resulted in significantly improved animal survival of 92% from 58% in air-treated controls. CO also significantly reduced mRNA for proapoptotic Bax, while it up-regulated anti-apoptotic Bcl-2. These changes in CO-treated grafts correlated with well-preserved CD31(+) vascular endothelial cells, less frequent apoptosis/necrosis in intestinal epithelial and capillary endothelial cells, and improved graft tissue blood circulation. Protective effects of CO in this study were mediated via soluble guanylyl cyclase, because 1H-(1,2,4)oxadiazole (4,3-alpha) quinoxaline-1-one (soluble guanylyl cyclase inhibitor) completely reversed the beneficial effect conferred by CO. Perioperative CO inhalation at a low concentration resulted in protection against ischemia/reperfusion injury to intestinal grafts with prolonged cold preservation.

Figure 1.

Effect of CO on animal survival after 6 hours of cold preservation of intestinal grafts. Fourteen-day survival of air-treated recipients (58.3%, 7 of 12) significantly (P < 0.05) improved with CO to 91.7% (11 of 12).

Figure 2.

Laser Doppler flowmeter measurements of graft blood flows of marginal artery (A) and intestinal wall (B). A: Blood flows of intestinal marginal artery were similar in all groups. B: CO inhalation significantly increased intestinal graft blood flow to 23.2 ± 7.0 ml/minute/100 g compared to <4 ml/minute/100 g in air-treated grafts. Intestinal microcirculation of normal and ODQ-treated unoperated animals was ∼40 ml/minute/100 g. ODQ (20 m/kg) administration to recipient 30 minutes before CO treatment completely abrogated CO effect and intestinal blood flow was 6 ml/minute/100 g. n = 3 to 4 for each group. *, P < 0.05. NM, normal.

Figure 3.

mRNA expression in intestinal grafts. A: mRNA for PAI-1 was up-regulated during I/R injury in air-treated controls with a peak at 6 hours. CO treatment tended toward a reduction in the expression of PAI-1 but statistical significance was not achieved. Cytokine and inflammatory mediator mRNA expressions of ICAM-1 (B), IL-6 (C), TNF-α (D), iNOS (E), and COX-2 (F) were evaluated in CO versus air-treated grafts. These mediators, except for TNF-α, were significantly reduced with CO inhalation. Of note, COX-2 was reduced with CO to <30% of control. n = 4 to 6 for each group. *, P < 0.05.

Figure 4.

Analysis of serum IL-6 (A) and nitrate/nitrite (B). A: Serum IL-6 rapidly elevated and peaked at 6 hours in air-treated controls. Similarly to mRNA expression, CO treatment significantly decreased serum IL-6. B: Serum nitrate/nitrite gradually increased for 24 hours during I/R injury in air-treated animals, which was reduced by CO with statistical significance (P < 0.05) at 3 and 6 hours.

Figure 5.

ODQ reverses CO-induced anti-inflammatory effects. Reduction of mRNA for IL-6 and iNOS by CO treatment was abrogated in animals treated with ODQ. ODQ administration to CO-treated animals restored IL-6 and iNOS mRNA expression comparable to air control grafts 3 hours after reperfusion. ODQ did not alter mRNA expression of these mediators in unoperated normal animals. n = 3 for each group. *, P < 0.05. NM, normal.

Figure 6.

Severity of mucosal injury assessed by routine histopathology. Intestinal cold I/R injury associated with intestinal epithelial cell loss and mucosal erosion (A), and villous congestion observed in air-treated control grafts (B). CO-treated grafts showed significantly less severe injury. n = 6 for each group. *, P < 0.05.

Figure 7.

CD31 expression of vascular endothelial cells. a and d: Immunofluorescent stain of CD31 revealed an abundant expression of CD31 on the vascular endothelial cells in normal intestinal lamina propria. F-actin, which indicates intestinal epithelial microvilli, is also well visualized on the entire villi of normal intestine. b and e: CD31 expression was faint and interrupted in air-treated intestinal grafts after cold I/R injury, and F-actin stain was either disrupted in the upper half of the villi or weak in the remaining villi (air-treated grafts, 1 hour after reperfusion). c and f: CD31-positive vascular endothelial cells were preserved in the lamina propria of CO-treated grafts with F-action stain revealing normal intestinal microvilli preservation (CO-treated graft, 1 hour after SITx). Green, CD31; red, F-actin; blue, nuclei. Original magnifications, ×400.

Figure 8.

Apoptosis-related molecules in intestinal grafts. A: There is a twofold increase of anti-apoptotic Bcl-2 in CO-treated grafts compared to air-treated controls at 1 hour after reperfusion. B: Bax, a proapoptotic gene, was significantly up-regulated 1 hour in air control (P < 0.05), which was not observed in CO-treated grafts. C: Numbers of activated caspase 3-positive cells increased in crypt epithelial cells of air control. CO significantly (P < 0.05) reduced caspase 3-stained apoptotic crypt epithelial cells. NM, normal intestine.

Figure 9.

Transmission electron microscopy of intestinal grafts. a: Numerous vacuolization (white arrows) and apoptotic bodies (black arrows) are present in epithelial cells of air exposed control grafts. b: CO-treated grafts contain minimum apoptotic bodies and considerably less vacuolization. c: In the air-exposed controls, vascular endothelial cells in the lamina propria showed karyolitic nuclei with disorganized internal architecture (arrow). d: Endothelial cells in the CO-treated grafts were well maintained and demonstrated a normal intracellular architecture (arrow). a–d: 1 hour after reperfusion. L, lumen of blood vessels. Scale bar, 0.5 μm. Original magnifications: ×1500 (a, b); ×5000 (c, d).

Figure 10.

Scanning electron microscopy of graft vascular cast. a: Normal intestine showed typical hollow arrangement of vessels that comprise the villi vasculature (arrowhead, afferent artery reaching the apex of the villous and giving rise to the villous capillary). b: Severe leakage (collection of methyl methacrylate, black arrow) with interruption of vascular cast (white arrow) was evident in the internal sections of the villi (air-treated, 1 hour). c: Nearly normal villous vasculature was obtained without leakage (CO-treated, 1 hour). Scale bar, 20 μm.

Figure 11.

HO-1 mRNA (A) and protein (B) levels during intestinal I/R injury with and without CO inhalation. A: The real-time PCR for HO-1 showed an up-regulation of HO-1 mRNA expression in air- and CO-treated grafts with peaks at 3 and 6 hours after transplantation. B: Western blot showed a gradual increase of HO-1 protein expression 1 hour after reperfusion. CO treatment did not alter HO-1 protein expression. NM, normal.

Figure 12.

Antioxidant power in intestinal grafts. Intestinal I/R injury resulted in a significant reduction of antioxidant power in the air-treated control. In contrast, the CO-treated group showed preserved antioxidant power. *, p < 0.05.