Publications by authors named "Aurelia Bihari"

24 Publications

  • Page 1 of 1

Carbon monoxide-releasing molecule-3 (CORM-3) offers protection in an in vitro model of compartment syndrome.

Microcirculation 2019 10 19;26(7):e12577. Epub 2019 Jul 19.

Division of Orthopaedic Surgery, Department of Surgery, The University of Western Ontario, London, Ontario, Canada.

Objective: Limb compartment syndrome (CS), a complication of trauma, results in muscle necrosis and cell death; ischemia and inflammation contribute to microvascular dysfunction and parenchymal injury. Carbon monoxide-releasing molecule-3 (CORM-3) has been shown to protect microvascular perfusion and reduce inflammation in animal models of CS. The purpose of the study was to test the effect of CORM-3 in human in vitro CS model, allowing exploration of the mechanism(s) of CO protection and potential development of pharmacologic treatment.

Methods: Confluent human vascular endothelial cells (HUVECs) were stimulated for 6 h with serum isolated from patients with CS. Intracellular oxidative stress (production of reactive oxygen species (ROS)) apoptosis, transendothelial resistance (TEER), polymorphonuclear leukocyte (PMN) activation and transmigration across the monolayer in response to the CS stimulus were assessed. All experiments were performed in the presence of CORM-3 (100 μM) or its inactive form, iCORM-3.

Results: CS serum induced a significant increase in ROS, apoptosis and endothelial monolayer breakdown; it also increased PMN superoxide production, leukocyte rolling and adhesion/transmigration. CORM-3 completely prevented CS-induced ROS production, apoptosis, PMN adhesion, rolling and transmigration, while improving monolayer integrity.

Conclusion: CORM-3 offers potent anti-oxidant and anti-inflammatory effects, and may have a potential application to patients at risk of developing CS.
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http://dx.doi.org/10.1111/micc.12577DOI Listing
October 2019

Compartment syndrome-induced muscle injury is diminished by the neutralization of pro-inflammatory cytokines.

OTA Int 2018 Dec 18;1(3):e011. Epub 2018 Dec 18.

Division of Orthopaedic Surgery, University of Western Ontario.

Objectives: Compartment syndrome (CS) is one of the most devastating consequences of musculoskeletal trauma. The pathophysiology of CS includes elevation of intracompartmental pressure (ICP), causing damage to the microcirculation, decreased oxygen delivery, tissue anoxia, and cell death. CS is a combined ischemic and inflammatory condition that induces the systemic inflammatory cascade. In complete ischemia, within the first hour of reperfusion, a peak in the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α) has been previously reported. The purpose of this study was to examine the suspected systemic inflammatory cytokine/chemokine release in response to CS, and to evaluate the microvascular dysfunction, tissue injury, and inflammatory response following the neutralization of pro-inflammatory cytokines TNF-α and/or interleukin-1 beta (IL-1β).

Methods: Twenty-eight male Wistar rats were randomly assigned into 5 groups: Sham (no CS), CS (with isotype control), CS+TNF-α neutralizing antibody (NA), CS+IL-1β NA, CS+Combo (both TNF-α and IL-1β NA). CS was induced by elevation of ICP above 30 mm Hg through an infusion of isotonic saline into the anterior compartment of the hind limb for 2 hours; NA were administered just prior to fasciotomy. Microvascular perfusion, cellular tissue injury, and inflammatory response within the extensor digitorum longus muscle were assessed using intravital video microscopy for 45 minutes after fasciotomy. Systemic levels of 24 different cytokines/chemokines were also measured, using the xMAP Luminex technology.

Results: Of the 24 cytokines/chemokines sampled, 6 were significantly elevated from their baseline levels, and included the pro-inflammatory cytokines TNF-α, IL-1β, growth-related oncogene/keratinocyte chemoattractant (GRO/KC), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), and the anti-inflammatory cytokine IL-10. CS resulted in a significant decrease in microvascular perfusion, from 75 ± 2% continuously perfused capillaries in the sham to 31 ± 4% in CS (.001), a significant increase in tissue injury (0.33 ± 0.4 versus 0.04 ± 0.01 in sham) and leukocyte activation (14 ± 2 adherent leukocytes/1000 μm versus 2 ± 1 adherent leukocytes/100 μm in sham, .001). CS-associated tissue injury was significantly decreased with TNF-α neutralization (.05), both when administered alone or in combination with IL-1β (.05). Additionally, TNF-α neutralization blocked CS-associated leukocyte activation (.05); IL-1β neutralization also diminished leukocyte adhesion (.05). Perfusion remained virtually unchanged in CS animals treated with NA (36 ± 4%, 32 ± 3% and 30 ± 2% in CS+TNF-α, CS+IL-1β and CS+Combo groups, respectively).

Conclusion: The results of this study indicate that CS induces a systemic inflammation, as evidenced by upregulation of inflammatory cytokines/chemokines in circulation. Neutralization of TNF-α led to a significant reduction in tissue injury; however, it had no effect on the CS-induced microvascular dysfunction. This suggests a distinct role of TNF-α in the pathophysiology of muscle injury in CS.
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http://dx.doi.org/10.1097/OI9.0000000000000011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953480PMC
December 2018

Systemic Administration of Carbon Monoxide-Releasing Molecule-3 Protects the Skeletal Muscle in Porcine Model of Compartment Syndrome.

Crit Care Med 2018 05;46(5):e469-e472

Division of Orthopaedic Surgery, Department of Surgery, London Health Sciences Centre, London, ON, Canada.

Objectives: Acute limb compartment syndrome, a complication of musculoskeletal trauma, results in muscle necrosis and cell death. Carbon monoxide, liberated from the carbon monoxide-releasing molecule-3, has been shown protective in a rat model of compartment syndrome. The purpose of this study was to test the effect of carbon monoxide-releasing molecule-3 in a preclinical large animal model of compartment syndrome, with the ultimate goal of developing a pharmacologic adjunct treatment for compartment syndrome.

Design: Animal research study.

Setting: Basic research laboratory in a hospital setting.

Subjects: Male Yorkshire-Landrace pigs (50-60 kg).

Interventions: Pigs underwent 6 hours of intracompartmental pressure elevation by infusing fluid into the anterior compartment of the right hind limb. Carbon monoxide-releasing molecule-3 was administered systemically (2 mg/kg, IV) at fasciotomy, followed by 3-hour reperfusion.

Measurements And Main Results: Muscle perfusion, inflammation, injury, and apoptosis were assessed in the skeletal muscle. Systemic leukocyte activation was assessed during compartment syndrome and reperfusion. Elevation of hind limb intracompartmental pressure resulted in significant microvascular perfusion deficits (44% ± 1% continuously perfused capillaries in compartment syndrome vs 76% ± 4% in sham; p < 0.001), increased tissue injury (ethidium bromide/bisbenzimide of 0.31 ± 0.07 in compartment syndrome vs 0.17 ± 0.03 in sham; p < 0.05), apoptosis (fluorescence in vivo/bisbenzimide of 0.26 ± 0.06 in compartment syndrome vs 0.13 ± 0.03 in sham; p < 0.05), and systemic leukocyte activation (14.7 relative luminescence units/10 polymorphonuclear leukocytes in compartment syndrome vs 1.0 ± 0.1 in baseline; p < 0.001). Systemic application of carbon monoxide-releasing molecule-3 at fasciotomy increased the number of continuously perfused capillaries (68% ± 3%; p < 0.001), diminished tissue injury (ethidium bromide/bisbenzimide of 0.13 ± 0.04; p < 0.05), apoptosis (fluorescence in vivo/bisbenzimide of 0.12 ± 0.03; p < 0.05), and blocked systemic leukocyte activation (3.9 ± 0.3 relative luminescence unit/10 polymorphonuclear leukocytes; p < 0.001).

Conclusions: Administration of carbon monoxide-releasing molecule-3 at fasciotomy offered protection against compartment syndrome-induced microvascular perfusion deficit, tissue injury, and systemic leukocyte activation. The data suggest the potential therapeutic application of carbon monoxide-releasing molecule-3 to patients at risk of developing compartment syndrome.
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http://dx.doi.org/10.1097/CCM.0000000000002998DOI Listing
May 2018

Carbon monoxide and hydrogen sulphide reduce reperfusion injury in abdominal compartment syndrome.

J Surg Res 2018 02 28;222:17-25. Epub 2017 Oct 28.

Division of Orthopedic Surgery, Department of Surgery, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada.

Background: Carbon monoxide (CO)- and hydrogen sulphide-releasing molecules (CORM-3 and GYY4137, respectively) have been shown to be potent antioxidant and antiinflammatory agents at the tissue and systemic level. We hypothesized that both CORM-3 and GYY4137 would reduce the significant organ dysfunction associated with abdominal compartment syndrome (ACS).

Material And Methods: Randomized trial was conducted where ACS was maintained for 2 hours in 27 rats using an abdominal plaster cast and intraperitoneal CO insufflation at 20 mmHg. Three experimental groups underwent ACS and received an experimental molecule at the time of decompression: inactive CORM-3, active CORM-3, and GYY4137, whereas three groups underwent no ACS to serve as a sham. Sinusoidal perfusion, inflammatory response and cell death were quantified in exteriorized livers. Respiratory, liver, and renal dysfunction was assessed biochemically.

Results: Hepatocellular death and the number of activated leukocytes within postsinusoidal venules were significantly increased in rats with ACS (16-fold increase, 17-fold leukocyte activation, respectively, P < 0.05). Administration of CORM-3 or GYY4137 resulted in a significant decrease of both parameters (P = 0.03 and P = 0.009). ACS resulted in an increase in markers of renal and liver injury; CORM-3 or GYY4137 partially restored levels to those seen in sham animals. Myeloperoxidase was significantly elevated in the ACS group in lung, liver, and small intestine (P = 0.0002, P = 0.01, and P = 0.08, respectively). CORM-3 treatment, but not GYY4137, was able to completely block the response (65 ± 11 U/ml and 92 ± 18 U/ml, respectively versus 110 ± 10U/ml in the ACS group, lung tissue).

Conclusions: We have demonstrated the effect of two molecules, CO and hydrogen sulphide, on tempering the reperfusion-associated metabolic and organ derangements in ACS. CORM-3 demonstrated a greater effect than GYY4137 and was able to restore most of the measured parameters to levels comparable to sham.
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http://dx.doi.org/10.1016/j.jss.2017.09.028DOI Listing
February 2018

Systemic application of carbon monoxide-releasing molecule 3 protects skeletal muscle from ischemia-reperfusion injury.

J Vasc Surg 2017 12 17;66(6):1864-1871. Epub 2017 Feb 17.

Division of Orthopaedic Surgery, University of Western Ontario, London, Ontario, Canada; Centre for Critical Illness Research, Lawson Health Research Institute, London, Ontario, Canada; Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada.

Objective: Ischemia-reperfusion (IR) is a limb- and life-threatening complication of acute limb ischemia and musculoskeletal trauma. Carbon monoxide-releasing molecules (CORMs) have recently been shown to protect microvascular perfusion and to reduce inflammation and injury in various ischemic animal models. The purpose of this study was to examine the effects of water-soluble CORM-3 on the extent of IR-induced muscle injury.

Methods: Wistar rats were randomized into three groups: sham (no ischemia), IR + CORM-3 (10 mg/kg intraperitoneally), and IR + inactive CORM-3 (iCORM-3; 10 mg/kg intraperitoneally). No-flow ischemia was induced by the application of a tourniquet to the hind limb for 2 hours; tourniquet release commenced the reperfusion phase. Both CORM-3 and iCORM-3 were injected immediately after tourniquet release. Temporal changes in microvascular perfusion, cellular tissue injury (ethidium bromide and bisbenzimide staining), and inflammatory response (leukocyte recruitment) within the extensor digitorum longus muscle were assessed using intravital video microscopy every 15 minutes for a total of 90 minutes after initiation of reperfusion. Systemic levels of tumor necrosis factor-α were also measured.

Results: Hind limb IR resulted in (1) a significant no-reflow phenomenon followed by progressive increase in microvascular perfusion deficit (21% ± 2% continuously perfused capillaries in IR vs 76% ± 4% in sham [P < .001]; 52% ± 8% nonperfused capillaries in IR vs 13% ± 2% in sham at 90 minutes of reperfusion [P < .001]), (2) tissue injury (ethidium bromide and bisbenzimide staining of 0.52 ± 0.07 in IR vs 0.05 ± 0.03 in sham at 90 minutes of reperfusion [P < .001]), (3) leukocyte recruitment (13.7 ± 0.9 adherent leukocytes/30 seconds/1000 μm in IR vs 1.8 ± 0.5 adherent leukocytes/30 seconds/1000 μm in sham at 90 minutes of reperfusion [P < .001]), and (4) an increase in circulating tumor necrosis factor-α levels. Systemic administration of CORM-3 (but not of iCORM-3) effectively reduced the IR-associated skeletal muscle perfusion deficits, tissue injury, and inflammatory activation.

Conclusions: CORM-3 displays potent protective and anti-inflammatory effects in an experimental model of hind limb IR, suggesting a potential therapeutic application of CORMs in treatment of ischemic conditions.
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http://dx.doi.org/10.1016/j.jvs.2016.11.065DOI Listing
December 2017

Hepatic microvascular changes in rat abdominal compartment syndrome.

J Surg Res 2015 Aug 21;197(2):398-404. Epub 2015 Apr 21.

Centre for Critical Illness Research, Lawson Health Research Institute, London, Ontario, Canada; Division of Orthopaedic Surgery, The University of Western Ontario, London, Ontario, Canada.

Background: Abdominal compartment syndrome (ACS) is associated with an increased rate of multiple organ dysfunction and is an independent marker for mortality. Our objective was to develop an animal model to study the mechanisms of tissue and microvascular injury associated with ACS at the microscopic level.

Materials And Methods: ACS was established in rats with CO₂ insufflation at 20 mm Hg for 2 h, with an abdominal cast. Sinusoidal perfusion, inflammatory response, and cell death were quantified in exteriorized livers. Respiratory and renal dysfunction were assessed biochemically and morphologically. Myeloperoxidase levels, a marker of neutrophil activation, were measured in the liver, lung, and small intestine.

Results: Continuously perfused sinusoids were significantly lower in the ACS group (81.4 ± 2.2% versus 99.6% ± 0.50), with an increase in nonperfused and intermittently perfused sinusoids (P < 0.05). Hepatocellular death and the number of activated leukocytes in postsinusoidal venules showed 7- and 18-fold increases, respectively, in the ACS group (P < 0.05). A significant increase in blood urea nitrogen levels in experimental rats was also observed. Myeloperoxidase levels were found to be 8-fold higher in lungs of ACS rats relative to control (P < 0.05), as well as statistically significant increase in the pCO₂ and decrease in pH of ACS rats.

Conclusions: We have successfully developed a model of ACS with documented evidence of renal and respiratory dysfunction. In addition, we have microscopy-confirmed evidence of early inflammatory changes and perfusion deficits in the liver with a concomitant increase in cell death in the ACS group.
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http://dx.doi.org/10.1016/j.jss.2015.04.049DOI Listing
August 2015

The severity of microvascular dysfunction due to compartment syndrome is diminished by the systemic application of CO-releasing molecule-3.

J Orthop Trauma 2014 Nov;28(11):e263-8

*Division of Orthopaedic Surgery, University of Western Ontario, London, Ontario, Canada; †Centre for Critical Illness Research, Lawson Health Research Institute, London, Ontario, Canada; and ‡Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada.

Objectives: To examine the protective effects of carbon monoxide (CO), liberated from a novel CO-releasing molecule (CORM-3), on the function of compartment syndrome (CS)-challenged muscle in a rodent model, thus providing for a potential development of a pharmacologic adjunctive treatment for CS.

Methods: Wistar rats were randomized into 4 groups: sham (no CS), CS, CS with inactive CORM-3 (iCORM-3), and CS + CORM-3 (10 mg/kg intraperitoneally). CS was induced by elevation of intracompartmental pressure to 30 mm Hg through an infusion of isotonic saline into the anterior compartment of the hind limb for 2 hours. Both CORM-3 and iCORM-3 were injected immediately after fasciotomy. Microvascular perfusion, cellular tissue injury, and inflammatory response within the extensor digitorum longus muscle were assessed using intravital video microscopy 45 minutes after fasciotomy. Systemic levels of tumor necrosis factor alpha (TNF-α) were also measured.

Results: Elevation of intracompartmental pressure resulted in significant microvascular perfusion deficits (23% ± 2% continuously perfused capillaries in CS vs. 76% ± 4% in sham, P < 0.0001; 55% ± 2% nonperfused capillaries in CS vs. 13% ± 2% in sham, P < 0.0001), significant increase in tissue injury (ethidium bromide/bisbenzimide of 0.31 ± 0.05 in CS vs. 0.05 ± 0.03 in sham, P < 0.0001) and adherent leukocytes (13.7 ± 0.9 in CS vs. 1.8 ± 0.5 in sham, P < 0.0001), and a progressive rise in systemic TNF-α. CORM-3 (but not iCORM-3) treatment restored the number of continuously perfused capillaries (57% ± 5%, P < 0.001), diminished tissue injury (ethidium bromide/bisbenzimide of 0.07 ± 0.01, P < 0.001), reversed the CS-associated rise in TNF-α, and decreased leukocyte adherence (0.6 ± 0.3, P < 0.001).

Conclusions: CORM-3 displays a potent protective/anti-inflammatory action in an experimental model of CS, suggesting a potential therapeutic application to patients at risk of developing CS.
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http://dx.doi.org/10.1097/BOT.0000000000000097DOI Listing
November 2014

Compartment syndrome-induced microvascular dysfunction: an experimental rodent model.

Can J Surg 2011 Jun;54(3):194-200

Division of Orthopaedic Surgery, University of Western Ontario, London, Canada.

Background: Acute compartment syndrome (CS) is a limb-threatening disease that results from increased intracompartmental pressure. The pathophysiologic mechanisms by which this occurs are poorly understood. This study was designed to measure the effects of increased intracompartmental pressure on skeletal muscle microcirculation, inflammation and cellular injury using intravital videomicroscopy (IVVM) in a clinically relevant small animal model.

Methods: We induced CS in 10 male Wistar rats (175-250 g), using a saline infusion technique. Intracompartmental pressure was controlled between 30 and 40 mm Hg and maintained for 45 minutes. After fasciotomy, the extensor digitorum longus muscle was visualized using IVVM, and perfusion was quantified. We quantified leukocyte recruitment to measure the inflammatory response. We measured muscle cellular injury using a differential fluorescent staining technique.

Results: The number of nonperfused capillaries increased from 12.7 (standard error of the mean [SEM] 1.4 ) per mm in the control group to 30.0 (SEM 6.7) per mm following 45 minutes of elevated intracompartmental pressure (CS group; p = 0.031). The mean number of continuously perfused capillaries (and SEM) decreased from 78.4 (3.2) per mm in the control group to 41.4 (6.9) per mm in the CS group (p = 0.001). The proportion of injured cells increased from 5.0% (SEM 2.1%) in the control group to 16.3% (SEM 6.8%) in the CS group (p = 0.006). The mean number of activated leukocytes increased from 3.6 (SEM 0.7) per 100 μm(2) in the control group to 8.6 (SEM 1.8) per 100 μm(2) in the CS group (p = 0.033).

Conclusion: Early CS-induced microvascular dysfunction resulted in a decrease in nutritive capillary perfusion and an increase in cellular injury and was associated with a severe acute inflammatory component.
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http://dx.doi.org/10.1503/cjs.048309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104305PMC
June 2011

Carbon monoxide liberated from CO-releasing molecule (CORM-2) attenuates ischemia/reperfusion (I/R)-induced inflammation in the small intestine.

Inflammation 2010 Apr;33(2):92-100

Centre for Critical Illness Research, Lawson Health Research Institute, 800 Commissioners Rd., London, ON, N6A4G5, Canada.

CORM-released CO has been shown to be beneficial in resolution of acute inflammation. The acute phase of intestinal ischemia-reperfusion (I/R) injury is characterized by oxidative stress-related inflammation and leukocyte recruitment. In this study, we assessed the effects and potential mechanisms of CORM-2-released CO in modulation of inflammatory response in the small intestine following I/R-challenge. To this end mice (C57Bl/6) small intestine were challenged with ischemia by occluding superior mesenteric artery (SMA) for 45 min. CORM-2 (8 mg/kg; i.v.) was administered immediately before SMA occlusion. Sham operated mice were injected with vehicle (0.25% DMSO). Inflammatory response in the small intestine (jejunum) was assessed 4 h following reperfusion by measuring tissue levels of TNF-alpha protein (ELISA), adhesion molecules E-selectin and ICAM-1 (Western blot), NF-kappaB activation (EMSA), along with PMN tissue accumulation (MPO assay) and leukocyte rolling/adhesion in the microcirculation of jejunum (intravital microscopy). The obtained results indicate that tissue levels of TNF-alpha, E-selectin and ICAM-1 protein expression, activation of NF-kappaB, and subsequent accumulation of PMN were elevated in I/R-challenged jejunum. The above changes were significantly attenuated in CORM-2-treated mice. Taken together these findings indicate that CORM-2-released CO confers anti-inflammatory effects by interfering with NF-kappaB activation and subsequent up-regulation of vascular pro-adhesive phenotype in I/R-challenged small intestine.
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http://dx.doi.org/10.1007/s10753-009-9162-yDOI Listing
April 2010

Hindlimb ischemia/reperfusion-induced remote injury to the small intestine: role of inducible nitric-oxide synthase-derived nitric oxide.

J Pharmacol Exp Ther 2009 Jun 6;329(3):919-27. Epub 2009 Mar 6.

The Centre for Critical Illness Research, Lawson Health Research Institute, London, ON N6A 4G5, Canada.

Systemic inflammatory response syndrome, as a consequence of ischemia/reperfusion (I/R), negatively influences the function of the affected organs. The objective of this study was to assess the role of nitric oxide (NO) in remote intestinal inflammatory response elicited by hindlimb I/R. To this end, C57BL/6 (wild type; WT) and inducible nitric-oxide synthase (iNOS)-deficient mice were subjected to bilateral hindlimb ischemia (1 h) followed by 6 h of reperfusion. Some WT mice were injected with iNOS inhibitor N-[3-(aminomethyl)benzyl] acetamidine (1400W) (5 mg/kg s.c.) immediately before reperfusion, and proinflammatory response was assessed 6 h later. Hindlimb I/R resulted in dysfunction of the small intestine as assessed by the increase in permeability [blood-to-lumen clearance of Texas Red-dextran (molecular mass 3 kDa)] and an increase in the luminal levels of tumor necrosis factor (TNF)-alpha protein and nitrate/nitrite (NO(2)(-)/NO(3)(-)). The above-mentioned changes were accompanied by up-regulation of the proinflammatory phenotype in the mucosa of small intestine with respect to 1) an increase in TNF-alpha and iNOS protein expression, 2) leukocyte accumulation, 3) formation of edema, 4) an increase in leukocyte rolling/adhesion in the submucosal microvasculature, and 5) activation of transcription factor nuclear factor-kappaB and up-regulation of adhesion molecule expression. Interestingly, the most profound changes with respect to intestinal dysfunction were found in jejunum and ileum, whereas duodenum was affected the least. Interfering with iNOS activity (1400W and iNOS-deficient mice) significantly attenuated hindlimb I/R-induced inflammatory response and dysfunction of the small intestine with respect to the above-mentioned markers of inflammation. The obtained results indicate that hindlimb I/R induces remote inflammatory response in the small intestine through an iNOS-derived NO-dependent mechanism.
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http://dx.doi.org/10.1124/jpet.108.148460DOI Listing
June 2009

Low-dose inhaled carbon monoxide attenuates the remote intestinal inflammatory response elicited by hindlimb ischemia-reperfusion.

Am J Physiol Gastrointest Liver Physiol 2009 Jan 16;296(1):G9-G14. Epub 2008 Oct 16.

Dept. of Medical Biophysics and Surgery, Univ. of Western Ontario, Centre for Critical Illness Research, London Health Sciences Centre, Victoria Hospital, Rm A6-132, London, Ontario, Canada N6A 4G5.

Heme oxygenase (HO) represents the rate-limiting enzyme in the degradation of heme into carbon monoxide (CO), iron, and biliverdin. Recent evidence suggests that several of the beneficial properties of HO, may be linked to CO. The objectives of this study were to determine if low-dose inhaled CO reduces remote intestinal leukocyte recruitment, proinflammatory cytokine expression, and oxidative stress elicited by hindlimb ischemia-reperfusion (I/R). Male mice underwent 1 h of hindlimb ischemia, followed by 3 h of reperfusion. Throughout reperfusion, mice were exposed to AIR or AIR + CO (250 ppm). Following reperfusion, the distal ileum was exteriorized to assess the intestinal inflammatory response by quantifying leukocyte rolling and adhesion in submucosal postcapillary venules with the use of intravital microscopy. Ileum samples were also analyzed for proinflammatory cytokine expression [tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta] and malondialdehyde (MDA) with the use of enzyme-linked immunosorbent assay and thiobarbituric acid reactive substances assays, respectively. I/R + AIR led to a significant decrease in leukocyte rolling velocity and a sevenfold increase in leukocyte adhesion. This was also accompanied by a significant 1.3-fold increase in ileum MDA and 2.3-fold increase in TNF-alpha expression. Treatment with AIR + CO led to a significant reduction in leukocyte recruitment and TNF-alpha expression elicited by I/R; however, MDA levels remained unchanged. Our data suggest that low-dose inhaled CO selectively attenuates the remote intestinal inflammatory response elicited by hindlimb I/R, yet does not provide protection against intestinal lipid peroxidation. CO may represent a novel anti-inflammatory therapeutic treatment to target remote organs following acute trauma and/or I/R injury.
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http://dx.doi.org/10.1152/ajpgi.90243.2008DOI Listing
January 2009

Carbon monoxide liberated from carbon monoxide-releasing molecule CORM-2 attenuates inflammation in the liver of septic mice.

Am J Physiol Gastrointest Liver Physiol 2008 Jan 8;294(1):G184-91. Epub 2007 Nov 8.

Centre for Critical Illness Research, Lawson Health Research Institute, London, Ontario, N6A 4G4, Canada.

Recent studies suggest that exogenously administered CO is beneficial for the resolution of acute inflammation. In this study, we assessed the role of CO liberated from a systemically administered tricarbonyldichlororuthenium-(II)-dimer (CORM-2) on modulation of liver inflammation during sepsis. Polymicrobial sepsis in mice was induced by cecal ligation and perforation (CLP). CORM-2 (8 mg/kg iv) was administered immediately after CLP induction, and neutrophil [polymorphonuclear leukocyte (PMN)] tissue accumulation, activation of transcription factor, NF-kappaB, and changes in adhesion molecule ICAM-1 expression (inflammation-relevant markers) were assessed in murine liver 24 h later. In addition, the effects and potential mechanisms of CORM-2-released CO in modulation of vascular endothelial cell proinflammatory responses were assessed in vitro. To this end, human umbilical vein endothelial cells (HUVEC) were stimulated with LPS (1 microg/ml) in the presence or absence of CORM-2 (10-100 microM) and production of intracellular reactive oxygen species (ROS), (DHR123 oxidation) and NO (DAF-FM nitrosation) and subsequent activation of NF-kappaB were assessed 4 h later. In parallel, expression of ICAM-1 and inducible NO synthase (iNOS) proteins along with PMN adhesion to LPS-challenged HUVEC were also assessed. Induction of CLP resulted in increased PMN accumulation, ICAM-1 expression, and activation of NF-kappaB in the liver of septic mice. These effects were significantly attenuated by systemic administration of CORM-2. In in vitro experiments, CORM-2-released CO attenuated LPS-induced production of ROS and NO, activation of NF-kappaB, increase in ICAM-1 and iNOS protein expression and PMN adhesion to LPS-stimulated HUVEC. Taken together, these findings indicate that CO released from systemically administered CORM-2 provides anti-inflammatory effects by interfering with NF-kappaB activation and subsequent downregulation of proadhesive vascular endothelial cell phenotype in the liver of septic mice.
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http://dx.doi.org/10.1152/ajpgi.00348.2007DOI Listing
January 2008

Role of heme oxygenase in the protection afforded skeletal muscle during ischemic tolerance.

Microcirculation 2006 Mar;13(2):71-9

Centre for Critical Illness Research, Lawson Health Research Institute, London, Ontario, Canada.

Objective: Ischemic tolerance (IT) is known to improve resistance to ischemia/reperfusion (I/R)-induced injury; however, the mechanisms remain unknown. The authors hypothesized that induction of heme oxygenase (HO), a heat shock protein, would provide anti-inflammatory benefits during IT, thereby preventing leukocyte-derived I/R injury.

Methods: Male Wistar rats were randomly assigned to sham (n = 4), I/R (n = 9), preconditioning (PC)+I/R (n = 7), chromium mesoporphyrin, to inhibit HO (CrMP; n = 4), or PC+I/R+CrMP (n = 6) groups. PC consisted of 5 cycles of I/R, each lasting 10 min, induced by tightening a tourniquet placed above the greater trochantor of the hindlimb. Twenty-four hours later, the hindlimb underwent 2 h of no-flow ischemia followed by intravital microscopy during 90 min reperfusion to assess capillary perfusion (#/mm), tissue injury (ratio of ethidium bromide to bisbenzimide labeled cells/100 microm2), leukocyte rolling (Lr, #/1000 microm2), and adhesion (La, #/1000 microm2) in postcapillary venules of the extensor digitorum longus (EDL) muscle.

Results: In the I/R group, Lr was significantly increased (7.1 +/- 0.4) compared to sham (3.1 +/- 0.4). PC+I/R increased Lr (10.8 +/- 0.72), which was further exacerbated by the removal of HO (14.2 +/- 1.3). La (7.8 +/- 2.0) was significantly increased compared to sham (2.4 +/- 0.9), while PC returned La back to sham levels (1.9 +/- 0.7). Removal of HO activity, via CrMP, had no significant effect on La (3.9 +/- 0.7). However, CrMP removed the protection to microvascular perfusion (I/R = 9.4 +/- 1.1, PC = 16.6 +/- 1.8, sham = 20.5 +/- 2.8, PC+CrMP+I/R = 12.3 +/- 2.3) and prevented protection from ischemia-induced tissue injury.

Conclusion: The data suggest that HO is an important protective mechanism during IT in skeletal muscle, but such protection was by mechanisms other than altered leukocyte-endothelial cell interaction.
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http://dx.doi.org/10.1080/10739680500466228DOI Listing
March 2006

Heme oxygenase modulates small intestine leukocyte adhesion following hindlimb ischemia/reperfusion by regulating the expression of intercellular adhesion molecule-1.

Crit Care Med 2005 Nov;33(11):2563-70

Medical Biophysics, University of Western Ontario, London, ON, USA.

Objective: Heme oxygenase is the rate-limiting enzyme in the degradation of heme into carbon monoxide, iron, and bilirubin. Recent evidence suggests that the induction of heme oxygenase-1 is associated with potent anti-inflammatory properties. The objectives of this study were to determine the temporal, regional, and cellular distribution of heme oxygenase-1 within the small intestine and its role in modulating remote intestinal leukocyte recruitment following trauma induced by hindlimb ischemia/reperfusion.

Design: Randomized, controlled, prospective animal study.

Setting: Hospital surgical research laboratory.

Subjects: Male C57BL/6 mice.

Interventions: Mice underwent 1 hr of bilateral hindlimb ischemia, followed by 3, 6, 12, or 24 hrs of reperfusion.

Measurements And Main Results: Heme oxygenase-1 messenger RNA, heme oxygenase-1 protein, and heme oxygenase activity were measured using reverse transcription polymerase chain reaction, Western blot, immunohistochemistry, and spectrophotometric assay, respectively. The jejunum was also exteriorized to quantify the flux of rolling and adherent leukocytes and R-Phycoerythrin conjugated intercellular adhesion molecule-1 monoclonal antibody fluorescence intensity in submucosal postcapillary venules with the use of intravital microscopy. Ischemia/reperfusion led to a significant increase in heme oxygenase-1 messenger RNA in the jejunum and ileum 3 hrs following limb reperfusion, with a subsequent increase in heme oxygenase-1 protein and heme oxygenase activity at 6 hrs. Ischemia/reperfusion also led to a significant 1.4-fold increase in leukocyte rolling, whereas inhibition of heme oxygenase via injection of tin protoporphyrin IX (20 micromol/kg intraperitoneally) resulted in a three-fold increase in leukocyte adhesion, compared with ischemia/reperfusion alone. This increase in adhesion was significantly reduced to baseline in mice treated with intercellular adhesion molecule-1 monoclonal antibody before heme oxygenase inhibition (40 microg/mouse), whereas inhibition of heme oxygenase activity following ischemia/reperfusion also led to a significant increase in R-Phycoerythrin intercellular adhesion molecule-1 monoclonal antibody fluorescence intensity.

Conclusions: Our data suggest that remote trauma induced by hindlimb ischemia/reperfusion leads to an increase in heme oxygenase activity within the small intestine, which modulates intercellular adhesion molecule-1 dependent intestinal leukocyte adhesion.
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http://dx.doi.org/10.1097/01.ccm.0000186765.61268.fcDOI Listing
November 2005

Inhalation of carbon monoxide prevents liver injury and inflammation following hind limb ischemia/reperfusion.

FASEB J 2005 Jan 28;19(1):106-8. Epub 2004 Oct 28.

Victoria Research Lab, 6th Floor, Rm. A6-105, 800 Commissioners Rd., London, ON, Canada N6A 4G4.

The induction of heme oxygenase (HO), the rate limiting enzyme in the conversion of heme into carbon monoxide (CO) and biliverdin, limits liver injury following remote trauma such as hind limb ischemia/reperfusion (I/R). Using intravital video microscopy, we tested the hypothesis that inhaled CO (250 ppm) would mimic HO-derived liver protection. Hind limb I/R significantly decreased sinusoidal diameter and volumetric flow, increased leukocyte accumulation within sinusoids, increased leukocyte rolling and adhesion within postsinusoidal venules, and significantly increased hepatocyte injury compared with naive animals. Inhalation of CO alone did not alter any microcirculatory or inflammatory parameters. Inhalation of CO following I/R restored volumetric flow, decreased stationary leukocytes within sinusoids, decreased leukocyte rolling and adhesion within postsinusoidal venules, and significantly reduced hepatocellular injury following hind limb I/R. HO inhibition did not alter microcirculatory parameters in naive mice, but did increase inflammation, as well as increase hepatocyte injury following hind limb I/R. Inhalation of CO during HO inhibition significantly reduced such microcirculatory deficits, hepatic inflammation, and injury in response to hind limb I/R. In conclusion, these results suggest that HO-derived hepatic protection is mediated by CO, and inhalation of low concentrations of CO may represent a novel therapeutic approach to prevent remote organ injury during systemic inflammatory response syndrome, or SIRS.
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http://dx.doi.org/10.1096/fj.04-2514fjeDOI Listing
January 2005

Remote liver injury is attenuated by adenovirus-mediated gene transfer of heme oxygenase-1 during the systemic inflammatory response syndrome.

Microcirculation 2004 Oct-Nov;11(7):587-95

Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada.

Objectives: Adenovirus-mediated gene therapy is being investigated with increasing success for future treatment of autoimmune diseases. However, the use of adenoviruses is still limited by inflammatory and immune responses in the target organ. Previous work by the authors' laboratory established that the adenovirus encoding inducible heme oxygenase (Ad-HO-1) does not elicit the acute hepatic inflammation normally caused by adenoviruses, inviting further investigation in models of severe inflammation. Concurrently, there is increasing evidence for an endogenous protective role for heme oxygenase (HO) in the liver during the systemic inflammatory response syndrome (SIRS). Building on our previous results, this study investigated the effect of Ad-HO-1 pretreatment on remote liver injury during normotensive SIRS, induced by bilateral hind limb ischemia and reperfusion.

Methods: Microvascular perfusion and hepatocyte death were quantified using established intravital videomicroscopy techniques. Hepatocellular injury and liver function were assessed using blood-borne indicators.

Results: Microvascular perfusion deficits and increased hepatocyte death occurred following limb ischemia and 3 h of reperfusion in vehicle-pretreated animals; however, Ad-HO-1 pretreatment prevented these deficits. In contrast, the increase in serum alanine transaminase levels was unaffected by Ad-HO-1 pretreatment. Serum bilirubin levels were increased during systemic inflammation, predominantly in the conjugated form; and, this increase was prevented by administration of Ad-HO-1.

Conclusions: These data indicate that gene transfer of inducible HO is an effective method to protect the liver during SIRS, providing incentive for further investigation into gene therapy strategies exploiting this anti-inflammatory enzyme.
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http://dx.doi.org/10.1080/10739680490503384DOI Listing
March 2005

Endogenous heme oxygenase induction is a critical mechanism attenuating apoptosis and restoring microvascular perfusion following limb ischemia/reperfusion.

Surgery 2004 Jul;136(1):67-75

Department of Medical Biophysics, University of Western Ontario, and the London Health Science Centre and Lawson Health Research Institute, London, Ontario, Canada.

Background: A protective role for endogenous heme oxygenase (HO) in the initiation of remote liver injury after limb ischemia/reperfusion has been established. This study expands on our previous work by investigating the role of endogenous HO on hepatocellular injury, hepatocyte death (necrotic and apoptotic), and microvascular perfusion at protracted post-reperfusion times.

Methods: Remote liver injury was studied after 1 hour of bilateral hind limb ischemia and 3, 6, or 24 hours of reperfusion in male C57BL6 mice. Inhibition of HO was achieved with the use of chromium mesoporphrin (CrMP). Established intravital videomicroscopy techniques were used to evaluate microvascular perfusion and hepatocyte death. Hepatocellular injury was quantified by serum alanine transaminase. Apoptosis was measured by using DNA laddering, Cell Death ELISA, and caspase-3 activity.

Results: Although significant perfusion deficits and hepatocellular injury/death occurred after 3 hours, progression of hepatocellular death beyond 6 hours was not observed. A transient increase in apoptosis was observed at 6 hours. By 24 hours, microvascular perfusion was completely restored. This lack of progression correlated with increased HO activity, observed throughout the protocol. Administration of CrMP reduced HO activity to sham nonstressed levels, and caused increased microvascular perfusion deficits, hepatocellular injury, and hepatocyte death over 24 hours. The transient increase in apoptosis was increased in duration and magnitude in CrMP-treated animals.

Conclusions: These results suggest that endogenous HO activity prevents the progression of remote liver injury after limb ischemia/reperfusion.
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http://dx.doi.org/10.1016/j.surg.2003.11.002DOI Listing
July 2004

Heme oxygenase modulates hepatic leukocyte sequestration via changes in sinusoidal tone in systemic inflammation in mice.

Microvasc Res 2004 Jul;68(1):20-9

Klinik und Poliklinik für Anästhesiologie, Julius-Maximilians-Universität, Würzburg, Germany.

Heme oxygenase (HO) modulates the accumulation of leukocytes within the liver during the early stages of a systemic inflammatory response syndrome (SIRS), but the anti-inflammatory mechanism(s) remain to be tested. The influence of HO on the adhesion molecule expression within the liver and on circulating leukocytes was assessed. In addition, the effect of HO and nitric oxide synthase (NOS) on the liver microcirculation was tested. Mice were subjected to 1 h bilateral hindlimb ischemia followed by 3 h of reperfusion, at which time blood samples and the liver were harvested and adhesion molecule expression determined (ICAM-1, CD49d and CD11b). Direct measures of sinusoidal diameter and estimates of volumetric blood flow were obtained using intravital microscopy. HO was specifically induced and inhibited by hemin and chromium mesoporphyrin (CrMP), respectively, whereas NOS was inhibited by N-nitro-L-arginine methyl ester (L-NAME). ICAM-1 expression was increased following hindlimb ischemia-reperfusion. Hemin caused only a modest, but significant decrease in ICAM-1 expression, whereas inhibition of HO had no effect. However, HO inhibition significantly reduced sinusoidal diameters and volumetric flow and such vessels were correlated with significantly increased numbers of stationary leukocytes. Inhibition of NOS had no effect on sinusoidal diameter or volumetric flow. In conclusion, the anti-inflammatory benefits afforded by HO activity within the liver appear to involve the control of sinusoidal diameter and volumetric blood flow rather than altered adhesion molecule expression during the early stages of SIRS.
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http://dx.doi.org/10.1016/j.mvr.2004.03.003DOI Listing
July 2004

Protective mechanisms during ischemic tolerance in skeletal muscle.

Free Radic Biol Med 2004 Feb;36(3):371-9

Department of Medical Biophysics, The University of Western Ontario, and The Lawson Health Research Institute, London, Ontario, Canada.

The purpose of this study was to test specific mechanisms of protection afforded the rat extensor digitorum longus (EDL) muscle during ischemic tolerance. Two days following five cycles of 10 min ischemia and 10 min reperfusion, heme oxygenase (HO) and calcium-dependent nitric oxide synthase (cNOS) activities were increased 2- and 2.5-fold (p <.05), respectively. Interestingly, calcium-independent NOS (iNOS) activity was completely downregulated (p <.05). The levels of superoxide dismutase (SOD) and catalase were increased 2-fold (p <.05), while glutathione peroxidase activity remained unchanged from non-preconditioned controls. Using intravital microscopy combined with chromium mesoporphyrin (CrMP), a selective HO inhibitor, and l-NAME, a NOS inhibitor, the roles of HO and cNOS were evaluated. Ischemic tolerance in the EDL muscle, 48 h after the preconditioning stimulus, was characterized by complete protection from both microvascular perfusion deficits and tissue injury after a 2-h period of ischemia. Removal of NOS activity completely removed the benefit afforded microvascular perfusion, while inhibition of HO activity prevented the parenchymal protection. These data suggest that ischemic tolerance within skeletal muscle is associated with the upregulation of specific cytoprotective proteins and that the benefits afforded by cNOS and HO activity are spatially discrete to the microvasculature and parenchyma, respectively.
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http://dx.doi.org/10.1016/j.freeradbiomed.2003.11.015DOI Listing
February 2004

Role of the mesolimbic cholinergic projection to the septum in the production of 22 kHz alarm calls in rats.

Brain Res Bull 2003 May;60(3):263-74

Department of Anatomy and Cell Biology, University of Western Ontario, London, Ont., Canada N6A 5A5.

The role of the ascending cholinergic projection from the laterodorsal tegmental nucleus (LDT) to septum in the production of 22 kHz ultrasonic vocalization was studied in adult rats, using behavioral-pharmacological and anatomical tracing methods. Direct application of carbachol, a muscarinic agonist, into the lateral septal region induced species-typical 22 kHz alarm calls. The septum receives cholinergic input from LDT, thus, activation with glutamate of predominantly cholinergic neurons of the LDT induced comparable 22 kHz alarm calls in the same animals. This glutamate-induced response from LDT was significantly reduced when the lateral septum was pretreated with scopolamine, a cholinergic antagonist. To investigate the localization of the cell groups projecting to septum, the fluorescent retrograde tracer, fluorogold, was pressure injected into the lateral septum and sections from these brains were also immunostained against choline acetyltransferase (ChAT) to visualize cholinergic cell bodies. Several ChAT-fluorogold double-labeled cells within the boundaries of the LDT were found, while other fluorogold-labeled regions did not contain double-labeled cells. These results provide both direct and indirect evidence that at least a part of the mesolimbic ascending cholinergic projection from LDT to septum is involved in the initiation of the 22 kHz vocalization. It is concluded that the septum is an integral part of the medial cholinoceptive vocalization strip and the 22 kHz alarm vocalization is triggered from septum by the cholinergic input from the LDT.
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http://dx.doi.org/10.1016/s0361-9230(03)00041-8DOI Listing
May 2003

Endothelin-a receptor in rat skeletal muscle microvasculature.

Microvasc Res 2002 Jul;64(1):179-85

Departmente of Pharmacology and Toxicology, The University of Western Ontario, London, Ontario, N6A 5C1, Canada.

Although the effect of endothelin-1 (ET-1) on vascular tone has been studied extensively at the arterial/arteriolar level, little is known about the direct effect of ET-1 at the level of the capillary. Using intravital microscopy, we determined capillary red blood cell velocity and arteriolar diameter responses to ET-1, ET(A)-receptor blocker BQ-123, and ET(B)-receptor blocker BQ-788 applied locally on capillaries in rat extensor digitorum longus (EDL) muscle. Using immunohistochemistry, we examined capillaries in this muscle and microvascular endothelial cells isolated from this muscle for immunoreactivity with ET(A)-receptor antibody. ET-1 (10(-9) to 10(-5) M in micropipette) caused quick reductions (i.e., within several seconds), whereas BQ-123 (10(-8) to 10(-4) M) and BQ-788 (10(-6) and 10(-4) M) caused quick increases, in both velocity and diameter. Capillaries and endothelial cells showed ET(A)-receptor immunoreactivity. We conclude that the microvasculature of the rat EDL muscle is sensitive to ET-1 and its receptor blockers and that the ET(A) receptor may be present in the capillary wall of this muscle, including the endothelium.
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http://dx.doi.org/10.1006/mvre.2002.2427DOI Listing
July 2002

Functional inhibition of constitutive nitric oxide synthase in a rat model of sepsis.

Am J Respir Crit Care Med 2002 May;165(10):1426-32

A. C. Burton Vascular Biology Laboratory, Department of Medicine, University of Western Ontario and London Health Sciences Centre, London, Ontario, Canada.

Induction of inducible nitric oxide synthase (iNOS) expression is likely important in the pathogenesis of sepsis. However, the sepsis-mediated induction of iNOS is associated with a decrease in constitutive NO synthase (cNOS) activity (which is reversible following acute but not chronic sepsis). Whether this decreased cNOS activity is due to functional inhibition of cNOS by the high concentrations of NO produced by iNOS or to downregulation of cNOS expression is not clear. Thus, we tested the hypothesis that sepsis produces a reversible iNOS/NO-mediated inhibition of cNOS activity. Using a rat cecal ligation and perforation (CLP) model of sepsis, we examined the time course of the changes in iNOS and cNOS activities in lung and thoracic aortae. Reversibility of the sepsis-induced decrease in cNOS activity was assessed in vitro by enzyme activity determination following selective inhibition of iNOS. iNOS and endothelial cNOS protein concentrations were determined by Western blotting. In all septic tissues, cNOS activity was depressed at 6, 12, 24, and 48 hours post-CLP. Inhibition of the increased iNOS activity with aminoguanidine, in vitro, partially restored cNOS activity following acute (6-12 hours) but not chronic sepsis (24-48 hours post-CLP). Consistent with the irreversible depression of cNOS activities in tissues following chronic sepsis, endothelial NOS protein concentrations declined progressively during the time course of sepsis. We have demonstrated the restoration of cNOS activity following in vitro inhibition of iNOS, early, and the downregulation of endothelial NOS, later, in a rat CLP model of sepsis. This suggests that further study is required before iNOS-selective inhibition can be considered in human sepsis.
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http://dx.doi.org/10.1164/rccm.2011144DOI Listing
May 2002

Inhibition of haem oxygenase activity increases leukocyte accumulation in the liver following limb ischaemia-reperfusion in mice.

J Physiol 2002 May;540(Pt 3):1013-21

Klinik für Anaesthesiologie, Julius-Maximilians-Universität Würzburg, Germany.

The role of haem oxygenase (HO) in the hepatic accumulation of leukocytes in mice during the initiation of remote organ injury following normotensive limb ischaemia-reperfusion (I-R) was investigated. Remote organ injury was initiated by 1 h bilateral hindlimb ischaemia followed by either 1 or 1.5 h reperfusion (I-R) in male C57BL/6 mice. Mice were randomly assigned to either sham (no I-R, n = 4), I-R (n = 4 for both time points), I-R plus chromium mesoporphyrin (CrMP, n = 4) to inhibit HO or I-R plus haemin (n = 4) to increase HO. Leukocyte accumulation and leukocyte-endothelial interaction were directly measured using fluorescence intravital microscopy. Leukocytes were labelled via an injection of rhodamine 6G. In sinusoids the total number and the number of stationary leukocytes were assessed. In postsinusoidal venules the number of adherent and rolling leukocytes and the velocities of both red blood cells and leukocytes were measured. The total number of leukocytes increased in sinusoids of I-R mice reaching a plateau within 1 h compared with sham animals, while the number of stationary leukocytes progressively increased over the entire study period. Stationary leukocytes in sinusoids increased after 1 and 1.5 h of I-R following CrMP, while they were significantly reduced following haemin treatment compared to animals treated with I-R only. In postsinusoidal venules a progressive increase in adherent leukocytes also occurred. As observed in sinusoids, CrMP significantly increased, while haemin significantly reduced leukocyte adhesion. The number of rolling leukocytes increased after CrMP in both I-R groups (1 and 1.5 h). The velocities of rolling leukocytes declined following 1.5 h of I-R compared with sham. Haemin treatment of 1.5 h I-R animals restored the velocities back to sham levels. The calculated wall shear rates in postsinusoidal venules were significantly lower in all I-R groups in comparison to sham animals. Combination of 1.5 h I-R with CrMP resulted in the lowest shear rates of all I-R groups. The number of stationary leukocytes within sinusoids and adherent leukocytes in postsinusoidal venules were correlated to the corresponding alanine aminotransferase (ALT) levels. In conclusion, endogenous HO reduces leukocyte-endothelial interactions within the liver. Thus, endogenous HO activity provides an important mechanism controlling the hepatic inflammatory response during the initiation of remote organ injury following normotensive limb ischaemia-reperfusion.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2290293PMC
http://dx.doi.org/10.1113/jphysiol.2001.015446DOI Listing
May 2002

The role of endogenous heme oxygenase in the initiation of liver injury following limb ischemia/reperfusion.

J Hepatol 2002 May;36(5):624-30

Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada N6A 4G5.

Background/aims: Heme oxygenase (HO) derived liver protection was tested in mice following 1 h bilateral hindlimb ischemia and either 1.5 or 3 h reperfusion.

Methods: Groups consisted of limb ischemia/reperfusion (I/R), sham (no I/R), I/R+chromium mesoporphyrin (I/R+CrMP;40 micromol/kg, i.p.), or I/R+hemin (10 mg/kg, i.p.). The vital dye propidium iodide (PI), was used to measure hepatocellular death (#/0.1 mm(3)), while the number of sinusoids perfused by red blood cells (SP(RBC)) were measured from the periportal (Pp) and pericentral (Pc) zones of liver acini using intravital microscopy. Whole organ injury was estimated from serum alanine aminotransferase (ALT).

Results: SP(RBC) reduced within 1.5 h with no further decline following 3 h. CrMP resulted in a dramatic loss of SP(RBC) following 3 h only. Hemin restored perfusion in both zones. Hepatocellular death and organ injury increased at 1.5 and 3 h. At 1.5 h, CrMP further increased cell death in the Pc zone, as well as whole organ injury, while hemin restored cell viability. Increased HO mRNA, protein and activity suggested induction within 3 h.

Conclusions: HO does not protect perfusion during the early stage (1.5 h), but becomes increasingly important in preserving liver perfusion and cell viability during the later stage (3 h) of liver injury.
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http://dx.doi.org/10.1016/s0168-8278(02)00025-9DOI Listing
May 2002
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