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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 28, 2004 as doi:10.1096/fj.04-2514fje. |
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,,1
* Department of General Surgery and
Vascular Surgery, London Health Sciences Centre, London, Ontario, Canada;
The Centre for Critical Illness Research, Lawson Health Research Institute, London, Ontario, Canada; and
Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburg School of Medicine, Pittsburgh, Pennsylvania, USA4
1Correspondence: Victoria Research Lab, 6th Floor, Rm. A6-105, 800 Commissioners Rd., London, ON, Canada N6A 4G4. E-mail: rpotter{at}uwo.ca
SPECIFIC AIMS
In our previous studies we showed significant reductions in microvascular perfusion and hepatocellular injury in the liver after a systemic inflammatory response (SIRS) generated by hind limb ischemia/reperfusion (I/R). We have demonstrated that induction of heme oxygenase in the liver during the early stages of SIRS is an important endogenous protective mechanism. In the present study we use inhaled carbon monoxide (CO) at low concentration (250 ppm) to determine whether remote benefits could be realized within the liver in the setting of SIRS and to test the role of CO as the mechanism of heme oxygenase-derived protection.
PRINCIPAL FINDINGS
1. Heme oxygenase activity and protein expression
There were no significant differences in HO activity between naive, CO alone, or I/R plus CO groups. However, I/R in room air resulted in a significant increase in HO activity compared with naive animals, suggesting that inhalation of CO during I/R may have reduced HO activity. As expected, treatment with CrMP, a selective inhibitor of HO, significantly decreased HO activity in all groups compared with untreated animals; such activity was not altered by inhalation of CO. There were no statistically significant differences in HO protein expression between naive, CO alone, and CrMP groups. There was a trend to increased HO protein expression in animals undergoing hind limb I/R while inhalation of CO prevented such protein induction.
2. CO restores sinusoidal perfusion after remote I/R injury
Hepatic sinusoidal diameter did not differ between naive and CO groups (7.31±0.14 µm and 7.28±0.37 µm, respectively). However, hind limb I/R in room air caused a significant decrease in sinusoidal diameter compared with naive (5.57±0.14 µm, P<0.05). Exposure to CO after hind limb I/R partially restored sinusoidal diameter compared with the I/R group (6.44±0.27 µm, P<0.05). Inhibition of HO activity by CrMP in naive animals did not alter sinusoidal diameter (7.56±0.06 µm). When CrMP was administered to the I/R group, however, sinusoidal diameter was significantly decreased compared with naive group (5.75±0.14 µm) but did not differ from the I/R alone group. Inhalation of CO to the I/R plus CrMP group significantly increased sinusoidal diameter compared with the I/R plus CrMP group (7.25±0.11 µm), thereby restoring sinusoidal diameters to naive levels.
Similar to results obtained for sinusoidal diameter, there was no difference observed in volumetric flow within hepatic sinusoids between naive and CO alone groups (10.66±0.16 
lsec–1 and 9.33±0.98 lsec–1, respectively). However, I/R in room air resulted in a significant reduction in volumetric flow (2.62±0.29 lsec–1, P<0.05) compared with naive animals whereas inhalation of CO significantly increased volumetric flow (6.18±0.82 lsec–1, P<0.05). Inhibition of HO activity by application of CrMP did not significantly alter sinusoidal volumetric flow in the naive group (10.24±0.33 lsec–1). Application of CrMP to the I/R group significantly decreased volumetric flow but no more than observed with I/R alone (2.50±0.10 lsec–1). Inhalation of CO in the I/R plus CrMP group significantly increased volumetric flow compared with I/R or I/R plus CrMP groups (9.60±0.64 lsec–1, P<0.05), an increase that returned flow to near naive levels.
3. CO limits hepatic inflammation after remote I/R injury
No differences in the number of stationary leukocytes within hepatic sinusoids were observed between naive, CO, and CrMP groups (0.42±0.15, 0.39±0.09, and 0.83±0.23 leukocytes/sinusoidal view, respectively), but a significant increase in number of stationary leukocytes within the sinusoidal bed occurred in animals that underwent hind limb I/R (3.0±0.54 leukocytes/sinusoidal view) vs. the naive group. Inhalation of CO during I/R returned leukocyte accumulation within sinusoids back to baseline levels (1.0±0.17 leukocytes/sinusoid). Inhibition of HO activity by CrMP in the I/R group resulted in a significant increase in the number of stationary leukocytes (5.7±0.29 leukocytes/sinusoidal view) within sinusoids compared I/R alone, whereas inhalation of CO blocked such leukocyte accumulation (1.37±0.22 leukocytes/sinusoidal view).
The pattern of inflammatory changes observed in postsinusoidal venules was similar to that measured within the sinusoidal bed. There was no significant difference in leukocyte rolling or sticking within postsinusoidal venules in naive vs. the CO group (2.9±1.47 and 6.14±1.86, respectively). However I/R significantly increased leukocyte rolling (19.9±2.2) compared with the naive or CO alone groups. Exposure to CO during I/R resulted in a significant decrease in leukocyte rolling (9.8±1.8) compared with the I/R alone group. Inhibition of HO by CrMP in naive animals resulted in significantly increased leukocyte rolling (34.4±6.23) compared with naive animals. The number of rolling leukocytes was significantly increased in the I/R plus CrMP group compared with I/R alone. Addition of exogenous CO in animals with I/R plus CrMP did not alter the number of rolling leukocytes compared with the I/R plus CrMP group (23.5±4.43).
Leukocyte adhesion within postsinusoidal venules did not differ between naive and CO groups (Fig. 1
). I/R resulted in a highly significant increase in leukocyte adhesion (4.6±0.74, Fig. 1
) compared with naive or CO groups. However, when CO was added to the I/R group, the increased leukocyte adhesion after I/R was abolished (Fig. 1)
. Treatment of naive animals with CrMP significantly increased the number of adherent leukocytes within the postsinusoidal venule (6.4±1.82) compared with naive, CO, or I/R groups, but inhalation of CO significantly reduced leukocyte adhesion after I/R plus CrMP (Fig. 1)
.
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4. CO limits hepatocellular injury after remote I/R
Hepatocellular injury was almost nonexistent in naive and CO groups (12.1±3.3 and 18.97±1.8, respectively). The increased hepatocellular injury after I/R (168.2±36.87) was eliminated if the I/R group was exposed to CO (20.1±3.9, Fig. 2
). Use of CrMP alone resulted in a trend toward increased propidium iodide (P.I.) labeled cells, an index of cell death. Application of CrMP to the I/R group resulted in a highly significant increase in cellular injury/death, which was prevented by inhalation of CO (Fig. 2)
.
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CONCLUSIONS AND SIGNIFICANCE
The current study provides the first direct in vivo evidence of the ability of inhaled CO to improve hepatic microvascular dysfunction, inflammation, and hepatocellular injury/death after remote injury (Fig. 3
). CO alone appears to be the active by-product of HO activity in restoring hepatic perfusion; both products likely play a role in limiting hepatocellular injury. Inhalation of a low dose of CO (250 ppm) is able to alter sinusoidal accumulation of leukocytes while decreasing the adhesion of leukocytes within the postsinusoidal venules after hind limb I/R. These findings provide evidence to suggest low-dose inhaled CO may represent a novel therapeutic approach to preserve hepatic integrity following SIRS produced by remote trauma.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2514fje;
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