Hyperbaric Oxygen Inhibits Ischemia-reperfusion–induced Neutrophil Cd18 Polarization by a Nitric Oxide Mechanism

Ischemia reperfusion (IR) injury can be a serious consequence of diseases and procedures where blood flow is interrupted, including myocardial infarction, cerebral ischemic stroke, transplantation, replantation, and free tissue transfer. Restoration of blood flow to ischemic tissue initiates the inflammatory cascade which increases the injury. The prevention, diagnosis and treatment of IR injury are important in order to improve survival and function of affected tissues. Hyperbaric oxygen (HBO) treatment in myocardial infarction,1,2 ischemic stroke 3 and skeletal muscle ischemia 4 have shown a positive effect on tissue survival, however, the absence of an elaborated mechanism of action has hindered wide acceptance of HBO treatment for IR injury. Ischemia and reperfusion lead to injury of the microvasculature resulting in cell damage and death.5,6 Early efforts to explain this injury, based theories on the deleterious effects of oxygen free radicals produced by xanthine oxidase.7,8 Later experiments demonstrated the pathophysiologic role of free radicals produced from activated neutrophils.9,10 A morphologic analysis of skeletal muscle during IR showed an increase in the number of rolling and adherent neutrophils to the endothelium of the postcapillary venules compared with nonischemic controls.6 These adherent neutrophils release free radicals that damage the endothelium and provide a nidus for platelet aggregation. Attachment of the neutrophil to venular endothelium is dependent upon the CD18 protein complex. This complex needs to be functionally present for neutrophil attachment to the intercellular adhesion molecule-1 (ICAM-1) found on endothelial cells.11 Larson, et. al.12 reported that neutrophils exposed to IR tissue show an up-regulation of CD18 protein on the neutrophil surface. 12 More importantly, IR induced CD18 molecule aggregates become polar by concentrating the surface protein to one area of the neutrophil making the cell more apt to attach to the venular endothelium.13 It has been reported that clustering of the adhesion molecules is closely associated with firm adhesion of leukocytes through the surface membrane CD18 integrin.14–16 Hyperbaric oxygen (HBO) treatment has been shown to decrease edema and necrosis in ischemic skeletal muscle.17–19 With microcirculation studies, researchers found a reduction in the number of leukocytes adherent to venular endothelium in groups treated with HBO during and immediately following the ischemic event.6 Further studies showed that HBO treatment prevented the neutrophil CD18 surface protein polarization and, therefore, the neutrophil attachment to the venular endothelium.20 Important molecules in cell signal transduction during IR include nitric oxide synthase (NOS), cGMP, and guanyl cyclase. Nitric oxide (NO) has been shown to decrease neutrophil-endothelial adhesion and improve survival rate when L-Arginine (L-Arg), a NO precursor, was infused into ischemic rectus femoris and gracilis muscles.21 Conversely, when ischemic rectus femoris was infused with L-nitro-aminomethylamine (L-NAME), a nitric oxide synthase inhibitor, the percent necrosis was unchanged from ischemic conditions.21,22 Studies by Elayan23 showed an increase in NO formation in the brain with HBO treatment. Hyperbaric oxygen has also been shown to decrease PMN cGMP24, and may interfere with guanylate cyclase interference of CD18 function.25 Nitric oxide, cGMP and guanylate cyclase have all been implicated in cytoskeleton rearrangement.24–26 Integrating the progression of the HBO and NO studies led to the purpose of this study which is to evaluate the role of nitric oxide in HBO mediated inhibition of CD18 surface polarity, and neutrophil adhesion, in vitro. The in vitro polarization and adhesion assays, with activated plasma from in vivo HBO treated animals, were used with and without carboxy-PTIO, a NO scavenger, to evaluate the role of nitric oxide in HBO mediated inhibition of CD18 surface polarity and neutrophil adhesion. Nitric Oxide synthase inhibitors (L-NAME, 7-NI, 1400W), were infused into HBO treated animals to assess the involvement of NOS isoforms in the HBO effect on CD18 polarization and adhesion.