Purpose Ischemia reperfusion injury (IR) is characterized by the excessive adherence of activated neutrophils to the endothelial wall, leading to tissue necrosis and death. Hyperbaric Oxygen (HBO) ameliorates the effects of IR injury by decreasing the amount of adherent neutrophils during reperfusion. Previous studies have shown that NOS inhibitors and NO scavengers both reverse the effects of HBO and that vascular endothelial growth factor (VEGF) induces angiogenesis via a pathway that increases NOS activity. They have also shown that while HBO treatment upregulates VEGF mRNA, it does not upregulate NOS mRNA. The purpose of this experiment is to determine whether HBO inhibits neutrophil adhesion via a NOS dependent mechanism that is concurrent with the actions of VEGF by evaluating the effect of anti-VEGF (a monoclonal antibody) on CD18/11b polarization and adhesion of neutrophils.
Methods The right gracilis muscle flap was raised in 4 groups of male Wistar rats. (1) SHAM (n=5), (2) IR alone (n=5), (3) IR+HBO (n=3) (4) IR+HBO+Anti VEGF (n=5). The femoral vessels were clamped to induce ischemia. IR was characterized by 4 hours of ischemia followed by 15 minutes of reperfusion. HBO was administered during the last 90 minutes of ischemia. Anti-rat VEGF was infused 30 minutes before HBO treatment. Following IR, blood was drawn from the gracilis flap pedicle vein to obtain activated plasma. ADHESION ASSAY: Normal neutrophils and activated plasma were exposed to an ICAM coated coverslip and incubated at room temperature. The number of adherent neutrophils was counted via light microscopy and the percent adherence was calculated. POLARIZATION ASSAY: Normal neutrophils, activated plasma and s-ICAM were incubated. The cells were labeled with FITC Anti CD11b and viewed via confocal microscopy. The first 20 single cells from each reaction were photographed and the cell surface was analyzed as three equal areas. Polarization was defined as one area of the cell membrane having fluorescent intensity two times the sum of the intensities of the remaining two areas.
Results Neutrophil adherence and CD18/11b polarization were significantly increased in the IR group. HBO treatment significantly decreased adhesion and polarization, while Anti-VEGF infusion significantly reverses the HBO effect.
Conclusion Infusion of Anti-VEGF in IR+HBO treated animals appears to increase polarization of neutrophils suggesting that the beneficial HBO effect may be through a VEGF pathway or mechanism.