Measurement of neutrophil content in brain and lung tissue by a modified myeloperoxidase assay.

Abstract

Myeloperoxidase (MPO) activity is assessed for the quantification of neutrophil accumulation in tissues. In particular, it may be used to support in vivo data on leukocyte kinetics obtained by intravital microscopy and to clarify whether phenomena observed on the organ surface reflect the situation of the whole organ microcirculation. Previous measurements of MPO activity were limited by interference with other peroxidases and by inhibition of MPO activity by specific enzymes. To circumvent these limitations, a modified assay was devised that combined a two-step tissue homogenization technique with heat incubation in a continuous photometric measurement. MPO activity was quantified in neutrophils isolated from rat and rabbit whole blood, rat brain and rabbit lung and compared with intravital microscopic data on leukocyte accumulation. The modified assay is characterized by high reproducibility, strong correlation of MPO activity with number of neutrophils and full recovery of neutrophils added to tissue homogenate. MPO activity per neutrophil was 342.9 +/- 11.7 mU/10(6) cells in rats and 40.3 +/- 0.8 mU/10(6) cells in rabbits. MPO activity in tissue was significantly lower in rat brains (18.9 +/- 29.7 mU/g) as compared to rabbit lungs (741 +/- 67 mU/g). Whereas global cerebral ischemia/reperfusion did not increase MPO activity in rat brain (18.1 +/- 26.1 mU/g), intravenous infusion of cobra venom factor (1,447 +/- 407 mU/g) or endotoxin (1,439 +/- 285 mU/g), enhanced MPO activity in rabbit lung. These results parallel microcirculatory data from the organ surface. Therefore they supplement the intravital microscopic observations by demonstrating that these are indeed representative of deeper parenchymal tissue areas.