TIME-RELATED CHANGES IN MYELOPEROXIDASE ACTIVITY AND LEUKOTRIENE B-4 RECEPTOR-BINDING REFLECT LEUKOCYTE INFLUX IN CEREBRAL FOCAL STROKE

In previous studies, we have used histological methods to characterize cellular changes, and validated the use of the myeloperoxidase (MPO) activity assay to quantitate increased neutrophil infiltration in isch emic stroke. We also identified increased leukotriene B-4 (LTB(4)) bin ding sites as a potential marker for neutrophil infiltration into foca l ischemic tissue. However, these studies were conducted at only one t ime-point, 24 h after ischemia. In the present study, we examined the full time-course of MPO activity and LTB(4) receptor binding following middle cerebral artery occlusion (MCAO) made permanently (PMCAO) or t ransiently (160 min followed by reperfusion; TMCAO) in spontaneously h ypertensive rats, and compared the results to previously characterized histologic changes in these models. Ischemic and contralateral (contr ol) cortical tissue samples were assayed for MPO (U/g wet wt) and [H-3 ]LTB(4) receptor binding (fmol/mg protein). Following PMCAO, MPO activ ity significantly increased as early as 12 h and continued to increase over the next 5 d (p < 0.05). Following TMCAO, MPO activity was signi ficantly elevated already after only 6 h of reperfusion and also conti nued to increase over the next 5 d of reperfusion (p < 0.05). LTB(4) r eceptor binding and MPO activity were highly correlated during periods when both measures increased together (i.e., 0.5-5 d; p < 0.01). Howe ver, by 15 d post-MCAO, LTB(4) receptor binding remained elevated afte r MPO activity levels had returned to normal. This persistent LTB(4) b inding was associated with the significant gliosis that was characteri zed previously to persist in these models. The time-course of increase d MPO activity and initially increased LTB(4) binding post-MCAO corres pond very well to our previous histological data that characterized th e time-course for leukocyte infiltration under these conditions. There fore, the increased MPO activity over time was associated with initial neutrophil and later mononuclear cell infiltration into ischemic tiss ue in these models. In addition, the present studies utilized histoche mical analysis to demonstrate peroxidase activity in macrophages withi n the cerebral infarct following MCAO, thus validating that MPO activi ty originates from the later infiltrating mononuclear cells in additio n to the early infiltrating neutrophils that had been previously chara cterized in the same manner. TMCAO produces a significantly larger and earlier increase in ischemic cortex MPO activity and a similar later increase in MPO activity compared to PMCAO treatment. Clearly, reperfu sion of cerebral tissue following ischemia greatly exacerbates the deg ree of cerebral tissue inflammation. These biochemical assays, especia lly the MPO activity assay, have now been validated for quantitating t he early and late phases of the cerebral inflammatory reaction to tiss ue injury.