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 ischemic stroke. We also identified increased leukotriene B-4 (LTB(4)) binding sites as a potential marker for neutrophil infiltration into focal ischemic tissue. However, these studies were conducted at only one time-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 transiently (160 min followed by reperfusion; TMCAO) in spontaneously hypertensive rats, and compared the results to previously characterized histologic changes in these models. Ischemic and contralateral (control) cortical tissue samples were assayed for MPO (U/g wet wt) and [H-3]LTB(4) receptor binding (fmol/mg protein). Following PMCAO, MPO activity significantly increased as early as 12 h and continued to increase over the next 5 d (p < 0.05). Following TMCAO, MPO activity was significantly elevated already after only 6 h of reperfusion and also continued to increase over the next 5 d of reperfusion (p < 0.05). LTB(4) receptor binding and MPO activity were highly correlated during periods when both measures increased together (i.e., 0.5-5 d; p < 0.01). However, by 15 d post-MCAO, LTB(4) receptor binding remained elevated after MPO activity levels had returned to normal. This persistent LTB(4) binding was associated with the significant gliosis that was characterized previously to persist in these models. The time-course of increased MPO activity and initially increased LTB(4) binding post-MCAO correspond very well to our previous histological data that characterized the time-course for leukocyte infiltration under these conditions. Therefore, the increased MPO activity over time was associated with initial neutrophil and later mononuclear cell infiltration into ischemic tissue in these models. In addition, the present studies utilized histochemical analysis to demonstrate peroxidase activity in macrophages within the cerebral infarct following MCAO, thus validating that MPO activity originates from the later infiltrating mononuclear cells in addition to the early infiltrating neutrophils that had been previously characterized 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, reperfusion of cerebral tissue following ischemia greatly exacerbates the degree of cerebral tissue inflammation. These biochemical assays, especially the MPO activity assay, have now been validated for quantitating the early and late phases of the cerebral inflammatory reaction to tissue injury.