Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: Myeloperoxidase activity assay and histologic verification

Abstract

Two different techniques were utilized to identify the infiltration of polymorphonuclear leukocytes (PMN) into cerebral tissue following focal ischemia: histologic analysis and a modified myeloperoxidase (MPO) activity assay. Twenty-four hours after producing permanent cortical ischemia by occluding and severing the middle cerebral artery of male spontaneously hypertensive rats, contralateral hemiparalysis and sensory-motor deficits were observed due to cerebral infarction of the frontal and parietal cortex. In hematoxylin-and-eosin-stained histologic sections, PMN, predominantly neutrophils, were identified at various stages of diapedesis from deep cerebral and meningeal vessels at the periphery of the infarct, into brain parenchyma. When MPO activity in normal brain tissue was studied initially, it could not be demonstrated in normal tissues extracted from non-washed homogenates. However, if tissue was homogenized in phosphate buffer (i.e., washed), MPO activity was expressed upon extraction. Utilizing this modified assay, MPO activity was significantly increased only in the infarcted cortex compared to other normal areas of the brain. This was observed in non-perfused animals and after perfusion with isotonic saline to remove blood constituents from the vasculature prior to brain removal. The increased PMN infiltration and MPO activity were not observed in forebrain tissue of sham-operated control rats. Also, MPO activity was not increased in the ischemic cortex of MCAO rats perfused immediately after middle cerebral artery occlusion, indicating that blood was not trapped in the ischemic area. By using a leukocyte histochemical staining assay, activity of peroxidases was identified within vascular-adhering/infiltrating PMN in the infarcted cortex 24 hr after focal ischemia. An evaluation of several blood components indicated that increased MPO activity was selective for PMN. The observed increase of approximately 0.3 U MPO/g wet weight ischemic tissue vs. nonischemic cerebral tissues probably reflects the increased vascular adherance/infiltration of approximately 600,000 PMN/g wet weight infarcted cortex 24 hr after focal ischemia. This combined biochemical and histological study strongly suggests that PMN adhere within blood vessels and infiltrate into brain tissue injured by focal ischemia and that the associated inflammatory response might contribute to delayed progressive tissue damage in focal stroke. This modified MPO assay is a useful, quantitative index of PMN that can be utilized to elucidate the potential deleterious consequences of neutrophils infiltrating into the central nervous system after cerebral ischemia, trauma, or other pro-inflammatory stimuli.

Dual Functionality of Myeloperoxidase in Rotenone-Exposed Brain-Resident Immune Cells

“…MPO is mainly present in neutrophils, monocytes, and macrophages; thus, it has been used as a marker for infiltrated neutrophils at sites of injury. 49,50 Recently, MPO was reported to be present in brain-resident cells under pathological conditions. 18,23 The results of the present study using primary glial cell cultures strongly support the hypothesis that glial cells are among the MPO-expressing cells of the brain.…”

Preclinical Studies in the mdx Mouse Model of Duchenne Muscular Dystrophy with the Histone Deacetylase Inhibitor Givinostat

“…We therefore measured the MPO activity in muscles of mdx mice killed at the end of the treatment with different doses of givinostat, compared with control vehicle-treated mdx mice. MPO is an enzyme produced by neutrophils, monocytes and macro phages, and MPO activity can be used to quantitate the magnitude of inflammation associated with muscle degeneration in muscular dystrophies (18,19). Once again, a significant reduction of MPO was observed in muscles from mdx mice exposed to 5 and 10 mg/kg/d doses of givinostat ( Figure 6).…”

Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats through its anti-oxidative effect and modulation of NF-kappa B activation

“…Myeloperoxidase (MPO) activity is used to quantitatively assess leukocyte infiltration into ischemic tissue [23]. Briefly, brain tissues were homogenized in 1.5 ml of potassium phosphate buffer (PPB, 50 mM, pH 6).…”