Time-related changes in myeloperoxidase activity and leukotriene B4 receptor binding reflect leukocyte influx in cerebral focal stroke

Abstract

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 B4 (LTB4) 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 LTB4 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 [3H]LTB4 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). LTB4 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, LTB4 receptor binding remained elevated after MPO activity levels had returned to normal. This persistent LTB4 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 LTB4 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Inflammatory Cell Infiltration after Endothelin-1-Induced Cerebral Ischemia: Histochemical and Myeloperoxidase Correlation with Temporal Changes in Brain Injury

“…From the results obtained, using both immunohistochemistry and the biochemical assay, we observed that neutrophil infiltration increased over time with the progression of the ischemic injury, which is in accordance with previous findings but with different methods of producing focal ischemia (Barone et al, 1995;Feuerstein et al, 1998;Matsuo et al, 1994). Results from the biochemical assay in the present study indicated that MPO activity was 20% lower than values obtained by Barone et al (1991), suggesting that the type of model used to induce cerebral ischemia may partially determine the inflammatory response observed in the rats (Beray-Berthat et al, 2003b;Emerich et al, 2002;Hayward et al, 1996;Macrae, 1992).…”

“…The increase and peak in MPO activity at 5 days seen by Barone et al (1995) has previously been demonstrated histologically to be the result of a dramatic increase in monocyte and macrophage accumulation within the ischemic brain, and corresponds to a time when neutrophils were reported to be decreasing (Clark et al, 1993(Clark et al, , 1994. Barone et al (1995) unequivocally demonstrated that macrophages are a source of MPO, and suggested that macrophages may have acquired additional MPO activity through phagocytized neutrophils in the tissue or alternatively, that MPO is available after the lysis of neutrophils. It is likely that both of these events are occurring, however, the first event is most likely the predominant mechanism, with macrophages having been shown previously to engulf apoptotic neutrophils in the periphery and can also induce neutrophil apoptosis before phagocytizing them.…”

“…Similar correlations can be made using MPO, between neutrophils and volume of damage, this however is only true over the first 3 days after cerebral ischemia (unpublished observations). Previous studies have demonstrated that 3 days after ischemia, MPO activity increases dramatically, peaking at 5 days, before decreasing to preischemic values by 15 days (Barone et al, 1995). The peak in MPO activity at 5 days, because of monocyte-macrophage accumulation (Feuerstein et al, 1998), and the relationship between MPO and infarct volume is no longer maintained, and cannot be related to neutrophil accumulation.…”

Immune Cell Infiltration in Malignant Middle Cerebral Artery Infarction: Comparison with Transient Cerebral Ischemia

“…Previous studies of experimental stroke in rats using histochemical approaches identified neutrophils to be one of the earliest cell types infiltrating the ischemic brain, contributing to inflammation as early as 12 hours after pMCAO. 19 Furthermore, pharmacological inhibition (at 1.5 hours) or genetic deletion of neutrophil elastase was reported to reduce infarct volume in mice after tMCAO but not pMCAO, suggesting that reperfusion may be necessary to allow neutrophils to access and elicit damage to vulnerable ischemic tissue. 20 However, here our comprehensive analyses of whole ischemic hemispheres utilising flow cytometry have revealed that neutrophils are B3-fold as numerous in the ischemic hemisphere at 24 hours after pMCAO compared with 1 or 2 hours of tMCAO, despite a comparable infarct volume after 2-hour tMCAO and the pMCAO models.…”

Interrelation between inflammation, thrombosis, and neuroprotection in cerebral ischemia

“…Leukocytes and, after a few hours post-injury, microglia secrete proinfl ammatory cytokines and chemokines, the severity of which play detrimental roles in the pathophysiology of stroke (Morganti -Kossmann et al, 2001 ;Wang et al , 2007 ;Suzuki et al , 2009 ). Signifi cant leukocyte infl ux into cerebral parenchyma and tissue remodeling are characteristics of cerebral ischemia/reperfusion (Barone et al , 1995 ;Wang et al , 2007 ). Interestingly, infarct volume has been shown to be reduced signifi cantly through the inhibition of neutrophil infi ltration, as it is evident that neutrophils wield the most damage to ischemic lesions once reperfusion is undertaken (Connolly et al , 1996 ;Guha and Mackman , 2001 ;Wang et al , 2007 ).…”