Spatio-temporal properties of 5-lipoxygenase expression and activation in the brain after focal cerebral ischemia in rats

Abstract

The role of 5-lipoxygenase (5-LOX) in brain injury after cerebral ischemia has been reported; however, the spatio-temporal properties of 5-LOX expression and the enzymatic activation are unclear. To determine these properties, we observed post-ischemic 5-LOX changes from 3 h to 14 days after reperfusion in rats with transient focal cerebral ischemia induced by 30 min of middle cerebral artery occlusion. We found that the expression of 5-LOX, both mRNA and protein, was increased in the ischemic core 12–24 h after reperfusion, and in the boundary zone adjacent to the ischemic core 7–14 days after reperfusion. The increased 5-LOX was primarily localized in the neurons in the ischemic core at 24 h, but in the proliferated astrocytes in the boundary zone 14 days after reperfusion. As 5-LOX metabolites, the level of cysteinyl-leukotrienes in the ischemic brain was substantially increased 3 h to 24 h, near control at 3 days, and moderately increased again 7 days after reperfusion; whereas the level of LTB₄ was increased mildly 3 h but substantially 7–14 days after reperfusion. Thus, we conclude that 5-LOX expression and the enzymatic activity are increased after focal cerebral ischemia, and spatio-temporally involved in neuron injury in the acute phase and astrocyte proliferation in the late phase.

Introduction

5-Lipoxygenase (5-LOX, EC 1.13.11.34) is a key enzyme metabolizing arachidonic acid to produce leukotriene B₄ (LTB₄) and cysteinyl-leukotrienes (CysLTs, including LTC₄, LTD₄ and LTE₄) (Samuelsson et al., 1987, Funk, 2001). The importance of 5-LOX in stroke has been proven by the reports indicating that the gene encoding 5-LOX activating protein (ALOX5AP) confers risk of stroke (Helgadottir et al., 2004, Helgadottir et al., 2005, Lohmussaar et al., 2005). 5-LOX expression is increased and leukotriene contents are elevated in the ischemic brain (Ohtsuki et al., 1995, Ciceri et al., 2001, Shishido et al., 2001, Tomimoto et al., 2002, Zhang et al., 2003), indicating a role of 5-LOX in cerebral ischemia. However, as discordant evidence, no difference in ischemic infarcts has been found between 5-LOX-deficient and wild type mice with focal cerebral ischemia (Kitagawa et al., 2004). Therefore, the involvement of 5-LOX needs to be further investigated.

Ischemic brain injury can be separated into 3 serial phases: metabolic stress and excitotoxicity (acute, minutes to hours), inflammation and apoptosis (subacute, hours to days), and repair and regeneration (chronic, days to months) (Dirnagl et al., 2003, Fagan et al., 2004). Neuron injury, including necrosis and apoptosis, is the main lesion in the acute or subacute phase; the formation of a glial scar due to reactive gliosis (astrogliosis, mainly consisting of proliferated astrocytes) is one chronic change (Fawcett and Asher, 1999, Silver and Miller, 2004). Gliosis in the infarct boundary (Persson et al., 1989) may be a physico-biochemical barrier to the regeneration of axons (Fawcett and Asher, 1999). Whether 5-LOX plays a role in acute and subacute/chronic phases is still unclear.

We have found that 5-LOX was activated after oxygen-glucose deprivation (OGD)-induced in vitro ischemic injury in PC12 cells (Song et al., 2004) and cultured rat cortical neurons (Ge et al., 2006). Moreover, 5-LOX inhibitors attenuate arachidonic acid-induced in vitro neuron death in cortical neuron culture (Kwon et al., 2005) and OGD-induced neuron death in hippocampal slice culture (Arai et al., 2001). On the other hand, 5-LOX facilitates the growth of some tumor cells (Ye et al., 2004, Hoque et al., 2005) and proliferation of pulmonary artery endothelial cells (Walker et al., 2002). 5-LOX metabolites, CysLTs, also induce the proliferation of epithelial cells, fibroblasts, and smooth muscle cells (Baud et al., 1987, Leikauf et al., 1990, Brink et al., 2003). CysLTs also increase astrocyte proliferation by the stimulating CysLT₁ receptor (one of the two cloned cysteinyl leukotriene receptors), which may contribute to the gliosis (Ciccarelli et al., 2004). A CysLT₁ receptor antagonist, pranlukast, inhibits glial scar formation in mouse brain 10 weeks after focal cerebral ischemia (Yu et al., 2005a). From these findings, we hypothesize that 5-LOX may be involved in neuron injury and astrocyte proliferation after cerebral ischemia. To test this hypothesis, in the present study we investigated the spatio-temporal properties of 5-LOX expression and enzymatic activation as well as post-ischemic injury for a period of 14 days after ischemia/reperfusion. We defined 3–24 h after reperfusion as acute phase, and 3–14 days as late phase (including subacute and chronic phases).