Expression And Functional Modulations Of 5-lipoxygenase After Ischemic Brain Injury

5-Lipoxygenase (5-LOX, EC 1.13.11.34) is a key enzyme metabolizing arachidonic acid to produce leukotrienes, including leukotriene B₄ (LTB₄) and cysteinyl leukotrienes (CysLTs, namely LTC₄, LTD₄ and LTE₄). Leukotrienes are potent inflammatory mediators, and they can induce smooth muscle contraction, microvascular leakage and other pathophysiological changes. 5-LOX was initially found in the peripheral organs, thus earlier studies have focused on their roles in the peripheral diseases, such as asthma and allergic rhinitis. Recently, 5-LOX has been reported to be also expressed in the brain, and may be involved in various central nervous diseases, such as cerebral ischemia, trauma, tumor and epilepsy. After cerebral ischemia, 5-LOX expression is increased and the contents of its metabolites, leukotrienes, are elevated in the ischemic brain. Moreover, the inhibitors of 5-LOX, such as AA861, MK-886 and nordihydroguaiaretic acid (NDGA), exert protective effects against ischemic brain injury, indicating the involvement of 5-LOX in cerebral ischemia. The importance of 5-LOX in stroke has been proven by the reports that the gene encoding 5-LOX activating protein (ALOX5AP) confers risk of stroke. However,as discrepant evidence, no difference in ischemic infarcts has been found between 5-LOX-deficient and wild type mice with focal cerebral ischemia. 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). Neuron injury, including necrosis and apoptosis, is the main lesion in the acute or subacute phase; while the formation of a glial scar due to reactive gliosis (astrogliosis, mainly consisted of proliferated astrocytes) is one chronic change. Whether 5-LOX plays a role in acute, subacute and chronic phases is still unclear and needs further investigation.To further investigate the role of 5-LOX in cerebral ischemia, in the present study, we at first determined the spatio-temporal properties of 5-LOX expression and enzymatic activation as well as post-ischemic injury during 14 days after focal cerebral ischemia induced by middle cerebral ischemia occlusion (MCAO) in Sprague-Dawley (SD) rats. 3-24 h after reperfusion was defined as early phase (including acute and subacute phases), and 3-14 days as late phase (including subacute and chronic phases). Then, the involvement of 5-LOX in acute neuronal damage and late astrocyte proliferation were observed respectively, after focal cerebral ischemia in vivo induced by MCAO in SD rats using 5-LOX inhibitor caffeic acid. Moreover, the role of 5-LOX was observed in oxygen-glucose deprivation (OGD)-induced in vitro ischemic-like injury using 5-LOX inhibitors in a neural cell line, PC12 cells.Part I Spatio-temporal properties of 5-LOX expression and enzymatic activation in the brain after focal cerebral ischemia in ratsAIM: To determine the spatio-temporal properties of 5-LOX expression andenzymatic activation in the brain after focal cerebral ischemia. METHODS: 5-LOX changes were observed from 3 h to 14 days after reperfusion in rats with transient focal cerebral ischemia induced by 30-min MCAO. 5-LOX expression was detected by RT-PCR and Western blot analyses. 5-LOX localization was visualized by immunohistochemistry and double immunofluorescence. The levels of CysLTs and LTB4 in brain tissue were measured by enzyme immunoassay. RESULTS: (1) The expressions of 5-LOX mRNA and protein were increased in the ischemic core 12-24 h after reperfusion; the expressions were also increased in the boundary zone adjacent to the ischemic core 7-14 days after reperfusion. (2) In the ischemic core, 5-LOX-positive cells were significantly increased from 12 h to 3 days after reperfusion with the maximum at 24 h; the increased 5-LOX was primarily localized in the neurons. In the boundary zone, 5-LOX-positive cells were not increased within 24 h, but gradually increased from 3 to 14 days after reperfusion; the increased 5-LOX was primarily localized in the proliferated astrocytes. (3) As 5-LOX metabolites, the level of CysLTs in the ischemic brain was largely increased 3 h to 24 h, returned 3 days, and moderately increased again 7 days after reperfusion; whereas the level of LTB4 was increased mildly 3 h but largely 7-14 days after reperfusion. CONCLUSION: (1) The expressions of 5-LOX mRNA and protein are increased during 14 days after transient focal cerebral ischemia, and 5-LOX spatio-temporally relates to post-ischemic neuron injury in the ischemic core (in early phase) and astrocyte proliferation in the boundary zone (in late phase). (2) The level of CysLTs is abruptly increased at 3 h and maintained up to 24 h, but shows only a relatively smaller peak at 7 days after reperfusion. In contrast, LTB4 level is mildly increased at 3 h in the acute phase, but largely increased 7 and 14 days in the late phase.Part II Roles of 5-LOX in ischemic neuronal injuryAIM: According to the result of Part I, the increased 5-LOX was primarily localized in the neurons in the ischemic core 24 h after brain ischemia. To further determine whether 5-LOX is involved in the early ischemic injury, we investigatedthe effects of 5-LOX inhibitors on the in vivo injury induced by focal cerebral ischemia in rats, and on in vitro ischemia-like injury in neural cell line, PC12 cells. METHODS: Focal cerebral ischemia was induced by 30-min MCAO and 24-h reperfusion in rats. Caffeic acid (10 and 50 mg/kg) was intraperitoneally injected 30 min before MCAO and 0, 1, 2 h after reperfusion. On the other hand, the effect of 5-LOX inhibitors, caffeic acid and zileuton, was observed on PC12 cell injury induced by 2-h OGD and 24-h reperfusion. MTT reduction assay was used to evaluate PC12 cell viability; Hoechst 33258/PI staining was used to detect the cell death. RESULTS: (1) MCAO for 30 min and reperfusion for 24 h induced neurological dysfunction, brain edema, neuron death in the ischemia core and brain infarct. Caffeic acid (50 mg/kg) attenuated neurological dysfunction, increased the density of neurons in the ischemic core, and reduced brain infarct volume 24 h after ischemia. In addition, it reduced the production of leukotrienes (5-LOX metabolites) in the ischemic hemispheres 3 h after ischemia. (2) MTT assay showed that 2-h OGD and 24-h reperfusion decreased the viability of PC12 cells. Hoechst 33258/PI staining indicated that in OGD/reperfusion-induced death of PC12 cells, about 40% was apoptotic, while about 13% was necrotic. Caffeic acid (0.01, 0.1, 1μM) increased the viability of PC12 cells; the apoptotic cells were reduced to 20% and necrotic cells to 3%. Zileuton (0.1, 1, 10, 100μM) also increased the viability of PC12 cells; the apoptotic cells were reduced to 15% and necrotic cells to 3%. CONCLUSION: 5-LOX inhibitor caffeic acid protects the neurons against the injury 24 after focal cerebral ischemia in rats. 5-LOX inhibitors (caffeic acid and zileuton) protect PC12 cells against OGD-induced ischemic-like injury. These results indicated that 5-LOX is involved in the neuronal injury after cerebral ischemia.Part III Roles of 5-LOX in astrocyte proliferationAIM: According to the result of Part I, increased 5-LOX was primarily localized in the proliferated astrocytes in the boundary zone 14 d after brain ischemia. To further determine whether 5-LOX is involved in the astrocyte proliferation afterfocal cerebral ischemia, we observed the effect of 5-LOX inhibitor caffeic acid on the astrocyte proliferation in the late phase in rats. METHODS: Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 30 min followed by reperfusion for 14 d. Caffeic acid (10 and 50 mg/kg) was intraperitoneally injected 30 min before MCAO and 0, 1, 2 h after reperfusion in the first day, and twice daily in the 2nd to 5th day. After 14-day reperfusion, we observed the delayed brain injury, especially the astrocyte proliferation in the boundary zone. RESULTS: After 30-min MCAO and 14-d reperfusion, in the ischemic core, the infarct tissue and brain atrophy were found. In the boundary zone, the number of GFAP-positive astrocytes was greatly increased and the intense astrocytic fibers surrounded the ischemic core. Caffeic acid (50 mg/kg) decreased infarct volume, attenuated brain atrophy, and inhibited the astrocyte proliferation in the boundary zone. In addition, it reduced the production of leukotrienes (5-LOX metabolites) in the ischemic hemispheres 7 d after brain ischemia. CONCLUSION: 5-LOX inhibitor caffeic acid protects the brain tissue against the delayed brain injury and inhibited the astrocyte proliferation in the boundary zone after focal cerebral ischemia in rats, indicating that 5-LOX is involved in the astrocyte proliferations after cerebral ischemia.