Arachidonate 5-lipoxygenase Modulates Traumatic Brain Injury And Neurite Outgrowth

5-Lipoxygenase is a key enzyme (EC1.13.11.34, 5-LOX) metabolizing arachidonic acid (AA) to form inflammatory mediators, leukotriene B₄ (LTB₄) and cysteinyl leukotrienes (CysLTs, including LTC₄, LTD₄ and LTE₄). Recent studies of 5-LOX in molacular biology and cell biology suggested that 5-LOX may also exert non-enzymatic effects as a constutive protein in the cells. Moreover, 5-LOX is widely expressed in the central nervous system and involved in multiple neurophysiological or neuropathological processes.Traumatic brain injury is a common cause for the mortality and disability in adults. Traumatic brain injury produces sequential consequences that can be divided into early and late (or chronic) phases. In the early phase, the main changes include excitotoxicity, oxidative stress, free radical production, apoptosis, inflammation and degeneration. In the late phase, the main changes include angiogenesis, neurogenesis, regeneration of neurocircuits, gliosis and glial scar wall formation. Some of the late changes (angiogenesis, neurogenesis and regeneration of neurocircuits) are benefit for brain reconstruction, but some others (gliosis and glial scar wall formation) are deleterious.In the present study, we focus on (1) the expression patterns of 5-LOX in human brain with traumatic brain injury; (2) the expression property of 5-LOX in mouse brain after cryoinjury; (3) the neuroprotective effects of 5-LOX inhibitor caffeic acid on mouse cryoinjury, especially in the late phase astrogliosis and glial scar formation; (4) effects of 5-LOX in nerve growth factor (NGF)-induced PC12 cell neurite outgrowth. Our purposes are elucidating the possible modulating roles of 5-LOX inearly and late phase of traumatic brain injury and neural regeneration, and exploring its possible neurophysiological implications.Part 1Expression patterns of 5-lipoxygenase in human brain with traumaticinjury and astrocytomaIt has been reported that 5-LOX is widely expressed in the brain. The levels of 5-LOX metabolites, leukotrienes, are increased after brain injury and tumors, and 5-LOX inhibition provides neuroprotective effects. However, there is still no information available for the expression patterns of 5-LOX in human brain with trauma and astrocytoma. Thus, we investigated its expression patterns by immunohistochemistry. We found that 5-LOX was normally expressed in the neurons and glial cells. In the neurons, it was expressed in two patterns: both in the cytosol and nucleus or only in the cytosol. In traumatic brain injury, 5-LOX expression was increased in the glial cells and the neutrophils. Double-labeling immunohistochemistry showed that part of the 5-LOX positive glial cells were GFAP positive astrocytes. 5-LOX was not expressed in microvessel endothelia in the relatively normal brain, but highly expressed in the regenerated endothelia of a patient 8-days following brain trauma. Furthermore, 5-LOX expression was increased and showed a granular pattern in high-grade (grade III/TV) astrocytoma. These results indicate that 5-LOX has multiple expression patterns, and can be induced by brain injury, which imply that 5-LOX might play pathophysiological roles in the human brain.Part 2Expression of 5-lipoxygenase and protective effects of its inhibitorcaffeic acid in mouse brain with cryoinjuryTraumatic brain injury induces neuron damage in early phase, and astrogliosis and the formation of the glial scar in late phase. Recent studies have shown that 5-LOX may take part in the processes in brain injury and neurodegeneration. 5-LOX inhibitor caffeic acid (3, 4-dihydroxycinnamic acid), one of the natural phenolic compounds, exerts neuroprotective effects on ischemic brain injuries. In this part, to elucidate the possible roles of 5-LOX and the effect of caffeic acid on traumatic brain injury, we induced cryoinjury, one of traumatic brain injury, in mouse brain. We found that 5-LOX was expressed in neurons and the activated astrocytes 14 day after cryoinjury. 5-LOX inhibitor caffeic acid (10 and 50 mg/kg, i.p., for 7 days after cryoinjury) reduced the lesion area and attenuated the neuron loss around the lesion core 1 to 28 days; however, it attenuated the neuron loss in the lesion core only 1 day after cryoinjury. Moreover, caffeic acid attenuated astrocyte proliferation, glial scar wall formation and GFAP protein expression in the late phase of cryoinjury (7 to 28 days). Caffeic acid also inhibited the reduced superoxide dismutase activity and the increased malondialdehyde content in the brain 1 day after cryoinjury, indicating its anti-oxidant ability. These findings suggesting that 5-LOX is involved in the pathophysiological processes in the early and late phases of cryoinjury. Caffeic acid may represent a new prototype compound of potential neuroprotective agents in the treatment of early and late traumatic brain injuries.Part 35-Lipoxygenase is involved in nerve growth factor-induced PC12 cellneurite outgrowthUnlike axons in the peripheral nervous system the axons are unable to regenerate spontaneously in the adult central nervous system (CNS). Developing strategies topromote axonal regeneration is therapeutically attractive for various CNS diseases such as traumatic brain injury, stroke and Alzheimer's disease. Cell biology researches for 5-LOX have shown that 5-LOX can interact with several key proteins in growth factor receptor signal pathways, and 5-LOX may exert non-enzymatic effects in cells as a constitutive protein. In this part, to elucidate the possible of 5-LOX in neuronal differentiation, we induced neurite outgrowth by NGF in a neural cell line, PC12 cells. We transfected 5-LOX gene into PC12 cells by stable transfection and monocloning technique, to prepare the cells stably expressing 5-LOX-GFP fusion protein or GFP (named as PC12-5-LOX or PC12-GFP cells). The percentage of NGF-induced neurite-positive cells was significantly higher in PC12-5-LOX-GFP cells than in wide type PC12 and PC12-GFP cells. 5-LOX protein expression was down-regulated about 40% in wide type PC12 cells treated with 5-LOX antisense oligonucleic acid. This down-regulation decreased NGF-induced neurite-positive cell percentage. However, lipoxygenase inhibitor NDGA, 5-LOX inhibitor caffeic acid, 12-LOX inihibitor baicalein and CysLT₁ receptor antagonist pranlukast did not attenuate NGF-induced neurite outgrowth. These results indicate that 5-LOX is involved in NGF-induced neurite outgrowth, which might be mediated through a 5-LOX non-enzymatic signal pathway.Summary1. 5-LOX is expressed in the relatively normal human brain, in traumatic brain, and in astrocytoma in different patterns. 5-LOX may be responsible for the damages in the early stages injury and in the reconstruction in the late stages of traumatic brain as well as in the tumorgenesis of astrocytoma,.2. 5-LOX is expressed in mouse brain after cryoinjury, and closely relates to the late astrocyte proliferation around the lesion 14 days after cryoinjury. 5-LOX inhibitor caffeic acid attenuates the lesion and neuron loss after cryoinjury, and especially blocks the activation of astrocytes resulting in attenuation of their proliferation and glial scar formation in the late phase of cryoinjury in mice. 5-LOX modulates cryoinjury in the early and late phases, and 5-LOX inhibitors are potential therapeutic agents for the treatments of brain injury.3. In the PC12 cells stably expressing 5-LOX-GFP, over-expression of 5-LOX facilitates NGF-induced neurite outgrowth, but down-regulation of 5-LOX expression attenuates NGF-induced neurite outgrowth. Moreover, 5-LOX may modulate neurite outgrowth through a non-enzymatic signal pathway.