Spatio-temporal properties of 5-lipoxygenase expression and activation in the brain after focal cerebral ischemia in rats
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).
Section snippets
Measurements of physiological variables
A total of 196 male Sprague-Dawley rats weighing 250–300 g (Experimental Animal Center, Zhejiang Academy of Medicine Sciences) were used in this study. Animals were housed under a controlled temperature (22 ± 1 °C), 12 h light/dark cycle and allowed free access to food and water. All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
Rats were anesthetized with an intraperitoneal injection of chloral hydrate
Physiological variables
Mean arterial blood pressure, arterial blood pH, PaO2, PaCO2 and glucose were not changed before and after the surgery, and there were no differences between sham operation and ischemia groups. In all the ischemic rats, rCBF of the MCA territory was reduced by approximately 50–60% during 30-min MCAO, and recovered to nearly baseline levels 15 min after reperfusion (Table 1).
Ischemic injury
Neurological deficit scores gradually increased from 3 h, reached the maximum at 24 h, thereafter decreased and
Discussion
In the present study, we found that 5-LOX is spatio-temporally expressed and activated in rat brain during 14 days of reperfusion after transient focal cerebral ischemia. The evidence is that both 5-LOX expression (mRNA and protein) and enzymatic activity were increased and spatio-temporally related to post-ischemic neuron injury (in acute phase) and astrocyte proliferation (in late phase). Therefore, our findings confirm the involvement of 5-LOX in cerebral ischemic injury as reported in
Conclusion
5-LOX expression (mRNA and protein) and enzymatic activity are increased during 14 days after transient focal cerebral ischemia, and spatio-temporally related to post-ischemic neuron injury in the ischemic core (in acute phase) and astrocyte proliferation in the boundary zone (in late phase). Our findings suggest that 5-LOX may act as a modulator in the brain injuries after cerebral ischemia, and contribute to the investigation of pathophysiology and development of therapeutics for ischemic
Acknowledgments
This study was supported by grants from the National Natural Science Foundation of China (No. 30371637) and the Scientific Foundation of Education Ministry of China (20050335105).
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