Consistent with a damaging role for increased LOX

Consistent with a damaging role for increased 12/15-LOX activity in stroke, systemic administration of the 12/15-LOX inhibitor, LB1, 2 h after permanent focal cerebral ischemia significantly reduced infarct size 24 h after the ischemia, and the treatment group demonstrated improved behavioral and health parameters. Finally, we investigated the effects of a combination therapy of lipoxygenase inhibitor with tPA in FeCl3-induced transient ischemia-reperfusion model. LB1 treatment applied 4 h after ischemia onset which was followed by thrombolysis with tPA, significantly decreased infarct volume and hemorrhage area accompanying with better NSS results and reduced weight loss. It might demonstrate that 12/15-LOX inhibition provides benefits when coadministered with tPA after stroke. Yigitkanli et al. also supports our results with the data made 2 h after ischemia onset in filament-induced proximal MCAO model (Yigitkanli et al., 2013). It was previously shown that application of intraperitoneal LB1 treatment either 2, 4 or 6 h after filament-induced focal cerebral ischemia decreased infarct volume (Yigitkanli et al., 2013), and our results here suggest a similar time window for this distal model. In this study it was shown that lipoxygenase activity contributes to the stroke pathophysiology and inhibition of this activity decreases infarct volume in acute distal MCA ischemia model. This study and previous studies also showed that lipoxygenase inhibitors are safe against hemorrhage after stroke when administered alone or even with tPA treatment (Liu et al., 2017, Yigitkanli et al., 2013). This safe time window is 4 h after stroke yet we did not investigate the effects beyond this time window. In addition, inhibition of 12/15-LOX activity strengthens the effect of tPA in accordance with decreased infarct volume and better NSS results. This effect might result from the decrease in the blood-brain barrier permeability through application of the 12/15 LOX inhibitor after stroke (Jin et al., 2008). Recently it was shown that not only tPA-induced hemorrhage, but also warfarin-associated hemorrhagic Actinomycin D synthesis was significantly reduced using a novel lipoxygenase inhibitor with and without tPA administration (Liu et al., 2017). These results show that 12/15-LOX inhibitors may be a good option to prevent thrombolysis-induced side effects of acute stroke treatment. Also these inhibitors might be used in human acute stroke patients without tPA in the ambulance as it is safe against hemorrhage and effectively decreases infarct volume. It is noteworthy to mention the limitations of this study like tPA plus LB1 treatment beyond 4 h after stroke was not investigated. Especially 6 or 8 h after stroke could be studied in future projects for translational purposes. Second drawback might be that we performed the experiments in different strains of mice within different stroke models. In permanent MCAO model, C57BL6 mice were used and in transient ischemia/ thrombolysis MCAO groups, CD1 mice were used. Here our purpose was to show the applicability of FeCl3-induced MCA thrombosis model in different strains, so that different stroke laboratories would prefer this model in their experiments due to the fact that this model is easy to learn, cheap and it also results in consistent infarct volume (Karatas et al., 2011). In addition, permanent and transient ischemia models would be feasible to use with different concentrations of FeCl3 application.
Experimental procedures
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Acknowledgement
Introduction The lipoxygenase (LOX) superfamily is abundant in plants, fungi, and animals, and catalyzes the formation of a single specific hydroperoxide derivative from polyunsaturated fatty acids [1]. The nomenclature of LOXs is based on the substrate carbon where oxygenation is catalyzed, and largely the chain length of common substrates determines the specificity of the enzyme. Whereas in plants 18-carbon fatty acids (such as linoleate and linolenate) are the dominant LOX substrates, in animals arachidonate (20-carbon fatty acid) is more common, corresponding to plant 13-LOX and animal 15-LOXs, respectively. Mammalian LOXs oxygenate arachidonic acid to hydroperoxyeicosatetraenoic acids (HPETEs) that are subsequently reduced to their corresponding hydroxyeicosatetraenoic acids (HETEs).