br Conclusion br Acknowledgement This

Conclusion
Acknowledgement This study was supported by NIH/NINDSR01 NS036812-16.
Introduction Stroke is the leading cause of disability and the fifth leading cause of death in the United States (Writing Group et al., 2016). On average, every 4 min a human dies of stroke (Lackland et al., 2014, Mozaffarian etal., 2016). Together with cardiovascular diseases, its economic burden is higher than the other diagnostic groups including cancer. The annual direct and indirect cost of cardiovascular diseases and stroke is an estimated $316.6 billion in the United States (Writing Group et al., 2016). Despite these catastrophic effects of stroke, the only FDA-approved drug treatment option for acute stroke is the application of tissue plasminogen activator (tPA) which has many drawbacks including the narrow time window, low rate of reachable patients, and severe side effects including hemorrhage (Kaur et al., 2004, Lees et al., 2010, National Institute of Neurological Disorders and stroke rt-PA Stroke Study Group, 1995, Wang et al., 2004, Yepes et al., 2009). New treatment options are thus of great interest in stroke management, and screening for novel drugs in animal models is an important drug development approach. Besides STAIR advices to use different animal stroke models both permanent and transient and also with reperfusion and with thrombolysis (Fisher et al., 2009). The great majority of ischemic strokes are due to an occlusion in a cerebral artery by a thrombus especially in the middle cerebral artery (MCA) in human (Hossmann, 2012). So it is feasible to choose a model that mimics human stroke like FeCl3-induced MCA occlusion model to test novel drugs (Denorme et al., 2016). Stroke damages the neurovascular unit, causes massive cell death and activates several oxidative stress-related pathways like lipid peroxidation, during its acute phase (Hardingham and Lipton, 2011, Lo et al., 2003, Lo et al., 2005, Moskowitz et al., 2010, Niizuma et al., 2009). Lipoxygenases play one of the major roles in stroke related oxidative stress. Especially 12/15-lipoxygenase, the dominant isoform in the brain, is increased in neurons and endothelial ikk inhibitor in the peri-infarct area, contributing to delayed cell death in the penumbra, weakening of the blood–brain barrier, and resulting in edema formation (Jin et al., 2008, van Leyen et al., 1998, van Leyen et al., 2006, van Leyen et al., 2014). Therefore, lipoxygenase inhibitors are in scope of acute stroke treatment research, and among them LOX Block-1 (LB-1) is a new candidate considering its potential (Yigitkanli et al., 2013). The current study was designed to investigate the effects of the 12/15-LOX inhibitor, LOXBlock-1 (LB1) in mice using a FeCl3-induced distal MCAO model, and to test its utility in conjunction with subsequent thrombolysis with tPA, which is to date the only FDA-approved drug for acute stroke treatment. This study will help us to improve our knowledge about the effect of LB-1 in different models of stroke other than previously used reperfusion models.
Results
Discussion Within this study, as proven using the immunohistochemical methods, we have demonstrated that lipoxygenase activation has a vital contribution in the pathophysiology of ischemia in the FeCl3-induced distal MCAO model. Previously, Khanna et al. (Khanna et al., 2005, Park et al., 2011) and our group have reported the involvement of 12/15-LOX in proximal MCAO models (Jin et al., 2008, van Leyen et al., 2006, Yigitkanli et al., 2013). However, considering a distal MCAO model, this is the first report demonstrating an increased LOX immunoreactivity in the ischemic cortex and in the peri-infarct area. This immunoreactivity was colocalized with an oxidative stress marker, malondialdehyde (MDA2) (Fig. 1A–C). Malondialdehyde is a breakdown product formed by the oxidation of arachidonic acid. Increased LOX immunoreactivity was also coincidental with the AIF immunopositivity in the peri-infarct area (Fig. 1J–L). AIF is known to be increased following ischemia (Zhao et al., 2004), and the staining pattern closely resembles what we have found in our previously published data, where 12/15-LOX and AIF were both increased in the peri-infarct region (Pallast et al., 2010). This immunoreactivity was also found in human stroke patients in the peri-infarct area (Yigitkanli et al., 2013).