ICH induced striatal lesion produced a reduction
ICH-induced striatal lesion produced a reduction of EAAT1 expression analogous to the decreased glutamate uptake at 6 h. The combined reduction of excitatory amino 235 6 transporters and of glutamate uptake activity might explain the well-known glutamate excitotoxicity following brain ischemia (Rao et al., 1998; van Landeghem et al., 2006; Yi and Hazell, 2006). Although the relationship between glial glutamate transporters and their clearance capacity in the pathogenesis of ICH is only now being described, this is the first demonstration of ICH-induced changes in glutamate transporters. Interestingly, it was recently shown that thrombin, an important contributor of hemorrhagic stroke, causes a decrease in the expression of EAAT1 and compromises glutamate uptake (Piao et al., 2015). Additionally, thrombin might potentiate N-methyl-d-aspartate (NMDA) receptor function and enhance glutamate release under ischemic-like conditions, aggravating the risk of excitotoxicity and oxidative damage (Chen et al., 2013; Gingrich et al., 2000; Vázquez-Juárez et al., 2009). However, in present study, at least acutely (6 h post-ICH), reduced ROS production was observed accompanied by the increase of GPx activity and GSH content. GPx catalyzes the reduction of hydroperoxides using GSH, and glutathione metabolism is an essential antioxidant defense against oxidative damage (Matés et al., 1999). Distinct to 6 h post-injury time, 24 h after ICH there was an increase of ROS production and lipid peroxidation demonstrating an increased oxidative stress. Experimental studies have indicated that thrombin formation, red blood cell lysis and iron toxicity are the main contributors in ICH-induced oxidative damage (Duan et al., 2016; Hua et al., 2007; Wang et al., 2002; Wu et al., 2002). Nonetheless, under excitotoxic conditions, excessive release of glutamate into the extracellular space is followed by an overstimulation and pronounced activation of NMDA receptors. This cause a massive calcium influx and a subsequent rise in the production of ROS (Chen et al., 2013; Gingrich et al., 2000; Ha and Park, 2006). Overproduction of ROS subsequent to NMDA receptor stimulation leads to reduced ability of astroglial cells to regulate glutamate turnover via inhibition of GS activity (Chen et al., 2013; Eid et al., 2012; Fernandes et al., 2011; Ha and Park, 2006). In fact, our data demonstrate a decrease of GS activity 24 h after ICH, corroborating previous data. Enzymatic antioxidant catalase exerts its defense through the decomposition of H2O2 in H2O and O2. Here, however, the increased catalase activity was not sufficient to overcome the high oxidative stress generated after collagenase-induced ICH. 72hs after ICH, EAAT2 expression decline was accompanied by the massive increase of oxidative stress as well as by the intensification of the antioxidative defense system (SOD, CAT, GPx and GSH). This decreased EAAT2 expression may represent an increased vulnerability to oxidative stress. Previous studies suggest that glutamate transporters are vulnerable to reactive oxygen species, resulting in reduced uptake and neuronal damage (Rao et al., 2003; Trotti et al., 1998), and oxidative stress plays an important role in the secondary brain injury after ICH (Aronowski and Zhao, 2011). The increased response of SOD, CAT, GPx and GSH substantiate the direct evidence of the peaked increase of oxidative damage produced at this time point. However, to the best of our knowledge, this is the first study showing this temporal dynamic of glial excitatory amino acid transporters and the oxidative profile during ICH progression. Surprisingly, 7 days after ICH-induced striatal lesion there was a decrease of EAAT1 content and an increase of EAAT2 expression and glutamate uptake. The differential expression of EAAT1 and EAAT2 at 7 days suggest an interplay between them to better deal with glutamate clearance. Moreover, different modulators are thought to act differently in both, EAAT1 and EAAT2, favoring the distinguishing pattern of expression observed in this study. For example, the coexpression of glycogen synthase kinase 3 beta (GSK3β) with EAAT2 results in the up-regulation of the transporter activity, whereas under the same experimental conditions EAAT1 is down-regulated (Jiménez et al., 2014). Similarly, TNF-α treatment induced a decrease in EAAT1 and an increase in EAAT2 content in cortical astroglial cultures (Dumont et al., 2014). The increased EAAT2 expression linked to the increased glutamate uptake represent a compensatory response in an attempt to restore the astrocytic function of uptake of the excess of glutamate after ICH. Additionally, at 7 days, ICH continued to produce ROS, as well as induce an inhibition of GS activity. The increased ROS production, despite of minor intensification, may have been responsible of inhibit GS activity, since GS is sensitive to oxidative stress, as previously discussed (Fernandes et al., 2011).