Furthermore we found up regulated protein and mRNA levels

Furthermore, we found up-regulated protein and mRNA levels of adenosine kinase in mouse retina with TON. The up-regulation of ADK was previously demonstrated in diabetic retinopathy (Pang et al., 2010). Greater levels of ADK in activated macrophages and microglial cells abrogate the immune response. Earlier research showed that adenosine level dramatically increase in extracellular in ischemic condition (Hagberg et al., 1987), and also in the rat model of transient ischemia the concentration of adenosine in the cerebrospinal fluid was increased four-fold (Meno et al., 1991). Previous reports have shown that a beneficial role of elevated adenosine in ischemic condition, and the increased adenosine accumulation appears to be protective in Quinacrine hydrochloride hydrate cell injury (Phillis et al., 1991, Tatlisumak et al., 1998). However, adenosine kinase influences adenosine reuptake by converting into AMP. During ischemic or traumatic condition cells need more adenosine but other side adenosine kinase inhibits adenosine production. In current study ABT-702 significantly reduced the adenosine kinase protein and mRNA expression. Thus, the inhibition of adenosine kinase seems beneficial for the adenosine signaling. Our results are in agreement with others where adenosine kinase inhibition with selective ADK inhibitors showed increased adenosine level in brain cells and retinal inflammation (White, 1996, Tatlisumak et al., 1998, Elsherbiny et al., 2013). In addition, increased ENT1 was previously demonstrated in human aortic smooth muscle cells by hyperglycemia (Leung et al., 2005). Similar to this study, we found increased ENT1 protein and mRNA levels in mouse retina with TON (Fig. 3). AKI treatment attenuated its expression level in retinal tissue. ENT1 transports adenosine from intercellular to extracellular and vice versa. Previously it was reported that ENT1 expression was increased in high glucose (Liou, 2010), which may affect the availability of adenosine for its receptor to work as anti-inflammatory in diabetes. Thus, we may say that in TON ENT1 up-regulation is pathologically implicated and causes low concentration of adenosine by reuptake intercellular, but its low expression may be beneficial in TON. This result is supported by our recent work where we have shown that ABT-702 markedly decreased ENT1 expression in diabetic retinopathy (Elsherbiny et al., 2013). Further, we investigated the effect of ABT-702 treatment on TON induced oxidative stress. We previously demonstrated increased oxidative stress in mice retina with TON (Ahmad et al., 2013). Here, we studied the effect of ABT-702 treatment on superoxide anion, iNOS/nNOS and nitrotyrosine levels in mice retina with TON. Nitration of tyrosine residues is evident in several retinal inflammatory and neurodegenerative diseases (Gouder et al., 2004), which occurs due to reaction of tyrosine with reactive nitrogen species such as peroxynitrite (Pacher et al., 2007). These reactive nitrogen species are formed by reaction of superoxide anion and Nitric oxide (NO), thus, serving as a likely indicator simultaneous generation of NO and superoxide (El-Remessy et al., 2003). However, It is reported that retinal ganglion cell loss during retinal hypoxia regulated by NO (Kaur et al., 2006). Under pathological conditions, NO is synthesized by the inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). Studies reported the expression of nNOS and iNOS in glial cells, infiltrating leukocytes and in RGCs in hypoxic retina (Kashiwagi et al., 2003, Kaur et al., 2006). The produced NO from nNOS and iNOS contributes to neurotoxicity resulting in cell death and axonal damage (Kaur et al., 2008). Report suggests that NO triggered several pathways including N-methyl-d-aspartate (NMDA)-mediated intracellular Ca influx and CREB-mediated apoptotic proteins which results to neuronal death (Mishra et al., 2002). Increased NO production is shown to mediate MAPKinase activation during hypoxia in cerebral cortical nuclei of newborn piglets (Mishra et al., 2004). In the current study, we found increased levels of iNOS/nNOS expression in TON retinal tissue, and increased superoxide anion and nitrotyrosine levels in mouse retinal sections with TON. Treatment with ABT702 attenuated TON- induced oxidative and nitrative stress in mouse retina with TON. These findings suggested that ABT 702 attenuated TON induced activated microglia production of NO and reactive oxygen species by augmenting adenosine signaling.