br Material and methods br Results br Discussion

Material and methods
Results
Discussion To develop a novel regenerative and/or neuroprotective therapy for optic neuropathy, including glaucoma, numerous studies have tested new candidates favorable to RGC survival and axon regeneration. Among the many candidate drugs, AdoR modulators are of special interest because of their neuroprotective and IOP reduction effects in animal and human eyes (Crosson, 1995; Avila et al., 2001, 2002; Oku et al., 2004; Avni et al., 2010; Galvao et al., 2015; Madeira et al., 2016). This study showed that AdoR modification is also able to contribute to RGC axon regeneration, which may be used for the treatment of neurodegenerative diseases. First, the distribution of AdoR in the sensory retina showed that most AdoRs were located in the inner retina, including the RGC layers, in accordance with previous in situ hybridization studies, which showed that A1, A2, and A3 receptor mRNAs were present in the inner retinal layers (Kvanta et al., 1997; Zhang et al., 2006a). Our results showed immunoreactivity in the inner retinal layers of all AdoR types tested (A1, A2A, A2B and A3 receptors), which was consistent with the aforementioned mRNA studies. Taken together, these results suggest that adenosine may induce biological effects, including in RGCs, through activation of the identified AdoRs in the inner retina. Second, the outgrowth of RGC neurites was determined, to identify the AdoR subtype responsible for the induction of neurite elongation effects by specific agonists for each AdoR. The results showed that among the tested agonists, only 2-Cl-IB-MECA, an A3 receptor agonist, was able to cause significant neurite outgrowth of cultured RGCs in a dose-dependent manner. Next, we evaluated the neurite-promoting effects of the A3 receptor agonist in an in vivo glaucoma model. Our results showed that the number of regenerating Cy7 NHS ester (non-sulfonated) was significantly increased in eyes treated with 2-Cl-IB-MECA compared to control eyes at >500 μm from the optic nerve crush site. The axon-regenerating effects of 2-Cl-IB-MECA may be comparable with other drug-induced axon promotion by the ROCK or Caspase 8 inhibitors, which are well-known axon-regenerating candidates (Lingor et al., 2007; Monnier et al., 2011). Previous studies have reported adenosine-induced positive neurite outgrowth in goldfish RGCs (Benowitz et al., 1998). Adenosine also enhanced the effects of nerve growth factor-induced neurite outgrowth in PC12 cells (Muroi et al., 2004). However, no study has determined whether adenosine induces outgrowth in mammalian RGCs, and no report has determined which AdoR subtype is important in neurite outgrowth. A2A receptor stimulation induced positive neurite outgrowth in cerebral cortical neurons (Ribeiro et al., 2016), and the A2B receptor interacted with netrin-1 to promote positive neurite outgrowth in dorsal spinal cord axons (Corset et al., 2000). However, to the best our knowledge, there has been no report on whether AdoR modulation causes neurite outgrowth in RGCs. This study is the first to show that the A3 receptor is a specific receptor for the neurite-inducing effects of adenosine-treated RGCs. We also measured cAMP levels, because adenosine induces activation of AC as a second messenger, which in turn synthesizes cAMP and activates the PKA/CREB pathway (Montminy and Bilezikjian, 1987; Kim et al., 2002). During the embryonic stage, cAMP promotes neurite outgrowth in the central nervous system, and cAMP levels have been reported to decrease after birth (Cai et al., 2001). It has also been reported that intravitreal injection of dibutyryl-cAMP, a cAMP analogue, promotes neural regeneration in the optic nerve of mammals (Qiu et al., 2002; Monsul et al., 2004). In addition, soluble AC was shown to promote neurite outgrowth in RGCs via activation of the cAMP signaling pathway (Corredor et al., 2012). However, our results showed that 2-Cl-IB-MECA decreased cAMP levels in cultured RGCs. Phosphorylation of CREB, the cAMP downstream signaling component, also decreased after treatment with 2-Cl-IB-MECA. Thus, the neurite outgrowth-inducing activities of the A3 receptor agonist may be independent of the cAMP-PKA/CREB pathway.