br Materials and methods br Results br

Materials and methods
Results
Discussion According to the literature, zaprinast is a high-affinity agonist of GPR35 (Berlinguer-Palmini et al., 2013, Divorty et al., 2015, Taniguchi et al., 2006, Zhao et al., 2010). GPR35 Meleagrin has been identified within discrete regions of the nervous system, including the spinal cord and DRG (Alkondon et al., 2015, Wang et al., 2006, Ohshiro et al., 2008). GPR35 has been shown to be expressed in a subpopulation of small-medium sensory neurons but also in larger-sized neurons that convey non-nociceptive information (Ohshiro et al., 2008). Importantly, in light of our research, GPR35 is also expressed on glia (Alkondon et al., 2015). The activation of GPR35 by zaprinast, even in nanomolar concentrations, may successfully decrease glutamate release and the extracellular concentration in the central nerve system (Carpenedo et al., 2001, Cosi et al., 2011). Recently, Resta et al. (2016) have shown that zaprinast treatment leads to reduced excitability of small DRG neurons and causes dose-dependent analgesia in a phosphodiesterase 2-induced hypersensitivity model. Importantly, zaprinast is also known as a relatively potent phosphodiesterase inhibitor and is most commonly reported as an inhibitor of phosphodiesterase 5 and phosphodiesterase 6 (Beavo, 1995, Loughney et al., 1998), as well as phosphodiesterase 1, phosphodiesterase 9, phosphodiesterase 10 and phosphodiesterase 11 (Fawcett et al., 2000, Fisher et al., 1998, Fujishige et al., 1999, Nakamizo et al., 2003, Yuasa et al., 2000). Pentoxifylline/propentofylline, a non-specific phosphodiesterase inhibitor, has been shown to diminish hypersensitivity (Mika et al., 2007, Sweitzer et al., 2001, Raghavendra et al., 2004) and post-operative pain in patients (Szczepanik et al., 2004, Wordliczek et al., 2000). Our study provides the first evidence that even single intrathecal administration of zaprinast induces an antinociceptive effect, which is quite unique under conditions of neuropathy. Moreover, the effect of zaprinast is most pronounced when the hypersensitivity symptoms are fully developed, on day 7–14 in our model, at the same time point that microglia cell activation is at its strongest (Rojewska et al., 2014a, Rojewska et al., 2014b). There is a rapidly growing body of evidence indicating that signalling from microglia plays a role in the pathogenesis of neuropathy (Mika et al., 2013; Wen et al., 2011; Zhuo et al., 2011). Interestingly, repeated intrathecal administration of zaprinast reduced spinal microglia activation evoked by nerve injury. Importantly, the data obtained from animal studies revealed that substances that diminished microglial activation, such as fluoxetine (Zychowska et al., 2015), gabapentin (Staal et al., 2017), SB203580 (Jin et al., 2003; Tsuda et al., 2004), FR167653 (Wen et al., 2007), CNI-1493 (Milligan et al., 2003), pentoxifylline (Mika et al., 2007) and minocycline (Mika et al., 2007), prevented neuropathic pain development. In agreement with our data, the research conducted by Pifarré et al. (2010) indicates that zaprinast also diminishes microglia activation after traumatic brain injury. However, our data show that zaprinast enhanced astrocyte activity. The role of astrocytes in neuropathy remains unclear, and the data suggest that astrocytes have both pronociceptive (Garrison et al., 1991, Ji et al., 2014, Hashizum et al., 2000, Gwak and Hulsebosch, 2009) and antinociceptive (Milligan and Watkins, 2009, Hansen and Malcangio, 2013) properties. Many studies suggest a protective role of astrocytes in different pathologies (Faulkner et al., 2004, Chen et al., 2001). In light of the latest literature, these seemingly contradictory results are not surprising; distinct astrocytic phenotypes may develop sequentially during different phases of a pathological process: in the first phase, reactive astrocytes may produce proinflammatory factors; in the second phase, astrocytes promote antiinflammatory and neuroregenerative functions (Pekny et al., 2016, Zamanian et al., 2012, Ponath et al., 2018). We believe that zaprinast may promote antinociceptive and neuroregenerative function of astrocytes; however, this hypothesis needs to be studied in the future.