br Materials and methods br Results br Discussion This study

Materials and methods
Results
Discussion This study demonstrates that treatment with a selective adenosine kinase inhibitor ABT-702, thereby enhancing extracellular adenosine, can provide partial protection from age-related hearing loss in C57BL/6J mice. Chronic treatment with ABT-702, commencing at the age of 3months or 6months, improved auditory thresholds, suprathreshold responses and hair cell survival in ageing (9-month-old) mice. This implies that ABT-702 enhances the survival of both sensory hair PAC-1 mg and spiral ganglion neurones in the ageing mouse cochlea. The treatment appears to be more effective in the apical, lower frequency range, compared with the more basal high frequencies. Given there was no difference in the distribution of ADK along the cochlea, this difference in response may indicate differential injury processes along the cochlea, or perhaps differences in the distribution of adenosine receptors or ABT-702. Oxidative stress may be a prevalent mechanism causing the age-related degeneration in the low-mid frequency region. However, structural abnormalities of hair cell tip-links, which facilitate the opening of hair cell mechanical transduction channels, could be the principal mechanism leading to hair cell degeneration in the high-frequency region, which would render this region less susceptible to manipulation of adenosine receptor signalling. Potential base-to-apex gradients in the distribution of adenosine receptors may also result in differential sensitivity to ADK inhibition in the high- and low-frequency regions of the cochlea. It should also be noted that with systemic drug administration, differential drug access to the cochlea and the distribution within the cochlea can be expected (Sha et al., 2001). Age-related hearing loss (ARHL) is the most common sensory deficit in senescence, and yet the prevention of ARHL is a relatively novel research field (Bielefeld et al., 2010). To date, several strategies have been proposed to mitigate ARHL: increasing the anti-oxidant defence system (Finkel and Holbrook, 2000), caloric restriction (Sweet et al., 1988, Seidman, 2000, Someya et al., 2010), induction of heat shock proteins (Mikuriya et al., 2008) and electrical stimulation to restore the endocochlear potential (Schmiedt, 1993). The cochlear anti-oxidant system can be strengthened endogenously in the enhanced acoustic environment (Tanaka et al., 2009) or exogenously using a variety of anti-oxidant treatments such as vitamin C and E (Seidman et al., 2000) and lecithin (Seidman et al., 2002). In addition, animals with genetic deletion of anti-oxidant enzymes such as superoxide dismutase 1 and 2 (SOD1, SOD2) or glutathione peroxidase show accelerated ARHL (McFadden et al., 1999, Ohlemiller et al., 2000b, Keithley et al., 2005). Caloric restriction (CR) delays the onset of many age-related diseases in animal models, and it was postulated that the delayed ARHL could be a consequence of the prolonged lifespan (Bielefeld et al., 2010). It is more likely, however, that CR suppresses apoptotic cell death in the ageing mammalian cochlea by down-regulating stress-related intracellular signalling pathways and up-regulating molecular pathways essential for mitochondrial function and DNA repair (Someya et al., 2007). Based on the demonstrated tight inverse correlation of ADK expression levels and the ambient tone of adenosine (Li et al., 2008, Pignataro et al., 2007, Pignataro et al., 2008, Theofilas et al., 2011) we postulate that increased endogenous adenosine levels in cochlear fluids resulting from ADK inhibition leads to the better preservation of the sensory cells and delayed onset of hearing loss in the mid-frequency region. It is not clear from this study whether treatment with ABT-702 has any off-target effects, and how these effects could contribute to the improvement of hearing thresholds observed in this study. As for the role of adenosine kinase in tissue protection, previous studies have demonstrated that ADK may have a pivotal role in the brain response to injury (Boison, 2006, Boison, 2008). Transient down-regulation of ADK after acute brain injury protects the brain from further seizures and cell death, whilst up-regulation of ADK is associated with epileptogenesis and neuronal cell loss (Li et al., 2008). Regulation of adenosine levels by ADK may also have a key role in brain protection from ischemic injury (Pignataro et al., 2008). Pharmacological inhibition of ADK is effective therapeutically with an improved side-effect profile compared with A1 receptor agonists (Kowaluk and Jarvis, 2000, Jarvis et al., 2002, Gouder et al., 2004). ABT-702 is a new generation ADK inhibitor (McGaraughty et al., 2005) lacking the off-target effects of classical ADK inhibitors such as 5′-amino-5′-deoxyadenosine or 5′-deoxy-5-iodotubercidin. ABT-702 was shown to readily cross the blood brain barrier and brain levels of ABT-702 are approximately one third of plasma levels (Suzuki et al., 2001). This orally active drug has an exceptional potency in thermal hyperalgesia (Jarvis et al., 2000, Lee et al., 2001) and neuropathic pain (Suzuki et al., 2001). In our study, no toxic effects or overt behavioural changes were associated with chronic administration of ABT-702 over the period of 3–6months. Body weight of mice increased evenly in ABT-702-treated and control groups during the measurement period.