Abstract
Purinergic signaling regulates important physiological processes and the homeostatic response to stress in the cochlea via extracellular nucleosides (adenosine) and nucleotides (ATP, UTP). Using a previously established organotypic culture model, the current study investigated the effect of purinergic P1 (adenosine) and P2 (ATP) receptor activation on the survival of the sensory hair cell population in the cochlea exposed to the ototoxic aminoglycoside neomycin. Organ of Corti explants were obtained from C57BL/6 mice at postnatal day 3 (P3) and maintained in normal culture medium (with or without purine receptor agonists or analogs) for 19.5 h prior to neomycin exposure (1 mM, 3 h) followed by a further incubation for 19.5 h in culture medium. The cochlear explants were then fixed in 4% paraformaldehyde (PFA) and sensory hair cells labeled with Alexa 488-phalloidin. Neomycin induced a substantial loss of the sensory hair cells, mostly in the middle segment of the cochlea. This neomycin-induced ototoxicity was unaffected by the addition of P2 receptor agonists (ATP and UTP) in the culture medium, whilst the addition of their slowly-hydrolyzable analogs (ATPγS, UTPγS) aggravated neomycin-induced sensory hair cell loss. In contrast, the activation of P1 receptors by adenosine or adenosine amine congener (ADAC) conferred partial protection from neomycin ototoxicity. This study demonstrates a pro-survival effect of P1 receptor stimulation, whilst prolonged activation of P2 receptors has an opposite effect. Based on these findings, we postulate that P1 and P2 receptors orchestrate differential responses to cochlear injury and that the balance of these receptors is important for maintaining cochlear homeostasis following ototoxic injury.
Highlights
Aminoglycoside antibiotics are used in treatment of tuberculosis and serious Gram-negative bacterial infections such as bacterial endocarditis, urinary tract infections, and pneumonia (Rybak and Ramkumar, 2007)
Inner hair cells (IHCs) exposed to neomycin demonstrated significantly lower survival rates in the middle (47.1 ± 8.8%, p = 0.023) and the basal (45.6 ± 6.6%, p = 0.017) segment compared to the apical segment (81.0 ± 8.8%, Figure 1C), resulting in ‘Lshaped’ cochleogram
Neomycin-Texas Red conjugate (NTR) fluorescence was significantly reduced in both IHC and outer hair cells (OHCs) with the addition of ATPγS to the culture medium (p < 0.05, oneway ANOVA, Figure 4B). These findings demonstrate that the addition of ATPγS to the culture medium did not increase NTR uptake in the cochlear hair cells, but instead significantly reduced NTR uptake in the basal segment IHC and OHC (Figure 4B)
Summary
Aminoglycoside antibiotics are used in treatment of tuberculosis and serious Gram-negative bacterial infections such as bacterial endocarditis, urinary tract infections, and pneumonia (Rybak and Ramkumar, 2007). A common side-effect associated with aminoglycoside use in humans is damage to the sensory hair cells of the inner ear leading to their death and a significant loss of Purinergic Signaling and Aminoglycoside Ototoxicity hearing and vestibular function (Schacht et al, 2012). The formation of an aminoglycoside-iron complex can react with electron donors, such as arachidonic acid, to form reactive oxygen species (ROS) (Poirrier et al, 2010). The superoxide, hydroxyl radical, and hydrogen peroxide in turn activate stressactivated protein kinase JNK, which translocates to the nucleus to activate downstream genes involved in the cell death pathways (Poirrier et al, 2010; Karasawa and Steyger, 2011). As the mammalian auditory hair cells do not regenerate, their loss and the resulting hearing loss are permanent
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