| Part Ⅰ Localization of a2-adrenergic Receptors in Rat CochleaObjectives/Hypothesis:α2-adrenergic receptors (α2-ARs) are important components of the adrenergic receptors (ARs). Although it’s expression in cochlea has been demonstrated by some functional studies, such as pharmacological studies, using α2-ARs agonist and selective α2-AR antagonist, morphologic data about the existence of α2-ARs in the cochlea are absence. The present study investigated the localization of three subtypes:α2a-, α2b-α2c-ARs in the cochlea of rat in the early postnatal period.Methods:Characterization of α2-ARs subtypes expression in the post-natal day7(P7) rat cochlea was undertaken by immunohistochemisty staining from at least five animals. Polycolnal antibodies were applied for subtypes α2a-, α2c-ARs and monoclonal antibody for subtype α2b-AR to paraffin sections. For positive controls, immunoreactivity was determined in regions of rat kidney. Dilutions that provided optimal staining with the positive control tissues were used for incubating the cochlear sections.Results:We found that the expression of α2-ARs in cochlea was very extensive, including the organ of corti, spiral ganglion neurons (SGNs), spiral ligament, stria vassularis etc.. In the corti, hair cells, inner sulcus (epithelial) cells, outer sulcus cells and Deiters’s cells exhibited three α2-AR subtypes immunolabeling. The apical and basal poles of peri-nuclear compartment of hair cells were stained prominently and the intensity was invariable. Furthermore, the cellular locations of the α2-ARs between different receptors subtypes had diversity:α2a-receptor mainly targeted to nuclear, peri-nuclear compartment; The α2b-and α2c-receptors exhibited predominantly in plasma membrane and peri-nuclear compartment. Little difference of expression was observed among the three cochlear turns.Conclusions:Expression of αa2-ARs in SGNs may indicate that α2-ARs might modulate SGNs function and development. The widely expression of α2-ARs in supporting cells of the organ of corti indicated these cells could provide hair cells not only with mechanical support, but also with metabolic support. α2-ARs expressed in stria vascularis suggested that α2-ARs might modulate particular ion concentration gradients and generation of endolymph. Overall, the extensive expression of α2-ARs establishes a significant foundation for the exploration of the function of α2-ARs in cochlea. Part Ⅱ α2-adrenergic Receptors in Spiral Ganglion Neurons May Mediate Protective Effects of Brimonidine Against Glutamate and Hydrogen Peroxide ToxicityObjectives/Hypothesis:Brimonidine, a highly selective α2-adrenergic receptor agonist, is thought to be neuroprotective in some types of neurons via the activation of α2-adrenergic receptors (α2-ARs). Studies have shown that brimonidine can alleviate glutamate-induced neurotoxicity, prevent an ischemia-induced rise in extracellular glutamate, and it can completely abolish reactive oxygen species generation, attenuate cellular impairment induced by hydrogen peroxide (H2O2), thereby preventing cell from oxidative stress-induced apoptosis. It is well known that excitotoxicity injury and oxidative damage to spiral ganglion neurons (SGNs) are basic mechanisms for many types of sensorineural hearing disorders, so we hypothesize that brimonidine can protect cochlear SGNs from glutamate and H2O2injury, which may represent a novel strategy for the treatment of sensorineural hearing disorders. The prerequisite for this hypothesis is the existence of α2-ARs in SGNs. However, it is still unknown whether the α2-ARs exist in cochlear SGNs, and neuroprotection by brimonidine on SGNs insults has never been reported to date. The authors aimed to demonstrate the presence and localization of α2-ARs in rat cultured SGNs and to investigate the effects of brimonidine on glutamate-and H2O2-induced damage in the primary cultured rat SGNs. if so, the possible mechanisms underlying the actions of such stresses.Methods:Rats at age less than5postnatal days (majority at day3) were used to obtain cultured SGNs. Primary SGNs were identified by anti-NSE antibody. The expression of a2-ARs was determined by RT-PCR, Western blot analysis and immunofluorescence. The location of a2-ARs was also investigated by immunofluorescence. Then SGNs were exposed to glutamate or H2O2respectively with or without brimonidine. Cell viability was measured by MTT assay. Apoptosis was determined by acridine orange (AO) and Hoechst33342/propidium iodide (PI) double staining. To investigate the possible underlying mechanisms, the protein expressions of a2-ARs, Bax, Bcl-2, Caspase-9, Caspase-3, p-ERK1/2, iNOS, and artemin were determined by Western blot respectively.Results:we demonstrated that all three subtypes were expressed in SGNs. The trends of mRNA expressions apparently mismatch that of protein expressions of a2-ARs. Furthermore, the location of these three subtypes of α2-ARs in SGNs was obviously different as indicated by immunocytochemistry, i.e., a2a-receptor mainly exhibited in nuclear, intracellular, or peri-nuclear compartment, while, a2b-and a2c-AR predominantly localized in peri-nuclear compartment and plasma membrane. The cell viability was markedly reduced after exposure of glutamate (1mM) or H2O2(300μM) to SGNs:After completing24h of cultivation in the presence of glutamate (1mM) alone,76.45±2.76%of the SGNs survived compared to controls, In the presence of 300μM H2O2, cellular viability was reduced to62.84±5.16%compared to the control group. Our data also showed that300μM H2O2-induced apoptotic lesions were more prominent than the changes induced by1mM glutamate. AO fluorescence staining and Hoechst33342/PI staining showed significant morphological changes of SGNs after exposure to glutamate and H2O2: apoptotic cells with nuclear fragmentation, cytoplasmic contraction, chromatin condensation and apoptotic body were present in some cells. Treatment with brimonidine protected SGNs against glutamate-or H2O2-induced cell damage, enhanced SGNs survival:In the presence of1μM of brimonidine, cellular viability was92.97±3.43%(P<0.001); In the H2O2group, cellular viability was85.48±5.84%for brimonidine concentrations of1μM (P<0.001). Brimonidine alse decreased the elevation of Bax, Caspase-9, Caspase-3, p-ERK1/2, and artemin triggered by glutamate or H2O2(P<0.005), and altered the expressions of Bcl-2and iNOS(P<0.005). These protective effects of brimonidine can be reversed by yohimbine(P<0.005).Conclusions:Overall, the study describes the localization of a2-ARs in rat cultured SGNs and indicates that brimonidine, which may work directly via interaction with a2-ARs, attenuates glutamate-and H2O2-induced damage in SGNs by Caspase-dependent modes as well as Caspase-independent modes. Brimonidine can be potentially used for prevention or treatment of sensorineural hearing disorders. |
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