Salicylate Induced GABAAR Internalization by Dopamine D1-Like Receptors Involving Protein Kinase C (PKC) in Spiral Ganglion Neurons.

BACKGROUND Sodium salicylate (SS) induces excitotoxicity of spiral ganglion neurons (SGNs) by inhibiting the response of γ-aminobutyric acid type A receptors (GABAARs). Our previous studies have shown that SS can increase the internalization of GABAARs on SGNs, which involves dopamine D1-like receptors (D1Rs) and related signaling pathways. In this study, we aimed to explore the role of D1Rs and their downstream molecule protein kinase C (PKC) in the process of SS inhibiting GABAARs. MATERIAL AND METHODS The expression of D1Rs and GABARγ2 on rat cochlear SGNs cultured in vitro was tested by immunofluorescence. Then, the SGNs were exposed to SS, D1R agonist (SKF38393), D1R antagonist (SCH23390), clathrin/dynamin-mediated endocytosis inhibitor (dynasore), and PKC inhibitor (Bisindolylmaleimide I). Western blotting and whole-cell patch clamp technique were used to assess the changes of surface and total protein of GABARγ2 and GABA-activated currents. RESULTS Immunofluorescence showed that D1 receptors (DRD1) were expressed on SGNs. Data from western blotting showed that SS promoted the internalization of cell surface GABAARs, and activating D1Rs had the same result. Inhibiting D1Rs and PKC decreased the internalization of GABAARs. Meanwhile, the phosphorylation level of GABAARγ2 S327 affected by PKC was positively correlated with the degree of internalization of GABAARs. Moreover, whole-cell patch clamp recording showed that inhibition of D1Rs or co-inhibition of D1Rs and PKC attenuated the inhibitory effect of SS on GABA-activated currents. CONCLUSIONS D1Rs mediate the GABAAR internalization induced by SS via a PKC-dependent manner and participate in the excitotoxic process of SGNs.

Exposure to sodium salicylate disrupts VGLUT3 expression in cochlear inner hair cells and contributes to tinnitus.

To examine whether exposure to sodium salicylate disrupts expression of vesicular glutamate transporter 3 (VGLUT3) and whether the alteration in expression corresponds to increased risk for tinnitus. Rats were treated with saline (control) or sodium salicylate (treated) Rats were examined for tinnitus by monitoring gap-pre-pulse inhibition of the acoustic startle reflex (GPIAS). Auditory brainstem response (ABR) was applied to evaluate hearing function after treatment. Rats were sacrificed after injection to obtain the cochlea, cochlear nucleus (CN), and inferior colliculus (IC) for examination of VGLUT3 expression. No significant differences in hearing thresholds between groups were identified (p>0.05). Tinnitus in sodium salicylate-treated rats was confirmed by GPIAS. VGLUT3 encoded by solute carrier family 17 members 8 (SLC17a8) expression was significantly increased in inner hair cells (IHCs) of the cochlea in treated animals, compared with controls (p<0.01). No significant differences in VGLUT3 expression between groups were found for the cochlear nucleus (CN) or IC (p>0.05). Exposure to sodium salicylate may disrupt SLC17a8 expression in IHCs, leading to alterations that correspond to tinnitus in rats. However, the CN and IC are unaffected by exposure to sodium salicylate, suggesting that enhancement of VGLUT3 expression in IHCs may contribute to the pathogenesis of tinnitus.

Suppression of aspirin-mediated eosinophil activation by prostaglandin E2: Relevance to aspirin and nonsteroidal anti-inflammatory drug hypersensitivity.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by severe, sometimes life-threatening reactions to nonsteroidal anti-inflammatory drugs (NSAIDs). Mechanisms driving the disease include overproduction of leukotrienes and loss of anti-inflammatory prostaglandin E2 (PGE2) production. Many cell types contribute to the disease; however, eosinophils are markedly elevated and are important drivers of pathologic findings. OBJECTIVE: To investigate the capacity of aspirin and NSAIDs to drive eosinophil activation and the ability of PGE2 to inhibit this activation. METHODS: Eosinophils were purified from blood of healthy individuals without AERD and stimulated with lysine aspirin, ketorolac, or sodium salicylate. The role of PGE2 in altering activation was determined by incubating eosinophils with increasing doses of PGE2 before lysine aspirin stimulation. Specific PGE2 receptor use was determined by incubating eosinophils with receptor agonists and antagonists before aspirin stimulation. Cysteinyl leukotrienes (CysLTs), leukotriene B4 (LTB4), and eosinophil-derived neurotoxin (EDN) were quantified by enzyme-linked immunosorbent assay. RESULTS: Stimulation of eosinophils with lysine aspirin, ketorolac, or sodium salicylate resulted in secretion of CysLTs and LTB4 in the absence of EDN release. Low doses of PGE2 inhibited LTB4 and CysLT release, an effect lost at higher PGE2 concentrations. Use of butaprost, an EP2 receptor agonist, suppressed lysine aspirin stimulation. This mechanism was supported by blocking activity of the EP1 and EP3 receptors. CONCLUSION: Eosinophils can be directly activated by NSAIDs via cyclooxygenase-independent pathways to produce CysLTs and LTB4. This effect can be inhibited by PGE2 acting through the EP2 receptor. The recognized loss of EP2 receptor expression combined with low PGE2 levels explains in part the sensitivity to NSAIDs.

Sodium salicylate alters temporal integration measured through increasing stimulus presentation rates.

OBJECTIVE: High doses of sodium salicylate (SS) are known to induce tinnitus, general hyperexcitability in the central auditory system, and to cause mild hearing loss. We used the auditory brainstem response (ABR) to assess the effects of SS on auditory sensitivity and temporal processing in the auditory nerve and brainstem. ABRs were evoked using tone burst stimuli varying in frequency and intensity with presentation rates from 11/s to 81/s. DESIGN: ABRs were recorded and analysed prior to and after SS treatment in each animal, and peak 1 and peak 4 amplitudes and latencies were determined along with minimal response threshold. STUDY SAMPLE: Nine young adult CBA/CaJ mice were used in a longitudinal within-subject design. RESULTS: No measurable effects of presentation rate were found on ABR threshold prior to SS; however, following SS administration increasing stimulus rates lowered ABR thresholds by as much as 10 dB and compressed the peak amplitude by intensity level functions. CONCLUSIONS: These results suggest that SS alters temporal integration and compressive nonlinearity, and that varying the stimulus rate of the ABR may prove to be a useful diagnostic tool in the study of hearing disorders that involve hyperexcitability.

Effect of Long-Term Sodium Salicylate Administration on Learning, Memory, and Neurogenesis in the Rat Hippocampus.

Tinnitus is thought to be caused by damage to the auditory and nonauditory system due to exposure to loud noise, aging, or other etiologies. However, at present, the exact neurophysiological basis of chronic tinnitus remains unknown. To explore whether the function of the limbic system is disturbed in tinnitus, the hippocampus was selected, which plays a vital role in learning and memory. The hippocampal function was examined with a learning and memory procedure. For this purpose, sodium salicylate (NaSal) was used to create a rat animal model of tinnitus, evaluated with prepulse inhibition behavior (PPI). The acquisition and retrieval abilities of spatial memory were measured using the Morris water maze (MWM) in NaSal-treated and control animals, followed by observation of c-Fos and delta-FosB protein expression in the hippocampal field by immunohistochemistry. To further identify the neural substrate for memory change in tinnitus, neurogenesis in the subgranular zone of the dentate gyrus (DG) was compared between the NaSal group and the control group. The results showed that acquisition and retrieval of spatial memory were impaired by NaSal treatment. The expression of c-Fos and delta-FosB protein was also inhibited in NaSal-treated animals. Simultaneously, neurogenesis in the DG was also impaired in tinnitus animals. In general, our data suggest that the hippocampal system (limbic system) may play a key role in tinnitus pathology.

Enhancement of Endocannabinoid-dependent Depolarization-induced Suppression of Excitation in Glycinergic Neurons by Prolonged Exposure to High Doses of Salicylate.

The Dorsal Cochlear Nucleus (DCN) is a region which has been traditionally linked to the genesis of tinnitus, the constant perception of a phantom sound. Sodium salicylate, a COX-2 inhibitor, can induce tinnitus in high doses. Hyperactivity of DCN neurons is observed in several animal models of tinnitus, including salicylate-induced tinnitus. The DCN presents several forms of endocannabinoid (EC)-dependent synaptic plasticity and COX-2 can also participate in the oxidative degradation of ECs. We recently demonstrated that short-term perfusion of sodium salicylate and other inhibitors of both oxidative and hydrolytic EC degradation did not affect depolarization-induced suppression of excitation (DSE), a form of EC-dependent short-term synaptic plasticity. Here, we show that prolonged incubation with high doses of sodium salicylate (1.4 mM) enhances DSE of synapses onto glycinergic DCN interneurons but not those innervating glutamatergic DCN fusiform neurons. This effect was not reproduced with lower doses of salicylate (140 µM) or with ibuprofen, another inhibitor of COX-2. This effect was not observed in the presence of AM251, an antagonist/inverse agonist of cannabinoid CB1 receptors, showing that it was dependent on EC release. Finally we demonstrated that incubation with salicylate potentiated the increase in intracellular calcium during the depolarization. Our results point to an increased inhibition of DCN inhibitory CW neuron during depolarizations, probably by an enhanced EC release during the depolarizations, which is potentially significant for DCN hyperactivity and tinnitus generation.

A Conditioned Behavioral Paradigm for Assessing Onset and Lasting Tinnitus in Rats.

Numerous behavioral paradigms have been developed to assess tinnitus-like behavior in animals. Nevertheless, they are often limited by prolonged training requirements, as well as an inability to simultaneously assess onset and lasting tinnitus behavior, tinnitus pitch or duration, or tinnitus presence without grouping data from multiple animals or testing sessions. To enhance behavioral testing of tinnitus, we developed a conditioned licking suppression paradigm to determine the pitch(s) of both onset and lasting tinnitus-like behavior within individual animals. Rats learned to lick water during broadband or narrowband noises, and to suppress licking to avoid footshocks during silence. After noise exposure, rats significantly increased licking during silent trials, suggesting onset tinnitus-like behavior. Lasting tinnitus-behavior, however, was exhibited in about half of noise-exposed rats through 7 weeks post-exposure tested. Licking activity during narrowband sound trials remained unchanged following noise exposure, while ABR hearing thresholds fully recovered and were comparable between tinnitus(+) and tinnitus(-) rats. To assess another tinnitus inducer, rats were injected with sodium salicylate. They demonstrated high pitch tinnitus-like behavior, but later recovered by 5 days post-injection. Further control studies showed that 1): sham noise-exposed rats tested with footshock did not exhibit tinnitus-like behavior, and 2): noise-exposed or sham rats tested without footshocks showed no fundamental changes in behavior compared to those tested with shocks. Together, these results demonstrate that this paradigm can efficiently test the development of noise- and salicylate-induced tinnitus behavior. The ability to assess tinnitus individually, over time, and without averaging data enables us to realistically address tinnitus in a clinically relevant way. Thus, we believe that this optimized behavioral paradigm will facilitate investigations into the mechanisms of tinnitus and development of effective treatments.

Expression of immediate-early genes in the inferior colliculus and auditory cortex in salicylate-induced tinnitus in rat.

Tinnitus could be associated with neuronal hyperactivity in the auditory center. As a neuronal activity marker, immediate-early gene (IEG) expression is considered part of a general neuronal response to natural stimuli. Some IEGs, especially the activity-dependent cytoskeletal protein (Arc) and the early growth response gene-1 (Egr-1), appear to be highly correlated with sensory-evoked neuronal activity. We hypothesize, therefore, an increase of Arc and Egr-1 will be observed in a tinnitus model. In our study, we used the gap prepulse inhibition of acoustic startle (GPIAS) paradigm to confirm that salicylate induces tinnitus-like behavior in rats. However, expression of the Arc gene and Egr-1 gene were decreased in the inferior colliculus (IC) and auditory cortex (AC), in contradiction of our hypothesis. Expression of N-methyl d-aspartate receptor subunit 2B (NR2B) was increased and all of these changes returned to normal 14 days after treatment with salicylate ceased. These data revealed long-time administration of salicylate induced tinnitus markedly but reversibly and caused neural plasticity changes in the IC and the AC. Decreased expression of Arc and Egr-1 might be involved with instability of synaptic plasticity in tinnitus.

Phase I trial of sodium salicylate in patients with myelodysplastic syndromes and acute myelogenous leukemia.

Sodium salicylate is an inexpensive, readily available anti-inflammatory agent which inhibits NF-κB in in vitro models. We examined whether it was possible to safely achieve and maintain salicylate levels known to inhibit NF-κB in vitro in 11 patients with MDS or AML taking sodium salicylate. Most patients achieved the target blood salicylate level (20-30mg/dL) with acceptable toxicity, including reversible grade 1/2 elevations of hepatic transaminases (n=4) and ototoxicity (n=4). One patient had grade 3/4 elevations in AST/ALT. This study suggests that sodium salicylate may be safely combined with conventional chemotherapy regimens which are not associated with significant ototoxicity or hepatotoxicity.

Spontaneous reversibility of damage to outer hair cells after sodium salicylate induced ototoxicity.

BACKGROUND: High sodium salicylate doses can cause reversible hearing loss and tinnitus, possibly due to reduced outer hair cell electromotility. Sodium salicylate is known to alter outer hair cell structure and function. This study determined the reversibility and cochlear recovery time after administration of an ototoxic sodium salicylate dose to guinea pigs with normal cochlear function. STUDY DESIGN: Prospective experimental investigation. METHODS: All animals received a single 500 mg sodium salicylate dose, but with different durations of action. Function was evaluated before drug administration and immediately before sacrifice. Cochleae were processed and viewed using scanning electron microscopy. RESULTS: Changes in outer hair cell function were observed to be present 2 hours after drug administration, with recovery of normal anatomy beginning after 24 hours. Subsequently, derangement and distortion of cilia reduced, with effects predominantly in row three. At 168 hours, cilia were near-normal but with mild distortions which interfered with normal cochlear physiology. CONCLUSIONS: Ciliary changes persisted for up to 168 hours after ototoxic sodium salicylate administration.

[The state of lipid peroxidation and antioxidant protection system in parietal cells under experimental chronic atrophic gastritis development].

The lipid peroxidation state and the system functioning of antioxidant protection in parietal cells under rat chronic atrophic gastritis development was investigated. It was detected that the compensatory increase of superoxide dismutase and catalase activity did not affect the lipoperoxidation process and this resulted in accumulation of toxic TBA reactive substances and diene conjugates during the whole stages of the experimental pathology development. It was shown that the reserved power of the glutathione antioxidant system is sufficient to provide adoptable response in the acute period of the disease owing to increasing intracellular found of the reduced glutathione, but it is insufficient to prevent its decreasing in parietal cells in case of the chronic atrophic gastritis development. Our findings suggest that glutathione system is involved in processes of gastric atrophy. The obtained results testify about considerable system dysfunctions of lipid peroxidation and the antioxidant protection in processes of the rat experimental atrophic gastritis development.

Sodium salicylate reduces inhibitory postsynaptic currents in neurons of rat auditory cortex.

Sodium salicylate (SS) is a medicine for anti-inflammation and for chronic pain relief with a side effect of tinnitus. To understand the cellular mechanisms of tinnitus induced by SS in the central auditory system, we examined effects of SS on evoked and miniature inhibitory postsynaptic currents (eIPSCs and mIPSCs) recorded from layer II/III pyramidal neurons of rat auditory cortex in a brain slice preparation with whole-cell patch-clamp techniques. Both eIPSCs and mIPSCs recorded from the auditory cortex could be completely blocked by bicuculline, a selective GABA(A) receptor antagonist. SS did not change the input resistance of neurons but was found to reversibly depress eIPSCs in a concentration-dependent manner. SS reduced eIPSCs to 82.3% of the control level at 0.5 mM (n=7) and to 60.9% at 1.4 mM (n=12). In addition, SS at 1.4 mM significantly reduced the amplitude of mIPSCs from 24.12+/-1.44 pA to 19.92+/-1.31 pA and reduced the frequency of mIPSCs from 1.34+/-0.23 Hz to 0.89+/-0.13 Hz (n=6). Our results demonstrate that SS attenuates inhibitory postsynaptic currents in the auditory cortex, suggesting that the alteration of inhibitory neural circuits may be one of the cellular mechanisms for tinnitus induced by SS in the central auditory region.

Salicylate blocks L-type calcium channels in rat inferior colliculus neurons.

To investigate the effects of the tinnitus inducer sodium salicylate on L-type voltage-gated calcium channels, we studied freshly dissociated inferior colliculus neurons of rats by the whole-cell voltage clamp method. Salicylate's blocking of L-type calcium channels was concentration dependent, and the IC(50) value of salicylate was estimated to be 1.99 mM. An amount of 1 mM salicylate significantly shifted the steady-state inactivation curve of L-type calcium channels about 9 mV in the hyperpolarizing direction and significantly delayed calcium channel recovery. Our results suggest that salicylate's blocking of L-type calcium channels may contribute to salicylate-induced tinnitus by decreasing GABA release in the inferior colliculus.

Auditory nerve fibre responses to salicylate revisited.

Ototoxicity of salicylate is accompanied by a temporary hearing loss and tinnitus and has therefore been used to study tinnitus in animal models. Salicylate induced elevated central auditory activity has been interpreted as a correlate of tinnitus. Whether this elevated activity in the central auditory system is due to an increased activity in the auditory nerve is still under discussion. To explore this issue, we recorded the activity of single auditory nerve fibres in anaesthetised gerbils following systemic injection of salicylic acid. Firstly, compound action potential (CAP) thresholds were determined at 5-0 min intervals. Fifteen to 30 min after 200 mg/kg salicylic acid, threshold loss developed in the high frequency range. At 2 h CAP threshold loss reached a plateau amounting to 15-20 dB above 16 kHz, 0-5 dB below 2 kHz. An almost immediate start of threshold loss was observed after 400 mg/kg salicylic acid. A plateau of threshold loss was reached after 1.5 h with values of 25 dB in the high, 5-10 dB in the low frequency range. Secondly, responses of single auditory nerve fibres were studied after administration of 200 mg/kg salicylic acid. Frequency tuning curves and rate intensity (RI) functions at characteristic frequency (CF) were measured. Two hours and more after application, single fibre thresholds were elevated by about 20 dB at all CFs. Sharpness of tuning was reduced. Mean spontaneous rate was significantly reduced at CFs below 5 kHz (mean: 44 vs 28 AP/s). At CFs above 5 kHz mean spontaneous rate remained unchanged. In RI functions no change in maximum discharge rate was observed. The altered response properties can be interpreted by the known effects of salicylate on the prestin mediated active process of the outer hair cells. The elevated activity in the central auditory system after salicylate intoxication thus cannot be caused by cochlear nerve hyperactivity.

Intraperitoneal administration of salicylate dose-dependently prevents stress-induced ulcer formation in rats.

Stress has an important role in the induction of gastroduodenal injury. It was reported that oxygen free radicals played a role in the pathogenesis of this injury. Although some other antioxidant compounds and calcium channel blockers were examined in ulcer models, salicylate has not been tested for its gastroprotective effect in ulcer models by now. In the present study, intraperitoneal administration of 10, 25 and 50 mg/kg of salicylate dose-dependently prevented ulcer formation in obligatory immobilization model in rats. This protective effect of salicylate was found more potent than that of ranitidine for all doses tested. As expected, peroral (by gavage) administration of salicylate at 50 mg/kg exacerbated the ulcer score, in comparison with the control.

Effects of nimodipine on salicylate ototoxicity.

The purpose of this study was to examine the effects of nimodipine on salicylate ototoxicity in guinea pigs. The compound action potential (CAP) was recorded at the round window, and the cochlear blood flow (CBF) was measured simultaneously from the lateral wall of the basal turn of the cochlea by laser Doppler flowmetry. After administration of salicylate (100 mg/kg), the CAP thresholds were significantly elevated, by 5 to 20 dB (p < .05), and the CBF was significantly decreased (p < .05). After administration of nimodipine (2 mg/kg), the CAP thresholds were unchanged, but the CBF had increased significantly (p < .05), while systemic blood pressure had decreased significantly (p < .05). Simultaneous administration of both salicylate (100 mg/kg) and nimodipine (2 mg/kg) resulted in significant elevation of the CAP thresholds (p < .05), while the CBF did not decrease. These results suggest that nimodipine prevents the decrease in CBF induced by salicylate, but that nimodipine does not prevent the deterioration in the CAP.

Neurophysiologic mechanisms of tinnitus.

Research over the past decade has provided new insights into the neural mechanisms likely to produce the false percepts of sound associated with tinnitus. These insights have emerged mainly as a result of electrophysiologic studies, examining changes in brain activity, and behavioral studies, examining changes in perception, in animals that have been treated with well-known tinnitus inducers such as salicylates, quinine, and intense sound. The available evidence, based on electrophysiologic studies, suggests that tinnitus is associated with disturbances in spontaneous neural activity in the auditory system. These abnormalities include increases in spontaneous activity (hyperactivity), changes in the timing of neural discharges (i.e., the temporal firing properties of neurons), and an increase in bursting activity of neurons. Parallel studies using behavioral testing methods have demonstrated that agents, which produce these neural changes, also cause tinnitus in animals. This article reviews the literature concerned with both behavioral evidence for tinnitus in animal models and the associated changes that occur at peripheral and central levels of the auditory system.

[Effects of salicylate on electrically evoked otoacoustic emissions in the guinea pig].

Clinically, salicylates have been known to have a ototoxic side effect of reversible hearing loss and to reduce the voltage-dependent length change (electromotility) in experiments on isolated outer hair cells (OHC). In order to clarify how the reduction of OHC electromotility due to salicylates contributes to cochlear dysfunction in vivo, we observed compound action potentials (CAP) threshold as well as the outputs of the electrically evoked otoacoustic emissions (EEOAE) before and after intravenous administration of 500 mg/kg sodium salicylate in a guinea pig model. A silver ball electrode placed on the round window membrane of the animal was used for both recording of the CAP and stimulation to elicit the EEOAE. The CAP threshold to tone bursts with frequencies from 2 to 10 kHz elevated by 13 to 22 dB, and outputs of the EEOAE decreased approximately 4 dB for 5 kHz stimulation, and 12 dB for 8 kHz after salicylate administration. These results suggested that systemically administered salicylate also reduced the electromotility of the OHC, and caused the hearing impairment in vivo.

Repair of rat gastric mucosa: effect of 16,16-dimethyl prostaglandin E2.

Prostaglandins protect the gastric mucosa against a variety of injurious agents and may accelerate the recovery of the gastric mucosa following damage. In previous studies prostaglandins were given prior to the injurious agent, so it was not possible to distinguish their potential effects on accelerating repair or reducing initial damage. We have investigated the effect of 16,16-dimethyl prostaglandin E2 (dmPGE2) on the repair of the gastric mucosa after injury induced by several injurious agents. dmPGE2 was given orally 15 min prior to aspirin or sodium salicylate, or 30 min after aspirin, sodium salicylate, or ethanol. dmPGE2 delivered prior to injury reduced the aspirin-induced fall in mucosal potential difference (PD), but had no effect on that induced by sodium salicylate. dmPGE2 administered after ASA injury significantly increased recovery of PD (P < 0.05), but did not alter the rate of recovery of PD with other damaging agents. Histological damage was decreased in rats treated with dmPGE2 after aspirin compared to aspirin-only-treated rats (P < 0.02). Exogenous dmPGE2 protects and restores gastric mucosal integrity after aspirin damage but has no effect on the repair of sodium salicylate and ethanol injured mucosa, suggesting that repair of the gastric mucosa after aspirin damage is enhanced by dmPGE2 due to its ability to prevent ongoing damage, rather than directly enhancing repair processes.

Effect of corticosteroid treatment on salicylate ototoxicity.

Our previous studies showed that salicylate ototoxicity is associated with decreased levels of prostaglandins (PGs) and elevated levels of leukotrienes (LTs) in the perilymph. The purpose of this study was to determine whether or not pretreatment with corticosteroid, which suppresses both PGs and LTs in arachidonic acid metabolism, prevents salicylate ototoxicity. Salicylate ototoxicity was induced in chinchillas with or without treatment with dexamethasone. Hearing thresholds were measured by auditory brain stem response, and perilymph samples were assayed by high-performance liquid chromatography. Dexamethasone pretreatment, given by either systemic or local round window membrane application, partially prevented salicylate-induced hearing loss. Prevention of salicylate ototoxicity by dexamethasone seems to be correlated with increased PG levels and decreased LT levels in the perilymph. This is another piece of evidence that salicylate ototoxicity may be mediated by abnormal arachidonic acid metabolism in the inner ear.