HDAC2 Is Associated With Glucocorticoid Responsiveness In Guinea Pigs With LPS-induced Sudden Hearing Loss

Objective It has been 60 years since glucocorticoid (GC) was first used for hearing and balance disorders, such as sudden idiopathic sensorineural hearing loss(SSNHL), autoimmune inner ear disease and Meniere’s disease. Moreover, GC has been also used to control inflammation in the inner ear induced by otitis media, bacterial meningitis and sepsis-associated hearing loss. GC exerts a great suppression of inflammation and immune responses in the cochlear and vestibular organs. Despite numerous benefits of GC treatments for hearing and vestibular dysfunction, some clinical reports and reviews indicated that some patients with these disorders did not respond to GCs. In other words, these patients had GC insensitivity or resistance. Recently, some studies indicated that reduction of histone deacetylase 2 (HDAC2) played a critical role in GC insensitivity or resistance. Our previous research showed that the effectiveness of GC in treating SSNHL were associated with HDAC2 levels. However, some studies demonstrated that inflammation might impair HDAC2 activity to enhance the inflammatory response, which contributes to the relative GC insensitivity. Our aim of this study was to demonstrate the relationship between HDAC2 and GC responsiveness and to explore the effect of aminophylline (AMI) in restoring GC sensitivity by increasing HDAC2 level in the cochlea of guinea pigs in an animal model of sudden hearing loss induced by lipopolysaccharide (LPS)Methods Fifty guinea pigs were randomly divided into five groups control/artificial perilymph (AP) group (n=10), model (LPS) group (n=10), dexamethasone (LPS+DEX) group (n=10), aminophylline (LPS+AMI) group (n=10), dexamethasone+aminophylline (LPS+DEX+AMl) group (n= 10). Choose normal hearing of guinea pigs by auditory brain stem response (ABR) test before drugs treatment. Both the ears of control group were administrated 5μl sterile artificial perilymph fluid by means of drilling the scala tympani of the cochlear basal turn. 5μl 5mg/ml LPS was transferred into the cochlea of both the ears of the others group in the same way. Intravenous injection of AMI 20mg/kg and DEX lmg/kg were administrated before and after surgery. ABRs were recorded in all guinea pigs in 48 hours after surgery. The changes of cochlea were observed by HE staining. The fluorography immunohistochemistry test was used to observe the situation of HDAC2 express and distribution in inner ear. The HDAC2 levels in the cochlea of guinea pigs were detected by ELISA test.Results ABR showed that hearing loss was obvious in LPS group, espacially the high frequency were more obvious deteriorate (the threshold shift about 60dB SPL). Compared to AP group, hearing loss of LPS group was statistically significant (p<0.01). The improvement of hearing was obvious in frequency 16k,32k in group of LPS+DEX and LPS+AMI (p<0.05). The effect of LPS+DEX+AMI group for GC treatment was most remarkable at all frequencies compared with single drug treatment, especially at 16kHz (p<0.01). HE staining results showed a serious morphologically damage of cochlea in LPS group. The thickness of stria vascularis was decreased, the spiral ligament was separated from with the lateral wall of cochlea and the spiral ganglion cell nuclei was shrinking. Compared with LPS Group, the damage of LPS+DEX or LPS+AMI group was milder. Moreover, the cochlear structure in the LPS+DEX+AMIgroup was roughly in normal.The fluorography immunohistochemistry test in the inner ear showed a positive expression of HDAC2 in the cochlea of guinea pigs including in the area of inner and outer hair cells, stria vascularis, spiral ganglion neurons. It indicated that HDAC2 had widely distributed in the inner ear and located in cell nucleus. The correlation analysis showed negative correlations between the expression of HDAC2 and threshold shifts of ABR in 8kHz,16kHz, and 32kHz. (p<0.05)Conclusion In the present study, when 5 μl LPS (5mg/ml) was directly infused into the tympanic scale, sensorineural hearing loss was detected in all animals at all frequencies, especially in the high frequency region. Campared with AP group, the level of HDAC2 in LPS group was significantly decreased, which indicated that inflammation in the inner ear may impair HDAC2 activity. The effect of LPS+DEX group was not obvious compared with LPS group, which had statistical significances at 16,32 kHz (p<0.5). However, the effect of combined intervention group for GC +AMI treatment was most remarkable. Some studies demonstrated that low concentrations of theophylline could restore GC sensitivity by enhancing HDAC2 activity through inhibition of PI3K-mediated expression of inflammatory genes. In our study, the level of HDAC2 in LPS+AMI+DEX group was restored and the effection of it also was most dramatic,whicn indicated theophylline can restore GC sensitivity by enhancing HDAC2 activity. It was effective for GC and aminophylline treatment in LPS-induced hearing loss of guinea pigs.Aminophyline could improve effect of GC, which indicated HDAC2 played an important role in restoring GC sensitivity in the inner ear.