clinical use of aminoglycoside antibiotics is complicated by ototoxicity , a condition characterized by damage to cochlear hair cells , cochlear spiral ganglion cells , and the auditory center . understanding the molecular events behind the ototoxicity of aminoglycoside antibiotics and developing novel , non - toxic treatments are of great clinical importance . neurotrophin-3 ( nt-3 ) , a member of the neurotrophin family , is widely expressed in the inner ear and auditory center . nt-3 has been reported to play an important role in the structural and functional regulation of the nervous system . specifically , nt-3 is essential for the development , survival , growth , and differentiation of auditory epithelial cells and neurons ; nt-3 also plays an important role in neuronal repair after injury and in the plasticity of remnant neurons . additionally , it has also been shown that nt-3 expression can protect the inner ear and mitigate hearing loss through various mechanisms . thus , increasing the local expression of nt-3 in auditory nerve cells may represent a promising approach to protecting against ototoxicity . acupuncture , a traditional chinese therapy , has been found to yield beneficial effects on deafness and to promote hearing recovery . , most studies examining this issue have focused on the effects of acupuncture on the inner ear and cochlear spiral ganglion [ 810 ] . to the best of our knowledge , it has not been reported whether the auditory benefits of acupuncture involve the auditory nuclei . therefore , in the present study , we aimed to assess the preventive effect of acupuncture on gentamicin - induced hearing impairment by measuring auditory brainstem responses ( abrs ) before and after treatment . in addition , we sought to determine whether the underlying protective mechanism may involve nt-3 in the cochlear and inferior colliculus nuclei . a total of 24 healthy sprague - dawley rats ( 250350 g ) with normal reflection of the auricle were provided by the animal laboratory of xian jiaotong university . all rats were housed in an air - conditioned room with a 12-h light / dark cycle and provided free access to standard rodent chow diet and water . after acclimation for 1 week , rats were randomly divided into the following 4 groups : control group , gentamicin group , neitinggong group , and tinggong group . for 14 consecutive days , control group rats received an intraperitoneal injection of saline each morning ( 2.5 ml / kg ) , while rats in the other groups received an intraperitoneal injection of gentamicin ( 100 mg / kg tianjin pharmaceuticals group co. , ltd . ) each morning . starting on the first day of intraperitoneal injection , rats in the neitinggong and tinggong groups also underwent acupuncture at the neitinggong or tinggong acupoints , respectively , once every 2 days for 20 days . the neitinggong and tinggong acupoints are located in the middle ear and adjacent to the antilobium , respectively . for rats in the neitinggong group , filiform needles ( 0.35100 mm ) were acupunctured around the umbo of the tympanic membrane using an electric auriscope , at a depth of about 2 mm , which was adjacent to the promontory of the tympanum . for rats in the tinggong group , filiform needles ( 0.3535 mm ) were inserted ~4 mm subcutaneously , then manipulated by the reinforcing - reducing method every 10 min for 30 s each time . for both the neitinggong and tinggong groups , the needles were removed 30 min after application . all rats were anesthetized by intraperitoneal injection with a 10% ( w / v ) chloral hydrate solution ( 0.3 ml per 100 g body mass ; shanghai mingbo biological technology co. , ltd ) every afternoon prior to testing the abr . abr was determined using brainstem - evoked potentiometer detection ( denmark ics chartr ep ) . the potentiometer was set to generate a stimulatory clicking sound every 100 s ( 15-ms scanning time , 1003000-hz filtering range , and 1024 overlaps ) . electrodes were made from 0.3550 mm platinum acupuncture needles and were inserted subcutaneously at the vertex ( active ) and ventrolaterally to the right ( reference ) and left ( ground ) ears . recording electrodes were placed at the intersections of the cranial sagittal midline and connected with the external auditory canal , reference electrodes were placed in retroauricular subcutaneous tissue , and grounding electrodes were placed in the nasion . abrs were averaged by the computer and displayed on the screen on a fixed scale . the threshold was obtained by first reducing the sound pressure level ( spl ) in 10-db steps , then fine tuning the level in 5-db steps up and down to identify the lowest level at which an abr pattern could be recognized . the minimal spl intensity able to induce wave iii of the abr waveform was considered to be the abr threshold . after the experiment , rats were anesthetized by a 10% chloral hydrate solution , and then secured on an animal operating table . a perfusion cannula was inserted into the aorta through which 250 ml of saline solution was perfused quickly , followed by 2000 ml of phosphate - buffered saline perfused slowly over 12 h. rats were killed rapidly by decapitation and the bilateral cochlear nucleus and inferior colliculus were removed . after paraffin embedding , antigen retrieval , and blocking , sections were incubated overnight at 4c with a rabbit anti - rat nt3 antibody ( boster biological technology , china ) . after washing with pbs , the sections were incubated with a biotinylated secondary antibody ( boster biological technology , china ) for 30 min at room temperature . sections were washed thoroughly , mounted onto gelatin - coated slides , dehydrated , and cover - slipped before being examined under a microscope operated in the bright field mode . images ( 40 magnification ) from 5 non - overlapping horizons were taken for each slice . the average optical density value was measured by image - pro plus ( version 6.0 ) . one - way analysis of variance ( anova ) was used for statistical analysis ( spss17.0 software ) . a total of 24 healthy sprague - dawley rats ( 250350 g ) with normal reflection of the auricle were provided by the animal laboratory of xian jiaotong university . all rats were housed in an air - conditioned room with a 12-h light / dark cycle and provided free access to standard rodent chow diet and water . after acclimation for 1 week , rats were randomly divided into the following 4 groups : control group , gentamicin group , neitinggong group , and tinggong group . for 14 consecutive days , control group rats received an intraperitoneal injection of saline each morning ( 2.5 ml / kg ) , while rats in the other groups received an intraperitoneal injection of gentamicin ( 100 mg / kg tianjin pharmaceuticals group co. , ltd . ) each morning . starting on the first day of intraperitoneal injection , rats in the neitinggong and tinggong groups also underwent acupuncture at the neitinggong or tinggong acupoints , respectively , once every 2 days for 20 days . the neitinggong and tinggong acupoints are located in the middle ear and adjacent to the antilobium , respectively . for rats in the neitinggong group , filiform needles ( 0.35100 mm ) were acupunctured around the umbo of the tympanic membrane using an electric auriscope , at a depth of about 2 mm , which was adjacent to the promontory of the tympanum . for rats in the tinggong group , filiform needles ( 0.3535 mm ) were inserted ~4 mm subcutaneously , then manipulated by the reinforcing - reducing method every 10 min for 30 s each time . for both the neitinggong and tinggong groups , the needles were removed 30 min after application . all rats were anesthetized by intraperitoneal injection with a 10% ( w / v ) chloral hydrate solution ( 0.3 ml per 100 g body mass ; shanghai mingbo biological technology co. , ltd ) every afternoon prior to testing the abr . abr was determined using brainstem - evoked potentiometer detection ( denmark ics chartr ep ) . the potentiometer was set to generate a stimulatory clicking sound every 100 s ( 15-ms scanning time , 1003000-hz filtering range , and 1024 overlaps ) . electrodes were made from 0.3550 mm platinum acupuncture needles and were inserted subcutaneously at the vertex ( active ) and ventrolaterally to the right ( reference ) and left ( ground ) ears . recording electrodes were placed at the intersections of the cranial sagittal midline and connected with the external auditory canal , reference electrodes were placed in retroauricular subcutaneous tissue , and grounding electrodes were placed in the nasion . abrs were averaged by the computer and displayed on the screen on a fixed scale . the threshold was obtained by first reducing the sound pressure level ( spl ) in 10-db steps , then fine tuning the level in 5-db steps up and down to identify the lowest level at which an abr pattern could be recognized . the minimal spl intensity able to induce wave iii of the abr waveform after the experiment , rats were anesthetized by a 10% chloral hydrate solution , and then secured on an animal operating table . a perfusion cannula was inserted into the aorta through which 250 ml of saline solution was perfused quickly , followed by 2000 ml of phosphate - buffered saline perfused slowly over 12 h. rats were killed rapidly by decapitation and the bilateral cochlear nucleus and inferior colliculus were removed . after paraffin embedding , antigen retrieval , and blocking , sections were incubated overnight at 4c with a rabbit anti - rat nt3 antibody ( boster biological technology , china ) . after washing with pbs , the sections were incubated with a biotinylated secondary antibody ( boster biological technology , china ) for 30 min at room temperature . sections were washed thoroughly , mounted onto gelatin - coated slides , dehydrated , and cover - slipped before being examined under a microscope operated in the bright field mode . images ( 40 magnification ) from 5 non - overlapping horizons were taken for each slice . the average optical density value was measured by image - pro plus ( version 6.0 ) . one - way analysis of variance ( anova ) was used for statistical analysis ( spss17.0 software ) . there was no significant difference in the general condition of the rats in the control group before versus after the experiments , and their body weights increased steadily . in contrast , rats in the other 3 groups exhibited behaviors characteristic of poor health , such as decreased spontaneous activity , loss of appetite , shaggy hair , and arrested weight gain . these rats also exhibited an absence of the auricle reflex to differing extents . under the otoendoscope , we observed small tympanic membrane perforations around the handle of malleus in rats from the neitinggong group ; these perforations were partially healed , and did not exhibit discharge or infection . the integrity of tympanic membranes in rats from the other experimental groups was not affected . as shown in table 1 , on day 14 , the average abr threshold values in rats from the gentamicin , neitinggong , and tinggong groups were significantly increased compared to those of the control group , indicating that their hearing had been impaired . compared with the values for the gentamicin group , the average threshold abr values for the neitinggong and tinggong groups appeared to be slightly , though not significantly , decreased . on day 20 , the average threshold abr value for the gentamicin group was significantly higher than that of the control group . the average threshold abr values for the neitinggong and tinggong groups were significantly decreased compared with that of the gentamicin group . as shown in figure 1 , nt-3 immunopositivity ( yellow ) nt-3 expression in the cochlear nucleus did not differ significantly among the groups ( table 2 , p>0.05 ) . as shown in figure 2 , nt-3 was also observed in the cytoplasm of cells in the inferior colliculus . as shown in figure 2 and table 2 , the expression of nt-3 in the inferior colliculus was significantly decreased by gentamicin treatment ( p<0.01 ) , compared with that in the control group . of note , expression of nt-3 in the inferior colliculus in the neitinggong and tinggong groups was significantly higher than that in the gentamicin group ( p<0.01 ) , showing a restoration trend toward physiological levels . these results indicate that acupuncture at the neitinggong or tinggong acupoints suppressed the inhibitory effect of gentamicin on nt-3 expression in the inferior colliculus ( p<0.01 ) . there was no significant difference in the general condition of the rats in the control group before versus after the experiments , and their body weights increased steadily . in contrast , rats in the other 3 groups exhibited behaviors characteristic of poor health , such as decreased spontaneous activity , loss of appetite , shaggy hair , and arrested weight gain . these rats also exhibited an absence of the auricle reflex to differing extents . under the otoendoscope , we observed small tympanic membrane perforations around the handle of malleus in rats from the neitinggong group ; these perforations were partially healed , and did not exhibit discharge or infection . the integrity of tympanic membranes in rats from the other experimental groups was not affected . as shown in table 1 , on day 14 , the average abr threshold values in rats from the gentamicin , neitinggong , and tinggong groups were significantly increased compared to those of the control group , indicating that their hearing had been impaired . compared with the values for the gentamicin group , the average threshold abr values for the neitinggong and tinggong groups appeared to be slightly , though not significantly , decreased . on day 20 , the average threshold abr value for the gentamicin group was significantly higher than that of the control group . the average threshold abr values for the neitinggong and tinggong groups were significantly decreased compared with that of the gentamicin group . as shown in figure 1 , nt-3 immunopositivity ( yellow ) was readily observed in the cytoplasm of cells in the cochlear nucleus area . nt-3 expression in the cochlear nucleus did not differ significantly among the groups ( table 2 , p>0.05 ) . as shown in figure 2 , nt-3 was also observed in the cytoplasm of cells in the inferior colliculus . as shown in figure 2 and table 2 , the expression of nt-3 in the inferior colliculus was significantly decreased by gentamicin treatment ( p<0.01 ) , compared with that in the control group . of note , expression of nt-3 in the inferior colliculus in the neitinggong and tinggong groups was significantly higher than that in the gentamicin group ( p<0.01 ) , showing a restoration trend toward physiological levels . these results indicate that acupuncture at the neitinggong or tinggong acupoints suppressed the inhibitory effect of gentamicin on nt-3 expression in the inferior colliculus ( p<0.01 ) . specifically , on day 20 the average threshold abr values of rats treated with neitinggong or tinggong acupuncture were much lower than that of the gentamicin group . these results are consistent with the prior observation of ma et al . that neitinggong acupuncture attenuated gentamicin - induced ototoxicity . previous results from clinical trials and animal experiments have demonstrated that acupuncture can improve inner ear microcirculation and the hemorheology index , release blood vessel contraction of the inner ear , promote the regeneration and repair of hair cells , and influence brainstem response , all of which may contribute to the attenuation of sensorineural deafness [ 9,1316 ] . together , these data suggest that acupuncture may serve as a promising treatment of auditory impairment induced by various factors . regarding the mechanism by which acupuncture may counter gentamicin - induced ototoxicity , there are several possible mechanisms to consider . first , previous studies have shown that acupuncture can protect against hair cell damage directly by improving inner ear microcirculation and energy metabolism , stabilizing the mitochondria and lysosomes , scavenging oxygen free radicals , preventing inner ear lesions , increasing afferent signals , reducing the damage to the inferior colliculus nucleus , and inducing neuroplastic changes . second , acupuncture could stimulate the auditory center directly to produce nt-3 , which exerts neurotrophic effects on the inferior colliculus nucleus neurons . third , acupuncture could produce these effects indirectly , perhaps by improving renal blood flow and glomerular filtration , thereby alleviating renal pathological damage , which in turn could promote the excretion of gentamicin . in this work we found that acupuncture directed at the neitinggong or tinggong acupuncture site increased nt-3 expression selectively in the inferior colliculus nucleus in rats . for example , chen et al . found that nt-3 expression in the spared l6 dorsal root ganglion ( drg ) in cats that received electro - acupuncture was markedly higher than that of other groups . evaluated the effect of electro - acupuncture in cats subjected to dorsal rhizotomy , and found that nt-3-positive neurons were increased in lamina ii on the acupunctured side . several studies have shown altered nt-3 expression levels in the auditory center after hearing damage . stem auditory nuclei after unilateral cochlear ablation , and found that nt-3 levels were depressed by 2244% on day 3 , elevated transiently by 28124% on day 7 , and then returned to normal levels by day 60 . in mice injected with gentamicin for 15 days , li et al . and song found that the nt-3 expression in the cochlea , the cochlear nucleus , and the inferior colliculus nucleus was significantly decreased . however , in the mice that received early treatment with herbal compounds for reinforcing kidneys , activating blood , and arousing consciousness ( hcraa ) , hearing was obviously improved and the expression of nt-3 was elevated . in the present study , we found that the expression of nt-3 in the inferior colliculus nucleus was decreased significantly in the gentamicin group , which may be partially explained by direct transport of gentamicin to the inferior colliculus nucleus through blood circulation where it caused hyposecretion of nt-3 . in the present study , nt-3 expression in the cochlear nucleus this observation could be due to some nt-3 in the cochlear nucleus coming from the cochlea through axonal transport , which may mask reductions in nt-3 expression in the cochlear nucleus . alternatively , it could be due to expression of nt-3 being related to an increase in synaptic plasticity , given that cochlear injuries may influence nt-3 expression through regulation of synaptic plasticity in auditory nuclei . these results suggest that acupuncture is a simple and effective way to increase nt-3 expression in auditory nuclei , maintain auditory neuron survival , and promote the repair of hearing loss . however , the kinetics of nt-3 expression in auditory brain stem nuclei are not clear . further studies are needed that focus on the dynamics of nt-3 expression in different parts of the auditory nerve nuclei and under different conditions , such as acupuncture treatment alone ( as a positive group ) . such experiments would provide more direct evidence for the efficacy of acupuncture treatment for hearing loss and pave the way for elucidating the mechanism of the protective effects of acupuncture . in this study , acupuncture at the neitinggong or tinggong acupoints was shown to cure sensorineural deafness caused by gentamicin in rats . acupuncture therapy increased the expression of nt-3 in the inferior colliculus nucleus , which plays a key role in maintaining the integrity of the hearing pathway and mediates , partially , the benefits of acupuncture on auditory repair . increasing nt-3 expression in the inferior colliculus nucleus may represent a new strategy for preserving auditory capabilities .