Impact Of Auditory Deprivation On Rats And Expression Of Growth Associated Protein-43 In Auditory Cortex

Objective:To explore the molecular mechanism of neural plasticity in the development and reconstitution following impairment of auditory system,we studied the changing levels of growth associated protein-43(GAP-43) expression in the auditory cortex of the developing rats and in ototoxicity deafened rats before and after electrical intracochlear stimulation.The changes and plasticity of GAP-43 expression in the developing auditory cortex were also investigated,aiming at providing theoretical evidence for early discovery and prevention of hearing loss in children.Methods:A total of 130 Sprague-Dawley(SD) rats were assessed in this study.They were healthy and aged 1 week at the beginning of this study.All animals were divided randomly into six groups:(1) auditory deprivation of Aminoglycoside ototoxicity group(OAD)(n=30):Amikacin sulphate(500 mg/kg body weight/day) were injected subcutaneously from postnatal day 7(P_7d) to P_16d,and they were bred to postnatal week 3,4,8.(2) auditory deprivation and early electrical intracochlear stimulation group(EIS₁)(n=30):Stimulation wire was introduced into cochlear at 3 weeks of age,and electrical stimulation was given daily for a continuous duration of 3 hours over a period of 7 days.(3) auditory deprivation and late electrical intracochlear stimulation group(EIS₂)(n=30):Same operation was performed at 7 weeks of age,and animals were subjected to the same stimulation as in the early group.(4) normal control of Aminoglycoside ototoxicity group(NC₁) (n=30):Equal amount of Sodium Chloride was injected subcutaneously and same treamments were given to animals.(5) conductive auditory deprivation group(CAD)(n=5):Bilateral external ear canals were pluged and obstructed with sound-attenuating foam anthropo-earplugs from P_14d to P_28d.(6) normal control of conductive auditory deprivation group(NC₂) (n=5):The normal hearing rats at P_2W were bred for 2 weeks. Auditory brain stem response(ABR) measurements were performed to rats of OAD,NC₁,CAD and NC₂ group to evaluate hearing function.We removed brains of OAD,NC₁, EIS₁ and EIS₂ group and made a single coronal cut to generate a flat surface anteriorly to the auditory cortex(OAC),next to the Bregma in our immunohistochemistry experiments.RT-PCR was performed to do semiquantitative analysis and to define the AC to rats of OAD,NC₁ group.Results:(1) Aminoglycoside antibiotics results in severe to profound sensorineural hearing loss,the threshold readings are more than 103 dB nHL.(2) Conductive auditory deprivation induced a slight hearing loss.The threshold of the CAD group (35.00±17.35 dB nHL) raised compared with the NC₂ group (3.64±3.23 dB nHL,P＜0.05).(3) Immunohistochemistry: In the immediate postnatal stage(P_3W),the AC was found to contain generous GAP-43-positive neurons(111.50±4.90/HP). With the development,a continuous reduction of GAP-43 immunoreactivity occurs in the AC.By 4 weeks of age(P_4W), we found a significant reduction of positive neurons (84.17±3.24/HP),which was about 74%of P_3W.By the age of 8 weeks(P_8W),lower staining of GAP-43 was shown in the AC (66.67±4.17/HP),and the positive neurons decreased by about 40%in contrast to P_3W.Five days after the AC of auditory deprivation by ototoxic drug(OAD P_3W),the expression of GAP-43 reaches to a peak(138.00±4.53/HP),and the positive neurons was about 1.3-fold compared to the same age control (NC₁ P_3W).By deafened 12 days(OAD P_4W),the GAP-43-positive neurons(99.75±4.21/HP) declined by 29%, but increased by about 18%over non-administration.By deafened 40 days(OAD P_8W),the positive neurons were reduced dramatically(75.33±3.50/HP),just about 55%of group deafened 5 days,and rose by 14%from the control.The early electrical intracochlear stimulation elevated GAP-43 level(EIS₁, 120.00±5.59/HP) in ipsilateral AC instead of in the contralateral AC compared with the non-stimulation group(OAD_4W, 93.25±4.30/HP),and the positive neurons were rather more than the control(NCl_4W,72.75±3.34/HP).After the late electrical intracochlear stimulation,similar to the early group,the staining level of GAP-43(EIS₂,102.50±4.22/HP) in the AC was also higher than the unaffected group(OAD_8W,81.67±3.76/HP),and sharply rose above the control(NCl_8W,69.33±3.48/HP). Summarily,the early and late electrical stimulation both induced upregulation of GAP-43 immunoreactivity,and the amount of the positive neurons demonstrated a 20%above increase contrast to the group before stimulation.(4) RT-PCR:In the newborn rats AC(P_3W),we observed a high level expression of GAP-43 mRNA.With the development,a gradual decline of GAP-43 mRNA presents in the NC₁ group.By P_4W,GAP-43 mRNA was at a low level.And by P_8W,it expressed weakly. The expression of GAP-43 mRNA in the AC of deafened rats was more than that in the control group,and diminished gradrually likewise.Conclusions:In the immediate postnatal stage,AC was found to contain high levels of GAP-43.Over the first few postnatal weeks,a continuous reduction of GAP-43 occurred, and declined persistently.Shortly after the acoustic deprivation by ototoxicity,GAP-43 was dramaticly boosted in the AC.After electrical intracochlear stimulation,GAP-43 levels obviously upregulated in the ipsilateral AC.Together,GAP-43 has close correlation with the growth and plasticity of the AC,which can be a significant marker for auditory plasticity.