The Effect Of Kanamycin On Outer Hair Cells Regulataed By Acetylcholine

IntroductionAminoglycoside antibiotics(aminoglycoside antibiotics,AmAn)are widely used, but there are ototoxicity,renal toxicity and other side effects.Study has confirmed that AmAn damage the inner ear receptors and cause hearing impairment and vestibular dysfunction.Thereby the clinical application of these antibiotics are limited.Many studies found that the most obvious damage of AmAn is in outer hair cells(OHCs)of mammalian cochlear.OHCs were mainly dominated by medial olivocochlear(MOC)efferent nerve,and the OHCs and efferent nerve endings formate synaptic connections.Its main neurotransmitter is acetylcholine(ACh).The current study shows that acetylcholine receptor(AChR)has a mixture of N,M receptor sensitivity of the pharmacological characteristics.Homologous expression of a9 receptor can be non-competitively blocked by aminoglycoside antibiotics.Therefore ototoxicity of these drugs are possibly because that the combination of these drugs with acetylcholine receptor impede combination of acetylcholine with acetylcholine receptor, weaken the protective effect of efferent system,so that outer hair cells are damaged.In this study,through the establishment of kanamycin ototoxicity in guinea pig model,combinations of electrophysiology,morphology,cytology,we observed the effects of kanamycin on outer hair cells regulataed by acetylcholine.Methods1,Experimental groups and preparation of ototoxicity model: Guinea pigs were randomly divided into three groups:â‘ control group,â‘¡kanamycin administered 3 days group,â‘¢kanamycin administered 6 days group. Physiological saline was daily injected in intramuscular dosing of 2.5ml/kg in normal control group,kanamycin was daily injected in intramuscular dosing of 400mg/kg. Measurement of body weight was daily to adjust the dose. 2,ABR threshold test:ABR threshold of guinea pigs was measured in each group before treatment and 1 day after treatment separately through auditory evoked potential response system.The intensity begin from 95dB SPL.Threshold is judged according to wave P3.3,Surface preparation of cochlea basement membrane and silver nitrate staining of outer hair cells:Guinea pigs were decapitated and the bulla was removed and opened.The cochlea was exposed and the injection of 0.5%AgNO₃ solution 5ml was gently pushed into the cochlear inner for 15min.Fix the cochlea with 4%formaldehyde solution for more than 4h,while exposuring for 1-1.5h under sunlight.Then basement membrane was segregated and placed on the slide.4,Immunohistochemical detection of cochlear AChE: Guinea pigs were decapitated and the bulla was removed and opened.The cochlea was expose and the injection of 4 formaldehyde solution was gently pushed into the cochlear inner to fix.Decalcification was done with 10%EDTA solution.Then the cochlea was made into the paraffin-embedded sections.Slices were done with AChE immunohistochemistry staining.The average gray value of AChE positive reaction was measured using microscopic image analysis system. 5,Outer hair cells isolated: Guinea pigs were quickly decapitated,the bulla was removed and opened in the artificial perilymph.Bone implant shell was remove,cochlear was cut off and placed into calcium-free artificial perilymph solution containing trypsin 0.25%to digest 10min at 20-25℃,then washed three times by artificial perilymph to terminate the digestion. Cells were gently agitated to disperse.OHCs were placed at 4℃to preserve.6,Fluorescence measurement of calcium:OHCs were devided according to the following experimental groups:â‘ control group,â‘¡ACh group,â‘¢ACh+Kanamycin group.Outer hair cells were incubated by Fluo-3/AM 30min.Fluo-3 fluorescence intensity change was detected by micro-fluorescence measurement system.Fluorescence images were recorded in the computer and analysis fluorescence intensity changes useing supporting analysis software. Fluorescence intensity indicated intracellular free calcium concentration. 7,Statistical Analysis:All datas are Expressed by mean±standard deviation(x^—±s).t-test was used with excel software for statistical analysis.Results1,Guinea pig ABR threshold detectionABR threshold was no significant difference in each group before treatment (P＞0.05).ABR threshold was no significant difference between kanamycin administered 3 days group and control group after administration(P＞0.05).Knamycin administered 6 days group increased significantly ABR threshold compared with, compared with the control group there was significant difference(P＜0.01).2,Changes in outer hair cellsCochlear outer hair cells were three platoons,no significant deficiencies in control group.Outer hair cells slightly decrease in kanamycin administered 3 days group,but were no significant difference compared with the control group.Outer hair cell lost significantly in kanamycin administered 6 days group.3,The expression of AChECompared with the control group,the average gray value of AChE at the organ of Corti in Kanamycin administered 3 days group decreased.In kanamycin administered 6 days group,the average gray value of AChE further decreased.4,Image of isolated OHCOHC was like a test-tube,nucleus located at the bottom.The residual synapses could be seen at the bottom of cells.Fluorescence intensity in the nucleus was higher than in the cytoplasm and the concentration of Ca²⁺at the bottom of was higher than at the top.5,Change of intracellular free Ca²⁺concentrations.Ca²⁺fluorescent intensity in OHCs remained basically unchanged in control group, increased 2.32±0.32 times(n=6)in ACh Group,and increased 1.51±0.41 times(n=6)in kanamycin+ACh Group.There is significant difference between the two groups(P＜0.01).The results show that Kanamycin can inhibit the increasing of Ca concentration in OHCs caused by ACh. Conclusion1,Kanamycin increase ABR threshold and damage outer hair cells in guinea pigs.2,Kanamycin increase the expression of acetylcholinesterase at the organ of Corti.3,Kanamycin inhibit the increasing of Ca²⁺concentration in outer hair cells caused by ACh.