| Introduction and ObjectiveWith the growth of the national economy and development of the overall living standards,the definition of health improves to a new different level:not only for a longer survival time but also for a better quality of life.Under such background,the danger of hearing lost to human health is attached great importance.Two important reasons of hearing loss are drug and senescence.The hearing loss by using aminoglycoside antibiotics is the main cause of deaf in our country.Being the main reason of sensorineural hearing loss(SNHL),the mechanism of aminoglycoside antibiotic's ototoxicity has been researched in many aspects,such as morphology, protein expression,gene regulation,and electrophysiological etc.But,most of those researches are focus on inner/outer hair cell,cochlear striae vascularis and spiral ganglion neuron(SGN).Ribbon synapse,which is the first afferent nerve synapse of the process in which audio information transfer to central nervous system,has not been given more concern in aminoglycoside antibiotic's ototoxicity.The cochlea,the mammalian hearing organ,codes sounds over wide ranges of frequencies and intensities.Outer hair cells(OHCs) confer to its high sensitivity and the selectivity of its sound frequency,whereas inner hair cells(IHCs) are the genuine sensory cells transmitting information of sound to the central nervous system.IHCs transmit sound wave to the electric signal and form potential in IHCs which stimulate neurotransmitter of ribbon synapses released to the corresponding SGN.Hearing loss is generally considered to be related with the damage of IHCs/OHCs and the degeneration of SGNs.Ribbon synapse,formed between IHCs and SGNs,was important to sound signal transmitting but not was attended to. Much research proved that efficiency of synapse transmitting was not changeless. In the progress that presynaptic neuron was excited,which caused postsynaptic potential changing by the release of neurotransmitters,and then electrical signal was transferred,the function and form of synapses could change.This kind of change may be the synapse transmission potency enhancement,or synapse transmission potency weakening;may be in the short-term(several seconds to several minutes);or in the long-term(several hours to several weeks).Kinds of synapse transmission potency's changes are called as the synapse plasticity.Because the sound information is transmitted into the brain through auditory nerve's action potential,therefore the ribbon synapse's structure will influence the sound code by rearrangement and/or the degeneration.In a particular location of the cochlea,signaling transfer damaged between afferent nerve and inner hair cell will affect language understanding which the location corresponding to.For these reasons,the drug-induced hearing loss in mice,the form and the function of ribbon synapses in IHCs will change.Because ribbon synapses in IHCs are at deep location and in small quantity, research progress about them had been slow.In our country,the system research about ribbon synapse has not yet to be reported.In recent years,along with the research technology and experimental condition's development,researchers overseas got certain achievement in form,molecular dissection,electricity physiology etc in normal physiology condition which enables us to research ribbon synapses' structure and the function under the pathological state.Observed through electron microscope,typical ribbon synapse is sphere or ellipse body of electron density and attached with single-layer synaptic vesicles around.With the hearing threshold's difference,ribbon synapse's quantity and form also change.For example,each inner hair cell of mouse has 16.8±2.4 ribbon synapses,but after the hearing threshold increased,ribbon synapse's quantity reduces,and the volume increases.We can obtain accurate quantity of ribbon synapses by reconstruction of electron microscope ultra-thin serial section.These research methods and the findings of ribbon synapse in normal physiology situation have built the good foundation for further research under pathology condition.In this study,the model of SNHL was made by intraperitoneal injection of gentamicin and the ribbon synapse plasticity was investigated in the early period of SNHL including the number and function changes of ribbon synapses.The number of ribbon synapses was analyzed by immune-fluorescence,using both an antibody against RIBEYE and an antibody against glutamate receptor subunit 2 and 3(GluR2-3) associated with a confocal laser scanning microscope,three-dimensional reconstruction. Semi-quantity analysis was carried on using immunofluorescence and western blotting. Because otoferlin is important in the process of neurotransmitter exocytosis in ribbon synapse,the change of expression of otoferlin can embody function of ribbon synapses.The research about the number and functional changes of IHCs ribbon synapse when drug-induced hearing loss,builds the good foundation for the further pathogenic mechanisms' study of SNHL caused by other reasons,for example:senescence;noise. Further research can use electron microscopy to observe microstructure changes of ribbon synapse,or use physiological methods to study function of ribbon synapses.In brief,research about ribbon synapse's plasticity is new supplement to SNHL pathogenesis mechanism,and has the extensive research and application prospect.Material and MethodsIn this experiment,120 C57 mice were selected(provided by the China Medical University testing animal center),with the exception of auricle and middle ear disease, 2 months old and weighing 25±1.23 g.ABRs were used to assess hearing threshold. The hearing threshold is normal before the model was made,and the hearing threshold was higher than 30dB after the model was made.The mice were divided into 2 parts. Each part was divided into 6 groups.Each group had 10 mice.Mice in 5 groups were intraperitoneally injected with gentamicin(100mg/kg) separately for 4days(the 4d group),7days(the 7d group),10days(the 10d group),14days(the 14d group),21 days (the 21d group).Another one group was normal control group(Od group). The cochlea basilar membranes were separated,one part was carries on double-label immunofluorescence of the basilar membrane,then use confocal laser scanning microscope to carry on serial scanning of the basilar membrane.Three dimensional reconstructions were carried on with 3ds max 8.0 software to analyze quantity of ribbon synapse.We used immunofluorescence method to examination otoferlin expression in basilar membranes,and western blotting to carry on the semi-quantitative analysis of otoferlin expression in the other part.Results1.To make three dimension reconstructions and count the number of IHCs ribbon synapse of 10 serial scanning,the number of ribbon synapses in each IHC was(?)±SD=16.10±1.0322.Using 3d max 8.0software to analyze the number of ribbon synapses in each group,the number of ribbon synapses contained in each inner hair cell showed with (?)±SD in every group.The number of ribbon synapses was most at the 7th days. Significant differences existed in 6 groups(P＜0.01).In the 7d group,the quantity of ribbon synapses was obviously more than other 5 groups.3.Using immunofluorescence method,otoferlin labeled by FITC was green, located at the bottom of the inner hair cells.Western blotting was used to detect the expression of otoferlin:at the location of about 140.5kD otoferlin was detected and semi-quantitative analyzed.Before gentamicin was injected,the expression of otoferlin was few.At the 4th days,expression quantity increased.The expression quantity was highest at the 7th day.The absorbance ratio of otoferlin expression had obvious differences between the groups(P＜0.01).Conclusion1.As the ribbon synapses were not present in the same plane,for quantitative analysis of the ribbon synapses,double-labeled immunofluorescence was used to mark the synaptic structure protein and the postsynaptic receptor protein,combined with series scanning of confocal laser scanning microscope,three-dimensional reconstruction by 3d max 8.0software.The accurate number of ribbon synapses could be obtained.2.In early period of gentamicin effect,when the number of hair cells had not changed,the number changes of IHC ribbon synapses had taken place.At the 7th day gentamicin injected,the number of ribbon synapses reached a peak,and then decreased. Through the number of ribbon synapses changing,IHCs increased or decreased connections with SGNs.3.In early period of gentamicin effect,when the number of hair cells had not changed,the expression change of otoferlin had taken place.At the 4th day with gentamicin effect,otoferlin expression increased and got to the peak at the 7th day,then declined.This kind of change took place possibly because ribbon synapse increased vesicle fusion and neurotransmitter releasing to make compensation for gentamicin ototoxicity in early period.After the 7th day,the expression of otoferlin reduced possibly because gentamicin ototoxicity influenced metabolism of IHCs,which enabled ribbon synapses not to maintain the normal form and function.
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