The Research Of D-Methionine Protects Against Chemotherapeutic Agent Damage To The Cochlear Hair Cell

Objective: The vestibular system is one of primary organs which to keep balance.A lot of disease of inner ear always cause deafness and damage vestibular system,for example aminoglyc- oside antibiotics,antitumor chemotherapeutic agent,and neurot- oxin et al.Most studies of animal and clinical have reported that anticancer druges could cause damage of hair cell in the inner ear and vestibular,thereby resulting hearing loss and disequilibr- ium,induceing vertigo and losing act capability.But vestibular system is an important organ in charge of balance,and the hair cell is not regenerate in inner ear.Accordingly,its an bottleneck how to attenuate ototoxicity drugs-induced vestibulotoxicity, thus more and more physician of otolaryngology pay attention to the study.D-Methionine is an essential amino acid of contain- ing sulphur,its main function is to synthesis protein,and also have effect of detoxication.It have reported D-Methionine's protection of hair cell in cochlea.But we have not reported that D-Methionine protect vesti- bulum auris internae endorgan up to now.This study is to form animal model of carboplatin's ototoxicity,by injecting carboplat- in into Kunming mouse.Then we inject D-Methionine by peritoneal injection.Then we observe the protection to hair cell of D-Methionine in the recovery of microstructure and ultrastru- cture,in order to provide theory for developing new drugs for protecting hair cell in vestibular sensory end-organ,and provide new thinkings about treatment of antcancer drugs induced otot- oxicity.Methods: Light microscop,scanning electron microscope and hair cell counting were used to observe the protection of D-Methionine for injury of cochlea and vestibular hair cells which is carboplatin induced.The specific procedure as following:1.Animal preparations:sixteen healthy Kuming mouse of both sexes were randomly divided into 4 groups(n=4 each).As follows:the normal group:not giving carboplatin(A);the experi- mental group:giving D-Methionine treatment after deaf (carbop- latin 50mg/Kg intraperitoneal injection for 14 days; D-Methion- ine 3.96mg/Kg intraperitoneal injection for 14 days)(B);the control group 1:not giving D-Methionine treatment after deaf (carboplatin 50mg/Kg intraperitoneal injection for 14 days; Sodium Chloride 3.96mg/Kg intraperitoneal injection for 14 days)(C);the control group 2:not giving D-Methionine treatment after deaf,and not giving Sodium Chloride (carboplatin 50mg/Kg intraperitoneal injection for 14 days)(D).Tthe animals were deeply anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg) and then decapitated.2. The preparation and observation of specimens 2.1 Light microscope specimens: Following decapitation, the temporal bones were quickly removed and opened to expose the otic capsule. The primary fixative was 10% formaldehy- de,the stain was 0.5% silver nitrate cream,for 3-5 times respe- ctive.A small hole in the otic capsule was hand drilled beneath the first turn with a three sided,sharpened pick.In vitro perfusion was performed intermittently within 5 min of death through the small hole in scala tympani,allowing the fluid to exit through the opened oval window.After perfusion fixation, the round window membrane was removed and the cochleae immersed then placed in the 10% formaldehyde (at 4°C) for 6 hours.Under the dissect- ing microscope,the bony capsule of the cochlea was carefully removed.The tissue was then stretched on the slide,and was viewed through a light microscope and photographed.2.2 Scanning electron microscopy:The animals were killed by decapitation while under general anesthesia and cochleae perfused with fixative through the perilymphatic spaces for 3-5 times.The primary fixative was 2.5% glutaraldehyde at 4°C in 0.1M phosphate buffer (pH 7.4).A small hole in the otic capsule was hand drilled beneath the first turn with a three sided, sharpened pick.In vitro perfusion was performed intermittently within 5 min of death through the small hole in scala tympani, allowing the fluid to exit through the opened oval window.After perfusion fixation,the round window membrane was removed and the cochleae immersed then placed in the refrigerator overnight.After overnight fixation in glutaraldehyde,the cochle- ae were rinsed in 0.1 M phosphate buffer and immersed then placed in the edathamil fixation fluid (EDTA 0.35mol/L pH 7.4) for 24 hours.Cochleae were then rinsed in buffer 3 times.After rinsing,Under the dissecting microscope,the bony capsule of the cochlea was carefully removed.The tissue was then serially dehydrated in 50%, 70%, 80%, 90% and 3×100% EtOH.When the dehydration was over,we followed it by drying by the critical point method of CO2,in which the material was deprived from water.After being fixed in the appropriate specimen holder, the material was recovered in a vacuum chamber with gold vapors and examined under a Hitachi S-3500 scanning electron microscopy and photographed.3. Statistics:Data were analysed by completely randomized one-factor analysis of variance test (one-way ANOVA)and least significant difference test (LSD-t)using SAS 8.0 software.P<0.05 was considered significant.Results:1. The comparison of outer hair cell(OHC) and inner hair cell(IHC) respectively:There was a significant difference in IHC of four groups (F=128.55,P=0.000).The data was analysed by LSD-t between two groups,B,C,D groups were significant difference to A group(P<0.01 respectively).C,D groups were significant diff- erence to A group(P<0.01 respectively).There was no differe- nce between C and D groups(P>0.05).There was a significant difference in OHC of four groups (F=55.84,P=0.000).The data was analysised by LSD-t between two groups,B,C,D groups were significant difference to A group(P<0.01 respectively).C,D groups were significant diff- erence to A group(P<0.01 respectively). There was no differe- nce between C and D groups(P>0.05).2. The observation by light microscope:The normal control animal had essentially normal IHC and OHC hair cell counts,and the hair cell arrange in order,and was no degeneration and deterioration.The hair cells of B,C,D groups had necrosis and abscission.Especially,the IHC was lossed seriously than OHC lossing.The B group was protected by D-Methionine,so its injury of hair cell was slighter than C,D groups'.The hair cells of macula sacculi,macula utriculi and crista ampullaris which dissected from the animals of normal control arranged in order,and had high density.But the B,C,D groups had a lower density of hair cells,and not arranged in order, especially C and D groups had which D-Methionine did not applicated.The main lossing of hair cells happened at striola area in macula sacculi and macula utriculi,but the lossing of hair cells in perim was lesser than striola area.The lossing of hair cells in crista ampullaris was mainly showed the loss of TypeⅠhair cell,but the quantity of TypeⅡhair cell did not obviously changed.But the loss of macula utriculi of B group was not obvious change compared to C,D. 3. The observation by scanning electron microscope:The normal control animal had essentially normal IHC and OHC hair cell counts,and the hair cell arrange in order,and was no lodging,disperative replication,swelling,accretion and missi- ng et al.The cilium of IHC was arranged in straighten cluster,the three rows OHC arranged in pattern of"v".but we also observed the ciliary of B,C,D groups which was treated by carboplatin distortion with"v"pattern disarrangement,with folded hairs or partial absence.The injury of OHC was gently than IHC's.The injury of cilium of C and D groups which was not treated by D-Methionine was serious than B group's which was treated by D-Methionine.The hair cells of macula sacculi,macula utriculi and crista ampullaris which dissected from the animals of normal control arranged in order,and were well-distributed without lodgi- ng,confused of hair bundle,and pllarity disappearing.The hair cells of B,C,D groups which was treated by carboplatin were damaged,and the injury of macula sacculi and crista ampullaris of C and D groups were seriously than B group.But the hair cells of B group which were treated by D-Methionine was not repaired compared to C,D groups.Conclusion:Through the observation of injury of IHC and OHC on basal membrane,which dissected from mouse that were treated by carboplatin,we found that carboplatin had vestibulot- oxicity and ototoxicity,in other words,carboplatin could cause the symptoms of hearing loss,tinnitus and/or dizzy and so on. The injury of IHC,OHC and hair cell of vestibular endorgan were slight in the mouse which were treated by D-Methionine than that were not.This certificated that the injury of hair cell induced by carboplatin partially recovered after D-Methionine application.Therefore,its extrapolated that the symptoms of hearing loss,tinnitus and dizzy induced by carboplatin were able to partially recovered,in other words,D-Methionine attenuated cisplatin-induced ototoxicity.For this reason,the study provided new thingking for therapy of anticancer drugs induced ototoxici- ty.