Optogenetic Transfection Of Cochlear Spiral Ganglion Neurons On The Big Animals

Cochlear implant(CI)remains the most effective treatment for people with severe-profound sensorineural hearing loss(SNHL),which have helped more than 500,000 patients with sensorineural hearing loss get a useful representation of sounds in the environment and help them to understand speech.However,most of the CI users show poor speech recognition ability in noisy environments compared to people with normal hearing for the coding limitations of current CIs.People with normal hearing can distinguish2,000 kinds of sound frequency;however,the current CIs mediate electrical stimulation of spiral ganglion neurons(SGNs)via an array of 12-24 electrodes,which impede the recognition of music,especially the perception of trebles and tones.Optogenetic applications have brought hope to the dilemma.The opsins,which show high temporal precision and spatial revolution,can enhance frequency and intensity resolution in auditory activities by regulating the SGNs accurately.As a result,patients with severe hearing loss can have a better hearing experience.In 2014,Moser,a German scholar,transduced opsins to the cochlear SGNs of the mice and evoked auditory potential successfully using blue light.Wrobel et al.restored the auditory-driven behavior of some deaf gerbils in 2018 by optogenetic stimulation.These important findings show that optogenetic techniques are promising in both refinements of current CI technology and the treatment of deafness.The most commonanimal models for optogenetic auditory research are rodents.We believe that these research results will be more significant for thetranslational research of optical cochlear implants(oCIs)and optogenetic treatment in SNHL once they are verified with big animal models as their hearing system more closely resembles human condition.Here,we established an optogenetic big deaf animal model by AAV-mediated expression of opsin Channelrhodopsin-2(ChR2)in SGNs of albino deaf Rongchang pigs,Investigate the mechanisms how ChR2 regulates the spiral ganglion neurons(SGNs)in optogenetic big deaf animal models.Provide experimental and theoretical evidence for the development of oCIs and clinical treatment of SNHL.PART I Optogenetic Technology Verification on Guinea PigsObjective: Establish optogenetic adult mammalian models.Verify animal safety in virus transfection and microsurgery.Explore the optogenetic stimulation parameters for adult mammals.Methods: 20 adult guinea pigs weighing 350 to 400 grams were divided into two groups randomly.One group was introduced AAV2/8-hSyn-ChR2(H134R)-mCherry to cochlea through round window to establish an optogenetic adult mammal model.The other group was introduced saline to cochlea served as the control group.Record and analyze the threshold,latency and amplitude of Click-ABR before and after the transduction.Observe the expression of opsins on cochlear SGNs using confocal laser microscope after 3 weeks of transfection.470 nm blue laser was applied to evoke optically compound action potential(oCAP)by irradiating the spiral ganglion neurons of transfected guinea pigs’ cochleae.Results: Optogenetic adult mammal models were successfully constructed with adult guinea pigs.The ABR threshold of the guinea pigs has increased by about 6 dB after the transduction,which demonstrated lightdamage in hearing function.The expression of opsins in guinea pig SGNs was detected by immunofluorescence staining after three weeks of transfection.By irradiating the SGNs of transfected guinea pig with a 470 nm blue laser,oCAP was recorded successfully.OCAP can be evoked when the laser intensity is higher than 3.7 mW,and the amplitude increases as the laser intensity increases.The maximum oCAP was evoked by a laser intensity of 5.8 mW;the amplitude of it is 0.78μv,which is close to the CAP(amplitude of 0.7μv)evoked by an acoustic intensity of 40 dB SPL.Conclusions: Optogenetic adult mammal models were successfully constructed with adult guinea pigs.The hearing function was lightly affected,which proved that the transfection method introduced by round window was safe.The expression of opsins on the cochlear SGNs of adult guinea pigs was verified by immunohistochemical staining,and oCAP was recorded successfully by electrophysiological approch,which indicated that the optogenetic stimulation parameters are reliable,which laid a foundation for exploring the optogenetic transfection on SGNs of large mammalian.PART II Hearing Function and Inner Ear Morphology Study inRongchang PigsObjective: Investigate the hearing function and inner ear morphological characteristics of Rongchang pigs and determine specific objects to establish optogenetic big animal models.Methods: Record and analyze the threshold,latency and amplitude of Click-ABR at different stages of Rongchang pigs(P1,P7,P14,and P28).Remove cochleae after recording,combine whole-mount dissection and frozen section with immunofluorescence staining to observe the hair cells,stria vascularis,and SGNs,etc.Results: The Albino Rongchang pigs have shown severe sensorineural hearing loss in both ears since P1.The ABR threshold of normal Rongchang pigs is 35-40 dB,consisting of seven primary waveform components as humans.The latency and amplitude of each waveform change with age and tend to be stable after P14.There was no significant difference in the numbers and morphology of hair cells and cilia between albino Rongchang pigs and normal ones at P1.The OHCs began degenerate,and some cilia became clustered,collapsed,and lost from P7,while the IHCs still survived at P28.SGNs did not show degeneration until P28.At P28,the number of SGNs was 23.72 % less than P1 and 24.94 % less than normal pigs of the same age.Stria vascularis of albino pigs were thinner than normal ones at P1 and became thinner and thinner with age.Conclusions: It is the first time to identify P14-P28 as an important period for pathological progress in albino Rongchang pigs by analyzing the hearing function and the morphological characteristics of Rongchang pigs,indicating that treatment research for albino deaf Rongchang Pig should start before P14 for better outcomes.PART III The Optogenetic Research on Albino Deaf Rongchang PigsObjective: Establish an optogenetic big deaf animal model,clarify parameters for optogenetic stimulation in big deaf animals,investigate the mechanisms how opsin regulates the spiral ganglion neurons(SGNs)on optogenetic big deaf animal models,provide experimental and theoretical evidence for the development of optical cochlear implants(oCIs)and the treatment of sensorineural deafness,and contribute to reveal the pathological mechanisms of Waardenburg syndrome and provide new ideas for its treatment.Methods: Six 14-day-old Rongchang pigs were randomly divided into two groups.One group was used to establish optogenetic big animal models,and the other group served as the saline control group.The threshold,latency and amplitude of Click-ABR were measured before and after the transductionn.Immunofluorescence staining was performed to verify the expression of opsinon cochlear SGNs of Rongchang pigs.Eleven 14-day-old albino deaf Rongchang pigs were employed,eight of them were used to establish optogenetic big deaf animal models by transfecting SGNs with AAV2/8-hSyn-ChR2(H134R)-mCherry injection through round window,and the left served as the saline injection control group.470 nm blue laser was applied to evoke oCAP by irradiating the spiral ganglion neurons of transfected cochleae.Immunofluorescence staining was performed to verify the expression of opsinon SGNs of transfected cochleae.Results: AAV2/8-hSyn-ChR2(H134R)-mCherry was successfully delivered to the cochlea through the round window in Rongchang pigs.The ABR threshold of normal Rongchang pigs was increased by about 8 dB after the transduction,which demonstrated a light damage in hearing function.The immunofluorescence co-localization revealed that opsins were expressed in the SGNs of both normal and albino deaf Rongchang pigs.However,no oCAP was recorded by irradiating the cochlear SGNs of transfected albino deaf pigs with a 470 nm blue laser.Conclusions: It is the first time to transfect cochlear SGNs of the big deaf animals with an optogenetic approach in the world.For powerful experimental evidence in developing oCIs,deaf animal models will be further employed for oCAP recording,while the temporal,frequency,and intensity resolution of signal coding under laser stimulation will be deeplyexplored.Albino Rongchang pigs are natural models for human Waardenburg syndrome type 2A disease.The research will also contribute to reveal the pathological mechanisms of Waardenburg syndrome and provide new ideas for its treatment.