Experimental Study Of Protective Effects For NGB Gene Transfect Into Guinea Pigs Through Gentamicin-Induced Deafness

Objective:Neuroglobin(neuroglobin,NGB) is the oxygen globulin which presents in human and mouse brain.It is reported that it has high oxygen affinity and can combine oxygen reversibly.The eukaryotic expression vector (pEGFP-NGB)with the reporter gene EGFP was constructed in pre-trial.The liposome-plasmid complex was injected into guinea pig cochlea through the round window,then the guinea pigs were treated by gentamicin.We used auditory brainstem response(ABR) audiometry to record the hearing threshold of guinea pigs.We Observed the morphology changes of the hair cells and the spiral ganglion cells through techniques of stretched preparation,scanning electron microscopy;and observed the expressions of NGB protein in the pig cochleas through immunohistochemistry.Then verify the protective effects of cationic liposome-mediated NGB gene transfected into deaf guinea pig cochlea induced by gentamicin.Methods:Each guinea pig experienced by ABR to remove the hearing threshold more than 40dB SPL,then 120 healthy guinea pigs were employed in experiment and divided randomly into five groups,24 in each group(part of them died during the experiment and 21 survived from each group had statistical significance):groupâ… :blank control group(without administration);groupâ…¡: artificial perilymph control group(artificial perilymph was injected into left ear without gentamicin administration);groupâ…¢:artificial perilymph experimental group(artificial perilymph was injected into left ear and then gentamicin administration);groupâ…£:empty plasmid transfection group(empty plasmid pEGFP-C₁ was injected into left ear and then gentamicin administration);group V:NGB gene transfection group(pEGFP-NGB was injected into left ear and then gentamicin administration).Gentamicin was injected with 120mg/kg/d for 14 days,then observed the animals for 14 days.All guinea pigs were measured by auditory brainstem response,then we picked 6 samples from each group and observed morphological changes of the hair cells on basement membrane in cochlear with techniques of stretched preparation;picked 6 samples from each group and observed surface structures of the hair cells on basement membrane in cochlear through SEM;picked 9 samples from each group and detected the expression of NGB protein through immunohistochemistry.Results:Auditory brainstem response showed that the threshold of all the guinea pigs in blank control group and artificial perilymph control group was less than 40dB SPL,the threshold of each guinea pig in artificial perilymph experimental group and empty plasmid transfection group was higher than 110dB SPL,the threshold range of each guinea pig in NGB gene transfection group was 70-110dB SPL;the stretching of cochlear basement membrane and scanning electron microscopy showed that both the inside and outside hair cells of blank control group and artificial perilymph control group arranged neatly and have no defects.The hair cells of artificial perilymph experimental group and empty plasmid transfection group were lossing substantially,the hair cells almost vanished corresponding with the ABR threshold more than 135dB SPL. NGB gene transfection group of some missing hair cells damaged lightly,and mainly the outer hair cell;immunohistochemistry showed that NGB protein expressed on cochlear hair cells and spiral ganglion cells of NGB gene transfection group increased significantly,and all the other groups nearly had no obvious positive expression.Conclusion:The ABR threshold of NGB gene transfection group increased much less than groupâ…¢and groupâ…£,the listening function damage was reduced in a certain extent;the stretching of cochlear basement membrane and scanning electron microscopy showed that the hair cells pathological changes of NGB gene transfection group were lighter than the other groups; immunohistochemistry showed that NGB gene had been transfected into cochlea hair cells and spiral ganglion cells,the expression of NGB protein increased significantly.This study verified successfully that cationic liposome-mediated NGB gene transfection could provide effective protection to the guinea pigs treated with gentamicin in a certain degree.