noise - induced hearing loss ( nihl ) has now become one of the most prevalent occupational injuries reported .
currently , it has been estimated that about 500 million individuals suffer from this hazard in the world .
nihl is a preventable deficit , but it is difficult to reverse it once it occurs since the lost mammalian sensory cells can not regenerate [ 36 ] ; therefore , understanding its pathogenesis has become a very important task for researchers .
moreover , there have been great efforts to clarify the molecular and biochemical mechanisms involved in nihl .
studies have shown that acoustic overstimulation could lead to the pathogenesis and biochemical changes that result in hearing loss [ 7 , 8 ] . the continued and evolving research involving nihl has determined that there is a close relationship between the occurrence of noise - induced deafness and changes in some genes , cell metabolism , cell apoptosis , and so forth .
the pathogenesis of nihl is very complicated , and the exact mechanism is unknown . generally , it is the outcome of the interaction between genetic and environmental factors [ 911 ] . in spite of many efforts ,
the research progress of nihl has been slow , and it is difficult to study nihl in humans .
no heritability studies have been performed because families exposed to identical noise conditions are almost impossible to collect .
gene modified mice are good models for studying the mechanism of nihl . in recent years
, some genes have been found to affect the susceptibility to noise in animal models [ 13 , 14 ] .
several of the knockout mouse lines that have been developed , including pjvk , pmca2 , p2rx2 , and cdh23 , were determined to be more sensitive to noise than their wild type controls .
meanwhile , more studies are beginning to search for new nihl susceptibility genes , and hundreds of single nucleotide polymorphism ( snp ) loci have been found in genes involved in different pathways of the inner ear .
an extended analysis of 644 snps in 53 candidate genes was performed in two independent ( swedish and polish ) populations ; and two snps ( rs667907 and rs588035 ) in myh14 resulted in a positive association in the polish sample set and significant interaction with noise exposure level in the swedish sample set [ 19 , 20 ] .
this result suggested that myh14 is likely to be a nihl susceptibility gene [ 4 , 19 ] .
myh14 , also known as nonmuscle ii heavy chain ( nmhcii - c ) , together with two other nonmuscle chains ( myh9 and myh10 ) , is a member of the myosin family , which have been implicated in many motile processes such as ion - channel gating , organelle translocation , and cytoskeleton rearrangement [ 21 , 22 ] .
myh14 has been shown to play roles in neuritogenesis and maintenance of apical cell junctions in epithelial cells within the cochlea [ 23 , 24 ] .
however , the relationship between myh14 and nihl is still obscure , and the role they play in nihl needs further analysis . in our study , we applied the crispr / cas9 technology to establish a myh14 knockout mouse line ( myh14 ) using the cba / caj background strain .
then , we investigated the hearing threshold and morphological changes in these mice under normal conditions or under noise exposure .
we found that myh14 mice did not exhibit significant hearing loss until five months of age .
moreover , myh14 mice were more vulnerable to high intensity noise compared to control mice .
more significant outer hair cell ( ohc ) loss was observed in myh14 mice after acoustic trauma .
these data indicate that the absence of myh14 may increase susceptibility to nihl and that myh14 may play a beneficial role in the protection of the cochlea after acoustic stimulation in the cba / caj mouse line .
the use of animals in this study and the experimental procedures were approved by the animal ethics review committee of shandong university .
animal management was performed strictly in accordance with the standards of the animal ethics committee of shandong university ( permit number : ecaesdusm 20123004 ) .
myh14-deficient mice were generated using the crispr - cas9 genome - editing technology and were maintained on the cba / caj background .
both px330 and pst1374 were obtained from addgene ( plasmid i d : # 42230 , # 44758 , resp . ) .
the crispr - cas9 genome - editing technology in mice was used as previously described [ 25 , 26 ] . in brief , a pair of oligonucleotides for the target sequence ( 5-cctgaagaaagagcgcaata-3 ) was annealed and ligated to px330 digested with bbsi . then sgrna was produced by in vitro transcription ( t7 as promoter ) using the megashortscript kit ( ambion , am1354 , usa ) .
the hcas9 mrna was derived from pst1374-n - nls - flag - linker - cas9 , synthesized using the mmessage mmachine t7 kit ( ambion , am1345 , usa ) , and polyadenylated with a polya tailing kit ( life technologies , usa ) .
both the sgrna and the cas9 mrna were purified using the megaclear kit ( ambion , am1908 , usa ) and eluted in rnase - free water .
cba / caj female mice were superovulated and mated with cba / caj male mice . then
, the female mice were sacrificed and the fertilized eggs were removed from the oviducts . the purified sgrnas ( 50  ng/l ) and hcas9 mrnas ( 100  ng/l ) were coinjected into the cytoplasm of pronuclear stage eggs . following the injected pronuclear stage
the genomic dna fragment around the grna target site was amplified by pcr using two sets of primers : myh14 forward , 5-acctcgtgcttgttcag-3 , and myh14 reverse 5-tgtcttcagcagggtgt-3. the pcr products obtained were sequenced directly or cloned using the t / a cloning method and then sequenced to identify the mutation .
off - target effects may exist in crispr / cas9 technology . in response to this possibility
, we used the crispr design tool available on the web ( http://crispr.mit.edu ) , to search for the off - target locus .
the primers for the off - target analysis were as follows : sin3b 5-gccaggaggtatatgagaac-3 and 5-gaaatttccccaggaatggactgaca-3 ; pcdh9 5-gtgtacttatagcactcacc-3 and 5-ctaacgcggaaacacctcac-3 ; patl1 5-ccactgagcctttcctaccttc-3 and 5-gtaaatttagaaattttatttt-3 ; arhgap27 5-gaagctaggccagcgggcgaaag-3 and 5-ctgcccatgggcggggctg-3 ; and olfr1333 5-gcgctatactgtcatcctcaac-3 and 5-gcaagccaagctacgcactg-3. the genomic dna fragment from the newborn pups was amplified using the pcr primers mentioned above .
then , the pcr products obtained were sequenced directly or cloned using the t / a cloning method and sequenced to identify the mutation .
mice were decapitated ; the cochlea were quickly removed from the skull and homogenized in ice - cold cell lysis buffer ( 10  mm tris , ph = 7.4 , 1% triton x-100 , 150  mm nacl , 1  mm edta , and 0.2  mm pmsf ) , then lysed for 30  min on ice , and centrifuged at 10,000  g at 4c for 30  min
. the supernatant was then collected , and the protein concentration was measured using a bca kit .
the samples were mixed with loading buffer and heated at 100c for 5  min and stored at 20c .
protein samples were separated by a 10% sodium dodecyl sulfate ( sds ) polyacrylamide gel and transferred onto a pvdf membrane . the membrane was blocked in 5% nonfat dry milk in tbs - t at room temperature for 1  h and incubated with primary antibodies at 4c overnight . after washing with tbs - t ( three times for 10  min each at room temperature )
, membranes were incubated with an anti - rabbit hrp - conjugated secondary antibody ( 1  :  8000 , cell signaling , usa ) diluted in 5% nonfat dry milk in tbs - t at room temperature for 1  h. next , membranes were washed in tbs - t ( three times for 10  min each ) and bands were detected using ecl western blot detection kit ( thermo , usa ) .
membranes were incubated with the following primary antibodies diluted in 5% bovine serum albumin ( bsa ) : rabbit anti - myosin - iia ( 1  :  1000 , cell signaling , usa ) , rabbit anti - myosin - iib ( d8h8 ) ( 1  :  1000 , cell signaling , usa ) , rabbit anti - myosin - iic ( d4a7 ) ( 1  :  1000 , cell signaling , usa ) , and rabbit anti--actin ( 1  :  5000 , bioworld , china ) .
mice were deeply anesthetized with pentobarbital sodium ( 50  mg / kg body weight ) by intraperitoneal injection and the body temperature was maintained at 37c using a heat pad .
three needle electrodes were inserted subcutaneously in the anesthetized mice : one was inserted between the ears at the forehead , one was underneath the left external ear , and one was at the back near the tail . click and tone burst stimuli at frequencies of 4 , 8 , 16 , and 32  khz were generated and responses were recorded using a tucker - davis technologies system ( tdt , usa ) workstation running the siggen32 software ( tdt , usa ) .
auditory thresholds ( db spl ) were defined by reducing the sound intensity in5  db steps from 90  db to 10  db .
the abr threshold was defined as the lowest sound intensity sufficient to elicit the first wave clearly . in parallel experiments ,
total rna was extracted from mouse cochleae at a defined time point after noise exposure using trizol reagent ( invitrogen ) according to the provided directions .
all samples had a260/280 ratios of 1.92.1 and showed two sharp peaks corresponding to the 18s and 28s rna on electropherograms .
then cdna was synthesized using primescript rt reagent kit with gdna eraser ( takara ) .
quantitative pcr was performed on a bio - rad real - time thermal cycling system with power sybr green pcr master mix ( takara ) .
the amplification reaction mixture ( 20  l ) contained 800  nmol of each primer in the sybr green system .
the quality control for the mrna quantification was performed using three integrated control assays in the pcr array : a reverse transcription control , a positive pcr control , and a genomic dna control .
the primers utilized in this study are shown as follows : myh9 f : acaatggaggccatgagaat , myh9 r : gagatgacccgcagcaag , myh10 f : ggaggacaccctagacacca , and myh10 r : ccacttcctgctcacgtttt mice were kept awake and placed in a stainless steel wire cage in the center of an open - field acoustic chamber .
then , they were exposed to 210  khz band noise at an intensity of 105  db spl for 4  h to induce a temporary change in auditory threshold shifts .
the sound was generated by a noise generator ( sf-06 , random noise generator , rion , usa ) , amplified by a power amplifier ( cdi 1000 power amplifier , crown , usa ) , and delivered to microphones .
the noise sound files were created and equalized with audio editing software ( audacity portable ) .
sound levels were calibrated at multiple locations within the sound chamber to ensure uniformity of the stimulus .
cochlea were fixed in 4% formaldehyde in 10  mm phosphate - buffered saline ( pbs ) at 4c overnight and decalcified in 10% edta in 10  mm pbs at room temperature for at least one day . for sectioning ,
the cochleae were dehydrated with 15% sucrose for 2  h and then 30% sucrose overnight at 4c .
samples were embedded in tissue - tek oct compound and frozen in liquid nitrogen and then sectioned into 10  m thick slices . for whole - mount immunostaining ,
the organ of corti 's sensory epithelium was isolated from the cochleae and divided into apical , middle , and basal turn sections to then permeabilize the samples in 0.5% triton x-100 in pbs at room temperature for 15  min . the sections or cochlea samples were washed in pbs and then blocked in 10% goat serum in pbs at 37c for 30  min .
the samples were incubated with a primary antibody at 4c overnight . after washing with pbs , followed by further incubation with an anti - rabbit tritc - conjugated secondary antibody diluted in pbs at 37c for 1  h , followed by alexa fluor 488-conjugated phalloidin ( sigma - aldrich , usa ) at 37c for 30  min and 4,6-diamidino-2-phenylindole ( dapi ) at 37c for 10  min ,
cochleae were incubated with the following primary antibodies diluted in pbs : rabbit anti - myosin - iia ( 1  :  100 , cell signaling , usa ) , rabbit anti - myosin - iib ( d8h8 ) ( 1  :  100 , cell signaling , usa ) , rabbit anti - myosin - iic ( d4a7 ) ( 1  :  50 , cell signaling , usa ) , rabbit anti - zo-1 ( 1  :  400 , invitrogen , usa ) , and rabbit anti - e - cadherin ( 1  :  200 , cell signaling , usa ) .
goat anti - rabbit tritc - conjugated secondary antibodies ( 1  :  200 ) were from invitrogen , usa . cochlea samples were fixed and decalcified using a procedure similar to that used for the immunostaining assay , dehydrated by an ethanol series ranging from 30% to 100% , and embedded in paraffin to then be sectioned at a thickness of 10  m .
sections were deparaffinized by an ethanol series ranging from 100% to 30% , stained with hematoxylin and eosin ( h&e ) , and viewed under light microscopy ( nikon ys100 , japan ) .
mice were sacrificed 2 weeks after noise exposure and the cochlear epithelia were immunostained for hc counts .
briefly , the apical , middle , and basal parts of the basilar membrane were counted and labeled with alexa fluor 488-conjugated phalloidin to outline hcs and their stereocilia for quantitative assessment .
the numbers of missing ohcs were counted and the ratio of missing ohcs was expressed as a percentage .
repeated - measures anova was performed to evaluate the difference in abr threshold shifts , one - way anova or t - test was selected to test the myh14 expression and cochlear hearing loss , and statistical analysis was performed using graphpad software .
for all tests , a value of p < 0.05 was considered to be statistically significant .
to investigate the functions of myh14 in hearing , the crispr / cas9 genome - editing technology was used to destroy the myh14 gene in mice . in brief , a single guide rna ( sgrna ) containing a 20  nt target sequence and the endonuclease cas9 are essential for successful targeting .
when the sgrna guides cas9 to the target sequence , a double - strand break will be generated .
the double - strand break produces indels ( insertions and deletions ) , which then produce frameshift mutations .
exon 9 of the myh14 gene is the crispr - amenable target , as shown in figure 1(a ) .
the cas9 mrna and the sgrna , which were produced by in vitro transcription , were microinjected into the pronuclear stage of mouse embryos .
16 pups contained the mutation , as determined by genotyping sequence analysis , and surprisingly , all of them had heterozygous mutations .
then , ta clones of the pcr products were analyzed by dna sequencing , where a total of three types of mutations were shown ( figures 1(b ) and 1(c ) ) .
we chose the third - type mutation as our research subject , as this type of mutation can cause a frameshift mutation and a stop codon will appear eight amino acids later ( figure 1(d ) ) .
concurrently , we performed the off - target analysis using the mouse line mentioned above .
the results showed no off - target effect in our experiment ( data not shown ) . in order to produce a myh14 homozygous mutant , f0 male mice with a 1  bp insertion
then , the f1 heterozygous mice were inbred for one generation , obtaining a mouse strain homozygous for the myh14 gene . to confirm that the myh14 protein in the myh14 mutant mice was abolished , we performed a western blot analysis on the cerebellum ( where myh14 expression is high ) using specific antibodies .
as showed in figure 2(a ) , myh14 protein expression in the cerebellum was completely abolished in the homozygous mutants . finally , we performed immunocytochemistry experiments on cochlea whole mounts ( figure 2(b ) ) .
it has been reported that myh14 is primarily expressed in or near the reticular lamina . in our study , myh14 immunoreactivity in the apical junctional complexes ( ajcs ) was completely abolished in myh14 homozygous mutants .
these results consistently show the successful generation of a myh14 knockout mouse using the cba / caj strain .
tracking analysis of myh14 mice was performed to determine that there were no significant differences in the appearance of myh14 mutant mice and wild type mice .
the cochleae of three - month old myh14 mice showed no differences ( figure 3 ) .
no abnormalities were seen in either the hcs or the sgn ( spiral ganglion ) .
wild type mice ( n = 10 ) and myh14 mice ( n = 10 ) showed no statistical differences in abr thresholds until five months of age ( figure 4(a ) ) .
tone burst abr showed that 5-month - old myh14 mice have high - frequency hearing loss , and the statistics show that there are significant differences .
consistent with the abr results , immunofluorescence results showed no loss of hc and no change of sgn at three months ; however , a 4%10% hc loss was found in the cochlea basal turn of 5-month - old mutants .
sporadic ohcs were also lost in the middle and apical turn of the cochleae ( figures 4(b ) , 4(c ) , and 4(d ) ) .
the small effect of myh14 loss on hearing thresholds raised the possibility that other related genes might compensate for the absence of myh14 .
evidence shows that myh14 and myh10 are colocalized in cell - cell junctions and that myh14 shares great similarities with both myh9 and myh10 in structure ; therefore , they may have complementary effects in auditory function .
an immunoblot analysis was performed to clarify changes in the expression of myh10 and myh9 .
results showed that the expression of myh10 in the cochleae of myh14 mice was prominent increased , but changes in the expression of myh9 were not obvious ( figures 5(a ) and 5(b ) ) .
immunofluorescence results further verified the results of the immunoblot analysis ( data not shown ) .
to investigate the response of myh14 in mouse cochleae ( 2 months old ) to noise exposure , western blot analysis and immunofluorescent staining ( n = 3 for each group ) were performed to evaluate myh14 protein levels at different time points : before ( 2  h ) , during , and after ( 2 and 4  h , 1 , 2 , and 7 days ) noise exposure ( figure 6 ) .
we found that myh14 protein expression was largely dependent on noise exposure ( figures 7(a ) and 7(b ) ) .
the expression levels of myh14 were significantly upregulated by acoustic stimuli , and they reached the highest peak 2  h after noise treatment .
the results of immunofluorescence also showed that the upregulation of myh14 was most obvious 2  h after noise treatment in the hcs of the cochlea ( figure 7(c ) ) .
however , this upregulation gradually disappeared until seven days after noise exposure , when the expression of myh14 was restored to basal levels ( before noise exposure ) .
all these results showed that myh14 is upregulated after noise exposure and that this change is time - dependent . to determine the effect of myh14 knockout on nihl , we examined the response to acoustic trauma in myh14 mice in comparison to wild type controls ( 4 months of age , n = 8 for each group ) .
the results showed that abr thresholds were almost comparable between the two genotypes before acoustic stimuli .
then , animals were exposed to 210  khz band noise at 105  db spl for 4  h. abr recordings 2  h after exposure showed that acoustic overstimulation induced great hearing loss ( temporary threshold shift , tts ) in both myh14 mice and control groups ; moreover , there were no significant differences between the two genotypes .
the abr test showed that wild type mice fully recovered two weeks after acoustic trauma .
however , the hearing of mutant mice failed to recover two weeks after the acoustic trauma .
abr thresholds of the mutants were significantly increased compared to the controls two weeks after the high - level noise exposure ( figures 8(a ) , 8(b ) , 8(c ) , 8(d ) , and 8(e ) ) .
these results suggest that myh14 mice are less capable of recovering from nihl than controls . in order to compare the degree of damage to hcs after noise exposure in myh14 mice and control groups , immunofluorescence staining ( phalloidin ) was performed to compare hc losses of cochleae two weeks after noise exposure .
the results showed that the hc loss mainly happened in the ohcs , and loss of ohcs was more significant in myh14 mice compared to controls ( figures 9(a ) and 9(b ) ) .
however , the number of inner hair cells ( ihcs ) showed no obvious differences between the two groups of mice .
additionally , we did not observe defects in the spiral ganglion of either myh14 or control mice ( data not shown ) .
because myh14 is positioned at the apical junctions in mouse hcs , we examined the expression of tight - junction proteins , zo1 and e - cadherin , in myh14 mice and controls after noise exposure .
the immunofluorescence staining assay showed that the expressions of zo1 and e - cadherin were not altered ( data not shown ) .
immunoblot analysis and qrt - pcr were performed to examine if there is change in the expression level of myh9 and myh10 .
the results showed that the expression of myh10 in the cochleae of noise - exposed myh14 mice was significantly increased compared with the wild type mice , while there was no obvious change in the expression of myh9 ( figures 10(a ) and 10(b ) ) . in summary ,
myh14 mice are less capable of recovering from noise exposure , and more hcs are lost after acoustic trauma .
our data indicate that myh14 mice are more vulnerable to high - level noise exposure and that myh14 plays a protective role in noise - induced damage of ohcs .
according to the world health organization ( who ) survey results , nihl has become a very serious problem ; thus , numerous efforts are being focused on understanding the mechanism of nihl and on the treatment of hearing loss ( updated february 2015 ) . a relationship between nihl and various genes has been established . in recent years , many nihl susceptibility candidate genes , including myh14 , have been discovered .
myh14 , which belongs to the myosin family , is required for normal hearing in humans . to this date , many mutations of the human myh14 gene have been reported to cause dfna4-type hearing impairment .
however , the specific roles of myh14 in auditory function and nihl are not fully understood . in our current study
, we generated a myh14 mice strain to clarify the role of myh14 in the auditory system and in nihl .
caused by exposure to high - level noise , nihl is one of the most common forms of sensorial hearing loss .
several experimental animal studies have reported morphological and physiological changes in the inner ear of mice after excessive noise exposure .
great progress has been made in nihl - related gene research . currently , there are two main ways to study nihl susceptibility genes : one is mass screening of population and the other is animal experiments .
mass screening is a commonly used method for the study of susceptibility genes in nihl .
in addition , to perform a study of nihl susceptibility genes in a population , it is necessary to carry out a large - scale field survey to identify nihl - susceptible persons . compared with human groups , knockout mice are a good model for studying the mechanism of nihl . in the past years , various animal knockout models have been developed .
however , the establishment of traditional gene - knockout animal models must be based on traditional stem - cell methods .
it is impossible to avoid using 129 and c57bl/6 background mice , which have a problem of age - related hearing loss ( ahl ) [ 20 , 28 ] .
evidence shows that ahl and nihl have common molecular mechanisms [ 29 , 30 ] , and both c57bl/6 and some 129 mice tend to be more susceptible to nihl than cba / caj mice .
c57bl/6 mice lose hair cells and show significant hearing loss by 6 months of age , and several 129 substrains except for 129sv / ev also show age - related hearing loss [ 33 , 34 ] .
therefore , c57bl/6 and some 129 mice are not suitable strains for studying delayed hearing loss and nihl .
in contrast , cba / caj mice retain normal hearing up to 18 months of age , and there is no ahl in this strain .
consequently , the cba / caj mouse becomes one of the most ideal mouse strains for performing hearing - related research in the 80 inbred strains .
the crispr / cas9 technology developed in the past three years makes the manipulation of cba / caj mouse genes possible . in our current study , we combined the crispr / cas9 technology in the cba / caj mouse strain to generate the myh14 knockout model , thus avoiding the influence of an adverse genetic background on the experimental results . a previous study by ma et al .
they found that there were no obvious differences between myh14 mice and wild type mice [ 23 , 36 ] . in our current study , we generated a myh14 mouse using the cba / caj background . in this model ,
no obvious differences in appearance were found ; the cochleae of myh14 mice develop normally .
however , our myh14 mice have high - frequency hearing loss at five months ; furthermore , moderate hc loss was also found in the cochlea from the basal turn of the mutants , which also started during this time period .
it is understandable that these two mouse models may have different phenotypes ; a possible explanation is that the mouse strain used in this study is not the same as the one used in the previous study , and the susceptibility to nihl is different between strains [ 37 , 38 ] .
myosin is a superfamily of proteins related to the movement of molecules that are present in all eukaryotic cells ; it binds to actin and uses atp to move along the actin filament .
the myosin superfamily is divided into two major categories : traditional myosin ( nonmuscle myosin ii ; nm ii ) and nonconventional myosin ( myosins i and iii 
the distribution of the myosin families in the cochlea is different , and there is a difference in their normal physiological functions in the cochlea .
nm ii is thought to be involved in mediating epithelial tissue morphogenesis and tensional homeostasis , regulating force within epithelial apical junctions [ 3941 ] .
there are three isoforms of nm ii in the cochlea , encoded by myh9 , myh10 , and myh14 in mice .
therefore , the slight effect of myh14 loss on hearing thresholds raised the possibility that the other two genes may compensate for its absence .
the immunoblot analysis result showed that only the expression of myh10 was significantly increased , and there was no obvious change in the expression of myh9 .
a previous study reported that myh10 is the major isoform regulating schaffer collateral inputs ; thus , it is likely that the increase in myh10 , but not myh9 , can compensate for most of the functions of myh14 in cochlea .
the affected individuals exhibited progressive hearing impairment beginning in the 1st decade of life with profound hearing loss in the 4th decade .
this is to contrast with our myh14 mice where onset of progressive hearing loss began at 5 months of age .
this milder phenotype observed with our myh14 mice could be explained by a more pronounced compensatory effect of myh10 on myh14 in the murine model .
however , myh10 can not completely replace the function of myh14 because loss of hcs in myh14 mice was still observed from five months .
moreover , we can not exclude the possibility that other unidentified molecules may compensate for myh14 .
evidence from several mouse mutants showed that the pathogenesis of ahl and nihl is highly similar in most cases [ 17 , 18 ] . in our study ,
myh14 mice were used to investigate the role of myh14 in the mechanism of nihl .
we found that abrogation of myh14 increased the mouse susceptibility to acoustic trauma . in the wild type mice ,
105  db spl noise exposure for 4  h caused a tts , and their abr thresholds recovered two weeks after noise exposure .
in addition , more hc loss ( mainly ohcs ) was observed in mutant mice compared to controls two weeks after noise exposure .
consistent with previous reports [ 4345 ] , our results indicated that ohcs are more vulnerable to noise compared to ihcs . failed recovery of abr thresholds in mutant mice
in addition , we found no obvious differences in the spiral ganglion and lateral wall ( two other places easily affected by noise exposure ) between myh14 and control mice after noise exposure .
thus , our results indicated that myh14 plays an indispensable role in hcs , when compared to that in other parts of the cochlea .
together , these findings suggest that myh14 may play a beneficial role in the protection of the cochlea after acoustic stimulation in the cba / caj mouse line .
because myh14 is positioned at the apical junctions in the mouse hcs , we studied the expression of zo-1 and e - cadherin cell junction proteins in myh14 mice after noise exposure using immunofluorescence analysis to then compare them to the control group .
we found that there were no statistically significant differences between both of these proteins in the myh14 mice when compared to the controls .
a possible explanation is that loss of myh14 can damage the permeability of ions in a noisy environment . under normal conditions
, myh10 can partially compensate for the function of myh14 , but the absence of myh14 makes it easier for mutant mice to be damaged in ajcs compared to the controls in a noisy environment .
ajcs with long - term damage may cause changes in ion concentration , which eventually induced more hc loss in myh14 mice [ 46 , 47 ] .
we used the crispr / cas9 technology in the cba / caj mouse strain to generate a myh14 knockout model .
 myh14 is a member of the myosin family , which has been implicated in many motile processes such as ion - channel gating , organelle translocation , and the cytoskeleton rearrangement . 
 mutations in myh14 lead to a dfna4-type hearing impairment . 
 further evidence also shows that myh14 is a candidate noise - induced hearing loss ( nihl ) susceptible gene . 
 however , the specific roles of myh14 in auditory function and nihl are not fully understood . in the present study , we used crispr / cas9 technology to establish a myh14 knockout mice line in cba / caj background ( now referred to as myh14/ mice ) and clarify the role of myh14 in the cochlea and nihl . 
 we found that myh14/ mice did not exhibit significant hearing loss until five months of age . 
 in addition , myh14/ mice were more vulnerable to high intensity noise compared to control mice . 
 more significant outer hair cell loss was observed in myh14/ mice than in wild type controls after acoustic trauma . 
 our findings suggest that myh14 may play a beneficial role in the protection of the cochlea after acoustic overstimulation in cba / caj mice . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion