one eye of the guinea pigs was randomly selected and treated with  7.00d lenses .
the method of cycloplegia was induced with three drops of tropicamide , and the refraction status was measured by means of streak retinoscopy in hand - held , awake animals .
the axial length of the eyes was measured by an a - scan ultrasound while the animals were anesthetized with ketamine ( 80 mg / kg ) by intramuscular injection .
ocular refraction and axial length were performed before the experiment and 1 , 2 , and 3 weeks after minus lens intervention .
the measurement was repeated at least three times for each eye , and the refraction and axial length of each guinea pig at every measurement were averaged .
anova , with repeated measures design , was used to investigate the influence of the intervention on the axial length and refraction .
a one - way anova , followed by student 's unpaired t - test , with bonferroni correction for multiple testing , was used to analyze ocular parameters between groups .
this study was complied with the tenets of the association for research in vision and ophthalmology statement for the use of animal in ophthalmic and vision research .
after the refraction and axial length measurements at 1 , 2 , and 3 weeks of minus lens intervention , five guinea pigs were randomly selected and were deep anesthetized with 0.03 ml / kg chloral hydrate by intraperitoneal injection . with the animals under deep anesthesia
, both eyes were enucleated at a similar time point ( between 4:00 and 6:00 pm ) to minimize the effect of diurnal variation on gene expression .
a circumferential incision was made along the limbus , followed by removal of the cornea , crystalline lens , and vitreous body .
the entire retina was separated from the choroid while the sample was soaked in iced neutral buffer formaldehyde solution .
twenty - four hours later , the retina was embedded in paraffin to detect expression of mfn1 with immunohistochemistry ( streptavidin - perosidase ( sp ) three steps ) .
before the intervention , there were no statistically significant differences between the two groups in terms of the refraction ( p = 0.860 ) and axial length data ( p = 0.115 ) . however , repeated measures anova [ tables 1 and 2 ] revealed a statistically significant effect of minus lens intervention and a significant minus lens intervention by time interaction for the axial length and refraction from baseline ( p = 0.000 ) . the lens - induced myopia ( lim ) eyes became more myopic by 4.70d and had an increase of axial length by 0.46 mm after lens induction for 3 weeks [ table 3 ] .
the average increase of axial length was 0.46 mm in lens - induced eyes and 0.18 mm in the normal control eyes [ table 4 ] .
the results of axial length analysis by anova ( mm ) the results of refraction analysis by anova the refraction data measured in the two eyes the results of axial length in both eyes by t - test the results of protein expression [ figs .
mfn1-positive cells could be observed in the retina of both eyes , mainly in the cytoplasm and cell membrane of the ganglion cells ; mfn1-positive cells appeared brownish or yellow . in the lim eyes
, the immunopositive cells were staining deep , and more positive cells could be observed ; however , mfn1-positive cells scattered expressed in the ganglion cell layer in the control eyes . furthermore , mfn1-positive expression could be seen mainly in ganglion cells at the 1 week of treatment ; with the extension of lens induction time , many mfn1-positive cells also appeared in the bipolar cell layer and the rod - cone cell layer , and this phenomenon could not be found in normal control eyes .
( a and b ) one week after treatment  mitochondrial fusion protein 1-positive cells were observed in the retina of both eyes , mainly in the cytoplasm and cell membrane of the ganglion cells , and the positive cells seem staining deep in the lens - induced eyes ( a and b ) two weeks after treatment  the positive cells in the lens - induced eyes were staining deep , abundant - positive cells could be observed in the ganglion cell layer , and many mitochondrial fusion protein 1-positive cells could also be seen in the bipolar cell layer and rod - cone cell layer ; however , positive cells only scattered in the ganglion cell layer in the control eyes ( a and b ) three weeks after treatment  mitochondrial fusion protein 1-positive cells scattered expressed in the ganglion cell layer and the bipolar cell layer of the control eyes , while many positive cells appeared in the rod - cone cell , the bipolar cell and ganglion cell layer in the lens - induced eyes
to the best of our knowledge , this is the first study that describes the mfn1 gene expression and myopia . in a previous british study ,
five single nucleotide polymorphism ( snp ) loci around the mfn1 gene were found strongly associated with myopia ( including high , medium , and low myopia ) . especially , the snp locus of rs6794192 and rs7618348 showed lower p values . in our recent study , we genotyped rs3976523 which was linked with rs6794192 ( r = 0.942 ) in the myopia population , and no correlation was found between rs3976523 and myopia . for rs7618348 , we found that the p values in the allele difference between controls and myopia patients were more than 0.05 .
however , we also genotyped another two other loci in the mfn1 gene ( rs6762399 and rs13098637 ) in our previous study .
interestingly , we found that rs13098637 locus located in an intron at the center of the mfn1 gene was significantly correlated to myopia .
although human studies have revealed mfn1 as a candidate gene of myopia , the relationship between the mfn1 expression and myopia has not been investigated . in this study
, we further investigated the expression of mfn1 in the retina , so as to get a better understanding about its possible involvement in the occurrence or development in the mammalian myopia . guinea pig is a kind of mammals , the eyeball structure is similar to the human 's , and myopia animal model of guinea pig has successfully been established .
all guinea pigs were raised with a monocular 7.00d lens , and the fellow eye was untreated and served as the self - control group . during the 1 week , both axial length ( 0.04 mm in the control eyes and 0.11 mm in the experimental eyes ) and refraction data changed in both groups ( 0.25d in the control eyes and 0.43d in the experimental eyes ) , but the refraction data did not show any statistically significant difference ( p = 0.388 ) whereas the axial length showed a statistically significant difference ( p = 0.008 ) .
the axial length in the lens - induced eyes increased rapidly when compared to control group , owing to the minus lens intervention .
the refraction data are the combined effect of axial length and the refractive components of the eye , but we suspect that the refractive components did not change to compensate for the axial length change during the 1 week in this research
. this could be the reason for the refraction data did not show a significant difference between the two groups during the 1 week while the axial length showed a significant difference . over the time of intervention , about 2 weeks later , the refraction data and axial length were both significantly different between the two groups .
three weeks after the intervention , the lens - induced eyes became more myopic , and the mean refraction was 1.78d in lim eyes whereas it was + 1.05d in control eyes , which indicated that myopia animal model had been successfully established by a 7.00d minus lens .
in the current study , mfn1-positive cells could be observed in the retina of both eyes . in the lim eyes
, the immunopositive cells were staining deeper and more positive cells could be observed while mfn1-positive cells only scattered expressed in the ganglion cell layer of the control eyes .
in addition , in the lim eyes , 1 week after minus lens intervention , we could find that mfn1 expression positive cells mainly located in the ganglion cell layer , with the intervention time extension , more and more positive cells occurred in the bipolar cell layer and the cone - rod cell layer , especially in the cone - rod cell layer , abundant - positive cells could be seen and the dyeing color was very deep .
these results indicated that mfn1 overexpression in the retina of the experimental eyes might be associated with the development of lim . from both the mfn1 expression results and refraction data
, we also speculated that minus lens intervention may interrupt the emmetropization in experimental eyes of animals and also the physiological control of gene expression .
, the investigation of mfn1 expression in the retina had not been extensively studied ; in addition , the sample size in this experiment was a little small , and so this investigation could only be looked as a preliminary exploratory research .
unfortunately , in the present study , only according to the images , we only could qualitatively describe this interesting phenomenon on mfn1 expression .
hence , mfn1 expression in the retina should be quantitatively studied and analyzed in future research .
we found some rough alterations of mfn1 expression in the lim eyes of the guinea pigs . by combining our previous study which found that rs13098637 locus within the mfn1 gene was related to myopia
, we speculated that mfn1 genetic variants might be likely to influence the development of myopia and that the relationship between myopia and mfn1 should be worthy of further investigation .
this study was supported by the national nature science foundation of china ( 81341105 ) and by the research funds from the health department of sichuan province ( 120442 ) .
this study was supported by the national nature science foundation of china ( 81341105 ) and by the research funds from the health department of sichuan province ( 120442 ) .
 purpose : the aim of this study is to preliminarily investigate the expression of mitochondrial fusion protein 1 ( mfn1 ) in a lens - induced animal myopia ( lim ) model and to explore the relationship between mfn1 and the visual development.materials and methods : mfn1 gene expression in guinea pigs was examined during the development of minus lim , 15 tri - colored guinea pigs were obtained , and one eye of each pig was randomly selected and treated with 7.00d lenses . 
 ocular refraction and axial length were collected before intervention and 1 , 2 , and 3 weeks after intervention . 
 after the refraction and axial length measurements at 1 , 2 , and 3 weeks of lens intervention , five guinea pigs were randomly selected . 
 mfn1 expression in the retina of both eyes was tested by immunohistochemistry technique.results:mfn1-positive cells could be observed in the retina of both eyes . 
 the positive cells in the lim eyes were staining deeper , and much more positive cells could be observed . 
 furthermore , mfn1-positive expression could be seen mainly in ganglion cells after 1 week of minus lens intervention , and with time extension , more and more positive cells appeared in the rod - cone cell and bipolar cell layer , and this phenomenon could not be found in the normal control eyes.conclusion:this study suggested that mfn1 might be correlated to the development of myopia . 
Materials and Methods
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