| Myopia is the factor that leads to the highest elimination rate in the pilot recruitment work,and is also one of the main reasons that lead to the suspension of flight students.At present,it is considered that increasing outdoor activities and using low-concentration atropine are effective means to prevent myopia in adolescents.However,according to the survey of the students in the Teenagers Aviation School,even if they pass the strict visual examination when enrolled and ensure sufficient outdoor activities and exercise during their study,more than 20%of the students still develop myopia after three years of high school study,suggesting that the effect of the existing commonly used measures for myopia prevention and control is still not ideal.Adolescent myopia is mainly axial myopia.Ocular axial length of eye is closely related to diopter.When axial length of eye increases by 1mm,diopter increases by 3.0D.The remodeling of sclera,the outermostly tough tissue of the eye,is an important link that affects the development of the eyeball and the changes of the ocular axis.Abnormal visual signals can lead to remodeling changes of scleral collagen and other related molecules,resulting in changes in axial length of the eye and thereby affecting the diopter.Therefore,intervention of scleral remodeling is a hotspot in the prevention and control of myopia in adolescents.It is generally believed that the true myopia caused by the growth of ocular axis cannot be recovered or reversed,but the report on the deep space mission found the phenomenon of flattening of the eyeball at posterior pole,shortening of the ocular axis,and hypermetropic displacement of the refractive state on some astronauts who underwent the long-term space flight,indicating that the changes of stress environment can affect the remodeling mechanism of sclera.According to such special phenomenon,we reckon that the change of stress environment can affect the plasticity of sclera and then participate in the prevention and development of myopia.If active exercise can be used to promote the blood transfer to the head and increase the hydrostatic pressure gradient of head and eye tissues,it may enable the local sclera to remodel toward shortening ocular axis or the slower trend of ocular axis growth.Once the above objectives can be achieved through exercise,it could provide a new idea for the prevention and control of juvenile myopia.Because of the small eyeball size of rats and mice,it is difficult to accurately measure the changes in the axial length of the eyes by clinical means,and the optometric method is affected by human and lens（±0.25D error）.In order to meet the requirements of this study,we first explored and established the measurement method of the eye axis length of small animals,meanwhile compared and observed the relationship between the eye axis and body mass of different strains of animals.On this basis,the effects of postural changes on the axis of eyes in rats with form deprivation or congenital static night blindness were observed.ObjectiveCombined with environmental factors and genetic factors,we explored whether postural changes could affect the scleral remodeling process.Materials and Methods1.Research on the establishment and application of measuring method of axial length of eye in small animalsUsing the ophthalmic optical biometrics instrument（SW-9000,Tianjin Sauver Electronic Technology Co.,LTD.）,we explored and established the method of measuring the axial length of the eyes in rats and mice,and identified the normal development trend and differences of the axial length of the eyes in normal rats or mice and in animals with different retinal degeneration.Eighty-two SPF-grade SD rats and 76 C57BL/6 mice of different body weights were purchased from the Experimental Animal Center of Air Force Medical University as normal controls.The spontaneous congenital stationary night blindness（CSNB）rats（76 rats）,HL rats（42 rats）,retinal cone dysfunction were selected for self-established treatment.RCD)rats（50）and Cone photoreceptor function loss 1（Jackson Laboratory,USA）cpfl1)mice（25）,Cone photoreceptor function loss 3（cpfl3）mice（11）and congenital stationary night blindness（No b-wave 2,Nob2）mice（22）were observed.The animals were divided into different groups according to body weight（rats were divided into 10 groups at 50g intervals and mice were divided into two groups at 10g interval）.The ocular axis of the rats and mice was measured to clarify the basic development of the ocular axis of each strain of rats and mice.Following that,male CSNB+/+and RCD+/+rats,retinal were selected to establish a family of mice+/+and Nob2 mice+/+with normal animals.The F1 generation littermates were then inbred to obtain F2 generation littermates,and the eye axis length was observed according to ERG phenotype distinguishing two groups of phenotypically positive（abnormal ERG waveform）homozygous+/+animals（Positive group,PC）from phenotypically negative（normal ERG waveform）wild-type+/-and heterozygous+/+animals（Negitive group,NC）,whose axial length was observed.2.Effect of body position changes on sclera remodeling in visual form-deprived rats48 SPF grade SD rats（male,4 weeks old）were randomly divided into complete control group（Control）,postural change group（SUS）,form deprivation group（FDM）and postural change+form deprivation group（SUS+FDM）,with 12 rats in each group.The left eye was taken as the model eye for inter-group control.Axial myopia of rats was induced by form deprivation,whose body position changes were achieved by tail suspension.Form deprivation and body position changes were performed simultaneously for 6 weeks.The intraocular pressure（IOP）,axial length and diopter of rats in each group were measured at0,2,4 and 6-week time point after the experiment.The structure of sclera was observed by HE staining,while the changes of Collagen I,MMP-2,TIMP-2 and other related proteins in rats sclera were observed by Western Blotting and immunohistochemical staining.Among them,animals at the 6-week time point first underwent slit lamp,fundus photography and ERG examination,and then observed other indicators to determine whether the 6-week postural changes caused other injuries of eyes.3.Effects of 4-week body position changes on sclera shaping in visual form-deprived ratsIn the above same way,24 SPF grade SD rats（male,4 weeks old）were randomly divided into complete control group（Control）,postural change group（SUS）,form deprivation group（FDM）and postural change+form deprivation group（SUS+FDM）,with6 rats in each group.Among them,the SUS+FDM group received body position changes intervention for 4 weeks after 2 weeks of form deprivation,while the start time and duration of body position changes in SUS group were the same.At the time point of 0,2,4 and 6weeks after the experiment,the indexes same as above were observed.4.Effects of body position changes on sclera remodeling in rats with congenital stationary night blindnessIn the experiment,spontaneous CSNB model rats（cacna1f gene mutation,sex-linked recessive inheritance）were selected to establish the research family,method is same as above,and F2 generation rats were selected as the research objects.According to ERG phenotypes,they were divided into complete control group(wild type-/-or heterozygote+/-)（Control group）and experimental group(homozygote+/+)（SUS group）,and underwent 4-week postural intervention.At the time point of 0,2,4 and 6 weeks after the experiment,the axial length and diopter were observed.The scleral tissue structure was observed by HE staining,and the expressions of Collagen I,MMP-2,TIMP-2 and other related proteins in the scleral tissues of rats were observed by Western Blotting and immunohistochemical staining.Among them,animals at the 4-week time point underwent slit lamp,fundus photography and ERG examination before other indicators were observed to determine whether the postural changes at the 4-week time point caused other eye injuries.Results1.Relationship between axial length of eye and body mass in different strains of animals.The ophthalmic optical biometrics instrument（SW-9000,Tianjin Sauver Electronic Technology Co.,LTD.）could be used to measure the ocular axes of rats and mice with different body weights,and the coefficient of variation was less than 5%,indicating that the method was of high precision in the extraction and could sensitively reflect the changes of the ocular axes of small animals.The ocular axis of rats and mice grew with the increasing of body weight,and there were differences between different strains.The axial length of CSNB rats with 100-150g body weight was greater than that of SD rats（P<0.05）.The ocular axis length of RCD rats with body weight of 150～200g was larger than that of SD rats（P<0.01）,but with no statistical difference between SD and CSNB rats;the ocular axis of HL and RCD rats with body weight of 200～250g,450～500g and 500～550g was longer than that of SD rats（P<0.05）,but with no statistical difference between SD and CSNB rats;the ocular axis of HL,RCD and CSNB strain rats with 250～300g and 300～350g body weight was longer than that of SD rats（P<0.01）;the ocular axis of CSNB and RCD rats with350～400g body weight was longer than that of SD rats（P<0.01）,but with no statistical difference between SD rats and HL rats;the ocular axis of the four strains of rats with body weight of 400～450g changed with no statistical difference;the ocular axis of HL rats with body weight of 550～600g was greater than that of SD rats（P<0.05）with no statistical difference between CSNB and SD rats.The length of ocular axis of C57BL/6 mice with full body weight was shorter than that of cpfl1,cpfl3 and Nob2 mice（P<0.01）.The axial length of eyes in F2 normal CSNB and RCD rats was shorter than that in control homozygous rats（P<0.05,P<0.01 at some time points）after each strain of rats or mice was established with healthy rats or mice.At 4 and 6 weeks of birth,the axial length of the eyes of the rdf mice with normal phenotype was lower than that of the control pure wok（P<0.01 at 4 weeks and P<0.01 at 6 weeks）,and the axial length of the eyes of the Nob2 mice with normal phenotype was shorter than that of the homozygote at 6 weeks of age（P<0.05）.2.The changes of sclera shaping in visual form-deprived rats after 6 weeks of body position changesThe change of body position has a certain effect on the intraocular pressure of rats.The IOP fluctuation range of rats in Control group was 10.00～12.00 mm Hg during the period from 4 to 10 weeks of age,with FDM group 10.00～12.00 mm Hg,SUS group 10.75～13.25mm Hg,and SUS+FDM group 10.75～14.00 mm Hg.The body position changes had a greater impact on IOP,and there were statistical differences in individual time points（P<0.01）but within the physiological range.The axial length of each experimental group gradually increased with time,in which the diopter of rats in FDM group and SUS+FDM group gradually changed to the direction of myopia（P<0.01）compared with Control group at the time point of 4 weeks after the experiment and their axial length became shorter（P<0.01）at the time point of 6 weeks after the experiment but with no difference between the two groups（P>0.05）.Except that,the axial length of SUS group was lower than that of Control group at 6 weeks（P<0.05）.HE staining results showed that the sclera structure of rats in FDM or SUS+FDM group was significantly changed,with the relatively thin sclera thickness,the sparse and not compact collagen fibers arranged in disorder in each layer,and the increased space between fibers,while the sclera structure of SUS group was not abnormal.The expression levels of Collagen I and TIMP-2 protein in the sclera of rats in the experimental group（SUS group,FDM group,SUS+FDM group）were higher than those in the Control group at each time point（Partial time point showed P<0.05）,while the expression of MMP-2 protein showed the opposite changes.The expression of related proteins in FDM group and SUS+FDM group showed statistical difference from that in Control group after 2 weeks experiment.After 6 weeks,the expression level of Collagen I and TIMP-2 protein in SUS+FDM group was higher than that in FDM group,while the expression level of MMP-2 protein was lower than that in FDM group（P<0.05）.According to slit lamp and fundus photography,there was no obvious damage to the ocular surface lesion,lens and retina of rats in each group,while the ERG b wave of rats in SUS group and SUS+FDM group decreased significantly after 6 weeks of body position changes（P<0.01）.3.The changes of sclera shaping in visual form-deprived rats after 4 weeks of body position changesSince the ERG function of rats was significantly decreased at 6 weeks after the postures were changed,the study was conducted at the time point of 4 weeks after the postures were shortened.The result shows that the axial length and diopter of FDM group and SUS+FDM group changed towards the direction of myopia after 6-week experiment（P<0.01）,with no statistical difference between the two groups（P>0.05）.There was no significant difference in the axial length and diopter between SUS group and Control group at the point of 4 weeks after experiment（P>0.05）.In HE staining section,the sclera structure of the eyes of rats in FDM group and SUS+FDM group changed significantly,also with the relatively thin sclera thickness,the sparse and not compact collagen fibers arranged in disorder in each layer,and the increased space between fibers,but there was no difference between the two groups and the sclera tissue of rats in Control group and SUS group did not change significantly.The results of Western Blotting showed that the levels of Collagen I and TIMP-2 protein in FDM group were lower than those in Control group,while MMP-2 was the opposite（P<0.05）,also with higher MMP-2 level than Control group in SUS+FDM group（P<0.05）.It also showed that the expression level of TIMP-2 in SUS group,FDM group and SUS+FDM group was higher than that in Control group（SUS group and SUS+FDM group P<0.05,FDM group P<0.01）,with no statistical difference between FDM group and SUS+FDM group（P>0.05）.According to slit lamp and fundus photography,there was no obvious damage to the ocular surface,lens and retina of rats in each group,and there was no abnormal change in ERG waveform.4.Effect of postural changes on scleral remodeling in rats with congenital static night blindnessThe ocular axis length of rats in SUS group was longer than that in Control group（P<0.05）before the experiment,but with slower growth rate than Control group during the experiment.The refraction of rats in the Control group and SUS group was both hyperopia before the experiment,but the development speed of emmetropization in the SUS group was faster than that in the Control group with statistical difference from 2 weeks to 4 weeks after the experiment（P<0.01）,which showed the closing to emmetropization in the SUS group and slight hyperopia in control group in 4 week after the experiment.In HE section,there was no significant changes in the sclera structure of rats in both groups.And in westernblot test,compared with the control group,the experimental group showed lower expression level of Collagen I protein（P<0.05）and slightly lower expression level of MMP-2 and TIMP-2（P>0.05）in the sclera of rats.There was no damage to the ocular surface,lens and retina of rats in the control group and experimental group by visual safety inspection.ConclusionsThe experiment has successfully established a method to observe the changes of the length of the eye axis in rats and mice by using the biometrics.The close relationship between the eye axis of rats and mice and body mass was observed.It was found that the eye axis of rats and mice was still growing in adulthood,which was different from that of humans.The axis of eyes of rats and mice with various degenerative retinopathy was longer than that of normal control animals with the same body mass.After 6 weeks of postural change,the axial length of eyes in normal rats tended to shorten,but the axial length of eyes in shape deprivation rats was not significantly affected.4 weeks of continuous postural changes had no significant effect on axial length of eyes in normal rats and shape deprivation rats.It has certain effect on rats with congenital static night blindness.Postural changes at6 or 4 weeks had certain effects on the expression of sclera related collagen in normal rats,shape deprivation rats or congenital static night blindness rats.Under the conditions of this experiment,no clear effects of body position change on the axial length,diopter and organizational structure of the rats were observed,and there were clear effects on the proteins related to scleral remodeling.This has certain scientific value for further exploring the prevention and control methods of myopia in adolescents,and also for exploring a more appropriate animal model and research paradigm in the future.It provides a new research idea to compare the influence of different sports on the prevention and control of myopia. |
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