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Objective:To observe the prevention and control effect of 1% atropine on the progression of form deprivation myopia (FDM) in guinea pigs and the potential biological mechanism.Methods:Sixty-nine 3-week-old tricolor guinea pigs with normal refraction were randomly divided into a normal control group ( n=19), a FDM group ( n=19), a FDM+ atropine group ( n=19), and an atropine group ( n=12). No intervention was given to guinea pigs in normal control group.The FDM model was established by covering the right eye of guinea pigs with a semitransparent latex facemask for 4 weeks in FDM and FDM+ atropine groups.For the FDM+ atropine group, 1% atropine gel was topically administered to the form-deprived right eyes once a day for 4 weeks.For the atropine group, the right eye was treated with 1% atropine gel once a day for 4 weeks.Refraction and axial length of guinea pigs were measured by retinoscopy and ophthalmic A-scan ultrasonography respectively at baseline, experiment week 2 and week 4.In experiment week 4, eyeballs were enucleated to make sections via the paraffin wax processing procedure, and the microstructural and ultrastructural changes of the sclera were observed under the light microscope and transmission electron microscope, respectively.The isobaric tags for relative and absolute quantitation labeling combined with liquid chromatography-tandem mass spectrometry were used to identify the differentially expressed proteins.Use and care of the animals complied with the Regulation for the Administration of Affairs Concerning Experiment Animals by State Science and Technology Commission.The study protocol was approved by the Institutional Animal Care and Use Committee of Tianjin Medical University (No.TJYY2020111028). Results:There were statistically significant differences in the diopter of guinea pigs at different time points among the four groups ( Fgroup=138.892, P<0.001; Ftime=167.270, P<0.001). Compared with normal control group, the diopter of guinea pigs in FDM group at experiment weeks 2 and 4, and FDM+ atropine group at experiment week 4 developed toward myopia, showing statistically significant differences (all at P<0.001). Compared with FDM group, the diopter of guinea pigs in FDM+ atropine group at experiment weeks 2 and 4 developed toward hyperopia, showing statistically significant differences (both at P<0.001). There were statistically significant differences in the axial length of guinea pigs at different time points among the four groups ( Fgroup=32.346, P<0.001; Ftime=353.797, P<0.001). The axial lengths of FDM group at experiment weeks 2 and 4 and FDM+ atropine group at experiment week 4 were longer than those of normal control group, and the axial lengths in FDM+ atropine group at experiment weeks 2 and 4 were shorter than those in FDM group, and the differences were statistically significant (all at P<0.001). The collagenous fibers of posterior sclera of guinea pigs were loose and disordered in FDM group, and were regular in FDM+ atropine group.The posterior scleral thickness of normal control group, FDM group, FDM+ atropine group and atropine group was (141.74±16.98), (101.46±9.15), (112.74±6.24) and (134.30±18.19) μm, respectively, with a statistically significant difference ( F=6.709, P=0.005). The posterior sclera was significantly thinner in FDM group than in normal control group and FDM+ atropine group (both at P<0.05). The diameter of posterior scleral collagen fiber gradually increased from inside to outside in normal control group, FDM+ atropine group and atropine group, and the diameters of the inner, middle and outer posterior scleral collagen fibers were smaller in FDM group than in normal control group.Proteomic analysis revealed 85 differentially expressed proteins (fold change>1.30) between FDM group and normal control group, FDM+ atropine group and FDM group, of which 38 were up-regulated and 47 were down-regulated after atropine treatment.Gene Ontology enrichment analysis showed that biological processes mainly involved were biological regulation, cell process, localization and metabolic process.Molecular function mainly involved were binding, catalytic activity, molecular function regulator, structural molecule activity and transporter activity.Cell components mainly involved were in cellular anatomical entity, intracellular and protein-containing complex. Conclusions:Atropine can increase the diameter of scleral collagen fibers in guinea pigs of FDM model, improve the arrangement of scleral collagen fiber, inhibit scleral thinning.The mechanism of atropine to control myopia progression is closely related to the tight junction between scleral cells, cytoskeleton and extracellular matrix remodeling.
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AIM: To investigate the effect of the intravitreal injection of vascular endothelial growth factor-A165(VEGF-A165)on the scleral remodeling of guinea pigs with form-deprivation myopia(FDM).METHODS: A total of 120 tricolor guinea pigs, aged three weeks, were randomly divided into 6 groups, with 20 in each group. The blank group did not undergo any intervention. In the FDM group, only the FDM model was established. In the phosphate buffer saline(PBS)group, 2.5 μL of PBS was injected into the vitreous cavity before establishing the FDM model. In the 1ng group, 5ng group, and 10ng group, VEGF-A165 was injected into the vitreous cavity at concentrations of 1, 5 and 10ng, respectively, before the establishment of the FDM model. The FDM model was established by covering the right eyes of guinea pigs with translucent balloons for 14d. The diopter and axial length of the right eyes were measured before and after covering. After 14d, the content of dopamine(DA)in retina was measured by high performance liquid chromatography. Additionally, the mRNA and protein expression levels of matrix metalloproteinase-2(MMP-2), tissue inhibitor of matrix metalloproteinase-2(TIMP-2), transforming growth factor(TGF)-β1, TGF-β2 and α-smooth muscle actin(α-SMA)in sclera were detected by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.RESULTS: Before covering, there were no significant differences in the diopter and axial length of the right eyes of guinea pigs in all groups(P>0.05). After 14d of modeling, when compared with the blank group, FDM group showed an increase in the degree of myopia in the right eye, a prolongation of the axial length, a decrease in the content of DA in the retina, and an increase in the expression of MMP-2, TGF-β2 and α-SMA in the sclera. Conversely, the expression of TIMP-2 and TGF-β1 were decreased(P<0.01). However, in comparison to the FDM group, the degree of myopia in the 1ng, 5ng, and 10ng groups of guinea pigs decreased, the growth trend of axial length slowed, the content of DA in the retina increased, and the expression of MMP-2, TGF-β2 and α-SMA in the sclera decreased. Furthermore, the expression of TIMP-2 and TGF-β1 in the sclera increased(P<0.01). As the concentration of intravitreal injection of VEGF-A165 increased, the degree of myopia in the right eye of guinea pigs gradually increased, and the axial length gradually prolonged. The content of DA in the retina gradually decreased, the expression of MMP-2, TGF-β2, and α-SMA in the sclera gradually increased, while the expression of TIMP-2 and TGF-β1 decreased gradually.CONCLUSION: Intravitreal injection of VEGF-A165 can increase the content of DA in the retina of FDM guinea pigs, affect the expression of MMP-2, TIMP-2, TGF-β1, TGF-β2 and α-SMA in the sclera, and inhibit scleral remodeling of guinea pigs. Notably, the VEGF-A165 at the concentration of 1ng showed the most significant efficacy.
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AIM: To investigate the effects of modified Zhujing Pill on the mitochondrion structure and dynamin-related protein of retinal pigment epithelial cells(RPEs)in mice with form deprivation myopia.METHODS: 3-week-old C57BL/6J mice were randomly divided into control group, model group and Chinese medicine group, with 10 mice in each group. Myopia model of the right eye of mice was established by means of form deprivation in model and Chinese medicine groups. After 4wk, the Chinese medicine group were given intragastric administration of modified Zhujing Pill suspension 0.546g/(kg·d)(0.2mL/d)for 4wk, and same amount of saline was given to mice in other groups at the same time of modeling. The axial length and diopter of the right eye of the mouse were measured before and after the experiment by A-ultrasound and a strip retinoscope respectively. At the end of the experiment, the mitochondrial ultrastructure of RPEs was observed by transmission electron microscope. Western blot, and real-time fluorescent quantitative PCR(q-PCR)were used to detect quantitative and gene expression of mitofusin 1(MFN1), optic atrophy 1(OPA1), and dynamin-related protein 1(DRP1)in retinal tissues respectively.RESULTS: At the beginning of the experiment, there was no statistically significant difference in axial length and diopter of the right eye of the mouse in control, model and Chinese medicine groups(P>0.05). At the end of the experiment, compared with the control group, the mice in the model group and the Chinese medicine group had lower diopter and continuously prolonged axial length(all P<0.05), while the mice in the Chinese medicine group had significantly shorter axial length and higher diopter than the model group(all P<0.05). Western blot and q-PCR results showed that the relative expression of MFN1 and OPA1 decreased and DRP1 increased in both the model group and the Chinese medicine group compared with the control group(all P<0.05), and the relative expression of MFN1 and OPA1 increased in the Chinese medicine group compared with the model group(all P<0.05). The electron microscopic results showed that the mitochondria in the right retina of the mice were only mildly swollen in the Chinese medicine group, while the mitochondria in the model group were obviously swollen and disordered and empty.CONCLUSION: Modified Zhujing Pill could protect the retinal mitochondria by regulating the key proteins of mitochondrial dynamics(MFN1, OPA1, and DRP1), and it has a protective effect on the retina of axial myopic mice.
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Objective:To investigate the effects of different intensity of lighting on normal refractive development and form deprivation myopia (FDM) in guinea pigs.Methods:A total of 108 healthy 3-week-old guinea pigs were divided into normal refractive development guinea pigs ( n=54) and FDM guinea pigs ( n=54). FDM models were prepared in FDM animals by occlusion of the left eyes using an opaque mask, and the bilateral eyes were open in the normal refractive development guinea pigs.The guinea pigs were randomized to low (20 lx), normal(300 lx), and high intensity-lighting (5 000 lx) groups with a 12-hour light/12-hour dark cycle for 6 consecutive weeks under LED light.The ocular biometry was performed in a two-week interval.Axial length (AL) and dilated diopter were measured by A-scan ultrasonography and retinoscopy, respectively, and were compared after different lighting durations, and the change trends of them in normal refractive development and FDM guinea pigs were evaluated. Results:The AL values were not significantly different among low, normal and high intensity-lighting groups ( Fgroup=0.365, P=0.697), and the AL was gradually prolonged over the lighting duration ( Ftime=353.750, P<0.001). The diopters showed a statistically significant difference among different intensity-lighting groups ( Fgroup=3.576, P=0.034). The diopter in high intensity-lighting for 4 weeks was (+ 2.75±2.15) D, which was significantly higher than (0.41±3.07) D in the normal refrective development guinea pigs ( P<0.001). In the FDM guinea pigs, both AL and diopter were not significantly different among low, normal and high intensity-lighting groups ( Fgroup=0.105, P=0.900; Fgroup=0.973, P=0.387), and significant differences were seen in AL and diopter among three groups ( Ftime=408.302, 27.407; both at P<0.001). The diopter in FDM eyes of low intensity-lighting for 2 weeks was (+ 2.35±1.95) D, which was higher than (+ 1.90±0.97) D before lighting, with no statistically significant difference between them ( P>0.05). The AL was shortest and the AL change was smallest in normal refractive development guinea pigs of high intensity-lighting group.The diopter change in FDM guinea pigs of the low intensity-lighting group was significantly smaller than that in the normal intensity-lighting group ( P<0.001), with a transient hyperopia drift. Conclusions:The 5 000 lx lighting can slow down the development toward myopia in the normal refractive development eyes, and 20 lx lighting tends to delay the progression FDM eyes with a hyperopic shift after lighting for 2 weeks.
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Objective:To investigate the role and mechanism of retinal Sigma-1 receptor antagonist N, N-diethyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethanaminehydrochloride (NE-100) in the formation of form deprivation myopia (FDM) in guinea pigs.Methods:Eighty-five 21-day-old guinea pigs were selected, and 36 of them were randomly divided into normal control group, occluded 14-day group and occluded 11-day group, with 12 in each group.The right eyes of guinea pigs in the occluded 14-day group were covered with translucent eye shield for consecutive 14 days, and guinea pigs in the occluded 11-day group were treated in the same way for consecutive 11 days plus 3 days without cover, and guinea pigs in the normal control group were not covered.The other 49 guinea pigs were randomly divided into FDM group ( n=10), FDM+ NE-100 6 μg group ( n=12), FDM+ NE-100 60 μg group ( n=10), FDM+ NE-100 600 μg group ( n=9), and FDM+ saline group ( n=8). The right eyes in each group received 100 μl peribulbar injection of NE-100 6 μg, 60 μg and 600 μg or saline once a day according to grouping.Ocular refraction and axial dimensions were measured using eccentric infrared photorefractor and A-scan ultrasonography, respectively.Corneal curvature was measured with keratometer.Immunohistochemical staining and Western blot were used to detect the expression levels of Sigma-1 receptor protein, and retinal dopamine content was evaluated by high-performance liquid chromatography with electrochemical detection.This study was approved by an Ethics Committee of the Department of Laboratory Animal Science of Central South University (No.2020sydw0084). The use and care of experimental animals followed the Regulations for the Administration of Affairs Concerning Experimental Animals in China. Results:There were statistical significant differences in diopter and axial length among the normal control group, occluded 14-day group and occluded 11-day group ( F=147.81, 160.10; both at P<0.01). Compared with the normal control group, the relative myopia was the deepest and the axial length was the longest in the occluded 14-day group, then the occluded 11-day group, showing significant differences between them (all at P<0.05). In the normal control group, Sigma-1 protein was mainly expressed in retinal ganglion cells (RGCs), photoreceptor inner segment and the inner nuclear layer.In the occluded 14-day group, Sigma-1 protein staining was enhanced in RGCs and photoreceptor inner segment.Sigma-1 staining positive cells in the inner nuclear layer were increased significantly and were also seen in the inner and outer plexiform layers, especially in Müller cells, in which the expression levels of Sigma-1 receptor protein were significantly increased.Compared with the occluded 14-day group, the Sigma-1 receptor protein expression levels in the retina of the occluded 11-day group was significantly decreased ( P<0.01). The diopters of guinea pigs in the FDM+ NE-100 6 μg, 60 μg and 600 μg groups were lower than those in the FDM group, and the diopters of FDM+ NE-100 60 μg and 600 μg guinea pigs were lower than those in the FDM+ NE-100 6 μg group, and the differences were statistically significant (all at P<0.05). The dopamine content in the retina of the FDM+ NE-100 60 μg group was (0.74±0.09) ng/mg, which was significantly higher than (0.57±0.10) ng/mg in the FDM group, with a significant difference between them ( t=15.18, P<0.01). Conclusions:Sigma-1 receptor antagonist inhibits FDM formation, which may be associated with the elevation of dopamine content in retina.
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@#AIM: To investigate the effect of modified Zhujing pill on retinal thickness and apoptosis in form deprivation myopia(FDM)mice.<p>METHODS: Totally 72 C57BL/6J mice aged 3-week-old were randomly divided into control group 1, model group 1, intervention group 1, control group 2, model group 2 and intervention group 2, with 12 mice in each group. The first three groups were tested for 3wk and the last three groups were tested for 6wk, except for the groups of control 1 and control 2, all the mice used translucent goggles to cover their right eyes for form deprivation. The mice of intervention 1 and intervention 2 were respectively given intragastric administration modified Zhujing pill suspension 0.546g/(kg·d)(0.1mL/d)for 3wk at the beginning and after 3wk of the experiment. Same amount of saline was given to mice in other groups at the same time of modeling. The eye axis was measured before and after the experiment. At the end of the experiment, the eye of mice was taken for HE staining to observe the thickness changes of each layer of retina. Immunohistochemistry(IHC)and western blotting(WB)were used to detect Bcl-2 and Caspase3 expression of protein.<p>RESULTS: At the end of the experiment, the axis of model group 1 was significantly higher than that of control group 1(<i>P</i><0.01), the axis of intervention group 1 was significantly lower than that of model group 1(<i>P</i><0.01), and the axis of model group 2 was significantly higher than that of control group 2(<i>P</i><0.01), the eye axis of intervention group 2 was significantly lower than that of model group 2(<i>P</i><0.01); HE staining showed that the thickness of NFL and ONL of model group 1 was significantly thinner than that of control group 1(<i>P</i><0.01). The thickness of INL of model group 1 was significantly thinner than that of control group 1(<i>P</i><0.05), and the thickness of NFL, INL and ONL of intervention group 1 was significantly higher than that of model group 1(<i>P</i><0.05); The thickness of NFL, INL and ONL model group 2 was significantly thinner than that of control group 1(<i>P</i><0.01); IHC testing showed that the average optical density of Bcl-2 protein in model group 1 was significantly lower than that of control group 1(<i>P</i><0.05), which in intervention group 1 was significantly higher than that of model group 1(<i>P</i><0.01), and which in the average optical density of Bcl-2 protein of model group 2 was significantly lower than that of control group 2(<i>P</i><0.01), which in intervention group 2 was significantly higher than that of model group 2(<i>P</i><0.01); Caspase 3 protein average optical density of model group 1 was significantly higher than that of control group 1(<i>P</i><0.01), which in intervention group 1 was significantly lower than that of model group 1(<i>P</i><0.01), which in model group 2 was significantly higher than that of control group 2(<i>P</i><0.05), which in intervention group 2 was significantly lower than that of model group 2(<i>P</i><0.01); WB test proved that the relative expression level of Bcl-2 protein in model group 1 was significantly lower than that of control group 1(<i>P</i><0.01), which in intervention group 1 was significantly higher than that of model group 1(<i>P</i><0.01), and which in model group 2 was significantly lower than that of control group 2(<i>P</i><0.01), which in intervention group 2 was significantly higher than that of model group 2(<i>P</i><0.01); The relative expression level of Caspase3 protein in model group 1 was significantly higher than that of control group 1(<i>P</i><0.01), which in intervention group 1 was significantly lower than that of model group 1(<i>P</i><0.01), the intervention group 2 was significantly lower than that of model group 2. <p>CONCLUSION: The results show that the modified Zhujing pill can interfere with the pathological changes of retinal thickness thinning in the process of myopia and formed myopia mice by regulating the expression of apoptosis-related proteins Bcl-2 and Caspase3, and alleviating the apoptosis of retinal cells in myopia formation and myopia mice.
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Objective To study the effect of optimized atropine administration regimen on myopia in guinea pigs. Methods Forty six 21-day old guinea pigs were used for this study. Six were randomly selected as blank control, and the remaining 40 were randomly divided into 5 intervention groups: 1% atropine group, 0.01% atropine group, optimized group 1, optimized group 2, and saline group. One eye of the guinea pig in the intervention groups was randomly selected as the model eye and given form deprivation, and the contralateral eye was the self-control. The duration of intervention was 4 weeks. The diopter and axial length of guinea pig eyes were measured before the experiment and at each weekend. Choroid and sclera were measured after the experiment. Results The diopter of the model eyes in the 0.01% atropine group decreased rapidly. There was a significant difference before and after the experiment [(2.82±1.35)D vs (−0.64±0.20)D, P<0.01]. The diopter of model eyes decreased in 1% atropine group and optimized group 1, and the difference was statistically significant [(3. 50±1.14)D vs (1.38±1.15)D, P<0.05; (3.55±1.85)D vs (0.95±1.90)D, P<0.01]. In optimized group 2, the diopter of model eyes decreased, and there was no significant difference before and after the experiment [(1.36±1.61)D vs (2.93±1.42)D, P>0.05). After form deprivation, the axial length in 1% atropine group did not change significantly (P>0.05). The axial length in other intervention groups was extended to varying degrees. The thickness of choroid and sclera in 1% atropine group, optimized group 1 and optimized group 2 were greater than that in 0.01% atropine group. Conclusion The two optimized dosing regimens worked better than 0.01% atropine in inhibiting myopia in guinea pigs with form deprivation, and were similar to 1% atropine.
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@#Myopic population of China is already nearly 600 million, the rate of teenager myopic occupies the first place in the world, myopia hsa already became one of the main diseases that endangers our adolescent's health. Dopamine is the main catecholamine in retina. Many studies have found that increasing the content of dopamine can effectively inhibit the development of myopia. Form-deprivation myopia is a classical method of myopia modeling. By observing the influence of dopamine and its receptors on the development of form-deprivation myopia, its role in the development of myopia can be reflected, and it is of great significance to guide and control the occurrence and development of myopia. In this paper, the effects of dopamine and its receptors on the development of form-deprivation myopia were reviewed in order to provide reference for the prevention and treatment of myopia.
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Objective@#To investigate the effects of scleral crosslinking using genipin on ocular biological parameters and scleral biomechanics of form-deprivation myopia rabbits.@*Methods@#Sixty healthy New Zealand rabbits of 14 days old were collected.The right eyes were selected as experimental eye.The rabbits were randomly divided into three groups: control group with no treatment; myopia model group with eyelid suture procedure performed on the right eye; genipin injection group with eyelid suture procedure performed on the right eye combined with subconjunctival injection of genipin.The suture was removed 60 days after the eyelid suture procedure.The diopter, length of vitreous cavity, and axial length were measured.The sclera at 1: 00 and 7: 00 position of the experimental eye was used to make a scleral strip.The thickness, elastic modulus, creep rate, ultimate stress and ultimate strain of the sclera were measured.This study was approved by the animal experimental Ethics Committee of Bethune International Peace Hospital (2018-ky-09).@*Results@#The diopters of genipin injection group, myopia model group and control group were (2.50±1.38), (0.33±0.52) and (2.08±0.52)D, respectively, the axial lengths of the three groups were (15.33±0.82), (15.83±0.41) and (15.00±0.43)mm, respectively; the changes in vitreous cavity lengths were (1.50±0.79), (2.59±0.83) and (1.48±0.66)mm, respectively; and the ratios of vitreous cavity length to axial length were 0.46±0.02, 0.51±0.02 and 0.47±0.02, respectively.The diopter in myopia model group was significantly lower than those in control group and genipin injection group, the axial length in myopia model group was significantly longer than that in control group, the change in vitreous cavity lengths and ratio of vitreous cavity length to axial length in myopia model group were significantly higher than those in control group and genipin injection group, the axial length in myopia model group was significantly longer than that in control group, the differences were statistically significant (all at P<0.05). The elasticity modulus was (9.10±3.12)MPa and ultimate stress was (1.42±0.57)MPa in genipin injection group, which were significantly higher than (6.98±3.30)MPa and (1.15±0.57)MPa in control group, and (6.25±3.17)MPa and (0.82±0.38)MPa in myopia model group (all at P<0.05). The ultimate strain was (20.99±5.60)% in genipin injection group, which was significantly lower than (25.08±6.35)% in control group and (27.78±8.20)% in myopia model group, with significant differences between them (both at P<0.05).@*Conclusions@#Posterior sub-Tenon capsule injection of genipin for collagen crosslinking in the sclera increases the biomechanical strength of sclera and effectively prevents the development of form-deprivation myopia in rabbits, which provides a new idea for the prevention and treatment of myopia in the future.
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Objective To study the expression of collapsin response mediator protein 2 (CRMP-2) in the visual cortex of monocular form deprivation amblyopia rats.Methods Sixty-four 14-day-old rats were randomly divided into monocular deprivation amblyopia group and normal control group by random number table method.Right eyelid margin suture was performed at 14 days after birth in the monocular deprivation amblyopia group.Eight rats in the monocular deprivation amblyopia group and the normal control group were observed at 14,21,45 and 120 days after birth,respectively.Flash visual evoked potential (F-VEP) was used to dectect the latency and amplitude of P1 wave.The expression of CRMP-2 in visual cortex was observed by immunohistochemical method.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.This study protocol was approved by Ethic Committee of the University of South China (No.20140228).Results F-VEP results showed that the amplitudes of P1 were decreased and latent periods of P1 were prolonged in the monocular deprivation amblyopia group compared with the normal control group (t=16.760,P =0.000;t =-22.919,P =0.000).CRMP-2 expression levels in the visual cortex of monocular deprivation amblyopia groups and normal control groups were compared at different time points after birth,and the differences were statistically significant (Fgroup =8.855,P =0.010;Ftime =63.091,P =0.000).Compared with normal control groups,the expressions of CRMP-2 at the postnatal 21,45 and 120 days were obviously decreased in the monocular deprivation amblyopia groups,the differences were statistically significant (all at P＜0.05).Conclusions CRMP-2 may be involved in the occurrence and development of amblyopia.
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Objective To observe the dopamine(DA)concentrations in the lateral geniculate nucleus in guinea pigs with flickering light-induced and form deprivation myopia,and to compare and analyze the pathogenetic mechanisms in the centers of vision of these two different myopia models. Methods Twenty-four two-week-old guinea pigs were randomly assigned to three groups(n=8):flickering light-induced myopia(FLM)group, form deprivation myopia(FDM)group and control group. All the groups were fed for 8 weeks. The refraction and axial length(AL)were measured before and af-ter modeling. After eight-week modeling,the contents of dopamine in the left lateral geniculate nucleus were detected by high performance liquid chromatography with electrochemical detection(HPLC-ECD). Results Before modeling,no sig-nificant difference was found in refraction and AL among the three groups. After eight-week modeling,in contrast with the control group,significant differences were found in changes of both refraction(P<0.001)and AL(P<0.05)in the right eyes of FLM and FDM groups,indicating that the two myopic models were successfully established. The result of HPLC-ECD showed that the contents of DA in the left lateral geniculate nucleus in FLM group were significantly higher than the control group(P=0.01),while in the FDM group it was lower than the control group(P=0.021). The average contents of DA were as follows:(37.04 ± 1.18)pg/μL in the control group;(24.27 ± 3.46)pg/μL in the FDM group; and (45.58 ± 1.98)pg/μL in the FLM group. Conclusions The content of DA in the lateral geniculate nucleus is increased in FLM,while decreased in FDM,indicating that the expression of DA in LGN and the mechanisms of formation of these two experimental myopia are different.
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Objective To preliminarily explore the feasibility of tree shrew as a new kind of animal model in research of amblyopia,to discuss the primary visual cortex plasticity mechanism of form deprivation in tree shrew,and to provide a theoretical basis for further understanding the mechanism of amblyopia formation and recovery.Methods Sixty 30-days old tree shrews were divided into five groups,12 in each group:the group A had the right eye sutured for 1 month;the group B had the right eye sutured for 2 months;the group C had the left eye sutured for 1 month and then opened and the righ eye was sutured for 1 month,in other words,the group C was performed by alternating suture;the tree shrews of control group 1(D1) were in the same age as the the group A,but fed in normal breedingenvironment;the tree shrews of control group 2(D2) were at the same age of groups B and C,but fed with a normal diet.Samples of the visual cortex were taken after the completion of modeling,and were processed to observe the histology and ultrastructure of the visual cortex,the neuron apoptosis,and the c-fos protein expression in the tree shrews of different groups.Results Damages to different degrees were found by histological and electron microscopic examination of the visual cortex in each experimental group,and they were more obvious in the group sutured for 2 months.TUNEL staining showed that there were no significant differences between the apoptosis in the experimental and control groups.The expression of c-fos mRNA and protein in the experimental groups was decreased,and it was the lowest in the group sutured for 2 months.There was a small increase in the c-fos expression after the alternate suture,and no significant difference of c-fos expression was found in the control groups.Conclusions Different degrees of deprivation amblyopia lead to different histopathological changes.There is a plasticity in the neurons affected by amblyopia.Tree shrew can be used as an ideal animal model for the studies of form deprivation amblyopia.
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Objective To observe the changes of S-opsin expression in guinea pigs with flickering light-induced and form-deprived myopia,and to investigate the causes.Methods Thirty-six two-week-old healthy guinea pigs were randomly assigned to three groups:Flickering light-induced myopia group(FLM group,n=13),form-deprived myopia group(FDM group,n=12) and control group(n=11).For the FLM group,the cages were equipped with astroboscope(0.5 Hz),and LEDs were used as the light source.The right eyes of the guinea pigs in FDM group wore translucent goggles which did not interfere with the normal activity of their eyelids.No special treatment was given to the guinea pigs in the normal groups.All measurements were performed prior to and then after 6 weeks of treatment.The first measurement day was recorded as 0 week.Biological parameters,such as the refraction,axial length(AL) and corneal radius of curvature(CRC),were measured and fundus photography is performed before and after 6 weeks of the treatment.The expression of S-opsin was observed and analyzed by immunofluorescence technique and image analysis system.Results Before the treatment,no significant difference was found in three biometric measurements including refraction,AL and CRC between the groups at 0 week(P>0.05).After the treatment for 6 weeks,significant differences were found in changes of both the biometric measurements between the FLM and control groups,and between the FDM and control groups(P0.05).Expression of S-opsin differed in the FLM and FDM groups.For the mean gray values of green channel,compared with the control group respectively,significant differences were found in both the FLM and FDM group(P<0.001).The mean gray value of green channel of the FLM group was higher,however the mean gray value of green channel of the FDM group was lower.Conclusions Both guinea pig models of flickering light-induced and form-deprived myopia can be established successfully.S-opsin is increased in the flickering light-induced myopia model and decreased in the form-deprived myopia model,indicating that the mechanisms of formation of these two experimental myopia models may be different.
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Background Sclera remodeling process in axial elongation is one of the main pathological mechanisms of axial myopia progression.Studies confirmed that transforming growth factor-β1(TGF-β1) participates in the sclera remodeling process,and Smad3 is one of TGF-β1 downstream signal gene transcriptive factors,so to explore its role in sclera remodeling process of myopic eyes is of great significance for pathogenesis and prevention research of myopia.Objective This study was to investigate the expressions of type Ⅰ collagen and Smad3,a TGF-31 downstream target,in sclera of form deprivation myopic (FDM) eyes and explore the impact of TGF-β1-Smad3-type Ⅰ collagen signaling pathway on collagen remodeling in myopic sclera.Methods Seventy-five 1-week-old guinea pigs were randomly divided into normal control group (25 guinea pigs) and FDM group (50 guinea pigs).Monocular FDM was induced by occluding the left eyes of guinea pigs in FDM group with translucent latex balloons for 2,4,6 weeks,respectively,and consecutive occluding for 4 weeks followed by uncovering for 1 week (4/-1 weeks).The refractive power was detected by retinoscopy and axial length was measured with A-type ultrasound.Immunohistochemistry and reverse transcription-PCR were employed to detect the dynamic expressions of type Ⅰ collagen and Smad3 protein ad mRNA in the sclera of guinea pigs with emmetropia and experimental myopia,ard the relationship between collagen Ⅰ and Smad3 levels was analyzed.Results The refraction was hypermetropic in both normal control group and FDM group before occluding of eyes (P＞0.05),and the hypermetropic power was gradually reduced over time in the normal control group.In the FDM group,the refractive power was gradually changed from (+2.09 ± 0.31)D before occluding to (-1.23±0.69),(-4.17±0.59),(-7.07±0.56) and (-4.30±0.58)D,and the axial length was increased from (5.93-±0.39)mm to (6.62±0.36),(7.30±0.34),(7.99--0.32),and (7.21 ±0.36) mm at weeks 2,4,6,and 4/-1 after occluding,respectively,indicating significant differences in refractive power and axial length over time in the FDM group from normal control group and self-control group (all at P＜0.05).The expressions of Smad3 and type Ⅰ collagen protein and mRNA in the sclera of the FDM group was significantly lower than those of the control group and self-control group in various time points (all at P＜0.05).The positive correlation were found in the expression of Smad3 on the myopic sclera with that of type Ⅰ collagen in both protein and mRNA levels (protein:r=0.993,P＜0.05;mRNA:r=0.954,P＜0.05).Conclusions The myopic power and ocular axis increase dependent upon occluding time,and the expressions of Smad3 and type Ⅰ collagen in the sclera are correspondingly weakened in FDM eyes.A consistent expression trend is found between Smad3 and type Ⅰ collage,suggesting Smad3 and type Ⅰ collagen participate in the regulation of sclera remodeling in myopia by TGF-β1-Smad3-Collagen Ⅰ signaling pathway.
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Myopia is one of the most common ocular problems,which causes visual dysfunction.The prevalence of myopia is increasing,while the prevention and treatment have made no breakthrough because the pathogenesis and mechanism of myopia remains unclear.Certain kinds of molecules were reported to play a role in myopia,such as dopamine,retinoic acid,glucagon,ZENK (Zif269、EGR-1、NGFI-A or Krox-24) etc.As a derivative of vitamin A,retinoic acid is the final metabolite of retinaldehyde,which combines to opsin in the photoreceptor cells on retina.Previous studies demonstrated that retinoic acid from choroid and retina plays significant roles in the development of myopia,which affects different molecules related to myopia development,including transforming growth factor-β,glycosaminoglycan,matrix metalloproteinase (MMP)-2 and tissue inhibitor of metalloproteinase (TIMP)-2.Based on the synthesis,mechanism and effects of the retinoic acid in the eye,this paper describes the new progress of retinoic acid in experimental myopia,including form deprivation myopia,defocus induced myopia and monochromatic myopia,hence offers new clues for the research on the mechanism of myopia.
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Objective To evaluate the inhibitory effect of highly selective M4 receptor antagonist MT3 on the form deprivation myopia in guinea pigs and its potential mechanism. Methods Thirty?two healthy male guinea pigs were ran?domly divided into three groups:control group, form deprivation group, and form deprivation + MT3 group, 8 animals in each group. Refraction was measured by retinoscopy after cycloplegia before and after the experiment. The ocular biological dimensions were measured by A?scan ultrasound. RT?PCR was used to detect the relative expression of TGF?β2 mRNA in the retina and choroid. Results Compared with the right eyes of control group, the right eyes of form deprivation + MT3 group developed relative myopia of -1?44 ± 0?50 D (right?left eye) (P =0?001). The vitreous chamber depth and axial length of the right eyes were significantly prolonged by 0?10 ± 0?02 mm and 0?14 ± 0?07 mm (P<0?001, P<0?001), respectively, but the increases of myopia and axial length were significantly smaller than that of the form deprivation group (P<0?001, P<0?001, P<0?001). Down?regulation of relative mRNA expression of TGF?β2 in retina and choroid was found in the form deprivation group (P<0?001, P =0?014) compared with the right eyes of the control group, while up?regulation of relative mRNA expression of TGF?β2 in retina and choroid was found in the form deprivation + MT3 group ( P<0?001, P<0?001). Conclusions MT3 can inhibit the development of form deprivation myopia in guinea pigs, which may play an important role by the regulation of TGF?β2 mRNA level in the retina and choroid.
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Background JAK/ signal transducer and activator of transcription 3 (STAT3) signal pathway plays a critical role during the sclera remodeling of experimental myopia.As a tyrosine kinase inhibitor,AG490 can inhibit the activation of this pathway.But whether AG490 plays a role in delaying the development of myopia is not completely clear.Objective This study was to investigate the inhibition of AG490 to activation of STAT3 signaling pathway and the sequential arresting effect on the sclera remodeling in form-deprived myopia (FDM) models.Methods Forty guinea pigs were randomly divided into the normal control group,model control group,PBS control group and AG490 treatment group.FDM models were established by the occlusion of the right eyes of guinea pigs for consecutive 4 weeks using translucent goggles in the model control group,PBS control group and AG490 treated group,and 25 μl PBS or AG490 were respectively injected into vitreous since the first day of modeling in two-day interval till the fourth week in the PBS control group and AG490 treated group.Refractive state and axial length were examined with retinoscopy and A-scan ultrasonography before and 4 weeks after experiment.The experimental eyes were extracted in the fourth week,and the expressions of scleral STAT3,p-STAT3,metal matrix proteinase-2 (MMP-2) proteins and STAT3 mRNA,MMP-2 mRNA were detected by immunocytochemstry and semi-quantitative reverse transcription PCR (RT-PCR) respectively.The use and care of experimental animals followed ARVO.Results Compared to the normal control group,the negative refraction power and axial length were significantly increased in the model control group,PBS control group and AG490 treated group,and the axial length in the AG490 treated group was smaller than those in the model control group and PBS control group,showing significant differences among the 4 groups (refraction:F =89.063,P =0.000;axial length:F =96.145,P =0.000).The expressions of STAT3,MMP-2 and p-STAT3 in scleral tissue were weaker in the normal control group.The expressional values (A values) of STAT3,p-STAT3 and M MP-2 were 0.064 ± 0.016,0.019 ± 0.002 and 0.155 ± 0.052 in the AG490 treated group,which were lower than 0.129±0.008,0.071 ±0.021,0.425 ±0.004 of the model control group and 0.130±0.004,0.069±0.002,0.421 ±0.042 of the PBS control group (STAT3:t =4.641,9.364,both at P＜0.01;p-STAT3:t =4.638,4.488,both at P＜ 0.05;MMP-2:t =9.123,9.029,both at P ＜ 0.05),however,these expressions were still higher than those of the normal control group (t =2.674,2.251,2.682,all at P ＜0.05).The expressional levels (A values) of STAT3 mRNA and MMP-2 mRNA in the AG490 treated group were 0.295±0.032 and 0.569±0.019,which were significantly lower than 0.547±0.015 and 0.782±0.051 in the model group as well as 0.544±0.015 and 0.779±0.048 in the PBS control group (STAT3 mRNA:t =10.115,11.703,both at P＜0.01;MMP-2 mRNA:t =9.218,9.494,both at P＜0.01).The expressional levels (A values) of STAT3 mRNA and MMP-2 mRNA in the AG490 treated group were still higher than those in the normal control group (t=2.576,3.565,both at P＜0.05).Conclusions AG490 can ultimately inhibit the development of axial myopia by arresting the activation of STAT3 signaling pathway in the FDM eyes and further regulating the expression of MMP-2 in sclera and delaying the remodeling of sclera.
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Objective To observe whether form-deprivation myopia/amblyopia could protect retinal microvascular system and postpone the development of diabetic retinopathy. Methods The form-deprivation myopia/amblyopia was established combined with hyperglycemia mouse model,and the retinal vascular endothelial cells and pericytes were counted. Results Endothelial cells/ pericytes ratio in hyperglyce-mia group is significantly lower than that in normal group and form-deprivation myopia/amblyopia combined with hyperglycemia group. Con-clusion Form-deprivation myopia/amblyopia combined with hyperglycemia mice have more pericytes than hyperglycemia mice.
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Background It is well known that sclera remodeling occurs during axial elongation in myopia under the control of growth hormone or its downstream effectors.The role of transforming growth factor-β (TGF-β) in myopia has been determined in previous studies.Bone morphogenetic protein (BMP) is one of members of the TGF-β superfamily,but if it plays an important role in the genesis and development of myopia is not completely clear.Objective This study was to identify the presence of BMPs in normal guinea pigs sclera and investigate the change of BMPs in the sclera in form-deprivation myopia (FDM) of guinea pigs.Methods Thirty young guinea pigs were randomized into normal control group and experimental group using table of random number.FDM models were established by occluding unilateral eyes of guinea pigs with a translucent lens for 14 days in the experimental group,and the fellow eyes served as the controls.Diopter of all eyes was tested by retinoscopy optometry,and ocular axial length was measured by A-sonography before and after modeling.Posterior sclera tissue of the animals was obtained on 14 days,and the relative expression level of BMPs mRNA and protein were assayed by reverse transcription PCR (RT-PCR) and Western blot.The use and care of the animals complied with ARVO Statement.Results On 14 days after occluding of unilateral eyes,the refraction diopter of the experimental group was (-0.48±0.51) D,and that of the fellow eyes was (3.22 ±0.34) D,showing a significant difference between them (t =-12.814,P =0.000).Also,a significant difference in the diopter was seen between the experimental group and normal control group ([-0.48±0.51]D vs.[2.97±0.70]D,t =-11.878,P=0.000).Axial length was (8.30 ± 0.05) mm in the experimental group,(8.11 ±0.06) mm in the fellow eyes and (8.06±0.06) mm in the normal control group,showing a significant increase in the experimental group compared with the fellow eyes and normal control group (t =7.230,P =0.000 ; t =9.084,P=0.000).The expressions of BMP-2 mRNA,BMP-4 mRNA,BMP-5 mRNA in posterior sclera were detected in the normal guinea pigs.Fourteen days after the induction of myopia,the relative levels of BMP-2 mRNA and BMP-5 mRNA in sclera were 0.41 ± 0.11 and 0.65 ± 0.06 in the experimental eyes,which were significantly lower than 0.62 ± 0.07 and 0.84 ± 0.03 in the fellow eyes with the descent range of 34.48％ and 23.67％ respectively (t=2.838,P=0.017; t=2.524,P=0.028).The relative values of BMP-2 protein and BMP-5 protein were 0.44±0.06 and 0.70±0.05 in the experimental eyes,and those of the fellow eyes were 0.61±0.05 and 0.82±0.03,showing significant decline in the experimental eyes with the lowing range of 23.42％ and 15.21％,respectively (t =2.465,P =0.030;t =2.445,P=0.031).No significant differences were found in the expression of BMP-4 mRNA and protein in posterior sclera between the experimental eyes and the normal control eyes (mRNA:t =0.704,P=0.460;protein:t=0.987,P=0.365).Conclusions The expressions of the BMP-2 and BMP-5 in sclera down-regulate significantly in FDM eyes,which suggest that BMP-2 and BMP-5 participate in sclera remodeling during myopia induction.
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Background Chondroitin sulphate proteoglycans (CSPGs) can cause the termination of ocular dominance plasticity in the visual cortex.Recently,protein tyrosine phosphatase σ (PTPσ) has been identified as a receptor that inhibits CSPGs.However,whether PTPσ and its downstream molecules participate in the reactivation of ocular dominance plasticity in adult visual cortex has not been studied.Objective The present study was to investigate the changes in the expression of the PTPσ,probabilistic neural networks (PNNs),and molecules downstream of PNN,such as N-cadherin/β-catenin,after the reactivation of adult visual cortical plasticity.Methods Fifty-four SPF Long Evans rats were grouped according to different postnatal week (PW) as the PW1 (6 rats),PW3 (6 rats),PW5 (6 rats),PW7 (24 rats),and PW9 (12 rats) groups,and the upper and lower eyelids were sutured in the 12 rats from the PW7 group for 14 days to establish the binocular plasticity reactivation models.Expression of PTPσ and PNNs in the rat visual cortex was detected using immunochemistry,and changes of PTPσ mRNA,N-cadherin mRNA and β-catenin mRNA expression in the rat visual cortex with plasticity reactivation were assessed by real-time fluorescence quantitative PCR (RT Q-PCR).The use of animals followed the Regulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results The expression level of PTPσ mRNA was significantly higher in the PW9 group than that of the binocular plasticity reactivation models and the PW7 group (t =1.965,3.526,P＜0.01).The staining of the rat visual cortex for PTPσwas localized to the cellular membrane,cytoplasm and axon.Cell densities of the PW9 group in the Ⅱ-Ⅲ layer,Ⅳ layer and Ⅴ-Ⅵ layer of the visual cortex were elevated in the PW9 rats compared with the PW7 rats (t =24.593,23.444,13.556,P＜0.01) and rats from the binocular plasticity reactivation model (t =44.111,43.000,16.556,P＜0.01).Cell densities for PNNs in the Ⅳ and Ⅴ-Ⅵ layers were significantly increased in the PW9 rats in comparison with the PW7 rats (t=1.926,P＜0.01 ;t=1.370,P＜0.05),but the cell density in the Ⅱ-Ⅱ layer has no statistical significance (t=0.889,P＞0.05).However,cell densities for PNNs in the Ⅱ-Ⅲ and Ⅳ layers in the binocular plasticity reactivation models were lower than those of the PW9 rats (t =2.556,4.585,P＜0.01).Compared with PW1 rats,the expression levels of the N-cadherin mRNA in the PW3,PW5,PW7,PW9 rats were lower (t =28.932,28.988,27.083,28.908,P＜0.01),but those in the PW7 rats were enhanced in comparison with the PW3 rats,PW5 rats and PW9 rats (t =1.848,1.904,1.825,P＜0.01).No significant difference was seen in the expression of the N-cadherin mRNA between the PW9 rats and rats from the binocular plasticity reactivation model (t =0.072,P＞0.05).A statistically significant increase was found in the β-catenin mRNA expression in the PW1 rats compared with the PW3,PW5,PW7 and PW9 rats (t =3.918,3.534,2.645,4.652,P＜ 0.0 1),as well as between rats from the binocular plasticity reactivation model and the PW9 rats (t =0.570,P＜0.01).Conclusions PTPr,PNNs and β-catenin are involved in the reactivation of ocular dominance plasticity in the adult visual cortex.