Form-deprivation Myopia Eyes Research Articles

Myopia Development in Tree Shrew Is Associated with Chronic Inflammatory Reactions

In this study, we aimed to investigate whether chronic retinal inflammation is involved in the pathogenesis of form-deprivation myopia (FDM) using tree shrews as an animal model. Twenty-one tree shrews were randomly divided into 7-day/14-day FDM (FDM7/FDM14) groups and their corresponding 7-day/14-day control groups. Refraction and axial length were measured. To determine the effects of form deprivation on inflammation, we used real-time polymerase chain reaction (PCR) and immunohistochemistry to assess the expression levels of several proinflammatory cytokines. At day 0, the eyes in the FDM and control groups were hyperopic. However, after 7 and 14 days of form deprivation, the refractive error of the eyes in the FDM7 and FDM14 groups shifted from +6.6 ± 0.3 diopters (D) to +4.0 ± 0.5 D and from +6.4 ± 0.3 D to +5.0 ± 0.3 D, respectively. The levels of tumor necrosis factor-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and nuclear factor κB were increased in the FDM eyes, compared with those in the control eyes. The increase in matrix metalloproteinase-2 expression was greater in the FDM eyes than in the contralateral and control eyes, whereas collagen type I expression was downregulated. In conclusion, chronic inflammation may play a crucial pathogenic role in form-deprivation myopia in tree shrews.

Endoplasmic Reticulum Stress Regulates Scleral Remodeling in a Guinea Pig Model of Form-Deprivation Myopia.

Purpose This study aimed to investigate the role of endoplasmic reticulum (ER) stress in scleral remodeling in a guinea pig model of form-deprivation myopia (FDM). Methods Guinea pigs were form deprived to induce myopia. ER ultrastructural changes in the sclera were examined by transmission electron microscopy (TEM). The protein levels of ER stress chaperones, including GRP78, CHOP, and calreticulin (CRT), were analyzed by western blotting at 24 hours, 1 week, and 4 weeks of FD. Scleral fibroblasts from guinea pigs were cultured and exposed to the ER stress inducer tunicamycin (TM) or the ER stress inhibitor 4-phenylbutyric acid (4-PBA). CRT was knocked down by lentivirus-mediated CRT shRNA transfection. The expression levels of GRP78, CHOP, TGF-β1, and COL1A1 were analyzed by qRT-PCR or western blotting. Results The sclera of FDM eyes exhibited swollen and distended ER at 4 weeks, as well as significantly increased protein expression of GRP78 and CRT at 1 week and 4 weeks, compared to the sclera of the control eyes. In vitro, TM induced ER stress in scleral fibroblasts, which was suppressed by 4-PBA. The mRNA expression of TGF-β1 and COL1A1 was upregulated after TM stimulation for 24 hours, but downregulated for 48 hours. Additionally, change of TGF-β1 and COL1A1 transcription induced by TM was suppressed by CRT knockdown. Conclusions ER stress was an important modulator which could influence the expression of the scleral collagen. CRT might be a new target for the intervention of the FDM scleral remodeling process.

The Role of the RhoA/ROCK Signaling Pathway in Mechanical Strain-Induced Scleral Myofibroblast Differentiation.

Biomechanical properties changes and α-smooth muscle actin (α-SMA) overexpression are involved in myopia scleral remodeling. However, interactions between altered tissue biomechanics and cellular signaling that sustain scleral remodeling have not been well defined. We determine the mechanisms of mechanotransduction in the regulation of α-SMA expression during myopia scleral remodeling. Guinea pigs were used to establish a form-deprivation myopia (FDM) model. Protein profiles in myopic sclera were examined using tandem mass spectrometry. Ras homolog gene family member A (RhoA) and α-SMA expressions were confirmed using quantitative (q) RT-PCR and Western blotting. Scleral fibroblasts were cultured and subjected to 4% cyclic strain. Levels of RhoA, rho-associated protein kinase-2 (ROCK2), myocardin-related transcription factor-A (MRTF-A), serum response factor (SRF), and α-SMA were determined by qRT-PCR and Western blotting in groups with or without the RhoA siRNA or ROCK inhibitor Y27632. MRTF-A and α-SMA were evaluated by confocal immunofluorescent microscopy and myofibroblasts were enumerated using flow cytometry. mRNA and protein levels of RhoA and α-SMA were significantly increased in the FDM eyes after 4 weeks of form-deprivation treatment. The 4% static strain increased expressions of RhoA, ROCK2, MRTF-A, SRF, and α-SMA as well as nuclear translocalization of MRTF-A in scleral fibroblasts compared to those without strain stimulation. Additionally, the percentage of myofibroblasts increased after strain stimulation. Conversely, inhibition of RhoA or ROCK2 reversed the strain-induced α-SMA expression and myofibroblast ratio. Mechanical strain activated RhoA signaling and scleral myofibroblast differentiation. Strain also mediated myofibroblast differentiation via the RhoA/ROCK2-MRTF-A/SRF pathway. These findings provided evidence for a mechanical strain-induced RhoA/ROCK2 pathway that may contribute to myopia scleral remodeling.

Crosslinking Enzyme Lysyl Oxidase Modulates Scleral Remodeling in Form-Deprivation Myopia

ABSTRACTPurpose: Scleral remodeling causes the excessive ocular elongation that underlies myopia. Lysyl oxidase (LOX), a copper-containing amine oxidase, can catalyze collagen and elastin crosslinking. The purpose of this study was to investigate the role of LOX in scleral remodeling in form-deprivation myopia (FDM).Methods: Seventy-five guinea pigs were randomly divided into five groups as follows: a normal control group, an FDM group, an FDM plus β-aminopropionitrile (BAPN) group, an FDM plus TGF-β1 (TGF-β1) group, and an FDM plus vehicle group. A translucent diffuser was used to induce FDM, and intravitreal injection was used to administer BAPN, TGF-β1 or vehicle. The scleral LOX and collagen gene and protein levels and the posterior scleral ultrastructure and biomechanics were measured.Results: In the FDM group, both the scleral LOX and collagen gene and protein levels were significantly lower than those in the control eyes. The collagen fibril diameters were significantly decreased in the FDM group compared with the diameters in the control group. A significant decrease in LOX gene and protein expression was observed after BAPN injection, and an increase was observed after TGF-β1 treatment compared with the levels in the FDM group. Additionally, the scleral collagen fibrils were significantly decreased in the BAPN-treated eyes but increased in the TGF-β1-treated eyes compared with the FDM eyes. The ultimate stress and Young’s modulus of the sclera were lowest in the BAPN group, followed by the FDM group and the TGF-β1 group. The ultimate strain (%) of the sclera was lowest in the TGF-β1 group, followed by the FDM group and the BAPN group.Conclusion: LOX expression was significantly lowered in myopic sclera. Modulating LOX expression induced a change in both the scleral collagen fibril diameter and the scleral biomechanics. Therefore, LOX may play a key role in the myopia scleral remodeling procedure.

Effects of Constant Flickering Light on Refractive Status, 5-HT and 5-HT2A Receptor in Guinea Pigs

PurposeTo investigate the effects of constant flickering light on refractive development, the role of serotonin (i.e.5-hydroxytryptamine, 5-HT)and 5-HT2A receptor in myopia induced by flickering light in guinea pigs.MethodsForty-five guinea pigs were randomly divided into three groups: control, form deprivation myopia (FDM) and flickering light induced myopia (FLM) groups(n = 15 for each group). The right eyes of the FDM group were covered with semitransparent hemispherical plastic shells serving as eye diffusers. Guinea pigs in FLM group were raised with illumination of a duty cycle of 50% at a flash frequency of 0.5Hz. The refractive status, axial length (AL), corneal radius of curvature(CRC) were measured by streak retinoscope, A-scan ultrasonography and keratometer, respectively. Ultramicroscopy images were taken by electron microscopy. The concentrations of 5-HTin the retina, vitreous body and retinal pigment epithelium (RPE) were assessed by high performance liquid chromatography, the retinal 5-HT2A receptor expression was evaluated by immunohistofluorescence and western blot.ResultsThe refraction of FDM and FLM eyes became myopic from some time point (the 4th week and the 6th week, respectively) in the course of the experiment, which was indicated by significantly decreased refraction and longer AL when compared with the controls (p<0.05). The concentrations of 5-HT in the retina, vitreous body and RPE of FDM and FLM eyes were significantly increased in comparison with those of control eyes (both p<0.05). Similar to FDM eyes, the expression of retinal 5-HT2A receptor in FLM eyes was significantly up-regulated compared to that of control eyes (both p<0.05). Western blot analysis showed that retinal 5-HT2A receptor level elevated less in the FLM eyes than that in the FDM eyes. Moreover, the levels of norepinephrine and epinephrine in FDM and FLM groups generally decreased when compared with control groups (all p<0.05).ConclusionsConstant flickering light could cause progressive myopia in guinea pigs. 5-HT and 5-HT2A receptor increased both in form deprivation myopia and flickering light induced myopia, indicating that 5-HT possibly involved in myopic development via binding to5-HT2A receptor.

Form-deprivation myopia induces decreased expression of bone morphogenetic protein-2, 5 in guinea pig sclera.

To identify the presence of various bone morphogenetic proteins (BMPs) and their receptors in normal sclera of human, rat and guinea pigs, and to determine whether their expression changed with form-deprivation myopia (FDM) in guinea pig sclera. The expression of BMPs and BMP receptors were detected using reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence. Two-week-old guinea pigs were monocularly form-deprived with a translucent lens. After fourteen days induction of FDM, total RNA was isolated and subjected to RT-PCR to examine the changes of BMPs and BMP receptors in tissues from the posterior sclera. Western blotting analysis was used to investigate their changes in protein levels. Human sclera expressed mRNAs for BMP-2, -4, -5, -7, -RIA, -RIB and BMP-RII. Conversely, rat sclera only expressed mRNA for BMP-7 and BMP-RIB, while the expression of BMPs and BMP receptors in guinea pigs were similar to that of humans. Human sclera also expresses BMP-2, -4, -5,-7 in protein level. Fourteen days after the induction of myopia, significant decreased expressions for BMP-2 and BMP-5 in the posterior sclera of FDM-affected eyes (P<0.05 vs internal control eyes). Various BMPs were expressed in human and guinea pig sclera. In the posterior sclera, expressions of BMP-2 and BMP-5 significantly decreased in FDM eyes. This finding indicates that various BMPs as components of the scleral cytokines regulating tissue homeostasis and provide evidence that alterations in the expression of BMP-2 and BMP-5 are associated with sclera remodeling during myopia induction.

胶原相关整合素β1在形觉剥夺性近视诱导及恢复期中调节巩膜重塑作用的研究

目的：探讨豚鼠形觉剥夺性近视(form-deprivation myopia，FDM)模型中巩膜胶原相关整合素β1在巩膜重塑中的作用。方法：2~3周龄三色豚鼠128只，分为实验组(n = 64)和正常对照组(n = 64)。实验组采用头套法遮盖右眼进行形觉剥夺，左眼则为自身对照，正常对照组双眼不予任何处理。实验组依据形觉剥夺l周、2周、4周不同时间建立不同近视诱导和进展过程中的动物模型。并通过对形觉剥夺4周动物去除遮盖物，依据去遮盖时间l天、3天、5天、7天及14天建立不同时间点FDM恢复期的动物模型。在各个时间点对各组动物分别进行检测：带状光检影测量屈光度值；游标卡尺测量眼轴长度；苏木素–伊红(HE)染色观察后极部巩膜组织形态学变化；实时荧光定量聚合酶链反应(QRT-PCR)、免疫组织化学方法检测后极部巩膜胶原相关整合素β1 mRNA及蛋白表达情况。结果：形觉剥夺2周、4周后，实验组右眼分别诱导出(−3.00 ± 0.50) D及(−5.50 ± 1.08) D的相对近视，眼轴分别拉长(0.22 ± 0.06) mm及(0.49 ± 0.11) mm，与自身对照组及正常对照组相比，组间差异均有统计学意义(P均 0.05)。去遮盖后，豚鼠右眼重新正视化，与自身对照、正常对照眼相比，去遮盖7天时屈光度差值差异无显著性(P > 0.05)；右眼眼轴长度缩短，14天时眼轴长度差值差异无显著性(P > 0.05)。巩膜形态学观察见形觉剥夺性近视眼豚鼠后极部巩膜胶原纤维排列紊乱，巩膜变薄；QRT-PCR及免疫组织化学染色显示，实验组右眼形觉剥夺l周后眼球后部巩膜整合素β1 mRNA及蛋白表达明显减少，差异有统计学意义(P 0.05)。自身对照组的上述所有检测指标与正常对照组相比，均无明显差异(P > 0.05)。结论：单眼遮盖可以导致FDM的发生；2~3周龄豚鼠去除形觉剥夺后可以重新进行正视化，伴随眼轴长度缩短；去遮盖7天内为眼生物学参数恢复的主要时期；豚鼠形觉剥夺时，后极部巩膜整合素β1表达减少，去遮盖后表达上调，提示整合素β1可能参与了形觉剥夺性近视的发生，其影响巩膜重塑的机制有待进一步研究。 Objective: To investigate the role of collagen-binding integrin β1 in sclera remodeling process of the guinea pig form-deprivation myopia (FDM). Methods: 128 young guinea pigs, aged 2 to 3 weeks, were randomized into experimental group (n = 64) and normal control group (n = 64). FDM models were monocular deprived by placement of a facemask over the right eye in the experimental group, and the fellow eyes served as the contralateral control eyes. The guinea pigs in the normal control group were raised with both eyes untreated. The FDM eyes were monocular deprived for 1, 2 and 4 weeks, and recovered 1, 3, 5, 7, 14 days after occluding for 4 weeks. Optical measurements and axial length were accomplished at all of the time points. The histological analysis was applied to assess the posterior sclera changes, and immunohistochemical staining and quantitative real-time PCR (QRT-PCR) were applied to investigate the expression of integrin β1 protein and mRNA. Results: After 2 and 4 weeks, compared with the fellow eyes and the normal control group, the refraction diopter of the experimental group was (−3.00 ± 0.50) D and (−5.50 ± 1.08) D, and the axial length elongated (0.22 ± 0.06) mm and (0.49 ± 0.11) mm, showing significant differences respectively (P 0.05). After re-exposure, the right eyes of guinea pigs in the experimental group experienced re-normalization and the refraction was rapidly recovered in the initial 7 days (P > 0.05); the axial length gradually shortened, and it was statistically non-significant after 14-day recovery (P > 0.05). The morphological observation of sclera found that the posterior sclera collagen fiber arrangement of guinea pigs with FDM was disordered and the sclera became thinner. Scleral integrin β1 mRNA and protein expression in FDM eyes were significantly lower than those in the controls after monocular deprived 1 week (P 0.05). The difference of all the above statistics between contralateral control eyes and the normal control group was not remarkable (P > 0.05). Conclusion: Form-deprivation myopia was able to be induced by monocular covering. Guinea pigs aged 2 to 3 weeks with form-deprivation can experience re-normalization after recovery, along with the shortening of the axial length. The main recovery stage of ocular biometric parameters was in the initial 7 days. The expression of integrin β1 in FDM sclera was found to be lower, but could be restored during the recovery period. This suggests that collagen- binding integrin β1 may be involved in the pathogenesis of myopia, and further study needs to be done on the mechanism of the scleral remodeling in myopia.

Wnt signaling in form deprivation myopia of the mice retina.

BackgroundThe canonical Wnt signaling pathway plays important roles in cellular proliferation and differentiation, axonal outgrowth, cellular maintenance in retinas. Here we test the hypothesis that elements of the Wnt signaling pathway are involved in the regulation of eye growth and prevention of myopia, in the mouse form-deprivation myopia model.Methodology/Principal Findings(1) One hundred twenty-five C57BL/6 mice were randomly distributed into form-deprivation myopia and control groups. Form-deprivation myopia (FDM) was induced by suturing the right eyelid, while the control group received no treatment. After 1, 2, and 4 weeks of treatment, eyes were assessed in vivo by cycloplegic retinoscopic refraction and axial length measurement by photography or A-scan ultrasonography. Levels of retinal Wnt2b, Fzd5 and β-catenin mRNA and protein were evaluated using RT-PCR and western blotting, respectively. (2) Another 96 mice were divided into three groups: control, drugs-only, and drugs+FDM (by diffuser). Experimentally treated eyes in the last two groups received intravitreal injections of vehicle or the proteins, DKK-1 (Wnt-pathway antagonist) or Norrin (Wnt-pathway agonist), once every three days, for 4 injections total. Axial length and retinoscopic refraction were measured on the 14th day of form deprivation.Following form-deprivation for 1, 2, and 4 weeks, FDM eyes had a relatively myopic refractive error, compared with contralateral eyes. There were no significant differences in refractive error between right and left eye in control group. The amounts of Wnt2b, Fzd5 and β-catenin mRNA and protein were significantly greater in form-deprived myopia eyes than in control eyes.DKK-1 (antagonist) reduced the myopic shift in refractive error and increase in axial elongation, whereas Norrin had the opposite effect in FDM eyes.Conclusions/SignificanceOur studies provide the first evidence that the Wnt2b signaling pathway may play a role in the development and progression of form-deprivation myopia, in a mammalian model.

Expression of bone morphogenetic protein in sclera of form deprivation myopic eye

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. Key words: Transforming growth factor-β/bone morphogenetic protein; Form-deprivation myopia; Sclera

GABAA and GABAC (GABAA0r) Receptors Affect Ocular Growth and Form-Deprivation Myopia

Roles of γ-aminobutyric acid (GABA) antagonists on the chick model of form-deprivation myopia (FDM) were investigated. Bicuculline (GABAA) or TPMPA (GABAA0r) was injected intravitreally (1 or 10 mg/mL) into the right eyes of chicks with or without FDM for 13 days. The contralateral eyes served as the control. The eye weight (EW), equatorial diameter (ED), ocular length (OL), axial length (AL), and refraction (RFN) were measured. Histological sections of the retina and sclera were measured, and changes in tissue thickness were compared. The EW, ED, OL, and AL of the FDM eyes went up by 13.1±2.0% (n = 24, p < 0.001), 18.7±2.0% (n = 24, p < 0.001), 7.2±1.9% (n = 24, p < 0.001) and 5.1±1.5% (n = 11, p < 0.05), respectively. Bicuculline and TPMPA significantly reversed these changes (p < 0.05) but not the OL at either concentration used. The RFN measurements confirmed this (n = 2–8, p < 0.01). The drugs have no effect on the retinal thickness but significantly reduced the thickness of cartilaginous scleral layer of chicks with or without FDM (n = 9–120, p < 0.05). Bicuculline and TPMPA reduced form-deprived as well as normal ocular growth. GABAergic-mediated mechanism may directly influence the growth, shape, and refractive state of the developing eye.

Disturbance of Electrolyte Balance in Vitreous of Chicks With Form-Deprivation Myopia

Purpose: To investigate the changes in the electrolyte and protein concentrations in the vitreous of 3-week-old chicks with form-deprivation myopia (FDM). Methods: FDM was induced in 2-day-old male white leghorn chicks by covering the left eye with a translucent plastic goggle and leaving the right eye uncovered to serve as control. After 19 days the animals were euthanized, and the axial dimensions of the eyes were measured with a caliper in an unfixed condition. The liquid vitreous and aqueous humor were removed by paracentesis, and blood was collected from the jugular vein. Sodium, potassium, and chloride concentrations were determined using ion-selective electrodes. Calcium and phosphate concentrations were determined by colorimetric assays using orthocresol phthalein complexone and bacterial xanthine oxidase, respectively. Results: The concentrations of potassium and phosphate were decreased, whereas chloride concentration was increased in the vitreous of the FDM eyes ( P < .01). Sodium and calcium concentrations were similar to those in the control eyes. No significant changes in the concentration of electrolytes were observed in the aqueous humor. No significant differences were found in the protein concentrations in the liquid vitreous, gel vitreous, and aqueous humor. Conclusions: Form-deprivation induced a significant increase of the volume of the liquid vitreous in the eye of the FDM chick. The increased liquid vitreous of the myopic eye was accompanied by an alteration of the electrolyte balance, by a mechanism that has not yet been clarified.