Form-deprivation Myopia Group Research Articles

Retinal Neurotransmitter Alteration in Response to Dopamine D2 Receptor Antagonist from Myopic Guinea Pigs.

This study aimed to investigate the changes in retinal neurotransmitters and the role of the dopamine D2 receptor (D2R) pathway in regulating the myopic refractive state. Tricolor guinea pigs were randomly divided into two groups: the normal control group (NC) and the form-deprivation myopia group (FDM). Animals in the FDM group had their right eye covered with a balloon for 4 weeks. These two groups were further divided into two subgroups based on intravitreal injection with D2R antagonist sulpiride once a week for 3 weeks (NC, NC-Sul, FDM, and FDM-Sul groups). Ultrahigh-performance liquid chromatography-tandem mass spectrometry was used to quantitatively detect the changes in 17 retinal neurotransmitters. Compared to the NC group, the concentrations of dopamine (DA) and γ-aminobutyric acid (GABA) decreased, while those of glutamate (Glu), 3-methoxytyramine (3-MT), and glycine increased, accompanied by an increase in myopic refraction and axial length (AL) in the FDM group. In the FDM-Sul group, glycine and DA levels were upregulated, whereas 3-MT and Glu levels were downregulated, accompanied by a decrease in myopic refraction and AL. The ratio of Glu to GABA (RGG) represents the balance between excitatory and inhibitory neurotransmitters. Notably, RGG changes occurred with corresponding AL changes, which increased in the FDM group and decreased in the FDM-Sul group. Decreased retinal DA concentration, with an increase in Glu, may be involved in the myopia progression. D2R antagonists might effectively slow myopia progression by increasing retinal DA, regulating Glu concentration to match GABA, and maintaining the balance between excitatory and inhibitory neurotransmitters.

Widespread Involvement of Acetylation in the Retinal Metabolism of Form-Deprivation Myopia in Guinea Pigs.

Myopia has become the major cause of visual impairment worldwide. Although the pathogenesis of myopia remains controversial, proteomic studies suggest that dysregulation of retinal metabolism is potentially involved in the pathology of myopia. Lysine acetylation of proteins plays a key role in regulating cellular metabolism, but little is known about its role in the form-deprived myopic retina. Hence, a comprehensive analysis of proteomic and acetylomic changes in the retinas of guinea pigs with form-deprivation myopia was performed. In total, 85 significantly differential proteins and 314 significantly differentially acetylated proteins were identified. Notably, the differentially acetylated proteins were markedly enriched in metabolic pathways such as glycolysis/gluconeogenesis, the pentose phosphate pathway, retinol metabolism, and the HIF-1 signaling pathway. HK2, HKDC1, PKM, LDH, GAPDH, and ENO1 were the key enzymes in these metabolic pathways with decreased acetylation levels in the form-deprivation myopia group. Altered lysine acetylation of key enzymes in the form-deprived myopic retina might affect the dynamic balance of metabolism in the retinal microenvironment by altering their activity. In conclusion, as the first report on the myopic retinal acetylome, this study provides a reliable basis for further studies on myopic retinal acetylation.

The Mechanosensitive Piezo1 Channel Mediates Mechanochemical Transmission in Myopic Eyes.

To identify the expression of the mechanosensitive ion channel Piezo1 in the retina of guinea pigs with form deprivation myopia (FDM) and to investigate mechanisms by which Piezo1 channels might regulate myopia. Sixty 3-week-old guinea pigs were divided into four groups randomly: normal control, FDM, FDM + vehicle control (DMSO), and FDM + Piezo1 inhibitor (GsMTx4). Measurements of spherical equivalent (SE) and axial length (AL) of the guinea pig were taken using retinoscopy and A-scan ultrasound examination, respectively. Location of Piezo1 protein was determined using immunohistochemistry. The histological structure and thickness changes of the guinea pig retina were observed by hematoxylin and eosin. Expression of Piezo1 in the retina was detected using quantitative RT-PCR and Western blot. Reactive oxygen species (ROS) levels in the retina were measured using flow cytometry. After 4 weeks of form deprivation, the FDM group exhibited a significantly increased myopic degree and axial length compared with the normal control group (all P < 0.001), and had higher expression levels of Piezo1 and ROS than the normal control group (P < 0.001 and P = 0.002, respectively). Piezo1 protein expression was down-regulated in guinea pigs given GsMTx4 compared with the DMSO group (P = 0.037). Additionally, the GsMTx4 group showed lower myopic degree (P < 0.001) and lower ROS levels (P = 0.019) compared with the DMSO group. The Piezo1 channel may be activated in the retinas of FDM guinea pigs and be involved in the development of myopia by regulating intraocular ROS levels.

Changes of Melanopsin Expression in the Retina of Guinea Pig during Experimental Myopia and Recovery Period

Purpose To explore whether melanopsin is associated with the development of myopia. Methods Seventy-two guinea pigs (3 weeks older) were randomly assigned to 6 groups: the form-deprivation myopia (FDM) group (monocularly covering the right eye for 14 days, n = 15), the FDM recovery group (removing the eye mask for 3 days, n = 13), the lens-induced myopia (LIM) group (monocularly wearing a −4D lens for 3 days, n = 15), the LIM recovery group (removing the lens for 2 days, n = 13), and another 2 age-matched normal groups (n = 8 each). The diopter, the vitreous chamber depth (VCD), and the axial length (AXL) were measured to confirm the effect of the treatments. Immunofluorescence and western blotting methods were used to examine the expression of melanopsin in the retina. Results Immunofluorescent results showed that in the FDM group, the melanopsin intensity in the retina of experimental eyes significantly decreased compared to those of contralateral eyes, but no significant difference was observed during their recovery periods. Western blotting showed that the expression of melanopsin in the experimental eyes of the FDM group was lower than that of the contralateral eyes (fold: 1.00 versus 1.36). The expression of melanopsin in the experimental eyes increased 3 days after removing form deprivation, although a slight reduction in melanopsin expression compared to that of the contralateral eyes (fold: 1.41 versus 1.58). For the LIM group, immunofluorescent showed an obvious decreased intensity of melanopsin-labeled cells in the experimental eyes compared to the contralateral eyes. Western blotting showed that although melanopsin expression in the experimental eyes decreased compared to that of the contralateral eyes (fold: 1.00 versus 1.96), no differences were found between two eyes 2 days after lens removal (fold: 1.99 versus 2.00). Conclusion The decreased expression of melanopsin in the retina may potentially participate in the development of FDM and LIM.

Altered whole-brain gray matter volume in form-deprivation myopia rats based on voxel-based morphometry: A pilot study.

Myopia is one of the major public health problems worldwide. However, the exact pathogenesis of myopia remains unclear. This study proposes using voxel-based morphometry (VBM) to investigate potential morphological alterations in gray matter volume (GMV) in form-deprivation myopia (FDM) rats. A total of 14 rats with FDM (FDM group) and 15 normal controls (NC group) underwent high-resolution magnetic resonance imaging (MRI). Original T2 brain images were analyzed using VBM method to identify group differences in GMV. Following MRI examination, all rats were perfused with formalin, and immunohistochemical analysis of NeuN and c-fos levels was performed on the visual cortex. In the FDM group, compared to the NC group, significantly decreased GMVs were found in the left primary visual cortex, left secondary visual cortex, right subiculum, right cornu ammonis, right entorhinal cortex and bilateral molecular layer of the cerebellum. Additionally, significantly increased GMVs were found in the right dentate gyrus, parasubiculum, and olfactory bulb. Our study revealed a positive correlation between mGMV and the expression of c-fos and NeuN in the visual cortex, suggesting a molecular relationship between cortical activity and macroscopic measurement of visual cortex structural plasticity. These findings may help elucidate the potential neural pathogenesis of FDM and its relationship to changes in specific brain regions.

Effect of shape deprivation on retinal thickness in myopic mice using an OCT method.

The purpose of this study was to study in retina thickness changes in myopic mice using optical coherence tomography (OCT). There were 18 mice in the form-deprivation myopia (FDM) group，in which the left eye was not treated as a control;18 untreated mice served as a normal control group. The diopter of all mice was measured 21 days after birth (P21), before form deprivation. After 4 weeks of form deprivation (P49), the refraction, fundus, and retinal sublayer thickness of all mice were measured. After 4 weeks of form deprivation, the refractive power of the right eye in the FDM group was significantly higher than that in the left eye (p < 0.05). There was no significant change in the refractive power of the left eye in the FDM group compared with the normal control group. The retina, nerve fiber layer (NFL), inner nuclear layer (INL), and outer nuclear layer (ONL) in the right eye of the FDM group were significantly thinner than those of both the FDM and control groups (p < 0.05). There was no significant change in photoreceptor (PR). Our study highlights that the myopic mice have decreased R thickness, which might reflect the potential pathological mechanism of myopia.

Effects of exogenous retinoic acid on ocular parameters in Guinea pigs with form deprivation myopia.

Aim: Myopia is a common chronic eye disease, this study is to investigate the effects of exogenous retinoic acid (RA) on intraocular parameters, especially choroidal thickness (CT) and retinal thickness (RT), in guinea pigs with form deprivation myopia (FDM). Methods: A total of 80 male guinea pigs were divided randomly into 4 groups: Control, FDM, FDM + RA, and FDM + Citral groups. The FDM + RA group was given 24mg/kg RA dissolved in 0.4mL peanut oil; the FDM + Citral group was given citral 445mg/kg dissolved in 0.4mL peanut oil; The other two groups were given 0.4mL peanut oil. After 4weeks, the refractive error (RE), axial length (AL), and intraocular pressure (IOP) of all guinea pigs were measured, and the parameters of RT and CT were obtained using enhanced depth imaging optical coherence tomography (EDI-OCT). Results: After 4weeks, both the RE and AL in the FDM and FDM + RA groups were increased, and the RT and CT in both groups were smaller than those in the Control group (p < 0.05). Only the IOP of the right eye in the FDM + RA group increased significantly (p < 0.05). The RT of the right eye of the 4 groups was compared: Control group > FDM + Citral group > FDM group > FDM + RA group. Compared with the RT of the left eye and the right eye among the 4 groups, the RT of the right eye in the FDM and FDM + RA groups was significantly less than that in the left eye (p < 0.05). Moreover, the CT of the right eye in the Control group was greater than that in the other three groups (p < 0.0001). There was no significant difference in the CT among the FDM, FDM + RA, and FDM + Citral groups (p > 0.05). In contrast to the RT results, the CT results of the left and right eyes in the FDM + Citral group showed statistically significant differences (p < 0.05). Conclusion: RA participates in the progression of FDM as a regulatory factor. Exogenous RA can increase the RE, AL, and IOP of FDM guinea pigs, and might aggravate the retinal thinning of FDM guinea pigs. Citral can inhibit these changes, but RA might not affect the thickness of the choroid.

EFEMP1 is a potential biomarker of choroid thickness change in myopia.

To explore the possible molecular mechanism by which epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) regulates choroid thickness (CT) in the development of myopia. In total, 131 subjects were divided into the emmetropia (EM) group, non-high myopia (non-HM) group and high myopia (HM) group. Their age, refraction, intraocular pressure, and other ocular biometric parameters were collected. A 6 × 6 mm area centered on the optic disc was scanned by coherent optical tomography angiography (OCTA) to measure CT, and the tear concentrations of EFEMP1 were quantified using enzyme-linked immunosorbent assay (ELISA) analysis. Twenty-two guinea pigs were divided into the control group and the form-deprivation myopia (FDM) group. The right eye of the guinea pig in the FDM group was covered for 4 weeks, and the diopter and axial length of the right eye of the guinea pig were measured before and after the treatment. After the measurement, the guinea pig was euthanized, and the eyeball was removed. Quantitative reverse transcription polymerase chain reaction, western blotting assays and immunohistochemistry were used to assess the expression of EFEMP1 in the choroid. There were significant differences in CT among the three groups (p < 0.001). CT was positively correlated with age in HM (r = -0.3613, p = 0.0021), but no significant correlation with SE (p > 0.05) was observed. Furthermore, there were increased levels of EFEMP1 in the tears of myopic patients. After 4 weeks of covering the right eye of the FDM guinea pigs, there was a significant increase in axial length and a decrease in diopter (p < 0.05). The mRNA and protein expression of EFEMP1 was significantly increased in the choroid. Choroidal thickness was significantly thinner in myopic patients, and the expression level of EFEMP1 in the choroid increased during the development of FDM. Therefore, EFEMP1 may be involved in the regulation of choroidal thickness in myopia patients.

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.

The Effect of CD 90 Expression after Intrasclera Limbal Mesenchymal Stem Cell Injection in Form Deprivation Myopia Animal Model

Purpose: To investigate the capability of limbal mesenchymal stem cells (LMSCs) in increasing CD 90 expression as marker that become new fibroblast to potentially reduce myopia progression in form deprivation myopia animal model. Method: This study using cultured LMSC from rabbit (Oryctolagus cuniculus) and isolated using modification of semienzimatic methods. 24 animal model divided into three groups, consists of control group, form deprivation myopia group (FDM), and form deprivation myopia group with LMSCs injection (FDM+LMSCs). Myopia model was made by monocular occlusion for six weeks. LMSCs was injected intrasclera after fourth week of occlusion. Enucleation was performed in sixth week. CD 90 expression of posterior sclera were evaluated using immunohistochemistry. The results between groups were analyzed using Kruskal-Wallis and Mann-Whitney with 95% confidence interval (p<0,05). Result: After four weeks of deprivation, all eyes became more myopic. LMSCs injection was done 2,5 mm posterior from limbus and two week-evaluation showed LMSCs had migrated on posterior region of the sclera. CD 90 expression showed significant differences between control and FDM+LMSCs group also FDM and FDM+LMSCs group (p<0,05). Conclusion: LMSCs has potential role in myopia control in form deprivation eye animal model by increasing CD 90 expression and lead to additional new scleral fibroblast cells.

Mechanism of Hypoxia Inducible Factor-1α (HIF-1α) in Choroidal Neovascularization in Macular Lutea of High Myopia

To explore the changes in the expression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in the process of choroidal neovascularization (CNV) in the macular area of cases with form deprivation myopia (FDM), in this study, 36 healthy mice were selected as research subjects for analysis. Specifically, they were randomly divided into test group (n = 18) and control group (n = 18). The right eye of the test group mice underwent form deprivation for 6 weeks to induce FDM, also known as FDM group. Five mice were randomly selected from FDM group and control group at 6 time points (before photocoagulation, at 7 d, 14 d, 21 d, 28 d, and 35 d after photocoagulation), respectively, for fluorescein angiography and hematoxylin-eosin staining (HE) staining to observe differences in the retina. It was found that the retina of Control group mice was well-structured with clear stratification, while that of the test group mice was severely damaged with a disordered structure, manifested as edema and swelling and damaged Bruch membrane and retinal pigment epithelium (RPE) layer. Furthermore, macrophages and RPE cells were observed in the photocoagulation area, and there was a high-fluorescence area with a blurred margin in the fundus, indicating the formation of new blood vessels. The immunohistochemical experiment revealed that within three weeks after the surgery, HIF-1α was highly expressed in the test group, and the expression level was obviously higher than the Control group. The real-time quantitative polymerase chain reaction (PCR) results showed that the expression level of VEGF and HIF-α in the test group was obviously higher than the Control group within three weeks after the surgery. The Western blot experiment was performed 1 week after the surgery, and it was noted that the expression level of VEGF and HIF-1α were higher in the test group versus the Control group. In summary, HIF-1α is instrumental in ocular CNV, which is also associated with VEGF.

Expression of SCO1 and SCO2 after form-deprivation myopia in Guinea pigs.

The retina is a highly energy-consuming tissue associated with visual development, and the reduced quality of retinal imaging can be related to myopia. Synthesis of cytochrome c oxidase 1 (SCO1) and synthesis of cytochrome c oxidase 2 (SCO2) are involved in ATP (adenosine triphosphate) synthesis and energy metabolism. This study aimed to observe the morphologic changes and investigate the expression of SCO1 and SCO2 induced by form-deprivation myopia (FDM) in the retina and sclera of guinea pigs. Thirty-six 3-week-old male guinea pigs were randomly assigned to one of two groups: (1) the model group (n = 18), in which the right eyes were covered by a thin opaque balloon as FDM group, and the left eyes were uncovered and served as the contralateral control group; (2) the blank control group (n = 18), in which bilateral eye received no manipulation. Eyeballs were enucleated for histological analysis. The retina and sclera of the guinea pigs were separated to determine the protein and mRNA expression levels of SCO1 and SCO2, respectively. After four weeks of form deprivation (FD), the refractive degree and axial length increased significantly (P < 0.001). The retinal and scleral tissues were moderately thinner, and the ganglion cells and the cells of inner and outer nuclear layers in the retina became fewer. Compared with the contralateral control group (P < 0.001) and the blank control group (P < 0.001), the collagen content of the sclera became less in the FDM group. The protein and mRNA expression levels of SCO1 and SCO2 in the FDM group were significantly lower than those in the contralateral control group and the blank control group (P < 0.05). The morphologies of the retina and sclera were changed, and the expression of SCO1 and SCO2 at the protein and transcription levels was significantly reduced in the FDM group. Given these changes, SCO1 and SCO2 genes may be involved in myopic progression.

Dynamic Changes of AREG in the Sclera during the Development of Form-Deprivation Myopia in Guinea Pigs

ABSTRACTPurposeTo investigate amphiregulin (AREG) expression in the sclera during the development of form-deprivation myopia (FDM) and after the recovery of FDM in guinea pigs.MethodsSixty-four 2-week-old guinea pigs were randomly divided into the control and FDM groups. The right eyes of animals in FDM group were covered for 2 weeks (2 W) and 4 weeks (4 W), or were covered for 4 weeks and then uncovered for the subsequent 2 weeks (6 W). The diopters and axial lengths (AL) in the right eyes of guinea pigs were measured. Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence, and western blotting assays were used to detect the relative mRNA and protein expressions of AREG in the sclera of guinea pigs.ResultsBefore masking (0 W), the refraction and AL in the right eyes of guinea pigs in the control and FDM groups did not differ significantly (both p > .05). Myopic shift was induced in guinea pigs with the diopters gradually decreased and AL increased in the FDM group. While no significant difference was found in control group at different time points, the relative AREG mRNA and protein expression levels in the FDM group were significantly increased in 2 W and 4 W and then decreased after 4 weeks of covering followed by uncovering for 2 weeks (all p < .05).ConclusionsAREG was expressed in the sclera of guinea pigs. Moreover, the expression levels of AREG increased during the development of FDM and downregulated after recovery of FDM. Therefore, AREG may be involved in the regulation of scleral remodeling in myopia.

Alterações dinâmicas de periostina e colágeno I na esclera durante miopia progressiva em cobaias

To investigate periostin and collagen I expression during a scleral remodeling in myopic eyes and to determine their role in collagen remodeling of the myopic sclera. Fifty one-week-old guinea pigs were divided into the control and form-deprivation myopia (FDM) groups. The eyes of animals in the form-deprivation myopia group were covered for 2, 4, and 8 weeks, or were covered for 4 weeks and then uncovered for 2 weeks. The diopters and axial lengths in the eyes in each group of guinea pigs were measured. Immunohistochemistry and reverse transcription polymerase chain reaction were used to detect the relative protein and mRNA expressions of periostin and collagen I in the scleral tissues of guinea pig. Before masking, guinea pigs in the control and form-deprivation myopia groups were hypermetropic and did not differ significantly (p>0.05). Hypermetropic refraction in the control group gradually decreased. In guinea pigs from the form-deprivation myopia group, the refractive power gradually changed from +2.14 ± 0.33 D to -7.22 ± 0.51 D, and the axial length gradually changed from 5.92 ± 0.37 mm to 8.05 ± 0.34 mm from before until the end of masking. Before covering, no significant difference was observed in the relative collagen I and periostin mRNA and protein expression levels in the sclera of the guinea pig control and form-deprivation myopia groups (p>0.05). The relative collagen I and periostin protein and mRNA expression levels in the sclera of guinea pigs in the form-deprivation myopia group at 2, 4, and 8 weeks, and after covering the eyes for 4 weeks followed by uncovering for 2 weeks, were significantly lower than those in the control group (p<0.05). The collagen I and periostin mRNA expression levels were positively correlated with protein expression levels in the sclera of guinea pigs (protein: r=0.936, p<0.05; mRNA: r=0.909, p<0.05). Periostin was expressed in the myopic sclera of guinea pigs, and changes in periostin and collagen I expression were highly consistent. Periostin and collagen I may be involved in the regulation of scleral remodeling in myopia.

Decreased expression of gap junction delta-2 (GJD2) messenger RNA and connexin 36 protein in form-deprivation myopia of guinea pigs.

Background:More than ten genome-wide association studies have identified the significant association between the gap junction delta-2 (GJD2) gene and myopia. However, no functional studies have been performed to confirm that this gene is correlated with myopia. This study aimed to observe how this gene changed in mRNA and protein level in the form-deprivation myopia (FDM) animal model.Methods:Four-week-old guinea pigs were randomly divided into two groups: control and FDM groups (n = 12 for each group). The right eyes of the FDM group were covered with opaque hemispherical plastic lenses for 3 weeks. For all the animals, refractive status, axial length (AL), and corneal radius of curvature were measured at baseline and 3 weeks later by streak retinoscope, A-scan ultrasonography, and keratometer, respectively. Retinal GJD2 mRNA expression and connexin 36 (Cx36) levels in FDM and control groups were measured by quantitative real-time PCR and Western blot analyses, respectively. Those results were compared using independent t test, Mann-Whitney U test, or paired t test. A significance level of P < 0.05 was used.Results:Three weeks later, the FDM group (form-deprived eyes) showed about a myopic shift of approximately −6.75 (−7.94 to −6.31) D, while the control group remained hyperopic with only a shift of −0.50 (−0.75 to 0.25) D (Z = −3.38, P < 0.01). The AL increased by 0.74 (0.61–0.76) and 0.10 (0.05–0.21) mm in FDM and control groups, respectively (Z = −3.37, P < 0.01). The relative mRNA expression of GJD2 in the FDM group decreased 31.58% more than the control group (t = 11.44, P < 0.01). The relative protein expression of CX36 on the retina was lowered by 37.72% in form-deprivation eyes as compared to the controls (t = 17.74, P < 0.01).Conclusion:Both the mRNA expression of GJD2 and Cx36 protein amount were significantly decreased in the retina of FDM guinea pigs. This indicates that Cx36 is involved in FDM development, providing compensating evidence for the results obtained from genome-wide association studies.

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.

Expression and role of specificity protein 1 in the sclera remodeling of experimental myopia in guinea pigs.

To study the expression of collagen I and transcription factor specificity protein 1 (Sp1), a transforming growth factor-β1 (TGF-β1) downstream target, and reveal the impact of the TGF-β1-Sp1 signaling pathway on collagen remodeling in myopic sclera. Seventy-five 1-week-old guinea pigs were randomly divided into normal control, form deprivation myopia (FDM), and self-control groups. FDM was induced for different times using coverage with translucent latex balloons and FDM recovery was performed for 1wk after 4wk treatment; then, changes in refractive power and axial length were measured. Immunohistochemistry and reverse transcription-polymerase chain reaction were used to evaluate dynamic changes in collagen I and Sp1 expression in the sclera of guinea pigs with emmetropia and experimental myopia, and the relationship between collagen I and Sp1 levels was analyzed. In the FDM group, the refractive power was gradually changed (from 2.09±0.30 D at week 0 to -1.23±0.69 D, -4.17±0.59 D, -7.07±0.56 D, and -4.30±0.58 D at weeks 2, 4, 6, and 1wk after 4wk, respectively; P<0.05), indicating deepening of myopia. The axial length was increased (from 5.92±0.39 mm at week 0 to 6.62±0.36 mm, 7.30±0.34 mm, 7.99±0.32 mm, and 7.41±0.36 mm at weeks 2, 4, 6, and 1wk after 4wk; P<0.05). The mRNA and protein expression of Sp1 and collagen I in the sclera of the FDM group was lower than that of the control groups (P<0.05), and the reduction was eye-coverage time-dependent. Furthermore, correlation between Sp1 and collagen I down-regulation in the myopic sclera was observed. Our data indicate that transcription factor Sp1 may be involved in the regulation of type I collagen synthesis/degradation during myopic sclera remodeling, suggesting that TGF-β1 signaling plays a role in the development and progression of myopia.

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.

Scleral TGF-β1 and Smad3 expression is altered by TCM Bu Jing Yi Shi Tablets in guinea pigs with form-deprivation myopia

ObjectiveTo explore whether the traditional Chinese medicine (TCM) Bu Jing Yi Shi Tablets alters the expression of scleral TGF-β1 and Smad3 in guinea pigs with form-deprivation myopia (FDM). MethodsSixty-five guinea pigs were randomly divided into control, model, low-, medium-, and high-dose treatment groups. Except for the control group, FDM was induced by covering the right eye of each animal with opaque latex. The treatment groups were gavaged with different suspension concentrations of Bu Jing Yi Shi Tablets. Refraction and axial length were performed before and after myopia induction. At the end of the experiment, all right eyes were extracted, and scleral sections were prepared for staining and TGF-β1 and Smad3 immunohistochemistry. Scleral thickness and area, the scleral fibroblast quantity, and scleral TGF-β1 and Smad3 expressions were measured. ResultsThe 5 FDM groups had the same initial axial length and diopter, the final diopter and axial length of the model group were significantly increased compared with the control group and treatment groups (P < .01). The axial length of each treatment group decreased as the dose decreased compared with the model group (P < .01); the total scleral area (P < .05–.01) and the number of scleral fibroblasts (P < .01) in the model group were significantly lower than the treatment groups. Both the TGF-β1 and Smad3 integral optical densities in the model group were significantly lower than the control and medium- and high-dose treatment groups (P < .01). TGF-β1 and Smad3 mRNAs in the model group were decreased compared with the control group, but increased in expression after treatment. ConclusionBu Jing Yi Shi Tablets may restore the expression of scleral TGF-β1 and Smad3 in FDM guinea pigs and impede myopia development.

The sonic hedgehog signaling pathway induces myopic development by activating matrix metalloproteinase (MMP)-2 in Guinea pigs.

PurposeTo investigate whether the Sonic hedgehog (Shh) signaling induces myopic development by increasing the expression of matrix metalloproteinase (MMP)-2 in guinea pigs.MethodsA translucent diffuser was glued onto the right eye to induce form-deprivation myopia (FDM) in 10 guinea pigs. Four guinea pigs were served as a control group. The other 100 guinea pigs were subdivided into 5 groups (20 per group) and received a 10 µl intravitreal injection every 2 days for 4 times. Two groups were injected with 20 or 50 µg/ml Shh amino-terminal peptide (Shh-N) into the right eye and 0.1% bovine serum albumin into the other. FDM was induced in the right eyes of the three cyclopamine-treated groups and both eyes were injected with 50, 100, or 200 µg/ml cyclopamine. Retinoscopic refraction and eye dimensions were assessed on Day 14 of treatment. MMP-2 protein expression was determined in both scleras by western blotting.ResultsBoth concentrations of Shh-N stimulated myopic development and axial growth as compared with control eyes. Myopia and axial elongation were significantly greater in the 50 µg/ml than in the 20 µg/ml Shh-N group (P<0.001 and P = 0.0019, respectively). All three doses of cyclopamine significantly attenuated myopic development compared with the FDM group (P<0.0001). Cyclopamine at 100 or 200 µg/ml significantly reduced axial elongation compared with the FDM group (P = 0.044 and P = 0.001, respectively). FDM-induced myopia and axial elongation were significantly greater in the 50 µg/ml than in the 200 µg/ml cyclopamine group (P<0.0001 and P = 0.008, respectively). MMP-2 expression was significantly greater in Shh-N–treated eyes than in the control eyes, and was lower in the cyclopamine plus FDM groups than in the FDM group.ConclusionsThe Shh signaling pathway induces myopic development by activating MMP-2 in guinea pigs.