Unveiling the gut microbiota and metabolite profiles in guinea pigs with form deprivation myopia through 16S rRNA gene sequencing and untargeted metabolomics.

Aim: The aim of this study was to confirm the presence of the form deprivation myopia (FDM) guinea pig eye-gut axis and investigate the relationship between serum vasoactive intestinal peptide (VIP), lipopolysaccharides (LPS), specific gut microbiota and their metabolites. Method: 20 specific-pathogen-free (SPF) guinea pigs were divided into the FDM and the control(Con) group. Following model induction, serum levels of VIP and LPS were quantified. A combination of 16S ribosomal ribosomal Ribonucleic Acid (rRNA) gene sequencing, non-targeted metabolomics and bioinformatics analysis were employed to identify disparities in gut microbiota and metabolites between the two groups of guinea pigs. Result: Compared to the control group, FDM guinea pigs exhibited a significant trend towards myopia, along with significantly elevated concentrations of LPS and VIP (p < 0.0001). Furthermore, Ruminococcus_albus emerged as the predominant bacterial community enriched in FDM (p < 0.05), and demonstrated positive correlations with 10 metabolites, including l-Glutamic acid, Additionally, Ruminococcus_albus exhibited positive correlations with VIP and LPS levels (p < 0.05). Conclusion: The findings suggest that the Ruminococcus_Albus and glutamate metabolic pathways play a significant role in myopia development, leading to concurrent alterations in serum VIP and LPS levels in FDM guinea pigs. This underscores the potential of specific gut microbiota and their metabolites as pivotal biomarkers involved in the pathogenesis of myopia.

Synchronous myopia development induced by bilateral form deprivation in chicks.

Form deprivation (FD) is a widely employed experimental paradigm, typically used to induce unilateral myopia in animal models. This model is weakened by potential influence upon the FD eye from vision in the freely-viewing contralateral eye, which could be eliminated by imposing FD in both eyes; but while a few previous studies have explored the feasibility of inducing bilateral FD in chicks, substantial discrepancies in treatment outcomes were noted. Consequently, this study aimed to establish a bilateral FD myopia model in chicks, with validation by investigating the associated ocular growth patterns, feeding, and social behavior. Six-day-old chicks were treated with bilateral (n = 21) or unilateral (n = 10) FD for 12 days; the fellow untreated eyes in the unilateral FD group served as controls. Refractive error, corneal power, and ocular axial dimensions were measured at 4-day intervals after the onset of form deprivation, with a Hartinger refractometer, a custom-made videokeratography system, and a high-resolution A-scan ultrasonographer, respectively. Body weight was monitored to assess the chick's physical development. Our results showed that birds treated with bilateral FD grew as robustly as the unilaterally form-deprived chicks, with similar or slightly heavier body weights and mortalities. Unilateral FD induced significantly higher myopia in the treated eye, with stronger corneal power, deeper anterior and vitreous chambers, and longer axial length. Moreover, either bilaterally or unilaterally FD eyes developed similar refractive error (bilateral FD, left: -28.03 ± 9.06 D, right: -28.44 ± 9.45 D; unilateral FD: -29.48 ± 8.26 D) and ocular biometric changes; but choroidal thickness was thicker in bilaterally FD eyes, rather than thinner as in unilaterally FD eyes. In addition to the highly synchronized (symmetrical, parallel) development reported previously in bilateral FD, we found in this study that the correlations between bilaterally form-deprived eyes were highest for ocular biometric parameters directly contributing to myopia development, including corneal power (r = 0.74 to 0.93), anterior chamber depth (r = 0.60 to 0.85), vitreous chamber depth (r = 0.92 to 0.94), and axial length (r = 0.90 to 0.96). The remarkably synchronized growth pattern confirmed the feasibility of the bilateral FD paradigm for future research on myopia.

The role of vasoactive intestinal peptide (VIP) in atropine-related inhibition of the progression of myopia.

OBJECTIVE: This study aimed to investigate the potential involvement of vasoactive intestinal polypeptide (VIP) in myopia development and its contribution to the mechanism of action of the anti-myopia drug, atropine. METHODS: Thirty-three-week-old guinea pigs were randomly divided into normal control (NC, n = 10), monocularly form-deprived (FDM, n = 10), and FDM treated with 1% atropine (FDM + AT, n = 10) groups. The diopter and axial length were measured at 0, 2, and 4 weeks. Guinea pig eyeballs were removed at week four, fixed, and stained for morphological changes. Immunohistochemistry (IHC) and in situ hybridization (ISH) were performed to evaluate VIP protein and mRNA levels. RESULTS: The FDM group showed an apparent myopic shift compared to the control group. The results of the H&E staining were as follows: the cells of the inner/outer nuclear layers and retinal ganglion cells were disorganized; the choroidal thickness (ChT), blood vessel lumen, and area were decreased; the sclera was thinner, with disordered fibers and increased interfibrillar space. IHC and ISH revealed that VIP's mRNA and protein expressions were significantly up-regulated in the retina of the FDM group. Atropine treatment attenuated FDM-induced myopic shift and fundus changes, considerably reducing VIP's mRNA and protein expressions. CONCLUSIONS: The findings of elevated VIP mRNA and protein levels observed in the FDM group indicate the potential involvement of VIP in the pathogenesis and progression of myopia. The ability of atropine to reduce this phenomenon suggests that this may be one of the molecular mechanisms for atropine to control myopia.

Targeting scleral remodeling and myopia development in form deprivation myopia through inhibition of EFEMP1 expression.

The role of extracellular matrix (ECM) remodeling in the axial elongation associated with myopia has not been fully elucidated, although it is considered a significant factor. EFEMP1, a regulator of ECM, has been associated with various pathological conditions. This study aimed to examine the involvement of EFEMP1 in scleral remodeling during form deprivation myopia. The results indicate a progressive increase in EFEMP1 expression following prolonged form deprivation treatment, followed by a subsequent decrease upon recovery. To gain a deeper understanding of the mechanism of EFEMP1, we conducted transcriptome sequencing on primary scleral fibroblasts that were subjected to lentivirus-mediated overexpression of EFEMP1. Validation was performed using lentivirus-induced overexpression and shRNA targeting EFEMP1 in combination with LY294002, a PI3K inhibitor. Our findings suggest that EFEMP1 may be involved in the development of FDM by regulating the expression of the PI3K/AKT/MMP2 axis. The AAV-mediated injection of shEFEMP1 under Tenon's capsule in guinea pigs was observed to effectively delay the progression of myopia and posterior scleral remodeling. In contrast, the AAV-mediated overexpression of EFEMP1 exacerbated the development of myopia and resulted in further thinning of collagen fibers in the posterior sclera. In summary, adjusting EFEMP1 concentrations could potentially serve as a viable approach to prevent and treat myopia by influencing the remodeling process of the posterior sclera.

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

Purpose: The purpose of this study was to study in retina thickness changes in myopic mice using optical coherence tomography (OCT). Methods: 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. Results: 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). Conclusion: 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 24 mg/kg RA dissolved in 0.4 mL peanut oil; the FDM + Citral group was given citral 445 mg/kg dissolved in 0.4 mL peanut oil; The other two groups were given 0.4 mL peanut oil. After 4 weeks, 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 4 weeks, 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.

Identification of miR-671-5p and Its Related Pathways as General Mechanisms of Both Form-Deprivation and Lens-Induced Myopia in Mice.

Animal models have been indispensable in shaping the understanding of myopia mechanisms, with form-deprivation myopia (FDM) and lens-induced myopia (LIM) being the most utilized. Similar pathological outcomes suggest that these two models are under the control of shared mechanisms. miRNAs play an important role in pathological development. Herein, based on two miRNA datasets (GSE131831 and GSE84220), we aimed to reveal the general miRNA changes involved in myopia development. After a comparison of the differentially expressed miRNAs, miR-671-5p was identified as the common downregulated miRNA in the retina. miR-671-5p is highly conserved and related to 40.78% of the target genes of all downregulated miRNAs. Moreover, 584 target genes of miR-671-5p are related to myopia, from which we further identified 8 hub genes. Pathway analysis showed that these hub genes are enriched in visual learning and extra-nuclear estrogen signaling. Furthermore, two of the hub genes are also targeted by atropine, which strongly supports a key role of miR-671-5p in myopic development. Finally, Tead1 was identified as a possible upstream regulator of miR-671-5p in myopia development. Overall, our study identified the general regulatory role of miR-671-5p in myopia as well as its upstream and downstream mechanisms and provided novel treatment targets, which might inspire future studies.

Time-Serial Evaluation of the Development and Treatment of Myopia in Mice Eyes Using OCT and ZEMAX.

Myopia is a significant cause of visual impairment which may lead to many complications. However, the understanding of the mechanisms of myopia is still limited. In this paper, in order to investigate the development and the treatment of myopia, we analyzed the biological structure parameters of mice eyes, obtained from optical coherence tomography (OCT), and the optical performance of mice eyes calculated using ZEMAX software (ZEMAX Development Corporation, Kirkland, WA, USA) in which the optical model was built on the segment-by-segment optically corrected OCT 3D-images. Time-serial evaluation of three groups of mice eyes (form-deprivation myopia mice eyes, normal mice eyes, and atropine-treated myopia mice eyes) was performed. In addition to the biological structure parameters, imaging performance with the development of root-mean-square wavefront aberration at six filed angles was compared and analyzed. Results show that the biological structure parameters of the eye are closely related to the development of myopia. The peripheral defocus of the retina has a significant impact on inducing myopia, which verifies the new theory of myopia development. The delaying effect of atropine solution on myopia development is shown to verify the therapeutic effect of the medicine. This study provides technical support for the investigation of the myopia mechanism.

Effect of vascular endothelial growth factor-A165 on scleral remodeling of guinea pigs with form-deprivation myopia / 国际眼科杂志(Guoji Yanke Zazhi)

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&#x003E;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&#x003C;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&#x003C;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.

Effects of modified Zhujing Pill on mitochondrial dynamics related protein in mice with form deprivation myopia / 国际眼科杂志(Guoji Yanke Zazhi)

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&#x003E;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&#x003C;0.05), while the mice in the Chinese medicine group had significantly shorter axial length and higher diopter than the model group(all P&#x003C;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&#x003C;0.05), and the relative expression of MFN1 and OPA1 increased in the Chinese medicine group compared with the model group(all P&#x003C;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.

Quantitative Assessment of the Choroidal Vessel Diameter during the Recovery of Form-Deprivation Myopia in Guinea Pigs.

PURPOSE: The development and recovery (REC) of myopia is associated with changing of choroidal thickness (CT) in the model of guinea pigs. Nitric oxide synthase (NOS) is an enzyme which can affect choroidal vasodilatation. This study wants to investigate the changes of choroidal vessel diameter (CVD) and NOS during the REC of form-deprivation (FD) myopia in guinea pigs. METHODS: Forty-eight guinea pigs were randomly assigned to the normal control (NC) group, FD group (FD for 21 d), and four REC groups: REC1/2 group (removal the deprivation and re-exposure to the normal environment for 1/2 d), REC1 group (1 d), REC 2 group (2 d), and REC7 group (7 d). CT was measured by optical coherence tomography (OCT), and CVD of foveal choroid was quantitatively assessed on OCT angiography images using MATLAB software at each time point. NOS in choroid was measured using enzyme-linked immunosorbent assays. Measurements were compared between groups and correlations between CT, CVD, and NOS were assessed using regression analyses. RESULTS: CVD and CT in FD group were significantly smaller than in NC group (both p < .05), while the NOS significantly larger (p < .001). When deprivation was removed, CVD and NOS were significantly larger and reached a peak in the REC1 group, while CT reached the peak in the REC2 group, then all gradually decreased, and no significant differences were observed in NC and REC7 group (all p > .05). In the REC and NC groups, there was a significant positive correlation between CVD and NOS (p < .001), CVD and CT (p = .0092), but no correlation was found between NOS and CT (p > .05). CONCLUSIONS: This study indicated that the CVD in guinea pigs could be significantly dilated following myopia REC, and this change coincides with changes in NOS and CT.

Biomechanical changes in myopic sclera correlate with underlying changes in microstructure.

Myopia alters the microstructural and biomechanical properties of the posterior sclera, which is characterized as a layered structure with potentially different inter-layer collagen fibril characteristics. Scanning acoustic microscopy (SAM) has been used to investigate how the micron-scale bulk mechanical properties of the posterior sclera are affected by myopia. Other investigators have employed second harmonic generation (SHG) imaging to characterize the collagen microstructure of tissues. In the present study, SAM and SHG imaging were used to investigate the existence of biomechanically-distinct scleral layers and identify relationships between mechanical properties and tissue microstructure in myopic guinea pig (GP) eyes. Diffusers were worn over the right eyes of six, 1-week-old GPs for one week to induce unilateral form-deprivation myopia. GPs were euthanized, enucleated, and eyes were cryosectioned. Twelve-micron-thick adjacent vertical cryosections were scanned with SAM or SHG. SAM maps of bulk modulus, mass density, and acoustic attenuation were estimated. A fiber-extraction algorithm applied to SHG images estimated collagen fiber length, width, straightness, alignment, and number density. Results revealed that the posterior sclera may exhibit biomechanically distinct layers that are affected differently in myopia. Specifically, a layered structure was observed in the mechanical-parameter maps of control eyes that was less apparent in myopic eyes. Collagen fibers in myopic eyes had smaller diameters and were more aligned. Myopia-associated biomechanical changes were most significant in the outermost and innermost scleral layers. SAM-measured mechanical parameters were correlated with collagen fiber microstructure, particularly fiber length, alignment, and number density, which may imply the biomechanical parameters estimated from SAM measurements are related to tissue microstructure. Interestingly, some changes were greatest in more-peripheral regions, suggesting interventions to strengthen the sclera may be effective away from the optic nerve and efficacy may be achieved best when intervention is applied to the outermost layer.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Declines in PDE4B activity promote myopia progression through downregulation of scleral collagen expression.

Myopia is the most common cause of a visual refractive error worldwide. Cyclic adenosine monophosphate (cAMP)-linked signaling pathways contribute to the regulation of myopia development, and increases in cAMP accumulation promote myopia progression. To pinpoint the underlying mechanisms by which cAMP modulates myopia progression, we performed scleral transcriptome sequencing analysis in form-deprived mice, a well-established model of myopia development. Form deprivation significantly inhibited the expression levels of genes in the cAMP catabolic pathway. Quantitative real-time polymerase chain reaction analysis validated that the gene expression level of phosphodiesterase 4B (PDE4B), a cAMP hydrolase, was downregulated in form-deprived mouse eyes. Under visually unobstructed conditions, loss of PDE4B function in Pde4b-knockout mice increased the myopic shift in refraction, -3.661 ± 1.071 diopters, more than that in the Pde4b-wildtype littermates (P < 0.05). This suggests that downregulation and inhibition of PDE4B gives rise to myopia. In guinea pigs, subconjunctival injection of rolipram, a selective inhibitor of PDE4, led to myopia in normal eyes, and it also enhanced form-deprivation myopia (FDM). Subconjunctival injection of dibutyryl-cyclic adenosine monophosphate, a cAMP analog, induced only a myopic shift in the normal visually unobstructed eyes, but it did not enhance FDM. As myopia developed, axial elongation occurred during scleral remodeling that was correlated with changes in collagen fibril thickness and distribution. The median collagen fibril diameter in the FDM + rolipram group, 55.09 ± 1.83 nm, was thinner than in the FDM + vehicle group, 59.33 ± 2.06 nm (P = 0.011). Thus, inhibition of PDE4 activity with rolipram thinned the collagen fibril diameter relative to the vehicle treatment in form-deprived eyes. Rolipram also inhibited increases in collagen synthesis induced by TGF-ß2 in cultured human scleral fibroblasts. The current results further support a role for PDE enzymes such as PDE4B in the regulation of normal refractive development and myopia because either loss or inhibition of PDE4B function increased myopia and FDM development through declines in the scleral collagen fibril diameter.

[Gene expression profiles of myopic mouse scleral fibroblasts: a bioinformatics analysis based on single-cell RNA sequencing].

OBJECTIVE: To investigate the changes in gene expression profiles of mouse scleral fibroblasts after myopia using single-cell RNA sequencing technology and explore the mechanism of dysfunction of the scleral fibroblasts in myopia. METHODS: Normal healthy C57BL/6J mice were randomly divided into negative control group and myopia model group (n=6), and in the latter group, form deprivation myopia was induced in the right eye using translucent goggles. Single cell capture was performed in the right eye to obtain the scleral fibroblasts for RNA sequencing. The differentially expressed genes (DEGs) were screened and GO and KEGG analyses were carried out for functionally significant enrichment analysis. RESULTS: Comparison of the gene expression profiles identified a total of 169 DEGs between the myopia model group and the negative control group (P>0.05), including 112 up-regulated and 57 down-regulated genes. GO function analysis showed that the DEGs were involved in leukocyte aggregation, differentiation and adhesion and other inflammation-related terms; ATP metabolism and binding, redox process, oxidative stress response, oxidative phosphorylation and other GO terms related to hypoxia; protein folding, protein transport, negative regulation of protein metabolism, endoplasmic reticulum (ER), ER cavity, ER stress and other biological processes related to protein and ER stress. KEGG analysis analysis showed that the significantly enriched pathways of the DEGs involved mainly the TCA cycle, oxidative phosphorylation, PPAR signaling, oxidative phosphorylation and other pathways related to hypoxia; MAPK signaling pathway related to inflammation; leukocyte transendothelial transport; and protein-related Parkinson's disease, Huntington's disease, protein digestion and absorption pathways. CONCLUSION: The dysfunction of the scleral fibroblasts occurs in myopia through complex mechanisms involving inflammation, hypoxia, protein regulation, and ER stress-related gene expression and pathway regulation.

The effect of Wnt/ß-catenin pathway on the scleral remolding in the mouse during form deprivation.

BACKGROUND: Many reports have shown that Wnt/ß-Catenin pathway is associated with a variety of diseases, but its role in the pathogenesis of myopia is still unknown. In order to clarify the role of Wnt/ß-catenin pathway in the development of form deprivation myopia (FDM), this study investigated the expression of scleral Wls, ß-catenin and TCF4 in mice model of form deprivation (FD) myopia. METHODS: Three parallel experimental groups, including FD, monocular exposure (SC) and binocular exposure (NC) group, were designed to investigate the effects of Wnt/ß-Catenin pathway on scleral remodeling mouse during form deprivation in three-week-old C57BL/6 mice. Diopters and axial lengths (AL) in each sample were measured with an infrared eccentric refractometer or spectral-domain optical coherence tomography. The expression of scleral Wls, ß-catenin and TCF4 were detected with Western blot. Morphological changes of posterior sclera were observed with a transmission electron microscope (TEM). The above characterization and analysis were performed on the 0th, 7th, 14th, 21st and 28th days, respectively. RESULTS: The difference of diopter and AL between the three groups (SC, NC and FD group) gradually increased with the prolongation of FD time (except AL between SC and NC groups). The changes of diopter and AL gradually increased with the prolongation of FD time. Especially, the diopter and AL will increase sharply after FD lasts for a long time, such as the measurement on the 21st for diopter and 28th days for AL. Most notably, the AL of FD eyes significantly increased after 28 days of deprivation. Thinning and disordered arrangement of collagen fibers and a decrease of extracellular matrix were observed with TEM. The expression of scleral Wls, ß-catenin and TCF4 increased with age in the NC and SC group. In FD group, they increased significantly on the 7th, 14th and 21st days but decreased on the 28th day. CONCLUSIONS: The expression of Wls, ß-Catenin and TCF4 to FD were more sensitive indicators than that of diopter and AL. Within the first 7 days of FD, the expression of Wls, ß-Catenin and TCF4 in sclera increased sharply. With the extension of FD duration, it gradually decreased. It is suggested that the Wnt/ß-catenin pathway might be involved in the scleral remodeling induced in FDM mice.

Effect of modified Zhujing pill on retinal thickness and apoptosis in form deprivation myopia mice / 国际眼科杂志(Guoji Yanke Zazhi)

@#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.

Effects of latanoprost on the expression of TGF-ß1 and Wnt / ß-catenin signaling pathway in the choroid of form-deprivation myopia rats.

In this study, we investigated the effect of latanoprost on the expression of TGF- ß1 and Wnt / ß - Catenin signal pathway in the choroid of form-deprivation myopia model rats. Forty rats were randomly divided into two groups: the control group and the FDM model group. Each group had 20 rats. The FDM model group was established by feeding latanoprost daily for 28 days. 15 rats in each group were used to measure the length of the ocular axis and the level of TGF-ß1 in choroidal tissue; the remaining 5 rats in each group were used for choroidal fibroblast culture. After modeling, the rats were killed, the length of the ocular axis was measured with a vernier caliper, and the level of TGF - ß1 protein and mRNA in the choroidal tissue of each group were measured with RT-PCR method. Results showed that compared with the control group, there was a significant difference in the axial length of the FDM model group (P< 0.05). There was a significant difference in the expression of TGF- ß1 protein and mRNA between the two groups (P<0.05). The cultured cells were identified as choroidal fibroblasts by immunocytochemistry. There was no significant difference (P>0.05) in the comparison of GSK3 ß protein in choroidal fibroblasts of rats in each group. TGF-ß 1 and APC protein in FDM group were significantly lower than those in the control group (P<0.05), while dcl3, p21-gsk3 ß and ß - Catenin proteins were significantly higher (P<0.05), and there was no significant difference (P>0.05) in the ratio of various indexes protein in FDM + ddk1 group and the comparison of TGF - ß1 and APC protein in FDM + ddk1 group and FDM group The expression of dcl3, p21-gsk3 ß and ß - Catenin decreased significantly (P<0.05). There was no significant difference in the expression of GSK3 ß mRNA in the choroidal fibroblasts of each group (P>0.05). The expression of TGF - ß 1 and APC mRNA in FDM group was significantly lower than that in the control group (P<0.05), while the expression of dcl3, p21-gsk3 ß and ß-catenin mRNA in FDM + ddk1 group was significantly higher than that in the control group (P<0.05) >In FDM + ddk1 group, TGF-ß 1 and APC mRNA were significantly lower than those in FDM group (P<0.05), while dcl3, p21-gsk3 ß and ß-Catenin mRNA were significantly higher (P<0.05).

Relationship between Aquaporin-1 Protein Expression and Choroidal Thickness during the Recovery of Form-deprivation Myopia in Guinea Pigs.

Purpose: The purpose of this study was to investigate the relationship between aquaporin-1 (AQP-1) protein expression in the choroid and choroid thickness (CT) during the recovery of form-deprivation (FD) myopia in guinea pigs.Materials and Methods: Seventy-two guinea pigs were randomly assigned to the normal control (NC) group, FD 21 group (animals wore a latex facemask in the right eye for 21 days to induce FD myopia) and four recovery (REC) groups. Guinea pigs in the REC groups also wore the facemask for 21 days to induce myopia; then, the facemask was removed, and the eye was re-exposed to the normal environment for 12 hours (REC ½ group), 1 day (REC 1 group), 2 days (REC 2 group), and 7 days (REC 7 group). All animals underwent biometric measurements (refraction, axial length, and CT), and the protein expression of AQP-1 in the choroid was determined using western blotting.Results: The protein expression of AQP-1 and CT were significantly decreased in the FD 21 group as compared with those in the NC group (p = .007 and p < .001). Both AQP-1 protein expression and CT gradually increased and peaked in the REC 2 group. Additionally, there were significant differences in AQP-1 protein expression and CT between the REC 2 group and all other groups (all p < .05). We observed a complete recovery in the in REC 7 group as compared with the NC group (p > .05). AQP-1 protein expression was significantly associated with CT (p = .001) in all groups; however, there was a significant negative correlation (p = .029) between AQP-1 protein expression and axial length in the REC groups.Conclusions: AQP-1 protein expression in the choroid was upregulated following recovery of FD myopia in guinea pigs, and these changes correlated with alterations in CT and axial length.

Dynamic changes of activator protein 1 and collagen I expression in the sclera of myopia guinea pigs.

AIM: To investigate the dynamic changes of activator protein 1 (AP1) and collagen I expression in the sclera of form-deprivation myopic model in guinea pigs. METHODS: A form-deprivation myopic model in guinea pigs were established with the left eye covered for 2 to 6wk (FDM group). Normal control group (n=25) were untreated. Changes in refractive power and axial length (AL) were measured and recorded at different time points. Expressions of AP1 and collagen 1 of the sclera were measured with Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The relationship between AP1 and collagen I levels was analyzed. RESULTS: After 0, 2, 4, 6wk, and 4/-1wk of form-deprivation, the diopter in the FDM group was gradually changed (2.08±0.31, -1.23±0.68, -4.17±0.58, -7.07±0.55, and -2.67±0.59 D, respectively, P<0.05), and the AL was gradually increased (5.90±0.38, 6.62±0.37, 7.30±0.35, 7.99±0.31, and 6.97±0.32 mm, respectively, P<0.05). With the prolongation of covered time, the protein expressions of AP1 and collagen I in the FDM group were gradually down-regulated (all P<0.05); the mRNA expressions of them were also gradually down-regulated (all P<0.05); and there was positive correlation between them. The control group had no obvious change in each index (all P>0.05). CONCLUSION: AP1 may be an important transcription factor involved in the regulation of collagen I synthesis and degradation during myopic scleral remodeling.