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Background: To assess predictive value of short-term choroidal changes for future myopic shift in children., Methods: 577 eyes of 289 primary school children were prospectively followed for 2 years. Cycloplegic refractions at baseline, 1 year and 2 years, and choroidal measurements by optical coherence tomography at baseline and 3 months, were used for analyses. Myopic shift was defined as refraction change of at least -0.50 dioptre/year, at 2 years compared with baseline., Results: 228 participants (455 eyes) completed 2-year follow-up. Approximately 37.6% of 311 initially non-myopic eyes and 73.6% of 144 initially myopic eyes developed a myopic shift. Notably, at 3 months greater reductions were found in initially myopic eyes with myopic shift, than in those without myopic shift-in choroidal thickness (ChT), luminal area (LA), stromal area (SA) and total choroidal area (TCA), but no significant differences in any choroidal parameters were observed between non-myopic eyes, with and without myopic shift. Multivariable analyses showed that in myopic eyes, each percentage increase in ChT, LA, SA and TCA was associated with reduced odds of myopic shift (all p<0.001). Similar associations were observed in non-myopic eyes, with smaller effects than in myopic eyes. Adding a 3-month percentage change of each choroidal parameter to a basic model including age, gender, parental myopia and baseline refraction significantly improved the predictive performance in myopic eyes (area under the receiver operating characteristic curves increasing from 0.650 to approximately 0.800, all p<0.05), but not in non-myopic eyes., Conclusion: Short-term choroidal changes could act as early indicators for future myopic shift in children., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Extreme myopia (EM), defined as a spherical equivalent (SE) ≤ -10.00 diopters (D), is one of the leading causes of sight impairment. Known EM-associated variants only explain limited risk and are inadequate for clinical decision-making. To discover risk genes, we performed a whole-exome sequencing (WES) on 449 EM individuals and 9606 controls. We find a significant excess of rare protein-truncating variants (PTVs) in EM cases, enriched in the retrograde vesicle-mediated transport pathway. Employing single-cell RNA-sequencing (scRNA-seq) and a single-cell polygenic burden score (scPBS), we pinpointed PI16 + /SFRP4+ fibroblasts as the most relevant cell type. We observed that KDELR3 is highly expressed in scleral fibroblast and involved in scleral extracellular matrix (ECM) organization. The zebrafish model revealed that kdelr3 downregulation leads to elongated ocular axial length and increased lens diameter. Together, our study provides insight into the genetics of EM in humans and highlights KDELR3's role in EM pathogenesis., (© 2024. The Author(s).)

Background: The objective of this study is to illustrate the changes in the choroidal vasculature in individuals with diffuse chorioretinal atrophy (DCA, early-stage myopic maculopathy) and investigate the association between them., Methods: This study included 1418 highly myopic eyes from 720 participants aged 18 - 60 years from the Wenzhou High Myopia Cohort Study. These participants underwent comprehensive ophthalmic assessments. Myopic maculopathy classification followed the Meta-PM system, with pathological myopia defined as myopic maculopathy of DCA or severer. Eyes with myopic maculopathy categorized as no macular lesions (C0), tessellated fundus (C1), and DCA (C2) were enrolled in the analysis. Choroidal images were obtained from swept-source optical coherence tomography (SS-OCT), and the images were processed with a deep learning-based automatic segmentation algorithm and the Niblack auto-local threshold algorithm., Results: DCA was detected in 247 eyes (17.4%). In comparison to eyes with C0, those with C2 exhibited significant reductions in choroidal thickness (ChT), luminal area (LA), and stromal area (SA) across all evaluated regions (all P < 0.001). An increase in choroidal vascular index (CVI) was observed in all regions, except for the nasal perifoveal (N2) and inferior perifoveal (I2) regions (all P < 0.01). Multivariable logistic regression analysis revealed a negative association between the presence of DCA and increases in choroidal LA and SA (odds ratio ≤ 0.099, P < 0.001). Multivariable linear regression analysis showed that the mean deviation of the visual field test was positively associated with LA and SA at the vertical meridian (B = 1.512, P < 0.001 for LA; B = 1.956, P < 0.001 for SA). Furthermore, the receiver operating characteristic curve analyses showed the optimal ChT to diagnose pathological myopia was 82.4 µm in the N2 region, the LA was 0.076 mm 2 and the SA was 0.049 mm 2 , with area under the curves of 0.916, 0.908, and 0.895, respectively., Conclusions: The results of this study indicated that both the presence of DCA and visual function impairment were associated with reductions in choroidal perfusion and stromal components. Moreover, we established threshold values for choroidal parameters in diagnosing pathological myopia, offering valuable references for clinical diagnosis and management., (© 2024. The Author(s).)

Pathological myopia (PM) is the leading ocular disease for impaired vision worldwide. Clinically, the characteristics of pathology distribution in PM are global-local on the fundus image, which plays a significant role in assisting clinicians in diagnosing PM. However, most existing deep neural networks focused on designing complex architectures but rarely explored the pathology distribution prior of PM. To tackle this issue, we propose an efficient pyramid channel attention (EPCA) module, which fully leverages the potential of the clinical pathology prior of PM with pyramid pooling and multi-scale context fusion. Then, we construct EPCA-Net for automatic PM recognition based on fundus images by stacking a sequence of EPCA modules. Moreover, motivated by the recent pretraining-and-finetuning paradigm, we attempt to adapt pre-trained natural image models for PM recognition by freezing them and treating the EPCA and other attention modules as adapters. In addition, we construct a PM recognition benchmark termed PM-fundus by collecting fundus images of PM from publicly available datasets. The comprehensive experiments demonstrate the superiority of EPCA-Net over state-of-the-art methods in the PM recognition task. For example, EPCA-Net achieves 97.56% accuracy and outperforms ViT by 2.85% accuracy on the PM-fundus dataset. The results also show that our method based on the pretraining-and-finetuning paradigm achieves competitive performance through comparisons to part of previous methods based on traditional fine-tuning paradigm with fewer tunable parameters, which has the potential to leverage more natural image foundation models to address the PM recognition task in limited medical data regime., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lactylation, a post-translational modification (PTM) induced by lactate, has crucial roles in the regulation of transcription, DNA repair, and mitochondrial metabolism. Here, we discuss the role of lactate/lactylation signaling in regulating eye morphogenesis and retinal homeostasis, as well as various ophthalmic disorders, such as myopia, intraocular malignancies, and retinal angiogenesis., Competing Interests: Declaration of interests The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: To investigate genetic loci associated with ocular axial length (AL) in the Chinese population., Methods: A genome-wide association study meta-analysis was conducted in totalling 2644 Chinese individuals from 3 cohorts: the Guangzhou cohort (GZ, 537 high myopes and 151 hyperopes), Wenzhou cohort (334 high myopes and 6 hyperopes) and Guangzhou Twin Eye Study (1051 participants with normally distributed AL). Functional mapping was performed to annotate the significant signals, possible tissues and cell types by integrating available multiomics data. Logistic regression models using AL-associated SNPs were constructed to predict three AL status in GZ., Results: Two novel loci (1q25.2 FAM163A and 7p22.2 SDK1 ) showed genome-wide significant associations with AL, together explaining 29.63% of AL variance in GZ. The two lead SNPs improved the prediction accuracy for AL status, especially for hyperopes. The frequencies of AL decreasing (less myopic) alleles of the two SNPs were lowest in East Asians as compared with other populations (rs17370084: f EAS =0.03, f EUR =0.24, f AFR =0.05; rs73046501: f EAS =0.06, f EUR =0.07, f AFR =0.20), which was in line with the global distribution of myopia. The cerebral cortex and gamma-aminobutyric acidergic interneurons showed possible functional involvement in myopia development, and the galactose metabolic pathways were significantly enriched., Conclusion: Our study identified two population-specific novel loci for AL, expanding our understanding of the genetic basis of AL and providing evidence for a role of the nervous system and glucose metabolism in myopia pathogenesis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Near work is generally considered as a risk factor for myopia onset and progression. This study aimed to investigate the choroidal responses to a brief-period of near work in children and young adults., Methods: Thirty myopic medical students (aged 18-28 years) and 30 myopic children (aged 8-12 years) participated in this study. The submacular total choroidal area (TCA), luminal area (LA), stromal area (SA), choroidal vascularity index (CVI) and choriocapillaris flow deficit (CcFD), as well as subfoveal choroidal thickness (SFCT) were measured with swept-source optical coherence tomography/optical coherence tomography angiography (SS-OCT/OCTA) before and immediately after 20 min, 40 min, 60 min of near work at a distance of 33 cm., Results: In adults, 20 min of near work induced a significant reduction in SFCT (- 5.1 ± 6.5 μm), LA [(- 19.2 ± 18.6) × 10 3 μm 2 ], SA [(- 8.2 ± 12.6) × 10 3 μm 2 ] and TCA [(- 27.4 ± 24.9) × 10 3 μm 2 ] (all P < 0.01). After 40 min of near work, LA was still reduced [(- 9.4 ± 18.3) × 10 3 μm 2 ], accompanied with a decreased CVI (- 0.39% ± 0.70%) and an increased CcFD (0.30% ± 0.78%) (all P < 0.05). After 60 min of near work, CVI was still reduced (- 0.28% ± 0.59%), and CcFD was still increased (0.37% ± 0.75%) (all P < 0.05). In children, 20 min of near work induced a significant increase in CcFD (0.55% ± 0.64%), while 60 min of near work induced increases in SA [(7.2 ± 13.0) × 10 3 μm 2 ] and TCA [(9.7 ± 25.3) × 10 3 μm 2 ] and a reduction in CVI (- 0.28% ± 0.72%) (all P < 0.05). Children exhibited lower near work-induced LA and TCA reduction than adults, with a mean difference of - 0.86% and - 0.82%, respectively (all P < 0.05)., Conclusions: The temporal characteristics and magnitude of changes of choroidal vascularity and choriocapillaris perfusion during near work was not identical between children and adults. The initial response to near work was observed in choriocapillaris in children, whereas it was observed in the medium- and large-sized vessels in adults., Trial Registration: Clinical Trial Registry (ChiCTR), ChiCTR2000040205. Registered on 25 November 2020, https://www.chictr.org.cn/bin/project/edit?pid=64501 ., (© 2024. The Author(s).)

Myopia is characterized of maladaptive increases in scleral fibroblast-to-myofibroblast transdifferentiation (FMT). Scleral hypoxia is a significant factor contributing to myopia, but how hypoxia induces myopia is poorly understood. Here, we showed that myopia in mice and guinea pigs was associated with hypoxia-induced increases in key glycolytic enzymes expression and lactate levels in the sclera. Promotion of scleral glycolysis or lactate production induced FMT and myopia; conversely, suppression of glycolysis or lactate production eliminated or inhibited FMT and myopia. Mechanistically, increasing scleral glycolysis-lactate levels promoted FMT and myopia via H3K18la, and this promoted Notch1 expression. Genetic analyses identified a significant enrichment of two genes encoding glycolytic enzymes, ENO2 and TPI1. Moreover, increasing sugar intake in guinea pigs not only induced myopia but also enhanced the response to myopia induction via the scleral glycolysis-lactate-histone lactylation pathway. Collectively, we suggest that scleral glycolysis contributes to myopia by promoting FMT via lactate-induced histone lactylation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dopamine is a key neurotransmitter in the signaling cascade controlling ocular refractive development, but the exact role and site of action of dopamine D1 receptors (D1Rs) involved in myopia remains unclear. Here, we determine whether retinal D1Rs exclusively mediate the effects of endogenous dopamine and systemically delivered D1R agonist or antagonist in the mouse form deprivation myopia (FDM) model. Male C57BL/6 mice subjected to unilateral FDM or unobstructed vision were divided into the following four groups: one noninjected and three groups that received intraperitoneal injections of a vehicle, D1R agonist SKF38393 (18 and 59 nmol/g), or D1R antagonist SCH39166 (0.1 and 1 nmol/g). The effects of these drugs on FDM were further assessed in Drd1 -knock-out ( Drd1 -KO), retina-specific conditional Drd1 -KO ( Drd1 -CKO) mice, and corresponding wild-type littermates. In the visually unobstructed group, neither SKF38393 nor SCH39166 affected normal refractive development, whereas myopia development was attenuated by SKF38393 and enhanced by SCH39166 injections. In Drd1 -KO or Drd1 -CKO mice, however, these drugs had no effect on FDM development, suggesting that activation of retinal D1Rs is pertinent to myopia suppression by the D1R agonist. Interestingly, the development of myopia was unchanged by either Drd1 -KO or Drd1 -CKO, and neither SKF38393 nor SCH39166 injections, nor Drd1 -KO, affected the retinal or vitreal dopamine and the dopamine metabolite DOPAC levels. Effects on axial length were less marked than effects on refraction. Therefore, activation of D1Rs, specifically retinal D1Rs, inhibits myopia development in mice. These results also suggest that multiple dopamine D1R mechanisms play roles in emmetropization and myopia development. SIGNIFICANCE STATEMENT While dopamine is recognized as a "stop" signal that inhibits myopia development (myopization), the location of the dopamine D1 receptors (D1Rs) that mediate this action remains to be addressed. Answers to this key question are critical for understanding how dopaminergic systems regulate ocular growth and refraction. We report here the results of our study showing that D1Rs are essential for controlling ocular growth and myopia development in mice, and for identifying the retina as the site of action for dopaminergic control via D1Rs. These findings highlight the importance of intrinsic retinal dopaminergic mechanisms for the regulation of ocular growth and suggest specific avenues for exploring the retinal mechanisms involved in the dopaminergic control of emmetropization and myopization., (Copyright © 2023 the authors.)

Purpose: To evaluate whether pigmented guinea pigs with spontaneous myopia present characteristic changes of pathologic myopia., Methods: The fundus images of guinea pigs (3 weeks old) were graded according to fundus tessellation (FT) degree. Biometric parameters, including refraction, vitreous chamber depth (VCD), and axial length (AL), were measured at ages 21 and 43 days. Some of these animals were divided into three groups: hyperopic without FT (H w/o FT), myopic without FT (M w/o FT), and myopic with FT (M w/ FT). The horizontal and vertical radii of curvature of posterior sclera (RP-H and RP-V, respectively) and the radii of curvature and arc lengths of superior sclera (RS and LS, respectively), inferior sclera (RI and LI, respectively), nasal sclera (RN and LN, respectively), and temporal sclera (RT and LT) were evaluated by Fuji., Results: The fundi were graded as type A or type B (both without FT), type C (mild FT), or type D (severe FT). The prevalence of FT was correlated with myopic refraction, longer VCD, and longer AL. Eyes of M w/FT animals had shorter RP-H and RP-V, longer RS and RT, and longer LS and LT than eyes of H w/o FT or M w/o FT animals. Refractions shifted toward hyperopia in eyes lacking FT, but not in eyes having FT. The changes in VCD were consistent with the changes in refraction. This relatively myopic shift in refraction and shortening of VCD were found only in myopic eyes with FT, but not in myopic eyes without FT., Conclusions: Spontaneously myopic guinea pig eyes have a high prevalence of FT. Myopic eyes with FT presented characteristic signs of pathologic myopia.

In this study, we explored the predictive role of choroidal blood perfusion (ChBP) and choroidal thickness (ChT) on the development of myopia in guinea pigs. Optical Coherence Tomography Angiography (OCTA) was used to assess the baseline choroidal blood perfusion (ChBP) and choroidal thickness (ChT) in 4-week-old guinea pigs. Refraction and axial length (AL) were measured at baseline. Myopia was induced for one week using form-deprivation (FD) or negative lenses followed by measurements of refraction, axial length and choroidal parameters (ChT and ChBP). The correlations were evaluated between the baseline choroidal values and the magnitude of myopia induced, along with the magnitude of changes in ChT and ChBP after myopia induction. There was a significant correlation between the baseline choroidal parameters and ocular refraction. Myopia induction led to choroidal thinning and less ChBP as well as longer eyes. On the other hand, following exposure to the same non-obstructed visual induction period, the myopic shift was less, and it was associated with thicker choroids and more ChBP at baseline. One week of myopia induction also resulted in thinner choroids and less ChBP, and these declines also correlated with their baseline values. In conclusion, the present study shows that the changes in the baseline choroidal ChT and ChBP parameters are proportional to the magnitude of myopia development and axial elongation in guinea pigs. These significant correlations between baseline ChBP and ChT and myopia development suggest that they may be a viable predictor of this process in guinea pigs., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Despite receiving orthokeratology (ortho-k), the efficacy of retarding ocular elongation during myopia varies among myopic children. The current study aimed to investigate the early changes of choroidal vasculature at one month after ortho-k treatment and its association with one-year ocular elongation, as well as the role of such choroidal responses in predicting the one-year control efficacy of ortho-k treatment., Methods: A prospective cohort study was conducted in myopic children treated with ortho-k. Myopic children aged between 8 and 12 years who were willing to wear ortho-k lenses were recruited consecutively from the Eye Hospital of Wenzhou Medical University. Subfoveal choroidal thickness (SFCT), submacular total choroidal luminal area (LA), stromal area (SA), choroidal vascularity index (CVI), choriocapillaris flow deficit (CcFD) were evaluated by optical coherence tomography (OCT) and OCT angiography over a one-year period., Results: Fifty eyes from 50 participants (24 males) who finished one-year follow-ups as scheduled were included, with a mean age of 10.31 ± 1.45 years. The one-year ocular elongation was 0.19 ± 0.17 mm. The LA (0.03 ± 0.07 mm 2 ), SA (0.02 ± 0.05 mm 2 ) increased proportionally after one-month of ortho-k wear (both P < 0.01), as did the SFCT (10.62 ± 19.98 μm, P < 0.001). Multivariable linear regression analyses showed that baseline CVI (β = - 0.023 mm/1%, 95% CI: - 0.036 to - 0.010), one-month LA change (β = - 0.009 mm/0.01 mm 2 , 95% CI: - 0.014 to - 0.003), one-month SFCT change (β = - 0.035 mm/10 µm, 95% CI: - 0.053 to - 0.017) were independently associated with one-year ocular elongation during ortho-k treatment after adjusting with age and sex (all P < 0.01). The area under the receiver operating characteristic curve of prediction model including baseline CVI, one-month SFCT change, age, and sex achieved 0.872 (95% CI: 0.771 to 0.973) for discriminating children with slow or fast ocular elongation., Conclusions: Choroidal vasculature is associated with ocular elongation during ortho-k treatment. Ortho-k treatment induces increases in choroidal vascularity and choroidal thickness as early as one month. Such early changes can act as predictive biomarkers of myopia control efficacy over a long term. The utilization of these biomarkers may help clinicians identify children who can benefit from ortho-k treatment, and thus has critical implications for the management strategies towards myopia control., (© 2023. The Author(s).)

Purpose: The purpose of this study was to evaluate and explore the determinants of choroidal vascularity and choriocapillaris perfusion in a Chinese population aged 8 to 30 years old., Methods: Three hundred eighty eyes from 380 subjects aged 8 to 30 years were included in this cross-sectional study. Submacular choroidal thickness (ChT), total choroidal area (TCA), luminal area (LA), stromal area (SA), choroidal vascularity index (CVI), and choriocapillaris flow deficit (CcFD) were estimated using images obtained from optical coherence tomography (OCT)., Results: In this population, the mean ChT was 260.4 ± 63.3 µm, TCA was 1.56 ± 0.38 mm2, LA was 0.94 ± 0.25 mm2, and SA was 0.62 ± 0.15 mm2. The mean CVI was 60.25 ± 3.21% and CcFD was 11.95 ± 1.98%. Multivariable analyses showed that higher CVI and LA was associated with older age, thicker ChT, and shorter AL; and lower CcFD was associated with shorter AL. However, the associations were not uniformly rectilinear between CcFD and age. Specifically, CcFD was positively associated with age in subjects ≤19 years old and negatively associated with age in subjects >19 years old., Conclusions: Development of the choroidal medium- and large-sized vascular layers and choriocapillaris was different across patients aged 8 to 30 years old. Greater axial length was associated with attenuated choroidal circulation. Choroidal thickness correlated well with choroidal vascularity, but not with choriocapillaris perfusion. Further comprehensive and longitudinal assessment of choroidal vasculature and choriocapillaris perfusion will help greatly to understand the physiological and pathological mechanisms responsible for myopia.

The choroid is the richly vascular layer of the eye located between the sclera and Bruch's membrane. Early studies in animals, as well as more recent studies in humans, have demonstrated that the choroid is a dynamic, multifunctional structure, with its thickness directly and indirectly subject to modulation by a variety of physiologic and visual stimuli. In this review, the anatomy and function of the choroid are summarized and links between the choroid, eye growth regulation, and myopia, as demonstrated in animal models, discussed. Methods for quantifying choroidal thickness in the human eye and associated challenges are described, the literature examining choroidal changes in response to various visual stimuli and refractive error-related differences are summarized, and the potential implications of the latter for myopia are considered. This review also allowed for the reexamination of the hypothesis that short-term changes in choroidal thickness induced by pharmacologic, optical, or environmental stimuli are predictive of future long-term changes in axial elongation, and the speculation that short-term choroidal thickening can be used as a biomarker of treatment efficacy for myopia control therapies, with the general conclusion that current evidence is not sufficient.

RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These "positional rules" can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: To determine the role of calcipotriol, a vitamin D3 analogue, in myopia development and altering the expression of scleral α1 chain of type I collagen (Col1α1) in mice. We also aimed to identify if the signaling pathway mediating the above changes is different from the one involved in transforming growth factor β2 (TGF-β2)-mediated increases of COL1A1 in cultured human scleral fibroblasts (HSFs)., Methods: C57BL/6J mice were either intraperitoneally injected with calcipotriol and subjected to form deprivation (FD) or exposed to normal refractive development for 4 weeks. Scleral vitamin D receptor (Vdr) expression was knocked down using a Sub-Tenon's capsule injection of an adeno-associated virus-packaged short hairpin RNA (AAV8-shRNA). Refraction and biometric measurements evaluated myopia development. A combination of knockdown and induction strategies determined the relative contributions of the vitamin D3 and the TGF-β2 signaling pathways in modulating COL1A1 expression in HSFs., Results: Calcipotriol injections suppressed FD-induced myopia (FDM), but it had no significant effect on normal refractive development. AAV8-shRNA injection reduced Vdr mRNA expression by 42% and shifted the refraction toward myopia (-3.15 ± 0.99D, means ± SEM) in normal eyes. In HSFs, VDR knockdown reduced calcipotriol-induced rises in COL1A1 expression, but it did not alter TGF-β2-induced increases in COL1A1 expression. Additionally, TGF-β2 augmented calcipotriol-induced rises in COL1A1 expression. TGF-β receptor (TGFBRI/II) knockdown blunted TGF-β2-induced increases in COL1A1 expression, whereas calcipotriol-induced increases in VDR and COL1A1 expression levels were unaltered., Conclusions: Scleral vitamin D3 inhibits myopia development in mice, potentially by activating a VDR-dependent signaling pathway and increasing scleral COL1A1 expression levels.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease that results from degeneration of retinal ganglion cells (RGC). Mitochondrial ND4 11778G > A mutation, which affects structural components of complex I, is the most prevalent LHON-associated mitochondrial DNA (mtDNA) mutation worldwide. The m.11778G > A mutation is the primary contributor underlying the development of LHON and X-linked PRICKLE3 allele (c.157C > T, p.Arg53Trp) linked to biogenesis of ATPase interacts with m.11778G > A mutation to cause LHON. However, the lack of appropriate cell and animal models of LHON has been significant obstacles for deep elucidation of disease pathophysiology, specifically the tissue-specific effects. Using RGC-like cells differentiated from induced pluripotent stem cells (iPSCs) from members of one Chinese family (asymptomatic subjects carrying only m.11778G > A mutation or PRICKLE3 p.Arg53Trp mutation, symptomatic individuals bearing both m.11778G > A and PRICKLE3 p.Arg53Trp mutations and control lacking these mutations), we demonstrated the deleterious effects of mitochondrial dysfunctions on the morphology and functions of RGCs. Notably, iPSCs bearing only m.11778G > A or p.Arg53Trp mutation exhibited mild defects in differentiation to RGC-like cells. The RGC-like cells carrying only m.11778G > A or p.Arg53Trp mutation displayed mild defects in RGC morphology, including the area of soma and numbers of neurites, electrophysiological properties, ATP contents and apoptosis. Strikingly, those RGC-like cells derived from symptomatic individuals harboring both m.11778G > A and p.Arg53Trp mutations displayed greater defects in the development, morphology and functions than those in cells bearing single mutation. These findings provide new insights into pathophysiology of LHON arising from RGC deficiencies caused by synergy between m.11778G > A and PRICKLE3 p.Arg53Trp mutation., (© The Author(s) 2022. Published by Oxford University Press.)

The association between near work activities and myopia has not been clearly established. This study establishes a model for near work myopia (NWM) induced by short viewing distance in guinea pigs with a carefully controlled visual environment, and evaluates the effect of viewing distance in myopia development. Pigmented guinea pigs (3 weeks old) were randomly assigned to 3 groups: near work (NW)-, form-deprivation (FD)-, and -4D hyperopic-defocus (HD)-induced myopia. Animals in NW groups were kept in cylindrical cages with vertical square-wave gratings, providing short- (S, d = 18 cm), middle- (M, d = 44 cm), and long- (L, d = 88 cm) mean viewing distances, all at the same illuminance, during daily treatment for 14 days. Biometric parameters, including refraction, anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL), were measured at the beginning and end of 14 days' treatment. Choroidal thickness (ChT) and choroidal blood perfusion (ChBP) were measured by optical coherence tomography (OCT) and OCT-angiography (OCTA), respectively, at the end of treatment. Refraction was shifted towards myopia in the S-cage group, compared with the M- and L-cage groups; refractions in the L-, M- and S-cage groups were 5.19 ± 0.65 D, 4.30 ± 0.64 D, and 0.53 ± 0.61 D, respectively (p < 0.001). VCD and AL in the S-cage group increased in parallel with the myopic shift (L vs M vs S: VCD: 3.15 ± 0.02 mm vs 3.17 ± 0.02 mm vs 3.26 ± 0.02 mm, p < 0.001; AL: 7.99 ± 0.03 mm vs 8.03 ± 0.03 mm vs 8.15 ± 0.02 mm, p = 0.001). In FD and HD eyes, changes similar to those in the S-cage group (near-work group, NW) were seen in refraction (NW vs FD vs HD: 5.36 ± 0.82 D vs -5.78 ± 0.44 D vs -4.96 ± 0.54 D, p = 0.734), ACD, LT, VCD and AL. Also, ChT and ChBP were significantly less in the S-cage group than in the M- and L-cage groups after 14 days' treatment (L vs M vs S: ChT: 74.84 ± 3.27 vs 76.07 ± 3.49 vs 61.95 ± 3.31, P = 0.002; ChBP: 48.32 ± 2.23 vs 48.66 ± 2.30 vs 38.14 ± 2.06, p = 0.002). Rearing in S-cages induced myopia in guinea pigs and correspondingly decreased ChBP and ChT. The present study provides objective evidence that short viewing distance could be a risk factor for myopia, and describes a useful model for studying the underlying mechanisms., Competing Interests: Declaration of competing interest The authors have no proprietary interest in any materials or methods described within this article., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Reduced levels of retinal dopamine, a key regulator of eye development, are associated with experimental myopia in various species, but are not seen in the myopic eyes of C57BL/6 mice, which are deficient in melatonin, a neurohormone having extensive interactions with dopamine. Here, we examined the relationship between form-deprivation myopia (FDM) and retinal dopamine levels in melatonin-proficient CBA/CaJ mice. We found that these mice exhibited a myopic refractive shift in form-deprived eyes, which was accompanied by altered retinal dopamine levels. When melatonin receptors were pharmacologically blocked, FDM could still be induced, but its magnitude was reduced, and retinal dopamine levels were no longer altered in FDM animals, indicating that melatonin-related changes in retinal dopamine levels contribute to FDM. Thus, FDM is mediated by both dopamine level-independent and melatonin-related dopamine level-dependent mechanisms in CBA/CaJ mice. The previously reported unaltered retinal dopamine levels in myopic C57BL/6 mice may be attributed to melatonin deficiency., (© 2022. Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.)

Competing Interests: The authors declare that they have no conflict of interest.

Purpose: Scleral hypoxia is a key factor that induces hypoxia-inducible factor-1α (HIF-1α) upregulation, and this response contributes to myopia progression. Currently, we aim to determine if the different HIF subtypes, including HIF-1α and HIF-2α, mediate hypoxia-induced myopia development through promoting scleral MMP-2 expression and collagen degradation., Methods: Our study included: (1) time-course of scleral HIF-2α, MMP-2, and COL1α1 expression during form-deprivation myopia (FDM) development was determined in C57BL/6J mice. (2) The effect of silencing either HIF-1Α or HIF-2A on hypoxia-induced alterations in MMP-2 expression was analyzed in cultured human scleral fibroblasts (HSFs) under a hypoxic condition (i.e. 1% oxygen). (3) To knock-down either HIF-1α or HIF-2α expression in the sclera, we performed Sub-Tenon's capsule injection of an adeno-associated virus (AAV)8-packaged Cre overexpression vector (AAV8-Cre) in HIF-1αfl/fl or HIF-2αfl/fl mice. HIF-1α, HIF-2α, MMP-2, and COL1α1 expression were analyzed by Western blot or quantitative real-time PCR (qRT-PCR). In addition, the effects of scleral HIF-2α knock-down on normal refractive development and FDM development were evaluated., Results: The time-dependent increases in scleral HIF-2α mimicked the HIF-1α expression profiles as we previously described. Hypoxia significantly promoted MMP-2 expression in HSFs, and this upregulation was solely alleviated by HIF-2A rather than HIF-1A silencing. Scleral HIF-2α knockdown significantly inhibited form-deprivation (FD)-induced MMP-2 upregulation and declines in COL1α1 accumulation and myopia development. Although scleral HIF-1α knockdown also significantly suppressed FD-induced declines in COL1α1 accumulation, it did not abrogate scleral MMP-2 upregulation., Conclusions: HIF-2α rather than HIF-1α induces myopia development through upregulating MMP-2 and promoting collagen degradation in the sclera.

The process of eye axis lengthening in myopic eyes is regulated by multiple mechanisms in the retina, and horizontal cells (HCs) are an essential interneuron in the visual regulatory system. Wherein intracellular Ca 2+ plays an important role in the events involved in the regulatory role of HCs in the retinal neural network. It is unknown if intracellular Ca 2+ regulation in HCs mediates changes in the retinal neural network during myopia progression. We describe here a novel calcium fluorescence indicator system that monitors HCs' intracellular Ca 2+ levels during form-deprivation myopia (FDM) in mice. AAV injection of GCaMP6s, as a protein calcium sensor, into a Gja10-Cre mouse monitored the changes in Ca 2+ signaling in HC that accompany FDM progression in mice. An alternative Gja10-Cre/Ai96-GCaMP6s mouse model was created by cross mating Gja10-Cre with Ai96 mice. Immunofluorescence imaging and live imaging of the retinal cells verified the identity of these animal models. Changes in retinal horizontal cellular Ca 2+ levels were resolved during FDM development. The numbers of GCaMP6s and the proportion of HCs were tracked based on profiling changes in GCaMP6s + calbindin + /calbindin + coimmunostaining patterns. They significantly decreased more after either two days (P < 0.01) or two weeks (P < 0.001) in form deprived eyes than in the untreated fellow eyes. These decreases in their proportion reached significance only in the retinal central region rather than also in the retinal periphery. A novel approach employing a GCaMP6s mouse model was developed that may ultimately clarify if HCs mediate Ca 2+ signals that contribute to controlling FDM progression in mice. The results indicate so far that FDM progression is associated with declines in HC Ca 2+ signaling activity., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: The transparency of the ocular lens is essential for refracting and focusing light onto the retina, and transparency is controlled by many factors and signaling pathways. Here we showed a critical role of chromatin remodeler zinc finger HIT-type containing 1 (Znhit1) in maintaining lens transparency., Methods: To explore the roles of Znhit1 in lens development, the cre-loxp system was used to generate lens-specific Znhit1 knockout mice (Znhit1Mlr10-Cre; Znhit1 cKO). Morphological changes in mice lenses were examined using hematoxylin and eosin staining. RNA sequencing (RNA-seq) and assay for transposase accessible chromatin using sequencing (ATAC-seq) were applied to screen transcriptome changes. Immunofluorescence staining were performed to assess proteins distribution and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining were used for determining apoptosis. The mRNAs expression was examined by quantitative RT-PCR and proteins expression by Western blot., Results: Lens-specific conditional knockout mice had a severe cataract, microphthalmia phenotype, and seriously abnormal lens fiber cells differentiation. Deletion of Znhit1 in the lens resulted in decreased cell proliferation and increased cell apoptosis of the lens epithelia. ATAC-seq showed that Znhit1 deficiency increased chromatin accessibility of cyclin-dependent kinase inhibitors, including p57Kip2 and p21Cip1, and upregulated the expression of these genes in mRNA and protein levels. And we also showed that loss of Znhit1 lead to lens fibrosis by upregulating the expression of p21Cip1., Conclusions: Our findings suggested that Znhit1 is required for the survival of lens epithelial cells. The loss of Znhit1 leads to the overexpression of p21Cip1, further resulting in lens fibrosis, and impacted the establishment of lens transparency.

Postzygotic mutations are acquired in normal tissues throughout an individual's lifetime and hold clues for identifying mutagenic factors. Here, we investigated postzygotic mutation spectra of healthy individuals using optimized ultra-deep exome sequencing of the time-series samples from the same volunteer as well as the samples from different individuals. In blood, sperm, and muscle cells, we resolved three common types of mutational signatures. Signatures A and B represent clock-like mutational processes, and the polymorphisms of epigenetic regulation genes influence the proportion of signature B in mutation profiles. Notably, signature C, characterized by C>T transitions at GpCpN sites, tends to be a feature of diverse normal tissues. Mutations of this type are likely to occur early during embryonic development, supported by their relatively high allelic frequencies, presence in multiple tissues, and decrease in occurrence with age. Almost none of the public datasets for tumors feature this signature, except for 19.6% of samples of clear cell renal cell carcinoma with increased activation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway. Moreover, the accumulation of signature C in the mutation profile was accelerated in a human embryonic stem cell line with drug-induced activation of HIF-1α. Thus, embryonic hypoxia may explain this novel signature across multiple normal tissues. Our study suggests that hypoxic condition in an early stage of embryonic development is a crucial factor inducing C>T transitions at GpCpN sites; and individuals' genetic background may also influence their postzygotic mutation profiles., (Copyright © 2022 Beijing Institute of Genomics. Published by Elsevier B.V. All rights reserved.)

Myopia is the most common cause of refractive error worldwide. High myopia is a severe type of myopia, which usually accompanies pathological changes in the fundus. To identify high myopia susceptibility genes, DNA-pooling based genome-wide association analysis was used to search for a correlation between single nucleotide polymorphisms and high myopia in a Han Chinese cohort (cases vs. controls in discovery stage: 507 vs. 294; replication stage 1: 991 vs. 1,025; replication stage 2: 1,021 vs. 52,708). Three variants (rs10889602T/G, rs2193015T/C, rs9676191A/C) were identified as being significantly associated with high myopia in the discovery, and replication stage. rs10889602T/G is located at the third intron of phosphodiesterase 4B ( PDE4B ), whose functional assays were performed by comparing the effects of rs10889602T/T deletion of this risk allele on PDE4B and COL1A1 gene and protein expression levels in the rs10889602T/T del/del , rs10889602T/T del/wt , and normal control A549 cell lines. The declines in the PDE4B and COL1A1 gene expression levels were larger in the rs10889602T/T deleted A549 cells than in the normal control A549 cells (one-way ANOVA, p < 0.001). The knockdown of PDE4B by siRNA in human scleral fibroblasts led to downregulation of COL1A1. This correspondence between the declines in rs10889602 of the PDE4B gene, PDE4B knockdown, and COL1A1 protein expression levels suggest that PDE4B may be a novel high myopia susceptibility gene, which regulates myopia progression through controlling scleral collagen I expression levels. More studies are needed to determine if there is a correlation between PDE4B and high myopia in other larger sample sized cohorts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhao, Chen, Zhou, Reinach, Wang, Juo, Yang, Xue, Shi, Liang, Zeng, Qu and Zhou.)

Purpose: To learn more about the locations of dopamine D2 receptors (D2Rs) that regulate form-deprivation myopia (FDM), using different transgenic mouse models., Methods: One eye of D2R-knockout (KO) mice and wild-type littermates was subjected to four weeks of monocular FDM, whereas the fellow eye served as control. Mice in both groups received daily intraperitoneal injections of either the D2R antagonist sulpiride (8 µg/g) or vehicle alone. FDM was also induced in retina- (Six3creD2Rfl/fl) or fibroblast-specific (S100a4creD2Rfl/fl) D2R-KO mice. A subset of retina-specific D2R-KO mice and D2Rfl/fl littermates were also given sulpiride or vehicle injections. Refraction was measured with an eccentric infrared photorefractor, and other biometric parameters were measured by optical coherence tomography (n ≈ 20 for each group)., Results: FDM development was attenuated in wild-type littermates treated with sulpiride. However, this inhibitory effect disappeared in the D2R-KO mice, suggesting that antagonizing D2Rs suppressed myopia development. Similarly, the development of myopia was partially inhibited by retina-specific (deletion efficiency: 94.7%) but not fibroblast-specific (66.9%) D2R-KO. The sulpiride-mediated inhibitory effects on FDM also disappeared with retinal D2R-KO, suggesting that antagonizing D2Rs outside the retina may not attenuate myopia. Changes in axial length were less marked than changes in refraction, but in general the two were correlated., Conclusions: This study demonstrates that D2Rs located in the retina participate in dopaminergic regulation of FDM in mice. These findings provide an important and fundamental basis for further exploring the retinal mechanism(s) involved in dopamine signaling and myopia development.

Background: Myopia is the leading cause of refractive errors. As its pathogenesis is poorly understood, we determined if the retinal VIP-VIPR2 signalling pathway axis has a role in controlling signalling output that affects myopia development in mice., Methods: Association analysis meta-study, single-cell transcriptome, bulk RNA sequencing, pharmacological manipulation and VIPR2 gene knockout studies were used to clarify if changes in the VIP-VIPR2 signalling pathway affect refractive development in mice., Results: The SNP rs6979985 of the VIPR2 gene was associated with high myopia in a Chinese Han cohort (randomceffect model: p=0.013). After either 1 or 2 days' form deprivation (FD) retinal VIP mRNA expression was downregulated. Retinal single-cell transcriptome sequencing showed that VIPR2 was expressed mainly by bipolar cells. Furthermore, the cAMP signalling pathway axis was inhibited in some VIPR2 + clusters after 2 days of FD. The selective VIPR2 antagonist PG99-465 induced relative myopia, whereas the selective VIPR2 agonist Ro25-1553 inhibited this response. In Vipr2 knockout ( Vipr2 -KO) mice, refraction was significantly shifted towards myopia (p<0.05). The amplitudes of the bipolar cell derived b-waves in 7-week-old Vipr2 -KO mice were significantly larger than those in their WT littermates (p<0.05)., Conclusions: Loss of VIPR2 function likely compromises bipolar cell function based on presumed changes in signal transduction due to altered signature electrical wave activity output in these mice. As these effects correspond with increases in form deprivation myopia (FDM), the VIP-VIPR2 signalling pathway axis is a viable novel target to control the development of this condition., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

The global "myopia boom" has raised significant international concerns. Despite a higher myopia prevalence in Asia, previous large-scale genome-wide association studies (GWASs) were mostly based on European descendants. Here, we report a GWAS of spherical equivalent (SE) in 1852 Chinese Han individuals with extreme SE from Guangzhou (631 < -6.00D and 574 > 0.00D) and Wenzhou (593 < -6.00D and 54 > -1.75D), followed by a replication study in two independent cohorts with totaling 3538 East Asian individuals. The discovery GWAS and meta-analysis identify three novel loci, which show genome-wide significant associations with SE, including 1q25.2 FAM163A, 10p11.22 NRP1/PRAD3, and 10p11.21 ANKRD30A/MTRNR2L7, together explaining 3.34% of SE variance. 10p11.21 is successfully replicated. The allele frequencies of all three loci show significant differences between major continental groups (P < 0.001). The SE reducing (more myopic) allele of rs10913877 (1q25.2 FAM163A) demonstrates the highest frequency in East Asians and much lower frequencies in Europeans and Africans (EAS = 0.60, EUR = 0.20, and AFR = 0.18). The gene-based analysis additionally identifies three novel genes associated with SE, including EI24, LHX5, and ARPP19. These results provide new insights into myopia pathogenesis and indicate the role of genetic heterogeneity in myopia epidemiology among different ethnicities., Competing Interests: Conflict of interest The authors declare that they have no competing interests., (Copyright © 2021. Published by Elsevier Ltd.)

Purpose: The development of myopia in guinea pigs can be inhibited by attenuating scleral hypoxia by increasing choroidal blood perfusion (ChBP). In this study, we reduced ChBP through surgical and pharmacological methods to determine the effect on myopia development. We also determined whether ChBP was reduced by quinpirole, a drug that enhances form-deprivation myopia (FDM)., Methods: ChBP was reduced in the right eyes of guinea pigs via transection of the temporal ciliary arteries or daily injections of phenylephrine into the inferior peribulbar space for one week during normal ocular growth. Other guinea pigs were subjected to two weeks of monocular FDM-with facemasks, along with daily injections of quinpirole, a dopamine D2 receptor agonist, to enhance the FDM. Changes in refraction, axial length, ChBP, and choroidal thickness (ChT) were measured in both treated and fellow eyes of the treatment and control groups. Scleral hypoxia labeling with pimonidazole adducts and α-smooth muscle actin (α-SMA) protein were also measured., Results: Surgical and pharmacological reduction of ChBP induced myopia development in the treated eyes. These treatments rendered the scleral hypoxia and increased scleral α-SMA expression. Furthermore, quinpirole injections, which increased the magnitude of myopia, augmented the FDM-associated reductions in ChBP and ChT and increased the levels of scleral hypoxia and α-SMA protein., Conclusions: Decreased ChBP in guinea pigs leads to scleral hypoxia and scleral myofibroblast transdifferentiation with increased α-SMA expression, ultimately resulting in myopia development. In future clinical trials, ChBP reduction can serve as a potential biomarker for early detection of myopia development.

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-β2 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Myopia is a leading cause of visual impairment and blindness worldwide. However, a safe and accessible approach for myopia control and prevention is currently unavailable. Here, we investigated the therapeutic effect of dietary supplements of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on myopia progression in animal models and on decreases in choroidal blood perfusion (ChBP) caused by near work, a risk factor for myopia in young adults. We demonstrated that daily gavage of ω-3 PUFAs (300 mg docosahexaenoic acid [DHA] plus 60 mg eicosapentaenoic acid [EPA]) significantly attenuated the development of form deprivation myopia in guinea pigs and mice, as well as of lens-induced myopia in guinea pigs. Peribulbar injections of DHA also inhibited myopia progression in form-deprived guinea pigs. The suppression of myopia in guinea pigs was accompanied by inhibition of the "ChBP reduction-scleral hypoxia cascade." Additionally, treatment with DHA or EPA antagonized hypoxia-induced myofibroblast transdifferentiation in cultured human scleral fibroblasts. In human subjects, oral administration of ω-3 PUFAs partially alleviated the near-work-induced decreases in ChBP. Therefore, evidence from these animal and human studies suggests ω-3 PUFAs are potential and readily available candidates for myopia control., Competing Interests: The authors declare no competing interest.

Purpose: To investigate the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-linked MT-ND1 3460G>A mutation., Methods: Cybrid cell models were generated by fusing mitochondrial DNA-less ρ0 cells with enucleated cells from a patient carrying the m.3460G>A mutation and a control subject. The impact of m.3460G>A mutations on oxidative phosphorylation was evaluated using Blue Native gel electrophoresis, and measurements of oxygen consumption were made with an extracellular flux analyzer. Assessment of reactive oxygen species (ROS) production in cell lines was performed by flow cytometry with MitoSOX Red reagent. Assays for apoptosis and mitophagy were undertaken via immunofluorescence analysis., Results: Nineteen Chinese Han pedigrees bearing the m.3460G>A mutation exhibited variable penetrance and expression of LHON. The m.3460G>A mutation altered the structure and function of MT-ND1, as evidenced by reduced MT-ND1 levels in mutant cybrids bearing the mutation. The instability of mutated MT-ND1 manifested as defects in the assembly and activity of complex I, respiratory deficiency, diminished mitochondrial adenosine triphosphate production, and decreased membrane potential, in addition to increased production of mitochondrial ROS in the mutant cybrids carrying the m.3460G>A mutation. The m.3460G>A mutation mediated apoptosis, as evidenced by the elevated release of cytochrome c into the cytosol and increasing levels of the apoptotic-associated proteins BAK, BAX, and PARP, as well as cleaved caspases 3, 7, and 9, in the mutant cybrids. The cybrids bearing the m.3460G>A mutation exhibited reduced levels of autophagy protein light chain 3, accumulation of autophagic substrate P62, and impaired PTEN-induced kinase 1/parkin-dependent mitophagy., Conclusions: Our findings highlight the critical role of m.3460G>A mutation in the pathogenesis of LHON, manifested by mitochondrial dysfunction and alterations in apoptosis and mitophagy.

Purpose: The purpose of this study was to evaluate the role of the canonical Wnt signaling in the development of the myopia., Methods: Plasma from adult patients with myopia, myopic animal models including the adenomatous polyposis coli (APC) gene mutation mouse model, and the form deprivation (FD) induced mouse model of myopia were used. Niclosamide, a canonical Wnt pathway inhibitor, was orally administrated in animal models. Plasma levels of DKK-1 were determined by using enzyme-linked immunosorbent assay. Refraction, vitreous chamber depth (VCD), axial length (AL), and other parameters, were measured at the end of the FD treatment. Canonical Wnt signaling changes were evaluated by Western blot analysis and immunostaining analysis., Results: Plasma level of Wnt inhibitor DKK-1 was markedly decreased in patients with myopia. Meanwhile, the canonical Wnt pathway was progressively activated during myopia development in mice. Moreover, inhibition of canonical Wnt signaling by niclosamide in mouse models markedly reduced lens thickness (LT), VCD, and AL elongation, resulting in myopia inhibition., Conclusions: Dysregulation of canonical Wnt signaling is a characteristic of myopia and targeting Wnt signaling pathways has potential as a therapeutic strategy for myopia.

Purpose: The purpose of this study was to determine whether retinal gap junctions (GJs) via connexin 36 (Cx36, mediating coupling of many retinal cell types) and horizontal cell (HC-HC) coupling, are involved in emmetropization., Methods: Guinea pigs (3 weeks old) were monocularly form deprived (FD) or raised without FD (in normal visual [NV] environment) for 2 days or 4 weeks; alternatively, they wore a -4 D lens (hyperopic defocus [HD]) or 0 D lens for 2 days or 1 week. FD and NV eyes received daily subconjunctival injections of a nonspecific GJ-uncoupling agent, 18-β-Glycyrrhetinic Acid (18-β-GA). The amounts of total Cx36 and of phosphorylated Cx36 (P-Cx36; activated state that increases cell-cell coupling), in the inner and outer plexiform layers (IPLs and OPLs), were evaluated by quantitative immunofluorescence (IF), and HC-HC coupling was evaluated by cut-loading with neurobiotin., Results: FD per se (excluding effect of light-attenuation) increased HC-HC coupling in OPL, whereas HD did not affect it. HD for 2 days or 1 week had no significant effect on retinal content of Cx36 or P-Cx36. FD for 4 weeks decreased the total amounts of Cx36 and P-Cx36, and the P-Cx36/Cx36 ratio, in the IPL. Subconjunctival 18-β-GA induced myopia in NV eyes and increased the myopic shifts in FD eyes, while reducing the amounts of Cx36 and P-Cx36 in both the IPL and OPL., Conclusions: These results suggest that cell-cell coupling via GJs containing Cx36 (particularly those in the IPL) plays a role in emmetropization and form deprivation myopia (FDM) in mammals. Although both FD and 18-β-GA induced myopia, they had opposite effects on HC-HC coupling. These findings suggest that HC-HC coupling in the OPL might not play a significant role in emmetropization and myopia development.

Purpose: The purpose of this study was to evaluate the interocular differences in choroidal vasculature, choriocapillaris perfusion, and retinal microvascular network, and to explore their associations with interocular asymmetry in axial lengths (ALs) in children with anisomyopia., Methods: Refractive error, AL, and other biometric parameters were measured in 70 children with anisomyopia. Using optical coherence tomography (OCT) and OCT-angiography, we measured the submacular choroidal thickness (ChT), total choroidal area (TCA), luminal area (LA), stromal area (SA), choroidal vascularity index (CVI), choriocapillaris flow deficit (CcFD), retinal vessel density (VD), and foveal avascular zone (FAZ) area., Results: The mean interocular differences in spherical equivalent refraction and AL were -2.26 ± 0.94 diopters and 0.95 ± 0.46 mm, respectively. Submacular ChT, TCA, LA, SA, and CVI were all significantly lower in the more myopic (longer AL) eyes than in the less myopic (shorter AL) fellow eyes. In eyes with longer ALs, both the CcFD and FAZ areas were significantly greater, whereas the superficial and deep retinal VDs were significantly less. After adjusting for corneal power and intraocular pressure, interocular differences in LA (β = -0.774), SA (β = -0.991), and CcFD (β = 0.040) were significantly associated with interocular asymmetry in AL (all P < 0.05)., Conclusions: In pediatric anisomyopes, eyes with longer ALs tended to have lower choroidal vascularity and choriocapillaris perfusion than the contralateral eyes with shorter ALs. Longitudinal investigations would be useful follow-ups to test for a causal role of choroidal circulation in human myopia.

Abstract: The Eye Hospital of Wenzhou Medical University (the Eye Hospital) has found its ways to thrive within the contexts of China to become one of the world-leading institutes in providing ophthalmology and optometry training, education, research, and clinical care at the international standards within 2 decades. It is also the only eye institute in China that possesses 3 national-level research centers (National Clinical Research Center for Ocular Disease, State Key Laboratory of Ophthalmology, Optometry and Visual Science, and National Engineering Technology Research Center for Ophthalmology and Optometry). The hospital is instrumental to the unique medical program in which medical graduates could be both medical doctors and optometrists. Over 10% of the eye doctors in China were trained by the hospital. The SCI-indexed journal Eye and Vision, which is the top eye journal from China, was established by the Eye Hospital in 2015. Its recent endeavor entitled "Eye Valley" provides a one-stop platform for innovation and technology incubation, basic research to clinical trial, from US Food & Drug Administration (FDA) registration to drug production, etc. This article reviews the history of development of the Eye Hospital and elaborates its spirit of "making the impossible possible", which has guided it all the way through from an unknown regional to a renowned top-tier hospital with national and global recognition as a first-class institution., (Copyright © 2021 Asia-Pacific Academy of Ophthalmology. Published by Wolters Kluwer Health, Inc. on behalf of the Asia-Pacific Academy of Ophthalmology.)

Purpose: We used a mouse model to explore the role of the endoplasmic reticulum membrane protein complex subunit 3 (EMC3) in mammalian retinal development., Methods: The transcription pattern of Emc3 in C57BL/6 mice was analyzed by in situ hybridization. To explore the effects of EMC3 absence on retinal development, the Cre-loxP system was used to generate retina-specific Emc3 in knockout mice (Emc3flox/flox, Six3-cre+; CKO). Morphological changes in the retina of E13.5, E17.5, P0.5, and P7 mice were observed via hematoxylin and eosin staining. Immunofluorescence staining was used to assess protein distribution and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to assess apoptosis changes. Proteins were identified and quantified by Western blotting and proteomic analysis. Electroretinogram (ERG), fundus color photography, and optical coherence tomography were performed on 5-week-old mice to evaluate retinal function and structure., Results: The Emc3 mRNA was widely distributed in the whole retina during development. Loss of retinal EMC3 led to retinal rosette degeneration with mislocalization of cell junction molecules (β-catenin, N-cadherin, and zonula occludens-1) and polarity molecules (Par3 and PKCζ). Endoplasmic reticulum stress and TUNEL apoptosis signals were present in retinal rosette-forming cells. Although the absence of EMC3 promoted the production of photoreceptor cells, 5-week-old mice lost all visual function and had severe retinal morphological degeneration., Conclusions: EMC3 regulates retinal structure by maintaining the polarity of retinal progenitor cells and regulating retinal cell apoptosis.

Purpose: To explore the association of choroidal vascularity and choriocapillaris blood perfusion with myopic severity in anisomyopes., Methods: Refractive error, axial length (AL), and other biometric parameters were measured in 34 anisomyopic young adults. Macular choroidal thickness (ChT) and choroidal vascularity, including total choroidal area (TCA), luminal area (LA), stromal area (SA), and choroidal vascularity index (CVI), were determined from swept-source optical coherence tomography (SS-OCT) vertical and horizontal B-scans. The percentage of choriocapillaris flow voids (FV%) was obtained from en face SS-OCT-angiography., Results: The spherical equivalent refraction (SER) was -3.35 ± 1.25 diopters in the more myopic eyes and -1.25 ± 1.17 diopters in the less myopic eyes (P < 0.001). The interocular difference in SER was highly correlated with that in AL (P < 0.001). The macular ChT, TCA, LA, and SA were smaller in the more myopic eyes than in the less myopic eyes in both vertical and horizontal scans (all P < 0.001). Importantly, the CVIs in vertical and horizontal scans were smaller and the FV% was greater in the more myopic eyes (P < 0.05). In vertical scans, the interocular difference in CVIs was correlated with that in the SER, AL, and ChT (all P < 0.05). The interocular difference in FV% was correlated with that in SER, AL, and vertical and horizontal ChTs (all P < 0.05)., Conclusions: Choroidal vascularity and choriocapillaris blood perfusion were lower in the more myopic eyes of anisomyopic adults. These changes were correlated with the severity of myopia and choroidal thinning, indicating that choroidal blood flow is disturbed in human myopia.

Myopia is the leading cause of visual impairment worldwide. The lack of a "rapid predictive index" for myopia development and progression hinders the clinic management and prevention of myopia. This article reviews the studies describing changes that occur in the choroid during myopia development and proposes that it is possible to detect myopia development at an earlier stage than is currently possible in a clinical setting using choroidal blood perfusion as a "rapid predictive index" of myopia.

Form deprivation myopia (FDM) is characterized by loss of choroidal thickness (ChT), reduced choroidal blood perfusion (ChBP), and consequently scleral hypoxia. In some tissues, changes in levels of peroxisome proliferator-activated receptor γ (PPARγ) expression modulate hypoxia-induced pathological responses. We determined if PPARγ modulates FDM through changes in ChT, ChBP, scleral hypoxia-inducible transcription factor (HIF-1α) that in turn regulate scleral collagen type 1 (COL1) expression levels in guinea pigs. Myopia was induced by occluding one eye, while the fellow eye served as control. They received daily peribulbar injections of either the PPARγ antagonist GW9662, or the GW1929 agonist, with or without ocular occlusion for 4 weeks. Ocular refraction and biometric parameters were estimated at baseline, 2 and 4 weeks post-treatment. ChT and ChBP were measured at the 2- and 4-week time points. Western blot analysis determined the expression levels of scleral HIF-1α and COL1. GW9662 induced a myopic shift in unoccluded eyes. Conversely, GW1929 inhibited FDM progression without affecting the refraction in unoccluded eyes. GW9662 reduced both ChT and ChBP in unoccluded eyes, while GW1929 inhibited their declines in occluded eyes. Scleral HIF-1α expression rose in GW9662-treated unoccluded eyes whereas GW1929 reduced HIF-1α upregulation in occluded eyes. GW9662 downregulated scleral COL1 expression in unoccluded eyes, while GW1929 reduced their decreases in occluded eyes. Therefore, PPARγ modulates collagen expression levels and FDM through an inverse relationship between changes in PPARγ and HIF-1α expression levels., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Myopia, also known as short-sightedness or near-sightedness, is a very common condition that typically starts in childhood. Severe forms of myopia (pathologic myopia) are associated with a risk of other associated ophthalmic problems. This disorder affects all populations and is reaching epidemic proportions in East Asia, although there are differences in prevalence between countries. Myopia is caused by both environmental and genetic risk factors. A range of myopia management and control strategies are available that can treat this condition, but it is clear that understanding the factors involved in delaying myopia onset and slowing its progression will be key to reducing the rapid rise in its global prevalence. To achieve this goal, improved data collection using wearable technology, in combination with collection and assessment of data on demographic, genetic and environmental risk factors and with artificial intelligence are needed. Improved public health strategies focusing on early detection or prevention combined with additional effective therapeutic interventions to limit myopia progression are also needed.

Purpose: In guinea pigs, choroidal thickness (ChT) and choroidal blood perfusion (ChBP) simultaneously decrease in experimental myopia, and both increase during recovery. However, the causal relationship between ChBP and myopia requires further investigation. In this study, we examined the changes of ChBP with three different antimyopia treatments. We also actively increased ChBP to examine the direct effect on myopia development in guinea pigs., Methods: Experiment 1: Guinea pigs wore occluders on the right eye for two weeks to induce form-deprivation myopia (FDM). Simultaneously they received daily antimyopia treatments: peribulbar injections of atropine or apomorphine or exposure to intense light. Experiment 2: The vasodilator prazosin was injected daily into the form-deprivation eyes to increase ChBP during the two-week induction of FDM. Other FDM animals received appropriate control treatments. Changes in refraction, axial length, ChBP, ChT, and hypoxia-labeled pimonidazole adducts in the sclera were measured., Results: The antimyopia treatments atropine, apomorphine, and intense light all significantly inhibited myopia development and the decrease in ChBP. The treatments also reduced scleral hypoxia, as indicated by the decrease in hypoxic signals. Furthermore, actively increasing ChBP with prazosin inhibited the progression of myopia, as well as the increase in axial length and scleral hypoxia., Conclusions: Our data strongly indicate that increased ChBP attenuates scleral hypoxia, and thereby inhibits the development of myopia. Thus ChBP may be a promising target for myopia retardation. As such, it can serve as an immediate predictor of myopia development as well as a long-term marker of it.

Retinal bipolar cells (BCs) connect with photoreceptors and relay visual information to retinal ganglion cells (RGCs). Retina-specific deletion of Lhx4 in mice results in a visual defect resembling human congenital stationary night blindness. This visual dysfunction results from the absence of rod bipolar cells (RBCs) and the loss of selective rod-connecting cone bipolar cell (CBC) subtypes and AII amacrine cells (ACs). Inactivation of Lhx4 causes the apoptosis of BCs and cell fate switch from some BCs to ACs, whereas Lhx4 overexpression promotes BC genesis. Moreover, Lhx4 positively regulates Lhx3 expression to drive the fate choice of type 2 BCs over the GABAergic ACs. Lhx4 inactivation ablates Bhlhe23 expression, whereas overexpression of Bhlhe23 partially rescues RBC development in the absence of Lhx4. Thus, by acting upstream of Bhlhe23, Prdm8, Fezf2, Lhx3, and other BC genes, Lhx4, together with Isl1, could play essential roles in regulating the subtype-specific development of RBCs and CBCs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Spectral composition affects emmetropization in both humans and animal models. Because color vision interacts the effects of chromatic defocus, we developed a method to bypass the effects of longitudinal chromatic aberration by placing a spectral filter behind the optics of the eye, using genetic tools. Newborn C57BL/6J (B6) mice were reared in quasi-monochromatic red (410-510 nm) or blue (585-660 nm) light beginning before eye-opening. Refractive states and ocular dimensions were compared at 4, 6, 8, and 10 weeks with mice reared in normal white light. Cre recombinase-dependent Ai9 reporter mice were crossed with Chx10-Cre to obtain Chx10-Cre;Ai9 mice, expressing red fluorescent protein in retinal Cre-positive cells. Ai9 offsprings, with and without Cre, were reared under a normal visual environment. Refraction and axial components were measured as described above. Expression levels of M and S opsin were quantified by western blotting at 10 weeks. Compared with those reared in white light, B6 mice reared in red light developed relative hyperopia, principally characterized by flattening of corneal curvature. Emmetropization was not affected by blue light, possibly because the reduction in vitreous chamber depth compensated for the increase in corneal curvature. Compared with Cre-negative littermates, the refraction and axial dimensions of Chx10-Cre;Ai9 mice were not significantly different at the follow-up timepoints. M opsin levels were higher in Chx10-Cre;Ai9 mice at 10 weeks while S opsin levels were not different. Red light induced a hyperopic shift in mouse refractive development. Emmetropization was not impacted in mice with perturbed color vision caused by intrinsic red-fluorescent protein, suggesting that color vision may not be necessary in mouse emmetropization when other mechanisms are present.

Myopia is a leading cause of visual impairment worldwide. This sight-compromising condition is associated with scleral thinning, extracellular matrix remodeling, and inappropriate optical axial length elongation. Although macrophages are present in the sclera, their involvement in this condition is unknown. By using a form-deprivation myopia (FDM) mouse model, we found that both the scleral macrophage density and their matrix metalloproteinase-2 (MMP-2) expression levels increased in myopic eyes. Partial scleral macrophage depletion by clodronate shifted the refraction toward hyperopia in both the form-deprived and the untreated fellow eyes compared with their respective counterparts in the vehicle-injected control mice. However, this procedure did not alter susceptibility to FDM. FDM development was 59% less in the macrophage-specific Mmp2 deletion (LysM Cre Mmp-2 fl/fl ) mice than in their Cre-negative littermates (Mmp2 fl/fl mice). Moreover, the expression of scleral C-C motif chemokine ligand-2 (CCL2), which is a potent monocyte chemoattractant recruiting monocytes to tissue sites, was increased during myopia progression. However, the increase in the density of scleral macrophages and myopia development were suppressed in fibroblast-specific Ccl2 deletion mice. These declines suggested that the increase in scleral macrophage density in myopic eyes stems from the up-regulation of scleral Ccl2 expression in fibroblasts, which, in turn, promotes monocytes recruitment. In summary, scleral monocyte-derived macrophages contribute to myopia development through enhancing MMP-2 expression in mice., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease. X-linked nuclear modifiers were proposed to modify the phenotypic manifestation of LHON-associated mitochondrial DNA (mtDNA) mutations. By whole-exome sequencing, we identified the X-linked LHON modifier (c.157C>T, p.Arg53Trp) in PRICKLE3 encoding a mitochondrial protein linked to biogenesis of ATPase in 3 Chinese families. All affected individuals carried both ND4 11778G>A and p.Arg53Trp mutations, while subjects bearing only a single mutation exhibited normal vision. The cells carrying the p.Arg53Trp mutation exhibited defective assembly, stability, and function of ATP synthase, verified by PRICKLE3-knockdown cells. Coimmunoprecipitation indicated the direct interaction of PRICKLE3 with ATP synthase via ATP8. Strikingly, cells bearing both p.Arg53Trp and m.11778G>A mutations displayed greater mitochondrial dysfunction than those carrying only a single mutation. This finding indicated that the p.Arg53Trp mutation acted in synergy with the m.11778G>A mutation and deteriorated mitochondrial dysfunctions necessary for the expression of LHON. Furthermore, we demonstrated that Prickle3-deficient mice exhibited pronounced ATPase deficiencies. Prickle3-knockout mice recapitulated LHON phenotypes with retinal deficiencies, including degeneration of retinal ganglion cells and abnormal vasculature. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutations and X-linked nuclear modifiers.

Background: Myopia is a good model for understanding the interaction between genetics and environmental stimuli. Here we dissect the biological processes affecting myopia progression., Methods: Human Genetic Analyses: (1) gene set analysis (GSA) of new genome wide association study (GWAS) data for 593 individuals with high myopia (refraction ≤ -6 diopters [D]); (2) over-representation analysis (ORA) of 196 genes with de novo mutations, identified by whole genome sequencing of 45 high-myopia trio families, and (3) ORA of 284 previously reported myopia risk genes. Contributions of the enriched signaling pathways in mediating the genetic and environmental interactions during myopia development were investigated in vivo and in vitro., Results: All three genetic analyses showed significant enrichment of four KEGG signaling pathways, including amphetamine addiction, extracellular matrix (ECM) receptor interaction, neuroactive ligand-receptor interaction, and regulation of actin cytoskeleton pathways. In individuals with extremely high myopia (refraction ≤ -10 D), the GSA of GWAS data revealed significant enrichment of the HIF-1α signaling pathway. Using human scleral fibroblasts, silencing the key nodal genes within protein-protein interaction networks for the enriched pathways antagonized the hypoxia-induced increase in myofibroblast transdifferentiation. In mice, scleral HIF-1α downregulation led to hyperopia, whereas upregulation resulted in myopia. In human subjects, near work, a risk factor for myopia, significantly decreased choroidal blood perfusion, which might cause scleral hypoxia., Interpretation: Our study implicated the HIF-1α signaling pathway in promoting human myopia through mediating interactions between genetic and environmental factors., Funding: National Natural Science Foundation of China grants; Natural Science Foundation of Zhejiang Province., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: Cyclic adenosine monophosphate (cAMP) and peroxisome proliferator-activated receptor alpha (PPARα) levels mediate extracellular matrix (ECM) changes by altering the levels of hypoxia-inducible factor 1-alpha (HIF-1α) in various tissues. We aimed to determine, in the sclera of guinea pigs, whether a prostanoid receptor (EP2)-linked cAMP modulation affects PPARα and HIF-1α signaling during myopia., Methods: Three-week-old guinea pigs (n = 20 in each group), were monocularly injected with either an EP2 agonist (butaprost 1 µmol/L/10 µmol/L), an antagonist (AH6809 10 µmol/L/30 µmol/L) or a vehicle solution for two weeks during normal ocular growth. Separate sets of animals received these injections and underwent form deprivation (FD) simultaneously. Refraction and axial length (AL) were measured at two weeks, followed by scleral tissue isolation for quantitative PCR (qPCR) analysis (n = 10) and cAMP detection (n = 10) using a radioimmunoassay., Results: Butaprost induced myopia development during normal ocular growth, with proportional increases in AL and cAMP levels. FD did not augment the magnitude of myopia or cAMP elevations in these agonist-injected eyes. AH6809 suppressed cAMP increases and myopia progression during FD, but had no effect in a normal visual environment. Of the diverse set of 27 genes related to cAMP, PPARα and HIF-1α signaling and ECM remodeling, butaprost differentially regulated 15 of them during myopia development. AH6809 injections during FD negated such differential gene expressions., Conclusion: EP2 agonism increased cAMP and HIF-1α signaling subsequent to declines in PPARα and RXR mRNA levels, which in turn decreased scleral fibrosis and promoted myopia. EP2 antagonism instead inhibited each of these responses. Our data suggest that EP2 suppression may sustain scleral ECM structure and inhibit myopia development.