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Diabetic retinopathy (DR) is the leading cause of blindness and visual loss in people with diabetes. It has been suggested that the progression of DR is associated with chronic inflammation and oxidative stress. The aim of the present work was to evaluate the ability of the natural compound madecassic acid (MEA) to reverse the negative impact of streptozotocin (STZ) on retinal injury in rats. Diabetic rats induced by STZ were treated with MEA at the doses of 10 and 20 mg/kg bw for 8 weeks. The study compared the efficacy of the drug in controlling high blood sugar levels and its impact on therapeutic targets such as SOD, CAT, GPx, NF-κB, TNF-α, IL-6, IL-1β, VEGF, IGF, bFGF and Keap1/Nrf-2 pathway. The results showed that the treatment with MEA significantly restored the retinal SOD, CAT, and GPx levels in diabetic rats to the near-normal levels. Moreover, the level of inflammatory mediators (TNF-α, IL-1β, IL-6) and growth factors (VEGF, IGF, bFGF) was significantly lower in retinas of animals treated with MEA as compared to retinas of diabetic animals. The study also established that MEA administration reduced the NF-κB protein and altered the Nrf-2/Keap1 pathway thereby reducing oxidative stress and inflammation. Furthermore, the use of MEA prevented the progression of the retinal capillary basement membrane thickening. It has been found that MEA offers significant protection to the retina and therefore, the compound may be useful in the treatment of DR in humans., 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 Inc. All rights reserved.)

Joubert Syndrome (JBTS) is a neurodevelopmental ciliopathy defined by a highly specific midbrain-hindbrain malformation, variably associated with additional neurological features. JBTS displays prominent genetic heterogeneity with >40 causative genes that encode proteins localising to the primary cilium, a sensory organelle that is essential for transduction of signalling pathways during neurodevelopment, among other vital functions. JBTS proteins localise to distinct ciliary subcompartments, suggesting diverse functions in cilium biology. Currently, there is no unifying pathomechanism to explain how dysfunction of such diverse primary cilia-related proteins results in such a highly specific brain abnormality. To identify the shared consequence of JBTS gene dysfunction, we carried out transcriptomic analysis using zebrafish mutants for the JBTS-causative genes cc2d2aw38, cep290fh297, inpp5ezh506, talpid3i264 and togaram1zh510 and the Bardet-Biedl syndrome-causative gene bbs1k742. We identified no commonly dysregulated signalling pathways in these mutants and yet all mutants displayed an enrichment of altered gene sets related to central nervous system function. We found that JBTS mutants have altered primary cilia throughout the brain but do not display abnormal brain morphology. Nonetheless, behavioural analyses revealed reduced locomotion and loss of postural control which, together with the transcriptomic results, hint at underlying abnormalities in neuronal activity and/or neuronal circuit function. These zebrafish models therefore offer the unique opportunity to study the role of primary cilia in neuronal function beyond early patterning, proliferation and differentiation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

As a prominent complication of diabetes mellitus (DM) affecting microvasculature, diabetic retinopathy (DR) originates from blood-retinal barrier (BRB) damage. Natural polyphenolic compound chlorogenic acid (CGA) has already been reported to alleviate DR. This study delves into the concrete mechanism of the CGA-supplied protection against DR and elucidates its key target in retinal endothelial cells. DM in mice was induced using streptozotocin (STZ). CGA mitigated BRB dysfunction, leukocytes adhesion and the formation of acellular vessels in vivo. CGA suppressed retinal inflammation and the release of tumor necrosis factor-α (TNFα) by inhibiting nuclear factor kappa-B (NFκB). Furthermore, CGA reduced the TNFα-initiated adhesion of peripheral blood mononuclear cell (PBMC) to human retinal endothelial cell (HREC). CGA obviously decreased the TNFα-upregulated expression of vascular cell adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1), and abrogated the TNFα-induced NFκB activation in HRECs. All these phenomena were reversed by overexpressing type 1 TNF receptor (TNFR1) in HRECs. The CGA-provided improvement on leukocytes adhesion and retinal inflammation was disappeared in mice injected with an endothelial-specific TNFR1 overexpression adeno-associated virus (AAV). CGA reduced the interaction between TNFα and TNFR1 through binding to TNFR1 in retinal endothelial cells. In summary, excepting reducing TNFα expression via inhibiting retinal inflammation, CGA also reduced the adhesion of leukocytes to retinal vessels through decreasing VCAM1 and ICAM1 expression via blocking the TNFα-initiated NFκB activation by targeting TNFR1 in retinal endothelial cells. All of those mitigated retinal inflammation, ultimately alleviating BRB breakdown in DR., 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.)

The choroid is the thin, vasculature-filled layer of the eye situated between the sclera and the retina, where it serves the metabolic needs of the light-sensing photoreceptors in the retina. Illumination of the interior surface of the back of the eye (fundus) is a critical regulator of subretinal fluid homeostasis, which determines the overall shape of the eye, but it is also important for choroidal perfusion. Noted for having some of the highest blood flow rates in the body, the choroidal vasculature has been reported to lack intrinsic, intravascular pressure-induced (myogenic) autoregulatory mechanisms. Here, we ask how light directly regulates choroid perfusion and ocular fluid homeostasis, testing the hypothesis that light facilitates ocular fluid absorption by directly increasing choroid endothelial permeability and decreasing choroid perfusion. Utilizing ex vivo pressurized whole-choroid and whole-eye preparations from mice expressing cell-specific Ca 2+ indicators, we found that the choroidal vasculature has two intrinsically light-sensitive Ca 2+ -signaling mechanisms: One increases Ca 2+ -dependent production of nitric oxide in choroidal endothelial cells; the other promotes vasoconstriction through Ca 2+ elevation in vascular smooth muscle cells. In addition, we found that choroidal flow, or pressure, modulates endothelial and smooth muscle photosensitivity and trans-retinal absorption of fluid into the choroid. These results collectively suggest that the choroid vasculature exhibits an inverted form of autoregulatory control, where pressure- and light-induced mechanisms work in opposition to regulate blood flow and maintain fluid balance in response to changes in light and dark, aligning with the metabolic needs of photoreceptors., Competing Interests: Competing interests statement:The authors declare no competing interest.

CRISPR-Cas13 nucleases are programmable RNA-targeting effectors that can silence gene expression in a transient manner. Recent iterations of Cas13 nucleases are compact for adeno-associated virus (AAV) delivery to achieve strong and persistent expression of various organs in a safe manner. Here, we report significant transcriptomic signatures of Cas13bt3 expression in retinal cells and show all-in-one AAV gene therapy with Cas13bt3 can effectively silence VEGFA mRNA in human retinal organoids and humanized VEGF transgenic mouse (trVEGF029, Kimba) models. Specifically, human embryonic stem cells (hESC)-derived retinal pigment epithelium cells show high expression of Cas13bt3 from virus delivery corresponding to a significant reduction of VEGFA mRNA. We further show that intravitreal delivery of Cas13bt3 by AAV2.7m8 can efficiently transduce mouse retinal cells for specific knockdown of human VEGFA in the Kimba mouse. Our results reveal important considerations for assessing Cas13 activity, and establish the Cas13bt3 RNA editing system as a potential anti-VEGF agent that can achieve significant control of VEGFA for the treatment of retinal neovascularization., Competing Interests: Competing interests statement:The authors declare no competing interest.

Mutations in PRPH2 are a relatively common cause of sight-robbing inherited retinal degenerations (IRDs). Peripherin-2 (PRPH2) is a photoreceptor-specific tetraspanin protein that structures the disk rim membranes of rod and cone outer segment (OS) organelles, and is required for OS morphogenesis. PRPH2 is noteworthy for its broad spectrum of disease phenotypes; both inter- and intra-familial heterogeneity have been widely observed and this variability in disease expression and penetrance confounds efforts to understand genotype-phenotype correlations and pathophysiology. Here we report the generation and initial characterization of a gene-edited animal model for PRPH2 disease associated with a nonsense mutation (c.1095:C>A, p.Y285X), which is predicted to truncate the peripherin-2 C-terminal domain. Young (P21) Prph2Y285X/WT mice developed near-normal photoreceptor numbers; however, OS membrane architecture was disrupted, OS protein levels were reduced, and in vivo and ex vivo electroretinography (ERG) analyses found that rod and cone photoreceptor function were each severely reduced. Interestingly, ERG studies also revealed that rod-mediated downstream signaling (b-waves) were functionally compensated in the young animals. This resiliency in retinal function was retained at P90, by which time substantial IRD-related photoreceptor loss had occurred. Altogether, the current studies validate a new mouse model for investigating PRPH2 disease pathophysiology, and demonstrate that rod and cone photoreceptor function and structure are each directly and substantially impaired by the Y285X mutation. They also reveal that Prph2 mutations can induce a functional compensation that resembles homeostatic plasticity, which can stabilize rod-derived signaling, and potentially dampen retinal dysfunction during some PRPH2-associated IRDs., (© The Author(s) 2024. Published by Oxford University Press.)

Purpose: Diabetic retinopathy (DR) is a significant complication of diabetes mellitus that can lead to progressive visual impairment. This study aimed to elucidate the role of fibrinogen, a protein whose serum and intraocular concentrations are elevated in patients with diabetes and DR, in the pathogenesis of DR., Methods: The changes in the protein levels of the neuronal marker tubulin-β3 (TUBB3) and retinal response induced by the intravitreal injections of 1× phosphate-buffered saline, 40 mg/mL of fibrinogen, and 40 mg/mL of fibrinogen in combination with anti-intracellular adhesion molecule-1 (ICAM-1) antibody in normal mice were observed using immunofluorescence, western blotting, and electroretinography., Results: High concentrations of fibrinogen led to a decrease in the expression of TUBB3 in immunofluorescence and western blotting. The amplitudes of the positive scotopic threshold response and b-wave were notably reduced after the injection of fibrinogen, indicating potential damage to the retinal ganglion cells. The co-administration of anti-ICAM-1 antibody effectively mitigated these fibrinogen-induced changes, indicating that fibrinogen-induced damage is mediated via the ICAM-1 pathway., Conclusions: The present study underscores the significance of elevated intraocular fibrinogen levels as a pathogenic factor in DR. Involvement of the fibrinogen/ICAM-1 pathway presents new avenues for therapeutic intervention, especially in patients with treatment-resistant conditions.

The phototransduction cascade enables the photoreceptor to detect light over a wide range of intensities without saturation. The main second messenger of the cascade is cGMP and the primary regulatory mechanism is calcium feedback. However, some experimental data suggest that cAMP may also play a role in regulating the phototransduction cascade, but this would require changes in cAMP on a time scale of seconds. Currently, there is a lack of data on the dynamics of changes in intracellular cAMP levels on this timescale. This is largely due to the specificity of the sensory modality of photoreceptors, which makes it practically impossible to use conventional experimental approaches based on fluorescence methods. In this study, we employed the method of rapid cryofixation of retinal samples after light stimulation and subsequent isolation of outer segment preparations. The study employed highly sensitive metabolomics approaches to measure levels of cAMP. Additionally, PKA activity was measured in the samples using a western blot. The results indicate that when exposed to near-saturating but still moderate light, cAMP levels increase transiently within the first second and then return to pre-stimulus levels. The increase in cAMP activates PKA, resulting in the phosphorylation of PKA-specific substrates in frog retinal outer segments., (© 2024 Chernyshkova et al.)

The operation of the retina, like other brain circuits, is under modulatory control. One coordinator of changes in retinal function is dopamine, a neuromodulator released in a light-dependent way to adjust vision on a diurnal cycle. Here, we demonstrate that substance P is a similarly powerful retinal modulator that interacts with the dopamine system. By imaging glutamatergic synaptic transmission in larval zebrafish, we find that substance P decreases the contrast sensitivity of ON and OFF visual channels up to 8-fold, with suppression of visual signals being strongest through the "transient" pathway responding to higher frequencies. These actions are exerted in the morning, in large part by suppressing the amplification of visual signals by dopamine, but substance P is almost completely inactive in the afternoon. Modulation of retinal gain is accompanied by changes in patterns of vesicle release at the synapses of bipolar cells: increased gain shifts coding of stimulus strength from the rate of release events to their amplitude generated by a process of multivesicular release (MVR). Together, these actions of substance P reduce the flow of visual information, measured in bits, ∼3-fold. Thus, whereas dopamine "pushes" the retina to transmit information at higher rates in the afternoon, substance P acts in antiphase to suppress dopamine signaling and "pull down" information transmission in the morning., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In this issue of the Journal of Experimental Medicine, Cao et al. (https://doi.org/10.1084/jem.20240386) demonstrate that the connection between the eye and the brain goes beyond the impulses carried by the optic nerve and that in Alzheimer's disease (AD), the influx of toxic Aβ from the brain to the retina underlies AD-induced retinal degeneration., (© 2024 Walsh.)

The eye is closely connected to the brain, providing a unique window to detect pathological changes in the brain. In this study, we discovered β-amyloid (Aβ) deposits along the ocular glymphatic system in patients with Alzheimer's disease (AD) and 5×FAD transgenic mouse model. Interestingly, Aβ from the brain can flow into the eyes along the optic nerve through cerebrospinal fluid (CSF), causing retinal degeneration. Aβ is mainly observed in the optic nerve sheath, the neural axon, and the perivascular space, which might represent the critical steps of the Aβ transportation from the brain to the eyes. Aquaporin-4 facilitates the influx of Aβ in brain-eye transport and out-excretion of the retina, and its absence or loss of polarity exacerbates brain-derived Aβ induced damage and visual impairment. These results revealed brain-to-eye Aβ transport as a major contributor to AD retinopathy, highlighting a new therapeutic avenue in ocular and neurodegenerative disease., (© 2024 Cao et al.)

Photoreception is essential for the development of the visual system, shaping vision's first synapse to cortical development. Here, we find that the lighting environment controls developmental rod apoptosis via Opn4-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). Using genetics, sensory environment manipulations, and computational approaches, we establish a pathway where light-dependent glutamate released from ipRGCs is detected via a transiently expressed glutamate receptor (Grik3) on rod precursors within the inner retina. Communication between these cells is mediated by hybrid neurites on ipRGCs that sense light before eye opening. These structures span the ipRGC-rod precursor distance over development and contain the machinery for photoreception (Opn4) and neurotransmitter release (Vglut2 & Syp). Assessment of the human gestational retina identifies conserved hallmarks of an ipRGC-to-rod axis, including displaced rod precursors, transient GRIK3 expression, and ipRGCs with deep-projecting neurites. This analysis defines an adaptive retrograde pathway linking the sensory environment to rod precursors via ipRGCs prior to eye opening., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Progression of diabetic retinopathy resists arrest even after institution of intensive glycemic control, suggesting a "metabolic memory" phenomenon, but the mechanism responsible for this phenomenon is still elusive. Gene expression and biological processes can also be regulated by long noncoding RNAs (LncRNAs), the RNAs with >200 nucleotides and no open reading frame for translation, and several LncRNAs are aberrantly expressed in diabetes. Our aim was to identify retinal LncRNAs that fail to reverse after termination of hyperglycemia. Microarray analysis was performed on retinal RNA from streptozotocin-induced diabetic rats in poor glycemic control for 8 months, followed by in good glycemic control (blood glucose >400 mg/dL), or for 4 months, with four additional months of good glycemic control (blood glucose <150 mg/dL). Differentially expressed LncRNAs and mRNAs were identified through Volcano filtering, and their functions were predicted using gene ontology and pathway enrichment analyses. Compared with age-matched normal rats, rats in continuous poor glycemic control had >1479 differentially expressed LncRNAs (710 downregulated, 769 upregulated), and among those, 511 common LncRNAs had similar expression in Diab and Rev groups (139 downregulated, 372 upregulated). Gene Ontology/pathway analysis identified limited LncRNAs in biological processes, but analysis based on biological processes/molecular function revealed >350 genes with similar expression in Diab and Rev groups; these genes were mainly associated with stress response, cell death, mitochondrial damage and cytokine production. Thus, identifying retinal LncRNAs and their gene targets that do not benefit from termination of hyperglycemia have potential to serve as therapeutic targets to slow down the progression of diabetic retinopathy., (© 2024 The Author(s). Journal of Diabetes published by Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

This study demonstrated the functions and molecular mechanisms of the IL-33/ST2 axis in experimental optic neuropathy. C57BL/6J mice were used to establish an optic nerve crush (ONC) model. ONC mice were administered with IL-33 intraperitoneal injection, with PBS vehicle as control. Immunofluorescence, quantitative RT-PCR, and western blot techniques were utilized to assess the expression of the IL-33/ST2 axis. The electroretinography (ERG), optical coherence tomography (OCT), H&E, and luxol fast blue were used to assess the structural and functional changes. Western blot was employed to detect the activation of the mTOR/S6 pathway. The IL-33 expression level in the inner nuclear layer of the retina in ONC mice reached its peak on day 3, accompanied by a significant increase in IL-33 receptor ST2 expression. IL-33 treatment promoted the survival of retinal ganglion cells, restored the thickness of inner retina layer (IRL), alleviated the demyelination of the optic nerve, and recovered the decreased amplitude of b-wave in ONC mice. Furthermore, administration of IL-33 activated the mTOR/S6 signaling pathway in RGCs, which was significantly suppressed in the ONC condition. This study indicated that boosting the IL-33/ST2/mTOR/S6 pathway can protect against structural and functional damage to the retina and optic nerve induced by ONC. As a result, the IL-33/ST2 axis holds potential as a therapeutic option for treating various optic neuropathies., (Copyright © 2024. Published by Elsevier Ltd.)

Diabetic retinopathy, an ocular complication of diabetes, is an important cause of blindness in adults. Puerarin is considered to have promising potential for clinical use in treating diabetic retinopathy. In this study, we designed a novel puerarin-loaded poly(lactic acid) sustained-release microspheres suitable for ocular administration, and we assessed itsin vitro and in vivo properties. The preparation of puerarin-loaded microspheres was optimized by Box-Behnken response surface design. The encapsulation efficiency and drug loading of microspheres were 35.71% and 3.85%, respectively. The microspheres exhibited good dispersion and high safety, making it suitable for ocular drug delivery. In vitro release demonstrated that microspheres had a well-sustained release effectiveness, and its release behavior complied with the zero-order kinetic characteristics. The results of ocular tissue distribution revealed that the C max andAUC 0-∞ of the microspheres group in the retina and choroid were considerably higher than those of the solution group and the intravenous injection group. This research revealed that intravitreal injection of microspheres can significantly prolong the half-life of puerarin in eye tissues and achieve sustained drug release. Therefore, intravitreal injection of microspheres has positive implications for the treatment of diabetic retinopathy., 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.)

Glaucoma is a degenerative optic neuropathy in which the degeneration of optic nerve and blindness occur. The main cause is a malfunction of ciliary processes (protrusions of the ciliary bodies) resulting in increased intraocular pressure (IOP). Ocular hypertension (OHT) causes ischemic events leading to retinal ganglion cell (RGC) depletion and blindness. Histaminergic and nitrergic systems are involved in the regulation of IOP. Therefore, we developed novel hybrid compounds that target histamine H 3 receptor (H 3 R) with nitric oxide (NO) releasing features (ST-1989 and ST-2130). After H 3 R binding was proven in vitro, we investigated their effects in two OHT models in New Zealand White rabbits. Compound ST-1989 showed the highest NO elevation, together with antioxidative and anti-inflammatory features partly superior to the co-administered H 3 R antagonist (ciproxifan) and NO donor (molsidomine). This hybrid compound demonstrated IOP reduction in both OHT models induced by intravitreal injection of hypertonic saline and carbomer into the anterior chamber of the eye, respectively. Ocular perfusion and photoreceptor neuroprotection were evaluated in a model of ischemia/reperfusion (I/R) of the ophthalmic artery induced by repeated sub-tenon injections of endothelin-1 (ET-1), twice a week for six weeks. Compound ST-1989 counteracts retinal degeneration reducing ophthalmic artery resistance index and increasing photoreceptor responses, thus rescuing RGCs. Our results indicate that compound ST-1989 is a promising molecule with long-lasting hypotensive effects and good effectiveness in reducing inflammation, oxidative stress, and RGCs apoptosis. In conclusion, these hybrid compounds could be a novel strategy to combat glaucomatous blindness and RGC depletion for ocular diseases involving retinal damage., 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. Mohammad A. Khanfar reports financial support was provided by Alexander von Humboldt Foundation. Holger Stark reports financial support was provided by German Research Council. Laura Lucarini reports financial support was provided by Fondazione Cassa di Risparmio di Firenze. If there are other authors, they 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 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Retinitis pigmentosa (RP) is a complex spectrum of inherited retinal diseases marked by the gradual loss of photoreceptor cells, ultimately leading to blindness. Among these, mutations in PDE6A, responsible for encoding a cGMP-specific phosphodiesterase, stand out as pivotal in autosomal recessive RP (RP43). Unfortunately, no effective therapy currently exists for this specific form of RP. However, recent advancements in genome editing, such as base editing (BE) and prime editing (PE), offer a promising avenue for precise and efficient gene therapy. Here, it is illustrated that the engineered BE and PE systems, particularly PE, exhibit high efficiency in rescuing a target point mutation with minimal bystander effects in an RP mouse model carrying the Pde6a (c.2009A > G, p.D670G) mutation. The optimized BE and PE systems are first screened in N2a cells and subsequently assessed in electroporated mouse retinas. Notably, the optimal PE system, delivered via dual adeno-associated virus (AAV), precisely corrects the pathogenic mutation with average 9.4% efficiency, with no detectable bystander editing. This correction restores PDE6A protein expression, preserved photoreceptors, and rescued retinal function in Pde6a mice. Therefore, this study offers a proof-of-concept demonstration for the treatment of Pde6a-related retinal degeneration using BE and PE systems., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Endothelial cells (ECs) display organ- and tissue-specific heterogeneity. In the eye, the retinal and choroidal vascular beds are distinct networks with different molecular and morphological properties that serve location-specific functions, i.e., the former maintaining a tight barrier and the latter, a permeable fenestrated vasculature. Given that retinal health critically relies on the function of these vascular beds and that their dysfunction is implicated in a variety of retinal diseases, a molecular understanding of both physiological and pathophysiological characteristics of these distinct vasculatures is critical. Given their interspersed anatomic distribution among parenchymal cells, the study of EC gene expression, in vivo, has been hampered by the challenge of isolating pure populations of ocular ECs in sufficient quantities for large-scale transcriptomics. To address this challenge, we present a methodological and analytical workflow to facilitate inter-tissue comparisons of the in vivo EC translatome isolated from choroid, retina, and brain using the Cre-inducible NuTRAP flox construct and two widely-used endothelial Cre mouse lines: constitutive Tie2-Cre and tamoxifen-inducible Cdh5-CreERT2. For each Cre line, inter-tissue comparison of TRAP-RNAseq enrichment (TRAP-isolated translatome vs input transcriptome) showed tissue-specific gene enrichments with differential pathway representation. For each mouse model, inter-tissue comparison of the EC translatome (choroid vs brain, choroid vs retina, and brain vs retina) showed over 50% overlap of differentially expressed genes (DEGs) between the three paired comparisons, with differential pathway representation for each tissue. Pathway analysis of DEGs in the Cdh5-NuTRAP vs Tie2-NuTRAP comparison for retina, choroid, and brain predicted inhibition of processes related to myeloid cell function and activation, consistent with more specific targeting of ECs in the Cdh5-NuTRAP than in the Tie2-NuTRAP model which also targets hematopoietic progenitors giving rise to immune cells. Indeed, while TRAP enriches for EC transcripts in both models, myeloid transcripts were also captured in the Tie2-NuTRAP model which was confirmed using cell sorting. We suggest experimental/analytical considerations should be taken when selecting Cre-lines to target ECs., Competing Interests: Declaration of competing interest Ana J. Chucair-Elliott: none, Kevin Pham: none, Audrey C.A. Cleuren: none, Christopher M. Schafer: none, Courtney T. Griffin: none, Sarah R. Ocanas: none, Willard M. Freeman: none. Michael H. Elliott: none., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Reactive oxygen species (ROS) play a significant role in toxicity to the retina in a variety of diseases. N-acetylcysteine (NAC), N-acetylcysteine amide (NACA) and the dimeric di-N-acetylcysteine amide (diNACA) were evaluated in terms of protecting retinal cells, in vitro, in a variety of stress models. Three types of rat retinal cell cultures were utilized in the study: macroglial-only cell cultures, neuron-only retinal ganglion cell (RGC) cultures, and mixed cultures containing retinal glia and neurons. Ability of test agents to attenuate oxidative stress in all cultures was ascertained. In addition, capability of agents to protect against a variety of alternate clinically-relevant stressors, including excitotoxins and mitochondrial electron transport chain inhibitors, was also evaluated. Capacity of test agents to elevate cellular levels of reduced glutathione under normal and compromised conditions was also determined. NAC, NACA and diNACA demonstrated concentration-dependent cytoprotection against oxidative stress in all cultures. These three compounds, however, had differing effects against a variety of alternate insults to retinal cells. The most protective agent was NACA, which was most potent against the most stressors (including oxidative stress, mitochondrial impairment by antimycin A and azide, and glutamate-induced excitotoxicity). Similar to NAC, NACA increased glutathione levels in non-injured cells, although diNACA did not, suggesting a different, unknown mechanism of antioxidant activity for the latter. In support of this, diNACA was the only agent to attenuate rotenone-induced toxicity in mitochondria. NAC, NACA and diNACA exhibited varying degrees of antioxidant activity, i.e., protected cultured rat retinal cells from a variety of stressors which were designed to mimic aspects of the pathology of different retinal diseases. A general rank order of activity was observed: NACA ≥ diNACA > NAC. These results warrant further exploration of NACA and diNACA as antioxidant therapeutics for the treatment of retinal diseases, particularly those involving oxidative stress. Furthermore, we have defined the battery of tests carried out as the "Wood, Chidlow, Wall and Casson (WCWC) Retinal Antioxidant Indices"; we believe that these are of great value for screening molecules for potential to reduce retinal oxidative stress in a range of retinal diseases., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Somatic mitochondrial DNA (mtDNA) mutation accumulation has been observed in individuals with retinal degenerative disorders. To study the effects of aging and mtDNA mutation accumulation in the retina, a polymerase gamma (POLG) exonuclease-deficient model, the Polg D257A mutator mice (D257A), was used. POLG is an enzyme responsible for regulating mtDNA replication and repair. Retinas of young and older mice with this mutation were analyzed in vivo and ex vivo to provide new insights into the contribution of age-related mitochondrial (mt) dysfunction due to mtDNA damage. Optical coherence tomography (OCT) image analysis revealed a decrease in retinal and photoreceptor thickness starting at 6 months of age in mice with the D257A mutation compared to wild-type (WT) mice. Electroretinography (ERG) testing showed a significant decrease in all recorded responses at 6 months of age. Sections labeled with markers of different types of retinal cells, including cones, rods, and bipolar cells, exhibited decreased labeling starting at 6 months. However, electron microscopy analysis revealed differences in retinal pigment epithelium (RPE) mt morphology beginning at 3 months. Interestingly, there was no increase in oxidative stress and parkin-mediated mitophagy in the ages analyzed in the retina or RPE of D257A mice. Additionally, D257A RPE exhibited an accelerated rate of autofluorescence cytoplasmic granule formation and accumulation. Mt markers displayed different abundance in protein lysates obtained from retina and RPE samples. These findings suggest that the accumulation of mtDNA mutations leads to impaired mt function and accelerated aging, resulting in retinal degeneration., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Objective: Pathological retinal neovascularization is vision-threatening. In mouse oxygen-induced retinopathy (OIR) we sought to define mitochondrial respiration changes longitudinally during hyperoxia-induced vessel loss and hypoxia-induced neovascularization, and to test interventions addressing those changes to prevent neovascularization., Methods: OIR was induced in C57BL/6J mice and retinal vasculature was examined at maximum neovessel formation. We assessed total proteome changes and the ratio of mitochondrial to nuclear DNA copy numbers (mtDNA/nDNA) of OIR vs. control retinas, and mitochondrial oxygen consumption rates (OCR) in ex vivo OIR vs. control retinas (BaroFuse). Pyruvate vs. vehicle control was supplemented to OIR mice either prior to or during neovessel formation., Results: In OIR vs. control retinas, global proteomics showed decreased retinal mitochondrial respiration at peak neovascularization. OCR and mtDNA/nDNA were also decreased at peak neovascularization suggesting impaired mitochondrial respiration. In vivo pyruvate administration during but not prior to neovessel formation (in line with mitochondrial activity time course) suppressed NV., Conclusions: Mitochondrial energetics were suppressed during retinal NV in OIR. Appropriately timed supplementation of pyruvate may be a novel approach in neovascular retinal diseases., Competing Interests: Declarations Competing interests IRS and MG have financial ties to EnTox Sciences (Mercer Island, WA), manufacturer of BaroFuse. All other authors declare no conflicts of interest., (© 2024. The Author(s).)

Purpose: Ischemic retinopathy is the major cause of vision-threatening conditions. Inflammation plays an important role in the pathogenesis of ischemic retinopathy. Formyl peptide receptor 1 (FPR1) has been reported to be implicated in the regulation of inflammatory disorders. However, the role of FPR1 in the progression of ischemic retinal injury has not been fully explained., Methods: The activation of FPR1 was measured by real-time PCR and western blotting in the retina of OIR. The effect of FPR1 on the expression of inflammatory cytokines and relevant pro-angiogenic factors was assessed between wild-type and FPR1-deficiency OIR mice. The impact of FPR1 on retinal angiogenesis was evaluated through quantifying retinal vaso-obliteration and neovascularization between FPR1 +/+ and FPR1 -/- OIR mice. At last, the neuronal effect of FPR1 on the ischemic retina was investigated by ERG between wild-type and FPR1-deficient OIR mice., Results: The expression of FPR1 significantly increased in the retina of OIR. Furthermore, FPR1 deficiency downregulated pro-inflammatory and pro-angiogenic factors. Ablation of FPR1 suppressed the retinal pathological neovascularization and promoted reparative revascularization, ultimately improving retinal neural function after ischemic injury., Conclusion: In ischemic retinopathy, FPR1 aggravates inflammation and inhibits reparative angiogenesis to exacerbate neuronal dysfunction.

Retinal ischemia/reperfusion injury (RI/R) is a common pathological process in ophthalmic diseases, which can cause severe visual impairment. The mechanisms underlying RI/R damage and repair are still unclear. Scholars are actively exploring effective intervention strategies to restore impaired visual function. With the development of nucleic acid nanomaterials, tetrahedral framework nucleic acids (tFNAs) have shown promising therapeutic potential in various fields such as stem cells, biosensors, and tumour treatment due to their excellent biological properties. Besides, miRNA-22-3p (miR-22), as an important regulatory factor in neural tissue, has been proven to have positive effects in various neurodegenerative diseases. By stably constructing a complex of tetrahedral framework nucleic acids miR22 (tFNAs-miR22), we observed that tFNAs-miR22 had a positive effect on the repair of RI/R injury in retinal neural tissue. Previous studies have shown that tFNAs can effectively deliver miR-22 into damaged retinal neurons, subsequently exerting neuroprotective effects. Interestingly, we found that there was a certain synergistic effect between tFNAs and miR-22. tFNAs-miR22 can selectively activated the ERK1/2 signalling pathway to reduce neuronal apoptosis, accelerate cell proliferation, and restore synaptic functional activity. In this study, we established a simple yet effective small molecule drug for RI/R treatment which may become a promising neuroprotectant for treating this type of vision impairment disease in the future., (© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Adeno-associated viruses (AAVs) are intensively explored for gene therapies in general and have found promising applications for treating retina diseases. However, controlling the specificity (tropism) and delivery of AAVs to selected layers, cell types, and areas of the retina is a major challenge to further develop retinal gene therapies. Magnetic nanoparticles (MNPs) provide effective delivery platforms to magnetically guide therapeutics to target cells. Yet, how MNPs can deliver AAVs to transfect particular retina layers and cells remains elusive. Here, it is demonstrated that MNPs can be used to transport different AAVs through the retina and to modulate the selective transduction of specific retinal layers or photoreceptor cells in ex vivo porcine explants and whole eyes. Thereby, transduction is triggered by bringing the viruses in close proximity to the target cell layer and by controlling their interaction time. It is shown that this magnetically guided approach to transport AAVs to selected areas and layers of the retina does not require the cell-specific optimization of the AAV tropism. It is anticipated that the new approach to control the delivery of AAVs and to selectively transduce cellular systems can be applied to many other tissues or organs to selectively deliver genes of interest., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Biological processes throughout the body are orchestrated in time through the regulation of local circadian clocks. The retina is among the most metabolically active tissues, with demands depending greatly on the light/dark cycle. Most cell types within the rodent retina are known to express the circadian clock; however, retinal clock expression in humans has not previously been localized. Moreover, the effect of local circadian clock dysfunction on retinal homeostasis is incompletely understood. The current study indicated an age-dependent decline in circadian clock gene and protein expression in the human retina. An animal model of targeted Bmal1 deficiency was used to identify the circadian clock of the retinal Müller glia as essential for neuronal survival, vascular integrity, and retinal function. These results suggest a potential role for the local retinal circadian clock within the Müller glia in age-related retinal disease and retinal degeneration., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: The development of the zebrafish visual system is significantly influenced by exposure to monochromatic light, yet investigations into its effects during juvenile stages are lacking. This study evaluated the impacts of varying intensities and durations of red and blue monochromatic light on the visual system and dopamine pathways in juvenile zebrafish., Methods: Juvenile zebrafish were exposed to red (650 nm) and blue (440 nm, 460 nm) monochromatic lights over four days at intensities ranging from 500 to 10,000 lx, for durations of 6, 10, and 14 h daily. A control group was maintained under standard laboratory conditions. Post-exposure assessments included the optokinetic response (OKR), retinal structural analysis, ocular dopamine levels, and the expression of genes related to dopamine pathways (Th, Dat, and Mao)., Results: (1) OKR enhancement was observed with increased 440 nm light intensity, while 460 nm and 650 nm light exposures showed initial improvements followed by declines at higher intensities. (2) Retinal thinning in the outer nuclear layer was observed under the most intense (10,000 lx for 14 h) light conditions in the 440 nm and 650 nm groups, while the 460 nm group remained unaffected. (3) Dopamine levels increased with higher intensities in the 440 nm group, whereas the 460 nm group exhibited initial increases followed by decreases. The 650 nm group displayed similar trends but were statistically insignificant compared to the control group. (4) Th expression increased with light intensity in the 440 nm group. Dat showed a rising and then declining pattern, and Mao expression significantly decreased. The 460 nm group exhibited similar patterns for Th and Dat to the behavioral observations, but an inverse pattern for Mao. The 650 nm group presented significant fluctuations in Th and Dat expressions, with pronounced variations in Mao., Conclusions: Specific red and blue monochromatic light conditions promote visual system development in juvenile zebrafish. However, exceeding these optimal conditions may impair visual function, highlighting the critical role of dopamine pathway in modulating light-induced effects on the visual system., (© 2024. The Author(s).)

Background: Sex as a biological variable is not a common consideration in molecular mechanistic or preclinical studies of retinal diseases. Understanding the sexual dimorphism of adult RPE and retina under physiological conditions is an important first step in improving our understanding of sex-based physio-pathological mechanisms., Methods: Isobaric tags for relative and absolute quantitation (iTRAQ) were used for quantitative proteomics of male and female mouse retina and RPE (10 mice of each sex for each tissue type). Differentially expressed proteins were subjected to Gene Ontology (GO) analysis and Ingenuity Pathway Analysis (IPA)., Results: Differential expression analysis identified 21 differentially expressed proteins in the retina and 58 differentially expressed proteins in the RPE. Ingenuity pathway analysis identified the top canonical pathways differentially activated in the retina to be calcium transport I, nucleotide excision repair, molecular transport and cell death and survival. In the RPE, the top canonical pathways were calcium signaling, dilated cardiomyopathy signaling, actin cytoskeletal signaling and cellular assembly and organization., Conclusions: These results provide insights into sex differences in the retina and RPE proteome of mice and begin to shed clues into the sexual dimorphism seen in retinal diseases., (© 2024. The Author(s).)

The family of pro-inflammatory and pro-angiogenic chemokines including Interleukin-8 (IL-8, aka CXCL8) and its homologues (CXCL1,2,3,5,6, and 7) exhibit promiscuous binding and activation of several G-protein-coupled receptors (i.e., CXCR2, CXCR1, and the atypical chemokine receptor (ACKR1)). A high proportion of their biological activity is attributed to CXCR2 activation, thus many CXCR2 inhibitors are in clinical trials for several chronic diseases. However, CXCR2 inhibition is often only investigated acutely in these trials or in Cxcr2 -/- mice grown in gnotobiotic conditions. Since humans do not live in germ-free environments, our first goal is to highlight novel retinal and systemic observations in Cxcr2 -/- mice grown in non-gnotobiotic conditions that suggest potential harmful consequences of long-term CXCR2 deficiency or blockade. Beyond confirmation of circulating blood/immune cell-related phenotypes, we report novel findings in Cxcr2 -/- mice including: (1) delayed dye transit to the retinal vasculature, (2) alterations in the density and distribution of retinal vessels, astrocytes and microglia, (3) decreased electroretinogram a- and b-wave amplitudes, (4) reduced visual acuity, and (5) increased polymorphonuclear cell accumulation in vascular lumina abutting venular walls in the retina and in vital non-ocular tissues (lung and liver). Furthermore, PheWAS of CXCR2 CXCR1, and ACKR1 gene variants using data from UK Biobank participants suggest clinical associations with both retinal and vascular disease phenotypes. We conclude that chronic CXCR2 deficiency in mice contributes to functional damage to the retina and that the long-term safety of CXCR1/2 inhibitors designed for chronic use in humans should be explored before clinical adoption to safeguard sight and overall vascular health., (© 2024. The Author(s).)

Metabolic disorder significantly contributes to diabetic vascular complications, including diabetic retinopathy, the leading cause of blindness in the working-age population. However, the molecular mechanisms by which disturbed metabolic homeostasis causes vascular dysfunction in diabetic retinopathy remain unclear. O-GlcNAcylation modification acts as a nutrient sensor particularly sensitive to ambient glucose. Here, we observe pronounced O-GlcNAc elevation in retina endothelial cells of diabetic retinopathy patients and mouse models. Endothelial-specific depletion or pharmacological inhibition of O-GlcNAc transferase effectively mitigates vascular dysfunction. Mechanistically, we find that Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, are O-GlcNAcylated in diabetic retinopathy. We identify threonine 383 as an O-GlcNAc site on YAP, which inhibits its phosphorylation at serine 397, leading to its stabilization and activation, thereby promoting vascular dysfunction by inducing a pro-angiogenic and glucose metabolic transcriptional program. This work emphasizes the critical role of the O-GlcNAc-Hippo axis in the pathogenesis of diabetic retinopathy and suggests its potential as a therapeutic target., (© 2024. The Author(s).)

Age-related macular degeneration (AMD) is marked by a progressive loss of central vision and is the third leading cause of irreversible blindness worldwide. The exact mechanisms driving the progression of this macular degenerative condition remain elusive, and as of now, there are no available preventative measures for dry AMD. According to ancient records, ginseng affects the eyes by brightening them and enhancing wisdom. Modern pharmacological research shows that the active ingredients in ginseng, ginsenosides, may be used to prevent or improve eye diseases that threaten vision. Some articles have reported that ginsenoside Rg3 can treat diabetic retinopathy in mice, but no reports exist on its effects and mechanisms in AMD. Therefore, the role and mechanism of ginsenoside Rg3 in AMD warrant further study. This study aims to investigate the effects of Rg3 on AMD and its underlying molecular mechanisms. We established a mouse model of AMD to examine the impact of ginsenoside Rg3 on NaIO 3 -induced apoptosis in the retina and to explore the related intrinsic mechanisms. The in vivo results indicated that ginsenoside Rg3 prevents NaIO 3 -induced apoptosis in retinal pigment epithelial cells by inhibiting reactive oxygen species production and preventing the reduction in mitochondrial membrane potential. Additionally, we assessed the levels of protein expression within the apoptosis pathway. Ginsenoside Rg3 decreased the expression of Bax, cleaved caspase-3, and cleaved caspase-9 proteins. Additionally, it increased the expression of Bcl-2 by decreasing P-JNK levels. Moreover, our in vivo results showed that ginsenoside Rg3 enhanced retinal structure, increased the relative thickness of the retina, and decreased the extent of disorganization in both the inner and outer nuclear layers. Ginsenoside Rg3 may safeguard the retina against NaIO 3 -induced cell apoptosis by attenuating reactive-oxygen-species-mediated mitochondrial dysfunction, in which the JNK signaling pathway is also involved. These findings suggest that ginsenoside Rg3 has the potential to prevent or attenuate the progression of AMD and other retinal pathologies associated with NaIO 3 -mediated apoptosis.

How tissue-specific progenitor cells generate adult tissues is a puzzle in organogenesis. Using single-cell RNA sequencing of control and Six3 and Six6 compound-mutant mouse embryonic eyecups, we demonstrated that these two closely related transcription factors jointly control diverse target genes in multiple cell populations over the developmental trajectories of mouse embryonic retinal progenitor cells. In the Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) graph of control retinas, naïve retinal progenitor cells had two major trajectories leading to ciliary margin cells and retinal neurons, respectively. The ciliary margin trajectory was from naïve retinal progenitor cells in the G1 phase directly to ciliary margin cells, whereas the neuronal trajectory went through an intermediate neurogenic state marked by Atoh7 expression. Neurogenic retinal progenitor cells (Atoh7+) were still proliferative; early retinal neurons branched out from Atoh7+ retina progenitor cells in the G1 phase. Upon Six3 and Six6 dual deficiency, both naïve and neurogenic retinal progenitor cells were defective, ciliary margin differentiation was enhanced, and multi-lineage neuronal differentiation was disrupted. An ectopic neuronal trajectory lacking the Atoh7+ state led to ectopic neurons. Additionally, Wnt signaling was upregulated, whereas FGF signaling was downregulated. Notably, Six3 and Six6 proteins occupied the loci of diverse genes that were differentially expressed in distinct cell populations, and expression of these genes was significantly altered upon Six3 and Six6 dual deficiency. Our findings provide deeper insight into the molecular mechanisms underlying early retinal differentiation in mammals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ferrena et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Perivascular mural cells including vascular smooth cells (VSMCs) and pericytes are integral components of the vascular system. In the central nervous system (CNS), pericytes are also indispensable for the blood-brain barrier (BBB), blood-spinal cord barrier, and blood-retinal barrier and play key roles in maintaining cerebrovascular and neuronal functions. However, the functional specifications of pericytes between CNS and peripheral organs have not been resolved at the genetic and molecular levels. Hence, the generation of reliable CNS pericyte-specific models and genetic tools remains very challenging. Here, we report a new CNS pericyte marker in mice. This putative cation-transporting ATPase 13A5 ( Atp13a5 ) marker was identified through single-cell transcriptomics, based on its specificity to brain pericytes. We further generated a knock-in model with both tdTomato reporter and Cre recombinase. Using this model to trace the distribution of Atp13a5- positive pericytes in mice, we found that the tdTomato reporter reliably labels the CNS pericytes, including the ones in spinal cord and retina but not peripheral organs. Interestingly, brain pericytes are likely shaped by the developing neural environment, as Atp13a5- positive pericytes start to appear around murine embryonic day 15 (E15) and expand along the cerebrovasculature. Thus, Atp13a5 is a specific marker of CNS pericyte lineage, and this Atp13a5- based model is a reliable tool to explore the heterogeneity of pericytes and BBB functions in health and diseases., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Guo et al.)

Neovascular age-related macular degeneration (nAMD), a leading cause of blindness in older adults, presents a challenging pathophysiology involving choroidal neovascularization (CNV) and retinal degeneration. Current treatments relying on intravitreal (IVT) administration of anti-angiogenic agents are costly and of moderate effectiveness. Metformin, the common anti-diabetic drug, has been associated with decreased odds of developing AMD. Studies have shown that metformin can mitigate cellular aging, neoangiogenesis, and inflammation across multiple diseases. This preclinical study assessed metformin's impact on vessel growth using choroidal explants before exploring IVT metformin's effects on laser-induced CNV and light-induced retinal degeneration in C57BL/6J and BALB/cJ mice, respectively. Metformin reduced new vessel growth in choroidal explants in a dose-dependent relationship. Following laser induction, IVT metformin suppressed CNV and decreased peripheral infiltration of IBA1 + macrophages/microglia. Furthermore, IVT metformin protected against retinal thinning in response to light-induced degeneration. IVT metformin downregulated genes in the choroid and retinal pigment epithelium which are associated with angiogenesis and inflammation, two key processes that drive nAMD progression. These findings underscore metformin's capacity as an anti-angiogenic and neuroprotective agent, demonstrating this drug's potential as an accessible option to help manage nAMD.

Neuromyelitis optica spectrum disorder (NMOSD) is associated with pathological aquaporin-4 immunoglobulin G (AQP4-IgG), which cause brain damage. However, the impact of AQP4-IgG on retinal tissue remains unclear. Additionally, dysregulated complement anaphylatoxins C3a and C5a, known to modulate the endothelial barrier, are implicated in NMOSD. This study evaluates the susceptibility of human brain microvascular endothelial cells (HBMEC) and human retinal endothelial cells (HREC) to C3a- and C5a-mediated stress using real-time cell barrier analysis, immunocytochemical staining, qPCR and IgG transmigration assays. The findings reveal that C3a induced a concentration-dependent paracellular barrier breakdown and increased transcellular permeability in HBMEC, while HREC maintained barrier integrity under the same conditions. C5a attenuated C3a-induced disruption in HBMEC, indicating a protective role. Anaphylatoxin treatment elevated transcript levels of complement component C3 and increased C5 gene and protein expression in HREC, with no changes observed in HBMEC. In HBMEC, C5a treatment led to a transient upregulation of C3a receptor ( C3AR ) mRNA and an early decrease in C5a receptor 1 ( C5AR1 ) protein detection. Conversely, HREC exhibited a late increase in C5aR1 protein levels. These results indicate that the retinal endothelial barrier is more stable under anaphylatoxin-induced stress compared to the brain, potentially offering better protection against paracellular AQP4-IgG transport.

The development of fetal organs can be impacted by systemic changes in maternal circulation, with the placenta playing a pivotal role in maintaining pregnancy homeostasis and nutrient exchange. In clinical obstetrics, oxytocin (OXT) is commonly used to induce labor. To explore the potential role of OXT in the placental homeostasis of OXT, we compared OXT levels in neonatal cord blood among neonates (23-42 weeks gestation) whose mothers either received prenatal OXT or experienced spontaneous labor. Our previous research revealed that the oxytocin receptor (OXTR), essential in forming the blood-retina barrier, is expressed in the retinal pigment epithelium (RPE). We hypothesized that perinatal OXT administration might influence the development of the neural retina and its vasculature, offering therapeutic potential for retinal diseases such as retinopathy of prematurity (ROP). Plasma OXT levels were measured using a commercial OXT ELISA kit. Human fetal RPE (hfRPE) cells treated with OXT (10 µM) were assessed for gene expression via RNA sequencing, revealing 14 downregulated and 32 upregulated genes. To validate these differentially expressed genes (DEGs), hfRPE cells were exposed to OXT (0.01, 0.1, 1, or 10 µM) for 12 h, followed by RNA analysis via real-time PCR. Functional, enrichment, and network analyses (Gene Ontology term, FunRich, Cytoscape) were performed to predict the affected pathways. This translational study suggests that OXT likely crosses the placenta, altering fetal OXT concentrations. RNA sequencing identified 46 DEGs involved in vital metabolic and signaling pathways and critical cellular components. Our results indicate that the perinatal administration of OXT may affect neural retina and retinal vessel development, making OXT a potential therapeutic option for developmental eye diseases, including ROP.

To meet the prodigious bioenergetic demands of the photoreceptors, glucose and other nutrients must traverse the retinal pigment epithelium (RPE), a polarised monolayer of cells that lie at the interface between the outer retina and the choroid, the principal vascular layer of the eye. Recent investigations have revealed a metabolic ecosystem in the outer retina where the photoreceptors and RPE engage in a complex exchange of sugars, amino acids, and other metabolites. Perturbation of this delicate metabolic balance has been identified in the aging retina, as well as in age-related macular degeneration (AMD), the leading cause of blindness in the Western world. Also common in the aging and diseased retina are elevated levels of cytokines, oxidative stress, advanced glycation end-products, increased growth factor signalling, and biomechanical stress - all of which have been associated with metabolic dysregulation in non-retinal cell types and tissues. Herein, we outline the role of these factors in retinal homeostasis, aging, and disease. We discuss their effects on glucose, mitochondrial, lipid, and amino acid metabolism in tissues and cell types outside the retina, highlighting the signalling pathways through which they induce these changes. Lastly, we discuss promising avenues for future research investigating the roles of these pathological conditions on retinal metabolism, potentially offering novel therapeutic approaches to combat age-related retinal disease., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Context: Oxidative stress is an important factor for vitreomacular interface disease development in a theoretical model., Purpose: The aim of the study was to evaluate the correlation between oxidative stress in the human epiretinal membrane (ERM) and retinal morphological changes., Material and Methods: The study included patients scheduled for vitrectomy with epiretinal membrane removal. LogMAR best corrected visual acuity was assessed and optical coherence tomography was performed. Patients were divided into three groups: Type 1 - epiretinal membrane with premacular fibrosis; type 2 - epiretinal membrane with co-existing layer hole; and type 3 - ERM with co-existing full-thickness macular hole. During vitrectomy, epiretinal membranes were collected. Total oxidant status was determined by an automated colorimetric method in homogenates of epiretinal membrane., Statistical Analysis: The Mann-Whitney U test, Kruskal-Wallis test and Spearman linear correlation analysis were used. Statistical significance was set with a level of α = 0.05., Results: Twenty-one Caucasian women (60%) and 14 men (40%) were included in the study. The average age of participants was 74.7 years (95% CI: 71.13-75.45). The mean best corrected visual acuity LogMAR value in the group was 0.8 (95% CI: 0.9-0.7). The mean ratio of total oxidant status to protein level in the collected samples was 0.161 (95% CI: 0.08-0.23) µmol/mg of protein. No correlation was found between total oxidant status and the degree of morphological retinal changes., Conclusion: The study found no significant correlation between the level of oxidative stress in epiretinal membrane and retinal morphological changes., 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 © 2024 Wilczyński, Zalejska-Fiolka, Sapeta-Wieckowska, Sarnat-Kucharczyk and Rokicki.)

Calcium is involved in vision processes in the retina and implicated in various pathologies, including glaucoma. Rod cells rely on store-operated calcium entry (SOCE) to safeguard against the prolonged lowering of intracellular calcium ion concentrations. Zebrafish that lacked the endoplasmic reticulum Ca 2+ sensor Stim2 (stim2 knockout [KO]) exhibited impaired vision and lower light perception-related gene expression. We sought to understand mechanisms that are responsible for vision impairment in stim2 KO zebrafish. The single-cell RNA (scRNA) sequencing of neuronal cells from brains of 5 days postfertilization larvae distinguished 27 cell clusters, 10 of which exhibited distinct gene expression patterns, including amacrine and γ-aminobutyric acid (GABA)ergic retinal interneurons and GABAergic optic tectum cells. Five clusters exhibited significant changes in cell proportions between stim2 KO and controls, including GABAergic diencephalon and optic tectum cells. Transmission electron microscopy of stim2 KO zebrafish revealed decreases in width of the inner plexiform layer, ganglion cells, and their dendrites numbers (a hallmark of glaucoma). GABAergic neuron densities in the inner nuclear layer, including amacrine cells, as well as photoreceptors significantly decreased in stim2 KO zebrafish. Our study suggests a novel role for Stim2 in the regulation of neuronal insulin expression and GABAergic-dependent vision causing glaucoma-like retinal pathology., (© 2024. The Author(s).)

Introduction: Diabetic retinopathy (DR) is a microvascular complication of diabetes in which neurodegeneration has been recently identified as a driving force. In the last years, mesenchymal stromal cells (MSCs) and neurotrophins like Nerve Growth Factor (NGF), have garnered significant attention as innovative therapeutic approaches targeting DR-associated neurodegeneration. However, delivering neurotrophic factors directly in the eye remains a challenge. Hence, this study evaluated the effects of MSCs from human amniotic fluids (hAFSCs) and recombinant human NGF (rhNGF) delivered by human corneal lenticule (hCL) on a high glucose (HG) induced ex vivo model simulating the molecular mechanisms driving DR., Methods: Porcine neuroretinal explants exposed to HG (25 mM for four days) were used to mimic DR ex vivo . hCLs collected from donors undergoing refractive surgery were decellularized using 0.1% sodium dodecyl sulfate and then bioengineered with hAFSCs, microparticles loaded with rhNGF (rhNGF-PLGA-MPs), or both simultaneously. Immunofluorescence (IF) and scanning electron microscopy (SEM) analyses were performed to confirm the hCLs bioengineering process. To assess the effects of hAFSCs and rhNGF, bioengineered hCLs were co-cultured with HG-treated neuroretinal explants and following four days RT-PCR and cytokine array experiments for inflammatory, oxidative, apoptotic, angiogenic and retinal cells markers were performed., Results: Data revealed that HG-treated neuroretinal explants exhibit a characteristic DR-phenotype, including increased level of NF-kB, NOS2, NRF2 GFAP, VEGFA, Bax/Bcl2 ratio and decreased expression of TUBB3 and Rho. Then, the feasibility to bioengineer decellularized hCLs with hAFSCs and rhNGF was demonstrated. Interestingly, co-culturing hAFSCs- and rhNGF- bioengineered hCLs with HG-treated neuroretinal explants for four days significantly reduced the expression of inflammatory, oxidative, apoptotic, angiogenic and increased retinal markers., Conclusion: Overall, we found for the first time that hAFSCs and rhNGF were able to modulate the molecular mechanisms involved in DR and that bioengineered hCLs represents a promising ocular drug delivery system of hAFSCs and rhNGF for eye diseases treatment. In addition, results demonstrated that porcine neuroretinal explants treated with HG is a useful model to reproduce ex vivo the DR pathophysiology., Competing Interests: The authors ND, GC, MA, and TR are employed by Dompé Farmaceutici SpA. ND, MA, MN, AP, and DM are authors of European Patent n° 20179055.7-1109, 09/06/2020, “New Drug Delivery System for Ophtalmic Use”. The remaining 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Pelusi, Hurst, Detta, Pipino, Lamolinara, Conte, Mastropasqua, Allegretti, Di Pietrantonio, Romeo, El Zarif, Nubile, Guerricchio, Bollini, Pandolfi, Schnichels and Mandatori.)

Inherited retinal diseases (IRDs) represent a diverse group of genetic disorders leading to progressive degeneration of the retina due to mutations in over 280 genes. This review focuses on the various methodologies for the preclinical characterization and evaluation of adeno-associated virus (AAV)-mediated gene therapy as a potential treatment option for IRDs, particularly focusing on gene therapies targeting mutations, such as those in the RPE65 and FAM161A genes. AAV vectors, such as AAV2 and AAV5, have been utilized to deliver therapeutic genes, showing promise in preserving vision and enhancing photoreceptor function in animal models. Despite their advantages-including high production efficiency, low pathogenicity, and minimal immunogenicity-AAV-mediated therapies face limitations such as immune responses beyond the retina, vector size constraints, and challenges in large-scale manufacturing. This review systematically compares different experimental models used to investigate AAV-mediated therapies, such as mouse models, human retinal explants (HREs), and induced pluripotent stem cell (iPSC)-derived retinal organoids. Mouse models are advantageous for genetic manipulation and detailed investigations of disease mechanisms; however, anatomical differences between mice and humans may limit the translational applicability of results. HREs offer valuable insights into human retinal pathophysiology but face challenges such as tissue degradation and lack of systemic physiological effects. Retinal organoids, on the other hand, provide a robust platform that closely mimics human retinal development, thereby enabling more comprehensive studies on disease mechanisms and therapeutic strategies, including AAV-based interventions. Specific outcomes targeted in these studies include vision preservation and functional improvements of retinas damaged by genetic mutations. This review highlights the strengths and weaknesses of each experimental model and advocates for their combined use in developing targeted gene therapies for IRDs. As research advances, optimizing AAV vector design and delivery methods will be critical for enhancing therapeutic efficacy and improving clinical outcomes for patients with IRDs.

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1 -/- knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1 -/- mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod-specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Lenadogene nolparvovec is a gene therapy which has been developed to treat Leber hereditary optic neuropathy (LHON) caused by a point mutation in the mitochondrial NADH dehydrogenase 4 (ND4) gene. Clinical trials have demonstrated a significant improvement of visual acuity up to 5 years after treatment by lenadogene nolparvovec but, surprisingly, unilateral treatment resulted in bilateral improvement of vision. This contralateral effect - similarly observed with other gene therapy products in development for MT-ND4-LHON - is supported by the migration of viral vector genomes and their transcripts to the contralateral eye, as reported in animals, and post-mortem samples from two patients. In this study, we used an AAV2 encoding fluorescent proteins targeting mitochondria to investigate whether these organelles themselves could transfer from the treated eye to the fellow one. We found that mitochondria travel along the visual system (optic chiasm and primary visual cortex) and reach the contralateral eye (optic nerve and retina) in physiological conditions. We also observed that, in a rotenone-induced model of retinal damage mimicking LHON, mitochondrial transfer from the healthy to the damaged eye was accelerated and enhanced. Our results thus provide a further explanation for the contralateral beneficial effect observed during clinical studies with lenadogene nolparvovec., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Microphthalmia, mostly an autosomal dominant disorder, is a worldwide severe congenital ocular malformation that causes visual impairment. Our investigation unveiled a total of 30 genes associated with microphthalmia. Employing the CytoHubba and PPI network, we identified Bmp4 as the most pivotal hub gene. Subsequently, the conditional overexpression of Bmp4 in the retina caused highly distinctive microphthalmia, manifested by retinal disorganization with ganglion cell misalignment. Significant reduction in the number and abnormal distribution location of retinal cells in microphthalmia model mice. Elevated Bmp4 was associated with an increase in retinal apoptosis and a decrease in proliferating cells, which exacerbates the development of microphthalmia. Here we identify Bmp4 as an extremely important gene responsible for microphthalmia and the involved mechanisms. Overexpression of Bmp4 induces retinal cell ectopic expression and developmental defects, highlighting the importance of a well-balanced Bmp4 level in shaping the embryonic retina during early development., Competing Interests: Declaration of competing interest The author declares 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 © 2024. Published by Elsevier Inc.)

Aim: The aim of this study was to evaluate the potential protective effect of Qiju Granule in a rat model of age-related macular degeneration (AMD) and investigate the underlying mechanisms involved., Methods: Rats were injected intravenously with 40 mg/kg of sodium iodate (SI) to induce a dry AMD model. The rats in the treatment group received three different doses of Qiju Granule once a day via gavage, while the rats in the control group were given an equal volume of physiological saline. On day 14 and day 28 following the intervention, various methods were employed to evaluate retinal function and structure, including electroretinography (ERG), optical coherence tomography (OCT), and histological examination. The expression of glial fibrillary acidic protein (GFAP), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) was assessed via immunofluorescence. Beyond immunofluorescence, the mRNA levels of bFGF, BDNF, and CNTF were quantitatively determined using real-time polymerase chain reaction (qRT-PCR)., Results: Rats treated with Qiju Granule exhibited significant improvements in both retinal function and structure compared to the model group. The most noteworthy effects were observed at a high dose of Qiju Granule. Furthermore, the expression levels of bFGF, BDNF, and CNTF were significantly unregulated in the treated groups compared to the model group., Conclusions: Qiju Granule demonstrated a protective effect on the retina in the SI-induced rat model of AMD. The protective mechanism may be attributed to the upregulation of retinal neurotrophic factors expression., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests related to this study. There are no financial or personal relationships with other people or organizations that could influence the conduct or interpretation of the research., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: To investigate the therapeutic potential of visual stimulation (VS) and BDNF in murine experimental autoimmune uveoretinitis (EAU)., Main Methods: Mice were immunized by subcutaneous injection of interphotoreceptor retinoid-binding protein in Freund's complete adjuvant and intravenous injection of pertussis toxin, and were then exposed to high-contrast VS 12 h/day (days 1-14 post-immunization). EAU severity was assessed by examining clinical score, visual acuity, inflammatory markers, and immune cells in the retina. The transcriptome of activated retinal cells was determined by RNA-seq using RNA immunoprecipitated in complex with phosphorylated ribosomal protein S6. The retinal levels of protein products of relevant upregulated genes were quantified. The effect of BDNF on EAU was tested in unstimulated mice by its daily topical ocular administration (days 8-14 post-immunization)., Key Findings: VS attenuated EAU development and decreased the expression of pro-inflammatory cytokines/chemokines and numbers of immune cells in the retina (n = 10-20 eyes/group for each analysis). In activated retinal cells of control mice (n = 30 eyes/group), VS upregulated genes encoding immunomodulatory neuropeptides, of which BDNF and vasoactive intestinal peptide (VIP) also showed increased mRNA and protein levels in the retina of VS-treated EAU mice (n = 6-10 eyes/group for each analysis). In unstimulated EAU mice, BDNF treatment mimicked the protective effects of VS by modulating the inflammatory and stem cell properties of Müller cells (n = 5 eyes/group for each analysis)., Significance: VS effectively suppresses EAU, at least through enhancing retinal levels of anti-inflammatory and neuroprotective factors, VIP and BDNF. Our findings also suggest BDNF as a promising therapeutic agent for uveitis treatment., 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 Inc. All rights reserved.)

The prevalence of myopia is rapidly increasing, significantly impacting the quality of life of affected individuals. Prior research by our group revealed reactive gliosis in Müller cells within myopic retina, prompting further investigation of their role in myopia, which remains unclear. In this study, we analyzed protein expression changes in CD29+ Müller cells isolated from a form deprivation-induced rabbit model of myopia using magnetic activated cell sorting to investigate the role of these cells in myopia. As the principal glial cells in the retina, Müller cells exhibited significant alterations in the components of metabolic pathways, particularly glycolysis and angiogenesis, including the upregulation of glycolytic enzymes, such as lactate dehydrogenase A and pyruvate kinase, implicated in the adaptation to increased metabolic demands under myopic stress. Additionally, a decrease in the expression of proteins associated with oxygen transport suggested enhanced vulnerability to oxidative stress. These findings highlight the proactive role of CD29+ Müller cells in modifying the retinal environment in response to myopic stress and provide valuable insights into mechanisms that could help mitigate myopia progression., (© 2024. The Author(s).)

Background: miR-210 is one of the most evolutionarily conserved microRNAs. It is known to be involved in several physiological and pathological processes, including response to hypoxia, angiogenesis, cardiovascular diseases and cancer. Recently, new roles of this microRNA are emerging in the context of eye and visual system homeostasis. Recent studies in Drosophila melanogaster unveiled that the absence of miR-210 leads to a progressive retinal degeneration characterized by the accumulation of lipid droplets and disruptions in lipid metabolism. However, the possible conservation of miR-210 knock-out effect in the mammalian retina has yet to be explored., Results: We further investigated lipid anabolism and catabolism in miR-210 knock-out (KO) flies, uncovering significant alterations in gene expression within these pathways. Additionally, we characterized the retinal morphology of flies overexpressing (OE) miR-210, which was not affected by the increased levels of the microRNA. For the first time, we also characterized the retinal morphology of miR-210 KO and OE mice. Similar to flies, miR-210 OE did not affect retinal homeostasis, whereas miR-210 KO mice exhibited photoreceptor degeneration. To explore other potential parallels between miR-210 KO models in flies and mice, we examined lipid metabolism, circadian behaviour, and retinal transcriptome in mice, but found no similarities. Specifically, RNA-seq confirmed the lack of involvement of lipid metabolism in the mice's pathological phenotype, revealing that the differentially expressed genes were predominantly associated with chloride channel activity and extracellular matrix homeostasis. Simultaneously, transcriptome analysis of miR-210 KO fly brains indicated that the observed alterations extend beyond the eye and may be linked to neuronal deficiencies in signal detection and transduction., Conclusions: We provide the first morphological characterization of the retina of miR-210 KO and OE mice, investigating the role of this microRNA in mammalian retinal physiology and exploring potential parallels with phenotypes observed in fly models. Although the lack of similarities in lipid metabolism, circadian behaviour, and retinal transcriptome in mice suggests divergent mechanisms of retinal degeneration between the two species, transcriptome analysis of miR-210 KO fly brains indicates the potential existence of a shared upstream mechanism contributing to retinal degeneration in both flies and mammals., (© 2024. The Author(s).)

The retina plays a crucial role in processing and decoding visual information, both in normal development and during myopia progression. Recent advancements have introduced a library-independent approach for data-independent acquisition (DIA) analyses. This study demonstrates deep proteome identification and quantification in individual mice retinas during myopia development, with an average of 6,263 ± 86 unique protein groups. We anticipate that the use of a predicted retinal-specific spectral library combined with the robust quantification achieved within this dataset will contribute to a better understanding of the proteome complexity. Furthermore, a comprehensive mice retinal-specific spectral library was generated, encompassing a total identification of 9,401 protein groups, 70,041 peptides, 95,339 precursors, and 761,868 transitions acquired using SWATH-MS acquisition on a ZenoTOF 7600 mass spectrometer. This dataset surpasses the spectral library generated through high-pH reversed-phase fractionation by data-dependent acquisition (DDA). The data is available via ProteomeXchange with the identifier PXD046983. It will also serve as an indispensable reference for investigations in myopia research and other retinal or neurological diseases., (© 2024. The Author(s).)

BMI1, also known as B lymphoma Mo-MLV insertion region 1, is a protein in the Polycomb group that is implicated in various cellular processes, including stem cell self-renewal and the regulation of cellular senescence. BMI1 plays a role in the regulation of retinal progenitor cells and the renewal of adult neuronal cells. However, the presence, location, and quantification of BMI1 in the adult human eye have never previously been reported. In this study, we collected 45 frozen globes from eye banks, and ocular tissues were dissected. Protein was quantified by utilizing a custom electrochemiluminescence (ECL) assay developed to quantify the BMI1 protein. BMI1 was found in all ocular tissues at the following levels: the retina (1483.6 ± 191.7 pg/mL) and the RPE (296.4 ± 78.1 pg/mL). BMI1 expression was noted ubiquitously in the GCL (ganglion cell layer), the INL (inner nuclear layer), the ONL (outer nuclear layer), and the RPE (retinal pigment epithelium) via immunofluorescence, with higher levels in the inner than in the outer retinal layers and the RPE. These data confirm that BMI1 is expressed in the human retina. Further studies will illuminate the role that BMI1 plays in ocular cells. BMI1 levels are lower in aged retinas, possibly reflecting changes in retinal somatic and stem cell maintenance and disease susceptibility.