Melatonin prevents EAAC1 deletion-induced retinal ganglion cell degeneration by inhibiting apoptosis and senescence.

Normal tension glaucoma (NTG) is referred to as a progressive degenerative disorder of the retinal ganglion cells (RGCs), resulting in nonreversible visual defects, despite intraocular pressure levels within the statistically normal range. Current therapeutic strategies for NTG yield limited benefits. Excitatory amino acid carrier 1 (EAAC1) knockout (EAAC1-/- ) in mice has been shown to induce RGC degeneration without elevating intraocular pressure, mimicking pathological characteristics of NTG. In this study, we explored whether daily oral administration of melatonin could block RGCs loss and prevent retinal morphology and function defects associated with EAAC1 deletion. We also explored the molecular mechanisms underlying EAAC1 deletion-induced RGC degeneration and the neuroprotective effects of melatonin. Our RNA sequencing and in vivo data indicated EAAC1 deletion caused elevated oxidative stress, activation of apoptosis and cellular senescence pathways, and neuroinflammation in RGCs. However, melatonin administration efficiently prevented these detrimental effects. Furthermore, we investigated the potential role of apoptosis- and senescence-related redox-sensitive factors in EAAC1 deletion-induced RGCs degeneration and the neuroprotective effects of melatonin administration. We observed remarkable upregulation of p53, whereas NRF2 and Sirt1 expression were significantly decreased in EAAC1-/- mice, which were prevented by melatonin treatment, suggesting that melatonin exerted its neuroprotective effects possibly through modulating NRF2/p53/Sirt1 redox-sensitive signaling pathways. Overall, our study provided a solid foundation for the application of melatonin in the management of NTG.

PHD2 attenuates high-glucose-induced blood retinal barrier breakdown in human retinal microvascular endothelial cells by regulating the Hif-1α/VEGF pathway.

OBJECTIVE: Diabetic macular edema (DME) is one of the most frequent causes of severe vision loss. The pathogenesis of DME is still not fully understood; however, it is hypothesized to result from breakdown of the blood-retinal barrier (BRB) due to retinal inflammation by vascular endothelial growth factor (VEGF) secretion under hyperglycemic conditions. In this investigation, we discovered that Prolyl-4-hydroxylase 2 (PHD2), an upstream regulator of hypoxia-inducible factor 1 (HIF-1) modulates VEGF expression and thus preserves BRB function in the mouse retina. MATERIALS AND METHODS: Primary human retinal microvascular endothelial cells (hRMECs) were cultured in human endothelial serum-free growth medium and exposed to hyperglycemia. Changes in cell viability were investigated by an MTT assay. BRB function in each group was revealed by a paracellular permeability assay and trans-endothelial electrical resistance (TEER). Morphological changes in the BRB were investigated by immunofluorescence staining of occludin and zonula occludens-1 (ZO-1). The mRNA and protein levels of the tight junction proteins, PHD2, HIF-1α, and VEGF were measured by reverse transcription-quantitative PCR (RT-qPCR), western blot analysis and ELISA. RESULTS: Under hyperglycemic conditions, the viability of hRMECs was decreased, and PHD2 expression was downregulated, accompanied by increased paracellular permeability and decreased trans-endothelial electrical resistance. Additionally, HIF-1α and VEGF expression levels were increased, whereas the expression levels of tight junction proteins, including occludin and ZO-1, were decreased and BRB function was compromised. The PHD2 activator R59949 (diacylglycerol kinase inhibitor II), altered these pathological changes, and the PHD2 inhibitor dimethyloxalylglycine (DMOG) resulted in the opposite effects. CONCLUSION: These results demonstrated that PHD2 inhibited HIF-1 activity by inhibiting HIF-1α expression in hRMECs under hyperglycemic conditions, which led to the downregulation of the expression of the angiogenic factor VEGF, and thus helped to maintain the functions of hRMECs. Therefore, it is reasonable to propose that PHD2 could be a potential novel target for the treatment of DME or other diseases with a similar pathogenesis.

Melatonin prevents experimental central serous chorioretinopathy in rats.

Central serous chorioretinopathy (CSC) is a vision-threatening disease with no validated treatment and unclear pathogenesis. It is characterized by dilation and leakage of choroidal vasculature, resulting in the accumulation of subretinal fluid, and serous detachment of the neurosensory retina. Numerous studies have demonstrated that melatonin had multiple protective effects against endothelial dysfunction, vascular inflammation, and blood-retinal barrier (BRB) breakdown. However, the effect of melatonin on CSC, and its exact pathogenesis, is not well understood thus far. In this study, an experimental model was established by intravitreal injection of aldosterone in rats, which mimicked the features of CSC. Our results found that melatonin administration in advance significantly inhibited aldosterone-induced choroidal thickening and vasodilation by reducing the expression of calcium-activated potassium channel KCa2.3, and attenuated tortuosity of choroid vessels. Moreover, melatonin protected the BRB integrity and prevented the decrease in tight junction protein (ZO-1, occludin, and claudin-1) levels in the rat model induced by aldosterone. Additionally, the data also showed that intraperitoneal injection of melatonin in advance inhibited aldosterone-induced macrophage/microglia infiltration, and remarkably diminished the levels of inflammatory cytokines (interleukin-6 [IL-6], IL-1ß, and cyclooxygenase-2), chemokines (chemokine C-C motif ligand 3, and C-X-C motif ligand 1), and matrix metalloproteinases (MMP-2 and MMP-9). Luzindole, as the nonselective MT1 and MT2 antagonist, and 4-phenyl-2-propionamidotetraline, as the selective MT2 antagonist, neutralized the melatonin-induced inhibition of choroidal thickening and choroidal vasodilation, indicating that melatonin might exert the effects via binding to its receptors. Furthermore, the IL-17A/nuclear factor-κB signaling pathway was activated by intravitreal administration of aldosterone, while it was suppressed in melatonin-treated in advance rat eyes. This study indicates that melatonin could serve as a promising safe therapeutic strategy for CSC patients.

Anti-PANoptosis is involved in neuroprotective effects of melatonin in acute ocular hypertension model.

Acute ocular hypertension (AOH) is the most important characteristic of acute glaucoma, which can lead to retinal ganglion cell (RGC) death and permanent vision loss. So far, approved effective therapy is still lacking in acute glaucoma. PANoptosis (pyroptosis, apoptosis, and necroptosis), which consists of three key modes of programmed cell death-apoptosis, necroptosis, and pyroptosis-may contribute to AOH-induced RGC death. Previous studies have demonstrated that melatonin (N-acetyl-5-methoxytryptamine) exerts a neuroprotective effect in many retinal degenerative diseases. However, whether melatonin is anti-PANoptotic and neuroprotective in the progression of acute glaucoma remains unclear. Thus, this study aimed to explore the role of melatonin in AOH retinas and its underlying mechanisms. The results showed that melatonin treatment attenuated the loss of ganglion cell complex thickness, retinal nerve fiber layer thickness, and RGC after AOH injury, and improved the amplitudes of a-wave, b-wave, and oscillatory potentials in the electroretinogram. Additionally, the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells was decreased, and the upregulation of cleaved caspase-8, cleaved caspase-3, Bax, and Bad and downregulation of Bcl-2 and p-Bad were inhibited after melatonin administration. Meanwhile, both the expression and activation of MLKL, RIP1, and RIP3, along with the number of PI-positive cells, were reduced in melatonin-treated mice, and p-RIP3 was in both RGC and microglia/macrophage after AOH injury. Furthermore, melatonin reduced the expression of NLRP3, ASC, cleaved caspase-1, gasdermin D (GSDMD), and cleaved GSDMD, and decreased the number of Iba1/interleukin-1ß-positive cells. In conclusion, melatonin ameliorated retinal structure, prevented retinal dysfunction after AOH, and exerted a neuroprotective effect via inhibition of PANoptosis in AOH retinas.

The phase changes of M1/M2 phenotype of microglia/macrophage following oxygen-induced retinopathy in mice.

OBJECTIVE: Microglia/macrophage activation is previously reported to be involved in various ocular diseases. However, the separate role of M1/M2 phenotype microglia/macrophage in the pathological process of oxygen-induced retinopathy (OIR) remains unknown. In this research, we explored the role and regulatory mechanism of M1/M2 microglia/macrophage in OIR in C57BL/6J mice. Furthermore, we demonstrated the time phase of M1/M2 shifting of microglia/macrophage during the natural process of OIR, which is very essential for further investigations. MATERIALS AND METHODS: C57BL/6j pups were exposed to hyperoxia environment from postnatal 7(P7) to P12 then returned to normoxia. The mice were then euthanized, and the eyes were harvested at a series of time points for further investigation. The M1/M2 phenotype microglia/macrophage activity was presented by immunofluorescent staining and real-time quantitative polymerase chain reaction (qPCR). The NF-κb-STAT3 signaling and IL-4-STAT6-PPAR-γ signaling pathway activity was examined by western blot analysis. RESULTS: The microglia/macrophage were activated when the OIR model was set up after P12. The M1 microglia/macrophage activation was found in neovascularization (NV) tufts in both central and peripheral retina, which started from P12 when the mice were returned to normoxia environment and peaked at P17. During this period of time, the NF-κb-STAT3 signaling pathway was activated, resulting in the upregulated M1 phenotype microglia/macrophage polarization, along with the enhanced inflammatory cytokine expression including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß. Consequently, the NV tufts were observed from P12 and the volume continued to increase until P17. However, the M2 phenotype microglia/macrophage activity took over during the late phase of OIR started from P17. The IL-4-STAT6-PPAR-γ signaling activity was upregulated from P17 and peaked at P20, inducing M2 phenotype microglia polarization, which consequently led to the inhibition of inflammatory cytokines and spontaneous regression of NV tufts. CONCLUSIONS: Microglia/macrophage participate actively in the natural process of OIR in mice, and two phenotypes exert different functions. Treatment modulating microglia/macrophage polarize toward M2 phenotype might be a novel and promising method for ocular neovascular diseases such as retinopathy of prematurity (ROP), wet age-related macular degeneration (wAMD), and diabetic retinopathy (DR).

Melatonin protects inner retinal neurons of newborn mice after hypoxia-ischemia.

Retinopathy of prematurity is a vision-threatening disease associated with retinal hypoxia-ischemia, leading to the death of retinal neurons and chronic neuronal degeneration. During this study, we used the oxygen-induced retinopathy mice model to mimic retinal hypoxia-ischemia phenotypes to investigate further the neuroprotective effect of melatonin on neonatal retinal neurons. Melatonin helped maintain relatively normal inner retinal architecture and thickness and preserve inner retinal neuron populations in avascular areas by rescuing retinal ganglion and bipolar cells, and horizontal and amacrine neurons, from apoptosis. Meanwhile, melatonin recovered visual dysfunction, as reflected by the improved amplitudes and implicit times of a-wave, b-wave, and oscillatory potentials. Additionally, elevated cleaved caspase-3 and Bax protein levels and reduced Bcl-2 protein levels in response to hypoxia-ischemia were diminished after melatonin treatment. Moreover, melatonin increased BDNF and downstream phospho-TrkB/Akt/ERK/CREB levels. ANA-12, a TrkB receptor antagonist, antagonized these melatonin actions and reduced melatonin-induced neuroprotection. Furthermore, melatonin rescued the reduction in melatonin receptor expression. This study suggests that melatonin exerted anti-apoptotic and neuroprotective effects in inner retinal neurons after hypoxia-ischemia, at least partly due to modulation of the BDNF-TrkB pathway.

Molecular Engineering on MoS2 Enables Large Interlayers and Unlocked Basal Planes for High-Performance Aqueous Zn-Ion Storage.

Aqueous Zn-storage behaviors of MoS2 -based cathodes mainly rely on the ion-(de)intercalation at edge sites but are limited by the inactive basal plane. Herein, an in-situ molecular engineering strategy in terms of structure defects manufacturing and O-doping is proposed for MoS2 (designated as D-MoS2 -O) to unlock the inert basal plane, expand the interlayer spacing (from 6.2 to 9.6 Å), and produce abundant 1T-phase. The tailored D-MoS2 -O with excellent hydrophilicity and high conductivity allows the 3D Zn2+ transport along both the ab plane and c-axis, thus achieving the exceptional high rate capability. Zn2+ diffusion through the basal plane is verified by DFT computations. As a proof of concept, the wearable quasi-solid-state rechargeable Zn battery employing the D-MoS2 -O cathode operates stably even under severe bending conditions, showing great application prospects. This work opens a new window for designing high-performance layered cathode materials for aqueous Zn-ion batteries.

Andrographolide attenuates choroidal neovascularization by inhibiting the HIF-1α/VEGF signaling pathway.

Choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), leads to severe vision loss amongst the elderly in the developed countries. Currently, the premier treatment for AMD is anti-VEGF therapy, which has limited efficacy, and is still controversial. Previous studies have showed that Andrographolide (Andro) had various biological effects, including anti-angiogenesis, anti-inflammation, and antioxidant. However, the effect of Andro on the formation of CNV has not been studied thus far. Here our results showed that Andro reduced the expression levels of HIF-1α and VEGF in the RF/6A cells chemical hypoxia model and the laser-induced CNV mouse model. Moreover, Andro inhibited the tube formation activity of RF/6A cells under hypoxic conditions. Furthermore, intraperitoneal injection of Andro reduced the severity of choroidal vascular leakage and the size of CNV in the laser-induced CNV mouse model, indicating that Andro attenuated the development of CNV by inhibiting the HIF-1α/VEGF signaling pathway. These results suggest that Andro could be a potential novel therapeutic agent for AMD.

Melatonin attenuates choroidal neovascularization by regulating macrophage/microglia polarization via inhibition of RhoA/ROCK signaling pathway.

Choroidal neovascularization (CNV) is an important characteristic of advanced wet age-related macular degeneration (AMD) and leads to severe visual impairment among elderly patients. Previous studies have demonstrated that melatonin induces several biological effects related to antioxidation, anti-inflammation, and anti-angiogenesis. However, the role of melatonin in CNV, and its underlying mechanisms, has not been investigated thus far. In this study, we found that melatonin administration significantly reduced the scale and volume of CNV lesions, suppressed vascular leakage, and inhibited the capacity of vascular proliferation in the laser-induced mouse CNV model. Additionally, the results also show that the melatonin-treated retinal microglia in the laser-induced mice exhibited enhanced expression of M1-type markers, such as iNOS, CCL-3, CCL-5, and TNF-α, as well as decreased production of M2-type markers, such as Arg-1, Fizz-1, IL-10, YM-1, and CD206, indicating that melatonin switched the macrophage/microglia polarization from pro-angiogenic M2 phenotype to anti-angiogenic M1 phenotype. Furthermore, the RhoA/ROCK signaling pathway was activated during CNV formation, yet was suppressed after an intraperitoneal injection of melatonin. In conclusion, melatonin attenuated CNV, reduced vascular leakage, and inhibited vascular proliferation by switching the macrophage/microglia polarization from M2 phenotype to M1 phenotype via inhibition of RhoA/ROCK signaling pathway in CNV. This suggests that melatonin could be a novel agent for the treatment of AMD.

Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo.

choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), causes severe vision loss among elderly patients. TANK-binding kinase 1 (TBK1) is a ubiquitously expressed serine-threonine kinase and is found to induce endothelial cells proliferation, represent a novel mediator of tumor angiogenesis and exert pro-inflammatory effect. However, the role of TBK1 in choroidal neovascularization has not been investigated so far. In this study, we found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model. Silencing of TBK1 suppressed the proliferation and tube formation activity of RF/6 A cells. Intravitreal injection of anti-TBK1 monoclonal antibody ameliorates CNV formation. Taken together, these findings exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD.

Occurrence and distribution of antibiotic pollution and antibiotic resistance genes in seagrass meadow sediments based on metagenomics.

Seagrass meadows are one of the most important coastal ecosystems that provide essential ecological and economic services. The contamination levels of antibiotic and antibiotic resistance genes (ARGs) in coastal ecosystems are severely elevated owing to anthropogenic disturbances, such as terrestrial input, aquaculture effluent, and sewage discharge. However, few studies have focused on the occurrence and distribution of antibiotics and their corresponding ARGs in this habitat. Thus, we investigated the antibiotic and ARGs profiles, microbial communities, and ARG-carrying host bacteria in typical seagrass meadow sediments collected from Swan Lake, Caofeidian shoal harbor, Qingdao Bay, and Sishili Bay in the Bohai Sea and northern Yellow Sea. The total concentrations of 30 detected antibiotics ranged from 99.35 to 478.02 µg/kg, tetracyclines were more prevalent than other antibiotics. Metagenomic analyses showed that 342 ARG subtypes associated with 22 ARG types were identified in the seagrass meadow sediments. Multidrug resistance genes and RanA were the most dominant ARG types and subtypes, respectively. Co-occurrence network analysis revealed that Halioglobus, Zeaxanthinibacter, and Aureitalea may be potential hosts at the genus level, and the relative abundances of these bacteria were higher in Sishili Bay than those in other areas. This study provided important insights into the pollution status of antibiotics and ARGs in typical seagrass meadow sediments. Effective management should be performed to control the potential ecological health risks in seagrass meadow ecosystems.

An Ultrahigh Modulus Gel Electrolytes Reforming the Growing Pattern of Li Dendrites for Interfacially Stable Lithium-Metal Batteries.

Gel polymer electrolytes (GPEs) have aroused intensive attention for their moderate comprehensive performances in lithium-metal batteries (LMBs). However, GPEs with low elastic moduli of MPa magnitude cannot mechanically regulate the Li deposition, leading to recalcitrant lithium dendrites. Herein, a porous Li7 La3 Zr2 O12 (LLZO) framework (PLF) is employed as an integrated solid filler to address the intrinsic drawback of GPEs. With the incorporation of PLF, the composite GPE exhibits an ultrahigh elastic modulus of GPa magnitude, confronting Li dendrites at a mechanical level and realizing steady polarization at high current densities in Li||Li cells. Benefiting from the compatible interface with anodes, the LFP|PLF@GPE|Li cells deliver excellent rate capability and cycling performance at room temperature. Theoretical models extracted from the topology of solid fillers reveal that the PLF with unique 3D structures can effectively reinforce the gel phase of GPEs at the nanoscale via providing sufficient mechanical support from the load-sensitive direction. Numerical models are further developed to reproduce the multiphysical procedure of dendrite propagation and give insights into predicting the failure modes of LMBs. This work quantitatively clarifies the relationship between the topology of solid fillers and the interface stability of GPEs, providing guidelines for designing mechanically reliable GPEs for LMBs.

Design of Ultrathin Asymmetric Composite Electrolytes for Interfacial Stable Solid-State Lithium-Metal Batteries.

Ultrathin composite electrolytes hold great promise for high energy density solid-state lithium metal batteries (SSLMBs). However, finding an electrolyte that can simultaneously balance the interfacial stability of the lithium anode and high-voltage cathode is challenging. The present study utilized the both-side tape casting technique to fabricate ultrathin asymmetric composite electrolytes reinforced with polyimide (PI) fiber membrane, with a thickness of 26.8 µm. The implementation of this asymmetric structural design enables SSLMBs to attain favorable interfacial characteristics, such as exceptional resistance to lithium dendrite puncture and compatibility with high voltages. The suppression of lithium dendrite growth and the extension of the cycle life of lithium symmetric batteries by 4000 h are both experimental and theoretically demonstrated under the dual confinement of PI fiber membrane and Li7La3Zr2O12 ceramic fibers. Furthermore, the integration of multicomponent solid electrolyte interphase and cathode electrolyte interface interfacial layers into the lithium anode and high-voltage cathode enhance theirs cycling stability. With a gravimetric/volumetric energy density of 333.1 Wh kg-1/713.2 Wh L-1, the assembled LiNi0.8Co0.1Mn0.1O2 pouch cell demonstrates exceptional safety. The extensive application of this design concept to SSLMBs enables the resolution of electrode/electrolyte interface issues.

12.6 µm-Thick Asymmetric Composite Electrolyte with Superior Interfacial Stability for Solid-State Lithium-Metal Batteries.

Solid-state lithium metal batteries (SSLMBs) show great promise in terms of high-energy-density and high-safety performance. However, there is an urgent need to address the compatibility of electrolytes with high-voltage cathodes/Li anodes, and to minimize the electrolyte thickness to achieve high-energy-density of SSLMBs. Herein, we develop an ultrathin (12.6 µm) asymmetric composite solid-state electrolyte with ultralight areal density (1.69 mg cm-2) for SSLMBs. The electrolyte combining a garnet (LLZO) layer and a metal organic framework (MOF) layer, which are fabricated on both sides of the polyethylene (PE) separator separately by tape casting. The PE separator endows the electrolyte with flexibility and excellent mechanical properties. The LLZO layer on the cathode side ensures high chemical stability at high voltage. The MOF layer on the anode side achieves a stable electric field and uniform Li flux, thus promoting uniform Li+ deposition. Thanks to the well-designed structure, the Li symmetric battery exhibits an ultralong cycle life (5000 h), and high-voltage SSLMBs achieve stable cycle performance. The assembled pouch cells provided a gravimetric/volume energy density of 344.0 Wh kg-1/773.1 Wh L-1. This simple operation allows for large-scale preparation, and the design concept of ultrathin asymmetric structure also reveals the future development direction of SSLMBs.

Acrizanib as a Novel Therapeutic Agent for Fundus Neovascularization via Inhibitory Phosphorylation of VEGFR2.

Purpose: The present study aimed to evaluate the effect of acrizanib, a small molecule inhibitor targeting vascular endothelial growth factor receptor 2 (VEGFR2), on physiological angiogenesis and pathological neovascularization in the eye and to explore the underlying molecular mechanisms. Methods: We investigated the potential role of acrizanib in physiological angiogenesis using C57BL/6J newborn mice, and pathological angiogenesis using the mouse oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) models. Moreover, vascular endothelial growth factor (VEGF)-treated human umbilical vein endothelial cells (HUVECs) were used as an in vitro model for studying the molecular mechanism underlying acrizanib's antiangiogenic effects. Results: The intravitreal injection of acrizanib did not show a considerable impact on physiological angiogenesis and retinal thickness, indicating a potentially favorable safety profile. In the mouse models of OIR and CNV, acrizanib showed promising results in reducing pathological neovascularization, inflammation, and vascular leakage, indicating its potential efficacy against pathological angiogenesis. Consistent with in vivo results, acrizanib blunted angiogenic events in VEGF-treated HUVECs such as proliferation, migration, and tube formation. Furthermore, acrizanib inhibited the multisite phosphorylation of VEGFR2 to varying degrees and the activation of its downstream signal pathways in VEGF-treated HUVECs. Conclusions: This study suggested the potential efficacy and safety of acrizanib in suppressing fundus neovascularization. Acrizanib functioned through inhibiting multiple phosphorylation sites of VEGFR2 in endothelial cells to different degrees. Translational Relevance: These results indicated that acrizanib might hold promise as a potential candidate for the treatment of ocular vascular diseases.

Characterization of Vortex Vein Drainage System in Healthy Individuals Imaged by Ultra-Widefield Optical Coherence Tomography Angiography.

Purpose: The purpose of this study was to investigate the choroidal characteristics of vortex vein (VV) drainage systems in healthy individuals using ultra-widefield optical coherence tomography angiography. Methods: The mean choroidal thickness (ChT) and choroidal vascularity index (CVI) of each VV quadrant (24 × 20 mm2 scan mode; superotemporal [ST], superonasal [SN], inferonasal [IN], and inferotemporal [IT] quadrants) were calculated. Furthermore, intervortex venous anastomosis (IVA) was classified into temporal, superior, inferior, and nasal types. Results: A total of 207 healthy eyes were analyzed to find that the ST quadrant had the thickest choroidal layer and highest CVI (all P < 0.05). Among the four VV drainage quadrants, the mean ChT and CVI decreased in the sequence of ST, SN, IT, and IN (all P < 0.05). Moreover, men had a higher CVI than women in all VV quadrants (all P < 0.05). IVA was observed in all VV quadrants of 91 eyes (43.96%), and in the macular region of 33 eyes (15.94%). Conclusions: The ST drainage system was identified as the preferred VV drainage route in healthy eyes. Among the four VV drainage quadrants, the drainage system adhered to the ST-SN-IT-IN order of descending perfusion. Furthermore, age- and sex-related differences were noted in the choroidal VV drainage systems of healthy eyes. Additionally, almost half of the healthy eyes had IVA in their choroidal vessel networks. Translational Relevance: The VV drainage system may be considered a novel imaging biomarker for ocular diseases.

Melatonin ameliorates retinal ganglion cell senescence and apoptosis in a SIRT1-dependent manner in an optic nerve injury model.

Melatonin is involved in exerting protective effects in aged-related and neurodegenerative diseases through a silent information regulator type 1 (SIRT1)-dependent pathway. However, little was known about the impact of melatonin on retinal ganglion cell (RGC) senescence and apoptosis following optic nerve crush (ONC). Thus, this study aimed to examine the effects of melatonin on RGC senescence and apoptosis after ONC and investigate the involvement of SIRT1 in this process. To study this, an ONC model was established. EX-527, an inhibitor of SIRT1, was injected intraperitoneally into mice. And melatonin was administrated abdominally into mice after ONC every day. Hematoxylin & eosin staining, retina flat-mounts and optical coherence tomography were used to evaluate the loss of retina cells/neurons. Pattern electroretinogram (p-ERG) was performed to evaluate the function of RGCs. Immunofluorescence and western blot were used to evaluate protein expression. SA-ß-gal staining was employed to detect senescent cells. The results demonstrated that melatonin partially rescued the expression of SIRT1 in RGC 3 days after ONC. Additionally, melatonin administration partly rescued the decreased RGC number and ganglion cell complex thickness observed 14 days after ONC. Melatonin also suppressed ONC-induced senescence and apoptosis index. Furthermore, p-ERG showed that melatonin improved the amplitude of P50, N95 and N95/P50 following ONC. Importantly, the protective effects of melatonin were reversed when EX-527 was administered. In summary, this study revealed that melatonin attenuated RGC senescence and apoptosis through a SIRT1-dependent pathway after ONC. These findings provide valuable insights for the treatment of RGC senescence and apoptosis.

Sema4D Knockout Attenuates Choroidal Neovascularization by Inhibiting M2 Macrophage Polarization Via Regulation of the RhoA/ROCK Pathway.

Purpose: The aim of this study was to elucidate the role of Sema4D in the pathogenesis of senescence-associated choroidal neovascularization (CNV) and to explore its underlying mechanisms. Methods: In this study, we utilized a model of laser-induced CNV in both young (3 months old) and old (18 months old) mice, including those with or without Sema4D knockout. The expression and localization of Sema4D in CNV were assessed using PCR, Western blot, and immunostaining. Subsequently, the morphological and imaging examinations were used to evaluate the size of CNV and vascular leakage. Finally, the expression of M2 markers, senescence-related markers, and molecules involved in the RhoA/ROCK pathway was detected. Results: We found that Sema4D was predominantly expressed in macrophages within CNV lesions, and both the mRNA and protein levels of Sema4D progressively increased following laser photocoagulation, a trend more pronounced in old mice. Moreover, Sema4D knockout markedly inhibited M2 polarization in senescent macrophages and reduced the size and leakage of CNV, particularly in aged mice. Mechanistically, aging was found to upregulate RhoA/ROCK signaling, and knockout of Sema4D effectively suppressed the activation of this pathway, with more significant effects observed in aged mice. Conclusions: Our findings revealed that the deletion of Sema4D markedly inhibited M2 macrophage polarization through the suppression of the RhoA/ROCK pathway, ultimately leading to the attenuation of senescence-associated CNV. These data indicate that targeting Sema4D could offer a promising approach for gene editing therapy in patients with neovascular age-related macular degeneration.

Red Light-Triggered Anti-Angiogenic and Photodynamic Combination Therapy of Age-Related Macular Degeneration.

Choroidal neovascularization (CNV) is the key pathological event of wet age-related macular degeneration (wAMD) leading to irreversible vision loss. Currently, anti-angiogenic therapy with anti-vascular endothelial growth factor (VEGF) agents has become the standard treatment for wAMD, while it is still subject to several limitations, including the safety concerns of monthly intravitreal administration and insufficient efficacy for neovascular occlusion. Combined therapy with photodynamic therapy (PDT) and anti-angiogenic agents has emerged as a novel treatment paradigm. Herein, a novel and less-invasive approach is reported to achieve anti-angiogenic and photodynamic combination therapy of wAMD by intravenous administration of a photoactivatable nanosystem (Di-DAS-VER NPs). The nanosystem is self-assembled by reactive oxygen species (ROS)-sensitive dasatinib (DAS) prodrug and photosensitizer verteporfin (VER). After red-light irradiation to the diseased eyes, intraocular release of anti-angiogenic DAS is observed, together with selective neo-vessels occlusion by VER-generated ROS. Notably, Di-DAS-VER NPs demonstrates promising therapeutic efficacy against CNV with minimized systemic toxicity. The study enables an efficient intravenous wAMD therapy by integrating a photoactivation process with combinational therapeutics into one simple nanosystem.

GSK840 Alleviates Retinal Neuronal Injury by Inhibiting RIPK3/MLKL-Mediated RGC Necroptosis After Ischemia/Reperfusion.

Purpose: This study aimed to explore the impact of GSK840 on retinal neuronal injury after retinal ischemia/reperfusion (IR) and its associated mechanism. Methods: We established an in vivo mouse model of IR and an in vitro model of oxygen and glucose deprivation/reoxygenation (OGDR) in primary mouse retinal ganglion cells (RGCs). GSK840, a small-molecule compound, was used to specifically inhibit RIPK3/MLKL-dependent necroptosis. Retinal structure and function evaluation was performed by using hematoxylin and eosin staining, optical coherence tomography, and electroretinography. Propidium Iodide (PI) staining was used for detection of necroptotic cell death, whereas Western blot analysis and immunofluorescence were used to assess necroptosis-related proteins and inner retinal neurons. Results: RIPK3/MLKL-dependent necroptosis was rapidly activated in RGCs following retinal IR or OGDR. GSK840 helped maintain relatively normal inner retinal structure and thickness by preserving inner retinal neurons, particularly RGCs. Meanwhile, GSK840 ameliorated IR-induced visual dysfunction, as evidenced by the improved amplitudes of photopic negative response, a-wave, b-wave, and oscillatory potentials. And GSK840 treatment significantly reduced the population of PI+ RGCs after injury. Mechanistically, GSK840 ameliorated RGC necroptosis by inhibiting the RIPK3/MLKL pathway. Conclusions: GSK840 exerts protective effects against retinal neuronal injury after IR by inhibiting RIPK3/MLKL-mediated RGC necroptosis. GSK840 may represent a protective strategy for RGC degeneration in ischemic retinopathy.