Retinal Inflammation Research Articles

Intravitreal faricimab as an adjuvant in the management of tubercular uveitis.

To report the use of intravitreal faricimab used as an adjunct in the management of intraocular tuberculosis (IOTB) in a patient with occlusive retinal vasculitis (ORV) and choroiditis. A case report. A 27-year-old Asian male presented with bilateral choroiditis and ORV complicated by retinal neovascularization and vitreous hemorrhage in one eye. The patient had a positive tuberculin skin test, indeterminate interferon-gamma release assay, and Ghon's focus on chest radiography. With a diagnosis of IOTB, the patient was treated with anti-tubercular therapy. Due to the significant vitreous hemorrhage, he underwent pars plana vitrectomy in one eye. The patient received bilateral faricimab. Within a week of the injection, he had a near-complete resolution of the extensive choroidal and retinal vascular inflammation. Intravitreal faricimab, given its angiopoietin-2 (ang2) inhibition and anti-vascular endothelial growth factor action, maybe a helpful adjunct in IOTB. Whether ang2 inhibition provides any additional benefit needs further evaluation in extensive studies.

Empagliflozin targets Mfn1 and Opa1 to attenuate microglia-mediated neuroinflammation in retinal ischemia and reperfusion injury

BackgroundNeuroinflammation and mitochondrial dysfunction play crucial roles in retinal ischemia and reperfusion (IR) injury. Recent studies have identified mitochondrial function as a promising target for immunomodulation. Empagliflozin (EMPA), an anti-diabetic drug, has exhibited great potential as both an anti-inflammatory agent and a protector of mitochondrial health. This study aimed to assess the therapeutic efficacy of EMPA in retinal IR injury.MethodsTo evaluate the protective effects of EMPA, the drug was injected into the vitreous body of mice post-retinal IR. Single-cell RNA sequencing (scRNA-seq) analysis was conducted to uncover the underlying mechanisms, and the results were further validated through in vivo and in vitro experiments.ResultsEMPA effectively protected retinal ganglion cells (RGCs) from IR injury by attenuating local retinal inflammation. The scRNA-seq analysis revealed that EMPA downregulated the nucleotide-binding domain and leucine-rich repeat containing protein 3 (NLRP3) signaling pathway and restored mitochondrial dynamics by upregulating the expression of mitochondrial fusion-related genes, Mitofusin 1 (Mfn1) and optic atrophy 1 (Opa1). These findings were further corroborated by Western blotting. In vitro experiments provided additional insights, demonstrating that EMPA suppressed lipopolysaccharide (LPS)-induced cell inflammation and NLRP3 inflammasome activation. Moreover, EMPA enhanced mitochondrial fusion, neutralized mitochondrial reactive oxygen species (mtROS), and restored mitochondrial membrane potential (MMP) in BV2 microglia. Notably, genetic ablation of Mfn1 or Opa1 abolished the anti-inflammatory effects of EMPA.ConclusionsOur findings highlight the positive contribution of Mfn1 and Opa1 to the anti-inflammatory therapeutic effect of EMPA. By restoring mitochondrial dynamics, EMPA effectively mitigates microglia-mediated neuroinflammation and prevents RGC loss in retinal IR injury.

Roles of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor 2 in Endotoxin-Induced Acute Retinal Inflammation

ABSTRACT Purpose To investigate the roles of sphingosine kinases (SphKs) and sphingosine-1-phosphate receptors (S1PRs) in endotoxin-induced uveitis (EIU) mice. Methods EIU model was induced using an intraperitoneal injection of lipopolysaccharide (LPS). The expression of SphKs and S1PRs in the retina was assessed using quantitative polymerase chain reaction (qPCR) and immunofluorescence. The effects of S1PR antagonists on the expression of inflammatory cytokines in the retina were evaluated using qPCR and western blotting. Effects of leukocyte infiltration of the retinal vessels were evaluated to determine the effects of the S1PR2 antagonist and genetic deletion of S1PR2 on retinal inflammation. Results Retinal SphK1 expression was significantly upregulated in EIU. SphK1 was expressed in the GCL, IPL, and OPL and S1PR2 was expressed in the GCL, INL, and OPL. Positive cells in IPL and OPL of EIU retina were identified as endothelial cells. S1PR2 antagonist and genetic deletion of S1PR2 significantly suppressed the expression of IL-1α, IL-6, TNF-α, and ICAM-1, whereas S1PR1/3 antagonist did not. Use of S1PR2 antagonist and S1PR2 knockout in mice significantly ameliorated leukocyte adhesion induced by LPS. Conclusion SphK1/S1P/S1PR2 signaling was upregulated in EIU and S1PR2 inhibition suppressed inflammatory response. Targeting this signaling pathway has potential for treating retinal inflammatory diseases.

Engineered mesenchymal stem cell-derived small extracellular vesicles for diabetic retinopathy therapy through HIF-1α/EZH2/PGC-1α pathway

Diabetic retinopathy (DR) is a leading cause of blindness worldwide with limited treatment options. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) hold promise as a cell-free therapy for retinal diseases. In this study, we present evidence that the intravitreal injection of MSC-sEVs improved retinal function and alleviated retinal apoptosis, inflammation, and angiogenesis in both db/db mice and streptozotocin-induced diabetic rats. Mechanistically, hyperglycemia-induced activation of hypoxia-inducible factor-1α (HIF-1α) inhibited the tripartite motif 21 (TRIM21)-mediated ubiquitination and degradation of enhancer of zeste homologue 2 (EZH2), ultimately resulting in the downregulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) through EZH2-induced methylation modification. The presence of miR-5068 and miR-10228 in MSC-sEVs targeted the HIF-1α/EZH2/PGC-1α pathway. The blockade of miR-5068 and miR-10228 abolished the retinal therapeutic effects of MSC-sEVs. Additionally, we engineered MSC-sEVs with elevated levels of miR-5068 and miR-10228 to enhance retinal repair efficiency. Together, our findings provide novel insights into the mechanism underlying DR progress and highlight the potential of MSC-sEVs, especially engineered MSC-sEVs, as a therapeutic option for DR.

Scutellarin Alleviates Diabetic Retinopathy via the Suppression of Nucleotide-Binding Oligomerization Domain (NOD)-Like Receptor Pyrin Domain Containing Protein 3 Inflammasome Activation

Purpose Diabetic retinopathy, a prevalent complication of diabetes, represents the leading cause of vision loss and blindness among middle-aged and elderly populations. Recent research has demonstrated the ameliorating effects of scutellarin on diabetes-associated complications such as diabetic retinopathy and type 2 diabetic cardiomyopathy. However, investigations into its protective impact and underlying mechanisms on diabetic retinopathy are scant. This study aims to explore the therapeutic potential of scutellarin in diabetic retinopathy treatment. Methods Diabetic retinopathy was induced in rats through intraperitoneal injections of streptozotocin (STZ, 60 mg/kg) administered daily for three consecutive days. Following this, diabetic retinopathy rats received daily intragastric administration of scutellarin (40 mg/kg) for 42 days. Results Our findings suggest that scutellarin alleviates histological damage in the retinal tissues of streptozotocin-challenged rats. Furthermore, scutellarin effectively enhances total retinal thickness and increases the number of ganglion cell layer (GCL) cells in the retinal tissues of streptozotocin-treated rats. Scutellarin also demonstrated anti-inflammatory and antioxidant effects in the retinal tissues of STZ-induced rats, as indicated by reduced levels of tumor necrosis factor-α, interleukin-1β, and interleukin-6, and elevated levels of glutathione peroxidase, superoxide dismutase, and catalase. Additionally, scutellarin effectively inhibited the expression of NOD-like receptor pyrin domain containing protein 3 inflammasome-related markers in the retinal tissues of streptozotocin-administered rats. Conclusions Collectively, our results indicate that scutellarin significantly reduces streptozotocin-induced retinal inflammation, an effect that may be partially attributed to the suppression of NLRP3 inflammasome activation.

Mechanical Regulation of Retinal Vascular Inflammation and Degeneration in Diabetes.

Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy (DR), a major microvascular complication of diabetes. Past studies investigating these diabetes-induced retinal vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in diabetes are also mechanically regulated by the increase in retinal vascular stiffness caused by overexpression of collagen-crosslinking enzyme lysyl oxidase (LOX). Treatment of diabetic mice with LOX inhibitor β-Aminopropionitrile (BAPN) prevented the increase in retinal capillary stiffness, vascular ICAM-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal capillary degeneration, pericyte ghost formation, and the diabetes-induced loss of contrast sensitivity in these mice. Finally, our in vitro studies indicate that retinal capillary stiffening is sufficient to increase the adhesiveness and neutrophil elastase-induced death of retinal endothelial cells. By uncovering a link between LOX-dependent capillary stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer a new insight into DR pathogenesis that has important translational potential.

Pharmacological Modulation of β-Catenin Preserves Endothelial Barrier Integrity and Mitigates Retinal Vascular Permeability and Inflammation.

Compromised blood-retinal barrier (BRB) integrity is a significant factor in ocular diseases like uveitis and retinopathies, leading to pathological vascular permeability and retinal edema. Adherens and tight junction (AJ and TJ) dysregulation due to retinal inflammation plays a pivotal role in BRB disruption. We investigated the potential of ICG001, which inhibits β-catenin-mediated transcription, in stabilizing cell junctions and preventing BRB leakage. In vitro studies using human retinal endothelial cells (HRECs) showed that ICG001 treatment improved β-Catenin distribution within AJs post lipopolysaccharide (LPS) treatment and enhanced monolayer barrier resistance. The in vivo experiments involved a mouse model of LPS-induced ocular inflammation. LPS treatment resulted in increased albumin leakage from retinal vessels, elevated vascular endothelial growth factor (VEGF) and Plasmalemmal Vesicle-Associated Protein (PLVAP) expression, as well as microglia and macroglia activation. ICG001 treatment (i.p.) effectively mitigated albumin leakage, reduced VEGF and PLVAP expression, and reduced the number of activated microglia/macrophages. Furthermore, ICG001 treatment suppressed the surge in inflammatory cytokine synthesis induced by LPS. These findings highlight the potential of interventions targeting β-Catenin to enhance cell junction stability and improve compromised barrier integrity in various ocular inflammatory diseases, offering hope for better management and treatment options.

Dysregulated CD200-CD200R signaling in early diabetes modulates microglia-mediated retinopathy.

Diabetic retinopathy (DR) is a neurovascular complication of diabetes. Recent investigations have suggested that early degeneration of the neuroretina may occur prior to the appearance of microvascular changes; however, the mechanisms underlying this neurodegeneration have been elusive. Microglia are the predominant resident immune cell in the retina and adopt dynamic roles in disease. Here, we show that ablation of retinal microglia ameliorates visual dysfunction and neurodegeneration in a type I diabetes mouse model. We also provide evidence of enhanced microglial contact and engulfment of amacrine cells, ultrastructural modifications, and transcriptome changes that drive inflammation and phagocytosis. We show that CD200-CD200R signaling between amacrine cells and microglia is dysregulated during early DR and that targeting CD200R can attenuate high glucose-induced inflammation and phagocytosis in cultured microglia. Last, we demonstrate that targeting CD200R in vivo can prevent visual dysfunction, microglia activation, and retinal inflammation in the diabetic mouse. These studies provide a molecular framework for the pivotal role that microglia play in early DR pathogenesis and identify a potential immunotherapeutic target for treating DR in patients.

CCR1 mediates Müller cell activation and photoreceptor cell death in macular and retinal degeneration.

Mononuclear cells are involved in the pathogenesis of retinal diseases, including age-related macular degeneration (AMD). Here, we examined the mechanisms that underlie macrophage-driven retinal cell death. Monocytes were extracted from patients with AMD and differentiated into macrophages (hMdɸs), which were characterized based on proteomics, gene expression, and ex vivo and in vivo properties. Using bioinformatics, we identified the signaling pathway involved in macrophage-driven retinal cell death, and we assessed the therapeutic potential of targeting this pathway. We found that M2a hMdɸs were associated with retinal cell death in retinal explants and following adoptive transfer in a photic injury model. Moreover, M2a hMdɸs express several CCRI (C-C chemokine receptor type 1) ligands. Importantly, CCR1 was upregulated in Müller cells in models of retinal injury and aging, and CCR1 expression was correlated with retinal damage. Lastly, inhibiting CCR1 reduced photic-induced retinal damage, photoreceptor cell apoptosis, and retinal inflammation. These data suggest that hMdɸs, CCR1, and Müller cells work together to drive retinal and macular degeneration, suggesting that CCR1 may serve as a target for treating these sight-threatening conditions.

Early Depletion of Neutrophils Reduces Retinal Inflammation and Neovascularization in Mice with Oxygen-Induced Retinopathy.

Retinal inflammation is a central feature of ocular neovascular diseases such as diabetic retinopathy and retinopathy of prematurity, but the contribution of neutrophils to this process is not fully understood. We studied oxygen-induced retinopathy (OIR) which develops in two phases, featuring hyperoxia-induced retinal vaso-obliteration in phase I, followed by retinal neovascularization in phase II. As neutrophils are acute responders to tissue damage, we evaluated whether neutrophil depletion with an anti-Ly6G mAb administered in phase I OIR influenced retinal inflammation and vascular injury. Neutrophils were measured in blood and spleen via flow cytometry, and myeloperoxidase, an indicator of neutrophil activity, was evaluated in the retina using Western blotting. Retinal vasculopathy was assessed by quantitating vaso-obliteration, neovascularization, vascular leakage, and VEGF levels. The inflammatory factors, TNF, MCP-1, and ICAM-1 were measured in retina. In the OIR controls, neutrophils were increased in the blood and spleen in phase I but not phase II OIR. In OIR, the anti-Ly6G mAb reduced neutrophils in the blood and spleen, and myeloperoxidase, inflammation, and vasculopathy in the retina. Our findings revealed that the early rise in neutrophils in OIR primes the retina for an inflammatory and angiogenic response that promotes severe damage to the retinal vasculature.

Impairing Gasdermin D-mediated pyroptosis is protective against retinal degeneration

BackgroundInflammasome activation and the subsequent release of pro-inflammatory cytokines including Interleukin 1β (IL-1β) have been widely reported to contribute to the progression of retinal degenerations, including age-related macular degeneration (AMD), the leading cause of blindness in the Western World. The role of Gasdermin D (GSDMD), a key executioner of pyroptosis following inflammasome activation, however, is less well-established. In this study we aimed to characterise the role of GSDMD in the healthy and degenerating retina, and uncover its role as a conduit for IL-1β release, including via extracellular vesicle (EV)-mediated release.MethodsGSDMD mutant and knockout mice, in vitro models of inflammation and a well-established in vivo model of retinal degeneration (photo-oxidative damage; PD) were utilised to explore the role and pathological contribution of GSDMD in regulating IL-1β release and propagating retinal inflammation. RNA sequencing of whole retinas was used to investigate GSDMD-mediated inflammation during degeneration. The role of EVs in GSDMD-mediated IL-1β release was investigated using nanoparticle tracking analysis, ELISA and EV inhibition paradigms. Finally, the therapeutic efficacy of targeting GSDMD was examined using GSDMD-specific siRNA.ResultsWe identified in this work that mice deficient in GSDMD had better-preserved retinal function, increased photoreceptor survivability and reduced inflammation. RNA-Seq analysis revealed that GSDMD may propagate inflammation in the retina via NF-κB signalling cascades and release of pro-inflammatory cytokines. We also showed that IL-1β was packaged and released via EV in a GSDMD-dependent manner. Finally, we demonstrated that impairing GSDMD function using RNAi or blocking EV release was able to reduce IL-1β content in cell-free supernatant and EV.ConclusionsTaken together, these results suggest that pyroptotic pore-forming protein GSDMD plays a key role in the propagation of retinal inflammation, in particular via the release of EV-encapsulated IL-1β. Targeting GSDMD using genetic or pharmacological inhibitors may pose a therapeutic opportunity to dampen inflammatory cascades and delay the progression of retinal degeneration.

Protective effects and mechanisms of Momordica charantia polysaccharide on early-stage diabetic retinopathy in type 1 diabetes

Momordica charantia polysaccharide (MCP) is a potential drug for the prevention and alleviation of diabetes mellitus (DM) and diabetic retinopathy (DR). This study aimed to investigate the potential protective effects of MCP on early-stage DR and explore the underlying mechanisms. The model group (DM group) and treatment group (D+H group) were established by inducing type 1 DM using a single dose of streptozotocin (STZ) at 60 mg/kg. After modeling, the D+H group was orally administered a 500 mg/kg dose of MCP solution once daily for 12 weeks. Monitoring of systemic indicators (FBG, body weight, general condition) and retinal tissue inflammation and apoptosis (HE staining, IL-6, MCP-1, TNF-α, VEGF, NF-κB, Caspase-3) in this study demonstrated that MCP intervention alleviated both DM and DR. MCP improved the body weight and general condition of DM rats by reducing FBG levels. It also enhanced the anti-inflammatory and anti-apoptotic capabilities of retinal neurons and microvessels by modulating the actions of cytokines, thereby further regulating the inflammation and apoptosis of retinal neurons and microvessels. The underlying mechanisms may be associated with the downregulation of NF-κB and Caspase-3 pathway protein expression, as well as the downregulation of mRNA expression of NF-κB and Caspase-3 pathway genes. Further research is needed to elucidate the potential mechanisms underlying the protective effects of MCP on DR. MCP may emerge as a selective medication for the prevention and alleviation of DM and a novel natural medicine for the prevention and alleviation of DR.

Limited Hyperoxia-Induced Proliferative Retinopathy (LHIPR) as a Model of Retinal Fibrosis, Angiogenesis, and Inflammation.

The progression to fibrosis and traction in retinopathy of prematurity (ROP) and other ischemic retinopathies remains an important clinical and surgical challenge, necessitating a comprehensive understanding of its pathogenesis. Fibrosis is an unbalanced deposition of extracellular matrix components responsible for scar tissue formation with consequent tissue and organ impairment. Together with retinal traction, it is among the main causes of retinal detachment and vision loss. We capitalize on the Limited Hyperoxia Induced Retinopathy (LHIPR) model, as it reflects the more advanced pathological phenotypes seen in ROP and other ischemic retinopathies. To model LHIPR, we exposed wild-type C57Bl/6J mouse pups to 65% oxygen from P0 to P7. Then, the pups were returned to room air to recover until later endpoints. We performed histological and molecular analysis to evaluate fibrosis progression, angiogenesis, and inflammation at several time points, from 1.5 months to 9 months. In addition, we performed in vivo retinal imaging by optical coherence tomography (OCT) or OCT Angiography (OCTA) to follow the fibrovascular progression in vivo. Although the retinal morphology was relatively preserved, we found a progressive increase in preretinal fibrogenesis over time, up to 9 months of age. We also detected blood vessels in the preretinal space as well as an active inflammatory process, altogether mimicking advanced preretinal fibrovascular disease in humans.

The effects of a highly bioavailable curcumin PhytosomeTM preparation on the retinal architecture and glial reactivity in the GFAP-IL6 mice.

Uncontrolled, chronic inflammation in the retina can disturb retinal structure and function leading to impaired visual function. For the first time, in a mouse model of chronic neuroinflammation (GFAP-IL6), we investigated the impact of chronic glial activation on the retinal microglia population and structure. In addition, we tested a curcumin PhytosomeTM preparation with enhanced bioavailability to investigate the effects of a cytokine-suppressing anti-inflammatory drug on retinal architecture. Curcumin PhytosomeTM was fed to 3-month old GFAP-IL6 mice for 4 weeks and compared to their untreated GFAP-IL6 counterparts as well as wild type mice on control diet. Microglial numbers and morphology together with neuronal numbers were characterized using immunohistochemistry and cell reconstruction in the retina, using retinal wholemount and slices. GFAP-IL6 mice showed a significant increase in Iba1-labelled mononuclear phagocytes, including microglia, and displayed altered glial morphology. This resulted in a reduction in cone density and a thinning of the retinal layers compared to wild type mice. Curcumin PhytosomeTM treatment contributed to decreased microglial density, significantly decreasing both soma and cell size compared to control diet, as well as preventing the thinning of the retinal layers. This study is the first to characterize the impact of chronic retinal inflammation in the GFAP-IL6 mouse and the therapeutic benefit of enhanced bioavailable curcumin PhytosomeTM to significantly reduce microglia density and prevent neuronal loss. These data suggest that curcumin could be used as a complementary therapy alongside traditional treatments to reduce associated retinal inflammation in a variety of retinal diseases.

Autosomal dominant neovascular inflammatory vitreoretinopathy with CAPN5 c.731T > C gene mutation; clinical management of a family cohort and review of the literature

ABSTRACT Background To report a cohort of patients with clinically and genetically diagnosed autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) and showcase the spectrum of the disease utilizing multimodal imaging and genetic testing. Additionally, the utility of multimodal imaging in guiding treatment will also be illustrated. Materials/Methods Five patients from a single-family pedigree in Ohio with clinical signs of ADNIV were evaluated. Medical history, family history, and complete ocular examinations were obtained during regular clinic visits. Multimodal imaging including ocular coherence tomography, fluorescein angiography, wide-field fundus photographs, and Humphrey visual field testing was obtained for all five patients. Additionally, genetic testing for the Calpain-5 (CAPN5) gene was conducted on all patients. Results All five patients were noted to have a CAPN5 c.731T > C (p.L244P) mutation on genetic testing. Using multimodal imaging to supplement the clinical examination, pathologic changes such as retinal vascular inflammation, macular edema, and tractional retinal membranes were well illustrated and monitored over time. This allowed for earlier intervention when appropriate such as with intraocular steroid or systemic anti-inflammatory treatments. Conclusion Phenotypic presentation varied among patients in this series, but is consistent with the spectrum of pathologic changes previously described in patients with other CAPN5 gene mutations. Monitoring of patients with ADNIV utilizing multimodal imaging can help better assess progression of this disease and guide treatment decisions. Additionally, increased genetic testing in patients with inherited retinal diseases may reveal novel gene mutations that could serve as potential targets for future genetic treatment regimens.

Inhibition of cGAS–STING pathway alleviates neuroinflammation-induced retinal ganglion cell death after ischemia/reperfusion injury

Acute glaucoma is a vision-threatening disease characterized by a sudden elevation in intraocular pressure (IOP), followed by retinal ganglion cell (RGC) death. Cytosolic double-stranded DNA (dsDNA)—a damage-associated molecular pattern (DAMP) that triggers inflammation and immune responses—has been implicated in the pathogenesis of IOP-induced RGC death, but the underlying mechanism is not entirely clear. In this study, we investigated the effect of the inflammatory cascade on dsDNA recognition and examined the neuroprotective effect of the cyclic GMP-AMP (cGAMP) synthase (cGAS) antagonist A151 on a retinal ischemia/reperfusion (RIR) mouse model. Our findings reveal a novel mechanism of microglia-induced neuroinflammation-mediated RGC death associated with glaucomatous vision loss. We found that RIR injury facilitated the release of dsDNA, which initiated inflammatory responses by activating cGAS–stimulator of interferon genes (STING) pathway. Correspondingly, elevated expressions of cGAS and STING were found in retinal samples from human glaucoma donors. Furthermore, we found that deletion or inhibition of cGAS or STING in microglia transfected with poly(dA:dT) specifically decreased microglia activation and inflammation response. We also observed that A151 treatment promoted poly(dA:dT)--stimulated changes in polarization from the M1 to the M2 phenotype in microglia. Subsequently, A151 administered to mice effectively inhibited the cGAS–STING pathway, absent in melanoma 2 (AIM2) inflammasome and pyroptosis-related molecules. Furthermore, A151 administration significantly reduced neuroinflammation, ameliorated RGC death and RGC-related reductions in visual function. These findings provide a unique perspective on glaucomatous neuropathogenesis and suggest cGAS as an underlying target of retinal inflammation to provide a potential therapeutic for acute glaucoma.

Retinal ferroptosis as a critical mechanism for the induction of retinochoroiditis during ocular toxoplasmosis

Toxoplasmosis is a major infectious disease, affecting approximately one-third of the world's population; its main clinical manifestation, ocular toxoplasmosis (OT), is a severe sight-threatening disease. Nevertheless, the diagnosis of OT is based on clinical findings, which needs improvement, even with biochemical tests, such as polymerase chain reaction and antibody detections. Furthermore, the efficacy of OT-targeted treatment is limited; thus, additional measures for diagnosis and treatments are needed. Here, we for the first time report a significantly reduced iron concentration in the vitreous humor (VH) of human patients infected with OT. To obtain further insights into molecular mechanisms, we established a mouse model of T. gondii infection, in which intravitreally injected tracer 57Fe, was accumulated in the neurosensory retina. T. gondii-infected eyes showed increased lipid peroxidation, reduction of glutathione peroxidase-4 expression and mitochondrial deformity in the photoreceptor as cristae loss. These findings strongly suggest the involvement of ferroptotic process in the photoreceptor of OT. In addition, deferiprone, an FDA-approved iron chelator, reduced the iron uptake but also ameliorated toxoplasma-induced retinochoroiditis by reducing retinal inflammation. In conclusion, the iron levels in the VH could serve as diagnostic markers and iron chelators as potential treatments for OT.

Panax notoginseng saponins alleviate diabetic retinopathy by inhibiting retinal inflammation: Association with the NF-κB signaling pathway

Ethnopharmacological relevanceDiabetic retinopathy (DR) is a neurovascular disease that causes blindness in adults and is the most serious and common complication of diabetes mellitus. Retinal inflammation is an early stage of DR, and it is believed to play a crucial role in the development of DR. Panax notoginseng saponins (PNS) are the major active constituent in the main root of P. notoginseng, and they exhibit various biological activities, including anti-inflammatory, antioxidant, neuroprotective, and immunomodulatory functions. However, the protective effects and underlying mechanisms of PNS against DR remain unclear. Aim of the studyThis study aimed to investigate the alleviation effects of PNS on DR and the mechanisms involved. Furthermore, it intended to explore the major components that exert efficacy in vivo. Materials and methodsStreptozotocin (STZ) was administered intraperitoneally to Sprague Dawley rats, and PNS was administered orally for 1 month after 2 months of STZ injection. The morphological structure of the retina and retinal acellular capillaries were assessed via hematoxylin and eosin (H&E) staining assay. The disruption of the blood–retinal barrier (BRB) was detected through Evans blue dye leakage assay, and retinal leukocyte adhesion was achieved via fluorescein isothiocyanate-coupled concanavalin A lectin labeling assay. Immunofluorescence staining and Western blot assays were conducted to detect the expression of tight junction proteins, adhesion molecules, and the ionized calcium-binding adapter molecule-1 (Iba-1) in the retina. Enzyme-linked immunosorbent assay was performed to detect the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in serum. In addition, the protein expression levels of nuclear factor (NF)-κB p65, phosphorylated IκB kinase (p-IKK), phosphorylated NF-κB inhibitor (p-IκB), and phosphorylated NF-κB p65 (p-p65) were measured using Western blot assay. The ocular tissue distribution of PNS in normal and diabetic rats was determined through ultra-performance liquid chromatography–tandem mass spectrometry. The in vitro anti-inflammatory effects of PNS, notoginsenoside (NGR1), ginsenoside Rg1, Re, Rb1, and Rd (GRg1, GRe, GRb1, and GRd) were evaluated on human Müller (MIO-M1) cells. ResultsPNS increased the reduction in retinal inner nuclear layer thickness, reduced the increase in retinal acellular capillaries, and attenuated elevated BRB disruption by upregulating the decrease in protein expression of claudin-1 and occludin. Furthermore, PNS significantly abrogated microglial cell activation and reversed the increase in leukocyte adhesion by downregulating the increase in the protein expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. Moreover, PNS reduced the elevated levels of TNF-α, IL-6, and IL-1β in serum and inhibited the increased protein expression of p-IKK, p-IκB, and p-p65, and the nuclear translocation of p65. The tissue distribution results revealed that NGR1, GRg1, GRe, GRb1, and GRd were detected in the ocular tissue, while GRg1 and GRb1 were found at the highest levels compared with the other components. The cellular results showed that PNS, NGR1, GRg1, GRe, GRb1, and GRd suppressed the development of cellular inflammatory responses by inhibiting the activation of the NF-κB signaling pathway in MIO-M1 cells and that their anti-inflammatory effects were comparable. ConclusionPNS suppressed retinal inflammation by inhibiting the activation of the NF-κB signaling pathway, alleviating DR. GRg1 and GRb1 may be the primary components that exert anti-inflammatory effects in vivo.

Enhanced therapeutic effect of PEDF-loaded mesenchymal stem cell-derived small extracellular vesicles against oxygen-induced retinopathy through increased stability and penetrability of PEDF

BackgroundSeveral common retinal diseases that cause blindness are characterised by pathological neovascularisation accompanied by inflammation and neurodegeneration, including retinopathy of prematurity (ROP), diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinal vein occlusion (RVO). The current treatment strategies for these diseases have limited benefits. Thus, safer and more effective alternative approaches are required. In this study, we loaded small extracellular vesicles (sEVs) derived from mesenchymal stem cell (MSC) with pigment epithelium-derived factor (PEDF), and tested the therapeutic effect of PEDF-loaded sEVs (PEDF-sEVs) using an oxygen induced retinopathy (OIR) mouse model, aiming to establish a new therapy strategy for the treatment of retinal pathological angiogenesis.ResultsWe formulated PEDF-loaded sEVs (PEDF-sEVs) containing high concentrations of PEDF and evaluated their effects through in vivo and in vitro experiments. In OIR mice, PEDF-sEVs showed significantly better effects on retinal avascular areas, inflammation, and neuronal degeneration compared with the anti-vascular endothelial growth factor (VEGF) drug, which may indicate a possible advantage of PEDF-sEVs over anti-VEGF drugs in the treatment of pathological neovascularisation. In vitro, PEDF-sEVs greatly inhibited endothelial cell (EC) proliferation, migration, and tube formation by suppressing the VEGF-induced phosphorylation of extracellular signal-regulated kinase (ERK) and AKT (also known as Protein Kinase B). All experiments and analyses were performed in triplicate. PEDF-sEVs were more effective than PEDF or sEVs alone, both in vitro and in vivo. Furthermore, to determine the distribution of PEDF-sEVs, we used DiD-labelled sEVs and FITC-labelled PEDF to track the sEVs and PEDF, respectively. We found that PEDF-sEVs effectively reduced the degradation of PEDF.ConclusionsLoading PEDF on sEVs effectively enhanced the anti-angiogenic, anti-inflammatory, and neuroprotective effects of PEDF by increasing the stability and penetrability. These results suggest a potential role for PEDF-sEVs in retinal pathological neovascularisation.Graphical

Retinal inflammation in murine models of type 1 and type 2 diabetes with diabetic retinopathy

Aims/hypothesisThe loss of pericytes surrounding the retinal vasculature in early diabetic retinopathy underlies changes to the neurovascular unit that lead to more destructive forms of the disease. However, it is unclear which changes lead to loss of retinal pericytes. This study investigated the hypothesis that chronic increases in one or more inflammatory factors mitigate the signalling pathways needed for pericyte survival.MethodsLoss of pericytes and levels of inflammatory markers at the mRNA and protein levels were investigated in two genetic models of diabetes, Ins2Akita/+ (a model of type 1 diabetes) and Leprdb/db (a model of type 2 diabetes), at early stages of diabetic retinopathy. In addition, changes that accompany gliosis and the retinal vasculature were determined. Finally, changes in retinal pericytes chronically incubated with vehicle or increasing amounts of IFNγ were investigated to determine the effects on pericyte survival. The numbers of pericytes, microglia, astrocytes and endothelial cells in retinal flatmounts were determined by immunofluorescence. Protein and mRNA levels of inflammatory factors were determined using multiplex ELISAs and quantitative reverse transcription PCR (qRT-PCR). The effects of IFNγ on the murine retinal pericyte survival-related platelet-derived growth factor receptor β (PDGFRβ) signalling pathway were investigated by western blot analysis. Finally, the levels of cell death-associated protein kinase C isoform delta (PKCδ) and cleaved caspase 3 (CC3) in pericytes were determined by western blot analysis and immunocytochemistry.ResultsThe essential findings of this study were that both type 1 and 2 diabetes were accompanied by a similar progression of retinal pericyte loss, as well as gliosis. However, inflammatory factor expression was dissimilar in the two models of diabetes, with peak expression occurring at different ages for each model. Retinal vascular changes were more severe in the type 2 diabetes model. Chronic incubation of murine retinal pericytes with IFNγ decreased PDGFRβ signalling and increased the levels of active PKCδ and CC3.Conclusions/interpretationWe conclude that retinal inflammation is involved in and sustains pericyte loss as diabetic retinopathy progresses. Moreover, IFNγ plays a critical role in reducing pericyte survival in the retina by reducing activation of the PDGFRβ signalling pathway and increasing PKCδ levels and pericyte apoptosis.Graphical