Experimental eye research最新文献

Murine cytomegalovirus (MCMV) intracameral infection successfully models clinical manifestations resembling CMV-positive Posner-Schlossman Syndrome (PSS). We observed distinct phenotypes in infected C57BL/6 and BALB/c mice: C57BL/6 exhibited severe intraocular inflammation with corneal edema and persistent elevated intraocular pressure (IOP), while BALB/c showed acute IOP spikes with mild uveitis. Single-cell sequencing revealed significant upregulation of CD74 in intraocular CD45⁺ immunocytes of C57BL/6 mice. CD74, a receptor for macrophage migration inhibitory factor (MIF), is a key regulator of inflammatory pathways. This study found that C57BL/6 with MCMV intracameral infection exhibited increased CD74⁺ cell counts systemically and intraocularly, alongside elevated intraocular and systemic levels of MIF and various inflammatory cytokines. In vitro, recombinant MIF enhanced cytokine secretion (including IL-6, IL-1β, CCL2, CCL5and CXCL1) in primary peripheral blood mononuclear cells (PBMCs), while CD74 blockade partially inhibited MIF-induced cytokine production. The CD74-MIF interaction is indispensable for cytokine secretion in downstream inflammatory pathways. These findings suggest that the CD74-MIF axis drives ocular inflammation and uveitic damage following MCMV infection in C57BL/6 mice, highlighting its potential as a therapeutic target for immunosuppression in acute uveitis.

Purpose: Changes in choroidal thickness are currently used to predict future refractive error development but there is incomplete knowledge about the communication between choroid and sclera. We studied how choroidal thickness changes interact with scleral thickness changes and how the abundance of dopamine (DA)- and all-trans retinoic acid (atRA)-synthetizing choroidal cells varies when choroidal thickness is altered by drugs.

Methods: Changes in choroidal thickness were induced by a single intravitreal injection in the morning of the muscarinic antagonist atropine, the DA agonist apomorphine or the DA antagonist spiperone. Thickness of the choroid and the scleral layers was measured by spectral domain optical coherence tomography (SD-OCT). Immunocytochemistry was used to study the distribution of dopamine-synthetizing structures in the choroid and their colocalisation with retinaldehyde dehydrogenase 2 (RADLH2), the key synthetizing enzyme of atRA.

Results: (1) Both atropine and apomorphine increased choroidal thickness over the day while spiperone resulted in a decrease. (2) For apomorphine and spiperone, choroidal thickness changes were positively correlated with thickness changes in both the cartilaginous and fibrous layers of the sclera. With atropine, only the cartilaginous layer thickened. (3) DA was co-localized with RALDH2 in stromal cells in the choroid in a few cases but the numbers of double-stained cells increased massively after drug injections. (4) RALDH2-immunoreactivity (indicating atRA activity) increased, no matter whether the choroid and the sclera thickened or thinned.

Conclusions: Following drug injections, thickness changes of choroid and sclera were correlated and occurred without phase delay. Numbers of DA and RALDH2 co-expressing cells in the choroid increased. Choroidal dopaminergic cells that synthesize atRA appear to act as activators of scleral metabolic activity during both scleral growth stimulation and inhibition.

Experimental autoimmune uveoretinitis (EAU) shows degeneration of retinal neurons, including retinal ganglion cells (RGCs), already in its early phase. Based on our previous study demonstrating the attenuation of EAU by brain-derived neurotrophic factor (BDNF), whose retinal levels were increased by visual stimulation (VS), this study evaluated the effect of VS on BDNF protein expression in brain visual centers, its retrograde transport to the retina, and RGC survival in healthy and EAU mice. 14-day VS increased BDNF expression in the superior colliculus (SC) but not in the lateral geniculate nucleus and primary visual cortex in healthy and EAU mice compared to their unstimulated groups. Furthermore, VS increased numbers of BDNF-positive neurons and astrocytes in the retinorecipient superficial SC (sSC) in healthy and EAU mice, although stimulated EAU mice showed a modest reduction in BDNF-positive neurons compared to stimulated healthy mice. In contrast, unstimulated EAU mice exhibited a marked loss of sSC BDNF-positive neurons and astrocytes compared to unstimulated healthy mice. Additionally, VS promoted retrograde axonal transport of fluorescently labeled BDNF from the sSC to the retina, where it was detected in RGCs, inner retinal neurons, and Müller cells (MCs). These results suggest that VS-induced increases in BDNF expression in the sSC and its retrograde transport to the retina may directly affect multiple types of retinal neurons and MCs, on which BDNF can exert neurotrophic and protective effects. The overall attenuation of EAU histopathology and retinal inflammation, along with improved survival of RGCs in VS-treated EAU mice, is consistent with this suggestion.

The purpose of this study was to analyze and compare cytokine and growth factor levels in modified autologous conditioned serum (mACS) and autologous serum (AS) and to evaluate their therapeutic effects in a benzalkonium chloride (BAK)-induced murine dry eye model. Serum samples were obtained from twenty healthy volunteers and analyzed by ELISA. A dry eye model was established in twenty-four C57BL/6 mice by topical application of 0.2% BAK twice daily for seven days. The mice were evenly divided into three subgroups: saline-treated, 0.5% AS-treated, and 0.5% mACS-treated. The right eyes were treated, and the left eyes served as untreated controls. Eyeballs were harvested on days 7 and 14 for immunofluorescence staining. Results showed that neuroprotective factors (BDNF and fractalkine), pro-inflammatory cytokines (IL-1β, IL-6, MIF, TNF-α), and VEGF-A were significantly elevated in the mACS group, whereas PDGF-BB was significantly reduced. Furthermore, immunofluorescence analysis demonstrated a significantly greater recovery of central corneal nerve fibers in the mACS-treated group compared with the saline group at day 7 (p < 0.01). At day 14, the mACS-treated group continued to show a trend toward increased central corneal nerve regeneration, although this difference did not reach conventional statistical significance (p < 0.1). No significant differences were observed between the AS- and saline-treated groups. In conclusion, compared with AS, mACS demonstrates a cytokine profile suggestive of enhanced neuroprotective potential and may facilitate corneal nerve regeneration in the BAK-induced murine dry eye model.

Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious corneal blindness worldwide. Viral persistence and disease severity are strongly influenced by the virus's ability to modulate host immune responses; however, the mechanisms by which HSV-1 alters corneal immunity remain incompletely understood. In particular, the role of virus-encoded microRNAs (v-miRs) in shaping corneal immune responses during herpes simplex infection remains unclear. In a previous study, inhibition of selected HSV-1 v-miRs reduced viral replication and disease severity in a mouse model of ocular infection. Building on these findings, the present study investigated how v-miR inhibition affects corneal immune responses. Using corneal tissue RNA from HSV-1-infected mice, we performed an immune profiling PCR array by analyzing the expression of 88 genes associated with immune cell markers and polarization states. Topical inhibition of miR-H1-5p, miR-H3-3p, and miR-H6-3p resulted in distinct patterns of immune gene expression compared with the control treatment. Inhibition of these v-miRs altered 16, 31, and 57 immune-related genes, respectively, spanning both myeloid- and lymphoid-associated pathways. Notably, the anti-inflammatory genes Arg1 and Il10 were consistently upregulated across all v-miR inhibitor-treated groups. In parallel, increased expression of the pro-resolution enzymes 15-lox and Alox5 suggested enhanced engagement of resolution pathways. Mechanistic studies demonstrated that v-miRs directly target immune regulatory genes through binding sites within their untranslated regions. Together, these findings suggest that HSV-1 v-miRs contribute to corneal immunopathology by suppressing anti-inflammatory and pro-resolving immune pathways, and that targeted inhibition of v-miRs may promote immune resolution during HSV-1-induced keratitis.

Objective: To investigate the protective effects and molecular mechanisms of apigenin (API) on lipopolysaccharide (LPS)-induced corneal inflammation.

Methods: Immortalized human corneal epithelial cell line (HCECs) and primary human corneal epithelial cells (PHCECs) were used to establish LPS-induced inflammation models in vitro. IL-6 and IL-8 mRNA and protein levels were assessed by qRT-PCR and ELISA. Transcriptome sequencing and KEGG/GO enrichment analyses were performed to identify key pathways. Western blotting evaluated the activation of MAPK (ERK, JNK, P38) and NF-κB (P65, IκBα) pathway proteins, and immunofluorescence was used to examine P65 nuclear translocation. Furthermore, following the establishment of an inflammation model via intrastromal LPS injection in mice, API was administered to assess its impact on ocular inflammation and pro-inflammatory cytokine expression.

Results: API significantly suppressed the expression and secretion of key pro-inflammatory mediators in LPS-stimulated corneal epithelial cells including IL-6, IL-8, and COX-2. Transcriptomic analysis confirmed the MAPK and NF-κB pathways as critical components in its anti-inflammatory action. API inhibited the phosphorylation of key proteins in the MAPK-JNK/ERK and NF-κB signaling pathways, blocked the nuclear translocation of P65. In mice, API alleviated corneal edema and inflammatory cell infiltration and decreased IL-6 and TNF-α levels in corneal tissue.

Conclusion: API effectively attenuates LPS-induced corneal inflammation by inhibiting JNK/ERK and NF-κB signaling pathways and downstream effectors, thereby reducing the production of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α.

This study focuses on the challenge of pathogen detection in keratitis, aiming to develop a dual target detection strip using recombinant enzyme polymerase amplification (RPA) combined with lateral chromatography strip (LFS) technology, and to detect the specific DNA sequences of Herpes simplex virus type 1 (HSV-1) and Fusarium keratoplasticum (F. keratoplasticum). To test the detection system in different ocular surface samples for keratitis pathogens diagnosis. The RPA primers with good specificity for HSV-1 and F. keratoplasticum were designed and screened separately, and further modified into probes for a dual target RPA-LFS detection system. This LFS test strip can simultaneously detect HSV-1 and F. keratoplasticum, and distinguish them by the positive bands at different test lines. The detection limit of this test strip for two targets is 203.69 copies/reaction for HSV-1 and 84.91 copies/reaction for F. keratoplasticum. There is no cross reactivity with DNA of other common pathogenic microorganisms of ocular surface except for Candida albicans. In clinical sample testing, the dual target detection system has shown reliable detection performance for both eye swaps and cornea tissue. The dual targets RPA-LFS detection system in this study could be completed within 20 minutes with visualized results, the coincidence rate with qPCR was 93.3%, indicating its broad clinical application prospects for the clinical diagnosis and treatment of infectious keratitis.

The objective of this study was to explore the lipid metabolic changes in the active thyroid-associated ophthalmopathy (TAO) through tear lipidomics analysis, screen for biomarkers related to disease activity, and analyze their correlation with clinical features. The study included 32 patients with active TAO and 30 patients with inactive TAO. Liquid chromatography-mass spectrometry (LC-MS) was used to perform lipidomics analysis on tear samples to identify differential lipid molecules. Multivariate statistical analyses, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were conducted, and machine learning algorithms were employed to evaluate the predictive ability of lipid biomarkers. Additionally, clinical characteristics and blood indicators of the patients were collected to analyze their correlation with the lipid biomarkers. The study identified 247 significantly different lipids in the tears of patients with active TAO, of which 104 were upregulated, mainly involving sphingolipids, glycerophospholipids, and glycerolipids. Through machine learning, four lipids (BisMePA(36:6e), MGMG(38:0), PC(38:3), SM(d38:1)) were selected, which showed good predictive ability for TAO activity (AUC >0.8 for all). Moreover, these lipids were significantly correlated with blood lipid indicators, exophthalmos degree, Schirmer I test, and the area of the foveal avascular zone (FAZ) of the retina. This tear lipidomics analysis provides a new approach for screening biomarkers in the active phase of TAO. The identified lipid biomarkers are significantly correlated with clinical features and have potential clinical application value.

Phosphoinositides (PIs) are a family of seven low abundance membrane lipids, each with distinct signaling functions. The phosphoinositide kinase PIKfyve generates phosphoinositide-3,5-bisphosphate (PI(3,5)P₂) and PI5P. Emerging evidence implicates PIKfyve in key cellular processes, including autophagy, phagocytosis, endosomal trafficking, lysosomal maintenance, and melanosome formation. Complete loss of PIKfyve function is embryonic lethal in model organisms. In humans, heterozygous mutations in PIKFYVE are associated with Fleck corneal dystrophy and congenital cataracts. In this study, we investigate the role of PIKfyve in photoreceptors and the adjacent retinal pigment epithelium (RPE), host to dynamic endolysosomal pathways required for enduring the high oxidative stress environment, transporting metabolites and phototransduction components, and the breakdown of outer segment discs. To assess PIKfyve function in the retina and RPE in our zebrafish model, we employed CRISPR/Cas9-mediated gene editing and pharmacological inhibition using the specific PIKfyve inhibitor apilimod. Loss of PIKfyve activity leads to RPE expansion characterized by the accumulation of LC3- and LAMP1-positive vacuoles, along with defects in phagosome degradation and minor changes to melanosome biogenesis. Photoreceptors deprived of PIKfyve function develop a single large vacuole in the inner segment, while the OS remains largely intact over the timespan analyzed. Electroretinography (ERG) recordings revealed complete visual impairment in pikfyve crispant larvae and significantly reduced visual function in larvae treated with apilimod post embryogenesis. These findings highlight the critical role of PIKfyve in the development and homeostasis of the RPE and retina.