Reticulated Retinoic Acid Synthesis is Implicated in the Pathogenesis of Dry Eye in Aqp5 Deficiency Mice.

Purpose: Abnormalities in aquaporins are implicated in the pathological progression of dry eye syndrome. Retinoic acid (RA) regulates cellular proliferation, differentiation, and apoptosis in the cornea, thereby being associated with dry eye disease (DED). The objective of this study is to explore the underlying mechanisms responsible for RA metabolic abnormalities in corneas lacking aquaporin 5 (AQP5). Methods: Dry eye (DE) models were induced via subcutaneous scopolamine hydrobromide. Aqp5 knockout (Aqp5-/-) mice and DE mice were utilized to assess corneal epithelial alterations. Tear secretion, goblet cell counts, and corneal punctate defects were evaluated. The impact of Aqp5 on RA-related enzymes and receptors was investigated using pharmacological RA or SR (A JunB inhibitor), a transcription factor JunB inhibitor, treatment in mouse corneal epithelial cells (CECs), or human corneal epithelial cells (HCECs). The HCECs and NaCl-treated HCECs underwent quantitative real-time PCR (qRT-PCR), immunofluorescent, Western blot, and TUNEL assays. The regulation of transcription factor JunB on Aldh1a1 was explored via ChIP-PCR. Results: Aqp5 and Aldh1a1 were reduced in both CECs of DE mice and NaCl-induced HCECs. Aqp5-/- mice exhibited DE phenotype and reduced Aldh1a1. RA treatment reduced apoptosis, promoted proliferation, and improved the DE phenotype in Aqp5-/- mice. JunB enrichment in the Aldh1a1 promoter was identified by ChIP-PCR. SR significantly increased Aldh1a1 expression, Ki67, and ΔNp63-positive cells, and decreased TUNEL-positive cells in CECs and HCECs. Conclusions: Our findings demonstrated the downregulation of Aqp5 expression and aberrant RA metabolism in DE conditions. Knockout of Aqp5 resulted in reduced production of RA through activation of JunB, subsequently leading to the manifestation of DE symptoms.

AQP5 deficiency promotes the senescence of lens epithelial cells through mitochondrial dysfunction.

Cataract is lens opacity, which is a common blinding eye disease worldwide. Aquaporin 5 (AQP5) is expressed in the human and mouse lenses. This study aimed to investigate the underlying mechanisms of AQP5 in the senescence of lens epithelial cells (LECs). Primary LECs were isolated and cultured from Aqp5+/+ and Aqp5-/- mice. Western blot or immunofluorescence staining of p16, Ki67, MitoSOX, JC-1 and phalloidin was used in the experiments to evaluate the changes in the primary LECs. The primary Aqp5-/- LECs showed increased p16 expression and mitochondrial reactive oxygen species, decreased mitochondrial membrane potential and activity, and cytoskeletal disorders. When the cells were pretreated with Mito-TEMPO, the Aqp5-/- mice showed decreased p16 expression, reduced mitochondrial dysfunction and cytoskeletal disorders. Our results revealed that AQP5 deficiency promotes the senescence of primary LECs through mitochondrial dysfunction. This provides a new perspective for the treatment of cataracts by regulating AQP5 expression.

Regulation of Axon Guidance by Slit2 and Netrin-1 Signaling in the Lacrimal Gland of Aqp5 Knockout Mice.

Purpose: Dry eye disease (DED) is multifactorial and associated with nerve abnormalities. We explored an Aquaporin 5 (AQP5)-deficiency-induced JunB activation mechanism, which causes abnormal lacrimal gland (LG) nerve distribution through Slit2 upregulation and Netrin-1 repression. Methods: Aqp5 knockout (Aqp5-/-) and wild-type (Aqp5+/+) mice were studied. LGs were permeabilized and stained with neuronal class III ß-tubulin, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). Whole-mount images were acquired through tissue clearing and 3D fluorescence imaging. Mouse primary trigeminal ganglion (TG) neurons were treated with LG extracts and Netrin-1/Slit2 neutralizing antibody. Transcription factor (TF) prediction and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) experiments verified the JunB binding and regulatory effect on Netrin-1 and Slit2. Results: Three-dimensional tissue and section immunofluorescence showed reduced LG nerves in Aqp5-/- mice, with sympathetic and sensory nerves significantly decreased. Netrin-1 was reduced and Slit2 increased in Aqp5-/- mice LGs. Aqp5+/+ mice LG tissue extracts (TEs) promoted Aqp5-/- TG neurons axon growth, but Netrin-1 neutralizing antibody (NAb) could inhibit that promotion. Aqp5-/- mice LG TEs inhibited Aqp5+/+ TG axon growth, but Slit2 NAb alleviated that inhibition. Furthermore, JunB, a Netrin-1 and Slit2 TF, could bind them and regulate their expression. SR11302, meanwhile, reversed the Netrin-1 and Slit2 shifts caused by AQP5 deficiency. Conclusions: AQP5 deficiency causes LG nerve abnormalities. Persistent JunB activation, the common denominator for Netrin-1 suppression and Slit2 induction, was found in Aqp5-/- mice LG epithelial cells. This affected sensory and sympathetic nerve fibers' distribution in LGs. Our findings provide insights into preventing, reversing, and treating DED.

The role of neutrophils in corneal nerve regeneration.

BACKGROUND: To investigate the role of neutrophils in corneal nerve regeneration. METHODS: A mouse model simulating corneal nerve injury was established and samples from corneal scraping with and without neutrophil closure were collected. These samples were used for corneal nerve staining, ribonucleic acid sequencing, and bioinformatics. Differential expression analysis was used to perform enrichment analysis to identify any significant differences between these two groups. The differential genes were then intersected with neutrophil-associated genes and a protein-protein interaction network was constructed using the intersected genes. The immune infiltration between the two groups was examined along with the immune cell variation between the high and low gene expression groups. RESULTS: Neutrophil removal delays corneal epithelial and nerve regeneration. A total of 546 differential genes and 980 neutrophil-associated genes, with 27 genes common to both sets were obtained. Molecular Complex Detection analysis yielded five key genes, namely integrin subunit beta 2 (ITGB2), matrix metallopeptidase 9 (MMP9), epidermal growth factor (EGF), serpin family E member 1 (SERPINE1), and plasminogen activator urokinase receptor (PLAUR). Among these genes, ITGB2, SERPINE1, and PLAUR exhibited increased expression in the neutrophil-confined group, while MMP9 and EGF showed decreased expression, with MMP9 and EGF displaying a more significant difference. Immune infiltration was also observed between the two groups, revealing significant differences in the infiltration of M0 macrophages, activated mast cells, and neutrophils. Moreover, the neutrophil levels were lower in the groups with low MMP9 and EGF expressions and higher in the high-expression group. CONCLUSION: Neutrophil confinement might significantly affect the MMP9 and EGF expression levels. Strategies to inhibit MMP9 could potentially yield therapeutic benefits.

Aquaporin5 Deficiency Aggravates ROS/NLRP3 Inflammasome-Mediated Pyroptosis in the Lacrimal Glands.

Purpose: The pathogenesis of the lacrimal glands (LGs) is facilitated by inflammation mediated by the NACHT, LRR, and NLRP3 inflammasomes in dry eye disease. This research aimed to explore the protective effects of Aquaporin 5 (AQP5) on LGs by inhibiting reactive oxygen species (ROS) and the NLRP3 inflammasome. Methods: AQP5 knockout (AQP5-/-) mice were used to evaluate pathological changes in LGs. ROS generation was detected with a dichlorodihydro-fluorescein diacetate assay. Lipid metabolism was assessed by Oil Red O staining. The reversal of the mitochondrial membrane potential was detected using a JC-1 fluorescent probe kit. The effect of AQP5 on NLRP3/caspase-1/Gasdermin-D (GSDMD)-mediated pyroptosis was examined using pharmacological treatment of N-acetyl L-cysteine or MCC950. Results: AQP5 loss significantly increased ROS generation, lipid metabolism disorders, TUNEL-positive cells, and reversal of the mitochondrial membrane potential in the AQP5-/- LGs. NLRP3 upregulation, increased caspase-1 and GSDMD activity, and enhanced IL-1ß release were detected in the AQP5-/- mouse LGs and primary LG epithelial cells. MCC950 significantly suppressed NLRP3 inflammasome-related pyroptosis induced by AQP5 deficiency in LGs and primary LG epithelial cells. Furthermore, we discovered that prestimulating the AQP5-/- primary LG epithelial cells with N-acetyl L-cysteine decreased NLRP3 expression, caspase-1 and GSDMD activity levels, and IL-1ß release. Conclusions: Our results revealed that AQP5 loss promoted NLRP3 inflammasome activation through ROS generation. Inhibiting the ROS or NLRP3 inflammasome significantly alleviated the damage and pyroptosis of AQP5-deficient LG epithelial cells, which could provide new insights into dry eye disease.

Loss of aquaporin 5 contributes to the corneal epithelial pathogenesis via Wnt/ß-catenin pathway.

AQP5 plays a crucial role in maintaining corneal transparency and the barrier function of the cornea. Here, we found that in the corneas of Aqp5-/- mice at older than 6 months, loss of AQP5 significantly increased corneal neovascularization, inflammatory cell infiltration, and corneal haze. The results of immunofluorescence staining showed that upregulation of K1, K10, and K14, and downregulation of K12 and Pax6 were detected in Aqp5-/- cornea and primary corneal epithelial cells. Loss of AQP5 aggravated wound-induced corneal neovascularization, inflammation, and haze. mRNA sequencing, western blotting, and qRT-PCR showed that Wnt2 and Wnt6 were significantly decreased in Aqp5-/- corneas and primary corneal epithelial cells, accompanied by decreased aggregation in the cytoplasm and nucleus of ß-catenin. IIIC3 significantly suppressed corneal neovascularization, inflammation, haze, and maintained corneal transparent epithelial in Aqp5-/- corneas. We also found that pre-stimulated Aqp5-/- primary corneal epithelial cells with IIIC3 caused the decreased expression of K1, K10, and K14, the increased expression of K12, Pax6, and increased aggregation in the cytoplasm and nucleus of ß-catenin. These findings revealed that AQP5 may regulate corneal epithelial homeostasis and function through the Wnt/ß-catenin signaling pathway. Together, we uncovered a possible role of AQP5 in determining corneal epithelial cell fate and providing a potential therapeutic target for corneal epithelial dysfunction.

Small Incision Lenticule Extraction (SMILE) and Laser in Situ Keratomileusis (LASIK) Used to Treat Myopia and Myopic Astigmatism: A Systematic Review and Meta-analysis of Randomized Clinical Trials.

PURPOSES: The purpose of this meta-analysis is to systematically compare the safety, efficacy, and predictability of small incision lenticule extraction (SMILE) and laser in situ keratomileusis (LASIK). METHODS: This study covered the data searched from the PubMed, the EMBASE and the Cochrane Library. The Cochrane Handbook was also referred to as evaluating the quality of the included studies. In addition, this meta-analysis was performed using Revman 5.4 software. RESULTS: A total of 11 randomized controlled trails (RCTs) were included. The proportion of eyes with refraction within ±0.5D was higher in LASIK group compared with SMILE group (RR, 0.91; 95% CI, 0.83 to 0.99; p = .04). The spherical aberration (SA) was smaller in SMILE group compared with LASIK group (RR, -0.12; 95% CI, -0.23 to -0.01; p = .04). There were no significant differences between two groups with regard to final mean refractive spherical equivalent (SE) (MD, -0.04; 95% CI, -0.12 to 0.03; p = .22), proportion of eyes losing one or more lines of corrected distance visual acuity (CDVA) (RR, 1.14; 95% CI, 0.58 to 2.27; p = .70), proportion of eyes with uncorrected distance visual acuity (UCVA) of 20/20 or better (RR, 0.99; 95% CI, 0.94 to 1.05; p = .71), postoperative mean logMAR UCVA (MD, 0.01; 95% CI, -0.00 to 0.03; p = .13), postoperative refraction within ±1.0D (RR, 1.00; 95% CI, 0.98 to 1.02; p = .60), postoperative astigmatism within ±0.25, 0.5 and 1.0D (RR, 0.80, 0.99, 1.00; 95% CI, 0.35 to 1.83, 0.94 to 1.05, 0.98 to 1.02; p = .60, 0.86, 0.87), postoperative higher order aberrations (HOAs) (RR, 0.00; 95% CI, -0.16 to 0.16; p = .99). CONCLUSION: For predictability, LASIK was superior to SMILE. There were comparably safety and efficacy for the correction of myopia and myopic astigmatism in SMILE and LASIK. SA was smaller after SMILE than after LASIK.

Microstent Implantation with Phacoemulsification Versus Phacoemulsification Alone for Patients with Open Angle Glaucoma: A Systematic Review and Meta-Analysis of Randomised Clinical Trials.

PURPOSES: The purpose of this meta-analysis is to systematically compare the IOP-lowering effect of different microstents combined with phacoemulsification versus phacoemulsification for patients with OAG and cataract. METHODS: This work was done through the data searched from PubMed, EMBASE, and the Cochrane Library. The Cochrane Handbook was also used to evaluate the quality of the included studies. In addition, this meta-analysis was performed using Revman 5.4 software. RESULTS: A total of 8 randomized controlled trials (RCTs) were included. Compared with phacoemulsification alone, microstent implantation with phacoemulsification resulted in significant reduction in the postoperative IOP (MD = -1.66, 95%CI: [-2.25 to -1.06]). Patients with medication free and patients with beyond 20% IOP reduction were significantly increased in the microstent implantation with phacoemulsification group compared with phacoemulsification alone group (RR = 1.54, 95%CI: [1.34 to 1.77]; RR = 1.34, 95%CI: [1.24 to 1.45]). CONCLUSION: Both microstent implantation with concurrent phacoemulsification and phacoemulsification alone result in a significant reduction in IOP. In terms of both reductions, microstent implantation with phacoemulsification significantly outperforms phacoemulsification alone.

Lacrimal gland homeostasis is maintained by the AQP5 pathway by attenuating endoplasmic reticulum stress inflammation in the lacrimal gland of AQP5 knockout mice.

Purpose: AQP5-/- mice spontaneously exhibit dry eye symptoms. The purpose of this study was to assess the endoplasmic reticulum (ER) stress-mediated inflammation generated by a deficiency of aquaporin 5 (AQP5) in the lacrimal gland. Methods: Hematoxylin and eosin (H&E) staining, Oil Red O staining, and transmission electron microscopy (TEM) analysis were performed to identify structural changes in lacrimal gland epithelial cells because of AQP5 deficiency. Corneal epithelial defects were assessed with sodium fluorescein staining. The expression profiles of mRNA and proteins were determined by quantitative real-time reverse transcription PCR (qRT-PCR) and western blot. Mice in the quercetin group were injected intraperitoneally with 40 mg/kg of quercetin, and the control group was injected with an equal volume of dimethyl sulfoxide (DMSO) for 4 weeks. Results: Aqueous tear secretion fell at about 50% in 1- and 6-month-old AQP5-/- mice compared with that of AQP5+/+ mice. TEM showed that the ER structure was damaged. ER stress was significantly increased in the lacrimal gland of AQP5-/- mice. Lipid droplets accumulated in the matrix and acinar cells, and changes occurred in the lipid metabolism and gene expression levels for PPARα, CPT1α, and CPT2 in the AQP5-/- mice. Immune cell infiltration and increases in the gene expression levels of the chemokines CXCL1, CXCL2, and CCL5 were found in the lacrimal gland of AQP5-/- mice. Quercetin partially reversed ER stress levels, inflammation, and lipid accumulation, and it inhibited tear secretion. Conclusions: The study data indicated that a deficiency of AQP5 induced pathophysiological changes and functional decompensation of the lacrimal gland. Quercetin may improve the inflammation in the lacrimal glands of AQP5-/- mice by regulating the ER stress levels.

Modified ulcer debridement in the treatment of the superficial fungal infection of the cornea.

AIM: To evaluate the efficacy of modified corneal ulcer debridement in superficial fungal keratitis unresponsive to medications. METHODS: A total of 209 patients (209 eyes) with fungal keratitis, involving no more than 50% of the stromal depth and not responding to antifungal agents for 2wk, were recruited in this retrospective, noncomparative study. The patients were treated with modified corneal ulcer debridement. All visible corneal infiltrates were removed under an operating microscope to obtain a clean stromal bed and smooth incised edges. Antifungal drugs were used immediately after surgery. Healing time of the ulcers was recorded. Fungal recurrence, visual acuity, corneal thickness and risk factors for treatment failure were monitored. RESULTS: The follow-up was 13.6±5.8mo. The corneal ulcers healed in 195 of 209 eyes (93.3%), with a mean healing time of 8.4±6.8d. The other 14 eyes were further treated by penetrating keratoplasty (PK) (1 eye), anterior lamellar keratoplasty (LK) (7 eyes), conjunctival flap covering (4 eyes) or amniotic membrane transplantation (2 eyes). The best corrected visual acuity (BCVA) was ≥20/70 in 80.3% of the eyes, ≥20/40 in 56.9% of the eyes, and ≥20/25 in 27.3% of the eyes. The corneas at the lesions became thinner, but all in the safe range. No fungal recurrence or corneal ectasis developed during the follow-up. The risk of treatment failure was higher in patients with preoperative hypopyon (P=0.036) and ever using steroid (P=0.025). CONCLUSION: Modified surgical debridement is a simple and effective method for the treatment of superficial fungal infection of the cornea, with improved visual acuity and no recurrence. Such an intervention in time can rapidly control fungal infection and largely shorten corneal ulcer healing time.

A Chinese family with Axenfeld-Rieger syndrome: report of the clinical and genetic findings.

AIM: To describe a Chinese family affected by a severe form of Axenfeld-Rieger syndrome (ARS) and characterize the molecular defect in PITX2 in the family. METHODS: Patients presented with typical ARS from a Chinese family were investigated. We performed genome-wide linkage scan and exome sequencing to identify the pathogenic mutations. Candidate mutations were verified for co-segregation in the whole pedigree using Sanger sequencing. Real-time polymerase chain reaction (RT-PCR) and Western blotting were performed to verify the expression of the pathogenic gene. RESULTS: Genome-wide linkage and exome sequencing analyses showed PITX2 as the disease candidate gene. A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G) that co-segregated with the disease phenotype was discovered in the family. The PITX2 messenger ribonucleic acid and protein levels were about 50% lower in patients with ARS than in unaffected family members in the family. CONCLUSION: Our findings implicate the first intronic mutation of the PITX2 gene in the pathogenesis of a severe form of ARS in a Chinese family. This study highlights the importance of a systematic search for intronic mutation in ARS cases for which no mutations in the exons of PITX2 have been found.