Multi-omics profiling of retinal pigment epithelium reveals enhancer-driven activation of RANK-NFATc1 signaling in traumatic proliferative vitreoretinopathy.

During the progression of proliferative vitreoretinopathy (PVR) following ocular trauma, previously quiescent retinal pigment epithelial (RPE) cells transition into a state of rapid proliferation, migration, and secretion. The elusive molecular mechanisms behind these changes have hindered the development of effective pharmacological treatments, presenting a pressing clinical challenge. In this study, by monitoring the dynamic changes in chromatin accessibility and various histone modifications, we chart the comprehensive epigenetic landscape of RPE cells in male mice subjected to traumatic PVR. Coupled with transcriptomic analysis, we reveal a robust correlation between enhancer activation and the upregulation of the PVR-associated gene programs. Furthermore, by constructing transcription factor regulatory networks, we identify the aberrant activation of enhancer-driven RANK-NFATc1 pathway as PVR advanced. Importantly, we demonstrate that intraocular interventions, including nanomedicines inhibiting enhancer activity, gene therapies targeting NFATc1 and antibody therapeutics against RANK pathway, effectively mitigate PVR progression. Together, our findings elucidate the epigenetic basis underlying the activation of PVR-associated genes during RPE cell fate transitions and offer promising therapeutic avenues targeting epigenetic modulation and the RANK-NFATc1 axis for PVR management.

Deciphering the molecular landscape of ionising radiation-induced eye damage with the help of genomic data mining.

Even at low levels, exposure to ionising radiation can lead to eye damage. However, the underlying molecular mechanisms are not yet fully understood. We aimed to address this gap with a comprehensive in silico approach to the issue. For this purpose we relied on the Comparative Toxicogenomics Database (CTD), ToppGene Suite, Cytoscape, GeneMANIA, and Metascape to identify six key regulator genes associated with radiation-induced eye damage (ATM, CRYAB, SIRT1, TGFB1, TREX1, and YAP1), all of which have physical interactions. Some of the identified molecular functions revolve around DNA repair mechanisms, while others are involved in protein binding, enzymatic activities, metabolic processes, and post-translational protein modifications. The biological processes are mostly centred on response to DNA damage, the p53 signalling pathway in particular. We identified a significant role of several miRNAs, such as hsa-miR-183 and hsamiR-589, in the mechanisms behind ionising radiation-induced eye injuries. Our study offers a valuable method for gaining deeper insights into the adverse effects of radiation exposure.

A, B, C's of Trk Receptors and Their Ligands in Ocular Repair.

Neurotrophins are a family of closely related secreted proteins that promote differentiation, development, and survival of neurons, which include nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4. All neurotrophins signal through tropomyosin receptor kinases (TrkA, TrkB, and TrkC) which are more selective to NGF, brain-derived neurotrophic factor, and neurotrophin-3, respectively. NGF is the most studied neurotrophin in the ocular surface and a human recombinant NGF has reached clinics, having been approved to treat neurotrophic keratitis. Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 are less studied neurotrophins in the ocular surface, even though brain-derived neurotrophic factor is well characterized in glaucoma, retina, and neuroscience. Recently, neurotrophin analogs with panTrk activity and TrkC selectivity have shown promise as novel drugs for treating dry eye disease. In this review, we discuss the biology of the neurotrophin family, its role in corneal homeostasis, and its use in treating ocular surface diseases. There is an unmet need to investigate parenteral neurotrophins and its analogs that activate TrkB and TrkC selectively.

Long-term treatment with 3, 4-methylenedioxymethamphetamine caused retina damage in C57BL/6 mice / Tratamento a longo prazo com 3, 4-metilenedioximetanfetamina causando dano na retina em camundongos C57BL/6

ABSTRACT Purpose: As a class of psychostimulant drugs, amphetamines are widely abused for their stimulant, euphoric, and hallucinogenic properties. Many of these effects result from acute increases in dopamine and serotonin neurotransmission. Following the onset of these effects, 3,4 methylenedioxymethamphetamine produces persistent damage to dopamine and serotonin nerve terminals, resulting in long-lasting neurotoxicity. The purpose of this investigation was to assess the effects of treatment with low dose of methylenedioxymethamphetamine on retinal function of C57BL/6 mice and its underlying mechanisms. Methods: C57BL/6 mice were divided randomly into two groups (n=10): one group was treated with phosphate buffered saline by intraperitoneal injection daily; the other group was treated with 1 mg/kg methylenedioxymethamphetamine by intraperitoneal injection daily for three months. Electroretinography was used to test retinal function every month. H&E staining and terminal deoxynucleotidyl transferase assay were used to evaluate the retinal morphology and histology. Enzyme-linked immunosorbent assay assays were used to measure markers of oxidative stress and inflammatory factors. Gene and protein expression was detected by real-time PCR and western blot. Results: Three-month treatment with methylenedioxymethamphetamine induced significant retinal dysfunction via photoreceptor cell apoptosis by oxidative stress and inflammatory responses. Conclusions: These results suggest that long-term treatment with methylenedioxymethamphetamine increases inflammatory responses in photoreceptor cells resulting in retinal dysfunction in C57BL/6 mice. Thus, this investigation provides preclinical rationale for the retina damage caused by the methylenedioxymethamphetamine abuse.

RESUMO Objetivos: Como uma classe de drogas psicoesti mulantes, as anfetaminas são amplamente usadas por suas propriedades estimulantes, eufóricas e alucinógenas. Muitos desses efeitos resultam de aumentos agudos na neurotransmissão da dopamina e da serotonina. Após o início desses efeitos, a 3,4-metilenedioximetanfetamina produz danos persistentes nos terminais nervosos de dopamina e serotonina, resultando em neurotoxicidade duradoura. O objetivo desta investigação foi avaliar os efeitos do tratamento baixa dose de metilenedioximetanfetamina na função da retina em camundongos C57BL/6 e seus mecanismos subjacentes. Métodos: Camundongos C57BL/6 foram divididos aleatoriamente em dois grupos (n=10): um grupo foi tratado com solução salina tamponada de fosfato por injeção intraperitoneal diária; o outro grupo foi tratado com 1 mg/kg de metilenedioximetanfetamina por injeção intraperitoneal diária durante 3 meses. Eletroretinografia foi utilizada para testar a função da retina a cada mês. A coloração H&E e análise com deoxinucleotidil terminal transferase foram utilizados para avaliar a morfologia e histologia da retina. Testes de imunoabsorção enzimática foram utilizados para medir marcadores de estresse oxidativo e fatores inflamatórios. A expressão de genes e proteínas foi detectada por PCR em tempo real e western blot. Resultados: O tratamento de três meses com metilenedioximetanfetamina induziu disfunção de retina significativa por apoptose de células fotorreceptoras por estresse oxidativo e resposta inflamatória. Conclusões: Estes resultados sugerem que o tratamento a longo prazo com metilenedioximetanfetamina aumenta as respostas inflamatórias em células fotorreceptoras, resultando em disfunção de retina em camundongos C57BL/6. Assim, a investigação foence uma justificação pré-clínica para os danos na retina causados pelo abuso de metilenedioximetanfetamina.

Long-term treatment with 3,4-methylenedioxymethamphetamine caused retina damage in C57BL/6 mice.

PURPOSE: As a class of psychostimulant drugs, amphetamines are widely abused for their stimulant, euphoric, and hallucinogenic properties. Many of these effects result from acute increases in dopamine and serotonin neurotransmission. Following the onset of these effects, 3,4 methylenedioxymethamphetamine produces persistent damage to dopamine and serotonin nerve terminals, resulting in long-lasting neurotoxicity. The purpose of this investigation was to assess the effects of treatment with low dose of methylenedioxymethamphetamine on retinal function of C57BL/6 mice and its underlying mechanisms. METHODS: C57BL/6 mice were divided randomly into two groups (n=10): one group was treated with phosphate buffered saline by intraperitoneal injection daily; the other group was treated with 1 mg/kg methylenedioxymethamphetamine by intraperitoneal injection daily for three months. Electroretinography was used to test retinal function every month. H&E staining and terminal deoxynucleotidyl transferase assay were used to evaluate the retinal morphology and histology. Enzyme-linked immunosorbent assay assays were used to measure markers of oxidative stress and inflammatory factors. Gene and protein expression was detected by real-time PCR and western blot. RESULTS: Three-month treatment with methylenedioxymethamphetamine induced significant retinal dysfunction via photoreceptor cell apoptosis by oxidative stress and inflammatory responses. CONCLUSIONS: These results suggest that long-term treatment with methylenedioxymethamphetamine increases inflammatory responses in photoreceptor cells resulting in retinal dysfunction in C57BL/6 mice. Thus, this investigation provides preclinical rationale for the retina damage caused by the methylenedioxymethamphetamine abuse.

Permanent neuroglial remodeling of the retina following infiltration of CSF1R inhibition-resistant peripheral monocytes.

Previous studies have demonstrated that ocular injury can lead to prompt infiltration of bone-marrow-derived peripheral monocytes into the retina. However, the ability of these cells to integrate into the tissue and become microglia has not been investigated. Here we show that such peripheral monocytes that infiltrate into the retina after ocular injury engraft permanently, migrate to the three distinct microglia strata, and adopt a microglia-like morphology. In the absence of ocular injury, peripheral monocytes that repopulate the retina after depletion with colony-stimulating factor 1 receptor (CSF1R) inhibitor remain sensitive to CSF1R inhibition and can be redepleted. Strikingly, consequent to ocular injury, the engrafted peripheral monocytes are resistant to depletion by CSF1R inhibitor and likely express low CSF1R. Moreover, these engrafted monocytes remain proinflammatory, expressing high levels of MHC-II, IL-1ß, and TNF-α over the long term. The observed permanent neuroglia remodeling after injury constitutes a major immunological change that may contribute to progressive retinal degeneration. These findings may also be relevant to other degenerative conditions of the retina and the central nervous system.

Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation.

In the pond culture of Eriocheir sinensis, high limb-autotomy seriously affects the quality and culture's economic efficiency. Based on our previous studies, limb autotomy can induce the changes of hematological immune response in E. sinensis hemolymph. Eyestalk ablation can accelerate the regeneration of limbs after autotomy. To detect the important functional genes related to the hematological molecular immunity of E. sinensis, we compared and analyzed the hemolymph transcriptome data of the intact crab, left cheliped autotomized crabs and bilateral eyestalk ablation crabs with high-throughput sequencing techniques. The results showed that the three groups obtained 62 172 414, 68 143 682, and 67 811 618 clean reads, respectively. A total of 9567 differentially expressed genes were obtained by multiple comparison of the three groups' libraries. Gene ontology (GO) functional classification analysis shows that the differential genes belong to 42 categories of biological process, cellular components and molecular function. The differentially expressed genes in the three libraries were enriched to 344 specific KEGG metabolic pathways by KEGG enrichment analysis, such as the up-regulated gene (dual oxidase (Duox), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAQ)) in MAPK signaling pathway, the up-regulated gene (aldehyde dehydrogenase 1 (ALDH 1)) and down-regulated gene (UDP-glucuronosyltransferase 2 (UGT 2)) in metabolism of the xenobiotics by cytochrome P450 pathway, the down-regulated gene (actin gene (AG), heat shock protein 90 (HSP 90)) in fluid shear stress and atherosclerosis pathway. To verify the expression levels of DEGs identified by RNA-Seq, the above six hematological immune-related genes were selected for qRT-PCR validation, the qRT-PCR results were consistent with the DEGs results. Our research obtained abundant E. sinensis hemolymph transcriptome information by RNA-Seq, which provides multi-level information for the cloning of novel genes and the study of hemolymph molecular immunology mechanisms of E. sinensis.

Effects of the loss of conjunctival Muc16 on corneal epithelium and stroma in mice.

PURPOSE: To examine the role of conjunctival Muc16 in the homeostasis of the ocular surface epithelium and stroma using Muc16-null knockout (KO) mice. METHODS: We used KO mice (n = 58) and C57/BL6 (WT) mice (n = 58). Histology and immunohistochemistry were employed to analyze the phenotypes in the ocular surface epithelium. The expression of phospho-Stat3, AP-1 components, interleukin 6 (IL-6), and tumor necrosis factor-α (TNFα) in the cornea and conjunctiva was examined. The shape of the nuclei of corneal epithelial cells was examined to evaluate intraepithelial cell differentiation. Epithelial cell proliferation was studied using bromo-deoxyuridine labeling. Finally, the wound healing of a round defect (2-mm diameter) in the corneal epithelium was measured. The keratocyte phenotype and macrophage invasion in the stroma were evaluated after epithelial repair. RESULTS: The loss of Muc16 activated Stat3 signal, affected JunB signal, and upregulated the expression of IL-6 in the conjunctiva. Basal-like cells were observed in the suprabasal layer of the corneal epithelium with an increase in proliferation. The loss of Muc16 accelerated the wound healing of the corneal epithelium. The incidence of myofibroblast appearance and macrophage invasion were more marked in KO stroma than in WT stroma after epithelial repair. CONCLUSIONS: The loss of Muc16 in the conjunctiva affected the homeostasis of the corneal epithelium and stroma. The mechanism might include the upregulation of the inflammatory signaling cascade (i.e., Stat3 signal, and IL-6 expression in the KO conjunctiva). Current data provides insight into the research of the pathophysiology of dry eye syndrome.

Impairment of corneal epithelial wound healing in a TRPV1-deficient mouse.

PURPOSE: To examine whether the absence or blockage of an ion channel receptor, transient receptor potential vanilloid subtype 1 (TRPV1), affects the healing of an epithelial injury using an experimental model of an epithelial defect in animal cornea. METHODS: The expression of TRPV1 in the corneal epithelium was examined using immunohistochemistry in mice and rats. The migration of the corneal epithelium was examined in epithelium-debrided rat cornea in organ culture in the presence or absence of a TRPV1 agonist or its antagonist. Epithelial migration and cell proliferation following the debridement were examined in the cornea of a TRPV1-null mouse. Real-time RT-PCR was performed in samples of healing corneas to analyze the expression pattern of epithelial migration-related components (i.e., IL-6, substance P, and TGF-ß1). RESULTS: TRPV1 was detected mainly in the basal layer of mouse or rat corneal epithelium. Adding a TRPV1 receptor agonist to the culture medium enhanced epithelial healing in the rat cornea, and a TRPV1 antagonist retarded it in organ culture. The loss of TRPV1 did not affect the histology of the mouse cornea. In vivo analysis showed the loss of TRPV1-impaired re-epithelialization of the debrided area of the corneal epithelium by the suppression of both cell migration and proliferation. The lack of TRPV1 suppressed the expression of IL-6 and substance P but not of TGF-ß1 in response to epithelial debridement in mice. CONCLUSIONS: TRPV1 signal is required for the upregulation of IL-6 and substance P and the healing of debrided corneal epithelium in mice.

Conditional knockout of CTGF affects corneal wound healing.

PURPOSE: This study aimed to elucidate the role of connective tissue growth factor (CTGF) in healthy eyes and wounded corneas of mice and rabbits. Conditional knockout mice were used to determine the role of CTGF in corneal healing. METHODS: CTGF expression was determined using transgenic mice carrying CTGF promoter driven-eGFP, quantitative RT-PCR, and immunofluorescent staining. Mice that carried two floxed CTGF alleles and a Cre/ERT2 transgene under the control of human ubiquitin C (ubc) promoter were used to conditionally delete CTGF gene in a tamoxifen-inducible manner. Phototherapeutic keratectomy (PTK) was used to generate an acute corneal wound and corneal re-epithelialization was assessed by fluorescein staining. RESULTS: Connective tissue growth factor expression was found in multiple ocular tissues with relatively high levels in the corneal endothelium, lens subcapsular epithelium, and in the vasculature of the iris and retina. Wounded corneas responded with an immediate upregulation of CTGF in the epithelium at the wound margin and a sustained CTGF induction during re-epithelialization. At the onset of haze formation, CTGF protein becomes more focused in the basal epithelium. Deletion of the CTGF gene caused a 40% reduction (P < 0.01) in the cornea re-epithelialization rate in knockout mice compared with wild-type mice. CONCLUSIONS: Connective tissue growth factor is expressed in the naïve cornea, lens, iris, and retina, and is expressed immediately after epithelial injury. Loss of CTGF impairs efficient re-epithelialization of corneal wounds.

Heat shock protein 27 phosphorylation is involved in epithelial cell apoptosis as well as epithelial migration during corneal epithelial wound healing.

We reported the expression of phosphorylated HSP27 during epithelial wound healing in murine corneas (Jain et al., 2012) in July of 2012. This in vivo investigation demonstrated that the expression levels of phosphorylated HSP27 were greater in wounded corneal epithelial cells than in unwounded controls and that the localization of phosphorylated HSP27 was in the basal and superficial epithelia three days following corneal epithelial wounding. We suggested that phosphorylated HSP27 had a role in the early phase of corneal epithelial wound healing. The purpose of this study was to investigate the exact role of heat shock protein 27 (HSP27) phosphorylation for the wound healing of cultured human corneal epithelial cells (HCECs). HSP27-specific siRNAs and control-siRNAs, with no known homologous targets in HCECs, were created. The cultured HCECs were divided into two groups: Scrambled control-siRNA-transfected group vs. HSP27-specific siRNA-transfected group. The scratch-induced directional wounding assay, Western blotting, using antibodies against non-phosphorylated and phosphorylated HSP27, non-phosphorylated and phosphorylated Akt, and Bcl-2-associated X protein (Bax), immunofluorescence staining to determine the filament actin, flow cytometry to measure apoptosis, and proliferation assay were performed to determine the role of HSP27. Western blot assay showed that the expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding, compared with those in unwounded HCECs (all p < 0.05). Western blot assay also showed HSP27-specific siRNAs effectively blocked the expression of non-phosphorylated HSP27. The HSP27-specific siRNA-transfected group had more Bax expression, less phosphorylated Akt expression, and less non-phosphorylated and phosphorylated HSP27 expression (all p < 0.05). The scratch-induced directional wounding assay showed the HSP27-specific siRNA-transfected group with a less migrating cell number than the control-siRNA-transfected group (p < 0.05). Immunofluorescence staining showed that reorganization of actin cytoskeleton prominently decreased in the HSP27-specific siRNA-transfected group, compared with the control siRNA-tranfected group. Flow cytometry revealed that the HSP27-specific siRNA-transfected group had more HCEC apoptosis. Proliferation assay showed no difference between the two groups. In conclusion, the role of HSP27 in corneal epithelial wound healing can be epithelial cell apoptosis, as well as epithelial migration. HSP27 is involved in HCEC migration by the reorganization of actin cytoskeleton.

Regulation of Cdc42 expression and signaling is critical for promoting corneal epithelial wound healing.

PURPOSE: Cdc42, a member of Rho GTPases (guanosine triphosphatases), participates in cytokine- and growth factor-controlled biological functions in mammalian tissues. Here, we examined Cdc42 role in corneal epithelial wound healing and the influence of hepatocyte, keratinocyte, and epidermal growth factor (HGF, KGF, and EGF)-mediated signaling on Cdc42. METHODS: Epithelial wounds were created on the corneas of live rabbits by complete debridement and in rabbit corneal epithelial primary cultures through scratch injury. Cdc42 expression in cultures was suppressed using Cdc42 siRNA. Cdc42 activation was determined by pull-down assays with PAK-agarose beads. Cdc42 expression was analyzed by immunoblotting and immunofluorescence. Association of Cdc42 with cell-cycle proteins was identified by immunoprecipitation. RESULTS: In rabbit corneas, significant increase in Cdc42 expression that occurred 2 to 4 days after the injury coincided with wound closure, and by 8 days the expression reached near basal levels. Silencing of Cdc42 expression in cultures caused inhibition of wound closure as a result of 60% to 75% decrease in epithelial migration and growth. HGF, KGF, and EGF increased Cdc2 expression, activation, and its phosphorylation on ser71. Inhibition of growth factor-mediated PI-3K signaling resulted in the downregulation of Cdc42 expression and its phosphorylation. Increased association of cell-cycle proteins p27(kip) and cyclin-dependent kinase 4 (CDK4) with Cdc42; and phosphorylated Cdc42 with plasma membrane leading edges was also observed in the presence of growth factors. CONCLUSIONS: Cdc42 is an important regulator of corneal epithelial wound repair. To promote healing, Cdc42 may interact with receptor tyrosine kinase-activated signaling cascades that participate in cell migration and cell-cycle progression.

Novel serum proteomic signatures in a non-human primate model of retinal injury.

PURPOSE: To identify candidate protein biomarkers in sera indicative of acute retinal injury. METHODS: We used laser photocoagulation as a model of acute retinal injury in Rhesus macaques. In a paired-control study design, we collected serum from each animal (n=6) at 4 h, 1 day, and 3 days following a mock procedure and then again following retinal laser treatment that produced mild lesions. Samples were fractionated by isoelectric focusing, digested with trypsin, and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Spectral counting was used to determine relative protein abundances and identify proteins with statistically significant differences between control and treated sera. RESULTS: Mild retinal injury was confirmed by fundus photography and histological examination. The average number of total proteins detected by LC-MS/MS was 908±82 among samples from all three time points. Following statistical analysis and employing stringent filtering criteria, a total of 19 proteins were identified as being significantly more abundant in sera following laser-induced retinal injury, relative to control sera. Many of the proteins detected were unique to one time point. However, four proteins (phosphoglycerate kinase 1, keratin 18, Lewis alpha-3-fucosyltransferase, and ephrin receptor A2) showed differences that were significant at both 4 h and 1 day after laser treatment, followed by a decrease to baseline levels by day 3. CONCLUSIONS: A serum biomarker response to mild retinal laser injury was demonstrated in a primate model. Among the proteins detected with highest significant differences, most are upregulated within 24 h, and their appearance in the serum is transient. It is conceivable that a panel of these proteins could provide a means for detecting the acute-phase response to retinal injury. Further investigation of these candidate biomarkers and their correlation to retinal damage is warranted.

Pigment epithelium-derived factor gene therapy targeting retinal ganglion cell injuries: neuroprotection against loss of function in two animal models.

Lentiviral vectors are promising tools for the treatment of chronic retinal diseases including glaucoma, as they enable stable transgene expression. We examined whether simian immunodeficiency virus (SIV)-based lentiviral vector-mediated retinal gene transfer of human pigment epithelium-derived factor (hPEDF) can rescue rat retinal ganglion cell injury. Gene transfer was achieved through subretinal injection of an SIV vector expressing human PEDF (SIV-hPEDF) into the eyes of 4-week-old Wistar rats. Two weeks after gene transfer, retinal ganglion cells were damaged by transient ocular hypertension stress (110 mmHg, 60 min) and N-methyl-d-aspartic acid (NMDA) intravitreal injection. One week after damage, retrograde labeling with 4',6-diamidino-2-phenylindole (DAPI) was done to count the retinal ganglion cells that survived, and eyes were enucleated and processed for morphometric analysis. Electroretinographic (ERG) assessment was also done. The density of DAPI-positive retinal ganglion cells in retinal flat-mounts was significantly higher in SIV-hPEDF-treated rats compared with control groups, in both transient ocular hypertension and NMDA-induced models. Pattern ERG examination demonstrated higher amplitude in SIV-hPEDF-treated rats, indicating the functional rescue of retinal ganglion cells. These findings show that neuroprotective gene therapy using hPEDF can protect against retinal ganglion cell death, and support the potential feasibility of neuroprotective therapy for intractable glaucoma.

Detection of differentially expressed wound-healing-related glycogenes in galectin-3-deficient mice.

PURPOSE: A prior study showed that exogenous galectin-3 (Gal-3) stimulates re-epithelialization of corneal wounds in wild-type (Gal-3(+/+)) mice but, surprisingly, not in galectin-3-deficient (Gal-3(-/-)) mice. In an effort to understand why the injured corneas of Gal-3(-/-) mice are unresponsive to exogenous Gal-3, the present study was designed to determine whether genes encoding the enzymes that regulate the synthesis of glycan ligands of Gal-3 are differentially expressed in Gal-3(-/-) corneas compared with the Gal-3(+/+) corneas. METHODS: Glycogene microarray technology was used to identify differentially expressed glycosyltransferases in healing Gal-3(+/+) and Gal-3(-/-) corneas. RESULTS: Of approximately 2000 glycogenes on the array, the expression of 8 was upregulated and that of 14 was downregulated more than 1.3-fold in healing Gal-3(-/-) corneas. A galactosyltransferase, beta3GalT5, which has the ability to synthesize Gal-3 ligands was markedly downregulated in healing Gal-3(-/-) corneas. The genes for polypeptide galactosaminyltransferases (ppGalNAcT-3 and -7) that are known to initiate O-linked glycosylation and N-aspartyl-beta-glucosaminidase, which participates in the removal of N-glycans, were found to be upregulated in healing Gal-3(-/-) corneas. Microarray data were validated by qRT-PCR. CONCLUSIONS: Based on the known functions of the differentially expressed glycogenes, it appears that the glycan structures on glycoproteins and glycolipids, synthesized as a result of the differential glycogene expression pattern in healing Gal-3(-/-) corneas may lead to the downregulation of specific counterreceptors for Gal-3. This may explain, at least in part, why, unlike healing Gal-3(+/+) corneas, the healing Gal-3(-/-) corneas are unresponsive to the stimulatory effect of exogenous Gal-3 on re-epithelialization of corneal wounds.

Yin and yang in cytokine regulation of corneal wound healing: roles of TNF-alpha.

During wound healing, corneal tissue has to restore its transparency for proper vision. Various cytokines and growth factors are believed to orchestrate cellular behavior in a healing cornea. This review summarizes the roles of 1 such factor, the proinflammatory cytokine tumor necrosis factor (TNF)-alpha, in the process of wound healing in the cornea. Many studies have shown the anti-transforming growth factor-beta activity of TNF-alpha in cultured cell types. However, it remains unknown whether endogenous TNF-alpha has such an effect in the in vivo healing cornea. Recently, experiments that used TNF-alpha-deficient mice clearly showed that loss of TNF-alpha results in excess inflammation and fibrogenic reaction in response to external stimuli in lung and joint tissue. In the cornea, my group's experiments reveal that the lack of TNF-alpha potentiates pathogenic excess inflammation, fibrogenic response, and neovascularization in an alkali-burned mouse cornea. We uncovered the principal role of the lack of TNF-alpha in invaded macrophages, but not in corneal cells, in the development of this phenomenon by using Smad7 gene transfer, bone marrow transplantation, and cell culture experiments. These findings provide additional information for understanding the role of the cytokine network in corneal wound healing. Further studies are needed to determine if anti-TNF-alpha strategies might be effective in the treatment of ocular surface inflammatory or allergic disorders.

Regulation of corneal repair by particle-mediated gene transfer of opioid growth factor receptor complementary DNA.

OBJECTIVE: To determine whether molecular manipulation of the opioid growth factor receptor (OGFr) alters corneal reepithelialization following central corneal abrasion in rats. METHODS: The plasmid pcDNA3.1 + OGFr, carrying the rat OGFr complementary DNA in both the sense and antisense orientations, and empty vector (EV), were delivered by gene gun to the rat cornea. After 24 hours, corneas were abraded and reepithelialization was documented by fluorescein photography. Twenty-four hours after wounding, DNA synthesis (with bromodeoxyuridine) was examined. RESULTS: Eyes transfected with sense constructs of OGFr had corneal defects that were 24%, 52%, and 50% larger than the EV group at 16, 24, and 28 hours, respectively. Conversely, corneas transfected with antisense constructs of OGFr had corneal defects that were 56% and 48% smaller than the EV group at 16 and 24 hours, respectively. Bromodeoxyuridine labeling in the basal and suprabasal layers of the antisense group were increased 3.3- and 3.7-fold, respectively, in DNA synthesis from corresponding EV layers; DNA synthesis was comparable in the sense and EV groups. CONCLUSIONS: Excess OGFr delays reepithelialization, whereas attenuation of OGFr accelerates repair of the corneal surface. Clinical Relevance Inhibition of opioid growth factor action using gene therapy could be important in the treatment of corneal diseases such as nonhealing and recurrent erosions, diabetic keratopathy, and neurotrophic keratitis.

Genetic networks controlling retinal injury.

PURPOSE: The present study defines genomic loci underlying coordinate changes in gene expression following retinal injury. METHODS: A group of acute phase genes expressed in diverse nervous system tissues was defined by combining microarray results from injury studies from rat retina, brain, and spinal cord. Genomic loci regulating the brain expression of acute phase genes were identified using a panel of BXD recombinant inbred (RI) mouse strains. Candidate upstream regulators within a locus were defined using single nucleotide polymorphism databases and promoter motif databases. RESULTS: The acute phase response of rat retina, brain, and spinal cord was dominated by transcription factors. Three genomic loci control transcript expression of acute phase genes in brains of BXD RI mouse strains. One locus was identified on chromosome 12 and was highly correlated with the expression of classic acute phase genes. Within the locus we identified the inhibitor of DNA binding 2 (Id2) as a candidate upstream regulator. Id2 was upregulated as an acute phase transcript in injury models of rat retina, brain, and spinal cord. CONCLUSIONS: We defined a group of transcriptional changes associated with the retinal acute injury response. Using genetic linkage analysis of natural transcript variation, we identified regulatory loci and candidate regulators that control transcript levels of acute phase genes.

Transcriptional activation in lens epithelial cells following an ocular blunt trauma.

PURPOSE: To determine whether an ocular blunt trauma activates anterior ocular segment (cornea and lens) by examining the expression patterns of c-fos and c-jun mRNAs in these tissues of an eye of adult rat following a blunt trauma. SETTING: Department of Ophthalmology, Wakayama Medical University School of Medicine, Kimiidera, Wakayama, Japan. METHODS: Adult Wistar rats (n=36) were generally anesthetized by ether inhalation. One eye was hit with an iron sphere (30 gram) that fell to the eye from 1 m. After the procedure, the animals were killed and the affected eye was enucleated at 15, 30, 60, 120, and 180 minutes. In situ hybridization using radiolabeled oligoprobes was used to detect mRNAs of c-fos and c-jun in tissue. RESULTS: The c-fos and c-jun mRNAs were not detected in the epithelium of uninjured cornea and lens by in situ hybridization. The mRNAs for c-fos and c-jun were then detected in corneal epithelium from 15 to 60 minutes posttreatment, and were no longer observed thereafter. In lens epithelium, mRNA for c-fos or c-jun were transiently detected from 15 to 60 minutes or 30 minutes posttreatment, respectively. CONCLUSION: The c-fos and c-jun mRNAs were transiently expressed in corneal and lens epithelial cells after blunt trauma. Ocular blunt trauma activates corneal and lens epithelial cells without apparent corneal ablation or direct injury in the lens epithelium. Such activation in lens epithelium might be involved in cataractogenesis.

Patologías oftalmológicas más frecuentes en la consulta pediátrica del preescolar y escolar / More frequent ophthalmologic pathologies in the pediatric consultation of the preschool and school child

A continuación se revisarán de manera muy esquemática las patologías oculares más frecuentes en el preescolar y escolar, y también algunas patologías que si bien no son muy frecuentes, si son importantes diagnosticarlas y tratarlas precozmente, idealmente antes de los 8 años, es decir, antes de la maduración total del sistema visual, de lo contrario el pronóstico de ese niño se empobrece, su visión no alcanza una normalidad y va a terminar con una ambliopía. por lo tanto, es muy importante destacar el rol de la derivación temprana de aquellos pacientes que presentan alguna patología ocular importante como una ptosis palpebral, estrabismo o algún problema refractivo importante, entre otros, para prevenir el compromiso visual posterior.