Prostaglandin E(2) enhances proliferation, dedifferentiation and stem-like properties of rat retinal Müller glial cells in vitro.

AIMS: To investigate the differences of gene expression in rat retinal Müller glial cells after basic fibroblast growth factor (bFGF) treatment, and to explore the function of prostaglandin E(2) (PGE(2)) in the proliferation and dedifferentiation of rat retinal Müller glial cells. MATERIALS AND METHODS: A gene expression profile in rat retinal Müller glial cells after bFGF treatment, matching untreated cells, was conducted using gene array technology. The candidate gene selection was performed on GenMAPP/MAPPFinder. The functional effects of PGE(2) on the proliferation and dedifferentiation of Müller cells were further investigated. RESULTS: Gene array analysis identified that 298 genes were upregulated and 293 genes were downregulated. The GenMAPP/MAPPFinder results showed that the PG biosynthetic process had the highest correlation (Z score 8.803) with the stem cell characteristics of Müller cells. The quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) results showed that PGE(2) can significantly upregulate the expression of cyclin D(1) in Müller cells. Also, the bromodeoxyuridine and flow cytometry results showed that PGE(2) can significantly increase the proportion of Müller cells in S phase. Furthermore, qRT-PCR showed that the expressions of nestin (a marker of neural stem/precursor cells) and Pax6 (a marker of retinal stem/precursor cells) were significantly upregulated by PGE(2) stimulation, and similar results were obtained by immunofluorescence staining. CONCLUSION: PGE(2) may enhance the proliferation, dedifferentiation, and stem-like properties of rat retinal Müller glial cells in vitro.

Changes of retinal vessel density in low to moderate myopic eyes with orthokeratology evaluated by optical coherence tomography angiography.

AIM: To explore the effect of orthokeratology (OK) fitting on retinal vessel density in low to moderate myopia adolescents by using optical coherence tomography angiography. METHODS: Children aged 10 to 14y with a cycloplegic spherical equivalent refraction of -0.50 diopter (D) to -5.00 D and astigmatism with more than -1.50 D were recruited. The enrolled adolescents were divided into OK group and spectacle group. During regular follow-up, adolescents were measured respectively at pre-wear, 1, 3, and 6mo after treatment. The follow-up included uncorrected distance visual acuity (UDVA), axial length (AL), superficial capillary plexus density (SCPD), deep capillary plexus density (DCPD), central retinal thickness (CRT), foveal avascular zone area (FAZ-A), foveal avascular zone perimeter (FAZ-P) and foveal vessel density in a 300-µm-wide region around foveal avascular zone (FD-300). The collected data were analyzed using statistical methods. RESULTS: By one month, SCPD significantly increased in the fovea and superior retina, and DCPD significantly increased inferiorly in OK group compared to spectacle group (P<0.05). By three months, there were significant increases in SCPD in the fovea and inferior retina, and DCPD in the parafovea, superior, and inferior retina in OK group (P<0.05), while the increase in SCPD and DCPD in the fovea were observed by six months (P<0.05). The FD-300 significantly increased at every follow-up in OK group compared to spectacle group (P<0.05). No significant differences in the CRT, FAZ-A and FAZ-P and FD-300 were observed between two groups (P>0.05). OK group showed a significant improvement in UDVA after wearing OK, compared to spectacle group (P<0.01), while the AL did not show a significant difference between two groups (P>0.05). CONCLUSION: Short-term OK worn can increase local retinal vessel density in adolescents with low-to-moderate myopia.

Time-dependent matrix metalloproteinases and tissue inhibitor of metalloproteinases expression change in fusarium solani keratitis.

AIM: To investigate matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) expression during the progress of fusarium solani (F.solani) keratitis in a rat model. METHODS: A rat model of F.solani keratitis was produced using corneal scarification and a hand-made contact lens. MMPs and TIMPs expressiond were explored in this rat model of F.solani keratitis using real-time polymerase chain reaction (PCR) and DIF. GM6001 (400 µmol/mL) was used to treat infected corneas. The keratitis duration, amount and area of corneal neovascularization (CNV) were evaluated. RESULTS: MMP-3 expression was 66.3 times higher in infected corneas compared to normal corneas. MMP-8, -9, and -13 expressions were significantly upregulated in the mid-period of the infection, with infected-to-normal ratios of 4.03, 39.86, and 5.94, respectively. MMP-2 and -7 expressions increased in the late period, with the infected-to-normal ratios of 5.94 and 16.22, respectively. TIMP-1 expression was upregulated in the early period, and it was 43.17 times higher in infected compared to normal corneas, but TIMP-2, -3, and -4 expressions were mildly downregulated or unchanged. The results of DIF were consistent with the result of real-time PCR. GM6001, a MMPs inhibitor, decreased the duration of F.solani infection and the amount and area of CNV. CONCLUSION: MMPs and TIMPs contributed into the progress of F.solani keratitis.

Exogenous MD-2 confers lipopolysaccharide responsiveness to human corneal epithelial cells with intracellular expression of TLR4 and CD14.

In the present study, we aimed to investigate the responsiveness of human corneal epithelial cells (HCECs) to lipopolysaccharide (LPS) in vitro and to elucidate the underlying molecular mechanism(s) controlling the LPS responsiveness. The expression and subcellular localization of toll-like receptor 4 (TLR4) and CD14 and the expression of myeloid differentiation (MD)-2 were studied in SDHCEC1 cells, one HCEC cell line. Upon exposure to different concentrations of LPS, cell responses were evaluated by examining nuclear factor-kappaB (NF-κB) activation and the production of interleukin (IL)-8. The influence of soluble MD-2 on LPS responsiveness were assessed in SDHCEC1 cells pretreated with MD-2-containing conditioned medium before LPS challenge. SDHCEC1 cells expressed both TLR4 and CD14 intracellularly and had no detectable expression of MD-2 transcripts. Unresponsiveness to LPS at doses of up to 1,000 ng/ml was observed in SDHCEC1 cells, which was evidenced by no evident NF-κB activation and IL-8 production. The addition of MD-2 conditioned medium significantly induced NF-κB activation and enhanced the production of IL-8 as compared with the treatment with the control medium (p < 0.05). Meanwhile, the total mRNA amounts of TLR4 and CD14 and the surface expression of the two proteins were significantly (p < 0.05) increased by the pretreatment with MD-2 conditioned medium. LPS hyporesponsiveness of HCECs is largely due to deficient LPS receptor complex formation caused by lack of MD-2 expression. Exogenous MD-2 is capable of restoring the LPS responsiveness, at least partially, through promoting the surface expression of TLR4 and CD14.

Experimental model of Fusarium solani keratitis in rats.

AIM: To establish a repeatable rat model of Fusarium solani keratitis (F. solani keratitis) that mimicked fungal keratitis in humans. METHODS: Wistar rats' corneas were scratched on the superficial stroma after scraping the unilateral corneal epithelia. Then, the corneal surface was inoculated with different inoculum dose of F. solani spore suspension. Doses ranged from 10(6) to 10(9) colony-forming unit per milliliter (CFU/mL). The treated corneas were covered by contact lenses that were made of Parafilm M membrane. Negative controls were inoculated with sterile phosphate-buffered saline (PBS). For statistical analysis, corneas were evaluated daily on a 12-point scale to check the state of corneal inflammation. Furthermore, the pathological characteristics of this model were investigated. RESULTS: The rat model of F. solani keratitis was established by the combination methods of corneal trauma and parafilm-made contact lens and inoculation of fungus spore suspension. 10(6) and 10(7)CFU/mL of F. solani induced mild corneal infection, while 10(8)CFU/mL of F. solani was sufficient to induce moderate infection that was consistent with human keratomycosis. Dose of 10(9)CFU/mL of F. solani was excessive and led to perforated corneas. CONCLUSION: The rat model of F. solani keratitis, established by the combinational methods of corneal trauma, parafilm-made contact lens and the appropriate dose of inoculum, that imitates the developing processes of F. solani keratitis in human beings and provides a repeatable method of creating a rat model.