Clinical outcomes of amniotic membrane loaded with 5-FU PLGA nanoparticles in experimental trabeculectomy.

AIM: To evaluate the effect of amniotic membrane loaded with 5-fluorouracil poly (lactic-co-glycolic acid) (PLGA) nanoparticles (5-FU-NPs) in the surgical outcomes of experimental trabeculectomy in rabbits. METHODS: Thirty-two New Zealand white rabbits were randomly categorized into four groups with 8 rabbits in each group. Group 1, the control group, performed traditional trabeculectomy without adjuvant treatment. While the experimental groups performed compound trabeculectomy with different implantations including amniotic membrane (group 2), 5-FU-NPs (group 3) and amniotic membrane loaded with 5-FU-NPs (group 4). Clinical evaluations including IOP measurement and filtration bleb analysis were performed in all groups postoperatively. RESULTS: There is no significant difference of mean IOP in all groups at first 7d after surgery. While at P14, mean IOPs of experimental group 2 (9.8±2.1 mm Hg), groups 3 (8.9±2.8 mm Hg) and group 4 (7.6±2.3 mm Hg) were significantly reduced compared to control group (12.4±2.6 mm Hg; n=8, P<0.05). At P21, mean IOPs of groups 3 (11.7±3.2 mm Hg) and group 4 (9.9±1.6 mm Hg) were significantly decreased compare to control group (17.9±1.6 mm Hg) and group 2 (16.6±2.8 mm Hg; n=8, P<0.05). At P28, mean IOPs of groups 3 (13.8±3.3 mm Hg) and group 4 (10.6±2.0 mm Hg) were also significantly reduced compare to control group (19.4±2.3 mm Hg) and group 2 (18.5±2.4 mm Hg; n=8, P<0.05). Meanwhile mean IOP of group 4 is significantly decreased compared to group 3 at P28 (n=8, P<0.05). Survival analysis of functional filtration blub in all groups revealed the longest survival time in group 4 (24.9±5.1d) compared to that in group 3 (20.6±4.3d), group 2 (15.0±5.2d) and control group (10.1±5.7d). CONCLUSION: Amniotic membrane loaded with 5-Fu-NPs may function as an effective anti-scarring implant and provides improved long-term surgical outcomes for experimental trabeculectomy in rabbits.

Minocycline inhibits alkali burn-induced corneal neovascularization in mice.

The purpose of this study was to investigate the effects of minocycline on alkali burn-induced corneal neovascularization (CNV). A total of 105 mice treated with alkali burns were randomly divided into three groups to receive intraperitoneal injections of either phosphate buffered saline (PBS) or minocycline twice a day (60 mg/kg or 30 mg/kg) for 14 consecutive days. The area of CNV and corneal epithelial defects was measured on day 4, 7, 10, and14 after alkali burns. On day 14, a histopathological examination was performed to assess morphological change and the infiltration of polymorphonuclear neutrophils (PMNs). The mRNA expression levels of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMPs), interleukin-1α, 1ß, 6 (IL-1α, IL-1ß, IL-6) were analyzed using real-time quantitative polymerase chain reaction. The expression of MMP-2 and MMP-9 proteins was determined by gelatin zymography. In addition, enzyme-linked immunosorbent assay was used to analyze the protein levels of VEGFR1, VEGFR2, IL-1ß and IL-6. Minocycline at a dose of 60 mg/kg or 30 mg/kg significantly enhanced the recovery of the corneal epithelial defects more than PBS did. There were significant decreases of corneal neovascularization in the group of high-dosage minocycline compared with the control group at all checkpoints. On day 14, the infiltrated PMNs was reduced, and the mRNA expression of VEGFR1, VEGFR2, bFGF, IL-1ß, IL-6, MMP-2, MMP-9, -13 as well as the protein expression of VEGFR2, MMP-2, -9, IL-1ß, IL-6 in the corneas were down-regulated with the use of 60 mg/kg minocycline twice a day. Our results showed that the intraperitoneal injection of minocycline (60 mg/kg b.i.d.) can significantly inhibit alkali burn-induced corneal neovascularization in mice, possibly by accelerating corneal wound healing and by reducing the production of angiogenic factors, inflammatory cytokines and MMPs.

Inhibition of mouse alkali burn induced-corneal neovascularization by recombinant adenovirus encoding human vasohibin-1.

PURPOSE: To evaluate the activity of recombinant adenovirus encoding human vasohibin-1 (Ad-Vasohibin-1) on mouse corneal neovasularization induced by alkali burn. METHODS: For the treatment group, 50 mice each received subconjunctival injection (5 microl) of 10(9) plaque forming units of replication-defective Ad-Vasohibin-1. Control group mice received the same dosage of blank adenoviral vector (AdNull). Five days after injection, corneal neovascularization (CNV) was induced by placing 2.5 microl of 0.1 M NaOH on the right cornea for 30 s. Subsequently, CNV was observed and photographed every 3 days for a total duration of 9 days after the alkali burn. The percentage of neovascularized area was measured and compared with the AdNull control. The expression of human vasohibin-1 protein was detected by immunohistochemistry and western blotting at 5, 8, and 14 days after injection. The mRNA expression levels of murine vascular endothelial growth factor (Vegf), VEGF receptor 1 and 2 (Vegfr1, Vegfr2), and vasohibin-1 (Vash1) were analyzed and compared by real time quantitative reverse-transcription polymerase chain reaction. RESULTS: The percentage of neovascularized area within the cornea was significantly reduced in mice treated with Ad-Vasohibin-1 compared to mice treated with AdNull at every time point after alkali-induced injury (7.11%+/-3.91% and 15.48%+/-1.79% of corneal area in the treatment and control groups, respectively, on day 3; 31.64%+/-4.71% and 43.93%+/-6.15% on day 6, and 45.02%+/-9.98% and 66.24%+/-7.17% on day 9, all p<0.001). Human vasohibin-1 protein was detected at the injection sites on day 3 after corneal burn and was highly expressed in the central subepithelial stroma and co-localized with neovascularized vessels within the alkali-treated cornea on day 6. On day 9, the peripheral cornea exhibited a similar staining pattern as the central cornea, but a more intense vasohibin-1 immunostaining signal was detected in the deep stroma. Some of the vasohibin-1 stain signal diffused into the frontal and deep stroma of the central cornea and was not co-localized with new vessels. By contrast, in mice injected with AdNull or normal corneas, no vasohibin-1 stain signal was detected within the corneas. Vasohibin-1 protein expression within treated corneas was also further confirmed by western blotting on day 5. Expression appeared to peak by day 8 and was maintained at high levels until day 14. However, Vasohibin-1 protein was not detected in the corneas of normal mice or mice treated with AdNull. Real-time quantitative reverse-transcription polymerase chain reaction analysis showed that expression of Vegfr2 and endogenous Vash1 mRNA were significantly decreased in the treatment versus control group (t(1)=-2.161, p(1)=0.047; t(2)=-2.236, p(2)=0.041). In contrast, there were no significant differences in Vegf and Vegfr1 mRNA expression levels between the treatment and control groups (p>0.05 for both). CONCLUSIONS: Subconjunctival injection of Ad-Vasohibin-1 significantly reduces corneal neovascularization in alkali-treated mouse corneas. This effect of anti-neovascularization may be related to the downregulation of Vegfr2 expression.