Yuko Maruyama

The Maintenance of Lymphatic Vessels in the Cornea Is Dependent on the Presence of Macrophages

PURPOSE It has been shown previously that the presence in the cornea of antigen-presenting cells (APC), such as macrophages (MPS) and lymphangiogenesis, is a risk for corneal transplantation. We sought to determine whether the existence of lymphatic vessels in the non-inflamed cornea is associated with the presence of MPS. METHODS Flat mounts were prepared from corneas of untreated C57BL/6, CD11b(-/-), F4/80(-/-), and BALB/c mice, and after suture placement or corneal transplantation, observed by immunofluorescence for the presence of lymphatic vessels using LYVE-1 as a marker of lymphatic endothelium. Innate immune cells were detected in normal mouse corneas using CD11b, F4/80, CD40, as well as MHC-class II. Digital images of the flat mounts were taken using a spot image analysis system, and the area covered by lymphatic vessels was measured using NIH Image software. RESULTS The number of spontaneous lymphatic vessels in C57BL/6 corneas was significantly greater than in BALB/c corneas (P = 0.03). There were more CD11b(+) (P < 0.01) and CD40(+), MHC-class II (+) cells in the C57BL/6 corneas than in BALB/c mouse corneas. MPS depletion via clodronate liposome in C57BL/6 mice led to fewer spontaneous lymphatic vessels and reduced inflammation-induced lymphangiogenesis relative to control mice. Mice deficient in CD11b or F4/80 had fewer spontaneous lymphatic vessels and less lymphangiogenesis than control C57BL/6 mice. CONCLUSIONS C57BL/6 mouse corneas have more endogenous CD11b(+) cells and lymphatic vessels. The endogenous lymphatic vessels, along with pro-inflammatory MPS, account for the high risk of corneal graft rejection in C57BL/6 mice. CD11b(+) and F4/80(+) MPS appear to have an important role in of the formation of new lymphatic vessels.

The Effect of Podoplanin Inhibition on Lymphangiogenesis Under Pathological Conditions

PURPOSE Podoplanin has been shown to be a reliable marker of lymphatic endothelium, but its role in the lymphatic system has not been well investigated. The purpose of this study is to investigate the role of podoplanin in lymphangiogenesis and macrophage functions under inflammatory conditions. METHODS Mouse corneal suture and ear section models were used to induce lymphangiogenesis and macrophage infiltration. Antilymphatic vessel endothelial hyaluronan-1 (Anti-LYVE-1) antibody was used to visualize lymphatic vessels. Thioglycollate-induced macrophages (mps) were collected and cultured with lipopolysaccharide (LPS), IFN-γ, and anti-mouse podoplanin antibody (PMab-1). Podoplanin, NF-κB, and mitogen-activated protein kinase (MAPK) pathway expression were detected by Western blot analysis. The TNF-α secretion was measured by ELISA. RESULTS Administration of PMab-1, reduced lymphangiogenesis in the corneal suture and ear wound healing models. Also, PMab-1 suppressed mps infiltration at the site of wound healing. Moreover, administration of PMab-1 led to a significant suppression of the rejection reaction in the corneal transplantation model. Our in vitro experiments showed that PMab-1 suppressed TNF-α secretion from mps under inflamed conditions, especially secretion caused by LPS stimulation. We confirmed the effect of PMab-1 on mps under inflamed conditions with a Western blot experiment, which clearly showed that the phosphorylation signal of the MAPK and NF-κB pathways was suppressed by PMab-1. CONCLUSIONS Podoplanin neutralization resulted in inhibition of lymphatic growth associated with corneal and ear wound healing as well as mps inflammation. These data suggest that podoplanin is a novel therapeutic target for suppressing lymphangiogenesis and inflammation.

Stage-specific reference genes significant for quantitative PCR during mouse retinal development.

Developing mouse retina has been serving as an ideal model for investigating the molecular mechanism of neural development and angiogenesis, because several significant events associated with these physiological phenomena are drastically occurring in conjunction with retinal development. However, as many genes are influencing on each other to establish mature retina within 21 days from E10 to P12, we must carefully design the experiments, such as in the case of quantitating the amount of altered gene expression toward the establishment of retina by quantitative PCR. As we have seen considerable variations of quantitative results in different developmental stages of retina depending on the reference genes used for compensation, we here attempted to determine a reliable reference gene to accurately quantitate the target genes in each stage. According to the results of in silico prediction and comparison with a database of SAGE, we found that the most stable gene from early to late stages was Sdha, whereas one of the most popular housekeeping genes, Actb, was the one that could mislead the quantitative results even in the adult stage. Consequently, we pointed out the importance of selecting an appropriate reference gene, especially to quantitate the amount of gene expression in the developmental stages of a certain tissue.

Comparison between bimatoprost and latanoprost-timolol fixed combination for efficacy and safety after switching patients from latanoprost

Background The purpose of this study was to prospectively evaluate and compare intraocular pressure (IOP) reduction efficacy and safety between bimatoprost and latanoprost-timolol fixed combination (LTFC) in Japanese patients with open-angle glaucoma. Methods In this prospective, randomized, non-masked study, after enrolling 70 eyes of 70 Japanese open-angle glaucoma patients who had used latanoprost monotherapy for more than 4 weeks, the subjects were randomly divided into a bimatoprost group or an LTFC group. Both groups were switched from latanoprost to bimatoprost or LTFC for 12 weeks. IOP, conjunctival injection score, corneal epitheliopathy score (area density classification; AD score), tear film break-up time, heart rate, and blood pressure were evaluated at 0, 4, and 12 weeks after switching. The paired t-test and Mann–Whitney U-test were used for the statistical analysis. Results After 13 of the 70 patients dropped out, 57 were analyzed for IOP reduction and safety. There was a significant decrease in mean IOP at 4 weeks compared with week 0 in both groups (both P<0.0001). Comparisons between the two groups showed no statistically significant differences. The conjunctival injection score was higher in the bimatoprost group than in the LTFC group at 12 weeks (P=0.0091). There were no statistically significant differences between the two drugs in relation to AD score, tear film break-up time, heart rate, and blood pressure. Conclusion Bimatoprost and LTFC exhibited similar efficacy for reduction of IOP. Safety results indicated that only the conjunctival injection score at 12 weeks was higher in the bimatoprost group compared with the LTFC group.

Morphological analysis of age-related iridocorneal angle changes in normal and glaucomatous cases using anterior segment optical coherence tomography.

Purpose To analyze age-related morphological changes of the iridocorneal angle in normal subjects and glaucomatous cases, using anterior segment optical coherence tomography (AS-OCT). Methods This study involved 58 eyes of 58 open-angle glaucoma cases and 72 eyes of 72 age-matched normal-open-angle control subjects. Iridocorneal angle structures in nasal and temporal regions and anterior chamber depth (ACD) were measured using AS-OCT. Axial length and refractive error were measured by use of an ocular biometer and auto refractor keratometer. Angle opening distance (AOD), angle recess area (ARA), and trabecular-iris space area (TISA), measured at 500 μm (TISA500) and 750 μm (TISA750) distant from the scleral spur, were calculated, in the nasal and temporal regions. A new index, the peripheral angle frame index (PAFI), which represents the peripheral angle structure, was proposed, and was defined as (TISA750-TISA500)/TISA500. Results Refractive power in the glaucoma cases was less than in control cases (P<0.0001). Axial length (P<0.0001) and ACD (P=0.0004) were longer and deeper, respectively, in the glaucoma cases, compared with the control cases. In both control and glaucoma groups, ACD, AOD, ARA, and TISA decreased linearly in an age-dependent manner, while PAFI stayed at relatively constant values throughout the age distribution. AOD in the glaucoma group was longer than in the control group, in both the temporal and nasal regions; ARA and TISA were larger in the glaucoma than in the control group. However, no significant differences in nasal or temporal PAFI were found between the glaucoma and control groups. Conclusion The findings of this study show that AS-OCT is useful for the quantitative evaluation of age-related changes in peripheral angle structure in glaucoma and control cases.

Severe Corneal Disorders Developed After Brimonidine Tartrate Ophthalmic Solution Use

Purpose: The primary side effects associated with 0.1% brimonidine tartrate (BT) ophthalmic solution with sodium chlorite are allergic conjunctivitis, blepharitis, and conjunctival hyperemia. However, cornea-related side effects are rare. In this study, we report 2 similar cases in which corneal neovascularization, corneal infiltration, and corneal opacity developed after BT eye-drop use. Methods: Retrospective report of 2 cases of corneal infiltration after BT eye-drop use. Results: Case 1 involved a 78-year-old woman with follicular conjunctivitis, corneal neovascularization, and infiltration in her left eye after unilateral instillation of BT eye drops in that eye. Case 2 involved a 75-year-old woman with bilateral corneal neovascularization and infiltration after instillation of BT eye drops. In both cases, the corneal complications were deemed to be side effects of BT, so those eye drops were replaced with 0.1% fluorometholone eye drops. After replacement, blepharitis and corneal neovascularization successfully resolved; however, a layer of opacity remained across the transparent layer of the cornea in both cases. Conclusions: We encountered 2 cases of corneal and conjunctival complications that developed as side effects after BT eye-drop use, thus indicating that strict attention should be paid to the possibility of side effects after initiation of antiglaucoma eye-drop use.

amniotic membrane-assisted trabeculectomy for refractory glaucoma with corneal disorders

Purpose To report the cases of six consecutive patients who underwent amniotic membrane (AM)-assisted trabeculectomy (TLE) to treat refractory glaucoma with severe corneal disorders. Methods This study involved six patients (three males and three females, mean age: 69.5±15.8 years) with refractory glaucoma and severe corneal disorders. The surgical procedure for each patient involved trabecular tissue being excised, and human AM then being placed epithelial side up on the corneal surface, sutured at the limbal sclera, and flipped over onto the sclera to cover the TLE area. The remaining edge of the AM was then inserted into the subconjunctival space and sutured. Medical records of all cases were reviewed with regard to intraocular pressure (IOP), visual acuity, and condition of the filtering bleb and ocular surface. Results The mean observation period was 69.5±15.8 months, and mean IOP at presurgery and at 1, 3, and 7 years postoperative was 40.3±6.9, 23.0±12.1, 25.6±12.8, and 28.5±19.1 mmHg, respectively. Glaucoma medications decreased from 3.0±1.1 drugs (presurgery) to 0.8±1.0 drugs (7 years postoperative). However, in some cases, ocular surface conditions or visual acuity worsened during the follow-up period. Conclusion Using AM as an internal patch for TLE, moderately good IOP control was obtained initially for the refractory glaucoma with severe corneal disorders; however, ocular surface conditions required special care, and the long-term IOP control was limited in some cases.