In Vivo Human Lacrimal Gland Imaging Using an Ultrasound Biomicroscopy.

PURPOSE: In the present study, we introduce human lacrimal gland imaging using an ultrasound biomicroscopy (UBM) with a soft cover and show their findings. METHODS: The representative UBM findings of palpebral lobes in seven subjects (four with non-Sjögren dry eye syndrome, one with Sjögren syndrome, and two healthy subjects) were described in this study. To prolapse the palpebral lobe, the examiner pulled the temporal part of the upper eyelid in the superotemporal direction and directed the subject to look in the inferonasal direction. We scanned the palpebral lobes longitudinally and transversely using UBM. We used an Aviso UBM with a 50 MHz linear probe and ClearScan. RESULTS: In UBM of two healthy subjects, the echogenicity of the lacrimal gland was lower than that of the sclera and homogeneous. But the parenchyma of a patient with Sjögren dry eye syndrome was quite inhomogeneous compared to the healthy subjects. In two patients with dry eye syndrome, we were able to observe some lobules in the parenchyma. We could find excretory ducts running parallel at the surface of the longitudinal section in some subjects. In the longitudinal UBM scan of a subject, we observed a tubular structure at a depth of 1,500 µm that was considered a blood vessel. It ran from the superonasal to the inferotemporal direction. In a subject, we observed a large cyst beneath the conjunctiva. CONCLUSIONS: Lacrimal gland imaging using UBM has both advantages of optical coherence tomography and sonography, and could be useful for evaluating dry eye syndrome.

Stability of neodymium:YAG laser posterior capsulotomy in eyes with capsular tension rings.

AIM: To evaluate the stability of neodymium (Nd):YAG laser posterior capsulotomy in eyes with capsular tension rings (CTRs). METHODS: A total of 60 eyes that underwent cataract surgery and laser posterior capsulotomy postoperatively were included in this retrospective cohort study. To evaluate the safety and stability of capsulotomy, changes in the size of posterior capsulotomy and anterior chamber depth (ACD) in three groups: the group without CTR, the group with 12 mm CTRs, and the group with 13 mm CTRs, at 1wk, 3, 12, and 15mo after capsulotomy, were compared. RESULTS: In the group without CTR and the group with 12 mm CTR, there was no significant change in ACD at every post-laser follow-up. In the group with 13 mm CTR, the ACD change was significant until 3mo after capsulotomy. In all groups, there was a significant increase in the area of capsulotomy between 1wk and 3mo post-laser. Between 3 and 12mo post-laser, only the group with 13 mm CTR showed a significant increase in the area of capsulotomy (P<0.01). CONCLUSION: Laser posterior capsulotomy is safe in all three groups. The capsulotomy and ACD become stabilized and have not shown significant changes since 1y post-laser, even with larger CTRs. The maintenance of centrifugal capsular tension can last longer with larger CTRs, and the stability of the capsulotomy site can be reached about 12mo after capsulotomy in pseudophakic eyes with larger CTRs.

Aqueous humor analyses of diabetic macular edema patients with subretinal fluid.

We identified treatment-naïve diabetic macular edema (DME) patients with or without subretinal fluid (SRF). We compared their baseline characteristics: aqueous concentrations of interleukin (IL)-1ß, IL-2, IL-6, IL-8, IL-10, and IL-17, as well as tumor necrosis factor-α, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF). We also compared fundus and optical coherence tomography (OCT) findings, and responsiveness to anti-VEGF treatments. Of 67 DME patients, 18 (26.87%) had SRF. Compared to the no SRF group, the SRF group had significantly higher levels of IL-6, IL-8, VEGF, and PlGF in aqueous humor. After grouping according to diabetic retinopathy stage, non-proliferative diabetic retinopathy (NPDR) patients with SRF had higher aqueous levels of IL-6 and IL-8, compared to NPDR patients without SRF. Moreover, proliferative diabetic retinopathy (PDR) patients with SRF had higher aqueous levels of VEGF and PlGF, compared to PDR patients without SRF. Fundus and OCT analyses revealed that the SRF group had a greater proportion of patients with succinate or patch-shaped hard exudates involving the macula, and greater central subfield thickness (CST) at baseline. After 6 months of anti-VEGF treatments, the SRF group showed better responsiveness in terms of CST; however, visual acuity was not correlated with responsiveness. Considering higher aqueous levels of VEGFs and pro-inflammatory cytokines, SRF could be a biomarker related to diabetic retinopathy activity. DME patients with SRF showed better anatomical responsiveness to anti-VEGF treatments, but did not show better functional improvement on short-term evaluation compared to those of DME patients without SRF.

Antiangiogenesis Effect of Albendazole on the Cornea.

Purpose: To investigate the anti-(lymph)angiogenic and anti-inflammatory effects of albendazole and to study whether these effects are additive with bevacizumab therapy in a murine corneal suture model. Methods: Corneal neovascularization (NV) and lymphangiogenesis (LY) were compared in a corneal suture model after administration of a subconjunctival injection of albendazole, bevacizumab, dexamethasone, or phosphate-buffered saline (PBS). Immunohistochemical staining and analysis were performed in each group. Real-time polymerase chain reaction (RT-PCR) was performed to quantify the expression of inflammatory cytokines (tumor necrosis factor [TNF]-alpha and interleukin-6), vascular endothelial growth factor (VEGF)-A, VEGF-C, vascular endothelial growth factor receptor (VEGFR)-2, and VEGFR-3. To evaluate the additive effect of albendazole, corneal NV and LY were also analyzed in a combined group of albendazole and bevacizumab therapy and the additive effect was compared with that in the group of double dose of bevacizumab. Results: The albendazole group showed less NV and less LY compared with the PBS control group (P < 0.01). When albendazole was combined with bevacizumab therapy, a significant decrease in NV and LY was seen compared with bevacizumab treatment alone, and with albendazole alone (all P values <0.05). The combination group showed better antilymphangiogenesis effect than the group of double dose bevacizumab. The albendazole-treated group showed reduced expression of VEGF-A, VEGF-C, TNF-alpha, and VEGFR-2 compared with corneas from the PBS group (P value <0.05 in all respective comparisons). Conclusion: Albendazole significantly decreased NV and LY in the cornea. This beneficial effect is additively enhanced when combined with bevacizumab treatment.

Correction: Prognosis for ocular toxocariasis according to granuloma location.

[This corrects the article DOI: 10.1371/journal.pone.0202904.].

Prognosis for ocular toxocariasis according to granuloma location.

PURPOSE: To determine the prognosis for ocular toxocariasis (OT) according to the location of the granuloma and to identify factors associated with its recurrence within 1 year. METHODS: OT patients were classified according to the granuloma lesion. After grouping the patients as posterior or peripheral, we compared sex, age, intraocular pressure, best corrected visual acuity (BCVA), degree of inflammation, immunoglobulin E, eosinophil profiles, recurrence, and complications in each group. We also identified factors associated with recurrence within 1 year. RESULTS: A total of 29 (61.70%) patients had granuloma at the periphery, and 18 (38.30%) patients had granuloma around the posterior pole. There were no significant differences in ocular or systemic evaluations except the initial BCVA. The mean decimal BCVA of the posterior pole granuloma group was worse than that of the peripheral granuloma group (p = 0.042). After treatment, the mean BCVA of the posterior pole granuloma group improved significantly (p = 0.019), and the final mean BCVA was not significantly different between the groups (p = 0.673). Multiple logistic regression analyses revealed that recurrence within a year was associated with age at diagnosis (p = 0.007). CONCLUSIONS: The initial BCVA of OT patients differed according to the location of the granuloma, but the BCVA after treatment was not significantly different between the groups. Younger age was associated with recurrence within 1 year.

Effect of itraconazole on the cornea in a murine suture model and penetrating keratoplasty model.

AIM: To investigate the anti-(lymph)angiogenic and/or anti-inflammatory effect of itraconazole in a corneal suture model and penetrating keratoplasty (PK) model. METHODS: Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among itraconazole, amphotericin B, dexamethasone, phosphate buffered saline (PBS) and surgery-only groups following subconjunctival injection in mice that underwent PK and corneal suture. Immunohistochemical staining and analysis were performed in each group. Real-time polymerase chain reaction (RT-PCR) was performed to quantify the expression of inflammatory cytokines (TNF-alpha, IL-6) and vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGFR-2, and VEGFR-3. RESULTS: In the suture model, the itraconazole group showed less angiogenesis, less lymphangiogenesis, and less inflammatory infiltration than the PBS group (all P<0.05). The itraconazole group showed reduced expression of VEGF-A, VEGFR-2, TNF-alpha, IL-6 than the PBS group (all P<0.05). In PK model, the two-month graft survival rate was 28.57% in itraconazole group, 62.50% in dexamethasone group, 12.50% in PBS group, 0 in amphotericin B group and 0 in surgery-only group. Graft survival in the itraconazole group was higher than that in the amphotericin, PBS and surgery-only group (P=0.057, 0.096, 0.012, respectively). The itraconazole group showed less total angiogenesis and lymphangiogenesis than PBS group (all P<0.05). CONCLUSION: Itraconazole decrease neovascularization, lymphangiogenesis, and inflammation in both a corneal suture model and PK model. Itraconazole has anti-(lymph)-angiogenic and anti-inflammatory effects in addition to its intrinsic antifungal effect and is therefore an alternative treatment option in cases where steroids cannot be used.

Effect of sorafenib in a murine high risk penetrating keratoplasty model.

AIM: To evaluate the effect of sorafenib in murine high risk keratoplasty model. METHODS: Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among the sorafenib, dexamethasone, dimethyl sulfoxide (DMSO), and phosphate buffered saline (PBS) groups following subconjunctival injection in mice that underwent high risk penetrating keratoplasty (HRPK). Real-time polymerase chain reaction was performed to quantify the expression of inflammatory cytokines and vascular endothelial growth factor (VEGF)-A, VEGF-C, vascular endothelial growth factor receptor (VEGFR)-2, VEGFR-3. RESULTS: The two-month graft survival rate for HRPK was 42.86% in sorafenib group, 37.50% in dexamethasone group, 0 in DMSO group, and 0 in PBS group. Sorafenib significantly increased graft survival compared to the DMSO and PBS group (P<0.05). The sorafenib didn't show significant effect in decreasing neovascularization compared with dexamethsone, DMSO, and PBS group. The sorafenib showed less total lymphangiogenesis than the dexamethasone, DMSO, and PBS group (P=0.011, P<0.001, P<0.001, respectively). The sorafenib group showed reduced expression of VEGF-C, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, VEGFR-2 and VEGFR-3 compared with DMSO group and PBS group (all P<0.05). The sorafenib group didn't show difference in the expression of VEGF-A compared with DMSO, neither with PBS. The sorafenib group showed reduced expression of VEGFR-3 compared with dexamethasone (P=0.051). CONCLUSION: The subconjunctivally administered sorafenib shows significant anti-lymphangiogenic effect, resulting in increased transplant survival in a murine high risk keratoplasty model. We suggest that a close linkage between decreased VEGF-C/VEGFR-2 and -3 signaling and increased corneal graft survival by sorafenib seems to exist.

Effect of trapping vascular endothelial growth factor-A in a murine model of dry eye with inflammatory neovascularization.

AIM: To evaluate whether trapping vascular endothelial growth factor A (VEGF-A) would suppress angiogenesis and inflammation in dry eye corneas in a murine corneal suture model. METHODS: We established two groups of animals, one with non-dry eyes and the other with induced dry eyes. In both groups, a corneal suture model was used to induce inflammation and neovascularization. Each of two groups was again divided into three subgroups according to the treatment; subgroup I (aflibercept), subgroup II (dexamethasone) and subgroup III (phosphate buffered saline, PBS). Corneas were harvested and immunohistochemical staining was performed to compare the extents of neovascularization and CD11b+ cell infiltration. Real-time polymerase chain reaction was performed to quantify the expression of inflammatory cytokines and VEGF-A in the corneas. RESULTS: Trapping VEGF-A with aflibercept resulted in significantly decreased angiogenesis and inflammation compared with the dexamethasone and PBS treatments in the dry eye corneas (all P<0.05), but with no such effects in non-dry eyes. The anti-inflammatory and anti-angiogenic effects of VEGF-A trapping were stronger than those of dexamethasone in both dry eye and non-dry eye corneas (all P<0.05). The levels of RNA expression of VEGF-A, TNF-alpha, and IL-6 in the aflibercept subgroup were significantly decreased compared with those in the PBS subgroup in the dry eye group. CONCLUSION: Compared with non-dry eye corneas, dry eye corneas have greater amounts of inflammation and neovascularization and also have a more robust response to anti-inflammatory and anti-angiogenic agents after ocular surface surgery. Trapping VEGF-A is effective in decreasing both angiogenesis and inflammation in dry eye corneas after ocular surface surgery.

Comparison of postoperative corneal changes between dry eye and non-dry eye in a murine cataract surgery model.

AIM: To compare the effects of the surgical insult of cataract surgery on corneal inflammatory infiltration, neovascularization (NV) and lymphangiogenesis (LY) between the dry eye and non-dry eye in murine cataract surgery models. METHODS: We established two groups of animals, one with normal eyes (non-dry eye) and the second with induced dry eyes. In both groups, we used surgical insults to mimic human cataract surgery, which consisted of lens extraction, corneal incision and suture. After harvesting of corneas on the 9(th) postoperative day and immunohistochemical staining, we compared NV, LY and CD11b+ cell infiltration in the corneas. RESULTS: Dry eye group had significantly more inflammatory infiltration (21.75%±7.17% vs 3.65%±1.49%; P=0.049). The dry eye group showed significantly more NV (48.21%±4.02% vs 26.24%±6.01%; P=0.016) and greater levels of LY (9.27%±0.48% vs 4.84%±1.15%; P=0.007). In corneas on which no surgery was performed, there was no induction of NV in both the dry and non-dry group, but dry eye group demonstrated more CD11b+ cells infiltration than the non-dry eye group (0.360%±0.160% vs 0.023%±0.006%; P=0.068). Dry eye group showed more NV than non-dry eye group in both topical PBS application and subconjunctival PBS injection (P=0.020 and 0.000, respectively). CONCLUSION: In a murine cataract surgery model, preexisting dry eye can induce more postoperative NV, LY, and inflammation in corneal tissue.

Preoperative Mannitolization Can Decrease Corneal Endothelial Cell Damage After Cataract Surgery.

PURPOSE: To evaluate whether preoperative mannitolization can change ocular biometry and affect postoperative corneal endothelial cell density. METHODS: Bilateral sequential cataract surgery was performed in 38 patients. Preoperative mannitolization was done in one eye of each subject. We checked the change in preoperative ocular biometry before and after intravenous mannitolization. We compared the postoperative corneal endothelial cell density between eyes with mannitolization and without mannitolization at postoperative week 1, 2, 5, and 8. We evaluated the relationship between change in ocular biometry and change in postoperative corneal endothelial cells in eyes that underwent preoperative mannitolization. RESULTS: After mannitolization, eyes exhibited decreased intraocular pressure, axial length (AL), and vitreous chamber depth (VCD) and increased anterior chamber depth (ACD) and lens position (LP) compared to before mannitolization (p < 0.05). Preoperative mannitolization has a tendency to decrease the intraoperative use of phaco energy in eyes with moderate nucleosclerosis. Eyes with preoperative mannitolization showed less loss of postoperative corneal endothelial cells than eyes without preoperative mannitolization (p < 0.05). The ACD, LP, and AL changes by mannitolization were all negatively correlated with corneal endothelial cell loss (p < 0.05). CONCLUSION: Preoperative mannitolization can decrease postoperative loss of corneal endothelial cells. The protective effect of preoperative mannitolization on the corneal endothelium may be due to the decreased need for phaco energy and changes in ocular biometry such as ACD, AL, and LP.

The Association Between Aqueous Connective Tissue Growth Factor and the Severity of Age-related Cataracts as Graded by the Lens Opacities Classification System III.

PURPOSE: To evaluate the relationship between aqueous humor concentrations of connective tissue growth factor (CTGF) and the severity of age-related cataracts. MATERIALS AND METHODS: We conducted a prospective clinical study on 43 eyes of 43 patients with senile cataracts scheduled to undergo routine phacoemulsification surgery. Before surgery, all patients were graded for cataract severity using the Lens Opacities Classification System III in terms of four features: nuclear opalescence (NO), nuclear color (NC), cortical cataracts (C), and posterior sub-capsular cataracts (P). During surgery, aqueous humor samples were obtained from all patients, and sandwich enzyme-linked immunosorbent assays (ELISAs) were used to determine CTGF concentrations. To assess any relationship between cataract severity and CTGF levels of the aqueous humor, various correlation analyses and multiple linear regression were used. RESULTS: We found a positive correlation between the overall cataract grade and aqueous CTGF level (p < 0.05). In addition, four features of the cataract grade (nuclear opalescence, nuclear color, cortical cataract and posterior sub-capsular cataract) were positively correlated with the aqueous CTGF concentration (p < 0.05). The final regression model identified overall cataract grade as an independent predictor of increased CTGF levels in the aqueous humor (p < 0.05). CONCLUSIONS: CTGF tends to increase in the aqueous humor as the severity of age-related cataracts increases. Therefore, this cytokine may play an important role in the pathogenesis of age-related cataracts. Additional studies are required for clarification of this finding.

Dry eye predisposes to corneal neovascularization and lymphangiogenesis after corneal injury in a murine model.

PURPOSE: Dry eye disease is becoming recognized as an immune-inflammation mediated disorder. Surgical insults such as corneal incision or suture can aggravate dry eye. We sought to determine whether underlying dry eye aggravates corneal inflammatory infiltration, hemangiogenesis, and lymphangiogenesis (LY) induced by surgical injury in a murine model. METHODS: We used treatment arms; one, normal eye (non-dry eye) and the other, a scopolamine-induced dry eye model. We first compared the corneas of both groups on which no surgery was performed with confocal and fluorescent microscopy. In subgroups of each treatment arm, we made a corneal incision followed by 2 corneal sutures to approximate the wound. After harvesting the cornea on postoperative day 9 and immunohistochemical staining, we compared corneal neovascularization (NV), LY, and CD11b inflammatory cell infiltration between non-dry eye and dry eye groups. RESULTS: In corneas in which no surgery was performed, the dry eye group showed more CD11b cell infiltration than did the non-dry eye group (P < 0.05). With respect to corneas after injury, there was significantly more hemangiogenesis, LY, and inflammatory infiltration in the dry eye group than in the non-dry eye group (all P < 0.05). CONCLUSIONS: The underlying status of the cornea, whether it is dry or not, plays a significant role in the development of NV, LY, and inflammation after corneal injury. Dry eye can aggravate post-injury NV, LY, and inflammation.

Vascular Endothelial Growth Factor Receptor 1 morpholino increases graft survival in a murine penetrating keratoplasty model.

PURPOSE: This study sought to determine whether a Vascular Endothelial Growth Factor Receptor 1 (VEGFR1)-specific morpholino (MO) could decrease neovascularization, thereby enhancing murine cornea transplant survival, and if this effect is synergistic with steroid therapy. METHODS: Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among the VEGFR1_MO, STD MO and PBS groups following subconjunctival injection in mice that underwent normal risk penetrating keratoplasty (NR PK) and high-risk penetrating keratoplasty (HR PK). Graft survival, corneal neovascularization, and corneal lymphangiogenesis in groups treated with both VEGFR1_MO and steroid therapy were also analyzed in HR PK. RESULTS: In NR PK, the VEGFR1_MO decreased angiogenesis, lymphangiogenesis, and increased graft survival compared with the PBS group (P = 0.055, P = 0.003, P = 0.043, respectively). In HR PK, VEGFR1_MO decreased angiogenesis, lymphangiogenesis, and increased graft survival compared with the STD MO (P = 0.000, P = 0.000, P = 0.029, respectively) and PBS groups (P = 0.004, P = 0.002, P = 0.024). In HR PK, when the VEGFR1_MO was combined with steroid therapy, a significant increase in graft survival was seen compared with steroid treatment alone (P = 0.045). The 2-month graft survival rate for HR PK was 27% in the combination group compared with 0% in the triamcinolone only group. CONCLUSIONS: VEGFR1_MO decreased angiogenesis and lymphangiogenesis, resulting in increased graft survival in both NR PK and HR PK. This beneficial effect is synergistically enhanced with steroid treatment in HR PK.

Flt23k nanoparticles offer additive benefit in graft survival and anti-angiogenic effects when combined with triamcinolone.

PURPOSE: To determine if nanoparticles delivering plasmids expressing Flt23k (an anti-VEGF intraceptor) can enhance murine cornea transplant survival and whether their effect is synergistic with steroid therapy. METHODS: Biodegradable PLGA Flt23k loaded or blank nanoparticles were prepared using the emulsion solvent evaporation METHOD: Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among the Flt23k nanoparticles, blank nanoparticles, triamcinolone acetonide, and PBS groups following subconjunctival injection in mice that underwent penetrating keratoplasty. Graft survival, corneal neovascularization, and corneal lymphangiogenesis in a group treated with both nanoparticles and steroid therapy were also analyzed. RESULTS: The Flt23k nanoparticle group showed less neovascularization, lymphangiogenesis, and graft failure compared with the PBS control group (P < 0.01). The 2-month graft survival rate was 20% in the Flt23k nanoparticle group with no grafts surviving in the PBS group. When the Flt23k nanoparticle was combined with steroid therapy, a significant increase in graft survival was seen compared with both steroid treatment alone (P < 0.05) and steroid combined with blank nanoparticle treatment (P < 0.05). The 2-month graft survival rate was 91.6% in the combination group compared with 47.6% in the triamcinolone-only group and 42.4% in the triamcinolone plus blank nanoparticle group. CONCLUSIONS: Anti-VEGF nanoparticles (Flt23k) have a significant effect on decreasing neovascularization and lymphangiogenesis, resulting in increased graft survival in penetrating keratoplasty. This beneficial effect is synergistically enhanced with steroid treatment.