Efficacy and safety of combined UV-light corneal crosslinking and fine-needle diathermy to regress pathological murine corneal (lymph)angiogenesis in vivo.

PURPOSE: To compare safety and efficacy of isolated and combined UV-light corneal crosslinking (CXL) and fine-needle diathermy (FND) to regress pathological corneal vessels in vivo. METHODS: Mice with inflamed and pathologically vascularized corneas received CXL or FND as monotherapy or a combination of both treatments. Corneal pathological blood and lymphatic vessels, immune cells and the morphology of anterior segment structures were evaluated. RESULTS: All three approaches were able to regress blood and lymphatic vessels in mice. A comparative analysis of the three methods revealed that the FND monotherapy and the CXL + FND combination were significantly more effective than the CXL monotherapy, one and 2 weeks after therapy and especially in regressing lymphatic vessels. Furthermore, the combination therapy induced significantly less immune cell recruitment compared to the monotherapies. All three methods were safe to use in regards of corneal integrity. CONCLUSIONS: A combination of FND and CXL led to regression of pathological corneal lymphatic and blood vessels and reduced the infiltration of immune cells into inflamed murine corneas. This approach offers a new effective, safe and clinically usable strategy to treat eyes with mature pathological blood vessels and even more so for lymphatic vessels, for example prior to high-risk corneal transplantation.

Epithelial downgrowth after Descemet membrane endothelial keratoplasty.

PURPOSE: To describe a patient with epithelial downgrowth after Descemet membrane endothelial keratoplasty. METHODS: Case report. RESULTS: A 73-year-old woman underwent triple Descemet stripping automated endothelial keratoplasty for cataract and corneal edema secondary to Fuchs endothelial dystrophy in the left eye elsewhere. Three years later, Descemet membrane endothelial keratoplasty was performed at our department due to graft failure. One month after the operation, her vision improved to 20/32 and maintained stable. At the 14-month visit, her visual acuity decreased, and a routine examination revealed epithelial downgrowth at the posterior surface of the cornea and partly beneath the graft, accompanied by presumed graft rejection. Therefore, repeat Descemet membrane endothelial keratoplasty with epithelial scraping and intracameral injection of 5-fluorouracil was indicated. She recovered 20/25 vision by 1 month after the surgery. However, small sheet-like epithelial downgrowth recurred 1 month later. The epithelial downgrowth was limited to the peripheral margin of the Descemet membrane endothelial keratoplasty graft and did not affect the visual axis. Epithelial downgrowth showed "islands" with connection between epithelial downgrowth and clear corneal incision on anterior segment optical coherence tomography images. Histopathologic evaluation of the removed Descemet membrane endothelial keratoplasty graft confirmed conjunctival epithelium as the source. Under close observation at the current 4-year follow-up, the epithelial downgrowth remained stable and localized and her vision increased to 20/20. CONCLUSION: Epithelial downgrowth can occur after Descemet membrane endothelial keratoplasty. The limited progression of epithelial downgrowth in this patient suggests that this condition after Descemet membrane endothelial keratoplasty even in the recurrence stage may cause less damage than expected and may only need to be observed closely if no progression occurs.

VEGF TrapR1R2 Suspended in the Semifluorinated Alkane F6H8 Inhibits Inflammatory Corneal Hem- and Lymphangiogenesis.

Purpose: Semifluorinated alkanes (SFAs) are used at the ocular surface as lubricants or vehicles for drugs. The purpose of this study was to test the effect of vascular endothelial growth factor (VEGF) TrapR1R2 suspended in the SFA perfluorohexyloctane (Trap/F6H8) on corneal neovascularization. Methods: Suture placement was used to induce inflammatory corneal neovascularization in mice. Treatment groups were: Trap/F6H8, VEGF TrapR1R2 as aqueous formulation dissolved in phosphate buffer (Trap), F6H8, and phosphate buffer (controls). Eye drops were applied 3×/daily for 2 weeks. Afterward, corneas were stained with CD31 and LYVE-1 to analyze corneal hem- and lymphangiogenesis. To investigate the effect of on inflammatory cell recruitment, corneal CD45+ cells were quantified. In addition, epithelial wound closure after debridement was assessed by corneal fluorescein staining. Results: Trap/F6H8 was as effective as Trap in inhibiting corneal hemangiogenesis and lymphangiogenesis after 2 weeks of treatment. After 3 days of treatment, Trap/F6H8 was even more effective than Trap in inhibiting corneal hemangiogenesis. Both treatment groups (Trap/F6H8 and Trap) significantly reduced corneal CD45+ cell recruitment. Epithelial closure after debridement was unaffected by Trap/F6H8 or Trap. Conclusions: In this study, we demonstrate that F6H8 is a potential carrier for VEGF TrapR1R2 to topically treat corneal neovascularization. Our findings might open new treatment avenues for local anti-angiogenic therapy at the cornea, as F6H8 is already approved for the usage at the ocular surface. Translational Relevance: With this study we show for the first time that SFAs can serve as carriers for anti-angiogenic drugs at the ocular surface.

Risk factors for endothelial cell loss after Descemet membrane endothelial keratoplasty (DMEK).

This study aimed to identify the risk factors for endothelial cell density (ECD) loss after Descemet membrane endothelial keratoplasty (DMEK) and analyse whether donor tissues from cold versus organ culture differ in terms of ECD loss after DMEK. Consecutive DMEK cases from a prospective database for Fuchs' endothelial corneal dystrophy were retrospectively analysed between 2011 and 2016 at the University of Cologne, and the possible risk factors for ECD loss, including patient-related factors, type of tamponade (air or 20% sulphur hexafluoride gas), type of surgery (triple DMEK or DMEK alone), re-bubbling, immune rejection, and donor-related factors were determined. Eight hundred and forty-one eyes were selected. There was no significant difference in the best-corrected visual acuity (logarithm of the minimal angle of resolution) and corneal thickness (P = 0.540 and P = 0.667) between groups. Immune reactions were more common in cold cultures (P = 0.019), but ECD loss (1 year after DMEK) was greater in organ cultures (38.3 ± 0.8%) than in cold cultures (34.7 ± 1.4%) (P = 0.022). Only re-bubbling was significantly associated with ECD loss (P < 0.001). Re-bubbling was found to be a key factor for ECD loss at 1 year after DMEK.

Supplemental Anti Vegf A-Therapy Prevents Rebound Neovascularisation After Fine Needle Diathermy Treatment to Regress Pathological Corneal (LYMPH)Angiogenesis.

Fine needle diathermy (FND) is an effective method to destroy and regress pathologic corneal blood and lymphatic vessels. However, it is unknown whether FND itself causes a rebound corneal neovascularisation and whether that can be prevented by VEGF blockade. In female BALB/c mice, the suture-induced inflammatory corneal neovascularisation model was used to induce hem- and lymphangiogenesis. Thereafter, prevascularized mice were divided into 2 groups: the combination therapy group received FND cauterization and subsequent VEGF TrapR1R2 eye drops three times per day whereas the monotherapy group was treated only with FND. Three, 7 and 14 days after the treatment, corneas were collected and stained with FITC-conjugated CD31 and LYVE-1 followed by Cy3-conjugated secondary antibody to quantify corneal blood and lymphatic vessels. Relative mRNA expression of VEGF in the cornea was quantified by using qPCR. FND cauterization as monotherapy significantly obliterated (lymph)angiogenesis at early time points; however, this treatment led to secondary corneal hem- and lymphangiogenesis associated with significant upregulation of pro(lymph)angiogenic VEGF-A, VEGF-C, VEGF-D and infiltration of macrophages. Combining FND cauterization with VEGF TrapR1R2 treatment prevented the undesired effect of the FND procedure alone and significantly better regressed corneal blood and lymphatic vessels at 1 week after the treatment compared to monotherapy and control group (p < 0.01).

Immune reactions after modern lamellar (DALK, DSAEK, DMEK) versus conventional penetrating corneal transplantation.

In the past decade, novel lamellar keratoplasty techniques such as Deep Anterior Lamellar Keratoplasty (DALK) for anterior keratoplasty and Descemet stripping automated endothelial keratoplasty (DSAEK)/Descemet membrane endothelial keratoplasty (DMEK) for posterior keratoplasty have been developed. DALK eliminates the possibility of endothelial allograft rejection, which is the main reason for graft failure after penetrating keratoplasty (PK). Compared to PK, the risk of endothelial graft rejection is significantly reduced after DSAEK/DMEK. Thus, with modern lamellar techniques, the clinical problem of endothelial graft rejection seems to be nearly solved in the low-risk situation. However, even with lamellar grafts there are epithelial, subepithelial and stromal immune reactions in DALK and endothelial immune reactions in DSAEK/DMEK, and not all keratoplasties can be performed in a lamellar fashion. Therefore, endothelial graft rejection in PK is still highly relevant, especially in the "high-risk" setting, where the cornea's (lymph)angiogenic and immune privilege is lost due to severe inflammation and pathological neovascularization. For these eyes, currently available treatment options are still unsatisfactory. In this review, we will describe currently used keratoplasty techniques, namely PK, DALK, DSAEK, and DMEK. We will summarize their indications, provide surgical descriptions, and comment on their complications and outcomes. Furthermore, we will give an overview on corneal transplant immunology. A specific focus will be placed on endothelial graft rejection and we will report on its incidence, clinical presentation, and current/future treatment and prevention options. Finally, we will speculate how the field of keratoplasty and prevention of corneal allograft rejection will develop in the future.

Risk of Corneal Graft Rejection After High-risk Keratoplasty Following Fine-needle Vessel Coagulation of Corneal Neovascularization Combined With Bevacizumab: A Pilot Study.

BACKGROUND: Corneal neovascularization is considered an important risk factor for allograft rejection after corneal transplantation (keratoplasty). Therefore, the aim of this study was to determine whether preoperative reduction of corneal neovascularization by fine-needle thermal cauterization combined with bevacizumab reduces the incidence of allograft rejection after subsequent high-risk keratoplasty. METHODS: In this interventional uncontrolled clinical pilot study, 31 eyes of 31 patients with corneal neovascularization in at least one corneal quadrant were included. All eyes were treated by fine-needle thermal cauterization of corneal vessels and subconjunctival injection of bevacizumab. Both treatments were repeated in the cases of visible reperfusion of occluded vessels. Afterward, penetrating keratoplasty was performed. When corneal neovascularization was present on the day of keratoplasty, additional vessel cauterization and injection of bevacizumab was performed. Patients were then followed to determine the incidence of allograft rejection. RESULTS: In 18 eyes, vessel cauterization with bevacizumab injection was performed once before keratoplasty, whereas 13 eyes required retreatment before keratoplasty. No complications were observed. In 23 eyes, corneal neovascularization was present on the day of keratoplasty due to reperfusion of previously occluded vessels and simultaneous vessel cauterization with bevacizumab injection was performed. During follow-up (mean: 560 days; range: 59-1095 days), 4 graft rejection episodes in 4 eyes were observed. Estimated probabilities of corneal graft survival were 92.9% after 1 year (number at risk: 23), 78.4% after 2 years (number at risk: 9), and 78.4% after 3 years (number at risk: 3). CONCLUSIONS: Our initial results indicate that angioregressive treatment of pathological corneal vessels by fine-needle thermal cauterization combined with subconjunctival injection of bevacizumab before high-risk keratoplasty seems to result in graft survival rates comparable to survival rates seen in normal-risk keratoplasty. The findings of our pilot study warrant further controlled clinical trials with longer follow-up in a larger patient cohort.

Fine Needle-Diathermy Regresses Pathological Corneal (Lymph)Angiogenesis and Promotes High-Risk Corneal Transplant Survival.

Pathological corneal hem- and lymphangiogenesis are prime risk factors for corneal graft rejection. Fine needle-diathermy (FND) is an option to regress corneal blood vessels; however, whether this treatment besides clinically visible blood vessels also affects invisible lymphatic vessels is so far unknown. Here we test the hypothesis that FND destroys not only blood but also lymphatic vessels, thereby promotes corneal high-risk graft survival. The effect of FND was studied in vivo using BALB/c mice and the model of suture-induced corneal neovascularization. Mice were divided into three groups: FND, ANTI (anti-inflammatory therapy) and NON (control). Five, 7, 10 and 20 days after cauterization, corneas were harvested and stained with LYVE-1, CD31 to quantify (lymph)angiogenesis. The long-term survival of allografts was compared between the three groups. FND caused significant regression of both blood and lymphatic vessels compared to the control group at all time points (p < 0.05) with the most obvious effect at day 7 (p < 0.01). Graft survival was significantly prolonged when transplants were placed into the FND pretreated group (p < 0.0001). The effect of the anti-inflammatory therapy alone was less effective compared to FND (p < 0.05). This novel lymphangioregressive effect of FND can be used clinically to precondition high-risk recipients to promote graft survival.

UV light crosslinking regresses mature corneal blood and lymphatic vessels and promotes subsequent high-risk corneal transplant survival.

Immunologic graft rejection is the main complication after corneal transplant into pathologically prevascularized so-called high-risk eyes. The aim of this study was to evaluate whether ultraviolet (UV) light crosslinking can regress pathologic corneal blood and lymphatic vessels and thereby improve subsequent graft survival. Using the murine model of suture-induced corneal neovascularization, we found that corneal crosslinking with UVA light and riboflavin regressed both preexisting blood and lymphatic vessels significantly via induction of apoptosis in vascular endothelial cells. In addition, macrophages and CD45+ cell counts were significantly reduced. Consistently, corneal crosslinking reduced keratocyte density and corneal thickness without affecting corneal nonvascular endothelial cells, iris, and lens depending on the crosslinking duration. Furthermore, using the murine model of corneal transplant, long-term graft survival was significantly promoted (P < .05) and CD4+ CD25+ FoxP3+ T regulatory cells were upregulated (P < .01) in high-risk eyes preoperatively treated with crosslinking. Our results suggest UV light crosslinking as a novel method to regress both pathologic corneal blood and lymphatic vessels and to reduce CD45+ inflammatory cells. Furthermore, this study demonstrates for the first time that preoperative corneal crosslinking in prevascularized high-risk eyes can significantly improve subsequent graft survival and may become a promising novel therapy in the clinic.

Novel Method to Detect Corneal Lymphatic Vessels In Vivo by Intrastromal Injection of Fluorescein.

PURPOSE: Corneal lymphatic vessels are clinically invisible because of their thin walls and clear lymph fluid. There is no easy and established method for in vivo imaging of corneal lymphatic vessels so far. In this study, we present a novel approach to visualize corneal lymphatic vessels in vivo by injecting intrastromal fluorescein sodium. METHODS: Six- to eight-week-old female BALB/c mice were used in the mouse model of suture-induced corneal neovascularization. Two weeks after the suture placement, fluorescein sodium was injected intrastromally. The fluorescein, taken up by the presumed lymphatic vessels, was then tracked using a clinically used Spectralis HRA + OCT device. Immunohistochemistry staining with specific lymphatic marker LYVE-1 and pan-endothelial marker CD31 was used to confirm the indirect lymphangiography findings. RESULTS: By injecting fluorescein intrastromally, both corneal blood and lymphatic vessels were detected. While the lymphatic vessels were visible as bright vessel-like structures using HRA, the blood vessels appeared as dark networks. Fluorescein-labeled lymphatic vessels were colocalized with LYVE-1 in immunohistochemically stained sections of the same specimen. CONCLUSIONS: Corneal lymphatic vessels can be easily imaged in vivo in the murine model using intrastromal fluorescein injection.

Photodynamic Therapy Leads to Time-Dependent Regression of Pathologic Corneal (Lymph) Angiogenesis and Promotes High-Risk Corneal Allograft Survival.

Purpose: Pathologic corneal (lymph) angiogenesis is a known risk factor for immune-mediated allograft rejections after corneal transplantation. However, there is no established treatment to regress pre-existing pathological corneal blood and lymphatic vessels. This study assessed the possibility to regress both vessel types by photodynamic therapy (PDT) after intravenous (i.v.) verteporfin injection, the influence of timing of PDT after verteporfin injection, and the effect on graft survival in high-risk keratoplasty. Methods: BALB/c mice were used for suture-induced inflammatory corneal neovascularization to induce combined hem- and lymphangiogenesis. The treated group received PDT 3 minutes, 1 hour, and 24 hours after an i.v. verteporfin injection (control group: phosphate buffered saline). Corneal flatmounts were excised 3 days, 1 week, and 2 weeks after corneal PDT and stained with cluster of differentiation 31 (CD31) and lymphatic vessel endothelial hyaluronan receptor 1 antibodies (LYVE-1) to quantify hem- and lymphangiogenesis. Graft survival rates were compared between high-risk recipients with and without preoperative PDT. Results: Corneal blood vessels were significantly reduced when PDT was performed 3 minutes after i.v. verteporfin injection, whereas lymphatic vessels showed no significant difference. Both blood and lymphatic vessels were regressed when PDT was performed 1 hour or 24 hours after i.v. verteporfin application. Long-term allograft survival increased significantly in PDT-pretreated eyes when compared with controls. Conclusions: PDT after i.v. verteporfin injection can selectively regress pre-existing corneal blood vessels or both blood and lymphatic vessels depending on the timing of PDT after verteporfin injection. The pretreatment of recipients with PDT and i.v. verteporfin might be a promising new method to improve graft survival in high-risk eyes.