Corneal Vessels Research Articles

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

To compare safety and efficacy of isolated and combined UV-light corneal crosslinking (CXL) and fine-needle diathermy (FND) to regress pathological corneal vessels invivo. 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. 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. 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.

Adjunctive bevacizumab therapy in an equine corneal stromal invasive squamous cell carcinoma with a 53-months follow-up.

A 17-year-old Appaloosa mare was referred for evaluation of presumed refractory keratitis of the left eye. Gross examination revealed ocular discomfort and corneal neovascularization with a nasal focal opacification affecting approximately 40% of the corneal surface. On ophthalmic examination, extensive subepithelial to mid-stromal vascular branching accompanied by a homogeneous white, dense opacification, which affected up to 80% of the total corneal thickness, were apparent. Signs of concurrent uveitis were absent. Deep-stromal lamellar keratectomy with a conjunctival pedicle graft was performed under general anesthesia. Histopathology confirmed a poorly differentiated corneal stromal invasive squamous cell carcinoma (SI-SCC) with neoplastic cell extension to the surgical margins. Postoperatively, 4 topical mitomycin C 0.04% chemotherapy cycles combined with oral firocoxib therapy were initiated. Seven months after surgery, regrowth of the SI-SCC was clinically suspected. A total volume of 1 ml bevacizumab 2.5% was administered in the standing sedated horse via 3 mid-stromal corneal injections. Four weeks later, intrastromal bevacizumab injections (ISBIs) were repeated, however, this time the solution was injected directly into the main corneal vessel branches.Seven weeks after the second ISBIs, the left eye was comfortable and significant remission of corneal vascularization and opacity was recognized. No recurrence has been noted for a follow-up period of more than 53 months.Equine SI-SCC usually has a very poor prognosis for globe maintenance. To the authors' knowledge this is the first report of well-tolerated intrastromal antivascular endothelial growth factor adjunctive therapy with bevazicumab 2.5% and SI-SCC resolution after a multimodal treatment approach.

UV light-mediated corneal crosslinking as (lymph)angioregressive pretreatment to promote graft survival after subsequent high-risk corneal transplantation (CrossCornealVision): protocol for a multicenter, randomized controlled trial

BackgroundGood vision highly depends on the transparency of the cornea, which is the “windscreen” of the eye. In fact, corneal blindness due to transparency loss is the second most common cause of blindness worldwide, and corneal transplantation is the main cure. Importantly, the cornea is normally avascular but can secondarily be invaded by pathological (blood and lymphatic) vessels due to severe inflammation, and the survival prognosis of a corneal graft mainly depends on the preoperative vascular condition of the recipient’s cornea. Whereas transplants placed into avascular recipient beds enjoy long-term survival rates of > 90%, survival rates significantly decrease in pathologically pre-vascularized, so-called high-risk recipients, which account for around 10% of all performed transplants in Germany and > 75% in lower and middle-income countries worldwide.MethodsThis parallel-grouped, open-randomized, multicenter, prospective controlled exploratory investigator-initiated trial (IIT) intends to improve graft survival by preconditioning pathologically vascularized recipient corneas by (lymph)angioregressive treatment before high-risk corneal transplantation. For this purpose, corneal crosslinking (CXL) will be used, which has been shown to potently regress corneal blood and lymphatic vessels. Prior to transplantation, patients will be randomized into 2 groups: (1) CXL (intervention) or (2) no pretreatment (control). CXL will be repeated once if insufficient reduction of corneal neovascularization should be observed. All patients (both groups) will then undergo corneal transplantation. In the intervention group, remaining blood vessels will be additionally regressed using fine needle diathermy (on the day of transplantation). Afterwards, the incidence of graft rejection episodes will be evaluated for 24 months (primary endpoint). Overall graft survival, as well as regression of corneal vessels and/or recurrence, among other factors, will be analyzed (secondary endpoints).DiscussionBased on preclinical and early pilot clinical evidence, we want to test the novel concept of temporary (lymph)angioregressive pretreatment of high-risk eyes by CXL to promote subsequent corneal graft survival. So far, there is no evidence-based approach to reliably improve graft survival in the high-risk corneal transplantation setting available in clinical routine. If successful, this approach will be the first to promote graft survival in high-risk transplants. It will significantly improve vision and quality of life in patients suffering from corneal blindness.Trial registrationClinicalTrials.gov NCT05870566. Registered on 22 May 2023.

Multiple effects of angiostatins in injured cornea

Prolonged inflammation and excessive neovascularization of the cornea due to severe injury can impair optical clarity and lead to vision impairment. Plasminogen kringle (K) fragments, known as angiostatins (AS), play a well-established role as inhibitors of neovascularization by suppressing pro-angiogenic signaling­. However, AS effects in the cornea, beyond inhibiting the angiogenesis, are still unexplored. In this study, we estimate the protective effect of two AS variants (K1-3 and K5) against alkali burn injury induced in rabbit and rat corneas. AS K1-3 in the single doses of 0.075 or 0.75 μg (0.1 or 1.0 μM, respectively) or 0.3 μg of AS K5 (1.0 μM) were applied locally as eye drops daily for 14 days after the injury. A significant regression of corneal vessels in-growth in injured eyes treated with AS was revealed. Western blot analysis of corneal tissue lysates revealed that injury-induced overexpression of protein markers­ of hypoxia (HIF-1α), angiogenesis (VEGF), tissue remodeling and fibrosis (MMP-9), autophagy (beclin-1) and endoplasmic reticulum stress (GRP-78) was significantly reduced under AS treatment. Besides, the level of tight junctions protein ZO-1 was shown to be up-regulated after the treatment of the damaged cornea with AS K1-3. Summarizing, our study uncovered novel biological functions of the kringle-containing plasminogen fragments indicating its beneficial effects during corneal healing in the experimental model of alkali burn. The data obtained can be helpful for the development of novel efficient formulations to manage complications of ocular surface injuries.

Correlation Between Corneal Whorl-Like Nerve and Retinal Neurodegenerative Changes and Their Association With Microvessel Perfusion in Diabetes.

The purpose of this study was to compare the evolution of changes in the corneal nerves, retinal nerves, and cells and blood vessels at a single time point in early diabetic retinopathy (DR). Eighty participants (60 with diabetes and 20 nondiabetic controls) were examined. DR was graded according to the International Classification of Diabetic Retinopathy. Inferior whorl length (IWL), spiral orientation, central nerve fiber length (CNFL), retinal nerve fiber layer (RNFL) thickness, ganglion cell complex (GCC) layer thickness, global loss volume (GLV), focal loss volume (FLV) indices, superficial (sVD), and deep vessel densities (dVD) were examined. Compared with those of healthy controls, the IWL, CNFL, and FLV were decreased in the diabetic groups (P < 0.001). The IWL was significantly positively correlated with the RNFL and GCC thicknesses in the diabetic group (r = 0.248, P = 0.006 and r = 0.207, P = 0.023, respectively) and significantly negatively correlated with the FLV (r = -0.535, P < 0.001). The sVD was significantly positively correlated with the RNFL thickness (r = 0.314, P < 0.001) and negatively correlated with the GLV (r = -0.229, P = 0.012). Our findings suggest a correlation between corneal whorl-like nerve plexus and retinal nerve changes in the early stages of DR and that the IWL of the cornea may be able to indicate the extent of DR. Retinal nerve changes are associated with retinal microvessel perfusion, and nerve changes may precede vessel lesions.

Snowflakes and frosted branches – Laser frosted corneal vessels stop snowflaking the deposits

Snowflakes and frosted branches – Laser frosted corneal vessels stop snowflaking the deposits

Pre-Ophthalmoscopic Quantitative Biomarkers in Diabetes Mellitus.

The purpose of this study was to assess whether retinal microvascular or corneal nerve abnormalities occur earlier in diabetes mellitus (DM) and to identify imaging biomarkers in order to help prevent the subsequent irreversible retinal and corneal complications. The study comprised 35 eyes of 35 healthy volunteers and 52 eyes of 52 patients with type 1 and type 2 DM. Swept-source optical coherence tomography (OCT), OCT angiography, and in vivo corneal confocal microscopy were performed in both groups. Corneal sub-basal nerve plexus and vessel density (VD) of superficial capillary plexus (SCP) and deep capillary plexus (DCP) were evaluated. All corneal sub-basal nerve fiber parameters were decreased in patients with DM compared with healthy subjects and the difference was significant for each result except for nerve fiber width (P = 0.586). No significant correlation was obtained between any nerve fiber morphology parameters and disease duration or HbA1C. VD in SCP was significantly decreased in the superior (P < 0.0001), temporal (P = 0.001), and nasal quadrant (P = 0.003) in the diabetes group. In DCP, only superior VD (P = 0.036), decreased significantly in the diabetes group. Ganglion cell layer thickness in the inner ring showed a significantly lower value in patients with DM (P < 0.0001). Our results implicate a more pronounced and earlier damage to the corneal nerve fibers compared to the retinal microvasculature in patients with DM. In DM, an earlier and more pronounced damage to the corneal nerve fibers was observed compared to the retinal microvasculature.

Overactivation of Norepinephrine-β2-Adrenergic Receptor Axis Promotes Corneal Neovascularization.

To investigate the role of the sympathetic nervous system in corneal neovascularization (CNV) and to identify the downstream pathway involved in this regulation. Three types of CNV models were constructed with C57BL/6J mice, including the alkali burn model, suture model, and basic fibroblast growth factor (bFGF) corneal micropocket model. Subconjunctival injection of the sympathetic neurotransmitter norepinephrine (NE) was administered in these three models. Control mice received injections of water of the same volume. The corneal CNV was detected using slit-lamp microscopy and immunostaining with CD31, and the results were quantified by ImageJ. The expression of β2-adrenergic receptor (β2-AR) was stained with mouse corneas and human umbilical vein endothelial cells (HUVECs). Furthermore, the anti-CNV effects of β2-AR antagonist ICI-118,551 (ICI) were examined with HUVEC tube formation assay and with a bFGF micropocket model. Additionally, partial β2-AR knockdown mice (Adrb2+/-) were used to establish the bFGF micropocket model, and the corneal CNV size was quantified based on the slit-lamp images and vessel staining. Sympathetic nerves invaded the cornea in the suture CNV model. The NE receptor β2-AR was highly expressed in corneal epithelium and blood vessels. The addition of NE significantly promoted corneal angiogenesis, whereas ICI effectively inhibited CNV invasion and HUVEC tube formation. Adrb2 knockdown significantly reduced the cornea area occupied by CNV. Our study found that sympathetic nerves grow into the cornea in conjunction with newly formed vessels. The addition of the sympathetic neurotransmitter NE and activation of its downstream receptor β2-AR promoted CNV. Targeting β2-AR could potentially be used as an anti-CNV strategy.

Functional Peptide-Loaded Gelatin Nanoparticles as Eyedrops for Cornea Neovascularization Treatment.

Corneal neovascularization (NV) is a process of abnormal vessel growth into the transparent cornea from the limbus and can disturb the light passing through the cornea, resulting in vision loss or even blindness. The use of nanomedicine as an effective therapeutic formulation in ophthalmology has led to higher drug bioavailability and a slow drug release rate. In this research, we designed and explored the feasibility of a new nanomedicine, gp91 ds-tat (gp91) peptide-encapsulated gelatin nanoparticles (GNP-gp91), for inhibiting corneal angiogenesis. GNP-gp91 were prepared by a two-step desolvation method. The characterization and cytocompatibility of GNP-gp91 were analyzed. The inhibition effect of GNP-gp91 on HUVEC cell migration and tube formation was observed by an inverted microscope. The drug retention test in mouse cornea was observed by in vivo imaging system, fluorescence microscope, and DAPI/TAMRA staining. Finally, the therapeutic efficacy and evaluation of neovascularization-related factors were conducted through the in vivo corneal NV mice model via topical delivery. The prepared GNP-gp91 had a nano-scale diameter (550.6 nm) with positive charge (21.7 mV) slow-release behavior (25%, 240hr). In vitro test revealed that GNP-gp91 enhanced the inhibition of cell migration and tube formation capacity via higher internalization of HUVEC. Topical administration (eyedrops) of the GNP-gp91 significantly prolongs the retention time (46%, 20 min) in the mouse cornea. In chemically burned corneal neovascularization models, corneal vessel area with a significant reduction in GNP-gp91 group (7.89%) was revealed when compared with PBS (33.99%) and gp91 (19.67%) treated groups via every two days dosing. Moreover, GNP-gp91 significantly reduced the concentration of Nox2, VEGF and MMP9 in NV's cornea. The nanomedicine, GNP-gp91, was successfully synthesized for ophthalmological application. These data suggest that GNP-gp91 contained eyedrops that not only have a longer retention time on the cornea but also can treat mice corneal NV effectively delivered in a low dosing frequency, GNP-gp91 eyedrops provides an alternative strategy for clinical ocular disease treatment in the culture.

Topical 1% cyclosporine eyedrops for the treatment of crystalline corneal dystrophy in dogs.

Crystalline corneal dystrophy (CCD) is the most common type of corneal lipidic deposition in dogs. CCD is a primary metabolic disorder of the corneal fibroblast featuring an accumulation of extracellular and intracellular lipid deposits. Corneal lipid deposits create a corneal opacity and modify the interfibrillar collagen distance, inducing light scattering. Corneal vascularization is not usually associated with the disease, but, in case of chronicity, cell death may produce inflammation, and new corneal vessels are developed. To the best of the authors' knowledge, this is the first report of a medical approach for CCD treatment in veterinary medicine. To evaluate the efficacy of topical 1% cyclosporine eyedrops (1% CsA) for the treatment of CCD in dogs. Medical records of dogs with CCD were retrospectively reviewed (2009-2020). Corneal opacification description (COD) [size (mm), depth, and opacification degree (0-3)] was evaluated at 0, 3, 6, 9, 12, and 15 months postinitial diagnosis. Dogs were classified into three groups: the control group (G0), the group receiving topical 1% CsA once per day (G1), and the group receiving topical 1% CsA twice daily (G2). Ninety-two client-owned dogs (163 eyes) of different breeds, ages, and gender fulfilled the inclusion criteria. When compared to G0, where the eyes significantly increased COD (p < 0.001), G1 and G2 significantly decreased COD (p < 0.001). In fact, the probability of reducing COD was about three times higher in G2 than in G1, being nearly the same for the right [odds ratio (OR) = 2.94; 95% confidence interval (95% CI) = 0.55-15.78] and left eye (OR = 2.92; 95% CI = 0.49-17.26). In addition, for each additional month of treatment in G2, the probability of reducing COD increased significantly (OR = 1.12; 95%CI = 1.00-1.26 for the right eye and OR = 1.16; 95%CI = 1.02-1.32 for the left eye). Long-term treatment with topical 1% CsA eyedrops significantly improved CCD in dogs, being the probability of reducing COD higher when applying the treatment twice daily.

Insights into corneal re‐vascularization and vascular remodelling in the eye

AbstractWhile corneal neovascularization is a well‐known, sight‐threatening condition presenting a risk for corneal transplant rejection and failure, corneal revascularization has received much less attention. Although ocular revascularization is well studied in the context of regrowth of choroidal vessels following regular anti‐angiogenic injections for age‐related macular degeneration, the corneal context is less well documented. Here, the differences between initial and subsequent episodes of corneal neovascularization are explored, as well as the underlying changes at the mechanistic, structural and biochemical levels, through basic studies in the murine cornea. Repeat neovascularization is found to be more aggressive than the vessel ingrowth in an initial phase, pointing towards a memory effect in the corneal tissue. Furthermore, these regrown vessels are more resistant to treatment than initial vessels, which can have implications for the long‐term treatment and rebound of corneal vessels and prognosis for graft survival. We also highlight several factors that may be driving this aggressive revascularization response, with an eye towards future interventions aimed at preventing revascularization following initial treatments for corneal neovascularization.

Corneal Collagen Cross-Linking Inhibits Corneal Blood and Lymphatic Vessels Temporarily in Alkali-Burned Rabbits

Purpose This study aimed to explore whether corneal cross-linking (CXL) could regress corneal blood vessels (CBV) and corneal lymphatic vessels (CLV) in alkali-burned rabbits. Methods A total of 80 rabbits 2–3 months old weighing 1.5–2.0 kg were randomly divided into four groups: CXL7 group; CTL7 group; CXL14 group; and CTL14 group. Then, 3% sodium pentobarbital 1 ml/kg and tetracaine eye drop 5 g/L were administered before surgery. NaOH 2 mol/L was topically applied to the central cornea to establish the alkali burning model. Then CXL was administered within 2 h in groups CXL7 and CXL14. Corneal opacity and edema, CBV and CLV volume, cluster differentiation 31 (CD31), and lymphatic vessel endothelial receptor 1 (LYVE-1) expression levels were analyzed on days 7 and 14. Results CXL reduced cornea opacity, CNV, and CLV volumes on day 7 in alkali-burned rabbits. However, CNV and CLV volumes were increased on day 14. CXL also showed down- and upregulation of CD31 and LYVE-1 expression levels on days 7 and 14, respectively. Conclusions CXL effectively regulated CBV and CLV in alkali-burned rabbits. The transient angioregression and lymphangioregression induced by CXL may be potentially helpful in vascularized high-risk eyes.

Optical coherence tomography angiography in herpetic leucoma

BackgroundHerpes simplex virus (HSV) keratitis remains a leading infectious cause of blindness worldwide. Although all forms of HSV keratitis are commonly recurrent, the risk is greatest in stromal keratitis, which is the most likely to result in corneal scarring, thinning, and neovascularization. Recent studies showed the ability of Optical Coherence Tomography Angiography (OCTA) to detect and study vascular abnormalities in the anterior segment, including abnormal corneal vessels. This study intends to investigate the potential of OCTA device to image and describe quantitatively the vascularization in eyes diagnosed with herpetic leucoma and to discuss and review the usefulness of this technique in this pathology.MethodsA Cross-sectional study was made, including 17 eyes of 15 patients with leucoma secondary to herpetic keratitis. All eyes underwent anterior segment Slit-Lamp photography (SLP), and OCTA with en-face, b-scans and c-scans imaging. The vessel density (VD) was analyzed in the inferior, nasal and temporal corneal margin in all patients, and in the central area, in eyes with central corneal neovascularization (CoNV). The measurements were calculated after binarization with ImageJ software, using OCTA scans with 6 × 6 mm in a depth of 800 μm.ResultsPatients included had a mean age 53.267 ± 21.542 (years ± SD). The mean total vessel area was 50.907% ± 3.435%. VD was higher in the nasal quadrant (51.156% ± 4.276%) but there were no significant differences between the three analyzed areas (p = 0.940). OCTA was able to identify abnormal vessels when SLP apparently showed no abnormal vessels; OCTA was able to distinguish between larger and smaller vessels even in central cornea; OCTA scans allowed the investigation of several corneal planes and the relation of them with clinical findings.ConclusionsOCTA can be useful in both qualitative and quantitative follow-up of patients and may become a non-invasive alternative to objectively monitor treatment response in eyes with corneal vascularization due to herpetic infection.

Surgical outcomes of concomitantly performed penetrating keratoplasty with intrascleral haptic fixation.

Purpose:To describe the surgical results of concomitantly performed optical penetrating keratoplasty (PKP) with glued intrascleral haptic fixation (ISHF).Methods:Retrospective review of 18 patients (15–72 years) with best-corrected visual acuity (BCVA) of ≤1/60 subjected to unilateral concomitant optical PKP with ISHF and followed up for 13.11 ± 5.83 months (6–26 months) was undertaken.Results:The most common diagnoses were failed PKP (9/18, 50%) followed by aphakic bullous keratopathy (5/18, 27%). Preoperative glaucoma, peripheral anterior synechiae (PAS), and deep vascularization were present in 7/18 (38.88%), 12/18 (61.11%), and 5/18 (27.77%) patients, respectively. Intraoperatively, concomitant procedures such as pupilloplasty and intraocular lens explant were undertaken in 5/18 (27.277%) patients and 1/18 patients (5.55%) experienced suprachoroidal hemorrhage. At final follow-up, BCVA was ≥6/60 in 50% patients (mean astigmatism: 4.79 ± 1.68D), and 55.55% cases experienced graft failure (90% failed within one year of surgery). The most common causes of graft failure were glaucoma (50%), glaucoma with rejection (20%), rejection (10%), retinal detachment (10%), and suprachoroidal hemorrhage (10%). The ODDS ratio (OR) of having graft failure with the following factors was postoperative secondary interventions (OR: 6), postoperative complications (OR: 2.25), prior failed graft (OR: 1.8), preoperative PAS (OR: 1.75), intraoperative concomitant procedures (OR: 1.5), preoperative glaucoma (OR: 1.33), previous surgeries (OR: 1.24), and deep corneal vessels (OR: 0.66).Conclusion:All patients underlying PKP combined with glued ISHF must be counseled about suboptimal surgical outcomes. Emphasis is laid on appropriate case selection and stringent follow-up during the first year after surgery. Secondary interventions should be undertaken cautiously and judiciously in these patients.

Inhibition of corneal neovascularization by topical application of nintedanib in rabbit models.

To evaluate the potential efficacy and mechanisms of nintedanib in corneal neovascularization (NV) in rabbit models. Corneal NV was induced using 1 mol/L NaOH. Rabbits (n=21) were randomized to 3 groups: Group 1 were treated with 0.9% NaCl, Group 2 with Avastin (5 mg/mL), and Group 3 with nintedanib (1 mg/mL). All treatments started 1d after alkaline burns and were topically performed 3 times a day for 2wk. Photographs were taken on a slit lamp microscope on day 7 and 14. The NV area, the length of the vascularization and angiogenesis index (AI) were used to evaluate the corneal NV. On day 14, the immunohistochemical (IHC) studies of the cornea were examined. Western blot was performed to test the expression levels of vascular endothelial growth factor (VEGF), Akt, p-Akt, P38, p-P38, MMP-2 and MMP-9. The corneal NV area, vessel length and AI in Group 3 were significantly lower than Group 2, with both being lower than Group 1. IHC staining showed that VEGF was significantly overexpressed in the epithelium and stroma of cornea following alkaline burns. In contrast, the level of VEGF was significantly suppressed in both Group 2 and Group 3. Western blot results further confirmed that, compared with Group 1, Group 3 had significantly reduced expressions of VEGF, Akt, p-Akt, p-P38, MMP-2, and MMP-9 in corneal tissues. Trends of lower levels of MMP-2, AKT, and p-AKT in Group 3 than Group 2 were identified. Nintedanib and Avastin can effectively inhibit corneal NV, with P38 MAPK and AKT signaling pathways being possibly involved. Nintedanib seems more effective than Avastin and has the potential to be a novel therapy for preventing corneal NV.

Ocular drug delivery system &amp;#40;ODDS&amp;#41;: Exploration the challenges and approaches to improve ODDS

The remarkable life structures and physiology of the eye presents huge difficulties to researchers in the field of visual medication conveyance frameworks. Nearby infusion is the most fitting and proper medication organization technique for the treatment of foremost front sickness. There are two kinds of hindrances in ophthalmic medication conveyance frameworks: static boundaries and dynamic obstructions. Static lamellae contain corneal, dermal, retinal, and retinal vessels while dynamic lamellae contain placental blood stream, conjunctiva, tear evacuation, and lymphatic seepage. These limitations influence the bioavailability of the medication. This article examines the limits of customary ophthalmic practice and the central point affecting the pharmacokinetics of the eye. Likewise, eye salves, gels, prodrugs, intranasal infusions, thickeners, entrance energizers, liposomes, microparticles, nanoparticles, visual infusions, inserts, nanoparticles, nanostructures, microemulsions, gels and periocular infusions. It guarantees the bioavailability of the medication and the controlled and constant control of the medication in the foremost and back alveoli.

Anterior Segment Optical Coherence Tomography Angiography Assessment of Corneal Vascularisation After Combined Fine-Needle Diathermy with Subconjunctival Ranibizumab: A Pilot Study.

To describe anterior segment optical coherence tomography angiography (AS-OCTA) imaging to monitor corneal vascularisation (CoNV) and scar reduction after combined fine-needle diathermy (FND) with subconjunctival ranibizumab. Prospective clinical study of six eyes from six subjects with corneal scar and CoNV which underwent combined FND with subconjunctival ranibizumab. All eyes were imaged using slit-lamp photography (SLP) and AS-OCTA (Optovue, Inc., Fremont, CA, wavelength: 840nm) before and after the operation, with two independent masked assessors analysing all images. Main outcome measures were changes in median corneal scar area and vessel density (AS-OCTA) comparing pre- and postoperative imaging up to month 3 and 6. The mean age of the subjects was 60 ± 23years, with three males and three females. CoNV and corneal scarring involving the visual axis were present in all eyes, secondary to previous infective keratitis (n = 3), severe blepharokeratoconjunctivitis (n = 2), or chemical injury (n = 1). Follow-up time frame ranged from 2 to 6months postoperation. There was a reduction in median corneal scar area from 30.2 mm2 (IQR 18.7-38.5) before surgery to 14.8 mm2 (IQR 7.1-19.6) after surgery, with a median reduction of 37.1% (IQR = -3.1-86.9, p = 0.046). There was also a reduction in median cornea vessel density (AS-OCTA) from 20.8% (IQR 16.1-20.8) before surgery to 17.6% (IQR 14.0-17.6) after surgery, with a median reduction of 15.1% (IQR 13.2-15.1, p < 0.001). Combined imaging of SLP and AS-OCTA is useful for monitoring treatment response of corneal scarring and CoNV after combined FND with subconjunctival Ranibizumab.

Loss of Primary Cilia Protein IFT20 Dysregulates Lymphatic Vessel Patterning in Development and Inflammation.

Microenvironmental signals produced during development or inflammation stimulate lymphatic endothelial cells to undergo lymphangiogenesis, in which they sprout, proliferate, and migrate to expand the vascular network. Many cell types detect changes in extracellular conditions via primary cilia, microtubule-based cellular protrusions that house specialized membrane receptors and signaling complexes. Primary cilia are critical for receipt of extracellular cues from both ligand-receptor pathways and physical forces such as fluid shear stress. Here, we report the presence of primary cilia on immortalized mouse and primary adult human dermal lymphatic endothelial cells in vitro and on both luminal and abluminal domains of mouse corneal, skin, and mesenteric lymphatic vessels in vivo. The purpose of this study was to determine the effects of disrupting primary cilia on lymphatic vessel patterning during development and inflammation. Intraflagellar transport protein 20 (IFT20) is part of the transport machinery required for ciliary assembly and function. To disrupt primary ciliary signaling, we generated global and lymphatic endothelium-specific IFT20 knockout mouse models and used immunofluorescence microscopy to quantify changes in lymphatic vessel patterning at E16.5 and in adult suture-mediated corneal lymphangiogenesis. Loss of IFT20 during development resulted in edema, increased and more variable lymphatic vessel caliber and branching, as well as red blood cell-filled lymphatics. We used a corneal suture model to determine ciliation status of lymphatic vessels during acute, recurrent, and tumor-associated inflammatory reactions and wound healing. Primary cilia were present on corneal lymphatics during all of the mechanistically distinct lymphatic patterning events of the model and assembled on lymphatic endothelial cells residing at the limbus, stalk, and vessel tip. Lymphatic-specific deletion of IFT20 cell-autonomously exacerbated acute corneal lymphangiogenesis resulting in increased lymphatic vessel density and branching. These data are the first functional studies of primary cilia on lymphatic endothelial cells and reveal a new dimension in regulation of lymphatic vascular biology.

THE PHARMACOLOGICAL ACTION OF CHOLINERGIC SUBSTANCES ON NEW CORNEAL BLOOD VESSELS IN RATS

This study aimed to evaluate the existence of muscarinic receptors in the new corneal blood vessels, experimentally induced with ketamine in rat pups. The experimental model of neovascularization was performed on 15-day-old Wistar rats, in which 5 intraperitoneal injections of ketamine were administered at a dose of 150 mg/kg body weight at 5-day intervals. Examination of the reactivity of new corneal blood vessels was performed on day 45 of life. Each eye that developed neovascularization was evaluated with a Nikon stereomicroscope, coupled to a Mshot video camera, and the total magnification of the system was 400X. The vascular diameter was measured at a chosen point, the same point /points for each recording made to that eye, and the acquisition of images was performed at set time intervals, every 60 seconds for each eye to be examined, throughout the recording period. The parameters under investigation were the variations of the vascular diameter, and the processing of the obtained data was performed with the help of Microsoft Office Excel. Our results suggest that muscarinic receptors are present in the new corneal blood vessels because the administration of conjunctival instillations of acetylcholine caused statistically significant vasodilation, while atropine, a blocker of muscarinic receptors, antagonized this effect.

Experimental research in rats on the reactivity of new corneal blood vessels to adrenaline.

Aim: The purpose of this experimental study was to evaluate the existence of adrenergic receptors in ketamine-induced corneal blood vessels in rat pups. Methods: The study of corneal neovascularization motricity was performed on 45-day-old Wistar rats in which, starting from the 15th day of life, corneal blood vessels were obtained by injecting intraperitoneal ketamine at a dose of 150 mg/ kg body weight, a total of 5 successive doses. The examination of the neovascularization was done with the help of a Nikon stereomicroscope connected to a video camera and a computer, the total magnification being 400X. The reactivity of the new corneal blood vessels to the administration in conjunctival instillations of a 1.5 mmol/L adrenaline solution was tested. The parameters followed were represented by variations in the caliber of corneal blood vessels. The data were analyzed using Microsoft Office Excel.Results: Administration of distilled water did not produce statistically significant changes in corneal blood vessels, while adrenaline produced a statistically significant constriction of vascular diameter (p=0.01 at T9, p=0.004 at T10, p=0.019 at time T11 of examinations).Conclusions: The results showed that adrenaline produces vasoconstriction in the new corneal blood vessels, which allows us to assume that they contain α-adrenergic receptors. However, we cannot say that corneal pathological vessels do not contain β2-type adrenergic receptors, because the effect of adrenaline may be an algebraic sum between vasoconstriction produced by stimulating α-adrenergic receptors and vasodilation produced by stimulating β2-adrenergic receptors, but in which the vasodilating effect may be masked by the vasoconstrictor effect given by a higher density of α-adrenergic receptors.Abbreviations: A= adrenaline, DNM = non-measurable diameter, NA= noradrenaline, Std.Er.= Standard error