Bernhard Steger

In vivo confocal microscopic characterisation of the cornea in chronic graft-versus-host disease related severe dry eye disease

Aims To first describe in vivo confocal microscopic (IVCM) corneal findings in severe dry eye syndrome due to ocular chronic graft versus host disease (cGvHD) after allogeneic stem cell transplantation. Methods IVCM of the central cornea was performed in 12 prospectively recruited patients with severe ocular cGvHD associated dry eye syndrome and in six control patients with haematological malignancies without cGvHD. Within each examined corneal layer, at least three non-overlapping areas were selected for representative analysis. Results The number of sub basal nerve branches was markedly reduced in patients with cGvHD. Sub basal nerve morphology was characterised by increased tortuosity and reduced reflectivity. Accumulation of hyper-reflective extracellular matrix, significantly increased haze and increased keratocyte density were found in the anterior stroma of the study group. Conclusions IVCM findings of the cornea in patients with severe ocular cGvHD include a rarefaction of the sub basal corneal nerve plexus and dense accumulation of hyper-reflective extracellular matrix in the anterior stroma.

Corneal Indocyanine Green Angiography to Guide Medical and Surgical Management of Corneal Neovascularization.

Purpose: To illustrate the role of corneal angiography in the clinical assessment and surgical treatment of patients with complex corneal neovascularization (CoNV). Methods: A case series of 3 patients with CoNV is presented whose management was guided by indocyanine green (ICG) and fluorescein corneal angiography. In the first case, there was recurrent lipid exudation into an intrastromal cleft from CoNV; in the second, there was progressive exudation from CoNV at the graft–host interface; in the third, CoNV was associated with rejection after deep anterior lamellar keratoplasty. Results: In the first case, angiography helped to identify and treat the feeder vessels and stop further leakage. In the second case, it was possible using angiography to differentiate CoNV arising from iris and limbal vasculature enabling angiographic-guided fine-needle diathermy with cessation of exudation. In the third case, angiography revealed the location of CoNV in the host–graft interface after deep anterior lamellar keratoplasty, rather than within the corneal stroma. Conclusions: Corneal angiography is a useful diagnostic tool to guide medical and surgical management of CoNV by enabling the localization of vessel depth and topography.

A COL17A1 splice-altering mutation is prevalent in inherited recurrent corneal erosions

PURPOSE Corneal dystrophies are a genetically heterogeneous group of disorders. We previously described a family with an autosomal dominant epithelial recurrent erosion dystrophy (ERED). We aimed to identify the underlying genetic cause of ERED in this family and 3 additional ERED families. We sought to characterize the potential function of the candidate genes using the human and zebrafish cornea. DESIGN Case series study of 4 white families with a similar ERED. An experimental study was performed on human and zebrafish tissue to examine the putative biological function of candidate genes. PARTICIPANTS Four ERED families, including 28 affected and 17 unaffected individuals. METHODS HumanLinkage-12 arrays (Illumina, San Diego, CA) were used to genotype 17 family members. Next-generation exome sequencing was performed on an uncle-niece pair. Segregation of potential causative mutations was confirmed using Sanger sequencing. Protein expression was determined using immunohistochemistry in human and zebrafish cornea. Gene expression in zebrafish was assessed using whole-mount in situ hybridization. Morpholino-induced transient gene knockdown was performed in zebrafish embryos. MAIN OUTCOME MEASURES Linkage microarray, exome analysis, DNA sequence analysis, immunohistochemistry, in situ hybridization, and morpholino-induced genetic knockdown results. RESULTS Linkage microarray analysis identified a candidate region on chromosome chr10:12,576,562-112,763,135, and exploration of exome sequencing data identified 8 putative pathogenic variants in this linkage region. Two variants segregated in 06NZ-TRB1 with ERED: COL17A1 c.3156C→T and DNAJC9 c.334G→A. The COL17A1 c.3156C→T variant segregated in all 4 ERED families. We showed biologically relevant expression of these proteins in human cornea. Both proteins are expressed in the cornea of zebrafish embryos and adults. Zebrafish lacking Col17a1a and Dnajc9 during development show no gross corneal phenotype. CONCLUSIONS The COL17A1 c.3156C→T variant is the likely causative mutation in our recurrent corneal erosion families, and its presence in 4 independent families suggests that it is prevalent in ERED. This same COL17A1 c.3156C→T variant recently was identified in a separate pedigree with ERED. Our study expands the phenotypic spectrum of COL17A1 disease from autosomal recessive epidermolysis bullosa to autosomal dominant ERED and identifies COL17A1 as a key protein in maintaining integrity of the corneal epithelium.

Angiographic and In Vivo Confocal Microscopic Characterization of Human Corneal Blood and Presumed Lymphatic Neovascularization: A Pilot Study.

Purpose: To characterize corneal nevocoarization (CoNV) in vivo. Methods: Patients with CoNV were categorized into those with active disease defined by the presence of corneal infiltrates and/or edema and those with inactive disease, the latter divided into those with or without corneal red blood cell traffic (RBCT). CoNV were imaged using in vivo confocal microscopy (IVCM) and angiography. Leakage and uptake of dye from the extravascular to intravascular space were investigated using repeat angiography and digital subtraction analysis. Vessel parameters and the presence of RBCT were analyzed using custom-designed software. Results: Thirteen patients with CoNV were included: 4 had active and 9 had inactive disease with CoNV present for 3.5 ± 1.7 months and 30.2 ± 30.7 months, respectively. Using a combination of digital subtraction analysis and IVCM, presumed lymphatic vessels were detected only in patients with active CoNV. These vessels were characterized by an indistinct vessel wall, carrying nucleated cells, and on IVCM, it had a mean diameter of 19.7 ± 6.1 &mgr;m (11–30 &mgr;m). The ratio of the diameter of presumed lymphatic vessels to that of neighboring vessels was 0.6 ± 0.1 (0.5–0.6). Of the patients with inactive disease, 4 had CoNV with RBCT (mean age CoNV, 17.3 ± 7.6 months) and 5 (mean age CoNV, 40.6 ± 35 months) had CoNV without evidence of RBCT on biomicroscopy, but evident on angiography and IVCM. These represent perfused vessels with no or intermittent RBCT. Conclusions: CoNV can be characterized in vivo using a combination of IVCM and angiography. The vascular features vary according to the age of the CoNV and disease activity. Further improvements in angiographic image alignment, however, are needed.

Method for Angiographically Guided Fine-Needle Diathermy in the Treatment of Corneal Neovascularization.

Purpose: To describe a method to assess corneal neovascular (CoNV) complexes and identify feeder vessels for selective arterial fine-needle diathermy (FND). Methods: In patients with CoNV, color photography and corneal indocyanine green angiography (ICGA) and fluorescein angiography are performed. After injection of indocyanine green and sodium fluorescein dye, videography and single-frame images of the region of interest are recorded. Videography is used to measure the time to leakage to assess vessel maturity to guide medical treatment and to discern afferent from efferent vessels. Single-frame images are then selected to locate the number of afferent vessels for surgery, which are selectively cut with a 25-gauge marked needle for the application of FND. Results: Angiography using fluorescein and indocyanine green allows the characterization of CoNV based on assessment of both morphologic (ICGA) and functional (fluorescein angiography) parameters. The time to leakage of fluorescein dye provides important functional information on vessel maturity and helps discern whether medical treatment should be followed before surgical. ICGA allows the identification and delineation of afferent feeder vessels even in the presence of corneal opacities affecting biomicroscopic visibility. Colocalizing the afferent vessel to a visible venous landmark or branch is helpful for placement of the incision and application of FND. Using the described approach, angiographically identified feeder vessels can be selectively treated by FND with minimal thermal energy applied to the corneoscleral limbus. Conclusions: The described method for angiographically guided assessment of CoNV is a useful approach for guiding the medical and surgical treatment of CoNV.

Reliability of the Effect of Artificial Anterior Chamber Pressure and Corneal Drying on Corneal Graft Thickness.

Purpose: To investigate the effect of artificial anterior chamber (AAC) pressure and corneal drying on the graft thickness in preparation for Descemet stripping automated endothelial keratoplasty. Methods: Twenty-seven corneoscleral discs were placed in an AAC. The AAC pressure (15, 45, 92, 109, and 198 mm Hg) was controlled using the height of an infusion bottle and a roller clamp. The endothelium was removed in 1 subgroup. Corneas were exposed to room air or repeatedly dried using cellulose spears. Central corneal thickness was measured every 90 seconds for the first 15 minutes and again at 20 minutes using an ultrasound pachymeter (SP-100, Tomey). Results: There was a significant linear relationship between the corneal thickness and both AAC pressure and corneal drying. Very high coefficients of determination and narrow 95% confidence intervals were present, in particular for high pressures and drying. The rate of thinning increased with increasing pressure and drying to 1.6% per minute. At the maximum rate of thinning, a 10% reduction in corneal thickness occurred in 6 minutes or 100 &mgr;m in 8.8 minutes. Removal of the corneal endothelium reduced the rate of thinning to 0.3% per minute (R2 = 0.72). Conclusions: Increasing AAC pressure and corneal drying reduced the graft thickness at a very predictable rate. Adequate corneal thinning can be achieved by increasing the pressure in the AAC by closing the clamp followed by removal of the residual corneal epithelium and repeated drying with a cellulose spear for 5 to 10 minutes, depending on the initial corneal thickness. This method is simple and is both suitable for use in the eye bank and by the surgeon.

Gene-based antiangiogenic applications for corneal neovascularization

Corneal avascularity is maintained by angiogenic privilege, an active process involving the production of higher level of angiostatic factors to offset the effect of angiogenic factors. A wide range of pathological insults to the cornea can disrupt this intricate equilibrium and promote angiogenesis and corneal neovascularization with resultant visual impairment. Corneal neovascularization is also a major risk factor for graft failure after keratoplasty. Current treatment options for corneal neovascularization are restricted by limited efficacy, adverse effects, and a short duration of action. The unique anatomical position and relative immune privilege of cornea make it an ideal tissue for gene-based therapies. Gene transfer vectors have been used to deliver or target genes involved in the pathogenesis of corneal neovascularization in animal models. Several proangiogenic and antiangiogenic factors have been targeted and assessed in experimentally induced corneal neovascularization. Antisense oligonucleotides targeting corneal neovascularization have entered human clinical trials and have not required vector delivery systems. The emergence of these RNA-based strategies heralds a new era in the management of corneal neovascularization and ocular therapeutics.

Angiographic Evaluation of Inflammation in Atopic Keratoconjunctivitis.

ABSTRACT Purpose: Grading of disease activity in patients with atopic keratoconjunctivitis (AKC) is limited by intra- and inter-observer variability. The aim of this study was to identify angiographic parameters to help evaluate inflammation and disease activity. Methods: In 12 patients (4 with active, 2 with inactive AKC, and 6 controls), disease activity were assessed using a validated biomicroscopic grading scale. Imaging of upper tarsal conjunctiva was undertaken using color photography, fluorescein (FA) and indocyanine green angiography (ICGA). Results: Extravascular ICG leakage only occurred in patients with active disease (mean 84.6 ± 28.8 s) except in one patient with inactive disease but only after 6 min. Transepithelial leakage of fluorescein occurred in all patients with active AKC (mean 63.5 ± 17.8 s) but not in patients with inactive disease or controls. Conclusions: Conjunctival transepithelial leakage of fluorescein and extravascular interstitial accumulation of ICG may be useful markers of disease activity patients in AKC.

Femtosecond Laser-Assisted Lamellar Keratectomy for Corneal Opacities Secondary to Anterior Corneal Dystrophies: An Interventional Case Series.

Purpose: To report results of femtosecond laser–assisted lamellar keratectomy (FLK) for corneal opacities secondary to anterior corneal dystrophies. Methods: Patients with a clinical diagnosis of Reis–Bücklers corneal dystrophy, granular corneal dystrophy, lattice corneal dystrophy, and macular corneal dystrophy were treated. FLK was performed to remove a central corneal free cap of 9.5 mm in diameter at a depth of 110 to 140 &mgr;m on which histological analysis was undertaken. Preoperative and postoperative refraction, best spectacle-corrected visual acuity, corneal topography results, and color photographs were recorded. Postoperative in vivo confocal microscopy of the cornea was performed. Changes in uncorrected visual acuity and best spectacle-corrected visual acuity, keratometry, refractive error, corneal irregularity, residual or recurrent central corneal opacities, and corneal haze were used to assess the outcome. Results: Eight eyes of 6 patients were treated. The clinical diagnosis was confirmed histologically in all cases. Visual acuity improved significantly from 0.49 ± 0.2 logMAR to 0.14 ± 0.13 logMAR after a mean follow-up of 29 ± 14 (range 8–54) months. Residual central stromal opacities were noted in 5 of 8 eyes immediately postoperatively. Clinically significant recurrence of disease was noted in 1 eye. Keratometry and refraction remained stable, and no further surgical intervention was needed. Patients with stromal corneal dystrophies had worse outcome than those with Reis–Bücklers corneal dystrophy. Conclusions: In this case series, FLK provided both therapeutic and diagnostic intervention, delaying more invasive surgery. In vivo confocal microscopy showed signs of postoperative corneal stromal neuropathy.

Combined Use of Rituximab and Intravenous Immunoglobulin for Severe Autoimmune Cicatricial Conjunctivitis—An Interventional Case Series

Purpose: Despite the availability of systemic immunosuppressants, cicatricial conjunctivitis (CC) remains a potentially blinding ocular surface disease. We aim to describe the combined use of rituximab (RTX) and intravenous immunoglobulin (IVIg) for severe recalcitrant autoimmune CC. Methods: In this single-center retrospective interventional case series with follow-up between 32 and 65 months, 3 cases with mucous membrane pemphigoid (patients 1–3) and 1 case with linear IgA disease (patient 4) were included. Initial conventional immunosuppressive therapy regimens included systemic steroids, dapsone, and mycophenolate. At the time of initiation of RTX and IVIg, all patients had only one eye with good visual acuity or good visual potential. Treatment included 1 to 2 cycles of RTX (1000 mg twice at an interval of 2 weeks apart), and 2 to 9 monthly courses of IVIg (2 g/kg over 3 days). Outcome measures were blindness, as defined by best spectacle-corrected visual acuity <0.05 on a decimal scale, and clinical staging of cicatricial disease (Rowsey and Foster staging). Results: In 4 presented cases, progression of cicatricial disease was stopped as assessed by the Foster grading scale and visual acuity was stabilized in all patients. Conjunctival scarring was stabilized in 2 cases and continued to progress in 2 cases. One patient developed septicemia 6 weeks after RTX infusion, which was successfully treated. Conclusions: Combination therapy of RTX and IVIg is a potent treatment regimen for recalcitrant autoimmune CC. Further prospective controlled studies on efficacy and safety are warranted before widespread clinical application.