Rachael L. Niederer

Age-related differences in the normal human cornea: a laser scanning in vivo confocal microscopy study

Aims: To quantify and establish baseline normative data for age-related differences in cellular and innervation density in the normal, healthy, human cornea using laser scanning in vivo confocal microscopy. Methods: Cross-sectional study of 85 normal subjects assessed via corneal topography and laser scanning in vivo confocal microscopy. Results: Mean age was 38±16 years (range 18–87 years) and 60% of subjects were female. Anterior keratocyte density declined by 0.9% per year (r = −0.423, p<0.001), posterior keratocyte density declined by 0.3% per year (r = −0.250, p = 0.021) and endothelial cell density declined by 0.5% per year (r = −0.615, p<0.001). Sub-basal nerve fibre density declined by 0.9% per year (r = −0.423, p<0.001). No association was observed between age and basal epithelial cell density, or between age and central corneal thickness, corneal astigmatism or horizontal corneal diameter (p>0.05). No association was observed between subject gender and corneal cell or innervation density. Conclusions: Using laser scanning in vivo confocal microscopy this study highlights a significant, and relatively linear, reduction in keratocyte and endothelial cell density with increasing subject age. Interestingly, corneal sub-basal nerve fibre density also significantly decreases with increasing age. In vivo laser scanning confocal microscopy provides a safe, non-invasive method for the establishment of normative data and assessment of alterations in human corneal microstructure following surgery or disease processes.

Clinical in vivo confocal microscopy of the human cornea in health and disease.

Confocal microscopy enables microstructural analysis of the in vivo cornea, allowing fresh insight into corneal microstructure in health, and in inherited and acquired corneal disease. This method of corneal examination is evolving in an exponential fashion, with rapid advances in technology being mirrored by rapid growth in both research and clinical applications. Whilst initially the evidence base for in vivo confocal microscopy consisted largely of small case studies, in recent years there has been a trend towards collecting quantitative data in an effort to better delineate between heath and disease. Confocal microscopy has been utilised clinically to aid in the diagnosis of infectious keratitis, in particular Acanthamoeba and fungal keratitis, and has also established a role in the diagnosis and phenotyping of corneal dystrophies. This article reviews in vivo confocal microscopy of the human cornea in health and disease and examines clinical and research applications of this new technology.

Laser scanning in vivo confocal microscopy reveals reduced innervation and reduction in cell density in all layers of the keratoconic cornea.

PURPOSE The exact pathophysiological processes underlying keratoconus remain an enigma. In this study, laser scanning in vivo confocal microscopy (IVCM) was used to define further the microstructural abnormalities in the keratoconic cornea and to establish the relationship with disease severity. METHODS This was a prospective, cross-sectional study comparing 52 subjects with keratoconus and 52 age-matched control subjects. Assessment included demographics, history, slit lamp biomicroscopy, computerized corneal tomography, and laser scanning IVCM. RESULTS Significantly lower cell densities (in cells per square millimeter, mean +/- SD) were observed in keratoconus corneas than in normal ones: basal epithelial cells, 4340.6 +/- 595.2 vs. 5777.6 +/- 958.2 (P < 0.001), anterior keratocytes, 523.6 +/- 206.4 vs. 859.7 +/- 219.1 (P < 0.001), posterior keratocytes, 240.4 +/- 64.5 vs. 330.6 +/- 52.3 (P < 0.001), and endothelial cells 2412.2 +/- 339.5 vs. 2845.6 +/- 313.0 (P < 0.001). Subbasal nerve fiber density was 52.7% lower in keratoconus corneas than in the control (P < 0.001). Basal epithelial cell density (P = 0.001), subbasal nerve fiber density (P = 0.015), and anterior keratocyte density (P < 0.001) correlated with severity of disease. Lower subbasal nerve density also correlated with younger age at diagnosis (r = 0.397, P = 0.004). Severe disease was associated with diagnosis at a younger age (P = 0.023), a history of eye rubbing (P = 0.025), and Maori or Pacific Island ethnicity (P = 0.001). CONCLUSIONS Significant microstructural abnormalities were identified at every level of the keratoconic cornea and were related to disease severity. IVCM offers a potential insight into the pathophysiology of the microstructural changes in keratoconus.

In vivo confocal microscopy of subepithelial infiltrates in human corneal transplant rejection.

Purpose: Corneal allograft rejection is the leading cause of penetrating keratoplasty failure in the first year after surgery. We report 2 cases of subepithelial infiltrates in corneal transplant rejection imaged by in vivo confocal microscopy. Methods: Case report and review of relevant literature. Results: Two subjects with subepithelial infiltrates in previously clear penetrating corneal transplants were assessed. In vivo confocal microscopy revealed focal accumulations of hyperreflective dendritic-like particles, postulated to represent Langerhans cells, at the level of the basal epithelium and Bowman membrane. Altered keratocytes with visible cytoplasmic processes were observed posterior to these foci. Conclusions: To our knowledge, these are the first reported cases of in vivo confocal microscopy appearance of corneal allograft rejection in humans. In vivo confocal microscopy may provide a valuable clinical tool to aid in the diagnosis of early corneal transplant rejection and in the differential diagnosis of other inflammatory conditions of the cornea.

Infectious endophthalmitis: clinical features, management and visual outcomes

Aim:  To identify the clinical features and outcomes of infectious endophthalmitis in New Zealand.

Recurrence of keratoconic pathology in penetrating keratoplasty buttons originally transplanted for keratoconus

Purpose: Study aimed to examine buttons removed from patients originally grafted for KC (group 1) for signs of recurrence at a cellular level and compare them with buttons removed from patients originally grafted for other conditions (group 2). The study further aimed to compare buttons from group 1 exhibiting high astigmatism (group 3) with the other buttons in the study (group 4). Methods: Together with clinical data, corneal buttons were collected at repeat penetrating keratoplasty and labeled immunohistochemically with a panel of antibodies to structural proteins to assist microanatomical interpretation. Image analysis of montaged images of many individual sections was performed using custom software. The resulting data were analyzed statistically for significant differences between groups 1/2 and 3/4. Results: Little evidence of KC recurrence could be found despite statistically significant differences between groups 1/2 in corneal thinning at both graft-host junction (GHJ) (P = 0.035) and within the graft (P = 0.001), epithelial thickening at the GHJ only (P < 0.001), high astigmatism (P = 0.028), and history of high intraocular pressure (P = 0.032) or rejection (P = 0.002) and between groups 3/4 in corneal thinning at both GHJ (P = 0.002) and within the graft (P = 0.003), epithelial thickening at the GHJ only (P = 0.003), and high astigmatism (P < 0.001). Conclusion: This study has highlighted the rarity of recurrence of KC in transplanted donor corneas and the corresponding difficulty in detecting early signs of the disease.

Presumed late diffuse lamellar keratitis progressing to interface fluid syndrome

We report an unusual case of presumed late-onset unilateral diffuse lamellar keratitis of uncertain etiology in a 23-year-old man who presented with elevated intraocular pressure following uneventful laser in situ keratomileusis (LASIK). After treatment with topical corticosteroid therapy, the condition progressed to interface fluid syndrome. Isolated pockets of fluid were clearly demonstrated at the level of the LASIK flap interface on slitlamp biomicroscopy and in Pentacam Scheimpflug images.

Resisting susceptibility: bacterial keratitis and generations of antibiotics

istered to the eye separately, at least 5 min apart. 10 Although dual therapy provides the broad spectrum antimicrobial activity necessary for initial empirical therapy in bacterial keratitis, this treatment differs substantially from the conceptualized ideal antimicrobial regimen. Cephalosporins and aminoglycosides penetrate the cornea relatively poorly, and subsequently need to be used at fortified concentrations that are not available commercially. 10 Use of aminoglycosides at fortified concentrations has been associated with ocular surface toxicity and long-term use with delayed wound healing. 10

Early-onset Fuchs endothelial dystrophy with a novel pathological phenotype

Fuchs endothelial dystrophy (FED) is a relatively common, slowly progressive, often asymmetric but bilateral corneal dystrophy. It currently accounts for 4.4% of all penetrating keratoplasties (PK) performed in New Zealand; however, its incidence is set to rise with an ageing population and more intraocular surgeries being performed in older patients. We report the unusual pathological features of a cornea affected by an early-onset form of FED. This cornea belonged to a female patient with highly progressive form of FED who required bilateral PK by the age of 31. She had no known family history of FED and no genetic studies have been undertaken to identify a possible novel mutation. For comparison, six corneal buttons were obtained from older subjects (mean age, 74 6 years; range, 65–79) undergoing PK for FED. The corneas were processed, cut into multiple 30 mm sections, and immunolabelled with the antibodies listed in Table 1. All sections were examined with a Leica DMRA fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). Representative slides were further imaged using a Leica TCS-4D confocal laser scanning microscope system (Leica Microsystems, Heidelberg, Germany). In this early-onset form of FED, decreased stromal innervation and near absence of subbasal and subepithelial nerves were observed in the anterior cornea (Fig. 1a). In contrast, subbasal and subepithelial nerves were observed in all of the late-onset FED corneas stained with the antibody specific for acetylated alpha-tubulin (antiAAT) (Fig. 1b). Peculiarly, in the early-onset form of FED, anti-AAT also labelled numerous dendriform structures in the Descemet’s membrane (Fig. 1c,d). When examined with an antibody specific for laminin, early-onset FED exhibited extensive disruption to the epithelial basement membrane (BM) in the majority of the sections (Fig. 1c). In contrast, epithelial BM integrity was largely preserved in the late-onset FED, with occasional mild to moderate disruption observed only in a small proportion of sections (Fig. 1d). In both forms of FED, Descemet’s layer was severely thickened and had guttae extending from the Descemet’s layer towards the endothelial surface. In the early-onset FED, guttae tended to be larger (Fig. 2a,b) and were observed in most of the sections, whereas in the late-onset form had smaller guttae with patchy distribution (Fig. 2c). In both forms but more prominently in the early-onset FED, extensive laminin deposits were identified on the guttae surface. Corneal nerves play an important role in the regulation of corneal epithelial integrity, proliferation and wound healing. In our study, the subbasal and subepithelial regions of the early-onset FED cornea were nearly devoid of nerves; however, these regions have been shown to have the highest nerve density of all layers, both in normal and FED corneas. In the early-onset FED, the pathological BM secretion which characterize FED may be further exaggerated, resulting in more extensive guttae and laminin deposition. This process may also lead to structural abnormalities in the epithelial BM of the early-onset FED corneas, which may predispose the epithelial BM to disruption even with minimal oedema, accounting for a more severe disease both pathologically and clinically. Also specific to the early-onset FED, anti-AAT immunolabelling revealed dendriform structures in the Descemet layer. As the Descemet’s layer is not innervated in humans and given the intracellular nature of AAT, these may represent fibroblasts or myofibroblasts originating from the stroma or macrophages or dendritic cells originating from the posterior limbus. Although speculative, this raises interesting question about the role of inflammatory processes in earlyonset FED. These distinct pathological features seen in early-onset FED may characterize a novel subtype of the disease, as well as providing insight into the pathogenesis of FED.

In vivo confocal microscopy of climatic droplet keratopathy

We describe the corneal microstructural changes in a patient with spheroidal degeneration using in vivo confocal microscopy. Multiple hypo‐ and hyper‐reflective spherical lesions were observed in the anterior corneal stroma and Bowmans layer ranging from 45 to 220 μm in size. The corneal epithelium, posterior stroma and endothelium were otherwise unaffected. In vivo confocal microscopy demonstrates good correlation with excised histological samples in climatic droplet keratopathy. It provides a non‐invasive technique to examine the living cornea for degenerative disease and acts as a bridge between clinical and laboratory observations.