Dipika V. Patel

Contemporary in vivo confocal microscopy of the living human cornea using white light and laser scanning techniques: a major review.

In vivo confocal imaging of the cornea has evolved exponentially over the last few decades and it has increasingly emerged from the laboratory to be used in the clinical setting in relation to inherited corneal diseases, corneal infections, contact lens wear and the effects of corneal surgery. This evolution has led to significant enhancement of our knowledge of the living cornea in both its physiological and pathological states. A number of in vivo confocal microscope devices using white, and more recently coherent, light sources have been developed to provide non‐invasive assessment of the corneal microstructure at a lateral resolution of 1–2 µm. The fundamental principles of in vivo confocal microscopy and the key differences between these devices are highlighted in this review. By providing a systematic review of the extensive literature on the human cornea, this perspective paper aims to provide an overview of how in vivo confocal microscopy has contributed to our greater understanding of the human cornea in health, in disease, and following surgery, with a particular emphasis on quantitative data. The utility and limitations of available data are highlighted as are possibilities for the future development of this innovative technology.

In vivo confocal microscopy of human corneal nerves in health, in ocular and systemic disease, and following corneal surgery: a review

The exponential evolution of in vivo confocal microscopy (IVCM) has led to a significant enhancement in our knowledge of the living cornea in both its physiological and pathological states. Studies using white light and coherent light-based IVCM have enabled detailed quantitative analysis of sub-basal nerve parameters, and have also resulted in the elucidation of the two-dimensional architecture of the normal corneal sub-basal nerve plexus. However, accurate and repeatable methods for quantitative analysis of stromal nerves imaged by IVCM remain to be developed. The effect of corneal surgery on central corneal nerves has been well documented in many IVCM studies, and these studies provide an indication of the regenerative capacity of corneal nerves. IVCM has also clearly demonstrated the involvement of corneal nerves in diseases such as keratoconus, although it remains unclear whether these alterations are a cause of, or occur secondary to, the disease process. IVCM has also been proposed as non-invasive method of accurately diagnosing and assessing the progression of diabetic neuropathy, highlighting the potential application of this technique as an indicator of systemic disease. This review consolidates our knowledge of how IVCM has contributed significantly to our greater understanding of corneal nerves in the living human cornea in health and disease.

In Vivo Laser Scanning Confocal Microscopy Confirms that the Human Corneal Sub-basal Nerve Plexus Is a Highly Dynamic Structure

PURPOSE To add to findings in a prior study on the two-dimensional arrangement of the living human sub-basal corneal nerve plexus and determine whether it is a dynamic structure. METHODS Laser scanning in vivo confocal microscopy was performed on the left cornea of a healthy subject who had been examined with the same methodology 2 years earlier. Examinations were performed once a week for 6 weeks with the purpose of producing a two-dimensional reconstruction map of the living human sub-basal corneal nerve plexus at each session. A two-dimensional graphics program was used to arrange and map images obtained at each session into confluent montages. RESULTS The mean dimensions of the corneal areas mapped were 4.80 +/- 0.45 mm horizontally and 4.60 +/- 0.52 mm vertically. The nerve branching patterns observed 2 years earlier did not correspond with those in any recent maps. Over the 6-week period, the sub-basal nerve pattern appeared to migrate centripetally from the corneal periphery toward an inferocentral whorl. In the region of the whorl the nerves altered their generally centripetal direction of migration, undergoing clockwise rotation. The centripetal rate of migration decreased with proximity to the center of the whorl (5.6 +/- 3.4 microm/wk at 13 microm from the whorl, 13.9 +/- 5.5 microm/wk at 333 microm from the whorl, and 25.9 +/- 8.6 microm/wk at 698 microm from the whorl). CONCLUSIONS This study provides strong evidence that the living human sub-basal corneal nerve plexus is a highly dynamic structure, with continuous centripetal movement of identifiable branch points of up to 26 microm/wk, creating dramatic pattern changes in the plexus over a 6-week period.

Laser scanning in vivo confocal microscopy and quantitative aesthesiometry reveal decreased corneal innervation and sensation in keratoconus

PurposeThe purpose of this study was to quantitatively analyse laser scanning in vivo confocal microscopy images of the corneal epithelium and sub-basal nerve plexus in patients with keratoconus and to correlate these microstructural observations with corneal sensitivity.MethodsA total of 31 eyes of 31 normal human subjects, and 27 eyes of 27 subjects with an established diagnosis of keratoconus were recruited. Twelve subjects with keratoconus had never worn contact lenses (K-NCL). Fifteen subjects with keratoconus wore contact lenses routinely (K-CL). All eyes were examined using slit-lamp biomicroscopy, Orbscan topography, non-contact corneal aesthesiometry, and laser scanning in vivo confocal microscopy.ResultsCentral corneal sensation was significantly lower in K-CL compared to normal (P=0.028). However, there was no significant difference in corneal sensation between the normal and K-NCL groups (P=0.059). Both sub-basal nerve density (P<0.001) and basal epithelial density (P<0.001) were significantly lower than normal in all keratoconic subjects. Central corneal sensation was only significantly correlated with sub-basal nerve density (P=0.001) and was not significantly correlated with any of the basal epithelial parameters. Sub-basal nerve density showed significant positive correlation with basal epithelial density (P<0.001).ConclusionThis quantitative study reveals decreased corneal innervation, sensation, and basal epithelial density in keratoconus. The results of this study provide strong evidence that both the sub-basal nerves and the basal epithelium may be involved in the pathogenesis of keratoconus, although it is uncertain whether these are primary or secondary changes.

Corneal sensitivity and slit scanning in vivo confocal microscopy of the subbasal nerve plexus of the normal central and peripheral human cornea.

Purpose: To determine the subbasal nerve density and tortuosity at 5 corneal locations and to investigate whether these microstructural observations correlate with corneal sensitivity. Method: Sixty eyes of 60 normal human subjects were recruited into 1 of 3 age groups, group 1: aged <35 years, group 2: aged 35-50 years, and group 3: aged >50 years. All eyes were examined using slit-lamp biomicroscopy, noncontact corneal esthesiometry, and slit scanning in vivo confocal microscopy. Results: The mean subbasal nerve density and the mean corneal sensitivity were greatest centrally (14,731 ± 6056 μm/mm2 and 0.38 ± 0.21 millibars, respectively) and lowest in the nasal mid periphery (7850 ± 4947 μm/mm2 and 0.49 ± 0.25 millibars, respectively). The mean subbasal nerve tortuosity coefficient was greatest in the temporal mid periphery (27.3 ± 6.4) and lowest in the superior mid periphery (19.3 ± 14.1). There was no significant difference in mean total subbasal nerve density between age groups. However, corneal sensation (P = 0.001) and subbasal nerve tortuosity (P = 0.004) demonstrated significant differences between age groups. Subbasal nerve density only showed significant correlations with corneal sensitivity threshold in the temporal cornea and with subbasal nerve tortuosity in the inferior and nasal cornea. However, these correlations were weak. Conclusions: This study quantitatively analyzes living human corneal nerve structure and an aspect of nerve function. There is no strong correlation between subbasal nerve density and corneal sensation. This study provides useful baseline data for the normal living human cornea at central and mid-peripheral locations.

Biomechanical Responses of Healthy and Keratoconic Corneas Measured Using a Noncontact Scheimpflug-Based Tonometer

PURPOSE To examine the repeatability of measurements obtained using a noncontact Scheimpflug-based tonometer (Corvis ST) and investigate potential differences in these parameters between healthy and keratoconic (KC) corneas. METHODS Forty-five keratoconic eyes and 103 healthy eyes were examined using biomicroscopy, corneal tomography and the Corvis ST (CST). RESULTS Intraocular pressure and central corneal thickness (CCT) were highly repeatable (intraclass correlation [ICC] > 0.70, coefficient of variation [CV] < 0.20). Deformation amplitude (DA) and applanation-1 time (A1T) were fairly repeatable (ICC > 0.60, CV < 0.20). There was no association between DA and age, sex, or ethnicity in healthy eyes. There was a greater mean DA in the KC group compared with 46 age-matched healthy eyes (KC 1.37 ± 0.21 mm, healthy 1.05 ± 0.11 mm, P < 0.001). Multivariate analysis showed DA in KC was predicted by IOP, CCT, and the steepest simulated keratometry value (P = 0.03, P = 0.03, P = 0.001 respectively, R(2) = 0.75). A subgroup analysis of healthy and KC eyes with comparable mean CCT and IOP highlighted a statistically significant difference in mean DA (KC 1.25 ± 0.08 mm, thin healthy 1.13 ± 0.09 mm, P = 0.006). Receiver-operating characteristic analysis showed an area under the curve of 0.77 (95% CI 0.61-0.93, P = 0.006) but no ideal cutoff value for DA. CONCLUSIONS Key parameters assessed by the CST are repeatable. Keratoconus is associated with greater DA than in healthy eyes, even when controlled for CCT and IOP. Deformation amplitude may be a useful adjunct in keratoconus assessment and monitoring, but cannot solely discriminate between healthy and keratoconic corneas.

Quantitative analysis of in vivo confocal microscopy images: A review

In vivo confocal microscopy (IVCM) is a non-invasive method of examining the living human cornea. The recent trend towards quantitative studies using IVCM has led to the development of a variety of methods for quantifying image parameters. When selecting IVCM images for quantitative analysis, it is important to be consistent regarding the location, depth, and quality of images. All images should be de-identified, randomized, and calibrated prior to analysis. Numerous image analysis software are available, each with their own advantages and disadvantages. Criteria for analyzing corneal epithelium, sub-basal nerves, keratocytes, endothelium, and immune/inflammatory cells have been developed, although there is inconsistency among research groups regarding parameter definition. The quantification of stromal nerve parameters, however, remains a challenge. Most studies report lower inter-observer repeatability compared with intra-observer repeatability, and observer experience is known to be an important factor. Standardization of IVCM image analysis through the use of a reading center would be crucial for any future large, multi-centre clinical trials using IVCM.

In vivo confocal microscopy of posterior polymorphous dystrophy.

Purpose: This study was designed to delineate the morphologic features of posterior polymorphous dystrophy (PPD) using in vivo confocal microscopy. Methods: Six patients with clinically diagnosed PPD were examined by slit-lamp biomicroscopy, Orbscan II slit-scanning elevation topography, and in vivo confocal microscopy. Results: Endothelial cell densities ranged from 613 to 3,405 cells/mm2 and endothelial polymegathism was noted in all cases, whereas endothelial pleomorphism was not a prominent feature. Three cases exhibited bright endothelial nuclei. A variety of abnormal curvilinear and vesicular abnormalities were imaged by in vivo confocal microscopy, with lesions ranging between 6 and 159 μm in diameter. Abnormal endothelial cells were visible within some of these lesions. Six cases showed hyperreflectivity at the level of Descemets membrane around the lesions. Deep stromal keratocytes appeared to aggregate around, or were compressed by, the endothelial lesions in one case. Conclusions: We report the largest case series of PPD imaged by in vivo confocal microscopy. The ability of in vivo confocal microscopy to assess the living cornea over time enables monitoring of disease progression and thus the potential to identify and correlate development of, or changes in, microstructural features. As more data become available, these analyses may enable the formulation of prognostic and diagnostic criteria.

New therapeutic approaches in the treatment of diabetic keratopathy: a review.

The cornea is densely innervated, and the integrity of these nerve fibres is critical in maintaining the refractive and protective functions of the cornea. Many ocular and systemic diseases can adversely affect corneal sensory nerves and consequently impair their function, with vision loss being the inevitable consequence of severe corneal neurotrophic ulceration. However, current standard treatments regimens are often ineffective. Over the past three decades, the role of growth factors in maintaining the normal structure and function of the cornea, and in corneal epithelial healing, has become increasingly evident. Many preclinical and clinical trials have shown that growth factors and cytokines can significantly enhance epithelialization (epithelial proliferation and migration) and consequently accelerate wound healing. More recently, local/topical administration of insulin, naltrexone (opioid antagonist) and nicergoline (ergoline derivatives) were found to improve, and significantly increase, the corneal wound healing rate. This report reviews the major attributes of these growth factors and therapeutic agents that may be used in ameliorating impaired corneal wound healing, and presents a perspective on the potential clinical use of these agents as a new generation of ophthalmic pharmaceuticals for the treatment of diabetic keratopathy.The cornea is densely innervated, and the integrity of these nerve fibres is critical in maintaining the refractive and protective functions of the cornea. Many ocular and systemic diseases can adversely affect corneal sensory nerves and consequently impair their function, with vision loss being the inevitable consequence of severe corneal neurotrophic ulceration. However, current standard treatments regimens are often ineffective. Over the past three decades, the role of growth factors in maintaining the normal structure and function of the cornea, and in corneal epithelial healing, has become increasingly evident. Many preclinical and clinical trials have shown that growth factors and cytokines can significantly enhance epithelialization (epithelial proliferation and migration) and consequently accelerate wound healing. More recently, local/topical administration of insulin, naltrexone (opioid antagonist) and nicergoline (ergoline derivatives) were found to improve, and significantly increase, the corneal wound healing rate. This report reviews the major attributes of these growth factors and therapeutic agents that may be used in ameliorating impaired corneal wound healing, and presents a perspective on the potential clinical use of these agents as a new generation of ophthalmic pharmaceuticals for the treatment of diabetic keratopathy.

In Vivo Confocal Microscopy Analyses of Corneal Microstructural Changes in a Prospective Study of Collagen Cross-linking in Keratoconus

PURPOSE To use in vivo confocal microscopy (IVCM) to quantitatively analyze microstructural changes over time, after corneal collagen cross-linking for keratoconus. DESIGN Prospective cohort study. PARTICIPANTS A total of 38 eyes of 38 patients undergoing collagen cross-linking for keratoconus. METHODS Prospective, clinical cohort study of corneal collagen cross-linking in progressive keratoconus. Laser scanning IVCM performed preoperatively and at 1, 3, 6, and 12 months postoperatively. MAIN OUTCOME MEASURES Density of corneal sub-basal nerves, anterior and posterior keratocytes, and corneal endothelium. RESULTS Compared with baseline values, the mean sub-basal nerve density decreased significantly at 1, 3, and 6 months postoperatively (P < 0.01); however, this returned to preoperative values at 12 months (P = 0.57). One month postoperatively, there was complete absence of keratocyte nuclei in 86% of corneas. Anterior stromal edema with hyper-reflective cytoplasm and extracellular lacunae in a honeycomb-like appearance was observed and persisted at 3 months postoperatively. Scattered, presumed fragmented keratocyte nuclei, were observed at 1 and 3 months, but by 6 months, keratocyte repopulation of the anterior stroma was apparent. Quantitative analysis confirmed a significant decrease in the mean anterior keratocyte density 1, 3, and 6 months postoperatively (P ≤ 0.01) with return to baseline values at 12 months postoperatively (P = 0.57). The demarcation between treated and untreated corneal stroma appeared as a region where normal keratocytes transitioned into elongated, hyper-reflective, needle-like structures and then into large hyper-reflective stromal bands. There was no significant change in posterior keratocyte density or endothelial density at any postoperative time point. CONCLUSIONS This prospective IVCM study revealed complete loss of the sub-basal nerve plexus and loss of anterior stromal keratocytes in the early postoperative period, with complete regeneration of the sub-basal nerve plexus and keratocyte repopulation by 12 months postoperatively. The posterior stroma and corneal endothelium were unaffected.