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Review
In vivo confocal microscopy of human corneal nerves in health, in ocular and systemic disease, and following corneal surgery: a review
  1. D V Patel,
  2. C N J McGhee
  1. Department of Ophthalmology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
  1. Professor C N J McGhee, Department of Ophthalmology, Private Bag 92019, University of Auckland, Auckland, New Zealand; c.mcghee{at}auckland.ac.nz

Abstract

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.

https://doi.org/10.1136/bjo.2008.150615

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    Contemporary in vivo confocal microscopy (IVCM) is a non-invasive method of examining the living human cornea in healthy and pathological states, making it a powerful clinical and research tool.1 The non-invasive nature of this imaging technique has the advantages of: enabling examination of the human cornea in its physiological state, avoiding artefacts induced by ex vivo study and allowing multiple examinations of the same cornea over time. There are three classes of IVCM: laser scanning confocal microscopes (LSCM), slit-scanning confocal microscopes (SSCM) and tandem scanning confocal microscopes (TSCM). These microscopes differ in factors such as the type and intensity of illumination, magnification, image contrast and image resolution. However, all use the same basic principles in their design enabling safe optical sectioning of the living human cornea.1

    Corneal nerves are of great interest to both clinicians and scientists due to their important roles in regulating corneal epithelial integrity, proliferation and wound healing in addition to their protective functions.2 Since the mid-1990s, IVCM has increasingly been used to enhance our knowledge of corneal nerves in health and disease. This article reviews our current state of knowledge in this rapidly developing field.

    NORMAL CORNEAL NERVES

    Sub-basal corneal nerves

    The sub-basal corneal nerve plexus is located between the Bowman layer and the basal epithelium. These nerve bundles consist of both straight and beaded fibres, with the beaded fibres located in the periphery of the bundle. The beads have been identified as axonal efferent and sensory terminals,3 4 and have been shown to consist of accumulations of mitochondria and glycogen.5

    When imaged by IVCM, sub-basal nerve bundles appear as beaded, well-defined linear structures with homogeneous reflectivity (fig 1). Dichotomous branches (Y-shaped) and thinner connecting nerve fibre bundles (H-shaped) are observed.2

    Figure 1
    Figure 1

    (A) Laser scanning confocal microscope and (B) slit-scanning confocal microscope images of the central corneal sub-basal nerve plexus. Both images were taken from the same subject but not from exactly the same corneal location.

    The recent trend towards quantitative studies using in vivo confocal microscopy has led to the development of a variety of methods for quantifying sub-basal nerve plexus parameters. However, the manner in which nerve density is defined has been somewhat inconsistent. The majority of studies have defined sub-basal nerve density as the total length of nerves visible within a defined area (μm/mm2 or mm/mm2), but some investigators have only included nerve branches longer than 50 μm in their measurements.6 Others have analysed the total number of nerves within a frame, but the definitions for this vary from the number of long nerve fibre bundles,7 to the sum of nerve branches present.2 Such variations cause obvious difficulties when comparing the results from different studies (table 1).

    View this table:
    Table 1 Summary of the results of quantitative in vivo confocal microscopy studies of the healthy corneal sub-basal nerve plexus.

    Reported sub-basal nerve densities also vary depending on the type of in vivo confocal microscope used. Studies using LSCM have reported densities as high as 20.3 mm/mm217 and 21.7 mm/mm2,18 whereas studies using TSCM and SSCM have reported densities of 8.4 mm/mm26 and 11.1 mm/mm2,2 respectively. One explanation for such a wide variation are differences in image contrast between the microscopes, with fine nerve branches more visible with the LSCM, and the fact that TSCM and SSCM exhibit a decreased contrast towards the lateral edges of the image, and so this may mean that fewer sub-basal nerves are visualised in these regions (fig 1).19

    Measurements of sub-basal nerve diameter range from 0.5216 to 4.68 μm,13 and those for sub-basal nerve beading frequency vary from 90 beads/mm12 to 198 beads/mm15 in healthy subjects. A likely reason for these discrepancies has been demonstrated using LSCM. When imaged by this microscope, sub-basal nerves appear thinner, and beadings appear to be more prominent and numerous when the illuminating brightness is reduced compared with the unit’s automatic brightness setting (fig 2).19 This therefore indicates that for measurements of the diameter of thin highly reflective structures (such as sub-basal nerves), or beading frequency to be comparable, all images need to be acquired using a fixed illumination intensity, since illumination intensity affects the apparent thickness of corneal nerves particularly as they approach the limit of resolution. It may also be inferred that comparisons of the dimensions of reflective objects in images obtained by in vivo confocal microscopy are only valid when the same type of in vivo confocal microscope is used, and illumination intensity is constant.

    Figure 2
    Figure 2

    Laser scanning confocal microscope images of the same field of view at the level of the sub-basal nerve plexus using the automatic brightness setting (A) and manually reducing the brightness (B), making the nerve beadings (arrow) more prominent and the nerves appear thinner.

    Subjective2 and objective20 grading scales for nerve tortuosity have also been described as methods for categorising and quantifying nerve morphology.

    Manual analysis of sub-basal corneal nerve parameters is a laborious task that has a subjective element. These problems are likely to be overcome by the development of automated methods of analysis that do not require any user intervention. Scarpa et al have made the first step in this direction with the recent development of a fully automated algorithm for analysing sub-basal nerve length.21

    The effect of age on sub-basal corneal nerves remains uncertain. One study has demonstrated a decrease in nerve fibre density but not nerve fibre bundle diameter or beading frequency with age,16 while a more recent study identified no significant correlation between age and sub-basal nerve density.6

    A recent study using the LSCM has, for the first time, elucidated the architecture of the sub-basal nerve plexus using a novel mapping technique, demonstrating a radiating pattern of nerve fibre bundles converging towards an area approximately 1–2 mm inferior to the corneal apex in a whorl-like pattern (fig 3).18 This study was later extended by performing examinations once a week for 6 weeks in the same individual in order to produce a two-dimensional reconstruction map of the living, human, sub-basal corneal nerve plexus at each session. Over this period, the sub-basal nerve pattern appeared to migrate centripetally from the corneal periphery towards an inferocentral whorl. In the region of the whorl the nerves altered their generally centripetal direction of migration, undergoing clockwise rotation, providing strong evidence that the living human sub-basal corneal nerve plexus is a highly dynamic structure with nerves branches migrating centrally at up to 26 μm per week.22

    Figure 3
    Figure 3

    Montage of laser scanning confocal microscope images depicting the architecture of the normal, central corneal sub-basal nerve plexus.

    Subepithelial nerve plexus

    The subepithelial nerve plexus lies at the interface between the Bowman layer and the anterior stroma, and is quite separate from the sub-basal nerve plexus, which lies anterior to the Bowman layer.

    The subepithelial nerve plexus is sparse and patchy in distribution, with the network apparently limited to the mid-peripheral cornea, and possibly absent in the central cornea.23 These nerves are of low contrast, with a granular texture and irregular edges.23 Fifty per cent of these nerves exhibit varicosities or beads (fig 4).2 Elegant 3D reconstructions of LSCM images by Stachs et al24 have enabled clear visualisation of the anterior corneal nerves including the subepithelial nerve plexus.

    Figure 4
    Figure 4

    Laser scanning confocal microscope image of a subepithelial nerve (arrows) in the anterior stroma immediately posterior to the Bowman layer.

    Stromal nerves

    Stromal nerves are present in the anterior and mid stroma but cannot be visualised in the posterior stroma.2 When imaged by in vivo confocal microscopy, they appear as thick, reflective linear structures of various orientations, which branch in a dichotomous pattern,2 but no internal detail of stromal nerves is visible (fig 5).23

    Figure 5
    Figure 5

    Laser scanning confocal microscope image of a stromal nerve (arrow) exhibiting dichotomous branching in the mid-stroma.

    Quantitative analysis of stromal nerves imaged by IVCM remains controversial. Investigators have reported stromal nerve diameters ranging from 5.5 μm11 to 11.4 μm15 in the normal cornea (table 2). The wide range of results may be explained by the fact that stromal nerves commonly traverse obliquely, relative to the en face section of IVCM images. The cross-section is therefore not always through the centre of the nerve, and so all off-centre cross-sections will make the nerve appear falsely thinner.

    View this table:
    Table 2 Summary of the results of quantitative in vivo confocal microscopy studies of healthy corneal stromal nerves

    Attempts have also been made to analyse stromal nerve density from IVCM images. Oliveira-Soto and Efron reported stromal nerve densities of 3.7 to 4.2 mm/mm2 (mid stroma and anterior stroma respectively) in normal corneas using SSCM.2 Two investigators reported markedly lower stromal nerve densities of 0.31 mm/mm215 and 0.45 mm/mm2,11 also using SSCM. Such densities, however, would equate to only 71 μm of stromal nerve per frame (a figure inconsistent with published images which all demonstrate stromal nerves traversing the whole frame). Stromal nerve orientation is an important factor when considering nerve density analysis. Visible nerve length per frame area will vary with the path of the nerve through the imaged field (ie, a nerve traversing obliquely through the image will appear shorter than one whose path is parallel to the plane of the image). The visible nerve length per frame area will also depend on the axial resolution of the microscope used (ie, a greater length of nerve will be visible with a thicker optical section). Therefore, measuring the length of nerve per frame does not necessarily relate to the true stromal nerve density.

    CORNEAL NERVES POSTSURGERY

    Corneal transplantation

    Penetrating keratoplasty involves transection of all corneal nerves in both the host and donor cornea. IVCM provides a practical method for investigating the short- and long-term effects of this form of surgery on corneal nerves.

    In a 12-month longitudinal study of corneal nerves using SSCM postpenetrating keratoplasty, no sub-basal nerves were detected in the central cornea throughout the postop study period.25 In a smaller study, Hollingsworth et al26 observed presumed nerve components at the level of the Bowman layer at 12 months postoperatively. Both studies detected stromal nerves in the central cornea from 6 months postsurgery. The study with the longest follow-up period was performed by Richter et al,27 reporting reinnervation of the central cornea with sub-basal nerves and stromal nerves occurring at 2 years and 7 months postoperatively, respectively.

    Larger cross-sectional studies have yielded interesting results regarding the long-term effects of corneal transplantation on corneal nerves (table 3). Studies confirm that sub-basal nerve density is reduced and that these nerves appear tortuous and disordered, even 40 years after surgery.9 28 Interestingly, both major studies also noted significantly higher sub-basal nerve densities in corneal grafts performed for keratoconus when compared with those performed for other indications. The reasons for this difference are not known, although Niederer et al9 postulated that the peripheral corneal innervation in keratoconus, a disease that primarily affects the central cornea, is relatively intact and so may be responsible for the greater regeneration observed. They also found no association between recipient age and nerve regeneration.

    View this table:
    Table 3 Summary of the results of quantitative in vivo confocal microscopy studies of the sub-basal nerve plexus following corneal surgery

    Photorefractive keratectomy

    In vivo confocal microscopy has enabled investigators to examine changes in corneal innervation over time following photorefractive keratectomy (PRK). Sharply cut stromal nerve trunks are visible 10–15 min postoperatively.33 At 1–2 months, sprouting nerve fibres are visible in the anterior stroma at the margin of the wound and are directed toward the centre of cornea.33 34 At 3 months, single non-branched sub-basal nerve fibres are visualised in the central cornea.34 At 5–8 months postoperatively, the sub-basal plexus appears to be fully regenerated; however, abnormal branching and thin accessory fibres are observed.33 34 It has been reported that there are no further changes in nerve morphology 8 months post-PRK;33 however, while some authors have reported a return to preoperative sub-basal nerve morphology at 1 year,35 other researchers have noted a bizarre pattern, compared with controls, 2 years postoperatively.36 Erie et al29 reported a 59% decrease in sub-basal nerve density at 1 year postoperatively and observed that sub-basal nerve densities returned to preoperative levels 2 years after PRK. In a study at 5 years post-PRK, no significant differences in the number of central sub-basal nerve fibres were identified when compared with normal controls, and 71% of subjects exhibited a similar branching pattern to normal (fig 6).37

    Figure 6
    Figure 6

    Imaging the cornea using laser scanning confocal microscope in a subject 10 years following photorefractive keratectomy revealing the absence of the Bowman layer, with sub-basal nerves and keratocytes visible in the same plane.

    Laser in situ keratomileusis

    Although, long sub-basal nerve fibres are still visible in the central cornea 3 days post-LASIK,38 they are significantly reduced in number, or entirely absent by 1 week.14 38–42 The number of identifiable sub-basal nerve fibres subsequently increases gradually over time, returning to the central cornea by 6 months.14 31 40 Despite this, sub-basal nerve regeneration appears to remain incomplete for up to 5 years following laser in situ keratomileusis (LASIK).29 Studies have revealed strong correlations between central corneal sensitivity and sub-basal nerve morphology38 and density.30 43

    Laser epithelial keratomileusis

    Sub-basal nerve density and diameter decrease significantly following laser epithelial keratomileusis (LASEK) and do not return to preoperative levels at 6 months postoperatively.30–32 The sub-basal nerve tortuosity coefficient decreases significantly after LASEK but was noted to return to preoperative levels by 3 months postoperatively.32 There are no prospective or cross-sectional studies reporting sub-basal nerve parameters more than 6 months following LASEK. Two studies have compared sub-basal nerves following LASIK and LASEK with conflicting results. Darwish et al31 found no differences in any sub-basal nerve parameters at any time following the procedures, while Lee et al reported faster sub-basal nerve regeneration following LASEK compared with LASIK.30

    CORNEAL NERVES IN DISEASE STATES

    Keratoconus

    The majority of early in vivo data regarding alterations in the sub-basal nerve plexus in keratoconus have been limited to qualitative studies, with observations such as “sub-basal nerve fibres running in and out of the plane of the field of view in the central cornea.”44 Subsequent quantitative studies have revealed a significantly lower central sub-basal nerve density in keratoconic corneas compared with normal corneas (table 4).8 11 However, the effects of contact lens wear on the sub-basal nerve plexus in keratoconus remains uncertain. Simo Mannion et al11 demonstrated significantly reduced sub-basal nerve density in contact lens wearing keratoconic subjects compared with contact lens wearing controls. However, in contrast to the results of Patel et al,8 the former authors reported no significant difference in sub-basal nerve density in the non-contact lens wearing subjects in the keratoconus and control groups.

    View this table:
    Table 4 Summary of the results of quantitative in vivo confocal microscopy studies of the cornea in disease states

    A recent study has demonstrated grossly abnormal sub-basal nerve architecture in keratoconus compared with normal corneas. At the apex of the cone, a tortuous network of nerve fibre bundles was noted, and many of these bundles formed closed loops. At the topographic base of the cone, nerve fibre bundles appeared to follow the contour of the base, with many of the bundles running concentrically in this region (fig 7).47

    Figure 7
    Figure 7

    Electronic tracing of nerves from a wide-field montage, depicting the architecture of the sub-basal nerve plexus in a case of moderate keratoconus, superimposed, to scale, onto the patient’s anterior tangential corneal topography map.

    Diabetes mellitus

    In vivo confocal microscopy has revealed significantly fewer sub-basal nerve fibre bundles in patients with diabetic peripheral neuropathy compared with normal controls, and this was associated with reduced corneal sensitivity in patients with severe peripheral neuropathy.7 In a refinement of this study, Malik et al48 observed a significantly lower nerve fibre density, fibre length and branch density, in patients with diabetes compared with controls. These measurements were lower with increasing severity of peripheral neuropathy. Mocan et al10 also observed that patients with proliferative diabetic retinopathy had significantly lower sub-basal nerve densities compared with patients with diabetes with no retinopathy.

    Interestingly, a novel quantitative analysis of sub-basal nerve fibre tortuosity in these patients revealed greater tortuosity in patients with greater severity of peripheral neuropathy.20 49

    By demonstrating a significant correlation between corneal and dermal nerve degeneration in diabetic peripheral neuropathy, Quattrini et al have further strengthened the evidence that IVCM is a valuable tool in the diagnosis and assessment of diabetic neuropathy.50 The reliable, non-invasive nature of IVCM allows repeated assessment to determine progression or response to therapeutic intervention. In the case of pancreas transplantation in patients with type 1 diabetes mellitus, IVCM has been used to demonstrate significant regeneration of corneal sub-basal nerves within 6 months of surgery. This observation was attributed to a recovery of peripheral neuropathy due to normalisation of blood glucose levels in these patients.51

    Dry eyes

    The prevalence of peripheral and cranial neuropathy in Sjögren syndrome46 and the postulated link between corneal innervation and aqueous tear production has led investigators to use IVCM to analyse sub-basal nerves in cases of aqueous deficiency dry eye (table 4).

    Studies present conflicting results regarding the effect of dry eye on sub-basal nerve density. Some have observed a significantly reduced sub-basal nerve density in both Sjögren syndrome and non-Sjögren syndrome dry eye compared with healthy controls,15 45 while others have reported no significant differences in this parameter.12 13 46 One study even noted an increased number of nerves per frame in patients with Sjögren syndrome compared with controls.12 With one exception,13 all studies agree that sub-basal nerve tortuosity is significantly increased in Sjögren syndrome.12 15 45 Villani et al45 postulated that this may be due to the simultaneous action of “detrimental phenomena and nerve growth factors secreted over the course of the inflammatory process.”

    Significant correlations have been observed between the number of sub-basal nerves and the results of the Schirmer test,15 and rose Bengal staining of the cornea has been found to correlate positively with nerve density and negatively with beading.12

    CORNEAL NERVES IN CONTACT LENS WEAR

    Although associated with reduced corneal sensitivity,52 long-term contact lens wear does not appear to affect the number, distribution or morphology of corneal nerves.52 53 These results suggest that the decreased corneal sensitivity in these subjects is due to functional nerve changes rather than a structural alterations.

    CONCLUSIONS

    Despite the highly significant contribution of IVCM to our knowledge of corneal nerves in both healthy and diseased corneas, it is clear that a number of challenges remain. The first is to overcome the problem of poor topographic reproducibility. Accurate corneal localisation remains a difficulty despite attempts at using fixation targets. This is partially due to the inevitable involuntary movements of the subject and the small area under study. Future developments such as increased speed of imaging may help to overcome this problem. The value of studies in this field would be significantly enhanced by the use of consistent protocols and methods for quantitative analysis, thereby allowing comparison of different studies. To this end, the development of new objective methods of analysis to replace subjective grading scales is also encouraging. The quantification of stromal nerve parameters also remains a challenge that remains to be tackled effectively. However, by actively addressing these challenges, the remaining mysteries regarding the morphology of living corneal nerves may be resolved in the near future by IVCM.

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    Footnotes

    • Competing interests: None.

    • See Editorial, p 850

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