In Vivo Confocal Microscopy of Corneal Nerves in Health and Disease

Abstract

In vivo confocal microscopy (IVCM) is becoming an indispensable tool for studying corneal physiology and disease. Enabling the dissection of corneal architecture at a cellular level, this technique offers fast and noninvasive in vivo imaging of the cornea with images comparable to those of ex vivo histochemical techniques. Corneal nerves bear substantial relevance to clinicians and scientists alike, given their pivotal roles in regulation of corneal sensation, maintenance of epithelial integrity, as well as proliferation and promotion of wound healing. Thus, IVCM offers a unique method to study corneal nerve alterations in a myriad of conditions, such as ocular and systemic diseases and following corneal surgery, without altering the tissue microenvironment. Of particular interest has been the correlation of corneal subbasal nerves to their function, which has been studied in normal eyes, contact lens wearers, and patients with keratoconus, infectious keratitis, corneal dystrophies, and neurotrophic keratopathy. Longitudinal studies have applied IVCM to investigate the effects of corneal surgery on nerves, demonstrating their regenerative capacity. IVCM is increasingly important in the diagnosis and management of systemic conditions such as peripheral diabetic neuropathy and, more recently, in ocular diseases. In this review, we outline the principles and applications of IVCM in the study of corneal nerves in various ocular and systemic diseases.

Keywords: corneal dystrophies; corneal nerves; corneal sensitivity; corneal surgery; diabetes; in vivo confocal microscopy; keratoconus; pain; peripheral neuropathy.

Figure 1

Diagrammatic representation of human corneal nerves.

Figure 2

Corneal apical whorl-like pattern of the human corneal subbasal nerve plexus seen with laser in vivo confocal microscopy. A whorl of subbasal nerves is seen at the corneal apex towards the inferonasal paracentral area.

Figure 3

Schematic diagram of the principle of laser-scanning corneal in vivo confocal microscopy (IVCM). Images acquired by the laser-scanning in vivo confocal microscope (Heidelberg Retinal Tomograph 3/Rostock Cornea Module, Heidelberg Engineering) provide an 800-fold magnification of the corneal tissue and subbasal nerve architecture as represented by the confocal micrograph at the top right-hand corner.

Figure 4

In vivo confocal microscopy of corneal subbasal nerves and immune cells in health and disease. (A) Normal subbasal nerve plexus (SSCM; Confoscan4, Nidek Technology); (B) Subbasal nerve plexus in herpes simplex keratitis (HSK) (SSCM; Confoscan4, Nidek Technology). Note the decrease in total nerve count, length and branching associated with severe loss of corneal sensation; (C) Normal subbasal nerve plexus and immune cells (LSCM; HRTIII/RCM, Heidelberg Engineering) in same patient as panel A.; (D) Subbasal nerve plexus and immune cells in HSK associated with severe loss of corneal sensation and decrease in total corneal nerve count, length and branching (LSCM; HRTIII/RCM, Heidelberg Engineering) in same patient as panel B.

Figure 5

Normal age-related changes in the corneal subbasal nerve plexus. (A) 25 year old patient, (B) 60 year old patient, by laser scanning in vivo confocal microscopy (HRTIII/RCM).

Figure 6

Keratoconus. Laser scanning in vivo confocal microscopy (HRTIII/RCM) demonstrates a decrease in nerve density and increased tortuosity in keratoconus patients.

Figure 7

Infectious keratitis. Laser scanning in vivo confocal microscopy (HRTIII/RCM) demonstrates a decrease in nerve density and increase in dendritic cells.

Figure 8

Map Dot Fingerprint Dystrophy. Subbasal nerve plexus and basal epithelial membrane alterations by laser in vivo confocal microscopy (HRTIII/RCM).

Figure 9

Fuchs’ endothelial corneal dystrophy. (A) Corneal subbasal nerve plexus alterations and (B) guttae observed in the endothelium by laser in vivo confocal microscopy (HRTIII/RCM).

Figure 10

Morphology of central corneal sub-basal nerves in corneal allodynia using in vivo confocal microscopy (IVCM). Central corneal IVCM of patients with corneal allodynia revealed presence of multiple neuromas (A), increased nerve tortuosity (B), stark decrease in sub-basal nerve density (C), nerve beading (D), and increased reflectivity (E). After treatment of corneal allodynia with 20% autologous serum tears, in addition to self-reported symptomatic improvement, IVCM revealed nerve regeneration, and reduced tortuosity, beading, and reflectivity (F).

Figure 11

Corneal graft re-innervation following penetrating keratoplasty. (A) Incomplete re-innervation of the corneal graft center 1 month after transplantation, a thin, sub-basal nerve is observed in the graft center. (B) Incomplete corneal graft re-innervation of the center 12 months after transplantation. (C) Partially re-innervated corneal graft at 18 months. Images taken by laser in vivo confocal microscopy (HRTIII/RCM).

Figure 12

Subbasal corneal innervation after photorefractive keratectomy (PRK). Subbasal corneal nerve plexus regenerates to normal nerve density between 24–36 months after surgery. Images taken by laser in vivo confocal microscopy (HRTIII/RCM).

Figure 13

Sub-basal corneal re-innervation after laser-assisted in situ keratomileusis (LASIK). Regenerating nerve fibers in the central cornea, (A) two months, (B) six months and (C) eight months after LASIK. Images taken by laser in vivo confocal microscopy (HRTIII/RCM).