Abstract
Purpose
Evaluation of a new method for in vivo visualization of the distribution and morphology of human anterior corneal nerves.
Method
The anterior cornea was examined to a depth of 100 μm in four human volunteers with a confocal laser scanning microscope (CLSM) using a Rostock Cornea Module (developed in house) attached to a Heidelberg Retina Tomograph II (Heidelberg Engineering, Germany). Optical sections were digitally reconstructed in 3D using AMIRA (TGS Inc., USA). The scanned volumes had a greatest size of 300×300×40 µm and voxel size of 0.78×0.78×0.95 µm.
Results
The spatial arrangement of the epithelium, nerves and keratocytes was visualized by in vivo 3D-CLSM. The 3D-reconstruction of the volunteers’ corneas in combination with the oblique sections gave a picture of the nerves in the central human cornea. Thin nerves run in the subepithelial plexus aligned parallel to Bowman’s layer and are partially interconnected. The diameter of these fibres varied between 1.0 and 5 µm. Thick fibres rose out of the deeper stroma. The diameter of the main nerve trunks was 12±2 µm. Branches penetrating the anterior epithelial cell layer could not be visualized.
Conclusions
3D-CLSM allows analysis of the spatial arrangement of the anterior corneal nerves and visualization of the epithelium and keratocytes in the living human cornea. The developed method provides a basis for further studies of alterations of the cellular arrangement and epithelial innervation in corneal disease. This may help to clarify alterations of nerve fibre patterns under various clinical and experimental conditions.
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References
Cavanagh HD, Petroll WM, Jester JV (1993) The application of confocal microscopy to the study of living systems. Neurosci Biobehav Rev 17(4):483–498
Cavanagh HD, El-Agha MS, Petroll WM, Jester JV (2000) Specular microscopy, confocal microscopy, and ultrasound biomicroscopy: diagnostic tools of the past quarter century. Cornea 9(5):712–722
Eckard A, Stave J, Guthoff R (2006) In vivo Investigations of the corneal epithelium with a confocal laser scanning microscope. Cornea 25(2):127–131
Elder SD (1961) Cornea. In: Elder SD (ed) System of ophthalmology. Kimpton, London, pp 120–127
Grupcheva CN, Wong T, Riley AF, McGhee CN (2002) Assessing the sub-basal nerve plexus in living healthy human corneas by in vivo confocal microscopy. Clin Exp Ophthalmol 30:187–190
Guthoff RF, Wree A, Wienss H, Hahnel C (2005) Epithelial innervation of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy. Cornea 24(5):608–613
Hara M, Morishige N, Chikama T, Nishida T (2003) Comparison of confocal biomicroscopy and noncontact specular microscopy for evaluation of the corneal endothelium. Cornea 22:512–515
Harrison DA, Joos C, Ambrosio R (2003) Morphology of corneal basal epithelial cells by in vivo slit-scanning confocal microscopy. Cornea 22:246–248
Hosal BM, Ornek N, Zilelioglu G, Elhan AH (2005) Morphology of corneal nerves and corneal sensation in dry eye: a preliminary study. Eye 19(12):1276–1279
Knappe S, Stave J, Guthoff RF (2005) [Epidemic keratoconjunctivitis. In vivo representation of corneal histopathological structures with the confocal Rostocker laser scanning microscope (RLSM)]. Ophthalmologe 102(8):798–801
Linna T, Tervo T (1997) Real-time confocal microscopic observations on human corneal nerves and wound healing after excimer laser photorefractive keratectomy. Curr Eye Res 16(7):640–649
Linna TU, Perez-Santonja JJ, Tervo KM, Sakla HF, Alio y Sanz JL, Tervo TM (1998) Recovery of corneal nerve morphology following laser in situ keratomileusis. Exp Eye Res 66(6):755–763
Linna TU, Vesaluoma MH, Petroll WM, Tarkkanen AH, Tervo TM (2000) Confocal microscopy of a patient with irregular astigmatism after LASIK reoperations and relaxation incisions. Cornea 19(2):163–169
MacIver MB, Tanelian DL (1993) Structural and functional specialisation of A delta and C fiber free nerve endings innervating rabbit corneal epithelium. J Neurosci 13:4511–4524
Malik RA, Kallinikos P, Abbott CA, van Schie CH, Morgan P, Efron N, Boulton AJ (2003) Corneal confocal microscopy: a non-invasive surrogate of nerve fibre damage and repair in diabetic patients. Diabetologica 46:683–688
Marfurt CF, Ellis LC (1993) Immunohistochemical localization of tyrosine hydroxylase in corneal nerves. J Comp Neurol 336:517–531
Masters BR, Farmer MA (1993) Three-dimensional confocal microscopy and visualization of the in situ cornea. Comput Med Imaging Graph 17(3):211–219
Masters BR, Thaer AA (1994) In vivo human corneal confocal microscopy of identical fields of subepithelial nerve plexus, basal epithelial, and wing cells at different times. Microsc Res Tech 29(5):350–356
Masters BR, Bohnke M (2001) Three-dimensional confocal microscopy of the human cornea in vivo. Ophthalmic Res 33(3):125–135
Meek KM, Quantock AJ (2001) The use of X-ray-scattering techniques to determine corneal ultrastructure. Prog Retin Eye Res 20:95–137
Müller LJ, Pels L, Vrensen GF (1996) Ultrastructural organization of human corneal nerves. Invest Ophthalmol Vis Sci 37:476–488
Müller LJ, Marfurt CF, Kruse F, Tervo TM (2003) Corneal nerves: structure, contents and function. Exp Eye Res 76:521–542
Mustonen RK, McDonald MB, Srivannaboon S, Tan AAL, Doubrava MW, Kim CK (1998) In vivo confocal microscopy of Fuchs endothelial dystrophy. Cornea 17:493–503
Oliveira-Soto L, Efron N (2001) Morphology of corneal nerves using confocal microscopy. Cornea 20:374–384
Patel DV, McGhee CN (2005) Mapping of the normal human corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy. Invest Ophthalmol Vis Sci 46(12):4485–4488
Pawley JB (ed) (1995) Handbook of biological confocal microscopy, 2nd edn. Plenum, New York
Petran M (1985) The tandem scanning reflected light microscope. Part 1. The principle, and its design. Proc RMS 20:125–129
Petran M, Hadravsky M (1968) Tandem-scanning reflected-light microscope. J Opt Soc Am 58:661–664
Petroll WM, Cavanagh HD, Jester JV (1998) Clinical confocal microscopy. Curr Opin Ophthalmol 9:59–65
Rosenberg ME, Tervo TM, Müller LJ, Moilanen JA, Vesaluoma MH (2002) In vivo confocal microscopy after herpes keratitis. Cornea 21:265–269
Scheuerle AF, Schmidt E (2004) Atlas of laser scanning ophthalmoscopy. Springer, Berlin Heidelberg New York
Somodi S, Hahnel C, Slowik C, Richter A, Weiss DG, Guthoff RF (1996) Confocal in vivo microscopy and confocal laser-scanning fluorescence microscopy in keratoconus. Ger J Ophthalmol 5:518–525
Stachs O, Knappe S, Stave J (2005) In vivo three-dimensional confocal laser scanning microscopy of the corneal nerve structure. Invest Ophthalmol Vis Sci 46: 2742 Suppl. S
Stave J, Zinser G, Grummer G, Guthoff RF (2002) [Modified Heidelberg Retinal Tomograph HRT. Initial results of in vivo presentation of corneal structures] Ophthalmologe 99(4):276–280
Tervo T, Palkama A (1978) Ultrastructure of the corneal nerves after fixation with potassium permanganate. Anat Rec 190:851–860
Tervo T, Moilanen J, Rosenberg ME, Tuominen IS, Valle T, Vesaluoma MH (2002) In vivo confocal microscopy for studying corneal diseases and conditions associated with corneal nerve damage. Adv Exp Med Biol 506:657–665
Tervo T, Moilanen J (2003) In vivo confocal microscopy for evaluation of wound healing following corneal refractive surgery. Prog Retin Eye Res 22:339–358
Ueda S, del Cerro M, LoCascio JA, Aquavella JV (1989) Peptidergic and catecholaminic fibers in the human corneal epithelium. An immunohistochemical and electron microscopic study. Acta Ophthalmol Suppl 192:80–90
Webb RH, Hughes GW, Delori FC (1987) Confocal scanning laser ophthalmoscope. Appl Opt 26:1492–1499
Webb RH (1996) Rep Prog Phys 59:427–471
Zhivov A, Stave J, Vollmar B, Guthoff RF (2005) In vivo confocal microscopic evaluation of the Langerhans cell density and distribution on the normal human corneal epithelium. Graefes Arch Clin Exp Opthalmol 243(10):1056–1061
Zinser G, Harbarth U, Schröder H (1990) Formation and analysis of three-dimensional data with the Laser Tomographic Scanner (LTS). In: Nasemann JE, Burk ROW (eds) Scanning laser ophthalmoscopy and tomography. Quintessenz, Munich, pp 243-252
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Stachs, O., Zhivov, A., Kraak, R. et al. In vivo three-dimensional confocal laser scanning microscopy of the epithelial nerve structure in the human cornea. Graefe's Arch Clin Exp Ophthalmol 245, 569–575 (2007). https://doi.org/10.1007/s00417-006-0387-2
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DOI: https://doi.org/10.1007/s00417-006-0387-2