In vivo confocal microscopy morphometric analysis of corneal subbasal nerve plexus in dry eye disease using newly developed fully automated system

  • Giuseppe GiannaccareEmail author
  • Marco Pellegrini
  • Stefano Sebastiani
  • Fabiana Moscardelli
  • Piera Versura
  • Emilio C. Campos



To evaluate in vivo confocal microscopy (IVCM) features of corneal subbasal nerve plexus (SNP) in the setting of dry eye disease (DED) using fully automated software “ACCMetrics,” and to further investigate its diagnostic performance in discriminating DED patients.


IVCM exams of SNP in DED patients and matched control subjects were performed using Heidelberg Retina Tomograph with the Rostock Cornea Module. The following parameters were obtained with ACCMetrics: corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), corneal nerve total branch density (CTBD), corneal nerve fiber area (CNFA), corneal nerve fiber width (CNFW), and corneal nerve fractal dimension (CNFrD). The Mann–Whitney U test was used to compare variables. Receiver operating characteristic curves with calculations of the area under the curve (AUC) were used to describe the accuracy of IVCM parameters for discriminating DED patients from controls.


Thirty-nine DED patients and 30 control subjects were included. Significantly, lower values of CNFD, CNBD, and CNFL and higher value of CNFW were found in DED patients compared to controls (respectively, 20.5 ± 8.7 vs 25.4 ± 6.7 n/mm2; 25.6 ± 20.1 vs 37.6 ± 21.5 n/mm2; 12.6 ± 4.4 vs 14.5 ± 2.9 mm/mm2; 0.021 ± 0.001 vs 0.019 ± 0.001 mm/mm2; always p < 0.024). CNFW value had the highest diagnostic power in discriminating DED patients (AUC = 0.828). When the diagnosis of DED was made based on either CNFW or CNBD, the sensitivity was 97.4% and the specificity 46.7%.


The software ACCMetrics was able to rapidly detect SNP alterations occurring in the setting of DED and showed good diagnostic performance in discriminating DED patients.


Dry eye In vivo confocal microscopy Sub-basal nerve plexus Automated analysis ACCMetrics 



We thank Prof. Rayaz Malik and Collaborators from the University of Manchester (UK) for their kind permission to use the “ACCMetrics” software for the automated analysis of the in vivo confocal microscopy images in the present work.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.


  1. 1.
    Muller LJ, Marfurt CF, Kruse F, Tervo TM (2003) Corneal nerves: structure, contents and function. Exp Eye Res 76:521–542CrossRefGoogle Scholar
  2. 2.
    Craig JP, Nichols KK, Akpek EK, Caffery B, Dua HS, Joo CK et al (2017) TFOS DEWS II definition and classification report. Ocul Surf 15:276–283. CrossRefGoogle Scholar
  3. 3.
    Tuominen IS, Konttinen YT, Vesaluoma MH, Moilanen JA, Helintö M, Tervo TM (2003) Corneal innervation and morphology in primary Sjögren’s syndrome. Invest Ophthalmol Vis Sci 44:2545–2549CrossRefGoogle Scholar
  4. 4.
    Zhang M, Chen J, Luo L, Xiao Q, Sun M, Liu Z (2005) Altered corneal nerves in aqueous tear deficiency viewed by in vivo confocal microscopy. Cornea 24:818–824CrossRefGoogle Scholar
  5. 5.
    Benítez del Castillo JM, Wasfy MA, Fernandez C, Garcia-Sanchez J (2004) An in vivo confocal masked study on corneal epithelium and subbasal nerves in patients with dry eye. Invest Ophthalmol Vis Sci 45:3030–3035CrossRefGoogle Scholar
  6. 6.
    Hoşal BM, Ornek N, Zilelioğlu G, Elhan AH (2005) Morphology of corneal nerves and corneal sensation in dry eye: a preliminary study. Eye (Lond) 19:1276–1279CrossRefGoogle Scholar
  7. 7.
    Villani E, Galimberti D, Viola F, Mapelli C, Ratiglia R (2007) The cornea in Sjogren’s syndrome: an in vivo confocal study. Invest Ophthalmol Vis Sci 48:2017–2022CrossRefGoogle Scholar
  8. 8.
    Labbé A, Liang Q, Wang Z, Zhang Y, Xu L, Baudouin C et al (2013) Corneal nerve structure and function in patients with non-Sjogren dry eye: clinical correlations. Invest Ophthalmol Vis Sci 54:5144–5150. CrossRefGoogle Scholar
  9. 9.
    Giannaccare G, Buzzi M, Fresina M, Velati C, Versura P (2017) Efficacy of 2-month treatment with cord blood serum eye drops in ocular surface disease: an in vivo confocal microscopy study. Cornea 36:915–921. CrossRefGoogle Scholar
  10. 10.
    Petropoulos IN, Manzoor T, Morgan P, Fadavi H, Asghar O, Alam U et al (2013) Repeatability of in vivo corneal confocal microscopy to quantify corneal nerve morphology. Cornea 32:e83–e89. CrossRefGoogle Scholar
  11. 11.
    Belmonte C, Nichols JJ, Cox SM, Brock JA, Begley CG, Bereiter DA et al (2017) TFOS DEWS II pain and sensation report. Ocul Surf 15:404–437. CrossRefPubMedCentralGoogle Scholar
  12. 12.
    Wolffsohn JS, Arita R, Chalmers R, Djalilian A, Dogru M, Dumbleton K et al (2017) TFOS DEWS II diagnostic methodology report. Ocul Surf 15:539–574. CrossRefGoogle Scholar
  13. 13.
    Lemp MA (1995) Report of the National Eye Institute/Industry Workshop on clinical trials in dry eye. CLAO J 21:221–232Google Scholar
  14. 14.
    Van Bijsterveld OP (1969) Diagnostic tests in the sicca syndrome. Arch Ophthalmol 82:10–14CrossRefGoogle Scholar
  15. 15.
    Schiffman RM, Christianson MD, Jacobsen G, Hirsch JD, Reis BL (2000) Reliability and validity of the ocular surface disease index. Arch Ophthalmol 118:615–621CrossRefGoogle Scholar
  16. 16.
    Jensen MP, Karoly P, Braver S (1986) The measurement of clinical pain intensity: a comparison of six methods. Pain 27:117–126CrossRefGoogle Scholar
  17. 17.
    Kheirkhah A, Rahimi Darabad R, Cruzat A, Hajrasouliha AR, Witkin D, Wong N et al (2015) Corneal epithelial immune dendritic cell alterations in subtypes of dry eye disease: a pilot in vivo confocal microscopic study. Invest Ophthalmol Vis Sci 56:7179–7185. CrossRefPubMedCentralGoogle Scholar
  18. 18.
    Dabbah MA, Graham J, Petropoulos IN, Tavakoli M, Malik RA (2011) Automatic analysis of diabetic peripheral neuropathy using multi-scale quantitative morphology of nerve fibres in corneal confocal microscopy imaging. Med Image Anal 15:738–747. CrossRefGoogle Scholar
  19. 19.
    Dabbah MA, Graham J, Petropoulos I, Tavakoli M, Malik RA (2010) Dual-model automatic detection of nerve-fibres in corneal confocal microscopy images. Med Image Comput Comput Assist Interv 13:300–307PubMedCentralGoogle Scholar
  20. 20.
    Chen X, Graham J, Dabbah MA, Petropoulos IN, Tavakoli M, Malik RA (2017) An automatic tool for quantification of nerve fibers in corneal confocal microscopy images. IEEE Trans Biomed Eng 64:786–794. CrossRefGoogle Scholar
  21. 21.
    Chen X, Graham J, Petropoulos IN, Ponirakis G, Asghar O, Alam U et al (2018) Corneal nerve fractal dimension: a novel corneal nerve metric for the diagnosis of diabetic sensorimotor polyneuropathy. Invest Ophthalmol Vis Sci 59:1113–1118. CrossRefPubMedCentralGoogle Scholar
  22. 22.
    Shetty R, Sethu S, Deshmukh R, Deshpande K, Ghosh A, Agrawal A et al (2016) Corneal dendritic cell density is associated with subbasal nerve plexus features, ocular surface disease index, and serum vitamin D in evaporative dry eye disease. Biomed Res Int 2016:4369750. CrossRefPubMedCentralGoogle Scholar
  23. 23.
    Szalai E, Deak E, Modis L Jr, Németh G, Berta A, Nagy A et al (2016) Early corneal cellular and nerve fiber pathology in young patients with type 1 diabetes mellitus identified using corneal confocal microscopy. Invest Ophthalmol Vis Sci 57:853–8588. CrossRefGoogle Scholar
  24. 24.
    Brines M, Culver DA, Ferdousi M, Tannemaat MR, van Velzen M, Dahan A et al (2018) Corneal nerve fiber size adds utility to the diagnosis and assessment of therapeutic response in patients with small fiber neuropathy. Sci Rep 8:4734. CrossRefPubMedCentralGoogle Scholar
  25. 25.
    Belmonte C, Acosta MC, Gallar J (2004) Neural basis of sensation in intact and injured corneas. Exp Eye Res 78:513–525CrossRefGoogle Scholar
  26. 26.
    Tepelus TC, Chiu GB, Maram J, Huang J, Chopra V, Sadda SR et al (2017) Corneal features in ocular graft-versus-host disease by in vivo confocal microscopy. Graefes Arch Clin Exp Ophthalmol 255:2389–2397. CrossRefGoogle Scholar
  27. 27.
    Dehghani C, Pritchard N, Edwards K, Russell AW, Malik RA, Efron N (2014) Fully automated, semiautomated, and manual morphometric analysis of corneal subbasal nerve plexus in individuals with and without diabetes. Cornea 33:696–702CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Ophthalmology Unit, DIMES, S.Orsola-Malpighi University HospitalUniversity of BolognaBolognaItaly

Personalised recommendations