Comparative quantitative assessment of the human corneal sub-basal nerve plexus by in vivo confocal microscopy and histological staining

B. S. Kowtharapu, K. Winter, Carl Marfurt, S. Allgeier, B. Köhler, M. Hovakimyan, T. Stahnke, A. Wree, O. Stachs, R. F. Guthoff

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Abstract

Purpose This study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and immunohistochemical staining of ex vivo donor corneas. Methods Seven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by β-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared. Results Our experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm2), CNFD (1358.8 vs 277.3 nerve fibers/mm2), CNBD (847.6 vs 163.5 branches/mm2), CNFTo (0.095 vs 0.081 μm-1), and CNCP (49.4 vs 21.6 connections/mm2) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 μm). No significant difference was observed in CNFTh (2.22 vs 2.20 μm) between the two groups. Conclusions The results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.

Original languageEnglish (US)
Pages (from-to)481-490
Number of pages10
JournalEye (Basingstoke)
Volume31
Issue number3
DOIs
StatePublished - Mar 1 2017

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Nerve Fibers
Confocal Microscopy
Staining and Labeling
Cornea
Intravital Microscopy
Nerve
Microscopy
Tissue Donors
Tubulin
Fiber
Healthy Volunteers
Immunohistochemistry

ASJC Scopus subject areas

  • Ophthalmology
  • Arts and Humanities(all)

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Comparative quantitative assessment of the human corneal sub-basal nerve plexus by in vivo confocal microscopy and histological staining. / Kowtharapu, B. S.; Winter, K.; Marfurt, Carl; Allgeier, S.; Köhler, B.; Hovakimyan, M.; Stahnke, T.; Wree, A.; Stachs, O.; Guthoff, R. F.

In: Eye (Basingstoke), Vol. 31, No. 3, 01.03.2017, p. 481-490.

Research output: Contribution to journalArticle

Kowtharapu, BS, Winter, K, Marfurt, C, Allgeier, S, Köhler, B, Hovakimyan, M, Stahnke, T, Wree, A, Stachs, O & Guthoff, RF 2017, 'Comparative quantitative assessment of the human corneal sub-basal nerve plexus by in vivo confocal microscopy and histological staining', Eye (Basingstoke), vol. 31, no. 3, pp. 481-490. https://doi.org/10.1038/eye.2016.220
Kowtharapu, B. S. ; Winter, K. ; Marfurt, Carl ; Allgeier, S. ; Köhler, B. ; Hovakimyan, M. ; Stahnke, T. ; Wree, A. ; Stachs, O. ; Guthoff, R. F. / Comparative quantitative assessment of the human corneal sub-basal nerve plexus by in vivo confocal microscopy and histological staining. In: Eye (Basingstoke). 2017 ; Vol. 31, No. 3. pp. 481-490.
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abstract = "Purpose This study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and immunohistochemical staining of ex vivo donor corneas. Methods Seven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by β-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared. Results Our experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm2), CNFD (1358.8 vs 277.3 nerve fibers/mm2), CNBD (847.6 vs 163.5 branches/mm2), CNFTo (0.095 vs 0.081 μm-1), and CNCP (49.4 vs 21.6 connections/mm2) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 μm). No significant difference was observed in CNFTh (2.22 vs 2.20 μm) between the two groups. Conclusions The results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.",
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AU - Marfurt, Carl

AU - Allgeier, S.

AU - Köhler, B.

AU - Hovakimyan, M.

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AU - Wree, A.

AU - Stachs, O.

AU - Guthoff, R. F.

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N2 - Purpose This study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and immunohistochemical staining of ex vivo donor corneas. Methods Seven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by β-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared. Results Our experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm2), CNFD (1358.8 vs 277.3 nerve fibers/mm2), CNBD (847.6 vs 163.5 branches/mm2), CNFTo (0.095 vs 0.081 μm-1), and CNCP (49.4 vs 21.6 connections/mm2) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 μm). No significant difference was observed in CNFTh (2.22 vs 2.20 μm) between the two groups. Conclusions The results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.

AB - Purpose This study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and immunohistochemical staining of ex vivo donor corneas. Methods Seven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by β-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared. Results Our experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm2), CNFD (1358.8 vs 277.3 nerve fibers/mm2), CNBD (847.6 vs 163.5 branches/mm2), CNFTo (0.095 vs 0.081 μm-1), and CNCP (49.4 vs 21.6 connections/mm2) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 μm). No significant difference was observed in CNFTh (2.22 vs 2.20 μm) between the two groups. Conclusions The results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.

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