Research Article| Volume 268, P354-362, December 2021

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Histomorphometry in Peripheral Nerve Regeneration: Comparison of Different Axon Counting Methods

Published:August 14, 2021DOI:


      • Histomorphometry is not standardized, making it difficult to compare study results.
      • Many different sampled and automated techniques have been published.
      • These techniques yield different results versus full manual analysis (gold standard).
      • Our data suggest sampled manual analysis is more reliable than automated.
      • Regardless, standard, transparent methods are essential to allow for collaboration.



      Histomorphometry quantitatively evaluates nerve regeneration. Total myelinated fiber count (TMFC) is most accurately obtained manually across full nerve cross-sections, but most researchers opt for automated, sampled analysis. Few of the numerous techniques available have been validated. The goal of this study was to compare common histomorphometric methods (full manual [FM], sampled manual [SM], and sampled automatic [SA]) to determine their reliability and consistency.

      Material and methods

      Twenty-four rats underwent sciatic nerve (SN) repair with 20mm isografts; SNs distal to the graft were analyzed. TMFC was manually determined in each full cross-section. Counts were also extrapolated from sampled fields, both manually and automatically with ImageJ software. Myelinated fiber diameter, axon diameter, and myelin sheath thickness were measured manually in full and sampled fields; G-ratio was calculated. Repeated-measures MANOVA, Spearman correlation, and Wilcoxon signed-rank tests were performed. A systematic review of histomorphometry in rat SN repair was performed to analyze the variability of techniques in the literature.


      FM TMFC was 13,506 ± 4,217. Both sampled methods yielded significantly different TMFCs (SM:14.4 ± 13.4%, P< 0.001; SA:21.8 ± 44.7%, P = 0.037). All three methods strongly correlated with each other, especially FM and SM (rs = 0.912, P< 0.001). FM fiber diameter, axon diameter, and myelin sheath thickness did not differ from SM (P = 0.493, 0.209, and 0.331, respectively). 65% of papers used sampling; 78% utilized automated or semi-automated analysis. Software, sampling, and histomorphometric parameters varied widely.


      SM and SA analysis are reliable with standardized, systematic sampling. Transparency is essential to allow comparison of data; meanwhile, researchers must be cognizant of the wide variety of methodologies in the literature.


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        • Kemp SWP
        • Cederna PS
        • Midha R.
        Comparative outcome measures in peripheral regeneration studies.
        Exp Neurol. 2017; 287: 348-357
        • Hunter DA
        • Moradzadeh A
        • Whitlock EL
        • et al.
        Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve.
        J Neurosci Methods. 2007; 166: 116-124
        • Wood MD
        • Kemp SWP
        • Weber C
        • Borschel GH
        • Gordon T.
        Outcome measures of peripheral nerve regeneration.
        Ann Anat. 2011; 193: 321-333
        • Vleggeert-Lankamp CLAM.
        The role of evaluation methods in the assessment of peripheral nerve regeneration through synthetic conduits: a systematic review.
        J Neurosurg. 2007; 107: 1168-1189
        • Mezin P
        • Tenaud C
        • Bosson JL
        • Stoebner P.
        Morphometric analysis of the peripheral nerve: advantages of the semi-automated interactive method.
        J Neurosci Methods. 1994; 51: 163-169
        • Munro CA
        • Szalai JP
        • Mackinnon SE
        • Midha R.
        Lack of association between outcome measures of nerve regeneration.
        Muscle and Nerve. 1998; 21: 1095-1097<1095::AID-MUS20>3.0.CO;2-S
        • Giusti G
        • Shin RH
        • Lee J-Y
        • Mattar TG
        • Bishop AT
        • Shin AY.
        The influence of nerve conduits diameter in motor nerve recovery after segmental nerve repair.
        Microsurgery. 2014; 34: 646-652
        • Godinho MJ
        • Teh L
        • Pollett MA
        • et al.
        Immunohistochemical, ultrastructural and functional analysis of axonal regeneration through peripheral nerve grafts containing Schwann cells expressing BDNF, CNTF or NT3.
        PLoS One. 2013; 8
        • Tang P
        • Kilic A
        • Konopka G
        • Regalbuto R
        • Akelina Y
        • Gardner T.
        Histologic and functional outcomes of nerve defects treated with acellular allograft versus cabled autograft in a rat model.
        Microsurgery. 2013; 33: 460-467
        • da Silva APD
        • Jordão CER
        • Fazan VPS.
        Peripheral nerve morphometry: comparison between manual and semi-automated methods in the analysis of a small nerve.
        J Neurosci Methods. 2007; 159: 153-157
        • Ebneter A
        • Casson RJ
        • Wood JP
        • Chidlow G.
        Estimation of axon counts in a rat model of glaucoma: comparison of fixed-pattern sampling with targeted sampling.
        Clin Experiment Ophthalmol. 2012; 40: 626-633
        • Marina N
        • Bull ND
        • Martin KR.
        A semiautomated targeted sampling method to assess optic nerve axonal loss in a rat model of glaucoma.
        Nat Protoc. 2010; 5: 1642-1651
        • Zarei K
        • Scheetz TE
        • Christopher M
        • et al.
        Automated axon counting in rodent optic nerve sections with AxonJ.
        Sci Rep. 2016; 6: 26559
        • Auer RN.
        Automated nerve fibre size and myelin sheath measurement using microcomputer-based digital image analysis: theory, method and results.
        J Neurosci Methods. 1994; 51: 229-238
        • Bilego Neto APDC
        • Silveira FBC
        • Rodrigues Da Silva GA
        • Sanada LS
        • Fazan VPS
        Reproducibility in nerve morphometry: comparison between methods and among observers.
        Biomed Res Int. 2013; 2013: 1-7
        • Harman K
        • Katnick J
        • de la Torre JC.
        A quick and accurate line-sampling technique to quantify myelinated axons in peripheral nerve cross-sections.
        J Neurosci Methods. 1991; 38: 107-110
        • More HL
        • Chen J
        • Gibson E
        • Donelan JM
        • Beg MF.
        A semi-automated method for identifying and measuring myelinated nerve fibers in scanning electron microscope images.
        J Neurosci Methods. 2011; 201: 149-158
        • Jin J
        • Park M
        • Rengarajan A
        • et al.
        Functional motor recovery after peripheral nerve repair with an aligned nanofiber tubular conduit in a rat model.
        Regen Med. 2012; 7: 799-806
        • Jin J
        • Limburg S
        • Joshi SK
        • et al.
        Peripheral nerve repair in rats using composite hydrogel-filled aligned nanofiber conduits with incorporated nerve growth factor.
        Tissue Eng Part A. 2013; 19: 2138-2146
        • Geuna S
        • Gigo-Benato D
        • De Castro Rodrigues A.
        On sampling and sampling errors in histomorphometry of peripheral nerve fibers.
        Microsurgery. 2004; 24: 72-76
        • Geuna S
        • Varejão AS.
        Evaluation methods in the assessment of peripheral nerve regeneration.
        J Neurosurg. 2008; 109 (author reply 362): 360-362
        • Angelov DN
        • Guntinas-Lichius O
        • Wewetzer K
        • Neiss WF
        • Streppel M.
        Axonal branching and recovery of coordinated muscle activity after transection of the facial nerve in adult rats.
        Adv Anat Embryol Cell Biol. 2005;
        • Kusaba H
        • Terada-Nakaishi M
        • Wang W
        • et al.
        Comparison of nerve regenerative efficacy between decellularized nerve graft and nonwoven chitosan conduit.
        Biomed Mater Eng. 2016; 27: 75-85
        • Wakimura Y
        • Wang W
        • Itoh S
        • Okazaki M
        • Takakuda K.
        An experimental study to bridge a nerve gap with a decellularized allogeneic nerve.
        Plast Reconstr Surg. 2015; 136: 319e-327e
        • Hausner T
        • Pajer K
        • Halat G
        • et al.
        Improved rate of peripheral nerve regeneration induced by extracorporeal shock wave treatment in the rat.
        Exp Neurol. 2012; 236: 363-370
        • Wang W
        • Itoh S
        • Takakuda K.
        Comparative study of the efficacy of decellularization treatment of allogenic and xenogeneic nerves as nerve conduits.
        J Biomed Mater Res - Part A. 2016; 104: 445-454
        • Haastert-Talini K
        • Schaper-Rinkel J
        • Schmitte R
        • et al.
        In vivo evaluation of polysialic acid as part of tissue-engineered nerve transplants.
        Tissue Eng Part A. 2010; 16: 3085-3098
        • Wang W
        • Itoh S
        • Yamamoto N
        • Okawa A
        • Nagai A
        • Yamashita K.
        Enhancement of nerve regeneration along a chitosan nanofiber mesh tube on which electrically polarized β-tricalcium phosphate particles are immobilized.
        Acta Biomater. 2010; 6: 4027-4033
        • Wang W
        • Itoh S
        • Konno K
        • et al.
        Effects of Schwann cell alignment along the oriented electrospun chitosan nanofibers on nerve regeneration.
        J Biomed Mater Res - Part A. 2009; 91: 994-1005
        • Isaacs J
        • Mallu S
        • Batchelor M.
        Modification of commercially available image analysis software for semi-automated qualitative analysis of axon regeneration and myelination in the rat sciatic nerve.
        J Neurosci Methods. 2014; 233: 45-49
        • Kim CY
        • Rho S
        • Lee N
        • Lee C-K
        • Sung Y.
        Semi-automated counting method of axons in transmission electron microscopic images.
        Vet Ophthalmol. 2016; 19: 29-37
        • Teixeira LBC
        • Buhr KA
        • Bowie O
        • et al.
        Quantifying optic nerve axons in a cat glaucoma model by a semi-automated targeted counting method.
        Mol Vis. 2014; 20: 376-385
        • DeLeonibus A
        • Rezaei M
        • Fahradyan V
        • Silver J
        • Rampazzo A
        • Bassiri Gharb B
        A meta-analysis of functional outcomes in rat sciatic nerve injury models.
        Microsurgery. 2021; : 286-295