A New Computerized Morphometric Analysis for Peripheral Nerve Study

Abstract

The commonly used methods to quantify axon numbers and mean area include manual and semiautomated procedures. The authors introduce a new fully automated method of morphometric analysis using ImageJ and Paint.net software to improve efficiency and accuracy. A total of six rat sciatic nerves were examined for their axon numbers and mean axon area by comparing the manual method or semiautomated MetaVue method with the new ImageJ method. It was observed that the number of axons for manual counting and ImageJ were 4,630 ± 403 and 4,779 ± 352, respectively, and the difference was not statistically significant (p > 0.5, t-test). The mean axon area measured was 13.44 ± 2.62 µm² for MetaVue and 8.87 ± 0.78 µm² for ImageJ, respectively, and the difference was statistically significant (p < 0.01, t-test). The standard error and coefficient of variation of MetaVue were 1.07 and 0.195; and for ImageJ were 0.32 and 0.087. The authors conclude that their new approach demonstrates improved convenience, time efficiency, accuracy, and less operator error or bias.

Keywords

peripheral nerve - morphometric analysis - nerve fiber count - mean axon area - ImageJ

Note

Chase A. Tobin and Ziyi Wang made equal contributions to this study.

• References

• 1 Dellon AL, Mackinnon SE. Selection of the appropriate parameter to measure neural regeneration. Ann Plast Surg 1989; 23 (3) 197-202
• 2 Geuna S, Tos P, Guglielmone R, Battiston B, Giacobini-Robecchi MG. Methodological issues in size estimation of myelinated nerve fibers in peripheral nerves. Anat Embryol (Berl) 2001; 204 (1) 1-10
• 3 Hunter DA, Moradzadeh A, Whitlock EL , et al. Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve. J Neurosci Methods 2007; 166 (1) 116-124
• 4 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 (2) 229-238
• 5 Sathyanesan A, Ogura T, Lin W. Automated measurement of nerve fiber density using line intensity scan analysis. J Neurosci Methods 2012; 206 (2) 165-175
• 6 ImageJ (Fiji Distribution). http://fiji.sc . Accessed June 28, 2013
• 7 Paint.net. http://www.getpaint.net/ . Accessed June 28, 2013
• 8 Waxman SG. Determinants of conduction velocity in myelinated nerve fibers. Muscle Nerve 1980; 3 (2) 141-150
• 9 Sakaguchi T, Okada M, Kitamura T, Kawasaki K. Reduced diameter and conduction velocity of myelinated fibers in the sciatic nerve of a neurofilament-deficient mutant quail. Neurosci Lett 1993; 153 (1) 65-68
• 10 Kanaya F, Firrell JC, Breidenbach WC. Sciatic function index, nerve conduction tests, muscle contraction, and axon morphometry as indicators of regeneration. Plast Reconstr Surg 1996; 98 (7) 1264-1271 , discussion 1272–1274
• 11 Yan JG, Matloub HS, Yan Y, Agresti M, Zhang LL, Jaradeh SS. The correlation between calcium absorption and electrophysiological recovery in crushed rat peripheral nerves. Microsurgery 2010; 30 (2) 138-145
• 12 Mackinnon SE, Dellon AL, O'Brien JP , et al. Selection of optimal axon ratio for nerve regeneration. Ann Plast Surg 1989; 23 (2) 129-134
• 13 Fox IK, Brenner MJ, Johnson PJ, Hunter DA, Mackinnon SE. Axonal regeneration and motor neuron survival after microsurgical nerve reconstruction. Microsurgery 2012; 32 (7) 552-562
• 14 Mackinnon SE, Dellon AL, O'Brien JP. Changes in nerve fiber numbers distal to a nerve repair in the rat sciatic nerve model. Muscle Nerve 1991; 14 (11) 1116-1122
• 15 Mohammadi R, Amini K, Charehsaz S. Homeopathic treatment for peripheral nerve regeneration: an experimental study in a rat sciatic nerve transection model. Homeopathy 2012; 101 (3) 141-146
• 16 Geuna S, Gigo-Benato D, Rodrigues AdeC. On sampling and sampling errors in histomorphometry of peripheral nerve fibers. Microsurgery 2004; 24 (1) 72-76
• 17 Muratori L, Ronchi G, Raimondo S, Giacobini-Robecchi MG, Fornaro M, Geuna S. Can regenerated nerve fibers return to normal size? A long-term post-traumatic study of the rat median nerve crush injury model. Microsurgery 2012; 32 (5) 383-387
• 18 Hu W, Yang M, Chang J , et al. Laser doppler perfusion imaging of skin territory to reflect autonomic functional recovery following sciatic nerve autografting repair in rats. Microsurgery 2012; 32 (2) 136-143
• 19 da Silva AP, Jordão CE, Fazan VP. Peripheral nerve morphometry: Comparison between manual and semi-automated methods in the analysis of a small nerve. J Neurosci Methods 2007; 159 (1) 153-157
• 20 Lennertz RC, Medler KA, Bain JL, Wright DE, Stucky CL. Impaired sensory nerve function and axon morphology in mice with diabetic neuropathy. J Neurophysiol 2011; 106 (2) 905-914