title: Methodological aspects of 3D and automated 2D analyses of white matter neuronal density in temporal lobe epilepsy.
abstract: White matter neuronal density has been correlated with clinical outcome after temporal lobectomy for refractory epilepsy. Both morphometric 2D (two-dimensional) and stereological 3D (three-dimensional) analyses of neuronal density have been performed. 3D analyses are thought to be more accurate than 2D counts, but more time-consuming. We compared 3D and automated 2D measurements in the same specimens. Adjacent 20-microm (for 3D analyses) and 5-microm (for 2D analyses) sections from 10 temporal lobectomies were stained for NeuN immunohistochemistry. Analysis of 100% of a region of interest (ROI) in deep white matter was performed using an image analysis system (Histometrix, Kinetic Imaging, UK). 3D analyses were undertaken using x 63 magnification (6 h/case). Automated 2D analyses were undertaken using automatic neuronal identification at x 10 magnification with three to four repeats (1.5 h/case). The range of neuronal densities for 3D measurements was 2120-4910 neurones/mm(3), and for automated 2D measurements 17.4-47.1 neurones/mm2. There was a linear correlation between the two methods with an r2 of 0.58. [corrected] Count-recount variability was 1.4-9.9% for the 3D and 5.1-36.6% for the automated 2D measurements. We found a wide range of white matter neuronal densities using either analysis. The low agreement between methods, and the high count-recount variability for the automated 2D analyses, indicate that despite being more time-consuming, rigorous 3D stereological analyses have to be performed to obtain reliable results. These findings have implications for studies requiring neuronal counts in normal and disease states.
authors.affiliation: Department of Clinical and Experimental Epilepsy, National Society for Epilepsy, London, UK. s.eriksson@ion.ucl.ac.uk
authors.forename: S H
authors.initials: SH
authors.lastname: Eriksson
authors.forename: S L
authors.initials: SL
authors.lastname: Free
authors.forename: M
authors.initials: M
authors.lastname: Thom
authors.forename: L
authors.initials: L
authors.lastname: Martinian
authors.forename: S M
authors.initials: SM
authors.lastname: Sisodiya
mesh_terms: D000328:Adult; D001921:Brain / Q000473:pathology*; D004833:Epilepsy, Temporal Lobe / Q000473:pathology*; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D009474:Neurons / Q000473:pathology*; D010865:Pilot Projects; D015203:Reproducibility of Results