Quantifying optic nerve axons in a cat glaucoma model by a semi-automated targeted counting method.
OBJECTIVE
To describe and validate a semi-automated targeted sampling (SATS) method for quantifying optic nerve axons in a feline glaucoma model.


METHODS
Optic nerve cross sections were obtained from 15 cats, nine with mild to severe glaucoma and six with normal eyes. Optic nerves were dissected, fixed in paraformaldehyde and glutaraldehyde, and processed for light microscopy by resin embedding, sectioning, and staining of axon myelin sheaths with 1% p-phenylenediamine before axon quantification. Commercially available image analysis software was used as a semi-automated axon counting tool (SCT) and was first validated by comparison with a manual axon count (MAC). This counting tool was then used in a SATS method performed by three masked raters and in a semi-automated full count (SAFC) method performed by a single observer. Correlation was assessed between the SCT and MAC using a linear model and analysis of covariance (ANCOVA). Correlation between the SATS and SAFC methods was calculated and the bias, systematic errors, and variance component assessed. The intraclass correlation coefficient (ICC) was determined to establish inter-rater agreement. In addition, the time required to perform the SATS and SAFC methods was evaluated.


RESULTS
Correlation between the axon counts obtained by the SCT and MAC was strong (r = 0.9985). There was evidence of an overcounting of axons by the SCT compared to the MAC with a percentage error rate of 13.0% (95% confidence interval [CI] 11.0%, 15.1%). Both the correlation of SATS count (average per rater) to SAFC (r = 0.9891) and inter-rater agreement (ICC = 0.986) were high. The SATS method presented an overall positive counting error (p<0.001) when compared to the SAFC, consistent with a fixed percentage overestimation of 11.2% (95% CI 8.3%, 14.2%) of the full count. The average time required to quantify axons by the SATS method was 10.9 min, only 27% of that required to conduct the SAFC.


CONCLUSIONS
Our data demonstrate that the SATS method provides a practical, rapid, and reliable means of estimating axon counts in the optic nerves of cats with glaucoma.
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Madison-Wisconsin, Madison, WI ; McPherson Eye Research Institute, University of Madison-Wisconsin, Madison, WI.
Leandro B C
LB
Teixeira
Department of Biostatistics & Medical Informatics, University of Madison-Wisconsin, Madison, WI.
Kevin A
KA
Buhr
Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI.
Owen
O
Bowie
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Madison-Wisconsin, Madison, WI ; McPherson Eye Research Institute, University of Madison-Wisconsin, Madison, WI.
Felicia D
FD
Duke
McPherson Eye Research Institute, University of Madison-Wisconsin, Madison, WI ; Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI.
T Michael
TM
Nork
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Madison-Wisconsin, Madison, WI ; McPherson Eye Research Institute, University of Madison-Wisconsin, Madison, WI.
Richard R
RR
Dubielzig
McPherson Eye Research Institute, University of Madison-Wisconsin, Madison, WI ; Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI ; Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI.
Gillian J
GJ
McLellan
D000818:Animals; D001331:Automation; D001369:Axons / Q000473:pathology*; D002415:Cats; D002452:Cell Count / Q000379:methods*; D004195:Disease Models, Animal; D005901:Glaucoma / Q000473:pathology*; D009900:Optic Nerve / Q000473:pathology*; D013997:Time Factors