Estimating normal optic nerve axon numbers in non-human primate eyes

Purpose: The goal of the present study is to develop a semi-automated method to estimate accurately, with minimum variance, the total number of axons by counting a subset of the axons within a primate optic nerve. Methods: Using an imaging analysis system, axons in 50% of the area of crosssections of the retrobulbar optic nerve from five adult Rhesus monkeys were counted and extrapolated as an estimate of total axon number of the optic nerves. Both neural and non-neural areas were sampled. With the coordinates of the counts topographically registered, axon numbers within areas ranging from 1 to 50% were resampled. A Monte Carlo and theoretical estimate of the standard deviation of the total axon count for each sampled area was computed. Results: The mean cross-sectional area of the five optic nerves counted was 7.26 +/- 0.6 mm(2), and the mean total axon count of the optic nerve area was 1,304,8168 +/- 89,112. When sampling less than 8% of the optic nerve, the standard deviation within the individual of the total estimated axon number increased sharply. Conclusion: With this technique, the variance within each individual increased only slightly when the counting area was reduced from 50 to 8%, but increased sharply when the counted area became less than 8%. While counting less than 8% of the optic nerve area gives a good estimation of total axon count, the effect of a substantial increase in the standard deviation on the statistical power needed to differentiate group differences will depend on the study design.