To assess the effects of phenytoin on axonal density within the optic nerve, we performed axon counts using antibodies specific for phosphorylated (healthy myelinated axons) and nonphosphorylated (nonmyelinated and demyelinated axons) neurofilament proteins.
7 10 18 This combination of antibodies against both phosphorylated and nonphosphorylated forms ensured labeling of the total population of axons. Nonmyelinated axons can comprise 2% to 50% of the axons in the optic nerve, depending on the location of the section.
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Optic nerves were collected from glaucomatous ipsilateral eyes of rats fed base-vehicle (n = 8) or phenytoin-supplemented chow (n = 8), as well as from the contralateral eyes of phenytoin-treated rats (n = 8). At 8 weeks after induction of glaucoma, rats were deeply anesthetized with ketamine-xylazine and perfused intracardially with 0.01 M PBS (pH 7.4), and then with 4% paraformaldehyde in 0.14 M Sorensen’s phosphate buffer (pH 7.4). Optic nerves were carefully removed and cryoprotected overnight with 30% sucrose in PBS. Tissue from different treatment groups was processed simultaneously. Thin (12 μm) longitudinal and transverse cryosections (n = 5 sections per animal) were collected immediately proximal to the retina (within 1 mm). Sections were mounted on slides and incubated at room temperature in blocking solution (PBS containing 5% normal goat serum, 2% BSA, 0.1% Triton X-100, and 0.02% sodium azide) for 30 minutes; subtype-specific primary antibodies raised in mice against phosphorylated neurofilaments (SMI-31, 1:20,000; Sternberger Monoclonals, Lutherville, MD) and nonphosphorylated neurofilaments (SMI-32, 1:20,000; Sternberger Monoclonals), overnight at 4°C on a rotating shaker; PBS, 6 times for 5 minutes each; rabbit anti-mouse IgG-Cy3 (1:2000; Amersham) in block, 2 hours; and PBS, 6 times for 5 minutes each. Control experiments were performed without primary or secondary antibodies, which yielded only background levels of signal.
An observer performed quantitative analysis using blinded protocols. Axonal densities were determined within preselected fields of 1920 μm
2 within the center of the optic nerve, with location of the fields carefully conserved from group to group. Neurofilament-stained axons were manually counted from each field using application software (IPLab v3.0; Scanalytics, Fairfax, VA), as described previously.
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