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M.L. Archibald, T. LeVatte, B.C. Chauhan; Semi-Quantitative Method for Grading Optic Nerve Damage in Rats . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3346.
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Purpose: To evaluate the inter- and intra-rater reliability of a semi-quantitative method for evaluating optic nerve damage and to compare these scores to axon counts with electron microscopy. Methods: Brown Norway rats (n = 73) were surgically implanted with osmotic minipumps which delivered 0.2 µg/day of endothelin-1 to the optic nerve approx. 1 mm behind the globe while the fellow eye served as control. Animals were sacrificed after 10, 21, 42 or 84 days of endothelin delivery. The optic nerves were fixed in 2.5% gluteraldehyde, embedded in Epon and sectioned approximately 1 mm behind the globe. Thick sections (~1 µm) were stained with 1% p-phenylenediamine and axon damage graded from 0 (normal) to 10 (complete damage) under light microscopy. Each of 3 observers independently rated 7 sets of between 8 and 26 nerves and one set of nerves three times on different days to estimate inter- and intra-rater reliability respectively. After the reliability values were calculated, consensus agreement was determined in cases of disagreement. Axon density estimates were made in 39 animals by counting axons in electron micrographs of thin sections (100-130 nm) obtained in 4-5 equidistant locations from the centre of the nerve in each quadrant. Results: The mean (± sd) inter-rater reliability kappa was 0.66 (± 0.09), indicating "substantial" agreement while the intra-rater agreement for the observers was 0.80, 0.80 and 0.81 respectively, indicating "substantial" to "almost perfect" agreement. The median consensus grade in the treated animals was 2 (range 0-10), while 38 of 39 control eyes had a consensus grade of 0. There was a high correlation between the ratio of treated to control axon density to consensus grade (Spearman’s r = -0.68, P < 0.001). Conclusions: This quick and relatively easy method of grading optic nerve damage has high inter- and intra-rater agreement in addition to high correlation with axon density counts.
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