Optic nerves were dissected, processed, embedded in plastic, sectioned and stained with paraphenylenediamine (PPD) as previously described [76 ], except that the staining time was increased to 35 min and Embed 812 medium was used. PPD stains all myelin sheaths, but differentially stains the axoplasm of sick or dying axons darkly. Counts of normal-appearing axons were performed using established nonbiased counting methods. Prior to beginning axon counts, the optic nerve was outlined at 100× magnification, and its cross-sectional area was automatically calculated. Magnification of the same nerve section was increased to 1,000×, and a total of 20 fields at 1,000× were electronically collected. The fields were spaced in a regular fashion across the entire nerve, taking care to avoid field overlap so that the same area was not counted twice. The 20 collected pictures were stacked on the computer screen so that only the final picture was visible to the operator. For nerves with a large number of axons (mildly and moderately affected nerves), a rectangular box that contained a minimum of 200 axons was then drawn on the twentieth image. For nerves with severe axon loss, a larger box was drawn so that a significant proportion of the nerve could be counted. The software program then “cut” a rectangle centred at the same location in all 20 images. Since the operator could only see the top image, this removed the possibility of unconscious operator bias and made the selection of axons to be counted random. Axons were counted manually and marked using the computer. The program tracked the total area counted and the total axon count for all 20 images. The total counted area averaged 12.1%, 14.2%, and 20.5% of the total nerve area for mildly, moderately, and severly affected nerves, respectively. The final count was calculated and expressed as number of axons per optic nerve. With this approach, the nerves with 95% or more axon loss were selected for RGC counts by comparing the remaining axon number to the average for unaffected nerves of the same genotype.
Because of the large number of mice (approximately 50–70 mice of each genotype at each age), an optic nerve rating scale was used for the glaucoma progression study (seeFigure 5 ). The indicated damage levels are readily distinguishable upon inspection of the nerve without counting. Nevertheless, axon counts were performed on at least eight randomly selected nerves of each damage grade to provide quantitative information about these distinct stages of disease (see below). Two investigators (masked to genotype, age, and the damage level assigned by the other investigator) assigned a damage level to each nerve. The two investigators assigned the same grade more than 90% of the time (321 out of 355 nerves). For the nerves on which the initial two investigators differed, a third (masked) investigator was utilized. The third investigator's grade always agreed with one of the initial grades, and the most common assigned grade was used. The number of nerves of each genotype assessed at each age were as follows. For 10.5 mo, Bax+/+n = 49, Bax+/−n = 62, Bax−/−n = 58; for 12 mo, Bax+/+n = 71, Bax+/−n = 50, Bax−/−n = 65.
The damage levels and typical numbers of normal axons present at each stage (determined through axon counts by an investigator masked to damage grade) follow. The representative axon counts were determined for randomly selected nerves of each grade using the counting procedure described above. In mildly affected nerves, there was very mild or no damage, with healthy axons having a clear axoplasm and intact myelin sheath (average number of axons ± SEM: 50,504 ± 1,988; n = 8). In moderately affected nerves, darkly stained, degenerating axons were readily detectable, but the vast majority of axons appeared completely normal (average number of axons ± SEM: 31,410 ± 2,199; n = 8 [79 (link)]). In severely affected nerves, there was extensive axon damage throughout the optic nerve with obvious axon loss (average number of axons ± SEM: 7,970 ± 2,150; n = 17). The axon number was significantly different between optic nerves of each damage level (p < 0.001 for all comparisons, t-tests).
Because of the large number of mice (approximately 50–70 mice of each genotype at each age), an optic nerve rating scale was used for the glaucoma progression study (see
The damage levels and typical numbers of normal axons present at each stage (determined through axon counts by an investigator masked to damage grade) follow. The representative axon counts were determined for randomly selected nerves of each grade using the counting procedure described above. In mildly affected nerves, there was very mild or no damage, with healthy axons having a clear axoplasm and intact myelin sheath (average number of axons ± SEM: 50,504 ± 1,988; n = 8). In moderately affected nerves, darkly stained, degenerating axons were readily detectable, but the vast majority of axons appeared completely normal (average number of axons ± SEM: 31,410 ± 2,199; n = 8 [79 (link)]). In severely affected nerves, there was extensive axon damage throughout the optic nerve with obvious axon loss (average number of axons ± SEM: 7,970 ± 2,150; n = 17). The axon number was significantly different between optic nerves of each damage level (p < 0.001 for all comparisons, t-tests).
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