Estimation of the number of nerve fibers in the human vestibular endorgans using unbiased stereology and immunohistochemistry.

The objective of this study was to obtain estimates of the number of nerve fibers in the human crista ampullaris and utricular macula from normal individuals using unbiased stereology and immunohistochemistry. Vestibular endorgans with the attached vestibular nerve stump were microdissected from the temporal bones. Specimens were divided into two groups. The first group (group 1, N = 8, age range, 68-98 years old, mean = 87 years) was fixed with paraformaldehyde and post-fixed with osmium tetroxide. The second group (group 2, N = 5, age range, 80-98 years old, mean = 86.6 years) was fixed with paraformaldehyde, immunoreacted with monoclonal antibodies against neurofilaments, and post-fixed with osmium tetroxide. The endorgans of both groups were embedded in resin and 2-mum thick sections were made. Estimates of the number of nerve fibers were obtained using an unbiased stereological method, the fractionator. The diameter distribution of nerve fibers was also obtained. The average number of fibers in the horizontal, posterior and superior cristae of individuals in group 1 (N = 14 cristae) was 1424+/-320 (CV = 0.22). The average percentage of small (less than 3 microm), medium (between 3 and 5 microm) and large (more than 5 microm) size fibers was 22.4%, 51.5% and 26.1%, respectively. In group 2 (N = 12), there was an average of 1792+/-99 (CV = 0.05) nerve fibers. The average percentage of small, medium and large size fibers was 22%, 51.2% and 26.8%. In the macula utricle from group 1, there was an average of 3026 nerve fibers (N = 2, ages 80 and 96 years old). There was an average 30.75% small, 56% medium and 13.2% large size fibers. In the utricular macula from group 2 (N = 3, ages 84, 92 and 96 years old), there was an average of 3715 nerve fibers. The average percentage of small, medium and large size fibers was 33.2%, 51.7% and 15.1%. The nerve fiber number in both groups is within the range of previous studies, however, the number of fibers in group 2 was significantly higher than that in group 1 (p = 0.01). This difference is likely due to increased sensitivity gained by the immunohistochemical staining of the axoplasm of nerve fibers in group 2. Results from the present study demonstrate the use of unbiased stereology and immunohistochemistry in human vestibular endorgans, as a reliable and efficient method to estimate the number of nerve fibers. These methods can be applied for studies of normal aging and pathological conditions of the vestibular periphery.