A stereological method for estimating the total length and size of myelinated fibers in rat cerebral cortex

An efficient and unbiased stereological technique to estimate the total length and total volume of the myelinated fibers in rat cortex is described. Unbiased sampling and measuring principles enable to obtain an unbiased estimation of the true value. In the present design, six tissue blocks were obtained from the entire rat brain cortex in a uniform random fashion. Isotropic, uniform random (IUR) sections were ensured by the use of the isector technique. One section with the thickness of 60 nm was cut from the center of each epon block using an ultramicrotome. Five fields of vision were photographed using electron microscopy. The length density of the myelinated fibers in the cortex was estimated using an unbiased counting frame. The volume density of the myelinated fibers in the cortex was estimated with point counting. The total length and total volume of the myelinated fibers in the cortex were estimated by multiplying the volume of the cortex, obtained using the Cavalieri principle and the length density and volume density of the fibers in the cortex. The sampling variance introduced by the stereological estimation procedure was a small fraction of the observed total variance indicating an efficient sampling protocol.