Design-based estimation of neuronal number and individual neuronal volume in the rat hippocampus

Tools recently developed in stereology were employed for unbiased estimation of the neuronal number and volume in three major subdivisions of rat hippocampus (dentate granular, CA1 and CA3 pyramidal layers). The optical fractionator is used extensively in quantitative studies of the hippocampus; however, the classical optical fractionator design may be affected by tissue deformation in the z-axis of the section. In this study, we applied an improved optical fractionator design to estimate total number of neurons on 100 microm thick vibratome sections that had been deformed, in the z-dimension, during histological processing. For estimating cell number, it is necessary to randomize only the location of section planes. But, in the local stereological methods, like cell volume estimation, the orientation of sections must also be randomized. We present a detailed application of a method for making vertical sections from the hippocampus. The volume of hippocampal neurons was estimated using the rotator principle on 40 microm thick plastic vertical uniform random sections and corrected for tissue shrinkage. Application of the proposed new design should result in more accurate estimates of neuron number and neuronal volumes in tissue sections affected by homogenous non-uniform shrinkage.