Comparison of unbiased estimation of neuronal number in the rat hippocampus with different staining methods

Abstract

BackgroundNeuN and Nissl staining (toluidine blue, cresyl violet staining) are routinely used methods in unbiased stereological estimation of the total number of hippocampal neurons. New methodIn the present study, we stained serial frozen coronal sections from 5 normal adult male Sprague-Dawley rat brains with different methods, measured the deformation of hippocampal area in brain sections and estimated the total number of hippocampal neurons using the optical fractionator. ResultsThe deformation in x, y-axis was not obviously different with different staining protocols, but shrinkage in z-axis was significant after staining (p<0.001). NeuN staining produced significant higher estimate number than cresyl violet staining by 24% (p=0.002), however, NeuN and Cresyl Violet staining showed a high degree of correlation in quantification of total neuronal numbers and both methods are suitable for unbiased stereological estimation. Comparison with existing method (s)NeuN is more reliable but if time is limited or the number of animals used in experiments is high, cresyl violet staining may be a feasible method. ConclusionsCompared with previous estimates of the neurons number in rat hippocampus, our present data is reliable and the stereological analysis based on our system is a cost-effective unbiased method for estimation of neuron number.