Statistical parametric mapping of immunopositive cell density

Abstract

We developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. As an example, we compared Iba-1 (microglial marker) positive cell densities in rats with (n = 6) and without (n = 6) unilateral injection of 1-methyl-4-phenylpyridinium salt (MPP⁺). Immunopositive cells were automatically counted in each animal over a coronal section in the midbrain (bregma −5.9 mm) and a positive cell density map was created for each animal. After the positive cell density map was normalized to a template section from an atlas, positive cell densities of the two groups were compared in each pixel over the section and a statistical parameter (p-value from t-test) was mapped on each pixel. We were able to detect significant increases of microglias in the side of MPP⁺ injection not only in the substantia nigra pars compacta but also in adjacent white matter. We also applied the same analysis to tyrosine hydroxylase stained sections and detected significant decreases of dopamine neurons in the side of MPP⁺ injection. The new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections.