Basic NeuroscienceAutomated immunohistochemical method to quantify neuronal density in brain sections: Application to neuronal loss after status epilepticus

- Method for the automatic analysis of confocal images stained with DAPI and NeuN antibody.
- Neuronal densities are invariant with moderate changes in NeuN expression.
- Method detects neuronal loss in the rat limbic system after status epilepticus.

BackgroundTo study neurotoxic processes, it is necessary to quantify the number of neurons in a given brain structure and estimate neuronal loss. Neuronal densities can be estimated by immunohistochemical quantitation of a neuronal marker such as the protein NeuN. However, NeuN expression may vary, depending on certain pathophysiological conditions and bias such quantifications.New methodWe have developed a simple automatic quantification of neuronal densities in brain sections stained with DAPI and antibody to NeuN. This method determines the number of DAPI-positive nuclei also positive for NeuN in at least two adjacent sections within a Z-stack of optical sections.ResultsWe tested this method in animals with induced status epilepticus (SE) a state of intractable persistent seizure that produces extensive neuronal injury. We found that SE significantly reduced neuronal density in the piriform cortex, the amygdala, the dorsal thalamus, the CA3 area of the hippocampus, the dentate gyrus and the hilus, but not in the somatosensory cortex or the CA1 area. SE resulted in increases in the total density of cellular nuclei within these brain structures, suggesting gliosis.Comparison with existing methodsThis automated method was more accurate than simply estimating the overall NeuN fluorescence intensity in the brain section, and as accurate, but less time-consuming, than manual cell counts.ConclusionThis method simplifies and accelerates the unbiased quantification of neuronal density. It can be easily applied to other models of brain injury and neurodegeneration, or used to screen the efficacy of neuroprotective treatments.