A Comprehensive Stereology Method for Quantitative Evaluation of Neuronal Injury, Neurodegeneration, and Neurogenesis in Brain Disorders

Abstract

Neuronal injury, neurodegeneration, and neuroanatomical changes are key pathological features of various neurological disorders, including epilepsy, stroke, traumatic brain injury, Parkinson's disease, autism, and Alzheimer's disease. Accurate quantification of neurons and interneurons in different brain regions is critical for understanding the progression of neurodegenerative disorders in animal models. Traditional scoring methods are often superficial, biased, and unreliable for evaluating neuropathology. Stereology, a quantitative tool that uses 3-dimensional visualization of cells, provides a robust protocol for evaluating neuronal injury and neurodegeneration. This article presents a comprehensive and optimized stereology protocol for unbiased quantification of neuronal injury, neurodegeneration, and neurogenesis in rat and mouse models. This protocol involves precise counting of injured neurons, surviving neurons, and interneurons through immunohistochemical processing of brain sections for NeuN(+) principal neurons, parvalbumin (PV+) interneurons, doublecortin (DCX+) newborn neurons, and Fluoro-Jade B (FJB+)-stained injured cells. Predefined hippocampal and amygdala regions were identified and analyzed using a Visiopharm stereology software-driven compound microscope. Cell density and absolute cell numbers were determined using the optical fractionation method. Our stereology protocol accurately estimated 1.5 million total NeuN(+) principal neurons and 0.05 million PV(+) interneurons in the rat hippocampus, as well as 1.2 million total principal neurons and 0.025 million interneurons in the mouse hippocampus. FJB(+) counting provided a quantitative index of damaged neurons, and the stereology of DCX(+) neurons demonstrated the extent of neurogenesis. Overall, this stereology protocol enables precise, accurate, and unbiased counting of total neurons in any brain region. This offers a reliable quantitative tool for studying neuronal injury and protection in various models of acute brain injury, neurotoxicity, and chronic neurological disorders. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Stereological quantification of principal neurons, interneurons, and immature neurons in the hippocampus in rat brain sections Basic Protocol 2: Stereological quantification of principal neurons, interneurons, and immature neurons in the hippocampus in mouse brain sections Basic Protocol 3: Stereological quantification of injured or necrotized cells stained with Fluoro-Jade B in the hippocampus and amygdala in rats Basic Protocol 4: Stereological quantification of injured or necrotized cells stained with Fluoro-Jade B in the hippocampus and amygdala regions in mice Basic Protocol 5: Brain fixation and histology processing Basic Protocol 6: Immunochemistry of principal neurons, interneurons, and newborn neurons Basic Protocol 7: Fluoro-Jade B staining of injured neurons.

Keywords: Caliveri estimator; neurodegeneration; optical disector; optical fractionator; quantification of neuron; stereology.