Isolation of Distinct Types of Neurons from Fresh Brain Tissue Using Laser Microdissection in Combination with High-Performance Liquid Chromatography—Mass Spectrometry

Abstract

Humans age and the ageing process affects cells in all areas of the human body, including nerve cells within the brain. With advancing age there is also a rise in the probability of developing a neurodegenerative disorder such as, e.g., amyotrophic lateral sclerosis, Huntington’s disease, Parkinson’s disease, or Alzheimer’s disease. In all these age-related neurodegenerative disorders, distinct neuron populations within specific brain regions are primarily affected. For example, Parkinson’s disease is characterized by a slowly progressive degeneration of dopaminergic neurons in the substantia nigra whereas the entorhinal cortex is first affected in Alzheimer’s disease. In patients suffering from Huntington’s disease, neurons in both striatum and cortex undergo substantial cell loss and in amyotrophic lateral sclerosis the neurodegeneration arises from the spinal cord and the motor cortex. For the investigation of the differences in neuronal vulnerability, it is important to examine the protein expression pattern in these specific neural populations. By this, conclusions about the origination process of these diseases can be achieved. In order to obtain this objective, specific isolation of distinct neurons from the surrounding brain tissue is indispensable. However, discrimination as well as isolation of distinct types of neurons can be challenging, due to the brain tissue’s complexity. With traditional methods such as the homogenization of tissue samples, a specific isolation of single neuron populations is not feasible because homogenization results into a mixture containing all cell types. Laser microdissection can overcome this technical limitation. First, this method enables visualization of tissues via a microscopic unit and therefore an enhanced discrimination of different brain cells. Second, a laser device guarantees a contact-free and consequently a contamination-free separation of distinct neurons from the surrounding brain tissue. In the following, we present a detailed protocol that includes a workflow for the isolation and analysis of neurons from freshly frozen post mortem human brain tissue samples. During this procedure, the brain tissue is sectioned, stained, laser microdissected, and ultimately analyzed by high-performance liquid chromatography—mass spectrometry.