Simplified Procedure for Isolation and Culture of Neuronal Cells from Brains of Sickle Cell Mice.

Primary neuronal cultures from the brain are critical for investigating disease-specific cellular and molecular mechanisms in mouse models. Current methods for obtaining primary cultures require embryonic brains that are affected by embryonic lethality and genotypic characterization in severe disease models such as sickle cell disease (SCD). Furthermore, these neuronal cultures require about 14 days in vitro (DIVs) for neurite outgrowth to mature. We adapted and optimized a relatively simplified and reproducible method using brains from postnatal day 1 mouse pups for isolating and culturing hippocampal and cortical neurons. This approach produces viable neurons that attach, extend neurites, and express key synaptic markers by 7 DIV and also minimizes glial outgrowth. We successfully applied this approach to isolating and culturing hippocampal and cortical neurons from the brains of one-day-old (P1) pups of humanized transgenic homozygous BERK sickle cell and control mice. Morphological observations at 3, 7, and 14 DIVs demonstrated robust neuronal attachment, neurite outgrowth, and overall structural development in both male and female hippocampal and cortical neurons. Neurons in culture expressed key markers including neuronal nuclear protein (NeuN/Rbfox3), neurofilament 200 (NF200), microtubule-associated protein 2 (MAP2), vesicular glutamate transporter 1 (VGLUT1), postsynaptic density protein 95 (PSD 95), and glutamate N-methyl-D-aspartate receptor subunit 2B (GluN2B). Notably, male SCD hippocampal neurons evinced a higher density of PSD 95 puncta on dendritic spines compared to controls on 7 as well as 14 DIVs. Incubation of male hippocampal neurons in a sickle cell-like microenvironment with TNF-α and heme further increased the density of PSD 95 puncta and colocalization of GluN2B with PSD 95, supporting the utility of this culture system for examining disease-relevant structural and molecular responses. This optimized culture system provides a simplified and reproducible platform to investigate the mechanisms involving neuronal dysfunction in challenging mouse models of brain disorders.