Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson's disease

Abstract

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder afflicting 2% of the population older than 65 years worldwide. Recently, brain organotypic slices have been used to model neurodegenerative disorders, including PD. They conserve brain three-dimensional architecture, synaptic connectivity and its microenvironment. This model has allowed researchers a simple and rapid method to observe cellular interactions and mechanisms. In the present study, we developed an organotypic PD model from rat brains that includes all the areas involved in the nigrostriatal pathway in a single slice preparation, without using neurotoxins to induce the dopaminergic lesion. The mechanical transection of the nigrostriatal pathway obtained during slice preparation induced PD-like histopathology. Progressive nigrostriatal degeneration was monitored combining innovative approaches, such as diffusion tensor magnetic resonance imaging (DT-RMI) to follow fiber degeneration and mass spectrometry to quantify striatal dopamine content, together with bright-field and fluorescence microscopy imaging. A substantia nigra dopaminergic cell number decrease was observed by immunohistochemistry against rat tyrosine hydroxylase (TH) reaching 80% after 2 days in culture associated with a 30% decrease of striatal TH-positive fiber density, a 15% loss of striatal dopamine content quantified by mass spectrometry and a 70% reduction of nigrostriatal fiber fractional anisotropy quantified by DT-RMI. In addition, a significant decline of medium spiny neuron density was observed from days 7 to 16. These sagittal organotypic slices could be used to study the early stage of PD, namely dopaminergic degeneration, and the late stage of the pathology with dopaminergic and GABAergic neuron loss. This novel model might improve the understanding of PD and may represent a promising tool to refine the evaluation of new therapeutic approaches.

Keywords: 6-OHDA; 6-hydroxydopamine; BSA; DARPP32; DT-RMI; FA; H(2)O(2); Hank’s; Hank’s Balanced Salt Solution; IgG; LC–MS/MS; MEM; MFB; MRI; MSN; Minimum Essential Medium Eagle; NGS; NeuN; PBS; PBST; PD; Parkinson’s disease; RARE; ROS; SN; TH; bovine serum albumin; diffusion tensor imaging; diffusion tensor magnetic resonance imaging; dopamine; dopamine- and cAMP-regulated neuronal phosphoprotein; fractional anisotropy; hydrogen peroxide; immunoglobulin G; liquid chromatography–mass spectrometry; magnetic resonance imaging; mass spectrometry; medial forebrain bundle; medium spiny neuron; neuronal nuclei; nigrostriatal organotypic slices; normal goat serum; phosphate-buffered saline; phosphate-buffered saline containing 1% Triton; rapid acquisition with relaxation enhancement; reactive oxygen species; substantia nigra; tyrosine hydroxylase.