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Analysis of cell phenotypes in E13.5 primary embryonic mouse midbrain culture with immunofluorescence and high content image analysis

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Title: Analysis of cell phenotypes in E13.5 primary embryonic mouse midbrain culture with immunofluorescence and high content image analysis
Author(s): Pykälämäki, Matias
Contributor: University of Helsinki, Faculty of Pharmacy
Degree program: Master 's Programme in Pharmacy
Specialisation: Pharmacology
Language: English
Acceptance year: 2023
Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by the death of nigrostriatal dopaminergic neurons and formation of intraneuronal protein aggregates called Lewy bodies and Lewy neurites. These inclusions consist of a protein called α-synuclein (aSyn) but also of other proteins, lipids and cell organelles. Progressive cell death leads to nonmotor and motor symptoms. Current therapies for PD are symptomatic and do not modify the disease progression. Therefore, there is a need for the development of therapies attenuating the neurodegeneration. The pre-formed fibrils (PFF) model enables studying of aSyn aggregation and mechanisms behind inclusion formation. The PFF model is based on the exogenous aSyn fibrils’ tendency to result in formation of Lewy body -like inclusions when added in cell culture or in animals. Primary neuronal cultures of mice and rats have typically been used to model aSyn aggregation in vitro with the PFF model. Primary neuronal cultures provide practicality and are able to depict relevant features of dopaminergic neurons. To gain insight about the composition of E13.5 primary embryonic mouse midbrain culture and to enable adaptation of an existing protocol to study other cell types, this study identified and quantified several relevant cellular phenotypes in the micro island culture. The cells were fixed on day in vitro (DIV) 8 or DIV 22 and analysis was conducted using fluorescent immunocytochemistry combined with automated image analysis software, CellProfiler. On DIV 8, tyrosine hydroxylase -positive dopaminergic neurons represented 5 % of the total cells in the culture. Neuronal nuclear antigen -positive neurons resulted representing 30 % of the total cells. Gabaergic neurons were identified to be abundant in the culture and certain dopaminergic neurons were identified as immunoreactive for GABA. Choline acetyltransferase -positive cholinergic neurons were also identified to be present in the culture. The number of oligodendrocyte precursors (OPCs) was observed to be significantly smaller than the number of dopaminergic neurons. OPCs represented around 1 % of the culture on DIV 8. Glutaminergic neurons, parvalbumin-positive interneurons, microglia or astrocytes were not identified in the culture on DIV 8. The number of astrocytes was observed to increase as the incubation time was prolonged to DIV 22. Overall these findings provide valuable insights of the composition of cell phenotypes in E13.5 mouse midbrain culture. The results also provide additional validation for suitability of the original protocol to robustly produce midbrain dopaminergic cultures with minimal number of glial cells. Understanding more about the relevance and interplay of different cell phenotypes in PD pathophysiology can provide valuable insight for the development of potential therapeutic strategies.
Keyword(s): dopaminergic neuron Parkinson’s disease glial cell primary neuronal culture phenotype high-content image analysis

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