Abstract

Astrocytes, also known as astroglia, are important cells for the structural support of neurons as well as for biochemical balance in the central nervous system (CNS). In this study, the polymerization of dopamine (DA) to polydopamine (PDA) and its effect on astrocytes was investigated. The polymerization of DA, being directly proportional to the DA concentration, raises the prospect of detecting DA concentration from PDA optically using image-processing techniques. It was found here that DA, a naturally occurring neurotransmitter, significantly altered astrocyte cell number, morphology, and metabolism, compared to astrocytes in the absence of DA. Along with these effects on astrocytes, the polymerization of DA to PDA was tracked optically in the same cell culture wells. This polymerization process led to a unique methodology based on multivariate regression analysis that quantified the concentration of DA from optical images of astrocyte cell culture media. Therefore, this developed methodology, combined with conventional imaging equipment, could be used in place of high-end and expensive analytical chemistry instruments, such as spectrophotometry, mass spectrometry, and fluorescence techniques, for quantification of the concentration of DA after polymerization to PDA under in vitro and potentially in vivo conditions.

Highlights

  • Astrocytes are the glial cells in the central nervous system (CNS) which provide both structural and biochemical support to neurons

  • A unique image analysis technique, based on the hue of red, green and blue intensities obtained from images of media with PDA, was developed to calculate the dopamine concentrations that started the polymerization in the cellular microenvironments under study

  • One mL of solution for each concentration of dopamine was placed in cell culture suspension plates and left at room temperature for 3 h following the protocol discussed by Sheng et al in their study [14]

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Summary

Introduction

Astrocytes are the glial cells in the central nervous system (CNS) which provide both structural and biochemical support to neurons. A colorimetric assay estimation of DA concentration in the primary brain astrocyte cell culture microenvironment was studied This was done by a multivariate regression analysis of the images of the cell culture media and the observed shift in their color as the polymer (PDA) particles started populating the system. A unique image analysis technique, based on the hue of red, green and blue intensities obtained from images of media with PDA, was developed to calculate the dopamine concentrations that started the polymerization in the cellular microenvironments under study This methodology for estimation of dopamine may spare usage of high-end analytical instruments and techniques, and could improve experimental time constraints by the use of image analysis software as described in the results of the work presented here

Materials and Methods
Preparing Astrocyte Culture Media
Toxicity of Astrocytes to DA
Formation of PDA in the Presence of Bicarbonate Buffer at Basic pH
Morphology Changes of Astrocytes during Polymerization of DA to PDA
The μfull of DAPI stainedin astrocytes
Morphology and number astrocyte nuclei in presence of DA andwere
Metabolic
Effect of PDA on Astrocyte Microenvironment Prior to Plating of Cells
Morphological changesininastrocytes astrocytes related of of prior to cell
Estimation
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