New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells

Summary

A protocol for the neuroprotective application of low-dose atmospheric pressure plasma treatment on glucose deprivation-induced SH-SY5Y injuries.

Abstract

The atmospheric pressure plasma jet (APPJ) has attracted the attention of many researchers from multiple disciplines in recent years because its emissions include multiple types of reactive nitrogen species (RNS) and reactive oxygen species (ROS). Our previous study has shown the cytoprotective effect of the APPJ against oxidative stress-induced injuries. The aim of the present study is to provide a detailed in vitro treatment protocol regarding the neuroprotective applications of helium APPJs on glucose deprivation-induced injury in SH-SY5Y cells. The SH-SY5Y human neuroblastoma-derived cell line was maintained in RPMI 1640 medium supplemented with 15% fetal calf serum. The culture medium was then changed to RPMI 1640 without glucose before APPJ treatment. After a 1 h incubation in a cell incubator, cell viability was determined using Cell Counting Kit 8. The results showed that, compared to the glucose deprivation group, cells treated with APPJ exhibited significantly increased cell viability in a dose-dependent manner, with 8 s/well observed as an optimal dose. Meanwhile, helium flow had no effect on the glucose deprivation-induced cell impairment. Our results indicated that APPJ could be potentially used as a treatment method for the diseases in the central nervous system related to glucose deprivation. This protocol could also be used as a cytoprotective application for other cells with different impairments, but the cell culture and APPJ treatment conditions should be readjusted, and the treatment dose must be relatively low.

Introduction

The adult brain almost exclusively uses glucose as a substrate for energy metabolism under normal physiological conditions. The human brain constitutes only 2% of the body weight but consumes approximately 25% of the total glucose within the body¹. It is well documented that glucose metabolism dysfunction is one of the major pathological changes during ischemic stroke and various neurodegenerative diseases, including Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD)^2,3. The lack of glucose and either impaired glucose uptake or oxidative phosph....

Protocol

1. Preparation of the APPJ device

CAUTION: Please consult all relevant material safety data sheets (MSDS) before use. Please use appropriate safety practices when performing all the experiments, including the use of a fume hood and personal protective equipment (safety glasses, protective gloves, lab coat, etc.). The protocol requires standard cell handling techniques (sterilizing, cell recovery, cell passaging, cell freezing, cell staining, etc.).

1. Choose a quartz tube with an internal diameter of 1 mm and an external diameter of 3 mm. Smooth the cross-sections at both ends using a polishing foil.

Results

Data are expressed as the mean ± SD of at least three independent experiments. Group results were analyzed for variance using ANOVA. All analyses were performed using statistical analysis software Prism and p<0.05 was the threshold for statistical significance.

Cell viability was measured after 4 h of CCK-8 incubation. As shown in Figure 3, glucose deprivation reduced the viability of SH-SY5Y cells to 44.1 ± 2.6% compared to that of the control group (cells nor.......

Discussion

SH-SY5Y cells are a human neuroblastoma-derived cell line and are widely used as an appropriate cell model for in vitro studies on neurotoxicity or neuroprotection¹². The SH-SY5Y cell line was sensitive to glucose deprivation conditions. Cell viability decreased to nearly 50% after 1 h glucose deprivation, which is the optimal cell viability condition for studies of pharmacodynamics. Furthermore, CCK-8 reagent has no cytotoxicity to cells and cells were incubated with CCK-8 reagent for an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SH-SY5Y cell line	China Center for Type Culture Collection	3111C0001CCC000026
RPMI 1640 medium	Thermo Scientific	21875091	stored at 4 °C
RPMI 1640 medium no glucose	Thermo Scientific	11879020	stored at 4 °C
fetal calf serum	Thermo Scientific	16000044	stored at -20 °C
tripsin-EDTA solution	Solarbio	T1300	stored at 4 °C
96 wells plate	corning	3599
Cell Counting Kit-8 (CCK-8)	Dojindo Laboratories	CK04	stored at 4 °C
microplate reader	Tecan	M200 Pro	for measuring the absorbance at 450 nm
High – voltage Power Amplifier	Trek	PD06087	for amplifing the power
Function Signal Generator	MaZe Electronics Science&Technology	AT30120	for providing the specific signal
High – Voltage Probe	Tektronix	P6015A	for detecting high voltage
Digital Oscilloscope	Tektronix	DPO4104B	for displaying the signal