Name: new application of an atmospheric pressure plasma jet as a neuro-protective agent against glucose deprivation-induced injury of sh-sy5y cells
Uploaded: 2017-10-09T15:00:00.000+00:00
Duration: 6 min 34 s
Description: Watch this Scientific Journal Video about New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells at JoVE.com

This work was supported by the Innovation fund of Beijing Neurosurgical Institute (2014-11), National Natural Science Foundation of China (Nos. 11475019 and 81271286) and Beijing Natural Science Foundation (No. 7152027).

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&#160;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.).
<ol>
	<li>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.</li>
	<li>Use a Nickel-plated stainless-steel needle with a diameter of 1.0 mm as the high voltage electrode. Grind its tip to a radius of curvature of 0.05 mm.</li>
	<li>Wrap aluminum foil (2 mm in width) around the quartz tube at 1 cm from the quartz tube nozzle. Fix the stainless-steel needle point at 1 cm from the other end of the aluminum foil. Use the aluminum foil ring as the low voltage electrode.</li>
</ol>
2. Acquisition of Jets
<ol>
	<li>To provide an AC signal, connect the high-voltage power amplifier to the function signal generator that serves as a power supply. To record the waveforms of the applied voltage to the high voltage electrode, connect one end of the high-voltage probe to the digital oscilloscope and connect the other end to the power supply. To protect the circuit, use a 2 k&#8486; resistor as a protective resistor. Connect the circuit as shown in Figure 2. 
	CAUTION: Do not touch the high voltage lines.</li>
	<li>Continuously pass helium (volume fraction, 99.999%) over the quartz tube and control the gas flow rate at a stable 1.4 Standard Liter per Minute (SLM). 
	NOTE: We do not use a filter before treating cell cultures. The volume fraction of helium used in the experiment is 99.999%, and most microorganisms cannot live under these conditions.</li>
	<li>Turn on the power of the oscilloscope, signal generator, and high-voltage power amplifier. Rotate the frequency adjustment knob to 5 kHz. Gradually increase the voltage to a peak-to-peak value of 6 kV. 
	NOTE: The jet is long enough (approximately 3 cm) when the peak-to-peak value of the voltage applied in the needle is approximately 6 kV.</li>
</ol>
3. Preparation of SH-SY5Y Cells
<ol>
	<li>Grow the SH-SY5Y human neuroblastoma-derived cell line in a 25 cm2 flask in RPMI 1640 medium supplemented with 15% fetal calf serum (FBS). Maintain the cells in a humidified incubator containing 5% CO2 and 95% air at 37 &#176;C.</li>
	<li>When the cells reach 85% confluence, carefully aspirate the culture media and add 1 mL 0.25% trypsin + 0.1% EDTA to the cells.</li>
	<li>After 15 s incubation at room temperature, carefully aspirate the trypsin and add 2 mL RPMI 1640 containing 15% FBS to neutralize.</li>
	<li>Gently pipette up and down, washing the bottom of the well, until the SH-SY5Y monolayer is completely detached.</li>
	<li>Count the cells by means of the hemocytometer and adjust the cell concentration to 2 x 105 cells/mL by adding RPMI 1640 medium +15% FBS, and then transfer 100 &#181;L of cell suspension to each well of a 96-well plate.</li>
	<li>Allow the cells to attach for 12 h in a cell incubator before APPJ treatment.</li>
</ol>
4. APPJ treatment of SH-SY5Y
<ol>
	<li>Adjust the distance between the nozzle of the quartz tube and the platform where the 96-well plate will be placed to 3 cm. Ensure that the beam can touch the surface of the culture medium. 
	NOTE: The distance is not measured from the bottom of the plate. It is first adjusted to 3 cm between the nozzle of the quartz tube and the platform used to place 96-well plate.</li>
	<li>Before the APPJ treatment, change the culture medium in each well except the control wells to RPMI 1640 without glucose medium.</li>
	<li>Place the plate under the APPJ nozzle and ensure that the jets can shoot vertically into each well.</li>
	<li>Treat cells in separate wells with APPJ for 0 s, 1 s, 2 s, 4 s, 8 s, and 12 s. 
	NOTE: APPJ is generated by ionizing helium (Figure 1). The cells injured by glucose deprivation are treated by 4 s and 8 s helium flow to eliminate the effects of helium on cells. All the treatments should be performed in triplicate.</li>
</ol>
5. Cell Viability Assay
NOTE: Do not change the medium in this step.
<ol>
	<li>After the APPJ treatment, incubate the cells for 1 h in a cell incubator.</li>
	<li>Add 10 &#181;L of Cell Counting Kit-8 (CCK-8) solution to each well.</li>
	<li>Incubate the cells at 37 &#176;C for 4 h. 
	NOTE: The SH-SY5Y cell line is sensitive to glucose deprivation conditions12. Cell viability decreases to nearly 50% after 1 h glucose deprivation, which is the optimum cell viability condition for pharmacodynamics studies. CCK-8 has no cytotoxicity to cells and cells are incubated with CCK-8 reagent for another 4 h in the glucose deprivation conditions after APPJ treatment to check cell viability. After 8 h glucose deprivation and APPJ treatment, the protective effect of APPJ was significantly reduced because the long-duration of glucose deprivation led to severe damage of the SH-SY5Y cells. There was no evidence of living cells after 24 h glucose deprivation11.</li>
	<li>Measure the absorbance at 450 nm with a microplate reader.</li>
</ol>

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	<li>Yang, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+mitochondrial+electron+transfer+for+the+treatment+of+neurodegenerative+diseases+and+cancers:+Methylene+blue+connects+the+dots.">Alternative mitochondrial electron transfer for the treatment of neurodegenerative diseases and cancers: Methylene blue connects the dots.</a> Prog. Neurobiol. , (2015).</li><li>Bhat, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress,+mitochondrial+dysfunction+and+neurodegenerative+diseases;+a+mechanistic+insight.">Oxidative stress, mitochondrial dysfunction and neurodegenerative diseases; a mechanistic insight.</a> Biomed. Pharmacother. 74, 101-110 (2015).</li><li>Bullon, P., Newman, H. N., Battino, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity,+diabetes+mellitus,+atherosclerosis+and+chronic+periodontitis:+a+shared+pathology+via+oxidative+stress+and+mitochondrial+dysfunction?.">Obesity, diabetes mellitus, atherosclerosis and chronic periodontitis: a shared pathology via oxidative stress and mitochondrial dysfunction?.</a> Periodontol. 2000. 64 (1), 139-153 (2014).</li><li>Sutherland, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+for+ischaemic+stroke:+translation+from+the+bench+to+the+bedside.">Neuroprotection for ischaemic stroke: translation from the bench to the bedside.</a> International Journal of Stroke. 7 (5), 407-418 (2012).</li><li>Yan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+using+Cold+Atmospheric+Plasma+Stimulated+Media+for+Cancer+Treatment.">Principles of using Cold Atmospheric Plasma Stimulated Media for Cancer Treatment.</a> Scientific Reports. 5 (5), 18339 (2015).</li><li>Lu, X., Laroussi, M., Puech, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+atmospheric-pressure+non-equilibrium+plasma+jets+and+plasma+bullets.">On atmospheric-pressure non-equilibrium plasma jets and plasma bullets.</a> Plasma Sources Science &amp; Technology. 21 (3), 034005 (2012).</li><li>Lu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+species+in+non-equilibrium+atmospheric-pressure+plasmas:+Generation,+transport,+and+biological+effects.">Reactive species in non-equilibrium atmospheric-pressure plasmas: Generation, transport, and biological effects.</a> Phys. Rep. 630, 1-84 (2016).</li><li>Lu, X., Naidis, G. V., Laroussi, M., Ostrikov, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+ionization+waves:+Theory+and+experiments.">Guided ionization waves: Theory and experiments.</a> Phys. Rep. 540 (3), 123-166 (2014).</li><li>Di, M. S., Reed, T. T., Venditti, P., Victor, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ROS+and+RNS+Sources+in+Physiological+and+Pathological+Conditions.">Role of ROS and RNS Sources in Physiological and Pathological Conditions.</a> Oxid. Med. Cell Longev. 2016 (22), 1245049 (2016).</li><li>Contestabile, A., Ciani, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nitric+oxide+in+the+regulation+of+neuronal+proliferation,+survival+and+differentiation.">Role of nitric oxide in the regulation of neuronal proliferation, survival and differentiation.</a> Neurochemistry International. 45 (6), 903-914 (2004).</li><li>Yan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+atmospheric+pressure+plasma+on+oxidative+stress-induced+neuronal+injuries:+an+in+vitro+study.">Protective effect of atmospheric pressure plasma on oxidative stress-induced neuronal injuries: an in vitro study.</a> J. Phys. D: Appl. Phys. 50 (9), 095401 (2017).</li><li>Xie, H. R., Hu, L. S., Li, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SH-SY5Y+human+neuroblastoma+cell+line:+in+vitro+cell+model+of+dopaminergic+neurons+in+Parkinson's+disease.">SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease.</a> Chin. Med. J. 123 (8), 1086-1092 (2010).</li><li>Tsutsui, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+cardiac+disease+by+inhalation+of+atmospheric+pressure+plasma.">Treatment of cardiac disease by inhalation of atmospheric pressure plasma.</a> Japanese Journal of Applied Physics. 53 (6), 060309 (2014).</li><li>Graves, D. 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No conflicts of interest were declared in relation to this paper.

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 another 4 h in the glucose deprivation conditions after APPJ treatment to check cell viability. In the current study, we provide a detailed in vitro treatment protocol regarding the neuroprotective applications of APPJ on the glucose deprivation-induced injury of SH-SY5Y cells.
Modifications and troubleshooting 
The CCK-8 incubation time could be shorter if the color in each well significantly changed. But if SH-SY5Y cell density is below 1 x 104 cells per well, cells will be dead after glucose deprivation and APPJ treatment. It is also recommended to reduce the gas flow rate, while ensuring that the plasma beam can touch the surface of the culture medium. APPJ could also be used as a cytoprotective agent for other neuronal related cell lines (HT-22, neuro-2A, or even primary neurons) with different impairments (hypoxia, oxidative stress, etc.), but the cell culture and APPJ treatment conditions should be readjusted, and the treatment dose must be relatively low. We have tried to reduce distance in this APPJ generation parameter, and we found that the plasma jet could directly affect the attachment of SH-SY5Y cells which could result in cell injuries (SH-SY5Y cells were easily detached from their adherent state). We believe that the treatment distance should be based on the cell characteristics and the tolerance to the plasma jet treatment.
Limitations of the technique 
The current protocol only focused on the in vitro neuroprotective effect of APPJ on glucose deprivation-injured SH-SY5Y cells. Previous research has shown that inhalation of plasma could improve cardiac functions in a rat myocardial infarction model13. More work is still needed to investigate the in vivo treatment method for the brain protection.
Significance with respect to existing methods 
Previous research on plasma medicine paid more attention to inactivation capacities in bacteria, cancer cells, and tissues because of the oxidative and/or nitrative stress induced by APPJ treatment14. Our protocol used low-dose plasma treatment for neuroprotective applications without the consequences of excessive plasma-induced injuries, indicating that the APPJ treatment could be potentially used as a novel &#34;NO donor drug&#34; for the future research and even for clinical translation.
Critical steps within the protocol 
The most critical step in this protocol is to make sure the APPJ treatment dose is relatively low, since over treatment with APPJ will aggravate cell injuries and directly induce cell death. Another critical step is to control the glucose deprivation duration or the cells will die and the cytoprotective effect of APPJ will be significantly reduced. Pure helium, rather than helium mixed with a small amount of O2 or air, was used. When helium mixed with a small amount of O2 or air is used, the amount of ROS in the plasma will increase. It is very difficult to make a diagnosis when complicated plasma chemical reactions occur.
Future applications 
It is also worthwhile to note that the APPJ treatment was applied after induction of glucose deprivation on SH-SY5Y cells, indicating that APPJ could be potentially used as a treatment method for glucose deprivation-related diseases in the central nervous system, especially ischemic stroke. Therefore, it is necessary for future studies to evaluate the treatment conditions of the neuroprotective effect of APPJ both alone and in combination with other neuroprotective agents at different periods after glucose deprivation.

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&#39;s disease (AD), Huntington&#39;s disease (HD), and Parkinson&#39;s disease (PD)2,3. The lack of glucose and either impaired glucose uptake or oxidative phosphorylation can directly impact ATP production and further induce neural cell death, which may increase the risk of neuronal dysfunction, suggesting that maintaining cell viability or delaying cell injury after glucose deprivation might be a reasonable approach for treating these diseases. The investigation of neuroprotective effects via glucose modulation, focusing on anti-inflammatory agents, ion channel modulators, free radical scavengers, neurotrophic factors, etc. has been of interest. However, translation of these neuroprotective approaches from bench to clinical practice has not been successful4.
Atmospheric pressure plasma jets (APPJs) are a new kind of atmospheric low temperature gas discharge technology that has attracted the attention of many researchers from multiple disciplines in recent years. APPJs have been used for decades in various biomedical applications such as cancer cell treatment, bacterial inactivation, blood coagulation, wound healing, oral medicine, etc.5,6, due to its emissions of multiple types of reactive nitrogen species (RNS) and reactive oxygen species (ROS) (Figure 1)7. Previous plasma bio-medicine applications mainly focused on the oxidative and/or nitrative stress on bacteria, cells, and tissues8. However, the APPJ could also be a &#34;double-edged sword&#34; since RNS and ROS are important intracellular signaling molecules related to many physiological and pathophysiological processes9. Nitrous oxide (NO) controls a wide range of biological processes and plays a dual role in the human body, especially in central nervous system (CNS). Low levels of NO have shown their neuroprotective activities both in vitro and in vivo via multiple signal pathways10. Our previous study first reported that helium APPJ-induced NO production was involved in the neuroprotective effect of APPJ against oxidative stress-induced injuries11. However, the effects of APPJs on other injuries have not been reported. Therefore, the aim of the present study is to provide an in vitro treatment protocol regarding the neuroprotective applications of helium APPJ on glucose deprivation-induced injury in SH-SY5Y cells. Different from previous studies, our protocol used low-dose plasma treatment for neuroprotective applications without the consequences of excessive plasma-induced injuries, indicating that the APPJ treatment could be potentially used as a novel &#34;NO donor drug&#34; for future research and even for clinical translation. This protocol was also suggested to be used as a cytoprotective application for other cell types with different impairments, but the APPJ treatment conditions should be re-adjusted and the treatment dose must be relatively low.

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

new application of an atmospheric pressure plasma jet as a neuro-protective agent against glucose deprivation-induced injury of sh-sy5y cells

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.

Data are expressed as the mean &#177; 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&#60;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 &#177; 2.6% compared to that of the control group (cells normally cultured in RPMI 1640 medium containing 15% FBS). The APPJ treatment significantly increased cell viability in a dose-dependent manner at an optimal dose of 8 s/well, and the cell viability reached to 62.27 &#177; 3.1%. Gas flow had no effect on the glucose deprivation-induced cell impairment (Table 1).

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56323/56323fig1.jpg" /> 
Figure 1: Typical RNS and ROS reactions in APPJ emissions. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56323/56323fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56323/56323fig2.jpg" /> 
Figure 2: Schematic of the experimental setup. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56323/56323fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56323/56323fig3.jpg" /> 
Figure 3: Protective effect of APPJ on glucose deprivation-induced injury of SH-SY5Y cells. Cells were treated with APPJ and subjected to glucose deprivation for 1 h, after which the cell viability was determined using the CCK-8 assay. Error bars represent mean &#177; SD. ***P &#60;0.001 versus control; #P &#60;0.05 and ##P &#60;0.01 versus the glucose deprivation group (n = 3). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56323/56323fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Groups</td>
			<td></td>
			<td colspan="2">Cell viability (control %)</td>
		</tr>
		<tr>
			<td colspan="2">Control</td>
			<td></td>
			<td colspan="2">100 &#177; 3.7%</td>
		</tr>
		<tr>
			<td colspan="2">Glucose deprivation</td>
			<td></td>
			<td colspan="2">44.1 &#177; 2.6%***</td>
		</tr>
		<tr>
			<td colspan="2" rowspan="5">APPJ treatment + Glucose deprivation</td>
			<td>1 s</td>
			<td colspan="2">49.3 &#177; 2.8%</td>
		</tr>
		<tr>
			<td>2 s</td>
			<td colspan="2">53.0 &#177; 2.7%</td>
		</tr>
		<tr>
			<td>4 s</td>
			<td colspan="2">60.4 &#177; 2.3%#</td>
		</tr>
		<tr>
			<td>8 s</td>
			<td colspan="2">62.3 &#177; 3.1%##</td>
		</tr>
		<tr>
			<td>12 s</td>
			<td colspan="2">51.3 &#177; 2.7%</td>
		</tr>
		<tr>
			<td colspan="2" rowspan="2">He flow + Glucose deprivation</td>
			<td>4 s</td>
			<td colspan="2">45.4 &#177; 2.4%</td>
		</tr>
		<tr>
			<td>8 s</td>
			<td colspan="2">44.1 &#177; 3.1%</td>
		</tr>
	</tbody>
</table>
Table 1: Percent viability data of SH-SY5Y cells after glucose deprivation with or without APPJ treatment. ***P &#60;0.001 versus control; #P &#60;0.05 and ##P &#60;0.01 versus the glucose deprivation group (n = 3).

Watch this Scientific Journal Video about New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells at JoVE.com

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

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

The atmospheric pressure plasma jet (APPJ) has attracted the attention of many researchers from multiple disciplines in recent years because its emissions ...

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

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Environment

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, wound healing, root canal treatment, etc., forming a new research field named plasma medicine. Being a mixture of electrical, chemical, and biological reactive species, CAPs have shown their abilities to enhance nerve stem cells differentiation both in vitro and in vivo and are becoming a promising way for neurological disease treatment in the future. The much more exciting news is that using CAPs may realize one-step, and safely directed, differentiation of neural stem cells (NSCs) for tissue transportation. We demonstrate here the detailed experimental protocol of using a self-made CAP jet device to enhance NSC differentiation in C17.2-NSCs and primary rat neural stem cells, as well as observing the cell fate by inverted and fluorescence microscopy. With the help of immunofluorescence staining technology, we found both the NSCs showed an accelerated differential rate than the untreated group, and ~75% of the NSCs selectively differentiated into neurons, which are mainly mature, cholinergic, and motor neurons.

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

This protocol aims to provide detailed experimental steps of a cold atmospheric plasma treatment on neural stem cells and immunofluorescence detection for differentiation enhancement.

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Neuroscience

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Medicine

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in the efficacy and mechanism between the preparation combination and the component combination in order to explore the two herb combination strategy to treat injured PC12 cells. Cobalt chloride (CoCl2) combined with a glucose-free medium was employed to induce oxidative damage of PC12 cells. Then, the optimal combination of Astragalus mongholicus (Ast) and Erigeron breviscapus (Eri) injection was selected and combined following uniform design methods after screening their safe and effective concentration on PC12 cells. Further, the component combination screened comprises 10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid in two herbs. Then, MTT, Annexin V-FITC/PI, immunofluorescence, and Western blot analysis were used to evaluate the efficacy and mechanism of the preparation combination and the component combination on injured PC12 cells. The results showed that the optimal preparation combination for cell pro-survival was Ast injection and Eri capsule with a concentration of 6:1.8 (&#181;M). The component combination (10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid) was more effective than the preparation combination. Both combinations remarkably reduced apoptotic rate, the fluorescence intensity of caspase-3, and intracellular reactive oxygen species (ROS) level; meanwhile, they upregulated the expression levels of p-Akt/Akt, Bcl-2/Bax, and Nrf2. These effects were more evident in the component combination. In conclusion, both combinations can inhibit the injury induced by CoCl2 combined with a glucose-free medium on PC12 cells, thus promoting cell survival. However, the efficiency of the component combination over the preparation combination may be due to its stronger regulation of the PI3K/Akt/Nrf2 signaling pathway related to oxidative stress and apoptosis.

This protocol provides a combination strategy of two herbs to treat injured PC12 cells. The protocol provides a reference for optimizing the best application mode of traditional Chinese medicine (TCM).

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in ...

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

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