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

这项工作得到了北京神经外科研究所 (2014-11)、中国国家自然科学基金 (Nos. 11475019 和 81271286) 和北京自然科学基金会 (No. 7152027) 的创新基金会的支持。

1. APPJ 设备的准备 警告: 请在使用前查阅所有相关的材料安全数据表 (MSDS)。在执行所有的和 #160 时, 请使用适当的安全做法; 实验, 包括使用通风罩和个人防护设备 (安全眼镜、防护手套、实验室大衣、 等 )。该协议要求标准的细胞处理技术 (杀菌, 细胞恢复, 细胞传代, 细胞冻结, 细胞染色, 等 ). 
<ol> <li> 选择一个内径为 1 mm 的石英管, 外部直径为 3 mm. 使用抛光箔平滑两端的截面. </li>
	<li> 使用直径为1.0 毫米的镀镍不锈钢针作为高压电极。研磨其尖端的曲率半径为 0.05 mm. </li>
	<li> 将铝箔 (2 毫米宽) 环绕在石英管的1厘米处, 从石英管喷嘴。将不锈钢针点从铝箔的另一端固定在1厘米处。使用铝箔环作为低压电极. </li>
</ol> 2。获取 jet <ol> <li> 提供交流信号, 将高压功率放大器连接到用作电源的功能信号发生器。将所施加电压的波形记录到高压电极上, 将高压探头的一端连接到数字示波器, 并将另一端连接至电源。为了保护电路, 使用 2 k 和 #8486; 电阻作为保护电阻。连接电路, 如 图 2 所示. 
	注意: 请勿触摸高压线路. </li>
	<li> 在石英管上连续通过氦 (体积分数, 99.999%), 控制气体流速在稳定的1.4 标准升每分钟 (解运). 
	注意: 在处理单元格区域性之前, 我们不使用筛选器。实验中使用的氦体积分数为 99.999%, 大多数微生物在这种情况下无法生存. </li>
	<li> 打开示波器、信号发生器和高压功率放大器的电源。将频率调节旋钮旋转5赫。将电压逐渐增加到 6 kV 的峰值. 
	注: 当在针中施加的电压的峰值约为6伏时, 该射流足够长 (约3厘米). </li>
</ol> 3。SH-SY5Y 细胞的制备 <ol> <li> 在一个25厘米 2 SH-SY5Y 中, 在 RPMI 1640 培养基中, 以15% 胎小牛血清 (FBS) 为基质, 培养出具有神经母细胞瘤的人的神经干细胞。在含有 5% CO 2 和37和 #176 的95% 空气的湿恒温培养箱中维护电池; C. </li>
	<li> 当细胞达到85% 汇流时, 仔细吸取培养基, 并在细胞中加入1毫升0.25% 胰蛋白酶 + 0.1% EDTA. </li>
	<li> 十五年代在室温下孵化后, 小心地吸取胰蛋白酶, 并加入2毫升 RPMI 1640, 内含 15% FBS 以中和. </li>
	<li> 轻轻吸管向上和向下, 冲洗井底, 直到 SH-SY5Y 单层完全分离. </li>
	<li> 通过例计数单元格并将单元格浓度调整为 2 x 10 5 单元格/mL, 方法是添加 RPMI 1640 中等 +15% FBS, 然后转移100和 #181; L 对96孔板的每一个井进行细胞悬浮. </li>
	<li> 在 APPJ 治疗前允许细胞在细胞孵化器中附加12小时. </li>
</ol> 4。APPJ 处理 SH-SY5Y <ol> <li> 调整石英管喷嘴与平台之间的距离, 将96孔板放置在3厘米处. 确保光束能够触及培养基的表面. 
	注意: 距离不是从盘子的底部测量的。它是第一次调整到3厘米之间的喷嘴的石英管和平台用于放置96井板. </li>
	<li> 在 APPJ 处理前, 除控制井外, 将每井的培养基改变为 RPMI 1640, 无葡萄糖培养基. </li>
	<li> 将板放在 APPJ 喷嘴下, 确保射流可以垂直射入每个井. </li>
	<li> 在单独的井中处理单元格的 APPJ 为 0 s、1 s、2 s、4 s、8 s 和十二年代. 
	注意: APPJ 是由电离氦生成的 ( 图 1 )。通过 4 s 和 8 s 氦流来处理因葡萄糖剥夺而损伤的细胞, 以消除氦对细胞的影响。所有的治疗方法都要进行三份. </li>
</ol> 5。细胞活力测定方法 注意: 请不要在此步骤中更改介质. 
<ol> <li> 在 APPJ 处理后, 在细胞孵化器中孵育1小时的细胞. </li>
	<li> 添加10和 #181; 单元格计数 Kit-8 (CCK-8) 解决方案. </li>
	<li> 孵育37和 #176 的细胞; C 为 4 h. 
	注: SH-SY5Y 细胞系对葡萄糖剥夺情况敏感, 12 。1小时葡萄糖缺乏后, 细胞存活率降低到近 50%, 这是药效学研究的最佳细胞生存条件。CCK-8 对细胞没有细胞毒性, 细胞与 CCK-8 试剂在 APPJ 治疗后的葡萄糖剥夺条件下再孵育4小时, 以检查细胞的活力。在8小时葡萄糖剥夺和 APPJ 治疗后, APPJ 的保护作用明显降低, 因为长期的葡萄糖剥夺导致 SH-SY5Y 细胞的严重损害。在 24 h 葡萄糖剥夺后没有活细胞的证据 11 . </li>
	<li> 使用微读取器测量 450 nm 的吸光度. </li>
</ol>

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SH-SY5Y 细胞是人神经母细胞瘤衍生的干细胞系, 并广泛应用作为一个适当的细胞模型, 为体外神经系统毒性或脑保护的研究12。SH-SY5Y 细胞系对葡萄糖缺乏状态敏感。1小时葡萄糖缺乏后, 细胞活力降低到近 50%, 这是研究药效学的最佳细胞生存条件。此外, CCK-8 试剂对细胞没有细胞毒性, 在 APPJ 治疗后的葡萄糖剥夺条件下, CCK-8 试剂孵育4小时, 以检测细胞的活力。在目前的研究中...

在正常的生理条件下, 成年大脑几乎完全使用葡萄糖作为能量代谢的基质。人脑只占身体重量的 2%, 但在身体的总葡萄糖中消耗了大约25% 的1。众所周知, 葡萄糖代谢失调是缺血性中风和各种神经退行性疾病 (包括阿尔茨海默氏病 (AD)、亨廷顿氏病 (HD) 和帕金森病 (PD) 的主要病理变化之一2,3。葡萄糖的缺乏和糖的摄取受损或氧化磷酸化可以直接影响 ATP 的生产, 并进一步诱发神经细胞死亡, 这可能会增加神经元功能障碍的风险, 这表明维持细胞活力或延缓葡萄糖剥夺后的细胞损伤可能是治疗这些疾病的合理方法。通过葡萄糖调制对神经保护作用的研究, 着重于抗炎剂、离子通道调制器、自由基清道夫、神经营养因子、等。然而, 这些神经保护方法的翻译从长凳到临床实践没有成功4。
常压等离子体射流 (APPJs) 是一种新型的大气低温气体放电技术, 近年来引起了众多学科研究者的关注。APPJs 已用于几十年的各种生物医学应用, 如癌症细胞治疗, 细菌灭活, 凝血, 伤口愈合, 口服药物,等5,6, 由于其多种类型的排放活性氮种类 (RNS) 和活性氧 (ROS) (图 1)7。以前的等离子体生物医学应用主要集中在细菌、细胞和组织的氧化和/或 nitrative 压力上8。然而, APPJ 也可能是一把 "双刃剑", 因为 RNS 和 ROS 是重要的细胞内信号分子与许多生理学和病理生理学过程9。一氧化二氮 (no) 控制着广泛的生物过程, 在人体, 特别是中枢神经系统中扮演着双重角色。低水平的 NO 已经显示了他们的神经保护活动的体外和在体内通过多个信号通路10。我们先前的研究首次报告, 氦 APPJ 诱导 NO 的产生参与了 APPJ 抗氧化应激损伤的神经保护作用11。然而, 未报告 APPJs 对其他损伤的影响。因此, 本研究的目的是提供一个体外治疗方案, 关于氦 APPJ 在葡萄糖剥夺诱导的 SH-SY5Y 细胞损伤中的应用。与以往的研究不同, 我们的协议使用低剂量等离子治疗的神经保护应用, 而不会造成过多的血浆损伤, 表明 APPJ 治疗可能被用作一种新型的 "无供药物"为将来的研究, 甚至临床翻译。本协议也被建议作为细胞应用的其他细胞类型有不同的损伤, 但 APPJ 治疗条件应调整和治疗剂量必须相对较低。

<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

大气压力等离子体射流 (APPJ) 近年来引起了许多研究人员的关注, 因为它的排放包括多种类型的活性氮 (RNS) 和活性氧物种 (ROS)。我们先前的研究表明 APPJ 对氧化应激损伤的细胞作用。本研究的目的是提供一个详细的体外治疗方案, 关于神经保护应用的氦 APPJs 对葡萄糖剥夺诱导损伤 SH-SY5Y 细胞。SH-SY5Y 的人神经母细胞瘤的干细胞系维持在 RPMI 1640 培养基补充15% 胎小牛血清。在 APPJ 治疗前, 培养基被转化为 RPMI 1640, 无葡萄糖。在一个细胞培养器 1 h 孵化后, 细胞活力是确定使用细胞计数套件8。结果表明, 与葡萄糖剥夺组相比, APPJ 治疗的细胞表现出明显增加的细胞活力, 以剂量依赖性的方式, 8 s/良好观察作为最佳剂量。同时, 氦流动对葡萄糖剥夺诱导的细胞损伤无影响。我们的研究结果表明, APPJ 可能被用作治疗中枢神经系统中与葡萄糖缺乏相关的疾病的一种方法。本协议也可作为其他不同损伤细胞的细胞应用, 但细胞培养和 APPJ 处理条件应重新调整, 治疗剂量必须相对较低。

数据被表示为至少三独立实验的平均± SD。采用方差分析法对组结果进行方差统计。所有的分析都是用统计分析软件棱镜和 p 和 #60 进行的; 0.05 是统计意义的门槛。
CCK-8 孵育4小时后测定细胞活力。如图 3所示, 与对照组相比, 葡萄糖剥夺使 SH-SY5Y 细胞的生存能力降低到44.1 ± 2.6% (通常在 RPMI 1640 介质中含有 15% FBS)。APPJ 治疗显著增加了细胞生存能力的剂量?...

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

常压等离子体射流作为神经保护剂抗葡萄糖剥夺诱导 SH-SY5Y 细胞损伤的新应用

小剂量大气压等离子体治疗糖剥夺性 SH-SY5Y 损伤的神经保护应用协议

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

new-application-an-atmospheric-pressure-plasma-jet-as-neuro

Research

JoVE Journal

Engineering

9.0K 次观看.  Capital Medical University. 小剂量大气压等离子体治疗糖剥夺性 SH-SY5Y 损伤的神经保护应用协议

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

Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). We demonstrate in this video article how blast TBI-relevant impulsive pressurization is applied to the neuronal cells in vitro. This is achieved by using well-controlled pressure pulse created by a specialized Kolsky bar device, with complete pressure history within the cell pressurization chamber recorded. Pressurized neuronal cells are inspected immediately after pressurization, or further incubated to examine the long-term effects of impulsive pressurization on neurite/axonal outgrowth, neuronal gene expression, apoptosis, etc. We observed that impulsive pressurization at about 2 MPa induces distinct neurite loss relative to unpressurized cells. Our technique provides a novel method to investigate the molecular/cellular mechanisms of blast TBI, via impulsive pressurization of brain cells at well-controlled pressure magnitude and duration.

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.

创伤性脑损伤研究神经细胞的脉冲增压

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). ...

科研

生物工程

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.

A high-throughput cold physical plasma treatment protocol of liquids and cells is shown. It involves setting up different feed gas compositions for plasma ignition, measuring the plasma&#39;s emission spectra, and the subsequent analysis of liquids and cellular activity after plasma treatment.

等离子体医学基础研究--从液体到细胞的吞吐量方法

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

环境科学

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.

一步冷常压等离子体体外处理神经干细胞分化

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

工程学

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.

神经元共培养与单细胞精度的制备

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

神经科学

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.

缺氧缺糖再给氧作为<em&gt;在体外</em&gt;缺血再灌注损伤模型的研究血脑屏障功能障碍

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

医学

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.

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

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.

实验模型研究酸性后处理对脑缺血的神经保护作用

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.

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

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.

一种新的脑外伤体损伤模型

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).

探索两种草药组合策略治疗受损PC12细胞

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