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

이 작품은 베이징 신경외과 학회 (2014-11), 중국 (No. 11475019와 81271286)의 국가 자연과학 기초 및 베이징 자연 과학 재단 (No. 7152027)의 혁신 기금에 의해 지원 되었다.

1. APPJ 장치의 준비 주의: 사용 하기 전에 모든 관련 물질 안전 데이터 시트 (MSDS)를 참조 하십시오. 증기 두건 및 개인 보호 장비 (보호 안경, 보호 장갑, 랩 코트, 등)의 사용을 포함 하 여 모든 실험을 수행할 때 적절 한 안전 관행을 사용 하십시오. 프로토콜 처리 기술 (살 균, 세포 복구, 셀 뿌리고, 세포 동결, 셀 얼룩, 등) 표준 셀 필요.
<ol><li>1 m m 내부 직경 석 영 튜브를 선택 하 고 외부 직경 3 m m.의 부드러운 연마 호 일을 사용 하 여 양 끝 횡단면.</li>
	<li>1.0 m m의 직경을 가진 니켈 도금된 스테인리스 바늘을 사용 하 여 고전압 전극으로. 0.05 m m의 곡률 반경에 팁을 갈기.</li>
	<li>포장 알루미늄 호 일 (폭에서 2 m m) 주위에 1 cm에서 석 영 튜브 석 영 튜브에서 노즐. 알루미늄 호 일의 다른 쪽 끝에서 1 cm에서 스테인리스 스틸 바늘 포인트를 수정. 알루미늄 포 일 반지를 사용 하 여 낮은 전압 전극으로.</li>
</ol> 2. 제트기의 수집 <ol><li>는 AC 신호를 제공 하는 기능 신호 발생기 전원 공급 장치는 고압 전력 증폭기 연결. 높은 전압 전극에 인가 전압의 파형을 기록, 디지털 오실로스코프에 고전압 프로브의 한쪽 끝을 연결 하 고 전원 공급 장치를 다른 쪽 끝을 연결 합니다. 회로 보호 하려면 2 k &#8486; 보호 저항으로 저항. 그림 2와 같이 회로 연결. 
	주의: 높은 전압 라인을 만지지 마십시오.</li>
	<li>지속적으로 석 영 튜브를 통해 헬륨 (볼륨 분수, 99.999%)을 통과 하 고 안정적인 1.4 표준 리터 당 분 (SLM)에서 가스 유량 제어. 
	참고: 우리가 사용 하지 마십시오 필터 셀 문화를 치료 하기 전에. 실험에 사용 하는 헬륨의 볼륨 분수 99.999% 이며 대부분 미생물이이 조건 하에서 살 수 없다.</li>
	<li>는 오실로스코프, 신호 발생기, 고압 전력 증폭기의 전원을 켭니다. 5 kHz에 주파수 조절 손잡이 회전 합니다. 점차적으로 증가 6의 피크-피크 값 전압 kV. 
	참고: 제트기는 충분히 (약 3cm) 때 바늘에 적용 하는 전압의 피크-피크 값은 약 6 kV.</li>
</ol> 3. SH SY5Y 세포의 준비 <ol><li>RPMI 1640에서 25 cm 2 플라스 크에 성장 SH-SY5Y 인간 신경 파생 셀 라인 매체 15% 태아 종 아리 혈 청 (FBS)와 보충. 포함 된 37에서 5% CO 2와 95% 공기 습도 인큐베이터에 셀 유지 ° c.</li>
	<li>때 셀 85% 합류에 도달, 신중 하 게 문화 미디어를 발음 하 고 셀에 1 mL 0.25 %trypsin + 0.1 %EDTA 추가.</li>
	<li>후 15의 보육 실내 온도에, 신중 하 게는 트립 신을 발음 하 고 추가 2 mL 15% 포함 된 RPMI 1640 무력화 FBS.</li>
	<li>부드럽게 아래로, 플라스틱 SH SY5Y 단층 완전히 분리 될 때까지, 우물의 바닥 세척.</li>
	<li>는 hemocytometer를 사용 하 여 셀을 계산 하 고 RPMI 1640 중간 + 15를 추가 하 여 2 x 10 5 셀/mL로 세포 농도 조정 %FBS, 그리고 96 잘 접시의 각 음에 세포 현 탁 액의 전송 100 µ L.</li>
	<li>APPJ 치료 전에 셀 인큐베이터에 12 h에 대 한 연결을 세포 허용.</li>
</ol> 4. SH-SY5Y의 APPJ 치료 <ol><li>조정 석 영의 노즐 사이의 거리 튜브와 96 잘 접시 배치 될 위치에 3 cm. 플랫폼 빔 문화 매체의 표면을 만질 수 있는 보장. 
	참고: 거리는 플레이트의 하단에서 측정 하지 됩니다. 그것은 먼저 석 영 튜브의 노즐 및 96 잘 접시를 사용 하는 플랫폼 간의 3 cm 조정.</li>
	<li>APPJ 치료 하기 전에 변경 제어 웰 스 제외 하 고 각 잘 배양 RPMI 1640 포도 당 매체 없이.</li>
	<li>APPJ 노즐 아래 접시 놓고 제트기 각 우물에 수직으로 쏠 수 있다 확인 하십시오.</li>
	<li>0 APPJ와 함께 별도 우물에서 치료 세포 s, 1의 2 s, 4 s, 8, 및 12 s. 
	참고: APPJ ( 그림 1) 헬륨 이온화에 의해 생성 됩니다. 4 세포 포도 당 부족으로 부상 처리 됩니다 셀에 헬륨의 영향을 제거 하는 8 s 헬륨 흐름과 s. 모든 치료는 3 중에서 수행 되어야 합니다.</li>
</ol> 5. 세포 생존 능력 분석 결과 참고:이 단계에서 매체를 변경 하지 마십시오.
<ol><li>APPJ 치료 후 세포 배양 기에서 1 시간에 대 한 셀을 품 어.</li>
	<li>각 잘 셀 계산 키트-8 (CCK-8) 솔루션의 추가 10 µ L.</li>
	<li>품 어 4 h. 위해 37 ° C에서 셀 
	참고: SH-SY5Y 셀 라인은 포도 당 부족 조건 12에 민감합니다. 세포 생존 능력은 pharmacodynamics 연구에 대 한 최적의 세포 생존 조건이 1 시간 포도 당 부족 후 거의 50% 감소 합니다. CCK-8 셀에 아무 세포 독성 있으며 셀은 APPJ 치료 세포 생존 능력을 확인 후 CCK-8 시 포도 당 부족 조건에서 다른 4 h와 인 큐베이 팅. 8 h 포도 당 부족 APPJ 처리 후에, APPJ의 보호 효과 때문에 포도 당 부족의 긴 기간 SH SY5Y 세포의 심한 손상을 주도 크게 감소 되었다. 24 시간 포도 당 부족 11 후 살아있는 세포의 증거가 없었다.</li>
	<li>450에서 흡 광도 측정 microplate 리더 nm.</li>
</ol>

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SH SY5Y 세포는 인간의 신경 파생 셀 라인 고 neurotoxicity 또는 neuroprotection12에 생체 외에서 연구에 대 한 적절 한 셀 모델로 서 널리 이용 된다. SH SY5Y 셀 라인 포도 당 부족 조건에 민감 했다. 세포 생존 능력은 pharmacodynamics의 연구에 대 한 최적의 세포 생존 조건이 1 시간 포도 당 부족 후 거의 50% 감소. 또한, CCK-8 시 약은 아무 세포 독성 세포 및 셀 APPJ 치료 세포 생존 능력을 확...

성인 두뇌는 거의 독점적으로 정상적인 생리 적인 조건 하에서 에너지 대사에 대 한 기판으로 포도 당을 사용합니다. 인간의 뇌는 몸 무게의 2%만 하지만 바디1내 총 포도의 약 25%를 소비 한다. 그것은 잘 문서화 되어있다 그 포도 당 대사 장애는 주요 학적 변화 중 허 혈 성 뇌졸중 및 Alzheimer의 질병 (광고), 파 킨 슨 병 (PD) , 헌팅턴의 질병 (HD) 등 다양 한 신경 퇴행 성 질환 2,3. ATP 생산에 직접 영향을 줄 수 및 더 신경 부전, 제안 그 유지 세포 생존 능력의 위험을 증가 시킬 수 있습니다 신경 세포 죽음을 유도 포도 당 및 손상된 포도 당 통풍 관 또는 산화 인 산화의 부족 또는 지연 셀 부상 후 포도 당 부족이이 질병 치료에 대 한 합리적인 접근 될 수 있습니다. 포도 당 변조, 항 염증 제 대리인, 이온 채널 변조기, 자유 래 디 칼 청소부, neurotrophic 요인에 초점을 통해 신경 보호 효과의 조사 등 관심 되었습니다. 그러나, 임상 연습에 벤치에서 이러한 신경 접근의 번역 성공4되지 않았습니다.
대기압 플라즈마 제트 (APPJs)는 최근 몇 년 동안에서 여러 분야에서 많은 연구자의 관심을 끌고있다 대기 저온 가스 방전 기술의 새로운 종류. APPJs 암 세포 치료, 세균 비활성화, 혈액 응고, 상처 치유, 구두 약, 등5,6, 여러 종류의 그것의 방출 때문에 다양 한 생물 의학 응용 프로그램에 수십 년 동안 사용 되었습니다. 반응 질소 종 (RNS) 및 반응성 산소 종 (선생님) (그림 1)의7. 이전 플라즈마 바이오 의학 응용 프로그램은 주로 박테리아, 세포, 조직과8에 산화 그리고/또한 nitrative 스트레스에 집중 했다. 그러나, RNS과 선생님은 많은 생리 적 및 병 태 생리 과정9와 관련 된 중요 한 세포내 신호 분자는 APPJ 또한 "양날의 검" 될 수 있습니다. 질소 산화물 (NO) 생물학 과정의 넓은 범위를 제어 하 고 인체, 특히 중앙 신경 시스템 (CNS)에 이중 역할을 한다. 낮은 레벨의 그들의 신경 활동 모두 나타났습니다 생체 외에서 그리고 vivo에서 여러 신호 경로10통해. 우리의 이전 연구는 그 헬륨에 처음 보고 된 APPJ 유도 아니 생산 산화 스트레스 유발 부상11에 대 한 APPJ의 신경 보호 효과에 참여 했다. 그러나, 다른 상해에 APPJs의 효과 되지 보고 되었습니다. 따라서, 현재 연구의 목표 SH SY5Y 세포에 포도 당 부족 유발 부상에는 체 외에서 치료 프로토콜 헬륨 APPJ의 신경 보호 응용 프로그램에 대 한 제공 하는 것입니다. 이전 연구에서 다른, 우리의 프로토콜 부상의 과도 한 플라즈마 유도, APPJ 치료 잠재적으로 소설 "기증자 약으로 사용 될 수를 나타내는 결과 없이 신경 보호 응용 프로그램에 대 한 낮은 복용량 플라즈마 처리를 사용 "미래 연구 및 임상 번역에도. 이 프로토콜은 또한 다른 장애가 있는 다른 세포 유형 대 한 cytoprotective 응용 프로그램으로 사용할 수 제안 하지만 APPJ 치료 조건 다시 조정 해야 하며 치료 복용량 상대적으로 낮은 되어야 합니다.

<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

대기압 플라즈마 제트 (APPJ)는 자사의 배출량 반응성 질소 종 (RNS) 및 반응성 산소 종 (선생님)의 여러 종류를 포함 하기 때문에 최근 몇 년 동안에서 여러 분야에서 많은 연구자의 주목을 받고 있다. 우리의 이전 연구는 산화 스트레스 유발 부상에 대 한 APPJ의 cytoprotective 효과 보이고 있다. 현재 연구의 목표는 SH-SY5Y 세포에 포도 당 부족 유발 부상에 헬륨 APPJs의 신경 보호 응용 프로그램에 관한 자세한 생체 외에서 치료 프로토콜을 제공 하는입니다. SH SY5Y 인간 신경 파생 셀 라인 보완 15% 태아 종 아리 혈 청 RPMI 1640 매체에 유지 되었다. 문화 매체 APPJ 치료 전에 포도 당 없이 RPMI 1640 다음 변경 되었습니다. 셀 인큐베이터에서 1 h 부 화, 후 세포 생존 능력 셀 계산 키트 8를 사용 하 여 결정 했다. 결과, 보여주었다 포도 당 부족 그룹에 비해, APPJ로 치료 하는 세포 전시 크게 증가 세포 생존 복용량 의존 방식에서 8 s/잘 최적의 복용량으로 관찰. 한편, 헬륨 흐름이 했다 포도 당 부족-유도 된 세포 손상에 영향을 주지 않습니다. 우리의 결과 APPJ 잠재적으로 포도 당 부족에 관련 된 중추 신 경계에에서 질환에 대 한 치료 방법으로 사용 될 수 표시. 이 프로토콜도 사용할 수 cytoprotective 응용 프로그램으로 다른 장애가 있는 다른 셀에 대 한 하지만 세포 배양 및 APPJ 치료 조건을 재조정 해야, 그리고 치료 복용량은 상대적으로 낮은 되어야 합니다.

데이터는 적어도 3 개의 독립적인 실험의 평균 ± SD로 표현 됩니다. 그룹 결과 ANOVA를 사용 하 여 분산에 대 한 분석 했다. 모든 분석은 통계 분석 소프트웨어 프리즘 및 p를 사용 하 여 수행 했다 &#60; 0.05 통계적 의미에 대 한 임계값을 했다.
세포 생존 능력은 CCK-8 보육의 4 h 후 측정 했다. 그림 3에서 보듯이, 포도 당 부족 통제 그룹에 비해 44.1 ± 2.6%에 SH-SY5...

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

SH SY5Y 세포의 포도 당 부족 유발 부상에 대 한 신경 보호 요원으로 대기압 플라즈마 제트의 새로운 응용 프로그램

포도 당 부족 유발 SH-SY5Y 상해에 낮은 복용량 대기압 플라즈마 처리의 신경 보호 응용 프로그램에 대 한 프로토콜.

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 조회수.  Capital Medical University. 포도 당 부족 유발 SH-SY5Y 상해에 낮은 복용량 대기압 플라즈마 처리의 신경 보호 응용 프로그램에 대 한 프로토콜.

비디오: 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.

외상성 뇌 손상 연구를위한 Neuronal 셀의 충동 가압

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

연구

생체공학

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.

플라즈마 의학-세포 액체에서 처리량 접근 기초 연구

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

환경과학

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.

신경 줄기 세포 분화는 원스텝 차가운 대기 플라즈마 치료에서 체 외에 의해

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

공학

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.

단일 셀의 정밀도와의 연결을 공동 문화의 준비

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

신경과학

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.

같은 산소 포도당 박탈과 재산 소화<em&gt; 체외</em&gt; 허혈 - 재관류 손상 모델 공부 뇌 혈관 장벽 부전

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

의학

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.

사이토 반응성 및 확산에서 체 외에 허 혈 성 같은 조건 모니터링

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.

대뇌 허혈에 대한 산성 후행 조절의 신경 보호를 연구하기위한 실험 모델

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.

인간의 고속 스트레치 부상에 대 한 방법 유도 만능 줄기 세포 유래 신경 96 잘 형식

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.

모델을 새로운 시험관에 폭발 외상 성 뇌 손상의

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).

손상된 PC12 세포를 치료하기 위한 두 가지 허브 조합 전략 탐색

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