미국 심장 협회와 천 년 3 월에 의해 자금 지원.

이 프로토콜은 실험 동물의 사용을위한 건강 지침 국립 연구소에 의해 스탠포드 대학 abides에서 기관 애니멀 케어 및 사용위원회에 의해 승인되었습니다. 신생아 랫 히에 모델 <ol><li> 3-4 유도에 대한 %와 1-2 %; 포스트 나탈 일 (쿨러) 7 스프 라그 - 돌리 랫 새끼가 (찰스 리버 연구소, 주식 회사, 윌밍턴, MA) (Aerrane, 백스터, 디어 필드, IL isoflurane과 완벽하게 anesthetized 아르 유지 보수). </li><li> 작은 절개는 왼쪽 일반적인 경동맥의 동맥 (CCA)를 노출 (엑스트라 씬 아이리스 가위 # 14088-10, 좋은 과학 도구 병원, 포스터 시티, CA)를 목에 이루어집니다. </li><li> CCA는 5-0 실크 봉합사 (5-0 실크, Ethicon, 소머, 뉴저지)과 출혈도 잡았입니다. </li><li> 절개는 cyanoacrylate 접착제 (엘머스 제품 병원, 콜럼버스, OH)으로 닫힙니다. 절개는 테이프로 덮여 있으며, 마취가 중지됩니다. 새끼들은 댐에 반환하고 1-2시간에 대한 회복하다 수 있습니다. </li><li> 펍스 그러면 37 100 분 92% 질소와 균형을 8 % 산소를 포함하는 hypoxic 챔버 ° C.에 배치됩니다 8퍼센트 oxygen/92 %의 질소 가스 (Airgas, 새크라멘토, CA) 플라스틱 커버와 복 마우스 케이지에 튜브를 통해 흐름. 커버가 케이지에 맞게 만들어 직경 2cm의 구멍을 가지고있다 한 8 %의 산소 가스 탱크 및 다른 연결된 튜브를받을 수있는 중 가스 flowout 수 있습니다. 챔버는 물을 욕조에 배치되고 사용하기 전에 예열하고 그 내부에 온도계가 hypoxia의 시작하기 전에 37 ° C를 등록해야합니다. </li><li> hypoxia의 백분의 끝에, 새끼가 복구를 위해 자신의 댐을 다시 반환됩니다. </li><li> 24시간 hypoxia의 끝 후에 새끼는 isoflurane과 깊은 마취에 의해 euthanized 있습니다. 뇌 조직은 염분, pH를 7.4 (시그마 화학 CO, 세인트 루이스, MO) 버퍼 인산의 추위 1퍼센트 TTC의 해결책 다음, 차가운 정상적인 생리와 perfused 있습니다. TTC는 빛과 보호 보관해야합니다. </li><li> 두뇌가 제거하고 얼음에 1-2분, 4 코로나 섹션 간격 3mm (레벨 1-4)를 냉각하는 것은 bregma 1.8에 해당하는 opticchiasm과 infundibulum 수준에서 rostrally 시작 버렸어요의 ~ 2.0 mm 수준 성인 마우스 뇌. </li><li> 뇌 조각이 TTC 용액에 열중하고 4 % paraformaldehyde / PBS 하룻밤에 고정 다음, 8 분 ° C 37 incubated 수 있습니다. TTC는 빛을 구분하므로,이 시간 동안 빛으로부터 조각 거리 유지. </li><li> TTC 자국이 섹션은 스캔과 디지털됩니다. 경색 면적 (흠없는 지역), ipsilateral 북반구의 영역, 그리고 contralateral 북반구의 면적 : 이미지 J (NIH, 베데스다, MD), 다음은 각 수준에 대한 측정을 사용합니다. 1, 2, 3, 4 (경색 / ipsilateral 반구의 영역의 면적) 또는 : %는 슬라이스마다 지역으로 계산됩니다 infarcted. 경색의 퍼센트 볼륨의 합계 (A 1 + 2 + 3 + 4) * 3mm로 계산됩니다. 피질 또는 striatal 부상 분야도 별도로 비슷한 방식으로 계량하실 수 있습니다. </li></ol> 대표 결과 <img src="/files/ftp_upload/old/955_2008_9_2_2/955_Fig-01.png" alt="그림 1" width="660" height="190" /> 그림 1. TTC와 함께 대표 히에 코로나 레벨 1-4 얼룩. TTC 스테인드 코로나 섹션 경색의 영역 (흰색)을 보여줍니다.

<ol>
	<li>Rice, J. E., Vannucci, R. C., Brierley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+immaturity+on+hypoxic-ischemic+brain+damage+in+the+rat.">The influence of immaturity on hypoxic-ischemic brain damage in the rat.</a> Annals of neurology. 9 (2), 131-141 (1981).</li><li>Vannucci, R. C., Vannucci, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinatal+Hypoxic-Ischemic+Brain+Damage:+Evolution+of+an+Animal+Model.">Perinatal Hypoxic-Ischemic Brain Damage: Evolution of an Animal Model.</a> Dev Neurosci. 27 (2-4), 81-86 (2005).</li><li>Bona, E., Hagberg, H., Loberg, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+moderate+hypothermia+after+neonatal+hypoxia-ischemia:+short-+and+long-term+outcome.">Protective effects of moderate hypothermia after neonatal hypoxia-ischemia: short- and long-term outcome.</a> Pediatric research. 43 (6), 738-738 (1998).</li><li>Trescher, W. H., Ishiwa, S., Johnston, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hypothermia+and+hyperthermia+on+attentional+and+spatial+learning+deficits+following+neonatal+hypoxia-ischemic+insult+in+rats.">Effects of hypothermia and hyperthermia on attentional and spatial learning deficits following neonatal hypoxia-ischemic insult in rats.</a> Behavioural brain research. 151 (1-2), 209-209 (2004).</li><li>Mishima, K., Ikeda, T., Yoshikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+post-hypoxic-ischemic+hypothermia+markedly+delays+neonatal+brain+injury.">Brief post-hypoxic-ischemic hypothermia markedly delays neonatal brain injury.</a> Brain development. 19 (5), 326-326 (1997).</li><li>Young, R. S., Olenginski, T. P., Yagel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+graded+hypothermia+on+hypoxic-ischemic+brain+damage:+a+neuropathologic+study+in+the+neonatal+rat.">The effect of graded hypothermia on hypoxic-ischemic brain damage: a neuropathologic study in the neonatal rat.</a> Stroke; a journal of cerebral circulation. 14 (6), 929-929 (1983).</li><li>Bederson, J. B., Pitts, L. H., Germano, S. M., 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=Evaluation+of+2,3,5-triphenyltetrazolium+chloride+as+a+stain+for+detection+and+quantification+of+experimental+cerebral+infarction+in+rats.">Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats.</a> Stroke; a journal of cerebral circulation. 17 (6), 1304-1304 (1986).</li><li>Cai, J., Kang, Z., Liu, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+therapy+reduces+apoptosis+in+neonatal+hypoxia-ischemia+rat+model.">Hydrogen therapy reduces apoptosis in neonatal hypoxia-ischemia rat model.</a> Neuroscience letters. 441 (2), 167-167 (2008).</li><li>Grow, J., Barks, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+hypoxic-ischemic+cerebral+injury+in+the+term+infant:+current+concepts.">Pathogenesis of hypoxic-ischemic cerebral injury in the term infant: current concepts.</a> Clinics in perinatology. 29 (4), 585-585 (2002).</li><li>Aden, U., Halldner, L., Lagercrantz , 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=Aggravated+brain+damage+after+hypoxic+ischemia+in+immature+adenosine+A2A+knockout+mice.">Aggravated brain damage after hypoxic ischemia in immature adenosine A2A knockout mice.</a> Stroke; a journal of cerebral circulation. 34 (3), 739-739 (2003).</li></ol>

설치류 출생 후의 히에 모델 recapitulates 대뇌 hypoxia은 신생아에서 페리 - 산후 기간 동안 발생하는 설치 모델입니다. 광범위한 histological 및 immunocytochemical 특성화 연구 칠일 오래된 설치류은 세번째 임신 인간 태아의 7 모델 유사한 뇌의 성숙을 갖고 증명하고있다. 쥐 히에 모델은 저체온증과 같은 개입은 3 확인되었습니다 페리 - 나탈 hypoxia 2의 뇌 손상의 메커니즘을 이해하기위한 매우 유?...

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		<th>Material Name</th>
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<td>Sprague-Dawley Rat Pups</td>
<td>Animal</td>
<td>Charles River Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Isoflurane</td>
<td>Surgery</td>
<td>Aerrane,Baxter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>8% Oxygen/ 92% Nitrogen Gas</td>
<td>Surgery</td>
<td>Airgas</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2,3,5-triphenyl tetrazolium chloride </td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T8877</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline (PBS) pH 7.4 </td>
<td>Reagent</td>
<td>Gibco/Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Paraformaldehyde</td>
<td>Reagent</td>
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the hypoxic ischemic encephalopathy model of perinatal ischemia

Hypoxic - 허혈성 뇌증 (히에)는 출생시 질식 발생 체계의 결과이다. 히에과 neonates의 25 %는 정신 지체, 뇌성 마비 및 간질을 포함하여 심각한 및 영구 neuropsychological sequelae을 개발합니다. 히에의 결과는 히에와 신생아의 뇌 손상을 줄이기 위해 치료 전략을 파악하고 개발하는 것이 중요하고, 파괴 및 영구 있습니다. 이를 위해, hypoxic - 허혈성 뇌 손상에 대한 신생아 쥐 모델이 인간의 조건을 모델로 개발되었습니다. 히에 모델 처음 Vannucci 외 1 확인되었으며 이후 광범위하게 perinatal hypoxia - 국소 빈혈이로 인한 뇌 손상의 메커니즘을 파악하고 잠재적인 치료 중재에게 3,4를 테스트하는 데 사용되었습니다. 히에 모델은 두 단계 프로세스입니다 hypoxic 환경에 노출 다음 왼쪽 일반적인 경동맥의 결합을 포함한다. 출혈도 잡았 경동맥에 ipsilateral 반구의 대뇌 혈류 (CBF)이 있기 때문에 윌리스의 원을 통해 부수적인 혈류의 감소하지 않고 있지만 낮은 산소 긴장과 ipsilateral 북반구에서 CBF가 크게 감소하고 일방적 허혈성 부상 결과 . 허혈성 뇌 조직을 얼룩 및 식별 2,3,5 - triphenyltetrazolium 염화물 (TTC)의 사용은 원래 쥐 대뇌 국소 빈혈 5 성인 모델 개발되었고, 최대 72 초기 시간 지점에서 뇌성 infarctin의 범위를 평가하는 데 사용됩니다 허혈성 이벤트 6 시간 이후. 이 비디오에서는, 우리는 출생 후의 쥐 두뇌에있는 hypoxic - 허혈성 손상 모델 및 TTC의 얼룩을 사용하여 경색 크기의 평가를 보여줍니다.

Watch this Scientific Journal Video about The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia at JoVE.com

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

hypoxic - 허혈성 뇌 손상에 대한 출생 후의 쥐 모델은 인간의 신생아 hypoxic 허혈성 뇌증 (히에)의 잘 설립 모델입니다. 이 문서에서는, 우리는 포스트 나탈 쥐 새끼의 히에의 모델을 설명합니다.

Perinatal 국소 빈혈의 Hypoxic 허혈성 뇌증 모델

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

the-hypoxic-ischemic-encephalopathy-model-of-perinatal-ischemia

Research

JoVE Journal

Biology

35.5K 조회수.  Stanford University School of Medicine. hypoxic - 허혈성 뇌 손상에 대한 출생 후의 쥐 모델은 인간의 신생아 hypoxic 허혈성 뇌증 (히에)의 잘 설립 모델입니다. 이 문서에서는, 우리는 포스트 나탈 쥐 새끼의 히에의 모델을 설명합니다.

비디오: The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

컴퓨터가 만든 동물 모델 자극

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

연구

생물학

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Hindlimb 국소 빈혈의 Murine 모델

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

고환 세균 세포의 3차원 크로마틴 아키텍처를 보존하는 슬라이드 준비 방법

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

허혈 / 재관류에 대한 산소 결핍 - 기아 모델<em&gt; C. 엘레</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca2+ sensitivity affect intracellular Ca2+ homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na+, Ca2+ and K+ channels and the Na+-Ca2+ exchanger) and intracellular Ca2+ handling proteins (e.g., ryanodine receptors and Ca2+-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca2+ changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca2+ sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca2+ sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca2+ level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca2+ sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca2+ sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca2+ handling, and myofilament Ca2+ sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

Myofilament 칼슘의 평가<sup&gt; 2 +</sup&gt; 감도 근본적인 심장 여진 수축 커플 링

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

Hypoxic 조건 하에서 경작 하는 세포에서 내 생 적인 핵 단백질을 사용 하 여 공동 immunoprecipitation 분석 결과

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

ApoE에서 뒷다리 허혈의 변형 된 수술 모델-/- 소형 절개를 사용하여 마우스

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells (ECs) can protect cardiomyocytes (CMs) from reperfusion injury through cell-cell interactions. Co-cultures can help investigate the role of cell-cell interactions. A mixed co-culture is the simplest approach but is limited as isolated treatments and downstream analyses of single cell types are not feasible. To investigate whether ECs can dose-dependently attenuate CM cell damage and whether this protection can be further optimized by varying the contact distance between the two cell lines, we used Mouse Primary Coronary Artery Endothelial Cells and Adult Mouse Cardiomyocytes to test three types of cell culture inserts which varied in their inter-cell layer distance at 0.5, 1.0, and 2.0 mm, respectively. In CMs-only, cellular injury as assessed by lactate dehydrogenase (LDH) release increased significantly during hypoxia and further upon reoxygenation when the distance was 2.0 mm compared to 0.5 and 1.0 mm. When ECs and CMs were in nearly direct contact (0.5 mm), there was only a mild attenuation of the reoxygenation injury of CMs following hypoxia. This attenuation was significantly increased when the spatial distance was 1.0 mm. With 2.0 mm distance, ECs attenuated CM injury during both hypoxia and hypoxia/reoxygenation, indicating that sufficient culture distancing is necessary for ECs to crosstalk with CMs, so that secreted signal molecules can circulate and fully stimulate protective pathways. Our findings suggest, for the first time, that optimizing the EC/CM co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of ECs in CM-protection against simulated ischemia/reperfusion injury. The goal of this report is to provide a step-by-step approach for investigators to use this important model to their advantage.

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro

Spatial distance is a key parameter in assessing hypoxia/reoxygenation injury in a co-culture model of separate endothelial and cardiomyocyte cell layers, suggesting, for the first time, that optimizing the co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of endothelial cells in cardiomyocyte protection.

체외에서 심장 허혈/재관류를 모방하기 위한 세포 공동 배양 모델 개발

Ischemic heart disease is the leading cause of death and disability worldwide. Reperfusion causes additional injury beyond ischemia. Endothelial cells ...

Generation of Hook Ischemia-Reperfusion Model using a Three-Day Developing Chick Embryo

Ischemia and reperfusion (I/R) disorders, such as myocardial infarction, stroke, and peripheral vascular disease, are a few of the leading causes of illness and death. Many in vitro and in vivo models are currently available for studying the I/R mechanism in disease or damaged tissues. However, to date, no in ovo I/R model has been reported, which would allow for a better understanding of I/R mechanisms and faster drug screening. This paper describes I/R modeling using a spinal needle customized hook in a 3-day chick embryo to understand I/R development and treatment mechanisms. Our model can be used to investigate anomalies at the DNA, RNA, and protein levels. This method is simple, quick, and inexpensive. The current model can be used independently or in conjunction with existing in vitro and in vivo I/R models.

This paper describes ischemia-reperfusion (I/R) modeling in a 3-day chick embryo using a spinal needle customized hook to better understand I/R development and treatment. This model is simple, quick, and inexpensive.

3일간의 병아리 배아를 이용한 후크 허혈-재관류 모델의 생성

Ischemia and reperfusion (I/R) disorders, such as myocardial infarction, stroke, and peripheral vascular disease, are a few of the leading causes of ...