アメリカ心臓協会とダイムの月によって資金を供給。

このプロトコルは、実験動物の使用のための健康ガイドラインの国立研究所によってスタンフォード大学と遵守に動物実験用の委員会により承認された。 ラット新生HIEモデル<ol><li> 3から4誘導のための％とは1-2％、産後の日（PND）7のSprague - Dawleyの仔ラットは、（チャールズリバー研究所、（株）、ウィルミントン、MA）（Aerrane、バクスター、ディアフィールド、イリノイ州イソフルランで完全に麻酔するメンテナンス）。 </li><li>小切開は、左総頸動脈（CCA）を公開する（エクストラシンアイリスはさみ＃14088から10、ファイン科学ツール株式会社、フォスターシティ、カリフォルニア州）の首に行われます。 </li><li> CCAは、5から0絹縫合糸（5-0絹、エチコン、ゾマー、NJ）と連結する。 </li><li>切開は、シアノアクリレート系接着剤（エルマーズプロダクツ株式会社、コロンバス、オハイオ州）で閉じられます。切開は、テープで覆われていると麻酔が停止されます。子犬は、そのダムに戻り、1〜2時間のために静養に許可されています。 </li><li>子犬は、その後37℃で100分間、92％の窒素℃でバランス8％の酸素が含まれている低酸素室に置かれている8パーセントoxygen/92％の窒素ガス（Airgasの、サクラメント、カリフォルニア州）プラスチックカバーを装備したマウスのケージにチューブを経由して流れ。カバーがケージに合うように作られ、直径2cmの2つの穴を持っている、1つの8％の酸素ガスのタンクと別に接続されたチューブを受け取るためにそのうちにガスがflowoutすることができます。チャンバーは、水浴に配置され、使用前にウォームアップしていて、その内部に温度計は、低酸素の開始前に37℃を登録する必要があります。 </li><li>低酸素の100分の終わりに、子犬は​​回復のための彼らのダムに再び戻されます。 </li><li> 24時間低酸素状態の終了後、仔はイソフルランと深い麻酔により安楽死されています。脳組織は、生理食塩水、pH7.4（シグマケミカル共同、セントルイス、MO）リン酸緩衝の冷たい1パーセントTTCのソリューションに続く、冷生理食塩水で灌流される。 TTCは、光から保護しておく必要があります。 </li><li>脳を除去し、冷却した氷上で1〜2分、4つの冠状セクション離れて3ミリメートル（レベル1-4）のためにブレグマ1.8〜に対応する、opticchiasmと漏斗のレベルで吻方始め、カットされています〜2.0 mmのレベルを成体マウスの脳。 </li><li>脳スライスをTTC溶液に浸漬し、4％パラホルムアルデヒド/ PBSで一晩固定に続いて、8分間37℃でインキュベートする。 TTCは光に敏感なので、この時間の間に光からスライスを遠ざけること。 </li><li> TTC染色した切片はスキャンおよびデジタル化されています。使用してイメージJ（NIH、ベセスダ、MD）、以下はレベルごとに測定されています：梗塞の領域（未染色領域）、同側半球の面積、および対側半球の面積を。スライスごとのパーセント梗塞部位は次のように計算されます（同側半球の梗塞/地域の面積）または1、2、3、4。梗塞のパーセントの容積は、合計として計算される（A 1 + A 2 + A 3 + A 4）* 3ミリメートル。皮質や線条体損傷の領域も別々に同様の方法で定量することができる。 </li></ol>代表的な結果<img src="/files/ftp_upload/old/955_2008_9_2_2/955_Fig-01.png" alt="図1" width="660" height="190" /> 図1。 TTCと代表的なHIE冠状レベル1-4で染色。 TTC染色した冠状セクションでは、梗塞の領域（白）を示しています。

<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>

げっ歯類生後HIEモデルでは、反復する脳低酸素症は新生児の周囲出生期間に発生したことが確立されたモデルです。大規模な組織学的および免疫組織化学的特性の研究は、7日齢齧歯類は妊娠後期ヒトの胎児7をモデル化するために類似した脳の成熟度を持っていることを実証している。げっ歯類のHIEモデルでは、低体温などの介入が3を検証している周囲の出生低酸素症2、から脳損傷...

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		<th>Material Name</th>
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		<th>Comment</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>
<tr>
<td>Isoflurane</td>
<td>Surgery</td>
<td>Aerrane,Baxter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<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>
<td>Sigma-Aldrich</td>
<td>P6148</td>
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the hypoxic ischemic encephalopathy model of perinatal ischemia

低酸素性虚血性脳症（HIE）は、出生時に発生する全身性仮死の結果です。 HIEによる新生児の二五％は精神遅滞、脳性麻痺、てんかんを含め、厳しいと永続的な神経心理学的後遺症を開発する。 HIEの成果は、それが重要なHIEで新生児の脳損傷を軽減する治療戦略を特定し、開発すること、壊滅的と永続的です。そのために、低酸素性虚血性脳損傷のための新生児ラットモデルは、この人間の状態をモデル化するために開発されました。 HIEモデルは、最初バヌチら1によって検証され、以来広く周産期低酸素虚血2から生じる脳損傷のメカニズムを識別し、潜在的な治療介入の3,4をテストするために使用されています。 HIEモデルでは、2段階のプロセスであり、低酸素環境への曝露に続いて左総頸動脈の結紮を伴います。連結された頸動脈に同側半球の脳血流量（CBF）は、理由ウィリス動脈輪を介して側副血流量の減少しませんが低い酸素分圧で、同側半球のCBFは有意に減少し、一方的な虚血性傷害の結果。虚血脳組織を染色して識別するために、2,3,5 -塩化トリフェニルテトラゾリウム（TTC）の使用は、元来はげっ歯類の脳虚血5の成人モデルのために開発され、最大72までの早期の時点で脳infarctinの程度を評価するために使用されます虚血性イベント6時間後。このビデオでは、我々は生後ラット脳における低酸素性虚血性損傷モデル、TTC染色を用いて梗塞サイズの評価を示しています。

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

低酸素性虚血性脳損傷のための出生後のラットのモデルは、ヒト新生児低酸素性虚血性脳症（HIE）の十分に確立されたモデルです。この記事では、出生後の仔ラットのHIEのモデルについて説明します。

周産期虚血低酸素虚血性脳症モデル

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 ビュー.  Stanford University School of Medicine. 低酸素性虚血性脳損傷のための出生後のラットのモデルは、ヒト新生児低酸素性虚血性脳症（HIE）の十分に確立されたモデルです。この記事では、出生後の仔ラットのHIEのモデルについて説明します。

ビデオ: 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.

コンピュータによって生成された動物モデルの刺激

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

生物学

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.

後脚虚血症のマウスモデル

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).

精巣生殖細胞の三次元クロマチンアーキテクチャを保存するためのスライド調製方法

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.

における虚血/再灌流のための無酸素飢餓モデル<em&gt; C.虫</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.

筋フィラメントのCaの評価<sup&gt; 2+</sup&gt;感度基礎心臓興奮収縮連関

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.

低酸素の条件の下で培養された細胞から内因性の核蛋白質を用いた Co 免疫沈降試験

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.

EYFP タグ付き CENP-A (EYFP-CENP-A) のユビキタス性を同定するための質量分析分析

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.

ApoEにおける後肢虚血の改変外科モデル-/- ミニチュア切開を用いたマウス

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.

インビトロで心臓虚血/再灌流を模倣する細胞共培養モデルの開発

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.

3日間発生した雛胚を用いたフック虚血再灌流モデルの生成

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

Methods Article

周産期虚血低酸素虚血性脳症モデル