Amerikan Kalp Derneği ve March of Dimes tarafından finanse edilmektedir.

Bu protokol, deney hayvanlarının kullanımı için sağlık kuralları Ulusal Sağlık Enstitüleri tarafından, Stanford Üniversitesi ve uymaktadır Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylandı. Yenidoğan Rat HIE modeli <ol><li> Indüksiyonu için% 3-4 ve% 1-2; Post-natal gün (PND) 7 Sprague-Dawley sıçan yavrular (Charles River Laboratuvarları, Inc, Wilmington, MA), (Aerrane, Baxter, Deerfield, IL izofluran ile tam anestezi bakım). </li><li> Küçük bir kesi (Ekstra İnce Iris Makas # 14.088-10, Güzel Bilim Araçlar A.Ş., Foster City, CA), boyun sol karotis arter (CCA) ortaya çıkarmak için yapılır. </li><li> CCA, 5-0 ipek sütür (5-0 ipek, Ethicon, Somer, NJ) ile bağlandı. </li><li> Kesi siyanoakrilat yapıştırıcı (Elmers Products Inc, Columbus, OH) ile kapatılır. Kesi bant ile kaplanmıştır ve anestezi durdurulur. Yavrular baraj döndü ve iyileşmek için 1-2 saat izin verilir. </li><li> Pups sonra% 8, 37, 100 dakika boyunca% 92 azot ile dengeli oksijen içeren bir hipoksik odasına ° C yerleştirilir % 8 oxygen/92% azot gazı (Airgas, Sacramento, CA), plastik bir kapak ile donatılmış fare kafes içine tüp yoluyla akış. Kapak kafesine uygun yapılan ve 2 cm çapında iki delik vardır, biri,% 8 oksijen gaz tankı ve başka bir bağlı tüpü almak için hangi gaz flowout sağlar. Odasının bir su banyosuna yerleştirilir ve kullanmadan önce ısıtılır ve içinde bir termometre 37 ° C hipoksi başlamadan önce kaydetmeniz gerekir. </li><li> Hipoksi 100 dakika sonunda yavrular kurtarma için baraj tekrar iade edilir. </li><li> Hipoksi sona ermesinden sonra 24 saat, yavrular izofluran ile derin anestezi ile ötenazi. Beyin dokusu, tuzlu, pH 7.4 (Sigma Chemical Co, St Louis, MO) tamponlu fosfat soğuk% 1 TTC bir çözüm, soğuk serum fizyolojik ile perfüze. TTC ışıktan koruyarak saklanmalıdır. </li><li> Brain kaldırılır ve 1-2 dakika, buz ve dört koronal bölümler dışında 3 mm (seviye 1-4) soğutulur opticchiasm ve infundibulum bregma 1.8 &#39;e karşılık gelen düzeyde rostrally başlayarak kesilir ~ 2.0 mm seviyesinde yetişkin bir farenin beyin. </li><li> Beyin dilim TTC çözüm dalmış ve% 4 paraformaldehid / gecede PBS fiksasyonu takiben, 8 dakika 37 ° C inkübe. TTC ışığa duyarlı olduğundan, bu süre içinde, ışıktan dilimleri uzak tutun. </li><li> TTC boyanan kesitler taranır ve dijital. Enfarktüs alanı (lekesiz alan), ipsilateral yarımkürede alanında ve kontralateral yarımkürenin alanı: Resim J (NIH, Bethesda, MD), aşağıdaki her seviye için ölçülür. A 1, A 2, A 3, A 4 (infarkt alanı / ipsilateral yarımkürede alan) ya da yüzde dilim ortalama alanı olarak hesaplanır enfarkte. Enfarkt yüzde hacmi toplamı (A 1 + A 2 + 3 + 4) * 3mm olarak hesaplanır. Kortikal veya striatal yaralanma alanları da benzer bir şekilde ayrı ayrı belirlenebilir. </li></ol> Temsilcisi Sonuçlar <img src="/files/ftp_upload/old/955_2008_9_2_2/955_Fig-01.png" alt="Şekil 1" width="660" height="190" /> Şekil 1. TTC Temsilcisi HIE koronal seviyeleri 1-4 boyandı. TTC lekeli koronal bölümleri infarkt alanı (beyaz) göstermektedir.

<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>

Özetlediği serebral hipoksi yenidoğanlarda peri-natal dönemde meydana gelen kemirgen postnatal HIE modeli oluşturulmuş bir model. Kapsamlı histolojik ve immünositokimyasal karakterizasyon çalışmaları 7 gün eski kemirgen, üçüncü trimester insan fetus 7 modeli için analog beyin olgunluğa sahip olduğunu göstermiştir . Kemirgen HIE modeli böyle bir hipotermi gibi müdahaleler 3 valide edilmiş peri-natal hipoksi 2, beyin hasarı mekanizmaları anlamak için çok bilgilendirici olmuştur . ...

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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>
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<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

Hipoksik-İskemik Ensefalopati (HIE), doğum sırasında oluşan sistemik asfiksi sonucu. HIE olan yenidoğanlarda yirmi beş oranında zeka geriliği, serebral palsi, ve epilepsi dahil olmak üzere, ciddi ve kalıcı nöropsikolojik sekeller gelişir. HIE sonuçları HIE yeni doğanlarda beyin hasarı azaltmak için tedavi stratejileri belirlemek ve geliştirmek için kritik yapma, yıkıcı ve kalıcı değildir. Bu amaçla, hipoksik-iskemik beyin hasarı için yenidoğan sıçan modelinde bu insanlık durumunun model geliştirilmiştir. HIE modeli ilk Vannucci ve ark 1 doğrulanmış oldu ve o zamandan beri yoğun perinatal hipoksi-iskemi 2 kaynaklanan beyin hasarının mekanizmaları belirlemek ve potansiyel terapötik müdahaleler 3,4 test etmek için kullanılır olmuştur. HIE modeli, iki adımdan oluşan bir süreçtir ve hipoksik ortama maruz sol karotis arter ligasyonu içerir. Yarımkürede bağlanan karotis arteri ipsilateral serebral kan akımı (CBF), çünkü Willis daire ile kollateral kan akımı azalması değil; ancak düşük oksijen basıncı ile ipsilateral yarımkürede CBF önemli ölçüde azaltır ve tek taraflı iskemik hasar sonuçları . 2,3,5-triphenyltetrazolium klorür (TTC) iskemik beyin dokusu leke ve tanımlamak için kullanımı başlangıçta kemirgen serebral iskemi 5 yetişkin modeller için geliştirilen ve 72 adete kadar erken zaman noktalarında serebral infarctin ölçüde değerlendirmek için kullanılır. iskemik olay 6 saat sonra. Bu video, postnatal sıçan beyin hipoksik-iskemik yaralanma modeli TTC boyama kullanarak infarkt değerlendirilmesi göstermektedir.

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

Hipoksik-iskemik beyin hasarı için postnatal sıçan modelinde, insan yenidoğan hipoksik iskemik ensefalopati (HIE) köklü bir model. Bu makalede, doğum sonrası sıçan Yavrularda HIE modeli açıklanmaktadır.

Perinatal İskemi Hipoksik İskemik Ensefalopati Modeli

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

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35.6K Görüntüleme.  Stanford University School of Medicine. Hipoksik-iskemik beyin hasarı için postnatal sıçan modelinde, insan yenidoğan hipoksik iskemik ensefalopati (HIE) köklü bir model. Bu makalede, doğum sonrası sıçan Yavrularda HIE modeli açıklanmaktadır.

Video: 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.

Bilgisayar oluşturuldu Hayvan Modeli Uyaranlar

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

Biyoloji

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Embriyonik Kök Hücre Tedavi Hindlimb İskemi Türetilmiş Endotel Hücreleri

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

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.

Murin Model Hindlimb İskemi

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).

Testis Germ Hücrelerinin Üç Boyutlu Kromatin Mimarisini Korumak için Slayt Hazırlama Yöntemi

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.

Iskemi / reperfüzyon içinde bir anoksisi-açlık Modeli<em&gt; C. elegans</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 Ca Değerlendirilmesi<sup&gt; 2+</sup&gt; Duyarlılık temelinde Kardiyak Uyarma-kasılma Kaplin

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.

Co-immunoprecipitation tahlil hipoksik koşullarda kültürlü hücrelerden endojen nükleer proteinler kullanarak

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 etiketli CENP-A (EYFP-CENP-A) Ubiquitylation tanımlamak için Kütle Spektrometresi Analizi

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'de Hind Limb iskemisinin Modifiye Cerrahi Modeli-/- Minyatür Kesi Kullanan Fareler

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.

Kardiyak İskemi / Reperfüzyonu In Vitro Taklit Etmek için Bir Hücre Ko-Kültür Modelinin Geliştirilmesi

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