Name: generation of hook ischemia-reperfusion model using a three-day developing chick embryo
Uploaded: 2022-02-19T14:00:00
Description: Watch this Scientific Journal Video about Generation of Hook Ischemia-Reperfusion Model using a Three-Day Developing Chick Embryo at JoVE.com

我们要感谢Hari Shankar先生在摄像和编辑期间提供的重要投入，Baqer Hussain先生提供画外音，Asghar Rizvi先生负责视频编辑，Mohammad Haider先生负责录像，Mohammad Danish Siddiqui先生在实验期间提供协助。

Era勒克瑙医学院和医院的机构动物伦理委员会发布了一份书面豁免，指出根据动物实验控制和监督委员会（CPCSEA）进行这些实验不需要正式批准。然而，遵循标准操作程序以尽量减少胚胎痛苦的可能性。
1. 缓冲液制备（表1）
<ol><li>准备林格的解决方案<ol>
<li>为了制备林格溶液，将0.72克NaCl（123mM），0.017gCaCl2（1.53mM），0.037gKCl（4.96mM）溶解在70mL无菌蒸馏H2O</s...

<ol>
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S., 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=Neuroprotective+effect+of+naringenin+is+mediated+through+suppression+of+NF-&#954;B+signaling+pathway+in+experimental+stroke.">Neuroprotective effect of naringenin is mediated through suppression of NF-&#954;B signaling pathway in experimental stroke.</a> Neuroscience. 29 (230), 157-171 (2013).</li><li>Raza, S. S., 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=Hesperidin+ameliorates+functional+and+histological+outcome+and+reduces+neuroinflammation+in+experimental+stroke.">Hesperidin ameliorates functional and histological outcome and reduces neuroinflammation in experimental stroke.</a> Brain Research. 28 (1420), 93-105 (2011).</li><li>Raza, S. S., 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=Silymarin+protects+neurons+from+oxidative+stress+associated+damages+in+focal+cerebral+ischemia:+a+behavioral,+biochemical+and+immunohistological+study+in+Wistar+rats.">Silymarin protects neurons from oxidative stress associated damages in focal cerebral ischemia: a behavioral, biochemical and immunohistological study in Wistar rats.</a> Journal of the Neurological Sciences. 15 (1-2), 45-54 (2011).</li><li>Fan, L., Zhou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AG490+protects+cerebral+ischemia/reperfusion+injury+via+inhibiting+the+JAK2/3+signaling+pathway.">AG490 protects cerebral ischemia/reperfusion injury via inhibiting the JAK2/3 signaling pathway.</a> Brain and Behavior. 11 (1), 01911 (2021).</li><li>Wu, M. Y., 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=Current+Mechanistic+Concepts+in+Ischemia+and+Reperfusion+Injury.">Current Mechanistic Concepts in Ischemia and Reperfusion Injury.</a> Cellular Physiology and Biochemistry. 46 (4), 1650-1667 (2018).</li><li>Collard, C. D., Gelman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology,+clinical+manifestations,+and+prevention+of+ischemia-reperfusion+injury.">Pathophysiology, clinical manifestations, and prevention of ischemia-reperfusion injury.</a> Anesthesiology. 94 (6), 1133-1138 (2001).</li><li>Allen, D. D., 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=Cell+lines+as+in+vitro+models+for+drug+screening+and+toxicity+studies.">Cell lines as in vitro models for drug screening and toxicity studies.</a> Drug Development and Industrial Pharmacy. 31 (8), 757-768 (2005).</li><li>Schmeer, C., Gamez, A., Tausch, S., Witte, O. W., Isenmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statins+modulate+heat+shock+protein+expression+and+enhance+retinal+ganglion+cell+survival+after+transient+retinal+ischemia/reperfusion+in+vivo.">Statins modulate heat shock protein expression and enhance retinal ganglion cell survival after transient retinal ischemia/reperfusion in vivo.</a> Investigative Ophthalmology and Visual Science. 49 (11), 4971-4981 (2008).</li><li>Huang, K. Y., 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=A+systematic+review+and+meta-analysis+of+acupuncture+for+improving+learning+and+memory+ability+in+animals.">A systematic review and meta-analysis of acupuncture for improving learning and memory ability in animals.</a> BMC Complementary and Alternative Medicine. 16 (1), 297 (2016).</li><li>Sommer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+stroke:+Experimental+models+and+reality.">Ischemic stroke: Experimental models and reality.</a> Acta Neuropathologica. 133 (2), 245-261 (2017).</li><li>Yang, W., Chen, J., Meng, Y., Chen, Z., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+targets+for+treating+ischemia-reperfusion+injury+in+the+liver.">Novel targets for treating ischemia-reperfusion injury in the liver.</a> International Journal of Molecular Sciences. 19 (5), 1302 (2018).</li><li>Seabra, R., Bhogal, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+research+using+early+life+stage+models.">In vivo research using early life stage models.</a> In Vivo. 24 (4), 457-462 (2010).</li><li>Liu, 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=Adiponectin+peptide+alleviates+oxidative+stress+and+NLRP3+inflammasome+activation+after+cerebral+ischemia-reperfusion+injury+by+regulating+AMPK/GSK-3beta.">Adiponectin peptide alleviates oxidative stress and NLRP3 inflammasome activation after cerebral ischemia-reperfusion injury by regulating AMPK/GSK-3beta.</a> Experiments in Neurology. 329, 113302 (2020).</li><li>Aboutaleb, N., Jamali, H., Abolhasani, M., Pazoki Toroudi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lavender+oil+(Lavandula+angustifolia)+attenuates+renal+ischemia/reperfusion+injury+in+rats+through+suppression+of+inflammation,+oxidative+stress+and+apoptosis.">Lavender oil (Lavandula angustifolia) attenuates renal ischemia/reperfusion injury in rats through suppression of inflammation, oxidative stress and apoptosis.</a> Biomedicine and Pharmacotherapy. 110, 9-19 (2019).</li><li>Wallert, 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=alpha-Tocopherol+preserves+cardiac+function+by+reducing+oxidative+stress+and+inflammation+in+ischemia/reperfusion+injury.">alpha-Tocopherol preserves cardiac function by reducing oxidative stress and inflammation in ischemia/reperfusion injury.</a> Redox Biology. 26, 101292 (2019).</li><li>Ashafaq, 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=Catechin+hydrate+ameliorates+redox+imbalance+and+limits+inflammatory+response+in+focal+cerebral+ischemia.">Catechin hydrate ameliorates redox imbalance and limits inflammatory response in focal cerebral ischemia.</a> Neurochemical Research. 37 (8), 1747-1760 (2012).</li><li>Gallagher, S., Chakavarti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblot+analysis.">Immunoblot analysis.</a> Journal of Visualized Experiments. 20 (16), 759 (2008).</li><li>Abt, M. A., Grek, C. L., Ghatnekar, G. S., Yeh, E. S. <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+lung+metastasis+in+mouse+mammary+tumor+models+by+quantitative+real-time+PCR.">Evaluation of lung metastasis in mouse mammary tumor models by quantitative real-time PCR.</a> Journal of Visualized Experiments. (107), e53329 (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> Journal of Visualized Experiments. (62), e3923 (2012).</li><li>Wu, Y., 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=Cathelicidin+aggravates+myocardial+ischemia/reperfusion+injury+via+activating+TLR4+signaling+and+P2X(7)R/NLRP3+inflammasome.">Cathelicidin aggravates myocardial ischemia/reperfusion injury via activating TLR4 signaling and P2X(7)R/NLRP3 inflammasome.</a> Journal of Molecular and Cellular Cardiology. 139, 75 (2020).</li><li>Franke, 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=The+NLRP3+inflammasome+drives+inflammation+in+ischemia/reperfusion+injury+after+transient+middle+cerebral+artery+occlusion+in+mice.">The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice.</a> Brain Behaviour and Immunity. 92, 223 (2021).</li><li>Lawrence, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+factor+NF-kappaB+pathway+in+inflammation.">The nuclear factor NF-kappaB pathway in inflammation.</a> Cold Spring Harbor Perspectives in Biology. 1 (6), 001651 (2009).</li><li>Liu, 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=Pterostilbene+attenuates+astrocytic+inflammation+and+neuronal+oxidative+injury+after+ischemia-reperfusion+by+inhibiting+NF-kappaB+phosphorylation.">Pterostilbene attenuates astrocytic inflammation and neuronal oxidative injury after ischemia-reperfusion by inhibiting NF-kappaB phosphorylation.</a> Frontiers in Immunology. 10, 2408 (2009).</li><li>Prakash, R., 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=Sivelestat-loaded+nanostructured+lipid+carriers+modulate+oxidative+and+inflammatory+stress+in+human+dental+pulp+and+mesenchymal+stem+cells+subjected+to+oxygen-glucose+deprivation.">Sivelestat-loaded nanostructured lipid carriers modulate oxidative and inflammatory stress in human dental pulp and mesenchymal stem cells subjected to oxygen-glucose deprivation.</a> Materials Science and Engineering: C Materials for Biological Applications. 120, 111700 (2021).</li><li>Prakash, R., 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=Oxidative+stress+enhances+autophagy+in+stem+cells+through+Erk1/2+signaling+pathway+-+implications+for+neurotransplantations.">Oxidative stress enhances autophagy in stem cells through Erk1/2 signaling pathway - implications for neurotransplantations.</a> Stem Cell Reviews and Reports. , (2021).</li><li>Ahmad, A., 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=Gelatin-coated+polycaprolactone+nanoparticle-mediated+naringenin+delivery+rescue+human+mesenchymal+stem+cells+from+oxygen+glucose+deprivation-induced+inflammatory+stress.">Gelatin-coated polycaprolactone nanoparticle-mediated naringenin delivery rescue human mesenchymal stem cells from oxygen glucose deprivation-induced inflammatory stress.</a> ACS Biomaterials Science and Engineering. 5 (2), 683-695 (2019).</li><li>Guan, X., 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=The+neuroprotective+effects+of+carvacrol+on+ischemia/reperfusion-induced+hippocampal+neuronal+impairment+by+ferroptosis+mitigation.">The neuroprotective effects of carvacrol on ischemia/reperfusion-induced hippocampal neuronal impairment by ferroptosis mitigation.</a> Life Science. 235, 116795 (2019).</li><li>Jin, Z., Guo, P., Li, X., Ke, J., Wang, Y., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effects+of+irisin+against+cerebral+ischemia/+reperfusion+injury+via+Notch+signaling+pathway.">Neuroprotective effects of irisin against cerebral ischemia/ reperfusion injury via Notch signaling pathway.</a> Biomedicine and Pharmacotherapy. 120, 109452 (2019).</li><li>Wainrach, S., Sotelo, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscope+study+of+the+developing+chick+embryo+heart.">Electron microscope study of the developing chick embryo heart.</a> Zeitschrift fur Zellforschung und mikroskopische Anatomie. 55, 622-634 (1961).</li><li>Joshi, V. C., Wilson, A. C., Wakil, 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=Assay+for+the+terminal+enzyme+of+the+stearoyl+coenzyme+A+desaturase+system+using+microsomes.">Assay for the terminal enzyme of the stearoyl coenzyme A desaturase system using microsomes.</a> Journal of Lipid Research. 18 (1), 32-36 (1977).</li><li>Kain, K. 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缺血再灌注研究的目标是制定预防细胞死亡和促进恢复的治疗策略29，30。为了克服当前I/R研究的限制，我们设计了一个Hook I/R雏鸡胚胎模型，以产生可靠且可重复的I/R模型。据我们所知，除了研究应激信号（例如，氧化和炎症应激）之外，我们还是在3天雏鸡胚胎中创建的第一个I / R模型，用于常规I / R实验。鉴于发育第3天的大小和可及性的好处<sup class=...

缺血再灌注组织损伤与许多病理有关，包括心脏病发作、缺血性卒中、创伤和外周血管疾病1，2，3，4，5。这主要是由于缺乏对疾病进展的全面了解以及缺乏有效的研究模型。缺血性损伤发生在组织特定区域的血液供应被切断时。结果，缺血组织最终坏死，尽管速率因组织而异。因此，恢复血液供应可能有助于减轻损害。然而，已经观察到，在某些情况下，再灌注比单独的缺血造成的组织损伤更多6，7，8。因此，需要了解缺血再灌注的分子和细胞机制，以开发有效的治疗干预措施。目前，尚无针对 I/R 损伤的有效治疗方法。这种差异促使创建了新的实验模型，从体外到体内模型，以解决现有问题9，10，11，12，13。
雏鸡胚胎（Gallus gallus domesticus）因其易于获取，道德可接受性，相对较大的尺寸（与其他胚胎相比），低成本和快速生长而被广泛用于研究14。我们使用雏鸡胚胎在发育72小时时通过在脊髓针的帮助下闭塞和释放右维特林动脉来创建 卵内 I / R。我们将其命名为 Hook-I/R 缺血再灌注模型（图 1）。本研究中使用的模型能够准确模拟所有下游过程，包括氧化和炎症途径，这些途径通常与I / R损伤相关15，16，17。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(-80&#176;C) freezer</td>
			<td>Haier, China</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5mL Centrifuge tube</td>
			<td>TARSONS, India</td>
			<td>500010X</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm Petri dish (sterile)</td>
			<td>Tarsons, India</td>
			<td>460050</td>
			<td></td>
		</tr>
		<tr>
			<td>18G Needle (18G&#215;1.5 (1.25&#215;38mm)</td>
			<td>Ramsons, India</td>
			<td>13990</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL Syringe</td>
			<td>DISPO VAN</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>26G Needle (26G&#215;1/2 (10.45x13mm)</td>
			<td>DISPO VAN, india</td>
			<td>30722D</td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C egg incubator with adjustable percentage humidity</td>
			<td>Gentek, India</td>
			<td>GL-100</td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C laboratory incubator</td>
			<td>SCIENCE TECH, India</td>
			<td>CB 101-14</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Methyladenine (3-MA)</td>
			<td>Sigma Aldrich, USA</td>
			<td>M9281</td>
			<td></td>
		</tr>
		<tr>
			<td>3mL Pasture Pipette</td>
			<td>TARSONS, India</td>
			<td>940050</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Beaker</td>
			<td>TARSONS, India</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Syringe</td>
			<td>DISPO VAN, India</td>
			<td>IP53</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Merck Millipore, United States</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive tape/Cello tape</td>
			<td>Sunrise, India</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ambra1 primers</td>
			<td>Applied Biosystems, Foster city, USA</td>
			<td>Hs00387943_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG</td>
			<td>Cell Signaling Technology, USA</td>
			<td>7076S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rabbit IgG</td>
			<td>Jackson Immuno Research Laboratories, USA</td>
			<td>711-035-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Atg7</td>
			<td>R&#38;D Systems, USA</td>
			<td>MAB6608</td>
			<td></td>
		</tr>
		<tr>
			<td>Atg7 primers</td>
			<td>Applied Biosystems, Foster city, USA</td>
			<td>Hs00893766_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave Bag</td>
			<td>Tarsons, India</td>
			<td>550022</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave Machine</td>
			<td>Local made, Lucknow, India</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Beclin-1</td>
			<td>Proteintech, USA</td>
			<td>66665-1-Ig</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta Actin</td>
			<td>ImmunoTag, USA</td>
			<td>ITT07018</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Himedia, Mumbai, India</td>
			<td>TC194</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Himedia, Mumbai, India</td>
			<td>GRM534</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>ImmunoTag, USA</td>
			<td>ITT5155</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaning wipes</td>
			<td>Kimberly-Clark, India</td>
			<td>370080</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaved Caspase3</td>
			<td>ImmunoTag, USA</td>
			<td>ITT07022</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium hydrogen phosphate heptahydrate</td>
			<td>Himedia, Mumbai, India</td>
			<td>GRM39611</td>
			<td></td>
		</tr>
		<tr>
			<td>Doppler blood flowmeter</td>
			<td>Moors instrument, United Kingdom</td>
			<td>moorVMS-LDF1</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg rack</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg rack</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>ImmunoTag, USA</td>
			<td>M1000110</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH primers</td>
			<td>Applied Biosystems, Foster city, USA</td>
			<td>Hs02758991_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Himedia, Mumbai, India</td>
			<td>MB013</td>
			<td></td>
		</tr>
		<tr>
			<td>Kidney tray</td>
			<td>HOSPITO</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>LC3A/B</td>
			<td>Cell Signaling Technology, USA</td>
			<td>4108S</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Rankem laboratories, Mumbai, India</td>
			<td>M0252</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige, Japan</td>
			<td>M-152</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-L-cysteine (NAC)</td>
			<td>Sigma Aldrich, USA</td>
			<td>A7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Naringenin</td>
			<td>Sigma Aldrich, USA</td>
			<td>67604-48-2</td>
			<td></td>
		</tr>
		<tr>
			<td>NF-k&#946;</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>51-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>NLRP3</td>
			<td>ImmunoTag, USA</td>
			<td>ITT07438</td>
			<td></td>
		</tr>
		<tr>
			<td>Nose plier</td>
			<td>Local made, Lucknow, India</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular forceps</td>
			<td>Stoelting, Germany</td>
			<td>52106-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular iris</td>
			<td>Tufft Surgical Instruments, Jaipur, India</td>
			<td>Hard Age Vannas Micro Scissors Angled 8CM / 3 1/8&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>OHP marker pen</td>
			<td>Camlin, India</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>ORP-150</td>
			<td>ImmunoTag, USA</td>
			<td>ITT08329</td>
			<td></td>
		</tr>
		<tr>
			<td>Pointed sharp edge scissor</td>
			<td>Stoelting, Germany</td>
			<td>52132-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Himedia, Mumbai, India</td>
			<td>MB043</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic anhydrous</td>
			<td>Himedia, Mumbai, India</td>
			<td>MB050</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor</td>
			<td>Abcam, United States</td>
			<td>Ab65621</td>
			<td></td>
		</tr>
		<tr>
			<td>SOD-1</td>
			<td>ImmunoTag, USA</td>
			<td>ITT4364</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific, Mumbai, India</td>
			<td>27605</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate</td>
			<td>Himedia, Mumbai, India</td>
			<td>GRM886</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinal needle 25GA; 3.50 IN (90.51 X 90mm)</td>
			<td>Ramson, India</td>
			<td>GS-2029</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Zoom surgical microscope</td>
			<td>Olympus, Japan</td>
			<td>SZ2-STU3</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe discarder</td>
			<td>BIOHAZARD</td>
			<td>882210</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed forceps</td>
			<td>Stoelting, Germany</td>
			<td>52102-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>G Biosciences, United States</td>
			<td>RC1217</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Hydrochloric Acid</td>
			<td>Himedia, Mumbai, India</td>
			<td>MB030</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>G Biosciences, United States</td>
			<td>RC1227</td>
			<td></td>
		</tr>
		<tr>
			<td>White Leghorn Chicken 0-day eggs</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Z-Val-Ala-Asp(OMe)-FMK</td>
			<td>MP Biomedicals, LLC, USA</td>
			<td>FK009</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of hook ischemia-reperfusion model using a three-day developing chick embryo

缺血和再灌注 （I/R） 疾病（如心肌梗死、卒中和外周血管疾病）是疾病和死亡的一些主要原因。目前有许多 体外 和 体内 模型可用于研究疾病或受损组织中的I / R机制。然而，迄今为止，还没有报告 任何蛋内 I / R模型，这将允许更好地了解I / R机制和更快的药物筛选。本文描述了在3天雏鸡胚胎中使用脊柱针定制钩进行I / R建模，以了解I / R发育和治疗机制。我们的模型可用于研究DNA，RNA和蛋白质水平的异常。此方法简单，快速且价格低廉。当前模型可以独立使用，也可以与现有的 体外 和 体内 I / R模型结合使用。

多普勒血流成像技术用于评估我们模型的有效性。简而言之，我们将来自对照组的数据与来自RVA组的数据进行了比较，以确定我们创建的成功。 图4A 描绘了与对照动物相关的典型通量，而 图4B 描绘了从RVA获得的结果。数字 1-8 表示与 I/R 阶段关联的各种事件。简而言之，数字1-3对应于正常氧的相位，而3点和4点的通量急剧下降代表了与RVA中血流量减少?...

Watch this Scientific Journal Video about Generation of Hook Ischemia-Reperfusion Model using a Three-Day Developing Chick Embryo at JoVE.com

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

本文描述了使用脊髓针定制钩在3天雏鸡胚胎中的缺血再灌注（I / R）建模，以更好地了解I / R发展和治疗。该模型简单，快速且价格低廉。

使用三天发育中的雏鸡胚胎生成钩缺血再灌注模型

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

嗨，我是Syed Shadab Raza博士，干细胞和恢复性神经病学实验室的首席研究员，位于印度勒克瑙时代大学校园生物技术系。在我的实验室中，我们努力了解缺血再灌注障碍背后的病理生理机制以及可以逆转这些过程的干预措施。在这种情况下，我们经常采用缺血性中风的体外和体内模型。最近，我们在三天发育中的雏鸡胚胎中创建了缺血再灌注损伤模型。现在我的学生将向你们展示 如何在三天发育中的雏鸡胚胎中复制缺血再灌注损伤。现在，我们将向您展示如何在三天的雏鸡胚胎中产生缺血再灌注。我们的模型具有成本效益，时间有效且高度可重复。让我们开始吧。第一天。第一天的要求。程序。用薄纸巾用70%乙醇清洁零日鸡蛋。接下来，用OHP标记在鸡蛋上写下当天的日期，然后将鸡蛋放入温度为36至37摄氏度，湿度为60%至65%的鸡蛋孵化器中。在接下来的24小时内孵育卵。第二天。第二天的要求。程序。用70%乙醇擦拭手术剪刀或随后用高压灭菌器擦拭。孵化24小时后，将卵子从孵化器中取出以进行分层。接下来，将一小块长和宽约一英寸的Sellotape连接到卵的边缘，然后使用尖尖的边缘剪刀在蛋壳的边缘打一个小孔，然后以大约75度角插入五毫升注射器。将针头插入蛋黄袋后，慢慢取出五到六毫升白蛋白。取出白蛋白后，用Sellotape重新密封孔，然后将卵放回37摄氏度的鸡蛋培养箱中，持续48小时。第四天。第四天的要求。程序。按照本手稿中提供的表格准备林格氏溶液，0.9%生理盐水和一个X磷酸盐缓冲盐水。然后高压灭菌三种溶液。高压灭菌后，可以将相应的溶液置于室温下。接下来，从37度的鸡蛋孵化器中取出鸡蛋。现在，在切割外壳之前，用Sellotape覆盖窗户区域。现在在蛋壳上打一个小洞，开始切一个圆形的开口。此过程称为窗口化。确保圆形切口足够大，以便胚胎可以从任何方向轻松接近，因为可能需要根据胚胎的位置调整卵子的位置。现在，使用立体变焦手术显微镜，找到正确的生命线动脉或RVA。在这里，箭头表示胚胎的三天发育阶段。一旦RVA被定位，使用26 G针在RVA的左侧和右侧形成两个小孔。接下来，将多普勒血流成像探头调整到RVA上。多普勒血流成像探头应放置在距缺血部位5加减1毫米处，放置在RVA远端的三分之二处。根据需要，将通量读数进行30秒至2分钟。这将使正态氧相读数。现在，在鼻钳的帮助下，手动模塑脊柱针的边缘，使边缘呈钩状，然后是齿镊子。现在，使用微型机械手，将脊髓针插入右生命线动脉下方。您已准备好抬起脊柱针。现在在微机械手的帮助下，缓慢抬起动脉，直到多普勒血流通量显示动脉流量至少减少80%。一旦多普勒通量达到80%或更高的下降，将脊髓针向上抬起，拉动动脉五分钟。这将是缺血期。在此缺血时间五分钟后，在微机械手的帮助下缓慢释放动脉，以恢复正常的血流水平。现在，多普勒血流量计应显示与正常氧合期间观察到的读数相似的读数。这将是RVA中再灌注的时期。在I / R过程之后，将两到三滴1X PBS滴到胚胎上。最后，用Sellotape重新密封窗口，然后将鸡蛋放回鸡蛋孵化器中5小时55分钟。5小时55分钟后，从鸡蛋孵化器中取出鸡蛋，放到蛋盘上，然后重新打开窗口。治疗要求。程序。为了用药物，激活剂或抑制剂治疗动脉，应在I / R过程一小时后切除RVA。通过将胚胎从壳中取出到无菌的100毫米培养皿中，将其从壳中取出。一旦胚胎在培养皿中释放，在立体变焦手术显微镜的指导下使用眼部虹膜切除RVA。RVA的切除尺寸应为距躯干远端15加减1毫米，朝向躯干2加减1毫米。切除RVA后，用1X PBS在新的无菌培养皿中洗涤。对于所需的治疗，将动脉放入充满500微升Ringer溶液的灭菌1.5毫升离心管中。并将动脉放入离心管中后，放入37°C实验室培养箱中五小时。孵育五小时后，从37摄氏度的实验室培养箱中取出RVA并进行所需的处理。现在，展示结果。为了验证我们的模型在缺血性动脉缺血再灌注研究中的效用，我们首先研究了氧调节蛋白150或ORP150，细胞质超氧化物歧化酶1或SOD1和过氧化氢酶的活性。与对照组相比，I / R处理的RVA显示出ORP150，细胞质SOD1和过氧化氢酶的活性增加，然而，补充N-乙酰基-L-半胱氨酸或NAC（ROS淬灭剂）降低了I / R组中的氧化应激，如当前图所示。为了确定该模型在炎症研究中的效用，我们研究了I / R-1β和TNF-α在I / R处理的RVA与对照RVA中的表达。与对照组相比，发现两种白细胞介素在Hook-I / R组中均过度表达。补充柚皮素，一种抗炎药，显着降低了IL1-β和TNF-α的水平。接下来，我们通过蛋白质印迹检查了NLRP3和NF-kappa beta的表达。该分析发现了NLRP3 inflamasome激活的证据，以及NF-kappa beta响应RVA中产生的I / R的证据。与之前的结果类似，柚皮素降低了NLRP3和NF-kappa beta的表达。这些结果表明，我们的模型可用于研究炎症改变。接下来，我们研究了I / R对细胞凋亡和自噬途径的影响。首先，在图A中，我们访问了裂解的半胱天冬酶-3的表达。与对照组相比，我们观察到I / R组中切割的半胱天冬酶-3的表达增强，而接受ZVADFMK的I / R组显示半胱天冬酶-3的表达降低。与对照组相比，I / R组表现出高蛋白水平的LC3，自噬体标志物Beclin-1（哺乳动物细胞自噬的关键调节因子）和ATG-7（基础自噬所需的蛋白质），而接受自噬抑制剂3-ma的组显示上述三种自噬蛋白的表达降低， 从图B到D可以看出，图E证明了Hook介导的I / R对ambra-1和ATG-7 mRNA水平的影响。这些结果与先前的结果一致，即与各自的对照相比，在I / R处理的RVA中观察到ambra-1和ATG-7的mRNA表达增强。然而，当将3-ma添加到I / R组时，结果相反。目前的数字代表了我们的模型研究DNA变化的有效性。与对照组相比，我们在I / R治疗组中观察到强烈的DNA涂片。然而，对I / R RVA组补充NAC逆转了结果。为了研究我们的模型与其他模型相比的有效性，我们比较了本实验中 Hook-I/R 模型和 MCAO 模型生成的数据。简而言之，凋亡蛋白，裂解的半胱天冬酶-3，自噬蛋白Beclin-1和ATG-7以及炎症白细胞介素TNF和IFN的表达在I / R处理的RVA中必然高于对照RVA。有趣的是，Hook-I / R模型给出的结果与MCAO模型获得的结果非常相似，无论是炎症应激还是细胞死亡途径的分析。我们的模型可用于常规缺血再灌注实验，无论是单独使用还是与现有模型并行。然而，在模仿缺血再灌注期间，在RVA的右侧和左侧打孔时，以及在阻塞或释放RVA时，应采取最高的预防措施，使其不会损坏任何动脉或静脉。在常规分子生物学中，如蛋白质印迹，QRT-PCR分析仪，化学酸和成像技术可以即兴发挥，以将与三天发育中的雏鸡胚胎中的缺血再灌注相关的病理生理学联系起来。该模型可以有效地用于研究DNA，RNA和蛋白质水平的分子力学。正如我们向您展示的那样，I/ R建模在三天发育中的雏鸡胚胎中，我们相信我们的模型将在I / R研究领域开辟新的途径。通过这项工作，我祝愿你们的实验一切顺利。谢谢。

Research

JoVE Journal

Biology

Generation of Stable Transgenic C. elegans Using Microinjection

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Method for Culture of Early Chick Embryos ex vivo New Culture

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

双整装原位杂交技术在早期鸡胚

Method for Whole Mount Antibody Staining in Chick

整装在鸡的抗体染色方法

Assay for Neural Induction in the Chick Embryo

在鸡胚神经诱导的含量为

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

子宫 - 输卵管胚胎移植和输精管结扎术在小鼠模型

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

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

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

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

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