Name: isolation of retinal arterioles for ex vivo cell physiology studies
Uploaded: 2018-07-14T15:00:00
Duration: 12 min 42 s
Description: Watch this Scientific Journal Video about Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies at JoVE.com

このペーパーで説明したプロトコルの開発は、次の資金の機関からの助成金によって支えられた: BBSRC (BB/I026359/1)、視力 (1429 と 1822)、若年性糖尿病研究財団 (2-2003-525)、Wellcome の信頼 (074648/Z/04) のための戦い英国心臓財団 (PG/11/94/29169)、HSC の R & D 部門 (STL/4748/13) と MRC (MC_PC_15026)。

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	<li>Kohner, E. M., Patel, V., Rassam, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+blood+flow+and+impaired+autoregulation+in+the+pathogenesis+of+diabetic+retinopathy.">Role of blood flow and impaired autoregulation in the pathogenesis of diabetic retinopathy.</a> Diabetes. 44, 603-607 (1995).</li><li>Hafez, A. S., Bizzarro, R. L., Lesk, M. R. <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+optic+nerve+head+and+peripapillary+retinal+blood+flow+in+glaucoma+patients,+ocular+hypertensives,+and+normal+subjects.">Evaluation of optic nerve head and peripapillary retinal blood flow in glaucoma patients, ocular hypertensives, and normal subjects.</a> American Journal of Ophthalmology. 136, 1022-1031 (2003).</li><li>Curtis, T. M., Gardiner, T. A., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+lesions+of+diabetic+retinopathy:+clues+towards+understanding+pathogenesis?.">Microvascular lesions of diabetic retinopathy: clues towards understanding pathogenesis?.</a> Eye (London). 23, 1496-1508 (2009).</li><li>Pournaras, C. J., Rungger-Br&#228;ndle, E., Riva, C. E., Hardarson, S. H., Stefansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+retinal+blood+flow+in+health+and+disease).">Regulation of retinal blood flow in health and disease).</a> Progress in Retinal and Eye Research. 27, 284-330 (2008).</li><li>Kuwabara, T., Cogan, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+retinal+vascular+patterns.+I.+Normal+architecture.">Studies of retinal vascular patterns. I. Normal architecture.</a> Archives of Ophthalmology. 64, 904-911 (1960).</li><li>Scholfield, C. N., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+physiology+of+retinal+and+choroidal+arteriolar+smooth+muscle+cells.">Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.</a> Microcirculation. 14 (1), 11-24 (2007).</li><li>Puro, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinovascular+physiology+and+pathophysiology:+new+experimental+approach/new+insights.">Retinovascular physiology and pathophysiology: new experimental approach/new insights.</a> Progress in Retinal and Eye Research. 31 (3), 258-270 (2012).</li><li>Kur, J., Newman, E. A., Chan-Ling, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+physiological+mechanisms+underlying+blood+flow+regulation+in+the+retina+and+choroid+in+health+and+disease.">Cellular and physiological mechanisms underlying blood flow regulation in the retina and choroid in health and disease.</a> Progress in Retinal and Eye Research. 31 (5), 377-406 (2012).</li><li>Delaey, C., Van de Voorde, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+Mechanisms+in+the+Retinal+and+Choroidal+Circulation.">Regulatory Mechanisms in the Retinal and Choroidal Circulation.</a> Ophthalmic Research. 32 (6), 249-256 (2000).</li><li>Metea, M. R., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+dilate+and+constrict+blood+vessels:+a+mechanism+of+neurovascular+coupling.">Glial cells dilate and constrict blood vessels: a mechanism of neurovascular coupling.</a> Journal of Neuroscience. 26, 2862-2870 (2006).</li><li>Pournaras, C. J., Rungger-Br&#228;ndle, E., Riva, C. E., Hardarson, S. H., Stefansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+retinal+blood+flow+in+health+and+disease.">Regulation of retinal blood flow in health and disease.</a> Progress in Retinal and Eye Research. 27 (3), 284-330 (2008).</li><li>Hughes, S., Chan-Ling, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+smooth+muscle+cell+and+pericyte+differentiation+in+the+rat+retina+in+vivo.">Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo.</a> Investigative Ophthalmology &amp; Visual Science. 45 (8), 2795-2806 (2004).</li><li>Shepro, D., Morel, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericyte+physiology.">Pericyte physiology.</a> The FASEB Journal. 7 (11), 1031-1038 (1993).</li><li>Kornfield, T. E., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Blood+Flow+in+the+Retinal+Trilaminar+Vascular+Network.">Regulation of Blood Flow in the Retinal Trilaminar Vascular Network.</a> Journal of Neuroscience. 34 (34), 11504-11513 (2014).</li><li>Matsushita, K., Puro, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographical+heterogeneity+of+KIR+currents+in+pericyte-containing+microvessels+of+the+rat+retina:+effect+of+diabetes.">Topographical heterogeneity of KIR currents in pericyte-containing microvessels of the rat retina: effect of diabetes.</a> Journal of Physiology. 573, 483-495 (2006).</li><li>Needham, 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+role+of+K++and+Cl-+channels+in+the+regulation+of+retinal+arteriolar+tone+and+blood+flow.">The role of K+ and Cl- channels in the regulation of retinal arteriolar tone and blood flow.</a> Investigative Ophthalmology &amp; Visual Science. 55 (4), 2157-2165 (2014).</li><li>Hessellund, A., Jeppesen, P., Aalkjaer, C., Bek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+vasomotion+in+porcine+retinal+arterioles.">Characterization of vasomotion in porcine retinal arterioles.</a> Acta Ophthalmologicaogica Scandinavica. 81, 278-282 (2003).</li><li>Delaey, C., Van de Voord, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-induced+myogenic+responses+in+isolated+bovine+retinal+arteries.">Pressure-induced myogenic responses in isolated bovine retinal arteries.</a> Investigative Ophthalmology &amp; Visual Science. 41, 1871-1875 (2000).</li><li>Kur, J., 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=Role+of+ion+channels+and+subcellular+Ca2++signaling+in+arachidonic+acid-induced+dilation+of+pressurized+retinal+arterioles.">Role of ion channels and subcellular Ca2+ signaling in arachidonic acid-induced dilation of pressurized retinal arterioles.</a> Investigative Ophthalmology &amp; Visual Science. 55 (5), 2893-2902 (2014).</li><li>Kur, J., Bankhead, P., Scholfield, C. N., Curtis, T. M., McGeown, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca(2+)+sparks+promote+myogenic+tone+in+retinal+arterioles.">Ca(2+) sparks promote myogenic tone in retinal arterioles.</a> British Journal of Pharmacology. 168 (7), 1675-1686 (2013).</li><li>Fern&#225;ndez, J. A., McGahon, M. K., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaV3.1+T-Type+Ca2++Channels+Contribute+to+Myogenic+Signaling+in+Rat+Retinal+Arterioles.">CaV3.1 T-Type Ca2+ Channels Contribute to Myogenic Signaling in Rat Retinal Arterioles.</a> Investigative Ophthalmology &amp; Visual Science. 56 (9), 5125-5132 (2015).</li><li>McGahon, M. K., 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=TRPV2+Channels+Contribute+to+Stretch-Activated+Cation+Currents+and+Myogenic+Constriction+in+Retinal+Arterioles.">TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles.</a> Investigative Ophthalmology &amp; Visual Science. 57 (13), 5637-5647 (2016).</li><li>Guidoboni, G., 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=Intraocular+Pressure,+Blood+Pressure,+and+Retinal+Blood+Flow+Autoregulation:+A+Mathematical+Model+to+Clarify+Their+Relationship+and+Clinical+Relevance.">Intraocular Pressure, Blood Pressure, and Retinal Blood Flow Autoregulation: A Mathematical Model to Clarify Their Relationship and Clinical Relevance.</a> Investigative Ophthalmology &amp; Visual Science. 55 (7), 4105-4118 (2014).</li><li>Scholfield, C. N., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+cytosolic+calcium+regulation+among+different+microvascular+smooth+muscle+cells+of+the+rat+retina.">Heterogeneity in cytosolic calcium regulation among different microvascular smooth muscle cells of the rat retina.</a> Microvascular Research. 59 (2), 233-242 (2000).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> Journal of Biological Chemistry. 260, 3440-3450 (1985).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single Channel Recording, Second Edition. , 53-90 (1995).</li><li>Fern&#225;ndez, J. A., Bankhead, P., Zhou, H., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+detection+and+measurement+of+isolated+retinal+arterioles+by+a+combination+of+edge+enhancement+and+cost+analysis.">Automated detection and measurement of isolated retinal arterioles by a combination of edge enhancement and cost analysis.</a> PLoS One. 9, e91791 (2014).</li><li>McGahon, M. K., 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=Diabetes+downregulates+large-conductance+Ca2+-activated+potassium+beta+1+channel+subunit+in+retinal+arteriolar+smooth+muscle.">Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle.</a> Circulation Research. 100 (5), 703-711 (2007).</li><li>Curtis, T. M., Scholfield, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nifedipine+blocks+Ca2++store+refilling+through+a+pathway+not+involving+L-type+Ca2++channels+in+rabbit+arteriolar+smooth+muscle.">Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle.</a> Journal of Physiology. 532 (3), 609-623 (2001).</li><li>Tumelty, J., 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=Endothelin+1+stimulates+Ca2+-sparks+and+oscillations+in+retinal+arteriolar+myocytes+via+IP3R+and+RyR-dependent+Ca2++release.">Endothelin 1 stimulates Ca2+-sparks and oscillations in retinal arteriolar myocytes via IP3R and RyR-dependent Ca2+ release.</a> Investigative Ophthalmology &amp; Visual Science. 52 (6), 3874-3879 (2011).</li><li>Huynh, K. W., 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=Structural+insight+into+the+assembly+of+TRPV+channels.">Structural insight into the assembly of TRPV channels.</a> Structure. 22 (2), 260-268 (2014).</li><li>McGahon, M. K., Dawicki, J. M., Scholfield, C. N., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A-type+potassium+current+in+retinal+arteriolar+smooth+muscle+cells.">A-type potassium current in retinal arteriolar smooth muscle cells.</a> Investigative Ophthalmology &amp; Visual Science. 46 (9), 3281-3287 (2005).</li><li>McGahon, M. K., Zhang, X., Scholfield, C. N., Curtis, T. M., McGeown, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+downregulation+of+the+BKbeta1+subunit+in+diabetic+arteriolar+myocytes.">Selective downregulation of the BKbeta1 subunit in diabetic arteriolar myocytes.</a> Channels (Austin). 1 (3), 141-143 (2007).</li><li>Ljubimov, A. V., 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=Basement+membrane+abnormalities+in+human+eyes+with+diabetic+retinopathy.">Basement membrane abnormalities in human eyes with diabetic retinopathy.</a> Journal of Histochemistry and Cytochemistry. 44, 1469-1479 (1996).</li><li>Roy, S., Maiello, M., Lorenzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+basement+membrane+collagen+in+human+diabetic+retinopathy.">Increased expression of basement membrane collagen in human diabetic retinopathy.</a> Journal of Clinical Investigation. 93, 438-442 (1994).</li><li>Scholfield, C. N., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+physiology+of+retinal+and+choroidal+arteriolar+smooth+muscle+cells.">Cellular physiology of retinal and choroidal arteriolar smooth muscle cells.</a> Microcirculation. 14 (1), 11-24 (2007).</li><li>Hinds, K., Monaghan, K. P., Fr&#248;lund, B., McGeown, J. G., Curtis, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+control+of+arteriolar+diameter+in+the+rat+retina.">GABAergic control of arteriolar diameter in the rat retina.</a> Investigative Ophthalmology &amp; Visual Science. 54 (10), 6798-6805 (2013).</li><li>Torring, M. S., Aalkjaer, C., Bek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constriction+of+porcine+retinal+arterioles+induced+by+endothelin-1+and+the+thromboxane+analogue+U46619+in+vitro+decreases+with+increasing+vascular+branching+level.">Constriction of porcine retinal arterioles induced by endothelin-1 and the thromboxane analogue U46619 in vitro decreases with increasing vascular branching level.</a> Acta Ophthalmologica. 92 (3), 232-237 (2014).</li><li>P, S. k. o. v. . J. e. n. s. e. n. ., S, M. e. t. z. . M. a. r. i. e. n. d. a. l. . P. e. d. e. r. s. e. n., Aalkjaer, C., Bek, 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+vasodilating+effects+of+insulin+and+lactate+are+increased+in+precapillary+arterioles+in+the+porcine+retina+ex+vivo.">The vasodilating effects of insulin and lactate are increased in precapillary arterioles in the porcine retina ex vivo.</a> Acta Ophthalmologica. 94 (5), 454-462 (2016).</li></ol>

博士ジョアンナ Kur メドトロニック (ツイン都市、ミネソタ州、米国) の従業員は、今、彼女の現在の仕事は競合しないとここで紹介します。

上記で説明したプロトコルは、練習は必要最小限のトラブルシューティングで達成する必要があります。平均日の分離から 6-8 使用可能な動脈を取得し、3-4 実験の成功を達成するため。問題が発生した場合ただし、成功率を向上させるために取ることができるいくつかの手順があります。時折我々 とき発見した、特に若いラット (< 8 週間)、細動脈の収量が低いことができます。この問題を回避するためには、1.12 1.14 の手順で手順、および trituration のそれぞれの間の網膜組織 (500 x g で 10-30 s) を遠心分離をお勧めします。これはしばしば船の収量を向上させるのに役立ちますが、準備内細胞の残骸の量も増えます。
Cannulating 圧力図法研究血管分岐サイト近くに切断されている可能性があります側枝のために慎重に細動脈を確認することが重要です。これは、実験中に漏れの一般的な原因と圧力損失を表します。[Ca2 +]私のプロトコルで発生する主な問題は、染料のローディングのレベルです。貧しい染料の読み込みは、通常 Ca2 +ホメオスタシスの中断の結果をオーバー ロードしながら低信号対雑音比につながります。これらの問題のいずれかの可能性を減らすためには、網膜のホモジネートの小さい因数を削除ことができますと孤立した血管を読み込みプロトコル中に定期的にチェックします。事業 - パッチク ランプ実験、gigaseal 形成の成功率も高い方法に依存するとき、基底膜が消化されました。とき最初このプロトコルをテストまたは新しい多くの酵素を使用して酵素の消化力の濃度/期間を調整する必要があります。十分な基底を削除するための十分な消化は、内皮細胞および平滑筋層の分離を注意深く監視アクセス亂。慎重な監視も、血管が収縮するにつれてマニフェストすることができます過剰消化は避けるべきです。このような場合は平滑筋細胞、パッチ ・ クランプ記録のためあまりにも壊れやすい。酵素を適用すると、DNAse を含めることが重要だから解放された DNA の繊維の除去を確実に私は分離プロセスの間に損傷した細胞。DNA のフラグメントはべたべたして洗浄工程 (ステップ 4.4) の最終段階で、血管に付着する鉗子を引き起こす多くの場合容器の損失の結果します。容器の洗浄は技術的に難しいと最高の可能な先端の直径の閉じ鉗子の穏やかな抜本的な動きと高倍率 (20 X) で実行される最も。スイープ、間きれいラボで鉗子をロールバックします。
ハイライトとして以前、本稿に記載されているプロトコルの開発の後ろの主な動機は網膜血管中の血流が中断される理由を理解するためだった。糖尿病性網膜症28,33日に我々 の仕事のほとんどが集中しています。細動脈はセクション 1 で説明した方法を使用して糖尿病の実験齧歯動物モデルの網膜から分離できます。糖尿病から網膜細動脈分離の実験、密接に生体内で血管が経験, 高血糖状態を複製しようとすることが重要です。このため、分離 25 mm 実験的ソリューションで D-グルコース レベルを通常我々 引き上げます。血管基底膜の肥厚は糖尿病34,35中に網膜血管のよく知られている現象です。動物を使用すると、長期にわたる糖尿病 (> 1 ヶ月病悩期間)、増加酵素濃度または消化時間はしばしばパッチ ・ クランプ記録方法のアプリケーションを有効にする必要です。
前のヴィヴォを使用しての重要な制限事項が網膜血管生理学を勉強する網膜細動脈を分離し、病態は周囲の網膜の構造の損失。不在そして網膜の存在の血管作動性メディエーターに対する血管の反応が劇的に変化することができます網膜のグリアと神経細胞の除去は、細胞生理学の網膜血管平滑筋細胞へ簡単にアクセス可能組織。アデノシン 3 リン酸 (ATP) のアクションは、たとえば、このポイントのよい例を提供します。分離ラットの網膜細動脈, ATP トリガーの追加船36の堅牢な収縮、そのまま構造の存在中に、血管を広げる37。したがって、可能な限り、我々 は通常ください網膜全体-マウント前のヴィヴォと血管径と血流16,37の生体内測定を使用して私たちの孤立した細動脈の準備から主要な調査結果を検証するには.注記のうち、新しい方法最近小細動脈と38,39体外灌流全体のブタ網膜の毛細血管を研究するため浮上しています。このような準備は、網膜の神経網が網膜の細動脈や毛細血管のトーンに調整して網膜された血流動態の変化が網膜神経細胞の活動を調節する方法の私達の理解を改善する可能性があります。
またにおける内皮細胞の機能の研究を有効にするプロトコルも行っているが、本稿で説明している手順を理解細動脈の平滑筋細胞生理学研究用ラット網膜細動脈を当てている、これらの血管。予備的な作業で Ca2 +イメージング、ホルモンアッセイ法について実習研究の記録に適している実行可能な血管内皮細胞の管をもたらす網膜細動脈セグメントの酵素消化を変更することに成功しました。腔内配薬を有効にする、両端に孤立した細動脈の穿刺は、将来的にこれらの血管で調査される有効にする内皮依存性血管拡張反応をまたできた。

網膜の血流を制御する方法を理解することは重要な目標、異常な血流は、視力にかかわる各種の病態に関与している網膜疾患1,2,3、 4。内側の網膜神経細胞とグリア細胞の供給, 網膜の循環、網膜動脈、網膜静脈そして静脈5に毛細血管を通って細動脈からの血液のすべてと終わり動脈提携しています。網膜の血流が網膜動脈および細動脈のトーンだけでなく、網膜毛細血管・後毛細血管細静脈6,7,の壁にある血管の収縮の活動によって調節されています。8. 網膜血管音の制御は複雑であり血液循環システムおよび循環分子とホルモン、血液ガスを含む周囲の網膜組織からの入力の範囲によって変調され、血管作動性物質から解放、網膜血管内皮細胞と macroglia9,10,11。裏地の縦配置内皮細胞12,13,と網膜の細動脈は、網膜の小さな動脈枝血管平滑筋細胞の単一の層で構成されています14です。 これらの血管網膜循環内血管抵抗のメインサイトを形成し、したがって網膜血流の局所制御に重要な役割を果たします。網膜の細動脈は、拡張または収縮血管平滑筋収縮力10,1516,の変化を介したその内腔の直径によって網膜の毛細血管の血流を調節します。どの網膜細動脈調節を通じて網膜血流したがって必要です準備細動脈の平滑筋細胞をアクセスしてできるだけ生理的に近い条件で勉強できる分子機構を理解します。
まだその機能と基になる血管内皮細胞と相互接続性を維持しつつ、孤立した網膜血管の前のヴィヴォ準備は血管平滑筋細胞へのアクセスを提供します。孤立した船舶を使用してほとんどの研究が大きなウシまたはブタ動脈血管 (60-150 μ m) に焦点を当てた。これらは、市販のワイヤーや血管平滑筋細胞収縮メカニズム17,18の薬理学的尋問を有効にする圧力第 1 報システムでマウントできます。このような準備大きく通常の条件下での網膜血管生理学の知識に貢献します。いくつかの研究は、その小さい直径として実験小動物から分離された網膜細動脈を使用している (〜 8-45 μ m) 慣習的な図法システム19,20,21,22での使用を防ぐことができます。ただし、実験小動物から船舶を使用してのなる重要な利点、遺伝的変更された遺伝子組換え、網膜疾患モデルの幅広い可用性です。実験小動物は介入研究生体内で適しています。
ここで隔離し、圧力図法実験用ラット網膜細動脈の cannulating のための簡単なプロトコルについて述べる。また、Ca2 +イメージング、電気生理学のプロトコルこれらの血管を使用して詳細します。これらはさらに血管平滑筋収縮、網膜の血流の調節に洞察力を提供することができます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Beakers</td>
			<td>Fisherbrand</td>
			<td>15409083</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Curved Scissors</td>
			<td>Fisher Scientific</td>
			<td>50-109-3542</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Disposable plastic pipette/ transfer</td>
			<td>Sarstedt</td>
			<td>86.1174</td>
			<td>6mL is best but other sizes are acceptable; remove tip to widen aperture</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Brunel Microscopes LTD</td>
			<td></td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>World Precision Instruments</td>
			<td>Dumont #5 14095</td>
			<td>Any equivalent fine forceps</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Fisherbrand</td>
			<td>11546963</td>
			<td>Fire polished to reduce friction but not enough to narrow the tip</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sigma-Aldrich</td>
			<td>P7741</td>
			<td>Or any equilavent 10cm product</td>
		</tr>
		<tr>
			<td>Pipette teat</td>
			<td>Fisherbrand</td>
			<td>12426180</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Purified water supply</td>
			<td>Merek</td>
			<td>Milli-Q Integral Water Purification System</td>
			<td>Or any equilavent system</td>
		</tr>
		<tr>
			<td>Round bottomed test tube</td>
			<td>Fisherbrand</td>
			<td>14-958-10 B</td>
			<td>5 mL; any equilavent product</td>
		</tr>
		<tr>
			<td>Seratted forceps</td>
			<td>Fisher Scientific</td>
			<td>17-467-230</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Single edge blades</td>
			<td>Agar Scientific</td>
			<td>T585</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>Dow Corning</td>
			<td>184</td>
			<td>Or any pliable surface&#160;</td>
		</tr>
		<tr>
			<td>Testtube rack</td>
			<td>Fisherbrand Derlin</td>
			<td>10257963</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Pressure myography</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-axis mechanical manipulator</td>
			<td>Scientifica</td>
			<td>LBM-7&#160;</td>
			<td>x2 (or any equilavent product)</td>
		</tr>
		<tr>
			<td>3-way taps</td>
			<td>Cole-Parmer</td>
			<td>UY-30600-02</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Air table</td>
			<td>Technical Manufacturing Corporation&#160;</td>
			<td>Clean Bench</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Analysis software&#160;</td>
			<td>Image J</td>
			<td>https://downloads.imagej.net/fiji/</td>
			<td>Free imaging software</td>
		</tr>
		<tr>
			<td>Analysis plugin</td>
			<td>Myotraker&#160;</td>
			<td>https://doi.org/10.1371/journal.pone.0091791.s002</td>
			<td>Custom plugin for imageJ Freely available for download</td>
		</tr>
		<tr>
			<td>Cannulation pipette glass</td>
			<td>World Percision instruments</td>
			<td>TW150F-4</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Optiplex 7010</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Digital Thermometer</td>
			<td>RS</td>
			<td>206-3722</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Fluo-4-AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>F14201</td>
			<td>Make 1 mM stock in DMSO</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>World Precision Instruments</td>
			<td>Dumont #7 14097</td>
			<td>Any equivalent fine forceps</td>
		</tr>
		<tr>
			<td>Fura-2-AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>F1221</td>
			<td>Make 1 mM stock in DMSO</td>
		</tr>
		<tr>
			<td>Glass bottomed recording chamber</td>
			<td>Warner Instruments</td>
			<td>64-0759</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Helper pipette glass</td>
			<td>World Percision instruments</td>
			<td>IB150F-3</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>In-line heater</td>
			<td>Custom made</td>
			<td></td>
			<td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclypse TE 300</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Manometer</td>
			<td>Riester</td>
			<td>LF1459</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Microelectrode puller</td>
			<td>Sutter instruments</td>
			<td>P97</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Microforge +&#160; Olympus CX 31 microscope</td>
			<td>Glassworks</td>
			<td>Fine Point F-550</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter instruments</td>
			<td>MP-285</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Multi-channel delivery manifold&#160;</td>
			<td>Automate Scientific</td>
			<td>Perfusion Pencil</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Pipette filler&#160;</td>
			<td>BD Plastipak</td>
			<td>1mL</td>
			<td>Syringe heated and pulled to internal diameter of pipette</td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>Molecular Devices</td>
			<td>1-HL-U</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Suction pump</td>
			<td>Interpet</td>
			<td>AP1</td>
			<td>Converted aeration pump by reversing bellows</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Plastipak</td>
			<td>20mL Luer-lok</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Tungsten wire</td>
			<td>Advent</td>
			<td>W558818</td>
			<td>75&#956;m diameter</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>VWR</td>
			<td>miscellaneous sizes</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>USB camera</td>
			<td>&#160;Logitech&#160;</td>
			<td>HD Pro Webcam C920</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Video capture software</td>
			<td>Hypercam</td>
			<td>version 2.28.01</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td>Sub Aqua 12 Plus</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x20 lens</td>
			<td>Nikon</td>
			<td>20x/0.40 WD 3.8, &#8734;/0.17</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x4 lens</td>
			<td>Nikon</td>
			<td>4x/0.10, WD 30, &#8734;/-</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Y-connectors</td>
			<td>World Percision Instruments</td>
			<td>14012</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Patch clamping</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-way taps</td>
			<td>Cole-Parmer</td>
			<td>UY-30600-02</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>3-axis mechanical manipulator</td>
			<td>Scientifica</td>
			<td>LBM-7&#160;</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Air table</td>
			<td>Technical Manufacturing Corporation&#160;</td>
			<td>Clean Bench</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2411</td>
			<td>3 mg disolved daily in 50 &#956;L of DMSO (sonicate to dissolve)</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices</td>
			<td>Axopatch 200a</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Analogue digital converter</td>
			<td>Axon</td>
			<td>Digidata 1440A</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Collagenase Type 1A</td>
			<td>Sigma-Aldrich</td>
			<td>C9891&#160;</td>
			<td>0.1mg/mL in LCH</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Optiplex 7010</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Digital Thermometer</td>
			<td>RS</td>
			<td>206-3722</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Millipore</td>
			<td>260913</td>
			<td>Working stock 1 MU mL-1 (10 MU diluted in 10 mL LCH)&#160;</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Custom made</td>
			<td></td>
			<td>Any equilavent commerical product</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>No. 7</td>
			<td>Any superfine forcep</td>
		</tr>
		<tr>
			<td>Glass bottomed recording chamber</td>
			<td>Warner Instruments</td>
			<td>64-0759</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Headstage</td>
			<td>Molecular Devices</td>
			<td>CV203BU</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>In-line heater</td>
			<td>Custom made</td>
			<td></td>
			<td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclypse TE 300</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Microelectrode puller</td>
			<td>Sutter instruments</td>
			<td>P97</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Microforge +&#160; Olympus CX 31 microscope</td>
			<td>Glassworks</td>
			<td>Fine Point F-550</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter instruments</td>
			<td>MP-285</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Multi-channel delivery manifold&#160;</td>
			<td>Automate Scientific</td>
			<td>Perfusion Pencil</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Patching software</td>
			<td>Molecular Devices</td>
			<td>Pclamp v10.2</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Pipette filler&#160;</td>
			<td>BD Plastipak</td>
			<td>1mL</td>
			<td>Syringe heated and pulled to internal diameter of pipette</td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>Molecular Devices</td>
			<td>1-HL-U</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Protease Type XIV</td>
			<td>Sigma-Aldrich</td>
			<td>P5147&#160;</td>
			<td>0.01mg/mL in LCH</td>
		</tr>
		<tr>
			<td>Single channel pipette glass</td>
			<td>World Percision instruments</td>
			<td>IB150F-3</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Suction pump</td>
			<td>Interpet</td>
			<td>AP1</td>
			<td>Converted aeration pump by reversing bellows</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Plastipak</td>
			<td>20mL Luer-lok</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Tungsten wire</td>
			<td>Advent</td>
			<td>W557418</td>
			<td>50&#956;m diameter</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>VWR</td>
			<td>miscellaneous sizes</td>
			<td>Any equilavent product to fit</td>
		</tr>
		<tr>
			<td>USB camera</td>
			<td>&#160;Logitech&#160;</td>
			<td>HD Pro Webcam C920</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td>Sub Aqua 12 Plus</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Whole cell pipette glass</td>
			<td>Warner Instruments</td>
			<td>GC150TF-7.5</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x20 lens</td>
			<td>Nikon</td>
			<td>20x/0.40 WD 3.8, &#8734;/0.17</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x4 lens</td>
			<td>Nikon</td>
			<td>4x/0.10, WD 30, &#8734;/-</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x40 lens</td>
			<td>Nikon</td>
			<td>40x/0.55, WD 2.1, &#8734;/1.2</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Y-connectors</td>
			<td>World Percision Instruments</td>
			<td>14012</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Ca2+ imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-way taps</td>
			<td>Cole-Parmer</td>
			<td>UY-30600-02</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>3-axis mechanical manipulator</td>
			<td>Scientifica</td>
			<td>LBM-7&#160;</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Acquisition software&#160;</td>
			<td>Cairn Research Ltd.</td>
			<td>Acquisition Engine V1.1.5</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>Air table</td>
			<td>Technical Manufacturing Corporation&#160;</td>
			<td>Clean Bench</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Analysis software for confocal imaging</td>
			<td>Image J</td>
			<td>https://downloads.imagej.net/fiji/</td>
			<td>Free imaging software</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Optiplex 7010</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica Geosystems</td>
			<td>SP5&#160;</td>
			<td>Or any equilavent product; confocal</td>
		</tr>
		<tr>
			<td>Digital Thermometer</td>
			<td>RS</td>
			<td>206-3722</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>No. 7</td>
			<td>Any superfine forcep</td>
		</tr>
		<tr>
			<td>Glass bottomed recording chamber</td>
			<td>Warner Instruments</td>
			<td>64-0759</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>In-line heater</td>
			<td>Custom made</td>
			<td></td>
			<td>Commerical equilavent SH-27B Solution In-Line Heater from Harvard Apparatus</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TE2000</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>Monochromator&#160;</td>
			<td>Cairn Research Ltd.</td>
			<td>Optoscan</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>Multi-channel delivery manifold&#160;</td>
			<td>Automate Scientific</td>
			<td>Perfusion Pencil</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Pipette filler&#160;</td>
			<td>BD Plastipak</td>
			<td>1mL</td>
			<td>Syringe heated and pulled to internal diameter of pipette</td>
		</tr>
		<tr>
			<td>Software for confocal microscope</td>
			<td>Leica Geosystems</td>
			<td>LAS-AF version 3.3.</td>
			<td>Or any equilavent product; confocal</td>
		</tr>
		<tr>
			<td>Suction pump</td>
			<td>Interpet</td>
			<td>AP1</td>
			<td>Converted aeration pump by reversing bellows</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Plastipak</td>
			<td>20mL Luer-lok</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Tungsten wire</td>
			<td>Advent</td>
			<td>W557418</td>
			<td>50&#956;m diameter</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>VWR</td>
			<td>miscellaneous sizes</td>
			<td>Any equilavent product to fit</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td>Sub Aqua 12 Plus</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>x100 lens</td>
			<td>Nikon</td>
			<td>x100 N.A. 1.3 oil</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>x20 lens</td>
			<td>Nikon</td>
			<td>20x/0.40 WD 3.8, &#8734;/0.17</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>x20 lens</td>
			<td>Leica Geosystems</td>
			<td>HCX PL FLUOTAR, 20x/0.50, &#8734;/0.17/D</td>
			<td>Or any equilavent product; confocal</td>
		</tr>
		<tr>
			<td>x4 lens</td>
			<td>Nikon</td>
			<td>4x/0.10, WD 30, &#8734;/-</td>
			<td>Or any equilavent product; microfluorimetry</td>
		</tr>
		<tr>
			<td>x4 lens</td>
			<td>Leica Geosystems</td>
			<td>C PLAN, 4x/0.10, &#8734;/-/&#10013;</td>
			<td>Or any equilavent product; confocal</td>
		</tr>
		<tr>
			<td>x63 lens</td>
			<td>Leica Geosystems</td>
			<td>HCX PL APO, 63x/1.40 - 0.60 OIL CS &#8734;/0.17/E</td>
			<td>Or any equilavent product; confocal</td>
		</tr>
		<tr>
			<td>Y-connectors</td>
			<td>World Percision Instruments</td>
			<td>14012</td>
			<td>Or any equilavent product</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td></td>
			<td></td>
			<td>Specifications and settings for fabrication of pipettes for experimentation</td>
		</tr>
		<tr>
			<td>Helper pipette</td>
			<td>World Percision instruments</td>
			<td>IB150F-3</td>
			<td>Inner diameter: 0.86 mm 
			Ramp: 261 
			Pressure: 300 
			Heat: 280 
			Pull : 0 
			Velocity: 56 
			Time: 250 
			No of cycles: 4 
			Tip diameter: 0.5-2 &#956;m 
			Polishing: yes 
			Final Resistance: &#62;10 M&#937;</td>
		</tr>
		<tr>
			<td>Cannulation pipette</td>
			<td>World Percision instruments</td>
			<td>TW150F-4</td>
			<td>Inner diameter: 1.17 mm 
			Ramp: 290 
			Pressure: 300 
			Heat: 280 
			Pull : 0 
			Velocity: 58 
			Time: 150 
			No of cycles: 4 
			Tip diameter: 3-10 &#956;m 
			Polishing: no 
			Final Resistance: &#60;1 M&#937;</td>
		</tr>
		<tr>
			<td>On-cell patching</td>
			<td>World Percision instruments</td>
			<td>IB150F-3</td>
			<td>Inner diameter: 0.86 mm 
			Ramp: 261 
			Pressure: 300 
			Heat: 280 
			Pull : 0 
			Velocity: 56 
			Time: 250 
			No of cycles: 4 
			Tip diameter: 0.5-2 &#956;m 
			Polishing: yes 
			Final Resistance: &#62;5 M&#937;</td>
		</tr>
		<tr>
			<td>Whole-cell patching</td>
			<td>Warner Instruments</td>
			<td>GC150TF-7.5</td>
			<td>Inner diameter: 1.17 mm 
			Ramp: 296 
			Pressure: 200 
			Heat: 287 
			Pull : 0 
			Velocity: 50 
			Time: 250 
			No of cycles: 4 
			Tip diameter: 2-3 &#956;m 
			Polishing: helpful but not necessary 
			Final Resistance: 1-2 M&#937;</td>
		</tr>
	</tbody>
</table>

isolation of retinal arterioles for ex vivo cell physiology studies

網膜は、高い代謝活性の組織で実質的な血液供給を必要とします。網膜循環には、脈絡膜血管供給視細胞間内部の網膜がサポートしています。網膜の血流の変化、緑内障や網膜枝静脈閉塞糖尿病性網膜症を含む、多数の視力を脅かす障害に。網膜と眼疾患の中にどのようにこれらの変更を介して血流の調節に関わる分子機構の解明は、これらの条件の処置のための新しいターゲットの同定に 。網膜の細動脈は網膜の主要な抵抗血管であり、その結果、内腔の直径の変化を通じて網膜循環動態の調節に重要な役割を果たします。近年では、細胞生理学を含むアプリケーションの広い範囲のために適しているラット網膜の細動脈を分離する方法を開発しました。どのように血流が網膜内で制御して眼疾患中に発生する主な変更のいくつかを識別することができましたに新しい洞察力をもたらすこの準備が始まっています。この記事では、ラット網膜細動脈の隔離のための方法を説明およびパッチ ・ クランプ電気生理学、カルシウム イメージング、圧力図法の研究の使用のためのプロトコルが含まれます。これらの血管は PCR - 西部にしみが付くと免疫組織化学-ベースの研究での使用に適しています。

Watch this Scientific Journal Video about Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies at JoVE.com

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

本稿では、電気生理学的、カルシウム イメージングおよび圧力図法試験に使用することができますラット網膜の細動脈の隔離のための簡単なプロトコルについて説明します。

Ex Vivo細胞生理学研究のため網膜細動脈の隔離

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

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Biology

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

Mouse Adrenal Chromaffin Cell Isolation

マウス副腎髄質クロマフィン細胞分離

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

マウス網膜神経節細胞における遺伝子発現のための子宮内および生体外でのエレクトロポレーションで

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

In vivo-like Organotypic Murine Retinal Wholemount Culture

生体のような器官型マウス網膜Wholemountの文化で

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

レンチウイルスを介したノックダウン中生体外で</emヒト造血幹細胞の>赤血球

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

ザ ex vivoで</em血管反応性の調査のために>隔離された骨格微小血管の準備

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

An Ex vivo Culture System to Study Thyroid Development

An Ex vivo Culture System to Study Thyroid Development

アン<em&gt;生体外</em甲状腺の開発を研究する&gt;文化システム

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

運動の時空間マッピングで<em&gt;エクスビボ</em腸の&gt;準備

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

ラット精巣挙筋の細動脈における無細胞層の可視化と定量化

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

マウス生殖管自発的運動性を評価するためのEx Vivo法とデータ解析のためのMATLABベースの子宮運動追跡アルゴリズム

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

上頭頂皮細胞活性化に関与する経路を解析するエクスビボアッセイとしての糸球体成長

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...