Name: isolation of retinal arterioles for ex vivo cell physiology studies
Uploaded: 2018-07-14T15:00:00.000+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><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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Clean Bench</td><td>Or any equilavent product</td></tr><tr><td>Analysis software&amp;nbsp;</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&amp;nbsp;</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 +&amp;nbsp; 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&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</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&amp;mu;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>&amp;nbsp;Logitech&amp;nbsp;</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, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</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&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Air table</td><td>Technical Manufacturing Corporation&amp;nbsp;</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 &amp;mu;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&amp;nbsp;</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)&amp;nbsp;</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 +&amp;nbsp; 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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;mu;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>&amp;nbsp;Logitech&amp;nbsp;</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, &amp;infin;/0.17</td><td>Or any equilavent product</td></tr><tr><td>x4 lens</td><td>Nikon</td><td>4x/0.10, WD 30, &amp;infin;/-</td><td>Or any equilavent product</td></tr><tr><td>x40 lens</td><td>Nikon</td><td>40x/0.55, WD 2.1, &amp;infin;/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>Ca&lt;sup&gt;2+&lt;/sup&gt; 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&amp;nbsp;</td><td>Or any equilavent product</td></tr><tr><td>Acquisition software&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Cairn Research Ltd.</td><td>Optoscan</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>Multi-channel delivery manifold&amp;nbsp;</td><td>Automate Scientific</td><td>Perfusion Pencil</td><td>Or any equilavent product</td></tr><tr><td>Pipette filler&amp;nbsp;</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&amp;mu;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, &amp;infin;/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, &amp;infin;/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, &amp;infin;/-</td><td>Or any equilavent product; microfluorimetry</td></tr><tr><td>x63 lens</td><td>Leica Geosystems</td><td>HCX PL APO, 63x/1.40 - 0.60 OIL CS &amp;infin;/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>&lt;strong&gt;Pipettes&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Specifications and settings for fabrication of pipettes for experimentation&lt;/strong&gt;</td></tr><tr><td>Helper pipette</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;10 M&amp;Omega;</td></tr><tr><td>Cannulation pipette</td><td>World Percision instruments</td><td>TW150F-4</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 290&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 58&lt;br/&gt; Time: 150&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 3-10 &amp;mu;m&lt;br/&gt; Polishing: no&lt;br/&gt; Final Resistance: &amp;lt;1 M&amp;Omega;</td></tr><tr><td>On-cell patching</td><td>World Percision instruments</td><td>IB150F-3</td><td>Inner diameter: 0.86 mm&lt;br/&gt; Ramp: 261&lt;br/&gt; Pressure: 300&lt;br/&gt; Heat: 280&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 56&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 0.5-2 &amp;mu;m&lt;br/&gt; Polishing: yes&lt;br/&gt; Final Resistance: &gt;5 M&amp;Omega;</td></tr><tr><td>Whole-cell patching</td><td>Warner Instruments</td><td>GC150TF-7.5</td><td>Inner diameter: 1.17 mm&lt;br/&gt; Ramp: 296&lt;br/&gt; Pressure: 200&lt;br/&gt; Heat: 287&lt;br/&gt; Pull : 0&lt;br/&gt; Velocity: 50&lt;br/&gt; Time: 250&lt;br/&gt; No of cycles: 4&lt;br/&gt; Tip diameter: 2-3 &amp;mu;m&lt;br/&gt; Polishing: helpful but not necessary&lt;br/&gt; Final Resistance: 1-2 M&amp;Omega;</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 ...

isolation-of-retinal-arterioles-for-ex-vivo-cell-physiology-studies

Research

JoVE Journal

Biology

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

10.2K ビュー.  Queen's University of Belfast. 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。

ビデオ: Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

フローサイトメトリーによる一次マウス網膜神経節細胞（RGCs）の単離

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

生物工学

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

次世代シーケンシングのためのマウス網膜内皮細胞の単離

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

発生生物学

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

マウス脳血管の精製

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

神経科学

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

単離および脳実質細動脈のカニューレ挿入

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

マウス網膜細動脈Ex Vivoでの反応性の測定の準備手順

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

医学

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

無条件網膜色素上皮を用いた、オルガマウス網膜の長期無血清栽培

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

不拡散性網膜症を有するマウスの全型網膜における血管パラメータの定量化

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

網膜神経血管疾患研究のための ex vivo モデルとしての成体マウス網膜網膜の外植片

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

網膜の低温電子顕微鏡セクション全体-マウント、および微小血管の免疫組織化学的可視化の低張性分離血管準備

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

生物学

糖尿病性網膜症の病因研究のための新しい網膜保護アッセイ

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves the loss of protection from diabetes-induced damage to the retina, while the second phase centers on the accumulation of this damage. Traditional assays primarily focus on evaluating capillary degeneration, which is indicative of the severity of damage, essentially addressing the second phase of DR. However, they only indirectly provide insights into whether the protective mechanisms of the retinal vasculature have been compromised. To address this limitation, a novel approach was developed to directly assess the retina's protective mechanisms - specifically, its resilience against diabetes-induced insults like oxidative stress and cytokines. This protection assay, although initially designed for diabetic retinopathy, holds the potential for broader applications in both physiological and pathological contexts. In summary, understanding the pathogenesis of diabetic retinopathy involves recognizing the dual phases of protection loss and damage accumulation, with this innovative protection assay offering a valuable tool for research and potentially extending to other medical conditions.

著者スポットライト:網膜血管の回復力と疾患の進行を理解する

A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines.

マウスの糖尿病関連死からの網膜血管系の保護を検出するためのアッセイ

Diabetic retinopathy (DR) is a complex and progressive ocular disease characterized by two distinct phases in its pathogenesis. The first phase involves ...