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

이 문서에서 설명 하는 프로토콜의 개발은 다음 자금 지원 기관에서 교부 금에 의해 지원 되었다: BBSRC (BB/I026359/1), 시력 (1429와 1822), 소아 당뇨병 연구 재단 (2-2003-525), Wellcome 신뢰 (074648/Z/04)에 대 한 싸움 영국 심장 재단 (PG/11/94/29169), HSC R & D 부문 (STL/4748/13) 및 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>

박사 조 안 나 Kur Medtronic (트윈-도시, 미네소타, 미국)의 직원은 지금, 그녀의 현재 작업을 충돌 하지 않는 여기에 제시 된 그.

위에서 설명한 프로토콜 연습을 필요로 하지만 최소한의 문제 해결 달성 되어야 합니다. 평균 하루에 우리 6-8 사용할 arterioles 고립에서 얻을 것이 고 3-4 성공적인 실험을 달성. 그러나 문제가 발생 하는 경우, 성공률을 개선 하기 위해 취할 수 있는 몇 가지 단계가 있습니다. 때때로 우리는 발견, 어린 쥐 (< 8 주)를 사용 하는 경우에 특히 arterioles의 수익률 낮은 될 수 있습니다. 이 문제를 회피, 단계 1.12-1.14 단계는 분쇄의 각 사이 망막 조직 (500g x 10-30 s) centrifuging 좋습니다 것 이다. 이 종종 혈관의 수율을 개선 하는 데 도움이 하지만 준비 내 세포 파편의 양을 증가.
때 혈관 압력 myography 연구에 대 한 cannulating arterioles 수 분기 가까운 죽 습 되었습니다가지고 있는 모든 사이드 지점에 대 한 신중 하 게 확인 하기 중요 하다. 이 실험 동안 누설의 일반적인 원인 및 압력의 손실을 나타냅니다. [캘리포니아2 +]나 프로토콜 함께 발생할 수 있는 주요 문제는 염료 로드의 수준 이다. 불 쌍 한 염료 로드 일반 Ca2 + 항상성 장애에 결과 오버 로드 하는 동안 낮은 신호 대 잡음 비율, 리드. 이러한 문제 중 하나에의 가능성을 줄이기 위해, 망막 homogenate의 작은 약 수를 제거할 수 있습니다, 하 고 격리 된 선박 로드 프로토콜 동안 정기적으로 검사 합니다. 사업 패치 클램프 실험, gigaseal 형성의 성공률도 높은 방법에 의존 하는 경우 기저 lamina 소화 되어 있다. 때 처음이 프로토콜 테스트 또는 사용 하 여 새로운 많은 효소의 효소 소화 농도/기간 조정 필요할 수 있습니다. 충분히 기초를 제거 하는 충분 한 소화 보장 한다 내 피 및 평활 근 층의 분리를 신중 하 게 모니터링 액세스 laminal. 주의 깊은 모니터링 하는 것은 또한 혈관 수축 시작으로 매니 페스트 수 있습니다-소화를 방지 하는 데 필요한입니다. 이 경우, 평활 근 세포는 종종 패치 클램프 기록에 대 한 너무 약 합니다. 효소를 적용할 때 그것은 DNAse를 포함 하는 것이 중요 내가 DNA에서 해방의의 제거를 보장 하기 위해 손상 된 세포 격리 과정. DNA 파편은 끈 적 원인과 (4.4 단계), 청소 과정의 최종 단계는 되에 고착을 겸 종종 선박 손실의 결과로. 혈관 청소 기술적으로 어려운 이며 가장 높은 최고의 가능한 팁 직경의 닫힌된 집게의 부드러운 연소 움직임 확대 (20 X)에서 수행 됩니다. 스윕, 사이 랩 롤과 집게 청소.
강조,이 원고에 설명 된 프로토콜의 개발 뒤에 주요 동기 망막 혈관 질환 중 혈 중단 왜 이해를 했다. 날짜를 우리의 작품의 대부분은 당뇨병 안구 질환28,33에 집중 했다. Arterioles 섹션 1에에서 설명 된 방법을 사용 하 여 당뇨병의 실험적인 설치류 모델의 망막에서 격리 될 수 있습니다. 실험에서 당뇨병 동물에서 분리 된 망막 arterioles 때 그것은 밀접 하 게 비보에혈관에 의해 경험 hyperglycemic 조건 복제 하려고 하는 것이 중요. 이러한 이유로, 우리가 일반적으로 우리의 절연 및 25 m m 실험적인 솔루션에서 D-포도 당 수준을 인상 것입니다. 혈관 지하실 막 두껍게 당뇨병34,35동안 망막 혈관에 잘 인식 된 현상입니다. 동물을 사용 하 여 장기간 당뇨병 (> 1 개월 질병 기간) 때 증가 효소 농도 또는 소화 시간 종종 필요 패치 클램프 기록 방법의 응용 프로그램을 활성화 합니다.
비보 전 을 사용 하 여 중요 한 제한 절연 망막 arterioles 망막 혈관 생리학을 공부 하 고 이상 주변 망막 neuropile의 손실 이다. 망막 폐해 및 신경 세포의 제거는 세포 생리학 연구에 대 한 망막 혈관 평활 근 세포에 쉽게 액세스할 수 있습니다,. 비록 vasoactive 중재자에 대 한 혈관의 응답 부재 및 망막의 존재에서 극적으로 변경할 수 있습니다. 조직입니다. 아데노신 트라이-인산 염 (ATP)의 작업은 예를 들어이 포인트의 좋은 그림을 제공합니다. 고립 된 쥐 망막 arterioles, ATP 트리거의 추가 선박36의 강력한 수축, 그대로 neuropile의 존재에 혈관 팽창37. 따라서, 가능 하다 면, 우리가 일반적으로 시도 비보 전 망막 전체 마운트 및 선박 직경 및 혈액 흐름16,37 의 측정 vivo에서 를 사용 하 여 우리의 고립된 되 준비에서 주요 연구 결과 유효성을 검사 하 . 주의 새로운 방법을 최근 작은 arterioles38,39 비보 전전체 끼얹는다 돼지 망막 모세 혈관을 공부 하 고 대 한 나왔다. 이러한 준비 크게 망막 neuropile 망막 arteriolar 및 모 세관 톤을 조절 하는 방법 및 망막 haemodynamics에 변화 망막에 신경 활동을 조절 하는 어떻게의 우리의 이해를 향상 시킬 가능성이 있다.
이 문서에서 설명 하는 절차는 이해 arteriolar 부드러운 근육 세포 생리학에 대 한 고립 된 쥐 망막 arterioles의 사용에 초점을 맞추고 있다, 하지만 우리는 현재 프로토콜에 내 피 세포 기능 연구 활성화를 개발합니다 이러한 선박입니다. 예비 작업에서 우리는 캘리포니아2 + 이미징 및 연구 기록 patchclamp 의무가 가능한 내 피 세포 튜브를 망막 arteriolar 세그먼트의 효소 소화 수정에 성공 되었습니다. Intraluminal 배달, 약물의 사용, 양쪽 끝에 격리 arterioles cannulation 수도 미래에 이러한 선박에 조사를 사용 피 종속 vasodilatory 응답.

비정상적인 혈 시력을 위협 하는 다양 한의 병 인에 연루 되어 이후 중요 한 목표는 망막에 혈액 흐름을 제어 하는 방법을 이해 망막 질병1,2,3, 4. 제공 안 망막 신경 그리고 glial 세포, 망막 순환 arterioles 망막 venules 그리고 마지막으로 혈관5모 세관을 통과 하 고 망막 동맥에서 혈액의 모든 최종 동맥 배열을, 있다. 망막에 혈액 흐름의 망막 동맥, arterioles 톤 후 모 세관 venules6,7, 및 망막 모세 혈관의 벽에 있는 pericytes의 수축 성 활동에 의해 통제 된다 8. 망막 혈관 음색의 제어는 복잡 하 고 다양 한 분자와 호르몬, 순환 혈액 가스를 포함 한 주변 망막 조직 하 고 순환 시스템에서 입력에 의해 변조 및 vasoactive 물질에서 발표는 망막 혈관 내 피와 macroglia9,,1011. 망막 arterioles 망막의 작은 동맥 가지 단일 층의 혈관 평활 근 세포의 구성와의 내부 안 감 경도 배열 내 피 세포12,13, 14. 이러한 혈관 망막 순환 내 혈관 저항의 주요 사이트를 형성 하 고 따라서 망막 혈액 흐름의 로컬 제어에서 중요 한 역할. 망막 arterioles dilating 또는 변화 혈관 평활 근 수축10,15,16에 의해 중재 luminal 그들의 직경을 constricting에 의해 망막에 모 세관 혈액의 흐름을 조절 합니다. Arterioles 규제는 망막을 통해 망막 관류 필요 준비 arteriolar 평활 근 세포를 액세스할 수 있으며 가능한 생리 적 조건에 가깝게에서 공부 하는 분자 메커니즘을 이해.
고립 된 망막 혈관의 비보 전 준비 아직도 그들의 기능 및 기본 피와 상호를 유지 하는 동안 혈관 평활 근 세포에 대 한 액세스를 제공 합니다. 격리 된 용기를 사용 하 여 현재까지 대부분의 연구는 큰 소 또는 돼지 동맥 혈관 (60-150 µ m)에 집중 했다. 이러한 상용 와이어 또는 혈관 평활 근 세포 수축 메커니즘17,18의 약리 심문 수 있도록 압력 myograph 시스템에 장착할 수 있습니다. 이러한 준비 정상적인 조건에서 망막 혈관 생리학의 우리의 지식에 크게 기여 했다. 몇 가지 연구는 그들의 작은 직경으로 작은 실험실 동물에서 분리 된 망막 arterioles 사용 (~ 8-45 µ m) 기존의 myography 시스템19,20,,2122에 그들의 사용을 방지. 그러나 중요 한 이점,, 작은 실험 동물에서 혈관을 사용 하 여 유전자 변형, 유전자 변형 및 망막 질환 모델의 넓은 가용성 이다. 작은 실험실 동물 개입 연구 비보에 대 한 더 많은 의무가 있습니다.
여기 우리는 분리 하 고 압력 myography 실험 쥐 망막 arterioles cannulating에 대 한 간단한 프로토콜을 설명 합니다. 캘리포니아2 + 이미징 및 전기 생리학 프로토콜 사용 하 여 이러한 혈관도 자세히 나와 있습니다. 이러한 추가 혈관 평활 근 수축 및 망막에 혈액 흐름의 규칙으로 통찰력을 제공할 수 있습니다.

<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

망막은 상당한 혈액 공급을 필요로 하는 높은 metabolically 활성 조직. 망막 순환 안 배는 대뇌를 공급 하는 동안 내부 망막을 지원 합니다. 당뇨병 성 망막 증을 포함 하 여 수많은 시력을 위협 하는 장애에 기여 하는 망막 관류에 변경, 녹 내장 및 망막 정 맥 않기. 망막이 안구 질병 동안 변경 되는 방법을 통해 혈액 흐름의 제어에 관련 된 분자 메커니즘을 이해 이러한 조건의 치료에 대 한 새로운 목표의 식별 될 수 있습니다. 망막 arterioles, 망막의 주요 저항 혈관 이며 따라서 luminal 직경에서 변화를 통해 망막 hemodynamics 조절에 핵심적인 역할을 담당할. 최근 몇 년 동안, 우리는 다양 한 세포 생리학 연구를 포함 하는 응용 프로그램에 대 한 적당 한 쥐 망막에서 arterioles 격리 하기 위한 방법을 개발 했습니다. 이 준비는 이미 혈은 망막에 제어 방법과 눈 질병 중 발생 하는 주요 변경 사항 중 일부를 식별 하기 위해 우리 수 있다에 새로운 통찰력을 얻을 시작 됩니다. 이 문서에서는, 우리는 쥐 망막 arterioles의 고립 위한 방법을 설명 하 고 패치 클램프 전기 생리학, 칼슘 이미징 및 압력 myography 연구에 그들의 사용에 대 한 프로토콜을 포함. 이러한 혈관 PCR-서 부 럽 고 immunohistochemistry-기반 연구에 사용 하기 위해 의무가 있습니다.

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

이 원고는 arterioles, electrophysiological 칼슘 이미징 및 압력 myography 연구에 사용 될 수 있는 쥐 망막에서의 격리에 대 한 간단한 프로토콜을 설명 합니다.

Ex Vivo 세포 생리학 연구에 대 한 망막 Arterioles의 절연

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

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JoVE Journal

Biology

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

10.2K 조회수.  Queen's University of Belfast. 이 원고는 arterioles, electrophysiological 칼슘 이미징 및 압력 myography 연구에 사용 될 수 있는 쥐 망막에서의 격리에 대 한 간단한 프로토콜을 설명 합니다.

비디오: 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.

유동 세포 계측법에 의한 일차 망막 신경절 세포 (RGCs)의 분리

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

연구

생체공학

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.

차세대 염기서열분석을 위한 뮤린 망막 내피 세포의 분리

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

발생학

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.

마우스 뇌 혈관의 정화

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 ...

신경과학

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.

분리 및 대뇌 실질 세동맥의 캐 뉼러

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.

마우스 망막 Arterioles Ex Vivo 에서 반응의 측정을 위한 준비 단계

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

의학

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.

통가 경사진 마우스 망막의 장기, 세럼 없는 재배는 그대로 망막 색소 상피와 함께 익스처스

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.

비증식 및 증식 망막증을 가진 마우스의 전체 산 망막에 있는 혈관 매개변수의 정량화

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.

망막 신경혈관 질환 연구를 위한 생체 외 모델로서의 성인 마우스 망막의 망막 외 이식편

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.

망막 Cryo-섹션, Microvascular Pericytes의 Immunohistochemical 시각화에 대 한 전체-마운트 및 소형 고립 된 맥 관 구조

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

생물학

당뇨병성 망막병증 발병기전 연구를 위한 새로운 망막 보호 분석

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.

저자 스포트라이트: 망막 혈관 회복력과 질병 진행의 이해

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.

마우스의 당뇨병 관련 사망으로부터 망막 혈관 보호를 감지하기 위한 분석

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