Name: assessment and characterization of hyaloid vessels in mice
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この研究は、国立衛生研究所(NIH)の助成金(R01 EY024963およびEY028100)がJ.C.Z.W.に支援し、ナイツテンプラーアイ財団キャリアスターター助成金によって支援されました。本研究で説明したヒアロイド分離手順は、リチャード・ラング博士、栗原俊英博士、ロイス・スミス博士が寛大に共有したプロトコルからの改変に適応した。

すべての動物は、国立衛生研究所のガイドラインに従って、眼科および視力研究における動物の使用に関する視力と眼科研究協会(ARVO)の声明に従って治療された。ボストン小児病院の施設動物ケア・使用委員会(IACUC)が定める実験手順および規制に対する動物のケアと使用に関するNIH)。Lrp5-/-マウス (在庫なし 005823;ジャクソン研究所)とその野生型(WT)制御C57BL/6Jマウス(ストックNo...

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	<li>Lutty, G. A., McLeod, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+hyaloid,+choroidal+and+retinal+vasculatures+in+the+fetal+human+eye.">Development of the hyaloid, choroidal and retinal vasculatures in the fetal human eye.</a> Progress in Retinal and Eye Research. 62, 58-76 (2018).</li><li>Anand-Apte, B., Hollyfield, J., Besharse, J., Bok, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+anatomy+of+the+retinal+and+choroidal+vasculature.">Developmental anatomy of the retinal and choroidal vasculature.</a> The Retina and its Disorders. , (2011).</li><li>Hobbs, R. P., Hartnett, M. E., Hartnett, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2:+The+hyaloidal+vasculature+and+its+role+in+development.">Chapter 2: The hyaloidal vasculature and its role in development.</a> Pediatric Retina: Second Edition. , (2013).</li><li>Fruttiger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+retinal+vasculature.">Development of the retinal vasculature.</a> Angiogenesis. 10 (2), 77-88 (2007).</li><li>Saint-Geniez, M., D'Amore, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+pathology+of+the+hyaloid,+choroidal+and+retinal+vasculature.">Development and pathology of the hyaloid, choroidal and retinal vasculature.</a> The International Journal of Developmental Biology. 48 (8-9), 1045-1058 (2004).</li><li>Ito, M., Yoshioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regression+of+the+hyaloid+vessels+and+pupillary+membrane+of+the+mouse.">Regression of the hyaloid vessels and pupillary membrane of the mouse.</a> Anatomy and Embryology. 200 (4), 403-411 (1999).</li><li>Stahl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+retina+as+an+angiogenesis+model.">The mouse retina as an angiogenesis model.</a> Investigative Ophthalmology &amp; Visual Science. 51 (6), 2813-2826 (2010).</li><li>Liu, C. H., Wang, Z., Sun, Y., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ocular+angiogenesis:+from+development+to+pathologies.">Animal models of ocular angiogenesis: from development to pathologies.</a> FASEB Journal. 31 (11), 4665-4681 (2017).</li><li>Shastry, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+hyperplastic+primary+vitreous:+congenital+malformation+of+the+eye.">Persistent hyperplastic primary vitreous: congenital malformation of the eye.</a> Clinical &amp; Experimental Ophthalmology. 37 (9), 884-890 (2009).</li><li>Lobov, I. B., 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=WNT7b+mediates+macrophage-induced+programmed+cell+death+in+patterning+of+the+vasculature.">WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature.</a> Nature. 437 (7057), 417-421 (2005).</li><li>Kato, 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=Cbfa1-independent+decrease+in+osteoblast+proliferation,+osteopenia,+and+persistent+embryonic+eye+vascularization+in+mice+deficient+in+Lrp5,+a+Wnt+coreceptor.">Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor.</a> The Journal of Cell Biology. 157 (2), 303-314 (2002).</li><li>Xia, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+familial+exudative+vitreoretinopathy+caused+by+LPR5+mutations.">A model for familial exudative vitreoretinopathy caused by LPR5 mutations.</a> Human Molecular Genetics. 17 (11), 1605-1612 (2008).</li><li>Xu, Q., 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=Vascular+development+in+the+retina+and+inner+ear:+control+by+Norrin+and+Frizzled-4,+a+high-affinity+ligand-receptor+pair.">Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.</a> Cell. 116 (6), 883-895 (2004).</li><li>Ye, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norrin,+frizzled-4,+and+Lrp5+signaling+in+endothelial+cells+controls+a+genetic+program+for+retinal+vascularization.">Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization.</a> Cell. 139 (2), 285-298 (2009).</li><li>Ohlmann, A. V., Adamek, E., Ohlmann, A., Lutjen-Drecoll, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norrie+gene+product+is+necessary+for+regression+of+hyaloid+vessels.">Norrie gene product is necessary for regression of hyaloid vessels.</a> Investigative Ophthalmology &amp; Visual Science. 45 (7), 2384-2390 (2004).</li><li>Chen, 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=Retinal+expression+of+Wnt-pathway+mediated+genes+in+low-density+lipoprotein+receptor-related+protein+5+(Lrp5)+knockout+mice.">Retinal expression of Wnt-pathway mediated genes in low-density lipoprotein receptor-related protein 5 (Lrp5) knockout mice.</a> PLoS One. 7 (1), 30203 (2012).</li><li>Nayak, 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=Developmental+vascular+regression+is+regulated+by+a+Wnt/beta-catenin,+MYC+and+CDKN1A+pathway+that+controls+cell+proliferation+and+cell+death.">Developmental vascular regression is regulated by a Wnt/beta-catenin, MYC and CDKN1A pathway that controls cell proliferation and cell death.</a> Development. 145 (12), (2018).</li><li>Kurihara, T., 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=Astrocyte+pVHL+and+HIF-alpha+isoforms+are+required+for+embryonic-to-adult+vascular+transition+in+the+eye.">Astrocyte pVHL and HIF-alpha isoforms are required for embryonic-to-adult vascular transition in the eye.</a> The Journal of Cell Biology. 195 (4), 689-701 (2011).</li><li>Huang, T. Q., 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=Deletion+of+HIF-1alpha+partially+rescues+the+abnormal+hyaloid+vascular+system+in+Cited2+conditional+knockout+mouse+eyes.">Deletion of HIF-1alpha partially rescues the abnormal hyaloid vascular system in Cited2 conditional knockout mouse eyes.</a> Molecular Vision. 18, 1260-1270 (2012).</li><li>Yoshikawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regression+of+hyaloid+vasculature+is+triggered+by+neurons.">Developmental regression of hyaloid vasculature is triggered by neurons.</a> The Journal of Experimental Medicine. 213 (7), 1175-1183 (2016).</li><li>Garcia, C. 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+function+of+VEGF-A+in+lens+development:+formation+of+the+hyaloid+capillary+network+and+protection+against+transient+nuclear+cataracts.">The function of VEGF-A in lens development: formation of the hyaloid capillary network and protection against transient nuclear cataracts.</a> Experimental Eye Research. 88 (2), 270-276 (2009).</li><li>Hurskainen, 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=Abnormal+maturation+of+the+retinal+vasculature+in+type+XVIII+collagen/endostatin+deficient+mice+and+changes+in+retinal+glial+cells+due+to+lack+of+collagen+types+XV+and+XVIII.">Abnormal maturation of the retinal vasculature in type XVIII collagen/endostatin deficient mice and changes in retinal glial cells due to lack of collagen types XV and XVIII.</a> FASEB journal. 19 (11), 1564-1566 (2005).</li><li>Fukai, N., 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=Lack+of+collagen+XVIII/endostatin+results+in+eye+abnormalities.">Lack of collagen XVIII/endostatin results in eye abnormalities.</a> The EMBO Journal. 21 (7), 1535-1544 (2002).</li><li>McKeller, R. N., 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+Arf+tumor+suppressor+gene+promotes+hyaloid+vascular+regression+during+mouse+eye+development.">The Arf tumor suppressor gene promotes hyaloid vascular regression during mouse eye development.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (6), 3848-3853 (2002).</li><li>Hackett, S. F., Wiegand, S., Yancopoulos, G., Campochiaro, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiopoietin-2+plays+an+important+role+in+retinal+angiogenesis.">Angiopoietin-2 plays an important role in retinal angiogenesis.</a> Journal of Cellular Physiology. 192 (2), 182-187 (2002).</li><li>Chang, B., 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=Haploinsufficient+Bmp4+ocular+phenotypes+include+anterior+segment+dysgenesis+with+elevated+intraocular+pressure.">Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure.</a> BMC Genetics. 2, 18 (2001).</li><li>Wang, Z., 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=Pharmacologic+Activation+of+Wnt+Signaling+by+Lithium+Normalizes+Retinal+Vasculature+in+a+Murine+Model+of+Familial+Exudative+Vitreoretinopathy.">Pharmacologic Activation of Wnt Signaling by Lithium Normalizes Retinal Vasculature in a Murine Model of Familial Exudative Vitreoretinopathy.</a> The American Journal of Pathology. 186 (10), 2588-2600 (2016).</li><li>Gong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+an+Image-Guided+Laser-Induced+Choroidal+Neovascularization+Model+in+Mice.">Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice.</a> PLoS One. 10 (7), 0132643 (2015).</li><li>Kishimoto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+characteristics+of+regressing+vessels+in+the+hyaloid+vascular+system+of+neonatal+mice:+Novel+implication+for+vascular+atrophy.">Histochemical characteristics of regressing vessels in the hyaloid vascular system of neonatal mice: Novel implication for vascular atrophy.</a> Experimental Eye Research. 172, 1-9 (2018).</li><li>Lang, R. A., Bishop, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+are+required+for+cell+death+and+tissue+remodeling+in+the+developing+mouse+eye.">Macrophages are required for cell death and tissue remodeling in the developing mouse eye.</a> Cell. 74 (3), 453-462 (1993).</li><li>Riazifar, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+characterization+of+Bst+/-+mouse+retina.">Phenotypic and functional characterization of Bst+/- mouse retina.</a> Disease Models &amp; Mechanisms. 8 (8), 969-976 (2015).</li></ol>

ヒアロイド血管を評価し、特徴付ける技術は、動物モデルにおけるヒアロイド血管回帰を観察するために直感的かつ必要な手順であり、発達中の血管回帰の基礎となるメカニズムに関する研究を可能にする。生体内のレチナルイメージングは、同じ動物におけるヒアロイド回帰の縦方向の観察を可能にするが、OCTおよびFFAのためのげっ歯類眼球眼科イメージングシステムへのアクセスは、制?...

眼の血液供給は、網膜および周囲の眼組織の正常な発達を確保し、適切な視覚機能を備えるために不可欠である。眼には、網膜血管系、結状体、ヒアロイド血管の一過性胚循環ネットワークの3つの血管ベッドがあります。眼血管の発達は、胚発生および組織の成熟を通じて空間的および時間的な協調を必要とする。3つの血管床の中で、ヒアロイド血管系は、新たに形成された胚レンズおよび現像網膜に栄養および酸素を供給する最初の機能的な血液供給システムである。ヒヤロイド血管は、網膜血管が発達し、成熟すると同時に退行する。ヒアロイド血管系の回帰は、視覚機能の発達のための明確な視覚経路を可能にするために極めて重要である。したがって、この血管回帰プロセスは、静脈血管系の成長と同じくらい重要である。ヒアロイド回帰障害は眼疾患を引き起こす可能性がある。さらに、ヒアロイド血管の回帰は、血管回帰の調節に関与する細胞および分子機構を調べるためのモデルシステムを提供し、他の器官の血管新生調節にも影響を及ぼす可能性がある。
ヒアロイド動脈(HA)に由来するヒアロイド血管系は、バサ・ヒアロイデア・プロプリア(VHP)、チュニカ・バスクロサ・レンティス(TVL)、および瞳孔膜(PM)で構成される。これは、発生する網膜、一次静脈、および胚発生時のレンズに栄養を提供します2.HAから生じる、VHPはレンズへの静脈を通して前に分岐する。TVLはレンズカプセルの後面をカップし、アナストモスをPMに接続し、レンズ2、3の前面を覆い、PM内の血管のネットワークを形成する。3,4,5.興味深いことに、ヒアロイド血管系に静脈がなく、静脈の排水を達成するために、このシステムは甲状腺静脈を利用する。
ヒト胚では、ヒアロイド血管系は妊娠のおよそ9週目にほぼ完了し、最初の口骨血管が現れると、妊娠2の4ヶ月目に退行し始める。VHPの萎縮から始まり、TVLの毛細血管ネットワークの回帰、PM、そして最後に、HAはその後2、3が起こる。一方、一次静脈内引き込みおよび二次静脈は、コラーゲン繊維を含む細胞外マトリックス成分からなる形成を開始する。妊娠6ヶ月目までに、一次静脈は、視神経ディスクからレンズに伸びる小さな透明な運河に減少し、クロケットの運河またはヒアロイド管と呼ばれ、二次静脈は後部セグメントの主成分となる。2,3.ヒアロイド循環は、出産直前の妊娠35~36週でほとんど消滅し、3.
ヒアロイド血管系が出生時に完全に後退するヒトとは異なり、マウスヒアロイド血管系は出生後に後退し始める。マウス網膜が生まれ、網膜血管が生まれた後に発達するにつれて、ヒアロイド血管は出生後(P)4から同時に退行し、P21 6(図1)によってほとんど完全に後退する。PMはP10とP12の間で最初に消失し、VHPはP12とP16の間で消失し、少数のTVLおよびHA細胞はP16でも残り、P21によってヒヤロイド血管系回帰はほぼ完了する6である。その間、骨格は出生後に発症し始める。P7-P8では血管叢の表面層が完全に末梢網膜に広がり、P7-P12から深層(外プレキシフォーム層に位置する)が発達し、最後にP12とP15 7の間に中間プレクサスが発達する。.陰管血管が発達するにつれて、それは徐々に結び付け可能なヒアロイド血管の機能を置き換え、発達中の眼に栄養と酸素を提供する。マウスにおけるヒアロイド血管回帰の出生後の発生は、ヒアロイド血管系を観察し、研究するための容易にアクセス可能な実験モデルを提供し、生理学的および両方の下で血管回帰プロセスを支配する分子基盤を提供する。病理学的状態8.
ヒアロイド回帰の失敗は、胚学、原発性二頭状およびヒアロイド血管系の失敗または不完全な回帰に起因する眼の稀な先天性発達異常であるPHPVなどの疾患で見ることができる。ヒアロイド血管系の回帰過程を調節するメカニズムは複雑で広く研究されている。ヒアロイド血管の正常な回帰に不可欠な主要な分子経路の1つは、Wntシグナル伝達経路10であり、この経路における遺伝的変異がヒト9においてPHPVと関連している。実験的研究では、この回帰プロセスを仲介するために、現像眼のヒヤロイド血管の周りのマクロファージによって生成されるWntリガンドWnt7bを同定した。Wnt7bは、隣接する内皮細胞における受容体フリズルド4(FZD4)/LRP5との結合によりWntシグナル伝達を活性化し、細胞アポトーシスを開始し、ヒアロイド血管10の回帰を引き起こした。その結果、Wnt7b欠損マウスは、ヒアロイド血管10の持続性を示す。同様に、非従来型のWntリガンド、Norrin(Ndp遺伝子によってコードされる)も、開発中にヒアロイド血管回帰を誘導するためにFZD4/LRP5に結合する。Ndpy/-, Lrp5-/-およびFzd4-/-マウスはすべて、Wnt シグナル伝達11,12の重要な調節役割をサポートする延期されたヒヤロイド血管回帰を表示します。 13,14,15,16.さらに、別のWntコセプターLRP6は、ヒアロイド血管内皮細胞17におけるWntシグナル伝達経路を調節する上での機能においてLRP5と重なる。ヒアロイド回帰に寄与する可能性のある他の要因には、低酸素誘導因子18、19、血管内皮成長因子20、21、コラーゲン-1822、が含まれる。 23, Arf24, アンジオポエチン-25 , 骨形態形成タンパク質-426.本論文では、持続性ヒアロイド血管のモデルとしてLrp5-/-マウスを用いて、生体内法とex vivo法の両方を通じてヒヤロイド血管系を評価し特徴付ける技術を実証する。
生体内およびex vivoにおけるヒアロイド血管系の可視化は、ヒアロイド血管回帰のメカニズムを研究するために不可欠である。ヒアロイド血管を観察する現在の方法は、主にOCTおよびFFA画像、眼断面、およびヒアロイド平らなマウントを介して、VHPとHAを視覚化し、分析することに焦点を当てています。OCTおよびFFAは生体内イメージングツールに強力であり、目を開けた後に生きた動物の縦方向の観察を可能にする。さらに、隔離されたヒアロイドフラットマウントは、ヒヤロイド血管全体の可視化と容器数の正確な定量を達成するための手段を提供します。しかし、ヒアロイド血管の繊細で壊れやすい性質と、その分離の結果として生じる技術的な困難は、眼科研究における使用をいくらか制限している可能性があります10,17,27.本論文では、生体内の生きているレチナルイメージングとex vivo単離されたヒヤロイドフラットマウントの両方を組み合わせて、これらの技術の実現可能性を高めるために、ヒヤロイド血管の可視化の詳細なプロトコルを提供する。このプロトコルは、ライブファンダスおよびOCTイメージング28および単離されたヒヤロイドフラットマウント11のex vivo法に関する以前の出版物からの改変および拡張に適合している。

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			<td height="21" style="height:21px;width:355px;">AK-Fluor (fluorescein injection, USP)</td>
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			<td></td>
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			<td style="width:156px;">S2828</td>
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			<td height="21" style="height:21px;">Antifade Mounting Medium with DAPI</td>
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			<td height="21" style="height:21px;">Artificial tear eyedrop</td>
			<td style="width:191px;">Systane</td>
			<td style="width:156px;">N/A</td>
			<td></td>
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			<td height="21" style="height:21px;width:355px;">Bovine serum albumin (BSA)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">A2058</td>
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			<td height="21" style="height:21px;width:355px;">C57BL/6J mice</td>
			<td style="width:191px;">The Jackson Laboratory</td>
			<td style="width:156px;">Stock NO: 000664</td>
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			<td height="21" style="height:21px;width:355px;">Calcium chloride (CaCl2)</td>
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			<td style="width:156px;">N/A</td>
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			<td height="21" style="height:21px;width:355px;">Lrp5-/- mice</td>
			<td style="width:191px;">The Jackson Laboratory</td>
			<td style="width:156px;">Stock NO. 005823</td>
			<td>Developed by Deltagen Inc., San Mateo, CA</td>
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			<td height="21" style="height:21px;width:355px;">Micron IV and OCT</td>
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			<td style="width:156px;">N/A</td>
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			<td style="width:191px;">Tissue-Tek</td>
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			<td height="21" style="height:21px;width:355px;">Optimal cutting temperature compound</td>
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			<td style="width:156px;">15710</td>
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			<td height="42" style="height:42px;width:355px;">Peel-A-Way disposable embedding molds (tissue molds)</td>
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			<td style="width:156px;">12-20</td>
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			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS) buffer (10X)</td>
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			<td height="21" style="height:21px;width:355px;">Slide cover glass</td>
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			<td height="21" style="height:21px;width:355px;">Xylazine sterile solution</td>
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			<td style="width:156px;">NDC: 59399-110-20</td>
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assessment and characterization of hyaloid vessels in mice

眼では、胚性ヒアロイド血管は、網膜血管が発達すると、発達するレンズと網膜に栄養を与え、回帰する。ヒアロイド血管の持続的または失敗した回帰は、持続性過形成原発性静脈(PHPV)などの疾患で見られ、閉塞した光経路および視覚機能障害につながる。ヒアロイド血管回帰の根底にあるメカニズムを理解することは、血管回帰プロセスに関する新しい分子洞察と、持続的なヒアロイド血管を有する疾患を管理する潜在的な新しい方法につながる可能性があります。ここでは、光コヒーレンス断層撮影(OCT)と眼球フルオレセイン血管造影(FFA)を用いた生きたマウスにおけるヒヤロイドのイメージング手順と、定量分析のためのヒヤロイドを分離およびフラットマウントする詳細な技術プロトコルについて説明する。低密度リポタンパク質受容体関連タンパク質5(LRP5)ノックアウトマウスを、持続性ヒアロイド血管の実験モデルとして用い、その技術を例示した。これらの技術は、血管回帰の実験モデルとしてのヒアロイド血管の徹底的な評価と持続性ヒアロイド血管のメカニズムに関する研究を容易にする可能性がある。

生きたマウスにおけるヒアロイド血管の生体内イメージング 図 33ヶ月前のWTおよびLrp5-/-マウスにおける網膜およびヒアロイド組織のOCT画像の断面図を明らかにする。WT眼はヒヤロイド組織の不在を示し、Lrp5-/-眼は視神経頭部に由来する2つの持続性ヒアロイド血管を示す。図 3B?...

Watch this Scientific Journal Video about Assessment and Characterization of Hyaloid Vessels in Mice at JoVE.com

Assessment and Characterization of Hyaloid Vessels in Mice

このプロトコルは、生体イメージングとex vivoの両方の方法で、マウスの目の血管回帰モデルであるヒアロイド血管を完全に視覚化し、特徴付けする方法の両方について説明します。定量分析のためのヒヤロイドの分離および後続の平らな台紙。

マウスにおけるヒアロイド血管の評価と特徴付け

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...

我々のプロトコルは、血管回帰を研究するための有用な実験モデルとして、目の中のヒアルロイド血管系を視覚化し、特徴付けるためのin vivoとex vivoの両方の尺度を提供します。この技術は、生きたマウスにおけるヒヤロイド血管のインビボ縦断観察と、解剖された全ヒアロイド血管の正確な定量化とを組み合わせたものです。我々の技術は、持続性胎児血管系の病因および基本的メカニズムを研究するための実用的かつ実現可能な方法を提供する。この方法はまた、他の器官の血管新生研究に関連する可能性のある眼血管退縮の調節に関与する細胞および分子メカニズムに関する洞察を提供する。ヒヤロイドの分離手順は、初心者にとって技術的に困難な場合があります。練習と忍耐の多くは、解剖を成功させるために役立ちます。この方法を視覚化すると、より効率的にスキルを習得するのに役立つテクニックに関する詳細な実践的な情報とヒントが提供されます。光コヘレンス断層撮影またはOCTイメージングの場合、眼科画像の視野の中心に光神経ヘッドが配置されるようにマウスとプローブを調整し、画像を取得する前に、OCTイメージングソフトウェアで示された線の焦点と角度を調整して、持続性のヒアロイド血管を明らかにする。ヒアロイド血管の眼管フルオレセイン血管造影画像の場合、視神経ヘッドを視野の中央に配置するように調整し、顕微鏡フィルターを緑色の蛍光チャネルに変更します。次に、持続性ヒアロイド血管に焦点を当て、注入後1、3、5、および10分で複数の画像を得て、血管を観察するための信号対バックグラウンド比の最適なタイムポイントを決定します。ex vivoヒアロイド血管解剖および可視化では、マイクロサージャリー鉗子を使用して出生後8日目マウスのまぶたを開き、片目の軌道に地球の下に湾曲したマイクロ手術鉗子を置き、眼球を絞らずに視神経をつかむ。眼球を鉗子で軽く引っ張って取り外し、室温で4%パラホルムアルデヒドで目を固定します。30分後、解剖顕微鏡下で固定眼球を氷冷PBSに移し、50マイクロリットルのゼラチン溶液を、四肢の周りの4つの異なる等間隔の部位で眼球の膜炎体に注入する。その後、氷の上に眼球を30分間インキュベートし、注入したゼラチンを膜領域内で固めます。ゼラチンが硬化すると、眼球を解剖顕微鏡下でPBSのペトリ皿に移し、マイクロサージャスハサミを使用して手足を切開して角膜を取り除きます。切り取り、解剖顕微鏡の下で視神経を取り除きます。2組の鉗子を使用して、強膜、脈絡膜および網膜色素上皮層を剥離して捨て、続いて虹彩を除去する。レチナの視神経側が上を向き、レンズが下を向いている場合、レチナカップのすぐ下に50マイクロリットルのPBSを注入して、ゼラチン化された亜質体とレチナの間にPBSの蓄積を可能にする。マイクロサージャリー鉗子を使用して、生膜からレティナカップと毛様体を穏やかに剥がします。転写ピペットを用いて、PBSに浸漬したヒアロイド組織を含むゼラチンカップを顕微鏡スライドに移す。そして、レンズが上向きになるように残りの組織をひっくり返します。その後、レンズを持ち上げ、レンズチュニカ血管系レンティスと血管状血管の支柱との間の接続を穏やかに緩めてから、マイクロサージャリージャスハサミでヒャロイド動脈を切断してレンズチュニカ血管系レンティスを取り除きます。平らなマウントのためのヒアロイドカップのバサヒャロイデプロリア領域を保ちます。ヒアロイド容器サンプルの平らな取り付けのために、任意の解剖破片を除去するために、新鮮なPBSでヒアロイドカップを静かにすすいだ。PBSに浮かぶ組織では、マイクロサージャリード鉗子を使用して、ゼラチン化されたヒアロイドカップの位置を穏やかに配置し、カップの縁が下向きになるように調整します。ヒアロイドカップをPBSに浸してスライドを37度のスライドウォーマーに置きます。ゼラチンが溶けてヒアロイドが平らにした後、かろうじて乾燥しているときにスライドを取り除き、DAPIでアンチフェード実装媒体を一滴加えてヒアロイド血管の核を染色します。その後、ヒアロイドにカバースリップをそっと置き、フラットマウントを完成させます。DAPI染色を画像化するために、その取り付けられたサンプルを蛍光顕微鏡のステージに移し、中央のヒアロイド動脈が見えることを確認する。次に、ヒアロイド動脈から直接導出された血管枝を特定し、定量のために容器の枝の数を手動で数える。ここでは、3ヶ月前の野生型および低密度リポタンパク質受容体関連タンパク質5またはLRP5ノックアウトマウスにおける残膜およびヒアロイド組織のOCT画像の断面図が、持続性ヒアロイド血管を有する動物モデルである、図示されている。これらの眼管フルオレスセイン血管の持続的なヒアロイド血管の血管造影画像は、6週齢のノックアウトマウスの膜炎の体内で観察されるが、同じ年齢の野生型動物ではヒアルロイド血管の残骸は観察されない。これらのヒアロイド血管では、血管細胞および関連するマクロファージを平らに取り付け、それらの核DAPI染色によって同定することができる。そして、中央ヒアロイド動脈から分岐する細胞の各行は、血管の血管を表す血管の血管を表す。野生型マウスは出生後8日目に平均12のヒアロイド血管の枝を有し、年齢に合わせたLRP5ノックアウト子犬はヒアロイド血管の約25の枝を示し、ヒアロイド血管構造の著しく損なわれた退縮を示す。さらに、遅延および不完全なretina血管の発達は、LRP5ノックアウトの子犬にも観察される。実際、ヒアロイド血管は、出生後8日目にLRP5ノックアウト眼組織切片の断面で同定することができる。一方、野生のタイプの目はもはやこれらの容器を表示しません。覚えておくべきことの最も重要なことは、ヒアロイド系を眼部および他の眼組織から分離する際に、穏やかで忍耐強くないことです。異なる細胞マーカーを用いた免疫染色を行うヒアロイド血管を単離した後、ヒアロイド退縮を調節する細胞相互作用を明らかにすることができる。この技術は、眼血管新生研究における持続性胎児血管系の基礎となる細胞および分子メカニズムを探る。パラホルムアルデヒド、ケタミン、キシラジンは危険です。適切な個人用保護具を使用して、化学発煙フードにこれらの試薬を準備してください。

assessment-and-characterization-of-hyaloid-vessels-in-mice

Research

JoVE Journal

Medicine

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used techniques require killing of the animals and postmortem recovery of the meal1,2. This approach prevents longitudinal studies to determine changes in gastric emptying with age and progression of disease. The commonly used [13C]-octanoic acid breath test for humans3 has been modified for use in mice4-6 and rats7 and we previously showed that this test is reliable and responsive to changes in gastric emptying in response to drugs and during diabetic disease progression8. In this video presentation the principle and practical implementation of this modified test is explained. As in the previous study, NOD LtJ mice are used, a model of type 1 diabetes9. A proportion of these mice develop the symptoms of gastroparesis, a complication of diabetes characterized by delayed gastric emptying without mechanical obstruction of the stomach10.
This paper demonstrates how to train the mice for testing, how to prepare the test meal and obtain 4 hr gastric emptying data and how to analyze the obtained data. The carbon isotope analyzer used in the present study is suitable for the automatic sampling of the air samples from up to 12 mice at the same time. This technique allows the longitudinal follow-up of gastric emptying from larger groups of mice with diabetes or other long-standing diseases.

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.

[を使用した非肥満糖尿病マウスにおける胃排出能の評価<sup&gt; 13</sup&gt; C] - オクタン酸呼気テスト

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

医学

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

マウスの右心の心エコー評価

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

マウスの圧負荷モデルを用いた冠動脈の流れと冠血流準備の超音波ベースのアセスメント

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

マウスの弁膜症の包括的表現型の特徴のための心エコーアプローチとプロトコル

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

ダブル室容積脈波による意識下マウスにおける呼吸機能の評価

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

マウスで内がえし筋機能の非侵襲的評価

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

低酸素/SU5416モデルを用いたマウスにおける肺高血圧の誘導と特徴づけ

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

マウスにおける右心室系と低酸素誘発性肺動脈高血圧に対する侵襲的血行力学的評価

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

移植質評価のための化学バイオプシーの使用

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...