AbstractThe lymphatic system is a crucial contributor to allograft rejection after corneal transplantation. However, no surgical procedures for the central pathway where conjunctival lymphatic vessels converge have been investigated. Therefore, we aimed to establish a murine model of lymphatic vessel ligation and evaluate its inhibitory effect on corneal allograft rejection.

摘要淋巴系统是角膜移植后同种异体移植排斥反应的关键因素。然而，尚未研究结膜淋巴管汇合的中央通路的外科手术。因此，我们旨在建立淋巴管结扎的小鼠模型，并评估其对角膜同种异体移植排斥反应的抑制作用。

A tracer was used to visualise lymphatic vessels, and complications were evaluated. A surgical technique was developed to block the lymphatic vessels. Corneas from C57BL/6 mice were transplanted into BALB/c mice divided into two groups—one with and one without lymphatic vessel ligation, to evaluate their effects on allograft rejection.

示踪剂用于可视化淋巴管，并评估并发症。开发了一种阻断淋巴管的手术技术。将C57BL/6小鼠的角膜移植到BALB/c小鼠中，分为两组，一组有淋巴管结扎，另一组没有淋巴管结扎，以评估它们对同种异体移植排斥反应的影响。

Graft opacity scores were evaluated for 8 weeks, and immunohistochemistry was used to quantify angiogenesis and lymphangiogenesis. 20% trypan blue used as a tracer showed clear inflow with no complications. The two sutures and cyanoacrylate glue combination demonstrated a blocking effect after 25 days and was thus used for lymphatic ligation.

评估移植物不透明度评分8周，并使用免疫组织化学定量血管生成和淋巴管生成。用作示踪剂的20%台盼蓝显示出清晰的流入，没有并发症。两种缝合线和氰基丙烯酸酯胶组合在25天后表现出阻断作用，因此用于淋巴结结扎。

Three and nine out of fourteen eyes showed rejection at 8 weeks post-surgery in the lymphatic vessel ligation and control groups, respectively. Furthermore, neovascularisation and lymphangiogenesis significantly decreased in the lymphatic vessel ligation group. Overall, we present a novel therapeutic strategy for corneal transplantation..

在淋巴管结扎组和对照组中，14只眼中有3只眼和9只眼在术后8周分别出现排斥反应。此外，淋巴管结扎组的新血管形成和淋巴管生成显着减少。总的来说，我们提出了一种新的角膜移植治疗策略。。

IntroductionRecently, corneal transplantation has progressed to selectively replacing diseased corneal layers1. However, penetrating keratoplasty (PKP) remains a prevalent form of corneal transplantation for various indications such as infection, regraft, and keratoconus2. Although the graft survival rate after 1 year in patients with PKP exceeds 90%, the rates differ with indications.

引言最近，角膜移植已经发展到选择性替代患病角膜层1。然而，穿透性角膜移植术（PKP）仍然是角膜移植的一种普遍形式，用于各种适应症，如感染，再移植和圆锥角膜2。虽然PKP患者1年后移植物存活率超过90%，但适应症不同。

For example, the survival rate at 10 years is also > 90% in patients with keratoconus; however, the survival rate of a failed previous graft is approximately 30%.3,4 Graft failure is often caused by immunologic graft rejection, and developing therapeutic strategies to improve graft survival is essential.The lymphatic system is a key player in allograft rejection after corneal transplantation; however, excising cervical lymph nodes increases graft survival5.

例如，圆锥角膜患者的10年生存率也>90%；然而，先前失败的移植物的存活率约为30%[3,4]。移植物衰竭通常是由免疫性移植物排斥反应引起的，开发治疗策略以提高移植物存活率至关重要。淋巴系统是角膜移植后同种异体移植排斥反应的关键因素；然而，切除颈部淋巴结会增加移植物的存活率5。

Lymphatic vessels play a role in graft rejection, and alloantigen and antigen-presenting cell migration to lymph nodes via lymphatic vessels may trigger graft rejection6. Consequently, inhibiting lymphatic vessel function could reduce the risk of graft rejection. Previous studies have regulated the lymphatic pathway, including inhibition of vascular endothelial growth factor (VEGF) expression7.

淋巴管在移植物排斥反应中起作用，同种异体抗原和抗原呈递细胞通过淋巴管迁移到淋巴结可能引发移植物排斥反应6。因此，抑制淋巴管功能可以降低移植物排斥的风险。先前的研究已经调节了淋巴通路，包括抑制血管内皮生长因子（VEGF）的表达7。

However, no surgical procedures have been considered for the central pathway where conjunctival lymphatic vessels assemble. Therefore, in this study, we aimed to visualise the oculo-lymphatic pathway and demonstrate its blocking effect on allograft rejection after corneal transplantation using simple surgical techniques.ResultsVisualisation of lymphatic vessels and establishment of a lymphatic vessel ligation modelAlthough the Evans blue tracer was thoroughly rinsed, superficial punctate keratitis (SPK) was observed after administering a drop of.

然而，尚未考虑结膜淋巴管聚集的中央通路的外科手术。因此，在这项研究中，我们旨在可视化眼淋巴通路，并使用简单的手术技术证明其对角膜移植后同种异体移植物排斥反应的阻断作用。结果淋巴管可视化和淋巴管结扎模型的建立虽然伊文思蓝示踪剂被彻底冲洗，但在给予一滴后观察到浅表点状角膜炎（SPK）。

Data availability

数据可用性

Data availability; The datasets used and/or analysed during the current study availablefrom the corresponding author on reasonable request.

数据可用性；本研究期间使用和/或分析的数据集可根据合理要求从通讯作者处获得。

ReferencesZhou, Y., Wang, T., Tuli, S. S., Steigleman, W. A. & Shah, A. A. Overview of corneal transplantation for the nonophthalmologist. Transpl. Direct. 9, e1434 (2023).Article

参考文献Zhou，Y.，Wang，T.，Tuli，S.S.，Steigleman，W.A。＆Shah，A.A。非眼科医生角膜移植概述。运输。直接。9，e1434（2023）。文章

Google Scholar

谷歌学者

Pluzsik, M. T. et al. Changing trends in penetrating keratoplasty indications between 2011 and 2018 - histopathology of 2123 corneal buttons in a single Center in Germany. Curr. Eye Res. 45, 1199–1204 (2020).Article

Pluzsik，M.T.等人。2011年至2018年间穿透性角膜移植适应症的变化趋势-德国单个中心2123个角膜纽扣的组织病理学。货币。Eye Res.451199–1204（2020）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Sangwan, V. S., Ramamurthy, B., Shah, U., Garg, P. & Sridhar, M. S. Rao, G. N. Outcome of corneal transplant rejection: A 10-year study. Clin. Exp. Ophthalmol. 33, 623–627 (2005).Article

Sangwan，V.S.，Ramamurthy，B.，Shah，U.，Garg，P。＆Sridhar，M.S.Rao，G.N。角膜移植排斥反应的结果：一项为期10年的研究。临床。实验眼科。33623-627（2005）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Yamagami, S., Suzuki, S. & Tsuru, T. Risk factors for graft failure in penetrating keratoplasty. Acta Ophthalmol. Scand. 74, 584–588 (1996).Article

Yamagami，S.，Suzuki，S。＆Tsuru，T。穿透性角膜移植术中移植失败的危险因素。眼科学报。斯堪的纳维亚。74584-588（1996）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Yamagami, S. & Dana, M. R. The critical role of draining lymph nodes in corneal alloimmunization and graft rejection. Invest. Ophthalmol. Vis. Sci. 42, 1293–1298 (2001).PubMed

Yamagami，S。＆Dana，M.R。引流淋巴结在角膜同种免疫和移植物排斥中的关键作用。投资。眼科。可见。。421293-1298（2001）。PubMed出版社

Google Scholar

谷歌学者

Hou, Y., Bock, F., Hos, D. & Cursiefen, C. Lymphatic trafficking in the eye: Modulation of lymphatic trafficking to promote corneal transplant survival. Cells. 10, 1661 (2021).Article

Hou，Y.，Bock，F.，Hos，D。＆Cursiefen，C。眼睛中的淋巴运输：调节淋巴运输以促进角膜移植存活。细胞。101661（2021）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Cursiefen, C. et al. Inhibition of hemangiogenesis and lymphangiogenesis after normal-risk corneal transplantation by neutralizing VEGF promotes graft survival. Invest. Ophthalmol. Vis. Sci. 5, 2666–2673 (2004).Article

Cursiefen，C.等人。通过中和VEGF抑制正常风险角膜移植后的血管生成和淋巴管生成可促进移植物存活。投资。眼科。可见。。52666-2673（2004）。文章

Google Scholar

谷歌学者

Lohrberg, M. & Wilting, J. The lymphatic vascular system of the mouse head. Cell. Tissue Res. 366, 667–677 (2016).Article

Lohrberg，M。＆Wilting，J。小鼠头部的淋巴管系统。细胞。Tissue Res.36667–677（2016）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Maloveska, M. et al. Dynamics of Evans blue clearance from cerebrospinal fluid into meningeal lymphatic vessels and deep cervical lymph nodes. Neurol. Res. 40, 372–380 (2018).Article

Maloveska，M.等人。伊文思蓝从脑脊液清除到脑膜淋巴管和颈深淋巴结的动力学。。第40372-380号决议（2018年）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Plsková, J., Kuffová, L., Holán, V., Filipec, M. & Forrester, J. V. Evaluation of corneal graft rejection in a mouse model. Br. J. Ophthalmol. 86, 108–113 (2002).Article

Plsková，J.，Kuffová，L.，Holán，V.，Filipec，M。＆Forrester，J.V。评估小鼠模型中的角膜移植排斥反应。Br.J.眼科。86108-113（2002）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Hegde, S. & Niederkorn, J. Y. The role of cytotoxic T lymphocytes in corneal allograft rejection. Invest. Ophthalmol. Vis. Sci. 41, 3341–3347 (2000).PubMed

Hegde，S。＆Niederkorn，J.Y。细胞毒性T淋巴细胞在角膜同种异体移植排斥反应中的作用。投资。眼科。可见。。413341-3347（2000）。PubMed出版社

Google Scholar

谷歌学者

Wu, Y. et al. Organogenesis and distribution of the ocular lymphatic vessels in the anterior eye. JCI Insight. 5, e135121 (2020).Article

Wu，Y.等人。前眼淋巴管的器官发生和分布。。5，e135121（2020）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Subileau, M. et al. Eye lymphatic defects induced by bone morphogenetic protein 9 deficiency have no functional consequences on intraocular pressure. Sci. Rep. 10, 16040 (2020).Article

Subileau，M。等人。骨形态发生蛋白9缺乏引起的眼淋巴缺陷对眼压没有功能性影响。。代表1016040（2020）。文章

ADS

广告

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Dieterich, L. C., Tacconi, C., Ducoli, L. & Detmar, M. Lymphatic vessels in cancer. Physiol. Rev. 102, 1837–1879 (2022).Article

Dieterich，L.C.，Tacconi，C.，Ducoli，L。＆Detmar，M。癌症中的淋巴管。生理学。版本1021837-1879（2022）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Petrova, T. V. & Koh, G. Y. Biological functions of lymphatic vessels. Science. 369, eaax4063 (2020).Article

Petrova，T.V。＆Koh，G.Y。淋巴管的生物学功能。科学。369，eaax4063（2020）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Cursiefen, C. et al. Lymphatic vessels in vascularized human corneas:Iimmunohistochemical investigation using LYVE-1 and podoplanin. Invest. Ophthalmol. Vis. Sci. 43, 2127–2135 (2002).PubMed

Cursiefen，C。等人。血管化人角膜中的淋巴管：使用LYVE-1和podoplanin的免疫组织化学研究。投资。眼科。可见。。432127-2135（2002）。PubMed出版社

Google Scholar

谷歌学者

Ambati, B. K. et al. Corneal avascularity is due to soluble VEGF receptor-1. Nature. 443, 993–997 (2006).Article

Ambati，B.K。等人。角膜无血管是由于可溶性VEGF受体-1引起的。自然。443993-997（2006）。文章

ADS

广告

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Cursiefen, C. et al. VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J. Clin. Invest. 113, 1040–1050 (2004).Article

Cursiefen，C。等人。VEGF-A通过巨噬细胞募集刺激炎症性新血管形成中的淋巴管生成和血管生成。J、 临床。投资。1131040–1050（2004）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Amadio, M., Govoni, S. & Pascale, A. Targeting VEGF in eye neovascularization: What’s new? A comprehensive review on current therapies and oligonucleotide-based interventions under development. Pharmacol. Res. 103, 253–269 (2016).Article

Amadio，M.，Govoni，S。＆Pascale，A。靶向VEGF在眼部新血管形成中的作用：有什么新进展？目前正在开发的疗法和基于寡核苷酸的干预措施的全面综述。药理学。第103253-269号决议（2016年）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Albuquerque, R. J. C. et al. Alternatively spliced VEGF receptor-2 is an essential endogenous inhibitor of lymphatic vessels. Nat. Med. 15, 1023–1030 (2009).Article

Albuquerque，R.J.C.等人。剪接的VEGF受体-2是淋巴管的必需内源性抑制剂。《自然医学》第151023-1030页（2009年）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Zhang, W., Schönberg, A., Bock, F., Cursiefen, C. & Posttransplant VEGFR1R2 trap eye drops inhibit corneal (lymph)angiogenesis and improve corneal allograft survival in eyes at high risk of rejection. Transl Vis. Sci. Technol. 11, 6 (2022).Article

Zhang，W.，Schönberg，A.，Bock，F.，Cursiefen，C。＆Posttransplant VEGFR1R2 trap滴眼液抑制角膜（淋巴）血管生成并改善排斥高风险眼睛的角膜同种异体移植物存活。Transl Vis公司。。技术。11，6（2022）。文章

PubMed

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Dohlman, T. H. et al. VEGF-trap Aflibercept significantly improves long-term graft survival in high-risk corneal transplantation. Transplantation. 99, 678–686 (2015).Article

Dohlman，T.H.等人，VEGF-trap Aflibercept可显着改善高危角膜移植中移植物的长期存活率。移植。99678-686（2015）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Chen, L. et al. Vascular endothelial growth factor receptor-3 mediates induction of corneal alloimmunity. Nat. Med. 10, 813–815 (2004).Article

Chen，L。等人。血管内皮生长因子受体-3介导角膜同种异体免疫的诱导。《自然医学》10813-815（2004）。文章

PubMed

PubMed

Google Scholar

谷歌学者

Chen, G. L. et al. Evaluation of corneal graft survival in mice model. Int. J. Ophthalmol. 6, 578–583 (2013).PubMed

Chen，G.L.等人。小鼠模型中角膜移植物存活率的评估。国际眼科杂志。。PubMed出版社

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Ma, D. et al. Conditions affecting enhanced corneal allograft survival by oral immunization. Invest. Ophthalmol. Vis. Sci. 10, 1835–1846 (1998).

Ma，D。等人。通过口服免疫影响增强的角膜同种异体移植物存活的条件。投资。眼科。可见。。101835-1846（1998）。

Google Scholar

谷歌学者

Download referencesAcknowledgementsThe study is reported in accordance with ARRIVE guidelines.FundingThis research was supported by the Charitable Trust Fund for Ophthalmic Research in the Commemoration of Santen Pharmaceutical’s Founder and Japan Cornea Society.SPK, superficial punctate keratitis.Author informationAuthors and AffiliationsDivision of Ophthalmology, Department of Ophthalmology, Department of Visual Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo, 173-8610, JapanAmi Igarashi, Takahiko Hayashi, Toshiki Shimizu & Satoru YamagamiKikuna Yuda Eye Clinic, Yokohama, JapanKentaro YudaAuthorsAmi IgarashiView author publicationsYou can also search for this author in.

下载参考文献致谢根据ARRIVE指南报告研究。资助这项研究得到了纪念Santen Pharmaceutical创始人和日本角膜协会的眼科研究慈善信托基金的支持。SPK，浅表点状角膜炎。作者信息作者和附属机构日本大学医学院眼科，视觉科学系眼科，30-1 Oyaguchi kamicho，Itabashi ku，东京，173-8610，JapanAmi Igarashi，Takahiko Hayashi，Toshiki Shimizu＆Satoru Yamagamikuna Yuda Eye Clinic，横滨，JapanKentaro YudaAuthorsAmi IgarashiView作者出版物您也可以在中搜索这位作者。

PubMed Google ScholarTakahiko HayashiView author publicationsYou can also search for this author in

PubMed Google ScholarTakahiko HayashiView作者出版物您也可以在

PubMed Google ScholarToshiki ShimizuView author publicationsYou can also search for this author in

PubMed Google ScholarToshiki ShimizuView作者出版物您也可以在

PubMed Google ScholarKentaro YudaView author publicationsYou can also search for this author in

PubMed Google ScholarKentaro YudaView作者出版物您也可以在

PubMed Google ScholarSatoru YamagamiView author publicationsYou can also search for this author in

PubMed Google ScholarSatoru YamagamiView作者出版物您也可以在

PubMed Google ScholarContributionsAll authors reviewed the manuscript. A.I, and T.H. were writing, reviewing, and editing of the manuscript. T.S. was doing curation of the data and editing of the manuscript. K.Y. was reviewing and editing of the manuscript. S.Y. was responsible for supervision and validation.

PubMed谷歌学术贡献所有作者都审阅了手稿。A、 我和T.H.正在撰写，审阅和编辑手稿。T、 美国正在进行数据管理和手稿编辑。K、 Y.正在审阅和编辑手稿。S、 Y.负责监督和验证。

All authors critically checked the manuscript and approved its submission.Corresponding authorCorrespondence to.

所有作者都严格检查了稿件并批准了其提交。对应作者对应。

Takahiko Hayashi.Ethics declarations

林崇彦。道德宣言

Competing interests

相互竞争的利益

The authors declare no competing interests.

作者声明没有利益冲突。

Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Electronic supplementary materialBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Rights and permissions.

Additional informationPublisher的noteSpringer Nature在已发布地图和机构隶属关系中的管辖权主张方面保持中立。电子补充材料流是指向电子补充材料的链接。补充材料1补充材料2权利和许可。

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material.

开放获取本文是根据知识共享署名非商业性NoDerivatives 4.0国际许可证授权的，该许可证允许以任何媒介或格式进行任何非商业性使用，共享，分发和复制，只要您对原始作者和来源给予适当的信任，提供知识共享许可证的链接，并指出您是否修改了许可材料。

You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

根据本许可证，您无权共享源自本文或其部分的改编材料。本文中的图像或其他第三方材料包含在文章的知识共享许可证中，除非该材料的信用额度中另有说明。如果材料未包含在文章的知识共享许可中，并且您的预期用途不受法律法规的许可或超出许可用途，则您需要直接获得版权所有者的许可。

To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/..

要查看此许可证的副本，请访问http://creativecommons.org/licenses/by-nc-nd/4.0/..

Reprints and permissionsAbout this articleCite this articleIgarashi, A., Hayashi, T., Shimizu, T. et al. Inhibiting corneal transplantation rejection via lymphatic vessel ligation in a novel murine model.

转载和许可本文引用本文Igarashi，A.，Hayashi，T.，Shimizu，T。等人在新型小鼠模型中通过淋巴管结扎抑制角膜移植排斥反应。

Sci Rep 14, 25692 (2024). https://doi.org/10.1038/s41598-024-77160-9Download citationReceived: 18 May 2024Accepted: 21 October 2024Published: 28 October 2024DOI: https://doi.org/10.1038/s41598-024-77160-9Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

Sci Rep 1425692（2024）。https://doi.org/10.1038/s41598-024-77160-9Download引文接收日期：2024年5月18日接受日期：2024年10月21日发布日期：2024年10月28日OI：https://doi.org/10.1038/s41598-024-77160-9Share本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。。

Provided by the Springer Nature SharedIt content-sharing initiative

由Springer Nature SharedIt内容共享计划提供

KeywordsCorneal transplantationLymphangiogenesisLymphatic vessel ligationNeovascularisation

关键词腹膜移植淋巴管生成淋巴管结扎血管化