Lymphatic Vessel Ligation: A Novel Murine Model for Inhibiting Corneal Transplantation Rejection

Lymphatic Vessel Ligation: A Novel Murine Model for Inhibiting Corneal Transplantation Rejection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lymphatic Vessel Ligation: A Novel Murine Model for Inhibiting Corneal Transplantation Rejection Ami Igarashi, Takahiko Hayashi, Toshiki Shimizu, Kentaro Yuda, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4439625/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract The 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. 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. Graft opacity scores were evaluated for 8 weeks, and immunohistochemistry was used to quantify angiogenesis and lymphangiogenesis. Twenty percent 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. Three and nine out of fourteen eyes showed rejection at 8 weeks post-surgery in the lymphatic vessel ligation and allograft groups, respectively. Furthermore, neovascularisation and lymphangiogenesis significantly decreased in the lymphatic vessel ligation group. Overall, we present a novel therapeutic strategy for corneal transplantation. corneal transplantation lymphangiogenesis lymphatic vessel ligation neovascularisation Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Recently, corneal transplantation has progressed to selectively replacing diseased corneal layers. 1 However, penetrating keratoplasty (PKP) remains a prevalent form of corneal transplantation for various indications such as infection, regraft, and keratoconus. 2 Although the graft survival rate after 1 year in patients with PKP exceeds 90%, the rates differ with indications. 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 survival. 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 rejection. 6 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) expression. 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. RESULTS Visualisation of lymphatic vessels and establishment of a lymphatic vessel ligation model Although the Evans blue tracer was thoroughly rinsed, superficial punctate keratitis (SPK) was observed after administering a drop of the tracer. Therefore, the minimum concentration and method of tracer administration were evaluated. Table 1 shows the results of the confirmed influx into the superficial cervical and facial lymph nodes and complications at different concentrations. Table 1 Tracers used to visualise lymphatic vessels Number of confirmed inflows to the lymph nodes (eyes) Complications Evans blue 1% 3/3 SPK (one eye) 10% 3/3 SPK (two eyes) Trypan blue 10% 0/3 None 20% 3/3 None SPK, superficial punctate keratitis Figure 1 shows a schematic representation of the lymphatic vessel originating from the nasal aspect of the conjunctiva and culminating in cervical lymph nodes. The junction demarcated the peripheral and central pathways. The lymphatic vessels assembled from the limbus to the nasal side of the conjunctiva, and they were stained with trypan blue (Fig. 2 A). Figure 2 B shows the tracing lymphatic vessel with the tracer pooling into the cervical lymph nodes. The results of the period during which lymphatic vessels were obstructed are shown in Table 2 . Table 2 Lymphatic vessel inhibition Mean duration of lymphatic vessel inhibition (days) Complications (1) One suture with 10 − 0 nylon 3.0 None (2) Two sutures with 10 − 0 nylon 5.7 ± 1.9 None (3) Two sutures with 10 − 0 nylon and bipolar cauterisation 7.0 None (4) Two sutures with 8 − 0 silk 5.7 ± 1.9 None ( 5 ) Two sutures with 8 − 0 silk and bipolar cauterisation 14.0 None ( 6 ) Two sutures with 8 − 0 silk with cyanoacrylate glue 25.0 None The approaches of using two sutures with 8 − 0 silk (Fig. 2 C) and cyanoacrylate glue (Fig. 2 D) were selected because of their ability to block lymphatic vessels the longest. Clinical course of corneal allografts in a lymphatic vessel ligation model Corneal transparency was maintained for 8 weeks in the control and lymphatic ligation groups without corneal transplantation. Corneal rejection was observed in 3 of 14 eyes in the ligation/Ag group, whereas 9 eyes exhibited rejection in the allograft group at 8 weeks post-surgery. The ligation/Ag group had significantly better graft survival than the allograft group at 8 weeks (log-rank test, p 50% survival at 8 weeks after surgery, and the median survival time (MST) was undefined. The MST in the allograft group was 3.0 weeks (Fig. 3 ). Angiogenesis and lymphangiogenesis after corneal transplantation Neovascularisation and lymphangiogenesis were significantly increased in the corneas with rejection (Figure A, B) compared with those in the corneas without rejection (Figure C, D). Figure 4 E and 4 F presents graphs quantifying neovascularisation and lymphangiogenesis and comparing the allograft (n = 13), ligation (n = 14), and ligation/Ag (n = 14) groups. Quantitative assessment could not be conducted in one eye in the allograft group because of suboptimal tissue conditions. The allograft group had a higher rejection rate than the ligation/Ag group and demonstrated a significant increase in neovascularisation and lymphangiogenesis (Steel–Dwass test, p = 0.008 and p = 0.004, respectively). DISCUSSION In this study, we successfully visualised the oculo-lymphatic pathway—which contributes to allograft rejection—after corneal transplantation in mice. The results showed that 20% trypan blue is useful for detecting lymphatic vessels directly. Furthermore, blocking the oculo-lymphatic route using a simple surgical technique reduced the rate of allograft rejection after keratoplasty. Thus, we elucidated the anatomical structure of lymphatic flow in ophthalmology, developed a surgical technique to block a specific lymphatic vessel, and applied the technique to regulate allograft rejection after keratoplasty. The dense lymphatic network around the eye is connected to the cervical lymph nodes, and thus is consistent with the present study results. 8 Evans blue is used to determine the localisation of lymphatic flow; 9 however, in this study, Evans blue exerted superficial keratitis adverse effects in the mouse cornea. Therefore, other types of stains were considered for staining lymphatic vessels, and 20% trypan blue helped to successfully detect lymphatic vessels with equivalent visualisation. In this study, we also investigated the effects of blocking the oculo-lymphatic pathway on allograft rejection after corneal transplantation using a simple surgical technique. First, the central lymphatic duct was ligated using 10 − 0 nylon or 8 − 0 silk. The suturing technique alone was ineffective for more than 7 days. However, two sutures of the duct using 8 − 0 silk combined with cauterisation and cyanoacrylate glue use had a blocking effect for 14 and 25 days, respectively. Rejection is more pronounced in the early phase, approximately 2 weeks after surgery; therefore, 25 days of lymphatic occlusion can prevent rejection. 10, 11 To the best of our knowledge, this is the first study to develop a surgical procedure that consistently blocks a specific lymphatic system. Oculo-lymphatic vessels exist in the peripheral area of the conjunctiva. 12, 13 Lymphatic vessels are essential for maintaining organ function in humans and mice by draining liquids. The importance of lymphatic vessels has been investigated in medical fields such as oncology, immunology, and pathology. 14, 15 In ophthalmology, several studies have focused on the relationship between lymphatic vessels and inflammation during corneal transplantation, and inhibiting the lymphatic pathway and blocking lymph nodes can increase transplant survival. 7, 16 Theoretically, three factors are responsible for the oculo-lymphatic pathway after corneal transplantation in mice. The first factor is the cervical lymph nodes, which our group first introduced. 5 However, surgically removing lymph nodes has not been recommended for clinical use owing to its invasive nature. The second factor is blocking of peripheral lymphangiogenesis. Corneal transparency is maintained by anti-angiogenic factors such as soluble VEGF receptor 1 (R1) or soluble VEGF-R2. 17 VEGF plays a role in producing pathological blood vessels via VEGF-R1 and VEGF-R2. In contrast, VEGF contributes to producing lymphatic vessels via VEGF-R2 and VEGF-R3. 18–20 VEGF is responsible for physiological or pathological angiogenesis, and previous studies have attempted to regulate the peripheral route by inhibiting VEGF expression. A pharmaceutical blocking approach using VEGF TrapR1R2 was developed,7 followed by related approaches to regulate VEGF, which is known as a VEGF trap. 20–22 The third factor is blocking the central pathway of the oculo-lymphatic route, that connects the peripheral area of the cornea or conjunctiva to the draining lymph node. Although the critical role of draining lymph nodes has been demonstrated by evaluating the effect of surgical lymphadenectomy in the immunology of corneal transplantation, no study has focused on blocking the central pathway of the oculo-lymphatic route. Therefore, in this study, we developed an approach for corneal transplantation for the first time. In this study, the blocking approach using a ligation technique combined with cyanoacrylate glue resulted in reduced allograft rejection compared to control transplantation in normal-risk settings. Although further studies are required to evaluate the immune reaction in low- and high-risk settings in mice, the consistent blocking effect in the early phase after transplantation may have contributed to the significant improvement in allograft rejection in the sutured group compared with that in the control group. These results are in accordance with the findings of a previous study, which demonstrated that the pharmaceutical blocking approaches in corneal transplantation minimises allosensitisation. 23 In conclusion, this study demonstrated that the surgical approach using suturing had a consistent effect and significantly improved the rejection rate after experimental corneal transplantation. Therefore, blocking the central pathway of oculo-lymphatic flow may be a novel strategy for regulating allosensitisation. METHODS Mice and anaesthesia Animal experimental protocols were approved by the Animal Care Committee of Nihon University (approval nos. P22-MED-028-1 and AP22-MED-062-3). All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research as well as in accordance with the ARRIVE guidelines. Male BALB/c mice (8–12 weeks old; Clea, Tokyo, Japan) were used to establish the lymphatic vessel ligation model. For corneal transplantation, BALB/c male mice (H-2, I-A d , 8–12 weeks old; Clea) were used as recipients and C57BL/6 male mice (H-2, I-A b , 8–12 weeks old; Clea) were used as donors. The mice were anesthetised using a mixture of medetomidine (1 mg/mL; ZENOAQ, Fukushima, Japan), midazolam (5 mg/mL; Sandoz, Tokyo, Japan), and butorphanol tartrate (5 mg/mL; Meiji, Tokyo, Japan). The mice were euthanised through carbon dioxide-induced hypoxia, followed by cervical dislocation. A total of 96 mice were randomly assigned to 24 cages (n = 4 mice per cage). There were no exclusions in our study. Lymphatic vessel visualisation and evaluation Lymphatic vessels were traced using vital staining. The minimum concentration of vital stain that did not cause corneal disorders was determined by evaluating different concentrations of Evans blue (#05604061; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and trypan blue (#20421102; Fujifilm Wako Pure Chemical Corporation). The 12 eyes were randomly assigned to four groups (n = 3 per group): ( 1 ) 1% Evans blue, ( 2 ) 10% Evans blue, ( 3 ) 10% trypan blue, and ( 4 ) 20% trypan blue. Both vital stains were topically administered. The vital stains were thoroughly rinsed with phosphate-buffered saline (PBS) after 15 min. After shaving and disinfecting the surgical site with a 7.5% povidone-iodine surgical scrub, an incision was made in the skin at the maxillary region to confirm that the lymphatic vessels ran from the limbal to the nasal side. A longitudinal incision was made from the cervical region to the sternum after verifying the continuity of lymphatic vessels from the limbus to the maxillary region. Superficial cervical and facial lymph nodes were identified using a microscope, as previously reported. 5 The lymphatic vessels that were traced with vital staining ran from the limbus to the superficial cervical and facial lymph nodes. The cornea was observed once a week for 9 weeks using a slit-lamp biomicroscope to examine complications by applying a vital stain. Lymphatic vessel inhibition in a normal murine model After administering a drop of vital stain, 7.5% povidone-iodine was applied to the surgical site. The maxillary region was then partially skinned to reveal lymphatic vessels. Eighteen eyes were divided into six groups (n = 3 per group) to inhibit lymphatic vessels assembling on the nasal side of the conjunctiva: ( 1 ) one suture with 10 − 0 nylon (MANI Ophthalmic Suture, Mani, Tochigi, Japan), ( 2 ) two sutures with 10 − 0 nylon, ( 3 ) two sutures with 10 − 0 nylon and bipolar cauterisation, ( 4 ) two sutures with 8 − 0 silk (CROWNJUN, Kono Seisakusho, Chiba, Japan), ( 5 ) two sutures with 8 − 0 silk and bipolar cauterisation, and ( 6 ) two sutures with 8 − 0 silk and cyanoacrylate glue (AlonAlpha A; Sankyo, Tokyo, Japan). The 8 − 0 silk was used to close the maxillary region in all the treated eyes. The lymphatic vessels between the two ligated lymphatic vessels were cauterised. Additionally, a small amount of cyanoacrylate glue was added to the ligated area. The tracer was administered following the same procedure described above on days 3, 7, 14, 21, 25, and 28 post-treatment, and the cervical lymph nodes were examined. The endpoint was determined based on the inflow of tracer into the superficial cervical and facial lymph nodes. Evaluation of corneal rejection in the lymphatic vessel ligation model As staining limits simultaneous lymphatic ligation and corneal transplantation owing to poor visibility, corneal transplantation was performed 1 week after lymphatic ligation. Four experimental groups were created to evaluate the effect of lymphatic vessel ligation: ( 1 ) full-thickness allograft corneal transplantation (allograft group, n = 14), ( 2 ) lymphatic vessel ligation (ligation group, n = 14), ( 3 ) full-thickness allograft corneal transplantation after lymphatic vessel ligation (ligation/Ag group, n = 14), and ( 4 ) control group without lymphatic vessel ligation or corneal transplantation (control group, n = 10). Orthotopic corneal transplantation and defining graft rejection Full-thickness corneal transplantation was performed as previously described. 5 Briefly, the recipient host bed was marked with a 1.5-mm trephine (Inami, Tokyo, Japan) and excised with a pair of micro-scissors (Vannas; Storz Instruments, San Dimas, CA, USA). The donor cornea was excised using a 2.0-mm trephine and transplanted into the host corneal bed with eight interrupted 11–0 needle sutures (Mani) in the right eye. After surgery, a gentamicin ointment was applied. The corneal sutures were removed 7 days after surgery. Eyes with postoperative cataracts, infections, or anterior chamber loss were excluded from the study. Corneal grafts were observed twice a week for 8 weeks using a slit-lamp biomicroscope. Graft opacity was scored on a standardised scale from one to five. 24 Corneal rejection was defined as rejection with successive scores of 3. Corneal angiogenesis and lymphangiogenesis were evaluated using whole-mount immunofluorescence staining 8 weeks after corneal transplantation. Corneal flat mount immunostaining The corneal flat mounts were excised after perfusion fixation and rinsed with PBS. The tissues were incubated with 20 mM ethylenediaminetetraacetic acid for 30 min at 37°C to remove the corneal epithelium. The corneas were then rinsed thrice with PBS and lysed with 10% Triton X-100 (Sigma Chemical, St Louis, MO, USA) for 30 min at room temperature. The tissues were then blocked with 10% donkey serum albumin (#168665; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 30 min at room temperature. The tissues were incubated with purified rat anti-mouse CD31 (1:200; #557355; BD Biosciences Pharmingen, Franklin Lakes, NJ, USA) and goat anti-mouse LYVE-1 (1:100; #AF2125; R&D Systems, Minneapolis, MN, USA) overnight at 4°C. The tissues were then rinsed with PBS and stained with Alexa Fluor 647-conjugated donkey anti-goat IgG (1:200; #A21447; Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 488-conjugated donkey anti-rat IgG (1:200; #A21208; Thermo Fisher Scientific) secondary antibodies for 90 min at room temperature. Finally, corneal flat mounts were rinsed with PBS and covered with Vectashield mounting medium (Vector Laboratories, Newark, CA, USA). The immunofluorescence signals were evaluated using a fluorescence microscope (ZEISS Axio Imager Z2; Carl Zeiss Microscopy GmbH, Oberkochen, Germany). The area of the total cornea covered by blood or lymphatic vessels was analysed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Statistical analyses Kaplan–Meier analysis was used to construct survival curves, and the log-rank test was used to compare corneal graft survival. Immunostaining results for hemangiogenesis and lymphangiogenesis were compared between the groups using the Steel–Dwass test. Statistical significance was set at p < 0.05. All quantitative variables are expressed as mean ± standard deviation (SD). Statistical analyses were performed using the JMP Pro software v 15.0.0 (SAS Institute, Cary, NC, USA). Sample size was based on a previous study. 7 Declarations Acknowledgements The study is reported in accordance with ARRIVE guidelines. The authors thank Akiko Tomioka for the support in histological examination. Author contributions A.I, T.H.: writing, reviewing, and editing of the manuscript; T.S.: curation of the data and editing of the manuscript; K.Y.: reviewing and editing of the manuscript; S.Y.: supervision and validation; All authors critically checked the manuscript and approved its submission. Data availability: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Competing interests: The author(s) declare no competing interests. Funding This research was supported by the Charitable Trust Fund for Ophthalmic Research in the Commemoration of Santen Pharmaceutical’s Founder and Japan Cornea Society. References Zhou, Y., Wang, T., Tuli, S. S., Steigleman, W. A. & Shah, A. A. Overview of corneal transplantation for the nonophthalmologist. Transplant. Direct 9 , e1434 (2023). 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). 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). Yamagami, S., Suzuki, S. & Tsuru, T. Risk factors for graft failure in penetrating keratoplasty. Acta Ophthalmol. Scand. 74 , 584-588 (1996). 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). 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). 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). Lohrberg, M. & Wilting, J. The lymphatic vascular system of the mouse head. Cell Tissue Res . 366 , 667-677 (2016). 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). 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). Hegde, S. & Niederkorn, J. Y. The role of cytotoxic T lymphocytes in corneal allograft rejection. Invest. Ophthalmol. Vis. Sci. 41 , 3341-3347 (2000). Wu, Y. et al . Organogenesis and distribution of the ocular lymphatic vessels in the anterior eye. JCI Insight 5 , e135121 (2020). 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). Dieterich, L. C., Tacconi, C., Ducoli, L. & Detmar, M. Lymphatic vessels in cancer. Physiol. Rev. 102 , 1837-1879 (2022). Petrova, T. V. & Koh, G. Y. Biological functions of lymphatic vessels. Science 369 , eaax4063 (2020). Cursiefen, C. et al . Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin. Invest. Ophthalmol. Vis. Sci. 43 , 2127-2135 (2002). Ambati, B. K. et al. Corneal avascularity is due to soluble VEGF receptor-1. Nature 443 , 993-997 (2006). Cursiefen, C. et al . VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J. Clin. Invest. 113 , 1040-1050 (2004). 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). 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). 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). Dohlman, T. H. et al. VEGF-trap Aflibercept significantly improves long-term graft survival in high-risk corneal transplantation. Transplantation 99 , 678-686 (2015). Chen, L. et al. Vascular endothelial growth factor receptor-3 mediates induction of corneal alloimmunity. Nat. Med. 10 , 813-815 (2004). Sonoda, Y. & Streilein, J. W. Orthotopic corneal transplantation in mice--evidence that the immunogenetic rules of rejection do not apply. Transplantation 54 , 694-704 (1992). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 Oct, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 29 Jul, 2024 Reviews received at journal 25 Jul, 2024 Reviewers agreed at journal 15 Jul, 2024 Reviews received at journal 08 Jul, 2024 Reviewers agreed at journal 20 Jun, 2024 Reviews received at journal 30 May, 2024 Reviewers agreed at journal 30 May, 2024 Reviewers invited by journal 26 May, 2024 Editor assigned by journal 26 May, 2024 Editor invited by journal 22 May, 2024 Submission checks completed at journal 22 May, 2024 First submitted to journal 18 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4439625","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":309264849,"identity":"e621c7e9-a426-4d25-b26c-419896095143","order_by":0,"name":"Ami Igarashi","email":"","orcid":"","institution":"Nihon University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ami","middleName":"","lastName":"Igarashi","suffix":""},{"id":309264850,"identity":"4c93a86a-5e14-4c4a-977c-d1b6f2848226","order_by":1,"name":"Takahiko Hayashi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYFACNhDBLAciDzwgRgMPVIsxWEsCKVoSG0AUUVrs2Y8lfrpRYZ0+P+zwQ6AtdnK6DYRs4Uk7LJ1zJj134+00A6CWZGOzAwQdlt4gndt2OHfj7ASQlgOJ2whq4X/e/Dv33+F0w9npH4jUIpF2TDq34XCCvHQOsbbceJZmnXMs3XCDdE7BgQQDIvzC3p9mfDunxlpefnb65g8fKuzkCGqBAwOwSgNilYOAfAMpqkfBKBgFo2BEAQDr40TLAPcAeAAAAABJRU5ErkJggg==","orcid":"","institution":"Nihon University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Takahiko","middleName":"","lastName":"Hayashi","suffix":""},{"id":309264851,"identity":"d8705b9a-edde-400a-8f97-0d58b56b826a","order_by":2,"name":"Toshiki Shimizu","email":"","orcid":"","institution":"Nihon University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Toshiki","middleName":"","lastName":"Shimizu","suffix":""},{"id":309264855,"identity":"4e71da7e-24dc-420b-8fb5-8c79a1c33bc6","order_by":3,"name":"Kentaro Yuda","email":"","orcid":"","institution":"Kikuna Yuda Eye Clinic","correspondingAuthor":false,"prefix":"","firstName":"Kentaro","middleName":"","lastName":"Yuda","suffix":""},{"id":309264856,"identity":"10e92d2c-210f-496c-b58c-79e5b33c742d","order_by":4,"name":"Satoru Yamagami","email":"","orcid":"","institution":"Nihon University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Satoru","middleName":"","lastName":"Yamagami","suffix":""}],"badges":[],"createdAt":"2024-05-18 06:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4439625/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4439625/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-77160-9","type":"published","date":"2024-10-28T16:20:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57873885,"identity":"d29b70bc-5f21-42ae-9e72-f4b59e8ddaed","added_by":"auto","created_at":"2024-06-06 18:42:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":218889,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of lymphatic vessels. Lymphatic vessels from the conjunctival region assemble at the nictating membrane and flow from the nasal side of the conjunctiva into the superficial cervical and facial lymph nodes. The junction of lymphatic vessels divides into the peripheral and central pathways. LV: lymphatic vessel, Md: deep head of the masseter muscle Ms: superficial head of the masseter muscle, CLN: cervical lymph node.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4439625/v1/84bd86af9d88a1e374af7259.png"},{"id":57873886,"identity":"f6d4d73f-06de-4911-87e3-22ae61912934","added_by":"auto","created_at":"2024-06-06 18:42:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":729826,"visible":true,"origin":"","legend":"\u003cp\u003eImages of lymphatic vessels stained with trypan blue in the right mouse eye. \u003cstrong\u003eA.\u003c/strong\u003e The white triangle indicates peripheral conjunctiva. The white arrow shows the junction of the lymphatic vessels. \u003cstrong\u003eB.\u003c/strong\u003e The blue triangle indicates lymphatic vessels flowing into the lymph nodes. The blue arrow indicates the cervical lymph node. \u003cstrong\u003eC.\u003c/strong\u003e Images of the ligation technique. The lymphatic vessels were ligated at the junction in two places with 8-0 silk (red arrows). \u003cstrong\u003eD. \u003c/strong\u003eCyanoacrylate glue was used to fix the ligated area (blue circle).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4439625/v1/fa610b10df7a3d48c687c54c.png"},{"id":57873887,"identity":"4cf1d3e3-9d09-4724-b5ed-b68f14921382","added_by":"auto","created_at":"2024-06-06 18:42:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":33901,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan–Meier survival curves were obtained using the log-rank test to statistically compare the allograft and ligation/Ag groups. The ligation/Ag group had significantly improved graft survival compared with that of the allograft group at 8 weeks.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4439625/v1/e0e829adbee341c919a39f77.png"},{"id":57873888,"identity":"1a76e86a-6c82-4faa-b7bf-23ca6a2489ba","added_by":"auto","created_at":"2024-06-06 18:42:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":364004,"visible":true,"origin":"","legend":"\u003cp\u003eNeovascularisation and lymphangiogenesis images (\u003cstrong\u003eA–D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e CD31 (blood vessels) and \u003cstrong\u003eB.\u003c/strong\u003e LYVE-1 (lymphatic vessels) staining of the cornea exhibiting rejection in the allograft group. \u003cstrong\u003eC. \u003c/strong\u003eCD31 and \u003cstrong\u003eD. \u003c/strong\u003eLYVE-1 staining of the cornea without rejection in the ligation/Ag group. \u003cstrong\u003eE.\u003c/strong\u003e The graph shows a comparison of the percent area of CD31 staining among the groups. \u003cstrong\u003eF.\u003c/strong\u003e The graph represents a comparison of the percent area of LYVE-1 staining among the groups. * p \u0026lt; 0.01, *** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4439625/v1/4a6b2e9ef5ed5231678d1fa4.png"},{"id":68207345,"identity":"8cab7d59-9fdd-41ad-ae13-6996dfd06866","added_by":"auto","created_at":"2024-11-04 16:36:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2537224,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4439625/v1/d02960ff-5eb3-4d2e-b444-27d7d03c3478.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lymphatic Vessel Ligation: A Novel Murine Model for Inhibiting Corneal Transplantation Rejection","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRecently, corneal transplantation has progressed to selectively replacing diseased corneal layers.\u003csup\u003e1\u003c/sup\u003e However, penetrating keratoplasty (PKP) remains a prevalent form of corneal transplantation for various indications such as infection, regraft, and keratoconus.\u003csup\u003e2\u003c/sup\u003e Although the graft survival rate after 1 year in patients with PKP exceeds 90%, the rates differ with indications. For example, the survival rate at 10 years is also \u0026gt;\u0026thinsp;90% in patients with keratoconus; however, the survival rate of a failed previous graft is approximately 30%.\u003csup\u003e3,4\u003c/sup\u003e Graft failure is often caused by immunologic graft rejection, and developing therapeutic strategies to improve graft survival is essential.\u003c/p\u003e \u003cp\u003eThe lymphatic system is a key player in allograft rejection after corneal transplantation; however, excising cervical lymph nodes increases graft survival.\u003csup\u003e5\u003c/sup\u003e Lymphatic vessels play a role in graft rejection, and alloantigen and antigen-presenting cell migration to lymph nodes via lymphatic vessels may trigger graft rejection.\u003csup\u003e6\u003c/sup\u003e 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) expression.\u003csup\u003e7\u003c/sup\u003e 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.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eVisualisation of lymphatic vessels and establishment of a lymphatic vessel ligation model\u003c/h2\u003e \u003cp\u003eAlthough the Evans blue tracer was thoroughly rinsed, superficial punctate keratitis (SPK) was observed after administering a drop of the tracer. Therefore, the minimum concentration and method of tracer administration were evaluated. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the results of the confirmed influx into the superficial cervical and facial lymph nodes and complications at different concentrations.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTracers used to visualise lymphatic vessels\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of confirmed inflows to the lymph nodes (eyes)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eComplications\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEvans blue\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e1%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSPK (one eye)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSPK (two eyes)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTrypan blue\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e10%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e20%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eSPK, superficial punctate keratitis\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows a schematic representation of the lymphatic vessel originating from the nasal aspect of the conjunctiva and culminating in cervical lymph nodes. The junction demarcated the peripheral and central pathways. The lymphatic vessels assembled from the limbus to the nasal side of the conjunctiva, and they were stained with trypan blue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows the tracing lymphatic vessel with the tracer pooling into the cervical lymph nodes. The results of the period during which lymphatic vessels were obstructed are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLymphatic vessel inhibition\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean duration of lymphatic vessel inhibition (days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eComplications\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(1) One suture with 10\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(2) Two sutures with 10\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(3) Two sutures with 10\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon and bipolar cauterisation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(4) Two sutures with 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) Two sutures with 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk and bipolar cauterisation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) Two sutures with 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk with cyanoacrylate glue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe approaches of using two sutures with 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and cyanoacrylate glue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) were selected because of their ability to block lymphatic vessels the longest.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eClinical course of corneal allografts in a lymphatic vessel ligation model\u003c/h2\u003e \u003cp\u003eCorneal transparency was maintained for 8 weeks in the control and lymphatic ligation groups without corneal transplantation. Corneal rejection was observed in 3 of 14 eyes in the ligation/Ag group, whereas 9 eyes exhibited rejection in the allograft group at 8 weeks post-surgery. The ligation/Ag group had significantly better graft survival than the allograft group at 8 weeks (log-rank test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The ligation/Ag group showed\u0026thinsp;\u0026gt;\u0026thinsp;50% survival at 8 weeks after surgery, and the median survival time (MST) was undefined. The MST in the allograft group was 3.0 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAngiogenesis and lymphangiogenesis after corneal transplantation\u003c/h2\u003e \u003cp\u003eNeovascularisation and lymphangiogenesis were significantly increased in the corneas with rejection (Figure A, B) compared with those in the corneas without rejection (Figure C, D). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF presents graphs quantifying neovascularisation and lymphangiogenesis and comparing the allograft (n\u0026thinsp;=\u0026thinsp;13), ligation (n\u0026thinsp;=\u0026thinsp;14), and ligation/Ag (n\u0026thinsp;=\u0026thinsp;14) groups. Quantitative assessment could not be conducted in one eye in the allograft group because of suboptimal tissue conditions. The allograft group had a higher rejection rate than the ligation/Ag group and demonstrated a significant increase in neovascularisation and lymphangiogenesis (Steel\u0026ndash;Dwass test, p\u0026thinsp;=\u0026thinsp;0.008 and p\u0026thinsp;=\u0026thinsp;0.004, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we successfully visualised the oculo-lymphatic pathway\u0026mdash;which contributes to allograft rejection\u0026mdash;after corneal transplantation in mice. The results showed that 20% trypan blue is useful for detecting lymphatic vessels directly. Furthermore, blocking the oculo-lymphatic route using a simple surgical technique reduced the rate of allograft rejection after keratoplasty. Thus, we elucidated the anatomical structure of lymphatic flow in ophthalmology, developed a surgical technique to block a specific lymphatic vessel, and applied the technique to regulate allograft rejection after keratoplasty.\u003c/p\u003e \u003cp\u003eThe dense lymphatic network around the eye is connected to the cervical lymph nodes, and thus is consistent with the present study results.\u003csup\u003e8\u003c/sup\u003e Evans blue is used to determine the localisation of lymphatic flow;\u003csup\u003e9\u003c/sup\u003e however, in this study, Evans blue exerted superficial keratitis adverse effects in the mouse cornea. Therefore, other types of stains were considered for staining lymphatic vessels, and 20% trypan blue helped to successfully detect lymphatic vessels with equivalent visualisation.\u003c/p\u003e \u003cp\u003eIn this study, we also investigated the effects of blocking the oculo-lymphatic pathway on allograft rejection after corneal transplantation using a simple surgical technique. First, the central lymphatic duct was ligated using 10\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon or 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk. The suturing technique alone was ineffective for more than 7 days. However, two sutures of the duct using 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk combined with cauterisation and cyanoacrylate glue use had a blocking effect for 14 and 25 days, respectively. Rejection is more pronounced in the early phase, approximately 2 weeks after surgery; therefore, 25 days of lymphatic occlusion can prevent rejection.\u003csup\u003e10, 11\u003c/sup\u003e To the best of our knowledge, this is the first study to develop a surgical procedure that consistently blocks a specific lymphatic system.\u003c/p\u003e \u003cp\u003eOculo-lymphatic vessels exist in the peripheral area of the conjunctiva.\u003csup\u003e12, 13\u003c/sup\u003e Lymphatic vessels are essential for maintaining organ function in humans and mice by draining liquids. The importance of lymphatic vessels has been investigated in medical fields such as oncology, immunology, and pathology.\u003csup\u003e14, 15\u003c/sup\u003e In ophthalmology, several studies have focused on the relationship between lymphatic vessels and inflammation during corneal transplantation, and inhibiting the lymphatic pathway and blocking lymph nodes can increase transplant survival.\u003csup\u003e7, 16\u003c/sup\u003e Theoretically, three factors are responsible for the oculo-lymphatic pathway after corneal transplantation in mice. The first factor is the cervical lymph nodes, which our group first introduced.\u003csup\u003e5\u003c/sup\u003e However, surgically removing lymph nodes has not been recommended for clinical use owing to its invasive nature. The second factor is blocking of peripheral lymphangiogenesis. Corneal transparency is maintained by anti-angiogenic factors such as soluble VEGF receptor 1 (R1) or soluble VEGF-R2.\u003csup\u003e17\u003c/sup\u003e VEGF plays a role in producing pathological blood vessels via VEGF-R1 and VEGF-R2. In contrast, VEGF contributes to producing lymphatic vessels via VEGF-R2 and VEGF-R3.\u003csup\u003e18\u0026ndash;20\u003c/sup\u003e VEGF is responsible for physiological or pathological angiogenesis, and previous studies have attempted to regulate the peripheral route by inhibiting VEGF expression. A pharmaceutical blocking approach using VEGF TrapR1R2 was developed,7 followed by related approaches to regulate VEGF, which is known as a VEGF trap.\u003csup\u003e20\u0026ndash;22\u003c/sup\u003e The third factor is blocking the central pathway of the oculo-lymphatic route, that connects the peripheral area of the cornea or conjunctiva to the draining lymph node. Although the critical role of draining lymph nodes has been demonstrated by evaluating the effect of surgical lymphadenectomy in the immunology of corneal transplantation, no study has focused on blocking the central pathway of the oculo-lymphatic route. Therefore, in this study, we developed an approach for corneal transplantation for the first time.\u003c/p\u003e \u003cp\u003eIn this study, the blocking approach using a ligation technique combined with cyanoacrylate glue resulted in reduced allograft rejection compared to control transplantation in normal-risk settings. Although further studies are required to evaluate the immune reaction in low- and high-risk settings in mice, the consistent blocking effect in the early phase after transplantation may have contributed to the significant improvement in allograft rejection in the sutured group compared with that in the control group. These results are in accordance with the findings of a previous study, which demonstrated that the pharmaceutical blocking approaches in corneal transplantation minimises allosensitisation.\u003csup\u003e23\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrated that the surgical approach using suturing had a consistent effect and significantly improved the rejection rate after experimental corneal transplantation. Therefore, blocking the central pathway of oculo-lymphatic flow may be a novel strategy for regulating allosensitisation.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMice and anaesthesia\u003c/h2\u003e \u003cp\u003eAnimal experimental protocols were approved by the Animal Care Committee of Nihon University (approval nos. P22-MED-028-1 and AP22-MED-062-3). All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research as well as in accordance with the ARRIVE guidelines. Male BALB/c mice (8\u0026ndash;12 weeks old; Clea, Tokyo, Japan) were used to establish the lymphatic vessel ligation model. For corneal transplantation, BALB/c male mice (H-2, I-A\u003csup\u003ed\u003c/sup\u003e, 8\u0026ndash;12 weeks old; Clea) were used as recipients and C57BL/6 male mice (H-2, I-A\u003csup\u003eb\u003c/sup\u003e, 8\u0026ndash;12 weeks old; Clea) were used as donors. The mice were anesthetised using a mixture of medetomidine (1 mg/mL; ZENOAQ, Fukushima, Japan), midazolam (5 mg/mL; Sandoz, Tokyo, Japan), and butorphanol tartrate (5 mg/mL; Meiji, Tokyo, Japan). The mice were euthanised through carbon dioxide-induced hypoxia, followed by cervical dislocation. A total of 96 mice were randomly assigned to 24 cages (n\u0026thinsp;=\u0026thinsp;4 mice per cage). There were no exclusions in our study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eLymphatic vessel visualisation and evaluation\u003c/h2\u003e \u003cp\u003eLymphatic vessels were traced using vital staining. The minimum concentration of vital stain that did not cause corneal disorders was determined by evaluating different concentrations of Evans blue (#05604061; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and trypan blue (#20421102; Fujifilm Wako Pure Chemical Corporation). The 12 eyes were randomly assigned to four groups (n\u0026thinsp;=\u0026thinsp;3 per group): (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) 1% Evans blue, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) 10% Evans blue, (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) 10% trypan blue, and (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) 20% trypan blue. Both vital stains were topically administered. The vital stains were thoroughly rinsed with phosphate-buffered saline (PBS) after 15 min. After shaving and disinfecting the surgical site with a 7.5% povidone-iodine surgical scrub, an incision was made in the skin at the maxillary region to confirm that the lymphatic vessels ran from the limbal to the nasal side. A longitudinal incision was made from the cervical region to the sternum after verifying the continuity of lymphatic vessels from the limbus to the maxillary region. Superficial cervical and facial lymph nodes were identified using a microscope, as previously reported.\u003csup\u003e5\u003c/sup\u003e The lymphatic vessels that were traced with vital staining ran from the limbus to the superficial cervical and facial lymph nodes. The cornea was observed once a week for 9 weeks using a slit-lamp biomicroscope to examine complications by applying a vital stain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLymphatic vessel inhibition in a normal murine model\u003c/h2\u003e \u003cp\u003eAfter administering a drop of vital stain, 7.5% povidone-iodine was applied to the surgical site. The maxillary region was then partially skinned to reveal lymphatic vessels. Eighteen eyes were divided into six groups (n\u0026thinsp;=\u0026thinsp;3 per group) to inhibit lymphatic vessels assembling on the nasal side of the conjunctiva: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) one suture with 10\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon (MANI Ophthalmic Suture, Mani, Tochigi, Japan), (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) two sutures with 10\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon, (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) two sutures with 10\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon and bipolar cauterisation, (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) two sutures with 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk (CROWNJUN, Kono Seisakusho, Chiba, Japan), (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) two sutures with 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk and bipolar cauterisation, and (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) two sutures with 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk and cyanoacrylate glue (AlonAlpha A; Sankyo, Tokyo, Japan). The 8\u0026thinsp;\u0026minus;\u0026thinsp;0 silk was used to close the maxillary region in all the treated eyes. The lymphatic vessels between the two ligated lymphatic vessels were cauterised. Additionally, a small amount of cyanoacrylate glue was added to the ligated area. The tracer was administered following the same procedure described above on days 3, 7, 14, 21, 25, and 28 post-treatment, and the cervical lymph nodes were examined. The endpoint was determined based on the inflow of tracer into the superficial cervical and facial lymph nodes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of corneal rejection in the lymphatic vessel ligation model\u003c/h2\u003e \u003cp\u003eAs staining limits simultaneous lymphatic ligation and corneal transplantation owing to poor visibility, corneal transplantation was performed 1 week after lymphatic ligation. Four experimental groups were created to evaluate the effect of lymphatic vessel ligation: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) full-thickness allograft corneal transplantation (allograft group, n\u0026thinsp;=\u0026thinsp;14), (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) lymphatic vessel ligation (ligation group, n\u0026thinsp;=\u0026thinsp;14), (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) full-thickness allograft corneal transplantation after lymphatic vessel ligation (ligation/Ag group, n\u0026thinsp;=\u0026thinsp;14), and (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) control group without lymphatic vessel ligation or corneal transplantation (control group, n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOrthotopic corneal transplantation and defining graft rejection\u003c/h2\u003e \u003cp\u003eFull-thickness corneal transplantation was performed as previously described.\u003csup\u003e5\u003c/sup\u003e Briefly, the recipient host bed was marked with a 1.5-mm trephine (Inami, Tokyo, Japan) and excised with a pair of micro-scissors (Vannas; Storz Instruments, San Dimas, CA, USA). The donor cornea was excised using a 2.0-mm trephine and transplanted into the host corneal bed with eight interrupted 11\u0026ndash;0 needle sutures (Mani) in the right eye. After surgery, a gentamicin ointment was applied. The corneal sutures were removed 7 days after surgery. Eyes with postoperative cataracts, infections, or anterior chamber loss were excluded from the study. Corneal grafts were observed twice a week for 8 weeks using a slit-lamp biomicroscope. Graft opacity was scored on a standardised scale from one to five.\u003csup\u003e24\u003c/sup\u003e Corneal rejection was defined as rejection with successive scores of 3. Corneal angiogenesis and lymphangiogenesis were evaluated using whole-mount immunofluorescence staining 8 weeks after corneal transplantation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCorneal flat mount immunostaining\u003c/h2\u003e \u003cp\u003eThe corneal flat mounts were excised after perfusion fixation and rinsed with PBS. The tissues were incubated with 20 mM ethylenediaminetetraacetic acid for 30 min at 37\u0026deg;C to remove the corneal epithelium. The corneas were then rinsed thrice with PBS and lysed with 10% Triton X-100 (Sigma Chemical, St Louis, MO, USA) for 30 min at room temperature. The tissues were then blocked with 10% donkey serum albumin (#168665; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 30 min at room temperature. The tissues were incubated with purified rat anti-mouse CD31 (1:200; #557355; BD Biosciences Pharmingen, Franklin Lakes, NJ, USA) and goat anti-mouse LYVE-1 (1:100; #AF2125; R\u0026amp;D Systems, Minneapolis, MN, USA) overnight at 4\u0026deg;C. The tissues were then rinsed with PBS and stained with Alexa Fluor 647-conjugated donkey anti-goat IgG (1:200; #A21447; Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 488-conjugated donkey anti-rat IgG (1:200; #A21208; Thermo Fisher Scientific) secondary antibodies for 90 min at room temperature. Finally, corneal flat mounts were rinsed with PBS and covered with Vectashield mounting medium (Vector Laboratories, Newark, CA, USA). The immunofluorescence signals were evaluated using a fluorescence microscope (ZEISS Axio Imager Z2; Carl Zeiss Microscopy GmbH, Oberkochen, Germany). The area of the total cornea covered by blood or lymphatic vessels was analysed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eKaplan\u0026ndash;Meier analysis was used to construct survival curves, and the log-rank test was used to compare corneal graft survival. Immunostaining results for hemangiogenesis and lymphangiogenesis were compared between the groups using the Steel\u0026ndash;Dwass test. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All quantitative variables are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using the JMP Pro software v 15.0.0 (SAS Institute, Cary, NC, USA). Sample size was based on a previous study.\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003eThe authors thank Akiko Tomioka for the support in histological examination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.I, T.H.: writing, reviewing, and editing of the manuscript; T.S.: curation of the data and editing of the manuscript; K.Y.: reviewing and editing of the manuscript; S.Y.: supervision and validation; All authors critically checked the manuscript and approved its submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe author(s) declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Charitable Trust Fund for Ophthalmic Research in the Commemoration of Santen Pharmaceutical\u0026rsquo;s Founder and Japan Cornea Society.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhou, Y., Wang, T., Tuli, S. 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Rev.\u003c/em\u003e \u003cstrong\u003e102\u003c/strong\u003e, 1837-1879 (2022).\u003c/li\u003e\n\u003cli\u003ePetrova, T. V. \u0026amp; Koh, G. Y. Biological functions of lymphatic vessels. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e369\u003c/strong\u003e, eaax4063 (2020). \u003c/li\u003e\n\u003cli\u003eCursiefen, C. \u003cem\u003eet al\u003c/em\u003e. Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin. \u003cem\u003eInvest. Ophthalmol.\u003c/em\u003e \u003cem\u003eVis. Sci.\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 2127-2135 (2002).\u003c/li\u003e\n\u003cli\u003eAmbati, B. K. \u003cem\u003eet al.\u003c/em\u003e Corneal avascularity is due to soluble VEGF receptor-1. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e443\u003c/strong\u003e, 993-997 (2006). \u003c/li\u003e\n\u003cli\u003eCursiefen, C. \u003cem\u003eet al\u003c/em\u003e. VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 1040-1050 (2004). \u003c/li\u003e\n\u003cli\u003eAmadio, M., Govoni, S. \u0026amp; Pascale, A. Targeting VEGF in eye neovascularization: What\u0026apos;s new?: A comprehensive review on current therapies and oligonucleotide-based interventions under development. \u003cem\u003ePharmacol. Res\u003c/em\u003e. \u003cstrong\u003e103\u003c/strong\u003e, 253-269 (2016). \u003c/li\u003e\n\u003cli\u003eAlbuquerque, R. J. C. \u003cem\u003eet al.\u003c/em\u003e Alternatively spliced VEGF receptor-2 is an essential endogenous inhibitor of lymphatic vessels. \u003cem\u003eNat. Med.\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1023-1030 (2009). \u003c/li\u003e\n\u003cli\u003eZhang, W., Sch\u0026ouml;nberg, A., Bock, F. \u0026amp; Cursiefen, C. Posttransplant VEGFR1R2 trap eye drops inhibit corneal (lymph)angiogenesis and improve corneal allograft survival in eyes at high risk of rejection. \u003cem\u003eTransl. Vis. Sci. Technol.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 6 (2022).\u003c/li\u003e\n\u003cli\u003eDohlman, T. H. \u003cem\u003eet al.\u003c/em\u003e VEGF-trap Aflibercept significantly improves long-term graft survival in high-risk corneal transplantation. \u003cem\u003eTransplantation\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 678-686 (2015).\u003c/li\u003e\n\u003cli\u003eChen, L. \u003cem\u003eet al.\u003c/em\u003e Vascular endothelial growth factor receptor-3 mediates induction of corneal alloimmunity. \u003cem\u003eNat. Med.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 813-815 (2004). \u003c/li\u003e\n\u003cli\u003eSonoda, Y. \u0026amp; Streilein, J. W. Orthotopic corneal transplantation in mice--evidence that the immunogenetic rules of rejection do not apply. \u003cem\u003eTransplantation\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 694-704 (1992). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"corneal transplantation, lymphangiogenesis, lymphatic vessel ligation, neovascularisation","lastPublishedDoi":"10.21203/rs.3.rs-4439625/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4439625/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe 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. 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\u0026mdash;one with and one without lymphatic vessel ligation, to evaluate their effects on allograft rejection. Graft opacity scores were evaluated for 8 weeks, and immunohistochemistry was used to quantify angiogenesis and lymphangiogenesis. Twenty percent 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. Three and nine out of fourteen eyes showed rejection at 8 weeks post-surgery in the lymphatic vessel ligation and allograft groups, respectively. Furthermore, neovascularisation and lymphangiogenesis significantly decreased in the lymphatic vessel ligation group. Overall, we present a novel therapeutic strategy for corneal transplantation.\u003c/p\u003e","manuscriptTitle":"Lymphatic Vessel Ligation: A Novel Murine Model for Inhibiting Corneal Transplantation Rejection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-06 18:42:36","doi":"10.21203/rs.3.rs-4439625/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-29T05:26:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-25T21:11:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298873343658677146514470083273967831777","date":"2024-07-15T07:03:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-08T20:38:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"57755295365194085647576208478158090263","date":"2024-06-20T07:46:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-31T03:29:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182492787199504929185378125599187301288","date":"2024-05-31T00:52:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-26T23:53:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-26T23:49:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-22T04:13:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-22T04:11:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-18T06:04:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd9ad2f4-b55b-4b46-be9a-312bdf04a823","owner":[],"postedDate":"June 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-04T16:29:19+00:00","versionOfRecord":{"articleIdentity":"rs-4439625","link":"https://doi.org/10.1038/s41598-024-77160-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-10-28 16:20:36","publishedOnDateReadable":"October 28th, 2024"},"versionCreatedAt":"2024-06-06 18:42:36","video":"","vorDoi":"10.1038/s41598-024-77160-9","vorDoiUrl":"https://doi.org/10.1038/s41598-024-77160-9","workflowStages":[]},"version":"v1","identity":"rs-4439625","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4439625","identity":"rs-4439625","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}