Novel Method to Detect Corneal Lymphatic Vessels In

TECHNIQUES

Novel Method to Detect Corneal Lymphatic Vessels In Vivo by Intrastromal Injection of Fluorescein Viet Nhat Hung Le, MD,* Yanhong Hou, MD,* Jens Horstmann, PhD,*† Felix Bock, PhD,*‡ and Claus Cursiefen, MD*‡

Purpose: Corneal lymphatic vessels are clinically invisible because

of their thin walls and clear lymph fluid. There is no easy and established method for in vivo imaging of corneal lymphatic vessels so far. In this study, we present a novel approach to visualize corneal lymphatic vessels in vivo by injecting intrastromal fluorescein sodium.

Methods: Six- to eight-week-old female BALB/c mice were used in the mouse model of suture-induced corneal neovascularization. Two weeks after the suture placement, fluorescein sodium was injected intrastromally. The fluorescein, taken up by the presumed lymphatic vessels, was then tracked using a clinically used Spectralis HRA + OCT device. Immunohistochemistry staining with specific lymphatic marker LYVE-1 and pan-endothelial marker CD31 was used to confirm the indirect lymphangiography findings. Results: By injecting fluorescein intrastromally, both corneal blood and lymphatic vessels were detected. While the lymphatic vessels were visible as bright vessel-like structures using HRA, the blood vessels appeared as dark networks. Fluorescein-labeled lymphatic vessels were colocalized with LYVE-1 in immunohistochemically stained sections of the same specimen. Conclusions: Corneal lymphatic vessels can be easily imaged in vivo in the murine model using intrastromal fluorescein injection. Key Words: corneal lymphatic imaging, intrastromal fluorescein injection, indirect fluorescein lymphangiography (Cornea 2018;37:267–271) Received for publication September 5, 2017; revision received September 21, 2017; accepted September 24, 2017. Published online ahead of print November 9, 2017. From the *Department of Ophthalmology, University Hospital of Cologne, Cologne, Germany; †Cluster of Excellence: Cellular Stress Responses in Aging-associated Diseases, CECAD, University of Cologne, Cologne, Germany; and ‡Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany. Supported by German Research Foundation (DFG) FOR2240 “(Lymph)angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye”, Cu 47/ 4-2 (C. Cursiefen), Cu 47/6-1 (C. Cursiefen), Cu 47/9-1 (C. Cursiefen), and STE1928/4-1 (J. Horstmann) (www.for2240.de); EU COST BM1302 (F. Bock and C. Cursiefen; www.biocornea.eu); EU Horizon 2020 ARREST BLINDNESS (C. Cursiefen; www.arrestblindness.eu); Center for Molecular Medicine Cologne, University of Cologne (F. Bock and C. Cursiefen; www. cmmc-uni-koeln.de/home/), DAAD (German Academic Exchange Service: V. N. H. Le), Chinese Scholarship Council CSC (Y. Hou). The authors have no conflicts of interest to disclose. Reprints: Claus Cursiefen, MD, Department of Ophthalmology, University of Cologne, Kerpener Strasse 62, 50924 Cologne, Germany (e-mail: [email protected]). Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.

Cornea Volume 37, Number 2, February 2018

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he cornea is one of few human tissues devoid of blood and lymphatic vessels. Because of the avascularity and easily accessible location, the cornea becomes an ideal site to investigate mechanisms of hemangiogenesis and lymphangiogenesis.1 Furthermore, breach of “corneal (lymph)angiogenic privilege” renders a cornea high-risk in case of subsequent corneal transplantation.2 Lymphatic vessels, especially lymphatic capillaries, have a thin, discontinuous, or even absent basement membrane, drain clear and transparent lymph fluid, and are not surrounded by smooth muscle cells.3 These facts make detection of lymphatic vessels hard in the clinical setting and in experimental research, and there is a great unmet need for live in vivo imaging.4 That is the reason why the lymphatic system was unrecognized for many centuries, although already described in 1622 by Gasparo Aselli.5 With the discovery of several specific markers for lymphatic vessels in the late 20th century, such as vascular endothelial growth factor receptor-3 (VEGFR-3),6,7 podoplanin,8,9 and lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1),10 their structure and function could be well studied ex vivo. The lymphatic system complements the vascular system and is responsible for the tissue fluid and protein balance. Moreover, lymphatic vessels are of particular importance for induction and regulation of immune responses, therefore playing a crucial role in cancer metastasis, inflammatory conditions, and transplant rejection. More recently, Dietrich et al11 showed that lymphatic vessels are more important than blood vessels in immune rejection after corneal transplantation. Hence, lymphatics become a novel target in treatment strategies for corneal and ocular surface diseases.1,12 It is necessary to find a new technique replacing the conventional ex vivo immunohistochemical assays to image lymphatic vessels in vivo to easily translate that into the clinical setting. Over the last decade, lymphatic imaging modalities have been advanced. Several studies demonstrated the feasibility of in vivo live imaging methods to track and assess the lymphatic system in both animal and human. These included computed tomography,13 lymphoscintigraphy,14,15 positron emission tomography,16 magnetic resonance imaging,17,18 ultrasound,19 optical imaging,20 optical coherence tomography (OCT),21–23 laser speckle imaging,24 and photoacoustic flow cytometry techniques.25 However, in the field of ophthalmology, especially in corneal lymphatic imaging, there has still been a paucity of studies and imaging methods. In 2011, Yuen et al26 showed that corneal lymphatic vessels could be imaged by subconjunctival injection of large-molecular-weight www.corneajrnl.com |

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Le et al

fluorescein isothiocyanate-labeled dextran. At the same time, Steven et al27 and Peebo et al28,29 identified corneal lymphatic vessels at the cellular level using intravital 2-photon microscopy and in vivo confocal microscopy. In summary, the drawbacks of the above-mentioned techniques are the need for cell markers, high-cost, specific instruments, and time consumption. In this study, we present a novel method to visualize corneal lymphatic vessels by intrastromally injecting the most clinically used contrast agent—fluorescein sodium. Here we test the hypothesis that the excess intrastromal fluorescein dye will be taken up by corneal lymphatic vessels and thereby can be detected using commercially available and widely used fluorescein angiography devices.

Cigankova, Czech Republic). Sodium fluorescein 10% (Fluorescein ALCON; Alcon Pharma GmbH, Freiburg, Germany) was diluted with the ratio of 1:20, and 2 to 3 mL of this solution was subsequently injected into the pocket through the small hub removable needle and 25-mL Hamilton syringe (Hamilton Company, Reno, NV). The mouse was then placed in front of the HRA machine, and the camera lens was adjusted to get the proper focus on the nasal side of the cornea. The fluorescein angiography mode was chosen, and the images were taken every 10 seconds until the first fluorescent signal in the vessel-like structure was obtained. After that, we focused on the fluorescein-labeled vessels, and images were taken until the signal disappeared. During this experiment, the mouse cornea was moisturized using Methocel 2% (OmniVision GmbH, Puchheim, Germany).

MATERIALS AND METHODS Mice and Anesthesia All experimental procedures were approved by the local animal care and use committee and conformed to the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmology and Vision Research. Female BALB/c mice, purchased from Charles River Laboratories, Sulzfeld, Germany (aged 6–8 weeks), were used in the mouse model of suture-induced corneal inflammation and the neovascularization assay.30 Before surgery, mice were deeply anesthetized with an intraperitoneal injection of a mixture of Ketanest S (Pfizer Deutschland GmbH, Berlin, Germany) (8 mg/kg) and Rompun (Bayer Vital GmbH, Leverkusen, Germany) (0.1 mL/kg).

Suture-Induced Corneal Inflammation and Neovascularization Assay The mouse model of suture-induced inflammatory neovascularization was performed as previously described.30–32 One 11-0 nylon suture (Serag Wiessner, Naila, Germany) was placed intrastromally on the right eye on the nasal side of the cornea with 2 stromal incursions extending over 120 degrees of the corneal circumference each. The outer point of suture placement was chosen near the limbus, and the inner suture point was inserted near the center of the cornea equidistant from the limbus to obtain standardized angiogenic responses. The suture was left in place for 14 days and was then removed. One small suture was placed on the nasal side for marking the orientation of the cornea in the subsequent immunohistochemical wholemount staining.

Indirect Lymphangiography Procedure

Color Imaging Procedure

After finishing the fluorescein lymphangiography procedure, color images of this area were captured by a Nikon Digital Sight high-speed DS-Vi1 color microscope camera (Nikon Corporation, Japan) mounted on a Nikon SMZ800 stereomicroscope (Nikon Corporation). The cornea was moisturized during this procedure with Methocel 2%. These images were then used to differentiate the fluorescein-labeled lymphatic vessels from the blood vessels.

Immunohistochemical Staining of Corneal Flat Mounts The blood and lymphatic vessels in corneal wholemounts were double stained as described previously.4 After being rinsed 2 times in phosphate buffered saline (PBS), harvested corneas then were fixed in acetone for 30 minutes. Corneas were subsequently washed in PBS 3 times and blocked with 2% bovine serum albumin in PBS. These samples were stained overnight (in dark, at 4°C) with rabbit anti-mouse lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1) (1:200; AngioBio, Del Mar, CA) for lymphatic vessels and with a rat anti-mouse CD31/PECAM-1 antibody (1:100; Acris Antibodies, MA) for blood vessels. On the second day, LYVE-1 and CD31/ PECAM-1 were detected using a goat-anti-rabbit Alexa Fluor 488 secondary antibody (1: 2000; Invitrogen) and a goat-anti-rat Alexa Fluor 555 secondary antibody (1: 2000; Invitrogen), respectively. Double-stained wholemount images were taken at ·100 magnification with a fluorescence microscope (BX53; Olympus Optical Co, Hamburg, Germany). These photographs were then confronted with color images and lymphangiography images to validate the corneal lymphatic vessels.

For indirect fluorescein lymphangiography, a clinical Spectralis HRA + OCT device (Heidelberg Engineering, Heidelberg, Germany) with a 55-degree imaging lens was chosen. A combination of Ketanest S and Rompun was used to create deep anesthesia. After general anesthesia, Conjucain EDO eye drops (Dr. Mann Pharma and Bausch & Lomb GmbH, Berlin, Germany) were used for topical anesthesia. Subsequently, a small central intrastromal pocket was created using a 30-G needle (Ø 0.3 · 12 mm, B Braun Medical Praha,

Based on the concept that lymphatic vessels collect excess interstitial fluids,27,33 we injected fluorescein intrastromally and then tracked the fluorescent signal to identify corneal lymphatic vessels. In the early phase of this procedure,

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RESULTS Intrastromal Injection of Fluorescein Can Visualize Presumed Lymphatic Vessels



blood vessels appear as dark shadows in the fluorescein angiography mode, and no fluorescein-labeled vessels were detected. About 30 minutes after the injection, the fluorescent signals were detected in some tubular structures. However, from the 70th minute onward, we did not detect any fluorescein in the cornea, and the fluorescein-labeled vessels disappeared (data not shown). To differentiate blood and lymphatic vessels, color images were taken at the same location of presumed lymphatic vessels. The fluoresceinlabeled vessels were invisible in color images and considered as corneal lymphatic vessels (Fig. 1). Immunohistochemistry was used to confirm the indirect lymphangiography findings.

Fluorescein-Labeled Vessels in Live Corneas Expressed LYVE-1 After detecting the presumed lymphatic vessels on fluorescein indirect lymphangiography, the cornea of the mouse was harvested and double stained with CD31 and LYVE-1, the specific markers for blood and lymphatic vessels, respectively, as described above.30 Comparison of lymphangiography images with immunohistochemical staining images showed the colocalization of presumed

Novel Method to Detect Corneal Lymphatic Vessels

corneal lymphatic vessels with LYVE-1 (Fig. 2). Thereby, we could confirm that the fluorescent vessel-like structures are corneal lymphatic vessels.

DISCUSSION Corneal lymphatic vessels recently became a new target for treatment of corneal and ocular surface diseases because of their important functions in cancer metastasis, inflammatory conditions, and transplant rejection.1,11,34–36 Although corneal blood vessels are visible and easy to detect at the ophthalmic slit-lamp microscope, corneal lymphatic vessels appear invisible because of the transparency of lymph fluid, the thinness of their walls, and the lack of luminal cells.3,4 Imaging corneal lymphatic vessels, therefore, remains a challenge. Yuen et al26 in 2011 described a technique of live imaging of newly formed lymphatic vessels in the cornea by subconjunctivally injecting large-molecular-weight fluorescein isothiocyanate-labeled dextran. This was a potential method to visualize and evaluate the lymphatic vessels in vivo; however, it requires a new imaging system, which is not commonly available in the clinic. Hence, in this study, we provide a novel method to visualize corneal lymphatic vessels in vivo using

FIGURE 1. Visualization of the presumed lymphatic vessels with indirect fluorescein lymphangiography. The fluorescein-labeled vessels (arrows), clearly revealed under the fluorescein angiography mode, were undetectable under the ophthalmic slit-lamp microscope. Blood vessels (head arrows), however, were detectable in both fluorescein and color modes. Left panels: indirect fluorescein lymphangiography images; right panels: color images; asterisk: nasal marking suture. Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.

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FIGURE 2. Colocalization of fluorescein-labeled vessels with lymphatic specific marker LYVE-1. Left panel: lymphangiography image; right panel: immunohistochemical staining image (green: lymphatic vessels; red: blood vessels); head arrow: blood vessels; arrow: lymphatic vessels.

fluorescein sodium and a commonly and commercially available angiography device. Sodium fluorescein is also used in the cornea to confirm epithelial lesions and impaired epithelial wound healing (abrasion, ulcers, and herpetic infections) and in fluorescein angiography to detect retinal vascular disorders (macular degeneration, diabetic retinopathy, etc). Additionally, fluorescein angiography devices (here we used Heidelberg Spectralis HRA + OCT) are standard and available in almost every eye department. Therefore, it is easy to translate this technique into clinical practice. Ecoiffier et al37,38 indicated that the lymphatic vessels were distributed preponderantly on the nasal side of the cornea and the conjunctiva under both physiological and pathological conditions. Based on this finding, the nasal side was chosen to perform the suture-induced neovascularization assay. Also, the nasal canthus where the limbus is uncovered by the eyelids is easily accessible to examine the corneal vessels and the limbal arcade. To our knowledge, this is the first study using fluorescein sodium to visualize corneal lymphatic vessels. Because of the uptake of fluorescein from the interstitial tissue, corneal lymphatic vessels became brighter and detectable on indirect lymphangiography. In contrast, the blood vessels were still in dark shadow because fluorescein cannot get into these vessels. These findings were confirmed by immunohistochemical staining, showing the colocalization between fluoresceinlabeled vessels with lymphatic specific marker LYVE-1 and dark shadow vessels with CD31. This new technique, therefore, allows us to detect and evaluate both blood and lymphatic vessels at the same time. The location of lymphatic vessels was quickly determined based on the blood vessel landmarks when the subsequent specific antilymphatic treatment is performed under the ophthalmic microscope. Another advantage of this imaging method is that it allows for time course

evaluation of anti-(lymph)angiogenesis treatment in the same

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living cornea. A limitation of our approach is that the intrastromal injection technique without impacting blood vessels and the anterior chamber seems to be quite difficult in the mouse model. Also, because of steep corneal curvature in the mouse, many parts of corneal lymphatic networks were still out of focus and therefore affected the image quality. In our experiments, we used the suture-induced neovascularization assay on the nasal side and then continuously monitored only this side. Hence, we do not have any evidence to show the diffusion of fluorescein to the other part of the cornea. However, from our previous study, we know that Verteporfin injected into a stromal pocket can be drained by a high percentage of the corneal lymphatic vessels. Thereby these vessels could be destroyed specifically up to 50% by the photo dynamic therapy.12 Therefore, we believe that the fluorescein could be detected in the entire cornea. Further experimental and clinical studies should be implemented to improve this technique and the image quality. To sum up, we have presented a novel method based on simple and available clinical instruments to visualize not only the blood vessels but also the lymphatic vessels of the mouse cornea in vivo. This concept of lymphatic imaging with fluorescein sodium can be translated into the clinic for diagnostic and therapeutic indications.

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Novel Method to Detect Corneal Lymphatic Vessels

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