July 2015
Volume 56, Issue 8
Free
Anatomy and Pathology/Oncology  |   July 2015
Topography of Lymphatic Markers in Human Iris and Ciliary Body
Author Affiliations & Notes
  • Alexandra Kaser-Eichberger
    University Clinic of Ophthalmology and Optometry Research Program for Experimental Ophthalmology and Glaucoma Research, SALK, Salzburg, Austria
  • Falk Schrödl
    University Clinic of Ophthalmology and Optometry Research Program for Experimental Ophthalmology and Glaucoma Research, SALK, Salzburg, Austria
    Department of Anatomy, Paracelsus Medical University, Salzburg, Austria
  • Andrea Trost
    University Clinic of Ophthalmology and Optometry Research Program for Experimental Ophthalmology and Glaucoma Research, SALK, Salzburg, Austria
  • Clemens Strohmaier
    University Clinic of Ophthalmology and Optometry Research Program for Experimental Ophthalmology and Glaucoma Research, SALK, Salzburg, Austria
  • Barbara Bogner
    University Clinic of Ophthalmology and Optometry Research Program for Experimental Ophthalmology and Glaucoma Research, SALK, Salzburg, Austria
  • Christian Runge
    University Clinic of Ophthalmology and Optometry Research Program for Experimental Ophthalmology and Glaucoma Research, SALK, Salzburg, Austria
  • Karolina Motloch
    University Clinic of Ophthalmology and Optometry Research Program for Experimental Ophthalmology and Glaucoma Research, SALK, Salzburg, Austria
  • Daniela Bruckner
    University Clinic of Ophthalmology and Optometry Research Program for Experimental Ophthalmology and Glaucoma Research, SALK, Salzburg, Austria
  • Martin Laimer
    University Clinic of Dermatology, SALK, Salzburg, Austria
  • Simona L. Schlereth
    Department of Ophthalmology, University Cologne, Cologne, Germany
  • Ludwig M. Heindl
    Department of Ophthalmology, University Cologne, Cologne, Germany
  • Herbert A. Reitsamer
    University Clinic of Ophthalmology and Optometry Research Program for Experimental Ophthalmology and Glaucoma Research, SALK, Salzburg, Austria
  • Correspondence: Falk Schrödl, Department of Ophthalmology, SALK/PMU, Muellner Hauptstrasse 48, 5020 Salzburg, Austria; falk.schroedl@pmu.ac.at
  • Footnotes
     AK-E and FS contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science July 2015, Vol.56, 4943-4953. doi:10.1167/iovs.15-16573
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      Alexandra Kaser-Eichberger, Falk Schrödl, Andrea Trost, Clemens Strohmaier, Barbara Bogner, Christian Runge, Karolina Motloch, Daniela Bruckner, Martin Laimer, Simona L. Schlereth, Ludwig M. Heindl, Herbert A. Reitsamer; Topography of Lymphatic Markers in Human Iris and Ciliary Body. Invest. Ophthalmol. Vis. Sci. 2015;56(8):4943-4953. doi: 10.1167/iovs.15-16573.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Reports of lymphatics in the anterior human uvea are contradictory. This might be caused due to a certain topography, which has not been considered yet. Therefore, here we systematically analyze iris and adjacent ciliary body with immunohistochemistry by combining various lymphatic markers.

Methods: Human iris and ciliary body were obtained from cornea donors and prepared for cryosectioning. Cross sections of tissue blocks at 12/3/6/9 o‘clock position and at corresponding intersections (1:30/4:30/7:30/10:30) were processed for immunohistochemistry of LYVE-1, PDPN, PROX1, FOXC2, VEGFR3, and CCL21, and when necessary, these lymphatic markers were combined with CD31, α-smooth muscle-actin, CD68, and 4′,6-diamidino-2 phenylindole dihydrochloride (DAPI). Double, triple, and quadruple marker combinations were documented using confocal microscopy.

Results: Numerous podoplanin+ cells were mainly located at the anterior border of the iris while LYVE-1+ cells were distributed throughout the nonpigmented part. Both cell populations were PROX1/FOXC2/CCL21/VEGFR3−. Blood vessels, iris smooth muscles, and individual cells were VEGFR3+. While PDPN+ cells were rarely detected posteriorly of the iris root, many LYVE-1+ cells were present within the ciliary body muscle and villi. Within the muscle, occasionally PDPN+ vessel-like structures were detectable, but these were never colocalized with LYVE-1. Similar vessel-like structures were VEGFR3+/PROX1−/CCL21−, but CD31+. Further, ciliary muscle fibers and ciliary epithelium were immunoreactive for VEGFR3/CCL21, but were LYVE-1/PDPN−. A certain topography of structures at the various uvea-positions investigated was not obvious. The majority of LYVE-1+ cells displayed immunoreactivity for CD68.

Conclusions: Lymphatic vessels colocalizing for at least two lymphatic markers were not detectable. Therefore, if present, putative lymphatic channels of the anterior uvea might display a different marker panel than generally presumed.

Understanding the lymphatic system is important for many pathological aspects, notably inflammation or tumor dissemination; in the latter, understanding is most important for prognostic reasons. Research on lymphatics was long time hampered by the fact that a discrimination of small-caliber blood or lymphatic vessels even for the well-trained eye was nearly impossible in routine histologic sections.1 Therefore, for the confirmation of lymphatics, sophisticated techniques such as electron microscopy were used, but for obvious reasons this is not suitable for screening or large scale studies. With the introduction of markers considered specific for lymphatic endothelium such as the lymphatic vessel hyaluronate receptor 1 (LYVE-12; or the membrane bound glycoprotein podoplanin),3 this changed dramatically, and lymphatic research gained a tremendous boost. 
In this context, the eye has an exceptional position, because it is devoid of a lymphatic system, at least in its inner parts, and this has been counted for the “immunprivilege of the eye,” being an organ where foreign tissue grafts experience extended survival.4,5 Still, this is the nowadays general opinion in most of the ophthalmologic and anatomical textbooks.6 However, with the newly discovered lymphatic markers, it was possible to show that under inflammatory conditions a lymphatic system is indeed detectable in anterior parts of the eye, namely the cornea,7,8 and, further, also intraocularly if the scleral border is compromised by tumor or trauma.911 In this respect, the existence of lymphatics in deeper or internal layers of the eye, at least in physiological conditions, is controversially discussed: While in the human choroid, classical lymphatics were not detectable,12 lymphatic structures with diameters up to 25 μm13 were reported within the anterior uvea from one group, but these findings were not confirmed by others.14 However, in diseased conditions such as melanoma, lymphatic vessels have been reported in the ciliary body.9,15 
It has to be mentioned that in the eye, certain topographic peculiarities exist that highly differ from other nearby structures: For example, the fovea, located in the temporal retina as a point of high visual acuity, in its most simple aspect, structurally differs from other nearby retinal spots just by its rod–cone ratio. Other examples are the vortex vein system, concentrated on some particular spots of the globe but lacking on other positions,16 or the organization of corneal sensory nerve fibers, which differ at the various corneal layers (subbasal versus midstromal plexus).17,18 Therefore, to identify the aforementioned anatomical peculiarities, one needs to hit the right spot for analysis, and if missed, false negative results will be obtained as a consequence. In terms of lymphatics, a reason for the divergent reports in the anterior uvea might be the existence of a certain lymphatic topography that has been overlooked so far. Therefore, we systematically analyze the topography of lymphatics in the human anterior uvea in this study by applying a combination of various lymphatic markers followed by confocal microscopy. 
Methods
Specimens
In compliance with the Declaration of Helsinki, human tissue was obtained from the cornea-donor program of the University Eye Clinic Salzburg (Salzburg, Austria) and showed no signs of pathology (n = 7 from five different donors of both sex, 51–69 years; postmortem time 10–21 hours). After corneal removal, eyes were opened at the ora serrata and fixed by immersion in PBS containing 4% PFA (2 hours at room temperature [RT]). Iris and adjacent ciliary body were dissected free and tissue blocks from eight different sites were retrieved, at 12, 3, 6, and 9 o‘clock positions and at all corresponding intersections (i.e., 1:30, 4:30, 7:30, 10:30). These tissue blocks were rinsed in PBS (12 hours) and transferred into PBS containing 15% sucrose (12 hours at 4°C), embedded in tissue embedding medium (Slee Technik, Mainz, Germany) and frozen at −80°C by using liquid nitrogen-cooled methylbutane and stored at −20°C for further processing. 
Immunohistochemistry
Cross sections of these tissue blocks were prepared in a cryostat (HM 550; Microm, Walldorf, Germany) in a way that the continuous integrity from iris tip to ciliary body stayed preserved (Fig. 1; micrographs obtained via standard hematoxilin-eosin staining [HE]). Serial sections of 16 μm were collected on adhesion slides (Superfrost Plus; Thermo Scientific, Wien, Austria) and air-dried for 1 hour at RT. After a 5-minute rinse in tris-buffered saline (TBS; Roth, Karlsruhe, Germany) slides were incubated for 1 hour at RT in TBS containing 5% donkey serum (Sigma-Aldrich, Wien, Austria), 1% bovine serum albumin (BSA; Sigma-Aldrich), and 0.5% Triton X-100 (Merck, Darmstadt, Germany). Followed by a 5-minute rinse, slides were further incubated for double and triple immunohistochemistry of the markers listed in Table 1 (in TBS, containing 1% BSA and 0.5% Triton X-100, 12 hours at RT). After a rinse in TBS (four times, 5 minutes) binding sites of primary antibodies were visualized by corresponding Alexa488-, Alexa555-, or Alexa647-tagged antisera (1:1000; Invitrogen, Karlsruhe, Germany) in TBS, containing 1% BSA and 0.5% Triton X-100 (1 hour at RT) followed by another rinse in TBS (four times, 5 minutes). Some of the slides received an additional nuclear staining using 4′,6-diamidino-2 phenylindole dihydrochloride (DAPI). For that, slides were incubated for 10 minutes (1:4000, stock 1 mg/mL; VWR, Vienna, Austria), followed by a rinse in PBS (three times, 5 minutes). All slides were embedded in TBS-glycerol (1:1 at pH 8.6). All antibodies used were generated against human epitopes and prior to the experiments described here, the reliability of these antibodies has been tested in human skin samples and resulted in appropriate immunoreactivity (i.e., these detected lymphatic endothelial cells [PDPN, LYVE-1, VEGFR3, CCL21] or their nuclei [PROX1, FOXC2]). Further, to avoid possible cross reactions in experiments with two or more antibodies, interval incubations have been performed (i.e., first and second antibody for epitope one, followed by incubation with first and second antibody for epitope two, etc.). Additionally, to avoid misinterpretation due to unspecific background fluorescence caused by collagen or other autofluorescent signals, the same epitopes have been detected with different color-coded secondary antibodies. Negative controls were performed by omission of the primary antibodies during incubation and resulted in absence of immunoreactivity, and identical results were also obtained for isotype controls of the podoplanin antiserum. For each of the eight positions investigated, at least three consecutive sections have been evaluated. Because tissue samples derive from cornea donors, for which cornea with an adjacent scleral ring is removed prior to uveal analysis, Schlemms canal/trabecular meshwork were absent in these tissue samples and therefore were not analyzed. 
Figure 1
 
Main sites presented. (A) Sketch of the anterior segment of the eye in cross section with main sites presented (B, C, D1D3) in figures. (B) Cross section of the iris tip (HE; corresponds to Figs. 2A, 2B, 2H, 2I). (C) Cross section of the iris, middle part (HE; corresponds to Figs. 2C, 2E–G, 2I–K). (D) Cross section of the ciliary body (HE) with transition zone iris root-ciliary body ([D1]; corresponds to Figs. 2D, 3A, 3E, 3F, 3J), ciliary muscle ([D2]; corresponds to Figs. 3B–D, 3H, 3I, 3L), and ciliary body epithelium ([D3]; corresponds to Figs. 3G, 3K, 4A–F).
Figure 1
 
Main sites presented. (A) Sketch of the anterior segment of the eye in cross section with main sites presented (B, C, D1D3) in figures. (B) Cross section of the iris tip (HE; corresponds to Figs. 2A, 2B, 2H, 2I). (C) Cross section of the iris, middle part (HE; corresponds to Figs. 2C, 2E–G, 2I–K). (D) Cross section of the ciliary body (HE) with transition zone iris root-ciliary body ([D1]; corresponds to Figs. 2D, 3A, 3E, 3F, 3J), ciliary muscle ([D2]; corresponds to Figs. 3B–D, 3H, 3I, 3L), and ciliary body epithelium ([D3]; corresponds to Figs. 3G, 3K, 4A–F).
Figure 2
 
Iris. (A) In the iris tip, numerous PDPN-immunoreactive cells (red) are detectable anterior of the sphincter muscle (blue, α-SMA), while almost none are seen posterior of the muscle (P, posterior side of the iris). (B) LYVE-1–positive cells are interspersed in all parts of the iris tip, being larger and their amount lesser than the PDPN (red)-positive cells. Both cell populations do not overlap. (C) Middle part of the iris: PDPN-immunoreactive cells (red) are concentrated in a belt-like area of approximately 50-μm thickness at the anterior side. Within this belt like area, few LYVE-1–positive cells (green) are detectable, but these were also seen at posterior parts. (D) Iris root: The belt like area of PDPN-positive cells (red) at the anterior side of the iris suddenly ceases, while LYVE-1–positive cells (green) were still seen posterior of the iris root. Note the PDPN-immunoreactive cells are also concentrated at a spot anterior of ciliary muscle in the area of the trabeculum iridis (asterisk; CB denotes ciliary body). (E) High-resolution scan of PDPN (red)- and LYVE-1 (green)-positive cells at the anterior side of the iris: Individual cells do not colocalize for both markers (arrowheads), but are sometimes closely related to each other, resulting in yellow-mixed color (arrow). (F) PDPN-positive cells (red) in the iris do not colocalize for PROX1 (green) or FOXC2 (blue). Also, no individual structures immunoreactive for PROX1 or FOXC2 were detectable. (G) Close-up situation of single PDPN-positive cells (red) in the iris stroma. These cells do not display PROX-1 or FOXC2-positive nuclei. (H) Iris tip: VEGFR3-immunoreactivity (red) was detected in the sphincter muscle as well as in individual cells (arrowheads). Both muscle and cells were lacking PROX1-immunoreactivity (green). (I) Middle part of the iris: VEGFR3 immunoreactivity (red) was detected in the dilator muscle (open arrowheads) and in vessel-like structures within the iris stroma (arrowhead); again, PROX1-immunoreactivity (green) was absent. (J) Middle part of the iris: Vessel-like structures in the iris immunoreactive for VEGFR3 (red) are colocalized with CD31 (blue), but lacking α-SMA (green), thus representing vascular capillaries (arrowheads). P, posterior part of the iris with adjacent dilator muscle in green. (K) Middle part of the iris: Magnification of individual VEGFR3-immunoreactive cells in the stroma. These cells do not colocalize for PROX-1 (green) or CCL21 (blue). (L) Iris tip: The VEGFR3-immunoreactive sphincter muscle (green) is also colocalized with CCL21 (red), as seen by yellow-mixed color. Arrowhead indicates individual VEGFR3-positive blood vessel. P, posterior part of the iris.
Figure 2
 
Iris. (A) In the iris tip, numerous PDPN-immunoreactive cells (red) are detectable anterior of the sphincter muscle (blue, α-SMA), while almost none are seen posterior of the muscle (P, posterior side of the iris). (B) LYVE-1–positive cells are interspersed in all parts of the iris tip, being larger and their amount lesser than the PDPN (red)-positive cells. Both cell populations do not overlap. (C) Middle part of the iris: PDPN-immunoreactive cells (red) are concentrated in a belt-like area of approximately 50-μm thickness at the anterior side. Within this belt like area, few LYVE-1–positive cells (green) are detectable, but these were also seen at posterior parts. (D) Iris root: The belt like area of PDPN-positive cells (red) at the anterior side of the iris suddenly ceases, while LYVE-1–positive cells (green) were still seen posterior of the iris root. Note the PDPN-immunoreactive cells are also concentrated at a spot anterior of ciliary muscle in the area of the trabeculum iridis (asterisk; CB denotes ciliary body). (E) High-resolution scan of PDPN (red)- and LYVE-1 (green)-positive cells at the anterior side of the iris: Individual cells do not colocalize for both markers (arrowheads), but are sometimes closely related to each other, resulting in yellow-mixed color (arrow). (F) PDPN-positive cells (red) in the iris do not colocalize for PROX1 (green) or FOXC2 (blue). Also, no individual structures immunoreactive for PROX1 or FOXC2 were detectable. (G) Close-up situation of single PDPN-positive cells (red) in the iris stroma. These cells do not display PROX-1 or FOXC2-positive nuclei. (H) Iris tip: VEGFR3-immunoreactivity (red) was detected in the sphincter muscle as well as in individual cells (arrowheads). Both muscle and cells were lacking PROX1-immunoreactivity (green). (I) Middle part of the iris: VEGFR3 immunoreactivity (red) was detected in the dilator muscle (open arrowheads) and in vessel-like structures within the iris stroma (arrowhead); again, PROX1-immunoreactivity (green) was absent. (J) Middle part of the iris: Vessel-like structures in the iris immunoreactive for VEGFR3 (red) are colocalized with CD31 (blue), but lacking α-SMA (green), thus representing vascular capillaries (arrowheads). P, posterior part of the iris with adjacent dilator muscle in green. (K) Middle part of the iris: Magnification of individual VEGFR3-immunoreactive cells in the stroma. These cells do not colocalize for PROX-1 (green) or CCL21 (blue). (L) Iris tip: The VEGFR3-immunoreactive sphincter muscle (green) is also colocalized with CCL21 (red), as seen by yellow-mixed color. Arrowhead indicates individual VEGFR3-positive blood vessel. P, posterior part of the iris.
Figure 3
 
Ciliary body. (A) PDPN immunoreactivity was detected at the anterior margin of the ciliary muscle (α-SMA, blue) in the area of the trabeculum ciliare (asterisk). Here, an association with blood-vessels (CD31, green) was not obvious. More posteriorly, between muscle fibers of the ciliary body, CD31-positive blood vessels (green) were detectable, but podoplanin positive vessels were absent. (B) Occasionally, single PDPN-immunoreactive cells (red, arrowhead) were detectable in the connective tissue between muscle fibers (α-SMA, blue). These cells were not associated with CD-31 immunoreactive blood vessels (green). (C) Interspersed in the ciliary muscle, LYVE-1–positive cells (green) were detectable (arrowheads), these cells were PDPN-negative. Further, a PDPN immunoreactive signal was occasionally detectable on the margin of a cell-free lumen that appeared between bundles of muscle fibers (arrows). These observed immunoreactive signals were not colocalized with LYVE-1. (D) Similar podoplanin immunoreactive signals (red, arrows) were also detected surrounding cell-filled lumina. Again, these immunoreactive signals were not colocalized with LYVE-1 (green). Note a single LYVE-1+/PDPN− cell adjacent to the podoplanin-positive lumen (arrowhead). (E) Podoplanin-immunoreactive signals (red, arrows) were not associated with PROX-1 (green)-positive nuclei. (F) In double-labeling experiments applying VEGFR3 (red) and α-SMA (green), VEGFR3-immunoreactive cells were detectable anteriorly of the ciliary muscle (asterisk). (G) A VEGFR3-immunoreactive signal (red) was also detectable in the ciliary muscle, while any PROX-1–positive structures (green) were absent within muscle fibers as well as interspersed connective tissue fibers (green color in this figure represents background fluorescence). Note the VEGFR3-immunoreactive structures (arrowheads), most likely representing blood vessels, as detected in iris. (H) High-resolution images of the ciliary body revealed cell-free lumina bordered by a VEFGR-1–immunoreactive cells (red). These cells did not show a nuclear immunoreactivity for PROX-1 (green) or CCL21-immunoreactivity (blue). (I) VEGFR3-immunoreactive lumina (red, arrowheads) were also colocalized with CD31 (blue), and were detected in and between muscle fibers of the ciliary body (green, α-SMA), thus representing blood vessels. Note that also VEGFR3-immunoreactive cells are also present within the ciliary body (arrows). (J) VEGFR3-immunoreactive ciliary muscle fibers (green) are colocalized with CCL21 (red), resulting in yellow-mixed color. Note VEGFR3-immunoreactive blood vessels interspersed between the muscle fibers. (K) Very rarely, CCL21 immunoreactive cells (red, arrowhead) were detectable within the ciliary body. These cells were not colocalized with VEGFR3 (green). Yellow-mixed color here (open arrowhead) corresponds to autofluorescent collagen fibers. (L) Very rarely, LYVE-1–immunoreactive cells (red) were observed displaying a FOXC2-immunoreactive nucleus (blue, arrowhead), but lacking PROX1 (green).
Figure 3
 
Ciliary body. (A) PDPN immunoreactivity was detected at the anterior margin of the ciliary muscle (α-SMA, blue) in the area of the trabeculum ciliare (asterisk). Here, an association with blood-vessels (CD31, green) was not obvious. More posteriorly, between muscle fibers of the ciliary body, CD31-positive blood vessels (green) were detectable, but podoplanin positive vessels were absent. (B) Occasionally, single PDPN-immunoreactive cells (red, arrowhead) were detectable in the connective tissue between muscle fibers (α-SMA, blue). These cells were not associated with CD-31 immunoreactive blood vessels (green). (C) Interspersed in the ciliary muscle, LYVE-1–positive cells (green) were detectable (arrowheads), these cells were PDPN-negative. Further, a PDPN immunoreactive signal was occasionally detectable on the margin of a cell-free lumen that appeared between bundles of muscle fibers (arrows). These observed immunoreactive signals were not colocalized with LYVE-1. (D) Similar podoplanin immunoreactive signals (red, arrows) were also detected surrounding cell-filled lumina. Again, these immunoreactive signals were not colocalized with LYVE-1 (green). Note a single LYVE-1+/PDPN− cell adjacent to the podoplanin-positive lumen (arrowhead). (E) Podoplanin-immunoreactive signals (red, arrows) were not associated with PROX-1 (green)-positive nuclei. (F) In double-labeling experiments applying VEGFR3 (red) and α-SMA (green), VEGFR3-immunoreactive cells were detectable anteriorly of the ciliary muscle (asterisk). (G) A VEGFR3-immunoreactive signal (red) was also detectable in the ciliary muscle, while any PROX-1–positive structures (green) were absent within muscle fibers as well as interspersed connective tissue fibers (green color in this figure represents background fluorescence). Note the VEGFR3-immunoreactive structures (arrowheads), most likely representing blood vessels, as detected in iris. (H) High-resolution images of the ciliary body revealed cell-free lumina bordered by a VEFGR-1–immunoreactive cells (red). These cells did not show a nuclear immunoreactivity for PROX-1 (green) or CCL21-immunoreactivity (blue). (I) VEGFR3-immunoreactive lumina (red, arrowheads) were also colocalized with CD31 (blue), and were detected in and between muscle fibers of the ciliary body (green, α-SMA), thus representing blood vessels. Note that also VEGFR3-immunoreactive cells are also present within the ciliary body (arrows). (J) VEGFR3-immunoreactive ciliary muscle fibers (green) are colocalized with CCL21 (red), resulting in yellow-mixed color. Note VEGFR3-immunoreactive blood vessels interspersed between the muscle fibers. (K) Very rarely, CCL21 immunoreactive cells (red, arrowhead) were detectable within the ciliary body. These cells were not colocalized with VEGFR3 (green). Yellow-mixed color here (open arrowhead) corresponds to autofluorescent collagen fibers. (L) Very rarely, LYVE-1–immunoreactive cells (red) were observed displaying a FOXC2-immunoreactive nucleus (blue, arrowhead), but lacking PROX1 (green).
Table 1
 
Antibodies Used in This Study
Table 1
 
Antibodies Used in This Study
Documentation
In order to document double and triple label immunohistochemistry, a confocal laserscanning unit (Axio ObserverZ1 attached to LSM710; Zeiss, Göttingen, Germany; ×20 dry or ×40 and ×60 oil immersion objective lenses, with numeric apertures 0.8, 1.30, and 1.4, respectively; Zeiss) was used. Sections were imaged using the appropriate filter settings for Alexa555 (568-nm excitation, channel 1, coded red), Alexa488 (488-nm excitation, channel 2, coded green), Alexa647 (647-nm excitation, channel 3, coded blue), and DAPI (345-nm excitation, coded white) and up to four channels were detected simultaneously. All images presented here represent confocal images in single optical section mode. 
Results
Iris
In the iris, dense podoplanin-immunoreactivity was detected on the iris tip, anteriorly of the sphincter muscle (Fig. 2A), while the muscle itself was lacking podoplanin. Double immunohistochemistry with podoplanin and LYVE-1 revealed numerous LYVE-1–positive cells located in the sphincter muscle as well as in the podoplanin-positive areas (Fig. 2B). A belt-like positive area of approximately 50 μm in thickness was present on the anterior side of the iris midway between iris tip and iris root, still interspersed with LYVE-1–positive cells (Fig. 2C). The podoplanin-positive belt-like area ceased at the iris root, while LYVE-1–positive cells were also detected more posteriorly (Fig. 2D). High-resolution scans revealed that the podoplanin-positive area on the anterior side of the iris was caused by numerous cells with rather scarce cytoplasm and numerous processes (Fig. 2E), which were not colocalized with LYVE-1 (Figs. 2B–E), but closely surrounded by these LYVE-1–positive cells. Triple-labeling experiments applying podoplanin, PROX1, and FOXC2 revealed that all podoplanin-positive cells were lacking PROX1 and FOXC2 and, further, that no other cells were detectable expressing either PROX1 or FOXC2 in the nucleus (Figs. 2F, 2G). In double-labeling experiments with PROX1 and VEGFR3, a clear VEGFR3 immunoreactive signal was detected in the sphincter muscle (Fig. 2H) and in the dilator muscle (Fig. 2I) as well as in vessel-like structures within the iris stroma (Fig. 2I). Again, PROX1-positive nuclei were not detectable here. In triple-labeling experiments using VEGFR3, CD31, and α-smooth muscle actin (α-SMA), aforementioned VEGFR3+ vessel like structures were also colocalized with CD31, but lacking α-SMA, and were therefore identified as iris capillaries (Fig. 2J). Additionally, VEGFR3-immunoreactivity was also detected in few cells within the iris stroma (Figs. 2I–K). Again, these cells were not displaying a PROX1-positive nucleus nor were those cells immunoreactive for CCL21 (Fig. 2K). While CCL21-positive cells were not identified in the iris stroma (Figs. 2K, 2L), CCL21 immunoreactivity was detected in the muscles of the iris (sphincter and dilator), and was colocalized with VEGFR3 in these structures (Fig. 2L). 
Ciliary Body
In the posterior chamber, podoplanin immunoreactivity was detected on the anterior margin of the ciliary body, namely the irido-ciliary trabeculum, with a sharp stop at the anterior margin of the ciliary muscle fibers. This immunopositive signal could not be unequivocally attributed to a cellular structure, nor any CD31 positive- or any other luminal structure (Fig. 3A). Nevertheless, within the ciliary body, individual podoplanin immunoreactive cells were detectable in the connective tissue gaps of neighbored ciliary muscle fibers (Fig. 3B). Further, LYVE-1–positive cells were detected here (Fig. 3C) and these LYVE-1+ cells were lacking podoplanin (Figs. 3C, 3D). In the same set of experiments applying LYVE-1 and podoplanin, structures reminiscent on vessels (i.e., forming a true lumen or at least appear as double line of two adjacent endothelial cell layers in the sense of a collapsed vessel) were never found. Nevertheless, occasionally an immunopositive signal has been detected with pseudovessel appearance (i.e., forming a one-sided endothelial-like border around a cell free area, Fig. 3C; or cell-filled area, Fig. 3D). These pseudovessels were never colocalized with LYVE-1, nor were similar structures positive for LYVE-1–only detected; also, an association with PROX1-positive nuclei was never detected (Fig. 3E). Double-labeling experiments with VEGFR3 and α-SMA revealed numerous VEGFR3-positive structures at the iris root, just anteriorly of the ciliary muscle (Fig. 3E). In combination with a DAPI nuclear staining, these structures were identified as VEGFR3-positive cells, with round nucleus and cell size between 10 and 15 μm (Fig. 3F). Further, the ciliary muscle showed immunoreactivity for VEGFR3, but again a PROX1- immunoreactive nucleus was not detectable here (Fig. 3G). Within the ciliary muscle, structures were identified displaying VEGFR-immunoreactivity bordering a cell-free lumen, thus possibly representing a lymphatic vessel. However, these lumina were not associated with PROX1- nor CCL21-immunopositive signals (Fig. 3H). Instead, these structures displayed also immunoreactivity for CD31, and were thus identified as blood-vessels (Fig. 3I). Again, few VEGFR3-immunoreactive cells were present within the muscle (Fig. 3I). Combining VEGFR3 with CCL21 revealed that the latter was also expressed in ciliary muscle fibers (Fig. 3J). Occasionally, also CCL21-positive cells were detected within the ciliary body, being VEGFR3-negative (Fig. 3K). Further, few LYVE-1–positive cells were found, displaying a FOXC2-immunoreactive nucleus, but lacking a PROX1 nuclear signal (Fig. 3L). Because aforementioned cell populations were only occasionally found and their overall amount was very low, we resisted to further classify these cells. 
Ciliary Body Epithelium
LYVE-1–immunoreactive cells were detected within ciliary body villi, but these were not colocalized with podoplanin; also, LYVE-1 or podoplanin immunoreactivity was not detected in the ciliary epithelium (Fig. 4A). In experiments applying VEGFR3 and CD31, we could confirm VEGFR3-immunoreactive vessels in the stroma of ciliary villi, and further, VEGFR3-immunoreactivity was also detected in the nonpigmented ciliary epithelium (Fig. 4B). This VEGFR3-immunoreactive signal was colocalized with CCL21 (Fig. 4C), but CCL21 immunoreactivity ceased anteriorly in direction to the posterior part of the iris (Fig. 4D). 
Figure 4
 
Ciliary body epithelium (AD) and LYVE+ cells (EF). (A) PDPN-immunoreactivity (red) and LYVE-1 immunoreactivity (green) is lacking in the ciliary body epithelium, while LYVE-1 was detected in cells in ciliary body villi. Note that the observed red signal in the epithelium is autofluorescence. (B) VEGFR3-immunoreactivity (red) was detected in the ciliary epithelium, but was also found in supplying capillaries, as detected by colocalization of CD31 (blue, arrowheads) and absence of α-SMA (green). Note also the signal difference between epithelial autofluorescence (A) and specific signal here. (C) VEGFR3 immunoreactivity (red) in the epithelium is also colocalized with CCL21 (green), as seen by yellow-mixed color. Again, VEGFR3-immunoreactive capillaries are seen in the stroma of villi. (D) Colocalized CCL21 (red)- and VEGFR3 (green)-immunoreactivity in the ciliary body epithelium, as seen by yellow-mixed color (arrowheads) ceases when reaching the posterior side of the iris (arrows). (E) LYVE-1–immunoreactive cells (green) colocalize almost complete with CD68 (red), as seen by yellow-mixed color, thus representing macrophages. (F) While the majority of LYVE-1–immunoreactive cells (green) are colocalized with CD68 (red; arrow), individual cells display immunoreactivity for CD68 only, as seen by absence of yellow-mixed color (arrowhead).
Figure 4
 
Ciliary body epithelium (AD) and LYVE+ cells (EF). (A) PDPN-immunoreactivity (red) and LYVE-1 immunoreactivity (green) is lacking in the ciliary body epithelium, while LYVE-1 was detected in cells in ciliary body villi. Note that the observed red signal in the epithelium is autofluorescence. (B) VEGFR3-immunoreactivity (red) was detected in the ciliary epithelium, but was also found in supplying capillaries, as detected by colocalization of CD31 (blue, arrowheads) and absence of α-SMA (green). Note also the signal difference between epithelial autofluorescence (A) and specific signal here. (C) VEGFR3 immunoreactivity (red) in the epithelium is also colocalized with CCL21 (green), as seen by yellow-mixed color. Again, VEGFR3-immunoreactive capillaries are seen in the stroma of villi. (D) Colocalized CCL21 (red)- and VEGFR3 (green)-immunoreactivity in the ciliary body epithelium, as seen by yellow-mixed color (arrowheads) ceases when reaching the posterior side of the iris (arrows). (E) LYVE-1–immunoreactive cells (green) colocalize almost complete with CD68 (red), as seen by yellow-mixed color, thus representing macrophages. (F) While the majority of LYVE-1–immunoreactive cells (green) are colocalized with CD68 (red; arrow), individual cells display immunoreactivity for CD68 only, as seen by absence of yellow-mixed color (arrowhead).
An overview and semiquantitative summary of the results obtained is given in Table 2
Table 2
 
Semiquantitative Summary of the Results Obtained
Table 2
 
Semiquantitative Summary of the Results Obtained
Topography
In general, a topographical difference was not obvious in the iris/ciliary body segments with the markers investigated here. Consequently, the aforementioned results apply to all of the various segments. 
LYVE-1 Immunoreactive Cells
To further characterize LYVE-1 immunoreactive cells, double immunohistochemistry with CD68 has been performed. Three hundred randomly chosen cells displaying a DAPI+ nucleus and immunoreactivity for LYVE-1 have been evaluated out of three different donors, and 291 of these LYVE+ cells displayed an overlap with CD68 (97%), regardless of size, shape, or localization (Fig. 4E). In a second analysis, 300 CD68-positive cells displaying a DAPI-positive nucleus have been evaluated for LYVE-1. Of these, 218 cells displayed immunoreactivity for LYVE-1 (72.6%), while 82 cells showed immunoreactivity for CD68 only (27.3%). 
Discussion
In this study, various structures in the anterior uvea were immunoreactive for several lymphatic markers, while a classical lymphatic system was not detectable, and, further, a certain topography was absent with markers used. 
The term classical lymphatic system refers to structures displaying a lumen bordered by thin endothelial cells that eventually enter into larger vessels.19 In histologic sections, however, a lumen is not always detectable due to tissue, and hence lymphatic vessel compression, and further a lymphatic capillary or small lymphatic vessel is often not discernible from similar sized blood vessel, even for the well trained observer.20 Therefore, the introduction of various lymphatic markers allowed for better discrimination of blood and lymphatic vessels. “Better” in this sense means that despite the availability of several lymphatic markers, endothelial, cytoplasmatic and nuclear, a single exclusive marker for lymphatics does not exist up to now,2123 thus making an unequivocal identification of lymphatics challenging. Moreover, it is also known that lymphatic as well as vascular markers are differentially expressed at different sites of the vascular bed,1,14,21 and further available lymphatic endothelial markers are also expressed on structures/cells other than lymphatic endothelium.12,14,2426 For these aforementioned reasons, a single lymphatic marker identification seems problematic, especially for the detection of lymphatics in a hitherto alymphatic organ as within the eye. However, recently published guidelines could offer a possible solution in ocular lymphatic research.27 By the combination of at least two lymphatic markers for the detection of ocular lymphatics aforementioned drawbacks are minimized27 and the reliability of results obtained increases dramatically. While following these guidelines in this study, we were unable to detect a classical lymphatic system within the anterior uvea. We could also demonstrate that the diverging earlier studies reporting about lymphatics in the anterior uvea13,14 are not related to a certain putative lymphatic topography. Most likely, this divergence is caused due to technical difficulties (i.e., postmortal tissue alterations, fixation protocols, cryo versus paraffin embedding, use of divergent antibodies; see below). 
It is commonly accepted that a fluid drainage from the eye exists via several pathways (i.e., Schlemm's canal, vortex veins, and uveoscleral pathway)2830 still the existence of a true lymphatic drainage out of the eye remains enigmatic. While tumor metastasis in regional neck lymph nodes highlights the clinical importance of an ocular lymphatic pathway,31 the route from the eye into this regional lymph nodes is unknown.3234 In this sense, an important question is where this lymphatic system starts that should not compromise the immune privilege of the eye but nevertheless draining fluid. This is not an academic question, but has direct impact to therapeutic aspects (i.e., knowing the proper receptor composition may allow for pharmacological treatment)3537 as already seen in the treatment of high risk corneal transplant patients with respective receptor antibodies targeted to lymphatic epitopes.38,39 
Interpretation of Markers Applied
VEGFR3 is the target of VEGFC and D and thus responsible for lymphangiogenesis, and VEGFR3 expression was also described in some blood vessels during development,40 fenestrated capillaries41,42 and some stem cells.43 Interestingly, VEGFR3 also controls the transcription factor PROX1 in a feed-back loop,44 the latter one representing a marker for lymphatic endothelial nuclei1,4547 (despite reports of extra-nuclear PROX1 localization in cell types other than lymphatic endothelium).4850 
VEGFR3-immunoreactivity has been detected in iris vessels, single cells of the iris and ciliary body, and further in smooth muscle of the iris (sphincter and dilator) and ciliary body, as well as ciliary body epithelium. Earlier reports described VEGFR3-imunoreactivity in CD31+ blood vessels in the human dental pulp,51 matching with our results in iris. In this sense, it has to be mentioned that CD31 is not exclusively expressed in vascular endothelium, but also in lymphatic endothelium,23,5255 but because the here observed iris vessels were lacking PROX1+ nuclei, these were therefore considered vascular capillaries. Additionally, in none of our samples investigated, a typical arrangement of endothelial nuclei resulting in a pearl string–like pattern of PROX1-positivity55 was obvious. While VEGFR3-imunoreactivity was observed in CD68+ macrophages51 it is most likely that VEGFR3 +/PROX1−/CCL21− cells in the iris and also the VEGFR3-positive cells of similar size and shape in the ciliary body represent macrophages.51 On the other hand, it has been shown that VEGFR3-positive cells in mouse trachea do not represent macrophages,1 therefore the role/identity of the single VEGFR3-positive cells in the anterior uvea needs to be established in upcoming studies. Earlier studies reported VEGFR3 not only in vascular endothelium, but also in vascular smooth muscle cells in physiological as well as pathological conditions,56,57 and further also in nonvascular smooth muscle cells of the bovine uterus.56 Also, VEGFC and D have been detected in vascular smooth muscle cells, and their presence has been also mentioned in other smooth muscle cells42 without further explanation. While vascular smooth muscle cells of iris and ciliary body were lacking VEGFR3, sphincter, and iris dilator as well as ciliary muscle displayed immunoreactivity for VEGFR3, in line with above mentioned results. Because muscle receives a higher degree of vascularization compared with other tissue one could speculate that VEGFR3 might be involved in a certain barrier function between this vascularized environment and the inner compartment of the eye, in order to maintain the ocular immune privilege. Surprisingly, VEGFR3 has been detected in the nonpigmented epithelium of the ciliary body. However, a similar situation has been detected also for other epithelia in healthy conditions (e.g., in the human prostate,58 or esophageal mucosa59) the function of which is unknown. In summary, VEGFR3-immunoreactivity was widely distributed in the anterior uvea, despite earlier reports of VEGFR3 absence.14 Because the antibodies used in this study were generated against human epitopes and since our positive controls in human skin (control in this study) and also rat tissue55 revealed classical lymphatics, we consider presented results reliable. 
Podoplanin has been described in renal podozytes, alveolar type1 cells, mature osteoblasts fibroblastic reticular cells, but also in lymphatic endothelium60 and serves important functions during development, especially of the heart, lung, and lymphatic system. While its function in physiological conditions is not known, blocking podoplanin in an experimentally inflammation model inhibited lymphatic growth.61 Here, we demonstrate numerous podoplanin+ cells, mainly in the anterior iris, and mainly concentrated around the iris tip, while a homogeneous stromal distribution, as described earlier14 was not observed. Also, we were unable to detect an overlap of these podoplanin+ cells with LYVE-1, one of the most specific markers for lymphatic endothelium,62 as revealed by our high-resolution images. Nevertheless, LYVE-1–and podoplanin-positive cells were found to be in close proximity. While it seemed likely that the LYVE-1–positive cells by size and shape represent macrophages, as already described for the choroid12 and sclera26 we tested for this by colocalization with the macrophage marker CD68. Because indeed the vast majority of LYVE-1–positive cells colocalize for CD68, as we show here, these cells represent a subpopulation of macrophages. While the amount of CD68+ macrophages outnumbers the population of the LYVE-1+/CD68+ subset by one-third, of interest is the high concentration of macrophages detected in the anterior uvea. If and how these contribute to the immunological situation in the eye is unknown, but could be related to the high content of vitreous hyaluronan, as speculated earlier in a nonocular mouse model.63 Because podoplanin expression was also found in a subset of F4/80-positive macrophages,64 the here detected single podoplanin-positive cells therefore might also represent macrophages, however, due to antibody incompatibility, were unable to test for this. Regarding luminal structures with vessel-like character positive for podoplanin, these were very few and the immunoreactive signal was very weak compared with other signals. Because these vessel-like structures were not colocalized with LYVE-1, a feature that should be detected in lymphatic endothelium, and since further an association with PROX1+ nuclei was not detected (those were never observed in iris or ciliary body), these structures most likely do not represent lymphatics. While some vessel-like structures appeared densely packed with cells, an uncommon feature of lymphatics in noninflamed tissue, others were devoid of luminal cells, and could mimic lymphatics, but could also represent unbound antibody remnants in gaps of the ciliary muscle. In sight of aforementioned points, we interpret these results as staining artifacts. However, we find it necessary to present and discuss these results, that could lead to false positive interpretation, especially if a single marker strategy in combination with amplification methods is applied as it is the case in earlier studies.13 Thorough testing is needed to avoid false positive results due to inherent pitfalls in signal-amplification.65 
The chemokine ligand CCL21 is expressed in lymphatic endothelial cells as a target for the chemokine receptor CCR7 and CCR10 in activated dendritic cells, T and B cells. It is essential for dendritic cell intravasation66 but is also expressed within stromal cells of lymph nodes.67 It is strongly upregulated in lymphatic vessels upon exposure to TNFa.68 In a mouse diabetes model, CCL21 has been detected in vessels of Langerhans-islets, some of which where CD31-positive, and might represent blood vascular endothelium.69 In our study, CCL21-immunoreactivity has been localized in nonpigmented epithelium of the ciliary body. This finding matches with results in other secretory epithelia: in glandular epithelial cells of the human endometrium, CCL21-immunoreactivity and CCL21 mRNA, though at low levels has been observed, but was absent from endometrial stromal cells.70 While in the eye CCL19, the second ligand for CCR7, has been located in aqueous humor and was elevated in vitreous in proliferative diabetic retinopathy,71 to our knowledge CCL21 has not been described in vitreous/aqueous humor so far and levels of CCL21 need to be established. The here detected single CCL21 immunoreactive cells might represent leucocytes, as already stated earlier.72 Few cells being immunoreactive for CCL21 and podoplanin reported earlier in the anterior iris14 were not detected in our study, most likely caused due to different tissue handling conditions and/or antibodies used. The role of CCL21 in smooth muscle cells of the anterior uvea is unknown, nevertheless CCL21 has been found in vascular smooth muscle of the aorta.73 
When summarizing the presence of lymphatic markers used in this study and earlier reports, it is apparent that the use of a single lymphatic marker is inappropriate for the detection of lymphatics, especially in an unknown/alymphatic environment such as the anterior uvea. However, while classical lymphatics were not detectable in this study, this does not imply that these are indeed absent. Instead, they might express a hitherto unknown marker panel as, for example, recently reported for Schlemm's canal.24 The purely descriptive character of this study might appear as putative drawback. Nevertheless, it is the basis and represents an important step to properly understand pathological conditions9 where markers are considerably changed. Further, the individual cells expressing single lymphatic markers most likely representing macrophages (positive for LYVE-1 or podoplanin) or leucocytes (CCL21) need further characterization in upcoming studies, in physiological as well as pathological conditions. In conclusion, following recently established guidelines for the detections of ocular lymphatics,27 the current study demonstrates that the anterior uvea still has to be considered as an alymphatic organ in physiological conditions. 
Acknowledgments
Supported by grants from the Research Fund of the Paracelsus Medical University (R13-/01/042-KAS, AK-E; Salzburg, Austria), The Lotte Schwarz Endowment for Experimental Ophthalmology and Glaucoma Research (Salzburg, Austria), The Fuchs-Foundation for Research in Ophthalmology (Salzburg, Austria), and the German Research Foundation (FOR 2240 “[Lymph] Angiogenesis And Cellular Immunity In Inflammatory Diseases Of The Eye” [LMH], HE 6743/2-1, and HE 6743/3-1 [LMH], GEROK Program University Hospital of Cologne [SLS and LMH]; Cologne, Germany). 
Disclosure: A. Kaser-Eichberger, None; F. Schrödl, None; A. Trost, None; C. Strohmaier, None; B. Bogner, None; C. Runge, None; K. Motloch, None; D. Bruckner, None; M. Laimer, None; S.L. Schlereth, None; L.M. Heindl, None; H.A. Reitsamer, None 
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Figure 1
 
Main sites presented. (A) Sketch of the anterior segment of the eye in cross section with main sites presented (B, C, D1D3) in figures. (B) Cross section of the iris tip (HE; corresponds to Figs. 2A, 2B, 2H, 2I). (C) Cross section of the iris, middle part (HE; corresponds to Figs. 2C, 2E–G, 2I–K). (D) Cross section of the ciliary body (HE) with transition zone iris root-ciliary body ([D1]; corresponds to Figs. 2D, 3A, 3E, 3F, 3J), ciliary muscle ([D2]; corresponds to Figs. 3B–D, 3H, 3I, 3L), and ciliary body epithelium ([D3]; corresponds to Figs. 3G, 3K, 4A–F).
Figure 1
 
Main sites presented. (A) Sketch of the anterior segment of the eye in cross section with main sites presented (B, C, D1D3) in figures. (B) Cross section of the iris tip (HE; corresponds to Figs. 2A, 2B, 2H, 2I). (C) Cross section of the iris, middle part (HE; corresponds to Figs. 2C, 2E–G, 2I–K). (D) Cross section of the ciliary body (HE) with transition zone iris root-ciliary body ([D1]; corresponds to Figs. 2D, 3A, 3E, 3F, 3J), ciliary muscle ([D2]; corresponds to Figs. 3B–D, 3H, 3I, 3L), and ciliary body epithelium ([D3]; corresponds to Figs. 3G, 3K, 4A–F).
Figure 2
 
Iris. (A) In the iris tip, numerous PDPN-immunoreactive cells (red) are detectable anterior of the sphincter muscle (blue, α-SMA), while almost none are seen posterior of the muscle (P, posterior side of the iris). (B) LYVE-1–positive cells are interspersed in all parts of the iris tip, being larger and their amount lesser than the PDPN (red)-positive cells. Both cell populations do not overlap. (C) Middle part of the iris: PDPN-immunoreactive cells (red) are concentrated in a belt-like area of approximately 50-μm thickness at the anterior side. Within this belt like area, few LYVE-1–positive cells (green) are detectable, but these were also seen at posterior parts. (D) Iris root: The belt like area of PDPN-positive cells (red) at the anterior side of the iris suddenly ceases, while LYVE-1–positive cells (green) were still seen posterior of the iris root. Note the PDPN-immunoreactive cells are also concentrated at a spot anterior of ciliary muscle in the area of the trabeculum iridis (asterisk; CB denotes ciliary body). (E) High-resolution scan of PDPN (red)- and LYVE-1 (green)-positive cells at the anterior side of the iris: Individual cells do not colocalize for both markers (arrowheads), but are sometimes closely related to each other, resulting in yellow-mixed color (arrow). (F) PDPN-positive cells (red) in the iris do not colocalize for PROX1 (green) or FOXC2 (blue). Also, no individual structures immunoreactive for PROX1 or FOXC2 were detectable. (G) Close-up situation of single PDPN-positive cells (red) in the iris stroma. These cells do not display PROX-1 or FOXC2-positive nuclei. (H) Iris tip: VEGFR3-immunoreactivity (red) was detected in the sphincter muscle as well as in individual cells (arrowheads). Both muscle and cells were lacking PROX1-immunoreactivity (green). (I) Middle part of the iris: VEGFR3 immunoreactivity (red) was detected in the dilator muscle (open arrowheads) and in vessel-like structures within the iris stroma (arrowhead); again, PROX1-immunoreactivity (green) was absent. (J) Middle part of the iris: Vessel-like structures in the iris immunoreactive for VEGFR3 (red) are colocalized with CD31 (blue), but lacking α-SMA (green), thus representing vascular capillaries (arrowheads). P, posterior part of the iris with adjacent dilator muscle in green. (K) Middle part of the iris: Magnification of individual VEGFR3-immunoreactive cells in the stroma. These cells do not colocalize for PROX-1 (green) or CCL21 (blue). (L) Iris tip: The VEGFR3-immunoreactive sphincter muscle (green) is also colocalized with CCL21 (red), as seen by yellow-mixed color. Arrowhead indicates individual VEGFR3-positive blood vessel. P, posterior part of the iris.
Figure 2
 
Iris. (A) In the iris tip, numerous PDPN-immunoreactive cells (red) are detectable anterior of the sphincter muscle (blue, α-SMA), while almost none are seen posterior of the muscle (P, posterior side of the iris). (B) LYVE-1–positive cells are interspersed in all parts of the iris tip, being larger and their amount lesser than the PDPN (red)-positive cells. Both cell populations do not overlap. (C) Middle part of the iris: PDPN-immunoreactive cells (red) are concentrated in a belt-like area of approximately 50-μm thickness at the anterior side. Within this belt like area, few LYVE-1–positive cells (green) are detectable, but these were also seen at posterior parts. (D) Iris root: The belt like area of PDPN-positive cells (red) at the anterior side of the iris suddenly ceases, while LYVE-1–positive cells (green) were still seen posterior of the iris root. Note the PDPN-immunoreactive cells are also concentrated at a spot anterior of ciliary muscle in the area of the trabeculum iridis (asterisk; CB denotes ciliary body). (E) High-resolution scan of PDPN (red)- and LYVE-1 (green)-positive cells at the anterior side of the iris: Individual cells do not colocalize for both markers (arrowheads), but are sometimes closely related to each other, resulting in yellow-mixed color (arrow). (F) PDPN-positive cells (red) in the iris do not colocalize for PROX1 (green) or FOXC2 (blue). Also, no individual structures immunoreactive for PROX1 or FOXC2 were detectable. (G) Close-up situation of single PDPN-positive cells (red) in the iris stroma. These cells do not display PROX-1 or FOXC2-positive nuclei. (H) Iris tip: VEGFR3-immunoreactivity (red) was detected in the sphincter muscle as well as in individual cells (arrowheads). Both muscle and cells were lacking PROX1-immunoreactivity (green). (I) Middle part of the iris: VEGFR3 immunoreactivity (red) was detected in the dilator muscle (open arrowheads) and in vessel-like structures within the iris stroma (arrowhead); again, PROX1-immunoreactivity (green) was absent. (J) Middle part of the iris: Vessel-like structures in the iris immunoreactive for VEGFR3 (red) are colocalized with CD31 (blue), but lacking α-SMA (green), thus representing vascular capillaries (arrowheads). P, posterior part of the iris with adjacent dilator muscle in green. (K) Middle part of the iris: Magnification of individual VEGFR3-immunoreactive cells in the stroma. These cells do not colocalize for PROX-1 (green) or CCL21 (blue). (L) Iris tip: The VEGFR3-immunoreactive sphincter muscle (green) is also colocalized with CCL21 (red), as seen by yellow-mixed color. Arrowhead indicates individual VEGFR3-positive blood vessel. P, posterior part of the iris.
Figure 3
 
Ciliary body. (A) PDPN immunoreactivity was detected at the anterior margin of the ciliary muscle (α-SMA, blue) in the area of the trabeculum ciliare (asterisk). Here, an association with blood-vessels (CD31, green) was not obvious. More posteriorly, between muscle fibers of the ciliary body, CD31-positive blood vessels (green) were detectable, but podoplanin positive vessels were absent. (B) Occasionally, single PDPN-immunoreactive cells (red, arrowhead) were detectable in the connective tissue between muscle fibers (α-SMA, blue). These cells were not associated with CD-31 immunoreactive blood vessels (green). (C) Interspersed in the ciliary muscle, LYVE-1–positive cells (green) were detectable (arrowheads), these cells were PDPN-negative. Further, a PDPN immunoreactive signal was occasionally detectable on the margin of a cell-free lumen that appeared between bundles of muscle fibers (arrows). These observed immunoreactive signals were not colocalized with LYVE-1. (D) Similar podoplanin immunoreactive signals (red, arrows) were also detected surrounding cell-filled lumina. Again, these immunoreactive signals were not colocalized with LYVE-1 (green). Note a single LYVE-1+/PDPN− cell adjacent to the podoplanin-positive lumen (arrowhead). (E) Podoplanin-immunoreactive signals (red, arrows) were not associated with PROX-1 (green)-positive nuclei. (F) In double-labeling experiments applying VEGFR3 (red) and α-SMA (green), VEGFR3-immunoreactive cells were detectable anteriorly of the ciliary muscle (asterisk). (G) A VEGFR3-immunoreactive signal (red) was also detectable in the ciliary muscle, while any PROX-1–positive structures (green) were absent within muscle fibers as well as interspersed connective tissue fibers (green color in this figure represents background fluorescence). Note the VEGFR3-immunoreactive structures (arrowheads), most likely representing blood vessels, as detected in iris. (H) High-resolution images of the ciliary body revealed cell-free lumina bordered by a VEFGR-1–immunoreactive cells (red). These cells did not show a nuclear immunoreactivity for PROX-1 (green) or CCL21-immunoreactivity (blue). (I) VEGFR3-immunoreactive lumina (red, arrowheads) were also colocalized with CD31 (blue), and were detected in and between muscle fibers of the ciliary body (green, α-SMA), thus representing blood vessels. Note that also VEGFR3-immunoreactive cells are also present within the ciliary body (arrows). (J) VEGFR3-immunoreactive ciliary muscle fibers (green) are colocalized with CCL21 (red), resulting in yellow-mixed color. Note VEGFR3-immunoreactive blood vessels interspersed between the muscle fibers. (K) Very rarely, CCL21 immunoreactive cells (red, arrowhead) were detectable within the ciliary body. These cells were not colocalized with VEGFR3 (green). Yellow-mixed color here (open arrowhead) corresponds to autofluorescent collagen fibers. (L) Very rarely, LYVE-1–immunoreactive cells (red) were observed displaying a FOXC2-immunoreactive nucleus (blue, arrowhead), but lacking PROX1 (green).
Figure 3
 
Ciliary body. (A) PDPN immunoreactivity was detected at the anterior margin of the ciliary muscle (α-SMA, blue) in the area of the trabeculum ciliare (asterisk). Here, an association with blood-vessels (CD31, green) was not obvious. More posteriorly, between muscle fibers of the ciliary body, CD31-positive blood vessels (green) were detectable, but podoplanin positive vessels were absent. (B) Occasionally, single PDPN-immunoreactive cells (red, arrowhead) were detectable in the connective tissue between muscle fibers (α-SMA, blue). These cells were not associated with CD-31 immunoreactive blood vessels (green). (C) Interspersed in the ciliary muscle, LYVE-1–positive cells (green) were detectable (arrowheads), these cells were PDPN-negative. Further, a PDPN immunoreactive signal was occasionally detectable on the margin of a cell-free lumen that appeared between bundles of muscle fibers (arrows). These observed immunoreactive signals were not colocalized with LYVE-1. (D) Similar podoplanin immunoreactive signals (red, arrows) were also detected surrounding cell-filled lumina. Again, these immunoreactive signals were not colocalized with LYVE-1 (green). Note a single LYVE-1+/PDPN− cell adjacent to the podoplanin-positive lumen (arrowhead). (E) Podoplanin-immunoreactive signals (red, arrows) were not associated with PROX-1 (green)-positive nuclei. (F) In double-labeling experiments applying VEGFR3 (red) and α-SMA (green), VEGFR3-immunoreactive cells were detectable anteriorly of the ciliary muscle (asterisk). (G) A VEGFR3-immunoreactive signal (red) was also detectable in the ciliary muscle, while any PROX-1–positive structures (green) were absent within muscle fibers as well as interspersed connective tissue fibers (green color in this figure represents background fluorescence). Note the VEGFR3-immunoreactive structures (arrowheads), most likely representing blood vessels, as detected in iris. (H) High-resolution images of the ciliary body revealed cell-free lumina bordered by a VEFGR-1–immunoreactive cells (red). These cells did not show a nuclear immunoreactivity for PROX-1 (green) or CCL21-immunoreactivity (blue). (I) VEGFR3-immunoreactive lumina (red, arrowheads) were also colocalized with CD31 (blue), and were detected in and between muscle fibers of the ciliary body (green, α-SMA), thus representing blood vessels. Note that also VEGFR3-immunoreactive cells are also present within the ciliary body (arrows). (J) VEGFR3-immunoreactive ciliary muscle fibers (green) are colocalized with CCL21 (red), resulting in yellow-mixed color. Note VEGFR3-immunoreactive blood vessels interspersed between the muscle fibers. (K) Very rarely, CCL21 immunoreactive cells (red, arrowhead) were detectable within the ciliary body. These cells were not colocalized with VEGFR3 (green). Yellow-mixed color here (open arrowhead) corresponds to autofluorescent collagen fibers. (L) Very rarely, LYVE-1–immunoreactive cells (red) were observed displaying a FOXC2-immunoreactive nucleus (blue, arrowhead), but lacking PROX1 (green).
Figure 4
 
Ciliary body epithelium (AD) and LYVE+ cells (EF). (A) PDPN-immunoreactivity (red) and LYVE-1 immunoreactivity (green) is lacking in the ciliary body epithelium, while LYVE-1 was detected in cells in ciliary body villi. Note that the observed red signal in the epithelium is autofluorescence. (B) VEGFR3-immunoreactivity (red) was detected in the ciliary epithelium, but was also found in supplying capillaries, as detected by colocalization of CD31 (blue, arrowheads) and absence of α-SMA (green). Note also the signal difference between epithelial autofluorescence (A) and specific signal here. (C) VEGFR3 immunoreactivity (red) in the epithelium is also colocalized with CCL21 (green), as seen by yellow-mixed color. Again, VEGFR3-immunoreactive capillaries are seen in the stroma of villi. (D) Colocalized CCL21 (red)- and VEGFR3 (green)-immunoreactivity in the ciliary body epithelium, as seen by yellow-mixed color (arrowheads) ceases when reaching the posterior side of the iris (arrows). (E) LYVE-1–immunoreactive cells (green) colocalize almost complete with CD68 (red), as seen by yellow-mixed color, thus representing macrophages. (F) While the majority of LYVE-1–immunoreactive cells (green) are colocalized with CD68 (red; arrow), individual cells display immunoreactivity for CD68 only, as seen by absence of yellow-mixed color (arrowhead).
Figure 4
 
Ciliary body epithelium (AD) and LYVE+ cells (EF). (A) PDPN-immunoreactivity (red) and LYVE-1 immunoreactivity (green) is lacking in the ciliary body epithelium, while LYVE-1 was detected in cells in ciliary body villi. Note that the observed red signal in the epithelium is autofluorescence. (B) VEGFR3-immunoreactivity (red) was detected in the ciliary epithelium, but was also found in supplying capillaries, as detected by colocalization of CD31 (blue, arrowheads) and absence of α-SMA (green). Note also the signal difference between epithelial autofluorescence (A) and specific signal here. (C) VEGFR3 immunoreactivity (red) in the epithelium is also colocalized with CCL21 (green), as seen by yellow-mixed color. Again, VEGFR3-immunoreactive capillaries are seen in the stroma of villi. (D) Colocalized CCL21 (red)- and VEGFR3 (green)-immunoreactivity in the ciliary body epithelium, as seen by yellow-mixed color (arrowheads) ceases when reaching the posterior side of the iris (arrows). (E) LYVE-1–immunoreactive cells (green) colocalize almost complete with CD68 (red), as seen by yellow-mixed color, thus representing macrophages. (F) While the majority of LYVE-1–immunoreactive cells (green) are colocalized with CD68 (red; arrow), individual cells display immunoreactivity for CD68 only, as seen by absence of yellow-mixed color (arrowhead).
Table 1
 
Antibodies Used in This Study
Table 1
 
Antibodies Used in This Study
Table 2
 
Semiquantitative Summary of the Results Obtained
Table 2
 
Semiquantitative Summary of the Results Obtained
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