October 2015
Volume 56, Issue 11
Letters to the Editor  |   October 2015
Author Response: Sufficient Evidence for Lymphatics in the Developing and Adult Human Choroid?
Author Affiliations & Notes
  • Tailoi Chan-Ling
    Discipline of Anatomy & Histology, Bosch Institute, University of Sydney, Sydney, New South Wales, Australia;
  • Mark E. Koina
    Discipline of Anatomy & Histology, Bosch Institute, University of Sydney, Sydney, New South Wales, Australia;
    Department of Anatomical Pathology, ACT Pathology, The Canberra Hospital, Garran, Australia;
  • Frank Arfuso
    School of Anatomy, Physiology and Human Biology, Faculty of Science, University of Western Australia, Crawley, Western Australia, Australia;
  • Samuel J. Adamson
    Discipline of Anatomy & Histology, Bosch Institute, University of Sydney, Sydney, New South Wales, Australia;
  • Louise C. Baxter
    Discipline of Anatomy & Histology, Bosch Institute, University of Sydney, Sydney, New South Wales, Australia;
  • Ping Hu
    Discipline of Anatomy & Histology, Bosch Institute, University of Sydney, Sydney, New South Wales, Australia;
  • Michele C. Madigan
    School of Optometry, University of New South Wales, Sydney, New South Wales, Australia; and
    Save Sight Institute, University of Sydney, Sydney, New South Wales, Australia.
Investigative Ophthalmology & Visual Science October 2015, Vol.56, 6711-6713. doi:10.1167/iovs.15-18011
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      Tailoi Chan-Ling, Mark E. Koina, Frank Arfuso, Samuel J. Adamson, Louise C. Baxter, Ping Hu, Michele C. Madigan; Author Response: Sufficient Evidence for Lymphatics in the Developing and Adult Human Choroid?. Invest. Ophthalmol. Vis. Sci. 2015;56(11):6711-6713. doi: 10.1167/iovs.15-18011.

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

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In response to Heindl et al.,1 we thank the authors for their continued interest in our work. A number of issues raised by these authors require further consideration. 
Our study2 provided substantial ultrastructural and immunohistochemical evidence using lymphatic markers for the existence of early absorption lymphatic capillaries located on the external side of the fenestrated vessels of the choriocapillaris, an ideal juxtaposition for the recirculation of extracellular fluid, as well as being a strategic location for immunologic surveillance. The presence of the lymphatic-like system we described in the human choroid thus provides an anatomical basis for antigen presentation in the posterior segment of the eye, as well as a pathway from the eye to the sentinel lymph nodes, similar to that described for the anterior uveal lymphatic vessels.3 
Schroedl et al.4 proposed in their original consensus statement on the immunohistochemical detection of ocular lymphatic vessels that “the use of markers in ultrastructural analysis is recommended (except for ocular regions where existence of lymphatics is well established as in conjunctiva and inflamed cornea).” Further, Heindl et al.1 commented on our study of the developing and adult human choroid2 that “despite possible postmortem tissue alterations, numerous previous studies successfully applied different detection systems for ultrastructural investigations using ocular human donor tissue.” However, a recent paper from this group of authors investigating lymphatics in the human iris and ciliary body5 does not provide ultrastructural evidence, let alone immunogold transmission electron microscopy (TEM), to support their conclusion that “various structures in the anterior uvea were immunoreactive for several lymphatic markers, while a classical lymphatic system was not detectable.” 
The ultrastructural characteristics of lymphatics presented in our study are well defined and compelling. Recent studies have demonstrated, for the first time, the existence of lymphatics; in the central nervous system (CNS), citing our work and our criteria for ultrastructural characterization of lymphatics and similarly using the presence of anchoring filaments associated with lymphatic capillaries as the defining ultrastructural criteria for the identification of lymphatics.6 
Considering that the capillaries of the choroid are leaky, and a constant outflow of plasma proteins into the stroma of the choroid is expected, questions related to where this fluid goes to and how the interstitial (“lymph”) pressure within the choroid is managed are relevant for ocular homeostasis. As such, a major aim of our study was to use immunohistochemical and ultrastructural approaches to discover the most compelling evidence we could for lymphatic-like structures in the human choroid. Numerous studies to date have struggled to identify a system of “true lymphatics” in the anterior and posterior eye,79 although there is now functional1012 and anatomical evidence in support of lymphatic-like systems within the human anterior angle,13 iris, and ciliary body,14 and in our own study of the choroid.2 
Further, the relative scarcity of these lymphatic structures in normal human tissue, which has prevented their previous identification, indicates the value of detailed and extensive studies such as ours for exploring the presence of a lymphatic-like system in the choroid. 
A major point of contention relates to the concept of whether there are classical/typical lymphatics within the human choroid. The choroidal vasculature is known to be significantly different to the vasculature in the retina, where the choroidal vessels display a highly fenestrated phenotype, and the retinal vasculature displays barrier properties similar to those observed in the brain.15 
For example, there are three well-described and recognized types of vascular capillaries in the human body: continuous capillaries, which have no fenestrae, and microvesicles that transport macromolecules directly across the cell in either direction; fenestrated capillaries with no diaphragm, which allow macromolecules with a size up to 100 nm to have direct access to cells (as in the liver); and fenestrated capillaries with a diaphragm, which have transendothelial channels (diaphragms) that can allow passage of large and small molecules, as observed in the choroid.16 Their existence in contrast to the blood vessels with tight endothelial properties found in the human retina requires a system of fluid return as provided by lymphatics in the choroid. 
The expectation that lymphatics are always of a classical/typical appearance is inconsistent with observations from recent studies. For example, this is clearly evidenced by the work of Iliff et al.,17 who demonstrated the existence of a lymphatic-like system (referred to as the glymphatics) within the brain, which is dependent on the function of the water channel protein Aquaporin 4. 
The possibility of a nonclassical lymphatic endothelium that does not consistently display a set phenotype is also not without precedent, based on observations within the vascular system. A growing body of evidence supports the heterogeneity of endothelial cells in normal tissue and in tumors, providing support for this remarkable capacity for endothelium to express different markers depending on the tissue context and microenvironment.18 Nolan et al.19 reported tissue-specific molecular signatures for microvascular endothelium and clearly showed heterogeneity associated with maintenance and repair in various body organs. A similar situation for lymphatic endothelium has also been reported,20 indicating that a limited panel of phenotypic markers does not perhaps identify the biology of lymphatics (or vasculature). 
Taken together, these observations indicate the possibility that not only classical/typical lymphatics can occur and that a limited definition does not serve ongoing research assessing the presence of lymphatics within tissues previously considered devoid of lymphatic drainage. A growing body of evidence (as discussed) supports the likelihood of our observations in choroid being confirmed by other research groups. 
Baluk et al.21 note that the most useful markers for microscopic imaging of lymphatic vessels are lymphatic vessel endothelial hyaluronan receptor (LYVE)-1, VEGFR3, Prospero homeobox (Prox)-1, and podoplanin. In their letter, Heindl et al.1 state that, in our study, “the only lymphatic endothelial cell marker used in whole mounts was VEGFR-3.” This misrepresents our results, as we investigated other lymphatic endothelial surface markers, including D2-40 and LYVE-1 in choroidal whole mounts (our Figs. 3, 5). Further, we found endomucin immunolabeled vascular structures in the human choroid that were not consistent with the known structure of venous or capillary vessels. Because endomucin also labels lymphatic endothelial cells,22 we reasoned these structures must be endomucin+ lymphatic vessels. Two characteristics distinguish the presumed lymphatic VEGFR-3+ capillaries from the honeycomb-like lobular pattern of the choriocapillaris: the lymphatic VEGFR-3+ capillaries are located just external to the CD34+ choriocapillaris, and the lymphatic vessel lumens are slightly wider than the choriocapillaris, and their frequency is far rarer than choriocapillaris. 
A potential outcome envisaged from our study was to stimulate further investigations of the choroid with in vivo imaging techniques such as enhanced depth imaging-optical coherence tomography (EDI-OCT) and swept-source OCT. As the sensitivity of the imaging modalities improves, these can provide a foundation for further research into the role of the lymphatic system in posterior eye ophthalmic diseases, especially those where inflammation or edema are involved, such as diabetic retinopathy and age-related macular degeneration. Anecdotal evidence for the possibility of these vessels has been observed by ophthalmologists who have noted the presence/visualization of tube-like structures in the choroid that are not blood vessels in fluorescein angiography. 
Since the publication of our study, accumulating evidence of lymphatics in the central nervous system has also been reported.6 Louveau et al.6 cite our study, adopted the same ultrastructural criterion of anchoring filaments as being definitive for lymphatic endothelium, and noted a similar low frequency of occurrence where the lymphatics are embedded within the vasculature in the dura mater. Aspelund et al.23 have also recently shown a dural lymphatic vascular system that drains brain interstitial fluid and macromolecules in the mouse. 
Because the optic nerve is continuous with the dural-meningeal sheaths that encase the brain, and there is accumulating evidence of lymphatics in the anterior, mid-, and posterior eye (i.e., choroid), it is logical that this would require the existence of a lymphatic-like system in the human choroid that would drain into the dural sheath of the human optic nerve head. 
The nature of the CNS lymphatics, as we originally described in the human choroid, has since been confirmed by both Louveau et al.6 and Aspelund et al.,23 who demonstrated that the elusive lymphatic vessel in the “CNS hides in plain sight—as they are very small and tucked behind a major blood vessel.”24 
As recently as 2006, the literature claimed as “an undisputed anatomical fact,” that the brain is the only major organ that lacks a direct connection to the lymphatic system.25 We suggest that a similar fate will meet the long held idiom that the eye is uniquely immune privileged, due to the presumed absence of lymphatics in the posterior eye. 
We anticipate that as researchers are provided with further details of a lymphatic-like system in the human choroid and posterior eye, additional evidence for (or against) these structures will emerge, and we welcome ongoing dialogue with researchers in the ocular lymphatic and CNS glymphatic research community. Certainly, the letter of Heindl et al.1 does realize a primary aim of our study, namely to stimulate research discussion and activity regarding the presence and function(s) of lymphatics in the eye and surrounding tissues. 
Heindl LM, Kaser-Eichberger A, Schlereth SL, et al. Sufficient evidence for lymphatics in the developing and adult human choroid? Invest Ophthalmol Vis Sci. 2015 ; 56: 6709–6710.
Koina ME, Baxter L, Adamson SJ, et al. Evidence for lymphatics in the developing and adult human choroid. Invest Ophthalmol Vis Sci. 2015 ; 56: 1310–1327.
Yucel YH, Johnston MG, Ly T, et al. Identification of lymphatics in the ciliary body of the human eye: a novel “uveolymphatic” outflow pathway. Exp Eye Res. 2009 ; 89: 810–819.
Schroedl F, Kaser-Eichberger A, Schlereth SL, et al. Consensus statement on the immunohistochemical detection of ocular lymphatic vessels. Invest Ophthalmol Vis Sci. 2014 ; 55: 6440–6442.
Kaser-Eichberger A, Schrodl F, Trost A, et al. Topography of Lymphatic markers in human iris and ciliary body. Invest Ophthalmol Vis Sci. 2015 ; 56: 4943–4953.
Louveau A, Smirnov I, Keyes TJ, et al. Structural and functional features of central nervous system lymphatic vessels. Nature. 2015 ; 523: 337–341.
Schlereth SL, Neuser B, Herwig MC, et al. Absence of lymphatic vessels in the developing human sclera. Exp Eye Res. 2014 ; 125: 203–209.
Schroedl F, Brehmer A, Neuhuber WL, Kruse FE, May CA, Cursiefen C. The normal human choroid is endowed with a significant number of lymphatic vessel endothelial hyaluronate receptor 1 (LYVE-1)-positive macrophages. Invest Ophthalmol Vis Sci. 2008 ; 49: 5222–5229.
Xu H, Chen M, Reid D, Forrester J. LYVE-1-positive macrophages are present in normal murine eyes. Invest Ophthalmol Vis Sci. 2007 ; 48: 2162–2171.
Kim M, Johnston MG, Gupta N, Moore S, Yucel YH. A model to measure lymphatic drainage from the eye. Exp Eye Res. 2011 ; 93: 586–591.
Tam AL, Gupta N, Zhang Z, Yucel YH. Quantum dots trace lymphatic drainage from the mouse eye. Nanotechnology. 2011 ; 22: 425101.
Tam AL, Gupta N, Zhang Z, Yucel YH. Latanoprost stimulates ocular lymphatic drainage: an in vivo nanotracer study. Transl Vis Sci Technol. 2013 ; 2 (5): 3.
Park DY, Lee J, Park I, et al. Lymphatic regulator PROX1 determines Schlemm's canal integrity and identity. J Clin Invest. 2014 ; 124: 3960–3974.
Yücel YH, Johnston MG, Ly T, et al. Identification of lymphatics in the ciliary body of the human eye: a novel “uveolymphatic” outflow pathway. Exp Eye Res. 2009 ; 89: 810–819.
Chan-Ling T. The blood retinal interface: similarities and contrasts with the blood-brain interface. In: Dermietzel R Spray DC, Nedergaard M, eds. Blood-Brain Barriers - From Ontogeny to Artificial Interfaces. Weinheim, Germany: Wiley-VCH; 2006: 701–724.
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Iliff JJ, Wang M, Liao Y, et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci Transl Med. 2012 ; 4:147ra111.
Yu DY, Yu PK, Cringle SJ, Kang MH, Su EN. Functional and morphological characteristics of the retinal and choroidal vasculature. Prog Retin Eye Res. 2014 ; 40: 53–93.
Nolan DJ, Ginsberg M, Israely E, et al. Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration. Dev Cell. 2013 ; 26: 204–219.
Lee S, Choi I, Hong YK. Heterogeneity and plasticity of lymphatic endothelial cells. Semin Thromb Hemost. 2010 ; 36: 352–361.
Baluk P, McDonald DM. Markers for microscopic imaging of lymphangiogenesis and angiogenesis. Ann N Y Acad Sci. 2008 ; 1131: 1–12.
Samulowitz U, Kuhn A, Brachtendorf G, et al. Human endomucin: distribution pattern, expression on high endothelial venules, and decoration with the MECA-79 epitope. Am J Pathol. 2002 ; 160: 1669–1681.
Aspelund A, Antila S, Proulx ST, et al. A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med. 2015 ; 212: 991–999.
Friedman LF. This stunning discovery about the brain will have scientists rewriting textbooks. Business Insider Australia. June 4 2015. http://www.businessinsider.com.au/brain-immune-system-connection-lymphatic-vessel-2015-6. Accessed June 18, 2015.
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