Experimental Eye Research 196 (2020) 108049

Contents lists available at ScienceDirect

Experimental Eye Research
journal homepage: www.elsevier.com/locate/yexer

Functional, structural, and molecular identiﬁcation of lymphatic outﬂow
from subconjunctival blebs

T

Goichi Akiyamaa,b, Sindhu Saraswathya, Thania Bogarina, Xiaojing Panc, Ernesto Barrona,
Tina T. Wongd, Mika K. Kanekoe, Yukinari Katoe,f, Young Hongg, Alex S. Huanga,∗aDoheny Eye Institute and Department of Ophthalmology, David Geﬀen School of Medicine at UCLA, Los Angeles, CA, USA
Jikei School of Medicine, Tokyo, Japanc
Qingdao Eye Hospital, Shandong Eye Institute, Shandong First Medical University, Qingdao, ChinadSingapore National Eye Center and Singapore Research Institute, Singapore, SingaporeeTohoku University Graduate School of Medicine, Miyagi, Japanf
New Industry Creation Hatchery Center, Tohoku University, Miyagi, Japang
Division of Plastic and Reconstructive Surgery, Department of Surgery, Norris Comprehensive Cancer Center Keck School of Medicine, University of Southern California,
Los Angeles, CA, USAb

A R T I C LE I N FO

A B S T R A C T

Keywords:
Conjunctiva
Lymphatics
Aqueous outﬂow
Glaucoma surgery
Blebs

The purpose of this study is to evaluate outﬂow pathways from subconjunctival blebs and to identify theiridentity. Post-mortem porcine (n = 20), human (n = 1), and bovine (n = 1) eyes were acquired, and tracers(ﬂuorescein, indocyanine green, or ﬁxable/ﬂuorescent dextrans) were injected into the subconjunctival space tocreate raised blebs where outﬂow pathways were visualized qualitatively and quantitatively. Rodents withﬂuorescent reporter transgenes were imaged for structural comparison. Concurrent optical coherence tomography (OCT) was obtained to study the structural nature of these pathways. Using ﬁxable/ﬂuorescent dextrans,
tracers were trapped to the bleb outﬂow pathway lumen walls for histological visualization and molecular
identiﬁcation using immunoﬂuorescence against lymphatic and blood vessel markers. Bleb outﬂow pathways
could be observed using all tracers in all species. Quantitative analysis showed that the nasal quadrant had morebleb-related outﬂow pathways compared to the temporal quadrant (nasal: 1.9 ± 0.3 pathways vs. temporal:
0.7 ± 0.2 pathways; p = 0.003). However, not all blebs resulted in an outﬂow pathway (0-pathways = 18.2%;
1-pathway = 36.4%; 2-pathways = 38.6%; and 3-pathways = 6.8%). Outﬂow signal was validated as true
luminal pathways using optical coherence tomography and histology. Bicuspid valves were identiﬁed in the
direction of ﬂow in porcine eyes. Immunoﬂuorescence of labeled pathways demonstrated a lymphatic (Prox-1
and podoplanin) but not a blood vessel (CD31) identity. Therefore, subconjunctival bleb outﬂow occurs indiscrete luminal pathways. They are lymphatic as assessed by structural identiﬁcation of valves and molecular
identiﬁcation of lymphatic markers. Better understanding of lymphatic outﬂow may lead to improved eye carefor glaucoma surgery and ocular drug delivery.

1. Introduction
Native aqueous humor outﬂow (AHO) occurs in the eye via two
pathways (conventional and unconventional) (Huang and Weinreb,
2017; Johnson, 2006). However, an alternative pathway exists as aresult of glaucoma surgery to lower intraocular pressure (IOP). Thisoccurs in the subconjunctival space and is accessed during bleb-creating
surgeries such as trabeculectomies (Gedde et al., 2018), glaucoma
drainage devices (GDDs) (Gedde et al., 2018), and in some of the new

Minimally Invasive Glaucoma Surgeries (Green et al., 2018; Lenzhofer
et al., 2018a) (MIGS; Xen and Preserﬂo). While the subconjunctival
aqueous outﬂow pathway is artiﬁcially created during glaucoma surgery, given its important role in glaucoma treatment, it deserves study
as a way to improve AHO and to lower IOP.
Blebs are essentially ﬂuid ﬁlled blisters or sacs located under the
conjunctiva and above the scleral surface. Blebs can form naturally(termed chemosis) in certain pathological situations such as systemic
ﬂuid overload, post-surgical, or during ocular surface infection/∗
Corresponding author. Doheny Eye Institute, Department of Ophthalmology, David Geﬀen School of Medicine, University of California, Los Angeles, 1355 San
Pablo Street, Los Angeles, 90033, CA, USA.
E-mail address: ahuang@doheny.org (A.S. Huang).

https://doi.org/10.1016/j.exer.2020.108049
Received 14 January 2020; Received in revised form 5 April 2020; Accepted 27 April 2020
Available online 06 May 2020
0014-4835/ © 2020 Elsevier Ltd. All rights reserved.

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to determine their identity. By better understanding bleb biology, andin particular bleb outﬂow, improved glaucoma surgeries can be designed for enhanced IOP lowering outcomes.

inﬂammation (Glauser, 1974; Michael et al., 2016; Shoukath et al.,2017). Blebs can also be artiﬁcially created when medicines are injectedsubconjunctival to prophylax against infection after surgery and to treat
scleritis and macular edema in patients (Gower et al., 2017; Sohn et al.,2011; Thach et al., 1997). Surgically, blebs are crafted to create a lowresistance AHO pathway to decrease IOP in trabeculectomies (Gedde
et al., 2018), GDDs (Gedde et al., 2018), or subconjunctival MIGS
(Green et al., 2018; Lenzhofer et al., 2018a). We predict that for a blebto survive, a) the pathway for aqueous inﬂow must be stable, b) thebleb itself cannot scar down, and c) aqueous must have the ability toexit the bleb.
The major eﬀort in studying and improving blebs and bleb surgeries
has focused on the ﬁrst two requirements above. By using artiﬁcial
implants in GDDs and subconjunctival MIGS, the pathway from theanterior chamber to the bleb becomes stable. Then the introduction ofanti-ﬁbrotics (mitomycin-C [MMC] and 5-ﬂuorouracil [5FU]) (PalancaCapistrano et al., 2009) has been instrumental in enhancing bleb survival in both animal models and human surgeries. These agents nonspeciﬁcally ablate dividing cells to limit ﬁbroblastic activity and extracellular matrix deposition that result in a scarred bleb (Lama andFechtner, 2003). Unfortunately, research into more speciﬁc agents thattarget single regulatory points in scar-related molecular pathways, suchas TGF-β, have been less successful (Khaw et al., 2007). Nevertheless,despite these advances, all glaucoma surgeons have seen surgical cases
where the bleb appears structurally present, a cavity could even be
demonstrated using imaging tools such as optical coherence tomography (OCT), but IOP is still elevated. Thus, aqueous is entering thebleb but apparently cannot escape.
The biology of how ﬂuid exits a bleb in the subconjunctival space
has not been studied enough. It was noted decades ago that, after
subconjunctival hemorrhage, blood resolved with linear sausageshaped patterns (Grüntzig and Hollmann, 2019). Ink clearance fromscleral tattooing can do the same (Grüntzig and Hollmann, 2019). Injection of tracers directly under the conjunctiva have more clearly demonstrated these bleb outﬂow pathways. These experiments have been
performed in enucleated and live eyes in non-primate vertebrates (Yuet al., 2009), non-human primates (Guo et al., 2012; Yu et al., 2009),
and humans (Benedikt, 1976; Freitas-Neto et al., 2015). Based on theirappearance and by virtue of the known presence of lymphatics in thesubconjunctival space, these pathways have been hypothesized to belymphatic. This has not been proven though by structural or molecularstudy of the literal pathways that were observed. Alternatively, anotheroption is that local blood vessels represent these pathways and remove
agents delivered into the subconjunctival space.
Aqueous angiography is a tracer-based method designed to visualize
AHO from the conventional AHO pathways (Huang et al., 2016a, 2018;
Xie et al., 2019). Aqueous angiography was developed in the laboratory
using post-mortem animal and human eyes. Aqueous angiography wasvalidated as representing conventional AHO using immunoﬂuorescentand structural methods (Huang et al., 2017a; Saraswathy et al., 2016).
Aqueous angiography was translated to the operating room for living
non-human primates (Huang et al., 2017c) followed by imaging inhumans undergoing clinically-indicated cataract surgery (Huang et al.,2017b). Aqueous angiography has demonstrated segmental, pulsatile,and dynamic AHO behaviors (Huang et al., 2017b, 2017c). Aqueous
angiography has shown, in laboratory (Huang et al., 2016b, 2016c) andin live glaucoma patients undergoing trabecular MIGS (Huang et al.,2019), that surgery can lead to many types of angiographic AHO improvement.
Because of our tracer-based outﬂow research experience for conventional/trabecular outﬂow, we modify aqueous angiography to perform ocular surface lymphangiography (OSL) to study subconjunctival
AHO. We start with the previous hypothesis that subconjunctival outﬂow is lymphatic in nature. After tracer-identiﬁcation of these pathways in post-mortem eyes of various species, we speciﬁcally isolate andstudy these pathways using molecular and structural assessment tools

2. Methods
2.1. Study materials
Post-mortem and enucleated eyes were used for this study. Porcine(n = 20) and bovine (n = 1) eyes were obtained from SiouxPreme
Packing Company (Sioux Center, IA; within 48 h of death) and Manning
Beef LLC (Pico Rivera, CA; within 6 h of death), respectively. Of the 20
porcine eyes, 11 received indocyanine green (ICG) for counting blebrelated outﬂow pathways, 1 received ICG for optical coherence tomography (OCT), 3 received ﬂuorescein for videos or still photography,
and 5 received ﬁxable/ﬂuorescent dextrans for some combination of
OCT, excisional, luminal dimension measurements, or immunoﬂuorescent studies. The human eye (n = 1; 54-year-old male; right eye;cause of death: cardiac arrest) was obtained from OneLegacy Eye Bank
(Los Angeles, CA; within 36 h of death). Rodent eyes are described
below.
2.2. Ocular surface lymphangiography
Eyes (porcine/bovine/human) were procured, excess tissue wastrimmed, and the eyes were pinned to Styrofoam. Eyes with visible
damage to the conjunctiva near the limbus were excluded. Damage only
occurred in porcine eyes and represented less than 10% of eyes obtained from the abattoir. Tracers included ﬂuorescein, indocyanine
green, and ﬂuorescent dextrans. For ﬂuorescein, a 25% stock (Akorn;
Lake Forest, IL) was diluted at room temperature (RT) in balanced salt
solution (BSS) to 2.0%. ICG (Sigma I2633; St. Louis, MO) was dissolved
with deionized water into a 2% stock solution, and this stock was further diluted in BSS to 0.4%. Fluorescent FITC-dextrans (Thermo-Fisher;
Waltham, MA; D7136) were diluted to 2.5 mg/ml in BSS, and a 500 kDa
size was purposefully chosen given the large molecular weight andlesser chance for leakage out of lymphatics. The dextrans were additionally ﬁxable due to an attached lysine moiety so that they could betrapped to nearby tissue in the presence of an aldehyde ﬁxative. In eachcase, 0.12 forceps were used to raise the conjunctiva. Using a 30-gauge
needle, the conjunctiva was pierced just until the bevel and needle
lumen were fully buried, and 100 μL of the tracer were injected into thesubconjunctival space to create a bleb for imaging. In quantitative experiments, four separate ICG blebs (superior, inferior, nasal, and temporal) were created in eleven porcine eyes.
The imaging protocol utilized a 12 mega-pixel digital camera(Apple, Cupertino, CA) or the Heidelberg Spectralis (HRA + OCT;
Heidelberg Engineering, Heidelberg, Germany) for pictures and videos.
Videos were cropped and motion corrected (Vegas Pro 13, Sony; New
York, USA). Natively, ﬂuorescein is orange, and ICG is dark green.
Thus, ﬂuorescein images were taken with a digital camera. The ICG
images were taken using the confocal scanning laser ophthalmoscopic(CSLO) infrared function on the Spectralis. For ﬂuorescent dextrans, astandard pre-tracer background image was taken using the Spectralis
CSLO angiographic FITC setting with a sensitivity setting where thebackground appeared black. The FITC settings were: excitation wavelength = 486 nm and transmission ﬁlter set at > 500 nm. On theSpectralis, images were either taken with a 55-degree lens using a 25
diopter focus or the anterior segment lens.
2.3. Quantitative analyses
In eleven porcine eyes, 100 μL of ICG was injected subconjunctival
in diﬀerent locations (superior, inferior, nasal, and temporal). The
number of outﬂow pathways that were visualized were counted. To test
whether subconjunctival tracer injection in certain locations (superior/
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2.6. Histology and immunoﬂuorescenceinferior/temporal/nasal) were better or worse for accessing an outﬂow
pathway, a Kruskal-Wallis test (SAS, version 9.3) was performed. The
Kruskal-Wallis test is a non-parametric statistical test used to detectmean diﬀerences across groups. It was chosen in this analysis becauseof the relatively small sample size, which does not have enough statistical power to check the normal distribution assumption used in
ANOVA. Here, the null hypothesis was that the number of outﬂow
pathways across all quadrants was equal. Rejecting the null hypothesis
would mean that there was an overall statistically signiﬁcant diﬀerencein the number of bleb-related outﬂow pathways across quadrants. Since
this ended up being the case, to determine which quadrant had more or
fewer outﬂow pathways, the diﬀerent quadrants (inferior vs. temporal,
inferior vs. superior, inferior vs. nasal, temporal vs. superior, temporalvs. nasal, and superior vs. nasal) were compared using six t-tests. As
each eye generated data points for superior, inferior, nasal, and temporal quadrants, paired t-tests were used. To preserve an overall type-1
error rate of 0.05 and to statistically account for multiple comparisons,results were considered signiﬁcant across the 6 comparisons only if
p < 0.05/6 = 0.008 (Bonferroni correction; p < [acceptable pvalue]/[number of comparisons]). All quantitative data are shown asmean ± SEM.

After injection of ﬂuorescent and ﬁxable dextran and visualizationof the bleb-related outﬂow pathways, the entire region of porcine eyes(n = 3) that included the bleb and outﬂow pathways (from conjunctiva
to sclera) were carefully excised using scissors and placed in PFA for
15 min at RT followed by overnight at 4 °C to trap the tracer. The tissue
block was then marked for orientation and placed in Tissue-Tek(Sakura; Torrance, CA), frozen under liquid nitrogen, and 8-μm sections
cut (Thermo Scientiﬁc CryoStar NX70) onto Superfrost slides (Fisher;
Pittsburgh, PA). After blocking sections with 5% bovine serum albuminin phosphate buﬀered saline (PBS) and permeabilization with 0.3%
Triton X-100 in PBS, sections were incubated with primary antibody at
4 °C overnight. Antibodies included: a) mouse monoclonal anti-porcine
podoplanin (10 μg/ml; pMab-213 provided by Y. Kato (Kato et al.,2019)), b) rabbit polyclonal anti-mouse CD31 (1:20; Abcam ab28364,
Cambridge, UK), c) goat polyclonal anti-human Prox-1 (1:100; R&D
Systems AF2727, Minneapolis, MN), and d) anti-goat, anti-mouse, andanti-rabbit Texas red secondary antibodies (1:100; Thermo-Fisher).
Slides were mounted with 4′6-diamidino-2-phenylindole (DAPI) containing mounting medium (Vector Lab; Burlingame, CA) and viewed
under a Keyence BZ-X700 digital imaging microscope (Keyence; Chicago, IL). Sections were imaged using a 10x or 20x plan-ﬂuor lens. All
images were taken using identical settings for illumination and image
capture sensitivity (Keyence imaging software v.f1.51). For FITC-dextrans (EX BP 470/30, DM 495, EM BP 520/35), DAPI (EX BP 360/40,
DM 400, EM BP 460/50), and Texas Red (EX BP 560/40, DM 565, BP
630/75) appropriate ﬁlters were used. Identiﬁcation of above lymphatics followed consensus guidelines, including the use of the above
markers, more than one marker, structural analyses, and assessment ofblood vessel markers (Schroedl et al., 2014). This was done for additional assurance even though the guidelines exactly stated that thesesteps were not required for the conjunctiva since lymphatic presence
has already been well-documented (Schroedl et al., 2014).

2.4. Anterior segment optical coherence tomography (OCT)
Anterior segment OCT (anterior segment module, Scleral Mode
[Spectralis, Heidelberg Engineering; Germany]) was also conductedconcurrently with ocular surface lymphangiography. In angiographically positive regions, single line scans with a 15-degree scanangle (3.9-μm axial and 11-μm lateral resolution; ~4.5 mm) were takenwith oversampling (automated real time = 20). Size measurements
were made with in-built calipers. Spectralis comparisons between OCT
and CSLO images were performed using in-built rulers and sliders. The
accuracy of this cross-modality comparison between the Spectralis OCT
and CSLO has been reported at ~14 μm which is near the lateral resolution of the OCT itself (~14 μm) (Barteselli et al., 2013). This is dueto the Spectralis TruTrack system where the CSLO image is mapped atover 1000 points using one light beam during CSLO imaging andmapped as a reference to the second beam to acquire the OCT. This
mitigates eye motion artifact and ensures accurate point-to-point correlations between the OCT and CLSO images. Given such high accuracy
and as an example, retinal lesions can even be mapped and compared
between the CSLO and OCT images longitudinally with images acquired
over 1 year apart from the same subject (Huang et al., 2012).

2.7. Two-photon microscopy
In one case, whole-labeled porcine tissue was imaged by two-photon
microscopy on a Zeiss 710 NLO (Carl Zeiss, Ag; Oberkochen, Germany)equipped with a chameleon multiphoton laser (Coherent, Santa Clara;
CA, USA). Conjunctiva where the outﬂow pathway was located wasdissected from the underlying sclera and placed on a glass bottom dish
(P35G-1.5-14-C; MatTek corporation, Ashland, MA) along with a dropof PBS. A 10x Neoﬂuar lens NA 0.3 was used to focus an 850 nm wavelength laser to excite the ﬂuorescent dextrans. A 500–550 nm
bandpass ﬁlter was used to collect the ﬂuorescent signal. Images (1024x1024 pixels and a 16-bit grayscale resolution) were captured using the
Zeiss Zen software.

2.5. Rodent studies
To provide a comparison, adult mice and rats with ﬂuorescent reporter lymphatic genes were studied. Experiments complied with the
Association for Research in Vision and Ophthalmology (ARVO)
Statement for Use of Animals in Ophthalmic and Vision Research.
Approval was obtained from the Institutional Animal Care and Use
Committees (IACUC) at USC (PI: YK Hong) and UCLA (PI: Huang). Miceand rats were housed and raised in air-ﬁltered clear cages in a 12-h
light/dark cycle environment and fed ad libitum. Prox1-EGFP mouse(Choi et al., 2011) and Prox1-EGFP rat (Jung et al., 2017) were previously reported where lymphatic vessels could be conveniently visualized. All mice were maintained as outbred strains, whereas inbred
Prox1-EGFP rats (Sprague Dawley) were used for this study. One rat
and one mouse were euthanized by carbon dioxide asphyxiation followed by cervical dislocation, and the eyes were harvested. Eyes were
ﬁxed in 4% paraformaldehyde (PFA) at 4 °C for 2–4 h. The eyes were
cut in half and the lens and iris removed. The anterior half was thenﬂattened using radial incisions, placed on a slide, and mounted with acover slip. Fluorescent images were captured using a Zeiss ApoTome
Microscope (Zeiss AxioVision software).

3. Results
Various tracers were injected subconjunctival in enucleated postmortem eyes of several species (porcine, bovine, and human). Video
recordings from pig eyes showed that after bleb formation, distinct
tracer-based linear extensions could arise from some blebs (Clip 1; arrows) but not others (Clip 2). Comparing diﬀerent tracers, these extensions could be seen using ﬂuorescein (Fig. 1A), ICG (Fig. 1B/C), andﬂuorescent dextrans (Fig. 1D/E). Fluorescein and ICG have native coloration so that they could be visualized with standard photography or
confocal laser scanning ophthalmoscopy, without ﬂuorescent laser excitation. Fluorescein was also noted to leak, and this is consistent withits known small molecular weight (332 g/mol or daltons). ICG is
however larger (774 g/mol or daltons) and can even be protein boundto be yet larger. Therefore, ICG and very large ﬂuorescent dextrans(500 kDa) gave sharper and more deﬁned images of the outﬂow pathways. Across species (porcine [Fig. 1 A/B/D], bovine [Fig. 1C], and3

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Fig. 1. Distinct Outﬂow Pathways Can Be Visualized oﬀ Blebs Created Using Multiple Tracers in Multiple Species.
In all cases, 100 μL of a tracer are injected subconjunctival using a 30-gauge needle to create a bleb (asterisks). Distinct outﬂow pathways are seen arising oﬀ of theblebs. A/B/D) Porcine eyes. C) Bovine eye. E) Enucleated right eye from a 54-year-old male. Blue arrows demonstrate outﬂow pathways. White arrows showintervening regions in the outﬂow pathways with narrow bands of darker signal. F/G) Zoomed in images of D and E, respectively, showing the dark bands moreclearly (white arrows). Scale bars = 1 mm. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the Web version of this article.)

temporal (0.7 ± 0.2), and nasal (1.9 ± 0.3) (Fig. 2B). Regardless oflocation, across all blebs, zero-pathways were seen in 18.2% (8 of 44),
1-pathway was seen in 36.4% (16 of 44), 2-pathways were seen in
38.6% (17 of 44), and three-pathways were seen in 6.8% (3 of 44) ofblebs (Fig. 2C). To determine if access to outﬂow pathways was nonuniform around the limbus, a Kruskal-Wallis test was performed with
the null hypothesis that there was no overall mean diﬀerence among
the quadrants. The null hypothesis was rejected (H-statistic: 10.03;
p = 0.018). Thus, bleb-related outﬂow pathways were not uniform
around the limbus, and because of this pair-wise t-tests were performed
comparing all combinations. Given multiple comparisons, a Bonferroni
correction was made such that statistical signiﬁcance was only considered for p < 0.008. There was only a statistically signiﬁcant difference comparing nasal vs. temporal (p = 0.003). The results for theother comparisons were non-signiﬁcant: superior vs. temporal(p = 0.09), superior vs. nasal (p = 0.30), superior vs. inferior (p =0.64), nasal vs. inferior (p = 0.05), and temporal vs. inferior(p = 0.08).
To determine the nature of these outﬂow pathways, structural andhuman [Fig. 1E]), bleb-related outﬂow pathways were similar. Overall,these outﬂow pathways were qualitatively diﬀerent from veins whichtake typical linear and Y-shaped patterns (Gaasterland and Pederson,1983). Instead, bleb-related outﬂow pathways were non-linear and irregular. Also, careful observation showed a variegated signal where
along the outﬂow pathways, brighter regions were intermixed withdarker bands (Fig. 1D/E; white arrows). Zoomed-in images for Fig. 1D/E showed the darker bands (Fig. 1 F/G white arrows, respectively) moreclearly. Given greatest access to porcine eyes, most of the subsequent
quantitative, structural, and molecular studies focused on this species.
Supplementary video related to this article can be found at https://doi.org/10.1016/j.exer.2020.108049
The number of outﬂow pathways that arose from subconjunctival
blebs were quantiﬁed. Four blebs were created in diﬀerent locations(superior, inferior, temporal, and nasal) in eleven separate eyes. Eachbleb was created with 100 μL of ICG, and the total number of extending
outﬂow pathways were counted, showing a diﬀerent distribution ineach quadrant (Fig. 2A). On average, the number of pathways per bleb
location were: superior (1.5 ± 0.3; mean ± SEM), inferior (1.3 ± 0.2),

Fig. 2. Variation in Number of Outﬂow Pathways Oﬀ Blebs in Porcine Eyes.
Four separate (superior/inferior/temporal/nasal) 100 μL subconjunctival blebs were created in each of 11 porcine eyes (n = 44 blebs total), and the number ofoutﬂow pathways were counted and compared. A) Each quadrant had a diﬀerent distribution of total number of pathways seen. B) A statistically signiﬁcantdiﬀerence was only seen comparing the temporal to the nasal quadrant. B) The distribution of the number of pathways across all blebs was Zero (18.2%), One(36.4%), Two (38.6%), and Three (6.8%).
4

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Fig. 3. Outﬂow Pathways Oﬀ Blebs Are Located Within the Conjunctiva of a Porcine Eye.
A) A baseline ﬂuorescent bleb (asterisk) and ﬂuorescent outﬂow pathway (blue arrow) are apparent. B) To determine the location of this pathway, the conjunctiva
from this region was excised and pinned to Styrofoam. C) The residual scleral bed showed ﬂuorescent signal where the bleb was previously located (double asterisks).
However, the baseline ﬂuorescent outﬂow pathway was now gone. Instead a non-ﬂuorescent episcleral vein was seen (red arrow). D) The exact same piece of excised
conjunctiva (from B) was then imaged with ﬂuorescent excitation showing the bleb (asterisk) and now the ﬂuorescent outﬂow pathway (blue arrow) again. Scalebars = 1 mm. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the Web version of this article.)

OCT on the bleb and outﬂow pathway junction demonstrated a hole(159 x 34 μm) through which tracer could exit the bleb (Fig. 4I/J).
Structural studies supported a lymphatic identity. Given the ﬂuorescent signal, a small piece of the excised porcine conjunctiva with anoutﬂow pathway but away from the bleb (Fig. 1D area pointed out by
white arrows which is the tissue that was also shown in Fig. 3B) was
submitted for two-photon microscopy. Higher magniﬁcation imaging
again demonstrated irregular structures but now with blind ends
(Fig. 5A; white arrows) which may represent lymphatic capillary tips.
The outﬂow pathways then grew into larger extensions which could
represent pre-collector lymphatics followed by even bigger pathways
notable for bright ﬂuorescent regions (Fig. 5A) separated by dark bands(Fig. 5A; orange arrow). This qualitative appearance of dark bands
using two-photon microscopy was very similar to the results from theSpectralis above (Fig. 1D–G; white arrows). To further delve into this,these images were compared to lymphatic appearance from lymphatic
reporter mouse (Choi et al., 2011) and rat (Jung et al., 2017). Theserodents express GFP under a Prox-1 promoter (Fig. 5B/C), and thenatively ﬂuorescent subconjunctival lymphatics also show a variegatedsignal with bright and dark regions with focal bright spots known torepresent valves (Choi et al., 2011) (Fig. 5B/C). Comparing ocularsurface lymphangiography to the reporter rodents, the focal dark regions in ocular surface lymphangiography may correspond to the focal
bright region in the reporter rodents and thus be valves.
Thus, we tested for the presence of valves using OCT on ocularsurface lymphangiography with an emphasis on the focal dark regions.
In porcine eyes, the presence of bicuspid valves was conﬁrmed using
OCT (Fig. 5D–F). After creation of the bleb (Fig. 5D), tracer ﬂow exitedin the direction denoted by the sequence of arrows (Fig. 5E; red, blue,
and then yellow arrows). As this pathway was more linear, OCT was
performed to capture the entire pathway (Fig. 5F). The color arrows on
the OCT (Fig. 5F) corresponded to the exact location of the same color
arrows of the angiographic image (Fig. 5E). Thus, the OCT demonstrated conjunctival bicuspid valves that lined correctly with the direction of ﬂow (Fig. 5F). The location of the valves also matched that ofthe dark bands (Fig. 5D/F; white arrows). Lymphatics (including in the
conjunctiva) are known to have valves (Gong et al., 2018; SchulteMerker et al., 2011). Measuring valve leaﬂets (n = 6 leaﬂets; Fig. 5)
from the base to the valve tip, the average leaﬂet length was
151.7 ± 14.9 μm (mean ± SEM).
To determine the molecular characteristic of bleb-related outﬂow
pathways, immunoﬂuorescence was performed. However, the ﬁrst andcritical step was to be able to identify the exact outﬂow pathways seenon the angiographic image on a histological section. Thus, this experiment utilized blebs created by the ﬂuorescent and ﬁxable dextrans.
A lysine moiety, attached to the dextran, allowed for trapping of the
tracer to the luminal walls of the pathways that it ﬂowed through. After

molecular studies were performed. First, in one porcine eye, subconjunctival ﬂuorescent dextran was injected to create a bleb (Fig. 3A;
black asterisk) with a superiorly-directed outﬂow pathway (Fig. 3A;
blue arrow). Then the conjunctiva where the bleb and outﬂow pathway
was located was excised (Fig. 3B). The residual and underlying baresclera showed continued ﬂuorescence where the bleb was once located(Fig. 3C; black asterisks). However, the previous ﬂuorescent outﬂow
pathway was gone, and instead a black linear structure was seen butthat did not carry a ﬂuorescent signature (Fig. 3C; red arrow). This
could represent an artery or vein, not associated with ﬂuid ﬂow oﬀ thebleb. Focusing on the excised piece of conjunctiva (Fig. 3B), underﬂuorescent excitation (Fig. 3D), both the bleb (Fig. 3D; black asterisk)
and original outﬂow pathway (Fig. 3D; blue arrow) were re-visualized.
This demonstrated that the outﬂow pathway was located within theexcised conjunctival piece.
Concurrent optical coherence tomography (OCT) was then performed on the bleb-related outﬂow pathways. OCT, perpendicular to an
outﬂow pathway, showed a distinct and luminal structure that corresponded exactly to the angiographic outﬂow pathway location in theeye (Fig. 4A/B). OCT comparison to angiography is possible because of
the Spectralis dual-beam TruTrack system that ensures highly accurate
point-to-point correlations between the OCT and CLSO images(Barteselli et al., 2013). The bleb-related outﬂow pathway's relatively
superﬁcial location supported the results from the excisional experiment result (Fig. 3) and a conjunctival origin for these pathways. OCT
along a short-segment of an outﬂow pathway also showed a luminal
structure whose length exactly matched the extent of the ﬂuorescent
outﬂow pathway itself (Fig. 4C/D; blue and orange arrows on the angiographic image matched exactly the location of the same coloredarrows on the OCT). Other luminal pathways were seen as well. In somecases, OCT across an angiographic pathway showed the lumen (Fig. 4E/
F; yellow arrows) next to another lumen (Fig. 4E/F; red arrows) that did
not carry angiographic tracer signal. Note that this non-angiographic
lumen was smaller in size compared to the lumen associated with theangiographic signal. (Fig. 4F; red arrow vs. yellow arrow). This may
reﬂect increased intraluminal pressure which one would expect to behigher in the ﬂuorescent pathway since actual tracer was injected. Notethat in the excised piece of conjunctiva (Fig. 4G/H), OCT in the same
approximate location gave the same arrangement. There were two lumens, and one matched the angiographic signal but the other did not.
Further, both lumens were of approximately the same size after the
conjunctiva was excised. This may be expected as excising and opening
the conjunctiva would remove any pressure head initially driven bytracer injection. Across ﬁve eyes receiving ﬂuorescent dextran blebs, 14
pathways were visualized with OCT demonstrating luminal dimensionsof 215.9 ± 24.4 μm (mean ± SEM; range: 110 to 455 μm) horizontal
and 102.4 ± 7.8 μm (range: 37 to 136 μm) vertical. Lastly, detailed
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G. Akiyama, et al.

Fig. 4. Structural Evaluation of Outﬂow Pathways
Oﬀ Blebs of Porcine Eyes.
Blebs (asterisks) were created with various tracers in
porcine eyes, and anterior segment optical coherence
tomography (OCT; dotted green arrows) was performed. A/B) OCT transverse to an outﬂow pathway
oﬀ the bleb showed a structural lumen. C/D) OCT
longitudinal to an outﬂow pathway from a blebshowed that the extent of the OCT performed on theangiographic outﬂow pathway matched the extent of
the lumen seen on the OCT (blue arrow to orange
arrow). E/F) OCT transverse to another outﬂow
pathway from a bleb showed the luminal structure(yellow) arrow. However, next to it was another luminal pathway that did not correspond (red arrow)to an angiographic outﬂow pathway. G/H) OCT in
the exact same location (as E/F) but after the conjunctiva was excised showed the same outﬂow lumen(yellow arrow) and non-bleb-related outﬂow lumen(red arrow). I/J) OCT on the junction between thebleb and outﬂow lumen showed the luminal opening
in the bleb (yellow arrow) that could allow ﬂuid toexit from the bleb. White scale bars = 1 mm. Yellow
scale bars = 200 μm. (For interpretation of the references to color in this ﬁgure legend, the reader isreferred to the Web version of this article.)creation of the ﬂuorescent bleb and outﬂow pathway, 4% PFA wasgiven to trap the tracer in place. Histological section identiﬁed thelumen with tracer coating the lumen wall (Fig. 6A/D/G/J/M). Co-immunoﬂuorescence against Prox-1 and podoplanin demonstrated co-labeling of these lumens with lymphatic markers (Fig. 6B/E/H/K). Notethat other lymphatics could be seen nearby the bleb-related outﬂow
pathway that did not carry the ﬂuorescent dextran tracer (Fig. 6K/L;
white arrows). These lymphatics presumably drain nearby regions
outside of where the bleb was created. In some cases, dextran- andlymphatic marker-positive debris-like material could be seen within thelumens. Their identity is unclear and maybe valvular or vessel lumen
lining tissue that migrated into the lumen during sectioning steps. Then,
immunoﬂuorescence against blood vasculature (CD31 is highly expressed in blood vessels and only weakly in lymphatics (Baluk et al.,2007)) showed blood vessels in the section that did not co-localize withthe bleb-related ﬂuorescent dextran outﬂow pathway (Fig. 6M–O), indicating that blood vessels are not draining the blebs.

subconjunctival space) distinct outﬂow pathways have been observed(Benedikt, 1976; Freitas-Neto et al., 2015; Grüntzig and Hollmann,
2019; Yu et al., 2009). These pathways have long been hypothesized
and assumed to be lymphatics because a) subconjunctival lymphatics
exist (Guo et al., 2012) and because b) lymphatics should drain extracellular spaces in the body. However, while long-studied and potentially clinically important, the lymphatic identity of ﬂuid outﬂow
pathways from the subconjunctival space has not been deﬁnitively
proven. The results herein provide direct evidence of a lymphatic
identity using a multi-model approach starting with ﬂuorescent tracersthat can be visualized after subconjunctival injection. After identiﬁcation of the pathways, simultaneous anterior segment optical coherence
tomography (OCT) on these exact structures demonstrated lumens andin particular biscuspid valves in the direction of ﬂow. One of the hallmarks of lymphatics is that they have valves (Gong et al., 2018; SchulteMerker et al., 2011), and the valve leaﬂet size in this work (~150 μm)
matched well with published lymphatic valve leaﬂet measurements(~100 μm (Swartz, 2014)). Co-immunoﬂuorescence against two lymphatic markers further conﬁrmed a molecular lymphatic identity. Thus,
we not only observed outﬂow pathways for subconjunctival outﬂow,
but we simultaneously studied the identity of these pathways using
structural (OCT) and molecular (immunoﬂuorescence) methods.
The clinical implications of these ﬁndings are important and can bedirected at drug delivery or glaucoma surgery. Drug delivery that isbetter than topical drop application has long been sought. The

4. Discussion
Tracer-based studies where labeled substances were delivered into
the subconjunctival space have been performed for many years(Benedikt, 1976; Freitas-Neto et al., 2015; Grüntzig and Hollmann,
2019; Yu et al., 2009). Across many species (including live humans) andconditions (via glaucoma surgeries or direct injection into the6Experimental Eye Research 196 (2020) 108049

G. Akiyama, et al.

Fig. 5. Structural Characteristics of Outﬂow
Pathways Oﬀ Blebs Are Reminiscent of Lymphatics.
A) Two-photon microscopy image of ﬂuorescentdextrans trapped in porcine outﬂow pathways
showed structures that are similar to blind-end
lymphatics capillaries (white arrow) leading to apossible initial lymphatic trunk with signal variation
along the way which may represent valves (orange
arrow) (scale bar = 200 μm). This region came fromthe same tissue as Figs. 1D and 3B that included theoutﬂow pathway but not the bleb. Natively ﬂuorescent conjunctival lymphatics are seen from transgenic (B) rat and (C) mouse that expresses EGFP
under a Prox-1 promoter. B/C) These lymphatics
showed bright spots (white arrows) that are known
to represent lymphatic valves (scale bars = 1 mm).
D) In a porcine eye, a bleb was created with two
outﬂow
pathwaysarisingsuperior(scalebar = 1 mm). E) OCT was performed and placed on
the outﬂow pathway as denoted by the dotted green
arrow with the direction of ﬂow from red to blue toyellow horizontal arrows (scale bar = 1 mm). F)
Bicuspid valves (white arrows; that match white arrows in D) are seen in the direction of ﬂow (red toblue to yellow vertical arrows) (scale bar = 100 μm).
The red/blue/yellow arrows in (E) match the samecolored arrows in (F) and demonstrate a 2-dimensional representation of the outﬂow pathway oﬀ ofthe bleb. (For interpretation of the references to color
in this ﬁgure legend, the reader is referred to the
Web version of this article.)outﬂow with a lack of lymphatics being associated with failure. All of
this is important because glaucoma surgeons are familiar with cases ofbleb-related surgeries which successfully create an extraocular reservoir(implying adequate inﬂow), but IOP is not reduced (implying inadequate outﬂow). A complex relationship between bleb connective
tissue and lymphatics likely forms post-operative, and variability here
may inﬂuence surgical success. Therefore, future study of how theselymphatics respond to surgery may lead to strategies for improving IOPlowering surgical outcomes.
The qualitative and quantitative results of lymphatic bleb drainage
in this report provide important clues about how lymphatics may drain
the subconjunctival space. Qualitatively, the lymphatic structural evaluation in rodents showed numerous lymphatics while injection studies
only accessed 0–3 lymphatic pathways (Fig. 2). The structural result is
consistent with Guo et al.‘s results that showed numerous and widespread subconjunctival lymphatics in non-human primates (Guo et al.,2012). In tracer studies, since lymphatics only drain at their tips, only
the lymphatics that drain where the tracers are delivered are expected
to be visualized. Further, most bleb-related outﬂow pathways were seen
nasal, but this was only statistically signiﬁcant compared to the temporal quadrant. Overall, these ﬁndings also agree with preliminary datathat we have obtained in our mouse lymphatic reporter model(manuscript under review). Developmentally, the subconjunctival
lymphatics developed from a nasal direction before wrapping aroundthe limbus to end on the temporal side of the eyes, resulting in a ﬁnal
adult nasal > temporal lymphatic distribution. Further, when tracer
was injected under the conjunctiva (temporal vs. nasal side of the eye),the tracer cleared to a greater extent on the nasal side where there weremore lymphatics. This gives functional importance. In the future, studied in humans, with greater power, and with a focus on individual
variation, there may be locations with the greatest lymphatic density
and where bleb-related glaucoma surgeries could be best targeted.
Lastly, there was an uneven distribution of outﬂow pathways arisingfrom the blebs studied. Approximately, 36% of blebs showed one

disadvantages of topical drug delivery include the need for adherent
and persistent (Cairns, 1968) use, the need to bypass epithelial barriers(Pleyer et al., 2013), and the inability to maintain long-standing highconcentration drug depots. Current modern drug-delivery approaches
include intravitreal (Martin et al., 2011) and intracameral injections.
Limitations of intraocular injections include pain (Shiroma et al., 2017)
and the risk of endophthalmitis (Menchini et al., 2018). Thus, thesubconjunctival space has also been studied as an alternative drug delivery site. This space is highly accessible. Drugs have been proven toenter the eye from this space (Raghava et al., 2004; Weinreb, 2001) andcan even reduce IOP (Pitha et al., 2018). Special formulations havebeen proposed to enhance the longevity of drugs delivered there (Wonget al., 2014). Clinically, subconjunctival (Sohn et al., 2011) or subtenon(Thach et al., 1997) steroids are injected for inﬂammatory conditions.
After surgery, subconjunctival steroids and antibiotics can also be given(Gower et al., 2017). However, surgeons have long known that while ableb is initially formed by the drug injection, by the next morning thephysical bleb is gone. Thus, subconjunctival drugs must ﬂow out of andexit this space. Future strategies designed to limit lymphatics or totarget drug injections to where there are fewer lymphatics could enhance the subconjunctival space as a better drug delivery location.
Glaucoma surgery can also beneﬁt from a better understanding of
subconjunctival lymphatics and bleb outﬂow. The relationship between
lymphatics and blebs are in an early stage of research. It has beenshown that tracer ﬂow from Xen blebs move into lymphatic-likestructures (Yu et al., 2009). Xen blebs with luminal openings that look
like lymphatics seen in this report are associated with greater IOP reduction (Lenzhofer et al., 2018b). Bleb histology from failed trabeculectomy blebs show a complete lack of lymphatics compared to normal
conjunctiva (Bouhenni et al., 2016). Peri-failed bleb conjunctiva showintermediate lymphatic presence (Bouhenni et al., 2016). In trabeculectomy, better IOP lowering is also seen in cases when blebs show
lymphatic-like outﬂow pathways during tracer studies (Khoo et al.,2019). Thus, lymphatics seem to be associated with clinical bleb
7

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G. Akiyama, et al.

Fig. 6. Outﬂow Pathways Oﬀ Blebs Express
Lymphatic Markers in Porcine Eyes.
A/D/G/J/M) After injection of a ﬂuorescent dextran
bleb, outﬂow pathways were seen, ﬁxed, and sectioned. Trapped tracer was seen lining the outﬂow
pathway
lumens(whiteasterisks).
A/D)
Immunolabeling on the same section against (B/E)
Prox-1 showed (C/F) co-localization with the tracerlabeled outﬂow pathways. G/J) Immunolabeling on
the same section against (H/K) podoplanin showed(I/L) co-localization with the tracer-labeled outﬂow
pathways. K/L) Shows images with dextran and podoplanin co-localization but also another lymphatic(white arrow) that did not show tracer labeling. MO) Immunolabeling on the same section against a
blood vessel marker (CD31) did not show co-localization with the tracer-labeled outﬂow pathway but
instead showed distinct blood vessels elsewhere(white arrows). Scale bars = 100 μm.

VEGF-C has been found in the anterior segment of the eye (Schlereth
et al., 2019). Small molecules such as retinoic acid (RA) have also beenshown to promote lymphangiogenesis (Choi et al., 2012). After systemic RA delivery, surgical models of hind-limb lymphedema wereameliorated due to lymphatic in-growth (Choi et al., 2012). Thus,
various drugs and proteins could be injected into blebs to promote
lymphangiogenesis and aqueous outﬂow. This may be particularly important if anti-ﬁbrotics (such as mitomycin-C or 5-ﬂuorouracil) aretoxic to lymphatics. In this case, concurrent injection of anti-ﬁbrotics(to limit scarring) and pro-lymphatic agents (to increase outﬂow) may
simultaneously improve aqueous ﬂow into and out of a bleb.
In conclusion, combining careful and concurrent multi-modal approaches of ocular surface lymphangiography (OSL) with structuralassessment and molecular identiﬁcation, we have conﬁrmed the longstanding hypothesis that lymphatics drain the subconjunctival space.
The next steps are to better study subconjunctival outﬂow to identify
the features that allow lymphatic access from blebs. Then, tools need to
be developed to suppress or enhance lymphatic function. Applied todrug delivery and glaucoma surgeries, lymphatic manipulation may
become a new way to improve ophthalmic clinical care.

outﬂow lymphatic, while ~39% showed two, ~7% showed three, and~18% showed none. This is important because this means that not
every bleb investigated in this study accessed lymphatic outﬂow. Some
accessed a lot. The features that govern whether a bleb in a certain
location accesses lymphatics need to be better understood. If thesefeatures could be pre- or intra-operatively identiﬁed, surgical blebs
could be placed in just those locations, again for potentially better
outﬂow and success.
Lastly, lymphatic manipulation may assist bleb-related glaucoma
surgeries. Opposite to the drug delivery rationale, supporting blebs withincreased lymphatics could allow for stronger and longer-lasting blebfunction and IOP lowering. This could be pharmacologically induced.
Vascular endothelial growth factor (VEGF) is well-known in ophthalmology and vision science with biologics against it useful to combat
new blood vessel growth in various diseases such as macular degeneration (Martin et al., 2011). However, those biologics target VEGF-A
and VEGF-B while VEGF-C promotes relatively more lymphatic development (Helotera and Alitalo, 2007). In fact, loss-of-function VEGF-C
mutations are known to be associated with lymphedema (Nadarajah
et al., 2018). Individual VEGF-C mutations (C156S) (Joukov et al.,1998) even more speciﬁcally promote the growth of only lymphatics.
8

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G. Akiyama, et al.

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Funding for this work came from National Institutes of Health,Bethesda, MD (Grant Numbers K08EY024674 [ASH], R01EY030501[ASH] and R21EY026260 [YH]); AMED, Japan (Grant Number
JP19am0101078 [YK]; Glaucoma Research Foundation Shaﬀer Grant,
San Francisco, CA [ASH]; Research to Prevent Blindness Career
Development Award 2016 [ASH]; an unrestricted grant from Research
to Prevent Blindness [UCLA and USC] (New York, NY).
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