lable at ScienceDirect

Experimental Eye Research 93 (2011) 586e591

Contents lists avai

Experimental Eye Research

journal homepage: www.elsevier .com/locate/yexer

A model to measure lymphatic drainage from the eye

Minhui Kim a,b,c,d, Miles G. Johnston b,d, Neeru Gupta a,b,c, f, Sara Moore b,d, Yeni H. Yücel a,b,c,e,*aDepartment of Ophthalmology & Vision Sciences, University of Toronto, St. Michael’s Hospital, Canadab Laboratory Medicine & Pathobiology, University of Toronto, CanadacKeenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, CanadadBrain Sciences Program, Sunnybrook Health Sciences Centre, CanadaeOphthalmic Pathology Laboratory, University of Toronto, CanadafGlaucoma & Nerve Protection Unit, St. Michael’s Hospital, Canada

a r t i c l e i n f o

Article history:Received 3 June 2011Accepted in revised form 18 July 2011Available online 27 July 2011

Keywords:aqueous humorlymphaticlymph nodesuveoscleral outflowconventional outflowsheepglaucomaintraocular pressureinterstitial fluidalbumin

* Corresponding author. St. Michael’s Hospital, 30Toronto, Ontario M5B 1W8, Canada. Tel.: þ1 416 86864 5648.

E-mail addresses: yucely@smh.ca, yeni.yucel@utor

0014-4835/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.exer.2011.07.006

a b s t r a c t

Intraocular pressure (IOP) is the most important risk factor for glaucoma development and progression.Most anti-glaucoma treatments aim to lower IOP by enhancing aqueous humor drainage from the eye.Aqueous humor drainage occurs via well-characterized trabecular meshwork (TM) and uveoscleral (UVS)pathways, and recently described ciliary body lymphatics. The relative contribution of the lymphaticpathway to aqueous drainage is not known. We developed a sheep model to quantitatively assesslymphatic drainage along with TM and UVS outflows. This study describes that model and presents ourinitial findings.

Following intracameral injection of 125I-bovine serum albumin (BSA), lymph was continuouslycollected via cannulated cervical lymphatic vessels and the thoracic lymphatic duct over either a 3-h or5-h time period. In the same animals, blood samples were collected from the right jugular vein every15 min. Lymphatic and TM drainage were quantitatively assessed by measuring 125I-BSA in lymph andplasma, respectively. Radioactive tracer levels were also measured in UVS and “other” ocular tissue, aswell as periocular tissue harvested 3 and 5 h post-injection. Tracer recovered from UVS tissue was usedto estimate UVS drainage. The amount of 125I-BSA recovered from different fluid and tissue compart-ments was expressed as a percentage of total recovered tracer. Three hours after tracer injection,percentage of tracer recovered in lymph and plasma was 1.64% � 0.89% and 68.86% � 9.27%, respectively(n ¼ 8). The percentage of tracer in UVS, other ocular and periocular tissues was 19.87% � 5.59%,4.30% � 3.31% and 5.32% � 2.46%, respectively. At 5 h (n ¼ 2), lymphatic drainage was increased (6.40%and 4.96% vs. 1.64%). On the other hand, the percentage of tracer recovered from UVS and other oculartissue had decreased, and the percentage from periocular tissue showed no change. Lymphatic drainageincreased steadily over the 3 h post-injection period, while TM drainage increased rapidly e reachinga plateau at 30 min.

This quantitative sheep model enables assessment of relative contributions of lymphatic drainage, TMand UVS outflows, and may help to better understand the effects of glaucoma agents on outflowpathways.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Intraocular pressure (IOP) is a major risk factor for glaucomadevelopment, and disease progression (Wilson and Singh, 2009).Current pharmaceutical and surgical treatments preserve visual

Bond Street, 209 LKSKI 4094 6060x77594; fax: þ1 416

onto.ca (Y.H. Yücel).

All rights reserved.

function by lowering IOP e mainly by increasing aqueous outflowfrom the eye.

Aqueous humor leaves the anterior chamber by the well-characterized trabecular meshwork (TM) and uveoscleral (UVS)pathways (Gabelt and Kaufman, 1995). Tracer studies suggest thatabout 80% of total aqueous outflow passes through the TM into thegeneral circulation e with the remainder drained via the UVSpathway (Bill, 1966a, b). In the TM pathway, aqueous humor drainsthrough the TM, into the lumen of Schlemm’s canal, and then intoepiscleral veins and blood circulation. In the UVS pathway, aqueoushumor appears to flow across the base of the iris, through

Fig. 1. Experimental preparation in a dorsal recumbency position: After cannulation ofleft and right cervical lymphatic vessels (B) of thoracic lymphatic duct (C) forcontinuous collection of lymph, placement of catheter in right jugular vein forsampling of blood (E), IV line for fluids (D) and pulse oximeter (F). The sheep wasplaced in a sternal recumbent position e with their head on a head stand and chinresting on the bar, and 125I-BSA was injected into the anterior chamber of each eye (A).

M. Kim et al. / Experimental Eye Research 93 (2011) 586e591 587

interstitial tissue in the ciliary body, into suprachoroidal space, andthen through the sclera and perivascular spaces before beingreabsorbed outside the eye (Bill and Phillips, 1971).

While lymphatic vessels have awell-established role in drainageof extracellular fluid and solutes, they have long been believed to beabsent from the eye. Flow across the sclera has been considereda substitute for lymphatic vessels (Alm and Nilsson, 2009). Wechallenged this assumption by demonstrating the presence oflymphatics in human and sheep ciliary body, and showing themovement of aqueous humor and protein from the anteriorchamber to head and neck lymph nodes (Yucel et al., 2009).However, the contribution of the lymphatic pathway to aqueoushumor outflow, relative to the TM and UVS pathways, is not known.

Sheep offer several advantages in the current study. First, sheeplymphatic vessels are large and, therefore, relatively easy to can-nulate to quantify lymph flow rates (Boulton et al., 1998b). Second,aqueous secretion physiology (Gerometta et al., 2005) and TManatomy (Simones et al., 1996; Guyomard et al., 2008) are similar insheep and primates. Finally, a recently developed sheep model ofglaucoma (Gerometta et al., 2009) has already been used fordeveloping and testing therapeutics (Gerometta et al., 2010a, b).However, quantitative assessment of aqueous humor drainage insheep has not been reported.

The goal of the current study was to quantitatively assess thecontributions of lymphatic, TM and UVS pathways to aqueoushumor outflow in normal sheep. To do this, we employed tracermethodologies used to quantify lymphatic drainage in non-oculartissues (Boulton et al., 1998b) and radio-labeled albumin used tostudy TM and UVS drainage (Bill, 1965; Bill and Hellsing, 1965Gabelt and Kaufman, 1989).

2. Material and methods

2.1. Animals

Sixteen randomly bred sheep (25e35 kg; Hutchison Farms, ON,Canada) were used in experiments approved by the institutionalanimal care committee and conforming to guidelines set by theCanadian Council on Animal Care and the Animals for Research Actof Ontario.

2.2. Surgery

Sheep were anesthetized by induction with 20 mL thiopentalsodium (Hospira Healthcare Corp., Vaughan, ON, Canada) adminis-tered intravenously; deep general anesthesia was maintained underisoflurane (Abbott Laboratories,Montreal, QC, Canada). Aheatingpadwas placed under the belly of anesthetized animals, and percentageHbO2 and heart rate continuously monitored using a pulse oximeter(BensonMedical Instruments,Minneapolis,MN). Catheterplacementwas performed with animals in a dorsal recumbent position (Fig. 1).Cervical lymphatic vessels and the thoracic lymphatic duct, at itsjunctionwith the venous system,were cannulated using 1.5- and 1.7-mm clear vinyl tubes (Dural Plastics Eng. Pty Ltd., Dural, NSW,Australia), respectively, as previously described (Boulton et al.,1998b). Lymph was collected into 16 � 100 mm polystyrene tubes(Diamed Lab Supplies Inc., ON, Canada) containing about 15 U/mLheparin (Pharmaceutical Partners of Canada Inc, ON, Canada) toprevent coagulation; outflow ends of all lymphatic catheters werepositioned approximately level with the sheep’s olecranon and leftatrium. Lymph samples were checked for blood contamination;contaminated samples were excluded. In control experiments, rightprescapular lymphatic vessels were cannulated and lymph collectedto calculate filtration from plasma to lymph. In eight sheep(ID numbers 1, 2, 3, 4, 6, 8, 9, and 10), distinct right and left cervical

vessels were visible and cannulated. In two sheep (ID numbers5 and 7), distinct right and left cervical vessels could not be identified,and multiple cervical vessels that were too small to cannulate wereligatedwithnon-absorbable silk suture (Covidien Syneture, Norwalk,CT) to prevent transport of intracamerally injected tracer to plasmavia this route. For blood sampling, a right jugular vein line (ArgonMedical Devices Inc, Athens, TX) connected to a four-way stop cock(Smiths Medical ASD Inc, Dublin, OH) was inserted into the rightjugular vein. To replenish fluids lost from sampling and salivation, anintravenous line was placed in a cephalic vein for infusion of 5%dextrose saline infusion (10 mL/kg/h).

2.3. IOP measurements

With animals in a sternal recumbent position, IOP was measuredwith a tonometer (Tono-Pen XL, Colonial Medical Supply, Franconia,NH) before intracameral injection and 3 h after injectione at the endof the experiment. Average IOP values were determined from fourmeasurements (Passaglia et al., 2004). Paired t-testwasperformed tocompare IOP before injectionwith IOP at the end of the experiment.Average IOP values before and after injection were not significantlydifferent (right eye, 10.75 � 4.19 mmHg vs. 11.00 � 4.83 mmHg(p > 0.05); left eye: 10.50 � 3.70 mmHg vs. 10.00 � 3.46 mmHg(p > 0.05); mean � SD) (n ¼ 4, ID numbers 3, 6, 7 and 8).

2.4. Collection of 125I-BSA in lymph and plasma samples

With lymphatic and intravenous (IV) catheters in place, sheepwere placed in a sternal recumbent position e with their head on

Fig. 2. Model of the anterior chamber of the eye (aqueous humor) and the TM andlymphatic pathways. 125I-BSA is injected into the anterior chamber of each eye. Fromthe anterior chamber, there is transfer of the protein into the episcleral veins via theTM pathway. 125I-BSA concentration was measured for each plasma sample collectedevery 15 min. There is also drainage to the cervical lymphatics and the thoraciclymphatic duct. The lymphatics, which drain the anterior chamber, join with drainagefrom the cervical tissues to form the cervical tissue lymph. The cervical lymph flowrate and the concentration of tracer in this lymph were measured directly. To ensurethat all possible lymphatic drainage is accounted for, it is assumed that some lymphfrom the eye transports via other pathways to reach the thoracic duct. The thoracicduct flow rate and the concentration of tracer in this lymph were also measureddirectly. (A) Represents the filtration of tracer to the tissues and back into thelymphatic compartments. (B) Represents the filtration of tracer from blood into thevarious tissues of the body. Drainage to lymph and drainage to plasma were bothcorrected for filtration.

M. Kim et al. / Experimental Eye Research 93 (2011) 586e591588

a head stand and chin resting on the bar. The anterior chamber ofeach eye was then injected with 25 mL 125I-labeled bovine serumalbumin (BSA) (13.95 mg in 25 mL saline) (PerkineElmer, Waltham,MA), using a 250 mL Hamilton syringe (Hamilton Company, Reno,NV) and a 301/2 gauge BD needle (Becton Dickinson, Oakville, ON,Canada). When the injection was complete, the needle wasremoved and a cotton swab gently held at the site of injection for2e3 min.

Lymph from cervical vessels and the thoracic duct was collectedcontinuously at 15-min intervals for 3 (n ¼ 8) and 5 (n ¼ 2) hours,respectively. For cases where lymph was collected from both rightand left cervical lymph vessels (ID numbers 2, 3, 4, 9, and 10),measurements of the two samples were pooled, since there was nosignificant difference in radioactivity in right and left cervicallymphatics. In some cases, only one of the cervical vessels could beaccessed for lymph collection e due to technical problems(ID numbers 5, 7 and 8), clot formation in the collecting tube(ID numbers 1 and 6) or the small size of the cervical lymphatics(ID numbers 5 and 7). Two additional animals were used todetermine protein concentration in prescapular lymph.

Tomonitor plasma tracer levels, serial blood sampleswere takenevery 15 min for 3 (n ¼ 8) and 5 h (n ¼ 2). At each time point, twoconsecutive 3 mL samples were drawn. The first sample was putback in the jugular vein, followed by 3 mL of heparin/4% dextrosesaline (Hospira, Montreal, QC, Canada) to prevent blood clotformation in the tube. The second blood sample was placed ina plastic test tube with heparin (15 U/mL), centrifuged at 2400 rpmfor 15 min at room temperature, yielding an upper plasma layer,lower red blood cell layer and a thin white blood cell interface. Onemilliliter of upper plasma layer was pipetted into a separate tubefor radioactivity measurement.

2.5. Post-mortem harvesting of ocular and extraocular tissues

Three (ID numbers 1e8) and 5 h (ID numbers 9 and 10) afterintracameral tracer injection, sheep were euthanized by IV injec-tion of sodium pentothal (Euthanyl, Bimeda-MTC Animal HealthInc., Cambridge, ON, Canada), aqueous humor collected using a 30-gauge needle, and right and left periocular orbital tissue with eyesexenterated. Eyes were then dissected into uvea (ie, iris, ciliarybody and choroid) and sclera e as well as other ocular (cornea,retina, vitreous body) and periocular (conjunctiva, orbital tissueand optic nerve) tissues, and radioactivity measured. UVS drainagewas calculated as the quantity of tracer retained in the uvea andsclera during the specified time interval (Toris, 2008).

2.6. 125I-BSA measurements

Radioactivity in plasma, lymph, ocular tissue, and lymph nodesamples was measured using a gamma counter (Compu-gamma,LKBWallac, Turku, Finland), with appropriate window settings andbackground subtraction. To ensure that most of the measuredradioactivity in any sample was protein-bound, a second set ofaliquots was assayed after precipitation with 10% trichloroaceticacid; free or non-protein-bound 125I amounted to <15% of the totalradioactivity.

2.6.1. Lymph I 125I-BSA measurements and correctionfor filtration (Fig. 2)

The quantity of 125I-BSA measured in lymph was corrected fortracer filtration to lymph, which was estimated to be the traceramount in lymph collected from prescapular vessel, which drainsflank skin andmuscle of the forearm. The assumptionwas that tracerin prescapular lymph originated from intracameral tracer thatdrained via the TM pathway into plasma, then filtrated to interstitial

tissue, and finally to lymph; thus, lymph from prescapular vessel isrepresentative of that from any lymphatic vessel that does notdirectly drain intracameral tracer. We devised a mathematicalmethod based on protein concentrations in lymph, since the degreeofmacromolecule restriction of themicrovasculature in the drainagebasins of the lymphatics of interest vary depending on theiranatomical locations. First, prior to 125I-BSA injection, total proteinconcentration in lymph from prescapular vessel was determined(25.71 � 3.74 mg/mL, n ¼ 6), using a bicinchoninic acid (BCA) proteinassay kit (Thermo Scientific, IL). Following intracameral injection of125I-BSA, average tracer concentration in prescapular lymph wascalculated (ID numbers 9 and 10). To estimate filtered tracer inprescapular lymph as a function of protein concentration, totalradioactivity was divided by the product of the lymph volumecollected and the average protein concentration. To derive this value,weused thedata fromthefirst 90minpost-injection. For lymph fromcervical vessels, the total mass of protein over 3 h was calculated asthe product of volume and average protein concentration e or20.69 � 3.07 mg/mL (n ¼ 8). Radioactivity in cervical lymph due tofiltration from plasma was estimated as the product of the pre-scapular cpm/mg value and the mass of tracer as calculated above.This value was then subtracted from the actual measured radioac-tivity in cervical lymph (volume � radioactivity/mL) to give anestimate of tracer entry into the cervical vessels directly fromthe eye.

For lymph from thoracic duct, the total mass of protein over 3 hwas calculated as the product of volume and average protein

Fig. 3. Time course of lymphatic and plasma (TM) drainage assessed by monitoringradioactive tracer levels over180 min following intracameral injection of 125I-BSA.Lymphatic drainage at various time points was estimated as percentage of the totalamount radioactivity (cpm) in lymph e cervical lymphatics and thoracic duct e overthe 180 min. Plasma (TM) drainage at various time points was estimated as percentageof the total amount radioactivity (cpm) in plasma e sample from right jugular vein e

over the 180 min. While lymphatic drainage (A) increased steadily over the timeperiod, TM drainage (B) increased rapidly e plateauing at 30 min. The numbers on theright correspond to ID numbers of sheep.

M. Kim et al. / Experimental Eye Research 93 (2011) 586e591 589

concentration e or 40.17 � 7.22 mg/mL (n ¼ 8). Radioactivity inthoracic duct lymph due to filtration from plasma was estimated asthe product of the prescapular cpm/mg value and the mass oftracer as calculated above. This value was then subtracted from theactual measured radioactivity in thoracic duct lymph (volume �radioactivity/mL) to give an estimate of tracer entry into thoracicduct directly from the eye.

Based on these parameters, radioactivity per mg protein wasestimated for prescapular lymph and themass of filtered tracer wassubtracted from the measurement of lymph from vessels ofinterest.

2.6.2. Plasma 125I-BSA measurements and correctionfor filtration (Fig. 2)

The total quantity of tracer in plasma was used to estimate TMdrainage, which is known to drain to the general circulation (Gabeltand Kaufman, 1989). Radioactivity for each plasma sample collectedevery 15 min was tabulated and the data corrected for filtration oftracer from the plasma to various tissue components using theaverage rate of albumin elimination from plasma of 6.5% per hour(Boulton et al., 1997, 1998a, 1998b; 1999). Thus, 6.5% of the plasmaradioactivity was added back to the radioactivity recovered inplasma for each hour of the experiment. Plots of 125I-BSA concen-tration of the ten sheep over time showed that measurementsincreased rapidly before starting to plateau about 30 min afterinjection. The 125I-BSA concentration at the plateau for each sheepwas estimated by fitting a line to the data and determining the value180 min after tracer injection. To estimate the plateau value,each measurement after 15 min post-injection was converted tonatural log. Analysis of variance was performed using PROC GLM inSAS (SAS Institute, NC), and a model (measurement ¼ subject þ(subject� (time� 180))). By using the “no intercept” option in PROCGLM and subtracting 180 from time, the parameters estimated thelog of concentration at time ¼ 180 for each subject; 95% confidencelimits (95% CL) around plateau values were calculated, and plateauvalues back-converted to the original scale.

The plasma volume (mL) calculation, the product of 21.77 andthe animal’s weight (kg)þ 649.68, is based on previously publishedplasma volumes derived from 41 sheep against their weights(Boulton et al., 1997, 1998a, 1998b; 1999). The total quantity oftracer in plasma was calculated by multiplying the plateau value ofplasma concentration at 180 min after injection with plasmavolume, as calculated above.

Lymphatic, TM and UVS drainage were calculated as percentageof tracer in lymph, plasma and UVS tissue of total drainage,respectively. Percentage of tracer in other ocular tissue andpercentage of tracer in periocular tissues of total drainagewere alsocalculated. Total drainage was considered as the sum of tracerrecovered from lymph, plasma, UVS tissues, and other ocular andperiocular tissues.

To study the time course of lymphatic drainage and plasma (TM)drainage, we calculated radioactive tracer levels over180 minfollowing intracameral injection of 125I-BSA. Lymphatic drainage atvarious time points was estimated as percentage of the totalamount radioactivity (cpm) in lymph e cervical lymphatics andthoracic duct e over the 180 min. Plasma (TM) drainage at varioustime points was estimated as percentage of the total amountradioactivity (cpm) in plasma over the 180 min.

2.7. Impact of tracer drainage from conjunctiva-lacrimalductal pathway

It was important to determine whether tracer leaked during orafter intracameral injection and drained via the conjunctiva-lacrimal ductal pathway contributed to tracer drainage into the

plasma and lymph. To test this, 125I-BSAwas applied topically to theconjunctiva in two sheep (the same mass as injected into aqueoushumor), and then lymph and blood samples were collected andmeasured as described above. Over 3 h following topical 125I-BSAapplication to the conjunctiva, 0% and 0.06% of total recoveredtracer was detected in the lymph, and 8.70% and 3.82% in plasma.Based on these control experiments, we concluded that tracerleaked onto the conjunctival surface during or after intracameralinjection did not contribute significantly to tracer accumulation inlymph and plasma.

3. Results

Average lymph flow rates were determined for cervical vessels(2.88 � 0.55 mL/h; ID numbers 2, 3, 4, 9, and 10), thoracic duct(124.41 � 9.73 mL/h; ID numbers 1e10), and prescapular vessel(1.31 � 0.12 mL/h; ID numbers 9 and 10).

The percentage of 125I-BSA in lymph increased steadily duringthe 3 h following intracameral tracer injection (Fig. 3a). In contrast,plasma levels of 125I-BSA increased rapidly and reached a plateaulevel after 30 min (Fig. 3b).

Table 1 shows the distribution of radioactivity in lymphatic,tissue and plasma compartments as a percentage of the totalamount of recovered tracer. Data are for ten animals e eightanalyzed at 3 h post-intracameral tracer injection, and twoanalyzed at 5 h. At 3 h, mean lymphatic drainagewas 1.64%� 0.89%of the total. Mean TM drainage, estimated by the percentage of

Table 1Distribution of 125I-BSA in various fluid and tissue compartments 3 (n ¼ 8) and 5 (n ¼ 2) hours after intracameral injection.

Animal IDnumber

Lymph Oculareperiocular tissue Plasma %

Cervical % Thoracic % Total % Uveoscleral % Other intraocular % Periocular %

3 h1 0.02 0.50 0.52 16.31 3.27 3.19 76.712 0.17 2.02 2.19 18.16 5.76 12.11 61.793 0.08 1.41 1.50 11.07 1.93 1.84 83.664 0.06 1.28 1.34 16.87 2.44 2.06 77.295 e 0.87 0.87 18.70 8.18 3.58 68.676 0.07 1.02 1.09 25.88 7.13 4.99 60.927 0.22 2.29 2.51 24.45 6.04 3.82 63.198 0.22 2.92 3.14 27.53 7.84 2.84 58.66Lower CL 0.05 0.86 0.90 15.19 3.26 1.54 61.11Mean 0.12 1.54 1.64 19.87 5.32 4.30 68.86Upper CL 0.20 2.22 2.39 24.55 7.38 7.07 76.615 h9 0.13 6.28a 6.40a 14.35a 2.85a 3.44 72.9610 0.09 4.88a 4.96a 10.27a 2.59a 1.91 80.27a

CL: 95% confidence limits of the measurements at 3 h.Mean: Mean of measurements at 3 h.

a Indicates measurements at 5 h that are significantly different compared to corresponding 95% confidence limits of the measurements at 3 h.

M. Kim et al. / Experimental Eye Research 93 (2011) 586e591590

tracer in plasma, was much higher e 68.86% � 9.27%. The meanpercentage of tracer accumulated in UVS, other intraocularand periocular tissues was 19.87% � 5.59%, 5.32% � 2.46% and4.30% � 3.31%, respectively.

At 5 h post-injection, lymphatic drainage was increasedcompared to 3 h (6.40% and 4.96%, for the two animals examined at5 h, vs. 0.90%e2.39%, 95% confidence limits of the measurements at3 h (CL)) In contrast, tracer recovered at 5 h in UVS and other oculartissue was reduced (14.35% and 10.27% vs. 15.19%e24.55%; CL and2.85% and 2.59% vs. 3.26%e7.38%; CL), respectivelye or unchanged,in the case of periocular tissue (3.44% and 1.91% vs.1.54%e7.07%; CL).

4. Discussion

The present study provides, for the first time e in any species,a quantitative demonstration of the contribution of lymphatics toaqueous humor drainage. Continuous measurement of bothlymphatic and TM drainage in sheep showed that the contribution ofthe lymphatic pathway to aqueous humor outflow was significantlyless than the TM pathway. Several factors in our study are likely tohave contributed to anunderestimation of the lymphatic contributionto aqueous humor drainage. Experimental conditions e such asgeneral anesthesia (Quin and Shannon, 1975), immobility andmechanicalventilation (Negrini andMoriondo, 2011)emayhave leadto reductions in lymphatic flow. In addition, it is difficult to cannulateall lymph vessels in the head and neck (Zakharov et al., 2003).

UVS drainage was estimated by measurement of tracer retainedin uveal and scleral tissue harvested at the end of the experimentseeither 3 or 5 h after intracameral injection. The ratio of TM to UVSoutflow in sheepwas 69:20, which is comparable to ratios in speciessuch as Cynomolgus monkeys (Bill, 1971), Vervet monkeys (Bill,1971), mice (Aihara et al., 2003), and dogs (Barrie et al., 1985) e

whose ratios are 45:55, 62:38, 75:25, and 85:15, respectively. On theother hand, the 69:20 ratio in sheep is greater than in rabbits andcats e whose ratios are 92e97:3e8 and 97:3, respectively (Bill,1966a).

In our experiments, the percentage of tracer retained in UVStissue appeared to decrease between 3 and 5 h after injection, whilelymphatic drainage appeared to increase. Thus, ocular lymphaticsmay provide an exit route for fluid and proteins retained in UVStissue e in addition to exit routes through sclera (Bill, 1971) andemissary canals around the vortex veins (McMaster and Macri,1968; Green et al., 1977; Krohn and Bertelsen, 1997).

In this report, we assessed lymphatic drainage of aqueoushumor using BSA as a lymph flow marker (Boulton et al., 1997,1998a, 1998b). Our mathematical model assumes that there is noloss of tracer en route to the lymphatics during absorption into theinitial lymphatic vessels, since there is no evidence that this occurs.On the other hand, once tracer is in lymph and passing throughlymph nodes, it can be concentrated or diluted depending on thebalance of hydrostatic and oncotic forces (Adair and Guyton, 1985).However, this would not affect our analysis that depends on thetotal mass of tracer (product of flow and concentration), whichwould be unaffected. Particular attention was paid to the contri-bution of filtration to lymph and plasma in estimating drainage ofaqueous humor into lymphatics and plasma.

Detailed quantitative assessments of TM, UVS and lymphaticdrainage in sheep are particularly relevant since the recently devel-oped sheep model of glaucoma (Gerometta et al., 2009) has provenuseful in developing and testing new IOP-lowering drugs (Geromettaet al., 2010a, b). Lymphatic vessels respond to a wide variety ofbiochemical and pharmacological agents (Johnston, 1985) e

including prostaglandin analogs, which enhance lymphatic drainage(Franzeck et al.,1997; Sinzinger et al.,1997; Amann-Vesti et al., 2003)and have a profound IOP-lowering effect (Gabelt and Kaufman,1989). It is important to assess whether drugs that targetlymphatics, also enhance lymphatic drainage of aqueous humor andlower IOP. Thismodelmayallowa better understandingof the effectsof various glaucoma therapies on TM, UVS, and lymphatic drainagepathways from the eye.

Acknowledgments

This work was supported by Dorothy Pitts Research Fund (NG)and Henry Farrugia Research Fund (YHY). M.K. was supported byCIHR Frederick Banting and Charles Best Canada Graduate Schol-arship and Vision Science Research Program at University of Tor-onto. We thank Barbara Thompson, MSc for assistance withstatistical analysis and Steve Fish, PhD for his help with illustratingFig. 1.

References

Adair, T.H., Guyton, A.C., 1985. Lymph formation and its modification in thelymphatic system. In: Johnston, M.G. (Ed.), Experimental Biology of theLymphatic Circulation. Elsevier Science Publishers B.V., Amsterdam, pp. 13e44.

M. Kim et al. / Experimental Eye Research 93 (2011) 586e591 591

Aihara, M., Lindsey, J.D., et al., 2003. Aqueous humor dynamics in mice. Invest.Ophthalmol. Vis. Sci. 44 (12), 5168e5173.

Alm, A., Nilsson, S.F., 2009. Uveoscleral outflow e a review. Exp. Eye Res. 88 (4),760e768.

Amann-Vesti, B.R., Gitzelmann, G., et al., 2003. Isoprostane 8-epi-prostaglandin F2alpha decreases lymph capillary pressure in patients with primary lymphe-dema. J. Vasc. Res. 40 (1), 77e82.

Barrie, K.P., Gum, G.G., et al., 1985. Quantitation of uveoscleral outflow in normo-tensive and glaucomatous beagles by 3H-labeled dextran. Am. J. Vet. Res. 46 (1),84e88.

Bill, A., 1965. The aqueous humor drainage mechanism in the cynomolgus monkey(Macaca irus) with evidence for unconventional routes. Invest. Ophthalmol. 4,911e919.

Bill, A., 1966a. Formation and drainage of aqueous humour in cats. Exp. Eye Res. 5,185e190.

Bill, A., 1966b. The routes for bulk drainage of aqueous humour in rabbits with andwithout cyclodialysis. Doc. Ophthalmol. 20, 157e169.

Bill, A., 1971. Aqueous humor dynamics in monkeys (Macaca irus and Cercopithecusethiops). Exp. Eye Res. 11 (2), 195e206.

Bill, A., Hellsing, K., 1965. Production and drainage of aqueous humor in the cyn-omolgus monkey (Macaca irus). Invest. Ophthalmol. 4 (5), 920e926.

Bill, A., Phillips, C.I., 1971. Uveoscleral drainage of aqueous humour in human eyes.Exp. Eye Res. 12 (3), 275e281.

Boulanger, B., Yuan, Z., et al., 1999. Pericardial fluid absorption into lymphaticvessels in sheep. Microvasc. Res. 57 (2), 174e186.

Boulton, M., Flessner, M., et al., 1997. Lymphatic drainage of the CNS: effects oflymphatic diversion/ligation on CSF protein transport to plasma. Am. J. Physiol.272 (5 Pt 2), R1613eR1619.

Boulton, M., Armstrong, D., et al., 1998a. Raised intracranial pressure increases CSFdrainage through arachnoid villi and extracranial lymphatics. Am. J. Physiol. 275(3 Pt 2), R889eR896.

Boulton, M., Flessner, M., et al., 1998b. Determination of volumetric cerebrospinalfluid absorption into extracranial lymphatics in sheep. Am. J. Physiol. 274(1 Pt 2), R88eR96.

Franzeck, U.K., Spiegel, I., et al., 1997. Combined physical therapy for lymphedemaevaluated by fluorescence microlymphography and lymph capillary pressuremeasurements. J. Vasc. Res. 34 (4), 306e311.

Gabelt, B.T., Kaufman, P.L., 1989. Prostaglandin F2 alpha increases uveoscleraloutflow in the cynomolgus monkey. Exp. Eye Res. 49 (3), 389e402.

Gabelt, B.T., Kaufman, P.L., 1995. In: Kaufman, P.L., Alm, A. (Eds.), Aqueous HumorHydrodynamics. Adler’s Physiologyof the Eye, vol. 8.Mosby, St. Louis, pp. 237e289.

Gerometta, R.M., Malgor, L.A., et al., 2005. Cl-concentrations of bovine, porcine andovine aqueous humor are higher than in plasma. Exp. Eye Res. 80 (3), 307e312.

Gerometta, R., Podos, S.M., et al., 2009. Steroid-induced ocular hypertension innormal sheep. Invest. Ophthalmol. Vis. Sci. 50 (2), 669e673.

Gerometta, R., Alvarez, L.J., et al., 2010a. Effects of sildenafil and tadalafil on intra-ocular pressure in sheep: implications for aqueous humor dynamics. Invest.Ophthalmol. Vis. Sci. 51 (6), 3139e3144.

Gerometta, R., Spiga, M.G., et al., 2010b. Treatment of sheep steroid-induced ocularhypertension with a glucocorticoid-inducible MMP1 gene therapy virus. Invest.Ophthalmol. Vis. Sci. 51 (6), 3042e3048.

Green, K., Sherman, S.H., et al., 1977. Fate of anterior chamber tracers in the livingrhesus monkey eye with evidence of uveo-vortex outflow. Trans. Ophthalmol.Soc. U. K. 97 (4), 731e739.

Guyomard, J.L., Rosolen, S.G., et al., 2008. A low-cost and simple imaging techniqueof the anterior and posterior segments: eye fundus, ciliary bodies, iridocornealangle. Invest. Ophthalmol. Vis. Sci. 49 (11), 5168e5174.

Johnston, M.G., 1985. Involvement of lymphatic collecting ducts in the physiologyand pathophysiology of lymph flow. In: Johnston, M.G. (Ed.), ExperimentalBiology of the Lymphatic Circulation. Elsevier Science Publishers, B.V.,pp. 81e120.

Krohn, J., Bertelsen, T., 1997. Corrosion casts of the suprachoroidal space anduveoscleral drainage routes in the human eye. Acta Ophthalmol. Scand. 75 (1),32e35.

McMaster, P.R., Macri, F.J., 1968. Secondary aqueous humor outflow pathways in therabbit, cat and monkey. Arch. Ophthalmol. 79 (3), 297e303.

Negrini, D., Moriondo, A., 2011 Apr 11. Lymphatic anatomy and biomechanics.J. Physiol. [Epub ahead of print].

Passaglia, C.L., Guo, X., et al., 2004. Tono-Pen XL calibration curves for cats, cows andsheep. Vet. Ophthalmol. 7 (4), 261e264.

Quin, J.W., Shannon, A.D., 1975. The effect of anaesthesia and surgery on lymphflow, protein and leucocyte concentration in lymph of the sheep. Lymphology 8(4), 126e135.

Simones, P., De Geest, J.P., et al., 1996. Comparative morphology of the pectinateligaments of domestic mammals, as observed under the dissecting microscopeand the scanning electron microscope. J. Vet. Med. Sci. 58 (10), 977e982.

Sinzinger, H., Oguogho, A., et al., 1997. Isoprostane 8-epi-prostaglandin F2 alpha isa potent contractor of human peripheral lymphatics. Lymphology 30 (3),155e159.

Toris, C.B., 2008. Aqueous humor dynamics I. In: Civan, M.M. (Ed.), Current Topics inMembranes, the Eye’s Aqueous Humor, second ed., vol. 62. Academic Press, SanDiego, pp. 193e229.

Wilson, M.R., Singh, K., 2009. Intraocular pressure: does it measure up? OpenOphthalmol. J. 17 (3), 32e37.

Yucel, Y.H., Johnston, M.G., et al., 2009. Identification of lymphatics in the ciliarybody of the human eye: a novel "uveolymphatic" outflow pathway. Exp. EyeRes. 89 (5), 810e819.

Zakharov, A., Papaiconomou, C., et al., 2003. Lymphatic cerebrospinal fluidabsorption pathways in neonatal sheep revealed by subarachnoid injection ofmicrofil. Neuropathol. Appl. Neurobiol. 29 (6), 563e573.