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Originally published In Press as doi:10.1167/iovs.08-2501 on October 20, 2008
 (Investigative Ophthalmology and Visual Science. 2009;50:765-770.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-2501
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Enhanced Inflow and Outflow Rates Despite Lower IOP in Bestrophin-2-Deficient Mice

Youwen Zhang,1 Bryan R. Davidson,2 W. Daniel Stamer,1,3 Jennifer K. Barton,2,4 Lihua Y. Marmorstein,1,4 and Alan D. Marmorstein1,5

1From the Departments of Ophthalmology and Vision Science, 2Electrical and Computer Engineering, 3Pharmacology, and 4Physiology and the 5College of Optical Sciences, University of Arizona, Tucson, Arizona.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Bestrophin-2 (Best2), a putative Cl– channel is expressed in the nonpigmented epithelium (NPE). Disruption of Best2 in mice results in a diminished intraocular pressure (IOP). Aqueous humor dynamics were compared in Best2+/+ and Best2–/– mice, to better understand the contribution of Best2 to IOP.

METHODS. Measurements of IOP, episcleral venous pressure (EVP), conventional outflow facility (Ct), aqueous humor production (Fa), and anterior chamber volume (Va) were made using anterior chamber cannulation. Conventional (Fc) and uveoscleral outflow (Fu), and rate of aqueous humor turnover, were calculated from measured data. The anterior chamber was examined in live mice by optical coherence tomography (OCT) and postmortem by light microscopy.

RESULTS. IOP in Best2–/– mice was lower compared with Best2+/+ littermates. EVP was unchanged. Since Best2 is expressed in NPE cells, the hypothesis was that Best2 is involved in generating aqueous flow. However, Fa in Best2–/– mice was increased by ~73% compared with Best2+/+ mice. This was accompanied by increases in Fc and Fu. Aqueous humor turnover was enhanced more than twofold in Best2–/– mice. No evidence of developmental structural changes was noted.

CONCLUSIONS. Best2 appears to antagonize the formation of aqueous humor and cause an inhibition of both Fc and Fu, despite being expressed only in NPE cells. These data support the hypothesis that the inflow and outflow pathways communicate via soluble agents present in the aqueous humor and implicate Best2 as a critical mediator of that communication.

The production of aqueous humor by the ciliary epithelium requires the transepithelial movement of H2O and various ions. H2O transport by the nonpigmented epithelium (NPE) like that of the retinal pigment epithelium (RPE) and transporting epithelia in the colon, is thought to be coupled to Cl– transport.1 2 While there is evidence of the participation of as many as three distinct Cl– channels in aqueous humor formation, CLC-3 is the only candidate to date for which there is direct evidence of involvement.3 4

Bestrophins are a recently recognized family of proteins that have gained significant interest as potential Ca2+-activated Cl– channels (CaCCs), although this function is somewhat controversial.5 6 Recently, our laboratory demonstrated that mice in which the gene Best2, encoding bestrophin-2 (Best2) was disrupted exhibit a significantly lower IOP than Best2+/+ littermates.7 Best2 is uniquely expressed in the basolateral plasma membrane of the NPE in the eye, as well as in several other tissues, including the transporting epithelium of the colon.7 In addition to their putative CaCC activity, bestrophins have been proposed to function as voltage-dependent anion or bicarbonate channels,6 8 regulators of voltage-dependent Ca2+ channels,5 9 10 and regulators of Na+ dependent H+ transport (Zhang et al., unpublished observations, 2008). The diminished IOP observed in Best2–/– mice suggests that Best2 may serve as one of the Cl– channels responsible for driving aqueous humor formation.

To test the hypothesis that Best2 is a Cl– channel necessary for aqueous formation, we performed a comprehensive study of aqueous dynamics in Best2–/– mice, anticipating that aqueous formation would be diminished. To our surprise, we found that although IOP in these mice is lower in comparison to Best+/+ mice, the rate of aqueous formation, Fa, is significantly increased as is drainage through both the conventional (Fc) and uveoscleral (Fu) outflow pathways. Morphologic inspection of the anterior chamber did not identify any developmental or anatomic changes that would explain this phenomenon. Based on these data, we propose that Best2 does not participate in, but antagonizes the formation of aqueous humor, and that there is a communicative link between the ciliary epithelia and the outflow pathways that involves signaling via the only common component of the two, the aqueous humor. Finally, we conclude that these data continue a string of recent findings that are inconsistent with Bestrophins functioning in vivo as CaCCs,5 11 12 and are more consistent with the hypothesis that bestrophins are regulators of ion transport.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
IOP Measurements
Mice deficient in Best2 have been reported previously.7 All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research on mice aged 2 to 4 months. IOP was measured in mice by cannulation of the anterior chamber, as described previously,7 13 14 15 by using Avertin anesthesia (300 mg/kg injected intraperitoneally). In brief, the anterior chamber was cannulated with a borosilicate glass microneedle, filled with Hanks’ balance salt solution (HBSS) and connected to a pressure transducer (BLPR; World Precision Instruments, Sarasota, FL). The signal was amplified (Bridge8 amplifier; World Precision Instruments), converted from analog to digital (Iworx model 108 converter; CB Sciences, Dover, NH), and recorded (LabScribe software ver. 1.6; CB Sciences). IOP was recorded for a period of >90 seconds, and the IOP was determined from the average of each recording. Recordings were discarded if the variance during the recording period exceeded 1 mm Hg or did not immediately return to zero after withdrawal of the needle. All measurements were performed between 2 and 6 PM, to avoid diurnal pressure variation. The apparatus was calibrated by using a fluid reservoir the height of which could be adjusted to generate a series of known pressures.

Determination of Aqueous Humor Production
The rate of aqueous humor formation was determined by dilution of a fluorescent perfusate according to the method of Aihara et al.13 substituting 5 µg/mL FITC-Dextran (70 kDa) for rhodamine dextran. Perfusion pressure was maintained at EVP so that pressure-dependent outflow was reduced to zero. Fluorescence was determined on a multilabel counter (Wallac, Victor3 1420; PerkinElmer Life Sciences, Wellesley, MA), with excitation and emission wavelengths of 485 and 535 nm, respectively. Aqueous production (Fa) was calculated based on the aspiration rate (3 µL/min) and the ratio of the concentration of FITC-dextran in the perfusion outflow fluid (Co) to the concentration of FITC-dextran in the perfusion inflow fluid (Ci), according to Aihara et al13 :

Formula 1(1)

Measurement of Conventional Outflow Facility
Conventional outflow facility (Ct) was determined according to Aihara et al.,13 with the same infusion system used to measure IOP and aqueous production. All fluid within the infusion system was replaced with physiological saline. The measurement was based on measuring total outflow volume (Vt) for a period of 10 minutes at two different levels of IOP (25 and 35 mm Hg), maintained by altering the reservoir height. Ct was determined according to the following equation:

Formula 2(2)

Determination of Conventional and Uveoscleral Outflow
IOP, EVP, and Ct were measured and averaged for each genotype. Using these data, the conventional outflow (Fc) was calculated as:

Formula 3(3)
Uveoscleral outflow (Fu) was then calculated according to the modified Goldmann equation:

Formula 4(4)

Anterior Chamber Volume and Aqueous Humor Turnover Rate
Anterior chamber volume (Va) was determined by aspiration of the aqueous humor at a rate of 100 nL/s, according to Aihara et al.13 Aspiration was deemed complete when the central border of the iris was observed by dissecting microscope to make contact with the cornea. The turnover rate (% · min–1) was calculated as:

Formula 5(5)

Statistical Analysis
Data from Best2+/+ and Best2–/– mice were compared by using the two-tailed, homoscedastic t-test function (Excel 2004 for MAC; Microsoft, Redmond, WA).

Histology
Two-month-old mice were fixed by intracardiac perfusion with half-strength Karnovsky’s fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer [pH 7.2]). The eyes were enucleated and further fixed by immersion in half strength Karnovsky’s fixative for an additional 18 hours, after which they were transferred to 0.1 M cacodylate buffer (pH 7.2). The eyes were then postfixed with 1% osmium tetroxide, dehydrated in a graded series of alcohols, and embedded in Spurr’s resin. Semi-thin sections (0.5 µm) were cut on a microtome (Reichert Ultracut; Leica, Deerfield, IL) and stained with toluidine blue. Sections were inspected with a microscope (E-600; Nikon, Tokyo, Japan) and photographed with a color CCD camera.

Optical Coherence Tomography
Two- to 4-month-old mice were anesthetized with Avertin (250 mg/kg IP) and placed on a mechanical stage that permitted movement along two axes. The anterior chamber was imaged (OCP930SR Spectral Radar OCT Imaging System; Thorlabs, Newton, NJ) with a 930-nm center wavelength light source and an axial resolution 4.5 µm in tissue. Cross-sectional images were recorded in the nasal-temporal plane as well as the superior-inferior plane. Pixel images (500 x 512; x–z) were captured at maximum pupillary diameter and corrected for asymmetries in x–z spacing (Photoshop 7.01; Adobe Systems, San Jose, CA). Measurements of anterior chamber depth and corneal thickness were made from distortion-corrected images obtained along both the nasal-temporal and superior-inferior axis using Image J software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html).


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Effect of Best2 Disruption on IOP
In our previous study,7 we observed that IOP was diminished in Best2–/– mice in comparison to their Best2+/+ littermates. Since that initial report, we have increased the number of animals on which we have performed measurements of IOP. In the current study, The measured IOP in Best2+/+ was 11.70 ± 0.16 mm Hg (mean ± SE, n = 31) and for Best2–/– it was 10.22 ± 0.16 mm Hg (mean ± SE, n = 55), a significant (P < 0.0001) difference of 1.48 mm Hg (Fig. 1 , Table 1 ).


Figure 1
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  		FIGURE 1. Comparison of IOP in Best2+/+ and Best2–/– mice. IOP was measured via anterior chamber cannulation. Note that IOP is significantly (P < 0.0001) lower in Best2–/– mice. Data are presented as a box plot in which the line within the box marks the median IOP, and the boundaries of the box indicate the range covered by the middle 50% of measurements. Bars above and below the boxes indicate the 90th and 10th percentiles, respectively. Symbols outside of the box and bars are outliers. Best2+/+, n = 31; Best2–/–, n = 55.

 

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  		TABLE 1. Comparison of Aqueous Humor Dynamic Parameters of Best2+/+ and Best2–/– Mice

 
Measurement of Aqueous Humor Formation
According to the modified Goldmann equation, IOP = [(Fa – Fu)/Ct] + EVP. In our prior study7 we measured EVP in Best2+/+ and Best2–/– mice and found no difference with both groups having an EVP of 6.3 ± 0.3 mm Hg (Table 1) . Since EVP between Best2+/+ and Best2–/– mice are identical, the difference in IOP must arise from differences in Fa or Ct. Combined with our observation that Best2 is expressed only in NPE cells in the eye, we hypothesized that the diminished IOP is the result of a diminished rate of aqueous humor formation.

To test our hypothesis, we used a modification of the method of Aihara et al.,13 in which the anterior chamber of the eye is cannulated, clamped at EVP, and perfused with a physiologic salt solution containing a 70-kDa FITC-dextran. By observing dilution of the fluorescence emission of the perfusion outflow fluid versus that of the inflow fluid, we determined Fa. In Best2+/+ mice Fa was 0.160 ± 0.047 µL/min (mean ± SD, n = 9; Fig. 2A , Table 1 ), similar to the 0.18 ± 0.05 µL/min reported by Aihara et al. in NIH Swiss white mice. Unexpectedly, Fa in Best2–/– mice was 0.277 ± 0.069 µL/min (mean ± SD, n = 9; Fig. 2A , Table 1 ), a significant (P < 0.001) increase (73%) over the Best2+/+ mice.


Figure 2
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  		FIGURE 2. Comparison of Fa (A) and Ct (B) in Best2+/+ and Best2–/– mice. Fa is significantly (P < 0.001) elevated in Best2–/– mice compared with Best2+/+ mice (A). Ct, is also elevated in Best2–/– mice compared with Best2+/+ mice (B). Data are presented as a box plot in which the line within the box marks the median, and the boundaries of the box indicate the range covered by the middle 50% of measurements. Bars above and below the boxes indicate the 90th and 10th percentiles respectively. Symbols outside of the box and bars are outliers. Best2+/+, n = 9 in (A) and 23 in (B); for Best2–/– n = 9 in (A) and 23 in (B).

 
Measurement of Conventional Outflow Facility
Based on the modified Goldmann equation (equation 4) , for IOP to be diminished without altering EVP, either Fa must be diminished or Fu and Ct must be increased. Since Fa was significantly increased for Best2–/– mice, and Fu cannot be measured directly, we next measured Ct (Fig. 2B , Table 1 ). Again employing the methods of Aihara et al.13 we observed in Best2+/+ mice, Ct was 0.0050 ± 0.0015 µL · min–1/mm Hg (mean ± SD, n = 23, Fig. 2B , Table 1 ), nearly identical with the 0.0051 µL · min-1/mm Hg reported by Aihara et al. in NIH Swiss white mice. Of interest, in Best2–/– mice, Ct was 0.0085 ± 0.0026 µL · min–1/mm Hg (mean ± SD, n = 23, Fig. 2B , Table 1 ), a significant (P < 0.001) increase (70%) compared with Best2+/+ mice.

Having obtained measured values for IOP, EVP, Fa, and Ct, we calculated Fc and Fu by using equations 3 and 4 , respectively. As shown in Table 1 , Fc was 0.027 and 0.033 µL · min–1 in Best2+/+ and Best2–/– mice, respectively, a 22% increase in Fc for Best2–/– mice. Fu was calculated to be 0.133 and 0.211 µL · min–1 in Best2+/+ and Best2–/– mice respectively, a 59% increase in Best2–/– mice. These results indicate that both pressure-dependent and independent outflow appears to overcompensate for the increase in Fa in Best2–/– mice resulting in an IOP that is lower in Best2–/– mice than in Best2+/+ mice, despite the 73% increase in Fa in Best2–/– mice.

Anatomy of the Angle
The increase in Ct was perplexing, considering that Best2 is expressed only in the NPE and not by cells in drainage tissues. One potential explanation is anatomic differences or abnormalities resulting from a lack of Best2. A postmortem examination of the angles of Best2+/+ and Best2–/– mice identified no gross anatomic abnormalities in outflow structures; however, we occasionally observed an expanded TM and deposits of pigment in the TM of Best2–/– mice (Fig. 3) .


Figure 3
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  		FIGURE 3. Comparison of the angle in Best2+/+ and Best2–/– mice. Representative photomicrographs of toluidine blue-stained thick sections of the angle in Best2+/+ (A, C) and Best2–/– (B, D) mice. No significant differences were noted in the angle examined at low magnification (A, B). Inspection of Schlemm’s canal (sc) and the trabecular meshwork (tm) at higher magnification (C, D) indicated little difference as well, although tm tissue was often more expanded in the Best2–/– mouse, perhaps indicative of a higher rate of flow through drainage tissues.

 
In parallel studies, we examined the anterior chamber of live mice using OCT (Fig. 4) . No difference was observed in the thickness of the cornea (Table 2) . However, the anterior chamber depth (ACD) measured from the inner surface of the cornea to the anterior surface of the lens capsule was 496 µm in Best2+/+ mice (n = 12) but only 458 µm in Best2–/– mice (n = 15), a decrease of nearly 40 µm (Fig. 4 , Table 2 ).


Figure 4
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  		FIGURE 4. OCT imaging of the anterior chamber of Best2+/+ and Best2–/– mice. The anterior chambers of live Best2+/+ (A) and Best2–/– (B) mice were examined using OCT. Anterior chamber depth (ACD) and corneal thickness (CT) were measured as indicated from images. On average, the ACD of Best2–/– mice was shallower than that of Best2+/+ mice (see Table 2 ) with no significant difference in CT.

 

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  		TABLE 2. Structural Attributes of the Anterior Chamber of Best2+/+ and Best2–/– Mice

 
To determine anterior chamber volume (Va), we used the aqueous humor aspiration method of Aihara et al.13 Consistent with the change observed in ACD, the anterior chamber volume was decreased by 17% in Best2–/– mice (Table 1) . Based on Va and Fa, we calculated the turnover rate of aqueous humor by using equation 5 . Turnover in Best2+/+ and Best2–/– mice was 3.5% · min–1 and 7.4% · min–1 respectively; a 2.1-fold increase in the Best2–/– mice.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In our study, Best2–/– mice exhibited diminished IOP, despite a ~73% increase in Fa and a more than twofold increase in the rate of aqueous turnover. The increase in Fa was overcompensated for by enhanced drainage. Both Fc and Fu were increased, although a more significant portion of the aqueous flow drained via Fu than Fc in the Best2–/– mice. Based on the antagonistic effect of Best2 on Fa we conclude that Best2 is not one of the Cl– channels involved in aqueous humor formation, leaving us with the question of how Best2 antagonizes aqueous humor production.

The hypothesis that bestrophins function as CaCCs16 arose from the observation that individuals with Best vitelliform macular dystrophy (BVMD), due to mutations in BEST1, exhibit a diminished electrooculogram light peak.17 This response is generated by a Cl– conductance across the basolateral surface of the retinal pigment epithelium18 (RPE) where Best1 is normally expressed.19 Support for this idea came from heterologous expression studies showing bestrophin-specific CaCC activity that was diminished or absent in all mutant forms of Best1 tested.16 However, disruption of Best1 does not diminish the light peak in mice,11 and the diminished CaCC activity exhibited by mutant Best1 does not explain how different Best1 mutants cause four clinically distinct diseases.

A second hypothesis for bestrophin function is that this family of proteins functions as regulators of ion transport.5 6 9 10 11 We have shown that L-type voltage dependent Ca2+ channels (VDCCs) are required for the light peak,9 11 20 and that Best1 and Best1 mutants exert specific effects on the kinetics of VDCCs,9 11 a finding recently confirmed by Yu et al.,10 who also demonstrated a physical interaction of Best1 with the β3 subunit of VDCC channels. Thus, Best2 may play a role in regulating Ca2+ signaling, similar to what we have proposed for Best1.5 9 11 The NPE is in close physical contact with the pigment epithelium (PE), working in tandem to generate the aqueous flow. According to Mitchell et al.21 PE cells release ATP due to cell swelling, which results in activation of a hypothesized Ca2+ dependent negative feedback loop sending Cl– into the stroma.2 21 ATP stimulates P2Y2 receptors in PE cells to trigger Ca2+ release. In mice, disruption of Best1 results in an increase in [Ca2+]i compared with wild-type mice in response to extracellular ATP, suggesting that Best1 functions to regulate intracellular Ca2+ signaling. Although Best2 is in the NPE and not the PE, these cells communicate through an extensive network of GAP junctions.22 Perhaps, Best2 normally acts as the governor of the negative feedback loop altering Ca2+ signaling to promote aqueous resorption to control the desired rate of aqueous flow. In its absence this failure to properly regulate Ca2+ signaling could result in a diminished rate of Cl– recycling and thus enhanced aqueous production.

The increase in Fa observed in Best2–/– mice in the present study should logically have led to an increase in IOP. However, there was an overcompensating effect on the conventional outflow facility that resulted in a diminished IOP. How could this occur? We observed no obvious anatomic changes such as breaks in the inner wall of Schlemm’s canal or larger spaces between ciliary muscle bundles that would account for elevated drainage through either the conventional or uveoscleral pathways. However, we did observe TM tissues that appeared expanded, perhaps the result of enhanced outflow. Such a difference may be more obvious if eyes are fixed under pressure. If there is no dramatic developmental or structural abnormality in the angle, then the increase in Ct must have been functional and have been triggered by the increase in Fa. Others have proposed that the ciliary body communicates with the TM via the aqueous humor.23 24 25 The mechanism underlying this putative communication is unknown although bioactive peptides have been suggested as one possible mechanism.23 24 25 Changes in [Ca2+]i could have profound effects not only on ion transport properties of NPE cells, but may alter secretion of bioactive peptides,26 27 or perhaps alter the ionic composition,21 28 29 30 and/or the viscosity31 of the aqueous humor in a manner promoting enhanced outflow. Further studies are needed to test these hypotheses and to better understand the role of Best2 and how it can affect outflow.

It has been noted that there is an enormous variation in the ratio of drainage through the conventional and uveoscleral pathways in different species. In rabbits, Fu is <10% of Fa, whereas mice are at the other end of the spectrum with ~80% of drainage flowing through the uveoscleral pathway.13 31 Humans and other primates range from 4% to 60%,32 depending on age. In general, younger primates make greater use of the uveoscleral pathway than older ones. In the present study we have confirmed that mice are prolific in their use of uveoscleral drainage, with Fu constituting 83% of total outflow in our wild-type mice and 88% in the Best2–/– mice. Although Lindsey and Weinreb33 have qualitatively demonstrated that the mouse has a uveoscleral pathway, the small size of the mouse eye has stymied efforts to directly measure Fu. As a result, Fu in the mouse is calculated indirectly based on the assumption that drainage must be split between the conventional and uveoscleral pathways (see equation 4 ). The absence of Best2 causes a further increase in Fu such that it constitutes 88% of total drainage. However, this does not alter our hypothesis regarding how outflow is enhanced. Bioactive peptides, ion composition, and aqueous humor viscosity could all play roles in enhancing uveoscleral drainage.

In summary, we have shown that disruption of Best2 in mice results in a significant decrease in IOP despite a 73% increase in Fa. Therefore, Best2 is a potent antagonist of aqueous humor production. The enhanced production of aqueous humor is overcompensated for by an increase in both conventional and uveoscleral drainage. Since Best2 is expressed uniquely in the NPE and there is no obvious anatomic reason for increased outflow, these data imply a communicative link between the ciliary body and the outflow pathway that is modulated by Best2. Therefore, drugs that target Best2 function would appear to be novel candidates for the treatment of glaucoma.


   Acknowledgements
 
The authors thank Nicholas Delamere for helpful discussions.


   Footnotes
 
Supported by National Institutes of Health Grants EY13160 (ADM) and EY13847 (LYM), the Macular Vision Research Foundation (ADM), a Career Development Award (LYM), and an unrestricted grant to the Department of Ophthalmology and Vision Science at the University of Arizona from Research to Prevent Blindness.

Submitted for publication June 27, 2008; revised August 29 and October 1, 2008; accepted December 9, 2008.

Disclosure: Y. Zhang, None; B.R. Davidson, None; W.D. Stamer, None; J.K. Barton, None; L.Y. Marmorstein, None; A.D. Marmorstein, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Alan D. Marmorstein, University of Arizona, Department of Ophthalmology and Vision Science, 655 N. Alvernon Way, Suite 108, Tucson, AZ 85711; amarmorstein{at}eyes.arizona.edu.


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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