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Glaucoma  |   July 2015
Aqueous Humor Outflow Physiology in NOS3 Knockout Mice
Author Affiliations & Notes
  • Yuan Lei
    Research Centre Eye and ENT Hospital, Shanghai Medical College, Fudan University, China
    Key Laboratory of Myopia, Ministry of Health, Fudan University, China
    Shanghai Key Laboratory of Visual Impairment and Restoration, Eye and ENT Hospital, Shanghai Medical College, Fudan University, China
  • Xuejin Zhang
    Research Centre Eye and ENT Hospital, Shanghai Medical College, Fudan University, China
    Shanghai Key Laboratory of Visual Impairment and Restoration, Eye and ENT Hospital, Shanghai Medical College, Fudan University, China
  • Maomao Song
    Shanghai Key Laboratory of Visual Impairment and Restoration, Eye and ENT Hospital, Shanghai Medical College, Fudan University, China
    Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Shanghai Medical College, Fudan University, China
  • Jihong Wu
    Research Centre Eye and ENT Hospital, Shanghai Medical College, Fudan University, China
    Shanghai Key Laboratory of Visual Impairment and Restoration, Eye and ENT Hospital, Shanghai Medical College, Fudan University, China
  • Xinghuai Sun
    Key Laboratory of Myopia, Ministry of Health, Fudan University, China
    Shanghai Key Laboratory of Visual Impairment and Restoration, Eye and ENT Hospital, Shanghai Medical College, Fudan University, China
    Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Shanghai Medical College, Fudan University, China
    State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Fudan University, China
  • Correspondence: Xinghuai Sun, Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, 200031 China; xhsun@shmu.edu.cn
  • Footnotes
     YL and XZ contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science July 2015, Vol.56, 4891-4898. doi:10.1167/iovs.15-16564
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      Yuan Lei, Xuejin Zhang, Maomao Song, Jihong Wu, Xinghuai Sun; Aqueous Humor Outflow Physiology in NOS3 Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2015;56(8):4891-4898. doi: 10.1167/iovs.15-16564.

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

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Abstract

Purpose: To investigate the role of endothelial nitric oxide synthase (eNOS) on conventional outflow function using NOS3 knockout (KO) mice.

Methods: Intraocular pressure was measured in both NOS3 KO and wild type (WT) by rebound tonometry. Outflow facility was measured by perfusing enucleated mouse eyes at multiple pressure steps. A subset of eyes was sectioned and stained for histology. Mock aqueous humor ± the nitric oxide (NO) donors nitroprusside dihydrate (SNP) or S-Nitroso-N-Acetyl-D,L-Penicillamine (SNAP) was perfused into enucleated eyes. SNP and SNAP was administered topically at 0, 1, 2, and 3 hours while the contralateral eyes served as vehicle controls. Intraocular pressure was measured in both eyes before and after the last drug treatment.

Results: Intraocular pressure was higher in KO mice (18.2 ± 0.7 mm Hg vs. 13.9 ± 0.5 mm Hg, mean ± SEM, n = 30, P < 0.05), and pressure-dependent conventional drainage was significantly lower (0.0058 ± 0.0005 μL/min/mm Hg, mean ± SEM, n = 21) compared with WT mice (0.0082 ± 0.0013 μL/min/mm Hg, n = 23, P < 0.05). No obvious morphological differences in iridiocorneal angle tissues were observed in hematoxylin and eosin (H&E)-stained sections. SNP and SNAP significantly increased pressure-dependent drainage in KO animals (n = 12, P < 0.05). In WT mice, SNP and SNAP caused a significant increase in pressure dependent drainage (n = 12, P < 0.05) to a similar degree as in KO mice. Topical application of SNP significantly reduced IOP in WT and KO mice (n = 12, P < 0.05), but SNAP did not change IOP significantly (n = 19).

Conclusions: NOS3 KO mice have elevated IOP, which is likely the result of reduced pressure-dependent drainage. These findings are consistent with human data showing polymorphisms in the NOS3 gene associate with ocular hypertension and the development of glaucoma.

There are three major nitric oxide synthase (NOS) isotypes, NOS1-3 designations to neuronal nitric oxide synthase (nNOS), induced nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS [also called NOS1-3, respectively]). eNOS is an important regulator of IOP.1 In line with this observation, the risk of developing POAG was shown to be associated with NOS3 gene polymorphisms.24 Recently, it was also found that the common variations in eNOS are also involved in the pathogenesis of POAG.5 
Analysis of the aqueous humor from POAG patients showed that the production of nitric oxide (NO) was lower in glaucoma patients compared with control patients.6 NO was also lower in the plasma of POAG patient, which may lead to alteration in ocular perfusion pressure and cause retinal damage.7 Consistent with clinical findings, Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) diaphorase staining, which was an indicator for NOS activity, was less in the outflow tissues of POAG patients compared with age-matched control patients.810 
eNOS is an enzyme that converts L-arginine to L-citrulline and produces NO. Our previous work showed that eNOS overexpression resulted in lower IOP and increased conventional outflow.1 In the cardiovascular system, NO produced by eNOS is involved in the regulation of vascular tone by inhibiting smooth muscle contraction. In eyes, NO producing cells are enriched in major sites of outflow resistance, including trabecular meshwork, Schlemm's canal (SC) and collecting channels.7,8,1113 
NO donors and NO releasing drugs can increase outflow facility and reduce IOP.1417 NO can also be liberated from the conventional pathway upon pressure elevation.18 At the cellular level, NO decreases the volume of trabecular meshwork (TM)11,12 and SC cell,12,13 which correlated with increased outflow facility. It can also decrease contractility of TM cells, a process known to increase outflow facility.19 Previous studies showed that NO donating compounds significantly increased conventional outflow and lowered IOP in rabbits,14,16,20,21 pigs,22 dogs,14 monkeys,14,23 and in perfused postmortem human eyes.24,25 However, the relative contribution of eNOS versus other NOS enzymes to NO production and conventional outflow regulation is unknown. 
The aim of this study was to specifically investigate the role of eNOS on IOP and aqueous outflow function in NOS3 (the gene that encodes eNOS) gene knockout (KO) mice. 
Materials and Methods
Animals
All experiments were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. eNOS null mice B6.129P2-Nos3tm1Unc/J (NOS3 KO) were obtained from Jackson Laboratory through the Model Animal Research Center at Nanjing University. Wild-type (WT) C57BL/6Jlittermates were used as controls. Mice were bred and housed in clear cages covered loosely with air filters and containing white pine shavings for bedding. In this study the mice aged 8 to 10 weeks were used. 
Intraocular Pressure Measurements in Mice
We measured IOP in both mouse eyes using rebound tonometery (TonoLab; ICare Espoo, Finland). Intraocular pressure was measured without anesthesia at the same time of day (10–11 AM). During measurements, the mice were standing on a custom made restrainer according to ICare LAB's recommendation (in the public domain, http://www.youtube.com/watch?v=6Dx45Q5Ue3Y) with their body restrained using Velcro (3M, Shanghai, China). The pressures of both eyes were measured for three times and the average was counted as a single measurement. 
Histology
Selected eyes were fixed by immersion in 4% paraformaldehyde in PBS. The eyes were embedded in paraffin, sectioned (5 μm) and stained with hematoxylin and eosin (H&E). Sections were viewed by standard light microscopy and images were captured using a Leica imaging system (Leica, Wetzlar, Germany). 
Immunohistochemistry
Paraffin sections of the enucleated NOS3 KO and WT mouse eyes were stained for iNOS and nNOS. After deparaffinization of the sections, endogenous peroxidase activity was neutralized by incubating sections in 1% H2O2 in methanol for 20 minutes and washing with PBS. Nonspecific binding sites on cells were blocked in 1% BSA solution for 1 hour at room temperature and permeablized with 0.2% Triton-X100 for 5 minutes. Sections were then incubated with iNOS (Goat polyconal, 1:200; Abcam, Shanghai, China) and nNOS (rabbit monoclonal, 1:100; Cell Signaling Technology, Shanghai, China) IgGs overnight at 4°C. Sections were washed three times in PBS (10 minutes each) then incubated with rabbit anti-goat biotin conjugated secondary antibodies (1:1000 dilution; Abcam) in PBS for 30 minutes at room temperature. Sections were rinsed in PBS three times (10 minutes each), and incubated in streptavidin-biotin solution (1:500 dilution; Invitrogen, Shanghai, China) in PBS for 30 minutes at room temperature. After rinsing in PBS three times (5 minutes each), sections were incubated in DAB enhanced substrate system (Sigma-Aldrich, Shanghai, China) for 5 minutes, and rinsed three times again in PBS. 
Topical Drug Application
Mouse eyes were treated with sodium nitroprusside dihydrate (SNP; 4 × 2 μL drops, total dose 160 μg) and S-Nitroso-N-Acetyl-D,L-Penicillamine (SNAP; 4 × 2 μL drops, total dose 160 μg) by topical application at 0, 1, 2, and 3 hours. The drops were given 1 minute apart. For all eye drop administrations, the drug was administered to the central cornea. The contralateral eyes were treated with drug vehicle diluted in PBS. Intraocular pressure was measured in both eyes before drug treatment and 1 hour after the last drug treatment. 
Mouse Eye Perfusion
Perfusions were performed in male mice 8 to 10 weeks old at the time of experiments. After IOP measurements, the mice were euthanized by cervical dislocation. The eyes were enucleated within 5 minutes of death and were kept in Dulbecco's modified Eagle's medium (Hyclone, Shanghai, China) at room temperature until preparation for perfusion. The elapsed time from enucleation to perfusion was 1 to 4 hours. The experimental setup was developed by our laboratory and is described in detail elsewhere.26 Briefly, enucleated mouse eyes were cannulated with a 33-G beveled-tip needle (Nanofil; World Precision Instruments, Shanghai, China). The eye was kept moist with the gauze underneath the eye and drops of PBS on top of the cornea applied between perfusions. The cannulation needle was mounted on a micromanipulator, and was connected to a perfusion reservoir (25-μL syringe; Hamilton, Logan, UT, USA) via pressure tubing (inner diameter, 0.020 in, outer diameter 1/16 in; PEEK; Sigma-Aldrich, Poole, UK) and a T-junction (WPI, Shanghai, China). The outflow rate measurement method was as previously described.27 Briefly, after cannulation the eye pressure was stabilized using a perfusion reservoir containing PBS for 30 minutes, and the total infusion volume was recorded after a 10-minute interval. At each pressure, the measurement was repeated at least twice to ensure that the reading was stable. In some cases the initial measurement was repeated at the end of the experiment to ensure that the readings were consistent, but the repeat data was not included in the data analysis in the study for consistency across the experiments. The eye was perfused at four different levels of IOPs (8, 15, 22, and 30 mm Hg). To test the effect of the NO donor SNP (Sigma-Aldrich) and SNAP (Sigma-Aldrich), mouse eyes were perfused with 10−3 M SNP and 100 μM SNAP at 8, 15, 22, and 30 mm Hg. The mouse eyes were perfused with SNP or SNAP solution (in PBS) for 60 minutes before the measurements were taken to exchange the aqueous humor and ensure that the drug concentration was uniform throughout the experiment. The contralateral control eyes were treated in the same way but perfused with drug vehicle. The SNP solution was kept from light exposure as it was known to degrade when exposed to light. 
Total outflow facility (Ctotal) was calculated as  where F is total inflow rate. We assume that at equilibrium, the total inflow rate equals the total outflow rate. Conventional out flow facility (Ccon), unconventional flow rate (Fu) was calculated according Goldman's equation:    
Where Fu is the pressure-independent (unconventional) outflow rate, EVP is episcleral venous pressure, and Ccon is the conventional outflow facility. As the mouse eyes were enucleated at the time of the experiment, EVP equals zero. 
Statistics
For IOP measurement in WT and KO mice, the data was analyzed by the Mann-Whitney U test (SPSS 16 for Windows; IBM-SPSS, Chicago, IL, USA). In the experiments involving SNP or SNAP treatment, IOP and outflow data were analyzed by paired sample t-test. In all cases, differences were considered significant at P less than 0.05. 
Results
The goal of this study was to determine the impact of eNOS expression on IOP and outflow function. To accomplish our goal, we studied NOS3 KO mice as an experimental model. We first examined IOP in NOS3 KO mice and WT littermates using rebound tonometry. We found that IOP was significantly higher in the NOS3 KO mice (18.2 ± 0.7 mm Hg) compared with the WT control group (13.9 ± 0.5 mm Hg, mean ± SEM, n = 30, P < 0.05, Fig. 1A). To determine whether NOS3 KO affected the anatomic structure of the iridiocorneal angle tissues responsible for IOP generation, mouse eyes were sectioned and H&E stained. Similar to control mice, NOS3 KO mice displayed an open iridiocorneal angle plus normal gross morphology of the TM and SC inner wall endothelium (Fig. 1B). Mouse eye sagittal sections were labeled with iNOS or nNOS antibodies and visualized by immunohistochemistry. Staining with the omission of primary antibody resulted in no labeling (negative control [NC] Fig. 1C). Positive control (PC) was mouse retina which expresses iNOS and nNOS abundantly (PC; Fig. 1C). In KO and WT mice, the TM/SC tissue expressed a little iNOS or nNOS (Fig. 1C). 
Figure 1
 
Intraocular pressure and outflow tissue morphology of NOS3 KO mice. (A) Intraocular pressure measurements of NOS3 KO mice and WT controls. Intraocular pressure was significantly higher in the NOS3 KO mice (18.2 ± 0.7 mm Hg, mean ± SEM) compared with the WT control group (13.9 ± 0.5 mm Hg, P < 0.05, n = 30). (B) Hematoxylin and eosin–stained sections of mouse eye outflow tissue. Scale bar: 30 μm. (C) iNOS and nNOS staining of KO and WT mouse eye sections. NC is antibody negative control where the primary antibody was omitted, PC is positive control of mouse retina (n = 6).
Figure 1
 
Intraocular pressure and outflow tissue morphology of NOS3 KO mice. (A) Intraocular pressure measurements of NOS3 KO mice and WT controls. Intraocular pressure was significantly higher in the NOS3 KO mice (18.2 ± 0.7 mm Hg, mean ± SEM) compared with the WT control group (13.9 ± 0.5 mm Hg, P < 0.05, n = 30). (B) Hematoxylin and eosin–stained sections of mouse eye outflow tissue. Scale bar: 30 μm. (C) iNOS and nNOS staining of KO and WT mouse eye sections. NC is antibody negative control where the primary antibody was omitted, PC is positive control of mouse retina (n = 6).
Aqueous Humor Outflow in NOS3 KO Mice.
We then examined if/how NOS3 deletion impacted aqueous humor outflow profile compared with WT mice.1,26,27 The flow rate data was plotted as a function of IOP and compared the data between WT and KO mice (Fig. 2A). In WT control mice, the outflow rate was 0.1244 ± 0.008, 0.1576 ± 0.009, 0.2381 ± 0.0171, and 0.2978 ± 0.0296 μL/min at 8, 15, 22, and 30 mm Hg, respectively (mean ± SEM, n = 21). In NOS3 KO mice, the outflow rate was 0.0966 ± 0.0073, 0.1329 ± 0.0102, 0.1702 ± 0.0138, and 0.2132 ± 0.0152 μL/min at 8, 15, 22, and 30 mm Hg, respectively (n = 23). The outflow rate of KO mice was significantly lower than control at 22 and 30 mm Hg (P < 0.05). 
Figure 2
 
Relation between the mean flow rate and IOP in all perfused WT and NOS3 KO mouse eyes. Mouse eyes were perfused at sequential pressures of 8, 15, 22, and 30 mm Hg. (A) The outflow rate and pressure relationship of WT and NOS3 KO mice. The slope of the regression line (solid line) is the conventional outflow facility (Ccon). (B) The comparison of Ccon of WT and KO mice that Ccon of KO mice is significantly lower than WT mice (*P < 0.05).
Figure 2
 
Relation between the mean flow rate and IOP in all perfused WT and NOS3 KO mouse eyes. Mouse eyes were perfused at sequential pressures of 8, 15, 22, and 30 mm Hg. (A) The outflow rate and pressure relationship of WT and NOS3 KO mice. The slope of the regression line (solid line) is the conventional outflow facility (Ccon). (B) The comparison of Ccon of WT and KO mice that Ccon of KO mice is significantly lower than WT mice (*P < 0.05).
In order to determine the conventional outflow facility of the mouse eyes, a linear regression was performed on the flow rate versus pressure data. The conventional outflow facility (Ccon, which is the slope of the flow rate versus pressure graph) for the WT and KO mice was 0.0082 ± 0.0013 μL/min/mm Hg and 0.0058 ± 0.0005 μL/min/mm Hg, respectively. Ccon of KO mice was 29% lower than WT controls (P < 0.05, Fig. 2B). 
The Effect of No Donors on IOP.
Next, we investigated the effects of NO donors on the IOP in NOS3 KO and WT mice. We used two well-characterized NO donors SNP and SNAP, which were previously tested on monkey23 and mouse eyes (Chang JYH, et al. IOVS 2014;55:ARVO E-Abstract 2886). In KO mice, IOP of SNP-treated eye was significantly lower than contralateral vehicle treated eyes (mean ± SEM, 13.4 ± 0.7 vs. 18.8 ± 0.9 mm Hg, n = 19, Fig. 3A). However, NO donor SNAP did not significantly change IOP comparing the drug treated and contralateral vehicle treated eyes (16.7 ± 0.8 vs. 15.0 ± 0.8 mm Hg, n = 19, Fig. 3B). In WT mice, the IOP of the SNP-treated eyes was significantly lower than that of the contralateral vehicle-treated eyes (10.2 ± 1.1 vs. 15.2 ± 1.2 mm Hg, mean ± SEM, n = 12, Fig. 3C). In SNAP-treated eyes, the IOP was not significantly different from vehicle-treated eyes (14.2 ± 0.9 vs. 12.3 ± 0.7 mm Hg, mean ± SEM, n = 12, Fig. 3D). 
Figure 3
 
Intraocular pressure of NOS3 KO (A, B) and WT (C, D) mice treated with topical application of NO donors SNP and SNAP. SNP significantly reduced IOP from compared with vehicle treated eyes in both KO (A) and WT mice (C), however SNAP did not significantly affect IOP (B, D).
Figure 3
 
Intraocular pressure of NOS3 KO (A, B) and WT (C, D) mice treated with topical application of NO donors SNP and SNAP. SNP significantly reduced IOP from compared with vehicle treated eyes in both KO (A) and WT mice (C), however SNAP did not significantly affect IOP (B, D).
The Effect of NO Donors on Aqueous Outflow in NOS3 KO Mouse.
Following IOP measurements, we further investigated the effects of NO donors on aqueous humor outflow rate and facility. With the NO donor SNP, the outflow rates at 8, 15, 22, and 30 mm Hg were 0.0746 ± 0.0063, 0.1952 ± 0.0181, 0.2675 ± 0.0323, and 0.4086 ± 0.0346 μL/min, respectively. SNP-treated eyes showed a linear relationship in the flow-pressure graphs (Fig. 4A). At 8 mm Hg, SNP did not significantly change the outflow rate in NOS3 KO mouse eyes compared with PBS controls (n = 10). However at higher pressures of 15, 22, and 30 mm Hg, the outflow rate in SNP-treated eyes was significantly greater by 47%, 58%, 121%, respectively. The conventional outflow facility in SNP-treated and control eyes was 0.0147 ± 0.0014 and 0.0062 ± 0.0009 μL/min/mm Hg (mean ± SEM, n = 12). Data displayed in a box and whisker plot shows that SNP increased Ccon by 2.4-fold (Fig. 4B). 
Figure 4
 
NOS3 knockout eyes perfused with NO donor SNP and SNAP. (A) Comparison of flow rate in SNP-treated eyes and paired control eyes perfused with the drug vehicle (PBS). The outflow rate was significantly higher in SNP perfused eyes (n = 10, *P < 0.05, error bars are mean ± SEM.). (B) Conventional outflow facility was significantly higher in SNP treated NOS3 KO mice compared with vehicle controls (*P < 0.05, n = 12). Similarly, (C, D) show outflow rate data and conventional outflow facility comparison for SNAP. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values.
Figure 4
 
NOS3 knockout eyes perfused with NO donor SNP and SNAP. (A) Comparison of flow rate in SNP-treated eyes and paired control eyes perfused with the drug vehicle (PBS). The outflow rate was significantly higher in SNP perfused eyes (n = 10, *P < 0.05, error bars are mean ± SEM.). (B) Conventional outflow facility was significantly higher in SNP treated NOS3 KO mice compared with vehicle controls (*P < 0.05, n = 12). Similarly, (C, D) show outflow rate data and conventional outflow facility comparison for SNAP. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values.
Experiments with a second NO donor, SNAP were performed. One eye was perfused with SNAP and the contralateral eye served as a control and was perfused with the drug vehicle. The outflow data of each pair of eyes is illustrated in Figure 4C. The flow of SNAP-treated eyes was significantly higher than the paired control eyes at each pressure level (n = 6, Fig. 4C). Ccon was 3.4-fold higher in SNAP-treated eyes (0.0223 ± 0.0044 μL/min/mm Hg) compared with paired control (0.0067 ± 0.0010 μL/min/mm Hg, P < 0.05, Fig. 4D). 
The Effect of NO Donors on Aqueous Outflow on WT Mouse.
In SNP-treated eyes, the outflow rates at 8, 15, 22, and 30 mm Hg were 0.0954 ± 0.0212, 0.2645 ± 0.0551, 0.3750 ± 0.0604, and 0.6313 ± 0.1761 μL/min, respectively (mean ± SEM, Fig. 5A). In the paired control eyes, the outflow rates at the above pressures were 0.0563 ± 0.0065, 0.1080 ± 0.0161, 0.1690 ± 0.1231, 0.1844 ± 0.0543 μL/min/mm Hg, respectively. Ccon in SNP-treated and control eyes was 0.0190 ± 0.0022 and 0.0075 ± 0.0023 μL/min/mm Hg. SNP increased Ccon by 2.5 fold (P < 0.05, n = 6, Fig. 5B). 
Figure 5
 
Wild type mouse eyes perfused with NO donor SNP and SNAP. (A) Compares flow rate in SNP-treated eyes and paired control eyes perfused with the drug vehicle. (B) Box plot showed that the conventional outflow was significantly higher in SNP-treated eyes compared with paired controls (*P < 0.05, n = 12). Similar, (C, D) shows outflow rate data and conventional outflow facility comparison for SNAP. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values.
Figure 5
 
Wild type mouse eyes perfused with NO donor SNP and SNAP. (A) Compares flow rate in SNP-treated eyes and paired control eyes perfused with the drug vehicle. (B) Box plot showed that the conventional outflow was significantly higher in SNP-treated eyes compared with paired controls (*P < 0.05, n = 12). Similar, (C, D) shows outflow rate data and conventional outflow facility comparison for SNAP. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values.
In SNAP-treated eyes, the outflow rates at 8, 15, 22, and 30 mm Hg were 0.1624 ± 0.0324, 0.3875 ± 0.0919, 0.5601 ± 0.1501, and 0.6723 ± 0.1013 μL/min (Fig. 5C). In paired control eyes, the outflow rate at the above pressures was 0.0563 ± 0.0065, 0.1082 ± 0.1556, 0.1460 ± 0.4189, and 0.2455 ± 0.0650 μL/min/mm Hg. SNAP increased Ccon from 0.0070 ± 0.0006 to 0.0201 ± 0.0024 μL/min/mm Hg by 2.9-fold (P < 0.05, n = 6, Fig. 5D). 
Discussion
The main finding of this study was the following: firstly, NOS3 knockout resulted in elevation of IOP and corresponding decrease in conventional pressure dependent outflow in the mouse eyes. This suggest that eNOS plays an important role in regulating IOP. Secondly, NO donor SNP resulted in reduction of IOP by topical application suggesting that the NO produced could act on guanylate cyclase, bypassing NOS. So in effect the addition of SNP puts the IOP of KO mice almost back to levels seen in WT eyes. Thirdly, the outflow rate was higher in WT than in KO mice, suggesting that in the absence of eNOS, pressure still has the ability to increase flow rate. Fourthly, both SNP and SNAP increased conventional pressure dependent outflow facility in KO and WT mice when perfused into the anterior chamber. 
Intraocular pressure was higher and pressure-dependent outflow in NOS3 KO mouse was lower than WT control (Figs. 1, 2). It is consistent with results in WT mice perfused with NOS inhibitor L-NAME, which reduced Ccon by 36% and the eNOS inhibitor cavtratin reduced the Ccon in WT mice by 24%.28 The authors also showed that eNOS is only expressed by SC and that cavtratin an eNOS inhibitor knocks down outflow facility equivalent to L-NAME. This suggests that eNOS protein is an important regulator of conventional aqueous humor outflow. This is consistent with findings using eNOS selective immunocytochemistry which showed that eNOS is the primary isozyme expressed in both SC cells and ciliary muscle fibers, which by ciliary tendon contraction and relaxing regulate the opening and compressing of TM/SC and aqueous humor outflow.8,28 We previously demonstrated in a strain of eNOS overexpressed transgenic mice that an approximately 2-fold overexpression of eNOS resulted in 57% increase in conventional outflow facility.1 eNOS is present in the particulate and cytosolic fractions of the endothelial cells.29 It binds to caveolin-1 (CAV1) in the microdomains of the plasma membrane called caveolae. eNOS activity is inhibited by interaction with CAV1, which is regulated by Ca2+/calmodulin.30 In the eye, shear stress generated by aqueous humor outflow could either stimulate SC endothelial cells to release NO,31 or induce phosphorylation of Ser1177/1179 of eNOS through phosphatidylinositol-3 (PI3) kinase and the downstream serine/threonine protein kinase Akt (protein kinase B), resulting in enhanced NO synthesis without any increase in intracellular Ca2+.3234 However, with eNOS KO its regulatory role is significantly reduced as shown here in IOP and outflow facility (Figs. 1, 2). Our immunostaining results (Fig. 1) showed that in the eNOS KO mice there is still some expression of iNOS and nNOS in the TM and SC. Immunoreactivity against nNOS antibody in the epidermis showed that although eNOS was absent nNOS was still present in the tissue, and nNOS may play an compensatory role for eNOS in the brain35 and vasculature.36 Consistent with current literature findings in human donor eyes,24 our data confirmed that NO-donor SNP and SNAP induced a greater pressure-dependent drainage in both WT and KO mouse eyes compared with vehicle-treated eyes (Figs. 4, 5). In vivo study in monkey showed that NO donor SNP caused 77% increase in outflow facility.23 In mouse eyes, the increase was greater (2.5- and 2.4-fold in WT and KO mice, respectively). The smaller magnitude of increase in conventional outflow in monkey might be due to difference in drug concentration, species, and dosing method. In the monkey study, SNP was given topically as an eye drop to the central cornea, which was almost equivalent to a drug concentration of 10−3 M assuming 1% cornea penetration. We perfused mouse eyes using the same concentration as that of the monkey study of 10−3 M, and with this drug concentration, outflow rate increased significantly at pressures greater than 15 mm Hg but there was little affect at 8 mm Hg. NO has a short half-life in the circulation of approximately 2 minutes. It is rapidly distributed to the extracellular space and is cleared from intraerythrocytic reaction with hemoglobin. However, the aqueous humor is free of hemoglobin, and the half-life of SNP would be considerably longer. From the flow readings, it had no signs of degradation or metabolism during the experiment. The flow rate was measured three times at each pressure level for 10 minutes each time and the readings were generally consistent. The effect of NO donor SNP and SNAP on IOP is variable between species and studies. In our study, topical application of SNP reduced IOP, whereas SNAP did not have a significant IOP-lowering effect. This is consistent with findings in a monkey study.23 Kotikoski et al.37 showed that SNP lowered IOP in normotensive rabbits after either topical or intravitreal dosing, and the maximal IOP reduction was between 2 and 5 hours. However, others found that SNP had a tendency to increase IOP in anesthetized rabbit.38 Intravitreal or intracameral injection of the NO donors SNAP induced a marked IOP-lowering effect.20,21 Together with our results of SNAP increasing outflow in perfused eyes, it suggest that dosing method is important for SNAP and topical application might not give it enough corneal penetration to induce IOP changes. 
NO donors seemed to increase conventional outflow targeting both TM and SC cells. Although both TM39 and SC cells13 are responsive to NO, recent evidence seemed to point to a primary role of SC cells as the cellular targets for NO in IOP lowering. Ashpole et al.31 showed that shear stress of 10 dynes/cm2 increased NO levels by eightfold after 7 days of perfusion. This is comparable with human umbilical vein endothelial cells, and greater than TM cells, which appeared shear-insensitive. 
It was interesting that NOS3 KO mice were still able to respond to NO donors in regulating aqueous humor flow (Fig. 4). NO mediated several biological actions such as vasodilation, apoptosis, vascular tone, and inflammatory responses.40,41 The vasodilation action of NO is mediated through stimulation of soluble guanylate cyclase (sGC), which leads to elevation of intracellular cGMP levels. cGMP then interacts with various cyclic-nucleotide–gated channels, protein kinases, and protein phosphodiesterases. NO-cGMP signaling pathway is involved in homeostatic processes in the eye in regulating aqueous humor outflow. Topical application of cyclic GMP analog, 2′-O-(4-benzoyl)benzoylguanosine 3′,5′ cyclic monophosphate (BB-cGMP),42 significantly reduces IOP in normotensive and hypertensive eyes of rabbits. And 8-Br-cGMP increased aqueous humor outflow in normotensive rabbit eyes.16 Our data imply that although NOS3 was knocked out, the downstream pathways were still intact and functional. 
Preclinical data showed that NO-donating drugs had superior IOP-lowering effect than the equal molar concentration conventional compounds (without NO releasing). It includes three NO-donating analogs of prostaglandins or prostamides and a β-adrenoceptor blocker. The prostaglandin analogs or prostamides are NCX 139, a NO donating prostamide; NCX 125 and LBN (NCX 116), two NO donating prostaglandin F2 alpha analogs. Their IOP lowering has been demonstrated in multiple animal species. LBN, NCX 125, and NCX 139 were effective in decreasing IOP in a transiently hypertensive rabbit model, glaucoma dog model, and in laser-induced hypertensive nonhuman primates, while equimolar doses of the corresponding prostamide or prostaglandin alone were ineffective.14,17,43 Nipradilol, a β-adrenoceptor blocker with a NO donating nitroxy group, has the IOP-lowering ability in both normotensive and hypertensive rabbits, while latanoprost was without significant effect.42 
In summary, this study mechanistically confirmed the role of eNOS in regulating IOP, which is an important risk factor for glaucoma progression. The lack of eNOS expression resulted in a reduction in conventional outflow facility and a corresponding increase in IOP. 
Acknowledgments
The authors thank Shi Jufang and Xing Chao for their excellent assistant with the animal work. We are very grateful for the insightful comments of W. D. Stamer, PhD, on the manuscript. 
Supported by grants from National Science Foundation China (81100662, 81371015; Beijing, China), Shanghai Municipal Health Bureau Young Outstanding Scientist Program (XYQ2013083; Shanghai, China), 211 Project of Fudan University (EHF158351; Fudan, China), Scientific Research Foundation for Chinese Scholars returned from overseas (State Education Ministry, Beijing, China). 
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Figure 1
 
Intraocular pressure and outflow tissue morphology of NOS3 KO mice. (A) Intraocular pressure measurements of NOS3 KO mice and WT controls. Intraocular pressure was significantly higher in the NOS3 KO mice (18.2 ± 0.7 mm Hg, mean ± SEM) compared with the WT control group (13.9 ± 0.5 mm Hg, P < 0.05, n = 30). (B) Hematoxylin and eosin–stained sections of mouse eye outflow tissue. Scale bar: 30 μm. (C) iNOS and nNOS staining of KO and WT mouse eye sections. NC is antibody negative control where the primary antibody was omitted, PC is positive control of mouse retina (n = 6).
Figure 1
 
Intraocular pressure and outflow tissue morphology of NOS3 KO mice. (A) Intraocular pressure measurements of NOS3 KO mice and WT controls. Intraocular pressure was significantly higher in the NOS3 KO mice (18.2 ± 0.7 mm Hg, mean ± SEM) compared with the WT control group (13.9 ± 0.5 mm Hg, P < 0.05, n = 30). (B) Hematoxylin and eosin–stained sections of mouse eye outflow tissue. Scale bar: 30 μm. (C) iNOS and nNOS staining of KO and WT mouse eye sections. NC is antibody negative control where the primary antibody was omitted, PC is positive control of mouse retina (n = 6).
Figure 2
 
Relation between the mean flow rate and IOP in all perfused WT and NOS3 KO mouse eyes. Mouse eyes were perfused at sequential pressures of 8, 15, 22, and 30 mm Hg. (A) The outflow rate and pressure relationship of WT and NOS3 KO mice. The slope of the regression line (solid line) is the conventional outflow facility (Ccon). (B) The comparison of Ccon of WT and KO mice that Ccon of KO mice is significantly lower than WT mice (*P < 0.05).
Figure 2
 
Relation between the mean flow rate and IOP in all perfused WT and NOS3 KO mouse eyes. Mouse eyes were perfused at sequential pressures of 8, 15, 22, and 30 mm Hg. (A) The outflow rate and pressure relationship of WT and NOS3 KO mice. The slope of the regression line (solid line) is the conventional outflow facility (Ccon). (B) The comparison of Ccon of WT and KO mice that Ccon of KO mice is significantly lower than WT mice (*P < 0.05).
Figure 3
 
Intraocular pressure of NOS3 KO (A, B) and WT (C, D) mice treated with topical application of NO donors SNP and SNAP. SNP significantly reduced IOP from compared with vehicle treated eyes in both KO (A) and WT mice (C), however SNAP did not significantly affect IOP (B, D).
Figure 3
 
Intraocular pressure of NOS3 KO (A, B) and WT (C, D) mice treated with topical application of NO donors SNP and SNAP. SNP significantly reduced IOP from compared with vehicle treated eyes in both KO (A) and WT mice (C), however SNAP did not significantly affect IOP (B, D).
Figure 4
 
NOS3 knockout eyes perfused with NO donor SNP and SNAP. (A) Comparison of flow rate in SNP-treated eyes and paired control eyes perfused with the drug vehicle (PBS). The outflow rate was significantly higher in SNP perfused eyes (n = 10, *P < 0.05, error bars are mean ± SEM.). (B) Conventional outflow facility was significantly higher in SNP treated NOS3 KO mice compared with vehicle controls (*P < 0.05, n = 12). Similarly, (C, D) show outflow rate data and conventional outflow facility comparison for SNAP. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values.
Figure 4
 
NOS3 knockout eyes perfused with NO donor SNP and SNAP. (A) Comparison of flow rate in SNP-treated eyes and paired control eyes perfused with the drug vehicle (PBS). The outflow rate was significantly higher in SNP perfused eyes (n = 10, *P < 0.05, error bars are mean ± SEM.). (B) Conventional outflow facility was significantly higher in SNP treated NOS3 KO mice compared with vehicle controls (*P < 0.05, n = 12). Similarly, (C, D) show outflow rate data and conventional outflow facility comparison for SNAP. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values.
Figure 5
 
Wild type mouse eyes perfused with NO donor SNP and SNAP. (A) Compares flow rate in SNP-treated eyes and paired control eyes perfused with the drug vehicle. (B) Box plot showed that the conventional outflow was significantly higher in SNP-treated eyes compared with paired controls (*P < 0.05, n = 12). Similar, (C, D) shows outflow rate data and conventional outflow facility comparison for SNAP. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values.
Figure 5
 
Wild type mouse eyes perfused with NO donor SNP and SNAP. (A) Compares flow rate in SNP-treated eyes and paired control eyes perfused with the drug vehicle. (B) Box plot showed that the conventional outflow was significantly higher in SNP-treated eyes compared with paired controls (*P < 0.05, n = 12). Similar, (C, D) shows outflow rate data and conventional outflow facility comparison for SNAP. In the box plots, the bottom and top of the box is the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers are the minimum and maximum values.
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