September 2015
Volume 56, Issue 10
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Physiology and Pharmacology  |   September 2015
Strain and Age Effects on Aqueous Humor Dynamics in the Mouse
Author Affiliations & Notes
  • J. Cameron Millar
    North Texas Eye Research Institute (NTERI), University of North Texas Health Science Center, Fort Worth, Texas, United States
    Department of Cell Biology & Immunology, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Tien N. Phan
    North Texas Eye Research Institute (NTERI), University of North Texas Health Science Center, Fort Worth, Texas, United States
    Department of Cell Biology & Immunology, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Iok-Hou Pang
    North Texas Eye Research Institute (NTERI), University of North Texas Health Science Center, Fort Worth, Texas, United States
    Department of Cell Biology & Immunology, University of North Texas Health Science Center, Fort Worth, Texas, United States
    Department of Pharmaceutical Sciences, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Abbot F. Clark
    North Texas Eye Research Institute (NTERI), University of North Texas Health Science Center, Fort Worth, Texas, United States
    Department of Cell Biology & Immunology, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Correspondence: J. Cameron Millar, North Texas Eye Research Institute and Department of Cell Biology & Immunology, UNTHSC, Fort Worth, TX 76107, USA; Cameron.Millar@unthsc.edu
Investigative Ophthalmology & Visual Science September 2015, Vol.56, 5764-5776. doi:10.1167/iovs.15-16720
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      J. Cameron Millar, Tien N. Phan, Iok-Hou Pang, Abbot F. Clark; Strain and Age Effects on Aqueous Humor Dynamics in the Mouse. Invest. Ophthalmol. Vis. Sci. 2015;56(10):5764-5776. doi: 10.1167/iovs.15-16720.

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

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Abstract

Purpose: We evaluated differences in aqueous humor dynamics (AHD) among several mouse strains within younger and older age groups.

Methods: Albino (A/J, BALB/cJ) and pigmented (C3H/HeJ, C57-BL/6J) mice (young [2½–4½ months] and aged [10–12 months]) were studied. Intraocular pressure (IOP) was measured. In cannulated eyes, episcleral venous pressure (Pe) was assessed (blood reflux). Other AHD parameters (outflow facility [C], aqueous humor formation rate [Fin]) were assessed (constant flow infusion). Uveoscleral outflow rate (Fu) was obtained by calculation (Fu(calc)) using the modified Goldmann equation, and in additional eyes (for comparison), by FITC-dextran perfusion (Fu(FITC-dex)).

Results: Intraocular pressure was higher in pigmented strains, but did not exhibit age-dependence, except in the C57-BL/6J strain. Fu(calc) decreased with age in BALB/cJ (↓83.3%), C3H/HeJ (↓78.0%), and C57-BL/6J (↓85.0%) strains. In the A/J strain, Fu(calc) decreased with age (↓70.0%), but not significantly. Fin decreased with age in the C3H/HeJ (↓53.6%) strain. In C57-BL/6J and A/J strains, Fin decreased with age, but not significantly. C in the BALB/cJ strain increased with age (↑62.5%). In C3H/HeJ and C57-BL/6J strains, C increased with age, but not significantly. Episcleral venous pressure ranged from 6.0 to 6.6 mm Hg (albino strains) to 8.5 to 8.9 mm Hg (pigmented strains). Pe was not age dependent, but was higher in pigmented animals.

Conclusions: In mouse, Fu and Fin diminish with age. C tends to increase as animals progress to middle life. There are strain differences in Fu, IOP, C, Fin, and Pe. The current findings provide an important foundation for comparisons among different strains in different study reports.

In recent years, mice have been used extensively as an experimental model for POAG.120 The anterior segment of the mouse eye bears many similarities to the human eye in terms of structural anatomy, physiology, anterior segment blood supply and innervation, and pharmacologic receptor profile.1,21 In addition, the mouse eye conventional aqueous humor drainage pathway resembles that of the human and nonhuman primate (NHP) eye in that it possesses a trabecular meshwork with three distinct histologic layers (uveal, corneoscleral, and juxtacanalicular regions), although there are only three to four trabecular lamellae present in the mouse eye,22 compared with approximately 20 in the human.23 A true continuous Canal of Schlemm with an inner wall endothelium is also present, unlike the discontinuous anterior aqueous venous plexus found in most mammalian eyes. In addition, in common with human eyes but unlike the eyes of most other species, mouse eyes do not exhibit ‘washout' (defined as an increase in outflow facility with time following extended perfusion).24 Mouse eyes also resemble human and NHP eyes with their net of three-dimensional elastic structures that tether together the trabecular meshwork and inner wall of Schlemm's canal via the tendinous insertion of the ciliary muscle (CM).25 
Despite their small size, mice are amenable to studies of aqueous humor dynamics (AHD), and such studies have been undertaken in on-going attempts to further characterize the mouse as a suitable animal model for POAG. Aqueous humor dynamics parameters in both living mice and mouse eyes perfused ex vivo have been reported by several laboratories in various strains with various values2,57,21,2630 (Toris CB, et al. IOVS 2007;48:ARVO E-Abstract 39; results summarized in Table 1). Thus far there is no clear consensus. 
Table 1
 
Summary of Previously Published AHD Parameters in Mouse Eyes (Live and Ex Vivo Perfused)
Table 1
 
Summary of Previously Published AHD Parameters in Mouse Eyes (Live and Ex Vivo Perfused)
In a further study in mouse eyes obtained from BALB/cJ, C57-BL/6J, and CBA/J strains perfused ex vivo,31 it was reported that there are strain differences in aqueous outflow facility (C). C was lowest in eyes obtained from CBA/J animals (0.0113 ± 0.0031 μL/min/mm Hg), intermediate in eyes obtained from C57-BL/6J animals (0.0147 ± 0.0029 μL/min/mm Hg), and highest in eyes obtained from BALB/cJ animals (0.0164 ± 0.0059 μL/min/mm Hg). Interestingly, conscious IOP measurements obtained in these animals prior to euthanasia were: CBA/J (14.5 ± 0.9 mm Hg), C57-BL/6J (12.3 ± 1.0 mm Hg), and BALB/cJ (10.6 ± 1.8 mm Hg). Thus, the CBA/J animals with the highest conscious IOP had the lowest value for C, and the BALB/cJ animals with the lowest conscious IOP had the highest value for C. The C57-BL/6J animals were intermediate in both respects. 
We reasoned that such strain differences may also exist in the living mouse eye, and we hypothesized that strain differences may exist for other parameters of AHD, in addition to C. We further reasoned that age may also play a role. For example, in the human and NHP eye, advancing age is reported to correlate with decreases in aqueous humor formation rate and uveoscleral outflow.3235 
Recently, we reported a novel continuous flow infusion method for assessing all parameters of AHD in a single mouse in a single experimental session.7 We sought to use this methodology to investigate the effect of age and strain on AHD in this species. To our knowledge this is the first study of the effects of these factors on the complete profile of AHD in the living mouse. 
Materials and Methods
Animals
Female A/J, BALB/cJ, C3H/HeJ, and C57-BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA) were used. The animals were divided into two cohorts (young and aged). The young cohort consisted of animals aged between 2½ to 4½ months, weighing from 21 to 28 g. The aged cohort consisted of animals aged between 10 to 12 months, weighing from 28 to 36 g. Animals were fed standard chow and kept in 12-hour light/dark conditions (lights on at 6 AM). 
All experimental procedures were conducted in accordance with and adherence to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the University of North Texas Health Science Center (UNTHSC; Fort Worth, TX, USA) Institutional Animal Care and Use Committee (IACUC) Regulations and Guidelines. 
IOP Measurement
Baseline (BL) IOP was determined in behaviorally trained conscious and also anesthetized animals (30 minutes following induction of anesthesia) using a TonoLab rebound tonometer (Colonial Medical Supply, Franconia, NH, USA), following our previously published methodology.36 All IOP measurements were performed in both eyes and took place between 10 AM and 11:30 AM. 
Aqueous Humor Dynamics (AHD)
All parameters of AHD were established in living mice by constant flow infusion following our previously published methodology.7 Briefly, immediately following bilateral tonometry in anesthetized animals, both eyes received a drop of proparacaine HCl (0.5%) for topical anesthesia, and both anterior chambers were cannulated with a 30-G needle (one per eye) connected to previously calibrated (sphygmomanometer; Diagnostix 700, Hauppage, NY, USA) BLPR-2 flow-through pressure transducers (World Precision Instruments [WPI], Sarasota, FL, USA) for the continuous determination of pressure. A drop of PBS was also given to each eye topical ocular to prevent corneal drying. The opposing end of each transducer was connected to a 3-way valve, which in turn was connected to: (1) a 1-mL syringe filled with sterile PBS (filtered through a 0.2-μm HT Tuffryn Membrane Acrodisc Syringe Filter; PALL Gelman Laboratory, Port Washington, NY, USA) loaded into an SP101i microdialysis infusion pump (WPI); and (2), an open-ended, variable height manometer. Signals from the pressure transducers were passed via a TBM4M Bridge Amplifier (gain setting: 1000×; WPI) and a Lab-Trax analog-to-digital converter (WPI) to a computer. Data were recorded using LabScribe2 software (WPI). 
Aqueous Outflow Facility (C)
The manometer was closed to the circuit and eyes were infused at a flow rate of 0.1 μL/min. When pressure had stabilized (typically within 10–30 minutes, but occasionally after 50–60 minutes), pressure measurements were recorded, and flow rate was increased sequentially to 0.2, 0.3, 0.4, and 0.5 μL/min. Three stabilized pressures (spaced 5 minutes apart) at each flow rate were recorded. C in each eye of each animal was calculated as the reciprocal of the slope of a plot of mean stabilized pressure as ordinate against flow rate as abscissa. 
Episcleral Venous Pressure (Pe)
Pe was estimated using the blood reflux method as reported by Aihara et al.37 Briefly, following anterior chamber cannulation, the manometer was opened to the circuit, manometric pressure was set to equal precannulation (anesthetized) IOP, and then manometric pressure was lowered incrementally (at the rate of 1 mm Hg/min) until the point at which blood was seen (using a dissection microscope under ×30 magnification) to reflux into the scleral collector channels and then Schlemm's canal. The manometric pressure at which Schlemm's canal was seen to fill with refluxed blood was regarded as Pe. 
Calculated Uveoscleral Outflow Rate (Fu(calc))
Animals were euthanized by anesthetic overdose and, 20 minutes following euthanasia, C was measured again. Thus, values for Clive and Cdead were obtained. Following euthanasia, both aqueous humor formation rate (Fin) and Pe are equal to zero, and via algebraic rearrangement of the modified Goldmann equation {IOP = [(Fin − Fu)/C] + Pe}, the following expression was derived:  where Fp is Flow Rate set by perfusion pump and IOPp is stabilized pressure developed at Fp.  
Values for Fu(calc) were thus calculated for each individual Fp and corresponding IOPp. The mean of those resultant five values was reported. 
Direct Measure of Uveoscleral Outflow Rate Via Perfusion of FITC-Dextran (Fu(FITC-dex))
In separate cohorts of animals, studied for the purpose of comparison of Fu(calc) with Fu as measured by perfusion of FITC-Dextran (Fu(FITC-dex)), the anterior chamber was cannulated using a 30-G steel needle following anesthesia. The eyes were infused at a flow rate of 0.5 μL/min for 10 minutes with FITC-dextran (10−4 M; 7 μg/μL; Sigma-Aldrich Corp., St. Louis, MO, USA) dissolved in sterile PBS. Following 10 minutes of perfusion with FITC-dextran, animals were euthanized by exposure to 100% CO2. Eyes were then enucleated and promptly dissected to isolate the retina/choroid/iris–ciliary body/scleral shell, from which the cornea/trabecular meshwork, lens, and vitreous had been removed. Each retina/choroid/iris–ciliary body/scleral shell was placed in a 1.5-mL tube with 150 μL PBS and homogenized for 3 minutes with a Hand-Held Homogenizer (VWR catalog #47747-370; VWR, Radnor, PA, USA). Homogenates were then centrifuged at 2576g for 5 minutes using a Mini Centrifuge (catalog #05-090-100; Fisher Scientific, Pittsburgh, PA, USA). From each tube, 100 μL supernatant was decanted and placed in a cuvette (UV-Cuvette micro, 70 μL minimum volume, center height 8.5 mm; BRAND GMBH, Wertheim, Germany). Fluorescence was read using a QuantiFluor Portable Fluorometer (Promega BioSytems, Sunnyvale, CA, USA). Concentration of FITC-dextran recovered from each specimen was calculated via a standard curve. Based on the concentration of FITC-dextran in the PBS perfusate (7 μg/μL), and perfusion time (10 minutes), Fu(FITC-dex) was calculated using the following equation:  where the Conc. FITC-Dextran Recovered (μg/mL) is obtained from mean of three blank-corrected fluorescence values in combination with a standard curve; the eye tissues were suspended in 150 μL PBS; and the concentration of FITC-Dextran perfused through anterior chamber in PBS was 7000 ng/μL or 7 μg/μL. T is time of perfusion (10 minutes).  
Computation of Aqueous Humor Formation Rate (Fin)
By further algebraic rearrangement of the modified Goldmann equation, the following expression was derived:    
A value for Fin was calculated for each eye. Intraocular pressure as determined in anesthetized mice prior to initial anterior chamber cannulation was used in this case. 
Anesthesia
All anesthesia was achieved by intraperitoneal injection of a cocktail (dissolved in sterile PBS) of ketamine (100 mg/kg) and xylzine (10 mg/kg), given in a volume of 10 mL/kg (for induction). Maintenance doses (½ × induction dose) were given subsequently as required. A suitable plane of anesthesia was achieved when the animal failed to react to a toe-pinch with a flexor withdrawal response, failed to flinch upon receiving a tail-pinch, and failed to blink upon light touching of the cornea with the tip of a Merocel Spear/Sponge (Beaver Visitec International, Waltham, MA, USA). 
Statistical Analysis
Both eyes of all mice were perfused. Each individual eye was considered as N = 1 eye for statistical purposes. Young versus aged data within each strain were analyzed using the two-tailed unpaired Student's t-test. Multiple comparisons across and within strains were performed using two-way ANOVA followed by individual pairwise comparisons via Holm-Sidak post hoc analysis. P values of less than 0.05 were considered significant. All data are presented as mean ± SEM. 
Results
Conscious IOP
When comparing conscious IOP between young and aged animals within each strain, there was no significant difference, except in the C57-BL/6J animals, which showed a modest elevation as they aged (13.9 ± 0.2 vs. 15.4 ± 0.2 mm Hg, mean Δ = 1.5 mm Hg, n = 7, P < 0.001; Table 2). 
Table 2
 
Conscious IOP, Anesthetized IOP, and Pe in Four Mouse Strains (Young and Aged)
Table 2
 
Conscious IOP, Anesthetized IOP, and Pe in Four Mouse Strains (Young and Aged)
Conscious IOP varied significantly amongst both the younger and aged animals when comparing across strains (Supplementary Table S1). 
Anesthetized IOP
When comparing anesthetized IOP between young and aged animals within each strain, there was no significant difference, except in the C57-BL/6J animals, which showed a modest elevation as they aged (10.9 ± 0.1 vs. 11.3 ± 0.2 mm Hg, mean Δ = 0.48 mm Hg, n = 7, P < 0.05; Table 2). 
Anesthetized IOP varied significantly amongst both the younger and aged animals when comparing across strains (Supplementary Table S2). 
Episcleral Venous Pressure (Pe)
When comparing Pe between young and aged animals within each strain, no significant differences were found (Table 2). 
Pe varied significantly amongst both the younger and aged animals when comparing across strains (Supplementary Table S3). 
Calculated Uveoscleral Outflow (Fu(calc))
Fu as calculated using the modified Goldmann equation (Fu(calc)) was found to decrease with age in the BALB/cJ strain (0.066 ± 0.022 μL/min vs. 0.011 ± 0.007 μL/min, mean Δ = 0.055 μL/min [mean decrease of 83.3%], P < 0.01, n = 5–6), in the C3H/HeJ strain (0.109 ± 0.028 μL/min vs. 0.024 ± 0.009 μL/min, mean Δ = 0.085 μL/min [mean decrease of 78.0%], P < 0.05, n = 6), and in the C57-BL/6J strain (0.080 ± 0.022 μL/min vs. 0.012 ± 0.008 μL/min, mean Δ = 0.068 μL/min [mean decrease of 85.0%], P < 0.05, n = 7). In the A/J strain Fu(calc) decreased with age (0.12 ± 0.048 μL/min vs. 0.036 ± 0.031 μL/min, mean Δ = 0.084 μL/min [mean decrease of 70.0%], but the decrease was not statistically significant, P = 0.15; Fig. 1). 
Figure 1
 
Calculated uveoscleral outflow (Fu(calc)) versus age in each of four mouse strains. There was a substantial decrease in Fu(calc) in young (2½–4 months) versus aged (10–11 months) animals, which was statistically significant (unpaired t-test) in all cases except in the A/J strain (P = 0.15).
Figure 1
 
Calculated uveoscleral outflow (Fu(calc)) versus age in each of four mouse strains. There was a substantial decrease in Fu(calc) in young (2½–4 months) versus aged (10–11 months) animals, which was statistically significant (unpaired t-test) in all cases except in the A/J strain (P = 0.15).
Computed Aqueous Flow Rate (Fin)
Fin was found to significantly decrease with age in the C3H/HeJ strain (0.140 ± 0.027 vs. 0.065 ± 0.006 μL/min, mean Δ = 0.075 μL/min [mean decrease of 54%], P < 0.05). In the C57-BL/6J strain Fin tended to decrease with age but the decrease did not reach statistical significance (0.122 ± 0.021 vs. 0.069 ± 0.013 μL/min, mean Δ = 0.053 μL/min [mean decrease of 43%], P = 0.056). In the A/J strain Fin also tended to decrease with age but the decrease did not reach statistical significance (0.202 ± 0.035 vs. 0.116 ± 0.029 μL/min, mean Δ = 0.086 μL/min [mean decrease of 43%], P = 0.094). In the BALB/cJ strain Fin did not change with age (0.155 ± 0.018 vs. 0.157 ± 0.016 μL/min, P = 0.94; Fig. 2). 
Figure 2
 
Computed aqueous flow rate (Fin) versus age in each of four mouse strains. There was a significant decrease (unpaired t-test) in Fin in young (2½–4 months) versus aged (10–11 months) C3H/HeJ animals. Mean Fin was also trended toward a reduction in the aged A/J and C57-BL/6J animals versus the younger animals, but this did not reach significance (P = 0.094 and 0.056, respectively). In the BALB/cJ strain, there was no difference in Fin between the young and aged animals (P = 0.94).
Figure 2
 
Computed aqueous flow rate (Fin) versus age in each of four mouse strains. There was a significant decrease (unpaired t-test) in Fin in young (2½–4 months) versus aged (10–11 months) C3H/HeJ animals. Mean Fin was also trended toward a reduction in the aged A/J and C57-BL/6J animals versus the younger animals, but this did not reach significance (P = 0.094 and 0.056, respectively). In the BALB/cJ strain, there was no difference in Fin between the young and aged animals (P = 0.94).
Fin varied significantly amongst the aged animals (but not the younger animals) when comparing across strains (Supplementary Table S4). 
Fu(calc) Expressed as a Percentage of Aqueous Humor Flow Rate (Fin)
When expressed as a percentage of computed Fin, Fu(calc) was found to decrease with age in the BALB/cJ strain (37.4 ± 10.2% vs. 7.9 ± 4.9%, mean Δ = 29.5%, P < 0.05), in the C3H/HeJ strain (71.2 ± 8.6% vs. 34.4 ± 8.6%, mean Δ = 36.7%, P < 0.05), and in the C57-BL/6J strain (52.7 ± 13.7% vs. 14.2 ± 8.2%, mean Δ = 38.4%, P < 0.05). Fu(calc) expressed as a percentage of Fin also decreased with age in the A/J strain (53.9 ± 14.5% vs. 24.8 ± 11.9%, mean Δ = 29.2%), but the decrease was not statistically significant (P = 0.159; Fig. 3A). 
Figure 3
 
(A) Calculated uveoscleral outflow (Fu(calc)) expressed as percentage of aqueous humor flow rate (Fin) versus age in each of four mouse strains. There was a substantial decrease in Fu(calc) as a percentage of Fin in young (2½–4 months) versus aged (10–11 months) animals, which reached statistical significance (unpaired t-test) in all cases except in the A/J strain (P = 0.15). (B) Uveoscleral outflow as measured directly via perfusion of FITC-dextran (Fu(FITC-dex)) expressed as percentage of aqueous humor flow rate (Fin) versus age in each of four mouse strains. There was a substantial decrease in Fu(FITC-dex) as a percentage of Fin in young (2½–4 months) versus aged (10–12 months) animals, which reached statistical significance (unpaired t-test) in all strains except the A/J strain (P = 0.079). In this case, Fu(FITC-dex) values were derived from different individual animals than those from which Fin values were computed.
Figure 3
 
(A) Calculated uveoscleral outflow (Fu(calc)) expressed as percentage of aqueous humor flow rate (Fin) versus age in each of four mouse strains. There was a substantial decrease in Fu(calc) as a percentage of Fin in young (2½–4 months) versus aged (10–11 months) animals, which reached statistical significance (unpaired t-test) in all cases except in the A/J strain (P = 0.15). (B) Uveoscleral outflow as measured directly via perfusion of FITC-dextran (Fu(FITC-dex)) expressed as percentage of aqueous humor flow rate (Fin) versus age in each of four mouse strains. There was a substantial decrease in Fu(FITC-dex) as a percentage of Fin in young (2½–4 months) versus aged (10–12 months) animals, which reached statistical significance (unpaired t-test) in all strains except the A/J strain (P = 0.079). In this case, Fu(FITC-dex) values were derived from different individual animals than those from which Fin values were computed.
Uveoscleral Outflow as Measured Directly via Perfusion of FITC-Dextran (Fu(FITC-dex))
Perfusion of PBS incorporating FITC-dextran at a rate of 0.5 μL/min for 10 minutes resulted in a maximal IOP ranging from 29.2 to 35.7 mm Hg. 
Fu(FITC-dex) was found to significantly decrease with age in the A/J strain (0.055 ± 0.013 μL/min vs. 0.009 ± 0.002 μL/min, mean Δ = 0.046 μL/min [mean decrease of 83.6%], P < 0.01), in the BALB/cJ strain (0.051 ± 0.018 μL/min vs. 0.010 ± 0.007 μL/min, mean Δ = 0.041 μL/min [mean decrease of 80.4%], P < 0.05), and in the C57-BL/6J strain (0.044 ± 0.012 μL/min vs. 0.006 ± 0.003 μL/min, mean Δ = 0.038 μL/min [mean decrease of 86.4%], P < 0.05; Fig. 4). In the C3H/HeJ strain there was also a decrease but this did not reach statistical significance (0.054 ± 0.020 μL/min vs. 0.006 ± 0.003 μL/min, mean Δ = 0.048 μL/min [mean decrease of 88.9%], P = 0.058). 
Figure 4
 
Uveoscleral outflow as measured directly via perfusion of FITC-dextran (Fu(FITC-dex)) versus age in each of four mouse strains. There was a substantial decrease in Fu(FITC-dex) in young (2½–4½ months) versus aged (12 months) animals, which was statistically significant (unpaired t-test) in all cases except the C3H/HeJ animals (P = 0.058).
Figure 4
 
Uveoscleral outflow as measured directly via perfusion of FITC-dextran (Fu(FITC-dex)) versus age in each of four mouse strains. There was a substantial decrease in Fu(FITC-dex) in young (2½–4½ months) versus aged (12 months) animals, which was statistically significant (unpaired t-test) in all cases except the C3H/HeJ animals (P = 0.058).
Mean values obtained for Fu(FITC-dex) were consistently lower than corresponding mean Fu(calc) values (albeit in different animals) by the following percentages: A/J (Young) Fu(FITC-dex) = 45.83% of Fu(calc); A/J (aged) Fu(FITC-dex) = 25.00% of Fu(calc); BALB/cJ (Young) Fu(FITC-dex) = 77.27% of Fu(calc); BALB/cJ (aged) Fu(FITC-dex) = 90.91% of Fu(calc); C3H/HeJ (Young) Fu(FITC-dex) = 49.54% of Fu(calc); C3H/HeJ (aged) Fu(FITC-dex) = 25.00% of Fu(calc); C57-BL/6J (Young) Fu(FITC-dex) = 55.00% of Fu(calc); C57-BL/6J (aged) Fu(FITC-dex) = 50.00% of Fu(calc)
Fu(FITC-dex) Expressed as a Percentage of Aqueous Humor Flow Rate (Fin)
When expressed as a percentage of computed Fin, Fu(FITC-dex) was found to decrease with age in the BALB/cJ strain (35.7 ± 5.7% vs. 7.3 ± 4.5%, mean Δ = 28.4%, P < 0.005), in the C3H/HeJ strain (30.3 ± 8.4% vs. 9.3 ± 3.7%, mean Δ = 21.0%, P < 0.05), and in the C57-BL/6J strain (42.0 ± 9.1% vs. 8.8 ± 4.5%, mean Δ = 33.2%, P < 0.01). Fu(FITC-dex) expressed as a percentage of Fin also decreased with age in the A/J strain (28.3 ± 9.3% vs. 8.6 ± 2.9%, mean Δ = 19.7%), but the decrease was not statistically significant (P = 0.078; Fig. 3B). 
Outflow Facility (C)
Examples of pressure–flow rate relationships in individual young and aged C57-BL/6J animals while live and following euthanasia are shown in Figure 5. In the aged animal, pressure at any given flow rate was a little higher than in the young animal, consistent with the observation that Fu was significantly decreased with age. Following euthanasia, aqueous secretion from the ciliary epithelium and Pe were both reduced to zero and, thus, pressure generated at given flow rates was considerably less. 
Figure 5
 
Representative pressure-time traces obtained during actual C determinations in two individual (young and aged) C57-BL/6J animals. Both the young and the aged animal are shown live (A) and then euthanized (B). In the aged animal, pressure at any given flow rate was a little higher than in young animal, consistent with the observation that Fu was significantly decreased with age. Euthanized animals had no aqueous secretion from the ciliary epithelium, and episcleral venous pressure (Pe) was reduced to zero. Thus, pressure generated at given flow rates was less. ACC, stabilized pressure following anterior chamber cannulation.
Figure 5
 
Representative pressure-time traces obtained during actual C determinations in two individual (young and aged) C57-BL/6J animals. Both the young and the aged animal are shown live (A) and then euthanized (B). In the aged animal, pressure at any given flow rate was a little higher than in young animal, consistent with the observation that Fu was significantly decreased with age. Euthanized animals had no aqueous secretion from the ciliary epithelium, and episcleral venous pressure (Pe) was reduced to zero. Thus, pressure generated at given flow rates was less. ACC, stabilized pressure following anterior chamber cannulation.
C in the BALB/cJ animals was found to significantly increase with age (0.024 ± 0.002 vs. 0.039 ± 0.005 μL/min/mm Hg, mean Δ = 0.015 μL/min/mm Hg [mean increase of 62%], P < 0.05). In the C3H/HeJ strain, C tended to increase with age (0.014 ± 0.002 vs. 0.019 ± 0.002 μL/min/mm Hg, mean Δ = 0.005 μL/min/mm Hg [mean increase of 36%], P = 0.205), and also in the C57-BL/6J strain (0.020 ± 0.003 vs. 0.025 ± 0.006 μL/min/mm Hg, mean Δ = 0.005 μL/min/mm Hg [mean increase of 25%], P = 0.53), but in these strains the increase did not reach statistical significance. In the A/J strain, there was no change in C with age (0.023 ± 0.006 vs. 0.023 ± 0.004), P = 0.98; Fig. 6). 
Figure 6
 
C versus age in each of four mouse strains. There was no significant difference in C in young (2½–4 months) versus aged (10–11 months) animals, except in the BALB/cJ strain, which increased with age (unpaired t-test). In the C3H/HeJ and C57-BL/6J strains C also tended to increase with age (P = 0.205 and 0.53, respectively). In the A/J strain there was no change in C with age (P = 0.98).
Figure 6
 
C versus age in each of four mouse strains. There was no significant difference in C in young (2½–4 months) versus aged (10–11 months) animals, except in the BALB/cJ strain, which increased with age (unpaired t-test). In the C3H/HeJ and C57-BL/6J strains C also tended to increase with age (P = 0.205 and 0.53, respectively). In the A/J strain there was no change in C with age (P = 0.98).
C varied significantly amongst the aged animals (but not the younger animals) when comparing across strains (Supplementary Table S5). 
Eyes did not develop corneal opacities or lenticular cataracts during perfusion. 
Discussion
Since the first reports of measurement of C in mice,2,26 numerous laboratories have studied both this and/or other parameters of AHD in living animals using various techniques, such as: constant flow infusion,57 constant pressure infusion,27 and fluorophotometry (Toris CB, et al. IOVS 2007;48:ARVO E-Abstract 39; Toris CB, et al. IOVS 2014;55:ARVO E-Abstract 2901). In addition, measurements have also been performed on enucleated mouse eyes perfused ex vivo.21,2830 But values reported for various parameters of AHD in this species have been very variable (summarized in Table 1). However, studies performed to date have taken place using cohorts of different strains and ages. These differences may influence the outcome of such measurements, more especially when one considers that different mouse strains are known to exhibit different IOPs, which may point also to other differences in AHD. In support of this possibility, it was reported that there was an influence of genetic background on conventional outflow facility in a study in mouse eyes perfused ex vivo.31 
Thus, we were prompted to investigate potential interstrain variability upon all five parameters of AHD in this species. We chose the same strains and sex as those used in our earlier study.12 However, because earlier published studies used a variety of different age groups, we also compared younger versus aged animals. 
We chose not to investigate the effect of sex on AHD. Although previous studies have used exclusively male, exclusively female, or animals of both sexes (and in some studies, sex was not specified), it was reported that sex had no effect on IOP in normal laboratory strains.38 But there is a possibility, albeit tenuous, that sex may still play a role, particularly as animals age. Some human studies have found sex-specific differences, typically with higher IOP in females, and the magnitude of the difference increasing after 40 years of age.39,40 Female DBA/2J and AKXD-28/Ty mice develop elevated IOP (associated with a glaucoma phenotype that develops in these strains) at an earlier age than males.41,42 This may be of interest for future investigation. 
We found that conscious IOP was unaffected by age in all strains studied except the C57-BL/6J animals. In this strain, there was a small but statistically significant increase in mean IOP of 1.5 mm Hg with age. However, we found interstrain variability in IOP, in both the young and the aged animals, with the pigmented (C3H/HeJ and C57-BL/6J) animals exhibiting a higher IOP than the albino (A/J and BALB/cJ) animals. Interstrain variability in IOP in mice has been reported by others, with which the present data compare favorably.38,43 Anesthesia with ketamine and xylazine reduced IOP in all strains. Ketamine and xylazine anesthesia, and anesthesia with 2,2,2,-tribromoethanol has been reported to reduce IOP within 30 minutes of administration in mice.27,44,45 
We report that Fin as computed via the modified Goldmann equation significantly diminished with age in the C3H/HeJ animals, and trended toward a diminishment with age in the A/J and the C57-BL/6J animals. However, we found no difference with age in the BALB/cJ animals. In addition, while there was no significant interstrain difference in Fin within the young animals, among the aged animals there was (summarized in Supplementary Table S4). An effect of aging upon Fin has been noted in humans. In a study of 300 healthy volunteers, aged from 5 to 83 years, an average decline in Fin of 25% between the ages of 10 and 80 years, or approximately 3.6% per decade, was reported.35,46 
Fu(calc) diminished very sharply with age, both in terms of flow rate of aqueous through the uveoscleral pathway (by 70%–85%), and Fu(calc) expressed as a percentage of Fin (37%–71% in young animals to 8%–34% in aged animals). This sharp reduction in Fu(calc) with age parallels the situation seen in the human and NHP eye,34 although it is more severe. If we assume that the maximal lifespan of a mouse is approximately 24 months, and the maximal lifespan of a human is approximately 75 years, then the young cohort of animals were equivalent in age to humans of approximately 8 to 14 years (juvenile), while the aged cohort of animals were equivalent in age to humans of approximately 31 to 38 years (approaching early middle age). Fu in the juvenile living human eye may be approximately 30% or more of Fin,32,47 whereas in the living human eye, approaching middle age is reported to be approximately less than or equal to 20% to 25% of Fin.48 These values for living human eyes have been estimated by via tonometry, fluorophotometry, tonography, and episcleral venomanometry, and may not be entirely accurate. However, the situation seen in the NHP eye, in which Fu has been measured directly by both tracer perfusion techniques followed by euthanasia and dissection, or estimated by perfusion of 125I or 131I-labeled albumin with concomitant periodic blood draws and assessment of radioactivity present in the blood, has proven to be similar, ranging from 40% to 50% in the juvenile NHP, to 15% to 20% in the aged NHP.4953 
Thus, the high degree of variability for murine Fu (both in absolute terms and as a percentage of Fin) reported in the literature may be a function of the age of the animals used. For example, the very high values reported in very young (2–2¾ months) NIH Swiss White mice by Aihara et al.2 of 0.148 μL/min (representing 82% of Fin) may not be the case if middle aged animals were studied. But this figure compares reasonably well with the present calculated figure of 0.140 ± 0.027 μL/min (71% of Fin) reported for C3H/HeJ animals aged 2½ to 4 months. In our previous study on male BALB/cJ mice aged 7 to 9¾ months, we assessed Fu to be 0.012 ± 0.003 μL/min to 0.029 ± 0.005 μL/min (9%–21% of Fin).7 
Results obtained via direct measurement of Fu (Fu(FITC-dex)) using a technique of FITC-dextran perfusion7,54 albeit on different animals, compared favorably with the results obtained for Fu(calc), although mean values for Fu(FITC-dex) were 25% to 90.91% of corresponding mean values for Fu(calc), with five of eight values lying between 45.83% and 77.27%. While the data were not identical, they were within a similar order of magnitude, with absolute values of Fu(FITC-dex) ranging from 0.044 ± 0.012 μL/min to 0.055 ± 0.013 μL/min for young animals, compared with 0.006 ± 0.003 μL/min to 0.010 ± 0.007 μL/min for aged animals (representing a reduction with age of 80% to 89%, compared with a reduction with age of 70 to 85% as obtained via Fu(calc)). Fu(FITC-dex) values, however, even although directly measured, were not used in calculations of other AHD parameters. This was because all other measurements (C, Pe, IOP) and computational estimation of Fin were made on animals not perfused with FITC-dextran. Entirely separate animals were perfused with FITC-dextran, and underwent only this procedure, to compare Fu(FITC-dex) with Fu(calc) values. We did not perfuse animals in which we measured C with FITC-dextran because precise equilibration periods vary from one animal to another and would render standardization of time of FITC-dextran perfusion challenging. We did, however, calculate Fu as a percentage of Fin in all strains of animals in both age groups using both Fu(calc) and Fu(FITC-dex) values. Using Fu(calc) values, we computed values in the young cohort ranging from approximately 37% to approximately 71%, and in the aged cohort this was reduced to approximately 8% to approximately 34%. Corresponding calculations using Fu(FITC-dex) values in the young cohort ranged from approximately 28% to approximately 42%, and in the aged cohort this was reduced from approximately 7% to approximately 9%. The difference lies in the fact that Fu(calc) values were consistently higher than the Fu(FITC-dex) values. But the trend toward a reduction in age was still apparent, whichever way these calculations were performed. Fu(FITC-dex) measurements may have been less than the corresponding Fu(calc) values because of less than optimal recovery of fluorescent tracer from the scleral and ciliary tissues (and also because Fu(calc) estimates and Fu(FITC-dex) measurements were made on different individual animals). The perfusion rate of 0.5 μL/min for a period of 10 minutes would result in an intracameral delivery of 5 μL (with a corresponding maximal IOP of 29.2–35.7 mm Hg), which should be sufficient to fill the chamber. Based on Fu assessments, a total volume of 0.1 to 1.2 μL fluorescent tracer should be present in the ciliary and scleral tissues. But perhaps a longer perfusion time (say 20–30 minutes) would lead to a more complete saturation of the uveoscleral pathway, which may improve the correlation between Fu(FITC-dex) and Fu(calc). In accordance with the relative pressure-independence of the uveoscleral pathway, we have found that different flow rates (and hence maximal perfusion pressures) of FITC-dextran do not significantly affect Fu(FITC-dex) calculations (data not shown). Unlike other values for AHD, values for Fu, expressed either as Fu(calc) or as Fu(FITC-dex), did not exhibit significant interstrain variation, within either the young or aged cohorts. 
C showed a trend toward an increase with age in the C3H/HeJ and the C57-BL/6J strains (36% and 25%, respectively). The BALB/cJ strain also exhibited an increase, which was larger (62%) and achieved statistical significance. The reasons for such an increase with age are not immediately clear. However, in a similar anatomic arrangement to both the human and NHP eye, the longitudinal fibers of the mouse CM insert into the trabecular meshwork via elastic fibers. Contraction of the longitudinal fibers of the CM in the mouse via intracameral perfusion of 100 μM pilocarpine leads to an approximate doubling of outflow facility, via dilation of the intertrabecular spaces.25 One might speculate that as mice proceed from a juvenile to a middle-aged stage in life (as in the present study), the CM develops more tone, perhaps in relation to a marginal increase in the size of the eye that may occur over this period as the animals become, on average, physically larger and heavier. But the age range of the animals in the present study was limited to 2½ to 4, to 10 to 12 months. Studies conducted on older animals (≥ 14 months, equivalent to a ≥ 45-year-old human) may reveal a tendency for facility to decline toward senescence, concomitant with the loss of the ability of the CM to contract (and thus loss of ability of the longitudinal fibers of the CM to deform the trabecular meshwork via tone applied to the scleral spur) that is seen in the human55,56 and NHP5761 eye from midlife onward. 
Boussommier-Calleja and Overby31 reported that there are strain differences in C in murine eyes perfused ex vivo. In their study, in eyes derived from female animals at 4 to 7 months of age, C was lowest in eyes obtained from CBA/J animals (0.0113 ± 0.0031 μL/min/mm Hg), intermediate in eyes obtained from C57-BL/6J animals (0.0147 ± 0.0029 μL/min/mm Hg [compared with 0.020 ± 0.003 μL/min/mm Hg in the younger C57-BL/6J animals in the present study]), and highest in eyes obtained from BALB/cJ animals (0.0164 ± 0.0059 μL/min/mm Hg [compared with 0.039 ± 0.005 μL/min/mm Hg in the younger BALB/cJ animals in the present study]). We did not investigate the CBA/J strain. In living animals at 3 to 4 months of age they recorded IOP as: 10.6 ± 1.8 mm Hg (conscious) and 10.1 ± 2.3 mm Hg (anesthesia) for the BALB/cJ strain, and 12.3 ± 1.0 mm Hg (conscious) and 11.5 ± 2.4 mm Hg (anesthesia) for the C57-BL/6J strain. Their conscious IOP measurements were slightly lower than in the present study, but by only 0.75 mm Hg (BALB/cJ) and 1.6 mm Hg (C57-BL/6J). They also reported a lesser effect of anesthesia, but in their study isoflurane was used, and measurements took place within 5 to 6 minutes of initial exposure to anesthesia. 
We reported no change in Pe with age. However, we did see significant interstrain differences, with Pe being higher in the pigmented strains, which were consistent with strain differences in baseline IOP. Techniques for measuring Pe in larger species (Tambour, episcleral venomanometry, episcleral venous cannulation) are not at present practical for use in the small eye of the mouse.62 Thus, we employed the methodology first described by Aihara et al.2 and at present the only methodology for measuring Pe in the murine eye. But this method suffers from two drawbacks. When intracameral pressure is artificially slowly lowered until reaching the point at which Schlemm's canal fills with blood, the assumption that Pe is at the same value throughout the entire episcleral venous plexus is made, which may or may not be the case.62 Further, as intracameral pressure is slowly lowered, initially the scleral collector channels fill with blood refluxed from the episcleral venous plexus, but Schlemm's canal itself remains blood-free. Lowering of intracameral pressure by an additional 1 to 1.5 mm Hg will result in the canal itself filling with blood, and this is the point at which we routinely assume that intracameral pressure is equal to Pe. We choose this point because the scleral collector channels are more difficult to visualize, even when filled with blood and under illumination and ×30 magnification, especially in pigmented animals. Schlemm's canal by contrast is very much easier to see when filled with blood in either albino or pigmented animals. But obviously measurement of Pe via this method is to an extent dependent on definition. However, in the present study, values for Fu(calc) were computed based on our previous observation (data not shown) that Pe (as assessed by this method) drops to zero 10 to 15 minutes following euthanasia (we perfused eyes in life and then starting 20 minutes following death); therefore, measurement error in Pe will not affect our calculated value for this parameter. But, nevertheless, accuracy in measurement of Pe has relevance. For example, in AHD measurements made exclusively in life in other species, where a value for Pe factors in to the modified Goldmann equation, Sit and McLaren62 reported that an error of 1 mm Hg in Pe (if true Pe equals 9 mm Hg) can lead to a large error in calculated Fu, perhaps up to 69%, depending on specific values for other parameters of AHD. Equivalent calculations in living mice using our data would suggest a figure of approximately 36%. 
In previous mouse AHD studies, which we performed in BALB/cJ mice,7 we used a 3-mL syringe in the infusion pump. This was done in accordance with the recommendations of the manufacturer and the need to use syringe size sufficiently large to fill the perfusion tubing while having sufficient perfusate left in the syringe to conduct the perfusion itself. However, in consideration of the Pusher Advance per Half Step of the infusion pump model (WPI SP101i) of approximately 88 nm (as listed by the manufacturer), and a desire to examine possible confounding effects of pulsatile flow on the final calculated results, we switched to using a smaller syringe size (1 mL, 4.6-mm internal diameter) in the present study such that at the lowest flow rate of 0.1 μL/min the stepper rate would be 65.36 steps/min, or one step every 1.09 seconds (rising to one step every 0.22 seconds at the highest flow rate of 0.5 μL/min). However, we did not find that this led to any significant difference in the observed or calculated results. We have also (in subsequent unpublished mouse AHD studies) used 100-μL microsyringes (1.46-mm internal diameter; Hamilton Company, Reno, NV, USA) but once again, the end result did not change. Despite the theoretical improvement in the stepper rate, we have found no difference in chart recorder trace profiles, pressure oscillation, calculated final results, or variation. 
During perfusion in a live eye, pressure fluctuates from 1.25 to 2.5 mm Hg over a period of 2.5 to 10 minutes. Following euthanasia, pressure fluctuates from 1.25 to 1.66 mm Hg over the same period. We did not see any age effect on this pressure fluctuation however. We have run sham perfusions where no eye is connected to the system, and found that pressure and fluctuation are negligible at flow rates of 0.1 to 0.5 μL/min over a period of 30 minutes, regardless of syringe size used (3 mL, 1 mL, or 100 μL). Thus fluctuation, when seen, appears to be a function of the eye itself, and which is diminished somewhat by euthanasia. However, because of pressure fluctuation, we measure several pressures at each flow rate following the initial rise after flow rate is increased, and then compute the mean as the stabilized pressure.7 
Ramos and Stamer63 reported that pulsatile pressure oscillations of 2.7 mm Hg at a frequency of 1 per second significantly reduced C. This reduction in C started approximately 9 hours after commencement of IOP pulses in perfused human anterior segments, and approximately 15 hours after commencement of IOP pulses in perfused porcine anterior segments. The authors also noted very much smaller changes in outflow facility 7 hours after commencement of ocular pulses (human) and 1 hour after commencement of ocular pulses (porcine), but these changes were statistically insignificant. Our perfusion ran for a period of only approximately 2 hours, with a pressure fluctuation frequency of 1.25 to 2.5 mm Hg in some living eyes over a period of 2.5 to 10 minutes (1/150 to 1/600 × the pressure oscillation frequency reported by Ramos and Stamer63). It is unlikely that the pressure fluctuations we saw at such a low frequency, combined with the relatively brief period of perfusion, would have a significant effect on C. Also, in Ramos and Stamer's study,63 perfused anterior segments were used which, unlike the living eye, lack a blood supply, autonomic innervation, vascular, ciliary and iridial muscle tone, and circulating hormonal influences. Contractile tone in the trabecular meshwork may also be altered or absent in perfusion cultured anterior segments. 
From the perspective of AHD in the mouse, of all strains studied, the pigmented strains most closely resemble the human eye in terms of IOP and Pe. But all strains showed a larger decrease in Fu from young to middle life compared with humans. Like humans, mice appear to show a trend toward reduction in Fin with age, but do show a trend toward increased C from young to middle life, which may be different from humans. 
In conclusion, we report that, in all strains studied, Fu diminished very sharply with age, both in absolute terms (by 70%–89%) and as a percentage of Fin (from 30%–71% to 7%–34%). There was a tendency toward reduction of Fin with age (with the exception of the BALB/cJ animals, in which it was unaffected by age). Fin among the aged animals (but not the young animals) exhibited interstrain difference. C showed a tendency to increase with age (except in A/J animals, in which it was unaffected by age). C among aged animals (but not young animals) showed interstrain difference. Within each strain, baseline IOP did not change with age, except in C57-BL/6J animals, which showed a small but significant increase. Intraocular pressures did exhibit interstrain difference, being higher in the pigmented versus the albino strains. Within each strain, Pe was unaffected by age, but Pe did show interstrain difference in both young and aged animals, being higher in the pigmented versus the albino strains, consistent with the higher IOP in the pigmented strains. We did not measure systemic blood pressure in the present study, but a relationship (if any) between Pe and blood pressure in different strains of mice may merit future investigation. Future studies of murine AHD should take into account the age and strain of the subject animals, which may explain the different values reported in previous studies. 
Acknowledgments
Supported by the University of North Texas Health Science Center Institute of Aging and Alzheimer's Disease Research (Fort Worth, TX, USA) Grant #RI6071. 
Disclosure: J.C. Millar, None; T.N. Phan, None; I.-H. Pang, None; A.F. Clark, None 
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Figure 1
 
Calculated uveoscleral outflow (Fu(calc)) versus age in each of four mouse strains. There was a substantial decrease in Fu(calc) in young (2½–4 months) versus aged (10–11 months) animals, which was statistically significant (unpaired t-test) in all cases except in the A/J strain (P = 0.15).
Figure 1
 
Calculated uveoscleral outflow (Fu(calc)) versus age in each of four mouse strains. There was a substantial decrease in Fu(calc) in young (2½–4 months) versus aged (10–11 months) animals, which was statistically significant (unpaired t-test) in all cases except in the A/J strain (P = 0.15).
Figure 2
 
Computed aqueous flow rate (Fin) versus age in each of four mouse strains. There was a significant decrease (unpaired t-test) in Fin in young (2½–4 months) versus aged (10–11 months) C3H/HeJ animals. Mean Fin was also trended toward a reduction in the aged A/J and C57-BL/6J animals versus the younger animals, but this did not reach significance (P = 0.094 and 0.056, respectively). In the BALB/cJ strain, there was no difference in Fin between the young and aged animals (P = 0.94).
Figure 2
 
Computed aqueous flow rate (Fin) versus age in each of four mouse strains. There was a significant decrease (unpaired t-test) in Fin in young (2½–4 months) versus aged (10–11 months) C3H/HeJ animals. Mean Fin was also trended toward a reduction in the aged A/J and C57-BL/6J animals versus the younger animals, but this did not reach significance (P = 0.094 and 0.056, respectively). In the BALB/cJ strain, there was no difference in Fin between the young and aged animals (P = 0.94).
Figure 3
 
(A) Calculated uveoscleral outflow (Fu(calc)) expressed as percentage of aqueous humor flow rate (Fin) versus age in each of four mouse strains. There was a substantial decrease in Fu(calc) as a percentage of Fin in young (2½–4 months) versus aged (10–11 months) animals, which reached statistical significance (unpaired t-test) in all cases except in the A/J strain (P = 0.15). (B) Uveoscleral outflow as measured directly via perfusion of FITC-dextran (Fu(FITC-dex)) expressed as percentage of aqueous humor flow rate (Fin) versus age in each of four mouse strains. There was a substantial decrease in Fu(FITC-dex) as a percentage of Fin in young (2½–4 months) versus aged (10–12 months) animals, which reached statistical significance (unpaired t-test) in all strains except the A/J strain (P = 0.079). In this case, Fu(FITC-dex) values were derived from different individual animals than those from which Fin values were computed.
Figure 3
 
(A) Calculated uveoscleral outflow (Fu(calc)) expressed as percentage of aqueous humor flow rate (Fin) versus age in each of four mouse strains. There was a substantial decrease in Fu(calc) as a percentage of Fin in young (2½–4 months) versus aged (10–11 months) animals, which reached statistical significance (unpaired t-test) in all cases except in the A/J strain (P = 0.15). (B) Uveoscleral outflow as measured directly via perfusion of FITC-dextran (Fu(FITC-dex)) expressed as percentage of aqueous humor flow rate (Fin) versus age in each of four mouse strains. There was a substantial decrease in Fu(FITC-dex) as a percentage of Fin in young (2½–4 months) versus aged (10–12 months) animals, which reached statistical significance (unpaired t-test) in all strains except the A/J strain (P = 0.079). In this case, Fu(FITC-dex) values were derived from different individual animals than those from which Fin values were computed.
Figure 4
 
Uveoscleral outflow as measured directly via perfusion of FITC-dextran (Fu(FITC-dex)) versus age in each of four mouse strains. There was a substantial decrease in Fu(FITC-dex) in young (2½–4½ months) versus aged (12 months) animals, which was statistically significant (unpaired t-test) in all cases except the C3H/HeJ animals (P = 0.058).
Figure 4
 
Uveoscleral outflow as measured directly via perfusion of FITC-dextran (Fu(FITC-dex)) versus age in each of four mouse strains. There was a substantial decrease in Fu(FITC-dex) in young (2½–4½ months) versus aged (12 months) animals, which was statistically significant (unpaired t-test) in all cases except the C3H/HeJ animals (P = 0.058).
Figure 5
 
Representative pressure-time traces obtained during actual C determinations in two individual (young and aged) C57-BL/6J animals. Both the young and the aged animal are shown live (A) and then euthanized (B). In the aged animal, pressure at any given flow rate was a little higher than in young animal, consistent with the observation that Fu was significantly decreased with age. Euthanized animals had no aqueous secretion from the ciliary epithelium, and episcleral venous pressure (Pe) was reduced to zero. Thus, pressure generated at given flow rates was less. ACC, stabilized pressure following anterior chamber cannulation.
Figure 5
 
Representative pressure-time traces obtained during actual C determinations in two individual (young and aged) C57-BL/6J animals. Both the young and the aged animal are shown live (A) and then euthanized (B). In the aged animal, pressure at any given flow rate was a little higher than in young animal, consistent with the observation that Fu was significantly decreased with age. Euthanized animals had no aqueous secretion from the ciliary epithelium, and episcleral venous pressure (Pe) was reduced to zero. Thus, pressure generated at given flow rates was less. ACC, stabilized pressure following anterior chamber cannulation.
Figure 6
 
C versus age in each of four mouse strains. There was no significant difference in C in young (2½–4 months) versus aged (10–11 months) animals, except in the BALB/cJ strain, which increased with age (unpaired t-test). In the C3H/HeJ and C57-BL/6J strains C also tended to increase with age (P = 0.205 and 0.53, respectively). In the A/J strain there was no change in C with age (P = 0.98).
Figure 6
 
C versus age in each of four mouse strains. There was no significant difference in C in young (2½–4 months) versus aged (10–11 months) animals, except in the BALB/cJ strain, which increased with age (unpaired t-test). In the C3H/HeJ and C57-BL/6J strains C also tended to increase with age (P = 0.205 and 0.53, respectively). In the A/J strain there was no change in C with age (P = 0.98).
Table 1
 
Summary of Previously Published AHD Parameters in Mouse Eyes (Live and Ex Vivo Perfused)
Table 1
 
Summary of Previously Published AHD Parameters in Mouse Eyes (Live and Ex Vivo Perfused)
Table 2
 
Conscious IOP, Anesthetized IOP, and Pe in Four Mouse Strains (Young and Aged)
Table 2
 
Conscious IOP, Anesthetized IOP, and Pe in Four Mouse Strains (Young and Aged)
Supplement 1
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