December 2013
Volume 54, Issue 13
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Physiology and Pharmacology  |   December 2013
The Influence of Genetic Background on Conventional Outflow Facility in Mice
Author Notes
  • Department of Bioengineering, Imperial College London, London, United Kingdom 
  • Correspondence: Darryl R. Overby, Department of Bioengineering, Imperial College London, London SW7 2AZ, UK; d.overby@imperial.ac.uk
Investigative Ophthalmology & Visual Science December 2013, Vol.54, 8251-8258. doi:10.1167/iovs.13-13025
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      Alexandra Boussommier-Calleja, Darryl R. Overby; The Influence of Genetic Background on Conventional Outflow Facility in Mice. Invest. Ophthalmol. Vis. Sci. 2013;54(13):8251-8258. doi: 10.1167/iovs.13-13025.

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

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Abstract

Purpose.: Intraocular pressure (IOP) varies between genetically distinct strains of mice. The purpose was to test the hypothesis that strain-dependent differences in IOP are attributable to differences in conventional outflow facility (C).

Methods.: The IOP was measured by rebound tonometry in conscious or anesthetized BALB/cJ, C57BL/6J, and CBA/J mice (N = 6–10 per strain). Conventional outflow facility was measured by ex vivo perfusion of enucleated eyes (N = 9–10 per strain).

Results.: Conscious IOP varied between strains, being highest in CBA/J (14.5 ± 0.9 mm Hg, mean ± SD), intermediate in C57BL/6J (12.3 ± 1.0 mm Hg), and lowest in BALB/cJ (10.6 ± 1.8 mm Hg) mice. Anesthesia reduced IOP and eliminated any detectable differences between strains. Conventional outflow facility also varied between strains, but, in contrast to IOP, C was lowest in CBA/J (0.0113 ± 0.0031 μL/min/mm Hg) and highest in BALB/cJ (0.0164 ± 0.0059 μL/min/mm Hg). Like IOP, C was intermediate in C57BL/6J (0.0147 ± 0.0029 μL/min/mm Hg). There was a strong correlation between conscious IOP and outflow resistance (1/C) from individual eyes across all three strains, revealing that 70% of the variation in IOP was attributable to variation in outflow resistance.

Conclusions.: Differences in IOP among three genetically distinct murine strains are attributable largely to differences in conventional outflow facility. These results motivate further studies using mice to identify the morphologic and genetic factors that underlie IOP regulation within the conventional outflow pathway.

Introduction
The facility of aqueous humor drainage through the conventional or trabecular outflow pathway is a major determinant of IOP, and decreased conventional outflow facility (C) is the underlying cause of ocular hypertension associated with primary open-angle glaucoma. 1 Therefore, an improved understanding of aqueous outflow physiology may identify targetable mechanisms that may lower IOP more successfully for glaucoma therapy. 
Recently, there has been growing interest in mouse models of aqueous humor outflow. Despite having a thinner and less elaborate trabecular meshwork, the mouse conventional outflow pathway is similar to that of humans by having a lamellated trabecular meshwork and a continuous Schlemm's canal, 2 and mice exhibit segmental outflow in the trabecular meshwork. 3 The C in mice responds to compounds that affect outflow facility in humans, including TGF-β, 4,5 sphingosine 1-phosphate, 6,7 latanoprost, 812 steroids, 13,14 and prostaglandin EP4 receptor agonists. 7,15 Like humans, mice do not appear to exhibit “washout,” 16 which is the characteristic increase in C that occurs during prolonged ocular perfusion 17,18 in most nonhuman species including monkeys. 19,20 On the other hand, some studies 10,21,22 (but not all8) have suggested that the majority of aqueous humor outflow in mice passes through the unconventional or uveoscleral outflow pathway, unlike humans. Nevertheless, in light of the anatomic and functional similarities between mice and humans in regard to conventional aqueous humor outflow, there is growing interest in using mice as animal models to investigate the physiology of outflow regulation within the trabecular meshwork. 
In mice, IOP depends on genetic background and spans nearly a 2-fold range between strains (e.g., 11 vs. 19 mm Hg between BALB/cJ versus CBA/CaJ23,24). Because phenotypic differences often are linked to genomic polymorphisms, 25 there is an opportunity to exploit the natural phenotypic diversity of IOP to gain insight into the genetic basis of IOP regulation. According to Goldmann's equation, differences in IOP could be attributed to differences in any of four parameters describing aqueous humor dynamics: aqueous humor production rate (Fin ), episcleral venous pressure (EVP), C, and unconventional outflow rate (Fu ). While several studies have measured some or all of these parameters for a particular strain, 8,10,22 few studies have measured parameters across different strains, 26 and post hoc comparison between studies is complicated by technical differences or environmental conditions. For example, C measured in a single inbred strain (BALB/cJ) varies 3-fold among different reports 8,9 (0.0055 vs. 0.0180 μL/min/mm Hg). Thus, there is a need for a study that directly compares aqueous humor dynamics between different strains of mice. 
The aim of this project was to test the hypothesis that differences in IOP between different strains of mice are attributable to differences in C. To test this hypothesis, we measured IOP in BALB/cJ, C57BL/6J, and CBA/J mice, three inbred strains selected based on previous studies 23,24 as exhibiting low, medium, and high IOP. We then measured C ex vivo in enucleated eyes from the same cohort of animals, and examined the relationship between C and IOP as predicted by Goldmann's equation. 
Methods
All experiments were done in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research under UK Home Office Project License 70/7306. 
Animal Husbandry
This study used three inbred strains of mice chosen as strains typically exhibiting low, medium, and high IOP 23,24 : BALB/cJ (N = 13 mice), C57BL/6J (N = 13), and CBA/J (N = 15), respectively. Preliminary studies used CBA/CaJ (N = 10), but this strain did not exhibit as high an IOP as expected based on a prior report. 23 Therefore, we replaced CBA/CaJ with CBA/J. All mice were female and were purchased from Jackson Laboratories (Bar Harbor, ME) at 6 weeks of age. Mice were housed in individually ventilated cages, fed ad libitum, and maintained at 21°C with a 12-hour light (6 AM to 6 PM) and 12-hour dark cycle. The IOP measurements were attempted on 30 mice (10 per strain) aged between 3 and 4 months, while facility measurements were attempted on 30 mice aged between 4 and 7 months. Between IOP and facility measurements, there was an outbreak of pinworm in the room where our mice were housed. There was no evidence as to whether our mice had contracted the parasite, and fenbendazole prophylaxis was refused to avoid potential effects on facility. 
IOP Measurements
The IOP was measured noninvasively using a commercially available rebound tonometer (TonoLab; ICare, Helsinki, Finland) in conscious and anesthetized mice. This tonometer measures IOP as a function of the deceleration of a lightweight probe propelled against the central cornea. 24 All IOP measurements were performed on the right eye between 12 PM and 4 PM to minimize the influence of diurnal variations. 27 The tonometer was held by hand for consecutive rebound measurements used to calculate a single IOP value, following manufacturer's instructions, and tonometer readings were repeated if there was any sign of error or large variability between consecutive rebound measurements. More specifically, readings were repeated if the “dash” indicator on the tonometer display reported medium or high variability, or if IOP values were artificially elevated (typically >20 mm Hg) due to the mouse becoming restless or stressed during the measurement. 
The tonometer was calibrated in one eye of two mice from each strain to validate the tonometer readings and to account for potential interstrain differences in corneal biomechanics that may affect the rebound measurement. Calibration was performed on cadaveric eyes maintained in situ that were pressurized between 6 and 30 mm Hg via a cannula connected to an adjustable height reservoir, with the calibrated IOP determined based on the height difference between the limbus to the reservoir meniscus. The eyelids were removed and the cannula was inserted into the anterior chamber near the limbus so as to minimize interference with the tonometer probe that rebounds against the central cornea. At each of 10 pressure levels, the eye was hydrated with a drop of PBS and left to equilibrate for at least 3 minutes. The drop of PBS was removed with tissue paper before rebound measurements. There was no evidence of corneal damage after repeated IOP measurements. 
During the calibration, tonometric IOP was measured using two methods. First, following manufacturer's instructions, a single IOP value was read from the tonometer display following six consecutive rebound measurements. We refer to this as the “tonometer-reported IOP.” However, the manufacturer provides little information on the algorithm used to calculate the tonometer-reported IOP, and our separate calibration studies suggested that the tonometer-reported IOP is not a simple average of the six measurements, nor an average after excluding the highest and lowest measurements as suggested previously. 28 Because of the ambiguity in how the tonometer-reported IOP was calculated, we used a second method to obtain IOP by manually calculating the average of the first five rebound measurements as presented on the tonometer display after each rebound (the sixth rebound measurement was not actually displayed, but rather was replaced with the tonometer-reported IOP described above). We referred to this as the “manually-calculated IOP.” When applied to the same calibration data, both methods exhibited a linear relationship with manometric IOP, but the manually-calculated IOP yielded a larger R 2 value (0.96 vs. 0.91), indicating greater linearity and a smaller root-mean-squared error in relation to the true manometric pressure (2.2 vs. 1.5 mm Hg). For this reason, we used the manually-calculated IOP for all further IOP measurements in this study. 
For each strain, the tonometric pressure (IOPt) was expressed as a linear function of the true manometric pressure (IOPm) as determined by linear regression based on the following relationship:  where the slope (α) and intercept (β) values are given in Table 1. Following previous studies,29,30 there was no statistical difference in the parameters of the linear regression between strains (P > 0.48, 1-way ANOVA). Data from all strains, therefore, were lumped together to yield a single linear regression (Fig. 1, Table 1). All tonometric IOP values reported in this study were corrected by the following relationship:  where the units are in mm Hg and IOPt is the “manually-calculated IOP” described above. This correction amounts to a small increase of approximately 1% at physiologic IOP with the tonometer tending to slightly underestimate the true pressure.  
Figure 1
 
Calibration data from the rebound tonometer using cadaveric eyes in situ (two eyes per strain). Each eye was cannulated and manometric IOP m was controlled by a fluid reservoir, while tonometric IOP t was measured using the “manually-calculated” method (see Methods). The calibration relationship was not different between strains, and, therefore, data from all strains were lumped together to obtain the linear regression shown in the Figure. All tonometric IOP data in this study were corrected according to the inverse of this linear regression (Equation 2). Each data point refers to a single manually-calculated IOP measurement from a single eye.
Figure 1
 
Calibration data from the rebound tonometer using cadaveric eyes in situ (two eyes per strain). Each eye was cannulated and manometric IOP m was controlled by a fluid reservoir, while tonometric IOP t was measured using the “manually-calculated” method (see Methods). The calibration relationship was not different between strains, and, therefore, data from all strains were lumped together to obtain the linear regression shown in the Figure. All tonometric IOP data in this study were corrected according to the inverse of this linear regression (Equation 2). Each data point refers to a single manually-calculated IOP measurement from a single eye.
Table 1
 
Parameters of the Tonometer Calibration as Described by Equation 1
Table 1
 
Parameters of the Tonometer Calibration as Described by Equation 1
Strain α β, mm Hg R 2
BALB/cJ, N = 2 1.0 −0.9 0.97
C57BL/6J, N = 2 0.9 0.7 0.98
CBA/J, N = 2 1.1 −0.1 0.95
All strains, N = 6 1.0 −0.1 0.96
To investigate the effects of anesthesia, IOP measurements were attempted in 10 conscious mice of each strain and again in the same mice under anesthesia within one or two days. For conscious measurements, mice were placed in a customized restraining device and allowed to settle for 3 minutes before tonometry in the right eye only. Despite repeated training attempts, conscious IOP measurements were possible in only 21 mice (6–8 per strain) because either the mice did not remain still or IOP values were unreasonably large (>20 mm Hg). For anesthetized measurements, mice were exposed to 4% isoflurane (IsoFlo; Abbott Animal Health, Abbott Park, IL) and 2% oxygen for 3 minutes in a sealed box, followed by 3.5% isoflurane and 0.7% oxygen via a nose cone for 2 to 3 minutes. The IOP was measured in the right eye between 5 and 6 minutes after the initial exposure to anesthesia. Previous studies reveal that anesthesia lowers IOP in mice, 24 and that IOP measurements should be made within minutes following the onset of anesthesia to minimize the IOP-lowering effect. 9 Animals then were allowed to recover under a steady flow of oxygen without isoflurane. 
Conventional Outflow Facility (C) Measurements
Outflow facility was measured in ex vivo mouse eyes using a computer-controlled perfusion system modified from previous studies 7,16,18,31 to allow perfusion while the eye was submerged in a heated bath of isotonic saline (see below). On a typical experimental day, one eye was perfused from each strain, and the order of perfusion was randomized. Eyes were enucleated within 15 minutes of death by cervical dislocation, and eyes were stored in PBS at 4°C for no longer than 6 hours before perfusion. All mice were euthanized and their eyes enucleated at approximately the same time. Facility was measured in only one eye per mouse, typically the right eye (N = 20 mice) unless an unsuccessful perfusion required the left eye to be perfused (N = 8). Perfusion was unsuccessful in both eyes of two mice. 
For perfusion, the eye was mounted in a plastic tube trimmed from a 0.2 mL standard centrifuge tube that was filled with gauze, with the posterior sclera affixed to the tube using cyanoacrylate adhesive. Eyes were immersed to the limbus in a PBS bath and covered with tissue paper to provide thorough hydration. The PBS bath was heated indirectly with a custom-built temperature-controller to maintain bath temperature in the physiologic range of 35°C ± 1°C. The eye was cannulated using a 33-gauge needle that was inserted into the anterior chamber under a stereomicroscope using a micromanipulator. The needle was connected via rigid pressure tubing to a pressure transducer (142PC01G; Honeywell, Columbus, OH) and a 25 μL syringe (Gastight; Hamilton, Reno, NV) placed on a computer-controlled syringe pump (PhD Ultra; Harvard Apparatus, Holliston, MA). Custom-written software 18 was used to maintain IOP at a user-defined pressure by adjusting the variable flow rate of the syringe-pump. All eyes were perfused with Dulbecco's PBS including divalent cations and 5.5 mM glucose passed through a 0.22-μm filter (DBG). 
To measure C, eyes were perfused at sequential pressure steps of 4, 8, 15, and 25 mm Hg, typically 20 minutes at each step to allow at least 10 minutes of steady flow rate data (Fig. 2). The average flow rate (F) at each pressure step (Pp ) then was fit to a simplified version of Goldmann's equation:  where episcleral venous pressure and aqueous humor production are assumed to be zero for an enucleated eye. In Equation 3, perfusion pressure is defined as Pp to avoid confusion with IOP as measured in vivo. According to Equation 3, C is equal to the slope of the least-squares linear regression of F versus Pp measured during perfusion, and Fu is the zero-pressure intercept that often is interpreted (correctly or incorrectly) as unconventional or pressure-independent outflow. Equation 3 assumes that C and Fu are independent of Pp , and that F reaches a steady state at each pressure step. For a perfusion to be acceptable, the flow rate must achieve stable value, as indicated by a deviation of less than approximately 10%, in at least three of the four pressure steps. According to these criteria, C was measured successfully in 28 of 30 mice (9–10 per strain). Of the 28 mice where C was measured, 14 mice also had measurements of conscious IOP without anesthesia (3–6 per strain). Of these 14 mice, 11 mice had facility measured in the right eye (the same eye used for IOP measurements) and three mice had facility measured in the left eye.  
Figure 2
 
A typical perfusion tracing showing the flow rate (solid tracing, left axis) and pressure (dashed tracing, right axis) as a function of time for one enucleated eye of a C57BL/6J mouse. The pressure increases in steps of 4, 8, 15, and 25 mm Hg, while the flow rate increases in proportion to the increase in pressure. The spikes in flow rate are due to rapid adjustments of the syringe pump as it attempts to maintain the desired pressure. The average flow rate at each pressure step is calculated by averaging over at least 10 minutes of data, excluding the spike values.
Figure 2
 
A typical perfusion tracing showing the flow rate (solid tracing, left axis) and pressure (dashed tracing, right axis) as a function of time for one enucleated eye of a C57BL/6J mouse. The pressure increases in steps of 4, 8, 15, and 25 mm Hg, while the flow rate increases in proportion to the increase in pressure. The spikes in flow rate are due to rapid adjustments of the syringe pump as it attempts to maintain the desired pressure. The average flow rate at each pressure step is calculated by averaging over at least 10 minutes of data, excluding the spike values.
Statistical Analysis
The IOP was analyzed using a linear mixed model (LMM) to test for statistical differences between strains with and without anesthesia (SPSS Software; IBM, Armonk, NY). Two fixed effects were included in the LMM (strain and anesthesia) with IOP as the dependent variable. The maximum likelihood method was used to estimate the solutions to the LMM equations. Using a full-factorial design, the LMM analysis yielded three P values (corresponding to the probability of a type I error) describing the influence of strain, anesthesia, and interactions between strain and anesthesia on IOP. If a significant influence was detected in any one factor, the software automatically performed a post hoc Bonferroni test to evaluate pairwise differences between any two groups. To investigate further the effect of anesthesia in each strain, conscious and anesthetized IOP measurements from the same eye were compared using a paired Student's t-test (6–8 eyes per strain). To determine whether IOP differences between strains were masked by anesthesia, IOP was compared between strains for anesthetized mice only (N = 30) using a 1-way ANOVA. 
Differences in C, Fu , α, and β between strains were analyzed using a 1-way ANOVA, followed by a post hoc Bonferroni test for detecting pairwise differences between any two strains. Linear regression analysis was performed using Excel (Microsoft Corporation, Redmond, WA). A bivariate correlation analysis was performed to evaluate whether there was a statistically significant correlation between IOP and 1/C. More specifically, we computed the Pearson's product-moment correlation coefficient, and tested whether this quantity was significantly different from 0 using a 2-tailed t-test (SPSS; IBM). Data points were considered outliers in the linear regression if the Cook's distance was larger than 1.0 or if the probability of the studentized deleted residual was lower than 0.05/N, where N was taken as the number of data points used for the regression. 32 Data points were considered outliers from a population based on the Dixon's Q-test with a 95% confidence threshold. 33 Outlier analysis was performed separately on C and 1/C. The significance threshold was taken as 0.05 divided by the number of comparisons (e.g., 0.016 if comparing across three strains, per Bonferroni's correction). 
Results
IOP Measurements
The IOP was significantly different between strains (Fig. 3, P = 0.001, LMM). Consistent with prior reports, 23,24 IOP was lowest for BALB/cJ and highest for CBA/J, with an intermediate value of IOP for C57BL/6J (Table 2). According to a post hoc Bonferroni test, IOP was statistically different between BALB/cJ versus CBA/J, but not between BALB/cJ versus C57BL/6J or C57BL/6J versus CBA/J. Compared to IOP in conscious mice, anesthesia caused a reduction in IOP (Fig. 3, Table 2; P = 0.046, LMM) that eliminated any detectable differences between strains (P = 0.260, 1-way ANOVA). Comparing IOP within individual eyes with and without anesthesia, the IOP reduction was significant for CBA/J (26.0 ± 18.3%, P = 0.01, paired Student's t-test with Bonferroni correction), but smaller or insignificant for C57BL/6J (8.3 ± 11.0%, P = 0.14) and BALB/cJ (−0.9 ± 29.1%, P = 0.86). These results were consistent with prior reports 23,24 and demonstrated that anesthesia has an IOP-lowering effect that varies between strains or that has a stronger effect on mice that have a larger baseline IOP. 
Figure 3
 
IOP measured in conscious (white) and anesthetized (black) mice from BALB/cJ (N = 8 and 10), C57BL/6J (N = 6 and 10), and CBA/J (N = 7 and 10) strains. The conscious mice were a subset of the anesthetized mice, and were fewer because of difficulties obtaining IOP measurements in conscious animals (see text and Table 2 for further details). IOP was corrected as described in Equation 2. Error bars: SD.
Figure 3
 
IOP measured in conscious (white) and anesthetized (black) mice from BALB/cJ (N = 8 and 10), C57BL/6J (N = 6 and 10), and CBA/J (N = 7 and 10) strains. The conscious mice were a subset of the anesthetized mice, and were fewer because of difficulties obtaining IOP measurements in conscious animals (see text and Table 2 for further details). IOP was corrected as described in Equation 2. Error bars: SD.
Table 2
 
IOP, C, Conventional Outflow Resistance (R), and Fu for Each Murine Strain (Mean ± SD)
Table 2
 
IOP, C, Conventional Outflow Resistance (R), and Fu for Each Murine Strain (Mean ± SD)
Strain IOP, mm Hg C, μL/min/mm Hg R, mm Hg/μL/min Fu , μL/min
Conscious Anesthetized
BALB/cJ 10.6 ± 1.8, N = 8 10.1 ± 2.3, N = 10 0.0164 ± 0.0059, N = 10 62.1 ± 18.2, N = 9* −0.029 ± 0.041, N = 10
C57BL/6J 12.3 ± 1.0, N = 6 11.5 ± 2.4, N = 10 0.0147 ± 0.0029, N = 9 71.0 ± 16.6, N = 9 −0.031 ± 0.032, N = 9
CBA/J 14.5 ± 0.9, N = 7 12.1 ± 3.2, N = 10 0.0113 ± 0.0031, N = 9 96.6 ± 33.6, N = 9 −0.010 ± 0.022, N = 9
Conventional Outflow Facility (C) Measurements
Measurements of C were significantly different between strains (P = 0.05, 1-way ANOVA), as indicated by different slopes of the flow rate versus pressure relationship (Fig. 4). A post hoc Bonferroni test revealed that C was significantly larger in BALB/cJ compared to CBA/J (P = 0.049), with C57BL/6J having an intermediate value of C (Table 2). Importantly, the ranking of C between strains was opposite to the ranking of IOP, suggesting that strains that had a higher conscious IOP also had a lower C. There was no difference in the intercept of the flow-versus-pressure relationship (Fu ) between strains (P = 0.363, 1-way ANOVA). 
Figure 4
 
The average flow rate at each pressure step for BALB/cJ (N = 10), C57BL/6J (N = 9), and CBA/J (N = 9). Trend lines are plotted as the slope (C) and intercept (Fu ) averaged over all eyes that were perfused for each strain (data given in Table 2). Flow rate and perfusion pressure correspond to F and Pp as defined in equation 3. Error bars: SD.
Figure 4
 
The average flow rate at each pressure step for BALB/cJ (N = 10), C57BL/6J (N = 9), and CBA/J (N = 9). Trend lines are plotted as the slope (C) and intercept (Fu ) averaged over all eyes that were perfused for each strain (data given in Table 2). Flow rate and perfusion pressure correspond to F and Pp as defined in equation 3. Error bars: SD.
To investigate the relationship between IOP and C, conscious IOP was plotted against outflow resistance (the mathematical inverse of C, or 1/C) as measured within individual mice (Fig. 5A, see Discussion for rationale). The relationship between IOP and 1/C was highly significant (P = 0.0003, 2-sided test for bivariate correlation, SPSS; IBM), demonstrating that larger conscious IOP was associated with increased outflow resistance. One data point (filled triangle in Fig. 5A) was found to be an outlier (probability of studentized deleted residual was 0.00132 ≤ 0.05/N = 0.00357, see Methods) and was excluded from the regression, but the regression remained significant even if the outlier was retained (P = 0.006). The R 2 value of the regression in Figure 5A indicated that 70% of the variation in IOP between individual mice from three different strains can be attributed to variation in outflow resistance (excluding the one outlier). 
Figure 5
 
(A) Conscious IOP versus conventional outflow resistance (1/C) measured within individual mice for BALB/cJ (N = 6), C57BL/6J (N = 5), and CBA/J (N = 3). These data include only those mice where conscious IOP and C were measured in the same individual. The trendline and equation represents the linear regression of IOP versus 1/C excluding the one outlier (indicated by the filled triangle, see Equation 5). The slope of the regression provides an estimate of the conventional volumetric outflow rate (Fc = 0.068 ± 0.013 μL/min, see Equation 5), while the intercept provides an estimate of EVP (7.1 ± 1.0 mm Hg), assuming constant values for all mice. (B) The mean conscious IOP ( Image not available ) versus mean outflow resistance ( Image not available = the mean value of 1/C) measured for the entire set of BALB/cJ (N = 8 for Image not available and N = 9 for Image not available , with one outlier excluded), C57BL/6J (N = 6 and 9), and CBA/J (N = 7 and 9). These data include all mice of each strain where conscious IOP and C were measured, not necessarily in the same individual. The trendline and equation represents the linear regression of Image not available versus Image not available using only three data points (one per strain, see Equation 6). The population-based estimates of Fc (0.108 ± 0.022 μL/min) and EVP (4.2 ± 1.7 mm Hg) are not statistically different from the estimates based on individual eyes. Error bars: SD. See Discussion for further details.
Figure 5
 
(A) Conscious IOP versus conventional outflow resistance (1/C) measured within individual mice for BALB/cJ (N = 6), C57BL/6J (N = 5), and CBA/J (N = 3). These data include only those mice where conscious IOP and C were measured in the same individual. The trendline and equation represents the linear regression of IOP versus 1/C excluding the one outlier (indicated by the filled triangle, see Equation 5). The slope of the regression provides an estimate of the conventional volumetric outflow rate (Fc = 0.068 ± 0.013 μL/min, see Equation 5), while the intercept provides an estimate of EVP (7.1 ± 1.0 mm Hg), assuming constant values for all mice. (B) The mean conscious IOP ( Image not available ) versus mean outflow resistance ( Image not available = the mean value of 1/C) measured for the entire set of BALB/cJ (N = 8 for Image not available and N = 9 for Image not available , with one outlier excluded), C57BL/6J (N = 6 and 9), and CBA/J (N = 7 and 9). These data include all mice of each strain where conscious IOP and C were measured, not necessarily in the same individual. The trendline and equation represents the linear regression of Image not available versus Image not available using only three data points (one per strain, see Equation 6). The population-based estimates of Fc (0.108 ± 0.022 μL/min) and EVP (4.2 ± 1.7 mm Hg) are not statistically different from the estimates based on individual eyes. Error bars: SD. See Discussion for further details.
Discussion
In our study, we provided evidence that differences in IOP between three genetically distinct strains of mice are attributable largely to differences in C. Our data showed that the strain having highest IOP (CBA/J) had the lowest outflow facility, and, conversely, the strain having the lowest IOP (BALB/cJ) had the highest outflow facility. The strain having an intermediate value of IOP (C57BL/6J) had an intermediate value of outflow facility. Regression analysis applied to individual mice from all three strains revealed a highly significant correlation between conscious IOP and conventional outflow resistance (1/C), with 70% of the variability in IOP being attributable to differences in conventional outflow resistance as predicted by Goldmann's equation. 
Intraocular Pressure
Our IOP data showed clear differences among three inbred strains, in good agreement with prior reports. Using the data reported by Savinova et al., 23 IOP for BALB/cJ, C57BL/6J, and CBA/J under general isoflurane anesthesia is approximately 11, 13, and 16 mm Hg, respectively (read from Fig. 1 of the report of Savinova et al.23). Similarly, Wang et al. 24 reported IOP in conscious BALB/c, C57BL/6, and CBA as 10.6, 13.3, and 16.4 mm Hg. These values are in reasonable agreement with our conscious IOP measurements reported in Table 2. In regard to anesthesia, our results suggested that the IOP-lowering effect of isoflurane anesthesia varies between strains, with a larger IOP reduction observed in strains having a higher baseline IOP, consistent with prior reports. 9,24 Importantly, these data demonstrated that IOP differences between strains can be masked by anesthesia, and, thus, the anesthetic regimen is an important consideration for future studies examining aqueous humor dynamics in mice. 9  
Our preliminary studies examined IOP in a fourth strain, CBA/CaJ, chosen based on prior reports 23 suggesting that CBA/CaJ mice exhibit a high baseline IOP (19.3 mm Hg). In our hands, however, CBA/CaJ mice exhibited an intermediate IOP of 11.7 ± 1.3 mm Hg (N = 4) that was not dissimilar from IOP in C57BL/6J (12.3 ± 1.0 mm Hg), and, therefore, we replaced CBA/CaJ with CBA/J. It is unclear why our IOP measurements in CBA/CaJ were lower than reported by Savinova et al., 23 but this difference may be attributable to age because Savinova et al. 23 used mice between 6 and 12 months, compared to our study that measured IOP in mice between 3 and 4 months. Consistent with our results, another group also reported intermediate IOP values for CBA/CaJ mice as measured via telemetry (12.4 ± 2 mm Hg averaged over the entire light cycle from 6 AM to 6 PM), but age was not specified in that study. 34  
Conventional Outflow Facility (C)
Our data showed that CBA/J mice that have a greater IOP also have a lower C, while BALB/cJ mice that have a lower IOP also have a greater C, relative to C57BL/6J mice. Therefore, differences in C can account for a portion of the differences in IOP between strains (see quantitative analysis below). Our measurements of C also are in good agreement with published values for C57BL/6J (0.0105 μL/min/mm Hg 35 ) and BALB/c(J) (0.006–0.018 μL/min/mm Hg 8,9 ), although we noted that the literature values are rather ambiguous because they span a rather large 3-fold range, which makes comparison difficult. To the best of our knowledge, there are no prior reports of C in CBA/J mice. 
These data raised the interesting question of whether morphologic differences in the conventional outflow pathway between strains may underlie the differences in C. Savinova et al. 23 also examined this question in the context of strain-dependent differences in IOP, and found no obvious anatomic or pathologic differences in the drainage angle structures between strains. Other studies, however, have reported differences in gross ocular anatomy between strains, including anterior chamber depth, axial length, and corneal thickness. 36 We, ourselves, attempted a comparison of trabecular meshwork and Schlemm's canal between strains, but the histologic sections were of insufficient quality to allow a proper analysis. 
It is interesting that the albino BALB/cJ strain exhibited the lowest IOP and highest outflow facility compared to the pigmented strains CBA/J and C57BL/6J. Pigment typically accumulates in the trabecular meshwork and phagocytosed pigment granules are observed commonly in trabecular meshwork cells. 37 Heavy pigmentation of the trabecular meshwork occurs in pigmentary dispersion syndrome, and excess pigment load may lead to acute obstruction of the trabecular meshwork. 38 Thus, it is possible that differences in pigmentation or the cellular response to pigment may underlie the differences in outflow facility and IOP between pigmented and nonpigmented strains. Future studies including a larger number of strains would be needed to examine more thoroughly the relationship between pigmentation and outflow facility in mice. 
It should be noted that our measurements of C were done ex vivo using enucleated eyes, while most other studies measured C in vivo under anesthesia. Following enucleation, one safely may assume that aqueous production is eliminated (Fin = 0) and the episcleral veins are open to the atmosphere (EVP = 0 mm Hg). Enucleated eyes, therefore, provide a somewhat more direct means to measure C without possible confounding effects of Fin or EVP that may influence in vivo measurements. However, we must assume that C measured in enucleated eyes is similar to C in vivo. This assumption often is taken for granted in eyes of larger species, such as humans and rabbits, 39 where measurements of C in enucleated eyes are consistent with in vivo measurements of C by tonography or intracameral perfusion under anesthesia. For mice, however, to our knowledge no studies have yet compared C directly between enucleated and in vivo eyes. However, Millar et al. 8 have shown that C is unaffected by euthanization via anesthetic overdose in BALB/cJ mice perfused in situ without enucleation. 
Somewhat surprisingly, values of C measured in our study were significantly larger than reported previously by our own group 16,31 for enucleated C57BL/6 eyes (0.0062–0.0091 μL/min/mm Hg). These differences likely were caused by differences in temperature and hydration of the eye during perfusion. In our current study, the eye was immersed in a bath of isotonic saline at physiologic temperature. However, in our previous studies, 16,31 the eyes were perfused at room temperature outside of a bath, with the eye covered by tissue paper that was moistened with isotonic saline. In our earlier studies, 16,31 the facility at physiologic temperature was estimated by multiplying the slope of the flow-versus-pressure relationship measured at room temperature by a factor (∼1.4) proportional to the temperature-dependent change in perfusate viscosity. 40 However, it is possible that effects other than viscosity may influence outflow facility differences between room and physiologic temperature. Furthermore, the zero-pressure intercept of the flow-versus-pressure relationship (Fu in Equation 3) in this study was not significantly different from zero, in contrast with our previous studies, 16,31 where this intercept was significantly larger than zero (0.035–0.157 μL/min). This intercept often is interpreted as the quantity of outflow passing through the unconventional or pressure-independent pathway. 16,31 It is important to recognize that this intercept is an extrapolated value that is estimated based on flow rate measurements at nonzero pressures, and measurements at higher pressures may have significant leverage on the estimate of Fu . Osmotic differences between the intraocular and extraocular compartments also may influence the zero-pressure intercept by driving flow in the absence of an applied pressure gradient. Moreover, if the apparent intercept is, indeed, so strongly dependent upon hydration, temperature, and/or osmolarity, then the accuracy and interpretation of the extrapolated intercept (often taken to represent unconventional outflow) may be drawn into question, at least based on studies in enucleated eyes. Ongoing studies are examining how temperature, hydration, and osmolarity affect C and Fu in an attempt to resolve this important issue. 
Relationship Between IOP and C
Plotting IOP versus 1/C as shown in Figure 5A provides a physically meaningful interpretation, as can be seen using the modified Goldmann's equation:  where Fin is the aqueous production rate, EVP is episcleral venous pressure, and Fu is the outflow rate through the unconventional or pressure-independent pathway. All parameters in Equation 4 apply to conscious mice. Solving Equation 4 for IOP yields:  where Fc is the outflow rate through the conventional pathway (Fc = FinFu). Assuming that C measured in enucleated eyes represents C in vivo (see above), Goldmann's equation thereby predicts a linear relationship between IOP and conventional outflow resistance (1/C), with the slope of the relationship equal to Fc and the zero-IOP intercept equal to EVP. This method, therefore, allows indirect estimates of Fc and EVP based on measurements of C and IOP from a sample population of mice (or any species for that matter).  
If we assume that Fc and EVP are constant between C57BL/6J, BALB/cJ, and CBA/J mice, then the regression in Figure 5A predicts (excluding the outlier) that Fc = 0.068 ± 0.044 μL/min and EVP = 7.1 ± 3.2 mm Hg (mean ± SD). Whether Fc and EVP truly are constant between strains is an open question. However, the strong agreement between Equation 5 and our data (R2 = 0.70, N = 13 mice) suggests that Fc and EVP are reasonably consistent between C57BL/6, BALB/cJ, and CBA/J strains, or that any variation in Fc and EVP between strains has relatively minor influence (≤30%) on IOP compared to the principal dependence of IOP on 1/C. We point out that a portion of the remaining 30% influence is likely attributable to experimental uncertainty in the measurements themselves. The value of EVP estimated by linear regression (7.1 mm Hg) agrees fairly well, but tends to be slightly higher than published values from the same strains using the blood-reflux technique 21 (compare to 5.4 mm Hg for BALB/cJ 8 and 6.3 mm Hg for C57BL/6J35) that requires anesthesia where EVP may be depressed artificially. The value of Fc estimated by linear regression (0.068 μL/min) also fits within the published range for Fc that varies 3-fold between strains (0.032 μL/min in NIH Swiss White, 21 0.060 μL/min for C57BL/6, 35 and 0.111 μL/min for BALB/cJ8), which may be related to methodologic differences between studies or true physiologic differences between strains. It should be pointed out that Fc lumps effects of Fin and Fu , such that variations in Fin between strains may be masked by compensatory differences in Fu (or vice versa), and this approach, therefore, is unable to make inferences about Fin or Fu without additional data beyond C and IOP. We attempted to perform regression analysis on each strain individually, so as to estimate values of Fc and EVP for each strain, but the small numbers of mice (3–6 per strain) made this analysis unreliable. 
As an alternative to the regression analysis of individual eyes presented above, one also may examine a population-based analysis to investigate whether differences in the mean value of 1/C can account for differences in mean conscious IOP between strains. One advantage of the population-based analysis is that it uses all of our data for 1/C and conscious IOP (35 mice), not just data where 1/C and conscious IOP were measured in the same mouse (14 mice). One disadvantage, however, is that the number of data points in the population analysis is limited to the number of strains included in the study. Because Goldmann's equation essentially is a statement of the conservation of aqueous humor mass that is strictly true only for individual eyes (and not populations of eyes), Goldmann's equation must be expressed in terms of population averages (or expected values). Taking the expected value of Equation 5 and neglecting any covariance between 1/C and Fc yields:  where the overbars indicate population averages for a given strain, and Display FormulaImage not available represents the average conventional outflow resistance (average value of 1/C, Table 2). Assuming that Display FormulaImage not available and Display FormulaImage not available are the same for all strains, then Equation 6 predicts a linear relationship between Display FormulaImage not available and Display FormulaImage not available with a slope equal to Display FormulaImage not available and an intercept equal to Display FormulaImage not available . As shown in Figure 5B, Display FormulaImage not available and Display FormulaImage not available from the three strains do, indeed, exhibit a linear relationship with 96% of the variation in Display FormulaImage not available between strains attributable to the variation in Display FormulaImage not available . However, with only three data points, there is only one degree of freedom for a linear fit, and, thus, the R2 value (0.96) is almost certainly an overestimate of the true relationship between Display FormulaImage not available and Display FormulaImage not available (compare R2 = 0.70 as predicted based on individual eyes; Fig. 5A). For the same reason, the predicted values of Display FormulaImage not available (0.108 ± 0.022 μL/min) and Display FormulaImage not available (4.2 ± 1.7 mm Hg) based on the population analysis are likely inaccurate, but nevertheless are not statistically different from the values predicted based on regression analysis of individual eyes (P > 0.22). More strains would be needed in the population-based analysis to improve these predictions.  
In conclusion, our data suggest that differences in IOP between three genetically distinct strains of mice are attributable largely to differences in C, rather than to differences in aqueous inflow, episcleral venous pressure, or unconventional outflow. These data motivate further studies to identify the morphologic or genetic basis that underlie the strain-dependent differences in C to help illuminate the mechanisms of IOP regulation. 
Acknowledgments
The authors thank the donors of National Glaucoma Research, and C. Ross Ethier (Georgia Institute of Technology) for thoughtful advice and comments. 
Supported by grants from National Glaucoma Research, a Program of The BrightFocus Foundation (Formerly the American Health Assistance Foundation), the National Eye Institute (EY022359), Entente Cordiale Scholarship from the British Council, and the Santander Mobility Award from Santander Bank. 
Disclosure: A. Boussommier-Calleja, None; D.R. Overby, None 
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Figure 1
 
Calibration data from the rebound tonometer using cadaveric eyes in situ (two eyes per strain). Each eye was cannulated and manometric IOP m was controlled by a fluid reservoir, while tonometric IOP t was measured using the “manually-calculated” method (see Methods). The calibration relationship was not different between strains, and, therefore, data from all strains were lumped together to obtain the linear regression shown in the Figure. All tonometric IOP data in this study were corrected according to the inverse of this linear regression (Equation 2). Each data point refers to a single manually-calculated IOP measurement from a single eye.
Figure 1
 
Calibration data from the rebound tonometer using cadaveric eyes in situ (two eyes per strain). Each eye was cannulated and manometric IOP m was controlled by a fluid reservoir, while tonometric IOP t was measured using the “manually-calculated” method (see Methods). The calibration relationship was not different between strains, and, therefore, data from all strains were lumped together to obtain the linear regression shown in the Figure. All tonometric IOP data in this study were corrected according to the inverse of this linear regression (Equation 2). Each data point refers to a single manually-calculated IOP measurement from a single eye.
Figure 2
 
A typical perfusion tracing showing the flow rate (solid tracing, left axis) and pressure (dashed tracing, right axis) as a function of time for one enucleated eye of a C57BL/6J mouse. The pressure increases in steps of 4, 8, 15, and 25 mm Hg, while the flow rate increases in proportion to the increase in pressure. The spikes in flow rate are due to rapid adjustments of the syringe pump as it attempts to maintain the desired pressure. The average flow rate at each pressure step is calculated by averaging over at least 10 minutes of data, excluding the spike values.
Figure 2
 
A typical perfusion tracing showing the flow rate (solid tracing, left axis) and pressure (dashed tracing, right axis) as a function of time for one enucleated eye of a C57BL/6J mouse. The pressure increases in steps of 4, 8, 15, and 25 mm Hg, while the flow rate increases in proportion to the increase in pressure. The spikes in flow rate are due to rapid adjustments of the syringe pump as it attempts to maintain the desired pressure. The average flow rate at each pressure step is calculated by averaging over at least 10 minutes of data, excluding the spike values.
Figure 3
 
IOP measured in conscious (white) and anesthetized (black) mice from BALB/cJ (N = 8 and 10), C57BL/6J (N = 6 and 10), and CBA/J (N = 7 and 10) strains. The conscious mice were a subset of the anesthetized mice, and were fewer because of difficulties obtaining IOP measurements in conscious animals (see text and Table 2 for further details). IOP was corrected as described in Equation 2. Error bars: SD.
Figure 3
 
IOP measured in conscious (white) and anesthetized (black) mice from BALB/cJ (N = 8 and 10), C57BL/6J (N = 6 and 10), and CBA/J (N = 7 and 10) strains. The conscious mice were a subset of the anesthetized mice, and were fewer because of difficulties obtaining IOP measurements in conscious animals (see text and Table 2 for further details). IOP was corrected as described in Equation 2. Error bars: SD.
Figure 4
 
The average flow rate at each pressure step for BALB/cJ (N = 10), C57BL/6J (N = 9), and CBA/J (N = 9). Trend lines are plotted as the slope (C) and intercept (Fu ) averaged over all eyes that were perfused for each strain (data given in Table 2). Flow rate and perfusion pressure correspond to F and Pp as defined in equation 3. Error bars: SD.
Figure 4
 
The average flow rate at each pressure step for BALB/cJ (N = 10), C57BL/6J (N = 9), and CBA/J (N = 9). Trend lines are plotted as the slope (C) and intercept (Fu ) averaged over all eyes that were perfused for each strain (data given in Table 2). Flow rate and perfusion pressure correspond to F and Pp as defined in equation 3. Error bars: SD.
Figure 5
 
(A) Conscious IOP versus conventional outflow resistance (1/C) measured within individual mice for BALB/cJ (N = 6), C57BL/6J (N = 5), and CBA/J (N = 3). These data include only those mice where conscious IOP and C were measured in the same individual. The trendline and equation represents the linear regression of IOP versus 1/C excluding the one outlier (indicated by the filled triangle, see Equation 5). The slope of the regression provides an estimate of the conventional volumetric outflow rate (Fc = 0.068 ± 0.013 μL/min, see Equation 5), while the intercept provides an estimate of EVP (7.1 ± 1.0 mm Hg), assuming constant values for all mice. (B) The mean conscious IOP ( Image not available ) versus mean outflow resistance ( Image not available = the mean value of 1/C) measured for the entire set of BALB/cJ (N = 8 for Image not available and N = 9 for Image not available , with one outlier excluded), C57BL/6J (N = 6 and 9), and CBA/J (N = 7 and 9). These data include all mice of each strain where conscious IOP and C were measured, not necessarily in the same individual. The trendline and equation represents the linear regression of Image not available versus Image not available using only three data points (one per strain, see Equation 6). The population-based estimates of Fc (0.108 ± 0.022 μL/min) and EVP (4.2 ± 1.7 mm Hg) are not statistically different from the estimates based on individual eyes. Error bars: SD. See Discussion for further details.
Figure 5
 
(A) Conscious IOP versus conventional outflow resistance (1/C) measured within individual mice for BALB/cJ (N = 6), C57BL/6J (N = 5), and CBA/J (N = 3). These data include only those mice where conscious IOP and C were measured in the same individual. The trendline and equation represents the linear regression of IOP versus 1/C excluding the one outlier (indicated by the filled triangle, see Equation 5). The slope of the regression provides an estimate of the conventional volumetric outflow rate (Fc = 0.068 ± 0.013 μL/min, see Equation 5), while the intercept provides an estimate of EVP (7.1 ± 1.0 mm Hg), assuming constant values for all mice. (B) The mean conscious IOP ( Image not available ) versus mean outflow resistance ( Image not available = the mean value of 1/C) measured for the entire set of BALB/cJ (N = 8 for Image not available and N = 9 for Image not available , with one outlier excluded), C57BL/6J (N = 6 and 9), and CBA/J (N = 7 and 9). These data include all mice of each strain where conscious IOP and C were measured, not necessarily in the same individual. The trendline and equation represents the linear regression of Image not available versus Image not available using only three data points (one per strain, see Equation 6). The population-based estimates of Fc (0.108 ± 0.022 μL/min) and EVP (4.2 ± 1.7 mm Hg) are not statistically different from the estimates based on individual eyes. Error bars: SD. See Discussion for further details.
Table 1
 
Parameters of the Tonometer Calibration as Described by Equation 1
Table 1
 
Parameters of the Tonometer Calibration as Described by Equation 1
Strain α β, mm Hg R 2
BALB/cJ, N = 2 1.0 −0.9 0.97
C57BL/6J, N = 2 0.9 0.7 0.98
CBA/J, N = 2 1.1 −0.1 0.95
All strains, N = 6 1.0 −0.1 0.96
Table 2
 
IOP, C, Conventional Outflow Resistance (R), and Fu for Each Murine Strain (Mean ± SD)
Table 2
 
IOP, C, Conventional Outflow Resistance (R), and Fu for Each Murine Strain (Mean ± SD)
Strain IOP, mm Hg C, μL/min/mm Hg R, mm Hg/μL/min Fu , μL/min
Conscious Anesthetized
BALB/cJ 10.6 ± 1.8, N = 8 10.1 ± 2.3, N = 10 0.0164 ± 0.0059, N = 10 62.1 ± 18.2, N = 9* −0.029 ± 0.041, N = 10
C57BL/6J 12.3 ± 1.0, N = 6 11.5 ± 2.4, N = 10 0.0147 ± 0.0029, N = 9 71.0 ± 16.6, N = 9 −0.031 ± 0.032, N = 9
CBA/J 14.5 ± 0.9, N = 7 12.1 ± 3.2, N = 10 0.0113 ± 0.0031, N = 9 96.6 ± 33.6, N = 9 −0.010 ± 0.022, N = 9
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