duration of anesthesia affects intraocular pressure, but not outflow facility in mice

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INTRODUCTION

The leading risk factor for the development of glaucoma is elevated intraocular pressure (IOP) caused by increased resistance to aqueous humor outflow.1 Transgenic mice provide effective tools for exploring glaucoma and abnormalities in aqueous

humor drainage. Mice exhibit anatomical similarities to humans with the presence of both trabecular and uveoscleral drainage pathways.2–3 Therefore, the study of aqueous humor dynamics in mice offers exciting opportunities to understand the disease process and to develop new treatment strategies.

Outflow facility (C) is difficult to measure in the mouse due to the challenges of measuring minute changes in flow and/or pressure in such small eyes. Several methods have been described recently. A constant pressure perfusion technique measured the outflow of fluid draining through the eye over time

Current Eye Research, 35(9), 819–827, 2010Copyright © 2010 Informa Healthcare USA, Inc.ISSN: 0271-3683 print/ 1460-2202 onlineDOI: 10.3109/02713683.2010.494241

ORIGINAL ARTICLE

Duration of Anesthesia Affects Intraocular Pressure, But Not Outflow Facility in Mice

Lucinda J. Camras, Kari E. Sufficool, Carl B. Camras, Shan Fan, Hong Liu, and Carol B. Toris

Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA

ABSTRACT

Purpose: The study of aqueous humor dynamics (AHD) in mice is becoming more prevalent as more strains with elevated intraocular pressure (IOP) are developed. High IOP is usually associated with reduced outflow facility making this one of the more important AHD parameters to evaluate. Ocular measurements in mice require anesthesia that has profound effects on IOP but unknown effects on outflow facility. This study evaluates the effects of anesthesia duration and latanoprost treatment on outflow facility and IOP in BALB/c mice.Methods: IOPs were measured in conscious and anesthetized mice by tonometry. Outflow facility was evaluated in 15-min intervals at three pressure levels over two 45-min periods. Comparisons were made between latanoprost-treated eyes and untreated contralateral eyes. To determine the effect of anesthesia duration on IOP, a microneedle method was used to follow IOP for 120 min in separate mice.Results: IOP was 9.7 ± 0.3 mmHg (mean ± SEM) in conscious mice and 7.1 ± 0.02 within 10 min of anesthesia initiation (p < 0.01). IOP changed significantly between but not within assessment periods. IOP at 75 min was significantly (p = 0.004) reduced compared to IOP at 15 min after initial anesthesia. In control eyes, outflow facility did not change between the two 45-min assessment periods during the 120 min test (p = 0.80). In latanoprost-treated eyes, outflow facility increased compared with control eyes during both assessment periods (p = 0.03). A test of filters in series with known resistance found that the method was sensitive enough to detect a change in outflow facility of 0.001 μl/min/mmHg.Conclusions: Administration of ketamine/xylazine anesthesia for 120 min did not alter outflow facility or lessen the effect of latanoprost on outflow facility in mice as determined by a new analysis system. Accurate IOP measurements must be made within minutes of anesthesia administration but outflow facility measurements can be made with less haste.

KEYWORDS: Anesthesia; Intraocular pressure; Mouse; Outflow facility; Prostaglandin

Received 22 December 2009; accepted 13 May 2010

Correspondence: Carol B Toris, Ph.D., Department of Ophthalmology, 985840 Nebraska Medical Center, Omaha, NE 68198-5840. E-mail: [email protected]

22 December 2009

13 May 2010

© 2010 Informa Healthcare USA, Inc.

2010

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10.3109/02713683.2010.494241

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mailto:[email protected]

820 L. J. Camras et al.


at two target pressures to determine outflow facility.3–5 Another method infused fluid into the anterior chamber at three different flow rates determined by empirical data to maintain set levels of IOP.6 These techniques were sensitive enough to detect differences in outflow facility between wild-type and knockout mice4,6 and changes with ophthalmic drugs.5 The current study describes a method to measure outflow facility in mice using a fluid column perfusion system and a new analysis technique. The system and analy-sis technique were tested using varying numbers of Millipore filters connected in series to create different levels of resistance. BALB/c mice were used for the assessment. Both anesthetized and awake BALB/c mice demonstrate lower IOP than other strains of mice.7–10 Therefore, if the method successfully found changes in IOP and outflow facility in this strain, it should work in other strains with higher IOPs because the magnitude of an experimental change should be greater and easier to detect.

A serious confounding factor in the measure-ment of outflow facility in mice is anesthesia. Ketamine/xylazine anesthesia in mice rapidly decreases IOP2,11,12 but has unknown effects on outflow facility. The purpose of the current study was to determine the effect of anesthesia duration on IOP using microcannulation, and outflow facility using the fluid column perfusion system and our new analysis method. Additionally, the system was tested in eyes treated with latanoprost to evaluate whether anesthesia would attenuate the drug effect on outflow facility over time and whether the new analysis system is sensitive enough to detect a change.

METHODS

Perfusion System

Glass microneedles were made with a micropipette puller (Model p-87, Sutter Instrument Co, Novato, California, USA) and beveller (World Precision Instruments, Sarasota, Florida, USA). Each micronee-dle was placed in a micropipette manipulator (World Precision Instruments), which was attached to pres-sure tubing (0.05 inch inner diameter (ID) tube, Mall-inckrodt, Hazelwood, Missouri, USA) leading to a three-way stopcock. The stopcock was connected to a 10-mL syringe filled with balanced salt solution and to pressure tubing connected to a second three-way stopcock. A pressure transducer (Honeywell model 140 PC, Honeywell Sensing and Control, Freeport, Illinois, USA) and a vertical fluid column (Intramedics Polyethylene Tubing 0.58 mm ID, Becton Dickinson

and Co, Sparks, Maryland, USA) were connected to the second stopcock. The transducer was connected to a PowerLab data acquisition system (ML870/P PowerLab 8/30, ADInstruments, Colorado Springs, Colorado, USA), running PowerLab software on a computer (Figure 1). The height of the fluid in the column was adjusted using the attached fluid-filled syringe. Transducers were calibrated prior to each experiment to ensure accurate pressure measure-ments. The outflow facility was measured using a “constant pressure” perfusion method. The fluid height of the column set the pressure in the system. The fluid exiting the system was proportional to the decline in height of the column or slight decline in pressure over time, and thereby was used to deter-mine the flow rate based on the dimensions of the fluid column (3.59 μL/mmHg). Although the pressure declined during the perfusion at each pressure level, this method was still considered a constant pressure perfusion since the pressure did not decrease more than 1.0 mmHg over the 15-min interval in which it was assessed.

Perfusion System Assessment

To test the system, Millipore filters (Cellulose Acetate Syringe Filters, Sterlitech Corporation, Kent, Washington, USA) were arranged in series of 60, 70, 80, 85, 90, 95, and 100 filters to mimic a range of outflow facility values. Obeying Ohm’s law, filters arranged in series have an additive resistance (inverse of outflow facility). The filters were connected to the perfusion system and wetted to ensure there was no additional fluid absorption during outflow measurements (Figure 1). The fluid in the column was brought to three pressure levels (15, 25, and 35 mmHg), and the pressure decline over 15-min intervals was measured (Figure 2). The slope of pressure over time at each pressure level was converted to a flow rate using a conversion factor of 3.59 μL/mmHg. The correspond-ing pressures were averaged within the timeframe of the flow assessment. The linear fit of the flow rates plotted against their corresponding pressures was equivalent to the outflow facility of each series of filters based on Goldmann’s equation (Equation (1)):

C FIOP P

t

ev

=−

(1)

where IOP is intraocular pressure; C is outflow facility; Ft is trabecular outflow; and Pev is episcleral venous pressure. In this system of simulated outflow facility, Pev was zero and IOP was the pressure set by the fluid column (Pc). Additionally, Ft was the flow through

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Outflow Facility in Mice 821


the filters and was equivalent to the flow out of the column (Fc):

C FP

c

c

=

(2)

Based on these assumptions, outflow facility was determined from the slope of the flow versus pressure curve. Since the inverse of the outflow facility is equivalent to the resistance, a linear regression of the resistance of the filters in series and the number of filters was used to determine the sensitivity of the system.

Animal Husbandry

All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Oph-thalmic and Vision Research and regulations outlined by the Animal Care and Use Committee of the University of Nebraska Medical Center. BALB/c mice ranging in age from 8 to 24 months and weighing between 22 and 30 grams were used in this study. Housing rooms were kept at 21°C with a 12-hr light (6 AM to 6 PM) and 12-hr dark cycle. All measurements were performed between 12 PM and 7 PM in consideration of the mouse’s diurnal IOP rhythm. Anesthetized mice were kept warm throughout the measurements with a mouse heating pad set between 30 and 32°C.

Anesthesia

One 10 μL drop of topical proparacaine (Proparacaine-HCl 0.5%, Akorn, Inc, Buffalo Grove, Illinois, USA) was

applied to the cornea prior to all ocular measurements. Intraperitoneal injections of ketamine (100 mg/kg) and xylazine (9 mg/kg) were administered prior to invasive measurements. Supplemental doses were administered as needed. A sensor clip was placed on the mouse’s thigh to obtain oxygen saturation and heart rate (Mouse Ox™, Revision 5.1, STARR Life Sciences Corp, Oakmont, Pennsylvania, USA). These parameters were used to monitor the level of anesthesia for the duration of the experiment.

Outflow Facility Measurement

The time of the initial injection of anesthetics was considered time zero. Measurements were made over a 45-min period in one eye starting at approximately 15 min from time zero and in the contralateral eye starting at approximately 75 min from time zero (Figure 3A). The zero pressure reference point was acquired by placing the tip of the needle in the tear film. The microneedle was then placed in the anterior chamber and IOPs were set at pressures of 15, 25, or 35 mmHg. The pressure decline in the eye was monitored at 15-min intervals. At the end of the experiment, the needle again was placed in the tear-film to confirm the zero pressure reference point. The slope of the pressure drop (mmHg/min) during each time interval was mea-sured at three IOPs set by the fluid column. The eyes stabilized within the first 1 to 2 min in each interval. Therefore, the data collected during the first 5 min of each interval were excluded. The exact dimensions of the fluid column were used to calculate a conversion factor (3.59 µL/mmHg) to convert pressure drops

ComputerTransducer

Power Lab

Syringe

Water Column,0.58 mm ID

Mouse

Or

Millipore filters

FIGURE 1 Fluid column perfusion system used for measurement of outflow facility and intraocu-lar pressure in BALB/c mice or outflow facility in Millipore filters. A microneedle attached to pressure tubing is connected to a fluid column and syringe so that pressure levels can be set to perform a constant pressure perfusion. A transducer connected to PowerLab transmits the data to a computer interface running PowerLab software, where it can be recorded and analyzed. The filters were used to simulate a range of outflow facilities and test the system. Obeying Ohm’s Law, different numbers of filters arranged in series created an additive resistance. The inverse of the resistance produces an outflow facility that can be detected by the fluid column perfusion system.

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into flow rates (µL/min). The outflow facility was calculated from the Goldmann equation (Equation (1)) and knowledge that, when in equilibrium, the inflow of fluid into the eye is equivalent to outflow of fluid from the eye (Equation (3)):

F F F Ft uc a+ = + , (3)

where Fc is inflow from the fluid column; Fa is aqueous production; Ft is trabecular outflow; and Fu

is uveoscleral outflow. Equation (3) was rearranged as Equation (4) to determine trabecular outflow:

Ft c u a= − +F F F .

(4)

Equation (4) was substituted into Equation (1) to yield Equation (5):

F C P C P F Fc ev ac u= − + −( ) ( ) .

(5)

The inflow from the fluid column was equivalent to the flow rate (Fc) determined by the system for each IOP set by the fluid column (Pc). A linear fit method was used to determine outflow facility by plotting the three Fc measurements calculated from the pressure decline versus the three Pc measurements calculated from the average pressure for each interval. Based on the assumption that aqueous production (Fa), uveoscleral outflow (Fu), and episcleral venous pressure (Pev) remained constant during the time in which outflow facility was assessed, the slope of the linear fit was equivalent to the outflow facility (Equation (5)). A two-tailed paired t-test was used to determine the statistical significance of the duration of anesthesia on outflow facility between the two 45-min periods (15 to 60 and 75 to 120 min from initial injection of anesthetic).

Intraocular Pressure Measurement

Intraocular pressure was measured at the time of the outflow facility measurements and in a separate group of mice in which outflow facility was not assessed. Following the application of topical proparacaine to the cornea, IOP was measured non-invasively using rebound tonometry (TonoLab Tonometer TV02, Tiolat Oy, Helsinki, Finland) in both awake and anesthetized mice. Measurements were repeated five times and averaged to obtain one IOP value for each eye. Measurements before and after administration of a ketamine/xylazine cocktail were compared by two-tailed paired t-tests.

Intraocular pressure was measured invasively with the aid of a dissecting microscope. The cor-nea was anesthetized with topical proparacaine and a microneedle (connected to a transducer and mounted on a micromanipulator) was inserted into the anterior chamber of one eye. The mouse tear film was set as the zero pressure reference point for the system. Two different experiments were conducted (Figure 3). In one group of mice, after initial anesthe-sia injection, IOP was followed for 45 min starting at 15 min in one randomly chosen eye and at 75 min in the contralateral eye. After each 45-min assessment, the microneedle was removed from the anterior

A

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FIGURE 2 Calculation of outflow facility using linear fit analysis. The outflow facility in mice was measured in 15-min intervals at target pressures of 15, 25, and 35 mmHg using a constant pressure perfusion method. The fluid height in the column set the pressure in the system. The fluid exiting the system was proportional to the decline in height of the column (i.e., decline in pressure over time). (A) The slope of the pressure drop was measured at three target pressures and converted into flow rates. The flow rate at each set pressure was calculated by converting the pressure drop at each level into a volume displacement (3.59 μL/mmHg) during a given timeframe. This conversion factor is based upon the dimensions of the fluid column. (B) The flow rates were plotted against their corresponding pressures. Based on Goldmann’s equation and assuming aqueous produc-tion, uveoscleral outflow, and episcleral venous pressure remained constant during the 45 min in which outflow facility was assessed; the outflow facility was equal to the slope of the linear fit of flow rate versus pressure.

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chamber and placed in the tear-film to confirm the integrity of the system. The data were excluded if the pressure in the tear film was not zero. In a second group of mice, IOP in one randomly chosen eye was followed invasively for up to 120 min after initial anesthesia injection. Averaged IOPs were obtained for each of the six 15-min intervals. Single factor ANOVA and Bonferroni comparison post-hoc tests were performed to determine statistical significance between and within groups.

IOP was monitored during the time in which outflow facility was assessed. When there was no inflow of fluid from the column and the pressure measured by the transducer was the spontaneous IOP

of the mouse, Fc was equal to zero and IOP replaced Pc in Equation (5) as shown in (Equation 6):

0 .= − + −C IOP C P F Fev u( ) ( ) a (6)

Equation (6) was combined with Equation (5) to yield Equation (7):

F C P IOPc c= −( ).

(7)

This is the effect that IOP has on outflow facility during the assessment period. A change in the average IOP during each time interval could introduce an error in the outflow facility calculation and prove the

Assessment period 1

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press

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m of co

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Confirm

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in tea

rfilm

FIGURE 3 Timeline of outflow facility measurement. (A) IOP was taken with TonoLab while mice were awake and anesthetized. Once anesthetized, the needle was zeroed in the tear film and then placed in the anterior chamber of one eye. The IOP was set at 15, 25, and 35 mmHg for 15 min each. When the 45-min assessment period was done, the needle was removed, re-zeroed in the tear film, and then placed in the contralateral eye. The pressure increases were repeated in the same manner for the contralateral as the initial eye. (B) IOPs were measured in awake mice with the TonoLab. One 5 μL drop of latanoprost was then placed in one eye, while the contralateral eye remained untreated. Two hours after drug treatment, IOP measurements were repeated in awake and anesthetized mice. The outflow facilities of both eyes were assessed simultaneously in two 45-min assessment periods in the same manner as described in A.C

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assumption to be false that aqueous production, uveoscleral outflow, and episcleral venous pressure remained constant.

Latanoprost Effect on Outflow Facility

Outflow facility was measured during two 45-min assessment periods, beginning at approximately 15 min and 75 min after initial anesthesia administra-tion (Figure 3B). Latanoprost ophthalmic solution (4 µL of 0.005%, 200 ng) was applied between 10 AM and 5 PM to one randomly assigned eye, while the contralateral eye was used as an untreated control. Measurements commenced 2 hr after drug application, a time of peak effect.5 In conscious animals, IOPs were obtained by TonoLab tonometry at baseline and at 90 min after latanoprost application. Invasive IOP measurements were obtained approximately 15 min after initial anesthesia administration. Treated and control eyes were compared by two-tailed paired t-tests.

RESULTS

Perfusion System Assessment

Resistance of the millipore filters arranged in series increased in a linear manner as more filters were added to the system (correlation coefficient of 0.99 [Figure 4A]). The equivalent outflow facility values for each filter series is summarized in Figure 4B. Outflow facilities simulated by the filters were higher than those found in mice. It was unrealistic to attempt to simulate outflow facilities in the range of a mouse eye (0.005 μL/min/mmHg) as approximately 550 filters would have been needed in the test. Nevertheless, with our system we were able to detect changes of less than 0.001 μL/min/mmHg, providing strong evidence that the fluid column perfusion system is sensitive enough to detect small changes in outflow facility in mice.

Outflow Facility Measurements

Using a linear fit method, mean outflow facility was 0.0055 ± 0.0004 μL/min/mmHg during 15 to 60 min and 0.0051 ± 0.0003 µL/min/mmHg during 75 to 120 min after onset of ketamine/xylazine anesthesia. There was no statistically significant difference (p = 0.80, n = 8) in outflow facility between the two assessment periods.

Intraocular Pressure Measurements

Intraocular pressure was measured by TonoLab tonom-etry with and without anesthesia. The average IOP was 9.7 ± 0.3 mmHg, which dropped significantly (p = 0.006, n = 9) by 2.7 ± 0.3 mmHg at 10 min under anesthesia. Invasive IOP (microneedle method) was monitored during the time of the outflow facility assessment in two separate groups of mice. In Group 1, IOP was measured invasively for 45 min in one eye beginning 15 min after initial anesthesia administration and in the contralateral eye beginning 75 min after initial anesthesia administration (n = 8). This was done to sim-ulate the assessment intervals utilized in the outflow facility study. Invasive IOP was 6.9 ± 0.6 mmHg and 3.8 ± 0.7 mmHg for the assessment period beginning at approximately 15 min and 75 min after time zero (initial anesthesia administration), respectively. This difference in IOP was statistically significant (p = 0.004). However, the average IOPs during the three 15-min intervals within each 45-min period were not signifi-cantly different (15–60 min: p = 0.34; 75–120 minutes: p = 0.73) from each other. In Group 2, invasive IOP was

Number of Filters

Res

ista

nce

(mm

Hg/

µg/m

in)

5030

35

40

45

50A

B

60 70 80

y = 0.340x + 12.692

R2 = 0.99

90 100 110

Number ofFilters in Series

Resistance(mmHg/µL/min)

60 33.270 36.680 39.185 41.790 43.995 44.4

100 47.0

Outflow Facility(µL/min/mmHg)

0.03010.02730.02560.02400.02280.02250.0213

FIGURE 4 Sensitivity of perfusion system. (A) Millipore filters arranged in series (60, 70, 80, 85, 90, 95, and 100 filters) were used to simulate a range of resistance. The system detected a linear increase in resistance with additional filters (R2 = 0.99). (B) The system could detect changes of outflow facility of less than 0.001 µL/min/mmHg.

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assessed in a single eye for up to 120 min after initial anesthesia administration (n = 9). Three mice died during the second 45-min period (75–120 min after anesthesia). The average IOPs within the seven 15-min intervals were not significantly different (p = 0.81) from one another. The average IOP for each time interval in both groups was not significantly different (15–60 min: p = 0.90, n = 8 (Group 1), n = 9 (Group 2); 75–120 min: p = 0.93, n = 8 (Group 1), n = 6 (Group2)) in either 45-min period (Figure 5).

Latanoprost Effect on Outflow Facility

For the initial 45-min assessment period, approxi-mately 15 to 60 min after time zero, the average outflow

facility was significantly increased (p = 0.03, n = 9) from 0.0038 ± 0.0005 µL/min/mmHg in the control eye to 0.0078 ± 0.0013 µL/min/mmHg in the eye treated with latanoprost. Similarly, the average outflow facility dur-ing the second 45-min assessment period, approximately 75 to 120 min after initial anesthesia administration, was significantly increased (p = 0.03, n = 7) from 0.0032 ± 0.0007 µL/min/mmHg in the control eye to 0.0079 ± 0.0016 µL/min/mmHg in the latanoprost-treated eye (Fig-ure 6). The outflow facilities of the control (p = 0.44) and latanoprost-treated eyes (p = 0.82) did not change over time. No effect of latanoprost was found on IOP measured non-invasively, because the baseline IOPs (7.4 ± 0.33 mmHg, n = 9) were below the lower limits of detection by the TonoLab tonometer. When evalu-ated invasively, the IOP of the control eye was higher (6.6 ± 0.99 mmHg) than the contralateral eye treated with latanoprost (5.2 ± 0.57 mmHg); although this difference was not statistically significant (p = 0.33, n = 9).

DISCUSSION

In this study, a fluid column perfusion system and linear fit analysis method were used to determine

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FIGURE 5 Invasive measurements of IOP (mean ± SEM) in BALB/c mice. (A) Group 1: In 8 mice, IOP was monitored at 15-min intervals for 45 min beginning at 15 min after initial anes-thesia administration in one eye (black bars) and beginning at 75 min after initial anesthesia administration in the contralat-eral eye (white bars). The average pressure for the three 15-min intervals of each 45-min period was not significantly different (15–60 min: p = 0.91; 75–120 min: p = 0.82) from each other. (B) Group 2: In an additional group of mice, the effect of anesthesia duration on IOP was assessed invasively in one eye for 120 min (gray bars, n = 9). The average IOP within the six 15-min inter-vals did not change significantly (p = 0.72) over time. Overall, the average IOP for each time interval in both groups was not significantly different (15–60 min: p = 0.76, n = 9, n = 9; 75–120 min: p = 0.91, n = 9, n = 6) in either 45-min period.

Control

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flow

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lity

(µL/

min

/mm

Hg)

0.000

0.003

0.006

0.009

0.012

0.015

Latanoprost

Assessed 15-60 minsafter initial K/X doseAssessed 75-120 minsafter initial K/X dose

FIGURE 6 Effect of latanoprost on outflow facility (mean ± SEM) in BALB/c mice. Latanoprost was applied to one randomly selected eye, leaving the contralateral eye as an untreated control. Measurements commenced 2 hr later. During the initial 45-min assessment period, approxi-mately 15–60 min after initial anesthesia administration, outflow facility was significantly increased (p = 0.029, n = 9) from 0.0038 ± 0.0005 µL/min/mmHg in the control eye to 0.0078 ± 0.0013 µL/min/mmHg in the latanoprost-treated eye. Similarly, during the second 45-min assessment period, 75–120 minutes after initial anesthesia administration, out-flow facility was significantly increased (p = 0.034, n = 7) from 0.0032 ± 0.0007 µL/min/mmHg in the control eye to 0.0079 ± 0.0016 µL/min/mmHg in the latanoprost-treated eye. The IOPs of control (p = 0.44) and treated eyes (p = 0.82) of the two 45-min assessment periods were not significantly different. Overall, latanoprost increased outflow facility by 49 and 40% during the 15–60-min and 75–120-min assess-ment periods, respectively.

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outflow facility in mice with and without latanoprost treatment. The system could detect differences of less than 0.001 μL/min/mmHg in the Millipore filter tests. The outflow facility of BALB/c mice in this study was similar to values found in other strains of mice.3–6 The advantages of this method include its simple design and its sensitivity. Other methods to measure outflow facility in mice either: (i) apply a fixed pressure and measure fluid displacement; or (ii) perfuse eyes at a constant flow rate and measure IOP once stabilized. Our method allows the experimenter to characterize the pressure decline to differentiate when the eye is filling (an exponential decay) from when the eye has reached the desired IOP and begun its linear decrease. By using three pressure levels, a correlation value (see Figure 2B) can be assigned to the outflow facility to assess the linearity of the measurements. Poorly correlated data are easy to identify and exclude from further analysis. This method enables the experimenter to observe the expected linearity of the response and evaluate outflow dynamics in a time-dependent fashion.

In agreement with other studies, IOPs were sig-nificantly reduced shortly after ketamine/xylazine administration2,11,12 and were similar to values previ-ously documented in BALB/c mice.7–10 This suggests that outflow facility also might be affected by anesthe-sia. Since the measurement could not be made without anesthesia, the ketamine/xylazine effect on outflow facility could not be determined. Early changes in outflow facility could not be assessed because the measurement could not be made earlier than 15-min post-administration of anesthesia. However, the pro-longed effect of ketamine/xylazine anesthesia on out-flow facility could be evaluated between 15 and 75 min. After initial administration of anesthesia, additional doses were given, as needed, for up to 75 min. The number and amount of supplemental doses of anes-thesia were not significantly different between the two 45-min periods (starting at 15 and 75 min from time zero). Therefore, it was concluded that the outflow facility was not significantly influenced by the duration of ketamine/xylazine anesthetic within the timeframe in which it was assessed. The reduction in IOP dur-ing anesthesia in the mouse cannot be explained by a change in outflow facility.

In the current study, IOP was followed invasively over the same timeframe that the outflow facility was assessed. There was no significant difference in average IOP among each 15-min interval within the first 45-min period (15–60 min after time zero). Similar results were found in the second 45-min period (75–120 min after time zero). If the IOP remains unchanged during each 15-min assessment period, the variables that determine IOP (trabecular outflow, episcleral venous pressure, outflow facility) also could

be assumed to remain unchanged and not affect the gradient of pressure set by the fluid column. In other words, the pressures in the eye (15, 25, and 35 mmHg) set by the fluid column were not significantly altered during the outflow facility assessments. Although the outflow facility values during the first and second 45-min periods were not significantly different, the IOP during the second assessment period was significantly lower than during the first. Therefore, when evaluating the effectiveness of a drug to lower IOP in mice under ketamine/xylazine anesthesia, it is preferable to conduct these measurements beginning within 15 min from initial dosage of anesthesia.

Following latanoprost dosing, outflow facility was significantly increased when measured during both 45-min assessment periods when compared to contra-lateral untreated eyes. The method was sufficiently sen-sitive to detect this difference despite the confounding effect of anesthesia on IOP and the lack of a statistically significant reduction in IOP in latanoprost-treated eyes during the two assessment periods. The duration of anesthesia did not lessen the drug effect.

The average outflow facility values of the control eyes of BALB/c mice over the age of 12 months were lower than the values of mice between the ages of 8–12 months. It has been well established that aqueous humor dynamics change with age in many species.13–16 Even though the outflow facility was lower in the older group compared to the younger group, this was not significant. Further study with more well-defined age groups would be of interest. Certainly age should be considered when designing studies of outflow facility in mice.

There are several assumptions inherent in the current study that require discussion. It is assumed that there are no leaks in the system. First, although the system was tested prior to each outflow facility measurement, very small and undetectable leaks cannot be ruled out. Such leaks may lead to an overes-timation of outflow facility, especially considering that the flow rates measured by this system were less than 0.5 μL/min. Second, outflow facility was assumed to remain constant over the range of pressures set by the fluid column in order to fit a linear regression. At the higher set pressures, Schlemm’s canal com-pression might occur and the outflow facility might decrease.17–25 A third assumption is that uveoscleral outflow, aqueous flow, and episcleral venous pres-sure do not change during the measurement. This study showed minimal change in IOP between time intervals within each assessment period, providing support for the assumption.

Overall, the fluid column perfusion system provides a simple and sensitive means of measuring outflow facility and IOP in mice. Unlike other methods, this

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technique calculated the flow rate from the slope of the pressure decline at three starting pressures. Considering the pressure did not drop more than 1.0 mmHg at each level, this system simulated a constant pressure perfu-sion at each fluid height. Based on the IOP data, the assumptions that episcleral venous pressure, aqueous production, and uveoscleral outflow remain constant were not incorrect for the assessment period of outflow facility. The effect of anesthesia duration on the outflow facility measurement can be considered negligible, since it did not attenuate the effect of latanoprost on outflow facility and outflow facility was unchanged during the 120 min in which it was assessed.

ACKNOWLEDGMENTS

This work was supported by Research to Prevent Blindness.

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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duration of anesthesia affects intraocular pressure, but not outflow facility in mice

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