JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICSVolume 24, Number 2, 2008© Mary Ann Liebert, Inc.DOI: 10.1089/jop.2007.0114

Rebound Tonometry in Conscious, Conditioned Mice Avoids the Acute and Profound Effects of

Anesthesia on Intraocular Pressure

THOMAS V. JOHNSON, SHAN FAN, and CAROL B. TORIS

ABSTRACT

Aims: The aims of this study were to evaluate the accuracy, repeatability, and safety of mul-tiple intraocular pressure (IOP) measurements by a commercially available rebound tonome-ter in conscious, conditioned mice, and to characterize the acute and profound effects of anes-thesia on IOP in mice.

Methods: To test the accuracy of the tonometer, IOPs of CD-1 mice under ketamine/xylazineanesthesia were experimentally set and monitored with a water manometer/transducer sys-tem following transcorneal cannulation while simultaneously performing tonometry. Thelong- and short-term repeatability of the tonometer was tested in conscious, restrained mice,as measurements were taken once-daily in the afternoon for 4 consecutive days. On day 5,IOPs were measured in the same mice once every 4 min for 32 min. On 2 separate days, micewere administered ketamine/xylazine or 2,2,2-tribromoethanol anesthesia, in a crossover de-sign, and IOPs were measured once every 2 min for 32 min. Rebound tonometry was per-formed in conscious mice before and 1 hour after 1 drop of timolol maleate (10 �L of 0.5%)application to 1 eye.

Results: IOP measurements by rebound tonometry correlated well with manometry for pres-sures between 8 and 38 mmHg (y � 0.98x � 0.32, R2 � 0.94; P � 0.001). The average tonomet-ric IOP was invariant over 4 days (range, 11.7–13.2 mmHg). IOPs dropped significantly (P �0.05) within 6 min (ketamine/xylazine) or 10 min (2,2,2-tribromoethanol) postadministrationof anesthesia but not with conscious restraint. Timolol significantly (P � 0.001) lowered IOPfrom 12.8 � 0.3 (mean � standard error of the mean) to 10.1 � 0.6 mmHg, as measured by thetonometer.

Conclusions: Rebound tonometry can be used to obtain accurate IOP measurements in con-scious, restrained mice while avoiding the rapid and profound ocular hypotensive effects ofgeneral anesthesia. Small changes in IOP with an aqueous-flow suppressant are readily de-tectable with conscious restraint that may be missed with chemical restraint.

175

INTRODUCTION

GLAUCOMA IS A PROGRESSIVE optic neuropathycurrently ranked as one of the leading causes

of irreversible blindness worldwide.1 The only ef-fective treatment for glaucoma is to slow diseaseprogression through a reduction of intraocularpressure (IOP) either by pharmacologic or surgi-

Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE.This work was presented, in part, at the Annual Meeting of the Association for Research in Vision and Ophthal-

mology in Fort Lauderdale, FL., April 30–May 4, 2006.

cal means.2–4 Current research efforts are focus-ing on the causes of the IOP elevation and im-proving methods to reduce it to a clinically ac-ceptable level. The mouse is becoming anincreasingly valuable research tool in this en-deavor, owing to its important cellular and mol-ecular similarities to humans and the many waysin which glaucoma and/or ocular hypertensioncan be modeled in this animal,5–9 includingthrough genetic manipulation.10 The utility of themouse as an animal model depends upon theability to measure IOP noninvasively, accurately,and repeatedly in such a small eye. Previous stud-ies have relied on cannulation of the anteriorchamber to measure IOP, but these measure-ments are technically difficult to perform, mustbe carried out under general anesthesia, arehighly invasive, and are not repeatable over theshort term.11,12 Attempts to adapt a commerciallyavailable tonometer,13 indentation tonometry,6

and Goldmann applanation tonometry14 for non-invasive tonometry in mice have produced someencouraging results; however, characterization ofthe effects of repeated tonometric measurementsin a single group of mice, and their potential foruse in longitudinal studies using conscious ani-mals, is generally limited and heterogeneousacross instruments.

Relatively recently, an impact-induction (re-bound) tonometer was developed that deter-mines IOP by measuring several kinetic parame-ters of a magnetized probe as it contacts thecornea.15,16 The extremely small probe size allowsfor repeatable, noninvasive measurements in thesmall eyes of laboratory rodents. A limited num-ber of studies have assessed the accuracy of re-bound tonometry when compared to direct can-nulation of mouse and rat eyes both in vivo andex vivo.17–22 These studies also have investigatedthe IOP as measured by the tonometer in live ro-dents under anesthesia,17,22 among differentstrains of mice,22 including ocular hypertensiveanimals19 and in animals treated with IOP-low-ering drugs.21

The current study adds to this information bydirectly demonstrating the benefit of measuringIOP in conscious, restrained, and conditioned an-imals rather than in animals that are under gen-eral anesthesia. We assessed the stability of IOPin conscious mice, as measured by the tonometerin the same group of animals over a period of 4days. We also evaluated the IOP effect of condi-tioned restraint for a period of up to 32 min.

While some studies have suggested that IOP maybe affected by general anesthesia in themouse,12,23 a detailed time course for this effecthas never been investigated. As such, we demon-strated the rapidity and extent of ocular hy-potension following two different types of sys-temic anesthesia, ketamine/xylazine, a commonand effective anesthetic mixture in mice, and2,2,2-tribromoethanol, an anesthetic thought tohave less of an effect on cardiac function24,25 and,possibly, IOP. The effects of an aqueous-flow sup-pressant on IOP and multiple repeat measure-ments on corneal integrity also were evaluated inconscious mice.

METHODS

Animals

Adult (8–12 weeks old), male CD1 mice(Charles River Laboratories, Wilmington, MA)were housed in light- and temperature-controlledconditions where food and water were availablead libitum. Handling and experimental proce-dures were conducted in accordance with theguidelines set forth by the Institutional AnimalCare and Use Committee of the University of Ne-braska Medical Center and in compliance withthe ARVO Statement for the Use of Animals inOphthalmic and Vision Research.

Rebound tonometer

The TonoLab rebound tonometer for rodents(Colonial Medical Supply, Franconia, NH) wascalibrated for mice as per the manufacturer’s in-structions. A single probe was used for all mea-surements taken within a single day, and theprobe was changed at the beginning of each day.A 10-�L drop of proparacaine 0.5% (AllerganInc., Irvine, CA) was placed in the eye prior tomeasurement for the comfort of the animal andto standardize corneal hydration. For multiplemeasurements over the short term, proparacainewas added only when necessary to maintain avisible drop of fluid lying over the cornea (twiceor three times over a half hour). A single mea-surement consisted of six consecutive probe-to-cornea contacts, which were automatically aver-aged by the TonoLab device and displayed as asingle value. The TonoLab automatically calcu-lates a measure of variation within the six read-ings, and the device describes the variation semi-

JOHNSON ET AL.176

quantitatively as being normal, low (but higherthan normal), medium, or high. IOP measure-ments were repeated if any abnormal level ofvariance was indicated, which was seldom (lessthan once per five measurements). The use of adevice to stabilize and/or aim the TonoLab18,21,22

was found to be cumbersome and unnecessary.

Comparison of rebound tonometry to direct manometry

Ten (10) eyes of 7 mice were used in this ex-periment. After each mouse was anesthetizedwith 100 mg/kg of ketamine and 9 mg/kg of xy-lazine injected intraperitoneally (i.p.), the anteriorchamber of 1 randomly selected eye was cannu-lated by using a borosilicate glass microneedleconnected in series via PE-tubing to an electronicfluid-pressure transducer (#142PC05D; Honey-well International Inc., Plymouth, MN) and ver-tical water column; the entire system was filledwith balanced salt solution (BSS) and devoid ofair bubbles. The transducer, in turn, was con-nected to a PowerLab™ receiver (Model ML870;ADI Instruments Pty Ltd, Richardson, TX), whichtransmitted data to a computer running Power-Lab™ software (version 5.0; ADI Instruments PtyLtd). In a randomized fashion, one investigatorraised or lowered the level of BSS in the watercolumn to control the IOP between 6 and 38mmHg, and a second, masked investigator mea-sured the IOP by using the TonoLab tonometer.Data from the TonoLab were plotted against datafrom the pressure transducer, and a linear re-gression analysis was performed. To determine ifvariance in the TonoLab was related to IOP, thedifference in IOP, as measured by the two differ-ent techniques, was plotted against an averageIOP by the two methods in a Bland-Altman plot.

Restraint of live animals

All the remaining experiments were carried outlongitudinally on a single group of mice. Micewere gently restrained by placing them into aclear plastic rodent restraint bag (Harvard Ap-paratus, Holliston, MA) and then strapping theminto a specially crafted restraint device. Care wastaken to avoid placing pressure on the head orneck, which could raise IOP. Before any mea-surements were taken, the animals were accli-mated to the restraint device through a series ofat least five training sessions lasting 30 min eachand spaced over 3 days. Mice also were accli-

mated to the restraint for a period of 5 min im-mediately preceding each experiment.

Repeatability of TonoLab tonometry

Between 1:00 PM and 3:00 PM each day for 4 con-secutive days, conscious mice (n � 9) were placedin the restraint, given approximately 5 min to set-tle, and then the IOP was measured 3 times pereye (taken in an alternating manner) and aver-aged to determine IOP for each day. As no sig-nificant difference was found between the rightand left eyes (data not shown), values from botheyes were averaged, yielding a single IOP valueper mouse per day. The average of these IOP val-ues was compared for each of the 4 days by us-ing a one-way analysis of variance (ANOVA)with Bonferroni adjustments for multiple com-parisons.

Effect of restraint or general anesthesia on IOPmeasured by TonoLab

Tonometry was performed repeatedly for 30min during restraint or under anesthesia. First,IOP in each of the 9 mice was measured while inthe restraint. TonoLab measurements were takenonce per eye after a 5-min acclimation period(time t � 0 min), and then IOP was measuredonce every 4 min thereafter for a total of 32 min.On the following day, 5 of the mice were placedback in the restraint, as previously described. Af-ter a 5-min acclimation period, IOP was mea-sured in the conscious animals (time t � �5 min-utes). At time zero (t � 0 min), restrained animalswere injected i.p. with either 100 mg/kg keta-mine/9 mg/kg xylazine or 500 mg/kg 2,2,2-tri-bromoethanol (Sigma-Aldrich, St. Louis, MO).Starting 2 min later (time t � 2 min), when mostanimals had ceased struggling within the re-straint, IOP was measured every 2 min for a to-tal of 32 min. On the following day, the same micewere injected i.p. with the anesthetic, which hadnot been administered the previous day. IOPswere measured as before. As no significant dif-ference was found between the right and left eyes(data not shown), values from both eyes were av-eraged, yielding a single IOP value per mouse pertime point. For data of conscious, restrained mice,IOPs at various time points were comparedwithin treatment groups and IOPs at each timepoint were compared across treatment groups byusing one-way ANOVAs with Bonferroni correc-tions.

TONOMETRY IN CONSCIOUS MICE 177

Effect of unilateral timolol administration on IOPin mice measured by TonoLab

Following a 5-min acclimation period in therestraint, IOP was measured by taking 3 mea-surements per eye, in an alternating manner be-ginning in the right eye. This was followed by a10-�L drop of 0.5% timolol maleate (Timoptic;Merck & Co. Inc., Whitehouse Station, NJ) givento 1 randomly selected eye and artificial tears(Refresh Tears; Allergan Inc.) to the fellow eye.Mice were released from the restraint for 1 h,then placed back in the restraint, and their IOPswere assessed again by an investigator maskedto the identity of the drug- and vehicle-treatedeyes, as previously described. Treated eyes werecompared to the same eye at baseline and to un-treated eyes by using a one-way ANOVA with

Bonferroni corrections for multiple compar-isons.

Effect of repeated TonoLab measurements oncorneal integrity

Once-daily during the course of the study andprior to measurements, the corneas of each ofthe 9 mice were examined under a dissecting mi-croscope. Abnormalities were found in 2 ani-mals and were documented with digital micro-graphs. These animals were removed from thestudy. Data from these animals were includedin the analysis up until the point in which thecorneal integrity was compromised. All subse-quent data were derived from replacement ani-mals that also underwent the same training andacclimation procedures as the discarded mice.

JOHNSON ET AL.178

FIG. 1. The TonoLab (Colonial Medical Supply, Franconia, NH) measurement regimen for mice used in all experi-ments except the cannulation experiment. Each mouse underwent either 6 or 8 consecutive days of intraocular pres-suer measurements using the TonoLab. The number of measurements varied between days according to which ex-periment was being performed, but experiments were always performed in the same order. For the 4 animals thatdid not receive anesthesia, topical application of timolol occurred on the day immediately after the restraint trial.

Figure 1 outlines the regimen of the TonoLab ex-periments.

RESULTS

IOP measurements by the TonoLab andmanometry were highly correlated for IOPs be-

tween 8 and 38 mmHg (R2 � 0.94, P � 0.001, Fig.2A). The slope of the linear regression was 0.98 �0.02 (mean � standard error of the mean) and theY-intercept was �0.32 � 0.42 mmHg. The lowestreading that the TonoLab would provide withconsistency was 7 mmHg, even when the mano-metric IOP was set below 6 mmHg. Therefore, the lowest meaningful TonoLab reading was

TONOMETRY IN CONSCIOUS MICE 179

y = 0.98x � 0.32

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FIG. 2. Comparison of intraocular pressures (IOPs) by the TonoLab versus a pressure transducer. Ten (10) eyes of7 anesthetized mice were cannulated with a microneedle connected in series to an adjustable water column and pres-sure transducer. One investigator manually adjusted the IOP by raising or lowering the water column, while anotherinvestigator measured the IOP using the TonoLab; each investigator was masked to the IOP as measured by the otherinvestigator. (A) a linear regression between the two IOP measurement techniques (solid line). There was a highlysignificant (P � 0.001) correlation between the IOPs measured by the TonoLab and those measured by the transducer.(B) a Bland-Altman plot. The difference between IOPs measured by the TonoLab and the transducer was indepen-dent of the IOP itself. The mean difference (solid line) was 0.7 mmHg, and the upper and lower 95% limits of agree-ment (dotted lines) were 5.1 and �3.7 mmHg, respectively.

A

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8 mmHg. The agreement between the two meth-ods was independent of IOP, according to aBland-Altman plot of the data (Fig. 2B). On av-erage, the TonoLab IOP was 0.7 mmHg lowerthan the manometric IOP. The upper and lower95% limits of agreement were 5.1 and �3.7mmHg, respectively. Besides demonstrating thegeneral accuracy of the TonoLab, these resultsconfirm the validity of measurements made byhand, without the aid of stabilization or aimingdevices for the tonometer.

In conscious, restrained mice, the mean IOPwas relatively steady (measuring between 11 and14 mmHg over 4 consecutive days; Fig. 3) andstatistically similar (P � 0.61 for one-wayANOVA). The magnitude of the standard-errormeasurement of IOP measurements decreasedover time from 1.24 mmHg on day 1 to 0.48mmHg on day 4. Measured at 4-min intervals for32 min, IOPs ranged between 10.3 and 12.4mmHg in restrained, conscious animals (Fig. 4A).A one-way ANOVA demonstrated that mean IOPdid not vary with time (P � 0.88), indicating that

IOP was statistically steady over the 32-min pe-riod.

Baseline IOPs (t � �5 minutes) for the 32-minrestraint trial and the two anesthesia trials werestatistically similar (Fig. 4). In the first 10 min fol-lowing administration of both types of anesthe-sia, there was a sharp decline in IOP, which wassignificantly correlated to time (one-wayANOVA: P � 0.001 for both types of anesthesia;Fig. 4B and 4C). By 6 min, IOPs in animals anes-thetized with ketamine/xylazine demonstrated asignificantly (P � 0.05) lower IOP, when com-pared to baseline (9.2 � 2.2 vs. 12.7 � 0.8 mmHg,respectively, mean � standard deviation; Figure4B). Statistically significant ocular hypotensionincreased and persisted throughout the 32-minexperiment (Fig. 4B). Animals anesthetized with2,2,2-tribromoethanol required 10 min before thiseffect was significant (P � 0.01, 7.8 � 1.1 vs.11.0 � 3.2 mmHg, respectively; Figure 4C). Whencompared to IOPs measured under conscious re-straint at similar time points, the IOPs of animalsunder both types of anesthesia exhibited a sig-nificant (P � 0.05) difference beginning at 12 min.As quickly as 2 min following administration ofeither type of anesthesia, the variation in IOP ex-panded dramatically, as indicated by the en-larged standard-error bars at early time points,although the mean IOP was not significantly af-fected until later (Fig. 4B and 4C). The IOP in an-imals under both types of anesthesia eventuallydropped to below 8 mmHg, the previously es-tablished lower limit of accuracy (Fig. 4B and 4C).

Before treatment, no difference was found be-tween the eyes randomly selected to receive tim-olol (12.8 � 1.0 mmHg) and those that would re-ceive vehicle (11.8 � 1.5 mmHg; P � 0.13).Administration of vehicle to the control eye didnot result in a significant change in IOP (11.4 �2.7 mmHg; P � 0.65). Timolol significantly re-duced IOP (10.1 � 1.8 mmHg), compared to pre-treatment levels (P � 0.001) and to the contralat-eral untreated eyes (P � 0.02; Fig. 5). Importantly,

JOHNSON ET AL.180

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FIG. 3. Intraocular pressures (IOPs) in conscious, re-strained mice (n � 9) measured between 1:00 PM and 3:00PM for 4 consecutive days. (A) IOPs over four days. IOPsranged from a low of 11.7 mmHg to a high of 13.2 mmHg.Error bars are the standard error of the mean. No signif-icant differences were found between average IOPs onany of the days by one-way analysis of variance with post-hoc Bonferroni comparisons.

FIG. 4. Time course of the effect of restraint (n � 9, A) and two types of general anesthesia (ketamine/xylazine [B]or 2,2,2-tribromoethanol [C]; n � 5 for each) on intraocular pressure (IOP) in mice. The restraint had a minimal andinsignificant effect on IOP during the 32-min trial (P � 0.88; one-way analysis of variance [ANOVA]). Both types ofanesthesia caused a sharp decline in IOP, which began immediately, became significant at 6 (ketamine/xylazine) or10 min (2,2,2-tribromoethanol), and persisted for the remainder of the trial. Error bars are standard error of the mean.In (A) animals were placed in the restraint at time t � �5 min. In (B and C) animals were placed in the restraint attime t � �10 min and anesthesia was administered at time t � 0 min. *P � 0.05 comparing baseline IOP (time � �5min) to the time point indicated; ‡P � 0.01 comparing baseline IOP to the time point indicated; †P � 0.002 compar-ing baseline IOP to the time point indicated. This comparison also refers to the horizontal bars to the right of † andabove individual time points. All time-point comparisons were made using one-way ANOVAs with post-hoc Bon-ferroni corrections for multiple comparisons.

TONOMETRY IN CONSCIOUS MICE 181

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the mean final IOP in the timolol-treated eyes wasstill greater than the IOP observed in animals lessthan 6 min after the administration of either typeof anesthesia.

After 5 days of multiple measurements, whichended with the conscious restraint trial (Fig. 1), 3eyes of 2 mice (3 of 18 eyes in the study) displayedcorneal opacities (Fig. 6). Visual localization ofthe opacity to the cornea, rather than the lens, wasmade possible by examining the eye with a dropof BSS covering the cornea. The physical changein the corneal structure caused inaccurately highIOP measurements by the TonoLab. These ani-mals were removed from the study.

DISCUSSION

Previous studies have shown good correlationbetween IOP measured by the TonoLab and IOPmeasured by direct cannulation of the eye inanesthetized rats with26 or without20 ocular hy-pertension, enucleated and anesthetized mouseeyes,17,21 and anesthetized mice treated with top-ical prostaglandins.21 Likewise, the current studyfound good agreement between TonoLab mea-surements and manometric measurements in theanesthetized mouse with cannulated eyes at pres-sures between 8 and 38 mmHg. The general reli-ability of individual IOP measurements, usingthe TonoLab, has been well established.

Several investigators have asserted that meth-ods to stabilize and aim the TonoLab while mea-suring IOP are essential for accuracy and re-peatability. Wang and colleagues22 reportedutilizing a clamp connected to a ring stand to fixthe TonoLab in place, and Filippopoulos andcoauthors18 visualized alignment and placementof the probe with respect to the cornea under an

JOHNSON ET AL.182

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FIG. 6. Representative eye of 1 mouse showing an opac-ity of the central cornea after 5 days of repeated topicalproparacaine application and subsequent intraocularpressure measurements with the TonoLab. Magnificationis 40�.

FIG. 5. TonoLab measurements of intraocular pressure (IOP) in conscious, restrained mice (n � 9). IOP was signif-icantly reduced 1 h after a unilateral dose of timolol maleate 0.5%. The IOPs were statistically similar between eyesat baseline. The contralateral vehicle-treated control eye showed no change in IOP. Error bars are standard error ofthe mean. The horizontal bar with * indicates that *P � 0.025 when comparing timolol-treated eyes before and aftertreatment by one-way analysis of variance with post-hoc Bonferroni corrections for multiple comparisons.

operating microscope. Morris and coworkers21

adapted both approaches for their measurements.According to Kontiola and coauthors,20 initialprobe to cornea distances of 3–5 mm and angleswith respect to the visual axis of up to 25 degreesshould produce minimal effects on IOP mea-surements in rats. The current study employed ahandheld approach for making tonometry mea-surements in mice, and all IOP readings were vi-sualized directly. Still, we were able to attaingood agreement with direct manometric mea-surements using this method, indicating that,with experience, accurate TonoLab measure-ments can be made by hand.

The current study found that neither restraintnor repeated TonoLab measurements have a ma-jor effect on IOP, a finding that attests to the valueof the TonoLab in making repeated IOP mea-surements in a single group of mice over an in-definite period of time. Previously, it had beendemonstrated that rapid, successive reboundtonometric measurements (10 measurementswithin 20 sec) could significantly lower IOP inanesthetized mice.21 There are important differ-ences between the studies that may account forthese discrepant findings. In the current study,rebound tonometry measurements in unanes-thetized animals were separated by longer timeintervals (4 min between readings in the currentstudy, compared to 2 sec in the previous study),thus allowing time for reequilibration of IOP fol-lowing each measurement. While the amount oftime necessary to avoid the tonographic effect ofrebound tonometry appears to be no more than4 min, the lower limit has yet to be determined.Further, conscious, rather than anesthetized, an-imals were used in the current study. Consciousanimals may be able to maintain stable IOP in theface of repeated IOP measurements.

The current study demonstrates potential ben-efits of the training and acclimatization of animalsto restraint prior to obtaining IOP measurements.This is suggested by the decrease in standard-er-ror measurement of IOP values over consecutivedays. While most previous studies have assessedmouse IOP in an acute manner, this study uti-lized a single group of mice whose IOPs weremeasured many times over many consecutivedays. The fact that consistent readings were ob-tained longitudinally indicates that this noninva-sive form of tonometry will be beneficial for long-term studies in mice.

Tonometry by noninvasive means allows the

determination of IOP in conscious animals. Wangand colleagues22 obtained reliable TonoLab IOPmeasurements in restrained, conscious mice andrats, and demonstrated interstrain differences inthe average IOP of mice. They also investigatedthe ocular hypotensive effect of anesthesia, butonly through comparison of conscious IOP andIOP 10–15 min following the administration ofanesthesia, which the current study demonstratesis well after the hypotensive effects of anesthesiacause a change in IOP and near the time when thischange becomes significant. The IOP-lowering ef-fect of anesthesia has been investigated in rats27

and in mice,12,23 but the speed and severity of theeffect only now has been demonstrated. Our timecourse shows the early and sharp nature of the IOPdrop as well as the lower limit of instrument de-tection. The current data includes the effects of twodifferent anesthetics and found that general anes-thesia causes a quick decline in IOP; the effects ofketamine/xylazine seem to be more rapid thanthose of 2,2,2-tribromoethanol. It should be notedthat the current study’s temporal resolution was 2min, and, therefore, the hypotensive effect of anes-thesia may have become statistically significant atany time between measurement intervals.

The current study found that IOP began todrop as soon as 2 min following the adminis-tration of ketamine/xylazine or 2,2,2-tribromo-ethanol, though a statistically significant reduc-tion in IOP was not reached until later timepoints. We also found that IOP measurementstaken during this time were likely to have a largevariance, as the standard error of our measure-ments was largest during the earliest time pointsfollowing anesthetic administration (Fig. 4B and4C). This is the first study to measure IOP at veryearly time points following the administration ofan anesthetic. It also offers further substantiationthat the ability to measure IOP in unanesthetizedanimals is extremely valuable. IOP obtained justa few minutes after the start of anesthesia is notfree of artifact. A higher variance in IOP mea-surements is likely to occur soon after the anes-thetic takes effect, thereby making the acute ex-perimental manipulation and assessment of IOPmore difficult to surmise. Clearly, IOP measure-ments in conscious, conditioned animals are moreconsistent than those made in animals undergo-ing general anesthesia, even in the first few min-utes after the loss of consciousness.

This study detected a significant IOP reductioncaused by the topical administration of timolol in

TONOMETRY IN CONSCIOUS MICE 183

conscious mice. Such an effect might not havebeen observable if obscured by an already re-duced and highly variable baseline IOP causedby the hypotensive effects of anesthesia. Indeed,the final mean IOP of timolol-treated eyes in thecurrent study was 10.1 mmHg. The IOP was re-duced to this level in untreated eyes by generalanesthesia just 6 min after administration. Whileother studies were able to observe the hypoten-sive effect of a topical prostaglandin analog21 ortimolol12 administration, the mice in those stud-ies had a higher baseline IOP (16–18 mmHg) thanthose of the current study, when measured within7 min of anesthesia administration. The IOP re-duction observed in the previous studies mayhave been even more dramatic had tonometrybeen performed in unanesthetized animals.

There is a limit to the number of IOP mea-surements on an individual eye when using theTonoLab. Three (3) eyes of 2 mice developed se-vere corneal opacities after receiving approxi-mately 45 IOP measurements per eye with theTonoLab over a 5-day period. It is possible thatthe opacities developed from mechanical stressplaced on the cornea, resulting in damage to thecorneal epithelium and/or repeated topical treat-ment with proparacaine, which can be cyto-toxic.28,29 The possibility that corneal compro-mise was a result of tissue dehydration duringgeneral anesthesia was excluded, as the opacitiesformed on day 6, prior to the administration ofany anesthesia (Fig. 1). Thus, there may be an up-per limit to the frequency with which IOPs canbe measured in animals without compromisingthe physical integrity of the cornea and, therefore,the validity of further IOP measurements. Limit-ing the amount of topical anesthetic applied tothe eye, and standardizing corneal hydrationwith continuous saline application rather thananesthetic, may prevent this adverse event.

CONCLUSIONS

In summary, the current study demonstratesthat rebound tonometry is an advantageousmethod for measuring IOP in mice. It is accuratewhen compared to cannulated eyes of anes-thetized mice, even when handheld and aimedby direct observation. It can be used in restrained,conscious animals, which avoids the potentiallimitations imposed by general anesthesia. Nei-ther restraint of the animal nor the measurements

themselves affects the IOP, though we recom-mend a series of training sessions and a premea-surement acclimation period of at least 5 min tominimize measurement variation. The TonoLabhas enough sensitivity to detect a reduction inIOP caused by a topical aqueous-flow suppres-sant in conscious mice. A potential limitation ofthis tonometric method is indicated by thecorneal opacities that can develop from multiplemeasurements over a short time interval. Over-all, however, rebound tonometry in conscious an-imals is likely to prove extremely useful in futureinvestigations involving IOP in the mouse.

ACKNOWLEDGMENTS

This work was supported by an unrestrictedgrant from Research to Prevent Blindness, Inc.(New York, NY). The authors wish to thankCourtney Krohn for assistance in data collectionand Tyrone Moreno and Lisa Stapp for the re-straint device construction.

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Received: October 3, 2007Accepted: December 12, 2007

Reprint Requests: Carol B. TorisDepartment of Ophthalmology and Visual Sciences

985840 University of Nebraska Medical CenterOmaha, NE 68198-5840

E-mail: ctoris@unmc.edu

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