Intraocular Pressure in Lewis Rats Andre Mermoud*\ George Baerveldt* Don S. Minckler,* Martha B. Lee,% and Narsing A. Rao*

Purpose. To perform noninvasive measurements of intraocular pressure (IOP) in rats, the Tono-Pen-1 and Tono-Pen-2 were calibrated against direct manometry. Normal values and the long-term fluctuations of IOP in Lewis rats were established. Methods. For calibration, 24 eyes were cannulated and connected to a pressure transducer with a chart recorder. IOP was increased from 5 to 40 mm Hg in 5 mm Hg increments, and from 40 to 60 mm Hg in 10 mm Hg increments. After each incremental increase, IOP was measured with a Tono-Pen-1 and a Tono-Pen-2 tonometer. To determine normal IOP in Lewis rats, IOP was measured with a Tono-Pen-1 in 229 eyes of 115 rats, and a histogram of normal IOP was established. To ascertain long-term IOP fluctuations, the pressure in 52 eyes of 26 rats was measured every day between 8:30 and 9:30 AM for 7 consecutive days. Results. Plotting the mean Tono-Pen readings for each eye against the transducer IOP produced two regression formulas: y = 1.819 + 0.711 x (r2 = 0.92) for Tono-Pen-1, and y = -1.291 + 0.784 x (r2 = 0.97) for Tono-Pen-2. The normal IOP in rats was 17.30 ± 5.25 mm Hg (90% confidence interval: 7.28 and 26.98 mm Hg for the lower and upper limits of normal IOP). There was no long-term fluctuation in IOP (P = 0.55). Conclusions. IOP can be measured accurately in living rats with the Tono-Pen-1 or the TonoPen-2. Invest Ophthalmol Vis Sci. 1994;35:2455-2460.

I n experimental glaucoma research, it is highly desirable to move from expensive nonhuman primate models to smaller, more easily handled, and less expensive models, such as rats. Furthermore, the physiology and physiopathology of the rat have already been studied extensively, and a large number of immunologic substances are available for this animal, which may be used for studying optic nerve damage and the mechanisms of glaucoma associated with ocular inflammation. Any animal model for glaucoma research requires accurate and reproducible measurements of intraocular pressure (IOP). Until recently, noninvasive measurement of intraocular pressure in domestic or laboratory animals has been limited to the use of Perkins applanation tonomeFrom the Doheny Eye Institute and the Departments of *Ophthalmology and %Preventive Medicine, University of Southern California School of Medicine, Los Angeles, California, and the fHopital Ophtalmique Jules Gonin and the Department of Ophthalmology, University of Lausanne, Switzerland. Supported in part by a grant from Hopital Ophtalmique Universitaire Jules Gonin, Lausanne, Switzerland, and NIH grant EY03040, a core grant for vision research Submitted for publication June 7, 1993; revised December 6, 1993; accepted Decembers, 1993. Proprietary interest category: N. Reprint requests: Dr. George Baerveldt, Doheny Eye Institute, 1450 San Pablo Street, Los Angeles, CA 90033.

try or pneumotonometry; unfortunately, neither method is possible in small animals such as rats.1"3 Using the Tono-Pen (Bio Rad, Santa Ana, CA), a small handheld applanation tonometer, it is now possible to measure accurately the intraocular pressure in small eyes.4 To establish the normal range of pressures for Lewis rats, the Tono-Pen tonometer was calibrated and the long-term physiologic fluctuation in IOP of this animal was evaluated. MATERIALS AND METHODS All experiments were performed on Lewis rats (Charles River, Wilmington, MA). Rats were 2- to 3months-old and weighed 150 to 200 gm. Animals were kept at 21 °C in a normal 12 hour light: 12 hour dark cycle and fed laboratory chow ad lib. Experiments were performed in compliance with the ARVO Statement for the use of Animals in Ophthalmic and Vision Research. The anterior chamber of 24 normal eyes of 12 Lewis rats were cannulated at the limbus using a 25gauge needle. There was no leakage around the needle, and corneal deformation was minimal. The needle

Investigative Ophthalmology & Visual Science, April 1994, Vol. 35, No. 5 Copyright © Association for Research in Vision and Ophthalmology

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was connected via polyethylene tubing to a mercury manometer, a water manometer, and an electronic pressure transducer (Statham, Hato Rey, PR) connected to a strain gauge coupler (Type 9803, Beckman Instruments Inc, Chicago, IL). Pressure was then amplified with an amplifier (Type 474A, Beckman Instruments) and the measurement printed using a chart recorder (Type 506 RAM1, Beckman Instruments) (Fig. 1). The IOP could be increased or lowered by adding or removing air in the mercury manometer. A water reservoir was connected to the system to fill the tube and flush air bubbles. The intraocular pressure was measured in millimeters of mercury on the chart recorder which, at the beginning of measurement, was calibrated against the mercury column and checked for accuracy against the column of water. All animals were anesthetized by an intramuscular injection of 0.6 ml/kg of a solution of 5 ml ketamine (100 mg/ml), 4 mg xylazine (100 mg/ml), and 1 ml sterile water. A drop of 0.5% proparacaine hydrochloride was applied to each eye. The rats were then positioned on their left side for measurement of the IOP of the right eye, and on their right side for measurement of the IOP of the left eye. The head was adjusted so that the pupillary axis was in the vertical position. After the needle was introduced into the eye under microscopic control, IOP was transiently 0 mm Hg. Intraocular pressure was increased in 5 mm Hg increments from 5 to 40 mm Hg, and in 10 mm Hg increments from 40 to 60 mm of Hg. At each pressure level, after stabilization of the system for 3 minutes, IOP was recorded from two tonometers used in succession: a Tono Pen-1 and a Tono Pen-2, each of which had been previously calibrated. The Tono-Pen was oriented perpendicular to the cornea and was applied gently to the corneal epithelium, avoiding indentation of the cornea. Before performing the second measurement with the Tono-Pen-2, the IOP was checked and rnmH20

FIGURE l. Diagram of instrumentation used to cannulate the rat's eye and to adjust and measure intraocular pressure.

adjusted when necessary to be the same as for the measurement made with the Tono-Pen-1. With the Tono Pen-1, at least three accurate average readings were recorded after each incremental increase in pressure. When statistical reliability of the average measurement (SRAM), which represented the coefficient of variance of the measurements, was >10%, the reading was ignored and another measurement was taken. Ten consecutive readings at each pressure level were recorded with the Tono-Pen-2. The Tono-Pen-2 occasionally reported pressure just before the tip contacted the cornea (tear film meniscus), or just after the tip was removed from the cornea; these readings were inaccurate and were ignored, as were the periodic, automatically averaged readings. The eyes were kept moist by frequent administration of balanced salt solution throughout the test period. At the end of the experiment, the animals were sacrificed by intracardiac injection of 13 ml/kg of a solution of sodium pentobarbital (65 mg/ml). The needle became plugged by fibrin, iris, or lens in seven eyes during the experiments, and these were deleted from the study. The intraocular pressure measurements of the remaining 17 eyes were used for analysis. Each Tono-Pen measurement was plotted against corresponding manometric values. Coefficient of determination values (r2) were determined. Linear and polynomial regression analyses were performed to obtain the mathematical formulation of the relationship between tonometric measurements and the manometric values. Agreement of Tono-Pen-1 and Tono-Pen-2 readings with manometric values was determined using the method described by Bland and Altman.5 Normal Intraocular Pressure in Lewis Rats The intraocular pressures of 229 eyes of 115 Lewis rats were measured clinically to establish the normal IOP in this animal. All measurements were done between 8:30 and 9:30 AM to eliminate IOP fluctuations due to circadian rhythm. All animals weighed between 150 and 200 gm; 24 rats were male and 91 were female. Before the IOP measurements, one drop of 0.5% proparacaine hydrochloride was applied to each eye, after which the animal was placed in a carbon dioxide (CO2) chamber for 30 to 60 seconds until sedated. The rat was then positioned so that the pupillary axis was in the vertical position. To avoid artificial increase in IOP, no traction was exerted on the eyelid because the eyes were open. Three automatic averages of acceptable measurements displayed on the Tono Pen-1 were recorded when the statistical reliability of the average measurement (SRAM) was 5%, another measurement was taken. The 229 IOP measurements were analyzed by establishing a histogram. Mean IOP, with standard devia-

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To determine whether or not there are long-term fluctuations in the IOP of Lewis rats, we measured the IOP between 8:30 and 9:30 AM for 7 consecutive days in 52 eyes of 26 female Lewis rats weighing 150 to 180 gm. These IOP measurements were performed using the same technique as described above.

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A regression line was calculated by plotting each Tono-Pen reading against the corresponding manometric values, and the following formulas were determined: (y = 1.819 + 0.711 x) for Tono-Pen-1 and (y = -1.291 + 0.784 x) for Tono-Pen-2, where x represents the pressure measured with the pressure transducer and y represents the mean of Tono-Pen-1 or -2 readings. The correlation of Tono-Pen-1 (Fig. 2) and Tono-Pen-2 (Fig. 3) with the pressure transducer was high (r2 = 0.92 for Tono-Pen-1 and r2 = 0.97 for Tono-Pen-2). Comparison of the slope of both regression lines using the method of Draper6 showed a significant difference (P = 0.052). Compared with direct manometry, both tonometers underestimate IOP except when IOP is lower than 6.5 mm Hg measured with the Tono-Pen-1, where the Tono-Pen readings were slightly overestimated. For IOP measured with Tono-Pen-2 and IOP higher than 6.5 mm Hg mea-

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FIGURE 3. Diagram representing the regression line (solid line) of the calibration of the Tono-Pen-2; dotted-line represents the line of equality.

sured with Tono-Pen-1, the underestimation was linear and increased with increasing IOP. This underestimation is also supported by the method of Bland and Altman5 that compared at each pressure level the mean of the difference between transducer pressure and Tono-Pen-1 and Tono-Pen-2 readings (Figs. 4 and 5). A correction formula with 95% confidence intervals was generated for both tonometers (Table 1).

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FIGURE 2. Diagram representing the regression line (solid line) of the calibration of the Tono-Pen-1; dotted-line represents the line of equality.

4. Diagram representing the difference (transducer minus Tono-Pen-1, or y) against the mean (transducer and Tono-Pen-1, or x). The mean difference is 6.71 mm Hg, and 2 standard deviations is ± 11.01 mm Hg. Regression line formula is y = -1.230 + 0.303 x (r2 = 0.54). The slope of the regression line differs from zero, with t-statistic 4.833 (P = 0.0001). FIGURE

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FIGURE 5. Diagram representing the difference (transducer minus Tono-Pen-2, or y) against the mean (transducer and Tono-Pen-2, or x). The mean difference is 7.57 mm Hg, and with 2 SD it is ± 8.06 mm Hg. Regression line formula is y = 1.833 + 0.226 x (r2 = 0.67). The slope of the regression line differs from zero, with t statistic 13.644 (P = 0.0001).

Normal Intraocular Pressure The histogram of intraocular pressures measured in 229 normal eyes is represented in Figure 6. The mean Tono-Pen reading ± SD was 13.89 ± 4.22 mm Hg. Using the correction formula for Tono-Pen-1, the mean normal IOP in Lewis rats was 16.98 ± 5 . 1 7 mm Hg. The range of normal IOPs in Lewis rats, with a 90% confidence interval, was 7.28 mm Hg for the lower limit and 26.98 mm Hg for the higher limit. Intraocular pressures in the right and the left eyes were similar (16.86 ± 5.22 mm Hg and 17.09 ± 5.36 mm Hg for the mean IOP in the right and the left eyes, respectively; P = 0.77, Student's paired Mest). Long-Term Fluctuations of IOP The intraocular pressures of 52 eyes of 26 Lewis rats were recorded every day between 8:30 and 9:30 AM for 7 consecutive days. IOPs were constant within and between individuals, with a mean value of 16.67 ± 4.89 mm Hg. Detailed mean intraocular pressures for each day are shown in Table 2. Repeated measure design showed no statistical differences between days (P = 0.55). DISCUSSION Intraocular pressure in living rats was measured for the first time by Ohnesorge et al7 in 1968, who used an invasive continuous impression tonometer that did not report the absolute IOP. Direct cannulation of the anterior chamber and connection of the needle to a pressure transducer was described by Funk et al8 in

1985, who reported that the normal IOP in 20 anesthetized Wistar Kyoto rats (each weighing 350 to 400 gm) was 15.9 ± 0.4 mm Hg. Recently, Moore et al4 evaluated the Tono-Pen-2 tonometer for measuring IOP in living anesthetized Brown Norway rats. They plotted the mean Tono-Pen readings for each animal against a transducer IOP, with pressures ranging from 15 to 45 mm Hg, and reported the following regression line: y = 4.54 + 0.79 x (r = 0.98). In our study of Lewis rats using the same technique and a range of IOPs of 5 to 60 mm Hg, we obtained similar results concerning the slope of the regression line (0.784 for Tono-Pen-2, versus 0.79 in the study of Moore et al), but significantly different results regarding the intersection of the regression line with the y-axis (—1.291 for Tono-Pen-2, versus + 4.54 using Tono-Pen-2 in the study of Moore et al). Moore et al concluded that the Tono-Pen-2 overestimates low IOP (