Effect of General Anesthetics on IOP in Rats with Experimental Aqueous Outflow Obstruction

Abstract

purpose. To determine the effect of several common general anesthetics on intraocular pressure (IOP) after experimental aqueous outflow obstruction in the rat.

methods. A single episcleral vein injection of hypertonic saline was used to sclerose aqueous humor outflow pathways and produce elevated IOP in Brown Norway rats. Animals were housed in either standard lighting or a constant low-level light environment. Awake IOPs were determined using a TonoPen (Mentor, Norwell, MA) immediately before induction of anesthesia by either isoflurane, ketamine, or a mixture of injectable anesthetics (xylazine, ketamine, and acepromazine). For each anesthetic, IOPs were measured immediately after adequate sedation (time 0) and at 5-minute intervals, up to 20 minutes.

results. Awake IOPs ranged from 18 to 52 mm Hg. All anesthetics resulted in a statistically significant (P < 0.01) reduction in measured IOP at every duration of anesthesia when compared with the corresponding awake IOP. With increasing duration of anesthesia, measured IOP decreased approximately linearly for both the anesthetic mixture and isoflurane. However, with ketamine, IOP declined to 48% ± 11% (standard lighting) and 60% ± 7% (constant light) of awake levels at 5 minutes of anesthesia, where it remained stable. In fellow eyes, the SD of the mean IOP in animals under anesthesia was always greater than the corresponding SD of the awake mean. Anesthesia’s effects in normal eyes and eyes with elevated IOP were indistinguishable.

conclusions. All anesthetics resulted in rapid and substantial decreases in IOP in all eyes and increased the interanimal variability in IOPs. Measurement of IOP in awake animals provides the most accurate documentation of pressure histories for rat glaucoma model studies.

Many risk factors can influence glaucoma. Of these, elevated IOP is the best recognized and documented. Nearly all glaucoma therapy relies on lowering IOP. Understanding the mechanisms by which IOP damages optic nerve fibers is important for developing new, logical glaucoma treatments designed to protect the optic nerve directly. 

Fortunately, optic neuropathy similar to that occurring in human glaucoma can be produced in otherwise normal animals by experimental IOP elevation. ^{1 2 3 4 5} Such models offer the ability to reproduce effects solely due to elevated IOP and allow identification of early effects of elevated IOP, before extensive nerve damage. In addition, the affected retinas and optic nerves can be evaluated directly to obtain unequivocal answers regarding the extent of optic nerve damage and accompanying cellular alterations. 

Due to expense and logistic considerations, there has been an increased interest in producing such models in laboratory rats. ^{2 3 4 5} Successful use of such models requires careful correlation of the extent of IOP elevation with the resultant injury and accurate determination of the IOP responsible for the optic nerve damage it induces. 

The introduction of the TonoPen tonometer (Mentor, Norwell, MA) made it possible to measure IOP in the rat eye without direct, anterior chamber manometry. This instrument provides an accurate, reproducible, noninvasive assessment of IOP in the rat eye. ^{6 7} In addition, when used on awake Brown Norway rats, the TonoPen allows documentation of a natural circadian fluctuation of IOP of approximately 10 mm Hg, which is exaggerated during the dark phase of the circadian cycle after obstruction of aqueous humor outflow. ^{8 9} These fluctuations can be stabilized by housing the animals in constant low-level light, thereby simplifying the documentation of IOP exposure history. ¹⁰  

In spite of these observations, many laboratories measure IOP in rats with the aid of general anesthetics, particularly when using strains that are less tolerant of awake measurements than the Brown Norway rat. However, frequent use of general anesthetics may not be well tolerated by experimental animals, ⁷ and commonly used anesthetic agents, such as ketamine, may have neuroprotective properties, confounding experimental results. ^{11 12 13 14} Finally, various anesthetic agents may alter IOP in humans and several experimental animals, ^{7 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38} and the effects of these agents on eyes with experimentally elevated IOP are not well understood. 

In this study, we have combined the ability to measure IOP in awake Brown Norway rats with our model of aqueous outflow obstruction. ² The purpose was to determine the effect of several commonly used general anesthetics on measured IOP in the rat eye, because they would most likely be used in a routine laboratory setting, and learn the extent of their effect on eyes with experimentally elevated IOP. 

Methods

Glaucoma Model

All experiments complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Twenty-three male Brown Norway rats (Rattus norvegicus), weighing 300 to 400 g, were used in this study. 

All animals were housed initially in standard lighting with food and water provided ad libitum and the room temperature maintained at 21°C. The room was lit by fluorescent lights (330 lux) that were turned on and off automatically every 12 hours. The animals were weighed weekly to monitor their general health. 

A TonoPen XL tonometer was used to measure awake IOP, with one drop of 0.5% proparacaine hydrochloride applied to each eye, as described previously. ⁸ Briefly, the mean of 10 valid IOP readings was obtained from each experimental and fellow (control) eye for each awake or anesthetized IOP measurement. All animals were acclimated to daily handling for 1 week, and normal awake IOPs were confirmed before episcleral injection. 

Initially, one eye of each animal was injected with 50 μl of a 1.75-M hypertonic saline solution through an episcleral vein, as described previously. ² After the injection, animals were housed in either standard lighting conditions, as described, (12 animals, 12 injected eyes), or a constant low-level (40–90 lux) light environment (11 animals, 16 injected eyes). In five constant-light animals, the fellow eye was subsequently injected, so that more information regarding eyes with elevated IOP could be obtained from this group. Awake IOP was determined in both eyes daily, from the first day after injection throughout the experiment. 

Anesthesia Study

Three different anesthetics were evaluated in every animal: isoflurane, ketamine, and a mixture of injectable anesthetics. This mixture consisted of a solution of 5 ml ketamine (100 mg/ml), 2.5 ml xylazine (20 mg/ml), 1 ml acepromazine (10 mg/ml), and 1.5 ml sterile water. Every animal was evaluated with all three anesthetic agents. Anesthesia and IOP measurements were performed in the following manner. 

For isoflurane, animals were anesthetized by placing them in a shoebox with 3 maximum alveolar concentration (MAC) isoflurane (Forane; Anaquest, Madison, WI), and 0.5 l/min oxygen from an anesthesia cart (Ohmeda 8000; BOC Health Care, West Yorkshire, UK). ⁷ After sedation, animals were transferred to a respirator, and isoflurane was continually supplied at 2.5 MAC, the minimum required to maintain anesthesia. Ketamine (Sanofi Winthrop, New York, NY) and the anesthetic mixture were administered by intraperitoneal injection at a dose of 1.0 ml/kg (100 mg/ml) and 1.0 ml/kg, respectively. 

Awake IOPs were measured immediately before induction of anesthesia. The first postanesthesia IOP reading (time 0) was obtained as soon as the animals reached a depth of anesthesia determined by the absence of pain and palebral reflexes. Time from the administration of anesthesia to time 0 varied, but subsequent measurements were obtained at 5-minute intervals up to 20 minutes. IOP measurements in awake and anesthetized animals were performed in both eyes by the same examiner. All IOP measurements in anesthetized rats were obtained with identical animal handling and without restraint or eyelid manipulation. 

Statistical Analysis

For each measurement time point, average IOPs were determined for both the experimental and control eyes. Statistical analyses were performed by computer (Excel 97; Microsoft, Redmond, WA, and Prism 2.01; GraphPad, San Diego, CA, statistical software packages). 

Results

Awake IOPs in eyes with normal and experimentally elevated pressure ranged from 18 to 52 mm Hg. For animals in both lighting conditions and at every duration of anesthesia, all anesthetics produced a statistically significant (P < 0.01) reduction in measured IOP, compared with the corresponding awake IOP. The IOP data (in millimeters of mercury) for uninjected fellow eyes are illustrated in Figure 1 . The findings for eyes with experimentally elevated IOP are shown in Figure 2 , with values expressed as a percentage of the initial, awake IOP. 

Effects of Anesthesia on IOP in Normal Rat Eyes

Awake IOPs in uninjected normal Brown Norway rat eyes were approximately 20 and 29 mm Hg for rats housed in standard and constant light conditions, respectively. For animals in standard housing, all anesthetics lowered the measured IOP rapidly and dramatically to pressures of between 9 and 11 mm Hg within 5 minutes (Fig. 1A) . The IOP reduction in constant light–exposed eyes was similar (Fig. 1B) , although slightly less dramatic. In both lighting conditions, IOP after isoflurane and the anesthetic mixture continued to decrease throughout the period of observation. For ketamine, the IOP decrease appeared to stabilize at approximately 50% and 60% of the awake IOP level for animals housed in standard and constant lighting conditions, respectively. 

Effects of Anesthesia on IOP in Rat Eyes with Experimentally Elevated IOP

Figure 2 presents the effects of anesthetics on eyes with experimentally elevated IOP for animals housed in both lighting conditions. As with normal eyes, IOP reduction after isoflurane and the anesthetic mixture progressed throughout the experiment. After ketamine, IOP stabilized after 5 minutes at approximately 50% and 60% of the awake IOP in animals housed in standard lighting and constant light, respectively. For each of the three anesthetics, the pattern of anesthesia-induced IOP reduction over time was unaffected by the initial level of the awake IOP. 

Estimation of Awake IOP from IOPs under Ketamine Anesthesia

We used the observed 52% and 40% reductions in IOP (for standard and constant light, respectively) after 5 minutes of ketamine to extrapolate an estimated “awake” IOP from the measured IOPs. Figure 3 illustrates the correlation between actual awake IOP and these extrapolated pressures for all eyes in both lighting groups. The R ²s for these correlations were 0.61 and 0.67 for the standard lighting and constant light conditions, respectively. The average difference between the actual measured IOP and the extrapolated awake IOP was 4.3 mm Hg. The impact of this on measurement variability is best seen among the 12 uninjected, fellow eyes in standard lighting. Whereas the mean actual awake IOP for this group was 20.0 ± 0.50 mm Hg, the extrapolated mean was 21.4 ± 5.1 mm Hg, representing a 10-fold increase in the SD. 

Discussion

The reliability of animal models for understanding the mechanism of pressure-induced optic nerve damage relies heavily on our ability to measure accurately the level of IOP to which the eye and optic nerve are exposed. Previous studies strongly suggest that many general anesthetics can lower IOP in humans, ^{15 16 17 18 19 20 21 22} monkeys, ^{23 24 25 26 27 28 29 30} dogs, ^{31 32 33} cats, ^{24 34} and rabbits. ^{24 35} Others report that some agents, such as ketamine, can elevate IOP. ^{36 37 38}  

The present study provides a detailed, direct comparison between awake and anesthetized IOP measurements in rat eyes and encompasses a wide range of pressures after experimental outflow obstruction. The Brown Norway rat is well suited for measuring awake IOP (using only topical 0.5% proparacaine HCl), in part because of its docile nature and moderately prominent globes. 

The present study demonstrates that all tested general anesthetics significantly and dramatically reduced the measured IOP both in normal eyes and in those with aqueous humor outflow obstruction in the rat. The reduction in IOP with isoflurane and the anesthetic mixture was relatively linear with time, whereas that with ketamine stabilized after the first 5 minutes. Housing the animals in constant low-level light, which minimizes large circadian IOP oscillations in the rat, diminished the magnitude of the IOP reduction, but did not change the overall characteristics of the response to anesthetics. 

The use of general anesthetics resulted in a significant lowering of IOP and a large increase in the SD in these measurements, suggesting variable animal response to the anesthesia, even though all IOPs were measured in a consistent manner by the same individual. In addition, pressure measurements with both isoflurane and the anesthetic mixture continued to decline over time. Both of these observations are contrary to what would be expected if actual IOP were “uncovered” by the anesthetics. These considerations, as well as the fact that physiologic, circadian IOP oscillations can be measured in awake animals, ⁸ argue that awake IOP, rather than IOP under anesthesia, accurately reflects the actual IOP experienced by these eyes. 

Because IOP measured under ketamine stabilized after 5 minutes, its use would appear to be the most reliable in situations in which an anesthetic agent is unavoidable. However, the increased variability in IOP induced by anesthetics limits this strategy, as shown in Figure 3 . Although the correlation between actual awake IOP and estimated awake IOP extrapolated from pressures determined under ketamine was linear, it is only marginally accurate. This is dramatically illustrated by the group of normal, uninjected eyes in standard lighting, in which the measured actual awake IOP ranged from 19 to 21 mm Hg. In this group of eyes, there was a 10-fold increase in the SD of estimated awake IOPs when extrapolated from pressures obtained in animals under ketamine anesthesia, resulting in a range of 16 to 33 mm Hg. Similarly, if all the animals with an estimated awake IOP of approximately 40 mm Hg are considered, they corresponded to a very broad range in actual awake IOP from 28 to 48 mm Hg. 

Through careful comparison of IOP level and optic nerve damage, we have determined that damage is linearly correlated with IOP in the range of 30 to 40 mm Hg. ¹⁰ That IOP measurement under anesthesia produces estimated IOPs that can vary both above and below this range strongly suggests that the use of anesthetics for measuring IOP rats does not reliably represent the true IOP created in response to experimental aqueous humor outflow obstruction. 

In our experience, Brown Norway rats are easily acclimated to awake IOP measurement, which is rapid and can be performed as frequently as necessary. Avoiding general anesthesia eliminates the interanimal variability in response to anesthetics, making the measurement of IOP more reliable and accurate. It also minimizes concerns that anesthetics may mask normal circadian fluctuations in IOP and it avoids any confounding neuroprotective effects of the anesthetics themselves. ^{11 12 13 14} We have found that measuring awake IOP in Brown Norway rats provides the most reliable method for determining IOP both in normal eyes and in those with experimentally elevated pressures. 

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 1999.

 Supported by National Institutes of Health Grant EY10145; the Glaucoma Research Foundation; Alcon Laboratories, Fort Worth, Texas; and unrestricted funds from Research to Prevent Blindness.

 Submitted for publication January 24, 2000; revised April 3 and May 24, 2000; accepted June 8, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

IOP in awake animals and after administration of general anesthetics for untreated, control eyes of Brown Norway rats housed in standard lighting conditions (A) and in constant low-level light (B). Time 0 indicates the first possible postanesthetic pressure measurement. All values are expressed as absolute IOP readings to allow comparison of SD between the awake and anesthetized measurements. Note the large increase in measurement SD (error bars) at all time points after administration of general anesthetics.

Figure 2.

 

IOP in awake animals and after administration of general anesthetics in eyes with experimental outflow pathway sclerosis in rats housed in standard lighting (A) and constant low-level light (B). Time 0 indicates the first possible postanesthetic pressure measurement. Because of the range in baseline IOP for these eyes with outflow obstruction, IOPs are normalized for the graph by calculating each measured IOP under anesthesia as a percentage of the corresponding awake IOP.

Figure 3.

 

Extrapolation from IOP measured under ketamine anesthesia to an estimated “awake” IOP. Measured IOP under ketamine stabilized after 5 minutes at means of 48.2% and 59.5% of awake pressures for rats in standard and constant lighting conditions, respectively. The graph illustrates the correlation between estimated awake IOPs determined by using these average percentages for extrapolation compared with the actual measured awake IOPs. The R ²s were 0.61 and 0.67 for standard and constant lighting conditions, respectively.