Current Eye Research, 2014; 39(4): 365–369 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2013.845224

ORIGINAL ARTICLE

Effects of Three Commonly Used Anesthetics on Intraocular Pressure in Mouse

Department of Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China

ABSTRACT Purpose: To investigate the effects of three commonly used general anesthetics on intraocular pressure (IOP) in mouse. Methods: Fifteen 2–3-month-old C57BL/6J mice were randomly divided into three groups (each group, n = 5). A non-invasive TonoLab tonometer (Icare LAB, Icare Finland Oy, Espoo, Finland) was used to measure IOP at 0, 5, 10, 15, 20, 30 min after mice were anesthetized, respectively, by intraperitoneal injection of sodium pentobarbital (150 mg/kg), chloral hydrate (500 mg/kg) and a mixture of ketamine and xylazine (75 mg/kg and 13.6 mg/kg). IOP were obtained in the daytime and nighttime. Anterior segment was photographed and palpebral fissure height was measured offline. Results: Immediately after anesthesia, the averaged IOPs in the three groups were 17.2  1.5, 16.7  1.4 and 17.3  2.4 mmHg in the daytime and 19.3  2.1, 21.3  1.1 and 21.7  1.5 mmHg in the nighttime. Thereafter, the averaged IOPs in sodium pentobarbital and chloral hydrate groups showed a trend of decline. Then IOPs became stable at 10–15 min after anesthesia. In contrast, the IOPs of ketamine and xylazine injected group increased to 23.7–25.1 mmHg at 10–15 min in the daytime and 26.1–27.7 mmHg in the nighttime. Compared to chloral hydrate and sodium pentobarbital treated mice (2.4  0.1 mm, 1.7  0.0 mm), ketamine and xylazine injected animals had significantly increased palpebral fissure height (3.6  0.3 mm, p50.01). Conclusion: General anesthetics have a large impact on mouse IOP. Sodium pentobarbital and chloral hydrate reduce but the ketamine and xylazine mixture increases mouse IOP. IOP levels become stabilized at 10 to 15 min after anesthesia. The ketamine and xylazine cocktail mediated elevation of palpebral fissure height may be associated with an increasing of intraorbital pressure. Measurement performs at 10–15 min after anesthesia may obtain more reliable IOPs. Keywords: Chloral hydrate, intraocular pressure, ketamine and xylazine, mouse, sodium pentobarbital

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Yiguo Qiu*, Hongxia Yang* and Bo Lei

INTRODUCTION

extraocular compressive pressure including tonus from the extraocular musculature) also plays an essential part in regulating IOP.1 As one of the most important endpoints in ophthalmic research, IOP is a very useful parameter. Elevated IOP has been associated with progressive retinal ganglion cell (RGC) degeneration and is one of the most recognized and documented

Intraocular pressure (IOP) is defined as the pressure exerted by the components of the eye against the fibrous tunics of the eye. IOP level is determined by ocular volume that includes intraocular fluid volume, choroidal blood volume and vitreous volume. Ocular compliance (scleral rigidity and

*These authors contributed equally to this work Received 25 February 2013; revised 22 July 2013; accepted 11 September 2013; published online 11 November 2013 Correspondence: Bo Lei, MD, PhD, Department of Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, 1 You Yi Road, Yu Zhong District, Chongqing 400016, China. Tel: 86 23 89011851. Fax: 86 23 89011851. E-mail: [email protected]

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366 Y. Qiu et al. features of glaucoma.2,3 However, accurate assessment of IOP practically in mouse was not easy. One of the obstacles is that many factors including strain, gender, age, weight and circadian rhyme could impact on IOP. Furthermore, drugs with sympathetic or parasympathetic actions, such as anesthetics, can alter the IOP as well. To obtain accurate IOP, thoroughly understanding of the factors that affect the IOP in experimental animal is crucial. Previous studies indicated that many reagents for general anesthesia could affect the IOP in both human and animals.4–7 Since sodium pentobarbital, chloral hydrate and a mixture of ketamine and xylazine are the most commonly used anesthetics in rodents and it is unknown whether these anesthetics affect mouse IOP differently. It is reported recently that TonoLab tonometer (Icare LAB, Icare Finland Oy, Espoo, Finland) provide an accurate means to evaluate IOP in mouse.8–11 Here, we study the effects of systemic administration of the three anesthetics on IOP in mouse.

Measurement of IOP and Palpebral Fissure Height A TonoLab tonometer was used to measure the IOP in anesthetized mice. The TonoLab tonometer had been calibrated by the manufacturer. IOP measurements were performed according to the manufacturer’s instruction in both eyes by the same examiner with identical procedure. The time point when the animal was sedate was defined as 0 min. IOP was measured at 0, 5, 10, 15, 20 and 30 min. The IOP readings of each eye were averaged from three measurements. To compare the difference of IOP on day-time and night-time, measurements were made between 9 AM to noon and 9 PM to midnight. Ten minutes after the mice were anaesthetized, anterior segment photography was performed with a slit lamp microscope and the photos were digitalized with a computer. The palpebral fissure height was measured with Photoshop (Adobe Systems Incorporated, San Jose, CA) for the photos.

Data Analysis MATERIALS AND METHODS Animals Fifteen 2–3-month-old C57BL/6J mice were randomly divided into three groups with five animals in each group (n = 5). Mice (C57BL/6J) were obtained from the Jackson Laboratory (Bar Harbor, ME). All animal procedures performed in this study complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Chongqing Medical University. Mice were housed under the standard specific pathogen free conditions and were fed with standard laboratory food without limitation to access to water.

Anesthetic Agents Three different groups of anesthetic agents i.e. sodium pentobarbital (Bopei Biotech Co., Ltd, Chongqing, China), chloral hydrate (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) and a mixture of ketamine (Fujian Gutian pharmaceutical Co., Ltd, Ningde, Fujian, China) and xylazine (VEDCO Inc., St. Joseph, MO) were evaluated. Chloral hydrate, sodium pentobarbital and the mixture of ketamine and xylazine solutions were administered intraperitoneally at 150 mg/kg, 500 mg/kg, 75 mg/kg (ketamine) and 13.6 mg/kg (xylazine) respectively based on previous studies.12–14

Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). Results were expressed as mean  SD. Data were analyzed with two-way ANOVA followed by post Hoc Bonferroni correction. The statistical significance between daytime and nighttime of each time point for each group was analyzed by paired t-test. p Value50.05 was considered to be significantly different.

RESULTS Effects of General Anesthetics on IOP in Mouse Mice were sedate at 9 min after injection of sodium pentobarbital, 4 min after chloral hydrate and 2 min after the ketamine and xylazine cocktail. Immediately after anesthesia (time 0), the averaged IOP in the three groups were 17.2  1.5 mmHg, 16.7  1.4 mmHg and 17.3  2.4 mmHg in the daytime and were 19.3  2.1, 21.3  1.1 and 21.7  1.5 mmHg in the night time, respectively. There was no significant difference between chloral hydrate and sodium pentobarbital treated group at any time point in both daytime and nighttime. But IOPs in the ketamine and xylazine mixture treated group were higher than other two groups at all the time points except at the 0 min. (Figure 1, p50.001). Thereafter, the averaged IOPs in the three groups exhibited different changes: IOPs decreased in the sodium pentobarbital and the chloral hydrate groups, but increased in the Current Eye Research

Effects of Anesthetics on Intraocular Pressure

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FIGURE 1 The IOPs measured in mice anesthetized respectively with chloral hydrate, sodium pentobarbital and a mixture of ketamine and xylazine. IOPs measured in the daytime (A) and in the nighttime (B). (p50.001).

FIGURE 2 Comparison of IOPs measured in the day time and night time at 0, 5, 10, 15, 20 and 30 min after anesthetized with sodium pentobarbitol (A), chloral hydrate (B), and a mixture of ketamine and xylazine (C) in mice. No significant difference was found between the daytime and nighttime at each time point.

ketamine and xylazine mixture group. In all three groups, IOPs were stable from 10 to 15 min after anesthesia: IOPs were 11.0–11.6 (mean  SD:11.4  0.99, 95% IC: [10.99,11.81]) mmHg in the daytime and 10.8–12.2 (mean  SD: 11.7  2.7, 95% IC: [10.86,12.54]) mmHg in the nighttime in the sodium pentobarbital and the chloral hydrate groups, and were 23.7–25.1 (mean  SD: 24.4  4.6, 95% IC: [21.8, 27.0]) mmHg in the daytime and 26.1– 27.7 (mean  SD: 27.1  5.1, 95% IC: [24.87, 29.33]) mmHg in the night time in the ketamine and xylazine mixture injected group. In addition, IOPs showed higher variance among individuals in the ketamine and xylazine mixture group than the other two groups.

IOPs Measured at Daytime and Nighttime in Mice We compared the differences of IOP measured at daytime and nighttime in mice treated with sodium pentobarbital, chloral hydrate and the mixture of ketamine and xylazine, respectively, no significant difference among each time point were noted (Figure 2, p40.05). !

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Palpebral Fissure Height Compared to sodium pentobarbital (1.7  0.0 mm) and chloral hydrate (2.4  0.1 mm) treated mice, ketamine and xylazine mixture injected mice had significantly increased palpebral fissure height (Figure 3, 3.6  0.3 mm p50.01, p50.001).

DISCUSSION Mouse has been the most commonly used animals in eye researches. When IOP is taken as one of the parameters, the effects of anesthetics should not be neglected. However, such studies are limited. In present study, we compared the IOP in mice anesthetized with three commonly used general anesthetics using an accurate and noninvasive apparatus. Previous studies showed various anesthetics altered IOP in rodents in different ways.4–6 For instance, propofol, isoflurane reduced IOPs,5,6,15,16 while ketamine increased IOPs.7,17 Many factors including the dosage, route of administration, strain, procedure, apparatus may contribute to the discrepancies. In this study, we found that the three anesthetics change IOP in a time-dependent manner.

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368 Y. Qiu et al.

FIGURE 3 Anterior segment photographs of mice anesthetized with intraperitoneally injection of sodium pentobarbital, chloral hydrate and a mixture of ketamine and xylazine. Pictures were taken at 10 min after the animals were sedate. Mice anesthetized with ketamine and xylazine exhibited higher palpebral fissure. (**p50.01, ***p50.001).

Therefore, IOP obtained at different time intervals after anesthesia may also contribute to the IOP discrepancies. We found that sodium pentobarbital and chloral hydrate decreased IOP in the initial 5 min after the animals were sedated. Then, the IOP was stable from 10 to 15 min. IOP underwent 5–8 mmHg changes during this period. On the contrary, the mixture of ketamine and xylazine increased IOP in the initial 5 min after the animals were sedated. IOP underwent 7–8 mmHg changes during this period. Therefore, measurements made at different time intervals during the 30-min period may yield different results. We observed that the IOPs were relative stable from 10 to 15 min after sedate, we suggest the IOP measurements should be performed at this time window. Selection of this time window for IOP measurement may provide consistent results. The effects of ketamine on IOP are inconsistent in literature. Some reports showed that ketamine decreases IOP in human,18 monkeys,19 rats5 and mice,6 while others showed that ketamine increased IOP in human7 and rabbits.17 The mixture of ketamine and xylazine reduced the IOP in mice.10 A recent

study20 described a time-dependent change of IOP in mice anaesthetized with ketamine and xylazine. There was an initial increase of IOP followed by a decrease of IOP after the administration of ketamine and xylazine. After induction of general anesthesia, an increase phase of IOP occurred very fast (less than 3 min) followed by a decline of the IOP until it reaches a relatively stable level at 15–20 min after anesthesia. Our results are similar to these findings.20 We have also seen a rise in IOP (7–8 mmHg) in the initial 5 min after anesthesia. Although the mechanism is unknown, ketamine is believed to have sympathomimetic effects.21 Activation of sympathetic nerves by ketamine may be associated with the phenomenon. As mentioned before, intraocular fluid volume may have influence on IOP level. Activation of sympathetic nerve may increase secretion of aqueous humor and the resistance of aqueous humor out flow, thus increase the IOP. Since ketamine has the effect to activate sympathetic nerves, some studies supported our guess: antisympathetic drugs have been used to reduce intraocular pressure by decreasing the production of aqueous humor.22 Pupile dilation is primarily mediated by the sympathetic nervous system. Current Eye Research

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Effects of Anesthetics on Intraocular Pressure Pupile dilation will increase the resisitance of the aqueous humor out flow.23 Interestingly, we found the mixture of ketamine and xylazine resulted in significantly increase of palpebral fissure height than the other two anesthetics. Increasing of palpebral fissure height in mouse is helpful for some ocular examinations, such as fundus and electroretinogram. However, it may reflect an increasing of intraorbital pressure and may be a sign of increased IOP. The reasons the mixture of ketamine and xylazine enhances the palpebral fissure height is uncertain. Excitement of sympathetic nerves may be associated with the phenomenon. The sympathomimetic effects of ketamine may increase the muscular tone of extraocular muscle, increase secretion of aqueous humour or impede the out flow of aqueous humour,22,23 then consequently increase the palpebral fissure height. In summary, we demonstrated that sodium pentobarbital, chloral hydrate and a mixture of ketamine and xylazine anesthetics affected the IOP in a timedependent manner. Sodium pentobarbital and chloral hydrate reduce while the mixture increases mouse IOP. Measurement performs at 10–15 min after anesthesia may obtain more reliable IOPs.

DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported in part by National Natural Science Foundation of China grants (30973251, 81271033), a Ministry of Science and Technology grant (2011ZX09302-007-002) and Chongqing Key Laboratory of Ophthalmology (CSTC).

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Effects of three commonly used anesthetics on intraocular pressure in mouse.

To investigate the effects of three commonly used general anesthetics on intraocular pressure (IOP) in mouse...
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