Effect of general anesthetics on IOP in elevated IOP mouse model

Abstract

Elevated intraocular pressure (IOP) is the best recognized risk factor for the pathogenesis of glaucoma and the extent of retinal ganglion cell (RGC) degeneration in glaucoma is closely correlated with the extent of IOP elevation. Therefore, accurately and reliably measuring IOP is critical in investigating the mechanism of pressure-induced RGC damage in glaucoma. However, IOP is measured under general anesthesia in most studies using mouse models and many anesthetics affect the IOP measurements in both human and animals. In the present study, we used a noninvasive approach to measure the IOP of mice with normal and elevated IOP. The approach used mice that were awake and mice that were under general anesthesia. Our results demonstrate that not only the behavioral training enables IOP measurement from conscious mice without using a restrainer, it also significantly improves the consistency and reliability of the IOP measurement. In addition, we provide a direct comparison between awake and anesthetized IOP measurements as a function of time after the induction of general anesthesia with several commonly used anesthetic agents. We found that all tested general anesthetics significantly altered the IOP measurements both in normal eyes and in those with elevated IOP. Therefore, we conclude that behavioral training of mice can provide an approach to measure awake IOP that does not require general anesthesia and thus produces reliable and consistent results.

Fig 1. Behavioral training of mice significantly reduces the IOPs

During the second phase of behavioral training, ten daily measurements of IOP were taken from both eyes of 15 conscious mice (n = 30) under topical anesthesia. (A) IOP as a function of time of each eye (gray lines) and the average IOP of each day (solid line). The daily IOP measurement of each eye was averaged from 5 trials and each trial was averaged from 6 measurements. (B) Distributions of the IOP during the first 3 days (Day 1–3) and the last 3 days (Day 8–10) of the second phase behavioral training. The solid and dash vertical lines indicate the average IOP for the two groups. (C) Standard deviation (SD) among the 5 trials of IOP measurements of each eye as a function of time (gray) and the average of the SD of each day (solid line). (D) Average IOP and SD at the beginning (Day 1) and the end (day 10) of the second phase of behavioral training. In the panel D of this figure and all following figures, the error bars indicate standard error (SE). * indicates 0.05 > p > 0.01, ** indicates 0.01 > p > 0.001, *** indicates p < 0.0001. For details of statistical analyses of data presented in all figures, see results section.

Fig 2. General anesthetics significantly reduce the IOP in normal mice

IOP was measured from both eyes of 15 well-trained mice. Measurements were taken from mice that were awake and mice that were under general anesthesia. (A) IOPs immediately before induction of general anesthesia by isoflurane (Iso) and as a function of time after induction of general anesthesia of each eye (gray lines) and the average IOP (solid line). The first point indicates the IOP measured immediately before induction of general anesthesia. The time 0 indicates the time of induction of general anesthesia. (B) IOP immediately before induction of general anesthesia by avertin (Ave) and as a function of time after induction of general anesthesia of the same group of mice as shown in panel A. (C) IOP immediately before induction of general anesthesia by a mixture of ketamine and xylazine (K+Z) and as a function of time after induction of general anesthesia of the same group of mice as shown in panel A. (D) Normalized postanesthesia IOP of mice under general anesthesia by isoflurane (Iso), avertin (Ave), ketamine and xylazine (K+Z) as a function of time after induction of general anesthesia. The horizontal dash-line indicates the conscious IOP measured immediately before induction of general anesthesia.

Fig 3. General anesthetics significantly reduce the IOP in mice with elevated IOP

IOPs were measured from 22 microbead injected eyes with elevated IOP, 13 microbead injected eyes with “normal” IOP, and 11 saline injected eyes with normal IOP. Measurements were taken from mice that were awake or mice that were under general anesthesia. The elevated IOP model was generated by intraocular injection of microbeads into 35 eyes of 23 mice. (A) IOPs of individual eyes as a function of time before and after microbeads injection (gray lines) and the average IOPs as a function of time before and after microbead or saline injection of the eyes with elevated and “normal” IOP (solid lines). The arrows indicate the days for the tests of general anesthetics. The IOPs of day 0 are averaged from the measurements of 3 consecutive days prior to the microbead/saline injection. (B) IOPs measured immediately before induction of general anesthesia by isoflurane (Iso) and as a function of time after induction of general anesthesia of each eye and the average IOPs (solid line). (C) IOPs measured immediately before induction of general anesthesia by avertin (Ave) and as a function of time after induction of general anesthesia of individual eyes (gray lines) and the average IOPs (solid line) of the same group of mice as shown in panel B. (D) IOPs measured immediately before induction of general anesthesia by a mixture of ketamine and xylazine (K+Z) and as a function of time after induction of general anesthesia of individual eyes (gray lines) and the average IOPs (solid line) of the same group of mice as shown in panel B. (E) Normalized postanesthesia IOPs of mice under general anesthesia by isoflurane (Iso), avertin (Ave), ketamine and xylazine (K+Z) as a function of time after induction of general anesthesia.

Fig 4. The effects of general anesthetics on IOP is stronger in mice with elevated IOP

IOPs were measured from 22 microbead injected eyes with elevated IOP and 30 un-injected eyes with normal IOP. Measurements were taken from mice that were awake or mice that were under general anesthesia for 30 minutes. (A) Scatter plot of the IOPs measured immediately before (IOP awake) and 30 minutes after (IOP at 30 min) induction of general anesthesia by isoflurane (Iso) of mice with un-injected normal (Control) and elevated IOP (High IOP) eyes. Dash-line indicates a linear regression of data from mice with normal IOP. Solid line indicates a linear regression of data from mice with elevated IOP. R²1 indicates the correlation coefficient of the linear fitting of data from eyes with normal IOP. R²2 indicates the correlation coefficient of the linear fitting of data from eyes with elevated IOP. (B) Scatter plot and linear regression of the IOPs immediately before and 30 minutes after induction of general anesthesia by avertin (Ave) of the same two groups of eyes shown in panel A. (C) Scatter plot and linear regression of the IOPs immediately before and 30 minutes after induction of general anesthesia by ketamine and xylazine (K+Z) of the same two groups of eyes shown in panel A. (D) Normalized IOPs of eyes with normal and elevated awake IOP under general anesthesia by isoflurane (Iso), avertin (Ave), ketamine and xylazine (K+Z) for 30 minutes.