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Published in final edited form as: Exp Eye Res. 2021 Aug 12;210:108727. doi: 10.1016/j.exer.2021.108727

Effects of acute stress, general anesthetics, tonometry, and temperature on intraocular pressure in rats

Christina M Nicou a, Aditi Pillai a, Christopher L Passaglia a,b
PMCID: PMC8429265  NIHMSID: NIHMS1733806  PMID: 34390732

Abstract

Intraocular pressure (IOP) is important for eye health as abnormal levels can led to ocular tissue damage. IOP is typically estimated by tonometry, which only provides snapshots of pressure history. Tonometry also requires subject cooperation and corneal contact that may influence IOP readings. The aim of this research was to investigate IOP dynamics of conscious animals in response to stressors, common anesthetics, tonometry, and temperature manipulations. An eye of male Brown-Norway rats was implanted with a fluid-filled cannula connected to a wireless telemetry system that records IOP continuously. Stress effects were examined by restricting animal movements. Anesthetic effects were examined by varying isoflurane concentration or injecting a bolus of ketamine. Tonometry effects were examined using applanation and rebound tonometers. Temperature effects were examined by exposing anesthetized and conscious animals to warm or cool surfaces. Telemetry recordings revealed that IOP fluctuates spontaneously by several mmHg, even in idle and anesthetized animals. Environmental disturbances also caused transient IOP fluctuations that were synchronous in recorded animals and could last over a half hour. Animal immobilization produced a rapid sustained elevation of IOP that was blocked by anesthetics, whereas little-to-no IOP change was detected in isoflurane- or ketamine-anesthetized animals if body temperature (BT) was maintained. IOP and BT decreased precipitously when heat support was not provided and were highly correlated during surface temperature manipulations. Surface temperature had no impact on IOP of conscious animals. IOP increased slightly during applanation tonometry but not rebound tonometry. The results show that IOP is dynamically modulated by internal and external factors that can activate rapidly and last long beyond the initiating event. Wireless telemetry indicates that animal interaction induces startle and stress responses that raise IOP. Anesthesia blocks these responses, which allows for better tonometry estimates of resting IOP provided that BT is controlled.

Keywords: wireless telemetry, IOP, stress, anesthesia, tonometry, temperature, glaucoma

1. Introduction

Intraocular pressure (IOP) is necessary for maintaining the optical properties of the eye and for providing biomechanical support to internal tissues. Deviations from baseline can induce an assortment of vision problems depending on the magnitude, direction, and duration of IOP change. Ocular hypotension can cause retinal detachments (Fine et al., 2007), while ocular hypertension can lead to glaucomatous degeneration of the retina and optic nerve (Morrison et al., 2011). It is therefore important to identify and understand sources of IOP variation and physiological mechanisms of IOP regulation.

Determining baseline IOP and the impact of IOP deviations is not trivial. For one, many external and internal factors are reported to alter IOP on time scales of seconds to days. External sources of variation include posture (Malihi and Sit, 2012; Nelson et al., 2020; Turner et al., 2017), altitude (Bruttini et al., 2020; Van de Veire et al., 2008), ambient temperature (Shapiro et al., 1979; Van de Veire et al., 2008), and psychoactive agents like caffeine (Chandrasekaran et al., 2005), cannabis (Wang and Danesh-Meyer, 2021), and anesthetics (Ding et al., 2011; Jasien et al., 2019; Jia et al., 2000; Mikhail et al., 2017). Internal sources include saccades and blinks (Cooper et al., 1979; Downs et al., 2011; Turner et al., 2019a), body temperature (Shapiro et al., 1981), respiration (Cooper et al., 1979; Gökhan and Gökçe, 1975), blood pressure (Klein et al., 2005), cerebrospinal fluid pressure (Berdahl et al., 2008; Ficarrotta and Passaglia, 2020; Ren et al., 2011), circadian rhythms (Agnifili et al., 2015; Moore et al., 1996), and mental stress (Dinslage et al., 1998; Miyazaki et al., 2000; Turner et al., 2019b). Baseline IOP varies continuously as a result. For another, the precise effect of some factors is not certain because results are contradictory or confounded by the other factors. The method of measurement may be partially to blame since most studies used tonometry, which only gives a snapshot of IOP. Moreover, the snapshot may be influenced by psychosomatic responses to the act (Méndez-Ulrich et al., 2018). Baseline IOP is thus implicitly unknown and inferred from collected data. Tonometry also provides limited information about IOP fluctuations and the time course over which different sources of IOP variation exert their modulatory effects.

We have developed a wireless telemetry system for continually recording IOP in free-moving conscious rats. Similar systems are commercially available for larger animals and have been adapted to monitor IOP in rabbits and non-human primates (Downs et al., 2011; McLaren et al., 1996; Schnell et al., 1996). The aim of this study was to use the rat telemetry system to examine internal sources of IOP variation with minimal experimental bias and greater temporal detail than tonometry permits. Of particular interest are putative effects of acute stress, general anesthetics, tonometry, and temperature.

2. Material and methods

All experiments were conducted in accordance with the National Institutes of Health guide for the care and use of laboratory animals and compliance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of South Florida.

2.1. Animal preparation

Male retired-breeder Brown-Norway rats (300-400 g) were housed in a temperature-controlled room (22 °C) under a 12-h light (6 AM):12-h dark (6 PM) cycle with food and water available ad libitum. On the day of surgery, animals were anesthetized with an intraperitoneal bolus of ketamine hydrochloride (75 mg/kg) and xylazine (7.5 mg/kg) that was supplemented as needed. Animals were rested on an isothermal (37 – 38 °C) heat pad (T/Pump Pro, Stryker, Portage, MI) to maintain body temperature (BT). A polyimide microcannula (MicroLumen, Oldsmar, FL) was inserted in one eye and connected to the telemetry system, details of which have been described (Bello et al., 2017; Bello and Passaglia, 2017). In short, the cannula (100 μm inner diameter, 20 mm length) was guided subdermally through an incision in the skull to a translimbal hole in the eye and the tip was inserted in the anterior chamber. The cannula was sutured to the sclera and connected to a plastic coupler affixed to the skull with bone screws and cement. The coupler was connected with 16G PTFE tubing to the IOP sensor, which was fastened to the back of a custom-fit vest worn by the animal. The pressure line was filled with balanced salt solution, 3 mM moxifloxacin hydrochloride (Vigamox®, Alcon, Fort Worth, TX), 1.3 mM enoxaparin sodium (Lovenox®, Henry Schein, Melville, NY), and 2.2 mM triamcinolone acetonide (Triesence®, Alcon, Fort Worth, TX) to prevent microbial and fibrin buildup that can clog the cannula over time. After surgery, animals received an intramuscular bolus of carprofen analgesic (5 mg/kg) every 12 hours for 3 days and their status was monitored with a cage-mounted webcam. Figure 1 illustrates the IOP telemetry system, which wirelessly transmitted data round-the-clock at 0.25 Hz. Sensor calibration was checked by mercury manometry at placement and removal.

Figure 1.

Figure 1.

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Wireless telemetry system for continuous IOP recording in rats. The IOP sensor is worn on the back and connects to a fine cannula that is implanted in the eye via a coupler affixed atop the skull with bone screws. Inset shows a rat eye implanted with a cannula (arrowhead).

2.2. Experimental design

Implanted rats were transported during daytime hours (9A - 4P) from housing to a test room in the animal care facility. For sake of quantification, resting IOP, transport IOP, and baseline IOP were respectively defined as the 5-min average IOP immediately before the researcher entered housing, the 5-min average of peak IOP after exiting housing, and the 5-min average immediately before each experiment in the test room. To assess stress effects, animals were placed for 10 min in an anesthesia chamber perfused with oxygen at 2 L/min. The chamber (9 cm x 9 cm x 23 cm) was considered stress-inducing because it greatly constrained animal movement. Chamber temperature (CT) was monitored with a digital thermometer and pre-heated in some experiments by a warm gel pack (34 °C). IOP and CT readings were averaged in sequential 2-min intervals before, during, and after constraint. To assess anesthetic effects, animals were sedated in the chamber with 3% isoflurane in oxygen at the same flow rate. Upon sedation animals were placed belly down on the heat pad and anesthesia was maintained via isoflurane inhalation though a nose cone or an intraperitoneal injection of ketamine (75 mg/kg). BT was recorded every 60 s with a rectal thermometer until the animal waked. Saline drops were periodically instilled to keep corneas moist. In isoflurane experiments, anesthetic concentration was varied in 10-min intervals between 1, 3, and 5% and IOP and BT readings were averaged over the last 5-min of each interval. In ketamine experiments, IOP and BT readings were averaged in sequential 10-min intervals until the anesthetic wore off. To assess tonometry effects, isoflurane-anesthetized animals were placed belly down on the heat pad and IOP was measured via applanation (TonoPen XL, Medtronic, Sarasota, FL) and rebound (TonoVet, Icare USA, Raleigh, NC) tonometry. Ten tonometry readings were taken by hand and averaged. To assess temperature effects, isoflurane-anesthetized animals were placed belly down on the heat pad or a cool (20 °C) metal table. IOP and BT readings were averaged in sequential 2-min intervals as the animal was slid every 10 min between the two surfaces. Animals were returned to housing after experiments for testing on another day.

2.3. Data Analysis

Raw IOP records were processed with Matlab software (The Mathworks, Natick, MA) using a running median filter and lowpass filter with 80-s windows to remove spurious data. Filtered IOP records were subjected to statistical analysis using SigmaPlot software (Systat, San Jose, CA), with significance assessed at α of 0.05. Resting IOP was log-normal distributed across animals (Reina-Torres et al., 2019), so data of each experiment were referenced to baseline IOP and expressed as ΔIOP. IOP changes were evaluated with paired t-tests if normally distributed and reported as mean ± standard deviation. Otherwise, they were log transformed before t-test evaluation and reported as median with confidence intervals in brackets. Anesthetic concentration and time dependence were evaluated with a one-way repeated-measure ANOVA followed by a Holm-Sidak multiple-pairwise comparison. Animals were implanted for weeks so some were tested on different experiments and multiple times on the same experiment. No bias was noted of a particular rat dataset on the group for any experiment.

3. RESULTS

Continuous IOP recordings were performed on 32 rats. Median resting IOP was 12.7 [11.1, 14.2] mmHg. Figure 2A shows that IOP was rarely constant, varying even when animals were idle. Spontaneous fluctuations of ≥2 mmHg were observed in 34 of 51 video clips of animal inactivity, which implies that IOP is dynamically modulated by internal physiological processes. Figure 2B shows that these processes respond to environmental disturbances. Opening the animal housing door led to a transient IOP bump, which was prolonged if a person entered the room. Moreover, IOP bumps were synchronous across implanted rats. Figure 2C shows that transporting animals to the test room produced an even longer IOP perturbation in most instances (23 of 33 room transfers across 17 animals). IOP in the test room was therefore slightly higher than resting IOP in housing (peak ΔIOP = 3.7 ± 2.0 mmHg, p < 0.001, n = 23) for up to 50 mins.

Figure 2.

Figure 2.

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IOP variability in conscious free-moving rats. (A) Left, IOP recorded from a conscious rat during a period of animal inactivity. Inset images were acquired at times marked by arrowheads and show that the animal had not moved during a spontaneous bump in IOP (box). Right, histogram of spontaneous IOP bump amplitudes across 51 video clips of animal inactivity. (B) IOP recorded simultaneously from two conscious rats exhibited synchronous bumps (boxes) when the animal housing room door was momentarily opened (arrowheads) and when the investigator was in the room (bar). (C) Left, IOP recorded from conscious rats transferred on four instances from housing to the test room. Bars mark the period of housing room entry and cage transport (solid) and undisturbed waiting in the test room (dashes). Traces are shifted vertically for ease of comparison. Resting IOPs are 9.6, 13.6, 11.9, and 15.8 mmHg (top to bottom). Right, histogram of the duration of IOP elevation following 33 room transfers.

3.1. Effect of stress on IOP

The nature of the environmental disturbances suggests that IOP bumps may reflect a startle or stress response. However, disturbances often evoked a burst of motor activity as well. To exclude hyperactivity as an explanation, animals were put in a clear restrictive chamber. Figure 3A shows that the bumps cannot be attributed to animal motion since IOP still increased by varying amount and time course during chamber confinement. CT rose concurrently by ~1.5 °C due to animal body heat. Figure 3B plots the average time course of IOP and CT changes across 11 experiments on 4 animals. IOP went up 2.7 ± mmHg (p = 0.01) in the chamber and took around 40 min to return to near-baseline level. Since IOP remained high long after chamber removal, it cannot be attributed to ambient heating of sensor fluids or electronics. Figure 4 provides further confirmation of the stress response. IOP increased when conscious but not anesthetized animals were placed in a pre-heated chamber of roughly constant CT (n = 2).

Figure 3.

Figure 3.

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Effect of acute stress on IOP. (A) IOP of two conscious rats before, during, and after animal immobilization in a clear chamber, the ambient temperature of which (CT) was concurrently monitored. Selected records illustrate the range in IOP stress response amplitude and waveform. (B) Time-average change in IOP and CT across immobilization experiments relative to their baseline levels before chamber placement. Error bars give standard errors.

Figure 4.

Figure 4.

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Blocking IOP stress response with anesthesia. IOP and BT of a rat that was first placed conscious in the immobilization chamber and then put back in the chamber unconscious under isoflurane anesthesia. CT was concurrently monitored.

3.2. Effect of anesthesia on IOP

Effects of two commonly-used anesthetics on IOP were examined. Figure 5A shows the IOP record of a rat before, during, and after isoflurane anesthesia. Heat support was provided throughout the experiment to counter any anesthetic effect on BT. An IOP stress response can be seen upon animal placement in the isoflurane chamber, which subsides as anesthesia is induced and IOP returns to near-baseline level. Subsequent alterations in isoflurane concentration had no discernible effect on IOP or BT. Spontaneous IOP bumps were also seen in anesthetized animals on occasion. Figure 5B summarizes the results of 15 isoflurane experiments on 7 animals in which BT was held constant with heat support (BT = 37.5 ± 0.9 °C across concentrations, F = 2.39, p = 0.11). Mean IOP change was not significantly different from zero (F = 1.50, p = 0.23) under 1% (−0.3 ± 1.8 mmHg), 3% (0.2 ± 1.8 mmHg), or 5% (−0.6 ± 1.6 mmHg) isoflurane. Figure 5C shows the IOP record of a rat before, during, and after ketamine anesthesia. An IOP stress response can again be seen upon sedation and ketamine injection. IOP returned erratically to near-baseline level as anesthesia was induced and remained there for 30 min until the bolus wore off and the animal began waking. BT rose slightly after initiating heat support and held steady thereafter. Figure 5D summarizes the results of 16 ketamine experiments on 6 animals. BT was constant for the most part during ketamine anesthesia (BT = 38.3 ± 0.4, p > 0.05 for all comparisons except 5 versus 15 min). Mean IOP change was not significantly different from zero for the 5 min (−0.5 ± 1.9 mmHg, p = 0.31), 25 min (−0.5 ± 2.6 mmHg, p = 0.44), and 35 min (0.7 ± 2.8 mmHg, p = 0.27) post-induction intervals but was slightly lower for the 15 min interval (−1.9 ± 1.9 mmHg, p < 0.01).

Figure 5.

Figure 5.

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Effect of common anesthetics on IOP. (A) IOP and BT of a rat before, during, and after isoflurane anesthesia. Animal was sedated in the isoflurane chamber, placed on a heat pad, and anesthetic concentration was then varied via a nose cone. Spontaneous IOP fluctuations (box) still occurred in anesthetized animals. (B) Cumulative analysis of IOP and BT data across isoflurane concentration. (C) IOP and BT of a rat before, during, and after ketamine anesthesia. Animal was briefly sedated with isoflurane in the chamber, injected with ketamine just before waking, and placed on a heat pad when unconscious. (D) Cumulative analysis of IOP and BT time course across ketamine experiments. Brackets indicate time points that differ significantly. All IOP changes are relative to experimental baseline, and whiskers in box plots indicate minima and maxima.

3.3. Effect of tonometry on IOP

Possible effects of tonometry on IOP were also examined. Figure 6A shows the IOP record of an anesthetized rat while collecting applanation tonometry (AT) and rebound tonometry (RT) data. IOP continually crept higher during AT and was fairly steady during RT. Individual AT readings were highly variable and the average exceeded baseline. RT readings, on the other hand, scattered closely around baseline. Figure 6B summarizes the results of 9 experiments with each tonometer across 5 animals. IOP measured by AT and the system after AT differed significantly from baseline (median ΔIOP = 3.1 [1.0, 4.7] mmHg and 2.7 [1.4, 3.2] mmHg, respectively; p < 0.01 for both), while there was no measurable difference for RT or the system after RT (p = 0.44 and 0.97, respectively). IOP likely increased during AT because of repeated tapping of its much larger tip on the small rat cornea. Figure 6C shows the IOP record of a conscious rat placed in a large plexiglass enclosure with an open wall to perform RT when the animal was idle. Transient bumps can be seen when the experimenter entered housing and when the animal was placed in the enclosure. The latter bump was prolonged though the animal was not further handled. Individual RT readings were more variable in conscious animals and averaging does not capture the dynamic nature of IOP. Across 5 awake tonometry experiments, IOP was significantly higher than the resting level for both the system and RT (median ΔIOP = 3.0 [2.7, 5.8] mmHg and 4.9 [1.8, 7.2] mmHg, respectively; p < 0.05 for both). Again, no difference was detected between system and RT (p = 0.48).

Figure 6.

Figure 6.

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Effect of different tonometers on IOP. (A) IOP of an anesthetized rat before, during, and after applanation tonometry (AT, top) and rebound tonometry (RT, bottom). Bars indicate period when tonometer readings were made, circles indicate individual tonometer readings, and squares give the mean and standard deviation of those readings. (B) Cumulative analysis of IOP data measured with the telemetry system (S) and tonometers (T) for AT (top) and RT (bottom) experiments. IOP changes are relative experimental baseline, and whiskers indicate minima and maxima. (C) IOP of a free-moving conscious animal before, during, and after RT. Arrowhead marks the experimenter entering the housing room. Bar indicates period when animal was placed in a tabletop three-walled enclosure in the room and tonometer readings were made when the animal was idle. Circles indicate individual tonometer readings, and squares give the mean and standard deviation of 10 sequential readings.

3.4. Effect of temperature on IOP

It was noted during anesthesia experiments that BT influenced IOP. Figure 7A shows the IOP record of an anesthetized rat without heat support. IOP decreased steadily after anesthetic induction at −0.5 mmHg/min and returned to near-baseline level as the animal waked. The IOP decline was confirmed by RT and mirrored by a BT decline of −0.2 °C/min. Figure 7B shows the IOP and BT records of an anesthetized rat before, during, and after sliding the animal between a heat pad and cool table. Both initially crept higher on the heat pad, which was presumably warmer than resting BT, and steadily decreased on the cool table. Sliding the animal back on the heat pad incompletely reversed the decline, so IOP and BT declined further upon return to the cool table. The IOP changes were again confirmed by RT. Figure 7C summarizes the results of 11 temperature experiments on 5 animals. IOP and BT were highly correlated (R2 = 0.73).

Figure 7.

Figure 7.

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Effect of temperature on IOP. (A) IOP and BT of a rat before, during, and after isoflurane anesthesia with the animal resting on a cool table without heat support. Square symbols give mean IOP via RT. (B) IOP and BT of a rat before, during, and after isoflurane anesthesia with the animal moved between a heat pad and cool table. Square symbols give mean IOP via RT. (C) Time-average IOP and BT data across temperature manipulation experiments. IOP changes are relative to experimental baseline. Error bars give standard error.

Additional experiments were conducted to corroborate the temperature results. Figure 8A shows the IOP record of a conscious rat steered between the heat pad and cool table. IOP bumps can be seen when the animal was removed and returned to its cage, as well as during each surface transition when it was not directly handled. More importantly, IOP fluctuated about a baseline that was comparable to the resting level. Figure 8B shows that IOP readings from an euthanized rat were similarly unaffected by warm and cool surfaces. Temperature effects in anesthetized animals thus reflect inhibition of thermoregulation. Figure 8C shows IOP and BT records of an anesthetized rat in different recumbent positions. BT was steady except for slight dips on rotation from sternal to lateral recumbency. IOP was unchanged when the cannulated eye was rotated away from the heating pad and rose several mmHg when rotated toward the pad. IOP also increased when a warm gel pack was positioned near the cannulated eye while BT was unchanged. Effect of BT on IOP is thereby mediated in part or whole through changes in eye temperature.

Figure 8.

Figure 8.

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Control experiments for temperature effects on IOP. (A) IOP of a conscious rat moving between warm and cool surfaces. Animal was gently steered onto a heat pad and cool table using a large clear enclosure. (B) IOP of a euthanized rat laid on the warm and cool surfaces. (C) IOP and BT of an anesthetized rat in different recumbent positions. Animal rested either belly down or on its side with the cannulated eye up (CE↑) or down (CE↓). A warm gel pack was brought near the cannulated eye over the period indicated by the + symbol.

4. Discussion

In this study, IOP was found to spontaneously vary by several mmHg in conscious free-moving rats. The fluctuations persisted in idle and anesthetized animals and thereby reflect internal physiological processes that directly or indirectly modulate IOP. Some of these processes are keenly sensitive to environmental disturbances. Their sensory trigger was not investigated but auditory, tactile, and perhaps visual cues are certainly involved. Activation of these processes was generally associated with transient IOP increases but transient decreases were sometimes observed. IOP bumps lasted a few minutes if the disturbance was startling in nature or tens of minutes if more stressful. The fast dynamics cannot be captured by averaging AT or RT readings because data are too sparse and variable. Tonometry measurements were found to overestimate resting IOP by a few mmHg in conscious rats, and better estimates were obtained by attenuating the stress response with anesthetics. Tonometry measurements on anesthetized animals also had less variance, implying that the variability seen in conscious animals reflects spontaneous IOP fluctuations in addition to operator skill. Isoflurane and ketamine anesthetics both maintained IOP at near-baseline level if BT was regulated. Changes in BT, or specifically eye temperature, due to anesthetics or ambient environment had a rapid and pronounced impact on IOP. Care should be exercised during tonometry to ensure operator body heat does not artificially elevate readings.

4.1. Relation to prior work

Our results support and extend prior work and clarify contradictory reports on effects of stress, anesthetics, and temperature on IOP. Psychophysiological stress has long been associated with IOP elevation and glaucoma precipitation (Shily, 1987). Mental relaxation techniques that aim to reduce stress were recently shown to lower IOP, reduce stress biomarkers, promote anti-inflammatory gene expression, and improve the quality-of-life of glaucoma patients (Dada et al., 2018). Like this study, stress effects on IOP have been investigated experimentally by forcibly immobilizing animals. Rabbits immobilized for 1 hr in a clear plastic tube had elevated cortisol, adrenaline, and noradrenaline levels and tonometry readings were 2-3 mmHg higher compared to non-immobilized animals (Miyazaki et al., 2000). The rise time was not examined but the duration lasted over an hour after immobilization. An IOP elevation of comparable magnitude was also observed via wireless telemetry in immobilized monkeys (Turner et al., 2019b) and here in immobilized rats. The duration of elevation is unknown for the monkey study because anesthetic was injected during immobilization, which terminates the stress effect. It was similarly long-lasting in rats although the rabbit immobilization period was much longer. Prolonged effects on IOP do not, however, require forced immobilization since a rabbit telemetry study saw a 1-hr IOP bump after cage change (Dinslage et al., 1998). Anticipation of immobilization appears sufficient because IOP jumped simultaneously in implanted rats upon opening the housing room door and remained elevated following room transfers. An anticipatory bump was also observed in monkeys when someone was in the housing room even if the animal was not handled (Turner et al., 2019b). Stress from actual or anticipated handling and immobilization leads to variability and inaccuracy in tonometry estimates of resting IOP that can only be overcome by contactless telemetry systems. The IOP stress response is important to consider in the interpretation of tonometry data.

Unlike acute stress there is less consensus about the effect of general anesthetics on IOP, and the consensus that does exist may be faulty or misleading. Most studies report that isoflurane decreases IOP (Buehner et al., 2011; Craig and Cook, 1988; Ding et al., 2011; Jia et al., 2000; Mirakhur et al., 1990; Tsuchiya et al., 2021) but some have seen IOP increase (Chae et al., 2020) or not change (Ausinsch et al., 1975; Dear et al., 1987). Even more contradictory is ketamine, with numerous studies reporting an IOP decrease (Chae et al., 2020; Ding et al., 2011; Holve et al., 2013; Jia et al., 2000; Rajaei et al., 2017; Trim et al., 1985), increase (Antal et al., 1978; Ghaffari and Moghaddassi, 2010; Hofmeister et al., 2006; Qiu et al., 2014; Schutten and Van Horn, 1977), or no change (Blumberg et al., 2007; Drayna et al., 2012). An implicit and likely mistaken assumption of these tonometry studies is that IOP measured before anesthetic induction reflects resting IOP. Our and other telemetry (Dinslage et al., 1998) results show that animal handling induces a stress response that raises IOP long past the handling event. There was actually little-to-no effect of isoflurane or ketamine anesthesia on rat IOP, which agrees with a monkey telemetry study (Jasien et al., 2019). Hence, the IOP decrease reported by tonometry studies may instead reflect anesthetic inhibition of the stress response. In support of this interpretation, early tonometry studies noticed no significant IOP change in children that were already sedated (Ausinsch et al., 1975; Ausinsch et al., 1976). Another potential complication is the effect of anesthetics on thermoregulation (Lenhardt, 2010). Some tonometry studies report IOP decreases of 5 mmHg or more in mice and rats under isoflurane and ketamine anesthesia (Ding et al., 2011; Jia et al., 2000; Tsuchiya et al., 2021). Such large drops were recorded in this study only from implanted animals that were not provided heat support. None of the cited rodent studies monitored BT to confirm it was stable, one does not mention heat support, and one explicitly states a heat pad was not used. Reported IOP decreases may thereby be explained by a combination of stress-elevated baseline and BT loss and not necessarily a direct anesthesia effect. However, our results cannot reconcile reported IOP increases. IOP elevation is primarily seen for ketamine, which is known to increase heart rate, blood pressure, and vascular resistance (Reich and Silvay, 1989). Perhaps anesthetic effects on IOP depend on species, dosage amount, or delivery method (intravenous versus intramuscular).

There is comparatively less research on the effect of temperature on IOP. Two groups put individuals in a heated room and neither noticed changes in IOP (Shapiro et al., 1979; Van de Veire et al., 2008). One detected a few mmHg increase after 3 hrs that was attributed to a rise in BT since it was not observed in acclimated subjects (Shapiro et al., 1979; Shapiro et al., 1981). No IOP change was noted here either when conscious rats were exposed to different surface temperatures, presumably because thermoregulatory processes held BT constant. IOP decreases were though reported in humans and rabbits immersed to the head in warm water (Findikoglu et al., 2015; Nichols et al., 1984). They might not have been seen in implanted rats because the thermal challenge to the body is much greater than a heat pad, causing elevated heart rate and reduced blood pressure in immersed humans and elevated episcleral venous pressure and reduced aqueous humor formation in immersed rabbits. Our results are not only consistent with tonometry studies that demonstrate IOP increases upon warming the eye via prolonged eyelid closure and IOP decreases upon cooling with a cold mask or air stream (Fabiani et al., 2016; Orgül et al., 1995; Ortiz et al., 1988), but also reveal the fast dynamics of temperature-driven IOP changes.

4.2. Limitations of the work

Two limitations of the work should be noted. One is the external locus of the pressure sensor, which introduces non-physiological variability in IOP records due to hydrodynamic effects of head and body rotation (Bello and Passaglia, 2017). The extraneous noise is heightened during hyperactivity, such as when animals are stressed or waking from anesthesia. Another is that physiological mechanisms underlying observed effects were not investigated. While IOP startle and stress responses can be broadly attributed to the autonomic nervous system (McDougal and Gamlin, 2015) since they were blocked by anesthetics, the precise mode of action is uncertain. Autonomic signals could alter IOP in rats by changing ocular blood flow or components of aqueous humor dynamics (Ficarrotta et al., 2018). Also, IOP still fluctuated in anesthetized rats so some autonomic signals are not inhibited by isoflurane or ketamine. The autonomic nervous system could mediate temperature effects as well given that circadian BT and IOP rhythms are strongly coupled (Ostrin et al., 2019). A biophysical explanation cannot though be discarded since aqueous outflow depends on temperature-sensitive factors like fluid viscosity and metabolic activity (Boussommier-Calleja et al., 2015; Reina-Torres et al., 2020), which is suggested by the sensitivity of rat IOP to ocular heating. To address these limitations, a telemetry system that mounts to the skull and records additional parameters like BT, heart rate, and motor activity is being developed to reduce extraneous noise and better understand the mechanisms of IOP variation.

5. Conclusion

The principal findings of the study are that IOP is dynamically modulated in conscious rats by physical and physiological processes that are sensitive to temperature and animal stress but not anesthetics. The processes can act rapidly and produce IOP changes that last long after initiation. Continuous IOP telemetry is required to record these fast changes without disturbing the animal. Rebound tonometry gives accurate snapshots of IOP but animal handling elevates IOP in conscious rats. A better estimate of resting IOP may be obtained by anesthetizing animals while maintaining resting BT.

Highlights.

  • IOP was continuously recorded from conscious rats via wireless telemetry

  • IOP varied spontaneously, even in idle and anesthetized animals

  • Startling and stressful disturbances to animal rapidly and transiently raised IOP

  • Anesthesia blocked the stress response without altering baseline IOP

  • Loss of body heat during anesthesia caused a large and rapid decline in IOP

Acknowledgements

Authors thank Roger Kayeleh and Simon Bello for help with anesthesia and tonometry data collection.

Funding

The work was supported by the National Institutes of Health [grant R01 EY027037].

Abbreviations

AT

applanation tonometry

BT

body temperature

CE

cannulated eye

CT

chamber temperature

IOP

intraocular pressure

RT

rebound tonometry

Footnotes

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Declaration of competing interest

Authors have no relevant financial disclosures.

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