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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Anesthesiology. 2019 Jun;130(6):981–994. doi: 10.1097/ALN.0000000000002660

Disruption of rapid eye movement sleep homeostasis in adolescent rats after neonatal anesthesia

Nadia Lunardi 1, Ryan Sica 2, Navya Atluri 3, Kathryn A Salvati 4, Caroline Keller 5, Mark Beenhakker 6, Howard P Goodkin 7, Zhiyi Zuo 8
PMCID: PMC6520183  NIHMSID: NIHMS1520025  PMID: 30946702

Abstract

Background:

Prior studies suggest that rapid eye movement sleep rebound and disruption of rapid eye movement sleep architecture occur during the first 24 hours following general anesthesia with volatile anesthetics in adult rats. However, it is unknown whether rapid eye movement sleep alterations persist beyond the anesthetic recovery phase in neonatal rats. This study tested the hypothesis that rapid eye movement sleep disturbances would be present in adolescent rats treated with anesthesia on postnatal day 7.

Methods:

Forty-four neonatal rats were randomly allocated to treatment with anesthesia consisting of midazolam, nitrous oxide, and isoflurane or control conditions for 2 hours or 6 hours. Electroencephalographic and electromyographic electrodes were implanted and recordings obtained between postnatal days 26–34. The primary outcome was time spent in rapid eye movement sleep. Data were analyzed using two-tailed unpaired t-tests and two-way repeated measures analysis of variance.

Results:

Rats treated with midazolam, nitrous oxide and isoflurane exhibited a significant increase in rapid eye movement sleep three weeks later when compared to control rats, regardless of whether they were treated for 2 hours (174.0 ± 7.2 minutes in anesthetized, 108.6 ± 5.3 in controls, p <0.0001) or 6 hours (151.6 ± 9.9 minutes in anesthetized, 108.8 ± 7.1 in controls, p = 0.002).

Conclusions:

Treatment with midazolam, nitrous oxide and isoflurane on post-natal day 7 increases rapid eye movement sleep three weeks later in rats.

Introduction:

Numerous laboratories have documented widespread neuronal degeneration and persistent learning deficits in a variety of developing species following exposure to common inhaled and intravenous anesthetics1–7. Several retrospective clinical studies, especially of longer and repeated exposures, have also linked childhood anesthesia and surgery to cognitive impairment8–11. However, the clinical significance of these findings remains unclear, due to concerns regarding the translational value of the animal models used in bench research and the confounding factors associated with retrospective studies in humans. Though several clinical trials are ongoing,12,13 the results of three recent large-scale studies [General Anesthesia and Awake-Regional Anesthesia in Infancy (GAS)14, Pediatric Anesthesia Neurodevelopment Assessment (PANDA)15and Mayo Anesthesia Safety in Kids (MASK)16] suggest that anesthetic exposures of less than 1 hour do not lead to severe cognitive deficits in young children. While these clinical data are consistent with most animal studies that find little evidence for neurodevelopmental effects after brief exposures, it remains unclear whether longer anesthetics are safe.

To date, most preclinical and clinical studies of anesthetic-related neurotoxicity have focused on learning disabilities as the primary outcome, whereas very few have investigated the effects of anesthetic drugs on other functional, non-cognitive domains17,18. However, several lines of evidence support the idea that anesthetic drugs interfere with endogenous sleep pathways and can affect sleep-wake behavior in the short term. For example, adult rats exposed to 6 hours of isoflurane exhibit lower levels of slow wave sleep in the 18 hours following general anesthesia, compared to sham-controls19. Also, rapid eye movement (REM) sleep debt accrues in adult mice exposed to 6 hours of isoflurane or sevoflurane immediately after anesthesia20. Importantly, it is becoming increasingly evident that sleep is crucial for the assembly of new synaptic circuits during brain development, learning and memory. For example, Boyce et al recently demonstrated that optogenetic silencing of neurons activated during REM sleep after learning impairs fear-conditioned contextual memory (a test of hippocampus-dependent learning and memory), establishing direct causality between REM sleep and memory consolidation21. Li and colleagues also recently showed that pruning of new dendritic spines is impaired during development and motor learning in REM sleep-deprived mice22.

Thus, the aim of this study was to determine whether exposure to anesthetic drugs during a phase of intense brain plasticity interferes with subsequent sleep-wake behavior. We examined the effects of a combination of anesthetics on the sleep-wake behavior of adolescent rats following a single brief (2 hours) exposure on postnatal day 7 (Anesthesia 2h group). In keeping with previous studies from our laboratory that documented decreased synaptic density and disruption of presynaptic vesicle spatial organization after prolonged anesthetics2,3, we also assessed the effects of a 6 hour exposure to the same anesthetic protocol (Anesthesia 6h group). Our specific hypothesis was that REM sleep disturbance would be present in adolescent rats exposed to anesthesia at neonatal age. The primary outcome was time spent in rapid eye movement sleep.

Materials and Methods

Animals and Anesthesia:

We exposed Sprague-Dawley rat pups to anesthesia at postnatal day 7. Postnatal day 0 was considered the day of birth. Experimental rats were exposed to either 2 hours (Anesthesia 2h group, N=10) or 6 hours (Anesthesia 6h group, N=11) of anesthesia; sham-controls were exposed to 6 hours of sham anesthesia (N=11 sham-controls in Anesthesia 2h group, N=12 sham-controls in Anesthesia 6h group). An approximately equal number of male and female rats were used (mean weight: 16.03 g, Envigo, Indianapolis, IN). Pups from 9 different litters were employed to control for litter variability. Animals were allocated to experimental and sham-control groups the morning of anesthesia using the random Excel function. Experimental animals received intraperitoneal midazolam (9 mg/kg, Sigma-Aldrich Chemical, St. Louis, MO) freshly dissolved in dimethyl sulfoxide at a final concentration of 0.1% and were placed in a clear plexiglass anesthesia chamber kept inside a temperature chamber set at 37 ± 1 °C (Hotpack, Warminster, PA). Nitrous oxide (70%), isoflurane (0.75%) and oxygen (30%) were delivered into the anesthesia chamber by a calibrated flowmeter. Isoflurane was administered using an isoflurane-specific calibrated vaporizer. Animals were continuously observed during anesthesia, and the inspired gas concentrations monitored with an inline Datex Capnomac Ultima gas analyzer (Datex Ohmeda, Madison, WI). Sham-control animals received an equal volume of vehicle (0.1% dimethyl sulfoxide) intraperitoneally, and were kept in a separate chamber at room air. Temperature inside the chamber was maintained at 37 ± 1 °C via a heating pad. At the conclusion of anesthesia, pups were recovered until return of spontaneous movements and reunited with their mothers. In general, the animals tolerated the anesthetic exposure well.

Of note, midazolam 9 mg/kg does not cause loss of righting reflex (the behavioral endpoint of sedation) in rodents23,24 and the minimum alveolar concentration (MAC) that prevents movement to pain in 50% of subjects (MAC EC50, the behavioral endpoint of general anesthesia) is 2.3% for isoflurane25 and 175% for nitrous oxide26,27 in rats. In line with this, and as shown in Table 1, rats in the Anesthesia 2h group remained sedated through the 2 hour exposure (righting reflex scores of 1 and toe pinch scores above 1). Anesthesia 6h rats were sedated during the first 4 hours of exposure and achieved a surgical plane of anesthesia during the last 2 hours (scores of 1 for both righting reflex and toe pinch).

Table 1.

Monitoring of anesthesia depth.

Time point (h) Toe pinch score (1–5) Righting reflex score (1–5)
0 5 (5–5) 5 (5–5)
1 4 (4–3) 1 (1–1)
2 3 (3–2) 1 (1–1)
3 2 (2–2) 1 (1–1)
4 2 (2–1) 1 (1–1)
5 1 (1–1) 1 (1–1)
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A priori power calculations were based on two-tailed T-test of mean difference using preliminary data of total time spent in REM sleep in a 4 hour period in sham-control and anesthesia-exposed rats. We determined that a group size of n=22 (11 rats/group) would provide 80% power to detect a 30% change in REM sleep with the expected mean (SD) at 9 (3%). All studies were approved by the Institutional Animal Care and Use Committee at the University of Virginia (Charlottesville, VA) and conducted in accordance with the National Institutes of Health guidelines. Figure 1 is a schematic representation of the experimental design.

Figure 1. Schematic representation of the experimental design.

Figure 1.

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Median age of electroencephalographic (EEG) implant and EEG recording is indicated for sham-controls (o symbol) and anesthesia-exposed (■ symbol) rats for both Anesthesia 2h and Anesthesia 6h groups. (Anesthesia 2h group: Sham-controls: median EEG implant: postnatal day 18 (P18), median EEG recording: P29; Anesthesia-exposed: median EEG implant: P19, median EEG recording: P30. Anesthesia 6h group: Sham-controls: median EEG implant: P21, median EEG recording: P28.5; Anesthesia-exposed: median EEG implant: 21, median EEG recording: P29).

Assessment of anesthesia depth and physiologic monitoring:

The level of anesthesia depth was assessed by toe pinch and righting reflex scores at baseline (0 hour) and at 1, 2, 3, 4, 5 and 6 hours of anesthetic exposure (Table 1). A toe pinch test was performed first, and the response was scored on a scale from 1 to 5 as follows: 1=no spontaneous activity, no response to toe pinch; 2=no spontaneous activity, single limb withdrawal to pinch; 3=no spontaneous activity, purposeful movement to toe pinch; 4=moving spontaneously but slowly and irregularly; and 5=fully awake, moving regularly and spontaneously. The righting reflex was then evaluated by placing the infant rat in the supine position and observing whether and how it was able to turn prone. The righting reflex was scored on a scale from 1 to 5 as follows: 1=complete loss of righting reflex, no response to being placed in supine position; 2=uncoordinated movements and unable to return to prone position in 30 seconds; 3=uncoordinated movements initially but able to return to prone position in 30 seconds; 4=coordinated movements, returns to prone position in less than 10 seconds; and 5=awake, returns to prone position immediately. All depth of anesthesia assessments were performed by the same investigator. In addition, heart rate (HR), oxygen peripheral saturation (SpO2) and respiratory rate (RR) were monitored at baseline (0 hour) and at 1, 2, 3, 4, 5 and 6 hours of anesthetic exposure (see Figure 2). HR and SpO2 were measured using a PhysioSuite monitor (Kent Scientific, Connecticut). RR was determined via observation of abdominal movements for 30 seconds at each of the indicated time points. All RR measurements were performed by the same investigator.

Figure 2. Physiologic monitoring during anesthetic exposure.

Figure 2.

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Surgery:

Rats were surgically implanted with four electroencephalographic (EEG) and one electromyographic (EMG) electrodes for recording of sleep-wake states between postnatal days 18–24 (see Figure 1). Briefly, anesthesia was induced with 2.5–3% isoflurane and maintained with 1.5–2% isoflurane and 35% oxygen. Rats were placed into a stereotactic frame (Stoelting, Kiel, WI) and kept at a temperature of 37 ± 1 °C via a heating pad. Blunt ear bars were used to stabilize the animals’ head. The skin over the scalp was shaved and scrubbed with alcohol and povidone-iodine solution. A midline incision was performed to expose the skull and permit the drilling of four Burr holes (approximately 0.6 mm in diameter) to accommodate four epidural electrodes (left and right frontal cortex, and left and right parietal cortex, respectively), with an additional hole drilled over the right cerebellum (to accommodate a sinus ground electrode). Electrodes consisted of stainless steel screws affixed with stainless steel Teflon-insulated wire. In addition, one insulated stainless steel wire was sutured to the dorsal neck muscles to record nuchal EMG. All wires were capped with stainless steel, gold-plated socket contacts and inserted into a 6-channel, light plastic pedestal secured to the skull with dental acrylic. All materials were supplied by Plastics One (Roanoke, VA). Postoperative analgesia was provided with ketoprofene. We are not aware of any documented effects of ketoprofene on sleep.

EEG recording:

EEG recordings were obtained between postnatal days 26–34. Median interval time between EEG implant and EEG recording was 9 days for Anesthesia 2h group and 8 days for Anesthesia 6h group (see Figure 1). Each rat was subjected to several 24 hour EEG/EMG recordings within this time interval. One 24 hour recording was randomly chosen per each animal for data analysis (analysis range postnatal day 26–32). The decision to start recording at postnatal day 26 was based on the consideration that rats attain adult levels of REM sleep at about the end of their fourth postnatal week28. This age is estimated to correspond to adolescence in rats29. Rats were placed in an individual cage and their head stage tethered to a commutator via lightweight flexible cables. They were habituated to the EEG cables and cages for at least 24 hours prior to recording. Rats were free to navigate throughout the cage and were provided food and water ad libitum. Animals were maintained in a dedicated EEG room on a 12 hour light: 12 hour dark cycle. EEG and EMG waveforms were amplified and sampled at 200 Hertz (Hz) using a 16-channel extracellular differential amplifier (model 3500, A-M systems, Sequim, WA). High- and low- pass filters were set at 0.1 Hz and 100 Hz for EEG, and 1.0 Hz and 500 Hz for EMG, respectively.

Data were processed through a MatLab-based application (Mathworks, Torrance, CA) and LabChart software (Biopac Systems Inc., The Netherlands), and analyzed with SleepSign (Kissei, Japan) and MatLab-based software. States of REM sleep, non-rapid eye movement (NREM) sleep and wakefulness were first automatically scored by the software, then manually verified by an experienced scorer, and randomly sampled for scoring accuracy by a board certified sleep medicine physician (E.M.D.) on the basis of the predominant state within each 4 second epoch. Both scorers were blinded to experimental condition. There was a 95% overall agreement between double-scored recordings of sleep-wake states. Wakefulness was scored when low-amplitude, high-frequency, desynchronized EEG activity was combined with elevated muscular tone20,30–32. NREM sleep was scored when high-amplitude, low-frequency EEG activity was coupled with reduced motor tone compared to wakefulness20,30–32. REM sleep was scored when low-amplitude, high-frequency, synchronized EEG activity was coupled with minimal to absent muscular tone20,30–32. Sleep structure was evaluated by analyzing the number of bouts of each state per hour, their average duration, REM sleep and NREM sleep latency, and slow wave sleep. A bout was counted as one continuous episode in a state. Average bout duration was calculated as the total time spent in a state divided by the total number of bouts in that state. REM sleep and NREM sleep latency were measured as the time elapsed from light onset until the first bout of REM sleep and NREM sleep, respectively. EEG waveforms were fast Fourier transformed in 4 second epochs. Slow wave sleep or delta power, defined as the EEG power between 0.5 and 4 Hz during NREM sleep, was averaged over the 24 hour recording period and normalized by the total power for each animal.

Plasma corticotropin measurement:

We measured plasma adrenocorticotropic hormone (ACTH) in anesthesia-exposed and sham-control rats subjected to EEG implant and recording. Since blood collection was a terminal procedure (open thoracotomy and left ventricle puncture), blood was collected at the end of the last recording (postnatal days 33–35). Blood collection was performed between 10:00 and 12:00 for all animals. We used an enzyme-linked immunosorbent assay ACTH kit per manufacturer instructions (M046006, MD Bioproducts, St. Paul, MN). Briefly, whole blood was placed in EDTA tubes after collection. Plasma was immediately separated by centrifugation at 1000 g for 20 minutes at 4 °C and stored at - 80 °C for further analysis. Plasma samples were diluted 1:10. Standards and plasma samples from sham-control and anesthesia-exposed rats were simultaneously incubated with the enzyme-linked antibody and a biotin-coupled antibody in a streptavidin-coated microplate well. At the end of incubation, the microwells were washed to remove unbound components and the enzyme bound to the solid phase was incubated with the substrate tetramethylbenzidine. An acidic stopping solution was added to halt the reaction and convert the color to yellow. The intensity of the yellow color was directly proportional to the concentration of ACTH in the samples. Absorbance was read at 450 nanometers using an enzyme-linked immunosorbent assay microplate reader (model 680, Biorad, CA). A standard curve was plotted using absorbance unit versus concentration of standards and the samples’ absorbance values were interpolated on the standard curve to determine ACTH concentrations in the samples (ng/ml).

Statistical Analysis:

The percent of time that animals spent asleep (NREM+REM sleep) and awake was plotted for each of four time blocks [first 6 hours and second 6 hours of the light cycle, and first 6 hours and second 6 hours of the dark cycle] and analyzed with a two-way repeated measures ANOVA of sleep-wake state as a function of Treatment and Time. HR, SpO2 and RR were also analyzed with two-way repeated measures ANOVA. No post hoc testing was conducted. The total amount of time spent in REM sleep, NREM sleep and wakefulness was calculated over the 24 hour recording period and compared between anesthesia-treated and sham-control animals with a two-tailed unpaired T-Student test. Number of bouts per hour, mean bout duration, REM sleep and NREM sleep latency and normalized delta power were also compared between anesthesia-exposed and sham-control animals with a two-tailed unpaired T-Student test. ACTH plasma concentrations were compared between sham-control and anesthesia-treated animals using a two-tailed unpaired T-Student test. Statistical analyses were performed using the software package GraphPad Prism 7.0 (La Jolla, CA). The data were confirmed to be consistent with a Gaussian distribution. There were no missing data in the Anesthesia 2h group of rats. In the Anesthesia 6h group, one rat developed forelimb weakness two days after surgery. Data from this rat were not included in sleep-wake state analysis. All values are shown as mean ± SD. In all cases P values of less than 0.05 were considered statistically significant.

Results:

Raw EEG data of sleep-wake states

Figure 3 shows representative EEG/EMG tracings from sham-control (panel A) and anesthesia-treated (panel C and E) animals. Panels B, D and F are raster plots of sleep-wake states as a function of time of Anesthesia 2h rats and sham-controls. The sleep-wake raster plots of Anesthesia 6h rats and their sham-controls can be found in Figure, Supplemental Digital Content 1.

Figure 3. Raw electroencephalogram (EEG) data of sleep-wake states.

Figure 3.

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Panels A, C and E show representative EEG and electromyogram tracings. For each panel the top two rows of signals represent frontal and parietal EEG, respectively, and lower row represents electromyogram. Panels B, D, F: raster plots of sleep-wake states as a function of time for wakefulness (in blue: B), non-rapid eye movement (NREM) sleep (in black: D) and rapid eye movement (REM) sleep (in red: F). On the x axis: time of the day. On the y axis: animals that recordings were obtained from. Each vertical line represents a bout. Symbol indicates “light on”. Symbol • indicates “light off”. Yellow dotted line divides the 24 hour recording period in two 12 hour intervals (at the time of light off). A: tracings from an awake sham-control animal. Note the low amplitude, high frequency, desynchronized EEG activity combined with elevated muscular tone. B: wakefulness raster plot of sham-control and Anesthesia 2h rats. C: transition from awake to NREM sleep in an anesthesia-exposed rat. Note the change from low amplitude, high frequency EEG with elevated muscular tone to high amplitude, low frequency EEG activity with reduced motor tone. D: NREM sleep raster plot of sham-control and Anesthesia 2h rats. E: transition from NREM to REM sleep in an anesthesia-treated rat. Note the change from high amplitude, low frequency EEG activity of NREM sleep to low amplitude, high frequency, synchronized EEG activity coupled with minimal to absent muscle tone of REM sleep. F: REM sleep raster plots of sham-control and Anesthesia 2h animals. N=11 sham-control and 10 Anesthesia 2h rats.

Sleep-wake circadian rhythm is not affected by neonatal anesthesia

In order to assess for a potential influence of early life anesthesia on the brain’s circadian pacemaker, we evaluated patterns of sleep (NREM+REM sleep) and wakefulness in relation to light/dark cues and compared them between anesthesia-treated and sham-control rats. Notably, rodents are nocturnal animals, i.e. they are asleep when light is on and are active when light is off. As shown in Figure 4A, Anesthesia 2h and sham-control animals spent the majority of time asleep when lights were on (rest phase), and progressively less time asleep after lights were turned off (active phase). Conversely, they spent less time awake when lights were on, and progressively more time awake after lights were turned off (Figure 4B). Such sleep-wake behavior reflects proper timing of rest and activity with external light cues, and thus suggests appropriate function of the internal circadian pacemaker in both sham-control and anesthesia-treated rats. Two-way repeated measures ANOVA of sleep and wake as a function of Treatment and Time confirmed no differences between Anesthesia 2h and sham-control rats (Treatment: p=0.103; Time: p<0.0001; Interaction: p=0.987). Similarly, rats exposed to a 6 hour long anesthesia maintained intact their ability to time rest and activity with light cues. A two-way repeated measures ANOVA of sleep and wake as a function of Treatment and Time showed no differences between Anesthesia 6h and sham-control rats (Treatment: p=0.788; Time: p<0.0001; Interaction: p=0.681; see Figure, Supplemental Digital Content 2, representing patterns of sleep (NREM+REM sleep) and wakefulness in Anesthesia 6h and sham-control rats).

Figure 4. Effect of neonatal anesthesia on circadian rhythm.

Figure 4.

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Symbols represent mean of hourly values (n=6 hours) expressed as percent of recording time. Quiet (light) and active (dark) phase are delimited by a white and a black box, respectively, below the abscissa. A: the percentage of time spent in sleep (non-rapid eye movement + rapid eye movement sleep) was appropriately high during the quiet phase and progressively lower during the active phase. A two-way repeated measures ANOVA of sleep as a function of Treatment and Time confirmed no effect of anesthesia on sleep (Treatment: p=0.103; Time: p<0.0001; Interaction: p=0.987). B: the percent of time spent awake was appropriately low during the quiet phase and progressively higher during the active phase. N=11 sham-control and 10 Anesthesia 2h rats.

Early life anesthesia does not impact the quantity or structure of wakefulness

The quantity and structure of wakefulness exhibited no significant changes in anesthesia-exposed adolescent rats compared to sham-controls following anesthesia administered at a young age. Figure 5A shows that the total amount of time spent in wakefulness during the 24 hour recording period was not significantly different in Anesthesia 2h rats compared to sham-controls. Anesthesia 2h rats were awake on average for 651.9 ± 25.0 minutes versus 728.6 ± 29.9 in sham-controls (p=0.067). To assess for a potential detrimental effect of neonatal anesthesia on wakefulness structure, we analyzed the number and mean duration of wake bouts per hour. Figure 5B shows that the average number of wake bouts was not different in Anesthesia 2h rats compared to sham-controls (33 ± 2 in Anesthesia 2h rats and 34 ± 2 in sham-control rats, p=0.776). Figure 5C shows that the mean duration of wake bouts was also unaffected by anesthetics. In fact, it was 90.7 ± 20.9 seconds in Anesthesia 2h rats versus 102.9 ± 18.8 in sham-control animals (p=0.669). In line with the findings in the Anesthesia 2h group, there were no differences in quantity and structure of wakefulness between Anesthesia 6h and sham-control rats (see Figure, Supplemental Digital Content 3, representing total amount of time spent in wakefulness, number and mean duration of wake bouts in Anesthesia 6h rats compared to sham-controls).

Figure 5. Effect of neonatal anesthesia on wakefulness.

Figure 5.

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A: neonatal anesthesia did not affect the total amount of time spent awake in Anesthesia 2h animals compared to sham-controls (p= 0.067). B: the average number of wake bouts per hour was unchanged in Anesthesia 2h rats compared to sham-controls (p=0.776). C: the mean duration of wake bouts per hour was also unchanged in Anesthesia 2h rats compared to sham-controls (p=0.669). N=11 sham-control and 10 Anesthesia 2h rats.

Prolonged -but not brief- neonatal anesthesia causes increased propensity to enter deep sleep in adolescent rats

The quantity of NREM sleep of adolescent rats did not exhibit significant changes following early life anesthesia. Figure 6A shows that the total amount of time spent in NREM sleep during the 24 hour recording period was not different in Anesthesia 2h rats compared to sham-controls. Anesthesia 2h rats spent on average 614.1 ± 21.8 minutes in NREM sleep, compared to 602.8 ± 28.5 in sham-controls (p=0.760). To assess for a potential detrimental effect of neonatal anesthesia on NREM sleep structure, we analyzed the number of NREM sleep bouts per hour and their mean duration, as well as latency to enter NREM sleep and slow wave sleep. Figure 6B shows that the number of NREM sleep bouts was not different between Anesthesia 2h and sham-control rats (41 ± 3 in Anesthesia 2h rats and 36 ± 2 in sham-controls (p=0.205)). Figure 6C shows that the mean duration of NREM sleep bouts was also unaffected by anesthetics. In fact, average duration of NREM bouts was 40.5 ± 2.6 seconds in Anesthesia 2h rats and 42.8 ± 1.4 in sham-controls (p=0.428). When latency to enter NREM sleep was analyzed (Figure 6D), no difference was found in the time from light onset to first bout of NREM sleep in Anesthesia 2h rats compared to sham-controls (3.3 ± 0.8 minutes in Anesthesia 2h rats versus 5.9 ± 2.1 in sham-controls, p= 0.279). Last, to determine the propensity to enter deep sleep we performed a Fourier transformation of the NREM sleep electroencephalogram and computed the power in the delta frequency range (between 0.5 and 4 Hz). As shown in Figure 6E, we found no difference in delta power in Anesthesia 2h rats compared to sham controls (0.6205 ± 0.0212 in Anesthesia 2h rats versus 0.6287 ± 0.0187 in sham-controls, p=0.775). A significant increase in delta power was found instead in Anesthesia 6h rats compared to sham controls (0.6781 ± 0.0289 versus 0.6053 ± 0.0184; p=0.042; see Figure, Supplemental Digital Content 4, displaying total amount of time spent in NREM sleep, number and mean duration of NREM sleep bouts, latency to NREM sleep and delta power in Anesthesia 6h rats compared to sham-controls).

Figure 6. Effect of early life anesthesia on non-rapid eye movement (NREM) sleep.

Figure 6.

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A: early life anesthesia did not affect the total amount of time spent in NREM sleep in Anesthesia 2h animals compared to sham-controls (p=0.760). B: the number of NREM bouts per hour was unchanged in Anesthesia 2h rats compared to sham-controls (p=0.205). C: the mean duration of NREM bouts per hour was also unchanged in Anesthesia 2h rats compared to control condition (p=0.428). D: latency to enter NREM sleep was not significantly changed in Anesthesia 2h rats compared to sham-controls (p=0.279). E: propensity to enter deep NREM sleep (delta power) was not different in Anesthesia 2h rats compared to sham-controls (p=0.775). N=11 sham-control and 10 Anesthesia 2h rats.

Adolescent rats exhibit perturbation of REM sleep quantity and structure following neonatal anesthesia

The quantity and structure of REM sleep were significantly disrupted in adolescent rats after neonatal exposure to anesthetic drugs. Figure 7A shows that Anesthesia 2h rats exhibited a significant 40% increase in the total amount of time spent in REM sleep compared to sham-controls (p<0.0001). Specifically, they spent on average 65 more minutes in REM sleep than sham-control animals (174.0 ± 7.2 minutes versus 108.6 ± 5.3). To assess for a potential detrimental impact of neonatal anesthesia on REM sleep structure, we analyzed the number of REM sleep bouts per hour and their mean duration, as well as latency to enter REM sleep. We found that REM sleep structure exhibited significant abnormalities that were present several days after the initial anesthetic exposure, at an age that corresponds to adolescence in rats. When the number of REM bouts was quantified (Figure 7B), it was found to be significantly increased from 9 ± 1 in sham-controls to 17 ± 2 in Anesthesia 2h rats (p=0.001). Mean duration of REM sleep bouts was instead unaffected by the anesthetic treatment (28.9 ± 2.9 seconds in Anesthesia 2h rats versus 29.7 ± 3.5 in sham-controls, p=0.871, Figure 7C). Figure 7D displays details of the first two hours of REM sleep after light onset, taken from the REM sleep raster plots of sham-control (left) and Anesthesia 2h (right) rats. As shown in the figure, while the majority of Anesthesia 2h rats were in REM sleep within two hours from light onset, only six out of 11 sham-control animals had entered the first bout of REM sleep. When time from light onset to first REM sleep bout was quantified (Figure 7E), we found that Anesthesia 2h rats took half the time of sham-controls to enter REM sleep (24.8 ± 9.4 minutes in Anesthesia 2h rats versus 51.0 ± 6.3 in sham-controls, p=0.029). Of note, rats exposed to a 6 hour long anesthesia displayed very similar changes in quantity and structure of REM sleep (see Figure, Supplemental Digital Content 5, representing total amount of time spent in REM sleep, number and mean duration of REM sleep bouts and REM sleep latency in Anesthesia 6h rats compared to sham-controls). Collectively, these data indicate that i) the increase in total REM sleep time in anesthesia-treated rats is the result of increased consolidation of REM sleep bouts and shorter latency to first REM sleep bout and ii) even brief anesthetic exposures can evoke substantial electroencephalographic and sleep-wake behavior changes.

Figure 7. Effect of neonatal anesthesia on rapid eye movement (REM) sleep.

Figure 7.

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A: the total amount of time spent in rapid eye movement sleep across the 24 hour recording period was increased by 40% in Anesthesia 2h rats compared to sham-controls (p<0.0001). B: the number of REM bouts per hour was significantly increased in Anesthesia 2h rats compared to the control condition (p<0.001). C: neonatal anesthesia did not affect the average duration of REM bouts in Anesthesia 2h animals compared to sham controls (p=0.871). D: details of the first two hours of REM sleep after light onset, from the REM sleep raster plots of sham-control (left) and Anesthesia 2h (right) rats. Red vertical lines represent REM sleep bouts. Note that although the majority of Anesthesia 2h rats are in REM sleep, only about half of sham-control rats enter the first bout of REM sleep within two hours from light onset. E: Anesthesia 2h rats took half the time of sham-controls to enter REM sleep (p=0.029). N=11 sham-control and 10 Anesthesia 2h rats.

EEG implant surgery and recording did not affect corticotropin concentration in anesthesia-exposed and sham-control rats

Some investigations suggest that activation of the hypothalamus-pituitary-adrenal gland axis in response to biological stress can interfere with EEG sleep patterns33–36. Therefore, in order to rule out the possibility that stress may have been responsible for the sleep abnormalities we observed, we measured plasma ACTH concentrations in sham-control and Anesthesia 2h rats. Blood was collected between 10:00 and 12:00 on postnatal days 33–35 (the last day of EEG recording). As shown in Figure 8, we found no significant differences in ACTH plasma concentration in Anesthesia 2h rats compared to sham-controls (0.6 ± 0.2 ng/ml in Anesthesia 2h rats versus 0.8 ± 0.2 in sham-controls, p=0.414). Likewise, there were no differences in ACTH plasma concentrations between Anesthesia 6h and sham-control rats (see Figure, Supplemental Digital Content 6, representing ACTH plasma concentration in Anesthesia 6h rats compared to sham-controls). Therefore, we conclude that the sleep abnormalities we observed after exposure to anesthesia are likely due to the effects of the anesthetic drugs per se, rather than stress, on endogenous sleep pathways.

Figure 8. Electroencephalographic implant surgery and recording do not affect corticotropin concentration in anesthesia-exposed and sham-control rats.

Figure 8.

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Plasma concentration of adrenocorticotropic hormone (ACTH) did not differ between sham-control and Anesthesia 2h rats (p=0.414). N=7 sham-control and 7 Anesthesia 2h rats.

Discussion:

Our findings indicate that early life anesthesia causes a significant increase in REM sleep and disruption of REM sleep structure that are present in adolescent rats nearly three weeks after the initial anesthetic exposure, while leaving NREM sleep, wakefulness and circadian rhythm substantially unchanged.

Several lines of evidence support the notion that anesthetics may interfere with endogenous sleep19,20,29–31,37,38. Kelz et al20 reported that adult mice exposed to 6 hours of sevoflurane or isoflurane exhibited increased REM sleep and shortened latency to enter REM sleep in the first 18 hours after anesthesia, but no changes in the amount of NREM sleep or wakefulness. Similarly, sleep-deprived adult rats exposed to 6 hours of sevoflurane presented a significant increase in REM sleep during the first 12 hours post anesthesia30. Relatedly, selectively REM sleep-deprived adult rodents subjected to 4 hours of isoflurane displayed increased REM sleep in the 4 hours post-isoflurane31. Since a well-known manifestation of sleep deprivation is the ensuing rebound when animals are presented with the opportunity to recover sleep39, the REM sleep increase observed in these studies was interpreted as a “rebound”, i.e., a homeostatic response to a REM sleep deficit that accumulated during anesthetic exposure. Our anesthesia protocols were delivered to infant rats and caused a significant increase in REM sleep that was present during adolescence. Although it would be tempting to label such increase in REM sleep as a rebound in response to REM sleep deprivation accrued during anesthesia, a rebound is by definition limited in duration and should not be present several days after the anesthetic exposure. In other words, even if a REM sleep deficit accumulated during anesthesia at postnatal day 7, it would be expected to have dissipated three weeks after the initial exposure.

These considerations call for a different interpretation of our data. Importantly, REM sleep is present at high levels at birth in the rat (and humans), begins to decline steeply during the second week of age, and attains adult levels at about the end of the 4th week28. In light of this, a possible explanation of our findings is that the changes in REM sleep we observe may reflect a delay in the maturation of the neural circuits that regulate sleep induced by anesthetic drugs, such that the normal age at which adult REM sleep levels are attained becomes postponed, i.e., the length of the period during which REM sleep remains physiologically elevated is extended. Alternatively, anesthetic drugs administered during a phase of intense brain plasticity may permanently affect the assembly and function of the neural systems that regulate REM sleep. In other words, it is possible that early life anesthesia may cause rewiring of the REM sleep homeostat and that the REM sleep abnormalities we observe may reflect a controller that has recovered to a new set point. Given the crucial role of REM sleep for the normal development and function of synapses during brain growth and learning21,22,28, either scenario, whether a transient delay in REM sleep maturation or a permanent rewiring of REM sleep homeostatic regulation circuitry, could have profound implications for proper brain maturation and cognitive development. Clearly more studies are needed to further elucidate the mechanisms underlying the sleep changes we observe after early life anesthesia.

Our findings indicate a selective effect of anesthetics on quantity and structure of REM sleep, as NREM and wake quantity and structure were substantially unaffected by anesthetic exposure. In fact, the increase in REM sleep time we observed in our animals led to a decrease that was equally distributed between NREM sleep and wakefulness time. As a result, there were no significant differences in the total time spent in NREM sleep or wakefulness between anesthesia-exposed and sham-control rats. In this respect, our results are consistent with previous studies in which state-specific effects of anesthesia are documented19,20,30,31. This diversity of interaction between general anesthetics and sleep-wake states suggests that the brain structures that regulate REM sleep, NREM sleep and arousal may be distinct and utilize dedicated neural circuits.

The suprachiasmatic nucleus serves as the brain’s circadian clock. It drives a rhythm that alternates daily between rest and activity cycles and is synchronous with external light cues33,34. Our data suggest that early life anesthesia had no influence on the brain’s circadian pacemaker. In fact, percent of sleep was appropriately higher during the light cycle (the rest phase) and lower during the dark cycle (the active phase). Conversely, rats spent less time awake during the light cycle and were awake longer during dark hours. These patterns of sleep and wakefulness in anesthesia-treated and sham-control rats are identical to those described in the literature for wild type animals, suggesting proper timing of rest and activity with external light cues, and therefore normal function of the circadian pacemaker.

One consideration with our experimental design is that rats may have experienced increased stress, as a result of anesthesia exposure and/or implant surgery, leading to sleep alterations. To minimize this concern, a minimum of 9 days of postsurgical recovery was allowed between EEG probe placement and EEG measurement (range 9–14 days) in Anesthesia 2h rats. In addition, to rule out an effect of any stress on sleep, we collected blood from EEG-implanted animals and measured plasma concentration of ACTH. We found no differences in ACTH between sham-control and anesthesia-exposed animals, ruling out the possibility that stress may have been responsible for the sleep abnormalities we observed. Further, our physiologic monitoring data indicate that RR and HR were significantly different from baseline at the conclusion of the 6 hour exposure (RR: 0 hour vs 6 hour p=0.021; HR 0 hour vs 6 hour p=0.016). While it cannot be formally excluded that disturbances in RR and/or HR are responsible for the changes in REM sleep in the Anesthesia 6h group, the observation that anesthesia in the presence of physiological homeostasis reproduced identical REM sleep changes in the Anesthesia 2h group argues against this possibility. Rather, it supports the notion that anesthetic drugs per se, and not physiological derangements, are responsible for the sleep abnormalities we observed.

In this study we examined the effects of a brief (2 hours) exposure of infant rats to sub anesthetic doses of midazolam, nitrous oxide and isoflurane on subsequent sleep-wake behavior (Anesthesia 2h group). We also assessed the effects of a longer (6 hours) exposure to the same anesthetics (Anesthesia 6h group), in keeping with a substantial body of preclinical work that adopted the same duration of exposure to characterize anesthesia-related morpho-functional changes in the rodent central nervous system2–6. Our depth of anesthesia monitoring data indicate that Anesthesia 2h rats remained sedated throughout the exposure, while Anesthesia 6h rats were sedated for the first 4 hours and achieved a surgical plane of anesthesia during the last 2 hours of exposure. Of relevance here, nitrous oxide40,41, midazolam42,43 and halogenated gases44 (isoflurane and sevoflurane) at sub anesthetic doses are often used to provide sedation to pediatric patients for minor procedures, i.e., dental treatment, superficial wound repair, bone marrow biopsy, and diagnostic image acquisition. Building on these considerations, notwithstanding the challenges of translating time and behavior from rodents to humans, our Anesthesia 2h protocol can be taken to model the clinical scenario of procedural sedation, and the Anesthesia 6h protocol of prolonged postoperative sedation45,46. Regardless of time equivalences between rodents and humans, our data indicate that even a brief anesthetic exposure in rodents is sufficient to evoke substantial electroencephalographic and sleep-wake behavior changes.

Study Limitations:

There are limitations in our study. Since our experimental protocol consisted of a combination of anesthetics, we cannot draw conclusions on the contribution of each anesthetic agent to the sleep abnormalities we describe. However, the aim of this study was to test the novel hypothesis that administration of anesthesia during a critical period of brain plasticity may disrupt sleep, rather than to focus on the specific effects of each anesthetic agent on sleep. Further studies are needed to characterize the impact of each component of our anesthetic protocol on sleep-wake behavior. Additionally, although rodent models have significantly advanced the field of sleep research, our data regarding sleep-wake states in rodents will warrant verification in humans.

Conclusions and scientific/clinical implications:

We demonstrate that a brief exposure to a sub anesthetic combination of midazolam, nitrous oxide and isoflurane during a critical period of brain plasticity results in REM sleep increase and perturbation of REM sleep architecture that are present in adolescent rats several days after the anesthetic encounter. These results are significant for many reasons. First, given the association between REM sleep, synaptic plasticity and memory consolidation, REM sleep disruption could represent an unexplored mechanism contributing to the learning disabilities observed after neonatal anesthesia. Second, it may represent a novel marker of anesthetic effects on the developing brain and should be evaluated as relevant outcome in future studies of anesthetic-induced neurotoxicity. Third, the study of the electroencephalographic changes during and following anesthesia may provide insight into the mechanisms by which anesthetics modulate neuronal function.

Supplementary Material

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Acknowledgments:

Eric M. Davis, M.D., Assistant Professor, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Virginia Health System, Charlottesville, VA, USA. The authors wish to thank Eric Davis for his assistance with accuracy validation of electroencephalographic scoring in rats. Mark E. Smolkin, M.S., Senior Biostatistician, Department of Public Health Sciences, University of Virginia, Charlottesville, VA, USA. The authors wish to thank Mark Smolkin for his assistance with statistical analysis of sleep-wake data. Douglas A. Bayliss, Ph.D., Professor, Department of Pharmacology, University of Virginia, Charlottesville, VA, USA. The authors wish to thank Douglas Bayliss for his critical review of the manuscript. Bianca Ferrarese, M.D., Post-Doctoral fellow, Department of Anesthesiology, University of Virginia, Charlottesville, VA and Department of Anesthesiology, University of Padova, Padova, Italy. The Authors wish to thank Bianca Ferrarese for her help with assessment of anesthesia depth and physiologic monitoring during anesthesia.

Funding Statement:

This work was supported by funds through two grants (NIGMS-K08GM123321–01 to N.L. and R01 HD089999 to Z.Z.) from the National Institutes of Health, Bethesda, Maryland, a grant (GF13062 to N.L.) from the International Anesthesia Research Society, San Francisco, CA and research funds from the Department of Anesthesiology and Critical Care, University of Virginia, Charlottesville, VA, USA.

Footnotes

Prior Presentations:

AUA 2018 Annual Meeting, April 26–27, Chicago, IL

IARS 2018 Annual Meeting, April 28-May 1, Chicago, IL

Conflicts of Interest:

The Authors declare no competing interest.

Contributor Information

Nadia Lunardi, University of Virginia Health System, Department of Anesthesiology, Charlottesville, VA, USA

Ryan Sica, University of Virginia, School of Medicine, Charlottesville, VA, USA.

Navya Atluri, University of Virginia Health System, Department of Anesthesiology, Charlottesville, VA, USA

Kathryn A. Salvati, Department of Pharmacology and Neuroscience Graduate Program, University of Virginia, Charlottesville, VA, USA.

Caroline Keller, University of Virginia, Charlottesville, VA, USA.

Mark Beenhakker, University of Virginia, Department of Pharmacology, Charlottesville, VA, USA

Howard P. Goodkin, University of Virginia Health System, Department of Neurology, Charlottesville, VA, USA

Zhiyi Zuo, University of Virginia Health System, Department of Anesthesiology, Charlottesville, VA, USA

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